R. J. Sokol S. Hewitt Regional Blood Transfusion Centre Sheffield, England

Transcription

1 Volume 4, Issue AUTOIMMUNE HEMOLYSIS: A CRITICAL REVIEW Authors: R. J. Sokol S. Hewitt Regional Blood Transfusion Centre Sheffield, England Rcfercc: H,H, Gunson Regional Blood Transfusion Service Manchester. England I. INTRODUCTION Autoimmune hemolytic anemia (AIHA) is usually considered to be a well-defined clinical syndrome, but the more closely cases are examined, the more obvious it becomes that the anernia merely represents an extreme of a fundamental disturbance of immune homeostasis. As hemolysis may be present in the absence of anemia, we prefer the term autoimmune hemolysis (AIH), which refers to the situation when an antibody is produced which binds to autologous red cells and there is evidence that this causes a shortening of the red cell lifespan. Like all definitions, this one has its limitations, diagnosis depending on the sensitivity of in vitro and in vivo investigations in an often complicated situation, and on the awareness and skill of the attending physician; no doubt many mild, self-limiting cases pass unnoticed. In this article we will attempt to provide a critical in-depth review of recently published papers on AIH and evaluate them in terms of our own experience. ~--~ Needless to say, we have had to be selective and have chosen to concentrate on the following topics of current interest: development of autoimmunity, mechanisms of immune destruction of red cells, detection and measurement of ceil bound autoantibodies and complement fragments, treatment of AIH, and clinical studies. II. DEVELOPMENT OF AUTOIMMUNITY In spite of considerable study, the reasons why autoimmune diseases occur are incompletely understood; some of the various theories that have been put forward can be discussed in relation to AIH. A. Theory of Natural Occurrence of Autoantibodies A widely accepted concept suggests that all the factors necessary for the development of autoimmune disease (i.e., autoantigens and self-reactive B and T lymphocytes) are already present." Autoantibodies per se are not necessarily pathological and there is evidence that they occur in the absence of disease and are involved in the transportation and subsequent phagocytosis ( by macrophages) of undigested macromolecular self; this is essential for the cleansing of the organism. 7 Normally, some form of self-restraint (or self-tolerance) occurs and homeostasis is maintained. This self-restraint may be via clonal deletion, the silencing of the appropriate T-inducer cells by various types of T-suppressor cells, by the inability of the antigen-presenting cells to function, or by antiidiotypic antibodies reactive against relevant antigen receptors on B-cells. 6 Under certain circumstances the self-restraint mechanism may be overcome and an autoimmune state develops. 6 This hypothesis is shown in Figure 1.

2 126 CRC Critical Reviews in Oncology/Hemutology Autoantigen T suppressor lymphocytes [antigen specific, idiotype specific, non-specific) may be defective leading to development of autoimmune disease ~ Bypass (e.g. by adjuvants, polyclona! activators, new carriers) leadinq to develop. ment of the autoimmune disease T helper l ymphocytes [ B ly cytes T effector lymphocytes Autoa Autoimmune disease FIGURE I. Development of autoimmune disease.' 1. Alterations in Antigen or Antigen-Presenting Cells Changes in concentration of antigen or antigen-presenting cells can disturb immune homeostasis and lead to an imbalance in favor of overt autoimmunity. Increased expression of autoantigens (resulting from aging, infection, activation of retrovirus,or from incomplete catabolic degradation) w" may be normal or pathological, Exposure of antigens on red cell surfaces with age, and the subsequent attachment of IgG, is likely to be the normal mechanism of removal (by macrophages) of senescent red cells. 9,~~ A similar mechanism is thought to have been responsible for AIH in a patient with pulmonary infection: the microorganism produced neurarninidase which removed sialic acid from the red cell surface and exposed T antigens to the naturally occurring anti-t. ~j Self-tolerance can also be broken by new carrier determinants (provided by exogenous agents such as viruses, cross-reacting antigens, and drugs) bypassing the normal T-suppressor block and triggering appropriate helper/effector cells; the bypass (Figure I) can also be brought about by adjuvants, e.g., Freund's complete, or through direct triggering by po-

3 Volume 4, Issue lyclonal B.cell activators, e.g., Epstein-Barr virus. Two examples relating to the development of AIH will illustrate the bypass mechanism. First, during renal dialysis, traces of formaldehyde (used to sterilize the machines) may come into contact with red cells and interact with receptors for the MN blood groups to create a neoantigen; this induces the production of anti-n-like antibedies capable of destroying the cells which bear Ihe neoantigen. ~2~1"~ Second, erythrocyte autoantibodies can be produced in chimpanzees by injecting autologous erythrocyte stroma treated with methyldopa and diethyidithiocarbamate; this finding, which could be pertinent to the AIH seen in some patients on methyldopa therapy, probably results from the interaction of oxidized drug with certain unidentified peptides of the erythrocyte membrane. ~5 The role of antigen-presenting cells in the development of autoimmunity has received very little attention and is ill understood; no doubt it is of very great importance and will provide a fruitful area for future work Alterations in the Idioo,pe-Antiidio~.pe Network Autoimmune states may also result from disturbances of the idiotype-antiidiotype network normally involved in the control of the immune response. ~' For inst~mce, if an environmental agent (virus or parasite) stimulates alloantibodies having a cross-reacting idiotype shared with self-reactive lymphocytes, the latter may be triggered to produce an autoantibody response. Alternatively, an autoreactive lymphocyte could be stimulated if its receptors have a cross-reacting idiotype similar in shape to a determinant on the foreign antigen. The AIH due to anti-i which follows infection with Mycoplasma pneumoniae possibly results from this sort of reaction." A recent variation on this theory has suggested that autoantibodies are u~ttiidiotype antibodies to antiviral antibodies: ~6 antiviral antibodies are produced against thorpe viral structures which interact directly with the host; and the antiidiotype antibodies (which are elicited against these antiviral antibodies) also have the in~'idental property of being antibodies to host structures, i.e., they act as autoantibodies, j' It would be interesting to test this hypothesis in patients with AIH, since many cases appear to be dramatically linked with a preceding viral infection. 3. Alterations in T-Lymphocytes Normally, autoantibody responses are damped clown through T-suppressor interactions, and so it is likely that some fault in immune regulation is necessary before autoimmune states become established. T-suppressor lymphocytes may be antigen specific, idiotype specific, or nonspecific, and in spite of the difficulties in assessing the T-suppressor system, there is evidence that defects can occur in some or all of these cell types/' perhaps, for example, as a result of an antiidietype-antiviral reaction affecting the cell surface. TM Altered immune regulation has been reported in AIH. In humans, the results of early studies were apparently contradictory. In one report, the absolute number of peripheral blood TI cells was decreased, ~7 while in another series, high levels of activated T., lymphocytes were found i~1 a majority of patients, and over a 2-year period, the levels bore a direct relationship to the activity of the disease. ~K In neither of these studies was the T-cell subsets indentified. More recently, attempts have been made io differentiate T-helper and T-suppressor cells in patients with AIH using monoclonal antibodies (OKT4 and OKT8, respectively), but again results apparently conflict. In one study, (cited in Reference 19) an increased OKT4/ OKT8 ratio, attributed to loss of suppressor cells, was found in a large percentage of cases. However, anotller group" reported an increase in T-suppressor cells compared with T-helper cells in 8 out of 15 patients and concluded that not all forms of AIH were associated with a deficit of T-suppressor lymphocytes; but it should be noted that many of these patients

4 128 CRC Critical Reviews in Oncology/Hematology had received treatment which could itself tlave altered the distribution of the T-cell subsets. A further investigation of 40 cases found that while patients with cold lgm type AIH did not show major abnormalities of T-cell subset distribution, a large proportion of patients with warm IgG type disease had abnormal T-helper/T-suppressor (OKT4/OKT8) ratios, both higher and lower' than normal. -'~ Because of the effect of associated pathologies and treatment on T-cell subset distribution, no simple explanation could be given for these results in respect of the AIH. Further evidence for immuno-regulatory defects in the pathogenesis of AIH has been provided by studies of patients taking methyldopa. In vitro, T-lymphocyte suppression of lgg production was inhibited both in isolated T-cells incubated with the drug and in T cells from patients on therapy; in addition, the drug caused a reduction in the mitogen induced activation of T-lymphocytes. In vivo, it was thought that methyldopa altered the immune system by causing a persistent increase in lymphocyte cyclic AMP; this inhibited suppressor T-cell function and thus led to unregulated autoantibody production by B cells; in certain patients this resulted in overt hemolysis. "-2 In some interesting animal work mice developed AIH following immunization with crossreacting rat erythrocytes. The AIH was self-limiting in normal mouse strains due to the activity of specific T-suppressor cells, but in other strains of mice, where T-suppressor lymphocytes were deficient, the AIH persisted. -'-~ The importance of T-suppressor lymphocytes in preventing the spontaneous expression of AIH in New Zealand black mice was shown in a series of cell transfer experiments;" however, caution should be applied in this case, since a mutant strain of New Zealand black mouse has been described recently where T-suppressor cells were retained but AIH still developed, probably as a result of a defect in the B-cells. '--~ B. Theory of Somatic Mutation The above views are not universally accepted, and it has been argued that autoimmune disease results from somatic mutations in lymphocytes producing clones with autoreactive potential. -'5 It is thought that normal persons lack pathogenic clones capable of reacting with self-antigens, whereas other individuals are genetically predisposed to particular autoimmune diseases because they have "preforbidden clones" capable of somatic mutation into "forbidden clones. ''25 This view, which contrasts with the thesis that autoantibodies are physiologically necessary,'' explains the alterations in peripheral blood T-cell subsets merely a manifestation of the nonspecific effector phase of the imtnune respon:~e, and therefore unlikely to be a prime cause of disease. 25 The somatic mutation theory also offers an explanation for the discordance of autoimmune disease in monozygotic twins; it suggests that environmental triggers, particularly microorganisms bearing suitable cross-reactive antigens, alter the probability of particular mutational changes occurring. 25 Some evidence to support the somatic mutation theory comes from experiments in mice where AIH can be induced by exposure to a methylating carcinogen; it is thought that the age~..', has a direct effect on B-cell DNA at the level of the immunoregulato~ genes. 26 However, against the theory is the argument that since most autoantibodies are polyctonal, they are unlikely to have arisen as a result of lymphoid mutation. K C. Genetic Factors The presence of a strong genetic element in the development of autoimmune disease has been known for a long time, and family studies have been used to support the various theories of pathogenesis. For example, reports of AIH occurring in families, otten in association with other autoimmune diseases, -'7--~1 were thought to indicate that the patients had a generalized immune dysfunction, the hemolysis representing the "lip of the iceberg. ''-~~ Alternatively; the specific manner of transmission of autoimmune disease, s within families, together /

5 Volume 4, Issue with the restricted nature of autoreactivity, have been used to support the view that specific abnormalities of the immune response, rather than a generalized breakdown of tolerance mechanisms, were the cause of the disease. 2' With respect to the HLA system, it has been suggested that autoimmunity results from dez":pression of genes coding for the HLA-D antigens; this would allow their expression in the plasma membrane and thereby make the normal surface components potentially antigenic. 6 Another possibility is that the histocotr~patibility systems, both major and tninor, cause clonal deletion (at least in a functional sense) throughout life; this results i:n a dynamic immune response repertoire, and thus influences the probability of specific cones arising by somatic mutation. Such clonal deletion could have either a positive or a negative effect depending on whether the deleted clone was the precursor of a forbidden clone or that of a clone which would have acted as an arltiidiotype against the forbidden clone; this is the "Hgene theory of inherited autoimmune disease. ''25 Non-HLA genetic factors are also important. 32"-~3 A study of ten families where more than one member was affected with AIH, immune thrombocytopenia (ITP) or systemic lupus erythematosus (SLE) strongly pointed to an autosomal dominant factor that enhanced the predisposition both to overt disease, and to serological abnormalities..'~ This factor was possibly an enzyme deficiency,-~-~ which would be in keeping with the theory that autoimmune disease can result fi'om failed or incomplete catabolic degradation. 7 O. Conclusions It can be seen that the pathogenesis of AIH is complex and at this stage a full explanation of the reasons for its development is not possible. The subject is fascinating and undoubtedly further understanding will be gained in time, probably necessitating considerable revision of the points made here; for the present, we recommend that one retains an open mind and does not become too fervent a supporter of any particular theory MECHANISMS OF IMMUNE DESTRUCTION OF RED CELLS In patients with AIH there is strong evidence that the coating of autoantibodies per se, does not damage the ged cells but causes erythrocyte destruction by complement activation and/or by induch~g interactions with cells of the mononuclear phagocyte system. -~4"-~-~ Erythrocyte destruction may be extra- or intravascular. In most patients, hemolysis is extravascular and involves tnononuclear phagocytes reacting with erythrocytes coated with antibodies of lgg (and possibly lga) class and with C3b. Destruction of IgG coated cells usually occurs in the spleen, but when the coating is heavy it may occur anywhere in the mononuclear phagocyte system. 35.-~' Less frequently, destruction occurs intravascularly, either through the activation of complement, or more controversially, through the erythrocytes interacting with lymphoid and granulocytic cells. A. Intravascular Destruction of Erythrocytes by Complement Intravascular hemolysis is seen in less than 20% of patients with AIH; -~7 it occurs if complement activation proceeds to completion, when the resultant defect in the erythrocyte membrane causes osmotic lysis of the cell. Recently, deposition of the terminal C5b-9 complex on erythrocytes of patients with AIH has been demonstrated using a radioisotopic method. 3s Antibodies which trigger this process are called hemolysins; most are of IgM class though some, notably the Donath Landsteiner (DL) autoantibody, are lgg. Complement-induced lysis is tile most efficient method of red cell destruction and causes severe anemia; fortunately it is uncommon since the in viva activity of hemolysins is restricted by their optimal temperature of activity being well below 37~ 3s Hemolysis is never complete since regulatory inactivators operate at several levels of the c~mplement cascade. In patients

6 130 CRC Critical Reviews in Oncology/Hematology with AIH, inhibition occurs at the level of C3; and C3 can be detected on unlysed cells by the direct antiglob~lin test (DAGT). "'~s Erythrocytes coated with C3b can then be destroyed extravascularly by cells of the mononuclear phagocyte system. B. Erythrocyte.Mononuclear Phagocyte Interactions Erythrocyte-mononuclear phagocyte interactions in patients with AIH are of particular current interest. Their efficiency depends on several factors including the immunoglobulin class and subclass of the autoantibody, the number of antibody molecules bound per erythrocyte, the thermal range of the antibody, the ability of the antibody to activate complement, the amount of free IgG present in the surrounding medium and probably on the activity of the individual's macrophages. 37'39 In most patients, the erythrocytes are coated with noncomplement fixing IgG autoantibodies which induce red cell destruction by the attachment of the Fc portion of the IgG molecule to the macrophage surface at specific receptor sites. 35 This adherence leads to erythrocyte membrane damage by ADCC (antibody dependent cell-mediated cytotoxicity) and to erythrophagocytosis. 37 Whether ADCC or phagocytosis is the prime mechanism of erythrocyte destruction remains to be determined, though both may cause significant hemolysis in vivo. 37 The amount of antibody bound to the cell seems to be important: using blood monocytes and erythrocytes coated with anti-rhesus (Rh) D, it has been shown that the amount of phagocytosis is inversely proportional to the degree of sensitization. At low levels of sensitization, phagocytosis becomes more important than ADCC; the level at which this occurs is much lower with lgg3 than with lgg I antibodies. 3s Adherence to macrophages may also result in partial engulfment of the erythrocyte, leaving a nonphagocytosed portion to become a rigid spherocyte which is prematurely destroyed in the spleen. 3-~ In vivo, splenic rnacrophages and Kupffer cells are the main effectors of red cell destruction, the blood monocytes appearing to play only a minor role, 37 though they are the most convenient cells for laboratory use. In an investigation of methods suitable for routine laboratory assessment of red cetlmonocyte interactions, adherence, ADCC, and erythrophagocytosis were compared using erythrocytes coated with varying amounts of anti-rh(d). 4" Tests for adherence were the simplest to perform; following a brief incubation period, the number of monocytes with three or more attached erythrocytes (i.e., rosettes) were counted. ADCC was e~amined at a monocyte-erythrocyte ratio of 10:1, the amount of ~Cr released being measured after 3.5 hr. Erythrophagocytosis was determined microscopically and expressed as the percentage of monocytes that had ingested one or more erythrocytes. Testing for rosette formation took the least time and ADCC the most. Of the systems studied, erythrophagocytosis was much more sensitive than the others, possibly because it could be recognized at the single cell level and was concluded to be the method of choice. Erythrocytes coated with C3b can also bind to specific receptors on mononuclear phagocytes and be prematurely destroyed; in vivo, the main effector cells are macrophages. ~5 While IgG-coated cells are usually destroyed in the spleen, in situations where erythrocytes become coated with C3b alone, destruction (by phagocytosis and through the production of spherocytes) occurs mostly in the liver. The binding of C3b-coated erythrocytes to macrophages is very efficient 35 and a major factor in determining the rate of liver sequestration appears to be the rate of blood flow 4~ however, maximal adherence and phagocytic capacities (the latter being considerably less than the former) limit the amount of destruction. 3,s Although many erythrocytes are ingested, others are released back into the circulation; by this time the C3b has been cleaved further and C3d (which does not react with macrophages) can be detected on the nonphagocytosed cells; 42 interestingly, recent work in patients with cold hemagglutinin disease (CHAD) has shown that the final product of in vivo C3 activation is C3d,g rather than C3d. 43

7 Volume 4, Issue In patients with AIH due to anti-i or anti-i, red cells coated with C3d alone have a normal lifespan. -~ The erythrocytes are protected against premature destruction because C3b receptor and Ii antigen sites are in close proximity, the C3d sterically inhibiting binding of more autoantibody, and hence fixation of further active C3b, onto the cells; C3d protection does not extend to cases where the autoantibodies have other specificities. 4~ These C3b-mediated reactions are evidenced by a dual red cell survival curve in most patients with CHAD, a population of rapidly destroyed cells being present in conjunction with one having a normal lifespan ADCC The basic mechanisms of ADCC have been investigated using monocytes and 5~Cr-labeled red cells coated with lgg alloantibodies, the degree of cytotoxicity being expressed either as a percentage of S~Cr released or the number of tar'get cells lysed per monocyte. '~ Monocytes suitable for ADCC testing can be obtained by adhesion to plastic surfaces, and subsequently detaching the cells by lowering the temperarure to 4~ to 1500 molecules of lgg anti-rh(d) per erythrocyte are necessary to initiate ADCC, but thereafter the relationship between the quantity of antibody and ~lcr released is linear. 42 ADCC is inhibited by normal serum, immune complexes and platelets 46 and is significantly reduced in adult patients who have had a previous splenectomy, but only if all the splenic tissue has been removed. 47 ADCC is mediated by release of lysosomal enzymes from the mononuclear phagocytes, probably at sites of erythrocyte attachment, and is independent of phagocytosis/8 Cytochalasin B inhibits phagocytosis but enhances ADCC, whereas hydrocortisone and colchicine, which inhibit ADCC, have no effect on phagocytosis: 8 Exclusion of oxygen from the incubation medium halves ADCC, though has no effect on phagocytosis, and it has been suggested that lysis is partly dependent on the generation of hydroxyl radicals by the effector cells. 49 Inhibition of monocyte ADCC by low concentrations of free lgg occurs with erythrocytes coated with anti-rh(d) but not with cells bearing IgG anti-a (except when the coating is weak): ~ the vastly greater A and B antigen densities allow sufficiently high numbers of antibody molecules to be bound to overcome the inhibition. 4~ The number of adherent erythrocytes per monocyte also has a beating on the inhibition; increasing the number results in a decrease in the percentage inhibition, though only when relatively weakly coated red cells are used: ~ However, ADCC of anti-d-coated erythrocytes can be induced in the presence of free lgg if the cells are centrifuged to a pellet before incubation, and the effect is augmented by increasing the red cell-monocyte ratio, s* Splenic hemoconcentration produces similar conditions in vivo, and this helps to explain why the spleen is the main organ of red cell destruction in patients with AIH due to noncomplement fixing IgG autoantibodies. 5~ However, the effect of free lgg is probably less important in vivo where the prime effector cells are macrophages; compared with monocytes, macrophages show reduced lgg blocking of ADCC and generally have enhanced Fc receptor activity. 52 Although erythrocytes coated solely with C3b do not undergo ADCC, complement coating augments IgG-mediated lysis, signficantly reducing the number of IgG molecules necessary to initiate ADCC; the augmentation persists even after degradation of C3b to C3d Erythrophagocytosis In vitro, studies using monocytes showed that erytl'd-ophagocytosis was proportional to the concentration of the celi bound antibody. In the case of anti-rh(d), engulfment was observed with as few as 1000 to 2000 molecules lgg per cell; ingestion was markedly augmented by concurrent coating of the red cell with complement components, and a~,though free gg caused some degree of inhibition of phagocytosis, this was never complete..~

8 132 CRC Critical Reviews in Oncalogy/Hematolagy 3. Relationship between lgg Aumantibody Subclass and Er3.,throcyte Destruction Early studies showed that IgG receptor sites on mononuclear phagocytes were specific lbr antibodies of the lggl and lgg3 subclasses only ~-''s't and that these receptors could act independently or cooperatively with similar ones for C3b. -s'~ In a series of patients where only IgGl autoantibodies were present, the amcaunt of cell bound antibody (determined by flow cytofluorometry) was compared both with signs of increased hemolysis in vivo and with the cytotoxic acfiv'~y of normal monocytes to these erythrocytes in vitro. -~ The results showed that induction of hemolysis or cytotoxicity was predominantly determined by the amount of cell bound antibody. ~ The number of bound lggl molecules needed to cause erythrocyte destruction was well above that necessary to give a postive DAGT. 3-~ In a study of 22 healthy blood donors with a positive DAGT, nooe had more than 1000 molecules of lggl per cell, suggesting that the critical level for sequestration was above ttais. 56 In the case of IgG3 autoantibodies, fewer cell-bound molecules are needed to effect erythrocyte destruction than are necessary to produce a positive DAGT. IgG3 molecules are much more efficient in causing adherence to macrophages than IgGl molecules, probably because of a greater affinity for the Fc receptors, rather that~ two separate receptors being present. 35 An investigation of 271 patients with noncomplement-binding IgG autoantibodies co, m- pared subclass composition with the occurrence of hemolysis in vivo and with erythrocytemonocyte rosetting in vitro. Patients wt~ose ce)}s were co, ted wilh lgg3 were almost always hemolyzing, whereas only some of the 259 patients witt~ lggl coating showed evidence of hemolysis. Cases where lgg2 and/gg4 coated the cells (tvr patients in each group), showed no evidence of increased red cell destruction..st As an identical relationship was noted between subclass composition and the ability to form rosettes, it was suggested that the latter could be used to predict the likelihood of in vivo hemolysis. 5r 4. Studies of Erythrocyte-Mononuclear Phagocyte lntemcth~ns & Patients with Autointmune Hemolysis Erythrocytes from 16 patients with active AIH showed increased adherence and phagocytosis per monocyte compared with erythrocytes from normal subjects, at~d art immune etiology was suggested by similar findings in 7 out of I I patients with acquired hemolytic anemia and a negative DAGT. s8 Erythrocyte-monocyte reactions appeared to be fu~her enhanced in those patients where complement was detected on the red cells in addition to lgg autoantibody. 59 An assessment of the number of eryflwocytes bound or ingested by autologous monocytes was made by isotopic and morphological means in 17 patients with AIH, and the findings compared with those using control monocytes from normal subjects. ~' Increased patient-control ratios, seen in 12 patients with the morphological assay and in 15 with the isotopic method, suggested that enhanced monocyte activity oci:curred in certain patients, -~ probably as a result of increased numbers of Fc receptors."' Human peritoneal macrophages can also be used as effector cells, significant erythropbagocytosis being demonstrated in six patients with warm type All-I. ~'~ Methyldopa-induced autoantibodies of dubious clinical significance failed to promote erythrophagocytosis with either autologous or allogeneic monocytes. ~ In a study of six cases with a p~sitive 13AGT due to methyldopa therapy, increased monocyte-erythrocyte activity was only seen in the two patients who were hemolyzing; ~ when followed sequentially, such patients only showed significant erythrophagocytosis during hemolytic periods.~z When red cell adherence to a}}ogeneic monocytes was assessed in 42 patients with a positive DAGT due to noncomp}ement-binding IgG autoantibodies, rosette formation was seen in all 22 subjects who were actively hemolyzing, but did not occur in the 20 others where there was no evidence of hemolysis. '~7 Although rosette formatiot,,:,,as noted in untreated patients with AIH, it was usually absent in patients who were receiving more than 30 mg prednisolone per day; '~ a glucocorficoid-induce(!

9 Volume 4, Issue reduction in the number of monocyte receptors for lgg has been reported both in normal volunteers and in patients with AIH. ~ C. Role of lga Autoantib~dies in Autoirnmuue Hemolysis in about 2% of patients with warm type AIH, '" the red cells are coated solely or predominantly with autoantibotlies of IgA class: `"'~' il~ such cases, hemolysis is often severe. `'7 In our own unpublished studies, 39 patients (3%) were found wt~ere IgA coating was readily detectable; in all ir~stances autoantibodies of other classes or complement were present ir~ addition: when an enzyme-linked DAGT was used, increased amounts of cell bound l/ga could be demonstrated in about 8.5% of patients. Specific Fc receptors for lga (which is generally thought to be t~oacompiement binding) -~.~ have been demonstrated on monocytes.".'' and it is possible that red cell destruction occurs via the mononuclear phagocyte system in a manner similar to that induced by lgg autoantibodies. '~ Spherocytosis was seen in patients with IgA-coated red ceils, and splenic sequestration occurred when such cells were labeled with 5~Cr and used tbr survival studies. -~~ However, the pathogenic sigrtificar~ce of erythrocyte bound IgA has not been proven; in vitro sensitization with secretory IgA alone did not induce ADCC of ox red cells, though it could significantly augment that due to lgg: TM and in several cases of AIH thought to be due to iga antibodies, small quantities of autoantibodies of other classes, which could have been responsible for the hemolysis, either were demons~ratetl using sensitive techniques '0"r or were not looked for. `'~ Clearly. further studies are needed to elucidate the role of cell bound IgA in All-t. D. Immune Deslruction of Erythrocytes Mediated by Lymphoid Cells and Granulocytes The role of lymphoid ceils it~ immune hemolysis is controversial. It is thought th~tl K or null cells, which possess receptors to IgG and C3b, can effect ADCC, 7~ and such reactions have been demonstrated in vitro using human K cells and erythrocytes sensitized with Rh antibodies. 7-' K-cell ADCC is almost as efficient as that obtained with monocytes 7~ and similarly is more effectively promoted by lgg3 than lggl antibodies. 7-~ K cells are capable of acting at effector-larget cell ratios of i:1 or less, and destruction occurs even when the red cells are weakly coated. 72 Since the reaction is not inhibited by free IgG. lysis of IgGcoated cells would not be restricted t~ the spleen. TM It has been suggested that significant lymphocyte-induced hemolysis may occur in patients with AIH, and compared with normal individuals, that such patients have more aggressive K-cell activity which could contribute to the severity of the disease. 72 Neutrophils, which also carry receptors for the Fc portion of lgg, normally show very little cytotoxicity towards antibody-coated red cells. However, considerable activity was demonstrated by granulocytes from a patient with an infectious disease, and this may in part explain the increased severity of AIH seen during some infections. -'.' E. Conclusions The mechanisms involved in immune-mediated erythrocyte destructiota are an important and fascinating subject, and future developments in this field are awaited with interest, No doubt assessment of lgg subclass and erythrocyte-mononuclear phagocyte reactions (or even possibly erythrocyte-k cell reactions) will shortly become routine in the investigatior~ of patients with suspected AIH. IV. DETECTION AND MEASUREMENT OF CELL BOUND AUTOANTIBODIES AND COMPLEMENT FRAGMENTS ReccnOy, the quantitation of autoantibody and r on erylhrocytes has become increasingly fini~rtant in investigating and monitoring the clinical progress of patients with

10 134 CRC Critical Reviews in Oncoiogy/Hematology AIH. Such methods are particularly useful in those rare i~atients with Ihe clinical,,~jgm~ia of AIH, but in whom a negative DAGT is found because too few fnolecules are attached to the erythrocytes to be detected by conventional agglutination techniques. 7-~ One sensitive method, capable of detecting lgg on eryzhrocytes from normal individuals and differentiating young from old cells, involves augmenting the DAGT with polyvinyl pyrrolidone in an autoanalyser system. 7r Another method uses concentrated eluates to demonstrate small amounts of cell bound autoantibodies; TM in six patients with AIH and a negative DAGT, concentrated eluates, prepared from 50 to 200 m~ of blood, were able to sensitize red cells of common phenotype (but not Rh null cells) to give positive anr reactions. 7~':''~ We use much smaller quantities of blood to make concentrated eluates, the method being both simple and practical, 2'~ and IgG antibodies could be readily detected in five patients with evidence of AIH but a negative DAGT. In the complement-fi antibody consumption test, a known nm~ber of patients' red cells are incubated with a predetermined amount of rabbit-amihuman!gg. The amount of IgG remaining after absorption is measured (by quantitative complement fixation), and by reference to a standard curve the number of lgg molecules per red cell can be determined. TM By this method, normal individuals were found to have 35 or fewer molecules IgG per red cell, whereas 14 patients with A[H, in whom the DAGT was negative, had 70 to 434 molecules per cell, ~-' Similarly, in a patient with a relapsing hemolytic anemia during pregnancy and a negative DAGT, complement-fixing antibody consumption demonstrated 212 molecules of IgG per erylhrocyte, The hemolysis, which responded partially to prednisolone during pregnancy, resolved post-panum and repeat testing showed a marked reduction in the number of cell bound lgg molecules. ~r More recently, radioisotopes have been used to assess the amount of lgg or complement bound to erythrocytes. Such methods are very sensitive and increased numbers of cell bound antibody molecules can be readily demonstrated in patients with warm type AIH where the standard DAGT is I~egative. ~j Using ~:sl labeled anti-lgg, and by suitable absorption and correction for nonspecific binding, a reproducible and accurate method of quantitating erythrocyte IgG has been developed: the number of IgG molecules on normal cells ranged from 5 to 90, with an average of 39, xz a value similar to that obtained using complement-fixing antibody consumption. 7s In four patients with warm AIHA where the standard DAGT was positive, between 2700 and 6000 molecules of IgG per red cell were found. -~6 However, in 16 cases of AIHA where the standard DAGT was negative, the highest number of IgG molecules per cell was 260 and the lowest 10; in 11 of the cases it was less than 100, and it was conlcuded that the anemia was not simply due to the number of cell bound IgG molecules, and other factors, e.g., IgG subclass, needed te be taken into account. ~.~ The latter point is illustrated by the occurrence of positive DAGTs in healthy blood donors; in these cases, although the number of lgg molecules per red cell ranged from 110 to 850, the autoantibodies were of IgGl or IgG4 subclass. 5~' Two patients with cold AIHA had normal quantities of IgG per erythrocyte. -~6 A method using ~2Sl labeled protein A to detect erythrocyte bound IgG has also been described. ~4 The method does not seem to have any advantage over radiolabeled anti-lgg; furthermore, protein A is relatively expensive and does not bind with IgG3, an immunoglobulin subclass of great importance in the development of AiH. Isotopic methods also readily lend themselves to quantitation of erythrocyte bound complement. In earlier studies, a radiolabeled polyclonal anti-c3d was used. One group reported that normal red cells had 97 to 557 molecules of bound C3d, ~5"~6 while another found 50 to 160 molecules per cell; 8~ generally, there was no variation with age or sex, K7 although cord erythrocytes had fewer C3d molecules than adult cells, 8f' individual levels remained stable over a!0- to 12-week period of study. 8v Moderately elevated amounts of erythrocyte C3d were found in just over 30% of hospital patients, 86-~a and although these patients generally

11 Volume 4, Issue did not have AIHA, they did suffer from diseases where complement was likely to be activated. Patients with markedly elevated levels (up to 31,000 molecules of C3d per cell) usually had florid AIHA; hemolysis could occur when only moderate increases were present, the critical level being about 1100 molecules; in individual cases, tile level correlated with the severity of the disease and its reponse to treatment. ~' More recently, a method has been developed using an ~25I labeled monoclonal antibody directed against a C3d determinant on all forms of cell bound C3;"" by this means, normal individuals were found to have 420 +_ 140 molecules C3 per cell, whereas three patients suffering from CHAD and active hemolysis had between 16,000 and 52,000 molecules per celt. "/'he advantage of using a monoclonal antibody is that only one C3 antigen site is recognized, as compared with polyclonal antibodies which can recognize several sites and thus give less reliable results due to variations in antisera specificity and binding ratios. Because few cases were tested with the monoclonal antibody, it is difficult to determine the number of C3 molecules necessary to cause erythrocyte destruction, but two blood donors, with no evidence of henloly,,,is had 3500 and a300 molecules C3 per cell; s.' this conflicts with the previously quoted critical figure of I100 molecules, ~s and more work is urgently needed to resolve this difference. Enzyme-linked antiglobu!in tests have also been used to detect and quantitate antibodies bound to erythrocytes. ';''-'j-' Such methods are reported to be as sensitive as radioisotopic techniques and have the advantages of a long shelf-life for the reagents, avoidance of radioactive materials, and simplicity of measurement."" In early developmental studies. erythrocytes were sensitized with various blood group antibodies, washed, and incubated with an anti-lgg-alkaline phosphatase conjugate: after further washing, enzyme substrate was added and the optical density of the product was measured; this was found to be linearly proportional to the concentration of the antibody and the results were reproducible.'"' Recently, it has been suggested that the test can be improved by using glucose oxidase-linked antiglobulin. '~3 However, using the alkaline phosphatase method, and by reference ~o a standard curve derived from reactions of anti-rh(d) with Rh(D)-positive cells, normal subjects were found to have less than 54 molecules of lgg per red ceil. ~'2 In four patients with warm AIHA and a positive DAGT by agglutinatiorl, up to 26,000 molecules lgg per cell were found; in two other cases of AIHA where the DAGT was negative, the erythrocytes were coated with 650 molecules per cell. "2 Our own experience (in conjunction with D. J. Booker) confirms the value of enzymelinked antiglobulin tests in the investigation and monitoring of patients with AIH. particularly where only a small amount of autoantibody is bound to the red cells. Several interesting findings have emerged from unpublished preliminary studies in I65 patients with obvious or suspected AIH: (1) in 15 patients, where only C3d was detected on the cells using the standard DAGT, the enzyme-linked method revealed increased amounts of lgm and lgg in 12 and 3 instances, respectively, the presence of IgM seemed to correlate with cold agglutinin activity at 30~ a reaction temperature reported to be critical for in vivo hemolysis."" (2) In 16 patients with suspected AIH but a negative DAGT, increased quantities of lgg (usually). iga, lgm, or combinations of these, were readily detected on the red cells. (3) in 22 patients with obvious warm type AIH and IgG coating of the cells, increased amounts of other immunoglobulin classes were found by the enzyme-linked method, but not by the standard DAGT; in 12 patients this was IgA, in 8 lgm and in 2 lga plus IgM. Currently, work is in progress to determine the significance of these findings. Methods for quantitating cell-bound immunoglobulins and complement are clearly at an exciting stage of development, and an understandh~g of these quantita,'ive results in relation to AIH will become important. No doubt the use of monoclonal antibodies, perhaps directed against IgG subclasses, will shed further light on the matter, and in the near future, such tests could well become routine. Whether radioisotopic or enzyme-linked techniques will dominate is not known, the method chosen could well depend on the preference of the individual laboratory.

12 136 CRC Critical Reviews in Oncology/Hematology V. TREATMENT OF AIH In the absence of any recent therapeutic breakthroughs, corticosteroids (usually prednisolone), blood transfusion, immunosuppressive agents (usually azathioprine or cyclophosphamide, but occasionally chlorambucil) and splenectomy are still the mainstays of treatment for patients with AIH. When and how these should be used varies with the individual patient, and the skill of the physicia~t remains an important aspect of case management, in some instances, particularly children where the disease is rapidly self-limiting, no treatment may be needed other than general care, warmth, food, and adequate hydration. 2'~ On the whole, current treatment is effective and few patients die of hemolysis; those that succumb usually do so of associated disorders or, very rarely, from the side effects of treatment, e.g., severe infections during steroid therapy, postsplenectomy complications, '~ or possibly lymphoma developing with prolonged use of chlorambucil in patients with CHAD. '~' Any underlying condition ~ should be treated; in the case of malignancy and collagen disease, this is often more than adequate therapy for the AIH. High dose i.v. gammaglobulin (400 mg/kg) has been recommended in the treatment of autoimmune diseases, particularly ITP and immune neutropoenia. '7-j~ Hopes that such therapy would also be useful in patients with AIH appear to be unfounded, and in some instances, it may be detrimental; r~ in four patients there was no obvious effect, '~7"~2"~-~ but in a fifth, signs of clinical deterioration and intensified hemolysis occurred, je'-~ It was suggested that the gammaglobulin, by nonspecifically coating red cells, competitively inhibited macrophage binding of other cell types; thus in patients with AIH, immunoglobulin therapy might exacerbate hemolysis, or at best, have no effect.,o2 This hypothesis is supported in patients with ITP both by evidence of mild compensated immune hemolysis following immunoglobulin treatment, and also by a rise in platelet count in some Rh(D)-positive patients after the injection of anti-d immunoglobulin, t~ Treatment of AIH by selective injury to macrophages with vinca-ioaded platelets has been recommended where standard treatment is unsatisfactory."~ Platelets (either autologous "~'"'~' or from ABO compatible donors ~~ were incubated with vinca alkaloids, usually vinblastine, and ITP plasma, and the resulting complex was infused into the patients. In three splenectomized subjects, good responses were obtained, characterized by prompt prolongation of red cell survival, correction of anemia, gradual decrease in cell bound antibody, and remission from hemolysis, ranging from several months to 3 years; t~ a fourth case improved considerably though a degree of compensated chronic hemolysis remained. ~~ One patient who relapsed was treated again; when incubation with ITP plasma was omitted, hematological deterioration resulted, but when this step was included, the hemolysis again responded. ~'~"~'~ However, a further patient, who had initially refused splenectomy and required high maintenance doses ofsteroids, did not respond to vinblastine-antibody-platelet complexes, though subsequently did so to splenectomy. ~ Reports of benefit from infusions of vinblastine by itself are conflicting; in one patient, the drug alone was ineffective but the hemolysis responded to treatment with drug-antibody-platelet complexes; "j6 while in another two patients (refractory to predisolone and splenectomy), vinblastine infusions successfully controlled the anemia. JoT The conc.~pt of selective damage to mononuclear phagocytes in patients with AIH is attractive. Whether a vinca-platelet-antibody system is the best is not known; a simpler and therefore probably better method would be to use therapeutic substances which bind to erythrocytes directly; bleomycin is such an agent j~ and its use in patients with AIH in this context deserves consideration. Alternatively, mononuclear phagocyte function could be suppressed by developing antimacrophage monoclonal antibodies suitable for clinical use. Plasma exchange may sometimes be helpful. In patients with warm IgG-type AIH, clinical benefit and stability can be achieved in about half the cases, though sometimes this is only

13 Volume 4, Issue temporary. ~c~,j Plasma exchange appears to be most useful where the hemolysis is fulminant, in order to gain time for other therapy to act. "~''~lc~ A rapid and complete rernission followed a series of plasma exchanges in a 16-year-old female with AIH whose clinical condition continued to deteriorate in spite of treatment with hydrocortisone, cyclophosphamide, azathioprine, and blood transfusion. JcJ3 Similarly, plasma exchange apparently played a major part in controlling a severe AIH, due to anti-a, which developed in a renal transplant recipient; TM and a spectacular response to plasmapheresis combined with exchange transfusion was observed in a patient with hyperacute AIH unresponsive to steroids and azathioprine. ~j2 In patients with cold lgm-type AIH, plasma exchanges may give short-term stability, but durable or significant control of hemolysis is not seen; ~t~9"~l~ on occasions it may be unhelpful clinically and fraught with technical difficulties due to autoagglutination. ~4 In two patients with advanced lymphoma and chronic CHAD refractory to conventional therapy, plasma exchange resulted in improvement in the compatibility tests and tolerance to transfusion in one individual, but were of no benefit in the other. 1~5 These findings are disappointing; igm autoantibodies are confined intravascularly and plasma exchange should, theoretically, be more efficient than in cases where lgg is involved. However, the response to plasma exchange may depend on the type of disease; CHAD, due to monoclonal IgM, is only mildly responsive, but patients with the much rarer mixed cryoglobulin-type CHAD show significant improvement, t~(,. ~7 In patients with mixed AIH, autoantibody levels can be effectively lowered resulting in clinical benefit;'* in one patient, where hemolysis was particularly severe and the patient's condition was deteriorating, plasma exchanges stabilized the situation giving time for antilymphoma therapy to take effect. 4 In another patient, who had failed to respond to steroids, immunosuppressives, blood transfusion, and splenectomy, plasma exchange resulted in a rise in hematocrit and platelet count.' ~ Although the benefits of plasma exchange are difficult to assess because of the effect of other concurrent treatment, we feel that the procedure is a useful adjunct to chemothcrapy, 9articularly at periods of hemolytic crisis, and also during times of chronic hemolysis in the hope of reducing the dosage of maintenance therapy. Certainly, in patients with mixed AIH, we recommend that plasma exchange should be tried before splenectomy is cgnsidered. ~ One new form of therapy, specific immunoadsorption, has been reported recently in a patient with warm AIH associated with chronic lymphocytic leukemia (CLL). l l'~ The patient was experiencing progressive hemolysis despite treatment with chlorambucil, steroids, and blood transfusion; coronary artery disease precluded splenectomy. Extracorporeal immunoadsorption of plasma lgg was carried out with a cell separator using protein A (from Staphyloccus aureas) as the immunoadsorbent. The procedure was well tolerated and a good response, as judged by increases in hemoglobin (Hb) and platelets, was seen after two treatments. The specificity of the procedure was shown by a decrease in the serum IgG and by eluting igg from the protein A. ~L,~ Specific immunoadsorption appears to be more efficient and selective than plasma exchange and also has the advantage that replacement of fluid and other plasma factors is not necessary; future development in this field is awaited with interest. Currently, the use of specific immunoregulatory agents in the treatment of patients with AIH is theoretical and speculative, and as far as we are aware, no work on their potential has been carried out. No doubt, once the immunoregulatory disturbances causing AIH are better understood, such therapeutic modalities could become the treatment of choice, having considerable advantage over macrophage destruction and general immunosuppression. At present, immunoregulatory agents are ill defined and the preparations crude; nevertheless, reports are appearing of their use in human disease with an immune basis. For example, six patients with idiopathic aplastic disorders, where there was an absolute decrease in T-

14 138 CRC Critical Reviews in Oncology/Hematology helper cells and a relative predominence of T-suppressor cells, were treated with lhymostimulin-tp-1, r2~ After treatment, the T-helper:T-suppressor imbalance was corrected in all cases, a selective increase in the absolute number of T-heIper cells having occurred; clinical and hematological recovery followed in three patients, t-'~' in other studies, thyrnostimulin appeared to induce the return of T-celt competence to certain untreated patients with Hodgkin's disease ~2' and to increase peripheral blood T celts to normal levels in patients with melanoma. ':2 In the future, monoclonal antibodies acting against specific parts of the immune response, and thus modifying it, may also be useful in the treatment of AIH; one interesting report has documented remarkable tumor regression and clinical improvement following treatment with monoclonai antiidiotype antilx~dy in a patient with advanced B-cell lymphoma. '~-'~ Vl. CLINICAL STUDIES This part of the review is based on our experience of 1223 patients with AIH. The clinical findings from a study of 865 patients I have been updated with a further 358 cases and compared with those of other major series and recent case reports. Classification, incidence, autoantibody specificity, and Alia induced by drugs arid by cold autoantibodies are discussed briefly. AIH associated with DL antibodies, AIH of mixed type, AIH occurring in pregnancy, and AIH in childhood are cortsidered in more depth. The 1223 patients, drawn from a population of approxinlately 5 million people living in an area of 6500 square miles (I6,828 ~q kin), have been classified by disease association and divided into groups depending on the optimunt ten~perature of ~,utoantibody activity (Table I). The present classification ~ differs from the more IraditionaL one of fairly clearcut warm and cold antibody-associated groups in that a separate category of mixed A1H is included, it has ~he advantage that allowances are made h~r the variable pattern of serology in AIH, and for the fact th~tt particular diseases are not always associated with either warm or cold groups (Table t). The concept of secondary A[H is complicated. It may mean tt~at there is an association with the other disease with a greater frequency than can be explained by chance alone, or that the AIH resolves when the associated disease is corrected (e.g., ovarian neoplasms) or that the two disorders are different parts of a complex imntunologically mediated multisystem disorder. :' ~ -'4 ~ "-s Although 56.8% of the eases in Table I are shown as being secondary, the above considerations should be borne in,hind: for example, is there significance in the occasional association of AIH with the relatively common disorders of pernicious anemia, rheumatoid arthritis, thyroid disease, diabetes metlitus, and certain infections, or should this be regarded as a chance occurrence'? The incidence of AIH per 100,000 of the population at risk, related to the age of presentation, is shown in Table 2. The incidence ir~creases dramatica)ly after 50 years of age and continues to rise thereafter; the childhood peak of cases of cold AIH is due mainly to patients with DL hemolysis. The figures in Table 2 are similar to the l.in 80,000 previously quoted for the minimum annual incidence of AIHA, ~z6 but are more delailed. In all groups, except patients with DL hemolysis, there is a slight female predominance, the male-femrnale ratio varying from l:t. 15 to 1:1.33. ~ In other studies, an equal sex ratio overall has been found; ~'~' this was attributed to the sex ratio of the underlying disorders; idiopathic AIHA and that associated with SLE were more common in women, and AIHA associated with lymphoma and CLL were more common in men. Although in many of our patients the autoantibodies showed evidence of blood group specificity, this was rarely simple, and, in nearly every case, pan-reacting antibodies could be delected by at least one method. ~ Warm autoantibodies usually expressed aclivity within the Rh syslem, auto anti-e being lhe most common, Specificity was more obvious in patients with cold AIH; anti-i was most frequent, followed by anti-"not It" and then by anti-i;

15 Volume 4, Issue Table 1 DISTRIBUTION OF 1223 PATIENTS WITH AIH BASED ON DISEASE ASSOCIATION AND SEROLOGICAL FINDINGS CoM AIH... ~..... Condition Warm AIH CHAD PCH Mixed AIH Idiopathic AIH ,5 47 Drug-related AIH Neoplasia Lymphoid neoplasms Non-Hodgkin's lymphoma Angioimmunoblastic lymphadenopathy I Chronic lymphocytic leukemia Myeloma Macroglobulinaemia Thymoma Hodgkin's disease i l Ovarian tumors 4 I 0 2 Carcinoma (non-ovarian) Non-lymphoid leukemias Myelolibrosis Infection Pneumonia ~ mycoplasma Pneumonia -- "viral" and unspecified 2 t Respiratory infections -- unspeci fled I 14 7! "Flu-like' 'illness Infectious mononucleosis 0 II 1 O Infectious hepatitis 2 l 0 0 Chicken pox 0 0 I 0 Meningitis 0 0 I 0 Tuberculosis Infection -- unspecified 2 5 I l Collagen diseases Syslemic lupus erythematosus I6 Rheumaloid arthritis Polyarteritis nodosa Wegener's granulomatosis! Collagen disease -- unspecified I Other immune based and miscellaneous disorders Thyrotoxicosis I I 0 1 Myxoedema Chronic active hepatitis Pernicious anaemia Diabetes mellitus Sarcoidosis l i 0 1 Ulcerative colitis i Myasthenia gravis Multiple autoimrnunity Marrow hypoplasia! AIH associated with pregnancy AIH associated with dialysis AIH associated with PNH i I 0 0

16 140 CRC Critical Reviews in Ontology~Hematology Table 2 AGE AND INCIDENCE OF AIH PER 100,000 OF POPULATION AT RISK (BASED ON 865 CASES) _Inc_ide_nce (per_100,000 of population). Age (years) Warm AIH Cold AIH Mixed AIH o o. 19 o o o others, such as anti-n and anti-a, were rare: DL antibodie'~ showed reactivity within the P system. J These findings were similar to those of e~.:rlier reports. ~27 More recent studies have emphasized the complexity of autoantibodies apparently showing specificity within the Rh ~ystem ~2~ and have drawn attention to an occasional depression of Rh antigen expression in patients with warm AIH; '-''jl-~c' in addition, some case reports of autoantibodies showing interesting specificities have appeared lately, the following blood groups were involved: A (often associated with severe hemolysis), '' '.~-~t-~33 Sdx, TM Kell, '3~'13~ Kidd, ~-t7 Duffy, ~as Ge, ~-~'~ M, 14~ and Pr3. TM Drug-induced AIH was well reviewed in ~4'- In our experience cases are always associated with warm autoantibodies; usually lgg alone coats the red cells but in a few cases a complement component is also present, and rarely the AIH is of mixed type (Table i). All the present cases (with one exception where mefenamic acid was implicated), were secondary to treatment with methyldopa.! Only those patients with auto- and pan-reacting antibodies have been included in Table 1 ; cases with a positive DAGT but without circulating antibody are excluded as the consensus opinion is that these do not hemolyze. Occasionally, other conditions that may be associated with AIH were also present, but in these subjects it was thought that the drug was the main etiological agent. Despite a positive DAGT and the presence of circulating antibody, some patients taking methyldopa have no evidence of a hemolytic anemia; they may have mild compensated hemolysis, but occasionally the red cell lifespan is normal. ~42 Interestingly, in a recent study of ten such patients, the red cell autoantibodies disappeared despite continuation of the drug.,4.~ Perhaps a distinction between hemolyzing and nonhemolyzing methyldopa cases can be made serologicatly; hemolyzing patients were reported to have high-affinity, warm reacting IgM autoantibodies coating their cells in addition to the IgG autoantibodies.,.~4 The IgM autoantibodies, Which were probably monomeric, were thought to cause the hemolysis by activating complement; such IgM autoantibodies were not present on cells from nonhemolyzing cases. ~4'~ AIH associated with cold autoantibodies has a much more variable picture than the usually portrayed characteristic clinical syndrome with high titer cold agglutinins of wide thermal amplitude. ~ Roughly 50% of our patients with cold AIH had these features; the remainder showed varying degrees of hemolysis associated with low titer cold agglutinins. Other studies have produced similar findings. 94'145"147 In CHAD, hemolysis depends on lhe ability of the cell bound autoantibody to initiate complement activation; this ability is modified by antibody concentration, thermal amplitude, and the presence of complement on the cell affecting further antibody fixation, m The importance of thermal amplitude in the development of hemolysis must be emphasized; as a practical test, in vitro autoantibody activity at 30~ or above in albumen correlates well with the occurrence of hemolysis in vivo. '94"145"147

17 Volume 4, Issue 2 141[ Several interesting reports of patients with CHAD have been published recetltly; these include: fatal hemolysis with autoantibodies of low titer and high thermal amplitude; ~ ~'~ an IgG cold agglutinin showing anti-pr specificity; ~'~'~ the association of CHAD with severe anemia, reticulocytopoenia, and an erythroid marrow; z~~ massive intravascular hemagglutination causing pulmonary embolism; ~-~ CHAD associated with I.,egionnaires' disease ~-s2 and with systemic leishmaniasis; ~-~3 the problems associated with intraoperative Iiypothermia; t54-~5~' the development of mild AIH due to auto anti-n in patients with renal failure who used formaldehyde to sterilize their dialysers ~'~2'j4 and a study of 78 patients, which concluded that conditions with persistent cold agglutinins represented a spectrum of B-cell disorders ranging from "benign" autoimmune CHAD to malignant lymphoma. ~-~7 A. AIH Associated with Donath Landsteiner Antibodies In patients with AIH due to cold autoantibodies, a small but distinctive group with paroxysmal cold hemoglobinuria (PCH) can be separated on the basis of a positive test for DL hemolysins. Very little has been published on this condition of late and, as far as we are aware, the most recent report of a series of cases is our publication in 1982, presenting 14 patients.:' This study has now been updated with the findings in a further five cases. Patients with DL autoantibodies can be divided into acute and chronic groups on the basis of the clinical progression of the disease; a change has occurred over the years, and whereas previously the classical form of chronic PCH predominated, nowadays most cases are acute episodes following viral infections in children. Because these episodes are usually nonrecurrent and not clearly related to exposure to cold, we (and others) IS'~ prefer the term DL hemolysis, reserving the term PCH for the chronic disorder; 18 of our patients were in the a~'ute group, only one of these being an adult (aged 62 years); one had chronic PCH. DL hemolysis is rare, accounting for only 1.6% of all patients with AIH (Table l). However, in AIH occurring in childhood, 40% of cases are in the DL g~'oup; 5 younger children are most at risk, 14 of our patients presenting before their fifth birthday; in this age group, the male-female ratio was 2.5:1 and incidence figures of 1 in and I ip , respectively, were calculated for age and sex-matched populations. -~ The frequency of DL hemolysis in childhood has been noted previously but the male predominance has not been stressed, and on occasion stated not to exist; 1~9 however, a review of reports of 20 patients less than 10 years old, and of patients with chronic PCH secondary to syphilis, confirmed male predominance.:' Infection is an important factor in the development of acute Dr, hemolysis and may offer an explanation for the clustering of cases sometimes seen; 2,~Ss.i~.t6j previously syphilis was the predominant cause of chronic PCH, but nowadays, with the decline in incidence of the congenital and late forms of syphilis, PCH is more rare and is usually idiopathic. No reference could be found of syphilis ever causing acute DL hemolysis,-" and although false-positive Wasserman reactions were seen on occasion, none of our patients had a luetic infection. Evidence of infection was present in the majority of acute cases; most commonly these were of viral type and involved the upper respiratory tract. Frequently, no microorganisms were isolated, though chicken pox, adenovirus type 2, influenza A, cytomegalovirus (CMV), measles, mumps, and infectious mononucleosis have been implicated in individual cases. 2.:' Although one authority ~'16:' feels that most, if not all, nonsyphilitic examples are associated with viruses, nonviral infections, e.g., H. influenzae, E. coli, Mycoplasma, and Klebsie/la have been reported to cause DL hemolysis on occasicn, a,,158.~63 The clinical behavior of the acute and chronic disorders differs. Typically in the acute cases, there is a hislory of a recent infection from which recovery or part-recovery has been made; this is followed by a fairly sudden onset of malaise and hemoglobinuria; extreme pallor and jaundice are notable features, the latter usually being mild, tachycardia, occasionally with an ejection systolic murmur, is a frequent finding and hepatomegaly and

18 142 CRC Critical Reviews in Ontology~Hematology splenomegaly are sotnetimes found. Raynaud's phenomenon is not seen.-' Rarely, no history of a previous infection can be elicited,-" and in some cases, even hemoglobinuria is absent. :~''1~'4 In the acute cases, hemoglobinuria following exposure to cold has only been reported infrequently,"- possibly because it has not been tested for, since it could be induced in one patient by immersing the hand in cold water. Hemolysis is marked, and a fall in Hb to less than 5.0 g/de is common, the rate of fall being greater than 3.0 g/dc/day on occasions. -''5 Such rates of fall may be partly due to the reticulocytopoenia fi'equently seen in the early stages of the illness,-" and attributed to either suppression of marrow erytt~ropoiesis or to the DL antibody having stronger activity against reticulocytes;~5~ the reticulocyte count may not reach its maximum tbr several days. 2 Blood film appearances are often spectacular with autoagglutination and erythrophagocytosis being prominent, z DL hemolysis is not usually associated with thrombocytopoenia or disordered coagulation. -~ In acute cases, the DAGT often demonstrates complement coating of the red cells; the strength of the reaction varies but is mostly weak; weak auto- and pan antibodies, acting mainly in saline at 18~ but occasionally also at 37~ with enzyme and indirect antiglobulin techniques, are usually present. :.~ DL antibodies show specificity within the P blood group system (though other specificities have been reported) in both acute and chronic forms of the disease;'- anti-p specificity, which has been regarded as essential for diagnosis, ~'~ was found in our patients where looked for. ~ In the majority of cases, acute DL hemolysis is self-limiting with rapid and complete recovery in a few days, though occasionally a further episode may occur within a short time; positive DL tests are often present for a longer period but usually become negative within a few weeks. However, most patients are very ill initially, and rarely, the illness is fatal.-' Treatment is frequently necessary, rest and warmth being important; blood transfusion should be witheld if possible but was required in about half of our cases.'- We transfuse with washed red cells, unseiected for the P group, and use a blood warmer. This view is in broad agreement with other authorities, though washing and warming of the blood has not always been carried out, apparently with little ill effect, (cited in Reference 2). Three patients were transfused with compatible blood of the very rare Tj (a-) group, with excellent results, j~'-~-s''' The benefit of steroids, which are sometimes prescribed empirically in cases of severe hemolysis,'~'7 are controversial, and in view of the self-limiting nature of the disease, we feel that if they are given then they should be stopped as soon as possible; however, in one unusual case where the DL antibody had an exceptionally high thermal amplitude, they were considered beneficial in controlling the hemolysis. ~'~ In another patient where the DL antibody also had a high thermal range, some success in reducing the degree of hemolysis was claimed for plasmapheresis,~nd immunosuppression. I,,4 Patients with chr6nic PCH are easy to diagnose with characteristic episodes of hemoglobinuria and systemic symptoms on exposure to cold. Our only patient in this group, a 26- year-old male, presented between periods of hemolysis when he was completely well; bouts of hemolysis continued to occur but the patient soon learned that warmth and avoidance of cold were all that were needed to maintain his well-being; he also found that the attacks could be reduced in severity by sitting in front of the fire as soon as prodromal symptoms occurred. 2 B. AIH of Mixed Type In patients with mixed AIH, both warm and cold autoantibodies are present and the serological reactions are such that both are considered capable of causing the hemolysis, i.e., the combined criteria for warm AIH and for CHAD are satisfied.,.4 Patients with mixed AIH should be distinguished from two other groups of patients; first, from patients with warm type AIH in whom abnormal, but clinically insignificant, cold agglutinins can be detected at 20~ (these findings are seen particularly in patients with SLE); and second,

19 Volume 4, Issue from patients in whom both IgM and IgG autoantibodies are present but where both autoantibodies are of cold type with a wide thermal range. ~ Patients with mixed AIH are considered rare, I~'*' and until 1981 were not classified as tt distinct group. ~ Our description of 25 illustrative cases" remains the largest published series. In adults, reports of only seven other well-defined c~.lses, I 8.1~''')-171 and one which was less well defined, ~72 could be found: and in children. 4 cases of mixed AIH were noted in a group of 42 patients. -~ About 8% of patients have AIH of mixed type (Table 1); it occurs in all age groups bul is more frequent in the over 60s (Table 2). The overall male-female ratio is i:1.5, 4 more females being affected in adulthood 4,1j~-'~'~-~7-" and more nlales in childhood. -~ Nearly 46c~, of our cases were idiopathic: in the secondary cases, SLE and lymphoprolifera ive disorders were most common (Table 1). In the other reports, five patients had lymphoproliferative disorders, ~''~7~ one was classified as Evans" syndrome, ~j~ one was idiopathic, ~''' and one probably followed a viral infection..7-~ The DAGT is always positive, usually with both IgG and complement coating the red cells, "~'..~'t(''~tv: though occasionally positive reactions with anti-iga and anti-igm are found in addition. ~'-~.~'' Both warm and cold auto- and pan-reacting antibodies are always presenl and their separate lgg and IgM nature can be confirmed by fractionation and 2-mercaptoethanol treatment. "~~t~~7"'~7~ The mixed nature is maintained thoughout the course of the illness, though at different times one or other antibody can dominate serologically: very few cases eventually change to a solely warm or cold type picture. ~ The warm atttoantibodies usually show no evidence of blood group specificity when either eluates or sera are tested, 4'~'l~s'~''~ though rarely some evidence of specificity within the Rh system has been noted, 4"~7~ and in one recent case, anti-i r was observed. 17~ The cold autoantibodies are active at 30~ or above in all patients?,~-~r and in about 75% hemolysins can also be demonstrated? Patients with mixed type AIH tend to be severely affected: Hb levels commonly fa~ling to below 6.0 g/d~'. 4.5.~'~)-~7~ Typically, the picture at presentation is dominated by the symptoms and signs of AIH, even in the secondary cases. ~.-'l ~,~.~,,,.~7: Several of our patients were thrombocytopoenic; where no other associated disorder was present, the thromobcyt..,,penia was thought to represent part of a fundamental disturbance in immune homeostas.i:s ~ and such cases are shown in Table I as being idiopathic. Most patients eve~tually respond well to conventional treatment, though the disease tends to run a chronic course with intermittent exacerbations, tlaus making overall m~,,aagerr~ent difficult; we have followed One patient with chronic hemolysis tar over 20 years. In such cases, the aim is to maintain good health with the minimal amount of therapy which suits that particular individual. "~ Death due to fulminating hemolysis can occur. "~-s~ ~~ In secondary cases, where treatment of the underlying disorder is immunosuppressive, e.g., in SLE or lymphomas, it is also effective in controlling hemolysis. ~,-~'~'~-~7~ On the whole, splenectomy does not appear to be helpful 4-1~' though perhaps it may be of benefit in some individuals, it,,, Plasma exchange effectively lowers autoantibody levels, with apparent clinical benefit. ~' ~"7 Blood should be transfused though a warmer, and where hemolysins are present, the cells should also be washed? C. AIH Occurring in Pregnancy The development of erythrocyte autoantibodies and AIH in pregnancy is rare; ~ less than 2% of patients with AIH having this association (Table 1). The subject is of particular interest to the present authors who recently presented their experiences of 20 such cases. -~ Since then, we have seen three more patients and reports have appeared of a further three cases ~37'17"x.174 and of a group of four Thai patients, tt-~ A distinction must be made between cases where AIH occur,; de nova in pregnancy (there presumably being a causal relationship), and cases where pregnancy occurs in patients known

20 144 CRC Critical Reviews in Oncology/Hematology to have AIH. 3 On average, erythrocyte autosensitization is seen in 1 in 50,000 pregnancies. -~ The possibility that the association is mere coincidence is most unlikely since AIH occurs at least four times more frequently during pregnancy than in a nonpregnant fernale population of the same age range. -a Whether all patients with erythrocyte autoantibodies are hemolyzing is not known, probably not in our view. Ten of the present group had proven hemolysis and the sera of three other patients (wllo were not fully investigated), contained anti-i reacting at 30~ or above. In all our patients, the serological abnormalities were discovered during pregnancy ot" in the immediate postnatal period; in eight of them no autoantibodies had been detected earlier in the pregnancy, and in a further six, none had been found on investigation prior to the pregnancy. The relative importance of other diseases or intercurrent infections is difficult to assess; presumably these reflect, or cause, disturbances in immune homeostasis which make the development of AIH in pregnancy even more likely. Five of our patients had long histories of ulcerative colitis (well controlled in all instances), which has a recognized association with AIH. ~.s71'.!77 Unfortunately, only one patient in tlais group had been investigated before becoming pregnant, at which time no evidence of AIH was demonstrated; however, in respect of the other four cases, the possibility remained that the antenatal testing merely detected preexisting serological abnormalities. These etiological problems are also illustrated by another patient who presented late in pregnancy with severe AIH which did not respond until further treatment was given for Hodgkin's disease, which had apparently been in remission for more than 30 months. J7~ Although AIH associated with pregnancy can be severe, it is more often mild, usually does not require active treatment, and frequently is discovered during routine antenatal testing. 3 Only three of the present patients developed severe AIH (lowest Hb 5 to 8 g/de); all three were treated with steroids with excellent results and all produced live children; two of the patients had further pregnancies with no problems. Overall, 36 pregnancies have been followed by the present authors; 31 infants, including two sets of twins, were born alive and well. Immune hemolysis due to maternal autoantibody crossing the placenta was seen in four cases; in all instances the disease in the infant was mild and required no treatment; as expected, the maternal autoantibodies were of IgG class. The relationship between fetal loss (seen in seven patients) and red cell autoantibodies is difficult to assess; other possible causative autoantibodies were present, patients had been inadequately investigated, and there had been previous miscarriages at times when no serological abnormalities were evident. However, an immune mechanism could have been responsible in some cases si~lce three of the patients subsequently gave birth to normal infants following immunosuppressive therapy during pregnancy. Reports of 16 patients in whom AIH was diagnosed during pregnancy have been reviewed previously; 3 briefly, 15 live births occurred with 8 infants being affected with hemolytic disease; complete recovery occurred in all instances though treatment was required in three cases; a similar pattern was noted in 7 subsequent pregnancies. The three cases of infant loss were thought to be. due to maternal anemia or to obstetric complications rather than to hemolytic disease in the child. In more recent reports, seven patients who developed AIH during pregnancy produced six live il~fants I-~7.~73"17~ and one stillbirth; ~7~ one baby was severely affected with hemolytic disease and needed four exchange transfusions before making a complete recovery; TM one infant was mildly affected but required no treatment, 173 and four were unaffected, j37. ~75 The stillbirth, which occurred in a patient where lgm coated the red cells, was tikely to have resulted from the profound drop in Hb during the last month of pregnancy. 175 Our findings disagree with those of earlier studies which concluded that the outlook was grave, life-threatening anemia (Hb <5.0 g/de) being seen in nearly 50% of patients and stillbirth or postpartum immune hemolysis occurring in 35 to 40% of infants, its However,

21 Volume 4, Issue 2 14S Table 3 AIH IN CHILDHOOD AND ADOLESCENCE: DISTRIBUTION OF CASES s Number of patients Cold AIH Age and sex Warm AIH CHAD DL Mixed AIH years Male 7 2 I0 I Female ~9 years Male I I I 0 Female 2 I I I years Male Female 0 I ~' 1 0 Autoanlibody of lgg class. this conclusion was reached without a distinction being made between patients where AIH developed in pregnancy and patients with AIH who became pregnant; a recent study has found that the outlook is much worse in the latter gl'otlp; 175 also, it should be remembered that earlier reports favored the extreme of the condition where the patients were severely affected. More recent papers have confirmed our views and have recognized that mild disease can occur and be discovered on routine antenatal testing, t-~7.tt-~ and that even when patients are severely affected, the response to therapy is usually very good D. AIH in Childhood About 3.4% of patients with AIH are children or adolescents. In this section, the finding in 42 of our patients who presented before their 16th birthday s are compared with those of other major series 179~x5 and with reports of individual cases. Table 3 shows the distribution of the 42 patients in respect to age, sex, and serological findings, This classification is considered to be more informative than those where patients were placed into acute or chronic categories ~~ (or even into a subacute one ~8-~) depending on clinical progress and the length of time before recovery. Some differences from the other studies are e',i,jent in respect of the type of case seen; in our patients DL hemolysis accounts for 40% of the series (Table 3), whereas other groups found that warm type AIH with IgG coating the cells predominated, *~t-i~-'~ and that no more than 5% of cases had DL hemolysis; r~~ this presumably reflects population differences between the various studies. AIH can. occur at any age and cases as young as 2 weeks have been reported; rarely is there is a familial occurrence (cited in Reference 5). Over 65% of our patients presented in the first 5 years of life; in this age group, males predominated 2.5:i, but in the older patients the sex ratio was I' 1 (Table 3). Taking the populations at risk, incidence figures of I in 267 x 103, 1 in 1027 x 103, and 1 in 1780 x 103 were calculated for the respective quinary age bands 0 to 4, 5 to 9, and 10 to 14 years. ~ A higher proportion of males in the younger children and male-female ratios of the same order as our patients, were also seen in most earlier studies; ~t~~ however, two groups reported that slightly more females than males were affected. 1~14,185

22 146 CRC Critical Reviews in Oncology/Hematology In most patients, whatever the type of hemolysis (Table 3), there is evidence of a current or very recent infection: these are frequently mild, often involve the upper respiratory tract, ~'-~'~7'-'j'~J'-~ and are particularly noted where the hemolytic episodes are of the acute and transient type, I~~ DL hemolysis being a good example. The organisms are not always identified. ~ In our experience, -~ CMV infection is not as important a factor in the etiology of childhood AIH as previously suggested; ~79"js~' again, tb.is may reflect populatiotl differences, since many of these previous patients '7'.1~" had varying degrees of immune deficiency, a factor known to predispose towards CMV infection. Differences in populations may also account for the varying incidence of AIH associated with other disorders: we only saw two such patients (both with collagen diseases) ~ whereas other groups reported that AIH secondary to immunodeficiency, collagen, or lymphoproliferative disorders made up nearly 40% of their cases: ~'~ neither did we see an association with Hodgkin's disease ~4-~7-j'~c~ or giant cell hepatitis. TM Although the patients with collagen diseases, and a further patient who developed chronic persistent hepatitis, could be considered to have some degree of immune dysfunction, none of our patients was obviously immunodeficient, though perhaps in retrospect, full investigation of immune status should have been carried out in two cases with lifelong histories of frequent infections. -~ Well-defined congenital immune deficiency syndromes featured prominently in some of the earlier series jt'-~sp'~x-~'~-s and case reports (cited in Reference 5); and recently AIH has been observed in a child with acquired immune deficiency syndrome (AIDS). 1'~2 The characteristic presenting features are sudden pallor and malaise (often following an intercurrent infection), with slight jaundice, splenomegaly, and hepatomegaly being [bund in about half of the patients; where the AIH is secondary, signs and symptoms of the associated disorder can predominate initially. ~,t''~~85 In our cases, hemoglobinuria occurred in all patients, with DL hemolysis, occasionally in patients with cold autoantibodies, but not in patients with warm AIH; -~ however, in some of the studies, hemoglobinuria was commonly observed where the hemolysis was other than DL in type. ~8~''~t'~s5 The illness is usually severe with Hb level.,; falling to below 6.0 g/dr in more than 50% of cases. ~'~s'~'~8-~ The rate of fall can be quite spectacular, especially in patients with DL hemolysis. 5 Although the reticulocyte response is generally brisk and commensurate with the degree of hemolysis, an initial delay is seen 5-J8~ in over 20% of cases, ~3.~-~ and may, ir~ part, be responsible for the dramatic falls in Hb level. In all our patients, the DAGT was positive, -s though occasional cases have been observed where the result is negative. ~29'~3~.~94 In one patient, the serology was unusual in that the lgg autoantibodies reacted much better at room temperature than at 37~ (Table 3); a few other examples of cold autoantibodies of IgG class causing hemolysis in children have been reported. ~9~.~96 The autoantibodies occasionally showed evidence of blood group specificity, 5 the pattern being similar to that in adult cases. Although platelet counts are normal in the majority of cases, and high values are occasionally seen during periods of active hemolysis, ~ a variable degree of thrombocytopenia, presumably immune in origin, is present in some patients ~'~32,~8~ and can lead to fatal bleeding. ~:~ The larger series suggest that thrombocytopenia occurs in about 14 to 32% of cases, 5,~79"~8~,~83"~85 If treatment is needed, and it should be noted that many patients recover completely with either no treatment or just folic acid and/or antibiotics, 5 then steroids and blood transfusion are considered to be effective by most authorities, particularly in,~he acute cases, with treatment of any underlying disorder as appropriate. ~'~s~ If the initial treatment fails, a good response can be obtained from splenectomy in some cases, 5.ts~ though not in others. ~.~4 In children with Evans' syndrome, treatment is reported to be generally unsatisfactory. 198 Immunosuppressive agents or, more dramatically, thymectomy, have been rec-

23 Volume 4, Issue ommended in cases of severe and progressive hemolysis, though none of our patients needed to be considered for such measures. 5 Most patients eventually recover completely, -~'~c~~x-~ usually within 6 months in our experience, "~ though in other studies, the proportion of cases recovering within this interval ranged from 36 to 77%, t~~ again presumably reflecting population differences. Even when hemolysis continues for longer periods, full recovery is the rule. S'~'~~ Two of our patients (5%) died; 5 this is a lower mortality rate than in other series where figures of 10 to 31% were noted, jt'j-~"~ Most deaths are due to underlying chronic disorders, ~~ though postsplenectomy sepsis was a significant cause in one study, ~84 and severe hemolysis could itself be fatal. ~.~.~3 However, in spite of the mortality risk we feel that a cautiously optimistic prognosis can be given for children with AIH, particularly where there is no underlying chronic disorder. REFERENCES 1. Sokol, R. J., Hewitt, S., and Stamps, B. K., Autoimmune haemolysis: an 18-year sludy of 865 cases referred to a regional transfusion centre, B,~'. Med, J.. 282, 2023, Sokol, R. J., Hewitt, S., and Stamps, B. K., Autoimmune haemolysis associated with Donath-Landsteiner antibodies, Acta Haematol.. 68,268, Sokol, R. J., itewitt, S., and Stamps, II. K., Erythrocyte autoantibodies, autoimmune haemotysis and pregnancy, Vo.t Sang.. 43, Sokol, R. J., llewitt, S.~ a~ltl Slamps, B. K,, Autoimmune haemolysis: mixed warm and cold.'mtibody type, Acta Haematol.. 69, 266, 1983, 5. Sokol, R. J., ltewitt, S., Stamps, II. K., and ltitchea, P. A., Autoitnmune haenlolysis in childhood :~nd adolescence, Acta Haematol., 72, 245, Cooke, A.; Lydyard, P. M., anti Roilt, I. M., Mechanisms of aut~immunity: a role for cross-reactive idiotypes, llntnt~ltol. Today. 4, 170, Grabar~ P., Autoantibodies and the physiological role of immunoglobulins, hmnunol. Todto'. 4, Mackay, 1. R., Natural autoanlibodies to the fore -- lbrbidden clones to the rear, lmmunol. 7~d~o', 4, 340, Kay, M, M. B., Mechanisms of removal of senescent cells by human macrophages in situ. l-'roc'. Natl. Acad. Sci. U.S.A.. 72, , I0. Khansari, N. and Fudenherg, It. H,, Phagocytosis of senescent erythrocytes by autologou:; monocytes: requirement of membrane-specific autologous lgg for immune elimination of aging red blood cells, Cell. {mmmlol,, 78, 114, Moores, P., Pudlfin, D., and I~atel, P. L., Severe hemolytic anemia it~ atx adult associated with anti-t, Transfusion. 15, Fasshinder, W., Seidl, S., and Koch, K. M., The role of formaldehyde in the fomlation of haemodialysisassociated anti-n-like antibodies, VoxSang., 35, 41, Sharon, R., Relationship between tormaldehyde-related antibodies and cross-reacting anti-n-like antibt~ties in patients undergoing chronic haemodialysis, J. Ciip~. Pathol., 34, 41, Fassbinder, W. and Koch, K. M., A specific immunohaemolytic anaemia irjduced bv Ibrmaidehyde sterilization of dialysers, Contrib. Nephrol.. 36, 5t, Owens, N. A., Hui, H. L., and Green, F. A., Induction of direct coombs positivity with alpha-methyldopa in chimpanzees, J. Med,. 13,473, Plotz, P. H., Autoantibodies are anti-idiotypic antibodies to antiviral antibodies, Ltttwet, , Kruger, J., Rahman, A., Mqt~;, K.,U., and Mueller-Eckhardt, C., T cell deficiency in patients with autoirnmune hemolytic anemia ("warm type"), Vox Sang.. 31, i, Parker, A. C., Sluarl, A. E., and Dewar, h. E., Activated T-cells in autoin~mune haemolytic anaemia, Br. J. Haematol., "/. 19. Bach, M.,A. and Bach, j-f., The use of monoclonal anli-t cell anlibodies to study T cell imbalances in human diseases. Clin. Exp. Immlmol.. 45, 449, 198t. 20. Molaro, G., Santtni, G., de Paoli, P., and da Ponte, C., T lymphocyte subsets and autoimmu~e hemolylic diseases, Haematologica, 68, 167, 1983.

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25 Volume 4, Issue Fleer, A., van Schaik, M. L. J., yon dem Borne, A. E. (;. K., and Engelfriet, C. P., Oe~tructitm ~t sensitized erythmcytes by human inonocytes in vitro: effects of cytochalasin B, hydroctmisone and ct~lchicine, Scand, J. Itpttnt#lol., 8.5 } Borregaard, N, and Kragballe, It., Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils, J, Clin, hn'est., 66, 676, Fleer, A., van der Meulen, F. W., Linthout, E., yon dem Borne, A. E. G. K., and Engelfriet. C. ~,, Destruction of lgg-sensitized erythrocytes by human bioc~d monocytes: modulation of inhibition by lgg, Br..I. ttaemawl., Kurlander, R, J. and Rosse, W. F,, Monocyte-medialed destruction in the presence of serum tff red cells coated with antibody, Blood, 54, Wuest, D., Crane, R., and Rinehart, J, J., Enhancement of Fc receptor lul~clion during human monoc~, le differentiatiott in vitro, J. Reticuloemtothel. Sot'., 31[) Ahramson, N,, Gelfand, E. W., Jandl, j. tt., and Rosen, F. S.,, The interaction between human monocytes and red cells. ~,pecificily for lgg subclasses and IgG fragments. J. Exp. Med., Huber, H., [)ouglas, S. D., Nusbacher, J., Kochvca, S., and Rosenfield, R. E., lgg subclass specificity of human monocyte receptor sites. Nature (London), , 1971, 55. van tier Meuien, F, W., de.bruin, H. G., Godson, P. C. M., Bruynes, E. ~S, E., Joustca-Ma~,~. C. J,, Telkamp, H, G., yon dem Borne, A. E. G. K., and Engelfriel, C. P., Quantita0ve aspects ~Jt" the destruction of red cells sensitized with lggl autoanlihodies: an ;lpp/icaiicm of flow cytoouorometry. Br. J. Haematol,, 46, Stratlon, F., Rawlinson, V. i,, Merry, A. H., and Thomson, E. E., Po.,,itive direct antiglobulin test it, normal individuals, I1. Cihl. Lab. Haematol., 5, 17, van der Meulen, F. W., van der Hart, M., Fleet, A., yon dem Borne, A. E. G. K,, Engeffriet, C. P,, and van Loghem, J. J., The role of adherence to human mononuclear phagocytes in the destruction of red ceils sensitized with non-complement binding lgg antibodies. Br. J. Haematol , , 58. Gallagher, M. T., Branch, D, R., Mien, A., and Petz, L. D,, Evaluation of reticuloend~atheliai functi,.m in autoimmune hemolytic anemia using an in vitro assay of monocyte-macrophage interaction with erythrocyles, E.V). Hematol.,!1.82, , Kay, N. E. and Douglas, S. D., Monocyle-erythrocyte ]mer~ction i~7 vilro in immune hemolytic anemia.,,. Blood, 50, 889, , Fries, L. F., Brickman, C. M., and Frank, M. M., Monocyte receptors for the Fc portion of lgg increase in number in autoimmune hemolytic anemia and other hemolytic states and are decreased by gluc~corticosd therapy. J. immunol, 131.!240, Schanfield, M. S., Stevens, J. O., and Bauman, D., The detection of clinically significant erythmcyte al/oantibodies using a human mononuclear phagocyte assay, Transfusion. 21., 571, 198 I. 62. Branch, D, R,, Gallagher, M. T., Schulman, I. A., Mison, A. P., Sy Siok Hian. A. L., and Petz, L. D., Reticutoendothelial cell ftinction in a/pha-mechyldopa-induced hemolytic anemia, Vox, Sang, , Brojer, E., Zupanska, B., and Michalewska, B., Adherence to hun'ta~t trtoa,acytes of red cel[~ from autoimmune haemolytic anaemia and red cells sensitized with alioantin)dies. Haematolog& Petz, L. D. and Garratly, G., Acquired Immune Hemolytic A~lemias, Churchill Livingstone. New York. 1980, Suzuki, S., Amano, T., Milsunaga, M., Yagyu, i,'., and Ofuji, T., Autoimmune hemolytic anemia associated with lga autoantibody, Clin. hnmunol. Immumq~athol., 2 l, 247, Wdf, C. F. W., Wolf, D. J., Pelerson, P., Brandsetter, R. D., and Hanson, D. E,, Autoimmune hemolytic anemia with predominance of IgA autoanitbody. Tratl~siotl, , , Kemppinen, E., Vuol/io, P., Sandstrom, R., and W~ger, O., Significance of mont~specific antisera in the diagnosis and prognosis of autoi/nmune haemolytic anaemia, Antt. Clitt. Res., Fanger, M. W,, Shen, L., Pugh, J., and Bernier, G, M., Subpopulations of human peripheral granulocytes and monocytes express receptors for lga, Proc. Natl. Acad. Sci. U.S,A., 77, Fanger, M. W., Pugh, J., and Bernier, G. M., The specificity of receptors for lga o~ humim peripheral polyrnorphonuclear cells and mot~ocytes. Cell. l,vnmnol.. 60, Short, L. and Fanger, M. W., Secretory IgA antibodies synergize with igg in promoting ADCC by human polymorphonuclear cells, monocytes, and lymphocytes, Cell lnmvunol., Pelz, L. D. and Garratty, G., Acquired lmmt~ne Hemoh'tic Anemias, Churchill Livingstone. New York, 1980, Urbaniak, S, J., Lymphoid cell dependent IlK-cell) lysis of human erylhrocytes sensitized with Rhesus ailoantibodies, Br. J. Haematol,. 33, 409, Urbaniak, S. J. and Greiss, M. A,, ADCC (K-ceil)/ysis of human erythrocytes sensitized with Rhesus alloantibodies. Ill. Comparison of igg anti-d agglutinating and lyric (ADCC) activity and the role of lgg subclasses, Br. J. Haematol., , 1980.

26 150 CRC Critical Reviews in Oncology/Hematology 74. Poston, R. N. and Morgan, R. S., Interactions between soluble igg, complement and cells in lymphocyte and nlonoeyte ADCC, Immunology, 50, 461, 1983, 75. Gillllnnd, B,, Coombs-negative immune hemolytic anemias, Semin. Hematol., 13,267, Syzmanski,!. O,, Odgren, P, R., Fortler, N. L., and Snyder, M., Red blood cell associated IgG in normal and pathological states, Blood, 55, 48, Petz, L. D. and Garratty, G., Acquired hnntune Hemoh'tic Anemias, Churchill L.ivingstone, New York, 1980, 307, 78, Gilliland, B. C,, Baxter, E., and Evans, R, S., Red-cell antibodies in acquired hemolytic anemia with negative antiglobulin serum test, N. Engi, J. Med.. 285,252, , Petz, L. D. and Garratty, G,, Acquired Immune Hemolytic Anemias, Churchill Livingstone, New York, 1980, Yam, P., Wilkinson, L., Petz, L. D., and Garratty, G., Studies on hemolytic anemia in pregnancy witl~ evidence for autoimmunization in a patient with a negative direct antiglobulin (Coombs') test, Attt, J. Hemato/., 8, 23, t Schnaitz, N., Djibey, L., Kretschmer, V., Mahn, 1., aud Mueller-Eekhardt, C., Assessment of red cell autoantibodies in autoimmune hemolytic anemia of the warm type by a radioactive anti-lgg test, Vox Sang, 4 I, 224, 198 i. 82, Merry, A. Ho, Thomson, E. E., Rawlinson, V, 1., and Stratton, F., A quantitative antiglobulin test for IgG for use in blood transfusion serology, Clin. Lab. Haematol., 4, 393, Merry, A. H., Thomson, E, E., Rawlinson, V. I., and Stratton, F., Quantitation of IgG on erythrocytes: correlation of number of lgg molecules per cell with the strength of the direct and indirect antiglobulin tests, Vox Sang., 47, 73, Kaplan, A. V. and Quimby, F. W., A radiolabelled staphylococcal protein A assay tbr detection of antierythrocyte IgG in warm agglutinin autoimmune hemolytic anemia of dogs and man, Vet, hzonuttol, h~munopathol,, 4, 307, Freedman, J. and Massey, A., Complement componenls detected on normal red blood cells taken into EDTA and CPD. Vox Sang., 37, 1, , Freedman, J. and Barefoot, C., Red blood cell-bound C3d in normal subjects and in random hospital patienls, Transfusion, 22, 51 I, Chaplin, H., Nasongkla, M., and Monroe, M. C,, Quantitation of red blood cell-bound C3d in normal subjects and random hospitalized patients, Br. J, Haematcl., 48, 69, Freedman, J., Ho, M,, and Barefoot, C., Red blood cell-bound C3d in selected hospital patients, 7'ran.~ion, 22, Merry, A. H., Thomson, E. E., Rawlinson, V. I., and Stratton, F., The quantification of C3 fragments on erythrocytes: estimation of C3 fragments on normal cells, acquired haemolylic anaemia cases and correlation with agglutination of sensitized cells, Clin. Lab. Haematol., 5, 387, Leikola, J. and Perkins, H. A., E~zyme-linked antiglobulin test: an accurate and simple method to quantify red cell antibodies, Transfusion, 20,! 38, Bessos, H. and Yule, A., Direct comparisons between a radio-immune test, an enzyme linked antiglobulin test, and a haemagglutination assay: application to the screening of anti-rbc sera and monoclonal antibodies, Vox Sang., 44, 289, Bodensteiner, D., Brown, P., Skikne, B., and Plapp, F., The enzyme-linked immunosorbent assay: accurate detection of red blood celt antibodles in autointmune hemolytic anemia, Ara, J. C/in, Pa{hol,, 79, 182, 1983, 93. Rigal, D., Monestier, M., Lafont, S., Raffin, T., Got, A., Meyer, F., and Jouvenceaux, A., hnprovemerit of enzyme-linked antiglobulin tes! by using an ~ntiglobulin linked to glucose oxidase: description of the lechnique, Vox Sang., 46, 349, Garratty, G., Petz, L, D., and Hoops, J. K., The correlation of cold agglutinin titrations in saline and albumin with haemolytic anaemia, Br. J. Haematol., 35,587, 1977, 95. Zupanska, B., Sylwestrowiez, T., and Pawelski, S., The results of prolonged treatment of autoimmune haemolytic anaemia, Haematologia, 14, 425, Chaplin, H., Lymphoma in primary chronic cold hemagglutinin disease treated with chlorambucil, Arch. Intern. Med., 142, 2119, Fehr, J., Hofmann, V., and Kappeler, U., Transient reversal of thrombocytopenia in'idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin, N, Eng, J. Med., 306, 1255, Newland, A. C., Treleaven, J, G., Minehinton, R. M., and Waters, A. H,, High-dose intravenous lgg in adults with autoimmune thrombocytopenia, Lancet, 1, 84, Bussel, J. B., Kimberly, R. P., inman, R. D., Schulman, L., Cunningham-Rundles, C., Cheu_ng, N., Smithwick, E. M., O'Malley, J.,Barundun, S., and Hilgartner, M. W., Intravenous gammaglobulin treatmem of chronic thrombocy~openic purpura, Blood, 62, 480~ 1983, IO0, Bussel, J. B. and ililgartner, M. W., The useand meci~anism of action of intravenous immunoglobulin in the treatment of haematologic disease, Br.,I. Haematol., 56, I, 1984.

27 Volume 4, Issue Pollack, S., Cunningham-Kundles, C., Smilhwick, E. M., Barundun, S., and Good, R. A., High. dose intravenous gamma globulin for auloimmune neulmpenia, N. Eng. J. Med.. 307, 253, Salama, A., Mueller-Eckhardt, C., and Kiefel, V., Effect of intravenous immunnglobulin in immune thrombocytopenia, Lancet. 2, 193, Kutti, J., Wadenvik, H., Safai-Kuttt, S., Bjorkander, d., Hanson, L, A., Westberg, (;., Johnsen, S. A., and Larsson, B., Successful treatment ot' refractory autoimmune haemolytic anaemia by plasmapheresis, Stand. J, Haematoh. 32, Ahn, Y. S., Byrnes, J. J., Brunskill, D. E., Moritz, J., Harrlngton, W. J., and Schmale, J. D., Selective injury to macrophages: a new treatment for idiopathic autoimmune hemolytic anemia, Clin, ICes., 26, 340A, Gertz, M. A., Petitt, R. M., Alvaro, A., Pineda, A. A., Wick, M. R., and Burgstaler, E. A., Vinblastine.loaded plate/ets for autoimmune hemolytic anemia, Ann. Intern. Med., 95, 325, Ahn, Y. S., ltarrington, W. J,, Byrtles, J, J., Pall, L., and MeCrainie, J., Treatment ot" autoimmune hemolytic anemia with vinca-loaded platelets, JAMA. 249, 2189, Medeilin, P. L., Patten, E., and Weiss, G, B., Vinblastine tbr autoimmune hemolytic anemia, 4tin. Intern. Med., 96, Lynch, W. E., Sartiano, G. P., and Ghaffar, A., Erythrocytes as carriers ol ~ chemotherapeutic agents lbr targeting ttle reliculoendothelial system, Am. J. Hematol.. 9, 249, Brooks, B. D., Steane, E. A., Sheehan, R. G., and Frenkel, E. P., Therapeutic plasma exchange in the immune hemolytic anemias and immunological thrombocytopenic purpura. Prog. Ciin. Biol. Res.. 106, 317, I10. Bernstein, M. L., Schneider, B. K., and Naiman, J. L., Plasma exchange in refractory acute atttoimmune hemolytic anemia, J. Pediat.. 98,774, 1981, 111. Lundgren, G., Asaba, H., Bergstrom, J,, Groth, C-G., Magnusson, G., Moiler, E,, Strindberg, J., and Wehle, B., Fulminating anti-a autoimmune hemolysis with anuria in a renal transplant recipient: therapeutic role of plasma exchange, Clin. Nephrol., 16, il2. Garelli, S., Mosconi, L., Valbonesi, M., Schieppali, G., and Navassa, G., PhJsma exchange fi~r a hemolytic crisis due to autoimmune hemolytic anemia of the lgg warm type. Blut , OrUn, J. B., Berkman, E. M., Matloff, D. S., and Marshall, M. K., Primary biliary cirrllosis and cold autoimmune hemolytic anemia: effect of partial plasma exchange, Gastroenterology. 78,576, Sddon, M., lsbister, J. P., Raik, E., and Biggs, J. C., A fatal case of cold autoimmune hemolytic anemia, Am. J, Clin. Pathol.. 73, Taft, E. G., Propp, R. P., and Sullivan, S. A,, Plasma exchange for cold agglutinin hemolytic anemia, 'rran,~lsion, , Rosenfield, R. E., Current treatment of the immune hemolytic anemias, Sangr~,, 25, 883, Rosenfield, R. E. and Diamond, S. H., Diagnosis and treatment of the imnlune hemolytic anemias, Haematologia. 14, 247, 1981.!18. Patten, E. and Reuter, F. P., Evans' syndrome: possible benefit from plasma exchange, Transfusion, , Besa, E. C., Ray, P. K.~ Swami, V. K., ldiculla, A., Rhoads, J. E., Bassett, J. G., Joseph, R. R., and Cooper, D. R., Specific immunoadsorption of IgG antibody in a patient with chronic lymphocytic leukemia and autoimmune hemolytic anemia, Am. J. Meal.. 7[, f035, I Guglielmo, P., Giustolisi, R., Cacciola, E., and Milone, G., lmmunomodulating treatment in patients with aplastic anemia, N. Eng. J. Med., , Martelli, M. F., Velardi, A., Rambotti, P., Cernetti, C., Berlolto, A., Spinozzi, F., Bracaglia, A. M., Falini, 11., and Davis, S., The in vivo effect of a thymic factor (thymostimulin) on immunologic parameters of patients with untreated Hodgkin's disease. Cam'er, 50, 490, t Bernengo, M. G., Cappella, G., de Matteis, A., Tovo, P. A., ar.j Zina, G., The in vitro effect of a calf thymus extract on the peripheral blood lymphecytes of sixty-six melanoma patients, Clin. E.W. lmmunol., 36, 279, Miller, R, A., Maloney, D. G., Warnke, R., and Levy, R., Treatment of a B-cell lymphoma with monoclonal anti-~diotype antibody, N. Engt. J. Med.. 306, 517, , Payne, D., Muss, H, B., Homesley, H. D., Jobson, V. W., and Baird, F. G., Autoimmune hemolytic anemia and ovarian dermoid cysts; case report and review of the literature, Cancer, 48, 721, Carreras.Veseio, L. A., l'oblli, J. E., Rey, J. A., Assal', M. E., de Maria,!!. E., and Marietta, J., Autoimmune hemolytic anemia associated with an ovarian neoplasm, Medicina (Buenos Aires), 43, 415, Pirolltsy, B., Clinical aspects of autoimmune hemolytic anemia, Semin Hematol.. 13, Worrledge, S. M., Immune haemolytic anaemias, in Blood and ~ts Disorders. Hardisty, R. M. and Wcatherall, D. J., Eds., Blackwell Scientific, 1974, chap Issltt, P. D. and Pavone, B., Critical ~'e-examination of the specificity of auto-anti-rh antibodies in patients with a positive direct antiglobulin test, Br. J. Haematol.. 38, 63, 1978.

28 152 CRC Critical Reviews in Ontology~Hematology 129. van't Veer, M. B., Van Wieringen, P. M. V., van Leeumen, 1., Overbeeke, M. A. M., yon dem Borne, A. E. (;. K., and Engelfriet, C. I'., A negative direct antiglobulin test with strong lgg red cell autoantibodies present in the serum of a patient with autoimmune haemolytic anaemia, Br. Y, Haemalvl., , lssitt, P. D., Gruppo, R. A,, Wilkinson, S, L., and lssitt, C. It., Atypial presentation of acute phase, antibody induced haemolytic anaemia in an infant, Br. J. Haematol,, 52, Syzmanski, 1. O., Roberts, P. L,, and Rosenfield, R. E., Anti-A aumantibody with severe intravascular hemolysis, N. Engl. J. Med.. 294,995, Parker, A. C., Willis, C., Urbaniak, S. J., and Innes, E. M., Autoimmune haemolytic anaemia with anti-a autoantibody, Br. Med. J., 1, 26, Castella, A., LaBarge, B, P., Lauenslein, K. J., and Davey, F. R., Auto-anti-A antibody in a patient with metastatic adenocarcinoma, Transfusion. 23,339, Oenegri,,I, F., Nanji, A. A., Sinclair, M., and Stillwell, G,, Autoimmune hemolytic anemia due to immunogk~blin G with anti-sdx specificity. Acta Haematol , Marsh, W. L., Oyen, R., Alicea, E., Linter, M., and Horlon, S., Autoimmune hemolytic a~emia and the Kell blood groups, Am. J. Hematol.. 7. t55, Manny, N., Levene, C., Sela, R., Johnson, C. L., Mueller, K. A., and Marsh, W. L., Autoimmunity and the Kell blood groups, Vox Sang., , , Ellisor, S. S., Reid, M. E., O'Day, T., Swanson,,i., Papenfus, L., and Avoy, D. R., Autoantibodies mimicking anti-jkb plus anti-jk3 associated with autoimmune hemolytic anemia in a primipara who delivered an unaffected child. Vo.r Sang., 45, 53, van't Veer, M. B., van Leeuwen, 1., Itaas, F. J. L. M., Smelt, M., Overbeeke, M. A, M., and Engelfriet, C. P., Red-cell auto-antibodies mimicking anti-fyb specificity, Vo.r Saltg., 47, 88, Reynolds, M. V., Vengelen-Tyler, V., and Morel, P. A., Autoimmune hemolytic anemia assoclat,:d with auto anti-ge, Vox Sang., 41, 61, Chapman, J., Murphy, M. F., and Waters, A, H., Chronic cold haemagglutinin disease due to an anti- M-like autoantibody, Vox Sang., , Birgens, H. S., Dybkjaer, E., and Roelcke, O., Identification of a cold agglutinin with anti-pr3 specificity, Stand. J. Haematol., 29, 207, Petz, L. D,, Drug-induced immune haemolytic anaemia. C/hz. HaemawL, 9, 455, Habibi, B., Disappearance of alpha-methyldopa induced red celt autoantibodies despite continuation of the drug, Br. J. Haematol., 54, 493, Lalezari, P., Louie, J. E., and Fadlailah, N., Serological profile of alphamethyldopa-induced hemolytic anemia: correlation between cell-bound lgm and hemolysis, Blood, 59, 61, Pruzanski, W. and Shumak, K. H., Biological activity of cold-reacting autoantibodies, I, N. Eng. J. Med.. 297, 538, Pruzanski, W. and Shumak, K. H., Biological activity of cold.reacting autoantibodies. 11., N. Engl. J. Med,, 297,583, Sehreiber, A. D., Herskovitz, B. S., and Goldwein, M., Low-titer cold-haemagglutinin disease: mechanism of hemolysis and response to corticosteroids, N. Engt. J. Med.. 296, /490, Rosse, W. F. and Adams, J. P., The variability of hemolysis in the cold agglutinin syndrome, Blood, 56, 409, l)ell~gi, g., Brouet, J. C., Schenmetzler, C., and Praloran, V., Chronic hemolytic anemia due to a monoclonal IgG cold agglutinin with anti-pr specificity, Blood, , Cazzola, M., Barosi, 13., and Ascari, E., Cold haemagglutinin disease with severe anemia, reticulocytopenia and erythroid bone marrow, Stand. J. Haentatol., 30, 25, Murayama, A., Ayabe, T., Toyama, K., and Nakamura, Y., Pulmonary embolism and hemolytic anemia in a patient with cold agglutinin disease, Jpn. Circ. J , King, J. W. and May, J. S., Cold agglutinin disease in a patient with legionnaires' disease, Arch. Intern. Med.. 140, 1537, Kokkini, G,, Vrionis, G., Liosos, G., and Papaefstathiou, J., Cold agglutinin syndrome and haemophagocytosis in systemic leishmanniasis, Stand. J. Haematol., 32, 441, Guena. L., Addei, K. A., and Brook, S., intraoperative hypothermia in a patient with cold agglutinin disease, J. Natl. Med. Assoc., 74, 691, Blumberg, N., Hicks, G., Woll, J., Nusbacher, J., Cox, M. T., Wilbur, O., Schwedfeger, O., and McMican, A., Successful cardiac bypass surgery in the presence of a potent cold agglutinin without plasma exchange, Transfusion, 23, 363, 1983, 156. Leach, A. B., van Hasselt, G. L., and Edwards, J. C., Cold agglutinins and deep hypothermia, Anaesthesia. 38, 140, Crisp, I). and Pruzanski, W., B-cell neoplasms with homoger~ous cold reacting antibodies (cold agglutinins), Am. j, Med.. 72, 915, 1982.

29 Volume 4, Iss,ae Wolach, B., Heddle, N., Barr, R. D., Zipursky, A., Pal, K. It. M., and Blajchman, M. A., Transient Donath-Landsteiner haemolytic anaemia, Br. J. H~wtmJtol Petz, L. D. and Garratty, G., Acquired hnmune Hemr Anemias. Churchill Livingstone, New York. 1980, 55. ~69. Bird, G, W. G,, Wiagham, J., Martin, A. J., Rit:hardson, S. G. N., Cole, A. P., Payne, R. W., and Savage, B. F., Idiopathic non-syphililic paroxsysmal cold haemoglobinuria in children, ]. Clin. Pathol.. 29, 215, Johnsezt, H. E., Bromstrom, K., and Madsen, M., Paroxsysmal cold haemoglobinuria in children: 3 cases encountered within a period of 7 months, Stand. J. Haematol., I62. Bird, G. W. G., Paroxysmal cold hae~noglobinuria, b;r. J. H,em,tol., , Lau, P., Sererat, S.,/Hoore, V., McLeish, K., and Alousi, M., Paroxsysmal cold hemog, iobinuria in a ~atient with Klebsiella pneunwnia, Vox Sang.. 44, 167, Andersen, E., Skov, F., and Hippe, E., A case o1" cold haetut~gt~3binuria ~ith ~ater sarcuidosis: treatnlent with plasmapheresis and immunosuppressiva, &'and. J. Haem,lol., Rausen, A. R., LeVine, R., Hsu, T. C. S., and Rosenfield, R. E., Compatible transfusion therapy for paroxsysmal cold hemog, lobinura, Pedialrics. 55, 275, Silvergleid, A. J., Wells, R. F., Hafleigh, E. B., Korn, G., Kellner, d. J., and Grumet, F. C., Compatibility test using ~Chromium-labclled red blood cells in crossma~ch posidve patients. TransJitsiott, 18, Petz, L. D. and Garratty, G., Acquired Immune Hemolytic Anemias. Churchill Livingstone, New York, 1980, Ries, C. A., Garratty, G., Petz, L. D., anti Fudenberg, it. H., Paroxsysmai cold hemoglobinuria: repor~ of a case with an exceptionally high thermal range Donath-Lardsteiner antibody, Blood. 38, I Petz, L. D. and Garratty, G., Acquired Immune Hemolvth' Anemias. Churchill Livingstone, New York, 1980, Crookston, J. H., Hemolytic anemia with lgg and igm autoantibodies and alloantibodies. Arch. Intern. Med.. 135, 1314, Freedman, J., Cheng, L. F., Murray, D., and Myers, R., AJ~ unusual autoimmune hemolytic anemia in a patient with immunoblastic.sarcoma. Atn. J. Hem~m)l Moake, J. L. and Schultz, D. R., Hemolytic anemia associated with multiple autoantibodies and low serum complement. Am. J. Med., 58,431, Sachs, A. S., Platt, L. D., and Johnson, C. S., Autoimmune hetuolytic disease during pregnancy, Aoz. J. Obstet. Gynecol,. 140, Lawe, J. E., Successful exchange transfusiorl of an infant ['or AIHA developing late in mother's pregnancy, Tran.~fitsion , Issaragrisil, S. and Kruatrachue, M., An association of pregi~ancy and autoimmune haemolytic anaenlia. Stand. J. Haematol.. 31, 63, Shash~ty, G. G., Rath, t2. E., and Britt, E, J., Autoimmune hemolytic anemia associated with ulcerative colitis. Am. J. Hematol.. 3, 199, Bell, D, W., Urban, E., Sears, D. A., Walder, A. 1., and Ostrower, V. S., Ulcerative colitis comt~licated by autoimmune hemolytic anemia, Soutt~. Med. J.. 74,359, 1981, 178. Petz, L. D. and Garratty, G., Acquired Immune Hemolytic Aneotias, Churchill Livingstone, New York, 1980, Zuelzer, W, W., Mastrangelo, R., $tulberg, C. S., Poulik, M. D., Page, R. It., and Thompson, R. 1., Autoimmune hemolytic znemia: rmtural history and viral-immui~ologic interactions in chi!dhood. Am. J. Med., 49, 80, Habibi, B., Homherg, J-C., Schaison, G., and Salmon, C., Autoimmune hemolytic anemia in children: a review of 80 cases, Am. J. Med., 56, 61, I81. Buchanan, G. R., Boxer, L. A., and Nathan, D. G., The acute and transient nature of idiopathic immune hemolytic anemia in childhood, J, Pediatr.. 88, 780, Zupanska, B., Lawkowiez, E., Gorska, B., Kozlowska, J., Ochocka, M., Rokicka-Milewska, R., Derulska, D., and Ciepielewska, D., Autoimmune haemolytic anaemia in children, Br. J. Haematol.. 34, 51 I, Carapella de Luca, E., Casadei, A. M., di Piero, G., Midulla, M., Bi~omini, C., and Purpura, M,, Autoimmune hemolytic anemia in childhood: fo~jow.tzp in 29 cases, Vox Sang.. 36, Heisel, M. A. and Ortega, J. A., Factors influencing prognosis in childhood autoimmune hemolytic anemia, Ant. J. Pediatr. Hematol. Om'ol.. 5, 147, Miyazaki, S., Nakayama, K., Akabane, T., Taghuchi, N., Akatsuka, J., Nagao, 'If'., Tsujino, G., and Nakagawa, T., Follow-up study of 34 children with autoimmune hemolytic anemia, Acta Haematol. Jpn.. 46, 6, Zudzer~ W. W., Stulberg, C. S., Page, R. II., Teruya, J., and Brough, A. J., Etiology and pathogenesis of acquired hemolytic anemia, Transfusion, 6,438, 1966.

30 154 CRC Critical Reviews in Oncology/Hematology 187. Chu, J-Y., MrEIfresh, A. E., and Waeltermann, R. M., Autoimmunr hemtflytir anemia as a prescn{ing manifestation (:f l-lodgkin's disease. J. Pedialr., 89, May, R. B. and Bryan, J. H., Autt)immtme hemolylic anemia and Hodgkin disease. J. Pedi,tr., Carpentieri, U., Daeschner, C. W,, and Haggard, M. E., lmmunohcmtjlylic anemia ai~d Hodgkm disease, Pediatrir 70, (1. Chu, J-Y., Autoimmtmc hetu()lytic anemia in childhood t-lodgkin's disease, Am. J. Pcdhltr. Hem,to/. Om~/.. 4, Bernard, O., Hadchouel, M., Scolto, J,, Odievre, M., and Alagille, I)., Severe giajr cell hepaliti.,, with auloimmune henlolylic anemia in early childhood. J. Pediatr, ~ Ammann, A. J., Cowan, M. J., Warn, D. W., Weintraub, P., Dritz, S., Goldman, H., and Perkins, It. A., Acquired immunodeficieacy in an infant: possible transmission by,1cans of blood pro,jucls. Lam'c,t, I. 95~ It)3. (;reenberg, J., Curtis-Cohen, M., Gill, F. M,, and Cohen, A., Prolonged reticuh)cytopenia in autoimmune hemolytic anemia of childhood. J. Pedhttr., , 194. Ghosh, M. L. and Harris-Jones, J. N., Coombs-negalive autoimmunc hemolylic anemia and ilnmunogh~btflin deficiency. Am. J. ClhJ. P,tht)l., 62, , 195. Roeicke, D., Anstee, D. J., Jungfer, H., Nutzenadef, W., and Webb, A. J., lgg-typr cold agglutinin~ in children and correspondin~ antigens. Detection of a new Pr antigen: Pr,. Vox Sottg,. 2( , Moore, J. A, and Chaplin, H., Autoimmunr hen~olytic anemia associated with an lgg cold incomplete antibody, Vo.v Sung,, It)73. 1~)7. Johnson, C. A. and Ahildgaard, C. F., Treatment of idiopathic aul,immune hemolytic" anemia in children. A('t, Paediatr. S('and., 65, Pui, C-tt., Wilimas, J., and Wang, W., Evans syndrome in t.'hildtlood..i. Pediatr., 97,754, 19l~(I Bikowski, M. R. and Mitchell,,I. E., Autoimmune hemolylic anemia in children. Am. F,m. Phys.. 20, 131, (/0. Sokol, r. J. and Itewitl, S., unpublished ob,~ervation.