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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,
vi
Preface to the Second Edition
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
viii
Preface to the First Edition
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!
C
H
A
P
T
E
R
1
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
2
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.)
4
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
6
Immune Hemolytic Anemias
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
7
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.)
8
Immune Hemolytic Anemias
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.
12
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