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CHURCHILL LIVINGSTONE An Imprint of Elsevier The Curtis Center Independence Square West Philadelphia, Pennsylvania 19106 Copyright © 2004, 1980, Elsevier Inc. (USA). All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Health Sciences Rights Department in Philadelphia, USA: phone: (+1)215 238 7869, fax: (+1)215 238 2239, email: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com, by selecting “Customer Support” and then “Obtaining Permissions.” CHURCHILL LIVINGSTONE and the the Sail Boat Design are trademarks of Harcourt, Inc., registered in the United States of America and/or other jurisdictions. Library of Congress Cataloging-in-Publication Data Petz, Lawrence D. Immune hemolytic anemias / Lawrence D. Petz, George Garratty.—2nd ed. p. ; cm. Rev. ed. of: Acquired immune hemolytic anemias. 1980. Includes bibliographical references. ISBN 0-443-08559-5 1. Hemolytic anemia, Autoimmune. I. Garratty, George. II. Petz, Lawrence D. Acquired immune hemolytic anemias. III. Title. [DNLM: 1. Anemia, Hemolytic, Autoimmune. WH 170 P513i 2004] RC641.7.H4P47 2004 616.152—dc21 2003043767 Printed in United States of America Last digit is the print number: 9 8 7 6 5 4 3 2 1 Notice Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the Publisher nor the editor assumes any liability for any injury and/or damage to persons or property arising from this publication. The Publisher

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CHURCHILL LIVINGSTONE An Imprint of Elsevier The Curtis Center Independence Square West Philadelphia, Pennsylvania 19106 Copyright 2004, 1980, Elsevier Inc. (USA). All Rights Reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elseviers Health Sciences Rights Department in Philadelphia, USA: phone: (+1)215 238 7869, fax: (+1)215 238 2239, email: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com, by selecting Customer Support and then Obtaining Permissions. CHURCHILL LIVINGSTONE and the the Sail Boat Design are trademarks of Harcourt, Inc., registered in the United States of America and/or other jurisdictions. Notice Medicine is an ever-changing eld. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the Publisher nor the editor assumes any liability for any injury and/or damage to persons or property arising from this publication. The Publisher Library of Congress Cataloging-in-Publication Data Petz, Lawrence D. Immune hemolytic anemias / Lawrence D. Petz, George Garratty.2nd ed. p. ; cm. Rev. ed. of: Acquired immune hemolytic anemias. 1980. Includes bibliographical references. ISBN 0-443-08559-5 1. Hemolytic anemia, Autoimmune. I. Garratty, George. II. Petz, Lawrence D. Acquired immune hemolytic anemias. III. Title. [DNLM: 1. Anemia, Hemolytic, Autoimmune. WH 170 P513i 2004] RC641.7.H4P47 2004 616.152dc21 2003043767

Printed in United States of America Last digit is the print number: 9 8 7 6 5 4 3 2 1

Preface to the Second EditionWe thought we might have set a record for the longest time between editions of a book, since the rst edition of this book, entitled Acquired Immune Hemolytic Anemias, was published 24 years ago. However, our mentor, Professor Sir John Dacie, published the third edition of Autoimmune Haemolytic Anaemias (Volume 3 of The Haemolytic Anemias) in 1992, just 30 years after the publication of the previous edition of that volume! We have been attered during these long years by a number of physicians, immunohematologists, and blood bankers who insist that they still use the rst edition and have continued to press us for the second. As with the rst edition, this book is intended primarily as a useful source of information for those who care for patients who have immune hemolytic anemias, that is, clinicians with patient care responsibility and blood bank professionals, including physicians and technical staff. However, this purpose cannot be properly served without an adequately detailed scientic background, and we have endeavored to supply this. We have attempted to be rather comprehensive, but we do not intend this book to be only a reference volume and have therefore included practical aspects of the evaluation and management of patients with hemolysis. Patients with immune hemolytic anemias are sufciently common as to constitute an important problem, but, on the other hand, they are sufciently unusual that it is difcult for many individuals outside of referral centers to acquire adequate experience to feel at ease in managing the multitude of problems such patients may present. We earnestly hope that sharing our experiences through the medium of this book will be of value to others who confront such problems less commonly. During the years between editions of this text, medical disciplines that were rather early in their developmental stages, such as hematopoietic cell and solid organ transplantation, have emerged to be major components of health care and have contributed to the emergence of entirely new causes of immune hemolysis. Also, new generations of drugs have been developed, one of the consequences of which is an expansion of the causes of drug-induced immune hemolytic anemias. Molecular biology and DNA technology have evolved to become a part of our everyday scientic lives and are being utilized in hematology as in all other disciplines. We have attempted to bring our rst edition up to date while not ignoring important earlier contributions. We have added chapters on Historical Concepts of Immune Hemolytic Anemias, Hemolytic Disease of the Fetus and Newborn, Immune Hemolysis Associated with Transplantation, and Hemolytic Transfusion Reactions. As we emphasized in the preface to the rst edition, one of the important aspects of diagnosis and management of patients with immune hemolytic anemias is that the care of such patients depends on a knowledge of some aspects of both clinical and laboratory medicine. Although this is true throughout clinical medicine, a problem of particular magnitude is created by the need for clinicians to be able to interpret such unusual laboratory tests as the direct antiglobulin test with monospecic antiglobulin sera and the specicity and thermal range of allo- and autoantibodies. Similarly,

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laboratory personnel should be able to assist clinicians in the interpretation of important data, as when transfusion is indicated for a patient whose serum reacts with all RBCs in compatibility tests. Accordingly, one of the primary purposes of this book is to present both the clinical and laboratory aspects of immune hemolytic anemias in a single volume. We strongly feel that neither laboratory personnel (including physicians) nor clinicians can optimally contribute to the care of patients with immune hemolytic anemias without a rm understanding of both aspects of the subject. Lawrence D. Petz George Garratty

Preface to the First EditionThis book is intended to be a useful source of information for those who care for patients who have immune hemolytic anemias, i.e., clinicians with primary responsibility for patient management, physicians concerned with laboratory medicine, including blood bank directors, and the technical staff of such laboratories. It is not intended as an encyclopedic review or as a tour de force exposition of facts that are of interest primarily to those with extensive background and a highly specialized interest in the eld. Patients with immune hemolytic anemias are sufciently common as to constitute an important problem but, on the other hand, are sufciently unusual that it is difcult for many individuals outside of referral centers to acquire adequate experience to feel at ease in managing the multitude of problems such patients may present. We have had a special interest in these disorders and we earnestly hope that sharing our experiences through the medium of this book will be of value to others who confront such problems less commonly. We include previously unpublished data concerning our experiences with various phases of the diagnosis and management of more than 300 patients, as well as a review of relevant information available in the medical literature. Although the primary purpose of this book is, therefore, to be a source of information that will be of value in management of patients, this purpose cannot be adequately served merely by a supercial exposition of practical facts, and we do not intend this book to be a manual of patient care. We trust that the interested reader would demand an adequately detailed scientic background to make meaningful the recommended laboratory procedures and their clinical interpretation. For example, the knowledge that the direct antiglobulin (Coombs) test performed on red cells from patients with cold agglutinin syndrome is invariably positive using anit-C3d antiglobulin serum and invariably negative using anti-IgG antiglobulin serum is of some clinical value (Ch. 6). When such information is augmented by an understanding of pertinent aspects of the serum complement system (Ch. 3) and the mechanisms of immune hemolysis (Ch. 4), one then has a basis for understanding such facts and their clinical signicance. Writing this book presents a unique problem. That is, one of the important aspects of diagnosis and management of patients with immune hemolytic anemias is that the care of such patients depends upon a knowledge of some aspects of both clinical and laboratory medicine. Although this is true throughout medicine, a problem of unusual magnitude is created by the fact that most clinicians have very little exposure to immunohematology. Results of direct antiglobulin tests with monospecic antiglobulin sera and the characterization of serum antibody specicity and thermal range is information that is difcult or impossible for most practicing physicians to utilize. This problem is augmented by the fact that laboratory personnel are faced with difcult technical tasks, and, in the very best of hands, uncertainties may remain. For example, in regard to blood transfusion (Ch. 10), what is the probability of not detecting a red cell alloantibody in the serum of a patient with autoimmune hemolytic anemia when the serum reacts with all donor cells tested, and what is the risk of transfusion of blood that is incompatible because of the presence of an autoantibody? One of the prime purposes

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of this book, and one of the more difcult tasks we faced in writing it, is to present both the laboratory and clinical aspects of immune hemolytic anemias in a single volume in a manner that is understandable by those in both elds. Neither laboratory personnel (including physicians) nor clinicians can optimally contribute to the care of patients with immune hemolytic anemias without an understanding of both aspects of the subject. Therefore, it is our rm opinion that, with few exceptions (e.g., some sections concerning technical details which may justiably be ignored by clinicians, and some aspects of therapy which may not be essential knowledge for technologists), the information herein is important to those in both clinical and laboratory medicine for proper management of patients with immune hemolytic anemias. Lawrence D. Petz George Garratty

AcknowledgmentsAs indicated in the rst edition, we are both indebted to Professor Sir John Dacie for the privilege of working in his laboratory at the Royal Postgraduate Medical School and Hammersmith Hospital in London. His teachings served as a foundation for our work and, moreover, we have attempted to emulate his dedication and precision in scientic investigation. Grateful acknowledgment is also due to the numerous physicians and technologists who were kind enough to refer interesting and challenging clinical and laboratory problems to us. Without this continued support it would have been impossible to acquire the experience and data necessary to compile this volume. In addition, we appreciate the collaboration of our colleagues at the City of Hope Medical Center, Duarte, California, and the University of California Los Angeles Medical Center (Dr. Petz) and American Red Cross Blood Services, Southern California Region (Dr. Garratty). We would especially like to thank some extraordinary medical technologists who were not just a pair of hands in the laboratory but were innovative contributors to the design and results of our studies: Donald Branch (now the proud possessor of a PhD); Alana (Loni) Calhoun, Patricia Arndt, Regina Leger, Sandra Nance, and Nina Postoway. Their relevant roles were obvious from our publications mentioned throughout the book. Dr. Garratty would especially like to thank Ann Tunick, his administrative assistant (since 1978), who typed multiple error-free drafts of material, found and formatted references, and dealt imperturbably with all problems that arose. Without her help Dr. Garrattys contributions would never have appeared in this book! Both of us would like to acknowledge the tremendous support of our wives (Thelma Petz and Eileen Garratty), who put up with our working every weekend and many evenings without too many grumbles!

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Historical Concepts of Immune Hemolytic Anemias

Immune hemolysis is a shortening of red blood cell (RBC) survival due, directly or indirectly, to antibodies. These antibodies may be autoantibodies or alloantibodies. This chapter will deal mainly with historical aspects of autoimmune hemolytic anemia (AIHA), followed by a brief discussion of historical aspects of hemolytic transfusion reactions. AIHA is an acquired immunologic disease in which the patients RBCs are selectively attacked and destroyed (hemolysed) by autoantibodies produced by the patients own immune system. Shortened RBC survival is frequently associated with the presence of a reticulocytosis, spherocytes in the peripheral blood lm, autoantibodies in the patients serum, and occasionally splenomegaly, hemoglobinemia, and hemoglobinuria. Although these facts are common knowledge now, it was not always so. Reviewing how these concepts developed over the centuries by observation and clinical and laboratory experimentation is both fascinating and instructive. It is evident that concepts that collectively led to our present understanding of AIHA required knowledge of the existence of RBCs, understanding the possibility of anemia without blood loss, distinguishing hemoglobinuria from hematuria, understanding the mechanism by which hemoglobinuria occurs, recognizing the

process of agglutination, understanding the distinction between congenital and acquired disorders, understanding that premature destruction of RBCs can cause anemia and jaundice, recognizing spherocytes and abnormal osmotic fragility of RBCs and determining their signicance in patients with hemolysis, recognizing reticulocytes, determining that serum antibodies may cause destruction of foreign cells and also autologous cells, developing means to measure RBC survival, developing diagnostic assays for antibodies, refuting the concept of horror autotoxicus, and understanding the role of the spleen and splenectomy. The discoveries that led to the development of our knowledge about these concepts are herein reviewed in the approximate order in which the relevant observations were made. Here, then, is how our knowledge of AIHA came to be. The development of this short review was aided signicantly by previous reviews on various aspects of hemolysis and AIHA.1-9

THE LESSONS OF HISTORYEveryone who studies the stories of discovery in what has come to be called the eld of hematology will recognize the early gropings in the midst of profound ignorance and the difculties that confronted the investigators. We have gained an understanding of biology that could hardly have been dreamed of only a short time ago, let alone at the time of the rst 1

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Immune Hemolytic Anemias

tentative forays into the unknown. Moreover, understanding has been crowned by tangible benets for humanity. It is worthwhile to consider how such great progress comes about and why. How is knowledge achieved, and what can we learn from the process by which important discoveries were made?10 The rst lesson to be learned of history is that the path of progress is anything but straight. The course of research has been likened to the ow of a stream that ultimately becomes a rushing torrent whose importance is obvious. This certainly has been the history of research in hematology. It certainly does not follow that, because a concept is plausible and is in accord with the understanding of the time, it is necessarily correct. The following pages provide many examples of misinterpretations resulting from such an assumption. Furthermore, because they have been plausible, such views often have endured and have stood in the way of acceptance of observations and interpretations that proved to be the correct ones. Discovery begins with an observation or the posing of a question. But observation is not as simple as it sounds. Indeed, many look but few see. It is the exceptional person who recognizes the unusual event or manifestation. Still fewer pursue it to new understanding. Many may ask questions but few have the imagination, the energy, and the overpowering drive to persist in the search for an answer, especially when this must be done in the face of difculties and failures and even despite scorn from their peers. Imagination and industry alone, however, have not sufced. Means have had to be devised to explore the questions that were posed. When these were provided, it is impressive to see what the introduction of a new technique made possible for an area of inquiry. A simple example, described later, is the introduction of the antiglobulin test, which very rapidly led to a much clearer distinction between immune and nonimmune hemolytic anemias. Progress depends on the contributions of many. Moreover, scientic discipline has beneted from developments in other elds, progress in one eld spurring another, and vice versa. As knowledge has grown, it has become impossible for a single human being to encompass the whole, and the discovery and growth of understanding have become more and more dependent on interchange among scientic disciplines. Still another aspect of the progress of understanding is worth noting. It is not generally appreciated how often curiosity concerning an observation made at the bedside by clinicians has led to far-reaching investigations. An example is the observation of hemoglobinuria, which led to the understanding of destruction of RBCs and to the early delineation of certain clinical syndromes (e.g., paroxysmal cold hemoglobinuria [PCH], paroxysmal nocturnal hemoglobinuria [PNH], and march hemoglobinuria) characterized by hemoglobin in the urine. Investigators have not always been farseeing and logical, moving steadily and directly to their goal, nor

did they fail to make mistakes. Indeed, incorrect theories have hampered the advance of knowledge, especially when these theories were widely disseminated and were pronounced by eminent authorities. A number of such examples appear in the following pages. It follows that authorities must be humble and novices skeptical.

EARLIEST DESCRIPTIONS OF POSSIBLE ACQUIRED HEMOLYTIC ANEMIAThe rst written description of what may have been an acquired hemolytic anemia, albeit not of an immune nature, was Galens description in 150 AD of a person bitten by a viper whose skin turned the color of a ripe leek.1,4,11 Galens understanding of physiology was such that he implicated the spleen as leading to the skin discoloration, an association of the spleen and hemolysis that was not conrmed until the late nineteenth century.1 PCH may have been described as early as 1529 by Johannes Actuarius, a court physician in Constantinople. In his work, De Urinis, Acturarius described a condition in which the urine is azure & livid as well as black in patients being of melancholic humor and complaining of loss of strength, after an exposure to cold.4 Further mention of PCH seems, however, to be absent for nearly 300 years, until the latter half of the nineteenth century.

EARLY EXPERIMENTAL INVESTIGATION OF BLOODDescription of Red Blood Cells. The development of the scientic method led to the seminal discoveries of the circulation of blood by Harvey in the early sixteenth century and the cardinal experiments with transfusion of blood by Lower in England and Denis in Paris in the mid-seventeenth century. Despite this interest in blood, the discovery of the RBCs had to await the appearance of the microscope around 1650. The rst observation of an RBC was likely made by Malpighi in 1661, when he described the circulation of RBCs in the capillaries, and this was followed in 1663 by Swammerdans description of minute globules in the blood of a frog. A decade later, human RBCs were described in detail by van Leeuwenhoek (Fig. 1-1),12 who also established their size at about 13000 of an inch by comparing an RBC with a grain of sand of known size. John Huxham, in 1770, described the changing shapes of degenerating RBCs and, importantly, recognized that such cells were the origin of hemoglobin.4 Anemia without Blood Loss. In 1843, Andral (Fig. 1-2) described a spontaneous anemia, which arises without any prior blood loss.13 He quantied red blood globules in healthy patients and reported

Historical Concepts of Immune Hemolytic Anemias

3

FIGURE 1-1. Antonj van Leeuwenhoek (16321723). (From Wintrobe MM: Milestones on the path of progess. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:131.)

FIGURE 1-2. Gabriel Andral (17971876). (From Wintrobe MM: Milestones on the path of progress. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:131.)

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Immune Hemolytic Anemias

16 early case of anemia. Although he provided no other information concerning the patients condition, what is important in relation to hemolytic anemia is the observation of anemia without prior blood loss. Hemoglobinuria. Vogel, in 1853,14 stated that the matter in the urine is the same as that in the blood and suggested that the matter in the urine consists of a decomposition of blood discs. He suggested that the degree of blood decomposition can readily be ascertained by the degree of coloration in the urine, and he indicated a connection between fevers, colored urine, decomposition of blood discs, and anemia. This represents one of the early examples of the association between a decreased RBC count and the term anemia. It also represents early evidence suggesting that anemia may be secondary to infections.

RED BLOOD CELL AGGLUTINATIONThe description of the phenomenon of RBC agglutination and its development as a tool in elucidating blood groups took place in the last 30 years of the nineteenth century in Germany and Austria, and were reviewed in depth in 2002 by Hughes-Jones and Gardner.15 The discoveries were largely the work of three people: Adolf Creite, a medical student in Gttingen, Germany; Leonard Landois, Director of the Physiological Institute at the University of Greifswald, Germany; and Karl Landsteiner, working in the Pathological Anatomy Institute in Vienna, Austria.15 Adolph Creite. Creites (Fig. 1-3) almost unknown contribution was published in 1869 under the title Investigations concerning the properties of serum proteins following intravenous injection.16 His work is quite remarkable in that it showed that serum proteins had the property of both dissolving and bringing about clustering of red cells, that is, lysis and agglutination in present-day terms, anticipating the discovery of antibodies by a quarter of a century. Creite injected sera from calf, pig, dog, sheep, cat, chicken, duck, and goat into rabbits. The rst three had little or no effect on the recipient, but the sera of the latter ve almost always resulted in the appearance of blood-stained urine, general malaise, and death of the animal. He noted that the urine was free of intact RBCs. He concluded that serum contains agents that are able to dissolve red cells directly. He performed additional experiments in which he removed protein from the serum before its injection and observed that all of the urine samples examined until the evening of the following day are normal. Accordingly, he concluded that the most likely active ingredients were serum proteins, but added, However, I cannot say how they function. He also performed in vitro experiments and provided a remarkably clear account of what is probably the rst description of agglutination. He reported, If you add blood serum from any of the animals with which I have carried out my experiments to a drop of

FIGURE 1-3. Adolf Creite, about 1920. (From Hughes-Jones NC, Gardner B: Red cell agglutination: The rst description by Creite (1869) and further observations made by Landois (1875) and Landsteiner (1901). Br J Haematol 2002;119:889893.)

fresh rabbit blood, then you observe under the microscope that in the regions where the foreign serum mixes with the rabbit red cells, the cells suddenly ow together in a peculiar way forming different shaped drop-like clusters with irregular branches. I believed that I had found an explanation for the appearance of blood in the urine, as it was possible that some blood cells had dissolved completely. Leonard Landois. RBC agglutination and lysis were put on an even rmer basis by Landois, who published an extensive monograph on the subject of transfusion,17 which included a section describing his in vitro experiments. In his experiments, Landois was successful in demonstrating both lysis and agglutination. (It should be noted that the terms lysis and agglutination were not in use until the end of the nineteenth century. For lysis, both Creite and Landois used a German word meaning dissolve; for agglutination, words translatable as accumulation, ball formation, or sticky clumps were used.) Landois also distinguished agglutination from rouleaux, for which he used the term, like rolls of coins. Landois added 4 to 5 mL of clear serum into a test tube and then added fresh debrinated blood. He incubated the mixture at 37C to 38C or at room temperature and observed the initiation of the RBC lysis.

Historical Concepts of Immune Hemolytic Anemias

5

Sooner or later the mixture becomes completely clear and transparent and the cells are no longer visible. I observe the whole process of the lysis and the changes in red cell shape under the microscope. Commenting on another experiment on the mixing of cells and serum, Landois described the changes in shape of RBCs and added, The cells develop the ability to stick to neighboring cells and form larger or smaller clumps. Karl Landsteiner. At the turn of the century, there was a considerable amount of disagreement and confusion about the occurrence and signicance of agglutination in both health and disease.15 It was at this point that Landsteiner (Fig. 1-4) entered the eld.17a,b The rst suggestion of the existence of serum agglutinins and red cell antigens within what would nally be known as the ABO blood group system is to be found as a footnote in a publication by Landsteiner in 1900.18 In it he states, The serum of healthy individuals not only have an agglutinating effect on animal red cells but also on human red cells from different individuals. It remains to be decided whether this phenomenon is due to individual differences or to the inuence of injuries or bacterial infection. In a detailed paper in 1901, he reported that he obtained sera and red cells from 29 different people, including himself and four medical colleagues, to study agglutination reactions. The reason that Landsteiner was successful in elucidating the mechanism underlying intraspecies agglutination where others had failed arose from the nature of Landsteiners experimental design. He used all of the sera against all of the samples of RBCs, using checkerboard blocks of ve or six different sera and RBCs in 144 combinations. He found that certain sera would agglutinate the RBCs of certain other people. This discovery of isoagglutination became the basis of human blood-group classication, which would subsequently be found to have relevance for autoantibody specicity in AIHA. In his characteristically brief but data-lled paper of 1901,19 Landsteiner further noted and pointed out that the blood isoagglutinins retained their activity after drying and redissolving. Also, he observed agglutination with serum extracted after 14 days from blood dried on a cloth. The reaction may be suited to establish the identity or more correctly the non-identity of a blood specimen. This predicted the value of Landsteiners discovery to forensic medicine in the future. The closing statement in his paper was, Finally, it might be mentioned that the reported observations may assist in the explanation of various consequences of therapeutical blood transfusions. In three pages, Landsteiner compressed knowledge that would ll thousands of pages in the future.20 On November 8, 1930, Karl Landsteiner was awarded the Nobel Prize (Fig. 1-5). The lecture given by Landsteiner at the conferment of his Noble Prize was based on the differences in the blood of human

individuals. More than a century later, his theories about isoantigens are accepted and are a fundamental part of the theoretical basis of immunology, tissue transplantation, forensic medicine, and population genetics.21,22

FIRST DESCRIPTION OF HEMOLYTIC ANEMIAThe concept that premature destruction of RBCs might lead to a disease state and jaundice was rst suggested in 1871 by Vanlair and Masius.1,23 These observers described a patient with anemia and marked splenomegaly without hepatomegaly. The patient suffered acute attacks of left upper quadrant pain and jaundice without acholia, and passed reddish brown urine. Morphologic evidence of an RBC abnormality was suggested by nding spherical dwarf cells in the peripheral blood that they called microcytes. The authors postulated that clinical jaundice could result from two different mechanisms: mechanically by reabsorption or liver induced and paradoxical icterus. The latter group included the blood induced icterus, where excessive amounts of colorant material is released from the blood cells and followed by the formation of bile which is deposited in the tissues. More explicitly, they stated that there are at least a certain number of non-mechanical types of icterus which are caused by the exaggerated destruction of red cells and the transformation to bilirubin of released hematin. This concept was essentially correct, but little attention was paid to this remarkable publication and, for almost 30 years, hepatic disease, jaundice, and hemolytic anemia became hopelessly intertwined.1

THE DISTINCTION BETWEEN CONGENITAL AND ACQUIRED HEMOLYTIC ANEMIASAt the turn of the twentieth century, Hayem24 (Fig. 1-6) and Minkowski25 showed that the jaundice associated with hemolytic anemia was distinct from that of hepatic diseases. Hayem made the distinction between congenital and acquired hemolytic anemias, whereas Minkowski described only a hereditary condition. Hayem has repeatedly been said to be the rst to describe acquired hemolytic anemia, although he did not name it that, but, instead, coined the term chronic infectious splenomegalic icterus.24 Minkowski is credited with the rst clear recognition of icterus due to hemolytic anemia (chronic hereditary acholuric icterus) separate from obstructive jaundice; he associated the anemia with urobilinuria and splenomegaly and postulated that RBC destruction was attributable to lesions in the spleen.25

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A

B

C

D

FIGURE 1-4. Karl Landsteiner at various times in his life. (A) Landsteiner at about the age of 5 (c. 1873), posing in a Husara riding costume on the photographers papier-mach rocks. (B) Photograph of Landsteiner probably taken at the Institute for Pathological Anatomy, where he worked from 1897 to 1907. (C) Landsteiner and his coworker, Emil Prsek from Belgrade, December 1913. The two worked together on the chemical manipulation of the specicity of serum albumin. (D) Landsteiner at about the time he left Europe for the United States. (From Mazumbar MH: Species and Specicity. An Interpretation of the History of Immunology. Cambridge, UK: Cambridge University Press, 1995.)

Historical Concepts of Immune Hemolytic Anemias

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FIGURE 1-5. The Noble Prize certicate for Karl Landsteiner in 1930. (From Tagarelli A, Piro A, Lagonia P, Tagarelli G: Karl Landsteiner: A hundred years later. Transplantation 2001;72:37.)

DESCRIPTION OF SPHEROCYTES AND ANALYSIS OF THEIR SIGNIFICANCEVanlair and Masius23 described the case of a young woman who developed icterus, recurrent attacks of

left upper quadrant abdominal pain, and splenomegaly shortly after giving birth. The patients mother and sister were also icteric, and the sisters spleen was enlarged. The most remarkable aspect of this paper lies in their description of the blood ndings. Although they made no mention of anemia and had no concept of hemolysis as a pathological process, they unmistakably described RBCs that we now recognize as spherocytes with remarkable clarity (Fig. 1-7). The authors noted that some of the RBCs, which they called microcytes, were smaller than normal RBCs, 3 to 4 m in diameter, spherical in shape, and the contours were completely smooth. They concluded, The jaundice of our patient appears to be a peculiar type of icterus. The fact that the patients mother and sister had a slight jaundice and that the sister had an enlarged spleen may indicate that this condition is one disease entity. Naegli is often credited with rst use of the term spherocyte. However, according to Crosby26 (Fig. 1-8), two British army ofcers, Christophers and Bentley, were the rst. They were assigned to India to study

FIGURE 1-6. Georges Hayem. (From Packman CH: The spherocytic haemolytic anaemias. Br J Haematol 2001;112:888899.)

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blackwater fever and made very careful descriptions of spherocytes in a monograph published in 1909. Naegli also proposed that the spherocyte was pathognomonic of congenital hemolytic icterus, an observation that

FIGURE 1-8. William H. Crosby. (From Wintrobe MM: Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:XVIII. Reproduced with permission of The McGraw-Hill Companies.)

constricted thinking about hemolytic icterus for the next 15 or 20 years. In fact, many authorities began to doubt the existence of an acquired type of hemolytic icterus, regarding the disease as a variation on the congenital form.

OSMOTIC FRAGILITY OF RED BLOOD CELLSDuring the rst decade of the twentieth century, a number of signicant studies of the osmotic fragility of RBCs were conducted. Chaufford27 (Fig. 1-9) noted that RBCs of several patients, but not those of normal subjects, were hemolysed by hypotonic saline. He developed an osmotic fragility test, in which RBCs were placed in a series of tubes containing successively decreasing concentrations of saline. The osmotic fragility was expressed as the concentration of saline at which hemolysis began and at which hemolysis was complete (Fig. 1-10). Chauffard recognized that the liver was not at fault and that the disorder was a result of hemolysis. He wrote, Perhaps after this clinical and hematologic inquiry, the cause of the hemolytic theory could be considered as won. This observation nally enabled physicians to distinguish hepatic and hemolytic jaundice, as Ribbierre had recently (in 1903) demonstrated that the cells from patients with hepatic jaundice are resistant to osmotic stress.7

FIGURE 1-7. A reproduction of part of the tinted lithograph illustrating the paper by Vanlair and Masius (1871) entitled De la microcythmie. I is a drawing of the patients blood. II is a drawing of control normal blood. (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:211255.)

Historical Concepts of Immune Hemolytic Anemias

9

Of course, Chauffard and coworkers27 had discovered the in vitro pathophysiological expression of the spherical microcytes described by Vanlair and Massius23 almost 40 years earlier. However, they were probably unaware of the work of these early investigators and they certainly made no association between microcytic spherical cells and increased osmotic fragility. That correlation was noted much later by Haden.28

RETICULOCYTESAbout 1 year after his description of increased osmotic fragility in congenital hemolytic icterus, Chauffard and Fiessinger29 and Chauffard30 stained RBCs from patients with hemolytic icterus with Pappenheims31 (Fig. 1-11) solution and noted large numbers of cells containing a peculiar basophilic granulation or reticulum, which they called granular degeneration. Ehrlich had rst described this special staining method in 18817 and noted increased numbers of reticular cells in anemic patients. Vaughan,32 in 1903, noted these granular cells constituted about 1% of the RBCs in normal subjects. Chauffard had hoped to explain the anatomical lesion that underlay the increased fragility of the RBCs. What he actually discovered, or rediscovered, was the reticulocytosis that is now a hallmark of hemolytic anemia. Chauffards drawing30 of a blood smear stained with Pappenheim stain from a patient with familial hemolytic icterus is shown in Figure 1-12.

FIGURE 1-9. Anatole Chauffard (18551932). (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGrawHill Book Company, 1980:211255.)

7,5. Diamtre moyen des hmatics 5 89 Diamtre maxima 4. minima Hmolyse totale....

Hmolyse trs nette

Hmolyse nette....

Hmolyse lgre....

Pas dhmolyse.... Nombre de gouttes de la solution....

70 68 66 64 62 60 58 56 54 52 50 48 46 44 42 40 38 36 34 32 Rsistance globulaire (Solution de NaCI 0.70%)

FIGURE 1-10. The gure illustrates the precocious and prolonged lysis in hypotonic saline of the red cells of a patient suffering from ictre congnital de ladulte (hereditary spherocytosis). (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:211255.)

10

Immune Hemolytic Anemias

FIGURE 1-11. Artur Pappenheim (18701916). (From Lajtha LG: The common ancestral cell. In: Wintrobe MM (ed): Blood, Pure and Eloquent. McGraw-Hill Book Company, 1980:8195. Reproduced with permission of The McGraw-Hill Companies.)

THE CONCEPTS OF IMMUNE HEMOLYSIS AND HORROR AUTOTOXICUSIn an impressive series of studies commencing in 1899,33 Paul Ehrlich (Fig. 1-13) and Julius Morgenroth sought to identify the constituents and to dene the mechanisms involved in the phenomenon of immune hemolysis, which Jules Bordet had only recently described.34 Such studies involved the immunization of animals with foreign RBCs, a procedure resulting in an immune serum whose thermostable antibody would collaborate with a

thermolabile substance (variously termed complement, alexin, or cytase) to cause the specic destruction in vitro of the erythrocyte species used for immunization.8 During the course of these studies, Ehrlich and Morgenroth attempted repeatedly to induce the animal to form hemolytic antibodies to its own cells. These attempts to elicit the formation of autoantibodies were uniformly unsuccessful, and, at best, they were only able to produce antibodies able to agglutinate or to hemolyse the RBCs of certain other members of the same species. Ehrlich had postulated, in his landmark paper of 1897, that antibody formation was part of the normal

Historical Concepts of Immune Hemolytic Anemias

11

FIGURE 1-12. Drawing of a blood smear (Pappenheim stain) as seen by Chauffard (1908). The granular appearing cells are reticulocytes from a patient with familial haemolytic icterus. (From Packman CH: The spherocytic haemolytic anaemias. Br J Haematol 2001; 112:888899.)

physiological process of cellular digestion and so might theoretically be stimulated by autochthonous as well as by foreign substances.8 Nevertheless, he pointed out, It would be dysteleologic in the highest degree, if under these circumstances self-poisons of the parenchymaautotoxinswere formed.35,35a Thus, we might be justied in speaking of a horror autotoxicus of the organism.36

THE FIRST DESCRIPTION OF AN AUTOIMMUNE HEMOLYTIC ANEMIAThe rst AIHA in which clinical and diagnostic laboratory ndings were clearly described is PCH.37 This appears, at rst, to be surprising because PCH is the least common type of AIHA. Its early recognition is due to the fact that hemoglobinuria is a striking symptom, a fact that also explains the early recognition of march hemoglobinuria and PNH. It is also true that PCH was much more common than it is at present because a majority of cases recorded in the early medical literature were associated with late stage syphilis or congenital syphilis. In the early 1900s, over 90% of patients with chronic PCH had a positive test for syphilis and approximately 30% showed clinical evidence of the disease.38 With the effective treatment of syphilis and the virtual elimination of the congenital form, classic syphilitic PCH is now an extremely rare disorder, as is chronic PCH. It was in patients with the chronic form of PCH that exposure to cold resulted in a paroxysm of hemoglobinuria.39,40

In the latter part of the nineteenth century, there were a number of reports of PCH. Dressler41 is generally credited with being the rst (in 1854) to give a clear description. His patient was a 10-year-old boy who may have had congenital syphilis. After exposure to cold, he passed red urine that gradually paled to clear to a natural color. Microscopic examination of the urine showed dirty brown pigment but no blood corpuscles. PCH, however, seems also likely to have been the diagnosis in the patient described by Elliotson in The Lancet in 18323,42 who had heart disease and cold ts and passed bloody urine whenever the east wind blew. Subsequently, several excellent clinical accounts were published during the 1860s.3 The authors realized that exposure to cold precipitated that attacks and that the urine contained blood pigment, but no blood cells. Wiltshire43 described an infant, perhaps the youngest such patient ever recorded, who passed bloody urine, free from RBCs in the sediment, when the weather was particularly inclement. The term hemoglobinuria seems to have been used rst by Secchi in 1872, but it is not clear whether the patient he described had PCH.44 In 1879, Stephen Mackenzie, at the London Hospital, elaborated on the pathophysiology of PCH.45 He described a young boy who had a sallow complexion and yellow eyes and whose urine was black. The microscopic examination and spectroscopic analysis of the urine showed it contained abundant hemoglobin but no RBCs. He suggested that the discolored urine was due to blood solution or disintegration (hemolysis) and stated that it must take place in some part of the organism. He believed that the hemolysis occurred in the genito-urinary apparatus, most probably the kidney. Kuessner, in 1879, made the important observation that serum obtained by cupping a patient during an attack of hemoglobinuria was tinged red.46 This probably was the rst direct evidence derived from observations in humans that indicated that the hemoglobin in the urine was being derived from hemoglobin liberated in the plasma, rather than being, in some mysterious way, of renal origin. Indeed, Mackenzie modied his previous theory of erythrocyte destruction, suggesting that the role of the kidney is in fact passive, and that the corpuscle solution, or hemolysis, occurs in the vasculature.47

EARLY DIAGNOSTIC TESTS FOR PAROXYSMAL COLD HEMOGLOBINURIAAlthough there were many clinical descriptions of PCH in the nineteenth-century medical literature documenting the relationship of acute attacks to exposure to cold and the fact that the urine contained blood pigment but no blood corpuscles, the pathophysiology was not understood.

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Immune Hemolytic Anemias

FIGURE 1-13. Paul Ehrlich (18541915) in his study.* (From Wintrobe MM: Milestones on the path of progress. In: Wintrobe MM: (ed): Blood, Pure and Eloquent. New York: McGrawHill Book Company, 1980:131.)

A diagnostic test described in 1879 was based on the development of hemoglobinuria after immersion of the patients feet in ice water.39,40 A test producing less discomfort to the patient was described in 1881 by Ehrlich, who showed that if a ligature was placed around a nger that was then chilled in ice water, serum subsequently obtained from the nger would contain hemoglobin.48 Although these tests helped to diagnose the disorder, they did not elucidate the mechanism by which exposure to cold resulted in hemolysis.

THE DONATH-LANDSTEINER DISCOVERY, 1904: THE FIRST DESCRIPTION OF AN AUTOANTIBODY AND OF AN AUTOIMMUNE HUMAN DISEASEThe greatest single step forward in understanding the pathogenesis of PCH was provided by the work of Donath and Landsteiner whose famous report was published in 1904.49 Julius Donath (19701950) was an assistant at the University of Vienna First Medical Clinic, and Karl Landsteiner (18681943) became a giant in the annals of immunology.50 These investigators demonstrated that hemolysis was due to an autolysin that reacted with the patients RBCs at low

*This is the authors favorite photograph, indicating that ofces circa 1899 were not necessarily neater than those of the present day. It certainly seems as though Ehrlich maintained enough reading material in his ofce.

temperatures and that labile serum factors (complement) caused lysis of the sensitized cells if the temperature was subsequently raised. Their interpretation of their observations are particularly noteworthy because they were published during the era of widespread acceptance of Ehrlichs dictum of horror autotoxicus. Here then was the rst report that appeared to contradict Ehrlichs concept.35a This bithermic procedure for the diagnosis of PCH was the rst immunohematologic test ever to be described51 and remains the diagnostic test for the disorder (see Chapter 5). Further, this discovery has been widely acclaimed as the rst description of an autoantibody and of an autoimmune human disease.8 The test is referred to as the Donath-Landsteiner (DL) test and the antibody thus detected as the DL antibody. Even after the passage of a century since the report of Donath and Landsteiner, the accuracy of their observations and the usefulness of the DL test persist. Primacy of Discovery of Biphasic Autoantibodies in Paroxysmal Cold Hemoglobinuria. It is of interest that similar and apparently independent observations were described by Eason. Easonss two papers,52,53 published in 1906, were based on his MD thesis. His experiments, which had been carried out in 1903, had been the subject of a communication read at a meeting of the Galenian Society, Edinburgh, in January 1904. He stated that ten months after the results had been communicated by me the most important of them were conrmed by Donath and Landsteiner whose research on these lines had been conceived independently of mine. These collaborators furthermore proved that it is the process of anchoring of the intermediary body to the red corpuscles which requires the low temperature.

Historical Concepts of Immune Hemolytic Anemias

13

Dr. Eason was awarded a Gold Medal and the MilnerFothergill Medal in Therapeutics by Edinburgh University for his thesis.37 However, Donath and Landsteiner contested the priority for their discovery with Eason and stated that Eason joined [himself] to our interpretation of the mechanism of hemolysis.8,54 They further stated that the development of autotoxic substances, which are bound to the organisms own cells, can be related to the process of antibody formation, a possibility which, so far as we know, has not previously been discussed.8,54 In a much more recent publication, Goltz55 maintained that Donath and Landsteiner did not actually discover the rst autoantibody because nowhere did they use the accepted terms antibody, ambozeptor, antigen, or even immune. Rather they used such apparently nonspecic terms as hemolysin, toxin, and poison. However, as reviewed in depth by Silverstein,8 numerous contemporary authors used the term toxin when they meant specic antibody, and the term did not imply some sort of nonimmunologic toxic action. Even if Landsteiners language might be misinterpreted at a later period, his contemporaries surely understood him. For example, Ehrlich in 1906 already referred to Donath and Landsteiner as observing hemolytic autoamboceptors.8,33 Further, Rssle, in 1909,8,56 while discussing the general evidence of the existence of autoantibodies, stated that there are also cases, however, in which direct evidence for the presence of autoamboceptor is splendid. The best known instance concerns paroxysmal hemoglobinuria. Even in their rst report, Donath and Landsteiner called our attention to the possibility that such a substance might be the result of self-immunization. Also in 1909, Meyer and Emmerich published an extensive report on paroxysmal hemoglobinuria.57 They concluded their paper with the statement that In [our] four cases of typical paroxysmal cold hemoglobinuria, the autohemolysin found by Donath and Landsteiner was observed. It is evident from the foregoing that Donath and Landsteiner as well as their contemporaries did, indeed, understand from the outset that they were describing an autoantibody and an immunological process, despite the curious terminology they used.8 The Original Experimental Protocol of Donath and Landsteiner. Excerpts from the original report in 1904 by Donath and Landsteiner58 are illustrated in Figures 1-14 and 1-15. A translation of the original protocol is provided in Figure 1-16. In essence, they demonstrated that sera from patients with PCH would cause hemolysis in vitro of RBC of normal individuals and of patients if the serum and cells were held (incubated) for 12 hour at 5C and then held at 37C. As controls, they used serum from normal individuals. They concluded that the serum of the hemoglobinuric patients contains a lytic substance that is effective against the patients and other human blood corpuscles.

Their article describes further experiments in which two aliquots of a patients blood were obtained. One aliquot was incubated at 0C, and the other was incubated at room temperature. Then the plasmas were removed and exchanged, mixed, and incubated at 37C. After 2 hours, the aliquots that were cooled had undergone much lysis, but no lysis occurred in the other aliquot. This nding indicates that red cells take up in the cold an effective substance from the plasma, and that neither red cells nor white blood cells give hemolytic substance into the serum. In an additional experiment, oxylated blood of the patient was cooled in ice water and centrifuged in the cold, and then the plasma that had been removed in the cold was mixed with a new aliquot of red cells of the patient. This mixture was then cooled and subsequently incubated at 37C. However, no hemolysis occurred, thus indicating that the hemolysin had been absorbed by the cells. Red cells that are cooled with serum or plasma of hemoglobinuric patients, whether the patients own or others red cells, take up substances that by this absorption develop the capability to hemolyse in the serum of hemoglobinuric patients and other human serum. The hemolysis is caused by the aid of factors in the serum described as complement (alexin, cytase, etc.).

FURTHER STUDIES ON THE MECHANISMS OF HEMOLYTIC ANEMIA AND OBSERVATIONS ON THE DISTINCTION BETWEEN CONGENITAL AND ACQUIRED FORMSChauffard was among several French scientists who explored the mechanisms of hemolytic anemia in the early years of the twentieth century.4 Chauffard (1907),27 along with Trosier (1908)59 and Vincent (1909),60 described autohemolysins in patients with acute acquired hemolytic jaundice. These authors described patients whose serum had the capability of hemolysing RBC, and they termed the condition hemolytic icterus; it was acute in course and associated with hemoglobinuria. The reports of hemolysins, although incomplete and to some extent unsatisfactory, were pioneer ones well in advance of their time, and the idea that hemolytic anemia could occur apparently spontaneously in humans in consequence of the development of abnormal agglutinins or hemolysins remained controversial for the next 30 years or so.3 Chauffard also standardized the osmotic fragility test, described reticulocytes and their increased numbers in congenital hemolytic icterus (later to be known as hereditary spherocytosis), and drew attention to the microcytic nature of the RBCs in some hemolytic anemias.27 Between 1908 and 1912, Widal, Abrami, and Brule61,62 introduced the term acquired hemolytic anemia. These investigators described hemolytic

14

Immune Hemolytic Anemias

FIGURE 1-14. The original report published in 1904 by Dr. Julius Donath and Dr. Karl Landsteiner describing their current theories of the pathogenesis of paroxysmal cold hemoglobinuria and the development of the biphasic lysis test that remains the diagnostic laboratory procedure for the disorder. A translation of portions of the text follows (a more complete translation has been published by Bibel50). About Paroxysmal Hemoglobinuria Different theories have been proposed to explain the pathogenesis of paroxysmal hemoglobinuria, a peculiar illness whose attack under the inuence of cold leads to hemoglobinuria and removal of blood pigment through the urine. Other, older explanations state that hemoglobinuria is caused by the destruction of blood corpuscles in the kidney. But after Kessner showed that hemoglobinemia is present during such paroxysms, the cause was located in the blood. The hemolysis itself was thought to be dependent on various factors. The original belief that cold would destroy the red cells that are sensitive in this disease is in opposition to the commonly acknowledged fact that the blood of these patients in vitro is not more sensitive to cold than the blood of normal individuals. Therefore one had to look for other causes of the hemolysis. Recent extensive studies on blood toxins have suggested that this disease is caused by hemolysins. Authors have spoken for the hemolytic effect of these toxic substances. But numerous efforts to nd the toxic agents did not succeed exactly, or even to nd a test system that allows one to study the hemolytic procedure during the period of hemolysis. (From Donath J, Landsteiner K: Uber paroxysmale Haemoglobinurie. Munchen Med Wschr 1904;51:1590.)

FIGURE 1-15. The original protocol of the experiments performed by Donath and Landsteiner and their interpretation. A translation of the protocol is given in Figure 1-16. (From Donath J, Landsteiner K: Uber paroxysmale Haemoglobinurie. Munchen Med Wschr 1904;51:1590.)

Hayem 10 years earlier. The patients exhibited reticulocytosis, but the alterations in the fragility test were less marked than in the congenital form. Hence, at this time, the two types of hemolytic anemia were well dened: the congenital form of Minkowski and Chauffard and the acquired form of Hayem and Widal (Figs. 1-6 and 1-17).

icterus that was apparently neither congenital nor familial, that could appear gradually or suddenly during the course of various diseases, or that could be unassociated with any underlying disease. These cases were considered similar to those described by

THE ROLE OF THE SPLEEN AND THE EFFECT OF SPLENECTOMYThe above-cited brilliant studies clearly distinguished hepatic jaundice and the jaundice resulting from

Historical Concepts of Immune Hemolytic AnemiasHeld for 1/2 hr at 5, then 21/2 hr at 37 Ruby red Red Red Red Ruby red Ruby red Ruby red Ruby red Ruby red Ruby red Red Ruby red 0 0 0 Red tinged 0 0 0 0 0 0 0 0

15

Serum Patient K (hemoglobinuria) 4 Drops Patient R (hemoglobinuria) 10 Drops Patient N (hemoglobinuria) 7 Drops B.W. 6 Drops

Blood Cells 3 Drops Patient K B.W. Ch.G. A.R. Patient R B.W. Ch.G. A.R. Patient N B.W. Ch.G. A.R. B.W. Patient R Patient N Ch.G. Ch.G. Patient K Patient N Patient K Patient N Patient R B.W. Ch.G.

Held 3 hours at 37 0 0 0 0 Trace of red Trace of red Trace of red Trace of red 0 0 0 0 0 0 Trace of red Clear distinct red 0 0 0 0 0 0 0 0

Ch.G. 7 Drops

A.R. 6 Drops

FIGURE 1-16. It is shown with this sequence of experiments that the blood corpuscles of other individuals are hemolyzed by the serum of patients with hemoglobinuria, although to a lesser degree than their own blood corpuscles; however, in the same series of experiments, the blood corpuscles of the hemoglobinuric patients which have been cooled with other serum do not lyse when they are warmed afterwards. (Serum B. W. had a normal isolytic activity against the red cells of Ch.G. and N which was not increased by cooling.) Therefore, the unusual composition of the blood of the hemoglobinuric patients which is causing the lysis lies in the serum (respectively plasma), although the red cells may be easier to lyse (as shown in our Case K). The serum (plasma) of the hemoglobinuric patients contains a lytic substance that is effective against the patients and other human blood corpuscles. This lysis cannot be demonstrated directly by mixing the serum of the hemoglobinuric patient with his own or other red cells; however, one must consider the dependence of its effects on temperature.

not generally regarded as a signicant site of RBC destruction. Therefore, it is not surprising that in 1911, Micheli, in Turin, performed the rst splenectomy for acquired hemolytic anemia.64 Banti65 (Fig. 1-18),66 in 1912, conducted an extensive investigation into splenic pathology and introduced the term hemolytic splenomegaly, when he observed that the spleens of animals undergoing hemolysis were enlarged and congested.65 He also noted that heteroimmune hemolytic serum, when transfused into splenectomized animals, led to less and slower hemolysis than that seen with normal animals. He implicated the splenic endothelial cells as erythrophagocytes and described agglutinated erythrocytes within the splenic pulp. Banti similarly showed that the Kupffer cells of the liver could have an erythrophagocytic function when intense hemolysis was present. Thus, Banti effectively described the reticuloendotheilal system and its function in RBC hemolysis.4 He recognized the importance of the spleen to the disease, but stressed that it was not the only, nor even the prime, site of RBC destruction. The combined activities of Micheli and Banti entrenched the recommendation of splenectomy as a treatment for hemolytic anemia, representing the rst specic therapy for AIHA. Despite the widespread acceptance of the benets of splenectomy, however, some, such as Antonelli, in 1913 refuted Bantis hemolytic splenomegaly as a separate disease, pointing out that it did not differ from acquired hemolytic anemia.4

FURTHER CHARACTERIZATION OF HEMOLYTIC ANEMIASWorld War I brought a halt to investigation and case reports of hemolytic icterus.7 By the 1920s, the prevailing understanding of the mechanism behind RBC destruction was that it resulted from autoagglutinininduced agglutination, the rst step in hemolysis. However, publications after World War I indicated the degree to which much of the knowledge discovered at the beginning of the century had been lost. Lederer (1925)67,68 and Brill (1926)69 described a number of cases of transfusion-responsive acute hemolytic anemia associated with infectious diseases. Because much of the prior French work had been forgotten, Lederers descriptions were thought to be of a new disease, in spite of the extensive review of hemolytic icterus by Tileston70 just 3 years earlier. Such cases became known as Lederers anemia or Lederer-Brill anemia, but it is likely that they were examples of AIHA.1 The hiatus in studies concerning hemolytic anemias obliterated the clear distinction between congenital and acquired forms of hemolytic anemia established by the French investigators. Indeed, Dacie71 states that it was generally assumed at that time in England that hemolytic anemia occurring in the adult was a latent form of hereditary spherocytosis. The lack of specic diagnostic procedures, the presence of spherocytes in

premature and excessive destruction of erythrocytes. The hemolytic process was further differentiated to include both congenital and acquired forms. Although the phenomenon of agglutination had been well described in the latter part of the nineteenth century, it was Widal, Abrami, and Brul who observed autoagglutination of erythrocytes,4 and their work as well as others was summarized at the twelfth session of the Congrs Franais de Medicina, which took place in Lyon in 1911.63 The topic was the role of hemolysins in pathology, and papers were presented by many of the foremost physicians and pathologists of the day. By now the role of the spleen was widely accepted as being the major site of hemolysis, and the liver was

16

Immune Hemolytic Anemias

FIGURE 1-17. Fernand Widal. (From Packman CH: The spherocytic haemolytic anaemias. Br J Haematol 2001;112:888899.)

both forms of hemolytic anemia and the unavailability of serologic testing made such a conclusion inevitable.1 In 1938 and 1940, important contributions were made by Dameshek (Fig. 1-19) and Schwartz.72-74 These workers published a remarkable review of

acquired hemolytic icterus in 1940 that was 96 pages in length with 380 references. They identied 81 articles that described cases tting their concept of acute (acquired) hemolytic icterus. Based on their own clinical observations of hemolysins in some patients,

FIGURE 1-18. Guido Banti (18521925) was one of the rst physicians who might properly be called a hematologist. A contemporary of Osler, he worked at a time when the methods and laws of biological research were just developing. Medical discovery was commonly a consequence of clinical insight aided only by physical examinations and necropsy. The titles of Bantis earliest publications give the direction of his lifelong interests: Splenic anemia and Enlargement of the spleen with cirrhosis of the liver. His efforts to dene these conditions as entities came to nothing, but the discussions about them did much to demonstrate the essentialness of method in clinical research. (From Crosby WH: The spleen. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:97138. Reproduced with permission of The McGraw-Hill Companies.)

Historical Concepts of Immune Hemolytic Anemias

17

FIGURE 1-19. William Dameshek (19001969), one of the most eminent of American hematologists of his era, was a strong proponent of the concept of autoimmunity at a time that others were reluctant to accept that a patient could produce autoantibodies. His extensive writings and teachings had a major inuence on the gradual acceptance of an autoimmune etiology for some types of acquired hemolytic anemias. (From Crosby WH: The Spleen. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:97138. Reproduced with permission of The McGraw-Hill Companies.)

cases reported in the literature, including those of Chauffard and coworkers, and their own experiments involving injection of varying amounts of hemolytic serum into guinea pigs, they proposed that all cases of hemolytic icterus were a result of hemolysins. The differences in clinical manifestations, ranging from mild congenital cases to fulminant acute hemoglobinurias, were accounted for by the dose of hemolysin.7 Dameshek and Schwartzs general thesis that hemolysins were responsible for the development of many cases of acquired hemolytic anemia was correct. However, they were incorrect in extrapolating their concept of the role of hemolysins to congenital hemolytic jaundice (hereditary spherocytosis) and in concluding that that disorder might be caused by the more or less continued action of an hemolysin. These studies reawakened interest in acquired hemolytic anemia and laid the broad outline for our modern concepts of the clinical and serologic implications of AIHA.1 However, the difculty in ascribing cases of acquired hemolytic anemia to the development of hemolysins was that they could not be demonstrated in the vast majority of cases by the serologic techniques then available.

Thereafter, during subsequent decades, the classication and serological characteristics of the various AIHAs were delineated, in large part through the extensive and meticulous work of Sir John Dacie in London.5,74a

MEASUREMENTS OF RED BLOOD CELL SURVIVALIn a review in 1923, Payton Rous (Fig. 1-20) discussed the question of whether the RBCs had a denite, as opposed to an almost indenite, sojourn in the blood, and, if nite, how long was their life span.75 In fact, he did not doubt that their life span was limited, and he listed a number of cogent arguments in favor of this view. For example, he cited the continuous activity of broadly distributed hematopoietic tissue and the daily excretion through the bile of a pigment nearly if not precisely identical with one of the pigmented derivatives of hemoglobin.75 The question as to how long RBCs circulate before undergoing destruction had been a vexing question for many years. A variety of methods and calculations had been used to come up with some answers, ranging from observations of

18

Immune Hemolytic Anemias

FIGURE 1-20. Peyton Rous (18791970). (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:211255.)

the time it took for the RBC count in a hypertransfused animal to be restored to normal to calculations based on bile excretion. The conclusions drawn from these studies were, however, erroneous. Data of Winifred Ashby. The conclusions of only one observer stood out in striking contrast to the above observationsthose of Winifred Ashby (Fig. 1-21), whose rst papers76,77 were published in 1919 (reviewed by Dacie3). Ashby described in her rst paper how she had transfused group IV (type O) blood into seven group II (type A) recipients who were suffering from various anemias and how she had been able to count the free (unagglutinated) type O RBCs by making suspensions of posttransfusion blood in an anti-A serum (Fig. 1-22). She concluded that transfused RBCs live a long time, 30 days or longer, and that the benecial results of blood transfusion are not due to the stimulation of the bone marrow (a view held by some

at the time) but to the functioning of the transfused RBCs. By 1921, Ashby78 was able to report on more than 100 patients. In four patients who were followed until the elimination of the transfused RBCs was complete or almost complete, this did not take place until 83 to 100 days after transfusion. One of the difculties inherent in Ashbys work, which she could not circumvent, was that she was not measuring the life span of the RBCs in their own environment. This raised the question of whether the foreign cells might have a different survival than those of the host, a point that she was unable to resolve. Additional Studies Using Differential Agglutination. In 1928 differential agglutination was also used in the reverse way by Landsteiner, Levine (Fig. 1-23) and Janes79 and Wiener.80 Wiener reported that he had detected blood group M (or N) cells, using anti-M (or anti-N) sera, in the circulation of N (or M)

Historical Concepts of Immune Hemolytic Anemias

19

FIGURE 1-21. Winifred Ashby (18791975). (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGrawHill Book Company, 1980:211255.)

recipients for between 80 and 120 days after transfusion. Wiener also used the Ashby method, using antiM (or anti-N) sera to agglutinate the recipients RBCs, and observed that between one third and one fourth of the transfused RBCs disappeared each month; he remarked that this continuous decrease in numbers was to be expected on the assumption that all the cells had approximately the same life span. He concluded, Curiously enough, despite all this work, most textbooks still give the life of the erythrocyte as thirty days. Ashbys data and conclusions are now known to be generally correct. But she was ahead of her time; her papers remained on library shelves largely unread and her technique was relatively unused until the late 1930s. In Oslo, Dedichen81 conceived the idea that it might be possible to obtain evidence by transfusion experiments as to which of the two current theories about the pathogenesis of ictere hemolytique (hereditary spherocytosis) was correct; hyperactivity of the organs of hemolysis or production of cells with less than normal resistance. However, for technical reasons, his experiments were unsuccessful, and more than a decade was to pass before similar (but more successful and decisive) experiments were again undertaken. Intrinsic and Extrinsic Mechanisms of Hemolysis. Dacie (Fig. 1-24) and Mollison (Fig. 1-25)82 rst applied Ashbys technique in patients with

FIGURE 1-22. Reproduction of one of Ashbys original gures. (A) A suspension of group II (type A) red cells in an anti-A serum. Relatively few cells are free and unagglutinated. (B) A similar preparation after the transfusion of group IV (type O) red cells. Many of the cells are now free and unagglutinated, the great majority being transfused cells. (From Dacie JV: The life span of the red blood cell and circumstances of its premature death. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:211255.)

hemolytic anemia over 20 years after her publication. They were able to show that normal RBCs transfused into patients with familial hemolytic anemia survived normally, for approximately 100 to 120 days. The survival curves from their paper are shown in Figure 1-26. In sharp contrast, Loutit and Mollison83 noted that normal RBCs transfused into patients with acquired hemolytic anemia exhibited markedly reduced survival. Loutit and Mollison83 also transfused RBCs from patients with congenital and acquired hemolytic icterus into normal recipients and followed their survival. The RBCs from patients with congenital acholuric jaundice, including those from a patient who had undergone splenectomy, exhibited short survival. The tracing by differential agglutination, as introduced by Ashby, demonstrated a clear distinction

20

Immune Hemolytic Anemias

FIGURE 1-23. Philip Levine. (From Diamond LK: The story of our blood groups. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:691717. Reproduced with permission of The McGraw-Hill Companies.)

between the two major groups of cases. In one group, transfused blood survived normally, and in another group of patients, it was destroyed along with the patients own blood. These observations supported the idea that there might be intrinsic and extrinsic mechanisms for increased hemolysis. Later, the distinction was used as a rational basis for classication of the hemolytic anemias.

THE ANTIGLOBULIN (COOMBS) TESTA major diagnostic advance was the development of the antiglobulin test, the discovery of which is an interesting aspect of the history of AIHA. The events leading to its discovery have been documented by Dr. Robin R. A. Coombs84,85 (Fig. 1-27). He points out that immunology in the 1940s was somewhat elementary, unsophisticated, and phenomenologic. The real nature of antibodies was still uncertain, but seemed to be associated with the serum globulins. After graduating in veterinary medicine in 1943, he joined an investigation on the serodiagnosis of Pfeifferela mallei infection, which causes a very

serious disease in horses and humans and for which there was no cure at that time. He later continued his work at Cambridge in the University Department of Pathology. Two eminent serologists, Robert Race (see Fig. 1-25B) and Arthur Mourant, were working in the department at that time. Race86 and Weiner,87 working separately, had by this time concluded that there were two types of Rh antibody: one that bound to the RBC surface and caused agglutination (the complete antibody) and another that absorbed to the RBC surface but did not cause agglutination (the incomplete antibody). Coombs, reminiscing in 1998,84 states, At coffee one day, discussion turned to Robs so-called blocking or incomplete antibody. What was the nature of this antibody, if indeed it was an antibody? Rob stressed that there was a real need for a better test (than his blocking test) to measure these so-called incomplete antibodies. The next step occurred on a late-night ill-lit train from London back to Cambridge. I was pondering on how to measure these incomplete antibodies on red cells with pictures in my head of Ehrlichs side-chain theory. In a ash I could see the globulin antibody on the red cells, and these red cells should be agglutinated with an anti-

Historical Concepts of Immune Hemolytic Anemias

21

FIGURE 1-24. Professor Sir John Dacie laid the foundation for the investigation of hemolytic anemias. His persistence and experimental approach enabled him to demonstrate the vast complexity of the factors involved in the anemias due to hemolysis, and for this he has justiably been considered a pioneer.81a He was also responsible for training many hematologists from numerous countries, including the present authors. (From Wintrobe MM: Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:XVIII. Reproduced with permission of The McGraw-Hill Companies.)

body to serum globulin, i.e., an antiglobulin. All the necessary thinking had been done! Coombs obtained some very crude [rabbit] antihuman globulin serum from a coworker and the very rst experimental protocols with Race and Mourant showed quite clearly that the procedure was going to work. They absorbed the antiglobulin serum (AGS) with human group AB Rh-positive RBCs and then incubated Rh-positive RBCs in sera known to contain incomplete Rh antibodies. The sensitized cells agglutinated in the antiglobulin serum and the appropriate controls were negative. The rst account of what we now call the indirect antiglobulin test was published by Coombs, Mourant, and Race in 1945.88 The authors were bold enough to state, This test may have useful applications in detecting ne degrees of sensitization in other antigen-antibody systems. . . . This has turned out to be an understatement, for quite apart from the tests on red cells and bacteria covering all the isotypes of antibody, an antiglobulin step or stage is a regular component in very many immunoassay procedures.85 A more substantial paper89 was published in the same year in the British Journal of Experimental Pathology, and just as the printers page proofs were on the point of dispatch back to the publisher, Mourant came across a paper in the German literature from 1908 by Carlo Moreschi90 (Fig. 1-28) that described enhancement of red cell agglutination with an antiserum to serum. An acknowledgment was

added to the proofs as an addendum. Coombs states, The lesson is that one should never refer to a discovery or a test as being new.84 Coombs, Mourant, and Race next went on to demonstrate RBC sensitization in babies with hemolytic disease of the newborn using the direct antiglobulin test (DAT).91 Cord RBCs from patients agglutinated when exposed to the antihuman antiglobulin reagent, but cells from healthy babies did not agglutinate. One of the positive tests they observed in newborns appeared at rst to be a false positive since there were no Rh antibodies in the mothers serum. However, Race went on to demonstrate the test was a true positive but that it was not caused by an Rh antibody. The mothers name was Kell, and this was the start of Races research on the Kell blood group system. In 1947, Coombs and Mourant92 demonstrated that the component in AGS that reacted with RBCs coated with Rh antibody was in all probability an antigamma globulin. They showed that the addition of a small amount of gamma globulin to the antiglobulin serum rendered it incapable of agglutinating cells coated with Rh antibody, whereas the addition of alpha globulin or beta globulin had only a slight effect, which could be ascribed to contamination with traces of gamma globlulin. An interesting phenomenon observed by Dacie93 was that the addition of gamma globulin to AGS produced a reagent that could discriminate between the RBCs of individual patients with AIHA. Thus, although in many instances the positive antiglobulin reaction was abolished by adding the gamma globulin, this was not true in all cases. It seemed clear that in those cases in which the reaction was inhibited, the autoantibody on the cell was itself a gamma globulin, but that when the reaction was not affected, the material on the RBC surface could not be gamma globulin. The nongamma protein was eventually shown to consist of components of complement xed to the cell as a result of antibody-antigen interaction.93,94 Use of the Antiglobulin Test to Distinguish Immune from Nonimmune Acquired Hemolytic Anemias. At the time of the discovery of the antiglobulin test, there was great difculty in distinguishing hemolytic anemia that was familial from that which was acquired. The only laboratory test available was the measurement of osmotic fragility, which was abnormal in familial hemolytic icterus (now called hereditary spherocytosis). However, Dameshek and Schwartz74 pointed out that spherocytes causing increased osmotic fragility could develop in cases that were clearly acquired hemolytic anemia. Barbara Dodd described the fact that she and Kathleen Boorman, who were working at the South London Transfusion Centre with the director, John Loutit, who was already an authority in the eld of anemias, were in a privileged position.95 They had visited Cambridge, where Race revealed to them the secrets of the antiglobulin test before it had appeared in print. Dodd states that, I shall never forget the gleam

22

Immune Hemolytic AnemiasFIGURE 1-25. (A) Patrick Mollison. (B) A 1947 photograph taken at the Lister Institute in London showing, from left to right: Louis K. Diamond whose research claried the pathogenesis of hemolytic disease of the fetus and newborn as well as the optimal management of that disorder; Patrick L. Mollison, a pioneer in the eld of blood transfusion and editor of ten editions of the famous text, Blood Transfusion in Clinical Medicine; Robert R. Race, an eminent immunohematologist who, along with his long-time collaborator, Ruth Sanger, made innumerable contributions to the eld of RBC genetics and serology; and Sir Ronald A. Fisher, a famous geneticist/biostatistician who, together with Race, devised a classication of the Rh blood group system that is still used. (Courtesy of Professor P. L. Mollison.)

A

B

100

FIGURE 1-26. Dacie and Mollison, using the Ashby technique, were the rst to demonstrate that normal RBCs survive normally in patients with familial hemolytic anemia. The gure shows survival of RBCs from normal donors after transfusion to six patients with familial hemolytic anemia. Case 3 was an Rh-negative patient who was later found to have developed an alloantibody to Rh, accounting for the shortened survival of transfused Rh-positive RBC. Although not shown in the gure, survival in cases 2 and 5 was followed to completion and found to exceed 100 days in each case. The dotted lines indicate the limits of survival in a group of normal recipients (Mollison, unpublished observations). (From Dacie JV, Mollison PL: Survival of normal erythrocytes after transfusion to patients with familial haemolytic anaemia (acholuric jaundice). The Lancet, volume i, May 1, 1943, pp 550552.)

80 Percentage survival

60

5

2

40Ca

Casse

e1

20

4

Ca se 3

Case0 0 20 40 60 80 Days after transfusion 100

6120

Historical Concepts of Immune Hemolytic Anemias

23

FIGURE 1-27. Robin R. A. Coombs. (Photograph by Lawrence E. Young M.D., Fellows Garden, Kings College, Cambridge University, 1950. From Packman CH: The spherocytic haemolytic anaemias. Br J Haematol 2001;112:888899.)

FIGURE 1-28. A photographic portrait of Carlo Moreschi. (From Coombs RR: Historical note: Past, present and future of the antiglobulin test. Vox Sang 1998;74:6773.)

in his eye when we returned from Cambridge with a description of the new test! They quickly collected the RBCs of 17 patients with familial hemolytic anemia and 5 others with hemolytic anemia of the acquired type. It was enormously exciting then, but no surprise now, to nd that the 5 patients having acquired type had positive DATs, whereas the 17 familials were negative. They concluded (correctly) that the agglutination tests will discriminate the congenital from the acquired form [of hemolytic icterus], and that it indicates that the acquired form is due to a process of immunization, whereas the congenital form is not. Thus, not only had they found a test that would distinguish between the familial and acquired forms of hemolytic anemia, but they had also demonstrated a difference in their etiology. A Note about Carlo Moreschi. Carlo Moreschi was deep in immunological research at Pavia at the turn of the twentieth century. He published two particularly interesting papers90,96 describing enhancement of agglutination with antiserum to serum (i.e., with antiglobulin) (Table 1-1). However, incomplete antibodies were unknown at the time and general acceptance or use of this procedure never resulted. Dr. Coombs paid tribute to Moreschi and his researches in a lecture to the Italian Association of Medical Analysts and Pathologists entitled Moreschi and Some Recent Developments in Agglutination. There seemed to be little

interest in the agglutination or in Moreschi himself. However, 6 months after the lecture was published in the Italian medical journal lInformatore Medico,97 Dr. Coombs received a letter from Dr. Pietro de Ruggieri, who was a steroid chemist in Milan and who was a nephew of Carlo Moreschi. He was delighted with the reference to his long-since-dead uncle.

THE CONCEPT OF AUTOIMMUNE HEMOLYTIC ANEMIAIn 1951, Young and associates98 were the rst to coin the term autoimmune hemolytic anemia. It was theorized that the production of an autoantibody was the result of a breakdown in the regulatory contrivances, thus leading to autoimmunization. However, the concept that a patient could produce autoantibodies was vigorously resisted by some. Witebsky,99 in particular, was reluctant to draw the conclusion that the RBC coating material demonstrated by the antiglobuin test was a true autoantibody. He considered it unproved that the RBC could be involved in autoimmunization, with the implied breaking of the principle of horror autotoxicus. This reluctance to accept the autoimmune nature of antiglobulin testpositive hemolytic anemias led to the use for a time of the noncommittal term antiglobulin-positive hemolytic anemia.100

24

Immune Hemolytic Anemias

TABLE 1-1. TRANSLATED FROM MORESCHI (1908), DEMONSTRATING THE PRINCIPLE OF THE ANTI-GLOBULIN (COOMBS) REACTIONGoat Immune Serum or Goat Normal Serum 0.005 mL 0.005 mL 0.005 mL 0.005 mL 0.005 mL 0.005 mL 0.005 mL 0.01 mL Cells centrifuged and washed with normal saline Rabbit Precipitating Serum 0.0001 mL 0.005 mL 0.001 mL 0.005 mL 0.01 mL 0.05 mL 0.1 mL 0.1 mL 2 hr room temperature Agglutination with Immune Serum 0 Scant Marked Very marked Very marked Very marked Very marked 0 0 Normal Serum 0 0 0 0 0 0 0 0 0

Rabbit RBCs 1 1 1 1 1 1 1 1 1 2 mL mL mL mL mL mL mL mL mL hr room temperature

Rabbit RBCs were incubated with goat immune serum, washed, and incubated with rabbit antibody to goat serum (precipitating serum). The RBCs agglutinated in a dose-dependent manner. The controls, lacking either goat immune serum or rabbit precipitating serum, showed no agglutination. Reproduced with modication from Packman CH: The spherocytic haemolytic anaemias. British Journal of Haematology 112:888899.

Through the extensive writings and teaching of such eminent physicians as Dameshek, the concept of an autoimmune etiology for some types of acquired hemolytic anemias gradually obtained general recognition and application.1

RADIOACTIVE CHROMIUM (51CR) AND DF32PThe rst studies using 51Cr were reported by Gray and Sterling101 in 1950 from Boston. They found that the labeled RBCs lost radioactivity at a rate more rapid than could be predicted from the known normal life span of dog RBCs and, consequently, did not recognize the potential usefulness of the method in determining long-term RBC survival.102 Later, Ebaugh and coworkers103 labeled normal blood with 51Cr and transfused it into normal human volunteers. Subsequently, the amount of radioactivity per milliliter of RBCs was quantitated and a simultaneous evaluation was made of the RBC survival by the Ashby differential agglutination technique. They found that the two curves reached extinction point at the same time. Calculations of the two curves were consistent with the hypothesis that chromium was leaking from the RBCs in an exponential fashion with a mean half-life of 77 12 days. Correcting for this leakage, the curve for the two techniques approximated that determined by the straightline Ashby differential agglutination survival curve.103 The value of the isotope as a harmless label of RBCs was soon conrmed in many centers throughout the world, and because the 51Cr could be used to label patients own RBCs and to study their survival in their own circulation, as well as to label transfused blood, Ashbys elegant but laborious technique, with its inherent limitations and technical difculties soon became obsolete. 51Cr is still widely used in studies of RBC life span and in the measurement or blood volume, although it

is not an ideal label because of the elution of the label from the RBCs. The nearest rival to 51Cr is DF32P, which was rst reported in 1954 to be a potentially a satisfactory label for RBCs.104 The DF32P technique has the advantage over 51Cr in that once attached to the RBCs, it is not eluted. The elimination curve of normal RBCs in a healthy recipient, as demonstrated by the Ashby method or by the use of DF32P, is virtually a straight line, and this is consistent with the concept of gradually increasing senescence rather than of random elimination in which the cells would be destroyed indiscriminately regardless of age. Indeed, the analysis of survival curves has contributed most signicantly to the understanding of the pathogenesis of increased hemolysis.3

COLD AGGLUTININ SYNDROME (CAS)Cold agglutinins were initially demonstrated by Landsteiner in animal blood in 1903105 and in human blood by Mino in 1924,106 but their signicance in human disease was not accurately appreciated until several decades later. The rst determination of titers in an acute postpneumonic cold agglutinin disease was made by Clough and Richter in 1918.107 A recognition of the relationship between cold agglutinins, hemolytic anemia, Raynauds phenomenon, and hemoglobinuria began to emerge with the case reports of Iwai and Mei-Sai in 1925 and 1926.108,109 Their rst patient was a 36-year-old Chinese man giving a 6-year*As mentioned in Chapter 2, describing the skin manifestations in cold agglutinin syndrome as Raynauds phenomena is, strictly speaking, incorrect.110 Raynauds disease, the consequence of vasoconstriction, leads in sequence of three phenomena: First, the affected part becomes white and perhaps numb; then it becomes swollen, stiff and livid; and nally, when the vasoconstriction passes off, the part becomes red due to reactive hyperemia. In CAS the changes, which preferably are termed acrocyanosis, or literally blue extremity, differ from those of Raynauds disease in the absence of an initial white phase because there is no

Historical Concepts of Immune Hemolytic Anemias

25

history of Raynauds disease.* His serum contained a cold agglutinin that reacted to a titer of 1,000 at 0C and reacted up to 30C against normal RBCs as well as those of the patient. They demonstrated that the circulation of the patients blood through ne tubes was impeded when the blood was cooled to 5C and suggested that the Raynauds phenomenon might be related to mechanical obstruction by autoagglutinated RBCs. In their second patient, a woman aged 78, they showed that cooling of the ngers was associated with breaking of the column of blood in the capillaries of the nail bed. However, in neither case did the authors describe hemoglobinuria or anemia. Druitt,113 writing from Madras in 1873, described in detail the history of a doctor, aged 51 years, who over a period of at least 6 years had experienced attacks of numbness of the feet and a purplish blue discoloration of the hands on exposure to cold. These attacks might be followed by the passage of hematinuria. The patient obtained relief from