Immune Hemolytic Anemias || Historical Concepts of Immune Hemolytic Anemias

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C H A P T E R 1

Immune hemolysis is a short-ening of red blood cell (RBC)survival due, directly or indi-rectly, to antibodies. These anti-bodies may be autoantibodiesor alloantibodies. This chapterwill deal mainly with historicalaspects of autoimmune hemo-lytic anemia (AIHA), followedby a brief discussion of histori-

cal aspects of hemolytic transfusion reactions.AIHA is an acquired immunologic disease in which

the patient’s RBCs are selectively attacked anddestroyed (hemolysed) by autoantibodies producedby the patient’s own immune system. Shortened RBCsurvival is frequently associated with the presence ofa reticulocytosis, spherocytes in the peripheral bloodfilm, autoantibodies in the patient’s serum, and occa-sionally splenomegaly, hemoglobinemia, and hemo-globinuria. Although these facts are commonknowledge now, it was not always so. Reviewing howthese concepts developed over the centuries by obser-vation and clinical and laboratory experimentation isboth fascinating and instructive.

It is evident that concepts that collectively led to ourpresent understanding of AIHA required knowledge ofthe existence of RBCs, understanding the possibility ofanemia without blood loss, distinguishing hemoglo-binuria from hematuria, understanding the mechanismby which hemoglobinuria occurs, recognizing the

process of agglutination, understanding the distinctionbetween congenital and acquired disorders, under-standing that premature destruction of RBCs can causeanemia and jaundice, recognizing spherocytes andabnormal osmotic fragility of RBCs and determiningtheir significance in patients with hemolysis, recogniz-ing reticulocytes, determining that serum antibodiesmay cause destruction of foreign cells and also autolo-gous cells, developing means to measure RBC survival,developing diagnostic assays for antibodies, refutingthe concept of horror autotoxicus, and understanding therole of the spleen and splenectomy.

The discoveries that led to the development of ourknowledge about these concepts are herein reviewedin the approximate order in which the relevant obser-vations were made. Here, then, is how our knowledgeof AIHA came to be. The development of this shortreview was aided significantly by previous reviews onvarious aspects of hemolysis and AIHA.1-9

THE LESSONS OF HISTORY

Everyone who studies the stories of discovery in whathas come to be called the field of hematology will rec-ognize the early gropings in the midst of profoundignorance and the difficulties that confronted theinvestigators. We have gained an understanding ofbiology that could hardly have been dreamed of onlya short time ago, let alone at the time of the first

Historical Concepts of Immune HemolyticAnemias

2 Immune Hemolytic Anemias

tentative forays into the unknown. Moreover, under-standing has been crowned by tangible benefits forhumanity. It is worthwhile to consider how such greatprogress comes about and why. How is knowledgeachieved, and what can we learn from the process bywhich important discoveries were made?10

The first lesson to be learned of history is that thepath of progress is anything but straight. The courseof research has been likened to the flow of a streamthat ultimately becomes a rushing torrent whoseimportance is obvious. This certainly has been thehistory of research in hematology.

It certainly does not follow that, because a conceptis plausible and is in accord with the understanding ofthe time, it is necessarily correct. The following pagesprovide many examples of misinterpretations result-ing from such an assumption. Furthermore, becausethey have been plausible, such views often haveendured and have stood in the way of acceptance ofobservations and interpretations that proved to be thecorrect ones.

Discovery begins with an observation or the posingof a question. But observation is not as simple as itsounds. Indeed, many look but few see. It is the excep-tional person who recognizes the unusual event ormanifestation. Still fewer pursue it to new under-standing. Many may ask questions but few have theimagination, the energy, and the overpowering driveto persist in the search for an answer, especially whenthis must be done in the face of difficulties and fail-ures and even despite scorn from their peers.

Imagination and industry alone, however, have notsufficed. Means have had to be devised to explore thequestions that were posed. When these were provided,it is impressive to see what the introduction of a newtechnique made possible for an area of inquiry. Asimple example, described later, is the introduction ofthe antiglobulin test, which very rapidly led to a muchclearer distinction between immune and nonimmunehemolytic anemias.

Progress depends on the contributions of many.Moreover, scientific discipline has benefited fromdevelopments in other fields, progress in one fieldspurring another, and vice versa. As knowledge hasgrown, it has become impossible for a single humanbeing to encompass the whole, and the discovery andgrowth of understanding have become more and moredependent on interchange among scientific disciplines.

Still another aspect of the progress of understand-ing is worth noting. It is not generally appreciatedhow often curiosity concerning an observation madeat the bedside by clinicians has led to far-reachinginvestigations. An example is the observation of hemoglobinuria, which led to the understanding ofdestruction of RBCs and to the early delineation ofcertain clinical syndromes (e.g., paroxysmal coldhemoglobinuria [PCH], paroxysmal nocturnal hemo-globinuria [PNH], and march hemoglobinuria) char-acterized by hemoglobin in the urine.

Investigators have not always been farseeing andlogical, moving steadily and directly to their goal, nor

did they fail to make mistakes. Indeed, incorrecttheories have hampered the advance of knowledge,especially when these theories were widely dissemin-ated and were pronounced by eminent authorities. Anumber of such examples appear in the followingpages.

It follows that authorities must be humble andnovices skeptical.

EARLIEST DESCRIPTIONS OF POSSIBLEACQUIRED HEMOLYTIC ANEMIA

The first written description of what may have beenan acquired hemolytic anemia, albeit not of animmune nature, was Galen’s description in 150 AD ofa person bitten by a viper whose “skin turned thecolor of a ripe leek.”1,4,11 Galen’s understanding ofphysiology was such that he implicated the spleen asleading to the skin discoloration, an association of thespleen and hemolysis that was not confirmed until thelate nineteenth century.1

PCH may have been described as early as 1529 byJohannes Actuarius, a court physician in Constan-tinople. In his work, De Urinis, Acturarius described acondition in which the urine is “azure & livid as wellas black” in patients being of melancholic humor andcomplaining of loss of strength, after an exposure tocold.4 Further mention of PCH seems, however, to beabsent for nearly 300 years, until the latter half of thenineteenth century.

EARLY EXPERIMENTAL INVESTIGATION OF BLOOD

Description of Red Blood Cells. The development ofthe scientific method led to the seminal discoveries ofthe circulation of blood by Harvey in the early sixteenthcentury and the cardinal experiments with transfusionof blood by Lower in England and Denis in Paris in themid-seventeenth century. Despite this interest in blood,the discovery of the RBCs had to await the appearanceof the microscope around 1650. The first observation ofan RBC was likely made by Malpighi in 1661, when hedescribed the circulation of RBCs in the capillaries, andthis was followed in 1663 by Swammerdan’s descrip-tion of minute globules in the blood of a frog. A decadelater, human RBCs were described in detail by vanLeeuwenhoek (Fig. 1-1),12 who also established theirsize at about 1⁄3000 of an inch by comparing an RBC witha grain of sand of known size.

John Huxham, in 1770, described the changingshapes of degenerating RBCs and, importantly, recog-nized that such cells were the origin of hemoglobin.4

Anemia without Blood Loss. In 1843, Andral (Fig. 1-2) described a spontaneous anemia, whicharises without any prior blood loss.13 He quantifiedred blood globules in healthy patients and reported

Historical Concepts of Immune Hemolytic Anemias 3

FIGURE 1-1. Antonj van Leeuwenhoek (1632–1723). (FromWintrobe MM: Milestones on the path of progess. In: WintrobeMM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill BookCompany, 1980:1–31.)

FIGURE 1-2. Gabriel Andral (1797–1876). (From Wintrobe MM:Milestones on the path of progress. In: Wintrobe MM (ed): Blood,Pure and Eloquent. New York: McGraw-Hill Book Company,1980:1–31.)


16 early case of anemia. Although he provided noother information concerning the patients’ condition,what is important in relation to hemolytic anemia isthe observation of anemia without prior blood loss.

Hemoglobinuria. Vogel, in 1853,14 stated that thematter in the urine is the same as that in the blood andsuggested that the matter in the urine consists of a“decomposition of blood discs.” He suggested thatthe degree of blood decomposition can readily beascertained by the degree of coloration in the urine,and he indicated a connection between fevers, coloredurine, decomposition of blood discs, and anemia. Thisrepresents one of the early examples of the associationbetween a decreased RBC count and the term anemia.It also represents early evidence suggesting thatanemia may be secondary to infections.

RED BLOOD CELL AGGLUTINATION

The description of the phenomenon of RBC agglutina-tion and its development as a tool in elucidating bloodgroups took place in the last 30 years of the nineteenthcentury in Germany and Austria, and were reviewed indepth in 2002 by Hughes-Jones and Gardner.15 The dis-coveries were largely the work of three people: AdolfCreite, a medical student in Göttingen, Germany;Leonard Landois, Director of the Physiological Instituteat the University of Greifswald, Germany; and KarlLandsteiner, working in the Pathological AnatomyInstitute in Vienna, Austria.15

Adolph Creite. Creite’s (Fig. 1-3) almost unknowncontribution was published in 1869 under the title“Investigations concerning the properties of serumproteins following intravenous injection.”16 His workis quite remarkable in that it showed that serum pro-teins had the property of both “dissolving” and bring-ing about “clustering” of red cells, that is, lysis andagglutination in present-day terms, anticipating thediscovery of antibodies by a quarter of a century.

Creite injected sera from calf, pig, dog, sheep, cat,chicken, duck, and goat into rabbits. The first threehad little or no effect on the recipient, but the sera ofthe latter five almost always resulted in the appear-ance of “blood-stained urine,” general malaise, anddeath of the animal. He noted that the urine was freeof intact RBCs. He concluded that serum containsagents that are able to dissolve red cells “directly.” Heperformed additional experiments in which heremoved protein from the serum before its injectionand observed that “all of the urine samples examineduntil the evening of the following day are normal.”Accordingly, he concluded that the most likelyactive ingredients were serum proteins, but added,“However, I cannot say how they function.”

He also performed in vitro experiments and pro-vided a remarkably clear account of what is probablythe first description of agglutination. He reported, “Ifyou add blood serum from any of the animals withwhich I have carried out my experiments to a drop of

fresh rabbit blood, then you observe under the micro-scope that in the regions where the foreign serummixes with the rabbit red cells, the cells suddenly flowtogether in a peculiar way forming different shapeddrop-like clusters with irregular branches. I believedthat I had found an explanation for the appearance ofblood in the urine, as it was possible that some bloodcells had dissolved completely.”

Leonard Landois. RBC agglutination and lysis wereput on an even firmer basis by Landois, who pub-lished an extensive monograph on the subject oftransfusion,17 which included a section describing hisin vitro experiments. In his experiments, Landois wassuccessful in demonstrating both lysis and agglutina-tion. (It should be noted that the terms lysis and agglu-tination were not in use until the end of the nineteenthcentury. For lysis, both Creite and Landois used aGerman word meaning “dissolve”; for agglutination,words translatable as “accumulation,” “ball forma-tion,” or “sticky clumps” were used.) Landois alsodistinguished agglutination from rouleaux, for whichhe used the term, “like rolls of coins.”

Landois added 4 to 5 mL of clear serum into a testtube and then added fresh defibrinated blood. Heincubated the mixture at 37°C to 38°C or at room tem-perature and observed the initiation of the RBC lysis.

FIGURE 1-3. Adolf Creite, about 1920. (From Hughes-Jones NC,Gardner B: Red cell agglutination: The first description by Creite (1869)and further observations made by Landois (1875) and Landsteiner(1901). Br J Haematol 2002;119:889–893.)


“Sooner or later the mixture becomes completely clearand transparent and the cells are no longer visible. Iobserve the whole process of the lysis and the changesin red cell shape under the microscope.” Commentingon another experiment on the mixing of cells andserum, Landois described the changes in shape ofRBCs and added, “The cells develop the ability tostick to neighboring cells” and “form larger or smallerclumps.”

Karl Landsteiner. At the turn of the century, therewas a considerable amount of disagreement and con-fusion about the occurrence and significance ofagglutination in both health and disease.15 It was atthis point that Landsteiner (Fig. 1-4) entered thefield.17a,b The first suggestion of the existence ofserum agglutinins and red cell antigens within whatwould finally be known as the ABO blood groupsystem is to be found as a footnote in a publication byLandsteiner in 1900.18 In it he states, “The serum ofhealthy individuals not only have an agglutinatingeffect on animal red cells but also on human red cellsfrom different individuals. It remains to be decidedwhether this phenomenon is due to individual differ-ences or to the influence of injuries or bacterial infec-tion.” In a detailed paper in 1901, he reported that heobtained sera and red cells from 29 different people,including himself and four medical colleagues, tostudy agglutination reactions. The reason thatLandsteiner was successful in elucidating the mecha-nism underlying intraspecies agglutination whereothers had failed arose from the nature ofLandsteiner’s experimental design. He used all of thesera against all of the samples of RBCs, using“checkerboard” blocks of five or six different sera andRBCs in 144 combinations. He found that certain serawould agglutinate the RBCs of certain other people.This discovery of isoagglutination became the basisof human blood-group classification, which wouldsubsequently be found to have relevance for autoan-tibody specificity in AIHA.

In his characteristically brief but data-filled paperof 1901,19 Landsteiner further noted and pointed outthat the blood isoagglutinins retained their activityafter drying and redissolving. Also, he observedagglutination with serum extracted after 14 daysfrom blood dried on a cloth. “The reaction may besuited to establish the identity or more correctly thenon-identity of a blood specimen.” This predictedthe value of Landsteiner’s discovery to forensic med-icine in the future. The closing statement in his paperwas, “Finally, it might be mentioned that thereported observations may assist in the explanationof various consequences of therapeutical bloodtransfusions.” In three pages, Landsteiner com-pressed knowledge that would fill thousands ofpages in the future.20

On November 8, 1930, Karl Landsteiner wasawarded the Nobel Prize (Fig. 1-5). The lecture givenby Landsteiner at the conferment of his Noble Prizewas based on the “differences in the blood of human

individuals.” More than a century later, his theoriesabout isoantigens are accepted and are a fundamentalpart of the theoretical basis of immunology, tissuetransplantation, forensic medicine, and populationgenetics.21,22

FIRST DESCRIPTION OF HEMOLYTIC ANEMIA

The concept that premature destruction of RBCsmight lead to a disease state and jaundice was firstsuggested in 1871 by Vanlair and Masius.1,23 Theseobservers described a patient with anemia andmarked splenomegaly without hepatomegaly. Thepatient suffered acute attacks of left upper quadrantpain and jaundice without acholia, and passedreddish brown urine. Morphologic evidence of anRBC abnormality was suggested by finding spheri-cal dwarf cells in the peripheral blood that theycalled microcytes. The authors postulated that clini-cal jaundice could result from two different mecha-nisms: “mechanically by reabsorption or liverinduced” and “paradoxical icterus.” The lattergroup included the “blood induced icterus,” whereexcessive amounts of colorant material is releasedfrom the blood cells and followed by the formationof bile which is deposited in the tissues. More explic-itly, they stated that “there are at least a certainnumber of non-mechanical types of icterus whichare caused by the exaggerated destruction of redcells and the transformation to bilirubin of releasedhematin.” This concept was essentially correct, butlittle attention was paid to this remarkable publica-tion and, for almost 30 years, hepatic disease, jaun-dice, and hemolytic anemia became hopelesslyintertwined.1

THE DISTINCTION BETWEENCONGENITAL AND ACQUIREDHEMOLYTIC ANEMIAS

At the turn of the twentieth century, Hayem24 (Fig. 1-6)and Minkowski25 showed that the jaundice associatedwith hemolytic anemia was distinct from that ofhepatic diseases. Hayem made the distinction betweencongenital and acquired hemolytic anemias, whereasMinkowski described only a hereditary condition.Hayem has repeatedly been said to be the first todescribe acquired hemolytic anemia, although he didnot name it that, but, instead, coined the term chronicinfectious splenomegalic icterus.24 Minkowski is creditedwith the first clear recognition of icterus due tohemolytic anemia (chronic hereditary acholuric icterus)separate from obstructive jaundice; he associated theanemia with urobilinuria and splenomegaly and pos-tulated that RBC destruction was attributable to lesionsin the spleen.25


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 pho-tographer’s papier-maché rocks. (B) Photograph of Landsteiner probably taken at the Institute for Pathological Anatomy, where he worked from 1897to 1907. (C) Landsteiner and his coworker, Emil Prás

∨ek from Belgrade, December 1913. The two worked together on the chemical manipulation of the

specificity of serum albumin. (D) Landsteiner at about the time he left Europe for the United States. (From Mazumbar MH: Species and Specificity. AnInterpretation of the History of Immunology. Cambridge, UK: Cambridge University Press, 1995.)

C D

A B


DESCRIPTION OF SPHEROCYTES ANDANALYSIS OF THEIR SIGNIFICANCE

Vanlair and Masius23 described the case of a youngwoman who developed icterus, recurrent attacks of

left upper quadrant abdominal pain, and spleno-megaly shortly after giving birth. The patient’smother and sister were also icteric, and the sister’sspleen was enlarged. The most remarkable aspect ofthis paper lies in their description of the bloodfindings. Although they made no mention of anemiaand had no concept of hemolysis as a pathologicalprocess, they unmistakably described RBCs that wenow recognize as spherocytes with remarkable clarity(Fig. 1-7). The authors noted that some of the RBCs,which they called microcytes, were smaller thannormal RBCs, 3 to 4 μm in diameter, spherical inshape, and the contours were completely smooth.They concluded, “The jaundice of our patient appearsto be a peculiar type of icterus. The fact that thepatient’s mother and sister had a slight jaundice andthat the sister had an enlarged spleen may indicatethat this condition is one disease entity.”

Naegli is often credited with first use of the termspherocyte. However, according to Crosby26 (Fig. 1-8),two British army officers, Christophers and Bentley,were the first. They were assigned to India to study

FIGURE 1-6. Georges Hayem. (From Packman CH: Thespherocytic haemolytic anaemias. Br J Haematol2001;112:888–899.)

FIGURE 1-5. The Noble Prize certificate for Karl Landsteiner in 1930.(From Tagarelli A, Piro A, Lagonia P, Tagarelli G: Karl Landsteiner: Ahundred years later. Transplantation 2001;72:3–7.)


blackwater fever and made very careful descriptions ofspherocytes in a monograph published in 1909. Naeglialso proposed that the spherocyte was pathognomonicof congenital hemolytic icterus, an observation that

constricted thinking about hemolytic icterus for thenext 15 or 20 years. In fact, many authorities began todoubt the existence of an acquired type of hemolyticicterus, regarding the disease as a variation on the con-genital form.

OSMOTIC FRAGILITY OF RED BLOOD CELLS

During the first decade of the twentieth century, anumber of significant studies of the osmotic fragilityof RBCs were conducted. Chaufford27 (Fig. 1-9)noted that RBCs of several patients, but not those ofnormal subjects, were hemolysed by hypotonicsaline. He developed an osmotic fragility test, inwhich RBCs were placed in a series of tubes contain-ing successively decreasing concentrations of saline.The osmotic fragility was expressed as the concen-tration of saline at which hemolysis began and atwhich hemolysis was complete (Fig. 1-10). Chauffardrecognized that the liver was not at fault and that thedisorder was a result of hemolysis. He wrote,“Perhaps after this clinical and hematologic inquiry,the cause of the hemolytic theory could be consid-ered as won.” This observation finally enabled physi-cians to distinguish hepatic and hemolytic jaundice,as Ribbierre had recently (in 1903) demonstrated thatthe cells from patients with hepatic jaundice areresistant to osmotic stress.7

FIGURE 1-7. A reproduction of part of the tinted lithograph illustratingthe paper by Vanlair and Masius (1871) entitled De la micro-cythémie. I is a drawing of the patient’s blood. II is a drawing of controlnormal blood. (From Dacie JV: The life span of the red blood cell andcircumstances of its premature death. In: Wintrobe MM (ed): Blood,Pure and Eloquent. New York: McGraw-Hill Book Company,1980:211–255.)

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


Of course, Chauffard and coworkers27 had discov-ered the in vitro pathophysiological expression of thespherical microcytes described by Vanlair andMassius23 almost 40 years earlier. However, theywere probably unaware of the work of these earlyinvestigators and they certainly made no associationbetween microcytic spherical cells and increasedosmotic fragility. That correlation was noted muchlater by Haden.28

RETICULOCYTES

About 1 year after his description of increasedosmotic fragility in congenital hemolytic icterus,Chauffard and Fiessinger29 and Chauffard30 stainedRBCs from patients with hemolytic icterus withPappenheim’s31 (Fig. 1-11) solution and noted largenumbers of cells containing a peculiar basophilicgranulation or reticulum, which they called “granu-lar degeneration.” Ehrlich had first described thisspecial staining method in 18817 and noted increasednumbers of reticular cells in anemic patients.Vaughan,32 in 1903, noted these granular cells consti-tuted about 1% of the RBCs in normal subjects.Chauffard had hoped to explain the anatomical lesionthat underlay the increased fragility of the RBCs.What he actually discovered, or rediscovered, was thereticulocytosis that is now a hallmark of hemolyticanemia. Chauffard’s drawing30 of a blood smearstained with Pappenheim stain from a patient withfamilial hemolytic icterus is shown in Figure 1-12.

FIGURE 1-9. Anatole Chauffard (1855–1932). (From Dacie JV: The lifespan 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, 1980:211–255.)

70 68 66 64 62 60 58 56 54 52 50 48 46 44 42 40 38 36 34 32

Résistance globulaire — (Solution de NaCI à 0.70%)

Pas d’hémolyse....Nombre de gouttes de la

solution....

Hémolyse légère....

Hémolyse nette....

Hémolyse très nette

Hémolyse totale....

Diamètre moyen des hématics 5 89 Diamètre maxima 7,5.— minima 4.μ

FIGURE 1-10. The figure illustrates the “precocious and prolonged” lysis in hypotonic saline of the red cells of a patient suffering from ictère con-génital de l’adulte (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, 1980:211–255.)


THE CONCEPTS OF IMMUNE HEMOLYSISAND HORROR AUTOTOXICUS

In an impressive series of studies commencing in1899,33 Paul Ehrlich (Fig. 1-13) and JuliusMorgenroth sought to identify the constituents andto define the mechanisms involved in the phenome-non of immune hemolysis, which Jules Bordet hadonly recently described.34 Such studies involved theimmunization of animals with foreign RBCs, a pro-cedure resulting in an immune serum whose ther-mostable antibody would collaborate with a

thermolabile substance (variously termed comple-ment, alexin, or cytase) to cause the specific destruc-tion in vitro of the erythrocyte species used forimmunization.8 During the course of these studies,Ehrlich and Morgenroth attempted repeatedly toinduce the animal to form hemolytic antibodies to itsown cells. These attempts to elicit the formation ofautoantibodies were uniformly unsuccessful, and, atbest, they were only able to produce antibodies ableto agglutinate or to hemolyse the RBCs of certainother members of the same species.

Ehrlich had postulated, in his landmark paper of1897, that antibody formation was part of the normal

FIGURE 1-11. Artur Pappenheim (1870–1916). (From Lajtha LG: The common ancestral cell. In: Wintrobe MM (ed): Blood, Pure and Eloquent.McGraw-Hill Book Company, 1980:81–95. Reproduced with permission of The McGraw-Hill Companies.)


physiological process of cellular digestion and somight theoretically be stimulated by autochthonousas well as by foreign substances.8 Nevertheless, hepointed out, “It would be dysteleologic in the highestdegree, if under these circumstances self-poisons ofthe parenchyma⎯autotoxins⎯were formed.”35,35a

Thus, “we might be justified in speaking of a horrorautotoxicus of the organism.”36

THE FIRST DESCRIPTION OF ANAUTOIMMUNE HEMOLYTIC ANEMIA

The first AIHA in which clinical and diagnostic labora-tory findings were clearly described is PCH.37 Thisappears, at first, to be surprising because PCH is theleast common type of AIHA. Its early recognition is dueto the fact that hemoglobinuria is a striking symptom,a fact that also explains the early recognition of marchhemoglobinuria and PNH. It is also true that PCH wasmuch more common than it is at present because amajority of cases recorded in the early medical litera-ture were associated with late stage syphilis or congen-ital syphilis. In the early 1900s, over 90% of patientswith chronic PCH had a positive test for syphilis andapproximately 30% showed clinical evidence of thedisease.38 With the effective treatment of syphilis andthe virtual elimination of the congenital form, “classic”syphilitic PCH is now an extremely rare disorder, as ischronic PCH. It was in patients with the chronic formof PCH that exposure to cold resulted in a paroxysm ofhemoglobinuria.39,40

In the latter part of the nineteenth century, there werea number of reports of PCH. Dressler41 is generallycredited with being the first (in 1854) to give a cleardescription. His patient was a 10-year-old boy whomay have had congenital syphilis. After exposure tocold, he passed red urine that gradually paled to clearto a natural color. Microscopic examination of theurine showed “dirty brown pigment” but no bloodcorpuscles. PCH, however, seems also likely to havebeen the diagnosis in the patient described byElliotson in The Lancet in 18323,42 who had heartdisease and cold “fits” and passed bloody urine“whenever the east wind blew.”

Subsequently, several excellent clinical accountswere published during the 1860s.3 The authors real-ized that exposure to cold precipitated that attacksand that the urine contained blood pigment, but noblood cells. Wiltshire43 described an infant, perhapsthe youngest such patient ever recorded, who passedbloody urine, free from RBCs in the sediment, whenthe “weather was particularly inclement.”

The term hemoglobinuria seems to have been usedfirst by Secchi in 1872, but it is not clear whether thepatient he described had PCH.44

In 1879, Stephen Mackenzie, at the LondonHospital, elaborated on the pathophysiology ofPCH.45 He described a young boy who had a sallowcomplexion and yellow eyes and whose urine wasblack. The microscopic examination and spectro-scopic analysis of the urine showed it containedabundant hemoglobin but no RBCs. He suggestedthat the discolored urine was due to blood solution ordisintegration (hemolysis) and stated that it musttake place in some part of the organism. He believedthat the hemolysis occurred in the “genito-urinaryapparatus,” most probably the kidney.

Kuessner, in 1879, made the important observationthat serum obtained by “cupping” a patient during anattack of hemoglobinuria was tinged red.46 This prob-ably was the first direct evidence derived from obser-vations in humans that indicated that the hemoglobinin the urine was being derived from hemoglobinliberated in the plasma, rather than being, in somemysterious way, of renal origin. Indeed, Mackenziemodified his previous theory of erythrocyte destruc-tion, suggesting that the role of the kidney is in factpassive, and that the corpuscle solution, or hemolysis,occurs in the vasculature.47

EARLY DIAGNOSTIC TESTS FORPAROXYSMAL COLD HEMOGLOBINURIA

Although there were many clinical descriptions ofPCH in the nineteenth-century medical literaturedocumenting the relationship of acute attacks to expo-sure to cold and the fact that the urine containedblood pigment but no blood corpuscles, the patho-physiology was not understood.

FIGURE 1-12. Drawing of a blood smear (Pappenheim stain) as seenby Chauffard (1908). The granular appearing cells are reticulocytesfrom a patient with familial haemolytic icterus. (From Packman CH: Thespherocytic haemolytic anaemias. Br J Haematol 2001;112:888–899.)


A diagnostic test described in 1879 was based on thedevelopment of hemoglobinuria after immersion ofthe patient’s feet in ice water.39,40 A test producing lessdiscomfort to the patient was described in 1881 byEhrlich, who showed that if a ligature was placedaround a finger that was then chilled in ice water,serum subsequently obtained from the finger wouldcontain hemoglobin.48 Although these tests helped todiagnose the disorder, they did not elucidate themechanism by which exposure to cold resulted inhemolysis.

THE DONATH-LANDSTEINER DISCOVERY,1904: THE FIRST DESCRIPTION OF ANAUTOANTIBODY AND OF ANAUTOIMMUNE HUMAN DISEASE

The greatest single step forward in understanding thepathogenesis of PCH was provided by the work ofDonath and Landsteiner whose famous report waspublished in 1904.49 Julius Donath (1970–1950) was anassistant at the University of Vienna First MedicalClinic, and Karl Landsteiner (1868–1943) became agiant in the annals of immunology.50 These investiga-tors demonstrated that hemolysis was due to anautolysin that reacted with the patient’s RBCs at low

temperatures and that labile serum factors (comple-ment) caused lysis of the sensitized cells if the temper-ature was subsequently raised. Their interpretation oftheir observations are particularly noteworthy becausethey were published during the era of widespreadacceptance of Ehrlich’s dictum of horror autotoxicus.Here then was the first report that appeared to contra-dict Ehrlich’s concept.35a

This bithermic procedure for the diagnosis of PCHwas the first immunohematologic test ever to bedescribed51 and remains the diagnostic test for the dis-order (see Chapter 5). Further, this discovery has beenwidely acclaimed as the first description of an autoan-tibody and of an autoimmune human disease.8 Thetest is referred to as the Donath-Landsteiner (DL) testand the antibody thus detected as the DL antibody.Even after the passage of a century since the report ofDonath and Landsteiner, the accuracy of their obser-vations and the usefulness of the DL test persist.

Primacy of Discovery of Biphasic Autoantibodiesin Paroxysmal Cold Hemoglobinuria. It is of interestthat similar and apparently independent observationswere described by Eason. Easons’s two papers,52,53

published in 1906, were based on his MD thesis. Hisexperiments, which had been carried out in 1903, hadbeen the subject of a communication read at a meetingof the Galenian Society, Edinburgh, in January 1904. Hestated that “ten months after the results had beencommunicated by me the most important of them wereconfirmed by Donath and Landsteiner whose researchon these lines had been conceived independently ofmine. These collaborators furthermore proved that it isthe process of anchoring of the intermediary body to thered corpuscles which requires the low temperature.”

FIGURE 1-13. Paul Ehrlich (1854–1915) in hisstudy.* (From Wintrobe MM: Milestones onthe path of progress. In: Wintrobe MM: (ed):Blood, Pure and Eloquent. New York: McGraw-Hill Book Company, 1980:1–31.)

*This is the authors’ favorite photograph, indicating that officescirca 1899 were not necessarily neater than those of the presentday. It certainly seems as though Ehrlich maintained enoughreading material in his office.


Dr. Eason was awarded a Gold Medal and the Milner-Fothergill Medal in Therapeutics by EdinburghUniversity for his thesis.37

However, Donath and Landsteiner contested thepriority for their discovery with Eason and stated that“Eason joined [himself] to our interpretation of themechanism of hemolysis.“8,54 They further stated that“the development of autotoxic substances, which arebound to the organism’s own cells, can be related to theprocess of antibody formation, a possibility which, sofar as we know, has not previously been discussed.”8,54

In a much more recent publication, Goltz55 main-tained that Donath and Landsteiner did not actuallydiscover the first autoantibody because nowhere didthey use the accepted terms “antibody,” “ambozep-tor,” “antigen,” or even “immune.” Rather they usedsuch apparently nonspecific terms as “hemolysin,”“toxin,” and “poison.” However, as reviewed in depthby Silverstein,8 numerous contemporary authors usedthe term toxin when they meant “specific antibody,”and the term did not imply some sort of nonimmuno-logic toxic action.

Even if Landsteiner’s language might be misinter-preted at a later period, his contemporaries surelyunderstood him. For example, Ehrlich in 1906 alreadyreferred to Donath and Landsteiner as observing“hemolytic autoamboceptors.”8,33 Further, Rössle, in1909,8,56 while discussing the general evidence of theexistence of autoantibodies, stated that “there are alsocases, however, in which direct evidence for the pres-ence of autoamboceptor is splendid. The best knowninstance concerns paroxysmal hemoglobinuria.” “Evenin their first report, Donath and Landsteiner called ourattention to the possibility that such a substance mightbe the result of self-immunization.” Also in 1909,Meyer and Emmerich published an extensive report onparoxysmal hemoglobinuria.57 They concluded theirpaper with the statement that “In [our] four cases oftypical paroxysmal cold hemoglobinuria, the autohe-molysin found by Donath and Landsteiner wasobserved.”

It is evident from the foregoing that Donath andLandsteiner as well as their contemporaries did,indeed, understand from the outset that they weredescribing an autoantibody and an immunologicalprocess, despite the curious terminology they used.8

The Original Experimental Protocol of Donathand Landsteiner. Excerpts from the original report in1904 by Donath and Landsteiner58 are illustrated inFigures 1-14 and 1-15. A translation of the originalprotocol is provided in Figure 1-16.

In essence, they demonstrated that sera frompatients with PCH would cause hemolysis in vitro ofRBC of normal individuals and of patients if theserum and cells were held (incubated) for 1⁄2 hour at 5°C and then held at 37°C. As controls, they usedserum from normal individuals. They concluded thatthe serum of the hemoglobinuric patients contains alytic substance that is effective against the patients’and other human blood corpuscles.

Their article describes further experiments in whichtwo aliquots of a patient’s blood were obtained. Onealiquot was incubated at 0°C, and the other was incu-bated at room temperature. Then the plasmas wereremoved and exchanged, mixed, and incubated at 37°C. After 2 hours, the aliquots that were cooled hadundergone much lysis, but no lysis occurred in theother aliquot. “This finding indicates that red cellstake up in the cold an effective substance from theplasma, and that neither red cells nor white bloodcells give hemolytic substance into the serum.”

In an additional experiment, “oxylated blood” ofthe patient was cooled in ice water and centrifuged inthe cold, and then the plasma that had been removedin the cold was mixed with a new aliquot of red cellsof the patient. This mixture was then cooled and sub-sequently incubated at 37°C. However, no hemolysisoccurred, thus indicating that the hemolysin had beenabsorbed by the cells.

“Red cells that are cooled with serum or plasma ofhemoglobinuric patients, whether the patient’s own orother’s red cells, take up substances that by this absorp-tion develop the capability to hemolyse in the serum ofhemoglobinuric patients and other human serum. Thehemolysis is caused by the aid of factors in the serumdescribed as complement (alexin, cytase, etc.).”

FURTHER STUDIES ON THEMECHANISMS OF HEMOLYTIC ANEMIAAND OBSERVATIONS ON THEDISTINCTION BETWEEN CONGENITALAND ACQUIRED FORMS

Chauffard was among several French scientists whoexplored the mechanisms of hemolytic anemia in theearly years of the twentieth century.4 Chauffard(1907),27 along with Trosier (1908)59 and Vincent(1909),60 described autohemolysins in patients withacute acquired hemolytic jaundice. These authorsdescribed patients whose serum had the capability ofhemolysing RBC, and they termed the condition“hemolytic icterus”; it was acute in course and associ-ated with hemoglobinuria. The reports of hemolysins,although incomplete and to some extent unsatisfac-tory, were pioneer ones well in advance of their time,and the idea that hemolytic anemia could occur appar-ently spontaneously in humans in consequence of thedevelopment of abnormal agglutinins or hemolysinsremained controversial for the next 30 years or so.3

Chauffard also standardized the osmotic fragilitytest, described reticulocytes and their increasednumbers in congenital hemolytic icterus (later to beknown as hereditary spherocytosis), and drew atten-tion to the microcytic nature of the RBCs in somehemolytic anemias.27

Between 1908 and 1912, Widal, Abrami, andBrule61,62 introduced the term acquired hemolyticanemia. These investigators described hemolytic


icterus that was apparently neither congenital norfamilial, that could appear gradually or suddenlyduring the course of various diseases, or that couldbe unassociated with any underlying disease. Thesecases were considered similar to those described by

Hayem 10 years earlier. The patients exhibitedreticulocytosis, but the alterations in the fragility testwere less marked than in the congenital form.

Hence, at this time, the two types of hemolyticanemia were well defined: the congenital form ofMinkowski and Chauffard and the acquired form ofHayem and Widal (Figs. 1-6 and 1-17).

THE ROLE OF THE SPLEEN AND THE EFFECT OF SPLENECTOMY

The above-cited brilliant studies clearly distinguishedhepatic jaundice and the jaundice resulting from

FIGURE 1-14. The original report published in 1904 by Dr. JuliusDonath and Dr. Karl Landsteiner describing their current theories ofthe pathogenesis of paroxysmal cold hemoglobinuria and the develop-ment of the biphasic lysis test that remains the diagnostic laboratoryprocedure for the disorder. A translation of portions of the text follows(a more complete translation has been published by Bibel50).

About PParoxysmal HHemoglobinuria

Different theories have been proposed to explain the pathogenesisof paroxysmal hemoglobinuria, a peculiar illness whose attack underthe influence of cold leads to hemoglobinuria and removal of bloodpigment through the urine.

Other, older explanations state that hemoglobinuria is caused bythe destruction of blood corpuscles in the kidney. But after Küessnershowed that hemoglobinemia is present during such paroxysms, thecause was located in the blood. The hemolysis itself was thought to bedependent on various factors. The original belief that cold woulddestroy the red cells that are sensitive in this disease is in oppositionto the commonly acknowledged fact that the blood of these patients invitro is not more sensitive to cold than the blood of normal individuals.Therefore one had to look for other causes of the hemolysis. Recentextensive studies on blood toxins have suggested that this disease iscaused by hemolysins. Authors have spoken for the hemolytic effect ofthese toxic substances. But numerous efforts to find the toxic agentsdid not succeed exactly, or even to find a test system that allows oneto study the hemolytic procedure during the period of hemolysis. (FromDonath J, Landsteiner K: Uber paroxysmale Haemoglobinurie.Munchen Med Wschr 1904;51:1590.)

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


premature and excessive destruction of erythrocytes.The hemolytic process was further differentiated toinclude both congenital and acquired forms. Althoughthe phenomenon of agglutination had been welldescribed in the latter part of the nineteenth century, itwas Widal, Abrami, and Brulé who observed autoag-glutination of erythrocytes,4 and their work as well asothers was summarized at the twelfth session of theCongrès Français de Medicina, which took place inLyon in 1911.63 The topic was the role of hemolysins inpathology, and papers were presented by many of theforemost physicians and pathologists of the day.

By now the role of the spleen was widely acceptedas being the major site of hemolysis, and the liver was

not generally regarded as a significant site of RBCdestruction. Therefore, it is not surprising that in 1911,Micheli, in Turin, performed the first splenectomy foracquired hemolytic anemia.64 Banti65 (Fig. 1-18),66 in1912, conducted an extensive investigation into splenicpathology and introduced the term hemolytic spleno-megaly, when he observed that the spleens of animalsundergoing hemolysis were enlarged and congested.65

He also noted that heteroimmune hemolytic serum,when transfused into splenectomized animals, led toless and slower hemolysis than that seen with normalanimals. He implicated the splenic endothelial cells aserythrophagocytes and described agglutinated erythro-cytes within the splenic pulp. Banti similarly showedthat the Kupffer cells of the liver could have an ery-throphagocytic function when intense hemolysis waspresent.

Thus, Banti effectively described the reticuloendo-theilal system and its function in RBC hemolysis.4 Herecognized 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 ofMicheli and Banti entrenched the recommendation ofsplenectomy as a treatment for hemolytic anemia, rep-resenting the first specific therapy for AIHA. Despitethe widespread acceptance of the benefits of splenec-tomy, however, some, such as Antonelli, in 1913 refutedBanti’s hemolytic splenomegaly as a separate disease,pointing out that it did not differ from acquiredhemolytic anemia.4

FURTHER CHARACTERIZATION OF HEMOLYTIC ANEMIAS

World War I brought a halt to investigation and casereports of hemolytic icterus.7 By the 1920s, the prevail-ing understanding of the mechanism behind RBCdestruction was that it resulted from autoagglutinin-induced agglutination, the first step in hemolysis.However, publications after World War I indicated thedegree to which much of the knowledge discovered atthe beginning of the century had been lost. Lederer(1925)67,68 and Brill (1926)69 described a number of casesof transfusion-responsive acute hemolytic anemia asso-ciated with infectious diseases. Because much of theprior French work had been forgotten, Lederer’sdescriptions were thought to be of a new disease, inspite of the extensive review of hemolytic icterus byTileston70 just 3 years earlier. Such cases became knownas “Lederer’s anemia” or “Lederer-Brill anemia,” but itis likely that they were examples of AIHA.1

The hiatus in studies concerning hemolytic anemiasobliterated the clear distinction between congenital andacquired forms of hemolytic anemia established by theFrench investigators. Indeed, Dacie71 states that it wasgenerally assumed at that time in England thathemolytic anemia occurring in the adult was a latentform of hereditary spherocytosis. The lack of specificdiagnostic procedures, the presence of spherocytes in

Patient K(hemoglo-binuria)4 Drops

Patient R(hemoglo-binuria)10 Drops

Patient N(hemoglo-binuria)7 Drops

B.W.6 Drops

Ch.G.7 Drops

A.R.6 Drops

Patient KB.W.Ch.G.A.R.

Patient RB.W.Ch.G.A.R.

Patient NB.W.Ch.G.A.R.

B.W.Patient RPatient N

Ch.G.

Ch.G.Patient KPatient N

Patient KPatient NPatient R

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Ruby redRedRedRed

Ruby redRuby redRuby redRuby red

Ruby redRuby red

RedRuby red

000

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000

00000

0000

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hr at 37°Held 3 hours

at 37°

Trace of redTrace of redTrace of redTrace of red

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00

Trace of redClear distinct red

000

00000

FIGURE 1-16. It is shown with this sequence of experiments that theblood corpuscles of other individuals are hemolyzed by the serum ofpatients with hemoglobinuria, although to a lesser degree than theirown blood corpuscles; however, in the same series of experiments,the blood corpuscles of the hemoglobinuric patients which have beencooled with other serum do not lyse when they are warmed after-wards. (Serum B. W. had a normal isolytic activity against the redcells of Ch.G. and N which was not increased by cooling.) Therefore,the unusual composition of the blood of the hemoglobinuric patientswhich is causing the lysis lies in the serum (respectively plasma),although the red cells may be easier to lyse (as shown in our CaseK). The serum (plasma) of the hemoglobinuric patients contains alytic substance that is effective against the patient’s and otherhuman blood corpuscles. This lysis cannot be demonstrated directlyby mixing the serum of the hemoglobinuric patient with his own orother red cells; however, one must consider the dependence of itseffects on temperature.


both forms of hemolytic anemia and the unavailabilityof serologic testing made such a conclusion inevitable.1

In 1938 and 1940, important contributions weremade by Dameshek (Fig. 1-19) and Schwartz.72-74

These workers published a remarkable review of

acquired hemolytic icterus in 1940 that was 96 pagesin length with 380 references. They identified 81 arti-cles that described cases fitting their concept of acute(acquired) hemolytic icterus. Based on their ownclinical observations of hemolysins in some patients,

FIGURE 1-17. Fernand Widal. (From Packman CH: The spherocytic haemolytic anaemias. Br J Haematol2001;112:888–899.)

FIGURE 1-18. Guido Banti (1852–1925) was one of the first physicians who might prop-erly be called a hematologist. A contemporary of Osler, he worked at a time when themethods and laws of biological research were just developing. Medical discovery was com-monly a consequence of clinical insight aided only by physical examinations and necropsy.The titles of Banti’s earliest publications give the direction of his lifelong interests: “Splenicanemia” and “Enlargement of the spleen with cirrhosis of the liver.” His efforts to definethese conditions as entities came to nothing, but the discussions about them did much todemonstrate the essentialness of method in clinical research. (From Crosby WH: Thespleen. In: Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw-Hill BookCompany, 1980:97–138. Reproduced with permission of The McGraw-Hill Companies.)


cases reported in the literature, including those ofChauffard and coworkers, and their own experi-ments involving injection of varying amounts ofhemolytic serum into guinea pigs, they proposedthat all cases of hemolytic icterus were a result ofhemolysins. The differences in clinical manifesta-tions, ranging from mild congenital cases to fulmi-nant acute hemoglobinurias, were accounted for bythe dose of hemolysin.7 Dameshek and Schwartz’sgeneral thesis that hemolysins were responsible forthe development of many cases of acquiredhemolytic anemia was correct. However, they wereincorrect in extrapolating their concept of the role ofhemolysins to congenital hemolytic jaundice (hered-itary spherocytosis) and in concluding that that dis-order might be caused by the “more or lesscontinued action of an hemolysin.”

These studies reawakened interest in acquiredhemolytic anemia and laid the broad outline for ourmodern concepts of the clinical and serologic implica-tions of AIHA.1 However, the difficulty in ascribingcases of acquired hemolytic anemia to the develop-ment of “hemolysins” was that they could not bedemonstrated in the vast majority of cases by the sero-logic techniques then available.

Thereafter, during subsequent decades, theclassification and serological characteristics of thevarious AIHAs were delineated, in large part throughthe extensive and meticulous work of Sir John Daciein London.5,74a

MEASUREMENTS OF RED BLOOD CELL SURVIVAL

In a review in 1923, Payton Rous (Fig. 1-20) discussedthe question of whether the RBCs had a definite, asopposed to an almost indefinite, sojourn in the blood,and, if finite, how long was their life span.75 In fact, hedid not doubt that their life span was limited, and helisted a number of cogent arguments in favor of thisview. For example, he cited the “continuous activity ofbroadly distributed hematopoietic tissue” and the“daily excretion through the bile of a pigment nearlyif not precisely identical with one of the pigmentedderivatives of hemoglobin.”75 The question as to howlong RBCs circulate before undergoing destructionhad been a vexing question for many years. A varietyof methods and calculations had been used to comeup with some answers, ranging from observations of

FIGURE 1-19. William Dameshek (1900–1969), one of themost eminent of American hematologists of his era, was astrong proponent of the concept of autoimmunity at a time thatothers were reluctant to accept that a patient could produceautoantibodies. His extensive writings and teachings had amajor influence on the gradual acceptance of an autoimmuneetiology for some types of acquired hemolytic anemias. (FromCrosby WH: The Spleen. In: Wintrobe MM (ed): Blood, Pure andEloquent. New York: McGraw-Hill Book Company, 1980:97–138.Reproduced with permission of The McGraw-Hill Companies.)


the time it took for the RBC count in a hypertrans-fused animal to be restored to normal to calculationsbased on bile excretion. The conclusions drawn fromthese studies were, however, erroneous.

Data of Winifred Ashby. The conclusions of only oneobserver stood out in striking contrast to the aboveobservations⎯those of Winifred Ashby (Fig. 1-21),whose first papers76,77 were published in 1919 (re-viewed by Dacie3). Ashby described in her first paperhow she had transfused group IV (type O) blood intoseven group II (type A) recipients who were sufferingfrom various anemias and how she had been able to count the free (unagglutinated) type O RBCs bymaking suspensions of posttransfusion blood in ananti-A serum (Fig. 1-22). She concluded that transfusedRBCs live a long time, 30 days or longer, and that thebeneficial results of blood transfusion are not due to thestimulation of the bone marrow (a view held by some

at the time) but to the functioning of the transfusedRBCs. By 1921, Ashby78 was able to report on morethan 100 patients. In four patients who were followeduntil the elimination of the transfused RBCs was com-plete or almost complete, this did not take place until 83 to 100 days after transfusion.

One of the difficulties inherent in Ashby’s work,which she could not circumvent, was that she was notmeasuring the life span of the RBCs in their own envi-ronment. This raised the question of whether theforeign cells might have a different survival than thoseof the host, a point that she was unable to resolve.

Additional Studies Using Differential Aggluti-nation. In 1928 differential agglutination was alsoused in the reverse way by Landsteiner, Levine (Fig. 1-23) and Janes79 and Wiener.80 Wiener reportedthat he had detected blood group M (or N) cells, usinganti-M (or anti-N) sera, in the circulation of N (or M)

FIGURE 1-20. Peyton Rous (1879–1970). (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, 1980:211–255.)


recipients for between 80 and 120 days after transfu-sion. Wiener also used the Ashby method, using anti-M (or anti-N) sera to agglutinate the recipient’s RBCs,and observed that between one third and one fourthof the transfused RBCs disappeared each month; heremarked that this continuous decrease in numberswas to be expected on the assumption that all thecells had approximately the same life span. He con-cluded, “Curiously enough, despite all this work,most textbooks still give the life of the erythrocyte asthirty days.”

Ashby’s data and conclusions are now known to begenerally correct. But she was ahead of her time; herpapers remained on library shelves largely unreadand her technique was relatively unused until the late1930s. In Oslo, Dedichen81 conceived the idea that itmight be possible to obtain evidence by transfusionexperiments as to which of the two current theoriesabout the pathogenesis of “ictere hemolytique”(hereditary spherocytosis) was correct; hyperactivityof the organs of hemolysis or production of cells withless than normal resistance. However, for technicalreasons, his experiments were unsuccessful, and morethan a decade was to pass before similar (but moresuccessful and decisive) experiments were againundertaken.

Intrinsic and Extrinsic Mechanisms of Hemo-lysis. Dacie (Fig. 1-24) and Mollison (Fig. 1-25)82

first applied Ashby’s technique in patients with

hemolytic anemia over 20 years after her publication.They were able to show that normal RBCs transfusedinto patients with familial hemolytic anemia sur-vived normally, for approximately 100 to 120 days.The survival curves from their paper are shown inFigure 1-26. In sharp contrast, Loutit and Mollison83

noted that normal RBCs transfused into patientswith acquired hemolytic anemia exhibited markedlyreduced survival.

Loutit and Mollison83 also transfused RBCs frompatients with congenital and acquired hemolytic icterusinto normal recipients and followed their survival. TheRBCs from patients with congenital acholuric jaundice,including those from a patient who had undergonesplenectomy, exhibited short survival.

The tracing by differential agglutination, as intro-duced by Ashby, demonstrated a clear distinction

FIGURE 1-21. Winifred Ashby (1879–1975). (From Dacie JV: The lifespan 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, 1980:211–255.)

FIGURE 1-22. Reproduction of one of Ashby’s original figures. (A) Asuspension of group II (type A) red cells in an anti-A serum. Relativelyfew cells are free and unagglutinated. (B) A similar preparation afterthe transfusion of group IV (type O) red cells. Many of the cells are nowfree and unagglutinated, the great majority being transfused cells.(From Dacie JV: The life span of the red blood cell and circumstancesof its premature death. In: Wintrobe MM (ed): Blood, Pure andEloquent. New York: McGraw-Hill Book Company, 1980:211–255.)


between the two major groups of cases. In one group,transfused blood survived normally, and in anothergroup of patients, it was destroyed along with thepatient’s own blood. These observations supportedthe idea that there might be “intrinsic” and “extrinsic”mechanisms for increased hemolysis. Later, the dis-tinction was used as a rational basis for classificationof the hemolytic anemias.

THE ANTIGLOBULIN (COOMBS’) TEST

A major diagnostic advance was the development ofthe antiglobulin test, the discovery of which is aninteresting aspect of the history of AIHA. The eventsleading to its discovery have been documented by Dr.Robin R. A. Coombs84,85 (Fig. 1-27).

He points out that immunology in the 1940s wassomewhat elementary, unsophisticated, and pheno-menologic. The real nature of antibodies was stilluncertain, but seemed to be associated with the serumglobulins. After graduating in veterinary medicine in1943, he joined an investigation on the serodiagnosisof Pfeifferela mallei infection, which causes a very

serious disease in horses and humans and for whichthere was no cure at that time. He later continued hiswork at Cambridge in the University Department ofPathology. Two eminent serologists, Robert Race (seeFig. 1-25B) and Arthur Mourant, were working in thedepartment at that time. Race86 and Weiner,87 workingseparately, had by this time concluded that there weretwo types of Rh antibody: one that bound to the RBCsurface and caused agglutination (the “complete”antibody) and another that absorbed to the RBCsurface but did not cause agglutination (the “incom-plete” antibody). Coombs, reminiscing in 1998,84

states, “At coffee one day, discussion turned to Rob’sso-called ‘blocking’ or ‘incomplete’ antibody. Whatwas the nature of this antibody, if indeed it was anantibody? Rob stressed that there was a real need fora better test (than his blocking test) to measure theseso-called incomplete antibodies. The next stepoccurred on a late-night ill-lit train from London backto Cambridge. I was pondering on how to measurethese incomplete antibodies on red cells with picturesin my head of Ehrlich’s side-chain theory. In a flash Icould see the globulin antibody on the red cells, andthese red cells should be agglutinated with an anti-

FIGURE 1-23. Philip Levine. (From Diamond LK: The story ofour blood groups. In: Wintrobe MM (ed): Blood, Pure andEloquent. New York: McGraw-Hill Book Company,1980:691–717. Reproduced with permission of The McGraw-Hill Companies.)


body to serum globulin, i.e., an antiglobulin. All thenecessary thinking had been done!”

Coombs obtained some “very crude [rabbit] anti-human globulin serum” from a coworker and the “veryfirst experimental protocols with Race and Mourantshowed quite clearly that the procedure was going towork.” They absorbed the antiglobulin serum (AGS)with human group AB Rh-positive RBCs and thenincubated Rh-positive RBCs in sera known to containincomplete Rh antibodies. The sensitized cells aggluti-nated in the antiglobulin serum and the appropriatecontrols were negative. The first account of what wenow call the indirect antiglobulin test was published byCoombs, Mourant, and Race in 1945.88 The authorswere bold enough to state, “This test may have usefulapplications in detecting fine degrees of sensitization inother antigen-antibody systems. . . .” This has turnedout to be an understatement, for quite apart from thetests on red cells and bacteria covering all the isotypesof antibody, an antiglobulin step or stage is a regularcomponent in very many immunoassay procedures.85

A more substantial paper89 was published in thesame year in the British Journal of ExperimentalPathology, and just as the printer’s page proofs wereon the point of dispatch back to the publisher,Mourant came across a paper in the German literaturefrom 1908 by Carlo Moreschi90 (Fig. 1-28) thatdescribed enhancement of red cell agglutination withan “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 discov-ery or a test as being new.”84

Coombs, Mourant, and Race next went on to demon-strate RBC sensitization in babies with hemolyticdisease of the newborn using the direct antiglobulintest (DAT).91 Cord RBCs from patients agglutinatedwhen exposed to the antihuman antiglobulin reagent,but cells from healthy babies did not agglutinate.

One of the positive tests they observed in newbornsappeared at first to be a false positive since there wereno Rh antibodies in the mother’s serum. However,Race went on to demonstrate the test was a true posi-tive but that it was not caused by an Rh antibody. Themother’s name was Kell, and this was the start ofRace’s research on the Kell blood group system.

In 1947, Coombs and Mourant92 demonstrated thatthe component in AGS that reacted with RBCs coatedwith Rh antibody was in all probability an anti-gamma globulin. They showed that the addition of asmall amount of gamma globulin to the antiglobulinserum rendered it incapable of agglutinating cellscoated with Rh antibody, whereas the addition ofalpha globulin or beta globulin had only a slighteffect, which could be ascribed to contamination withtraces of gamma globlulin.

An interesting phenomenon observed by Dacie93

was that the addition of gamma globulin to AGSproduced a reagent that could discriminate betweenthe RBCs of individual patients with AIHA. Thus,although in many instances the positive antiglobulinreaction was abolished by adding the gamma globulin,this was not true in all cases. It seemed clear that inthose cases in which the reaction was inhibited, theautoantibody on the cell was itself a gamma globulin,but that when the reaction was not affected, the mate-rial on the RBC surface could not be gamma globulin.The “nongamma protein” was eventually shown toconsist of components of complement fixed to the cellas a result of antibody-antigen interaction.93,94

Use of the Antiglobulin Test to Distinguish Immunefrom Nonimmune Acquired Hemolytic Anemias. Atthe time of the discovery of the antiglobulin test, therewas great difficulty in distinguishing hemolyticanemia that was familial from that which wasacquired. The only laboratory test available was themeasurement of osmotic fragility, which was abnor-mal in familial hemolytic icterus (now called hereditaryspherocytosis). However, Dameshek and Schwartz74

pointed out that spherocytes causing increasedosmotic fragility could develop in cases that wereclearly acquired hemolytic anemia.

Barbara Dodd described the fact that she andKathleen Boorman, who were working at the SouthLondon Transfusion Centre with the director, JohnLoutit, who was already an authority in the field ofanemias, were in a privileged position.95 They hadvisited Cambridge, where Race revealed to them thesecrets of the antiglobulin test before it had appeared inprint. Dodd states that, “I shall never forget the gleam

FIGURE 1-24. Professor Sir John Dacie laid the foundation for theinvestigation of hemolytic anemias. His persistence and experimentalapproach enabled him to demonstrate the vast complexity of thefactors involved in the anemias due to hemolysis, and for this he hasjustifiably been considered a pioneer.81a He was also responsible fortraining many hematologists from numerous countries, including thepresent authors. (From Wintrobe MM: Blood, Pure and Eloquent. NewYork: McGraw-Hill Book Company, 1980:XVIII. Reproduced withpermission of The McGraw-Hill Companies.)


A

B

FIGURE 1-25. (A) Patrick Mollison. (B) A 1947 photograph taken at theLister Institute in London showing, from left to right: Louis K. Diamondwhose research clarified the pathogenesis of hemolytic disease of thefetus and newborn as well as the optimal management of that disorder;Patrick L. Mollison, a pioneer in the field of blood transfusion and editor often 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 tothe field of RBC genetics and serology; and Sir Ronald A. Fisher, a famousgeneticist/biostatistician who, together with Race, devised a classificationof the Rh blood group system that is still used. (Courtesy of Professor P. L. Mollison.)

Case 1

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5

FIGURE 1-26. Dacie and Mollison, using the Ashby technique,were the first to demonstrate that normal RBCs survive nor-mally in patients with familial hemolytic anemia. The figureshows survival of RBCs from normal donors after transfusionto six patients with familial hemolytic anemia. Case 3 was anRh-negative patient who was later found to have developed analloantibody to Rh, accounting for the shortened survival oftransfused Rh-positive RBC. Although not shown in the figure,survival in cases 2 and 5 was followed to completion andfound to exceed 100 days in each case. The dotted lines indi-cate the limits of survival in a group of normal recipients(Mollison, unpublished observations). (From Dacie JV, Mollison PL: Survival of normal erythrocytes after transfusionto patients with familial haemolytic anaemia (acholuric jaun-dice). The Lancet, volume i, May 1, 1943, pp 550–552.)


in his eye when we returned from Cambridge with adescription of the new test!” They quickly collected theRBCs of 17 patients with familial hemolytic anemiaand 5 others with hemolytic anemia of the acquiredtype. “It was enormously exciting then, but no surprisenow, to find that the 5 patients having acquired typehad positive DATs, whereas the 17 familials were neg-ative.” They concluded (correctly) that the agglutina-tion tests “will discriminate the congenital from theacquired form [of hemolytic icterus], and that it indi-cates that the acquired form is due to a process ofimmunization, whereas the congenital form is not.”Thus, not only had they found a test that would distin-guish between the familial and acquired forms ofhemolytic anemia, but they had also demonstrated adifference in their etiology.

A Note about Carlo Moreschi. Carlo Moreschi wasdeep in immunological research at Pavia at the turn ofthe twentieth century. He published two particularlyinteresting papers90,96 describing enhancement ofagglutination with antiserum to serum (i.e., with anti-globulin) (Table 1-1). However, incomplete antibodieswere unknown at the time and general acceptance oruse of this procedure never resulted. Dr. Coombs paidtribute to Moreschi and his researches in a lecture to theItalian Association of Medical Analysts and Patholo-gists entitled “Moreschi and Some Recent Develop-ments in Agglutination.” There seemed to be little

interest in the agglutination or in Moreschi himself.However, 6 months after the lecture was published inthe Italian medical journal l’Informatore Medico,97

Dr. Coombs received a letter from Dr. Pietro deRuggieri, who was a steroid chemist in Milan and whowas a nephew of Carlo Moreschi. He was delightedwith the reference to his long-since-dead uncle.

THE CONCEPT OF AUTOIMMUNEHEMOLYTIC ANEMIA

In 1951, Young and associates98 were the first to cointhe term autoimmune hemolytic anemia. It was theorizedthat the production of an autoantibody was the resultof a breakdown in the “regulatory contrivances,” thusleading to autoimmunization. However, the conceptthat a patient could produce autoantibodies wasvigorously resisted by some. Witebsky,99 in particular,was reluctant to draw the conclusion that the RBCcoating material demonstrated by the antiglobuin testwas a true autoantibody. He considered it unprovedthat the RBC could be involved in autoimmunization,with the implied breaking of the principle of horrorautotoxicus. This reluctance to accept the autoim-mune nature of antiglobulin test−positive hemolyticanemias led to the use for a time of the noncommittalterm “antiglobulin-positive hemolytic anemia.”100

FIGURE 1-27. Robin R. A. Coombs. (Photograph by Lawrence E. YoungM.D., Fellows’ Garden, Kings College, Cambridge University, 1950.From Packman CH: The spherocytic haemolytic anaemias. Br JHaematol 2001;112:888–899.)

FIGURE 1-28. A photographic portrait of Carlo Moreschi. (FromCoombs RR: Historical note: Past, present and future of the anti-globulin test. Vox Sang 1998;74:67–73.)


Through the extensive writings and teaching ofsuch eminent physicians as Dameshek, the concept ofan autoimmune etiology for some types of acquiredhemolytic anemias gradually obtained general recog-nition and application.1

RADIOACTIVE CHROMIUM (51CR) AND DF32P

The first studies using 51Cr were reported by Gray andSterling101 in 1950 from Boston. They found that thelabeled RBCs lost radioactivity at a rate more rapidthan could be predicted from the known normal lifespan of dog RBCs and, consequently, did not recognizethe potential usefulness of the method in determininglong-term RBC survival.102 Later, Ebaugh and cowork-ers103 labeled normal blood with 51Cr and transfused itinto normal human volunteers. Subsequently, theamount of radioactivity per milliliter of RBCs wasquantitated and a simultaneous evaluation was madeof the RBC survival by the Ashby differential aggluti-nation technique. They found that the two curvesreached extinction point at the same time. Calculationsof the two curves were consistent with the hypothesisthat chromium was leaking from the RBCs in an expo-nential fashion with a mean half-life of 77 ± 12 days.Correcting for this leakage, the curve for the two tech-niques approximated that determined by the straight-line Ashby differential agglutination survival curve.103

The value of the isotope as a harmless label of RBCswas soon confirmed in many centers throughout theworld, and because the 51Cr could be used to labelpatients’ own RBCs and to study their survival intheir own circulation, as well as to label transfusedblood, Ashby’s elegant but laborious technique, withits inherent limitations and technical difficulties soonbecame obsolete.

51Cr is still widely used in studies of RBC life spanand in the measurement or blood volume, although it

is not an ideal label because of the elution of the labelfrom the RBCs. The nearest rival to 51Cr is DF32P,which was first reported in 1954 to be a potentially asatisfactory label for RBCs.104 The DF32P technique hasthe advantage over 51Cr in that once attached to theRBCs, it is not eluted.

The elimination curve of normal RBCs in a healthyrecipient, as demonstrated by the Ashby method or bythe use of DF32P, is virtually a straight line, and this isconsistent with the concept of gradually increasingsenescence rather than of random elimination in whichthe cells would be destroyed indiscriminately regard-less of age. Indeed, the analysis of survival curves hascontributed most significantly to the understanding ofthe pathogenesis of increased hemolysis.3

COLD AGGLUTININ SYNDROME (CAS)

Cold agglutinins were initially demonstrated byLandsteiner in animal blood in 1903105 and in humanblood by Mino in 1924,106 but their significance inhuman disease was not accurately appreciated untilseveral decades later. The first determination of titersin an acute postpneumonic cold agglutinin diseasewas made by Clough and Richter in 1918.107 A recog-nition of the relationship between cold agglutinins,hemolytic anemia, Raynaud’s phenomenon, andhemoglobinuria began to emerge with the case reportsof Iwai and Mei-Sai in 1925 and 1926.108,109 Their firstpatient was a 36-year-old Chinese man giving a 6-year

TABLE 1-1. TRANSLATED FROM MORESCHI (1908), DEMONSTRATING THE PRINCIPLE OF THE ANTI-GLOBULIN (COOMBS) REACTION

Goat Immune Serum or Rabbit PrecipitatingAgglutination with

Rabbit RBCs Goat Normal Serum Serum Immune Serum Normal Serum

1 mL 0.005 mL 0.0001 mL 0 01 mL 0.005 mL 0.005 mL Scant 01 mL 0.005 mL 0.001 mL Marked 01 mL 0.005 mL 0.005 mL Very marked 01 mL 0.005 mL 0.01 mL Very marked 01 mL 0.005 mL 0.05 mL Very marked 01 mL 0.005 mL 0.1 mL Very marked 01 mL – 0.1 mL 0 01 mL 0.01 mL – 0 02 hr room temperature Cells centrifuged and washed with normal saline 2 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 adose-dependent manner. The controls, lacking either goat immune serum or rabbit precipitating serum, showed no agglutination.Reproduced with modification from Packman CH: The spherocytic haemolytic anaemias. British Journal of Haematology 112:888–899.

*As mentioned in Chapter 2, describing the skin manifestations incold agglutinin syndrome as Raynaud’s phenomena is, strictlyspeaking, incorrect.110 Raynaud’s disease, the consequence ofvasoconstriction, leads in sequence of three phenomena: First, theaffected part becomes white and perhaps numb; then it becomesswollen, stiff and livid; and finally, when the vasoconstrictionpasses off, the part becomes red due to reactive hyperemia. InCAS the changes, which preferably are termed acrocyanosis, orliterally “blue extremity,” differ from those of Raynaud’s diseasein the absence of an initial white phase because there is no


history of Raynaud’s disease.* His serum contained acold agglutinin that reacted to a titer of 1,000 at 0°Cand reacted up to 30°C against normal RBCs as well asthose of the patient. They demonstrated that the cir-culation of the patient’s blood through fine tubes wasimpeded when the blood was cooled to 5°C and sug-gested that the Raynaud’s phenomenon might be re-lated to mechanical obstruction by autoagglutinatedRBCs. In their second patient, a woman aged 78, theyshowed that cooling of the fingers was associatedwith breaking of the column of blood in the capillariesof the nail bed. However, in neither case did theauthors describe hemoglobinuria or anemia.

Druitt,113 writing from Madras in 1873, described indetail the history of a doctor, aged 51 years, who over aperiod of at least 6 years had experienced attacks ofnumbness of the feet and a purplish blue discolorationof the hands on exposure to cold. These attacks might befollowed by the passage of “hematinuria.” The patientobtained relief from his symptoms when he went to livein a warm climate (India). Druitt believed that thenervous system and the blood were involved and sug-gested that the blood was undergoing “a hemolysis, adecomposition or necrosis of the blood globules.”

Roth, in 1935, reported a 59-year-old man whosuffered from Raynaud’s phenomenon affecting hishands, feet, and nose when exposed to mild degreesof cold.114 More severe chilling produced hemoglobin-uria. The author noted that the patient’s blood under-went rapid autoagglutination after withdrawal, whichwas reversed by warming.

In the same year, Ernstene and Gardner115 reporteda 38-year-old man who had attacks of hemoglobinuriaand Raynaud’s phenomenon on exposure to cold.Autoagglutination of his blood was noted at roomtemperature, red blood cell counts were difficult toperform, the cold agglutinin titer was 1280, and hewas anemic with a hemoglobin of 10.5 g/dL.

Despite these early reports, CAS did not receivewide recognition and the pathogenetic role of coldagglutinins was not well accepted. Indeed, as late as1943, Stats and Wasserman116 published a review inwhich they stated that in the great majority of casescold hemagglutination was innocuous, although “insome cases” of hemolytic anemia, PCH, Raynaud’ssyndrome, and peripheral gangrene, the cold hemag-glutination is of pathogenetic significance. Accuratedescriptions of the syndrome and features that distin-guished it from other forms of AIHA appeared duringthe 1950s.117

The hemolytic activity of serum of patients with coldagglutinin disease had not been well recognizedbecause the pH of blood rapidly rises to pH 8 and

higher in vitro following the loss of CO2, and the anti-bodies do not cause optimal lysis at alkaline pH.Dacie118 demonstrated the presence of cold hemolysinsin sera containing cold agglutinins by adding a trace ofhydrochloric acid to produce a slightly acid pH value.However, he still used the two-step temperaturearrangement in the classic Donath-Landsteiner test. In1953 Schubothe pointed out that hemolysis caused bythe cold agglutinins does not have a bithermic mode ofaction but takes place monothermically.119,120 He intro-duced the term cold hemagglutinin disease to separate thedisorder from other acquired hemolytic anemias.

In the 1950s it ultimately became apparent that thereexisted an obscure and rather unusual syndrome,which affected almost exclusively elderly subjects, thatwas characterized by mild to moderate hemolyticanemia and by the presence in the patient’s serum ofcold agglutinins at high titers, so that massive andrapid autoagglutination took place if their blood, afterwithdrawal, was allowed to cool to room temperature.In cold weather the patients suffered from what wasoften described as Raynaud’s phenomenon, affectingthe fingers, toes, and earlobes, and sometimes this ledto local gangrene. Hemoglobinuria, too, often devel-oped in cold weather. This is the condition we nowrefer to as cold CAS.

Discovery of Blood Group Specificity of PathologicCold Agglutinins. Early studies of the specificity of thecold agglutinin in patients with CAS demonstrated noblood group specificity. Mino121 is usually quoted ashaving introduced the concept of the “nonspecific”nature of cold agglutinins; he concluded that all humanRBCs shared a common receptor and that no distinc-tion could be made with regard to reactivity betweencells of different ABO groups. However, Wiener and hiscoworkers122 reported in 1956 that they had tested aserum derived from a patient with CAS against 22,964blood samples! Five samples only, as well as thepatient’s own RBCs, were not agglutinated at roomtemperature. The insensitive RBCs were designated “i”or “I-negative,” and the serum was said to contain“anti-I” (“I” for individuality). Thus started the unrav-eling of the complex Ii blood group system (seeChapter 6). By far the most common type of high-titercold antibody reacts with the I antigen, a small minor-ity with the i antigen, and a few antibodies react withantigens other than I and i (see Chapter 7).

The Physical Nature of Cold Agglutinins. The anti-bodies also have been studied by physical means. First,the use of the ultracentrifuge showed that in sera containing large amounts of a cold autoantibody, thiswould separate as a high-density protein and mightalso be visualized as a distinct sharp peak in the beta-gamma region on simple paper electrophoresis.123

Subsequently, when methods of immunoelectro-phoresis became available, it was clearly shown that notonly were these protein peaks composed of macro-globulin (IgM) but that they were also monoclonal. Inthat respect CAS is analogous to Waldenström’smacroglobulinemia in that the basis of both disorders is

vasoconstriction, and in that the blue cyanotic phase is moreintense; the affected part may in fact become deep purple. Thereis, too, no final hyperemic phase. Marshal et al.111 and Hillestad112

showed that the blood flow reactions to chilling are quite distinctfrom those in Raynaud’s disease proper. No evidence of anabnormal vascular response could be obtained. Both processescan, however, lead to local gangrene.


the formation by the patient of large amounts of an IgMparaprotein.

Subsequently, numerous case reports and detailedreviews of clinical findings, laboratory features, sero-logic and immunochemical characterization of theantibodies, and the pathogenesis of CAS have beenpublished (see Chapter 3).

MORE RECENT EVENTS

The investigators who, in the early days, contributed toour understanding of AIHA as we know it today wereclinicians in the true sense of the word. They studied atthe bedside and in clinical laboratories, using theirminds, hands, eyes, and ears; their most sophisticatedinstrument was a microscope. Information transmittaland retrieval were rudimentary at best; if the journalswere available, the language was more probablyforeign to the reader than not, either French, German,or English. They made errors, but they also identifiedand corrected them, so as to lay a foundation for themore sophisticated studies that were to come.

The second half of the twentieth century broughtimportant new insights into the diagnosis, pathogene-sis, and management of AIHA. Important advancesoccurred concerning the roles of RBC structure and bio-chemistry, the specificity of autoantibodies and theirmolecular structure, the molecular nature and reactionmechanism of serum complement, the concept of drug-induced immune hemolytic anemias including drug-induced AIHA, mechanisms of hemolysis, RBCstructure, and its genetic regulation. Future years willundoubtedly bring new understandings of pathogene-sis at the molecular and genetic level, and new meansof treatment, possibly involving the sciences of stemcell transplantation and gene replacement therapy.

HISTORICAL NOTES REGARDINGHEMOLYTIC TRANSFUSION REACTIONS

The fascinating history of blood transfusion has beenreviewed in a number of publications124-127 and,among descriptions of the early attempts at transfu-sion therapy, are dramatic accounts of hemolytictransfusion reactions. This is to be expected becausetransfusions were carried out long before there wasknowledge of blood groups or current good manufac-turing practices.

The Early History of Blood as a TherapeuticMeasure. Blood, in one form or another, was men-tioned as a possible therapeutic measure throughoutancient times. The Egyptians were said to advocateblood baths for purposes of recuperation and rejuve-nation. As late as the fifteenth century, blood was rec-ommended to remedy a variety of ailments, such aslunacy, fits, palsy, melancholia, and bad disposition,but not for blood loss or anemia, as would haveseemed more logical.

There is an apocryphal story that when PopeInnocent VIII was on his deathbed in 1492, a last des-perate attempt at his survival was made on the recom-mendation of an unknown physician. He received theblood of three youths supposedly via transfusion,although more likely as a draught. The fact is thatshortly thereafter he passed on, to Heaven, doubtlessly.The prescribing physician wisely and quickly disap-peared⎯in which direction is not recorded.125

Early Suggestions for Transfusions. Up to the seven-teenth century, blood must have been given only bymouth. Direct transfusion into the circulation had toawait the discovery that there was a circulation. Thebeginnings of transfusion therapy date from the mid-seventeenth century following Harvey’s momentousdiscovery of the circulation of the blood. He announcedin a monumental treatise, De Motu Cordis, that bloodcirculated within the body in a closed system, main-tained by the heart acting as a pump, and that the bloodwas sent to the limbs through the arteries and returnedthrough the veins, whose valves did not oppose itscourse that way. This stimulated actual experimenta-tion with injections into the bloodstream.125

FIRST RECORD OF TRANSFUSIONS

The first well-documented transfusions were carriedout by two widely separated investigators, oneEnglish, the other French. Because both individualand national priorities were at stake, considerablecontroversy was engendered and numerous publica-tions resulted as to who should be accredited withdoing the first transfusion.128-131

In England, a young physiologist and physician,Richard Lower (Fig. 1-29), of Oxford, participated inexperiments of injecting opiates, emetics, and othermedicines into the veins of living animals. As he statedin letters then and in a book published later, this stimu-lated ideas about injecting large quantities of bloodfrom different animals. In February 1665, he developedthe needed surgical skill and performed his first suc-cessful transfusion, from the cervical artery of one doginto the jugular vein of another, previously almost ago-nally exsanguinated. The recipient animal waspromptly restored to a healthy active state. There wasno untoward reaction, for dogs do not have naturalisoagglutinins, although they do vary in blood groupantigens. Lower’s experiments were recorded in theJournal des Savants of January 31, 1667.124

THE FIRST RECORDS OF HEMOLYTICTRANSFUSION REACTIONS

In France, a philosopher-mathematician and physician,Jean-Baptiste Denis (Fig. 1-30), performed the firsttransfusion of a human on June 15, 1667. His patientwas a boy of 15, a sufferer from a prolonged febrileillness and profound lethargy. He had been subjected


to, and had somehow managed to survive, 20 phle-botomies. Denis succeeded in transfusing him withabout 9 ounces of sheep’s blood and actually “cured”him of his ailment. Encouraged by this success, Denistried his good fortune again. This time he used ahealthy paid volunteer who received 20 ounces ofsheep’s blood without recorded difficulties except forfeeling “very great heat” along the vein in his arm andlater voiding “black urine.” Although the black urinestrongly suggests a hemolytic transfusion reaction, hewas otherwise asymptomatic and was so little dis-turbed that he proceeded to butcher the sheep and thenwent off on a drinking bout with companions.125

A third subject, a Swedish nobleman already mori-bund, did not fare so well and died soon after anattempted transfusion.

Next Denis treated a man who had episodes ofviolent maniacal behavior. The transfusion was onDecember 19, 1667, with 5 or 6 ounces of blood from thefemoral artery of a “gentle calf,” which “might dampenhis spirits.” The patient seemed to improve. A few days

later the procedure was repeated. This time, theredeveloped all the signs now recognized as typical of asevere hemolytic transfusion reaction. Denis’s descrip-tion can be considered a medical classic132:

As soon as the blood began to enter into his veins, hefelt the heat along his arm and under his armpits. Hispulse rose and soon after we observed a plentiful sweatover all his face. His pulse varied extremely at thisinstant and he complained of great pains in his kidneys,and that he was not well in his stomach, and that hewas ready to choke unless given his liberty. He wasmade to lie down and fell asleep, and slept all nightwithout awakening until morning. When he awakenedhe made a great glass full of urine, of a color as black asif it had been mixed with the soot of chimneys.124

Denis recounted that the following morning thesubject also manifested hemoglobinuria and had epis-taxis. However, by the third day his urine cleared, andhe improved his mental status and returned to hiswife. Denis attributed the color of the urine to a “black

FIGURE 1-29. Richard Lower(1631–1691). Oil painting by JacobHuysmans. (From Moore P: Blood andJustice. Chichester, England: John Wiley &Sons Ltd., 2003.)


choler” that had been retained in the body and hadsent vapors to the brain that caused the subject’smental disturbance.132 Several months later thepatient again became violent and irrational and hiswife insisted on yet another transfusion. Denisattempted this but without success because the manwas violent and would not cooperate. He died the fol-lowing night.

By this time, Lower had also initiated transfusion inhumans. On November 23, 1667, he and his skilledassociate Edmund King performed their first humantransfusion before The Royal Society. The patient was a22-year-old member of the clergy who was “somewhatunbalanced, whose brain was considered a little toowarm.” It was hoped that the operation would alter hischaracter. Accordingly, he was bled from his antecu-bital vein for 6 or 7 ounces and then he was connected

via silver tubes and quills to a sheep’s carotid artery. Itwas surmised that during 2 minutes, 9 to 10 ounces ofblood were so transferred. The patient afterward“found himself very well” and 6 days later gave thesociety a talk in Latin telling how much better he felt.Nowhere was any comment recorded about the effectof the transfusion on the patient’s temperament or his“too warm brain.”125

NATIONAL AND INTERNATIONALCONTROVERSY

In an action that presages modern medicine, the wifeof the patient who was transfused by Denis suedhim, charging that the transfusion had killed herhusband.124,125,127 Considerable furor was raisedamong Parisian physicians, but at the trial thedefense was successful in proving that the man hadbeen poisoned with arsenic by his wife. AlthoughDenis was thus exonerated, the Paris Society ofPhysicians declared itself against such experimentsand persuaded the criminal court in Paris on April17, 1668, to forbid further transfusions withoutapproval from the Faculty of Medicine of Paris,known to be bitterly opposed to the procedure. Tenyears later, an edict of Parliament prohibited transfu-sion experiments on humans. Soon thereafter, theRoyal Society in England disapproved transfusionpractices, as did the magistrates in Rome. Thiseclipse of overt interest in transfusion therapy lasted150 years.125

In the meantime, an international debate had beeninitiated as to who and which country should be cred-ited with the first transfusion. Throughout 1667 and1668, many around Europe contributed to the debate inthe form of letters and published pamphlets. Most fellneatly into pro-Denis or anti-Denis camps, although afew were prepared to express an open mind. The con-troversy is reviewed in detail by Moore.127

England’s claim was based on Lower’s thoroughlydocumented dog-to-dog transfusions in 1665. TheFrench claimed that the idea had been proposed 10 years earlier and that human transfusions werefirst done by Denis. National prestige seemed to be atstake even though the treatment was admittedly lessthan uniformly successful. A considerable exchangeof letters between Denis and Henry Oldenburg,133

the secretary of the Royal Society, took place in late1667 and 1668 with publication in the Proceedings ofthe Society. Denis had sent a letter to the PhilosophicalTransactions, in London, the official publication of theRoyal Society, describing his first transfusion andthis was actually printed dated July 22, 1667.However, its publication did not take place untilSeptember because the editor, Oldenberg, was incar-cerated in the Tower of London on suspicion oftreason. Fortunately, he was declared innocent. (Feweditors can claim so valid an excuse for delays inpublication.125)

FIGURE 1-30. Jean-Baptiste Denis (From Moore P; Blood and Justice.Chichester, England: John Wiley & Sons Ltd., 2003.)


Nevertheless, considering that Lower did notperform his first human transfusion until November ofthat year, there seems little question that Denis was the first to perform transfusion of a human being.124

The best that Oldenburg could contend was that the“English might well have been first if they had not beenso tender in hazarding the life of man, “a post hoc solic-itude with no foundation in fact.125 The controversyregarding priority long remained in doubt and was notreally resolved satisfactorily. It finally seemed to beaccepted that Lower, of England, deserved the creditfor doing and fully describing the first animal transfu-sions, whereas Denis, of France, was credited with thefirst successful transfusions in humans.125 Denis shouldalso be credited with the first accurate and detaileddescription of a hemolytic transfusion reaction!

It was not until the late 19th century that successfultransfusions were reported, again by an English obste-trician, William Blundell. Transfusion did not becomecommonly used until almost a decade followingLandsteiner’s discovery of the ABO blood groups.124–126

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Immune Hemolytic Anemias || Historical Concepts of Immune Hemolytic Anemias