THE BETHESDA
HAND BOOK

OF CLINICAL
HEMATOLOGY

THIRD EDITION

EDITORS

GRIFFIN P. RODGERS, MD, MACP

Chief, National Institute of Diabetes and Digestive and Kidney Diseases Chief, Molecular and Clinical Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

NEAL S. YOUNG, MD, MACP

Chief, Hematology Branch, National Heart, Lung, Blood Institute and National Institutes of Health Bethesda, Maryland

Senior Executive Editor: Jonathan W. Pine, Jr.

Product Managers: Grace Caputo, Emilie Moyer

Manufacturing Manager: Benjamin Rivera

Marketing Manager: Alexander Burns

Design Coordinator: Holly McLaughlin

Production Service: Integra Software Services Pvt. Ltd.

© 2013 by Lippincott Williams & Wilkins, A Wolters Kluwer business

© 2010 by LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business

© 2005, First Edition, Lippincott Williams & Wilkins

2001 Market Street, Philadelphia, PA 19103 USA

LWW.com

All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form by any means, including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above- mentioned copyright.

Printed in China

Library of Congress Cataloging-in-Publication Data

The Bethesda handbook of clinical hematology / editors, Griffin P. Rodgers, MD, MACP, director, National Institute of Diabetes, Digestive, and Kidney Disorders, chief, Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health Bethesda, Maryland, Neal S. Young, MD, MACP, chief, Hematology Branch National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland. — Third edition.

pages cm

Includes bibliographical references and index.

ISBN 978-1-4511-8270-5

1. Blood—Diseases—Handbooks, manuals, etc. 2. Hematology—Handbooks, manuals, etc. I. Rodgers, Griffin P., editor of compilation. II. Young, Neal S., editor of compilation. III. Title: Handbook of clinical hematology.

RC633.B49 2013

616.1’5—dc23

2012049314

Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of the information in a particular situation remains the professional responsibility of the practitioner.

The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug.

Some drugs and medical devices presented in the publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in their clinical practice.

To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. International customers should call (301) 223-2300.

Visit Lippincott Williams & Wilkins on the Internet: at LWW.com. Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6 pm, EST.

1 0 9 8 7 6 5 4 3 2 1

For our children, with love: Chris and Gregory Rodgers Andrea, Max, and Giorgio Young

PREFACE

Life is short, the art long

—Hippocrates c. 460–357 BC

The accessibility of blood and bone marrow has made hematology historically the engine of basic research in internal medicine. Hematology has thrived at the National Institutes of Health because of this close relationship with the research laboratory, and investigators from the various institutes in Bethesda have contributed to the knowledge of blood diseases from the study of individual patients with sometimes rare diseases and to the development of clinical protocols for the rigorous assessment of diagnostic criteria or treatments, both established and novel. Our hematology fellowship programs have fostered a scientific approach to hematology, not only to assess outcomes but also to advance the experimental basis of our understanding of blood diseases and the application of laboratory insights to their treatment in practice. The collegial relationships among local institutions and individuals in the greater Washington area who share training and patients have greatly furthered these efforts.

HANDBOOKS are intended to be highly accessible, both literally and figuratively, so as to be useful. Our HANDBOOK should be carried in the white coat pocket of the student, resident, and fellow on a hematology or oncology service and in the briefcase of the internist, hospitalist, family practitioner, and pediatrician whose practice includes patients with blood diseases. We have purposely combined authors who are recognized experts in their fields with senior fellows who have had current experience of learning hematology and daily care of hematology patients, and encouraged a thoughtful approach to the presentation of the core knowledge, using tables, algorithms, meaningful figures, and bulleted text structures[CE3]. The HANDBOOK is organized according to disease categories and hematological problems of importance to the consulting and treating hematologist, and additional chapters are provided to acquaint the reader with familiar and new laboratory methodologies that underlie modern clinical approaches to diagnosis and treatment.

For the opportunity to publish a third edition of the BHOCH, we thank our readers, who have supported our efforts with both kind comments and constructive criticisms and, very importantly, concrete purchases of the book, more remarkable in an age of Internet-based medical information. Indeed, the plan is to have this BHOCH available as a handheld application. The HANDBOOK remains focused on providing the practitioner at every level of training practical, authoritative, and current guidance to the diagnosis and treatment of blood diseases and to consultative problems in hematology. Many aspects of hematology, as the field advances, have entered the domain of internal medicine but remain complex and challenging—from new anticoagulants to the mundane management of once fatal diseases like chronic myeloid leukemia and aplastic anemia.

All chapters have been revised and updated. We look forward to your responses to our authors’ efforts.

Griffin P. Rodgers, MD, MACP

Neal S. Young, MD, MACP

Disclaimer: Dr. Rodgers’ and Dr. Young’s work as editors and authors was performed outside the scope of their employment as U.S. government employees. Their work represents their personal and professional views and not necessarily those of the U.S. government.

CONTRIBUTORS

Jame Abraham, MD Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia

Georg Aue, MD Staff Clinician, Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

A. John Barrett, MD Chief, Allogeneic Stem Cell Transplantation Section, Hematology Branch, National Heart, Lung and Blood Institute National Institutes of Health, Bethesda, Maryland

Minoo Battiwalla, MD, MS Staff Clinician, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Charles D. Bolan, Jr. Professor, Department of Medicine, Uniformed Services University of the Health Sciences; Program Director, Hematology Fellowship, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health—Clinical Center, Bethesda, Maryland

Richard W. Childs, MD Chief, Section of Transplantation Immunotherapy, Hematology Branch, National Heart, Lung and Blood Institute National Institutes of Health—Clinical Center, Bethesda, Maryland

Michael Craig, MD Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia

Ronan Desmond, MD Staff Clinician, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Bogdan Dumitriu, MD Clinical Fellow, Hematology National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Cynthia E. Dunbar, MD Senior Investigator, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Mohammed Z.H. Farooqui, D.O Hematologist, Hematology Branch, Hatfield Clinical Research Center, National Heart, Lung, and Blood Institute, National institutes of Health, Bethesda, Maryland

Thomas A. Fleisher, MD Chief, Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland

Patrick F. Fogarty, MD Director, Penn Comprehensive Hemostasis and Thrombosis Program, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania

Peiman Hematti, MD Associate Professor of Medicine, Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health, University of Wisconsin Carbone Cancer Center, Madison, Wisconsin

Matthew M. Hsieh, MD Staff Clinician, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Elizabeth A. Jaben, MD Assistant Professor, Laboratory Medicine and Pathology, Mayo Clinic; Medical Director, Transfusion Medicine, Laboratory Medicine and Pathology, Mayo Clinic Hospital, Phoenix, Arizona

Elaine S. Jaffe, MD National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Abraham S. Kanate, MD Fellow, Blood and Marrow Transplant Program, Stanford University Hospital, Stanford, California

Harvey G. Klein, MD Senior Investigator and Chief, Transfusion Medicine, National Institutes of Health, Bethesda, Maryland

Jeffrey K. Klotz, MD Chief, Hematology/ Oncology, Madigan Army Medical Center, Tacoma, Washington

C. Ola Landgren, MD Senior Investigator, Chief Multiple Myeloma Section, National Cancer Institute, Bethesda, Maryland

Susan F. Leitman, MD Chief, Blood Services Section, Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland

Richard F. Little, MD, MPH, Senior Investigator, Clinical Investigations Branch, National Cancer Institute, Bethesda, Maryland

Johnson M. Liu, MD, FACP Professor, Department of Pediatrics, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York; Attending Physician, Department of Pediatrics, Steven & Alexandra Cohen Children’s Medical Center of NY, New Hyde Park, New York

Jay N. Lozier, MD, PhD, FACP Senior Physician, Department of Laboratory Medicine, NIH Clinical Center, Bethesda, Maryland

Jaroslaw P. Maciejewski, MD, PhD, FACP Staff Physician, Cleveland Clinic Department of Hematologic Oncology and Blood Disorders, Department Chair, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Professor of Medicine, Cleveland Clinic

Lerner College of Medicine, Case Western Reserve University, Cleveland, Ohio

Harry L. Malech, MD Chief, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health; Senior Investigator, Attending Physician, National Institutes of Health Clinical Center, Bethesda, Maryland

Vera Malkovska, MD, MRCP, FRCPath Associate Research Professor of Pediatrics, George Washington University, Washington, DC; Director of Hematology, Washington Cancer Institute, Medstar Washington Hospital Center, Washington, DC

Elisabet E. Manasanch, MD Clinical Fellow, Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Jeffery L. Miller, MD Tenured Investigator, Chief, Section on Molecular Genomics and Therapeutics, Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Raul C. Braylan, MD Chief, Hematology Laboratory, Department of Laboratory Medicine Clinical Center, National Institutes of Health, Bethesda, Maryland

Pierre Noel, MD Professor of Medicine, Hematology-Oncology, Mayo College of Medicine, Scottsdale, Arizona

Matthew J. Olnes, MD, PhD, FACP Special Volunteer, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; Service Chief, Hematology and Medical Oncology, Alaska Native Tribal Health Consortium, Anchorage, Alaska

Patricia A. Oneal, MD Assistant Professor, Medicine, Division of Hematology and Oncology, Howard University; Washington, DC

Ankur R. Parikh, D.O Special Volunteer, Hematology Branch, National Institutes of Health, Bethesda, Maryland; Medical Oncologist/Hematologist, Medical Oncology, Cancer Treatment Centers of America, Philadelphia, Pennsylvania

Griffin P. Rodgers, MD, MACP Director, National Institute of Diabetes, Digestive, and Kidney Diseases; Chief, Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Institute National Institutes of Health, Bethesda, Maryland

Bartlomiej Przychodzen, BSc Translational Hematology and Oncology Research, Cleveland Clinic, Taussig Cancer Institute, Cleveland, Ohio

Roger J. Kurlander, MD Staff Clinician, Hematology Section, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland

Geraldine P. Schechter, MD Professor, Department of Medicine, George Washington University; Hematologist, Hematology Section, Medical Service, Washington Veterans Affairs Medical Center, Washington, DC

Phillip Scheinberg, MD Special Volunteer, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; Chief, Hematology Service, Oncology Center, Hospital  São José, Beneficência Portuguesa, São Paulo, Brazil

Nirali N. Shah, MD Research Fellow, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Ramaprasad Srinivasan, MD, PhD Urologic Oncology Branch, National Cancer Institute, Bethesda, Maryland

Louis M. Staudt, MD, PhD Deputy Chief, Metabolism Branch, National Cancer Institute-Center for Cancer Research, National Institutes of Health, Bethesda, Maryland

Nishant Tageja, MD Clinical Fellow, Medical Oncology Branch, National Cancer Institute, Bethesda, Maryland

John F. Tisdale, MD Senior Investigator, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Danielle M. Townsley, MD, MSc Staff Clinician, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Alan S. Wayne, MD Clinical Director, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Adrian Wiestner, MD, PhD Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Wyndham H. Wilson, MD, PhD Chief, Lymphoid Therapeutics Section, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland

Fang Yin, MD Clinical Fellow, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Neal S. Young, MD, MA Chief, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

Agnes S. M. Yong, MBBCh, MRCP(UK), FRCPath, FRCPA, PhD Clinical Senior Lecturer, School of Medicine, Faculty of Health Sciences,University of Adelaide, Adelaide, Australia; Senior Consultant Hematologist, Division of Hematology, IMVS, SA Pathology, Royal Adelaide Hospital, Adelaide, Australia

1

Iron Deficiency

Bogdan Dumitriu, Jeffrey Miller and Griffin P. Rodgers

Iron deficiency is the most common cause of anemia throughout the world, with over 1 billion people being affected.¹ In the United States 10% of women of childbearing age and young children are iron deficient.² Functional iron deficiency is more common in elderly patients, who have a high frequency of anemia resulting from inadequate utilization of iron stores.³ Clinical presentation includes fatigue, weakness, headaches, pallor, stomatitis, and glossitis. Presenting symptoms may also include appetite for nonnutritional or unusual food items (pica), restless leg syndrome,⁴ or beeturia.⁵ While described in cases of severe iron deficiency, Plummer-Wilson syndrome (dysphagia, esophageal webs, atrophic glossitis with iron deficient anemia), koilonychias (spoon nails), chlorosis (greenish color of skin), or blue sclerae are very rarely found at presentation in modern industrialized countries.

ABSOLUTE VERSUS FUNCTIONAL IRON DEFICIENCY

Iron deficiency anemia is caused by:

  Decrease in total body iron (“absolute” iron deficiency)

  Inadequate utilization of iron stores (“functional” iron deficiency)

IRON METABOLISM

Approximately half of the 3 to 4 g of total body iron is contained in the hemoglobin of circulating red cells (Fig. 1.1). Nonerythroid iron is contained in reticuloendothelial system (RES), myoglobin, and the liver. Intracellular iron is stored in ferritin, and the circulating level of ferritin normally correlates closely with the intracellular iron stores.⁶

The average daily requirement of iron to support erythropoiesis is 20 mg. Most of the daily iron requirement is supplied by recovery of erythroid iron through phagocytosis of senescent erythrocytes by RES. Daily, 1 to 2 mg of iron is obtained from dietary intake to compensate for losses in sweat, urine, and feces.⁷ For women during the childbearing years, there are additional losses due to menstruation (average of 0.3–0.5 mg iron/day).⁸ To balance these losses:

  Adult males must absorb about 1 mg of iron each day from their diet, and menstruating females require about twice this much.

  During pregnancy and periods of rapid growth, iron balance must be positive in order to support increased production of hemoglobin and myoglobin.

  Negative iron balance results from increased loss of iron (nearly always due to bleeding), inadequate dietary intake, and increased utilization of iron (Table 1.1).

FIGURE 1.1 The iron cycle. RES, reticuloendothelial system.

Dietary iron is present in ferric (Fe⁺³) salts in meat and vegetables and in heme in meat.⁷ Heme iron is the most bioavailable because it is soluble at the alkaline pH of the duodenum, where it is absorbed as an intact iron–porphyrin complex. In contrast, ferric iron is not soluble at alkaline pH and is not absorbable by the duodenal mucosa. To be absorbed, it must be solubilized in the acidic stomach where it is loosely complexed with small molecules such as amino acids. Ferric reductase in the duodenal mucosa reduces the iron to its divalent state, which can be transported into enterocytes.⁹ The enhancing effect of ascorbate on iron absorption results from increased solubilization of ferric iron, as well as increased ferric reductase activity.¹⁰ In contrast, absorption of ferric iron is impaired by achlorhydria and by foods containing chelators of iron, such as tannins and phytates, which are prevalent in tea and cereals. Although medicinal iron is in ferrous state, thus presumably unaffected by these factors, it is still recommended to have a 2-hour delay in taking anything that might impair absorption.

Ferrous iron is released from duodenal enterocytes via the exporter ferroportin, which is regulated by the hormone hepcidin, and becomes oxidized to ferric iron before binding to transferrin.⁹ Hepcidin binding to ferroportin induces internalization and degradation of ferroportin, thus decreasing cellular iron export.¹¹ The same export mechanism exists in macrophages and hepatocytes.¹²

Once released in circulation, iron binds transferrin.⁹ Each transferrin molecule can bind one or two iron atoms. Diferric transferrin is taken up by developing red cells more easily than monoferric transferrin and delivers twice as much iron per molecule.¹³ Therefore, the concentration of diferric transferrin is critical to the support of erythropoiesis. Steady state erythropoiesis requires a serum concentration of diferric transferrin that is achieved when the transferrin saturation is at least ~16%.¹⁴

ABSOLUTE IRON DEFICIENCY

A negative iron balance depletes body iron stores before iron deficient erythropoiesis occurs.¹⁵ Multiple laboratory parameters associated with iron depletion state precede anemia (Table 1.2).

Table 1.1  Causes of Absolute Iron Deficiency

Increased Loss of Iron

Bleeding

Menorrhagia

Gastrointestinal

Surgery

Trauma

Childbirth

Excessive phlebotomy

Blood donations

Factitious

Hemodialysis

Hematuria

Chronic hemoglobinuria

Mechanical heart valve hemolysis

Paroxysmal nocturnal hemoglobinuria

Decreased Intake of Iron

Dietary deficiency

Limited meat

Malabsorption

Achlorhydria

Gastric atrophy

Partial gastrectomy

Gastric bypass

Proton pump inhibitors

Helicobacter pylori gastritis

Inflammatory bowel disease

Celiac disease

Increased Utilization of Iron

Pregnancy or lactation

Rapid growth

  Stainable marrow iron (RES hemosiderin) and serum ferritin are the primary markers of a negative iron balance. Serum ferritin accurately reflects body iron stores. Thus a bone marrow biopsy is rarely needed. Serum ferritin below ~30 ng/dL is indicative of absolute iron deficiency, while in the presence of inflammation or liver disease the cutoff is higher (~100 ng/mL). ¹⁶

  Reticulocyte hemoglobin is reported as part of the automated profile of reticulocytes. Due to the long half-life of mature erythrocytes in the circulation, reduced hemoglobin content in reticulocytes may be useful in cases of acute iron deficiency or for monitoring response to iron repletion therapy. In the absence of thalassemia, reticulocyte hemoglobin values below 26 pg per reticulocyte indicate early iron deficiency. ¹⁷

  As storage iron becomes depleted and the iron supply to red cells becomes limiting, an increase in circulating transferrin receptors was reported.¹⁸ Elevated serum soluble transferrin receptor concentration is not specific for iron deficiency and can be associated with erythroid hyperplasia. While a recent prospective multicenter trial suggested an added benefit of identifying absolute iron deficiency when anemia of chronic disease was also present,¹⁹ it is not generally recommended for use in clinical practice.¹⁸

  When the storage iron becomes depleted, serum iron and transferrin saturation begin to drop while the transferrin concentration usually rises.

  When transferrin saturation reaches ~16%, the supply of iron to developing red cells becomes rate limiting, and the red cell count begins to decrease.

  The new iron deficient red cells are smaller than the older ones, and therefore the red cell distribution width (RDW) begins to increase.

  When microcytes become more numerous, the mean cell volume (MCV) falls below the normal range, typically when the hemoglobin reaches ~10 g/dL.

FUNCTIONAL IRON DEFICIENCY

Hypoferremia despite seemingly adequate iron stores due to increased erythropoietic activity can be driven by endogenous erythropoietin stimulation as for patients recovering from absolute iron deficiency if the rate of supply of iron from their stores limits the rate of red cell production.²⁰ During pregnancy, iron requirements increase to 5 to 7 mg/day, so iron supplementation is needed to prevent the depletion of iron stores.²¹ Erythropoietin-stimulating agents (ESA) administration in patients with chronic kidney disease (CKD) also causes increased erythroid iron demand, although iron sequestration also plays a role. These patients may have adequate iron stores, but their response to ESA is blunted until they are given iron supplementation.²² Thalassemia major leads to increased iron absorption and ultimately to iron overload due to high erythropoietic activity as well as pathological mechanisms caused by ineffective erythropoiesis.²³

Anemia of chronic inflammatory states or anemia of chronic disease (ACD) accounts for most of iron sequestration syndromes but rare causes like hepcidin-producing adenomas, copper deficiency, and iron refractory iron deficiency anemia (IRIDA) have been described.²⁴ ACD develops in patients with chronic infectious, inflammatory, or neoplastic diseases. The anemia associated with functional iron deficiency is usually mild and asymptomatic.²⁵ Although usually normocytic, the MCV is often on the low end of normal and may be in the microcytic range. The serum iron concentration and transferrin saturation often suggest absolute iron deficiency, but the transferrin concentration is not elevated and may be low.²⁵ Furthermore, there is evidence of storage iron in the form of an elevated serum ferritin, as well as stainable iron in the bone marrow.

Patients with chronic illnesses can also have absolute iron deficiency, which can be particularly difficult to diagnose because of the effects of inflammation on the laboratory parameters of iron status. Chronic inflammation, for example, can suppress transferrin and elevate serum ferritin even in the true absence of storage iron.²⁶

Hepcidin biology is being aggressively developed for iron-related diseases. Diagnostic²⁷ and therapeutic28,29 applications have been proposed (Table 1.3). More robust assays are needed before hepcidin levels become generally available in the clinic.³⁰ However, research studies have already shown that serum hepcidin levels are very low or at undetectable levels in patients with absolute iron deficient anemia.³¹ In contrast, iron administration upregulates hepcidin in healthy volunteers.³² Importantly, hepcidin is increased by the inflammatory cytokines such as interleukin-6.³³ Therefore, patients with increased inflammatory states possess a wide range of serum hepcidin levels (100–4,000 ng/mL) when compared with healthy volunteers (5–350 ng/mL).³¹

TREATMENT OF IRON DEFICIENCY ANEMIA

Dietary Iron

Dietary review and counseling is needed for all patients evaluated for iron deficiency. Iron malabsorption or inhibition of absorption by other substances needs to be evaluated. Non-vegan patients should be encouraged to increase red meat or liver in their diet, as well as vitamin C, known to increase iron absorption. Because the heme of meat is so readily absorbed and without gastrointestinal side effects, it is an excellent source of iron. The presence of heme in the diet also increases the absorption of inorganic iron. Patients presenting with anemia usually require more than just diet supplementation.³⁴

Oral Iron Therapy

Several oral iron formulations are available, all containing iron sulfate, gluconate, or fumarate (Table 1.4). Most of them are tablets, non–enteric coated, enteric-coated, or slow release, but they can also be elixirs, usually containing less elemental iron.

Slow release or enteric-coated formulations of iron are touted to cause fewer gastrointestinal side effects, but they also often contain less iron per dose and are considerably more expensive than the nonenteric salts (see Table 1.4). Furthermore, they may release their iron below the duodenum, too distal for significant absorption.

A daily supplement of ~200 mg of elemental iron taken in a fasting state provides the marrow with enough iron to raise the blood hemoglobin concentration up to 0.25 g/dL/day in severely anemic patients.³⁵ Oral iron, however, causes nausea or constipation in some patients. Because these symptoms correlate with the amount of iron ingested, the dose should be lowered until tolerable or the medication should be stopped altogether until the symptoms resolve and then restarted at a lower dose. Using an elixir of iron allows doses as low as 10 to 20 mg of elemental iron, and multivitamins often contain even smaller amounts. Low doses of iron can be therapeutic; the response is just slower.³⁶ Patients should be prescribed stool softeners as needed. Often they can avoid nausea by taking their iron with food. This practice reduces iron absorption, but it usually does not make patients refractory to iron. Alternatively, bedtime dosing may be used to increase the tolerability to oral formulations.

A variety of medications can reduce oral iron absorption (Table 1.5) and should not be taken within several hours of iron tablets. Conversely, oral iron supplements can hinder the absorption of other medications (Table 1.6).

Oral iron absorption testing may be considered for patients suspected of malabsorption.³⁷ An 8 to 12 hour fasting serum iron is compared with the iron serum level 1 hour after ingestion of 65 mg of elemental iron (325 mg tablet of ferrous sulfate). An increase in serum iron of over 100 µg/dL from baseline demonstrates adequate absorption. In case of malabsorption, gastrointestinal consultation should be sought to identify and treat reversible etiologies. Parenteral iron treatment may be considered in cases of iron malabsorption (post-gastric bypass surgery, celiac disease, autoimmune gastritis, and Helicobacter pylori infection).³⁸

Table 1.6  Medications Malabsorbed When Co-administrated with Iron

Quinolone antibiotics

Thyroxine

Biphosphonates

Penicillamine

Cefdinir

Mycophenolate mofetil

Levodopa, carbidopa, methyldopa

Zinc or copper salts

Data from Lexicomp.com.

Iron supplements should be taken until the anemia resolves, which may only require a few weeks. Additional supplements are required to replenish iron stores. Several algorithms can be used to decide the length of the therapy (Table 1.7). The rate of iron absorption becomes slower once the patient is no longer anemic,³⁷ so serum ferritin levels may be followed to determine when iron stores are replenished. Once the anemia is reversed, a serum ferritin of 40 to 50 µg/L should be reached before the supplements are discontinued.³⁹

Intravenous Iron Therapy

CKD and dialysis patients receiving ESA require intravenous (IV) iron therapy.⁴⁰ When compared with oral iron treatment, administration of 100 mg of IV elemental iron twice weekly required 46% less erythropoietin to maintain same hematocrit goal.⁴¹ Other inflammatory states associated with anemia benefit from the combination of ESA and IV iron, including inflammatory bowel disease (IBD), rheumatoid arthritis, and malignancy.²⁴ Other indications include patients that cannot tolerate an adequate dose of oral iron, such as during pregnancy, or when they have severe and recurrent gastrointestinal or uterine hemorrhage.

Four formulations of parenteral iron are currently marketed in the United States: iron dextran (DexFerrum™, INFeD™), sodium ferric gluconate in sucrose (Ferrlecit™), iron sucrose (Venofer™), and Ferumoxytol (Feraheme™) (Table 1.8). Until 1999 when FDA approved Ferrlecit™ for treatment of anemia in renal failure patients, iron dextran formulations were the only available option. Iron dextran is now the only formulation that requires a test dose and premedication due to reported anaphylactic reactions. Because fewer adverse events were reported with the non-dextran formulations,⁴² iron dextran is being replaced in clinical practice. For all preparations, the infusion can be repeated weekly depending on the iron deficit magnitude. Increased amount of elemental iron per dose with newer formulations allows for more rapid correction of the iron deficit, with fewer hospital visits, as well as increased compliance.⁴³ The clinical effects of the oxidative stress and other inflammatory changes reported with parenteral iron treatment are not fully understood.⁴⁰

Red Blood Cell Transfusion

Red blood cell transfusion is reserved for acute presentation in hemodynamically unstable patients. The iron content of packed red blood cells (PRBCs) is around 1.0 mg of heme iron per 1.0 mL of packed erythrocytes. After one unit of PRBCs is transfused, the expected increase in hemoglobin and hematocrit is 1 g/dL and 3%, respectively.⁴⁴ It may take 2 to 3 weeks for the effects of transfused PRBCs to be realized in the iron parameters.⁴⁵

RESPONSE TO IRON THERAPY

When iron is given orally in full doses or parenterally to otherwise healthy individuals,

  within 3 or 4 days, peripheral blood reticulocytes increase

  within the first week, the hemoglobin begins to rise

A failure to observe a rise in hemoglobin after 1 to 2 weeks can be due to an incorrect diagnosis of iron deficiency, continued bleeding (in which case reticulocytes will increase despite no improvement in the anemia), noncompliance with the therapy, malabsorption for oral iron therapy, or a combination of these factors.

TREATMENT OF FUNCTIONAL IRON DEFICIENCY

Increased iron requirements during stress erythropoiesis can be addressed with either oral or parenteral iron administration. In pregnancy, parenteral iron may be required due to intolerance to oral formulations. ESA treatment in patients with CKD or end stage renal disease (ESRD) requires adequate iron stores. Iron supplementation for dialysis patients is usually parenteral.⁴⁰

In iron sequestration syndromes, the only truly satisfactory solution is adequate treatment of their underlying cause. Although the anemia is typically mild, treatment with iron supplements should be attempted for those patients with more severe anemia who are being considered for transfusion therapy. Parenteral iron supplementation may be helpful due to decreased oral absorption.²⁵ Although giving both erythropoietin and iron can eliminate the need for red cell transfusions, the effect of this combination on a patient’s distribution of iron is the same as that of transfusions: iron accumulates in inaccessible stores. Long-term safety studies are needed to determine the iron dosing schedules and limits in this clinical setting.

Treatment of Anemia in Patients with Advanced Malignancy

Advanced malignancies, as well as their treatment with chemotherapy, puts patients at a higher risk of anemia. In this setting, support with parenteral iron and ESA is the standard approach. Oral iron has been shown to be inferior to parenteral iron in patients receiving ESA for chemotherapy-induced anemia.⁴⁶ Multiple studies in recent years have shown a decreased survival in patients with different malignancies when receiving ESA.⁴⁷ Increased risks of thromboembolic disease, pro-angiogenic effects, as well as elevated blood pressure are proposed mechanisms.⁴⁷ The Food and Drug Administration (FDA) recently issued a black box warning for cancer patients receiving ESA. A mandatory risk assessment online tool was also provided at www.esa-apprise.com. Further recommendations for ESA usage are addressed in this handbook’s appendix.

Treatment of Anemia Associated with Chronic Inflammatory Diseases

The anemia of rheumatoid arthritis has been reported to respond to parenteral iron alone, as well as to ESA alone, with elevations of hemoglobin from ~11.5 to ~12.5 g/dL in both instances.48,49 An additive effect was reported when adding parenteral iron to ESA in one series.⁵⁰ In IBD, (1) absolute iron deficiency is common, (2) the anemia typically responds to iron alone, (3) parenteral iron is usually required because of gastrointestinal intolerance, and (4) erythropoietin can magnify the erythroid response.⁵¹ In contrast, anemic patients with chronic infections, including HIV, should receive iron only if they have absolute iron deficiency, because of concern that an increased iron supply may promote the growth of certain microorganisms that are siderophoric like Yersinia enterocolitica or Klebsiella pneumoniae.52,53

SUMMARY

Iron deficiency anemia remains one of the most prevalent health problems in United States and worldwide despite improved understanding of its pathophysiology and the availability of more oral and parenteral supplementation options. In addition to assessment of the hematological status and iron parameters, effort should always be made to determine the cause of absolute or functional iron deficiency. In cases of hemorrhage or nutritional iron deficiency, the diagnosis and case management are usually accomplished in the primary care setting.

Specialized care is indicated when no cause is identified or the patient does not respond to oral therapy. In some cases, parenteral iron formulations may be required. Based on improved safety profiles, administration of parenteral iron may be provided in the outpatient setting. Iron replacement regimens should be designed to correct the anemia and to additionally replenish iron stores. Rapid advances in iron and hepcidin biology are predicted to improve future diagnostic and therapeutic approaches to this disease.

References

  1.  Rastogi T, Mathers C. Global Burden of Iron Deficiency Anaemia in the year 2000. WHO report; 2002:1-13.

  2.  Centers for Disease Control and Prevention. Recommendations to prevent and control iron deficiency in the United States. MMWR Morb Mortal Wkly Rep. 1998;47(RR3):1-29.

  3.  Vanasse GJ, Berliner N. Anemia in elderly patients: an emerging problem for the 21st century. Hematology Am Soc Hematol Educ Program. 2010;2010:271-275.

  4.  Silber MH, Richardson JW. Multiple blood donations associated with iron deficiency in patients with restless legs syndrome. Mayo Clin Proc. 2003;78(1):52.

  5.  Tunnessen WW, Smith C, Oski FA. Beeturia: a sign of iron deficiency. Am J Dis Child. 1969;117(4):424.

  6.  Heeney MM, Andrews NC. Iron homeostasis and inherited iron overload disorders: an overview. Hematol Oncol Clin North Am. December 2004;18(6):1379-1403.

  7.  Bothwell TH, Baynes RD, MacFarlane BJ, MacPhail AP. Nutritional iron requirements and food iron absorption. J Intern Med. 1989;226:357-365.

  8.  Bothwell TH, Charlton RW. Iron Deficiency in Women. Washington, DC: The Nutrition Foundation; 1981.

  9.  Andrews NC. Understanding heme transport. N Engl J Med. 2005;353:2508-2509.

10.  McKie AT, Barrow D, Latunde-Dada GO, et al. An iron-regulated ferric reductase associated with absorption of dietary iron. Science. 2001;291:1755-1759.

11.  Nemeth E, Tuttle MS, Powelson J, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306(5704):2090-2093.

12.  Donovan A, Lima CA, Pinkus JL, et al. The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab. 2005;1(3):191-200.

13.  Huebers HA, Csiba E, Huebers E, Finch CA. Competitive advantage of diferric transferrin in delivering iron to reticulocytes. Proc Natl Acad Sci. 1983;80:300-304.

14.  Bainton DF, Finch CA. The diagnosis of iron deficiency anemia. Am J Med. 1964;37:62-70.

15.  Alleyne M, Horne MK, Miller JL. Individualized treatment for iron-deficiency anemia in adults. Am J Med. 2008;121(11):943-948.

16.  Lipschitz DA, Cook JD, Finch CA. A clinical evaluation of serum ferritin as an index of iron stores. N Engl J Med. 1974;290:1212-1216.

17.  Mast AE, Blinder MA, Dietzen DJ. Reticulocyte hemoglobin content. Am J Hematol. 2008;83(4):307-310.

18.  Mast AE, Blinder MA, Gronowski AM, Chumley C, Scott MG. Clinical utility of the soluble transferrin receptor and comparison with serum ferritin in several populations. Clin Chem. 1998;44(1):45-51.

19.  Skikne BS, Punnonen K, Caldron PH, et al. Improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: a prospective multicenter evaluation of soluble transferrin receptor and the sTfR/log ferritin index. Am J Hematol. 2011;86(11):923-927.

20.  Hillman RS, Henderson PA. Control of marrow production by the level of iron supply. J Clin Invest. 1969;48:454-460.

21.  Bothwell TH. Iron requirements in pregnancy and strategies to meet them. Am J Clin Nutr. 2000;72(1 suppl):257S-264S.

22.  Nurko S. Anemia in chronic kidney disease: causes, diagnosis, treatment. Cleve Clin J Med. 2006;73(3):289-297.

23.  Ginzburg Y, Rivella S. β-thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism. Blood. 2011;118(16):4321-4330.

24.  Goodnough LT, Nemeth E, Ganz T. Detection, evaluation, and management of iron-restricted erythropoiesis. Blood. 2010;116(23):4754-4761.

25.  Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med. 2005;352:1011-1023.

26.  Cash, JM, Sears, DA. The anemia of chronic disease: spectrum of associated diseases in a series of unselected hospitalized patients. Am J Med. 1989;87:638-644.

27.  Ganz T. Hepcidin and iron regulation, 10 years later. Blood. 2011;117(17):4425-4433.

28.  Sasu BJ, Cooke KS, Arvedson TL, et al. Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia. Blood. 2010;115(17):3616-3624.

29.  Hashizume M, Uchiyama Y, Horai N, Tomosugi N, Mihara M. Tocilizumab, a humanized anti-interleukin-6 receptor antibody, improved anemia in monkey arthritis by suppressing IL-6-induced hepcidin production. Rheumatol Int. 2010;30(7):917-923.

30.  Young B, Zaritsky J. Hepcidin for clinicians. Clin J Am Soc Nephrol. 2009;4(8):1384-1387.

31.  Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood. 2008;112(10):4292-4297.

32.  Pak M, Lopez MA, Gabayan V, Ganz T, Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity. Blood. 2006;108:3730-3735.

33.  Nemeth E, Rivera S, Gabayan V, et al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest. 2004;113:1271-1276.

34.  Crosby WH. The rationale for treating iron deficiency anemia. Arch Intern Med. 1984;144:471-472.

35.  Pritchard JA. Hemoglobin regeneration in severe iron-deficiency anemia. J Am Med Assoc. 1966;195:97-100.

36.  Radtke H, Tegtmeier J, Rocker L, Salama A, Kiesewetter H. Daily doses of 20 mg of elemental iron compensate for iron loss in regular blood donors: a randomized, double-blind, placebo-controlled study. Transfusion. 2004;44:1427-1432.

37.  Cook JD. Diagnosis and management of iron-deficiency anemia. Best Pract Res Clin Haematol. 2005;18:219-332.

38.  Hershko C, Hoffbrand AV, Keret D, et al. Role of autoimmune gastritis, Helicobacter pylori and celiac disease in refractory or unexplained iron deficiency anemia. Haematologica. 2005;90(5):585-595.

39.  Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor: a quantitative measure of tissue iron deficiency. Blood. 1990;75(9):1870-1876.

40.  National Kidney Foundation. KDOQI clinical practice guidelines and clinical practice recommendations for anemia in chronic kidney disease. Am J Kidney Dis. 2006;47:S11-S45.

41.  Silverberg DS, Iaina A, Peer G, et al. Intravenous iron supplementation for the treatment of the anemia of moderate to severe chronic renal failure patients not receiving dialysis. Am J Kidney Dis. 1996;27:234-238.

42.  Chertow GM, Mason PD, Vaage-Nilsen O, Ahlmén J. Update on adverse drug events associated with parenteral iron. Nephrol Dial Transplant. 2006;21:378-382.

43.  Gozzard D. When is high-dose intravenous iron repletion needed? Assessing new treatment options. Drug Des Dev Ther. 2011;5:51-60.

44.  Wiesen AR, Hospenthal DR, Byrd JC, Glass KL, Howard RS, Diehl LF. Equilibration of hemoglobin concentration after transfusion in medical inpatients not actively bleeding. Ann Intern Med. 1994;121(4):278-230.

45.  Hod EA, Brittenham GM, Billote GB, et al. Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non-transferrin-bound iron. Blood. 2011;118(25):6675-6682.

46.  Auerbach M, Ballard H, Trout JR, et al. Intravenous iron optimizes the response to recombinant human erythropoietin in cancer patients with chemotherapy-related anemia: a multicenter, open-label, randomized trial. J Clin Oncol. 2004;22:1301-1307.

47.  Unites States Food and Drug Administration. FDA Drug Safety Communication: Erythropoiesis-Stimulating Agents (ESAs): Procrit, Epogen and Aranesp. http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationfor
PatientsandProviders/ucm200297.htm

48.  Bentley DP, Williams P. Parenteral iron therapy in the anaemia of rheumatoid arthritis. Rheumatol Rehabil . 1982;21:88-92.

49.  Pincus T, Olsen NJ, Russell IJ, et al. Multicenter study of recombinant human erythropoietin in correction of anemia in rheumatoid arthritis. Am J Med. 1990;89:161-168.

50.  Nordstrom D, Lindroth Y, Marsal L, et al. Availability of iron and degree of inflammation modifies the response to recombinant human erythropoietin when treating anemia of chronic disease in patients with rheumatoid arthritis. 1997;17:67-73.

51.  Gasche C, Lomer MCE, Cavill I, Weiss G. Iron, anaemia, and inflammatory bowel disease. Gut. 2004;53:1190-1197.

52.  Henry DH, Beall GN, Benson CA, et al. Recombinant human erythropoietin in the treatment of anemia associated with human immunodeficiency virus (HIV) infection and zidovudine. Ann Intern Med. 1992;117:739-748.

53.  Maynor L, Brophy DF. Risk of infection with intravenous iron therapy. Ann Pharmacother. September 2007;41(9):1476-1480.

2

Deficiencies of Vitamin B12 and Folate

Danielle M. Townsley and Griffin P. Rodgers

Besides iron deficiency, shortages of vitamin B12 (cobalamin) and folate are the most common nutritional causes of anemia. The frequencies of these deficiencies are highly dependent upon the population under study. Because vitamin B12 deficiency usually develops as a consequence of insidious malabsorption that occurs over many years, it becomes more prevalent with advancing age. Because folate deficiency is largely a consequence of inadequate dietary folate, it is most prevalent in populations at risk for malnutrition or areas where the food is unfortified.

VITAMIN REQUIREMENTS, SOURCES, AND STORES

To avoid clinically apparent ill effects, the daily adult requirement for vitamin B12 is 1 to 3 µg and for folic acid ~ 200 µg (Table 2.1).1,2 However, recent information indicates that between 4 and 7 µg/day of B12 is necessary to prevent biochemical changes secondary to a limiting supply of the vitamin.³ This suggests that the current recommended dietary allowance (RDA) of 2.4 µg/day may be insufficient. The daily requirement for folate has been more easily met since 1996, when the U.S. Food and Drug Administration mandated that all grains be fortified with the vitamin in order to reduce the risk of neural tube defects in developing fetuses.⁴

Bacteria in the gut of herbivorous animals synthesize vitamin B12 and supply it to their hosts, who in turn supply it to humans in the form of meat. There is no vitamin B12 in plant products other than that attributable to bacterial contamination. Plants synthesize folic acid and provide it to man directly in fruits and vegetables and indirectly in meat from herbivores.

The human body normally stores 2 to 3 months’ supply of folic acid, although marginally nourished patients, such as chronic alcoholics, may have stores that can be depleted much sooner.⁵ In contrast, body stores of vitamin B12 are normally sufficient for 5 to 10 years (Table 2.1).

METABOLIC ROLES OF FOLATE AND VITAMIN B12

The metabolic roles of folate and B12 are closely interrelated (Fig. 2.1). Folate derivatives are essential cofactors in thymidylate synthesis, which is a rate-limiting step in the synthesis of DNA. RNA synthesis, however, is not dependent upon folate. Therefore, deficiency of folate limits gene transcription but not RNA translation, retarding cell division but not cytoplasmic protein synthesis. This leads to the typical cytonuclear dissociation of maturation characteristic of megaloblastic hematopoiesis. Because cobalamin supports the recycling of folate, vitamin B12 deficiency causes megaloblastic changes by restricting the folate supply. This restriction can be at least partially overcome by increasing dietary folate, allowing the hematopoietic effects of cobalamin deficiency to be ameliorated by high doses of folic acid. In contrast, the hematopoietic effects of folate deficiency cannot be overcome by treatment with vitamin B12.

FIGURE 2.1 Metabolic pathways involving folic acid and vitamin B12 (cobalamin).

Cobalamin is also necessary in the pathway leading to the synthesis of S-adenosyl-methionine, which is the only donor of methyl groups for numerous reactions in the brain involving proteins, membrane phospholipids, and neurotransmitters.⁶ Presumably this explains the frequent neuropsychiatric signs and symptoms associated with vitamin B12 deficiency.⁷ Folate is not involved in these reactions and cannot reverse the neuropsychiatric deficits caused by vitamin B12 deficiency. However, methyltetrahydrofolic acid is the methyl donor for the synthesis of methionine, the precursor of S-adenosyl-methionine. Therefore, folate deficiency may restrict the synthesis of S-adenosyl-methionine and produce some neuropsychiatric effects as well, although this is rare.⁸

DEVELOPMENT OF VITAMIN B12 DEFICIENCY

Cobalamin deficiency is rarely caused by inadequate intake or increased utilization of the vitamin (Table 2.2). This is in part due to the pronounced enterohepatic circulation of cobalamin. Although strict vegetarians become depleted of vitamin B12, vegetables often contain sufficient bacteria to provide a marginally adequate supply. A developing fetus shunts cobalamin from its mother, placing her at risk of deficiency, particularly if her baseline stores are low. Rarely, intestinal parasites can induce deficiency; for example the fish tapeworm, Diphyllobothrium latum, competes with the host for cobalamin.⁹ Acutely, cobalamin metabolism can be disrupted by nitrous oxide anesthesia and can induce a rapid, usually transient, megaloblastic anemia.¹⁰ However, fatalities and severe neuropsychiatric damage have been associated with chronic administration in patients and recreational use of nitrous oxide.¹¹ There is recent evidence that prolonged use of metformin in diabetic patients is associated with reduced serum B12 levels, although the mechanism is unknown.¹²

Far more commonly, defects in any of the three levels of the gastrointestinal tract can lead to vitamin B12 malabsorption: the fundus of the stomach, the pancreas, or the small bowel. Obviously surgical removal or bypass of any of these regions leads to B12 malabsorption.¹³ Bariatric surgery is becoming more prevalent and subsequently an important risk factor for developing B12 deficiency. Otherwise the etiology is inflammatory.

  Stomach: In the stomach, food (protein)-bound vitamin B12 must be freed by digestion with pepsin and bound to “R-proteins,” which is a generic term for proteins that bind B12.¹ The parietal cells in the fundus secrete both the acid necessary for this digestion and intrinsic factor, the protein to which cobalamin is later transferred in the alkaline duodenum. Therefore, any process that damages the parietal cells can lead to vitamin B12 malabsorption and eventually deficiency. The most common cause is autoimmune atrophic gastritis, which increases in prevalence with age and is sometimes associated with other autoimmune diseases, such as thyroiditis. Helicobacter pylori, however, which typically causes antral gastritis, can occasionally also infect the fundus.14,15 Proton pump inhibitors induce chronic hypochlorhydria but rarely cause clinically significant B12 malabsorption. Parodoxically, the hypersecretion of acid in the Zollinger-Ellison syndrome leads to B12 malabsorption by acidifying the small bowel, which must remain alkaline for the transfer of B12 from the R-binders to intrinsic factor. Antibodies to intrinsic factor, as in the case of pernicious anemia, will also lead to a reduction of vitamin B12-intrinsic factor complexes necessary for absorption in the small bowel.

  Pancreas: Deficiency of pancreatic enzymes impairs the digestion of R-binders in the small bowel and therefore the release of B12 to intrinsic factor. Although pancreatic insufficiency causes cobalamin malabsorption, it rarely is significant enough to become clinically apparent.

  Small bowel: Vitamin B12–intrinsic factor complexes are endocytosed by the mucosa of the terminal ileum. Inflammatory bowel disease or particularly extensive celiac or tropical sprue will interfere with this process.¹⁶ Bacterial overgrowth in the small bowel, especially common in the elderly, competes for B12 and makes it less available for absorption.¹⁷ HIV infection is sometimes also associated with B12 malabsorption, especially in the presence of chronic diarrhea.

DEVELOPMENT OF FOLIC ACID DEFICIENCY

A diet poor in fresh vegetables is a major cause of folic acid deficiency (Table 2.3). Cooked vegetables and meat are less satisfactory sources because cooking destroys much of the folate. (This is less of a problem for vitamin B12.) The other major causes are gastrointestinal diseases that affect the jejunum, where folic acid is absorbed, and conditions such as pregnancy that increase folate requirements. Ethanol abuse and several chronic medications (Table 2.3) lead to folate deficiency by interrupting folate metabolism or inhibiting its absorption

PATIENT POPULATIONS AT RISK

Vitamin B12 deficiency due to lack of intrinsic factor (“pernicious anemia”) is sometimes believed to be limited to elderly patients of European descent. In this population the median age at presentation is almost 70 years.

  Nevertheless, intrinsic factor deficiency may be almost as prevalent in African Americans and Latinos, who tend to present with vitamin B12 deficiency a decade earlier. 18,19

  Older patients are actually more likely to become B12 deficient from achlorhydria or small bowel bacterial overgrowth than from lack of intrinsic factor.15,20

Whereas cobalamin deficiency is generally found in older age groups,²¹ folic acid deficiency is likely to occur in any patient who has an inadequate diet or who has an increased need for the vitamin, e.g., during pregnancy or hemolytic anemia (Table 2.3). Folate deficiency secondary to poor nutrition is generally rare in the United States since the introduction of mandatory folate fortification of cereal grains. A recent population study among centenarians indicates that less than 6% of the very old have low RBC folate levels.²²

CLINICAL PRESENTATION

The clinical presentation of vitamin B12 and folic acid deficiency covers a wide range (Table 2.4). The investigation of any new neuropsychiatric changes should include an evaluation of folate and evaluation of vitamin B12 and folate status even in the absence of hematologic signs of a deficiency.⁷ Changes in the tongue mucosa and mouth angle stomatitis may be the earliest sign of folate deficiency on physical exam.²³

LABORATORY EVALUATION

Hematologic Abnormalities

Macrocytosis develops before anemia when either folic acid or vitamin B12 is limiting (Table 2.5).5,24 With the advent of automated blood cell analyzers, isolated macrocytosis has become a typical presentation of deficiencies of either vitamin, although other causes of macrocytosis are more common (Table 2.6).²⁵ An unexplained rise in mean cell volume (MCV) of 5 fL or more even within the normal range should also attract suspicion. However, the macrocytosis may be masked if the patient is also iron deficient or has a thalassemic trait. At hemoglobin concentrations below ~10 g/dL, B12 or folate deficiency leads to elevations of serum lactate dehydrogenase, which can become quite high.5,24 This results from marked intramedullary death of developing red cells and a shortening of the circulating red cell life span but can mislead the clinician to suspect metastatic disease or a primary hemolytic anemia.

The earliest change in the peripheral blood caused by folate or vitamin B12 deficiency is hypersegmentation of the neutrophils, which can be easily overlooked unless a blood smear is carefully examined. Finding even 5% of neutrophils with five lobes or just 1% with six lobes is highly suggestive of a deficiency, although this could also be seen with myelodysplasia. In advanced deficiencies characterized by severe anemia, pancytopenia can develop.

There is rarely if ever a need to perform a bone marrow examination in the evaluation of vitamin B12 and folic acid status. The megaloblastic changes in the marrow are identical in both deficiencies and are variable in intensity. A marrow examination cannot rule out myelodysplasia or even a smoldering leukemic process until cobalamin and folate deficiencies have been excluded first.

Serum Vitamin Concentrations

When vitamin B12 and folate deficiency is suspected, a common diagnostic starting point is measurement of the serum concentrations of the vitamins, which should be done after the patient has been fasting. The results, however, can be difficult to interpret (Table 2.7).

Serum folic acid does not reliably reflect the body’s supply of the vitamin, unless it is consistently below ~3 ng/mL, and even then it does not distinguish between negative balance and actual tissue deficiency.²⁶ In general, the serum folate level reflects recent folate intake, and red cell folate is a better measurement of tissue folate stores. Because red cell folate is packaged at the time the cell is made and remains in the cell throughout its 3 to 4 month lifespan, the measured mean value may fail to reflect relatively recent reductions in dietary folate. Furthermore, the reproducibility of assays for red cell folate is relatively poor so that borderline values can be misleading.26,27 To complicate matters further, red cell folate can be reduced by vitamin B12 deficiency and lead to an erroneous diagnosis (Table 2.7).

Interpreting serum vitamin B12 levels can also be problematic because occasionally serum B12 can be decreased in rare cases of folate deficiency.¹ A more frequent problem, similar to that with folic acid, relates to uncertainties in the physiologic levels of the vitamin. The “normal range” of serum cobalamin typically extends down to 200 pg/mL, because healthy, non-anemic donors occasionally have B12 concentrations that are this low. However, patients who are truly deficient in vitamin B12 can have serum cobalamin levels as high as 300 pg/mL and maybe higher.26,28 The reason for this discrepancy is that the total cobalamin is measured, rather than just the vitamin B12 bound to transcobalamin, which is the metabolically available B12 but represents only ~20% of the total serum vitamin. Therefore, individuals who have relatively low concentrations of transcobalamin 1, which binds the other ~80% of the serum vitamin B12, can have alarmingly low B12 levels (<100 pg/mL) without any harmful effect because they have adequate amounts of B12 bound to transcobalamin 2, which is necessary for transfer of B12 to hematopoietic precursors. Occasionally, healthy patients have been described with low transcobalamin 1 levels.²⁹ Transcobalamin 1 can also be low in patients with multiple myeloma.³⁰ Such patients will be easier to identify when tests become available to measure B12 specifically bound to transcobalamin rather than total serum B12.³¹

Serum Methylmalonic Acid and Homocysteine

Measurements of methylmalonic acid (MMA) and homocysteine (Hcy), though they are more expensive than the vitamin assays, answer the question of vitamin deficiency more reliably.³² Although these metabolites can be elevated for other reasons (Table 2.8), if these causes are excluded, these metabolites become specific reflections of vitamin B12 and folate depletion at the tissue level.19,32 Usually MMA and Hcy both become elevated when B12 is the limiting factor, whereas only Hcy becomes elevated when folate is limiting. However, in 1% to 2% of cases of vitamin B12 deficiency, only Hcy will be elevated, whereas in ~10% of cases of folate deficiency MMA will be high. Therefore, elevated Hcy alone does not always differentiate B12 and folate deficiency but makes folate deficiency more likely.

Although normal levels of MMA and Hcy were reported to exclude a metabolic effect from B12 deficiency, the negative predictive value of these assays has been questioned because of reports of patients with normal metabolites who clearly respond to vitamin B12.19,33

Therapeutic Trial

If the laboratory evaluation is impossible because resources are lacking or evaluation is inconclusive, a therapeutic trial can be diagnostic if a single vitamin is given at a time. Vitamin B12 should be given first because it will do nothing for folic acid deficiency, whereas replacement with folic acid will improve the anemia secondary to B12 deficiency but not the neuropathic changes. The response to treatment can be judged by following the reticulocyte count and hemoglobin (see below). A more expensive way is to remeasure Hcy or MMA levels 2 to 5 days after one or the other of the vitamins has been administered. The metabolites will fall only in response to replacement of the deficient vitamin.³²

DETERMINING THE CAUSE OF B12 OR FOLATE DEFICIENCY

  The etiology of folate deficiency must always be determined because virtually all causes are either preventable or treatable. If the patient appears to have an adequate diet, a gastrointestinal evaluation is indicated to search for the underlying causes shown in Table 2.3.

  In contrast, if vitamin B12 is deficient and the patient is not a vegan and there are no symptoms of gastrointestinal disease, an argument can be made to take the evaluation no further and simply to treat the individual with the vitamin.

If the patient and/or his physician feels compelled to confirm that the pathology lies in the stomach, however, a test for anti-intrinsic factor antibodies should be done.³⁴ A positive test is diagnostic of pernicious anemia and is found in half the cases. Anti-parietal cell antibodies are more common but are also found in a small percentage of normal individuals. Demonstrating an elevation in serum gastrin also strongly supports the diagnosis of gastric atrophy.³⁵

If the patient is unusually young to have achlorhydria or pernicious anemia (e.g., <50 years old or younger if African American), a gastrointestinal evaluation is indicated (Table 2.2).¹⁸ In the past, the Schilling test was used in an attempt to detect vitamin B12 malabsorption, but Schilling tests are no longer performed because of lack of a commercial source for radiolabeled cobalamin and the realization that this test also often gives misleading results. Because the incidence of many malignancies, especially gastric cancer, is slightly higher in patients with pernicious anemia than in age- and sex-matched controls, following the patient for any signs of gastrointestinal blood loss is wise, although more aggressive surveillance is not indicated.³⁶

TREATMENT/RESPONSE

There are two treatment goals: replacement of the deficient vitamin and correction of the cause of the deficiency. The first goal is always achievable; the second may not be. Treatment should always be given if the clinical presentation is suspicious, even if the laboratory data are confusing, because the laboratory data are not totally sensitive and the consequences of undertreating can be devastating.32,33 The current evidence indicates that there is no benefit to further supplementation with vitamin B12 or folate if a deficiency is nonexistent. For example, elevated levels of Hcy have been associated with an increased risk of vascular thrombosis, however, there is no role for supplementing vitamin B12 or folate in a sole attempt to decrease Hcy levels and subsequent vascular events.37,38

In the past, vitamin B12 was always given intramuscularly in North America, although the oral route was used in Sweden.³⁹ Recently, however, the effectiveness of oral B12 was demonstrated in two randomized, controlled clinical trials.40,41 This works even in patients with pernicious anemia because approximately 1% of any oral dose of vitamin B12 is absorbed by simple diffusion across the mucosa. So the recommended daily dose of 1 to 2 mg vitamin B12 results in the absorption of ~ 10 to 20 µg, which is much more than the daily requirement. A caveat, however, is that 1 mg tablets of vitamin B12 are sometimes difficult to find in hospital pharmacies.

Intramuscular B12, of course, is perfectly acceptable if it is more practical for a patient, and this route would still be preferred by most physicians to treat a patient with neurologic symptoms. Although there are a variety of accepted regimens, daily injections of 50 to 100 µg should be given for a week, followed by weekly injections for a month, and then monthly injections of 1 mg. For most patients with vitamin B12 deficiency, lifelong treatment is required because the underlying cause is not reversible.

The usual dose of oral folic acid is 1 mg/day (Table 2.1). This is ample even during pregnancy or chronic hemolysis. If the deficiency is dietary, replacement with folic acid should continue until the diet has become adequate. A month of daily folic acid should be sufficient to replenish body stores. If the etiology of the deficiency is small bowel dysfunction, higher doses for longer periods of time may be necessary. It is important to rule out concomitant vitamin B12 deficiency prior to replenishing folate because anemia may improve but the neurologic symptoms due to vitamin B12 deficiency can progress and the diagnosis missed.

The response to correction of vitamin B12 or folate deficiency is the same:

  Mental changes and tongue soreness improve almost immediately after starting to replace the deficient vitamin. ¹

  After 4 to 5 days, reticulocytosis appears and may elevate the MCV even further.

  Soon thereafter, the hemoglobin concentration begins to rise.

  Neuropathic abnormalities, such as paresthesias, improve slowly, over several months, but may never disappear entirely if they have been long-standing.

If the hematologic response is blunted, additional etiologies for the anemia should be sought. It is not unusual for iron deficiency to accompany folate or vitamin B12 deficiency. An underlying anemia of chronic disease is always a possibility.

References

  1.  Chanarin I. The Megaloblastic Anaemias. 3rd ed. Oxford: Blackwell; 1990.

  2.  Tighe P, Ward M, McNulty H, et al. A dose-finding trial of the effect of long-term folic acid intervention: implications for food fortification policy. Am J Clin Nutr. 2011;93(1):1-2.

  3.  Bor MV, von Castel-Roberts KM, Kauwell GP, et al. Daily intake of 4 to 7 microg dietary vitamin B-12-related biomarkers in a healthy young population. Am J Clin Nutr. 2010;91:571-577.

  4.  Jacques PF, Selhub J, Bostom AG, Wilson PWF, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. 1999;340:1449-1454.

  5.  Lindenbaum J, Allen RH. Clinical spectrum and diagnosis of folate deficiency. In: Bailey LB, ed, Folate in Health and Disease. New York, NY: Marcel Dekker; 1995:43-74.

  6.  Reynolds EH, Carney MWP, Toone BK. Methylation and mood. Lancet. 1984;2:196-198.

  7.  Lindenbaum J, Healton EB, Savage DG, et al. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Eng J Med. 1988;318:1720-1728.

  8.  Carmel R, Green R, Rosenblatt DS, Watkins D. Update on cobalamin, folate, and homocysteine. Hematology Am Soc Hematol Educ Program. 2003:62-81.

  9.  Tanowitz HB, Weiss LM, Wittner M. Tapeworms. Curr Infect Dis Rep. 2001;3(1):77-84.

10.  Hathout L, El-Saden S. Nitrous oxide-induced B-12 deficiency myelopathy: perspectives on the clinical biochemistry of vitamin B-12. J Neurol Sci. 2011;301(1-2):1-8.

11.  Flippo TS, Holder WD Jr. Neurologic degeneration associated with nitrous oxide anesthesia in patients with vitamin B12 deficiency. Arch Surg. 1993;128(12):1391-1395.

12.  de Jager J, Kooy A, Lehert P, et al. Long term treatment with metformin in patients with type 2 diabetes and risk of vitamin B-12 deficiency: randomized placebo controlled trial. BMJ. 2010;340:c2181.

13.  Skroubis G, Sakellaropoulos G, Pouggouras K, Mead N, Nikiforidis G, Kalfarentzos F. Comparison of nutritional deficiencies after Roux-en-Y gastric bypass and after biliopancreatic diversion with Roux-en-Y gastric bypass. Obes Surg. 2002;12:551-558.

14.  Kaptan K, Beyan C, Ural AU, et al. Helicobacter pylori—is it a novel causative agent in vitamin B12 deficiency? Arch Intern Med. 2000;160:1349-1353.

15.  Hershko C, Ronson A, Souroujon M, Maschler I, Heyd J, Patz J. Variable hematologic presentation of autoimmune gastritis: age-related progression from iron deficiency to cobalamin depletion. Blood. 2006;107:1673-1679.

16.  Dahele A, Ghosh S. Vitamin B12 deficiency in untreated celiac disease. Am J Gastroenterol. 2001;96:745-750.

17.  Haboubi NY, Montgomery RD. Small-bowel bacterial overgrowth in elderly people: clinical significance and response to treatment. Age Aging. 1992;21:13-19.

18.  Carmel R, Johnson CS. Racial patterns in pernicious anemia. N Engl J Med. 1978;298:647-650.

19.  Savage DG, Lindenbaum J, Stabler SP, Allen RH. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J Med. 1994;96:239-246.

20.  Dharmarajan TS, Adiga GU, Norkus EP. Vitamin B12 deficiency, recognizing subtle symptoms in older patients. Geriatrics. 2003;58:30-38.

21.  Johnson MA, Hausman DB, Davey A, et al. Vitamin B12 deficiency in African American and white octogenarians and centenarians in Georgia. J Nutr Health Aging. 2010;14(5):339-345.

22.  Hausman DB, Johnson MA, Davey A, et al. The oldest old: red blood cell and plasma folate in African American and white octogenarians and centenarians in Georgia. J Nutr Health Aging. 2011;15(9):744-750.

23.  Bjorkegren K, Svardsudd K. Reported symptoms and clinical findings in relation to serum cobalamin, folate, methylmalonic acid and total homocysteine among elderly Swedes: a population-based study. J Intern Med. 2003;254(4):343-352.

24.  Stabler SP, Allen RH, Savage DG, Lindenbaum J. Clinical spectrum and diagnosis of cobalamin deficiency. Blood. 1990;76:871-881.

25.  Savage DG, Ogundipe A, Allen RH, Stabler SP, Lindenbaum J. Etiology and diagnostic evaluation of macrocytosis. Am J Med Sci. 2000;319:343-352.

26.  Klee GG. Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B12 and folate. Clin Chem. 2000;46:1277-1283.

27.  Pfeiffer CM, Zhang M, Lacher DA, et al. Comparison of serum and red blood cell folate microbiologic assays for national population surveys. J Nutr. 2011;141(7):1402-1409.

28.  Tucker KL, Rich S, Rosenberg I, et al. Plasma vitamin B-12 concentrations relate to intake source in the Framingham Offspring Study. Am J Clin Nutr. 2000;71:514-522.

29.  Carmel R. A new case of deficiency of the R binder for cobalamin, with observations on minor cobalamin-binding proteins in serum and saliva. Blood. 1982;59:152-156.

30.  Hansen OP, Drivsholm A, Hippe E. Vitamin B12 metabolism in myelomatosis. Scand J Haematol. 1977;18:395-402.

31.  Ulleland M, Eilertsen I, Quadros EV, et al. Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum. Clin Chem. 2002;48:526-532.

32.  Lindenbaum J, Savage DG, Stabler SP, Allen RH. Diagnosis of cobalamin deficiency II: relative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentrations. Am J Hematol. 1990;34:99-107.

33.  Solomon LR. Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing. Blood. 2005;105:978-986.

34.  Fairbanks VF, Lennon VA, Kokmen E, Howard FM. Tests for pernicious anemia: serum intrinsic factor blocking antibody. Mayo Clin Proc. 1983;58:203-204.

35.  Lindgren A, Lindstedt G, Kilander AF. Advantages of serum pepsinogen A combined with gastrin or pepsinogen C as first-line analytes in the evaluation of suspected cobalamin deficiency: a study in patients previously not subjected to gastrointestinal surgery. J Intern Med. 1998;244:341-349.

36.  Schafer LW, Larson DE, Melton LJ, et al. Risk of development of gastric carcinoma in patients with pernicious anemia: a population-based study in Rochester, Minnesota. Mayo Clin Proc. 1985;60:444-448.

37.  SEARCH Collaborative Group, Armitage JM, Bowman L, et al. Effects of homocysteine-lowering with folic acid plus vitamin B12 vs placebo on mortality and major morbidity in myocardial infarction survivors: a randomized trial. JAMA. 2010;303(24):2486-2494.

38.  Løland KH, Bleie O, Blix AJ, et al. Effect of homocysteine-lowering B vitamin treatment on angiographic progression of coronary artery disease: a Western Norway B Vitamin Intervention Trial (WENBIT) substudy. Am J Cardiol. June 2010;105(11):1577.

39.  Hvas A-M, Nexo E. Diagnosis and treatment of vitamin B12 deficiency. An update. Haematologica. 2006;91:1506-1512.

40.  Kuzminski AM, Giacco EJD, Allen RH, Stabler SP, Lindenbaum J. Effective treatment of cobalamin deficiency with oral cobalamin. Blood. 1998;92:1191-1198.

41.  Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barutca S, Senturk T. Oral versus intramuscular cobalamin treatment in megaloblastic anemia: a single-center, prospective, randomized, open-label study. Clin Ther. 2003;25:3124-3134.

3

Hemolytic Anemia

Patricia A. Oneal, Geraldine P. Schechter, Griffin P. Rodgers and Jeffery L. Miller

Many diseases share the clinical feature of red blood cell (RBC) hemolysis. Hemoglobinopathies and immune-mediated hemolysis are the most common causes (see discussions in Chapters 4 and 24, respectively). Very rare inherited or acquired diseases may also directly or indirectly result in increased red cell destruction.¹ Understanding the mechanisms that lead to hemolysis assists with the diagnosis, prognosis, and consideration of the most appropriate therapy. In this post-genomic era, correlations between genotype and phenotype are being pursued in cases of inherited hemolytic syndromes. Genetics-based discoveries are being translated into new clinical tools in anticipation of mechanism-specific therapies.

Hemolytic anemia is defined as decreased levels of erythrocytes in circulating blood because of their accelerated destruction. All circulating erythrocytes are subject to physiologic stresses such as turbulence in blood flow, endothelial damage, and age-related catabolic changes. Normally, damaged RBCs are removed from the circulation by the reticuloendothelial system. In hemolytic syndromes, erythrocyte clearance by the reticuloendothelial system may be increased (extravascular hemolysis) or the cells may be lysed within the circulation (intravascular hemolysis). As a result, RBC survival is generally shortened to less than 100 days (normal survival is approximately 120 days). When sufficient numbers of erythrocytes are destroyed, oxygen delivery to tissues is impaired. Tissue hypoxia leads to increased release of erythropoietin, which signals the bone marrow to produce more RBCs.

A hallmark of hemolytic anemia is an elevated number of immature erythrocytes (reticulocytes) in the peripheral blood. In low-level hemolysis, erythrocyte production may adequately compensate for blood cell destruction and minimize the anemia. Alternatively, patients with acute hemolysis or with underlying defects in hematopoiesis may present with pronounced anemia without reticulocytosis. Hence, the evaluation of suspected hemolysis requires consideration of the hemolysis itself as well as the marrow’s ability to compensate. The diagnostic strategy usually begins with a search for common causes of hemolysis and proceeds toward rare etiologies. The extent of diagnostic studies should be guided by the magnitude of hemolysis and the available therapeutic options. With the information contained here, practicing clinicians should be able to develop a clinical approach, differential diagnosis, and therapeutic plan for patients with suspected hemolysis.

ETIOLOGY AND DIFFERENTIAL DIAGNOSIS

Grouping the various causes of the disease generates a differential diagnosis for hemolysis. As shown in Figure 3.1, hemolysis results from pathology intrinsic or extrinsic to the erythrocytes. Intrinsic hemolysis may be categorized further according to hemoglobin, membrane, or enzyme-based factors. Alternatively, the patient’s immune status or infectious agents can lead to hemolysis in the absence of intrinsic defects. Other chemical or physical features of the erythrocyte environment can also cause hemolysis. A more complete differential organized according to these categories is shown in Table 3.1.

FIGURE 3.1 Intrinsic and extrinsic causes of hemolysis.

Most intrinsic causes for hemolysis are inherited, while the extrinsic causes of hemolysis are typically acquired. In some cases, such as paroxysmal nocturnal hemoglobinuria (PNH) or glucose-6-phosphate dehydrogenase (G6PD) deficiency, both intrinsic and extrinsic factors may contribute to the hemolytic picture. Consideration of the primary site of hemolysis (intravascular versus extravascular) may also be helpful in determining the origin of erythrocyte destruction.⁶

To complete the differential diagnosis, diseases or events that may in part mimic a typical hemolytic episode should be considered. The laboratory evaluation may be normal with the exception of a single variable such as hemoglobin, absolute reticulocyte count (ARC), or unconjugated bilirubin. For instance, the compensatory reticulocytosis that occurs after an acute hemorrhagic event may be mistaken for evidence of hemolysis. In the absence of other clinical or laboratory abnormalities, artifactual reticulocytosis may be caused by a malfunction in the automated cell counter. While hypersplenism may be associated with increased red cell clearance and anemia, the abnormal RBC morphology seen in patients with asplenia is usually not associated with hemolysis. Finally, patients with chronic idiopathic unconjugated hyperbilirubinemia (Gilbert’s syndrome) are sometimes erroneously referred to a hematologist to rule out hemolysis.⁷

CLINICAL APPROACH TO PATIENTS WITH SUSPECTED HEMOLYSIS

As with most diseases, the approach to hemolysis involves a combination of clinical and laboratory investigations directed by the judgment and skills of the clinician (Fig. 3.2).

Low-level or chronic hemolysis should be suspected in all patients with unexplained anemia. A detailed history and physical examination should be the cornerstone of each patient’s evaluation.

History

Onset/duration (hereditary versus acquired)

History of fatigue

History of jaundice

Abdominal pain/cholelithiasis (chronic hemolysis)

Medications (may exacerbate enzyme deficiencies)

Travel (consider infection)

History of recent or current infection

Vascular/cardiac surgery

Blood loss or sequestration (increases reticulocytes in the absence of hemolysis)

Discolored urine (intravascular hemolysis)

Complete family history (jaundice, gallbladder disease, splenectomy, hereditary anemia, or other inherited diseases)

Physical

Pallor

Increased temperature

Rapid pulse

Jaundice (chronic hemolysis)

Mechanical click from heart valves

Splenomegaly

FIGURE 3.2 A clinical approach to hemolytic anemia. LDH, lactate dehydrogenase; PNH, paroxysmal nocturnal hemoglobinuria; RBC, red blood cell.

The laboratory evaluation is performed to confirm the suspected diagnosis, provide insight regarding the underlying mechanism, and gauge a therapeutic response. The complete blood count (CBC) usually confirms the diagnosis of anemia. Reticulocytosis increases the mean cell volume (MCV) and red cell distribution width (RDW). A critical test in the evaluation of all patients with suspected hemolysis is the reticulocyte count. An increased number of reticulocytes are present in hemolysis unless erythropoiesis is suppressed. Stressed erythropoiesis associated with acute hemolysis also causes the release of large polychromatic reticulocytes with a decreased area of central pallor into the circulation, called shift cells, which can be appreciated by evaluating the peripheral blood smear.⁸ Reticulocytes are also identified by their RNA content, so automated detection of RNA in the cells provides an accurate alternative to manual inspection. Normal values for reticulocytes in newborn infants range from 2.5% to 6.5% and fall to less than 2% by the second week of life. In adults, reticulocytes comprise 0.5% to 1.5% of circulating erythrocytes in the absence of anemia, consistent with the normal turnover of 1% of normal red cell mass per day in adults. Percentages above the normal range are usually detected in the setting of hemolysis due to increased erythropoiesis. However, in the setting of anemia an uncorrected reticulocyte percentage may also reflect the prolonged survival of stress reticulocytes and the lower total number of circulating RBC. Therefore, an ARC more accurately measures the compensatory response than does the uncorrected reticulocyte percentage.⁹

Absolute reticulocyte count (ARC) = Reticulocyte percentage/100 × RBC count/µL

The normal ARC ranges between 25,000 and 75,000/µL. In patients with hemolysis, the ARC is usually elevated to levels greater than 100,000/µL. If hemolysis is acute, a rise in reticulocytes may be delayed by 3 to 5 days.

While a bone marrow examination is generally not required to determine the etiology of uncomplicated hemolysis, the peripheral blood smear should never be overlooked. This simple test is rapid, inexpensive, and can provide important clues regarding the mechanism of hemolysis (Table 3.2).

ACUTE INTRAVASCULAR HEMOLYSIS

The clinical syndrome associated with acute intravascular hemolysis deserves special attention because of its potential catastrophic consequences. Its recognition can lead to rapid institution of specific therapies and prevention of acute renal failure and death. Diagnosis and treatment of Clostridium perfringens sepsis or thrombotic thrombocytopenia purpura may be triggered by a hemolysis workup. Intravascular hemolysis is almost exclusively caused by extrinsic mechanisms, which may have the potential to be rapidly modified or reversed (Table 3.1).

Examination of several key laboratory values may also be used to assess the severity of intravascular hemolysis. Lactate dehydrogenase is released from hemolyzed RBCs. Small amounts of hemoglobin released into the circulation are metabolized in the liver after binding and clearance by haptoglobin. With robust intravascular hemolysis, a rapid decrease of serum haptoglobin to undetectable levels occurs. Free hemoglobin not bound to haptoglobin can be oxidized to methemoglobin or bound to transport proteins such as hemopexin or albumin, which the liver will then remove from the circulation. Free hemoglobin at levels of 100 to 200 mg/dL can be detected by visual examination of plasma or serum. The capacity of renal tubular cells to reabsorb free hemoglobin is limited resulting in hemoglobinuria. Because renal tubular cells slough, iron staining can identify the tubular epithelium containing hemosiderin in the urine sediment. Cessation of hemolysis leads to a rapid recovery of the haptoglobin levels, but urine hemosiderin is detectable for longer periods (Fig. 3.3). Urine hemosiderin in the absence of urine hemoglobin provides clinical evidence for subacute or chronic intravascular hemolysis. In the absence of cirrhosis, reduced levels of haptoglobin (<28 mg/dL) provide 92% sensitivity and 98% specificity for predicting hemolysis.¹⁰

SPECIAL CONSIDERATION OF ENZYME AND MEMBRANE DEFECTS

Once the more obvious causes of hemolysis are ruled out, the clinician must consider those etiologies less frequently encountered in daily practice, including enzyme or membrane defects. The laboratory evaluation can be confusing because of the numerous etiologies and the diversity of tests available. Therefore, the extent of diagnostic testing is dictated by the magnitude of hemolysis and the impact of a specific diagnosis on therapy. PNH is diagnosed by flow cytometry because of the associated absence of glycosylphosphatidylinositol-anchored proteins (e.g., CD59) on the plasma membranes of hematopoietic cells (see Chapter 6). General evaluation of the erythroid cytoskeleton abnormalities can usually be assessed by peripheral blood smear evaluation. In the case of enzymopathies, specific functional assays are available from reference laboratories.

FIGURE 3.3 Indicators of acute intravascular hemolysis. (From Hillman RS, Finch CA. Red Cell Manual. 7th ed. Philadelphia, PA: F. A. Davis; 1996, with permission.)

ERYTHROID ENZYMOPATHIES

Enzyme deficiencies are most often associated with congenital nonspherocytic hemolytic anemia. Inheritances of G6PD and phosphoglycerate kinase (PGK) deficiencies are chromosome X-linked. Because the other red cell enzyme abnormalities exhibit an autosomal recessive mode of inheritance, they may be suspected in cases of unexplained hemolysis during infancy or childhood. While G6PD deficiency may be the most common enzyme deficiency in humans,¹¹ the other enzymopathies associated with hemolysis are rarely diagnosed. Based on their low incidence, laboratory evaluation of suspected enzymopathies requires assays performed at specialized or research laboratories (e.g. Mayo Medical Laboratories, Rochester, MN), which measure the functional properties of each enzyme. In the setting of acute hemolysis, however, the magnitude of the functional deficit may be underestimated because of the generally higher levels of enzyme activity in reticulocytes and other “young” erythrocytes. As the clinical application of information contained in the human genome improves, genetic testing may become more practical for these enzymopathies. The success of clinical genotyping in this regard will depend upon the number of mutations identified, as well as the strength of correlation between genotype and phenotype. A genome-based profile of the known hemolysis-related enzymes is available on the Internet (http://fmp-8.cit.nih.gov/hembase/index.php).

Enzyme deficiencies most commonly associated with hemolysis are linked to the prevention of oxidative damage or the generation of energy (ATP) in RBCs. Glutathione reduction (hexose monophosphate shunt) is necessary for the prevention of oxidative damage from hydrogen peroxide to cellular proteins including hemoglobin. Glycolysis (Embden-Meyerhof pathway) provides the sole source of energy to the RBCs once they lose their mitochondria. Below is a brief synopsis of the enzymopathies associated with hemolysis (organized according to the involved metabolic pathway).

Enzymes Involved in Glutathione Metabolism

G6PD deficiency is the most common RBC enzyme disorder associated with hemolysis. As an X-linked disorder, it is far more common in males, but females do present with the disease due to mosaicism of the X chromosome as well as compound heterozygosity of inheritance. It has been estimated that this disorder affects 400 million people throughout the world, with the highest frequencies occurring in populations from the Mediterranean region, Africa, and China.¹¹ Clinical classification is made according to the magnitude of the enzyme deficiency and the severity of hemolysis.

Severe enzyme deficiency (less than 10% of normal activity) with chronic or intermittent hemolysis (Mediterranean and Asian populations).

Moderate enzyme deficiency (10%–60% of normal) with intermittent hemolysis usually associated with infection or drugs. Approximately 10% to 15% of African American males are moderately deficient in G6PD activity.

Mild enzyme deficiency (greater than 60% activity) without significant hemolysis.

Importantly, the severity of hemolysis among all G6PD-deficient patients depends on two major variables: G6PD protein and oxidative stress. G6PD deficiency is defined at the genetic level by mutations that cause either reduced synthesis of functional G6PD (quantitative defect) or production of abnormal G6PD (qualitative defect). The Johns Hopkins University (http://omim.org/entry/305900)has catalogued the known 400 G6PD variants. The neonatal hyperbilirubinemia in G6PD-deficient infants is caused by increased bilirubin production from erythrocyte breakdown and inadequate clearance by an immature liver. Neonates with severe G6PD deficiency are at greatest risk of developing neonatal hyperbilirubinemia.

The second major factor in determining the level of hemolysis is the level of intracellular oxidative stress. G6PD acts to catalyze the conversion of glucose-6-phosphate to 6-phosphogluconate. That biochemical reaction is coupled to the production of NADPH and subsequent reduction of glutathione. Erythrocytes that are exposed to oxidants or oxidative stresses become depleted of reduced glutathione (GSH). Once GSH is depleted, oxidation of other red blood cell sulfhydryl-containing proteins (including hemoglobin) occurs. Oxidation of hemoglobin leads to the formation of sulfhemoglobin and hemoglobin precipitates called Heinz bodies. Heinz body inclusions are generated during acute, drug-induced hemolytic episodes. Patients with moderate G6PD deficiency are generally asymptomatic in the steady state. They present episodically with acute hemolytic anemia due to oxidative stress from infections such as acute viral hepatitis and pneumonia. Hemolysis is also associated with the ingestion of fava beans, which contain pyrimidine aglycones (divicine and isouramil). Favism is most commonly associated with the G6PD variant in Mediterranean populations. Certain drugs and chemicals (Table 3.3) that increase the risk of hemolysis in G6PD-deficient patients should be avoided.¹² Hemolysis occurs 1 to 3 days after ingestion of these drugs or fava beans, with resolution usually within one week of cessation.

Testing for suspected G6PD deficiency can be performed by simple qualitative or quantitative fluorescence tests that measures the production of NADPH. During episodes of acute hemolysis, measured levels of enzyme activity may be increased due to the loss of older erythrocytes with the least activity. A more definitive diagnosis of G6PD deficiency requires genetic testing of the involved patient or family. Treatment of a G6PD-deficient individual depends on the degree of hemolysis. Potentially harmful foods and drugs should always be avoided by the patient, as well as nursing mothers of infants with G6PD deficiency. Patients with infection should be carefully monitored for early signs of increased hemolysis. Blood transfusion may be life-saving during acute hemolytic episodes. While controversial, splenectomy may be considered in cases of G6PD deficiency presenting with severe hemolysis that do not respond to other measures.

γ-Glutamylcysteine synthetase is the rate-limiting enzyme in glutathione biosynthesis. Hemolytic anemia is associated with low activity of this enzyme and normal glutathione synthetase levels. These rare patients present with a history of lifelong anemia, intermittent jaundice and spinocerebellar degeneration in adulthood.¹³

Glutathione peroxidase (GSH-Px) is primarily responsible for the elimination of hydrogen peroxide from erythrocytes. Production of this protein is dependent upon adequate nutritional levels of selenium.¹⁴ Moderate deficiencies in GSH-Px activity may result in the formation of Heinz bodies and nonspherocytic hemolytic anemia in infants. Like G6PD deficiency, oxidizing agents should be avoided in these patients.

Glutathione reductase is the enzyme that reduces oxidized GSH in the presence of flavin adenine dinucleotide. Deficiency of glutathione reductase causes increased susceptibility to drug-induced hemolysis. Glutathione reductase activity increases with dietary supplementation of riboflavin, and a subset of these patients respond well to riboflavin dietary supplements. In some cases of glutathione reductase deficiency, the enzymatic activity cannot be restored by riboflavin supplementation due to a 2,246 base pair deletion found in the gene encoding for glutathione reductase.¹⁵

Glutathione synthetase deficiency is caused by autosomal recessive inheritance of mutations of the glutathione synthetase gene with subsequently low levels of glutathione in RBCs. The disease is marked by accumulation of the metabolite oxyproline in the urine.¹⁶ The patients present with the clinical triad of hemolysis, metabolic acidosis, and mental deterioration. Treatment includes vitamin C, vitamin E, bicarbonate, and avoidance of oxidative drugs.

Table 3.3  Common Drugs and Chemicals to Avoid in Patients with Glucose-6-Phosphate Dehydrogenase Deficiency

Dapsone

Methylene blue

Nitrofurantoin

Phenazopyridine (Pyridium)

Primaquine

Rasburicase

Toluidine blue

Enzymes Involved in Glycolysis

Pyruvate kinase (PK) deficiency is associated with over 150 genetic mutations.¹⁷ PK deficiency is the second most common enzymopathy associated with congenital nonspherocytic hemolytic anemia with a prevalence of approximately 1:20,000 in Caucasian populations. PK converts phosphoenolpyruvate to pyruvate, simultaneously generating adenosine triphosphate (ATP) from adenosine diphosphate (ADP). PK activity decreases during RBC aging, as the enzyme is gradually denatured. The eventual result is failure of glycolysis as PK activity falls below a critical level. Because glycolysis is the sole source of ATP synthesis in the mature RBC, ATP depletion and hemolysis follow glycolytic failure.

Consistent with several other inherited causes of hemolysis, PK deficiency is postulated to provide some protection from malarial infection.¹⁸ Selection for variations of this gene may involve other factors as well, because PK deficiency is less common in Africa and other malarial-endemic regions. Most patients are compound-heterozygotes for the two most common mutant forms of the enzyme. Approximately one-third of the cases present with jaundice during the newborn period, and one-third of those cases are severe enough to require transfusion. Death during the neonatal period may result from severe anemia. In individuals with milder forms of the enzymopathy, the anemia is less severe and the diagnosis may not be established until later in childhood. Unfortunately, poor correlation between PK activity and the severity of clinical hemolysis confounds the accuracy of prognosis.¹⁷ There is currently no reliable method to predict success of splenectomy for individual cases.

Glucose phosphate isomerase (GPI) deficiency is the third most common glycolytic enzyme deficiency associated with hemolytic anemia. GPI catalyzes the production of fructose-6-phosphate from glucose-6-phosphate. It is found in all ethnic groups, but is prevalent in individuals of European descent. Over two-dozen genetic variants have been identified to date with considerable variability in disease severity. In severe cases, anemia and hyperbilirubinemia are evident at birth. In addition to chronic hemolysis and hyperbilirubinemia, acute hemolytic crises can occur with viral and bacterial infections.¹⁹

Aldolase A deficiency has been found to cause moderately severe lifelong hemolytic anemia, sometimes requiring transfusions during acute hemolytic crises. Aldolase catalyzes the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Abnormal expression of the aldolase variant causes hemolysis and myopathy.²⁰ Other congenital anomalies include short stature, mental retardation, delayed puberty, and a distinct facial appearance.

2,3-Diphosphoglycerate mutase (DPGM) deficiencies of greater than 50% cause a compensated hemolytic anemia. DPGM converts 1,3-biphosphoglycerate to 2,3-diphosphoglycerate (2,3-DPG). Deficiencies of this enzyme may lead to a combination of hemolysis and polycythemia because of the resulting 2,3-DPG deficiency.²¹

Enolase is the enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate. Case studies have shown a decrease in the enzyme activity in patients with a mild spherocytic hemolytic anemia.²²

Hexokinase deficiency causes a rare congenital hemolytic anemia, predominantly in persons of northern European ancestry. Hexokinase acts at the initial enzymatic step in glycolysis, catalyzing the conversion of glucose to glucose-6-phosphate. Hexokinase activity in reticulocytes is considerably higher than in mature cells. Anemia is associated with a reduction of this enzyme activity to 25% that of normal erythrocytes.²³

Phosphofructokinase deficiency (also called Tarui disease) results in a glycogen storage disorder characterized by hemolysis and myopathy. Phosphofructokinase is an allosteric enzyme that catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-diphosphate. Most affected individuals have exhibited exertional myopathy resulting in weakness, easy fatigability, muscle cramps on exercise, and myoglobinuria. Hemolysis is caused by decreased erythrocyte deformability from leakage of calcium ions.²⁴

PGK deficiency results in a moderate-to-severe nonspherocytic hemolytic anemia. PGK converts 1,3 biphosphoglycerate to 3-phosphoglycerate. PGK deficiency is the only X-linked disorder involved in glycolysis. The disease phenotype is unusually pleomorphic and may include varying degrees of hemolytic anemia, mental retardation, and myopathy.²⁵

Triosephosphate isomerase (TPI) deficiency is a rare disorder characterized by severe hemolytic anemia and increased susceptibility to infection. In addition, progressive neurological deterioration is a hallmark of the associated disease. Deficiencies usually become evident during infancy, with spasticity, motor retardation, hypotonia, weakness, and seizures.²⁶

Other Enzymopathies Associated with Hemolysis

Pyrimidine 5′ nucleotidase (uridine 5′ monophosphate hydrolase) deficiency leads to accumulation of high concentrations of pyrimidine nucleotides within the erythrocytes that precipitate and cause basophilic stippling. Diagnosis is confirmed by a decrease in the nucleotide OD260:OD280 ratio and measurement of enzyme activity. Disease severity is variable, but the patients typically manifest lifelong hemolysis with the expected sequelae.²⁷

Adenosine deaminase (ADA) is a purine catabolic enzyme that converts adenosine to inosine. ADA deficiency causes inherited severe combined immunodeficiency. In contrast, elevations in ADA cause hemolytic anemia. Studies show that ADA amplification in reticulocytes results from increased translation of ADA mRNA.²⁸

Heme oxygenase-1 is an enzyme involved in the conversion of heme to bilirubin. Heme oxygenase-1 further provides protection against certain oxidative stresses. Two pediatric patients were described with severe growth retardation, asplenia, an abnormal coagulation/fibrinolysis system, and persistent hemolytic anemia. This enzyme deficiency may cause the unique findings of erythrocyte fragmentation and intravascular hemolysis in the absence of hyperbilirubinemia or decreased haptoglobin potentially due to a defect in the macrophage’s ability to catabolize heme.²⁹

Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in lipoprotein metabolism. Deficiencies of LCAT result in erythroid membrane defects caused by excess unesterified cholesterol. The patients also develop renal disease with proteinuria, corneal opacifications, and their serum lipid profiles include decreased serum HDL levels (55–10% normal).³⁰

ERYTHROID MEMBRANE DEFECTS

The RBC membrane comprises integral and peripheral proteins distributed in the context of a lipid bilayer. Integral membrane proteins interact to form a lattice-like structure (cytoskeleton) at the cytoplasmic surface of the lipid bilayer that is responsible for the strength and deformability of the red blood cell. Band 3, a protein that functions as an anion exchanger (AE1), is the major protein that physically links the lipid bilayer to the underlying membrane cytoskeleton. The cytoskeleton proteins include spectrin, ankyrin, actin, band 3, band 4.1, and band 4.2. Other red cell membrane proteins serve roles in maintaining osmotic equilibrium or have adhesive properties. The exact function of a number of erythroid membrane proteins remains vague.

Immune-mediated hemolysis results from antibodies directed toward erythrocyte membrane proteins. Approximately 24 proteins are largely responsible for transfusion-related alloimmunity (see Chapter 24). Nonimmune hemolysis may also be due to rare erythroid phenotypes involving those proteins. Nonimmune hemolysis due to the Rh-null phenotype is generally mild and well compensated with a reticulocyte count below 10%. The red cell morphology may be stomatocytic or spherocytic, and the RBC osmotic fragility is increased.³¹ Weak expression of the Kell blood group results in the so-called “McLeod phenotype.” This phenotype is X-linked and occurs with relatively high frequency in individuals who have chronic granulomatous disease (CGD). Neurodegeneration and acanthocytosis are the characteristic features of the McLeod phenotype. The associated hemolysis is mild with slightly elevated reticulocyte counts.³²

The first indication that a patient may have a membrane abnormality as a cause for hemolysis usually comes from microscopic examination of the peripheral blood smear. As shown in Table 3.2, the presence of spherocytes, elliptocytes, stomatocytes, acanthocytes, or pyropoikilocytes may be the primary alert for an underlying membrane defect.

Hereditary spherocytosis (HS) is the most common hereditary anemia among people of northern European descent, occurring at a frequency of 1 in 5,000. It is commonly caused by mutations in the genes that encode the components of the erythroid cytoskeleton (α- or β-spectrin, ankyrin, band 3, protein 4.2). Autosomal recessive or dominant patterns of inheritance have been identified, and a positive family history is gathered in over half of the cases. Patients are usually diagnosed in childhood with the clinical triad of anemia, jaundice, and splenomegaly. Spherocytes are identified by their small size (low MCV) and absence of the central pallor seen in normal erythrocytes. Immune-based spherocytosis is ruled out by negative DAT testing.

In most cases, the clinical presentation and hematological parameters are sufficient to make the diagnosis.³³ If the diagnosis is subtle or complicated, confirmation of the diagnosis using assays of membrane fragility, electrophoretic quantitation of membrane proteins, or genetic analyses may be considered. The osmotic fragility test for HS has been used to detect hemolysis by measuring the fraction of total hemoglobin released from red cells at progressively more dilute salt concentrations. Hemolysis occurs in circulating HS spherocytes at salt concentrations that do not affect normal RBC. A cryohemolysis test may also be used to detect increased hemolysis in HS erythrocytes. Red cells are suspended in a hypertonic solution, briefly heated to 37°C, and then cooled to 4°C for 10 minutes. A widely separated degree of hemolysis between spherocytes and normal cells is seen with the cryohemolysis test, and asymptomatic disease carriers may also be identified.³⁴ Although interesting historically, membrane fragility tests lack sensitivity and specificity, and therefore should be ordered conservatively as they add little to the evaluation of a well-prepared blood smear.

Hereditary elliptocytosis (HE) is endemic in areas of Africa and Asia. The disease also results from mutations in the α-spectrin, β-spectrin, and band 4.1 genes. In the homozygous state, hemolysis may be lifelong and exacerbated by acute or chronic illnesses.³⁵ In the heterozygous state, people with HE have no clinical syndrome, with slight reticulocytosis and the characteristic abnormalities of the red blood cell morphology providing the only clues toward diagnosis.

Hereditary pyropoikilocytosis is a severe type of HE resulting from a mutation in either protein 4.1 or α-spectrin. The usual presentation involves mild to moderate hemolytic anemia with evidence of marked poikilocytosis. The spectrin in these abnormal cells has an increased sensitivity to thermal denaturation, and the cells exhibit mechanical fragility. As a result, the red cell volume distribution is broad, and a striking number of fragmented cells and microspherocytes are observed on the peripheral smear.³⁶

Hereditary stomatocytosis is identified by a pinched rather than circular area of central pallor in erythrocytes. While all the patients share the common clinical feature of stomatocytes, research in defining the underlying cause of the morphological change has resulted in more specific clinical descriptions including dehydrated hereditary stomatocytosis (xerocytosis), overhydrated hereditary stomatocytosis, and cryohydrocytosis. The common features include hemolysis and red cell cation leaks and variable severity of hemolysis.³⁷ Most importantly, splenectomy is not helpful and may result in a high risk of thromboembolic disease.³⁸

Acanthocytosis on the peripheral smear may be caused by abnormal lipids in hepatic cirrhosis, or by other abnormalities in lipids or band 3. Abetalipoproteinemia is a rare genetic disorder resulting in hypolipidemia, acanthocytosis, malabsorption of fat, retinitis pigmentosa, and ataxia. Infants with this autosomal recessive disorder are normal at birth but soon develop steatorrhea, abdominal distension, and growth failure. Retinitis pigmentosa and ataxia appear between ages 5 and 10 years and are progressive. Therefore, acanthocytosis seen on the peripheral smear of children in the absence of hemolysis or liver disease should alert the clinician to consider associated neurodegenerative conditions.³⁹

TREATMENT OPTIONS FOR CONFIRMED HEMOLYSIS

Therapeutic strategies for hemolytic anemia are determined by the underlying cause of red cell destruction, the magnitude of the anemia, and cardiopulmonary status of the patient.

For extrinsic causes, the treatment plan usually becomes obvious at the time of diagnosis. Immune-mediated hemolysis may require immunoglobulin infusion, corticosteroids, or other immunosuppressive therapies. Transfusion therapy (packed RBCs) should be avoided unless absolutely necessary. However, RBC transfusion should not be withheld if a severely compromised cardiopulmonary status exists even when compatibility of the donor cells is incomplete. In those rare cases, the department of transfusion medicine involved should be asked to identify the most compatible product available, and the transfusion should be closely monitored. Infections are treated with antimicrobials. For thrombotic thrombocytopenic purpura, plasmapheresis is specifically indicated along with immunosuppressive therapy to reverse the depletion of ADAMTS-13 that results in excessive high molecular weight multimers of von Willebrand factor triggering disseminated platelet thrombi. Prevention of immune- or G6PD-mediated hemolysis involves discontinuation or avoidance of the associated medications. Monitoring urine hemoglobin and hemosiderin levels can determine the response to intravascular hemolysis therapy.

One acquired intrinsic red cell disorder, PNH, can be effectively treated by eculizumab, an anti-C5 antibody that can control the intravascular hemolysis by converting it to a milder extravascular hemolysis (Chapter 6). Eculizumab has also been shown to reduce the thrombosis risk that accompanies the intravascular hemolysis. Immunization with anti-meningococcal vaccination is required before eculizumab is prescribed.40,41 Eculizumab has also been reported to reverse thrombotic microangiopathy and renal failure in patients with atypical hemolytic uremic syndrome.⁴²

Despite the considerable advances in defining these genetic diseases at the molecular level, equally specific treatment regimens for other intrinsic causes of hemolysis are lacking. In general, intrinsic causes of hemolysis are inherited, and they are present during infancy or childhood. In those cases, prognosis and treatment are complex, as the hemolytic picture may change over time. The first question to ask is whether treatment is necessary. Chronic hemolysis may only require a yearly clinical evaluation of the CBC, ARC, and blood smear to determine if the patient is able to maintain adequate levels of erythropoiesis. Parvovirus infections in these patients may result in acute worsening of their anemia due to a sudden decrease in their erythropoiesis. Folic acid should be given to all patients with chronic hemolysis (1 mg/day) because this vitamin is consumed with the accelerated production of erythrocytes. Whether folic acid supplementation is still necessary in the United States where the fortification of food folate has occurred since the mid-1990s has not been evaluated. Transfusion regimens should be tailored for individual patients, and iron overload should be anticipated. Even in the absence of transfusion, iron overload may result from ineffective erythropoiesis. The increased metabolism of heme also leads to a significant increase in pigmented gallstone formation.

Treatments such as splenectomy or bone marrow transplantation should be reserved for marked hemolysis producing life-threatening anemia. As stated above, splenectomy should be discouraged for patients with hereditary stomatocytosis syndromes.³⁸ When severe hemolysis is due to other membrane defects, splenectomy may be beneficial and indicated. In children, splenectomy should be delayed until the age of 6 (if possible) due to the increased risk of sepsis. In general, splenectomy risks must be compared to those associated with lifelong transfusion. After a splenectomy is performed, special care must be taken to compensate for the loss of splenic function. The spleen is responsible for the clearance of encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis. The combined use of pneumococcal polysaccharide immunization and early empiric antibiotic therapy offers a high level of protection for post-splenectomy patients. It is estimated that sepsis is fatal in 40% to 50% of all splenectomized patients. Within that group, children with thalassemia and sickle cell syndromes have the highest risk of death.⁴³ In all cases, patients must be informed that the asplenic state carries a significant risk of overwhelming and life-threatening infection.

CONCLUSION

A broad range of genetic and acquired diseases are manifested by hemolysis. The differential diagnosis is useful in developing diagnostic and therapeutic strategies and should be thought of in terms of intrinsic or extrinsic causes of erythrocyte damage. A careful search for the cause of hemolysis should be pursued because treatments are so different. When a common cause of hemolysis is not found, an underlying enzyme or membrane defect should be sought.

Clinical severity in all cases of hemolysis is determined by the rate of red cell destruction, and the host’s ability to compensate by producing fresh erythrocytes. Disease can vary from a subtle and clinically silent syndrome to hemolysis of sufficient intensity to dominate the clinical picture and even cause death if left untreated. Every therapeutic plan should be designed for both the severity of disease as well as the cause of hemolysis.

HELPFUL INTERNET SITES

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM

http://fmp-8.cit.nih.gov/hembase/index.php

References

  1.  Lichtman MA, Beutler E, Kipps TJ, et al. Williams Hematology. 8th ed. New York, NY: McGraw-Hill Publishers; 2010.

  2.  Dacie JV. The Haemolytic Anaemias: Secondary or Symptomatic Haemolytic Anaemias. 3rd ed. Vols. 1–5. New York, NY: Churchill Livingstone; 1985–1999.

  3.  Nathan DG, Orkin SH, Oski FA. Hematology of Infancy and Childhood. 6th ed. Philadelphia, PA: WB Saunders; 2003.

  4.  Foerster J, Lee RG. Wintrobe MM, eds. Wintrobe’s Clinical Hematology. 10th ed. Philadelphia, PA: Williams & Wilkins; 2008.

  5.  Hoffman, R, Benz EJ, Shattil, SJ, et al. Hematology: Basic Principles and Practice. 4th ed. Philadelphia, PA: Elsevier; 2005.

  6.  Beutler E, Luzzatto L. Hemolytic anemia. Semin Hematol. 1999;36:38-47.

  7.  Strassburg CP. Hyperbilirubinemia syndromes (Gilbert-Meulengracht, Crigler-Najjar, Dubin-Johnson, Rotor syndrome). Best Pract Res Clin Gastroenterol. 2010;5:555-571.

  8.  Finch CA, Hillman RS. Red Cell Manual. 7th ed. Philadelphia, PA: F.A. Davis; 1996.

  9.  Riley RS, Ben-Ezra JM, Tidwell A, et al. Reticulocytes and reticulocyte enumeration. J Clin Lab Anal. 2001;15:267-294.

10.  Körmöczi GF, Säemann MD, Buchta C, et al. Influence of clinical factors on the haemolysis marker haptoglobin. Eur J Clin Invest. 2006;36:202-209.

11.  Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64-74.

12.  Youngster I, Arcavi L, Schechmaster R, et al. Medications and glucose-6-phosphate dehydrogenase deficiency: an evidence-based review. Drug Saf. 2010;33:713-726.

13.  Hamilton D, Wu JH, Alaoui-Jamali M, et al. A novel missense mutation in the γ-glutamylcysteine synthetase catalytic subunit gene causes both decreased enzymatic activity and glutathione production. Blood. 2003;102:725-730.

14.  Gondo H, Ideguchi H, Hayashi S, et al. Acute hemolysis in glutathione peroxidase deficiency. Int J Hematol. 1992;55:215-218.

15.  Mojzikova R, Dolezel P, Pavlicek J, et al. Partial glutathione reductase deficiency as a cause of diverse clinical manifestations in a family with unstable hemoglobin (Hemoglobin Haná, β63(E7) His-Asn). Blood Cells Mol Dis. 2010; 45:219-222.

16.  Ristoff E, Larsson A. Inborn errors in the metabolism of glutathione. Orphanet J Rare Dis . 2007;2:16-24.

17.  Zanella A, Fermo E, Bianchi P, et al. Red cell pyruvate kinase deficiency: molecular and clinical aspects. Br J Haematol . 2005;130:11-25.

18.  Wang C, Chiarelli LR, Bianchi P. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001;98(10):3113-3120.

19.  Kugler W, Lakomek M. Glucose-6-phosphate isomerase deficiency. Baillière’s Clin Haematol. 2000;13:89-101.

20.  Yao DC, Tolan DR, Murray MF, et al. Hemolytic anemia and severe rhabdomyolysis caused by compound heterozygous mutations of the gene for erythrocyte/muscle isozyme of aldolase, ALDOA. Blood. 2004;103:2401-2403.

21.  Jacobasch G, Rapoport SM. Hemolytic anemias due to erythrocyte enzyme deficiencies. Mol Aspects Med. 1996;17:143-170.

22.  Boulard-Heitzmann P, Boulard M, Tallineau C. Decreased red cell enolase in 40 year old woman with compensated haemolysis. Scand J Haemotol. 1984;33:401-404.

23.  Kanno H. Hexokinase: gene structure and mutations. Baillière’s Clin Haematol. 2000;13:83-88.

24.  Sabina RL, Waldenström A, Ronquist G. The contribution of Ca²†-calmodulin activation of human erythrocyte AMP deaminase (isoform E) to the erythrocyte metabolic dysregulation of familial phosphofructokinase deficiency. Haematologica. 2006;91:652-655.

25.  Beutler E. PGK deficiency. Br J Haematol. 2007;136:3-11.

26.  Oláh J, Orosz F, Keserü GM, et al. Triosephosphate isomerase deficiency: a neurodegenerative misfolding disease. Biochem Soc Trans. 2002;30:30-38.

27.  Zanella A, Bianchi P, Fermo E, et al. Hereditary pyrimidine 5′-nucleotidase deficiency: from genetics to clinical manifestations. Br J Haematol. 2006;133:113-123.

28.  Chottiner EG, Cloft HJ, Tartaglia AP, et al. Elevated adenosine deaminase activity and hereditary hemolytic anemia: evidence for abnormal translational control of protein synthesis. J Clin Invest. 1987;79:1001-1005.

29.  Fraser ST, Midwinter RG, Berger BS, et al. Heme oxygenase-1: a critical link between iron metabolism, erythropoiesis, and development. Adv Hematol. 2011;2011:473709.

30.  Hovingh GK, de Groot E, van der Steeg W, et al. Inherited disorders of HDL metabolism and atherosclerosis. Curr Opin Lipidol. 2005;16:139-145.

31.  Nash R, Shojania AM. Hematological aspect of Rh deficiency: a case report and review of literature. Am J Hematol. 1987;24:267-275.

32.  Walker RH, Danek A, Uttner I, et al. McLeod phenotype without the McLeod syndrome. Transfusion. 2007;47:299-305.

33.  Bolton-Maggs PH, Stevens RF, Dodd NJ, et al. Guidelines for the diagnosis and management of hereditary spherocytosis. Br J Haematol. 2004;126:455-474.

34.  Iglauer A, Reinhardt D, Schöter W, et al. Cryohemolysis test as a diagnostic tool for hereditary spherocytosis. Ann Hematol. 1999;78:555-557.

35.  Delaunay J. The molecular basis of hereditary red cell membrane disorders. Blood Rev. 2007;21:1-20.

36.  Ramos MC, Schafernak KT, Peterson LC. Hereditary pyropoikilocytosis: a rare but potentially severe form of congenital hemolytic anemia. J Pediatr Hematol Oncol. 2007;29:128-129.

37.  Delaunay J. The hereditary stomatocytoses: genetic disorders of the red cell membrane permeability to monovalent cations. Semin Hematol. 2004;41:165-172.

38.  Stewart GW, Amess JA, Eber SW, et al. Thrombo-embolic disease after splenectomy for hereditary stomatocytosis. Br J Haematol. 1996;93:303-310.

39.  Jung HH, Danek A, Walker RH. Neuroacanthocytosis syndromes. Orphanet J Rare Dis. 2011;25:68.

40.  Brodsky RA, Young NS, Antonioli E, et al. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Blood. 2008;111:1840–1847.

41.  Parker CJ. Management of paroxysmal nocturnal hemoglobinuria in the era of complement inhibitory therapy. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29.

42.  Nurnberger J, Witzke O, Opazo Saez A, et al. Eculizumab for atypical hemolytic-uremic syndrome. N Engl J Med. 2009;360:542-544.

43.  Bisharat N, Omari H, Lavi I, et al. Risk of infection and death among post-splenectomy patients. J Infect. 2001;43:182-186.

4

Hemolytic Anemia: Thalassemias and Sickle Cell Disorders

Matthew M. Hsieh, John F. Tisdale and Griffin P. Rodgers

Normal hemoglobin within red blood cells is comprised of two α and two β chains, with α to β synthesis ratio of 1 to 1. Thalassemias are a group of quantitative disorders with insufficient production of α or β chains, leading to an imbalanced accumulation of β or α chains, respectively. In contrast, hemoglobinopathies (or abnormal hemoglobin structural variants) are a separate group of qualitative disorders, with abnormal β or α chains in normal quantity, of which sickle cell disease (SCD) is best recognized. While these two disorders share features of hemolytic anemia and transfusion-related complications, they differ in their pathophysiology, clinical manifestations, and management (Table 4.1). The thalassemias and hemoglobinopathies are commonly encountered in areas where malaria is endemic because abnormal genes offer protection against malaria.¹

PATHOPHYSIOLOGY

Thalassemias

Normally there are four copies of α-globin gene, two copies on each chromatid of chromosome 16 (Table 4.2). α-Globin chains are essential in the synthesis of both fetal and adult hemoglobin. α-Thalassemia syndromes result from deletions of a large α-globin gene segment from unequal crossover or recombination, and less frequently from mutations. The deleted segments of DNA vary in size and can involve one (−+, same as α+ or trans) or both (−−, same as α0 or cis) alleles on the same chromatid. A deletion of one gene (−+/++) confers a silent carrier. A two-gene deletion (−−/++ or −+/−+) is commonly referred as α-thalassemia minor or trait with microcytosis, hypochromia, but little or no anemia (Fig. 4.1). The deletion of three genes (−−/−+) leads to Hb H (β4), which is an unstable form of hemoglobin. Hb H disease is manifested by hypochromia, moderately severe hemolytic anemia, and splenomegaly. The absence of all four genes leads to hydrops fetalis with Hb Bart (γ4). Hb Bart transports O₂ poorly, causes profound tissue hypoxia, leads to heart and liver failure, and is almost always incompatible with life without in utero red cell transfusion.

Both α-globin gene deletion haplotypes, (−+) and (−−), occur equally in Southeast Asians, whereas the (−−) haplotype is much less common in Mediterraneans and rare in Africans. Hence all the α-thalassemic syndromes are seen in Southeast Asians, but hydrops fetalis is uncommon to rare in Mediterraneans and Africans. In addition to α-globin gene deletions, there are α-globin structural variants, which may occur alone or in combination with α-gene deletions, and lead to further reduction of α-globin synthesis. The best characterized α-globin variant is Hb Constant Spring.

In contrast to α-globin genes, there are only two β-globin genes, one on each chromatid of chromosome 11. Whereas there are close to 200 mutations described, only about 20 mutations account for the majority of ß-thalassemic individuals. Mutations are grouped by regional ethnic locations: Mediterranean basin, Southeast Asia, Africa, and Asian India. All disease-causing mutations alter β-globin gene mRNA transcription, processing, or translation. Some mutations decrease β-globin production by as little as 10% and some by as much as 90%. Homozygosity or heterozygosity of variably affected alleles explains the wide range of β-thalassemic syndromes.

Patients with one abnormal allele have β-thalassemia minor or trait: the synthesis of the β chain is reduced by about one-half. Although normal Hb A (α2β2) is mildly decreased, there is no accumulation of excess α-chains. There is hypochromia and microcytosis, but no clinically significant anemia, hemolysis, or ineffective erythropoiesis. Thalassemia intermedia refers to the condition in patients with a lesser degree of hemolytic anemia, usually secondary to compound heterozygosity of two mild β-thalassemia alleles, δ- and β-thalassemia, Hb E and β-thalassemia, β-thalassemia with hereditary persistence of fetal hemoglobin (HPFH), or co-existence of α- and β-thalassemia. The phenotype of two severe β-chain alleles is referred to as β-thalassemia major or Cooley’s anemia: β-chain synthesis and Hb A are virtually absent, with α-chains in great excess and consequently leading to severe hemolytic anemia. The compensatory increase in Hb A₂ and F is inadequate to offset the lack of β chain production.

FIGURE 4.1 Diagram for α-thalassemia gene deletion and corresponding phenotypes. Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.

Hemoglobinopathies: Hemoglobin Structural Variants

Hemoglobin variants of α or β chains, or the hemoglobinopathies, are most commonly caused by point mutations. The nomenclature of hemoglobinopathies employs alphabetic letters (S, C, or E), and sometimes occurs with locations of first discovery (OArab or DPunjab) or name of the index case (Lepore or Constant Spring), then followed by the chain, location, and amino acid substitution in that hemoglobin chain (β⁶ Glu→Val).

Hemoglobin S

Sickle hemoglobin (Hb S) is the best characterized hemoglobinopathy. SCD is an inherited disorder in which normal glutamic acid is substituted by valine in the 6th codon of β-globin chain (β6 Glu→Val), which favors bonding of hemoglobin molecules. As a result, Hb S is less soluble when deoxygenated (in the normal oxygenation–deoxygenation cycle), precipitates and polymerizes quickly in red cells, and causes a morphologic change to a crescent shape. These rigid sickle cells lead to hemolytic anemia and vaso-occlusion, which together cause all the complications of SCD.

The lifespan of sickle cells is about 10 to 20 days, compared to 120 days for normal red cells. In the absence of clinically significant pain episodes, there is a chronic hemolytic anemia with mean hemoglobin of 6 to 8 g/dL, despite compensatory reticulocytosis of greater than 5% or 150 k/µL. Most sickle erythrocytes are removed in the spleen; some are destroyed intravascularly by mechanical forces or oxidative stress. Hemolysis has been implicated to activate inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), thrombin, and platelet-activating factor.² Leukocytosis is common at baseline, and higher leukocyte counts are often associated with more frequent pain crises, stroke, and a shorter life expectancy in homozygous sickle disease (Hb SS) patients. Free hemoglobin, released by hemolysis, can consume nitric oxide and participate in endothelial dysfunction to promote vasoconstriction.

Other Hemoglobinopathies (E, C, Lepore, D, OArab, Constant Spring)

Hb E is a common hemoglobin variant, present in about 15% to 30% of the individuals in southern China and Southeast Asia. Hb E results from replacement of the normal glutamic acid to lysine in the 26th amino acid of the β-chain (β26 Glu→Lys), and leads to only 50% of mRNAs being spliced normally. Individuals with heterozygous and homozygous Hb E have mild anemia, hypochromia, and microcytosis. When Hb E is combined with β-thalassemia, the clinical features resemble those of β-thalassemia intermedia.

Hb C results from substitution of the normal glutamic acid to lysine in the 6th amino acid of β-chain. Hb C is found mostly in individuals of African descent and is the second most common hemoglobinopathy in the United States and third most common worldwide. Carriers of Hb C are asymptomatic; homozygous individuals (Hb CC) exhibit mild hemolytic anemia but are largely asymptomatic. Hb C combined with β-thalassemia produces mild to moderate hemolytic anemia with some features of β-thalassemia major. Compound heterozygosity with Hb C and S (Hb SC) leads to milder anemia with fewer leg ulcers, pain crises, and osteonecrosis than with homozygous SCD (Hb SS); there is also a slightly lower risk of infection from encapsulated organisms. Retinal proliferative disease, avascular necrosis, and splenomegaly, however, manifest earlier and more frequently in Hb SC disease.

Hb Lepore is a fused globin chain, which consists of the N-terminal half of the δ-chain and the C-terminal half of the β-chain; it is produced at very low levels (2.5%) compared with normal β-chains. Although typically seen in Greeks or Italians, this variant can occur in many ethnic groups of northern European descent. Hb Lepore can occur alone or in combination with other β-thalassemic mutations, leading to symptoms similar to β-thalassemia major. Hb D (same as Hb Los Angeles or Hb Punjab), another β-chain variant, is seen in the Asian Indian population. When combined with β-thalassemia or SCD, the anemia is mild. Hb OArab, a rare β-chain variant, when combined with SCD (Hb SOArab), behaves similar to severe SCD.

Hb Constant Spring, present in 5% to 10% of Southeast Asians, is caused by a point mutation in the stop codon of α-chain mRNA, leading to an elongated α-chain (αCS). Because synthesis of αCS is much reduced (to about 1%), Hb Constant Spring behaves like an α-chain deletion. When αCS is combined with a cis α-thalassemic defect, it resembles Hb H disease (−−/αCS α). Fortunately αCS is typically coupled with a normal α-chain gene (αCS α) on the same allele, and hydrop fetalis has not been observed.

DIAGNOSIS AND SCREENING

The diagnosis of SCD and thalassemias is now accomplished by neonatal or prenatal testing. The goal of postnatal testing is to identify α- or β-thalassemia carriers, Hb S, C, E, and other clinically important hemoglobinopathies. The process typically begins with a complete blood count (CBC). When red cell indices are suggestive (Table 4.3), peripheral blood smear and high performance liquid chromatography (HPLC) provide a provisional diagnosis. HPLC has largely replaced traditional electrophoresis because it reliably quantitates the fraction of hemoglobin A₂, F, and S. Hemoglobinopathies are confirmed by isoelectric focusing or gel electrophoresis under alkaline (separates Hb S from Hb D/G) or acidic (separates Hb C, E, and OArab) conditions. Specific thalassemia mutations require polymerase chain reaction (PCR) based DNA testing. Blood count indices vary widely and may deviate from typical values if there is concurrent iron deficiency or compound heterozygosity of other hemoglobinopathies. Any transfusion would also alter the hematologic parameters commonly found in each syndrome.

α-Thalassemia Trait and Disease

α-Thalassemia trait can been suspected with elevated total red blood cell number, normal or borderline Hb A₂, mean corpuscular volume (MCV) less than 78 fL, and mean corpuscular hemoglobin (MCH) less than 25 pg. The peripheral blood smear in Hb H disease can be stained with cresol blue to show Hb H precipitates in erythrocytes and reticulocytes.

β-Thalassemia Trait and Major

β-Thalassemia trait can be suspected from an elevated total red blood cell number, MCV less than 78 fL, MCH less than 27 pg, and normal or slightly low hemoglobin. On HPLC, there is a characteristic elution pattern of variably elevated Hb F (higher in the Mediterranean variant, and lower in the African variant), normal Hb A, and >4% Hb A₂. However, Hb A₂ may be normal (less than 3%) in individuals with concurrent iron deficiency or α-thalassemia, compound heterozygous δ- and β-thalassemia, or those with certain β-chain mutations. With β-thalassemia major, the MCV is usually less than 70 fL, MCH is less than 25 pg, there is variably low hemoglobin (5–9 g/dL), and no Hb A on HPLC.

Sickle Cell Trait and Disease

Sickle cell trait (SCT) or disease can be diagnosed by the combination CBC, HPLC, and the sickle solubility test. SCT has near-normal red cell indices; hemoglobin may be slightly low to normal and the MCH and red cell distribution width (RDW) may be slightly elevated. Hb S will comprise 35% to 40% on HPLC. On the other hand in SCD, hemoglobin will range from 6 to 8 g/dL, and sickle erythrocytes will be seen on peripheral blood smear. Hb S will comprise 80% to 90% of total hemoglobin and slightly elevated Hb A₂ on HPLC (due to co-elution of Hb S with Hb A₂). Trait or SCD is then confirmed by sickle solubility test and acidic or alkaline gel electrophoresis to screen for other concurrent hemoglobinopathies, such as Hb D or G. Sickle SC disease is easily distinguished by HPLC. Additionally, the peripheral smear in Hb SC disease shows fewer sickle cells, more spherocytes and target cells, and an uneven distribution of hemoglobin among red blood cells.

CLINICAL SYNDROMES AND TREATMENT OF SICKLE CELL DISEASE

Vaso-Occlusive Episodes (Pain Crises)

Vaso-occlusive episode (VOE) is the most frequent clinical manifestation of SCD,³ and can occur spontaneously or precipitated by infection, stress, dehydration, or changes in weather/temperature. Frequent assessment and adjustment of pain therapy, involvement of a pain management service, and other consultations are important to address the complex etiology of pain in SCD. Evaluation begins by obtaining a full history of current and prior VOEs. Physical examination and vital signs identify any signs or symptoms related to a pain episode. Acute pain can affect multiple sites: bones, joints, the cardiopulmonary system, CNS, or abdominal visceral organs. Chronic pain is typically confined to leg ulcers and the skeletal system.

Mild acute pains are often managed in the outpatient setting with a combination of nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, and/or an oral opioid. Moderate to severe acute pain typically requires intervention in a day-hospital or emergency department, which begins with rapid assessment of the pain, hydration using 5% dextrose in half normal saline (D5 ¹/2 NS) and 20 mEq KCl, not exceeding 1¹/2 times maintenance, and an opioid analgesic (typically morphine, hydromorphone, or fentanyl). The choice, dose, and frequency of medication depend on the patient’s outpatient drug regimen and prior responses. Severe pain is managed by inpatient bolus and continuous infusion of an opioid analgesic, often through patient-controlled delivery (PCA) pumps. In those with poor intravenous access, subcutaneous injection is an acceptable short-term alternative; however, intramuscular injection should be avoided because the absorption varies. Parenteral meperidine should not be used as first-line treatment because the metabolite, normeperidine, has a long half-life and increases the risks of mood disturbances and seizures. Opioid agonists are metabolized by the liver and excreted variably by the kidney, and dose reduction may be necessary in those with hepatic impairment. Meperidine and morphine have active metabolites, and should be used with great caution in patients with renal impairment. The common side effects of all opioids—nausea, vomiting, pruritus, constipation, and respiratory depression—should be monitored and treated accordingly.

Non-opioid analgesics such as acetaminophen and NSAIDs have a ceiling effect and are often used with an oral opioid agonist. The total acetaminophen dose should not exceed 4 grams daily in adults with normal hepatic function. Gastrointestinal, renal, and hematologic toxicities should be monitored. Benzodiazepines, antidepressants, anti-emetics, and opioid agonist-antagonists (such as pentazocine, nalbuphine, and butorphanol), are useful adjuncts to opioid agonists and potentiate their analgesic effects. Gabapentin and tricyclic antidepressants can be useful for neuropathic pain.

In a recent review of chronic opioid therapy,⁴ failure to achieve desired analgesia may result from (i) opioid tolerance, where the number of opioid receptors is reduced; (ii) opioid-induced hypersensitivity, where an individual experiences increased tenderness from known noxious stimuli; or (iii) worsening pain from progressive tissue damage. Prolonged, high dose opioid therapy is associated with testosterone deficiency and suppression of immunity. Opioid dose de-escalation every 6 to 8 weeks or opioid rotation with a period of opioid abstinence may improve desired analgesia and minimize adverse effects in long-term analgesic therapy (Table 4.4).

Red cell transfusions are not routinely administered during typical VOEs, but are important for concurrent complications, such as acute chest syndrome (ACS), stroke, or other organ ischemia and damage.

Infections

Because of functional asplenia, SCD patients are at an increased risk of infection with encapsulated organisms: Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Thus, fever should be evaluated and managed promptly as a potential sepsis event, and empiric antibiotics administered while awaiting blood or urine culture and chest radiograph results. Neonatal diagnosis enables prompt initiation of penicillin prophylaxis and family education about vigilant monitoring for infections. In a placebo-controlled clinical trial,⁵ prophylactic penicillin prevented 84% of life-threatening S. pneumoniae infections. Vaccinations against S. pneumoniae should begin concurrently with penicillin prophylaxis. Penicillin may be discontinued in those older than 5 years of age who have completed vaccination, because there was no statistically significant additional benefit compared to placebo. Patients allergic to penicillin can receive azithromycin (10 mg/kg, up to 250 mg/day).

Human parvovirus B19 is commonly spread among school-age children. B19 infects erythroid progenitors and causes transient red cell aplasia. While there is a wide range of clinical severity, influenzalike symptoms, fever, pain, and splenic sequestration can accompany an acute infection. Laboratory testing may reveal acute anemia, reticulocytopenia, and IgM antibody to parvovirus. Milder forms of SCD, e.g., Hb SC or Hb S/β⁺ thalassemia, hydroxyurea treatment, or chronic transfusions, do not protect SCD individuals from developing severe complications related to B19.⁶ Parvovirus infection is also known as Fifth Disease, but in patients suffering transient aplastic crisis, the characteristic facial rash is absent. B19 can cross the placenta and cause hydrop fetalis and stillbirths; therefore, pregnant staff should be strictly isolated.

Central Nervous System and Eye Disease

CNS (Stroke)

Stroke is a major complication more frequently observed in Hb SS than in Hb SC. Children tend to have thrombotic strokes, and adults hemorrhagic strokes. Because the incidence of stroke is 11% up to 20 years of age, children with Hb SS should be screened with transcranial Doppler (TCD) every 6 to 12 months from age 2 to 18 years. For primary stroke prevention, the Stroke Prevention (STOP) trial showed that children (age 2 to 16 years old) who had a TCD velocity greater than 200 cm/sec in the internal carotid or middle cerebral artery had improvement in the TCD velocity and a much lower incidence of brain infarction, when managed on long-term transfusions to maintain Hb S less than 30%, compared to supportive care (penicillin prophylaxis, vaccinations, folate supplementation, treatment of acute crises, and transfusions as needed).⁷ A follow-up study to the original STOP trial showed that when long-term transfusions were discontinued, TCD velocity quickly became abnormal and a few children developed acute strokes.⁸ Individuals 18 years and older tend to have lower transcranial velocities, thus TCD may not be a good screening tool.

Children with suspected stroke or transient ischemic attacks (TIAs) are evaluated promptly, and hydration, therapy for hypoxia or hyperthermia, and blood pressure stabilization should follow immediately. Tissue plasminogen activator (t-PA) has not been extensively used in children and therefore is not routinely recommended. The use of antiplatelet agents, aspirin, or clopidogrel, is uncertain, but may be appropriate in selected circumstances. If a thrombotic stroke is present, exchange transfusions are initiated to reduce the Hb S level to less than 30%. If a hemorrhagic stroke is present, the source and the extent of bleeding are identified and the treatments are individualized; exchange transfusions may be indicated to reduce the Hb S level to less than 30%. If imaging studies do not identify any abnormality, the next steps may involve observation, simple transfusions, and/or participation in clinical trials.

As the recurrence rate for thrombotic strokes is high, long-term transfusion therapy to maintain Hb S less than 30% until 16 to 18 years of age should be planned. Long-term transfusions can also be considered in hemorrhagic stroke or vasculopathy (aneurysm, arterial stenosis, or moyamoya disease). Allogeneic stem cell transplantation should be encouraged in those with HLA-matched siblings. A multicenter Stroke with Transfusion Changing to Hydroxyurea (SWiTCH) trial that compared long-term transfusions + iron chelation with hydroxyurea + phlebotomy for secondary prevention of stroke was stopped early. The results showed a non-statistically significant difference: no stroke in 66 patients in the transfusion arm and 7 strokes in 60 patients in the hydroxyurea arm.⁹ An international Silent Infarct Transfusion (SIT) trial that compares long-term transfusion with no transfusions in children who had silent infarct on brain MRI with normal TCD velocities is ongoing.

Adults with acute strokes or TIAs are managed similarly to children.³ If a thrombotic stroke is identified, t-PA, antiplatelet therapy, and/or exchange transfusions can be considered. If a hemorrhagic stroke is identified, treatment is based on the source and the extent of bleeding; exchange transfusion to reduce the Hb S level to less than 30% may be indicated. For long-term therapy or secondary prophylaxis, antiplatelet therapy may be continued, with or without chronic transfusions to maintain the Hb S level less than 30%. Warfarin or dipyridamole may be added to or may substitute antiplatelet therapy for patients with recurrent strokes. In general for secondary stroke prevention in adults, blood transfusions should be continued; switching to hydroxyurea should be done ideally in a clinical trial. Those with HLA-matched siblings should be encouraged to pursue allogeneic stem cell transplantation.

There is currently no single best screening method to identify adults who are at high risk for stroke. MRI/MRA of brain can be considered in those who have general risk factors of thrombotic strokes (age, prior TIAs, and systemic hypertension) or risk factors specific to SCD (prior history of ACSs, dactylitis, severe anemia, and leukocytosis).

Eye Disease

Neovascularization results from repetitive vaso-occlusive episodes within the eye and leads to visual impairment. These proliferative changes are often asymptomatic early in the disease process; clinically detectable retinal changes are typically discovered between 15 and 30 years of age. Patients with Hb SC and sickle-thalassemia are disproportionately more prone to develop clinically significant ophthalmologic problems. Annual eye examinations starting in adolescence, carefully evaluating visual acuity, papillary reactivity, and anterior and posterior structures are important. In stage I eye disease, there is peripheral arteriolar occlusion; in stages II and III, vascular remodeling and neovascularization; in stage IV, vitreous hemorrhage; and in stage V, retinal detachment.

In patients with SCD or SCT, direct eye trauma that causes bleeding into anterior chamber requires urgent evaluation, because RBCs can occlude the trabecular channels, increase intraocular pressure, and cause acute glaucoma.

Cardiovascular Manifestations

Individuals with SCD have lower blood pressures compared to individuals with other types of chronic anemia. Renal sodium wasting is postulated as one possible cause although other mechanisms may be present. Blood pressures in SCD correlate with age, hemoglobin, and body-mass index. When systolic or diastolic blood pressures approach those of age-, sex-, and race-matched normal individuals, the risk of stroke and mortality increases.

Other cardiac manifestations include frequent systolic flow murmurs, typically related to the degree of anemia. A loud P₂ may suggest elevated right-sided pressure or pulmonary hypertension. On echocardiograms, small amount of pericardial effusions are found in approximately 10% of all studies; cardiac output, cardiac chamber size, and myocardial wall thickness are increased to improve the stroke volume without increasing the heart rate. With long-term and consistent increases in cardiac output, the ability to perform physical work is reduced by half in adults and by one-third in children. Congestive heart failure is uncommon, except in individuals with transfusional iron overload, leading to dilated cardiomyopathy. Myocardial infarction due to coronary arterial occlusion is rare, but damage from small vessel diseases may occur. Sudden death due to unexplained arrhythmia or autonomic dysfunction also has been described in adults with SCD, presumably from excess iron interfering with in the cardiac conduction system.

Pulmonary Complications

Acute Complications

ACS typically presents with cough, chest pain, and other respiratory symptoms, and is confirmed by a temperature higher than 38.5°C, a new pulmonary infiltrate on chest radiograph, and rales on auscultation (often in multiple lobes).¹⁰ Children tend to have more respiratory symptoms (wheezing, cough, and fever), while adults report musculoskeletal pain and dyspnea, and have a more severe course. Risk factors for ACS are Hb SS, low Hb F, high baseline hemoglobin (11 g/dL or greater), high white blood cell count (greater than 15 k/µL), and prior episodes of ACS. ACS is a frequent cause of death in both children and adults with SCD, the second leading cause of hospitalization, and the most frequent complication following surgery and anesthesia. Complications from ACS include CNS injury (anoxia, infarct, or hemorrhage), seizure, or respiratory compromise with or without multi-organ failure. Frequent ACS episodes are associated with shortened survival.

Only less than half of ACS events have identifiable causes. These include pneumonia, pulmonary infarction from vaso-occlusion within pulmonary vasculature, fat embolism, or pulmonary thromboembolism. Microbiologic culture of sputum may reveal a variety of atypical organisms (chlamydia or mycoplasma), viruses (respiratory syncytial virus), and bacteria (Staphylococcus aureus, S. pneumoniae, or H. influenzae ); up to 30% of microbiologic cultures are negative.¹¹ Systemic fat embolism syndrome is rare, and occurs when there is infarcted/necrotic bone marrow and fat being released and lodged in the pulmonary vasculature. Fat embolization can precipitate or develop concurrently with ACS; multi-organ failure may result. Risk factors for developing systemic fat embolism syndrome are Hb SC genotype, pregnancy, and prior corticosteroid treatment.

Treatment for ACS includes oxygen, antibiotics coverage for atypical organisms, simple or exchange transfusions to improve oxygen saturation, bronchodilators (as airway hyperreactivity often accompanies ACS), and analgesia for pain. All these efforts are aimed at reducing the percentage of sickle erythrocytes and minimizing sickle polymerization. After an episode of ACS is successfully managed, strategies to prevent future episodes may include vaccinations (especially against S. pneumoniae), hydroxyurea, continuing transfusions, or allogeneic stem cell transplantation.

Chronic Complications

Pulmonary hypertension is multifactorial in its pathogenesis including sickle cell-related vasculopathy, pulmonary damage from recurrent ACS, high blood flow from anemia, chronic thromboembolic disease, and chronic hemolysis. Clinically, pulmonary hypertension can manifest as dyspnea, clubbing, loud second heart sound (P₂), an enlarged right side of the heart on chest radiograph, and 95% or less oxygen saturation on room air at rest. Pulmonary hypertension can initially be screened with an echocardiogram showing a tricuspid regurgitation jet velocity (TRV) of ≥2.9 m/sec and subsequently confirmed by right heart catheterization with a mean pulmonary arterial pressure of ≥25 mmHg.¹²

There is currently no single preferred treatment for patients with pulmonary hypertension. Simple transfusion to maintain hemoglobin at approximately 9 g/dL can reduce pulmonary pressure in some, but the possible development of red cell antibodies limits the wide applicability of this approach. Hydroxyurea reduces the frequency and severity of vaso-occlusive crises and ACS, but may only delay the onset of pulmonary hypertension. Improving anemia, ruling out left-sided heart failure, sildenafil, anticoagulation with warfarin (target INR 2-3), prostacyclin analogs, endothelin receptor antagonists, and nitric oxide are adjunctive agents to consider; continuous or nocturnal oxygen can be considered.

Gall Bladder, Hepatic, and Splenic Manifestations

SCD can affect the hepato-biliary and splenic systems in multiple ways (Table 4.5). Hyperbilirubinemia (typically less than 4 mg/dL of unconjugated bilirubin) from chronic hemolysis is common. Other factors that increase total bilirubin level include: cholesterol intake, presence of Gilbert’s syndrome, and cephalosporin antibiotic use. Biliary sludge and cholelithiasis can occur as early as 2 to 4 years of age, can have similar clinical manifestations, and are managed similarly as those without SCD. Elevation in the direct or conjugated bilirubin from baseline of 0.1 to 0.4 mg/dL can indicate sickle-related liver disease.

Acute splenic sequestration is caused by trapping of erythrocytes, and presents with weakness, pallor, tachypnea, an acute drop in hemoglobin (typically 2 g/dL or greater than 20% from baseline), or abdominal fullness from acute splenomegaly (2 cm increase in palpated spleen size). This syndrome can be precipitated by infections, and is typically seen in children under age 5 with Hb SS, older children with Hb SC, or in a few adults with Hb S/β⁺ thalassemia. Children tend to have recurrent and severe episodes that require immediate attention with transfusion; a long-term plan of chronic transfusion therapy and/or splenectomy should be discussed. While splenic sequestration in children tends to be more severe, in adults it is typically self-limited requiring only supportive care and observation.

Renal Abnormalities

There are several sickle-related manifestations in the kidneys. There is supranormal creatinine secretion in the proximal tubules, which explains lower serum creatinine (near 0.5 mg/dL). A serum creatinine approaching 1.0 mg/dL can indicate subtle renal insufficiency. The renal medulla is composed of tubules and blood vessels collectively called the vasa recta; this region is chronically in an acidic, hypoxic, and hypertonic environment, and is thus very susceptible to Hb S polymerization. Over time, gradual loss of the vasa recta leads to an inability to concentrate urine. Hyposthenuria develops early in childhood, is frequently associated with nocturia, and is the chief reason why SCD individuals are so susceptible to dehydration. Hb S sickling can also lead to papillary necrosis and hematuria. Other renal manifestations include proteinuria from chronic glomerular damage, which in some individuals can progress to renal insufficiency or failure; angiotensin converting enzyme (ACE) inhibitors can ameliorate this progression. A decrease in hemoglobin from baseline due to renal insufficiency can be treated by erythropoietin injection (overlapped with hydroxyurea) and/or transfusion. Although serum erythropoietin levels are already elevated in renal failure, they are relatively lower when corrected for the degree of anemia.

Priapism

Priapism is a sustained painful erection that is stuttering (duration <3 hours and spontaneous resolution) or prolonged (>3 hours). Priapism begins early in puberty, and as many as 80% of men with SCD would have experienced at least one episode by 20 years of age. Priapism is caused by vaso-occlusion of the venous drainage of the penis, and physical examination will reveal a hard penis with a soft glans. Oral hydration and analgesia should be instituted at the onset of priapism. A prolonged priapism represents an urologic emergency and requires urgent evaluation, intravenous hydration, and analgesia. If not improved within 1 hour, blood aspiration and irrigation with dilute epinephrine to the corpus cavernosum under local anesthesia is needed.¹³ Simple or exchange transfusions are sometimes used.

Recurrent priapism can lead to impotence and fibrosis. No treatment has been well studied. A shunting procedure between the glans and distal corpus cavernosum (Winter’s procedure), α-adrenergics (e.g. pseudoephedrine), and medications reducing the frequency of erection, tricyclic antidepressants, β-blockers, or leuprolide, have all shown variable success. Simple or exchange transfusions can be considered. Recent studies have grouped patients into two potentially distinct phenotypic presentations: one group that has priapism and leg ulcers and the other that has frequent vaso-occlusive crises and osteonecrosis.

Skeletal Complications and Leg Ulcers

Repetitive vaso-occlusion in marrow sinusoids eventually causes bone infarction. Osteonecrosis ensues when ischemic necrosis of juxta-articular bone in femur, humerus, or tibia occurs. In children, prior to bone maturation, osteonecrosis is treated conservatively with analgesia, NSAIDs, and protected weight bearing. In adults, secondary degenerative arthritis can compound osteonecrosis, and the usual conservative treatment is ineffective. Core decompression and osteotomy with aggressive physical therapy have been reported to offer temporary relief of pain and increased joint mobility¹⁴; joint replacement is reserved for those with severe symptoms or advanced disease.

Dactylitis or “hand-foot” syndrome in infants and young children presents with pain or swelling in one or more extremities (hands or feet). Plain radiograph may show periosteal elevation and a motheaten appearance. This syndrome usually requires hydration and analgesia, but transfusions or antibiotics are not necessary. There are no associated long-term sequelae.

Bacteremia can lead to osteomyelitis or septic arthritis. Both present with warmth, tenderness, and edema caused by vaso-occlusion in bones; fever in the acute phase, increase in white blood cell count, and positive blood cultures help to distinguish infection from pain crisis. A positive microbial culture from aspiration of the bone or joint is diagnostic. Both of these infections are treated with surgical drainage and short-term (2–6 weeks) intravenous antibiotics. Additionally, temporary joint mobility with exercises to improve the range of motion in the convalescent phase may be indicated for septic arthritis.

Leg ulcers are seen in 10% to 20% of individuals with Hb SS, and much less in individuals with Hb SC or Hb S/β⁺-thalassemia. The frequency of leg ulcers increases with age and is associated with a lower mean hemoglobin (6 g/dL or less) and a higher LDH. Their exact etiology is unclear, but trauma, chronic vaso-occlusion, edema, and hemolysis have all been implicated.¹⁵ Ulcers tend to locate in the dorsum of the feet, ankles, or tibia; other sites are rare. They begin as small hyperpigmented areas with edema, pain, and dysesthesia, subsequently appearing as denuded and “punched out” ulcers. Ulcers are usually infected locally, and osteomyelitis is rare. Two principles are important in promoting wound healing: reduction of local edema by elevation and/or pressure dressing, and debridement of ulcers by frequent wet-to-dry dressing changes to maximize granulation. There are many topical treatments available, but no single therapy works uniformly. Topical or systemic antibiotics are generally not helpful and eventually select for drug-resistant organisms. Ulcers smaller than 4 cm usually heal in weeks; larger ones may require consultation with wound care service and plastic surgery for skin flaps. Transfusions can be considered for recurrent or persistent ulcers. The use of hydroxyurea in leg ulcer is controversial. There are reports of hydroxyurea causing leg ulcers in individuals with SCD and myeloproliferative disease. But in the Multicenter Study of Hydroxyurea (MSH), hydroxyurea did not appear to change the incidence of leg ulcers.¹⁶ Other reports, however, suggest that Hb F elevation is associated with reduced rates of leg ulcers.

THERAPY

Transfusions

Transfusions are an important therapy for SCD (Table 4.6), and are commonly used acutely, but also chronically for primary or secondary prevention of a specific complication. Transfusions can be separated into simple episodic, simple chronic, or exchange. It is important to notify blood bank of the type (simple or exchange), indication, and duration (episodic or chronic). A detailed transfusion history should be maintained that includes the total number of prior red cell units, presence of any red cell antibodies, percent Hb S, and the target hemoglobin or hematocrit.

Red cell antigen difference between sickle cell patients and blood donors (mostly Caucasians) is the major reason for the high rate of alloimmunization. Alloimmunization can be minimized by typing for other Rh (D, E/e, and C/c), and Kell (K) antigens, in addition to the usual ABO typing. In developing countries, nonroutine use of leukocyte filters is another reason for the high rate of alloimmunization. Prestorage leukocyte depletion is now commonly employed in blood banks. When a patient has a prior transfusion history, other minor antigens (Kidd, S, and Duffy) should also be typed. Other potential complications include the usual transfusion reactions that can occur in non-SCD patients: volume overload, acute/delayed hemolytic reactions, transfusion-transmitted infections, and iron overload.

Fetal Hemoglobin (Hb F) Induction

The beneficial effect of Hb F was first recognized from the observations that neonates with Hb SS do not develop SCD-related symptoms in the first 6 months, and patients with co-inheritance of SCD and HPFH, such as in Saudi Arabia and India, have milder symptoms. Currently, hydroxyurea is the only Food and Drug Administration (FDA)-approved drug for Hb F induction. Hydroxyurea is a cell cycle (S-phase)-specific agent that blocks the conversion of ribonucleotides to deoxyribonucleotides. Its primary clinical impact is Hb F induction, which inhibits Hb S polymerization, but other benefits may include reduced leukocyte and platelet counts, lessened hemolysis, decreased bone marrow cellularity, and generation of nitric oxide.

The MSH study was a randomized placebo-controlled clinical trial that confirmed the beneficial effects and the safety of hydroxyurea. The 150 hydroxyurea-treated patients had fewer pain episodes and ACS, required fewer transfusions, and experienced minimal toxicity.¹⁶ Approximately 70% of SCD patients are likely hydroxyurea-responsive: steady state Hb F should increase two fold from baseline or approximately 10% to 15%, total hemoglobin should increase by 1 to 2 g/dL, or there should be a subjective reduction in the severity and frequency of pain (Table 4.7). Whether hydroxyurea will prevent or reverse end organ damage is under active investigation.

Hydroxyurea can be started at 10 to 15 mg/kg daily, and adjusted by 5 mg/kg/day increments every 6 to 8 weeks, to a target of approximately 25 mg/kg daily. Compromised hepatic or renal function may require lower dosing. Within a week of therapy, Hb F-containing reticulocytes will rise; at the end of 2 to 3 weeks, Hb F-containing red cells will increase. Other hematologic effects include increase in MCV (to >100) and decrease in leukocytes (mostly neutrophils), platelets, and reticulocytes. Two to 3 months are usually required before the effects on Hb F and blood counts are stabilized; a trial of 6 to 12 months is adequate to assess clinical benefit. Hydroxyurea in several cohorts of children has shown to be safe and efficacious in reducing pain and sickle-related complications.²¹

There are other inducers of fetal hemoglobin available in research studies: 5-azacytadine, decitabine, and HDAC inhibitors. These drugs aim to “reactivate” gamma globin expression that has been silenced in the postnatal hemoglobin switching process.

Other Drugs

Agents that modify the pathophysiology of SCD are based on their ability to reduce Hb S polymerization in vitro. These include Hb S modifiers (urea, organic compounds), inhibitors of Gardos channel (clotrimazole, ICA 17403), chloride and cation channel blockers (dipyridamole, magnesium pidolate, NS-3623), and anti-adherence agents (Poloxamer 188, pentoxifylline). Erythropoietin, at doses 2 to 5 times those used in renal failure, in combination with hydroxyurea, can increase total hemoglobin.

SPECIAL TOPICS

Contraception and Pregnancy

Hydroxyurea is a teratogen in animal models, therefore both male and female patients on hydroxyurea should use contraception, and discontinue the drug if pregnancy is planned. Recent long-term updates from the MSH investigators indicated that neonates delivered from women receiving hydroxyurea therapy throughout pregnancy or from men taking hydroxyurea did not have any teratogenic effects,²² but any unplanned pregnancy should be discussed. Hydroxyurea is also secreted into breast milk, and breast feeding should be avoided.

Hydroxyurea is considered as an oral chemotherapeutic drug, thus it is not surprising that there is a perceived risk of developing malignancy. While there are case reports of SCD patients who have developed leukemia while receiving hydroxyurea, this rate is probably similar to the general population. No definitive risk increase has been directly attributed to hydroxyurea for individuals with myeloproliferative diseases or cyanotic heart disease.

Pregnant women with SCD are at an increased risk of miscarriage, preeclampsia, sickle pain crises, acute anemia or hemolysis, or infections.23,24 Maternal mortality rate is low and the overall outcome of the pregnancies is favorable, but infants tend to be born prematurely (average at 34–37 weeks) and are small for gestation age (less than tenth percentile). Hb SS women tend to have more frequent or severe complications than Hb SC women. Transfusions are usually reserved for pregnancies complicated by progressive anemia, increased pain episodes, preeclampsia, prior pregnancy loss, or multiple gestations; prophylactic transfusions are generally discouraged.

Anesthesia and Surgery

Surgery and general anesthesia have higher morbidity and mortality in SCD compared to the general population. The risk is higher in those with Hb SS or Hb S/β⁰ thalassemia than those with Hb SC or Hb S/β⁺ thalassemia.¹⁷ Complications may be as frequent as in those receiving regional anesthesia. ACS and postoperative infection are the most common complications, followed by pain crisis and stroke (Table 4.8).

Table 4.8  Perioperative Considerations for Sickle Cell Patients

Preoperative

1.  Simple transfusions to achieve hemoglobin of 10 g/dL in Hb SS and Hb S/β⁰ thalassemia. Individuals with Hb SC may require exchange transfusion, especially prior to abdominal surgery.

2.  Blood typing for additional antigens, such as C, E, Kell, Kidd (Jk), S, and Duffy (Fy), to minimize alloimmunization.

3.  Hydration.

Postoperative

1.  Hydration, oxygen, and monitoring respiration and peripheral perfusion.

2.  Monitoring for acute chest syndrome, infection, pain crisis, or stroke.

SICKLE CELL TRAIT (SCT)

Approximately 8% of African-Americans have SCT. Under physiologic conditions, vaso-occlusion does not occur. Carriers have normal life expectancy and many participate successfully in competitive sports or rigorous military training. There are exercise-related mortalities from rhabdomyolysis, which can be minimized by avoiding heat stress, dehydration, and sleep deprivation. Current controversy exists whether to implement universal sickle cell screening in competitive collegiate sports. However, most medical societies (including hematologists and pediatricians) agree that proper education, efforts to prevent dehydration, and vigilant monitoring for rhabdomyolysis, as currently employed by the US military services, are better than universal screening of athletes to prevent morbidity and mortality in trait carriers.

Compared to the general population, persons with SCT have normal risk of developing heart disease, stroke, leg ulcers, or arthritis. They are not more likely to develop complications from anesthetic agents. However, SCT is associated with increased risk for traumatic eye injury, hyposthenuria, hematuria, splenic infarction, pulmonary embolism, and proteinuria.²⁵ If traumatic eye injury occurs with hemorrhage into anterior chamber (hyphema), erythrocytes may clog the trabecular outflow channels, increase intraocular pressure, and this may lead to acute glaucoma, requiring urgent evaluation and treatment. Pregnant women with SCT are at an increased risk for urinary tract infections.

CLINICAL SYNDROMES AND TREATMENT OF THALASSEMIAS

While the thalassemic syndromes are highly variable, severity is directly related to the imbalance of α to β chain ratio: higher the imbalance, more severe the phenotype where excess unpaired α or β chains precipitate in erythrocyte precursors, resulting in their early death and ineffective erythropoiesis. Excess unpaired α or β chains also denature intracellular hemoglobin, promoting splenic sequestration and hemolysis, and eventual splenomegaly and anemia. In β-thalassemia major, any genetic conditions that reduce α-chain excess (co-inheritance of α-thalassemia, or increase in δ- or γ-chain production) or preserve some β-chain synthesis (a mild or silent β-thalassemic allele) ameliorate the severity of β-thalassemia.

Without regular red cell transfusions, chronic hemolytic anemia and tissue hypoxia stimulate bone marrow expansion and produce skeletal and metabolic derangements: bony deformities, fractures, extramedullary hematopoiesis, and increased gastrointestinal iron absorption. In addition, red cell membrane damage, activation of platelets and endothelium, and abnormal levels of coagulation inhibitors (anti-thrombin III, protein C and S) all contribute to increased risk of thromboembolism.

Other complications of thalassemia include leg ulcers, gallstones, and folate deficiency. There are also rare forms of α-thalassemia with mental retardation and developmental abnormalities (mutations in the ATRX gene), and β-thalassemia minor with thrombocytopenia (mutation in GATA1).

Transfusions and Splenomegaly

Transfusions and iron chelation are the mainstay of therapy, and have improved the quality of life and extended life expectancy in thalassemia individuals. After the diagnosis in infancy or childhood, the decision to start transfusion depends on the degree of impact anemia has on the child: fatigue, reduced growth velocity, skeletal dysmorphism, poor weight gain, or organomegaly. Once started, the target hemoglobin of 9 to 10 g/dL is reasonable, although others have used a higher target. Transfusions are maintained every 2 to 4 weeks and continued through adulthood. Improvements in the clinical signs and symptoms can be seen in adequately transfused individuals. Red cell alloimmunization can be minimized as in SCD by typing for major and minor blood group antigens (ABO, Rh, Kell, Kidd, and Duffy) and prestorage leukocyte depletion.

Splenomegaly can be seen in those with inadequate transfusions or red cell alloimmunization, and is associated with worsening anemia, leukopenia, and thrombocytopenia. Hemoglobin will typically drop about 1.5 g/dL/week in non-splenectomized individuals; in hypersplenism, this rate of decline will be higher and eventually there will be an inadequate rise in post-transfusion hemoglobin. Splenectomy will improve these hematologic parameters and transfusion effectiveness, but should be performed after vaccination for encapsulated organisms (S. pneumoniae, H. influenzae, and N. meningitidis), and in children older than 5 years of age. Post-splenectomized transfusions should produce a 1 g/dL/week decrease in hemoglobin. Penicillin prophylaxis is appropriate. Post-splenectomy thrombocytosis can be variable, but generally does not require antiplatelet therapy.

Iron Overload and Chelation

Chronic transfusions and early iron chelation have shifted the major thalassemic complications from those secondary to the hemolytic anemia to the sequelae of iron overload. Iron chelation should start when ferritin is approaching 1,000 ng/L, approaching 20 units of red cells, or within 18 months from the start of chronic red cell transfusion. Excess iron from cumulative transfusions overwhelms the transferrin system and accumulates in the liver, heart, and various endocrine organs. The most serious result is nonuniform iron deposition in cardiac myocytes that eventually leads to heart failure and sudden unpredictable arrhythmia, which previously accounted for the majority of deaths in thalassemic individuals. MRI with T2* sequences is quickly becoming the standard method of cardiac evaluation: 5 to 10 ms is seen in severe overload, 10 to 20 ms in moderate overload, and >20 ms in normal non-overload states.²⁶ Excess iron also accumulates in the liver, producing hepatic inflammation, dysfunction, and fibrosis. While MRI with T2* sequences, superconducting susceptometry (SQUID), and serum ferritin are helpful in estimating the amount of iron in liver, biopsy remains the standard. Furthermore, iron overload also affects endocrine organs and can cause reduced growth velocity in children, hypothyroidism, hypogonadism with pubertal delay or arrest, hypoparathyroidism leading to hypocalcemia and osteoporosis, and diabetes. All are managed in collaboration with endocrinologists.

The toxic effects of excess iron can be minimized by long-term maintenance of iron chelation. Deferoxamine (Desferal) can be administered as early as 21/2 years of age at 20 to 60 mg/kg (or 1.5–4.0 g per adolescent or adult) per day, delivered by subcutaneous or intravenous injection over 8 to 12 or 24 hours,27,28 and typically for at least 5 days a week. Twice a day subcutaneous bolus injection may also be efficacious.²⁹ Side effects of deferoxamine are infrequent; they include impaired vision or hearing, motorsensory neuropathy, changes in renal or pulmonary function, joint pain, metaphyseal dysplasia or growth retardation. Oral deferasirox (Exjade) should be titrated to a target dose of 30 mg/kg/day, and 40 mg/kg/ day for those with cardiac iron deposition. Side effects include abdominal pain, diarrhea, rash, arthralgia, and mild increases in liver enzymes and serum creatinine. Oral deferiprone has recently been approved and has side effects such as gastrointestinal discomfort, joint pain, and agranulocytosis. These three chelators individually have been shown to reduce iron burden in the liver or heart; many hematologists prefer combination therapy for those with severe cardiac iron to quickly remove iron. Eye and audiology evaluations should be performed prior to iron chelation and yearly while on transfusions and chelation.

Fetal Hemoglobin Induction

The major endpoint for Hb F induction in thalassemia is an increase in total hemoglobin. Unfortunately, hydroxyurea has not been able to achieve this goal in most thalassemia major patients receiving chronic transfusions, possibly due to a loss of Hb F response with transfusion or due to certain mutations that are resistant to Hb F induction. Hydroxyurea, however, has had some effect in Hb Lepore/β-thalassemia, Hb E/β-thalassemia, and β-thalassemia intermedia. Erythropoietin also can be used with hydroxyurea but the response is variable. Other inducers of Hb F (such as decitabine) can be considered.

THERAPY WITH CURATIVE INTENT

Hematopoietic Stem Cell Transplantation

Myeloablative hematopoietic stem cell transplantation (HSCT) is currently the only cure for SCD and thalassemia,30,31 with the best outcome from HLA-matched sibling donors. In SCD, HSCT is typically recommended for patients less than 17 years of age, those non-responsive to hydroxyurea, or those who had prior SCD-related organ damage (e.g., stroke, ACS, frequent pain crises, and multiple sites of osteonecrosis). For thalassemia, HSCT is also typically recommended for those aged less than 17 years with signs of liver dysfunction or fibrosis from iron damage (Pesaro class II or III). The disease-free survival can be as high as 90% to 95% with a 10% risk of graft versus host disease (GVHD). Often the GVHD is easily treated, and most of the children can gradually discontinue immunosuppression. Post transplant they enjoy improved quality of life and growth velocity. The preexisting organ damage from the underlying disease and the long-term effects from HSCT (small increase in secondary cancer, reduction in gonadal hormones/sterility, changes in thyroid function) are closely monitored periodically.

Recent encouraging clinical data showed that non-myeloablative HSCT can achieve mixed donor and host hematopoiesis with successes approaching those of myeloablative HSCT.³² This non-ablative approach, with less toxicity from conditioning regimen, is a reasonable alternative for young or older adults who otherwise meet criteria for a standard myeloablative HSCT or for those with severe organ dysfunction. For patients without matched sibling donors, umbilical cord blood transplantation can be considered for pediatric patients. Approaches using matched unrelated donors for Caucasians and haploidentical donors for African Americans are currently being tested and optimized.

Gene Therapy

Autologous transplantation following the insertion of a normal or therapeutic globin gene into hematopoietic stem cells is continually being refined for SCD and thalassemia.³³ Significant advances have been made toward this goal using lentiviral vector based on the human immunodeficiency virus. Therapeutic correction of murine models of both β-thalassemia and SCD has been achieved using this approach. There is additional progress in achieving moderate levels of engraftment of genetically modified cells in the nonhuman primate autologous transplant model. Clinical human gene therapy trials have begun and the results of the first reported patient with β-thalassemia/Hb E demonstrate the therapeutic potential of this approach.³⁴ Further progress is clearly necessary as the therapeutic effect in this patient was derived from an equal contribution of endogenous Hb F, Hb E, and the therapeutic transgene. Summaries for SCD and thalassemias are provided in Tables 4.9 and 4.10.

References

  1.  Gong L, Maiteki-Sebuguzi C, Rosenthal PJ, et al. Evidence for both innate and acquired mechanisms of protection from Plasmodium falciparum in children with sickle cell trait. Blood. 2012;119:3808-14.

  2.  Ataga KI, Cappellini MD, Rachmilewitz EA. Beta-thalassaemia and sickle cell anaemia as paradigms of hypercoagulability. Br J Haematol. 2007;139:3-13.

  3.  National Heart, Lung, and Blood Institute, National Institutes of Health. The Management of Sickle Cell Disease; 2002. 4th edition, NHLBI NIH Pub. 02-2117.

  4.  Ballantyne JC, Mao J. Opioid therapy for chronic pain. N Engl J Med. 2003;349:1943-1953.

  5.  Gaston MH, Verter JI, Woods G, et al. Prophylaxis with oral penicillin in children with sickle cell anemia. A randomized trial. N Engl J Med. 1986;314:1593-1599.

  6.  Smith-Whitley K, Zhao H, Hodinka RL, et al. Epidemiology of human parvovirus B19 in children with sickle cell disease. Blood. 2004;103:422-427.

  7.  Adams RJ, McKie VC, Hsu L, et al. Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography. N Engl J Med. 1998;339:5-11.

  8.  Adams RJ, Brambilla D. Discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease. N Engl J Med. 2005;353:2769-2778.

  9.  Ware RE, Helms RW. Stroke with transfusions changing to hydroxyurea (SWiTCH). Blood. 2012;119:3925-32.

10.  Platt OS. The acute chest syndrome of sickle cell disease. N Engl J Med. 2000;342:1904-1907.

11.  Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and outcomes of the acute chest syndrome in sickle cell disease. N Engl J Med. 2000;342:1855-1865.

12.  Parent F, Bachir D, Inamo J, et al. A hemodynamic study of pulmonary hypertension in sickle cell disease. N Engl J Med. 2011;365:44-53.

13.  Mantadakis E, Ewalt DH, Cavender JD, Rogers ZR, Buchanan GR. Outpatient penile aspiration and epinephrine irrigation for young patients with sickle cell anemia and prolonged priapism. Blood. 2000;95:78-82.

14.  Mukisi-Mukaza M, Manicom O, Alexis C, et al. Treatment of sickle cell disease’s hip necrosis by core decompression: a prospective case-control study. Orthop Traumatol Surg Res. 2009;95:498-504.

15.  Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood. 2006;107:2279-2285.

16.  Charache S, Terrin ML, Moore RD, et al. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. N Engl J Med. 1995;332:1317-1322.

17.  Vichinsky EP, Haberkern CM, Neumayr L, et al. A comparison of conservative and aggressive transfusion regimens in the perioperative management of sickle cell disease. The Preoperative Transfusion in Sickle Cell Disease Study Group. N Engl J Med. 1995;333:206-213.

18.  Koshy M, Burd L, Wallace D, Moawad A, Baron J. Prophylactic red-cell transfusions in pregnant patients with sickle cell disease. A randomized cooperative study. N Engl J Med. 1988;319:1447-1452.

19.  Miller ST, Wright E, Abboud M, et al. Impact of chronic transfusion on incidence of pain and acute chest syndrome during the Stroke Prevention Trial (STOP) in sickle-cell anemia. J Pediatr. 2001;139:785-789.

20.  Neumayr L, Koshy M, Haberkern C, et al. Surgery in patients with hemoglobin SC disease. Preoperative Transfusion in Sickle Cell Disease Study Group. Am J Hematol. 1998;57:101-108.

21.  Wang WC, Ware RE, Miller ST, et al. Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG). Lancet. 2011;377:1663-1672.

22.  Ballas SK, McCarthy WF, Guo N, et al. Exposure to hydroxyurea and pregnancy outcomes in patients with sickle cell anemia. J Natl Med Assoc. 2009;101:1046-1051.

23.  Villers MS, Jamison MG, De Castro LM, James AH. Morbidity associated with sickle cell disease in pregnancy. Am J Obstet Gynecol. 2008;199:e121-125.

24.  Yu CK, Stasiowska E, Stephens A, Awogbade M, Davies A. Outcome of pregnancy in sickle cell disease patients attending a combined obstetric and haematology clinic. J Obstet Gynaecol. 2009;29:512-516.

25.  Key NS, Derebail VK. Sickle-cell trait: novel clinical significance. Hematology Am Soc Hematol Educ Program. 2010;2010:418-422.

26.  Pennell DJ, Porter JB, Cappellini MD, et al. Continued improvement in myocardial T2* over two years of deferasirox therapy in beta-thalassemia major patients with cardiac iron overload. Haematologica. 2011;96:48-54.

27.  Giardina PJ, Grady RW. Chelation therapy in beta-thalassemia: an optimistic update. Semin Hematol. 2001;38:360-366.

28.  Davis BA, Porter JB. Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk beta-thalassemia. Blood. 2000;95:1229-1236.

29.  Franchini M, Gandini G, de Gironcoli M, et al. Safety and efficacy of subcutaneous bolus injection of deferoxamine in adult patients with iron overload. Blood. 2000;95:2776-2779.

30.  Angelucci E. Hematopoietic stem cell transplantation in thalassemia. Hematology Am Soc Hematol Educ Program. 2010;2010:456-462.

31.  Hsieh MM, Fitzhugh CD, Tisdale JF. Allogeneic hematopoietic stem cell transplantation for sickle cell disease: the time is now. Blood. 2011;118:1197-1207.

32.  Hsieh MM, Kang EM, Fitzhugh CD, et al. Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. N Engl J Med. 2009;361:2309-2317.

33.  Arumugam P, Malik P. Genetic therapy for beta-thalassemia: from the bench to the bedside. Hematology Am Soc Hematol Educ Program. 2010;2010:445-450.

34.  Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature. 2010;467:318-322.

5

Porphyrias

Peiman Hematti

The porphyrias are a diverse group of uncommon metabolic disorders caused by inherited deficiencies of the enzymes involved in the heme biosynthetic pathway,¹ except for one recently described porphyria syndrome due to a gain-of-function mutation. Mutations in the genes of all these heme-synthesizing enzymes have been identified at the molecular level. An exception to the genetic origin for these disorders is porphyria cutanea tarda (PCT), in which the enzyme deficiency in most cases is acquired. In all these ecogenic disorders, it is the interaction of genetic, physiologic, and environmental factors that causes disease in affected individuals. Each defective enzyme results in a characteristic clinical phenotype of porphyria, although disease mechanisms are not fully understood. Any patient with a long history of undiagnosed abdominal pain and/or atypical neuropsychiatric symptoms should have porphyria in their differential diagnosis, as simple tests can be used to confirm diagnosis in symptomatic patients and to screen family members to recognize asymptomatic carriers.² Counseling about avoidance of precipitating factors can decrease clinical manifestations in the latter group.

EPIDEMIOLOGY

PCT is the most prevalent of the porphyrias, both genetic and acquired combined, but acute intermittent porphyria (AIP) is the most common of the genetic porphyrias. AIP has an estimated incidence of 5 in 100,000 in the United States and northern European countries. Approximately 90% of patients with this inherited enzyme deficiency remain symptom free throughout their life. In contrast, only six cases of δ-aminolevulinic acid dehydratase (ALAD) deficiency have been thus far reported.

PATHOPHYSIOLOGY

Heme is a complex of an iron atom and protoporphyrin IX. Heme is produced in a multistep biosynthetic pathway that functions mostly in the erythroid bone marrow and hepatocytes. Approximately 85% of the heme produced in the body is synthesized in erythroid cells to provide for hemoglobin formation; most of the remainder is produced in the liver to provide heme for cytochrome P-450 and other enzymes. Eight enzymes are involved in this tightly regulated biosynthetic pathway that sequentially converts glycine and succinyl CoA into heme (Fig. 5.1). In eukaryotic cells, the first and the last three steps of this pathway localize in mitochondria, while the others are cytoplasmic. Sequences of the genes for all these enzymes and their molecular defects have been well characterized.

The first enzyme active in the pathway, δ-aminolevulinic acid synthase (ALAS), is coded by two genes: ALAS1, which is expressed ubiquitously in all cells, and ALAS2, which is expressed only in erythroid cells. So far, no mutation has been identified in ALAS1, and until very recently all reported pathogenic mutations of ALAS2 were loss of function and resulted in recessive X-linked sideroblastic anemia, the only non-porphyria syndrome due to abnormalities in the heme biosynthetic pathway. Recently, gain-of-function mutations have been reported in the ALAS2 gene in eight families causing X-linked dominant protoporphyria (XLDPP). In general, mutations of these enzymes result in porphyria syndromes because of overproduction of metabolic precursors and intermediates and/ or their accumulation in tissues. All of these intermediate products are potentially toxic, and their overproduction causes the neurovisceral and/or photocutaneous symptoms characteristic of porphyria syndromes.

FIGURE 5.1 Classification of porphyrias based on their corresponding enzymatic deficiencies, mode of inheritance, major symptoms, and biochemical abnormalities.

Despite the characterization of these disorders at the molecular level, the exact pathophysiologic mechanisms responsible for specific organ manifestations are not fully understood.³ Porphyrias are heterogeneous at the molecular level, with numerous mutations found for each gene. There is a significant interaction between specific inherited genetic defects and acquired or environmental factors that result in a spectrum of clinical manifestations in affected patients. Patients with gene mutations for the acute hepatic forms of porphyrias may remain asymptomatic unless they are exposed to certain medications (Table 5.1) or hormones or are stressed by starvation, infection, surgery, or other intercurrent disorders. Under these environmental circumstances, affected patients develop characteristic neurologic disturbances. Photocutaneous hypersensitivity and skin damage occurs after exposure to ultraviolet light. When porphyrins absorb light of this wavelength, they produce free radicals that can induce oxidant tissue damage. Consequently, avoidance of precipitating factors is key in the therapy of porphyrias.⁴

CLASSIFICATION AND CLINICAL MANIFESTATIONS

For clinical purposes, porphyrias can be classified into hepatic and erythropoietic types depending on the major tissue site of production and accumulation of the heme precursors. The major manifestations of the hepatic porphyrias are neurovisceral symptoms, including abdominal pain, neurologic symptoms, and psychiatric disorders, whereas the erythropoietic porphyrias usually present primarily with cutaneous photosensitivity and hemolytic anemia. Porphyrias can be also classified according to their clinical presentations into a) acute porphyrias presenting with life-threatening neurovisceral manifestations and b) nonacute (or cutaneous) porphyrias characterized by photosensitivity syndromes, but there can be some overlap in clinical manifestations. However, because the porphyrias are well characterized at the molecular genetic level, they are better specifically classified by their unique enzyme deficiencies.5,6

DIAGNOSIS

Many symptoms of the porphyrias are nonspecific, and diagnosis requires a high index of suspicion. However, although the porphyrias are often suspected in a patient with vague and unexplained complaints, actual diagnosis is rare. A useful first step is to determine which one of the three major manifestations of the porphyrias—neurovisceral symptoms, photosensitivity, or hemolytic anemia—is present.7,8

  Neurovisceral symptoms are present in ALAD deficiency porphyria (ADP), AIP, hereditary coproporphyria (HCP), and variegate porphyria (VP).

  Photosensitivity is present in congenital erythropoietic porphyria (CEP), PCT, hepatoerythropoietic porphyria (HEP), HCP, VP, erythropoietic protoporphyria (EPP), and XLDPP.

  Neurovisceral symptoms and photosensitivity are present in HCP and VP.

  Hemolytic anemia is present in CEP, HEP, and EPP.

Laboratory testing is then required to confirm or exclude the various types of porphyrias. The diagnosis is made initially by detection of the metabolite(s) produced and/or excreted in excess in red blood cells, plasma, urine, and/or feces.⁷ Porphyrin precursors in urine and total porphyrins in plasma are the initial diagnostic tests for acute and cutaneous porphyrias, respectively. Today the diagnosis of many of porphyrias can be confirmed by measuring the enzymatic activity in the appropriate tissue directly or by specific molecular genetic testing. Family screening is advisable to prevent acute attacks in presymptomatic stages, with DNA analysis for the identification of the mutations being the gold standard.

SPECIFIC TYPES OF PORPHYRIAS

X-linked dominant protoporphyria

This most recently described syndrome is the only porphyria not caused by enzyme deficiency but by gain-of-function deletions in ALAS2.⁹ ALAS2 gain of function leads to excessive production of protoporphyrin in red blood cells and leads to a clinical presentation similar to EPP. Patients in these eight families all had photosensitivity; five of them also had overt liver disease, but they did not have anemia. Supportive and preventive interventions for these patients are similar to those for erythropoietic porphyrias.

δ-Aminolevulinic Acid Dehydratase Deficiency Porphyria

ADP is an autosomal-recessive porphyria caused by markedly deficient activity of ALA dehydratase, the second enzyme in the heme biosynthetic pathway. The diagnosis has been unequivocally confirmed only in a few cases. Clinical manifestations are primarily neurovisceral and their treatment and prevention are the same as for other acute porphyrias. Lead poisoning should be excluded, because it also diminishes activity of ALA dehydratase, may present as a clinical phenocopy, and is far more common.

Acute Intermittent Porphyria

AIP is inherited as an autosomal dominant condition resulting from a partial deficiency of porphobilinogen deaminase (PBGD) activity, the third enzyme of the pathway. Approximately 90% of heterozygotes remain biochemically normal and clinically asymptomatic throughout life. Clinical expression of the disease is usually the result of exposure to factors such as endogenous and exogenous corticosteroid hormones, a low-calorie diet, certain drugs (barbiturates and sulfonamide antibiotics are the most commonly implicated), alcohol ingestion, and stresses such as intercurrent illnesses, infection, and surgery. Symptoms usually develop after puberty and are more frequent in women. The pathophysiologic hallmark of the disease is neurologic dysfunction affecting peripheral, autonomic, and/or central nervous systems occurring as intermittent acute attacks. The most common symptom is acute abdominal pain (in 90% of cases), which may be generalized or localized, but tenderness, fever, and leukocytosis are absent because the symptoms are neurologic in origin, from the visceral autonomic nervous system involvement. Gastrointestinal manifestations also include abdominal distention, nausea, vomiting, diarrhea, or constipation. Peripheral sensory or motor neuropathy is another common feature of AIP. Psychiatric symptoms including hysteria, anxiety, apathy, depression, phobia, psychosis, agitation, disorientation, hallucinations, and schizophrenic-type behaviors can be the only manifestations of the disease. Acute attacks may be accompanied by seizures, either a manifestation of the porphyria itself or caused by hyponatremia (from inappropriate secretion of antidiuretic hormone), which also commonly occur during attacks. Sympathetic hyperactivity results in tachycardia (in 80% of cases), hypertension, tremors, and sweating. Because of the nonspecific nature of symptoms and signs, the use of highly sensitive and specific laboratory tests is essential to the diagnosis.

During acute attacks, symptomatic treatment may include narcotic analgesics, phenothiazines, low-dose benzodiazepines, and propranolol for hypertension and tachycardia. Although intravenous glucose (at least 300 g/day) can be effective in acute attacks of porphyria, intravenous heme is now considered the treatment of choice to reduce excretion of porphyrins. Infusion of heme should be initiated as soon as possible after onset of an attack, but the rate of recovery depends on the degree of neuronal damage and may take days to months. Human heme solution stabilized with arginine (Normosang) is widely available except in the United States, where the lyophilized form (PanHaematin) is FDA approved.⁶ Any intercurrent infection or disease should also be treated immediately. Identification and avoidance of precipitating factors is also essential for prevention. Cyclical attacks in some women associated with fluctuations in estrogen and progestins can be prevented with a long-acting gonadotropin-releasing hormone analogue.

Congenital Erythropoietic Porphyria

CEP, an autosomal-recessive disorder also known as Gunther’s disease, is caused by deficient activity of uroporphyrinogen III cosynthase (the fourth enzyme of the pathway) and is associated with hemolytic anemia and cutaneous lesions. Severe cutaneous photosensitivity usually begins in early infancy as blistering of sun-exposed areas of the skin. Recurrent vesicles, bullae, and secondary infection can lead to cutaneous scarring and deformities. Porphyrin deposition may also occur in bones, leading to brownish discoloration of teeth. Protecting skin from sunlight is essential.

Mild to severe hemolytic anemia and secondary splenomegaly are features of CEP, and anemia can be severe. Transfusion is effective but results in iron overload if chronic. Splenectomy may reduce hemolysis and decrease the transfusion requirement. In transfusion-dependent children, allogeneic stem cell transplantation can be considered.

Porphyria Cutanea Tarda

PCT, the most common of the porphyrias, is caused by acquired or inherited deficiency of uroporphyrinogen decarboxylase (the fifth enzyme of the pathway). The disease occurs worldwide but its exact incidence is not known. The disease can be sporadic (noninherited or type I, most common) or familial (types II and III), although these subtypes are not distinguishable clinically. The frequency of disease varies in relation to risk factors such as alcohol use, smoking, and hepatitis C and human immunodeficiency virus (HIV) infection. The hallmark of PCT is cutaneous photosensitivity presenting as chronic blistering lesions on sun-exposed areas of skin without neurologic manifestations.⁸ Chronic changes including cutaneous thickening, scarring, and calcification can mimic systemic sclerosis. Also common are facial hypertrichosis and hyperpigmentation. PCT is almost always associated with abnormalities in liver function tests, and the risk of developing hepatocellular carcinoma is significantly increased in this disease.

Alcohol ingestion, estrogens, iron supplements and, if possible, any other drugs that may exacerbate the disease, and sun exposure should be avoided. A complete response can usually be achieved by repeated phlebotomy to reduce hepatic iron and is still considered standard treatment. Low-dose chloroquine or hydroxychloroquine are also effective, especially when phlebotomy is not indicated. Chloroquine slowly mobilizes the porphyrins from the liver and increases their excretion into the urine. In contrast, similar skin lesions in VP, HCP, CEP, and HEP are unresponsive to these therapeutic interventions.

Hepatoerythropoietic Porphyria

This rare form of porphyria has been recently described. HEP is clinically indistinguishable from CEP and is caused by homozygous or compound heterozygous defects of the same enzyme involved in PCT. Patients usually present after birth with dark urine in the diapers followed by severe photosensitivity with blistering skin lesions and scleroderma-like scarring. Hemolytic anemia is often present with splenomegaly. The avoidance of sunlight is essential.

Hereditary Coproporphyria

HCP is an autosomal dominant porphyria resulting from deficiency of coproporphyrinogen oxidase (the sixth enzyme of the pathway). The neurovisceral symptoms and other manifestations as well as the precipitating factors are virtually identical to those of AIP but photosensitivity similar to PCT may also occur in one-third of the patients. Avoidance of precipitating factors as in AIP is important. Neurologic symptoms are treated as in AIP but in contrast to PCT, phlebotomy or chloroquine is not effective for cutaneous lesions.

Variegate Porphyria

This hepatic porphyria, the result of a mutation of the protoporphyria oxidase gene (the seventh enzyme in the pathway), is transmitted as autosomal dominant disorder and is particularly common in South African whites (prevalence of 3 in 1,000) because of a genetic founder effect from a couple who emigrated from Holland to South Africa in the late 1600s. The disease was termed variegate because it can present with either neurovisceral symptoms, cutaneous photosensitivity, or both. Neurovisceral symptoms are very similar to those of AIP and are provoked by the same precipitates. Acute attacks are treated with glucose and heme infusion as in AIP. Occurrence of skin manifestations is usually separate from the neurovisceral symptoms, and avoiding sun exposure is the only effective preventative measure for cutaneous photosensitivity.

Erythropoietic Protoporphyria

EPP, also known as protoporphyria, results from deficiency of ferrochelatase activity, the last enzyme in the heme biosynthetic pathway. EPP is the most common erythropoietic porphyria and the third most common porphyria in general. Skin photosensitivity beginning in childhood is typical of the disease but the skin lesions are different from other porphyrias. Erythema, burning, and itching accompanied by swelling can develop within minutes of sun exposure, but sparse vesicles and bullae are seen in only a minority of the cases. Chronic skin changes may occur but severe scarring is rare. Treatment involves avoidance of sun exposure and the use of topical sun screens. Oral β-carotene (120 to 180 mg/day) can be effective in many patients with EPP, in contrast to those with photosensitivity from other forms of porphyria. The mechanism of action of β-carotene is not clear but is attributed to its antioxidant effect. In some patients, accumulation of protoporphyrin causes chronic liver disease that can progress to hepatic failure and death. Neurovisceral symptoms are seen only in patients with severe hepatic complications. Protoporphyrin-rich gallstones may occur. Mild anemia is sometimes seen in patients with EPP, but hemolysis is either infrequent or very mild. Splenectomy may be helpful when the disease is accompanied by hemolysis and significant splenomegaly. Caloric restriction, drugs, and exogenous sex hormones should be avoided. Intravenous heme therapy is sometimes beneficial. Liver transplantation has been performed but the protoporphyrin-induced damage can recur in the donor liver.¹⁰

References

  1.  Sassa S. Hematologic aspects of the porphyrias. Int J Hematol. January 2000;71(1):1-17.

  2.  Sassa S. Modern diagnosis and management of the porphyrias. Br J Haematol. November 2006;135(3):281-292.

  3.  Foran SE, Abel G. Guide to porphyrias. A historical and clinical perspective. Am J Clin Pathol. June 2003;(119 suppl):S86-S93.

  4.  Dombeck TA, Satonik RC. The porphyrias. Emerg Med Clin North Am. August 2005;23(3):885-899.

  5.  Kauppinen R. Porphyrias. Lancet. January 2005;365(9455):241-252.

  6.  Puy H, Gouya L, Deybach JC. Porphyrias. Lancet. March 2010;375(9718):924-937.

  7.  Anderson KE, Bloomer JR, Bonkovsky HL, et al. Recommendations for the diagnosis and treatment of the acute porphyrias. Ann Intern Med. March 2005;142(6):439-450.

  8.  Poblete-Gutierrez P, Wiederholt T, Merk HF, Frank J. The porphyrias: clinical presentation, diagnosis and treatment. Eur J Dermatol. May–June 2006;16(3):230-240.

  9.  Whatley SD, Ducamp S, Gouya L, et al. C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload. Am J Hum Genet. September 2008;83(3):408-414.

10.  Seth AK, Badminton MN, Mirza D, Russell S, Elias E. Liver transplantation for porphyria: who, when, and how? Liver Transpl. September 2007;13(9):1219-1227.

6

Bone Marrow Failure Syndromes: Aplastic Anemia, Acquired and Constitutional; Paroxysmal Nocturnal Hemoglobinuria; Pure Red Blood Cell Aplasia; and Agranulocytosis

Phillip Scheinberg, Neal S. Young and Johnson M. Liu

ACQUIRED BONE MARROW FAILURE SYNDROMES

The bone marrow failure syndromes are characterized by inadequate blood cell production leading to low red blood cell, white blood cell, and/or platelet counts in the peripheral blood. Marrow failure can be acquired or constitutional, and may affect all three blood cell lines, resulting in pancytopenia, or only a single lineage. In most, the bone marrow shows a simple deficiency of the related precursor cells, but marrow failure can also occur with relatively cellular marrows, presumably due to ineffective hematopoiesis, and be associated with cytogenetic abnormalities (see the chapter on myelodysplastic syndromes (MDSs)) or a genetically altered cell, as in paroxysmal nocturnal hemoglobinuria (PNH), discussed in this chapter because of its intimate relationship to aplastic anemia (AA).¹ Even the paradigmatic syndrome of AA clinically and pathophysiologically shows overlap with related diseases (Fig. 6.1).

ACQUIRED APLASTIC ANEMIA

Acquired AA is characterized by pancytopenia with a hypocellular, often empty, bone marrow. AA is uncommon in the West: its incidence in Europe is about two new cases per million of population. However, the disease is two- to three fold more frequent in East Asia and probably elsewhere in the developing world. In most series, patients are young with the majority presenting between 15 and 25 years of age. Historically, chemicals (benzene) and medical drugs (chloramphenicol) were implicated as causative, but without satisfactory mechanisms of pathogenesis. The most important current associations are with nonsteroidal anti-inflammatory drugs, antithyroid drugs, penicillamine, allopurinol, and gold (Table 6.1).² Nonetheless, most AA is idiopathic, and it is usually not possible to assign an environmental cause in an individual patient. One objective association is with prior seronegative hepatitis, present in 5% to 10% of patients in most case series.

FIGURE 6.1 Venn diagram of the relationship among bone marrow failure syndromes. AA, aplastic anemia; AML, acute myeloid leukemia; DKC, dyskeratosis congenita; MDS, myelodysplasia; PNH, paroxysmal nocturnal hemoglobinuria; SDS, Shwachman-Diamond syndrome.

Table 6.1  Drugs Associated with Aplastic Anemia in the International Aplastic Anemia and Agranulocytosis Study

Nonsteroidal analgesics

Butazones

Indomethacin

Piroxicam

Diclofenac

Antibiotics

Sulfonamides†

Antithyroid drugs

Cardiovascular drugs

Furosemide

Psychotropic drugs

Phenothiazines

Corticosteroids

Penicillamine

Allopurinol

Gold

†Other than trimethoprim-sulfonamide combination.

Etiology and Pathophysiology

  Hematopoiesis is severely reduced in all AA, as observed in bone marrow specimens, CD34 cell counts, magnetic resonance imaging, or in colony culture assays of progenitors.

  Clinical and laboratory studies suggest that most acquired AA is secondary to immunologically mediated destruction of hematopoietic cells by cytotoxic lymphocytes (CTL) and their cytokine products, especially interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α).

  Marrow failure rarely can follow infectious mononucleosis (Epstein-Barr virus [EBV] infection) and is a component of the stereotypical post-hepatitis AA syndrome. EBV and the putative agent of seronegative hepatitis likely behave as triggers for immune system activity. In contrast, parvovirus B19 directly infects and kills erythroid progenitor cells and causes transient red cell aplasia and occasionally chronic pure red cell aplasia (PRCA), but not AA.

  Direct killing of marrow cells by cytotoxic agents occurs following cancer chemotherapy, producing transient marrow aplasia, but is probably unusual as a mechanism of idiosyncratic drug-associated AA.

Clinical Features

  Anemia leads to fatigue, weakness, lassitude, headaches, and in older patients dyspnea and chest pain and these symptoms are most commonly responsible for the clinical presentation.

  Thrombocytopenia produces mainly mucosal bleeding: petechiae of the skin and mucous membranes, epistaxis, and gum bleeding are frequent and early complaints. Bleeding is not brisk from low platelets unless in the presence of accompanying physical lesions, as in gastritis and fungal infection of the lungs. The most feared complication of thrombocytopenia is intracranial hemorrhage.

  Infection is unusual at presentation.

  Dark urine suggests PNH.

  Occasionally, moderate cytopenias are identified serendipitously by routine blood work or at preoperative evaluation.

  Constitutional symptoms (malaise, anorexia, and weight loss) should be absent.

Physical findings range from a well appearing patient with minimal findings to an acutely ill patient with signs of systemic toxicity. Cachexia, lymphadenopathy, and splenomegaly are not seen and suggest an alternative diagnosis.

  Thrombocytopenia results in petechiae, ecchymoses, gingival oozing, epistaxis, and subconjunctival and retinal hemorrhage.

  Anemia is reflected in pallor of the skin, mucous membranes, and nail beds.

  Constitutional AA is suggested by areas of hyper- or hypopigmentation of the skin, abnormal hands and thumbs, short stature (Fanconi anemia [FA]), and nail dystrophy and oral leukoplakia (dyskeratosis congenita [DKC]).

Diagnosis and Differential Diagnosis

At diagnosis:

  Marked pancytopenia or reduction in two of three or, less commonly, one of three cell lines

  Peripheral blood smear shows reduced platelets and neutrophils, normal red cells

  Microspherocytes and giant platelets suggestive of peripheral destruction are not present

  Bone marrow markedly hypocellular on biopsy (1 cm core); mainly residual lymphocytes, plasma cells, mast cells on aspirate smear

  Overall marrow biopsy cellularity is low (<30%, excluding lymphocytes), but there may be pockets

of cellularity, so-called hot-spots.

  Myeloblasts should not be increased

  Megakaryocytes are almost always absent

  Marrow cytogenetics should be normal, but some authorities accept cytogenetic abnormalities such as trisomy 6 or 8, loss of Y or del20q as consistent with AA in the absence of significant dysplastic marrow findings.

In secondary marrow failure, the degree of pancytopenia is usually moderate, and the underlying illness is usually obvious from history and physical examination (e.g., stigmata of alcohol liver disease, presence of other autoimmune disease or infection). However, pancytopenia has many causes, of which AA is not the most common (Table 6.2).

Table 6.2  Differential Diagnosis of Pancytopenia

Pancytopenia with Hypocellular Bone Marrow

Acquired aplastic anemia

Inherited aplastic anemia

Some myelodysplastic syndromes

Rare aleukemic leukemia

Some acute lymphoblastic leukemia

Some lymphomas of the bone marrow

Pancytopenia with Cellular Bone Marrow

Primary Bone Marrow Diseases

Myelodysplasia syndromes

Paroxysmal nocturnal hemoglobinuria

Myelofibrosis

Hairy cell leukemia

Some aleukemic leukemia

Myelophthisis

Bone marrow lymphoma

Secondary to Systemic Diseases

Systemic lupus erythematosus, Sjögren’s syndrome

Hypersplenism

Vitamin B12, folate deficiency (familial defect)

Overwhelming infection

Alcoholism

Brucellosis

Ehrlichiosis

Sarcoidosis

Tuberculosis and atypical mycobacteria

Hypocellular Bone Marrow with or without Cytopenia

Q fever

Legionnaire’s disease

Toxoplasmosis

Mycobacteria

Tuberculosis

Anorexia nervosa, starvation

Hypothyroidism

Most important is to distinguish among primary marrow diseases (Table 6.3, Fig. 6.2):

  Constitutional AA presenting in adults. A family history is highly suggestive. FA pedigrees often have instances of leukemias and myelodysplasia (MDS); telomeropathy families often include not only malignant hematologic diseases but also pulmonary fibrosis and hepatici cirrhosis. There may be no or only subtle physical stigmata. Patients under 40 years of age (or older if the history or examination are suggestive) should be tested for FA. Although phenotypic abnormalities have been classically described in both FA and DKC, patients with adult onset constitutional AA may have subtle signs on routine physical examination or no characteristic findings at all. ³

  MDS is hypocellular in about 20% of cases. Dysplastic changes in AA when present are mild and limited to erythrocytes. In MDS, megaloblastic changes are more extreme; megakaryocytes are preserved and can be aberrantly small and mononuclear; and myeloid precursors may be increased, left shifted, and poorly granulated. Chromosomal analysis of bone marrow cells is almost always normal in AA, while MDS is often associated with cytogenetic abnormalities. Nonetheless, the distinction may be so difficult that some patients are best labeled AA/MDS.

  PNH/AA. Small PNH expanded clones are common—in as many as 50% of cases at presentation—in the setting of marrow failure now that flow cytometry has replaced the Ham test. Growth of clone size over time may lead to clinical hemolysis. Thrombosis is rare.

  Acute lymphocytic leukemia in children and acute myeloid leukemia in the elderly can occasionally present with pancytopenia and marrow hypocellularity.

  Myelofibrosis has a characteristic leukoerythroblastic blood picture, marrow is dry tap (rather than watery, as in AA), and hepatosplenomegaly is common.

  Large granular lymphocytosis is characterized by prolonged neutropenia, less frequently anemia or thrombocytopenia, and increased numbers of large granular lymphocytes in the peripheral blood. Marrow is usually cellular; diagnosis rests on flow cytometry or molecular evidence of rearrangement of the T cell receptor.

Severe AA is defined by two of the following three peripheral blood count (Camitta) criteria

  absolute neutrophil count (ANC) <500/µL

  platelet count <20,000 /µL

  reticulocyte count (automated) <60,000/µL

Definitive Treatments

Definitive therapy of AA consists of allogeneic hematopoietic stem cell transplantation (HSCT) or immunosuppression; both have dramatically changed the natural course of this illness, with 5 year survival of 75% in patients undergoing either treatment.⁴ Support with growth factors alone or in combination with transfusion is of unproven long-term benefit and is unlikely to address the underlying pathophysiology of the disease. The main distinctions between immunosuppression and HSCT are shown in Table 6.4.

FIGURE 6.2 Differential diagnosis of cytopenias. MDS, myelodysplastic syndrome; BM, bone marrow; PNH, paroxysmal nocturnal hemoglobinuria.

  HSCT cures AA. Most transplants are performed using a histocompatible sibling donor, and most recipients are young. Overall long-term survival is about 70% to 80%, with better results observed in children.5,6 HSCT is preferred in children to about age 20 who have an appropriate donor. With current cyclophosphamide-based conditioning, major toxicities are related to graft-versus-host disease (GVHD) and infection (not always easily separable). GVHD and mortality risk increases with recipient age.⁵ The source of donor cells may be important: in recent retrospective studies, granulocyte colony stimulating factor (G-CSF)-mobilized stem cells produced a higher rate of chronic GVHD and mortality in younger patients than did bone marrow cells.7,8 Modest numbers of erythrocyte and platelet transfusions do not appear to increase the risk of graft rejection, especially with leukocyte-depleted products.⁹

  Seventy percent of patients will lack a suitable matched sibling donor. Transplantation from matched unrelated donors (MUD) is now more feasible with the development of huge donor registries and an effective network. Overall results have been about half as good as with human leukocyte antigen (HLA)-matched family members but likely to improve with modifications of conditioning regimens and high-resolution molecular typing for donor selection10,11; in some small studies, outcomes with alternative donor sources approximate standard transplant outcomes.12,13 Donor searches should be initiated early for younger patients who might be eligible later for a MUD. Of note, the success rate for identifying a suitable matched unrelated donor in the United States decreases in non-Caucasian patients such as in the African American and Hispanic ethnic groups. For unclear reasons, umbilical cord blood transplantation has been associated with poor results in marrow failure states.¹⁴ In current practice, unrelated transplant is offered for children who have failed a single course of immunosuppression and to adults who are refractory to multiple courses of antithymocyte globulin (ATG) and alternative therapies such as androgens.

  Immunosuppression using regimens combining ATG and cyclosporine (CsA) is standard therapy. About 2/3 of patients improve to transfusion-independence and, overall survival rates at 5 years are comparable to HSCT. Immunosuppression is almost always preferred in older patients, especially if the neutrophil count is not severely decreased. One frequently used protocol for horse ATG is 40 mg/kg/day for 4 days. Rabbit ATG is administered at 3.5 mg/kg/day for 5 days Corticosteroids, such as methylprednisolone at 1 mg/kg, are administered during the first 2 weeks to ameliorate serum sickness. In a recent randomized study, hematologic response rate at 6 months was inferior with rabbit ATG (37%) compared to horse ATG (68%) when given as first therapy.¹⁵ This large difference in response translated in worst survival at 3 years after rabbit ATG (70%) as compared to horse ATG (94%).¹⁶ Thus, horse ATG is the preferred initial immunosuppressive treatment in SAA.

In patients who are refractory to initial horse ATG, hematologic recovery can be achieved with a second course of rabbit ATG¹⁷; in our institutional experience, about 30% of patients showed a hematologic response.¹⁸ Alemtuzumab also has activity in SAA primarily in the relapse and refractory settings.¹⁹ In relapse, alemtuzumab (without CsA) produced hematologic response in 55% of cases, which is comparable to the reported rates of rabbit ATG in this setting; in refractory SAA, the response rate to alemtuzumab was 37%, which was similar to that of rabbit ATG (33%) in direct comparison.¹⁹ As first therapy, alemtuzumab fared poorly, with a response rate of only 19% in a prospective randomized study.¹⁹ Thus, alemtuzumab may be an option in relapsed and refractory SAA, especially in those intolerant to ATG and/or CsA, or in older patients.

Major toxicities of ATG include immediate allergic reaction, serum sickness, and transient blood count depression. Anaphylaxis is rare but has been fatal and may be predictable by skin testing. Treatment of ATG allergy is mainly symptomatic: intravenous hydration, antihistamines (for urticaria) and meperidine (for rigors), and increased doses of corticosteroids (for symptomatic serum sickness). CsA is begun at 10 mg/kg in adults and 12/mg/kg in children, with dose adjustments to maintain blood levels about 200 ng/mL. We administer CsA for 6 months following ATG. It is common for CsA to be tapered after 6 to 12 months with limited data to support this practice. A retrospective Italian study suggested that a CsA taper may be of benefit in preventing relapse, but in our experience in about 70 responders who had their CsA tapered prospectively from 2003 to 2010, we did observe a delay in the occurrence of relapse but did not ultimately prevent it when compared to historical control.15,20 Renl and liver function monitoring is required to avoid nephrotoxicity; hypertension, gingival hypertrophy, and tremulousness which are common side effects. Alemtuzumab is usually well tolerated with infusion-related toxicities more manageable than with ATG. In AA, while immunosuppression (ATG, alemtuzumab) reactivates latent herpes virus infection and increases EBV and cytomegalovirus (CMV) virus in the circulation, disease is rare and routine prophylactic or preemptive antiviral therapy is not required.19,21

Prognosis is strongly correlated to hematologic response at 3 months, especially the robustness of blood count recovery defined by absolute reticulocyte and platelet.²² Pre-therapy, the absolute reticulocyte has also been correlated with a better response rate and survival.23,24 In a recent analysis, the neutrophil count did not correlate with hematologic response outcomes but associated to short-term mortality.²⁵ Despite the lack of progress in developing more efficacious immunosuppressive regimens, survival in SAA has improved over the years, especially among non-responders to an initial course of horse ATG, with survival in 5 years increasing nearly three fold among this group (from 23% in the 1990s to 57% in recent years).²⁵ This improvement has been attributed to better supportive care (with antifungals) and more effective salvage therapies with repeat immunosuppression and salvage HSCT. Rates of graft rejection and survival have also improved over time with better transplantation and supportive care protocols, less immunogenic blood products, and closer HLA matching between unrelated donors and recipients with high-resolution tissue typing.13,26-28 However, rates of acute and chronic GVHD have remained steady over the years. A bone marrow source of stem cells (compared to mobilized peripheral blood CD34+ cells) and conditioning with alemtuzumab have been associated with lower rates of GVHD.7,8,29

Even after hematologic response to ATG, blood counts may fall, especially on withdrawal of CsA. Reinstitution of CsA usually suffices, but retreatment with ATG may be necessary and relapse is sometimes irreversible and fatal. Evolution to a clonal hematologic disease occurs in about 15% of patients over the decade after initial therapy, manifesting as a dysplastic bone marrow or cytogenetic abnormalities, especially monosomy 7. Pre-treatment telomere length may identify increased risk for clonal evolution long-term. In a recent report, patients with the shortest telomere length (adjusted for age) had a three fold higher risk of acquiring a new cytogenetic abnormality, and five- to six fold higher for monosomy 7 and complex cytogenetics, when compared to those with longer pre-treatment telomere length. These differences translated into a survival disadvantage associated with short telomeres (66%) compared to long (84% in 6 years).

  Other therapies that are occasionally successful include growth factor combinations (erythropoietin and G-CSF), androgens, and high dose cyclophosphamide (controversial due to the prolonged neutropenia that it induces).³⁰ Our approach to treatment is shown in Figure 6.3.

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

PNH is a rare clonal disease of the bone marrow, which can produce a clinical triad of (1) hemolysis, (2) venous (and rarely arterial) thrombosis, and (3) AA.³¹

Etiology and Pathophysiology

  A somatic mutation in a gene called PIG-A occurs in a hematopoietic stem cell

  Leads to deficient synthesis of a glycolipid moiety called the glycosylphosphoinositol anchor (GPI).

FIGURE 6.3 Treatment of severe aplastic anemia. ANC, absolute neutrophil count; ATG, antithymocyte globulin; BM, bone marrow; CsA, cyclosporine A; HSCT, hematopoietic stem cell transplantation; MUD, matched unrelated donor.

  Lack of cell surface presentation of a large family of GPI-linked proteins

  Absence of one of these proteins, CD59, on the cell surface of erythrocytes leads to their susceptibility to complement and to intravascular hemolysis

  PIG-A mutant cells are probably present in normal adult marrow, but clonal expansion of these cells is unusual—except especially in AA (about 50% of cases) and in MDS, where they may range in size from small to large.

  Which GPI-anchored proteins are important in permitting clonal expansion in AA and MDS and in the thrombotic proclivity is unknown.

Clinical Features

  Intravascular hemolysis, classically as periodic bouts of dark urine in the morning but also as continu-

ous red cell destruction and without hemoglobinuria.

  Venous thrombosis in unusual sites, especially hepatic, mesenteric, and portal veins and intracranial veins.

  Marrow failure, frank AA or poor marrow function despite a relatively cellular histology.

Diagnosis

In general, intravascular hemolysis is unusual (see Chapter 3), and PNH should be considered in the setting of a suggestive history, hemoglobinuria, and elevated LDH. There may be accompanying iron deficiency and neutropenia/thrombocytopenia.

PNH patients can present with abdominal pain due to Budd-Chiari syndrome or symptoms of stroke.

PNH clonal expansion should be sought in patients with AA and MDS. Peripheral blood Flow cytometry provides evidence of a PNH clonal expansion through quantification of GPI-anchored proteins on erythrocytes and granulocytes (especially the latter in the transfused patient). However, severe hemolysis and thrombosis typically occur only in patients with large clones (>50% erythrocytes).

Treatment

The course is highly variable where clone size may range from small and inconsequential clinically to large and associated with clinical hemolysis. AA patients post-immunosuppressive therapy with expanded clones may be asymptomatic; modest and intermittent hemolysis managed with transfusions alone is consistent with long survival; conversely, PNH can be associated with catastrophic thrombotic events. Clones may spontaneously disappear in some patients.

  Transfusion to maintain hemoglobin levels consistent with full activity. Use of washed erythrocytes is not necessary.

  Iron supplementation may be required; loss of hemoglobin as a result of intravascular destruction prevents secondary hemochromatosis.

  Corticosteroids, usually in moderate doses (30 to 50 mg prednisone on alternate days) have been employed to control hemolysis but never rigorously tested. A short trial in a patient with continuous red cell destruction may be warranted.

  Marrow failure presenting as frank AA with associated PNH should be treated with HSCT or immunosuppression (see above).

  Most patients in Western series die of thrombotic complications, and thromboses, once they occur, may be refractory to anticoagulation. An uncontrolled trial has suggested that coumadin prophylaxis is effective, but the relative risk of hemorrhage secondary to chronic anticoagulation for years, even decades, in this population remains unclear. ³²

  HSCT is the only curative therapy, but may carry a higher risk in PNH due to comorbid conditions; nonmyeloablative conditioning regimens may offer improved survival.⁹ Transplant can be considered in younger patients with severe marrow failure or thrombotic complications.

  Eculizumab (a monoclonal antibody directed to the active component of C5) has been approved by the FDA for patients with PNH and is marketed as Soliris. In a large prospective multicenter trial, the drug blocked intravascular hemolysis, which translated to a clinically significant improvement in anemia, transfusion requirements, and in quality of life measures.³³ Eculizumab also appeared to dramatically reduce the risk of clinical thromboses in patients with PNH, which is the major cause of morbidity and mortality in this disease.³⁴

PURE RED CELL APLASIA

PRCA is defined as anemia with absent reticulocytes and marrow erythroid precursor cells.³⁵ This rare aregenerative anemia has a number of clinical associations and is also usually responsive to treatment.

Etiology and Pathophysiology

  Constitutional PRCA is Diamond-Blackfan anemia (DBA) and is secondary to inherited mutations in ribosomal protein genes.

  Acquired PRCA often behaves as an immunologically mediated disease. Clinical associations include thymoma (but probably <10% of PRCA cases), collagen-vascular syndromes, myasthenia gravis, chronic lymphocytic leukemia, and large granular lymphocytic leukemia.

  PRCA may also be seen in MDS, especially with 5q-syndrome. The phenotype is due to acquired loss of a ribosomal protein gene on chromosome 5, as occurs in the germline in DBA.

  Parvovirus B19 infection causes erythema infectiosum (fifth disease) in children and transient aplastic crisis in patients with underlying hemolysis. Virus infection is ordinarily terminated by production of neutralizing antibodies. Persistence of parvovirus results from failure to mount a neutralizing antibody response, leading to chronic erythroid precursor destruction and anemia. Persistence of parvovirus B19 can occur in an immunodeficient host: in congenital immunodeficiencies (Nezelof ’s syndrome), iatrogenic (immunosuppressive drugs and cytotoxic chemotherapy), and HIV infectioninduced immunodeficiency.

Clinical Features and Diagnosis

Reticulocytes are very low or absent; erythroid precursor cells are usually absent but a few normoblasts may be present in the marrow. There are morphologic clues: giant pronormoblasts signal parvovirus; uninuclear micromegakaryocytes, 5q-syndrome. Other blood counts are normal, as are cytogenetics (except for PRCA associated with MDS).

Thymoma should be excluded by CT scan.

In persistent parvovirus infection and PRCA, antibodies to virus are usually absent, or only IgM may be observed; virus can be detected in the blood by DNA hybridization.

Treatment

  For DBA, corticosteroids are standard; patients may be dependent on exquisitely low doses, and relapse may not always be responsive to reinstitution of treatment.

  For acquired PRCA, corticosteroids in moderate doses are usually first therapy, followed by either other immunosuppressives such as CsA, ATG, or cytotoxic drugs such as moderate doses of azathioprine or cyclosphosphamide, administered orally. In a few case reports the monoclonal antibodies to CD20 (rituximab) and CD25 (daclizumab) have been shown effective and more recently (in a few case reports) or cyclophosphamide. ³⁶

  Thymomas should be excised as they are locally invasive, but surgery does not necessarily resolve the anemia.

  Persistent parvovirus B19 infection responds to intravenous immunoglobulins at 0.4 g/kg daily for 5 to 10 days. Patients with large viral loads, especially in the acquired immunodeficiency syndrome, may relapse and require periodic retreatment.

AGRANULOCYTOSIS

Severe neutropenia with either complete or partial absence of myeloid precursor cells is agranulocytosis.

Etiology and Pathophysiology

  Most agranulocytosis is drug-associated (Table 6.5). Idiopathic pure white cell aplasia (without exposure to a suspicious drug) is exceedingly rare (and like PRCA may also be associated with thymoma). 37,38

  Mechanisms of drug destruction of granulocyte precursors include direct effects (as with thorazine) and immune (antibody)-mediated (as with dipyrone) (Table 6.6).

Table 6.5  Drugs Associated with Agranulocytosis

Heavy metals

Gold

Arsenic compounds

Analgesics

Aminopyrine, dipyrone

Butazones

Indomethacin

Ibuprofen

Acetaminophen

Para-aminosalicylic acid

Sulindac

Antipsychotics, sedatives, antidepressants

Phenothiazines

Tricyclics

Chlordiazepoxide

Barbiturates

Serotonin reuptake inhibitors

Anticonvulsants

Phenytoin

Ethosuximide

Carbamazepine

Antithyroid drugs

Propylthiouracil

Methimazole

Cardiovascular drugs

Procainamide

Captopril

Nifedipine

Quinidine

Propranolol

Methyldopa

Propafenone

Aprinidine

Sulfa drugs

Thiazide diuretics like spironolactone and

acetazolamide

Oral hypoglycemics

Sulfasalazine

Dapsone

Sulfa antibiotics

Antibiotics

Sulfa antibiotics

Pyrimethamine

Penicillins

Cephalosporins

Macrolides

Vancomycin

Clindamycin

Aminoglycosides

Antituberculosis agents

Levamisole

Antimalarials

Mebendazole

Antifungals

Fluconazole

Antiviral

Zidovudine

Antihistamines

Cimetidine

Ranitidine

Chlorpheniramine

Miscellaneous

Isotretinoin

Omeprazole

Colchicine

Allopurinol

Aminoglutethimide

Metoclopramide

Ticlopidine

Tamoxifen

Penicillamine

Insecticides

Hair dye

Chinese herbal medicines

Diagnosis and Treatment

  The patient is usually older with a history of clear exposure to an incriminated agent, usually with introduction of the drug in the preceding 6 months. Absent neutrophils on smear should lead to a confirmatory bone marrow examination.

  Classic presentation is fever and sore throat.

  Recovery occurs spontaneously but over a highly variable time period, from a few days to several weeks. G-CSF or granulocyte monocyte colony-stimulating factor (GM-CSF) is almost always administered without clear evidence of efficacy.

  Fever and signs of infection require prompt administration of broad spectrum antibiotics by a parenteral route.

  Mortality remains substantial (about 10%) due to the combination of patient age, comorbid conditions, and lethal sepsis.

CONSTITUTIONAL BONE MARROW FAILURE SYNDROMES

Among the constitutional disorders that present with AA, it is important to consider FA and DC. Genes mutated in FA and in DC have been identified and are important in the functions in the cell; they have a key role in genomic stability and the maintenance of telomeres, respectively. An algorithm for laboratory testing to exclude FA and DC is presented below.

FANCONI ANEMIA

  Autosomal recessive or X-linked inheritance; most common of the constitutional syndromes, seen in all races; diagnosed on the basis of positive chromosome breakage test (see below)

  Mutations in any of these 15 genes: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO, FANCP

  Chief criteria of pancytopenia, hyperpigmentation, malformation of the skeleton, small stature, and hypogonadism

  Malformations of the eye, ear, genitourinary and gastrointestinal tracts, and cardiopulmonary and central nervous systems can occur.

  FA is notoriously heterogeneous in the degree and number of clinical manifestations, and patients presenting solely with either congenital malformations or hematologic abnormalities may either be misdiagnosed or go unrecognized entirely.

Clinical Features

  The diagnosis is suggested when a child presents with hyper- or hypo-pigmented skin lesions; short stature (poor growth); anomalies of the upper limb or thumb; male hypogonadism; microcephaly; characteristic facial features, including a broadened nasal base, epicanthal folds, and micrognathia; and structural renal abnormalities. When this constellation of physical anomalies is accompanied by bone marrow failure (which may often trigger initial medical evaluation), confirmation of the diagnosis can be made by standard DEB or MMC chromosome breakage analysis (see below).

  The mean age at diagnosis 8 to 9 years.

Diagnostic Tests

  Chromosome breakage test with diepoxybutane (DEB) or mitomycin C (MMC), performed on peripheral blood cells.

  Based solely on definition by the DEB test, nearly 40% of patients may be free of major physical anomalies. These FA patients with normal appearances may go unrecognized unless there is a high index of suspicion for familial disease.

  A challenge is the diagnosis of FA in older patients. Although the mean age of diagnosis is in the first decade of life, FA has been described in the 5th and 6th decades of life.

Hematologic Presentations and Cancer Predisposition

  The symptoms and signs of FA typically relate to the hematologic presentation of cytopenias from marrow failure. Often, thrombocytopenia or leukopenia is noted before full pancytopenia; furthermore, the pancytopenia typically worsens with time. Almost all FA patients will develop hematologic abnormalities in their lifetime. Erythropoiesis is usually macrocytic.

  Classically, the bone marrow is hypocellular and fatty, indistinguishable from that of acquired AA. Microscopic examination of the marrow may show dyserythropoiesis and dysplasia. Some patients may develop or even present with a morphologically defined MDS or frank acute myeloid leukemia (AML).

  The crude risk of leukemia (exclusive of MDS) is ∼5% to 10%, while the cumulative incidence of leukemia is about 10% by age 25. Less commonly recognized is the probability of developing MDS, approximately 5%, which appears also to correlate with a poor prognosis for FA patients. Clonal karyotypic abnormalities, identical to those seen in non-FA MDS and secondary AML, are frequently found in FA patients, whether or not they meet marrow morphologic criteria for a defined MDS. The prognostic significance of these clonal chromosomal abnormalities in FA patients is not entirely clear, however, since cytogenetic changes can fluctuate over time. Certain clonal abnormalities may be associated with poor prognosis, such as gains of chromosome 3q.

  Solid organ malignancies occur frequently, with a crude risk of 5% to 10% of patients overall (the risk increases with age, as those patients who have survived into adulthood develop solid tumors). Particularly common are vulvar, esophageal, and head and neck cancers. In addition to these (presumed) de novo tumors, a subset of long-term survivors of SCT will develop secondary malignancies, typically head and neck.

  For FANCD1/BRCA2 and FANCN/PALB2, as has been demonstrated thus far, affected family members with monoallelic mutations are predisposed to breast cancer, whereas those with biallelic mutations present with an FA phenotype associated with childhood cancers and leukemia.

Stem Cell Transplantation and Supportive Care

  Allogeneic SCT from an HLA-matched sibling donor is the only curative therapy for the hematologic manifestations of FA (aplasia or MDS). Typically, decreased doses of cyclophosphamide and irradiation must be used in order to avoid severe toxicity due to the chemo- and radio-sensitivity of FA patients. Transplantation centers, which generally adopted this modified conditioning regimen with or without thoracoabdominal irradiation, have reported good results for FA patients that did not present with leukemia or pre-leukemic transformation.

  Umbilical cord blood transplantation from related donors has also been successfully applied to a small number of FA patients. A few FA patients have also undergone successful HSCT with cord blood from unrelated donors.

  Clearly, young patients with an HLA-compatible sibling should be treated by HSCT at the earliest stages of marrow failure in preference to other therapies. However, most patients do not have an HLA-identical donor and are dependent upon the identification of a suitably matched nonsibling relative or unrelated donor. A smaller number of FA patients have undergone HSCT from such alternative sources (matched unrelated and haploidentical family donors) to treat either aplasia or MDS, with or without clonal chromosomal abnormalities. The results from these alternative donor transplants have generally been inferior to those from matched sibling donor transplants, but are improving.

Patients lacking a suitable HLA-compatible donor (either sibling or matched unrelated) may benefit from chronic administration of androgens or hematopoietic growth factors, which may serve as temporizing measures.

Androgens

  Androgens have been shown to induce hematologic responses in approximately 50% of FA patients although their effectiveness in raising blood counts may be neither durable nor complete in all lineages. Typically, androgen therapy is initiated when the platelet count is consistently below 30,000/µL and/or the hemoglobin less than 7 gm/dL. Orally administered oxymetholone, at a dose of 2 to 5 mg/ kg/day, is usually combined with prednisone, 5 to 10 mg every other day, in order to counterbalance the anabolic properties of oxymetholone with catabolic actions of corticosteroids.

  Androgen therapy is associated with liver toxicities including transaminase enzyme elevation, cholestasis, peliosis hepatitis, and hepatic tumors.

Hematopoietic Growth Factors

  Levels of most growth factors are markedly increased in FA as they are in acquired AA, likely as a compensatory physiologic response.

  One worrisome aspect of chronic growth factor administration is the theoretical risk of stimulating a leukemic clone, particularly in patients prone to developing MDS or AML, or speeding the process of stem cell exhaustion.

  Chronic administration of G-CSF may have transient beneficial effects on multiple hematopoietic lineages in some patients.

DYSKERATOSIS CONGENITA

  Classical DKC is an inherited bone marrow failure syndrome characterized by the muco-cutaneous triad of abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia.

  It has been observed in many races and estimated prevalence of DKC is approximately 1 per 1,000,000 persons.

  X-linked recessive (mutations in DKC1), autosomal dominant (some cases due to heterozygous mutations in the RNA component of telomerase, TERC), and autosomal recessive (some cases due to NOP10 gene mutations) forms of the disease are recognized. Heterozygous mutations in the enzymatic component of telomerase (TERT) as well as in TERC can lead to variable phenotypes.

  A variety of other (dental, gastrointestinal, genitourinary, hair greying/loss, immunological, neurological, ophthalmic, pulmonary, and skeletal) abnormalities have also been reported.

  Bone marrow failure is the principal cause of early mortality with an additional predisposition to malignancy and fatal pulmonary complications

  Clinical manifestations in DKC often appear during childhood although there is a wide age range. The skin pigmentation and nail changes typically appear first, usually by the age of 10 years. BM failure usually develops below the age of 20 years; 80% to 90% of patients will have developed BM abnormalities by the age of 30 years. In some patients the BM abnormalities may appear before the muco-cutaneous manifestations and can lead to an initial diagnosis of idiopathic AA.

  The clinical features of these disorders are very heterogeneous and this makes diagnosis based on clinical criteria alone difficult and unreliable. In many pedigrees, disease only manifests in adulthood, hematologic manifestations can range from severe AA and AML to mild macrocytosis, and affected members may only have lung or liver disease.

  Oxymetholone can produce durable hematological responses in more than >50% of DKC and FA patients, but patients have to be monitored carefully for side effects. Androgens may function by increasing TERT transcription.

  The current definitive treatment is allogeneic HSCT. In both DKC and FA patients, low-intensity transplant protocols are producing prompt engraftment, reduced toxicity, and have the potential to reduce the risk of secondary malignancies.

Diagnostic Testing for Aplastic Anemia with Short Telomeres

Telomeres are the tips of chromosomes and are maintained by a complex that includes the enzyme telomerase reverse transcriptase (TERT), its RNA component (TERC), the protein dyskerin, and other associated proteins (NHP2, NOP10, and GAR1). Telomere length measurement is now commercially available and should be a screening test in most cases of severe AA, especially with a typical clinical history (moderate, slowly progressive AA) or if the family history suggests an inherited syndrome.

AA with short telomeres is typical in DKC patients. Since the genes mutated in the X-linked recessive (DKC1) and other (TERC and others) DKC subtypes are now known, it is possible to substantiate the diagnosis in a significant proportion of DKC patients. In particular, it is appropriate to screen for the DKC1 gene if patients are male and have two out the following: abnormal skin pigmentation, nail dystrophy, leukoplakia, or bone marrow failure.

Besides patients with a typical DKC pedigree, however, there are also patients with acquired AA with short telomeres who can carry mutations in TERT and TERC and not have the physical abnormalities observed in DKC or have a family history suggestive for telomeropathy. Overall, mutations in telomerase genes (TERC or TERT but not DKC1) appear to explain the short telomeres detected in about 10% of patients with AA with some responding to conventional immunosuupressive therapy as is applied in acquired AA cases. The failure of organs other than the bone marrow, including the liver and the lung, may also be associated with TERT mutations.

Marrow Failure Involving a Single Lineage

DIAMOND-BLACKFAN ANEMIA

  Probably the second most common constitutional marrow failure syndrome after FA

  Most patients present with anemia in the neonatal period or in infancy.

  Approximately 30% of affected children present with a variety of associated physical anomalies. Thumb and upper limb malformations and craniofacial abnormalities are common. Other defects: atrial or ventricular septal defects, urogenital anomalies, and prenatal or postnatal growth retardation.

  A moderately increased risk of developing MDS and solid organ malignancies

  Cases are sporadic, with an equal sex ratio, but 10% to 25% of patients have a positive family history for the disorder.

  Heterozygous mutations in the ribosomal protein genes RPS19, RPS24, RPS17, RPS6, RPS10, RPS26, RPL5, RPL11, and RPL35A account for some cases.

Hematological Findings

  Minimal diagnostic criteria for DBA: normochromic anemia in infancy (<2 years), low reticulocyte counts, absent or decreased bone marrow red cell precursors (<5% of nucleated cells), and a normal chromosome breakage test (to rule out FA)

  Additional features: presence of malformations, macrocytosis, elevated fetal hemoglobin (HbF), and elevated erythrocyte adenosine deaminase (eADA) level

  Some patients identified after the age of 2 years after a more severely affected family member first diagnosed

  Anemia usually severe at the time of diagnosis (usually macrocytic)

  The bone marrow aspirate is usually normocellular, but erythroblasts are markedly decreased or absent. The other cell lines are normal, but mild to moderate neutropenia, thrombocytopenia, or both may occur later in the course.

  Progression of the single-lineage erythroid deficiency of DBA into pancytopenia and AA is rare but may occur.

  Differential diagnosis includes transient erythroblastopenia of childhood (TEC). Both TEC and DBA show similar marrow morphology, but TEC is self-limited, with a recovery within 5 to 10 weeks.

Treatment

  Initial treatment in DBA is transfusions, but long-term administration of red cells may cause secondary hemochromatosis.

  Corticosteroids are the mainstay of treatment, and at least 50% of patients respond. There is no known predictor of steroid responsiveness, and later relapses occur. During treatment, some patients may recover sensitivity to corticosteroids or even proceed to a spontaneous remission.

  Allogeneic BMT is a treatment option for DBA in steroid-resistant patients.

  Hematopoietic growth factor therapy with interleukin-3 (IL-3) or EPO has been attempted for DBA.

SHWACHMAN-DIAMOND SYNDROME

  Probably the third most common constitutional marrow failure syndrome after FA

  Autosomal recessive disorder usually manifest in infancy and characterized by exocrine pancreatic insufficiency, short stature, and bone marrow dysfunction

  Mutations in the SBDS gene account for about 90% of cases

  The SBDS protein appears to be involved in multiple functions, including ribosome maturation

  Additional clinical features include: metaphyseal dysostosis, epiphyseal dysplasia, immune dysfunction, liver disease, growth failure, renal tubular defects, insulin-dependent diabetes mellitus, and psychomotor retardation.

  Hematological manifestations: neutropenia, raised HbF levels, anemia, thrombocytopenia, impaired neutrophil chemotaxis

  Predilection for malignant myeloid transformation and MDS

Clinical Diagnostic Criteria

Clinical Diagnosis

Fulfill the combined presence of hematological cytopenia of any given lineage (most often neutropenia) and exocrine pancreas dysfunction

Hematologic abnormalities may include:

  Neutropenia <1.5 × 109/L on at least two occasions over at least 3 months

  Hypoproductive cytopenia detected on two occasions over at least 3 months

Pancreatic dysfunction may be diagnosed by the following:

  Reduced levels of pancreatic enzymes adjusted to age [fecal elastase, serum trypsinogen, serum (iso) amylase, serum lipase]

Tests that support the diagnosis but require corroboration:

  Abnormal 72 hours fecal fat analysis

  Reduced levels of at least two fat-soluble vitamins (A, D, E, K)

  Evidence of pancreatic lipomatosis (e.g., ultrasound, CT, MRI, or pathological examination of the pancreas by autopsy)

Molecular Diagnosis: Biallelic SBDS Gene Mutation

Positive genetic testing for SBDS mutations known or predicted to be deleterious, for example, from protein modeling or expression systems for mutant SBDS

Treatment

  Treatment of hematologic manifestations (neutropenia, bone marrow failure) may involve Hematopoietic growth factor therapy with G-CSF. For neutropenia unresponsive to G-CSF, SAA, MDS, or leukemia, hematopoietic HSCT may be considered.

SEVERE CONGENITAL NEUTROPENIA AND CYCLIC NEUTROPENIA

  Originally described as an autosomal recessive disorder (Kostmann syndrome) but majority of SCN cases due to dominant-acting point mutations in the neutrophil elastase (ELANE or ELA2) gene. Mutations in the protooncogene GFI1 that target and repress ELANE have also been implicated.

  Characterized by severe neutropenia and an early stage maturation arrest of myelopoiesis, leading to bacterial infections from early infancy

  >90% of these patients respond to G-CSF (filgrastim, lenograstim) with ANC that can be maintained around 1.0 × 10⁹/L. Adverse events include mild splenomegaly, moderate thrombocytopenia, osteoporosis, and malignant transformation into MDS/leukemia. Development of additional genetic aberrations (G-CSF-receptor or RAS gene mutations, monosomy 7) during the course of the disease indicates an underlying genetic instability.

  Hematopoietic SCT is still the only available treatment for patients refractory to G-CSF.

  Cyclic neutropenia (CN), which is also caused by mutations in ELANE, is an autosomal dominant disorder (sporadic or inherited) characterized by regular oscillations of neutrophils from near normal to severely low levels, generally with a 21-day periodicity. ANC nadirs are associated with fever, mouth ulcers, pharyngitis, sinusitis, or more serious infections. CN usually presents early in childhood but be asymptomatic, and transformation to MDS and AML has not been reported. Symptomatic CN is responsive to G-CSF, which typically shortens nadir duration and increases ANC but usually does not ablate cycling.

CONGENITAL AMEGAKARYOCYTIC THROMBOCYTOPENIA

  Characterized by severe thrombocytopenia due to a lack of megakaryocytes in the bone marrow from birth

  Diagnosis based mainly on the exclusion of other forms of congenital thrombocytopenia with ineffective megakaryopoiesis such as FA.

  Molecular basis for this autosomal recessive disorder may be homozygous or compound heterozygous mutations in the c-mpl gene coding for the thrombopoietin receptor.

  At time of diagnosis, the bone marrow of congenital amegakaryocytic thrombocytopenia (CAMT) patients is normocellular with a normal representation of all hematopoietic lines except for megakaryocytes. During the course of CAMT, the disease usually evolves into AA.

  SCT has been shown to be the only curative therapy.

References

  1.  Young NS, Calado RT, Scheinberg P, et al. Current concepts in the pathophysiology and treatment of aplastic anemia. Blood. 2006;108:2511-2521.

  2.  Kaufman DW, Kelly JP, Levy M, et al. The Drug Etiology of Agranulocytosis and Aplastic Anemia. New York, NY: Oxford; 1991.

  3.  Fogarty PF, Yamaguchi H, Wiestner A, et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet. 2003;362:1628-1630.

  4.  Bacigalupo A, Brand R, Oneto R, et al. Treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy—the European Group for Blood and Marrow Transplantation experience. Semin Hematol. 2000;37:69-80.

  5.  Gupta V, Eapen M, Brazauskas R, et al. Impact of age on outcomes after transplantation for acquired aplastic anemia using HLA-identical sibling donors. Haematologica. 2010. doi: haematol.2010.026682.

  6.  Locasciulli A, Oneto R, Bacigalupo A, et al. Outcome of patients with acquired aplastic anemia given first line bone marrow transplantation or immunosuppressive treatment in the last decade: a report from the European Group for Blood and Marrow Transplantation (EBMT). Haematologica. 2007;92:11-18.

  7.  Eapen M, Le Rademacher J, Antin JH, et al. Effect of stem cell source on outcomes after unrelated donor transplantation in severe aplastic anemia. Blood. 2011;118:2618-2621.

  8.  Schrezenmeier H, Passweg JR, Marsh JC, et al. Worse outcome and more chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia. Blood. 2007;110:1397-1400.

  9.  Srinivasan R, Takahashi Y, McCoy JP, et al. Overcoming graft rejection in heavily transfused and allo-immunised patients with bone marrow failure syndromes using fludarabine-based haematopoietic cell transplantation. Br J Haematol. 2006;133:305-314.

10.  Passweg JR, Perez WS, Eapen M, et al. Bone marrow transplants from mismatched related and unrelated donors for severe aplastic anemia. Bone Marrow Transplant. 2006;37:641-649.

11.  Margolis DA, Casper JT. Alternative-donor hematopoietic stem-cell transplantation for severe aplastic anemia. Semin Hematol. 2000;37:43-55.

12.  Kennedy-Nasser AA, Leung KS, Mahajan A, et al. Comparable outcomes of matched-related and alternative donor stem cell transplantation for pediatric severe aplastic anemia. Biol Blood Marrow Transplant. 2006;12:1277-1284.

13.  Maury S, Balere-Appert ML, Chir Z, et al. Unrelated stem cell transplantation for severe acquired aplastic anemia: improved outcome in the era of high-resolution HLA matching between donor and recipient. Haematologica. 2007;92:589-596.

14.  Peffault de Latour R, Purtill D, Ruggeri A, et al. Influence of nucleated cell dose on overall survival of unrelated cord blood transplantation for patients with severe acquired aplastic anemia: a study by eurocord and the aplastic anemia working party of the European group for blood and marrow transplantation. Biol Blood Marrow Transplant. 2011;17:78-85.

15.  Scheinberg P, Nunez O, Scheinberg P, Weinsten B, Wu CO, Young NS. Cyclosporine taper does not prevent relapse in severe aplastic anemia. ASH Annual Meeting Abstracts. 2011;117:2406.

16.  Scheinberg P, Nunez O, Weinstein B, et al. Horse versus rabbit antithymocyte globulin in acquired aplastic anemia. N Engl J Med. 2011;365:430-438.

17.  DiBona E, Coser P, Bruno B, et al. Rabbit ATG (r-ATG) plus cyclosporin (CYA) and granulocyte colony stimulating factor (G-CSF) is an effective treatment for aplastic anemia (AA) patients (pts) unresponsive to a first course with horse ALG (h-ALG) therapy. Bone Marrow Transplant. 1999;23(suppl 1):S28-S28.

18.  Scheinberg P, Nunez O, Young NS. Retreatment with rabbit anti-thymocyte globulin and cyclosporine for patients with relapsed or refractory severe aplastic anemia. Br J Haematol. 2006;133:622-627.

19.  Scheinberg P, Nunez O, Weinstein B, Scheinberg P, Wu CO, Young NS. Activity of alemtuzumab monotherapy in treatment-naive, relapsed, and refractory severe acquired aplastic anemia. Blood. 2012;119:345-354.

20.  Saracco P, Quarello P, Lori AP, et al. Cyclosporin A response and dependence in children with acquired aplastic anaemia: a multicentre retrospective study with long-term observation follow-up. Br J Haematol. 2008;140:197-205.

21.  Scheinberg P, Fischer SH, Li L, et al. Distinct EBV and CMV reactivation patterns following antibody-based immunosuppressive regimens in patients with severe aplastic anemia. Blood. 2007;109:3219-3224.

22.  Rosenfeld S, Follman D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe aplastic anemia. Association between hematologic response and long-term outcome. J Am Med Assoc. 2003;289:1130-1135.

23.  Scheinberg P, Wu CO, Nunez O, Young NS. Predicting response to immunosuppressive therapy and survival in severe aplastic anaemia. Br J Haematol. 2009;144:206-216.

24.  Afable MG II, Shaik M, Sugimoto Y, et al. Efficacy of rabbit anti-thymocyte globulin in severe aplastic anemia. Haematologica. 2011;96:1269-1275.

25.  Valdez JM, Scheinberg P, Nunez O, Wu CO, Young NS, Walsh TJ. Decreased infection-related mortality and improved survival in severe aplastic anemia in the past two decades. Clin Infect Dis. 2011;52:726-735.

26.  Perez-Albuerne ED, Eapen M, Klein M, et al. Outcome of unrelated donor stem cell transplantation for children with severe aplastic anemia. Br J Haematol. 2008;141:216-223.

27.  Viollier R, Socie G, Tichelli A, et al. Recent improvement in outcome of unrelated donor transplantation for aplastic anemia. Bone Marrow Transplant. 2008;41:45-50.

28.  Bacigalupo A, Socie’ G, Lanino E, et al. Fludarabine, cyclophosphamide, antithymocyte globulin, with or without low dose total body irradiation, for alternative donor transplants, in acquired severe aplastic anemia: a retrospective study from the EBMT-SAA working party. Haematologica. 2010;95:976-982.

29.  Marsh JC, Gupta V, Lim Z, et al. Alemtuzumab with fludarabine and cyclophosphamide reduces chronic graft-versus-host disease after allogeneic stem cell transplantation for acquired aplastic anemia. Blood. 2011;118:2351-2357.

30.  Tisdale JF, Dunn DE, Geller N, et al. High-dose cyclophosphamide in severe aplastic anemia: a randomized trial. Lancet. 2000;356:1554-1559.

31.  Rosse WF, Nishimura J. Clinical manifestations of paroxysmal nocturnal hemoglobinuria: present state and future problems. Int J Hematol. 2003;77:113-120.

32.  Hall C, Richards S, Hillmen P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2003;102:3587-3591.

33.  Hillmen P, Hall C, Marsh JC, et al. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Eng J Med. 2004;350:552-559.

34.  Hillmen P, Muus P, Duhrsen U, et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007. doi: blood-2007-2006-095646.

35.  Kang EM, Tisdale J. Pure red cell aplasia. In: The Bone Marrow Failure Syndromes, Neal S. Young. Philadelphia, PA: W.B. Saunders; 2000;135-155.

36.  Sloand EM, Scheinberg P, Maciejewski J, Young NS. Successful treatment of pure red-cell aplasia with an anti-interleukin-2 receptor antibody (Daclizumab). Ann Intern Med. 2006;144:181-185.

37.  Young NS, Young NS. Agranulocytosis. In: The Bone Marrow Failure Syndromes, Neal S. Young. Philadelphia, PA: W.B. Saunders; 2000;156-182.

38.  Andersohn F, Konzen C, Garbe E. Systematic review: agranulocytosis induced by nonchemotherapy drugs. Ann Intern Med. 2007;146:657-665.

7

The Myelodysplastic Syndromes

Jeffrey Klotz, Ankur R. Parikh and Minoo Battiwalla

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis and a variable tendency to progress to acute myelogenous leukemia (AML). Increasingly, MDS is diagnosed incidentally when modestly abnormal blood counts trigger a bone marrow examination.

MDS is a disease of older adults; the median age is in the mid-60s. Estimates of its incidence range from 4 to 160 per 100,000 people, and in the elderly the rate may be 10-fold higher, making MDS a relatively common hematologic disease.¹⁻³ In a well-characterized population study that included bone marrow biopsies for all subjects, the incidence of MDS in males 80 years or older was 35 per 100,000 people.⁴ Death due to MDS occurs from the complications of cytopenias and/or progression to AML, but many patients will succumb first to comorbidities of the elderly.

Myelodysplasia of the marrow can also be seen in aplastic anemia, especially as a late event after immunosuppressive treatment, in the course of Fanconi anemia, and with paroxysmal nocturnal hemoglobinuria (PNH) and T-large granular lymphocyte lymphoproliferative (T-LGL) disorders, and preceding AML (Fig. 7.1).

ETIOLOGY AND PATHOGENESIS

MDS is related to the accumulation of somatic mutations in a hematopoietic stem cell. In the majority of cases (85%), MDS is a de novo phenomenon with no definitive antecedent cause. In secondary MDS (15%), prior chemotherapy (alkylating agents and topoisomerase inhibitors) and ionizing radiation are clearly etiologic; the latency period between exposure and the development of secondary MDS is typically 2 to 10 years. Radiation has been implicated in the marrow failure syndromes historically reported in occupationally and accidentally exposed individuals and in atomic bomb victims; solvents and smoking are also associated. For most MDS, age is the dominant risk factor. Indeed, childhood MDS is exceedingly rare (incidence rate = 0.01/100,000); it can be seen de novo or in patients with a history of acquired or constitutional aplastic anemia, especially Fanconi anemia.

The bone marrow is typically hypercellular, implying that ineffective hematopoiesis rather than absence of stem cells results in the cytopenias. In general, early MDS (refractory anemia) is characterized by an increased susceptibility to apoptosis, while late MDS (in transition to leukemia) is associated with reduced apoptosis. Although the principal defect is in the hematopoietic stem cells, immunological factors and the bone marrow microenvironment contribute to the bone marrow failure. There are significant abnormalities in apoptosis, cytokine profiles, angiogenesis, and the T-cell repertoire. Specific mutations, in particular, abnormalities in chromosome 7 and a complex karyotype, predispose to leukemic transformation. In contrast, 5q-, del 20q, and -Y are recurrent chromosomal abnormalities not associated with a high risk of transformation.

FIGURE 7.1 Myelodysplastic syndromes. AML, acute myelogenous leukemia; FA, Fanconi anemia; MDS, myelodysplastic syndrome; SAA, severe aplastic anemia;T-LGL,T-cell large granular lymphocytic leukemia. Suggested schema for management of MDS.

Advances continue to be made in refining the understanding of the molecular mechanisms underlying specific MDS subtypes. In addition to the identification of haploinsufficiency of the RPS14 gene for the phenotype of del(5q), the importance of cyclin D1 in trisomy 8, and the high frequency of uniparental disomy by single nucleotide polymorphism (SNP) arrays in patients with normal metaphase cytogenetics, more recent insights include the frequent identification of specific somatic mutations in approximately 50% of MDS patients including the majority of patients with normal cytogenetics, the strong association of SF3B1 mutations in MDS syndromes characterized by ringed sideroblasts, and recurrent mutations in DNMT3A implicating epigenetic alterations in the pathogenesis of MDS.⁵⁻¹¹

CLINICAL FEATURES

Patients present with symptoms due to cytopenias, usually anemia. Approximately 17% of patients with unexplained anemia aged 65 or older (“Anemia of the Elderly”) have peripheral blood count abnormalities consistent with MDS.12,13 Lymphadenopathy and splenomegaly are absent. The clinical course is variable: patients may be asymptomatic or have mild anemia progressing to transfusion dependence over many years while others have an aggressive course with multilineage involvement and rapid evolution to acute leukemia.

DIAGNOSTIC STUDIES

Minimum diagnostic criteria for MDS require unexplained persistent cytopenia(s), and either evidence of clonality (such as with a cytogenetic abnormality) or unambiguous dysplastic marrow morphology (dysplasia in at least 10% of the cells of one of the myeloid lineages or excess blasts).14,15

  Peripheral blood smear typically shows macrocytosis; hypogranular neutrophils; sometimes with Pelger-Huet nuclei and other abnormal nuclear patterns; and circulating micromegakaryocytes. Significant numbers of large granular lymphocytes should raise suspicion of a T-LGL/MDS overlap syndrome.

  Bone marrow biopsy is frequently hypercellular but may be frankly hypocellular or fibrotic in about 20% of MDS. Moderate to severe bone marrow fibrosis, while variably characterized histologically, is an important adverse prognostic feature .16,17 Abnormal localization of immature precursors (ALIPs) near bony trabeculae is characteristic. Increase in myeloblasts and dysplastic morphology in the white cell and/or the megakaryocytic lineages can be seen on the aspirate smear. Mononuclear, small, or dysplastic megakaryocytes are evidence of MDS. Erythroid dysplasia alone is less specific, but large numbers of ringed sideroblasts identify a specific MDS subtype.

  Chromosome analysis of marrow cells is critical; abnormal cytogenetics strongly influence prognosis. Even in the absence of conclusive morphologic dysplasia, a presumptive diagnosis of MDS can be made in the presence of specific recurring chromosomal abnormalities including -5 and -7.¹⁵ However, approximately 50% of MDS patients will have a normal karyotype by routine metaphase cytogenetics.¹⁸ Karyotyping should be repeated periodically as chromosome patterns can evolve. Fluorescent in situ hybridization (FISH) analysis may provide more subtle information than karyotyping alone. A chromosome breakage test for Fanconi anemia is recommended for younger patients even if physical examination is normal. SNP array karyotyping and somatic mutation testing, while currently used only in the research setting, may become more widely available as the prognostic significance of specific molecular abnormalities is clarified.

  Flow cytometry has limited utility; blasts enumeration, critical to prognosis, can be assessed by routine morphology. Nevertheless, expert flow cytometry can be highly specific in diagnosing MDS, and may offer useful phenotypic information. ¹⁹

  HLA typing is needed to evaluate younger patients for allotransplantation and may provide predictive information for responsiveness to immunosuppression.

CLASSIFICATIONS

  Accurate classification and prognosis for this highly heterogeneous disorder is necessary to individualize therapy.

  The first validated classification, the French-American-British (FAB; expanded in 1982) was based on morphology (Table 7.1). This scheme recognized that the risk of leukemic progression was proportional to the blast count in the marrow.

  The International Prognostic Scoring System (IPSS; Table 7.2), derived from analyses of outcomes in large series of patients, combined information from cytogenetics, cytopenias, and blast count to generate a prognostic score.²⁰ These values separate out median survivals for patients with low risk (5.7 years), intermediate-1 (3.5 years), intermediate-2 (1.2 years), and high-risk (0.4 years) MDS. A revised IPSS (IPSS-R) has recently been proposed by the International Prognostic Working Group for Prognosis in MDS (IWG-PM) that places greater emphasis on poor-risk cytogenetics, more precisely characterizes cytopenias, and places patients in five clinical risk groups.

  The WHO classification (Table 7.3) attempts to better define risk and separate individual syndromes.¹⁵ Some of the deficiencies of the FAB are addressed by including the prognostic relevance of cytopenias and karyotypic information in addition to blast counts. (Table 7.3). RAEB-t is discarded and the threshold for defining AML is lowered to 20% blasts. However, this distinction may be misleading, as older adults with 20% to 30% blasts (WHO-AML) had superior outcomes compared with conventional care when treated with azacitidine, an MDS therapy. ²¹

  5q- syndrome is one of several specific MDS syndromes. Deletion of 5q, between bands q31 and q33, is separate in the WHO classification. 5q- usually manifests as anemia, with or without mild neutropenia and platelet counts either preserved or elevated. The prognosis is relatively good. Several cytokines, growth factors, and their receptors are found at the 5q locus; Haploinsufficiency of ribosomal gene RPS14 has been identified as potentially causative. Lenalidomide (Celgene Corporation, Summit, NJ), a thalidomide analog, is especially efficacious in 5q- syndrome.

  Hypocellular MDS, while not categorized in any schema, may be easily confused with aplastic anemia, and patients may respond more favorably to immunosuppression with ATG.

  Idiopathic cytopenia of undetermined significance (ICUS) and idiopathic dysplasia of uncertain significance (IDUS) are two recently described syndromes characterized by meaningful cytopenias and dysplasia, respectively, which do not meet the minimum diagnostic criteria for MDS.²² While the natural history of these conditions is not yet well characterized, in some cases they may precede a myeloid malignancy and warrant serial follow-up.

  Chronic myelomonocytic leukemia (CMML) is biologically distinct, now classified as a myelodysplastic/myeloproliferative neoplasm by the WHO, which is discussed in other chapters.

  Therapy-related (or secondary) MDS is an important subtype, constituting about 15% of cases in most series. This subtype has the highest rate of progression (75%) to acute leukemia, is difficult to treat, and rapidly fatal. Almost all patients have recurrent chromosomal abnormalities: deletions in chromosomes 5 and/or 7 occur at a mean interval of 4 to 5 years after exposure to alkylating agents, and 11q23 abnormalities follow in a shorter time period after topoisomerase II inhibitors. A very high frequency of therapy-related MDS is seen in patients who have undergone high-dose chemotherapy with autologous stem cell rescue (up to 19% at 10 years), more likely due to the cumulative prior therapy, especially alkylating agents, rather than the autotransplant itself. Overall, median survival is only 9 months.

  MDS can be associated with large granular lymphocytosis (LGL). Significant numbers of circulating T-LGLs should prompt suspicion of this overlap syndrome; the diagnosis is confirmed by a clonal pattern of T-cell receptor gene rearrangement. Cases of T-LGL/MDS may have hematological responses to therapy directed against the T-LGL component, such as cyclosporine (CsA) or monoclonal antibody to CD52 (Campath); HLA-DR4 is a strong predictor of responsiveness.

  Pediatric MDS is unusual and should lead to evaluation for genetic syndromes, such as Fanconi anemia, MonoMAC syndrome, Bloom syndrome, neurofibromatosis type 1, Schwachman syndrome, Pearson disease, Kostmann syndrome, familial monosomy 7, and constitutional chromosomal abnormalities.

THERAPY

Therapeutic strategies combine supportive care, suppression of the MDS clone and its leukemic progeny, efforts to improve bone marrow function, and curative attempts with allogeneic stem cell transplantation. Optimum management often requires the application of some or all of these approaches, preferably in the context of a research protocol (Table 7.4). Evidence-based decisions may be constrained by the clinical heterogeneity of MDS and the paucity of adequate data from clinical trials.

Supportive Care

Cytopenias are the single most important contributor to mortality.

Supportive care to maintain adequate peripheral counts and to prevent or treat infections is critical to the patient with MDS. Even moderate degrees of anemia may not be well tolerated by the elderly, especially in the presence of cardiopulmonary disease, and maintenance of higher hemoglobin levels (>9 g/dL) can improve the quality of life without altering transfusion frequency. Iron chelation should be considered in patients who are younger, without serious comorbidities, and who are in favorable diagnostic categories.

  Leukodepletion of blood products and single-donor platelet transfusions reduce the risk of eventual alloimmunization to platelets. If a prophylactic regimen is adopted, 10,000/µL is usually an adequate platelet transfusion threshold. Aminocaproic acid may be a useful adjunct in patients who are refractory to platelet transfusions although this has not been studied in clinical trials.

  Neutrophils may be dysfunctional in MDS. Infections in the setting of neutropenia must be treated promptly and aggressively.

  Growth factors are frequently used in MDS and are used at the lowest doses that maintain a response.²³ Combinations of erythropoietin and granulocyte colony-stimulating factor (G-CSF) are synergistic, with hematological improvements in 40% of low-grade MDS patients.²⁴ Growth factor combinations can be effective even when individual factors have failed to improve blood counts. Patients requiring fewer than 2 units of red blood cells per month and a serum erythropoietin level less than 500 U/L have a higher probability of response (>70%) to erythropoietin plus G-CSF according to an established predictive model.²⁴⁻²⁶ Erythropoietin and G-CSF therapy do not appear to hasten leukemic progression, but there is also little evidence for a positive impact on survival. Thrombopoietin agonists are in clinical trials in thrombocytopenic MDS patients but have raised concerns of leukemogenesis. Romiplostim (Amgen, Thousand Oaks, CA) was used in early clinical trials in patients with MDS and severe thrombocytopenia but an increased rate of progression to AML was observed. Eltrombopag (GlaxoSmithKline, Philadelphia, PA) is currently being used in clinical trials in patients with low and high-risk MDS.

Table 7.4  Therapeutic Strategies for Myelodysplastic Syndrome

Supportive care

Transfusion

Antimicrobials

Iron chelation

Treatments aimed at improving bone marrow function

Growth factors (erythropoietin, G-CSF, GM-CSF)

Immunosuppression (ATG, cyclosporine, alemtuzumab)

Anti-cytokine approaches (lenalidomide)

Anti-apoptosis (ON 01910.Na)

Treatments directed at the abnormal clone

Low intensity chemotherapy (hydroxyurea, low dose ara-C,VP-16)

Intermediate intensity chemotherapy (clofarabine)

Induction regimens (anthracycline/ara-C combinations, FLAG, ADE)

DNA methyltransferase inhibitors (5-azacitidine, decitabine)

Histone deacetylase inhibitors

Farnesyl transferase Inhibitors

Curative attempts (stem cell transplantation)

Myeloablative transplantation

Reduced intensity conditioning

ADE, Ara-C daunorubicin etoposide; ATG, antithymocyte globulin; FLAG, Fludarabine Ara-C G-CSF; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte monocyte colony-stimulating factor.

Stem Cell Transplant

Allotransplant is the only curative therapy. Favorable transplant outcomes are more likely in younger patients, those with a short interval between diagnosis and transplant, and patients with HLA identical siblings.²⁷ Generally, patients with IPSS risk of Int-2 or High would benefit from allogeneic transplant as soon as a donor is identified whereas those with IPPS risk of Low of Int-1 would benefit from waiting till progression.²⁸ Survival outcomes after transplant from HLA-matched unrelated donors have been similar to conventional allotransplant from matched siblings; the improvement is in part attributed to use of high-resolution HLA typing to screen for HLA disparity at the allele level.²⁷ Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) document that survival rates decrease precipitously in advanced stage MDS; survival is approximately 30% for both HLA-matched related and unrelated transplants, which is comparable or slightly worse than transplant outcomes for a similar age group with AML. The IPSS score also predicts relapse and survival; patients with low-risk disease (low risk/Int-1 by IPSS) have significantly lower relapse rates (13% vs. 43%) and better disease-free survival (55% vs. 28%) than do patients with high-risk MDS.²⁹ Therefore, the decision for transplantation requires balancing the probability of disease progression and complications against the morbidity and mortality incurred with transplant (highest in the first year following the procedure). While allotransplant remains the treatment of choice in younger patients, until recently transplant was not feasible for older adults because of higher treatment-related mortality with advanced age; as most patients with MDS are diagnosed in their 60s, the age limit eliminated the only curative option. With the advent of reduced intensity regimens (RIC) that employ less myelosuppression with more intense immunosuppression, older patients with MDS are now being transplanted. RIC regimens offer less treatment-related mortality but at the expense of more disease relapse.

Specific Therapies: DNA Methyltransferase Inhibitors

The DNA methyltransferases function to hypermethylate the CpG promoter regions of many tumor suppressor genes and decrease their gene expression. Hypermethylation is one of many epigenetic modifications that can influence gene expression. In malignancy (such as in MDS), acquired hypermethylation of tumor suppressor genes downregulates expression, increasing the potential for dysplastic growth. Two agents that inhibit hypermethylation are 5-azacitidine (Vidaza, Celgene Corporation, Summit, NJ) and its active metabolite 5-aza-2′-deoxyazacitidine (Decitabine or Dacogen, Eisai Inc, Woodcliff Lake, NJ). At low doses they induce cellular differentiation by inhibiting DNA methyltransferase, while at higher doses these analogs of cytidine can be incorporated into DNA (decitabine) or both RNA and DNA (azacitidine) to exert a direct cytostatic effect.

Azacitidine is currently approved for use for patients with all FAB subtypes. The landmark trial that demonstrated efficacy of 5-azacitidine, CALGB 9221, was a randomized trial of 5-azacitidine versus best supportive care. The data from this trial was re-analyzed using the WHO classification for MDS and the International Working Group response criteria.³⁰ Overall response rate was 47%.³¹ Although the complete and partial remission rates were low (10% and 1%, respectively), there was a significant improvement in overall survival, time to leukemia progression, and quality of life.32,33 In responders, median time to first response was three cycles and 90% responded by the sixth cycle. While worsening cytopenias were seen in many patients, there was no increase in infection or bleeding. A definitive phase III trial (Aza-001) compared Azacitidine (75 mg/m²/day × 7 days q4 weeks) versus a choice of three conventional regimens (best supportive care, low dose Ara-C or Ara-C + Daunorubicin) for Int-2 to High IPSS risk patients.³⁴ Patients were treated until disease progression. A significant survival benefit for azacitidine treatment was seen over conventional care regimens (24.5 months vs. 15 months median overall survival). Clinical trials exploring the efficacy of oral azacitidine in MDS are ongoing.³⁵

Decitabine is FDA approved for the treatment of IPSS scored intermediate-1 or higher risk patients. At a dose of 15 mg/m² given intravenously inpatient every 8 hours for 3 days every 6 weeks, a phase III trial demonstrated a statistically significant overall response rate (17% vs. 0%) and improvement in quality of life compared to supportive care alone but only a nonstatistically significant trend toward improved overall survival or time to leukemogenesis.³⁶ An alternative outpatient dosing schedule of 20 mg/m² given intravenously once daily for 5 consecutive days every 4 weeks demonstrated similar efficacy.³⁷ A subsequent EORTC trial comparing the 3-day dosing schedule versus best supportive care in intermediate or high-risk MDS demonstrated improvements in progression-free survival and AML transformation but no impact on overall survival.³⁸

Dose and schedule optimization of the demethylating agents are ongoing. If tolerated, patients should receive an extended course of therapy (e.g., up to six cycles) before deeming it ineffective. Maintenance therapy is important and hematological responses are not a precondition for a survival benefit. Demethylating agents should now be considered a standard of care in Int-2 and high IPSS patients who are not transplant candidates or as a bridge to allogeneic BMT.²⁶

Specific Therapies: Immunomodulatory Agents

Lenalidomide is an oral analog of thalidomide with far greater potency, superior safety, and established efficacy in MDS. Lenalidomide is approved for use in patients with transfusion-dependent anemia and low or intermediate-1 risk MDS with deletion 5q with or without other cytogenetic abnormalities. A landmark clinical trial in MDS patients demonstrated rapid responses (median time to response 4.6 weeks) including cytogenetic response and complete transfusion independence in 67% of patients with isolated deletion 5q.³⁹ A confirmatory randomized phase III study comparing lenalidomide to best supportive care in low or intermediate-1 risk MDS with deletion 5q demonstrated similar results.⁴⁰ Approximately 50% of patients with deletion 5q experienced grade 3 or 4 neutropenia or thrombocytopenia early in the course of treatment. Those patients who had a greater platelet and neutrophil decline while on therapy had an increased transfusion independence response which suggests a direct cytotoxic effect of lenalidomide specific to the deletion 5q clone.⁴¹ Fifty percent of patients will have a clinical and cytogenetic relapse after 2 to 3 years of treatment. A recent study suggests that there may be a small deletion 5q stem cell population that persists despite treatment with lenalidomide accounting for the relapse rate.⁴² Lenalidomide is indicated in patients with lower risk MDS with deletion 5q and transfusion dependence. Patients who lack deletion 5q demonstrated a 26% transfusion-independence response rate.⁴³

Immunosuppression

Horse antithymocyte globulin (hATG) at 40 mg/kg/day × 4 days produces hematological responses in about one-third of patients with low-risk MDS.⁴⁴ Subjects who are less than 50 years of age, with a shorter duration of red cell transfusion dependence and who are HLA-DR15+ are most likely to respond to immunosuppression.⁴⁵ In a retrospective analysis of 129 MDS patients treated with antithymocyte globulin (ATG) and/or CsA, younger age was the most significant factor favoring response to therapy.⁴⁶ Other favorable factors affecting response were HLA-DR15 positivity and combination ATG plus CsA treatment. CsA (5 mg/kg/day for 3 months, tapered to a low maintenance dose thereafter) may be effective, especially in T-LGL/MDS patients who are HLA-DR4 positive. Alemtuzumab (Campath) is an alternative choice for immunosuppressive therapy, which was recently shown to improve blood counts and induce cytogenetic remissions in selected patients with intermediate-1 MDS.⁴⁷

Specific Therapies: Histone Deacetylase Inhibitors, Multi-Kinase Inhibitors

Histone Deacetylase (HDAC) inhibitors inhibit deacetylation of histone lysine tails, resulting in relaxation of chromatin and decreased transcription of the involved DNA. The compounds have activity in AML and MDS. HDAC inhibitors currently under investigation include valproic acid, suberoylanilide hydroxamic acid (vorinostat), depsipeptide, phenylbutyrate, MGCD0103, MS-275, and LBH589. HDAC inhibitors as single-agent therapy do not have a strong favorable impact on disease, but early results from phase I/II trials suggest a synergistic response with the combination of DNA methyltransferase inhibitors and HDAC inhibitors. Dosing regimens, toxicity, and impact on disease are under ongoing investigation.⁴⁸

ON 01910.Na (Onconova) is a multi-kinase inhibitor that selectively induces mitotic arrest leading to apoptosis in cancer cells and blasts. Recent phase I/II trials have shown encouraging responses in high-risk MDS patients no longer responding to DNA methyltransferase inhibitors.⁴⁹ A phase III trial is currently ongoing (NCT01241500 from ClinicalTrials.gov).

Failure of DNA Methyltransferase Inhibitor-Based Therapy

Although the DNA methyltransferase inhibitors (azacitidine and decitabine) represent the standard of care for transplant-ineligible high-risk MDS patients, not all patients will respond and most responders will experience disease progression within 2 years of response.³⁴ In this situation, the prognosis is poor with no definitive standard salvage option.⁵⁰ Clinical trials are preferred for such patients and current studies using various agents including ezatiostat (TLK199), ON 01910.NA, clofarabine, and alemtuzumab are ongoing.⁵¹ Switching hypomethylating agents may be considered if there is no response or disease progression after initial response but this approach has not yet been validated in significant patient numbers.⁵²

Chemotherapy

Many guidelines have suggested a role for standard intensive induction chemotherapy with regimens typically used for AML to eliminate the neoplastic clone. However, no prospective studies show long-term survival benefit and this approach should be discouraged outside the context of a clinical trial. While advanced MDS often demonstrates high response rates to induction chemotherapy, it is usually followed by the virtual certainty of relapse (up to 90%). Likely futile efforts to eradicate an MDS clone have to be balanced against the risk of further reduction of the marrow reserve. Low intensity chemotherapy (hydroxyurea or etoposide) may be useful for cytoreduction with a palliative goal once MDS transforms, especially in the aged patient.

SUMMARY

The past decade has seen a significant growth in treatment options for MDS. When evaluating a patient, many factors need to be considered in the decision to treat, including age, comorbidities, karyotype, HLA status, prognostic-risk assessment, and availability of sibling and unrelated donor matches (Fig. 7.2). Targeted therapies have not yet made a clear long-term impact on survival. Growth factors should be tried on low-risk patients with MDS if erythropoietin level is low. Lenalidomide can be used in patients with deletion 5q and hypomethylating agents should be reserved for patients with higher risk MDS or as a bridge to stem cell transplant. Clinical and laboratory research in MDS is robust and may continue to yield therapeutic advances. When possible, patients should always be referred to a clinical trial to further define the heterogenous nature of MDS and improve therapeutic options for disease subsets.

FIGURE 7.2 Risk-adjusted schema for management of MDS. When deciding upon treatment of MDS, a careful prognostic assessment is essential. Age and comorbidities also influence treatment options that are realistic. Note that 5-azacitidine is approved for use in patients with all FAB types and IPSS scores. Decitabine is approved for patients with IPSS scores that are intermediate-1 or higher.

References

  1.  Rollison DE, Howlader N, Smith MT, et al. Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001-2004, using data from the NAACCR and SEER programs. Blood. 2008;112:45-52.

  2.  Cogle CR, Craig BM, Rollison DE, List AF. Incidence of the myelodysplastic syndromes using a novel claims-based algorithm: high number of uncaptured cases by cancer registries. Blood. 2011;117:7121-7125.

  3.  Goldberg SL, Chen E, Corral M, et al. Incidence and clinical complications of myelodysplastic syndromes among United States Medicare beneficiaries. J Clin Oncol. 2010;28:2847-2852.

  4.  Neukirchen J, Schoonen WM, Strupp C, et al. Incidence and prevalence of myelodysplastic syndromes: data from the Dusseldorf MDS-registry. Leuk Res. 2011;35:1591-1596.

  5.  Ebert BL, Pretz J, Bosco J, et al. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 2008;451:335-339.

  6.  Maciejewski JP, Mufti GJ. Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood. 2008;112:965-974.

  7.  Sloand EM, Pfannes L, Chen G, et al. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins. Blood. 2007;109:2399-2405.

  8.  Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364:2496-2506.

  9.  Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid cancers. N Engl J Med. 2009;360:2289-2301.

10.  Papaemmanuil E, Cazzola M, Boultwood J, et al. Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med. 2011;365:1384-1395.

11.  Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia. 2011;25:1153-1158.

12.  Guralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004;104:2263-2268.

13.  Steensma DP, Tefferi A. Anemia in the elderly: how should we define it, when does it matter, and what can be done? Mayo Clin Proc. 2007;82:958-966.

14.  Valent P, Horny HP, Bennett JM, et al. Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: consensus statements and report from a working conference. Leuk Res. 2007;31:727-736.

15.  Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937-951.

16.  Della Porta MG, Malcovati L. Myelodysplastic syndromes with bone marrow fibrosis. Haematologica. 2012;96:180-183.

17.  Della Porta MG, Malcovati L, Boveri E, et al. Clinical relevance of bone marrow fibrosis and CD34-positive cell clusters in primary myelodysplastic syndromes. J Clin Oncol. 2009;27:754-762.

18.  Bejar R, Levine R, Ebert BL. Unraveling the molecular pathophysiology of myelodysplastic syndromes. J Clin Oncol. 2011;29:504-515.

19.  Westers TM, Ireland R, Kern W, et al. Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European leukemianet working group. Leukemia. 2012;26(7):1730-1741.

20.  Greenberg P, Cox C, LeBeau MM, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood. 1997;89:2079-2088.

21.  Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast count acute myeloid leukemia. J Clin Oncol. 2010;28:562-569.

22.  Valent P, Bain BJ, Bennett JM, et al. Idiopathic cytopenia of undetermined significance (ICUS) and idiopathic dysplasia of uncertain significance (IDUS), and their distinction from low risk MDS. Leuk Res. 2012;36:1-5.

23.  Rizzo JD, Brouwers M, Hurley P, Seidenfeld J, Somerfield MR, Temin S. American society of clinical oncology/american society of hematology clinical practice guideline update on the use of epoetin and darbepoetin in adult patients with cancer. J Oncol Pract. 2010;6:317-320.

24.  Jadersten M, Malcovati L, Dybedal I, et al. Erythropoietin and granulocyte-colony stimulating factor treatment associated with improved survival in myelodysplastic syndrome. J Clin Oncol. 2008;26:3607-3613.

25.  Hellstrom-Lindberg E, Gulbrandsen N, Lindberg G, et al. A validated decision model for treating the anaemia of myelo-dysplastic syndromes with erythropoietin + granulocyte colony-stimulating factor: significant effects on quality of life. Br J Haematol. 2003;120:1037-1046.

26.  Greenberg PL, Attar E, Bennett JM, et al. NCCN Clinical Practice Guidelines in Oncology: myelodysplastic syndromes. J Natl Compr Canc Netw. 2011;9:30-56.

27.  Runde V, de Witte T, Arnold R, et al. Bone marrow transplantation from HLA-identical siblings as first-line treatment in patients with myelodysplastic syndromes: early transplantation is associated with improved outcome. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant. 1998;21:255-261.

28.  Cutler CS, Lee SJ, Greenberg P, et al. A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed transplantation for low-risk myelodysplasia is associated with improved outcome. Blood. 2004;104:579-585.

29. de Witte T, Hermans J, Vossen J, et al. Haematopoietic stem cell transplantation for patients with myelo-dysplastic syndromes and secondary acute myeloid leukaemias: a report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol. 2000;110:620-630.

30. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood. 2006;108:419-425.

31. Silverman LR, McKenzie DR, Peterson BL, et al. Further analysis of trials with azacitidine in patients with myelodysplastic syndrome: studies 8421, 8921, and 9221 by the Cancer and Leukemia Group B. J Clin Oncol. 2006;24:3895-3903.

32. Kornblith AB, Herndon JE 2nd, Silverman LR, et al. Impact of azacytidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol. 2002;20:2441-2452.

33. Silverman LR, Demakos EP, Peterson BL, et al. Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol. 2002;20:2429-2440.

34. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol. 2009;10:223-232.

35. Garcia-Manero G, Gore SD, Cogle C, et al. Phase I study of oral azacitidine in myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia. J Clin Oncol. 2011;29:2521-2527.

36. Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer. 2006;106:1794-1803.

37. Steensma DP, Baer MR, Slack JL, et al. Multicenter study of decitabine administered daily for 5 days every 4 weeks to adults with myelodysplastic syndromes: the alternative dosing for outpatient treatment (ADOPT) trial. J Clin Oncol. 2009;27:3842-3848.

38. Lubbert M, Suciu S, Baila L, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate- or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol. 2011;29:1987-1996.

39. List A, Dewald G, Bennett J, et al. Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med. 2006;355:1456-1465.

40. Fenaux P, Giagounidis A, Selleslag D, et al. A randomized phase 3 study of lenalidomide versus placebo in RBC transfusion-dependent patients with Low-/Intermediate-1-risk myelodysplastic syndromes with del5q. Blood. 2011;118:3765-3776.

41. Sekeres MA, Maciejewski JP, Giagounidis AA, et al. Relationship of treatment-related cytopenias and response to lenalidomide in patients with lower-risk myelodysplastic syndromes. J Clin Oncol. 2008;26:5943-5949.

42. Tehranchi R, Woll PS, Anderson K, et al. Persistent malignant stem cells in del(5q) myelodysplasia in remission. N Engl J Med. 2010;363:1025-1037.

43. Raza A, Reeves JA, Feldman EJ, et al. Phase 2 study of lenalidomide in transfusion-dependent, low-risk, and intermediate-1 risk myelodysplastic syndromes with karyotypes other than deletion 5q. Blood. 2008;111:86-93.

44. Molldrem JJ, Leifer E, Bahceci E, et al. Antithymocyte globulin for treatment of the bone marrow failure associated with myelodysplastic syndromes. Ann Intern Med. 2002;137:156-163.

45. Saunthararajah Y, Nakamura R, Nam JM, et al. HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome. Blood. 2002;100:1570-1574.

46. Sloand EM, Wu CO, Greenberg P, Young N, Barrett J. Factors affecting response and survival in patients with myelodysplasia treated with immunosuppressive therapy. J Clin Oncol. 2008;26:2505-2511.

47. Sloand EM, Olnes MJ, Shenoy A, et al. Alemtuzumab treatment of intermediate-1 myelodysplasia patients is associated with sustained improvement in blood counts and cytogenetic remissions. J Clin Oncol. 2010;28:5166-5173.

48. Gore SD. New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematology Am Soc Hematol Educ Program. 2011;2011:550-555.

49. Seetharam M, Fan AC, Tran M, et al. Treatment of higher risk myelodysplastic syndrome patients unresponsive to hypomethylating agents with ON 01910.Na. Leuk Res. 2012;36:98-103.

50. Prebet T, Gore SD, Esterni B, et al. Outcome of high-risk myelodysplastic syndrome after azacitidine treatment failure. J Clin Oncol. 2011;29:3322-3327.

51. Kadia TM, Jabbour E, Kantarjian H. Failure of hypomethylating agent-based therapy in myelodysplastic syndromes. Semin Oncol. 2011;38:682-692.

52. Borthakur G, Ahdab SE, Ravandi F, et al. Activity of decitabine in patients with myelodysplastic syndrome previously treated with azacitidine. Leuk Lymphoma. 2008;49:690-695.

8

The Myeloproliferative Neoplasms: Polycythemia Vera, Essential Thrombocythemia, and Primary Myelofibrosis

Ankur R. Parikh and Matthew J. Olnes

The chronic myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell diseases characterized by overproduction of one or more blood cell lines that were first recognized by William Dameshek in 1951.¹ Unlike myelodysplasia, the MPNs are associated with normal maturation and effective hematopoiesis (Fig. 8.1). Organomegaly is common and often symptomatic. Varying degrees of extramedullary hematopoiesis and leukemic transformation are also seen.

A diagnostic marker important to Philadelphia chromosome-negative MPNs was identified in 2005 in Janus Kinase 2 (JAK2) V617F, a tyrosine kinase in the JAK-STAT pathway responsible for erythropoietin (EPO) receptor signaling. Somatic mutation JAK2V617F is a valine to phenylalanine substitution at codon 617 on chromosome 9. This mutation is present in the majority of polycythemia vera (PV) and to varying degrees in essential thrombocytosis (ET) and primary myelofibrosis (PMF) as well as other myeloid malignancies (Table 8.1).2,3 Other mutations including MPL, LNK, CBL, TET2, ASXL1, IDH, IKZF1, EZH2, and DNMT3A have also been identified in subsets of MPN patients, but their pathogenic role is unclear at present.

Although algorithms have been devised for PV, ET, and PMF, diagnosis may remain problematic in some instances because of significant overlap of hematological manifestations. This chapter focuses on PV, ET, and PMF. Chronic myelogenous leukemia (CML) and systemic mastocytosis are also included in the category of MPNs but are discussed in other chapters. Chronic myelomonocytic leukemia (CMML) is included as well, although it is placed in a separate disease category (myeloproliferative/myelodysplastic neoplasms) by the World Health Organization (WHO).⁴

POLYCYTHEMIA VERA

PV was first described by Vaquez in 1892. In the early 1900s, Osler recommended phlebotomy as treatment for PV, and Dameshek classified PV as a myeloproliferative disorder in 1951.¹ In 1967, Wasserman organized the Polycythemia Vera Study Group (PSVG) designed to define the natural history of PV and to determine optimal therapeutic management.

FIGURE 8.1 Bone marrow hematopoiesis. Baso, basophil; CBL, chronic basophilic leukemia; CEL, chronic eosinophilic leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; CNL, chronic neutrophil leukemia; Eos, eosinophil; ET, essential thrombocytosis; Mono, monocytes; PMN, polymorphonuclear leukocytes; P. vera, polycythemia vera; RBC, red blood cells.

Epidemiology

The incidence of PV is 2 per 100,000. Rare familial cases have been described.⁵ The median age at presentation is 60 years, and there is a slight male predominance of the disease. The median survival of untreated symptomatic PV is 6 to 18 months from diagnosis, 3.5 years for PV treated with phlebotomy, and 7 to 12 years for PV treated with myelosuppression. The incidence of leukemic transformation is 5% to 10% in the first 15 years after diagnosis.

Pathophysiology

PV is a clonal stem cell disorder with trilineage myeloid involvement. Some studies suggest that PV involves the B lymphocytes as well. PV is characterized by growth factor-independent erythroid proliferation producing an elevated red cell mass; in vitro, endogenous erythroid colony growth means that progenitors form colony forming units-erythrocyte-derived (CFU-E-derived) and burst forming units-erythrocyte derived (BFU-E-derived) colonies in the absence of EPO. PV may evolve from a proliferative phase of increased marrow activity and splenomegaly to a spent phase characterized by a leukoerythroblastic blood smear and extramedullary hematopoiesis producing massive hepatosplenomegaly, known as fibrotic transformation.

The JAK2V617F mutation was identified in 96% of patients with PV. In ET and PMF the JAK2V617F mutation is heterozygous, while in PV this mutation is homozygous.⁵ A higher JAK2V617F mutant allele burden is associated with fibrotic transformation and pruritus in patients with PV.⁶ In a minority of patients with PV that lack JAK2V617F, there are frameshift or point mutations in exon12 of JAK2. These patients have erythrocytosis without thrombocytosis or leukocytosis, a low serum EPO level, and marrow erythroid hyperplasia without megakaryocyte or granulocyte abnormalities.7,8 While a few cases of congenital polycythemia caused by abnormal expression of a truncated form of the EPO receptor have been described,⁹ there is no evidence that EPO receptor mutations are involved in the pathogenesis of PV. Splicing defects in the EPO receptor RNA in some patients with PV are of unclear significance.¹⁰

At diagnosis, 10% to 20% of patients with PV have abnormal cytogenetics, including trisomy 8, trisomy 9, and deletion 20q. Loss of heterozygosity at chromosome 9p24, undetectable on routine cytogenetics, is found in 33% of patients. The frequency of chromosomal abnormalities increases with disease progression.⁴

Clinical Features

The elevated red cell mass in PV may result in a myriad of clinical signs and symptoms including:

Hypertension

Thrombosis, venous or arterial

Pruritis

Erythromelalgia (a sudden, severe burning pain in the hands or feet, usually accompanied by a reddish or bluish coloration of the skin)

Ulceration of fingers and toes

Joint pain

Epigastric pain

Weight loss

Headache

Weakness

Paresthesias

Visual disturbances

Vertigo

Tinnitus

Ruddy cyanosis

Conjunctival plethora

Pruritis aggravated by bathing is a distinctive feature of PV and is present in almost 50% of patients. PV is the most common cause of erythromelalgia, which often responds to aspirin therapy. Increased cellular turnover in PV may result in gout or kidney stones. Palpable splenomegaly is found in 70% of patients.

Both bleeding and thrombosis can occur in PV. Less than 10% of patients experience major bleeding episodes, and hemorrhage is the cause of death in only 2% to 10% of PV. A variety of platelet defects are detectable and acquired von Willebrand disease exists in 33% of patients.

Thrombotic events (coronary events, cerebral vascular accidents, deep venous thrombosis (DVT), pulmonary embolism (PE), mesenteric thrombosis, and many others) are a major complication of PV. They likely arise from abnormalities in blood viscosity, platelets, and leukocytes.¹¹ Multiple series have documented the incidence of major thrombosis to be 34% to 39% at diagnosis; 66% of these are arterial events and one third are venous.¹¹⁻¹³ Increased risk of thrombosis is associated with age >65 years, hematocrit >45%,¹⁴ leukocytosis of ≥15 × 10⁹/L,¹⁵ and a history of thrombosis. Patients at high risk of thrombosis and thrombocytosis (i.e., older individuals, patients with a history of thrombosis or atherosclerotic disease), should be treated with hydroxyurea to lower platelet counts to <400,000 cells/µL.¹⁶

While erythrocytosis distinguishes PV from the other MPN, only 20% of PV patients present with erythrocytosis alone, while 40% have trilineage hyperplasia at the onset of disease. PV can also present with isolated leukocytosis or thrombocytosis. Laboratory abnormalities include elevated leukocyte alkaline phosphatase, lactate dehydrogenase (LDH), uric acid, and elevated serum B12 (in 40% of patients). Secondary causes of an elevated red cell mass should also be excluded. Typical bone marrow findings in PV include hypercellularity, atypical megakaryocyte hyperplasia and clustering, and decreased stainable iron.

The risk of transformation to acute leukemia is 1.5% in patients treated with phlebotomy alone. Patients with PV have a 10% to 25% risk of fibrotic transformation at 10 and 25 years of follow-up, respectively. Fibrotic transformation is characterized by normalization of the red cell mass associated with cytopenias, increasing splenomegaly due to extramedullary hematopoiesis, progressive reticulin deposition, and collagen fibrosis of the bone marrow.

Diagnostic Criteria

The WHO criteria for the diagnosis of PV are based on clinical and laboratory characteristics (Table 8.2).⁴ A bone marrow biopsy is not mandatory to make a diagnosis of PV in a patient who otherwise fulfills the WHO criteria. In the 2008 WHO guidelines, an elevated red cell mass is not an absolute requirement for diagnosis, while a JAK2V617F or a similar mutation can be used to diagnose PV. While erythrocytosis distinguishes PV from the other MPNs, not all patients with PV have elevated hematocrits and not all patients with elevated hematocrits have PV. Although dehydration can cause spurious elevation of the hematocrit, resulting in apparent erythrocytosis, a hematocrit greater than 60% in men or 55% in women is usually caused by an elevated red cell mass. Direct determination of blood volume and red cell mass is usually needed. Conversely, erythrocytosis may be masked by expanded plasma volume secondary to splenomegaly or by occult blood loss. Iron deficiency can also cause a decrease in the hematocrit in patients with PV. Secondary erythrocytosis caused by elevation of serum EPO must also be excluded.

Conditions associated with physiologically appropriate production of EPO caused by hypoxemia as well as diseases associated with inappropriate EPO production that result in erythrocytosis are listed in Table 8.3.

Laboratory studies that may be useful in evaluation of erythrocytosis are:

Arterial blood gas measurement

Iron studies

Serum EPO level

Liver and kidney function studies

Abdominal ultrasound or computed tomography (CT) scan

Bone marrow aspirate and biopsy

Red cell mass

Table 8.4 shows clinical findings and assay results other than JAK2 mutational status that can be useful for distinguishing secondary polycythemia from PV. Gene expression profiling and mutational analysis are being investigated to help discriminate PV from secondary polycythemia.3,16,17 For example, a polymerase chain reaction (PCR)-based assay for overexpression of PRV1 (CD177) mRNA in peripheral granulocytes is positive in most patients with PV but not in secondary erythrocytosis.¹⁸ Reduced thrombopoietin (TPO) receptor (c-MPL) levels have been described in PV megakaryocytes and platelets and in some patients with ET and PMF. Production of endogenous erythroid colonies in vitro is seen in PV but not in secondary erythrocytosis.¹⁹ When it is impossible to make a definitive diagnosis, laboratory evaluation should be repeated in 3 months.

Staging and Prognostic Features

In untreated PV, median survival is only 6 to 18 months; death most frequently results from thrombosis.¹² Age greater than 65 years and a previous history of thrombosis are the major risk factors for thrombosis.¹⁶ Other causes of mortality include transformation to acute leukemia or fibrotic transformation.

Table 8.3  Conditions Related to Erythropoietin Production

EPO Overproduction Secondary to Hypoxia

Lung disease

High altitude

Smoking (carboxyhemoglobin)

Cyanotic heart disease

Methemoglobinemia

High oxygen affinity hemoglobin

Cobalt

EPO Overproduction

Tumors—renal, brain, hepatoma, uterine fibroids, pheochromocytoma

Renal artery stenosis

Neonatal

Inappropriate EPO secretion

Bartter syndrome

Renal cysts, hydronephrosis

Other Causes

EPO receptor hypersensitivity

Congenital erythrocytosis

Androgen therapy

Adrenal tumors

Autotransfusion (blood doping), self injection of EPO

Polycythemia vera*

*EPO levels in PV may be either low or normal; high EPO levels are not consistent with PV.

Treatment

Treatment goals are to (i) relieve the clinical symptoms that result from an elevated red cell mass, (ii) decrease thrombotic risk, and (iii) slow or prevent leukemic transformation. The efficacy of therapies must be balanced against their toxicities.

The international Polycythemia Vera Study Group (PVSG) began to organize large randomized trials in 1967. Patients were randomly assigned to phlebotomy, chlorambucil, or P32. The thrombosis rate for patients treated with phlebotomy alone was 37.3%, significantly higher than for those treated with chlorambucil or P32. However, there were an excess number of deaths secondary to leukemia in the chlorambucil and P32 arms of the study. The PVSG08 study showed that hydroxyurea significantly lowered the risk of thrombosis compared with phlebotomy alone, but patients who received hydroxyurea exhibited a trend toward increase in the risk of leukemic transformation. The European Collaboration on Low-Dose Aspirin in PV (ECLAP) trial followed a cohort of 518 patients with PV without a contraindication to aspirin therapy who received low-dose aspirin and were undergoing phlebotomy; major thrombosis was decreased by 60% in this cohort compared to controls, without a significant increase in bleeding.¹⁵

Therapy in PV is based on risk of thrombohemorrhagic complications. Current risk stratification is outlined below:

Low Risk

Age <60 years, and

No history of thrombosis

Low Risk with Extreme Thrombocytosis

Low risk with platelet count >1 million/µL

High Risk

Age ≥60 years, or

Previous history of thrombosis

Phlebotomy is the treatment of choice for most patients. The hematocrit should be maintained at <45% in men, 42% in women, and <37% in late pregnancy. Additionally, for patients aged 60 years or older, myelosuppression is recommended to decrease the thrombotic risk. Paradoxically, the initiation of phlebotomy is transiently associated with an increase in thrombotic risk and is greatest in the elderly. Interferon-α has also been used for cytoreduction in younger patients, and during pregnancy. Busulfan or P32 may be used in the elderly who may be unable to tolerate hydroxyurea. A suggested treatment algorithm is seen in Figure 8.2.

Additional therapies may be required for other complications related to PV. Low-dose aspirin appears effective for alleviation of microvascular sequelae including headache, vertigo, visual disturbances, distal paresthesias, and erythromelalgia. The safety and benefits of low-dose aspirin in PV have been investigated in a multicenter project (ECLAP)10,20: aspirin lowered the risk of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, and total mortality; treatment nonsignificantly increased major bleeding. Aspirin should not be used in patients with a history of hemorrhage. All PV patients with extreme thrombocytosis (more than 1 million platelets/µL) should be assessed for acquired von Willebrand syndrome with a ristocetin cofactor activity level, and aspirin should be avoided in those with ristocetin cofactor levels of <30%.

Pruritis is a problem in 40% to 50% of patients with PV. Variably effective measures include reduction of water temperature and the use of antihistamines. Other agents of uncertain efficacy for these symptoms include cholestyramine, psoralen + UVA (PUVA), and interferon-α. The selective serotonin reuptake inhibitors paroxetine (20 mg every day) or fluoxetine (10 mg every day) have been shown to provide relief in many patients suffering from prurits.²¹

Patients with PV undergoing surgery are at high risk of postoperative complications. Elective procedures should be postponed until the hematocrit has normalized for more than 2 months.

Fibrotic transformation occurs on average 10 years after the initial diagnosis and is heralded by the development of cytopenias and splenomegaly. Hydroxyurea and interferon-α may alleviate cytopenias due to splenomegaly. Although splenectomy may provide some relief from these symptoms, hepatomegaly secondary to extramedullary hematopoiesis may be a consequence. Low-dose splenic irradiation usually provides only short-term relief.

FIGURE 8.2 Treatment algorithm for polycythemia vera. *Phlebotomy HCT goals are <45% in men, <42% in women, and <37% in 3rd trimester pregnancy.

Stem cell transplantation remains an option for advanced PV and can be curative. Outcomes are more favorable in those transplanted in fibrotic transformation than in those after evolution to acute leukemia.²²

ESSENTIAL THROMBOCYTOSIS

ET was first described by Epstein and Goedel in 1934 and called hemorrhagic thrombocythemia. Dameshek classified it as one of the myeloproliferative disorders in 1951.

Epidemiology

The annual incidence of ET is estimated at 1 to 2.5 per 100,000. Most patients are between age 50 and 60 at presentation, and there is no gender predilection. A second peak occurs around age 30 when females are more often affected. Prevalence is higher in women than men 1.5–2:1. The median survival of ET is more than 10 years.⁴ Most patients with ET have a normal life expectancy without disease-related complications. The etiology of the disease is unknown.

Pathophysiology

Although ET has been traditionally described as a clonal disorder, X-chromosome inactivation studies suggest polyclonal hematopoiesis in some patients.²³ JAK2V617F is found in 55% of patients with ET; the mutation is associated with elevated hemoglobin and neutrophil counts, lower EPO levels, and increased progression to polycythemia.²⁴ One percent of patients with ET have a mutation in the gene encoding the TPO receptor (c-MPL 515), and in many cases this is found with the JAK2 mutation. Patients with ET tend to have normal to high TPO levels and many have low TPO receptor (c-MPL) levels.²⁵ The rate of clonal cytogenetic abnormalities in ET is approximately 5%.

Clinical Features

As many as half of the patients are asymptomatic at presentation. Vasomotor symptoms occur in approximately 40% of patients and include visual disturbances, light-headedness, headaches, palpitations, atypical chest pain, erythromelalgia, livedo reticularis, and acral paresthesias. Thrombosis occurs in 15% of cases at presentation and in 10% to 20% during the course of the disease. Associated thrombotic events include DVT and PE, digital ischemia, portal vein thrombosis, and cerebrovascular and coronary ischemia. Major hemorrhage occurs in 5% to 10% of patients during the disease course. Other disease associations include recurrent first trimester abortions greater than the unaffected population and palpable splenomegaly, which is present in less than 50% of patients. The risk of leukemic transformation is low in the first decade after diagnosis but increases with each subsequent decade, but overall it is less than for other MPNs.

Diagnostic Testing

ET is characterized by persistent nonreactive thrombocytosis. WHO diagnostic criteria are shown in Table 8.2. The differential diagnosis includes reactive thrombocytosis and other MPNs, as well as chronic myeloid disorders. Causes of thrombocytosis other than ET are listed below:

Asplenia

Acute hemorrhage

Infections

Hemolysis

Postthrombocytopenic rebound

Cancer

Inflammatory states (infection, collagen vascular disorders)

Iron deficiency

Pregnancy

MPN (Note that most of the MPN may present with isolated thrombocytosis.)

Often a careful patient history will exclude reactive thrombocytosis. In addition to JAK2 mutational analysis, other laboratory testing to assist in diagnosis include:

Iron studies to exclude iron deficiency.

C reactive protein (CRP), erythrocyte sedimentation rate (ESR), and fibrinogen to rule out an occult inflammatory or malignant process.

Blood smear: Howell-Jolly bodies indicate anatomic or functional asplenia.

Bone marrow morphology.

Cytogenetics including fluorescence in situ hybridization (FISH) or PCR for BCR/ABL to exclude chronic myeloid leukemia (CML).

Decreased megakaryocyte c-MPL expression and increased granulocyte PRV1 and endogenous erythroid colony formation can be seen in both PV and ET and do not distinguish between them.²⁶ When a definitive diagnosis is not initially possible, later periodic evaluation may be revealing.

Treatment

The decision to treat must be based on risk-based management because life expectancy in this disease is nearly normal. High-risk patients are those over 60 years or older or those who have a history of thrombosis. Low-risk patients are younger than 60 years old with no history of thrombosis and may have extreme thrombocytosis (platelet count >1 million/µL).

Choice of therapy is based on efficacy and toxicity.¹⁶ Therapeutic options include mechanical reduction of counts using plateletpheresis (in acute situations), myelosuppressive agents (alkylating agents, hydroxyurea, or radiophosphorus), maturation modulators (interferon-α or anagrelide), or antiplatelet agents (Table 8.5). Treatment should be focused on a platelet count goal of less than 400,000/µL.

Table 8.6 Essential Thrombocytosis Treatment Algorithm

Low risk: Low-dose aspirin therapy

Low risk with extreme thrombocytosis: Low-dose aspirin for patients in whom von Willebrand syndrome has been excluded

High risk: Low-dose aspirin and cytoreductive therapy

The treatment algorithm for ET is shown in Table 8.6. All patients older than age 60 are high risk. All ET patients with extreme thrombocytosis (more than 1 million platelets/µL) should be tested for acquired von Willebrand syndrome with a ristocetin cofactor activity level. Low-dose aspirin can be used if ristocetin cofactor level is greater than 30%. Women of childbearing age not using birth control should be treated with interferon-α based on anecdotal evidence of safety in pregnancy.

Low-risk patients (with or without extreme thrombocytosis) should be observed and not treated with cytoreductive therapy unless they develop high risk features. Cytoreductive therapy should be administered to high-risk patients. Hydroxyurea is usually the first choice in high-risk patients, with interferon-α or busulfan used as the second line.

As in PV, low-dose aspirin is safe, and may even lower thrombotic complications in patients who do not have a significant bleeding risk.27,28 Aspirin is efficacious for treating vasomotor symptoms. Aspirin is contraindicated in patients who have experienced bleeding episodes, and in those with acquired von Willebrand syndrome.

Alkylating agents are generally avoided because of the risk of leukemia but are useful in the very elderly whose comorbidities make them intolerant to other therapies.

Hydroxyurea decreases thrombotic complications in patients with ET¹⁶ but can cause bone marrow suppression. Questions remain as to its leukemogenic potential in the absence of controlled randomized clinical trials. Hydroxyurea is contraindicated in women of childbearing age.

Anagrelide acts by interfering with platelet maturation but is associated with toxicities including fluid retention, headache, and palpitations and is extremely expensive compared to hydroxyurea. Most side effects abate within 2 to 4 weeks after initiation of therapy, so it is prudent to slowly titrate the dose. Anagrelide should be avoided in patients with cardiovascular comorbidities because of its side effect profile. In a trial where randomized patients received aspirin plus hydroxyurea or anagrelide, there were lower rates of venous thromboembolism in the anagrelide arm, but arterial thrombosis, hemorrhage, and marrow fibrosis were increased.²⁹

Interferon-α is effective in reducing platelet count but is associated with significant side effects including flu-like symptoms and depression.

Plateletpheresis is used in emergent thrombosis where abrupt decrease in platelet count is mandated.

All patients with ET should be instructed to avoid smoking and to avoid nonsteroidal anti-inflammatory drugs.

PRIMARY MYELOFIBROSIS

Myelofibrosis was first described in 1879 by Hueck and was first included as one of the myeloproliferative diseases by Dameshek in 1951.¹

Epidemiology

The annual incidence of PMF is 0.5 to 1.5 per 100,000. The median age at presentation is 67 years. The male to female ratio is 1:1. PMF has the worst prognosis among the MPNs, with a median survival of 3 to 5 years. The etiology of the disease is unknown, but a familial occurrence has been reported in rare kindreds.⁴ A high incidence of PMF was observed in individuals exposed to radiation at Hiroshima.

PMF that develops in late-stage PV or ET is referred to as postpolycythemic metaplasia (PPMM) or postthrombocythemic myeloid metaplasia (PTMM), respectively. De novo PMF is referred to as idiopathic myelofibrosis.

Pathophysiology

PMF is characterized by marrow fibrosis and extramedullary hematopoiesis. The marrow fibroblasts in PMF are not derived from the abnormal clone. Increased levels of platelet derived growth factor (PDGF), transforming growth factor (TGF)β, and other cytokines produced by megakaryoctyes may be responsible for the marrow fibrosis. Cytogenetic abnormalities are seen in approximately 50% of patients and include 13q, 20q, 12p, trisomy 8, and trisomy 9. High levels of CD34⁺ cells and of hematopoietic colony forming cells are typical in the circulation of patients with PMF and appear to correlate with the extent of myeloproliferation.³⁰

The JAK2V617F mutation is found in 65% of patients. The MPL 515 mutation, also identified in ET, is present in approximately 5% of these patients. Other somatic mutations currently being investigated are LNK, TET2, ASXL1, IDH1/IDH2, EZH2, DNMT3A, CBL, IKZF1, TP53, and SF3B1.3,16

Clinical Features

Approximately one third of patients are asymptomatic at diagnosis. Presenting complaints include profound fatigue, symptoms of anemia, abdominal discomfort, early satiety, or diarrhea caused by splenomegaly, bleeding, weight loss, and peripheral edema. The constitutional symptoms of fever and night sweats occur in most patients during the course of the disease. Splenomegaly is common in PMF and may be marked. Episodic left upper quadrant pain can occur secondary to splenic infarction. Palpable hepatomegaly is found in the majority of cases. Extramedullary hematopoiesis may occur in almost any organ.

Laboratory abnormalities in patients with PMF may include leukocytosis or leukopenia, and thrombocytosis or thrombocytopenia. The classic blood smear shows leukoerythroblastosis but bone marrow morphologic findings vary from mild to marked fibrosis.³¹ Osteosclerosis and periostitis can cause severe bone pain. Elevations of LDH, serum B12, and alkaline phosphatase are commonly seen. Transformation to acute leukemia occurs in approximately 20% of patients during the first decade after diagnosis.

Diagnostic Testing

The WHO diagnostic criteria for PMF are listed in Table 8.2. The bone marrow is often inaspirable, a “dry tap.” The classic peripheral smear shows teardrop-shaped red cells, nucleated red cells and granulocyte precursors (leukoerythroblastosis). However, other marrow infiltrative processes can cause a similar picture and must be excluded (Table 8.7). Absence of splenomegaly should make the diagnosis of PMF suspect. Many benign and malignant conditions mimic PMF, including metastatic cancer, granulomatous disease, connective tissue disease, lymphoma, systemic mast cell disease, hypereosinophilic syndrome, and other myeloid disorders. Both ET and PV can transform to PMF. Cytogenetics and FISH or PCR for BCR/ABL should be performed to exclude fibrotic CML.

Staging and Prognostic Features

PMF often progresses to marrow failure. Features associated with decreased survival include the following:

Advanced age

Hypercatabolic symptoms*

Anemia (hemoglobin less than 10 g/dL)**

Leukopenia (white cell count <4,000/mm³)**

Leukocytosis (white cell count >30,000/mm³)

Abnormal cytogenetics or the presence of circulating granulocyte precursors or blasts*

(*May be an indication for splenectomy or **transplantation.)

Splenic irradiation may provide short-term improvement in patients with symptoms referable to organomegaly who are not surgical candidates.

Median survival in high-risk patients is less than 2 years, while patients with low-risk features have median survivals of over 10 years. Up to 30% of patients may progress to acute myeloid leukemia (AML), and this is thought to be more common after splenectomy.

A number of prognostic scoring systems have been developed for PMF. The International Prognostic Scoring Scale (IPSS) developed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) is based on five adverse prognostic features noted on multivariate analysis: presence of constitutional symptoms, age greater than 65 years, hemoglobin less than 10 g/dL, leukocyte count greater than 25,000/µL, and circulating blast greater than or equal to 1%.³² Each category received one point and subjects with zero (low risk), one (intermediate risk-1), two (intermediate risk-2), or greater than or equal to 3 (high risk) at presentation had median survivals of 135, 95, 48, and 27 months, respectively. A dynamic IPSS (DIPSS) score has been developed, which can be used at any time during the disease course, and a more recent scoring system called DIPSS-plus incorporates the previous factors plus platelet count, red cell transfusion need, and unfavorable karyotype.³³

Treatment

Treatment for PMF is largely palliative. Approximately 30% of patients with anemia will show improvement with a combination of androgen (oxymethalone 50 mg 4 times per day or fluoxymesterone 10 mg 3 times per day) and prednisone (30 mg/day) therapy. Responses are usually brief in duration. EPO is most often ineffective. In patients with a more favorable prognosis who require transfusions for symptomatic anemia, timely initiation of chelation therapy is warranted.

Hydroxyurea, busulfan, interferon, or melphalan may be used to control thrombocytosis, leukocytosis, or organomegaly. Lower doses of hydroxyurea are used in PMF than in ET or PV (start at 20 to 30 mg/kg two or three times per week). None of these agents is effective in preventing disease progression or improving survival. Anagrelide and imatinib are not effective. Thalidomide and prednisone may treat anemia and lenalidomide can be used if there is a deletion 5q abnormality.³⁴ JAK2 inhibitors have shown significant reduction in splenic size and relief of symptoms in some patients. The JAK2 inhibitor ruxolitinib has been approved for the treatment of intermediate and high-risk myelofibrosis.³⁵ There are ongoing studies using mTOR kinase inhibitors and proteasome inhibitors.

Allogeneic stem cell transplantation remains the only treatment with curative potential for patients with PMF.³⁶ Debate continues as to the value of splenectomy prior to transplantation. Concerns about graft failure because of marrow fibrosis have proven unwarranted and, in fact, successful transplantation is associated with resolution of marrow fibrosis. In both the European multicenter cooperative studies and Seattle single institution trials, overall survival after myeloablative transplantation was 60%. Reduced intensity conditioning is under exploration for older patients and for those who are not candidates for myeloablative protocols.

CHRONIC MYELOMONOCYTIC LEUKEMIA

Epidemiology

The annual incidence of CMML is estimated at 4 cases per 100,000. There is a male predominance of 1.5–3:1. The median age at presentation is 70 years. Median survival is estimated at 12 to 18 months. The etiology of the disease is unknown.

Pathophysiology

The WHO classification places CMML in the category labeled myelodysplastic/myeloproliferative, which is appropriate because the marrow cells in this disease show dysplastic features, and there are many characteristics of myeloproliferation as well. The spleen, liver, and lymph nodes are the most common sites of extramedullary involvement. Clonal cytogenetic abnormalities are present in 20% to 40% of CMML and include trisomy 8, deletion 7q, and translocations involving 5q31–35; the latter activate the platelet derived growth factor receptor β (PDGFRβ) and are associated with eosinophilia4,37,38 Mutational spectrum analysis has demonstrated the heterogeneity of CMML with various mutations in TET2, ASXL1, CBL, IDH1/2, KRAS, NRAS, JAK2V617F, UTX, DNMT3A, and EZH2. A recent study of 72 patients with CMML found at least 1 mutation in 86% of cases.³⁹ Studies evaluating prognostic significance are currently ongoing.

Clinical Features

CMML frequently presents with fatigue, fever, weight loss, or night sweats. There is risk of infection because of neutropenia and of bleeding secondary to thrombocytopenia. In approximately 50% of patients, the white count at presentation may be normal or decreased, while in the remainder it is elevated. In all cases there is persistent peripheral blood monocytosis, the defining feature of the disease. Progression to acute leukemia occurs in 15% to 30% of cases.⁴⁰

Diagnostic Testing

WHO diagnostic criteria include the following:

Persistent peripheral blood monocytosis (greater than 1 × 10⁹ per liter for more than 3 months)

Absence of the Philadelphia chromosome or BCR/ABL fusion gene

Less than 20% blasts in the blood or bone marrow

Dysplasia of one or more myeloid lineages

Clonal cytogenetic abnormality

If dysplasia is absent, the diagnosis can be made if there is a clonal abnormality and no other causes of monocytosis.

Staging and Prognostic Features

Based on peripheral blood leukocyte counts, the French American British (FAB) group proposed dividing CMML into a dysplastic and a proliferative form with a white count over 13,000/mm³. Attempts to evaluate the prognostic value of these distinctions have yielded disparate results. Recent analysis of CMML diagnosed based on FAB classification identified the following factors as independently associated with shorter survival: hemoglobin <12 g/dL; lymphocyte count >2,500/mm³; medullary blast count 10% or more, and presence of circulating immature myeloid cells. Median survival was 12 months.⁴¹ A recent investigation of 414 patients revealed an abnormal karyotype was associated with poorer overall survival and a higher risk of leukemic transformation. Low-risk category included a normal karyotype or loss of the Y chromosome as single anomaly; high-risk patients had trisomy 8, abnormalities of chromosome 7, or complex karyotype. All other abnormalities were intermediate risk. 5-year overall survival for low, intermediate, and high-risk cytogenetics were 35%, 26%, and 4%, respectively.⁴²

Treatment

Treatment approaches are all experimental, and none has proven effective in modifying the natural course of the disease. Evaluation of treatment responses of patients with CMML specifically is difficult, because they have historically been grouped under the myelodysplastic syndromes. Growth factors have been used in an attempt to treat cytopenias and low-dose chemotherapy during the pre-leukemic phase of the disease. Hydroxyurea is effective in controlling cell counts in the proliferative phase. Although many patients respond initially to chemotherapy, complete responses are rare and remissions generally short lived. A variety of low-dose chemotherapeutic agents including cytarabine, topotecan, fludarabine, oral idarubicin, and oral etoposide have showed little success in altering long-term survival rates. Imatinib mesylate is effective in the rare CMML patients who have PDGFRβ translocations.43,44 Hypomethylating agents can induce complete or partial remissions in subsets of patients. Stem cell transplantation has proved successful in a small number of cases and remains the only option for cure.

References

  1.  Dameshek W. Some speculations on the myeloproliferative disorders. Blood. 1951;6:372-375.

  2.  Levine RL, Pardanani A, Tefferi A, Gilliland DG. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer. 2007;7:673-683.

  3.  Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA. New mutations and pathogenesis of myeloproliferative neoplasms. Blood. 2011;118:1723-1735.

  4.  Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: Rationale and important changes. Blood. 2009;114:937-951.

  5.  Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054-1061.

  6.  Passamonti F, Rumi E, Pietra D, et al. A prospective study of 338 patients with polycythemia vera: The impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications. Leukemia. 2010;(24):1574-1579.

  7.  Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med. 2007;356:459-468.

  8.  Passamonti F, Elena C, Schnittger S, et al. Molecular and clinical features of the myeloproliferative neoplasm associated with JAK2 exon 12 mutations. Blood. 2011;117:2813-2816.

  9.  Kralovics R, Indrak K, Stopka T, et al. Two new EPO receptor mutations: truncated EPO receptors are most frequently associated with primary familial and congenital polycythemias. Blood. September 1997;90(5):2057-2061.

10.  Spivak JL, Barosi G, Tognoni G, et al. Chronic myeloproliferative disorders. Hematology Am Soc Hematol Educ Program. 2003:200-224.

11.  Tefferi A, Elliot M. Thrombosis in myeloproliferative disorders: prevalence, prognostic factors, and the role of leukocytes and JAK2V617F. Semin Hematol. 2007;33:313-320.

12.  Gruppo Italiano Studio Policitemia. Polycythemia vera: the natural history of 1213 patients followed for 20 years. Ann Intern Med. 1995;123:514-515.

13.  Marchioli R, Finazzi G, Landolfi R, et al. Vascular and neoplastic risk in a large cohort of patients with polycythemia vera. J Clin Oncol. 2005;23:2224-2232.

14.  McMullin MF, Bareford D, Campbell P, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis. Br J Haematol. 2005;130:174-195.

15.  Landolfi R, Di GL, Barbui T, et al. Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera. Blood. 2007;109:2446-2452.

16.  Tefferi A, Vainchenker W. Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol. 2011;29:573-582.

17..  Goerttler PS, Kreutz C, Donauer J, et al. Gene expression profiling in polycythaemia vera: overexpression of transcription factor NF-E2. Br J Haematol. 2005;129:138-150.

18.  Klippel S, Strunck E, Temerinac S, et al. Quantification of PRV-1 mRNA distinguishes polycythemia vera from secondary erythrocytosis. Blood. 2003;102:3569-3574.

19.  Streiff MB, Smith B, Spivak JL. The diagnosis and management of polycythemia vera in the era since the Polycythemia Vera Study Group: a survey of American Society of Hematology members’ practice patterns. Blood. 2002;99:1144-1149.

20.  Landolfi R, Marchioli R, Kutti J, et al. Efficacy and safety of low-dose aspirin in polycythemia vera. N Engl J Med. 2004;350:114-124.

21.  Diehn F, Tefferi A. Pruritus in polycythaemia vera: prevalence, laboratory correlates and management. Br J Haematol. 2001;115:619-621.

22.  Fruchtman SM. Transplant decision-making strategies in the myeloproliferative disorders. Semin Hematol. 2003;40:30-33.

23.  Harrison CN, Gale RE, Machin SJ, Linch DC. A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications. Blood. 1999;93:417-424.

24.  Campbell PJ, Scott LM, Buck G, et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet. 2005;366:1945-1953.

25.  Cerutti A, Custodi P, Duranti M, Noris P, Balduini CL. Thrombopoietin levels in patients with primary and reactive thrombocytosis. Br J Haematol. 1997;99:281-284.

26.  Kralovics R, Buser AS, Teo SS, et al. Comparison of molecular markers in a cohort of patients with chronic myeloproliferative disorders. Blood. 2003;102:1869-1871.

27.  Landolfi R, Marchioli R. European Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP): a randomized trial. Semin Thromb Hemost. 1997;23:473-478.

28.  van Genderen PJ, Mulder PG, Waleboer M, et al. Prevention and treatment of thrombotic complications in essential thrombocythaemia: efficacy and safety of aspirin. Br J Haematol. 1997;97:179-184.

29.  Harrison CN. Essential thrombocythaemia: challenges and evidence-based management. Br J Haematol. 2005;130:153-165.

30.  Barosi G. Myelofibrosis with myeloid metaplasia. Hematol. Oncol Clin North Am. 2003;17:1211-1226.

31.  Tefferi A. Myelofibrosis with myeloid metaplasia. N Engl J Med. 2000;342:1255-1265.

32.  Cervantes F, Dupriez B, Pereira A, et al. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood. 2009;113:2895-2901.

33.  Gangat N, Caramazza D, Vaidya R, et al. DIPSS plus: a refined Dynamic International Prognostic Scoring System for primary myelofibrosis that incorporates prognostic information from karyotype, platelet count, and transfusion status. J Clin Oncol. 2011;29:392-397.

34.  Arana-Yi C, Quintás-Cardama A, Giles F, et al. Advances in the therapy of chronic idiopathic myelofibrosis. Oncologist. 2006;11:929-943.

35.  Verstovsek S, Kantarjian H, Mesa RA, et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med. 2010;363:1117-1127.

36.  Guardiola P, Anderson JE, Bandini G, et al. Allogeneic stem cell transplantation for agnogenic myeloid metaplasia: a European Group for Blood and Marrow Transplantation, Societe Francaise de Greffe de Moelle, Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson Cancer Research Center Collaborative Study. Blood. 1999;93:2831-2838.

37.  Gunby RH, Cazzaniga G, Tassi E, et al. Sensitivity to imatinib but low frequency of the TEL/PDGFRbeta fusion protein in chronic myelomonocytic leukemia. Haematologica. 2003;88:408-415.

38.  Magnusson MK, Meade KE, Brown KE, et al. Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia. Blood. 2001;98:2518-2525.

39.  Jankowska A, Makishima H, Tiu R, et al. Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood. October 2011;118(14):3932-3941.

40.  Cortes J. CMML: a biologically distinct myeloproliferative disease. Curr Hematol Rep. 2003;2:202-208.

41.  Onida F, Kantarjian HM, Smith TL, et al. Prognostic factors and scoring systems in chronic myelomonocytic leukemia: a retrospective analysis of 213 patients. Blood. 2002;99:840-849.

42.  Such E, Cervera J, Costa D, et al. Cytogenetic risk stratification in chronic myelomonocytic leukemia. Haematologica. 2011;96:375-383.

43.  Apperley JF, Gardembas M, Melo JV, et al. Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med. 2002;347:481-487.

44.  Magnusson MK, Meade KE, Nakamura R, et al. Activity of STI571 in chronic myelomonocytic leukemia with a platelet-derived growth factor beta receptor fusion oncogene. Blood. 2002;100:1088-1091.

9

Neutrophil Disorders and Neutropenias

Matthew M. Hsieh and Harry L. Malech

Neutrophils or polymorphonuclear (PMN) cells are 5 μm in diameter with a distinctive multilobed nucleus and many small granules. Neutrophil maturation begins with myeloblasts in the bone marrow. Myeloblasts differentiate into promyelocytes, which are characterized by the appearance of primary (azurophilic) granules containing myeloperoxidase (MPO), followed by differentiation into myelocytes, characterized by the formation of secondary granules containing lactoferrin and gelatinase, and progress through metamyelocytes, band forms, and finally mature neutrophils. This process of differentiation typically occurs over 10 to 14 days but may be accelerated in the setting of infection, in some cases leading to mature forms retaining large azurophilic granules (toxic granulation). Once neutrophils exit bone marrow, they circulate for about 6 to 12 hours. At sites of infection or inflammation, neutrophils adhere to and migrate between post-venule endothelial cells in order to exit blood vessels into the tissues, where they remain for 1 to 3 days. In the absence of overt infection, most neutrophils in the circulation ultimately undergo apoptosis and are taken up by macrophages in the spleen. Even without infection, there is a baseline rate of neutrophil migration into the mouth and gastrointestinal tract where, together with the barrier function of the mucosa, they prevent entry of bacteria into tissues at local sites. In the setting of severe neutropenia, the gastrointestinal tract is often the first site of invasive bacterial infection.

Neutrophils circulate in a metabolically quiescent state. When stimulated by inflammation, infection-related cytokines, or chemotactic factors, they exit the circulation by adherence to endothelial cells and migrate to sites of inflammation. Neutrophils are among the first cells to migrate to sites of inflammation and thus represent the first line of defense against microbes. They internalize microbial particles by phagocytosis via Fc receptors and complement C3, and granule contents and reactive oxidants are released into phagosomes to kill microbes. Increased numbers of life-threatening bacterial infections occur in association with inherited or acquired disorders characterized by abnormal granule formation, poor neutrophil adherence, failure to produce microbicidal oxidants, or where there is very low production or increased destruction of neutrophils.¹ Table 9.1 provide an overview of neutrophil disorders and Abbreviated List of Common Drugs Causing Neutropenia.

NEUTROPHIL DISORDERS

Leukocyte Adhesion Deficiency

The β₂ integrins in neutrophils are particularly important for normal neutrophil egress from blood at postcapillary venules, for migration through tissues, and for complement-mediated phagocytosis.

There are three leukocyte β₂ integrin adhesion molecules which share the CD18 protein antigen as a common subunit; CD11a/CD18 (lymphocyte function associated antigen-1), CD11b/CD18 (macrophage 1 antigen), and CD11c/CD18 (also known as p150/95). Mutations in the gene encoding CD18, leading to near absent CD18 protein expression, are responsible for a disease known as leukocyte adhesion deficiency 1 (LAD-1). LAD-2 and -3 have been described in a handful of individuals, and they are due to an abnormality of fucose glycosylation (ligand required for selectin binding) and FERMT3 protein-mediated integrin activation, respectively.³ In general, “LAD” used without the numerical identification refers to LAD-1, the defect responsible for the great majority of cases. LAD has an autosomal recessive inheritance pattern, affecting only a few individuals per million. LAD is associated with recurrent life-threatening infections and other characteristic clinical manifestations. Diagnosis is usually made by flow cytometry measurement of the amount of CD11b or CD18 on the surface of neutrophils using specific antibodies. The severity of disease manifestations, including risk of early death from infection, appears to be correlated with the amount of β₂ integrins present.

Moderate phenotype has 1% to 10% of normal levels of β₂ integrins; while the severe phenotype is associated with presence of less than 1% detectable β₂ integrins. On the cellular level, there is poor neutrophil adhesion to endothelial and other immune cells, and neutrophils do not egress from vasculature and migrate to sites of inflammation. Baseline peripheral blood neutrophil count, even in the absence of infection, is characteristically about 2 to 3 times normal; with infection, neutrophil counts can exceed 60 k/µL and mimic a leukemic condition. Despite the very high circulating levels of neutrophils, there may be only mild erythema or pain at sites of infection, and the patients fail to form pus (called “tissue neutropenia”). One of the hallmarks of severe LAD is delayed separation of the umbilical cord, indicating a role for neutrophils in providing the proteases and hyaluronidases required for that event. A prominent manifestation of severe LAD is recurrent infections with large non-healing ulcers of the skin, particularly over the lower abdomen, perineum, and legs. Patients also suffer recurrent infections of the oral cavity (gingivitis, periodontitis with early loss of primary and secondary teeth), respiratory tract (sinusitis, otitis media, and pneumonia), gastrointestinal tract, and genital mucosa. Infection of the wall of the small bowel or colon complicated by perforation is a particular risk that often leads to a fatal outcome. Infections are commonly caused by Staphylococcus aureus, enteric organisms, Candida, and Aspergillus species. In patients with milder forms of LAD, who are not transplanted and survive past the first decade, large non-healing ulcers of the lower extremities and groin become chronic and are very difficult to control or treat. Therapies include bacterial prophylaxis with trimethoprim/sulfamethoxazole (TMP/SMX), supportive antibiotics during acute infections, surgical debridement of skin, and skin grafting. There is a high mortality rate (∼75%) of severe LAD in the first year of life. Successful bone marrow transplant is curative and should be considered for all patients with severe LAD.⁴

Myeloperoxidase Deficiency

MPO is the most abundant protein in neutrophil granules. MPO resides in the primary (azurophilic) granules, and has antimicrobial functions (catalyzes the production of hypochlorous acid from chloride and the hydrogen peroxide product of the phagocyte NADPH oxidase). MPO deficiency is the most common neutrophil abnormality, with an incidence of about one per 2,000 for partial deficiency and one per 4,000 for complete deficiency.⁵ Most individuals with MPO deficiency do not manifest clinically though ex vivo assays of bacterial and fungal killing demonstrate a defect associated with MPO deficiency. MPO deficiency is inherited in an autosomal recessive pattern, but MPO deficiency can also appear as an acquired abnormality associated with leukemia or myelodysplasia. Specific mutations in the MPO gene have been identified, which may affect transcription, translation, and/or insertion of the heme group. Neutrophils with MPO deficiency mature, migrate, and phagocytose normally, but as noted there are defects in microbial killing. Some individuals may have a mildly increased frequency of bacterial infections, and in the setting of cofactors like diabetes there may be particular difficulty in clearance of candida species (albicans, tropicalis, stelatoidea, and krusei ) infections. Diagnosis of MPO deficiency may be made by measurement of peroxidase activity using flow cytometry or using certain types of automated blood count devices that use peroxidase activity to perform differential counts of blood leukocytes. Since most affected patients have mild disease, antimicrobial and supportive therapy is sufficient. Prophylactic antibiotics should be limited to those with recurrent infections or with another disorder predisposing to infections, such as diabetes.

Chronic Granulomatous Disease

Chronic granulomatous diseases (CGDs) are a group of closely related inherited disorders characterized by defective phagocyte NADPH oxidase manifested by a failure of stimulated neutrophils, monocytes, eosinophils, and macrophages to produce superoxide and hydrogen peroxide.⁶ CGD affects approximately 5 individuals per million, equally affecting all ethnic groups. CGDs are caused by mutations in any of 5 subunit components of the phagocyte NADPH oxidase. The clinically most severe is the X-linked, gp91phox subunit deficient form of CGD, usually associated with total absence of any oxidant production and affecting almost 70% of patients with CGD. The other 4 types of CGD are inherited in an autosomal recessive pattern and consist mostly of p47phox deficient CGD patients (25% of CGD patients) with the remainder comprised of the much less common p67phox, p40phox, or p22phox deficient forms of CGD. Clinical manifestations of CGDs involve both recurrent infections and formation of inflammatory granulomas, for which severity and individual manifestations can vary widely. The average age of diagnosis of X-linked CGD is 3 years, but the average age of diagnosis of females with the p47phox form of CGD is 9 years of age. Thus, some patients with no family history may reach young adulthood before the disease is recognized.

Unlike patients with severe neutropenia or LAD who get infected primarily with commensal organisms (such as enteric bacteria normally found in the gastrointestinal tract), CGD patients are generally not susceptible to commensal organisms such as E. coli. Patients are particularly prone to infection by environmental organisms that are catalase positive: the usual bacterial pathogens are Staphylococcus aureus, nocardia, Burkholderia cepacia (and other Burkholderia species), and Serratia marcescens . Fungal pneumonia and other fungal infections are primarily aspergillus species, with Aspergillus nidulans being a problem particular to CGD patients. However, infections with paecilomyces, and other fungi including dematiaceous molds are an increasing problem; they need to be considered because they may be resistant to voriconazole but sensitive to posaconazole. CGD patients do not seem to be particularly susceptible to Candida albicans, though infections with other candida species such as C. glabrata occur. While infections are usually recurrent, prolonged, and episodic, CGD patients who are on appropriate effective prophylaxis may have many months or even years between severe infections.

In infancy, S. marcescens osteomyelitis or soft tissue infection is a very common first presenting infection leading to diagnosis. In older children and adults with CGD, the most common life-threatening infections are bacterial or fungal pneumonias, even if local soft tissue infections and lymph node infections are more common. Other tissues can be infected, including sites as diverse as osteomyelitis or brain abscess. After pneumonia, the most common severe infections are liver abscesses. It is noteworthy that in CGD patients taking TMP/SMX as daily prophylaxis, severe staphylococcal deep tissue infections are relatively uncommon, yet almost 90% of liver abscesses are caused by S. aureus. Methicillin-resistant S. aureus liver abscess is an increasing problem. A liver abscess is generally not an easily drained pustular lesion but often consists of a solid granulomatous mass with micro-abscesses. In the recent past, the standard of care often involved surgical extirpation of the infected granulomatous mass together with prolonged antibiotic therapy for most effective cure. More recently, a period of use of high dose corticosteroids together with prolonged antibiotic therapy directed at the liver abscess pathogen have been demonstrated to effectively treat liver abscess in CGD without the need for surgical intervention in most cases. The steroids likely reduce the granulomatous inflammation and fibrotic reaction, allowing improved penetration of antibiotic into the infected mass in the liver.

In some CGD individuals, granuloma formation in the absence of active infection may predominate over recurrent infections; in some cases the granulomatous inflammation can cause gastroesophageal junction or gastric outlet obstruction, bladder outlet obstruction, or chronic abdominal pain with diarrhea. A gastrointestinal granulomatous process may be indistinguishable from Crohn’s disease and even respond to similar treatments such as those used for Crohn’s disease. CGD granulomas are different from granulomas of autoimmune diseases, as they are responsive to tapering doses of corticosteroids and controllable in the long term by very low dose alternate day prednisone. Autoimmune disorders of the Th1 cytokine pattern type (Crohn’s disease, rheumatoid arthritis, lupus, sarcoidosis) occur with increased frequency in CGD. Whether this association is triggered by recurrent infections, by the hyperinflammation associated with CGD, or an intrinsic characteristic of CGD lymphocytes is yet to be determined.

CGDs should be suspected in patients with a family history of unexplained deaths in infant or young boys, or in boys or girls infected with specific organisms characteristic of CGD infections (serratia osteomyelitis in an infant is almost diagnostic of CGD), and in children with pneumonia that does not rapidly resolve with conventional therapy. Diagnosis is made by a dihydrorhodamine flow cytometry, demonstrating defective oxidase activity in neutrophils, and confirmed by quantitative assays of superoxide production. Acute infections are managed with antibiotics and supportive therapy. Because of the propensity of CGD patients to infections with unusual organisms such as nocardia or aspergillus, aggressively seeking a pathogenic organism is essential to achieve correct antimicrobial therapy. When infections resolve, prophylaxis is implemented with good oral hygiene using chlorhexidine and/ or peroxide based mouthwash, daily oral TMP/SMX (5–6 mg/kg/day TMP equivalent), daily oral itraconazole (4–5 mg/kg/day), and three-times-a-week subcutaneous injections of recombinant interferon gamma (0.05 mg/m²). Surgical intervention may be necessary to identify pathogens, debride devitalized tissues, or accelerate recovery and response to therapy. Granulomatous processes can occur with or without infections, so appropriate microbial cultures are an important part of the evaluation. Gastrointestinal or genitourinary granulomas not associated with any pathogen can be treated with 0.5 to 1 mg/kg of prednisone for 2 weeks, followed by gradual taper, though some patients require 0.1 to 0.25 mg/kg prednisone long term for control of (GI) and/or (GU) lesions. Some CGD patients may have difficulty with dehiscence of surgical wounds, particularly on the abdomen or neck, and paradoxically they may require a course of low-dose corticosteroid to suppress granuloma formation in the wound to allow healing. Bone marrow or other source hematopoietic stem cell transplant may be considered for some patients with severe disease and/or many recurrent infections who have an HLA-matched sibling donor.⁷ Gene therapy appears to be a promising alternative for those who are not eligible for stem cell transplantation.⁸

Chédiak-Higashi Syndrome

Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder caused by LYST gene mutations, leading to abnormal intracytoplasmic protein transport and vacuole formation.8,9 The genetic defect results in the fusion of intracellular granules and uneven distribution of giant granules in the cytoplasm of neutrophils and many other cells, such as platelets, melanocytes, renal tubular cells, Schwann cells, thyroid follicular cells, and mast cells. Cells containing giant granules have impaired function and manifest as recurrent bacterial infections; bleeding or easy bruising; hypopigmentation of skin, eyes, and hair; recurrent infections; peripheral nerve defects (neuropathy, nystagmus); or abnormal natural killer cell functions. Diagnosis is made by detecting large granules in neutrophils on the peripheral blood smear. Treatment includes supportive therapy and bacterial prophylaxis with TMP/SMX. Not all patients appear to have recurrent infections; major problems include a progressive peripheral neuropathy that manifests during the third decade of life and the risk of developing a lymphoma-like condition, which can be fatal. Vitamin C was shown to partially reverse some of the cellular defect observed in vitro, leading to its use in patients, but whether it reduces infection or alters the course of the disease is not clear. Bone marrow transplantation, immunosuppression, or rituximab can be considered for those who develop an “accelerated phase” with lympho-proliferative lymphoma-like syndrome.

Specific Granule Deficiency

Neutrophil secondary (or specific) granules contain a variety of proteases and other antimicrobial molecules. These proteins perform important normal functions in infection control and possibly also in wound healing. Specific granule deficiency (SGD) occurs as a very rare inherited disorder, or more commonly appears associated with leukemia or myelodysplasia.¹⁰ Acute burn injury has also been noted to result in neutrophils deficient in specific granules, perhaps secondary to degranulation. Inherited SGD can result from a mutation in the gene encoding a key regulatory factor required for late events during myeloid differentiation (CCAAT/enhancer binding protein ε). Failure of function of this DNA-binding differentiation factor protein results in the inability to produce the specific granule itself, the contents of the specific granule, as well as the inability to produce other proteins normally made during the late phase of myeloid differentiation. Clinically, SGD patients have recurrent bacterial infections starting in early childhood. Common sites of infections are skin (cellulitis) and respiratory tract (sinusitis, pneumonia, otitis media). Similar to LAD, there is no erythema or pus at the site of infections, and recurrent large non-healing ulcers are a chronic problem. The presence of non-healing ulcers in both SGD and in LAD probably points to an important role of neutrophils not only for infection control but possibly also in wound healing. Treatment includes antibiotics for acute infections and prophylaxis with daily TMP/ SMX and itraconazole.

NEUTROPENIAS

Neutropenia is typically defined as an absolute neutrophil count (ANC) less than 1.5 × 10⁹/L (or <1,500/µL). Neutropenia is characteristic of some specific hereditary syndromes, and it can result from infections, drugs or toxins, or autoimmune disorders. The risk of infection from neutropenia depends on three factors: the ANC, the neutrophil reserve in the bone marrow, and the duration of neutropenia. This risk is increased with neutrophil counts of 0.5 to 1.0 × 10⁹/L (500–1,000/µL) and the greatest with less than 0.5 × 10⁹/L (<500/µL). A falling neutrophil count or a significant decrease over steady-state levels, with a failure to increase neutrophil counts in the setting of infection or other bone marrow stress carries a higher risk of complication than a stable chronically low neutrophil count over many months or years that rises significantly in response to infection.

Acquired Neutropenias

Drug-Induced Neutropenia

Drugs can cause neutropenia by one or more of the following mechanisms: direct cytotoxic effect to rapidly dividing bone marrow cells, immune mediated, or other non-immune mediated neutrophil destruction. A recent review indicated that the duration of drug exposure to onset of neutropenia can vary from less than 1 week to 60 days.¹¹ The degree of neutropenia can be severe (ANC less than 0.1 × 10⁹/L or 100/µL) but usually requires only that the sensitizing drug be discontinued. Neutrophil counts usually begin to recover within 5 to 10 days after the offending drug is stopped. Re-administration of the sensitizing drug may decrease neutrophil counts abruptly. While some drugs (Table 9.2) have more often been cited as a cause of drug-related neutropenia, severe immune-mediated, drug-induced neutropenia can occur with any drug, including unlikely agents such as aspirin or acetaminophen.

Infection-Related Neutropenia

Neutropenia following infections is common, from one or more of the following mechanisms: destruction, margination, sequestration, or marrow suppression. Neutropenia from viral infections can be seen as early as a few days, and can persist for the duration of viremia. The degree and duration of virus-induced neutropenia is usually mild and short, but neutropenia from Epstein-Barr virus, hepatitis, and HIV can be severe and protracted. Gram-negative bacterial infections can cause neutropenia in those with impaired marrow neutrophil reserve, such as neonates, the elderly, and the chronically immunosuppressed. Protozoal (Leishmania) and rickettsial (RMSF and Ehrlichia) infections can also cause neutropenia, often with accompanying anemia and/or thrombocytopenia.

Immune-Related Neutropenia

This form of neutropenia is typically associated with specific antibodies directed to neutrophil antigens (not to be confused with anti-nuclear antibodies). These antibodies may occur with or without autoimmune disorders. Many syndromes are clinically similar and will be briefly discussed below.

In alloimmune (or isoimmune) neonatal neutropenia,¹² maternal IgG antibodies are directed toward paternal antigens on fetal neutrophils causing moderate neutropenia that is self-limiting, lasting only a few weeks to a few months. These neonates have an increased risk of infections and can develop pulmonary, skin, or urinary tract infections from gram-positive or -negative organisms. The treatment is supportive with antibiotics, IVIg, and sometimes G-CSF.

Table 9.2  Abbreviated List of Common Drugs Causing Neutropenia

Antiplatelet agents: ticlodipine

Sulfa-containing drugs: sulfasalazine, dapsone

Anti-thyroid agents: methimazole, propylthiouracil

Calcium dobesilate

Antimicrobials: particularly penicillins, cephalosporins, carbapenems

NSAIDs: dipyrone, indomethacin

Tricyclic antidepressants: clomipramine

Cardiac medications: antiarrhythmics agents, digoxin, diuretics, ACE inhibitors

Reflux/ulcer agents: cimetidine, ranitidine

Antipsychotic agents: clozapine, chlorpromazine

Antiviral agents (against HIV, HSV, CMV)

Rheumatoid arthritis agents: penicillamine, gold compounds

Chemotherapy

ACE, angiotensin converting enzyme; CMV, cytomegalovirus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; NSAIDs, non-steroidal anti-inflammatory drugs.

From Palmblad JE, von dem Borne AE. Idiopathic, immune, infectious, and idiosyncratic neutropenias. Semin Hematol. 2002;39:113-120.

Autoimmune neutropenia of infancy/childhood¹³ is typically seen in those less than 2 years of age. The degree of neutropenia is variable, and infections in the oropharynx, ear, sinus, and upper respiratory tract can occur. The neutropenia may resolve spontaneously over many months or years, and typically does not require treatment. Antibiotics and G-CSF are given during acute infections, and TMP/SMX is often given for prophylaxis.

Large granular lymphocytosis or leukemia (LGL)¹⁴ is caused by abnormally expanded T or NK cells infiltrating the bone marrow, spleen, and liver, resulting in variable degrees of pancytopenia and splenomegaly. LGL may be an oligoclonal or monoclonal disease, and in its more aggressive form is considered a form of leukemia. LGL is usually diagnosed in individuals about 60 years old. Laboratory evaluation shows multiple abnormalities: 80% of affected individuals will have lymphocytosis >2 × 10⁹/L, 80% with ANC <1.5 × 10⁹/L, 50% with hemoglobin <11 g/dL, 20% with platelet <150 × 10⁹/L. LGL can also occur with other autoimmune disorders (rheumatoid arthritis most commonly), myeloid and B cell malignancies, or solid tumors. Bone marrow examinations are also variable, but the majority will have hypercellular marrow. No treatment is necessary until there are recurrent infections, severe neutropenia, or symptomatic anemia.¹⁵ Corticosteroids, methotrexate, cyclophosphamide, and other immunosuppressive therapy have been used with generally good response rates. However, aggressive monoclonal LGL disease should be considered a form of leukemia requiring specific chemotherapies appropriate to control the disease.

Congenital Neutropenias

Severe Congenital Neutropenia (Kostmann Syndrome and Autosomal Dominant Forms)

Dr. Kostmann in 1956 described severe neutropenia associated with recurrent bacterial infections in several families in northern Sweden. This syndrome was later observed in other geographic locations. Kostmann syndrome is an autosomal recessive form of severe congenital neutropenia that is a rare clinical entity with an incidence rate of about 1 to 2 per million.¹⁶ Neutrophil elastase (ELA 2 or ELANE) mutations are responsible for almost half of the individuals with the autosomal dominant or sporadic forms of severe congenital neutropenia. Mutations in ELA 2 have been hypothesized to cause defective signal transduction and to cause programmed cell death (apoptosis) at the myelocyte level.¹⁷ These effects may be a result of cellular mechanisms that detect protein misfolding. There are additional abnormalities that can be acquired, which may lead to myelodysplasia and/or acute myeloid leukemia: G-CSF receptor mutation, RAS oncogene mutation, or chromosome 7 monosomy. An autosomal dominant form of neutropenia has been reported to result from heterozygous mutations in the GFI1 gene that may affect ELA 2.¹⁸ Recent studies have shown that Kostmann syndrome can also be caused by mutations in the HAX1,¹⁹ G6PC3,²⁰ or other genes.

Clinically, individuals are infected as early as 2 to 3 months of age by gram-positive or gram-negative bacteria in one or more of the following sites: skin, ears, oral or gastrointestinal mucosa, upper or lower respiratory tract, urinary tract, or blood. Blood counts usually reveal neutrophils less than 500/µL (<0.5 × 10⁹/L) with compensatory monocytosis and eosinophilia. Bone marrow biopsies show maturation arrest at the promyelocyte-myelocyte level and absent band forms or mature neutrophils. Treatment includes supportive therapy and antibiotics for acute infections. G-CSF between 3 and 10 µg/ kg increases the neutrophil count and reduces the frequency of infections. A minority of individuals will require a dose in excess of 30 µg/kg/day. G-CSF is not currently thought to be associated with acquisition of G-CSF mutations and is not itself thought to be a cause of the leukemia associated with this disorder. However, individuals requiring longer duration or high cumulative doses of G-CSF may have a more severe form of the disorder, and therefore a higher risk of malignant transformation to leukemia. Toxicities of chronic G-CSF administration include bone pain from marrow expansion, osteopenia or osteoporosis, and splenomegaly. Bone marrow transplant is a curative option for those with HLA-matched sibling donors.

Cyclic Neutropenia

The incidence of inherited cyclic neutropenia is not known,17,21 nor is the etiology completely understood, although neutrophil elastase 19p13.3 mutations are associated with this disorder and have been hypothesized to cause neutrophil apoptosis and thus to initiate the cycling. Clinically, neutrophil count oscillates predictably between very low or agranulocytic to low normal range; the average cycle length is 21 days with neutropenic duration of 3 to 6 days. The nadir neutrophil count can be zero or as low as 200/µL (0.2 × 10⁹/L). Platelet, reticulocyte, lymphocyte, and monocyte counts may also “counter-cycle” between normal to high range, either coinciding or not coinciding with the neutrophil cycles. Serial bone marrow examinations will appear normal, when the neutrophil count is high, and show decreased myeloid precursors in the neutropenic phase. Individuals with cyclic neutropenia may be asymptomatic during periods of normal neutrophil count, and may have fever, lymphadenopathy, mild skin infections, and/or oral mucosal ulcers during periods of neutropenia. Mild skin infections and/or mouth ulcers are treated symptomatically. G-CSF, at 2 to 3 µg/kg per one or two days, appears to improve the neutrophil nadir, shorten the cycles, and thus reduce infections. GM-CSF does not effectively treat inherited cyclic neutropenia.

Other Inherited Disorders Associated with Clinically Significant Neutropenia

There are a number of inherited disorders where clinically significant neutropenia is observed, but where neutropenia is not considered the most prominent feature of the inherited syndrome. Three examples are provided. A specific mutation responsible for Wiskott-Aldrich syndrome is associated with neutropenia.²² Patients with WHIM syndrome, which is caused by inherited C-terminal truncations in CXCR4 suffer from clinically significant neutropenia that is responsive to treatment with G-CSF.²³ A subset of patients with CD40 ligand deficiency (X-linked Hyper-IgM syndrome) have clinically significant neutropenia.²⁴

Other Neutropenias

Idiopathic Neutropenia

Idiopathic neutropenia, or chronic idiopathic neutropenia, affects about 2 to 4 individuals per million, and can be seen in both children and adults.²⁵ Clinically, it behaves very similar to autoimmune neutropenia, except anti-neutrophil antibodies are not detected and other studies are non-diagnostic. Majority of these individuals have moderate neutropenia with mild symptoms. Pro-inflammatory cytokines that promote neutrophil apoptosis and myelosuppression from activated lymphocytes have been proposed as possible mechanisms. There is a small subset of individuals that have severe neutropenia, recurrent fevers, oropharyngeal infections (mucosal ulcers, gingivitis), or severe systemic infections. Treatments are largely tailored for symptomatic relief and antibiotics dictated by sites of infection. G-CSF, 1 to 3 μg/kg per dose weekly or on alternate day, is used in those with severe clinical syndromes. Development of myelodysplastic syndrome or leukemias is very rare. Generally, patients who increase their neutrophil count with infection or with other stress do well clinically.

Benign Ethnic Neutropenia

Benign ethnic neutropenia (BEN) is a condition seen mostly in individuals of African descent, including African-Americans, Yemenite Jews, and certain populations in the Caribbean and Middle East. Prior studies showed that up to 25% of non-US individuals of African descent and about 4% of African-Americans have neutrophil counts between 1.0 and 1.5 × 10⁹/L.²⁶ The cause for this observation is unknown, but several investigators earlier have excluded stem cell disorder, excessive margination, and differentiation defect, suggesting that this may be a normal population-based variant. The physiologic mechanisms controlling the normal set point for circulating levels of neutrophils is unknown, but there is accumulating evidence that the Duffy antigen and receptor for chemokine (DARC) is associated with those of African descent with lower leukocyte/neutrophil counts.27,28 CXCR4 chemokine receptor for the SDF-1 chemokine may also play a role in egress of neutrophils from the marrow and, at least theoretically, differences in expression or function of this cytokine/cytokine receptor could affect this set point. Perhaps normal variants in this or other receptors may be responsible for these population differences observed in average circulating neutrophil counts. Clinically, individuals with this ethnically based neutropenia variant are asymptomatic, without recurrent oral, skin, or systemic infections. When these individuals acquire typical viral or bacterial infections, these infections are not more severe and do not need longer periods of treatment. Laboratory evaluations will show many blood counts that are abnormal over many years, and the bone marrow examinations will be normal. Other than usual symptomatic treatment and antibiotics as needed (as for a normal healthy adult), no additional treatment is required, but it is important to note this variation to avoid unnecessary medical evaluation.

References

  1.  Lekstrom-Himes JA, Gallin JI. Immunodeficiency diseases caused by defects in phagocytes. N Engl J Med. 2000;343:1703-1714.

  2.  van de Vijver E, Maddalena A, Sanal O, et al. Hematologically important mutations: leukocyte adhesion deficiency (first update). Blood Cells Mol Dis. 2012;48:53-61.

  3.  Qasim W, Cavazzana-Calvo M, Davies EG, et al. Allogeneic hematopoietic stem-cell transplantation for leukocyte adhesion deficiency. Pediatrics. 2009;123:836-840.

  4.  Lanza F. Clinical manifestation of myeloperoxidase deficiency. J Mol Med. 1998;76:676-681.

  5.  Malech HL, Hickstein DD. Genetics, biology and clinical management of myeloid cell primary immune deficiencies: chronic granulomatous disease and leukocyte adhesion deficiency. Curr Opin Hematol. 2007;14:29-36.

  6.  Kang EM, Marciano BE, DeRavin S, et al. Chronic granulomatous disease: overview and hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2011;127:1319-1326.

  7.  Kang EM, Malech HL. Gene therapy for chronic granulomatous disease. Methods Enzymol. 2012;507:125-154.

  8.  Kaplan J, De Domenico I, Ward DM. Chediak-Higashi syndrome. Curr Opin Hematol. 2008;15:22-29.

  9.  Gombart AF, Koeffler HP. Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon). Curr Opin Hematol. 2002;9:36-42.

10.  Andersohn F, Konzen C, Garbe E. Systematic review: agranulocytosis induced by nonchemotherapy drugs. Ann Intern Med. 2007;146:657-665.

11.  Palmblad JE, von dem Borne AE. Idiopathic, immune, infectious, and idiosyncratic neutropenias. Semin Hematol. 2002;39:113-120.

12.  Audrain M, Martin J, Fromont P, et al. Autoimmune neutropenia in children: analysis of 116 cases. Pediatr Allergy Immunol. 2011;22:494-496.

13.  Bareau B, Rey J, Hamidou M, et al. Analysis of a French cohort of patients with large granular lymphocyte leukemia: a report on 229 cases. Haematologica. 2010;95:1534-1541.

14.  Lamy T, Loughran TP Jr. How I treat LGL leukemia. Blood. 2011;117:2764-2774.

15.  Welte K, Zeidler C, Dale DC. Severe congenital neutropenia. Semin Hematol. 2006;43:189-195.

16.  Horwitz MS, Duan Z, Korkmaz B, et al. Neutrophil elastase in cyclic and severe congenital neutropenia. Blood. 2007;109:1817-1824.

17.  Person RE, Li FQ, Duan Z, et al. Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2. Nat Genet. 2003;34:308-312.

18.  Klein C, Grudzien M, Appaswamy G, et al. HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease). Nat Genet. 2007;39:86-92.

19.  Boztug K, Rosenberg PS, Dorda M, et al. Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: novel genotypes and phenotypic variability in severe congenital neutropenia. J Pediatr. 2012;160(4):679-683.

20.  Dale DC, Welte K. Cyclic and chronic neutropenia. Cancer Treat Res. 2011;157:97-108.

21.  Devriendt K, Kim AS, Mathijs G, et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat Genet. 2001;27:313-317.

22.  Hernandez PA, Gorlin RJ, Lukens JN, et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet. 2003;34:70-74.

23.  Winkelstein JA, Marino MC, Ochs H, et al. The X-linked hyper-IgM syndrome: clinical and immunologic features of 79 patients. Medicine (Baltimore). 2003;82:373-384.

24.  Palmblad J, Papadaki HA. Chronic idiopathic neutropenias and severe congenital neutropenia. Curr Opin Hematol. 2008;15:8-14.

25.  Hsieh MM, Everhart JE, Byrd-Holt DD, et al. Prevalence of neutropenia in the U.S. population: age, sex, smoking status, and ethnic differences. Ann Intern Med. 2007;146:486-492.

26.  Nalls MA, Couper DJ, Tanaka T, et al. Multiple loci are associated with white blood cell phenotypes. PLoS Genet. 2011;7:e1002113.

27.  Reiner AP, Lettre G, Nalls MA, et al. Genome-wide association study of white blood cell count in 16,388 African Americans: the continental origins and genetic epidemiology network (COGENT). PLoS Genet. 2011;7:e1002108.

28.  Klempner MS, Malech HL. Phagocytes: normal and abnormal neutrophil host defenses. In: Gorbach SL, Bartlett JG, Blacklow NR, eds. Infections Diseases. 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2004:24.

10

Childhood Hematologic Diseases

Hema Dave and Alan S. Wayne

Despite some overlap with disorders encountered in adults, many congenital and acquired hematologic diseases manifest primarily during childhood. In addition, pediatric hematology is distinguished by developmental differences in normal physiology and blood parameters.¹ The purpose of this chapter is to highlight unique features in the evaluation, diagnosis, and treatment of common pediatric hematologic conditions. The reader is referred to other chapters in this edition for additional details of the management of specific disorders.

ANEMIA

Normal red blood cell (RBC) values vary with age and are affected by factors such as race, sex, and altitude (Table 10.1). The RBC count is highest at birth and continues to decrease gradually to a physiologic nadir at 2 to 4 months (earlier for premature infants), at which point erythropoiesis is stimulated. Anemia is defined as an overall reduction in red cell mass or hemoglobin (Hb) concentration, 2 standard deviations below the mean normal value for the specific population.

Pediatric anemia is commonly classified according to RBC size (Table 10.2). Microcytic anemias account for the majority of cases of anemia in early childhood (Table 10.3). Current recommendations by the American Academy of Pediatrics include screening for anemia testing Hb and serum transferrin receptor between the ages of 9 to 12 months with additional screening between the ages of 1 and 5 years for patients at risk and adolescent females post-puberty (Table 10.4).²⁻⁵

The initial diagnostic evaluation of a child with anemia should consist of a detailed history and physical examination and the following minimal laboratory testing: complete blood count (CBC), reticulocyte count, and examination of the peripheral blood smear. Consideration of the physiologic basis for anemia can be helpful in guiding further investigation (Table 10.5).

Microcytic Anemias

Iron deficiency is the most common cause of anemia during childhood and may result from a combination of low stores at birth, high requirements because of growth and blood volume expansion, inadequate nutrition, and poor bioavailability of dietary iron. Iron deficiency due to blood loss is usually a result of gastrointestinal tract irritation and occult hemorrhage associated with introduction of cow’s milk before the first year of life or menstruation during adolescence. On history, additional risk factors for iron deficiency may include prematurity, limited or prolonged breast-feeding, non-iron-fortified formula or excessive intake of whole milk (generally more than 1 quart per day). Early iron deficiency may result only in a low ferritin level. Falling iron stores leads to decreased serum iron and transferrin saturation and an increase in total iron-binding capacity (TIBC) and free erythrocyte protoporphyrin (FEP). With frank deficiency, there is hypochromia, microcytosis, and anisocytosis on the blood smear. The platelet count also may be increased. A response to a trial of elemental iron (3 mg/kg/day) is often helpful in differentiating iron deficiency from thalassemia trait: a Hb increase of more than 1 g/dL at 1 month and reticulocyte peak at 10 to 14 days is diagnostic. Iron supplementation (3 mg/kg/day for mild anemia, 6 mg/kg/day for moderate to severe anemia) should be provided for 3 to 6 months.²⁻⁶ The most common reason for a lack of response to oral iron therapy is lack of compliance. If anemia persists, other causes of microcytic anemia should be considered (Table 10.2).

Table 10.4  Recommendations for Anemia Screening

American Academy of Pediatrics (AAP) Recommendations⁴

Option 1: Universal screening (communities with high-risk populations) 9–12 mo, 15–18 mo

Adolescents: Males at peak growth, Females at routine exams

Option 2: Selective screening (communities with low-risk populations) Screen high-risk patients at 9–12 mo, 15–18 mo, yearly until 5 Adolescents: Males at peak growth, Females at routine exams

Center for Disease Control (CDC)⁵

High-risk populations: 9–12 mo, 15–18 mo, yearly until 5

Adolescent females every 5–10 yr, yearly if risk factors

Adolescent males at peak growth

Lead toxicity often coexists with iron deficiency in at-risk populations and may further inhibit gastrointestinal absorption of iron. Lead poisoning should be suspected if there is a history of pica or exposure to lead-based paint, particularly in children with developmental delay or autism as these can be comorbid conditions. An elevated FEP and basophilic stippling on peripheral smear may be seen. Therapy should include oral treatment with succimer or, in severe cases, parenteral treatment with dimercaprol (BAL) or calcium-sodium ethylenediaminetetraacetic acid (EDTA).⁷ Center for Disease Control (CDC) recommendations for management of children with elevated lead levels can be found in Table 10.6.

Thalassemia syndromes are common causes of microcytic anemia in childhood. They are classified into α and β thalassemias based on the affected globin chain. The α-thalassemias present in utero or at birth, while the β-thalassemias are not evident until 6 months of age, when β-globin synthesis becomes predominant. Thalassemia trait is often mistaken for iron deficiency.⁸ In contrast to iron deficiency, β-thalassemia trait is associated with a normal red cell distribution width (RDW), basophilic stippling and targeting on the blood smear, and an elevated Hb A2 on electrophoresis. α-thalassemia trait is associated a normal Hb electrophoresis outside of the newborn period, although Hb Bart’s (γ₄) is present on newborn screening samples. In the evaluation of thalassemias, ethnic heritage is often suggestive and microcytosis should be present in at least one parent. Thalassemia trait (heterozygous β-1 and 2 gene deletion α-thalassemias) requires no therapy. In contrast, in thalassemia major, aggressive packed RBC transfusion should be initiated early in life to eliminate the increased erythropoietic drive and allow normal linear growth and bone development. In utero transfusion has been used to prevent hydrops fetalis in 4-gene deletion α-thalassemia (Hb Bart’s disease), which is otherwise fatal. Care should be given to address iron overload and chelation therapy in transfusion-dependent children to prevent end-organ damage later in life. As an alternative to life-long transfusion and chelation therapy, allogeneic hematopoietic stem cell transplantation (SCT) is a curative approach for children with thalassemia major who have human leukocyte antigen (HLA)-matched sibling donors. Prenatal diagnosis of thalassemia can be made as early as the 10th week of gestation using chorionic villus sampling.⁹

Normocytic Anemias

Anemia is a common manifestation of numerous systemic conditions in pediatrics. The anemia of acute inflammation and chronic disease is frequently mild and usually normocytic, although the mean cell volume (MCV) is occasionally low. Transferrin is frequently diminished (Table 10.3). Treatment is aimed at the primary condition. Viral infection is the most common cause of transient bone marrow suppression in children and may result in both anemia and leukopenia. The hallmark of viral suppression is failure of the reticulocyte count to increase in the face of anemia. Usually, only close observation is required because the bone marrow suppression is self-limited.

Transient erythroblastopenia of childhood (TEC) is an acquired pure red cell aplasia that can also follow viral illness in previously healthy children.¹⁰ The median age for presentation is 2 years, in contrast to congenital pure red cell aplasia, which commonly presents in infancy. Reticulocytopenia and, occasionally, leukopenia and thrombocytopenia are seen. Most children with TEC recover in 1 to 2 months. Observation alone is usually sufficient, although short-term transfusion therapy may be required for cardiovascular compromise associated with severe anemia.

Hemolysis usually results in normocytic anemia. There are a large number of congenital and acquired hemolytic conditions of childhood, including membrane disorders, hemoglobinopathies, metabolic defects, enzymopathies, and immune-mediated hemolysis. Immune-mediated hemolysis, either isoimmune or alloimmune, may present in neonates; autoimmune hemolytic anemia is seen in older children. Hemoglobinopathies such as sickle cell disease, enzyme deficiencies such as glucose-6-phosphate dehydrogenase deficiency, and membrane disorders such as hereditary spherocytosis (HS) should be considered in the differential diagnosis of hemolysis. Microangiopathic hemolysis may be seen in hemolytic uremic syndrome (HUS) and disseminated intravascular coagulation (DIC). Laboratory features consistent with hemolysis include reticulocytosis, elevated lactate dehydrogenase (LDH), indirect hyperbilirubinemia, decreased serum haptoglobin, and, in severe cases, hemoglobinuria. A positive direct Coomb’s test indicates immune-mediated hemolysis. Examination of the peripheral smear may reveal characteristic red cell morphology. Thrombocytopenia and renal impairment are additional features of HUS. Treatment of hemolysis should be directed toward the underlying cause, with transfusions reserved for severe anemia and cardiovascular compromise. Immune hemolysis often requires corticosteroids and/or other immunosuppressive medications.

The diagnosis of sickle cell disease is usually made during routine newborn screening by Hb electrophoresis. Children diagnosed with sickle cell disease should be cared for by practitioners with specific expertise in the management and prevention of its complications.11,12 Consultation with a pediatric hematologist is strongly recommended. One early presentation of sickle cell crisis in pediatric patients is dactylitis, due to vaso-occlusive crisis of the hands and feet. Children should receive influenza vaccine annually starting at six months of age, the 7-valent conjugated pneumococcal vaccine series in the first year of life, and 23-valent polysaccharide pneumococcal vaccine and meningococcal vaccine at 2 years. Penicillin prophylaxis should begin at diagnosis and continue until age five years and completion of the pneumococcal vaccination series. Of importance, the risk of pneumococcal sepsis is lifelong and individuals with sickle cell disease require immediate medical attention for fever or signs of infection. Because of the risk of cerebrovascular accident and the difficulty of clinical diagnosis in the pediatric population, children should undergo annual transcranial Doppler ultrasound (TCD) starting at the age of 2 years.¹¹ Chronic blood transfusions can prevent stroke in children with abnormal TCD (velocities ≥200cm/sec)11,13,14 Hydroxyurea increases fetal Hb and reduces vaso-occlusive pain crises, acute chest syndrome and need for transfusion.¹⁵ Annual echocardiogram to detect pulmonary hypertension is recommended starting at 15 years and annual eye examination at 10 years to evaluate for retinopathy.¹²

Macrocytic Anemias

Vitamin B12 deficiency causes megaloblastic changes in the bone marrow.¹⁶ In infants, B12 deficiency may be the result of maternal depletion and decreased stores at birth. In older children and adolescents, etiologies include pernicious anemia, malabsorption, dietary deficiency, and inborn errors of metabolism. Unrecognized, severe deficiency early in life may cause failure to thrive and even permanent neurologic damage. Symptoms in older children may include anorexia, weight loss, diarrhea, constipation, weakness, glossitis, peripheral neuropathy, ataxia, and dementia. Anemia is commonly accompanied by neutropenia, hypersegmented neutrophils, and thrombocytopenia. A low serum B12 level and a response to replacement therapy are confirmatory.

Folate deficiency also results in a megaloblastic bone marrow.¹⁶ The newborn infant has increased demands for folate. Risk factors for early deficiency include prematurity, low levels in maternal breast milk, and a predominance of goat’s milk intake. In older children, folate deficiency is usually the result of malnutrition, although it may also be caused by certain medications, chronic hemolysis, malabsorption, and inborn errors of metabolism. Serum and erythrocyte folate levels will be low and the anemia should respond to small replacement doses of folic acid.

Diamond-Blackfan anemia (DBA) or congenital pure red cell aplasia is usually noted soon after birth or during the first year of life. The main competing entity in the differential diagnosis is TEC, but TEC more commonly presents after the first year of life. Twenty-five percent of patients with DBA have associated anomalies, such as short stature and/or abnormalities of the head, face, and upper limbs. Laboratory features include reticulocytopenia, high MCV (often only mildly elevated), increased Hb F, elevated adenosine deaminase activity, normal or decreased white blood cell (WBC) count, and normal or increased platelet count. Mutations in genes that encode ribosomal proteins have been found in about 50% of patients,¹⁷ and large deletions or changes in regulatory regions likely account for the remainder. The bone marrow shows erythroid hypoplasia. In considering the differential diagnosis, a normal CBC in the past supports TEC and an abnormal chromosomal breakage study confirms Fanconi anemia (FA). The majority of children with DBA respond to corticosteroids. Prednisone is begun at a dosage of 2 mg/kg/day with a response usually seen within 1 month. Once the Hb has reached a satisfactory level, steroids should be tapered to the lowest possible dose (ideally on an alternate day schedule). Although spontaneous remissions have occurred, corticosteroid dependence is the rule and chronic transfusion and chelation therapy should be considered for those with associated toxicity. Allogeneic SCT can be curative.¹⁸

FA can often be differentiated from acquired aplastic anemia by characteristics such as impaired growth and/or anomalies of the thumbs, radii, kidneys, head, eyes, ears, skin, and/or genitourinary system. Inheritance is autosomal recessive and the family history may be positive for marrow failure and leukemia. There is a 10% to 35% risk of developing leukemia or myelodysplastic syndrome.¹⁹ Although the mean age of diagnosis is between eight and nine years, the first hematologic signs of FA may develop in infancy and often includes macrocytosis, elevated Hb F and/or mild cytopenia(s). Severe pancytopenia usually develops later in life. The differential includes other familial or acquired bone marrow failure syndromes. Abnormal chromosomal breakage analysis or FA genotyping confirms the diagnosis. Anemia is frequently responsive to androgen therapy. Only SCT is curative for the hematologic manifestations of FA, but a modified pretransplant conditioning is critical to avoid severe toxicity caused by chemotherapy and radiation sensitivity.

BLEEDING

Many congenital and acquired disorders of hemostasis, including platelet abnormalities, present in infancy and childhood. Hemorrhagic disorders in infancy may manifest as bleeding from the umbilicus, circumcision site, unusually large cephalohematomas, and more serious but fortunately rare intracranial hemorrhages. The normal ranges for coagulation assays are age dependent and differ greatly from the neonatal period to infancy and later childhood (Table 10.7). Most coagulation proteins increase in parallel with gestational age. Because physiologic levels of many clotting factors are low at birth, it is often difficult to diagnose disorders of hemostasis in newborns.

Acquired Factor Deficiencies

Hemorrhagic disease of the newborn (HDN) is a complication of physiologically low levels of vitamin K-dependent factors in the newborn.²⁰ Classic HDN presents on days 2 to 7 of life in otherwise healthy, full-term infants and occurs in 1/10,000 live births without vitamin K prophylaxis. Risk factors include poor placental transfer of vitamin K, marginal levels in breast milk, inadequate milk intake, and the sterile newborn gut. Although rarely necessary, diagnosis can be confirmed by screening coagulation tests and vitamin K-dependent factor levels. Determination of decarboxylated forms of vitamin K-dependent factors or protein induced by vitamin K antagonists also may be helpful. HDN should be prevented in all newborns by prophylactic administration of vitamin K at birth with a single dose of 0.5 to 1 mg intramuscularly (preferred route) or an oral dose of 2 to 4 mg, followed by continued supplementation in breast-fed infants.

Vitamin K deficiency can also be seen in children with liver disease, with chronic antibiotic use, and due to inadequate intake or disorders that interfere with vitamin K absorption, such as chronic diarrhea, cystic fibrosis, or other fat malabsorption syndromes. Therapy should include vitamin K administration as well as measures directed to the underlying disease.

DIC can be differentiated from vitamin K deficiency and liver disease by assaying coagulation factor levels. DIC decreases all clotting factors due to consumption. In contrast, factor VIII, the only clotting protein not synthesized solely in the liver, is normal or elevated in liver disease. Therapy should be directed at the underlying cause, although supportive measures may include treatment with fresh-frozen plasma (FFP).

Inherited Factor Deficiencies

Hemophilias A and B often present in early childhood. Newborns with hemophilia can bleed with circumcision and, rarely, may suffer an intracranial hemorrhage after delivery. In the absence of a family history, the diagnosis of hemophilia is most often made when a child with moderate to severe factor deficiency begins to crawl or walk. Common symptoms include easy bruising, hemarthrosis in weightbearing joints, and deep intramuscular hemorrhage. Central nervous system (CNS) bleeding is the usual cause of early mortality. Laboratory evidence for hemophilia includes a prolonged partial thromboplastin time (PTT), which corrects on mixing studies. An abnormally low factor VIII or IX level confirms the diagnosis. Care should be taken to evaluate and treat hemarthroses aggressively in children to prevent the later development of chronic arthropathy. The treatment of hemophilia in children is similar to that in adults and includes factor replacement dosed according to the site, type, and severity of hemorrhage.²¹ The availability of recombinant factor concentrates has increased the safety and feasibility of prophylaxis in children with frequent hemorrhagic episodes. For dose determination of factor concentrates, factor VIII levels will typically increase by approximately 2% for every 1 unit/kg given and factor IX levels by about 1% for every 1 unit/kg administered. In patients with mild hemophilia A, desmopressin is often effective for short-term management of mild bleeding. As in adults, routine screening for inhibitors should be conducted.

Von Willebrand disease (vWD) usually presents with less severe bleeding, primarily mucocutaneous, as compared to hemophilia. Because recurrent bruising and epistaxis are relatively common in children, history should be directed toward the presence of prolonged, unusual, or severe bleeding. A careful family history may reveal similar symptoms in parents or siblings. The diagnosis is confirmed by abnormal assays for factor VIII, von Willebrand factor (vWF) antigen and activity, and multimer analysis. Factor VIII and vWF are acute phase reactants and, in children, falsely elevated levels caused by interval illnesses may obscure the diagnosis. Thus, repeat testing should be considered if the diagnosis of vWD is suspected. Therapy is as in adults.²²

Platelet Disorders

Neonatal alloimmune thrombocytopenia (NAIT) results from the placental transfer of maternal alloantibodies against paternally inherited antigens (most commonly HPA-1a) on fetal platelets. Newborns present with transient, isolated but severe thrombocytopenia that must be distinguished from other causes, including maternal immune thrombocytopenic purpura (ITP), severe infection, DIC, hypersplenism, and Kasabach-Merritt syndrome. Approximately 15% of affected neonates experience intracranial hemorrhage, either in utero or in the immediate postnatal period. Unlike Rh disease of the newborn, prior sensitization is not required, and thus NAIT may occur with the first pregnancy. A normal platelet count in the mother helps to differentiate NAIT from maternal ITP. Immunophenotyping of maternal and paternal platelets is useful to confirm the diagnosis. The treatment of choice in severe NAIT is transfusion of maternal platelets. When they are not readily available, platelets obtained from a known HPA-1a-negative donor or from random donors may be used for active bleeding. Intravenous immunoglobulin (IVIG) or corticosteroids may also be used as a temporary measure either in the antenatal or postnatal periods, with dosing as in ITP.

ITP affects approximately 1 in 10,000 children annually in the United States. In contrast to the disease in adults, ITP in childhood is usually a self-limited, benign condition. Children typically present under the age of 10. Eighty percent have spontaneous resolution within 6 months. Infants and older children are more likely to have prolonged thrombocytopenia. The typical presentation in acute ITP is the abrupt onset of mucosal bleeding, petechiae, and bruising in healthy children, often preceded by a viral illness. Most children have severe thrombocytopenia (platelet counts less than 20,000/µL) but an otherwise normal CBC. Large platelets are usual on the peripheral blood smear. Although acute ITP is a diagnosis of exclusion, otherwise healthy children with no significant medical history or findings on physical examination rarely require extensive laboratory testing. Testing for human immunodeficiency virus (HIV) infection can be considered. The diagnostic utility of bone marrow examination in suspected acute ITP is low. Evaluation for chronic ITP should include bone marrow studies and testing for immunodeficiency and autoimmune disease. The need for treatment in acute ITP is often debated; current guidelines recommend therapy for significant bleeding (Table 10.8).²³ Although the risk of intracranial hemorrhage is small, precautions should be taken to prevent head trauma and helmets are recommended for toddlers just learning to walk.

Inherited platelet disorders may be qualitative or quantitative; they are a rare cause of thrombocytopenia in infancy and childhood. The variety of qualitative disorders includes Glanzmann thrombasthenia (GT), Bernard-Soulier syndrome (BSS), platelet-type pseudo-vWD, and platelet storage granule defects. Quantitative defects are seen in congenital amegakaryocytic thrombocytopenia, thrombocytopenia-absent radii (TAR), X-linked thrombocytopenia, Wiskott-Aldrich syndrome (WAS), and May-Hegglin anomaly. Children with these disorders present with petechiae, easy bruising, or mucocutaneous bleeding. Rarely, gastrointestinal or intracranial bleeding may occur. Screening for qualitative disorders requires platelet aggregation studies. Characteristic features of specific disorders should be sought, such as forearm deformities in TAR, immunodeficiency in WAS, and macrothrombocytes in May-Hegglin anomaly. Treatment for bleeding is usually supportive. Platelet transfusions should be avoided if possible in patients with BSS and GT because of the risk of developing alloantibodies to the missing platelet antigens GPIb–IX and αIIb-β3, respectively.

Table 10.8  Treatment Regimens for Childhood ITP

IVIG: 0.8–1 g/kg × 1 d OR

Prednisone 2 mg/kg/d × 14 d with taper OR

Anti-D: 50–75 µg/kg × 1 d (Rh positive, non-splenectomized patients only)

Modified from Neunert C, Lim W, Crowther M, et al.The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood. 2011;117(16):4190-4207.

THROMBOSIS

As with coagulation factor levels, normal ranges for endogenous antithrombotic proteins are age- and gestation-dependent (Table 10.7). Venous thromboembolic events (TEE) are less common in children than in adults, with the exception of specific at-risk patient populations.²⁴ Unless risk factors are identified, arterial thrombosis is extremely infrequent.

Anticoagulant and thrombolytic therapy should be dosed according to age and weight (Tables 10.9 and 10.10). The duration, monitoring, efficacy, and long-term effects of anticoagulation in the management of TEE in children require further study. The treatment of children with oral anticoagulants is complicated by the risk of bleeding complications. As in adults, caution is required when instituting warfarin therapy. To avoid paradoxical thrombosis, heparinization should be continued until the international normalized ratio (INR) is in the therapeutic range.

Congenital Prothrombotic Disorders

Children who are homozygous or compound heterozygous for deficiencies of anticoagulant proteins usually present in the neonatal or early childhood period. In the absence of additional risk factors, however, individuals who are heterozygous for thrombophilic conditions infrequently experience their first TEE early in life. In general, evaluation for possible inherited deficiency is recommended for children with a family history of congenital thrombophilia, and if thromboses are unexplained, occur in unusual sites, are particularly severe, and/or are recurrent.

Protein C and S deficiencies, in the homozygous state, classically present as purpura fulminans within hours or days of birth. Purpura fulminans is more common with protein C deficiency and is characterized by acute DIC with rapidly progressive hemorrhagic necrosis of the skin and other thrombotic/hemorrhagic complications, including death. Homozygous infants usually have undetectable levels of protein C or S, and their parents have heterozygous deficiency. Both functional and immunologic assays for protein C and S should be utilized. Acquired causes of protein C and S deficiency, such as liver disease and sepsis, should be excluded. Purpura fulminans should be treated with FFP and, if available, purified protein C concentrate. Warfarin-induced skin necrosis has been described in children with heterozygous protein C and S deficiency, and extreme caution is required when converting such individuals from heparin to warfarin anticoagulation.

Other inherited thrombophilic states including antithrombin III deficiency, factor V Leiden, prothrombin G20210A mutations, and homocysteinemia also have been associated with recurrent thromboembolism in children and adolescents.²⁴ The incidence of deep vein thrombosis related to those conditions is low and the value of an extensive evaluation of the first TEE has been questioned.

Acquired Prothrombotic Disorders

As in adult patients, thromboembolism in children is usually secondary; central venous catheters are the most common cause. Neonates are at particularly high risk, and the use of umbilical lines may be associated with portal venous system thrombosis. Other risk factors include malignancy, surgery, trauma, pregnancy, congenital heart disease, Kawasaki disease, nephrotic syndrome, and systemic lupus erythematosus (SLE). Complete evaluation for possible underlying conditions should be undertaken. The laboratory examination should be guided by clinical findings and risk factors, and in most cases should include lupus anticoagulant or antiphospholipid antibody assay.

NEUTROPENIA

Normal neutrophil counts vary with age and are affected by race and other factors (Table 10.1). For example, the lower limit of normal in blacks may be 200 to 600/µL less than in whites.

Neutropenia is commonly encountered in pediatrics, most often caused by viral suppression. Children, like adults, are at increased risk for life-threatening bacterial infection when the absolute neutrophil count (ANC) falls below 500/µL. Common pyogenic infections seen in association with neutropenia include cellulitis, superficial or deep abscesses, pneumonia, sepsis, and recurrent or chronic otitis media and sinusitis.

General management recommendations include aggressive monitoring for and treatment of infection, judicious use of antibiotics, rapid institution of empiric broad-spectrum parenteral antibiotics for fever or possible infection, and maintenance of good skin and oral hygiene. Granulocyte-colony stimulating factor (G-CSF, filgrastim) is often effective in increasing the rate of ANC recovery in certain disorders.

Viral infections are the most common cause of transient neutropenia in childhood. Neutropenia usually develops during the first 24 to 48 hours of illness and commonly lasts up to a week or longer. Neutropenia may also occur with serious bacterial infections, especially in neonates.

Autoimmune neutropenia of childhood is the most common cause of chronic neutropenia in pediatrics, primarily affecting children younger than 3 years of age. The ANC at presentation is usually below 250/µL. Associated monocytosis is common and antineutrophil antibodies can be detected in most patients.²⁵ Other causes of neutropenia should be excluded, such as immunodeficiency, drug-related, transient postinfectious, and congenital neutrophil disorders. Although the ANC is often extremely low or absent, most children experience only minor infections and thus, this condition is sometimes referred to as chronic benign neutropenia of childhood. Nonetheless, empiric, broad-spectrum parenteral antibiotics are recommended for the first few episodes of fever. If a child appears to have a benign course, subsequent febrile episodes might be managed more routinely unless there is documented infection or signs of sepsis. Daily trimethoprim/sulfamethoxazole may be useful in preventing recurrent minor bacterial infections. G-CSF is usually effective in low doses (1 to 2 µg/kg/day) and should be considered for children with recurrent, severe neutropenic complications. Spontaneous remission within the first few years of diagnosis is common, especially in young children.

Cyclic neutropenia is characterized by periodic oscillations in the ANC. Cycles commonly occur every 21 days and the nadir is usually below 200/µL. Symptoms typically begin in the first year of life and commonly include recurrent fever, gingivitis, stomatitis with oral aphthous ulcers and pharyngitis. Diagnosis is confirmed by monitoring serial CBCs twice per week for 6 to 8 weeks to establish the periodicity of the neutropenia, which may also be accompanied by asymptomatic oscillations in other blood counts. Cyclic neutropenia is an autosomal dominant disorder caused by mutation in the gene encoding neutrophil elastase (ELANE). Parental history and/or CBCs may be helpful. Although in most cases cyclic neutropenia is a benign condition, serious infectious complications may occur and treatment with G-CSF is sometimes indicated. Low doses of G-CSF often suffice, and doses of 2 to 3 µg/kg daily or every other day can be titrated to maintain the neutrophil count above 500/µL.

Severe congenital neutropenia (Kostmann disease) is a disorder associated with severe, chronic neutropenia from birth. The ANC is usually less than 200/µL and recurrent bacterial infections are common, including those seen in more benign neutropenic conditions, as well as life-threatening sepsis, meningitis, and gastrointestinal tract infection. Neonates may present with omphalitis. Bone marrow examination reveals neutrophil developmental arrest. Inheritance is most often due to autosomal recessive mutations in ELANE, although other genetic defects and inheritance patterns are well described.²⁶ Standard therapy consists of daily G-CSF, but high doses may be required and patients undergoing long-term therapy are at risk for myelodysplastic syndrome and acute myelogenous leukemia. SCT should be considered as a curative approach.

Shwachman-Diamond syndrome and Chédiak-Higashi syndrome are both autosomal recessive constitutional neutropenias. Shwachman-Diamond, caused by compound heterozygous or homozygous mutations in the SBDS gene, is characterized by progressive marrow failure, pancreatic exocrine insufficiency, short stature, skeletal deformities, and predisposition to myelodysplastic syndrome and acute myeloid leukemia. Two-thirds of patients have a moderate neutropenia that can be intermittent and responsive to G-CSF. Chédiak-Higashi is also a multiorgan disease that includes oculocutaneous albinism, recurrent bacterial infection, a mild bleeding disorder due to platelet dysfunction, and neuropathy. The accumulation of giant granules in neutrophils leads to premature destruction. This disorder is caused by homozygous or compound heterozygous mutations in the lysosomal trafficking regulator gene (LYST or CHS1). Affected individuals commonly progress to an accelerated phase characterized by hemophagocytic lymphohistiocytosis, which is often fatal. Therapy is mostly supportive and SCT is the only known cure for the hematologic manifestations.

Other causes of neutropenia include drug-induced, inborn errors of metabolism, nutritional deficiency, or bone marrow infiltration. Treatment should be directed at the underlying etiology.

LEUKOCYTOSIS

Leukocytosis refers to an increase in total WBC count for age. Neutrophilia is an increase in the ANC above 7,500/µL, but the upper limit of normal may be higher in newborns and infants (Table 10.1). Neutrophilia may result from increased production, mobilization from the bone marrow, or peripheral demargination. In children, acute neutrophilia is most often due to bacterial or viral infection. Absolute lymphocytosis usually indicates an acute or chronic viral process. Evaluation of leukocytosis should include a detailed history and physical examination directed to symptoms and signs of infection and for lymphadenopathy and hepatosplenomegaly. Examination of the peripheral blood smear is essential to distinguish normal from atypical and malignant WBCs.

Leukocyte adhesion deficiency type I (LAD I) is a disorder of impaired phagocyte adhesion, chemotaxis, and ingestion, caused by partial or total deficiency of CD18-related surface glycoproteins due to deficiency of the gene encoding the beta-2 integrin chain (ITGB2).²⁷ The hallmark of this disorder is the occurrence of repeated, severe bacterial or fungal infections in the absence of pus despite persistent neutrophilia. Infants typically present in the newborn period with omphalitis or delayed umbilical cord separation. The diagnosis of LAD can be confirmed by flow cytometry, which reveals deficiency of surface expression of the CD18/CD11 molecules. Treatment is supportive, consisting primarily of prophylaxis against and treatment of infection. SCT may be curative, and more recently investigational gene therapy has been studied.

Infectious mononucleosis (IM) is classically associated with atypical lymphocytosis and is caused by infection with Epstein-Barr virus (EBV). In adolescents and young adults, a prodrome of fatigue and anorexia usually precedes development of fever, lymphadenopathy, exudative pharyngitis, and hepatosplenomegaly. Young children commonly present with a mild respiratory illness only. A muco-cutaneous eruption can occur, especially after treatment with penicillin or ampicillin. Hematologic complications, including immune-mediated hemolytic anemia, thrombocytopenia, aplastic anemia, and hemophagocytosis can be seen, as many other rare complications of CNS involvement, myocarditis, orchitis, and splenic rupture. Children with acquired or congenital immunodeficiency states can develop EBV-associated lymphoproliferative syndrome, which can evolve into non-Hodgkin’s lymphoma. EBV in boys with X-linked lymphoproliferative syndrome, results in fulminant IM, hemophagocytic lymphohistiocytosis, lymphoma, and/or other severe EBV-associated complications that are fatal in the majority of cases.²⁸

Atypical lymphocytosis and early immunologic evidence of EBV infection, either by heterophil or EBV-specific antibody testing, are the most consistent laboratory findings. Nonspecific heterophil antibody tests are often negative in children younger than 4 years of age. Other infections, such as cytomegalovirus (CMV), pertussis, and cat-scratch disease, should be excluded in the setting of EBV-negative IM.

Therapy of IM is supportive. Rarely, short course corticosteroids are used to manage life-threatening manifestations, such as upper airway obstruction from tonsillar/adenoidal hypertrophy. To reduce the risk of splenic rupture, contact sports should be avoided until splenomegaly resolves.

HEMATOLOGIC MANIFESTATIONS OF SYSTEMIC CONDITIONS

Many systemic conditions can result in secondary hematologic abnormalities. Evaluation of the CBC and peripheral smear may also provide important clues during the evaluation of a diagnostic dilemma. Systemic disorders that have prominent hematologic findings and present predominantly in childhood are detailed below.

Lysosomal storage diseases are caused by a deficiency in specific enzymes of the lysosomal metabolic pathway and result in pathologic accumulation of normal substrate, leading to dramatic changes in the central nervous and hematologic systems as well as enlargement of organs that comprise the reticuloendothelial system, including the liver and spleen. Vacuolated lymphocytes and hypergranulated neutrophils in the peripheral blood and lipid-laden macrophages (“foam” or “storage” cells) in the bone marrow are commonly observed. Other characteristic cell types include the sea-blue histiocyte in Niemann-Pick disease and the Gaucher cell. Specific enzyme replacement therapy is available for some of these disorders, while therapy is only supportive for others and SCT curative in certain conditions.²⁹

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of early childhood caused by defective lymphocyte apoptosis.³⁰ Symptoms include lymphadenopathy, splenomegaly, autoimmunity, and an increased risk of lymphoid malignancy. Autoimmune cytopenias are common. The presence of increased numbers of double negative (CD4−/CD8−) T-cells on flow cytometry supports the diagnosis. A number of molecular defects have been identified, most commonly mutations in Fas (TNFRSF6, CD95). Therapy is mainly supportive, although immunosuppressive drugs may be needed to manage complications of autoimmunity and lymphoproliferation. SCT can be curative.

Collagen vascular diseases commonly have hematologic manifestations, most often anemia of chronic illness and/or autoimmune-mediated cytopenias. Aplastic anemia has been described in SLE. Patients with autoimmune disorders are at increased risk for developing antiphospholipid antibodies, although such lupus anticoagulants result in prolongation of prothrombin time (PT) and PTT, they predispose to thromboembolism rather than bleeding.

TRANSFUSION SUPPORT

The indications for transfusion in infants and children are similar to those in adults. Patient size, blood volume, and underlying condition mandate special precautions in regard to dosing and risks. In all cases, careful consideration should be given to the indication for, appropriate dose of, and potential toxicities of the specific blood product. Formulas to calculate pediatric transfusion requirements are detailed in Table 10.11.

Packed red blood cells (PRBCs) should be transfused in children according to age-specific blood volumes and target Hb levels. Unless rapid replacement is required for shock or rapid loss, the recommended infusion rate is 2 to 4 mL/kg/hour or 10 to 15 mL/kg aliquots over 4 hours. With volume intolerance, gradual correction can be achieved by infusing small aliquots (5 to 10 mL/kg) over 4 to 6 hours. Diuretics may be helpful. When rapid correction is required but is limited by fluid intolerance, partial exchange transfusion can be utilized with whole blood removed in small aliquots and replaced with equal volumes of PRBCs.

Platelets transfused at a dose of 0.1 U/kg are expected to increase the platelet count by approximately 50,000/µL in most infants and children. The target post-transfusion platelet count varies with the clinical scenario. In general, the aim should be to raise the count to a level at which bleeding stops. Values of around 50,000/µL usually suffice, although normal counts should be maintained for life-threatening circumstances, such as CNS, vascular, or surgical hemorrhage. In the setting of myelosuppression without additional risk factors for severe bleeding, prophylactic platelet transfusion is recommended at a level of 10,000/µL. To minimize the risk of bleeding in newborns, standard recommendations are to maintain the platelet count above 30,000/µL for full-term and 50,000/µL for premature infants. Prophylactic transfusions are not indicated in the setting of ITP and other antibody-mediated forms of platelet destruction where no transfusion-related increment is expected; rather, transfusions should be reserved for life-threatening bleeding. To decrease the risk of alloimmunization in children who require multiple transfusions, single-donor (apheresis) and leukocyte-depleted platelets should be used whenever possible.

FFP is recommended for children with coagulopathy, as evidenced by prolonged PT and/or PTT, who have active bleeding or to prevent hemorrhage in those at high risk (e.g., preoperatively). FFP should be used to replace clotting factors for which specific concentrates are not available and a dose of 10 to 15 mL/kg usually raises the clotting activity by approximately 20%. Multiple doses may be required if there is ongoing consumption. The rate of transfusion is limited by citrate toxicity, and vital signs and ionized calcium levels should be monitored closely when large or rapid infusions are used.

Cryoprecipitate is used primarily to combat bleeding with hypofibrinogenemia. A dose of 0.3 U/kg will increase the fibrinogen level by approximately 200 mg/dL.

Specialized Blood Products to Prevent Toxicities

WBC removal by leukofiltration should be performed for recipients of blood products who require multiple transfusions in order to decrease the risk of sensitization to leukocyte antigens. Leukodepletion also decreases the risks of febrile reactions and CMV transmission. Irradiation of cellular blood products with 2,500 cGy should be used to prevent transfusion-associated graft-versus-host disease in the following situations: (i) potential immunocompromised host, including very low birth weight infants, immunodeficiency, malignancy, marrow or organ transplantation; (ii) blood from first-degree family members or HLA-matched donors; and (iii) all granulocyte transfusions.

References

  1.  Orkin SH, Fisher DE, Nathan DG, Ginsburg D, Look AT, eds. Nathan and Oski’s Hematology of Infancy and Childhood. 7th ed. Philadelphia, PA: Elsevier Health Sciences; 2009.

  2.  Baker RD, Greer FR; Committee on Nutrition American Academy of Pediatrics. Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age). Pediatrics. 2010;126(5):1040-1050.

  3.  Kohli-Kumar M. Screening for anemia in children: AAP recommendations—a critique. Pediatrics. 2001;108(3):E56.

  4.  Iron. In: Kleinman RE, ed. Pediatric Nutrition Handbook. 6th ed. Elk Grove Village, IL: American Academy of Pediatrics, Committee on Nutrition; 2009:403-422.

  5.  Recommendations to prevent and control iron deficiency in the United States. MMWR 1998;47(No. RR-3).

  6.  Arcara K, Tschudy M, eds. The Harriet Lane Handbook: A Manual for Pediatric House Officers. 19th ed. Philadelphia, PA: Elsevier Mosby Publishers; 2011.

  7.  Woolf AD, Goldman R, Bellinger DC. Update on the clinical management of childhood lead poisoning. Pediatr Clin North Am. 2007;54(2):271-294.

  8.  Earley A, Valman HB, Altman DG, Pippard, MJ. Microcytosis, iron deficiency, and thalassaemia in preschool children. Arch Dis Child. 1990;65:610-614.

  9.  Rachmilewitz E and Giardina P. How I treat thalassemia. Blood. 2011;118(13):3479-3488.

10.  Cherrick I, Karayalcin G, Lanzkowsky P. Transient erythroblastopenia of childhood. Prospective study of fifty patients. Am J Pediatr Hematol Oncol. 1994;16:320-324.

11.  The Management of Sickle Cell Disease. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, National Heart, Lung, and Blood Institute. NIH Publication No. 02-2117. 4th ed. 2002. http://www.nhlbi.nih.gov/guidelines/current.htm

12.  Section on Hematology/Oncology and Committee on Genetics. Health supervision for children with sickle cell disease. Pediatrics. 2002;109:526-535.

13.  Adams RJ, McKie VC, Hsu L, et al. Prevention of first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial doppler ultrasonography. N Engl J Med. 1998;339:5-11.

14.  Scothorn DJ, Price C, Schwartz D, et al. Risk of recurrent stroke in children with sickle cell disease receiving blood transfusion therapy for at least five years after initial stroke. J Pediatr. 2002;140(3):348-354.

15.  Wang WC, Ware RE, Miller ST, et al. Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG). Lancet. 2011;377(9778):1663-1672.

16.  Rosenblatt DS, Whitehead VM. Cobalamin and folate deficiency: acquired and hereditary disorders in children. Semin Hematol. 1999;36:19-34.

17.  Boria I, Garelli E, Gazda HT, et al. The ribosomal basis of Diamond-Blackfan Anemia: mutation and database update. Hum Mutat. 2010;31(12):1269-1279.

18.  Vlachos A, Federman N, Reyes-Haley C, Abramson J, Lipton JM. Hematopoietic stem cell transplantation for Diamond Blackfan anemia: a report from the Diamond Blackfan Anemia Registry. Bone Marrow Transplant. 2001;27:381-386.

19.  Rosenberg PS, Greene MH, Alter BP. Cancer incidence in persons with Fanconi anemia. Blood. 2003;101:822-826.

20.  Sutor AH, von Kries R, Cornelissen EA, McNich AW, Andrew M. Vitamin K deficiency bleeding (VKDB) in infancy. ISTH Pediatric/Perinatal Subcommittee. International Society on Thrombosis and Haemostasis. Thromb Haemos. 1999;81:456-461.

21.  Report of Joint WHO/WFH Meeting on the Control of Haemophilia: Delivery of Treatment for Haemophilia. World Health Organization, 2002. http://www.who.int/genomics/publications/reports/en/index.html

22.  The Diagnosis, Evaluation, and Management of von Willebrand Disease. U.S. Department Of Health And Human Services, National Institutes of Health, National Heart, Lung, and Blood Institute. NIH Publication No. 08-5832. 2007. http://www.nhlbi.nih.gov/guidelines/current.htm

23.  Neunert C, Lim W, Crowther M, et al. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood. 2011;117(16):4190-4207.

24.  Goldenberg NA. Thrombophilia states and markers of coagulation activation in the prediction of pediatric venous thromboembolic outcomes: a comparative analysis with respect to adult evidence. Hematology Am Soc Hematol Educ Program. 2008:236-244.

25.  Kobayashi M, Nakamura K, Kawaguchi H, et al. Significance of the detection of antineutrophil antibodies in children with chronic neutropenia. Blood. 2002;99:3468-3471.

26.  Ward AC, Dale DC. Genetic and molecular diagnosis of severe congenital neutropenia. Curr Opin Hematol. 2009;16(1):9-13.

27.  Etzioni A. Defects in the leukocyte adhesion cascade. Clin Rev Allergy Immunol. 2010;38(1):54-60.

28.  Rezaei N, Mahmoudi E, Aghamohammadi A, Das R, Nichols KE. X-linked lymphoproliferative syndrome: a genetic condition typified by the triad of infection, immunodeficiency and lymphoma. Br J Haematol. 2011;152(1):13-30.

29.  Boelens JJ, Prasad VK, Tolar J, Wynn RF, Peters C. Current international perspectives on hematopoietic stem cell transplantation for inherited metabolic disorders. Pediatr Clin North Am. 2010;57(1):123-145.

30.  Oliveira JB, Bleesing JJ, Dianzani U, et al. Revised diagnostic criteria and classification for the autoimmune lymphoproliferative syndrome (ALPS): report from the 2009 NIH International Workshop. Blood. 2010;116:e35-40.

11

Acute Myeloid Leukemia

Fang Yin and Vera Malkovska

Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation of myeloid progenitor cells that gradually replace normal hematopoiesis in the bone marrow. The genetic changes arising in the neoplastic clone lead to cascades of molecular events that cause abnormal proliferation and aberrant differentiation of the malignant cells and ultimately result in inhibition of normal hematopoiesis.

Characterization of transforming genetic events is becoming increasingly important in establishing diagnosis, defining prognosis, and planning therapy in AML. Aggressive chemotherapy with optimal supportive treatment has improved outcomes in younger patients with AML. Most of these patients achieve a complete remission (CR) but many relapse and their 5-year survival remains below 50% in large studies.¹ Patients over the age of 60 have a median survival of less than a year and long-term survival rates below 10%. Both the unfavorable biology of AML and poor tolerance of chemotherapy in the elderly are to blame.² The current challenge is to improve the understanding of the molecular mechanisms of AML and design leukemia-specific treatments that are effective in chemotherapy-resistant disease and applicable to older patients.

EPIDEMIOLOGY

The age-adjusted incidence rate of AML in the United States is 3.5 per 100,000, accounting for approximately 10,000 deaths per year. AML accounts for about 15% to 20% of acute leukemias in children and adolescents and 80% in adults. The incidence of AML rises rapidly after the age of 60 and the median age at diagnosis is 67 years (Fig. 11.1).¹⁻³

ETIOLOGY

The molecular origins of AML are unknown. The pathophysiologic mechanisms are multiple, act in concert, and are distinct in different types of AML. Inherited genetic predisposition and environmental mutagens such as radiation, drugs, and other toxins all play a role in the development of AML.⁴ Genetic causes are suggested by the increased incidence of AML in identical twins, as well as the known association of AML with a variety of congenital disorders. Genetic predisposition is probably important in the development of AML in children and young adults while it has not been shown to play a major role in older adults. A large European population-based study found no significant familial aggregation for AML or myelodysplastic syndromes (MDS).⁴ These findings suggest that in most patients with AML/MDS environmental factors play a more important role than germline mutations. AML arising from pre-existing hematologic disorders, most commonly MDS or myeloproliferative disorders, have inferior prognosis. AML following exposure to chemotherapy and radiation is characterized by resistance to treatment and short survival.

FIGURE 11.1 The age-related incidence of AML in the United States.

Known risks factors for the development of AML are shown in Table 11.1.

PATHOGENESIS

At the molecular level, pathogenesis of AML is a complex multistep process that results from the interaction of two different classes of mutations. The first class of mutations impairs cell differentiation resulting in clonal expansion of myeloid progenitors. The second class causes abnormal cell proliferation by constitutive activation of cellular proto-oncogenes including FLT3 tyrosine kinase, RAS, c-KIT, and others. The silencing of tumor suppressor genes also contributes to the pathogenesis. Some molecular genetic alterations in AML are highlighted by distinct chromosomal changes including translocations, inversions, and deletions while others can be only identified by molecular analysis. The common mutations found in AML that are thought to cooperate in the malignant transformation are shown in Table 11.2.⁵ The analysis of these cytogenetic and molecular changes is used to predict clinical outcomes and to formulate treatment paradigms in AML. Studies are underway to understand the downstream molecular pathways triggered by these mutations that lead to the unrestricted growth of leukemic cells and suppression of normal hematopoiesis by the malignant clone. Whole genome sequencing of AML cells and their normal counterparts opens the possibility of identifying all pathogenetic mutations and using them for disease classification and therapy.

CLINICAL FEATURES

Patients with AML usually present with bone marrow failure that causes symptoms of anemia, bleeding from thrombocytopenia, and neutropenic infections. Tissue infiltration with leukemic blasts involving gums, skin, meninges, and other organs is most commonly associated with monocytic morphology. Striking bruising and life-threatening hemorrhage should raise suspicion of disseminated intravascular coagulation (DIC) frequently seen in acute promyelocytic leukemia (APL). However, DIC can occur in any type of AML. Leukostasis and hyperviscosity causing organ dysfunction usually occur with blast cell counts over 100,000/µL. Manifested by confusion, visual impairment, and shortness of breath, leukostasis can also lead to hemorrhage in the retina, brain, lungs, and other organs. Rare but striking manifestations of AML include the Sweet syndrome, a skin rash with neutrophilic infiltrates in the dermis, and chloromas, tumors of myeloid blasts. Extramedullary leukemia portends a worse prognosis.

Table 11.1  Risk Factors for Development of Acute Myeloid Leukemia

Environmental Exposures

Benzene

Ionizing radiation

Smoking

Genetic Disorders

Down syndrome

Bloom syndrome

Fanconi anemia

Dyskeratosis congenita

Ataxia telangiectasia

Li-Fraumeni syndrome

Kostmann syndrome

Klinefelter syndrome

Pre-Existing Hematologic Disorders

Myelodysplastic syndromes (MDS)

Myeloproliferative disorders

Aplastic anemia

Treatment-Associated

Alkylating agents: AML usually arises from MDS, after a 3–10-yr latency period and is associated with deletions involving chromosomes 5 or 7.

Topoisomerase II inhibitors: AML lacks preceding myelodysplasia, has a shorter latency (1–3 yr), exhibits monocytic morphology, and is associated with changes involving the long arm of chromosome 11 (11q23).

Radiotherapy alone or in combination with chemotherapy

Symptoms and signs on presentation include the following:

Marrow failure

  Fatigue

  Shortness of breath

  Fever

  Focal bacterial infections

  Petechiae

  Bruising

  Bleeding (if severe, suspect promyelocytic leukemia)

Tissue involvement

  Bone pain, tenderness

  Moderate splenomegaly

  Gingival hyperplasia

  Central nervous system (CNS) and cranial nerve dysfunction

  Visual changes (retinal involvement, hemorrhage, papilledema)

Rare manifestations

  Sweet syndrome

  Chloromas

LABORATORY FINDINGS

The most common laboratory findings in AML include anemia, thrombocytopenia, neutropenia, and myeloid blasts on the blood smear. The blasts have distinct immunophenotypes detected by flow cytometry. In aleukemic leukemia, blasts are seen only in the bone marrow. Coagulopathy resulting from DIC is common in promyelocytic leukemia. Hyperuricemia from high cell turnover is often seen on presentation and worsens during chemotherapy. Rapidly rising serum levels of uric acid, potassium, and phosphate with decreasing calcium herald a tumor lysis syndrome that can result in acute renal failure. Renal tubular dysfunction caused by muramidase released from leukemic blasts can add to the electrolyte abnormalities commonly seen in AML. Lactic acidosis tends to occur with leukostasis while high lactate dehydrogenase (LDH) is associated with CNS involvement. High numbers of leukemic blasts in blood samples can lead to spurious hypoglycemia, hypoxemia, hypokalemia, and other abnormalities resulting from cellular metabolic activity in vitro. Rapid processing of anticoagulated blood samples avoids this artifact.

In addition to routine chest X-rays, imaging studies including computerized tomography (CT) and magnetic resonance imaging (MRI) scans directed according to symptoms can reveal leukemic infiltrates, hemorrhage, or infection.

Laboratory findings in AML include the following:

Hematologic

  Increased white blood cell count with blasts in peripheral blood

  Anemia

  Granulocytopenia

  Thrombocytopenia

  DIC

Chemistry

  Hyperuricemia

  Elevated blood urea nitrogen and creatinine (urate nephropathy)

  High LDH

  Hypokalemia (tubular dysfunction)

  Lactic acidosis (leukostasis)

  Hypercalcemia, rarely hypocalcemia

  Spurious hypoxemia, hypoglycemia, hyperkalemia or hypokalemia

Imaging studies

  Intracranial hemorrhage (often with hyperviscosity) (CT)

  Thickened nerve sheaths (MRI)

  Lung infiltrates (CT)

CLASSIFICATION

The classification of AML has evolved from the mainly morphology-based French-American-British (FAB) to the more comprehensive World Health Organization (WHO) classification. The older FAB classification based on morphology, cytochemical staining, and immunophenotype of the predominant cells divides AML into eight subtypes (M0–M7) (Tables 11.3 and 11.4).

The WHO classification is based on the concept that different subgroups of AML can be defined as unique diseases through correlation of morphology, cytochemistry, immunophenotype, genetics, and clinical features.⁶ In this classification, diseases associated with specific genetic abnormalities, namely AML with t(8;21)(q22;q22), AML with inv(16)(p13,1q22) or t(16;16)(p13.1;q22), and APL with t(15;17) (q22;q12), can be diagnosed as AML regardless of the blast cell count. In all other entities within the category of AML with recurrent genetic abnormalities, 20% or more blasts must be present in the blood or bone marrow to establish the diagnosis of AML. The category “AML with myelodysplasia-related changes” includes disease that evolves from previously documented MDS, AML with myelodysplasiaassociated cytogenetic abnormalities (e.g., −7 or −5), or AML that exhibits dysplasia in 50% or more of the cells in two or more myeloid lineages.⁶

Table 11.4  World Health Organization Classification of Acute Myeloid Leukemias (AMLs)

AML with Recurrent Cytogenetic Translocations

AML with t(8;21)(q22;q22); RUNX1-RUNX1T1

AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB- MYH11

APL with t(15;17)(q22;q12); PML-RARA

AML with t(9;11)(p22;q23); MLLT3-MLL

AML with t(6;9)(p23;q34); DEK-NUP214

AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1

AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1

AML with mutated NPM1

AML with mutated CEBPA

AML with Myelodysplasia-Related Changes

Therapy-Related Myeloid Neoplasms

AML, not Otherwise Specified

AML with minimal differentiation

AML without maturation

AML with maturation

Acute myelomonocytic leukemia

Acute monoblastic/monocytic leukemia

Acute erythroid leukemia

Acute megakaryoblastic leukemia

Acute basophilic leukemia

Acute panmyelosis with myelofibrosis

Myeloid Sarcoma

Myeloid Proliferations Related to Down syndrome

Transient abnormal myelopoiesis

Myeloid leukemia associated with Down syndrome

Acute Leukemias of Ambiguous Lineage

APL, acute promyelocytic leukemia.

Modified from Vardiman JW,Thiele J, Arber DA, et al.The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937-951.

The WHO classification of AML continues to evolve and incorporate newly detected molecular mutations with a defined impact on disease biology. The hope is that continuing refinement of the classification into biologically distinct entities will define prognosis and help guide the type and intensity of therapy.

DIAGNOSTIC EVALUATION

Leukemic myeloblasts are usually seen on the blood smear and are always found in the bone marrow biopsy. According to the WHO consensus, the diagnosis of AML requires at least 20% myeloid blasts in the peripheral blood or bone marrow. The evaluation of a patient with AML at diagnosis includes the following:

  Blood count and inspection of blood smear

  Bone marrow aspirate and biopsy

  Morphology with Wright-Giemsa stain

  Immunophenotyping by multiparameter flow cytometry

  Cytogenetics

  Molecular analysis

  Lumbar puncture (after blasts cleared from blood): if CNS symptoms, monocytic morphology or high blast count

The morphologic diagnosis of AML can be supported by the presence of Auer rods in the cytoplasm, positive cytochemistry with Sudan black, and staining for myeloperoxidase and esterases. Immunophenotyping with a panel of monoclonal antibodies is particularly useful for distinguishing AML from acute lymphoblastic leukemia (ALL) and for identification of the subtypes, including AML with minimal differentiation, erythroleukemia, and megakaryoblastic leukemia. Cytogenetic and molecular abnormalities associated with the morphologic subtypes can further support the diagnosis and help predict treatment outcomes (Table 11.5).

PROGNOSTIC FACTORS

The most powerful prognostic factors that have been established over decades including age over 60, cytogenetics, prior MDS, and treatment-related AML have been recently supplemented by molecular genetic abnormalities.⁶ The independent prognostic variables associated with treatment outcomes are listed below:

Clinical prognostic factors

  Age

  AML arising from pre-existing MDS

  Treatment-related AML

  Performance status

  Extramedullary disease

  Comorbid conditions

Laboratory-based prognostic factors

  White blood cell count >20,000/μL at presentation

  Cytogenetics

  Molecular genetic changes (mutations in nucleophosmin 1 (NPM1), FLT3, CCAAT enhancer binding protein alpha (CEBPA), KIT)

  CD34 positive blasts

  Multidrug resistance

The best predictors of long-term outcome next to age are chromosomal and molecular genetic findings in leukemic cells (Tables 11.6 and 11.7).6,7 Cytogenetic analysis is the basis of stratification of patients into three risk groups with different responses to chemotherapy (Table 11.3). For example, patients in the better-risk group with core-binding factor mutations are most likely to obtain a long remission after consolidation with high-dose cytosine arabinoside while patients with poor-risk cytogenetics do not benefit from this treatment. Approximately 40% to 50% of all patients have a normal karyotype at the time of diagnosis and most of them fall into the intermediate-risk category (Table 11.3). The molecular genetic changes in cytogenetically normal AML that correlate with prognosis have been used to subdivide this group and create more refined treatment algorithms.⁷ The frequency and prognostic impact of gene mutations in AML with normal karyotype are listed in Table 11.7.7,8 Some of these mutations including NPM1, FLT3, CEBPA, and KIT already impact therapy while the rest are under investigation. The best-studied mutations, the internal tandem duplications (ITD) of the FLT3 gene which codes for a tyrosine kinase receptor, are found in approximately 20% to 30% of AML patients. Several retrospective studies have demonstrated that patients harboring such mutations respond poorly to treatment and have significantly shorter disease-free survival (DFS) and overall survival (OS).⁷ By contrast, in the absence of FLT3 mutations patients with NPM1 and CEBPA mutations have improved CR rates and OS (Table 11.6).⁷ These mutations provide substantial insights into the pathogenesis of AML and help identify molecular targets for treatment. Studies of wholegenome sequences, mircoRNA, and gene expression in AML cells may alter prognostic categories in the near future.7,9

The outcome of younger patients with AML has markedly improved over the last three decades, due to advances in both chemotherapy and supportive care. Over 50% of patients under the age of 60 in the favorable prognostic category can be cured with current chemotherapy. The cure rates of younger patients without better-risk chromosomal and molecular markers are about 30% to 40%. Unfortunately, little progress has been made in the long-term survival of older adults with AML. These patients have more poor-risk disease characteristics, higher frequency of comorbid conditions, and poor tolerance of toxic therapy. Novel treatment strategies are needed for the majority of older patients.2,10

TREATMENT

Because AML is strikingly heterogeneous, its treatments must be individualized. Treatment plans in AML patients depend on age, performance status, and prognostic factors detected by conventional cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction (PCR).7,8 The first example of a targeted treatment directed against a specific mutation is the successful use of all-trans-retinoic acid (ATRA) for APL, which is discussed separately in this chapter.

The treatment for AML is generally divided into two phases: remission induction and postremission therapy. The goal of the former is to achieve a CR defined by the following criteria: less than 5% blasts in a bone marrow that is 20% or more cellular, absent extramedullary leukemia, a neutrophil count greater than 1,000/μL, and a platelet count greater than 100,000/μL. The achievement of CR as defined by these simple criteria translates into improved survival. Disappearance of karyotypic or molecular abnormalities is not required for the definition of CR. It is therefore not surprising that clinical trials confirm an almost 100% risk of relapse when patients receive only induction chemotherapy. Long-term survival requires postremission treatment. Intensive chemotherapy given after achievement of CR (similar to that given during induction) is termed consolidation therapy.

Initial Management

The early management of AML patients should be well organized and implemented by an experienced team.

The initial evaluation should include the following:

  History and physical exam

  Complete blood count with differential

  Examination of the peripheral blood smear

  Coagulation studies: prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimer

  Serum chemistries with uric acid, calcium, and phosphorus

  Evaluation of renal and liver functions

  Hepatitis B and C, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella, and human immunodeficiency virus (HIV) serologies

  Pregnancy test in females of reproductive age

  Bone marrow aspirate for morphology, cytochemistry, cytogenetics, molecular genetic studies, and flow cytometry

  Bone marrow biopsy

  Human leukocyte antigen (HLA) typing of patients who are candidates for hematopoietic stem cell transplant

  Lumbar puncture delayed until blasts cleared from blood, in patients at high risk for CNS involvement (CNS symptoms, elevated leukocyte count, extramedullary disease, and monocytic morphology FAB M4 or M5)

  Chest radiograph and electrocardiogram

  Evaluation of cardiac function by echocardiogram or multigated acquisition scan (MUGA) in selected patients

  Central venous-access catheter placement

The workup should be followed by an unhurried discussion with the patient about the diagnosis, prognosis, side effects of therapy, probable impact on life style, and anticipated requirements for support by family and friends. In frail elderly patients, a decision to give only supportive treatment without chemotherapy may be reached jointly by the patient and the attending physician.

The final outcome depends not only on the choice of chemotherapy but also on close monitoring, prevention, and meticulous management of complications. Many events are associated with AML therapy and their timing is predictable. For example, hyperleukocytosis, tumor lysis, and DIC tend to occur early while marrow aplasia with resulting complications can be expected from the second week of chemotherapy. Patients should be monitored for side effects such as cardiotoxicity due to anthracyclines or neurotoxicity from high doses of cytosine arabinoside. Infection prophylaxis includes meticulous care for indwelling central venous catheters and prevention of mucositis. Empirical broad spectrum intravenous antibiotics should be administered immediately if the patient becomes febrile during neutropenia. Cultures should be obtained before administration of antibiotics. Prompt treatment or prophylaxis of oral and perianal herpetic ulcerations prevents discomfort and bacterial superinfection. Prophylaxis against herpes viruses can prevent these complications and decrease the severity of mucositis. Prophylactic antifungal therapy may lead to decrease in fungal infections. Transfusion support with irradiated and leukodepleted blood products should be provided to prevent symptoms of severe anemia and to maintain the platelet count above 10,000/μL. Transfusions of blood products in AML are guided by the same principles as described for ALL (Chapter 12).

Induction Therapy

The most commonly used chemotherapy regimen consists of one or two cycles of a combination of cytarabine 100 to 200 mg/m² given by continuous intravenous (IV) infusion over 7 days and 3 days of an anthracycline (e.g., daunorubicin 60–90 mg/m², idarubicin 10–13 mg/m², or mitoxantrone 10–12 mg/m² by IV bolus). This “3 + 7” regimen results in CR rates of approximately 70% to 80% in patients younger than 55 to 60 years.¹⁰⁻¹² If leukemia persists in the bone marrow at day 14 to 21, a second identical or modified course of chemotherapy is usually given.

Several recent studies demonstrated that high-dose daunorubicin at 90 mg/m²/day for 3 days is well tolerated and results in improved CR rates and a significantly higher OS, especially in younger adults under the age of 50 or 65 years and those with better-risk or intermediate-risk cytogenetics. Therefore, high-dose daunorubicin is now considered a new standard care in the initial treatment of patients <65 years of age with a good performance status and adequate cardiac function.¹² Idarubicin could be an alternative to daunorubicin, because a recent study showed no difference in outcome between high-dose daunorubicin and standard-dose idarubicin.¹³

Elderly patients with no comorbidities have a better outcome with intensive chemotherapy induction than with supportive care.14,15 Clofarabine is active as a single agent and is currently explored as part of induction regimens for elderly patients.¹⁶ In older patients unlikely to benefit from chemotherapy, agents such as azacitidine, decitabine, and lenalidomide, may prolong survival even in the absence of CR. Novel agents including tipifarnib, sapacitabine, and lenalidomide are being tested in this population. The goals and end points of therapy for elderly patients are therefore shifting.

Further strategies to intensify induction chemotherapy include the addition of high-dose cytarabine, etoposide, 6-thioguanin, double induction and timed sequential therapy, priming and growth-factor support. Randomized trials demonstrate benefit in DFS but not in OS with the above approaches that may cause additional toxicities.

The anti-CD33 immunoconjugate, gemtuzumab ozogamicin, used in combination therapy shows promising outcome in de novo AML.17,18 In a recent prospective phase III trial, patients were randomized to receive a traditional induction 3 + 7 regimen or to receive the same regimen in combination with fractionated small doses of gemtuzumab. The trial demonstrated no significant difference in fatal adverse events between the two arms, and a highly significant advantage in event-free survival and DFS at 2 years for patients who were treated on the gemtuzumab arm. This translated into a 10-month OS advantage for the combination arm.¹⁷ Another recent randomized study from the UK showed similar results in AML patients age greater than 60.¹⁸ Though gemtuzumab was withdrawn from the US market in June 2010 due to concerns of toxicity, the above results are encouraging. In the United States, further studies can be conducted with gemtuzumab as an investigational new drug (IND).

Postremission Treatment

Nearly all patients in CR after induction therapy have residual disease that, without further treatment, leads to relapse. The main strategies to prevent relapse involve postremission treatment with high-dose cytarabine and allogeneic or autologous hematopoietic stem cell transplantation (HSCT). Maintenance therapy is not part of current standard treatment for AML. However, maintenance therapies including immunotherapy, demethylating agents, and targeted therapies are currently being tested in this disease with the intention of achieving leukemia cure.

Intensive consolidation treatment improves survival in younger patients with AML. A dose- dependent response to cytarabine has been shown in randomized controlled trials.¹¹ Consolidation with high-dose cytarabine (HDAC)-based therapy using daily doses of 1 to 6 g/m² (e.g., 2–3 g/m² twice daily on days 1, 3, and 5 or twice daily for 6 days) is the standard for patients younger than 60 years. HDAC appears to be most beneficial to younger patients with better-risk cytogenetics, especially those with core-binding factor mutations (i.e., t(8;21) and inv (16)). Patients over the age of 60 years do not benefit from HDAC consolidation.¹⁹ The optimal number of courses of HDAC-based consolidation has not been determined, but the evidence suggests that 1 to 4 courses are reasonable.20,21 For older patients, there are also studies that suggested no particular value in intensifying postremission therapy beyond a single course of consolidation.21,22 The optimal cumulative dose of cytarabine in the initial therapy of younger patients with AML is still an open issue. The reduction in cytarabine doses (cumulative dose not beyond 12 g/m² within first consolidation) during conventional chemotherapy did not appear to worsen treatment outcome.14,23 For younger AML patients, the choice between consolidation chemotherapy and allogeneic HSCT should be based on the risk of relapse.

Hematopoietic Stem Cell Transplantation

High doses of marrow-ablative chemotherapy and total body radiation followed by autologous or allogeneic stem cell rescue have been widely used in AML. Autologous HSCT (auto-HSCT) requires stem cell collection from the patient while in CR. Allogeneic stem cells are obtained from HLA-matched siblings, unrelated donors, and cord blood or more recently haploidentical donors. Allogeneic HSCT (allo-HSCT) confers an additional immune-mediated antileukemic activity, the so-called graft-versus-leukemia effect.

Multiple prospective randomized trials comparing standard consolidation chemotherapy with HSCT have demonstrated that allo-HSCT provides the best antileukemic therapy with the lowest risk of recurrence, followed by auto-HSCT, which in some studies is superior to conventional postremission chemotherapy (Table 11.8). Therefore, in patients lacking an available HLA-matched donor, auto-HSCT can offer a benefit over conventional consolidation chemotherapy. However, the OS has not improved in AML patients undergoing auto-HSCT compared to patients receiving intensive consolidation chemotherapy because of more opportunities for salvage with second-line chemotherapy and stem cell transplantation in patients relapsing on the chemotherapy arm.26,27

The powerful antileukemic activity of allo-HSCT has not always translated into better OS, due to the high treatment-related mortality (TRM) rate associated with allo-HSCT (Table 11.9). The availability of a donor has been used as a surrogate for randomization in these studies. These donor–no donor comparisons may be confounded by limited application of the assigned therapy, and the results of comparative studies have not always been consistent. Recent analyses demonstrate superior OS after allo-HSCT for AML patients excluding those with better-risk cytogenetics, and this benefit is most pronounced in younger patients.³⁰ A large meta-analysis of 24 prospective studies including over 6,000 patients with AML in first CR (CR1) compared the role of HSCT and non-HSCT treatments. Overall, 3,638 patients were analyzed by cytogenetic risk category, including 547 better-risk, 2,499 intermediate-risk, and 592 poor-risk patients. Compared with non-allogeneic therapy, the hazard ratio of relapse or death with an allo-HSCT for patients in CR1 was 0.80 (95% confidence interval 0.74–0.86). When the analysis was broken down by risk category and outcome, there was a significant relapse-free survival and OS benefit for allo-HSCT during CR1 in intermediate-risk and poor-risk AML patients, but not for better-risk AML patients.³¹

The role of myeloablative allo-HSCT is limited in older patients due to lower tolerance of intensive chemotherapy and significant comorbidities. Reduced-intensity conditioning (RIC) regimens have broadened the applicability of allotransplants to elderly patients and to younger patients with comorbidities who would not tolerate standard myeloablative conditioning regimens. Studies reveal that more than one-third of patients can achieve long-term survival with RIC transplantation.³² The best insight into the differences in outcome comes from large registry-based retrospective studies demonstrating that RIC is associated with a reduction in TRM but an increased risk of relapse. Patients undergoing RIC stem cell transplantation in remission have similar DFS and OS compared to myeloablative conditioning.³²

In the absence of an HLA-matched donor, alternative donors such as unrelated umbilical cord blood (UCB) or haploidentical donors are increasingly used. Both strategies have important advantages such as shorter time to transplant, which is particularly relevant to patients requiring urgent transplantation, and the availability of mismatched donors for virtually all patients. Recent studies have demonstrated similar leukemia-free survival after UCB transplantation compared to HLA-matched HSCT in patients with acute leukemia.33,34 The European Bone Marrow Transplant Registry (EBMTR) study also indicated that the haplo-HSCT results for poor-risk AML in CR are similar to those reported for unrelated donor transplants.³⁵ As both the transplantation technology and chemotherapy continue to improve, the best treatment approach remains a moving target. Since AML is a heterogeneous malignancy, the most appropriate therapy will be ultimately determined by the cytogenetic and molecular characteristics of the disease. With progress in understanding of the disease biology and identification of new molecular markers, current practice will continue to evolve.

Risk-Based Approach to Acute Myeloid Leukemia Treatment in Younger Patients

Better-Risk Acute Myeloid Leukemia

Patients with a better-risk karyotypes (core-binding factor AML with t(8;21) or inv(16), normal karyotype with an NPM1 mutation but no FLT3-ITD), or (with a CEBPA mutation) do well with either intensive consolidation chemotherapy or autologous HSCT (Table 11.6). Long-term DFS of 60% to 70% can be achieved with these approaches in younger patients. Allo-HSCT, with its higher TRM, is generally not used in this subset of patients outside clinical trials.10,31

Poor-Risk Acute Myeloid Leukemia

Patients with poor-risk cytogenetics or patients with normal karyotype and FLT3-ITD as well as patients with secondary AML have very poor prognosis (Table 11.6). If an HLA-matched donor is available, these patients should be evaluated for HSCT as soon as possible. The outcome remains dismal with conventional consolidation or autologous HSCT in this group of patients. Allo-HSCT from unrelated donors matched by high-resolution HLA-typing has comparable outcomes to transplants from HLA-matched family donors.¹⁹ Younger and healthier patients without an HLA-matched donor should be considered for allo-HSCT from a haploidentical family member, or UCB transplant. Patients with significant comorbidity who achieve CR can be offered allo-HSCT with RIC. New therapeutic agents given on clinical trials should be offered to the majority of patients without a donor. Patients with FLT3-ITD should be considered for clinical trials with FLT3 tyrosine kinase inhibitors such as midostaurin, lestaurtinib, sorafenib, and quizartinib.7,36

Intermediate-Risk Acute Myeloid Leukemia

Treatment decisions are particularly complex in the largest and most heterogeneous prognostic group of patients with normal cytogenetics, no molecular markers, or markers of unknown impact on therapy (e.g., IDH1 and IDH2, RUNX1, TET2, and DNMT3A mutations) (Tables 11.6 and 11.7). Allo-HSCT, consolidation chemotherapy, and auto-HSCT are considered of equivalent benefit. Recent meta-analysis revealed allogeneic HSCT has significant relapse-free survival and OS benefit for intermediate-risk AML in CR1 compared with non-allogeneic HSCT therapies.³¹ However, novel genetic lesions detected by new technologies, such as whole genome analyses, RNA and microRNA profiles continue to refine AML risk. Until outcome data are available for these new subgroups, treatment decisions should be carefully individualized based on patient age, comorbidity, type of transplant, and risk of relapse. If an HLA-matched family donor is available, allo-HSCT should be offered to patients less than 55 to 60 years of age with good performance status, given the superior antileukemic activity of this therapy. Auto-HSCT, following intensive consolidation for in vivo purging, can be offered to patients without an HLA-matched donor. In general, the benefit of allo-HSCT over auto-HSCT or HDAC consolidation will be seen in subgroups of younger healthier patients in whom the improvement in relapse risk outweighs the higher procedural mortality.

Treatment of Acute Promyelocytic Leukemia

APL is a well-defined disease entity with a distinct epidemiology, characteristic morphology, and potentially life-threatening coagulopathy at presentation but overall better prognosis than other AML subgroups. It is the first example of leukemia in which therapy directed against the leukemogenic event, the t(15;17) resulting in the PML-RARα fusion transcript, leads to improved outcome. With better management of the associated coagulopathy and the introduction of the differentiating agents, ATRA and arsenic trioxide (ATO), APL now represents the most curable subtype of AML with cure rates of >90% quoted in large clinical trials.37,38 The most widely tested treatment for APL consisted of a combination of ATRA and anthracycline-based chemotherapy for induction, followed by at least two courses of anthracycline-based chemotherapy and ATRA for consolidation, and maintenance therapy with intermittent ATRA alone (15 days every 3 months) or combined with 6-mercaptopurine and methotrexate. Accumulating data from historically controlled and randomized trials suggest a benefit for ara-C during induction and/or consolidation in both low-risk (WBC count < 10,000/μL) and high-risk patients.37,38

It is now established that ATO is a more effective agent in APL than ATRA. In particular, single-agent ATO can cure APL much more frequently than single-agent ATRA.³⁸ ATO-based initial therapy can be used as an alternative for patients who cannot tolerate anthracycline therapy. The North American Intergroup Trial incorporated ATO into the consolidation schema aiming to decrease toxicity. Patients randomized to receive two courses of 25 days of ATO (5 days a week for 5 weeks) had a significantly better event-free and OS than those who received only two courses of ATRA plus chemotherapy.³⁹

Despite the introduction of ATRA and improvement in the treatment of coagulopathy, bleeding remains an important cause of death during initial management. Therefore, APL should be treated as a medical emergency. ATRA and aggressive supportive measures should be started as soon as the diagnosis is suspected, even before the confirmatory genetic tests are available. The coagulopathy is treated with fresh-frozen plasma, fibrinogen, and platelets with the goal of maintaining fibrinogen level above 150 mg/dL and platelets well above 30,000/μL until the resolution of coagulopathy.38,39 The APL differentiation syndrome emerged as a major toxicity associated with both ATRA and ATO. It is characterized by pleural and pericardial effusions, weight gain, edema, dyspnea, fever, episodic hypotension, and pulmonary infiltrates. It should be promptly treated with intravenous administration of dexamethasone at a dose of 10 mg twice daily. The incidence of this complication is reduced in patients receiving concomitant chemotherapy during induction. Patients receiving ATO should be also closely monitored for prolongation of QT interval and their electrolytes should be maintained within normal range.38,39 The goal of induction and consolidation therapy should be the attainment of PCR negativity for the PML-RARα rearrangement, as the persistence of such minimal residual disease (MRD) predicts relapse. Patients treated in molecular relapse may have better outcome compared with those treated at frank hematologic relapse.⁴⁰ Novel formulations of the same targeted agents such as the oral formulation of ATO and an oral synthetic retinoid (Tamibarotene) designed to overcome ATRA resistance may be a promising therapeutic option for patients with relapsed disease.

Treatment of Relapsed and Refractory Acute Myeloid Leukemia

Unfortunately, AML relapses in the majority of patients. Moreover, approximately 25% of younger patients are refractory to standard induction chemotherapy. Patients with recurrent AML experience lower CR rates with reinduction chemotherapy compared to initial treatment. If second CR is achieved, it tends to be shorter. Therefore, patients with relapsed and refractory AML are candidates for clinical trials exploring innovative therapeutic strategies. Best supportive care is an option for those who do not wish to pursue intensive treatment.

If a suitable HLA-matched donor is available, the first choice of therapy is an allo-HSCT.¹⁹ Patients transplanted at the time of relapse have similar outcomes to those treated in second CR. If a suitable donor is not available, management should be guided by the duration of CR1. For patients with CR greater than 12 months, reinduction with HDAC-containing regimen is reasonable since it can achieve a CR rate of 50% to 60% and a 5% to 10% long-term DFS. For patients with shorter CR durations, the priority is treatment on a clinical trial. Novel agents currently being investigated include clofarabine, farnesyltransferase inhibitors (FTIs, FLT3 inhibitors, and other agents (see Table 11.11). Clinical trials should be considered in relapsed and refractory patients since response rates with currently available agents remain dismal.

Hematopoietic Growth Factors

Colony-stimulating factors (CSFs) can shorten the duration of neutropenia during AML treatment and have the potential to improve outcomes. Both granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) have been shown to accelerate neutrophil recovery after induction chemotherapy. G-CSF and GM-CSF have also been used to sensitize blasts to chemotherapy by recruiting cells into the cell cycle. However, numerous clinical trials have failed to show a reproducible survival benefit of such approach. Studies to date demonstrate that the benefit of CSFs is limited to reduction of neutropenic and febrile (FTI) days. The addition of CSFs to chemotherapy yielded no difference in all-cause mortality, CR, or relapse rates in patients with AML.⁴¹ Growth factors may be considered in the elderly after chemotherapy is complete. Their use may confound interpretation of the bone marrow biopsy. Patient should be off G-CSF and GM-CSF for a minimum of 7 days before obtaining bone marrow to document remission. Myeloid growth factors should not be used in APL.

Acute Myeloid Leukemia in Older Patients

Older adults have a dismal prognosis that has not changed significantly over the last three decades. Care of these patients is challenging due to the higher rates of poor-risk prognostic features including comorbidities, poor-risk cytogenetics, and multidrug resistance of leukemic blasts.2,10 Older age per se, however, should not be a reason to withhold intensive therapy.

Studies suggest that remission-induction chemotherapy provides better quality of life and longer survival than supportive care only.¹⁵ Patients over the age of 60 years without significant comorbidities can be treated with standard “3 + 7” induction therapy. Their outcomes worsen with advancing age and decreasing performance status (Table 11.10). Patients who benefit most from standard chemotherapy can be reasonably selected by the following criteria: age 60 to 69 years, no secondary AML or pre-existing MDS, good performance status, no pretreatment infection, and normal bilirubin and creatinine.² Results from MD Anderson Cancer Center in this small subgroup of patients show a 70% CR rate and a median survival of 14 months.² Choice of post remission therapy for older patients remains problematic. In contrast to younger patients, no studies with HDAC-based therapy have demonstrated a survival advantage in patients over 60 years of age. As this population represents a majority of patients with AML, new strategies are needed to improve their outcomes. For the majority of elderly patients, clinical trials with novel agents should be considered at the time of diagnosis due to poor results with conventional therapy. Several modestly successful options are available outside clinical trials including azacitidine, decitabine, low-dose cytarabine, and RIC stem cell transplantation. The regimen of azacitidine 75 mg/m²/day for 7 days every 4 weeks used as outpatient therapy in older adult patients with low marrow blast count (20% to 30%) significantly prolongs OS and improves several morbidity measures compared with palliative treatment.⁴² Data from recently published randomized trials suggest that the efficacy of treatment with hypomethylating agents including decitabine may be comparable to that of intensive chemotherapy and superior to that of other palliative treatment approaches.42,43

Azacitidine has become an upfront option for patients who cannot tolerate intensive induction or for whom clinical trials are unavailable. Low-dose decitabine has also been used in high-risk MDS and elderly AML patients with CR rates of 14% to 28% and is better tolerated than standard induction.42,43 Low-dose cytarabine (20 mg subcutaneously twice daily for 10 days every 4 to 6 weeks) also produced better OS than did supportive care and hydroxyurea in an earlier randomized trial.⁴⁴ Clofarabine is active as a single agent and is currently explored as part of induction regimens for elderly patients.¹⁶ Choice among these agents is based on individual preference because comparisons of efficacy or toxicity are problematic across different studies. In many patients, especially those with poor performance status, abnormal organ function, or active infection, palliative care may be the treatment of choice.

Acute Myeloid Leukemia in Pregnancy

The prevalence of leukemia during pregnancy is low, roughly one in 75,000 to 100,000 pregnancies. AML accounts for two-thirds of cases.⁴⁵ Acute leukemia is usually reported during the second and third trimesters of pregnancy. This could be a result of a selection bias of unreported cases that occurred early and resulted in termination of pregnancy. The management of pregnant patients with AML poses major challenges. Patients are at high risk for pregnancy-associated complications due to the bleeding and infection.

Acute leukemia needs immediate treatment irrespective of gestational stage because delay or modification of therapy results in inferior maternal outcome. A recent systematic review identified 87 AML patients (88 pregnancies) treated with systemic therapy during the course of pregnancy. With few exceptions, patients were electively treated following the end of the first trimester.⁴⁶ Nearly 50% of those who were exposed to chemotherapy during the first trimester had poor fetal outcomes. Chemotherapeutic agents and target therapies, such as ATRA, should be avoided during the first trimester. ATRA, a highly effective agent in APL, is associated with substantial toxicity to the fetus when used in the first trimester, including CNS and cardiovascular malformations. Hence, women diagnosed early in gestation should be offered a termination of pregnancy. Administration of chemotherapy and ATRA in the second and third trimesters is less likely to result in teratogenesis, although it increases the risk of intrauterine growth retardation.⁴⁵ In summary, management of AML in pregnancy should focus on survival of the mother, while minimizing treatment-related fetal toxic effects.

Evaluation of Minimal Residual Disease

Postremission assessments of MRD by using PCR and multiparametric flow cytometry techniques are rapidly becoming part of the standard of care in AML. The recent development of real-time quantitative PCR allows for monitoring of patients with known genetic markers with the sensitivity of detecting one leukemic cell in up to 10⁵ normal cells. Flow cytometry can be also used to detect and quantify low numbers of AML cells with a unique leukemia-associated immunophenotype. It is applicable to more than 85% of AML. Most laboratories can detect the leukemic cells with a sensitivity of between 10⁻⁴ and 10⁻⁵ by using the combination of 10-color flow cytometry.⁸

Extensive data support the prognostic value of MRD detection in AML patients after therapy. The quantification of residual AML cells at defined time points permits individualization of postremission therapy. It can also serve as a surrogate end point for the evaluation of new treatments. The goal of MRD detection is to identify patients at higher risk for relapse and assign them to different therapeutic approaches. MRD has been best studied in APL, where the persistence of PML-RARA fusion transcript at the end of consolidation therapy or subsequent recurrence of PCR positivity after a molecular remission precedes overt relapse. In other types of AML, MRD can be an early predictor of clinical relapse, but the optimal timing and frequency of MRD monitoring, and the optimal intervention based on MRD remains to be determined. Caution should be taken in applying MRD detection techniques to clinical practice outside well-designed prospective studies.

Novel Therapeutic Targets in Acute Myeloid Leukemia

Standard treatment for AML is based on aggressive cytotoxic chemotherapy given in repetitive cycles in order to eradicate disease. Only a minority of patients is cured by this approach. The success of ATRA and ASO in APL demonstrates that more selective and less toxic drugs can substantially increase cure rates of AML. The concept of targeting leukemic cells and sparing normal ones from the broad attack of chemotherapy is now under investigation in other types of AML. With improved understanding of the mechanisms underlying leukemogenesis, novel classes of drugs have entered clinical trials (Table 11.11). These include antibody-based therapies and other immunotherapeutic approaches, FLT3 inhibitors and other tyrosine kinase inhibitors, FTIs, clofarabine, and inhibitors of methylation (Table 11.11). Most of these drugs have limited activity as single agents and their full potential may be only realized in combination therapy.

Gemtuzumab ozogamicin (Mylotarg) is the first monoclonal antibody-directed chemotherapy targeting the CD33 epitope. It was withdrawn from the US market in June 2010 due to concerns of toxicity, though it may be of benefit for patients with better-risk cytogenetics or APL. In two recent randomized phase III trials, small doses of gemtuzumab in combination with the traditional 3 + 7 regimen demonstrated no significant difference in fatal adverse events compared to chemotherapy alone, and a highly significant advantage in DFS and OS for the combination arm.17,18 Both trials suggest that gemtuzumab warrants further investigation.

FLT3 kinase inhibitors have generated interest because activating FLT3 mutations in AML confers poor prognosis. Specific inhibitors of FLT3 such as sorafenib, lestaurtinib (CEP-701), and quizartinib (AC220) have been studied in relapsed or refractory AML. A phase III randomized trial of salvage chemotherapy with or without lestaurtinib failed to demonstrate a general benefit.³⁶ The target inhibition of FLT3 was achieved in only 58% of patients receiving lestaurtinib but it did correlate with higher remission rate and longer OS. This suggests that FLT3 inhibition remains a promising approach and inhibitors with improved pharmacokinetic and pharmacodynamic properties should be studied. Sorafenib was demonstrated to slow down disease progression in relapse but did not induce second remission.⁵¹ The results of quizartinib large-scale phase III trials in relapsed AML are still pending.³⁶

FTIs represent a new class of small-molecule inhibitors that selectively inhibit farnesylation of a number of intracellular proteins such as Ras. Single-agent tipifarnib has shown antileukemic activity in patients with MDS and refractory/poor-risk AML.⁵² However, a phase III study comparing single-agent tipifarnib to best supportive care including hydroxyurea in patients 70 years of age or older with untreated AML failed to demonstrate a survival advantage.⁵³ Subsequently a phase I trial combining tipifarnib and etoposide in elderly poor-risk AML patients led to an improved CR rate of 25% across multiple dose levels of both drugs compared with 14% for single-agent tipifarnib.⁴⁷ High RASGRP1 and low APTX gene expression was found to predict clinical response to tipifarnib and etoposide.⁴⁷

Efforts are also under way to optimize the delivery of ara-C, for example, via conjugation to the lipid moiety elaidic acid, which allows bypassing the nucleoside transporter protein hENT1. Another novel formulation, CPX-351, fixes a 5:1 ratio of cytarabine to daunorubicin within a liposomal carrier. Novel topoisomerase II inhibitors are also under development. There is a plethora of new agents and drug combinations that awaits testing in AML. All patients with AML including the elderly should be offered therapy on well-designed clinical trials whenever possible.

SUMMARY

AML represents a genetically, morphologically, and clinically heterogeneous group of hematopoietic malignancies characterized by a rapid growth of myeloid blasts and suppression of normal hematopoiesis. The initiating genetic events and the pathways involved in the pathogenesis of AML are the subject of intense investigations. These events determine the type of AML, response to therapy, and to some extent, the final outcome. The well-established prognostic factors including age and cytogenetic changes are continuously expanded by molecular characteristics. Mutation testing is now incorporated into prognostic models and used to identify treatment targets. Unfortunately, less than half of younger patients and less than 10% of patients over 60 years are cured of their disease with current therapies. In younger patients, the longest DFS can be achieved with repeated cycles of intensive chemotherapy containing anthracyclines and cytarabine. Postremission consolidation therapy is crucial while maintenance is not necessary with the exception of APL. HDAC is the consolidation of choice for younger patients with better-risk cytogenetics. Patients with intermediate- or poor-risk disease who have an HLA-matched donor and good performance status benefit from allo-HSCT. Although questions remain about the optimal drugs for induction and about the numbers of consolidation cycles, it is unlikely that further modifications of standard chemotherapy will result in dramatic improvement in survival. Aggressive chemotherapy is not suitable for older patients with comorbidities and decreased performance status. These patients can derive modest benefit from hypomethylating agents or low-dose cytarabine. New targeted treatments including antibody-based therapies, tyrosine kinase inhibitors and other drugs targeting signal transduction, and vaccines have shown some activity and acceptable toxicity in clinical studies. Combinations of these agents based on studies of cell biology should lead to better outcomes than unselective cytotoxic therapy.

References

  1.  Howlander N, Noone AM, Krapcho M, et al., eds. SEER Cancer Statistics Review, 1975-2008. Bethesda, MD: National Cancer Institute., http://seer.cancer.gov/csr/1975_2009_pops09/, based on November 2010 SEER data submission, posted to the SEER web site; 2011.

  2.  Appelbaum FR, Gundacker H, Head DR, et al. Age and acute myeloid leukemia. Blood. 2006;107:3481-3485.

  3.  Dores GM, Devesa SS, Curtis RE, et al. Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood. 2012;119:34-43.

  4.  Goldin LR, Kristinsson SY, Liang SX, et al. Familiar aggregation of acute myeloid leukemia and myelodysplastic syndromes. J Clin Oncol. 2011;30:179-183.

  5.  Bernasconi P. Molecular pathways in myelodysplastic syndromes and acute myeloid leukemia: relationship and distinctions—a review. Br J Haematol. 2008;142:695-708.

  6.  Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937-951.

  7.  Dohner H, Gaidzik VI. Impact of genetic features on treatment decisions in AML. ASH Education Book. Hematology Am Soc Hematol Educ Program. 2011;2011:36-42.

  8.  Buccisano F, Maurillo L, Del Principe MI, et al. Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Blood. 2012;119(2):332-341.

  9.  Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363(25):2424-2433.

10.  Döhner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood. 2010;115:453-474.

11.  Mayer R, Davis R, Schiffer C, et al. Intensive postremission chemotherapy in adults with acute myeloid leukemia, Cancer and Leukemia Group B. N Engl J Med. 1994;331:896-903.

12.  Dombret H, Gardin C. An old AML drug revisited. N Engl J Med. 2009;361(13):1301-1303.

13.  Pautas C, Merabet F, Thomas X, et al. Randomized study of intensified anthracycline doses for induction and recombinant interleukin-2 for maintenance in patients with acute myeloid leukemia age 50 to 70 years: results of the ALFA-9801 study. J Clin Oncol. 2010;28(5):808-814.

14.  Löwenberg B, Pabst T, Vellenga E, et al. Cytarabine dose for acute myeloid leukemia. N Engl J Med. 2011;364(11):1027-1036.

15.  Juliusson G, Antunovic P, Derolf Å, et al. Age and acute myeloid leukemia: real world data on decision to treat and outcomes from the Swedish Acute Leukemia Registry. Blood. 2009;113(18):4179-4187.

16.  Kantarjian HM, Erba HP, Claxton D, et al. Phase II study of clofarabine monotherapy in previously untreated older adults with acute myeloid leukemia and unfavorable prognostic factors. J Clin Oncol. 2010;28(4):549-555.

17.  Castaigne S, Pautas C, Terre C, et al. Fractionated doses of Gemtuzumab Ozogamicin (GO) combined to standard chemotherapy (CT) improve event-free and overall survival in newly-diagnosed de novo AML patients aged 50-70 years old: a prospective randomized phase 3 trial from the Acute Leukemia French Association (ALFA). ASH Annual Meeting Abstracts. Blood. 2011;118:Abstract 6.

18.  Burnett AK, Hills RK, Hunter AE, et al. The addition of gemtuzumab ozogamicin to intensive chemotherapy in older patients with AML produces a significant improvement in overall survival: results of the UKNCRI AML16 randomized trial. ASH Annual Meeting Abstracts. Blood. 2011;118:Abstract 582.

19.  Tallman MS, Gilliland G, Rowe JM. Drug therapy for acute myeloid leukemia. Blood. 2005;106:1154-1163.

20.  Byrd JC, Ruppert AC, Mrózek K, et al. Repetitive cycles of high-dose cytarabine benefit patients with acute myeloid leukemia and inv(16)(p13q22) or t(16;16)(p13;q22): results from CALGB 8461. J Clin Oncol. 2004;22:1087-1094.

21.  Grimwade D, Walker H, Harrison G, et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood. 2001;98(5):1312-1320.

22.  Goldstone AH, Burnett AK, Wheatley K, et al. Attempts to improve treatment outcomes in acute myeloid leukemia (AML) in older patients: the results of the United Kingdom Medical Research Council AML11 trial. Blood. 2001;98(5):1302-1311.

23.  Schaich M, Rollig C, Soucek S, et al. Cytarabine dose of 36 g/m2 compared with 12 g/m2 within first consolidation in acute myeloid leukemia: results of patients enrolled onto the prospective randomized AML96 study. J Clin Oncol. 2011;29:2696-2702.

24.  Zittoun RA, Mandelli F, Willemze, et al. Autologous or allogeneic bone marrow transplantation compared to intensive chemotherapy for acute myeloid leukemia in first remission. EORTC and GIMEMA Leukemia Cooperative Groups. N Engl J Med. 1995;332:217-223.

25.  Burnett AK, Goldstone AH, Stevens RM, et al. Randomized comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukaemia in first remission: results of MRC AML 10 trial. Lancet. 1998;351:700-708.

26.  Cassileth PA, Harrington DP, Appelbaum FR, et al. Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission. N Engl J Med. 1998;339:1649-1656.

27.  Vellenga E, Van Putten W, Ossenkoppele GJ, et al. Autologous peripheral blood stem cell transplantation for acute myeloid leukemia. Blood. 2011;118(23):6037-6042.

28.  Harousseau JL, Cahn JY, Pignon B, et al. Comparison of autologous bone marrow transplantation and intensive chemotherapy as postremission therapy in adult acute myeloid leukemia. The Groupe Ouest Est Leucemies Aigues Myeloblastiques (GOELAM). Blood. 1997;90:2978-2986.

29.  Suciu S, Mandelli F, de Witte T, et al. EORTC and GIMEMA Leukemia Groups. Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 46 years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMA AML-10 trial. Blood. 2003;102:1232-1240.

30.  Cornelissen JJ, Van Putten WLJ, Verdonck LF, et al. Results of a HOVON/SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? Blood. 2007;109:3658-3666.

31.  Koreth J, Schlenk R, Kopecky KJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta-analysis of prospective clinical trials. JAMA. 2009;301:2349-2361.

32.  Horwitz ME. Reduced intensity versus myeloablative allogeneic stem cell transplantation for the treatment of acute myeloid leukemia, myelodysplastic syndrome and acute lymphoid leukemia. Curr Opin Oncol. 2011;23(2):197-202.

33.  Eapen M, Rocha V, Sanz G, et al. Effect of graft source on unrelated donor haemopoietic stem-cell transplantation in adults with acute leukaemia: a retrospective analysis. Lancet Oncol. 2010;11:653-660.

34.  Brunstein CG, Gutman JA, Weisdorf DJ, et al. Allogeneic hematopoietic cell transplantation for hematologic malignancy: relative risks and benefits of double umbilical cord blood. Blood. 2010;116:4693-4699.

35.  Ciceri F, Labopin M, Aversa F, et al. A survey of fully haploidentical hematopoietic stem cell transplantation in adults with high-risk acute leukemia: a risk factor analysis of outcomes for patients in remission at transplantation. Blood. 2008;112:3574-3581.

36.  Levis M, Ravandi F, Wang ES, et al. Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Blood. 2011;117:3294-3301.

37.  Sanz MA, Montesinos P, Rayon C, et al. Risk-adapted treatment of acute promyelocytic leukemia based on all-trans retinoic acid and anthracycline with addition of cytarabine in consolidation therapy for high-risk patients: Further improvements in treatment outcome. Blood. 2010;115:5137-5146.

38.  Estey EH, Hutchinson F. Newly diagnosed acute promyelocytic leukemia: arsenic moves front and center. J Clin Oncol. 2011;29(20):2743-2746.

39.  Powell BL, Moser B, Stock W, et al. Arsenic trioxide improves event free and overall survival for adults with acute promyelocytic leukemia: North American Leukemia Intergroup Study C9710. Blood. 2010;116:3751-3757.

40.  Esteve J, Escoda L, Martin G, et al. Outcome of patients with acute promyelocytic leukemia failing to front-line treatment with all-trans retinoic acid and anthracycline-based chemotherapy (PETHEMA protocols LPA96 and LPA99): benefit of an early intervention. Leukemia. 2007;21(3):446-452.

41.  Gurion R, Belnik-Plitman Y, Gafter-Gvili A, et al. Colony-stimulating factors for prevention and treatment of infectious complications in patients with acute myelogenous leukemia. Cochrane Database Syst Rev. September 2011;9:CD008238.

42.  Fenaux P, Mufti GJ, Hellström-Lindberg E, et al. Azacitidine prolongs overall survival compared with conventional care regimes in elderly patients with low bone marrow blast count acute myeloid leukemia. J Clin Oncol. 2010;28:562-569.

43.  Lübbert M, Suciu S, Baila L, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate- or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol. 2011;29(15):1987-1996.

44.  Burnett AK, Milligan D, Prentice AG, et al. A comparison of low-dose cytarabine and hydroxyurea with or without ATRA for acute myeloid leukemia and high risk MDS in patients not considered fit for intensive treatment. Cancer. 2007;109:1114-1124.

45.  Brenner B, Avivi I, Lishner M. Haematological cancers in pregnancy. Lancet. 2012;379(9815):580-587.

46.  Azim HA Jr, Pavlidis N, Peccatori FA. Treatment of the pregnant mother with cancer: a systematic review on the use of cytotoxic, endocrine, targeted agents and immunotherapy during pregnancy. Part II: hematological tumors. Cancer Treat Rev. 2010;36(2):110-121.

47.  Karp JE, Vener TI, Raponi M, et al. Multi-institutional phase 2 clinical and pharmacogenomic trial of tipifarnib plus etoposide for elderly adults with newly diagnosed acute myelogenous leukemia. Blood. 2012;119(1):55-63.

48.  Giles F, Vey N, DeAngelo D, et al. Phase 3 randomized, placebo-controlled, double-blind study of high-dose continuous infusion cytarabine alone or with laromustine (VNP40101M) in patients with acute myeloid leukemia in first relapse. Blood. 2009;114:4027-4033.

49.  Blum W, Klisovic RB, Becker H, et al. Dose escalation of lenalidomide in relapsed or refractory acute leukemias. J Clin Oncol. 2010; 28:4919-4925.

50.  Van Tendeloo VF, Van de Velde A, Van Driessche A, et al. Induction of complete and molecular remissions in acute myeloid leukemia by Wilms tumor 1 antigen-targeted dendritic cell vaccination. Proc Natl Acad Sci USA. 2010;107(31):13824-13829.

51.  Sharma M, Ravandi F, Bayraktar UD, et al. Treatment of FLT3-ITD-positive acute myeloid leukemia relapsing after allogeneic stem cell transplantation with sorafenib. Biol Blood Marrow Transplant. 2011;17:1874-1877.

52.  Lancet JE, Gojo I, Gotlib J, et al. A phase 2 study of the farnesyltransferase inhibitor tipifarnib in poor-risk and elderly patients with previously untreated acute myelogenous leukemia. Blood. 2007;109(4):1387-1394.

53.  Harousseau JL, Martinelli G, Jedrzejczak WW, et al. A randomized phase 3 study of tipifarnib compared with best supportive care, including hydroxyurea, in the treatment of newly diagnosed acute myeloid leukemia in patients 70 years or older. Blood. 2009;114(6):1166-1173.

12

Acute Lymphoblastic Leukemia

Nirali N. Shah and Alan S. Wayne

Approximately 5,000 new cases of acute lymphoblastic leukemia (ALL) are diagnosed each year in the United States, more than half of them in children. There has been significant progress in the development of curative therapy, such that currently children and adults with ALL have expected disease-free survival (DFS) rates of about 80% and 50%, respectively.1,2

EPIDEMIOLOGY

ALL represents the most common pediatric malignancy, accounting for approximately 25% of childhood cancer. The peaks in prevalence of ALL occur between the ages of 2 and 5 years and after age 50. There is a slight male predominance, and Caucasians have a twofold increased risk compared with African-Americans, while the highest incidence is seen in Hispanic children.³

ETIOLOGY AND RISK FACTORS

Certain conditions predispose to ALL, most notably trisomy 21 (Down syndrome) in which the relative risk is increased 15-fold. Other predisposing conditions include immunodeficiency and chromosomal breakage syndromes, but most often no such underlying disorder is found. Epstein-Barr virus (EBV) infection is implicated in a minority of cases of mature B-cell ALL. Environmental exposure risks have been suggested, but with the exception of ionizing radiation, few have been shown to be causal. Acquired chromosomal abnormalities confined to lymphoblasts are found in more than 90% of cases, including aneuploidy (most commonly hyperdiploidy) and/or translocations that in some cases are prenatal in origin. The genes involved in leukemogenesis are frequently transcription factors expressed in hematopoietic tissues.⁴

CLINICAL FEATURES

Presenting signs and symptoms are almost always caused by lymphoblast infiltration of the bone marrow with resultant blood count abnormalities (Table 12.1). Other organs may also be involved, most commonly the central nervous system (CNS) and the testes. T-cell ALL frequently presents with bulky adenopathy, mediastinal mass, pleural effusion, and/or hyperleukocytosis. Gastrointestinal presentation due to Peyer’s patch involvement, usually ileocecal intussusception, is almost always confined to mature B-cell ALL. There are a number of life- or organ-threatening presentations that require emergent intervention (Table 12.2).

LABORATORY FEATURES

The diagnosis is readily confirmed by the demonstration of lymphoblasts in the blood and/or bone marrow. Blast morphology can be classified into three categories (L1, L2, L3) according to the French-American-British (FAB) system (Table 12.3). Only the latter is of clinical and prognostic significance, because L3 morphology is indicative of mature B-cell or Burkitt-type ALL. Routine hematopathologic analysis, immunohistochemistry, flow cytometry, and cytogenetics are used to define the subtype and further identify prognostic factors. The majority of ALL is of precursor B-cell (pre-B) phenotype (CD10, CD19, CD22, HLA-DR, TDT+), 10% to 20% is T-cell (CD2, CD7+), and less than 5% is mature B-cell or Burkitt-type (CD20, surface-IgM κ or λ+). Certain cytogenetic abnormalities are not apparent on routine karyotyping, and thus molecular testing may be required, most notably for t(12;21) seen in about 25% of cases in children (Table 12.4). Lumbar puncture is required to evaluate the possibility of meningeal leukemia.

Table 12.3  Classification

FAB Morphology

L1: homogeneous blasts, minimal cytoplasm

L2: increased nuclear heterogeneity, prominent nucleoli

L3: basophilic cytoplasm with prominent vacuolization

Bone Marrow

M1: <5% blasts

M2: 5%-25% blasts

M3: >25% blasts

Cerebrospinal Fluid Cytology

CNS-1: no blasts

CNS-2: WBC < 5/μL with blasts

CNS-3: WBC ≥ 5/μL with blasts

or symptomatic CNS involvement (e.g., cranial nerve palsy)

CNS, central nervous system; FAB, French-American-British; WBC, white blood cell.

PROGNOSTIC FACTORS

Clinical and biologic features, as well as initial response to therapy, are used to determine risk-directed treatment for individuals with pre-B ALL (Table 12.5).⁵ Age is a strong prognostic determinant, and outcome is inferior in infants and adults in comparison to children. T-cell and mature B-cell disease have historically had lower DFS rates than pre-B ALL; however, stratified treatment has minimized this difference. Recently, genomic analysis has revealed molecular alterations and profiles associated with poor outcome, thus allowing further discrimination of diagnostic subtype, risk classification, and treatment-response prediction.⁴

TREATMENT

Many chemotherapeutic regimens are effective for children and adults with ALL. Therapy is stratified based on clinicopathologic features, and treatment should be directed by physicians familiar with subtype-specific regimens. The following core recommendations are based on results of large cooperative group clinical trials.1,2,6-9

  Therapy should be instituted as soon as possible after diagnosis.

  Treatment is based on phenotype and prognostic factors and includes the following phases: induction, consolidation, CNS sterilization, and maintenance for a total of 2 to 3 years (Table 12.6). Initial induction therapy often consists of 3 to 5 drugs given as a 28-day cycle, although there are alternative approaches.⁸ A variety of consolidation and intensification regimens are commonly employed, some of which are detailed below. Multiple consolidation/intensification blocks are often advised for high-risk patients. A late reinduction phase, also known as delayed intensification, improves DFS for children who are slow early responders (SER).¹⁰ Randomized trials of various intensification blocks for adults have shown mixed results.11,12 The application of pediatric regimens to adult ALL has improved the DFS rates especially for adolescents and young adults, but at the expense of toxicity in older individuals.2,9,13-15 Prolonged maintenance with a total treatment duration of 24 to 36 months improves DFS for both adults and children.

Table 12.4  Common Chromosomal Translocations

t(12;21): TEL/AML1 (25% childhood ALL)

t(1;19): E2A/PBX1

t(9;22): BCR/ABL p190 fusion (25% adult ALL)

11q23: MLL, multiple fusion partners (70% infant ALL)

14q11 or 7q35: TCR,T-cell phenotype

t(8;14), t(8;22), t(2;8): c-myc/Ig, mature B-cell (Burkitt) phenotype

ALL, acute lymphoblastic leukemia.

Table 12.6  Common Treatment Regimens for Pre-B-cell Acute Lymphoblastic Leukemia and T-cell Acute Lymphoblastic Leukemia*

Induction Regimen (Weeks 1–4)

3-Drug

•  Prednisone 40–60 mg/m²/d or dexamethasone 6 mg/m²/d in divided doses PO × 21–28 d (days 0–28)

•  Vincristine 1.5 mg/m²/dose (maximum dose 2 mg) IV weekly × 4 doses (days 0, 7, 14, 21)

•  Escherichia coli l-asparaginase 6,000–10,000 IU/m²/dose IM × 6–9 doses, QOD, 3 d/wk × 2–3 wk

•  IT methotrexate: (or triple IT therapy with methotrexate, hydrocortisone and cytarabine)

•  CNS-1: Every 2 wk × 2 doses (days 0, 14)

•  CNS-2 or CNS-3:Weekly × at least 4 doses and until 2 successive CNS-1 (days 0, 7, 14, 21)

4-Drug: Add the following to above

•  Doxorubicin 25–30 mg/m²/dose or daunorubicin 25–45 mg/m²/dose IV weekly × 4 doses (days 0, 7, 14, 21) or IV daily × 2–3 doses (days 0, 1, +/-2)

5-Drug: Add the following to above

•  Cyclophosphamide 800–1,200 mg/m²/dose IV × 1 dose (day 0)

Response evaluation

Day 14 bone marrow

•  M1: Rapid early responder

•  M2 or M3: Slow early responder

Day 28 bone marrow

•  M1: Remission, continue as below. If MRD is present, may be allocated to a higher-risk group and receive intensified therapy.

•  M2 or M3: Induction failure, salvage reinduction required

Post-induction Regimens

Pretreatment criteria

•  ANC ≥ 750/μL, platelets ≥75,000/μL

•  ALT < 2 times the upper limit of normal, direct bilirubin normal for age

•  Serum creatinine normal for age

•  No active infection or life-threatening organ dysfunction

Consolidation (Week 5)

Standard Berlin-Frankfurt-Munster Study Group (BFM)

•  Cyclophosphamide 1,000 mg/m²/dose IV × 2 doses (days 0, 14)

•  Mercaptopurine (6-MP) 60 mg/m²/dose PO once daily (administer at bedtime on an empty stomach to improve absorption) × 28 d (days 0–27)

•  Vincristine 1.5 mg/m²/dose (maximum dose 2 mg) IV × 4 doses (days 14, 21, 42, 49)

•  Cytarabine 75 mg/m²/dose IV or SQ (days 1–4, 8–11, 15–18, 22–25)

•  IT methotrexate weekly × 4 doses (days 1, 8, 15, 22)

Augmented BFM

•  Cyclophosphamide 1,000 mg/m²/dose IV × 2 doses (days 0, 28)

•  Mercaptopurine (6-MP) 60 mg/m²/dose PO once daily (administer at bedtime on an empty stomach to improve absorption) × 28 d (days 0–13, 28–41)

•  Vincristine 1.5 mg/m²/dose (maximum dose 2 mg) IV × 4 doses (days 14, 21, 42, 49)

•  Cytarabine 75 mg/m²/dose IV or SQ (days 1–4, 8–11, 29–32, 36–39)

•  E. coli l-Asparaginase 6,000 IU/ m²/dose IM × 12 doses, every other day 3 doses per week (days 14, 16, 18, 21, 23, 25, 42, 44, 46, 49, 51, 53)

•  IT methotrexate weekly × 4 doses (days 1, 8, 15, 22)

High-dose methotrexate with leucovorin rescue

•  Refer to protocol-specific dosing, administration, and leucovorin rescue guidelines

Capizzi

•  Cytarabine (Ara-C) 3,000 mg/m²/dose IV over 3 h every 12 h × 4 doses, weekly × 2 (days 0, 1 and days 7, 8)

•  L-Asparaginase 6,000 IU/m²/dose IM at hour 42 following Ara-C (3 h after the completion of the fourth Ara-C infusion on days 1 and 8)

Ifosfamide/etoposide

•  Etoposide (VP-16): 100 mg/m²/dose IV × 5 doses (days 1–5)

•  Ifosfamide: 1.8 gm/m²/dose IV × 5 doses (days 1–5). Begin immediately upon completion of VP-16 infusion

•  Mesna: 360 mg/m²/dose IV prior to ifosfamide and every 3 h × 8 doses/d (days 1–5)

Interim Maintenance

•  Commonly employed between consolidation and delayed intensification/reinduction courses

Delayed Intensification/Reinduction

•  Dexamethasone 10 mg/m²/d in divided doses PO × 14–28 d (day 0–)

•  Vincristine 1.5 mg/m²/dose (maximum dose 2 mg) IV weekly × 5 doses (days 0, 14, 21, 42, 49)

•  Doxorubicin 25–30 mg/m²/dose IV weekly × 3 doses (days 0, 7, 14)

•  Cyclophosphamide 1,000 mg/m²/dose IV (day 28)

•  6-Thioguanine (6-TG) 60 mg/m²/dose PO once daily × 14 d (days 28–41)

•  Cytarabine 75 mg/m²/dose IV or SQ (days 29–32, 36–39)

•  IT methotrexate × 2 doses (days 29, 36)

With or without:

•  E. coli l-asparaginase 6,000 IU/m²/dose IM × 6 – 12 doses (days 3, 5, 7, 10, 12, 14 +/- 42, 44, 46, 49, 51, 53)

Maintenance/Continuation Regimen

Repeat cycles to complete 24–36 mo of total treatment.

•  Prednisone 40–60 mg/m²/d or dexamethasone 6 mg/m²/d in divided doses PO × 5 d every 28 d

•  Vincristine 1.5 mg/m²/d (maximum dose 2 mg) IV every 4 wk

•  Mercaptopurine (6-MP) 75 mg/m²/dose† PO once daily (administer at bedtime on an empty stomach to improve absorption)

•  Methotrexate 20 mg/m²/dose† PO once weekly

•  IT methotrexate every 4–12 wk for 1–2 yr of treatment

*A BCR/ABL kinase inhibitor should be incorporated into the treatment regimen for individuals with Philadelphia chromosome–positive ALL.

†6-MP and methotrexate doses should be adjusted to maintain the ANC between 750–1,500/μL and the platelet count >75,000/μL. IM, intramuscular; IT, intrathecal; IV, intravenous; PO, oral; SQ, subcutaneous; MRD, minimal residual disease.

  Allogeneic stem cell transplantation (SCT): Although relapse rates are lower after allogeneic SCT compared with chemotherapy, treatment-related mortality rates are higher after transplantation.¹⁶ Thus, SCT is rarely used for children in first remission (CR1) except within the context of clinical trials for individuals with extremely poor prognostic features such as induction failure.17,18 Given the relatively poor results of chemotherapy in older individuals, some groups recommend allogeneic SCT in CR1 for adults with human leukocyte antigen (HLA)-matched sibling donors.² When SCT is employed in the treatment of ALL, the conditioning regimen typically utilizes total body irradiation (TBI), which has been shown to decrease the relapse risk. ¹⁸

  Risk-group assignment for B-precursor ALL: Although there is protocol-specific variability in the approach to risk-adapted therapy, in general, age, white blood cell (WBC) count, CNS involvement, DNA index, and phenotype are used for the initial risk-group assignment (Table 12.5).1,2,5,9 Subsequently, the risk group may be elevated based on cytogenetics and response to therapy, the latter of which is defined by morphologic blast reduction (in peripheral blood on day 7 or bone marrow on day 7 or 14) and further quantified by minimal residual disease determination (MRD) by flow cytometry or polymerase chain reaction amplification.1,2,9 Recent studies have demonstrated the prognostic value of MRD determination at various treatment phases for adults and children with ALL. 19,20

  Infant ALL: Children younger than 1 year at diagnosis should be treated on age-specific protocols with certain agents dosed by weight to decrease the risk of severe toxicity.

  T-ALL: Patients with T-cell phenotype are treated similarly to higher risk group pre-B ALL. Improved outcome has been associated with the use of intensified therapy that commonly includes high-dose methotrexate and intensified l-asparaginase. 21,22

  Mature B-ALL: Patients with mature B-cell phenotype ALL should be treated with Burkitt lymphoma regimens and should include the anti-CD20 monoclonal antibody rituximab.²³

  CNS-directed therapy: All patients require CNS sterilization. Intensive intrathecal chemotherapy in combination with systemic agents that have good CNS penetration, most notably dexamethasone and high-dose methotrexate, are highly effective (Table 12.7). To minimize neurotoxicity, radiation is usually reserved for those with active meningeal leukemia or at very high risk of CNS relapse (Table 12.8). ²⁴

  Testicular leukemia: Males with testicular involvement have historically received radiation to both testes, although recent studies suggest that radiation might be spared when testicular involvement resolves completely during initial induction.²⁵

DOSE MODIFICATION

Improved outcome is associated with greater drug exposure, and every attempt should be made to administer protocol-specified doses unless toxicity precludes their delivery. Importantly, 6-mercaptopurine (6-MP) and methotrexate dosing should be increased during maintenance to achieve a targeted degree of myelosuppression (Table 12.6). In the event of significant chemotherapy-related toxicity, individual agents should be dose-reduced or discontinued as clinically indicated. Specific agents may require dose adjustment for renal or hepatic dysfunction. Patients with thiopurine S-methyltransferase deficiency (approximately 1:300 incidence) require significant dose reductions of 6-MP to avoid severe toxicity. Individuals with Down syndrome tolerate methotrexate poorly and require elimination or dose reduction of that agent.

EXTRAMEDULLARY LEUKEMIA

Current chemotherapy regimens are associated with low rates of extramedullary relapse in both the CNS and testes. Importantly, patients with isolated extramedullary relapse also require systemic therapy. Radiation is currently reserved primarily to treat overt CNS leukemia (Table 12.8).

NEW TREATMENT APPROACHES

Molecularly targeted agents have been applied to the treatment of ALL with improved outcomes. For example, the BCR/ABL tyrosine kinase inhibitors, imatinib mesylate (Gleevec), dasatinib, and nilotinib have been successfully utilized in combination with chemotherapy to improve results in Philadelphia chromosome-positive ALL.26,27 Monoclonal antibody-based therapies that target differentiation antigens expressed on the surface of lymphoblasts (e.g., CD19, CD20, CD22, CD52) have also shown promise in the setting of ALL.8,23,28,29

MANAGEMENT OF RELAPSE

The chance of cure decreases substantially after relapse. Attaining a second remission is critical and often times can be achieved with standard four- or five-drug reinduction regimens (Table 12.6).³⁰ The likelihood of prolonged DFS with standard regimens varies based in large part on the duration of the initial complete remission and the site of relapse. For those with bone marrow relapse and CR1 durations of more than 18–36 months, approximately 35% will achieve prolonged DFS with intensive re-treatment. Outcome is guarded with shorter CR1 durations, multiple relapses, or induction failure.³⁰ Curative salvage using standard chemotherapy and radiation is more likely in the setting of isolated extramedullary relapse.31,32

Allogeneic Stem Cell Transplantation

For patients with HLA-matched sibling donors, allogeneic SCT in second CR is standard care.¹⁶⁻¹⁸ The risks of transplant-related morbidity and mortality are increased with alternative donors (unrelated and HLA-mismatched related), with such transplants being consequently commonly reserved for short CR1 durations or subsequent relapses.

SUPPORTIVE CARE

Aggressive monitoring and supportive care are essential throughout all phases of treatment.

Antiemetics

Nausea and vomiting, common during induction, consolidation, intensification, and CNS-directed therapy, are managed with routine antiemetic prophylaxis and treatment.

Tumor Lysis Syndrome

Rapid blast lysis can result in life-threatening metabolic complications. Tumor lysis syndrome is usually seen within the first few hours to days of initiation of induction chemotherapy. Patients with WBC higher than 100,000/μL, elevated serum lactate dehydrogenase (LDH), and/or elevated uric acid are at increased risk and those with mature B-cell ALL (L3 or Burkitt-type) are at extreme risk. Tumor lysis precautions should be started as soon as possible after diagnosis and at least 6 to 12 hours prior to the start of induction. Prophylaxis and monitoring should continue until disease burden is reduced, peripheral blasts are clear, and it is apparent that no tumor lysis has developed, usually for 3 to 7 days. The following measures are indicated for all patients around initial induction³³:

  Allopurinol: 100 mg/m² per dose orally, three times daily. Urate oxidase (rasburicase) is an alternative for management of extreme hyperuricemia, especially in the setting of renal insufficiency. Rasburicase can cause severe hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, and thus should be avoided in such patients.

  Hydration: Intravenous fluids at a rate of at least twice the maintenance requirements (120 mL/m²/ hour) should be titrated to maintain a urine specific gravity of 1.010 or less and normal urine output. Because of the risk of hyperkalemia, potassium should be avoided.

  Frequent serial laboratory monitoring is required during initiation of induction chemotherapy. Complete blood count (CBC), potassium, phosphorous, calcium, creatinine, blood–urea nitrogen (BUN), and uric acid should be assayed every 4 to 6 hours for the first 24 to 48 hours, then less frequently once stable.

Transfusions

Blood transfusion should be used to prevent complications related to severe cytopenias. To decrease the risk of transfusion-associated complications, specialized products should be used.

  Platelets: To prevent bleeding, platelet counts should routinely be maintained above 10,000/μL. Higher levels are recommended for management of bleeding, prior to invasive procedures such as lumbar puncture, and to reduce the risk of CNS hemorrhage related to leukostasis in the setting of hyperleukocytosis. Single donor platelets are recommended whenever possible to decrease donor exposure and the risk of HLA-alloimmunization.

  Red blood cells (RBCs): Concomitant anemia partially offsets the hyperviscosity associated with severe hyperleukocytosis. Thus, RBC transfusion should be avoided if possible when the WBC is higher than 100,000/μL. If transfusion is necessary, the hemoglobin and hematocrit should be increased slowly using small aliquots of packed RBCs until the peripheral blast count is reduced.

  Irradiation: To reduce the risk of transfusion-associated graft-versus-host disease, all cellular blood products should be irradiated.

  Leukodepletion: Platelets and red cells should be leuko-reduced to decrease the risk of febrile reactions, HLA-alloimmunization with subsequent platelet-refractoriness, and transmission of cytomegalovirus (CMV) infection.

Infection Prophylaxis

Aggressive surveillance, prophylaxis, and treatment for bacterial, fungal, viral, and opportunistic infections are essential to prevent morbidity and mortality.

  Pneumocystis jiroveci pneumonia (PCP): Patients should receive PCP prophylaxis with trimethoprim/ sulfamethoxazole continuing until 6 months after chemotherapy is completed.

  Neutropenic fever: Patients with an absolute neutrophil count (ANC) less than 500/μL and temperature 38.3°C or higher should be evaluated for possible infection and treated empirically with parenterally administered broad-spectrum antibiotics. Antifungal therapy should be initiated for neutropenic fever that persists for 5 to 7 days. Antibiotics should be continued until the ANC rises to above 500/μL, fever resolves, cultures are negative, and any suspected infection is fully treated.

  Intravenous immunoglobulin (IVIG): Hypogammaglobulinemia is common during treatment for ALL. Immunoglobulin G (IgG) levels should be assayed for those with recurrent infections, and if low, IVIG supplementation should be considered (approximately 500 mg/kg every 4 weeks as needed to maintain an IgG level of 500 mg/dL).

  Myeloid growth factors: Granulocyte colony-stimulating factor (G-CSF) during induction has been shown to improve outcome for adults, but no benefit was demonstrated in a pediatric study. Myeloid growth factor support should be employed during treatment of mature B-cell ALL (Burkitt or L3) in both children and adults.

Chemotherapy Prophylaxis

Agent-specific prophylaxis should be utilized as clinically indicated. For example, gastritis prophylaxis is recommended during corticosteroid administration. Leucovorin rescue is indicated to prevent severe toxicity after high-dose methotrexate. To reduce the risk of conjunctivitis associated with high-dose Ara-C, corticosteroid ophthalmic solution should be administered during and for 24 to 48 hours after treatment. Mesna should be used in an attempt to prevent hemorrhagic cystitis associated with high-dose ifosfamide and cyclophosphamide.

Nutritional Support

Nutritional status should be monitored and supplementation provided as indicated. Routine folic acid use should be avoided with methotrexate administration because it may counteract the therapeutic efficacy of folate antagonism.

Psychosocial Support

Multidisciplinary support for the patient and family is an important part of successful treatment.

EVALUATIONS

Serial evaluations to monitor for response, relapse, complications, and therapy-associated toxicity should be conducted throughout all treatment phases.

Evaluations during Treatment

  History, physical examination, and routine laboratory assessments including CBC and chemistry panel should be performed regularly throughout treatment.

  Bone marrow aspiration should be obtained at the following times:

  Induction day 7 or 14 to assess early response.

  Induction day 28 to assess remission status. If indeterminate, repeat every 1 to 2 weeks until recovery in order to confirm remission or induction failure.

  End of therapy.

  Suspected relapse.

  Flow cytometry, cytogenetics, and/or molecular genetic studies can be used to monitor minimal residual disease, which is prognostic.

  CSF cell count and cytology should be performed at the time of all intrathecal chemotherapy administrations. Lumbar puncture should also be performed if CNS relapse is suspected.

Evaluations after Treatment

  Follow-up evaluations to include history, physical examination, and routine laboratory studies (CBC, chemistry panel) should be conducted to monitor for toxicity and recurrent disease until at least 5 years after completion of treatment on the following schedule (or as clinically indicated):

  Every 1 to 2 months during the first year.

  Every 2 to 3 months during the second year.

  Every 3 to 4 months during the third year.

  Every 6 months during the fourth year.

  Yearly thereafter.

Late Effects

  Life-long follow-up to monitor for a variety of possible late complications of treatment is recommended.³⁴ The following are among the most frequent late effects:

  Cardiomyopathy: To decrease the risk of cardiotoxicity, cumulative anthracycline doses are usually limited to less than 400 mg/m². Echocardiograms for left ventricular function determination should be performed at baseline, at completion of treatment, every 1 to 2 years after treatment until serial studies remain normal, and as clinically indicated.

  Neurologic toxicity: Children are at especially high risk of neurotoxicity from chemotherapy and radiation. All patients should be monitored for neurologic toxicity including neurodevelopmental dysfunction.

  Endocrinologic dysfunction: Patients should be monitored for endocrinopathies including growth retardation, hypothyroidism, and infertility.

  Osteonecrosis: Corticosteroids, especially dexamethasone, are associated with a high incidence of osteonecrosis.

  Secondary malignancy: Patients should be monitored for secondary malignancies because these continue to develop even in the second decade after treatment.

References

  1.  Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med. 2006;354:166-178.

  2.  Bassan R, Hoelzer D. Modern therapy of acute lymphoblastic leukemia. J Clin Oncol. 2011;29:532-543.

  3.  Smith MA, Ries LA, Gurney JG, et al. Leukemia. In: Ries LA, Smith MA, Gurney JG, et al., eds. Cancer incidence and survival among children and adolescents: United States SEER Program 1975-1995. Bethesda, MD: National Cancer Institute, SEER Program; 1999. NIH Pub.No. 99-4649:17-34.

  4.  Mullighan CG. New strategies in acute lymphoblastic leukemia: translating advances in genomics into clinical practice. Clin Cancer Res. 2011;17(3):396-400.

  5.  Smith M, Arthur D, Camitta B, et al. Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia. J Clin Oncol. 1996;14:18-24.

  6.  Pui CH. Acute lymphoblastic leukemia. In: Pui CH., ed. Childhood Leukemias. 3rd ed. Cambridge, UK: Cambridge University Press; 2012:332-366.

  7.  Silverman LB, Stevenson KE, O’Brien JE, et al. Long-term results of Dana-Farber Cancer Institute ALL Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1985-2000). Leukemia. 2010;24(2):320-334.

  8.  Thomas DA, O’Brien S, Faderl S, et al. Chemoimmunotherapy with a modified hyper-CVAD and rituximab regimen improves outcome in de novo Philadelphia chromosome-negative precursor B-lineage acute lymphoblastic leukemia. J Clin Oncol. 2010;28(24):3880-3889.

  9.  Stock W. Adolescents and young adults with acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program. 2010;2010:21-29.

10.  Nachman JB, Sather HN, Sensel MG, et al. Augmented post-induction therapy for children with high-risk acute lymphoblastic leukemia and a slow response to initial therapy. N Engl J Med. 1998;338:1663-1671.

11.  Richards S, Burrett J, Hann I, et al. Improved survival with early intensification: combined results from the Medical Research Council childhood ALL randomised trials, UKALL X and UKALL XI. Leukemia. 1998;12:1031-1036.

12.  Durrant IJ, Prentice HG, Richards SM. Intensification of treatment for adults with acute lymphoblastic leukemia: results of U.K. Medical Research Council randomized trial UKALL XA. Br J Haematol. 1997;99:84-92.

13.  Huguet F, Leguay T, Raffoux E, et al. Pediatric-inspired therapy in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia: The GRAALL-2003 study. J Clin Oncol. 2009;27: 911-918.

14.  Barry E, DeAngelo DJ, Neuberg D, et al. Favorable outcome for adolescents with acute lymphoblastic leukemia treated on Dana-Farber Cancer Institute Acute Lymphoblastic Leukemia Consortium Protocols. J Clin Oncol. 2007;25:813-819.

15.  Pui CH, Pei D, Campana D, et al. Improved prognosis for older adolescents with acute lymphoblastic leukemia. J Clin Oncol. 2011;29(4):386-391.

16.  Barrett AJ, Horowitz MH, Pollock BH, et al. Bone marrow transplants from HLA-identical siblings as compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission. N Engl J Med. 1994;331:1253-1258.

17.  Pulsipher MA, Peters C, Pui CH. High-risk pediatric acute lymphoblastic leukemia: to transplant or not to transplant? Biol Blood Marrow Transplant. 2011;17(1 suppl):S137-S148.

18.  Hahn T, Wall D, Camitta B, Davies S, et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of acute lymphoblastic leukemia in children: an evidence-based review. Biol Blood Marrow Transplant. 2005;11:823-861.

19.  Brüggemann M, Gökbuget N, Kneba M. Acute lymphoblastic leukemia: monitoring minimal residual disease as a therapeutic principle. Semin Oncol. 2012;39(1):47-57.

20.  Campana D. Should minimal residual disease monitoring in acute lymphoblastic leukemia be standard of care? Curr Hematol Malig Rep. 2012;7(2):170-7. PubMed PMID: 22373809.

21.  Reiter A, Schrappe M, Ludwig WD, et al. Intensive ALL-type therapy without local radiotherapy provides a 90% event-free survival for children with T-cell lymphoblastic lymphoma: a BFM group report. Blood. 2000;95:416-421.

22.  Amylon MD, Shuster J, Pullen J, et al. Intensive high-dose asparaginase consolidation improves survival for pediatric patients with T cell acute lymphoblastic leukemia and advanced stage lymphoblastic lymphoma: a Pediatric Oncology Group study. Leukemia. 1999;13:335-342.

23.  Thomas DA, Faderl S, O’Brien S, et al. Chemoimmunotherapy with hyper-CVAD plus rituximab for the treatment of adult Burkitt and Burkitt-type lymphoma or acute lymphoblastic leukemia. Cancer. 2006;106:1569-1580.

24.  Pui CH, Campana D, Pei D, et al. Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med. 2009;360(26):2730-2741.

25.  Hijiya N, Liu W, Sandlund JT, et al. Overt testicular disease at diagnosis of childhood acute lymphoblastic leukemia: lack of therapeutic role of local irradiation. Leukemia. 2005;19:1399-1403.

26.  Bassan R, Rossi G, Pogliani M, et al. Chemotherapy-phased imatinib pulses improve long-term outcome of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: Northern Italy Leukemia Group protocol 09/00. J Clin Oncol. 2010;28:3644-3652.

27.  Schultz K, Bowman WP, Aledo A, et al. Improved early event-free survival with imatinib in Philadelphia chromosome-positive acute lymphoblastic leukemia: a Children’s Oncology Group study. J Clin Oncol. 2009;27: 5175-5181.

28.  Kantarjian H, Thomas D, Wayne AS, O’Brien S. Monoclonal antibody-based therapies: a new dawn in the treatment of acute lymphoblastic leukemia. J Clin Oncol. 2012;30(31):3876-83.

29.  Hoelzer D, Gokbuget N. Chemoimmunotherapy in acute lymphoblastic leukemia. Blood Rev. 2012;26:25-32.

30.  Ko RH, Ji L, Barnette P, Bostrom B, et al. Outcome of patients treated for relapsed or refractory acute lymphoblastic leukemia: a Therapeutic Advances in Childhood Leukemia Consortium study. J Clin Oncol. 2010;28:648-654.

31.  Buchanan GR, Boyett JM, Pollock BH, et al. Improved treatment results in boys with overt testicular relapse during or shortly after initial therapy for acute lymphoblastic leukemia. A Pediatric Oncology group study. Cancer. 1991;68(1):48-55.

32.  Ritchey AK, Pollock BH, Lauer SJ, et al. Improved survival of children with isolated CNS relapse of acute lymphoblastic leukemia: a pediatric oncology group study. J Clin Oncol. 1999;17(12):3745-3752.

33.  Howard SC, Jones DP, Pui CH. The tumor lysis syndrome. N Engl J Med. 2011;364(19):1844-1854.

34.  Oeffinger KC, Mertesn AC, Sklar CA, et al. Chronic health conditions in adult survivors of childhood cancer. New Engl J Med. 2006;355:1572-1582.

13

Chronic Myelogenous Leukemia

Agnes S.M. Yong and A. John Barrett

Although chronic myelogenous leukemia (CML) is rare, it has achieved great prominence in medical literature because its biologic basis has been elucidated in unprecedented detail. As a result, CML became the model for the development of effective molecularly targeted and immune-based treatments for leukemia. Nowell and Hungerford reported the unique, unusually small G group chromosome in patients with CML in 1960 and named it the Philadelphia (Ph) chromosome¹ This was the first association of a human malignant disease with a consistent chromosomal marker. In 1973, the Ph chromosome was identified as the truncated chromosome 22 consequent to a reciprocal translocation involving chromosome 9.² It was not until the 1980s that the fusion partners of the translocation were identified as the ABL1 oncogene on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22.3,4 The BCR-ABL1 oncoprotein was found to have tyrosine kinase activity, and when the gene was inserted into mouse stem cells it induced leukemia in recipient animals.⁵ Until the 1990s, allogeneic stem cell transplantation (SCT) was the preferred first-line treatment in eligible CML patients as the disease is highly susceptible to a graft-versus-leukemia effect from transplanted donor lymphocytes.⁶ The advent of imatinib mesylate (Gleevec), the first of a new class of small molecule drugs designed specifically to block the BCR-ABL1 tyrosine kinase, has supplanted SCT for the majority of patients as these drugs confer durable disease control, particularly in the earlier stages of CML.⁷ Second-generation tyrosine kinase inhibitors (TKIs) dasatinib and nilotinib, which are more potent pharmacologically, have recently been shown to be more efficient in rapidly reducing the leukemic load compared to imatinib, and are now advocated as first-line treatment for CML.8,9 Despite progress in CML biology and treatment, fundamental questions about its origin remain unanswered. Evidence suggests that a predisposition to develop CML precedes the clonal expansion of BCR-ABL1 translocated stem cells,¹⁰ and the discovery of very low levels of BCR-ABL1 in the blood of normal individuals who do not develop CML raises the possibility that the BCR-ABL1 translocation alone is not sufficient to cause leukemia.¹¹ The emergence of drug resistance to TKIs in the era of molecular targeting for CML has driven the development of third-generation TKIs such as ponatinib. However, only a proportion of TKI-resistant CMLs are attributable to mutations of the BCR-ABL1 kinase domain. Drugs targeting alternate non-kinase dependent and stem cell pathways are also being pursued. Unfortunately, advanced phases of CML still are largely refractory to available treatments.

EPIDEMIOLOGY

Rare: incidence of 1.5 in 100,000

Represents 10% to 15% of all leukemias

Incidence increases with age (median age of diagnosis = 65); exceedingly rare in children Male predominance (1.5:1)

Worldwide distribution: no sociogeographic preponderance

Ionizing radiation is the only known causative factor, leukemia occurs usually within 6 to 8 years of exposure

No known genetic factors determine susceptibility to CML

PATHOPHYSIOLOGY

Leukemic Hematopoiesis Originates in a Multipotent Stem Cell

BCR-ABL1 translocation is found in all cells of the myeloid lineage (erythroid and granulocyte precursors and megakaryocytes) as well as in B cells but not in T cells.¹² There are two major hypotheses for this observation: first, the acquisition of BCR-ABL1 may occur in a multipotent stem cell with little or no T differentiation capacity; second, T cells bearing BCR-ABL1 may be systematically eliminated. Unregulated proliferation of BCR-ABL1-positive stem cells is responsible for massive expansion, primarily in granulocyte production, that leads to leukocytosis.

Clonal Dominance

The BCR-ABL1-positive clone outcompetes normal hematopoiesis. At diagnosis it is common to find a mixed population of Ph-positive and Ph-negative cells in the bone marrow. With time, normal stem cells are progressively replaced by CML stem cells. CML CD34+ progenitor cells require less hematopoietic growth factors than do normal progenitors for survival and proliferation, a characteristic that may be partially due to the presence of an autocrine production of hematopoietic growth factors by CML cells.¹³

Molecular Basis of Chronic Myelogenous Leukemia in the BCR-ABL1 Translocation¹⁴

The BCR-ABL1 oncoprotein is a constitutively activated tyrosine kinase that phosphorylates intermediate molecules in several important pathways, affecting proliferation, maturation, resistance to apoptosis, and cell adhesion, ultimately resulting in the typical leukemic phenotype.

Genomic Instability

CML is characterized by progression to refractory acute leukemia. CML usually starts as a relatively benign disorder that evolves to an accelerated phase (AP), when the leukemia is much more difficult to control and additional chromosomal abnormalities appear, followed by a progressive increase in blast cells in blood and marrow, termed the blastic phase (BP) or blast crisis, when the disease transforms to an acute myeloid or B lymphocytic leukemia.¹⁵ Clonal evolution, which is matched by increasing malignant behavior of the leukemia, has a variable pace but is inevitable.

PRESENTATION

Classic Presentation

CML presents with an insidious history of increasing fatigue, lassitude, weight loss, night sweats, massive splenomegaly, and gout. Some patients have leukocyte counts greater than 300 × 10⁹/L and experience symptoms of leukostasis with headache, focal neurologic deficits, and priapism.

Typical Presentation in the Developed World

Overt symptoms and signs are rarely encountered because the diagnosis is made earlier. Commonly, patients present with fatigue, with or without moderate weight loss, abdominal discomfort, and early satiety from an enlarged spleen, or simply due to the chance observation of an elevated leukocyte count. CML should be considered in the differential diagnosis of a patient at any age presenting with splenomegaly and with white blood cell count.

Rare Presentations

Rare presentations include chloroma, petechiae, and bruising. These features suggest progression of CML to an accelerated or blastic phase. Unlike other leukemias, CML seldom if ever presents with bacterial or fungal infection because neutrophil function is preserved.

DIAGNOSIS

While blood and bone marrow examinations share features with other myeloproliferative disorders, the typical presentation with high leukocyte count and a hypercellular marrow with basophilia is pathognomonic of CML. Chromosome and molecular analysis confirm the presence of a BCR-ABL1 translocation.

Blood Count

Leukocyte number varies between being slightly elevated to over 200 × 10⁹/L; counts as high as 700 × 10⁹/L are occasionally encountered. The platelet count is normal or elevated and there is often a mild normochromic normocytic anemia.

Blood Film

Blood film is of great diagnostic value, because many of the typical features of CML are unique—a left shift with circulating myeloblasts, myelocytes, metamyelocytes, and band forms. The hallmark of CML is basophilia with basophil counts often exceeding 1 × 10⁹/L. Sustained basophilia is almost never encountered outside CML and some cases of mastocytosis. Eosinophilia and occasional nucleated red blood cells are also common findings. Platelet morphology is usually normal but giant forms can be seen.

Bone Marrow

The aspirate shows cellular spicules and the biopsy is hypercellular with almost complete effacement of the fat spaces. There is granulocytic hyperplasia of the neutrophil, eosinophil, and basophil series. Megakaryocytes are normal or increased and may show reduced numbers of nuclei. Sea-blue histiocytes are commonly seen in CML marrows. Fibrosis of the marrow is a feature of accelerated phase CML, as is an increase in blasts over 10%. Blastic phase shows more than 20% blasts.

Chromosome Analysis

The typical karyotype of CML shows the reciprocal translocation t(9;22)(q34;q11) (Fig. 13.1). Variants include three-way translocations between chromosomes 9, 22 and 11, or 19. An additional chromosome abnormality or Ph chromosome duplication usually indicates a more advanced disease stage. Fluorescence in situ hybridization (FISH) is a rapid and sensitive technique to detect the Ph chromosome directly in blood or marrow since it does not rely on dividing cells.

Molecular Diagnosis

More than 95% of patients presenting with the clinical and morphologic features of CML will have Ph chromosomes in the marrow. Of the 5% who are Ph-negative, half have a cryptic BCR-ABL1 transcript detected by polymerase chain reaction (PCR). The remaining patients are described as atypical Ph-negative CML. A few such cases are morphologically indistinguishable from Ph-positive CML but most have atypical features on careful examination and are classified under myelodysplastic/myeloproliferative neoplasms.¹⁶ Molecular analysis provides further information of the precise transcript. Depending on the BCR breakpoint, four common BCR-ABL1 transcript variants are possible: e13a2 and e14a2 (formerly b2a2 and b3a2), both encoding the 210 kD BCR-ABL1 oncoprotein (p210); e1a2, which is more common in Ph-positive acute lymphoblastic leukemia, encoding the 190 kD BCR-ABL1 oncoprotein (p190); and e19a2, which encodes the 230 kD BCR-ABL1 oncoprotein of chronic neutrophilic leukemia. No prognostic or diagnostic significance is attached to either the e13a2 or the e14a2 variant in CML. Rarely, unusual transcript variants, such as e1a3 or e6a2, have also been described.¹⁷

FIGURE 13.1 The Philadelphia chromosome. G-banded metaphase preparation showing the diminutive Ph-positive chromosome and extra chromosomal material on the long arm of chromosome 9.

DIFFERENTIAL DIAGNOSIS

The diagnosis of CML is made in three stages (Fig. 13.2):

  Persisting leukocytosis without any obvious infective cause suggests a myeloproliferative neoplasm, prompting further examination of the blood and bone marrow.

  Morphology and blood count will show either typical features of CML (basophilia being especially significant) or suggest other myeloproliferative neoplasms (high platelet counts, essential thrombocythemia; high red cell count, polycythemia vera; teardrop red cells, myelofibrosis). Dysplasia suggests a hyperproliferative myelodysplastic syndrome.

  Definitive diagnosis requires chromosome analysis of the bone marrow. Cytogenetics will identify the Ph-positive chromosome and the BCR-ABL1 translocation in all but a small percentage of patients with a morphologic diagnosis of CML. Confirmation of the presence of BCR-ABL1 transcripts by PCR is advised as it aids in disease monitoring following treatment¹⁸ (Fig. 13.3).

COURSE OF CHRONIC MYELOGENOUS LEUKEMIA

CML is a multistage disease that progresses from chronic phase (CP) to AP and then to BP (Fig. 13.4).

Chronic Phase

Untreated patients in CP show a gradual rise in the leukocyte count with emergence of splenomegaly and ultimately the full picture of a myeloproliferative neoplasm with B symptoms, weight loss, and hyperleukocytosis.

FIGURE 13.2 Differential diagnosis of CML and related disorders.

Duration of CP is highly variable: some patients progress within months of diagnosis directly to AP and BP, while others can remain for more than a decade in stable CP. Sometimes patients present in AP or BP without a clear preceding CP; in this circumstance, it is important to distinguish CML presenting as acute leukemia from de novo (Ph-negative) acute leukemia since the treatment approaches are distinct.

Median time to progression from CP to AP has increased in the era of TKIs, and because the diagnosis of CML tends to be made earlier.

FIGURE 13.3 PCR for BCR-ABL1 transcripts to monitor treatment in CML. (Courtesy S Branford, IMVS/SA Pathology, Adelaide, Australia.)

FIGURE 13.4 Course and clonal evolution of CML.

Accelerated Phase

AP is characterized by one or more of the following¹⁹ (Table 13.1):

Clonal evolution by a further mutation. Patients may acquire new chromosomal abnormalities such as a second Ph chromosome.

Escape of the blood counts from treatment control.

Organomegaly.

Leukocytosis, basophilia, thrombocytosis, or thrombocytopenia in a patient previously well controlled with therapy.

Myelofibrosis with teardrop cells in the blood smear and increased marrow reticulin.

Chloromas in external soft tissues, the retroperitoneal spaces, paraspinal areas (leading to nerve root compression) and in intramedullary spaces.

Blastic Phase

Signs and symptoms of acute leukemia: bone pains, weight loss, and B symptoms, increasing numbers of blasts in the blood and marrow.

Marrow failure: decreasing red cell count and platelets. (Neutrophil counts are better conserved.)

Clonal evolution: further chromosomal abnormalities.

Characterization of Acute Leukemia in Blastic Phase

Approximately 60% of patients develop myeloid BP resembling acute myeloid leukemia (AML); the remainder has lymphoid BP reminiscent of acute lymphoblastic leukemia (ALL). In both phenotypes, blasts are poorly differentiated. Auer rods are not seen and the lymphoid or myeloid origin of the leukemia is only reliably determined by cytochemical stains and surface phenotype, revealing either a pre-B ALL (PAS block-positive, TdT-positive, CD10⁺, CD19⁺, CD33±, CD34±) or an undifferentiated AML (peroxidase weak-positive, CD33⁺, CD34⁺, CD13±). A peculiar feature of CML is the variability of its subsequent evolution. Patients achieving remission from AML can re-enter CP only to relapse again with ALL or vice versa.

PROGNOSTIC FACTORS

Poor prognosis (tendency to rapid progression to BP):

High leukocyte counts (>100 × 10⁹/L).

Massive splenomegaly and constitutional symptoms.

Patients of African origin.

High basophil counts.

Of predictive scores using patient characteristics at diagnosis which were validated in the preimatinib era to determine outcome and survival,20,21 the Sokal score still appears to be prognostic in patients who are treated with imatinib.7,22,23

TREATMENT

Treatment of CML involves diverse, evolving approaches¹⁸ outlined in the algorithm in Figure 13.5. The drugs commonly used to treat CML are detailed in Table 13.2.

CML treatment is guided by disease monitoring using regular blood counts and bone marrow examination to document hematologic changes, chromosome analysis of marrow or FISH analysis of blood or marrow to detect response or progression at the karyotypic level, and PCR for BCR-ABL1 mRNA transcripts in the blood to quantify response at the molecular level (see Fig. 13.3). The degree of disease bulk reduction determines the appropriate monitoring approach, and the degree of response is defined as hematologic response, cytogenetic response, and molecular response or complete molecular remission¹⁸ (Fig. 13.6).

Treating Newly Diagnosed Chronic Myelogenous Leukemia in Chronic Phase

The great majority (>80%) of CML patients is diagnosed in CP. Initial treatment is aimed at reducing disease bulk and obtaining hematologic remission (normalization of blood counts). Subsequent therapy is tailored toward achieving either a “cure” or “minimal residual disease” (MRD).

Imatinib, 400 mg daily.

Add hydroxyurea 0.5 to 2.5 g daily for patients with leukocyte counts over 100 × 10⁹/L or with massive splenomegaly.

Allopurinol, 300 mg daily until blood counts normalize.

Monitoring Response to Imatinib18,24

Full blood counts should be monitored every 2 weeks until complete hematologic response (HR), equivalent to normalization of blood counts, is achieved. Complete HR should be confirmed on two subsequent occasions.

Bone marrow aspiration every 6 months to assess cytogenetic response, analyzing at least 20 metaphases for the Ph chromosome. Patients who achieve complete cytogenetic response (0% Ph) have a prolonged period without disease progression (see definitions in Fig. 13.6). The cytogenetic response improves over time in responding patients, and once complete cytogenetic response is achieved and confirmed on two subsequent occasions, bone marrow examinations for cytogenetics can be performed every 12 months to detect possible onset of dysplasia or clonal changes in the Ph-negative cells.²⁵

Quantitative PCR for BCR-ABL1 transcripts in the blood should be performed at least every 3 months. Serial BCR-ABL1 measurements are clinically useful to document if patients are responding to treatment with declining transcripts, have stable (plateau) levels of transcripts, or are losing their response as signaled by rising transcripts. Reduction of BCR-ABL1 transcripts by 3 or more logs below a standardized baseline value for untreated patients (major molecular response) is associated with particularly good outcome.

FIGURE 13.5 Treatment algorithm for CML.

FIGURE 13.6 Disease monitoring in CML.

Achieving Minimal Residual Disease

Administer imatinib at the maximum tolerated dose (up to 800 mg daily). Continue treatment indefinitely, unless loss of response occurs (see below).

Over 85% of CML-CP patients treated with imatinib from diagnosis achieve a complete cytogenetic response (0% Ph) and of these, 80% have a 3 log reduction in BCR-ABL1 transcripts by the 4-year follow-up.⁷ This MRD status is associated with longer survival. Upfront treatment with imatinib may reduce the number of leukemic progenitors at risk of clonal evolution and disease progression. CP-CML patients who are treated with imatinib from diagnosis and achieve complete cytogenetic response appear to have decreasing annual rates of disease progression to AP or BP with longer follow-up.²⁴ Although the ultimate duration of imatinib treatment is still unclear, current recommendations are to continue treatment until relapse or progression of disease.¹⁸ A complete molecular remission (undetectable BCR-ABL1 transcripts in the blood by PCR) is achieved by less than 10% of patients in complete cytogenetic remission. Imatinib has been discontinued in patients with complete molecular remission for a minimum of 2 years in two clinical trials in France and Australia26,27 and found that up to 60% of patients relapse within a few months of cessation, suggesting that imatinib does not totally eradicate CML in most patients. Monitoring of patients who have not yet relapsed is ongoing, but some patients have ceased imatinib for up to 5 years without relapse, reflecting the heterogeneity of either disease biology or immune control.

Suboptimal Response or Loss of Response to Imatinib Treatment

Failure to achieve a hematologic remission with a combination of imatinib and hydroxyurea is uncommon unless the disease has already progressed to AP.

Failure of imatinib treatment¹⁸

  No HR in 3 months

  No cytogenetic response (Ph >95%) in 6 months

  Less than partial cytogenetic response (Ph >35%) in 12 months

  No complete cytogenetic response (any Ph detected) in 18 months

  Loss of previously achieved responses—for example, loss of complete HR, or complete cytogenetic response.

Suboptimal response to imatinib treatment¹⁸

  No cytogenetic response (Ph >95%) in 3 months

  Less than partial cytogenetic response (Ph >35%) in 6 months

  No complete cytogenetic response (any Ph detected) in 12 months

  No major molecular response in 18 months

  Loss of previously achieved responses: loss of major molecular response; additional chromosomal abnormalities in Ph+ cells in serial bone marrow examinations.

Patients who lose an initial response to imatinib may have developed drug resistance due to point mutations of the BCR-ABL1 gene, which result in amino acid changes in the catalytic domain of the BCR-ABL1 protein (“kinase domain mutation”), resulting in impaired imatinib binding.²⁸ Alternatively, CML may have progressed to AP or BP. A bone marrow aspirate is indicated to determine the status of the disease, and analysis for kinase domain mutations should be undertaken.

When stable MRD is not achieved or disease progression not responding to higher doses of imatinib is encountered, treatment with second-generation inhibitors of BCR-ABL1 tyrosine kinase such as dasatinib or nilotinib is indicated.¹⁸

Patients who have not achieved a satisfactory cytogenetic response or who have progressed after an initial response to TKIs should be offered an allogeneic SCT from a human leukocyte antigen (HLA)-identical sibling or a well-matched unrelated donor.

For patients unsuitable for SCT or without a matched donor, cytosine arabinoside (ARA-C) and interferon-α (IFN-α) improve the degree of response in a proportion of cases. Some patients may benefit from enrolling in clinical trials investigating the efficacy of combining TKIs with ARA-C or IFN-α, or exploring the use of SCT from the alternative donor sources of cord blood or haploidentical SCT. More experimental approaches with novel TKIs, aurora kinase inhibitors, autologous SCT, and peptide vaccines are also being evaluated.

Allogeneic Stem Cell Transplantation:Treatment with Curative Intent

SCT from an HLA-matched sibling in CP within a year of diagnosis achieves long-term disease control and survival of 70% (approximately 60% for patients with CP who undergo transplantation more than a year from diagnosis). Age has a major impact on outcome, results being especially favorable for the minority pediatric CML population, while patients older than 40 years of age have lower disease-free survival (DFS). Disease stage is the other major variable affecting transplantation success. Both transplant-related mortality (TRM) and relapse are higher in transplants performed for AP and BP (Fig. 13.7); however, patients who achieve a second CP have a better chance of DFS and results have improved in the imatinib era. Most reported results analyze survival in the first 5 years. Longer-term follow-up indicates that late relapses and deaths from chronic graft-versus-host disease (GVHD) continue to cause late mortality many years after transplant. In evaluating outcome after transplant for CML, measuring DFS underestimates the final cure rate because donor lymphocyte infusions can cure relapsed disease. In summary, the long-term, allogeneic SCT from a matched sibling results in cure in approximately 65% of individuals in CML CP. In the imatinib era where SCT is usually a second-line option for imatinib resistance, DFS of greater than 50% can be expected using reduced intensity SCT and applicable to patients up to the age of 75.

Unrelated Donor Transplants

There is now a large experience in transplants for CML using unrelated volunteer donors. Age, timing of transplant (early or late CP, more advanced disease), and degree of matching each strongly affects the success of the transplant. For low-risk patients (defined as younger than 40 years, in first CP, less than 1 year from diagnosis and with an HLA-matched unrelated donor) DFS of approximately 60% can be achieved; poorer results can be anticipated from patients with less favorable presentations. However, reduced intensity transplants have improved the outlook for older patients. Thus it is appropriate to offer lower intensity SCT to patients with CML up to the age of 70 who have no significant comorbidities.

FIGURE 13.7 Probability of survival after HLA-identical sibling donor transplants for CML from 1998 to 2009 by disease status and year.

Selecting Patients for Allogeneic Transplantation

Gratwohl et al.²⁹ described a simple scoring system to predict the chance of a successful transplant outcome (Table 13.3). SCT is no longer recommended as primary therapy for CP-CML for the majority of patients.³⁰ However, as SCT has a definite curative potential with long-term survival documented over many decades, it is still appropriate to recommend it for young patients under the age of 30 who are expected to have a particularly low morbidity and mortality, and also in circumstances in which there is difficulty obtaining TKIs due to economic reasons. For other patients, imatinib is the first-line treatment, followed by other TKIs, and SCT is reserved for patients who fail to respond, progress, or present with CML beyond CP.

Treatment of Accelerated Phase

Allogeneic SCT is the most substantiated curative therapy for CML AP and should be offered to patients with a fully or partially matched HLA-identical donor. However, up to 40% of patients with CML AP respond to imatinib 600 mg daily.¹⁶ Furthermore, second-generation TKIs are effective in imatinib-resistant disease. Alternatively interferon in combination with ARA-C can achieve disease control. Experimental treatment approaches include SCT from a mismatched-related donor and high-dose chemotherapy or radiation followed by autologous SCT.

Treatment of Blastic Phase

The first step in managing CML BP is to determine whether the leukemia has developed into lymphoid or myeloid BP. The average survival upon progression to BP is between 6 and 10 months, slightly longer for lymphoid BP.³¹ Patients who have not already developed resistance to imatinib should be treated immediately with 800 mg daily. Many patients treated de novo with imatinib will have a complete or at least a partial response. Imatinib-refractory patients may benefit from second-generation BCR-ABL inhibitors such as dasatinib or nilotinib, but those with rapidly progressing leukemia require induction chemotherapy with standard regimens—ALL-like for lymphoid BP: daunorubicin 45 mg/m² days 1 and 2, vincristine 2 mg/m² weekly, prednisolone 60 mg/m² daily for 3 weeks, and AML-like for patients with myeloid BP: daunorubicin 50 mg/m² daily for 2 to 3 days with ARA-C 200 mg/m² daily for 5 to 7 days. Lymphoid BP patients also require prophylactic central nervous system treatment to prevent meningeal leukemia. Up to 40% of patients achieve a second CP but most relapse rapidly. Allogeneic SCT, although considered salvage therapy and associated with significant TRM in patients who have progressed to BP, offers the only chance of cure and should be offered to eligible patients. A significant number of patients have prolonged cytopenia after successful eradication of blasts; for this reason moderate intensity remission induction chemotherapy is often used (such as “2 + 5” for myeloid BP, and avoidance of high-dose ARA-C regimens such as hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone) for patients with lymphoid BP). Sometimes further remissions can be induced, especially when the relapse occurs in the alternate lineage. Patients who have few clinical options may benefit from entering clinical trials of investigational agents.

SPECIAL ISSUES IN CHRONIC MYELOGENOUS LEUKEMIA MANAGEMENT

Treatment of Relapse after Transplantation

Durable molecular remissions after donor lymphocyte infusion (DLI) are achieved between 3 and 12 months after DLI in as many as 80% of patients relapsing in CP and in more than 90% of molecular relapses. Predictably, the occurrence of GVHD results in a much higher probability of leukemic response, and the antileukemic effect of DLI is greatest in the absence of immunosuppression. While DLI is often effective, it may cause bone marrow failure and lethal GVHD. Bone marrow failure is a greater risk in patients with no detectable residual donor marrow cells at relapse. Marrow aplasia in these patients can be prevented or treated by infusing more donor stem cells. Despite concerns that DLI in the setting of unrelated donor transplants would result in excessive toxicity, response and durable remission rates are similar to those seen after matched-sibling DLI. GVHD remains a hazard but does not appear to be more frequent or more severe than that encountered after matched-sibling DLI. Imatinib has been combined successfully with DLI in treatment of relapse.³²

  Leukostasis is an uncommon problem in CML and only occurs in a minority of patients with high leukocyte counts (over 300 × 10⁹/L). In patients with priapism or neurologic deficit, emergency leukapheresis can be effective but may require several large-volume apheresis sessions to lower the leukocyte count significantly. At presentation such patients should receive high-dose hydroxyurea (up to 4 g daily) or ARA-C 1 g/m² daily for 2 to 3 days with allopurinol 300 mg daily, adequate hydration, and monitoring of blood chemistry. Imatinib may be started once control of the leukocyte count has been achieved.

  Splenic infarcts usually occur when the disease is uncontrolled. Treatment is symptomatic, while attempts to lower the blood count are made. Splenectomy is not generally indicated.

  Myelofibrosis causing significant cytopenias can be treated by splenectomy but this maneuver is frequently followed by increasing symptomatic hepatomegaly. Myelofibrosis is not a contraindication for allogeneic SCT and diminishes after successful SCT.

  Chloromas often respond poorly to chemotherapy and are best treated with local radiotherapy.

Psychological Responses of Patients with CML

Psychological responses of patients with CML to their disease. Patients presenting with CML are often asymptomatic and may have difficulty accepting that they have a potentially lethal disease. Perhaps for this reason some will explore alternative treatments and attempt psychosomatic techniques to control their leukemia. The complexity of disease evolution and the dilemmas of treatment in CML make it essential to educate patients about their leukemia, in order to provide them an informed basis for making treatment decisions.

EVOLVING STANDARD OF CARE

Second-generation Tyrosine Kinase Inhibitors or Imatinib in Combination with Interferon-α as First-line Treatment

There is currently debate regarding whether CML patients diagnosed in CP should have dasatinib or nilotinib as first-line therapy rather than imatinib. Two recent trials8,9 have shown a more rapid achievement of therapeutic endpoints with upfront dasatinib or nilotinib compared with imatinib. However, survival benefit has not been demonstrated, and these second-generation TKIs are generally more costly than imatinib. In view of the shorter clinical experience with second-generation TKIs, their long-term side effects with prolonged therapy are also unknown. There are ongoing clinical trials accruing to address these questions. In favor of earlier switching to second-line treatment is a recent study which found that assessment of BCR-ABL1 transcript levels at 3 months is the only requirement for predicting outcome for patients treated with any TKI.³³

Combination therapy of imatinib with IFN-α has been shown to achieve better molecular responses compared to imatinib alone in some recent clinical trials³⁴⁻³⁶ but not in others.³⁷ Survival benefit has not been demonstrated in patients receiving combination therapy, who generally had more side effects than those on imatinib alone.

Third-Generation Tyrosine Kinase Inhibitors

BCR-ABL1 kinase domain mutations such as T315I which are highly resistant to imatinib, dasatinib, and nilotinib in many patients herald the onset of advanced phase CML. Ponatinib, a third-generation TKI with activity against T315I mutations, has shown very promising results in clinical trials, with a good balance of efficacy and acceptable side-effect profile compared to other emerging therapies for resistant CML.

References

  1.  Nowell PC, Hungerford DA. A minute chromosome in human chronic granulocytic leukemia. Science. 1960;132:1497-1497.

  2.  Rowley JD. Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293.

  3.  Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984;36:93-99.

  4.  Heisterkamp N, Stephenson JR, Groffen J, et al. Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia. Nature. 1983;306:239-242.

  5.  Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 1990;247:824-830.

  6.  Kolb HJ. Graft-versus-leukemia effects of transplantation and donor lymphocytes. Blood. 2008;112:4371-4383.

  7.  Druker BJ, Guilhot F, O’Brien SG, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med. 2006;355:2408-2417.

  8.  Kantarjian H, Shah NP, Hochhaus A, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2010;362:2260-2270.

  9.  Saglio G, Kim DW, Issaragrisil S, et al. Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med. 2010;362:2251-2259.

10.  Raskind WH, Ferraris AM, Najfeld V, Jacobson RJ, Moohr JW, Fialkow PJ. Further evidence for the existence of a clonal Ph-negative stage in some cases of Ph-positive chronic myelocytic leukemia. Leukemia. 1993;7:1163-1167.

11.  Biernaux C, Loos M, Sels A, Huez G, Stryckmans P. Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals. Blood. 1995;86:3118-3122.

12.  Takahashi N, Miura I, Saitoh K, Miura AB. Lineage involvement of stem cells bearing the Philadelphia chromosome in chronic myeloid leukemia in the chronic phase as shown by a combination of fluorescence-activated cell sorting and fluorescence in situ hybridization. Blood. 1998;92:4758-4763.

13.  Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine production and action of IL-3 and granulocyte colony- stimulating factor in chronic myeloid leukemia. Proc Natl Acad Sci U S A. 1999;96:12804-12809.

14.  Quintas-Cardama A, Cortes J. Molecular biology of bcr-abl1-positive chronic myeloid leukemia. Blood. 2009;113:1619-1630.

15.  Perrotti D, Jamieson C, Goldman J, Skorski T. Chronic myeloid leukemia: mechanisms of blastic transformation. J Clin Invest. 2010;120:2254-2264.

16.  Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937-951.

17.  Melo JV. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood. 1996;88:2375-2384.

18.  Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol. 2009;27:6041-6051.

19.  Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood. 2002;100:2292-2302.

20.  Hasford J, Pfirrmann M, Hehlmann R, et al. A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. Writing Committee for the Collaborative CML Prognostic Factors Project Group. J Natl Cancer Inst. 1998;90:850-858.

21.  Sokal JE, Cox EB, Baccarani M, et al. Prognostic discrimination in “good-risk” chronic granulocytic leukemia. Blood. 1984;63:789-799.

22.  Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med. 2003;349:1423-1432.

23.  White D, Saunders V, Lyons AB, et al. In-vitro sensitivity to imatinib-induced inhibition of ABL kinase activity is predictive of molecular response in de-novo CML patients. Blood. 2005;106:2520-2526.

24.  Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108:28-37.

25.  Hochhaus A, O’Brien SG, Guilhot F, et al. Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia. Leukemia. 2009;23:1054-1061.

26.  Mahon FX, Rea D, Guilhot J, et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11:1029-1035.

27.  Ross DM, Branford S, Seymour JF, et al. Patients with chronic myeloid leukemia who maintain a complete molecular response after stopping imatinib treatment have evidence of persistent leukemia by DNA PCR. Leukemia. 2010;24:1719-1724.

28.  Weisberg E, Manley PW, Cowan-Jacob SW, Hochhaus A, Griffin JD. Second generation inhibitors of BCR-ABL for the treatment of imatinib-resistant chronic myeloid leukaemia. Nat Rev Cancer. 2007;7:345-356.

29.  Gratwohl A, Hermans J, Goldman JM, et al. Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow transplantation. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Lancet. 1998;352:1087-1092.

30.  Gratwohl A, Heim D. Current role of stem cell transplantation in chronic myeloid leukaemia. Best Pract Res Clin Haematol. 2009;22:431-443.

31.  Wadhwa J, Szydlo RM, Apperley JF, et al. Factors affecting duration of survival after onset of blastic transformation of chronic myeloid leukemia. Blood. 2002;99:2304-2309.

32.  Savani BN, Montero A, Kurlander R, Childs R, Hensel N, Barrett AJ. Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant. 2005;36:1009-1015.

33.  Marin D, Ibrahim AR, Lucas C, et al. Assessment of BCR-ABL1 transcript levels at 3 months is the only requirement for predicting outcome for patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. J Clin Oncol. January 2012;30(3):232-238.

34.  Palandri F, Castagnetti F, Iacobucci I, et al. The response to imatinib and interferon-alpha is more rapid than the response to imatinib alone: a retrospective analysis of 495 Philadelphia-positive chronic myeloid leukemia patients in early chronic phase. Haematologica. 2010;95:1415-1419.

35.  Preudhomme C, Guilhot J, Nicolini FE, et al. Imatinib plus peginterferon alfa-2a in chronic myeloid leukemia. N Engl J Med. 2010;363:2511-2521.

36.  Simonsson B, Gedde-Dahl T, Markevarn B, et al. Combination of pegylated IFN-alpha2b with imatinib increases molecular response rates in patients with low- or intermediate-risk chronic myeloid leukemia. Blood. 2011;118:3228-3235.

37.  Hehlmann R, Lauseker M, Jung-Munkwitz S, et al. Tolerability-adapted imatinib 800 mg/d versus 400 mg/d versus 400 mg/d plus interferon-alpha in newly diagnosed chronic myeloid leukemia. J Clin Oncol. 2011;29:1634-1642.

14

Chronic Lymphocytic Leukemia

Mohammed Z.H. Farooqui, Adrian Wiestner and Georg Aue

BACKGROUND

Chronic lymphocytic leukemia (CLL) is a disorder of morphologically mature but immunologically incompetent B lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues.¹ CLL accounts for about 25% of all leukemia and is the most common leukemia in Western countries. Incidence increases with age from <1/100,000 individuals younger than 40 years of age to >20/100,000 in persons over age 65 and it is almost twice as frequent in men as in women. CLL is predominantly found in Caucasians and is rare in Hispanics and Asians. In 2011 there were 14,570 new cases of CLL in the United States, causing 4,380 deaths.²

ETIOLOGY AND PATHOGENESIS

CLL has historically been viewed as a disease of clonal B cells that have a low proliferation rate and a defect in apoptosis.¹ CLL cells express the B cell receptor for antigen and antigenic stimulation of the clonal cells appears to be a major pathogenic driver. Recent work highlights the importance of proliferation that occurs primarily in the tissue microenvironment of the bone marrow and lymph nodes. Key pathways promoting CLL cell proliferation and survival in these tissues are activation of the B cell receptor and NF-kB pathways.³

A positive family history is one of the strongest risk factors for the development of CLL. There is an increased incidence of CLL with exposure to Agent Orange. Otherwise, no clear environmental factors predisposing to CLL have been identified.

CLINICAL MANIFESTATIONS

The most common initial manifestation of CLL is the incidental detection of lymphocytosis on a routine blood test or the presence of asymptomatic lymphadenopathy. Abdominal fullness, fatigue, reduced exercise tolerance, or other constitutional symptoms can also be presenting complaints. Symptoms can precede the onset of anemia or clinically manifest organomegaly. With advanced stage, patients may have recurrent infections, weight loss, or symptoms related to anemia and thrombocytopenia.

CLL can cause most of the signs or symptoms of non-Hodgkin lymphoma, specifically those related to B symptoms (night sweats, fevers, weight loss). The pace of disease is slower than in aggressive lymphomas, and the sudden onset of new symptoms, especially in previously untreated patients, should prompt efforts to exclude other diagnoses. Lymphadenopathy is typically nontender but lymph node enlargement can cause abdominal discomfort, fullness, and malaise. Even bulky lymphadenopathy rarely leads to obstruction or organ impairment. Splenomegaly is frequent and hepatomegaly due to CLL infiltration of the liver can occur. Extranodal involvement is not uncommon and can manifest as skin lesions or pulmonary nodules. Pleural infiltrations leading to effusions or gastrointestinal (GI) tract involvement leading to GI bleeding have been reported. Central nervous system involvement is unusual and neurologic symptoms should be investigated to determine other etiologies, especially infections. While night sweats or low-grade fevers can be symptoms of CLL, it is important to also consider infectious etiologies. Additionally, in CLL patients infections may lead to an exaggerated, albeit transient, increase in lymphadenopathy or splenomegaly, which needs to be differentiated from transformation into high-grade lymphoma.

DIAGNOSIS AND LABORATORY STUDIES

According to the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria a diagnosis of CLL requires the presence of >5 × 10⁹ clonal B lymphocytes/L (5,000/µL) with a typical immunophenotype in the peripheral blood. Immunophenotyping or immunohistochemistry are required for diagnosis (see Immunophenotyping and Flow Cytometry). The World Health Organization classification of hematopoietic neoplasias describes CLL as a leukemic, lymphocytic lymphoma, being distinguishable from small lymphocytic lymphoma (SLL) only by its leukemic appearance.⁴ The diagnosis of SLL requires the presence of lymphadenopathy and/or splenomegaly but by definition clonal B lymphocytes in the peripheral blood are <5 × 10⁹/L. While histopathologic evaluation of a lymph node biopsy may be the standard diagnostic test for SLL, the presence of circulating clonal B cells of typical immunophenotype may suffice in cases with an indolent presentation.

The presence of less than 5 × 10⁹ clonal B lymphocytes per liter of blood in the absence of lymphadenopathy or organomegaly (as defined by physical examination or computerized tomography), cytopenias, or disease-related symptoms is defined as monoclonal B lymphocytosis (MBL). CLL is often preceded by MBL which progresses to CLL requiring treatment at a rate of around 1% to 2% per year.⁵ For the purposes of this chapter, the term “CLL” encompasses both CLL and SLL, and excludes MBL.

Complete Blood Count and Blood Smear

CLL cells are small, mature-appearing lymphocytes with round nuclei, clumped chromatin, and scant cytoplasm. Smudge cells, bare nuclei that appear squashed, are a classic feature. Prolymphocytes that are medium-sized cells with prominent nucleoli make up less than 10% of the lymphocytes in typical cases but may increase in proportion in rapidly progressive disease. Cases with more than 55% prolymphocytes are recognized as a distinct diagnostic entity called prolymphocytic leukemia (PLL). In advanced disease, anemia or thrombocytopenia is common, most often due to replacement of the bone marrow by tumor cells, with a possible contribution of hypersplenism or autoimmune mechanisms.

Immunophenotyping and Flow Cytometry

Flow cytometry is the single most informative diagnostic study in CLL. CLL cells are B cells (CD19 positive) that coexpress CD5 and CD23. These cells typically have weak expression of CD20, CD22, surface immunoglobulin, and are negative for FMC7, CD10, and CD103.

Bone Marrow Biopsy

Bone marrow is always involved in CLL and in the vast majority of SLL. Distinct patterns of infiltration, which have some prognostic value, are recognized: nodular, interstitial, diffuse, or mixed. Advanced disease is often associated with a diffuse pattern of infiltration. Bone marrow biopsy, outside of clinical studies, may be reserved for cases presenting diagnostic difficulties or those that have depressed peripheral blood counts. Immunohistochemistry on bone marrow or lymph node specimens can be used to assist in the diagnostic evaluation.

Lymph Node Biopsy

A lymph node biopsy can distinguish between CLL and other lymphomas or may be necessary to exclude transformation in patients with rapidly enlarging nodes, especially if the growth preferentially affects a single nodal area. The lymph node architecture in CLL is effaced by an abundance of small lymphocytes with clumped chromatin. Mitotic activity is typically low.

DIFFERENTIAL DIAGNOSIS

Morphology, flow cytometry, immunohistochemistry, and cytogenetics (see Prognostic Factors) are the tests with the best diagnostic yield. Briefly summarized in Table 14.1 are the main distinguishing features of closely related entities.

STAGING AND NATURAL HISTORY

The clinical course and the prognosis of CLL are extremely variable. Two staging systems have been used to risk-stratify CLL: the Rai classification (Table 14.2), utilized in North America, and the Binet classification commonly used in Europe. Both are based on clinical and laboratory parameters and confer prognostic information.

The Binet classification distinguishes three stages: stage A is characterized by fewer than 3 areas of lymphoid involvement, where the spleen counts as one lymphoid area; stage B involves 3 or more lymphoid areas; stage C is characterized by anemia and/or thrombocytopenia, regardless of the extent of lymphoid involvement.

IMAGING AND OTHER LABORATORY EVALUATIONS

Computed tomography (CT) is the imaging method of choice to determine the extent of lymph node involvement in CLL and to evaluate response to treatment. CT can identify Rai stage 0 patients with visceral lymphadenopathy, who have a clinical course similar to patients with Rai stage I.⁷ CLL lymph nodes typically have only weak metabolic activity on positron emission tomography (PET) even in extensive disease. While PET is not helpful in the diagnosis of CLL, it can add valuable information in advanced stage or relapsed disease when transformation into high-grade lymphoma is a consideration.

Other Laboratory Evaluations

  Direct antiglobulin test (DAT) should be obtained prior to treatment and in patients with anemia. Conversion of the DAT from negative to positive may herald the onset of autoimmune hemolytic anemia (AIHA).

  Serum immune globulins typically decrease with disease duration. Occasionally, a small M spike may be present.

  Beta-2-microglobulin (B2M) may be elevated and often increases with disease bulk. High B2M levels (>3 mg/L) have been associated with inferior response to treatment.⁸ It is important to consider that renal insufficiency may also increase B2M.

  LDH is typically normal. Elevated LDH is seen with AIHA and may be modestly increased in rapidly progressive disease. High LDH may be a sign of disease transformation.

  A mild elevation of alkaline phosphatase is common. Elevated transaminases should trigger evaluation for viral hepatitis, especially if treatment with rituximab is considered.

PROGNOSTIC FACTORS

Several molecular markers have been identified that hold important prognostic information independent of clinical stage and are therefore particularly valuable in assessing early-stage patients. These markers primarily predict the pace of disease progression in untreated patients and overall survival (OS). In contrast, factors that predict response to treatment are, at present, incompletely defined9,10 but include del 17p, high B2M, and advanced disease.

Immunoglobulin Variable Region Heavy Chain Gene Mutation

The immunoglobulin (Ig) expressed by B cells is composed of light and heavy chains encoded by distinct genes. The presence or absence of somatic mutations in the variable region of the heavy chain gene (VH) distinguishes between two disease subsets: patients whose CLL cells express unmutated immunoglobulin variable region heavy chain (IGHV) genes (Ig-unmutated CLL) have a shorter survival than patients whose CLL cells express mutated genes (Ig-mutated CLL). Ig-mutated CLL can have a median survival of 20 or more years, typically a stable or slow progressive course, and may never require treatment. In contrast, Ig-unmutated CLL is more rapidly progressive, typically requires treatment within a few years from diagnosis, and is associated with a shorter median survival of 8 to 10 years.¹¹

ZAP-70

The tyrosine kinase ZAP-70 is essential for T cell receptor signaling in response to antigen. ZAP-70 is highly expressed in T and NK cells and may be expressed in activated B cells. Relatively higher expression of ZAP-70 is typically seen in Ig-unmutated CLL and infrequent in Ig-mutated CLL.¹² ZAP-70 expression can be assessed by clinical flow cytometry and higher expression predicts more rapid disease progression as well as shorter OS. Time to treatment (TTT), measured as the time from diagnosis to treatment, is approximately 3 years in ZAP-70-positive CLL and reaches 9 years for ZAP-70-negative disease. Median OS in the two subtypes is about 9 years and can be as long as 25 years, respectively.13,14 ZAP-70 is a cytoplasmic protein that is expressed at levels lower in CLL B cells than in T cells, making the test technically challenging. Results close to the threshold should therefore be interpreted with caution.

Cytogenetics

Due to the low mitotic rate of CLL metaphase chromosomal studies using G banding are often not informative. To evaluate cytogenetic abnormalities in CLL, it is therefore necessary to use fluorescence in situ hybridization (FISH). FISH can identify abnormalities in approximately 80% of cases. In a pivotal study, chromosome 13q deletion was present in 55%, deletion of 11q in 18%, trisomy 12 in 16%, and deletion 17p in 7%. No karyotypic abnormality was detected in 18%. Several other abnormalities were found less frequently, and 29% of patients had two or more aberrations.¹⁵ These lesions are not CLL specific and their detection is more informative for prognosis than diagnosis. Currently considered a research test, whole genome sequencing so far has identified several genes that are mutated in up to 20% of untreated CLL patients including TP53, ATM, MYD88, NOTCH1, SF3B1, ZMYM3, MAPK1, FBXW7, DDX3X.¹⁶

Deletions of chromosome 17p (p53 locus) or 11q (ATM locus) are associated with inferior outcome: median survival in these patients has been estimated at 32 months for 17p deletion and 79 months for 11q deletion. However treatment-naive patients in whom tumor cells with 17p deletions make up <25% of the clone have a rate of progression that is not significantly different from patients without this abnormality.¹⁷ The longest OS of 133 months has been associated with an isolated 13 q deletion.9,15 Because many patients may have more than one abnormality, a hierarchical model of genetic subgroups has been proposed, which assigns patients to the genetic subgroup of the prognostic dominant abnormality. For example, a combination of 13q deletion and 17p deletion is assigned to the prognostic group of 17p deletion; 17p deletions are relatively infrequent in newly diagnosed patients but are more common in relapsed or refractory disease. 17p also correlates with poor treatment response to chemotherapy.⁹

CD38

Increased CD38 expression on the cell surface of CLL cells as measured by flow cytometry correlates with inferior outcome.¹⁸ CD38 expression is a partial surrogate for the Ig-gene mutation status and/or ZAP-70 expression in some studies but not others.

Lymphocyte Doubling Time

A lymphocyte doubling time (LDT) of less than 12 months indicates more rapidly progressive disease and is associated with decreased survival independent of stage. LDT < 6 months is a feature of active disease and may be an indication to consider treatment.

TREATMENT

When? The Paradigm of Watchful Waiting

Most patients with early-stage CLL are asymptomatic and have a relatively good long-term prognosis. Several randomized trials investigating immediate versus deferred chlorambucil-based treatment in early-stage patients showed a slightly inferior survival with immediate chemotherapy.¹⁹ Deferral of treatment or “watchful waiting” has therefore become the standard of care for patients with early-stage CLL. Periodic evaluation including basic laboratory testing at 3-to-6-month intervals is a reasonable strategy for asymptomatic patients with relatively stable disease. Treatment is reserved for symptomatic or rapidly progressive disease.²⁰ Guidelines notwithstanding, over the past decade it appears that patients are increasingly treated at earlier stages of disease, despite the absence of data demonstrating that this approach is of long-term benefit.

According to the IWCLL criteria⁴, active disease should meet the following:

  Constitutional symptoms due to CLL (fevers, night sweats, weight loss)

  Symptomatic/massive (>10 cm) lymphadenopathy

  Symptomatic/massive splenomegaly (>6 cm below costal margin)

  Progressive marrow failure: worsening anemia and/or thrombocytopenia

  Rapidly progressive lymphocytosis (lymphocyte doubling time <6 months)

  Autoimmune cytopenias (ITP, AIHA, PRCA) poorly responsive to corticosteroid treatment

How? An Increasing Choice of Active Agents

Alkylating Agents

Prior to the introduction of purine analogues, oral chlorambucil was the drug of choice. Response rates between 40% and 80% were achieved.¹⁸ However, complete remissions to chlorambucil are rare and the response duration is short. It may still be considered in older patients with comorbidities when aggressive combination therapy is not an option.²¹ Cyclophosphamide is frequently used in combination therapies.8,10 Side effects include myelosuppression, nausea, and fatigue. Bendamustine has single agent activity in untreated CLL.²² Toxicities are similar to other alkylators.

Purine Analogues

The purine analogue fludarabine induced more complete responses and increased progression-free survival compared to chlorambucil in a randomized trial.²³ There was no significant difference in OS, probably due to the crossover design of the study and the high response rate to fludarabine in patients who failed chlorambucil. Myelosuppression, with a nadir between days 12 and 16, lymphopenia, opportunistic infections, and the precipitation of autoimmune hematologic complications are among the main toxicities of fludarabine. Prolonged or repeated use of purine analogues can lead to severe suppression of hematopoiesis and protracted cytopenias. Other purine analogues, such as pentostatin and cladribine, are also effective in CLL, but these agents have been less extensively studied. Patients with no response to or relapse within 6 months of a fludarabine-containing regimen are considered fludarabine refractory.

Monoclonal Antibodies

Rituximab, a humanized anti-CD20 monoclonal antibody, is primarily used in combination regimens. Rituximab as a single agent at 375 mg/m² weekly for 4 weeks can induce relatively short partial responses in about 50% of treatment-naive patients.²⁴ Rituximab has more limited single agent activity in refractory or relapsed disease. Dose escalation studies found only minor increases in responses that remained short lived. The first infusion of rituximab often induces a systemic cytokine release syndrome that can be life threatening due to severe hypotension and bronchospasm. Premedication with antihistamines, slow infusion, and careful monitoring, especially during the first infusion have been helpful. Rituximab can be combined safely with chemotherapy. There is a slight increase in infectious complications; of special concern, treatment-related cases of progressive multifocal leukoencephalopathy (PML) caused by JC virus and reactivation of viral hepatitis have occurred.²⁵ Ofatumumab is a second generation anti-CD20 monoclonal antibody that is fully humanized. The drug is approved for patients who are refractory to fludarabine and alemtuzumab. The pivotal ofatumumab trial had overall response rates of 58% in fludarabine- and alemtuzumab-refractory patients and 47% in fludarabine-refractory patients with bulky lymph nodes.²⁶ Ofatumumab is now also being incorporated into combination therapy.

Alemtuzumab, a humanized anti-CD52 monoclonal antibody, is FDA approved for fludarabine-refractory and treatment-naive CLL. The pivotal trial found a response rate of 33% in fludarabine-refractory patients, mainly partial responses.²⁷ Importantly, alemtuzumab was equally effective in patients with or without 17p deletions. However, the antibody appears to be less active in patients with bulky lymphadenopathy (>5 cm). Side effects include cytokine release syndrome, neutropenia, and pronounced lymphopenia, with an increased risk of opportunistic infections, making antimicrobial prophylaxis necessary. Alemtuzumab is associated with CMV reactivation and monitoring for CMV is recommended.

Purine Analogue Combination Regimens

The combination of fludarabine with rituximab induced more responses (84%) including more complete responses (CRs) (38%) than did fludarabine monotherapy in previously untreated patients.²⁸ The combination of cyclophosphamide with fludarabine yielded superior response rates and better progression-free survival (PFS) than did fludarabine as a single agent.29,30 The addition of rituximab to cyclophosphamide and fludarabine (FCR, CLL8 trial) resulted in better PFS and overall response rates than cyclophosphamide and fludarabine alone.8,10 However, the combination with rituximab was more often associated with myelosuppression. There are other chemotherapies that are combined with rituximab, such as pentostatin and cyclophosphamide (PCR), or cladribine; however these options have not been shown to improve on FCR. Cyclophosphamide-containing combination therapies are associated with an increased risk of chemotherapy-related myeloid neoplasias.³¹ Therefore a case can be made to reserve cyclophosphamide for those patients who have deletion 11q, a group that is less responsive to fludarabine or FR regimens but responds very well to FCR.¹⁰ Patients with deletion 17p typically respond to FCR but responses are relatively shorter lived.

Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation is potentially curative because of a potent graft-versus-leukemia effect. Non-myeloablative conditioning regimens are preferred over myeloablative conditioning, mostly because the first approach has less acute toxicity and is better tolerated in the elderly. Most studies report a transplant-related mortality rate of about 20% at 1 year and a long-term disease-free survival in the range of 50%.³² Allogeneic transplantation, especially for biologically fit patients with adverse disease features, such as fludarabine refractoriness or 17p deletion, should be considered and discussed early in the course of treatment and not considered only a last resort.

Treatment Considerations

Table 14.3 summarizes outcomes for different treatments in CLL. Comparisons between different studies are difficult as patient selection and disease stage affect outcome. Clearly there are better responses in earlier stage patients than in patients with advanced disease,8,30 and therefore lead time bias is an important confounder when comparing the efficacy of different treatment regimens outside of randomized studies. In addition, data on survival in many randomized trials is still too preliminary to allow firm conclusions. The effect of more intense treatment regimens on survival for all cytogenetic subgroups is therefore unproven. Treatment considerations should include patient fitness, comorbidities, and the side-effect profile of the chosen regimen. Commonly used first-line treatment choices for fit CLL patients are two or three agent combinations of fludarabine, rituximab, and cyclophosphamide: FR and FCR are typically repeated every 28 days for up to six cycles. High-risk patients (17p deletion) should be considered for aggressive chemoimmunotherapy or a clinical trial using new agents followed by allogeneic transplant if this is an option. Patients who are not suitable candidates for chemoimmunotherapy can be considered for oral chlorambucil.²¹

Patients with relapsed or refractory disease should be referred to clinical trials. In general, CLL patients benefit from being cared for by a hematologist/oncologist with CLL expertise, as OS has been shown to be almost 2 years longer for patients cared for by a CLL-hematologist than for patients seeing a hematologist/oncologist with expertise primarily in other areas.³⁶ Patients who enjoyed a long remission with a fludarabine-based regimen may respond well again to a similar regimen, but repeated exposure to purine analogues carries an increased risk of severe myelosuppression. Fludarabine-refractory disease is defined by lack of response to purine analogue therapy or relapse within 6 months of achieving a response to such therapy.³⁷ Such patients have poor survival prospects and may have adverse cytogenetic features (del17p or del11q) and/or have acquired p53 mutations. Additional effective treatment options include bendamustine, alemtuzumab, ofatumumab, allogeneic transplantation, high-dose methylprednisolone, and investigational agents such as B cell receptor signaling inhibitors (PCI 32765 or GS1101) or lenalidomide (Table 14.4).

Minimal residual disease (MRD) refers to small numbers of CLL cells that persist after treatment in patients who achieved a remission by standard criteria. MRD has recently become the focus of many clinical trials due to its predictive power for PFS. The eradication of MRD has been associated with longer PFS and, in some studies, OS.

Supportive Care

Supportive care in advanced CLL will often focus on cytopenias and infections. Intravenous immune globulin (IVIG) replacement infusions have some benefit to reduce incidence and severity of recurrent bacterial infections and are typically reserved for patients with frequent infectious episodes. The use of IVIG, however, has not been shown to improve OS.⁴¹ The protection conferred by vaccinations may be limited because of the immune defect in advanced disease. Live vaccines, such as, for example, the new herpes zoster vaccine, are contraindicated. Anti-infective prophylaxis against pneumocystis and/or herpes infections is not typically necessary with single-agent fludarabine but often part of combination regimens.⁸

COMPLICATIONS OF CHRONIC LYMPHOCYTIC LEUKEMIA AND THEIR TREATMENT

Autoimmune Manifestations

Autoimmune hematologic manifestations occur quite frequently in advanced disease or during treatment with purine analogues. AIHA and immune thrombocytopenia (ITP) are more common than pure red cell aplasia (PRCA) and autoimmune neutropenia is rare. These autoimmune complications often respond to prednisone or cyclosporine. Rituximab appears a particularly useful and directed therapy in patients in whom autoimmune disease arises during purine analogue treatment.⁴² Retreatment with purine analogues is generally not recommended because of the risk of recurrence and the potentially fatal outcome, especially with AIHA.

Infections

Infections from bacterial, viral, and fungal agents are the most important cause of morbidity and mortality in CLL. The most common infections are due to S. pneumonia, Haemophilus influenzae, and Herpes zoster. Immunosuppression secondary to chemotherapy and biologic agents, in particular alemtuzumab but also fludarabine, contribute to the increased incidence of infections. Prophylactic use of antibiotics in neutropenic or hypogammaglobulinemic patients is not recommended. Prophylaxis for pneumocystis and herpes infections is typically administered to patients receiving alemtuzumab or combination chemoimmunotherapies and may be especially beneficial in relapsed disease.²⁷ G-CSF may be useful to reduce the duration of neutropenia associated with fludarabine combination regimens but should be used with caution as it may mask marrow toxicity of fludarabine that can result in persistent myelosuppression.

Richter Transformation

Transformation to an aggressive, high-grade, large B cell lymphoma or to a Hodgkin lymphoma is called Richter syndrome. Risk factors for the occurrence of Richter syndrome remain incompletely defined but its incidence does not seem to be increased as a result of prior treatment with fludarabine. Characteristic findings include systemic symptoms, rapid lymph node enlargement, elevation of LDH, and paraproteinemia. PET can be helpful in the diagnosis of Richter transformation. Treatment for Richter transformation is similar to high-grade lymphoma but the response to treatment is usually short lived.

SELECT INTERNET RESOURCES

Ongoing clinical trials: http://www.clinicaltrials.gov

Ongoing NHLBI clinical trials: http://patientrecruitment.nhlbi.nih.gov/Lymphoma.aspx

Education and patient information: http://www.clltopics.org

Research funding, education, and patient information: http://www.lymphoma.org

Patient run volunteer support groups: http://listserv.acor.org/archives/cll.html

Reference

  1.  Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8):804-815.

  2.  SEER Stat Fact Sheets: Chronic Lymphocytic Leukemia, National Cancer Institute Web Site, http://seer.cancer.gov/statfacts/html/clyl.html

  3.  Herishanu Y, Perez-Galan P, Liu D, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood. 2011;117(2):563-574. PMCID: 3031480.

  4.  Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111(12):5446-5456. PMCID: 2972576.

  5.  Marti G, Abbasi F, Raveche E, et al. Overview of monoclonal B-cell lymphocytosis. Br J Haematol. 2007;139(5):701-708.

  6.  Wierda WG, O’Brien S, Wang X, et al. Prognostic nomogram and index for overall survival in previously untreated patients with chronic lymphocytic leukemia. Blood. 2007;109(11):4679-4685.

  7.  Muntanola A, Bosch F, Arguis P, et al. Abdominal computed tomography predicts progression in patients with Rai stage 0 chronic lymphocytic leukemia. J Clin Oncol. 2007;25(12):1576-1580.

  8.  Keating MJ, O’Brien S, Albitar M, et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol. 2005;23(18):4079-4088.

  9.  Grever MR, Lucas DM, Dewald GW, et al. Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997. J Clin Oncol. 2007;25(7):799-804.

10.  Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010;376(9747):1164-1174.

11.  Hamblin TJ, Davis Z, Gardiner A, et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 1999;94(6):1848-1854.

12.  Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. 2003;101(12):4944-4951.

13.  Orchard JA, Ibbotson RE, Davis Z, et al. ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet. 2004;363(9403):105-111.

14.  Rassenti LZ, Huynh L, Toy TL, et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004;351(9):893-901.

15.  Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-1916.

16.  Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med. 2011;(26):2497-506.

17.  Tam CS, Shanafelt TD, Wierda WG, et al. De novo deletion 17p13.1 chronic lymphocytic leukemia shows significant clinical heterogeneity: the M. D. Anderson and Mayo Clinic experience. Blood. 2009;114(5):957-964.

18.  Krober A, Seiler T, Benner A, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood. 2002;100(4):1410-1416.

19.  CLL Trialists’ Collaborative Group. Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials. J Natl Cancer Inst. 1999;91(10):861-868.

20.  Gribben JG. How I treat indolent lymphoma. Blood. 2007;109(11):4617-4626.

21.  Eichhorst BF, Busch R, Stilgenbauer S, et al. First-line therapy with fludarabine compared with chlorambucil does not result in a major benefit for elderly patients with advanced chronic lymphocytic leukemia. Blood. 2009;114(16):3382-3391.

22.  Knauf WU, Lissichkov T, Aldaoud A, et al. Phase III randomized study of bendamustine compared with chlorambucil in previously untreated patients with chronic lymphocytic leukemia. J Clin Oncol. 2009;27(26):4378-4384.

23.  Rai KR, Peterson BL, Appelbaum FR, et al. Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. N Engl J Med. 2000;343(24):1750-1757.

24.  Hainsworth JD, Litchy S, Barton JH, et al. Single-agent rituximab as first-line and maintenance treatment for patients with chronic lymphocytic leukemia or small lymphocytic lymphoma: a phase II trial of the Minnie Pearl Cancer Research Network. J Clin Oncol. 2003;21(9):1746-1751.

25.  Gea-Banacloche JC. Rituximab-associated infections. Semin Hematol. 2010;47(2):187-198.

26.  Wierda WG, Padmanabhan S, Chan GW, et al. Ofatumumab is active in patients with fludarabine-refractory CLL irrespective of prior rituximab: results from the phase 2 international study. Blood. 2011;118(19):5126-5129.

27.  Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood. 2002;99(10):3554-3561.

28.  Byrd JC, Rai K, Peterson BL, et al. Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011. Blood. 2005;105(1):49-53.

29.  Eichhorst BF, Busch R, Hopfinger G, et al. Fludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of younger patients with chronic lymphocytic leukemia. Blood. 2006;107(3):885-891.

30.  Flinn IW, Neuberg DS, Grever MR, et al. Phase III trial of fludarabine plus cyclophosphamide compared with fludarabine for patients with previously untreated chronic lymphocytic leukemia: US Intergroup Trial E2997. J Clin Oncol. 2007;25(7):793-798.

31.  Smith MR, Neuberg D, Flinn IW, et al. Incidence of therapy-related myeloid neoplasia after initial therapy for chronic lymphocytic leukemia with fludarabine-cyclophosphamide versus fludarabine: long-term follow-up of US Intergroup Study E2997. Blood. 2011;118(13):3525-3527. PMCID: 3186330.

32.  Dreger P, Dohner H, Ritgen M, et al. Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial. Blood. 2010;116(14):2438-2447.

33.  Fischer K, Cramer P, Busch R, et al. Bendamustine combined with rituximab in patients with relapsed and/or refractory chronic lymphocytic leukemia: a multicenter phase II trial of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol. 2011;29(26):3559-3566.

34.  Castro JE, Sandoval-Sus JD, Bole J, et al. Rituximab in combination with high-dose methylprednisolone for the treatment of fludarabine refractory high-risk chronic lymphocytic leukemia. Leukemia. 2008;22(11):2048-2053.

35.  Wierda WG, Kipps TJ, Mayer J, et al. Ofatumumab as single-agent CD20 immunotherapy in fludarabine-refractory chronic lymphocytic leukemia. J Clin Oncol. 2010;28(10):1749-1755.

36.  Shanafelt TD, Kay NE, Rabe KG, et al. Hematologist/oncologist disease-specific expertise and survival: lessons from chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Cancer. 2012;118(7):1827-37.doi:10.1002/ cncr.26474.Epub 2011 Aug 26.

37.  Montserrat E, Moreno C, Esteve J, et al. How I treat refractory CLL. Blood. 2006;107(4):1276-1283.

38.  Chanan-Khan A, Miller KC, Musial L, , et al. Clinical efficacy of lenalidomide in patients with relapsed or refractory chronic lymphocytic leukemia: results of a phase II study. J Clin Oncol. 2006;24(34):5343-5349.

39.  Ferrajoli A, Lee BN, Schlette EJ, et al. Lenalidomide induces complete and partial remissions in patients with relapsed and refractory chronic lymphocytic leukemia. Blood. 2008;111(11):5291-5297.

40.  Friedberg JW, Sharman J, Sweetenham J, et al. Inhibition of Syk with fostamatinib disodium has significant clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood. 2010;115(13):2578-2585. PMCID: 2852362.

41.  Intravenous immunoglobulin for the prevention of infection in chronic lymphocytic leukemia: a randomized, controlled clinical trial. Cooperative Group for the Study of Immunoglobulin in Chronic Lymphocytic Leukemia. N Engl J Med. 1988;319(14):902-907.

42.  Hegde UP, Wilson WH, White T, Cheson BD. Rituximab treatment of refractory fludarabine-associated immune thrombocytopenia in chronic lymphocytic leukemia. Blood. 2002;100(6):2260-2262.

15

Hodgkin Lymphoma

Abraham S. Kanate, Michael Craig, Jame Abraham, Wyndham H. Wilson and Elaine S. Jaffe

Hodgkin lymphoma (HL) is named after Thomas Hodgkin, who first described some neoplastic disorders of the lymphatic system in 1832. The 2008 World Health Organization (WHO) classification of hematologic neoplasms divides HL into two distinct types. Classical Hodgkin lymphoma (CHL) is characterized by the presence of multinucleated giant cells of B-cell origin, known as Reed-Sternberg (RS) cells, in a background of numerous reactive lymphocytes. HL is one of the few malignancies for which there is effective combination therapy with chemotherapy and/or radiation, which has translated to over 80% cure rates and a steady decline in mortality. The high cure rate for HL has led to more emphasis on regimen-related long-term toxicities in the recent years. CHL is considered a distinct clinicopathalogic entity from nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL).

EPIDEMIOLOGY

Hodgkin lymphoma is among the most common cancer of young adults. HL constitutes approximately 1% of all malignancies and 18% of all lymphomas. In 2012, it is estimated that 9,060 new cases of HL will be diagnosed with 1,190 patient deaths in the United States.¹ In Europe and North America, there is a bimodal age distribution, with an increasing frequency between the second and third decades, and a second peak after the fifth decade.

PATHOLOGIC CLASSIFICATION

Table 15.1 lists the World Health Organization Revised European American Lymphoma classifications, in comparison to older historical schemes.²

  Classic HL.

  Nodular sclerosis classic Hodgkin lymphoma (NSCHL).

  Mixed cellularity classical Hodgkin lymphoma (MCCHL).

  Lymphocyte-depleted classical Hodgkin lymphoma (LDCHL).

  Lymphocyte-rich classical Hodgkin lymphoma (LRCHL).

  Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL).

The immunophenotypes for CHL and NLPHL are described in Table 15.2.³

PATHOLOGY

Hodgkin lymphoma is unique among the malignant lymphomas in that the RS cells and variants, the malignant cells, constitute the minority of cells present in the tumor mass (Fig. 15.1). The neoplastic cells in CHL are associated with a rich inflammatory background of lymphocytes, eosinophils, neutrophils, histiocytes, and plasma cells in varying proportions. New molecular studies have provided evidence for the B-cell origin of the neoplastic cell in both CHL and NLPHL, as the cells have undergone somatic mutations in the immunoglobulin variable region. However, the tumor cells down regulate the B-cell program and are typically negative for most B-cell associated markers,⁴ and they show constitutive activity of nuclear factor-kappa beta (NF-kB).

NSCHL requires the presence of a nodular growth pattern, broad bands of fibrosis, and a characteristic variant of the RS cell known as a lacunar cell. The lacunar cell has abundant clear cytoplasm with a sharply demarcated cell membrane. In formalin-fixed tissue a characteristic artifact often occurs; the cytoplasm of the cell retracts, leaving a clear space or lacuna. NSCHL is graded according to the proportion of neoplastic cells and the presence of necrosis, as well as depletion of normal lymphocytes. Two grades of NSCHL are identified in the WHO classification.² This is the most common form of CHL, comprising 60% to 70% of cases, and has an equal distribution in males and females. NSCHL often involves mediastinal, supraclavicular, and cervical lymph nodes.

MCCHL is characterized by numerous classical RS cells in a rich inflammatory background, with fine reticular fibrosis but with the absence of distinct fibrous bands. MCCHL is more common in males and is the second most common type of CHL, affecting. MCCHL is frequently associated with disseminated disease at presentation, and B symptoms are also common. It is one of the variants of CHL that occurs with HIV infection and is the subtype most often positive for Epstein-Barr virus (EBV) sequences.²

FIGURE 15.1 Diagnostic Reed-Sternberg cell seen in classic types of Hodgkin lymphomas (mixed cellularity, nodular sclerosis, lymphocyte depletion). Neoplastic cells in nodular lymphocyte-predominant Hodgkin lymphomas are termed popcorn cells or L and H cells (lymphocytic or histiocytic predominance). Reed-Sternberg cells of the classic type generally are not seen in a nodular lymphocyte-predominant Hodgkin lymphoma.

LDCHL is the rarest CHL, constituting approximately 1% of cases. Epidemiologically, it is seen in resource-limited regions of the world, and with increased frequency in HIV-infected individuals. LDCHL may represent a further evolution of MCCHL, with more frequent malignant cells, a depletion of normal lymphocytes, and greater fibrosis, which is often diffuse and reticular in nature. Patients can be elderly and present with B symptoms and at an advanced stage.

LRCHL is characterized by a cellular milieu of abundant normal lymphocytes, and a paucity of malignant cells that have the immunophenotype of classic RS cells. LRCHL may have a diffuse or nodular growth pattern, and especially in its nodular form may be mistaken for nodular lymphocyte-predominant HL. It tends to present in older individuals, often with isolated peripheral lymphadenopathy.

NLPHL differs from CHL in its immunophenotype, histologic characteristics, and clinical behavior.³ Classical RS cells are not seen. The neoplastic cells initially referred to as lymphocytic and histiocytic (L & H) cells are now called LP cells or more informally “popcorn cells.” They have a lobulated nuclear contour, dispersed chromatin, and inconspicuous nucleoli, and generally cluster within nodules associated with lymphocytes and histiocytes. Initially, the background lymphocytes are predominantly of B-cell phenotype, but T cells may predominate in later stages. The neoplastic cells, the LP cells, are positive for CD20, generally negative for CD15, and negative or weakly positive for CD30. NLPHL affects patients of all ages, and occurs more often in males than females. B symptoms are infrequent, and patients tend to have an indolent course but with multiple relapses over time, similar to low-grade non-Hodgkin lymphoma.

ETIOLOGY AND RISK FACTORS

EBV has been linked to many cases of CHL, but is absent in NLPHL. EBV is most commonly found in MCCHL and LDCHL but rarely in NSCHL. Infectious mononucleosis appears to be a predisposing risk factor for subsequent EBV-positive CHL but not EBV-negative CHL.⁵ NSCHL is most common in North America and is more prevalent among individuals of higher socioeconomic status, whereas MCCHL and LDCHL are seen in resource-limited regions of the world. The risk of CHL is increased 5- to 10-fold in HIV-positive patients. Familial cases of CHL have been reported, and siblings of CHL patients are at slightly increased risk. There is a weak association with certain HLA types.

CLINICAL FEATURES

Over 80% of patients have cervical lymph node enlargement, and more than 50% will have mediastinal adenopathy. Lymph nodes are usually nontender, firm, and rubbery.

  Constitutional symptoms (“B” symptoms):

  Unexplained fever (temperature >38°C, which may be cyclical).

  Drenching night sweats.

  Unexplained weight loss (>10% of body weight, over 6 months before the diagnosis).

  Other symptoms include fatigue, weakness, anorexia, alcohol-induced nodal pain, and pruritus.

Staging (Ann Arbor/AJCC and Cotswold) is outlined in Table 15.3.⁶⁻⁸

PRETREATMENT EVALUATION

  Excisional biopsy (and not needle biopsy) of a prominent node is highly recommended for diagnosis.

  Detailed history with attention to unexplained fever, night sweats, and significant weight loss (constitutional symptoms).

  Complete physical examination, including the lymph node examination and evaluation for hepatosplenomegaly.

  Laboratory tests include

  Complete blood count (CBC), erythrocyte sedimentation rate (ESR).

  Biochemical tests of liver function, renal function, serum lactate dehydrogenase (LDH), and serum uric acid. Pregnancy test should be included for all women of child-bearing age. HIV testing is recommended if risk factors exist.

  Radiologic studies

  Chest radiograph and computed tomography (CT) scan of the chest, abdomen, and pelvis.

  The use of positron emission tomography (PET) scan, often combined with CT has emerged as a powerful tool and is recommended in the initial staging and subsequent follow-up. ⁹

  Bone marrow biopsy of the posterior iliac crest for those with abnormal CBC or clinical stage IIB, III, or IV.

  Pulmonary function tests (PFT) with diffusion capacity for carbon monoxide and cardiac evaluation for the ejection fraction is useful prior to initiating therapy.

  Adequate counseling for semen/oocyte cryopreservation should be considered for certain treatment regimens and pelvic radiation therapy.

Prognostic Features

Unfavorable prognostic features for stage I/II disease include

  Sedimentation rate of 50 or more in the absence of B symptoms.

  Age greater than 50.

  Presence of B symptoms (fever and loss of weight).

  Greater than 3 sites of nodal involvement.

  More than 1 area of extranodal involvement.

  Bulky adenopathy including mediastinal disease more than one-third of the chest diameter on chest x-ray or tumor size >10 cm on CT scan.

The International Prognostic Factors Project on Advanced Hodgkin lymphoma prognostic score (for advanced disease only, 1 point for each positive factor):

  Albumin level of less than 4.0 g/dL.

  Hemoglobin level of less than 10.5 g/dL.

  Male sex.

  Age of 45 years or older.

  Stage IV disease.

  White blood cell count greater than 15,000/mm³.

  Absolute lymphocyte count of less than 600/mm³ or a lymphocyte count less than 8% of the total white cell count.

The 5-year progression-free survival according to the international prognostic score are as follows: 0 factors 84%; 1 factor 77%; 2 factors 67%; 3 factors 60%; 4 factors 51%; and 5 or greater factors 42%.¹⁰

NLPHL histology portends a more indolent disease course. The presence of increased tumor-related CD68+ (>5%) macrophages has recently been shown to be associated with poorer disease-specific survival in multivariate analysis.¹¹

MANAGEMENT OF NEWLY DIAGNOSED CLASSICAL HODGKIN LYMPHOMA

The goal of therapy for CHL is cure while limiting the early and delayed therapy related toxicity without compromising outcomes. Advances in the systemic treatment of CHL have dramatically improved the response rate and survival. This improvement is mainly due to careful staging, understanding of the pattern of spread, and advances in radiation and chemotherapy. In general, combination chemotherapy with or without radiation is used for most patients with CHL.¹² Therapy with radiation consists of treating regions of known disease (especially if bulky) +/− adjacent nodal groups. Treatment selection is influenced by stage, prognostic factors, and short- and long-term toxicity. Response criteria for lymphoma, incorporating PET scan results, have been updated by a consensus panel.⁹

Radiation Therapy

Because of the availability of effective chemotherapy regimens, radiation as a single mode of treatment is rarely used in HL, except in some cases of early-stage NLPHL. Involved field radiation therapy (IFRT) was shown to have similar freedom from treatment failure and overall survival (OS), with better tolerabiltiy and toxicity profile compared to extended field radiation therapy (EFRT, including adjacent clinically negative nodal sites).¹³ Classically, radiation therapy was delivered to three major fields: the mantle, paraaortic, and pelvic or inverted-Y fields (Fig. 15.2). The appropriate dose of radiation is traditionally described as 25 to 30 Gy to clinically uninvolved sites, and 30 to 40 Gy to regions of initial nodal involvement. Newer treatment strategies/clinical trials aim to reduce or eliminate radiation exposure. Long-term sequelae of radiation include cardiopulmonary disease, lung cancer (especially in smokers), and breast cancer.

Chemotherapy Regimens

With combination chemotherapy long-term disease-free remissions are the norm, even in advanced CHL. The first “curative regimen” was mechlorethamine, Oncovin (vincristine), procarbazine, prednisone (MOPP), which resulted in about 70% complete remission in stage III–IV patients. Historically, MOPP was considered the standard treatment for advanced CHL. Subsequently many regimens that included Adriamycin (doxorubicin) were developed. The first of these was doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and hybrids of MOPP and ABVD. In a randomized study, the Cancer and Leukemia Group B (CALGB) compared three regimens (Table 15.4), the results showing that ABVD alone or MOPP/ABVD was superior to MOPP alone, in terms of remission, freedom from progression, and survival.¹⁴ An intergroup study of ABVD versus MOPP-ABV showed equivalency but higher hematologic and pulmonary toxicity with MOPP-ABV. Studies have consistently shown that ABVD is less toxic and more effective than MOPP, with better rates of freedom from progression and OS. At 10 years, the risk of developing treatment-related leukemia with the MOPP regimen is 2% to 3%, whereas it is 0.7% with ABVD.¹⁵ ABVD is considered the standard treatment option in North America.

The German Hodgkin Study Group (GHSG) developed the bleomycin, etoposide, Adriamycin (doxorubicin), cyclophosphamide, Oncovin (vincristine), procarbazine, and prednisone (BEACOPP) regimen to improve treatment outcomes in HL. The HD9 clinical trial for patients with advanced-stage CHL using COPP/ABVD, BEACOPP, or increased-dose BEACOPP, and consolidative radiation therapy to sites of initial bulky (5 cm or larger) or residual disease, showed a 5-year OS of 83% for COPP/ ABVD, 88% for BEACOPP, and 91% for increased-dose BEACOPP.¹⁶ The actuarial rate of secondary acute leukemia 5 years after diagnosis of HL was 0.4% for COPP/ABVD, 0.6% for BEACOPP, and 2.5% for increased-dose BEACOPP (P = 0.03). Although this study suggests that dose intensity improves the survival of Hodgkin lymphoma, the improvement must be balanced against the increased toxicity and risk of leukemia.¹⁷ BEACOPP is considered unsuitable for patients over age 65, associated with significant noted, the primary end point of this trial was freedom from first progression and secondary end points included OS and freedom from second progression and EFS after initial therapy.

Results: BEACOPP superior to ABVD in freedom from first progression, however when salvage therapy with auto-transplant is included BEACOPP and ABVD where equal with regards to OS and freedom from second progression, suggesting patients who relapse after ABVD can be effectively salvaged with HDT and auto-HCT, shielding the majority of patients from the toxicity profile of using upfront BEACOPP.

The abbreviated 12-week chemoradiotherapy regimen, Stanford V, was developed to increase chemotherapy dose intensity by alternating myelosuppresive and non-myelosuppressive treatments weekly, and is given in combination with radiation to sites of disease.¹⁹ Radiation therapy (to nodal sites >5 cm and macroscopic splenic disease) is an integral part of the regimen. The Eastern Cooperative Oncology Group clinical trial E2496 (published in abstract form) compared ABVD to Stanford V. Disease-specific and survival outcomes were similar in both groups at 5 years. There was more grade 3 lymphopenia and sensory neuropathy in the Stanford V arm.²⁰ Another randomized trial showed Stanford V to be inferior to ABVD if the prescribed radiation treatment is modified.²¹

FIGURE 15.2 Radiation therapy fields used in treating Hodgkin disease.When the fields shown in C and D are combined, this is commonly called total nodal irradiation (TNI). (From Haskell CM. Cancer Treatment. 4th ed. Philadelphia, PA: WB Saunders; 1995:965)

Commonly used regimens include

  ABVD: doxorubicin + bleomycin + vinblastine + dacarbazine.

  BEACOPP: bleomycin + etoposide + doxorubicin + cyclophosphamide + vincristine + procarbazine + prednisone.

  COPP/ABVD: cyclophosphamide + vincristine + procarbazine + prednisone/doxorubicin + bleomycin + vinblastine + dacarbazine.

  MOPP: mechlorethamine + vincristine + procarbazine + prednisone.

  MOPP/ABV hybrid: mechlorethamine + vincristine + procarbazine + prednisone/doxorubicin + bleomycin + vinblastine.

  Stanford V: doxorubicin + vinblastine + mechlorethamine + etoposide + vincristine + bleomycin + prednisone. Radiation therapy is added to the involved field.

CHOOSING A TREATMENT REGIMEN

For treatment selection, patients may be divided into three major groups:

  Stages IA and IIA without the poor prognostic factors detailed above are considered “favorable early stage” and are at low risk for recurrence. Cure rate is >90%.

  Stages IB–IIB and stages I–II with poor-risk features are considered “unfavorable early disease.” Cure rate is >80%.

  Stages III and IV are considered “advanced stage” and are at significant risk for recurrence. Cure rate is about 60% to 70%.

Treatment Recommendations for Classical Hodgkin Lymphoma

In addition to the following information, Table 15.5 details commonly used treatment regimens.

Table 15.5  Commonly Used Treatment Regimen

ABVD

Doxorubicin, 25 mg/m² per dose IV push for two doses, days 1 and 15 (total dose/cycle, 50 mg/m²)

Bleomycin, 10 U/m² per dose IV push for two doses, days 1 and 15 (total dose/cycle, 20 U/m²)

Vinblastine, 6 mg/m² per dose IV push for two doses, days 1 and 15 (total dose/cycle, 12 mg/m²)

Dacarbazine, 375 mg/m² per dose IV infusion for two doses, days 1 and 15 (total dose/cycle, 750 mg/m²)

Treatment cycle repeats every 28 d

MOPP

Mechlorethamine, 6 mg/m² per dose IV push for two doses, days 1 and 8 (total dose/cycle, 12 mg/m²)

Vincristine, 1.4 mg/m² per dose IV push for two doses, days 1 and 8 (total dose/cycle, 2.8 mg/m²)

Procarbazine, 100 mg/m²/d orally for 14 doses, days 1–14 (total dose/cycle, 1400 mg/m²)

Prednisone, 40 mg/m²/d orally for 14 doses, days 1–14 (cycles 1 and 14 only) (total dose/cycle, 560 mg/m²)

Treatment cycle repeats every 28 d.

Alternating MOPP/ABVD

Alternate MOPP and ABVD cycles by 28 d

MOPP/ABV Hybrid

Mechlorethamine, 6 mg/m² IV push day 1 (total dose/cycle, 6 mg/m²)

Vincristine, 1.4 mg/m² IV push day 1 (total dose/cycle, 1.4 mg/m²; maximal dose, 2 mg)

Procarbazine, 100 mg/m²/d orally for 7 doses, days 1–7 (total dose/cycle, 700 mg/m²)

Prednisone, 40 mg/m²/d orally for 14 doses, days 1–14 (total dose/cycle, 560 mg/m²)

Doxorubicin, 25 mg/m² IV push day 8 (total dose/cycle, 25 mg/m²)

Hydrocortisone, 100 mg IV day 8, before bleomycin (total dose/cycle, 100 mg)

Bleomycin, 10 U/m² IV push day 8 (total dose/cycle, 10 U/m²)

Vinblastine, 6 mg/m² IV push day 8 (total dose/cycle, 6 mg/m²)

Treatment cycle repeats every 28 d

BEACOPP Standard Dose

Bleomycin, 10 mg/m² (day 8); etoposide, 100 mg/m² (days 1–3); doxorubicin, 25 mg/m² (day 1); cyclophosphamide, 650 mg/m² (day 1); vincristine, 1.4 mg/m² (day 8); procarbazine, 100 mg/m² (days 1–7); and prednisone 40 mg/m² (days 1–14)

Regimen was repeated on day 22

The maximum dose of vincristine is 2 mg

Increased-Dose BEACOPP

Bleomycin, 10 mg/m² (day 8); etoposide, 200 mg/m² (days 1–3); doxorubicin, 35 mg/m² (day 1); cyclophosphamide, 1250 mg/m² (day 1); vincristine, 1.4 mg/m² (day 8); procarbazine, 100 mg/m² (days 1–7); and prednisone 40 mg/m² (days 1–14)

Regimen was repeated on day 22

G-CSF starting on day 8 until count recovery

The maximum dose of vincristine is 2 mg

Stanford V

Mustard, 6 mg/m² IV weeks 1, 5, 9

Vincristine, 1.4 mg/m² IV weeks 2, 4, 6, 8, 10, 12 (maximal dose, 2 mg)

Prednisone, 40 mg/m²/d orally every other day, weeks 1–9, taper

Doxorubicin, 25 mg/m² IV weeks 1, 3, 5, 7, 9, 11

Bleomycin, 5 U/m² IV weeks 2, 4, 6, 8, 10, 12

Vinblastine, 6 mg/m² IV weeks, 1, 3, 5, 7, 9, 11

Etoposide, 60 mg/m² IV daily for 2 d, weeks 3, 7, 11

—The maximum dose of vincristine is 2 mg

—All drugs are administered on day 1, except for VP-16, which is given on days 1 and 2

—Taper prednisone by 10 mg of the total dose qod (every other day) on weeks 10 and 11

—Reduce the dose of vinblastine to 4 mg/m² on weeks 9 and 11 for patients over the age of 50 yr

—Reduce the dose of vincristine to 1 mg on weeks 10 and 12 for patients over the age of 50 yr

—If mustard is unavailable, cyclophosphamide 650 mg/m² IV on weeks 1, 5, 9 may be substituted

ABVD, doxorubicin (Adriamycin), bleomycin, vinblastin, and dacarbazine; BEACOPP, bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone; G-CSF, granulocyte colony-stimulating factor; IV, intravenous; MOPP, mechlorethamine, Oncovin, procarbazine, and prednisone.

Favorable Early Disease

  Combined modality therapy with chemotherapy (ABVD 2–4 cycles) + radiation was considered standard. Abbreviated therapy with 2 cycles of ABVD followed by 20 Gy IFRT is equally effective and far less toxic in suitable patients.²² The exclusion criteria for the trial were bulky mediastinal mass, large splenic involvement, >2 sites of disease, and elevated ESR.

  In a randomized controlled trial, 4 cycles of ABVD without radiation therapy were recently shown to have superior 12-year DFS and OS, compared to radiation therapy as a treatment strategy.²³ Notwithstanding the radiation protocol in the trial, which involved administering 35 Gy of subtotal nodal irradiation (no longer considered standard), chemotherapy alone with ABVD alone likely represents an effective alternative. The ongoing HD16 trial by GHSG compares ABVD for 2 cycles with or without radiation (20 Gy IFRT), guided by functional PET/CT imaging. Chemotherapy alone may be considered for patients with early-stage disease.

Unfavorable Early Disease

  ABVD 4–6 cycles followed by IFRT. Restaging PET/CT after 2 cycles of ABVD can guide therapy, 2 additional cycles after a complete response is considered followed by consolidative IFRT 30 to 36 Gy. Alternatively, patients may be treated with ABVD × 6 cycles without radiation.

  Massive mediastinal disease (defined as a mediastinal mass width greater than one-third of the maximum chest diameter or 10 cm mass) is usually treated with combined modality therapy. However, chemotherapy alone is curative in over half of patients; clinical trials are now assessing if radiation can be omitted in patients who have negative FDG-PET scans post-chemotherapy. Regimens such as ABVD should be considered for this patient group.

Advanced Disease

  ABVD for 6 cycles is the current standard. Treatment is usually continued 2 cycles after resolution of disease is confirmed by imaging studies.

  Increased-dose BEACOPP for patients with or without radiation treatment for poor prognosis disease (IPS score >4) may be considered in younger patients, but will likely not impact OS.

  Addition of IFRT is usually considered, particularly for bulky disease. Some evidence suggests that there may be no need to add radiation, if a complete response can be achieved with combination chemotherapy.

  Stanford V, if administered in the described manner, is a viable alternative in selected patients.

Role of Positron Emission Tomography Scan

PET scanning has emerged as a powerful tool in the management of HL. Integrated PET/CT scanning is invaluable in the initial evaluation and staging of the disease. Interim imaging with PET scan after 2 cycles of chemotherapy that is negative is a strong, positive prognostic factor implying better disease-free survival (DFS) and OS in multivariate analysis.²⁵ Incorporating interim PET results to guide further therapy—example: dose reduction of chemotherapy or withholding IFRT in PET-negative patients after 2 cycles of therapy—are under evaluation. Posttreatment PET/CT is useful in identifying residual disease versus fibrosis/necrosis, but the standard for evaluation of residual/progressive disease remains biopsy and pathologic evaluation.

Lymphocyte-Predominant Hodgkin Lymphoma

This subtype is associated with the propensity for multiple relapses, even up to 15 years after presentation. Early stages of NLPHL without risk factors are treated with radiation alone or, in some cases, with observation following complete surgical resection. Advanced stages are rare at diagnosis and may have a poor prognosis. While such cases are usually treated like CHL, the unique biology of NLPHL raises the question of whether it should be treated like an aggressive B-cell lymphoma.³ Indeed, phase II trials show high single agent activity of rituximab in NLPHL, the tumor cells of which are almost always CD20 positive. The use of rituximab combined with chemotherapy in the frontline setting is still considered investigational and affected patients should be referred for trials due to the rarity of the disease.

COMPLICATIONS OF THERAPY

While the disease-specific mortality in HL has improved greatly due to the advances made in combination chemotherapy, therapy-related long-term toxicity is quite significant. It is extremely important that patients be followed by an oncologist even years after the completion of therapy.

Radiation Therapy Early Complications

  Mantle field radiation may cause mouth dryness, pharyngitis, cough, and dermatitis.

  Subdiaphragmatic radiation may cause anorexia and nausea.

  Radiation can cause myelosuppression or thrombocytopenia.

Late Complications

  Hypothyroidism.

  Pericarditis and pneumonitis.

  Lhermitte sign: 15% of the patients receiving mantle radiation may experience electric shock sensation radiating down the back of the legs when the head is flexed, 6 to 12 weeks after the treatment. The pathophysiology may be transient demyelinization of the spinal cord, and the syndrome usually resolves spontaneously.

  Coronary artery disease (CAD): Increased risk in patients who receive cardiac radiation. Patients should be monitored and evaluated for other risk factors for CAD.

  Secondary neoplasms (lung, breast, stomach, and thyroid).

  Lung cancer: Twofold to eightfold increase in lung cancer is observed more than 5 years after the radiation treatment and persists through the second decade. The increased risk is greatest in inhaled tobacco users.

  Breast cancer risk is inversely proportional to the age at radiation treatment. The relative risk (RR) is 136 if the patient is younger than 15 years. RR is 19 for age group 15 to 24 years. RR is 7 for age group 24 to 29 years. The high risk is restricted to women irradiated before age 30 years. Average interval between radiation and diagnosis of breast cancer is 15 years. Breast examination should be part of follow-up for women at risk; routine mammography should begin about 8 years after completion of the radiation.

Chemotherapy

Early Complications

  Nausea and vomiting

  Alopecia

  Myelosuppression

  Infection

Late Complications

  Sterility (primarily with MOPP-based regimens)

  Neuropathy (primarily with vincristine)

  Cardiomyopathy (doxorubicin)

  Pulmonary fibrosis (bleomycin)

  Secondary leukemia (esc. BEACOPP, MOPP ± radiation)

RELAPSED/REFRACTORY HODGKIN LYMPHOMA

If the relapse is due to inadequate initial treatment, retreatment with chemotherapy or radiation is considered. Based on the results of two randomized trials that showed improved DFS and no difference in OS, high-dose chemotherapy followed by autologous hematopoietic cell transplantation (AHCT) is considered standard. About 60% to 65% of patients can be salvaged with AHCT, but time to relapse <12 months, primary refractory disease, extranodal site of relapse, and B symptoms each portends a worse outcome. Cytoreduction with salvage chemotherapy regimen has important ramifications on the outcome of AHCT. Chemo-sensitive disease to second-line chemotherapy tends to have better outcome with AHCT. Brentuximab vedotin (SGN35) is an antibody-drug conjugate targeting CD30 + cells that was recently approved for use in relapsed HL after AHCT.²⁴ AHCT is considered investigational therapy but may be employed in clinical research trials in refractory/progressive disease.

SALVAGE CHEMOTHERAPY REGIMENS

Non–Anthracycline-Containing Regimens

  ESHAP (etoposide, methylprednisolone, high-dose cytarabine, and cisplatin).

  ICE (ifosfamide, carboplatin, and etoposide).

  DHAP (dexamethasone, high-dose cytarabine, and cisplatin).

  EIP (etoposide, ifosfamide, and cisplatin).

Anthracycline-Containing Regimens

  Dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin).

  EVA (etoposide, vincristine, doxorubicin).

  ASHAP (doxorubicin, cisplatin, high-dose cytarabine, and methylprednisolone).

PALLIATIVE TREATMENT

Palliative treatment includes investigational treatment, radiation treatment, sequential single-agent chemotherapy (gemcitabine or vinblastine).

References

  1.  Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. January 2012;62(1):10-29.

  2.  Jaffe ES, Harris, NL, Stein H, Vardiman J. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. World health Organization Classification of Tumours. Lyon, France: IARC Press; 2001;237-254.

  3.  Nogova L, Rudiger T, Engert A. Biology, clinical course and management of nodular lymphocyte-predominant Hodgkin lymphoma. Hematology Am Soc Hematol Educ Program. 2006;(3):266-272.

  4.  Mathas S. The pathogenesis of classical Hodgkin’s lymphoma: a model for B-cell plasticity. Hematol Oncol Clin North Am. 2007;21:787-804.

  5.  Hjalgrim H, Askling J, Rostgaard K, et al. Characteristics of Hodgkin’s lymphoma after infectious mononucleosis. N Engl J Med. 2003;349:1324-1332.

  6.  Lister TA, Crowther D, Suteliffe SB, et al. Report of a committee convened to discuss the evaluation and staging of patients with Hodgkin’s disease: cotswolds meeting. J Clin Oncol. 1989;7:1630-1636.

  7.  Mauch P, Larson D, Osteen R, et al. Prognostic factors for positive surgical staging in patients with Hodgkin’s disease. J Clin Oncol. 1990;8:257-265.

  8.  Urba WJ, Longo DL. Hodgkin’s disease. N Engl J Med. 1992;326:678-687.

  9.  Cheson B, Pfistner B, Juweid M, et al. Revised response criteria for malignant lymphoma. J Clin Oncol. 2007;25:579-586.

10.  Hasenclever D, Diehl V. A prognostic score for advanced Hodgkin’s disease. N Engl J Med. 1998;339:1506-1514.

11.  Steidl C, Lee T, Shah SP, et al. Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med. March 2010;362(10):875-885.

12.  Conners J. State-of-the-art therapeutics: Hodgkin’s lymphoma. J Clin Oncol. 2005;23:6400-6408.

13.  Engert A, Schiller P, Josting A, et al. Involved-field radiotherapy is equally effective and less toxic compared with extended-field radiotherapy after four cycles of chemotherapy in patients with early-stage unfavorable Hodgkin’s lymphoma: results of the HD8 trial of the German Hodgkin’s Lymphoma Study Group. J Clin Oncol. October 2003;21(19):3601-3608.

14.  Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy of advanced Hodgkin’s disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med. 1992;327:1478-1484.

15.Swerdlow AJ, Douglas AJ, Hudson GV, et al. Risk of second primary cancers after Hodgkin’s disease by type of treatment: analysis of 2846 patients in the British National Lymphoma Investigation. BMJ. 1992;304:1137-1143.

16.  Diehl V, Franklin J, Pfreundschuh M, et al. U The German Hodgkin’s Lymphoma Study Group Standard and Increased-dose BEACOPP chemotherapy compared with COPP–ABVD for advanced Hodgkin’s disease. N Engl J Med. 2003;348:2386-2395.

17.  Federico M, Luminari S, Iannitto E, et al. ABVD compared with BEACOPP compared with CEC for the initial treatment of patients with advanced Hodgkin’s lymphoma: results from the HD2000 Gruppo Italiano per lo Studio dei Linfomi Trial. J Clin Oncol. February 2009;27(5):805-811.

18.  Viviani S, Zinzani PL, Rambaldi A, et al. ABVD versus BEACOPP for Hodgkin’s lymphoma when high-dose salvage is planned. N Engl J Med. July 2011;365(3):203-212.

19.  Horning SJ, Hoppe RT, Breslin S, Bartlett NL, Brown BW, Rosenberg SA. Stanford V and radiotherapy for locally extensive and advanced Hodgkin’s disease: mature results of a prospective clinical trial. J Clin Oncol. 2002;20:630-637.

20.  Gordon LI, Hong F, Fisher RI, et al. A randomized phase III trial of ABVD vs. Stanford V +/2 radiation therapy in locally extensive and advanced stage Hodgkin lymphoma: an intergroup study coordinated by the Eastern Cooperative Oncology Group (E2496). Blood (ASH Annual Meeting Abstracts). 2010;116:Abstract 415.

21.  Chisesi T, Bellei M, Luminari S, et al. Long-term follow-up analysis of HD9601 trial comparing ABVD versus Stanford V versus MOPP/EBV/CAD in patients with newly diagnosed advanced-stage Hodgkin’s lymphoma: a study from the Intergruppo Italiano Linfomi. J Clin Oncol. 2011;29:4227-4233.

22.  Engert A, Plütschow A, Eich HT, et al. Reduced treatment intensity in patients with early-stage Hodgkin’s lymphoma. N Engl J Med. August 2010;363(7):640-652.

23.  Meyer RM, Gospodarowicz MK, Connors JM, et al. ABVD alone versus radiation-based therapy in limited-stage Hodgkin’s lymphoma. N Engl J Med. February 2012;366(5):399-408.

24.  Younes A, Bartlett NL, Leonard JP, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med. November 2010;363(19):1812-1821.

25.  Gallamini A, Hutchings M, Rigacci L, et al. Early interim 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography is prognostically superior to international prognostic score in advanced-stage Hodgkin’s lymphoma: a report from a joint Italian-Danish study. J Clin Oncol. August 2007;25(24):3746-3752.

16

Non-Hodgkin Lymphomas

Richard F. Little and Wyndham H. Wilson

INTRODUCTION

The non-Hodgkin lymphomas (NHL) are a heterogeneous group of lymphoid tumors that have distinctive clinical and biologic behaviors. Currently tumor classification by the World Health Organization (WHO) establishes specific designations within the various lymphomas, whereas older classification systems did not do this. For example, in the older Revised European-American Lymphoma Classification or earlier systems such as the Kiel or the Lukes-Collins systems, tumors were not differentiated biologically. B- and T-cell tumors were not classified separately. Consequently, many findings from older clinical trials reflect outcomes based on the inclusion of divergent entities with markedly different clinical behaviors. Comparing newer trial data to older “historic control” trial data is generally not valid for this reason, among others. Current clinical trial design, inclusion criteria, and interpretation of findings must take these advances in classification into account.

This chapter focuses on the major disease entities of NHL while mentioning the spectrum of lymphoid diseases in order to emphasize the imprecision of the term non-Hodgkin lymphoma. This overview is intended to impress that accurate diagnosis of the specific NHL subtype is critical to interpretation of clinical trials data, to foster improved therapeutics in the future, and to understand individual patient management.

Refinement in diagnostic resolution is an evolving science. Recent advances have led to resolution of clinically relevant molecular-based distinctions among lymphomas. For example, gene transcription signatures enable distinction of multiple subtypes of diffuse large B-cell lymphoma (DLBCL). While some of these methods are not suited for everyday clinical practice, emerging algorithms to classify lymphomas based on immunohistochemistry may facilitate incorporation of important differences into clinical practice in the future.

EPIDEMIOLOGY

A steady increase in the age-adjusted incidence of lymphomas per 100,000 persons has been documented with 11.1 cases in 1976, 19.0 in 2000, and 22.7 by 2008. Approximately one-third of the increase may be attributed to a combination of iatrogenic immunosuppression and the AIDS epidemic. In more recent years, NHL cases attributable to HIV infection have decreased consequent to effective combination antiretroviral therapy (cART). Other potential causes include increased exposure to environmental carcinogens. Most, though not all, NHL types occur more commonly in males, and whites are affected more than blacks.

PATHOPHYSIOLOGY

A major risk factor for NHL is an abnormality of immune function (either immune deficiency or dysregulation). Examples include HIV infection, iatrogenic immune suppression, autoimmune diseases, and congenital immune deficiencies (e.g., Wiskott-Aldrich, X-linked lymphoproliferative disorder). Also, oncogenic viruses have been implicated. The gamma herpes viruses are linked to certain NHL subtypes, especially lymphomas associated with immune deficiency states. These include Epstein-Barr virus (EBV), which is highly associated with African Burkitt lymphoma (BL) and AIDS-related DLBCL; the Kaposi sarcoma-associated herpes virus (KSHV) (also known as human herpes virus-8 or HHV-8) is etiologically linked to primary effusion lymphomas (PELs) and multicentric Castleman disease, a rare lymphoproliferative disorder associated with increased risk of developing aggressive B-cell lymphoma. These latter two conditions are primarily seen in individuals with HIV infection. Other oncogenic viruses include the human retroviruses and RNA viruses. Human T-lymphotropic virus Type 1 (HTLV-1) is causative of adult T-cell leukemia/lymphoma (ATLL), and the hepatitis C virus (HCV) is associated with splenic marginal zone lymphoma. In addition to infectious agents, environmental and occupational exposures, especially organic compounds such as organophosphate insecticides have been linked to lymphoma risk.

CLASSIFICATION

The current WHO classification utilizes immunophenotypic, molecular, genetic, and clinical elements to distinguish NHL subtypes. New high-throughput technologies such as gene expression profiling (GEP), comparative genomic hybridization, single nucleotide polymorphism arrays, micro RNAs, methylation, acetylation, and tissue microarrays foster mechanistic understanding of lymphoma biology that may eventually inform targeted therapeutics. These technologies are not well suited to routine clinical application owing to expense and complicated performance characteristics of the tests. Efforts to develop simpler algorithms will be important for clinical application. Yet, currently available laboratory procedure should be applied in the clinical setting to classify lymphoid tumors appropriately. The text of this chapter presents the lymphoid tumors in the order of the 2008 WHO classification of lymphoid tumors (Table 16.1). Tables 16.2 to 16.5 provide summaries of molecular and immunophenotypic characteristics of selected tumors encountered in clinical practice.

NHLs are broadly classified as B-cell or T-cell lymphomas, depending on the lymphocyte lineage giving rise to the tumor. B-lymphocytes give rise to B-cell NHL, 88% of all NHL. T-lymphocytes give rise to T-cell NHL, 12% of NHL. Expression (or its lack thereof) of cell surface antigens and immunoglobulin proteins is dependent on the type of lymphocyte and its stage of differentiation. Analysis of these proteins in tumor cells is diagnostically useful as well as for determining tumor histogenesis. Importantly, no single immunophenotypic marker is specific for any tumor, and a constellation of features is required for specific diagnosis.

There is an increasing appreciation of the relationship between tumor tissue origin and clinical behavior. For example, those DLBCL with an activated B-cell-like gene signature (termed ABC-DLBCL) can be distinguished from those that have a germinal center-like signature (termed GCB-DLBCL), and the latter have a more favorable prognosis.

The WHO recognizes four major categories of lymphoid neoplasms: (1) B-cell neoplasms; (2) T and natural killer (NK) cell neoplasms; (3) Hodgkin lymphoma; and (4) immunodeficiency- associated lymphoproliferative disorders. The latter are considered distinct diagnostic entities, though they share similar characteristics with tumors arising in immunocompetent hosts. A cell of origin is postulated for each neoplasm, though this may not necessarily be the cell in which the initial transforming event occurs. Thus, the designated cell of origin represents the state of differentiation of the tumor cells that are seen in the tissues.

It can also be helpful to consider lymphoid neoplasms broadly in terms of the arm of the immune system from which the tumor arises: the innate or adaptive immune response. The innate immune responses are a first line of mucocutaneous defense not requiring antigen-presenting cells to initiate the immune response. Included in the innate immune systems are NK cells, CD3+ CD56+ T-cells or NK-like T-cells, and γδ T-cells. The innate immune system cells are mainly extranodal, and lymphomas arising from these cells thus tend to be extranodal. The adaptive immune response is more complex and specific for particular pathogens and capacity to develop memory. The complexity of the adaptive immune response not only gives rise to the molecular heterogeneity of many B-cell lymphomas, but also helps to determine the type of lymphoma as it relates to a particular B-cell stage of development. Most B-cell neoplasms tend to reflect the stages of normal B-cell differentiation and the lymph node architecture is highly relevant to this process: nodal neoplasms are more likely to be seen.

Table 16.1  2008 WHO Classification of Lymphoid Tumors

Precursor B- and T-Cell Neoplasms

Precursor B lymphoblastic leukemia/lymphoma

B lymphoblastic leukemia/lymphoma, NOS

B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities

B lymphoblastic leukemia/lymphoma with T(9;22) (q34; q11.2; BCR-ABL1)

B lymphoblastic leukemia/lymphoma with t(v;11Q23); MLL rearranged

B lymphoblastic leukemia/lymphoma with t(12;21) (p13;22);TEL_AML1,(ETV 6-RUNX1)

B lymphoblastic leukemia/lymphoma with hyperdiploidy

B lymphoblastic leukemia/lymphoma with hypodiploidy (hypodiploid ALL)

B lymphoblastic leukemia/lymphoma with t(5;14) (q31;q32); IL-3-IGH

B lymphoblastic leukemia/lymphoma with t(1;19) (q23; p13.3);E2A-PBX1 (TCF3-PBX1)

Precursor T lymphoblastic leukemia/lymphoma

Mature B-Cell Neoplasms

Chronic lymphocytic leukemia/small lymphocytic lymphoma

B-prolymphocytic leukemia

Splenic marginal zone lymphoma

Hairy cell leukemia variant

Splenic B-cell lymphoma/leukemia, unclassifiable

Splenic diffuse red pulp small B-cell lymphoma

Hairy cell leukemia variant

Lymphoplasmacytic lymphoma/Waldeström macroglobulinemia

Heavy chain disease

γ Heavy chain disease

μ Heavy chain disease

α Heavy chain disease

Plasma cell myeloma

Solitary plasmacytoma of bone

Extraosseous plasmacytoma

Extranodal marginal zone lymphoma (MALT lymphoma)

Nodal marginal zone lymphoma

Pediatric nodal marginal zone lymphoma

Follicular lymphoma

Pediatric follicular lymphoma

Mantle cell lymphoma

DLBCL, NOS

T-cell/histiocyte-rich large B-cell lymphoma

Primary DLBCL of CNS

Primary cutaneous DLBCL, leg type

DLBCL associated with chronic inflammation

Lymphomatoid granulomatosis

Mediastinal (thymic) large B-cell lymphoma

Intravascular large B-cell lymphoma A

LK-positive large B-cell lymphoma

Plasmablastic lymphoma

Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease

Primary effusion lymphoma

Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features intermediate between diffuse and large B-cell lymphoma and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features intermediate between diffuse and large B-cell lymphoma and classical Hodgkin lymphoma

Mature T-Cell and NK-Cell Neoplasms

T-cell prolymphocytic leukemia

T-cell large granular lymphocytic leukemia

Chronic lymphoproliferative disorder of NK cells

Aggressive NK-cell leukemia

Systemic EBV T-cell lymphoproliferative disease of childhood

Hydroa vacciniforme-like lymphoma

Adult T-cell leukemia/lymphoma

Extranodal NK/T-cell lymphoma nasal type

Enteropathy-type T-cell lymphoma

Hepatosplenic T-cell lymphoma

Subcutaneous panniculitis-like T-cell lymphoma

Mycosis fungoides

Sézary syndrome

Primary cutaneous CD30⁺ T-cell lymphoproliferative disorders

Primary cutaneous anaplastic T-cell lymphoma (ALCL)

Lymphomatoid papulosis

Primary cutaneous γδ T-cell lymphoma

Primary cutaneous CD8⁺ aggressive epidermotropic cytotoxic T-cell lymphoma

Primary cutaneous CD4 positive aggressive small to medium-size T-cell lymphoma

Peripheral T-cell lymphoma, NOS

Angioimmunoblastic T-cell lymphoma

Anaplastic large cell lymphoma, ALK⁺

Anaplastic large cell lymphoma, ALK−

Hodgkin Lymphoma

Nodular lymphocyte-predominant Hodgkin lymphoma

Classical Hodgkin lymphoma

Nodular sclerosis classical Hodgkin lymphoma

Mixed cellularity classical Hodgkin lymphoma

Lymphocyte-rich classical Hodgkin lymphoma

Lymphocyte-depleted classical Hodgkin lymphoma

Immunodeficiency-Associated Lymphoproliferative Disorders

Lymphoproliferative diseases associated with primary immune disorders

Lymphomas associated with HIV infection

Post-transplant lymphoproliferative disorders (PTLD)

Early lesions

Plasmacytic hyperplasia

Infectious mononucleosis-like

PTLD Polymorphic

PTLD Monomorphic PTLD (B and T/NK cell types)

Classical Hodgkin lymphoma type PTLD

ALCL, anaplastic large lymphoma; ALL, acute lymphoblastic leukemia; CNS, central nervous system; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; HHV8, human herpes virus-8; MALT, mucosa-associated lymphoid tissue; NK, natural killer; PTLD, post-transplant lymphoproliferative disorders.

Following are the specific disease entities in order of presentation of the 2008 WHO Classification of lymphoid tumors.¹

Precursor B- and T-Cell Neoplasms

Specific entities are B lymphoblastic leukemia/lymphoma that are (a) not otherwise specified, and (b) those with recurrently genetic abnormalities. Those with t(9;22)(q34;q11.2); BCR-ABL1 have improved prognosis in the era of imatinib and later-generation tyrosine kinase inhibitors. T lymphoblastic leukemia/ lymphoma is termed lymphoma when the disease is confined to nodal masses and does not involve the peripheral blood and marrow. T-ALL comprises 25% of adult ALL. See Chapter 20 for discussion and treatment.

Mature B-Cell Neoplasms

These tumors range broadly in clinical behavior. The very indolent tumors have low curative potential but median survivals measured in years to decades. The aggressive tumors have varying curative potential but without treatment can have a rapidly fatal clinical course.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

The term small lymphocytic lymphoma is used for non-leukemic cases with tissue morphology and immunophenotype of chronic lymphocytic leukemia (CLL). There must be adenopathy and no cytopenias due to bone marrow infiltration. See Chapter 20 for discussion and treatment.

B-Prolymphocytic Leukemia

B-Prolymphocytic leukemia (BPLL), formerly considered a variant of CLL, is now recognized as a distinct aggressive poor-prognosis tumor with a median survival of about 3 years. Purine analogues such as fludarabine, cladribine, and pentostatin are associated with approximately 50% response rates (RRs), including some complete responses. Allogeneic stem cell transplant may offer curative potential in selected patients.

Splenic Marginal Zone Lymphoma

Splenic marginal zone lymphoma comprises <2% lymphoid neoplasms, but may account for the majority of otherwise unclassifiable CLL that are CD5−. It appears to be associated with hepatitis C infection. Indolent cases may be observed without therapy. Splenectomy can be effective. Systemic alkylating or purine analog therapy with rituximab may be used.

Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a rare indolent lymphoid leukemia. Treatments include cladribine and pentostatin. Rituximab is also useful. Immunotherapy directed against CD22 is of interest. Targeted therapy exploiting the recent finding of BRAF mutations in HCL may prove effective, though limited data currently exists.

Splenic B-Cell Lymphoma/Leukemia, Unclassifiable

These include splenic diffuse red pulp small B-cell lymphoma, and hairy cell leukemia variant (HCLv). Both are indolent and may respond to splenectomy. HCLv may respond to immunotherapy directed at CD20 and/or CD22.

Lymphoplasmacytic Lymphoma

This tumor is the most frequent cause of Waldenström macroglobulinemia and is associated with marrow and nodal involvement with paraproteinemia (usually IgM). It is chemotherapy sensitive, but novel agents such as bortezomib, lenalidomide, bendamustine, azacitidine, and vorinostat have reported activity. Combined with rituximab, these agents are of interest and may prove to be useful. Some experts suggest autologous stem cell transplantation (ASCT), but this may not be suitable for some patients. Recent identification of a point mutation in the MYD88 gene leading to NF-κB activation may provide rationale for more targeted therapy in this disease, including use of bortezomib.

Heavy Chain Diseases

Heavy chain diseases (HCD) are characterized as gamma, alpha, or mu heavy chains. The gamma form ranges from indolent to aggressive behavior; mu HCD is slowly progressive; alpha HCD involves an immunoproliferative small intestinal disease that may remit with antibiotic therapy in early stages. Transformation to DLBCL and a fatal outcome is frequent.

Plasma Cell Neoplasms

Monoclonal gammopathy of undetermined significance, multiple myeloma, and other forms are increasingly appreciated to represent distinct entities with clonal evolution over the natural history of the diseases. For more details, see Chapter 30.

Extranodal Marginal Zone Lymphoma of Mucosa-associated Lymphoid Tissue

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is an indolent extranodal tumor comprised of morphologically heterogeneous small B-cell and scattered immunoblasts and centroblast-like cells. It comprises up to 50% of primary gastric lymphoma and is associated with Helicobacter pylori. Antibiotic therapy eradication of H. pylori can induce gastric MALT lymphoma remission in cases demonstrated to be associated with this infection. Other localized sites and cases not associated with H. pylori can be treated with surgery or low-dose radiation.

Nodal Marginal Zone Lymphoma

Nodal marginal zone lymphoma (NMZL) is a primary nodal B-cell neoplasm morphologically similar to MALT lymphoma. It is essential to rule out MALT, Hashimoto thyroiditis, or Sjögren syndrome.

Follicular Lymphoma

Follicular lymphoma (FL) arises from germinal center B-cells and comprises about 20% of all lymphomas, primarily affecting adults in the 6th decade. Cells express BCL2 protein related to t(14;18) (q32;q21), the genetic hallmark of FL. Widespread disease at diagnosis and bone marrow involvement is common. FL is graded by determining the content of centroblasts per high-power field (1–5 in grade 1, 5–15 in grade 2, and >15 n grades 3A and 3B. 3B reflects sheets of centroblasts without detectable centrocytes seen in 3A). This grading system has been criticized for lack of clinical relevance and poor interobserver reproducibility. Localized disease can be treated with radiation therapy alone. Asymptomatic disseminated disease may be watched. Symptomatic disease or disease-causing psychological distress can be managed in a variety of ways (as discussed below).

Primary Cutaneous Follicle Center Lymphoma

Primary cutaneous follicle center lymphoma (CFCL) generally presents on the head or trunk. This is distinguished from primary cutaneous DLBCL, leg type in that it has a variable number of centrocytes/ centroblasts as in FL. Rituximab and other immunotherapeutics are commonly used; disease control is variable.

Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) generally presents with stage III or IV disease and peripheral blood involvement is common. Cell cycle protein cyclin D1 overexpression is almost always present. MCL generally affects older adult males. Although it behaves somewhat aggressively, this lymphoma appears to be incurable, though perhaps there is a curative potential with allogeneic transplantation in selected patients.

Diffuse Large B-Cell Lymphoma

DLBCL is a constellation of heterogeneous disease entities each with distinct morphologic, biologic, and clinical characteristics. These differences are associated with molecular and immunophenotypic findings that help to segregate the various entities into various subgroups.

DLBCL, not otherwise specified comprises around 30% of adult NHL. It can arise de novo or consequent to progression of other lymphoid malignancies such as CLL/SLL, FL, marginal zone lymphoma, or nodular lymphocyte-predominant Hodgkin lymphoma. Morphologic variants include centroblastic, immunoblastic, and anaplastic subtypes. Molecular subgroups include germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Adjusted for clinical features such as the international prognostic index (IPI—see below), the 5-year overall survival (OS) for the GCB-like subset is superior to that of the ABC-like subset, establishing these as distinct clinicobiologic entities. Most are CD20+, a therapeutic target exploited to benefit survival with the use of monoclonal antibodies, particularly rituximab.

Other Diffuse Large B-cell Lymphoma Subtypes

  T cell/histiocyte–rich large B-cell lymphoma accounts for <10% of DLBCL; it is found mainly in middle-aged males. The large B-cells are surrounded by abundant T-cells and histiocytes.

  Primary DLBCL of the central nervous system (CNS) comprises <1% of NHL and approximately 2% of brain tumors; these occur mainly in older persons (median age 60 years) or those with immunosuppression. The latter are frequently associated with EBV in the tumor cells. Neurologic and psychiatric presentation is not uncommon. High-dose methotrexate forms the basis of therapy. Whole-brain radiation may prolong progression-free survival (PFS), though randomized study results show no survival advantage in comparison to high-dose methotrexate-based therapy alone. Radiotherapy induced late-term neurotoxicity can lead to severe cognitive deficit.

  Primary cutaneous DLBCL, leg type occurs mainly in women with median age in the 70s. Presentation is generally an ulcerative red or bluish tumor on the leg, though other sites can be involved. Five-year survival is approximately 50%. Rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is often used; relapse is common. Radiotherapy, immunotherapy, stem cell transplant are reported as being used in these patients.

  EBV-positive DLBCL of the elderly generally occurs at age >50 years unassociated with prior immunodeficiency and can have an aggressive course.

  DLBCL associated with chronic inflammation, as the name implies, arises in the setting of long-standing chronic inflammation, such as pyothorax or in chronic osteomyelitis. The inflammation appears to be etiologically related.

  Lymphomatoid granulomatosis is a proliferation of EBV-positive B-cells with reactive T-cells in the setting of a poorly defined T-cell immune-impaired state. Advanced disease can involve brain, kidneys, lung, liver, and skin. Early lower-grade disease may be cured with interferon therapy.

  Primary mediastinal (thymic) large B-cell lymphoma (PMBL) arises in the mediastinum and is of putative thymic B-cell origin. PMBL has distinctive clinical, immunophenotypic, and genotypic characteristics. Presenting features include localized disease, and signs and symptoms related to a large anterior mediastinal mass. Dissemination to multiple organs can occur. CD19 and CD20 are present, while CD10 and CD5 are absent. GEP indicates PMBL is a molecular type of DLBCL distinct from ABC- and GCB-like tumors. Phase II data suggests favorable outcome with dose-adjusted EPOCH-R chemotherapy.

  Intravascular large B-cell lymphoma (IVLBCL) is a rare extranodal lymphoma. Growth within the lumina of capillaries and other smaller vessels, generally widely disseminated to virtually any organ and bone marrow is characteristic. Lymph nodes are not usually involved. This is an aggressive tumor poorly responsive to chemotherapy. Rituximab may have activity.

  ALK-positive large B-cell lymphoma is also known as ALK-positive plasmablastic B-cell lymphoma, and comprises <1% of lymphomas. Advanced-stage presentation is typical; so few cases have been reported that there is limited information on outcomes, though short median survival of less than 12 months is reported.

  Plasmablastic lymphoma (PBL) is seen as diffuse proliferations of immunoblastic appearing cells and has the immunophenotype of plasma cells. Originally thought to involve predominantly the oral cavity, it is now appreciated to also involve other extranodal sites. It is uncommon and mainly seen in HIV-infected males. The tumor has a high proliferation rate. The presence of EBV by EBER can help establish the diagnosis. CD20 expression is generally weak. The clinical course is aggressive. Occasional patients have long-term survival. Some physicians have used abbreviated cycles of EPOCH chemotherapy with involved field radiotherapy for limited disease with encouraging results, though the data are anecdotal.

  Large B-Cell lymphoma arising in HHV8-associated multicentric Castleman disease is usually associated with HIV infection; the cells have appearance of plasma cells, and should be distinguished from PBL. Other HHV-8-associated neoplasms are sometimes seen (e.g., Kaposi sarcoma) at presentation.

  PEL is a viral associated tumor (HHV-8) and presents mainly as serous effusions in the body cavities and is associated with HIV infection. All the tumor cells contain HHV-8 in 100% of cases and in 70% of cases are also EBV positive. Generally B-cell markers are absent, though they express CD45, and may express CD30. Immunoglobulin genes are clonally rearranged and hypermutated. Occasional patients have good outcome, but in general the prognosis is very poor.

Burkitt Lymphoma

BL is a rapidly growing lymphoma often involving extranodal sites and can present as an acute leukemia. The “starry sky” pattern on histopathology examination results from macrophages that have injected the apoptotic tumor cells. The endemic form found in equatorial Africa is generally EBV associated and is the most common childhood tumor there. The sporadic type represents 1% to 2% of lymphomas in developed countries. Immunodeficiency-associated BL is seen primarily in those with HIV infection, and can be the initial AIDS-defining condition. Short course intensive chemotherapy with rituximab is generally recommended (for adults), and cure rates are quite high for standard-risk disease. Poor-risk features (involvement of bone marrow, CNS, tumor mass >10 cm, and high serum LDH) do not rule out curative intent, but this outcome may be achieved in only 60% of such patients. Preliminary data suggests that dose-adjusted EPOCH-R chemotherapy (given with additional rituximab doses) is very effective in BL and a national clinical trial is being undertaken to better define this regimen for BL.

B-Cell Lymphoma, Unclassifiable, with Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma

As the name suggests these aggressive tumors have morphologic features of both DLBCL and BL. Extranodal and leukemic presentation may occur along with mass lesions. CD19, CD20, CD22 are positive. BCL2 positivity in a case that would otherwise be classified as BL suggests a double hit lymphoma with both MYC and BCL2 translocations. The cellular proliferation is high. The standard of care is not established, but outcome with R-CHOP is reportedly poor.

Mature T- and NK-Cell Neoplasms

T-Cell Prolymphocytic Leukemia

The median age is 65 years and T-cell prolymphocytic leukemia (T-PLL) comprises <2% of mature lymphocytic leukemia. HTLV-1 is negative. Therapeutics includes alemtuzumab and allogeneic stem cell transplant. These are reported to benefit selected patients, but 3-year OSs are less than 30%.

T-Cell Large Granular Lymphocytic Leukemia

T-cell large granular lymphocytic leukemia (T-LGL) generally presents with a history of persistent, increased peripheral blood large granular lymphocytes of uncertain cause. Most cases are in those over 40 years of age, but the disease is rare (<2% of mature lymphocytic leukemia). It is important to distinguish T-LGL from a restricted clonal proliferation that sometimes occurs following allogeneic hematopoietic cell transplant. An indolent course is typical. Severe neutropenia is frequent, though thrombocytopenia is not generally seen. Along with the association of inflammatory comorbid conditions, immunophenotype suggests a chronic antigen-driven process and informs the use of immunosuppressive therapy for T-LGL.

Chronic Lymphoproliferative Disorders of NK Cells

Characterized by >6 months increased peripheral blood NK cells without a clear etiology, this rare condition presents equally in both genders with a median age of 60 years. The course is indolent in most cases. Spontaneous remission or aggressive transformation can occur.

Aggressive NK-Cell Leukemia

Nearly always EBV associated, with a median age of 42 years of age, it affects Asian ethnic groups predominantly. The immunophenotype is identical to that of extranodal NK/T-cell lymphoma except that CD16 is frequently positive.

Epstein Barr Virus-positive T-Cell Lymphoproliferative Disorders of Childhood

Two forms occur. The systemic form is a life-threatening clonal proliferation of EBV-infected T-cells that occurs shortly after primary acute EBV infection or in the setting of chronic active EBV infection. Rapid progression with multi-organ failure ensues over days to weeks. The hydroa vacciniforme-like lymphoma is a cutaneous T-cell lymphoma (CTCL) occurring in children and is associated with sun sensitivity. After a period of recurrent skin lesions over a period of up to 15 years, a more aggressive systemic progression may occur.

Adult T-Cell Leukemia/Lymphoma

Caused by the human retrovirus HTLV-1, ATLL is endemic in Southwestern Japan, the Caribbean basin, and parts of Central Africa where HTLV-1 prevalence is high. There are several clinical variants: acute, lymphomatous, chronic, and smoldering. The most common is the acute form with elevated white blood count, skin rash, lymphadenopathy, and hypercalcemia. Treatment with zidovudine and interferon-α (IFN-α) may prolong survival, but survival remains poor at a median of 9 months. Recent retrospective analysis supports using chemotherapy in sequence with zidovudine/IFN-α. Of interest, HTLV-1 also causes non-hematologic disease: tropical spastic paraparesis (TSP), also known as HTLVassociated myelopathy or chronic progressive myelopathy. HTLV-1 infection of the spinal cord results in paraparesis and weakness of the legs.

Extranodal NK/T-Cell Lymphoma, Nasal Type

This extranodal, often EBV-associated lymphoma results in vascular damage and often shows a cytotoxic T-cell phenotype (hence the NK/T designation). Upper aerodigestive involvement is characteristic, though it can involve other areas of the body. Nodular skin lesions, perforating intestinal lesions, or other sites are also seen. Prognosis is variable, but may be improved with upfront combination chemoradiotherapy. Bone marrow transplantation may be curative in selected patients.

Enteropathy-associated T-Cell Lymphoma

This is an intestinal lymphoma (mainly of the jejunum or ileum) composed of large lymphoid cells with an inflammatory background. It appears to be associated with celiac disease; early prevention with gluten-free diet appears to protect against lymphoma. There is a monomorphic variant occurring in regions where celiac disease is rare, and this is probably a different disease entity. Once lymphoma has developed, there is poor response to therapy and prognosis is unfavorable.

Hepatosplenic T-Cell Lymphoma

The T-cells of this extranodal and systemic lymphoma are generally derived from the γδ T-cell receptor type. The peak incidence is in adolescents and young adults and accounts for <1% of all lymphomas. There is an association with long-term iatrogenic immunosuppression. Initial response to chemotherapy is followed by relapse and median survival is generally less than 2 years.

Subcutaneous Panniculitis-like T-Cell Lymphoma

The median age with respect to subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is 35 years, and up 20% may have associated autoimmune disease, such as systemic lupus erythematosus. Patients often present with multiple subcutaneous nodules, particularly in the extremities and trunk. Cytopenias and elevated liver function tests are common. The neoplastic cells are usually CD8-positive.

They express βF1 and are negative for CD56: this finding helps distinguish from subcutaneous γδ T-cell lymphoma (which has a worse prognosis). If hemophagocytic syndrome develops, prognosis is poor. Otherwise, 80% survive 5 years or longer. Combination chemotherapy is used, but reports of immune suppressive therapy alone suggest activity and may help in treatment choices.

Mycosis Fungoides

Mycosis fungoides (MF) is a primary CTCL indicating the classical presentation of evolving skin patches, plaques, and tumors or the variants that show a similarly evolving clinical course. It accounts for 50% of CTCL. There is a wide age range, but most are older adults. In advanced disease, nodal, organ, and bone marrow involvement may be seen. The indolent clinical course slowly progresses over years to decades. Clinically it is staged I–IV. Disease confined to the skin with no lymph nodes is stage I and when there is a high blood Sézary cell count of >10,000/μL and or extensive lymph node involvement it is stage IV. Stage II has some lymph node involvement and stage III has skin erythroderma with or without lymph node and/or low Sézary cell count. Traditionally Sézary syndrome (SS) has referred to the leukemic form (see below). Early-stage disease does not benefit from multi-agent chemotherapy, and dermatologic skin-directed therapy is generally most appropriate.

Sézary Syndrome

SS is defined by the triad of erythroderma, generalized lymphadenopathy, and the presence of clonally related neoplastic T-cells with cerebriform nuclei (Sézary cells) in skin, lymph nodes, and peripheral blood. In addition, the absolute Sézary count must be >1,000/μL, there must be an expanded CD4+ T-cell population resulting in CD4/CD8 ratio of more than 10 and/or loss of one or more T-cell antigens. This is an aggressive disease with less than 20% survival at 5 years. Patients often succumb to opportunistic infections.

Primary Cutaneous CD30-Positive T-Cell Lymphoproliferative Disorders

Primary cutaneous CD30-positive T-cell lymphoproliferative disorders (PCALCL) account for 30% of CTCL; types are primary cutaneous anaplastic large lymphoma (C-ALCL), lymphoid papulosis (LyP), and borderline cases (where clear distinction between C-ALCL and LyP is not possible). Though prognosis is favorable, systemic lymphoma may develop, thus warranting ongoing surveillance. In PCALCL, surgical excision and radiotherapy are most commonly used for solitary tumors, whereas chemotherapy is given for multifocal disease. UV light phototherapy and low-dose methotrexate are commonly used therapies for LyP. There is interest in using the CD30-directed monoclonal immunoconjugate brentuximab vedotin for these tumors.

Primary Cutaneous Peripheral T-Cell Lymphomas, Rare Subtypes

These include the primary cutaneous γδ T-cell lymphoma with activated γδ T-cells having a cytotoxic phenotype and the CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, both with an aggressive course; the CD4-positive small/medium T-cell types have a more favorable prognosis with 80% survival at 5 years.

Peripheral T-Cell Lymphoma, Not Otherwise Specified

These account for about 30% of peripheral T-cell tumors in the Western world and are seen mainly in adults, with a male: female ratio of 2:1. Lymph node involvement is typical, but any site can be affected, including peripheral blood. Most patients present with advanced disease and B symptoms. CD30 expression is found in some cases. A variant, lymphoepitheliod (Lennert lymphoma) can be admixed with inflammatory and EBV+ Reed-Sternberg-like cells. The follicular variant can appear similar to nodular lymphocyte-predominant Hodgkin lymphoma while the T-zone variant can be mistaken for benign hyperplasia. Owing to CD30 expression, there is interest in brentuximab vedotin therapeutically.

Angioimmunoblastic T-Cell Lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) occurs in middle age and in the elderly, and accounts for 15% to 20% of peripheral T-cell lymphomas. It is nearly always EBV associated, though the neoplastic cells are EBV negative. Generally AITL presents with generalized lymphadenopathy, hepatosplenomegaly, systemic symptoms, and polyclonal hypergammaglobulinemia. Pruritic skin rash is common. Effusions, arthritis, circulating immune complexes, hemolytic anemia and EBV-positive B-cell expansion are common. Median survival is <3 years. The associated immune dysfunction renders aggressive chemotherapy administration infeasible in many cases. GEP suggests deregulation of vascular endothelial growth factor as part of the pathogenesis, and has created interest in targeting this pathway therapeutically.

Anaplastic Large Cell Lymphoma , ALK-Positive

Anaplastic large cell lymphoma (ALCL), ALK-positive, is a CD30+ T-cell lymphoma that has a translocation involving the anaplastic lymphoma kinase (ALK) gene and expression of the ALK protein. It is important to distinguish this tumor from primary cutaneous ALCL and other lymphomas with anaplastic features. ALCL, ALK+ accounts for about 3% of adult and up to 20% of childhood lymphomas. Most cases present with advanced-stage disease with peripheral and/or abdominal adenopathy and bone marrow infiltration. High fevers are common. These tumors have a better prognosis than the ALCL, ALK-negative counterpart. The overall 5-year survival is about 80% with standard CHOP chemotherapy. Brentuximab vedotin is approved for relapsed ALCL.

Anaplastic Large Cell Lymphoma, ALK-Negative

Morphologically similar to ALCL, ALK+ and also CD30+, but lacking ALK protein expression, this entity was given a provisional categorization in the 2008 WHO classification system. The ALK-negative occurs mainly in adults over 40 years of age, and has a poorer prognosis compared to the ALCL, ALK+. Less than 20% OS at 5 years is expected. Brentuximab vedotin is approved for relapsed ALCL.

Hodgkin Lymphoma

Hodgkin lymphoma is covered in Chapter 20.

Immunodeficiency-associated Lymphoproliferative Disorders

Lymphoproliferative Disease Associated with Primary Immune Disorders

The most common lymphoproliferative disease (LPD) in this setting are ataxia telangiectasia, Wiskott-Aldrich syndrome, common variable immunodeficiency (CVID), severe combined immunodeficiency, X-linked lymphoproliferative disorder, Nijmegen breakage syndrome, hyper IgM syndrome, and autoimmune lymphoproliferative syndrome. In addition to LPD, other neoplasms occur at high rates in affected persons. Except for CVID, children are affected most. EBV is frequently involved.

Lymphomas Associated with HIV Infection

DLBCL (including primary brain), Burkitt, PEL, and PBL are AIDS-indicator conditions in those with HIV infection. Since the advent of cART, there has been a marked epidemiologic shift in the occurrence and outcomes of these tumors. Rituximab-based chemotherapy is the standard for DLBCL and Burkitt. Many experts recommend R-EPOCH in this setting. CHOP should not be used for HIV-Burkitt. Primary brain lymphoma (PCNSL) is rarely seen in the cART era. However, delays in diagnosis impair outcome. The standard diagnostic algorithm for HIV-PCNSL established early in the AIDS epidemic is no longer justified. Mass brain lesions should be approached in this population with the same degree of urgency and using the same diagnostic wherewithal as used in the HIV-unrelated setting, especially in patients likely to respond to cART. In particular, empiric antibiotic therapy to assess treatment failure as a means of establishing a malignant diagnosis should be relegated to the history books. When surgical biopsy is not feasible, CSF should be assessed for EBV by PCR in conjunction with FDG-PET of the brain. If both tests are positive, the positive predictive value for lymphoma is 100%, and specific therapy can be commenced. Patients with advanced HIV disease not amenable to treatment because of resistant HIV are becoming rarer with improvement in HIV therapeutics. Thus, if palliative approaches are deemed most appropriate based on the underlying HIV, it is essential to obtain expert HIV assessment in order to justify this decision.

Post-transplant Lymphoproliferative Disorders

These occur after solid organ or hematopoietic cell transplant. There is a range of pathology from those that are EBV-driven polyclonal proliferations (the majority of cases) to those that are EBV-positive or EBV-negative B- and T-cell lymphomas similar to those seen in the immunocompetent setting.

Other Iatrogenic Immunodeficiency-associated Lymphoproliferative Disorders

These LPD arise in patients treated with immunosuppressive drugs and appear as a spectrum from polymorphic proliferations as seen in PTLD at one end to DLBCL at the other end of the spectrum. EBV is variably associated depending on the underlying cause for iatrogenic immune suppression and the LPD presentation itself.

CLINICAL MANAGEMENT

Staging

Pretreatment staging evaluation for systemic NHL follows.

I.  Diagnostic confirmation by tissue biopsy

A.  Sufficient material is critical in order to conduct the studies needed to ensure accurate diagnosis

B.  Needle biopsies generally yield inadequate tissue for these studies and should be avoided for primary diagnosis

C.  Important studies for diagnostic confirmation often include the following:

1.  Assessment of clonality

2.  Immunophenotypic, cytogenetic, and molecular studies

3.  Markers of histogenesis (B- versus T-cell origin; in DLBCL, determination of germinal center versus non-germinal center histogenesis are not yet part of standard care, but are increasingly important in developing novel therapeutics)

4.  Oncogene rearrangement can be diagnostically useful

a.  t(8;14) or MYC in Burkitt lymphoma

b.  t(14;18) or bcl-2 in FL

c.  t(2;5) or ALK in ALCL

d.  t(11;14) or bcl-1 in MCL

e.  Trisomy 3 or trisomy 18 (marginal zone lymphoma)

D.  Some tumors (e.g., T-cell–rich B-cell lymphoma or lymphomatoid granulomatosis) have excess reactive T-cells that may obscure the minority of diagnostic malignant B-cells if inadequate tissue is obtained

II.  History and physical examination

III.  Viral testing if indicated by risk or lymphoma subtype

A.  HIV serology in all aggressive NHL

B.  HTLV-1 serology

C.  Hepatitis B and C serology

IV.  Clinical and laboratory assessment of organ function

A.  Include CD4 cell count if HIV-positive

B.  In addition to routine blood tests

1.  Lactate dehydrogenase (LDH; indirect measure of tumor burden and prognosis)

2.  Serum β₂ microglobulin

3.  Serum α-fetoprotein or β-human chorionic gonadotropin in young males with an isolated mediastinal mass where the differential diagnosis includes mediastinal germ cell tumor

VI.  Chest x-ray, computed tomography scans of chest, abdomen, and pelvis, [18F]fluorodeoxyglucose (FDG). Positron emission tomography (PET) for potentially curable lymphomas such as Burkitt, DLBCL, and HL is incorporated into guidelines for staging and response assessment.

VII.  Bone marrow biopsy

VIII.  Lumbar puncture with cytology should be performed in patients at risk for CNS disease:

A.  DLBCL with elevated LDH and more than one extranodal site and/or lymphomatous involvement in the bone marrow

B.  All BL

C.  Some investigators recommend that all ARL cases (regardless of bone marrow and extranodal sites or histologic subtype) be evaluated for CNS disease.

The Ann Arbor Staging System, initially developed for patients with Hodgkin lymphoma, is used in NHL. This system does not apply to lymphoblastic leukemia/lymphoma or to MF (Table 16.6).

Posttherapy Restaging for Response Evaluation

At completion of therapy, repeat all restaging studies. Generally restaging after four cycles is indicated in aggressive lymphomas (repeat all abnormal tests). In indolent lymphomas, response to therapy may be slower; restaging can be performed less frequently. The rate of response may reflect tumor sensitivity to treatment and may have prognostic value. However, early PET in DLBCL has not consistently been shown to predict survival. Other novel imaging methods of interest include diffusion-weighted MRI scanning and may yield similar early response characteristics as FDG-PET.

Disease progression or no response implies extremely poor prognosis. Biopsy of residual masses after therapy may be required to determine whether viable tumor is present. Routine PET surveillance is not recommended after completion of treatment restaging has been performed.

PROGNOSTIC FEATURES

Prognostic features are related to disease and to the individual patient. Disease-related features include tumor bulk, stage, number of extranodal disease sites, and histologic type and tumor histogenesis (or tumor biology). Patient-related factors include age and performance status, and whether or not there are comorbid conditions present that may affect ability to administer therapy.

Prognostic assessment and modeling strategies have been developed to predict the outcome based on clinical presentation. The most commonly used model is IPI (Table 16.6). The IPI was initially developed for aggressive NHL, but is applicable or has been adapted to other NHL subtypes. For example (Table 16.7), the follicular IPI (FLIPI) and the mantle cell IPI (MIPI) are adaptations of the IPI and have prognostic value for the respective histologic NHL types (Table 16.8). In the IPI, 1 point is assigned for each of the following:

  Age

  For IPI and FLIPI age over 60, 1 point

  For MIPI, 1 point for age 50 to 59, 2 points for age 60 to 69, 3 points for age ≥ 70

  Eastern Cooperative Oncology Group (ECOG) performance status 2 or more

  For IPI, 1 point

  For MIPI, 2 points

  LDH above normal

  For IPI and FLIPI, 1 point

  For MIPI 0.67 to 0.99 1 point; 1.0−1.49, 2 points, ≥1.5, 3 points

  Extranodal sites

  For IPI, two or more, 1 point

  For FLIPI, more than four, 1 point

  Stage III or IV disease

  For IPI and FLIPI, 1 point

  Hemoglobin level

  For FLIPI < 120 g/L, 1 point

  WBC, 10⁶/L

  For MIPI 6.7–9.999, 1 point; 1.0–14,999, 2 points; ≥15,000, 3 points

Based on adding points from the various clinical characteristics, risk can be assigned. In age-adjusted IPI for patients younger than 60 years of age, 1 point each is assigned for

  Performance status 2 or more

  LDH above normal

  Stage III or stage IV disease

In ARL, the primary prognostic determinant has traditionally been the CD4 cell count. However, among those whose HIV is sensitive to cART, the IPI and lymphoma-specific features appear to be of relatively greater prognostic importance. Low CD4 cell count alone does not confer poorer outcome to curative intent therapy. However, very low CD4 cell count increases the risk of death due to other AIDS-related causes and unless the patient has an increase in CD4 cells, this risk persists after successful completion of lymphoma therapy.

TREATMENT PRINCIPLES

Treatment of NHL is guided by clinical behavior, and this is in large part dependent on the specific disease entity. Clinical behavior is generally described as indolent, aggressive, or highly aggressive. Conventional treatment now includes chemotherapy, radiotherapy, immunotherapy, or a combination of these modalities. Novel treatments, including immunoconjugates, immune modulators, and molecularly targeted agents are now in everyday practice or the subject of current investigations. Ongoing clinical research will refine how newer treatments are used and combined to augment or supplant current standards of care.

Principles of Therapy: Indolent B-Cell and T-Cell Lymphomas

The natural history is one of a relatively slow-growing lymphoma with low potential for cure but with median survival measured in years to decades. For disseminated symptomatic disease, many experts recommend single agent rituximab or rituximab combined with CHOP or bendamustine. ASCT increases complete responses but not cure. Maintenance rituximab improves PFS. Other approaches that may also be used in patients with disease progression include fludarabine (combined with rituximab and or mitoxantrone or cyclophosphamide) and yttrium 90 ibritumomab tiuxetan radioimmunotherapy. Novel agents such as lenalidomide are of interest. Grade 3B lymphomas are often treated differently than the other grades, using therapy as in DLBCL. FL can progress with transformation to a DLBCL, and this nearly always requires treatment. Generally the prognosis is poor after transformation. See the section on DLBCL for treatment of transformed disease.

Individual prognostic features must be considered in treatment planning. Those with the most favorable prognosis may benefit less from early therapy whereas those with poorer prognosis may derive greater benefit from early therapy. It is important to consider whether the planned therapy will improve or detract from a patient’s well-being. If the toxicity of therapy creates symptoms where there are none, watchful waiting may be a better strategy.

Rituximab as a single agent in previously untreated FL yields up to 75% RRs. Maintenance rituximab may prolong remission (at 3 years of median follow-up, duration of remission was 23 versus 12 months, favoring rituximab maintenance group receiving 375 mg/m² every 2 months for 4 doses post induction). As a single agent in previously treated FL, rituximab can yield responses in 50% to 60% of cases, with median response duration of 6 to 16 months. Rituximab with CHOP induces complete responses in up to 95% of previously untreated FLs with a median response duration not reached at 50 months of follow-up. Rituximab combined with fludarabine yields results similar to CHOP plus rituximab (though fludarabine is profoundly immunosuppressive). Rituximab combined with bendamustine has been reported to have superior RR, PFS, and less toxicity compared to CHOP combined with rituximab, though these findings have as yet not been reported in peer review form.8,9 Following initial therapy, 2 years of rituximab maintenance may improve PFS. Additionally, some advocate use of radioimmunotherapy for initial disease, utilizing ¹³¹I-tositumomab. Radioimmunotherapy for relapsed disease includes yttrium 90-ibritumomab tiuxetan (Zevalin), which is Food and Drug Administration (FDA)-approved and well tolerated. In a randomized trial, Zevalin resulted in statistically and marginally clinically significant higher objective response rate (ORR) and complete response (CR) but not response duration compared with rituximab alone in relapsed or refractory low-grade, follicular, or transformed B-cell NHL. Tositumomab and iodine-131 tositumomab (Bexxar) are approved by the FDA for the treatment of patients with CD20-positive, follicular NHL, in cases with and without transformation, when disease is refractory to rituximab and has relapsed after chemotherapy. Thus, there are a substantial number of therapeutic choices (Table 16.9).

Indolent stage I B-cell lymphomas may be curable with 10-year disease-free survival (DFS) of approximately 50% with radiation alone. Because of the long natural history, this is a difficult disease to study. For example, a large phase II trial of more than 100 patients was initiated in 1984 but was not completed and published until 2003. A 10-year DFS of 76% was reported, suggesting that combined radiation and chemotherapy may be superior to radiotherapy alone in stage I and II disease. Large retrospective databases indicate that the observational strategy does not compromise survival compared to early intervention.

For CLL/small lymphocytic lymphoma treatment options include bendamustine, fludarabine, and rituximab, given concurrently or sequentially. Alemtuzumab is approved for fludarabine-refractory disease, with RRs of approximately 30%. Cladribine may also be used. Novel therapeutics with agents such as PI3Kδ inhibitors, lenalidomide, and Bruton tyrosine kinase inhibition may transform therapy for this disease in the future.

For lymphoplasmacytoid lymphoma/Waldenström macroglobulinemia treatment options include conventional therapies with alkylating agents (especially chlorambucil), with or without corticosteroids. CHOP is sometimes used as well. Purine analogues such as fludarabine or cladribine are also active. RR to first-line therapy ranges from 38% to 85%. RR to fludarabine in previously treated patients ranges from 30% to 50%. Initial therapy with rituximab has produced overall RRs of 20% to 40%, with risk of IgM paraprotein flare. Preliminary data suggests a role for agents such as alemtuzumab, bortezomib. There is interest in thalidomide and analogs, particularly lenalidomide and pomalidomide.

For marginal zone lymphoma effective eradication of H. pylori infection can result in lymphoma regression and likely cure, a finding that strongly supports this bacterial etiology of the tumor. When associated with autoimmune disease (such as Sjögren syndrome or Hashimoto thyroiditis), chemotherapy with or without rituximab may be useful. Local therapy such as surgery or regional irradiation may yield relatively long-term disease control. Splenectomy may be indicated for splenic marginal zone lymphoma. Cases associated with HCV infection may regress with effective HCV therapy.

Principles of Therapy: Aggressive B-Cell Lymphomas

Mantle Cell Lymphoma

Most patients with MCL present with advanced-stage disease. Unlike other aggressive lymphomas, it is incurable and has a short median survival of approximately 6 years. The blastic variant may be more aggressive with a propensity for CNS involvement (25%) and shorter survival. There may be a survival advantage in younger patients with stage IA or IIA treated with radiation therapy. Therapy with rituximab-based CHOP or more intensive regimens such as the Hyper-CVAD regimen are used, sometimes followed with autologous bone marrow transplantation. Large cooperative group studies generally have not confirmed the more intensive therapy to benefit the general population with MCL as many subjects were unable to complete the planned therapy. Bortezomib is approved for treatment of relapsed MCL. Molecular targeted therapies, including mTOR inhibitors, and immune modulator drugs (e.g., lenalidomide) are of interest.

Diffuse Large B-Cell Lymphoma

R-CHOP is the standard of care for curative intent in DLBCL (Table 16.10). For most CD20+ lymphomas, addition of rituximab improves the outcome, and this is certainly the case for DLBCL in which rituximab improves the cure rate.² For localized disease, many experts recommend limited cycles of chemotherapy combined with involved field radiotherapy. Owing to late toxicity and recurrent disease, the OS benefit of this approach is not clear. Salvage therapy following relapse is most effective for those whose disease remains chemotherapy sensitive. Following treatment with regimens such as rituximab, ifosfamide, carboplatin, and etoposide, ASCT offers curative potential for approximately 50% of patients. However, in the rituximab era, patients relapsing within 12 months after initial R-CHOP therapy appear to represent a particularly poor prognostic group. Median event-free survival following ASCT may be less than 12 months for this group.

Probability of cure with initial therapy can be estimated using prognostic models, such as the IPI. Addition of rituximab to CHOP appears to “equalize” various regimens to some extent. For example, the advantage of CHOEP (etoposide added to CHOP) over CHOP alone in young patients is no longer seen when rituximab is added to either regimen.

For stage I to II disease, three cycles of CHOP plus involved-field radiotherapy results in 5-year PFS of 77% and OS of 82%, better than with 8 cycles of CHOP alone (64% and 72%, respectively). However, R-CHOP now is commonly used in early-stage disease without radiation.

In advanced-stage disease, the OS and PFS are approximately 50% and 32%, respectively, at 5 years with CHOP. Randomized trials show that addition of rituximab on day 1 of each cycle of CHOP or CHOP-like cycle resulted in improved event-free survival, PFS, and OS in all patient groups. Maintenance rituximab therapy in DLBCL shows no clear advantage when rituximab is administered concomitantly with chemotherapy. Possible alternatives to R-CHOP as front-line therapy, or as salvage therapy are listed in Table 16.11.

For HIV-associated DLBCL, an underpowered randomized trial of CHOP versus R-CHOP was reported; there was no rituximab benefit in these patients owing to increased infection-related death. However, excess infection was restricted to those with CD4 cells under 50/mm³. Most experts recommend all HIV+ patients receive rituximab if curative intent therapy is planned. Antibiotic prophylaxis is recommended for those with low CD4 cell counts. A randomized phase II trial of R-EPOCH compared to EPOCH followed by rituximab favored concomitant R-EPOCH, and this is a standard of care recommended by many experts treating AIDS-related lymphoma.

TREATMENT PRINCIPLES: HIGHLY AGGRESSIVE B-CELL LYMPHOMA

Burkitt Lymphoma/B-Cell ALL

Intensive short duration therapy forms the basic treatment strategy for curative intent (Table 16.12). Treatment may provoke tumor lysis syndrome and prophylaxis should be used: alkalinize the urine with D5W plus 100 mEq sodium acetate at 100 to 150 mL/h, add allopurinol, 600 mg, orally daily for 2 days, then 300 mg/day orally until resolution of the tumor lysis syndrome. Aggressive chemotherapy is potentially curative in these tumors. For adults, rituximab is now considered a standard part of the treatment. This has not yet been shown to benefit children. For adults, randomized trials have not informed therapy. The CODOX-M/IVAC risk-stratified regimen is based on phase II data.5,6 The regimen consists of three cycles of CODOX-M for low-risk disease (Tables 16.13 to 16.15). (All of the following: normal LDH, WHO performance status 0 or 1, Ann Arbor stage I to II, and no tumor mass 10 cm or larger). For high-risk disease (e.g., do not meet criteria for low risk) four cycles of alternating CODOX-M and IVAC are used. An alternative approach is the hyper-CVAD regimen with addition of rituximab.⁴ Additionally, preliminary data suggest EPOCH-R is effective in BL. As yet there is no sufficient data to recommend this treatment routinely outside of a research protocol, but favorable toxicity profile may justify use if a given patient cannot tolerate more established aggressive dose-intensive therapy. The dose-adjusted EPOCH-R regimen is the focus of a national clinical trial to define its efficacy in Burkitt and C-MYCpositive DLBCL.

Treatment of Recurrent and Refractory B-Cell Lymphoma

Many patients with NHL require additional therapy because of disease recurrence or refractoriness to therapy. Although grades I and II FL are not curable, high-dose chemotherapy followed by autologous transplant improves PFS. In aggressive NHL, approximately 40% to 50% of patients fail to achieve remission with conventional chemotherapy. Among those who do achieve a CR, 30% to 40% will relapse. These patients may benefit from salvage therapy (see Table 16.11).³

Principles of Salvage Therapy

Conventional wisdom promotes the use of non–cross-resistant chemotherapy such as (cisplatin, cytarabine, and dexamethasone), ESHAP (etoposide, methyl prednisolone, cytarabine, and cisplatin), and ICE (ifosfamide, carboplatin, etoposide). However, evolving understanding of cellular apoptotic response to chemotherapeutic stimuli suggests that true non–cross-resistance drugs may not exist—as mechanisms for tumor resistance may not be entirely drug-specific—because of the intrinsic high apoptosis thresholds in refractory tumors. Both in vitro and empiric clinical data provide evidence that tumor resistance can be overcome by using drugs already administered, but with different infusion schedules (e.g., by prolonged infusion regimens such as dose-adjusted EPOCH-R). In addition, other agents such as in ICE or ESHAP have also shown utility in the setting of relapsed and refractory NHL.

High-dose chemotherapy and ASCT may confer curative advantage in some patients whose disease is responsive to salvage chemotherapy. ASCT achieves long-term survival in up to 50% of patients with chemotherapy-sensitive relapsed DLBCL, and some prospective randomized studies have documented the superiority of ASCT over salvage chemotherapy for relapsed DLBCL. Patients with low-risk IPI are most likely to benefit. However, in the rituximab era, those that do relapse following rituximab-inclusive therapy appear to be selected for the poorest-prognosis patients, and salvage is less successful in many cases owing to this.

Allogeneic transplantation remains investigational. Nonmyeloablative or reduced intensity stem cell transplantation (RIST) attempts to exert immunologic effects against the tumor without the risk of high-dose chemotherapy. High-dose chemotherapy does not appear to overcome tumor resistance in the majority of cases. Graft engineering to enhance graft-versus lymphoma benefit and to decrease graft-versus-host complications remains an active area of investigation. Studies have not consistently shown strong graft-versus-lymphoma effects in the majority of patients.

Patients with well-controlled HIV with relapsed NHL should not be routinely excluded from consideration for ASCT.

Acquired Immune Deficiency Syndrome-related Lymphoma (Systemic) Treatment Considerations

In the current era of effective cART for HIV infection, most HIV-infected patients with lymphoma should be treated similar to their HIV-unrelated counterparts. One exception is that some experts recommend CNS prophylaxis for all systemic ARL (Table 16.16). Prior to cART, low-dose chemotherapy was recommended for HIV-infected patients. This should rarely, if ever, be used in the current era. Possible exceptions may be considered for patients who have advanced AIDS with essentially untreatable HIV infection. Standard-dose chemotherapy has supplanted low-dose therapy in the CART era. See the sections above on DLBCL and Burkitt for therapy. Phase II data support use of EPOCH-R and CODOX/M-IVAC in HIV-infected patients.⁷

The use of concurrent CART is unsettled. There are inadequate data to make evidence-based recommendations. With currently available cART, it should be possible to avoid clinically apparent toxicity when combining lymphoma and HIV therapy. However, there is a possibility for clinically subtle yet important pharmacokinetic interactions when cART and anticancer therapeutics are combined. For example, ritonavir appears to increase toxicity associated with vinka alkaloids through the cytochrome P450 3A4 metabolic pathway. This can result in decreasing doses of cancer agents and possibly undermining curative potential of the regimen. Certainly a case can be made to suspend cART for some patients. For example, a patient with Burkitt and tumor lysis syndrome should be started on definitive Burkitt therapy and cART can wait until the patient’s physiological condition permits consideration of administering lifelong chronic disease HIV therapy. Many oncologists recommend continuing CART if a patient is already on a stable well-tolerated regimen, but delaying its initiation until after lymphoma therapy in those who are not.

T-Cell Lymphoma:Treatment Principles

T-cell lymphomas tend to have a poorer PFS and OS than aggressive B-cell lymphomas. Systemic ALCL is an exception and is among the most curable subtypes with doxorubicin-based treatment. Some T-cell subtypes have no potential for cure and should be approached palliatively, as in ATL and primary cutaneous anaplastic lymphoma. Other T-cell subtypes, including angioimmunoblastic and PTL, have low curative potential with conventional dose treatment and should be considered for trials targeting high-risk patients.

Corticosteroids, alkylating agents, and bexarotene are available for topical use and are frequently utilized. In addition to topical therapies, ultraviolet radiation, either UVA or UVB, and total skin electron beam therapy (TSEBT) are utilized in limited-stage CTCL. Psoralen combined with ultraviolet A (PUVA) is associated with a CR rate exceeding 90%, and a prolonged disease-free interval, in early-stage disease. For advanced-stage disease, bexarotene, denileukin difitiox, and more recently histone deacetylase inhibitors (vorinostat and romidepsin are FDA approved for this indication in previously treated patients) all have activity. Systemic multi-agent chemotherapy is of limited use and reserved primarily for patients with advanced disease which has recurred and are not responding following other interventions.

Approval of histone deacetylase inhibitors for previously treated CTCL has advanced systemic therapeutic approaches. Both vorinostat and romidepsin are agents in this class with activity in CTCL.

Adult T-Cell Leukemia/Lymphoma

ATLL cannot be cured. Ameliorative treatments include interferon and doxorubicin-based combination chemotherapy. Retrospective data suggests that sequential combination chemotherapy followed by zidovudine with IFN-α improves survival substantially.

References

  1.  Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: World Health Organization; 2008.

  2.  Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med. 2002;346:235-242.

  3.  Friedberg JW. Relapsed/refractory diffuse large B-cell lymphoma. ASH Education Program Book. Hematology Am Soc Hematol Educ Program. 2011;2011:498-505.

  4.  Thomas DA, Cortes J, O’Brien S, et al. Hyper-CVAD program in Burkitt’s-type adult acute lymphoblastic leukemia. J Clin Oncol. 1999;17:2461-2470.

  5.  Magrath I, Adde M, Shad A, et al. Adults and children with small non-cleaved-cell lymphoma have a similar excellent outcome when treated with the same chemotherapy regimen. J Clin Oncol. 1996;14:925-934.

  6.  Mead GM, Sydes MR, Walewski J, et al. An international evaluation of CODOX-M and CODOX-M alternating with IVAC in adult Burkitt’s lymphoma: results of United Kingdom Lymphoma Group LY06 study. Ann Oncol. 2002;13:1264-1274.

  7.  Little RF, Pittaluga S, Grant N, et al. Highly effective treatment of acquired immunodeficiency syndrome-related lymphoma with dose-adjusted EPOCH: impact of antiretroviral therapy suspension and tumor biology. Blood. 2003;101:4653-4659.

  8.  Cheson BD, Wendtner CM, Pieper A, et al. Optimal use of bendamustine in chronic lymphocytic leukemia, non-Hodgkin lymphomas, and multiple myeloma: treatment recommendations from an international consensus panel. Clin Lymphoma Myeloma Leuk. 2010;10:21-27.

  9.  Rummel MJ, Niederle N, Maschmeyer G, et al. Bendamustine plus rituximab is superior in respect of progression free survival and CR rate when compared to CHOP plus rituximab as first-line treatment of patients with advanced follicular, indolent, and mantle cell lymphomas: final results of a randomized phase III study of the StiL (Study Group Indolent Lymphomas, Germany). ASH Annual Meeting Abstracts 2009;114:405.

17

Multiple Myeloma

Elisabet E. Manasanch, Nishant Tageja and C. Ola Landgren

EPIDEMIOLOGY AND RISK FACTORS

Multiple myeloma is the second most common hematologic malignancy in the United States, with an annual age-adjusted incidence rate of 6 per 100,000 and a prevalence of 70,000 as of January 1, 2009.¹ Myeloma is diagnosed predominantly in the elderly, with a median age at presentation of about 69 years. Men are more affected than women. African American men have the highest incidence rate at 14.3 per 100,000, twice the rate of their Caucasian counterparts.

Most cases are preceded by an asymptomatic plasma cell dyscrasia termed monoclonal gammopathy of undetermined significance (MGUS)² and smoldering multiple myeloma (SMM).

According to data from the Mayo Clinic, 3% of the general white population older than 50 have MGUS with an average multiple myeloma progression rate of 1% per year³; SMM has a 10% annual risk of progression to multiple myeloma.

Known risk factors are age, race, male sex, obesity, exposure to pesticides, and a family history of MGUS or multiple myeloma. Both environmental and genetic factors (single nucleotide polymorphisms, SNPs, at loci in chromosomal regions 2p, 3p, and 7p) may play a role in the development of multiple myeloma and its precursors.

Precursor States

The International Myeloma Working Group (IMWG) guidelines published in 2010, define MGUS as a serum M-protein <30 g/L, <10% clonal plasma cells in the bone marrow, and absence of end-organ damage (hypercalcemia, renal insufficiency, anemia, bone lesions (CRAB) criteria; Table 17.1) that can be attributed to the plasma cell proliferative disorder.⁴ SMM requires an M protein level of ≥30 g/L or ≥ 10% clonal plasma cells in the bone marrow, but no end-organ damage. After diagnosis, patients should be risk stratified for progression to multiple myeloma. Clinical follow-up depends on the risk of progression to symptomatic disease based on risk stratification (Table 17.2).

An ongoing randomized phase III trial is currently evaluating the role of early treatment with lenalidomide and high-dose dexamethasone in high-risk SMM patients. Preliminary results suggest delayed time to progression in the treatment arm.⁵ At this time, new phase II trials are being launched to assess whether early treatment can delay/prevent progression to frank multiple myeloma. The 2012 treatment standard for MGUS and SMM is to “watch and wait.” High-risk SMM patients may be treated only after enrollment in clinical trials.

Pathophysiology

Multiple myeloma is a heterogeneous disease and classified as one of the paraproteinemias. It is characterized by the proliferation and infiltration of the bone marrow by abnormal clonal terminally differentiated B lymphocytes (plasma cells) that produce antibodies. Deregulation of plasma cell biology as a result of somatic cell mutations drives the transformation from asymptomatic states such as MGUS and SMM to multiple myeloma and plasma cell leukemia.⁶

Multiple genetic abnormalities have been identified that contribute to the genesis and progression to multiple myeloma including inherited SNP variations, translocations (mostly involving the immunoglobulin heavy chain (IGH) locus in chromosome 14), trisomies of odd numbered chromosomes (hyperdiploidy), mutations, epigenetic changes, and microRNA abnormalities. Hyperdiploidy and translocations of IGH are the most common primary genetic events in multiple myeloma which can risk stratify the disease into standard, intermediate, and high risk and have prognostic information which correlates with overall survival (Table 17.2).⁶²

Secondary genetic events have been proposed to confer a selective advantage to the clonal plasma cell, which accumulates and interacts with the bone marrow microenvironment (stromal cells, osteoclasts, osteoblasts, and vasculature). Ultimately, there is a decrease in the number of normal plasma cells, high levels of circulating inflammatory cytokines, hypercoagulability, immunosuppression, myelosuppression, lytic bone lesions, and hypercalcemia.

Clinical Features

Multiple myeloma has different clinical presentations: it does not represent a single disease entity but rather a spectrum of clonal plasma cell disorders. Table 17.1 depicts the most common clinical abnormalities at diagnosis as well as type of monoclonal protein involvement.⁷

The most common symptom at diagnosis is bone pain (up to 70% of patients in most case series) affecting the lumbar and rib areas and causing pathologic fractures. Up to 90% of patients develop osteolytic lesions during the course of their disease. Increased risk of infections, weight loss, fatigue (anemia), and malaise are common. Around 10% to 20% of patients can present with hypercalcemia (somnolence, confusion, constipation, nausea) or renal insufficiency. Most patients (>95%) do not have symptoms of hyperviscosity (uncommon if serum viscosity level is not four times the upper limit of normal) or amyloidosis. About 1% of patients have extramedullary disease (EMD) at the time of presentation and about 8% develop EMD in their disease course.

Initial Evaluation

Recommended

  Complete history and physical examination.⁸

  Hematology Complete blood counts with differential and peripheral blood smear review.

  Chemistries Serum electrolytes, BUN, creatinine, calcium, magnesium, phosphorus, uric acid, β₂ microglobulin, serum albumin, C-reactive protein, and serum lactate dehydrogenase (LDH).

  Serum protein electrophoresis (SPEP) with serum immunofixation and the serum-free light chain (FLC) nephelometric assay. About 20% of multiple myeloma patients have a light-chain myeloma (abnormal FLC ratio in the absence of an M-spike). About 1% of multiple myeloma patients have nonsecretory disease without evidence of an M-spike and without an abnormal FLC ratio.

  Routine urine analysis, 24-hour collection for proteinuria, electrophoresis, and immunofixation.

  Quantitative immunoglobulins.

  Bone marrow aspirate plus trephine core biopsy. Standard metaphase cytogenetics and fluorescent in situ hybridization (FISH) for common chromosomal abnormalities found in multiple myeloma [t(11;14), t(4;14), t(14;16),t(6:14), t(14;20), hyperdiploidy, deletion 17p and 13q, gain of 1q].

  Gene expression profiling (usually for research).

Imaging

  Radiographic skeletal survey including the spine, pelvis, skull, humeri, and femurs is still the gold standard imaging technique; however, it has some limitations since it reveals a lytic lesion only after >30% to 50% of the trabecular bone is lost. ⁹

  Whole-body low-dose multidetector-row computed tomography (MDCT) is very sensitive at detecting small lytic lesions that may be negative on skeletal survey, but delivers a 1.3 to 3.0 times higher radiation dose. In some centers, MDCT has replaced conventional radiography for diagnosis and follow-up of multiple myeloma patients and it may be used if available.

  Magnetic resonance imaging (MRI) is particularly useful to rule out cord compression if spinal symptoms are present. Whole-body MRI is more sensitive than MDCT without radiation exposure. It is the preferred method to assess and follow osseous and extraosseous solitary bone plasmacytoma (SBP), with the suggestion that MRI should be included in the initial evaluation of SBP since it may reveal occult lesions elsewhere and lead to upstaging. MRI is recommended in patients with normal conventional radiography as part of the initial workup of multiple myeloma.

  Whole-body fluorodeoxyglucose/positron emission tomography (FDG/PET) imaging is not recommended for routine use outside of clinical trials.

  Technetium bone scans cannot be used for the evaluation of MGUS/SMM or multiple myeloma since

osteolytic lesions do not have increased uptake and up to 50% of bone lesions can be missed.

Diagnostic Criteria

International Myeloma Working Group Criteria

The diagnosis of multiple myeloma must include the following10,11:

1.  Clonal bone marrow plasma cells ≥10% (bone marrow involvement can be patchy and several random biopsies may be needed) and

2.  Presence of serum and/or urinary monoclonal protein (except in patients with true nonsecretory multiple myeloma) and

3.  Evidence of end-organ damage that can be attributed to the underlying plasma cell proliferative disorder:

a.  Hypercalcemia: serum calcium ≥11.5 mg/100 mL or

b.  Renal insufficiency: serum creatinine >1.73 mmol/L (2 mg/dL) or

c.  Anemia: normochromic normocytic with a hemoglobin value of >2 g/100 mL below the lower limit of normal or hemoglobin value <10 g/100mL or

d.  Bone: lytic lesions, severe osteopenia, or pathologic fractures

Staging

In 1975, the Durie-Salmon Staging System (DS SS) for multiple myeloma was proposed based on the mathematical correlation of individual clinical, laboratory, and X-ray features with multiple myeloma tumor burden.¹² Depending on the severity of anemia, levels of M-protein, hypercalcemia, and lytic lesions patients were classified as being stage I, II, or III. Patients could be further subclassified in stage A (creatinine < 2 mg/100 mL) or B (creatinine ≥ 2 mg/100 mL) (Table 17.3). The DS SS was widely adopted, however because the number and size of lytic lesions on plain X-ray is observer-dependent (subjective) a more objective staging system was proposed in 2005 by the IMWG using only laboratory values of β₂ microglobulin and serum albumin. This correlated well with overall survival (Table 17.3).¹³ Both staging systems continue to be used clinically and in research trials.

Criteria to Assess Response, Progression, and Relapse

Since 2006 the IMWG uniform response criteria have been used to categorize patient responses and to report efficacy of new agents in clinical trials (Table 17.4). Major advantages of these criteria include the ability to report on deeper responses, interpretation of response using serum FLC in patients with lack of measurable disease (no serum or urine M-protein), and the addition of the Very Good Partial Response (VGPR) category.¹⁴

The current definitions of progressive disease and relapse as per the IMWG are as follows:

1.  Progressive disease: requires any of the following

a.  Increase of ≥ 25% from baseline in

i.  serum M-component and/or (absolute increase must be ≥ 0.5 g/dL)

ii.  Urine M-component and/or (absolute increase must be ≥200 mg/24h)

iii.  Only in patients without measurable serum and urine M-protein levels: the difference between the involved and uninvolved FLC levels. The absolute increase must be > 10 mg/dL.

iv.  Bone marrow plasma cell percentage: the absolute % must be ≥ 10% (or ≥5% if relapse from CR) v. Definite development of new bone lesions or soft tissue plasmacytomas or definite increase in the size of existing bone lesions or soft tissue plasmacytomas

vi.  Development of hypercalcemia (corrected serum calcium >11.5 mg/dL or 2.65 mmol/L) that can be attributed solely to the plasma cell proliferative disorder.

2.  Clinical relapse: requires one or more of direct indicators of increasing disease (CRAB features)

a.  Development of new soft tissue plasmacytomas or bone lesions

b.  Definite increase in the size of existing plasmacytomas or bone lesions, defined as a 50% (at least 1 cm) increase as measured serially by the sum of the products of the cross-diameters of the measurable lesions

c.  Hypercalcemia (>11.5 mg/dL)

d.  Decrease in hemoglobin of ≥ 2g/dL

e.  Rise in serum creatinine by 2 mg/dL or more

3.  Relapse from CR: any one or more of the following

a.  Reappearance of serum or urine M-protein by immunofixation electrophoresis (IFE), or electrophoresis

b.  Development of ≥ 5% plasma cells in the bone marrow

c.  Appearance of any other sign of progression (i.e., new plasmacytoma, lytic bone lesion, or hypercalcemia).

Differential Diagnosis

Multiple myeloma must be accurately distinguished from other plasma cell disorders in order to offer adequate evaluation, treatment, and follow-up.

Immunoglobulin Light Chain Amyloidosis

Amyloidosis is caused by the accumulation of β-sheet secondary structure proteins that aggregate in tissues in the form of nonbranching fibrils causing organ-specific dysfunction (most notably the heart, kidneys, skin, peripheral nerves, autonomic nerves, and liver). In AL systemic primary amyloidosis the precursor protein is a bone marrow plasma cell derived immunoglobulin light chain (AL) (2:1, λ to κ). Treatment is directed toward eradicating the clonal plasma cell, however regimens derived from treatment of multiple myeloma are always more toxic if used in AL amyloidosis. Diagnosis must include all the following:

  Presence of an amyloid-related systemic syndrome

  Positive amyloid staining by Congo red in any tissue (fat aspirate, bone marrow biopsy, or involved

organ biopsy)

  Evidence that the amyloid is light-chain related (via mass spectrometry-based proteomic analysis, or immune-electronmicroscopy)

  Evidence of a monoclonal plasma cell proliferative disorder (serum or urine M-protein, abnormal FLC ratio, or clonal plasma cells in the bone marrow). ¹⁵

Waldenstrom Macroglobulinemia

Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma with IgM monoclonal protein that causes anemia, thrombocytopenia, hepatosplenomegaly, and lymphadenopathy. Patients can often be observed before treatment is indicated. Diagnosis must include the following:

  IgM monoclonal gammopathy

  ≥ 10% bone marrow lymphoplasmacytic infiltration by small lymphocytes that exhibit plasmacytoid or plasma cell differentiation and a typical immunophenotype (surface IgM+, CD5±, CD10−, CD19+, CD20+, CD23−) that excludes Mantle cell lymphoma and chronic lymphocytic leukemia

  Evidence of anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder.

Solitary Plasmacytoma

Solitary plasmacytoma (SP) is a mass of monoclonal plasma cells in either the bone (SBP) or the soft tissue (extramedullary plasmacytoma, EMP) without evidence of disease that can be attributed to multiple myeloma. It usually presents in the axial skeleton (bone) or head and neck (soft tissue). The goal of treatment is cure with local radiation therapy; however, while most patients with SBP (60%) progress to multiple myeloma in the years after diagnosis, most patients with EMP (65%) do not. Diagnostic criteria must include all of the following:

  Biopsy-proven solitary lesion of the bone or soft tissue with evidence of clonal plasma cells

  Normal bone marrow with no evidence of clonal plasma cells

  Normal skeletal survey and MRI of the spine and pelvis (except for the primary solitary lesion)

  Absence of end-organ damage such as CRAB that can be attributed to a plasma cell disorder.

Other Systemic Plasma Cell Dyscrasias

These include

  The rare paraneoplastic polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes (POEMS)¹⁶ syndrome

  The recently recognized telangiectasias, erythrocytosis with elevated erythropoietin levels, monoclo-nal gammopathy, perinephric-fluid collections, and intrapulmonary shunting (TEMPI) syndrome.17,18

It is worth noting that an elevated plasma VEGF level of 200 pg/mL has been reported to be 95% specific and 68% sensitive for POEMS syndrome.¹⁹

Miscellaneous

Metastatic carcinoma, hyperparathyroidism—Brown tumor, infection, primary bone tumors can all present with lytic lesions. If there is no evidence of a monoclonal gammopathy other diagnoses should be excluded before considering multiple myeloma

Treatment

The treatment of multiple myeloma has undergone a dramatic shift over the past decade with new agents that have been shown to be more effective and less toxic than conventional chemotherapy, which has translated into an improvement in overall survival.²⁰ There have also been major advances in risk stratification and classification of this disease, and risk-adapted therapy has been proposed as an approach to treatment.¹⁵

High-Risk Smoldering Multiple Myeloma

Recently, the first randomized phase III study designed to treat asymptomatic high-risk multiple myeloma with lenalidomide and dexamethasone was initiated with encouraging preliminary results. In the future, high-risk SMM individuals may have therapeutic options before they develop full-blown symptomatic multiple myeloma in an attempt to gain better and more prolonged control of the disease and maybe a possibility to cure.²¹

At this time, treatment of these patients outside of a clinical trial is not recommended due to the lack of long-term follow-up data.

Multiple Myeloma

High-dose conventional chemotherapy (HDT) with stem cell rescue is still at the cornerstone of treatment of multiple myeloma. However, major benefit has been added with the introduction of the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide as well as the proteasome inhibitor, bortezomib. Steroids continue to be an important part of therapy and bisphosphonates (BPs) have been seen to improve overall survival and are thought to have direct anti-myeloma effects.²²

Allogeneic stem cell transplantation (SCT) results in high treatment-related mortality (at least 10%– 20%) and graft-versus-host disease (GVHD) rates which have limited its clinical use to investigational trials. Autologous stem cell transplant (ASCT) followed by allogeneic SCT was not found to be more effective than tandem ASCT in a randomized controlled trial.²³

In standard clinical practice, patients with multiple myeloma are classified as high risk or low risk based on the ISS and chromosomal abnormalities (Table 17.5). The single most important factor in the frontline treatment of multiple myeloma is to ascertain whether the patient is eligible for high-dose chemotherapy with stem cell rescue. This is usually limited to patients younger than 70 to 75 years of age with good performance status and organ function; thus a significant proportion of newly diagnosed multiple myeloma patients are not found to be good candidates.

The recommended newly diagnosed anti-myeloma therapies are listed in Table 17.6. A flow chart with treatment options for multiple myeloma is shown in Figure 17.1.

Frontline Therapy for Patients Eligible for Autologous Stem Cell Transplant

Patients who are eligible for ASCT undergo two to four cycles of induction therapy before stem cell collection. Thereafter, patients can elect to undergo frontline ASCT or resume induction therapy with delay of ASCT until relapse. An ongoing clinical trial (DFCI-IFM RCT 2009) is investigating the role of upfront versus delayed ASCT. Preliminary data show no statistical significance observed in terms of overall survival and progression-free survival, however final results are pending. Alkylating agents (melphalan) and IMiDs (lenalidomide) can decrease the yield of stem collection due to their damaging properties on stem cells. As such, melphalan and prolonged lenalidomide treatment should be avoided as frontline therapy in patients who are eligible for SCT.

In the 1990s and early 2000s conventional chemotherapy with VAD (V = vincristine, A = adriamycin, D = dexamethasone) was used as the standard induction treatment before ASCT. However, with the advent of IMiDs and proteasome inhibitors induction regimens have changed drastically while achieving higher tumor reduction, better response rates, and improvement in progression-free survival and overall survival.³⁴⁻³⁶ Thus, VAD is no longer preferred as frontline therapy.

Different combinations of bortezomib, lenalidomide, or thalidomide with dexamethasone were effective and safe in multiple myeloma with high response rates. Three or four drug combination regimens were studied to assess whether higher response rates could be achieved without added toxicity. Bortezomib was added to the combination treatment consisting of thalidomide (Thal) and dexamethasone (Dex) (VTD) and was seen to be superior to ThalDex alone.³⁷ It was also superior to aggressive regimens combining conventional cytotoxic chemotherapy such as VBMCP (V = vincristine, M = melphalan, BCNU/carmustine, cyclophosphamide, prednisone), and vincristine, bortezomib, adriamycin, dexamehtasone (VBAD) (A= adriamycin).³⁸

ThalDex was less active and more toxic than lenalidomide-based regimens and is not recommended as frontline therapy. Lenalidomide combined with low-dose dexamethasone (Rd) [40 mg po weekly] had an overall survival advantage with less toxicity than used with high-dose dexamethasone (RD).³⁹

All patients treated with lenalidomide should receive deep venous thrombosis (DVT) prophylaxis with aspirin, low molecular weight heparin, or Coumadin.⁴⁰

Table 17.5  Risk Stratification of Multiple Myeloma by Mayo Clinic Model²⁴

Standard risk (overall survival 6–7 yr)

Hyperdiploidy

t(11;14)

t(6;14)

Intermediate risk

t(4;14)

Deletion 13 or hypodiploidy by conventional karyotyping

High risk (overall survival 2–3 yr)

17p deletion

t(14;16)

t(14;20)

High-risk gene expression profiling signature

FIGURE 17.1. Current treatment algorithm for patients with multiple myeloma.

* Maybe dose reduced to 140 or 100 mg/m2 in selected patients who would otherwise not tolerate 200 mg/m2. MPT, melphalan, prednisone, thalidomide; Rd, lenalidomide plus low-dose dexamethasone; VCD, bortezomib, cyclophosphamide, dexamethasone; VCR, bortezomib, cyclophosphamide, dexamethasone; VMP, bortezomib, melphalan, prednisone; VRD, bortezomib, lenalidomide, dexamethasone.

Bortezomib combination regimens including bortezomib, lenalidomide, and dexamethasone (VRD)⁴¹ and bortezomib-cyclophosphamide-dexamethasone (VCD)⁴² have high activity as induction treatments. Treatment with bortezomib seems to overcome the poor prognosis conferred by cytogenetic abnormalities.⁴³ Neurotoxicity from bortezomib can be significantly decreased by administering the drug subcutaneouosly.⁴⁴

Maintenance Therapy

In the past, corticosteroids and interferon alpha were used as maintenance agents. However, they failed to consistently provide improvement in overall survival and had increased toxic effects. Thalidomide was first seen to provide a consistent modest benefit with increased event-free and overall survival following autologous transplantation but was poorly tolerated.45,46

Very recently, three randomized trials have shown improved progression-free survival with lenalidomide.⁴⁷⁻⁴⁹ Only one of the trials showed better overall survival, perhaps due to the length of follow-up. A small but meaningful risk of increased secondary malignancies was reported in all three studies.⁵⁰ It is unclear whether maintenance with lenalidomide should be used in all patients post ASCT. An informed discussion with the patient, taking into account risks and benefits in each individual case is the preferred approach.

Other agents, including bortezomib and the IMiD pomalidomide, are currently being tested in the maintenance setting and seem to have activity in patients who are refractory to lenalidomide.

Frontline Therapy for Patients Not Eligible for Autologous Stem Cell Transplant

Before the era of novel agents, the combination of oral melphalan and prednisone (MP) used to be the main treatment regimen for those ineligible for ASCT due to its activity and tolerability.⁵¹ Currently, several combinations are available with improved overall survival when compared with MP:

  Melphalan, prednisone, and thalidomide (MPT)52,53

  Bortezomib, melphalan, prednisone (VMP)⁵⁴

  Lenalidomide-low-dose-dexamethasone (Rd)⁵⁵

An ongoing clinical trial is comparing Rd versus MPT. MPT had high rates of DVT (20%) in the absence of thromboprophylaxis with higher grades of neuropathy in the VMP combination.

Relapsed or Refractory Disease

Almost all patients who respond to their initial treatment will relapse. Responses in the relapse/ refractory setting tend to be short lived and decrease with each added line of therapy. The median overall survival for patients with relapsed myeloma that is refractory to lenalidomide and bortezomib is less than 12 months.⁵⁶ Alkylating agents, corticosteroids, and thalidomide are possible options for treatment (either alone or in combination); however, combinations of highly active regimens (VRd, VTD) have the best chance of response.⁵⁷ Liposomal doxorubicin added to bortezomib significantly improved overall survival in a randomized phase III trial and may be used in this setting.⁵⁸

Newer agents with promising activity in this setting are carfilzomib and pomalidomide with overall response rates as high as 50% in non-randomized trials.

Treatment of Bone Disease in Multiple Myeloma

More than 80% of patients with multiple myeloma will develop osteolytic bone lesions at some point during their disease course. This can result in increased morbidity due to skeletal-related events (SRE) (compression fractures) resulting in hypercalcemia, cord compression, severe pain, and the need for surgical intervention or radiation therapy.

Osteolytic bone disease (OBD) is thought to be the result of generalized osteoclast activation coupled with osteoblast inhibition. BPs are the standard of care for treatment of OBD due to multiple myeloma and are thought to augment osteoclast apoptosis and may inhibit tumor growth in vivo. Treatment with either pamidronate or zoledronic acid has shown to reduce pain due to bone disease and prevent SRE, however pamidronate is the recommended agent in multiple myeloma.59,60

Serious adverse events with BP use include osteonecrosis of the jaw and renal failure. Patients should have a dental evaluation prior to starting BP use and be advised against dental procedures (especially tooth extraction) while on BP treatment. Currently several trials are trying to answer whether using BP less frequently or at decreased doses can maintain efficacy with less toxicity. Different treatment guidelines and recommendations are available for the use of BP in multiple myeloma. Overall, pamidronate and clodronate (not available in the United States) have the lowest risk of osteonecrosis of the jaw: pamidronate 1% to 2% within the first 2 years of treatment and 0% to 0.5% for clodronate. The risk with zoledronic acid is double that of pamidronate. Renal failure is seen in less than 10% of patients and regular monitoring of urinary protein and serum creatinine should be instituted. If osteonecrosis of the jaw occurs, BP should be discontinued.

Supportive Care

Anemia

Normochromic, normocytic anemia is commonly part of the presentation of newly diagnosed patients (>70%) and can be attributed to multiple myeloma itself or due to treatment causing cytopenias. Erythropoiesis-stimulating agents (ESAs) are recommended for symptomatic anemic patients (Hb < 10 g/dL) and associated renal impairment. Treatment should be discontinued after Hb ≥ 12 g/dL or if there is no benefit after 6 to 8 weeks of treatment (rise in Hb or decrease in transfusion requirements).

Hemostasis and Thrombosis

Paraproteinemia in multiple myeloma has been associated with acquired von Willebrand Disease (VWD) and AL amyloidosis with factor X deficiency, with increased risk of bleeding. Prothrombin complex and recombinant factor VIIa are used successfully for management of factor X deficiency while desmopressin, intravenous immunoglobulins, and factor VIII/VW are used for VWD.

IMiDs in combination with dexamethasone substantially increase the risk of VTE (50%–70%) and patients treated with these regimens should receive prophylaxis with aspirin, heparin, or Coumadin which reduces the risk significantly (1%–2%).

Skeletal-related Events

Local radiation therapy can alleviate pain for skeletal disease and palliative soft tissue disease. Vertebroplasty (percutaneous injection of polymethacrylate bone cement in the vertebral body) or kyphoplasty (percutaneous insertion of a small inflatable balloon into the vertebral body which is then removed and the space filled with bone cement) are alternative options for controlling pain associated with vertebral collapse. They are best carried out soon after collapse and carry a small risk of cement leakage leading to pulmonary embolus and neural compromise.

Peripheral Neuropathy

The cause of peripheral neuropathy in multiple myeloma patients is multifactorial (disease, chemotherapy, comorbidities) with a potential risk of worsening due to treatment. Management of peripheral neuropathy requires identification and management of B12 deficiency and comorbid conditions with minimization of agents that worsen or induce neurotoxicity. The use of opioid drugs with pain-modulating agents (gabapentin, pregabalin) is recommended.

Osteonecrosis of the Jaw

Management is supportive with good oral care (daily irrigation/oral antimicrobial [0.12% chlorhexidine gluconate]) and antibiotics, however sometimes debridement of the bone is needed. Biopsies should not be routinely performed as additional bone trauma may further delay wound healing. Clinical follow-up and referral to an experienced oral surgeon is recommended.

Hyperviscosity Syndrome

The clinical manifestations of hyperviscosity include neurologic abnormalities, vision changes, renal insufficiency, and mucosal bleeding. Hyperviscosity occurs most frequently in IgM dyscrasias, but it can be seen in all Ig subtypes. It is thought to be present when viscosity levels reach 4 to 5 cp (reference range 1.4–1.8 cp); however, this does not usually correlate well with symptoms. Thus, treatment (aggressive hydration, diuresis, plasmapheresis, chemotherapy/high-dose dexamethasone) should be instituted and guided by symptomatology.

Solitary Plasmacytoma and Extramedullary Plasmacytoma

Plasma cell neoplasms can present as a single lesion, most frequently in bone (SP) but also in soft tissues (EMP). Some patients can have a small monoclonal protein, usually IgA which then disappears following treatment. Radiation therapy is the treatment of choice (40–50 Gy over a 4-week period). The role of adjuvant radiation is unclear if complete surgical resection was achieved as primary treatment. Most patients treated for SP will still progress to multiple myeloma (up to 85% at 10 years). The rate of progression to multiple myeloma for EMP is lower than in SP (up to 30% at 10 years).

Plasma Cell Leukemia

Plasma cell leukemia (PCL) is a rare, aggressive form of multiple myeloma characterized by high levels of circulating plasma cells in the peripheral blood. It occurs in less than 5% of patients with multiple myeloma. It can present de novo or, more frequently, from a secondary leukemic transformation of multiple myeloma. Diagnosis requires an absolute plasma cell count > 2,000 plasma cells/μL or 20% of peripheral blood white cells. These criteria are arbitrary and diagnosis should be considered whenever plasma cells are detected on a complete blood count. Median overall survival has improved recently from < 12 months to reported 24 months in a single institution study, due to the introduction of IMiDs and bortezomib.⁶¹ Given the rarity of this disease and grim prognosis patients should be enrolled in clinical trials when feasible.

References

  1.  SEER Stat Fact Sheets: Myeloma. National Cancer Institute Web Site. http://seer.cancer.gov/statfacts/html/mulmy.html. Accessed May 25, 2012.

  2.  Landgren O, Kyle RA, Pfeiffer RM, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood. 2009;113(22):5412-5417.

  3.  Kyle RA, Therneau TM, Rajkumar SV, et al. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med. 2006;354(13):1362-1369.

  4.  Kyle RA, Durie BG, Rajkumar SV, et al. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management. Leukemia. 2010;24(6):1121-1127.

  5.  Mateos MV, López-Corral L, Hernández M, et al. Smoldering multiple myeloma (SMM) at high-risk of progression to symptomatic disease: a phase III, randomized, multicenter trial based on lenalidomide-dexamethasone (Len-Dex) as induction therapy followed by maintenance therapy with Len alone vs no treatment. Presented at the 53rd Annual Meeting of the American Society of Hematology; December 13, 2011; San Diego, CA. https://ash.confex.com/ash/2011/webprogram/Paper40382.html. Accessed May 25, 2012.

  6.  Morgan GJ, Walker BA, Davies FE. The genetic architecture of multiple myeloma. Nat Rev Cancer. 2012;12(5):335-348.

  7.  Kyle RA, Gertz MA, Witzig TE, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc. 2003;78(1):21-33.

  8.  Palumbo A, Sezer O, Kyle R, et al. International Myeloma Working Group guidelines for the management of multiple myeloma patients ineligible for standard high-dose chemotherapy with autologous stem cell transplantation. Leukemia. 2009;23(10):1716-1730.

  9.  Dimopoulos M, Terpos E, Comenzo RL, et al. International myeloma working group consensus statement and guidelines regarding the current role of imaging techniques in the diagnosis and monitoring of multiple Myeloma. Leukemia. 2009;23(9):1545-1556.

10.  Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol. 2003;121(5):749-757.

11.  Rajkumar SV, Kyle RA. Multiple myeloma: diagnosis and treatment. Mayo Clin Proc. 2005;80(10):1371-1382.

12.  Durie BG, Salmon SE. A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival. Cancer. 1975;36(3):842-854.

13.  Greipp PR, San Miguel J, Durie BG, et al. International staging system for multiple myeloma. J Clin Oncol. 2005;23(15):3412-3420.

14.  Durie BG, Harousseau JL, Miguel JS, et al. International uniform response criteria for multiple myeloma. Leukemia. 2006;20(9):1467-1473.

15.  Rajkumar SV. Multiple myeloma: 2012 update on diagnosis, risk-stratification, and management. Am J Hematol. 2012;87(1):78-88.

16.  Dispenzieri A. How I treat POEMS syndrome. Blood. 2012;119(24):5650-5658. doi:10.1182/blood-2012-03-378992.

17.  Sykes DB, Schroyens W, O’Connell C. The TEMPI syndrome—a novel multisystem disease. N Engl J Med. 2011;365(5):475-477.

18.  Kwok M, Korde N, Landgren O. Bortezomib to treat the TEMPI syndrome. N Engl J Med. 2012;366(19):1843-1845.

19.  D’Souza A, Hayman SR, Buadi F, et al. The utility of plasma vascular endothelial growth factor levels in the diagnosis and follow-up of patients with POEMS syndrome. Blood. 2011;118(17):4663-4665.

20.  Kumar SK, Rajkumar SV, Dispenzieri A, et al. Improved survival in multiple myeloma and the impact of novel therapies. Blood. 2008;111(5):2516-2520.

21.  Smoldering multiple myeloma (SMM) at high-risk of progression to symptomatic disease: a phase III, randomized, multicenter trial based on lenalidomide-dexamethasone (Len-Dex) as induction therapy followed by maintenance therapy with len alone vs no treatment. 2011. http://myeloma.org/ArticlePage.action?tabId=0&menuId=0&articleId=3589&a Tab=-1&tBack=&tDisplayBack=true. Accessed June 2012.

22.  Morgan GJ, Davies FE, Gregory WM, et al. Effects of induction and maintenance plus long-term bisphosphonates on bone disease in patients with multiple myeloma: the Medical Research Council Myeloma IX Trial. Blood. 2012;119(23):5374-5383.

23.  Krishnan A, Pasquini MC, Logan B, et al. Autologous haemopoietic stem-cell transplantation followed by allogeneic or autologous haemopoietic stem-cell transplantation in patients with multiple myeloma (BMT CTN 0102): a phase 3 biological assignment trial. Lancet Oncol. 2011;12(13):1195-1203.

24.  Kumar SK, Mikhael JR, Buadi FK, et al. Management of newly diagnosed symptomatic multiple myeloma: updated Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) consensus guidelines. Mayo Clin Proc. 2009;84(12):1095-1110.

25.  Cavo M, Pantani L, Petrucci MT, et al. Bortezomib-thalidomide-dexamethasone is superior to thalidomide-dexamethasone as consolidation therapy following autologous hematopoietic stem-cell transplantation in patients with newly diagnosed multiple myeloma. Blood. 2012;120(1):9-19. doi:10.1182/blood-2012-02-408898.

26.  Garderet L, Iacobelli S, Moreau P, et al. Superiority of the triple combination of bortezomib-thalidomide-dexamethasone over the dual combination of thalidomide-dexamethasone in patients with multiple myeloma progressing or relapsing after autologous transplantation: the MMVAR/IFM 2005-04 randomized phase III trial from the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol. 2012;30(27):3429.

27.  Moreau P, Avet-Loiseau H, Facon T, et al. Bortezomib plus dexamethasone versus reduced-dose bortezomib, thalidomide plus dexamethasone as induction treatment before autologous stem cell transplantation in newly diagnosed multiple myeloma. Blood. 2011;118(22):5752-5758; quiz 982.

28.  Khan ML. A comparison of lenalidomide/dexamethasone (RD) versus cyclophosphamide/lenalidomide/ dexamethasone (CRD) versus cyclophosphamide/bortezomib/dexamethasone (CyborD) in newly diagnosed multiple myeloma (MM). 2010. http://www.asco.org/ascov2/Meetings/Abstracts?&vmview=abst_detail_view&conf ID=74&abstractID=42951. Accessed June 25, 2012.

29.  Roussel M. Bortezomib, lenalidomide, and dexamethasone (VRD) consolidation and lenalidomide maintenance in front-line multiple myeloma patients: updated results of the ifm 2008 phase ii vrd intensive program. 2011. http://myeloma.org/ArticlePage.action?articleId=3617. Accessed June 15, 2012.

30.  Jakubowiak AJ, Dytfeld D, Griffith KA, et al. A phase 1/2 study of carfilzomib in combination with lenalidomide and low-dose dexamethasone as a frontline treatment for multiple myeloma. Blood. 2012;120(9):1801-1809. doi:10.1182/ blood-2012-04-422683.

31.  Palumbo A, Bringhen S, Caravita T, et al. Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial. Lancet. 2006;367(9513):825-831.

32.  Mateos MV, Richardson PG, Schlag R, et al. Bortezomib plus melphalan and prednisone compared with melphalan and prednisone in previously untreated multiple myeloma: updated follow-up and impact of subsequent therapy in the phase III VISTA trial. J Clin Oncol. 2010;28(13):2259-2266.

33.  Kolb B. Phase I/II study of carfilzomib plus melphalan-prednisone (CMP) in elderly patients with de novo multiple myeloma. 2012. http://www.asco.org/ASCOv2/Meetings/Abstracts?&vmview=abst_detail_view&confID=114&abstrac tID=96240. Accessed June 25, 2012.

34.  Dexamethasone + Thalidomide (Dex/Thal) compared to VAD as a pre-transplant treatment in newly diagnosed multiple myeloma (MM): a randomized trial. 2006. http://www.cancereducation.com/CancerSysPagesNB/abstracts/
mmrf/72/abbr5.pdf. Accessed June 2012.

35.  Cavo M, Zamagni E, Tosi P, et al. Superiority of thalidomide and dexamethasone over vincristine-doxorubicindexamethasone (VAD) as primary therapy in preparation for autologous transplantation for multiple myeloma. Blood. 2005;106(1):35-39.

36.  Rajkumar SV, Rosinol L, Hussein M, et al. Multicenter, randomized, double-blind, placebo-controlled study of thalidomide plus dexamethasone compared with dexamethasone as initial therapy for newly diagnosed multiple myeloma. J Clin Oncol. 2008;26(13):2171-2177.

37.  Cavo M, Tacchetti P, Patriarca F, et al. Bortezomib with thalidomide plus dexamethasone compared with thalidomide plus dexamethasone as induction therapy before, and consolidation therapy after, double autologous stem-cell transplantation in newly diagnosed multiple myeloma: a randomised phase 3 study. Lancet. 2010;376(9758):2075-2085.

38.  Thalidomide/dexamethasone (TD) vs. bortezomib (Velcade®)/thalidomide/dexamethasone (VTD) vs. VBMCP/VBAD/ Velcade® as induction regimens prior autologous stem cell transplantation (ASCT) in younger patients with multiple myeloma (MM): first results of a prospective phase III PETHEMA/Gem Trial, 2008. http://www.cancereducation.com/CancerSysPagesNB/abstracts/mmrf/
113/abdz30.pdf. Accessed May 2012.

39.  Rajkumar SV, Jacobus S, Callander NS, et al. Lenalidomide plus high-dose dexamethasone versus lenalidomide plus low-dose dexamethasone as initial therapy for newly diagnosed multiple myeloma: an open-label randomised controlled trial. Lancet Oncol. 2010;11(1):29-37.

40.  Palumbo A, Cavo M, Bringhen S, et al. Aspirin, warfarin, or enoxaparin thromboprophylaxis in patients with multiple myeloma treated with thalidomide: a phase III, open-label, randomized trial. J Clin Oncol. 2011;29(8):986-993.

41.  Richardson PG, Weller E, Lonial S, et al. Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. Blood. 2010;116(5):679-686.

42.  Reeder CB, Reece DE, Kukreti V, et al. Cyclophosphamide, bortezomib and dexamethasone induction for newly diagnosed multiple myeloma: high response rates in a phase II clinical trial. Leukemia. 2009;23(7):1337-1341.

43.  van Rhee F, Szymonifka J, Anaissie E, et al. Total Therapy 3 for multiple myeloma: prognostic implications of cumulative dosing and premature discontinuation of VTD maintenance components, bortezomib, thalidomide, and dexamethasone, relevant to all phases of therapy. Blood. 2010;116(8):1220-1227.

44.  Moreau P, Pylypenko H, Grosicki S, et al. Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, non-inferiority study. Lancet Oncol. 2011;12(5):431-440.

45.  Attal M, Harousseau JL, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood. 2006;108(10):3289-3294.

46.  Spencer A, Prince HM, Roberts AW, et al. Consolidation therapy with low-dose thalidomide and prednisolone prolongs the survival of multiple myeloma patients undergoing a single autologous stem-cell transplantation procedure. J Clin Oncol. 2009;27(11):1788-1793.

47.  Attal M, Lauwers-Cances V, Marit G, et al. Lenalidomide maintenance after stem-cell transplantation for multiple myeloma. N Engl J Med. 2012;366(19):1782-1791.

48.  Palumbo A, Hajek R, Delforge M, et al. Continuous lenalidomide treatment for newly diagnosed multiple myeloma. N Engl J Med. 2012;366(19):1759-1769.

49.  McCarthy PL, Owzar K, Hofmeister CC, et al. Lenalidomide after stem-cell transplantation for multiple myeloma. N Engl J Med. 2012;366(19):1770-1781.

50.  Ludwig H, Durie BG, McCarthy P, et al. IMWG consensus on maintenance therapy in multiple myeloma. Blood. 2012;119(13):3003-3015.

51.  Combination chemotherapy versus melphalan plus prednisone as treatment for multiple myeloma: an overview of 6,633 patients from 27 randomized trials. Myeloma Trialists’ Collaborative Group. J Clin Oncol. 1998;16(12):3832-3842.

52.  Facon T, Mary JY, Hulin C, et al. Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial. Lancet. 2007;370(9594):1209-1218.

53.  Hulin C, Facon T, Rodon P, et al. Efficacy of melphalan and prednisone plus thalidomide in patients older than 75 years with newly diagnosed multiple myeloma: IFM 01/01 trial. J Clin Oncol. 2009;27(22):3664-3670.

54.  San Miguel JF, Schlag R, Khuageva NK, et al. Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med. 2008;359(9):906-917.

55.  Jacobus S, Callander N, Siegel D. Outcome of elderly patients 70 years and older with newly diagnosed myeloma in the ECOG randomized trial of lenalidomide/high-dose dexamethasone (RD) versus lenalidomide/low-dose dexamethasone (Rd). Haematologica. 2010;95:149.

56.  Kumar K, Blade J, Crowley J, et al. Outcome of patients with myeloma relapsing after IMiD and bortezomib therapy: a multicenter study from the international myeloma foundation working group. Haematologica. 2010;95(suppl 2):151.

57.  Pineda-Roman M, Zangari M, et al. VTD combination therapy with bortezomib-thalidomide-dexamethasone is highly effective in advanced and refractory multiple myeloma. Leukemia. 2008;22(7):1419-1427.

58.  Orlowski RZ, Nagler A, Sonneveld P, et al. Randomized phase III study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression. J Clin Oncol. 2007;25(25):3892-3901.

59.  Durie BG. Use of bisphosphonates in multiple myeloma: IMWG response to Mayo Clinic consensus statement. Mayo Clin Proc. 2007;82(4):516-517; author reply 7-8.

60.  Lacy MQ, Dispenzieri A, Gertz MA, et al. Mayo clinic consensus statement for the use of bisphosphonates in multiple myeloma. Mayo Clinic Proc. 2006;81(8):1047-1053.

61.  Lebovic D, Zhang L, Alsina M, et al. Clinical outcomes of patients with plasma cell leukemia in the era of novel therapies and hematopoietic stem cell transplantation strategies: a single-institution experience. Clin Lymphoma Myeloma Leuk. 2011;11(6):507-511.

62.  Korde N, Kristinsson SY, Landgren O. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM): novel biological insights and development of early treatment strategies. Blood. 2011;117(21):5573-5581.

18

Hematopoietic Stem Cell Transplantation

Richard W. Childs and Ramaprasad Srinivasan

Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapeutic modality widely used in the management of some hematologic malignancies and a variety of nonmalignant disorders. Autologous HSCT typically involves administration of high-dose chemotherapy followed by infusion of hematopoietic stem cells procured from the recipient prior to ablative therapy. On the other hand, allogeneic HSCT involves infusion of hematopoietic stem cells from a related or unrelated human leukocyte antigen (HLA)-compatible donor, following myeloablative or reduced intensity conditioning (RIC) of the recipient.

AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION

Autologous hematopoietic stem cell transplantation (auto-HSCT) was developed to overcome the lethal hematopoietic toxicity associated with high-dose chemotherapy used to treat dose-responsive malignancies.1,2 A role for auto-HSCT has been clearly established in the management of multiple myeloma and aggressive non-Hodgkin lymphoma (NHL). Initial enthusiasm for this approach in solid tumors such as metastatic breast, ovarian, and lung cancer has been tempered by the failure of prospective randomized trials to demonstrate benefit over conventional treatments. The role of auto-HSCT continues to be explored in neuroblastoma and Ewing sarcoma.

General Considerations

Most auto-HSCTs are performed using peripheral blood stem cells (PBSCs) collected after mobilization with granulocyte colony-stimulating factor (G-CSF), with or without chemotherapy priming. Curative potential resides solely in the ability of high-dose chemotherapy to eradicate the underlying malignancy; no immune-mediated graft-versus-tumor (GVT) effects are generated. The high-dose chemotherapeutic regimen utilized is tailored to the malignancy being treated based on its chemosensitivity profile; for instance, melphalan (200 mg/m²) is the most widely used high-dose conditioning agent in patients with multiple myeloma undergoing auto-HSCT.

Infections related to chemotherapy-induced neutropenia and immunosuppression as well as extramedullary toxicities from high-dose chemotherapeutic agents account for the majority of complications occurring after auto-HSCT. There is a lower risk of treatment-related mortality (TRM) compared with allogeneic HSCT, typically less than 5% in most series.

Contamination of the stem cell product by malignant cells may limit the beneficial effects of high-dose chemotherapy. Efforts to purge tumor cells contaminating hematopoietic grafts by CD34⁺ cell selection or by in vitro incubation of the stem cell graft with cytotoxic drugs remain investigational and have met with only moderate success.

Results of Autologous Hematopoietic Stem Cell Transplantation

Autologous Hematopoietic Stem Cell Transplantation in Hematologic Malignancies

Multiple Myeloma Large phase II trials have demonstrated high response rates (complete response [CR] 30%–50%) and impressive disease-free survival (DFS) and overall survival (OS) rates (median, more than 5 years). Randomized phase III trials in relatively young patients (less than 65 years of age) have shown superior response rates, DFS, and OS for auto-HSCT versus conventional chemotherapy.³

Consecutive or tandem auto-HSCTs have been compared with single auto-HSCT in several randomized prospective studies. While some studies suggest improved DFS (but not OS) with the tandem approach, other large multicenter trials also indicated a survival benefit for tandem auto-HSCT. Subgroup analyses suggest that the survival benefit is most pronounced in patients who do not achieve a CR or a very good partial response following the first auto-HSCT.

Tandem auto-HSCT has also been compared with auto-HSCT followed by reduced intensity or nonmyeloablative allogeneic HSCT, with one randomized study demonstrating superior OS and progression-free survival (PFS) with the latter approach. However, a recent randomized trial conducted through the Clinical Trials Network (CTN) which enrolled over 700 subjects reported no difference in 3-year PFS and OS between patients receiving a tandem auto-HSCT versus a tandem auto/allotransplant, with significantly higher TRM being reported in the latter group.⁴

Auto-HSCT is currently considered standard first-line therapy for younger patients (up to age 65) with myeloma and results in long-term CR rates of 5% to 10%. Melphalan (200 mg/m²) is the most commonly used preparative regimen. The role of tandem auto-HSCT remains to be clearly defined, although some subgroups of patients appear to benefit from this approach. Due to recent data showing no clear survival benefit with an upfront auto/allogeneic transplant approach, as well as the advent of newer drugs such as lenalidomide, bortezomib, and carfilzomib which are highly active against myeloma, the role of allogeneic transplant in this disease remains investigational. Indeed, these agents have led to a reevaluation of the role of auto-HSCT in the management of this disease. Several ongoing studies are attempting to determine whether

  newer anti-myeloma agents can supplant auto-HSCT as the standard of care in most patients;

  integration of these agents in the management algorithm can potentiate or extend the benefits derived from auto-HSCT.

Emerging data suggest that bortezomib-based pretransplant induction therapy as well as post-transplant consolidation using a variety of strategies might improve overall outcome associated with auto-HSCTs.⁵

Lymphoma Lymphomas are among the most common indications for auto-HSCT. The benefit of auto-HSCT has been most clearly observed in chemosensitive Hodgkin’s disease and in intermediate- and high-grade NHL. Auto-HSCT results in improved event-free survival (EFS) and OS in patients with relapsed chemosensitive intermediate-/high-grade NHL, compared with conventional salvage therapy.⁶ Results from a randomized study suggest that initial treatment with auto-HSCT may benefit some patients with intermediate-/high-grade NHL, compared to standard chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP).

Among Hodgkin’s disease patients, those with chemosensitive disease on first relapse have an improved EFS with auto-HSCT compared with standard salvage chemotherapy. Patients with primary progressive disease (those who progress on first-line chemotherapy) may also benefit from auto-HSCT as part of a salvage regimen.

Incorporation of therapeutic antibodies such as rituximab (anti-CD20) in first-line treatment regimens has significantly improved our ability to treat several subsets of NHL. The role of auto-HSCT in the era of B-cell–targeted monoclonal antibodies remains to be determined.

ACUTE MYELOID LEUKEMIA Auto-HSCT has been used both as postremission therapy in acute myeloid leukemia (AML) in first complete remission (CR1) and as therapy after relapse. Phase III studies in patients in CR1 suggest an improvement in DFS but not in OS, compared with conventional postremission therapy. Further studies are required to clarify which, if any, prognostic subgroups are likely to benefit from auto-HSCT.

Autologous Hematopoietic Stem Cell Transplantation in Solid Tumors

The knowledge that some malignancies exhibited dose-dependent responses to chemotherapy led to the investigation of high-dose chemotherapy followed by auto-HSCT in the treatment of solid tumors. Based on negative results from phase III trials, auto-HSCT has been largely abandoned in the management of some malignancies (particularly metastatic breast cancer) but remains under investigation in several other solid tumor types such as rhabdomyosarcoma.

Breast Cancer While promising results from phase II studies in patients with metastatic breast cancer paved the way for randomized phase III studies of auto-HSCT, these later larger studies failed to demonstrate an unequivocal benefit. At least seven large trials have compared auto-HSCT with standard chemotherapy in patients with metastatic breast cancer. Six demonstrated superior EFS with auto-HSCT, but none showed an OS advantage.⁷ Similar results were obtained in patients with high-risk breast cancer undergoing adjuvant auto-HSCT. Because of a lack of survival benefit and higher toxicity, there is little enthusiasm for further investigation of auto-HSCT in breast cancer.

Germ Cell Tumors Phase II trials of auto-HSCT in relapsed or refractory germ cell tumors have yielded response rates of 40% to 65% and long-term survival rates of 15% to 40%. Patients with progressive disease or human chorionic gonadotropin levels greater than 1,000 IU/L at transplantation, mediastinal primaries, and those refractory to cisplatin-based therapy have a worse outcome and may not benefit from auto-HSCT. An interim report from a European Group for Blood and Marrow Transplantation study suggested no advantage for auto-HSCT over standard salvage chemotherapy in patients failing cisplatin-based chemotherapy.

Auto-HSCT is considered an option for salvage of selected patients in first or subsequent relapse, although randomized phase III studies have failed to demonstrate unequivocal benefit for high dose over standard chemotherapy in this setting.⁸

There is no role for auto-HSCT in the initial management of germ cell tumors; several randomized phase III studies have demonstrated similar outcomes for conventional chemotherapy and auto-HSCT in patients with germ cell tumors with high-risk features.⁹

Other Tumors Available data do not suggest a clear benefit for auto-HSCT in ovarian and lung cancers. When administered after an initial course of standard induction chemotherapy, auto-HSCT appears to improve short-term DFS in high-risk neuroblastoma compared with conventional dose maintenance chemotherapy. However, a large randomized study was unable to demonstrate a survival advantage for patients undergoing auto-HSCT.

Some patients with Ewing sarcoma/primordial neurectodermal tumor and other soft tissue sarcomas may benefit from high-dose chemotherapy. In the absence of evidence to support a survival benefit, patients should be treated with auto-HSCT only in the setting of a clinical trial.

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Allogeneic HSCT can cure patients with advanced chemotherapy-resistant hematologic malignancies.¹⁰⁻¹² The first successful allogeneic HSCTs in humans were reported in 1968. These transplants were performed in children with congenital immune deficiencies, using donor stem cells from HLA-compatible sibling donors. In the early years after the advent of allogeneic HSCT, immune deficiency syndromes and disorders of hematopoiesis constituted the major indications for the procedure. With a better understanding of the graft-versus-leukemia (GVL) effect and its curative potential, hematologic malignancies have now become the most common indication for an allogeneic HSCT. More than 25,000 allogeneic HSCTs are performed worldwide annually for a wide array of both malignant and nonmalignant disorders.

Indications for Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic HSCT has been used as a potentially curative treatment modality in both malignant and nonmalignant diseases (Table 18.1). Currently, the most common indication for an allogeneic HSCT (over 75%) is an underlying hematologic malignancy (acute and chronic myelogenous leukemia, acute lymphocytic leukemia, and NHL being the most common). Nonmalignant conditions that are potentially curable by HSCT include disorders of hematopoiesis (e.g., aplastic anemia [AA]), immunodeficiency syndromes (e.g., Chediak-Higashi disease and severe combined immunodeficiency syndrome), congenital disorders of erythropoiesis (e.g., thalassemias), and inborn errors of metabolism (e.g., mucopolysaccharidoses).13,14 The advent of new treatment options for certain diseases (such as the tyrosine kinase inhibitors targeting bcr/abl, which are effective in the treatment of chronic myelogenous leukemia) and improvements in the toxicity profile associated with HSCT are likely to alter the indications for allogeneic transplantation.

Antileukemic Potential of Allogeneic Hematopoietic Stem Cell Transplantation: Underlying Principles

In the 1960s and 1970s, allogeneic HSCT was largely viewed as a means of ensuring immunohematopoietic reconstitution or replacement after administration of high doses of chemotherapy with or without radiation. This premise still applies to the treatment of nonmalignant conditions, for which the major goal is to provide normal cellular components to replace or rectify an underlying deficiency. For hematologic malignancies, the dose-intensive preparative or conditioning regimen was initially considered to be critical for the eradication of the underlying malignancy, with HLA-matched donor stem cells used merely to reverse the accompanying fatal bone marrow (BM) ablation. However, over the last two decades, it has become increasingly evident that the ability to generate a donor immune-mediated antimalignancy effect, termed GVL or GVT, is critical for the successful eradication of malignancies after allogeneic HSCT. The following clinical observations have provided incontrovertible evidence for the existence of GVL and highlight the role of donor T lymphocytes in mediating this effect10-12,15-17:

  Decreased risk of leukemia relapse in patients experiencing chronic graft-versus-host disease (GVHD).

  Increased risk of leukemia relapse in patients undergoing T-cell–depleted transplants.

  Increased risk of leukemia relapse in recipients of syngeneic as opposed to non-twin sibling donor allografts.

  Ability of immunosuppression withdrawal and/or donor lymphocyte infusions (DLIs) to induce sustained remission in patients with chronic myeloid leukemia (CML) who relapse after transplantation.

Recognition of GVL has led to the development of RIC or nonmyeloablative conditioning regimens, in which efficacy is largely dependent on the generation of donor immune-mediated antitumor responses.

Planning Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic HSCT is a complex procedure requiring careful planning and a multidisciplinary approach to patient management. Factors including patient age, performance status, underlying disease, and donor availability need to be considered before decisions regarding the type of transplantation to be performed (e.g., conventional myeloablative versus nonmyeloablative) and GVHD prophylaxis regimen are made.

Evaluation of Transplant Recipients

The degree of donor–host HLA compatibility is one of the most important factors affecting outcome after HSCT. Evaluation of the transplant recipient begins with HLA testing and a search for an appropriate donor. The initial donor search focuses on identifying a sibling matched at the allele level for HLA-A, HLA-B, and HLA-DR loci. If a suitable sibling donor is not available, a search for an HLA-compatible unrelated donor or unrelated cord blood unit can be made through the National Marrow Donor Program (NMDP).

Table 18.1  Indications for Hematopoietic Stem Cell Transplantation

Acute Leukemias

Acute lymphoblastic leukemia (ALL)

Acute myelogenous leukemia (AML)

Chronic Leukemias

Chronic myelogenous leukemia (CML)

Chronic lymphocytic leukemia (CLL)

Juvenile chronic myelogenous leukemia (JCML)

Juvenile myelomonocytic leukemia (JMML)

Myelodysplastic Syndromes

Refractory anemia (RA)

Refractory anemia with ringed sideroblasts (RARS)

Refractory anemia with excess blasts (RAEB)

Refractory anemia with excess blasts in transformation (RAEB-T)

Chronic myelomonocytic leukemia (CMML)

Stem Cell Disorders

Aplastic anemia (severe)

Fanconi anemia

Paroxysmal nocturnal hemoglobinuria

Pure red cell aplasia

Myeloproliferative Disorders

Acute myelofibrosis

Agnogenic myeloid metaplasia (myelofibrosis)

Polycythemia vera

Essential thrombocythemia

Lymphoproliferative Disorders

Non-Hodgkin lymphoma

Hodgkin disease

Phagocyte Disorders

Chediak-Higashi syndrome

Chronic granulomatous disease

Neutrophil actin deficiency

Reticular dysgenesis

Inherited Metabolic Disorders

Mucopolysaccharidoses (MPS)

Hurler syndrome (MPS-IH)

Scheie syndrome (MPS-IS)

Hunter syndrome (MPS-II)

Sanfilippo syndrome (MPS-III)

Morquio syndrome (MPS-IV)

Maroteaux-Lamy syndrome (MPS-VI)

Sly syndrome, β-C glucuronidase deficiency (MPS-VII)

Adrenoleukodystrophy

Mucolipidosis II (I-cell disease)

Krabbe disease

Gaucher disease

Niemann-Pick disease

Wolman disease

Metachromatic leukodystrophy

Histiocytic Disorders

Familial erythrophagocytic lymphohistiocytosis

Histiocytosis-X

Hemophagocytosis

Inherited erythrocyte abnormalities

β-Thalassemia major

Sickle cell disease

Inherited Immune System Disorders

Ataxia-telangiectasia

Kostmann syndrome

Leukocyte adhesion deficiency

DiGeorge syndrome

Bare lymphocyte syndrome

Omenn syndrome

Severe combined immunodeficiency (SCID)

SCID with adenosine deaminase

Absence of T & B cells SCID

Absence of T cells, normal B cell SCID

Common variable immunodeficiency

Wiskott-Aldrich syndrome

X-linked lymphoproliferative disorder

Other Inherited Disorders

Lesch-Nyhan syndrome

Cartilage-hair hypoplasia

Glanzmann thrombasthenia

Osteopetrosis

Inherited Platelet Abnormalities

Amegakaryocytosis/congenital thrombocytopenia

Plasma Cell Disorders

Multiple myeloma

Plasma cell leukemia

Waldenstrom macroglobulinemia

Other Malignancies

Breast cancer

Ewing sarcoma

Neuroblastoma

Renal cell carcinoma

Adapted from the list of transplant indications provided by the National Marrow Donor Program (NMDP).

Thorough history and physical examination, with emphasis on the underlying diagnosis and its treatment, concomitant medical problems, performance status, transfusion history, and any history of opportunistic (particularly fungal) infections should be done. Assessment of major organ function, including pulmonary function testing and full cardiac evaluation, should be undertaken. Serologic testing to detect prior exposure to cytomegalovirus (CMV), herpes virus, Epstein-Barr virus (EBV), hepatitis viruses, HIV, toxoplasmosis, and varicella is also needed. Counseling is required to focus on the potential benefits and risks of transplantation, need for a dedicated caregiver, and, when appropriate, fertility prospects.

Identification of a Suitable Donor

  Matched related donor: approximately one-third of patients screened will have a suitable HLA- compatible sibling donor. While donors with a less than complete (6/6) HLA match can be used, greater HLA disparity increases the risk of both graft rejection and GVHD.

  Syngeneic donor: identical twin transplants are rarely performed because high degrees of histocompatibility (including for minor histocompatibility antigens) minimize clinically meaningful GVL effects. Syngeneic donors are most desirable in the transplantation of acquired nonmalignant diseases (such as severe aplastic anemia [SAA]) where GVL is not required.

  Matched unrelated donor (MUD): There are more than 15 million donors in worldwide registries. Searches conducted through the NMDP in general will identify a suitable donor for approximately two-thirds of Caucasians, but for some minority groups, due to increased HLA diversity, it is much harder to find a match. The process should be initiated as early as possible because the time to transplant once a search is initiated is typically 3 to 4 months. For a given degree of donor–host HLA disparity, the risk of GVHD and graft rejection is higher with unrelated donors compared to related donors.

  Haploidentical donor: most patients have a sibling, parent, or child with one matched HLA haplotype who could serve as donor. Transplants from haploidentical donors are associated with a higher incidence of GVHD, necessitating T-cell depletion in most circumstances to prevent life-threatening GVHD. Impaired immune reconstitution is common and leads to a high incidence of opportunistic infections in recipients of haploidentical transplants. Recent studies utilizing posttransplant cyclophosphamide as a method to induce in vivo T-cell depletion following the transplantation of either haploidentical BM or PBSCs have shown encouraging results, with one retrospective report of extremely low rates of acute GVHD and chronic GVHD with DFS and OS rates comparable to dual cord blood transplants.¹⁸ Based on these promising results, a randomized trial comparing cord blood transplantation with haploidentical BM transplantation with posttransplant cyclophosphamide has recently been initiated through the CTN.

  Umbilical cord cells: blood collected from the placenta at the time of childbirth can be used as a source of hematopoietic stem cells. While umbilical cord transplants are associated with a lower incidence of GVHD (even with HLA mismatching), their widespread use is limited by an increased risk of graft failure due to the small numbers of stem cells harvested. Umbilical cord transplants were largely restricted to children and young adolescents. However, recent studies have established that umbilical cord transplants in adults are feasible. The use of dual cord units and ex vivo expansion of cord progenitor cells are some of the strategies being explored to boost the stem cell dose available for transplant. As of 2012, it is estimated that more than 600,000 cord blood units are available in the worldwide registry for public use.

Procurement of Hematopoietic Stem Cells

The majority of hematopoietic stem cells reside within the BM, which traditionally served as the source of the allograft. However, the availability of G-CSF, which mobilizes hematopoietic stem cells into the circulation, has led to the widespread use of PBSC allografts.

Obtaining stem cells from the BM involves multiple aspirations from the iliac crests, a relatively safe procedure performed under general anesthesia. Mobilized stem cells are typically collected from the peripheral blood by apheresis after administration of G-CSF (10 to 15 μg/kg/day) for 4 to 6 days. G-CSF mobilized PBSC grafts usually contain higher numbers of CD34⁺ progenitor cells as well as T cells (CD3⁺ cells) compared with BM grafts.

Compared with BM stem cells, transplants using PBSCs are associated with faster neutrophil and platelet engraftment, a reduction in transfusion requirements, and a similar incidence of acute GVHD, although chronic GVHD occurs with higher frequency with the use of PBSC compared with BM transplants.¹⁹ Some studies have shown that in patients with an underlying hematologic malignancy, PBSC transplants from HLA-identical siblings are associated with a survival advantage compared with BM transplants. However, a recent multicenter trial conducted through the CTN that randomized more than 500 patients with hematologic malignancies to either a BM or a PBSC transplant from an unrelated donor reported no difference in relapse, DFS, or OS between the two approaches with recipients of PBSC transplant having a significantly higher incidence of debilitating extensive chronic GVHD.²⁰ These data suggest that for patients with hematologic malignancies undergoing a MUD transplant, BM may be the preferred stem cell source.

Nevertheless, given the ease of stem cell collection from both the practitioner and the donor perspectives, the higher progenitor cell yield, earlier engraftment, and a lower risk of graft failure, the majority of allogeneic HSCTs worldwide in adults currently employ mobilized peripheral blood as a source of hematopoietic stem cells.

Conditioning Regimen

A variety of conditioning regimens have been used in allogeneic HSCT. The choice of conditioning regimen for a given patient is dictated by the underlying disease, the age of the patient, the presence of medical comorbidity, and donor characteristics (especially the degree of HLA compatibility). Table 18.2 lists some commonly used conditioning regimens.

Conventional or Myeloablative Conditioning

Myeloablative conditioning regimens serve a dual purpose:

  High doses of chemotherapy with or without radiation provide tumor cytoreduction, usually accompanied by eradication or ablation of host hematopoietic function.

  Suppression of the host’s immune system, a prerequisite for preventing rejection of the transplant.

The burden of tumor eradication in conventional transplants rests on both the transient cytoreductive properties of the conditioning agents and on more durable GVL effects mediated by donor immune cells.

The two most commonly used regimens are cyclophosphamide in combination with either total body irradiation (TBI) or busulfan. TBI-based regimens have a higher incidence of secondary malignancies, growth retardation, thyroid dysfunction, and cataracts, while non-TBI regimens, particularly those containing busulfan (oral or intravenous), are associated with more veno-occlusive disease (VOD) and mucositis. The advent of intravenous busulfan, with its more predicable pharmacokinetic profile, has allowed more consistent exposure to busulfan and a reduction in the incidence of VOD. The underlying condition often dictates the optimal conditioning regimen. For example, patients with acute lymphoblastic leukemia (ALL) appear to have a lower risk of relapse with TBI-based regimens, which are therefore preferentially used for this indication.

Reduced Intensity Conditioning Reduced intensity preparative regimens were devised in an effort to minimize conditioning-related morbidity associated with conventional transplants, while retaining the host immunosuppression necessary to ensure engraftment. Reduced intensity preparative regimens that do not eradicate host hematopoiesis are also referred to as nonmyeloablative conditioning regimens.

The burden of tumor eradication in reduced intensity transplants rests mainly on the donor immune-mediated GVL effect. These regimens are associated with a lower incidence of some conditioning-related toxicities (VOD, mucositis, prolonged neutropenia, etc.).

Reduced intensity regimens are better tolerated by older patients (up to 70 years) and by those with medical comorbidities and have allowed allo-HSCT to be extended to these populations. Several transplant centers are currently evaluating reduced intensity transplantation in patients with hematologic malignancies, solid tumors, and nonmalignant hematologic disorders. Although the risk of TRM appears lower with RIC regimens, several retrospective studies have suggested the risk of disease relapse in malignant diseases such as myeloma, and myelodysplastic syndrome (MDS) may be higher with this approach compared with conventional myeloablative transplants.

Results of Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic HSCT is the only curative option for many patients with hematologic malignancies: 85% to 90% of all allogeneic transplants in the United States are undertaken for this indication.

Chronic Myeloid Leukemia

CML is a myeloproliferative disorder characterized by the presence of a characteristic t(9;22) (q34;q11) translocation, the Philadelphia chromosome. The natural history consists of a relatively indolent chronic phase with progression to the more aggressive accelerated phase and blast crisis. Although allogeneic HSCT is the only proven curative therapy for this condition, the introduction of targeted agents with remarkable efficacy (such as imatinib mesylate, dasatinib, and nilotinib) has led to the acceptance of these agents as standard initial therapy for patients with chronic phase CML. Consequently, allo-HSCT is usually reserved for patients with accelerated phase or blast crises CML and for chronic phase patients who have failed agents targeting the abl kinase.

Among chronic phase patients undergoing transplantation from an HLA-compatible sibling, 65% to 80% are cured; similar results are now being reported in patients undergoing MUD transplantation. Early results in patients receiving RIC are promising, but prospective studies are needed to determine if this approach is equivalent to conventional allo-HSCT. Transplantation is much less effective in accelerated phase or blast crisis (where cure rates are 10%–20%).

Younger patients and patients who undergo transplantation within a year of diagnosis have the best outcomes. Chronic phase CML is sensitive to GVL effects and a single DLI can reinduce remission in 70% of patients who relapse after transplantation, as can withdrawal of immunosuppression (such as cyclosporine) used for GVHD prophylaxis/treatment. Chronic phase CML patients who fail to achieve a cytogenetic remission with imatinib can be successfully salvaged with allo-HSCT, as shown in a recent study.

Acute Myeloid Leukemia

The indication and timing for transplantation in AML and outcome after allogeneic HSCT depend on the risk category.

  Patients with intermediate or poor prognosis AML as determined by cytogenetics are at high risk for relapse after chemotherapy and should be evaluated for allogeneic HSCT in CR1 when an HLA-matched sibling donor is available. Recent studies have also shown that patients with normal cytogenetics who have an FLT3-ITD, or a wild-type NPM1 or CEBPA without an FLT3-ITD, are also at increased risk for relapse and may benefit from an allogeneic transplant in CR1. ²¹

  Patients transplanted in CR1 have a 45% to 60% probability of long-term DFS.

  Patients transplanted in first relapse or after induction of second complete remission (CR2) have only a 22% to 40% chance of long-term DFS.

  Outcomes after transplantation in first relapse or CR2 are comparable.

  Allogeneic HSCT in good-prognosis AML is usually reserved for CR2 or first relapse, because the risk of TRM outweighs the benefits from early transplantation (CR1) in this group.

  Less than 20% of patients with primary induction failure or those beyond CR2 have durable leukemia remission after allogeneic HSCT.

Acute Lymphoblastic Leukemia

While a significant proportion of childhood ALL is curable with chemotherapy, the majority (60%–70%) of adults with this disease relapse following initial chemotherapy. Patients older than 60 years of age, those with a leukocyte count higher than 30,000/μL, or with high-risk cytogenetics [t(4;11), t(1;19), t(8;14), or t(9;22)] have a particularly poor prognosis. Traditionally, allogeneic HSCT in CR1 has been recommended for adult patients with ALL with poor prognostic features (DFS rates in the range of 40%–60%), reserving transplant for patients without adverse factors for CR2 (DFS rates of approximately 40%). However, a recent randomized trial showed patients with standard-risk ALL who received upfront allogeneic transplant in CR1 had a survival advantage compared with patients who received consolidative chemotherapy reserving transplant for CR2.²² Therefore, allogeneic transplant in CR1 is a reasonable strategy to prevent disease relapse in adults with either standard-risk or high-risk ALL.

Myelodysplastic Syndrome

Allogeneic HSCT offers a 30% to 40% probability of long-term DFS in patients with MDS. The two most important factors predicting outcome after transplantation are blast percentage and cytogenetic risk group. Accordingly, patients with few blasts (refractory anemia or refractory anemia with ringed sideroblasts) have 50% to 75% long-term DFS, while more advanced stages (e.g., refractory anemia with excess blasts) are associated with a 30% DFS. Similarly, patients with good-risk cytogenetics have an approximately 50% probability of DFS compared with 10% or less for those with poor-risk cytogenetics. Nonetheless, allogeneic HSCT remains the only curative therapy for MDS and should be considered a potential definitive therapy. Reduced intensity HSCT is associated with a higher risk of relapse in MDS patients than is conventional HSCT and should be reserved for patients who are not candidates for myeloablative HSCT or performed as part of well-designed trials.

Non-Hodgkin’s Lymphoma

Low-Grade Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia Experience with allogeneic HSCT in low-grade lymphomas and CLL is largely restricted to patients undergoing the procedure late in the course of their disease after multiple chemotherapeutic options have been exhausted; 50% to 65% of patients will achieve long-term DFS. The typically indolent disease course and profound susceptibility to GVL makes low-grade lymphomas and CLL amenable to management and cure using nonmyeloablative conditioning approaches. Remarkably, several studies have shown that more than

40% of patients with 17p deletion CLL, who have the worst prognosis with conventional chemotherapy, can obtain long-term DFS with a reduced intensity transplant. These findings have led to an increased use of upfront transplantation in this high-risk CLL cohort.²³

Aggressive Non-Hodgkin Lymphoma The role of allogeneic HSCT in patients with intermediate- and high-grade lymphomas is unclear. Most studies have reported a high incidence of TRM with myeloablative transplantation in this group. As a consequence, allogeneic HSCT that uses RIC is generally reserved for those patients in whom potentially curative autologous HSCT has failed or for those unlikely to benefit from an autologous transplant (patients with chemotherapy-resistant disease).

Multiple Myeloma

TRM rates in the range of 50% have discouraged the use of conventional myeloablative transplantation for multiple myeloma. Nevertheless, there is evidence that donor immune-mediated graft-versus-myeloma effects can be curative. Recently, RIC has been explored as a safer transplant approach to treat multiple myeloma. TRM has been reported to be significantly lower (less than 25%) compared with historic myeloma cohorts undergoing myeloablative conditioning; importantly, graft-versus-myeloma effects resulting in durable disease remission can be induced after reduced-intensity transplants. Autologous transplantation as myeloma cytoreduction followed by nonmyeloablative allogeneic transplantation as immunotherapy to eradicate minimal residual disease appears promising, with DFS of more than 50% in some studies. However, a recent randomized trial conducted through the CTN group which enrolled over 700 subjects reported no difference in 3-year PFS and OS between patients receiving a tandem auto transplant versus a tandem auto/allotransplant with significantly higher TRM being reported in the latter group.⁴

Aplastic Anemia

Allogeneic HSCT can cure SAA.²⁴ Early studies of allogeneic HSCT in patients with SAA showed a high incidence of graft rejection (up to 35% in some early series) and GVHD. Sensitization to histocompatibility antigens as a result of multiple transfusions and the use of cyclophosphamide alone as pretransplant conditioning accounted for these high rejection rates. Subsequent approaches added antithymocyte globulin to cyclophosphamide to minimize graft rejection while preventing severe and potentially lethal GVHD. Additionally, the routine use of leukocyte-depleted and irradiated blood products has decreased the risk of graft rejection to less than 5% in most studies. A combination of cyclosporine A (CSA) and methotrexate is generally used as GVHD prophylaxis with delayed and gradual withdrawal of immunosuppression to minimize the risk of GVHD. Patients under the age of 40 years receiving an allogeneic HSCT from an HLA-matched sibling have an excellent chance for cure, with long-term survival rates approaching 90% in children. Several studies indicate that transplant outcomes are better in patients with AA when BM is used as the primary graft source, rather than PBSCs, since the latter is associated with an increased incidence of chronic GVHD.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation

Complications of allogeneic HSCT are most commonly related to preparative regimen toxicities, infections occurring as a consequence of immunosuppression, or acute or chronic GVHD.

Conditioning-related Toxicities

Conditioning-related toxicities vary depending on the type and dosage of agents used in the preparative regimen. Nausea, vomiting, and mucositis occur commonly with myeloablative preparative regimens. Busulfan tends to be associated with more severe mucositis.

Hemorrhagic cystitis occurring early in the course of transplantation is usually associated with preparative regimens containing high-dose cyclophosphamide. In contrast, hemorrhagic cystitis more than 72 hours after conditioning is typically viral (polyoma virus BK or adenovirus). Attention to hydration and the routine use of 2-mercaptoethanesulfonate has virtually eliminated cyclophosphamide- associated hemorrhagic cystitis.

Opportunistic infections occur with conditioning-related neutropenia. Bacteria and fungi that are normally present in the skin, gastrointestinal (GI) tract, or respiratory tract cause the majority of these infections.

Damage to gut mucosa and indwelling venous catheters serve as the portal of entry for most life-threatening gram-negative or aerobic gram-positive organisms. The use of Prophylactic oral antibiotics such as quinolones for gut decontamination has decreased the incidence of gram-negative bacteremia without impacting survival. Routine use of these agents should be weighed against the increased risk of gram-positive bacteremia and emergence of resistant gram-negative strains.

Candida and Aspergillus fungal infections occur commonly during conditioning-induced neutropenia. Prophylactic fluconazole appears to protect against sensitive Candida. In a recent phase III randomized trial, prophylactic voriconazole was associated with a trend toward a lower incidence of invasive fungal infection compared with fluconazole, although OS was similar in the two groups.²⁵

VOD is characterized by the triad of jaundice, tender hepatomegaly, and ascites occurring early posttransplant. Risk factors include

  Advanced age.

  Conditioning with busulfan, with up to 30% of patients receiving oral busulfan developing this complication. The advent and preferential use of IV busulfan appears to have led to a decrease in the incidence of VOD.

  Preexisting liver disease.

  Development of acute GVHD.

  Transplants from matched unrelated and haploidentical donors.

Prophylaxis with oral ursodiol may protect against this complication. VOD can be severe and life threatening in approximately 25% of patients developing this complication. Treatment remains largely supportive, although defibrotide and recombinant tissue plasminogen activator have each been used with some success in severe VOD. In a recently reported randomized phase III study in pediatric patients undergoing HSCT, defibrotide prophylaxis was associated with a lower incidence of VOD.²⁶

Graft-versus-Host Disease

GVHD is one of the most common complications of allogeneic HSCT. GVHD is a consequence of allogeneic donor T cells damaging normal recipient tissues. Based on the time of onset, clinical features, and pathophysiology, GVHD is classified as either acute or chronic.²⁷

Acute Graft-versus-Host Disease Acute GVHD typically commences during the first 100 days after transplantation. Of HLA-matched sibling donor transplant recipients, 20% to 50% experience acute GVHD; the incidence is higher in transplants utilizing unrelated or partial HLA-matched related donors. The extent of donor–host HLA disparity, recipient age, T-cell content of the graft, intensity of the conditioning regimen, and the type of GVHD prophylaxis regimen utilized all influence the incidence and severity of GVHD.

The skin, GI tract, and liver are the most common targets of alloreactive donor T cells causing GVHD. The following clinical and laboratory features should arouse suspicion of GVHD:

  Skin: erythematous maculopapular rash frequently involving the palms and soles. Severe cases can present with skin desquamation.

  GI: crampy abdominal pain, and large-volume watery diarrhea characterize GVHD of the colon and distal small bowel. In severe cases, bloody diarrhea or ileus may occur. Anorexia, dyspepsia, weight loss, and nausea and vomiting are characteristic of upper GI GVHD.

  Hepatic: elevated alkaline phosphatase and direct bilirubin with or without elevations in transaminases characterize acute GVHD of the liver.

Definitive diagnosis can be difficult because a variety of other conditions (such as drug-induced skin rash, viral colitis) can present with similar features. Biopsy and histopathologic examination of involved tissue is considered the gold standard for diagnosing GVHD.

GVHD is a major contributor to TRM; strategies directed at preventing this complication are an important aspect of transplant planning. Pharmacologic prophylaxis and graft T-cell depletion are established methods that effectively reduce the incidence and severity of GVHD. CSA or tacrolimus combined with methotrexate or mycophenolate mofetil are commonly used for GVHD prophylaxis. Effective T-cell depletion of the allograft can be achieved in vitro by CD34⁺ cell selection or in vivo pharmacologically (alemtuzumab or posttransplant cyclophosphamide). However, T-cell–depleted transplants are associated with a higher risk of graft failure, leukemia relapse, and opportunistic viral infections. Selective depletion of alloreactive T cells and T-cell–depleted transplants with scheduled T-cell add back 30 or more days following transplantation have been shown to reduce the incidence of GVHD without compromising GVL.

Treatment of established GVHD depends on the type and severity of involved organs (for grading of acute GVHD, see Table 18.3). While mild (grade I) skin GVHD can be managed effectively with topical corticosteroids, visceral GVHD and more severe forms of cutaneous GVHD require systemic immunosuppressive therapy. Glucocorticoids (methylprednisone, typically at doses of 1 to 3 mg/kg/day) are the mainstay of therapy and are given in conjunction with cyclosporine or tacrolimus, with doses titrated to maintain therapeutic serum levels. Unfortunately, only approximately 50% of patients demonstrate durable responses to this form of therapy (for treatment of established acute GVHD, see Table 18.4).

Nonresponding or steroid-refractory patients have a poor outcome, with mortality rates more than 80%. The majority developing steroid-refractory GVHD die from infectious complications or organ damage related to relentless immune attack. Comprehensive management of steroid-refractory GVHD patients with immunosuppressive agents such as daclizumab or infliximab accompanied by targeted infectious prophylaxis against enteric bacteria and Aspergillus appears to be a promising strategy that deserves further study (see Table 18.4).²⁸

Chronic Graft-versus-Host Disease The onset of chronic GVHD is usually between 100 days and 2 years after transplantation. It affects 20% to 50% of recipients of allogeneic BM transplants and up to 80% receiving an allogeneic PBSC transplant. Risk is increased by

  Prior history of acute GVHD.

  Older patient age.

  Use of HLA-mismatched or unrelated donors.

  DLI administration.

  Use of PBSC (as opposed to BM) allografts.

Table 18.4  Treatment of Acute Graft-versus-Host Disease: The National Heart, Lung, and Blood Institute Approach

Initial Management

Grade I GVHD (Stage 1–2 Skin)

Topical corticosteroid therapy

Grade II–IV GVHD

•  High-dose methylprednisolone 1–10 mg/kg/day up to a maximum of 500 mg/day IV 3–6 d and

•  IV cyclosporine or IV tacrolimus

•  Steroids tapered once response is evident over 10–14 d

•  All patients receiving ≥1 mg/kg of methylprednisolone undergo routine surveillance blood cultures every 3 d

•  All patients with ≥grade III GI GVHD receive prophylactic antibiotic therapy against enteric organisms (e.g., ampicillin–sulbactam)

Management of Steroid-Refractory GVHD

(GVHD not responsive to 6 or more days of continuous therapy with ≥1 mg/kg methylprednisolone)

(A) Treatment

•  Rapid taper of methylprednisolone to ≤1 mg/kg

•  Daclizumab (monoclonal antibody to interleukin-2 receptor-α) 1 mg/kg on days 1, 4, 8, 15, 22

•  Infliximab (monoclonal antibody to tumor necrosis factor-α) 10 mg/kg on days 1, 8, 15, 22

(B) Supportive care

•  All patients with GI GVHD are maintained NPO

•  All patients with ≥grade III GI GVHD receive prophylactic antibiotic therapy against enteric organisms (e.g., ampicillin-sulbactam)

•  All patients with steroid-refractory GVHD and those patients who receive ≥1 mg/kg methylprednisolone for more than 6 d receive prophylaxis against Aspergillus (e.g., liposomal amphotericin, 5 mg/kg/day, or voriconazole)

•  All patients receiving ≥1 mg/kg of methylprednisolone undergo routine surveillance blood cultures every 3 d

GI, gastrointestinal; GVHD, graft-versus-host disease; IV, intravenously; NPO, nothing by mouth.

Patients may present with a myriad of clinical features including lichenoid or sclerodermatous skin changes, elevated liver function tests, xerostomia, dry eyes, diarrhea, weight loss, bronchiolitis obliterans, and thrombocytopenia with or without pancytopenia.

Most clinicians use a two-stage staging system: limited GVHD, representing localized skin involvement and extensive GVHD, which includes patients with more diffuse skin involvement or involvement of other target organs. Investigators at the National Institute of Health (NIH) have recently proposed a system for the diagnosis and classification of chronic GVHD based on histopathologic, clinical, laboratory, and radiologic features which rates chronic GVHD as mild, moderate, or severe.²⁹

Therapy typically consists of cyclosporine or tacrolimus given in conjunction with low-dose corticosteroids. Alternative agents include mycophenolate mofetil, thalidomide, photochemotherapy with oral methoxypsoralen therapy followed by ultraviolet-A photophoresis, and monoclonal antibodies directed against T or B lymphocytes or cytokines implicated in pathogenesis.

A high risk of bacterial infections in patients with chronic GVHD warrants routine use of antibiotic prophylaxis against encapsulated bacteria and opportunistic pathogens.

Pulmonary Complications

Pulmonary complications may occur both early and late after transplantation, and they may be infectious or noninfectious in etiology.

Pulmonary Complications Attributable to Infections Fungi (Aspergillus and other agents) as well as viruses (CMV, respiratory syncytial virus, influenza, parainfluenza, etc.) can cause life-threatening pneumonia in the posttransplant setting. Early diagnosis, prophylactic or preemptive therapy (e.g., ganciclovir or foscarnet for CMV antigenemia), and prompt institution of definitive therapy when available are the major principles guiding management of these complications. The risk of Pneumocystis jiroveci pneumonia is greatest in the first 6 months after transplantation, particularly in patients receiving T-cell–depleted grafts or those suffering from chronic GVHD; prophylaxis with sulfa/trimethoprim or inhaled pentamidine virtually eliminates this complication.

Idiopathic interstitial pneumonitis usually occurs early after transplantation and is characterized by fever, hypoxia, and diffuse pulmonary infiltrates. TBI or drugs with pulmonary toxicity (i.e., busulfan) in the preparative regimen increase the risk of this complication. An infectious etiology as well as diffuse alveolar hemorrhage should be excluded before a diagnosis of idiopathic interstitial pneumonitis can be rendered. Corticosteroids and tumor necrosis factor–α antagonists have been used to treat this condition with modest results.

Diffuse alveolar hemorrhage is a relatively infrequent but often fatal complication of allogeneic HSCT. It is characterized by the rapid onset of dyspnea, cough, and hypoxia with diffuse bilateral infiltrates on radiography. High-dose corticosteroids and recombinant activated factor VII (Novo7) may be of therapeutic benefit, although the condition is lethal in 40% to 80% of cases.

Infectious Complications

Recipients of allogeneic HSCT continue to be at risk for infections beyond the period of conditioning-related neutropenia, with viral and fungal pathogens and encapsulated bacteria posing the greatest hazard.³⁰ Factors influencing infectious risk include the presence of acute or chronic GVHD, the extent of immunosuppressive pharmacotherapy in the posttransplant period, T-cell depletion of the graft, and the use of cord blood transplants or partial HLA-mismatched or unrelated donors.

Bacterial Infections

Gram-negative bacteremia associated with GI GVHD and venous catheter-related infections (predominantly gram-positive pathogens) occur with greatest frequency during the first 3 to 4 months after transplantation.

Recurrent sinus and pulmonary infections are associated with chronic GVHD. Antibiotic prophylaxis against encapsulated organisms, using penicillin or an appropriate alternative, reduces the risk of these infections.

Patients with recurrent infections and low serum immunoglobulin levels may benefit from prophylactic intravenous immunoglobulin (IVIG) infusions.

Fungal Infections

Fungal infections constitute a major cause of mortality after allogeneic HSCT: 60% to 70% of patients developing invasive fungal infections die despite antifungal therapy.

Yeast (Candida species) and molds (Aspergillus) account for the majority of opportunistic fungal infections in the posttransplant period.

Candida infections typically occur early in the course of transplantation, often near the end of the neutropenic phase. Candidal infections can manifest as mucocutaneous candidiasis, candidemia, or with visceral involvement (the liver and spleen are most commonly involved). Routine prophylaxis with fluconazole or echinocandins offers protection against sensitive strains of Candida.

Invasive Aspergillus infections typically involve the lungs, paranasal sinuses, and the central nervous system (CNS), although dissemination to other visceral organs has been described. Predisposing factors are

  Corticosteroids.

  Severe GVHD.

  The use of non–HLA-identical and unrelated stem cell donors.

  Transplantation in rooms lacking laminar air flow.

Invasive fungal infections are difficult to eradicate. Fluconazole or echinocandins may be effective against sensitive Candida strains (like Candida albicans). Amphotericin B, lipid formulation amphotericin, echinocandins (caspofungin, micafungin, etc.), voriconazole, and posaconazole have demonstrated efficacy against Aspergillus and a wide spectrum of Candida species.

The diligent application of preventive measures such as avoiding the indiscriminate use of corticosteroids remains the most effective strategy for minimizing mortality related to invasive fungal infections.

Viral Infections

Cytomegalovirus While advances in screening and preventive therapy have reduced CMV-related mortality, CMV infection continues to be a major contributor to posttransplant morbidity. CMV is a DNA virus belonging to the herpes virus family. Posttransplant CMV infection is most often a consequence of viral reactivation in patients with prior exposure to CMV and is observed in 50% to 70% of CMV seropositive recipients. Reactivation typically occurs in the first 100 days after transplantation. Primary infection in CMV-seronegative recipients can follow transplantation from a seropositive donor. Acquisition of primary infection from CMV-positive transfusion products has been all but eliminated with routine use of leukocyte-depleted or CMV-negative blood products.

GVHD, the use of T-cell–depleted allografts, cord blood transplants, and the use of immunosuppressive agents such as alemtuzumab, corticosteroids, or calcineurin inhibitors increase the risk of CMV reactivation.

Interstitial pneumonitis is the most common and serious manifestation of CMV disease, followed by enteritis/colitis. Other manifestations include febrile episodes and marrow suppression resulting in thrombocytopenia with or without neutropenia. Mortality rates with CMV pneumonitis range from 65% to 85%. Ganciclovir or foscarnet given in conjunction with IVIG improves outcome associated with CMV disease (Table 18.5). Early detection methods utilize polymerase chain reaction (PCR) for viral DNA in the blood which predicts the subsequent development of CMV disease.

Preemptive therapy with ganciclovir or foscarnet (see Table 18.5) begun when CMV reactivation is first detected (either PCR or immunofluorescence assay) has dramatically reduced the incidence of CMV pneumonitis/enteritis and consequently CMV-related mortality. Newer approaches to the treatment of or prophylaxis against CMV disease include adoptive transfer of ex vivo expanded CMVspecific cytotoxic T lymphocytes.

Table 18.5   Surveillance and Management of Cytomegalovirus:The National Heart, Lung, and Blood Institute Approach

Surveillance

(CMV PCR)

•  Weekly posttransplant until day 100

•  Continue surveillance beyond day 100 in the event of late CMV reactivation, continued immunosuppressive therapy, or if clinically indicated

Management of CMV Antigenemia*

Induction: Ganciclovir 5 mg/kg IV q12h or foscarnet 90 mg/kg IV q12h or valganciclovir 900 mg PO twice daily × 7 d,* followed by

Maintenance: Ganciclovir 5 mg/kg IV every day 5 times/week (M–F) or foscarnet 90 mg/kg IV every day 5 times/week (M–F) or daily valganciclovir 900 mg PO every day × 7 d

Management of CMV Disease†

Induction: Ganciclovir or foscarnet IV (at induction doses) q12h × 14 d and IVIg 500 mg/kg IV QOD × 14–21 d

Maintenance: Ganciclovir 5 mg/kg IV every day × 30 d

*If PCR shows two consecutive increases in copy number, continue induction and consider other treatment options (e.g., switching from ganciclovir to foscarnet) until PCR turns negative.

†Foscarnet may be the drug of choice in patients who have cytopenias and CMV reactivation or CMV disease.

CMV, cytomegalovirus; IV, intravenously; IVIg, intravenous immunoglobulin; PCR, polymerase chain reaction; PO, orally.

Epstein-Barr Virus–Associated Lymphoproliferative Disorder

EBV-related lymphoproliferative disorder is a B-cell malignancy arising as a consequence of impaired T-cell immunity against EBV. It is a relatively rare complication, affecting approximately 1% of all allogeneic transplant recipients, although certain transplants (especially T-cell depleted or umbilical cord blood) are associated with a significantly higher risk. The natural course of untreated EBV-related lymphoproliferative disorder is rapid progression, culminating in death. Allograft T-cell depletion, transplants from HLA-mismatched and unrelated donors or cord blood transplants, and the use of immunosuppressive agents predispose to the development of this malignancy.

Treatment with a monoclonal antibody to CD20 (rituximab), DLIs, withdrawal of posttransplant immunosuppression, or adoptive infusion of EBV-specific cytotoxic T cells are all effective in eradicating this disease, particularly when combined with withdrawal of immunosuppression.

Other Viral Infections Patients undergoing allogeneic HSCT are at risk for infectious complications associated with the herpes viruses, varicella zoster, and various respiratory viruses (respiratory syncytial virus, influenza, and parainfluenza). Adenoviruses or polyoma virus BK may appear clinically as hemorrhagic cystitis. Because cellular immunity is impaired in the posttransplant setting, otherwise self-limiting viral infections can have fatal sequelae. Recently, the investigational agent CMX-001 has shown promising activity in patients with systemic adenoviral infection.

Graft Failure

Graft failure is the inability to achieve (primary) or maintain (secondary) persistent donor hematopoiesis. Graft failure mediated by the recipient immune system is referred to as graft rejection. Graft failure is relatively uncommon in patients undergoing transplantation from an HLA-identical sibling donor (less than 2%). T-cell depletion, the use of HLA-mismatched or unrelated donor grafts, cord blood transplants, and pretransplant HLA alloimmunization caused by repeated transfusions are factors that increase the risk of graft rejection.

In myeloablative HSCT, primary graft failure presents as persistent pancytopenia (more than 3 to 4 weeks) after conditioning and is associated with a high mortality rate. Secondary graft failure is characterized by initial recovery of blood counts followed by a later loss of donor hematopoiesis.

Up to 50% of patients with graft rejection can be salvaged by repeat conditioning or immunosuppression (e.g., OKT3 plus corticosteroids) followed by reinfusion of a T-cell–replete allograft. Graft failure can also result from infections, drugs, or chronic GVHD. Patients can often be salvaged with hematopoietic growth factors (e.g., G-CSF) with or without additional stem cells.

Late Sequelae of Transplantation

Secondary Malignancies In addition to EBV-related lymphomas, leukemias and solid tumors can complicate allogeneic HSCT. The risk of solid tumors in transplant survivors at 10 years increases eightfold compared to age-matched controls. Melanomas, tumors of the oral cavity, bone, liver, CNS, and thyroid are some commonly encountered secondary malignancies. Younger patient age at transplantation and TBI-based conditioning regimens predispose to the development of secondary solid tumors.

Other Late Complications Growth retardation, infertility, restrictive pulmonary disease, cataracts, endocrine dysfunction, avascular necrosis of bones, osteopenia, and neurocognitive defects are other delayed sequelae of allogeneic HSCT.

Reduced Intensity and Nonmyeloablative Hematopoietic Stem Cell Transplantation

The high risk of TRM with conventional transplants and the appreciation that GVL effects can cure some hematologic malignancies provided the impetus for the development of reduced intensity and nonmyeloablative conditioning regimens.31,32 The basic principles underlying these regimens include the following:

  RIC to induce adequate host immunosuppression for donor allograft “take” while minimizing toxicities related to dose-intensive conditioning.

  Manipulation of posttransplant immunosuppression and administration of DLIs to promote rapid transition to complete donor immunohematopoiesis.

  Reliance on the GVL effect for eradication of the underlying malignancy.

A variety of different conditioning regimens have been used, and the cumulative experience from various transplant centers has led to the following observations:

  Toxicities such as VOD and mucositis are absent or mild compared to myeloablative transplants.

  TRM is substantially lower (7%–20%) than the 25% to 40% mortality associated with standard or myeloablative transplants.

  The improved toxicity profile has expanded the eligibility of allogeneic transplantation to older patients (up to 70 years of age) and patients with comorbid medical conditions.

GVL effects against several hematologic malignancies including AML, CML, ALL, CLL, NHL, and myeloma have been observed. However, at least in some malignancies (e.g., MDS, myeloma), the risk of relapse following RIC appears to be higher than that following myeloablative transplantation. Prospective randomized comparisons are required to determine if relapse risk and outcome in individual malignancies can be adversely affected by using RIC.

Pilot trials of nonmyeloablative HCT in solid tumors have shown for the first time the ability of the GVT effect to induce disease regression in treatment-refractory metastatic solid tumors. Renal cell carcinoma provides the best example of a tumor that may be susceptible to GVT effects³³; GVT effects have also been described in other solid tumors including breast, pancreatic, colon, and ovarian carcinoma.³⁴

Alternative Donor Transplantation

Transplantation from Matched Unrelated Donors

A 6/6 HLA-matched sibling donor can be identified for less than a third of patients evaluated for an allogeneic HSCT. Suitable volunteer donors registered with the NMDP can be identified for many patients who do not have a matched sibling donor but are otherwise considered candidates for an allogeneic HSCT. It is estimated that up to 70% of Caucasians will have at least one HLA-matched donor available. It may be more difficult to find appropriately matched donors for patients belonging to some racial/ethnic subgroups.

Outcome following matched unrelated HSCT has improved significantly with the introduction of routine molecular typing of HLA loci (as opposed to serologic typing) to identify donor–host compatibility. Transplantation using donors who are HLA-identical at the HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ loci by high resolution molecular typing (i.e., a 10/10 match) leads to outcomes approaching that following matched sibling transplants; however, GVHD remains a problem in unrelated donor transplants and contributes to worse overall outcome.

Transplantation from Mismatched Related Donors

Siblings with mismatches at one or more HLA loci can be used as donors for allogeneic HSCT. However, the incidence of both graft failure and GVHD is higher in recipients of partially matched sibling transplants.

Haploidentical transplants utilize parents, siblings, or children who share one haplotype with the recipient as donors. The high risk of lethal GVHD accompanying haploidentical transplantation mandates extensive T-cell depletion of grafts (either ex vivo of the allograft itself before infusion or in vivo using T-cell–depleting agents such as alemtuzumab or posttransplant cyclophosphamide). In addition, strategies such as the use of high CD34⁺ cell dose and/or nonmyeloablative conditioning have been utilized in attempts to improve outcome in transplants using partially matched, related donors. Donor– host killer immunoglobulin-like receptor (KIR) incompatibility may affect outcome following haploidentical transplantation. Specifically, transplants in which recipient cells do not express HLA molecules that can inhibit donor KIR are associated with a lower risk of GVHD and disease relapse, notably in patients with myeloid malignancies.³⁵ Current evidence suggests that natural killer cell alloreactivity may mediate both the heightened GVL effects and reduced GVHD incidence in KIR-incompatible haploidentical transplants. This observation may allow selection of donors who are KIR mismatched in a bid to maximize NK alloreactivity.

Umbilical Cord Transplants

Cord blood, collected from peripartum placenta, contains stem cells with remarkable proliferative capability and is being used increasingly as an alternative source of stem cells for allogeneic HSCT. Both their proliferative potential and their relatively immature lymphocyte content (which would be predicted to lead to a lower incidence of GVHD) are viewed as advantages over other alternative stem cell sources such as haploidentical and unrelated donors. Furthermore, since umbilical cord allografts are derived from previously collected and stored cord blood, they are more readily available than unrelated donor grafts which involve donor preparation and collection of stem cells once a suitable donor is identified. The major limitation of cord blood as a source of hematopoietic stem cells (particularly in adults) is the relatively small number of cells that can be obtained from single cord blood units. A minimum of 2.5 × 10⁷ nucleated cells/kg and/or ≥1.2 × 10⁵ CD34⁺ cells/kg are required to obtain acceptable engraftment rates.

Early studies of cord blood transplants in children established the feasibility of this procedure, with acceptable engraftment rates (85% in one study) and low rates of acute GVHD (<10% in matched cord blood transplants).³⁶ Retrospective analyses comparing cord blood transplants to unrelated donor marrow transplants have since been undertaken and indicate the following37,38:

  Cord blood transplants are typically associated with delayed hematopoietic recovery (median neutrophil recovery time of 27 days and median platelet recovery time of 60 days in one study), leading to a higher risk of infectious complications.

  A lower incidence of acute GVHD as well as chronic GVHD is encountered following cord blood transplants.

  TRM, disease relapse rates, and DFS following cord blood transplants are comparable to that seen in transplants using mismatched unrelated donor marrow.

The simultaneous use of multiple cord blood units (from different donors), the co-infusion of cord units with CD34⁺ selected haploidentical stem cells, and ex vivo expansion of cord blood stem cells are being explored as means of overcoming the limitations imposed by low stem cell dose and it is hoped that these approaches will allow more adults to undergo this procedure.39,40

References

  1.  Blume KG, Thomas ED. A review of autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2000;6:1-12.

  2.  Thomas ED. Bone marrow transplantation: a review. Semin Hematol. 1999;36:95-103.

  3.  Attal M, Harousseau JL, Stoppa AM, et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med. 1996;335:91-97.

  4.  Krishnan A, Pasquini MC, Logan B, et al. Autologous haemopoietic stem-cell transplantation followed by allogeneic or autologous haemopoietic stem-cell transplantation in patients with multiple myeloma (BMT CTN 0102): a phase 3 biological assignment trial. Lancet Oncol. 2011;12:1195-1203.

  5.  Cavo M, Rajkumar SV, Palumbo A, et al. International Myeloma Working Group consensus approach to the treatment of multiple myeloma patients who are candidates for autologous stem cell transplantation. Blood. 2011;117: 6063-6073.

  6.  Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med. 1995;333: 1540-1545.

  7.  Antman KH. Randomized trials of high dose chemotherapy for breast cancer. Biochim Biophys Acta. 2001;1471: M89-98.

  8.  Pico JL, Rosti G, Kramar A, et al. A randomised trial of high-dose chemotherapy in the salvage treatment of patients failing first-line platinum chemotherapy for advanced germ cell tumours. Ann Oncol. 2005;16:1152-1159.

  9.  Voss MH, Feldman DR, Motzer RJ. High-dose chemotherapy and stem cell transplantation for advanced testicular cancer. Expert Rev Anticancer Ther. 2011;11:1091-1103.

10.  Childs RW. Allogeneic hematopoietic cell transplantation. In: De Vita VJ, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. London: Lippincott Williams and Wilkins; 2011;2244-2261.

11.  Mathe G, Amiel JL, Schwarzenberg L, et al. Adoptive immunotherapy of acute leukemia: experimental and clinical results. Cancer Res. 1965;25:1525-1531.

12.  Storb R. Allogeneic hematopoietic stem cell transplantation—yesterday, today, and tomorrow. Exp Hematol. 2003;31:1-10.

13.  Boelens JJ, Prasad VK, Tolar J, et al. Current international perspectives on hematopoietic stem cell transplantation for inherited metabolic disorders. Pediatr Clin North Am. 2010;57:123-145.

14.  Smith AR, Gross TG, Baker KS. Transplant outcomes for primary immunodeficiency disease. Semin Hematol. 2010;47:79-85.

15.  Antin JH. Stem cell transplantation-harnessing of graft-versus-malignancy. Curr Opin Hematol. 2003;10:440-444.

16.  Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75:555-562.

17.  Riddell SR, Berger C, Murata M, et al. The graft versus leukemia response after allogeneic hematopoietic stem cell transplantation. Blood Rev. 2003;17:153-162.

18.  Brunstein CG, Fuchs EJ, Carter SL, et al. Alternative donor transplantation after intensity conditioning: results of parallel phase 2 trials using partially HLA-mismatched related bone marrow or unrelated double umbilical cord blood grafts. Blood. 2011;118:282-288.

19.  Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med. 2001;344:175-181.

20.  Claudio Anasetti BRL, Lee SJ, Waller EK, et al. Increased incidence of chronic graft-versus-host disease (GVHD) and no survival advantage with filgrastim-mobilized peripheral blood stem cells (PBSC) compared to bone marrow (bm) transplants from unrelated donors: results of blood and marrow transplant clinical trials network (BMT CTN) protocol 0201, a phase III, prospective, randomized trial. Blood. (ASH Annual Meeting Abstracts) 2011;118:1.

21.  Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358:1909-1918.

22.  Goldstone AH, Richards SM, Lazarus HM, et al. In adults with standard-risk ALL, the greatest benefit is achieved from a matched sibling allogeneic transplantation in CR1, and an autologous transplantation is less effective than conventional consolidation/maintenance chemotherapy in all patients: final results of the International ALL Trial (MRC UKALL XII/ ECOG E2993). Blood. 2008;111:1827-1833.

23.  Dreger P, Dohner H, Ritgen M, et al. Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial. Blood. 2010;116:2438-2447.

24.  Doney K, Leisenring W, Storb R, et al. Primary treatment of acquired aplastic anemia: outcomes with bone marrow transplantation and immunosuppressive therapy. Seattle Bone Marrow Transplant Team. Ann Intern Med. 1997;126: 107-115.

25.  Wingard JR, Carter SL, Walsh TJ, et al. Randomized, double-blind trial of fluconazole versus voriconazole for prevention of invasive fungal infection after allogeneic hematopoietic cell transplantation. Blood. 2010;116:5111-5118.

26.  Corbacioglu S, Cesaro S, Faraci M, et al. Defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial. Lancet. 2012;379:1301-1309.

27.  Vogelsang GB, Lee L, Bensen-Kennedy DM. Pathogenesis and treatment of graft-versus-host disease after bone marrow transplant. Annu Rev Med. 2003;54:29-52.

28.  Srinivasan R, Chakrabarti S, Walsh T, et al. Improved survival in steroid-refractory acute graft versus host disease after non-myeloablative allogeneic transplantation using a daclizumab-based strategy with comprehensive infection prophylaxis. Br J Haematol. 2004;124:777-786.

29.  Shulman HM, Kleiner D, Lee SJ, et al. Histopathologic diagnosis of chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. Pathology Working Group Report. Biol Blood Marrow Transplant. 2006;12:31-47.

30.  Leather HL, Wingard JR. Infections following hematopoietic stem cell transplantation. Infect Dis Clin North Am. 2001;15:483-520.

31.  Anagnostopoulos A, Giralt S. Critical review on non-myeloablative stem cell transplantation (NST). Crit Rev Oncol Hematol. 2002;44:175-190.

32.  Storb RF, Champlin R, Riddell SR, et al. Non-myeloablative transplants for malignant disease. Hematology Am Soc Hematol Educ Program. 2001:375-391.

33.  Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med. 2000;343:750-758.

34.  Childs RW, Srinivasan R. Allogeneic hematopoietic cell transplantation for solid tumors. In: Blume KG, Forman S, Appelbaum FR, eds. Thomas’ Hematopoitic Cell Transplantation. 4th ed. Malden, MA: Blackwell Science; 2009;958-969.

35.  Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097-2100.

36.  Wagner JE, Kernan NA, Steinbuch M, et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet. 1995;346:214-219.

37.  Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med. 2004; 351:2265-2275.

38.  Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med. 2004;351:2276-2285.

39.  Brunstein CG, Gutman JA, Weisdorf DJ, et al. Allogeneic hematopoietic cell transplantation for hematologic malignancy: relative risks and benefits of double umbilical cord blood. Blood. 2010;116:4693-4699.

40.  Brunstein CG, Miller JS, Cao Q, et al. Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics. Blood. 2011;117:1061-1070.

19

Thrombocytopenia

Patrick F. Fogarty and Cynthia E. Dunbar

PLATELET BIOLOGY

  Platelets are anucleate blood cells that participate in primary hemostasis, the formation of a platelet plug at sites of vascular injury.

  Platelets are produced from megakaryocytes, multinucleate hematopoietic cells located in the bone marrow. Cytokines such as thrombopoietin are necessary for normal platelet maturation and release.

  Once released into the circulation, the average life span of a platelet is 7 to 10 days. Platelets are removed from circulation when they are activated and utilized at sites of vascular injury or as they become senescent.

  At any given time, up to one-third of the platelet mass is stored in the spleen, providing a reserve of platelets that may be released during periods of physiologic stress.

  The normal platelet concentration in the blood is 150,000 to 400,000/μL as measured in most hospital laboratories.

ETIOLOGY AND CLINICAL FEATURES OF THROMBOCYTOPENIA

Thrombocytopenia may occur due to

  Decreased production of platelets

  Increased consumption of platelets

  Increased sequestration of platelets

  Any combination of these mechanisms (Table 19.1)

Regardless of the cause of thrombocytopenia, “platelet-type” bleeding is typically mucocutaneous and is characterized by petechiae, ecchymoses, epistaxis, and gingival and conjunctival hemorrhages. Less commonly, severe thrombocytopenia may lead to gastrointestinal, genitourinary, or central nervous system bleeding.

Spontaneous bleeding or bruising normally does not occur until the platelet count has fallen below 10,000 to 20,000/μL. The rate of decline of the platelet count may also influence the likelihood of unprovoked bleeding, presumably due to compensatory processes in remaining platelets that may occur over time with persistent thrombocytopenia. Patients with dysfunctional platelets may bleed with higher platelet counts. Patients with thrombocytopenia and platelet counts greater than 20,000 to 30,000/μL without bleeding usually do not require immediate treatment to increase the platelet count. A platelet count of 80,000 to 100,000/μL is generally adequate for hemostasis during most invasive procedures, including surgery (Table 19.2).

Table 19.1  Causes of Thrombocytopenia

Disorders Characterized by Decreased Production of Platelets

Bone marrow failure syndromes

Congenital (amegakaryocytic thrombocytopenia, Fanconi anemia, dyskeratosis congenita, Schwachmann-Diamond syndrome, thrombocytopenia–absent radii syndrome,Wiskott-Aldrich syndrome) Acquired (aplastic anemia, amegakaryocytic thrombocytopenia)

Myelodysplasia

Marrow infiltration (neoplastic, infectious)

Chemotherapy-induced

Irradiation-induced

Cyclic thrombocytopenia (some cases)

Immune thrombocytopenia

Folate, B12, or iron (advanced cases) deficiency

Ethanolism

Disorders or Conditions Characterized by Increased Clearance of Platelets

Immune thrombocytopenia

Heparin-induced thrombocytopenia

Thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome

Disseminated intravascular coagulation (HELLP syndrome)

Posttransfusion purpura

Neonatal alloimmune thrombocytopenia

Von Willebrand disease, type IIB

Cyclic thrombocytopenia (most cases)

Mechanical destruction (aortic valvular dysfunction; extracorporeal bypass)

Disorders Characterized by Increased Sequestration of Platelets

Hypersplenism (see Table 19.5)

Other Conditions

Artifactual (pseudothrombocytopenia)

Drug-induced (see Table 19.6)

Gestational thrombocytopenia

HIV-associated thrombocytopenia

Infection- and sepsis-related thrombocytopenia

Hemophagocytosis

Qualitative platelet disorder-related (Bernard-Soulier disease, gray platelet syndrome, May-Hegglin anomaly)

HIV, human immunodeficiency virus.

DISORDERS CHARACTERIZED BY DECREASED PRODUCTION OF PLATELETS

Bone Marrow Failure

  Congenital disorders, such as Fanconi anemia or dyskeratosis congenita, typically present early in life; these syndromes often cause depression of other blood cell lineages (i.e., white cells and red cells) in addition to the platelet count.

  Other congenital disorders such as congenital amegakaryocytic thrombocytopenia and the thrombocytopenia with absent radius (TAR) syndrome are characterized by isolated thrombocytopenia.

  Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder featuring thrombocytopenia, eczema, and immunodeficiency. Thrombocytopenia may improve with splenectomy, but allogeneic hematopoietic stem cell transplantation alone is potentially curative for the disorder.

  Adult patients with acquired amegakaryocytic thrombocytopenia initially may appear to have immune thrombocytopenia (ITP) (see ensuing section), but the bone marrow reveals markedly reduced or absent megakaryocytes. The disorder may progress to aplastic anemia.

  Patients with acquired aplastic anemia rarely present with isolated thrombocytopenia. Marked bone marrow hypocellularity with decreased megakaryocytes would suggest this diagnosis (see Chapter 6).

Myelodysplasia

  Mild thrombocytopenia with macrocytosis, with or without anemia or neutropenia, in an older individual is a typical presentation of myelodysplasia (MDS). Isolated severe thrombocytopenia (less than 20,000/μL) without any other blood count abnormalities is not typical.

  Bone marrow aspirate and blood smear may show megakaryocytic dysplasia (including small and mononuclear “micromegakaryocyte” forms) and maturation abnormalities of erythrocytic and granulocytic precursor cells. Concurrent cytogenetic abnormalities may be present (see Chapter 7).

  For treatment of thrombocytopenia due to MDS, see Chapter 7. Note that thrombopoietin receptor agonists may be contraindicated in MDS due to a potential acceleration in transformation to acute leukemia.

Marrow Infiltration

Infiltration of the bone marrow by malignant cells may cause thrombocytopenia, but usually only after massive replacement of the marrow space by tumor cells or immature hematologic precursor cells has occurred. Examination of the bone marrow biopsy and aspirate is required.

  The acute and chronic leukemias, myeloma, and lymphoma are the most common tumors resulting in cytopenias due to neoplastic marrow infiltration and direct suppression of normal hematopoiesis with some tumor types.

  Certain infections (such as tuberculosis and ehrlichiosis) can result in formation of granulomas in the bone marrow that supplant the normal marrow architecture.

  Effective treatment of the underlying condition should be expected to restore a low platelet count to the normal range, but platelet transfusions may be required initially if bleeding is present or invasive procedures are planned (Table 19.2).

Irradiation and Chemotherapy

  Irradiation and/or myelotoxic chemotherapy induce thrombocytopenia via direct toxicity to megakaryocytes or more immature hematopoietic stem and progenitor cells. The degree and duration of thrombocytopenia depends on the intensity and the type of the myelotoxic regimen.

  Chemotherapy-induced thrombocytopenia typically resolves more slowly than does neutropenia and/or anemia, especially following repetitive cycles of treatment.

  Platelet transfusions may be given if required. Trials of novel platelet growth factors for thrombocytopenia due to specific chemotherapeutic regimens are ongoing.

Cyclic Thrombocytopenia

This exceedingly rare disorder is characterized by episodes of thrombocytopenia that occur cyclically, typically every 3 to 6 weeks. The thrombocytopenia is frequently severe and may be associated with significant bleeding. Treatment with oral contraceptives (female patients), androgens, immunosuppressive agents (such as azathioprine), or thrombopoietic growth factor has led to responses in some cases.

Nutritional Deficiencies

Folate deficiency (commonly associated with alcoholism) and vitamin B₁₂ deficiency may cause decreased megakaryocytopoiesis and thrombocytopenia, usually in conjunction with anemia. In contrast, thrombocytosis is typical in cases of significant iron deficiency; in very severe iron deficiency, however, thrombocytopenia may also occur. In any of these situations replacement of the deficient vitamin or mineral corrects the thrombocytopenia.

DISORDERS CHARACTERIZED BY INCREASED CLEARANCE OF PLATELETS

Immune Thrombocytopenia

ITP is an acquired autoimmune disorder of increased platelet destruction and decreased platelet production, causing thrombocytopenia that may lead to bleeding.

  Epidemiology. The annual incidence of ITP in adults has been estimated to be about 2 to 4 cases per 100,000 persons and increases with age. ¹

  Pathophysiology.² Pathogenic antiplatelet antibodies can be identified in approximately 75% of patients with ITP and are directed against the platelet glycoprotein complexes IIb/IIIa and/or Ib/IX. The antibody-coated platelets are cleared by reticuloendothelial macrophages in the liver and/or spleen, decreasing the platelet life span from approximately 7 days to less than 2 days. Platelet production also is impaired in ITP, possibly because of antiplatelet antibody binding to bone marrow megakaryocytes. Primary adult-onset ITP is generally idiopathic and becomes chronic, whereas secondary ITP occurs in association with disorders of lymphoproliferation (lymphoma or chronic lymphocytic leukemia (CLL)) or immune dysregulation (systemic lupus erythematosus, human immunodeficiency virus (HIV) infection).³ In contrast, ITP in children often follows a viral infection and frequently resolves spontaneously without specific therapy.

  Presentation. Typically, new-onset severe ITP (platelets <30,000/μL) manifests with petechial bruising and bleeding from mucous membranes, including conjunctival hemorrhages, gingival bleeding, and epistaxis. Milder disease (platelet count >50,000/μL) often presents as an asymptomatically low platelet count on routine blood work.

  Diagnosis. ITP is a diagnosis of exclusion. New-onset, isolated thrombocytopenia with no other readily apparent cause (including medication-related) in an otherwise asymptomatic adult generally may be regarded as sufficient for the diagnosis of ITP and subsequent initiation of medical therapies (if appropriate based on the degree of thrombocytopenia; see below). ⁴

  The presence of other cytopenias, age greater than 60 years, or failure of primary therapy (corticosteroids for a trial of 1 week) should prompt bone marrow examination. The presence of abnormal or decreased numbers of megakaryocytes or abnormal marrow cellularity should redirect the diagnostic evaluation away from ITP.

  All patients should be screened for hepatitis B and hepatitis C virus (HBC/HCV) and HIV infection (see below) and undergo evaluation of the blood smear, direct antiglobulin test (DAT) (Coombs test), and blood type (for Rh status).

  Helicobacter pylori testing, antiphospholipid antibodies, and antinuclear antibodies may be useful in selected patients.

  Treatment. Individuals with mild or moderate thrombocytopenia (platelets >30,000/μL) who do not require a higher platelet count for surgery or active bleeding should not receive treatment. Rather, they may be observed at regular intervals for disease progression. Adults with platelet counts of <20,000 to 30,000/μL or those with significant bleeding generally should be treated.⁴

  Initial treatment (Table 19.3) generally consists of a short course of corticosteroids (prednisone 1 mg/kg/day for 7–10 days with subsequent rapid tapering or “pulse” dexamethasone cycles of 40 mg daily for 4 days). A significant increase in the platelet count should be seen within 3 to 7 days. In the event of a platelet response, prednisone may be tapered rapidly to a dose of 20 mg/day; thereafter, tapering should proceed more slowly (by dose decrements of no more than 5 mg/adjustment, no more frequently than once every 2 to 3 weeks). Dexamethasone cycles may be given every other week for four cycles or monthly for up to 6 months.

  For patients with serious active bleeding and/or very severe thrombocytopenia (<5,000–10,000/ μL), intravenous immune globulin (IVIg; 1 gm/kg/day for 2 days) or anti-D (WinRho®, 75 μg/kg/ dose; appropriate for non-splenectomized, Rh blood type-positive, non-anemic patients only) can be administered in addition to corticosteroids in order to decrease clearance of antibody-coated platelets. Responses are generally seen within 3 to 5 days of IVIg or anti-D administration.

  Platelet transfusions may be administered if the presentation is complicated by serious (intracranial) bleeding. Transfused platelets are expected to be cleared very rapidly in the presence of antiplatelet antibodies, but they may improve hemostasis temporarily.

  Immunize against encapsulated bacterial organisms (pneumococcus, Haemophilus influenzae, meningococcus) before prolonged immunosuppressive therapy in preparation for splenectomy if required at a later time point.

  Second-Line Treatment (Table 19.3). Despite a high initial response rate (60%–75%), the majority of adults with ITP experience relapse and develop chronic thrombocytopenia once initial treatments are reduced or discontinued. Treatment is appropriate for patients with platelet counts <30,000/μL or clinically significant bleeding.⁴ The selection of specific therapies should take into account patient preference; some individuals prefer sequential medical therapies prior to undergoing splenectomy, but splenectomy may be preferable in cases of very severe thrombocytopenia associated with bleeding because of a typically rapid postoperative increase in the platelet count in most responding patients.

  IVIg and anti-D (see above) typically must be readministered every 2 to 3 weeks in most instances.

  The thrombopoietin receptor agonists eltrombopag (starting dose 50 mg orally daily; 25 mg daily in individuals of Asian descent) or romiplostim (starting dose 1 μg/kg SC weekly) produce platelet responses (≥50,000/μL) in approximately 70% of patients with chronic ITP, and they generally are well tolerated.5,6 Potential complications include reticulin deposition in the bone marrow, thrombocytosis, and thrombosis.

  The monoclonal anti-B cell antibody rituximab (anti-cd20), given at a dose of 375 mg/m² weekly for 4 weeks, induces initial and long-term responses in about 50% and 25%, respectively, of adults with severe, chronic ITP. ⁷

  Splenectomy (preferably laparoscopic) yields an immediate response rate of 70% to 75% and durable response rates of 60% to 70%. All patients must receive immunization against encapsulated bacterial organisms (pneumococcus, H. influenzae, meningococcus) several weeks prior to splenectomy if possible. ⁴

  Secondary ITP. A variety of autoimmune, infectious, inflammatory, or malignant conditions may underlie a presentation of ITP.³ Treatment of the underlying predisposing condition may be required in some cases, in addition to management of the thrombocytopenia using accepted interventions (Table 19.3)

  Pregnancy-Associated ITP. Pregnant women with platelet counts <30,000/μL during the second or third trimester, or with platelet counts <10,000/μL, or bleeding in any trimester, should be treated. Intermittent infusions of IVIg or moderate-dose oral prednisone (commonly given on an every-otherday schedule) are standard. Splenectomy during the first or second trimester may be considered for women whose ITP has failed treatment with IVIg and corticosteroids and who have platelet counts <10,000/μL with associated bleeding. Platelets may be administered prophylactically prior to cesarean section in women who have platelet counts <10,000/μL or mucocutaneous bleeding near the time of delivery. A platelet count of >50,000/μL generally is regarded as adequate prior to cesarean section or vaginal delivery.

Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that results in platelet activation and clearance. Although the disorder produces thrombocytopenia, patients with HIT paradoxically are at high risk for thrombosis. If HIT is suspected, all forms of heparin should be discontinued immediately, and, if appropriate, alternative anticoagulation administered.⁸

  Epidemiology. HIT occurs in approximately 3% and <1% of patients who are exposed to unfractionated heparin or low molecular-weight heparin, respectively. As many as half of these individuals will develop thrombosis. ⁸

  Pathophysiology. The pathogenesis of HIT begins with binding of the heparin molecule to platelet factor 4 (PF4), a platelet alpha granule chemokine. The heparin-PF4 complex stimulates formation of an IgG antibody (HIT antibody) that binds both to the heparin-PF4 complex (via its Fab portion) and to platelet Fc receptors (via its Fc portion). Binding of the HIT antibody to platelets activates them, resulting in release of procoagulant microparticles, platelet clearance, and subsequent thrombocytopenia. PF4 also binds to polysaccharides (heparan sulfate) on the endothelial surface; recognition of these PF4-polysaccharide complexes by HIT antibodies may lead to endothelial damage, expression of tissue factor, and a prothrombotic state.

  Presentation. The typical presentation of HIT involves a hospitalized patient who develops thrombocytopenia within 5 to 10 days of receiving heparin.

  A decline of 50% or more from the baseline value in a heparin-treated patient may signify HIT. Platelet counts generally do not fall below 20,000/μL.

  Spontaneous bleeding (including petechiae) is not typical.

  Venous (upper or lower limb, dural sinus) or arterial (lower limb, CVA, MI, other locations) thromboses may accompany detection of thrombocytopenia and occur in up to 50% of untreated cases. In a minority of patients with HIT, thrombosis is the presenting clinical sign. The risk of HIT-related thrombosis persists for at least 30 days after the discontinuation of heparin if anticoagulation is not administered.

  Other presentations of HIT are possible:

  Rapid-onset HIT occurs within 1 to 3 days of re-exposure to heparin in patients who have received heparin usually within the prior 30 days and have preexisting HIT antibodies. An acute systemic reaction characterized by fever, chills, hypotension, and/or cardiovascular compromise immediately after re-exposure to heparin is typical.

  Delayed-onset HIT describes new thrombocytopenia and venous or arterial thrombosis that occurs up to 14 days after completion of an uneventful course of heparin therapy. Laboratory markers of disseminated intravascular coagulation (DIC) may be positive. Thrombocytopenia and thrombosis typically worsen if heparin is administered.

  Diagnosis. Strictly, the diagnosis of HIT requires both an appropriate clinical context and confirmatory laboratory testing (e.g., demonstration of HIT antibodies). Due to the limited immediate availability of HIT-specific laboratory assays, however, any patient in whom the clinical probability for the disorder is intermediate or high should be managed HIT, even if the results of diagnostic tests are pending or immediately unavailable.

  Clinical probability. Factors that make a diagnosis of HIT more likely are reviewed above. A variety of clinical prediction models, such as the 4Ts,⁹ that have been developed to assist in determination of pretest probability have not undergone external validation and may overestimate diagnoses.⁸ Review of the hospital chart (including nursing notes) may be necessary to document the extent and duration of exposure to heparin, especially if its use was transient (heparin flushes) or covert (heparin-impregnated catheters).

  Laboratory diagnosis. All patients with suspected HIT ideally should undergo testing with two types of assays, immunologic and functional. The enzyme-linked immunosorbent assay (ELISA) for PF4-heparin-associated antibody (immunologic test) has a sensitivity of >90% but a limited specificity. Platelet activation assays (functional test) measure activation of donor platelets in the presence of the patient’s serum and a high and low concentration of heparin. These assays are much more specific for HIT than the immunologic test, but are less widely available and have a slow turnaround time (days). A positive result on both tests or the immunologic assay alone indicates high and intermediate, respectively, probability for HIT.

  Treatment. All forms of heparin, including low-molecular weight preparations, must be discontinued immediately. In patients in whom laboratory testing for HIT eventually proves negative or in whom an alternative explanation for thrombocytopenia has been found, heparin may be subsequently restarted.

Doppler ultrasound of the lower extremities should be performed to rule out subclinical deep vein thrombosis.

Because (1) HIT patients rarely bleed and (2) transfused platelets may worsen the already increased thrombotic risk by providing substrate for HIT antibodies, platelet transfusions are rarely indicated.

Warfarin is contraindicated as initial treatment of clinically proven or suspected HIT, due to its propensity to exacerbate hypercoagulability by reduction of plasma levels of proteins C and S (see Chapter 23).

Because of the high rate of serious thrombosis among HIT patients, an alternative anticoagulant such as a direct thrombin inhibitor (DTI; Table 19.4; see also Chapter 23) is required in all cases of suspected (intermediate or high clinical probability) or proven HIT.

The alternative anticoagulant should be continued at least until significant recovery of the platelet count has occurred or for approximately 5 days, whichever is longer.

  Longer-term anticoagulation. Due to the extended risk of thrombosis up to 1 month following a diagnosis of HIT, patients without concurrent thrombosis require at least 30 days of anticoagulation. Warfarin therapy is appropriate in most patients, and the DTI should be continued until therapeutic anticoagulation with warfarin has been achieved. (Because Argatroban raises the INR, a special approach is required for transitioning from this DTI to warfarin; see Chapter 23.) Patients with HIT and thrombosis should receive anticoagulation with warfarin for duration of 3 to 6 months at an INR of 2.0 to 3.0.

  Thrombolysis/thromboembolectomy. Low-dose or very low-dose thrombolytic agents may be indicated in acute limb ischemia or life-threatening pulmonary embolism caused by HIT-associated thrombi. Surgical removal of large-vessel arterial thrombi may be required if the limb is threatened and other treatments have failed. Patients managed by either medical or surgical means require concomitant use of an alternative anticoagulant, regardless of the degree of thrombocytopenia.

  Retreatment with heparin. HIT antibodies probably do not persist beyond 100 days from the initial episode of HIT,⁸ in which case very transient use of heparin for cardiac or vascular surgery at least 100 days beyond an initial episode may be considered safe, if immunologic (ELISA) and functional (SRA) tests are both negative. If the immunologic test is positive but the functional test is negative, either the surgery can be delayed until the functional test becomes negative or a DTI can be used.

Thrombotic Microangiopathies

The thrombotic microangiopathies (TMAs) comprise thrombotic thrombocytopenic purpura (TTP) and the related disorder, the hemolytic-uremic syndrome (HUS), and feature microangiopathic hemolytic anemia (MAHA) due to the formation of platelet-rich thrombi in the arterial and capillary microvasculature and thrombocytopenia. Acquired (or “spontaneous” or “classical”) and congenital forms of TTP are recognized, and endemic (or “typical”) and atypical forms of HUS may occur (Table 19.5). In addition, some forms of TMA have been recognized in association with surgery, pregnancy, exposure to certain medical drugs, and bone marrow transplantation. Importantly, early aggressive intervention with plasma exchange is crucial in cases of classical TTP due to its extremely high mortality rate.

  Epidemiology. The incidence of classical TTP is approximately 3 to 4 cases/100,000 persons; there is a slight female predominance. Most cases of endemic or “typical” HUS occur in young children and are related to infection with enteropathogenic bacteria. TMA occurs at an increased rate during pregnancy and in the peri-partum period.

  Pathophysiology. The TMAs are thought to arise from factors that directly or indirectly cause platelet aggregation and/or endothelial cell damage, leading to the formation of microvascular thrombi and ischemia in involved organs. These factors include toxins, cytokines, drugs, or deficiencies in the function of the von Willebrand factor cleaving protease (VWFCP or ADAMTS-13). Red cells are sheared as they negotiate thrombotic obstructions and fibrin strands in the microvasculature, leading to hemolytic anemia. Consumption of platelets results in thrombocytopenia and bleeding.

  In classical TTP, an acquired deficiency of VWFCP results from production of an autoantibody against the VWFCP¹⁰ leading to an accumulation of ultra large VWF (ULVWF) in the plasma. The VWFCP, or ADAMTS-13, is a metalloproteinase whose normal function is to cleave newly synthesized, ULVWF multimers released in the plasma into multimers of smaller size. ULVWF multimers bind to platelets more avidly than smaller VWF molecules and may incite platelet aggregation.

  Patients with congenital TTP have decreased activity of the VWFCP due to an inherited deficiency.

  In many cases of endemic HUS, Shiga toxin from Escherichia coli (especially type 0157:H7) is thought to promote platelet aggregation by damaging endothelial cells or by other mechanisms.

  Patients with atypical HUS may have genetic defects in proteins that regulate complement activity, such as factor H. ¹¹

  Pregnancy-associated TTP-HUS may stem from decreased levels of the VWFCP that naturally occur in the second and third trimesters; in some cases an antibody to VWFCP is present. ¹²

  Drugs such as cyclosporine, quinine, ticlopidine, clopidogrel, mitomycin C, and bleomycin may cause TMA by endothelial cell injury and/or pro-aggregatory effects on platelets. Antibodies inhibiting the VWFCP have been described in patients who received some of these medications.TMA in the setting of cancer, hematopoietic stem cell transplantation, or HIV infection has not been linked to abnormalities of VWFCP, but effects on endothelial cells or platelets may be responsible.

  Presentation. All patients with TMA have MAHA. Varying degrees of neurologic impairment (more typical of classical TTP) or symptoms related to renal failure (predominant in HUS) may also be present (Table 19.4). MAHA, thrombocytopenia, fever, renal insufficiency, and neurologic system abnormalities (the classic pentad of TMA) occur in only about 25% of patients with TTP. Most patients with typical HUS have a recent or current diarrheal illness.

  In adults, TTP and HUS are often difficult to distinguish due to the overlap of symptoms, but if renal dysfunction predominates, the syndrome usually is classified as HUS.

  Manifestations of renal insufficiency may include elevated creatinine, azotemia, proteinuria, hematuria, and/or oliguria.

  Neurologic impairment (from microthrombi in the cerebral vasculature) occurs in about 75% and 30% of patients with TTP and HUS, respectively, and includes headache, somnolence, confusion, seizures, and (less commonly) paresis and coma.

  Diagnosis. The presence of new-onset MAHA and thrombocytopenia (and/or renal failure) in the absence of any other plausible explanation will suffice for the diagnosis.

  MAHA is essential to the diagnosis of TTP-HUS and is defined by anemia with positive markers of intravascular hemolysis (elevated LDH, elevated indirect bilirubin, decreased haptoglobin, and reticulocytosis) and negative DAT (Coombs test). The blood smear shows >3 schistocytes per high-power microscopic field, although fewer schistocytes may be present if the disorder is caught early.

  Depending on the type of TMA, thrombocytopenia may be variable.

  Clinical features such as an antecedent bloody diarrheal illness and/or renal insufficiency (more typically associated with HUS), or neurologic abnormalities with or without fever (more typically associated with TTP), or recent or current pregnancy, or treatment with associated drugs, or cancer, or recent hematopoietic stem cell transplantation are diagnostically corroborative.

  Stool culture for E. coli 0157:H7 or assays for antibody against Shiga or Shiga-like toxins, or against specific bacterial lipopolysaccharide may be positive in patients with endemic HUS.

  Assays for VWFCP activity typically are abnormal in congenital and classical TTP; activity is less than 5% in classical TTP.

  The prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen are within the normal range in TMA.

  Treatment. Without plasma exchange, the mortality rate of classical TTP exceeds 90%. Plasma exchange must be instituted expeditiously. An important exception is children or adults with endemic (E. coli diarrhea–associated) HUS, who generally recover with supportive care within 3 weeks, without plasma exchange.

  Plasma exchange should begin once appropriate vascular access has been obtained. It should be performed once daily until the LDH has normalized and the platelet count has returned to the preexisting baseline (if known) for at least 2 or 3 days. Failure to respond to once-daily therapy requires twice-daily treatments; once the LDH and platelet count indicate response, once-daily treatments can be resumed until these parameters have normalized for 2 or 3 days. Whether treatments then may be discontinued or continued for a number of weeks in a tapering fashion is controversial, but many clinicians prefer to gradually increase the interval between treatments instead of abrupt termination of therapy.

  Platelet transfusions generally are contraindicated in the treatment of TMAs due to possible propagation or new formation of platelet-rich microthrombi. If CT- or MRI-documented intracranial bleeding or other life-threatening bleeding is present, however, platelets may be transfused slowly, ideally after plasma exchange is initiated.

  Packed red cells may be transfused commensurate with the pace of the MAHA and degree of bleeding.

  In the event that plasma exchange is unavoidably delayed, infusion of fresh frozen plasma (FFP) may be helpful as a temporizing measure. Plasma infusion as a sole treatment of TMA, however, generally is regarded as substandard due to (1) the possible role of plasma exchange in removing offending drugs, cytokines, bacterial proteins, ULVWF multimers, or antibodies to the VWFCP and (2) the volume overload frequently incurred when the required large volumes of FFP are infused. An exception is familial relapsing TMA, wherein a congenital deficiency in the VWFCP can be corrected merely by infusion of smaller volumes of plasma.

  Refractory and relapsing TMA. If remission is not achieved with aggressive plasma exchange, second-line treatments should be considered. These include the addition of steroids or IVIG to plasma exchange, vincristine, cyclophosphamide, cyclosporine (in select cases of sporadic TTP), and splenectomy. The monoclonal antibody rituximab has also achieved responses in a limited number of refractory cases.¹³ Up to a third of patients with classical TMA relapse after discontinuation of plasma exchange. In these cases, plasma exchange should be reinitiated according to the guidelines above, and, if ineffective, immunosuppressive treatments should be considered. Selected patients with atypical HUS may respond to infusion of the monoclonal antibody anti-C5 inhibitor eculizumab. ¹⁴

  Hemodialysis. More than half of all patients with HUS (and a minority with TTP) require hemodialysis. Approximately half of these patients will attain durable restoration of renal function while 25% develop chronic renal failure. The remainder experience variable degrees of permanent renal insufficiency.

Disseminated Intravascular Coagulation

Thrombocytopenia in DIC occurs as a result of uncontrolled activation of coagulation in the circulation. Platelets participate in these reactions, leading to their consumption. If bleeding is present, platelet transfusions may be administered to reach a platelet count target of 20,000 to 30,000/μL (most cases) or >50,000/μL (if there is intracranial or life-threatening hemorrhage). Thrombocytopenia and the other clinical and laboratory manifestations of DIC are expected to resolve with effective treatment of the underlying, inciting disorder. (For a full discussion of DIC, see Chapter 21.)

Posttransfusion Purpura

Posttransfusion purpura (PTP) is characterized by the unexplained, sudden appearance of thrombocytopenia in an otherwise asymptomatic individual who recently has received a blood transfusion (red cells, platelets, or plasma) within 1 week prior to development of thrombocytopenia. The precipitating events are unknown, but more than 90% of individuals with PTP display antibodies against the human platelet antigen PlA1.

  Most patients with PTP are postmenopausal females who are either multiparous or who have received prior transfusions; they commonly present with severe thrombocytopenia and bleeding.

  If untreated, thrombocytopenia typically persists for up to 2 to 3 weeks, concurrent with a mortality rate of 10% from bleeding; hence, IVIg (1 gm/kg/day for 2 days) should be administered as soon as the diagnosis is suspected. Most patients will respond, but in the case of relapse, IVIg may be administered as a second course. Plasma exchange, adjunctive corticosteroids, and splenectomy are alternative treatments for refractory cases. Because transfused platelets are thought to be as susceptible to binding by antigen-antibody complexes as the patient’s own platelets, platelet transfusion is generally not performed unless severe bleeding is present; in this case, HLA antigen-matched platelets are preferred. Future transfusions should be administered judiciously, with washed or PlA1-negative blood products.

Neonatal Alloimmune Thrombocytopenia

  Neonatal alloimmune thrombocytopenia (NAIT) is a cause of severe thrombocytopenia in neonates. It occurs when fetal platelet antigens cross the placenta and trigger formation of maternal alloantibodies that can then enter the fetal circulation, bind platelets, and induce thrombocytopenia. Antibodies commonly have specificity for human platelet antigen [HPA]-1a, also known as PlA1. The presence of certain maternal platelet phenotypes (such as the homozygous HPA-1b state) appears to influence the risk of the disorder, especially if the fetus inherits a different, paternal platelet phenotype.

  The thrombocytopenia is typically severe and a high prevalence of intracranial hemorrhage (ICH) during or following delivery is observed, resulting in neonatal death in 5% of cases of NAIT. Thrombocytopenia typically resolves by 2 to 3 weeks of age.

  IVIg, with or without corticosteroids, is recommended for any neonate with platelet counts <20,000 to 25,000/μL. Random donor or (ideally) irradiated maternally antigen-matched platelets are often administered in cases of ICH. Subsequent pregnancies are regarded as high risk for recurrent NAIT.

Von Willebrand Disease,Type 2B

This type of von Willebrand disease (VWD) is characterized by an abnormal von Willebrand factor (VWF) that has increased affinity for its platelet receptor, glycoprotein Ib. Due to the bridging action of VWF, platelets aggregate in vivo and are cleared, typically resulting in a mild thrombocytopenia. VWD is discussed in detail in Chapter 21.

Extracorporeal Circulation-Related Thrombocytopenia

Passage of the blood for prolonged periods outside the body in an artificial circuit (such as used for cardiac bypass surgery) typically results in platelet activation and clearance. Thrombocytopenia generally is not severe. Other common causes of thrombocytopenia in the postsurgical patient (such as HIT, DIC, and sepsis-related and drug-induced thrombocytopenia) concomitantly must be considered.

DISORDERS CHARACTERIZED BY INCREASED SEQUESTRATION OF PLATELETS

  Hypersplenism results in sequestration of blood cells (including platelets) in an enlarged or abnormal spleen. Mild to moderate thrombocytopenia is most commonly observed, but if the bulk of the platelet mass is contained within a massively enlarged spleen, thrombocytopenia can be severe.

  Splenomegaly with hypersplenism is almost always an acquired condition, and there are many possible underlying disorders (Table 19.6).

  If adequate production of platelets can be documented and significant splenomegaly with thrombocytopenia is present, splenectomy may be considered in some cases. Splenic embolization and splenic irradiation are alternatives to removal of the spleen that generally do not result in maximal platelet responses. They may be considered, however, in patients with significant hypersplenism and disorders such as CLL or lymphoma who cannot tolerate surgery.

OTHER CAUSES OF THROMBOCYTOPENIA

Pseudothrombocytopenia

For reasons that are unclear, the calcium chelation induced by the anticoagulant ethylenediaminetetraacetic acid (EDTA) (present in blood-collecting tubes) causes changes on the platelet membranes of certain patients that expose cryptic antigens to which preformed, otherwise non-pathogenic agglutinating antibodies may bind; the result is artifactual platelet clumping. Typically, automated cell counters (such as those that are present in most hospital laboratories) will report a falsely low platelet count; examination of the blood smear reveals platelet clumps. Normalization of the platelet count upon automated determination from a blood specimen collected in citrate anticoagulant and /or disappearance of platelet clumps on a blood smear obtained from a finger stick source yields a correct assessment of platelet number and confirms the presence of this benign phenomenon.

Table 19.6  Selected Causes of Splenomegaly

Lymphoproliferation

Lymphoma

Chronic lymphocytic leukemia

Collagen vascular disease (Felty syndrome, systemic lupus)

Autoimmune lymphoproliferative disorder

Myeloproliferation

Myeloid leukemia

Polycythemia vera

Essential thrombocythemia

Inborn Errors of Metabolism

Gaucher disease

Niemann-Pick disease

Congestion

Cirrhosis

Heart failure

Hemolysis

Hereditary spherocytosis

Paroxysmal nocturnal hemoglobinuria

Thalassemia

Infection

Viral (CMV, EBV, hepatitis)

Parasitic (malaria, babesiosis)

Immunodeficiency

Common variable immunodeficiency

Drug-Induced Thrombocytopenia

  By definition, drug-induced thrombocytopenia develops after initiation of a given drug, resolves when the offending medication is discontinued and may recur if the agent is reintroduced.¹⁵ The mechanisms by which many drugs may lead to a low platelet count, however, have not been elucidated.

  Chemotherapeutic agents are clearly linked to decreased platelet production.

  Quinine purpura is a type of drug-induced immune thrombocytopenia (DITP), in which there is antibody-mediated destruction of platelets after exposure to a given drug. Quinine is thought to induce a conformational change in the platelet membrane allowing exposure of an otherwise cryptic antigen; circulating antibodies then bind the antigen, but only in the presence of the drug. Patients with DITP present with severe thrombocytopenia (<20,000/μL) and mucocutaneous bleeding, including purpura and ecchymoses. Thrombocytopenia should resolve within days to weeks of discontinuing the agent. In cases of severe bleeding, IVIg and platelet transfusions appear to be more effective than steroids in inducing responses.

  Other medications commonly associated with thrombocytopenia are listed in Table 19.7.

Gestational Thrombocytopenia

The blood volume increases by as much as 40% to 45% over baseline during pregnancy, causing a progressive hemodilution. Cytopenias result, though production of blood cells is normal or increased. Approximately 10% and less than 1% of pregnant women experience platelet counts <100,000/μL and <50,000/μL by the third trimester, respectively; the incidence of ITP is thought to be even lower. Severe thrombocytopenia in pregnancy (<50,000/μL) should prompt investigation to rule out a preexisting condition, preeclampsia, or a pregnancy-related TMA; if negative, the etiology may be presumed to be ITP and treated accordingly (see Immune Thrombocytopenia on page 272).

Table 19.7  Drugs Associated with Thrombocytopenia

Antimicrobials

Amphotericin

Ampicillin

Isoniazid

Rifampin

Methicillin

Piperacillin

Sulfisoxazole

Trimethoprim-sulfamethoxazole

Linezolid

Vancomycin

Antiplatelet Agents

Anagrelide

Abciximab

Eptifibatide

Ticlopidine

Tirofiban

Analgesics/Anti-Inflammatory Agents

Acetominophen

Diclofenac

Ibuprofen

Sulindac

H₂ Blockers

Cimetidine

Ranitidine

Cardiovascular Agents

Amiodarone

Captopril

Digoxin

Hydrochlorothiazide

Procainamide

Quinidine

Neuropsychiatric Agents

Carbamazepine

Chlorpromazine

Diazepam

Haloperidol

Lithium

Methyldopa

Phenytoin

Other

Gold

Heparin

Mycophenolate mofetil

Interferon-α

Quinine

Most chemotherapeutic drugs

Data from DeLoughery T. Hemorrhagic and thrombotic disorders in the intensive care setting. In: Kitchens C, Alving BM, Kessler C, eds. Consultative Hemostasis and Thrombosis. Philadelphia, PA: W.B. Saunders Company; 2002:493-513; George, JN, Raskob, GE, Shah, SR, et al. Drug-induced thrombocytopenia: a systematic review of published case reports. Ann Intern Med. 1998;129:886.

Human Immunodeficiency Virus-Related Thrombocytopenia

Thrombocytopenia in HIV infection may result both from immune-mediated phenomena leading to increased clearance of platelets and ineffective platelet production, possibly due to direct infection of megakaryocytes with HIV. Improvement or resolution of thrombocytopenia after initiation of antiretroviral therapy in newly diagnosed patients is commonly observed. If the thrombocytopenia proves refractory, therapies commonly used in the treatment of ITP (IVIg, anti-D, steroids, splenectomy, others) are employed, but the potentially immunosuppressive effects of some of these approaches need to be taken into consideration.

Infection- and Sepsis-Related Thrombocytopenia

  Thrombocytopenia in the setting of infection or sepsis is common. DIC is often implicated in critically ill patients, but other causes, such as megakaryocyte-specific effects or increased clearance due to fever or splenic enlargement, may be responsible.

  Transient thrombocytopenia is commonly observed in the setting of many viral infections; certain bacterial infections, such as ehrlichiosis, Rickettsial disease, and dengue characteristically produce thrombocytopenia. A corroborative travel history and directed microbiologic testing are usually necessary to make the diagnosis.

  If the platelet count does not return to baseline with effective antimicrobial treatment or after resolution of the infection, an alternative etiology should be sought.

Hemophagocytosis

  Hemophagocytosis is a process in which bone marrow macrophages (histiocytes) engulf cellular components of the marrow. The phenomenon is considered to be nonspecific if it is found only sporadically within an aspirate smear, but the observation of abundant histiocytes with intracytoplasmic white cells, red cells, or platelets in the setting of peripheral cytopenias indicates a pathogenic process.

  In adults, sepsis or EBV-related infection or malignancy can drive T cells to produce cytokines that mediate hemophagocytosis, leading to thrombocytopenia. In these cases, the treatment is principally immunosuppressive, but the disorder often is aggressive and unresponsive to treatment.

  Familial hemophagocytic lymphohistiocytosis is a rare, autosomal recessively inherited disorder featuring hemophagocytosis, fever, organomegaly, and hypertriglyceridemia or hypofibrinogenemia; it presents at a young age. The only curative treatment is allogeneic hematopoietic stem cell transplantation.

Qualitative Disorders

Several heritable platelet anomalies of structure or function (including the May-Hegglin anomaly and the Bernard-Soulier syndrome) are typically associated with a mild thrombocytopenia. These are discussed in more detail in Chapter 21.

References

  1.  Abrahamson PE, Hall SA, Feudjo-Tepie M, Mitrani-Gold FS, Logie J. The incidence of idiopathic thrombocytopenic purpura among adults: a population-based study and literature review. Eur J Haematol. 2009;83(2):83-89.

  2.  Semple JW, Provan D, Garvey MB, Freedman J. Recent progress in understanding the pathogenesis of immune thrombocytopenia. Curr Opin Hematol. 2010;17(6):590-595.

  3.  Cines DB, Bussel JB, Liebman HA, Luning Prak ET. The ITP syndrome: pathogenic and clinical diversity. Blood. 2009;113(26):6511-6521.

  4.  Provan D, Stasi R, Newland AC, et al. International consensus report on the investigation and management of primary immune thrombocytopenia. Blood. 2010;115(2):168-186.

  5.  Cheng G, Saleh MN, Marcher C, et al. Eltrombopag for management of chronic immune thrombocytopenia (RAISE): a 6-month, randomised, phase 3 study. Lancet. 2011;377(9763):393-402.

  6.  Kuter DJ, Rummel M, Boccia R, et al. Romiplostim or standard of care in patients with immune thrombocytopenia. N Engl J Med. 2010;363(20):1889-1899.

  7.  Godeau B, Porcher R, Fain O, et al. Rituximab efficacy and safety in adult splenectomy candidates with chronic immune thrombocytopenic purpura: results of a prospective multicenter phase 2 study. Blood. 2008;112(4):999-1004.

  8.  Cuker A. Heparin-induced thrombocytopenia: present and future. J Thromb Thrombolysis. 2011;31(3):353-366.

  9.  Crowther MA, Cook DJ, Albert M, et al. The 4Ts scoring system for heparin-induced thrombocytopenia in medicalsurgical intensive care unit patients. J Crit Care. 2010;25(2):287-293.

10.  Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998;339(22):1585-1594.

11.  Dragon-Durey MA, Blanc C, Garnier A, Hofer J, Sethi SK, Zimmerhackl LB. Anti-factor H autoantibody-associated hemolytic uremic syndrome: review of literature of the autoimmune form of HUS. Semin Thromb Hemost. 2010;36(6):633-640.

12.  Lattuada A, Rossi E, Calzarossa C, Candolfi R, Mannucci PM. Mild to moderate reduction of a von Willebrand factor cleaving protease (ADAMTS-13) in pregnant women with HELLP microangiopathic syndrome. Haematologica. 2003;88(9):1029-1034.

13.  Caramazza D, Quintini G, Abbene I, et al. Relapsing or refractory idiopathic thrombotic thrombocytopenic purpurahemolytic uremic syndrome: the role of rituximab. Transfusion. 2010;50(12):2753-2760.

14.  Nurnberger J, Philipp T, Witzke O, et al. Eculizumab for atypical hemolytic-uremic syndrome. N Engl J Med. 2009;360(5):542-544.

15.  Aster RH, Curtis BR, McFarland JG, Bougie DW. Drug-induced immune thrombocytopenia: pathogenesis, diagnosis and management. J Thromb Haemost. 2009;7(6):911-918.

20

Disorders of Hemostasis I: Coagulation

Patrick F. Fogarty

APPROACH TO THE BLEEDING PATIENT

Abnormalities of the activity of coagulation proteins and related molecules, decreased platelet function, or disruption of the vasculature (as by surgery or trauma) can lead to bleeding. Careful assessment of both the clinical history and laboratory testing are necessary to establish the reason for bleeding.

  Initial laboratory studies in a patient with new-onset or recent bleeding include a platelet count, activated partial thromboplastin time (aPTT), prothrombin time (PT), and fibrinogen. If the bleeding is moderate to severe, a hemoglobin level and specimen for red cell cross-matching should also be sent.

  The character, timing, and location of the bleeding should be considered. Is the bleeding spontaneous, or associated only with invasive procedures or trauma? If peri-procedural, is the bleeding immediate or delayed? Mucocutaneous bleeding (epistaxis, gingival hemorrhage, petechiae/ecchymoses, gastrointestinal, and urinary tract bleeding) are more characteristic of a defect in the activity of platelets, whereas soft-tissue bleeding or hemarthrosis suggests a deficiency in the activity of coagulation factors.

  The clinical context is very important in determining the reason for bleeding. Hemorrhage in a patient who has been receiving heparin or warfarin may indicate excess anticoagulation or presence of a previously undetected lesion. A lifelong history of excessive bleeding may occur due to an inherited disorder of hemostasis. Bleeding in an individual who is in septic shock may point to disseminated intravascular coagulation (DIC). New-onset, diffuse bleeding in a patient who is pregnant or post-partum may signify the HELLP syndrome or other entities. Postsurgical bleeding may stem from a number of causes, but an initial consideration should be deficient hemostasis due to a traumatized, bleeding vessel, as well as a coagulation factor defect or deficits.

  A family history of bleeding raises the clinical suspicion for heritable disorders such as hemophilia A or B (X-linked recessive inheritance) or von Willebrand disease (VWD) (autosomal dominant inheritance in most cases).

  Importantly, bleeding does not necessarily indicate an intrinsic abnormality of hemostasis. Individuals with normal coagulation and platelet function will bleed given a sufficient hemostatic challenge (trauma, surgery, invasive malignancy).

THE COAGULATION SYSTEM

Coagulation Factors: Background

  Coagulation factors (clotting factors) are synthesized in the liver.

  Factors II, VII, IX, X, XI, and XII are serine proteases that are inactive as synthesized and acquire enzymatic capability when cleaved (activated) by other proteins. A post-synthetic step in the production of factors II, VII, IX, and X and the natural anticoagulant proteins C and S requires the activity of a vitamin K–dependent carboxylase that modifies the amino-terminus of each factor, enabling it to function.

  Tissue factor (TF) and factors V and VIII serve as cofactors for coagulation reactions.

  The activity of all clotting factors culminates in a principal event: the generation of thrombin at sites of vascular injury. Thrombin activates platelets (primary hemostasis) and cleaves fibrinogen to form fibrin (secondary hemostasis) at sites of blood vessel compromise.

  The normal laboratory range of factor activity levels is ˜50% to 150% and is derived from plasma activity as observed in a reference pool of normal donors. The hemostatic level of a given clotting factor (the level of factor necessary to maintain normal hemostasis) typically is much lower. For instance, 5% activity of factor VIII is well below the laboratory reference range but usually is sufficient to prevent spontaneous bleeding.

The Coagulation Cascade

The coagulation cascade illustrates the activation of coagulation factors in the formation of a fibrin clot. It comprises the tissue injury (also known as extrinsic), contact (also known as intrinsic), and common pathways of coagulation (Fig. 20.1). The pathways within the coagulation cascade probably best reflect the activity of clotting factors in vitro, whereas in vivo the pathways not only interact at multiple points but also function in concert with the activation and aggregation of platelets to achieve hemostasis.

FIGURE 20.1. The coagulation cascade.The tissue injury pathway of coagulation begins with the binding of activated factor VII (VIIa) to tissue factor (TF), which is provided by cell membranes.VIIa converts X to Xa.The prothrombinase complex, formed by the binding of Xa to Va in the presence of PL and Ca²⁺, converts II (prothrombin) to IIa (thrombin).The contact pathway of coagulation begins with the activation of factor XII to XIIa by kallikrein. XIIa cleaves XI to XIa; XIa cleaves IX to IXa. IXa forms a complex with VIIIa in the presence of PL and Ca²⁺ (tenase complex), and converts X to Xa. Xa, in the presence of Va, PL, and Ca²⁺, then cleaves II (prothrombin) to IIa (thrombin).The common pathway involves the cleavage of II by prothrombinase to yield thrombin, and thrombin cleavage of fibrinogen to form fibrin, which is then cross-linked via the action of XIIIa. Activation of coagulation usually begins with the tissue injury system, which provides feedback to the contact system by IIa-mediated activation of factor XI.There are additional points of interaction between the pathways (not indicated).

  Tissue injury pathway. The tissue injury pathway of coagulation begins with the binding of activated factor VII (VIIa) to TF. The TF-VIIa complex mediates the conversion of X to Xa. The prothrombinase complex, formed by the binding of Xa to Va on a phospholipid (PL) surface (usually platelet membranes) in the presence of Ca²⁺, converts II (prothrombin) to IIa (thrombin).

  Contact pathway. Activation of contact factors at the site of vascular injury leads to the conversion of Factor XII to XIIa, and the sequential conversion of XI to XIa and IX to IXa. IXa complexes with VIIIa, PL, and Ca²⁺, forming the tenase complex, which converts X to Xa. Xa, in a complex with Va, PL, and Ca²⁺ then cleaves II (prothrombin) to IIa (thrombin). The TF-VIIa complex can also activate IX leading to the subsequent formation of the tenase complex.

  Common pathway. The tissue injury and contact pathways converge in the common pathway, where X is converted to Xa, and prothrombin (II) is cleaved to form thrombin. Thrombin then cleaves fibrinogen to form fibrin, which is then cross-linked via the action of XIII.

Common Coagulation Tests

An understanding of the basic laboratory tests for coagulation assists in the evaluation of bleeding disorders.

  The PT is performed by adding thromboplastin (TP), composed of crude or recombinant TF plus Ca²⁺, to plasma that has been anti-coagulated with citrate, and the time to formation of a fibrin clot is measured. Because the PT comprises reactions of coagulation that occur in the tissue injury and common pathways of coagulation, deficiencies in the activity of II, V, VII, X, or fibrinogen may prolong the PT.

  The International Normalized Ratio (INR) was developed to standardize the reporting of PT values in warfarin-anticoagulated patients. Standardization is necessary because commercially available TP reagents have varying potencies that directly impact the PT; one TP may yield a different PT result than another when the same sample is tested. The potency of a given TP is expressed in terms of the International Sensitivity Index (ISI).

  Because the INR was developed to report factors measured by the PT that are decreased by warfarin impairment of vitamin K–mediated synthesis (the INR is not standardized for abnormalities of factor V and fibrinogen), the INR should be used only to describe anticoagulation in patients who are receiving warfarin. In all other patients (such as patients with liver disease), the PT should be referenced.

  The formula for the INR is (PTpatient/PTmean normal)ISI.

  The aPTT begins with the addition of a contact activating agent to citrate-anticoagulated plasma. PL and Ca²⁺ are added, and the time for formation of a fibrin clot is measured. Because the aPTT reflects reactions of coagulation that occur in the contact and common pathways of coagulation, deficiencies in the activity of factors II, V, VIII, IX, X, XI, or XII may prolong the aPTT. A deficiency of other contact factors, such as prekallikrein or high molecular weight kininogen (HMWK), may also prolong the aPTT. Isolated abnormalities of fibrinogen rarely impact the aPTT.

  The long-incubation aPTT is performed by incubating the sample with activating agents for 10 minutes prior to the addition of PL and Ca²⁺. If the contact factor prekallikrein is deficient, this extra incubation time allows activation of factor XII and correction of the aPTT.

  Mixing studies are performed using a mixture of 50% patient plasma and 50% normal control plasma; the PT or aPTT is then performed as usual. Correction of a prolonged PT or aPTT with mixing generally implies a qualitative or quantitative abnormality of one or more clotting factors in the patient plasma. In contrast, failure of the PT or aPTT to correct completely upon mixing suggests the presence of an inhibitor in the patient plasma that neutralizes a component of the patient and normal plasma. Both lupus anticoagulants (LAs; see below) and inhibitors to specific clotting factors can result in a prolonged aPTT or PTT that does not correct upon mixing.

  When evaluating a prolonged aPTT, an aPTT is performed on the mixture, then the mixture is allowed to incubate for an hour and the aPTT is repeated; some inhibitors of factor VIII are maximally inhibitory at an hour or more post-mix. For instance, the aPTT on a 1:1 mixture of normal and patient plasma containing a factor VIII inhibitor may show correction initially but demonstrate a prolongation at 1 hour.

  Occasionally, a weak LA may produce a prolonged aPTT or PT that corrects on mixing.

  The bleeding time (BT) involves making a controlled incision in soft tissue (usually at a site on the forearm) and measuring the time to cessation of bleeding. Anemia and abnormalities of coagulation factors, platelets, or the vasculature may prolong the BT. The BT does not correlate with risk of surgical bleeding in most patients,¹ and is no longer widely used or recommended.

  The thrombin time (TT) involves the addition of exogenous thrombin to patient plasma, inducing cleavage of fibrinogen to fibrin and the formation of a fibrin clot.

  The most common cause of a prolonged TT is the presence of heparin in the sample, which can be confirmed by documentation of a normalization of the TT when the test is repeated using a heparin-binding agent such as protamine or Heparsorb®.

  Abnormalities of fibrinogen and circulating heparin-like anticoagulants also cause a prolonged TT.

  The reptilase time is also used to assess abnormalities of fibrinogen (reptilase cleaves fibrinogen to fibrin). Unlike thrombin, however, reptilase is not inhibited by the presence of heparin. Thus, a prolonged TT in conjunction with a normal reptilase time usually indicates heparin contamination, whereas prolongation of both tests indicates a qualitative abnormality of fibrinogen.

  The functional fibrinogen assay assesses fibrinogen concentration by addition of an excess of thrombin to a sample of diluted plasma.

Specialized Coagulation Tests

  The anti-Xa assay provides information about the degree of anticoagulation that has occurred in the patient plasma due to the effect of heparin (unfractionated or low molecular weight) on factor Xa in the sample. By convention, samples should be drawn 4 to 6 hours after low molecular weight heparin (LMWH) administration to estimate anticoagulation.

  Tests for LAs distinguish an inhibitor of a specific clotting factor from an LA as the cause of a prolongation in the aPTT that does not correct with mixing. Most tests for LAs involve addition of excess PLs to the reaction system to neutralize the LA and result in a correction of a prolonged clotting time. One such test is the Russell’s viper venom time; other systems for the diagnosis of LAs are available. ²

  The Bethesda Assay is a special type of mixing study that involves incubation of dilutions of patient plasma with normal (control) plasma to assess the potency of an inhibitor (generally, to factor VIII) in the patient’s plasma. After a 2-hour incubation phase, a factor VIII assay (or other appropriate factor assay, as indicated) is performed on each dilution (and on samples used to create a control curve); as the proportion of patient plasma in the mixture decreases, the effect of the inhibitor decreases, and the factor assay clotting time shortens. The Nijmegen modification to the Bethesda assay includes slightly different buffers to stabilize the proteins during the incubation period.³

  The potency of the inhibitor is expressed in Bethesda units (BU). The reciprocal of the dilution of the mixture of patient and normal control plasma that contains ˜50% of normal factor VIII activity is the inhibitor titer in BU. For instance, if 50% inhibition of normal FVIII activity occurred at a 1:40 dilution, the inhibitor titer would be said to be 40 BU.

  Assays for specific clotting factors. Factor activity levels can be assessed by clot-based reaction, which employ modifications of the aPTT or PT, and some factors by chromogenic systems.

  Factor activity levels are generally reported as percentages (of “normal” activity) or in units per milliliter (U/mL), with 1 U/mL corresponding to 100% of the factor found in 1 mL of normal plasma.

  Usually, levels of 25% to 40% are necessary to prolong the PT or aPTT. Mild or moderate deficiencies of a given clotting factor may lead to an elevated PT or aPTT, but may be adequate for hemostasis.

  The euglobulin clot lysis time (ECLT) measures time for dissolution of a fibrin clot; a shortened ECLT indicates activation of the fibrinolytic system. The most common cause of a shortened ECLT is DIC, in which fibrinolysis is activated in response to an activation of coagulation. Deficiencies in the activity of plasminogen activator inhibitor or alpha-2-antiplasmin also shorten the ECLT (see below).

  Thromboelastography is measurement of various parameters of clot formation in whole blood, which captures functional data on the activity of both platelets and coagulation factors.⁴ This test is used clinically mostly in cardiovascular surgical settings although other applications have been explored. ⁵

DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION TESTS

Conditions that predispose to bleeding or produce abnormal coagulation test results can be divided into those entities that prolong the aPTT, PT, or both (Table 20.1 and Figs. 20.2, 20.3, and 20.4).

Conditions Associated with a Prolonged aPTT

  LAs² are a very common cause of a prolongation in the aPTT that does not correct completely on mixing.

  LAs were so named due to their frequent presence in patients with systemic lupus erythematosus and tendency to prolong coagulation tests by interacting with PL in the test sample. In contradistinction to their name, however, LAs are not physiologic anticoagulants; additionally, over one-half of patients with LAs do not have connective tissue disease (see Chapter 22).

  LAs are diagnosed using the methods described above.

  Hemophilia A and B. More frequently than with any other factor essential to the aPTT reaction, a deficiency of factor VIII causes a prolongation in the aPTT that corrects completely on mixing. Congenital factor VIII deficiency is referred to as hemophilia A. (Factor VIII is also decreased in moderate and severe VWD; see below.) Congenital deficiency of factor IX is hemophilia B.

  Hemophilia A is estimated to occur in one per 5,000 to 10,000 live male births; hemophilia B is about a fifth as common. The disorders are inherited in an X-linked recessive fashion: males are affected, whereas females are carriers and are generally not affected unless significant lyonization has occurred favoring the X chromosome bearing the abnormal copy of the FVIII gene. There is no race predilection.

  The presentation of the disease relates to the level of residual factor activity in the plasma. Severe hemophilia (<1% factor activity) typically presents in infancy with bleeding at circumcision, or in early childhood with spontaneous bleeding into soft tissues (muscles) or joints, and intracranial, gastrointestinal, or urinary bleeding. Moderate hemophilia (1%–5% factor activity) is typified by less severe bleeding than that observed in severe disease, whereas individuals with mild hemophilia (>5% activity) usually do not experience spontaneous bleeding but may bleed upon significant hemostatic challenges such as trauma or surgery.

FIGURE 20.2. Laboratory diagnostic algorithm for a prolonged aPTT and normal PT. aPTT, activated partial thromboplastin time; LA, lupus anticoagulant.

* Some LAs escape detection, even after performing two tests. If all relevant coagulation factor activities are demonstrated to be hemostatic, an LA may be the explanation for the aPTT prolongation.

† Occasionally, weak LAs can cause a prolongation in the aPTT that corrects completely on mixing. In this scenario, factor assays may be indicated in addition to LA testing, especially if demonstration of hemostatic factor levels is regarded as important (e.g., in a preoperative patient).

  Factor concentrates are the mainstay of treatment (Table 20.2); both plasma-derived and recombinant products are commercially available. For acute major bleeding (e.g., intracranial) or prophylaxis prior to major surgery, doses of 50 U/kg (FVIII) or 100 to 120 U/kg (FIX) are administered by IV bolus infusion every 8 to 12 hours for 1 to 14 days, depending on the anatomic location and severity of the bleeding.⁶ Longer-acting FVIII and FIX molecules are in clinical development.⁷ Less severe bleeding (hemarthrosis) or prophylaxis prior to moderately invasive procedures (such as endoscopy with biopsy) may be addressed with lower doses of factor. Patients with mild hemophilia A may respond to infusion of DDAVP (0.3 mcg/kg/dose),⁸ but a trial should be done in the non-bleeding state to document a rise of the FVIII activity level into the hemostatic range.

FIGURE 20.3. Laboratory diagnostic algorithm for a prolonged PT and normal aPTT. LA, lupus anticoagulant; PT, prothrombin time; TT, thrombin time.

* Decreased functional fibrinogen in conjunction with a normal immunologic fibrinogen indicates an abnormal fibrinogen (dysfibrinogenemia), whereas decreased functional and immunologic assays are typical of hypofibrinogenemia.

† Occasionally, LAs can cause a prolongation in the PT that corrects on mixing.

  The oral antifibrinolytic agent aminocaproic acid (Amicar®), given at a dose of 1 to 2 g every 4 to 6 hours, may be useful for patients with mucosal or oral bleeding, or bleeding associated with dental procedures.

  Prophylactic factor replacement therapy (twice or thrice weekly infusions) is routinely used as a means of preventing the morbidity incurred from recurrent joint bleeding.⁹ It is more common among children than adults; generally, the practice is begun by the age of 4 years.

  Inhibitors. Patients with congenital hemophilia (usually with severe disease) who have received factor concentrates as treatment for bleeding are at risk for inhibitor formation. Over 25% of patients with hemophilia A and less than 5% of patients with hemophilia B will develop inhibitors to factor VIII or IX, respectively. The potency of the inhibitor is expressed in BU. Low-titer inhibitors (<5 BU) often may be overcome by increasing the dose or frequency of infused factor concentrate. It is usually not possible to overcome high-titer inhibitors (>5 BU) using this approach, however, and administration of activated prothrombin complex concentrates and/or recombinant factor VIIa is necessary (Table 20.2). ¹⁰

  Acquired hemophilia occurs at an incidence of approximately 1 per million per year, typically in elderly persons or in those with underlying lymphoproliferative conditions, cancer, autoimmunity, or prior pregnancy. Anti-FVIII IgG antibodies neutralize FVIII, leading to a prolonged aPTT that does not correct upon mixing. The clinical presentation commonly features extensive ecchymoses and soft-tissue hematomas. Bypassing agents (see Table 20.2) are used to treat acute bleeding, whereas a variety of immunosuppressive agents, typically incorporating corticosteroids initially, may produce a response. The mortality of this condition is considerable.¹¹

FIGURE 20.4. Laboratory diagnostic algorithm for a prolonged aPTT and PT. aPTT, activated partial thromboplastin time; DIC, disseminated intravascular coagulation; FDP, fibrin degradation products; LA, lupus anticoagulant; PT, prothrombin time.

* Co-existing conditions, such as vitamin K deficiency (leading to a prolonged PT) and a concomitant LA (leading to an elevated aPTT), are possible.

† Rarely, co-inheritance of deficiencies in multiple coagulation factors (such as factors V and VIII) can occur.

  VWD, due to the lack of adequate VWF to bind and protect circulating factor VIII from clearance, can lead to low VIII levels and a prolongation in the aPTT (see Chapter 21, Disorders of Hemostasis II). The prolongation in the aPTT corrects on mixing.

  Factor XI deficiency (sometimes called hemophilia C) typically results in a prolonged aPTT that corrects on mixing. It is inherited in an autosomal recessive manner and is most prevalent among individuals of Ashkenazi Jewish descent. It typically causes a mild bleeding tendency that is worsened by trauma or surgery. Levels of factor XI do not correlate well with bleeding symptoms.¹² Fresh frozen plasma (FFP) may be used prophylactically or for treatment of bleeding, and adjunctive aminocaproic acid decreases unmitigated fibrinolysis, making it useful for chronic prophylaxis, oral bleeding, dental work, or minor surgical procedures. In some locales, a plasma-derived FXI concentrate is available.

  Factor XII deficiency and deficiencies of prekallikrein and HMWK. Although they may lead to a prolonged aPTT, these conditions do not cause bleeding.

  Acquired inhibitors to coagulation proteins. Occasionally, adults without a prior history of hemophilia develop high-titer inhibitors to factor VIII; not infrequently, a concomitant lymphoproliferative or immune disorder is present. Immunosuppressive treatment with corticosteroids or chemotherapy usually is effective. ¹³

  Heparin contamination. The presence of heparin in the sample used for the aPTT determination may be verified by documenting normalization of the aPTT after the test is repeated using a heparin-binding agent.

  Warfarin may mildly prolong the aPTT due to depletion of factors II, X, or XI.

  Occasionally, traumatic venipuncture may cause the aPTT to be prolonged, due to the direct activation of coagulation at the site of venipuncture, leading to depletion of crucial coagulation proteins in the collected specimen. The blood should be redrawn with careful technique during phlebotomy and the aPTT repeated to document normalization. (Traumatic venipuncture may also lead to a shortening of the aPTT due to small amounts of thrombin generation.)

Conditions Associated with a Prolonged Prothrombin Time

  Vitamin K deficiency can cause an elevated PT that typically corrects completely on mixing. The vitamin K–dependent factors that are measured by the PT are II, VII, and X.

  Malabsorption or deficient dietary intake of vitamin K (from green leafy vegetables such as cabbage, cauliflower, and spinach, cereals, soybeans, and other foods) or decreased production by intestinal bacteria (which may be destroyed by antibiotics) may lead to vitamin K deficiency.

  For treatment, vitamin K (phytonadione) may be administered via parenteral or oral routes. Intravenous administration (1 mg/day) results in faster normalization of a prolonged PT than subcutaneous dosing, but occasionally has been associated with anaphylaxis; therefore, intravenous doses should be administered slowly (over 30 minutes) while the patient is monitored. Subcutaneous administration should be avoided due to erratic absorption. ¹⁴

  At least partial correction of the PT is expected within 24 hours after parenteral administration of vitamin K if vitamin K deficiency is the only reason for the prolongation in the PT.

  Coagulopathy of liver disease. Hepatic insufficiency leads to decreased synthesis of clotting factors, such as the vitamin K–dependent factors and, with more severe disease, factors V, VIII, XI, XII, and fibrinogen, resulting in a prolonged PT (and with severe disease, aPTT) that correct(s) on mixing.

  In contradistinction to coagulopathy due to isolated vitamin K deficiency, liver disease may feature a reduced factor V level in addition to decreased levels of factors II, VII, IX, and X.

  Patients with prolonged clotting times due to liver disease paradoxically may be prone to thrombosis.¹⁵

  Warfarin. Warfarin inhibits the vitamin K–dependent carboxylase that is important for the synthesis of factors II, VII, IX, and X. Decreased functional levels of factors II, VII, and X may prolong the PT and produce an elevated INR.

  Supratherapeutic INRs that are not associated with bleeding generally are managed by temporary withholding of warfarin to allow the INR to descend into the desired range, and restarting the warfarin at a lower dose.

  Critically elevated INRs (>9) may be addressed with temporary discontinuation of warfarin, plus administration of vitamin K¹⁴ or FFP if the patient is felt to be at very high risk for bleeding.

  For treatment of warfarin-associated major bleeding,¹⁶ the warfarin should be discontinued and FFP (4 units), prothrombin complex concentrate (such as Bebulin®, 35 units/kg/dose), or rhVIIa (NovoSeven®; 15–90 μg/kg/dose ) should be administered along with intravenous or oral vitamin K.

  LAs can cause a mild prolongation in the PT (see above).

  Hypofibrinogenemia/Dysfibrinogenemia. Quantitative or qualitative abnormalities of fibrinogen typically produce a long TT and reptilase time (see above), but the PT also may be prolonged (Fig. 20.3). The PT is much more sensitive to hypofibrinogenemia/dysfibrinogenemia than is the aPTT.

  Defects in the function of fibrinogen are more commonly acquired (as with cirrhosis or active liver disease) than congenital. For instance, DIC characteristically produces a consumptive hypofibrinogenemia.

  Replacement of fibrinogen in a bleeding patient with hypofibrinogenemia/dysfibrinogenemia may be accomplished by administration of cryoprecipitate or a plasma-derived fibrinogen concentrate; for major surgery or bleeding, a plasma fibrinogen level of 80 to 100 mg/dL is targeted.

  Deficiencies of individual clotting factors. Congenital deficiencies of isolated coagulation factors (for example, VII) leading to a prolonged PT are extremely rare and typically are inherited in autosomal recessive pattern.

  Antibodies to bovine factor V and thrombin may develop after exposure to topical bovine thrombin (used in orthopedic, neurologic, and vascular surgery). The antibodies cross react with human factor V and/or thrombin, resulting in prolongation of the PT and bleeding in some patients. ¹⁷

Conditions Associated with a Prolonged Activated Partial Thromboplastin Time and Prothrombin Time

  Coagulopathy of liver disease. If hepatic insufficiency is extreme, multiple factor deficiencies can result in a prolonged PT and aPTT.

  Deficiencies of individual clotting factors. Isolated deficiencies of factors II, V, or X are rare,¹⁸ but may prolong both the PT and aPTT.

  DIC. Depletion of coagulation factors via diffuse activation of coagulation may cause a prolongation in both the PT and aPTT (see Chapter 21).

  LAs can prolong both the PT and aPTT (discussed previously).

Conditions Associated with Bleeding and Normal Coagulation Tests

  Factor XIII deficiency. Activated factor XIII cross-links fibrin strands, stabilizing the fibrin clot. Individuals with a deficiency of factor XIII characteristically develop delayed bleeding several hours to days following surgery or trauma. Traumatic soft tissue and joint bleeds, recurrent pregnancy loss, and spontaneous intracranial hemorrhages also have been described. ¹²

  Clot lysis or enzymatic assays and sequencing of either of the two genes that encode the molecule may be diagnostic.

  Treatment of bleeding consists of infusion of cryoprecipitate or FFP.

  Alpha-2-antiplasmin deficiency or plasminogen activator I (PAI-1) deficiency leads to accelerated digestion of fibrinogen and fibrin clots and (in some patients) increased bleeding.¹⁹ Infusion of FFP may be clinically useful.

  Congenital and acquired abnormalities of the vasculature and integument can cause increased fragility of blood vessels and bruising or bleeding, despite normal coagulation, fibrinolysis, and platelet function.²⁰ Such conditions include hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease), heritable defects of collagen (Ehlers-Danlos syndrome, osteogenesis imperfecta), acquired collagen-associated conditions (scurvy, prolonged glucocorticoid administration, the normal aging skin), and other anomalies (Marfan syndrome, amyloidosis, vasculitis). There is no effective treatment for bruising associated with the congenital disorders; preventative measures to reduce the risk of trauma should be followed. Repletion of vitamin C (scurvy) and reduction of corticosteroids (glucocorticoid excess) ameliorate bruising associated with those acquired processes.

References

  1.  Thomas S, Katbab H, abu Fanas SH. Do preoperative cutaneous bleeding time tests predict the outcome of intraoral surgical bleeding? Int Dent J. 2010;60(4):305-310.

  2.  Pengo V, Tripodi A, Reber G, et al., Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphspholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost. 2009;7(10):1737-1740.

  3.  Towfighi F, Gharagozlou S, Sharifian RA, et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. Acta Haematol. 2005;114(2):84-90.

  4.  Chitlur M, Sorensen B, Rivard GE, et al. Standardization of thromboelastography: a report from the TEG-ROTEM working group. Haemophilia. 2011;17(3):532-537.

  5.  Young G, Zhang R, Miller R, Yassin D, Nugent DJ. Comparison of kaolin and tissue factor activated thromboelastography in haemophilia. Haemophilia. 2010;16(3):518-524.

  6.  Hermans C, De Moerloose P, Fischer K, et al. Management of acute haemarthrosis in haemophilia A without inhibitors: literature review, European survey and recommendations. Haemophilia. 2011;17(3):383-392.

  7.  Fogarty PF. Biological rationale for new drugs in the bleeding disorders pipeline. Hematology Am Soc Hematol Educ Program. 2011;2011:397-404.

  8.  Franchini M, Zaffanello M, Lippi G. The use of desmopressin in mild hemophilia A. Blood Coagul Fibrinolysis. 2010;21(7): 615-619.

  9.  Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med. 2007;357(6):535-544.

10.  Berntorp E. Importance of rapid bleeding control in haemophilia complicated by inhibitors. Haemophilia. 2011;17(1):11-16.

11.  Huth-Kuhne A, Baudo F, Collins P, et al. International recommendations on the diagnosis and treatment of patients with acquired hemophilia A. Haematologica. 2009;94(4):566-575.

12.  Gueguen P, Galinat H, Blouch MT, et al. Biological determinants of bleeding in patients with heterozygous factor XI deficiency. Br J Haematol. 2012;156(2):245-251.

13.  Shetty S, Bhave M, Ghosh K. Acquired hemophilia a: diagnosis, aetiology, clinical spectrum and treatment options. Autoimmun Rev. 2011;10(6):311-316.

14.  Crowther MA, Douketis JD, Schnurr T, et al. Oral vitamin K lowers the international normalized ratio more rapidly than subcutaneous vitamin K in the treatment of warfarin-associated coagulopathy. A randomized, controlled trial. Ann Intern Med. 2002;137(4):251-254.

15.  Lippi G, Targher G, Favaloro EJ, Franchini M. Venous thromboembolism in chronic liver disease. Semin Thromb Hemost. 2011;37(1):66-76.

16.  Ansell J, Hirsh J, Hylek E, Jacobson A, Crowther M, Palareti G. Pharmacology and management of the vitamin K antagonists: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest. 2008;133 (6 suppl):160S-198S.

17.  Ofosu FA, Crean S, Reynolds MW. A safety review of topical bovine thrombin-induced generation of antibodies to bovine proteins. Clin Ther. 2009;31(4):679-691.

18.  Peyvandi F, Favaloro EJ. Rare bleeding disorders. Semin Thromb Hemost. 2009;35(4):345-347.

19.  Iwaki T, Tanaka A, Miyawaki Y, et al. Life-threatening hemorrhage and prolonged wound healing are remarkable phenotypes manifested by complete plasminogen activator inhibitor-1 deficiency in humans. J Thromb Haemost. 2011;9(6):1200-1206.

20.  De Paepe A, Malfait F. Bleeding and bruising in patients with Ehlers-Danlos syndrome and other collagen vascular disorders. Br J Haematol. 2004;127(5):491-500.

21

Disorders of Hemostasis II

Patrick F. Fogarty

INTRODUCTION

In addition to thrombocytopenia (Chapter 19) and primary deficiencies in the activity of coagulation proteins (Chapter 20), disseminated intravascular coagulation (DIC), von Willebrand disease, and qualitative abnormalities of platelets can also result in bleeding.

DISSEMINATED INTRAVASCULAR COAGULATION

Although it frequently manifests as bleeding, DIC begins as a result of an uncontrolled local or systemic activation of coagulation due to an underlying disorder. DIC may be acute or chronic, limited or diffuse, and accompanied by hemorrhage or (less commonly) thrombosis. Conditions that are associated with DIC are listed in Table 21.1.

Pathophysiology

The inciting events are numerous but generally involve either overwhelming release of tissue factor (see Chapter 20) by cellular, vascular, or hypoxemic injury, or the presence of endogenously or exogenously derived procoagulant molecules (bacterial lipopolysaccharide proteins produced by neoplastic cells).¹ As coagulation is inappropriately and systemically activated, clotting factors and platelets are consumed, leading to bleeding. If activation of coagulation is chronic and low grade, however, clotting factors and platelets may be replenished and hypercoagulability may occur, manifesting as thrombosis (as in Trousseau syndrome).

Presentation

The appearance of DIC always indicates a serious underlying condition. A typical presentation of DIC involves a patient who has been hospitalized due to another disorder (Table 21.1) when unexplained bleeding and/or abnormalities in routine coagulation are observed.

  The hemorrhage of DIC is typically diffuse and may involve bleeding at sites of surgical incisions or vascular access catheters, as well as urinary, gastrointestinal, pulmonary, central nervous system, or cutaneous hemorrhage. Acral cyanosis and petechial and ecchymotic lesions may also occur. Widespread DIC-associated truncal and extremity bruising (Purpura fulminans) usually is limited to children or follows a viral infection. ²

  Severe systemic DIC may lead to widespread tissue hypoxia and multiorgan dysfunction; hepatic, neurologic, cardiac, renal impairment may occur. The development of multiorgan dysfunction is associated with a high mortality rate.

Diagnosis

Typically, acute DIC is suspected when a patient with a predisposing condition (Table 21.1) develops bleeding or thrombosis and/or a perturbation in laboratory tests indicative of DIC. DIC is a dynamic condition, especially in the acutely ill patient; considerable variation in laboratory markers from timepoint to timepoint is possible, and analysis of trends rather than isolated values is crucial. Laboratory parameters may show the following:

  Increased (prolonged) activated partial thromboplastin time (aPTT), prothrombin time (PT), or thrombin time—due to consumption of clotting factors and/or fibrinogen (most patients).

  Decreased fibrinogen (compared to baseline)*—due to consumption of fibrinogen.

  Increased products of fibrinogen and fibrin degradation (FDPs; D-dimer assay)—due to plasminmediated cleavage of fibrinogen and fibrin. The D-dimer assay measures fibrin products that have been cross-linked by activated factor XIII.

  Decreased platelet count (compared to baseline)*—due to clearance resulting from activation and aggregation at the sites of local prothrombotic reactions (most patients). DIC rarely produces a platelet count less than 20,000/μL. Patients with thrombosis and chronic DIC due to malignancy may have a normal or even elevated platelet count.

  Fragmented red cells (schistocytes) on peripheral blood smear—due to microvascular hemolysis (25%–50% of patients with DIC).

Treatment

The clinical and laboratory manifestations of DIC are expected to resolve with correction of the inciting disorder. This might entail effective administration of antimicrobials to a patient with sepsis, treatment of malignancy, surgery to repair an aneurysmal dilatation, removal of conceptus and placenta, or another intervention as dictated by the clinical scenario. If the DIC is severe enough to have eventuated in multiorgan dysfunction, management in an intensive care unit is required.

  Blood products should not be administered to patients with acute DIC unless clinically significant bleeding is present or if the risk of bleeding is felt to be high (as with thrombocytopenia in a patient who has sustained major trauma); there is, however, no reason to withhold blood products for fear of “fueling the fire.”

  If bleeding is present, platelet transfusions may be administered to stop clinical bleeding; a target platelet count of 20,000 to 30,000/μL (most cases) or >50,000/μL (intracranial or life-threatening hemorrhage) is reasonable. Higher target ranges may be desired for patients who are to undergo invasive procedures such as major surgery, but the consumptive process may make achieving the goal difficult.

  Cryoprecipitate may be administered for bleeding in the setting of fibrinogen levels that are consistently less than 80 to 100 mg/dL. Fresh frozen plasma (FFP) should be given only to patients with significant bleeding and a prolonged PT and aPTT.

  Due to its potential to exacerbate hemorrhage, heparin should be considered in acute DIC only in cases of bleeding, when DIC is ongoing despite appropriate treatment (blood product infusion). Heparin should not be administered unless the platelet count can be supported to 50,000/μL or higher and there is no central nervous system or diffuse gastrointestinal bleeding. If heparin is to be used, a low-dose infusion (6–10 U/kg/h) with no bolus dose is recommended. An improving platelet count and fibrinogen concentration signifies that the treatment is effective. Heparin is contraindicated in patients with placental abruption or other obstetric conditions that will require surgical management, because the anticoagulation is likely to complicate the curative treatment.

  Fibrinolysis inhibitors may have a role in patients with profuse bleeding who have failed to respond to other management, in whom FDPs are felt to be inhibiting platelets.

  Due to questionable efficacy and worsened bleeding in some subjects, activated protein C concentrate (APC, drotrecogin alfa) is no longer recommended for patients with severe sepsis and DIC.³ Use of antithrombin concentrates is controversial. ⁴

  Laboratory parameters (PT, PTT, fibrinogen, and platelet count) should be monitored at least every 6 hours in the acutely ill patient with DIC, and clinical bleeding should be followed to assess efficacy of therapeutic measures.

HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) affects women in the peri-partum period and produces clinically significant hemolytic anemia, hepatocellular injury, and low platelets. It may be difficult initially to distinguish the disorder from thrombotic thrombocytopenia purpura (TTP). Hepatic dysfunction (leading to elevated transaminases) may distinguish the diagnosis from TTP, which also may complicate pregnancy (see Chapter 19). Introduction of placental proteins into the maternal circulation has been thought to be etiologic; potential biomarkers have been identified.⁵ Gross hemoglobinuria with renal dysfunction and hypotension is common; the mortality rate is high. Management must include evacuation of the uterus, either by delivery of a term or near-term infant, or by dilatation and curettage to remove retained placental or fetal tissue.

Acute promyelocytic leukemia (APL) is frequently accompanied by DIC, potentially due to procoagulant molecules (tissue factor and others) contained within circulating promyelocytes. Bleeding commonly occurs in the lungs and brain and is frequently fatal. In addition to the appropriate use of blood products (FFP, cryoprecipitate, platelets) upon detection of APL-associated DIC, emergent initiation of treatment with all-trans-retinoic acid (ATRA) is recommended (see Chapter 11).⁶

Trousseau syndrome is a form of chronic DIC in which recurrent episodes of venous thromboembolism (VTE) complicate an underlying malignancy, especially adenocarcinomas. Experience in the management of the disorder has suggested that anticoagulation with warfarin is not effective in preventing further VTE; instead, subcutaneous low molecular weight heparin in therapeutic doses is usually necessary to prevent recurrence of thromboembolism (see Chapter 23).⁷

VON WILLEBRAND DISEASE

Epidemiology

von Willebrand disease (VWD) is the most common inherited bleeding disorder.⁸

Pathophysiology and Classification

Von Willebrand factor (VWF) is an extremely large multimeric glycoprotein that is synthesized in endothelial cells and megakaryocytes. Binding of VWF to its receptor, platelet glycoprotein Ib (GPIb), tethers platelets to one another and to the subendothelial collagen matrix, localizing them to the site of injury. This interaction is especially important to assure primary hemostasis in vessels such as arterioles, where a “high shear” state is present (Fig. 21.1). VWF also binds factor VIII (FVIII) in the circulation, protecting it from clearance.

  Type 1 (quantitative defect in VWF) includes approximately 75% to 80% of patients, the majority of whom do not have an identified causal mutation in the VWF gene, which is located on chromosome 12. Patients may have mild or moderate bleeding. Autosomal dominant inheritance is typical.

  Type 2 (qualitative defect in VWF) includes four subtypes; patients usually have moderate bleeding symptoms and present before adulthood. Type 2A (10% to 15% of VWD) involves mutations in VWF that cause either a defect in intracellular transport (2A, type 1) or render the molecule more susceptible to proteolysis (2A, type 2). Laboratory testing (Table 21.2) typically shows a marked decrease in VWF activity relative to antigen (a ratio of ≤0.6 is typical). Type 2B (5% of VWD) mutations result in an abnormal structure in the binding site for platelet GPIb (A1 domain of VWF), and are responsible for a “gain-of-function” defect that allows spontaneous binding of the abnormal VWF to platelets in the circulation. Patients typically have thrombocytopenia due to removal of VWF-bound platelet aggregates. The ristocetin-induced platelet aggregation (RIPA) (Table 21.2) shows an increase in platelet aggregation to low concentrations of ristocetin.† Type 2N (uncommon) features mutations in VWF that decrease its ability to bind and protect FVIII from clearance, resulting in decreased FVIII levels in the plasma and a phenotype similar to hemophilia A. Soft tissue and joint bleeding are common. The presence of affected females in the family is an important clue to consider this diagnosis. Laboratory studies show decreased FVIII (2% to 10%), and normal VWF function and antigen. Type 2M (very uncommon) results from mutations affecting the A1 domain in an area different from those mutations in type 2B. These result in decreased binding of platelets to VWF.

  Type 3 VWD (rare) is caused by a variety of mutations of the VWF molecule, including larger deletions; patients may be homozygous for a given mutation or double heterozygotes. Severe bleeding manifests in childhood. FVIII is usually about 5%, and VWF levels usually are too low to detect.

Presentation

Bleeding symptoms usually involve mucous membranes. Epistaxis, oral bleeding, menorrhagia, and gastrointestinal bleeding are common. Individuals with marked abnormalities of VWF usually present earlier in life with bleeding at the time of mucous membrane-related procedures (tooth extractions, tonsillectomy), or at menarche.

FIGURE 21.1. Primary hemostasis. A. Normal conditions. Under physiologic conditions, platelets do not interact with the endothelium. B. Adhesion. Upon disruption of the blood vessel wall, subendothelial collagen and fibronectin are exposed, leading to platelet adhesion. In the arterial/arteriolar circulation, subendothelial von Willebrand factor (VWF) assists in the adherence of platelets to the site of injury via binding to the platelet glycoprotein (GP) Ib receptor. C. Aggregation. Tissue factor interacts with factor VIIa, present locally, to catalyze the formation of thrombin.Thrombin, collagen, and other molecules bind to receptors on the platelet membrane, leading to platelet activation. Fibrinogen cross-links platelets via their GPIIb/IIIa receptors, promoting formation of an occlusive plug that prevents additional blood loss through the break in the vessel wall.VWF also bridges between platelets, via their GPIb and GPIIb/IIIa receptors.

Diagnosis

The diagnosis of VWD is based on a typical history of bleeding (i.e., mucous membrane-related) and confirmatory laboratory testing.⁹ The diagnosis can be difficult, given the large number of individuals (estimated up to 1% of the population) whose VWF levels fall below the laboratory reference range, many of whom do not experience abnormal bleeding.

  The personal and family history of bleeding should be carefully documented.

  A complete blood count and routine coagulation studies should be performed, to exclude other diagnoses and assess for anemia.

  Initial testing for VWD (Table 21.2). A VWF antigen level (by ELISA) and a VWF activity (by ristocetin cofactor assay) should be performed. The latter involves addition of ristocetin at 1.2 mg/mL to a mixture of patient plasma (the VWF source) and washed normal platelets. Ristocetin binds VWF, allowing it to bind GPIb on the platelet membrane, causing platelet aggregation. The factor VIII activity may be abnormal.

  Secondary testing. A VWF multimer study detecting the distribution of multimers is performed once a diagnosis of VWD has been made, to assess for type 2 VWD.

  VWF levels <30% are regarded by most clinicians as diagnostic of VWD. If borderline results are obtained, testing may need to be repeated up to three times to exclude the diagnosis. Menstruating females generally have the lowest VWF levels in the first 4 days of menstruation. Exogenous estrogens increase VWF levels, and testing may need to be repeated off of the hormones. Testing of family members also may aid in the diagnosis of patients with borderline results.

  “Low VWF.” This classification was developed to encompass VWF levels in the 30% to 50% range, which are too high to be considered a definitive criterion for diagnosis but may indicate a tendency for bleeding in selected patients. Hemostatic agents (see “Treatment” ) may be used if bleeding (or a high risk of bleeding) is present.

Treatment

The patient’s type of VWD, past response to bleeding challenges, current medications, and general medical condition should be considered (Table 21.3).⁹

  DDAVP (desmopressin acetate) indirectly causes release of VWF and factor VIII from storage sites (primarily the endothelium). After intravenous administration, levels of both factors are increased 2 to 7-fold for about 6 to 12 hours. Prior to use of DDAVP for clinically significant bleeding or as prophylaxis prior to invasive procedures, patients should undergo a therapeutic trial to document responsiveness to the medication (as assessed by increased VWF levels into the hemostatic range and lack of worsening thrombocytopenia [type 2B patients]). More than two doses, given 12 to 24 hours apart, generally should be avoided in a 24 to 48-hour period, as tachyphylaxis and serious hyponatremia (due to fluid retention) can occur after repeated doses. Nonsteroidal anti-inflammatory (NSAIDs) agents may aggravate this latter effect.

  VWF concentrates are used when bleeding is not controlled with DDAVP, or as prophylaxis prior to a major invasive procedure, or for clinically significant bleeding in patients who are less likely to be responsive to DDAVP (type 3 and some type 1 and 2 patients). Humate-P®, Alphanate, and Wilate are antihemophilic factors that contain VWF. Cryoprecipitate generally is not recommended because of its lack of viral inactivation.

  Antifibrinolytic agents such as epsilon aminocaproic acid (Amicar®) and topical agents (including topical thrombin, Gelfoam, and fibrin sealant) are used adjunctively, especially in cases of mucosal bleeding (e.g., dental).

Pregnancy and Von Willebrand Disease

All pregnant women with VWD should be managed in consultation with a hematologist and should deliver at a specialized center for bleeding disorders. VWF levels increase 2 to 3-fold during the last two trimesters of pregnancy; type 1 VWD patients whose VWF levels have reached the normal range during the third trimester may not require treatment during delivery. In more severely affected patients, VWF concentrates can be administered prophylactically, beginning usually after the onset of labor. Peripartum DDAVP use warrants caution due to the risk for hyponatremia and seizures. The risk of postpartum hemorrhage may persist for up to a month after delivery.¹⁰

QUALITATIVE PLATELET DISORDERS

Introduction

Most disorders of platelet function are acquired. Heritable qualitative platelet disorders are individually rare (occurring in 0.01–1/100,000 population) but in aggregate may not be uncommon.¹¹ Because of redundancy of biochemical and receptor pathways that mediate the function of platelets, certain defects may be detectable only on laboratory testing, whereas other qualitative defects characteristically produce clinically significant bleeding.

Review of Hemostasis and the Role of Platelet Biochemistry

Primary hemostasis describes the formation of a platelet plug at the site of vascular injury (Fig. 21.1). In a variety of reactions that are not entirely sequence-specific, individual circulating platelets must adhere to the denuded endothelial surface, undergo activation through receptor-ligand interactions, release the contents of their granules (i.e., platelet secretion), and aggregate to form a physical barrier to continued blood loss.¹²

Adhesion

Subendothelial molecules such as VWF, collagen, and fibronectin mediate adhesion of platelets to the exposed subendothelial matrix at sites of vessel wall compromise. In “high-shear” states such as arterioles, VWF is especially important, because it tethers the platelet to the endothelial surface via interaction with its receptor, GPIb.

Activation

Subendothelial collagen activates platelets; thrombin, which has been generated locally in reactions following the interaction of factor VIIa and tissue factor (provided by the membranes of cells), also activates platelets by binding to receptors on the platelet surface and initiating a series of signal transduction events.

Secretion

Agonists such as collagen, thrombin, Adenosine Diphosphate (ADP), and epinephrine bind to their receptors on the platelet membrane and induce a series of biochemical events that cause platelets to release the contents of their granules (Table 21.4), which act to promote further activation and aggregation.

Aggregation

Binding of agonists also promotes a conformational change in the platelet GP IIb/IIIa receptor, exposing its binding sites for fibrinogen and VWF; these molecules can then bridge between individual platelets at the site of vascular injury, promoting the formation and stability of the platelet plug.

Participation in Coagulation Reactions

The platelet membrane is rich in phospholipid, which is a required component for reactions involving clotting factor complexes.

Platelet Function Testing

Platelet Aggregation Studies (Platelet-Rich Plasma System)

According to the classical method, platelets in a suspension of platelet-rich plasma (PRP) impede transmission of light. When any of a variety of agonists (collagen, thrombin, ADP, epinephrine) is added, aggregation occurs, consolidating the platelets and allowing the passage of light through the plasma. The increase in light transmission as aggregation occurs is plotted as a function of time (Fig. 21.2).

  Ideally, the waveform shows two physiologic processes: a primary wave represents initial aggregation as platelet receptors are activated and become available to bind proaggregatory molecules such as fibrinogen.

  A secondary wave indicates further aggregation that is stimulated by the release of platelet granule contents.

  Routinely, secretion of platelet granule contents (Table 21.4) is assessed in tandem with platelet aggregation; per one methodology, after stimulation of platelets with an agonist, release of adenosine triphosphate into solution is measured through a chemiluminescence procedure, and plotted as a function of time.

Platelet Function Analyzer

The Platelet Function Analyzer (PFA-100™, Dade Behring, Inc.) device assesses the formation of a platelet plug after citrated whole blood is aspirated through an aperture in a collagen-impregnated membrane, leading to platelet activation and aggregation; the time to occlusion of the aperture is measured and compared with a normal range. Though it may be helpful for assessing aspirin-associated platelet inhibition,¹³ the test lacks sufficient sensitivity and specificity to be used in screening for inherited platelet disorders.¹⁴

Measurement of Granule Contents (Rarely Indicated)

Centrifugation of PRP produces a platelet pellet; the platelet membranes are then disrupted, liberating intracellular/intragranular proteins into the lysate. The molecule of interest is then assessed (VWF, by ristocetin co-factor assay, for intra-granular VWF).

Acquired Disorders

Drugs

The most common acquired qualitative platelet disorders are caused by the use of medications that directly or indirectly impair platelet function; of these, aspirin and the NSAIDs are most frequently responsible (Table 21.5). Patients who present with bruising or platelet-type bleeding and whose platelet function testing shows abnormal aggregation or secretion should be questioned regarding current medications, especially recently initiated drugs including over-the-counter, naturopathic, and herbal agents. Treatment of clinically significant bleeding due to drug-induced platelet dysfunction first involves discontinuation of the offending agent, and may require additional measures (Table 21.6).

FIGURE 21.2. Platelet aggregation studies. Platelet aggregation studies involve the addition of agonists (collagen, thrombin, ADP, arachidonic acid, or epinephrine) to a suspension of platelet-rich plasma (PRP); the agonist induces aggregation of platelets and allows transmission of light through the plasma component of the PRP. A. In the normal scenario, the binding of an agonist to its platelet receptor initiates a shape change that temporarily decreases light transmission; subsequently, a primary wave of platelet aggregation is recorded (as increased light transmission) as fibrinogen binds its receptor, GPIIb/IIIa, and begins to cross-link platelets. Unlike the other agonists, collagen does not induce a primary wave. A secondary wave occurs as signal transduction events (resulting from platelet activation) eventuate in augmented binding of GPIIb/ IIIa by fibrinogen and release of platelet granules, whose contents are able to induce further aggregation. B. In storage pool disease (SPD), platelet aggregation to ADP and other agonists typically shows an initial wave of aggregation, but the aggregates subsequently dissociate due to reduced or absent release of platelet granule contents. Because release of granules is largely dependent on thromboxane, the aspirin effect produces a similar platelet aggregation profile to that of SPD when ADP or epinephrine is used, but stronger agonists such as thrombin and collagen can circumvent the thromboxane pathway and produce a normal aggregation curve. C. Due to lack of GP IIb/IIIa expression on the platelet surface, platelets from patients with Glanzmann thrombasthenia show absent aggregation to all agonists except ristocetin.

  Aspirin irreversibly inhibits the platelet cyclooxygenase (COX-1) enzyme, which is responsible for the conversion of membrane-associated arachidonic acid to thromboxane A₂ (TxA₂); the inhibition is constant for the entire life span of the platelet (7–10 days). Platelet aggregation studies (Fig. 21.2) show decreased reactivity to most agonists, including low concentrations of thrombin and collagen, and normal aggregation with high concentrations of thrombin and collagen. Using the PFA-100 system, aspirin-induced platelet dysfunction is evident in an increased time to aperture occlusion with the epinephrine/collagen reagent, while that of the ADP/collagen reagent is unaffected. ¹³

  NSAIDs reversibly inhibit platelet COX-1; their inhibitory effect persists only as long as the drug is present in the circulation. Selective inhibitors of COX-2 do not bind or impair platelet COX-1.

  Platelet glycoprotein IIb/IIIa inhibitors are used in the management of patients with acute coronary syndromes or before or following percutaneous coronary intervention, frequently in conjunction with heparin. Eptifibatide is a small molecule that binds the GP IIb/IIIa receptor, inhibiting the binding of its ligands, fibrinogen, VWF, and others, hindering platelet aggregation. Abciximab is a monoclonal antibody against GP IIb/IIIa that also inhibits binding of these proaggregatory ligands. Abciximabbound platelets can be cleared at an accelerated rate due to interaction between the Fc portion of the antibody and Fc receptors on reticuloendothelial macrophages in the liver and spleen, producing thrombocytopenia that in some cases (<1.0%) is severe.

Table 21.5  Substances Associated with Platelet Dysfunction*

Platelet-Directed Agents

Aspirin

NSAIDS (except COX-2 inhibitors)

Dipyridamole (Aggrenox ®)

Clopidogrel (Plavix ®)

Ticlopidine (Ticlid ®)

Prasugrel (Effient ®)

Abciximab (ReoPro ®)

Eptifibatide (Integrilin ®)

Tirofiban (Aggrastat ®)

Anesthetics

Dibucaine

Procaine

Halothane

Antibiotics

Penicillins (penicillin G, ticarcillin, nafcillin, piperacillin, methicillin, ampicillin)

Cephalosporins (cefazolin, cefotaxime)

Nitrofurantoin

Chemotherapeutic Drugs

Car-mustine

Daunorubicin

Mithramycin

Psychiatric Medications

Selective serotonin reuptake inhibitors (e.g., fluoxetine, paroxetine, sertraline)

Tricyclic antidepressants (e.g., imipramine, amytriptyline, nortriptyline)

Other Agents

Nitrates

Antihistamines (diphenhydramine, chlorpheniramine)

Ethanol

Omega-3 fatty acids (eicosapentaenoic acid)

“Wood ear” mushrooms

Radiographic contrast dye

* Most of these agents have been reported to cause abnormalities in platelet aggregation or the bleeding time, rather than bleeding. Adapted from George JN, Shattil SJ. Acquired disorders of platelet function. In: Hoffman R, Benz EJ, Shattil SJ, et al., eds. Hematology: Basic Principles and Practice. 3rd ed. New York, NY: Churchill Livingstone; 2000:2174.

  Ticlopidine, clopidogrel, and prasugrel irreversibly inhibit the binding of ADP to its receptor on the platelet membrane, impairing the ADP-dependent binding of fibrinogen to GP IIb/IIa, decreasing platelet aggregation. Neutropenia and aplastic anemia have been reported with increased frequency in patients taking ticlopidine; TTP has been described with use of ticlopidine and less frequently with clopidogrel. ¹⁵

  Dipyridamole is used to prevent recurrent stroke or transient ischemic attack, usually in conjunction with aspirin. It inhibits ADP- and collagen-induced platelet aggregation via an effect on intracellular cyclic Adenosine Monophosphate (AMP).

  Other substances. Serotonin reuptake inhibitors may impair the function of platelets by reducing the serotonin content of platelet-dense granules.¹⁶ Omega-3 fatty acids may disrupt the phospholipid membrane of the platelet and interfere with reactions of coagulation that normally take place on the platelet surface. ¹⁷

Myelodysplasia/Myeloproliferative Diseases

The platelets that are produced in myelodysplasia and the myeloproliferative disorders (chronic myeloid leukemia, essential thrombocythemia, polycythemia vera, and idiopathic myelofibrosis) may show abnormal receptor-ligand interactions, ineffective signal transduction, or decreased secretion of platelet granule contents; in a minority of patients these abnormalities lead to bleeding.¹⁸

Renal Failure/Uremia

Platelets from individuals with impaired kidney function frequently show abnormalities upon aggregation testing. Although plasma urea itself may not be causative, other factors, such as dysfunctional VWF and increased levels of nitric oxide and cyclic GMP, may lead to clinically important bleeding, especially gastrointestinal. DDAVP (standard doses), cryoprecipitate, and high-dose estrogens (Premarin, 50 mg single dose) have been suggested to be of benefit in uremia-related bleeding.¹⁹ Because the presence of adequate numbers of intravascular red cells may facilitate interaction of platelets with the vessel wall, red cell transfusions or erythropoietin are recommended in patients with anemia related to renal failure who are bleeding, to keep the hematocrit above 30%.¹⁹ Platelet transfusion may be beneficial temporarily if other measures fail and bleeding persists. If a dialyzable substance in the uremic plasma is responsible for the defect in platelet function, hemodialysis also may be beneficial, albeit temporarily.¹⁹

Cardiac Bypass

Cardiac bypass causes defects in both platelet number and function.²⁰ As platelets pass through the extracorporeal oxygenating circuit, they contact the artificial surfaces of the system and are activated; they also are fragmented by distortional trauma. Both phenomena lead to their accelerated clearance. Following bypass, a decrease in the platelet count, abnormalities in platelet morphology on the blood smear, and impaired in vitro platelet aggregation are observed in most patients; but these effects typically persist for 24 to 48 hours following bypass. Platelet transfusion may be given for serious bleeding.

Inherited Disorders

Inherited disorders of platelet function are rare and produce varying degrees of platelet-type bleeding, usually beginning within the first decade of life.¹¹ Heritable disorders of platelet function may also remain clinically silent until unmasked by a significant hemostatic challenge. Prophylaxis prior to invasive procedures or treatment of significant hemorrhage (Table 21.6) typically involves transfusion of normal platelets. Early use of antifibrinolytic agents may decrease transfusion requirements. DDAVP may be effective in platelet disorders characterized by normal dense granules, whereas recombinant factor VIIa may be helpful as only an adjunctive measure to platelet transfusion when transfusions alone have been ineffective (Table 21.6).

Bernard-Soulier Syndrome

Bernard-Soulier syndrome (BSS) comprises a triad of large platelets, moderate thrombocytopenia, and a prolonged bleeding time; individuals with this disorder have reduced or abnormal expression of platelet glycoprotein Ib/IX (the receptor for VWF) on the surface of their platelets. BSS is autosomal recessively inherited. Platelet aggregation studies are normal with all agonists except ristocetin. BSS is distinguished from VWD in that the reduced RIPA in BSS is corrected by the addition of normal platelets whereas in VWD, it is corrected by the addition of normal plasma (which contains adequate VWF). The diagnosis can be confirmed by platelet flow cytometry.

Glanzmann Thrombasthenia

Glanzmann thrombasthenia is a recessively inherited qualitative or quantitative abnormality in GP IIb/IIIa expression on the platelet surface.²¹ Without adequate functional IIb/IIIa to bind fibrinogen and VWF (both of which cross-link platelets), platelet aggregation is markedly impaired (Fig. 21.2). Patients may present with mucocutaneous bleeding in infancy. In pregnant patients, the condition is associated with maternal and fetal hemorrhage, and a third of women experience primary postpartum hemorrhage (up to 20 days after delivery).²² Isoantibodies against the missing or defective platelet integrin may neutralize transfused platelets in some cases, suggesting a role for rVIIa as a platelet-sparing measure.²³ The diagnosis can be confirmed by platelet flow cytometry.

Storage Pool Disease

Storage pool disease (SPD) is characterized by abnormalities in number or content of platelet granules.²⁴ Defects in alpha-granules, dense granules, or both (Table 21.4) may be present. Platelet aggregation to ADP (Fig. 21.2) typically shows an initial wave of aggregation, but the aggregates subsequently dissociate due to reduced or absent release of granule contents, which reinforce the aggregatory response. Most patients have a prolonged bleeding time. A variable bleeding diathesis results. More commonly, patients may have release defects wherein granules are present, but signaling necessary for release of granule contents is defective.

  Albinism-associated SPD occurs in the context of disorders characterized by oculocutaneous albinism, such as the Hermansky-Pudlak and Chediak-Higashi syndromes. Impaired biogenesis of dense granules, lysosomes, and melanosomes may be responsible for the reduced number of dense granules in these patients.

  Non-albinism-associated SPD occurs in a variety of other conditions (TAR syndrome, Ehlers-Danlos syndrome, Wiskott-Aldrich syndrome, Osteogenesis imperfecta). Defects in dense granules may relate more to granular content rather than number and may occur in conjunction with alpha-granule abnormalities (alpha-delta SPD).²⁵ Decreased or empty dense granules can be visualized on electron microscopy.

  The Gray Platelet Syndrome: The gray platelet syndrome is a rare, inherited disorder characterized by abnormalities of platelet alpha granules, thrombocytopenia, and fibrosis in the bone marrow; consanguinity is common. A lifelong history of mild to moderate mucocutaneous bleeding usually is present. Review of the blood smear typically shows agranular platelets that appear “gray” on Wright staining due to a lack of azurophilic granules. In contrast to dense granule deficiency, platelet aggregation to epinephrine, ADP, and arachidonic acid is often normal, while thrombin and collagen produce variable results. The diagnosis is confirmed with electron microscopy.

  Quebec Platelet Disorder²⁶: The Quebec platelet disorder is an extremely rare disorder that is characterized by abnormal alpha-granule content and mild thrombocytopenia, leading to a moderate bleeding diathesis. Bleeding is unresponsive to platelet transfusion. Increased intraplatelet urokinase-type plasminogen activator results from a tandem duplication of the PLAU gene.

Scott Syndrome

Scott syndrome is an extremely rare disorder characterized by spontaneous bleeding due to a defect in scramblase, which is necessary for adequate expression of phosphatidylserine on the outer leaflet of the platelet membrane and normal binding of coagulation factors.²⁷ Patients may show a loss-of-function mutation in TMEM16F.²⁸

Congenital Disorders of Signal Transduction

These include defects in receptor-agonist interactions, G-protein activation, platelet enzymatic activity, and phosphorylation of signaling proteins.¹²

Isolated Laboratory-Specific Defects

Individuals with phenotypically normal hemostasis occasionally demonstrate reduced or (less frequently) absent aggregation to one or more agonists on platelet aggregation testing. These abnormalities, which may be genetically determined, probably reflect inter-individual differences in the reactivity of platelets to certain ligands and do not necessarily indicate an increased risk for spontaneous or traumainduced hemorrhage, unless a tendency to bleed has been previously demonstrated.

Other Conditions

The May-Hegglin anomaly²⁹ features mild to moderate thrombocytopenia, large platelets, and characteristic leukocyte azurophilic inclusions (Dohle bodies). While the large size of the platelets implies a qualitative abnormality, patients generally do not bleed excessively and aggregation studies are normal. An autosomal dominantly inherited disorder, it is a manifestation of mutated non-muscle myosin heavy chain IIA, which has been implicated in the related disorders Sebastian syndrome, Fechtner syndrome, and Epstein syndrome; these feature varying degrees of sensorineural hearing loss, nephritis, cataracts, and leukocyte inclusions. Eltrombopag has been used in some patients with severe thrombocytopenia with a resulting decrease in bleeding manifestations.³⁰

References

  1.  Levi M. Disseminated intravascular coagulation: a disease-specific approach. Semin Thromb Hemost. 2010;36(4):363-365.

  2.  Levi M, Schultz M, van der Poll T. Disseminated intravascular coagulation in infectious disease. Semin Thromb Hemost. 2010;36(4):367-377.

  3.  Thachil J, Toh CH, Levi M, Watson HG. The withdrawal of Activated Protein C from the use in patients with severe sepsis and DIC [Amendment to the BCSH guideline on disseminated intravascular coagulation]. Br J Haematol. 2012; 157(4):493-494.

  4.  Wiedermann CJ, Kaneider NC. A systematic review of antithrombin concentrate use in patients with disseminated intravascular coagulation of severe sepsis. Blood Coagul Fibrinolysis. 2006;17(7):521-526.

  5.  Stenczer B, Molvarec A, Szabo G, et al. Circulating levels of thrombospondin-1 are decreased in HELLP syndrome. Thromb Res. 2012;129(4):470-473.

  6.  Chang H, Kuo MC, Shih LY, et al. Clinical bleeding events and laboratory coagulation profiles in acute promyelocytic leukemia. Eur J Haematol. 2012;88(4):321-328.

  7.  Streiff MB. The National Comprehensive Cancer Center Network (NCCN) guidelines on the management of venous thromboembolism in cancer patients. Thromb Res. 2010;125(suppl 2):S128-133.

  8.  Favaloro EJ. Diagnosis and classification of von Willebrand disease: a review of the differential utility of various functional von Willebrand factor assays. Blood Coagul Fibrinolysis. 2011;22(7):553-564.

  9.  Nichols WL, Hultin MB, James AH, et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia. 2008;14(2):171-232.

10.  James AH, Kouides PA, Abdul-Kadir R, et al. Von Willebrand disease and other bleeding disorders in women: consensus on diagnosis and management from an international expert panel. Am J Obstet Gynecol. 2009;201(1):12 e11-18.

11.  Lambert MP. What to do when you suspect an inherited platelet disorder. Hematology Am Soc Hematol Educ Program. 2011;2011:377-383.

12.  Rao AK, Gabbeta J. Congenital disorders of platelet signal transduction. Arterioscler Thromb Vasc Biol. 2000;20(2):285-289.

13.  Crescente M, Di Castelnuovo A, Iacoviello L, Vermylen J, Cerletti C, de Gaetano G. Response variability to aspirin as assessed by the platelet function analyzer (PFA)-100. A systematic review. Thromb Haemost. 2008;99(1):14-26.

14.  Hayward CP, Harrison P, Cattaneo M, Ortel TL, Rao AK. Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J Thromb Haemost. 2006;4(2):312-319.

15.  Zakarija A, Kwaan HC, Moake JL, et al. Ticlopidine- and clopidogrel-associated thrombotic thrombocytopenic purpura (TTP): review of clinical, laboratory, epidemiological, and pharmacovigilance findings (1989–2008). Kidney Int Suppl. 2009(112):S20-24.

16.  Meijer WE, Heerdink ER, Nolen WA, Herings RM, Leufkens HG, Egberts AC. Association of risk of abnormal bleeding with degree of serotonin reuptake inhibition by antidepressants. Arch Intern Med. 2004;164(21):2367-2370.

17.  Cohen MG, Rossi JS, Garbarino J, et al. Insights into the inhibition of platelet activation by omega-3 polyunsaturated fatty acids: beyond aspirin and clopidogrel. Thromb Res. 2011;128(4):335-340.

18.  Vladareanu AM, Vasilache V, Bumbea H, Onisai M. Platelet dysfunction in acute leukemias and myelodysplastic syndromes. Rom J Intern Med. 2011;49(1):93-96.

19.  Hedges SJ, Dehoney SB, Hooper JS, Amanzadeh J, Busti AJ. Evidence-based treatment recommendations for uremic bleeding. Nat Clin Pract Nephrol. 2007;3(3):138-153.

20.  Flaujac C, Pouard P, Boutouyrie P, Emmerich J, Bachelot-Loza C, Lasne D. Platelet dysfunction after normothermic cardiopulmonary bypass in children: effect of high-dose aprotinin. Thromb Haemost. 2007;98(2):385-391.

21.  Nurden AT, Fiore M, Nurden P, Pillois X. Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models. Blood. 2011;118(23):5996-6005.

22.  Siddiq S, Clark A, Mumford A. A systematic review of the management and outcomes of pregnancy in Glanzmann thrombasthenia. Haemophilia. 2011;17(5):e858-869.

23.  Fiore M, Firah N, Pillois X, Nurden P, Heilig R, Nurden AT. Natural history of platelet antibody formation against alphaI-Ibbeta3 in a French cohort of Glanzmann thrombasthenia patients. Haemophilia. 2012;18(3):201-209.

24.  Sandrock K, Zieger B. Current strategies in diagnosis of inherited storage pool defects. Transfus Med Hemother. 2010;37(5):248-258.

25.  White JG, Keel S, Reyes M, Burris SM. Alpha-delta platelet storage pool deficiency in three generations. Platelets. 2007;18(1):1-10.

26.  Paterson AD, Rommens JM, Bharaj B, et al. Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene. Blood. 2010;115(6):1264-1266.

27.  Flores-Nascimento MC, Orsi FL, Yokoyama AP, et al. Diagnosis of Scott syndrome in patient with bleeding disorder of unknown cause. Blood Coagul Fibrinolysis. 2012;23(1):75-77.

28.  Lhermusier T, Chap H, Payrastre B. Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome. J Thromb Haemost. 2011;9(10):1883-1891.

29.  Filanovsky K, Shvidel L, Vorst E, Berrebi A, Shtalrid M. The May-Hegglin anomaly. Eur J Haematol. 2009;83(4):390.

30.  Pecci A, Gresele P, Klersy C, et al. Eltrombopag for the treatment of the inherited thrombocytopenia deriving from MYH9 mutations. Blood. 2010;116(26):5832-5837.

*Especially in early [Q21]DIC, the platelet count and fibrinogen may be reduced from the baseline value but still remain within the normal laboratory reference range.

† Pseudo or platelet type VWD is caused by a defect in the platelet GPIb molecule, allowing it to bind to the patient’s normal VWF with increased avidity and leading to a type 2B clinical phenotype. Mixing studies using a modified RIPA (patient’s platelets and control plasma) distinguish it from type 2B VWD.

22

Venous Thromboembolism

Elisabet E. Manasanch and Jay N. Lozier

Venous thromboembolism (VTE) is a major health problem in the United States with more than 900,000 estimated cases annually.¹ The average yearly incidence is 117 cases per 100,000 population, with higher rates in women of childbearing age, males over 45 years old, and the elderly (where rates are up to five-fold higher). Pulmonary embolism (PE) occurs about 60% as often as deep venous thrombosis (DVT) and has a high mortality; the incidence may be higher since the diagnosis is often missed in hospitalized patients.2,3 Chronic VTE-induced pulmonary hypertension is a late complication of PE in 1% to 4% of cases after 4 years of follow-up, with all cases occurring before 2 years.4,5 Most cases of DVT occur in the lower extremities, but virtually any venous vascular bed can be involved. Upper extremity DVTs represent 1% to 5% of the total and are usually associated with long-term indwelling central venous access devices, thrombophilia, and/or cancer.⁶ When upper extremity DVTs occur, the potential for subsequent pulmonary embolization is estimated to be as high as 36% in some trials,⁷ though fatal pulmonary embolism is less common than for DVT of the lower extremities.

DEEP VENOUS THROMBOSIS AND PULMONARY EMBOLISM

DVT of the extremity is often the precursor of PE, though both may become symptomatic at the same time, and it is possible that PE may occur without symptomatic DVT, due to complete embolism of nascent thrombus before complete occlusion of the vein occurs. One autopsy series indicates that a substantial number of patients with PE have no pathologic evidence for DVT.⁸ The signs and symptoms of DVT and PE are listed in Table 22.1.7,9

The postthrombotic syndrome (PTS) is an important complication of DVT. It is caused by venous hypertension from outflow obstruction and valvular injury and varies from mild edema with little discomfort to incapacitating limb swelling with pain and ulceration. The severity of PTS can be assessed using the Villalta scale,¹⁰ which is based on the cumulative rates of signs and symptoms characteristic of the syndrome (Table 22.2).

The reported incidence of PTS after an acute episode of DVT has been reported to range from 23% to 60%¹¹; severe, disabling PTS with skin breakdown and ulceration is seen much less commonly, perhaps in <10% of cases, while mild symptoms probably are experienced in the majority of DVT cases. Surprisingly, contralateral extremities may also develop postthrombotic manifestations without prior evidence of overt DVT¹¹; perhaps occult obstruction of the inferior vena cava (IVC) may be to blame. Sized-to-fit graded compression stockings (typically 20–40 mm pressure) should be applied shortly after diagnosis of DVT to prevent acute dilatation that may result in permanent damage to the valves. If there is poor circulation in the leg due to complete obstruction of venous outflow, compression stockings should be used cautiously or withheld if the increased compression threatens to stop blood flow altogether. After definitive therapy (thrombolysis or mechanical clot extraction) and venous flow is restored to some extent, compression stockings may be reconsidered to mitigate acute symptoms and eventual PTS. A sized-to-fit compression stocking can reduce the rate of PTS by about 50%.¹² Compression stockings do not substitute for adequate anticoagulation but are useful adjuncts to exercise-based rehabilitation programs. Stockings that are not fitted to provide 20 to 40 mm Hg compression do not confer equivalent benefits as seen from fitted stockings. A poorly fitted stocking can actually be detrimental, for instance, when the upper parts of the stocking roll down and form a loose “tourniquet” and thereby impair return blood flow from the leg. Unfortunately, compression stockings or sleeves for upper extremity DVTs have not proven to be as beneficial, perhaps due to the lesser hydrostatic pressures involved with the upper extremity.

Distal DVT is defined as thrombus found below the popliteal vein trifurcation, and occurs most commonly (71% of the time) in the peroneal vein.¹³ The risk for PE is almost negligible, whether treated with anticoagulation, or not; propagation to other distal calf veins does occur in about half of all patients, and propagation to more proximal deep veins is seen in ~5% of patients.13,14 Lysis of isolated calf DVT typically occurs within 3 months.¹³ The risk of anticoagulation under these circumstances (0%–6% bleeding risk) is approximately equal to the benefits realized (less propagation), so it remains controversial whether anticoagulation is necessary. A recent systematic review suggests that anticoagulation or serial imaging with noninvasive methods may be equally valid strategies.¹⁵ PTS occurs in ~5% of patients in the long term but is not characterized by severe changes such as skin ulceration.¹⁵

Table 22.2  The Villalta Scale for Evaluation of Postthrombotic Syndrome (PTS)

Symptoms

Pain

Cramps

Heaviness

Paresthesia

Pruritus

Clinical Signs

Edema

Venous dilatation/ectasia

Hyperpigmentation

Erythema

Lipodermatosclerosis (skin induration)

Pain during calf compression

Characteristics of PTS. Scoring is based on the cumulative rating of the signs and symptoms, with each rated a 0 (absent), 1 (mild), 2 (moderate), 3 (severe).Total score: 0–4, no PTS; 5–14, mild to moderate PTS; ≥15 or presence of a venous ulcer, severe PTS.

IMAGING OF DEEP VENOUS THROMBOSIS

Venography remains the standard for diagnostic imaging of DVT, but it is used less commonly than ultrasonography since it is an invasive procedure that uses radiocontrast dye and requires a skilled operator to perform the injection. Further, there is the need to bring a patient to a fluoroscopy suite, which may not be feasible in the acutely ill patient in an intensive care unit with other comorbidities. In contrast, ultrasonography is noninvasive, portable, and does not use contrast to which the patient may be allergic. Doppler ultrasound instruments are typically portable and can be brought to the bedside even in the most acutely ill patients. Venography retains an advantage for diagnosis of small distal DVTs that are not well imaged by ultrasound, as well as thrombosis of the vena cava or iliac veins of the pelvis that are not accessible to ultrasound examination because they are obscured by bowel gas. Venography may also be useful in instances where ultrasound is not feasible or an unequivocal diagnosis of DVT must be made. Ultrasonography is considerably more sensitive for the detection of proximal DVT than for distal DVT.

Sensitivity of compression ultrasound with venous imaging ranges from 89% to 96% when DVT is diagnosed by a combination of direct visualization of an occlusive thrombus and noncompressibility of a vein. The specificity of this finding for DVT ranges from 94% to 99%, but, unfortunately, the sensitivity may be substantially diminished (47%–62%) in patients with asymptomatic DVT.¹⁶ Serial ultrasound testing (which has little risk to the patient in contrast to venography) improves sensitivity, as a previously undiagnosed distal DVT may declare itself by proximal propagation. Furthermore, an ultrasound can accurately diagnose certain conditions that occasionally mimic DVT, such as Baker cyst. Impedance plethysmography may be useful in differentiating between a new or recurrent DVT, especially if the previous DVT has not resolved.

Magnetic resonance venography (MRV) and computed tomography venography (CTV) can diagnose DVT in a noninvasive manner. Prospective studies comparing CTV with venous ultrasound for diagnosis of DVT reported sensitivity rates of 100% and specificity of 96% to 100%.17,18 CTV can be easily combined with computed tomography angiography in patients suspected to have PE. However, it always requires the administration of IV contrast. One prospective blinded study reported the sensitivity to be >94% and the specificity to be >90% for the diagnosis of DVT using noncontrast MRV direct thrombus imaging.¹⁹ A major advantage for CTV and MRV is that deep abdominal, pelvic, and calf veins can be imaged. Furthermore, MRV can be successfully used avoiding contrast and its risks (such as gadolinium-associated systemic fibrosis in patients with chronic kidney disease20,21). Major disadvantages include cost, availability, expert reading, and possible need for IV contrast use when compared to ultrasound techniques.

PULMONARY EMBOLISM DIAGNOSIS: ECHOCARDIOGRAPHY, ELECTROCARDIOGRAPHY, AND X-RAY

PE is thought to be the consequence of clot breaking free from a lower extremity DVT. Upper extremity DVT embolization is less common, but the increasing use of central venous access devices for cancer chemotherapy or other long-term parenteral treatment may increase its frequency. Less commonly, PE may originate in the IVC (particularly in association with renal cell carcinoma) or the right ventricle of the heart from mural thrombus. There is intriguing evidence from the study of acute trauma patients⁸ that pulmonary “embolism” can be seen without concurrent DVT, raising the possibility that some pulmonary emboli may actually be in situ pulmonary thrombosis. Regardless of the mechanism, imaging approaches to diagnose pulmonary embolism include pulmonary angiography (the standard), CT angiography, ventilation/perfusion scanning, and more recently MRI. Echocardiographic confirmation of right ventricular hypertension may assist in the decision regarding thrombolysis with right heart failure. Both echocardiography and spiral CT have a low sensitivity for PE located in peripheral pulmonary vessels. In severe cases chest x-ray findings may include a Hampton hump (a wedge-shaped opacity with apex pointing to the hilum) or a focal paucity of blood vessel perfusion. Ancillary diagnostic chest radiography, ECG, and echocardiographic findings suggestive of PE are listed in Table 22.3.

LABORATORY DIAGNOSIS OF VENOUS THROMBOEMBOLISM

The D-dimer is a quantitative assay with good reproducibility that is often automated and can be used as an adjunct to imaging studies to exclude the presence of PE or DVT.²²⁻²⁴ When a thrombus is degraded by plasmin, D-dimers and other fibrin split products are formed from cross-linked chains of the fibrin clot—as such, they are markers for fibrin turnover. The use of D-dimer is preferable to the semi-quantitative fibrin split products that are measured by latex agglutination methods at varying dilutions of patient plasma. If a patient has a low pretest probability of VTE and the sensitive ELISA test for D-dimers is negative, as a practical matter VTE can be excluded. The low specificity for VTE requires that further diagnostic testing be conducted in the event of an elevated D-dimer to elucidate its etiology. Many other conditions can cause elevation of the D-dimer, including cancer, pregnancy, sepsis, sickle cell crisis, acute myocardial infarction, cardiopulmonary resuscitation, excessive bleeding, trauma, and recent surgery. Measurement of D-dimers may also guide the duration of anticoagulation for VTE, since the optimal course of therapy for such patients has not been established.²⁵

The PROLONG Study indicated that patients with positive D-dimers 1 month after the completion of at least 3 months anticoagulation with vitamin K antagonists (VKAs) for idiopathic VTE had a significantly higher risk for recurrent VTE, which was mitigated by prolonged anticoagulation. Patients with abnormal D-dimers who did not resume anticoagulation experienced a 15% incidence of rethrombosis over the 18-month observation period compared to 2.9% if anticoagulation was restarted.²⁶ The adjusted hazard comparing the rates of recurrence was 4.26 (95% CI 1.23–14.6, p = 0.02). A follow-up study, the PROLONG II, assessed the utility of repeated D-dimer testing in patients with a first unprovoked episode of VTE with normal D-dimer 1 month after stopping VKAs. D-dimer was tested at study initiation and every 2 months thereafter with a follow-up period of 13 months; 14% of patients with an initially negative D-dimer had a positive test at month 3 of evaluation. Furthermore, the D-dimer became abnormal at each subsequent time point in about 10–15% of patients up to 9 months of follow-up, at which time it decreased to 8–10%. An abnormal D-dimer at the first measurement or at day 30 usually remained abnormal over time in the majority of cases, and this pattern was associated with an increased risk of rethrombosis. The rates of recurrence for patients with an abnormal D-dimer at 3 months was 22.6% (95% CI 10%–41%) compared to 4.6% (95% CI 2%–9%) if D-dimer was normal (p = 0.003).²⁷ Repeat D-dimer testing may help identify the subgroup of patients at lower risk for recurrence, in whom anticoagulation could be stopped. A multicenter prospective study (DULCIS, http://clinicaltrials.gov:NCT00954395) is currently underway to clarify this issue.

Table 22.3  Ancillary Findings Diagnostic for Pulmonary Embolism

Chest Radiography

Hampton hump

Focal paucity of blood vessel perfusion (Westermark sign)

Dilated pulmonary artery proximal to the thrombus

Atelectasis

Pleural effusion

Elevated diaphragm

Electrocardiography

New right bundle branch block

S1Q3T3 pattern (sign of acute cor pulmonale)

Supraventricular arrhythmias

Echocardiography

Right ventricular dilatation, often with myocardial hypokinesis

Pulmonary artery dilatation

Right ventricular mural thrombi

Tricuspid regurgitation

Loss of inspiratory collapse of the inferior vena cava

DEEP VENOUS THROMBOSIS IN SITES OTHER THAN THE DISTAL VEINS OF THE LOWER EXTREMITIES

Aside from typical presentations involving the lower extremities, DVT can occur in other sites such as veins of the upper extremities (particularly in conjunction with central venous catheters commonly used in cancer patients for chemotherapy), or in the veins of the chest or abdomen. Bilateral upper extremity DVTs are uncommon and should prompt a search for malignancy. The effects of DVT in these sites can be devastating, even in the absence of PE. Perioperative DVT of the splanchnic veins is common, occurring more frequently in laparoscopic procedures than with open ones. Unprovoked DVT of the splanchnic vessels should prompt a search for underlying abnormalities of hemostasis, such as deficiency of anticoagulant proteins, undiagnosed cancer, hematologic diseases such as myeloproliferative disorders (MPD), or paroxysmal nocturnal hemoglobinuria (PNH).

Portal vein thrombosis can commonly occur as a complication of surgical procedures, especially splenectomy, or during pregnancy, or with peritonitis. In cases that are not associated with a precipitating factor, antiphospholipid antibodies, deficiency of protein C, protein S, or (less commonly) anti-thrombin III may be found; the factor V Leiden and prothrombin 20210 gene polymorphisms may also be seen in patients at rates greater than in the general population (as is true in any group of patients with pathologic thrombosis). Clonal V617F point mutation in the Janus 2 kinase (JAK2V617F) tyrosine kinase gene occurs in a large proportion of MPD (particularly polycythemia rubra vera), and is found in 45% of BCS and 34% of portal vein thrombosis. Table 22.4 indicates the prevalence of various prothrombotic states in patients with portal or hepatic vein thrombosis.²⁸⁻³⁰

Treatment options for splanchnic vein thrombosis include anticoagulation, thrombolysis, and in the most extreme cases consideration should be given to orthotopic liver transplantation for those with liver failure. In addition, acute reduction of red blood cell mass and/or platelet count may be beneficial if the splanchnic vein thrombosis is due to polycythemia vera (PV) or essential thrombocythemia.

ACQUIRED THROMBOPHILIC STATES

Heparin-Induced Thrombocytopenia and Heparin-Induced Thrombocytopenia with Thrombosis

Heparin-induced thrombocytopenia (HIT) is a prothrombotic state caused by a drug reaction to unfractionated heparin (UFH) and less commonly to low molecular-weight heparin (LMWH), which can also exacerbate HIT due to cross reactivity with UFH. Even small exposures to UFH, including flushing of intravenous lines, may precipitate HIT and even thrombosis, in patients with antibodies. Antibodies of the IgG class are formed that produce a strong activation of platelets through their FcγIIa receptors. They recognize large multimolecular complexes of platelet factor 4 bound to heparin (PF4/H), although only about 10% of all anti-PF4/H antibodies have platelet-activating properties.³¹ This activation promotes thrombosis in vivo in both venous and arterial sites. HIT is a “clinical-pathologic syndrome,” requiring both a compatible clinical picture and positive laboratory test results. In general, platelet counts begin to decrease 5 to 9 days after the initiation of heparin. Thrombocytopenia and thrombosis may occur earlier in patients primed by heparin administration in the prior 100 days. Clinical scoring systems have been implemented to evaluate the pretest probability of HIT: 4Ts³² and most recently the HIT Expert Probability (HEP) score³³ (Tables 22.5 and 22.6).

Low scores usually indicate a <2% probability of having a positive platelet activation assay. Two types of assays are used to establish diagnosis: enzyme immunoassays, such as an ELISA that detects PF4-heparin antibodies, and platelet activation/aggregation assays which detect spontaneous aggregation of platelets induced by the addition of heparin to the patient’s platelet rich plasma. Sensitivity of platelet aggregation assays can be increased when patient’s platelets are “loaded” with radioactive serotonin; serotonin release from platelets is detected as a marker of platelet activation in vitro, after the addition of heparin. The most sensitive test is the serotonin-loaded platelet aggregation assay. False positive results are common with the ELISA test, and its specificity varies depending on the pretest clinical probability. A negative result usually excludes HIT.

Two different classes of anticoagulants are used for the treatment of HIT in the presence or absence of thrombosis: direct thrombin inhibitors, lepirudin, bivalirudin, and argatroban, which are approved in the United States for the treatment of HIT and the factor Xa inhibitor fondaparinux, which is currently approved for DVT prophylaxis and treatment, but has growing evidence in literature to be an effective treatment for HIT.³⁴ Fondaparinux binds anti-PF4 antibodies but does not activate platelets, unlike traditional LMWH preparations such as enoxaparin, dalteparin, and tinzaparin, which crossreact with the PF4-heparin antibodies and can activate platelets. Fondaparinux binds irreversibly to factor Xa and has a long active half-life (17 hours), which may prevent rebound hypercoagulability, but cannot be readily reversed if bleeding ensues. Dosing needs to be adjusted for impaired renal function. Lepirudin and argatroban require intravenous continuous infusions due to their short half-lives (<2 hours), and need PTT monitoring. Lupus anticoagulants (LAC) and/or high factor VIII levels may result in prolonged or shortened clotting times, respectively, thereby complicating monitoring. Algorithms exist for infusions of argatroban without monitoring when LAC interfere with monitoring.³⁵ Levels of argatroban or other direct thrombin inhibitors can be monitored more directly by use of assays of the ecarin clotting time; ecarin is a snake venom that converts fibrinogen to fibrin but is not influenced by LAC or levels of factor VIII.³⁶ Lepirudin is antigenic, and antibody formation results in excessive anticoagulation. Lepirudin is contraindicated in the presence of renal insufficiency. Bivalirudin (approved in HIT specifically for patients undergoing percutaneous coronary intervention) and argatroban are both hepatic-excreted, nonantigenic, and short-acting, and they should be used cautiously in patients with liver disease. Initiation of warfarin should be postponed at least until the platelet count is above 150,000/μL, since warfarin use during acute HIT is a major risk factor for venous limb gangrene, perhaps on the basis of rapid depletion of protein C, a vitamin-dependent anticoagulant protein with a short half-life (6–7 hours).

Nephrotic Syndrome

Patients with nephrotic syndrome are at higher risk for VTE, arterial thromboembolism (ATE), and renal vein thrombosis than is the general population [annual incidence, 9.85% (VTE) and 5.52% (ATE)].³⁷ Membranous glomerulonephritis confers the highest risk of VTE. Hypercoagulability appears due to alterations in plasma levels of proteins involved in coagulation and fibrinolysis. The anticoagulant proteins antithrombin III and protein S are lost in the urine, perhaps due to their relatively small size compared to procoagulant factors V, VIII, von Willebrand factor, and fibrinogen, which may be retained by the kidney.³⁸ Further, fibrinogen, factor VIII, and von Willebrand factor are acute-phase reactants, and their levels may be increased by inflammation.³⁹ The routine use of prophylactic anticoagulation for patients with nephrotic syndrome has not been established by randomized controlled trials, but some experts advocate this when additional risk factors such as membranous glomerulonephritis or the antiphospholipid syndrome are present. LMWH must be used with extreme caution in patients with renal insufficiency in addition to nephrotic syndrome.

Antiphospholipid Antibody Syndrome

Antiphospholipid antibodies (APA) may be associated with thrombosis on the basis of various mechanisms,⁴⁰ summarized in Table 22.7. Anticardiolipin antibodies are APA associated with infections such as syphilis, but these are transient and not usually associated with thrombosis.

Tests for APA include specific assays to quantify levels directly by ELISA or indirectly through the detection of antiβ2-GPI antibodies. The APA that prolong phospholipid-dependent clotting assays (primarily the aPTT) are designated as LAC. Other, less commonly used assays that are affected by LAC include the dilute Russell viper venom time (DRVVT), the dilute prothrombin or tissue thromboplastin inhibition (TTI) test, and the kaolin clotting time (KCT). Confirmatory tests for LAC are performed by adding a source of phospholipids to a clotting reaction that is prolonged by the lupus anticoagulant to see if the abnormal clotting time is corrected. Phospholipids may include platelets (in the platelet neutralization procedure, or PNP), or more recently phospholipids with defined physical properties, such as hexagonal-phase lipids that are used in some commercial tests. The hematologist consultant should be aware of the specific screening and confirmatory tests that are used in the local laboratory when diagnosing APA syndromes or LAC. Also important is that LAC tests may be positive at the time of an acute thrombotic event but often are negative at subsequent time points only a few weeks later. In a study of 30 patients with lower extremity DVT that were treated with tPA, 19 of the 30 were initially positive for LAC at the time of presentation, but 11 of these were documented to be negative 6 months later (or sooner), three remained positive at later time points, and there were six lost to follow-up and one with an indeterminate value at later time points (unpublished observations on patients described in Refs. 41 and 42).

Table 22.7  Mechanisms of Thrombosis Associated with Antiphospholipid Antibodies (APA)

Binding of APA to β₂-glycoprotein I (β₂-GPI)/phospholipid complex exposed on the surface of injured or activated endothelial cells and monocytes

Overproduction of tissue factor by monocytes and endothelial cells

Activation of platelets that increases expression of glycoprotein IIb-IIIa and synthesis of thromboxane A2

Interaction with regulatory proteins such as annexin V, prothrombin, factor X, protein C, and plasmin

Two positive tests for APA (LAC, anticardiolipin antibody IgG or IgM, and anti-β2-glycoprotein I antibody IgG or IgM) obtained at least 12 weeks apart are required to fulfill the laboratory criteria for the APA syndrome. Clinical criteria for the diagnosis of the APA syndrome include detection of venous and/or arterial thrombotic events, autoimmune thrombocytopenic purpura, marantic endocarditis, multiple spontaneous abortions before the 10th week of gestation, or unexplained death of a morphologic normal fetus after the 10th week of gestation. Thrombosis can occur in virtually any vascular bed, and patients may present with a catastrophic syndrome with thrombosis in multiple vascular sites, including cerebrovascular accidents and DVT, with more than 50% mortality.

Treatment strategies should focus on modification or elimination of risk factors, such as smoking and oral estrogen contraceptives. For a thrombotic complication, systemic anticoagulation should be initiated. To prevent VTE recurrence, a randomized, double-blind, prospective study showed that dosing warfarin to an international normalized ratio (INR) of 2.0 to 3.0 was equally effective as higherintensity warfarin regimens to achieve an INR of 2.5 to 3.5.⁴⁰ However, patients with recurrent VTE or arterial thrombosis may need more aggressive treatment with an INR goal 3.0 to 4.0 or combined antithrombotic therapy (warfarin plus low-dose aspirin). This is based on the observation that the rate of recurrent thrombosis was low with an INR >3.0, and is not endorsed by all experts.⁴³

Hypercoagulability of Malignancy

VTE frequently complicates malignancy and results in significant morbidity and mortality. The estimated prevalence of VTE in patients with cancer is 10% to 15% and can be as high as 28% to 30% in pancreatic cancer or malignant gliomas.⁴⁴ Certain sites have been associated with VTE associated with malignancy: intra-abdominal and bilateral lower extremity DVT (p < 0.05), which may justify cancer screening that otherwise might not be performed.⁴⁵ Malignancy promotes thromboses through a variety of mechanisms: release of tissue factor, activation of factor X by cancer procoagulants(s), endotheliumtumor cell interactions, and platelet activation. Hypercoagulability associated with malignancies is designated Trousseau syndrome and manifests as disseminated intravascular coagulation, nonbacterial thrombotic endocarditis, PE, DVT, and arterial thromboses. Occasionally, chemotherapy agents promote thrombosis, possibly through direct injury to the vascular endothelium. Of equal importance is the observation that adjuvant therapy with selective estrogen receptor modulators (SERMS), such as tamoxifen, or antiangiogenic medications, such as thalidomide and lenalidomide for the treatment of multiple myeloma, and bevacizumab for the treatment of breast, colon, brain, or lung malignancies, can all increase the potential thrombogenicity of the cancer type. Central venous indwelling catheters often complicate cancer care because of thrombus formation in the catheter itself and the vessel into which it has been inserted.⁴⁶

Treatment of cancer-associated thrombosis is challenging. Warfarin, the current mainstay of long-term anticoagulation, may be difficult to manage due to concomitant medications and thrombocytopenia from chemotherapy or radiation; further, rethrombosis is common despite correct warfarin dosing. LMWH is the agent of choice for anticoagulation in cancer patients. Dalteparin is approved for the extended treatment of symptomatic VTE in patients with cancer based on the large clinical CLOT trial that showed a reduction in objectively confirmed, symptomatic DVT and/or PE during the 6-month study period when compared with oral anticoagulation (15.7% versus 8%).⁴⁷ Dalteparin appeared to reduce the incidence of rethrombosis most significantly in the first month of treatment compared to warfarin with a statistically significant (p = 0.03) improvement in overall survival (a secondary endpoint in the study) in patients with nonmetastatic disease. A similar trial randomized 138 patients to receive enoxaparin 1.5 mg/kg/day followed by warfarin for 3 months or enoxaparin 1.5 mg/kg/day for 3 months. There was a trend toward a decrease in VTE recurrence or major bleeding with enoxaparin versus warfarin (21.1% versus 10.5%; p = 0.09).⁴⁸ LMWH may exert an antineoplastic effect through interference with tumor cell adhesion, invasion, metastasis formation, and angiogenesis, all of which are needed for tumor progression. However, it is unclear whether this is true for all tumor types and further studies are needed.⁴⁹

Paroxysmal Nocturnal Hemoglobinuria

PNH causes intravascular hemolysis, bone marrow failure, and thrombotic events. Diagnosis can be made rapidly by detection of CD55- and CD59-deficient erythrocytes and neutrophils by flow cytometry. Patients with clones comprising greater than 50% of cells carry a high risk of thrombotic events (44% in 10 years), including unusual sites like the hepatic vain (Budd-Chiari syndrome) being a frequent manifestation.⁵⁰ Thrombosis is the most common cause of death in PNH. The etiology of thrombosis may involve release of free hemoglobin (which activates the endothelium), complement-mediated damage of GPI-deficient erythrocytes, and/or deficiency of GPI-anchored fibrinolytic factors such as urokinase/plasminogen activator receptor.⁵¹ The humanized monoclonal antibody eculizumab targets the C5 terminal complement component and is FDA approved for treatment of PNH. Eculizumab significantly reduced the rate of VTE in eculizumab-treated patients in a non-randomized trial when compared with the same patients pretreatment [1.07/100 patient years, versus 7.37/100 patient-years (P < 0.001)].⁵²

Surgery as a Risk for Acquired Thrombosis

Surgery is a major risk factor for thrombosis. Trauma to tissue results in endothelial injury, activation of the coagulation cascade (through the release of tissue factor), and platelet activation. The risk is modified by time of anesthesia, patient age, the presence of underlying heritable or acquired hypercoagulable states, and the nature of the surgical procedure. VTEs most frequently occur with hip or knee arthroplasty, hip fracture surgery, spinal cord injury, major trauma, and any surgery performed in the context of malignancy. Patients undergoing these procedures should receive thromboprophylaxis. Use of graduated pneumatic compression stockings plus LMWH, adjusted dose heparin, fondaparinux, and oral anticoagulation with warfarin to achieve an INR goal of 2 to 3 are all reasonable options. DVT prophylaxis should be individualized depending on bleeding risk, history of previous thrombosis, history of HIT, presence of renal insufficiency, and type of surgery; published guidelines can assist in management.⁵³ Outpatient surgical procedures performed in patients younger than 40 years who can be made readily ambulatory do not require prophylactic anticoagulation. Prolonged prophylactic anticoagulation up to 30 days after surgery may be indicated for patients undergoing total hip replacement (at least until the patient is mobile) and for those in whom a malignancy persists.

Laparoscopic surgery has become increasingly popular as a substitute for conventional open surgical procedures. Although there is less tissue damage, shorter procedure times, and quicker recovery, patients may be subjected to induction of pneumoperitoneum and prolonged use of the reverse Trendelenburg position to visualize and manipulate internal organs that may result in venous stasis and increased risk of thrombosis in some patients.⁵⁴ The American College of Chest Physicians’ (ACCP) Clinical Practice Guidelines (8th edition) recommend against routine prophylaxis for those undergoing laparoscopic surgery without additional thromboembolic risk factors, but recommends mechanical or pharmacologic prophylaxis in patients with any risk factors.⁵³ The Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) guidelines for DVT prophylaxis during laparoscopic surgery stratifies inpatients into low, moderate, and high risk groups for thrombosis on the basis of a risk score imputed from the type of procedure and patient risk factors.55,56 Procedure-related risk factors include procedure lasting over 1 hour and pelvic procedures. Patient-related factors include age > 40 years, immobility, malignancy, thrombophilic states (protein C, protein S, or ATIII deficiency), obesity, peripartum state (or use of estrogens), heart failure, renal failure, varicose veins, inflammatory states, or infection. In the lowest risk group (procedure < 60 minutes in patients with no risk factors) elastic stockings and early ambulation suffice, and UFH or LMWH is optional. In the moderate-risk group (one patient risk factor in a procedure of less than 60 minutes, or any procedure >60 minutes with no patient risk factors) pneumatic compression devices or prophylactic heparin or LMWH are recommended. In the high-risk group (two or more risk factors in procedures >60 minutes) a combination of serial compression devices and prophylactic UFH or LMWH are recommended.55,56

Myeloproliferative Disorders

MPDs are paradoxically associated with increased risks for both hemorrhage and thrombosis, but thrombosis is the most common cause of death in MPDs. The thrombotic risk of PV is often exacerbated by the hyperviscosity produced by a markedly increased red cell mass. Treatments for PV include serial phlebotomy to decrease red cell volume/hyperviscosity and cytotoxic agents, such as hydroxyurea (HU), to reduce erythrocyte, leukocyte, and platelet production, with the ultimate goal to minimize the risk of thrombosis.

³²P or alkylators such as chlorambucil or busulfan are rarely used, except in the elderly where long-term leukemia risk is not as concerning as for other patients. The goal of PV therapy by phlebotomy is to keep the hematocrit below 45% in males and below 42% in females, so as to prevent stroke, heart attack, or DVT, including the Budd-Chiari syndrome.⁵⁷ PV-associated erythromelalgia is usually treated with low-dose aspirin 81 mg daily. Higher aspirin doses may be associated with thrombosis.

Therapy to prevent thrombosis is usually considered for patients with essential thrombocythemia who are older than 60 years of age or have a prior history of thrombosis. Guidance is derived from a randomized trial evaluating low-dose aspirin plus anagrelide versus HU. HU plus aspirin was associated with a lower risk of arterial thrombosis, serious hemorrhage, and transformation to myelofibrosis than anagrelide, a specific platelet-lowering noncytotoxic, and non-chemotherapy agent. VTE incidence was higher in the HU group; however, the totality of the data when analyzed for composite endpoints favored treatment with HU.⁵⁸ Blood counts should be followed closely during therapy, and HU is contraindicated in pregnant women or for women who desire to become pregnant.

Inherited Hypercoagulable States

Most inherited hypercoagulable states associated with an increased risk of VTE are deficiencies of one or more anticoagulant proteins or defects in coagulation factors that increase their level of expression or make them no longer subject to inhibition or regulation by anticoagulant proteins. There may also be metabolic problems such as homocysteinemia where toxic levels of homocysteine may be toxic to the endothelium and promote thrombosis. Inherited hypercoagulable states are listed in Table 22.8.

Persistent elevations of coagulation factors VIII, IX, or XI have also been implicated as inherited hypercoagulable states.⁵⁹⁻⁶³ Extremely rare inherited hypercoagulable states include dysfibrinogenemias or other deficiencies of the fibrinolytic system (plasminogen deficiency). Screening for a thrombophilic hypercoagulable state should be considered for a young patient with a positive family history of thrombosis, unprovoked thrombosis, or recurrent thrombosis. The timing of testing is critical. Elevations of factor VIII or fibrinogen can occur at the time of an acute thrombotic event, perhaps from inflammation, and only a persistently increased level is likely to be the cause for thrombosis. Anticoagulant medications can interfere with measurements of proteins C and S (warfarin) or antithrombin (heparin), and testing should be done when the patient is not taking these medications to ensure an accurate assessment of levels. Pregnancy is a time when levels of factor VIII rise, and free protein S decreases, so the baseline assessment of hypercoagulability is best done when a woman is not pregnant.

Activated Protein C Resistance (Factor V Leiden)

A polymorphism in the gene for coagulation factor V (Arg506Gln; factor V Leiden) results in a factor V protein that is not inactivated by activated protein C. The factor V Leiden polymorphism is seen in ˜5% of the Caucasian population and is the most common known inheritable risk factor for DVT or PE.⁶⁴ There are other rare factor V polymorphisms associated with resistance to activated protein C in Caucasians (factor V Cambridge, Arg306Thr; Ref. 65) and also in Chinese (factor V Hong Kong, Arg306Gly, Ref. 66) that will be missed by the DNA test for factor V Leiden. Most patients who carry the mutation do not develop thrombosis; however, additive risk factors, including estrogen use and the coexistence of the prothrombin mutation, greatly increase the risk of VTE. Laboratory testing includes direct detection of the characteristic mutation in the factor V gene, accomplished by the polymerase chain reaction (PCR) on peripheral blood leukocyte DNA. Indirect testing for inability of activated protein C to prolong the PTT (resistance to activated protein C) is done less commonly now, but may be useful if there is suspicion that a polymorphism other than the factor V Leiden may be present.

Table 22.8  Inherited Hypercoagulable States

Activated protein C resistance (factor V Leiden gene polymorphism, 506R→Q)

Prothrombin gene 20210 A→ T polymorphism

Protein C deficiency

Protein S deficiency

Antithrombin deficiency

Hyperhomocysteinemia

Prothrombin Mutation (G20210A)

The second most common gene mutation responsible for congenital hypercoagulability in Caucasians is the prothrombin G20210A polymorphism. As with factor V Leiden, VTE is more commonly seen than arterial thrombosis. This mutation is associated with ˜20% greater than normal prothrombin levels, seen almost exclusively in Caucasians, and was found to have a crude 2.8 odds ratio for the development of thrombosis.⁶⁷ The polymorphism apparently leads to more stable factor II mRNA, and therefore greater prothrombin expression. G20210A can be detected directly by PCR analysis of the target site in the factor II gene; measurements of factor II are not particularly specific for the polymorphism, and should not be relied upon to make the diagnosis.

Deficiencies of Proteins S and C and Antithrombin (Antithrombin III)

Proteins C and S and antithrombin (previously known as antithrombin III) are synthesized in the liver and serve to modulate reactions of blood coagulation. Protein C is a serine protease, and when activated by thrombin cleavage has the ability to catalytically cleave and inactivate factors V and VIII, thereby shutting down further synthesis of thrombin. It is a vitamin K-dependent protein, and its activity level goes down in patients who are on warfarin. Protein S is a nonenzyme cofactor for activated protein C, and is also a vitamin K-dependent protein that is inactivated in patients on warfarin. Protein S binds to (complement) C4b-binding protein,⁶⁸ which may mediate a decrease in protein S activity during acute inflammation. Antithrombin (previously known as antithrombin III) is a serine protease inhibitor that neutralizes thrombin, and activated forms of other serine proteases such as factors Xa, XIa, IXa, and XIIa.⁶⁹ Antithrombin’s activity as a serine protease inhibitor is accelerated by binding to heparin, a complex, sulfated polysaccharide that is a normal component of various tissues (lung, liver, intestine) and used in purified form as an anticoagulant medication. Deficiency of antithrombin is probably the most serious risk factor for thrombosis, especially VTE in association with surgery or invasive procedures.

Hyperhomocysteinemia

Homocysteine is an amino acid that does not appear in proteins, but is an intermediate in the metabolism of methionine, a sulfur-containing, essential amino acid. Reactions of the metabolic pathways in which homocysteine participates require folic acid, cyanocobalamine (vitamin B12), or pyridoxine (vitamin B6) as cofactors. Deficiencies of these vitamins may cause accumulation of homocysteine to high levels in the bloodstream. Extremely high levels of homocysteine are seen in homocystinuria, and in methylene tetrahydrofolate reductase gene defects. Homocysteine may be toxic to endothelial cells,⁷⁰ and accelerated atherosclerosis can be a feature of the disease homocystinuria.70,71 Accordingly, many investigators have sought evidence for an association between elevated homocysteine and thrombosis in people who are not suffering from the extreme manifestations of homocystinuria. Evidence that elevated homocysteine levels (>95th percentile) is associated with VTE was found in a study comparing patients with first time DVT with matched controls, where the odds ratio for elevated homocysteine was 2.5.⁷² It is not clear if lowering homocysteine levels by vitamin therapy can alter the risk for VTE, however. Despite lowering homocysteine levels in the treatment group of the 5,222-patient HOPE-2 intervention trial, there was no difference in the incidence of VTE.⁷³ A meta-analysis of 31 published studies indicated that presence of the homozygous TT form of the methylene tetrahydrofolate reductase (MTHFR) gene at amino acid 677 was only a very weak risk factor for thrombosis.⁷⁴ Therefore, it is our practice not to routinely seek expensive molecular studies of polymorphisms in the MTHFR gene as risk factors for VTE, but instead to measure homocysteine levels in serum.

TREATMENT

Prophylaxis

The American College of Physicians (ACP) recently published guidelines for VTE prophylaxis in medical patients and patients with an acute stroke.⁷⁵ Careful assessment of the risk for bleeding and thrombosis need to be performed prior to starting prophylaxis with heparin. The ACP guidelines recommend starting prophylaxis with heparin (either LMWH or UFH) in medical (including stroke) patients when the benefit outweighs the risk of bleeding. Pooled data from 18 trials showed that heparin prophylaxis was associated with a borderline statistically significant reduction in risk for mortality compared to no heparin prophylaxis (RR 0.93, CI 0.86–1.00), a statistically significant reduction in the risk of PE (RR 0.70, CI 0.56– 0.87), at the expense of an increase in bleeding events, which was also statistically significant (RR 1.28, CI 1.05–1.56). Interestingly, no improvements in clinical outcomes were seen in three studies of mechanical prophylaxis in patients with stroke, but more patients had lower extremity skin damage (RR 4.02, CI 2.34–6.91) an increase of 39 events per 1,000 patients treated. As such, the ACP recommends against the use of mechanical prophylaxis with graduated compression stockings for the prevention of VTE.⁷⁵

The 9th edition guidelines of the American College of Chest Physicians address prophylactic treatment of VTE in nonorthopedic and orthopedic surgical patients.⁷⁶ Recommendations for or against anticoagulation or mechanical prophylaxis in general surgery patients are based on the type of surgery and individual thrombotic and bleeding risk assessment. Major orthopedic surgery has an estimated risk of symptomatic VTE of 4.3% in 35 days in patients receiving no prophylaxis.⁷⁶ Therefore, patients undergoing total hip arthroplasty, total knee arthroplasty, or hip fracture surgery who are not at increased risk of bleeding, should receive either thromboprophylaxis with LMWH (or alternative pharmacologic anticoagulation) or mechanical prophylaxis. Extended use of LMWH in the outpatient setting (up to 35 days post surgery) is recommended based on data from three systematic reviews including seven controlled trials showing a decrease in symptomatic VTE of 9 per 1,000 patients without an appreciable increase in major bleeding.⁷⁶

Initial Treatment for Acute Venous Thromboembolism

Anticoagulation is the essential primary treatment and prophylaxis for VTE. Mechanical barrier devices (IVC filters) may be used in certain circumstances in lieu of anticoagulation, or as an adjunctive measure. Fibrinolytic therapy may also be beneficial in select patients as an adjunct to anticoagulation.

The 9th ACCP Guidelines for anticoagulation and treatment of VTE⁷⁷ suggest that a DVT or PE should initially be treated with LMWH, fondaparinux, or rivaroxaban, that LMWH and fondaparinux are preferable to UFH, thrombolytic therapy may be advisable in the presence of hemodynamic compromise, and anticoagulation should be utilized for at least 3 months, in the setting of a provoked event, and longer for unprovoked events. The purpose of anticoagulation is to prevent additional clots from developing, to immediately stop further propagation of the existing clot, and to permit endogenous fibrinolysis to begin to dissolve the clot physiologically. If warfarin is used, it should be started after initiation of heparin, LMWH, or fondaparinux, typically at a 5-mg daily dose for most adults. A double-blind randomized study, however, showed that warfarin at 10 mg for 2 days followed by dose adjustment determined by a nomogram was also safe and effective.⁷⁸ Parenteral anticoagulation is continued until there are successive INR values of between 2 and 3 after adjustment of the warfarin dose. In the absence of PE, or with PE that is not complicated by cardiovascular compromise, or other reasons to admit the patient, this can typically be accomplished as an outpatient. Subcutaneous (SQ) UFH versus LMWH was evaluated in the FIDO study, a randomized trial of 708 patients, showing that fixed dose SQ UFH is as effective and safe as LMWH in patients with acute VTE and is suitable for outpatient treatment.⁷⁹ LMWH and fondaparinux are less likely than UFH to cause HIT, however. LMWH requires monitoring of anti-Xa levels in patients at high or low extremes of weight, or who have renal failure, or are pregnant. LMWH and fondaparinux are relatively contraindicated in renal failure and should be used with extreme caution (using dose reduction and monitoring), as the kidneys excrete LMWH. UFH may be preferred in a patient at risk for bleeding, due to the ability to neutralize with protamine and its shorter duration of effect. See Chapter 23 for discussion of new oral anticoagulant medications.

Extended Treatment to Prevent Recurrent Venous Thromboembolism

Warfarin has been the only oral anticoagulant available for long-term anticoagulation until recently. Despite the introduction of new oral anticoagulants such as rivaroxaban, dabigatran, and apixaban that have indications to prevent DVT or stroke in atrial fibrillation, it remains a useful drug due to its lower cost and established risk-benefit profile. Dalteparin sodium is FDA approved for the extended treatment and subsequent prevention of recurrent symptomatic VTE in patients with cancer. Unique risks of long-term LMWH use include osteopenia and HIT (the latter risk is uncommon in this setting).

The ideal intensity of warfarin anticoagulation for long-term (indefinite duration) therapy to prevent recurrent VTE has been addressed in two well-designed trials yielding conflicting results. The PREVENT trial concluded that low intensity anticoagulation with the target INR of 1.5–2.0 was successful in substantially reducing recurrent VTE risk.⁸⁰ In a randomized, two-arm study of standard warfarin anticoagulation in Canada to achieve an INR between 2 and 3 versus a low-intensity INR arm with target INR between 1.5 and 2.0, the standard dosing regimen was over 60% more effective (p = 0.03) than low-intensity warfarin anticoagulation in reducing the cumulative probability of recurrent thromboembolism.⁸¹ There was no difference in bleeding complications between the two dosing intensities. The differences between the two studies may be related to trial design. For instance, the Canadian trial, in contrast to the PREVENT study, included cancer patients, who more likely would experience warfarin resistance.

At least 3 months of anticoagulation are recommended for idiopathic (unprovoked) DVT, and if there is no contraindication to anticoagulation, it should be continued indefinitely.⁷⁷ Similarly, life-threatening PE requires indefinite anticoagulation, unless the risk of life-threatening bleeding on anticoagulation exceeds the risk of a fatal PE; in this rare circumstance an IVC filter may be utilized. The PREVENT trial corroborated that patients with idiopathic VTE have a high incidence of recurrent VTE and benefit from long-term anticoagulation.⁸⁰ As previously discussed, the APA syndrome requires prolonged anticoagulation. Individuals with cancer remain hypercoagulable as long as the malignancy is present. Patients with transient risk factors (i.e., trauma) usually require anticoagulation for 3 to 6 months. Patients with increased D-dimers, elevated FVIII activities, or evidence of significant residual DVT at 1 month after discontinuing anticoagulation are at increased risk for rethrombosis and consideration should be given to resumption of long-term anticoagulation. The D-dimer can also be measured intermittently after discontinuation of therapy to assess for the risk of rethrombosis.²⁷

Inferior Vena Cava Filters in the Treatment of Deep Venous Thrombosis or Pulmonary Embolus

IVC filters are placed to prevent large clots in the lower extremities from embolizing to the pulmonary circulation. Major reasons for the placement of an IVC filter include strong contraindications to the use of anticoagulants, intolerance to or noncompliance with anticoagulants, and recurrent PE despite adequate systemic anticoagulation. All of these factors are relative indications for use of an IVC filter, and the decision to place one is not to be taken lightly. A randomized study revealed that, in the short term, an IVC filter decreases the incidence of pulmonary embolus from 4.8% to 1.1%, but by 2 years the rate of recurrent DVT was 20.8% in the IVC filter group versus 11.6% in the nonfilter group, and overall mortality was no significantly different.⁸² Retrievable filters have the advantage that they may be removed after the risk of PE has passed, but they may be more prone to migration than permanent filters.

Fibrinolytic Therapy

Fibrinolytic therapy has typically been reserved for patients with massive PE associated with hemodynamic compromise.⁷⁷ Patients with systolic BP < 90 mm Hg, or a BP drop > 40 mm Hg for > 15 minutes, not caused by cardiac arrhythmias, sepsis, or hypovolemia, may benefit from thrombolytic therapy with improved survival.⁸³ Clinical studies do not show a survival advantage for thrombolytic agents in PE. Currently, tPA is the only fibrinolytic agent commonly available, and it has the advantage of a short half-life, and relative specificity for fibrin clot (as opposed to fibrinogen) when compared to urokinase or streptokinase. Fibrinolytic therapy is being investigated in the ongoing ATTRACT⁸⁴ and recently reported CAVENT⁸⁵ trials, in selected patients with massive iliac-femoral DVT. The CAVENT trial randomized 209 patients with iliofemoral DVT to anticoagulation with (101 patients) or without (108 patients) additional catheter-directed thrombolysis (CDT) with tPA. At 24 months, the patients given CDT had 41% incidence of PTS, versus 56% of the control group (p = 0.047). Patency of the iliofemoral system was seen in 66% of CDT patients versus 47% of the control group (p = 0.012). There was additional bleeding associated with CDT that included three major and five clinically significant bleeding episodes, consistent with typical estimates of bleeding risk from fibrinolytic therapy of ˜8%.⁸⁶

At this time, catheter-directed fibrinolytic therapy with low doses of tPA may be considered for use in patients with DVT that is not responding to standard anticoagulation, and adjunctive measures such as stenting or balloon dilatation of venous segments with strictures may also be considered, but this is not yet standard practice.41,42,77

References

  1.  The Surgeon General’s Call to Action to Prevent Deep Venous Thrombosis and Pulmonary Embolism. 2008. http://www.surgeongeneral.gov/topics/deepvein/index.html

  2.  Silverstein M, Heit J, Mohr D, et al. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Arch Int Med. 1998;158:585-593.

  3.  Heit J. The epidemiology of venous thromboembolism in the community. Arterioscler Thromb Vasc Biol. 2008;28:370-372.

  4.  Pengo V, Lensing AW, Prins MH, et al. Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med. 2004;350:2257-2226.

  5.  Becattini C, Agnelli G, Pesavento R, et al. Incidence of chronic thromboembolic pulmonary hypertension after a first episode of pulmonary embolism. Chest. 2006;130:172-175.

  6.  Isma N, Svensson PJ, Gottsäter A, et al. Upper extremity deep venous thrombosis in the population-based Malmö thrombophilia study (MATS). Epidemiology, risk factors, recurrence risk, and mortality. Thromb Res. 2010;125(6):e335-338.

  7.  Prandoni P, Polistena P, Bernardi E, et al. Upper extremity deep vein thrombosis: risk factors, diagnosis, and complications. Arch Intern Med. 1997;157:57-62.

  8.  Velmahos GC, Spaniolas K, Tabbara M, et al. Pulmonary embolism and deep venous thrombosis in trauma. Are they related? Arch Surg. 2009;144:928-932.

  9.  Anderson FA, Wheeler HB, Goldberg RJ, et al. A population-based perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism. The Worchester DVT Study. Arch Intern Med. 1991;151:933-938.

10.  Kahn SR. Measurement properties of the Villalta scale to define and classify the severity of the post-thrombotic syndrome. J Thromb Haemost. 2009;7:884-888.

11.  Ashrani A, Heit J. Incidence and cost burden of post-thrombotic syndrome. J Thromb Thrombolysis. November 2009;28(4):465-476.

12.  Brandjes DP, Büller HR, Heijboer H, et al. Randomised trial of effect of compression stockings in patients with symptomatic proximal-vein thrombosis. Lancet. 1997;349(9054):759-762.

13.  Masuda EM, Kessler DM, Kistner RL, et al. The natural history of calf vein thrombosis: lysis of thrombi and development of reflux. J Vasc Surg. 1998;28:67-74.

14.  Lohr JM, James KV, Deshmukh RM, Hasselfeld KA. Calf vein thrombi are not a benign finding. Am J Surg. 1995;170:86-90.

15.  Masuds EM, Kistner RL, Musikasinthorn C, et al. The controversy of managing calf vein thrombosis. J Vasc Surg. 2012;55:550-561.

16.  Segal J, Eng J, Tamariz L, et al. Review of the evidence on diagnosis of deep venous thrombosis and pulmonary embolism. Clinical practice guideline. American College of Physicians. Ann Fam Med. 2007;5:63-73.

17.  Loud PA, Katz DS, Klipoenstein DL, et al. Combined CT venography and pulmonary angiography in suspected thromboembolic disease: diagnostic accuracy for deep venous evaluation. Am J Roentgenol. 2000;174:61-65.

18.  Cham MD, Yankelvitz DF, Shaham D, et al. Deep venous thrombosis: detection by using indirect CT venography. The pulmonary angiography-indirect CT venography cooperative group. Radiology. 2000;216:744-751.

19.  Fraser DGW, Moody AR, Morgan PS, et al. Diagnosis of lower-limb deep venous thrombosis: a prospective blinded study of magnetic resonance direct thrombus imaging. Ann Intern Med. 2002;136:89-98.

20.  Ono A, Murase K, Taniguchi T, et al. Deep venous thrombosis: diagnostic value of non-contrast-enhanced MR venography using electrocardiography-triggered three-dimensional half-Fourier FSE. Magen Reson Med. 2010;64(1):88-97.

21.  Kay J, Czijak L. Gadolinium and systemic fibrosis: guilt by association. Ann Rheum Dis. 2010;69:1895-1897.

22.  Aguilar C, Martinez A, Martinez A, et al. Diagnostic value of d-dimer in patients with a moderate pretest probability of deep venous thrombosis. Br J Haematol. 2002;118:275-277.

23.  Kulstad EB, Kulstad CD, Lovel EO. A rapid quantitative turbimetric D-dimer assay has high sensitivity for detection of pulmonary embolism in the ED. Am J Emerg Med. 2004;22:111-114.

24.  Rathbun SW, Whitsett TL, Raskob GE. Negative D-dimer result to exclude recurrent deep venous thrombosis: a management trial. Ann Int Med. 2004;141:839-846.

25.  Verhovsek M, Douketis JD, Yi Q, et al. Systematic review: D-dimer to predict recurrent disease after stopping anticoagulant therapy for unprovoked venous thromboembolism. Ann Int Med. 2008;149(7):481-490.

26.  Palareti G, Cosmi B, Legnani C, et al. D-dimer testing to determine the duration of anticoagulation therapy. N Engl J Med. 2006;355:2797.

27.  Cosmi B, Legnani C, Tosetto A, et al. Usefulness of repeated D-dimer testing after stopping anticoagulation for a first episode of unprovoked venous thromboembolism: the PROLONG II prospective study. Blood. January 2010;115(3):481-488.

28.  Amitrano L, Brancaccio V, Guardascione MA, et al. Inherited coagulation disorders in cirrhotic patients with portal vein thrombosis. Hepatology. 2000;31:345-348.

29.  Denninger M-H, Chait Y, Casadevall N, et al. Cause of portal or hepatic venous thrombosis in adults: the role of multiple concurrent factors. Hepatology. 2000;31:587-591.

30.  Jansen HLA, Meinardi JR, Vleggar FP, et al. Factor V Leiden mutation, prothrombin gene mutation, and deficiencies in coagulation inhibitors associated with Budd-Chiari syndrome and portal vein thrombosis: results of a case-control study. Blood. 2000;96:2364-2368.

31.  Warkentin T. How I diagnose and manage HIT. ASH Education Book. Hematology Am Soc Hematol Educ Program. 2011;143-149.

32.  Warkentin TE. Heparin-induced thrombocytopenia: pathogenesis and management. Br J Haematol. 2003;121:535-555.

33.  Cuker A, Arepally G, Crowther MA, et al. The HIT Expert Probability (HEP) Score: a novel pre-test probability model for heparin-induced thrombocytopenia based on broad expert opinion. J Thromb Haemost. 2010;8:2642-2650.

34.  Papadopoulos S, Flynn JD, Lewis DA. Fondaparinux as a treatment option for heparin-induced thrombocytopenia. Pharmacotherapy. 2007;27:921-926.

35.  Pendleton R, Wheeler MM, Rodgers GM. Argatroban dosing of patients with heparin-induced thrombocytopenia and an elevated aPTT due to antiphospholipid antibody syndrome. Ann Pharmacother. 2006;40:972-976.

36.  Gosselin RC, King JH, Janatpour KA, et al. Comparing direct thrombin inhibitor using aPTT, ecarin clotting times, and thrombin inhibitor management testing. Ann Pharmacother. 2004;38:1383-1388.

37.  Singhal R, Brimble KS. Thromboembolic complications in the nephrotic syndrome: pathophysiology and clinical management. Thromb Res. 2006;118:397-407.

38.  Mahmoodi BK, ten Kate MK, Waanders F, et al. High absolute risks and predictors of venous and arterial thromboembolic events in patients with nephrotic syndrome: results from a large retrospective cohort study. Circulation. 2008;117:224-230.

39.  Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999;340:448-454.

40.  Ruiz-Irastorza G, Crowther M, Branch W, Khamashta MA. Antiphospholipid syndrome. Lancet. October 2010;376(9751): 1498-1509.

41.  Chang R, Horne MK, Shawker TH, et al. Low-dose, once-daily, intraclot injections of alteplase for treatment of acute deep venous thrombosis. J Vasc Interv Radiol. 2011;22:1107-1116.

42.  Lozier JN, Cullinane A, Nhgiem K, et al. Biochemical dynamics relevant to the safety of low-dose, intraclot alteplase for deep vein thrombosis. Trans Res. January 2012;160:217-222. In press

43.  Ruiz-Irastorza G, Cuadrado MJ, Ruiz-Arruza I, et al. Evidence-based recommendations for the prevention and long-term management of thrombosis in antiphospholipid antibody-positive patients: Report of a Task Force at the 13th International Congress on Antiphospholipid Antibodies. Lupus. 2011;20:206-218.

44.  Saltzman HA, Alavi A, Greenspan RH, et al. Value of the ventilation/perfusion in acute pulmonary embolism: results of the Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED). JAMA. 1990;263:2753-2759.

45.  Tafur AJ, Kalsi H, Wysokinski WE, et al. The association of active cancer with venous thromboembolism location: a population-based study. Mayo Clin Proc. 2011;86:25-30.

46.  Heit JA. The epidemiology of venous thromboembolism in the community: implications for prevention and management. J Thromb Thrombolysis. 2006;21:23-29.

47.  Lee AYY, Levine MH, Baker RI, et al. Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med. 2003;349:146-153.

48.  Meyer G, Marjanovic Z, Valcke J, et al. Comparison of low molecular weight heparin and warfarin for the secondary prevention of venous thromboembolism in patients with cancer: a randomized controlled study. Arch Intern Med. 2002;162(15):1729-1735.

49.  Kuderer NM, Ortel TL, Francis CW. Impact of venous thromboembolism and anticoagulation on cancer and cancer survival. J Clin Oncol. 2009;27:4902-4911.

50.  Hall C, Richards S, Hillmen P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2003;102:3587-3591.

51.  van Bijnen ST, van Heerde WL, Muus P. Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria. J Thromb Haemost. November 2011. doi: 10.1111/j.1538-7836.2011.04562.x. [Epub ahead of print]

52.  Hillmen P, Muus P, Duhrsen U, et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007;110:4123-4128.

53.  Geerts WH, Bergquvist D Pineo GF, et al. Prevention of venous thromboembolism. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest. 2008;133:381S-453S.

54.  Ageno W, Squizzato A, Garcia D, et al. Epidemiology and risk factors of venous thromboembolism. Semin Thromb Hemost. 2006;32:651-658.

55.  SAGES Guidelines for deep venous thrombosis prophylaxis during laparoscopic surgery. October 2006. http://www.sages.org/publication/id/C/

56.  SAGES Guidelines Committee. Surg Endosc. 2007;21:1007-1009.

57.  Spivak JL. Polycythemia vera: myths, mechanisms, and management. Blood. 2002;117:47.

58.  Harrison CN, Campbell PJ, Buck G, et al. Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia. NEJM. 2005;353:33-45.

59.  Kamphuisen PW, Eikenboom JCJ, Bertina RM. Elevated factor VIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol. 2001;21:731-738.

60.  Rosendaal FR. High levels of factor VIII and venous thrombosis. Thromb Haemost. 2000;83:1-2.

61.  Lowe GDO, Woodward M, Vessey MP, et al. Thrombotic variables and risk of idiopathic venous thromboembolism in women aged 45–64 years: relationships to hormone replacement therapy. Thromb Haemost. 2000;83:530-535.

62.  Daly E, Vessey MP, Hawkins MM, et al. Risk of venous thromboembolism in users of hormone replacement therapy. Lancet. 1996;348:977-980.

63.  Meijers JCM, Tekelenburg WLH, Bouma BN, et al. High levels of coagulation factor XI as a risk factor for venous thrombosis. N Engl J Med. 2000;342:696-701.

64.  Ridker PM, Miletich JP, Hennekens CH, Bring JE. Ethnic distribution of factor V Leiden in 4047 men and women. Implications for venous thromboembolism screening. JAMA. 1997;277:1305-1307.

65.  Williamson D, Brown K, Luddington R, et al. Factor V Cambridge: a new mutation (Arg306→Thr) associated with resistance to activated protein C. Blood. 1998;91:1140-1144.

66.  Chan WP, Lee CK, Kwong YL, Lam CK, Liang R. A novel mutation of Arg 306 of factor V gene in Hong Kong Chinese. Blood. 1998;91:1135-1139.

67.  Poort SR, Rosendaal FR, Reitsma PH, et al. A common genetic variation in the 3N-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88:3698-3703.

68.  Dahlback B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci U S A. 1981;78:2512-2516.

69.  Zwicker J, Bauer KA. Thrombophilia. In: Kitchens CS, Alving BM, Kessler CM, eds. Consultative Thrombosis and Hemostasis. Philadelphia, PA: W.B. Saunders Company; 2002:181-196.

70.  Rees MM, Rodgers GM. Homocysteinemia: association of metabolic disorder with vascular disease and thrombosis. Thromb Res. 1993;71:337-359.

71.  Mudd SH, Skovby F, Levy HL. The natural history of homocystinuria due to cystathinonine β-synthase deficiency. Am J Hum Genet. 1985:37:1-35.

72.  Den Heijer M, Koster T, Blom HK, et al. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 1996;334:759-762.

73.  Ray JG, Kearon C, Yi Qilong, et al. Homocysteine-lowering therapy and risk for venous thromboembolism: a randomized trial. Ann Intern Med. 2007;146:761-767.

74.  Ray JG, Shmorgun D, Chan WS. Common C677T polymorphism of the methylenetetrahydrofolate reductase gene and the risk of venous thromboembolism: meta-analysis of 31 studies. Pathophysiol Haemost Thromb. 2002;32:51-58.

75.  Qaseem A, Chou R, Humphrey LL, et al. Venous thromboembolism prophylaxis in hospitalized patients: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2011;155:625-632.

76.  Guyatt GH, Akl EA, Crowther M, et al. Antithrombotic therapy and prevention of thrombosis, 9th ed: American college of chest physicians evidence-based clinical practice guidelines. Chest. 2012;141:7S-47S.

77.  Kearon C, Akl EA, Comerata AJ, et al. Antithrombotic therapy for VTE disease antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012; 141(2)(suppl):e419S-e494S.

78.  Kovacs MJ, Roger M, Anderson DR, et al. Comparison of 10-mg and 5-mg warfarin initiation nomograms together with low-molecular-weight-heparin for outpatient treatment of acute venous thromboembolism. A randomized, double-blind, controlled trial. Ann Intern Med. 2003;138:714-719.

79.  Kearon C, Ginsberg JS, Julian JA. Comparison of fixed-dose weight-adjusted unfractionated heparin and low-molecular-weight heparin for acute treatment of venous thromboembolism. JAMA. 2006;296:935-942.

80.  Ridker PM, Goldhaber SZ, Danielson E, et al. Long-term, low intensity warfarin for the prevention of recurrent venous thromboembolism. N Engl J Med. 2003;348:1425-1434.

81.  Kearon C, Ginsberg JS, Kovacs MJ, et al. Comparison of low-intensity warfarin therapy with conventional-intensity warfarin therapy for long-term prevention of recurrent venous thromboembolism. N Engl J Med. 2003;349:631-639.

82.  Decousus H, Leizorovicz A, Parent F, et al. A clinical trial of vena caval filters in the prevention of pulmonary embolism in patients with proximal deep-vein thrombosis. N Engl J Med. 1998;338:409-415.

83.  European Society of Cardiology: Guidelines on the diagnosis and management of acute pulmonary embolism. Eur Heart J. 2000;21:1301-1336.

84.  Comerota AJ. The ATTRACT Trial: Rationale for early intervention for iliofemoral DVT. Perspect Vasc Surg Endovasc Ther. 2009;21:221-225.

85.  Enden T, Haig Y, Klow N-E, et al. Long-term outcome after additional catheter-directed thrombolysis versus standard treatment for acute iliofemoral deep vein thrombosis (the CaVenT study): a randomised controlled trial. Lancet. 2012;379:31-38.

86.  Vedantham S, Millward SF, Cardella JF, et al. Society of Interventional Radiology position statement: treatment of acute iliofemoral deep vein thrombosis with use of adjunctive catheter-directed intrathrombus thrombolysis. J Vasc Interv Radiol. 2006;17:613-616.

23

Consultations in Anticoagulation

Fang Yin and Jay N. Lozier

This chapter provides guidelines for the treatment of venous thromboembolism (VTE) in patients who require special consideration, such as those who have underlying cancer, malignancy, or who are pregnant. We also discuss use of inferior vena cava (IVC) filters, the prevention, diagnosis, and treatment of postthrombotic syndrome, and anticoagulant drugs with novel mechanisms that are now in development.

PROPHYLAXIS AND TREATMENT OF VENOUS THROMBOEMBOLISM IN THE PATIENT WITH CANCER IN SPECIFIC CLINICAL SETTINGS

Patients with malignancy have an increased risk for VTE due to multiple factors: hypercoagulability resulting from increased production and release of microparticles containing procoagulants such as tissue factor; vessel wall damage, impaired blood flow (stasis) from extrinsic compression; prolonged immobility, anticancer therapy including cytotoxic chemotherapy, certain antiangiogenic agents, or hormonal therapy, and the increasing use of long-term indwelling devices, such as central venous catheters. Tumor angiogenesis, progression, growth, and the metastatic process are enhanced by, and depend on activation of blood coagulation. P-selectin, a cell adhesion molecule has also been identified as a risk factor for recurrent VTE and can be used as a predictive parameter for development of VTE in cancer patients.¹ As long as the cancer is active, the increased risk for VTE is present. Cancers most commonly associated with VTE are carcinomas of the pancreas, stomach, kidney, lung, ovary, and bladder, certain hematologic malignancies, cancers of the testis, and gliomas of the brain. Adenocarcinomas appear to be associated with a higher risk than squamous cell cancers.

Studies performed during the current decade have demonstrated that low-molecular-weight heparin (LMWH) is more effective than oral anticoagulants in reducing the risk of recurrent VTE without increasing the risk of bleeding in patients with cancer and acute VTE. Products such as dalteparin, enoxaparin, nadroparin, and tinzaparin as well as the synthetic factor Xa inhibitor, fondaparinux are approved by the Food and Drug Administration (FDA) for the prophylaxis and treatment of VTE. In general, unfractionated heparin (UFH), LMWH, fondaparinux, and oral anticoagulants are the mainstay of therapy. Since LMWH undergoes renal excretion, patients with kidney impairment who receive LMWH should be monitored by measurement of the anti-factor Xa activity. The creatinine clearance should be estimated (or calculated) before initiation of LMWH in elderly patients, since they may have renal dysfunction despite having normal creatinine values. Specific dosing recommendations for enoxaparin in patients with severe renal insufficiency and/or low body weight are presented in Table 23.1. Monitoring of anti-factor Xa levels should also be used in severely obese patients (BMI ≥ 40) who receive therapeutic doses of LMWH and should be considered for those who are obese (BMI ≥ 30), especially if the patient has moderate to severe renal insufficiency (creatinine clearance less than 60 mL/min).²

The target peak anti-factor Xa levels (measured 4 hours after injection) for patients who are treated with LMWH vary according to the product. The target therapeutic range for enoxaparin is 0.6 to 1.2 U/mL for twice-daily dosing and 1 to 2 U/mL for once-daily dosing; for dalteparin the target range is 0.5 to 1.5 U/mL (Table 23.2). Since fondaparinux is produced by complete chemical synthesis and its structure is completely defined, it is dosed on the basis of mass rather than anti-factor Xa activity. The assay for fondaparinux is often reported in terms of its concentration in mass/volume (e.g., mg/L).

Primary Prophylaxis in Cancer Patients Undergoing Surgical Intervention

VTE is a common complication of cancer surgery and the most common cause of death at 30 days after surgery in the @RISTOS prospective observational study of cancer surgery patients.³ Prophylaxis should now be provided routinely to postoperative surgical patients, especially those with underlying cancer. Two randomized controlled trials have demonstrated that extending deep venous thrombosis (DVT) prophylaxis from 1 to 4 weeks reduces the incidence of VTE.4,5 Extended (up to 4 weeks) VTE prophylaxis is recommended for high-risk cancer surgery patients with a previous episode of VTE, anesthesia times longer than 2 hours, advanced stage disease, perioperative bed rest ≥4 days, and patient age ≥ 60 years.³

Laparoscopic surgery is rapidly becoming a common method for tumor resection. It is unclear how the recommendations developed for typical, open procedures should be applied to laparoscopic surgical procedures. Laparoscopic surgery offers the advantage of less tissue disruption, quicker recovery times, and shorter periods of postoperative immobilization. Intuitively the reduction in tissue damage and the possibility of faster mobilization predicts lower risk of thromboembolic complications. Conversely, patients undergoing laparoscopic procedures are subjected to increased venous stasis as a result of the induction of pneumoperitoneum and prolonged use of the reverse Trendelenburg position to visualize and manipulate internal organs.⁶ The American College of Chest Physicians’ (ACCP) Clinical Practice Guidelines (8th edition) recommend against routine prophylaxis (other than early and frequent ambulation) in patients undergoing laparoscopic surgery without thromboembolic risk factors, and recommends mechanical or pharmacologic prophylaxis in patients with any thromboembolic risk factors.⁷ The Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) guidelines for DVT prophylaxis during laparoscopic surgery stratifies inpatients into low, moderate, and high risk groups for thrombosis on the basis of a risk score imputed from the type of procedure and patient risk factors.8,9 Procedure-related risk factors include procedure lasting over 1 hour, and pelvic procedures. Patient-related factors include age > 40 years, immobility, malignancy, thrombophilic states (protein C, protein S, or ATIII deficiency), obesity, peripartum state (or use of estrogens), heart failure, renal failure, varicose veins, inflammatory states, or infection. In the lowest risk group (procedure < 60 minutes in patients with no risk factors) elastic stockings and early ambulation is all that is recommended, and UFH or LMWH is optional. In the moderate-risk group (one patient risk factor in a procedure of less than 60 minutes, or any procedure > 60 minutes with no patient risk factors) pneumatic compression devices or prophylactic heparin or LMWH are recommended. In the high-risk group (two or more risk factors in procedures >60 minutes) a combination of serial compression devices and prophylactic UFH or LMWH are recommended.8,9

Once-daily LMWH appears to be as safe and effective as multiple daily injections of UFH and provides convenience as well as a better quality of life for the patient.3,4 In the Clinical Center at the National Institutes of Health, enoxaparin is the LMWH most commonly employed. However, fondaparinux or other LMWHs, such as nadroparin, dalteparin, ardeparin, tinzaparin, and reviparin, may be considered equivalent. The administration of warfarin at a low, fixed dose (e.g., 1 mg/day) has not been shown to be of value for VTE prophylaxis, and is not recommended.

Primary Venous Thromboembolism Prophylaxis in Cancer Patients Receiving Chemotherapy, Hormonal, and/or Antiangiogenic Treatment

Patients with cancer who are undergoing treatment should be considered for VTE prophylaxis if they have one or more of the following: a history of VTE, a large mass compressing a major vessel, or treatment which includes tamoxifen/raloxifene, diethylstilbestrol, or chemotherapy, especially use of bevacizumab, thalidomide- or lenalidomide-based combination regimens, particularly those given in combination with high-dose dexamethasone.¹⁰ A recent clinical trial of patients with advanced cancer of lung, gastrointestinal, pancreatic, breast, ovarian, or head and neck undergoing chemotherapy (PROTECHT trial) showed a statistically significant (P = 0.02) decrease in thromboembolic events from 3.9% to 2.0% in the groups receiving prophylactic LMWH (such as nadroparin) or placebo, respectively.¹¹ VTE prophylaxis in cancer patients undergoing treatment should be individualized; if prophylaxis is chosen, LMWH (enoxaparin 40 mg/day) or UFH (low dose) should be considered (Table 23.3). Aspirin prophylaxis (81–325 mg daily) is an option for patients receiving thalidomide or lenalidomide for multiple myeloma.¹⁰ In chronic lymphocytic leukemia (CLL) patients treated with lenalidomide it was shown that TNFα, C-reactive protein, factor VIII, thrombomodulin, and sVCAM1 were significantly increased from baseline after initiation of treatment (P < 0.001), and TNFα and sVCAM levels were more elevated in patients who subsequently had DVTs, suggesting inflammation and endothelial cell dysfunction played an important role in VTE risk.¹² Thus, anti-inflammatory effects of aspirin may contribute to prophylaxis against VTE in addition to antiplatelet effects otherwise not thought to be important for VTE prophylaxis.

Primary Prophylaxis in Immobilized/Hospitalized Cancer Patients

VTE is a fairly common event in hospitalized cancer patients. A retrospective study of over 66,000 hospitalized neutropenic adult cancer patients showed that 3% to 12% of these patients, depending on the type of malignancy, experienced VTE during their first hospitalization.¹³ Primary prophylaxis is effective in hospitalized medical patients, who undergo a three-fold reduction in VTE when treated with enoxaparin at a daily dose of 40 mg, compared to control patients receiving no treatment. This conclusion is derived from the MEDENOX trial,¹⁴ a double-blind randomized study of 1,102 patients with acute medical illnesses who received prophylaxis against VTE (14.9% of these patients had cancer or a history of cancer). Patients were randomized to one of three groups that would receive for 6 to 14 days subcutaneous daily administration of 40 mg of enoxaparin, 20 mg of enoxaparin, or a placebo. The primary outcome was VTE during the ensuing 3 months. The data favored prophylactic treatment with subcutaneous enoxaparin at a dose of 40 mg daily. Adverse events, which included hemorrhage, local reaction, thrombocytopenia, and death from any cause, were not different between the groups receiving enoxaparin and placebo. The weakness of this study was that a more appropriate control group would have received UFH. Therefore, it has been recommended that all hospitalized patients with cancer should receive anticoagulation therapy in the absence of contraindications. A subsequent randomized trial, the LIFENOX Trial, indicates, however, that all-cause mortality is unchanged in medical patients undergoing thromboprophylaxis with LMWH.¹⁵ UFH is the agent of choice for thromboprophylaxis in hospitalized patients with a creatinine clearance of < 30 mL/min. A reduced enoxaparin dose of 30 mg daily can also be used in this situation and is preferred if prolonged use is required. Occasional monitoring of anti-factor Xa levels may be appropriate in the setting of renal failure to prevent overdosage and bleeding.

Primary Prophylaxis in Patients with Brain Metastases and Primary Brain Tumors

The risk of VTE in patients with primary or metastatic brain tumors is increased for various reasons including expression of tissue factor¹⁶ and PAI-1¹⁷ by gliomas, immobility due to paresis of limbs affected by the brain tumor or metastasis. Additionally, the use of antiangiogenic agents such as Avastin™ (bevacizumab) may further increase the risk of arterial thrombosis and ironically increase the risk of bleeding.18,19 The challenge in using anticoagulation is balancing the risk of thrombosis with the risk of precipitating intracranial hemorrhage. Studies have shown both increased risk as well as benefit with the use of LMWH prophylaxis in the nonsurgical setting.²⁰

For patients undergoing neurosurgery, the recommended prophylaxis is to initiate LMWH or low-dose UFH 24 hours postoperatively, in combination with mechanical thromboprophylaxis, such as graduated compression stocking and/or intermittent pneumatic compression. This is associated with minimal risk for bleeding.²¹ Initiation of prophylaxis before neurosurgery in patients with brain tumors may be associated with increased risk for intracranial hemorrhage, as shown in one such study terminated early due to increased bleeding.²²

Treatment of Venous Thromboembolism in Patients with Primary Brain Tumors or Brain Metastases

Patients with primary brain tumors or metastases who develop VTE can be treated will full doses of UFH or LMWH.²³ Here the use of UFH has the potential advantage over LMWH of a short half-life and the ability to administer protamine to neutralize it in the event of hemorrhage or overdosage. It may be advisable to forego the use of a bolus at the outset of treatment and simply start an infusion and increase the rate with frequent monitoring to prevent inadvertent overdosage. We prefer the use of anti-factor Xa activity monitoring (rather than the aPTT) due to the possibility that the aPTT is not as accurate in its ability to predict anticoagulant effects of heparin. Lupus anticoagulants or increased factor VIII levels may result in spuriously long or short values, respectively. A screening noncontrast head computed tomography (CT) may be considered to exclude recent intracranial bleeding before the initiation of anticoagulation,²³ especially in patients with certain types of brain metastases associated with high rates of spontaneous hemorrhage, such as with thyroid cancer, melanoma, renal cell carcinoma, and choriocarcinoma. Evidence of recent spontaneous bleeding is generally considered a contraindication to anticoagulation. IVC filters have a role in this situation, but their placement is often proposed on the mistaken assumption that the patient cannot be anticoagulated, and may be overused.

Treatment of Patients with Trousseau Syndrome

Trousseau syndrome is the constellation of venous and arterial thromboembolic disorders predating or associated with a malignancy.24,25 Patients with this syndrome, even if anticoagulated with warfarin with a therapeutic international normalized ratio (INR), may nevertheless have recurrent thrombi. Other clinical characteristics of Trousseau syndrome include microangiopathy, chronic, low-grade disseminated intravascular coagulation (DIC), and nonbacterial thrombotic endocarditis. UFH, LMWH, and fondaparinux are more effective than warfarin in the treatment of Trousseau syndrome. The dosing of anticoagulation varies depending on the clinical setting. For example, a patient with an acute DVT will require enoxaparin at therapeutic doses, whereas DIC may be controlled with lower doses. Treatment is administered indefinitely (or for as long as tumor persists).

General Approach in Treating Venous Thromboembolism in Cancer Patients

In general, treatment of VTE in patients with cancer consists of acute therapy with LMWH or UFH for at least 5 to 7 days’ duration in patients without contraindications to anticoagulation followed by LMWH or warfarin for at least 3 months. The CLOT trial²⁶ showed an 8% absolute risk reduction without an increase in major bleeding when cancer-related VTE was treated with an LMWH, for example, dalteparin for 6 months compared with warfarin. Chronic therapy with LMWH is associated with superior outcomes in cancer patients with VTE.

Cancer patients with VTE should be treated for a minimum of 3 months, while patients with PE should be treated for at least 6 months, ideally with LMWH. Anticoagulation for an indefinite duration may be considered in patients with active cancer or persistent risk factors who may be bedridden, critically ill, and/or malnourished. Extended anticoagulation therapy with an LMWH may require dosage reduction after an initial period. For example, in the CLOT study, the dalteparin dosing was lowered from 200 units/kg every day to 150 units/kg every day after 1 month.

In the event that warfarin will be used for chronic therapy (due to cost or patient’s preference), there should be a transition phase of at least 5 days during which the acute parenteral anticoagulant (e.g., UFH, LMWH, or fondaparinux) is overlapped with warfarin until an INR of 2.0 or more is achieved. Clinicians should be aware that the warfarin modulation of anticoagulation intensity can be clinically challenging due to drug-drug interactions with commonly used chemotherapeutics, antimicrobials, and other new drugs such as those undergoing testing in Phase I clinical trials.

Most institutions have nomograms for dosing and monitoring of UFH. Anti-factor Xa activity instead of the aPTT has been more frequently used to monitor UFH because of the observation of dissociation between the aPTT and heparin levels measured by anti-factor Xa activity, suggesting heparin resistance. Heparin resistance usually occurs in patients with elevations in factor VIII or von Willebrand factor, antithrombin III (AT) deficiency, increased heparin clearance, elevations in heparin-binding proteins, and use of fibrinogen. Factor VIII, von Willebrand factor, and fibrinogen are acute-phase proteins and elevated factor VIII levels shorten the aPTT.²⁷⁻²⁹ When UFH is used, the target therapeutic anti-factor Xa level should be 0.3 to 0.7 U/mL.

Anticoagulation Options in Cancer Patients

In patients who develop recurrent VTE despite adequate anticoagulation with warfarin (INR 2.0–3.0) the etiology may be cancer-related hypercoagulability such as the Trousseau syndrome, anatomic causes such as extrinsic vascular compression, and acquired or familial thrombophilia. Treatment can be changed to heparin (LMWH preferred) or fondaparinux. The use of heparin is preferred to use of vitamin K antagonists in the setting of cancer.³⁰ Switching to heparin therapy is an option following failure of fondaparinux to prevent VTE recurrence and vice versa. Twice-daily dosing of enoxaparin is an option for patients exhibiting recurrent VTE while receiving once-daily therapy with am LMWH,³¹ and escalating the dose of LMWH can be effective for treating cases that are resistant to standard, weight-adjusted doses of LMWH³² If thrombocytopenia occurs during anticoagulation, chemotherapy-induced thrombocytopenia, DIC, heparin-induced thrombocytopenia (HIT), antiphospholipid antibody syndrome (APS), thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, bone marrow failure, and folate or vitamin B12 deficiency should all be part of the differential diagnosis. Thrombocytopenia does not protect against thrombosis. Anticoagulation therapy should not be withheld because of relative thrombocytopenia alone. The management of antithrombotic therapy in patients with thrombocytopenia requires individualized assessments of the risk of bleeding and the risk of thrombosis.³³ Low-dose enoxaparin (< 1 mg/kg/day) may be considered safe at a platelet count in the range of 20 and 55 × 10⁹/L in stem cell transplantation patients who weigh >55 kg.³⁴ On the other hand, thrombocytopenia in APS and HIT may indicate increased disease activity and increased thrombotic potential and aggressive antithrombotic therapy may be warranted.³⁵ Clinical suspicion of HIT should be high when recurrent VTE is observed in a cancer patient receiving or recently exposed to heparinbased therapy. In the typical presentation, platelet counts fall by more than 50% from baseline 5 to 8 days after exposure to heparin. The drop in platelet count can occur even sooner if the patient has been primed by treatment with heparin prior to the current exposure. A major difficulty in diagnosis of HIT is that cancer patients often have multiple reasons for thrombocytopenia, including myelosuppressive drugs, radiation therapy, and infections. An algorithm for calculating pretest probability of HIT includes clinical elements such as presence of thrombocytopenia, timing of drop in platelet count, other causes for thrombocytopenia, and the presence of thrombosis³⁶ and has been modified since its introduction to improve the accuracy of pretest probability estimation.³⁷ Testing for antiplatelet factor 4 antibodies with a sensitive ELISA method can exclude HIT if negative. In cases where the result is positive, the possibility of a false positive can be ruled out by the more specific test for release of ¹⁴C serotonin from labeled platelets in the presence of patient serum and heparin. Once the diagnosis of HIT is established, all heparin must be discontinued (even apparently trivial catheter flushes), and an alternative anticoagulant must be started. Alternative anticoagulants that are useful in this setting include direct thrombin inhibitors such as argatroban, bivalirudin, or lepirudin. Long-term anticoagulation with warfarin may be initiated after the platelet count has recovered, and there must be at least 5 days of overlap with the alternative anticoagulant before its discontinuation. If warfarin is initiated while the patient is still in the acute phase of HIT, further acute thrombosis can be precipitated as protein C levels fall upon initiation of warfarin. The highest risk of HIT is seen with use of UFH, and lower risk is seen with LMWH with shorter glucosaminoglycan chains such as enoxaparin. Fondaparinux is manufactured by a total chemical synthesis of the core pentasaccharide chain that is the minimal essential element required for anti-factor Xa anticoagulant activity, and has the least tendency to provoke antiplatelet factor 4 antibodies. Further, it can be used as an alternative to heparin in patients diagnosed with HIT.³⁸

Thrombosis and Venous Access Devices

A majority of cancer patients have, at one time or another, central venous catheters placed for various amounts of time to administer chemotherapy, antibiotics, drugs, or blood products. A frequent complication of long-term central venous catheters is thrombosis that may involve the catheter tip, the entire length of the catheter, or the lumen of the vein in which the catheter resides.³⁹ It would be desirable to prevent such thrombosis not only to preserve central venous access but also to prevent morbidity from obstruction of the large veins of the arm or chest, and to prevent further extension or embolization. Although many strategies for prophylaxis of catheter-related clots have been proposed, a recent Cochrane Database Systematic Review shows that there is no statistically significant effect of prophylactic heparin or low-dose warfarin to prevent catheter-related DVT in patients with cancer.⁴⁰ Therefore, for cancer patients with indwelling central venous catheters, neither prophylactic doses of LMWH nor “mini-dose” warfarin are recommended to prevent catheter-related thrombosis.

For those patients who have developed a catheter-related DVT, treatment can be initiated with LMWH for 5 to 7 days, followed by warfarin (INR 2–3), or enoxaparin at a dose of 1.5 mg/kg/day for the life-time of the catheter for a total duration of therapy of at least 3 months, (whichever is longer). If the catheter is required but DVT symptoms persist or the clot progresses despite anticoagulation, the catheter should be removed. Thrombolytic therapy with relatively small doses of tPA (one or two treatments of less than 10 mg) has been successfully employed to clear venous occlusions and maintain patency of the vessel without any observed bleeding.⁴¹ Patients treated with tPA should have subsequent anticoagulation as above, in the absence of contraindication(s) to prevent repeat thrombosis. In some cases, dilatation of stenotic venous segments may be required when multiple catheters have been inserted and removed for therapy. Low doses of tPA (2 mg) can be used to open clotted catheter lumens.

Indications for Inferior Vena Cava Filters in a Cancer Patient

IVC filters should not be used as a routine method for prevention of pulmonary embolism (PE) in patients with DVT. Rather, their employment should be predicated on genuine contraindications to anticoagulation and/or documented failure of adequate anticoagulation. Patients with baseline cardiac or pulmonary dysfunction severe enough to make any new or recurrent PE life threatening or documented multiple PE and chronic thromboembolic pulmonary hypertension may also be candidates for an IVC filter. Patients with DVT who receive IVC filters are initially protected against PE. However, they are at increased risk for future recurrent DVT as well as future IVC thrombosis or postthrombotic changes in the lower extremities, presumably from increased resistance to venous outflow from the lower extremities. A randomized trial of use of IVC filters compared to non-use of IVC filters for DVT showed that the absolute risk of PE in the first 12 days (symptomatic or not) was reduced from 4.8% to 1.1%, but at 2 years the rate of recurrent DVT was 20.8% in the IVC filter group versus 11.6% in the nonfilter group; there was no difference in mortality between the two groups.⁴² There are retrievable as well as permanent IVC filters; however, retrievable filters are more prone to migration after placement than permanent filters, and they may, rarely, embolize. If the retrievable filter is not removed within the time frame indicated by the manufacturer, it can become technically difficult to retrieve, and the vascular surgeon or interventional radiologist may recommend that it remain permanently in place.

Results from a recent systemic review of the use of retrievable IVC filters showed that the average retrieval rate was 34%; most filters in fact became permanent devices.⁴³ Serious complications, including strut fracture, with or without embolization or filter migration, vena cava perforation, or vena cava occlusion occur with increasing incidence after prolonged implantation times (> 30 days). In one retrospective series that evaluated 80 patients with retrievable filters placed between 2004 and 2009, a strut fracture rate of 16% was observed, in some cases with embolization of filter components to the heart.⁴⁴ At the present time, the objective comparison data of different filter designs do not support superiority of any particular design.

PREVENTION, DIAGNOSIS, AND TREATMENT OF VENOUS THROMBOEMBOLISM IN PREGNANCY

Although mortality is rare in pregnant women in developed countries, pregnancy-associated PE remains one of the most frequent causes of the occasional deaths that do occur. Thrombosis during pregnancy and puerperium (the 6-week period following delivery) is attributable to venous stasis in the lower extremities caused by the gravid uterus as well as alterations in hemostasis, such as the progressive increase in fibrin turnover, increased levels of coagulation factors, decreased fibrinolytic activity, and decreased free protein S levels. In addition, the presence of inherited thrombophilias and the APS, as well as a previous history of thrombosis, may accentuate/increase the risk for DVT during pregnancy and the postpartum period.⁴⁵ Although DVT incidence seems to be evenly distributed throughout the three trimesters, PE is disproportionately found in the puerperium. Poor obstetric outcomes (including preeclampsia, placental abruption, intrauterine growth delay, and fetal loss) may be associated with thrombophilia.⁴⁶

Diagnosis of Venous Thromboembolism during Pregnancy

In pregnant women, signs and symptoms such as lower extremity edema, back pain, and/or chest pain may be attributed to pregnancy rather than to a possible VTE. Since levels of D-dimer increase during pregnancy, especially during the last trimester, the D-dimer assay may not be helpful in establishing a diagnosis of VTE in pregnant women. Radiologic studies have to be used judiciously, and considered with attention to potential risks to the fetus, and mother, and are usually contraindicated. Compression ultrasonography of the whole leg is preferred as the initial test for suspected DVT or PE in symptomatic patients and does not pose significant risks to the fetus. If the ultrasound is positive for DVT in patients presenting with symptoms suggestive of PE, an indication for anticoagulation is established, the diagnosis of PE is inferred, and no further imaging is required to start treatment.⁴⁵ If pelvic DVT is a consideration, duplex ultrasonography rather than compression alone will increase sensitivity. If results are equivocal or an iliac vein thrombosis is possible, then magnetic resonance venography should be considered, since it does not carry radiation risks and is reliable, though issues remain, for example, the use of gadolinium and availability of the test. When using ventilation/ perfusion (V/Q) lung scan, the perfusion scan should be completed first and, if normal, PE is excluded, and there is no need for additional radioisotope exposure from the ventilation assessment of the study. If abnormal, a ventilation scan should be performed to confirm mismatch. Those with an indeterminate V/Q scan should have CT pulmonary angiography (CTPA). CTPA is also preferred in the presence of hemodynamic compromise. The fetal radiation exposure, even with current multidetector CT scanning instruments, varies according to trimester of pregnancy, with potentially greater exposure later in gestation if the scanner is programmed to increase the amount of scanner current and radiation to compensate for greater tissue mass.⁴⁷ It is best to discuss the diagnostic needs of the CT scan with the radiologist so as to minimize fetal radiation through physical measures (shielding), selection of imaging programs appropriate for the pregnant woman at her stage of gestation, and procedural measures (in particular, stopping the study once a diagnosis has been made). The risk to the fetus for developing a later malignancy after a CT imaging procedure has been estimated at one excess cancer per 1,000 abdominal/pelvic CT procedures, which is a small, but not negligible risk.⁴⁸ In addition to fetal risk, the female breast is radiosensitive and CTPA is associated with an increased lifetime risk of breast cancer of 13.6% with a historic background risk of 1/200.48,49 Reducing radiation to the maternal breast favors use of perfusion scanning,⁵⁰ and avoiding ventilation scanning when possible.

Management of Pregnant Women at Increased Risk for Venous Thromboembolism

Risk factors for thrombosis include a personal history of VTE, known inherited or acquired thrombophilic mutations/polymorphisms, obesity, advanced maternal age, high parity, and prolonged bedrest. Based on safety data for the fetus, heparin compounds are preferred over warfarin for the prevention and treatment of VTE in pregnancy because UFH and LMWH do not cross the placenta. LMWH is preferred because it has better bioavailability, a longer plasma half-life, a more predictable dose response, and a better safety profile than UFH. The risks of HIT and osteoporosis appear lower with LMWH than with UFH.28,51 Warfarin is generally avoided in pregnant women, due to the risk of teratogenecity when administered between 6 and 12 weeks of gestation. After parturition, either warfarin or heparin anticoagulants can be safely used; since they do not appear in breast milk, they can be given to mothers who are nursing. The prophylactic anticoagulants should be continued for at least 6 weeks postpartum. The therapeutic anticoagulants should be given for a minimum total duration of 6 months.

Tables 23.4 and 23.5⁵¹ offer guidance on management in defined clinical settings. A complete description of thrombotic conditions, and appropriate treatments during pregnancy as well as during the post partum period, can be found there.

As parturition approaches, women who are receiving LMWH should plan to discontinue treatment 24 hours before delivery. This is particularly important for those in whom epidural or spinal anesthesia is to be used, due to the known additional risk for spinal hematoma that is conferred by use of LMWH. The drug can be resumed 24 hours after delivery. UFH, which has a half-life of 90 minutes, may well be preferable for women who require anticoagulation but whose time of delivery is not predictable.

RECOGNITION OF POSTTHROMBOTIC (“POSTPHLEBITIC”) SYNDROME

Postthrombotic syndrome occurs within 1 to 2 years of an episode of DVT in as many as 20% to 50% of patients.⁵² In some patients, it may take several months for the initial pain and swelling associated with acute DVT to resolve, so a diagnosis of postthrombotic syndrome should be deferred until after the acute phase has passed. Postthrombotic syndrome results from venous hypertension, which is due to obstruction and damage to the venous valves caused by DVT. The syndrome manifests as chronic pain, tingling, and edema in the affected leg, as well as hyperpigmentation of the skin, and painless ulcers on the medial malleolar surface in the worst cases. The diagnosis is primarily clinical; duplex scanning can be used if the symptoms increase in severity and if surgery is contemplated.

Long-term use of graduated compression stockings (GCS) after symptomatic proximal DVT has been proved to reduce the risk of any postthrombotic syndrome.⁵³ Application of GCS has been recommended to be initiated within 2 to 3 weeks after the first DVT and continued for at least 2 years. However, a recent trial reported that beyond an initial 6-month period use, there was no incremental benefit in prolonging compression therapy for an additional 18 months.⁵⁴ Although GCS are unlikely to cause harm, they are difficult to apply, uncomfortable, expensive, and require replacement every few months. Based on the current state of evidence on the use of GCS to prevent postthrombotic syndrome, GCS should be applied to patients who have residual leg pain or swelling after proximal or distal DVT, and continued for as long as the patient derives symptomatic benefit or is able to tolerate them.

RECOMMENDATIONS FOR MANAGEMENT OF POSTTHROMBOTIC SYNDROME

For minimally symptomatic postthrombotic syndrome, graduated stockings with moderate compression (15 to 20 mm Hg) or leg elevation at the end of the day may be palliative. An effective way for the patient to reverse or relieve the symptoms of venous hypertension is to elevate both legs above the level of the heart for 30 minutes three or four times daily. During the night, leg elevation can be achieved by elevating the foot of the bed (using blocks placed under the bed if necessary). In the presence of mild to moderate symptoms (edema, aching, heavy legs) graduated stockings with firm compression, 20 to 30 mm Hg, can be used; extra firm is also available (30 to 40 mm Hg) with or without nighttime pneumatic device. Patients who have experienced ulcer formation should wear stockings daily throughout the day. Nonelastic stockings that are comprised of multiple layers attached by Velcro (CircAid™) can also be used and may be easier to apply. Intermittent pneumatic compression in combination with sustained graduated compression has demonstrated improved outcome in patients with venous leg ulcers.⁵⁵

Pharmacologic Approach to Postthrombotic Syndrome

Horse chestnut seed extract, such as aescin was found to be effective for short-term treatment of chronic venous insufficiency symptoms such as leg pain and edema compared to a placebo. A short-term (up to 3 weeks) trial of twice-daily horse chestnut seed extract (available at natural product stores) may be suggested to patients whose postthrombotic symptoms are not adequately controlled by GCS. However, larger and more rigorous trials are needed to assess its long-term effectiveness and safety.⁵⁶ In patients with venous leg ulcers, pentoxifylline and rutosides are recommended in addition to local care and compression and/or intermittent pneumatic compression by ACCP guidelines.⁵⁷

Table 23.6  Approaches to Prevent and Treat Postthrombotic Syndrome

Prevention

•  Prevent the occurrence of DVT with the use of thromboprophylaxis in high-risk patients and settings, as recommended in evidence-based consensus guidelines.

•  Prevent DVT recurrence by providing anticoagulation of appropriate intensity and duration for the initial DVT and by use of thromboprophylaxis in high-risk patients and settings if long-term anticoagulation is discontinued.

•  Use knee-length, 30 to 40 mm Hg elastic compression stockings for 2 yr or more after DVT; optimal duration is uncertain.

•  The role of thrombolysis of acute DVT to prevent PTS is not yet established. Catheter-directed thrombolytic techniques require further evaluation in properly designed trials before being endorsed as effective and safe to reduce the risk of PTS.

Management

•  Use elastic compression stockings to reduce edema and improve PTS symptoms like leg heaviness.

•  Consider the use of intermittent pneumatic compression units and/or VenoWave™ device for severe symptomatic PTS.

•  Consider the short-term use of venoactive agents such as aescin (horse chestnut extract) or rutosides, which appear to improve some PTS symptoms; large controlled trials addressing long-term effectiveness and safety are needed.

•  Compression therapy, skin care, and topical dressings are used to treat venous ulcers.

•  Providing patient support and ongoing follow-up is an important component of PTS management.

DVT, deep venous thrombosis; PTS, postthrombotic syndrome.

Adapted from Kahn SR.The post-thrombotic syndrome. Hematology Am Soc Hematol Educ Program. 2010:216-220.

There is no evidence that diuretics are effective for the treatment of postthrombotic-related edema, or that nonsteroidal anti-inflammatory drugs improve symptoms of postthrombotic syndrome beyond their analgesic effects.

Surgical Management of Postthrombotic Syndrome

Surgical correction of superficial venous reflux in addition to compression bandaging was not shown to improve ulcer healing but did reduce recurrent ulceration compared with compression therapy alone in a randomized trial.⁵⁸ The available strategies to prevent and treat postthrombotic syndrome is summarized in Table 23.6.⁵⁹

ANTIPHOSPHOLIPID SYNDROME

APS is an acquired thrombophilic condition characterized by arterial or venous thrombosis or pregnancy morbidity in patients with persistent positive lupus anticoagulants (LAs), anticardiolipin antibodies, or anti-beta-2 glycoprotein I for at least 12 weeks apart. Antiphospholipid antibodies (APA) promote activation of endothelial cells, monocytes, and platelets, and overproduction of tissue factor and thromboxane A2. Complement activation might have a central pathogenic role. Of the different APA, the LA is the strongest predictor of thrombotic outcomes related to APS.⁶⁰

Prevention of thrombosis is a major goal in patients with APA. The 13th International Congress on APA recommended that all APA carriers receive primary thromboprophylaxis with usual doses of LMWH in high-risk situations, such as surgery, prolonged immobilization and puerperium, and indefinite anticoagulation at an INR of 2.0 to 3.0 for patients with APS presenting with first venous events (secondary thromboprophylaxis). APS patients with arterial disease or recurrent events, or both, may need a more aggressive treatment, such as warfarin with a target INR of more than 3.0 or combined antithrombotic therapy with aspirin or other antiplatelet medications. In cases of first venous event, patients with a single positive test for APA (anticardiolipin or anti-β2-glycoprotein 1), and a known transient precipitating factor, anticoagulation could be limited to 3–6 months. In patients with difficult management due to recurrent thrombosis, fluctuating INR levels, major bleeding or a high risk for major bleeding, alternative therapies could include long-term LMWH, hydroxychloroquine, or statins.60,61

NEW ANTICOAGULANTS

Warfarin therapy is affected by changes in diet and interactions with other drugs, requiring ongoing laboratory monitoring. Further, the onset of action is delayed, and the full anticoagulant effect may not be achieved for several days, necessitating concomitant use of parenteral anticoagulants with rapid onset of action (e.g., LMWH) while the dose is titrated. These limitations stimulated the search for alternative oral anticoagulants. The new oral direct thrombin inhibitor (dabigatran) and oral factor Xa inhibitors (apixaban and rivaroxaban) have good bioavailability and reliable anticoagulant activity, and are promising new medications. In 2010, the FDA approved dabigatran (Pradaxa™) 150 mg twice daily for the reduction of the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. Approval was based on a multicenter, active-control trial, the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY), in which 18,113 patients were randomly assigned to receive 150 mg of dabigatran, 110 mg of dabigatran, or warfarin.⁶² Dabigatran was given twice daily; warfarin was titrated to a target INR of 2.0 to 3.0. Both dabigatran regimens were noninferior to warfarin, but the 150-mg regimen was significantly superior to warfarin and to the 110-mg dabigatran regimen. Both regimens have been considered safe and effective if studied alone in comparison with warfarin, although the noninferiority finding for the 110-mg dose was less compelling, and only the 150-mg daily-dosing regimen was approved by the FDA.⁶³ Although it is not approved for treatment of DVT in the Unites States, a Phase III trial (RE-COVER) comparing 150 mg dabigatran BID with standard warfarin showed noninferiority of dabigatran to warfarin.⁶⁴ Likewise, trials of for prophylaxis of VTE in the setting of total hip⁶⁵ or total knee replacement66,67 have shown noninferiority of dabigatran to warfarin for this indication.

In 2011, the FDA approved rivaroxaban (Xarelto™) 10 mg once daily for 35 days following hip replacement and for 12 days following knee replacement for prevention of DVT in patients undergoing joint replacement surgery. The approval is based on testing of rivaroxaban’s safety and efficacy in the four Regulation of Coagulation in Orthopedic Surgery to Prevent Deep Venous Thrombosis and Pulmonary Embolism (RECORD) trials.⁶⁸⁻⁷¹ The data from the RECORD trials showed significantly greater efficacy of rivaroxaban, both in head-to-head comparison with enoxaparin and when comparing extended-duration (5 weeks) of rivaroxaban with short-duration enoxaparin (2 weeks), followed by a placebo. In these trials, rivaroxaban and enoxaparin demonstrated similar safety profiles, including low rates of major bleeding. Also in 2011, the FDA approved rivaroxaban 20 mg once daily to reduce the risk of stroke in patients with nonvalvular atrial fibrillation. Supportive evidence came primarily from Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF), in which 14,264 patients were randomly assigned in a double-blind fashion to either 20 mg of rivaroxaban once daily or warfarin therapy targeting an INR of 2.0 to 3.0. In this study, rivaroxaban was noninferior to warfarin for the prevention of stroke or systemic embolism. There was no significant between-group difference in the risk of major bleeding, although intracranial and fatal bleeding occurred less frequently in the rivaroxaban group. ⁷² In 2012 the FDA approved the use of rivaroxaban for treatment of DVT or PE based on the results of the EINSTEIN⁷³ and EINSTEIN-PE⁷⁴ trials, respectively, which showed rivaroxaban to be non-inferior to standard therapy with enoxaparin/warfarin.

The other oral factor Xa inhibitor, apixaban is also under FDA consideration for the prevention of stroke in subjects with atrial fibrillation. In the Apixaban for reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) trial, apixaban at a dose of 5 mg twice daily was compared with warfarin (target INR 2.0 to 3.0) in 18,201 patients with atrial fibrillation and at least one additional risk factor for stroke. Apixaban was found to be superior to warfarin in preventing stroke or systemic embolism, caused less bleeding, and resulted in lower mortality in patients with atrial fibrillation.⁷⁵ Anticoagulant medications with novel mechanisms or important features that promise advantages over our current armamentarium are reviewed in Ref. 66. and Ref. 77.

Certain disadvantages remain for all of the new oral anticoagulant medications that are in advanced stages of clinical testing. There currently are no reliable ways to reverse or neutralize these medications in the event of overdose or bleeding, in contrast to warfarin, for which reversal with vitamin K or fresh frozen plasma is readily accomplished. The rapid onset of anticoagulation, a benefit for most applications, is matched by an equally rapid decrease in the anticoagulant effect in contrast to the gradual onset/slow decrease of effect seen for warfarin. A quick onset of action may be problematic when a patient is poorly compliant and frequently misses doses of a drug that disappear rapidly from the circulation, particularly under conditions of high thrombotic risk. Although laboratory testing for anticoagulant effects can be accomplished by use of the ecarin clotting time, for instance, in the case of the direct antithrombin drugs, these are not as yet commonly available in contrast to the prothrombin time and INR. These drugs are all significantly more expensive than generic warfarin, and none are as yet given FDA approval for treatment of VTE (secondary prophylaxis), and a place for warfarin will likely remain even after FDA approval, in patients whose warfarin dose is stable and are not difficult to manage, or who cannot afford the more expensive novel agents.

These concerns aside, a new era of easier-to-use oral anticoagulants for the prevention and treatment of VTE is now beginning, which is a welcome development.

References

  1.  Ay C, Simanek R, Vormittag R, et al. High plasma levels of soluble P-selectin are predictive of venous thromboembolism in cancer patients: results from the Vienna Cancer and Thrombosis Study (CATS). Blood. 2008;112:2703-2708.

  2.  O’Shea SI, Ortel TL. Issues in the utilization of low molecular weight heparins. Semin Hematol. 2002;39:172-178.

  3.  Agnelli G, Bolis G, Capussotti L, et al. A clinical outcome-based prospective study on venous thromboembolism after cancer surgery. The @RISTOS project. Ann Surg. 2006;243:89-95.

  4.  Bergqvist D, Agnelli G, Cohen AT, et al. Duration of prophylaxis against venous thromboembolism with enoxaparin after surgery for cancer. N Engl J Med. 2002;346:975-980.

  5.  Rasmussen MS, Jorgensen LN, Wille-Jorgensen P, et al. Prolonged prophylaxis with dalteparin to prevent late thromboembolic complications in patients undergoing major abdominal surgery: a multicenter randomized open-label study. J Thromb Haemost. 2006;11:2384-2390.

  6.  Ageno W, Squizzato A, Garcia D, et al. Epidemiology and risk factors of venous thromboembolism. Semin Thromb Hemost. 2006;32:651-658.

  7.  Geerts WH, Bergquvist D, Pineo GF, et al. Prevention of venous thromboembolism. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest. 2008;133:381S-453S.

  8.  SAGES Guidelines for deep venous thrombosis prophylaxis during laparoscopic surgery. October 2006. http://www.sages.org/publication/id/C/

  9.  SAGES Guidelines Committee. Surg Endosc. 2007;21:1007-1009.

10.  Palumbo A, Rajkumar SV, Dimopoulos MA, et al. on behalf of the International Myeloma Working Group. Prevention of thalidomide- and lenalidomide-associated thrombosis in myeloma. Leukemia. 2008;22:414-423.

11.  Agnelli G, Gussoni G, Bianchini C, et al.; on behalf of the PROTECHT Investigators. Nadroparin for the prevention of thromboembolic events in ambulatory patients with metastatic or locally advanced solid cancer receiving chemotherapy: a randomised, placebo-controlled, double-blind study. Lancet Oncol. 2009;10:943-949.

12.  Aue G, Lozier JN, Tian X, et al. Inflammation, TNFα and endothelial dysfunction link lenalidomide to venous thrombosis in chronic lymphocytic leukemia. Am J Hematol. 2011;86:835-840.

13.  Khorana AA, Francis CF, Culakova E, et al. Thromboembolism in hospitalized neutropenic cancer patients. J Clin Oncol. 2006;24:484-490.

14.  Samama MM, Cohen AT, Darmon JY, et al. A comparison of enoxaparin with placebo for the prevention of venous thromboembolism in acutely ill medical patients. Prophylaxis in medical patients with enoxaparin study group. N Engl J Med. 1999;341:793-800.

15.  Kakkar AK, Cimminiello C, Goldhaber SZ, et al. for the LIFENOX investigators. Low molecular weight heparin and mortality in acutely ill medical patients. N Engl J Med. 2011;365:2463-2472.

16.  Hamada K, Kuratsu J, Saitoh Y, Takeshimo H, Nishi T, Ushio Y. Expression of tissue factor correlates with grade of malignancy in human glioma. Cancer. 1996;77:1877-1883.

17.  Sawaya R, Ligon L. Thromboembolic complications associated with brain tumors. J Neurooncol. 1994;22:173-181.

18.  Bevacizumab package insert. December 2011. http://www.gene.com/gene/products/information/pdf/avastin-prescribing.pdf

19.  Norden AD, Young GS, Setayash K, et al. Bevacizumab for recurring malignant gliomas: Efficacy, toxicity, and patterns of recurrence. Neurology. 2008;70:779-787.

20.  Agnelli G, Piovella F, Buoncristiani P, et al. Enoxaparin plus compression stockings compared with compression stockings alone in the prevention of venous thromboembolism after elective neurosurgery. N Engl J Med. 1998;339:80-85.

21.  Goldhaber SZ, Dunn K, Gerhard-Herman M, et al. Low rate of venous thromboembolism after craniotomy for brain tumor using multimodality prophylaxis. Chest. 2002;122:1933-1937.

22.  Dickinson LD, Miller LD, Patel CP, et al. Enoxaparin increases the incidence of postoperative intracranial hemorrhage when initiated preoperatively for deep venous thrombosis prophylaxis in patients with brain tumors. Neurosurgery. 1998;43:1074-1081.

23.  Gerber DE, Grossman SA, Streiff MB. Management of venous thromboembolism in patients with primary and metastatic brain tumors. J Clin Oncol. 2006;24(8):1310-1318. Review.

24.  Levine MN, Lee AYY, Kakkar AK. From Trousseau to targeted therapy: new insights and innovations in thrombosis and cancer. J Thromb Haemost. 2003;1:1456-1463.

25.  Walsh-McMonagle D, Green D. Low-molecular-weight heparin in the management of Trousseau’s syndrome. Cancer. 1997;80:649-655.

26.  Lee AYY, Levine MH, Baker RI, et al. Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med. 2003;349:146-153.

27.  Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999;340:448-454.

28.  Hirsh J, Rascke R. Heparin and low-molecular weight heparin. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest. 2004;126:188S-203S.

29.  Levine MN, Hirsh J, Gent M, et al. A randomized trial comparing activated thromboplastin time with heparin assay in patients with acute venous thromboembolism requiring large daily doses of heparin. Arch Intern Med. 1994;154:49-56.

30.  Akl EA, Barba M, Rohilla S, et al. Low-molecular-weight heparins are superior to vitamin K antagonists for the long term treatment of venous thromboembolism in patients with cancer: a Cochrane Systemic Review. J Exp Clin Cancer Res. 2008;27:21-31.

31.  Merli G, Spiro TE, Olsson C-G, et al. Subcutaneous enoxaparin once or twice daily compared with intravenous unfractionated heparin for treatment of venous thromboembolic disease. Ann Int Med. 2001;134:191-202.

32.  Carrier M, Le Gal G, Cho R, et al. Dose escalation of low molecular weight heparin to manage recurrent venous thromboembolic events despite systemic anticoagulation in cancer patients. J Thromb Haemost. 2009;7:760-765.

33.  Arnold D, Lim W. A rational approach to the diagnosis and management of thrombocytopenia in the hospitalized patient. Semin Hematol. 2011;48:251-258.

34.  Ibrahim RB, Peres E, Dansey R, et al. Safety of low-dose low-molecular-weight-heparins in thrombocytopenic stem cell transplantation patients: a case series and review of the literature. Bone Marrow Transplant. 2005;35:1071-1077.

35.  Lim W. Antiphospholipid antibody syndrome. Hematology Am Soc Hematol Educ Program. 2009:233-239. Review

36.  Warkentin TE. Heparin-induced thrombocytopenia: pathogenesis and management. Br J Haematol. 2003;121:535-555.

37.  Cuker A, Arepally G, Crowther MA, et al. The HIT Expert Probability (HEP) Score: a novel pre-test probability model for heparin-induced thrombocytopenia based on broad expert opinion. J Thromb Haemost. 2010;8:2642-2650.

38.  Papadopoulos S, Flynn JD, Lewis DA. Fondaparinux as a treatment option for heparin-induced thrombocytopenia. Pharmacotherapy. 2007;27:921-926.

39.  Horne MK III, Chang R. Thrombosis related to venous access devices. In: Kitchens C, Alving B, Kessler C, eds. Consultative Hemostasis and Thrombosis. 2nd ed. Philadelphia, PA: W.B. Saunders; 2007:553-559.

40.  Akl EA, Kamath G, Yosuico V, et al. Thromboprophylaxis for patients with cancer and central venous catheters (Cochrane Database Systematic Review). Cancer. 2011;112:2483-2492.

41.  Chang R, Horne MK, Shawker TH, Kam et al. Low-Dose, once-daily, intraclot injections of alteplase for treatment of acute deep venous thrombosis. J Vasc Interv Radiol. 2011;22:1107-1116.

42.  Decousus H, Leizorovicz A, Parent F, et al. A clinical trial of vena caval filters in the prevention of pulmonary embolism in patients with proximal deep-vein thrombosis. N Engl J Med. 1998;338:409-415.

43.  Angel LF, Tapson V, Galgon R, et al. Systematic review of the use of retrievable inferior vena cava filters. J Vasc Interv Radiol. 2011;22:1522-1530.

44.  Nicholson W, Nicholson WJ, Tolerico P, et al. Prevalence of fracture and fragment embolization of bard retrievable vena cava filters and clinical implications including cardiac perforation and tamponade. Arch Int Med. 2010;170:1827-1831.

45.  Marik PE, Plante LA. Venous thromboembolic disease and pregnancy. N Engl J Med. 2008;359:2025-2033.

46.  Battinelli EM, Bauer KA. Hematologic disorders in pregnancy. Hematol Oncol Clin North Am. 2011;25:323-333.

47.  Gilet AG, Dunkin JM, Fernandez TJ, et al. Fetal radiation dose during gestation estimated on an anthropomorphic phantom for three generations of CT scanners. Am J Radiol. 2011;196:1133-1137.

48.  Kalra MK, Maher MM, Toth TL, et al. Strategies for CT radiation dose optimization. Radiology. 2004;230:619-628.

49.  Remy-Jardin M, Remy J. Spiral CT angiography of the pulmonary circulation. Radiology. 1999;212:615-636.

50.  Shahir K, Goodman LR, Tali A, et al. Pulmonary embolism in pregnancy: CT pulmonary angiography versus perfusion scanning. Am J Roentgenol. 2010;195:W213-W220.

51.  Bates SM. Pregnancy-associated venous thromboembolism: prevention and treatment. Semin Hematol. 2011;48:271-284.

52.  Kahn SR, Ginsberg J. Relationship between deep venous thrombosis and the postthrombotic syndrome. Arch Intern Med. 2004;164:17-26.

53.  Kolbach D, Sandbrink M, Hamulyak K, et al. Non-pharmaceutical measures for prevention of post-thrombotic syndrome. Cochrane Database of Syst Rev. 2003, Issue 3. Art. No: CD004174.

54.  Aschwanden M, Jeanneret C, Koller MT, et al. Effect of prolonged treatment with compression stockings to prevent post-thrombotic sequelae: a randomized controlled trial. J Vasc Surg. 2008;47:1015-1021.

55.  Comerota AJ. Intermittent pneumatic compression: Physiologic and clinical basis to improve management of venous leg ulcers. J Vasc Surg. 2011;53:1121-1129.

56.  Pittler MH, Ernst E. Horse chestnut seed extract for chronic venous insufficiency. Cochrane Database Syst Rev. 2012, Issue 11. Art. No: CD003230.

57.  Kearon C, Kahn SR, Agnelli G, et al. Antithrombotic therapy for venous thromboembolic disease. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest. 2008;133:454S-545S.

58.  Gohel MS, Barwell JR, Taylor M, et al. Long term results of compression therapy alone versus compression plus surgery in chronic venous ulceration (ESCHAR): randomised controlled trial. Br Med J. 2007;335:83-88.

59.  Kahn SR. The post-thrombotic syndrome. Hematology Am Soc Hematol Educ Program. 2010:216-220.

60.  Ruiz-Irastorza G, Crowther M, Branch W, et al. Antiphospholipid syndrome. Lancet. 2010; 376:1498-1509.

61.  Ruiz-Irastorza G, Cuadrado MJ, Ruiz-Arruza I, et al. Evidence-based recommendations for the prevention and long-term management of thrombosis in antiphospholipid antibody-positive patients: report of a Task Force at the 13th International Congress on Antiphospholipid Antibodies. Lupus. 2011;20:206-218.

62.  Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361:1139-1151.

63.  Beasley BN, Unger EF, Temple R. Anticoagulant options—why the FDA approved a higher but not a lower dose of Dabigatran. N Engl J Med. 2011;364:1788-1790.

64.  Schulman S, Kearon C, Kakkar KC, et al. RE-COVER Study Group. Dabigatran versus warfarin in the treatment of acute venous thromboembolism. N Engl J Med. 2009;361:2342-2352.

65.  Eriksson BI, Dahl OE, Rosencher N, et al. Dabigatran etexilate versus enoxaparin for prevention of venous thromboembolism after total hip replacement: a randomized, double-blind, non-inferiority trial. Lancet. 2007;370:949-956.

66.  Eriksson BI, Dahl OE, Rosencher N, et al. Oral dabigatran etexilate vs. subcutaneous enoxaparin for the prevention of venous thromboembolism after total knee replacement: the RE-MODEL randomized trial. J Thromb Haemost. 2007;5:2178-2185.

67.  Ginsberg JS, Davidson BL, Comp PC, et al. Oral thrombin inhibitor dabigatran etexilate vs. North American enoxaparin regimen for prevention of venous thromboembolism after knee arthroplasty surgery. J Arthoplasty. 2009;24:1-9.

68.  Eriksson BI, Borris LC, Friedman RJ, et al. Rivaroxaban versus enoxaparin for thromboprophylaxis after hip arthroplasty. N Engl J Med. 2008;358:2765-2775.

69.  Lassen MR, Ageno W, Borris LC, et al. Rivaroxaban versus enoxaparin for thromboprophylaxis after hip arthroplasty. N Engl J Med. 2008;358(26):2776-2786.

70.  Kakkar AK, Brenner B, Dahl OE, et al. Extended duration rivaroxaban versus short-term enoxaparin for the prevention of venous thromboembolism after total hip arthroplasty: a double-blind, randomized controlled trial. Lancet. 2008;372:31-39.

71.  Turpie AG, Lassen MR, Davidson BL, et al. Rivaroxaban versus Enoxaparin for thromboprophylaxis after total knee arthroplasty (RECORD4): a randomised trial. Lancet. 2009;373:1673-1680.

72.  Patel MR, Mahaffey KW, Garg J, et al. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011;365:883-891.

73.  The EINSTEIN Investigators. Oral rivaroxaban for symptomatic venous thromboembolism. N Engl J Med. 2010;363: 2499-2510.

74.  The EINSTEIN-PE Investigators. Oral rivaroxaban for symptomatic pulmonary embolism. N Engl J Med. 2012;366: 1287-1297.

75.  Granger CB, Alexander JH, McMurray JJV, et al. Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2011;365:981-992.

76.  Eikelboom JW, Weitz JI. New anticoagulants. Circulation. 2010;121:1523-1532.

77.  Liesenfeld K-H, Schafer HG, Troconiz IF, et al. Effects of the direct thrombin inhibitor dabigatran on ex vivo coagulation time in orthopaedic surgery patients: a population model analysis. Br J Clin Pharmacol. 2006;62:527-537.

24

Blood Transfusion

Ronan Desmond and Harvey G. Klein

BLOOD CELL ANTIGENS

Red blood cell (RBC) antigens are classified according to their biochemical, phenotypic, and immunologic characteristics. Based on these characteristics, they have been separated into blood group systems. Currently 30 major blood group systems are recognized, the most clinically relevant being ABO, Rh, Kell, Kidd, and Duffy. Clinically important alloantibodies (specific for antigens not found on the individuals red cells) can cause destruction of transfused red cells or are implicated in hemolytic disease of the newborn (HDN).

The antiglobulin or Coombs test (see below) is used to detect antibodies to red cell antigens and in crossmatching compatible blood units for transfusion. When a clinically important alloantibody is present in a recipient’s serum, antigen-negative blood must be selected. If the alloantibody is against a very high frequency antigen (present in greater than 90% of individuals) or when multiple alloantibodies are present, procurement of compatible blood may be difficult or impossible. Occasionally, the presence of red cell autoantibodies in the recipient makes all units appear incompatible. Further investigations are necessary in these cases to rule out an underlying alloantibody.

“Naturally occurring” antibodies such as anti-A and/or anti-B are present in the absence of prior sensitizing stimulus, whereas development of most other alloantibodies requires prior sensitization by exposure to the corresponding red cell antigen in a previous transfusion or pregnancy.

  Blood group A individuals have naturally occurring anti-B

  Blood group B individuals have naturally occurring anti-A

  Blood group O individuals have naturally occurring anti-A and anti-B

  Blood group AB individuals have neither anti-A nor anti-B

LABORATORY DETERMINATION OF MAJOR BLOOD GROUPS

An individual’s blood group is determined by performing a forward and reverse grouping (ABO typing):

  In forward grouping, reagents of known antibody specificity (anti-A or anti-B) are added to the patient’s RBCs of unknown phenotype (A, B, AB, or O) and the mixtures are examined for visible agglutination; the absence of agglutination on combining the patient’s cells with anti-A or anti-B reagent indicates that the patient’s cells do not have the corresponding antigen. For example, red cells of group O will not agglutinate in the presence of anti-A and/or anti-B.

  In reverse grouping, the patient’s serum is added to reagent cells of known phenotype (A, B, or O) and the mixtures are examined for visible agglutination; the presence of agglutination on combining the patient’s serum with reagent cells of A or B phenotype, indicates that the patient’s serum contains the corresponding antibody. For example serum containing anti-A and anti-B (blood group O) will agglutinate in the presence of both group A and group B red cells.

The forward and reverse groups must be consistent. A specific blood group may not be assigned with certainty until an ABO discrepancy is resolved.

Some common causes of apparent ABO discrepancies are¹ presence of A or B subgroups,² missing/ weakly reacting antibodies (can occur with newborns, the elderly, or in hypogammaglobulinemic states) or weak expression/absence of expected antigens (can occur with lymphoproliferative disorders and post hematopoietic stem cell transplantation, HSCT),³ presence of unexpected or nonspecific antibodies such as cold reactive alloantibodies or autoantibodies,⁴ interfering substances such as Wharton jelly from the umbilical cord of a newborn, or⁵ hyperproteinemic states causing Rouleaux formation.

ANTIGLOBULIN TEST

The antiglobulin test uses antibodies with specificity for immunoglobulins or complement to detect the presence of antibody (or complement) on the RBC surface or in a patient’s serum.

The direct antiglobulin test (DAT), or the direct Coombs test, detects the presence of antibody or complement coating RBC and may be positive in a variety of settings, including those listed below:

  Hemolytic transfusion reactions

  HDN (usually strongly positive)

  Autoimmune hemolytic anemias (AIHA)

  With some pharmacologic agents (penicillins, cephalosporins)

  After administration of intravenous immunoglobulin (IVIG) or plasma (passively acquired)

  Post marrow or organ transplant (passenger lymphocyte syndrome)

  Autoimmune diseases

  Some normal individuals

A positive DAT does not necessarily indicate in vivo hemolysis or shortened RBC survival. Falsepositive results may occur when Rouleaux formation is mistaken for agglutination.

The indirect antiglobulin test (IAT), or indirect Coombs test, is used to screen for antibodies when looking for compatible blood for transfusion (“type and screen”) and in the serologic cross-match (patient serum and donor/ reagent red cells). The IAT detects antibody present in the serum, but not bound to the RBC. When the IAT is positive, the antibody must be identified and the corresponding antigen always avoided in transfusions. A negative IAT does not necessarily indicate absence of alloantibodies: the titer of antibody may be below the level of detection or the antibody might be directed against a low incidence antigen (present in less than 1% of individuals) not present on reagent testing cells. A negative IAT does not guarantee that blood is compatible, nor does a weak IAT indicate that hemolysis is likely to be mild. A positive IAT always requires further investigation.

In AIHA, in which the antibody may be present in the serum and on the RBC of the patient, both the DAT and IAT may be positive.

BLOOD COMPATIBILITY

In general, blood components that contain more than 2 mL of RBC must be compatible with the patient’s plasma. Particular attention is paid to Rh type because less than 1 mL of RBC, a volume found in most platelet concentrates, is sufficient to sensitize an Rh-negative patient.¹ Plasma-containing components, including platelet preparations, should be ABO compatible with the patient’s RBC when possible to prevent passive immune hemolysis from antibodies in the plasma (Table 24.1).

The most basic practical application of blood group serology involves the selection of compatible blood. The absence or presence of blood cell antigens can have important biological and clinical implications. Compatible blood takes time to prepare.

  In an emergency, group O Rh-negative RBC may be released uncrossmatched with the consent of the requesting physician; testing will be completed after release. Group-specific red cells and an abbreviated crossmatch can be prepared in 15 minutes.

  Fully tested red cells can be prepared in 45 to 60 minutes; cryopreserved RBC and fresh-frozen plasma (FFP) may take longer.

In the setting of transfusion or pregnancy in the previous 3 months a pretransfusion sample cannot be more than 3 days old.⁶

ABO INCOMPATIBILITY IN TRANSPLANT SETTINGS

Optimal results in HSCT rely on human leukocyte antigen (HLA) compatibility, so that selection of the recipient-donor pair is determined by similarity of HLA potentially at the expense of ABO compatibility. Because HLA and ABO genes are inherited independently, some (20%–40%) of these transplants will be ABO incompatible.¹

Whereas ABO incompatibility does not appear to impact graft failure, potential complications of mismatches include acute or delayed hemolysis and delayed RBC engraftment.²⁻⁵

Minor Incompatibility

  Donor’s serum contains antibodies against RBC antigens of the recipient (e.g., recipient blood group A, B, or AB, and donor blood group O)

  Prior to infusion of the stem cell preparation, plasma containing anti-A and anti-B can be removed to prevent immediate postinfusion hemolysis of the recipient’s RBC (plasma reduction)

  Of minor ABO-incompatible transplant recipients, 10% to 15% may experience abrupt onset of hemolysis 5 to 15 days posttransplant when immune-competent B lymphocytes in the graft mount a response against the recipient RBC antigens (passenger lymphocyte syndrome)

  Hemolysis may be severe or even fatal unless recognized promptly

Major Incompatibility

  Recipient’s serum contains antibodies against the RBC antigens of the donor (e.g., recipient group O and donor group A, B, or AB; recipient group A or B and donor group AB).

  Hemolysis of RBC in the stem cell preparation upon infusion may occur if the graft is not processed to remove RBC prior to infusion (red cell reduction).

  Post transplant, the recipient may produce antibodies against donor red cell antigens for months, especially with nonmyeloablative regimens.

  RBC engraftment and erythropoiesis may be delayed, resulting in red cell aplasia.5,6

Minor and major (bidirectional) incompatibility between the donor and recipient occurs when each has antibodies against the ABO blood group antigens of the other (combination of group A donor and B recipient, or vice versa).

Table 24.2 describes appropriate transfusion management during transplantation. All transfusion components must be irradiated.

Rh Incompatibility

Rh incompatibility occurs in 10% to 15% of stem cell transplants. Transfusion practice is analogous to that of major and minor ABO incompatibility, but the consequences are less severe.

For Rh-negative recipients of Rh-positive hematopoietic stem cell preparations

  Transplant product should be red cell reduced to decrease risk of alloimmunization similar to major ABO incompatibility

For Rh-positive recipient from Rh-negative donor previously alloimmunized to the Rh antigen

  Monitor the patient for signs of delayed hemolysis (as in minor incompatibility).

BLOOD COMPONENTS AND DERIVATIVES

Blood Components and Transfusion Therapy

Blood components can be separated from whole blood by centrifugation or by apheresis. Approximately 29 million blood components (RBCs, platelets, plasma, cryoprecipitate) are transfused annually in the United States.

Storage and infusion of blood products

  Blood components should be infused through standard 170 to 260 μm infusion filters to remove any clots that form during storage. ⁶

  An approved infusion pump may be used for strict control of transfusion rate. Nonapproved pumps may damage or hemolyze cells.

  Bedside leukoreduction filters may be used when leukoreduction is indicated for whole blood, packed RBCs, and platelets that have not been leukocyte reduced prior to storage.

  Hypotensive reactions have been associated with bedside leukoreduction, especially in patients receiving angiotensin converting enzyme (ACE) inhibitors.

  Allow blood to filter by gravity.

  Granulocyte concentrates must never be infused through leukoreduction filters.

  Whole blood and other cellular blood components may be infused with isotonic solutions: USP 0.9% NaCl (normal saline) and certain Food and Drug Administration (FDA) approved electrolyte solutions.

  Cellular blood products must never be infused with hypertonic or hypotonic solutions, for example, solutions containing glucose or calcium, such as D5W (5% dextrose in water) or lactated Ringer solution as hemolysis, clotting, or agglutination of RBC may result.

  Medications should never be added to blood components.

  Never store blood components in unmonitored refrigerators in nursing units or surgical suites; the risk for administration of blood components to the wrong patient increases in these cases.

  Return blood to storage (or blood bank) if transfusion is not started within 30 minutes of issue.

  Warming devices with internal monitors are available for blood products to avoid transfusion of large volumes of cold fluid.

  Blood components should never be warmed in uncertified devices (like microwave ovens or water baths) as hemolysis can result and can be lethal.

Most adverse transfusion reactions occur in the first 15 minutes:

  Administration of blood products should start slowly and under close observation.

  The time of transfusion should not exceed 4 hours as the risk of bacterial growth increases with time at room temperature.

  If transfusion is anticipated to take longer, the transfusion service can divide the unit into smaller aliquots.

See Table 24.3 for blood component administration.

See Table 24.4 for indications for additional modifications to blood components.

Storage conditions for different blood components vary and are designed to maximize preservation and effectiveness:

  Red cells are refrigerated (at 1–6°C) for up to 42 days.

  Platelets are stored at room temperature and expire in 5 days.

  Plasma components are stored frozen for a year (at −18°C) or more (at −65°C), but must be thawed before use and therefore are not immediately available.

As with any medical treatment, blood transfusion requires informed consent:

  Patients must be advised of the indications and common adverse events as well as any potential alternatives to allogeneic transfusion.

Whole Blood

A unit of whole blood typically has a volume of 450 to 500 mL and a hematocrit of 35% to 45%. Whole blood is rarely available and infrequently used.

Indications: Acute hypovolemia with red cell loss, massive transfusion, and exchange transfusion Not indicated: Chronic anemia (in which blood volume is often increased)

Whole blood is not a source of functional platelets or granulocytes, which deteriorate in less than 24 hours at refrigerator temperatures.

Red Blood Cells

RBC (packed red cells) are separated from whole blood by centrifugation. A unit of RBC contains approximately 200 mL and a hematocrit of 60% to 80%. In general, 1 unit of packed RBC will increase the hemoglobin (Hb) by 1 g/dL in an average-sized adult. In the average pediatric patient, transfusion of 8 to 10 mL/kg of RBC is expected to increase hemoglobin by 3 g/dL. The decision to transfuse should be based on assessment of symptoms, coexisting or underlying medical conditions, and the cause of the anemia and patients should not be transfused based solely on their Hb level. The single adequately powered prospective study (ICU patients) and numerous observational studies indicated that patients with cardiovascular disease are more sensitive to anemia and do better at a higher Hb level.7,8

Indications: Treatment of symptomatic anemia.

While it is generally accepted that patients with Hb <6 g/dL should be transfused and that transfusion is rarely required when it exceeds 10 g/dL, the interval between these values is an area of controversy. Practice guidelines support an Hb level of less than 7 g/dL as generally acceptable for the initiation of RBC transfusion in asymptomatic patients.8,9 Patients at particular risk for bleeding (thrombocytopenia, recent hemorrhage) should be maintained at a higher Hb.

Not indicated: RBC should not be transfused for volume expansion or nutritional purposes Transfusion is rarely indicated in otherwise treatable anemia, including anemia associated with Vitamin B₁₂, iron, or folate deficiency; if symptoms are severe, these patients may benefit from a single-unit transfusion as the underlying cause is corrected.

Platelets

Platelets may be separated from whole blood shortly after collection (“random donor” or “whole blood-derived” platelet concentrates) or collected by apheresis (“single donor” or “apheresis platelets”). A therapeutic dose of platelets for an adult is 1 unit of platelets (5.5 × 10¹⁰ platelets) per 10 kg of body weight, which should increase the platelet count in an average-sized adult by approximately 5,000/μL. Each apheresis (single-donor) platelet product is expected to contain approximately 3 × 10¹¹ platelets, roughly equivalent to 4 to 6 units of random donor platelets. Indications for use are the same for both preparations. Alloimmunized refractory patients may require single-donor HLA-matched platelets. Single-donor platelets also offer the additional advantage of decreased donor exposure and a lower risk of bacterial sepsis.

Indications: Prevention and treatment of hemorrhage in patients with thrombocytopenia or platelet function defects.

Not indicated: Bleeding unassociated with thrombocytopenia (in the absence of a clinically significant platelet function defect), other defects in hemostasis (such as factor deficiencies).

Platelets are usually contraindicated in thrombotic thrombocytopenic purpura (TTP),¹⁰ because they could potentially precipitate thrombosis, but this has been recently challenged.¹¹ However, patients with TTP who develop life-threatening hemorrhage may benefit from a cautious trial of platelets.

The threshold for prophylactic platelet transfusion varies based on the patient’s underlying condition and likelihood of hemorrhage:

  A threshold of 10,000/μL is effective in preventing morbidity and mortality from bleeding in stable oncology patients undergoing chemotherapy.

  A platelet count greater than 50,000/μL is desirable prior to invasive procedures and in the immediate postprocedure period.

  Platelet counts closer to 100,000/μL may be prudent for patients at high risk for intracranial hemorrhage, such as those with cerebral leukostasis, or when undergoing neurosurgical or ocular procedures.

  Stable chronically thrombocytopenic patients, such as those with aplastic anemia or myelodysplasia, may tolerate platelet counts as low as 5,000/μL in the absence of complicating factors including fever, infection, and additional defects in hemostasis. ¹

More aggressive support is indicated for patients who are unstable—febrile, infected, receiving multiple medications—especially if the platelet counts are decreasing.1,12

Platelet transfusions should be monitored by a 1 to 24 hour posttransfusion platelet or complete blood count (CBC) to assess response and guide subsequent transfusion therapy. A corrected count increment (CCI) may be used to determine the increase in platelet count in an individual post platelet transfusion:

*Posttransfusion platelet count, expressed per microliter, is best obtained 15 minutes to 1 hour posttransfusion

**Body surface area = the square root of [(height in cm × weight in kg)/3,600], expressed in meters squared

***Expressed as multiples of 1 × 10¹¹

An absolute posttransfusion increment of 10,000/μL or greater (approximately 2,000/μL per unit of random donor platelets) in an average-sized adult corresponds to a CCI of 5,000.

Platelet Refractoriness

Patients who respond poorly to repeated platelet infusions are termed refractory. Posttransfusion platelet counts (performed at 1 and 24 hours) are useful tests to determine refractoriness. The CCI should also be calculated and failure to achieve a CCI of 5,000 or greater is cause to suspect platelet refractoriness. Refractoriness may be immune or nonimmune mediated. Immune-mediated refractoriness indicates alloimmunization to HLA or human platelet antigens (HPA).

  Nonimmune-mediated causes of platelet refractoriness: fever, infection, splenomegaly, disseminated intravascular coagulation (DIC), massive bleeding, and medications that enhance platelet destruction; more likely to affect the 24-hour posttransfusion count.

  Immune-mediated platelet refractoriness caused by alloimmunization to HLA and human platelet antigens usually associated with multiparity or exposure to nonleukocyte reduced platelet transfusions; more likely to affect the 1 hour posttransfusion count.

In practice, the distinction between immune- and nonimmune-mediated platelet refractoriness becomes less clear as alloimmunized patients often have multiple medical issues predisposing to nonimmune refractoriness. When immune-mediated platelet refractoriness is suspected and CCI is less than 5,000 after each of two platelet transfusions the following steps should be taken:

  ABO-compatible fresh (less than 72 hours in storage) platelets should be used for two subsequent transfusions

  If CCI still does not exceed 5,000, HLA antibody screen to detect alloantibodies, or commercial platelet compatibility tests should be performed

  When alloantibodies with broad specificity are found (for HLA A and B loci) platelets from HLA-matched donors are indicated

  Crossmatch compatible platelets may be beneficial when HLA antibody status of the recipient cannot be determined, HLA-matched platelets cannot be obtained, or when the patient is refractory to HLA-matched platelets (up to 40% to 50% of cases)

  Corticosteroids, washed platelets, or IVIG have not proved useful in the treatment of refractoriness

Granulocytes

Granulocytes are collected by apheresis for specific patients, from donors who are mobilized prior to collection with corticosteroids and/or granulocyte colony-stimulating factor [G-CSF].

  Granulocytes can be stored at room temperature for only up to 24 hours post collection but optimally should be administered within 6 hours of collection.

  Granulocyte collections have a volume of 250 mL and contain plasma, approximately 30 mL RBC, and variable amounts of mononuclear leukocytes and platelets.

  Granulocyte concentrates should be ABO, Rh, and RBC crossmatch compatible.

  Products should be irradiated because of the presence of viable lymphocytes in the collection.

  The minimal therapeutic dose is 1 × 10¹⁰ granulocytes/unit however, increments are unlikely to be seen unless greater than 3 to 4 × 10¹⁰ granulocytes/unit are infused. ¹

Indications: Patients with absolute neutrophil counts of less than 0.5 × 10⁹/L and documented bacterial or fungal infection refractory to antimicrobials. Recipients must have a reasonable expectation of achieving hematopoietic recovery or engraftment (endogenous granulocyte production). Infants with bacterial sepsis, whose granulocyte counts are less than 3 × 10⁹/L with postmitotic neutrophils comprising less than 10% of their nucleated marrow cells, may benefit from granulocyte transfusions.¹

A 1 to 6 hour posttransfusion CBC with differential for determination of ANC may help assess efficacy. A 6-hour posttransfusion increment may be higher than a 1-hour posttransfusion ANC because granulocytes travel to the lungs before equilibrating in peripheral blood. If the patient’s ANC fails to reach expected levels or if a reaction occurs, an HLA antibody screen and tests for antibodies to human neutrophil antigens (HNA) are indicated to look for an immunologic cause.

Not indicated: Patients whose bone marrow function is not likely to recover. Contraindicated in patients with prior severe pulmonary reactions to HLA or HNA antibodies or HLA or HNA alloimmunization.

  Alloimmunized patients may develop chills, fever, rigors, shortness of breath, wheezing, pulmonary infiltrates, cyanosis, and hypotension6,13; rigors and fever may respond to intravenous meperidine.

  Pulmonary toxicity may be exacerbated when granulocytes and amphotericin B are administered in close temporal proximity.¹⁴ At NIH, amphotericin B administration and granulocyte transfusions are separated by at least 4 hours.

  Granulocyte transfusion therapy should be evaluated after an initial course of four infusions and then periodically.

  Granulocyte concentrates may contain leukocyte-associated pathogens such as cytomegalovirus (CMV), which may be a particular concern for immunosuppressed stem cell transplant recipients, solid organ transplant recipients, neonates undergoing extracorporeal membrane oxygenation, and low birth weight and premature infants.

  While granulocyte transfusions decrease the length of bacterial infection, proof that granulocyte transfusions decrease mortality in any situation has been elusive. 15,16

Fresh-Frozen Plasma

Plasma separated from whole blood or collected by apheresis and frozen within 8 hours is labeled FFP. FFP contains plasma proteins at the time of thaw in about the same concentrations as at the time of collection. The volume of a unit of plasma is approximately 200 mL.

  By convention, 1 mL of FFP is expected to provide 1 unit of activity of all factors (except labile factors V and VIII). In practice, individual units may vary in content.

  The dose used is 10 to 20 mL/kg in adults (equivalent to approximately 4 to 6 units of FFP) to increase coagulation factor levels by 20%.

Indications: Correction of multiple clotting factor deficiencies in patients who are bleeding or prior to an invasive procedure, replacement of factors in consumptive coagulopathy, coagulation factor deficiencies caused by liver disease, dilutional coagulopathy after massive transfusion, replacement fluid for plasma exchange in the treatment of TTP, rapid reversal of warfarin (Coumadin) effect, replacement of single congenital factor deficiencies when no virus-safe fractionated product is available (mostly applies to factor V deficiency).

  A PT greater than 1.5 normal or APTT ratio greater than 2.0 in the presence of microvascular bleeding is a guide to consider treatment. ⁹

  Note, in the setting of life-threatening bleeding associated with Coumadin therapy where rapid reversal is necessary or in overanticoagulated patients with volume overload a more appropriate therapy is Prothrombin Complex Concentrate (PCC). See section Blood Derivatives.

Not indicated: Volume expansion, protein replacement in nutritional deficiencies.

Cryoprecipitate

Cryoprecipitate (cryo) is the cold-insoluble portion of plasma which contains factor VIII, fibrinogen, von Willebrand factor, factor XIII, and fibronectin. Ordinarily stored frozen, cryo can be kept at room temperature for up to 6 hours; on pooling it must be transfused within 4 hours.

  Compatibility testing is unnecessary.

  A unit of cryo is usually less than 15 mL of plasma and contains more than 80 international units (IU) of factor VIII and more than 150 mg of fibrinogen.

  One unit of cryo can increase fibrinogen in an average adult by 5 to 10 mg/dL.

  A therapeutic dose for an adult is 80 to 150 mL of cryo (8 to 10 units pooled).

  Indications: Treatment of congenital fibrinogen deficiency, dysfibrinogenemia, factor XIII deficiency, DIC (if fibrinogen <1.0 g/L).

  Note a pathogen-inactivated fibrinogen concentrate has recently become available in the United States for treatment of congenital fibrinogen deficiency.

Cryo has also been used to correct the platelet defect of uremic bleeding, although with variable success.

The dosage of cryo depends on the underlying deficiency and on the plasma volume of the patient. To determine the number of bags of cryo to replace fibrinogen

*The plasma volume for an average adult = (1 {{{727}}} percent hematocrit/100) × patient weight in kg × 70 mL/kg. For infants and children under 40 kg, the plasma volume = (1{{{727}}}percent hematocrit/100) × patient weight in kg × 80 to 85 mL/kg.

Not indicated: Factor VIII deficiency and von Willebrand disease for which more specific and safer products now exist.

Hematopoietic Stem and Progenitor Cells

Optimal outcomes in HSCT depend on the successful procurement of cells from patients (autografts) or donors (allografts). Several recent developments in this field have improved clinical outcomes making this approach a safe and effective therapy for a variety of malignant and nonmalignant disorders.

Stem cell sources now include related and unrelated bone marrow, peripheral blood and umbilical cord blood. The vast majority of HSCT now uses mobilized peripheral blood although guidelines for management of aplastic anemia still recommend using a bone marrow source if possible.¹⁷

Historically, mobilization of stem cells into peripheral blood for autografting was done using myelosuppresive chemotherapy such as cyclophosphamide as increased numbers of cells entered the circulation during the recovery phase of the bone marrow. In the late 1980s G-CSF and GM-CSF became available and were used either alone or in combination with chemotherapy as mobilizing agents. G-CSF is now the standard for this indication.

A significant proportion of patients do not mobilize with G-CSF with or without chemotherapeutic agents. Factors that have been shown to predict for poor mobilization include increasing numbers of cycles of prior chemotherapy, prior radiation therapy, and the presence of marrow metastasis.¹⁸⁻²⁰ Fludarabine is particularly toxic to stem cells and this should be avoided in patients for whom a stem cell collection for autograft is planned. It has also been shown that there is a correlation between the number of circulating CD34+ cells and the probability of obtaining an adequate collection for transplant.²¹

Until recently poorly mobilizing patients had few options. However, an agent originally developed to treat HIV, plerixafor, has recently been shown to have significant activity in these patients. In contrast to G-CSF which needs multiple injections over several days a single dose of plerixafor mobilizes stem cells into the periphery beginning at 1 hour and peaking at 10 hours.²² In addition the quality of the stem cell graft mobilized by plerixafor may be superior. Compared to G-CSF mobilized cells, the plerixafor-mobilized cells were more primitive and therefore more quiescent and produced superior engraftment in both NOD/SCID mice and human recipients.²³ The combination of G-CSF and plerixafor has also been studied and produces greater increases in CD34+ cells than with either agent alone.²⁴

Standard collections of allogeneic peripheral blood stem cell (PBSC) involve 3 to 4 hours per apheresis procedure, during which approximately 10 L of blood are processed. At least two collections are usual, but large volumes of 25 to 30 L are more efficient and used increasingly to allow complete collections with a single procedure.²⁵ Donor demographic and laboratory predictors such as platelet count and CD34+ mononuclear cell count can be used to customize the length of the collection procedure.²⁶

As G-CSF is still the standard agent used, complications seen with stem cell mobilization are often related to this cytokine. Bone pain, headache, fatigue, insomnia, and gastrointestinal disturbances are usually mild and respond to administration of acetaminophen or NSAIDs. Spleen size increases in almost all individuals on G-CSF and this has been associated with splenic rupture. Donors should be advised to refrain from contact sports for a few weeks after the last mobilization.²⁷ Vascular complications and citrate toxicity are not unlike those experienced in other long apheresis procedures (see below).

PBSC grafts are infused as fresh collections or stored frozen with the cryoprotectant dimethyl sulfoxide (DMSO) in liquid nitrogen. Thawed cells infused with DMSO may cause nausea, vomiting, fever, dyspnea, hypotension, and anaphylaxis. Reactions are dose dependent and may be lessened by prophylactic antihistamines. PBSCs carry the risk of transfusion-transmitted infectious agents and are tested in the same manner as are other blood components. However, given their highly specialized use and their life-saving potential, exceptions are made to donor selection criteria normally used for allogeneic blood collections, with the concurrence of the treating physician and the recipient.

Adequate cell dose for engraftment depends on whether the procedure is an autograft, a related, or an unrelated allograft. Cell dose, cell source, and patient characteristics are all important variables. The dose of stem cells for unrelated donors (National Marrow Donor Program) is 2 to 4 × 10⁸ nucleated cells per kilogram of recipient weight, with 2 to 4 × 10⁶ CD34+ cells per kilogram of recipient weight deemed as an adequate dose for transplantation and doses of greater than 5 × 10⁶ CD34+ cells per kilogram of recipient weight associated with more rapid engraftment.²⁸ Lower doses may be adequate in related donor settings, but engraftment of both leukocytes and platelets correlates with CD34 cell content.

Cord blood is being used increasingly as a stem cell source. Less graft-versus-host disease (GVHD) is seen as grafts have less alloreactivity even when mismatched as compared with PBSC or bone marrow grafts. In addition because mismatching is better tolerated there is a better chance of finding a suitable donor. However, because of lower numbers of progenitor cells, hematopoietic reconstitution is delayed and as a result recipients of umbilical cord transplants have a higher risk of developing fatal infections. Cells are obtained from the placenta during the third stage of delivery or postdelivery, with the consent of the mother, and stored in liquid nitrogen. The component volume is usually 50 to 100 mL and may be further reduced by removing red cells and plasma. The small volumes and yields of CD34+ progenitor cells currently make cord blood most suitable for children and smaller adults, although simultaneous infusion of two and even three cord blood units have resulted in successful engraftment for larger adults.²⁹ PBSCs stored in liquid nitrogen likely remain stable for many years, but maximum safe storage periods have not been determined. Recipients of minor mismatched cord blood seem less likely to make red cell alloantibodies than do recipients of mismatched marrow or PBSC.

Blood Derivatives

Derivatives or blood products are produced commercially by fractionation of plasma and include colloids such as albumin and plasma protein fraction, immune globulins, coagulation factor concentrates, and a variety of orphan proteins such as α-1-antitrypsin and antithrombin.

Prothrombin Complex Concentrate

PCCs are derived from pooled human plasma. They were initially developed for the prophylaxis and treatment of patients with factor IX deficiency. The two products available in the United States (Profilnine SD and Bebulin VH) are termed 3 factor-PCCs as they contain low levels of factor VII.

  All PCCs contain the vitamin K dependent factors II, IX, and X while 4 factor-PCCs used in Europe contain higher levels of factor VII.

  Thromboembolism is a potential adverse effect and may occur less in 3-factor than 4-factor products.³⁰

Indications: Emergency treatment of bleeding due to overanticoagulation with warfarin. May be useful when volume overload with FFP a risk. Should always be used in combination with Vitamin K therapy.³¹

See Table 24.5 for more blood derivatives and Table 24.6 for coagulation factor preparations.

Rh Immune Globulin

Rh immune globulin (RhIg) is available in intramuscular (IM) form and intravenous (IV) form.

Indications: Prevention of alloimmunization of Rh-negative recipients exposed to Rh-positive RBC, immune thrombocytopenic purpura (ITP).

  Prevention of Rh immunization in Rh-negative women with Rh-positive fetuses and subsequent HDN or after blood transfusion with Rh-positive blood.

  Greater than 99% success in preventing Rh alloimmunization in pregnancy³²; failure is usually because of missed or insufficient injections.

  Treatment of ITP in Rh-positive patients only (intravenous RhIg).

Intramuscular RhIg is used in Rh-negative females of childbearing age after exposure to small- volume Rh-positive RBC (with platelet transfusions or the accidental transfusion of an Rh-positive unit of RBC). RhIg IV is used for large volume exposures.

Use of RhIg in Rh-negative males and females without childbearing potential is controversial but may protect from complications of future transfusions.

In a pregnancy where the mother is Rh negative and the father Rh positive, the fetus may be Rh positive and therefore there is a risk of Rh immunization of the mother. In these cases a prophylactic dose of 300 μg of anti-D is given at 28 weeks gestation.

A full dose containing 300 μg of anti-D to cover an exposure of 15 mL of Rh-positive RBC is also given:

  To Rh negative women at risk of Rh immunization within 72 hours of delivery.

  After amniocentesis and chorionic villus sampling, with manipulations such as external cephalic version, ectopic pregnancy, abortion, and abdominal trauma after 20 weeks gestation.

A minidose containing 50 μg of anti-D to cover an exposure of 2.5 mL of Rh-positive RBC can be given if these events occur before 12 weeks although 300 μg is often given as it is more readily available.

Erroneous transfusions:

  RhIg dose should be calculated based on the RBC volume transfused.

The half-life of RhIg is 21 days. Additional RhIg should be administered in the following situations:

  Expected ongoing risk of fetomaternal hemorrhage.

  Nonobstetric cases with additional transfusion of products containing Rh-positive RBC 21 days or more after the last dose of RhIg.

RhIg should be given within 72 hours but if not feasible it should still be administered as soon as the need is recognized for up to 14 days. Some authorities maintain that it may be useful up to 28 days.³³

Not indicated: Rh-positive individuals, previously immunized Rh-negative individuals, Rh-negative females with known Rh-negative fetuses or newborns, or in Rh-negative patients with ITP.

Derivative and Recombinant Coagulation Factors

Recombinant and plasma-derived coagulation factors provide a concentrated source of the desired factor for prevention and treatment of bleeding episodes in patients with factor deficiencies. Recombinant factors contain no other human-derived products and no risk of viral disease transmission (Table 24.6).

TRANSFUSION REACTIONS AND ADVERSE SEQUELAE

Any adverse response to blood component transfusion is considered a transfusion reaction. Most reactions occur at the beginning or during transfusion and are termed acute. Others, including development of alloantibodies, iron overload, and some parasitic and viral infections, do not become apparent for weeks, months, or years and are termed chronic.

  Because most transfusion reactions occur within 15 minutes, close monitoring of vital signs and status at the beginning of the transfusion may prevent more severe reactions.

  If a reaction is suspected, the infusion should be halted, the transfusion service notified, appropriate samples collected, and the patient monitored.

Transfusion reactions can be classified as hemolytic versus nonhemolytic and acute versus delayed. Hemolytic reactions may be immune or nonimmune-mediated (Table 24.7).

Acute Hemolytic Transfusion Reaction

Acute hemolytic transfusion reactions may be severe and fatal and may occur with as little as 10 mL of incompatible blood. Most result from ABO blood group incompatibility between patient plasma and donor RBC, and are usually the result of the incorrect transfusion of a unit (or several units) of blood intended for another patient.

Presentation: Fever, chills, flank/back pain, dyspnea, chest pain, anxiety which can progress to hypotension, renal failure, shock, and death if severe

Mechanism: ABO incompatibility resulting from destruction of transfused RBC by preformed, naturally occurring IgM anti-A and/or anti-B isohemagglutinins in the recipient’s plasma.

  Intravascular hemolysis is caused by complement fixation by IgM antibodies.

  Hemoglobinemia occurs followed by hemoglobinuria.

  Complement activation promotes histamine and serotonin release which causes wheezing, chest pain or tightness and gastrointestinal symptoms.

  Acute hemolysis results in anemia.

  Cytokine release contributes to renal failure, hypotension, shock, and DIC.

Evaluation

  Clerical check of component bag and compare with patient identification

Submit to the blood bank:

  The infusion set, the implicated unit, and any units transfused within 4 hours of the reaction.

  Blood specimens from the patient for repeat ABO group and Rh type determination, crossmatch, and DAT (usually positive); assessment of other parameters such as hematocrit (decreased), lactate dehydrogenase (increased), haptoglobin (decreased), and bilirubin levels (increased usually by 6 hours)¹ as indices of hemolysis.

  First posttransfusion-voided urine to examine for hemoglobinuria.

  Transfusion reaction report detailing the events, signs/symptoms, and documenting the patient’s pretransfusion and posttransfusion vital signs.

Management: Mainly supportive, transfusion must be stopped and disconnected at the hub of the needle and intravenous access maintained with physiologic saline.

  Support of blood pressure and renal blood flow with fluids and pressors if necessary, and induction of diuresis to maintain urine output at greater than 1 mL/kg/h. 6,34

  Withhold further transfusion until the cause of the reaction is determined.

  Coagulation status should be monitored and DIC treated if present.

Prevention: Meticulous clerical check of the blood unit and patient identification. In case of error in patient identification, immediate steps must be taken to insure that a second patient does not receive the wrong unit (companion error).

Hemolysis Associated with Passive Antibody Infusion

  Severe acute hemolysis can occur if large volumes of ABO-incompatible plasma (usually group O FFP or apheresis platelets into group A patient) are infused.

  Recognition and management of acute anemia is ordinarily sufficient.

Sickle Cell Hemolytic Transfusion Syndrome

Patients with sickle cell anemia are at increased risk from hemolytic transfusion reactions. A fall in Hb after transfusion of red cells is suggestive of the hyperhemolytic syndrome. This entity is poorly understood but occurs when both the patient’s and the transfused red cells are destroyed.1,35 Transfusion-associated hemolysis may mimic a severe posttransfusion pain crisis which is mediated by factors such as decreasing hemoglobin, complement activation, and increased oxygen consumption with fever. Stopping transfusion is imperative as additional transfusion may result in exacerbation of the syndrome. In the United States, inherent differences in RBC antigen phenotypes between patients with sickle cell anemia (almost exclusively of African descent) and the majority of blood donors (primarily non-African) place patients with sickle cell anemia at increased risk of alloantibody formation and immune hemolysis. In addition, patients with sickle cell disease are often chronically and heavily transfused. Extended red cell phenotyping of patients with sickle cell anemia in the early stages of transfusion therapy reduces the risk of alloimmunization.36,37

Delayed Hemolytic Transfusion Reaction

Delayed hemolytic transfusion reaction (DHTR) occurs within days, weeks, or even months after transfusion in patients who have been previously immunized by transfusion or by pregnancy (primary immunization). These reactions rarely, if ever, occur as a result of primary immunization and are usually a result of second transfusions. Because its manifestation may be mild and symptoms delayed in onset, DHTR may not be immediately recognized and this complication is likely underreported.³⁴ Serious sequelae are rare. The importance of recognizing these reactions is to document antibody formation and prevent hemolytic reactions with future transfusions.

Presentation: Fever with or without chills, and symptoms of anemia. Jaundice may be present.

  Subtle decrease in hemoglobin may be the only clinical manifestation.

  DAT is usually positive.

  Hemoglobinuria is rare because hemolysis is extravascular.

Mechanism: Repeat stimulation and accelerated (anamnestic) appearance of the antibody in a previously alloimmunized patient upon reexposure to the offending antigen.

Management: Close monitoring of the patient’s Hb for evidence of continuing hemolysis and supportive therapy.

Prevention: Future transfusions must be antigen-negative for the corresponding implicated antibody even if antibody is no longer detectable.

  Notification of the patient to prevent future reactions (as by antibody card or bracelet).

Other Causes of Hemolysis

Other causes of hemolysis temporally associated with transfusion that may mimic hemolytic transfusion reactions include drug-induced hemolysis, mechanical and thermal hemolysis, and hemolysis related to bacterial contamination of the RBC unit.

  Drug-induced hemolysis which may be either intra- or extravascular may present with anemia, positive DAT, elevated LDH and bilirubin.

  Hemolysis may result from administration of blood with hypotonic solutions (D5W, hypotonic saline, distilled water) or medications.

  Mechanical hemolysis may result from prosthetic heart valves and other intravascular devices and transfusion through small-bore catheters or with the use of roller pumps.

  Thermal hemolysis results from exposure of red cells to cold (ice, temperatures below 1–6°C, use of unmonitored refrigerators) or to temperatures above 42°C (malfunction of blood warmers, or unmonitored unconventional blood warming methods); the DAT should be negative in these cases; some of these reactions have been fatal.

Anaphylactic Transfusion Reactions

Anaphylactic transfusion reactions usually occur at the start of transfusion when small amounts of blood containing plasma are transfused. Although rare, anaphylactic reactions may be rapidly fatal.

Presentation: Sudden onset of urticaria, flushing, chills, vomiting, diarrhea, initial hypertension followed by hypotension, angioedema, coughing, stridor, laryngeal edema, and progression to respiratory distress and shock. Fever is not a feature of anaphylaxis.

Mechanism: IgE-mediated response to transfused proteins.

IgA-deficient patients with antibodies to IgA are particularly susceptible to anaphylaxis on receiving plasma containing IgA.

Management: Discontinue transfusion and employ standard measures for anaphylaxis: Epinephrine, corticosteroids, circulatory support.

Anti-IgA or antibody to a subspecies must be demonstrated to confirm whether patient is IgA deficient.

Prevention: Usually not predictable. Subsequent transfusions to IgA-deficient patients should come from IgA-deficient donors.

If IgA-deficient blood components are not available, patients may benefit from receiving blood products that are depleted of plasma, such as washed RBC and platelets or frozen-deglycerolized RBC.

Transfusion-Related Acute Lung Injury

Transfusion-related acute lung injury (TRALI) is characterized by noncardiogenic pulmonary edema and is associated with transfusion of plasma-containing blood components. Previously believed to be underreported,³⁴ it has an estimated frequency of between 1:1,300 and 1:5,000 transfusions and is the leading cause of transfusion-associated deaths reported to the FDA with a mortality rate of 6% to 10%.1,6

Presentation: Acute respiratory insufficiency, tachycardia, dyspnea, hypotension, oxygen desaturation (O2 saturation <90% on room air), chills, rigors, fever with 1°C to 2°C temperature increase, and a bilateral pulmonary infiltration by chest X-ray in the absence of heart failure or elevated central venous pressure, with no other causes of acute lung injury evident.34,38

  Occurs during or within 6 hours (most commonly after 2 hours) of transfusion.

  Hypoxemia may require intubation (70%–75% of cases).³⁴

  Symptoms subside rapidly; chest X-ray normal within 96 hours; clinical recovery in 48 to 96 hours in 80% of patients. ⁶

Mechanism: Reaction of antineutrophil antibodies and/or anti-HLA class I and II antibodies, usually donor derived, to corresponding recipient antigens; destruction occurs in the pulmonary vasculature and results in endothelial damage.

  Multiparous females are more likely to be HLA/HNA alloimmunized, as are donors who have themselves been transfused.

  Antibodies from the recipient against cells in the donor plasma implicated in approximately 5% of cases.

  In 10% of cases no donor or recipient antibodies are identified.

A second possible pathophysiologic mechanism of TRALI is the “two-hit” theory, which suggests an interaction between primed pulmonary neutrophils in patients with underlying illness (in proinflammatory states) and biologically active response modifiers (lipids, cytokines) introduced by transfusion.³⁹

Management: Discontinue transfusion and provide respiratory and circulatory supportive care; steroids and diuretics are not useful.

Prevention: A patient who has experienced TRALI is not necessarily at increased risk for development of TRALI with future transfusions, unless receiving blood from the same donor. Implicated donors are ordinarily deferred indefinitely. Many blood centers now draw plasma preferentially from male donors to reduce the risk from multiparous female donors.⁴⁰

Transfusion-Associated Circulatory Overload

Transfusion-associated circulatory overload (TACO) can present in a similar fashion to TRALI but is much more commonly seen. Unlike TRALI, circulatory overload is associated with central venous pressure elevation and cardiac failure. Pulmonary edema in TACO is cardiogenic in origin and may result in the development or exacerbation of congestive heart failure. In the absence of other complicating factors, circulatory overload is rarely fatal. Children, the elderly, those with compromised cardiac, renal, or pulmonary function, and patients in states of plasma volume expansion (normovolemic chronic anemia, thalassemia major, and sickle cell disease) are at particular risk.6,34

Presentation: Cough, dyspnea, cyanosis, orthopnea, chest discomfort, rales, headache, distension of jugular veins, and tachycardia.

Management: Discontinue transfusion and administer supportive care (oxygen, diuresis, phlebotomy), if necessary.

Prevention: Patients at risk should receive smaller aliquots of blood infused at slower rates (1–4 mL/ kg/h) in as concentrated a form as possible.

Transfusion-Associated Graft-versus-Host Disease

Transfusion-associated GVHD (TA-GVHD) is a rare but severe adverse outcome of transfusion. Those at risk include the immunocompromised, patients with certain malignancies such as lymphoma, neuroblastoma, and sarcoma, patients receiving directed donations (from family members or relations), and premature infants. Patients with HIV and aplastic anemia do not need irradiated blood products1,6,34 (see Table 24.4).

Presentation: Rash, diarrhea, hepatitis, mucositis, and pancytopenia; almost universally fatal with rare reports of survival.1,34

Mechanism: Immunocompetent lymphocytes from the donor engraft recognize the patient’s antigens as foreign and initiate an immune response.

Management: Supportive; no specific measures have proved effective. Prevention: Irradiation of blood components.

  Irradiation dose of 2,500 cGy.

  Leukoreduction does not prevent TA-GVHD.

Bacterial Contamination and Sepsis

The initial symptoms of bacterial contamination occur shortly after the start of transfusion and usually within 2 hours. These include chills and fevers. Note that temperature increase can be less marked in patients premedicated with antipyretics or receiving corticosteroids. In addition mild septic reactions may be initially obscured by underlying conditions that predispose the patient to fever or manifest similar signs and symptoms.6,34

Presentation: Rigors and shaking chills, fever, nausea, vomiting, abdominal cramp, bloody diarrhea, severe hypotension, and rapid progression to circulatory compromise, renal failure, shock, and DIC.

  Not all contaminated blood products result in clinically detectable sepsis, and fewer yet are fatal.

Mechanism: Common sources of bacterial contamination are subclinical bacteremia in the donor or skin contaminants at the phlebotomy site.

  Commonly implicated in RBC contamination are bacteria that survive the cold storage conditions, such as Yersinia and Pseudomonas.

  Platelets stored at room temperature are the component most susceptible to bacterial growth.

  Contamination occurs in approximately 1:5,000 collections, sepsis in 1:50,000 transfusions, and death in 1:500,000 transfusions. ⁴¹

  More than half of bacterial contaminations of platelets are caused by skin flora, Staphylococci, Streptococci, Propionibacterium; many species are not implicated in serious transfusion reactions.6,34, 41

  Currently, the residual risk of septic transfusion reactions from platelets is estimated to be more than twice as high for whole blood derived platelets, not tested by culture method, than for culture- negative apheresis platelets, 1:33,000 versus 1:75,000.⁴²

  The highest mortality is usually associated with blood components contaminated with endotoxinproducing gram-negative bacteria.

Evaluation and management: Transfusion must be stopped and broad spectrum antibiotics commenced.

  All transfusion units and blood component bags transfused within 4 hours should be returned to the blood bank for culture.

  Blood samples from both the blood component unit(s) and the patient should be sent for culture.

Prevention: Strict hygienic practice from collection to processing, storage, and administration of the component and transfusion of blood components within the allotted 4 hours.

Mild Allergic/Urticarial Transfusion Reactions

Allergic transfusion reactions are relatively common, do not generally progress to anaphylaxis, are rarely lethal, and do not necessarily recur with subsequent transfusions.

Presentation: Localized erythema, pruritus, flushing, and urticaria, usually near the IV site.

  Severe urticaria and pruritus may be the initial signs of anaphylaxis.

Mechanism: Release of histamine and other anaphylotoxins.

Evaluation and management: Mild allergic reactions generally resolve when transfusion is temporarily stopped, and symptoms improve with administration of oral or parenteral antihistamines.

  Mild allergic reactions (hives only): Transfusion with the same unit may be resumed, at a slower rate and with close monitoring of the patient.

  Transfusion reaction evaluation generally not necessary.

Prevention: Mild allergic reactions are considered atopic reactions and generally unpredictable.

  No method to prescreen for all possible offending antigens.

  Antihistamines are effective in treating, but not in preventing, allergic reactions.

Posttransfusion Purpura

Posttransfusion purpura (PTP) is a profound thrombocytopenia that may occur after transfusion with any blood component but is usually associated with transfusion of red cells or whole blood.

Presentation: Abrupt decline in the platelet count within days to 2 to 3 weeks posttransfusion (mean 9 days).⁶

  Usually self-limiting: resolves within 2 to 3 weeks without treatment.

Mechanism: Not well understood; antibodies against platelet-specific antigens to which the patient may have become sensitized as a result of pregnancy or prior transfusion destroy both transfused and autologous platelets (innocent bystander effect).

  Most commonly anti-human platelet antigen 1a (HPA-1a).

Management: IVIG is the current treatment of choice.

Although PTP almost never recurs antigen-negative components should be transfused in patients with previously documented PTP.

  Family members provide a good source of donors if antigen-negative blood is otherwise not available.

  Severe PTP that does not resolve spontaneously and if refractory to high-dose IVIG may respond to plasmapheresis. 1,34

Hypotension Associated with Transfusion

Mechanism: Transient isolated hypotension that resolves after discontinuation of transfusion may be caused by activation of bradykinin and is associated with use of bedside leukoreduction filters and apheresis procedures (especially with albumin replacement).³⁴

  Patients receiving ACE inhibitors are at particular risk since ACE inhibitors block normal metabolism of bradykinin.

Evaluation and Management: Severe transfusion reactions such as anaphylaxis, acute hemolytic transfusion reaction, TRALI, and bacterial contamination (sepsis) must be excluded.

Prevention: Avoid bedside leukoreduction (especially for patients taking ACE inhibitors).

Febrile Nonhemolytic Transfusion Reaction

Febrile nonhemolytic transfusion reactions (FNHTR) are defined by a greater than 1°C increase in temperature for which no other cause is identifiable. FNHTR is a diagnosis of exclusion, made after consideration of acute hemolytic transfusion reaction, TRALI, and sepsis and determination that the symptoms are not related to the patient’s underlying medical condition or medications. The incidence of FNHTR varies among patient populations and depends on the age and type of blood component and a variety of donor and recipient factors. Platelets are more likely to be implicated than are RBC or FFP, and older blood components more than fresh, leukocyte-reduced components (since cytokines accumulate during storage). The incidence is higher in patients who have received multiple transfusions.³⁴

Presentation: Chills, fever, rigors which may be preceded by headache, and nausea; the patient may also experience tachycardia, tachypnea, and general discomfort.

  Patients who sustain significant fever with transfusion are likely to have repeated reactions.

Mechanism: Interaction of antibodies in the recipient against donor leukocytes or platelets and subsequent cytokine release; accumulated cytokines in blood component bag during storage that are passively transferred from the donor to the recipient during transfusion.

Evaluation and management: Same as with a hemolytic reaction.

  Antipyretics may be administered.

Prevention: Prestorage leukoreduction, removal of plasma in extreme situations, and premedication with antipyretics.

Hemosiderosis

Hemosiderosis or iron overload occurs in chronically transfused patients (usually a cumulative dose of 50–100 red cell transfusions).

Presentation: Classical symptoms rarely seen nowadays but include “bronzing” of skin, hepatomegaly, hepatic fibrosis and malfunction, diabetes and other endocrine gland dysfunction, and cardiac failure.

Mechanism: Accumulation of iron in the skin, liver, heart, and endocrine organs

Evaluation and management: Iron studies and iron chelation or phlebotomy when appropriate. Prevention: Consider chelation for more than 50 unit red cell burden; red cell exchange using apheresis helps delay iron accumulation in patients with sickle cell disease.⁶

Transfusion-Transmitted Infections

Current testing of donor blood prior to release of blood components includes the following:

  Antibodies to human immunodeficiency virus (HIV) types 1 and 2 (anti-HIV-1/2); hepatitis C virus (HCV) (anti-HCV); hepatitis B core antigen (anti-HBc); human lymphotrophic virus types I and II (anti-human T-cell leukemia virus [HTLV] I/II); Treponema pallidum.

  Surface antigen: Hepatitis B surface antigen (HBsAg).

  Nucleic acid testing (NAT) for HIV; HCV; West Nile virus (WNV).

With current mandated testing, the estimated risk of transmission of viral infections per transfusion is reported in Table 24.8.42,43 Blood is also tested by enzyme-linked immunoassay for the detection Trypanosoma cruzi parasite, the causative agent for Chagas disease.

Other infectious agents and diseases transmissible by transfusion44,45 for which the blood supply is not currently routinely tested include the following:

  Hepatitis A virus, parvovirus B19, and dengue virus

  Parasitic diseases, not as common in the United States, including malaria, babesiosis, and leishmaniasis

  The protozoal disease toxoplasmosis which affects mainly immunocompromised patients

  Prions (protein particles) responsible for the transmission of variant Creutzfeldt-Jakob disease (vCJD). ⁴⁶

Alternatives to Allogeneic Blood Transfusion

Alternatives to the use of blood component therapy are available and may be particularly useful for bleeding patients who refuse allogeneic blood component transfusions (usually for religious beliefs) or bleeding patients unresponsive to appropriate transfusion therapy.

Patients with religious concerns about blood transfusion must be informed of any human-derived contents in products that may be administered, allowing them to make an informed decision.

Examples of alternatives to allogeneic blood transfusion6,47 are listed in Table 24.9.

Indications for use of some pharmaceutical hemostatic agents1,48 are summarized in Table 24.10.

Massive Transfusion

Massive transfusion is the administration of blood components over a 24-hour period in amounts that equal or exceed the total blood volume of the patient (10 or more units of whole blood or 20 units of packed RBC in an adult). After transfusion of one or more blood volumes, an abbreviated RBC cross-match (see general concepts) is performed to provide RBC more rapidly. Group O, Rh-negative, or Rh-positive blood (acceptable in male patients or postmenopausal females) may be released initially and once a specimen is received by the transfusion lab ABO-compatible blood can be provided.

  Adequate intravascular blood volume and blood pressure may be maintained initially with colloids (albumin, plasma protein fraction) or crystalloids (lactated Ringer solution or normal saline).

  Transfusion of packed RBC may become necessary after a loss of more than 25%–30% of blood volume, depending on the rate of blood loss, tissue perfusion, and oxygenation status of the patient. ⁴⁹

  Transfusion of blood components based on fixed ratios or algorithms should be avoided.

Adverse Sequelae of Massive Transfusion

  Dilution and/or consumption of hemostatic constituents of blood. Platelet count, prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen levels should be determined frequently.

  Replacement therapy is warranted if platelet count is less than 50, PT/PTT greater than 1.5, and fibrinogen concentration less than 100 mg/dL.

  Hypothermia, acidosis, hypocalcemia, and other biochemical disturbances may occur and electrolytes, particularly potassium and calcium, should be monitored. 1,49

  Hypocalcemia secondary to citrate accumulation may occur when large volumes of blood are administered at rapid rates (more than 100 mL/min), especially in the presence of liver and renal dysfunction. 1,49

Disseminated Intravascular Coagulation

DIC probably complicates massive transfusion less often than suspected, but DIC is associated with shock, independent of blood loss or transfusion.

  Laboratory coagulation test results are consistent with a consumptive coagulopathy.

  Treatment is correction of the underlying disorder while transfusion therapy is supportive.

  Administer cryoprecipitate when fibrinogen levels are below 80 to 100 mg/dL.

  Other components, such as platelets may be necessary, especially if bleeding is severe.

  If multiple factors are consumed, plasma factor levels of above 30% can be achieved with an FFP dose of 10 to 20 mL/kg. 1,49

IMMUNOHEMATOLOGIC DISORDERS

Hemolytic Disease of the Newborn

Hemolytic Disease of the Newborn (HDN) is the destruction of fetal erythrocytes by maternal IgG antibodies that cross the placenta and react with a paternally derived antigen present on the fetal RBC. Although traditionally associated with Rh antibodies (anti-Rh D), other antibodies including anti-A, anti-B, and anti-K:1 have been implicated and may cause significant HDN.

  Mild cases: The newborn is asymptomatic and laboratory findings of a positive DAT and mild hyperbilirubinemia are the only abnormalities.

  Severe cases: May result in intrauterine death (hydrops fetalis, erythroblastosis fetalis). There is a high risk of kernicterus caused by high unconjugated bilirubin.

Treatment: Intrauterine RBC transfusion (in severe cases) using antigen compatible (with the mother’s antibody), irradiated, CMV-negative, sickle-negative RBC suspended in 5% albumin or FFP.

  Usually a two-blood-volume red cell exchange removes approximately 25% of excess bilirubin, provides albumin to which excess bilirubin can bind, and removes antibody and approximately 70% of RBC coated with antibody.

  Additional exchange transfusions may be necessary if level of bilirubin continues to rise.

Neonatal Alloimmune Thrombocytopenia and Maternal Immune Thrombocytopenic Purpura

Neonatal Alloimmune Thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) is the destruction of platelets that carry paternally derived antigens by maternal antibodies that cross the placenta. As with HDN, NAIT may vary in severity

from very mild and asymptomatic thrombocytopenia to life-threatening bleeding, and it may occur in utero or in the neonatal period. The vast majority of NAIT is associated with antibody (IgG) against the common platelet antigen HPA-1a (PLA1), especially in the presence of HLA DRw52a phenotype.¹

NAIT is usually self-limiting and resolves within 2 to 3 weeks. If NAIT is suspected, often as a result of a previously affected pregnancy, cordocentesis to determine platelet counts may be performed in conjunction with administration of compatible platelets (maternal platelets or platelets known to be negative for the implicated antigen).

  In utero NAIT: IVIG is given with or without weekly corticosteroid administration to the mother (1 g/kg) until delivery.

  If there is high risk of intracranial hemorrhage, platelet transfusion is performed immediately prior to delivery.

  When compatible platelets are unavailable, high-dose IVIG has been administered to the neonate with variable effectiveness.

  An increase in platelet counts within 24 to 48 hours may be seen in patients who respond to IVIG.⁶

Maternal Immune Thrombocytopenic Purpura

In maternal ITP, antibodies against maternal platelets can cross the placenta and cause thrombocytopenia in the fetus.

  Degree of thrombocytopenia is milder than that associated with NAIT with a lower risk of fetal or neonatal intracranial hemorrhage.

  Maternal platelets and random donor platelets are generally not required but may be needed in about 44% of newborns. ⁵⁰

  IVIG may also be beneficial.

  Maternal ITP usually resolves in days to weeks (upon clearance of maternal antibodies from the neonate’s circulation).

Autoimmune Hemolytic Anemias

AIHA are characterized by the presence of antibodies against the individual’s own RBC antigens (autoantibodies), resulting in accelerated destruction of RBC. AIHA may be associated with autoimmune disorders, infections, medications or malignancies, or it may be primary. The laboratory hallmark is the positive DAT, indicating the presence of antibody or complement-coated red cells. Antibody may also be present in the serum such that positive DAT and IAT may coexist, making identification of underlying alloantibodies and compatibility testing difficult.

Warm Autoimmune Hemolytic Anemia

The majority of AIHA are caused by warm reacting antibodies. The implicated antibody is usually IgG and reacts with all cells, although occasionally a warm autoantibody will appear to have specificity against Rh antigens and several others. Patients with compensated warm AIHA require no specific treatment but should be investigated for an underlying condition such as systemic lupus erythematosus or a lymphoproliferative disorder. In children, viral illness may be accompanied by transient AIHA. Medications, particularly purine nucleoside analogues, are commonly associated with warm AIHA. Warm-reacting autoantibodies may be present only as a laboratory finding, or they may cause severe, even life-threatening hemolysis; these antibodies react optimally at 37°C in vitro. Patients are often totally asymptomatic, but may present with fatigue, jaundice, or mild anemia. Moderate splenomegaly occurs in about one-third to one-half of the cases and hepatomegaly in one-third of the patients. Hemolysis is usually not severe and is mainly extravascular.⁵¹

Laboratory findings include a positive DAT, spherocytes on the blood smear, elevated unconjugated bilirubin and LDH as indices of cell turnover, and a high reticulocyte count. Rarely, reticulocytopenia may be seen, either because of inadequate bone marrow response or because the autoantibody reacts with red cell precursors as well as with mature cells.

Red cell alloantibodies as a result of previous transfusions or pregnancies, found in approximately one-third of patients with AIHA, are capable of causing severe hemolytic transfusion reactions. Broadly reactive autoantibody may mask underlying alloantibodies and make procurement of compatible blood difficult.

Occasionally hemolysis may result in severe symptomatic anemia. Transfusion should not be delayed even when compatible blood cannot be obtained. The term “least incompatible” has not been adequately defined, does not correlate to clinical events, and would be best abolished.⁵¹

Oral glucocorticoids (prednisone at 1 mg/kg/day) are the current standard of care and about 50% of patients will respond. Splenectomy is effective in approximately half of those who are refractory to steroids. Immunosuppressive agents and IVIG may benefit selected patients.⁵¹ Refractory AIHA, especially when associated with lymphoproliferative disease, may respond to the monoclonal antibody rituximab (see below).

Cold Agglutinin Syndrome

Cold-reacting antibodies are common and usually of no significance, but some cold agglutinins, especially those with very high titer at 4°C but broad thermal amplitude (reactivity up to 30°C) may result in cold agglutinin syndrome (cold hemagglutinin disease). IgM is the immune globulin classically implicated and the DAT is almost always positive for C3d alone. Cold agglutinin syndrome may be primary (idiopathic) or secondary, often to a viral infection or lymphoproliferative disorder. Acute cold agglutinin syndrome may be associated with Mycoplasma pneumoniae and Epstein-Barr virus, is seen mostly in children and young adults, and tends to be transient and self-limited. Chronic cold agglutinin syndrome is most often seen in the elderly and may be associated with lymphoma, chronic lymphocytic leukemia, and Waldenstrom macroglobulinemia. Patients may present with acrocyanosis and hematuria precipitated by cold, and/or severe pain in the nose, ears, and distal extremities upon cold exposure. Severe anemia is rare in the chronic form.

Transfusion is rarely necessary, but, when performed, the typing specimen must be kept at body temperature from the time of phlebotomy through the testing procedure. Up to 50% of transfused cells may be destroyed by autoantibodies of the patient even when blood warmers are used.

Treatment with corticosteroids and splenectomy is not effective, and most patients do well simply by avoiding exposure to the cold. Rituximab, the anti-CD20 monoclonal antibody, appeared useful when administered as four weekly infusions in a small number of patients.⁵²

Paroxysmal Cold Hemoglobinuria

Paroxysmal cold hemoglobinuria (PCH) is a rare form of AIHA that results from a biphasic IgG antibody (Donath-Landsteiner antibody). Originally associated with untreated syphilis, it is now found most often with viral infections in children. The Donath-Landsteiner antibody binds to the RBC at cold temperatures and causes intravascular hemolysis as complement is fixed at warmer temperatures, accounting for the paroxysms of hemoglobinuria. Anemia associated with PCH is usually transient and self-limiting over 2 to 3 weeks. If transfusion support becomes necessary, crossmatch-compatible blood may be found if the antibody is not reactive at temperatures above 4°C. Unavailability of compatible RBC should not preclude transfusion in life-threatening anemia associated with hemolysis, despite shortened survival of the transfused RBC.1,6,51

Therapeutic Apheresis in the Management of Immunohematologic Disorders

Apheresis is the process by which selected components or substances in blood are removed from the circulation and the remainder of the blood returned to the patient. Apheresis is used to collect routine blood components (platelets, plasma, stem cells) from donors for transfusion. It also has therapeutic utility in many disorders where a pathologic substance is found in the blood. It is also used to remove an excess of cells, normal or abnormal, in the blood, known as cytapheresis. See Table 24.11 for commonly accepted indications for therapeutic apheresis for hematologic disorders endorsed by the American Society for Apheresis (ASFA).⁵³

  The kinetics of most intravascular substances indicate that exchange of 1 to 1.5 plasma volumes results in the highest efficiency removal with progressively decreased efficiency and added toxicity with each additional consecutive exchange.

  The volume of blood processed in order to attain the desired apheresis effect depends on the nature of the specific component, including its intravascular distribution and concentration in the particular patient.

Table 24.11  Recommendation for Therapeutic Apheresis in Hematologic Disorders

Category I: Accepted as Standard First-line or Primary Therapy

Babesiosis-severe (red cell exchange)

Cryoglobulinemia (plasma exchange)

Cutaneous T-cell lymphoma (photopheresis)

Hyperviscosity in monoclonal gammopathies (plasma exchange)

Hyperleukocytosis (cytapheresis)

Sickle cell disease with acute stroke (red cell exchange)

Thrombotic thrombocytopenic purpura (plasma exchange)

Polyneuropathy with IgM, with or without Waldenstrom macroglobulinemia (plasma exchange)

Hemolytic uremic syndrome—atypical (plasma exchange)

Category II: Generally Accepted as Adjunctive or Supportive Therapy

ABO-incompatible hematopoietic progenitor cell/marrow transplantation (plasma exchange recipient)

Graft-versus-host disease—skin (photopheresis)

Malaria—severe (red cell exchange)

Myeloma with acute renal failure (plasma exchange)

Red cell alloimmunization in pregnancy* (plasma exchange)

Sickle cell disease—primary/secondary prophylaxis/iron overload prevention (red cell exchange)

Thrombocytosis—symptomatic (cytapheresis)

Pure red blood cell aplasia (plasma exchange)

Category III: Optimal Role of Apheresis Therapy Not Established

Aplastic anemia (plasma exchange)

Erythrocytosis or polycythemia vera (phlebotomy/cytaphersis)

Warm autoimmune hemolytic anemia (plasma exchange)

Graft-versus-host disease—nonskin (photopheresis)

Multiple myeloma with polyneuropathy (plasma exchange)

Thrombocytosis—prophylactic (cytapheresis)

Posttransfusion purpura (plasma exchange)

*If fetus <20 wk gestation and previous severely affected pregnancy.

Adapted from Szczepiorkowski ZM,Winters JL, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice-Evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher. 2010;25(3):83-177.

  The patient’s total blood volume determines the safe extracorporeal blood volume (which should not exceed 15% of blood volume).

  Small patients may require that the machine be primed with saline or blood.

Apheresis is generally safe, especially for normal component donors. Complications mainly relate to vascular access, hemodynamic changes (especially for patients with cardiovascular disease), and a variable loss of blood components. Risks associated with apheresis are usually associated with a patient’s underlying disease.

  Plasma exchange may result in a 30% or more decrease in platelet counts.⁵⁴

  Platelet transfusion may be required for patients with low platelet counts and hemostatic problems.

  Cellular blood component counts return to normal after a few days and proteins and electrolytes re-equilibrate within hours, although fibrinogen may remain below baseline levels after 72 hours. ⁵⁵

  Hypotension may occur as a result of volume shifts and bradykinin activation from blood contact with plastic6,56; withhold ACE inhibitors, which potentiate this effect, from patients for 24 to 48 hours prior to an apheresis procedure.

  Plasma exchange may reduce blood levels of certain medications, especially those bound to plasma proteins or those with a long plasma half-life. ⁵⁶

  Citrate is used to prevent coagulation of blood in the circuit and may result in citrate toxicity: binding of calcium, decreased levels of ionized calcium, and potentially symptomatic hypocalcemia.

  Hypocalcemia may present with mild perioral tingling and discomfort, chest tightness, and tetany in severe cases; if symptoms do not subside with adjustment of citrate and whole blood flow rates, administration of oral calcium (as chewable tablets) or intravenous calcium gluconate replace the bound calcium and prevent the accompanying syndromes. ⁵⁷

Thrombocytapheresis

Increased platelet counts, particularly in myeloproliferative disorders in which platelets are also qualitatively abnormal, may be associated with bleeding or thrombosis.

Patients who are bleeding (e.g., with chronic myelogenous leukemia [CML]) may obtain immediate benefit from therapeutic cytapheresis. Generally plateletpheresis is a first-line therapy for thrombocytosis (platelet counts greater than 500,000/μL) in symptomatic patients.

  Each procedure will lower the count 30% to 50%.

  Cytoreductive chemotherapy should be initiated simultaneously, since plateletpheresis is not effective long term. 53,56

Leukocytapheresis (Leukapheresis)

Malignant leukocytosis or hyperleukocytosis (immature white blood cell counts of greater than 100,000/μL), in association with some leukemias, can result in leukostasis in the central nervous system, kidneys, and lungs. Symptoms may occur with rapidly rising blast counts less than 100,000/μL, especially in AML and CML.

  Changes in mentation, dizziness, blurred vision, hypoxia, or respiratory symptoms constitute a medical emergency where rapid cytoreduction is imperative.

  Therapeutic leukapheresis can reduce the leukocyte count by 30% to 50% in hours.

  Symptoms may abate promptly.

  Reduction of the white cell count permits cytoreductive chemotherapy and reduces the risk of developing tumor lysis syndrome.

  Chemotherapy with hydroxyurea (if myeloid malignancy) or a similar agent should be initiated concurrently since repeated leukocytapheresis may not control hyperleukocytosis.

Photopheresis (Extracorporeal Photochemotherapy)

Photopheresis is the separation of the patient’s leukocytes by apheresis for extracorporeal treatment with the chemotherapeutic agent 8-methoxy-psoralen (8-MOP) and photoactivation by ultraviolet A (UVA) light for subsequent reinfusion to the patient.

  It has some efficacy in the treatment of refractory cutaneous T-cell lymphoma, allograft rejection, refractory acute and chronic GVHD, scleroderma, and other autoimmune diseases.

  Mechanism of action is not fully understood; possibly related to apoptosis of pathogenic T lymphocytes and antigen-presenting cells or anti-idiotype cytotoxic T-cell response 1,56

  Use of 8-MOP is contraindicated in patients with light-sensitive disorders such as xeroderma pigmentosa, albinism, and certain porphyrias. ⁵⁶

Erythrocytapheresis/Red Cell Exchange

Red cell exchange involves the removal of abnormal red cells.

  Patient’s RBC are replaced with normal donor RBC in patients with sickle cell disease.

  Erythrocytapheresis may be used to reduce red cell mass acutely in symptomatic (visual disturbances, confusion, lethargy, hemorrhage, threatened stroke, thrombosis of abdominal vasculature) patients with excessive polycythemia. 53,56

  In Polycythemia Rubra Vera (PRV) saline or colloid volume replacement is administered to maintain isovolemia.

Red Cell Exchange and Sickle Cell Anemia

Red cell exchange may be used acutely to treat some complications of sickle cell disease⁵³ including acute chest syndrome, stroke, retinal infarction, priapism, and hepatic crisis, or as protracted or chronic

treatment for the prevention of complications such as stroke and recurrent severe painful crises, and for reduction of iron overload secondary to transfusion.⁵⁶

  In the perioperative setting, simple transfusion or a single red cell exchange prevents morbidity associated with sickle cell disease.

  The goal is to achieve hemoglobin S of less than 30%.

  Transfusion and exchange have been used to treat sickle complications during pregnancy but routine use is not warranted. Exchange transfusion can raise hemoglobin A to levels difficult to achieve with simple transfusion and may benefit patients in the third trimester for preeclampsia, sepsis, and preoperative management. ⁵⁶

Red Cell Exchange and Parasitemia

Red cell exchange has been used as antiparasitic treatment in malaria to decrease the circulating parasite load when it exceeds 5%.⁵⁸

Plasmapheresis

Plasmapheresis may be used to collect plasma for transfusion or manufacturing of plasma derivatives, or to remove undesirable substances from the circulation. Colloids or saline (plasma with TTP) are administered to maintain isovolemia. See Tables 24.11 and 24.12 for common indications for therapeutic plasmapheresis.⁵³

Thrombotic Microangiopathies

TTP and hemolytic uremic syndrome (HUS) belong to a spectrum of thrombotic microangiopathies: TTP may be associated with prominent neurologic symptoms; HUS presents with a more prominent renal component. Characteristic findings of TTP include fever, renal impairment, neurologic symptoms such as change in mental status, seizures, or coma, thrombocytopenia (platelet counts usually less than 30,000/μL), and microangiopathic hemolytic anemia with schistocytes.

TTP results from the accumulation of ultra-large von Willebrand factor multimers caused by congenital absence of or inhibitory antibodies to the vWF-cleaving metalloprotease ADAMTS13.1,56 When TTP and HUS-like syndromes are associated with immunosuppressive agents (typically vinca alkaloids, mitomycin, bleomycin, BL22, cisplatin, tacrolimus, and cyclosporin A), post bone marrow transplant or cancer they do not respond well to therapeutic plasma exchange (TPE).

  TPE is first-line therapy for the treatment of TTP and generally not effective for typical HUS.

  TPE should be performed as soon as TTP is suspected.

  The effectiveness of TPE in TTP depends on the removal of ultra-large vWF multimers and reduction of the IgG antibodies against vWF-cleaving protease.

  Plasma (FFP) is the fluid replacement of choice in TPE for TTP and also replaces the vWF-cleaving protease.

  TPE is often done daily, then tapered until platelet counts stabilize at more than 100,000/μL for 2 consecutive days.

  Response should be monitored by clinical assessment and laboratory measurements (platelet count, LDH, extent of schistocytosis).

Platelet transfusion is generally discouraged as this is thought to potentially precipitate thrombosis. This dogma has recently been challenged.¹¹ However, platelet transfusion may be necessary in the event of life-threatening hemorrhage.⁶

Dysproteinemias

Complications of paraproteinemias of multiple myeloma, Waldenstrom macroglobulinemia, and cryoglobulinemia respond to TPE.

  Hyperviscosity syndrome with mental status changes, mucosal and gastrointestinal bleeding, retinopathy, and hypervolemia constitutes a medical emergency.

  Hyperviscosity responds to even small volume exchanges, but procedures need to be repeated until the paraprotein is controlled with chemotherapy. ⁵⁶

References

  1.  Klein HG, Anstee DJ. Mollison’s Blood Transfusion in Clinical Medicine. 11th ed. Oxford: Blackwell; 2005.

  2.  Mielcarek M, Leisenring W, Torok-Storb B, Storb R. Graft-versus-host disease and donor-directed hemagglutinin titers after ABO-mismatched related and unrelated marrow allografts: evidence for graft-versus-plasma cell effect. Blood. 2000;96:1150-1156.

  3.  Worel N, Greinix HT, Keil F, et al. Severe immune hemolysis after minor ABO-mismatched allogeneic peripheral blood progenitor cell transplantation occurs more frequently after nonmyeloablative than myeloablative conditioning. Transfusion. 2002;42:1293-1301.

  4.  Griffith LM, McCoy JP, Bolan CD, et al. Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation. Br J Haematol. 2005;128:668-675.

  5.  Bolan CD, Leitman SF, Griffith LM, et al. Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation. Blood. 2001;98:1687-1694.

  6.  Roback JD, ed. Technical Manual. 17th ed. Bethesda, MD: AABB Press; 2011.

  7.  Hébert PC, Wells G, Blajchman MA, et al. A multicenter randomized, controlled clinical trial of transfusion requirements in critical care. N Engl J Med. 1999;340:409-417.

  8.  Majdpour C, Spahn DR, Weiskopf RB. Anemia and perioperative red blood cell transfusion: a matter of tolerance. Crit Care Med. 2006;34:S102-S108.

  9.  Practice Guidelines for perioperative blood transfusion and adjuvant therapies: An updated report by the American Society of Anesthesiologists task Force on Perioperative Blood Transfusion and Adjuvant Therapies. Anesthesiology. 2006;105:198-208.

10.  Fontana S, Kremer Hovinga JA, Lämmle B. Treatment of TTP. Vox Sang. 2006;90:245-254.

11.  Swisher KK, Terrell DR, Vesely SK. Clinical outcomes after platelet transfusions with TTP. Transfusion. 2009;49:873-887.

12.  Schiffer CA, Anderson KC, Bennett CL, et al. Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Oncology. J Clin Oncol. 2001;19:1519-1538.

13.  Stroncek DF, Leonard K, Eiber G, Malech HL, Gallin JI, Leitman SF. Alloimmunization after granulocyte transfusions. Transfusion. 1996;36:1009-1015.

14.  Wright DG, Robichaud KJ, Pizzo PA, Deisseroth AB. Lethal pulmonary reactions associated with the combined use of amphotericin B and leukocyte transfusions. N Engl J Med. 1981;304:1185-1189.

15.  Safdar A, Hanna HA, Boktour M, et al. Impact of high-dose granulocyte transfusions in patients with cancer with candidemia. Cancer. 2004;101:2859-2865.

16.  Price TH. Granulocyte transfusion: current status. Semin Hematol. 2007;44:15-23.

17.  Marsh JC, Ball SE, Cavenagh J. Guidelines for the diagnosis and management of aplastic anaemia. Br J Haematol. 2009;147(1):43-70.

18.  Carral A, de la Rubia J, Martin G, et al. Factors influencing the collection of peripheral blood stem cell in patients with acute myeloblastic leukemia and non-myeloid malignancies. Leuk Res. 2003;27:5-12.

19.  Koumakis G, Vassiolmanolakis M, Hatzichristou H, et al. Predictive factors affecting mobilization and peripheral blood stem cell collection using single apheresis for rescuing patients after high-dose chemotherapy in various malignancies. Bone Marrow Transplant. 1996;18:1065-1072.

20.  Noach EJK, Ausema A, van Os R, Akkerman I, Koopal S, Weesing E. Chemotherapy prior to autologous bone marrow transplantation impairs long-term engraftment. Exp Hematol. 2003;31(6):528-534.

21.  Fruehauf S, Schmitt K, Veldwijk MR, et al. Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported cytotoxic chemotherapy: an update on 100 patients. Br J Haematol. 1999;105:786-794.

22.  Devine SM, Flomenberg N, Vesole DH, et al. Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol. 2004;22:1095-1102.

23.  Hess D, Wirthlin L, Craft T, et al. Human CD34+ cells mobilized by AMD3100 demonstrate enhanced NOD/SCID repopulating function compared to CD34+ cells mobilized by granulocyte colony stimulating factor. Blood. 2005;106:1962.

24.  Liles WC, Rodger E, Broxmeyer HE, et al. Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist. Transfusion. 2005;45: 295-300.

25.  Bolan CD, Carter CS, Wesley RA, et al. Prospective evaluation of cell kinetics, yields and donor experiences during a single large-volume apheresis versus two smaller volume consecutive day collections of allogeneic peripheral blood stem cells. Br J Haematol. 2003;120:801-807.

26.  Vasu S, Leitman SF, Tisdale JF. Donor demographic and laboratory predictors of allogeneic peripheral blood stem cell mobilization in an ethnically diverse population. Blood. 2008;112(5):2092-2100.

27.  Stroncek D, Shawker T, Follmann D, Leitman SF. G-CSF-induced spleen size changes in peripheral blood progenitor cell donors. Transfusion. 2003;43:609-613.

28.  Snyder EL, Haley NR, eds. Cellular Therapy: A Physician’s Handbook. 1st ed. Bethesda, MD: AABB Press; 2004:11-23.

29.  Brunstein CG, Wagner JE. Cord blood transplantation for adults. Vox Sang. 2006;91:195-205.

30.  Dentali F, Marchesi C, Giorgi Pierfranceschi M, et al. Safety of prothrombin complex concentrates in patients requiring rapid reversal of anticoagulant treatment with the vitamin K antagonists: a systematic review and a meta-analysis of the literature. 52nd ASH Annual Meeting; December 4–7, 2010; Orlando, FL. Poster #1113.

31.  Ansell J, Hirsch J, Hylek E, et al. Pharmacology and management of the vitamin K antagonists. American College of Chest Physicians evidence-based clinical practice guidelines (8th edition). Chest. 2008;133(suppl):160s-198.

32.  Bowman JM. The prevention of Rh immunization. Transfus Med Rev. 1988;2(3):129.

33.  ACOG Practice Bulletin. Prevention of RhD alloimmunization. Number 4, May 1999 (replaces educational bulletin Number 147, October 1990). Clinical management guidelines for obstetrician-gynecologists. American College of Obstetrics and Gynecology. Int J Gynecol Obstet. 1999;66:63-70.

34.  Popovsky MA, ed. Transfusion Reactions. 3rd ed. Bethesda, MD: AABB Press; 2007.

35.  Petz LD, Calhoun L, Shulman IA, Johnson C, Herron RM. The sickle cell hemolytic transfusion reaction syndrome. Transfusion. 1997;37:382-392.

36.  Cohen AR. Transfusion therapy for sickle cell disease. In: Capon SM, Chambers LA, eds. New Directions in Pediatric Hematology. Bethesda, MD: AABB Press; 1996:39-88.

37.  Smith-Whitley K. Alloimmunization in patients with sickle cell disease. In: Hermann JN, Manno CS, eds. Pediatric Transfusion Therapy. Bethesda, MD: AABB Press; 2002:240-282.

38.  Toy P, Popovsky MA, Abraham E, et al. Transfusion-related acute lung injury: definition and review. Crit Care Med. 2005;33:721-726.

39.  Silliman CC, Boshkov LK, Mehdizadehkashi Z, et al. Transfusion-related acute lung injury: epidemiology and a prospective analysis of etiologic factors. Blood. 2003;101:454-462.

40.  Eder AF, Herron R, Strupp A, et al. Transfusion-related acute lung injury surveillance (2003-2005) and the potential impact of the selective use of plasma from male donors in the American Red Cross. Transfusion. 2007;47:599-607.

41.  Eder AF, Kennedy JM, Dy BA, et al. Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004-2006). Transfusion. 2007;47:1134-1142.

42.  Zou S, Dorsay KA, Notari EP, et al. Prevalence, incidence, and residual risk of human immunodeficiency virus and hepatitis C virus infections among United States blood donors since the introduction of nucleic acid testing. Transfusion. 2010;50:1495-1504.

43.  Zou S, Stramer SL, Notari EP, et al. Current incidence and residual risk of Hepatitis B infection among blood donors in the United States. Transfusion. 2009;49(suppl 2):1S-235S.

44.  Dodd RY. Transmission of parasites by blood transfusion. Vox Sang. 1998;74(suppl 2):161-163.

45.  Alter HJ, Stramer SL, Dodd RY. Emerging infectious diseases that threaten the blood supply. Semin Hematol. 2007;44:32-41.

46.  Health Protection Agency Press Statement: fourth case of transfusion-associated vCJD infection in the United Kingdom. Euro Surveill. January 18, 2007.

47.  Santrach P, ed. Standards for Perioperative Autologous Blood Collection and Administration. 4thed. Bethesda, MD: AABB Press; 2009.

48.  Bolan CD, Klein HG. Transfusion medicine and pharmacologic aspects of hemostasis. In: Kitchens C, Kessler C, Alving BM, eds. Consultative Hemostasis and Thrombosis. 2nd ed. New York, NY: Harcourt Health Sciences; 2007.

49.  Spence RK. Transfusion in surgery. In: Mintz PD, ed. Transfusion Therapy Clinical Principles and Practice. Bethesda, MD: AABB Press; 2011:265-292.

50.  Webert KE, Mittal R, Sigouin C. A retrospective 11-year analysis of obstetric patients with idiopathic thrombocytopenic purpura. Blood. 2003;102:4306-4311.

51.  Petz LD, Garratty G, eds. Immune Hemolytic Anemias. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2004.

52.  Schöllkopf C, Kjeldsen L, Bjerrum OW. Rituximab in chronic cold agglutinin disease: a prospective study of 20 patients. Leuk Lymphoma. 2006;47(2):253.

53.  Szczepiorkowski ZM, Winters JL, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice-Evidence-based approach from the apheresis applications committee of the American Society for Apheresis. J Clin Apher. 2010;25(3):83-177.

54.  Rogers RL, Johnson H, Ludwig R, et al. Efficacy and safety of plateletpheresis by donors with low-normal platelet counts. J Clin Apher. 1995;10:194-197.

55.  Flaum MA, Cuneo RA, Appelbaum FR, et al. The hemostatic imbalance of plasma-exchange transfusion. Blood. 1979;54:694-702.

56.  McLeod BC, ed. Apheresis: Principles and Practice. 3rd ed. Bethesda, MD: AABB Press; 2010.

57.  Bolan CD, Cecco SA, Wesley RA, et al. Controlled study of citrate effects and response to i.v. calcium administration during Allogeneic peripheral blood progenitor cell donation. Transfusion. 2002;42:935-946.

58.  Griffith KS, Lewis LS, Mali S, Parise ME. Treatment of malaria in the United States: a systematic review. JAMA. 2007;297(20):2264-2277.

Suggested Reading

Gottschall J. Blood Transfusion Therapy: A Physician’s Handbook. 10th ed. Bethesda, MD: AABB Press; 2011.

McLeod BC. Therapeutic Apheresis: A Physician’s Handbook. 3rd ed. Bethesda, MD: AABB Press; 2010.

Osby MA, Saxena S, Nelson J, et al. Safe handling and administration of blood components: review of practical concepts. Arch Pathol Lab Med. 2007;131:690-694.

Roseff SD. Pediatric Transfusion: A Physician’s Handbook. 3rd ed. Bethesda, MD: AABB Press; 2009.

Snyder EL, Haley NR, eds. Cellular Therapy: A Physician’s Handbook. Bethesda, MD: AABB Press; 2004.

Szczepiorkowski ZM, Winters JL, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice-Evidence-based approach from the apheresis applications committee of the American Society for Apheresis. J Clin Apher. 2010;25(3):83-177.

Zou S, Dorsay KA, Notari EP, et al. Prevalence, incidence, and residual risk of human immunodeficiency virus and hepatitis C virus infections among United States blooddonors since the introduction of nucleic acid testing. Transfusion. 2010;50:1495-1504.

Zou S, Stramer SL, Notari EP, et al. Current incidence and residual risk of Hepatitis B infection among blood donors in the United States. Transfusion. 2009;49(suppl 2):1S-235S.

25

Hemochromatosis

Susan F. Leitman and Charles D. Bolan

EPIDEMIOLOGY

Classic hereditary hemochromatosis, also known as HFE-hemochromatosis, is an autosomal recessive disorder caused by inappropriate dietary absorption of iron and abnormal iron cycling. It is characterized by progressive accumulation of iron in tissues, particularly the liver, pancreas, heart, endocrine organs, and skin, which may lead to end-stage organ damage, usually during or after middle age.¹⁻³ It is one of the most common single-gene disorders in Caucasians of northern European descent, with an incidence of 1 in 200 and a carrier rate of 1 in 10 persons. However, the clinical penetrance of the disorder is highly variable, and only a minority of affected persons develops severe or life-threatening organ dysfunction.4,5

Genetic Basis for Classic Hemochromatosis: HFE Mutations

  Mutations in HFE, an MHC class-I like gene on chromosome 6, are found in nearly 90% of people with the clinical phenotype and 100% of affected people with a strong family history of the disorder. 6,7

  Substitution of tyrosine for cysteine at amino acid 282 of the HFE gene product (C282Y) is the founder mutation; by linkage disequilibrium analysis, the mutation originated recently, within the past 2000 years. C282Y occurs with highest frequency in northwestern European populations, reaching 14% in areas of Great Britain (Table 25.1). Allele frequency decreases in a north-to-south and west-to-east direction across Europe, and the ancestral haplotype may have been of Viking or Celtic origin; the haplotype is extremely rare in African and Asian populations. Homozygosity for C282Y is seen in 64% to 96% of persons with clinical hemochromatosis.

  A second HFE mutation, replacement of histidine by aspartate at residue 63 of the HFE protein (H63D) is frequently found on the non-C282Y-containing chromosome of individuals with clinical hemochromatosis who are heterozygous for C282Y.⁶ H63D is an older mutation with a wider population distribution, having an allele frequency of 5% to 14% throughout Europe and Asia. It appears to be a genetic polymorphism without much clinical impact in the absence of another genetic or environmental factor. Compound heterozygosity for C282Y/H63D is seen in 4% to 7% of persons with a hemochromatosis phenotype.

  Seventeen additional polymorphisms in HFE have been described. Of these, only the S65C mutation appears to have clinical impact, and it may cause mild iron overload when the individual is compound heterozygous for C282Y or H63D.

PATHOPHYSIOLOGY

Since iron excretion in the gut is fixed at 1 mg/day, normal iron balance must be maintained by meticulous control of iron absorption in the intestine and iron release from macrophages. These are modulated in response to body iron stores and the erythropoietic demand for iron.

Hepcidin: Key Regulator of Iron Homeostasis

Hepcidin, a liver-derived peptide hormone, is a key negative regulator of iron release into the plasma by intestinal enterocytes, macrophages, hepatocytes, and placental cells.⁸ It binds to and causes internalization and degradation of the cell surface iron exporter, ferroportin.⁹ Hepcidin excess decreases intestinal iron absorption and macrophage iron release, and causes anemia. Hepcidin deficiency promotes intestinal iron absorption and leads to tissue iron overload. Hepcidin gene expression is enhanced by iron overload and inflammation, and suppressed by anemia and hypoxia. Although hepcidin is ordinarily induced by dietary iron loading, its expression is inappropriately reduced in all forms of inherited hemochromatosis.10,11

Iron Overload Disorders and Hepcidin Deficiency

Hepcidin deficiency plays a central role in the pathogenesis of the inherited hemochromatosis disorders, including those due to mutations in the HFE gene, the hemojuvelin gene (HJV), the transferrin receptor 2 gene (TfR2), and hepcidin itself (HAMP) (Table 25.2). Hemojuvelin acts as a coreceptor in the bone morphogenetic protein (BMP) pathway, interacting with BMP ligands and BMP type I and II receptors to generate an active signaling complex.¹⁰ This complex activates a Smad receptor signaling cascade and translocation of a Smad complex to the nucleus, where it increases HFE transcription. HJV and HAMP mutations are critical components in the same common pathway; their negative effect on hepcidin expression is associated with severe iron loading in childhood, or juvenile hemochromatosis.

HFE Localization and Function

HFE is highly expressed in Kupffer cells of the liver and in tissue macrophages. Binding to β2 microglobulin (β2m) allows expression of HFE/β2m on the cell surface,¹² where it forms a stable complex with transferrin receptor 1 (TfR1). The C282Y mutation prevents formation of a disulfide bond in HFE, disabling β2m binding, and preventing cell surface expression. Disruption of the HFE/β2m/TfR1 complex, and mutations in TfR2, are associated with adult-onset iron overload. HFE and TfR2 may regulate hepcidin expression by enhanced iron transport into the cell (endocytosis of diferric transferrin), by upstream regulation of hepcidin, or as weak coreceptors for BMP-SMAD signaling. According to this model, it might be possible to treat hemochromatosis by hepcidin replacement.

The most common form of secondary hemochromatosis is transfusional iron overload: 1 mL of red cells contains about 1 mg of iron. Inappropriate absorption of iron in the gut may also occur in association with ineffective erythropoiesis. In this case, the erythropoietic stimulus to decrease hepcidin levels overrides the effect of iron overload on increasing hepcidin expression.

Iron Homeostasis

The distribution of body iron is shown in Table 25.3, with a comparison of iron stores in the normal state and in subjects with hemochromatosis.

  Excess iron and tissue injury. When the capacity for iron storage is exceeded, excess tissue iron causes cellular damage by catalyzing the formation of oxyradicals.¹³ Oxidative damage to lipids, proteins, carbohydrates, and DNA may lead to widespread impairment in cell function and integrity. In particular, lipid peroxidation may result in impaired membrane-dependent mitochondrial and lysosomal function. Oxidative injury to DNA, particularly in hepatocytes, may predispose to mutagenesis and cancer.

  Nontransferrin-bound iron (NTBI): This represents “free iron in serum.” NTBI enters cells freely, independent of receptor-mediated uptake. NTBI levels are low or undetectable at transferrin saturation (TS) below 40%, and increase linearly with TS levels above 40% to 50%. NTBI and its intracellular labile iron counterpart may be the direct mediators of oxidant stress. ¹³

CLINICAL FEATURES AND DIAGNOSIS OF HFE-HEMOCHROMATOSIS

Prior to the availability of biochemical and genetic screening tests, HFE-hemochromatosis was identified by damage to the liver, pancreas, heart, and joints, and diagnosed by demonstrating increased iron stores on liver biopsy. The “classic triad” of cirrhosis, diabetes, and skin pigmentation appeared in many publications and textbooks.¹ Patients typically presented with the following symptoms:

  Severe liver disease due to hepatic fibrosis or cirrhosis

  Cardiac failure and refractory arrhythmias

  Polyendocrine failure: insulin-dependent diabetes and hypogonadotrophic hypogonadism

  Debilitating symmetric polyarthritis

  Grayish skin pigmentation

It is now recognized that this severe clinical phenotype is relatively rare and only develops in 1% to 4% of untreated C282Y homozygotes over their lifetime.⁴ Between 40% and 60% of C282Y homozygote males and 60% to 80% of homozygote females will remain asymptomatic or have minimal clinical manifestations throughout their lives; of the 40% to 50% who do develop symptoms that affect quality of life, arthritis, fatigue, and sexual dysfunction are the most common complaints (Table 25.4).14,15

New Diagnostic Definition

In the current era of molecular testing, recognizing that clinical penetrance can be highly variable, the diagnosis of hemochromatosis is established by the detection of two mutated HFE alleles. This definition does not require active symptoms or signs of illness or the presence of iron overload. Four stages of the disorder are recognized¹⁶:

1.  Genetic predisposition with no other abnormality (age 0–20, 0–5 g tissue iron storage)

2.  Iron overload without symptoms (age >20, >5 g iron storage)

3.  Iron overload with early symptoms (age >30, >8 g iron storage)

4.  Iron overload with organ damage (age >40, 10–20 g iron storage)

Common Clinical Presentation

The most common clinical presentation of hereditary hemochromatosis is with nonspecific symptoms, and therefore practitioners should have a low threshold for ordering serum TS and ferritin studies in patients with unexplained chronic fatigue, arthralgia, or arthritis, sexual dysfunction, hepatomegaly, or elevated liver function values (alanine aminotransferase [ALT]). Since symptoms are easily overlooked, the single most common event currently leading to a diagnosis of hemochromatosis is the incidental detection of an abnormal laboratory test result, either an elevated TS, serum ferritin, or ALT. In hemochromatosis subjects diagnosed with fatigue on presentation, screening laboratory tests to evaluate possible concomitant thyroid disease should be obtained.

Typical findings related to the most common clinical signs and symptoms of hemochromatosis are shown in Table 25.5. It is difficult to assign a frequency to these symptoms since there is a continuum of increasing frequency with increasing age, and with male versus female sex.¹⁶ Arthritis is the clinical feature with the greatest impact on quality of life.¹⁷ In contrast to significant cardiac abnormalities described in hemochromatosis patients who presented with very high iron prior to the advent of more frequent screening and the availability of a genetic test, heart disease now is generally absent or clinically insignificant in newly diagnosed, asymptomatic subjects.¹⁸

The considerable variability in clinical penetrance of C282Y homozygosity, both in rate of accumulation of iron stores and appearance of organ dysfunction, may be due to environmental, lifestyle, and genetic factors (Table 25.6).

LABORATORY TESTING

Once the clinical suspicion of hemochromatosis is raised, the diagnosis should be confirmed by laboratory testing, including those listed below:

Confirmatory Laboratory Tests

  Serum iron, transferrin, and TS: Transferrin is the major iron transport protein in plasma. Several assay methods for TS exist: the most accurate is direct colorimetric analysis of serum iron (SI) combined with nephelometric assay of transferrin, wherein TS = molar concentration of iron divided by twice the molar concentration of transferrin. Less expensive but also less robust methods include chemical analyses of total serum iron binding capacity (TIBC) and unbound iron capacity (UIBC). Saturation of serum iron binding capacity is measured by dividing the serum iron by either TIBC (SI/TIBC) or by the sum of iron and UIBC [(SI)/(SI+UIBC)]. Normal range for TS is 15% to 45%.

  Serum ferritin: This is a major intracellular iron storage protein and is measured immunologically. This estimates the degree of iron overload and size of mobilizable iron stores (1 μg/L ferritin = 7–8 mg stored iron; for example, 1,000 μg/L ferritin = 7,000–8,000 mg stored iron). It is used to determine pace of initial phlebotomy therapy. Normal levels are < 350 μg/L in men and < 120 μg/L in women.

  HFE genotype: Definitive diagnostic test, assesses predisposition to serious illness, useful for family counseling.

Ancillary Laboratory Tests

  ALT: to assess degree of liver injury

  Complete Blood Count: obtain baseline hemoglobin and red cell mean corpuscular volume (MCV), which can be monitored during therapy (decrease in MCV is an indicator of iron-limited erythropoiesis).

  Blood glucose and electrolytes

  Total and free testosterone: as indicated by symptoms

  Thyroid function tests: as indicated by symptoms

  Alpha fetoprotein: as baseline for subsequent monitoring for liver cancer

  Serologic tests for exposure to hepatitis B and C (HBsAg and anti-HCV): active viral hepatitis worsens liver injury; useful to guide vaccine administration

Role of Liver Biopsy

Liver biopsy is generally not required for diagnosis. Although it previously served as the “gold standard” for both diagnosis and prognosis, the diagnosis is now more safely and reliably made with use of the HFE genotype.¹⁶

Indications for Biopsy

1.  For prognostic purposes, to confirm high clinical suspicion of cirrhosis

a.  Ferritin >3,000 μg/L

b.  Hepatomegaly and/or signs of portal hypertension (large spleen, low platelets)

c.  ALT does not normalize with phlebotomy.

2.  If concomitant HBV or HCV infection present

3.  In diagnostic workup of elevated ferritin and ALT, with normal HFE genotype and absence of other genetic causes listed above.

Histologic Findings

1.  Marked increase in hepatocellular iron, with relative sparing of Kupffer cells. Iron is distributed in a decreasing gradient from the periportal to the centrilobular areas.

2.  With progressive damage, we may see portal fibrous expansion, bridging fibrosis with piecemeal necrosis, and macro or micronodular cirrhosis.

3.  Hepatic iron index (hepatic iron concentration/56 × age) >1.9 (in the absence of transfusional siderosis) strongly suggests iron overload is due to hemochromatosis rather than other causes.

Radiographic and Other Tests

  Skeletal films: performed to evaluate symptomatic joints

  Liver ultrasound: useful in workup of non-hemochromatosis causes of elevated ferritin; may show steatosis. Important in surveillance for liver cancer.

  CT and/or MRI of liver: not indicated diagnostically. Useful for suspected liver cancer.

  Superconducting quantum interference device (SQUID) assessment: provides most sensitive noninvasive quantitative assessment of iron stores; has limited availability.

POPULATION SCREENING

The clinical course of hemochromatosis meets the definition of a disorder for which population screening should be performed2,3:

1.  High prevalence in selected populations

2.  Burden of disease (clinical penetrance) high enough to warrant medical and public attention

3.  Prolonged presymptomatic phase, during which detection and treatment lead to reductions in morbidity and mortality (early detection prevents complications and improves outcomes)

4.  Availability of reliable, accurate, easily available, and inexpensive screening tests

5.  Treatment is effective, safe, inexpensive, and easily accessible.

Thus, the costs of widespread testing and preventive treatment are considered favorable (more effective and less expensive) than delaying until development of late symptoms—particularly as the early, presenting symptoms are nonspecific, often unrecognized as being caused by hemochromatosis, and are associated with a 5 to 10 year delay until accurate diagnosis.

Laboratory Screening

  Transferrin Saturation: The single best screening test is the serum TS: it is inexpensive, widely available, and highly sensitive and specific for the presence of the C282Y HFE allele.¹⁹ The decision threshold at which confirmatory testing should be initiated ranges from TS values of 45% to 62%, depending on whether sensitivity or specificity is preferred (Table 25.7). Since TS is affected by diet and diurnal variation, an elevated value should be confirmed by a second TS after an overnight fast, in the absence of oral iron supplements. Phenotype screening with TS is not advised until age 20 to 30, as iron burdens are generally low below this age.¹⁶ An algorithm for workup of persons detected through screening programs is shown in Figure 25.1.

  Ferritin screening: Ferritin is an acute phase reactant; levels rise with inflammation, infection, and non-hemochromatosis liver disease. Lack of sensitivity and specificity make it a less reliable screening test.

  Genotype screening: A 1998 consensus conference decided against widespread population screening using genetic tests. The high cost of genetic tests and variable clinical penetrance of hemochromatosis, coupled with concerns over stigmatization, discrimination, and insurability, led to rejection of this approach at the time. ²⁰

Screening of Family Members of C282Y Homozygotes

  Screening of children: The most cost-effective test is HFE genotype; biochemical screening is also acceptable; testing should be delayed until age 20 to 30. If more than two children are involved, the best approach may be genotyping of the other parent. ²¹

FIGURE 25.1 Decision tree for hemochromatosis population screening.

  Screening of siblings: All siblings should be counseled to undergo either genetic or phenotypic screening. The most cost-effective test is HFE genotype, but phenotypic screening with combination of TS and ferritin is also acceptable. ²¹

TREATMENT

Phlebotomy Therapy

  Phlebotomy: Periodic removal of one unit (500 mL) of whole blood. Safe, inexpensive, standard of care for the past 50 years.²² One unit of whole blood removes 200–250 mg of iron. Double red cell collection by apheresis removes 360 mL of packed red cells (400–420 mg of iron) and may be particularly useful in blood center setting.

  Controversy regarding treatment indications for subjects with modest iron burdens

  Patients generally desire treatment and are eager and willing to be blood donors.

  Therapy is safe, accessible, and prevents late organ damage.

  Referral to blood center shifts argument in favor of treatment (double benefit, to subject and to community; efficient care; no charge for procedure).

Guidelines for Phlebotomy Therapy

Phase I: Iron Depletion

  Pace: Initiate phlebotomy at 1 to 4 week intervals, depending on ferritin, hemoglobin, ALT, gender, and weight. As iron depletion approaches, decrease pace to monthly.

  Target of “de-ironing” therapy: several assays may be used

  Ferritin < 30 μg/L

  TS < 30%

  Decrease in red cell MCV to 3% to 5% below prephlebotomy level.²³

  Monitoring parameters

  Prephlebotomy fingerstick hemoglobin or hematocrit (+/− venous CBC) at each visit to avoid anemia

  Ferritin every 4 to 8 weeks initially, then ferritin +/− TS every 1 to 2 treatments once ferritin <100 μg/L.

  Safety guide: Threshold hemoglobin for therapeutic bleed ≥ 12.5 g/dL (hematocrit ≥ 38%). In general, do not bleed below this level; defer therapy for 1 to 4 weeks until hemoglobin recovers. Iron deficiency is not necessary during treatment; anemia should be avoided.

  General guide: For initial ferritin of 500 to 1,500, patients generally require 15 to 30 bleeds to achieve iron depletion. If initial ferritin is >2,000 μg/dL, it may require more than 40 to 50 bleeds.

Phase II: Preventing Reaccumulation (Maintenance)

  Pace: 500 mL removed every 8 to 26 weeks (mean 10–12 weeks), depending on gender, weight, age, and dietary habits. This is usually a lifelong requirement, although some subjects reaccumulate iron very slowly.

  Goals of maintenance therapy

  Ferritin 30 to 50 μg/L

  TS < 50%

  Hemoglobin > 12.5 g/dL

  Monitoring parameters

  Prephlebotomy fingerstick hemoglobin or hematocrit (+/− venous CBC) at each visit, and ferritin and/or TS every 1 to 2 treatments.

Evaluation of Anemia during Phlebotomy Therapy

Development of a hemoglobin <12.5 g/dL despite elevated ferritin levels may be due to occult bleeding, medications such as proton pump inhibitors,²⁴ or endocrine causes such as hypothyroidism (men and women), or decreased testosterone levels (men). If concomitant disorder of erythroid production is present (thalassemia, renal insufficiency) and urgent need for phlebotomy exists, weekly erythropoietin may be helpful. Anemia may also be due to development of liver cancer.

Arthritis, Endocrine Replacement,Vaccinations, and Cancer Surveillance

  Arthritis: Responds moderately well to nonsteroidal anti-inflammatory agents

Joint aspiration to exclude gout or pseudogout in acutely inflamed joints.

Orthopedic evaluation for joint replacement for severe chronic hip, knee, or ankle pain. Cumulative incidence of major joint replacement in C282Y +/+ hemochromatosis subjects is 30% by age 70.

  Testosterone replacement: Consider in males with symptomatic sexual dysfunction and low testosterone levels.

  HAV and HBV vaccination: Should be given as prophylaxis against future hepatic injury in unexposed patients.

  Alpha fetoprotein and liver ultrasound: Surveillance for hepatocellular cancer. Repeat every 6 months if cirrhosis is documented by biopsy.

Dietary and Lifestyle Counseling

  Avoid oral iron supplements.

  Limit alcohol intake to protect the liver.

  Red meat in moderation, but major change in dietary habits is not required. Iron stores are most efficiently controlled by adjusting frequency of bleeds rather than reducing intake of iron-rich foods.

  Avoid raw shellfish until iron depletion is achieved (avoid Vibrio vulnificus).

  If ALT elevated

  Discontinue alcohol intake until iron depletion completed and ALT normal

  Consider discontinuation of medications with potential hepatic toxicity.

PROGNOSIS AND RESPONSE TO THERAPY

If cirrhosis is not present, long-term survival is unchanged from the general population.²⁵ If cirrhosis is present, the risk of hepatic cancer is increased and persists for life: 18.5% of subjects with cirrhosis will develop liver cancer, which may not be detected until 5 to 10 years after iron depletion. Overall, the incidence of hepatic cancer is 100-fold greater in hemochromatosis than non-hemochromatosis subjects and accounts for 10% to 30% of hemochromatosis-related deaths. Progression of cirrhosis due to hemochromatosis is slower than in other types of cirrhosis (alcoholic, viral); however, hemochromatosis subjects undergoing liver transplant for end-stage liver disease or liver cancer have a higher than average peritransplant mortality.

The response to phlebotomy varies by tissue site (Table 25.8).

HEMOCHROMATOSIS SUBJECTS AS BLOOD DONORS

  Regulatory issues. FDA allows blood centers to obtain a “variance” from federal code to permit blood from hemochromatosis subjects to be made available for transfusion into others, even if collected more frequently than 56-day interval.

  FDA requirements. Phlebotomy must be performed:

  Under a physician’s direction

  Without charge regardless of whether subjects qualify as donors

  With periodic laboratory monitoring

  Logistics and safety²³

  Seventy-five percent of all hemochromatosis subjects meet allogeneic donor eligibility criteria

  Fifty-five percent of hemochromatosis subjects were blood donors prior to knowledge of their diagnosis

  Potential hemochromatosis-donor contribution estimated at 1 to 2 million red cell units per year in the United States

  Recent rapid increase in number of US blood centers with FDA-approved variances to allow hemochromatosis subjects to be blood donors (81 centers, May 2007)

  Hemochromatosis subjects documented to be safe, reliable donors

  Advantages of phlebotomy care in the blood center

  Treatment is free, consistent, accessible, and convenient

  Increased patient satisfaction: avoid frustration of knowing blood will be discarded

  Alleviate national blood shortages

NON-HFE IRON OVERLOAD

Ferroportin disease is the most common hereditary, non-HFE cause of iron overload²⁶; it arises from an autosomal-dominant mutation in a gene coding for ferroportin, which is the main iron export protein in mammals.²⁷ This condition is not restricted to Caucasians and is characterized by elevated ferritin levels despite low normal TS, iron accumulation in organs, and reticuloendothelial macrophages, and marginal anemia with poor tolerance of phlebotomy therapy.

Nonalcoholic fatty liver disease is a common acquired cause of iron overload, often associated with features of the metabolic syndrome such as obesity, hypertension, hypercholesterolemia, and elevated fasting glucose levels or type II diabetes. Subjects present with elevated liver transaminases and ferritin levels, without the degree of elevation in TS that accompanies classic HFE-associated hemochromatosis. Liver ultrasound may demonstrate findings consistent with fatty infiltration; diagnostic histopathologic features of this disorder are present on liver biopsy. Judicious phlebotomy therapy may decrease ferritin and transaminase levels, but the emphasis of patient management should be directed toward the patient’s underlying medical conditions.

FUTURE CHALLENGES

The process of molecular discovery is rapidly leading to a more comprehensive understanding of the role of HFE protein in iron homeostasis. At the same time, the availability of a genetic test has focused increased public and medical attention on hemochromatosis. Robust population screening studies are currently in progress to more accurately determine clinical penetrance, both for early as well as late complications. Increased emphasis on educational campaigns to foster prompt recognition of early symptoms by primary care providers will complement or perhaps even alleviate the need for targeted screening programs. Better appreciation of the advantages of referral to the blood center may improve the quality and accessibility of care and also confer a benefit to the general public health.

References

  1.  Bothwell TH, MacPhail AP. Hereditary hemochromatosis: etiologic, pathologic, and clinical aspects. Semin Hematol. 1998;35:55-71.

  2.  Bomford A. Genetics of hemochromatosis. Lancet. 2002;360:1673-1681.

  3.  Tavill AS. Diagnosis and management of hemochromatosis: AASLD practice guidelines. Hepatology. 2001;33:1321-1328.

  4.  Beutler E. Penetrance in hereditary hemochromatosis. The HFE Cys282Tyr mutation as a necessary but not sufficient cause of clinical hereditary hemochromatosis. Blood. 2003;101:3347-3350.

  5.  Ajioka R, Kushner JP. Clinical consequences of iron overload in hemochromatosis patients. Blood. 2003;101:3351-3354.

  6.  Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996;13:399-408.

  7.  Jazwinska EC, Cullen LM, Busfiled F, et al. Haemochromatosis and HLA-H. Nat Genet. 1996;14:249-251.

  8.  Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood. 2003;102:783-788.

  9.  Nemeth E, Tuttle M, Powelson J, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090-2093.

10.  Babitt JL, Huang FW, Wrighting DM, et al. Bone morphogenetic signaling by hemojuvelin regulates hepcidin expression. Nat Genet. 2006;38:531-539.

11.  Bridle KR, Frazer DM, Wilkins SJ, et al. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homeostasis. Lancet. 2003;361:669-673.

12.  Feder JN, Penny DM, Irrinki A, et al. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci U S A. 1998;95:1472-1477.

13.  Brissot P, Loreal O. Role of non-transferrin bound iron in the pathogenesis of iron overload and toxicity. In: Hershko C, ed. Iron Chelation Therapy. New York, NY: Kluwer Academic/Plenum Publishers; 2002:45-53.

14.  Olynyk JK, Cullen DJ, Aquilia S, et al. A population-based study of the clinical expression of the hemochromatosis gene. N Engl J Med. 1999;341:718-724.

15.  Bulaj ZJ, Ajioka RS, Phillips JD, et al. Disease-related conditions in relatives of patients with hemochromatosis. N Engl J Med. 2000;343:1529-1535.

16.  Adams P, Brissot P, Powell LW. EASL International Consensus Conference on Haemochromatosis—part II. Expert document. J Hepatol. 2000;33:487-496.

17.  Adams P, Speechley M. The effect of arthritis on the quality of life in hereditary hemochromatosis. J Rheumatol. 1996;23:707-710.

18.  Shizukuda Y, Bolan C, Tripodi D, et al. Left ventricular systolic function during stress echocardiography in subjects with asymptomatic hereditary hemochromatosis. Am J Cardiol. 2006:98:694-698.

19.  Bradley LA, Haddow JE, Palomaki GE. Population screening for hemochromatosis: expectations based on a study of relatives of symptomatic probands. J Med Screen. 1996;3:171-177.

20.  Burke W, Thomson E, Khoury MJ, et al. Hereditary hemochromatosis. Gene discovery and its implications for population-based screening. J Am Med Assoc. 1998;280:172-178.

21.  El-Serag HB, Inadomi JM, Kowdley KV. Screening for hereditary hemochromatosis in siblings and children of affected patients. Ann Int Med. 2000;132:261-269.

22.  Barton JC, McDonnell SM, Adams PC, et al. Management of hemochromatosis. Ann Intern Med. 1998;129:932-939.

23.  Leitman SF, Browning JN, Yau YY, et al. Hemochromatosis subjects as allogeneic blood donors: a prospective study. Transfusion. 2003;43:1538-1544.

24.  Adams PC, Barton JC. How I treat hemochromatosis. Blood. 2010;116:317-325.

25.  Niederau C, Fischer R, Purschel A, et al. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology. 1996;110:1107-1119.

26.  Pietrangelo A, Caleffi, Corradini E. Non-HFE hepatic iron overload. Semin Liver Dis. 2011;31:302-318.

27.  Pietrangelo A. The ferroportin disease. Blood Cells Mol Dis. 2004;32:131-138.

26

Consultative Hematology

Pierre Noel and Elizabeth A. Jaben

HEMATOLOGIC COMPLICATIONS OF PREGNANCY

Anemia in Pregnancy

During normal pregnancies, plasma volume increases by 40% to 60% and red cell mass by 20% to 40%. The hematocrit typically decreases 30% to 32%, and the lower limit of normal for hemoglobin declines to 11 g/dL in the first trimester and 10 g/dL in the second and third trimesters. The most common forms of anemia of pregnancy in North America are due to iron and folate deficiencies.

One thousand milligrams of additional iron are required during pregnancy. The normal 500 mg iron storage pool is insufficient, and iron deficiency anemia develops in the absence of iron supplementation throughout pregnancy. The recommended daily allowance for iron during pregnancy is 27 mg of elemental iron. The United States Centers for Disease Control recommends routine low-dose iron supplementation (30 mg of elemental iron daily) for all pregnant women, beginning at the first prenatal visit. Calculations of dosage for iron preparations should be based on the amount of iron in each preparation: Ferrous sulfate contains 20% elemental iron, 12% ferrous gluconate, and 33% ferrous fumarate. Low values of serum iron and ferritin are reliable indicators of iron deficiency in pregnancy. The consequences of maternal iron deficiency on the neonate are controversial. Mild-to-moderate maternal iron deficiency anemia is not associated with significant anemia in the fetus.

Folate needs are increased during pregnancy. Folate deficiency is associated with anemia, neural tube defects, and cleft palate. Neural tube closure occurs during the fourth week of pregnancy; therefore, folate supplementation is necessary prior to conception to prevent neural tube defects. Most prenatal vitamins contain both folate and iron.

Sickle Cell Disease in Pregnancy

Women with sickle cell anemia are in a high-risk pregnancy group. With modern obstetric and perinatal care, maternal mortality is less than 1% and perinatal mortality is less than 15%.

Prophylactic red cell transfusions are associated with fewer maternal painful episodes, but they have no impact on maternal morbidity, infant birth weight, gestational age, fetal distress, or perinatal mortality.

Maintenance transfusions should be administered to women who are symptomatic of vasoocclusive or anemia-related problems or when signs of fetal distress are present.

Thrombocytopenia in Pregnancy

Platelet count decreases by approximately 10% during pregnancy, mostly in the third trimester.

The most common cause of thrombocytopenia is incidental thrombocytopenia of pregnancy (75%), followed by thrombocytopenia complicating hypertensive disorders of pregnancy (20%) and finally immunological disorders of pregnancy (5%).

Thrombocytopenia of less than 100,000/μL in the first trimester of pregnancy is most typical for immune thrombocytopenic purpura. Thrombocytopenia of over 70,000/μL occurring late during the second trimester or during the third trimester, in the absence of hypertension or proteinuria, usually represents incidental thrombocytopenia of pregnancy. Platelet-associated IgG is elevated in both incidental thrombocytopenia of pregnancy and immune thrombocytopenic purpura.

It is important in any patient with thrombocytopenia to consider human immunodeficiency virus infection, systemic lupus erythematosus, and thrombocytopenia associated with antiphospholipid antibodies in the differential diagnosis.

Incidental Thrombocytopenia of Pregnancy

The platelet count in incidental thrombocytopenia generally remains above 100,000/μL. Incidental thrombocytopenia usually develops in the third trimester and is not associated with neonatal thrombocytopenia. The likelihood of a more serious cause of thrombocytopenia increases when the platelet count drops below 70,000/μL. The pathogenesis of incidental thrombocytopenia is not clearly defined but may involve a combination of hemodilution and decreased platelet half-life.

Incidental thrombocytopenia remains a diagnosis of exclusion. The diagnosis is made by the lack of other physical or laboratory abnormalities in patients who do not have an antecedent history of immune thrombocytopenia. Women with incidental thrombocytopenia should receive standard obstetrical care. A platelet count greater than 80,000/μL is felt to be sufficient for epidural anesthesia.

Immune Thrombocytopenic Purpura

Immune thrombocytopenia (ITP) is the most common cause of severe thrombocytopenia in the first trimester of pregnancy. An antecedent history of ITP or autoimmune disorder makes the diagnosis more likely. The nadir platelet count in ITP usually occurs in the third trimester.

Patients with platelet counts greater than 20,000 to 30,000/μL and no evidence of bruising or mucosal bleeding generally do not require treatment in the first two trimesters of pregnancy. A platelet count of greater than 50,000/μL is considered safe for normal vaginal delivery or cesarean section. Although there is no consensus, a platelet count greater than 80,000/μL is sufficient for epidural anesthesia. The bleeding time is not an accurate predictor of risk of bleeding in these situations.

Optimal first-line therapy for ITP in pregnant patients is controversial. Corticosteroids are the least expensive option, but they have been associated with pregnancy-induced hypertension, gestational diabetes, osteoporosis, excessive weight gain, and premature rupture of fetal membranes. The placenta metabolizes 90% of the administered dose of prednisone, and thus serious fetal side effects are unlikely. Prednisone is initiated at a dose of 1 mg/kg/day (based on the pre-pregnancy weight) and subsequently tapered to the minimum hemostatically effective dose. Intravenous immunoglobulin (IVIg) should be considered if the maintenance dose of prednisone is in excess of 10 mg/day. IVIg given at a dose of 1 g/kg (based on pre-pregnancy weight) is associated with a response in over 60% of patients, and response duration averages a month.

In patients refractory to corticosteroids and IVIg, splenectomy should be considered. Splenectomy is best performed in the second trimester of pregnancy. Splenectomy in the first trimester may induce labor, and splenectomy in the third trimester may be technically difficult. Splenectomy has been successfully performed laparoscopically during pregnancy. High-dose methylprednisolone and intravenous anti-D immunoglobulin have been used in small series of refractory patients.

Very little data are available regarding the safety and efficacy during pregnancy of thrombopoietin receptor mimetic agonists (romiplostim, eltrombopag). Experience with immunosuppressive and cytotoxic agents during pregnancy also is limited. Danazol and vinca alkaloids are best avoided. Interventions that raise maternal platelet count are not effective in augmenting the platelet count of the fetus.

The use of non-steroidal anti-inflammatory drugs should be avoided postpartum in patients with platelet counts less than 100,000/μL. Thromboprophylaxis should be considered in all women with a platelet count greater than 50,000/μL; if they have undergone surgical delivery, are immobilized for a prolonged amount of time, or have acquired or congenital thrombophilia.

Neonatal mortality is less than 1% in ITP. Five percent of neonates will have a platelet count of less than 20,000/μL; most hemorrhagic events in neonates occur 24 to 48 hours after delivery, at the nadir of the platelet count. There is no evidence that cesarean section is safer for the neonate than is vaginal delivery. The mode of delivery should be decided on the basis of routine obstetric indications.

Maternal platelet count, maternal platelet antibody levels, or a history of maternal splenectomy for ITP are not accurate predictors of neonatal platelet counts. The most accurate predictor of fetal thrombocytopenia is a history of thrombocytopenia at delivery in a prior sibling. Fetal scalp blood sampling and cordocentesis have been generally abandoned.

A cord platelet count should be determined following delivery in every neonate. Thrombocytopenic neonates should be followed closely following delivery, the platelet count nadir may not occur before 2 to 5 days. Neonates presenting with clinical bleeding or a platelet count less than 20,000 μL should be managed using IVIg at a dose of 1 g/kg. Life threatening bleeding can be treated with a combination of IVIg and platelet transfusions. Neonates with platelet counts less than 50,000/μL should undergo transcranial ultrasound to exclude intracranial hemorrhage.

Preeclampsia and HELLP Syndrome

Preeclampsia is defined as hypertension (systolic pressure greater than 140 mm of mercury or diastolic pressure greater than 90 mm of mercury) and proteinuria (greater than 300 mg of protein/24 hours) occurring after 20 weeks of gestation. Preeclampsia occurs in 5% of all pregnancies, and it accounts for 18% of maternal deaths in the United States. Predisposing factors include age below 20 or over 30, increased body mass index, chronic hypertension, and insulin resistance. Thrombocytopenia develops in 50% of patients with preeclampsia. Endothelial damage and activation of the coagulation system with thrombin generation may explain the thrombocytopenia. D-dimers and thrombin–anti-thrombin complexes are increased in patients with thrombocytopenia.

The criteria for HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) are as follows:

  microangiopathic hemolytic anemia

  increased transaminases

  lactic dehydrogenase greater than 600 units per milliliter

  thrombocytopenia (less than 100,000/μL).

HELLP occurs in up to 10% of women with severe preeclampsia. Proteinuria is present in 75% of patients with HELLP syndrome, but only 50% to 60% have hypertension. The syndrome usually occurs in white, multiparous women above the age of 25 years. Maternal mortality is 1% and fetal mortality is 10% to 20%. Fetal mortality is attributed to placental ischemia, abruption of the placenta, immaturity, and intrauterine asphyxia. Neonatal thrombocytopenia can occur in both preeclampsia and HELLP. The mechanism of neonatal thrombocytopenia remains unclear. There is a 3% risk of recurrence of HELLP in subsequent pregnancies.

The definitive treatment for eclampsia and HELLP is delivery of the fetus. Management focuses on stabilization of the patient and maturation of the fetal lung. The presence of multiorgan dysfunction, fetal distress, or a gestational age greater than 34 weeks warrants immediate delivery. Coagulopathy resulting from preeclampsia-associated diffuse intravascular coagulation (DIC) occurs in 20% of patients. The clinical manifestations of preeclampsia and HELLP resolve within a few days of delivery. Rarely, HELLP syndrome can present postpartum. If the manifestations worsen or persist after 1 or 2 days, plasma exchange is indicated.

Acute fatty liver of pregnancy occurs in the third trimester and is associated with hypertension and proteinuria in 50% of patients. Microangiopathic hemolytic anemia and thrombocytopenia are not prominent in this syndrome. Patients usually have a prolonged prothrombin time, a low fibrinogen, and low antithrombin levels.

Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome

Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) occur in only 0.004% of pregnancies. The following are the classic pentad of symptoms of TTP:

  Microangiopathic hemolytic anemia

  Thrombocytopenia

  Neurologic abnormalities

  Fever

  Renal dysfunction

However, the classic pentad is present in only 40% of patients. Pregnancy is a precipitating factor for TTP. The mean time of onset of TTP is at 23.5 weeks of pregnancy. Plasma exchange therapy is recommended for the management of the pregnant TTP patient, and delivery is indicated only for patients who do not respond to plasma exchange. Pregnancy termination is not considered therapeutic in TTP or HUS.

Ultralarge von Willebrand factor (VWF) multimers are found in TTP, thought to be secondary to the deficiency of a specific VWF-cleaving protease, identified as ADAMTS-13. ADAMTS-13 levels in TTP are typically less than 10%. ADAMTS-13 deficiency can be congenital or acquired. Acquired deficiency is associated with the presence of autoantibodies directed against ADAMTS-13. Reduced ADAMTS-13 levels are not specific for TTP; reduced levels are seen in the third trimester of pregnancy, in uremia, acute inflammation, malignancy, and in DIC. The majority of patients with TTP respond to plasma exchange. The role of corticosteroids remains controversial, mainly because of associated side effects. Patients who develop pregnancy-associated TTP are at high risk of recurrence with subsequent pregnancies.

The mean time of onset of HUS is 26 days following delivery. Patients with HUS present with microangiopathic hemolytic anemia and acute renal failure. VWF levels are usually elevated while multimer analysis may or may not show ultra-large multimers. Deficiency of VWF—cleaving protease is usually not associated with this syndrome.

Several women with a familial history of pregnancy-associated HUS have developed their first episode of HUS during pregnancy, and HUS has occurred in such patients with the use of oral contraceptives. Postpartum HUS is associated with a poor prognosis. Plasma exchange is less effective in reversing renal failure in pregnancy-associated HUS. Nevertheless, a trial of plasma exchange is indicated. Dialysis and other supportive-care measures may also need to be initiated (Table 26.1).

Diffuse Intravascular Coagulation

Placental abruption is the most common cause of DIC (Table 26.2). There is an increased incidence of placental abruption in cocaine addicts. The incidence of DIC complicating placental abruption and dead fetus syndrome has decreased with advances in ultrasonography and prenatal care.

Table 26.2  Causes of Obstetrical Disseminated Intravascular Coagulation

Placental abruption

Fetal death syndrome

Amniotic fluid embolism

HELLP syndrome

Clostridial sepsis

Sepsis

Major obstetrical hemorrhage

HELLP, hemolysis, elevated liver enzymes, and low platelets.

Fetal death syndrome is recognized by ultrasonography. Delivery of the dead fetus removes the source of tissue thromboplastin release. Blood component support and the use of antithrombin-3 have been useful in the management of the coagulopathy.

Placental abruption is managed with blood component support followed by delivery. Antithrombin-3 and activated protein C have been used with success in this disorder.

Transient DIC occurs in patients undergoing hypertonic saline abortions, but the DIC usually resolves once the fetus is delivered. Clostridial sepsis following abortions is associated with DIC and poor clinical outcome.

Venous Thromboembolism in Pregnancy

The per-day risk of venous thromboembolism (VTE) is increased 7- to 10-fold for antepartum VTE and 15- to 35-fold for postpartum VTE. The risk of VTE diminishes rapidly after delivery, returning to the antepartum risk level by 3 weeks postpartum and to the non-pregnant level after 6 weeks. Venous thrombi occur predominantly in the left leg, partly because of the compression of the left iliac vein by the right iliac artery as they cross.

Hemodynamic changes causing venous stasis and hypercoagulability play a role in the increased risk of VTE during pregnancy. Hypercoagulability is thought to be secondary to an increase in fibrinogen, factor VIII, and von Willebrand factor. Further, a decrease in protein S, the development of acquired protein C resistance, and reduced fibrinolytic activity from increased plasminogen activator inhibitor type 1 and 2 activity and decreased tissue plasminogen activator activity may contribute.

History of prior VTE, body mass index greater than 25, prolonged immobilization, inherited thrombophilias, antiphospholipid antibodies, and a family history of thrombosis all increase the risk of VTE during pregnancy.

Diagnosis of Venous Thromboembolism in Pregnancy

The diagnosis of VTE during pregnancy is complicated by the potential for fetal oncogenicity and teratogenicity due to ionizing radiation in diagnostic purposes.

Compression ultrasonography (CU) of the entire proximal venous system to the trifurcation should be performed as the initial test for suspected deep vein thrombosis (DVT) in pregnancy. A normal CU does not exclude a calf DVT. The CU needs to be repeated at day 2 and day 7 to exclude an extending calf-vein thrombosis. A limited venogram with fetal shielding can be used in equivocal cases. When iliac DVT is suspected, pulsed Doppler ultrasound should be used; if the results are negative or equivocal, magnetic resonance venography (MRV) or venography should be considered.

In patients with suspected pulmonary emboli during pregnancy, bilateral compression lower extremity ultrasounds should be performed. If the ultrasound is negative a ventilation/perfusion lung scan (V/Q) should be the next procedure. If the results of the V/Q scan are equivocal, computed tomography pulmonary angiography (CTPA) should be performed. However, should an isolated subsegmental defect be suggested by CTPA, additional testing is suggested because of the high false-positive rate.

D-dimer levels increase throughout pregnancy. The D-dimer test has a high sensitivity, relatively low specificity and very high negative predictive value.

Treatment of Venous Thromboembolic Disease in Pregnancy

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) do not cross the placenta; therefore, there is no risk of fetal bleeding or teratogenicity. Heparin-induced thrombocytopenia, bleeding, and heparin-induced osteoporosis are more common with UFH than with LMWH.

There are case reports describing the use of fondaparinux, argatroban, and lepirudin in pregnant women with heparin-induced thrombocytopenia. The three drugs are classified under United States Food and Drug Administration (FDA) Class B, indicating that animal studies have not shown harm in pregnancy, but there are no data from humans. Fondaparinux appears to cross the placenta in very low concentrations, but lepirudin and argatroban do not appear to cross the placenta. Coumarin derivatives do transit the placenta and have been associated with fetal bleeding and teratogenicity. Central nervous system abnormalities have occurred after the use of coumarin derivatives in every trimester of pregnancy: nasal hypoplasia and/or stippled epiphyses have been associated with these drugs employed between the sixth and twelfth week of pregnancy.

The activated partial thromboplastin time response to UFH is blunted in pregnancy because of increased factor VIII levels and increased heparin-binding proteins. This blunted response may lead to heparin overdosing. Measuring anti-activated factor (FXa) levels may obviate the problem. LMWHs have less non-specific binding to heparin-binding proteins, hence they have a more predictable doseresponse than UFH.

UFH and LMWH are not secreted in breast milk, nor are argatroban or bivalirudin. Fondaparinux, however, is present in breast milk. Clinical evidence suggests that warfarin sodium is not excreted in breast milk and that breast-feeding is safe when mothers are under warfarin sodium therapy.

The initial dose of LMWH is based on patient weight. Because of variation of weight and glomerular filtration rate during pregnancy, it is recommended to monitor anticoagulation by monthly anti-FXa levels. Dose reduction to ¾ dose after 3 to 4 weeks of full treatment appears safe and may obviate the need for continued anti-FXa monitoring. LMWH should be discontinued 24 hours prior to elective induction of labor and neuroaxial anesthesia. Intravenous UFH can be initiated in patients at high risk for thrombosis and discontinued 4 to 6 hours prior to the time of expected delivery. LMWH can usually be restarted within 12 hours of delivery.

UFH or LMWH should be continued for at least 4 days after initiation of warfarin, until the international normalized ratio has been therapeutic, 2.0 or more, for two consecutive days.

Graduated compression stockings providing a pressure of 30 to 40 mm Hg should be prescribed to decrease the incidence of post-phlebitic syndrome.

The placement of an inferior vena cava filter should be considered, if the VTE is diagnosed after 37 weeks of pregnancy. Discontinuation of anticoagulation for delivery without a filter in place is associated with high rates of morbidity and mortality.

Prophylactic Anticoagulation in Patients with a Previous History of Venous Thromboembolism

Prophylactic antepartum anticoagulation is indicated in patients with a history of unprovoked VTE. Patients with a previous provoked VTE secondary to a temporary risk factor, who do not have an identifiable thrombophilia, are at low risk of recurrence at the time of a subsequent pregnancy and do not require antepartum prophylactic anticoagulation. All women with prior VTE should receive prophylactic postpartum anticoagulation for 6 weeks.

Patients with no prior VTE who are heterozygous for Factor V Leiden or prothrombin gene mutation have a low risk of antepartum VTE without prophylaxis. Antepartum anticoagulation is warranted in patients with antithrombin III deficiency and in patients who are double heterozygotes for Factor V Leiden and prothrombin gene mutation (Table 26.3).

Patients with idiopathic VTE who are pregnant or plan to become pregnant should undergo screening for thrombophilias. Patients with history of fetal loss, abruption, preeclampsia, and intrauterine fetal growth retardation should also be screened for thrombophilias.

Cesarean section is not a risk factor for VTE. Pharmacologic or mechanical thromboprophylaxis is recommended in patients with one VTE risk factor. Combined pharmacologic and mechanical thromboprophylaxis are recommended in patients with multiple VTE risk factors. In high-risk patients, 6 weeks of thromboprophylaxis is recommended.

Table 26.3  Most Common Thrombophilias

Inherited

Factor V Leiden

Prothrombin G20210A mutation

4G/4G mutation of the plasminogen activator inhibitor gene (PAI-I)

Thermolabile variant of methylenetetrahydrofolate reductase, the most common cause of homocystinemia

Antithrombin III deficiency

Protein C deficiency

Protein S deficiency

Acquired

Antiphospholipid antibody

Thrombophilias and Recurrent Miscarriage

Recurrent miscarriage is defined as three consecutive, spontaneous abortions of an intrauterine pregnancy, each occurring at less than 20 weeks gestation. Anticardiolipin antibodies have been linked with recurrent miscarriage. There are insufficient data to include inherited thrombophilias in the evaluation of women with recurrent miscarriage.

Prednisone, low dose aspirin, UFH, LMWH, and IVIg have been used in the management of this problem. Prednisone was found to be equally effective as low-dose subcutaneous UFH in preventing pregnancy loss, but aspirin was associated with increased toxicities. UFH and aspirin have been shown to be superior to aspirin alone in preventing pregnancy loss. LMWH can be employed instead of UFH. The optimal dosages of UFH and LMWH remain to be defined.

HEMATOLOGICAL MANIFESTATIONS OF TROPICAL DISEASE

Malaria

Anemia is a serious complication of malaria, especially Plasmodium falciparum infection. The prevalence and degree of anemia depends on the nutritional and immune status of the patient. The degree of anemia cannot be explained entirely by intravascular rupture of parasitized red cells. Several mechanisms are involved in the anemia of malaria (Table 26.4). P. vivax, and P. ovale invade only reticulocytes, P. malariae invades only mature red cells, and P. falciparum invades red cells of all ages. The proportion of cells parasitized in P. vivax malaria rarely exceeds 1%, whereas as many as 50% of red cells may be parasitized in P. falciparum infections.

P. vivax uses the Duffy antigen as a receptor for junction formation during invasion. P. falciparum does not use the Duffy antigen as a receptor for invasion but rather sialic acid residues of glycophorin A and B. Certain inherited defects confer resistance to parasitization by malarial organisms (Table 26.5).

There are two major clinical patterns in malaria: (1) acute malaria in the nonimmune and (2) recurrent malaria. Acute malaria is associated with a rapid drop in hemoglobin. Recurrent malaria causes splenomegaly and less severe anemia, and there are only scanty asexual forms and some gametocytes on the peripheral blood smear (Table 26.6). In tropical areas, anemia tends to be more prevalent and most severe in children from 1 to 5 years of age and during pregnancy. Pregnant women who are not immune to P. falciparum develop severe malaria during pregnancy, and they have high rates of abortion, premature delivery, and perinatal and maternal mortality. In women who are immune, extravascular hemolysis and secondary folic acid deficiency play a major role in the pathogenesis of anemia. The extravascular hemolysis in immune women peaks during the second trimester and is accompanied by progressive splenomegaly.

Table 26.4  Causes of Anemia in Malaria

Intravascular rupture of parasitized red cells Hypersplenism

Autoimmune hemolysis (50% of patients have a positive direct Coombs)

Reticulocytopenia (Anemia of chronic disease) Dyserythropoiesis (Cytokine mediated)

Secondary bacterial, fungal, or viral infections

Nutritional anemias

Table 26.5  Protective Genetic Alterations

Southeast Asian ovalocytosis (Autosomal dominant, 27 base pair deletion in the band 3 gene).

Heterozygotes for beta-thalassemias (Protection against P. falciparum)

HbE, HbS

Hereditary persistence of fetal hemoglobin

Glucose-6-phosphate dehydrogenase deficiency

Duffy-null phenotype (The Duffy antigen receptor for chemokines serves as a receptor for red cell invasion by P. vivax. Individuals who are Duffy-null are resistant to vivax malaria.)

Glycophorin A deficient phenotypes [En(a-), Mk] (Glycophorins are important ligands for the attachment and invasion of P. falciparum merozoites).

Glycophorin B deficient phenotypes [S-s-U-]

CD35 (Knops antigen) variants (CD35 is involved in the resetting of P. falciparum-infected red cells with uninfected cells)

Hyperreactive malarial splenomegaly (HMS) is characterized by splenomegaly, hypersplenism, a polyclonal B-lymphocyte proliferation, high IgM levels, and raised titers of antibodies against the predominant species of malaria. Sickle cell trait is protective against HMS. Patients with HMS have a persistence of malaria-induced IgM lymphocytotoxic antibodies, which reduce the numbers of T-suppressor lymphocytes and permit the proliferation of B-lymphocytes. HMS has been associated with the development of splenic lymphoma with villous lymphocytes. Significant lymphocytosis will develop in 15% of patients with HMS, and may be mistaken for chronic lymphocytic leukemia.

Visceral Leishmaniasis

Visceral leishmaniasis (VL, Kala-Azar) is caused by one of three species of Leishmania donovani complex. L. donovani is transmitted by phlebotomine sand flies. VL can also be transmitted through sexual contact, blood transfusions, and vertically.

L. donovani infects macrophages throughout the reticuloendothelial system. Patients develop irregular patterns of fever, weight loss, hepatosplenomegaly, pancytopenia, and hypergammaglobulinemia. The pancytopenia is secondary to hypersplenism and is worsened by folic acid deficiency. Monocytosis and lymphocytosis are typically present.

Chronic VL infection can cause marrow hypoplasia, gelatinous transformation, dyserythropoiesis, and myelofibrosis.

African Trypanosomiasis (Sleeping Sickness)

African trypanosomiasis (AT or sleeping sickness) is endemic in sub-Saharan Africa. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are the etiologic agents. The tsetse fly is the vector. The infection is associated by the proliferation of macrophages and lymphocytes. Patients typically develop splenomegaly, pancytopenia secondary to hypersplenism, polyclonal hypergammaglobulinemia, monocytosis, and lymphocytosis.

Helminth Infections

Eosinophilia is present during the invasive migrating phase of hookworms, Strongyloides, and Ascaris. Hookworm is second in frequency only to malaria as an infectious cause of anemia. The daily loss of blood in the gut is 0.03 to 0.05 mL for each Necator americanus worm and 0.15 to 0.23 mL for each Ancylostoma duodenale worm. The development of iron deficiency is related to the dietary intake of iron, the size of the iron stores, and the hookworm load. Iron depletion is more common in women, during pregnancy, and in children. Less frequent causes of iron deficiency are outlined in Table 26.7.

CLONAL EOSINOPHILIC DISORDERS

Blood eosinophilia is defined as an eosinophil count superior to 450/μL. Eosinophils are much more abundant in tissues than in the peripheral blood. Sustained eosinophilia is associated with end-organ damage in a minority of patients (Table 26.8).

IL-5, IL-3, and GM-CSF all stimulate eosinophil production and inhibit eosinophil apoptosis. Eotaxin-1, eotaxin-2, and RANTES (regulated on activation T cell expressed and secreted) are chemotactic cytokines, causing eosinophils to migrate into tissues. Eosinophils are the source of multiple cytokines (IL-2, IL-3, IL-4, IL-5, IL-7, IL-13, IL-16, TNF-alpha, TGF-beta, and RANTES). They are also the source of cationic proteins such as eosinophil cationic protein, eosinophil peroxidase, major basic protein, eosinophil derived neurotoxin, and Charcot-Leyden crystal lysophospholipase.

Helminthic infections are the most common cause of eosinophilia worldwide, and atopic disorders are the most common cause in industrialized countries. Clonal eosinophilic disorders account for only a small proportion of all eosinophilias (Table 26.9).

Sustained hypereosinophilia, whether reactive or clonal, can damage tissue. The risk factors for end-organ damage are undefined.

The evaluation of a patient with eosinophilia is influenced by the patient’s geographical origin and travel history. Serial stool examinations for ova and parasites may need to be supplemented by endemically relevant serologies and occasionally tissue biopsies.

A clonal eosinophilic disorder needs to be investigated in patients without evidence of infectious or reactive causes of eosinophilia. Clonal eosinophilic disorders can be subdivided into (1) clonal T-cell disorders, (2) clonal myeloid disorders, (3) suspected clonality that cannot be proven [idiopathic hypereosinophilic syndrome (IHES)]. The number of patients classified as having IHES is decreasing as our diagnostic tools are improving (Table 26.10).

Table 26.9  Diseases Commonly Associated with Eosinophilia

Infectious (helminth, protozoa, fungi, HIV, HTLV-1)

Allergic diseases (asthma, atopic dermatitis, allergic rhinitis, urticarias, allergic drug reactions)

Respiratory tract disorders (hypersensitivity pneumonitis, Loeffler’s syndrome, allergic bronchopulmonary aspergillosis, tropical pulmonary eosinophilia)

Endocrinologic disorders (Addison’s disease)

Gastrointestinal disorders (inflammatory bowel disease, eosinophilic gastroenteritis)

Cutaneous and subcutaneous disorders (atopic dermatitis, eosinophilic cellulitis, scabies, episodic angioedema with eosinophilia, chronic idiopathic urticaria, recurrent granulomatous dermatitis, eosinophilic fasciitis)

Immunodeficiency syndromes

Connective tissue disease (Churg-Strauss and cutaneous necrotizing eosinophilic vasculitis)

Neoplastic (Lymphomas,T-ALL,T-cell lymphoproliferative disorders, solid tumors)

Myeloid leukemias and myeloproliferative disorders (acute eosinophilic leukemia, myelomonocytic leukemia with eosinophilia, chronic myelomonocytic leukemia with eosinophilia, chronic myeloid leukemia)

Idiopathic hypereosinophilic syndrome

Cytokines (IL-2, GM-CSF)

L-tryptophan and toxic oil syndrome

GM-CSF, granulocyte macrophage colony-stimulating factor; HIV, human immunodeficiency virus; HTLV-1, human T-cell leukemia virus; IL-2, interleukin 2;T-ALL,T-cell acute lymphoblastic leukemia.

Table 26.10  Clonal Hypereosinophilic Disorders

Clonal T-cell Disorders

•  T-ALL

•  T-cell lymphomas

•  Aberrant T-cell clones ([CD3+, CD4+, CD8−], [CD3+, CD4−, CD8+], [ CD3+, CD4−, CD8−], [CD3−, CD4+])

Clonal Myeloid Disorders

•  Acute leukemias (M2 AML with eosinophilia, M4 Eo AML with inv(16) (p13;q22), t(16;16) (p13;q22)

•  Chronic myelomonocytic leukemias with eosinophilia

•  Myeloproliferative disorders with eosinophilia (polycythemia vera, chronic myelogenous leukemia, essential thrombocytosis, agnogenic myeloid metaplasia)

•  Systemic mast cell disease with eosinophilia

FIP1L1-PDGFRα hypereosinophilic disorders

Clonal Hypereosinophilic Disorders

The evaluation of patients with suspected clonal hypereosinophilic disorders should include:

•  CBC-differential and peripheral blood smear

•  Chemistry group

•  Serum IgE

•  B12

•  Serum tryptase (Increased in mast cell disease with eosinophilia and the myeloproliferative variant of FIPILl-PDGFRa hypereosinophilic disorders.)

•  Peripheral blood flow cytometry (Used to identify an aberrant population of T-lymphocytes.)

•  T-cell receptor gene (beta) or (gamma) rearrangement studies

•  HIV serology

•  CT scans of the chest, abdomen, and pelvis

•  Bone marrow aspirate and biopsy (with reticulin and tryptase staining of the biopsy)

•  Bone marrow cytogenetics

•  PCR for FIP1L1-PDGFRA fusion gene and/or CHIC-2 fluorescent in situ hybridization

AML, acute myeloid leukemia; CBC, complete blood count; CT, computed tomography; HIV, human immunodeficiency virus; IgE, immunoglobulin E; PCR, polymerase chain reaction;T-ALL,T-cell acute lymphoblastic leukemia.

The clonality of eosinophils can be demonstrated by the expression of a single alloenzyme of glucose-6-phosphate dehydrogenase in purified eosinophils from female heterozygotes. Polymerase chain reaction amplification of the human androgen receptor gene locus (HUMARA) can also document clonality of eosinophils in female patients.

T-Cell Clonal Disorders

IL-5 overproduction by TH2 lymphocytes has been demonstrated in both clonal and reactive hypereosinophilic disorders. Aberrant clones of T–lymphocytes are present in 25% of patients with clonal hypereosinophilic disorders. The aberrant phenotypes are heterogeneous ([CD3+, CD4+, CD8−], [CD3+, CD4−, CD8+], [CD3+, CD4−, CD8−], [CD3−, CD4+]). In most cases, an activated T cell phenotype is present with expression of CD25 and HLA-DR. In 50% of patients, a clonal rearrangement of the T-cell receptor gene (beta) or (gamma) is found. T-cell lymphomas develop in a proportion of these patients.

Patients with aberrant CD4+, CD3− T cells producing high levels of IL-5, IL-4, and IL-13 typically present with skin manifestations, lack of severe end-organ involvement, and they have elevated IgE levels and polyclonal hypergammaglobulinemia.

The optimal treatment of patients with aberrant T-cell clones remains unclear. Corticosteroids have been associated with some responses. Interferon-alpha has in-vitro antiapoptotic effects on the clonal CD4+CD3− population and may increase the risk of lymphomatous transformation. The role of alemtuzumab in the management of CD52 positive clonal T-cell disorders is under evaluation.

Acute Leukemias

Acute eosinophilic leukemia is rare. Cyanide-resistant peroxidase can be used to identify eosinophilic blasts. Myelomonocytic leukemia (M4-Eo) with eosinophilia is associated with inv (16) (p13;q22) and t(16;16) (p13;q22). The core binding factor-beta is a transcription factor located at 16q22, and the smooth muscle myosin heavy chain is located at 16p13. The eosinophils in M4Eo frequently have a dysplastic appearance.

Chronic Myelomonocytic Leukemia with Eosinophilia

The two predominant subtypes of chronic myelomonocytic leukemia with eosinophilia involve, respectively, platelet-derived growth factor receptor beta (PDGFR-β) and fibroblast growth factor receptor 1 (FGFR1). In both subtypes, fusion oncoproteins are constitutively activated and are able to activate downstream stimulatory and antiapoptotic pathways.

Chronic Myelomonocytic Leukemia with Eosinophilia Subtypes

Platelet-derived growth factor receptor-β subtype:

  Age 50 to 60

  Male predominance (>90%)

  Monocytosis, eosinophilia, splenomegaly

  Most common chromosome abnormality is t(5;12)(p12;q31-32) [ETV6-PDGFR]

  Imatinib responsive

Fibroblast growth factor receptor 1 subtype (FGFR1):

  Median age: 32

  Male: female ratio (1.5:1)

  Associated with lymphoblastic lymphoma transformation (B and T)

  Associate with rearrangement of FGFR1 at the 8p11-12 locus

  Not responsive to imatinib

FIP1L1-PDGFR-α Hypereosinophilic Disorders

FIP1L1-PDGFR-α is a constitutively activated tyrosine kinase that was first described in a patient with hypereosinophilic syndrome, who genetically had an occult 800-kb interstitial deletion of chromosome 4q12. The FIP1L1-PDGFRα cannot be detected by conventional karyotyping; fluorescence in situ hybridization (CHIC-2) or reverse transcription polymerase chain reaction is required. The dose of imatinib (100 mg/day) required to inhibit FIP1L1-PDGFRα tyrosine kinase is less than the dose (400 mg/day) needed to block activity of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. The large majority of patients with the FIP1L1-PDGFR-α treated with imatinib obtain a molecular remission within 3 months of initiating therapy. The optimal dosage and duration of treatment remain to be defined, but discontinuation of imatinib leads to relapse. Molecular remissions can be reestablished by reinitiating imatinib therapy. Imatinib suppresses but does not eliminate the FIP1L1-PDGFR-α positive clone. Resistance to imatinib is associated with a T6741 mutation in PDGFR-α; this mutation occurs in the adenosine triphosphate (ATP)-binding region of PDGFR-α at the same position as the T3151 mutation in BCR-ABL. There is limited experience with the use of other tyrosine-kinase inhibitors (desatinib, nilotinib) in hypereosinophilia harboring the FIP1L1-PDGFR-α mutation.

Serum tryptase and vitamin B12 levels are increased in the majority of patients. Splenomegaly is reported in over 60% of cases.

A serum troponin and cardiac echocardiogram should be obtained prior to initiating imatinib. An increased level of serum cardiac troponin correlates with the presence of cardiomyopathy. Prophylactic use of corticosteroids during the first 7 to 10 days of treatment with imatinib is recommended

in patients with evidence of eosinophil-mediated cardiomyopathy and in patients with other cardiac comorbidities.

Idiopathic Hypereosinophilic Syndrome

Idiopathic hypereosinophilic syndrome is arbitrarily defined as eosinophilia in excess of 1,500/μL for more than 6 months with evidence of end-organ damage, without an evident primary or secondary cause of eosinophilia. Idiopathic hypereosinophilia is the term favored when end-organ damage is absent. Corticosteroids represent the first line therapy for idiopathic hypereosinophilic syndrome. Hydroxyurea, interferon-alpha, and monoclonal antibodies directed against interleukin-5 have been used in steroid-resistant patients. The role of imatinib as front-line therapy in patients without FIP1L1-PDGFR-α fusion proteins remains to be established.

NEUTROPENIA

Neutropenia is defined as a decrease in neutrophils below 1,500/μL. Severe neutropenia is defined as a decrease in neutrophils below 500/μL. In patients of African origin, the neutrophil count may normally be as low as 1,000/μL.

Neutropenias can be divided into intrinsic disorders of the hematopoietic system and secondary forms. The secondary forms are caused by extrinsic factors such as: immune causes, hypersplenism, infections, and drugs (Table 26.11).

Intrinsic Disorders

Congenital Neutropenias

Congenital neutropenias include Kostmann syndrome, cyclic neutropenia, congenital immunodeficiency syndromes, as well as several other rare syndromes that will not be discussed in this chapter.

Kostmann syndrome is an autosomal-dominant disorder presenting in the newborn. Characteristic findings include: neutrophils below 200/μL, monocytosis, anemia, thrombocytosis, splenomegaly, and maturation arrest in the marrow at the promyelocyte level. The accelerated apoptosis of neutrophilic precursors is secondary to a mutation of neutrophil elastase. Ninety percent of children with Kostmann syndrome respond to granulocyte colony stimulating factor (G-CSF). Evolution to myelodysplasia and acute leukemia occurs in some patients. It is unclear if G-CSF increases this risk.

Cyclic neutropenias can be congenital (autosomal-dominant congenital disorder) or acquired with clonal large granular lymphocyte syndrome. Congenital cyclic neutropenia is due to mutations at the enzyme active site of the neutrophil elastase gene, which leads to accelerated apoptosis of neutrophils. Clinically, patients present with cycles of neutropenia every 21 to 56 days. The neutropenia can be severe and last 3 to 6 days. Fever, mucosal ulcers, and lymphadenopathy can occur during the nadir of the cycles. G-CSF is useful in the management of cyclic neutropenia.

Table 26.11  Neutropenia Classification

Intrinsic Disorders

Congenital

Acquired

Extrinsic Disorders

Immune neutropenias

Neutropenia associated with autoimmune disorders

Neutropenia associated with large granular lymphocytes

Hypersplenism

Neutropenia associated with infectious diseases

Drug-related neutropenias

Nutritional deficiencies (B12, folate, copper)

Congenital immunodeficiency syndromes frequently associated with neutropenia, include: X-linked agammaglobulinemia, X-linked hyperimmunoglobulin M syndrome, and reticular dysgenesis.

Acquired Neutropenias

Acquired intrinsic disorders include leukemias, myelodysplastic syndromes, lymphoproliferative disorders, aplastic anemia, neutropenia of prematurity, and chronic idiopathic neutropenia.

Chronic idiopathic neutropenia occurs in both children and adults. The neutropenia in some patients can be severe. Patients have negative antineutrophil antibodies, normal marrow cytogenetics, and either normocellular marrows or marrows showing decreased postmitotic cells. The prognosis is excellent; patients do not progress to myelodysplasia or leukemia. A proportion of these patients may have autoimmune neutropenia, with undetectable antineutrophil antibodies. G-CSF is effective in increasing the neutrophil count.

Extrinsic Disorders

Immune Neutropenias

Five neutrophil-specific antigens carried on two different glycoproteins have been described. The NA antigens (NA1, NA2, SH) are expressed on FcμRIIIb (CD16), which is a low-affinity receptor for IgG1 and IgG3. The NB antigen is expressed on glycoprotein CD177. There are data suggesting that ANCA can be implicated in the pathogenesis of secondary AIN. The granulocyte immunofluorescence test (GIFT), the granulocyte agglutination test (GAT) and the monoclonal antibody immobilization of granulocyte antigens assay (MAIGA) can be used to detect antineutrophil antibodies. A combination of GIFT and GAT is recommended as the best approach.

Alloimmune neonatal neutropenia occurs when maternal antibodies cross the placenta and react with the infant’s neutrophils. In isoimmune neutropenia, the mother produces an antibody to the paternal CD16 isotype that is different from her own.

Primary autoimmune neutropenia (AIN) is diagnosed in patients with isolated neutropenia who have detectable antineutrophil antibodies. NA1 antibodies are detected in 35% to 40% of patients. The clinical course is usually benign, and spontaneous remissions are common.

Neutropenia Associated with Autoimmune Disorders

AIN is associated with common variable immunodeficiency. The condition should be excluded in patients with recurrent immune cytopenias and granulomatous disease. A high incidence of antineutrophil antibodies is found in patients with X-linked autoimmune lymphoproliferative syndrome (ALPS).

In systemic lupus erythematosus, Fas-mediated apoptosis of mature neutrophils and CD34 positive hematopoietic progenitor cells play an important role in the pathogenesis of neutropenia. Sjogren’s syndrome, systemic sclerosis, and primary biliary cirrhosis and Grave’s disease have all been associated with AIN.

Felty’s syndrome patients typically have deforming rheumatoid arthritis, splenomegaly, and elevated rheumatoid factor titers. The neutropenia in Felty’s is thought to be antibody mediated. In a proportion of patients with Felty’s syndrome, the neutropenia is secondary to the presence of clonal large granular lymphocytes.

Neutropenia Associated with Large Granular Lymphocyte Syndrome

Large granular lymphocyte (LGL) syndrome is caused by an expansion of either T-lymphocytes or natural killer cells (NK) cells. The NK-cell subtype is more aggressive and accounts for 15% of cases. Forty percent of LGL is associated with other diseases such as rheumatoid arthritis.

The T-cells in clonal LGL express the CD3-TCR complex and have rearranged T cell receptor genes. These cells are thought to represent in-vivo activated cytotoxic T cells. Clonal LGLs express high levels of Fas ligand. Normal neutrophil survival is regulated by the Fas-Fas ligand apoptotic system. The neutropenia in clonal LGL syndrome appears to be mediated by increased peripheral destruction of neutrophils secondary to immune complexes and bone marrow suppression of granulopoiesis by Fas ligand secretion.

Table 26.12  Mechanisms of Drug-Induced Isolated Neutropenia

Dose-dependent inhibition of granulopoiesis

•  β-lactam antibiotics, carbamazepine, valproic acid

Immune-mediated destruction of neutrophils and neutrophil precursors

•  Agent acts as hapten to induce antibody formation, complement fixation, and neutrophil destruction: Penicillin, gold, cephalosporins, antithyroid drugs

•  Immune complex related: Quinidine

Direct toxic effect on marrow granulocytic precursors

•  Sulfasalazine, captopril, phenothiazine, clozapine.

•  Chemotherapy drugs seldom cause isolated neutropenia

Neutropenia Associated with Infectious Diseases

The most common cause of acquired neutropenia is infection. Gram-negative septicemia, Staphylococcus aureus, typhoid fever, paratyphoid fever, tularemia, and brucellosis can cause neutropenia. Infectious hepatitis, influenza, measles, Colorado tick fever, mononucleosis, cytomegalovirus, Kawasaki disease, HIV, parvovirus B12 are included in the differential diagnosis of neutropenia associated with infectious diseases.

Parvovirus B19 is frequently associated with transient neutropenia and may cause protracted leucopenia in immunosuppressed patients. Neutropenia is seen in over 70% of patients with acquired immunodeficiency syndrome and can be associated with hypersplenism and the presence of antineutrophil antibodies.

Drug-Induced Neutropenia

The second most common cause of neutropenia is medication exposure: approximately 70% of agranulocytosis cases in the United States are attributed to medications. Procainamide, antithyroid drugs, and sulfasalazine are most commonly implicated. An exhaustive list of drugs causing neutropenia is beyond the scope of this chapter (see instead Kaufman DW et al. The Drug Etiology of Agranulocytosis and Aplastic Anemia, Oxford University Press, 1991).

Three pathogenetic mechanisms for isolated neutropenia include: dose dependent inhibition of granulopoiesis, immune-mediated destruction of neutrophils and their precursors, and direct toxic effect on marrow granulocytic precursors (Table 26.12).

The onset of neutropenia is rapid (1–2 days) in immune-mediated destruction of neutrophils and more variable with agents causing either direct toxic effect or dose-dependent inhibition. Immune-mediated destruction of neutrophils and their precursors occurs by two mechanisms. With hapten mediation, the agent acts as a hapten to induce antibody formation and needs to be present for neutropenia to occur. In the immune complex mechanism, once the complex is formed, continued drug presence is not required for neutrophil destruction.

Ipilimumab, fludarabine, and rituximab have been associated with AIN. Rituximab-associated neutropenia is delayed. Rituximab late onset neutropenia appears after a median of 38 to 175 days from the last rituximab dose, and its median duration is 5 to 77 days. The pathogenesis is not completely understood. The role of G-CSF remains controversial, and the decision as to its use should be made on an individual basis.

Suggested Reading

Abdalla SH, Pasvol G. Approach to the patient in the tropics with anemia. In: Guerrant RL, Walker DH, Weller PF, eds. Tropical Infectious Diseases: Principles, Pathogens and Practice. 3rd ed. Philadelphia, PA: Elsevier; 2011.

Akhtari M, Curtis B, Waller EK. Autoimmune neutropenia in adults. Autoimmun Rev. 2009;9:62-66.

Boztug K, Klein C. Genetic etiologies of severe congenital neutropenia. Curr Opin Pediatr. 2011;23:21-26.

Checkley AM, Chiodini PL, Dockrell DH, et al. Eosinophilia in returning travellers and migrants from the tropics: UK recommendations for investigation and initial management. J Infect. 2010;60:1-10.

Gotlib J. World Health Organization-defined eosinophilic disorders: 2011 update on diagnosis, risk stratification, and management. Am J Hematol. 2011;86:677-688.

Kaufman DW, Kelly JP, Levy M, Shapiro S. The Drug Etiology of Agranulocytosis and Aplastic Anemia. Oxford: Oxford University Press; 1991.

McCrae KR. Thrombocytopenia in pregnancy. Hematology Am Soc Hematol Educ Program. 2010;2010:397-402.

Noel P. Eosinophilic myeloid disorders. Semin Hematol. 2012;49:120-127.

Pels SG, Paidas MJ. Microangiopathic disorders in pregnancy. Hematol Oncol Clin North Am. 2011;25:311-322.

Pierangeli SS, Leader B, Barilaro G, et al. Acquired and inherited thrombophilia disorders in pregnancy. Obstet Gynecol Clin North Am. 2011;38:271-295.

Rodger M. Evidence base for the management of venous thromboembolism in pregnancy. Hematology Am Soc Hematol Educ Program. 2010;2010:173-180.

Szczepiorkowski ZM, Winters JL, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice: evidence-based approach from the apheresis. Applications Committee of the American Society for Apheresis. J Clin Apher. 2010;25:83-177.

Wilson ME, Weller PF. Eosinophilia. In: Guerrant RL, Walker DH, Weller PF, eds. Tropical Infectious Diseases: Principles, Pathogens & Practice. 3rd ed. Philadelphia, PA: Elsevier; 2011.

Wolach O, Bairey O, Lahav M. Late-onset neutropenia after rituximab treatment. Medicine. 2010;89:308-318.

Young NS. Agranulocytosis. In: Neal S Young eds. Bone Marrow Failure Syndromes. Philadelphia, PA: Saunders; 2000.

27

Interpretation of Standard Hematologic Tests

Charles D. Bolan, Roger J. Kurlander and Geraldine P. Schechter

CELLULAR ANALYSIS OF THE PERIPHERAL BLOOD AND BONE MARROW

The practice of hematology is characterized by safe and rapid access and analysis of the cellular elements of blood and bone marrow. Essential clinical information can be derived in less than an hour from a spun hematocrit and microscopic evaluation of peripheral blood or marrow using technology available for more than three quarters of a century,¹ while an automated complete blood count, including the hemoglobin concentration, hematocrit, white blood cell and platelet count, white cell differential, and estimates of red cell size, can be provided within minutes.²

Complete Blood Count

Red Cell Parameters

Using traditional methodology, the hematocrit, defined as the packed red cell volume or percent of the blood occupied by red blood cells, was first measured by direct visual inspection of a tube of blood following centrifugation. The hemoglobin concentration and red cell count were also directly measured, by spectrophotometry and use of a counting chamber, respectively. Other red cell indices were indirectly calculated, including the mean corpuscular volume, MCV (hematocrit divided by the red cell count), mean corpuscular hemoglobin, MCH (hemoglobin divided by the red cell count), and mean corpuscular hemoglobin concentration, MCHC (MCH divided by the MCV). Automated cell counters, which directly analyze and count individual cells by changes in impedance or light scatter, provide information for many of the same parameters provided by traditional methods. Automated methods differ in that the hematocrit is derived indirectly, by multiplying the directly measured red cell count and mean corpuscular volume. The hemoglobin and MCV are thus more reliable, reproducible assays than the hematocrit when measured from an automated counter. The MCV is of great clinical utility in evaluating anemia according to red cell size (macrocytic, microcytic, and normocytic anemias), while the RBC count is useful in comparison of iron deficiency and thalassemia. The MCH, which represents the average total hemoglobin content of the red cells, is highly dependent on cell size, while the MCHC represents the average total hemoglobin concentration or degree of “redness” of the red cells. These parameters have less clinical utility than the MCV and RBC count, although the MCHC is classically increased in hereditary spherocytosis.

The RDW, red cell distribution width, is a measure of the variability in size within the red cell population that is provided by automated cell counters. The precise mathematical equation used for calculation varies with the specific instrument, but in all cases elevated values indicate anisocytosis. For casual observers, the RDW is more reliable than is inspection of the blood film for detecting this characteristic.³ An elevated RDW is a valuable clue to abnormalities in red cell morphology; when the RDW is markedly increased, inspection of the blood film is essential. The RDW is sensitive to the presence of small subpopulations of large or small red cells, thus it may be more useful than the MCV for the early detection of nutritional deficiencies. The RDW often remains normal in thalassemia, and the combination of a high or normal RBC count, a low MCV, and a normal RDW is a common pattern in thalassemia trait.⁴ The use of the RDW in conjunction with other red cell indices has had limited acceptance in the classification of anemias because results may be unreliable in complex settings.

Erythrocytes newly released from the bone marrow contain intracellular RNA, which usually disappears within a day of circulation in the peripheral blood. These young red cells, termed reticulocytes based on the microscopic appearance of RNA-containing reticulum caused by supravital staining, are quantitated by microscopic examination of the peripheral smear. Optical and fluorescent cell-counting methods are also able to detect the uptake of RNA-binding dyes by reticulocyte red cells, and quantitation of the reticulocyte count by automated cell counters has largely replaced the older manual method. Although traditional microscopic and automated techniques produce similar values, the automated technique is more precise. Both methods are vulnerable to interference from intracellular organisms, basophilic stippling, and other artifacts; thus, microscopic inspection of a standard blood film, and/or of a reticulocyte preparation is warranted whenever a reticulocyte count seems inappropriately high for the clinical scenario.

The absolute reticulocyte count (absolute reticulocyte count = % reticulocytes X RBC count/100) is a more clinically useful parameter than the simple percentage of reticulocytes, which is influenced by patient total red cell count and hematocrit. Because a highly stimulated marrow produces larger, more polychromatophilic, RNA-rich reticulocytes (termed stress reticulocytes),⁵ which are released earlier from the marrow and stain with supravital dyes for up to 3 days after release into the peripheral circulation, the absolute reticulocyte count may overestimate the true rate of reticulocyte production in these clinical situations. Some automated counters may also provide the capability to detect reticulocyte subpopulations containing increased levels of RNA, designated as the immature reticulocyte fraction (IRF), which can indicate the early response to erythropoiesis-stimulating agents and early, pre-myeloid marrow recovery after chemotherapy or stem cell transplantation.⁶ Automated counters are also capable of selectively measuring the hemoglobin content of reticulocytes, which diminishes rapidly as accessible iron stores are exhausted, and this test may be of use in identifying early iron deficiency in blood donors undergoing repeated phlebotomy, iron-depleted dialysis patients receiving erythropoietin for the treatment of anemia, or iron-stressed women during the late stages of pregnancy.⁶

White Blood Cell Parameters

The automated white blood cell (WBC) count is measured using the same methods employed to count red cells and platelets. Since the peripheral blood in healthy individuals has approximately 1,000 fold more red cells and 40 fold more platelets than white cells, it is essential for the automated counter to distinguish among cell types. To facilitate the process, red cells are usually destroyed by osmotic lysis and residual red cells or platelets are excluded from the count based on size and granularity. Automated counting is faster, more reproducible, and usually more accurate than the older manual methods, particularly for leukopenic samples in which automated counters can quantitate WBC counts as low as 100 cells per μL.

Automated leukocyte differentials are quite accurate in characterizing white cells from normal controls or patients with qualitatively normal white cell morphology. In patients with hematologic disease or qualitative leukocyte abnormalities, automated differentials may still be accurate, but their reliability cannot be assumed. Thus the white cell differential of new patients with complex clinical findings should also be evaluated on the peripheral smear. The automated absolute granulocyte count plays a reliable and important role in evaluating the risk of infection in neutropenic patients, in grading chemotherapy-induced toxicity, and in following the recovery from hematopoietic stem cell transplantation.

Platelets

Similar to RBC and WBC counts, the automated platelet count is measured by impedance- and/ or optical-based techniques. Platelets are distinguished from red cells by size and in some cases by resistance to osmotic lysis. Automated counters are more accurate than older manual counting methods in monitoring thrombocytopenia, and they often can accurately count less than 10,000 platelets per microliter. On occasion, it may be difficult even with advanced optical techniques to distinguish true platelets from debris derived from precipitated proteins or cell fragments.

The mean platelet volume (MPV) is a parameter that is routinely measured by automated analyzers. The MPV has modest value as a measure of increased platelet turnover and/or activation due to the fact that young platelets are larger than older platelets. However, the MPV may increase rapidly during the first 2 hours after collection because of shape changes and swelling in EDTA, and the reference standards are not adequate for comparing values from different institutions.⁷

Some automated counters can measure the RNA content of new released platelets, expressed as the immature platelet fraction (IPF) or absolute immature platelet number (per cent IPF times the platelet count). These measurements may be useful clinical parameters in assessing marrow recovery after hematopoietic transplantation and marrow thrombopoiesis in cases of suspected idiopathic thrombocytopenic purpura (ITP).8,9

Sources of Artifact

Clinically relevant artifact at the point of sample collection may occur due to clot formation or due to dilution of blood with intravenous fluid.10,11 Although most automated cell counters are equipped to detect clot formation, some may not be detected, resulting in falsely lower platelet and WBC counts. In contrast, fluid dilution generally is apparent by some degree of change in all three cell lines (red cells, white cells, and platelets) as compared to a previous sample. Both clot formation and dilution may occur more frequently in samples drawn from indwelling catheters.

Artifact may also be introduced by factors associated with the storage of blood samples. When stored at room temperature, absolute RBC, WBC, and platelet counts are stable for up to 3 days, but the red cell MCV increases within 24 hours due to cell swelling, resulting in associated increases in the hematocrit and red cell distribution width.¹² After 2 days, the relative proportion of monocytes on the WBC differential also decreases, while relative leukocyte, lymphocyte, and eosinophil counts increase.¹²

Red cell agglutination within the test tube caused by cold or warm reactive antibodies or rouleaux formation can artifactually reduce the red cell count, while extreme leukocytosis, particularly with a WBC count of more than 500,000/μL, can falsely increase the RBC count and calculated hematocrit.¹¹ Similarly, RBC counts in patients with extremely small or fragmented red cells may be artifactually low. Falsely decreased platelet counts, or pseudothrombocytopenia, caused by platelet clumping and platelet satellitism induced by the EDTA anticoagulant, may occur in up to 0.1% of normal samples and may be present in up to 15% of patients referred for evaluation of a low platelet count.¹³ In most cases, pseudothrombocytopenia can be eliminated by complete blood count performed on samples collected into a tube containing citrate anticoagulant, in which instance the measured platelet count should be multiplied by 1.1. Platelet clumping may sometimes also falsely elevate the white blood cell count, while small white or red cell fragments, or protein precipitates may artifactually increase the platelet count.10,11

Peripheral Blood Film

One common use of review of the peripheral blood film is by laboratory personnel when values reported by automated counters are “flagged” on the device result print out, or by hematologists in clinical situations circumstances in which verification of the automated result is indicated. The automated value can be compared to a manual estimate obtained by counting the average number of platelets present in 5 to 10 oil immersion high power (1,000×) fields (HPF) and/or the number of leukocytes in a similar number of low power (100×) fields (LPF). The selected fields are chosen from representative regions that contain an even monolayer of cells, avoiding the edge of the film. The platelet count and leukocyte count are estimated by using the formulas:

Platelet count (platelets/mm³) = average platelets per HPF × 15,000
Leukocyte count (cells/mm³) = average leukocytes per LPF × 250

Evaluation of the peripheral blood smear is valuable in the evaluation of hematologic disease and should always be performed in conjunction with review of the bone marrow aspirate and core biopsy. Examination of the peripheral smear is not necessary in simple cases of anemia due to iron, vitamin B12, or folate deficiency. However, it can be very useful in cases that do not respond to therapy and in instances of suspected hemolysis or complex anemias, and it is essential in the evaluation of microangiopathic disorders such as thrombotic thrombocytopenic purpura (TTP). The presence of spherocytes, microspherocytes, schistocytes, targets, sickle cells, red cell membrane irregularities, polychromasia, and intracellular inclusions or parasites should be sought in cases of suspected hemolysis, while tear drop cells and nucleated red cells may be present in marrow fibrosis or tumor invasion. Evaluation of white blood cell abnormalities such as the presence of immature cells and blasts are indicated in cases of suspected malignancy, while other changes such as hypogranulated and hypersegmented granulocytes, Pelger-Huet cells, toxic granulation, and Döhle bodies may point towards a previously unsuspected underlying process.

Review of granulocyte morphology by review of the peripheral smear is superior to automated methods for evaluation of a shift to the left in the myeloid series, or an increased proportion of immature forms, which may be a common finding during the early response to stress or infection. The neutrophil band count is performed manually by grading 1,000 neutrophils on a Romanowsky-stained peripheral blood film, and expressed in percent or as an absolute number.

Routine inspection of platelet morphology is indicated in all patients with newly diagnosed thrombocytopenia in order to exclude platelet clumping and artifactual thrombocytopenia. Review of the blood film may also reveal morphologic abnormalities such as hypogranularity, as in gray platelet syndrome or myelodysplasia, or the presence of very large platelets in inherited syndromes such Bernard-Soulier disease, and May-Hegglin anomaly.

Bone Marrow Aspirate/Biopsy

Indications

Examination of the bone marrow aspirate and biopsy may establish or exclude diagnoses such as aplastic anemia, myelodysplastic syndrome, hemophagocytosis or marrow replacement by non-hematopoietic cells in patients with unexplained persistent or severe thrombocytopenia and granulocytopenia. Bone marrow evaluation is not indicated in cases of isolated anemia in which clinical information and review of the peripheral smear suffice to make the diagnosis. However, in patients with unexplained anemia, particularly if requiring transfusions, a bone marrow study is appropriate. Although marrow aspiration and biopsy are invaluable in hematologic malignancies, and in screening for metastatic spread of non-hematologic malignancies, radiologic or nuclear medicine techniques may be more sensitive methods for detecting metastases. Bone marrow can also be used to culture and identify infectious agents such as mycobacteria and fungi. Disorders such as Gaucher disease or amyloidosis may also be discovered, but biochemical tests for the former or less invasive biopsies for the latter are more useful.

Risks

Bone marrow aspirate biopsy can be performed with minimal discomfort using local anesthesia and is generally very safe when performed by an experienced operator. Informed consent should always be obtained, for the procedure as well as the performance of research studies or specialized laboratory tests. Attention should be paid to both patient comfort, as well as toward obtaining adequate sample volume—particularly of the core biopsy—to conduct the indicated analyses. Infiltration of the skin surface and periosteum with several milliliters of lidocaine provides sufficient anesthesia for most patients; conscious sedation should be reserved for those with significant anxiety and apprehension. Thrombocytopenia is not a contraindication provided adequate landmarks are identified and sufficient direct pressure is applied to the site post-procedure; some patients with platelet function defects or thrombocytopenia due to myelodysplasia may require platelet transfusion for control of bleeding. Anticoagulation should be discontinued or held for an adequate time prior to the procedure, and coagulation factor replacement be utilized for patients with serious factor deficits in order to avoid significant bleeding.

Hemorrhage and local infection occur rarely. In one study of more than 50,000 biopsies, there were 14 instances of serious hemorrhage with one death, and 6 instances requiring transfusion.¹⁴ Infection can be safely managed by the use of careful sterile technique and proper local care even in patients with neutropenia and compromised immune function.

Technique

In adults, the posterior iliac crest is the site of choice for most hematologists; it can be located using direct palpation with the patient in the prone position and generally provides adequate core and aspirate samples. The anterior iliac crest is a reasonable alternative when obesity, local irradiation, or local skin conditions preclude a posterior approach. Sternal aspiration may be justified when an iliac approach is not possible. However, the sternal approach cannot be used to obtain a core biopsy specimen, may be less well accepted by patients, and is more vulnerable to complications. Traumatic penetration or fracture of the sternum with damage to underlying structures is a rare but serious complication of particular concern when underlying structural damage secondary to malignancy or multiple myeloma is present.

Bone Marrow Aspirate

The bone marrow aspirate is obtained by advancing a specially designed needle fitted with an obturator through the cortex into the medullary space with use of negative pressure to withdraw cells into a syringe after removal of the obturator. The presence of individual marrow particles or particles is determined by visual inspection. The particles are spread on a glass slide, and they may be stained immediately to determine the presence and morphology of cellular marrow elements, such as megakaryocytes or blasts in the evaluation of suspected ITP or leukemia. Cell suspensions obtained from an aspirate may also be used in special studies such as cytogenetic and flow cytometric analyses, while the aspirate clot can also be fixed in a clot section, to be stained with hematoxylin and eosin, or other stains in the same way as a bone marrow biopsy. The marrow may be inaspirable in some patients due to hypercellularity, hypocellularity, fibrosis, or metastatic cancer cells. Aspirates are less reliable than biopsies in detecting marrow involvement with malignancy and not useful at all to detect myelofibrosis or granulomas.

Bone Marrow Biopsy

It is almost always possible to obtain a biopsy specimen, even when hematopoietic cells have been totally replaced by fibrous tissue or tumor. Hematoxylin and eosin-stained biopsy sections show less cytoplasmic and nuclear detail than Romanowsky-stained aspirate films, but provide other essential information about marrow architecture and cellularity. The bone marrow biopsy specimen, or core, is obtained by further advancing the bone marrow biopsy needle through the medullary space after removal of the obturator. In some situations, the core may be obtained from the same puncture site used to obtain the aspirate by advancing deeper into the marrow cortex after obtaining cells for the aspirate; this technique is convenient for the operator and patient but may be associated with artifactual hypocellularity, hemorrhage, and distorted architecture.¹⁵ Aspiration artifact may be reduced by obtaining a larger core sample size (1.5–2.5 cm), which may also enhance the evaluation of the marrow composition. The core biopsy sample is ejected from the needle by using a metal probe, fixed, decalcified, sectioned, and stained with hematoxylin and eosin, histochemical stains (for reticulin, collagen, iron), or a variety of immunohistochemical stains.

Marrow Cellularity

Marrow cellularity should be estimated from review of a large bone marrow core biopsy specimen. Because normal marrow cellularity decreases with age; normal percent cellularity can be estimated by subtracting patient age from 100. Inappropriately low cellularity suggests marrow damage, and high cellularity may be consistent with a proliferative disorder, dysplasia, stress reaction, or the use of growth factors.

Megakaryocyte Number and Appearance

A normocellular biopsy and aspirate specimen should contain multiple megakaryocytes per low power (100×) field. An increase in megakaryocytes is consistent with increased turnover secondary to peripheral destruction, inflammation, iron deficiency, or myeloproliferative/myelodysplastic disorders. Reduced megakaryocyte numbers may reflect a primary marrow disease such as aplastic anemia, amegakaryocytic thrombocytopenia, or suppression secondary to chemotherapy. Normal megakaryocytes appear as large cells with multilobated (three or more) attached nuclei. The presence of a substantial number of smaller megakaryocytes with less than three nuclear lobes indicates a shift to the left in megakaryocyte maturity due to increased platelet turnover or dysplasia; megakaryocytes with a single nucleus or multiple small separated nuclei and mature cytoplasm are particularly suggestive of myelodysplasia.

Myeloid to Erythroid Ratio

The myeloid to erythroid (M:E) ratio is quantitated by counting 300 to 500 cells from a marrow aspirate sample. In adults, the normal M:E ratio varies from 1:1 to approximately 3:1. The M:E ratio should be interpreted in the context of overall cellularity, the qualitative appearance of the affected cells, and the clinical setting. Erythroid hyperplasia, characterized by a low M:E ratio in a cellular marrow, suggests an erythroid response to anemia, (especially hemolytic anemia), myelodysplasia, or erythropoietin administration.

Erythroid hypoplasia may indicate red cell aplasia, decreased erythropoiesis due to autoimmunity, medication side effects, or major-ABO incompatible hematopoietic stem cell transplantation. Myeloid hyperplasia may be seen in response to physiologic stress, infection, exogenous growth factors, or in myeloproliferative disorders. Myeloid hypoplasia with a “maturation arrest” (an absence of myeloid precursors beyond the promyelocyte or myelocyte stage) may reflect drug-induced or autoimmune agranulocytosis.

Myeloid and Erythroid Cells

Myeloid and erythroid cell lines should demonstrate normal morphology, a normal distribution of maturation, and synchronous maturation of nucleus and cytoplasm. Disruption of the normal maturation sequence (such as a complete maturation arrest or a preponderance of immature cell forms, or a “shift to the left” of the differentiation pattern), the presence of excess numbers of blasts, or dysplastic changes affecting at least two of the three major hematopoietic cell-lines suggests a serious hematologic disorder. A mild shift to the left and mild megaloblastic changes are more nonspecific.

Lymphocytes and Plasma Cells

There is substantial normal variation in the number of lymphocytes encountered in a marrow specimen, and these elements may be distributed diffusely or in well-defined lymphoid aggregates. Paratrabecular lymphoid aggregates may occur in follicular lymphoma. Benign lymphoid aggregates typically contain more T cells than B cells when immunohistochemical stains are performed, while B cell-rich lymphoid collections are more likely due to clonal B cell lymphoproliferative disorders.

Plasma cells usually constitute less than 2% of marrow cells; increases may be noted in inflammatory disease, benign monoclonal gammopathy, and multiple myeloma. Distinguishing reactive from neoplastic plasma cells in a routine aspirate film may be difficult, as reactive plasmacytosis caused by inflammation or liver disease may reach 20% to 30%, and hypocellular marrows are often rich in plasma cells. Extensive multinuclearity or plasma cell aggregates of more than 5 to 10 cells are suspicious for malignancy, while conspicuous variation in cell size and immature nuclei with nucleoli may be present in myeloma. Immunohistochemical staining for intracellular kappa and lambda chains within plasma cells in the marrow biopsy can indicate the presence of a monoclonal process; distinguishing benign monoclonal gammopathy from multiple myeloma may require additional clinical and laboratory data. The presence of concentrated collections of plasmacytoid lymphocytes suggests Waldenström macroglobulinemia or lymphoplasmacytic lymphoma.

Other Cells Anomalous Present in the Marrow

Malignant cells of epithelial or mesenchymal origin adhere more tightly together and aspirate poorly, forming tightly clumped groups of unusually large cells with a very high nuclear-cytoplasmic ratio. Such clumps may be rare; therefore, when screening an aspirate for malignant cells, the whole slide should be scanned at low power including the leading edge of the film. Tumor cells can also be readily identified in marrow biopsies, and specialized immunohistochemical stains can sometimes help identify the site of origin.

Bone Marrow Iron Stores

When marrow iron is markedly increased, yellow granules of hemosiderin may be seen in routinely stained aspirate and biopsy preparations. The Prussian blue stain, which specifically detects iron, is necessary to evaluate lesser iron stores and iron granules in erythroid cells. Ringed sideroblasts are erythroid precursors containing coarse iron granules immediately surrounding at least half the nuclear circumference due to accumulation of iron within mitochondria. They are always abnormal and imply anomalous porphyrin synthesis secondary to a congenital abnormality, pyridoxine deficiency, exposure to toxins (such as lead or alcohol), medication, or myelodysplasia.

Although tissue iron stores can be assumed to be adequate when stainable marrow iron is demonstrated, this does not indicate that the stores can be mobilized for effective erythropoiesis. For example, when hepcidin levels are elevated by chronic inflammation, iron will be trapped in the macrophages and unavailable for hemoglobin synthesis. In contrast, the absence of stainable iron suggests depleted iron stores. The sensitivity of this method depends heavily on the quantity of sample obtained; for maximal yield, at least 7 separate particles should be evaluated for stainable iron.¹⁶ In addition, the absence of stainable marrow iron may incorrectly suggest iron depletion due to hypocellularity, technical error, or presence of cellular antigens binding to antibodies labeled with dyes.¹⁷

Additional Studies

Flow cytometry, which allows computerized digital analyses of cells according to size, granularity, and light scattered from colored dyes attached to specific cell markers, is an essential element in the analysis of hematologic malignancy, clonality, and other abnormalities, and can be performed on cells from peripheral blood and marrow.¹⁸

Because a very wide variety of possible analyses can be performed, the clinician should specify the clinical question and appropriate differential diagnosis to the laboratory prior to collection to facilitate proper labeling and use of appropriate device settings. As with the automated CBC, flow cytometry is subject to errors and artifacts due to clotting and cell clumping; thus, samples should be carefully collected into appropriately prepared tubes and rapidly and safely transported to the laboratory.

Molecular diagnostic studies such as cytogenetics and fluorescent in situ hybridization (FISH) have an important role in diagnosis and monitoring of hematologic disorders, especially myeloid malignancies, while immunohistochemical stains and molecular analysis of T and B cell receptor rearrangements are critical in documenting clonality in lymphoid malignancies.

SERUM TESTS TO EVALUATE NUTRITIONAL AND HYPOPROLIFERATIVE ANEMIAS

Serum Iron and Total Binding Capacity Assays

Serum iron is measured by automated chemical assays following dissociation from transferrin.¹⁹ Total iron binding capacity (TIBC), which is mainly binding to transferrin, can be measured by the addition of excess iron to the sample. Unbound iron is removed by absorption, and the iron bound to protein is again dissociated and measured by the serum iron assay. In many laboratories, unsaturated iron binding capacity is now measured directly by automated chemistry analyzers and the TIBC is calculated.¹⁹ Measurement of serum iron can be falsely elevated in specimens containing hemoglobin (hemolysed specimens) and also for many hours transiently after transfusion of older units of red blood cells due to clearance of red cells damaged in storage.²⁰ The percent saturation of transferrin by iron is calculated by dividing the serum iron by the total iron binding capacity and then multiplying by 100.

There is a marked diurnal variation of serum iron in healthy people, with highest levels in the morning; by midnight, serum iron levels can be very low and may fall into the iron deficiency range. Iron levels also vary with the menstrual cycle, with a 10% to 30% increase premenstrually and a similar decrease at the time of menstruation.¹⁹ Iron deficiency is characterized by low serum iron values and elevated serum total iron binding capacity and therefore, low iron saturation of transferrin. Serum iron levels also fall below normal in patients with the anemia of inflammation/chronic disease, but since total iron binding capacity also falls, percent iron saturation may remain normal. Levels of serum iron and the percent iron saturation in patients with severe chronic inflammation frequently overlap with those found in iron deficiency anemia.²¹ The high TIBC levels (more than 300 μg/dL) in patients with uncomplicated iron deficiency are helpful in distinguishing the two entities, but when iron depletion and inflammation coexist, the TIBC is frequently low, and serum ferritin and possibly transferrin receptor assays become necessary to help confirm the diagnosis of iron deficiency²¹ (see below).

Elevated serum iron levels and iron saturation occur in multiple inherited and acquired conditions (Table 27.1). Genetic hemochromatosis of many different types results in hepcidin deficiency, which leads to high gastrointestinal iron absorption despite high levels of iron. The iron overload of thalassemia major is mainly due to multiple transfusions but is also compounded by increased gastrointestinal iron absorption that is stimulated by hepcidin suppression by increased erythropoiesis²² The latter mechanism accounts for iron overload in patients with intermediate thalassemias and hemolytic anemias. Elevated serum iron levels also results from the failure of reutilization of iron, as in megaloblastic and sideroblastic anemias, and with pure red cell aplasia. Other causes include fulminant acute hepatitis due to severe hepatocyte injury and also chronic hepatitis, particularly hepatitis C. In patients with iron deficiency, a rise in serum iron to levels greater than 100 mcg/mL may occur 1 to 2 hours after ingestion of 325 mg of ferrous sulfate in the form of a tablet or elixir which indicates appropriate bioavailability and normal small bowel absorption.²¹

Soluble Transferrin Receptor

Soluble transferrin receptor, a truncated form of tissue transferrin receptor, is measured by a “sandwich type” enzyme-linked immunoassay. Serum transferrin receptor levels are elevated in states of increased erythropoiesis such as hemolytic anemias, megaloblastic anemia, thalassemia, and also in iron deficiency anemia. Transferrin receptor levels are decreased in conditions of reduced erythropoiesis such as aplastic anemia and renal insufficiency. The assay is reported to distinguish iron deficiency from the anemia of chronic inflammation/disease,21,23 but overlap in receptor levels between the two conditions is often noted. The ratio of the serum transferrin receptor level to the ferritin level or to log ferritin level may be more useful in distinguishing between the two entities in confirming when they coexist.²³ Elevated transferrin receptor levels observed in some patients with anemia of inflammation likely reflect limited availability of functional iron pools for erythropoiesis rather than depleted iron stores.²⁴ Automated assays are now available.23,25 As discussed previously, changes in reticulocyte hemoglobin content, which is a direct measure of new red cell hemoglobinization, may be the most dynamic and sensitive measure of functional iron availability during active hematopoiesis.

Table 27.1  Causes of High Serum Fe and Fe Saturation

Iron Overload States

Genetic hemochromatosis

HFE, TfR, HJV, hepcidin, ferroportin mutations

Transfusion hemosiderosis

Impaired Reutilization of Iron

Pure red cell aplasia

Aplastic anemia

Ineffective erythropoiesis

Nutritional megaloblastic anemias

Sideroblastic anemias

Thalassemia intermedia and major

Congenital dyserythropoietic anemias

Unknown or Multiple Mechanisms

Hepatitis C

Liver cirrhosis

Chronic alcoholism

Serum Ferritin

Serum ferritin can be measured by a variety of immunoassays.¹⁹ The level of serum ferritin generally correlates with body stores of iron. The ferritin assay is most useful clinically at the extremes: low values (<15 ng/mL) are very sensitive to storage iron depletion and very high levels (>1,000 ng/mL) usually indicate iron overload states. Since serum ferritin is an acute phase reactant, patients who have both iron deficiency and anemia of inflammation or liver disease often have serum ferritin levels in the normal range, usually between 40 and 70 ng/mL,²¹ and less than 7% of anemic patients with depleted iron stores exceeded a serum ferritin level of 100 ng/mL in one study.²⁶ The soluble transferrin receptor/log ferritin index was reported to diagnose iron deficiency in 25% of patients with anemia of inflammation and ferritin levels >100 ng/mL.²³

Very high levels of ferritin usually indicate iron overload states resulting from genetic hemochromatosis, liver disease, particularly chronic hepatitis C, or transfusion hemosiderosis. Patients with these disorders will have elevated serum iron levels. Ultrahigh levels above 5,000 ng/mL are often observed in severe inflammatory states, such as disseminated fungal or mycobacterial disease, or in disorders associated with macrophage activation, for example, Still’s disease or syndromes associated with hemophagocytosis. Table 27.2 lists the causes of high ferritin levels not associated with high levels of serum iron, which include genetic causes such as hyperferritinemia cataract syndrome²⁷ and apparently acquired causes such as the “metabolic” syndrome associated with diabetes, obesity and hepatosteatosis.²⁸ In the presence of concurrent inflammation or liver disease, ferritin levels may not accurately gauge the response to iron chelation therapy in patients with transfusion hemosiderosis and very high levels (>1,000 ng/mL).

Serum Vitamin B₁₂ (Cobalamin)

Serum vitamin B₁₂ is generally assayed by an enzyme-linked competitive binding assay most frequently based on binding to intrinsic factor.¹⁹ Serum levels below 100 pg/mL are almost invariably associated with cellular cobalamin deficiency, as reflected by elevated serum methylmalonic acid levels (see below).²⁹ Fifty percent of patients with cobalamin levels between 100 and 200 pg/mL and up to 10% of patients between 200 and 300 pg/mL have elevated methylmalonic acid levels indicating cellular deficiency. Above 300 pg/mL, only 0.1% of patients have cellular cobalamin deficiency. Low serum cobalamin in patients with normal methylmalonic acid levels may indicate early depletion of cobalamin stores or reduced levels of transcobalamin I, the major cobalamin-binding protein in the plasma, which however does not play a role in hematopoietic cell utilization of cobalamin. Clinical conditions where myeloid cells, the major producer of transcobalamin I, are severely depleted such as in aplastic anemia may result in low serum cobalamin levels. Patients with myeloma and those with HIV infection frequently have unexplained low levels of cobalamin, possibly due to a reduced myeloid cell mass. Patients whose levels remain low after parenteral treatment with cyanocobalamin are likely to have genetic transcobalamin I deficiency. Elevated cobalamin levels are seen after treatment with parenteral vitamin B₁₂, in patients with hepatic necrosis, or because of increased B₁₂ binding proteins associated with myeloproliferative disorders, particularly untreated chronic myeloid leukemia.¹⁹

Table 27.2  Causes of Hyperferritinemia without Increased Serum Fe Saturation

Inflammation

Infection

Metabolic syndrome

Malignancy

Macrophage activation syndrome

Ferroportin mutations (classical type)

Hyperferritinemia-cataract syndrome ( L ferritin gene (FTL) mutation)

Parenteral iron treatment of iron-refractory iron deficiency anemia (TEMPRSS6 mutation)

Serum Methylmalonic Acid

Methylmalonyl dehydrogenase is a cobalamin-dependent enzyme required for transformation of methylmalonate into succinate in mammalian cells. Methylmalonic acid is assayed by gas-liquid chromatography or mass spectrometry.¹⁹ Serum and urine methylmalonic acid increase in more than 95% of patients with cellular cobalamin deficiency. Most patients with normal methylmalonic acid levels and low serum cobalamin in the absence of macrocytosis or anemia are presumed to have depleted stores without cellular cobalamin deficiency. In renal insufficiency, reduced methylmalonic acid excretion can lead to elevated serum levels in the absence of cellular cobalamin deficiency. A diagnosis of cellular cobalamin deficiency can be confirmed by demonstrating a decrease in serum methylmalonic acid levels to normal after initiation of cobalamin treatment.

Serum Homocysteine

Deficiency of folic acid, cobalamin, or pyridoxine (vitamin B6) prevents the methylation of homocysteine to form methionine and leads to increased serum homocysteine levels, which can be measured by a number of methods.¹⁹ Cellular cobalamin deficiency usually causes elevated levels of both methylmalonic acid and serum homocysteine, but in 5% of cobalamin-deficient patients only serum homocysteine will be elevated. In usual practice, it is not cost effective to use homocysteine to confirm cellular deficiency of cobalamin or folate. Homocysteine levels are more often obtained for the clinical evaluation of arterial or venous hypercoagulability risk (see Chapter 22). Other causes of elevated levels of homocysteine include renal insufficiency and inherited abnormalities in the enzymes required for the folic acid cycle and sulfur-containing amino acid metabolism.

Serum Intrinsic Factor Antibody Assay

A positive result in this assay is highly specific for a diagnosis of malabsorption of cobalamin due to autoimmune depletion of intrinsic factor (pernicious anemia) but the sensitivity of the serum assay is less than 50%.

Serum and Red Cell Folate Assays

These levels are determined by a competitive receptor-binding assay.¹⁹ Serum folate levels reflect recent dietary intake while red cell folate levels reflect body folate stores at the time when the red cell was formed. Because cobalamin is required for cellular uptake of folate, reduced red cell folate levels are found with either folate or cobalamin deficiency; therefore, a serum cobalamin level is always required to interpret a low red cell folate level. Red cell folate is measured from a hemolysate prepared from whole blood and elevated levels of serum folate may therefore affect the red cell folate value. Elevated serum folate is found in cobalamin deficiency states and following treatment with folic acid. Testing for cobalamin levels is essential in the evaluation of patients with presumed folate deficiency, since folic acid replacement may improve the anemia but not the potentially irreversible neurologic complications of cobalamin deficiency. Folate deficiency in the United States has become rare, even in nutritionally deficient and alcoholic individuals, since cereal grain products were fortified with folate to reduce the risk of fetal neural tube defects. Therefore, since folic acid deficiency is infrequent in this country and so easily treated, it is likely that the assessment of serum and red cell folate levels in patients with anemia is no longer cost effective, and should be limited to those suspected of having malabsorption.

Serum Erythropoietin

In patients with refractory anemia due to marrow failure, markedly elevated erythropoietin levels (over 500–1,000 U/mL) usually predict failure of recombinant erythropoietin therapy. This immunoassay can therefore be useful in identifying a subset of patients unlikely to benefit from therapy with erythropoiesis-stimulating agents. On the other hand, it may not be worthwhile to assay erythropoietin levels in anemic patients with renal insufficiency, malignancy, or inflammation since erythropoietin levels in these conditions are commonly low (under 100 U/mL) and deficiency is treatable.

Erythropoietin assays which are sensitive to low levels are useful for distinguishing polycythemia vera from other causes of erythrocytosis. Patients with polycythemia vera have levels below the normal range, consistent with autonomous erythroid progenitor cell proliferation. Low erythropoietin levels may also be seen in rare patients with primary erythrocytosis due to genetic abnormalities in erythropoietin signaling pathway and in some patients in whom the cause cannot be identified (idiopathic erythrocytosis).³⁰ Erythropoietin levels in patients with secondary polycythemia are frequently within the normal range but will often become elevated following phlebotomy treatment.

TESTS FOR THE EVALUATION OF ABNORMAL HEMOGLOBINS AND HEMOLYTIC ANEMIAS

Hemoglobin Electrophoresis

Historical methods used to differentiate and quantitate abnormal hemoglobins and the minor hemoglobins included alkaline and acid electrophoresis, and have been replaced by cation high- performance liquid chromatography (HPLC), isoelectric focusing (IEF) and capillary electrophoresis in large clinical laboratories.³¹ Smaller clinical laboratories may continue to use alkaline cellulose acetate or agarose gel electrophoresis at pH 8.6 to screen patient samples and to identify the common hemoglobins A, S, and C. When aberrant hemoglobins are observed, levels are quantified by using densitometry (Fig. 27.1). The minor hemoglobins F and A₂ are also separated by this method, but their levels in adults cannot be measured accurately by densitometry. Since a number of G and D hemoglobins comigrate with Hb S, a solubility test is routinely used to confirm the presence of S (see below). Acid citrate agar electrophoresis at pH 6.0, also separates hemoglobins A, S, C, and F and is used routinely for confirmation of Hb S and C. It can distinguish the Hb D (containing mutated beta globin chains) and Hb G (containing mutated alpha globin chains) hemoglobins from Hb S. Acid citrate agar electrophoresis also separates Hb C from Hb E and Hb O Arab, variants that comigrate with C on alkaline electrophoresis and HLPC.³¹

IEF and HPLC are used in neonatal screening programs because they have greater power than do alkaline or acid agar electrophoresis in separating fetal hemoglobin from S and A; IEF and HPLC also are more expensive and require greater expertise for interpretation.¹⁹

Sickle Solubility Test

The insolubility of deoxygenated Hb S in a concentrated phosphate buffer can be exploited to confirm that a hemoglobin with the appropriate electrophoretic mobility is actually Hb S. Since the solubility test cannot distinguish between sickle trait and sickle cell disease, it is not useful to diagnose sickle cell disease in clinical settings.

FIGURE 27.1 Hemoglobin electrophoresis, cellulose acetate, pH 8.6.

Sickle Cell Prep

Red cells from sickle cell trait or homozygous S individuals will take on the sickle shape when deoxygenated. This test has been replaced by the sickle cell solubility test to confirm the presence of Hb S. It also does not distinguish sickle cell trait from sickle cell disease.

Hb A₂ Quantitation

HPLC is the method of choice for this measurement.³¹ Column chromatography is also frequently used but may be unreliable in the presence of Hb S. Elevated levels of Hb A₂ above 3.5%, will generally confirm a diagnosis of β thalassemia trait. The presence of iron deficiency may lower the Hb A₂ level into the normal range. Patients with α or δβ thalassemia trait have normal levels of Hb A2. When it is clinically necessary to confirm alpha thalassemia, DNA-based methods are necessary, for example, in the case of a Southeast Asian couple with microcytosis who are at risk to have a fetus with hydrops fetalis or Hgb H disease.³¹

Hb F Quantitation

Many clinical laboratories continue to use the alkali-denaturation test to quantitate the percentage of Hb F. This test exploits the persistent solubility of Hb F under alkaline conditions, which precipitate most other hemoglobins. After treatment with alkali, the residual Hb F can be separated by filtration and quantified spectrophotometrically. The assay is accurate, with samples containing as much as 10% to 15% Hb F but will often underestimate higher levels. HPLC-based methods are more accurate for the assay of higher levels of Hb F.

Hb F Cells

Hb F can also be measured immunologically to determine the amount of Hb F in red cells and distinguish high Hb F containing cells (“F cells”) from red cells containing low levels of Hb F. A uniform distribution of Hb F is found in the red cells of patients with hereditary persistence of fetal hemoglobin (see Chapter 4).

Tests for Unstable Hemoglobins

Some unstable hemoglobins such as Hb Zurich and Hb Koln can be recognized by their propensity to precipitate when hemolysates are exposed to heat (50°C) or to 17% isopropanol. Unstable hemoglobins may also be detected by the formation of Heinz bodies (denatured hemoglobin) in intact red blood cells after exposure to oxidizing conditions. These inclusions, located near the red cell membrane, are detected microscopically by their blue staining after incubating red cells with supravital stains such as brilliant cresyl blue or new methylene blue (see Chapter 3).

Glucose-6-Phosphate Dehydrogenase

Either qualitative or quantitative tests are used in clinical laboratories to detect G6PD deficiency. The assays depend on the generation of NADPH from NADP. The most common screening test is the fluorescent spot test, which depends on the intrinsic fluorescence of NADPH. Reticulocytes from individuals with the most common variant of G6PD deficiency seen in the United States (G6PD A-) have much higher quantities of enzyme than are present in mature red cells. Deficiency may not be diagnosed by these tests in G6PD A- individuals if reticulocytosis has developed in response to hemolysis produced by an oxidant chemical or drug. Heinz bodies (denatured hemoglobin, see above) may also be recognized in the red cells in this setting. Female heterozygotes may also escape detection by screening tests.³²

Serum Haptoglobin

This hemoglobin-binding protein can be assayed by nephelometric or turbidometric methods.¹⁹ Haptoglobin is an acute phase reactant, but its main usefulness is that very low levels are an indicator of acute or chronic hemolysis. Free hemoglobin bound to haptoglobin is cleared by the reticuloendothelial system in less than 30 minutes. In vivo hemolysis of as little as 50 mL of red blood cells will deplete the blood of haptoglobin. In the absence of continuing hemolysis, it will take at least 5 days to regenerate to normal levels. The normal range varies widely due to genetic differences in the alpha chains. Rare patients have very low haptoglobin levels on a genetic basis. Patients with severe liver disease may have decreased haptoglobin levels due to failure of hepatic synthesis.

Urine Hemosiderin

In patients with chronic intravascular hemolysis such as paroxysmal nocturnal hemoglobinuria or cardiac valve hemolysis, renal excretion of hemoglobin leads to uptake of heme, with subsequent accumulation of hemosiderin in the renal tubular cells. After staining the urine sediment with Prussian blue, microscopic evaluation of the sediment will demonstrate blue stained particles in renal casts indicating iron deposition in the tubular cells.

HEMOSTASIS AND COAGULATION ASSAYS

Activated Partial Thromboplastin Time

This assay measures the time required to initiate clotting after citrated plasma is incubated with calcium, a partial thromboplastin (a phospholipid source devoid of tissue factor) and a surface activating agent. Automated instruments detect clot initiation mechanically or based on turbidometric changes.³³ The activated partial thromboplastin time (aPTT) is particularly sensitive to deficiencies of factors VIII and IX, but will also be prolonged by less common deficiencies of factors XI, X, V, prothrombin, and fibrinogen. Mild reductions in factor VIII (>30%) and fibrinogen (>100 mg%), however, may not be detected by this assay.³³ Very rare deficiencies of factors not associated with bleeding, Factor XII, prekallikrein, and high molecular weight will also prolong the aPTT.

The aPTT is also commonly prolonged by anticoagulants (antithrombins such as heparin, hirudin, argatroban, bivalirudin, dabigatran), lupus anticoagulants and less commonly factor-specific antibodies (usually against factor VIII; antibodies against other factors are rare). Persistence of a prolonged aPTT in 1:1 mixing studies (50% patient plasma mixed with 50% normal plasma) suggests the presence of an antibody. Detection of antibodies against factor VIII may require incubation of the 1:1 mix with normal plasma for 1 hour at 37° to allow antibody to bind to Factor VIII. The presence of lupus anticoagulants can be confirmed by demonstrating correction of a prolonged aPTT by the addition of phospholipid (see below). Pan-inhibitors may rarely occur in patients with Waldenström macroglobulinemia or multiple myeloma due to M paraproteins inactivating multiple clotting factors. A rare cause of acquired severe factor IX and X and V deficiencies is absorption of the clotting factors on amyloid deposits of primary systemic amyloidosis.³³

Unfractionated heparin therapy is most frequently monitored using the aPTT to avoid subtherapeutic or supratherapeutic levels. A recurring problem is the variability of the sensitivity of the many available partial thromboplastins to heparin as well as differing results depending on the instruments used. A standard curve must be generated to compare PTT levels with heparin levels as measured by an anti-Xa assay in a group of patients receiving full dose heparin therapy, in order to confirm the therapeutic range of any new aPTT reagent and the instrument used. After testing a number of aPTT reagents in this manner, one study suggested that aPTT test to control ratio of 2.0 to 3.0 is generally a good target range for therapeutic heparin levels.³⁴ Due to concern regarding the variability of the sensitivity of aPTT reagents, many laboratories now monitor heparin levels solely by its capacity to inhibit anti-Xa. The aPTT is not sensitive to low molecular weight heparins (LMWH). When monitoring of LMWH is indicated, as in patients with renal insufficiency, obesity, pregnancy, or prior to elective surgery, an anti-Xa assay for the specific LMWH must be used.

Spurious results of the aPTT are usually due to poorly filled tubes, low plasma to citrate ratios due to high hematocrits, delays in delivery of samples to the laboratory, or contamination with intravenous fluids or heparin.

Prothrombin Time

The prothrombin time (PT) utilizes a more powerful thromboplastin to detect deficiencies of factors VII, V, X, prothrombin, and fibrinogen,³³ and therefore it is valuable in assessing liver function and monitoring warfarin therapy which lowers the Vitamin K dependent factors II, VII, IX, and X, which are synthesized by the liver. Inherited deficiencies of these factors and autoantibodies are rare. Inherited factor VII deficiency, the most common, will be recognized by the isolated prothrombin time prolongation. Deficiencies of factors X, V, prothrombin, and fibrinogen will prolong both the aPTT and the PT.

Some lupus anticoagulants may affect the PT as well as the aPTT; in rare instances, anti-prothrombin antibodies develop, which clear prothrombin from the circulation, causing severe deficiency and bleeding. Also rarely, exposure to bovine thrombin will trigger the development of anti-thrombin and anti-factor V antibodies.

The international normalized ratio (INR) has been useful in standardizing the control of warfarin therapy.³³ The INR is the ratio of the patient PT to the mean normal PT raised to the power of the International Sensitivity Index (ISI) of the thromboplastin reagent utilized. Commercial thromboplastins are calibrated and given an ISI value, which reflects their sensitivity to warfarinized plasma. It may be misleading to use the INR to describe the prolongation of the PT in a patient not receiving warfarin, particularly in patients with liver disease.³³ The PT is vulnerable to the same preanalytic artifacts described above for the aPTT assay.

Activated Clotting Times

Activated clotting times (ACT) are used on site for cardiac surgery and cardiac catheterization procedures.

Thrombin Time

This assay measures the time to initiate clotting by low concentrations of thrombin, which release only fibrinopeptide A and B. The thrombin time is prolonged by low fibrinogen levels or the presence of heparin, paraproteins, dysfibrinogens, or fibrin(ogen) split products.³³ The reptilase time is prolonged by similar molecules but is insensitive to heparin, since only fibrinopeptide A is released from soluble fibrinogen. Dysfibrinogenemia may be confirmed by comparing the antigenic level of fibrinogen with that determined by the thrombin time-based assay. It can be inherited or acquired usually secondary to liver disease.³³

Specific Factor Assays

Most specific factor assays are based on the ability of patient plasma to correct clotting times of specific factor-deficient plasma in PTT or PT-based assays (see Chapter 20). Deficiency of factor XIII, which is important to cross-link fibrin monomers, thereby increasing stability of the clot is not associated with abnormalities of aPTT, PT, or thrombin time. Factor XIII is measured immunologically or by the solubility of the clot formed in urea.³³

Fibrinogen

Fibrinogen levels are routinely determined by using a thrombin time-based assay using high concentrations of thrombin, but chemical or immunologic methods may also be used.³³ As fibrinogen is an acute phase reactant, levels are frequently raised in patients with inflammation and malignancy. Decreased levels are found with disseminated intravascular coagulation, the hemophagocytic syndrome, advanced liver disease, treatment with asparaginase, or rarely as an inherited condition.

Euglobulin Clot Lysis

Euglobulin, a plasma precipitate containing fibrinogen, plasminogen, and plasminogen inhibitor without most fibrinolysis inhibitors, is prepared from the patient sample, and clotted with thrombin; time required for clot lysis is then determined. Because euglobulins lack inhibitors, clot lysis normally occurs quite rapidly (within 90–300 minutes). Abnormally short lysis times occur in states of hyperfibrinolysis such as severe liver disease, but may also simply reflect poor clot formation due to hypofibrinogenemia.

D-Dimer

This immunologic assay detects fibrin split products that are cross-linked because of the action of thrombin and factor XIIIa. Elevated levels indicate extensive local fibrin formation (deep vein thrombosis, pulmonary emboli, pneumonia, postoperative states) or disseminated intravascular coagulation. The value of a positive value in predicting localized thrombosis is poor, particularly in the presence of comorbid conditions such as infection, inflammation, or malignancy. A negative high sensitivity or moderately sensitive D-dimer assay is more helpful in ruling out thrombosis.35,36 A positive D-dimer level has been also used to predict recurrence of thrombosis after discontinuing treatment of an initial unprovoked thrombosis,³⁷ but factors such as age and sex are also important to consider along with the D-dimer level.³⁸

Tests of Platelet Function

Mucocutaneous bleeding in the presence of a normal platelet count may indicate platelet dysfunction. Platelet aggregation in response to epinephrine, ADP, and collagen assessed by light transmission or impedance aggregometry is the most effective method of testing for platelet dysfunction. The availability of aggregometry assays is often limited. They require the patient to be present at the testing laboratory, since platelet-rich plasma or whole blood must be tested rapidly after venipuncture in order to prevent in vitro platelet activation and spurious results.

The bleeding time was frequently used in the past to screen for suspected platelet dysfunction. In the modified Ivy technique, a template is used to make two incisions on the volar surface of the forearm parallel to the antecubital fold, while a blood pressure cuff applied to the upper arm is inflated to 40 mm Hg. Blood is gently removed from the incisions every 30 seconds. The technique is affected by the depth of the incision, the skill of the technologist, and also the skin characteristics of the patient. For example, it may be abnormal in patients with Ehler Danlos disease, osteogenesis imperfecta, and scurvy.³³ Therefore, its reproducibility is a problem; nor has it been shown to be useful as a general screen for hemorrhagic risk.

In the last decade, automated instruments have been developed that may be more reliably used as a screening test for platelet function abnormalities.³³ Many of these instruments appear to be most useful in detecting aspirin use and some have been designed specifically for monitoring antiplatelet therapy, including aspirin and clopidogrel.³⁹ The PFA-100, the earliest instrument and the subject of most studies, measures the time that platelets aspirated at high shear rate through an aperture of a membrane coated with collagen and either epinephrine or ADP aggregate and obstruct further flow (the “closure” time). The PFA-100 is useful in screening for moderate and severe von Willebrand disease (VWD) and monitoring the effect of desmopressin therapy,⁴⁰ although its sensitivity and specificity in screening for milder VWD and rarer platelet secretion and storage function defects has been questioned.41,42 Abnormal findings need to be confirmed by specific testing for VWD disease (VWD antigen, ristocetin cofactor assay, and multimer analysis) or by platelet aggregometry or other specific assays.41,42 Of note, once medications have been excluded as the cause of closure time prolongation, 90% of abnormalities will be due to VWD and 10% due to other platelet defects.

The bleeding time or the automated instruments may also be of use in the initial screening of patients requiring an invasive procedure who have acquired disorders that affect platelet function such as severe renal insufficiency, myeloproliferative disorders, or drug-induced platelet dysfunction.³³

TESTS FOR HYPERCOAGULABILITY

Antithrombin, Protein C, and Protein S

Functional assays of these proteins are more sensitive to deficiency than are antigen-based assays. Patients heterozygous for an inherited defect in one of these factors typically have only modest reductions from the lower limit of normal in factor levels. Nevertheless, they may be at significant risk of venous hypercoagulability (particularly in patients with antithrombin deficiency). Acquired deficiency of antithrombin occurs in disseminated intravascular coagulation, liver disease, heparin therapy, and extensive thrombosis. Acquired deficiency of proteins C and S occurs with vitamin K deficiency, warfarin therapy, and extensive thrombosis. Free protein S deficiency occurs in patients with increased C4b binding protein secondary to inflammation. These assays should not be performed in the setting of acute venous thromboembolism.

Activated Protein C Resistance

Abnormality of this aPTT-based assay is mainly associated with the inherited polymorphism of factor V Leiden. This assay has been replaced by molecular assays for factor V Leiden.

Lupus Anticoagulants

These acquired antibodies to β₂-glycoprotein-1, a protein with a high affinity for phospholipid, can be associated with both arterial and venous hypercoagulability. On the other hand, positive assays are frequently seen in otherwise healthy individuals and may be transient. The functional assays, the dilute Russell viper venom test (DRVTT) and low phospholipid aPTT-based tests (with confirmation by high phospholipid or platelet neutralization tests), may be more sensitive to thrombosis risk than are the serologic tests for anticardiolipin antibodies. Russell viper venom directly activates factor X, bypassing factor VII and the earlier factors in the intrinsic pathway (see Chapters 20 and 22).

Factor V Leiden and Prothrombin G 20210A

The molecular assays for these common mutations, essentially polymorphisms, are not affected by the presence of acute venous thromboembolism.

TESTS FOR EVALUATION OF PATIENTS WITH HEMATOLOGIC MALIGNANCIES

Serum Protein Electrophoresis

Electrophoresis separates proteins mainly on the basis of electric charge, which can be performed by agarose gel electrophoresis or the automated capillary zone electrophoresis.¹⁹ When normal plasma proteins are electrophoresed on agarose films and stained with a protein-binding dye, six zones appear. Albumin, α₁, α₂, and two β globulins appear as 5 discrete bands, and γ globulins migrate as a more diffuse, electrophoretically heterogeneous band(Fig. 27.2 and Table 27.3). Monoclonal immunoglobulins (M paraproteins) are recognized because they form discrete bands in the β or γ globulin regions. The concentration of the proteins in normal or the paraprotein bands (“spikes”) can be determined by densitometry. When hyperglobulinemia is detected by routine total protein and albumin measurements, protein electrophoresis is essential to distinguish between reactive polyclonal from monoclonal processes. In order to confirm monoclonality, immunofixation studies are required to demonstrate that the suspected monoclonal band contains only a single heavy chain and a single light chain (Fig. 27.3). The capillary zone electrophoresis is usually more sensitive than agarose gel electrophoresis, particularly for small paraproteins, but false negative results have been reported.⁴³ Poor correlation with nephelometric immunoglobulin quantitation tests may alert to false negative results in capillary or agar electrophoresis when monoclonal proteins have atypical mobility due to temperature, or pH, or unknown factors. Falsepositive artifacts resembling spikes are more frequent in agarose electrophoresis, which will require immunofixation to be recognized.

The presence of a monoclonal immunoglobulin is consistent with, but not diagnostic of, the presence of a plasma cell or lymphoid malignancy or light-chain amyloidosis. Most monoclonal proteins with serum concentrations below 3 g/dL are not associated with clinical or pathologic evidence of malignancy, and they are referred to as monoclonal gammopathy of unknown significance (MGUS), because of the potential for future malignant transformation of the plasma cell clone. Occasional individuals exhibit two monoclonal proteins (“diclonal” gammopathy), which may represent the products of two separate clones. If both bands contain the same heavy and light chain, the two immunoglobulins may originate from a single clone despite differing electrophoretic mobilities, possibly due to multimer formation. Patients with marked polyclonal gammopathy, for example due to HIV infection or liver disease, may have multiple small discrete bands termed oligoclonal gammopathy. The majority of the γ globulin in these individuals is polyclonal.

FIGURE 27.2 Serum protein electrophoresis.

M paraproteins of greater than 3.0 g/dL usually reflect the presence of IgG or IgA multiple myeloma or Waldenström macroglobulinemia. The concentration of the M protein in the plasma (or urine) is a marker of tumor burden, and serial monitoring by electrophoresis is extremely important in assessing response to therapy. For this determination to be reliable, the M paraprotein must be quantitated separately from polyclonal immunoglobulins.

Table 27.3  Plasma Protein Migration Patterns on Standard Protein Electrophoresis

Albumin Zone

Albumin

Alpha₁ Zone

Alpha₁-antitrypsin

Alpha₁-lipoproteins (high density lipoproteins HDL)

Alpha₂ Zone

Alpha₂-macroglobulin

Haptoglobin

Ceruloplasmin

Beta Zone

β-Lipoprotein (low density lipoprotein)

Transferrin

C3 (complement)

Gamma Zone

Fibrinogen (in incompletely clotted specimens)

IgA

IgM

IgG

FIGURE 27.3 Immunofixation illustrating an IgG kappa monoclonal protein.The arrowhead points to the putative monoclonal band in the serum protein electrophoresis (SP lane) stained with a protein-binding dye. Staining in the remaining lanes indicates the presence of products reacting with specific antibodies directed against IgG (G), IgA (A), and IgM (M) heavy chains, and Ig kappa (κ) and lambda (λ) light chains. Small arrow on the right indicates origin.

Urine Protein Electrophoresis

Urinary light-chain excretion must be evaluated in patients with hypogammaglobulinemia or other findings suspicious of a plasma cell dyscrasia. Approximately 15% of myeloma patients excrete monoclonal light chains in the urine (Bence Jones proteinuria) in the absence of any detectable M protein in the serum. For screening, a random urine specimen is concentrated before electrophoresis. Discrete bands are then assayed by immunofixation to confirm whether they represent intact monoclonal immunoglobulins (due to “overflow” of the serum M protein) or free light chains. If a paraprotein is present, serial 24-hour urine collections are useful in monitoring tumor burden and response to therapy.

Immunofixation

Immunofixation replaced immunoelectrophoresis to confirm monoclonality of discrete bands noted in the β or γ globulin regions of the protein electrophoresis patterns. Antibodies against the γ, α, μ heavy chains, and κ and λ light chains are layered separately on membranes containing the electrophoresed samples. A monoclonal immunoglobulin will form immunofixation bands with antibodies against one heavy chain class and/or one light-chain type (Fig. 27.3). IgM and IgA proteins are more likely to be found close to the β globulin region; IgG proteins may be located in any area of the β and γ globulin zones. Free light chains are seen in the serum protein electrophoresis only in myeloma patients with severe renal failure or in instances where the light chains form spontaneous tetramers too large for renal clearance. The uncommon IgD and very rare IgE myelomas should be suspected when a serum paraprotein binds only an anti-light-chain antibody.

Serum Free Light Chains

This nephelometric automated immunoassay depends on the use of antibodies to κ and λ light chains that react with epitopes that are exposed in free light chains but hidden in the intact monoclonal immunoglobulins. Because of its superior convenience and comparable sensitivity to urine electrophoresis, it is being used frequently to monitor response to treatment of light-chain myeloma and amyloidosis.44,45 The assay also has diagnostic and prognostic utility in amyloidosis, monoclonal gammopathy, and smoldering multiple myeloma.⁴⁶ The concentration of serum free light chain over time have been noted to have a greater variability than M paraproteins do for monitoring of stable monoclonal gammopathy patients and therefore are less useful in detecting progression in these patients.⁴⁷

Quantitative Serum Immunoglobulins

Quantitative serum immunoglobulins are measured with automated nephelometric or turbidometric immunoassays; these assays are most useful for quantitating normal immunoglobulins rather than to detect M paraproteins. For example, IgA M proteins that form multimers and monoclonal IgM pentamers with a propensity to disassociate into smaller molecular weight species can produce erroneous results using this assay, since the immunoglobulin quantitation can be affected by changes in the molecular weight.

Serum Cryoglobulins

The most critical step in this test is the treatment of the specimen before it reaches the laboratory. The blood should be drawn into a preheated syringe or warm tube, transported to the laboratory at 37°C and kept at this temperature until the serum is separated from the clot. The serum is then refrigerated at 4°C and examined after 24 hours. A precipitate that dissolves when the tube is rewarmed to 37°C indicates the presence of a serum cryoglobulin. Electrophoresis and immunofixation of the separated and redissolved cryoprecipitate will reveal the immunoglobulins involved in the cryoglobulin formation. Cryoglobulinemia may due to (i) a monoclonal immunoglobulin, usually IgM, (ii) a monoclonal IgM with rheumatoid activity binding to polyclonal IgG (“mixed cryoglobulinemia”), or (iii) polyclonal IgM bound to polyclonal IgG. Mixed cryoglobulinemia (ii and iii) are associated with a variety of lymphoproliferative or autoimmune disorders and infections, particularly hepatitis C.⁴⁸

Serum Viscosity

The Ostwald viscosimeter measures viscosity by comparing the time required for serum and water to flow through a capillary tube at 37°C. Normal serum is 1.4 to 1.8 times more viscous than water. Symptoms resulting from hyperviscosity generally occur when the relative viscosity exceeds 6 but may occur with a relative viscosity as low as 3 or 4.

Serum β₂-Microglobulin

Serum β₂-microglobulin is a small protein noncovalently linked with class I human leukocyte antigen (HLA) molecules. Serum levels are measured by nephelometric immunoassay. Elevated levels may be found in inflammatory conditions, renal failure (due to failure of excretion) and in lymphoid and plasma cell malignancies. High serum β₂-microglobulin is an important prognostic feature indicating advanced disease and poor response to therapy in myeloma and certain lymphomas. In myeloma, elevated β₂-microglobulin levels predict early relapse following autologous transplantation. Serum levels are not useful however, to follow chemotherapy responsiveness in myeloma because of the lack of specificity.

Serum Lactic Dehydrogenase

This protein is measured by an automated enzyme activity-based assay. The normal range varies depending on the type of assay. Elevated serum levels reflect necrosis of cells rich in the enzyme. The most marked elevations (more than 5 times normal) usually are noted with severe megaloblastic anemia, intravascular hemolytic anemia, or hemophagocytic syndromes. Similar levels can be seen in acute leukemia and lymphomas often as harbingers of tumor lysis syndrome. An elevated serum level is a risk factor in the International Prognostic Index for non-Hodgkin lymphoma. Distinguishing the five isoenzymes of lactic dehydrogenase is seldom useful in diagnosing hematologic disorders.

Serum Uric Acid

Serum uric acid is measured by an automated enzyme assay. Elevated serum levels particularly over 10 mg/dL in patients with hematologic malignancies should raise concern for increased cell turnover (tumor lysis syndrome) and incipient renal compromise due to uric acid precipitation. Hypouricemia may occur in Hodgkin lymphoma. Bence Jones (light chain) proteinuria rarely can cause an interstitial nephritis with a proximal tubular defect resulting in hypouricemia due to marked hyperuricosuria along with other features including polyuria, glycosuria, and aminoaciduria, known as the Fanconi syndrome.

NEUTROPHIL EVALUATION

Marrow Granulocyte Reserve

Marrow granulocyte reserve may be assessed by the hydrocortisone stimulation test⁴⁹ or by the response to filgrastim.⁵⁰ In the hydrocortisone stimulation test, the absolute neutrophil concentration is measured before administering 200 mg hydrocortisone intravenously and again 3, 4, and 5 hours later. A failure of the neutrophil concentration to increase by at least 1,600 neutrophils per microliter indicates poor marrow granulocyte reserve. Similarly, failure of the absolute granulocyte level to rise above 5,000/μL 24 hours after a subcutaneous injection of 5 mcg/kg filgrastim indicates increased risk for chemotherapy-induced febrile neutropenia.

Leukocyte Alkaline Phosphatase

This assay is an inexpensive cytochemical test of peripheral blood neutrophils used to screen patients with leukocytosis suggesting a diagnosis of chronic myelogenous leukemia. The assay depends on the enzyme’s ability to cleave a dye, which then stains the cells. Individual neutrophils are scored by the intensity of staining as 0 to 4+, and the sum of the scores of 100 cells are tallied. Neutrophils from patients with chronic myelogenous leukemia or paroxysmal nocturnal hemoglobinuria have low levels of leukocyte alkaline phosphatase (<10 Kaplow units). Neutrophils from patients with reactive leukocytosis and polycythemia vera have elevated scores (>80 Kaplow units). Patients with other myeloproliferative disorders may have normal, low, or high levels. This assay has been largely replaced by the fluorescence in situ hybridization (FISH) assay for BCR-ABL in peripheral blood neutrophils or cytogenetic analysis of the bone marrow. The availability of highly effective therapy for chronic myelogenous leukemia warrants the use of the more specific, albeit expensive FISH assay⁵¹ (see Chapter 13).

References

  1.  Wintrobe MW. The principles and technic of blood examination. In: Wintrobe MW, ed. Clinical Hematology. Philadelphia, PA: Lea and Febiger; 1942:177-214.

  2.  Bourner G, Dhaliwal J, Sumner J. Performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4-way, side-by-side study. Lab Hematol. 2005;11(4):285-297.

  3.  Simel DL, Halvorsen RA Jr, Feussner JR. Erythrocyte anisocytosis. Visual inspection of blood films vs automated analysis of red blood cell distribution width. Arch Intern Med. 1988;148(4):822-824.

  4.  Bessman JD, Feinstein DI. Quantitative anisocytosis as a discriminant between iron deficiency and thalassemia minor. Blood. 1979;53(2):288-293.

  5.  Crouch JY, Kaplow LS. Relationship of reticulocyte age to polychromasia, shift cells, and shift reticulocytes. Arch Pathol Lab Med. 1985;109(4):325-329.

  6.  Piva E, Brugnara C, Chiandetti L, Plebani M. Automated reticulocyte counting: state of the art and clinical applications in the evaluation of erythropoiesis. Clin Chem Lab Med. 2010 Oct;48:1369-80.

  7.  Thompson CB, et al. The role of anticoagulation in the measurement of platelet volumes. Am J Clin Pathol. 1983;80(3):327-332.

  8.  Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol. 2004;126:93-99.

  9.  Barsam SJ, Psaila B, Forestier M, et al. Platelet production and platelet destruction: assessing mechanisms of treatment effect in immune thrombocytopenia. Blood. 2011;117:5723-5732.

10.  Zandecki M, Genevieve F, Gerard J, Godon A. Spurious counts and spurious results on haematology analysers, a review. Part I: platelets. Int J Lab Hematol. 2007;29:4-20.

11.  Zandecki M, Genevieve F, Gerard J, Godon A. Spurious counts and spurious results on haematology analysers, a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices, and reticulocytes. Int J Lab Hematol. 2007;29:4-20.

12.  Gulati GL, et al. Changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature. Arch Pathol Lab Med. 2002;126(3):336-342.

13.  Silvestri F, et al. Incidence and diagnosis of EDTA-dependent pseudothrombocytopenia in a consecutive outpatient population referred for isolated thrombocytopenia. Vox Sang. 1995;68(1):35-39.

14.  Bain BJ. Bone marrow biopsy morbidity and mortality. Br J Haematol. 2003;121(6):949-951.

15.  Islam A. Bone marrow aspiration before bone marrow core biopsy using the same bone marrow biopsy needle: a good or bad practice? J Clin Pathol. 2007;26:212-215.

16.  Hughes DA, Stuart-Smith SE, Bain BJ. How should stainable iron in bone marrow films be assessed? J Clin Pathol. 2004;57(10):1038-1040.

17.  Barron BA, Hoyer JD, Tefferi A. A bone marrow report of absent stainable iron is not diagnostic of iron deficiency. Ann Hematol. 2001;80(3):166-169.

18.  Nguyen D, Diamond LW, Braylan RC. Approach to flow cytometry: general considerations. In: Nguyen D, Diamond LW, Braylan RC, eds. Flow Cytometry in Hematopathology. A Visual Approach to Data Analysis and Interpretation. 2nd ed. Totowa, NJ: Humana Press; 2007:2-9.

19.  Burtis CA, Ashwood ER, Bruns DE, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed. Saint Louis: Elsevier Saunders Company; 2012.

20.  Hod EA, Brittenham GM, Billote GB, et al. Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non-transferrin-bound iron. Blood. 2011;118:6675-6682.

21.  Cook JD. Diagnosis and management of Iron deficiency. Best Pract Res Clin Haematol. 2005;18:319-332.

22.  Finberg KE. Unraveling mechanisms regulating systemic iron homeostasis. Hematology Am Soc Hematol Educ Program. 2011;2011:532-537.

23.  Skikne BS, Punnonen K, Caldron PH, et al. Improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: a prospective multicenter evaluation of soluble transferrin receptor and the sTfR/log ferritin index. Am J Hematol. 2011;86:923-927.

24.  Siebert S, Williams BD, Henley R, et al. Single value of serum transferrin receptor is not diagnostic for the absence of iron stores in anaemic patients with rheumatoid arthritis. Clin Lab Haematol. 2003;25:155-160.

25.  Pfeiffer CM, Cook JD, Mei Z, et al. Evaluation of an automated soluble transferrin receptor (sTfR) assay on the Roche Hitachi analyzer and its comparison to two ELISA assays. Clin Chim Acta. 2007;382:112-116.

26.  Guyatt GH, Patterson C, Ali M. Diagnosis of iron deficiency anemia in the elderly. Am J Med. 1990;88:205-209.

27.  Roetto A, Bosio S, Gramalglia E. Pathogenesis of hyperferritinemia cataract syndrome. Blood Cells Mol Dis. 2002;29:532-535.

28.  Brudevold R, Hole T, Hammerstrom J. Hyperferritinemis is associated with insulin resistance and fatty liver in patients without iron overload. PLoS One. 2008;3:e3547.

29.  Stabler SP, Allen RH, Savage DG, et al. Clinical spectrum and diagnosis of cobalamin deficiency. Blood. 1990;76:871-881.

30.  McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Hematol Educ Program. 2009;2009:629-635.

31.  Chui DHK, Steinberg, MH. Laboratory diagnosis of hemoglobinopathies and thalassemias. In: Hoffman R, et al., eds. Hematology: Basic Principles and Practices. Philadelphia, PA: Elsevier, Churchill Livingstone; 2009.

32.  Beutler E. Glucose-6-phosphate dehydrogenase deficiency. A historical perspective. Blood. 2008;111:16-24.

33.  Schmaier AH. Laboratory evaluation of hemostatic disorders. In: Hoffman R, et al., eds. Hematology: Basic Principles and Practices. Philadelphia, PA: Elsevier, Churchill Livingstone; 2009.

34.  Bates SM, Weitz JI, Johnston M, et al. Use of a fixed activated partial thromboplastin time ratio to establish a therapeutic range for unfractionated heparin. Arch Intern Med. 2001;161(3):385-391.

35.  Rathbun SW, Whitsett TL, Raskob GE. Exclusion of first-episode deep-vein thrombosis after-hours using D-Dimer. Blood Coagul Fibrinolysis. 2007;16(8):795-800.

36.  Bates SM, Jaeschke R, Stevens SM, et al. Diagnosis of DVT: Antithrombotic therapy and prevention of thrombosis. 9th ed: American College of Chest Physicians evidence based clinical practice guidelines. Chest. 2012;141(2 suppl): e351S-418S.

37.  Palareti G, Cosmi B, Legnani C, et al. D-Dimer testing to determine the duration of anticoagulation therapy. N Engl J Med. 2006;355:1780-1790.

38.  Baglin T, Palmer CR, Luddington R, Baglin C. Unprovoked recurrent venous thrombosis: prediction by D-Dimer and clinical risk factors. J Thromb Haemost. 2008;66:577-582.

39.  Chen F, Maridakis V, O’Neill EA, et al. A randomized clinical trial comparing point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel. Platelets [epub ahead of print]. September 2011.

40.  Chen F, Maridakis V, O’Neill EA, et al. A randomized clinical trial comparing point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel. Platelets. 2012;23:249-58.

41.  Favaloro EJ. Clinical utility of the PFA-100R. Semin Thromb Hemost. 2008;34:709-733.

42.  Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function. J Thromb Haemostas. 2006;4:312-319.

43.  Bossuyt X, Marien G. False-negative results in detection of monoclonal proteins by capillary zone electrophoresis: a prospective study. Clin Chem. 2001;47:1477-1479.

44.  Bradwell AR, Carr-Smith HD, Mead GP, et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem. 2001;47(4):673-680.

45.  Bradwell AR, Carr-Smith HD, Mead GP, et al. Serum test for assessment of patients with Bence Jones myeloma. Lancet. 2003;361(9356):489-491.

46.  Kyle RA, Rajkumar SV. Monoclonal gammopathy of unknown significance and smouldering multiple myeloma: emphasis on risk factors for progression. Br J Haematol. 2007;139:730-743.

47.  Katzmann JA, Snyder MR, Rajkumar SV, et al. Long-term biological variation of serum protein electrophoresis M-spike, urine M-spike, and monoclonal serum free light chain quantification: implications for monitoring monoclonal gammopathies. Clin Chem. 2011;57:1687-1692.

48.  Ferri C, Zignego AL, Pileri SA. Cryoglobulinemia. In: Young NS, Gershon SL, High KA, eds. Clinical Hematology. Mosby Elsevier, Philadelphia PA; 2006:625-636.

49.  Mason BA, Lessin L, Schechter GP. Marrow granulocyte reserves in black Americans. Hydrocortisone-induced granulocytosis in the “benign” neutropenia of the black. Am J Med. 1979;67:201-205.

50.  Hansen PB, Johnsen HE, Ralfkiaer E, et al. Blood neutrophil increment after a single injection of rhG-CSF or rhGM-CSF correlates with marrow cellularity and may predict the grade of neutropenia after chemotherapy. Br J Haematol. 1993;84:581-585.

51.  Tkachuk DC, Westbrook CA, Andreef M, et al. Detection of bcr-abl in chronic myelogenous leukemia by in situ hybridization. Science. 1990;250:559-562.

28

Basic Principles and Clinical Applications of Flow Cytometry

Thomas A. Fleisher and Raul C. Braylan

Flow cytometry is a technology used routinely in most hematology laboratories. Its entry into the mainstream of clinical laboratory analysis has been aided by the increasing availability of monoclonal antibodies that define cell surface and intracellular proteins as markers of cell lineage, differentiation, activation, and other biologic properties. Instrument design advances have yielded benchtop cytometers with fixed optics that, when linked with new developments in fluorochrome chemistry, enable a wide range of clinical applications. In addition, proficiency testing is now available in support of these clinical applications through the College of American Pathologists as mandated by the Clinical Laboratory Improvement Amendment of 1988. The major advantage that flow cytometry provides is its capacity to assess multiple measurements on large numbers of individual cells. Flow cytometric studies have extended the understanding of hematopoietic cell development, differentiation, activation, and apoptosis. In addition, they have provided important information regarding hematologic malignancies, insight into reconstitution after stem cell transplantation, and understanding of cell abnormalities that result in immune or hematologic deficiencies. As such, flow cytometry plays an important role in the diagnosis, characterization, and monitoring of a number of hematologic disorders.

The basic design of a flow cytometer involves four major elements: optics, fluidics, electronics, and a computer equipped with specialized software.1,2 The optical system utilizes one or more light sources, typically one or several lasers that produce monochromatic light and serve as the excitation beams. At the opposite side of the optical bench, light generated from the cells that have intersected the excitation beam is filtered, reflected by dichroic mirrors set in fixed locations, and finally collected by linked photodetectors to allow quantitation of the emitted light at specific wavelengths. To ensure that all cells analyzed experience consistent exposure to the excitation beam, the fluidic system must maintain the cells in a consistent location as they move sequentially through the beam. To accomplish this, the cell suspension is injected into a flowing stream of sheath fluid that hydrodynamically focuses the inner stream of cells within the outer sheath fluid stream.¹ The intersection of the cells with the excitation light beam(s) produces characteristic light scatter (nonfluorescent) signals; additional fluorescent signals are generated by fluorochromes that typically are linked to specific reagents that bind antigens present on or within the cells of interest. The various light signals (parameters) are collected by the optical bench, while instrument design determines the number of parameters collected per cell. The two reagent-independent (nonfluorescent) parameters are forward-angle light scatter, as a marker of cell size, and side-angle light scatter, as an index of cellular regularity/ granularity. The combination of these two parameters allows for an approximate discrimination among the three major types of leukocytes as well as evaluation of red blood cells and platelets in whole blood samples.³

The fluorescent data collected by a flow cytometer are the result of either cell surface or intracellular binding of antibodies or other specific ligands conjugated directly to fluorochromes or detected with secondary reagents conjugated to fluorochromes, as well as reagents that are inherently fluorescent. Fluorochromes are excited by light of a defined wavelength and emit light of lower energy (longer wavelength). There are currently many different fluorochromes used in clinical flow cytometry, including fluorescein isothiocyanate, phycoerythrin, peridin chlorophyll protein, and allophycocyanin. More recently, combinations of two fluorochromes linked to each other have been developed; they depend on the transfer of energy from the first fluorochrome to excite the second fluorochrome. These tandem fluorochromes extend the range of emission wavelengths available from one excitation beam. The availability of multiple fluorochromes that absorb light of the same wavelength but emit light at different wavelengths means that multiple reagents can be used simultaneously with a single light source to yield a multicolor (polychromatic) study. One or more additional light sources are present in most current clinical instruments to extend the range of multicolor studies. Extended multicolor studies require complex color compensation and data management processes that typically involve sequential data evaluation.

The clinical application of flow cytometry in hematology saw its earliest use as a supplement to the morphologic classification of leukemias and lymphomas, affording not only lineage information but also the state of differentiation and/or maturation,4,5 cell growth and apoptosis.⁶ In addition, flow cytometry provided the best prognosticator in human immunodeficiency virus (HIV) infection based on absolute CD4 T cell numbers.⁷ More recently, flow cytometry has proven important in characterizing hematopoietic stem cells, detecting minimal residual neoplastic disease, defining immune deficiencies, identifying certain red blood cell related disorders, measuring the number of contaminating leukocytes in plasma and red blood cell transfusions, evaluating platelets and characterizing other blood cells.⁸⁻¹² Flow cytometry also can be used to look inside the cell as well as to evaluate cell surface characteristics. Fixation and permeabilization facilitate intracellular entry of reagents to determine the presence of specific proteins and to assess functional characteristics.¹³ This chapter is directed at basic concepts of flow cytometry including data presentation and interpretation followed by a brief review of applications for hematologists.

DATA PRESENTATION AND INTERPRETATION

Current flow cytometers generally yield graphic displays of the cell frequency versus the light intensity for one or more parameters by means of specialized computer software. Figure 28.1, shows a single-parameter histogram that reflects the quantitative distribution of cells (y-axis) versus light signal strength (x-axis). Alternatively, the signal intensity of two correlated parameters can be plotted versus cell frequency, the latter displayed as dot density (dot plot, Fig. 28.2A) or a series of concentric lines (contour plot, Fig. 28.2B). When measuring multiple parameters (as colors), the data are typically evaluated by using dual-parameter displays. Typically, 10,000 to 20,000 events are collected to provide sufficient numbers of cells for meaningful data relative to the subpopulations of interest. However, when the cell or cells of interest are infrequent, such as evaluating hematopoietic stem cells (CD34⁺) in peripheral blood or detecting minimal residual disease (MRD) in leukemia, substantially larger total numbers of cells must be collected.8,14

Distinguishing a positive signal is usually based on defining background signals by evaluating either unstained cells (no monoclonal antibody added) or cells that have been incubated with a fluorochromeconjugated irrelevant antibody. By convention, the negative–positive discriminator is defined by the intensity of a signal that includes 99% (or 98%) of all cells based on one of the background conditions described above; cells that emit a signal above this discriminator are scored as positive for binding by the specific reagent(s) added to the cell suspension. This approach applies to well-defined populations with homogeneous antigen expression, but modifications or alternative interpretations may be necessary when the cell populations analyzed are heterogeneous or express dim fluorescence, which can lead to rather arbitrary calculations of percentages of positive cells.

The data generated by the computer are only as good as the instrument capabilities, settings, reagents, and cell preparation used. To prevent reporting invalid data, certain standards must be met.¹⁵ First, optimal instrument performance is integral and depends on a quality control program utilizing specialized software and methods. The use of validated reagents is also part of good laboratory practices, while the quality of the cell preparation can be assessed using the nonfluorescent parameters, forward- and side-angle light scatter, to confirm the presence of the population of interest. Each major blood cell type has distinctive features in this scatter plot. Platelets are obviously smaller than all other blood cells and heterogeneous in size, characteristics that can be confirmed in comparison to red blood cells. Erythrocytes have a characteristic appearance based on forward- and side-angle light scatter (Fig. 28.3A), which partially overlaps with that of lymphocytes. However, because of the large difference in frequency of circulating erythrocytes and leukocytes, there is no practical concern of lymphocytes contaminating erythrocytes (a collection of 10,000 erythrocytes normally would include less than 20 lymphocytes). In contrast, the presence of erythrocytes makes evaluation of lymphocytes virtually impossible. In part because of this reason, the study of lymphocytes in a whole blood sample or other specimens containing a significant amount of blood typically involves a red blood cell lysis step to eliminate erythrocytes (Fig. 28.3B). Following successful red cell lysis, a three-part differential is observed in peripheral blood with normal lymphocytes representing the smallest (forward-angle light scatter) and most regular/ agranular (side-angle scatter) cells, while granulocytes are slightly larger (higher forward-angle scatter) and show substantial granularity (high side-angle scatter), and monocytes fall between these two cell types (Fig. 28.3B). The two less prevalent granulocyte types differ in their location on a scatter plot, with eosinophils typically falling within the granulocyte population while basophils overlap with lymphocytes. Hematopoietic stem cells are normally found in the lymphocyte area of the scatter plot. It is important to recognize that the cell relationships noted above do not necessarily apply in cases of hematopoietic malignancy since neoplastic cells may exhibit altered light scatter properties or appear as a distinct population, separate from normal elements. Current standard practice for identifying the major leukocyte types, and particularly lymphocytes, includes using the pan-leukocyte monoclonal antibody CD45, alone or in combination with the monocyte-specific antibody CD14. CD45 is usually included in each staining combination as a specific lymphocyte identifier (characteristically bright staining) when red cell lysis is inadequate (Fig. 28.4) or substantial numbers of non-lymphocytes or debris contaminate the lymphocyte gate.¹⁶ Malignant cells also may differ from their normal counterparts in their staining characteristics with a variety of reagents, including CD45 expression (Figs. 28.5 and 28.6). The correlated analysis of side scatter and CD45 with or without appropriate sequential gating of subpopulations of interest is extremely helpful in recognizing hemopoietic and lymphoid neoplasia.¹⁷

FIGURE 28.1 Single-parameter histogram, a distribution plot of CD3 fluorescence intensity (x-axis) versus number of events/cells (y-axis) evaluating lymphocytes.

FIGURE 28.2 A. Dot plot of two-color (CD4 and CD8) staining evaluating lymphocytes. Frequency of events is reflected by the number of dots. B. Contour plot of two-color (CD4 and CD8) staining evaluating lymphocytes. Frequency of events is reflected by the contour levels.

FIGURE 28.3 (A) Dot plot of forward scatter (x-axis) versus side scatter (y-axis) on nonlysed whole blood sample (B) dot plot of forward scatter (x-axis) versus side scatter (y-axis) on lysed whole blood sample demonstrating a three-part leukocyte differential: lymphocytes, monocytes, and granulocytes.

FIGURE 28.4 Side scatter and CD45 analysis of normal peripheral blood demonstrates distinct clusters of granulocytes, monocytes, lymphocytes, and residual red cells.

The interpretation of fluorescent data based on antibody binding reflects the biology of the particular cognate cell surface protein. When the monoclonal reagent identifies exclusively one cell population, data interpretation is unambiguous, as for the pan-T-cell marker CD3 shown in Figure 28.1. In this example, when the evaluation is confined to lymphocytes using a lymphocyte gate, there clearly are two populations, CD3 negative cells, including B and natural killer (NK) lymphocytes, and CD3 positive T cells. In other situations, biologic variability in surface protein expression impacts data interpretation; examples are shown in Figures 28.7 and 28.8. In both histograms, there are at least three cell populations: cells negative for the marker, cells showing intermediate fluorescence, and cells that have bright fluorescence. In Figure 28.7, the CD8 intermediate cells are predominantly NK cells, while the bright staining cells are primarily CD8 positive T cells. In Figure 28.8A, the CD4 intermediate staining cells are monocytes, while the bright staining cells are T cells, with the former being present only in very small numbers with proper gating on lymphocytes (Fig. 28.8B). The finding of low-density CD4 expression on monocytes helps to explain HIV infection of this cell lineage.

Many monoclonal antibodies, individually or in combination, can serve to distinguish cells of a specific lineage (Table 28.1), and characteristic binding features can be used to direct a flow cytometry study to a specific cell population. As mentioned above, nonfluorescent parameters of forward-angle and side-angle scatter help distinguish among lymphocytes, monocytes, granulocytes, and platelets.³ Within the granulocyte population, neutrophils and eosinophils can be discriminated by the differential expression of the complement receptor CD16: neutrophils stain for CD16 while eosinophils are negative.¹¹ Cells of the erythroid lineage can be identified based on the expression of glycophorin. Within the lymphocyte population, lineage-specific antibodies differentiate various populations and subpopulations. Hematopoietic stem cells can be identified by the expression of the cell surface protein, CD34, a valuable marker that has enabled evaluation and ex vivo isolation of bone marrow or mobilized circulating hematopoietic stem cells for transplantation.⁸

FIGURE 28.5 Analysis of side scatter and CD45 of peripheral blood from a patient with acute myeloid leukemia demonstrates charcteristic blasts (myeloblasts).

FIGURE 28.6 (A) Side scatter and CD45 analysis of peripheral blood from a patient with chronic lymphocytic leukemia shows increased lymphocytes. A lymphocyte gate (oval selection) is used for further analysis (B) that demonstrates that the majority of the lymphocytes abnormally coexpress CD19 and CD5, which is a typical feature of chronic lymphocytic leukemia (CLL).

FIGURE 28.7 CD8 histogram evaluating lymphocytes.

Many of the monoclonal reagents used to evaluate hematopoietic elements detect antigens that are not exclusively expressed on one specific cell type, and interpretation of data must incorporate knowledge of different surface protein expression patterns. A combination of additional antibodies often clarifies the relative expression of different antigens on specific cell populations. Cell surface proteins may be altered under different circumstances during the life cycle of a cell, including preferential expression early and/or late during differentiation, expression in response to cell activation and/or in various states of cell-specific function. Protein upregulation implies a range of expression that could include cells transforming from negative to clearly positive, depending on the temporal pattern of expression. For example, the α chain of the interleukin-2 receptor (CD25) shows such a pattern on T cells (Fig. 28.9), but the interpretation of CD25 expression as an activation marker has been complicated by the identification of T regulatory cells among CD25 expressing CD4+ T cells.¹⁸ When the interpretation of positive and negative is visually less clear, consistent interpretation criteria are crucial for valid comparison of data between different studies. In some circumstances, isoforms of a specific protein are differentially expressed, and cells may express one or the other isoform or both (Fig. 28.10).

As mentioned above, sometimes the use of percentage positive for a specific marker is misleading, as shown in Figure 28.11, where the histogram for the unstained cells overlaps significantly with that of stained cells. The histogram overlay demonstrates that there is a shift in the stained cells that would not be adequately reflected by simply scoring cells as positive or negative. Currently, many laboratories typically note the geometric mean channel (GMC) fluorescence of the unstained and stained cells and then report the cells to be positive for the specific marker with an increased fluorescence of x-fold over background (based on the quotient of the GMC-stained cells divided by the GMC of unstained or irrelevant antibody-treated cells). These considerations are particularly relevant for many markers used to evaluate malignant cells. In fact, consensus groups have repeatedly emphasized that reporting percentage values when interpreting results in hematopoietic malignancies is generally unsatisfactory.19,20 These values may not be sufficiently informative to allow the detection of neoplastic cells and cannot adequately describe the phenotype of the malignant cells. For this reason, it is recommended that interpretation of flow cytometric results in hematopoietic and lymphoid malignancies be based on the visual examination of the plots for each of the antibodies used, and that the results be primarily descriptive, in a manner similar to the microscopic evaluation of cells or tissues. Numerical values are only used to indicate the fraction of neoplastic cells or other well-defined cell populations present in the sample.

Flow cytometry has also been applied to investigate intracellular characteristics and, specifically, the presence of proteins which may only be detected intracellularly¹³ or as well as those that ultimately are also expressed on the cell surface. In addition, there are a series of reagents that bind to DNA and allow assessment of cell-cycle status.²¹ More recently, intracellular flow cytometry has been applied to measure some functional properties of cells, including the detection of intracellular cytokines following cell stimulation and cell-activation-specific processes such as calcium flux, pH changes, and phosphorylation of intracellular signaling proteins,¹³ but these applications currently have limited applications in routine clinical laboratory practice. Certain intracellular proteins that are not expressed on the cell surface serve diagnostic or prognostic purposes in malignant conditions and are often used clinically; these include terminal deoxynucleotidyl transferase,²² bcl-2,²³⁻²⁵ and ZAP-70.26,27

FIGURE 28.8 A. CD4 histogram evaluating mononuclear cells (lymphocytes and monocytes). B. CD4 histogram evaluating only lymphocytes.

APPLICATIONS OF FLOW CYTOMETRY IN HEMATOLOGY

Flow cytometry is of value in the evaluation of numerous hematologic conditions, but no other diseases have benefited more from the use of this technology than hematopoietic and lymphoid neoplasias. Flow cytometry has revolutionized the manner in which we diagnose, classify, and monitor acute leukemia or lymphoproliferative disorders, and it is rare now that patients with these disorders are treated without including the flow cytometric data. The technology is rapid, quantitative, and can analyze simultaneously multiple antigens in a large number of cells, allowing the easy detection, characterization, and enumeration of malignant cells, even when admixed with normal elements. The ability of flow cytometry to recognize malignancy is based on its capacity to distinguish differences in antigen expression between normal and neoplastic cells. Normal hematopoietic cells originate from a stem cell in the marrow that subsequently gives rise to a progeny of different cell lineages. These cell progenitors traverse various developmental stages and ultimately evolve into mature elements in the circulation and other peripheral organs. As the hematopoietic cells develop and differentiate, they undergo changes in their surface or intracellular antigenic profile that is characteristic of their lineage and differentiation stage. Hematopoietic malignancies are clonal cell populations that express similar antigens to those of their non-neoplastic counterparts but usually with a different expression pattern that is unique for each type of neoplasia. This antigen expression can be increased, decreased, absent, asynchronous, or may be of a different cell lineage. Thus, knowledge of the immunophenotype of a cell together with its physical properties revealed by light scatter signals allows determination of not only their lineage and developmental stage but also, in most instances, their normal or neoplastic nature. Furthermore, in T- or B-cell lymphoproliferative disorders, the clonal nature of the lymphocytes can be established by recognizing the restriction of expression of immunoglobulin light chains²⁸ or T-cell receptor beta chains.²⁹ The identification of monoclonal lymphoid expansions simultaneously with other informative antigens has proven extremely useful in the differential diagnosis between benign and malignant lymphoid disorders³⁰ not only in marrow and blood³¹ but also in lymph nodes³² and extranodal lymphoid sites.³³ In conjunction with cytologic examination, this technology is especially helpful in samples obtained from fine-needle aspirates.³⁴⁻³⁶

Table 28.1  Commonly Used Leukocyte Antigens Used in Clinical Flow Cytometry Based on Cluster of Differentiation Designation

CD1a: Cortical thymocytes, dendritic cells, langerhans cells

CD2: T cells, thymocytes, NK-cell subset

CD3: T cells, thymocytes

CD4: T-cell subset, thymocyte subset, monocytes/macrophages

CD5: T cells, B-cell subset

CD7: Thymocytes,T cells, NK cells, early myeloid cells

CD8: T-cell subset, thymocyte subset, NK-cell subset

CD10: Early B cell, neutrophils, bone marrow stromal cells

CD11b: Monocytes, granulocytes, NK cells

CD11c: Myeloid cells, monocytes

CD13: Myelomonocytic cells

CD14: Monocytes, myelomonocytic cells

CD15: Granulocytes, monocytes, endothelial cells

CD16: NK cells, granulocytes, macrophages

CD19: B cells (from pre-B-cell stage to plasma cells)

CD20: Mature B cells

CD21: Mature B cells, follicular dendritic cells

CD22: Mature B cells

CD23: Activated B cells

CD25: Activated T cells, activated B cells, regulatory T cells

CD27: Memory B cells

CD30: Activated T, B, NK cells, monocytes, Reed Sternberg cells

CD33: Myeloid cells, myeloid progenitor cells, monocytes

CD34: Hematopoietic precursor cells, capillary endothelium

CD38: Most thymocytes, activated T cells, B-cell precursors, germinal center B cells, plasma cells, myeloid cells, monocytes, NK cells

CD36: Platelets, monocytes/macrophages

CD41: Megakaryocytes, platelets

CD42b: Megakaryocytes, platelets

CD45: Leukocytes

CD45RA:T-cell (naïve) subsets, B cells, monocytes

CD45RO:T-cell (memory) subsets, B-cell subsets, monocytes/macrophages

CD56: NK cells, NK T cells

CD57: NK cells, T-cell subsets, B cells, monocytes

CD61: Megakaryocyte platelets, megakaryocytes, macrophages

CD64: Mature neutrophils, monocytes

CD71: Erythroid precursors, proliferative cells

CD79a: B cells

CD95 (Fas): Lymphocytes (upregulated after activation), monocytes, neutrophils

CD103: Intestinal epithelial T lymphocytes

CD117: Myeloid blast cells, mast cells

CD138: Epithelial cells, plasma cells

CD, cluster of differentiation; NK, natural killer.

FIGURE 28.9 Single-parameter histogram of CD25 expression on lymphocytes (left panel) and contour plot of CD25 (y-axis) and CD3 (x-axis) expression.

The utilization of flow cytometry has recently extended to the assessment of myelodysplasia³⁷ and has also been found to be useful in plasma cell disorders.³⁸ In the latter, this analysis has relevance in the differential diagnosis between myeloma and other plasma cell disorders, and also provides prognostic utility based on the identification of high-risk monoclonal gammopathy of undetermined significance and smoldering myeloma.³⁹

FIGURE 28.10 Contour plot of CD45RA and CD45RO expression on CD4⁺ T cells.

FIGURE 28.11 Overlapping control and positively stained histograms.

Flow cytometry is a useful tool for the detection of MRD. Broader capabilities provided by technical advances, a larger selection of antibodies and fluorochromes, and new analytical approaches have resulted in an increased sensitivity in the identification of malignant cells. The presence of MRD has prognostic value for predicting acute leukemia relapse prior to or following curative therapy attempts such as allogeneic hematopoietic stem cell transplants,⁴⁰⁻⁴⁴ and is now a critical component of many therapeutic protocols. While genetic abnormalities in acute leukemic cells can be recognized by molecular genetic techniques, flow cytometry is an excellent alternative in cases where genetic markers are absent. In addition, in an era of curative attempts, MRD detection by flow cytometry has become a useful tool in chronic lymphoid leukemias.³¹

Many applications in nonmalignant diseases are also routinely performed in the clinical laboratory. This technology remains a critical tool in monitoring disease progression and therapy in HIV infection.⁷ There are specific lymphocyte findings characteristic of primary immunodeficiencies, including loss of cell populations or subpopulations, the absence of specific cell surface or intracellular proteins, and changes in normal immunologic processes that can be detected by flow cytometry (e.g., the development of memory T cells and/or B cells).¹³ Assessment of specific lymphocyte populations and subpopulations is being studied in a variety of disorders characterized by inflammation, with particular attention to expression of activation markers. Reconstitution of the immune system following intensive chemotherapy and hematopoietic stem cell transplantation can be monitored by flow cytometry, an approach that is even more significant due to the recent focus on immunotherapy and vaccines in experimental treatment protocols for malignancies.

Assessment of leukocytes other than lymphocytes is emerging in the clinical laboratory. Monocytes can be evaluated to define deficiencies associated with defective monocyte surface receptor expression.¹³ Granulocyte expression of critical adhesion molecules and their capacity to generate reactive oxygen species can be assessed by flow cytometry.¹³ In addition, granulocyte-specific autoantibodies can be detected. Flow cytometric methods are being used to characterize eosinophils in settings of increased production such as the hypereosinophilia syndrome,¹⁶ and basophils have been studied for intracellular cytokine production as well as activation of antigen expression ex vivo following specific antigen exposure.

Hematopoietic stem cell identification is dependent on flow cytometry, which is routinely used to characterize and quantitate stem cells in the transplantation setting, generally based on CD34 expression together with other cell surface markers.⁸ Separation techniques to purify stem cells from either bone marrow or peripheral blood typically utilize CD34 selection methods, and patients are followed by flow cytometric testing post transplantation in order to assess donor cell engraftment and in certain settings, donor–host cell chimerism.

The evaluation of erythrocytes by flow cytometry has been applied to cell surface proteins, autoantibodies in hemolytic anemia, and the detection of F-cells in fetomaternal hemorrhage and sickle cell anemia.9,45 The detection of glycosylphosphatidylinositol-anchored proteins on erythrocytes and leukocytes by flow cytometry is now the favored method to accurately diagnose paroxysmal nocturnal hemoglobinuria.⁴⁶

Flow cytometric evaluation of platelets has been described as a method to study these cells in whole blood, thus eliminating the need for platelet isolation and minimizing cell manipulation.¹⁰ This approach allows for the detection of platelet-associated immunoglobulin, assessment for states of platelet activation and aggregation, and detection of reticulated platelets. Flow cytometry is also a rapid and useful method to detect platelet defects in structural or functional glycoproteins, such as the abnormal expression of gpIIb/IIIa in Glanzmann thrombasthenia and gpIb in Bernard-Soulier disease.¹⁰

SUMMARY

Flow cytometry has evolved as integral in the laboratory assessment of many hematologic disorders. This technology provides a powerful tool to assess simultaneously cell surface and intracellular characteristics. The increasing range of reagents and expanded understanding of cell biology mean that flow cytometry will play an even larger role in the clinical evaluation of various cellular components of the hematologic system, both in benign and malignant conditions.

References

  1.  Givan AL. Principles of flow cytometry: an overview. Methods Cell Biol. 2001;63:19-50.

  2.  McCoy JP Jr. Basic principles of flow cytometry. Hematol Oncol Clin North Am. 2002;16:229-243.

  3.  Loken MR, Brosnan JM, Bach BA, Ault KA. Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry. 1990;11:453-459.

  4.  Szczepanski T, van der Velden V, van Dongen JJ. Flow-cytometric immunophenotyping of normal and malignant lymphocytes. Clin Chem Lab Med. 2006;44:775-796.

  5.  Kussick SJ, Wood BL. Using 4-color flow cytometry to identify abnormal myeloid populations. Arch Pathol Lab Med. 2003;127:1140-1147.

  6.  Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol. 2001;38:179-193.

  7.  Mandy F, Nicholson J, Autran B, Janossy G. T-cell subset counting and the fight against AIDS: reflections over a 20-year struggle. Cytometry. 2002;50:39-45.

  8.  Gratama JW, Kraan J, Keeney M, Sutherland DR, Granger V, Barnett D. Validation of the single-platform ISHAGE method for CD34+. Hematopoietic stem and progenitor cell enumeration in an international multicenter study. Cytotherapy. 2003;5:55-65.

  9.  Davis BH. Diagnostic utility of red cell flow cytometric analysis. Clin Lab Med. 2001;21:829-840.

10.  Linden MD, Frelinger AL III, Barnard MR, Przyklenk K, Furman MI, Michelson AD. Application of flow cytometry to platelet disorders. Semin Thromb Hemost. 2004;30:501-511.

11.  Gopinath R, Nutman TB. Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity. Cytometry. 1997;30:313-316.

12.  Khan SS, Solomon MA, McCoy JP Jr. Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry. Cytometry B Clin Cytom. 2005;64:1-8.

13.  Bleesing JJ, Fleisher TA. Cell function-based flow cytometry. Semin Hematol. 2001;38:169-178.

14.  Shulman HM, Wells D, Gooley T, Myerson D, Bryant E, Loken MR. The biologic significance of rare peripheral blasts after hematopoietic cell transplantation is predicted by multidimensional flow cytometry. Am J Clin Pathol. 1999;112:513-523.

15.  Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Quality assurance for polychromatic flow cytometry. Nat Protoc. 2006;1:1522-1530.

16.  Schnizlein-Bick CT, Mandy FF, O’Gorman MR, et al. Use of CD45 gating in three and four-color flow cytometric immunophenotyping: guideline from the National Institute of Allergy and Infectious Diseases, Division of AIDS. Cytometry. 2002;50:46-52.

17.  Borowitz MJ, Guenther KL, Shults KE, Stelzer GT. Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am J Clin Pathol. 1993;100:534-540.

18.  Raimondi G, Turner MS, Thomson AW, Morel PA. Naturally occurring regulatory T cells: recent insights in health and disease. Crit Rev Immunol. 2007;27:61-95.

19.  Braylan RC, Atwater SK, Diamond L, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: data reporting. Cytometry. 1997;30:245-248.

20.  Wood BL, Arroz M, Barnett D, et al. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia. Cytometry B Clin Cytom. 2007;72(suppl 1):S14-S22.

21.  Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol. 2001;38:179-193.

22.  Farahat N, Lens D, Morilla R, Matutes E, Catovsky D. Differential TdT expression in acute leukemia by flow cytometry: a quantitative study. Leukemia. 1995;9:583-587.

23.  Cook JR, Craig FE, Swerdlow SH. bcl-2 expression by multicolor flow cytometric analysis assists in the diagnosis of follicular lymphoma in lymph node and bone marrow. Am J Clin Pathol. 2003;119:145-151.

24.  Cornfield DB, Mitchell DM, Almasri NM, et al. Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20. Am J Clin Pathol. 2000;114:258-263.

25.  Laane E, Tani E, Bjorklund E, et al. Flow cytometric immunophenotyping including Bcl-2 detection on fine needle aspirates in the diagnosis of reactive lymphadenopathy and non-Hodgkin’s lymphoma. Cytometry B Clin Cytom. 2005;64:34-42.

26.  Slack GW, Wizniak J, Dabbagh L, Shi X, Gelebart P, Lai R. Flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia: correlation with immunocytochemistry and Western blot analysis. Arch Pathol Lab Med. 2007;131:50-56.

27.  Wang YH, Fan L, Xu W, Li JY. Detection methods of ZAP-70 in chronic lymphocytic leukemia. Clin Exp Med. 2012;12:69-77.

28.  Chizuka A, Kanda Y, Nannya Y, et al. The diagnostic value of kappa/lambda ratios determined by flow cytometric analysis of biopsy specimens in B-cell lymphoma. Clin Lab Haematol. 2002;24:33-36.

29.  Beck RC, Stahl S, O’Keefe CL, Maciejewski JP, Theil KS, Hsi ED. Detection of mature T-cell leukemias by flow cytometry using anti-T-cell receptor V beta antibodies. Am J Clin Pathol. 2003;120:785-794.

30.  Weisberger J, Wu CD, Liu Z, et al. Differential diagnosis of malignant lymphomas and related disorders by specific pattern of expression of immunophenotypic markers revealed by multiparameter flow cytometry review. Int J Oncol. 2000;17:1165-1177.

31.  Sanchez ML, Almeida J, Vidriales B, et al. Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation. Leukemia. 2002;16:1460-1469.

32.  Martinez A, Aymerich M, Castillo M, et al. Routine use of immunophenotype by flow cytometry in tissues with suspected hematological malignancies. Cytometry B Clin Cytom. 2003;56:8-15.

33.  Almasri NM, Zaer FS, Iturraspe JA, Braylan RC. Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens. Mod Pathol. 1997;10:650-656.

34.  Barrena S, Almeida J, Del CG-M, et al. Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas. Histopathology. 2011;58:906-918.

35.  Demurtas A, Accinelli G, Pacchioni D, et al. Utility of flow cytometry immunophenotyping in fine-needle aspirate cytologic diagnosis of non-Hodgkin lymphoma: a series of 252 cases and review of the literature. Appl Immunohistochem Mol Morphol. 2010;18:311-322.

36.  Meda BA, Buss DH, Woodruff RD, et al. Diagnosis and subclassification of primary and recurrent lymphoma. The usefulness and limitations of combined fine-needle aspiration cytomorphology and flow cytometry. Am J Clin Pathol. 2000;113:688-699.

37.  Kussick SJ, Fromm JR, Rossini A, et al. Four-color flow cytometry shows strong concordance with bone marrow morphology and cytogenetics in the evaluation for myelodysplasia. Am J Clin Pathol. 2005;124:170-181.

38.  Paiva B, Almeida J, Perez-Andres M, et al. Utility of flow cytometry immunophenotyping in multiple myeloma and other clonal plasma cell-related disorders. Cytometry B Clin Cytom. 2010;78:239-252.

39.  Perez-Persona E, Vidriales MB, Mateo G, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells. Blood. 2007;110:2586-2592.

40.  Elorza I, Palacio C, Dapena JL, Gallur L, Sanchez de TJ, az de HC. Relationship between minimal residual disease measured by multiparametric flow cytometry prior to allogeneic hematopoietic stem cell transplantation and outcome in children with acute lymphoblastic leukemia. Haematologica. 2010;95:936-941.

41.  ez-Campelo M, Perez-Simon JA, Perez J, et al. Minimal residual disease monitoring after allogeneic transplantation may help to individualize post-transplant therapeutic strategies in acute myeloid malignancies. Am J Hematol. 2009;84:149-152.

42.  Foster JH, Hawkins DS, Loken MR, Wells DA, Thomson B. Minimal residual disease detected prior to hematopoietic cell transplantation. Pediatr Blood Cancer. 2011;57:163-165.

43.  Sanchez J, Serrano J, Gomez P, et al. Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation. Br J Haematol. 2002;116:686-694.

44.  Venditti A, Maurillo L, Buccisano F, et al. Pretransplant minimal residual disease level predicts clinical outcome in patients with acute myeloid leukemia receiving high-dose chemotherapy and autologous stem cell transplantation. Leukemia. 2003;17:2178-2182.

45.  Chen JC, Davis BH, Wood B, Warzynski MJ. Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detection. Cytometry. 2002;50:285-290.

46.  Borowitz MJ, Craig FE, Digiuseppe JA, et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010;78:211-230.

29

Molecular Diagnostics in Hematology

Jaroslaw P. Maciejewski and Bartlomiej Przychodzen

BACKGROUND

The application of molecular biology and genetic techniques has greatly contributed to recent advances in hematology. Many new technologies have found utility in the clinical routine. This chapter illustrates the application of molecular techniques in the diagnosis of hematologic diseases and explains the principles and details of the most commonly used tests. The individual techniques are described in the context of specific applications; many of the methods are applied in a variety of diseases described in specific chapters of this handbook.

The most essential technologies of the next decades will include:

  Polymerase chain reaction (PCR) and its modification

  Sanger sequencing

  DNA microarray technologies

  Next generation sequencing (NGS)

In particular, these technologies are of fundamental importance for many derived diagnostic tests, and we provide here a short overview of their general principles, while subsequent paragraphs show specific diagnostic areas and modifications. Of note is that many indirect methods of sequence variant determination, such as melting curve analysis, restriction fragment polymorphism, PCR amplification with sequence-specific primers (SSP) and hybridization with a sequence-specific oligonucleotide probe (SSOP), will be increasingly replaced by direct sequencing.

Polymerase Chain Reaction

PCR revolutionized molecular diagnostics in hematology; various modifications of this technique exist. Both DNA and RNA reverse transcribed into cDNA can be used as a template. In the presence of forward and reverse DNA primers that bind to the sequence-specific regions of the target DNA, Taq polymerase extends both strands of the DNA. Repeated cycles of annealing, extension, and denaturation lead to the exponential amplification of the targeted DNA sequence with the specificity provided by the DNA primers (Fig. 29.1). PCR primers can be designed to distinguish polymorphic sequences; primers can be labeled to facilitate detection or quantitation. Various modifications of the basic PCR technologies have been described below in conjunction with specific applications.

FIGURE 29.1 Principle of polymerase chain reaction.Template consists of either DNA or cDNA generated by reverse transcription of mRNA. In the presence of forward and reverse DNA primers that bind to the sequence-specific region of the target DNA,Taq polymerase extends both strands of the DNA such that repeated cycles of annealing, primer extension, and denaturation lead to amplification and accumulation of the targeted newly synthesized DNA sequence with the specificity provided by DNA primers.

Traditional Sequencing

The most popular method uses modified chain-termination method and was introduced by Sanger in the early 1970s. Sanger sequencing relies on the use of color-labeled, dideoxynucleotide chain terminators (ddNTPs). Compared to a regular reaction mixture, it consists four, regular deoxynucleotides (dNTPs) mixed with color-labeled ddNTPs. In principle, when the ddNTP is added at the end of the fragment it restricts further elongation of the DNA chain. Since the addition of the dNTPs and ddNTPs is random, a whole array of fragments of different lengths will be produced. Each fragment will be end-labeled with only one dye that represents one of four different nucleotides. Because all the fragments have different sizes, they can be separated and visualized by capillary electrophoresis. In capillary electrophoresis, each color-labeled fragment migrates according to its length, shorter fragments migrating faster. At the end of the capillary, each fragment is analyzed by a laser beam and fluorescence detector. Since each fragment reaches the detector sequentially, nucleotide sequence is reconstructed from a wavelength chromatogram; in most cases, only a small portion of the gene of interest (usually 1 exon per reaction) is sequenced. Consequently, even single-gene sequencing is extremely labor and cost intensive, requiring multiple PCR reactions and site-specific sets of primers.

New Generation Sequencing

NGS overcomes the limitation of Sanger sequencing by using a massive, parallel sequencing approach (Fig. 29.2). DNA is randomly fragmented and millions of fragmented DNA molecules are physically arrayed and attached to universal adaptors on a solid surface. A set of universal primers is then used to massively amplify and ultimately sequence millions of fragments in parallel. Elongation of each of the small fragments is similar to conventional DNA sequencing. Four different color-labeled terminator nucleotides are added sequentially, elongating the DNA chain, unincorporated nucleotides being washed off. After one terminator nucleotide is successfully added, it is able to emit one of the four distinct colors when the laser beam is applied. With the advent of ultra-sensitive charged coupled device (CCD) cameras, added nucleotides can be distinguished. Since the process is done in parallel, “reading” of each of millions of sequenced fragments is accomplished. Finally, the cycle is finished with removal of reversible terminator. By repeating this cycle multiple times (until the template fragment is fully extended), the complete sequence of the fragment, one nucleotide at the time, is completed. The lengths of the fragments are determined by the number of cycles that are needed to completely sequence a given DNA fragment. The process produces millions of short fragments (reads) that need to be organized into meaningful, organized, continuous sequences corresponding to specific target genes or chromosomal regions or transcripts (if mRNA is the starting material). Computer algorithms and sequence aligners assemble the sequences for analysis.

FIGURE 29.2 Principle of next generation sequencing.The first step usually consists of DNA/RNA fragmentation. It is done either enzymatically (restriction enzyme) or mechanically (sonication). Subsequently all the fragments are arrayed by hybridization to universal adapters attached to a solid surface. Successful attachment along with proper dilution guarantees only one fragment per location.This enables each fragment to be sequenced in parallel. Usually fragments are being copied prior to sequencing. Sequencing proceeds in four steps. The first step is the addition of a reversible terminator that results in elongation by one nucleotide. Secondly, all the unincorporated nucleotides are removed. Subsequently the whole reaction surface with all the fragments is scanned using the laser beam and CCD camera.The last step involves modification of the termination moiety, and the cycle can start over.The number of cycles represents the length of millions of fragments sequenced.

DETECTION OF INDIVIDUAL GERMLINE MUTATIONS/ POLYMORPHISMS

Precise diagnosis of many hematologic diseases or detection of susceptibility to develop complications depends on the identification of mutated genes. Clinically applicable methods mostly involve detection of defined mutations occurring at specific sites within the genes. Currently, most protocols use PCR to amplify the involved gene fragments. For the identification of the presence of individual mutations, various methods can be used (Fig. 29.3). For example, they are applied in routine diagnosis of genetic hematologic diseases including thalassemia and other hemoglobinopathies, hereditary familial hemochromatosis (HFE) gene mutations such as C282Y and H63D, factor V Leiden, prothrombin gene mutations G20210A, and thermolabile C677T 5,10-methylenetetrahydrofolate reductase.1,2 Similar methods can be applied for the detection of other clinically relevant mutations or polymorphisms.

FIGURE 29.3 Application of PCR technology for the detection of a gene mutation.Various techniques based on PCR can be used for specific applications in hematology. Fluorescent primers can be used for determining the small differences in the size of the amplified product, a technique called genotyping. Fluorescent probes can also be selected to hybridize between the primer sequences of the template allowing design of real-time PCR. DNA amplicons generated in the process of PCR reactions can be used for restriction endonuclease digestion. If restriction sites for specific enzymes within the amplicon contain a mutation, restriction endonuclease digestion of the PCR product will result in fragments of different sizes, which can be resolved on either capillary or agarose gel electrophoresis. Finally, using specific fluorescent probes that hybridize to the amplified sequences, melting curves can be recorded to distinguish individual alleles.The presence of sequence differences between the probe and template results in different melting curves; these curves are recorded based on the emission of light induced by melting off the fluorescent probes from the template.

Restriction Fragment Length Polymorphism Analysis

Prior to the advent of PCR technology, traditional Southern blotting of genomic DNA followed by probe hybridization was used to detect changes in the endonuclease restriction patterns. Currently, restriction fragment length polymorphism (RFLP) analysis is used in conjunction with PCR amplification. Restriction digests can be performed either prior to or after amplification. If a mutation affects the restriction endonuclease digestion patterns, its presence can be easily demonstrated using RFLP analysis. After PCR amplification of a relevant gene fragment that carries a specific mutation, the resulting amplicons are subjected to restriction endonuclease cleavage. Using gel electrophoresis, changes in the fragment size can be demonstrated. Through comparison with a wild-type form, heterozygote and homozygote patterns can be easily distinguished. When a fluorochrome-labeled primer is used, capillary gel electrophoresis can be applied allowing for high sensitivity and throughput. Detection of HFE mutations by RFLP of PCR products serves as an example for this technique.

Melting Curve Analysis of Polymerase Chain Reaction Products

More recently, a light-cycler PCR method combined with melting curve analysis has been used for the detection of gene mutations allowing for a reduction in the workload and enabling automation. Melting curve analysis exploits the fact that even a single-nucleotide mismatch between the labeled probe and the targeted sequence significantly reduces the melting temperature. Consequently, amplicon/probe mismatches will melt off at lower temperatures different than that of matched target DNA. PCR amplification of specific gene fragments is performed in the presence of a fluorescent DNA probe or probes (anchor and sensor probe) that release light on hybridization to the internal portion of the amplicon containing the potential mutation. After completion of the reaction, the hybridized fragments are denatured; the release of the probe decreases the amount of the emitted fluorescence, a process recorded in the form of a melting curve. The shape of the melting curves identifies the presence of two normal alleles (singular curve), or heterozygotes (two peaks). For mutation homozygotes the curve is shifted, producing a singular characteristic peak. If multiple mutations are present in a gene, specific probes and primers must be applied to detect heterozygotes, homozygotes, and compound heterozygotes.

Allele-Specific Polymerase Chain Reaction

The tetra-primer amplification refractory mutation system (ARMS) PCR is one of the variants of allele-specific PCR; it allows for detection of single nucleotide polymorphisms (SNPs) as well as single-gene mutations. Two different allele-specific amplicons and a larger (non-allele-specific) control amplicon are generated by a pair of two common (outer) primers and by two allele-specific (inner) primers that have opposite orientation (allele 1–specific primer, antisense, and allele 2–specific primer, sense). Because the common primers are designed so that the mutation is located nearer one of them, the two allele-specific amplicons will have different lengths and thus easily separated by gel electrophoresis: the wild-type genotype generates two bands on gel electrophoresis, homozygous mutation generates two bands, and heterozygous mutation generates all three bands.

Direct Polymerase Chain Reaction–Amplified Product Sequencing

Alternative methods of mutation analysis include direct sequencing of PCR products. Both alleles can be easily identified, and the direct sequencing method has the advantage that it does not target a specific mutation and that all possible sequence differences can be detected within the sequenced gene region.

MOLECULAR DIAGNOSIS OF HEMOGLOBINOPATHIES

Hemoglobinopathies constitute a large group of inherited autosomal recessive hematologic disorders. While routine laboratory tests and clinical presentation are often sufficient for a proper diagnosis, molecular analysis is mandatory for the confirmation of the defect and precise characterization of the abnormal hemoglobin.³ For example, combinations of specific mutations may greatly affect the phenotype expected in the progeny. Thus, molecular diagnosis may have significant consequences for counseling affected patients, asymptomatic carriers, and prenatal diagnostics.

Traditionally, Southern blotting was used, but, recently, PCR-based methods are preferred. Allele-specific oligonucleotide (ASO) hybridization and allele-specific priming are the most commonly applied techniques. The first method relies on hybridization of ASO probes (wild type and mutant) to PCRamplified genomic DNA. In the dot-blot assay, ASO is labeled, while the reverse dot-blot technique utilizes labeled amplified DNA, allowing for simultaneous screening of multiple mutations. Allele-specific priming is based on the principle that a perfectly matched primer pair amplifies target DNA more efficiently than a mismatched pair. In the ARMS, genomic DNA is challenged with both wild-type and mutant primer sets. Multiple mutations may be simultaneously screened in a multiplex PCR assay using fluorescently labeled ARMS primers, producing products of different length that can be detected using an automated DNA analyzer. Large deletions of both the α and β globin gene may be screened using the gap-PCR, with primers complementary to the breakpoint sequences. However, for some deletion mutants, Southern blotting is still standard.

Combining all these approaches in the context of the ethnic- and region-specific distribution of globin mutations, successful molecular identification is possible in more than 90% of cases. Mutations remaining unknown after standard molecular screening may be investigated further by denaturing gradient gel electrophoresis or heteroduplex analysis; nevertheless, complete sequencing of the globin gene represents the best option to identify rare or unknown mutations.

CYTOGENETIC DIAGNOSTICS

Metaphase Karyotyping

Traditional cytogenetics, utilizing banding techniques, is performed on chromosomal metaphase spreads. Because mitotic activity is required, metaphase karyotyping is performed after cell culture in the presence of mitogens. For myeloid disorders, either lymphocyte-conditioned media or hematopoietic growth factors are most commonly used, while, for lymphoid malignancies, lectins are added. Various banding methods have been utilized for chromosome identification and resolution of individual chromosomal fragments, but G-banding is usual in clinical diagnostics. Characteristic bands result from the biochemical properties of chromatin such as AT and GC content.⁴⁻⁸

Cellularity and mitotic activity affect the diagnostic yield of the procedure, and the proportion of noninformative spreads varies from disease to disease. In myelofibrosis, marrow is often not aspirable. In aplastic anemia and myelodysplasia, noninformative results are frequent due to the lack of progenitor cells. In such cases, cytogenetic analysis may be also performed on blood specimens.

Approximately 330 chromosomal bands can be distinguished by routine karyotyping, and each band may contain as much as 10⁷ base pairs (bp) and a multitude of genes. Classic karyotyping can identify defects of approximately 5 Mb; thus, smaller defects and their locations may remain undetected (resolution). The sensitivity level depends on the number of analyzed cells; routinely 20 cells are counted with a detailed analysis of at least 2 cells. Analysis may be more complicated if several clones, each harboring a distinct defect, are present. Depending on the nature of the identified defect, the sensitivity limit is approximately 10% (i.e., identification of 2 abnormal cells) in 20 cells tested.

Both balanced and unbalanced translocations can be identified, but some defects may require a more intricate analysis. Some of the balanced translocations are highly diagnostic; examples are t(9;22) in chronic myelogenous leukemia (CML), t(15;17), inv 16 and t(8:21) in acute myeloid lymphoma (AML), t(15:17) in acute promyelocytic leukemia (APL), t(9:22) and t(12:21) in acute lymphoblastic leukemia (ALL), as well as t(14:18), t(11:14), t(11:18) in lymphomas. Once a specific defect is identified, metaphase karyotyping can be used for monitoring of therapy response (cytogenetic remission); however, the sensitivity of this method is limited.⁵⁻⁸

Fluorescence In Situ Hybridization

For the targeted detection of specific abnormalities, fluorescence in situ hybridization (FISH) is the most commonly applied method particularly helpful in the characterization of structural chromosomal abnormalities and identification of chromosomes of uncertain origin. However, FISH is not suitable for screening for unknown defects unless a high clinical suspicion exists. FISH does not require cell division and consequently cell culture, and is more sensitive than traditional cytogenetics. FISH provides a more accurate measure for the true frequency of abnormal cells and can be used for the monitoring of minimal residual disease (MRD). Identification of the donor versus recipient origin of the blood cell production following hematopoietic stem cell transplantation is another application of this technology (see below). The technique can be applied to blood, marrow, body fluids, tissue touch preparations as well as to paraffin-embedded tissues.5,9

In FISH, specific fluorescently labeled single-stranded DNA probes are hybridized to the nuclei of metaphase or interphase cells attached to glass slides. The use of probes labeled with different dyes allows for multicolor FISH on a single slide. Probes can also be designed to identify a specific chromosomal structure, hybridize to multiple chromosomal sequences, and to identify unique DNA sequences. Probes recognizing α-satellite sequences are chromosome specific; in diploid cells both chromosomes are labeled. Chromosome painting probes are derived from whole chromosomes (see also spectral karyotyping [SKY], discussed next). Probes can be derived from unique sequences cloned from specific regions of the genome. Finally, telomeric probes can be used to determine the telomere length based on the intensity of the hybridization.

For balanced translocations, probes spanning individual breakpoints are used. Dual-color/dual-fusion probes or single-fusion/dual-color FISH probes target sequences located at opposite ends of two breakpoints. In addition, two-color break-apart probes, recognizing DNA sequences from the 3´ and 5´ends of a single gene, can be applied. These probes yield combined yellow signal in the normal germline configuration while two colors are seen when target sequences are separated because of translocation. FISH is more reliable for the detection of duplication of chromosome fragments than deletions. In general, FISH is less sensitive than PCR, with detection limits of 1 of 100 cells. As a result of the false-positive rate, it is not clear whether sensitivity can be increased through routine counting of a higher number of cells.

FISH techniques have been widely applied for the detection of lymphoma-specific translocations, in the diagnosis of CML, myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) (Table 29.1).6,8-10 In addition, FISH is frequently used for intracellular detection of Epstein-Barr virus (EBV) in certain non-Hodgkin’s lymphomas, Hodgkin’s disease, and aggressive natural killer (NK) cell lymphomas (see later).

Spectral Karyotyping

SKY allows for the visualization of all 24 chromosomes and analysis of their structure based on hybridization with multicolor painting probes.¹¹ These probes are derived from individual chromosomes using degenerate primer-based PCR and differentially labeled with fluorochromes. After hybridization to metaphase spreads, a digital camera is used to record the complete emission spectra. As a result, each chromosome-specific probe is distinctively labeled and easily identified. SKY has a much higher precision than traditional cytogenetics and allows for identification of new, previously unidentified reciprocal translocations and defects that cannot be resolved by traditional banding. In one study, SKY allowed detection of new translocations in 35% of cases and resulted in confirmation of the previously known defects and refinement of 35% of diagnoses.

Comparative Genomic Hybridization and Single Nucleotide Polymorphism Arrays as Karyotyping Platforms

Array-Based Comparative Genomic Hybridization

Array-based comparative genomic hybridization (A-CGH) allows for cytogenetic analysis of chromosomal defects that can be applied in many clonal diseases. In this technique, genomic DNA from malignant cells and normal reference DNA are fragmented, differentially labeled with fluorescent dyes, and cohybridized to immobilized DNA probes. Chromosomal imbalances across the genome in tumor DNA can be detected, quantitated, and the position defined by analysis of the fluorescence intensity of the two different colors. Initially, this analysis was performed on metaphase chromosome preparations (M-CGH). However, the resolution of CGH as applied to metaphase spreads is limited by the standard cytogenetic resolution of approximately 5 Mb, and considerable cytogenetic expertise is required to accomplish such an analysis. Therefore, M-CGH has never become a widely utilized technique, and remained limited to specialist research applications.

Bacterial artificial chromosome was originally used to produce CGH arrays. The advent of oligoarray technology made CGH applicable to the study of genomic alterations in human disease. Sixty-mer oligonucleotide probes corresponding to individual SNPs cover the whole genome and include both coding and noncoding regions; thus an ordered array of DNA segments at high genomic resolution can be generated, circumventing the limitations associated with the use of metaphase preparations as the hybridization template. The fluorescence ratio of the two colors can be compared between different spots representing different genomic regions, providing a genome-wide molecular profile of the sample with respect to regions of the genome that are deleted or amplified (Fig. 29.4A). Resolution is dependent on a combination of the number, size, and map positions of the DNA elements within the array.

Single Nucleotide Polymorphism Array–Based Karyotyping

Recently introduced SNP arrays to study the genetic predisposition of diseases also can be applied for analysis of copy number variation and loss of heterozygosity (LOH). This technique utilizes arrays containing oligonucleotide probes corresponding to SNPs present throughout the human genome.³⁶ Test DNA is fragmented, ligated to universal linkers, amplified with primers corresponding to the linkers, and labeled (Fig. 29.4B). Unlike in CGH, there is no requirement for normal reference DNA. Following hybridization, fluorescence intensity is measured for each spot on the array. After bioinformatic analysis, genotyping calls are possible for each SNP in order to determine homozygosity or heterozygosity for each SNP. In addition to genotyping, the copy number for each locus (tagged by a specific SNP probe) can be deduced based on the fluorescence intensity that corresponds to hyperploid or hypoploid gene copy number. A whole variety of array designs (bead hybridization or array liquid phase hybridization) platforms are currently available with a density of >1 million markers covering 22 autosomes and the X chromosome. The average intermarker distance is approximately 10 Kb, resulting in a superior resolution. Initially designed for genotyping and whole genome association studies, SNP-A also can be used for karyotyping. As a karyotyping tool, SNP-A allows for a very high resolution (depending on the density of the SNP probes), does not require cell division (no culture and proliferation of cells is needed) but, due to the nature of the technology allows for detection of only balanced translocations. When compared to metaphase cytogenetics, SNP-A-based karyotyping allows for detection of clonal unbalanced chromosomal defects in a higher percentage of patients with hematologic malignancies including MDS, Multiple Myeloma (MM), AML, and chronic leukemia/lymphoma (CLL). The additional advantage over metaphase karyotyping and A-CGH is the ability to detect copy number neutral LOH (uniparental disomy), present in many solid tumors and myeloid malignancies. The sensitivity of SNP-A karyotyping is relatively low (as it depends on the proportion of clonal cells in the sample), comparable to metaphase karyotyping. In addition to whole genome arrays with various probe densities, customized SNP arrays are available or can be designed for specific applications: targeting non-synonymous SNPs, particular set of SNPs or specific regions of the genome (as for the human leukocyte antigen, HLA, locus).

Because of the high resolution of SNP-A and the convenient microarray format, these methods will likely be introduced into the clinical routine, especially for diseases in which unbalanced translocations are expected.¹²

FIGURE 29.4 A. Array-based comparative genomic hybridization (A-CGH). A-CGH consists of hybridization of tester and reference DNA samples that have been differentially labeled with fluorochromes to arrays of sequences corresponding to specific portions of chromosomes. Unbalanced translocations, such as deletion or duplication of individual genes (or portions of chromosomes) can be detected based on the disparity between fluorescent spectra emitted by the tester vs. reference DNA. Consequently, depending on the number of probes, a very intricate analysis of chromosomes can be performed with regard to the presence or absence of specific DNA segments. B. SNP array–based karyotyping. The principle of the method relies on amplification and labeling of fragmented genomic PCR products with subsequent hybridization to arrays containing oligonucleotide probes homologous to allelic variants of SNPs. Based on the density of the arrays and choice/location of SNPs to be detected, various resolution levels can be achieved. Bioinformatic analysis of hybridization results allows for detection of copy number changes (fluorescence intensity) for individual loci as well as loss of heterozygosity that can be a result of deletion or segmental uniparental disomy due to mitotic recombination.

QUANTITATION OF SOMATIC MUTATIONS AND TRANSLOCATIONS

Polymerase Chain Reaction–Based Analysis of Translocations

PCR has found a wide application in the diagnosis of malignant disorders associated with specific translocations of genetic material.5,7,8 The main advantage of PCR is the high specificity and sensitivity, but there is need for immaculate technique to avoid contamination. DNA primers are designed to flank the specific translocated region, producing a PCR product with a characteristic size, while in the absence of the specific translocation the amplification product is not generated. Appropriate controls can be incorporated into the reaction. Because of the higher template copy number per cell, reverse transcriptase (RT) real-time PCR may be a more sensitive modification of this technique. In real-time PCR, mRNA of the abnormal transcript is reverse transcribed and cDNA serves as template for the amplification reaction (Fig. 29.5). Sensitivity and specificity can be further improved by an additional round of amplification with a pair of internal primers, termed nested PCR. The sensitivity of this method approaches 1 malignant cell per 10⁶ normal cells, allowing for the assessment of MRD in various conditions.⁵

FIGURE 29.5 Real-time quantitative PCR. Either cDNA generated from mRNA or DNA can be used as a template.The principle of real-time PCR consists of target sequence amplification in the presence of fluorochrome-labeled probes. Such probes are designed to target the sequences between the forward and reverse primer that hybridize to the PCR product accumulated during amplification.The probes are usually labeled with a reporter fluorochrome at the 5´ and a quencher fluorochrome at the 3´ end. Probes are designed to have a higher melting temperature than the extension primer. As long as both fluorochromes are connected through the DNA sequence, no light is emitted. However, the 5´ to 3´ exonuclease activity of Taq polymerase degrades the probe and releases the fluorochromes. Consequently, progression of the reaction can be monitored by the detection of the fluorescent signal generated during the exponential phase of the reaction and the cycle number at which the reporter dye intensity rises above the background noise.This value, also referred to as the threshold number, is inversely related to the copy number of the target template.

Recently, introduction of the real-time light-cycler PCR assay (Fig. 29.5) has led to quantification of the numbers of cells up to one malignant cell per 10⁵ normal cells. The reference standard includes a single-copy gene. The principle of real-time PCR consists of target sequence amplification in the presence of fluorochrome-labeled probes. Such probes are designed to target the sequences between the forward and reverse primers. The probe is labeled at the 5´ end with a reporter fluorochrome (6-FAM) and a quencher fluorochrome (6-carboxytetramethyl-rhodamine [TAMRA]) at the 3´ end and are designed to have a higher melting temperature than the extension primers. As long as both fluorochromes are connected through the DNA sequence, no light is emitted. However, the 5´ to 3´ exonuclease activity of Taq polymerase degrades the probe and releases the fluorochromes. Consequently, progression of the reaction can be monitored by the detection of the fluorescent signal generated during the exponential phase of the reaction, and the cycle number at which the reporter dye intensity rises above the background noise. This threshold number is inversely related to the copy number of the target template. Measurement of the frequency of abnormal cells is based on standard curves and is generated with dilution of control cells or DNA/cDNA containing the targeted mutation. The results can be expressed as copy numbers of fusion transcripts per microgram of RNA or as the frequency of abnormal cells. Ubiquitously expressed housekeeping transcripts/genes are commonly incorporated, and the PCR cycle threshold number of the fusion gene is normalized to the value of the housekeeping gene.⁵

In clinical practice, PCR technology, including quantitative PCR, has proven predictive of therapy response and relapse. For certain hematologic malignancies with translocations coding for specific targets, cytogenetic and molecular remissions have been defined as specific end points of therapy. Risks of relapse associated with molecular versus cytogenetic remission have been determined. The most common applications of PCR in the detection of disease-defining translocations are for CML (bcr/abl), APL (PML/RARA), mantle cell and follicular lymphomas (cycD1/IgH and IgH/bcl2, respectively), and B-cell lymphoblastic leukemia (bcr/abl, rearranged IgH) (Table 29.2).

Clearly, separate screening for all described abnormalities is too expensive and time consuming to be applied as a global diagnostic battery. Multiple attempts have been made to adapt PCR technology for precise molecular diagnosis associated with individual leukemias and lymphomas. In multiplex PCR, multiple primer pairs are combined to enable detection of several translocations in a single PCR reaction. For example, primer mixtures have been designed to allow for the detection of 28 different translocations, including 80 breakpoints and splice variants, using a limited number of PCR reactions. The introduction of such technologies in clinical practice is hampered by the need for positive controls and limited sensitivity. However, the potential value of more comprehensive screening techniques has been demonstrated in cases that showed PCR positivity for PML/RARA transcripts despite M3 morphology or the cytogenetic presence of t(15:17), and cryptic translocations such as t(12:21) or t(4:11) in ALL and in AML.

Detection of Somatic Gene Mutations

Acquired mutations of individual genes can lead to the acquisition of a malignant phenotype and their presence or absence may have clinical significance.⁵ The principle of PCR-based detection of such mutations is similar to that used for germline mutations (see above). Methods include direct DNA PCR, if the mutation results in a length difference between the amplified fragments (e.g., internal tandem duplication of the FTL-3 gene¹³) wild-type and mutated amplicons will be clearly identified by electrophoresis. When a labeled primer is used for amplification, even small size differences can be resolved by capillary gel electrophoresis. Single-nucleotide changes can also be identified when the mutation produces a new restriction site or abolishes an existing one. Consequently, after amplification, PCR products are digested with appropriate restriction endonucleases and subjected to electrophoresis to identify the presence of the mutation (see above RFLP). Allele-specific PCR with primers designed to amplify either the mutated or wild-type allele is currently the most commonly employed method. Finally, exons affected by recurrent mutations can be amplified and directly sequenced.

Apart from canonical mutations or genes affected by a few invariant mutations, mutational testing may be very difficult for larger genes (multiple exons) affected by diverse mutations located in various exons. PCR-based site-targeted methods, including direct sequencing of the amplified PCR product may be difficult to implement for such genes due to the labor intensity involved. NGS sequencing may alleviate some of these problems.

In contrast to the detection of germline mutation, using PCR technologies, the proportion of mutated cells in the sample may affect the results of the test: even if 100% of cells carry the mutation, only 50% of the alleles will be affected. The current detection limits of PCR-based techniques can reach 10% of cells carrying the mutation.

Of note is that the choice of cellular material used for the detection of single-gene mutations may influence the results with regard to the status of homozygous mutations, such as Jak2V617F. In most methods beginning with unfractionated cells, the distinction between heterozygous and homozygous may not be possible due to contamination with wild-type cells.

Most recently, NGS technologies have been adopted for detection of somatic lesions. The diagnostic strategies can include unbiased approaches, in which whole genome, exome or individual chromosomes are sequenced and either known invariant mutations are identified and reported or a comparison is made with the parallel sequenced germline sample, all somatic mutations are identified. Various algorithms have been developed to distinguish false-positive results due to technologic errors and germline-encoded sequence alterations from true somatic events. Another NGS approach is targeted, deep sequencing, which can include enrichment steps for exons of selected or single genes. Because of the smaller overall size of the amplicon to be sequenced, a greater sequencing depth can be achieved (“deep sequencing”), which allows for assessment of mutational burden/clonal size. Such technologies may be used in the future to monitor the therapy effectiveness or MRD.

CLONALITY STUDIES

Where acquired defects (point mutations, translocations) have been identified, they can serve as a suitable marker. However, in many clinical situations such a marker is not available. Consequently, a number of clonality assays have been developed that allow for the diagnosis of oligoclonal or skewed hematopoietic function.

X Chromosome Inactivation Pattern Analysis

X chromosome inactivation pattern (XCIP) analysis is particularly useful in the analysis of disorders without a disease-specific clonality marker.¹⁴ XCIP clonality analysis can be informative in a large proportion of female patients. Clearly, clonal/oligoclonal XCIP does not secure the diagnosis of malignancy but may be complementary with other clinical signs and laboratory results.

XCIP is based on the inactivation of one X chromosome in female mammalian cells.¹⁴ The inactivation pattern is random, laid down early in embryogenesis and stably inherited by all daughter cells. The inactivation mechanism includes methylation of certain portions of the DNA. Based on the single-cell theory of malignant disorders, XCIP representation should change in the affected tissue, most significantly in blood. Consequently, the distinction between the two chromosomes made by using a polymorphic marker gene located on X chromosome and differentiation between the active and inactive X chromosomes underlie XCIP clonality analysis. While a disease-specific marker is not required, the pathologic change can be extrapolated from the expected pattern. The most commonly used polymorphic markers include human androgen receptors (HUMARA), phosphoglycerate kinase, or the fragile X (FRM1) genes. The heterozygosity rate for HUMARA is approximately 90%. Modern XCIP analysis techniques utilize PCR technology.

In the HUMARA assay, DNA is digested with a methylation-sensitive restriction endonuclease, amplified, and the electrophoretic pattern of the amplicons compared to the undigested amplicons. HpaII digests the unmethylated allele and leaves the methylated allele available for amplification. Comparison of the intensity of the bands allows extrapolation as to the skewing of the normally equally distributed amplicons of the methylated and unmethylated gene fragment.

In other common assays, single-base or short-tandem-repeat (STR) polymorphisms in the coding sequences of X chromosomal genes distinguish between the usage of the inactive or active chromosome by RNA-based techniques. RT-PCR is followed by RFLP of the amplicons. Interpretation of the results of the XCIP analysis must account for age-rated skewing of inactivation and also for a nonrandom pattern among normal cells that may be encountered in up to 25% of women.

ANALYSIS OF T-CELL RECEPTOR AND IMMUNOGLOBULIN REARRANGEMENT

During T- and B-cell ontogeny, rearrangement of VDJ genes of heavy (H) and light (L) Ig chains and α (A) and β (B) or δ (D) and γ (G) chains of T-cell receptor (TCR), respectively, provides the molecular basis for the heterogeneity of B- and T-cell recognition repertoire.¹⁵⁻¹⁷

For immunoglobulin (Ig) H-chain, there are at least 40 functional variable (VH) gene fragments, 27 diversity (DH) fragments, 6 functional junctional (JH) fragments, and several constant (CH) gene segments. The Igκ gene complex consists of 35 Vκ gene segments, 5 Jκ, and a single Cκ gene. The Igλ gene is generated through recombination of 30 Vλ segments and 4 Cλ segments, all preceded by a Jλ region.

The TCR is a homodimer consisting either of TCR α and β or TCR δ and γ chains. Similar to Ig genes, α and δ chains are encoded by recombined VDJ fragments (65 VB segments, 7 JB fragments, and 2 CB segments, each preceded by a DB region for β chain and 8 VD, 3 DD, and 4 JD gene segments, as well as a single CD region). TCR α and γ chain are generated through recombination of V and J regions. TCR α-chain gene complex consists of more than 50 VA, 61JA, and 1 CA gene segments. The TCR γ gene complex consists of 6 VG and 2 CG segments, each preceded by 2 or 3 JG1 or JG2 gene segments. Ig and TCR rearrangements are not lineage-restricted: B and T cells may contain complete or incomplete cross-lineage rearrangements that can be used for assessment of clonality. Clonal expansion results in overrepresentation of the specifically rearranged Ig or TCR configuration, a property of lymphocytes exploited for the diagnosis of T- and B-cell malignancies.

Immunoglobulin and T-Cell Receptor Junctional Region Polymerase Chain Reaction

PCR analysis of Ig and TCR gene segments is based on selective amplification of junctional regions. Amplification is only possible when the Ig or TCR gene are juxtaposed through rearrangement, because the distance between these gene segments in the normal germline configuration is too large for PCR amplification. In contrast to the Southern blot,¹⁸ PCR relies on both the combinatorial and the junctional diversity of the appropriate rearrangements. PCR can be easily applied to blood and tissue specimens including lymph nodes and skin (particularly helpful in the diagnosis of cutaneous T-cell lymphoma). For both IgH and TCR amplification, sets of consensus primers covering the full spectrum of the V regions combined with sets of JB primers are used in multiplex PCR. For B-cell populations, analysis of the IgH rearrangement is most informative, as the IgH locus rearranges first; a complete VH-JH rearrangement is usually investigated. However, as somatic hypermutation within the IgV gene hampers annealing and amplification, analysis of incomplete DH-JH rearrangement may also be helpful in identifying non-productive partial rearrangement in some immature B cells. Additionally, comprehensive Ig clonality assessment may include analysis of IgL chain, especially the Igκ locus. As discussed above, given the hierarchical rearrangement of IgL, all mature B cells have either a productive or a non-productive Igκ rearrangement (generally involving the κde element) if cells are Igλ+. Further analysis of the IgL genes may be added to increase the sensitivity of clonality assessment.

For T-cell clonality, analysis of the γ TCR locus has been the paradigm; the main advantage is that the rearrangement of the γ chain occurs early and is present in both α/β and δ/γ cells, and in some B cells. Furthermore, the number of primers required for all the possible combinations is small, and junctional diversity is limited compared to other TCR loci. Rearrangement of β genes is a very powerful tool for detection of α/β clonality; even considering the extreme combinatorial diversity, amplification of almost the entire repertoire may be obtained using a relatively limited number of reactions containing appropriate sets of primers. Both complete VB-JB and incomplete DB-JB rearrangements may be studied, increasing the sensitivity of the method; the extreme junctional diversity of the B locus gives high sensitivity to β TCR clonality, even if sophisticated analysis of the PCR product may be required (see below). Analysis of the D locus is relatively easy and may add information on immature T cells and G/D populations, but is usually not informative for A/B cells. In contrast, α genes are not too helpful given the extreme complexity of gene segments, and concomitant rearrangement of β locus.

Two modifications of PCR are currently used for the detection of clonality: heteroduplex electrophoresis¹⁹ and genescan analysis. In both methods, PCR amplicons are analyzed for fine discrimination and identification of identical products, utilizing different biologic properties of DNA.

Genotyping of rearranged TCR or Ig genes relies on the amplification of the gene fragments with primers that are labeled with a fluorochrome and detection of the labeled products using capillary gel electrophoresis.²⁰ Most capillary electrophoresis-based automated gene sequencers can be adopted for this technique, which allows for resolution of PCR products that usually vary in size by multiples of 3 bp. Under normal circumstances, the amplification products are polyclonal, showing a large number of distinct peaks on capillary electrophoresis tracing. Best results are seen with IgH and TCR B loci, which both have a high frequency of in-frame V-J rearrangements, while for other loci the spacing between peaks is not preserved because of out-of-frame (nonproductive) or incomplete rearrangements. If a monoclonal population is present, a distinct singular peak is present that corresponds to the immunodominant clone. An equivalent technique can be used for the identification of Ig rearrangement. The sensitivity of genotyping is around 5% of malignant (clonal) cells in a cell mixture. As with heteroduplex analysis, frequently a biallelic rearrangement can be detected.

As discussed previously, Ig and TCR clonality can be assessed regardless of the lineage restriction of a putatively clonal population; however, a classic genescan pattern with triplet spacing of peaks is limited to functional in-frame rearrangements. In this case, representation of productive rearrangements may be also obtained by RT-PCR starting from RNA.

Rearranged dominant junctional CDR3 sequences can be sequenced and used for the design of clonotypic PCR primers that uniquely amplify only the malignant clone. Using nested PCR and variable and J primers, further rounds of amplification with a clonotype specific primer (such as an internal J primer) can be added. Such an approach may increase the sensitivity and specificity of detection.

The application of TCR and Ig rearrangement analysis include determinations of B- and T-cell clonality in blood, marrow, lymph nodes, and skin lesions for the diagnosis of T-ALL, B-ALL, multiple myeloma, lymphomas, large granular lymphocyte (LGL) leukemia, and CLL. In CLL, Ig rearrangement analysis is a major prognostic marker (see also Chapter 14). Detailed sequencing of clonal IgH may reveal homology to the germline IgV gene. Somatic hypermutations of the IgV gene usually occur after antigen priming and, consequently, the IgV mutation status allows for distinction between pre- and postgerminal center CLL, entities with different clinical behavior.

MOLECULAR DIAGNOSIS OF INFECTIONS IN HEMATOLOGY

Molecular methods are increasingly supplementing serologic and histochemical methods in microbiology.²¹ Precise detection, localization, and quantitation of the nucleic acids of pathogenic viruses allows for better distinction of individual disease entities and are often used for therapeutic decisions. For instance, detection of EBV activation is essential for early diagnosis and therapy of posttransplant lymphoproliferative disorder. For DNA viruses such as herpes viruses, detection of mRNA transcripts by means of RT-PCR allows diagnosis of active infections, while DNA PCR is positive even when only latent virus in present. Molecular techniques are most often used for the diagnosis to EBV, cytomegalovirus (CMV), human herpes virus-6 (HHV-6), as well as retroviruses such as human T-cell leukemia virus (HTLV-1; Table 29.3).22,23

Epstein-Barr Virus

Both PCR and FISH can detect EBV in lymphoid neoplasms (Table 29.3). FISH probes complementary to EBV localize the virus to the malignant cells. Because of its sensitivity and lack of quantitation, DNA PCR may provide positive results without clinical relevance due to the frequency of latent EBV infection in the community. In contrast, quantitative light-cycler PCR assays provide highly precise copy numbers, but they are less sensitive methods. Both Taqman probes (single-stranded DNA probes that release light on degradation by Taq polymerase; see previous discussion) and beacon probes (DNA probes that release light on conformational change after hybridization to the internal segment of the amplicons) are used. TaqMan PCR is performed as described above for the detection of translocations. Sensitivity levels can be as low as 1 viral copy per 1 to 2 × 10⁵ cells.²⁴ Viruses are always cell-associated and DNA for analysis is extracted from blood leukocytes.

FISH performed with probes detecting EBV-encoded RNA (EBER) is the most commonly applied test for the detection of virus in malignant cells. The sensitivity of this method is related to the high copy number of these transcripts and the ability to detect EBV genome in its latent state (EBER transcription is not dependent on induction of a productive rival life cycle).

Cytomegalovius

Molecular methods have a wide application in the diagnosis of CMV disease and compete with traditional culture and histochemistry-based techniques. DNA PCR can be used for the detection of the CMV genome, but because of its high sensitivity, results may not be informative in seropositive individuals. CMV is strictly cell-associated and blood leukocytes are used as a source for DNA. Quantitative PCR utilizing the light-cycler technology is the usual method for detection of CMV viremia and titer quantitation. Both beacon and Taqman probes have been developed (as described for EBV). Based on standard curves with calibrated positive controls, the number of viral genome copies can be precisely calculated.²⁵ CMV can be detected at levels as low as one and as high as 5 × 10⁵ copies per milliliter, correlating well with the antigenemia measured per 2 × 10⁵ leukocytes.

B19 Parvovirus

Serologic methods are informative only in a minority of circumstances. Similar to CMV, B19 PCR may provide a high positivity rate that does not reflect clinically relevant viremia. Because of the extremely high copy number of virions during active infection, B19 can be detected and quantitated in serum using DNA hybridization methods without amplification. Dot-blot hybridization is most suitable to provide quantitation by serial dilution of positive sera with defined B19 genome copy numbers. Viral titer can be determined by comparison to the dilution standards.²¹

Other Viruses

In theory, all viruses of known nucleic acid sequence can be detected using PCR. In some clinical circumstances, viral detection may have diagnostic consequences (Table 29.3). For example, herpes virus-6 may be identified in primary effusion lymphomas; presence of adenovirus (type 11) and polyomavirus (BK, JC) DNA may be helpful for diagnosis of hemorrhagic cystitis after bone marrow transplantation.

MOLECULAR DETECTION OF DONOR/RECIPIENT CHIMERISM

Determination of the contribution of host versus recipient blood cell production after allogeneic stem cell transplantation has become a standard laboratory test with clinically relevant implications. Several techniques have been devised, including STR analysis, RFLP or FISH for X or Y chromosomes. Selection of various cell types for analysis allows for the separate determination of chimerism in individual hematopoietic lineages.

Polymerase Chain Reaction–Based Short-Tandem Repeat Analysis

Highly polymorphic microsatellite STRs exist for a large number of human loci. STR sequences show great variability in the number of repeats, their length is inherited, and their pattern may be individually specific.²⁶⁻²⁹ Examples of such loci include FGA, VWA, TH01, F13A1, and D21S11. A large number of primer pairs allow for amplification of STR from several loci that show biallelic deletion/insertion polymorphisms. Their size patterns enable forensic identity determination and paternity testing.

For bone marrow transplantation, STR for several loci can be amplified in a donor and recipient. Informative loci can be selected for the highest resolution of size differences between the donor and recipient pair. After transplantation, blood is sampled and DNA extracted. Following amplification of informative STR, gel electrophoresis is used to determine the presence of donor and/or recipient bands. When fluorochrome-labeled primers are used, PCR amplicons can be genotyped using capillary gel electrophoresis to precisely resolve the amplified products by size. The areas under the informative peaks are measured and the percentage of donor chimerism is calculated by the division of donor’s peak area by the sum of the areas of the recipient and donor. This calculation can be performed for several informative loci and averaged. The sensitivity of this type of STR analysis permits the detection of 5% of donor/recipient cells for all loci and around 1% for all patients and selected loci. Combining this method with techniques of cell separation allows determination of chimerism within lymphoid or myeloid cells compartments, adding useful information in the setting of nonmyeloablative transplantation.

Short-Tandem-Repeat Analysis by Real-Time (Quantitative) Polymerase Chain Reaction

Real-time PCR STR analysis is a more sensitive and highly quantitative method.³⁰⁻³² Two primer pairs, each specific for the donor and recipient STR alleles, are selected. STRs suitable for this analysis have biallelic polymorphism, with both alleles varying by at least two consecutive bases and showing a high level of heterozygosity. The sensitivity of this method can be as low as 0.1%, but there is need for a large selection of labeled primers and probes for identification of the most informative loci.

Restriction Fragment Length Polymorphism Analysis

Many loci within the human genome show significant allelic polymorphism, resulting in changes in endonuclease restriction sites. After enzyme digestion, DNA is electrophoresed. The resulting Southern blot is hybridized with tagged DNA probes derived from the polymorphic loci, resulting in the appearance of donor- and recipient-specific bands if there are allelic differences at the locus.

Fluorescence In Situ hybridization Analysis of Sex Chromosomes

Centromeric X and Y chromosome probes can be used for the detection and quantitation of donor and recipient cells. The assay is informative only in sex-mismatched transplants.

MOLECULAR HUMAN LEUKOCYTE ANTIGEN TYPING

Traditional serologic testing is increasingly replaced by molecular testing that allows for a higher precision and resolution of HLA alleles and polymorphisms.33,34 Molecular analysis of HLA loci resulted in the identification of a large number of new alleles, with new polymorphisms still being added. PCR-based methods and primers have been developed for intermediate resolution (IR) and high-resolution (HR) level typing. Serologic testing continues to be performed in many institutions for class I and II alleles, but elsewhere serologic testing has been abandoned for class II alleles.³⁵ Serologic testing retains some role, especially for the confirmation of null alleles after molecular testing.

Polymerase Chain Reaction–Based Human Leukocyte Antigen Testing

Two methods have dominated modern HLA testing technology: SSP and SSOP. Allele- or group-level typing is commonly performed using SSP. Group- and locus-specific primers can be used in the first stage of testing followed by allele-specific primers. Standardized primer sets have been developed for both IR and HR testing.³⁶ However, direct sequencing technologies will likely dominate the future of HLA testing.

SSOP testing can be used for the identification of individual alleles or for the detection of SNPs. In general, SSOP analysis includes differential hybridization of amplicons to specific probes that are designed to match nucleotide sequences at all polymorphic sites of exons 2 and 3. Standardized probe sets have been developed. Several modifications of SSOP hybridization are possible, including membrane- and bead-based fluorometric techniques. For flow cytometric approaches, such as using luminex technology, PCR amplification is performed in the presence of a fluorescently labeled primer pair. The probes are immobilized to polystyrene beads tagged with fluorochromes that can be characterized by a flow cytometric technology using their orange-red emission profile combined with the counterfluorescence of the tagged amplicons. The presence of specific alleles can be identified based on a specific double-fluorescence profile of beads that carry a probe complementary to the amplicon. This assay can be multiplexed to allow a wide screening for many alleles.

SNPs within individual HLA alleles can also be detected using sets of primers designed to investigate polymorphism. This method, also referred to as single nucleotide extension, can also be multiplexed, similar to SSOP.

Finally, for greatest resolution and detection of previously unknown or new polymorphisms, individual region of HLA genes can be PCR amplified and directly sequenced.

References

  1.  Lillicrap D. Molecular diagnosis of inherited bleeding disorders and thrombophilia. Semin Hematol. 1999;36:340-351.

  2.  Arcasoy MO, Gallagher PG. Molecular diagnosis of hemoglobinopathies and other red blood cell disorders. Semin Hematol. 1999;36:328-339.

  3.  Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev. 2003;17:43-53.

  4.  Spowart G. Mitotic metaphase chromosome preparation from peripheral blood for high resolution. In: Gosden JR, ed. Methods in Molecular Biology Chromosome Analysis Protocols. Vol 29. Totowa, NJ: Humana Press; 1994:1-10.

  5.  Hokland P, Pallisgaard N. Integration of molecular methods for detection of balanced translocations in the diagnosis and follow-up of patients with leukemia. Semin Hematol. 2000;37:358-367.

  6.  Rowley JD. Cytogenetic analysis in leukemia and lymphoma: an introduction. Semin Hematol. 2000;37:315-319.

  7.  Bernard OA, Berger R. Location and function of critical genes in leukemogenesis inferred from cytogenetic abnormalities in hematologic malignancies. Semin Hematol. 2000;37:412-419.

  8.  Ferrando AA, Look AT. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin Hematol. 2000;37:381-395.

  9.  Gozzetti A, Le Beau MM. Fluorescence in situ hybridization: uses and limitations. Semin Hematol. 2000;37:320-333.

10.  Kirsch IR, Reid T. Integration of cytogenetic data with genome maps and available probes: present status and future promise. Semin Hematol. 2000;37:420-428.

11.  Schrock E, Padilla-Nash H. Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumorspecific chromosomal aberrations. Semin Hematol. 2000;37:334-347.

12.  Lichter P, Joos S, Bentz M, Lampel S. Comparative genomic hybridization: uses and limitations. Semin Hematol. 2000;37:348-357.

13.  Murphy KM, Levis M, Hafez MJ, et al. Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay. J Mol Diagn. 2003;5:96-102.

14.  Gale RE. Evaluation of clonality in myeloid stem-cell disorders. Semin Hematol. 1999;36:361-372.

15.  Arstila TP, Casrouge A, Baron V, et al. Diversity of human alpha beta T cell receptors. Science. 2000;288:1135.

16.  Macintyre EA, Delabesse E. Molecular approaches to the diagnosis and evaluation of lymphoid malignancies. Semin Hematol. 1999;36:373-389.

17.  Butler JE. Immunoglobulin gene organization and the mechanism of repertoire development. Scand J Immunol. 1997;45:455-462.

18.  Beishuizen A, Verhoeven MA, Mol EJ, et al. Detection of immunoglobulin kappa light-chain gene rearrangement patterns by Southern blot analysis. Leukemia. 1994;8:2228-2236.

19.  Langerak AW, Szczepanski T, Van Der BM, et al. Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations. Leukemia. 1997;11:2192-2199.

20.  Plasilova M, Risitano A, Maciejewski JP. Application of the molecular analysis of the T cell receptor repertoire in the study of immune-mediated hematologic disease. Hematol J. 2003;8:173-181.

21.  Brown KE. Molecular diagnosis of viral disease in hematology patients. Semin Hematol. 1999;36:352-360.

22.  Precursor B-cell and T-cell neoplasms. In: Jaffe ES, Harris NL, Stein H, et al., eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:109-117.

23.  Mature B-cell neoplasms. In: Jaffe ES, Harris NL, Stein H, et al., eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissue. Lyon: IARCPress; 2001:119-187.

24.  Jebbink J, Bai X, Rogers BB, et al. Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons. J Mol Diagn. 2003;5:15-20.

25.  Li H, Dummer JS, Estes WR, et al. Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol. 2003;41:187-191.

26.  Brouha PC, Ildstad ST. Mixed allogeneic chimerism. Past, present, and prospects for the future. Transplantation. 2001;72:S36-S42.

27.  Kreyenberg H, Holle W, Mohrle S, et al. Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Tuebingen experience. Leukemia. 2003;17:237-240.

28.  Leclair B, Fregeau CJ, Aye MT, et al. DNA typing for bone marrow engraftment follow-up after allogeneic transplant: a comparative study of current technologies. Bone Marrow Transplant. 1995;16:43-55.

29.  Monaco AP. Chimerism in organ transplantation: conflicting experiments and clinical observations. Transplantation. 2003;75:13S-16S.

30.  Fernandez-Aviles F, Urbano-Ispizua A, Aymerich M, et al. Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeat-markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood. Leukemia. 2003;17:613-620.

31.  Nuckols JD, Rasheed BK, McGlennen RC, et al. Evaluation of an automated technique for assessment of marrow engraftment after allogeneic bone marrow transplantation using a commercially available kit. Am J Clin Pathol. 2000;113: 135-140.

32.  Alizadeh M, Bernard M, Danic B, et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood. 2002;99:4618-4625.

33.  Klein J, Sato A. The HLA system. First of two parts. N Engl J Med. 2000;343:702-709.

34.  Klein J, Sato A. The HLA system. Second of two parts. N Engl J Med. 2000;343:782-786.

35.  Cao K, Chopek M, Fernandez-Vina MA. High and intermediate resolution DNA typing systems for class I HLA-A, B, C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Rev Immunogenet. 1999;1:177-208.

36.  Tiu R, Gondek L, O’Keefe C, Maciejewski JP. Clonality of the stem cell compartment during evolution of myelodysplastic syndromes and other bone marrow failure syndromes. Leukemia. 2007;21:1648-1657.

30

Interpretation of Functional Genomics

Adrian Wiestner and Louis M. Staudt

The paradigm of “nature or nurture” juxtaposes genetically determined traits to the formative environment. When we consider gene expression in a given cell or organism, these apparent opposites converge. Genes required for cell lineage determination and genes required for cellular responses to environmental conditions are equally transcribed into RNA. Not all genes however are expressed in all cells at all times. Thus, the “transcriptome,” or the genes that are expressed in a given cell at a given time, is only a fraction of the genome. The transcriptome integrates cell lineage, cellular functions, activity of regulatory or oncogenic pathways, and response to external factors. In hematologic malignancies, the quantitative analysis of the transcriptome has refined disease classification and provided powerful prognostic information. In addition, profiling of the transcriptome revealed activation of distinct oncogenic pathways, the importance of which can be experimentally tested by targeted genetic interventions using short complementary RNAs that reduce expression of a specific gene. In some cases, these approaches have led to the discovery of oncogenic mutations, thus linking the “structural” genetic information to the “functional” genomic characteristic of the sample under study. In recent years, whole genome sequencing technologies have been widely applied in oncology and are rapidly generating a comprehensive map of tumor mutations. Functional genomics will likely continue to be a powerful tool to study the role of these mutations in tumor biology.

Here, we focus on discussing general concepts of functional genomic methods and illustrate their application with examples primarily from the study of lymphoid malignancies.

GENE EXPRESSION PROFILING TO CAPTURE THE TRANSCRIPTOME

Two major techniques are now available to capture the complement of genes expressed in a cell: DNA microarrays and RNA sequencing. DNA microarrays consist of solid supports onto which probes have been attached that detect the presence of a specific RNA. Each array consists of thousands of such probes and each probe specifically hybridizes to one distinct RNA. A type of microarray technology commonly used employs oligonucleotide probes attached to a solid support. Affymetrix GeneChip® arrays are commercially available oligonucleotide arrays that depending on the specific array type can quantify the expression of approximately 47,000 transcripts (Human Genome U133 Plus 2.0). Novel sequencing technologies have made it possible to determine the sequence of all RNAs in a given sample. In addition to the actual sequence information, this technology also provides a highly quantitative measure of the relative abundance of a given RNA in the sample.

GENE EXPRESSION SIGNATURES IN MOLECULAR DIAGNOSIS, OUTCOME PREDICTION, AND TARGETED CANCER THERAPY

Microarray experiments typically yield several thousand data points per sample. The amount of data generated in such studies can easily overwhelm the researcher and statistician alike and makes “eyeball” analysis of the data virtually impossible. A number of analytical techniques aid in the interpretation of microarray data.¹⁻⁴ In a so-called unsupervised analysis, statistical methods are used to visualize patterns of shared gene expression and to identify distinct groups of samples. This approach is independent of external data. “Supervised” approaches instead rely on statistical tests to relate gene expression characteristics to known biologic or clinical characteristics.

Unsupervised Analysis: Pattern Discovery by Hierarchical Clustering

One commonly used unsupervised strategy is called hierarchical clustering.¹ This analysis identifies genes that share a similar expression pattern across all samples. For example, hierarchical clustering will group genes together that are highly expressed in one group of samples and lowly expressed in a second group. Genes that are involved in the same cellular function are often coordinately expressed and thus form a distinct “gene expression signature” of a particular biologic process.⁵ Gene expression signatures capture biologic characteristics, including cell type, differentiation state, cellular functions, and activity of signaling pathways, and thereby provide a framework in which the complexity of microarray data can be related to the biology of the study samples. Hierarchical clustering is a valuable tool to discover such patterns of coordinately expressed genes. The strength of this analysis is the focus on distinct biologic functions, represented by sets of genes contributing to the same process rather than isolated genes. For example, to proliferate, a cell simultaneously expresses a set of hundreds of genes involved in cell cycle progression, DNA replication, and metabolism, which upon hierarchical clustering can be visualized as a proliferation signature. Gene expression studies often use a single array per sample. The apparent lack of replicates is sometimes felt to make such data inferior. However, signature-based analysis strategies intrinsically are based on numerous replicates, which are more valuably biologic as opposed to technical replicates.

Hierarchical clustering can not only identify genes with coordinate expression across samples but also group samples that share a common pattern of gene expression. Hierarchical clustering can thereby dissect the heterogeneity of tumor samples that may be very important clinically.⁶⁻⁸ Thus, hierarchical clustering is an especially useful tool for “question-driven” as opposed to “hypothesis-driven” analysis of a data set and can uncover unexpected associations.

Experimentally defined gene expression signatures are catalogued and made available for statistical analysis.3,5,9 Signature-based analysis algorithms can provide molecular classifications of cancer types, establish prognoses, identify cancer subtypes with sensitivity to specific pharmacologic interventions, establish optimal drug combinations, and facilitate the discovery of novel pathway inhibitors. Strategies that have proven particularly effective are gene set enrichment analysis (GSEA) and the connectivity map. GSEA provides a statistical measure of the probability that a set of genes contains a predefined functional signature.⁴ This method can test whether the gene expression difference expressed between two tumor types are due, for example, to differential activity of the nuclear factor kappa B signaling pathway; similarly, the effect of a drug can be related to a distinct signaling pathway. To link these characteristics of cancer and drug, the connectivity map was developed.³ In essence, the gene expression profile is used to match tumor biology with the mechanism of action of a pharmaceutical agent. Such methods aid in drug development and may guide the clinical use of cancer therapy by identifying patient populations likely to benefit from a given intervention.

Supervised Analysis: Building Molecular Predictors of Diagnosis, Prognosis, and Treatment Response

“Supervised” analytical methods use biologic or clinical data to search for gene expression differences that are most informative for diagnosis or prognosis. To derive a molecular predictor of survival, one can, for instance, use the Cox proportional hazard method to identify gene expression characteristics associated with a distinct outcome. This initial step may yield several hundred genes depending on the sample size and significant cutoffs chosen. To further organize data, hierarchical clustering can be used to identify specific gene expression signatures that reflect those biologic processes that impact on survival. The pattern of gene expression signatures represented by a molecular outcome predictor and the optimal number of genes vary between different diseases and analytical techniques. In a large study of diffuse large B-cell lymphoma, 17 genes representing several signatures related to differentiation, tumor proliferation, and tumor–host interactions were combined to form the best prognostic score.¹⁰ In chronic lymphocytic leukemia (CLL), in contrast, a single gene, ZAP-70, was the most differentially expressed gene between biologic and prognostic distinct subtypes of the disease.¹¹

CHALLENGES OF GENE EXPRESSION PROFILING

Not surprisingly, a method that yields quantitative data concerning many thousands of genes in hundreds of samples poses statistical obstacles. We focus briefly on three aspects; a recent review provides a more detailed discussion.¹²

Data Reproducibility: The Value of Training and Validation Sets

The large amount of data derived from gene expression studies increase the likelihood of finding chance associations between clinical variables and gene expression, increasing the likelihood that a model derived in one data set may not be reproducible in an independent data set. One approach against such overfitting problems is to randomly assign the cases in a study to two independent sets. The “training set” is used to derive the model while the “validation set” is used to test the general applicability of the model.

Multiple Testing Corrections: The Concept of False Discoveries

To analyze gene expression data, the traditional probability testing has to be corrected for the innumerable tests that can be performed on such large data sets. Because the p-value is designed to test a distinct hypothesis, a correction for multiple testing is necessary to avoid numerous false-positive calls. The false discovery rate (FDR) predicts the likely number of false-positive discoveries within a nominally significant set of variables. The FDR is computed as the number of expected chance findings at a given p-value divided by the number of observations at this significance cutoff.

Real and Apparent Discrepancies between Different Gene Expression Studies

A quality control study sponsored by the Food and Drug Administration involving different laboratories and different array platforms found excellent reproducibility of microarray measurements. While establishing, in principle, the robustness of the method, apparent and real discrepancies between reported studies can have many reasons, including faulty annotation of probes, lack of specificity of spotted array features, technical differences in hybridization and signal detection, and different analysis strategies that seemingly yield discrepant gene lists.¹³ The use of different platforms may result in gene lists that only partially overlap but nevertheless capture the same biologic characteristics, as a gene expression signature that identifies a distinct diagnostic entity or cellular process may be composed of several hundred genes, not all of which are equally represented and equally well measured on different platforms. Therefore, the rank order of these genes can substantially differ between different studies. Comparison of the entire set of differentially expressed genes, not only the top ranked genes, may be required to detect commonality.¹⁴ With the increasing standardization of technical platforms and more robust analysis algorithms, the reproducibility of microarray studies may already have surpassed the reproducibility of immunohistochemical or flow cytometric methods.

Genetic Interference Studies with Short Complementary RNAs to Identify Essential Pathways in Cancer Biology

The discovery that short (in the 20–30 bp range) RNAs can regulate the stability and translation of mRNAs has been transformed into a powerful screening tool for genes essential in cancer biology.¹⁵ Introduction of such short RNAs into a cell can effectively “knock-down” the activity of the target gene. It is therefore possible to assess the importance of a given gene for phenotype, proliferation, and survival of the transfected cell. Two major approaches are used: short RNAs are synthesized in vitro and transfected into cells and the RNA is integrated into a viral expression vector that is then used to transfect the cells. The later strategy has the advantage of providing stable expression of the short RNA while transfected RNA molecules typically show effects only in the first 2 to 3 days. The approach can be scaled so as to basically allow a functional screen across the whole genome. Some recent studies using this approach are listed in Table 30.1.

CLINICAL APPLICATION OF GENE EXPRESSION PROFILING

Gene expression profiling is in rapid transition from a research test to clinical application. Table 30.2 summarizes select informative studies, and recent reviews provide more detailed discussions. 5,38-40 Table 30.3 lists some prospective clinical trials incorporating gene expression profiling. Because of ready tissue availability, most clinical gene expression studies to date have been performed in malignancies. Many of the pioneering studies were retrospective, often based on archival material, and focused on diagnosis.

More recently, gene expression profiling has been used as a tool to capture dynamic changes in tumor biology. One study, for example, analyzed changes in CLL cells as a function of the location of the tumor in blood, lymph node, or bone marrow.²⁸ Gene expression profiling to analyze sequential tumor samples as a patient undergoes treatment provides a comprehensive pharmacodynamic assessment that can validate a hit on the intended target and characterizes the ensuing (stress) response in the tumor cell.29,33

References

  1.  Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 1998;95(25):14863-14868.

  2.  Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science. 1999;286(5439):531-537.

  3.  Lamb J, Crawford ED, Peck D, et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006;313(5795):1929-1935.

  4.  Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-15550.

  5.  Shaffer AL, Wright G, Yang L, et al. A library of gene expression signatures to illuminate normal and pathological lymphoid biology. Immunol Rev. 2006;210:67-85.

  6.  Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511.

  7.  Valk PJ, Delwel R, Lowenberg B. Gene expression profiling in acute myeloid leukemia. Curr Opin Hematol. 2005;12(1):76-81.

  8.  Zhan F, Huang Y, Colla S, et al. The molecular classification of multiple myeloma. Blood. 2006;108(6):2020-2028.

  9.  Nevins JR, Potti A. Mining gene expression profiles: expression signatures as cancer phenotypes. Nat Rev Genet. 2007;8(8):601-609.

10.  Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346(25):1937-1947.

11.  Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. 2003;101(12):4944-4951.

12.  Tinker AV, Boussioutas A, Bowtell DD. The challenges of gene expression microarrays for the study of human cancer. Cancer Cell. 2006;9(5):333-339.

13.  Sotiriou C, Piccart MJ. Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer. 2007;7(7):545-553.

14.  Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A. 2003;100(17):9991-9996.

15.  Ngo VN, Davis RE, Lamy L, et al. A loss-of-function RNA interference screen for molecular targets in cancer. Nature. 2006;441(7089):106-110.

16.  Davis RE, Ngo VN, Lenz G, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature. 2010;463(7277):88-92.

17.  Ngo VN, Young RM, Schmitz R, et al. Oncogenically active MYD88 mutations in human lymphoma. Nature. 2011;470(7332):115-119.

18.  Annunziata CM, Davis RE, Demchenko Y, et al. Frequent engagement of the classical and alternative NF-kappa B pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell. 2007;12(2):115-130.

19.  Shaffer AL, Emre NC, Lamy L, et al. IRF4 addiction in multiple myeloma. Nature. 2008;454(7201):226-231.

20.  Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med. 2004;350(16):1617-1628.

21.  Bullinger L, Dohner K, Bair E, et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med. 2004;350(16):1605-1616.

22.  Holleman A, Cheok MH, den Boer ML, et al. Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment. N Engl J Med. 2004;351(6):533-542.

23.  Yeoh E-J, Ross ME, Shurtleff SA, et al. Classification, subtype discovery and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell. 2002;1(2):133-143.

24.  Dave SS, Fu K, Wright GW, et al. Molecular diagnosis of Burkitt’s lymphoma. N Engl J Med. 2006;354(23):2431-2442.

25.  Hummel M, Bentink S, Berger H, et al. A biologic definition of Burkitt’s lymphoma from transcriptional and genomic profiling. N Engl J Med. 2006;354(23):2419-2430.

26.  Klein U, Tu Y, Stolovitzky GA, et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med. 2001;194(11):1625-1638.

27.  Rosenwald A, Alizadeh AA, Widhopf G, et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med. 2001;194(11):1639-1647.

28.  Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med. 2002;8:68-74.

29.  Dave SS, Wright G, Tan B, et al. Prediction of survival in follicular lymphoma based on molecular features of tumorinfiltrating immune cells. N Engl J Med. 2004;351(21):2159-2169.

30.  Rosenwald A, Wright G, Wiestner A, et al. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell. 2003;3(2):185-197.

31.  Shaughnessy JD Jr, Zhan F, Burington BE, et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1. Blood. 2007;109(6):2276-2284.

32.  Shaughnessy JD Jr, Qu P, Usmani S, et al. Pharmacogenomics of bortezomib test-dosing identifies hyperexpression of proteasome genes, especially PSMD4, as novel high-risk feature in myeloma treated with Total Therapy 3. Blood. 2011;118(13):3512-3524.

33.  Rosenwald A, Wright G, Leroy K, et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med. 2003;198(6):851-862.

34.  Savage KJ, Monti S, Kutok JL, et al. The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood. 2003;102(12):3871-3879.

35.  Bullinger L. Gene expression profiling in acute myeloid leukemia. Haematologica. 2006;91(6):733-738.

36.  Shaffer Iii AL, Young RM, Staudt LM. Pathogenesis of human B cell lymphomas. Annu Rev Immunol. 2012;30:565-610.

37.  Johnson SK, Heuck CJ, Albino AP, et al. The use of molecular-based risk stratification and pharmacogenomics for outcome prediction and personalized therapeutic management of multiple myeloma. Int J Hematol. 2011;94(4):321-333.

38.  Herishanu Y, Perez-Galan P, Liu D, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood. 2011;117(2):563-574.

39.  Rosenwald A, Chuang EY, Davis RE, et al. Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. Blood. 2004;104(5):1428-1434.

40.  Weniger MA, Rizzatti EG, Perez-Galan P, et al. Treatment-induced oxidative stress and cellular antioxidant capacity determine response to bortezomib in mantle cell lymphoma. Clin Cancer Res. 2011;17(15):5101-5112.

APPENDIX

Cytokines Approved for Clinical Use

Pierre Noel

ERYTHROPOIETIN (EPOIETIN ALFA, PROCRIT™, EPOGEN™)

Epoietin Alfa (EPO) Indications

  Anemia of chronic renal failure (CRF) patients (dialysis [+] or dialysis [–]).

  Anemia in zidovudine-treated human immunodeficiency virus (HIV)-infected patients.

  Anemia in patients undergoing chemotherapy.

  Reduction of allogeneic blood transfusions in surgical patients.

In adult patients with CRF, doses of 50 to 100 U/kg three times weekly are required to maintain a hematocrit in the mid- to high thirties. The intravenous (IV) route of administration is recommended in dialysis patients; the IV or subcutaneous (SC) administration routes can be utilized in patients with CRF not undergoing hemodialysis. Patients with CRF treated with erythropoietin (EPO) experienced an increased risk of serious cardiovascular disease when treated with target higher hemoglobin levels. The dose of EPO should be individualized to achieve and maintain hemoglobin levels between 10 and 12 g/dL and should be reduced in patients who respond rapidly (>1 g/dL increase in hemoglobin over a period of 2 weeks) to minimize the risk of serious cardiovascular events. In zidovudine-treated adult patients with HIV with a serum EPO of <500 mU/ mL who are receiving a dose of zidovudine <4,200 mg/week, the recommended starting dose is 100 U/kg as an IV or SC injection three times a week for 8 weeks. The dose of EPO should be titrated to avoid transfusions and not to exceed a hemoglobin level of 12 g/dL. Patients with endogenous EPO levels >500 mU/mL are unlikely to respond to EPO. In adult anemic cancer patients undergoing chemotherapy, the initial recommended dose of EPO is 150 U/kg SC three times a week or 40,000 U SC weekly. Therapy should not be initiated if the hemoglobin is >10 g/dL. Patients with serum EPO levels >200 mU/mL are unlikely to respond to EPO. In some recent clinical studies, erythropoiesis-stimulating agents (ESA) shortened overall survival and/or increased the risk of tumor progression or recurrence in patients with breast, nonsmall cell lung, head and neck, lymphoid, and cervical cancers; because of these findings, EPO use is not recommended in patients receiving myelosuppressive therapy when the anticipated outcome is cure. In adult surgical patients, the reduction of allogeneic transfusion can be achieved with an EPO dose of 300 IU/kg/day for 10 days prior to surgery, on the day of surgery, and for 4 days after surgery. An alternate dose schedule is 600 U/kg SC in once-a-week doses starting 3 weeks prior to surgery plus a fourth dose on the day of surgery. All patients should receive iron supplementation. The preoperative hemoglobin should be between 10 and 13 g/dL. In surgical patients not receiving prophylactic anticoagulation, EPO increased the rate of deep venous thrombosis. Deep venous thrombosis prophylaxis should be considered in this setting.

Prior to and during treatment with EPO, the patient’s iron stores should be evaluated. Most patients require iron supplementation during therapy with EPO. Blood pressure may rise during treatment of anemia with EPO; hypertension should be controlled before initiation of therapy. The hemoglobin levels need to be monitored regularly while patients are being treated with EPO.

Allergic reactions and antibody-mediated pure red cell aplasia have been associated with the use of EPO. EPO contains albumin; it carries a very small risk of transmission of viral diseases.

DARBEPOIETIN ALFA (ARANESP™)

Darbepoietin alfa differs from recombinant human EPO by having sialylated carbohydrate content increasing the molecular weight, prolonging its half-life and increasing its biologic in vivo activity.

Darbepoietin alfa is indicated for the treatment of anemia with CRF and the treatment of anemia associated with nonmyeloid malignancies because of chemotherapy. In adult patients with anemia associated with renal failure, the recommended starting dose is 0.45 μg/kg administered IV or SC once per week. Alternatively, in patients not undergoing hemodialysis an initial dose of 0.75 μg/kg may be administered SC once every 2 weeks.

Darbepoietin alfa has been associated with an increased risk of cardiovascular events in patients with CRF. The risk of cardiovascular events is higher in patients who have target hemoglobin of 14 g/dL compared with patients who have target hemoglobin of 10 g/dL. The dose should be titrated to achieve and maintain target hemoglobin of 10 to 12 g/dL. In patients with nonmyeloid cancer receiving chemotherapy, the recommended doses of darbepoietin alfa are 2.25 μg/kg SC weekly or 500 μg SC every 3 weeks. Therapy should not be initiated at hemoglobin levels >10 g/dL. The goal of darbepoietin alfa treatment in the cancer setting is to avoid transfusions.

Blood pressure may rise during treatment of anemia with darbepoietin alfa; hypertension should be controlled before initiation of therapy.

Seizures and serious cardiovascular events have been reported in patients who had a rapid rate of increase in hemoglobin; the dose of darbepoietin alfa should be reduced if the hemoglobin level increase exceeds 1.0 g/dL in any 2-week period.

Clinical studies in cancer patients treated with ESAs reported shortened overall survival and/or increase in the risk of tumor progression in patients with breast, nonsmall cell lung, head and neck, lymphoid, and cervical cancer; because of these findings, darbepoietin alfa use is not recommended in patients receiving myelosuppressive therapy when the anticipated outcome is cure.

Darbepoietin alfa is supplied in two formulations: one containing polysorbate 80 and another containing albumin. Albumin is associated with a small risk of transmitting viral diseases.

METHOXY POLYETHYLENE GLYCOL-EPOETIN BETA (MIRCERA™)

Methoxy polyethylene glycol-epoetin beta is a continuous erythropoietin receptor activator that can be administered monthly. It has an extended half-life of 130 hours and a low clearance as well as unique receptor-binding properties.

Methoxy polyethylene glycol-epoetin beta is indicated in the treatment of anemia associated with chronic renal disease. The recommended starting dose in patients not currently treated with an ESA is 0.6 μg/kg once every 2 weeks IV or SC.

The warnings and precautions that apply to all ESAs also apply to methoxy polyethylene glycol-epoetin beta (pure red cell aplasia, cardiovascular events, hypertension, effect on tumor growth).

GRANULOCYTE COLONY-STIMULATING FACTOR (FILGRASTIM, NEUPOGEN™)

Filgrastim regulates the production of neutrophils within the bone marrow and also impacts on their function.

Granulocyte Colony-Stimulating Factor Indications

  Nonmyeloid malignancies undergoing myelosuppressive chemotherapy with an expected risk of severe neutropenia and fever.

  Acute myeloid leukemia, following induction or consolidation therapy.

  Nonmyeloid malignancies undergoing myeloablative chemotherapy followed by marrow transplantation.

  Mobilization of peripheral blood hematopoietic cells.

  Severe chronic neutropenia.

Granulocyte colony-stimulating factor (G-CSF) can be administered SC or IV; the usual recommended dose is 5 μg/kg/day.

Patients treated with G-CSF have experienced allergic-type reactions, severe sickle-cell crises, bone pain, splenomegaly, and splenic rupture. G-CSF has the potential of stimulating the proliferation of myeloid leukemic cells.

PEGYLATED GRANULOCYTE COLONY-STIMULATING FACTOR (PEGFILGRASTIM, NEULASTA™)

Pegfilgrastim is produced by covalently conjugating a 20-kDa polyethylene glycol molecule to the N-terminus of filgrastim. The pegfilgrastim molecule is larger than the threshold for renal clearance, prolonging its half-life in circulation.

Pegfilgrastim is indicated to decrease the incidence of febrile neutropenia in patients with non-myeloid malignancies receiving myelosuppressive chemotherapy associated with a clinically significant incidence of febrile neutropenia. The adult recommended dose is 6 mg SC administered once per chemotherapy cycle.

Splenic rupture, adult respiratory distress syndrome, allergic reactions, severe sickle-cell crises, and proliferation of myeloid leukemic cells have been described in patients treated with filgrastim, which is the parent compound of pegfilgrastim.

GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (SARGRAMOSTIM, LEUKINE™)

Sargramostim induces a dose-dependent increase in neutrophils and, to a lesser extent, monocytes and eosinophils. When granulocyte-macrophage colony-stimulating factor (GM-CSF) is discontinued, the leukocyte counts decrease to pretreatment levels over 3 to 5 days.

Granulocyte-Macrophage Colony-Stimulating Factor Indications

  Following induction chemotherapy in patients with acute myeloid leukemia over the age of 55.

  Mobilization of peripheral blood hematopoietic progenitor cells.

  Myeloid reconstitution following autologous bone marrow transplantation for non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, and acute lymphoblastic leukemia.

  Myeloid reconstitution after allogeneic marrow transplantation.

  Allogeneic and autologous bone marrow transplantation engraftment failure or engraftment delay.

GM-CSF can be administered SC or IV. The usual daily recommended dose is 250 μg/m².

The use of GM-CSF is potentially associated with fluid retention, capillary leak syndrome, pleural and pericardial effusions, sequestration of granulocytes in the pulmonary circulation, supraventricular arrhythmias, as well as renal and hepatic dysfunction. GM-CSF has the potential to stimulate the proliferation of myeloid leukemic cells.

INTERLEUKIN-11 (OPRELVEKIN, NEUMEGA™)

Oprelvekin is a thrombopoietic growth factor that stimulates the proliferation of megakaryocytic progenitors and induces megakaryocyte maturation; it also promotes the integrity of gastrointestinal mucosal epithelial cells.

Oprelvekin stimulates platelet production in a dose-dependent manner with peak platelet counts 14 to 21 days following its administration.

Oprelvekin is indicated for the prevention of severe thrombocytopenia in patients with nonmyeloid malignancies undergoing myelosuppressive chemotherapy. Oprelvekin is not recommended for use following myeloablative chemotherapy.

The usual recommended dosage is 50 μg/kg given once a day SC. The use of Oprelvekin has been associated with allergic or hypersensitivity reactions, including anaphylaxis, papilledema, fluid retention, edema, arrhythmias, pleural effusions, and electrolyte imbalances.

ROMIPLOSTIM (NPLATE™)

Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune idiopathic thrombocytopenia who have had an inadequate response to corticosteroids, immunoglobulins, or splenectomy.

The recommended initial dose is 1 μg/kg once a week SC followed by weekly dose adjustments based on response.

It is recommended to use the lowest dose of Romiplostim to achieve and maintain a platelet count >50,000/μL as necessary to reduce the risk of bleeding.

Romiplostim is associated with an increased risk of marrow fibrosis secondary to reticulin fiber deposition.

INDEX

Page numbers followed by f and t denote figures and tables.

A

Abetalipoproteinemia, 33

Abnormal localization of immature precursors (ALIPs), 85

Absolute iron deficiency. See also Iron deficiency

causes of, 3t

laboratory abnormalities, 4t

laboratory parameters, 2

mean cell volume (MCV), 4

red cell count, 3

reticulocyte hemoglobin, 3

soluble transferrin receptor concentration, 3

Absolute neutrophil count (ANC), 116, 131, 166

Absolute reticulocyte count (ARC), 24

Acanthocytosis, 33

Accelerated phase (AP), 171

Acquired and constitutional. See Bone marrow failure syndromes

Acquired thrombophilic states

activated protein C resistance

Caucasian population, 321

characteristic mutation, 322

inherited hypercoagulable

states, 321t

polymorphism, 321

antiphospholipid antibody syndrome

antithrombotic therapy, 319

dilute Russell viper venom time (DRVVT), 318

kaolin clotting time (KCT), 318

mechanisms of, 318t

screening and confirmatory tests, 318

tissue thromboplastin inhibition (TTI), 318

treatment strategies, 319

heparin-induced thrombocytopenia (HIT)

enzyme immunoassays, 316

fondaparinux binds, 317

HEP, 316

LMWH, 316–317

lupus anticoagulants (LAC), 318

panel probability score, 317t

platelet aggregation assays, 317

platelet rich plasma, 316

pretest prediction of, 316t

PTT monitoring, 318

treatment of, 317

UFH, 316

vitamin-dependent anticoagulant, 318

hypercoagulability of malignancy

CLOT trial, 319

dalteparin, 319

nonmetastatic disease, 319

selective estrogen receptor modulators (SERMS), 319

thrombus formation, 319

tumor types, 320

vascular endothelium, 319

hyperhomocysteinemia, 322

inherited hypercoagulable states, 321

myeloproliferative disorders

blood counts, 321

hydroxyurea (HU), 321

platelet-lowering noncytotoxic, 321

thrombosis, 321

nephrotic syndrome, 318

paroxysmal nocturnal hemoglobinuria, 320

proteins S and C and antithrombin, 322

prothrombin mutation, 322

surgery as risk for

elastic stockings, 320

laparoscopic surgery, 320

peripartum state, 320

procedure of, 320

serial compression devices, 320

Activated B-cell-like (ABC), 218

Activated partial thromboplastin time (aPTT), 146, 298

Acute chest syndrome (ACS), 42

Acute graft-versus-host disease

grading of, 260t

treatment of, 261t

Acute intermittent porphyria, 60

acute abdominal pain, 61

central nervous systems, 60–61

cyclical attacks, 61

endogenous corticosteroid hormones, 60

exogenous corticosteroid hormones, 60

gastrointestinal manifestations, 61

hyponatremia, 61

narcotic analgesics, 61

neurologic dysfunction, 60–61

porphobilinogen deaminase (PBGD), 60

psychiatric symptoms, 61

tachycardia, 61

visceral autonomic nervous system, 61

Acute intravascular hemolysis. See also Erythrocyte morphology and associated pathology

associated pathology, 27–28t

clinical syndrome, 26

Clostridium perfringens, 26

erythrocyte morphology, 27–28t

haptoglobin, 26

indicators of, 28f

lactate dehydrogenase, 26

renal tubular cells, 26

tubular epithelium, 26

Acute lymphoblastic leukemia (ALL), 143, 176, 256

anti-CD20 monoclonal antibody rituximab, 163

antiemetics, 165

antiemetic prophylaxis, 165

CNS-directed therapy, 165

blood transfusion, 166

bone marrow aspiration, 167

Burkitt lymphoma regimens, 163

chemotherapeutic regimens, 160

chemotherapy prophylaxis

agent-specific prophylaxis, 166

corticosteroid ophthalmic solution, 166

cyclophosphamide, 166

hemorrhagic cystitis, 166

ifosfamide, 166

mesna, 166

classification, 160t

clinical features of, 158

CNS, 158

emergency presentations, 159t

hyperleukocytosis, 158

ileocecal intussusception, 158–159

mature B-cell ALL, 158–159

mediastinal mass, 158

Peyer’s patch involvement, 158–159

pleural effusion, 158

presenting features, 159t

clinicopathologic features, 160

CMV infection, 166

CNS-directed therapy, 163

CNS penetration, 163

common chromosomal translocations, 160t

common treatment regimens, 161–162t

CR1, 163

CSF cell count and cytology, 167

curative therapy, 158

cytogenetic abnormalities, 159

dasatinib, 164

delayed intensification, 163

DFS rate, 158

dose modification

drug exposure, 163

hepatic dysfunction, 163

6-MP, 163

myelosuppression degree, 163

severe toxicity, 163

thiopurine S-methyltransferase deficiency, 163

epidemiology, 158

etiology and risk factors

aneuploidy, 158

chromosomal abnormalities, 158

chromosomal breakage syndrome, 158

EBV infection, 158

hematopoietic tissues, 158

immunodeficiency syndrome, 158

mature B-cell ALL, 158

extramedullary leukemia, 164

FAB system, 159

genomic analysis, 160

graft-versus-host disease, 166

hematocrit, 166

hematopathologic analysis, 159

HLA-alloimmunization, 163, 166

imatinib mesylate, 164

infection prophylaxis

ANC, 166

antifungal therapy, 166

G-CSF, 166

hypogammaglobulinemia, 166

immunoglobulin G (IgG) level, 166

IVIG, 166

myeloid growth factors, 166

neutropenic fever, 166

PCP, 166

intensive intrathecal chemotherapy, 163

intrathecal chemotherapy, 164t, 167

late effects, 167

cardiomyopathy, 167

cumulative anthracycline doses, 167

endocrinologic dysfunction, 167

neurologic toxicity, 167

osteonecrosis, 167

secondary malignancy, 167

lumbar puncture, 159, 166

mature B-ALL, 163

meningeal leukemia, 159

molecular testing, 159

morphologic blast reduction, 163

nilotinib, 164

nutritional support

folate antagonism, 166

folic acid, 166

therapeutic efficacy, 166

peripheral blast count, 166

platelet counts, 166

prognostic factors, 159

psychosocial support, 167

radiation guidelines, 164t

RBCs, 166

reinduction phase, 163

relapse management, 165

risk-group assignment, 161t

SCT, 163

SER, 163

severe cytopenias, 166

severe hyperleukocytosis, 166

T-cell phenotype, 163

testicular leukemia, 163

therapy-associated toxicity, 167

tumor lysis syndrome

allopurinol, 165

BUN, 165

CBC, 165

hyperuricemia, 165

induction chemotherapy, 165

intravenous fluids, 165

LDH, 165

life-threatening metabolic complications, 165

mature B-cell ALL, 165

rapid blast lysis, 165

renal insufficiency, 165

uric acid, 165

urine specific gravity, 165

WBC count, 163

Acute myeloid leukemia (AML), 77, 83, 176

acute promyelocytic leukemia, 150

aleukemic leukemia, 140

ALL, 143

anthracycline-based chemotherapy, 150

anthracyclines, 146

anthracycline therapy, 150

aPTT, 146

ATO, 150

ATRA, 145, 150

bone marrow, 141

cardiotoxicity, 146

cellular metabolic activity, 140

chemistry, 140

chemotherapy, 137

clinical features of

APL, 139

bone marrow failure, 138

bruising, 139

CNS, 140

cranial nerve dysfunction, 140

DIC, 139

extramedullary leukemia, 140

life-threatening hemorrhage, 139

monocytic morphology, 138

neutropenic infections, 138

sweet syndrome, 140

CMV, 146

confirmatory genetic test, 150

consolidation therapy, 145

conventional cytogenetics, 145

CR, 137

CT, 140

cytosine arabinoside, 146

diagnostic markers, 143t

electrolyte abnormalities, 140

epidemiology, 137

age-related incidence, 138f

episodic hypotension, 150

etiology

congenital disorders, 137

genetic predisposition, 137

MDS, 137

myeloproliferative disorders, 137–138

pathophysiologic mechanisms, 137

risk factors, 139t

FAB, 141, 141t

fibrinogen level, 150

flow cytometry, 140

frank hematologic relapse, 150

fresh-frozen plasma, 150

genetic events, 137

hematopoietic growth factors

cell cycle, 151

CSFs, 151

G-CSF, 151

GM-CSF, 151

hematopoietic stem cell

transplantation

antileukemic therapy, 147

conventional postremission chemotherapy, 148

cytogenetic risk category, 148

EBMTR, 149

graft-versus-leukemia effect, 147

haploidentical donors, 149

hazard ratio, 148

immune-mediated antileukemic activity, 147

intensive consolidation chemotherapy, 148

marrow-ablative chemotherapy, 147

non-allogeneic therapy, 148

relapse rates, 148t

RIC, 148

transplantation technology, 149

trials evaluating allo-HSCT, 148t

TRM rate, 148

USB, 149

HIV, 146

HLA, 146

HSCT, 147

HSV, 146

induction chemotherapy, 145

induction therapy

adequate cardiac function, 146

anti-CD33 immunoconjugate, 147

azacitidine, 147

chemotherapy regimen, 146

decitabine, 147

gemtuzumab ozogamicin, 147

idarubicin, 146–147

IND, 147

lenalidomide, 147

sapacitabine, 147

tipifarnib, 147

infection prophylaxis, 146

intensive chemotherapy, 145

laboratory findings

chemistry, 140–141

hematologic, 140

imaging studies, 141

LDH, 140

leukemic myeloblasts, 142

liver functions, 146

malignant cells, 137

6-mercaptopurine, 150

methotrexate, 150

minimal residual disease

consolidation therapy, 152

genetic marker, 152

leukemic cell, 152

multiparametric flow cytometry technique, 152

PML-RARA fusion

transcript, 152

prognostic value, 152

therapeutic approach, 152

molecular mechanisms, 137

molecular relapse, 150

morphologic diagnosis of, 143

MRD, 150

MRI, 140

myeloid lineages, 141t

myeloid progenitor cells, 137

neutropenia, 140

novel therapeutic targets

antileukemic activity, 153

clinical experience, 154t

cytotoxic chemotherapy, 153

farnesylation, 153

FLT3 kinase inhibitors, 153

gemtuzumab ozogamicin, 153

immunotherapeutic approaches, 153

lestaurtinib, 153

lipid moiety elaidic acid, 153

methylation, 153

pharmacodynamic

properties, 153

pharmacokinetic

properties, 153

remission rate, 153

salvage chemotherapy, 153

single-agent tipifarnib, 153

toxic drugs, 153

tyrosine kinase inhibitors, 153

in older patients

abnormal organ function, 152

age and performance

status, 151t

azacitidine, 151

clofarabine, 152

conventional therapy, 151

decitabine, 151

intensive therapy, 151

marrow blast count, 151

outpatient therapy, 151

palliative care, 152

remission-induction chemotherapy, 151

oral synthetic retinoid, 150

pathogenesis

cytogenetic abnormalities and mutations, 139t

leukemic cells, 138

malignant

transformation, 138

molecular analysis, 138

myeloid progenitors, 138

tumor suppressor genes, 138

PCR, 145

perianal herpetic ulcerations, 146

pericardial effusions, 150

postremission therapy, 145, 147

in pregnancy

cardiovascular malformations, 152

chemotherapeutic agents, 152

fetal toxic effects, 152

intrauterine growth retardation, 152

prognostic factors, 143

cytogenetic analysis, 144

cytogenetic and molecular abnormalities, 144t

DFS, 144

gene expression, 144

gene mutations, 145t

ITD, 144

leukemic cells, 144

molecular genetic

abnormalities, 143

toxic therapy, 145

tyrosine kinase receptor, 144

promyelocytic leukemia, 140

prophylactic antifungal therapy, 146

PT, 146

pulmonary infiltrates, 150

remission induction, 145

renal tubular dysfunction, 140

risk-based approach

antileukemic activity, 149

better-risk karyotypes, 149

FLT3-ITD, 149

FLT3 tyrosine kinase inhibitors, 149

lestaurtinib, 149

midostaurin, 149

novel genetic lesions, 149

poor-risk cytogenetics, 149

quizartinib, 149

sorafenib, 149

thrombocytopenia, 140

treatment of

farnesyltransferase inhibitors, 151

induction chemotherapy, 150

refractory patients, 151

reinduction chemotherapy, 150

tumor lysis syndrome, 140

WHO, 141, 142t

Acute myeloid lymphoma (AML), 446

Acute promyelocytic leukemia (APL), 139, 299

Adenosine deaminase (ADA), 32

Adriamycin, 241

Adult T-cell leukemia (ATLL), 211

African trypanosomiasis (sleeping sickness), 397

Agranulocytosis. See also Bone marrow failure syndromes

diagnosis and treatment of, 75

drugs associated, 75t

etiology and pathophysiology

agranulocytosis, 75

thorazine, 75

thymoma, 75

immune versus toxic agranulocytosis, 76t

myeloid precursor cells, 74

Aldolase A deficiency, 31

Allele-specific oligonucleotide (ASO), 445

Allogeneic hematopoietic stem cell transplantation (AHSCT)

acute lymphoblastic leukemia

childhood ALL, 257

DFS rates, 257

high-risk cytogenetics, 257

acute myeloid leukemia

HLA-matched sibling donor, 257

poor prognosis, 257

transplantation, 257

alternative donor transplantation

cord blood, 266

haploidentical transplants, 265

HLA-matched sibling donor, 265

from matched unrelated donors, 265

from mismatched related donors, 265–266

retrospective analyses, 266

suitable volunteer donors, 265

umbilical cord transplants, 266

antileukemic potential, underlying principles

bone marrow (BM), 252

chronic myeloid leukemia (CML), 252

donor lymphocyte infusions (DLIs), 252

HLA-matched donor stem cells, 252

T-cell–depleted transplants, 252

complications

acute GVHD, 259

allogeneic PBSC transplant, 260

Aspergillus, 259

Candida, 259

chronic GVHD, 260

conditioning-related toxicities, 258

gastrointestinal (GI), 258

glucocorticoids, 260

graft-versus-host disease, 259

GVHD prophylaxis regimen, 259

hemorrhagic cystitis, 258

prophylaxis, 259

pulmonary complications, 261–262

steroid-refractory patients, 260

treatment of established GVHD, 260

two-stage staging system, 261

VOD, 259

graft-versus-leukemia (GVL), 252

hematopoietic stem cells, 266

indications for, 253t

congenital disorders of erythropoiesis, 252

hematologic malignancy, 252

hematopoiesis, 252

immunodeficiency syndromes, 252

malignant and nonmalignant diseases, 252

infectious complications

amphotericin B, 262

Aspergillus, 262

bacterial infections, 262

Candida infections, 262

CMV infection, 263

CMV-related mortality, 263

cytomegalovirus, 263

echinocandins, 262

Epstein-Barr virus–

associated lymphoproliferative disorder, 264

fluconazole, 262

fungal infections, 262–263

graft failure, 264

gram-negative bacteremia, 262

immunosuppressive agents, 263

interstitial pneumonitis, 263

molds, 262

other viral infections, 264

preemptive therapy, 263

surveillance and management of cytomegalovirus, 263t

T-cell–depleted allografts, 263

viral infections, 263

yeast, 262

late sequelae of transplantation

other late complications, 264

secondary malignancies, 264

lymphocyte content, 266

multiple myeloma

CTN group, 258

graft-versus-myeloma effects, 258

RIC, 258

TRM rates, 258

myelodysplastic syndrome

long-term DFS, 257

non-Hodgkin’s lymphoma

aggressive, 258

chronic lymphocytic leukemia, 257

long-term DFS, 257

low-grade, 257

low-grade lymphomas, 257

planning

conditioning regimen, 255

conventional or myeloablative conditioning, 256

donor–host HLA, 252

evaluation of transplant recipients, 252

factors including, 252

haploidentical donor, 254

HLA-identical siblings, 255

matched related donor, 254

matched unrelated donor, 254

mobilized stem cells, 254

non-TBI regimens, 256

obtaining stem cells, 254

PBSC allografts, 254

preparative regimens commonly used, 255t

procurement of hematopoietic stem cells, 254–255

reduced intensity conditioning, 256

suitable donor identification, 254

syngeneic donor, 254

TBI-based regimens, 256

umbilical cord cells, 254

reduced intensity and nonmyeloablative

basic principles, 265

GVL effects, 264

high risk of TRM, 264

nonmyeloablative HCT, 265

solid tumors, 265

resistant hematologic malignancies, 251

All-trans retinoic acid (ATRA), 145, 299

American Society for Apheresis (ASFA), 370

δ-Aminolevulinic acid dehydratase deficiency porphyria, 60

heme biosynthetic pathway, 60

Amplification refractory mutation system (ARMS), 445

Anaplastic lymphoma kinase (ALK), 222

Anemia

childhood anemia, 122t

ESAs, 244

evaluation of, 122t

hematologic parameters, 122t

hemoglobin (Hb) concentration, 121

macrocytic anemias

abnormal chromosomal breakage, 126

androgen therapy, 126

autosomal recessive, 126

chelation therapy, 126

chronic transfusion, 126

congenital pure red cell aplasia, 126

corticosteroid dependence, 126

DBA, 126

erythrocyte folate levels, 126

erythroid hypoplasia, 126

FA, 126

folate deficiency, 126

folic acid, 126

genitourinary system, 126

hematologic manifestations, 126

myelodysplastic syndrome, 126

pernicious anemia, 126

prednisone, 126

replacement therapy, 126

risk factors, 126

severe pancytopenia, 126

WBC, 126

microcytic anemias

blood volume expansion, 121

chelation therapy, 125

erythropoietic drive, 125

ferritin level, 121

frank deficiency, 123

gastrointestinal tract irritation, 121

globin chain, 123

β-globin synthesis, 123

iron deficiency, 121

microcytosis, 125

thalassemia syndrome, 123

α-thalassemia trait, 125

β-thalassemia trait, 123

utero transfusion, 125

venous blood lead levels, 124t

normochromic, 244

normocytic anemia, 244

normocytic anemias

acute chest syndrome, 126

acute inflammation, 125

autoimmune hemolytic anemia, 125

chronic blood transfusions, 126

Coomb’s test, 125

eye examination, 126

glucose-6-phosphate dehydrogenase deficiency, 125

hemolytic conditions, 125

hereditary spherocytosis (HS), 125

HUS, 125

hydroxyurea, 126

immune hemolysis, 125

immune-mediated hemolysis, 125

LDH, 125

membrane disorders, 125

meningococcal vaccine, 126

microangiopathic hemolysis, 125

pediatric hematologist, 125

pediatric patients, 125

penicillin prophylaxis, 126

pneumococcal sepsis, 126

pneumococcal vaccination series, 126

polysaccharide pneumococcal vaccine, 125–126

red cell morphology, 125

reticulocytopenia, 125

serum haptoglobin, 125

short-term transfusion therapy, 125

sickle cell disease, 125

TCD, 126

transient bone marrow, 125

vasoocclusive pain crises, 125–126

viral infection, 125

pathophysiology, 123t

physical examination, 121

physiologic nadir, 121

recommendations for, 123t

red cell mass, 121

serum transferrin receptor, 121

Anemia of chronic disease (ACD), 4

Angiotensin converting enzyme (ACE), 47

Antithymocyte globulin (ATG), 91

Aplastic anemia (AA). See Bone

marrow failure syndromes

Array-based comparative genomic

hybridization (A-CGH), 448, 449f

Arsenic trioxide (ATO), 150

Aspergillus, 262

Ataxia, 33

Autoimmune hemolytic anemia (AIHA), 189, 344

Autoimmune lymphoproliferative syndrome (ALPS), 133, 402

Autoimmune neutropenia (AIN), 402

Autologous hematopoietic stem cell

transplantation (AHSCT), 207, 240

general considerations, 249

contamination, 250

cytotoxic drugs, 250

G-CSF, 249

graft-versus-tumor (GVT), 249

infections, 249

PBSCs, 249

purge tumor cells, 250

treatment-related mortality (TRM), 249

in hematologic malignancies

lymphoma, 250–251

multiple myeloma, 250

lethal hematopoietic toxicity, 249

in solid tumors

breast cancer, 251

germ cell tumors, 251

other tumors, 251

B

B-cell acute lymphoblastic leukemia (B-ALL), 447

Bernard-Soulier disease, 408, 438

Bernard-Soulier syndrome (BSS), 129

Bleeding

acquired factor deficiencies

assaying coagulation factor levels, 127

breast-fed infants, 127

chronic diarrhea, 127

fat malabsorption syndromes, 127

FFP, 127

HDN, 127

prophylactic administration, 127

screening coagulation tests, 127

age-specific coagulation values, 128t

cephalohematomas, 127

and coagulation tests, conditions

acquired abnormalities, 295

acquired collagen, 295

alpha-2-antiplasmin

deficiency, 295

clot lysis, 295

congenital abnormalities, 295

cryoprecipitate, 295

Ehlers-Danlos syndrome, 295

enzymatic assays, 295

factor XIII deficiency, 295

glucocorticoid excess, 295

hereditary hemorrhagic telangiectasia, 295

heritable defects of collagen, 295

Osler-Weber-Rendu disease, 295

osteogenesis imperfecta, 295

plasminogen activator I, 295

recurrent pregnancy loss, 295

traumatic soft tissue, 295

hemorrhagic disorders, 127

inherited factor deficiencies

chronic arthropathy, 127

CNS, 127

PTT, 127

severe factor deficiency, 127

vWD, 127

vWF, 127

neonatal period, 127

physiologic levels, 127

platelet disorders

autoimmune disease, 129

BSS, 129

congenital amegakaryocytic thrombocytopenia, 129

GT, 129

HIV infection, 129

intracranial hemorrhage, 129

ITP, 129

IVIG, 129

May-Hegglin anomaly, 129

NAIT, 129

physical examination, 129

postnatal period, 129

severe thrombocytopenia, 129

TAR, 129

treatment regimens, 129t

WAS, 129

X-linked thrombocytopenia, 129

Blood components and derivatives

antithrombin, 354

α-1-antitrypsin, 354

cryoprecipitate

cold-insoluble portion, 352

fibrinogen, 352

fibronectin, 352

indications, 353

not indicated, 353

pathogen-inactivated fibrinogen, 353

von Willebrand factor, 352

fresh-frozen plasma

indications, 352

multiple clotting factor deficiencies, 352

not indicated, 352

granulocytes

alloimmunized patients, 352

cytomegalovirus (CMV), 352

G-CSF, 351

granulocyte transfusion therapy, 352

HNA, 352

indications, 351–352

minimal therapeutic, 351

not indicated, 352

pulmonary toxicity, 352

hematopoietic stem and

progenitor cells

acetaminophen, 354

allografts, 353

aplastic anemia, 353

autografts, 353

dimethyl sulfoxide (DMSO), 354

donor demographic, 354

G-CSF, 353

GM-CSF, 353

graft-versus-host disease

(GVHD), 354

laboratory predictors, 354

malignant and nonmalignant

disorders, 353

mobilizing agents, 353

National Marrow Donor Program, 354

NSAIDs, 354

PBSC grafts, 354

peripheral blood stem cell (PBSC), 353

plerixafor-mobilized

cells, 353

stem cells mobilization, 353

orphan proteins, 354

platelet refractoriness

alloimmunization, 351

B loci, 351

corticosteroids, 351

disseminated intravascular coagulation (DIC), 351

HLA A, 351

HLA matched donors, 351

human platelet antigens (HPA), 351

immune-mediated, 351

immune-mediated refractoriness, 351

nonimmune-mediated causes, 351

posttransfusion platelet counts, 351

platelets

alloimmunized refractory patients, 350

apheresis, 350

bacterial sepsis, 350

complete blood count (CBC), 350

corrected count increment (CCI), 350

indications, 350

not indicated, 350

posttransfusion increment, 351

prophylactic, 350

random donor, 350

single-donor, 350

stable chronically thrombocytopenic patients, 350

thrombotic thrombocytopenic purpura (TTP), 350

whole blood-derived, 350

prothrombin complex concentrate

3 factor-PCCs, 354

indications, 354

prophylaxis, 354

recombinant coagulation factors

plasma-derived coagulation factors, 358

recombinant factors, 358

red blood cells

by centrifugation, 349

hemoglobin (Hb), 350

indications, 350

numerous observational, 350

transfusion, 350

unit, 349

Rh immune globulin (RhIg)

erroneous transfusions, 358

immune thrombocytopenic purpura (ITP), 356

indications, 356

intramuscular (IM), 356

intravenous (IV), 356

nonobstetric cases, 358

not indicated, 358

RhIg IV, 356

Rh-positive RBC, 356

selected coagulation factor

preparations, 357t

selected blood derivatives, 355–356t

transfusion therapy

angiotensin converting enzyme, 347

cellular blood products, 347

Food and Drug Administration (FDA), 347

granulocyte concentrates, 347

hemolyze cells, 347

hypotensive reactions, 347

hypotonic solutions, 347

infusion, 347

isotonic solutions, 347

nonapproved pumps, 347

plasma components, 347

platelets, 347

storage, 347

whole blood, 347

whole blood

acute hypovolemia, 349

blood component administration per NIH practice, 348t

chronic anemia, 349

granulocytes, 349

hematocrit, 347

indications for additional modifications, 348–349t

Blood transfusion

ABO incompatibility in transplant settings

donor’s serum, 345

human leukocyte antigen (HLA), 345

major incompatibility, 345–346

minor incompatibility, 345

recipient-donor pair, 345

recipient’s serum, 345

Rh incompatibility, 346

transfusion in minor and major ABO, 346t

acute hemolytic transfusion reaction

ABO blood group, 358

management, 359

mechanism, 358–359

presentation, 358

prevention, 359

alternatives to allogeneic blood transfusion, 364

examples, 365t

pharmaceutical hemostatic agents, 365

selected pharmacologic hemostatic agents, 366–367t

anaphylactic transfusion reactions

anti-IgA, 361

IgA-deficient patients, 361

management, 361

mechanism, 361

occur, 360

presentation, 361

prevention, 361

antiglobulin test

AIHA, 344

direct antiglobulin test (DAT), 344

direct Coombs test, 344

IAT detects, 344

indirect antiglobulin test (IAT), 344

indirect Coombs test, 344

intravenous immunoglobulin (IVIG), 344

bacterial contamination and sepsis

cold storage conditions, 362

evaluation and management, 362–363

mechanism, 362

mild septic reactions, 362

presentation, 362

prevention, 363

blood cell antigens

antibodies, 343

antigen-negative blood, 343

antiglobulin, 343

Coombs test, 343

hemolytic disease of the newborn (HDN), 343

red blood cell (RBC), 343

blood compatibility

blood group serology, 345

fresh-frozen plasma (FFP), 345

group-specific red cells, 345

plasma-containing components, 344

RBC, 344

recipient blood with donor blood components, 345t

Rh-negative patient, 344

blood components and derivatives, 347–358

delayed hemolytic transfusion reaction

anamnestic, 360

management, 360

mechanism, 360

occurs, 360

presentation, 360

prevention, 360

primary immunization, 360

disseminated intravascular coagulation, 368

estimated risk of transfusion transmission, 365t

febrile nonhemolytic transfusion reaction

evaluation and management, 364

mechanism, 364

presentation, 364

prevention, 364

TRALI, 364

hemolysis associated with passive antibody infusion

ABO-incompatible plasma, 359

acute anemia, 359

hemosiderosis

evaluation and management, 364

mechanism, 364

presentation, 364

prevention, 364

hypotension associated with transfusion

apheresis procedures, 363

evaluation and management, 363

leukoreduction filters, 363

mechanism, 363

prevention, 363

immunohematologic disorders, 368–374

major blood groups, laboratory determination

ABO discrepancy, 344

anti-A, 343

anti-B, 343

antibody specificity, 343

autoantibodies, 344

cold reactive alloantibodies, 344

forward grouping, 343

group A, 343–344

group AB, 343

group B, 343–344

group O, 343–344

phenotype, 343

reverse grouping, 344

Rouleaux formation, 344

massive transfusion

ABO-compatible blood, 368

adverse sequelae, 368

RBC crossmatch, 367

Rh-positive blood, 368

mild allergic/urticarial transfusion reactions

evaluation and management, 363

mechanism, 363

presentation, 363

prevention, 363

other causes of hemolysis

drug-induced hemolysis, 360

hypotonic solutions, 360

mechanical, 360

mimic hemolytic transfusion, 360

RBC unit, 360

thermal, 360

posttransfusion purpura

high-dose IVIG, 363

management, 363

mechanism, 363

presentation, 363

reactions, 359t

reactions and adverse sequelae

acute versus delayed, 358

hemolytic, 358

hemolytic versus nonhemolytic, 358

immune and nonimmune-mediated, 358

sickle cell hemolytic transfusion syndrome

blood donors, 360

patients, 360

RBC antigen phenotypes, 360

red cell phenotyping, 360

transfusion-associated circulatory overload

cardiac failure, 361

central venous pressure elevation, 361

management, 362

plasma volume expansion, 362

presentation, 362

prevention, 362

pulmonary edema, 361

transfusion-associated graft-versus-host disease

aplastic anemia, 362

malignancies, 362

management, 362

mechanism, 362

patients with HIV, 362

presentation, 362

prevention, 362

transfusion-related acute lung injury

FDA, 361

management, 361

mechanism, 361

noncardiogenic pulmonary edema, 361

pathophysiologic mechanism, 361

plasma-containing blood, 361

presentation, 361

prevention, 361

two-hit theory, 361

transfusion-transmitted infections

antibodies, 364

nucleic acid testing, 364

surface antigen, 364

Trypanosoma cruzi, 364

Blue sclerae, 1

Bone marrow aspirate/biopsy

cells anomalous, 410

cell suspensions, 409

core biopsy, 409

eosin-stained biopsy, 409

fibrosis, 409

fibrous tissue, 409

fibrous tumor, 409

FISH, 411

hematopoietic cells, 409

hematoxylin, 409

histochemical stains, 409

hypercellularity, 409

hypocellularity, 409

immunohistochemical stains, 409, 411

indications

biochemical tests, 408

detecting metastases, 408

exclude diagnoses, 408

Gaucher’s disease amyloidosis, 408

nuclear medicine techniques, 408

iron stores of, 410–411

lymphocytes and plasma cells, 410

marrow cellularity, 409

megakaryocyte number and appearance, 409–410

metastatic cancer cells, 409

molecular diagnostic studies, 411

myeloid to erythroid ratio, 410

risks

anticoagulation, 408

core biopsy, 408

hemorrhage, 408

immune function, 408

sterile technique, 408

thrombocytopenia, 408

Romanowsky-stained aspirate films, 409

technique, 409

Bone marrow failure syndromes

absolute reticulocyte, 71

acute lymphocytic leukemia, 68

acute myelogenous leukemia, 68

alemtuzumab, 70

anaphylaxis, 70

antithymocyte globulin (ATG), 70

aplastic anemia (AA)

clinical features of, 66

diseases, 67t, 68t

differential diagnosis, 66

drugs associated with, 65t

etiology and pathophysiology, 65–66

treatments, 68–71

blood cell production, 64

bone marrow transplantation versus immunosuppression, 70t

chromosomal analysis, 67

clinical hemolysis, 68

corticosteroids, 71

cyclosporine (CsA), 70

cytogenetic abnormalities, 64, 71

cytopenias differential diagnosis, 69f

diagnostic testing

chromosomes, 79

telomeres, 79

dysplastic bone marrow, 71

Fanconi anemia (FA), 66, 67, 68t

granular lymphocytes, 68

growth factor combinations, 71

Ham test, 68

hematologic response rate, 70

hematopoiesis, 64

herpes virus infection, 71

histocompatible sibling donor, 68

immunosuppression, 70

immunosuppressive regimens, 71

liver function, 71

lymphadenopathy, 66

marrow hypocellularity, 68

megakaryocytes, 66

myeloblasts, 66

myelodysplasia (MDS), 67

myelofibrosis, 68

myeloid precursors, 67

nail dystrophy, 66

nephrotoxicity, 71

neutrophil count, 70

oral leukoplakia, 66

pancytopenia, 64, 67t

paradigmatic syndrome, 64

pathogenesis, 64

phenotypic abnormalities, 67

physical examination, 66

preemptive antiviral therapy, 71

prognosis, 71

serum sickness, 71

splenomegaly, 66

stem cell, 71

T cell receptor, 68

thrombocytopenia, 66, 68

transient blood count, 70

treatment of, 72f

umbilical cord blood transplantation, 70

white blood cell, 64

Bone marrow transplantation, 34

Bone morphogenetic protein (BMP), 378

Breakpoint cluster region (BCR), 170

Breast cancer

auto-HSCT, 251

Burkitt lymphoma (BL), 211

aggressive chemotherapy, 230

CODOX-M/IVAC regimen, 230t

CODOX-M regimen, 231t

high-risk patients, 230t

IVAC regimen, 231t

tumor lysis syndrome, 229

C

Carmustine, 241

Central nervous system (CNS), 127, 158, 218

chronic transfusions, 44

hemorrhagic stroke, 44

silent infarct transfusion (SIT), 44

Stroke with Transfusion Changing to Hydroxyurea (SWiTCH), 44

thrombotic stroke, 44

tissue plasminogen activator (t-PA), 44

transcranial Doppler (TCD), 44

transfusion therapy, 44

transient ischemic attacks (TIAs), 44

Chédiak-Higashi syndrome, 308

Childhood hematologic diseases

anemia. See Anemia

bleeding. See Bleeding

blood parameters, 121

hematologic manifestations

ALPS, 133

autoimmune cytopenias, 133

autoimmune disorders, 134

autoimmune-mediated

cytopenias, 133

collagen vascular diseases, 133

enzyme replacement therapy, 133

Gaucher cell, 133

lipid-laden macrophages, 133

lysosomal metabolic pathway, 133

lysosomal storage diseases, 133

molecular defects, 133

Niemann-Pick disease, 133

PT, 134

reticuloendothelial system, 133

systemic disorders, 133

leukocytosis

chronic viral process, 132

CMV, 133

congenital immunodeficiency, 133

EBV, 133

flow cytometry, 133

hematologic complications, 133

hepatosplenomegaly, 132

IM, 133

immune-mediated hemolytic anemia, 133

lymphadenopathy, 132

neutrophilia, 132

non-Hodgkin’s lymphoma, 133

physical examination, 132

X-linked lymphoproliferative syndrome, 133

neutropenia

acute myelogenous leukemia, 132

ANC, 131

asymptomatic oscillations, 132

autosomal dominant disorder, 132

bone marrow examination, 132

bone marrow infiltration, 132

Chédiak-Higashi syndrome, 132

congenital neutrophil disorders, 131–132

gastrointestinal tract infection, 132

genetic defects, 132

granulocyte colony stimulating factor, 131

hematologic manifestations, 132

hemophagocytic lymphohistiocytosis, 132

life-threatening sepsis, 132

mild bleeding disorder, 132

myelodysplastic syndrome, 132

neutrophil elastase, 132

pancreatic exocrine insufficiency, 132

periodic oscillations, 132

pyogenic infections, 131

severe neutropenic complications, 132

Shwachman-Diamond syndrome, 132

trafficking regulator gene, 132

transient postinfectious, 131–132

thrombosis. See Thrombosis

transfusion support

age-specific blood volume, 134

citrate toxicity, 135

cryoprecipitate, 135

fluid intolerance, 134

hypofibrinogenemia, 135

leukofiltration, 135

life-threatening bleeding, 135

pediatrics, 134t

pediatric transfusion, 134

platelets transfused, 134

PRBCs, 134

prophylactic platelet transfusion, 135

Chronic graft-versus-host disease (GVHD), 180

Chronic granulomatous disease (CGD), 32

Chronic kidney disease (CKD), 4

Chronic lymphocytic leukemia (CLL), 462

aggressive chemoimmunotherapy, 192

AIHA, 189

alkylating agents

cyclophosphamide, 191

myelosuppression, 191

oral chlorambucil, 191

response rates, 191

allogeneic stem cell transplantation

fludarabine refractoriness, 192

graft-versus-leukemia effect, 192

non-myeloablative conditioning regimens, 192

anti-infective prophylaxis, 194

asymptomatic patients, 190

autoimmune cytopenias, 191

autoimmune manifestations

AIHA, 194

cyclosporine, 194

ITP, 194

prednisone, 194

pure red cell aplasia (PRCA), 194

purine analogues, 194

B cell receptor, 192

bendamustine, 192

beta-2-microglobulin (B2M), 189

blood count and blood smear

autoimmune mechanisms, 187

prolymphocytes, 187

thrombocytopenia, 187

tumor cells, 187

bone marrow biopsy, 187

bone marrow, 188

infiltration diffuse pattern, 187

CD38 expression, 190

clinical flow cytometry, 189

clinical manifestations

asymptomatic lymphadenopathy, 186

blood test, 186

central nervous system, 187

GI tract, 187

lymph node, 187

neurologic symptoms, 187

non-Hodgkin lymphoma, 186

corticosteroid treatment, 191

CT, 188

cytogenetics

FISH, 190

genetic subgroups, 190

karyotypic abnormality, 190

mitotic rate, 190

cytoplasmic protein, 190

DAT, 189

disease progression, 189

disease-related symptoms, 187

etiology and pathogenesis

B cell receptor, 186

clonal B cells, 186

proliferation rate, 186

fludarabine combination regimens, 195

fludarabine-refractory

disease, 192

Haemophilus influenzae, 194–195

hematopoietic neoplasias, 187

Herpes zoster, 194–195

herpes zoster vaccine, 194

histopathologic evaluation, 187

hypogammaglobulinemic patients, 195

immune defect, 194

immunoglobulin (Ig), 189

immunophenotype of, 188, 188t

immunophenotyping and flow cytometry, 187

intense treatment regimens, 192

investigational treatment for, 194t

IVIG replacement infusions, 194

IWCLL, 187, 191

LDH, 189

LDT, 190

lymphadenopathy, 187

lymphatic tissues, 186

lymph node biopsy, 187, 188

MBL, 187

metabolic activity, 188

methylprednisolone, 192

monoclonal antibodies

alemtuzumab, 191

antimicrobial prophylaxis, 191

cytokine release syndrome, 191

JC virus, 191

lymphadenopathy, 191

lymph nodes, 191

ofatumumab, 191

PML, 191

rituximab, 191

severe hypotension, 191

MRD, 192

neutropenic, 195

periodic evaluation, 190

pneumocystis, 194

purine analogues

autoimmune hematologic complications, 191

complete responses (CRs), 192

cyclophosphamide, 192

fludarabine, 191

fludarabine monotherapy, 192

PFS, 192

purine analogue therapy, 192

Rai clinical staging system, 189t

Richter syndrome, 195

Richter transformation

B cell lymphoma, 195

Hodgkin lymphoma, 195

S. pneumonia, 194–195

side-effect profile, 192

SLL, 187

standard diagnostic test, 187

T cell receptor, 189

thrombocytopenia, 188

time to treatment (TTT), 190

treatment options, 193t

tyrosine kinase ZAP-70, 189

visceral lymphadenopathy, 188

World Health Organization, 187

Chronic myelogenous leukemia (CML), 95, 252, 446

accelerated phase

autologous SCT, 182

characteristics of, 176t

chloromas, 175

chromosomal abnormalities, 175

HLA-identical donor, 182

imatinib resistant disease, 182

marrow reticulin, 175

myelofibrosis, 175

thrombocytopenia, 175

treatment, 182

allogeneic stem cell transplantation

bone marrow transplantation, 181t

DFS, 180

GVHD, 180

HLA-identical sibling donor

transplants, 181f

low-risk patients, 180

lymphocyte infusions, 180

TRM, 180

BCR, 170

BCR-ABL1 oncoprotein, 170

BCR-ABL1 transcripts, 174f

BCR-ABL1 tyrosine kinase, 170

blastic phase

acute leukemia, 175

ALL, 176

AML, 176

auer rods, 176

BCR-ABL inhibitors, 182

clonal evolution, 175

cyclophosphamide, 182

cytochemical stains, 176

daunorubicin, 182

imatinib-refractory patients, 182

marrow failure, 175

prophylactic central nervous system, 182

red cell count, 175

salvage therapy, 182

treatment, 182

blood count, 172

blood film

basophilia, 172

eosinophilia, 172

metamyelocytes, 172

myeloblasts, 172

platelet morphology, 172

bone marrow examinations

blastic phase, 172

granulocytic hyperplasia, 172

sea-blue histiocytes, 172

chromosomal marker, 170

chromosome analysis, 173, 177

FISH, 172

Philadelphia chromosome, 173f

chronic phase

amino acid, 180

BCR-ABL1 protein, 180

BCR-ABL1 tyrosine kinase, 180

bone marrow aspiration, 177

cytogenetic response, 177

cytosine arabinoside, 180

hematologic remission, 177

HLA, 180

interferon-α(IFN-α), 180

leukocyte count, 173

MRD, 177

Ph-negative cells, 177

treatment, 177–180

cytogenetic response, 177

diagnosis of, 172–173

differential diagnosis of, 174f

disease bulk reduction, 177

disease monitoring, 179f

epidemiology, 170–171

focal neurologic deficits, 172

graft-versus-leukemia

effect, 170

hematologic response, 177

human malignant disease, 170

hyperproliferative myelodysplastic

syndrome, 173

imatinib mesylate, 170

karyotypic level, 177

leukocyte counts, 172

molecular diagnosis

acute lymphoblastic leukemia, 173

BCR-ABL1 transcript variants, 173

morphologic features, 172

myelodysplastic neoplasms, 172

PCR, 172

molecular level, 177

mouse stem cells, 170

myeloproliferative disorders, 172

neutrophil function, 172

pathophysiology, 171

Philadelphia (Ph) chromosome, 170

prognostic factors, 176

relapse treatment

antileukemic effect, 182

chloromas, 183

DLI, 182

leukemic response, 182

leukostasis, 182

marrow aplasia, 182

myelofibrosis, 183

neurologic deficit, 182

psychological responses, 183

psychosomatic techniques, 183

radiotherapy, 183

SCT, 170

stem cell pathways, 170

TKIs, 170

treatment algorithm, 178f

tyrosine kinase inhibitors

BCR-ABL1 kinase domain, 183

dasatinib, 183

first-line therapy, 183

IFN-α, 183

nilotinib, 183

Chronic myelomonocytic leukemia (CMML), 86, 95

acute leukemia, 108

chemotherapy, 109

clinical features of, 108

diagnostic testing, 108

epidemiology, 108

growth factors, 109

hypomethylating agents, 109

imatinib mesylate, 109

myelodysplastic syndromes, 109

pathophysiology, 108

clonal cytogenetic abnormalities, 108

eosinophilia, 108

lymph nodes, 108

marrow cells, 108

mutational spectrum analysis, 108

myeloproliferation, 108

stem cell transplantation, 109

thrombocytopenia, 108

Classical Hodgkin lymphoma (CHL), 197

Clinical trials network (CTN), 250

Clonal diseases, 448

Clonality studies

clinical situations, 452

skewed hematopoietic function, 452

XCIP analysis, 453

Clostridium perfringens sepsis, 26

Coagulation. See Hemostasis disorders

Colony-stimulating factors (CSFs), 151

Combination antiretroviral therapy (cART), 210

Common variable immunodeficiency (CVID), 222

Complete blood count

platelets

clinical parameters, 407

hematopoietic transplantation, 407

impedance-based technique, 406

IPF, 407

ITP, 407

marrow thrombopoiesis, 407

MPV, 407

optical-based technique, 406

RBC and WBC counts, 406

red cell parameters

absolute reticulocyte count, 406

automated cell counters, 405

automated counters, 406

basophilic stippling, 406

degree of redness, 405

hemoglobin concentration, 405

intracellular organisms, 406

IRF, 406

mathematical equation, 405

MCHC, 405

MCV, 405

microscopic inspection, 406

optical and fluorescent cell-counting methods, 406

RDW, 405–406

red cell count, 405

reticulocytes, 406

traditional methodology, 405

sources of artifact

platelet clumping, 407

pseudothrombocytopenia, 407

RBC, WBC, and platelet counts, 407

red cell agglutination, 407

WBC parameters

automated absolute granulocyte, 406

automated leukocyte differentials, 406

hematopoietic stem cell transplantation, 406

leukopenic samples, 406

osmotic lysis, 406

peripheral blood, 406

peripheral smear, 406

white cell morphology, 406

Complete blood count (CBC), 24, 121, 165, 200

Complete remission (CR), 137

Complete response (CR), 226

Compression ultrasonography (CU), 393

Computed tomography (CT), 140, 188, 201

Computed tomography pulmonary angiography (CTPA), 393

Congenital amegakaryocytic thrombocytopenia

autosomal recessive disorder, 81

thrombopoietin receptor, 81

Congenital erythropoietic porphyria

allogeneic stem cell transplantation, 61

autosomal-recessive disorder, 61

Gunther’s disease, 61

splenectomy, 61

Congenital neutropenia

autosomal dominant disorder, 81

autosomal recessive disorder, 81

Kostmann syndrome, 81

moderate thrombocytopenia, 81

osteoporosis, 81

Consultations in anticoagulation

anticoagulants

current armamentarium, 339

expensive novel agents, 340

laboratory testing, 340

medications of, 339

novel mechanisms, 339

oral factor Xa inhibitors, 339

randomized evaluation of long-term anticoagulation therapy (RE-LY), 339

reliable ways, 340

rivaroxaban group, 339

warfarin therapy, 339

antiphospholipid syndrome

aggressive treatment, 338

antiphospholipid antibodies (APA), 338

antithrombotic therapy, 339

fluctuating INR levels, 339

hydroxychloroquine, 339

lupus anticoagulants (LAs), 338

recurrent thrombosis, 339

inferior vena cava (IVC), 328

postthrombotic (“postphlebitic”) syndrome

graduated compression stockings (GCS), 337

management of, 337–338

patient derives symptomatic, 337

primarily clinical, 337

prolonging compression therapy, 337

venous hypertension, 337

postthrombotic syndrome, 328

prophylaxis and treatment of venous thromboembolism, cancer patient. See Venous thromboembolism treatment

venous thromboembolism (VTE), 328

Copper deficiency, 4

Cox proportional hazard method, 462

Cyclic neutropenia. See Congenital neutropenia

Cyclophosphamide, 241, 250

Cytogenetic diagnostics

fluorescence in situ hybridization (FISH)

B-ALL, 447

chromosome painting probes, 447

dual-color/dual-fusion probes, 447

EBV, 447

hematopoietic stem cell

transplantation, 446

Hodgkin’s disease, 447

MDS, 447

MRD, 446

natural killer (NK), 447

paraffin-embedded tissues, 446

single-fusion/dual-color probes, 447

T-ALL, 447

genomic hybridization and single nucleotide polymorphism arrays

A-CGH, 448, 449f

application of, 448

CGH arrays, 448

clonal diseases, 448

DNA segments, 448

fluorescence ratio, 448

intermarker distance, 448

M-CGH, 448

oligoarray technology, 448

SNP arrays, 448

unbalanced translocations, 448

metaphase karyotyping

AML, 446

banding techniques, 446

chromosomal bands, 446

CML, 446

G-banding, 446

spectral karyotyping (SKY)

emission spectra, 447

refinement of, 447

traditional cytogenetics, 447

translocations and deletions, 447t

Cytomegalovirus (CMV), 133, 146, 166, 254

Cytotoxic lymphocytes (CTL), 66

D

Data reproducibility, training and validation sets, 462

Deep venous thrombosis (DVT), 241, 393

anticoagulation risk, 313

compression threatens, 311

contralateral extremities, 311

definitive therapy, 311

distal veins of lower extremities

acute reduction of, 316

essential thrombocythemia, 316

myeloproliferative disorders

(MPD), 315

orthotopic liver transplantation, 315

paroxysmal nocturnal hemoglobinuria (PNH), 315

polycythemia vera (PV), 316

prevalence of, 315

splanchnic vein, 315

imaging of

computed tomography venography (CTV), 313

include cost, 313

magnetic resonance venography (MRV), 313

ultrasonography, 313

ultrasound techniques, 313

venography, 313

inferior vena cava (IVC), 311

lesser hydrostatic, 312

postthrombotic syndrome (PTS), 311

signs and symptoms of, 312t

skin ulceration, 313

Villalta scale for, 312t

DexFerrum. See Iron dextran

Diamond-Blackfan anemia

corticosteroids, 80

hematological findings

chromosome breakage test, 79

erythrocyte adenosine deaminase (eADA) level, 79

transient erythroblastopenia, 79

physical anomalies, 79

ribosomal protein genes, 79

treatment modalities, 80

ventricular septal defects, 79

Diamond-Blackfan anemia (DBA), 126

Dietary iron, 5. See also Absolute iron deficiency

Diferric transferrin, 2

Diffuse alveolar hemorrhage, 262

Diffuse intravascular coagulation (DIC)

activated protein C, 393

causes of, 393t

clostridial sepsis, 393

cocaine addicts, 392

dead fetus syndrome, 392

fetal death syndrome, 393

hypertonic saline abortions, 393

incidence of, 392

placental abruption, 392

pregnancy-associated microangiopathies, 392t

Diffuse large B-cell lymphoma (DLBCL)

antibiotic prophylaxis, 228

carboplatin, 228

field radiotherapy, 228

prognostic models, 228

rituximab therapy, 228

salvage regimens, 229t

salvage therapy, 228

standard therapy for, 228t

Diffuse large B-cell lymphoma (DLBCL), 210

Dilute Russell viper venom time (DRVVT), 318

2,3-Diphosphoglycerate mutase (DPGM) deficiencies, 31

Diphyllobothrium latum, 13

Direct antiglobulin test (DAT), 189, 344

Disease-free survival (DFS), 144, 158, 180, 206, 226, 250

Disseminated intravascular coagulation (DIC), 125, 139, 285

acral cyanosis, 297

aneurysmal dilatation, 298

APL, 299

aPTT, 298

ATRA, 299

blood products, 298

central nervous system, 297

clinical scenario, 298

conditions of, 298t

consumptive process, 299

cryoprecipitate, 299

ecchymotic lesions, 297

fetal tissue, 299

FFP, 299

fibrin degradation, 298

fibrinogen levels, 299

fibrinolysis inhibitors, 299

gastrointestinal, 297

gross hemoglobinuria, 299

HELLP syndrome, 299

hemolytic anemia, 299

hemorrhage, 297

heparin, 299

hepatic dysfunction, 299

laboratory manifestations, 279, 298

laboratory parameters, 298

laboratory tests, 298

microvascular hemolysis, 298

multiorgan dysfunction, 298

pathophysiology

bacterial lipopolysaccharide proteins, 297

hypoxemic injury, 297

neoplastic cells, 297

thrombosis, 297

tissue factor, 297

Trousseau syndrome, 297

platelet count, 298

platelet transfusions, 298

PT, 298

Purpura fulminans, 297

renal dysfunction, 299

severe sepsis, 299

surgical incisions, 297

thrombocytopenia, 279

tissue factor, 299

tissue hypoxia, 298

TTP, 299

vascular access catheters, 297

VTE, 299

warfarin, 299

Donor lymphocyte infusion (DLI), 182, 252

Donor/recipient chimerism

real-time PCR STR

analysis, 457

restriction fragment length polymorphism analysis, 457

sex chromosomes, 457

STR analysis

bone marrow transplantation, 456

cell separation, 456

fluorochrome-labeled primers, 456

myeloid cells, 456

Doxorubicin, 250

Drug-induced thrombocytopenia

associated with thrombocytopenia, 282t

chemotherapeutic agents, 281

mucocutaneous bleeding, 281

platelet transfusions, 281

quinine purpura, 281

Duffy antigen and receptor for chemokine (DARC), 119

Dyskeratosis congenita, 78

clinical manifestations, 78

hematologic manifestations, 78

mucocutaneous manifestations, 78

oxymetholone, 78

X-linked recessive, 78

E

Eastern Cooperative Oncology Group

(ECOG), 202, 225

Eculizumab, 34

Ehlers-Danlos syndrome, 295, 309

End stage renal disease (ESRD), 8

Enolase, 31

Eosinophilic disorders, 399t

blood eosinophilia, 397

clonal eosinophilic disorders, 397, 399t

end-organ damage, hypereosinophilia, 398t

diseases, 398t

helminthic infections, 397

human androgen receptor

gene locus, 399

idiopathic hypereosinophilic syndrome-(IHES), 397

iron deficiency associated, 397t

Epidemiology, 158, 197, 210

acute intermittent porphyria (AIP), 57

δ-aminolevulinic acid dehydratase (ALAD) deficiency, 57

bimodal age distribution, 197

cART, 210

classic hereditary hemochromatosis, 377

clinical penetrance of, 377

enzyme deficiency, 57

genetic factors, 171

HFE mutations

additional polymorphisms, 377

chromosome, 6, 377

compound heterozygous, 377

disequilibrium analysis, 377

frequency of, 378t

genetic polymorphism, 377

hemochromatosis phenotype, 377

Hispanic children, 158

HIV infection, 210

inappropriate dietary absorption of, 377

ionizing radiation, 171

leukemias, 170

organ dysfunction, 377

pediatric malignancy, 158

single-gene disorders, 377

Epstein-Barr virus (EBV), 133, 158, 199, 211, 254, 447

Erythrocyte morphology and associated pathology, 27–28t

Erythrocyte sedimentation rate (ESR), 200

Erythroid enzymopathies

acute hemolysis, 29

clinical application, 29

enzyme deficiencies, 29

enzymopathies associated with hemolysis, 32

glutathione metabolism

drugs and chemicals, 30t

γ-glutamylcysteine synthetase, 30

glutathione reductase, 30

glutathione synthetase, 30

G6PD deficiency, 29

peroxidase (GSH-Px), 30

glutathione reduction, 29

glycolysis, enzymes involved, 29

ADP and ATP, 31

aldolase A, 31

2,3-diphosphoglycerate mutase (DPGM), 31

enolase, 31

glucose phosphate isomerase (GPI), 31

hexokinase, 31

PGK deficiency, 31

phosphofructokinase deficiency, 31

PK and TPI deficiency, 31

hemolysis-related enzymes, 29

Erythropoiesis-stimulating agents (ESAs), 244

Erythropoietic protoporphyria

ferrochelatase activity, 62

heme biosynthetic

pathway, 62

intravenous heme therapy, 62

oral β-carotene, 62

protoporphyrin, 62

Erythropoietin-stimulating agents (ESA), 4

Essential thrombocythemia. See

Myeloproliferative

neoplasms

Ethylenediaminetetraacetic acid (EDTA), 123

European Bone Marrow Transplant Registry (EBMTR), 149

Extrinsic disorders

drug-induced neutropenia, 403

immune neutropenias

antineutrophil antibodies, 402

autoimmune neutropenia (AIN), 402

granulocyte agglutination test (GAT), 402

granulocyte immunofluorescence test (GIFT), 402

monoclonal antibody immobilization of granulocyte antigens assay (MAIGA), 402

neutrophil-specific antigens, 402

spontaneous remissions, 402

neutropenia associated with

brucellosis, 403

Colorado tick fever, 403

Fas-Fas ligand apoptotic system, 402

gram-negative septicemia, 403

immunodeficiency syndrome, 403

infectious hepatitis, 403

influenza, 403

large granular lymphocyte (LGL) syndrome, 402

mechanisms of, 403t

paratyphoid fever, 403

peripheral destruction, 402–403

Staphylococcus aureus, 403

T-cells, 402

tularemia, 403

typhoid fever, 403

neutropenia associated with autoimmune disorders

autoimmune lymphoproliferative syndrome (ALPS), 402

fas-mediated apoptosis, 402

Felty’s syndrome, 402

lupus erythematosus, 402

rheumatoid arthritis, 402

Eye disease

sickle-thalassemia, 44–45

vascular remodeling, 45

F

Fanconi anemia (FA), 126, 423

androgens, 78

anabolic properties, 78

hematologic responses, 78

oxymetholone, 78

clinical features of, 76

congenital malformations, 76

cyclophosphamide, 77

thoracoabdominal irradiation (TAI), 77

diagnostic tests, 77

diepoxybutane (DEB), 77

major physical anomalies, 77

hematologic abnormalities, 76

hematologic manifestations, 77

hematologic presentations and cancer predisposition

clonal karyotypic abnormalities, 77

erythropoiesis, 77

pancytopenia, 77

solid organ malignancies, 77

thrombocytopenia, 77

hematopoietic growth factors (HGFs), 78

MMC chromosome breakage analysis, 76

stem cell transplantation, 77

Felty’s syndrome, 402

Feraheme. See Ferumoxytol

Ferrlecit. See Sodium ferric gluconate in sucrose

Ferumoxytol, 7

Fibrinolytic therapy

catheter-directed thrombolysis (CDT), 324

iliofemoral system, 325

venous segments, 325

Flow cytometry, clinical applications

advantage of, 427

antigen expression, 437

Bernard-Soulier disease, 438

cell progenitors traverse, 436

cell surface markers, 427, 438

contour plot, 436f

control and positively stained histograms, 437f

data presentation and interpretation

bone marrow, 432

CD8 histogram evaluating lymphocytes, 433f

CD4 histogram evaluating mononuclear cells, 434f

cell-activation-specific processes, 433

cell preparation, 429

cell surface proteins, 433

contour plot, 429f

demonstrates myeloblasts, 432f

dot plot, 429–430f

erythrocytes, 429

GMC, 433

hematopoietic malignancies, 433

hematopoietic stem cells, 428, 430, 432

HIV infection, 431

homogeneous antigen expression, 428

intracellular characteristics, 433

intracellular proteins, 434

lineage-specific antibodies, 432

lymphocytes, 429

microscopic evaluation of, 433

minimal residual disease (MRD), 428

monoclonal antibodies, 431

natural killer (NK) lymphocytes, 431

negative–positive discriminator, 428

neutrophils stain, 432

pan-T-cell marker, 431

positive signal, 428

protein upregulation, 433

regular/agranular (side-angle scatter) cells, 430

side scatter and CD45 analysis, 431–432f

single-parameter histogram, 429f

validated reagents, 428

differentiation designation cluster, 435t

erythrocytes evaluation, 438

fine-needle aspirates, 436

fluorescein isothiocyanate, 428

fluorescent signals, 427

fluorochrome chemistry, 427

forward-angle light scatter, 427

functional characteristics, 428

Glanzmann thrombasthenia, 438

granulocyte expression, 437

hematology laboratories, 427

hematopoietic cells, 435

hematopoietic stem cell, 438

HIV, 428

hypereosinophilia syndrome, 437

immunophenotype of, 436

intracellular proteins, 427

lymphoproliferative disorders, 434

malignant cells, 435

monitor acute leukemia, 434

monochromatic light, 427

nonmalignant diseases, 437

peridin chlorophyll protein (PercCP), 428

phycoerythrin, 428

prognostic utility, 437

reagent-independent (nonfluorescent) parameters, 427

single-parameter histogram, 436

specific cell surface, 437

T cell numbers, 428

Fluorescence in situ hybridization (FISH), 85, 172

Folate deficiency

clinical presentation, 15

development of

autoimmune atrophic gastritis, 14

causes of, 13–14t

chronic medications, 14

etiology, 14

HIV infection, 14

prolonged use of, 14

elevated methylmalonic acid and

homocysteine, 18t

etiology of, 18

frequencies of, 11

gastrointestinal blood, 19

laboratory evaluation, 15–18

metabolic roles of

biology and dosing, 12t

metabolic pathways, 12f

methyltetrahydrofolic acid, 13

patient populations, 15

pernicious anemia, 19

requirements, sources, and stores

bacteria, 11

Food and Drug Administration standard, 11

plants role in, 11

treatment/response of

clinical presentation, 19

goals, 19

hematologic response, 19

intramuscular B12, 19

neuropathic abnormalities, 19

oral folic acid, 19

reticulocytosis, 19

Food and Drug Administration (FDA), 48, 226, 347, 394

Free erythrocyte protoporphyrin (FEP), 123

French-American-British (FAB), 141, 159

Fresh-frozen plasma (FFP), 127, 299, 345

Functional genomics, interpretation

gene expression profiling, 460

lymphoid malignancies, 460

Functional iron deficiency

chronic illnesses, 4

chronic inflammatory states, 4

CKD and IRIDA, 4

diagnostic tool and therapeutic target, 5t

ESA, 4

hepcidin biology, 4–5

robust assays, 4

serum hepcidin, 5t

treatment of

chronic inflammatory diseases, 8–9

patients with advanced malignancy, 8

G

Gall bladder manifestations

chronic hemolysis, 46

chronic transfusion therapy, 46

SCD manifestations, 46t

Gastrointestinal (GI), 187

Gaucher’s disease amyloidosis, 408

Gene expression profiling (GEP), 211

challenges of

data reproducibility, 462

genetic interference, 463

multiple testing corrections, 462

real and apparent discrepancies, 462–463

clinical application of

CLL cells, 463

functional genomic, hematologic malignancies, 464t

pharmacodynamic assessment, 463

prospective clinical trials, 465t

rapid transition, 463

Gene expression signature

microarray experiments, 461

supervised analysis

B-cell lymphoma, 462

biologic/clinical data, 461

chronic lymphocytic leukemia (CLL), 462

Cox proportional hazard method, 462

ZAP-70, 462

unsupervised analysis

connectivity map, 461

GSEA, 461

hierarchical clustering, 461

hypothesis-driven, 461

pharmaceutical agent, 461

question-driven, 461

Gene set enrichment analysis (GSEA), 461

Genetic interference, 463, 463t

Geometric mean channel (GMC), 433

Germ cell tumors

cisplatin-based therapy, 251

yielded response rates, 251

Germinal center B-cell-like, 218

Germline mutations/polymorphisms, detection

allele-specific polymerase chain reaction, 445

clinically applicable methods, 443

direct polymerase chain reaction, 445

hemoglobinopathies, molecular diagnosis

ASO hybridization, 445

α and β globin gene, 446

genomic DNA, 445–446

globin mutations, 446

molecular identification, 446

PCR-based methods, 445

Southern blotting, 445

hereditary familial hemochromatosis

(HFE), 444

melting curve analysis, 445

PCR technology application, 444f

precise diagnosis of, 443

restriction fragment length polymorphism (RFLP)

analysis of, 444

gel electrophoresis, 444–445

HFE mutations, 445

wild-type form, 445

Gestational thrombocytopenia

blood volume, 283

cytopenias result, 283

Gilbert’s syndrome, 24, 46

Glanzmann thrombasthenia (GT), 129

Glucose-6-phosphate dehydrogenase (G6PD), 23, 416. See also Erythroid enzymopathies

Glucose phosphate isomerase (GPI)

deficiency, 31

γ-Glutamylcysteine synthetase, 30

Glutathione peroxidase (GSH-Px), 30

Glutathione reductase, 30

Glutathione synthetase

deficiency, 30

Graft-versus-host disease (GVHD), 69, 240, 252

Granulocyte agglutination test (GAT), 402

Granulocyte colony-stimulating factor (G-CSF), 69, 88, 151, 166, 249, 351

Granulocyte-macrophage colony-stimulating factor (GM-CSF), 151

H

Helminth infections, 397

Hematologic complications

anemia in pregnancy

folate deficiency, 389

neural tube, 389

plasma volume, 389

prenatal vitamins, 389

red cell mass, 389

required iron, 389

sickle cell disease in pregnancy

anemia-related problems, 389

fetal distress, 389

gestational age, 389

infant birth weight, 389

maintenance transfusions, 389

red cell transfusions, 389

thrombocytopenia in pregnancy

anti-inflammatory drugs, 391

bruising, 390

congenital thrombophilia, 391

cord platelet, 391

cytotoxic agents, 390

danazol and vinca alkaloids, 390

diffuse intravascular coagulation, 392–393

epidural anesthesia, 390

excessive weight gain, 390

fetal membranes, 390

fetal scalp blood sampling, 391

gestational diabetes, 390

hemodilution, 390

hemolytic uremic syndrome (HUS), 392

hemostatically effective dose, 390

hypertensive disorders, 390

immune thrombocytopenia (ITP), 390

immune thrombocytopenic

purpura, 390

immunological disorders, 390

intracranial hemorrhage, 391

intravenous immunoglobulin (IVIg), 390

maternal platelet count, 391

mucosal bleeding, 390

neonatal mortality, 391

optimal first-line therapy, 390

osteoporosis, 390

patients refractory, 390

placenta metabolizes, 390

platelet count, 390

premature rupture, 390

pre-pregnancy weight, 390

splenectomy, 390

thromboprophylaxis, 391

thrombotic thrombocytopenic purpura (TTP), 392

transcranial ultrasound, 391

undergone surgical delivery, 391

venous thromboembolism in pregnancy

antiphospholipid antibodies, 393

argatroban, 394

bilateral compression, 393

body mass index, 393

breast-feeding, 394

central nervous system, 394

cesarean section, 394

clinical evidence, 394

common thrombophilias, 395t

compression ultrasonography (CU), 393

computed tomography pulmonary angiography (CTPA), 393

coumarin derivatives, 394

D-dimer levels, 393

deep vein thrombosis (DVT), 393

diagnosis of, 393

discontinuation of anticoagulation, 394

fondaparinux, 394

Food and Drug Administration (FDA), 394

glomerular filtration rate, 394

graduated compression stockings, 394

hemodynamic changes, 393

inherited thrombophilias, 393

lepirudin, 394

low molecular weight heparin

(LMWH), 394

magnetic resonance venography (MRV), 393

mechanical thromboprophylaxis, 394

morbidity and mortality, 394

pharmacologic thromboprophylaxis, 394

prolonged immobilization, 393

prophylactic antepartum, 394

pulmonary emboli, 393

thrombophilia, 394

thrombophilias and recurrent

miscarriage, 395

unfractionated heparin (UFH), 394

Hematologic malignancies, 249

immunofixation, 422

M paraproteins of, 421

quantitative serum immunoglobulins, 422–423

serum β2-microglobulin, 423

serum cryoglobulins

critical step, 423

cryoglobulinemia, 423

electrophoresis, 423

hepatitis C, 423

immunofixation, 423

serum free-light-chain assay, 422

serum lactic dehydrogenase, 423

serum protein electrophoresis, 421f

diclonal gammopathy, 420

electrophoresis separates, 420

immunofixation illustrating, 422

monoclonal immunoglobulin, 420

nephelometric immunoglobulin quantitation, 420

plasma protein migration patterns, 421t

polyclonal immunoglobulins, 422

poor correlation, 420

suspected monoclonal band, 420

serum uric acid

aminoaciduria, 423

cell turnover, 423

Fanconi syndrome, 423

glycosuria, 423

hypouricemia, 423

light chain, 423

polyuria, 423

proximal tubular, 423

tumor lysis syndrome, 423

serum viscosity, 423

urine protein electrophoresis, 422

Hematopoietic stem cell transplantation (HSCT), 68

allogeneic HSCT. See Allogeneic hematopoietic stem cell transplantation

autologous HSCT. See Autologous hematopoietic stem cell transplantation

RIC, 249

Heme iron, 2

Heme oxygenase-1, 32

Hemochromatosis

blood donors, 386

epidemiology, 377–378

future challenges, 387

HFE-hemochromatosis, clinical features, 379–380

laboratory testing

ancillary laboratory tests,

clinical and laboratory features of, 381

confirmatory laboratory tests, 382

factors influencing, 381

hemochromatosis, 380

liver biopsy role, 382

radiographic, 382–383

non-HFE iron overload, 387

pathophysiology, 378–379

population screening, 383–384

prognosis and response to therapy, 386

treatment of

phlebotomy therapy, 384–386

Hemoglobin electrophoresis

aberrant hemoglobins, 415

historical methods, 415

HPLC, 415

IEF, 415

separates hemoglobins, 415

Hemojuvelin gene (HJV), 378

Hemolysis. See also Erythrocyte

morphology and

associated pathology

associated pathology, 27–28t

clinical syndrome, 26

Clostridium perfringens, 26

erythrocyte morphology, 27–28t

haptoglobin, 26

indicators of, 28f

lactate dehydrogenase, 26

renal tubular cells, 26

tubular epithelium, 26

Hemolytic anemia

acute intravascular hemolysis, 26–28

anesthesia and surgery

sickle cell patients, 51t

β-thalassemia trait, 41

clinical approach, suspected hemolysis

bone marrow examination, 26

CBC, 24

chronic hemolysis, 24

history of, 24

MCV and RDW, 26

physical examination of, 24

reticulocytosis, 26

therapeutic response, 24

complete blood count (CBC), 40

contraception and pregnancy

chemotherapeutic drug, 50

cyanotic heart disease, 50

hydroxyurea, 50

teratogenic effects, 50

transfusions, 50

diagnosis of, 40

differential diagnosis of, 23–24t

enzyme and membrane defects

acute intravascular hemolysis, 28t

erythroid cytoskeleton, 28

glycosylphosphatidylinositol, 28

hematopoietic cells, 28

erythrocytes, 22

erythroid enzymopathies, 29–32

erythroid membrane defects, 32–33

etiology

ARC, 24

G6PD and PNH, 23

intrinsic and extrinsic causes, 23f

intrinsic hemolysis, 22

fetal hemoglobin (Hb F) induction, 48

gamma globin expression, 49

hematologic effects, 49

renal function, 48–49

reticulocytes, 49

gene therapy

autologous transplant model, 55

β-globin disorders, 53t

β-thalassemia, 55

health maintenance schedule, 54t

hematopoietic stem cells, 54

therapeutic effect, 55

hemoglobinopathies, 22, 37

high performance liquid chromatography (HPLC), 40

immune-mediated hemolysis, 22

iron deficiency, 40

leukocyte filters, 48

mechanism-specific therapies, 22

polymerase chain reaction (PCR), 40

prenatal testing, 40

quantitative disorder, 37

reticuloendothelial system, 22

sickle cell trait (SCT)

red cell distribution width (RDW), 41

solubility test of, 41

thalassemias, 37

α-thalassemia trait and disease, 40

mean corpuscular hemoglobin (MCH), 40

mean corpuscular volume (MCV), 40

sickle cell disease, 41t

tissue hypoxia, 22

transfusions, 48

indications for, 49t

transfusion-transmitted infections, 48

treatment of, 33–34

Hemolytic disease of the newborn (HDN), 343

Hemolytic uremic syndrome (HUS), 125, 276, 392. See also Thrombotic thrombocytopenic purpura (TTP)

Hemophagocytosis

bone marrow macrophages, 283

EBV-related infection, 283

familial hemophagocytic lymphohistiocytosis, 283

Hemorrhagic disease of the newborn (HDN), 127

Hemostasis and coagulation assays

activated clotting times (ACT), 418

activated partial thromboplastin time

anticoagulants, 417

anti-Xa assay, 417

intravenous fluids, 417

intravenous heparin, 417

low molecular weight heparins

(LMWH), 417

lupus anticoagulants, 417

PTT levels, 417

recurring problem, 417

D-dimer, 419

euglobulin clot lysis, 418

fibrinogen, 418

myeloproliferative disorders, 419

platelet function test

automated instruments, 419

bleeding time, 419

mucocutaneous bleeding, 419

platelet dysfunction, 419

von Willebrand disease (VWD), 419

prothrombin time (PT)

bovine thrombin, 418

commercial thromboplastins, 418

factors and autoantibodies, 418

fibrinogen, 417

INR, 418

ISI, 418

lupus anticoagulants, 418

thromboplastin, 417–418

warfarinized plasma, 418

renal insufficiency, 419

specific factor assays, 418

thrombin time, 418

Hemostasis and thrombosis

amyloidosis, 244

IMiDs, 245

paraproteinemia, 244

VWD, 244

Hemostasis disorders

abnormal coagulation tests

acquired hemophilia, 291–292

acquired inhibitors to coagulation proteins, 293

acute major bleeding, 290

aminocaproic acid, 291

aminocaproic acid decreases, 293

anti-FVIII IgG antibodies, 292

bleeding and normal coagulation tests, 295

bleeding episodes in hemophilia, 293t

bypassing agents, 292

causes of, 289t

deficiencies of prekallikrein and HMWK, 293

factor concentrates, 290

factor XI deficiency, 292

factor XII deficiency, 293

FIX molecules, 290

fresh frozen plasma (FFP), 293

FVIII gene, 289

hemophilia A, 289

hemophilia B, 289

hemophilia C, 292

hemostatic challenges, 290

heparin contamination, 293

high-titer inhibitors, 291

inhibitors, 291

laboratory diagnostic algorithms, 290–292f

low-titer inhibitors, 291

moderate hemophilia, 289

oral antifibrinolytic agent, 291

plasma-derived FXI, 293

prolonged activated partial thromboplastin time, 294–295

prolonged aPTT, 289

prolonged prothrombin time, 294

prophylactic factor replacement therapy, 291

prophylaxis prior, 290

soft-tissue hematomas, 292

thrombin generation, 293

tissue disease, 289

traumatic venipuncture, 293

warfarin, 293

X-linked recessive fashion, 289

activated partial thromboplastin time (aPTT), 285

bleeding reason, 285

citrate-anticoagulated plasma, 287

coagulation cascade

common pathway, 286f, 287

contact pathway, 286f, 287

tenase complex, 287

tissue injury pathway, 286f, 287

coagulation factors, 286

coagulation proteins, 285

coagulation system, 286

common coagulation tests

bleeding time (BT), 288

heparin-binding agent, 288

long-incubation aPTT, 287

reptilase time, 288

thrombin time (TT), 288

disseminated intravascular

coagulation (DIC)

acute promyelocytic leukemia (APL), 299

conditions, 298t

diagnosis, 298

HELLP syndrome, 299

pathophysiology, 297

presentation, 297–298

treatment, 298–299

Trousseau syndrome, 299

hemophilia A, 285

hemophilia B, 285

high molecular weight kininogen (HMWK), 287

lupus anticoagulants (LAs), 287

mucocutaneous bleeding, 285

postsurgical bleeding, 285

prekallikrein, 287

prothrombin time (PT), 285

qualitative platelet disorders

Bernard-Soulier syndrome (BSS), 308

cardiac bypass, 308

congenital disorders of signal transduction, 309

drug-induced, 304–307

Eltrombopag, treatment, 309

Epstein syndrome, 309

Fechtner syndrome, 309

Glanzmann thrombasthenia, 308

isolated laboratory-specific defects, 309

management of, 307t

May-Hegglin anomaly, 309

myelodysplasia/myeloproliferative diseases, 307

platelet biochemistry, 303–304

platelet function testing, 304

renal failure/uremia, 307–308

Scott syndrome, 309

Sebastian syndrome, 309

storage pool disease (SPD), 308–309

serine proteases, 286

specialized coagulation tests

anti-Xa assay, 288

Bethesda assay, 288

euglobulin clot lysis time (ECLT), 288

inhibitor decreases, 288

low molecular weight heparin

(LMWH), 288

Russell’s viper venom time, 288

specific clotting factors, 288

thromboelastography, 288

thromboplastin (TP), 287

Von Willebrand disease

diagnosis of, 301–302

epidemiology, 299

pathophysiology and

classification, 299–300

presentation, 300–301

pregnancy and, 303

treatment, 302–303

Heparin-induced thrombocytopenia (HIT)

alternative anticoagulant, 275

alternative anticoagulants in the treatment, 276t

antibody-mediated disorder, 274

diagnosis

clinical probability, 275

enzyme-linked immunosorbent assay (ELISA), 275

laboratory diagnosis, 275

PF4-heparin-associated antibody, 275

platelet activation assays, 275

epidemiology, 274

longer-term anticoagulation, 275

paradoxically, 274

pathophysiology

heparin-PF4 complex, 274

IgG antibody, 274

PF4-polysaccharide complexes, 274

platelet Fc receptors, 274

presentation

arterial, 274

delayed-onset HIT, 274

disseminated intravascular coagulation (DIC), 274

HIT-related thrombosis, 274

rapid-onset HIT, 274

venous, 274

retreatment with heparin, 275

thromboembolectomy, 275

thrombolysis, 275

treatment, 275

warfarin, 275

Hepatic manifestations, 46

acute splenomegaly, 46

chronic hemolysis, 46

chronic transfusion therapy, 46

Hepatitis C virus (HCV), 211

Hepatoerythropoietic porphyria, 62

coproporphyrinogen oxidase, 62

neurovisceral symptoms, 62

protoporphyria oxidase gene, 62

variegate porphyria, 62

Hepcidin-producing adenomas, 4

Hereditary elliptocytosis (HE), 33

Hereditary persistence of fetal hemoglobin (HPFH), 38

Hereditary pyropoikilocytosis, 33

Hereditary spherocytosis (HS), 32–33

Hereditary stomatocytosis, 33

Hermansky-Pudlak syndrome, 308

Herpes simplex virus (HSV), 146

Hexokinase deficiency, 31

HFE-hemochromatosis, clinical features

arthritis dysfunction, 380

biochemical and genetic screening tests, 379

cardiac failure and refractory arrhythmias, 379

classic triad, 379

clinical manifestations, 380

clinical presentation

alanine aminotransferase (ALT), 380

clinical signs and symptoms, 380

genetic factors, 380

debilitating symmetric polyarthritis, 380

fatigue and sexual dysfunction, 380

grayish skin pigmentation, 380

historic versus current, 380t

mutated HFE alleles, 380

polyendocrine failure, 380

severe liver disease, 379

skin pigmentation, 379

stages of disorder, 380

High-dose conventional chemotherapy (HDT), 240

Highly active antiretroviral therapy (HAART), 232

High-performance liquid chromatography (HPLC), 415

Histone deacetylase inhibitors

cancer cells, 91

single-agent therapy, 91

suberoylanilide hydroxamic acid, 91

switching hypomethylating agents, 91

valproic acid, 91

Hodgkin lymphoma (HL)

AHCT, 207

B-cell lymphoma, 206

biochemical test, 201

bone marrow biopsy, 201

bulky mediastinal mass, 205

CBC, 200t

chemo-sensitive disease, 207

chemotherapy regimens

bleomycin, 202, 204

CALGB, 202

curative regimen, 202

dacarbazine, 202

doxorubicin, 202

etoposide, 204

high-dose salvage therapy, 202

macroscopic splenic disease, 202

mechlorethamine, 202

ocovin, 202

prednisone, 202

procarbazine, 202

refractory disease, 202

response and survival, 204t

vinblastine, 202

vincristine, 204

CHL, 197

clinical features of

cervical lymph node, 200

mediastinal adenopathy, 200

CT scan, 201

epidemiology, 197

ESR, 200t

etiology and risk factors

CHL, 200

infectious mononucleosis, 200

excisional biopsy, 200t

FDG-PET scan, 206

hematologic neoplasms, 197

historical evolution of, 198t

immunophenotypic criteria for, 198t

laboratory test, 200t

LDCHL, 197

LDH, 201

liver function, 201

LRCHL, 197

lymphatic system, 197

MCCHL, 197

modality therapy, 206

neoplastic disorders, 197

NLPHL, 197

NSCHL, 197

palliative treatment, 208

pathology

B-cell program, 198

cervical lymph nodes, 198

cytoplasm, 198

diagnostic Reed-Sternberg cell, 199f

EBV, 199

eosinophils cell, 198

histiocytic cell, 199

HIV infection, 199

immunoglobulin variable region, 198

lacunar cell, 198

lymphocytic cell, 199

malignant cells, 198

malignant lymphomas, 198

mediastinal node, 198

neoplastic cells, 198

neutrophils cell, 198

nodular growth pattern, 198

nuclear factor-kappa beta (NF-kB), 198

plasma cell, 198

popcorn cells, 199

tumor cell, 198

tumor mass, 198

patient group, 206

pelvic radiation therapy, 201

PET, 201

PFT, 201

physical examination, 200t

positron emission tomography scan

DFS, 206

multivariate analysis, 206

progressive disease, 206

residual disease versus fibrosis, 206

prognostic features, 201

radiation therapy

breast cancer, 207

CAD, 207

cardiac radiation, 207

cardiopulmonary disease, 202

IFRT, 202

lhermitte sign, 207

lung cancer, 207

mammography, 207

mantle field radiation, 207

relative risk (RR), 207

subdiaphragmatic radiation, 207

thrombocytopenia, 207

toxicity profile, 202

radiation therapy fields, 203f

renal function, 201

RS cell, 197

salvage chemotherapy regimens

anthracycline-containing regimens, 208

cisplatin, 208

doxorubicin, 208

high-dose cytarabine, 208

ifosfamide, 208

methylprednisolone, 208

staging of, 200t

therapy-related complications, 206–207

treatment regimen, 204–206, 204t–205t

tumor cells, 206

WHO, 197

Homocysteine (Hcy), 18

Human immunodeficiency virus (HIV), 272, 283, 364, 428, 467

Human leukocyte antigen (HLA), 70, 125, 146, 163, 180, 249

Human T-lymphotropic virus type 1

(HTLV-1), 211

Hydroxyurea (HU), 321

Hyperviscosity syndrome

clinical manifestations, 245

occurs, 245

treatment, 245

I

Idiopathic cytopenia of undetermined significance (ICUS), 86

Idiopathic dysplasia of uncertain significance (IDUS), 86

Idiopathic thrombocytopenic purpura (ITP), 407

Immature platelet fraction (IPF), 407

Immature reticulocyte fraction (IRF), 406

Immune deficiency syndrome

dose-adjusted EPOCH, 232t

HAART, 232

lymphoma, 232

tumor lysis syndrome, 233

Immune-mediated hemolysis, 32

Immune thrombocytopenia (ITP), 129, 194, 271

diagnosis

bone marrow examination, 272

cytopenias, 272

direct antiglobulin test (DAT), 272

Helicobacter pylori testing, 272

hepatitis B virus, 272

hepatitis C virus, 272

HIV infection, 272

isolated thrombocytopenia, 272

megakaryocytes, 272

epidemiology, 272

intermittent infusions of IVIg, 274

pathophysiology

antibody-coated platelets, 272

chronic lymphocytic leukemia (CLL), 272

immune dysregulation, 272

lymphoproliferation, 272

platelet glycoprotein complexes, 272

primary adult-onset ITP, 272

pregnancy-associated ITP, 274

presentation

milder disease, 272

new-onset severe ITP, 272

secondary ITP, 274

therapy of, 273t

treatment, 272

anti-D, 273

corticosteroids, 273

dexamethasone cycles, 273

initial treatment, 273

intravenous immune globulin (IVIg), 273

monoclonal anti-B cell, 273

platelet transfusions, 273

rituximab, 273

romiplostim, 273

second-line treatment, 273

splenectomy, 274

thrombopoietin receptor, 273

Immunohematologic disorders

autoimmune hemolytic anemias

anemia associated, 370

cold agglutinin syndrome, 370

Donath-Landsteiner antibody, 370

hemolysis, 370

laboratory hallmark, 369

lymphoproliferative disorder, 369

oral glucocorticoids, 370

paroxysmal cold hemoglobinuria, 370

positive DAT, 369

RBC antigens, 369

red cell alloantibodies, 369

warm AIHA, 369–370

warm-reacting autoantibodies, 369

dysproteinemias

hyperviscosity syndrome, 374

paraproteinemias, 374

Waldenstrom macroglobulinemia, 374

hemolytic disease of newborn

mild cases, 368

Rh antibodies, 368

severe cases, 368

treatment, 368

maternal immune thrombocytopenic purpura, 369

neonatal alloimmune thrombocytopenia

with antibody (IgG), 369

with HDN, 368

HLA DRw52a phenotype, 369

IVIG, 369

therapeutic apheresis

ASFA, 370

cellular blood component, 371

citrate toxicity, 372

coagulation of blood, 372

erythrocytapheresis and red cell exchange, 372

hyperleukocytosis, 372

hypocalcemia, 372

hypotension, 371

leukocytapheresis, 372

myeloproliferative disorders, 372

parasitemia, 372

photopheresis, 372

plasma exchange, 371

plasmapheresis, 372

platelet transfusion, 371

recommendation, 371t

red cell exchange and sickle cell anemia, 372–373

routine blood components, 370

thrombocytapheresis, 372

ultraviolet A (UVA), 372

therapeutic plasma exchange

category I and II recommendations, 373

thrombotic microangiopathies

bone marrow transplant (BMT), 374

hemolytic uremic syndrome (HUS), 373–374

immunosuppressive agents, 374

platelet transfusion, 374

prominent neurologic symptoms, 373

therapeutic plasma exchange (TPE), 374

TTP, 373–374

Immunomodulatory drugs (IMiDs), 240

Indirect antiglobulin test (IAT), 344

Infection- and sepsis-related thrombocytopenia

transient thrombocytopenia, 283

Infectious mononucleosis (IM), 133

Inferior vena cava filters, 324

Inflammatory bowel disease (IBD), 7

Interferon-α (IFN-α), 220

Internal tandem duplications (ITD), 144

International blood and marrow transplant research (CIBMTR), 89

International Myeloma Working Group (IMWG), 234

International normalized ratio (INR), 130, 287

International Sensitivity Index (ISI), 287

International Working Group for Myelofibrosis Research and Treatment (IWG-MRT), 107

International Workshop on Chronic Lymphocytic Leukemia (IWCLL), 187

Intravascular large B-cell lymphoma (IVLBCL), 218

Intravenous immune globulin (IVIG), 194

Intravenous immunoglobulin (IVIG), 129, 166, 262

Intravenous iron therapy, 7. See also Absolute iron deficiency

Intrinsic disorders

acquired neutropenias, 402

congenital neutropenias

congenital immunodeficiency

syndromes, 402

granulocyte colony stimulating factor (G-CSF), 401

neutropenia classification, 401t

reticular dysgenesis, 402

X-linked hyperimmunoglobulin, 402

Investigational new drug (IND), 147

Involved field radiation therapy (IFRT), 202

Iron deficiency

absolute, 2–4

functional, 4–5

iron metabolism

diferric transferrin, 2

ferrous iron, 2

heme iron, 2

intracellular iron, 1

iron cycle, 2f

iron diet, 1–2

iron–porphyrin complex, 2

medicinal iron, 2

myoglobin, 1

reticuloendothelial system (RES), 1, 2f

medications malabsorbed, 6t

oral iron supplements, 5–6t

response to, 8

treatment of

dietary iron, 5

intravenous iron therapy, 7

oral iron therapy, 5

red blood cell transfusion, 7–8

Iron dextran, 7

Iron refractory iron deficiency anemia (IRIDA), 4

Iron sucrose, 7

Isoelectric focusing (IEF), 415

K

Kaolin clotting time (KCT), 318

Kaposi sarcoma-associated herpes virus (KSHV), 211

Kasabach-Merritt syndrome, 129

Killer immunoglobulin-like receptor (KIR), 265–266

L

Lactate dehydrogenase (LDH), 125, 140, 165, 201

Large granular lymphocytosis (LGL), 86, 454

Lecithin cholesterol acyltransferase (LCAT), 32

Lenalidomide, 86, 241, 244

Leukocyte adhesion deficiency type I (LAD I), 133

Leukocyte alkaline phosphatase

chronic myelogenous leukemia, 424

cytogenetic analysis of, 424

FISH assay, 424

leukocytosis, 424

myeloproliferative disorders, 424

neutrophils, 424

peripheral blood neutrophils, 424

polycythemia vera, 424

Liver biopsy role

histologic findings, 382

indications for, 382

Loss of heterozygosity (LOH), 448

Low molecular weight heparin (LMWH), 394

Lupus anticoagulants, 318

Lymphocyte-depleted classical Hodgkin lymphoma (LDCHL), 197

Lymphocyte doubling time (LDT), 190

Lymphocyte-rich classical Hodgkin lymphoma (LRCHL), 197

Lymphoma

and auto-HSCT, 250. See also specific subtypes of lymphoma

Lymphoproliferative disease (LPD), 222

M

Macrocytosis, 15

Magnetic resonance imaging (MRI), 140

Magnetic resonance venography (MRV), 393

Mantle cell lymphoma

blastic variant, 227

bone marrow transplantation, 227

bortezomib, 228

Hyper-CVAD regimen, 227

immune modulator drugs, 228

intensive regimens, 227

radiation therapy, 227

Matched unrelated donors (MUD), 70

Mature B-cell neoplasms

B-cell lymphoma, 217

B-prolymphocytic leukemia

allogeneic stem cell transplant, 216

purine analogues, 216

Burkitt lymphoma

AIDS-defining condition, 219

apoptotic tumor cells, 219

histopathology examination, 219

chronic lymphocytic leukemia

bone marrow infiltration, 216

tissue morphology, 216

cutaneous follicle center lymphoma (CFCL), 217

diffuse large B-cell lymphoma

ABC, 218

B-cell markers, 219

bluish tumor, 218

chronic inflammation, 218

CNS, 218

EBV, 218

GCB, 218

high-dose methotrexate, 218

international prognostic index, 218

IVLBCL, 218

lymphoid malignancies, 218

lymphomatoid granulomatosis, 218

marginal zone lymphoma, 218

morphologic variants, 218

multicentric Castleman disease, 219

PBL, 218

PFS, 218

PMBL, 218

severe cognitive deficit, 218

tumor cells, 218

viral associated tumor, 219

whole-brain radiation, 218

follicular lymphoma (FL)

BCL2 protein, 217

bone marrow involvement, 217

clinical relevance, 217

symptomatic disease, 217

hairy cell leukemia (HCL)

immunotherapy, 216

lymphoid leukemia, 216

rituximab, 216

heavy chain diseases (HCD), 217

lymphoplasmacytic lymphoma

ASCT, 217

novel agents, 217

paraproteinemia, 217

Waldenstrom macroglobulinemia, 217

mantle cell lymphoma, 217

mature T- and NK-cell neoplasms. See Mature T- and NK-cell neoplasms

mucosa-associated lymphoid tissue

centroblast-like cells, 217

gastric lymphoma, 217

Helicobacter pylori, 217

nodal marginal zone lymphoma (NMZL), 217

Hashimoto thyroiditis, 217

SjÖgren syndrome, 217

plasma cell neoplasms, 217

splenic B-cell lymphoma, 216

splenic marginal zone lymphoma

lymphoid neoplasms, 216

purine analog therapy, 216

Mature T- and NK-cell neoplasms

adult T-cell leukemia, 220

hypercalcemia, 220

IFN-α, 220

lymphadenopathy, 220

TSP, 220

white blood count, 220

aggressive NK-cell leukemia, 220

anaplastic large cell lymphoma

ALK gene, 222

bone marrow infiltration, 222

brentuximab vedotin, 222

angioimmunoblastic T-cell lymphoma (AITL), 221

chronic lymphoproliferative disorders, 220

enteropathy-associated T-cell lymphoma, 220

extranodal NK/T-cell lymphoma, 220

hemophagocytic syndrome, 221

hepatosplenic T-cell lymphoma, 220

Hodgkin lymphoma, 222

mycosis fungoides (MF), 221

positive T-cell lymphoproliferative

disorders, 220

Sézary syndrome, 221

SPTCL, 220

T-cell large granular lymphocytic leukemia

allogeneic hematopoietic cell transplant, 219

chronic antigen-driven process, 219

severe neutropenia, 219

thrombocytopenia, 219

T-cell lymphomas, 221

cytotoxic phenotype, 221

follicular variant, 221

low-dose methotrexate, 221

lymph node involvement, 221

lymphoepitheliod, 221

lymphoid papulosis, 221

Reed-Sternberg-like cells, 221

solitary tumors, 221

UV light phototherapy, 221

T-cell prolymphocytic leukemia, 219

May-Hegglin anomaly, 408

Mean cell volume (MCV), 15, 125

Mean corpuscular hemoglobin concentration (MCHC), 405

Mean corpuscular volume (MCV), 405

Measurements of methylmalonic acid (MMA), 18

Melphalan, 241, 244

6-Mercaptopurine (6-MP), 163

Microangiopathic hemolytic anemia (MAHA), 276

β2 microglobulin (β2m), 379

Minimal residual disease (MRD), 150, 177, 192

cytogenetic remission, 179

disease progression, 179

imatinib treatment, 179

leukemic progenitors, 179

molecular remission, 179

Mixed cellularity classical Hodgkin lymphoma (MCCHL), 197

Molecular diagnosis. See Gene expression signature

Molecular diagnostics in hematology

B19 parvovirus, 456

clonality studies, 452–453

cytogenetic diagnostics. See Cytogenetic diagnostics

cytomegalovius, 456

donor/recipient chimerism, 456–457

Epstein-Barr virus, 455

germline mutations/ polymorphisms, detection, 443–445

molecular human leukocyte antigen typing

intermediate resolution (IR), 457

polymorphisms, 457

SSOP hybridization, 457

SSOP testing, 457

NGS, 441

polymerase chain reaction (PCR), 441

primary effusion lymphomas, 456

sequence-specific oligonucleotide probe (SSOP), 441

sequence-specific primers (SSP), 441

somatic mutations and translocations. See Somatic mutations and translocations

Taqman probes, 455

T-cell receptor and immunoglobulin rearrangement, 453–455

viral pathogens, 455t

Monoclonal antibody immobilization of granulocyte antigens assay (MAIGA), 402

Monoclonal B lymphocytosis (MBL), 187

Monoclonal gammopathy of

undetermined significance (MGUS), 234

Multicenter Study of Hydroxyurea (MSH), 48

Multi-kinase inhibitors. See Histone deacetylase inhibitors

Multiple myeloma

allogeneic transplant approach, 250

bortezomib-based pretransplant induction therapy, 250

clinical features

clonal plasma cell disorders, 236

common symptom, 236

extramedullary disease (EMD), 236

hypercalcemia, 236

pathologic fractures, 236

symptoms of hyperviscosity, 236

clinical trials network (CTN), 250

criteria to assess response, progression, and relapse

clinical relapse, 239

IMWG uniform response, 237

international myeloma working group uniform response criteria, 238t

relapse from CR, 239

diagnostic criteria

International Myeloma Working Group criteria, 237

differential diagnosis

amyloidosis, 239

axial skeleton, 240

extramedullary plasmacytoma

(EMP), 240

hyperparathyroidism, 240

immunoglobulin light chain, 239

immunoglobulin light chain

amyloidosis, 239

metastatic carcinoma, 240

other systemic plasma cell dyscrasias, 240

plasma cell disorders, 239

POEMS syndrome, 240

solitary plasmacytoma, 240

TEMPI syndrome, 240

Waldenstrom macroglobulinemia, 239

disease-free survival (DFS), 250

epidemiology and risk factors

asymptomatic plasma cell, 234

environmental and genetic factors, 234

hematologic malignancy, 234

mayo clinic, 234

proliferative disorder, 234

initial evaluation

abnormal FLC ratio, 236

chemistries serum electrolytes, 236

free light chain (FLC), 236

hematology, 236

lactate dehydrogenase (LDH), 236

magnetic resonance imaging (MRI), 237

multidetector-row computed tomography (MDCT), 237

quantitative immunoglobulins, 236

radiographic skeletal survey, 236

routine urine-analysis, 236

serum protein electrophoresis (SPEP), 236

solitary bone plasmacytoma (SBP), 237

standard metaphase cytogenetics, 236

technetium bone scans, 237

whole-body fluorodeoxyglucose, 237

long-term CR rates, 250

melphalan, 250

overall survival (OS), 250

pathophysiology

bone marrow microenvironment, 236

clonal plasma cell, 236

genetic abnormalities, 236

heterogeneous disease, 236

hyperdiploidy, 236

inherited SNP variations, 236

paraproteinemias, 236

plasma cell biology, 236

plasma cell leukemia, 236

prognostic information, 236

somatic cell, 236

translocations, 236

plasma cell leukemia

diagnosis requires, 245

posttransplant consolidation, 250

precursor states, 234

CRAB criteria and frequency at presentation, 235t

high-dose dexamethasone, 234

high-risk SMM patients, 234

ongoing randomized phase, 234

risk stratification of monoclonal gammopathy, 235t

progression-free survival (PFS), 250

solitary plasmacytoma and extramedullary plasmacytoma

plasma cell neoplasms, 245

radiation therapy, 245

staging

Durie-Salmon Staging System (DSSS), 237

systems, 238t

supportive care

anemia, 244

hemostasis and thrombosis, 244

hyperviscosity syndrome, 245

osteonecrosis of the jaw, 245

peripheral neuropathy, 245

skeletal-related events, 245

tandem auto-HSCT, 250

treatment

alkylating agents, 241

anti-myeloma therapies, 241

autologous stem cell transplant (ASCT), 240

bisphosphonates (BPs), 240

bone disease, 244

bortezomib combination, 243

bortezomib- cyclophosphamide-dexamethasone, 243

corticosteroids, 243

current treatment algorithm for patients, 243t

dexamethasone, 241

DVT, 241

frontline therapy, 241

graft-versus-host disease (GVHD), 240

high-dose conventional chemotherapy (HDT), 240

high-risk smoldering multiple

myeloma, 240

IMiD pomalidomide, 243

immunomodulatory drugs (IMiDs), 240

interferon alpha, 243

lenalidomide, 241

liposomal doxorubicin, 244

maintenance therapy, 243

MPT, 244

neurotoxicity, 243

OBD, 244

oral melphalan, 243

prednisone, 243

progression-free survival, 243

Rd, 244

relapsed or refractory disease, 244

risk-adapted therapy, 240

risk stratification by mayo clinic model, 241t

selected frontline treatments, 242t

SRE, 244

stem cell transplantation (SCT), 240

ThalDex, 241

thalidomide, 241

VBMCP, 241

VMP, 244

zoledronic acid, 244

Multiple testing corrections, 462

Myelodysplastic syndromes (MDS), 137, 256, 447

acute myelogenous leukemia, 84f

aminocaproic acid, 88

aplastic anemia, 83

bone marrow biopsy, 85

bone marrow function, 88

cardiopulmonary disease, 88

chemotherapy, 91

chromosome analysis, 85

chromosome breakage test, 85

clinical features of

lymphadenopathy, 84

splenomegaly, 84

clinical heterogeneity, 88

clonal stem cell disorders, 83

cytogenetic abnormality, 84

cytopenias, 88

diagnostic criteria for, 84

DNA methyltransferase inhibitors, 89

azacitidine, 89

cytostatic effect, 89

hematological responses, 90

hypermethylation, 89

leukemogenesis, 89

tumor suppressor genes, 89

dysplastic marrow morphology, 84

erythroid dysplasia, 85

etiology and pathogenesis

bone marrow, 83

chromosomal abnormalities, 84

hematopoiesis, 83

hematopoietic stem cell, 83

immunological factors, 83

leukemic transformation, 84

refractory anemia, 83

single nucleotide polymorphism, 84

somatic mutations, 83

Fanconi anemia, 83

flow cytometry, 85

fluorescent in situ hybridization analysis, 85

French-American-British (FAB), 85t

genetic syndromes, 87

growth factors, 88

hematopoietic and lymphoid tissues, 87t

hypogranular neutrophils, 85

immunomodulatory agents

cytogenetic abnormalities, 90

cytotoxic effect, 90

lenalidomide, 90

thalidomide, 90

transfusion-dependent anemia, 90

transfusion-independence response rate, 90

immunosuppression

horse antithymocyte globulin (hATG), 90

international prognostic scoring system, 86t

iron chelation, 88

leukemic progeny, 88

leukemic progression, 85

leukodepletion, 88

marrow cells, 85

metaphase cytogenetics, 85

micromegakaryocytes, 85

myeloid malignancy, 86

myeloproliferative neoplasm, 86

paroxysmal nocturnal hemoglobinuria, 83

prophylactic regimen, 88

ribosomal gene, 86

serum erythropoietin level, 89

severe thrombocytopenia, 89

stem cell transplant

allotransplant, 89

myeloablative transplant, 89

T-cell receptor gene, 86

therapeutic strategies, 88t

T-large granular lymphocyte lymphoproliferative disorders, 83

transfusion frequency, 88

Myeloproliferative neoplasms

agents properties, 104t

allogeneic stem cell transplantation, 108

aspirin therapy, 97

bone marrow hematopoiesis, 96f

bone marrow suppression, 105

cardiovascular comorbidities, 105

chelation therapy, 107

chromosomal abnormalities, 97

clinical features of, 97, 102, 106

extramedullary hematopoiesis, 106

leukemic transformation, 103

osteosclerosis, 106

thrombocytosis, 106

thrombosis, 103

thrombotic events, 103

vasomotor symptoms, 103

clinical symptoms, 100

clonal hematopoietic stem cell diseases, 95

congenital polycythemia, 97

diagnostic criteria

apparent polycythemia, 101t

bone marrow biopsy, 98

computed tomography (CT) scan, 98

erythropoietin production, 100t

hypoxemia, 98

iron deficiency, 98

World Health Organization criteria for, 99t

diagnostic testing, 103

granulocyte precursors, 106

malignant conditions, 106

marrow fibrosis, 107t

myeloid disorders, 106

epidemiology, 96, 102, 105

erythrocyte sedimentation rate (ESR), 103

erythrocytosis, 97

erythromelalgia, 97

etiology, 105

extreme thrombocytosis, 101

fibrotic transformation, 97, 98, 101

granulocyte abnormalities, 97

hematocrit, 101

hydroxyurea, 98, 105

JAK2 inhibitor, 107

JAK2V617F mutation, 96t

lactate dehydrogenase (LDH), 98

lenalidomide, 107

leukemic transformation, 95

malignant process, 103

marrow erythroid hyperplasia, 97

marrow fibrosis, 105

megakaryocyte, 97

myeloablative protocols, 108

myeloablative transplantation, 108

myelofibrosis, 107

myelosuppression, 96

myelosuppressive agents, 103

optimal therapeutic management, 95

pathophysiology, 96, 102, 106

cytogenetic abnormalities, 106

cytokines, 106

marrow fibrosis, 106

myeloproliferation, 106

polyclonal hematopoiesis, 102

transforming growth factor (TGF), 106

X-chromosome, 102

phlebotomy, 100

plateletpheresis, 105

prednisone, 107

prognostic scoring systems, 107

ristocetin cofactor level, 105

ristocetin cofactor levels, 101

routine cytogenetics, 97

thalidomide, 107

thrombocytosis, 98

thrombohemorrhagic complications, 100

thrombotic complications, 105

thrombotic events, 97

treatment algorithm for, 102f

treatment algorithm of, 105t

vasomotor symptoms, 105

von Willebrand syndrome, 101

N

National Marrow Donor Program (NMDP), 254

Neonatal alloimmune thrombocytopenia (NAIT), 129

corticosteroids, 280

fetal platelet antigens, 280

human platelet antigen (HPA), 280

intracranial hemorrhage (ICH), 280

Neutropenia

extrinsic disorders. See Extrinsic disorders

extrinsic factors, 401

hematopoietic system, 401

intrinsic disorders, 401. See also Intrinsic disorders

secondary forms, 401

Neutrophil disorders and neutropenias

abnormal granule formation, 111

acute infections, 113

autoimmune disorders, 116

azurophilic granules, 111

barrier function, 111

benign ethnic neutropenia (BEN), 119

bone marrow examinations, 119

cytokine receptor, 119

physiologic mechanism, 119

SDF-1 chemokine, 119

stem cell disorder, 119

bone marrow, 111, 116

bone marrow transplant, 113

Chédiak-Higashi syndrome (CHS)

accelerated phase, 116

acute infections, 116

autosomal recessive disorder, 115

myeloid differentiation, 116

thyroid follicular cell, 115

chemotactic factors, 111

chronic granulomatous diseases (CGDs)

Aspergillus nidulans, 114

Aspergillus species, 114

autoimmune disorders, 115

autosomal recessive pattern, 114

bacterial pathogens, 114

bone marrow, 115

Burkholderia cepacia, 114

C. glabrata, 114

Candida albicans, 114

chronic abdominal pain, 115

commensal organisms, 114

Crohn’s disease, 115

dihydrorhodamine flow cytometry, 115

fibrotic reaction, 115

fungal pneumonias, 114

gastroesophageal junction, 115

gastrointestinal granulomatous process, 115

gene therapy, 115

granulomatous inflammation, 115

granulomatous process, 115

hematopoietic stem cell transplant, 115

hydrogen peroxide, 114

inflammatory granulomas, 114

pathogenic organism, 115

phagocyte NADPH oxidase, 114

Serratia marcescens, 114

Staphylococcus aureus, 114

clinical manifestations, 113

cyclic neutropenia

bone marrow examinations, 118

CD40 ligand deficiency, 118–119

myeloid precursors, 118

nadir neutrophil count, 118

oral mucosal ulcers, 118

disease manifestations, 113

drug-induced neutropenia

common drugs list, 117t

direct cytotoxic effect, 116

endothelial cells, 111

flow cytometry, 113

fucose glycosylation, 113

gastrointestinal tract, 111, 113

gelatinase, 111

idiopathic neutropenia, 119

inflammatory cytokines, 119

myelodysplastic syndrome, 119

oropharyngeal infections, 119

severe clinical syndromes, 119

severe systemic infections, 119

immune-related neutropenia

alloimmune neonatal neutropenia, 117

autoimmune disorders, 117

bone marrow examinations, 117

corticosteroids, 117

cyclophosphamide, 117

hypercellular marrow, 117

immunosuppressive therapy, 117

large granular lymphocytosis, 117

monoclonal disease, 117

oligoclonal, 117

symptomatic anemia, 117

urinary tract infections, 117

infection-related neutropenia

Epstein-Barr virus, 117

gram-negative bacterial infections, 117

marrow suppression, 116

inflammation, 111

lactoferrin, 111

leukocyte adhesion

deficiency, 111

leukocyte adhesion deficiency 1 (LAD-1), 113

macrophages, 111

metamyelocytes, 111

moderate phenotype, 113

myeloperoxidase deficiency

antimicrobial functions, 113–114

Candida species, 114

heme group, 114

MPO deficiency, 114

prophylactic antibiotics, 114

myeloperoxidase (MPO), 111

phagocytosis, 111

polymorphonuclear (PMN) cells, 111

promyelocytes, 111

reactive oxidants, 111

severe congenital neutropenia

acute myeloid leukemia, 118

bone marrow biopsies, 118

Kostmann syndrome, 118

lower respiratory tract, 118

malignant transformation, 118

myelocyte level, 118

osteopenia, 118

promyelocyte-myelocyte level, 118

severe phenotype, 113

severity of, 113t

specific granule deficiency

leukemia, 116

myelodysplasia, 116

Staphylococcus aureus, 113

steady-state level, 116

Neutrophil evaluation

marrow granulocyte reserve

febrile neutropenia, 424

granulocyte level, 424

hydrocortisone stimulation test, 424

leukocyte alkaline phosphatase, 424

neutrophil concentration, 424

Next generation sequencing (NGS)

computer algorithms, 443

DNA, 442

limitation of, 442

principle of, 443f

small fragments elongation, 442

starting material, 443

terminator nucleotide, 443

ultra-sensitive CCD cameras, 443

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), 197

Nodular sclerosis classic Hodgkin lymphoma (NSCHL), 197

Non-Hodgkin lymphomas (NHL), 249

aggressive B-cell lymphomas

Burkitt lymphoma. See Burkitt lymphoma

cisplatin, 230

diffuse large B-cell lymphoma. See Diffuse large B-cell lymphoma

high-dose chemotherapy, 230

immune deficiency syndrome. See Immune deficiency syndrome

mantle cell lymphoma. See Mantle cell lymphoma

methyl prednisolone, 230

RIST, 232

salvage therapy, 230

T-cell lymphoma. See T-cell lymphoma

treatment of, 230

Ann Arbor staging system, 224t

B- and T-cell tumors, 210

B-cell and T-cell lymphomas

autoimmune disease, 227

B-cell lymphomas, 226

Bruton tyrosine kinase inhibition, 227

cladribine, 227

CR, 226

DFS, 226

FDA, 226

fludarabine, 226

grade 3B lymphomas, 226

H. pylori infection, 227

indolent lymphoma treatment, 227t

iodine-131 tositumomab, 226

lenalidomide, 226, 227

lymphocytic lymphoma treatment, 227

marginal zone lymphoma, 227

novel agents, 226

ORR, 226

pomalidomide, 227

purine analogues, 227

radioimmunotherapy, 226

rituximab, 226

thalidomide, 227

tositumomab, 226

B-cell immunophenotype, 215t

B-cell lymphomas, 214t

B-cell neoplasms, 216

cell surface antigens, 211

clinical management, 223

comparative genomic hybridization, 211

conventional treatment, 226

DLBCL, 210

epidemiology, 210

GEP, 211

immune modulators, 226

immune response, 211

Immunodeficiency-associated lymphoproliferative disorders

AIDS epidemic, 222

AIDS-indicator conditions, 222

CVID, 222

diagnostic algorithm, 222

EBV-driven polyclonal proliferations, 223

hematopoietic cell transplant, 223

HIV infection, 222

iatrogenic immune suppression, 223

immunosuppressive drugs, 223

LPD, 222

mass brain lesions, 222

palliative approach, 222

polymorphic proliferations, 223

primary brain lymphoma, 222

rituximab-based chemotherapy, 222

immunoglobulin proteins, 211

immunophenotypic marker, 211

innate immune systems, 216

laboratory procedure, 211

lymphoid neoplasms, 211

lymphoid tumor, 211

lymphoid tumors, 210, 212–213t

mature B-cell neoplasms. See Mature B-cell neoplasms

nodal neoplasms, 216

pathophysiology

ATLL, 211

B-cell lymphoma, 211

BL, 211

EBV, 211

gamma herpes viruses, 211

HCV, 211

HTLV-1, 211

human retroviruses, 211

immune function, 211

KSHV, 211

lymphoproliferative disorder, 211

multicentric Castleman disease, 211

oncogenic viruses, 211

organophosphate insecticides, 211

PELs, 211

RNA viruses, 211

splenic marginal zone lymphoma, 211

posttherapy restaging, 224

precursor B- and T-cell neoplasms, 216

B lymphoblastic leukemia, 216

genetic abnormalities, 216

imatinib, 216

T lymphoblastic leukemia, 216

tyrosine kinase inhibitors, 216

prognostic features

comorbid conditions, 224

diffuse large B-cell lymphomas, 225t

ECOG, 225

follicular and mantle cell prognostic index, 225t

hemoglobin level, 225

IPI, 224

lymphoma therapy, 225

mantle cell, 224

patient-related factors, 224

T-cell immunophenotype, 215t

T-cell lymphomas, 214t

tumor tissue origin, 211

WHO, 210

Nonimmune hemolysis, 32

Nonsteroidal anti-inflammatory (NSAIDs), 302

O

Objective response rate (ORR), 226

Oral iron therapy, 5–7. See also Absolute iron deficiency

Osler-Weber-Rendu disease, 295

Osteolytic bone disease (OBD), 244

Osteonecrosis of jaw

biopsies, 245

Outcome prediction. See Gene expression signature

Overall survival (OS), 250

P

Packed red blood cells (PRBCs), 134

Parenteral iron treatment, 6

Paroxysmal nocturnal hemoglobinuria (PNH), 23, 64, 83

clinical features of, 73

diagnosis of, 73

Budd-Chiari syndrome, 73

flow cytometry, 73

iron deficiency, 73

PNH clonal expansion, 73

etiology and pathophysiology, 71

glycosylphosphoinositol anchor, 71

hematopoietic stem cell, 71

intravascular hemolysis, 71

GPI-anchored proteins, 73

treatment of

chronic anticoagulation, 73

stem cell transplantation (SCT), 73

Pathophysiology

acute hepatic, 59

acute porphyrias, 59t

biosynthetic pathway, 57

clonal dominance

autocrine production, 171

hematopoietic growth factors, 171

δ-aminolevulinic acid synthase (ALAS), 57

enzymatic deficiencies, 58f

erythroid bone marrow, 57

erythropoietic demand, 378

eukaryotic cells, 57

genomic instability

AP, 171

blast crisis, 171

B lymphocytic leukemia, 171

BP, 171

chromosomal abnormalities, 171

disease transforms, 171

heme, 57

heme biosynthetic pathway, 59

hemoglobinopathies, 39

α-chain gene, 40

amino acid, 40

β-thalassemia, 40

glutamic acid, 39

Hb Lepore, 40

homozygous sickle disease (Hb SS), 40

leukocytosis, 40

mechanical forces, 39

nitric oxide, 40

oxygenation–deoxygenation cycle, 39

platelet-activating factor, 40

retinal proliferative disease, 40

sickle erythrocytes, 39

sickle hemoglobin, 39

tumor necrosis factor-alpha (TNF-α), 39–40

hepcidin

hemochromatosis, 378

hepatocytes, 378

hepcidin gene expression, 378

intestinal enterocytes, 378

liver-derived peptide hormone, 378

macrophages, 378

placental cells, 378

HFE localization and function

β2 microglobulin (β2m), 379

C282Y mutation, 379

hepcidin expression, 379

inappropriate absorption, 379

secondary hemochromatosis, 379

iron excretion, 378

iron homeostasis

excess iron and tissue injury, 379

nontransferrin-bound iron (NTBI), 379

transferrin saturation (TS), 379

iron overload disorders

body iron distribution, 379t

bone morphogenetic protein (BMP), 378

classification of, 378t

hemojuvelin gene (HJV), 378

hepcidin expression, 379

transferrin receptor 2 gene (TfR2), 378

leukemic hematopoiesis

BCR-ABL1 translocation, 171

granulocyte production, 171

multipotent stem cell, 171

myeloid lineage, 171

mitochondria, 57

non-porphyria syndrome, 59

organ manifestations, 59

pathophysiologic mechanisms, 59

photocutaneous symptoms, 59

porphyrins, 59

thalassemias

α-globin gene, 37

α-thalassemia gene, 39f

β-chain alleles, 38

β-globin gene, 37

Cooley’s anemia, 38

general features of, 38t

hypochromia, 37

normal and variant hemoglobin, 38t

X-linked dominant protoporphyria (XLDPP), 59

X-linked sideroblastic anemia, 59

Peripheral blood stem cells (PBSCs), 249

Peripheral neuropathy

management, 245

opioid drugs, 245

Phlebotomy therapy

anemia evaluation, 385

arthritis, 385

cancer surveillance, 385

dietary and lifestyle counseling

in hemochromatosis, 386t

oral iron supplements, 385

raw shellfish, 385

endocrine replacement, 385

guidelines for

iron depletion, 384–385

preventing reaccumulation, 385

vaccinations, 385

Phosphofructokinase deficiency, 31

Phosphoglycerate kinase (PGK) deficiencies, 29. See also Erythroid enzymopathies

Plasmablastic lymphoma (PBL), 218

Plasma cell leukemia (PCL), 245

Platelet dysfunction, 419

Platelet function analyzer (PFA), 304

Platelet-rich plasma (PRP), 304

Platelets

disorders characterized by decreased production

acquired amegakaryocytic thrombocytopenia, 271

acute leukemia, 271

androgens, 272

aplastic anemia, 271

bone marrow aspirate, 271

bone marrow failure, 271

chronic leukemias, 271

congenital amegakaryocytic thrombocytopenia, 271

congenital disorders, 271

cyclic thrombocytopenia, 272

dyskeratosis congenita, 271

fanconi anemia, 271

folate deficiency, 272

immune thrombocytopenia (ITP), 271

immunosuppressive agents, 272

irradiation and chemotherapy, 271

isolated severe thrombocytopenia, 271

isolated thrombocytopenia, 271

lymphoma, 271

marrow infiltration, 271

micromegakaryocyte, 271

mild thrombocytopenia, 271

myelodysplasia, 271

myeloma, 271

myelotoxic regimen, 271

neutropenia, 271

normal marrow architecture, 271

nutritional deficiencies, 272

oral contraceptives, 272

platelet transfusions, 271

progenitor cells, 271

thrombocytopenia with absent radius (TAR) syndrome, 271

thrombocytosis, 272

thrombopoietic growth factor, 272

Wiskott-Aldrich syndrome (WAS), 271

X-linked recessive disorder, 271

disorders characterized by increased clearance

disseminated intravascular coagulation, 279

extracorporeal circulation-related thrombocytopenia, 280

heparin-induced thrombocytopenia, 274–276

immune thrombocytopenia, 272–274

neonatal alloimmune thrombocytopenia, 280

posttransfusion purpura, 279

thrombotic microangiopathies, 276–279

von Willebrand disease, type 2B, 280

disorders characterized by increased sequestration

hypersplenism, 280

lymphoma, 280

splenectomy, 280

splenic embolization, 280

splenic irradiation, 280

splenomegaly, 280

Plummer-Wilson syndrome, 1

Pneumocystis jiroveci, 262

Pneumocystis jiroveci pneumonia (PCP), 166

Polycythemia vera. See Myeloproliferative neoplasms

Polycythemia Vera Study Group (PSVG), 95

Polymerase chain reaction (PCR), 145, 172

amplification and accumulation of, 442f

DNA and RNA reverse, 441

modifications of, 441

primers of, 441

reverse transcription of, 442f

Population screening

accurate diagnosis, 383

hemochromatosis meets, 383

laboratory screening

ferritin screening, 383

genotype screening, 383

transferrin saturation, 383

preventive treatment, 383

screening of family members

decision tree, 384f

diagnostic yield of transferrin saturation (TS) screening, 383t

HFE genotype, 383

phenotypic screening, 384

siblings, 384

widespread testing, 383

Porphyria cutanea tarda

hepatic iron, 61

hepatocellular carcinoma, 61

liver function tests, 61

neurologic manifestations, 61

uroporphyrinogen decarboxylase, 61

Porphyrias

clinical manifestations, 57

enzyme deficiencies, 60

erythropoietic, 59

erythropoietic porphyrias, 59

hepatic, 59

molecular genetic level, 60

neurovisceral symptoms, 59

photosensitivity syndromes, 60

psychiatric disorders, 59

congenital erythropoietic porphyria (CEP), 60

ecogenic disorders, 57

enzyme deficiency, 57

erythropoietic protoporphyria (EPP), 60

heme biosynthetic pathway, 57

manifestations of, 60

metabolic disorders, 57

neuropsychiatric symptoms, 57

neurovisceral symptoms, 60

photosensitivity, 60

porphyria cutanea tarda (PCT), 57

X-linked dominant protoporphyria

enzyme deficiency, 60

protoporphyrin, 60

Positron emission tomography (PET), 188

Postthrombotic syndrome management

graduated compression, 337

intermittent pneumatic compression, 337

nonelastic stockings, 337

pharmacologic approach

chronic venous insufficiency symptoms, 337

nonsteroidal anti- inflammatory drugs, 338

pentoxifylline and rutosides, 337

prevent and treat postthrombotic syndrome, 338t

surgical management of, 338

Posttransfusion purpura (PTP)

adjunctive corticosteroids, 279

characterized by, 279

HLA antigen-matched platelets, 279

plasma exchange, 279

platelet transfusion, 279

splenectomy, 279

Prednisone, 241, 250

Preeclampsia and HELLP syndrome

acute fatty liver of, 391

chronic hypertension, 391

clinical manifestations, 391

criteria for, 391

D-dimers, 391

diffuse intravascular coagulation (DIC), 391

fetal distress, 391

fetal lung, 391

gestational age, 391

hypertension, 391

hypertension and proteinuria, 391

insulin resistance, 391

intrauterine asphyxia, 391

low fibrinogen, 391

multiorgan dysfunction, 391

neonatal thrombocytopenia, 391

prolonged prothrombin time, 391

proteinuria, 391

thrombin–anti–thrombin complexes, 391

Primary effusion lymphomas (PELs), 211

Primary mediastinal (thymic) large B-cell lymphoma (PMBL), 218

Primary myelofibrosis. See Myeloproliferative neoplasms

Progression-free survival (PFS), 218, 250

Progressive multifocal leukoencephalopathy (PML), 191

Prolonged activated partial thromboplastin time

coagulopathy of liver disease, 294

deficiencies of individual clotting factors, 294

depletion of coagulation factors, 295

Prolonged partial thromboplastin time (PTT), 127

Prolonged prothrombin time

antibodies to bovine factor V and thrombin, 294

associated with anaphylaxis, 294

coagulopathy of liver disease, 294

critically elevated INRs, 294

cryoprecipitate, 294

deficiencies of individual clotting factors, 294

dysfibrinogenemia, 294

erratic absorption, 294

hepatic insufficiency, 294

hypofibrinogenemia, 294

intestinal bacteria, 294

intravenous administration, 294

malabsorption, 294

mild prolongation, 294

phytonadione, 294

plasma-derived fibrinogen concentrate, 294

subcutaneous administration, 294

supratherapeutic INRs, 294

vitamin K deficiency, 294

vitamin K-dependent carboxylase, 294

warfarin

treatment of, 294

Prophylaxis

American College of Physicians (ACP), 323

controlled trials, 323

orthopedic surgical patients, 323

prophylactic treatment of, 323

thromboprophylaxis, 323

Prothrombin Complex Concentrate (PCC), 352

Prothrombin time (PT), 134, 146, 298

Pseudothrombocytopenia

ethylenediaminetetraacetic acid (EDTA), 280

finger stick source, 281

selected causes of splenomegaly, 281t

Pulmonary complications

acute complications

chlamydia, 45

H. influenze, 45

mycoplasma, 45

necrotic bone marrow, 45

respiratory symptoms, 45

S.taphylococcus aureus, 45

S.treptococcus pneumoniae, 45

systemic fat embolism syndrome, 45

vaso-occlusion, 45

viruses, 45

chronic complications

hydroxyurea, 46

nitric oxide, 46

vaso-occlusive crises, 46

Pulmonary embolism (PE). See also Deep venous thrombosis (DVT)

acute trauma patients, 313

chest radiography, 314

ECG, 314

echocardiographic confirmation, 314

echocardiographic findings, 314

Hampton hump, 314

IVC, 313

mural thrombus, 313

pulmonary angiography, 314

ventilation/perfusion scanning, 314

Pulmonary function tests (PFT), 201

Pure red blood cell aplasia. See Bone marrow failure syndromes

Pure red cell aplasia (PRCA)

clinical features and diagnosis of, 74

etiology and pathophysiology

chronic lymphocytic leukemia, 74

collagen-vascular syndromes, 74

erythema infectiosum, 74

ribosomal protein gene, 74

thymoma, 74

transient aplastic crisis, 74

treatment of, 74

azathioprine, 74

cyclosphosphamide, 74

cytotoxic drugs, 74

Pyrimidine 5´nucleotidase (uridine 5´monophosphate hydrolase) deficiency, 32

Pyruvate kinase (PK) deficiency, 31. See also Erythroid enzymopathies

Q

Qualitative platelet disorders

acquired disorders

cardiac bypass, 308

drug-induced, 304–307

myelodysplasia/myeloproliferative diseases, 307

renal failure/uremia, 307–308

Eltrombopag, treatment, 309

Epstein syndrome, 309

Fechtner syndrome, 309

inherited disorders

Bernard-Soulier syndrome (BSS), 308

Glanzmann thrombasthenia, 308

isolated laboratory-specific defects, 309

Scott syndrome, 309

signal transduction congenital disorders, 309

storage pool disease (SPD), 308–309

May-Hegglin anomaly, 309

platelet biochemistry, role of, 303, 304t

platelet function testing

adenosine triphosphate, 304

aggregation, 304, 305t

chemiluminescence procedure, 304

PFA, 304

platelet granules, 304, 304t

Sebastian syndrome, 309

treatment of, 307t

R

Red blood cell (RBC), 22, 24–26, 29, 31–33, 121, 343

Red blood cells (RBCs), 166

Red blood cell transfusion, 7

Red cell distribution width (RDW), 26, 123–125, 405

Reduced-intensity conditioning (RIC), 148, 249

Reduced intensity regimens (RIC), 89

Reduced intensity stem cell transplantation (RIST), 232

Reed-Sternberg (RS) cell, 197

Restriction fragment length polymorphism (RFLP), 444–445

Reticuloendothelial system (RES), 1, 2f

Retinitis pigmentosa, 33

Rheumatoid arthritis, 7

Ristocetin-induced platelet aggregation (RIPA), 300

Romanowsky-stained peripheral blood film, 408

S

Sanger sequencing, 442

Selective estrogen receptor modulators

(SERMS), 319

Sequence-specific oligonucleotide probe (SSOP), 441

Sequence-specific primers (SSP), 441

Serum haptoglobin

free hemoglobin bound, 416

hemoglobin-binding protein, 416

hemolysis of, 416

hepatic synthesis, 417

nephelometric method, 416

reticuloendothelial system, 416

turbidometric method, 416

Serum methylmalonic acid, 414

Serum tests, nutritional and

hypoproliferative anemias

red cell folate assays, 414

serum erythropoietin

assays, 415

erythropoiesis-stimulating agents, 414

low erythropoietin levels, 415

phlebotomy treatment, 415

refractory anemia, 414

secondary polycythemia, 415

serum ferritin

anemic patients, 413

chronic hepatitis C, 413

hyperferritinemia cataract syndrome, 413

indicate iron, 413

iron chelation therapy, 413

metabolic, 413

Still’s disease/syndromes, 413

transfusion hemosiderosis, 413

ultrahigh levels, 413

serum homocysteine, 414

serum intrinsic factor antibody assay, 414

serum iron and total binding capacity assays

diurnal variation, 411

inflammation/chronic disease, 411

iron deficiency, 411

serum iron levels, 411

serum iron measurement, 411

serum methylmalonic acid, 414

serum and red cell folate assays, 414

serum vitamin B12 (cobalamin), 413

soluble transferrin receptor

automated assays, 412

causes of, 412t

dynamic and sensitive measure of, 412

erythropoiesis, 412

levels of, 412

tissue transferrin receptor, 412

Short-tandem repeat (STR), 456–457

Shwachman-Diamond syndrome

clinical diagnostic criteria

biallelic SBDS gene mutation, 80

hematologic abnormalities, 80

hypoproductive cytopenia, 80

pancreatic dysfunction, 80

pancreatic lipomatosis, 80

clinical features of, 80

hematological manifestations, 80

malignant myeloid transformation, 80

treatment modalities, 80

Sickle cell disease (SCD), 37

clinical syndromes and treatment

acute infection, 43

cardiovascular manifestations, 45

central nervous system, 44

eye disease, 44–45

Hemophilus influenzae, 42

hyposthenuria, 47

Neisseria meningitides, 42

neonatal diagnosis, 42

oral hydration, 47

parvovirus infection, 43

penicillin prophylaxis, 42

priapism, 47

prophylactic penicillin, 42

pulmonary complications, 45–46

renal medulla, 47

S. pneumoniae, 42

serum creatinine, 47

serum erythropoietin levels, 47

skeletal complications, 47–48

Streptococcus pneumoniae, 42

transient aplastic crisis, 43

vasa recta, 47

vaso-occlusive episodes, 42

Sickle cell disorders. See Hemolytic anemia

Sickle cell trait

anesthetic agents, 51

rhabdomyolysis, 51

traumatic eye injury, 51

urinary tract infections, 51

Sickle solubility test

clinical settings, 415

electrophoretic mobility, 415

hemoglobin electrophoresis, 415f

phosphate buffer, 415

Single nucleotide polymorphism (SNP), 84

Single nucleotide polymorphisms (SNPs), 445

Skeletal complications

acute phase, 47

drug-resistant organisms, 48

etiology, 48

hand-foot syndrome, 47

hydroxyurea, 48, 50t

leg ulcers, 47

osteonecrosis, 47

physical therapy, 47

vaso-occlusion, 47

white blood cell count, 47

Skeletal-related events

klyphoplasty, 245

local radiation therapy, 245

vertebroplasty, 245

Skeletal-related events (SRE), 244

Slow early responders (SER), 163

Small lymphocytic lymphoma (SLL), 187

Smoldering multiple myeloma (SMM), 234

Sodium ferric gluconate in sucrose, 7

Somatic mutations and translocations

detection of

germline mutations, 452

NGS approach, 452

PCR products, 452

polymerase chain reaction

advantage of, 450

global diagnostic battery, 451

mantle cell, 451

multiple primer pairs, 451

primer mixtures, 452

real-time quantitative, 450f

reverse transcriptase (RT), 450

sensitivity and specificity, 450

sensitivity of, 450

specific translocation, 450

translocations diagnosed, 451

Specific granule deficiency (SGD), 116

Splenectomy, 34

Splenic manifestations

chronic hemolysis, 46

Standard hematologic tests, interpretation

abnormal hemoglobins and hemolytic anemias

glucose-6-phosphate dehydrogenase, 416

Hb A2 quantitation, 416

Hb F cells, 416

Hb F quantitation, 416

hemoglobin electrophoresis. See Hemoglobin electrophoresis

serum haptoglobin. See Serum haptoglobin

sickle cell prep, 416

sickle solubility test. See Sickle solubility test

unstable hemoglobins test, 416

urine hemosiderin, 417

hematologic malignancies evaluation test. See Hematologic malignancies

hemostasis and coagulation assays.

See Hemostasis and coagulation assays

hypercoagulability test

activated protein C resistance, 420

antithrombin, 419–420

DRVTT, 420

factor V Leiden and prothrombin, 420

lupus anticoagulants, 420

protein C and S, 419–420

neutrophil evaluation. See Neutrophil evaluation

nutritional and hypoproliferative anemias. See Serum test

peripheral blood and bone marrow, cellular analysis

Bernard-Soulier disease, 408

bone marrow aspirate/biopsy, 408–411

clinical information, 405

complete blood count. See Complete blood count

flagged, 407

granulocyte morphology, 408

gray platelet syndrome, 408

hematocrit, 405

hematologic disease, 407

hemoglobin concentration, 405

intracellular inclusions, 408

laboratory personnel, 407

leukocyte count, 407

marrow fibrosis, 408

May-Hegglin anomaly, 408

microangiopathic disorders, 408

microspherocytes, 408

myelodysplasia, 408

nucleated red cells, 408

peripheral blood smear, 407

peripheral smear, 408

platelet count, 405, 407

platelet morphology, 408

polychromasia, 408

red cell membrane irregularities, 408

Romanowsky-stained peripheral blood film, 408

schistocytes, 408

sickle cells, 408

spherocytes, 408

suspected hemolysis, 408

TTP, 408

tumor invasion, 408

white blood cell, 405

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL), 220

Systemic lupus erythematosus (SLE), 131

T

Targeted cancer therapy. See Gene expression signature

Tarui disease. See Phosphofructokinase deficiency

T-cell acute lymphoblastic leukemia (T-ALL), 447

T-cell lymphoma

bexarotene, 233

corticosteroids, 233

doxorubicin-based treatment, 233

primary cutaneous anaplastic lymphoma, 233

TSEBT, 233

ultraviolet radiation, 233

T-cell receptor and immunoglobulin rearrangement

α and γ chain, 453

gene of, 453

Ig and TCR clonality, 454

large granular lymphocyte (LGL), 454

major prognostic marker, 454

malignancies of, 453

ontogeny of, 453

PCR analysis, 453

receptor of, 453

somatic hypermutations, 454–455

V-J rearrangements, 454

Telomerase reverse transcriptase (TERT), 79

Thalassemias, clinical syndromes and treatment

erythrocyte precursors, 51

fetal hemoglobin induction, 53

Hb F response, 53

hydroxyurea, 53

iron overload and chelation

cardiac myocytes, 52

chronic transfusions, 52

endocrine organs, 52

oral deferasirox, 52

oral deferiprone, 52

pulmonary function, 52

toxic effects, 52

red cell membrane, 51

red cell transfusions, 51

thrombocytopenia, 51

thromboembolism, 51

transfusions and splenomegaly

hematologic parameters, 52

iron chelation, 52

penicillin prophylaxis, 52

splenomegaly, 52

Thrombocytopenia. See also Platelets

causes of, 270t

characterized by, 269

etiology and clinical features

central nervous system bleeding, 269

gastrointestinal, 269

genitourinary, 269

patients with dysfunctional platelets, 269

platelet count, 269

platelet-type bleeding, 269

spontaneous bleeding, 269

thrombocytopenia patients, 269

occur due to, 269

other causes

drug-induced thrombocytopenia, 281–282

gestational thrombocytopenia, 283

hemophagocytosis, 283

human immunodeficiency virus-related thrombocytopenia, 283

infection- and sepsis-related thrombocytopenia, 283

pseudothrombocytopenia, 280–281

qualitative disorders, 283

platelet biology

cytokines, 269

megakaryocytes, 269

normal platelet concentration, 269

physiologic stress, 269

primary hemostasis, 269

target platelet count values

commonly encountered clinical scenarios, 270t

Thrombocytopenia-absent radii (TAR), 129, 271

Thromboembolic events (TEE), 130

Thrombophilia, acquired conditions

activated protein C resistance

Caucasian population, 321

characteristic mutation, 322

inherited hypercoagulable states, 321t

polymorphism, 321

antiphospholipid antibody syndrome

antithrombotic therapy, 319

dilute Russell viper venom time (DRVVT), 318

kaolin clotting time (KCT), 318

mechanisms of, 318t

screening and confirmatory tests, 318

tissue thromboplastin

inhibition (TTI), 318

treatment strategies, 319

heparin-induced thrombo-

cytopenia (HIT)

enzyme immunoassays, 316

fondaparinux binds, 317

HEP, 316

LMWH, 316–317

lupus anticoagulants (LAC), 318

panel probability score, 317t

platelet aggregation assays, 317

platelet rich plasma, 316

pretest prediction of, 316t

PTT monitoring, 318

treatment of, 317

UFH, 316

vitamin-dependent

anticoagulant, 318

hypercoagulability of malignancy

CLOT trial, 319

dalteparin, 319

nonmetastatic disease, 319

selective estrogen receptor modulators (SERMS), 319

thrombus formation, 319

tumor types, 320

vascular endothelium, 319

hyperhomocysteinemia, 322

inherited hypercoagulable states, 321

myeloproliferative disorders

blood counts, 321

hydroxyurea (HU), 321

platelet-lowering non-

cytotoxic, 321

thrombosis, 321

nephrotic syndrome, 318

paroxysmal nocturnal

hemoglobinuria, 320

proteins S and C and anti-

thrombin, 322

prothrombin mutation, 322

surgery as risk for

elastic stockings, 320

laparoscopic surgery, 320

peripartum state, 320

procedure of, 320

serial compression devices, 320

Thrombophilia and recurrent miscarriage

anticardiolipin antibodies, 395

low dose aspirin, 395

optimal dosages, 395

prednisone, 395

Thrombosis

acquired prothrombotic disorders

antiphospholipid antibody assay, 131

central venous catheters, 131

congenital heart disease, 131

Kawasaki disease, 131

laboratory examination, 131

lupus anticoagulant, 131

nephrotic syndrome, 131

systemic lupus erythematosus (SLE), 131

anticoagulant therapy, 130

coagulation factor levels, 130

congenital prothrombotic disorders

hemorrhagic complications, 130

homozygous infants, 130

Purpura fulminans, 130

thrombophilic conditions, 130

warfarin anticoagulation, 130

heparin therapy, 130t

INR, 130

TEE, 130

thrombolytic therapy, 130

warfarin therapy, 130, 131t

Thrombotic microangiopathies (TMAs)

classical TTP, 278

congenital TTP, 278

cyclophosphamide, 279

cyclosporine, 279

diagnosis

activated partial thromboplastin time (aPTT), 278

assays for VWFCP activity, 278

clinical features, 278

E. coli 0157:H7, 278

new-onset MAHA, 278

prothrombin time (PT), 278

stool culture, 278

drugs

bleomycin, 278

clopidogrel, 278

cyclosporine, 278

mitomycin C, 278

quinine, 278

ticlopidine, 278

endemic HUS, 278

epidemiology

typical HUS, 276

features, 277t

hemodialysis, 279

hemolytic-uremic syndrome (HUS), 276

microangiopathic hemolytic anemia (MAHA), 276

pathophysiology

red cells, 277

von Willebrand factor cleaving protease (VWFCP), 277

pregnancy-associated TTP-HUS, 278

presentation

manifestations, 278

neurologic impairment, 278

refractory and relapsing TMA, 279

rituximab, 279

splenectomy, 279

thrombotic thrombocytopenic purpura (TTP), 276

treatment

fresh frozen plasma (FFP), 279

life-threatening bleeding, 279

mortality rate, 278

packed red cells, 279

plasma exchange, 278

plasma infusion, 279

platelet-rich microthrombi, 279

platelet transfusions, 279

vincristine, 279

VWF molecules, 278

Thrombotic thrombocytopenia purpura (TTP), 26, 276, 299, 408

cleaving protease, 392

multimer analysis, 392

plasma exchange, 392

postpartum of, 392

supportive-care measures, 392

symptoms of, 392

von Willebrand factor (vWF), 392

Tissue thromboplastin inhibition (TTI), 318

T-large granular lymphocyte, (T-LGL) leukemia, 219

Total body irradiation (TBI), 256

Total iron-binding capacity (TIBC), 123, 411

Total skin electron beam therapy (TSEBT), 233

Transcranial Doppler ultrasound (TCD), 126

Transferrin receptor 2 gene (TfR2), 378

Transient erythroblastopenia of childhood (TEC), 125

Triosephosphate isomerase (TPI) deficiency, 31

Tropical disease, hematological manifestations

acute leukemias

cyanide-resistant peroxidase, 400

dysplastic appearance, 400

eosinophilic leukemia, 400

chronic myelomonocytic leukemia with eosinophilia

antiapoptotic pathways, 400

eosinophilia subtypes, 400

fibroblast growth factor receptor 1 (FGFR1), 400

platelet-derived growth factor receptor beta (PDGFR-β), 400

predominant subtypes, 400

FIP1L1-PDGFR-β hypereosinophilic disorders

adenosine triphosphate (ATP), 400

cardiac echocardiogram, 400

eosinophil-mediated cardiomyopathy, 401

hypereosinophilia harboring, 400

prophylactic, 400

serum troponin, 400

serum tryptase, 400

idiopathic hypereosinophilic syndrome

corticosteroids, 401

front-line therapy, 401

monoclonal antibodies, 401

malaria

African trypanosomiasis, 397

causes of anemia, 395t

clinical patterns, 395

clonal eosinophilic disorders, 397–399

Duffy antigen, 395

helminth infections, 397

hyperreactive malarial splenomegaly (HMS), 395

Plasmodium falciparum, 395–396

protective genetic alterations, 396t

sickle cell trait, 396

significant lymphocytosis, 396

visceral leishmaniasis, 396–397

T-cell clonal disorders

interferon-alpha, 399

lymphomatous transformation, 399

optimal treatment of, 399

polyclonal hypergammaglobulinemia, 399

rearrangement of, 399

skin manifestations, 399

Tropical spastic paraparesis (TSP), 220

Tumor necrosis factor-α (TNF-α), 66

Tyrosine kinase inhibitors (TKIs), 170

U

Umbilical cord blood (UCB), 149

Unfractionated heparin (UFH), 394

Urine hemosiderin, 417

V

Vaso-occlusive episodes (VOE)

acute pain, 42

common opioid agonists, 43t

ceiling effect, 42

chronic opioid therapy, 42

chronic pain, 42

gabapentin antidepressants, 42

intramuscular injection, 42

neuropathic pain, 42

nonsteroidal anti-inflammatory drugs (NSAIDs), 42

pain management service, 42

patient-controlled delivery (PCA), 42

physical examination, 42

red cell transfusions, 42

skeletal system, 42

tricyclic antidepressants, 42

Veno-occlusive disease (VOD), 256

Venous thromboembolism treatment

antiangiogenic treatment

anti-inflammatory effects, 331

antiplatelet effects, 331

approaches to, 331t

chronic lymphocytic leukemia (CLL), 331

clinical trial, 330

thalidomide/lenalidomide, 330–331

thromboembolic events, 330

anticoagulation option, 332

activity of, 333

acute thrombosis, 333

clinical suspicion, 333

fondaparinux, 333

heparin-induced thrombocytopenia (HIT), 333

thrombocytopenia, 333

total chemical synthesis, 334

antithrombin III (AT) deficiency, 333

approaches, 329t

brain metastases and primary brain tumors

anticoagulation, 331

arterial thrombosis, 331

computed tomography (CT), 332

intracranial hemorrhage, 332

mechanical thromboprophylaxis, 332

neurosurgery, 332

renal cell carcinoma, 332

spontaneous bleeding, 332

thyroid cancer, 332

Xa activity, 332

chemical synthesis, 329

chronic therapy, 332

CLOT study, 332

diagnosis of

CT pulmonary angiography (CTPA), 335

CT scanning instruments, 335

D-dimer, 335

perfusion scanning, 335

reducing radiation, 335

ventilation/perfusion (V/Q), 335

ventilation scanning, 335

enoxaparin and fondaparinux dosing, 329t

fibrinogen, 333

fibrinolytic activity, 335

fondaparinux, 329

Food and Drug Administration (FDA), 328

immobilized/hospitalized cancer patients, 331

impaired blood flow (stasis), 328

inferior vena cava filters

filter components, 334

permanent devices, 334

pulmonary embolism (PE), 334

serious complications, 334

long-term indwelling devices, 328

low molecular-weight heparin (LMWH), 328

malignancy patients, 328

normal creatinine values, 328

obstetric outcomes, 335

poor obstetric outcomes, 335

pregnant women management

advanced maternal age, 335

antepartum management, 336t

osteoporosis, 335

parturition approaches, 337

postpartum management, 336t

risk factors, 335

spinal hematoma, 337

thrombotic conditions, 336

warfarin, 335

primary prophylaxis

ardeparin, 330

cancer surgery, 330

dalteparin, 330

deep venous thrombosis (DVT), 330

laparoscopic surgery, 330

postoperative immobilization, 330

reviparin, 330

Society of American Gastrointestinal and Endoscopic Surgeons (SAGES), 330

tinzaparin, 330

tumor resection, 330

thrombophilia, 335

thrombosis, 335

thrombosis and venous access devices

cancer patients, 334

catheter-related DVT, 334

central venous catheters, 334

Cochrane database systematic review, 334

contraindication, 334

Trousseau syndrome, 332

unfractionated heparin (UFH), 328

von Willebrand factor, 333

Venous thromboembolism (VTE), 299

acquired thrombophilic states. See Acquired thrombophilic states

laboratory diagnosis of

ancillary findings diagnostic, 314t

D-dimer, 314

good reproducibility, 314

hazard comparing, 314

multicentre prospective, 315

rethrombosis over, 314

vitamin K antagonists (VKAs), 314

pulmonary embolism (PE), 311

pulmonary hypertension, 311

treatment of

acute venous thromboembolism, 324

fibrinolytic therapy. See Fibrinolytic therapy

inferior vena cava filters. See Inferior vena cava filters

prophylaxis. See Prophylaxis

Vincristine, 241, 250

Visceral leishmaniasis

blood transfusions, 396

dyserythropoiesis, 397

gelatinous transformation, 397

hematologic manifestations, 396t

hypersplenism, 397

L. donovani, 397

myelofibrosis, 397

phlebotomine sand flies, 396

sexual contact, 396

Vitamin B12 and folate, deficiencies

clinical presentation, 15

determine cause of. See Folate deficiency

development of

autoimmune atrophic gastritis, 14

bariatric surgery, 14

causes of, 13t

cobalamin deficiency, 13

etiology, 14

HIV infection, 14

prolonged use of, 14

folic acid deficiency, development

causes of, 14t

chronic medications, 14

diet poor, 14

frequencies of, 11

laboratory evaluation, 15–18

metabolic roles of

biology and dosing, 12t

cobalamin, 13

metabolic pathways, 12f

methyltetrahydrofolic acid, 13

patient populations, 15

requirements, sources, and stores

bacteria, 11

Food and Drug Administration mandated, 11

Plants synthesize folic acid, 11

treatment/response of

clinical presentation, 19

goals, 19

hematologic response, 19

intramuscular B12, 19

neuropathic abnormalities, 19

oral folic acid, 19

reticulocytosis, 19

Von Willebrand disease (VWD), 127, 285

antifibrinolytic agents, 303

antihemophilic factors, 302

bleeding disorders, 303

bleeding symptoms, 300

desmopressin acetate, 302

diagnosis of, 301

epidemiology, 299

epsilon aminocaproic acid, 303

hemostatic range, 302

hyponatremia, 302

laboratory evaluation of, 301t

mucous membranes, 300

NSAIDs, 302

pathophysiology

endothelial cells, 299

gain-of-function defect, 300

megakaryocytes, 299

multimeric glycoprotein, 299

platelet glycoprotein Ib, 299

quantitative defect, 300

RIPA, 300

severe bleeding manifests, 300

subendothelial collagen matrix, 299

primary hemostasis, 300f

prophylaxis, 302

tachyphylaxis, 302

topical agents, 303

treatment of, 302t

type 1, 300

type 2, 280, 300

type 3, 300

W

White blood cell (WBC), 126, 163, 406

Wiskott-Aldrich syndrome (WAS), 118, 129, 271, 309

World Health Organization (WHO), 95, 141, 197

X

X chromosome inactivation pattern (XCIP), 453