Hematology: A Pathophysiologic Approach 9780323057097, 9780323036283, 9780323033916, 9780323034470, 9780323034463, 9780323036665, 9780323043113

Table of contents :
Hematology A Pathophysiologic Approach
Prelims
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Title page
Copyright page
Dedication
PREFACE
ACKNOWLEDGMENTS
Table of contents
Brief Overview of the Hematolymphoid System
Key Words and Concepts
Chapter 1: Hematopoiesis
Key Points
Key Words and Concepts
Technical Considerations
Chapter 2: Erythropoiesis and Oxygen Transport
Key Points
Key Words and Concepts
Oxygen Transport
Chapter 3: Iron, Heme, and Hemoglobin
Key Points
Key Words and Concepts
Iron Deficiency Anemia
Heme
Hemoglobin
Oxygen Affinity
Chapter 4: Hemoglobinopathy
Key Points
Key Words and Concepts
Non-Functional Hemoglobin Variants
Beta Globin Mutations
Thalassemia
Unstable Hemoglobin Variants
Chapter 5: Red Blood Cell Metabolism and Enzyme Defects
Key Points
Key Words and Concepts
Red Blood Cell Enzyme Defects
Chapter 6: Hemolytic Anemia
Key Points
Key Words and Concepts
Extrinsic Hemolytic Anemia
Intrinsic Hemolytic Anemia
Chapter 7: Aplastic Anemia and Related Disorders
Key Points
Key Words and Concepts
Anemias Associated with Non-Hematopoietic Organ Dysfunction
Chapter 8: Megaloblastic Anemia
Key Points
Key Words and Concepts
Chapter 9: Myeloid Cells
Key Points
Key Words and Concepts
Neutrophils
Eosinophils
Basophils and Mast Cells
Monocytes
Chapter 10: Immune System and Related Disorders
Key Points
Key Words and Concepts
Immunoglobulin Structure and Function
T CELL RECEPTOR SIGNALING
T Cell Subsets
Immunodeficiency
Lymphoid Tumors
Chapter 11: Genetic Basis of Hematologic Neoplasia
Key Points
Key Words and Concepts
Cell Proliferation
Apoptosis
Specific Genetic Defects in Hematologic Cancer
Molecular and Cytogenetic Techniques
Chapter 12: Leukemia and Related Disorders
Key Words and Concepts
Introduction
Acute Myeloid Leukemia
Acute Lymphoblastic Leukemia
Chronic Lymphoid Leukemia
Myelodysplastic Syndromes
Myeloproliferative Disorders
Myelodysplastic Syndromes/Myeloproliferative Diseases
Chapter 13: Lymphoma and Related Disorders
Key Points
Key Words and Concepts
B Cell Non-Hodgkin Lymphoma
T or Natural Killer Cell Non-Hodgkin Lymphoma
Hodgkin Lymphoma
Classic Hodgkin Lymphoma
Histiocytic and Dendritic Cell Neoplasms
Chapter 14: Blood Coagulation
Key Points
Key Words and Concepts
Blood Coagulation
Fibrinolysis
Bleeding Disorders
Hereditary Thrombophilia
Anticoagulation
Coagulation Testing
Chapter 15: Platelets
Key Points
Key Words and Concepts
Thrombocytopenia and Thrombocytosis
Platelet Function Defects
Chapter 16: Benign Conditions of Lymphoid Organs
Key Points
Key Words and Concepts
Spleen
Thymus
Chapter 17: Blood Transfusion and Stem Cell Transplantation
Key Points
Key Words and Concepts
Blood Transfusion
Blood Groups
Transfusion Reactions
Stem Cell Transplantation
Complications Of Allogeneic Stem Cell Transplantation
Human Leukocyte Antigens
Chapter 18: Cancer Chemotherapy
Key Points
Key Words and Concepts
Cell-Cycle Active Agents
Non-Cell-Cycle Active Agents
APPENDIX A: Complete Blood Count
Age and Pregnancy Related Hematologic Changes
Age and Pregnancy Related Coagulation Changes
Key Words and Concepts
APPENDIX B: Some Useful Immunophenotypic Markers for Hematologic Diagnosis
Index
Color plates

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Hematology: A Pathophysiologic Approach

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BLANKENSHIP: Neurophysiology (978-0-323001899-9) BLAUSTEIN et al: Cellular Physiology and Neurophysiology, 2nd Edition (978-0-323-05709-7) CLOUTIER: Respiratory Physiology (978-0-323-03628-3) JOHNSON: Gastrointestinal Physiology, 7th Edition (978-0-323-03391-6) KOEPPEN & STANTON: Renal Physiology, 4th Edition (978-0-323-03447-0) LEVY & PAPPANO: Cardiovascular Physiology, 9th Edition (978-0-323-03446-3) PORTERFIELD & WHITE: Endocrine Physiology, 3rd Edition (978-0-323-03666-5)

Hematology: A Pathophysiologic Approach n

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S. DAVID HUDNALL, MD Professor of Pathology and Laboratory Medicine Yale University School of Medicine Director of Hematopathology Yale-New Haven Hospital New Haven, Connecticut

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1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH Copyright © 2012 by Mosby, an imprint of Elsevier Inc.

ISBN: 978-0-323-04311-3

All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. International Standard Book Number: 978-0-323-04311-3

Acquisitions Editor: William Schmitt Developmental Editor: Barbara Cicalese Publishing Services Manager: Patricia Tannian Project Manager: Sarah Wunderly Design Direction: Steven Stave

Working together to grow libraries in developing countries Printed in the United States of America Last digit is the print number:  9  8  7  6  5  4  3  2  1

www.elsevier.com | www.bookaid.org | www.sabre.org

To my parents, Stanley and Marjorie Hudnall My wife, Amy And my children, Katie and Molly

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PREFACE n

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Hematology: A Pathophysiologic Approach is designed as an introductory hematology text for all students and trainees, including medical students, biomedical graduate students, pathology residents, and fellows in hematology/oncology and hematopathology. This book is based on my 26 years of experience practicing laboratory hematology and teaching hematology to medical students, residents, fellows, and graduate students at four medical schools in the United States. The idea of writing a textbook crystallized during my years as course director of an innovative 2-year, small group problem-based course in hematology at the University of Texas Medical Branch (UTMB) in Galveston. During that time I was disappointed by the choice of hematology textbooks suitable for medical students. Over the years we tried several texts and, while some were quite good, both faculty and students expressed dissatisfaction with one thing or another. The possibility of writing a textbook suddenly became a reality when I was approached by Mosby to write a hematologic physiology text for their Physiology Series—an opportunity that I undertook with gusto. It is important to state what this text is not. It is not a comprehensive textbook of clinical hematology. Clinical hematology is a vast, complex field that, to be thoroughly and expertly covered, requires many authors, many pages, and tracts of up-to-date references. To meet this demand, several excellent, large, and generously referenced 1000+ page textbooks written by multiple expert contributors are available. However, these textbooks are not appropriate as introductory texts. They are far too detailed, often presuming prior knowledge of basic anatomy and physiology of the hematologic system. The text you are about to read is radically different. It is written by a single author and contains no

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references. Although some expert advice was sought, I have for the most part been able to draw on my own experience as a course designer/director and teacher of hematology to present a single unified overview of the field. Because reference materials are readily available on the Internet, I decided not to clutter the text with references that are seldom used by students. Instead, the student interested in more depth of any topic can easily access the most recent literature online. This is a book that first and foremost approaches hematology from a pathophysiologic perspective, with mechanistic explanations of normal and abnormal function. Hematopoiesis, blood physiology, immunology, neoplasia, transplantation, and hemostasis are presented as interrelated subjects. Because the practice of hematology is highly dependent upon a relatively large number of diagnostic lab tests, fairly detailed descriptions of blood counting, histopathology, immunohistochemistry, flow cytometry, cytogenetics, and coagulation testing are provided. Numerous color photomicrographs of both normal and abnormal histology are also provided in full color. Over many years of teaching hematology and designing/directing a hematology course, I have formulated a good idea of what students need to know about this complex subject. But, with the primary goal of imparting an understanding of the subject by the student in mind, I have done my best to resist the temptation to assume that if I present the material, it will be understood. To this end, I have always encouraged students to question the material and contact me directly with their questions. Over the years, this has proven to be immensely valuable. Literally hundreds of insightful questions received from students have challenged me to provide more vii

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PREFACE

accurate, lucid explanations of difficult topics. In other cases, questions have led to my discovery that some facts we take for granted may be flawed, incomplete, or illogical. Based on this experience, I have tried to anticipate many of the issues that often lead to confusion and to provide a more explicit explanation than is usually offered. But as perfection is an elusive goal, I urge all readers of this text, whether expert or novice, to contact me with your questions, corrections, or

concerns. With your help, the text can be continually improved. I do hope you enjoy the book. S. David Hudnall, MD [email protected]

ACKNOWLEDGMENTS n

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I would like to thank some special people who have played important roles in furthering my understanding of the science and practice of hematology and in bringing this book to life. The list rightly begins with my late father-in-law Abner H. Levkoff, MD, who unselfishly shared his interest in physiology, read the first drafts of the text, and provided me with incisive critiques and sage advice. I am very grateful to my early mentors in immunology and hematology, Drs. Abul Abbas and Faramarz Naeim, for providing me with a strong foundation to build upon. Many thanks to my former colleagues at UTMB, Drs. Jack Alperin, David Bessman, Tarek Elghetany, and Frank Gardner, for their collective expertise regarding selected topics in hematology. I would also like to thank my good friends and colleagues Drs. Rolf Konig, Malcolm Brodwick, and Peter

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Rady for their invaluable opinions about academic life, biomedical science, and the joys and challenges of teaching medical and graduate students. And, of course, thanks go to the hundreds of second-year medical students at UTMB, who took the course in hematology during my tenure as a Course Director, for all your wonderful questions—questions that always begged for more lucid answers and sometimes highlighted the incompleteness of our knowledge. And finally, I would like to thank William R. (Bill) Schmitt, Barbara Cicalese, Kristin Saunders, Jeff Somers, and other members of the production staff at Elsevier for their patience and expertise over the past few years as the book progressed in fits and starts from a nascent idea to the final product. Without them the book simply would not have seen the light of day.

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CONTENTS n

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BRIEF OVERVIEW OF THE HEMATOLYMPHOID SYSTEM . . . . . . . 1

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CHAPTER

6

HEMOLYTIC ANEMIA . . . . . . . 37 CHAPTER

1

HEMATOPOIESIS . . . . . . . . . . 5

CHAPTER

2

ERYTHROPOIESIS AND OXYGEN TRANSPORT . . . . . . 15

CHAPTER

4

HEMOGLOBINOPATHY . . . . . 27

CHAPTER

5

RED BLOOD CELL METABOLISM AND ENZYME DEFECTS . . . . . . . . . 33

7

APLASTIC ANEMIA AND RELATED DISORDERS . . . . . . 41 CHAPTER

8

MEGALOBLASTIC ANEMIA . . . . . . . . . . . . . . . . 45

3

IRON, HEME, AND HEMOGLOBIN . . . . . . . . . . . 17

CHAPTER

CHAPTER

CHAPTER

9

MYELOID CELLS . . . . . . . . . . 49 CHAPTER

10

IMMUNE SYSTEM AND RELATED DISORDERS . . . . . . 59 CHAPTER

11

GENETIC BASIS OF HEMATOPOIETIC NEOPLASIA . . . . . . . . . . . . . . 75 xi

xii

CONTENTS

CHAPTER

12

LEUKEMIA AND RELATED DISORDERS . . . . . . 85

CHAPTER

14

BLOOD COAGULATION . . . . 115

CHAPTER

15

PLATELETS . . . . . . . . . . . . . 127

CHAPTER

16

BENIGN CONDITIONS OF LYMPHOID ORGANS . . . . . . 135

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BLOOD TRANSFUSION AND STEM CELL TRANSPLANTATION . . . . . . 141

13

LYMPHOMA AND RELATED DISORDERS . . . . . . 99

CHAPTER

CHAPTER

CHAPTER

18

CANCER CHEMOTHERAPY . . . . . . . . . 149 APPENDIX A COMPLETE BLOOD COUNT . . . . . . . . 153 APPENDIX B SOME USEFUL IMMUNOPHENOTYPIC MARKERS FOR HEMATOLOGIC DIAGNOSIS . . . . . . . . . . . 157

Happy is he who gets to know the reasons for things. Virgil (70-19 BCE)

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Hematology: A Pathophysiologic Approach

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BRIEF OVERVIEW OF THE HEMATOLYMPHOID SYSTEM n

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Hematology is the medical science that deals with all things blood. Blood is a non-Newtonian fluid composed of liquid plasma and suspended blood cells. The volume of blood in the normal adult human is approximately 5 L, of which about 55% (v/v) is plasma and 45% cells. Plasma is a slightly alkaline (pH 7.4) saline solution (0.9% sodium chloride) containing about 8% proteins, lipids, amino acids, glucose, hormones, and metabolic waste products, including carbon dioxide, urea, bilirubin, uric acid, and lactic acid. The four most abundant plasma proteins are albumin, gamma globulins, transferrin, and fibrinogen, in that order. Albumin maintains blood volume by contributing to the colloid oncotic pressure and serving as a carrier protein for a large variety of hydrophobic molecules, including fat-soluble hormones, vitamins, unconjugated bilirubin, and fatty acids. The gamma globulin fraction of serum protein is largely composed of immunoglobulin (antibody) (IgG > IgA > IgM > IgD > IgE). Transferrin is the major iron transport protein, and fibrinogen is the most abundant coagulation factor. Although most plasma proteins are produced by the liver, gamma globulins are produced by B cellderived plasma cells. Serum, the residual fluid obtained from clotted blood, is essentially plasma depleted of fibrinogen and other coagulation proteins. Peripheral blood obtained by venipuncture is usually drawn into glass tubes with or without anti­ coagulant (ethylenediaminetetraacetic acid [EDTA], citrate, or heparin). Blood drawn into tubes without anticoagulant forms a clot, leaving residual serum, the preferred substrate for most clinical laboratory tests. Blood drawn into tubes with anticoagulant does not clot, and provides a source of whole blood for lab tests such as complete blood count, erythrocyte

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sedimentation rate, and Coombs test; plasma (following high-speed centrifugation to remove all cellular elements) for coagulation tests; and platelet-rich plasma (following low-speed centrifugation to remove red cells and leukocytes) for platelet function testing. Blood volume is largely controlled by the kidneys in response to changes in renal arterial pressure. Specialized smooth muscle cells in the walls of the renal afferent arterioles termed juxtaglomerular cells secrete the enzyme renin in response to decreased arterial pressure. Renin converts plasma angiotensinogen to angiotensin I, which is rapidly converted to angiotensin II by pulmonary endothelial cells. Angiotensin II increases arterial pressure by inducing systemic arteriolar vasoconstriction and inhibiting salt and water excretion by the kidneys. Blood osmolarity is indirectly controlled by hypothalamic osmoreceptors that induce secretion of antidiuretic hormone (ADH, also known as vasopressin) from specialized neurons of the posterior hypothalamus in response to hyperosmolar (concentrated) extracellular fluid. ADH rapidly increases water reabsorption by the renal collecting ducts, thus increasing plasma volume and decreasing osmolarity. ADH secretion is also stimulated by decreased plasma volume, detected by atrial stretch receptors, and carotid, aortic, and pulmonary artery baroreceptors. Blood cell formation (hematopoiesis) begins in the embryonic yolk sac and later migrates to the liver and spleen in the developing fetus. By birth, hematopoiesis has moved to the bone marrow, a fatty, highly vascular tissue found in the spaces within spongy bone in the interior of the central skeleton. The marrow contains rare hematopoietic stem cells, their numerous progeny, and supportive stromal cells. Stromal cells include endothelial cells that line the vascular spaces, adventitial reticular cells that lay down 1

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

the extracellular reticulin fiber scaffolding of the marrow, adipocytes (fat storage cells), macrophages that serve both as scavengers and as iron-rich nurse cells for developing erythroid precursors, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells). Hematopoietic stem cells are pluripotential, and capable of proliferation and differentiation into erythroid, myelomonocytic, lymphoid, megakaryocytic, and stromal cell lineages. Proliferation, differentiation, and maturation of the various cell lineages occurs in response to small glycoproteins produced by a variety of cells and tissues termed hematopoietic growth factors (cytokines) that bind to cytokine receptorbearing marrow cells. The hematopoietic cytokines include stem cell factor, interleukin (IL) 3, IL-5, IL-7, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), and thrombopoietin (TPO). The most abundant blood cell (~5 million/µl of blood), is the red blood cell (RBC) or erythrocyte. Red blood cells (RBC) contain the cytoplasmic oxygenbinding protein hemoglobin that is responsible for oxygen delivery to the tissues. RBC production in the marrow is regulated by EPO, the renal synthesis of which is stimulated by low oxygen tension. The normal function of red blood cells depends upon the oxygencarrying capacity of heme, an iron-containing tetrapyrrole contained within the hemoglobin molecule. Dietary iron is absorbed by the duodenum and transferred to the marrow by the iron-binding protein transferrin. The second most abundant (~300,000/µl of blood), and smallest, blood cell is the platelet. Platelets are produced by cytoplasmic budding of marrow megakaryocytes. Platelets express receptors for adhesion to regions of vascular damage and, once activated, serve as the nidus for clot formation. Far less numerous but no less important are the leukocytes, or white blood cells (~5000/µl of blood). Unlike RBCs and platelets, most leukocytes spend only a small fraction of their lives in the blood, instead remaining in reserve within the marrow or spleen, or rapidly entering other tissues to mature and perform their allotted functions. Leukocytes are primed to respond to all variety of noxious materials, including infectious agents, foreign proteins (allergens), and cell debris. There are five leu-

kocyte types, each with a particular function: the neutrophil, the lymphocyte, the monocyte, the eosinophil, and the basophil, in that order of abundance in blood. One marrow leukocyte type, the mast cell, does not normally circulate in the blood. Mast cells are found in perivascular tissues, where they help maintain vascular integrity by producing heparin. Granulocytes, defined as those blood leukocytes with prominent cytoplasmic granulation (neutrophils, eosinophils, and basophils), enter the blood from marrow fully mature and ready for action. Granulocytes rapidly enter inflamed tissues and contribute to the inflammatory response to infection, trauma, and allergic reactions by phagocytosis and granule release. In contrast to the granulocytes, lymphocytes leave the marrow in an immature state. Immature T cells (precursor T lymphoblasts) released from the marrow rapidly migrate to the thymus, where they undergo selection and maturation into one of several T cell subsets—CD4+ T-helper cells, CD4+ suppressor T cells, CD8+ cytotoxic T cells, CD4/CD8− gammadelta T cells, etc.)—before migrating to lymphoid tissues as mature antigen-specific T cells. Unlike immature T cells, immature B cells that leave the marrow are antigen specific but naïve, never having responded to antigen in peripheral lymphoid tissues. These naïve B cells rapidly migrate from marrow to lymph nodes and spleen where, with help from follicular T-helper cells, they undergo antigendriven proliferation and affinity maturation in germinal centers. Following germinal center maturation, B cells differentiate into either antibody-secreting plasma cells or long-lived memory B cells. Similar to lymphocytes, monocytes leave the marrow as immature cells and rapidly enter peripheral tissues, where they undergo cytokine-mediated maturation into a variety of highly specialized phagocytic macrophages and antigen-presenting dendritic cells. Megakaryocytes are large cells with polyploid nuclei that normally reside only in the bone marrow. In response to the liver-derived cytokine TPO, megakaryocytes release small cytoplasmic fragments known as platelets into the circulation. Platelets maintain vascular integrity by binding to sites of vascular damage and, in conjunction with plasma coagulation factors, minimizing blood loss by forming blood clots.



BRIEF OVERVIEW OF THE HEMATOLYMPHOID SYSTEM

Blood coagulation is usually triggered by adhesion of platelets to subendothelial collagen and von Willebrand factor exposed by vessel wall damage, and release of tissue factor from damaged endothelial cells. Tissue factor release triggers a cascading series of enzymatic reactions (involving plasma coagulation factors produced mostly by the liver) that take place on the surface of activated platelets and endothelial cells, ultimately leading to fibrin deposition and clot formation. The mature clot is a platelet-fibrin meshwork that adheres to the site of vascular damage. Following vascular repair, enzymatic digestion of the clot by the thrombin-activated fibrinolytic protease plasmin leads to clot resorption.

KEY WORDS AND CONCEPTS

B cells Blood coagulation j Blood osmolarity j Blood volume j Bone marrow j Dendritic cells j Granulocytes j Hematopoiesis j Hematopoietic growth factors j Hematopoietic stem cells j Leukocytes j Macrophages j Megakaryocytes j Monocytes j Plasma j Platelet j Red blood cell (RBC) j Serum j Stromal cells j T cells j j

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K E Y 

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HEMATOPOIESIS n

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P O I N T S

Hematopoiesis begins in the embryonal yolk sac and aorto-gonado-mesonephros (AGM), migrates to the fetal liver and spleen, and then moves to the bone marrow by birth

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The non-hematopoietic marrow stroma provides a microenvironment conducive to growth and differentiation of hematopoietic cells

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Hematopoietic marrow stem cells are small undifferentiated cells capable of both self-renewal and pluripotential differentiation

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Growth and differentiating signals to hematopoietic cells are provided by both small protein cytokines released by stromal cells and by direct physical contact with marrow stroma

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Pluripotential stem cells differentiate into common myeloid progenitors (CMP) and common lymphoid progenitors (CLP) under the influence of specific cytokines

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Myeloid stem cells differentiate into seven lineages: erythroid, neutrophilic, eosinophilic, basophilic, monocytic, megakaryocytic, and mast cell

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luripotential hematopoietic stem cells first develop from endothelial cells within the embryonic yolk sac, where they form erythroid blood islands. Embryonic red cells are large, nucleated cells that contain embryonic hemoglobins Gower 1, Gower 2, and Portland. Embryonic erythropoiesis in the yolk sac is followed by the appearance of nonerythroid progenitors in both the yolk sac and the

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Lymphoid stem cells differentiate into three distinct lineages: B cell, T cell, and natural killer (NK) cell

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Hematopoietic cells released from the marrow into the blood include erythrocytes, neutrophils, eosinophils, basophils, monocytes, platelets, immature B cells, immature T cells, and NK cells

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Mast cells do not circulate in the blood

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Immature T and B cells rapidly migrate from the blood to extramedullary sites to complete the maturation process (T cell to the thymus, B cells to lymph nodes and spleen)

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Blood leukocytes are recruited to sites of inflammation by specific chemoattractant cytokines, known as chemokines, produced by a range of cell types

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Blood leukocytes enter peripheral tissues by binding to activated endothelial cells and migrating through the vascular wall in a process termed diapedesis

aorto-gonado-mesonephros (AGM) region of the embryo. Progenitor cells from the yolk sac and AGM colonize the hepatic cords of the fetal liver and later the red pulp of the spleen. Erythropoiesis predominates in the fetal liver and is associated with a switch from production of embryonic hemoglobin to fetal hemoglobin (hemoglobin F). The higher oxygen affinity of the embryonic hemoglobins and hemoglobin F 5

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

as compared with adult hemoglobin A likely facilitates oxygen transport in the placenta from maternal blood to fetal blood. Vascularization of intraosseous cartilage leads to formation of a well-vascularized cavity in the bones, known as the bone marrow. By week 20, hema­ topoietic cells from the fetal liver and spleen have migrated to the marrow. By birth, virtually all hematopoiesis takes place within the marrow, and hemoglobin F is steadily replaced by adult hemo­ globins A and A2. In young children, marrow production is found throughout the entire marrow space, including the long bones. In adults, marrow production is limited to the marrow space of the central axial skeleton, with the marrow space in peripheral bones of the extremities occupied primarily by fatty tissue. The marrow contains many non-hematopoietic cells and a specialized extracellular matrix that is collectively referred to as the marrow stroma. The stroma provides an environment conducive to stem cell growth and differentiation. Stromal cells include endothelial cells, adventitial cells, adipocytes, osteoblasts, osteoclasts, mast cells, and macrophages. The stromal extracellular matrix provides a physical site for binding of marrow stem cells. Stromal cells produce many of the growth factors required for marrow cell growth, including stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (Flt-3 ligand), interleukin 6 (IL-6) interleukin 11 (IL-11), granulocyte colony stimulating factor (G-CSF) and monocyte colony stimu­ lating factor (M-CSF). Growth factors produced by non-stromal cells include IL-1 (by monocytes and granulocytes); IL-3, IL-5, and granulocyte-monocyte colony stimulating factor (GM-CSF) (by T cells); erythropoietin (EPO) (by renal peritubular cells); and thrombopoietin (TPO) (by hepatocytes). These growth factors seldom act individually, instead acting synergistically to induce marrow cell growth and differentiation. Many growth factors bind to membrane receptors with inducible tyrosine kinase activity that trigger cell proliferation, activation, and differentiation. An example is SCF receptor (CD117, c-kit), which is expressed by all hematopoietic stem cells. Many of these growth factors act not only to stimulate proliferation but also to inhibit programmed cell death (apoptosis).

Not all cytokines act on marrow cells to promote growth and differentiation. Instead, some cytokines inhibit hematopoiesis. Examples of inhibitory cytokines include IL-1, tumor necrosis factor (TNF), transforming growth factor beta (TGFβ), and interferon gamma (IFNγ). These pro-inflammatory cytokines contribute to the marrow suppression seen in some chronic inflammatory conditions. Pathologic conditions caused by imbalances in hematopoietic cytokine production include anemia of renal failure due to EPO deficiency, anemia of chronic disease due to IL-1 and hepcidin excess, aplastic anemia due to IFNγ excess, and thrombocytopenia of hepatic failure due to TPO deficiency. Stem cells undergo a hierarchical process of stepwise differentiation from undifferentiated multipotential cells to differentiated unipotential cells. This process is largely controlled by differential binding of exogenous growth factors (secreted by numerous cell types, including marrow stromal cells, T cells, renal tubular cells, and hepatocytes) to growth factor receptors expressed by hematopoietic progenitor cells. Binding of growth factor ligands to cellular growth factor receptors leads to expression of nuclear transcription factors that activate lineage-specific gene expression. In addition to differentiation, growth factors (cytokines) acting at early stages of marrow cell differentiation induce cell proliferation. Thus, in general, progressive maturation is accompanied by a progressive increase in cell number. Stem cells under the influence of growth factors SCF and TPO differentiate into common myeloid progenitors (CMP) via the transcription factor Hox, while stem cells under the influence of IL-7 differentiate into common lymphoid progenitors (CLP) via the transcription factor Ikaros. CMP respond to cytokines GM-CSF and G-CSF, GM-CSF and M-CSF, SCF, or IL-5, by further differentiating into granulocytes, monocytes, mast cells, or eosinophils, respectively. Under the influence of cytokines IL-3 and TPO, CMP undergo differentiation into bilineal erythroid-megakaryocyte precursors that further differentiate into unilineal erythroid or megakaryocyte precursors induced by EPO or TPO, respectively. CLP respond to cytokines IL-2, IL-4, or IL-15 (among other factors), with further differentiation into T cell, B cell, or natural killer (NK) cell precursors, respectively (Figures 1-1 and 1-2).



Hematopoiesis

7

PSC SCF TPO

IL-7 ikaros

hox

CLP

CMP GM-CSF

SCF

baso/ mast

G-CSF N

GM

c-ebpa

IL-4 pax5, aml1

c-ebpa aml1 myb M-CSF egr1, myb

egr1 myb IL-5 c-ebpe Eo

IL-3 TPO

gata1 fog

B

IL-15 id2 NK

IL-2 ikaros, aml1 T

M E/MK EPO gata1 E

TPO fli1, aml1 MK

• Growth factor in caps • Transcription factor in lower case FIGURE 1-1   Hematopoietic cytokines and transcription factors.

Hematopoietic stem cells are rare cells with the ability to self-renew and give rise to multilineage and unilineage progenitor cells. Multilineage progenitor cells, such as colony forming unit–granulocyticerythroid-monocytic-megakaryocytic cells (CFUGEMM), can give rise to more than one type of lineage-committed precursor cell, whereas unilineage progenitor cells, such as colony forming unit–erythroid (CFU-E) cells, give rise to only one type of precursor cell. Stem cells and progenitor cells are primitive undifferentiated cells that display no identifiable morphologic features. Stem cells express specific cell surface proteins, including CD34, which mediates adhesion to marrow stroma; CD117 (c-kit), the SCF receptor that induces stem cell proliferation when bound by SCF (kit ligand, produced by endothelial cells); CD133, which induces development of cell

PSC . . Pluripotential stem cell CMP. . Common myeloid progenitor CLP . . Common lymphoid progenitor E/MK . Erythroid/megakaryocytic precursor GM. . . Granulocyte monocyte precursor N . . . . Neutrophil Eo . . . Eosinophil M . . . . Monocyte E . . . . Erythrocyte MK . . . Megakaryotype B . . . . B lymphocyte T . . . . T lymphocyte NK . . . NK lymphocyte

membrane protrusions; and c-mpl, the TPO receptor that promotes stem cell growth. In contrast, precursor cells display lineage-specific morphologic and phenotypic features. For example, erythroid precursor cells contain hemoglobin-rich cytoplasm, myeloid precursor cells contain myeloperoxidase (MPO)-positive cytoplasmic granules, and mega­karyocyte precursors display enlarged hyperlobated nuclei and cytoplasmic buds. Precursor cells also express lineage-specific molecules that can be exploited as phenotype markers when detected with monoclonal antibodies by flow cytometry, immunohistochemistry, or cytochemistry. Examples include glycophorin A, CD71 (transferrin receptor), and hemoglobin for erythroid precursors; CD13, CD33, MPO, and alpha naphthyl acetate esterase (ANAE) for myeloid precursors; CD41, CD61, and von Willebrand factor (VWF,

8

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH CTL Pre–T cell

CD3

Medullary thymocyte αβ

IL-7 αβ

Cortical thymocyte

CD7

CD4 γδ

KIR

Tγδ

Pre-DC

Plasmacytoid dendritic cell CD123

CD4

Pro–B cell Pre–B cell IL-7 CD19

Immature B cell

Naive B cell

IgM

Myeloblast CFU-G

Flt-3L

CD138 Plasma cell

Eosinophil

IL-5

Monocyte derived dendritic cell

Basophil

IL-3 SCF Mast cell

Myelomonoblast CFU-GM G-CSF

CD14 GM-CSF IL-4

M-CSF Monoblast

Promonocyte

Monocyte

Microglia Osteoclast

CD33 CFU-M

GM-CSF

CD14 Myeloid DC precursor Myeloid stem cell

IL-6

IgD

IL-8

IgG CD20

IgM

Neutrophil

SCF

B cell IL-6

node

Pre-IgM

Pluripotent stem cell IL-3

CD13

Th2

Th1

Pre-NK cell IL-12 NK cell IL-15 Lymphoid stem cell

Ts

CD8

BFU-Meg

CFU-Meg

Macrophage CD163 M-CSF CD11c

Megakaryoblast Megakaryocyte

Platelet

IL-6 TPO

CD41 EPO

CD61

BFU-E GlycoA

CFU-E

Basophilic erythroblast

TFR Hemoglobin A FIGURE 1-2  Hematopoiesis.

Normoblast

RBC

Langerhans cell/ interstitial dendritic cell



Hematopoiesis Arteriole

Adipocyte

Osteoblasts

9

Iron-rich macrophage

Erythroid colony Venule

Osteoclasts

Sinus

Trabecular bone

Larger immature myeloid cells

Adipocyte

structure.

Smaller mature myeloid cells

Megakaryocyte

Sinus endothelial cell

FIGURE 1-3    Bone marrow

Myeloid colony

factor VIII-related antigen) for megakaryocyte precursors; cytoplasmic CD3 (cCD3), CD7, and terminal deoxynucleotidyl transferase (TdT) for T cell precursors; CD19, paired box (PAX) protein 5 (PAX-5), cCD22, and TdT for B cell precursors; CD14, CD68, CD163, and alpha naphthyl butyrate esterase (ANBE) for monocyte precursors; and CD117 and mast cell tryptase for mast cell precursors. The normal bone marrow contains both stromal and hematopoietic elements (Figure 1-3). Bone marrow cellularity can be determined by examination of a bone marrow biopsy and aspirate. Bone marrow cellularity is calculated as the ratio of hematocellular marrow volume to fatty marrow volume. Normal iliac crest marrow cellularity in a newborn is 90%, with a steady reduction to 30%-40% in the elderly. The normal marrow is populated by myeloid and erythroid cells in approximately a 3 : 1 ratio (Image 1-1). Most myeloid cells are neutrophilic, with scattered eosinophils, basophils, and mast cells (Image 1-2). In the normal marrow most neutrophilic cells are mature (band neutrophils and segmented neutrophils) with lesser numbers of myelocytes and promyelocytes. Myeloblasts (and stem cells) are rare cells in the normal marrow, accounting for no more than 1%-3% of the marrow cell count. Lymphocytes account for about 10%-15% of the

marrow cellularity in adults, while in young children they may account for up to 50%. Monocytes and promonocytes account for 2%-3% of the marrow cellularity. Relatively few megakaryocytes (approximately 0.1%) are scattered throughout the normal marrow, often in proximity to vascular structures. Under normal circumstances only fully mature (enucleated) erythroid cells and myeloid cells are released into the bloodstream from the bone marrow (Image 1-3). Under stress conditions (infection, inflammation, blood loss, trauma, etc.) less mature cells are released into the blood. For example, acute bacterial infection leads to release of immature myeloid cells (band neutrophils, metamyelocytes, and myelocytes) and blood loss leads to release of reticulocytes and nucleated red cells. While mature erythrocytes remain in the bloodstream, myeloid cells (neutrophils, eosinophils, and basophils) and monocytes are recruited to inflamed tissues under the influence of a closely related group of chemoattractant cytokines known as chemokines. Chemokines are produced by a range of cell types, including endothelial cells, macrophages, T cells, fibroblasts, keratinocytes, and stromal cells. Under normal conditions, neutrophils migrate from the blood to bronchial and intestinal submucosa, where they serve as first responders to infection. Blood

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

neutrophils also rapidly migrate to localized sites of acute infection or injury. Intravascular neutrophils reside in two freely exchangeable pools: the circulating pool and the marginal pool. At any time, most intravascular neutrophils are not circulating and are instead marginating along capillary and venular walls (the marginal pool). In response to infection or inflammation, cells within the marginal pool rapidly enter the circulating pool. The total intravascular granulocyte pool (circulating and marginal) is supported by the marrow granulocyte reserve. This reserve, primarily composed of mature myeloid cells, is approximately 20 times larger than the blood granulocyte pool and capable of rapidly repleting the blood granulocyte pool in the face of infection or inflammation. The rapid migration of blood neutrophils into sites of inflammation is mediated by the chemokine IL-8 (CXCL-8) produced by activated macrophages. Eosinophils and basophils in blood migrate to and reside within the submucosa of the aerodigestive tract (Image 1-4) and, like neutrophils, enter other tissues in response to chemokines released, in turn, in response to infection or inflammation. Eosinophils are recruited to inflamed tissues by IL-5, eotaxin, and chemokine ligand 5 (CCL-5), while basophils are recruited by CCL-2 and CCL-5. Mast cells, unlike basophils, are not typically found in peripheral blood, instead homing to perivascular sites within a variety of connective tissues, including marrow stroma. But mast cells, like basophils, participate in allergic responses with release of the vasoactive factor histamine. Many blood monocytes, like neutrophils, reside in the marginal pool and rapidly enter tissues to undergo further differentiation into several specialized cell types of the mononuclear phagocyte system, including histiocytes (macrophages), dendritic cells, osteoclasts, and microglial cells. Blood monocyte-derived macrophages are particularly numerous in organs such as liver, spleen, lymph node, and lung that capture and process antigen (Image 1-5). To replenish macrophages in inflamed tissues, blood monocytes are recruited to areas of inflammation by the chemokine CCL-2 (also known as macrophage chemoattractant protein-1). Megakaryocytes remain in the marrow in the vicinity of vascular sinuses, producing platelets by cytoplasmic budding and releasing them directly into the bloodstream (Image 1-6).

In response to the chemokine IL-13 (CXCL-13), naïve immunoglobulin M (IgM) and/or IgD positive B cells enter peripheral lymphoid tissues via high endothelial venules and home in on lymphoid follicles in lymph nodes and spleen to await antigendriven germinal center maturation (Image 1-7). Naïve CD7+, CD3− T cells home to the thymic cortex to begin the complex process of T cell maturation (Image 1-8). NK cells are released into the bloodstream as fully mature and functional cells, homing primarily to lymphoid tissues and submucosal sites. Circulating NK cells also enter sites of inflammation in response to the cytokine IL-12 produced by activated macrophages.

TECHNICAL CONSIDERATIONS Bone marrow biopsy and aspiration is most often obtained from the posterior iliac crest. The cylindrical core biopsy is fixed in formalin and decalcified to allow for thin sectioning of the bony tissue. Typically, the biopsy sections are stained with hematoxylin and eosin (H&E) (Image 1-9). The aspirate, a liquid suspension of marrow cells, is smeared onto a glass slide or coverslip and stained with a Wright-Giemsa stain (Image 1-10). This stain renders nuclei and basophil or mast cell granules blue and hemoglobin and eosinophil granules red. The marrow aspirate is particularly useful for enumerating individual marrow cell types and detecting cytologic abnormalities such as dysplasia. As a single cell suspension, the marrow aspirate is also amenable to flow cytometry (see the next paragraph). The marrow biopsy is particularly useful for examining the in-situ architectural features of the marrow, including detection of lymphoid aggregates, granulomas, and fibrosis. In some cases, clotted marrow particles devoid of bone may be fixed and embedded for sectioning. This specimen, the clot section, is often useful as a biopsy surrogate in cases for which biopsy sections are inadequate. Flow cytometry is a technique that allows for antibody-mediated detection of specific cell types in cell suspensions. In this technique, aliquots of 106 cells in liquid suspension are incubated with fluorochromeconjugated monoclonal antibodies, rinsed to remove unbound antibody, and passed through a flow cytometry instrument. As the stream of cells passes through



Hematopoiesis

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Side scatter (SSC)

Forward scatter (FSC)

Cell

Laser Monochromatic light

Fluorochrome generated light

Granulocytes

Monocytes Lymphocytes

B

Forward scatter

Neutrophils and eosinophils Side scatter

Side scatter

A

Monocytes Basophils Lymphocytes CD45

FIGURE 1-4    A, Monochromatic laser light (ultraviolet) impacts each cell, generat-

ing several light signals. FSC light is a measure of cell size. SSC light is a measure of cell granularity. Fluorescent light emitted by fluorochrome-conjugated antibodies to cell-specific antigens is used to identify the cellular immunophenotype. Depending upon the number of fluorochrome-conjugated antibodies used, many different antigens can be simultaneously detected on each cell. B, Flow cytometry histograms of peripheral blood leukocytes. An FSC-SSC plot yields distinct clusters of lymphocytes, monocytes, and granulocytes (left). A CD45-SSC plot yields distinct clusters of lymphocytes, monocytes, granulocytes, and basophils (right).

the instrument, monochromatic laser light focused on the passing stream induces antibody-bound cells to fluoresce at specific wavelengths (Figure 1-4, A). The fluorescence is detected by a wavelength-selective detector and recorded for each cell. All cells, whether labeled or not, are identified by a light scatter detector that detects laser light deflected by each cell along the axis of the laser light beam. The intensity of the forward scatter (FSC) signal is directly proportional to

cell size. For example, as shown in Figure 1-4, B, lymphocytes are smaller cells than monocytes. Scattered light generated by each cell is also measured (at right angles to the incident laser light). The intensity of the side scatter (SSC) signal is directly proportional to the cytoplasmic complexity (i.e., cytoplasmic granularity). The FSC and SSC results for all cells are usually displayed as a dot plot histogram, with the twodimensional signal intensity of each cell displayed as a

12

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

single dot. Cells of similar type tend to form cell clusters. For example, lymphocytes, monocytes, and granulocytes nicely separate into distinct clusters when FSC and SSC results are plotted. Another commonly used technique for leukocyte display is to plot CD45 (common leukocyte antigen) expression against SSC (see Figure 1-4, B). Flow cytometry not only enumerates the relative percentages of each cell subset in a sample but also provides information regarding the relative fluorescence intensity of each cell, a measurement that is directly proportional to the antigen density expressed by each cell. Flow cytometry is applicable for samples that can be processed as single cell suspensions. Examples include blood, bone marrow, body fluids, and lymphoid tissues. Generally, given the difficulty of disaggregation of the tumor cells into a single cell suspension, most solid tumors cannot be processed for flow cytometry. Immunohistochemistry is a technique that allows for detection of specific cell types by light microscopy after staining formalin-fixed, paraffin-embedded (or fresh frozen) tissue sections with antigen-specific antibodies. Tissue sections on glass slides are first incubated with a monoclonal antibody made in a mouse (or a polyclonal affinity-purified antibody made in a rabbit or goat), rinsed to remove unbound antibody, incubated with an enzyme-conjugated secondary antimouse (or anti-rabbit or anti-goat) antibody, rinsed to remove unbound secondary antibody, and incubated with an enzyme substrate that yields an insoluble colorfast product that deposits at the site of the reaction. After counterstaining the tissue with a nonspecific cellular dye such as hematoxylin (which stains nuclei blue), the slides are examined under a light microscope to detect the labeled cells of interest (Image 1-11). Immunohistochemistry is particularly suitable for solid tissue samples that cannot be disaggregated into a single cell suspension for flow cytometry or for those tissues in which the in-situ distribution of the labeled cells is of particular interest. Marrow biopsy and clot sections can be incubated with a number of lineagespecific monoclonal antibodies for in-situ detection of glycophorin A/CD71+ erythroid cells, MPO/CD33+ myeloid cells, CD41/CD61+ megakaryocytes, CD3/ CD7+ T cells, CD20/CD79a+ B cells, CD56/CD57+ NK cells, CD14/CD68/CD163+ monocytes and macrophages, CD117/mast cell tryptase-positive mast

cells, CD34/CD117+ myeloblasts, and CD34/TdT+ lymphoblasts. Enzyme histochemistry is a colorimetric technique in which a fresh aliquot of cells in suspension is smeared (or pelleted) onto a glass slide, fixed, and incubated with an enzyme substrate that yields a colorfast product only within cells that express the desired enzyme. After counterstaining and coverslipping, the slide is examined with a microscope. A good example of enzyme histochemistry is the use of the dual esterase stain for simultaneous detection in marrow or blood of alpha naphthyl acetate esterase (ANAE)positive granulocytes (brown stain) and alpha naphthyl butyrate esterase (ANBE)-positive monocytes (blue stain) (Image 1-12). Fluorescence in-situ hybridization (FISH) is a technique that can be applied to freshly prepared smears (or pellets) of blood or marrow as well as sections of fixed tissue. The probes are small fluorescentlabeled single-stranded DNA oligonucleotides that bind to specific DNA sequences within cell nuclei. Some probes are used to detect amplification or deletion of disease-specific genes, while other probes can be used to detect disease-specific gene translocations (Image 1-13). Colorimetric in-situ hybridization is a related technique that can be applied to sections of formalin-fixed, paraffin-embedded tissue, including bone marrow biopsy and clot sections. The probes are labeled, singlestranded oligonucleotide probes designed to speci­ fically bind to nucleic acid target (RNA or DNA). Examples of probes used in hematopathology include the Epstein-Barr virus (EBV) early RNA (EBER) probe for detection of EBV infection in lymphoma (Image 1-14) and immunoglobulin light chain probes for detection of kappa and lambda light chain mRNA in plasma cells.

KEY WORDS AND CONCEPTS

  Aorto-gonado-mesonephros (AGM) region   Bone marrow j  Bone marrow biopsy and aspiration j  Chemokines j  Colorimetric in-situ hybridization j  Cytokines j j



  Embryonic yolk sac   Enzyme histochemistry j  Flow cytometry j  Fluorescence in-situ hybridization (FISH) j  Hematopoietic stem cells j  Hemoglobin Gower 1 j  Hemoglobin Gower 2 j  Hemoglobin Portland j  Hemoglobin F j  Hemoglobin A j  Hox

Hematopoiesis

  Ikaros   Immunohistochemistry j  Inhibitory cytokines j  Marginal pool j  Marrow stroma j  Multilineage progenitor cells j  Pluripotential hematopoietic stem cells j  Precursor cells j  Stem cell factor (SCF, kit ligand) j  SCF receptor (CD117, c-kit) j  Unilineage progenitor cells

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Red cell production is stimulated by EPO, a secreted glycoprotein produced by renal peritubular cells in response to hypoxia

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EPO binds to EPO receptor-bearing CFU-E cells in the marrow, inducing proliferation, maturation, and inhibition of apoptosis

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EPO deficiency in renal disease is associated with a hypoproliferative anemia

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EPO excess in patients with chronic hypoxia is associated with polycythemia

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Following enucleation, mature red cells are released into the circulation, with a half-life of 120 days

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Red cells newly released into the blood retain rough endoplasmic reticulum and are termed reticulocytes

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he rate of erythropoiesis is primarily controlled by the positive growth effect of the glycoprotein hormone erythropoietin (EPO) on erythroid progenitors in the bone marrow. EPO is produced in the fetus primarily by hepatocytes, while after birth, it is primarily produced by peritubular fibroblasts in the renal cortex. EPO production is stimulated by tissue hypoxia. The key regulator of EPO synthesis is hypoxiainducible factor 1 (HIF-1), a transcription factor that binds to hypoxia response elements not only in the EPO gene but also in genes involved in glucose metabolism, including glucose transporters and glycolytic enzymes. Plasma EPO levels rapidly increase (within 1 hour) after initiation of hypoxia, and peaks within 1-2 days. Marrow stem cells under the influence of a number of hematopoietic growth factors give rise to the earliest

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Under normal conditions, reticulocytes account for approximately 1% of all circulating RBCs

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Anemia due to marrow failure is characterized by an inappropriately low reticulocyte count, whereas anemia due to red cell destruction or loss is characterized by a high reticulocyte count.

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Anemia-induced hypoxia (assuming normal marrow function) stimulates increased EPO and consequent erythroid hyperplasia (marrow) and reticulocytosis (blood)

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Hypoxia-induced lactic acidosis and increased 2,3bisphosphoglycerate (2,3-BPG) production lead to a reduction in hemoglobin oxygen affinity and enhanced release of oxygen to hypoxic peripheral tissues

committed erythroid progenitors, the erythroid burst forming unit (BFU-E) cells, which give rise to CFU-E cells. CFU-E cells, which express the highest density of cell surface EPO receptors, are highly sensitive to the positive influence of EPO. EPO binding to a dimerized pair of EPO receptor molecules induces a variety of cytoplasmic signal transduction pathways that ultimately lead to the prevention of apoptosis of CFU-E cells. Under normal conditions, relatively few CFU-E cells survive to form mature red cells. In contrast, under hypoxic conditions, increased EPO levels allow for an increased number of CFU-E cells to survive, with each CFU-E capable of giving rise to 8-64 immature red cells. EPO further contributes to erythropoiesis by directly stimulating proliferation and maturation of pro­erythroblasts and normoblasts. 15

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Following enucleation, mature red cells are released into the circulation. A burst in EPO-induced erythropoiesis is followed 3-4 days later by an increased number of reticulocytes, immature red cells with a high content of rough endoplasmic reticulum, in peripheral blood. Given the normal red cell half-life of 120 days, approximately 1% of red cells are normally replaced each day with reticulocytes. Thus, under normal circumstances, reticulocytes account for approximately 1% of all red cells. In patients with anemia and a normal functional marrow reserve, hypoxia induces EPO production with consequent erythroid hyperplasia in the marrow, reticulocytosis in the blood, and eventual normalization of the red cell count, hemoglobin, and hematocrit.

OXYGEN TRANSPORT The average resting adult human consumes about 250 ml of oxygen per minute to fuel aerobic metabolism. The oxygen carrying capacity of red cells in blood is 200 ml/L, and normal cardiac output is 5 L/min. Thus, the total circulating red cell mass can deliver 1 L of oxygen per minute to tissues. At rest, extraction of only 25% of the available blood oxygen by peripheral tissues provides the necessary delivery rate of 250 ml of oxygen per minute. In contrast, exercise requires greater consumption of oxygen, a need that can be met by a combination of increased cardiac output and increased blood oxygen extraction rate. If the need for additional oxygen is not met, hypoxia develops. Tissue hypoxia develops as a consequence of inadequate oxygen delivery in a variety of settings including extreme physical activity, chronic anemia, decreased cardiac output, blood loss, or lung disease. Hypoxia leads to the rapid accumulation of lactic acid in tissues (lactic acidosis) due to the inability to efficiently operate the citric acid cycle. The lactic acidosis triggers an increased respiratory rate, leading to a compensatory respiratory alkalosis. Acidosis and increased production of red cell 2,3-bisphosphoglycerate (2,3-BPG) lead to a shift to the left of the oxygen dissociation curve, i.e., to decreased hemoglobin oxygen affinity. Decreased oxygen affinity allows for more rapid release of oxygen from red cells to hypoxic tissues. In acute (blood loss) anemia, vascular changes lead to shunting of blood to the gut, thus avoiding acute intestinal ischemia. On the other hand, in chronic anemia, blood is shunted to the skin and kidneys. Decreased oxygen in

blood triggers renal peritubular cells to increase their production of EPO some 1000-fold. EPO binds to and stimulates the growth and maturation of EPO receptorpositive erythroid precursors in the marrow, ultimately leading to increased production of red blood cells. EPO deficiency, caused both by loss of EPOproducing renal cells and inhibition of EPO synthesis by uremia-associated metabolic factors, is responsible for the anemia commonly seen in patients with chronic renal failure. EPO excess caused by chronic hypoxia from cyanotic heart disease, pulmonary insufficiency, or prolonged residence at high altitude leads to an increased number of red blood cells (polycythemia). This secondary form of polycythemia contrasts with primary polycythemia (polycythemia vera), a neoplastic myeloproliferative disease. The symptoms of polycythemia (headache, pruritus, plethora, paresthesia, and thrombosis) are due to a combination of increased blood viscosity, red cell mass, and blood volume. Normal erythropoiesis also depends upon an adequate dietary supply of iron, folic acid, and cobalamin (the active form of vitamin B12). Iron is necessary for a variety of cellular functions, including heme synthesis. Folic acid and cobalamin are necessary for normal DNA synthesis, and their deficiency leads to impaired proliferation of rapidly growing tissues, including bone marrow. Iron deficiency leads to an anemia due to the production of small (microcytic), hemoglobinpoor (hypochromic) red cells with reduced half-life. Deficiency of folic acid or cobalamin leads to anemia due to diminished production of abnormally enlarged (macrocytic) red cells with reduced half-life. KEY WORDS AND CONCEPTS

  2,3-bisphosphoglycerate (2,3-BPG)   BFU-E j  CFU-E j  Cobalamin j  EPO deficiency j  EPO excess j  Erythropoietin (EPO) j  Folic acid j  Hypoxia j  Hypoxia-inducible factor 1 (HIF-1) j  Iron j  Lactic acidosis j  Reticulocytes j j

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Dietary iron absorbed by the duodenum is transported to the bone marrow bound to transferrin and released to transferrin receptor-bearing erythroid cells

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Hereditary hemochromatosis is an iron-overload condition due to HFE gene mutations that lead to excess iron absorption by the duodenum

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Normal hemoglobin oxygen affinity primarily depends upon temperature, pH, and 2,3-bisphosphoglycerate (2,3-BPG) concentration

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Some abnormal hemoglobins manifest abnormal oxygen affinity, high-affinity hemoglobins presenting with polycythemia and low-affinity hemoglobins presenting with cyanosis

Heme is an iron-containing tetrapyrolle (protoporphyrin) that is synthesized in the mitochondria of developing erythroid cells

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Hemoglobin released from aged or damaged red cells is digested by hepatosplenic macrophages to yield amino acids (from globin), bilirubin (from heme), and iron

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Defective heme synthesis can lead to toxic accumulation of porphyrin intermediates (disease: porphyria) or ineffective erythropoiesis (disease: sideroblastic anemia)

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Unconjugated albumin-bound bilirubin is delivered to hepatocytes, where it is conjugated to glucuronic acid and excreted into the bile and urine

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Each molecule of hemoglobin is composed of a complex of four globin chains (two alpha and two non-alpha) and four heme molecules

An iron-poor diet or excessive bleeding (with iron loss) leads to inadequate hemoglobin production (anemia of iron deficiency)

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Each molecule of heme can reversibly bind one molecule of oxygen; thus, each molecule of hemoglobin can bind four molecules of oxygen

Chronic inflammation-mediated hepcidin excess coupled with inadequate EPO production leads to reduced erythropoiesis and inadequate delivery of iron to erythroid precursors (anemia of chronic disease)

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he human body contains about 3-4 g of iron, of which 70%-80% is complexed to heme, the oxygenbinding prosthetic group of hemoglobin and myoglobin. While iron plays a role in many enzymatic reactions, most non-heme iron is stored in the liver and marrow as ferritin and hemosiderin. A small amount (1/1000th) of total body iron circulates in blood plasma, mostly complexed to the iron transport

protein transferrin. Dietary iron absorption amounts to only 1-2 mg/day, just enough to balance the small physiologic iron loss from sloughed mucosal cells, desquamated epithelial cells, and menstrual bleeding. Iron and heme absorption occur at the apical membrane of duodenal and proximal jejunal enterocytes (Figure 3-1). Inorganic dietary iron, often in the poorly soluble ferric (Fe3+) form, is reduced in the gut lumen 17

18

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH Gut Cytochrome B lumen Fe3+ Fe2+

Transferrin receptor

Red blood cell

DMT-1 HFE

FIGURE 3-1    Iron transport.

–

Lysosome

+

Duodenal villous cell

Hemosiderin Fe2+

Hepcidin

Fe3+

Ferritin

–

Fe2+ Fe3+

Ferroportin

Heme

Transferrin Mitochondrion

Hephaestin Blood Transferrin

to ferrous (Fe2+) form by duodenal cytochrome b and transported across the apical membrane of enterocytes by divalent metal transporter 1 (DMT-1). Intracellular iron is transported across the enterocyte basolateral membrane by ferroportin, reoxidized to Fe3+ by membrane-bound hephaestin or plasma ceruloplasmin, and immediately bound to the major iron-binding plasma protein apotransferrin to form transferrin. Plasma transferrin binds to transferrin receptor-bearing cells, including erythroid cells, hepatocytes, and muscle cells, where it is endocytosed in clathrin-coated pits to form endosomes. Within the acidic environment of the endosome, Fe3+ is released from transferrin, reduced to Fe2+, and transported to the cytoplasm (probably by DMT-1) to be used in the synthesis of heme-containing proteins such as hemoglobin, cytochrome P450, and myoglobin. Apotransferrin and transferrin receptor recycle back to the cell surface. Apotransferrin is released into the circulation, and transferrin receptor is re-expressed on the cell surface. Iron that is not immediately used by the cell for protein synthesis or as low molecular weight chelates is stored as the iron-protein complex ferritin, the major form of storage iron. Ferritin is composed of a core of crystalline ferrihydrite enclosed within an apoferritin protein shell. Under normal circumstances, nearly all ferritin is found in the cytoplasmic compartment of cells, with a small soluble fraction in the blood. In most circumstances, the level of serum ferritin correlates closely with the level of intracellular

ferritin, thus providing a useful measure of total body iron stores. However, as an acute-phase reactant, serum ferritin is elevated in chronic inflammatory states and thus may not accurately reflect low storage iron in patients with both iron deficiency and chronic inflammation. In the marrow, most storage iron is present in macrophages as both ferritin and hemosiderin, an aggregated, iron-rich ferritin degradation product with very low apoferritin content. Approximately 20%-50% of normal marrow erythroid precursors are sideroblasts, nucleated erythroid cells that contain a few small, ferritin-rich lysosomes called siderosomes. Storage iron distribution in macrophages and sideroblasts can be visualized by staining of marrow smears or biopsy sections with the Prussian blue dye that stains ferritin granules and hemosiderin aggregates blue (Image 3-1). Even without Prussian blue iron staining, the golden-brown refractile aggregates of hemosiderin (but not ferritin) can often be seen by light microscopy (Image 3-2).

IRON DEFICIENCY ANEMIA The anemia of iron deficiency may occur as a result of an iron-deficient diet, inadequate intestinal iron absorption, chronic blood loss (menorrhagia or gastrointestinal) or intravascular hemolysis with hemoglobin loss in urine (hemoglobinuria). Iron deficiency in infants and children is most often due to an inadequate, iron-deficient diet. Cow’s milk, dairy



products, and non-supplemented infant formulas do not provide the necessary iron content. Iron deficiency in pregnant or lactating females is often due to inadequate dietary compensation for the increased iron requirements of fetal growth and milk production. Inadequate duodenal iron uptake is seen in patients with intestinal malabsorption (as in celiac disease) and the postgastrectomy state (due to achlorhydria with inadequate release of iron from organic matter). Menorrhagia in premenopausal females is the most common cause of iron deficiency. Gastrointestinal blood loss is the most common cause of iron deficiency in adult males and postmenopausal females. Lack of iron in erythroid cells reduces the rate of hemoglobin synthesis by interfering with the ratelimiting ferrochelatase step of heme synthesis. This reduction in hemoglobin synthesis leads to production of small, pale (microcytic hypochromic) hemoglobindeficient RBCs. The mildly reduced lifespan of hemoglobin-deficient red cells, uncompensated by a normal rate of production, leads to anemia, a condition marked by reductions in RBC count, hemoglobin concentration, and hematocrit (percentage of RBC volume per blood volume). In response to the hypoxia, EPO production by the kidney increases. However, since red cell production is hampered by the defect in heme synthesis, the marrow response is ineffective. This ineffective erythropoiesis is reflected by the inappropriate absence of an increase in young red cells (reticulocytes) in the peripheral blood, a situation referred to as reticulocytopenia. Clinical findings in patients with chronic iron deficiency may include pallor, fatigue, irritability, headache, glossitis, stomatitis, angular cheilitis, koilonychia, paresthesias, and restless leg syndrome. The diagnosis of iron deficiency anemia is confirmed by complete blood count (CBC) and serum iron measurements. CBC findings include anemia (low RBC, hemoglobin, and hematocrit), microcytosis (low mean corpuscular volume, MCV), hypochromia (low mean corpuscular hemoglobin concentration, MCHC), anisocytosis (high red cell distribution width, RDW), and decreased reticulocyte count. While iron deficiency itself is not typically associated with neutropenia or thrombocytopenia, patients with iron deficiency due to blood loss (bleeding) often present with thrombocytosis (increased platelet count). Peripheral smear reveals

Iron, Heme, and Hemoglobin

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small, hypochromic red cells, often accompanied by pencil-shaped elliptocytes (Image 3-3), and reduced numbers of polychromatophilic red cells (reticulocytes). Serum iron studies typically reveal decreased serum iron, increased total iron-binding capacity (largely due to transferrin), decreased percent iron (transferrin) saturation, and decreased serum ferritin. Of these tests, serum ferritin is generally considered to be the most useful because it most accurately reflects total body iron stores with the caveat that, as a positive acute-phase reactant, ferritin levels may be increased with inflammatory disorders, thus limiting its use as a marker of iron deficiency in this setting. Bone marrow examination reveals a normocellular marrow with absent stainable iron. Iron supplementation leads to a rapid regenerative erythroid hyperplasia in the marrow and a peripheral blood reticulocytosis, followed by eventual replacement of hypochromic microcytic red cells with normochromic, normocytic red cells in the peripheral blood.

Anemia may result from a variety of intrinsic red cell defects, as well as extrinsic causes (Figure 3-2). Based upon physiologic mechanism, the anemias can be broadly classified into those due to blood loss, decreased red cell production, or increased red cell destruction (Table 3-1). More simply, anemia can also be classified into two broad groups based upon the presence or absence of a compensatory reticulocyte response. Types of anemia marked by deficient erythropoiesis (and thus a low reticulocyte count) include those due to nutrient deficiencies and heme biosynthesis defects. Types of anemia marked by increased reticulocytes (reticulocytosis) include those due to blood loss and hemolysis. Under normal conditions, given the 120-day lifespan of red cells, about 1%-2% of red cells must be replaced each day. The replacements are reticulocytes, relatively large red cells newly released from the marrow that retain a loose reticulated network of rough endoplasmic reticulum that stains light blue (polychromatophilic) with the Wright stain (Image 3-4). Staining reticulocytes with RNA-binding dyes such as new methylene blue or the fluorochrome thiazole orange provides for improved accuracy in enumerating reticulocytes (Image 3-5).

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH DNA Synthesis defects • Folate deficiency • B12 deficiency

FIGURE 3-2    Etiology of anemia.

Erythropoietic defects • EPO deficiency • Aplastic anemia Hemoglobin defects • Sickle cell anemia • Hemoglobin C disease • Thalassemia • Low-affinity hemoglobin • High-affinity hemoglobin

Membrane defects • Hereditary spherocytosis • Hereditary elliptocytosis • Paroxysmal nocturnal hemoglobinemia

Nucleus

Cytoplasm

Metabolic enzyme defects • G6PD deficiency • Pyruvate kinase deficiency Heme deficiency • Iron deficiency • Porphyria Blood loss

Membrane

Extrinsic Hemolysis • Microangiopathic • Infection-mediated

HEME Heme, the oxygen-carrying prosthetic group of hemoglobin, is a Fe2+-containing tetrapyrolle that functions in electron exchange. Heme is incorporated as a prosthetic group into many proteins, including hemo­ globin, myoglobin, cytochrome P450, catalase, and peroxidase. Most heme synthesis (85%) takes place in bone marrow erythroid cells, where it is incorporated into hemoglobin; most of the remainder is produced in the liver and primarily incorporated into cytochrome P450. Steps in heme synthesis take place in both cytoplasm and mitochondria (Figure 3-3). The first step is conversion of glycine and succinyl coenzyme A to delta aminolevulinic acid by the enzyme aminolevulinic acid (ALA) synthase, with pyridoxine (vitamin B6) as a cofactor. The last (and rate-limiting) step is the addition of Fe2+ to protoporphyrin IX by the mitochondrial enzyme ferrochelatase to form heme (Figure 3-4). Enzyme defects in heme biosynthesis (excluding ALA synthase) lead to a group of disorders known collectively as the porphyrias. Most porphyrias are inherited autosomal dominant disorders. In porphyria, excess quantities of heme precursors formed by the liver, erythroid cells, or both accumulate in tissues and lead to variable degrees of cutaneous photosensitivity, abdominal pain, neuropathy, behavioral disturbances,

Mitochondrion

Glycine + succinyl CoA ALA synthase Aminolevulinic acid (ALA)

Protoporphyrinogen III

Porphobilinogen

Coproporphyrinogen III

Protoporphyrin IX Fe2+ Ferrochelatase HEME

Hemoglobin +Globin

FIGURE 3-3    Heme biosynthesis.

hemolytic anemia, and liver disease. Heme deficiency also plays a role in the neurologic dysfunction. Acute intermittent porphyria is due to mutations of the porphobilinogen deaminase gene. Only about 10% of patients who carry this mutation present with clinical disease, and attacks are often precipitated by a variety of medicinal drugs. During an acute intermittent porphyria attack, patients present with severe abdominal



Iron, Heme, and Hemoglobin

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T ABLE 3-1  Major Causes of Anemia DECREASED PRODUCTION (LOW RETICULOCYTE COUNT)

INCREASED DESTRUCTION OR LOSS (HIGH RETICULOCYTE COUNT)

Nutritional Deficiency

Blood Loss

Iron deficiency—hemoglobin production defect Folic acid deficiency—DNA production defect Cobalamin deficiency—DNA production defect

Menorrhagia Gastrointestinal bleeding Trauma

Bone Marrow Failure

Hemolytic Anemia (Intrinsic)

Fanconi anemia—autosomal recessive DNA repair defect Aplastic anemia—autoimmune or toxin mediated

Hemoglobinopathy Thalassemia (reduced alpha or beta globin synthesis) Production of mutant globin protein (hemoglobins S, C, D, E, etc.) Cell membrane defects (spectrin, ankyrin)—membrane instability Hereditary spherocytosis Hereditary elliptocytosis Enzyme deficiency—oxidative damage to cellular proteins Glucose-6-phosphate dehydrogenase deficiency Pyruvate kinase deficiency Paroxysmal nocturnal hemoglobinuria—glycosylphosphatidylinositol anchoring defect (PIG-A mutation) leading to loss of CD55 and CD57 and hypersensitivity to complement-mediated lysis

Red Cell Aplasia Diamond-Blackfan anemia—autosomal dominant Anemia of renal disease—EPO deficiency Parvovirus B19 infection Sideroblastic Anemia (Defective Heme Synthesis) Lead poisoning Ethanol toxicity Myelodysplasia Myelophthisis (Marrow Infiltration with Loss of Normal Hematopoietic Tissue) Granulomatous disease Metastatic cancer Leukemia Chronic Inflammation Anemia of chronic disease—hepcidin-mediated iron utilization defect

CH2 K

H C

CH

Hemolytic Anemia (Extrinsic) Autoimmune hemolytic anemia—immune-mediated hemolysis Microangiopathic hemolytic anemia—mechanical damage to red cells by microvascular thrombi, cardiac valve defects, thermal injury Hypersplenism—congestive and infiltrative diseases of the spleen Drug induced Non-immune (dapsone, sulfasalazine) Immune mediated (penicillin, cephalosporin, methyldopa, levodopa, quinidine)

CH3

H3C

CHKCH2 N

N CH

Fe

HC N CH2 HOOC W CH2

N C H

CH3 CH2 CH2 W COOH

FIGURE 3-4    Heme structure.

(neurovisceral) pain, motor paralysis, neuropsychiatric symptoms, or a combination of these. During an attack, clear, freshly voided urine slowly darkens when exposed to light as the porphyrinogens are oxidized to porphyrins. In chronic porphyria cutanea tarda,

porphyrins deposited in the epidermis induce bullous formation and hyperpigmentation of the skin following sun exposure. Patients with porphyria cutanea tarda may also develop cirrhosis due to deposition of porphyrins in the liver. Erythropoietic protoporphyria, due to mutations of the ferrochelatase gene, is characterized by severe cutaneous photosensitivity and chronic liver disease in later life. Some patients with erythropoietic protoporphyria also present with a mild microcytic anemia and ringed sideroblasts in the bone marrow, a process termed sideroblastic anemia. Sideroblastic anemia is due to a variety of inherited and acquired intra-mitochondrial defects in heme synthesis that lead to mitochondrial accumulation of iron within bone marrow erythroid precursors and abnormal erythroid maturation (dyserythropoiesis).

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

Iron staining of the bone marrow in sideroblastic anemia reveals numerous ringed sideroblasts, nucleated red cells with a perinuclear ring-shaped distri­ bution of iron-laden mitochondria (Image 3-6). The ringed sideroblasts fail to properly develop and undergo apoptotic death within the marrow. The hypercellular erythroid predominant marrow is due to EPO-driven dyserythropoiesis. The most common form of acquired sideroblastic anemia is idiopathic and is classified as a form of clonal myelodysplasia (refractory anemia with ringed sideroblasts), a disorder presumably due to as yet uncharacterized cytogenetic defects. Other causes of acquired sideroblastic anemia include drugs (e.g., cytotoxic chemothera­ peutics, isoniazid, and chloramphenicol), radiation therapy, chronic alcohol abuse, and lead poisoning. Lead poisoning leads to sideroblastic anemia by directly inhibiting multiple steps of heme synthesis. The abdominal pain and peripheral neuropathy seen in both lead poisoning and porphyria may be due to direct porphyrin deposition in affected tissues.

HEMOGLOBIN Hemoglobin, an oxygen-binding molecule produced only by erythroid cells, is composed of four globin chains (two alpha and two non-alpha, either beta or gamma) each of which binds one heme molecule (Figure 3-5). Since each heme molecule can reversibly bind one molecule of oxygen, each hemoglobin molecule can bind four molecules of oxygen. The three hemoglobin variants normally seen in healthy adults are hemoglobins A (α2β2), A2 (α2δ2), and F (α2γ2). Mutations affecting globin chain production lead to the thalassemia syndromes, while mutations affecting globin chain structure lead to a variety of conditions known broadly as the hemoglobinopathies. These structural mutations yield hemoglobins with poor solubility, altered oxygen affinity (high or low), and instability, all of which (except for high-affinity hemoglobins) lead to anemia. Aged and damaged red cells engulfed by splenic and hepatic macrophages are degraded within lysosomes to yield lipids, peptides, and heme. Aged (senescent) red cells that express high levels of phosphatidylserine (PS) in the outer leaflet of the plasma membrane are recognized and bound by PS receptor-positive macrophages. Normally, PS is maintained within the inner

β chain

Fe2+ Heme α chain FIGURE 3-5    Hemoglobin A structure. Note the four

globin peptide chains (two alpha and two beta), each harboring a single molecule of heme with an atom of Fe2+ and capable of binding an oxygen molecule (four molecules of oxygen bound per molecule of hemoglobin).

leaflet of the plasma membrane through the action of the adenosine triphosphate (ATP)-dependent enzyme flippase. During apoptosis, flippase is inactivated and the enzyme scramblase leads to redistribution of PS to the outer leaflet, where it is recognized by the PS receptor on macrophages. Heme is degraded within macrophages by heme oxidase to yield iron, carbon monoxide, and biliverdin. Iron is recycled back to immature erythroid cells in the marrow for heme synthesis. Carbon monoxide bound to hemoglobin as carboxyhemoglobin is eventually exhaled. Biliverdin (a green pigment) is reduced to unconjugated (fat soluble) bilirubin (a yellow pigment), transported to the liver bound to albumin, and converted to the conjugated water-soluble form, bilirubin diglucuronide. Conjugated bilirubin is then excreted in the bile, where it is converted to urobilinogen and stercobilin (a brown pigment) by colonic bacteria. Some urobilinogen is reabsorbed, converted to urobilin, and excreted in the urine, along with a small amount of bilirubin. Excess bilirubin deposited in skin and mucosa yields a yellowish discoloration termed jaundice (icterus). There is no normal physiologic mechanism for iron excretion, with minimal iron loss occurring by gastrointestinal mucosa cell loss, skin cell desquamation of skin cells, and in females, menstruation. Thus, iron balance is maintained primarily by the rate of intestinal absorption. The rate of intestinal absorption is



controlled by intestinal crypt epithelial cells that monitor the transferrin iron content of the interstitial fluid via the membrane-bound transferrin receptorHFE complex. HFE is a human leukocyte antigen (HLA) class I gene that associates in the plasma membrane with the transferrin receptor. In iron-deficient states, the low level of transferrin iron detected in the interstitial fluid by intestinal crypt cells triggers increased apical villous enterocyte expression of DMT-1 and ferroportin, leading to increased intestinal iron absorption. Also, since low serum iron leads to a reduction in hepcidin production by the liver, reduced hepcidin likely contributes to increased iron uptake by de-inhibiting ferroportin. Hemochromatosis is a family of clinical conditions associated with excessive accumulation of iron in parenchymal tissue. Classical hereditary hemochromatosis is an autosomal recessive disorder seen most often in northern Europeans that is due to mutations of the HFE gene. Only 1% of those with homozygous HFE mutations are symptomatic. HFE defects render the transferrin receptor-HFE complex on enterocytes unresponsive to high levels of interstitial fluid transferrin iron, preventing downregulation of DMT-1 and ferroportin and leading to excess iron absorption by the duodenum. Secondary (acquired) hemochromatosis may be seen in patients with a history of numerous red cell transfusions. In either form of hemochromatosis, excess iron accumulates in multiple tissues (skin, bone marrow, liver, gall bladder, pancreas, and myocardium) leading to tissue damage largely mediated by the highly reactive hydroxyl radical. Patients with hemochromatosis are at increased risk of infection, veno-occlusive disease, and liver disease. Anemia of chronic disease is a mild to moderate anemia associated with chronic inflammation or infection. This form of anemia is due to a combination of mildly decreased red cell survival and inadequate erythropoiesis. Inflammatory cytokine-mediated activation of splenic and hepatic macrophages and increased binding of antibody and complement to red cells leads to an increased rate of hemolysis. EPO production in anemia of chronic disease is blunted by inhibition of EPO synthesis by the inflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFα). Complicating the EPO production defect is a state of EPO resistance. EPO resistance

Iron, Heme, and Hemoglobin

23

is due to functional iron deficiency that prevents red cells from producing hemoglobin. The inflammatory cytokine IL-6 induces hepatic synthesis of the ironregulating hormone hepcidin. Hepcidin binding to the cell membrane iron transporter protein ferroportin leads to ferroportin degradation, blocking the release of iron by macrophages and intestinal cells and preventing the transfer of iron to red cell precursors in the marrow. While typically normochromic and normocytic, the anemia may be hypochromic, microcytic, or both hypochromic and microcytic in some cases. Serum iron and iron-binding capacity are decreased, and serum ferritin is increased. The decrease in ironbinding capacity is due to reduction in transferrin production by the liver. In contrast to blood levels of positive acute-phase reactants such as C-reactive protein, fibrinogen, and ferritin that rise in response to inflammation, blood levels of the negative acutephase reactant transferrin drop in response to inflammation. Examination of the marrow in anemia of chronic disease reveals numerous iron-laden macrophages and markedly decreased sideroblasts (nucleated red cells with particles of stainable iron) (Image 3-7).

OXYGEN AFFINITY The oxygen affinity of hemoglobin A primarily depends upon three factors—temperature, pH, and concentration of RBC 2,3-bisphosphoglycerate (2,3BPG) (also known as 2,3-diphosphoglycerate [2,3DPG]). Oxygen affinity declines with increasing temperature, decreasing pH, and increasing 2,3-BPG concentration. Oxygen affinity is usually expressed in terms of the P50—the oxygen tension (partial pressure) at which hemoglobin is half-saturated (Figure 3-6). It is important to keep in mind that the oxygen affinity and the P50 are inversely related; that is, a decrease in oxygen affinity translates to an increase in the P50. Because metabolic activity increases with increasing temperature, and tissues require more oxygen to meet these needs, it is advantageous to more rapidly deliver oxygen to the tissues with increasing temperature by reducing the oxygen affinity of hemoglobin. Similarly, a decrease in pH (acidosis) usually signifies hypoxia, a condition for which more rapid delivery of oxygen to tissues is highly advantageous. Thus, a decreasing pH triggers a decrease in hemoglobin

24

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH 100

KEY WORDS AND CONCEPTS

High oxygen affinity due to pH, temp, BPG Normal oxygen affinity

% Oxyhemoglobin

75

50 Low oxygen affinity due to pH, temp, BPG 25

0

0

25

50

75

100

Oxygen tension (mm Hg) FIGURE 3-6    Hemoglobin oxygen affinity.

oxygen affinity and more efficient delivery of oxygen to hypoxic tissues. Acidosis and hypoxia are both associated with increases in red blood cell 2,3-BPG concentration. 2,3-BPG stabilizes the structure of deoxyhemoglobin A by loosely and reversibly binding to the beta globin subunits of deoxyhemoglobin A, thereby reducing oxygen affinity. Some abnormal hemoglobins are characterized by increased or decreased oxygen affinity. Patients with high-affinity hemoglobin present with increased RBC mass (polycythemia). The reduced delivery of high-affinity hemoglobin–bound oxygen to tissues triggers the kidney to release high levels of EPO, consequently stimulating increased erythropoiesis. Low-affinity hemoglobins, on the other hand, are associated with cyanosis and mild anemia. Cyanosis is due to the rapid, inappropriate release of low-affinity hemoglobin–bound oxygen to tissues with enhanced conversion of bright red oxyhemoglobin to dark red (cyanotic) deoxyhemoglobin. The mild anemia is due to re-setting of the EPO set point to a lower hematocrit.

  Acute intermittent porphyria   Aminolevulinic acid (ALA) synthase j  Anemia of chronic disease j  Anemia of iron deficiency j  Apoferritin j  Apoptosis j  Bilirubin j  Biliverdin j  Carbon monoxide j  Ceruloplasmin j  Cytochrome P450 j  Delta aminolevulinic acid j  Divalent metal transporter 1 (DMT-1) j  Duodenal cytochrome b j  Dyserythropoiesis j  Elliptocytes j  EPO resistance j  Erythropoietic protoporphyria j  Ferrihydrite j  Ferritin j  Ferrochelatase j  Ferroportin j  Flippase j  Glycine j  Heme j  Hemochromatosis j  Hemoglobin j  Hemoglobinopathies j  Hemosiderin j  Hepcidin j  Hephaestin j  Hereditary hemochromatosis j  HFE j  High-affinity hemoglobin j  Inadequate, iron-deficient diet j  Interleukin 1 (IL-1) j  Iron j  Jaundice (icterus) j  Lead poisoning j  Low-affinity hemoglobin j  Oxygen affinity j  Phosphatidylserine (PS) j  Polychromatophilic j  Porphyria cutanea tarda j  Porphyrias j j



  Protoporphyrin IX   Pyridoxine j  Reticulocytes j  Ringed sideroblasts j  Scramblase j  Sideroblastic anemia j  Sideroblasts j  Siderosomes j  Stercobilin

Iron, Heme, and Hemoglobin

  Succinyl coenzyme A   Thalassemia syndromes j  syndromes j  Transferrin j  Transferrin receptor-HFE complex j  Tumor necrosis factor alpha (TNFα) j  Urobilin j  Urobilinogen

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Normal adult hemoglobins include hemoglobins A (97%), A2 (2%-3%), and F (95% hemoglobin A, 1%-3% hemoglobin A2, and 0%-2% hemoglobin F. Hemoglobin A is composed of two alpha and two beta globin chains, while hemoglobin A2 is composed of two alpha and two delta globin chains. Hemoglobins A and A2 bind to oxygen less avidly than hemoglobin F and thus deliver oxygen to tissues at a higher oxygen tension. Poorly controlled diabetes mellitus is characterized by increased amounts of hemoglobin A1c, a hemoglobin A variant formed by irreversible nonenzymatic glycation of the beta globin chain.

NON-FUNCTIONAL HEMOGLOBIN VARIANTS Trace amounts of non-functional hemoglobin are pre­ sent in normal blood. These include methemoglobin, 27

28

HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

carboxyhemoglobin, nitrosohemoglobin, and sulfhemoglobin. Methemoglobin, or oxidized hemoglobin, is formed from hemoglobin by oxidation of heme iron from the ferrous to the ferric form. In nearly all cases, methemoglobinemia is due to hemoglobin oxidation induced by drugs or toxins. Although methemoglobin itself does not bind oxygen reversibly, the interaction of methemoglobin with normal hemoglobin in the blood of patients with methemoglobinemia leads to increased oxygen affinity and consequent tissue hypoxia. Carboxyhemoglobin results from the binding of carbon monoxide to heme iron. Because carbon monoxide binds to hemoglobin 200 times more strongly than oxygen, exposure to very small amounts of carbon monoxide leads to a large amount of carboxyhemoglobin. Tissue hypoxia is due not only to the inability of carboxyhemoglobin to carry oxygen but also to the increased oxygen affinity of hybrid oxygen-carbon monoxide tetramers. Nitric oxide binds reversibly to hemoglobin to form nitrosohemoglobin. However, the physiologic and clinical significance of this interaction are unclear at this time. Sulfhemoglobin is formed by heme sulfation, most often by sulfur-containing drugs, leading to reduced oxygen affinity. Low hemoglobin oxygen affinity leads to more rapid oxygen delivery to tissues with reduced EPO production and mild anemia.

BETA GLOBIN MUTATIONS Nearly all common inherited disorders of hemoglobin are due to beta globin gene mutations that lead to single amino acid substitutions in the beta globin molecule. These disorders, known collectively as the beta hemoglobinopathies, are autosomal recessive conditions, with the symptomatic homozygous state often referred to as “disease” and the asymptomatic heterozygous form referred to as “trait”. Having said this, homozygous hemoglobin S (beta hemoglobin genotype SS) is best known as sickle cell anemia (rather than sickle cell disease), while heterozygous hemoglobin S (beta hemoglobin genotype AS) is known as sickle cell trait. In sickle cell anemia, hemoglobin S is the predominant hemoglobin, and no hemoglobin A is present. In sickle cell trait, hemoglobin A is the predominant hemoglobin, while hemoglobin S accounts for 35%-45% of total hemoglobin. Other common

inherited beta chain defects include hemoglobins C, D, and E. In every case, only the homozygous or mixed heterozygous condition (for example hemoglobin SC) is associated with anemia. Hemoglobin E is perhaps the most common hemoglobin variant worldwide, with the highest prevalence in Southeast Asia. Hemoglobin E disease is an asymptomatic condition associated with mild microcytosis. Hemoglobin D trait is an entirely asymptomatic condition, with the highest prevalence seen in people of northwest Indian descent. Hemoglobins S and C are poorly soluble hemoglobins that form intraerythrocytic crystalline precipitates in the deoxygenated state. Precipitated hemoglobin S forms rigid tactoids that induce red cell sickling (Image 4-1), while precipitated hemoglobin C forms discrete rhomboid structures that less significantly alter the shape of red cells (Image 4-2). In the case of sickle cell disease, the increased rigidity of sickle cells impedes their flow through small capillaries and splenic red pulp, leading to hemolysis, thrombosis, ischemia, and ultimately, end organ failure. Sudden episodes of severe bone pain due to ischemia in patients with sickle cell anemia are termed sickle cell crises. The multiple bouts of thrombosis and infarction of the spleen in patients with sickle cell disease ultimately lead to splenic atrophy and increased susceptibility to pneumococcal (Streptococcus pneumoniae) sepsis due to the loss of splenic function. Howell-Jolly bodies are discrete basophilic intraerythrocytic inclusions of DNA commonly seen on peripheral smears of patients with asplenia due to splenectomy or splenic atrophy (Image 4-3). The highest prevalence of the hemoglobin S allele is found in tropical Africa, ranging from 20%-40%. In African Americans, the frequency is about 8%. Hemoglobin S is also found in the Middle East, Greece, and India. Hemoglobin C disease, seen most often in people of West African descent, is a microcytic hypochromic anemia with numerous target cells on peripheral smear, and splenomegaly. Hemoglobin SC disease is a mild sickling disorder due to the coinheritance of a hemoglobin S allele and a hemoglobin C allele. Numerous target cells and relatively few plump sickle cells are seen on peripheral smears.



Hemoglobinopathy

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T ABLE 4-1  Thalassemia Syndromes CLINICAL VARIANT

GENOTYPE

COMPLETE BLOOD COUNT

HEMOGLOBIN VARIANTS

CLINICAL SEVERITY

Alpha Thalassemia Silent carrier Trait Hemoglobin H disease

−α/αα −α/−α − −/αα − −/−α

Normal Normal Hemoglobin H (β4)

Asymptomatic Asymptomatic—mild anemia Moderately severe anemia

Hydrops fetalis

− −/− −

Normal—mild microcytosis Microcytosis—minimal anemia Severe microcytic hypochromic anemia Severe microcytic hypochromic anemia

Hemoglobin Bart’s (γ4)

Severe anemia

Microcytosis, target cells

↑ Hemoglobin A2 Slight ↑ hemoglobin F

Asymptomatic

Severe microcytic anemia, targets, schistocytes, nucleated RBCs Microcytosis—normal

↑↑ Hemoglobin F ↓↓ Hemoglobin A

Severe anemia

↑↑ Hemoglobin F Hemoglobin A2 ↑↑ Hemoglobin F ↑ Hemoglobin Lepore

Asymptomatic

Beta Thalassemia Thalassemia minor

Thalassemia major

HPFH* Delta-beta thalassemia

β0/β β+/β β+/β+ β0/β0 β0/β+ β+/β+ δβ0/δβ0 δβ0/δβ δβ0/δβ0 δβ+/δβ+ δβ0/δβ δβ+/δβ

Microcytic hypochromic anemia, target cells

Mild-moderate anemia

*Other forms of HPFH not listed here are non-deletional, with point mutations upstream of the delta globin locus.

THALASSEMIA The thalassemias are another common group of hemoglobin defects due to autosomal co-dominant mutations or deletions in the alpha or beta globin gene region that lead to reduced synthesis of normal alpha or beta globin chains (Table 4-1). These defects lead to variable degrees of anemia due both to ineffective erythropoiesis (caused by increased red cell destruction in the marrow) and production of microcytic hypochromic red cells with a shortened life span. A tandem pair of alpha globin genes is inherited from each parent, yielding four alpha globin genes per diploid genome. In alpha thalassemia, one or more alpha globin genes are deleted. Reduced alpha globin synthesis leads to an imbalance in alpha and nonalpha (beta, gamma, delta) globin synthesis, leading to reduced hemoglobin synthesis and production of microcytic hypochromic red cells. Decreased production and shortened half-life of alpha thalassemic red cells (with consequent anemia) are due to formation

of unstable homotetramers of excess non-alpha globin chains leading to red cell destruction in the bone marrow (ineffective erythropoiesis) and spleen (extravascular hemolysis). Inheritance of four deleted alpha globin genes (genotype −−/−−) prevents production of all normal hemoglobins (hemoglobins F, A, and A2). In this situation, the major hemoglobin that forms in the fetus is hemoglobin Bart’s, a nonfunctional gamma chain homotetramer (γ4). Oxygen delivery to fetal tissues is markedly reduced, leading to a severe hypoxic condition incompatible with extrauterine life known as hydrops fetalis. Inheritance of three deleted alpha globin genes (−−/+−) leads to a moderate to severe hypochromic microcytic anemia known as hemoglobin H disease. In this condition, up to 25 hemoglobin Barts is present during infancy, and up to 30% hemoglobin H, the beta chain homotetramer (β4), is present after 1 year of age. Hemoglobin Bart’s and hemoglobin H are both highaffinity hemoglobin variants, avidly binding oxygen and releasing it only at a very low oxygen tension. As

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH

such, they do not function properly in tissue oxygenation. Both of these variant hemoglobins are easily detected by hemoglobin fractionation as differentially charged hemoglobin species. In contrast to the insoluble alpha chain tetramers formed in beta thalassemia, the gamma and beta chain tetramers (hemoglobins Bart’s and H) formed in alpha thalassemia are relatively soluble and form damaging intracellular precipitates only within aged red cells. Hemoglobin H precipitates can be identified in older red cells on peripheral blood smears only after staining with a redox dye, such as brilliant cresyl violet. Thus, in contrast to beta thalassemia, there is little destruction of developing red cells within the bone marrow in alpha thalassemia. Instead, the major causes of the anemia in alpha thalassemia are production of microcytic hypochromic red cells and extravascular (intra­ splenic) hemolysis of aged red cells. Inheritance of two deleted alpha globin genes (− −/+ + or + −/+ −) leads to the asymptomatic condition alpha thalassemia trait, characterized by pro­ duction of hypochromic microcytic red cells and target cells. Inheritance of a single deleted alpha gene (+−/++) leads to the asymptomatic condition alpha thalassemia silent carrier with no hematologic abnormalities. In beta thalassemia, reduced beta chain synthesis leads to decreased hemoglobin A production, while compensatory increased production of gamma and delta globins leads to increased levels of hemoglobins A2 and F, respectively. Excess alpha chains form insoluble alpha chain tetramers (α4) and poorly deformable red cells that are either destroyed in the bone marrow (ineffective erythropoiesis) or removed by the spleen (extravascular hemolysis). The alpha chain aggregates are not usually seen on routine blood smears but may be detected after exposure of smears to reducing agents. There are three clinical conditions—beta thalassemia major (homozygous), thalassemia intermedia, and beta thalassemia minor (heterozygous). Thalassemia major (homozygous β0/β0) is marked by severe microcytic hypochromic anemia with anisopoikilocytosis (significant variation in both size and shape) (Image 4-4). In this condition, no beta chain is produced at either allele (β0 signifying a non-functional gene) and nearly all the hemoglobin is hemoglobin F with a small fraction of hemoglobin A2. Patients with thalassemia major suffer from severe anemia requiring multiple red cell transfusions. Inadequate transfusion

therapy leads to childhood growth retardation, skeletal abnormalities, hepatosplenomegaly, hyperpigmenation, and high risk of infection. With transfusion therapy alone, by the second decade many patients develop endocrine and cardiac abnormalities due to deposition of excess iron (siderosis) in endocrine organs and heart—complications that can largely be avoided by iron chelation therapy. In contrast to thalassemia major, patients with thalassemia intermedia present with mild anemia that seldom requires transfusion therapy. In many cases, patients with homozygous β+ thalassemia (β+/β+), beta chain production is moderately reduced (β+ signifying a hypo-functional gene). Thus, patients with this condition produce less hemoglobin A, with increased amounts of hemoglobins F and A2. Thalassemia minor (the heterozygous condition) is marked by microcytosis, hypochromia, elevated RBC count (a compen­ satory mechanism), and numerous target cells (due to the reduction in red cell hemoglobin content) (Image 4-5). Coinheritance of a beta hemoglobinopathy such as sickle cell trait with thalassemia sometimes occurs. Sickle-alpha thalassemia, the sickle trait inherited with alpha thalassemia trait, results in a mild microcytic anemia, with an increased proportion of hemoglobin A and a reduced amount of hemoglobin S ( S), as well as the slightly increased hemoglobin A2, are highly characteristic of sickle cell trait (hemoglobin AS).

HbA

20%

HbS

10%

A2 F 0

0

1

2

3

4

5

HPLC: S heterozygote

KEY WORDS AND CONCEPTS

  Hemoglobin Lepore   Hemoglobin SC disease j  Hereditary persistence of fetal hemoglobin (HPFH) j  High-performance liquid chromatography (HPLC) 0 j  Homozygous δβ HPFH j  Howell-Jolly bodies j  Hydrops fetalis j  Ineffective erythropoiesis j  Isoelectric focusing j  Isopropanol solubility test j  Methemoglobin j  Nitrosohemoglobin j  Nondeletional HPFH j  Sickle cell anemia j  Sickle cell crises j  Sickle cell trait j  Sickle-alpha thalassemia j  Sickle-beta thalassemia j  Sulfhemoglobin j  Thalassemia intermedia j  Thalassemia major j  Thalassemia minor j  Thalassemias j  Unstable hemoglobin j j

  Acid elution test   Acid gel electrophoresis j  Alkaline gel electrophoresis j  Alpha chain tetramers j  Alpha thalassemia j  Alpha thalassemia silent carrier j  Alpha thalassemia trait j  Anisopoikilocytosis j  Beta thalassemia j  Carboxyhemoglobin j  Delta-beta thalassemia j  Extravascular hemolysis j  Heinz bodies j  Hemoglobin A j  Hemoglobin A1c j  Hemoglobin A2 j  Hemoglobin Bart’s j  Hemoglobin C j  Hemoglobin D j  Hemoglobin D trait j  Hemoglobin E j  Hemoglobin E disease j  Hemoglobin F (fetal hemoglobin) j  Hemoglobin H disease j j

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RBC energy is supplied entirely by the glycolytic pathway



Three auxiliary metabolic pathways branch from the glycolytic pathway: the hexose monophosphate (HMP) shunt, the 2,3-biphosphoglycerate (2,3-BPG) pathway, and the methemoglobin reductase pathway

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The HMP shunt produces glutathione, a reducing agent that protects red blood cells from oxidative damage

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Biphosphoglycerate produced by the 2,3-BPG pathway leads to decreased hemoglobin oxygen affinity and more rapid delivery of oxygen to hypoxic tissues

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The methemoglobin reductase pathway leads to greater activity of methemoglobin reductase dependent on the

F

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ollowing enucleation, RBC metabolism is maintained by mitochondria, a short supply of residual mRNA, and pre-formed enzymes. Glucose, which enters RBCs in an insulin-independent fashion, is catabolized via the anaerobic Embden-MeyerhofParnas glycolytic (EMP) pathway. Because the aerobic citric acid cycle does not function in the RBC, the end products of glycolysis (pyruvate and lactate) are released into the blood. Glycolysis yields a net energy gain of two molecules of ATP per molecule of glucose. This energy is primarily used by the red cell to maintain osmotic equilibrium and membrane integrity. Three important auxiliary metabolic pathways branch from the glycolytic pathway: the hexose monophosphate shunt, the 2,3-BPG pathway, and the methemoglobin reductase pathway (Figure 5-1).

reduced form of nicotinamide adenine dinucleotide (NADH) 

Methemoglobin reductase is an enzyme that converts non-functional methemoglobin (with ferric iron) to functional hemoglobin (with ferrous iron)

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Inherited defects in metabolic enzymes glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) lead to hemolytic anemia caused by either oxidative damage to hemoglobin (G6PD deficiency) or loss of red cell membrane integrity (PK deficiency)

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Methemoglobinemia, a cyanotic non-hemolytic condition, is most often seen as an acquired condition associated with exposure to oxidant drugs

The hexose monophosphate shunt, by converting nicotinamide adenine dinucleotide phosphate (NADP) to its reduced form, NADPH, replenishes the supply of glutathione, a sulfhydryl-containing tripeptide that serves as a protective reducing agent in red cells by inactivation of hydrogen peroxide and reduction of oxidized proteins, including hemoglobin. The first reaction in the hexose monophosphate shunt, the conversion of glucose-6-phosphate to 6-phosphogluconolactone, is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Under conditions of hypoxia or acidosis, oxygen delivery to tissues is enhanced by a reduction in hemoglobin oxygen affinity due to reversible binding of 2,3-bisphosphoglycerate (2,3-BPG) to hemoglobin. Conversion of 1,3-bisphosphoglycerate to 2,3-BPG 33

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH Blood glucose Insulin-independent uptake RBC glucose ATP Hexose monophosphate shunt NADP

ADP

10%

Glucose-6-phosphate G6PD* ATP 90% ADP

NADPH

Oxidized glutathione Glutathione

Fructose-1,6-diphosphate

Glyceraldehyde 3-phosphate NAD NADH

H2O2

H2O

1,3-Bisphosphoglycerate 2ADP 2ATP

Methemoglobin Methemoglobin reductase* Hemoglobin 2,3-Bisphosphoglycerate 2ADP

3-Phosphoglycerate

2ATP Oxyhemoglobin Deoxyhemoglobin Phosphoenol pyruvate + 2ADP O2 Pyruvate kinase* 2ATP Pyruvate NADH Lactate dehydrogenase* Lactate

NAD

FIGURE 5-1    Erythrocyte metabolism. Red cells need energy to maintain heme iron in

reduced Fe2+ form, to maintain reduced sulfhydryl groups in proteins, to preserve the osmotic gradient with ion pumps, and to prevent oxidation of membrane lipids. Asterisks mark those enzymes for which deficiency is associated with hemolytic anemia.

rather than to 3-phosphoglycerate leads to reduced hemoglobin oxygen affinity at the expense of one molecule of ATP. Methemoglobin, a nonfunctional hemoglobin variant formed by oxidation of heme iron from the ferrous to the ferric state, is normally reconverted to hemoglobin by NADH-dependent methemoglobin reductase—a complex of flavine adenine dinucleotide (FAD) and cytochrome b5. The source of the NADH (reduced NAD) is the glycolytic conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate.

RED BLOOD CELL ENZYME DEFECTS Several chronic hemolytic anemias result from inherited deficiency of red cell enzymes. By far, the most common inherited RBC metabolic disorder is deficiency of the hexose monophosphate shunt enzyme, G6PD. The gene for G6PD is located on the X chromosome; thus G6PD deficiency is a sex-linked disorder primarily affecting males. G6PD deficiency is sometimes seen in female carriers in whom a large



Red Blood Cell Metabolism and Enzyme Defects

proportion of the red cells derive from nucleated precursors expressing the mutated X chromosome. This disorder presents with episodic acute hemolysis triggered by a variety of oxidant stresses including infection, exposure to oxidant drugs, and ingestion of fava beans. The disease is most often seen in males of African and southern European descent. Infectionassociated hemolysis may be triggered by hydrogen peroxide produced by activated leukocytes. Fava beans contain substances that destroy reduced glutathione. Deficiency of G6PD prevents the conversion of oxidized glutathione to reduced glutathione, a reducing agent that converts (non-functional) oxidized hemoglobin (methemoglobin) to hemoglobin. In patients with G6PD deficiency, (homozygous or heterozygous) damaged hemoglobin rapidly accumulates to form insoluble precipitates within red cells called Heinz bodies. Heinz body-positive red cells are poorly deformable, become trapped within the splenic cords, and are destroyed in a process termed extravascular hemolysis. Bite cells, red cells with bite-like defects secondary to removal of Heinz bodies by splenic macrophages, may be seen on peripheral smears. In contrast, Heinz bodies are not visible on peripheral smears but can be identified following incubation with certain oxidant dyes, such as crystal violet (see Image 4-7). Laboratory features of hemolysis include increased indirect (unconjugated) bilirubin, decreased haptoglobin (hemoglobin carrier protein), and increased serum lactate dehydrogenase (LDH), a ubiquitous cytoplasmic enzyme released by damaged and dying cells. Unlike most cells, erythrocytes depend entirely upon glycolysis for energy production. Inherited deficiencies of glycolytic pathway enzymes lead to chronic hemolytic anemia caused by the inability to maintain energy (ATP)-dependent cell membrane integrity. Although quite rare, the most common anemia of this type results from pyruvate kinase (PK) deficiency. PK catalyzes the conversion of phosphoenolpyruvate to pyruvic acid, yielding two molecules of ATP. Thus, PK-deficient red cells are ATP deficient and unable to maintain cell membrane integrity. PK deficiency is an

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autosomal recessive disorder. Homozygous deficiency leads to a chronic non-spherocytic hemolytic anemia associated with jaundice, splenomegaly, and gallstones. The splenomegaly results from the massive sequestration and destruction of the abnormal red cells within the red pulp. The darkly pigmented gallstones are composed of insoluble calcium salts of unconjugated bilirubin derived from the chronic hemolysis. In contrast to the acute episodic hemolysis seen with G6PD deficiency, chronic hemolysis is seen in PK deficiency. Also, unlike G6PD deficiency, methemoglobinemia and Heinz body formation are not typical of PK deficiency. Methemoglobin, a brown, non-functional hemoglobin variant with heme iron in the oxidized (ferric) state, is normally reduced to hemoglobin by methemoglobin reductase (a complex of FAD and cytochrome b5). Methemoglobinemia is a cyanotic (non-hemolytic) condition with a variety of causes, including methemoglobin reductase deficiency, hemoglobin M (a hemoglobin variant in which heme iron cannot be maintained in a reduced state), and exposure to drugs with oxidizing properties (sulfonamides, nitrites, nitroglycerin, and lidocaine).

KEY WORDS AND CONCEPTS

  2,3-Bisphosphoglycerate (2,3-BPG)   Bite cells j  Embden-Meyerhof-Parnas (EMP) glycolytic pathway j  G6PD deficiency j  Glucose-6-phosphate dehydrogenase (G6PD) j  Glutathione j  Glycolysis j  Hexose monophosphate shunt j  Heinz bodies j  Hemoglobin M j  Methemoglobinemia j  Methemoglobin reductase j  Pyruvate kinase (PK) deficiency j j

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HEMOLYTIC ANEMIA n

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P O I N T S

Hemolytic anemia can be caused by intrinsic red cell defects (hemoglobin defects, enzyme defects, or membrane defects) or extrinsic causes (autoimmune, vascular defects, toxins, or heat)

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Intravascular hemolysis results from direct destruction of circulating red cells by extreme heat, toxins, infectious agents, complement, or shear forces

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Extravascular hemolysis results from phagocytosis of intact (but abnormal) red cells by macrophages of the spleen and liver

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Free hemoglobin released into the blood is rapidly bound by the serum protein haptoglobin and delivered

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emolysis, defined as shortened red cell survival, may result from any number of abnormalities, whether intrinsic or extrinsic to the red cell. Intrinsic abnormalities include membrane defects, abnormal hemoglobins, and enzyme defects. Extrinsic abnormalities include microangiopathy and anti-RBC antibody. Hemolysis is sometimes defined as intravascular or extravascular. Intravascular hemolysis occurs within the bloodstream, and extravascular hemolysis occurs in the marrow, spleen, or liver (Figure 6-1). Intravascular hemolysis is caused by destruction of circulating RBCs by extreme heat, toxins, infectious agents, complement, or shear forces (microangiopathic or mechanical heart valve induced). The loss of plasma membrane integrity rapidly destabilizes the red cell osmotic gradient, leading to rapid influx of sodium and water, cell swelling, and physical disintegration. Released hemoglobin bound to the plasma protein

to hepatic and splenic macrophages for degradation to peptides (from globin), iron, and bilirubin (from heme) 

Free heme released into the blood is rapidly bound to the serum protein hemopexin and delivered to splenic and hepatic macrophages for degradation to iron and bilirubin

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Unconjugated (hydrophobic) bilirubin is converted within macrophages to conjugated (hydrophilic) bilirubin (bilirubin diglucuronide) and is excreted in the bile and urine

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The most common inherited forms of hemolytic anemia (hereditary spherocytosis and hereditary elliptocytosis) result from intrinsic red cell membrane defects

haptoglobin and free heme bound to the plasma proteins hemopexin and albumin are both transported to the liver. In the liver, proteases convert globin into amino acids. In liver cells, heme protoporphyrin is oxidized by heme oxidase to biliverdin, reduced by biliverdin reductase to bilirubin, conjugated to form bilirubin diglucuronide, and excreted in the bile. Heme iron is transferred to plasma apotransferrin for transport to tissues, most prominently to the bone marrow. As a thrombogenic material, free red cell membrane phospholipid can initiate disseminated intravascular coagulation (DIC). Free hemoglobin can be deposited in the kidney and lead to renal tubular damage. Laboratory abnormalities indicative of intravascular hemolysis include increased serum LDH released from red cell cytoplasm, decreased serum haptoglobin (rapidly removed from the circulation upon binding to heme), and increased serum bilirubin. When bilirubin is 37

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HEMATOLOGY: A PATHOPHYSIOLOGIC APPROACH Intravascular Na+

H2O RBC

Heme

Urine

Extravascular

LDH

K+

RBC Nucleus

Hemoglobin Urine

Macrophage Heme hemoplexin complex

Hemoglobin Urine haptoglobin Hemosiderin complex

Phagolysosome Macrophage

FIGURE 6-1    Hemolysis. Albumin Unconjugated bilirubin + Storage iron (ferritin, hemosiderin) Urobilinogen uptake Urinary excretion

Intestinal lumen Fecal excretion

measured in the lab, a distinction is made between soluble (direct) and insoluble (indirect) forms of bilirubin. Hemolysis, whether intravascular or extravascular, is associated with an increase in unconjugated (indirect) bilirubin. Indirect bilirubin is converted to direct bilirubin by the liver and normally excreted in the bile. In the small bowel, gut bacteria reduce bilirubin to urobilinogen. A small amount of urobilinogen is absorbed by the small bowel and excreted in the urine. Extravascular hemolysis results from phagocytosis of RBCs by macrophages in the spleen, liver, and bone marrow. In the macrophage, heme is converted to bilirubin and transferred to the liver by albumin for conversion to bilirubin diglucuronide and excretion in the bile. A certain degree of extravascular hemolysis is normal, related to the necessary removal of aged RBCs from the circulation, primarily by splenic macrophages. The susceptibility of aged red cells to removal results from changes in cell membrane composition, with consequent decreased deformability, as well as from loss of terminal sialic acid from membrane glycoproteins. Several pathologic conditions are characterized by an abnormal degree of extravascular hemolysis leading to anemia. Although extravascular hemolysis often

Bile

Liver Conjugated bilirubin + Storage iron (ferritin, hemosiderin)

Iron (Fe3+)

Unconjugated bilirubin

Transferrin

Albumin

Bone marrow Hemoglobin

Liver Bile

Feces Urine

IgG

Phosphatidyl serine (PS)

FcR

PS receptor

accompanies intravascular hemolysis—for example, damaged red cells that escape intravascular lysis are phagocytosed and destroyed by splenic and liver macrophages—this term is more commonly applied to situations in which little to no intravascular hemolysis occurs. Examples of such situations include hypersplenism and autoimmune hemolytic anemia (AIHA). Laboratory abnormalities in hemolytic anemia of any cause may include normochromic normocytic anemia, elevated reticulocyte count, increased serum LDH, hyperbilirubinemia, decreased serum haptoglobin, increased plasma hemoglobin, hemoglobinuria, and hemosiderinuria. In severe cases, the anemia may be macrocytic, caused by marked reticulocytosis. Hemolytic anemia can be broadly classified into two etiologic types. Extrinsic hemolytic anemia, resulting from extracorpuscular factors, and intrinsic hemolytic anemia, resulting from RBC defects.

EXTRINSIC HEMOLYTIC ANEMIA The most common causes of extrinsic hemolytic anemia are microangiopathic, autoimmune, druginduced, and infection-mediated.



Microangiopathic hemolytic anemia results from traumatic red cell fragmentation during passage through abnormal arterioles laden with fibrin-platelet thrombi. The arteriolar thrombi are deposited as a result of coagulopathy induced by a variety of underlying conditions, including disseminated adenocarcinoma, (pre)eclampsia, malignant hypertension, DIC, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and immune-mediated vasculitis. Abnormalities noted on peripheral smear include fragmented red cells (schistocytes) (Image 6-1), increased polychromatophilic red cells (reticulocytes), and in severe cases, nucleated red cells (Image 6-2). Evidence of consumptive thrombotic coagulopathy (or DIC) may include prolonged prothrombin and activated partial thromboplastin times, increased fibrin split products, increased D dimer, decreased fibrinogen, and thrombocytopenia. Autoimmune hemolytic anemia (AIHA) results from hemolysis of antibody and/or complementbound red cells. The autoantibodies are directed against widely expressed red cell antigens, so-called public antigens, the most common being Rhesus (Rh) antigens. Extravascular hemolysis of antibody and/or complement-bound (opsonized) red cells occurs following Fc receptor-mediated phagocytosis by splenic and hepatic macrophages. Although the spleen is the primary site of phagocytosis, red cells coated with high levels of IgG and C3b also undergo Kupffer cellmediated phagocytosis in the liver. Opsonized red cells, especially those coated with IgM, a pentavalent immunoglobulin isotype with strong complementbinding properties, may also undergo intravascular complement-mediated hemolysis. AIHA is subclassified as either warm or cold on the basis of the optimal temperature at which the antibody binds to red cells in vitro (37° C or