Immune hemolytic anemia in children

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I M M U N E H E M O L Y T I C ANEMIA IN CHILDREN Authors:

Robert W. Warren Department of Pediatrics University of North Carolina Chapel Hill, North Carolina and Duke University Medical Center Durham, North Carolina

Myra L. Collins Department of Pathology University of North Carolina and Blood Bank Division of Hospital Laboratories North Carolina Memorial Hospital Chapel Hill, North Carolina Referee:

Campbell W. McMillan Department of Pediatrics University of North Carolina and North Carolina Memorial Hospital Chapel Hill, North Carolina

I. INTRODUCTION Although it occurs less frequently than in adults, profound anemia can result from immune destruction of red blood cells (RBC) in children. The RBC are coated with immunoglobin, complement, or both; they may be lysed intravascularly or engulfed by cells of the monocytemacrophage system. The immunoglobulins involved are almost always IgM or IgG. The immunoglobulins may be directed against native constituents, thus producing true autoimmune hemolytic anemia, or they may be directed against altered or foreign molecules in the red cell membrane. Immune hemolytic anemia (IHA) is separable into two types. "Warm-type" IHA is mediated by IgG or by IgG and complement while "cold-type" IHA is usually mediated by IgM and complement. These two types show significant differences in etiology, clinical characteristics, mechanism of hemolysis, and response to therapy. The purpose of this review is to describe the major clinical and laboratory features of the warm- and cold-types IHA, to present the most frequent etiologies, to review the basic pathophysiological mechanisms, and to outline a reasonable approach to therapy. II. L A B O R A T O R Y TESTING By definition, IHA requires that immunoglobulin, complement, or both be present on the circulating RBC. Before discussing the tests routinely done to demonstrate these molecules, some characteristics of IgM and IgG are reviewed. Since IgM molecules are large and pentameric, single molecules can bridge RBC and specifically agglutinate those cells possessing appropriate antigenic sites, even if the cells are suspended in a medium containing

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almost no protein. Hence, these IgM molecules are sometimes called complete agglutinins. Despite the fact that IgM molecules can agglutinate cells directly under some conditions, the avidity of each binding site for RBC antigens is not high, and the molecules tend to elute at temperatures near 37°C. Before the IgM molecules dissociate, they can stimulate the binding of complement components to the RBC membrane, as is detailed in the following. On the other hand, IgG molecules are monomeric, possess only two antigen-binding sites, and may not agglutinate cells directly; hence, they are called incomplete agglutinins. To agglutinate cells coated with IgG, a second antibody which recognizes and attaches j~elf to IgG is added to the test tube to form a bridge between the cells. To determine whether immunoglobulin and/or complement is bound to circulating RBC, a direct antiglobulin test (DAT or direct Coombs test) is done. Reagent antisera which contain antibodies directed against human immunoglobulins and complement are added to samples of the patient's RBC. The antisera may be monospecific, being able to recognize only IgG or complement, or it may have a broad spectrum of reactivity and be able to recognize both. IgM usually dissociates from the cells and is not directly detectable by this method; however, the complement components that remain may be detected by the direct antiglobulin test. If the RBC are coated by IgG, complement, or both, they are agglutinated by the reagent antisera, and the antiglobulin test is positive. As discussed in the following, the pathological process in a given patient depends on whether the RBC are coated by IgG only, complement only, or both IgG and complement. An indirect antiglobulin test (antibody screen) measures the ability of the patient's serum to agglutinate RBC from other individuals bearing known antigens. To increase the sensitivity of this test, a number of incubation conditions may be employed which consist of varying the temperature, ionic strength, and protein concentration of the reagents used. The conditions under which agglutination occurs suggest the class of immunoglobulin (IgM or IgG) present. Unlike the binding to RBC by IgG antibodies, which proceeds best at 37°C, IgM-mediated agglutination characteristically is more pronounced at cooler temperatures. In fact, it is common for sera from normal, asymptomatic individuals to agglutinate RBC at 4°C, but the titer or amount of antibody is relatively low (<64). During the development of IHA, serological studies may give confusing results. Thus, depending on the avidity of the antibody for RBC, the antibody screen may detect free antibody in the patient's serum either before or after the direct antiglobulin test reveals antibody bound to the patient's RBC. The antibody screening test is usually intially negative in patients whose cells are coated with IgG until the direct antiglobulin test is strongly positive. In other words, IgG antibody avidly binds to sites on the RBC, and only after those sites are covered does the antibody spill into the plasma. III. E T I O L O G I C A L A S S O C I A T I O N S IHA in children is almost always associated with one of the following factors: infection or immunization, drugs, underlying noninfectious disease, or unknown causes. A. Infection or Immunization

IHA follows viral infection or immunization much more often in children than in adults. IgM antibodies which fix complement and show a broad thermal amplitude may develop and react in vitro at temperatures from 10°C to near 37°C. A much less frequent but important type of hemolytic antibody that occurs after infection is the Donath-Landsteiner antibody which produces paroxysmal cold hemoglobulinuria (PCH). Donath-Landsteiner antibody is an unusual IgG which binds to RBC and fixes complement at low temperatures. Lysis results when the cells are wanned to 37°C. Significant intravascular hemolysis can result in IHA caused by IgM or the Donath-Landsteiner antibody if the child is exposed to cold.

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B. Drugs While exposure to any of several drugs may lead to the development of immune-mediated hemolysis in adults, this process occurs less frequently in children. The reason for this finding is probably that the common inducers of immune hemolysis, such as alpha-methyldopa, usually are not prescribed for children. When drug-induced IHA does occur, it is usually due to lgG antibodies and is associated with antibiotics such as penicillin. C. Underlying Noninfectious Disease While IHA associated with infections is usually acute and of short duration, the anemia associated with chronic diseases in childhood is often more prolonged. The antibody involved is almost always an IgG. The course of the anemia follows that of the underlying disease. Systemic lupus erythematosus (SLE) and other types of collagen vascular diseases are most commonly associated with immune hemolysis. Children with immunodeficiency or malignancy, especially malignancies of the lymphoreticular tissues, may have significant immune hemolysis. The prognosis of hemolytic anemia occurring in association with a chronic disease is much worse than that associated with infection. J The mortality rate is appreciable and is the same as that of the underlying disease. D. Unknown Causes There is always a group of patients who develop significant anemia associated with IgG bound to their RBC in whom the etiology is unclear. Long-term follow-up may reveal an associated condition. IV. M A J O R C L I N I C A L T Y P E S OF IHA The clinical features of IHA in children can be highly variable depending on the probable etiology and on whether IgG or IgM is responsible. In the following sections, the major features of the warm- and cold-types IHA are presented.

A. Warm-Type IHA The most common type of IHA in children is attributable to lgG antibodies which bind best at 37°C and which are directed against protein antigens in the RBC membrane. These antigens are often associated with the Rh blood group system. 2 Habibi et al. demonstrated IgG, or IgG and complement, on the RBC of 39 out of 80 children with IHA. 3 Almost all of these children had a chronic course, and in over half the anemia presented in association with an underlying disease, such as SLE, immunodeficiency, or a malignancy. Others have observed that children with warm-type IHA are older and that the ratio of girls to boys is increased. Nevertheless, these findings are not present in children with an acute course lasting less than 3 months in which the hemolytic anemia is associated with prior infection. 4-6 Cold-type IHA predominated in only one series; perhaps this anomalous finding was a consequence of atypical patient referral patterns. The patient with warm-type IHA may present with signs and symptoms of severe RBC destruction or may be relatively asymptomatic and have only mild anemia. The specific findings in a given case are a function of the IgG involved and the course of the underlying disease. The balance between RBC destruction and production is reflected in the laboratory findings of the peripheral blood. If the immune destruction is moderate to severe, many young RBC may be evident as the bone marrow responds with increased erythropoiesis. Reticulocytes and, occasionally, nucleated RBC are prominent. The percentage of reticulocytes may reach 70 or 80%. Spherocytes are common; they are hypothetically the result of removal of portions of RBC membrane by tissue macrophages. The anemia, as reflected in the measurements of hemoglobin and hematocrit, may be mild to severe. It should be

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noted that hematocrits obtained by centrifugation may be spuriously high since spherocytes and reticulocytes do not pack as well as normal RBC in capillary tubes. There are two major mechanisms by which the coating of RBC by IgG leads to hemolysis. In the first mechanism, mononuclear cells bearing receptors which recognize the Fc portion of IgG 3, and to a lesser extent of IgG~ and IgG2, phagocytose cells coated by these immunoglobulins. The greatest concentration of these phagocytes is located in the spleen; hence, the major site of RBC destruction in the warm-type IHA is usually in this organ. The second mechanism for RBC destruction depends on the binding of complement molecules stimulated by IgG. Again, IgG3 is the most active subclass. Since IgG is monomeric, at least two IgG molecules must bind to the RBC in close proximity to effect the binding of complement. Hence, the fixation of complement is also a function of antigen distribution and mobility in the RBC membrane. In fact, most IgG antibodies do not cause intravascular lysis by the complement mechanism. This is because IgG antibodies generally react with antigens which are immobile and too far apart in the RBC membrane for complement fixation. However, if the antibodyantigen configuration is such that two immunoglobulin Fc regions are close together, complement component C 1 may bind, and the complement cascade begins. If the membrane attack complex is formed, intravascular hemolysis can result. If hemolysis is rapid and massive, peptides will be released which may mediate several systemic effects of clinical significance (C3a, C5a). 8 In some patients, complement component C3b as well as IgG may bind to the RBC membrane. Binding of C3b is of clinical importance because RBC bearing both IgG and C3b may be destroyed by phagocytes which possess receptors for either or both of these molecules. The mechanism for phagocytosis by cells possessing the C3b receptor and for conversion of C3b to C3d is described in the discussion of cold-type IHA. The pathophysiology described previously is reflected in the immunohematological test results. The direct antiglobulin test may show IgG only or both IgG and C3d bound to the patient's RBC. The strength of the immunological reactions usually correlates with the severity of the anemia. As the anemia improves, the immunological reactions become weaker but may remain detectable even after the hematocrit has returned to a normal level. Examination of the bone marrow almost always shows erythroid hyperplasia. In some instances of chronic anemia, another pattern, such as hypoplasia, may be seen depending on the underlying disease, especially if the child has received antineoplastic chemotherapy. Other abnormal laboratory studies may include increased serum bilirubin and decreased plasma haptoglobin levels, especially if there has been intravascular release of hemoglobin. Gross hemoglobinuria is rare because the hemolysis is usually extravascular.

B. Cold-Type IHA The frequency of IgM-mediated IHA has been estimated to be 10 to 30% of the total cases of IHA in children. 3.9 When symptoms of anemia occur, they usually arise acutely and follow infection in more than 75% of the cases. 9 Multiple infectious agents have been implicated in the etiology of the acute form, including Mycoplasma pneumoniae, EpsteinBarr virus (EBV), mumps virus, and cytomegaiovirus.~° The hemolytic anemia is usually mild and rapidly resolving, but on occasion sudden and severe intravascular hemolysis may occur, resulting in hemodynamic instability and signs of heart failure. The immunoglobulin often involved is IgM, which recognizes the simple linear (i) or complex branched (I) polysaccharide chains of the Ii blood group, at The i structure is found on all human RBC but is more prevalent on RBC from the fetus and newborn. The branching I structure replaces i as the child ages. Both I and i represent multiple antigenic specificities, t2 Antibodies to these polysaccharide structures apparently develop because of cross-reactive specificity with unrelated foreign antigens or disordered immunoregulation. IHA which follows infection with mycoplasma is usually mediated by an IgM of anti-I specificity. IHA

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induced by infection with EBV is usually generated by anti-i antibodies. ~ The reasons for such differences of specificity following certain infections is unexplained at the molecular level. However, because of the known distribution of i and I antigens, it is not surprising that clinically significant IHA is more common following mycoplasma infection than following EBV infection. While cold-type IHA associated with infection is usually acute and self-limited, some children who have lymphoproliferative disease have chronic and recurrent anemia. The anti-RBC antibodies are usually specific for I. IgM antibodies bind to the RBC polysaccharide antigens best at low temperatures. The binding appears to be of low avidity, but IgM molecules vary in their binding strength and also in their temperature range of reactivity. ~3Thus, while some cold agglutinins may never be active at physiological temperatures, others may easily bind to RBC when they circulate to the cooler body surfaces. Presence of IgM on the RBC surface stimulates complement fixation, but IgM molecules also seem to vary in their ability to bind complement C1.8 Moreover, binding of human complement C4 is temperature dependent, with binding activity beginning at about 10°C. 14 Thus, some IgMs might produce marked RBC agglutination in vitro, but because they have a narrow temperature range of reactivity, a limited ability to bind C1, or both, they may produce little if any hemolysis in vivo. Following the early stages of complement activation on a membrane surface, bound immunoglobulin is no longer required for the complement cascade to continue. Indeed, much IgM is released at higher temperatures. Severe intravascular hemolysis is complement mediated, but it is also uncommon. Human RBC are relatively resistant to direct lysis by the terminal attack sequence of complement. 15 Thus, complement C3 inactivator (C3blNA) may inactivate C3b on these cells, producing C3d-coated RBC. It should be noted that such cells circulate and function normally and are resistant to intravascular hemolysis, apparently because C3d on the surface inhibits further binding by the specific IgM involved.~6 Alternatively, C3b-coated RBC may bind to C3b receptors on macrophages, particularly in the liver. These RBC may then be phagocytosed. C3d-coated RBC are less susceptible to this mechanism of extravascular hemolysis as well. Mechanisms in the pathophysiology of IgM-mediated cold-type IHA are also reflected by results of the relevant laboratory studies. If complement-mediated intravascular hemolysis is primarily responsible for the RBC destruction, spherocytosis will not be seen since the RBC are totally destroyed. On the other hand, extravascular removal of membrane from complement-coated RBC by the liver is a plausible mechanism for spherocyte formation. A characteristic finding in cold-type IHA is that the direct antiglobulin test is positive for complement binding but negative for IgG. IgM antibodies are of relatively low avidity and usually dissociate from antigenic sites after complement is bound. Thus, one would expect the immunoglobulin (IgM) to be present in the patient's serum and to be readily detected by the antibody screen. Since IgM preferentially reacts at lower temperatures and since IgMs associated with cold-type IHA have broad thermal amplitudes, antibody screens run at temperatures from 4°C to near body temperature may show a high titer of antibody. A titer between 1000 and 5000 is typical. Indeed, the most important aspect of the IgM molecule is its range of thermal reactivity or thermal amplitude. Those antibodies that react with RBC at temperatures slightly below body temperatures are more likely to be associated with disease than those that react only at temperatures below body surface temperature. C. PCI-I Unlike cold-type IHA described previously, PCH is a postinfectious hemolytic syndrome in children that is mediated by an IgG called the Donath-Landsteiner antibody. This antibody binds to the RBC membrane P antigen at low temperatures, such as are found near the skin surface, and fixes complement efficiently. As the RBC warm up when circulating to the inner regions of the body, the antibody elutes off, and bound complement mediates intra-

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vascular hemolysis. The hemoglobin that is released into the circulation is promptly excreted in the urine. Low-temperature binding of the antibody followed by warm-temperature hemolysis is characteristic of this biphasic hemolysin. IHA associated with the Donath-Landsteiner antibody is usually considered one of the rarest types. Petz and Garraty observed only 6 cases among 347 adults with IHA. 2 However, the incidence in children, especially in children less than 4 years old, is much higher. Habibi et al. saw 4 cases out of 80 children with IHA. 3 Nearly one half of the children with IHA (17 out of 42) reported by Sokoi et al. had PCH. 7 PCH is almost always associated with a prior infection. In the past, chronic PCH was seen as a consequence of congenital syphilis. Today it is most often associated with viral infections such as chickenpox, measles, mumps, or an influenza-like illness.17 Cases may occur after immunization or following episodes of fever of unknown origin. Acute hemolysis associated with a biphasic IgG with anti-P specificity was observed after Mycoplasma pneumonia in a 7-year-old boy ts and after Klebsiella pneumonia in a 60-year-old man. ~9 Patients with Donath-Landsteiner antibodies often present with malaise and pallor of sudden onset. A history of at least one episode of gross hemoglobinuria is common. Mild jaundice and headache are frequent. Anemia can be severe at presentation, and symptoms of high-output heart failure may necessitate emergency transfusions. Reticulocytosis is variable at presentation. The smear usually does not show marked anisocytosis since the RBC are lysed intravascularly. However, there may be some evidence of cell-mediated destruction since neutrophils containing RBC (erythrophagocytosis) are occasionally seen. 2° The direct antiglobulin test is typically positive if performed with a monospecific anticomplement reagent. It is uncommon to find IgG bound to RBC under the usual laboratory conditions in which the test is performed since the antibody dissociates from the cells at 37°C. To demonstrate the presence of the Donath-Landsteiner antibody requires that the antiglobulin test be performed in a specific manner. The inference that the antibody is present depends on the observation that RBC, which are incubated with the patient's serum at low temperature and then warmed, hemolyze if adequate complement is present, while cells that have been kept in the patient's serum at 37°C do not. A potent IgM anti-I can sometimes be difficult to distinguish from a true Donath-Landsteiner antibody, but a Donath-Landsteiner antibody should be nonagglutinating and possess anti-P rather than anti-I specificity. 2j The course of PCH in children is almost always acute. Typically, the anemia lasts from a few weeks to 3 months. The biphasic antibody may be detectable only during the phase of acute hemolysis, but the direct antiglobulin test using anticomplement reagent may remain positive longer. V. T H E R A P Y The differences in the pathophysiology of IgM-mediated IHA compared with IgG-mediated IHA underscore the need for different therapeutic approaches in children with each type.

A. Warm-Type IHA The therapeutic response of warm-type IHA associated with an underlying disease usually depends on giving the appropriate treatment for that disease, i Nevertheless, nonspecific therapy is very important. Steroids are the treatment of choice. About 80% of patients with warm-type IHA will have a prompt and dramatic reduction in the rate of RBC lysis on steroid therapy. 2 The primary mechanism of action is unknown. However, steroids may reduce the clearance of antibody-coated RBC; they may alter the avidity of the antibody for RBC antigens; and they may reduce the production of antibody. Some authors advocate that steroids be given intermittently as large-pulse doses in cases of severe IHA unresponsive to usual doses (see following). 22 In some instances, the anemia may be improved if antibody

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production is diminished by administration of other immunosuppressive agents. Physical removal of circulating antibody may be helpful; Bernstein et al. reported the successful treatment of life-threatening IHA by plasma exchange. 23 Since the spleen is commonly the site of sequestration and destruction of RBC in warmtype IHA, therapy directed at reducing splenic clearance of IgG-coated erythrocytes may be necessary in extremis. In such a crisis, the usual and often successful approach is splenectomy. Since high-dose i.v. immunoglobulin (i.v. IGG) may permit deferral of splenectomy for children with severe idiopathic thrombocytopenic purpura, 24.25 it may also be useful in treatment of severe, refractory warm-type IHA. Although the mechanisms of action of i.v. IGG in idiopathic thrombocytopenic purpura are probably complex, it has been theorized that a rapid, beneficial effect occurs via splenic Fc receptor blockade. 26 Case reports showing benefit of i.v. IGG in IHA have recently appeared, using total i.v. IGG doses greater than 2 g/kg. 27.28 In addition, we recently administered i.v. IGG to a severely alloimmunized patient dying of progressive anemia secondary to hemolysis of best-matched donor RBC, with prompt resolution of hemolysis. Nevertheless, the routine use of i.v. IGG therapy for warm-type IHA is inappropriate because no controlled study has yet been reported and because optimal dosing is undefined. In addition, the possible complications and expense of i.v. IGG are significant. If the anemia is so severe that the child becomes hemodynamically unstable and there are signs of heart failure, transfusion of RBC can be life-saving and should be given even though the RBC will likely be incompatible. Since the rate of destruction is a function of the number of circulating RBC, transfusion of the minimum amount necessary to stabilize the patient's cardiovascular system should be given. Transfusion should be given slowly and with careful monitoring. B. Cold-Type IHA Because of its self-limited course, postinfectious cold-type IHA can often be treated conservatively, but renal blood flow and urine output must be maintained to prevent deleterious renal effects of acute intravascular hemolysis. Maintaining patient warmth is imperative; all body surfaces should be covered with blankets or clothing, and only warm food and drink should be given. The importance of the last recommendation is emphasized by a 3-year-old patient observed by the authors. This child with PCH had a severe hemolytic episode while in the hospital which required blood transfusions after the parents gave him some ice cream, even though the room was 32°C and the child was fully covered. Blood transfusion should be given only if the patient has signs of hemodynamic instability or has symptoms of hypoxia. Since cold-reacting antibodies usually bind to ubiquitous RBC antigens, donor cells lacking these antigens are usually impossible to locate. Moreover, because antigen-beating donor RBC will not be coated with complement C3d, they may be more prone to lysis than those already circulating in the patient. It is of particular importance that the blood be warmed to 37°C and perhaps washed to remove the plasma which will serve as an additional source of complement. A blood warmer should be applied on the infusion line just before the blood enters the patient. The transfusing RBC must not be overheated since heating them above 37°C can significantly shorten their in vivo survival. Steroids in common doses are usually ineffective for cold-type IHA since at these doses they will not significantly change antibody levels or disturb complement function. On the other hand, since steroids inhibit monocyte-macrophage function, 29 they may reduce extravascular hemolysis mediated by the complement components bound to RBC. It is possible that steroids given in very high doses (such as methylprednisoione at a dose of 30 mg/kg/ day to a maximum of 1 g) may be more effective than the more commonly given lower doses for both types of IHA. 22 Steroids given in such intermittent-pulse doses are extremely effective at inhibiting monocyte-macrophage function and even may inhibit complement activation. 30

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Various cytotoxic drugs have been used in treatment of chronic cold agglutinin disease without significant favorable results. Cytotoxic drugs have no beneficial effect on acute intravascular hemolysis. Neither splenectomy nor high-dose i.v. gammaglobulin has a place in the therapy of cold-type IHA since the spleen is not the major site of extravascular hemolysis. Plasma exchange might be of value since the hemolytic IgM antibodies could perhaps be efficiently removed by this technique. The major treatment for chronic IHA, either the warm or the cold type, should be directed toward the underlying disease.

REFERENCES 1. Heisel, M. A, and Ortega, J. A., Factors influencing prognosis in childhood autoimmune hemolytic anemia, Am. J. Pediatr. Hematol. Oncol., 5, 147, 1983. 2. Petz, L. D. and Garraty, G., Acquired Immune Hemolytic Anemias, Churchill Livingstone, New York, 1980. 3. Habibi, B., Homberg, J. C., Schaison, G. et al., Autoimmune hemolytic anemia in children; a review of 80 cases, Am. J. Med., 56, 61, 1974. 4. Carpella de Luca, E., Casadei, A. M., di Hero, G., Midulla, M. et al., Autoimmune hemolytic anemia in childhood; follow-up in 29 cases, Vox Sang., 36, 13. 1979. 5. 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, 1976. 6. Zuelzer, W. W., Mastrangelo, R., Stulberg, C. S. et al., Autoimmune hemolytic anemia; natural history and viral-immunologic interactions in childhood, Am. J. Med.. 49, 80, 1970. 7. Sokol, R. J., Hewitt, S., Stamps, B. K. et al., Autoimmune hemolysis in childhood and adolescence, Acta Hematol.. 72, 245, 1984. 8. Logue, G. L. and Kurlander, R. J., Immunologic mechanisms of hemolysis in autoimmune hemolytic anemia, Pathobiol. Ann., 8, 61, 1978. 9. Zupanska, B., Lawkowicz, W., Gorska, B. et al., Autoimmune hemolytic anemia in children, Br. J. Haematol., 34, 511, 1976. 10. Burstein, Y. and Berns, L., Acquired immune hemolytic anemia in children, Pediatr. Ann.. I 1, 301, 1982. I I. Pruzanski, W. and Shumak, K. H., Biologic activity of cold-reacting autoantibodies, N. Engl. J. Med., 297, 538, 1977. 12. Feizi, T. and Kabat, E. A., lmmunochemical studies on blood groups, J. Exp. Med., 135, 1247, 1972. 13. Evans, R. S., Turner, E., and Bingham, M., Studies with radioiodinated cold agglutinins of ten patients, Am. J. Med., 38, 378, 1965. 14. Rosse, W., Adams, J., and Logue, G. L., Hemolysis by complement and cold-reacting antibodies; time and temperature requirements, Am. J. Hematol.. 2, 259, 1977. 15. Evans, R. S., Turner, E., and Bingham, M., Chronic hemolytic anemia due to cold agglutinins; the mechanism of resistance of red cells to cold hemolysis by cold agglutinins, J. Clin. Invest.. 46, 1461, 1967. 16. Frank, M. M., Schreiber, A. D., Atkinson, J. P. et al., Pathophysiology of immune hemolytic anemia, Ann. Intern. Med., 87, 210, 1977. 17. Bird, G. W. G., Wingham, J., Martin, A. J. et al., Idiopathic non-syphilic paroxysmal cold hemoglobinuria in children. J. Clin. Pathol., 29, 215, 1976. 18. Boceardi, V., D'Annibali, S., Di Natale, G. et al., Mycoplasma pneumoniae infection complicated by paroxysmal cold hemoglobinuria with anti-P specificity of biphasic hemolysin, Blut, 43, 211, 1977. 19. Lau, P., Sererat, S., Moore, V. et al., Paroxysmal cold hemoglobinuria in a patient with Klebsiella pneumonia, Vox Sang., 44, 167, 1983. 20. Hernandez, J. A. and Steane, S. M., Erythrophagocytosis by segmented neutrophils in paroxymal cold hemoglobinuria, Am. J. Clin. Pathol., 81, 787, 1984. 21. Mollison, P. L., Blood Transfusion in Clinical Medicine. 7th ed., Blackwell Scientific, Oxford, 1983, 454. 22. Jacobs, H. S. Pulse steroids in hematologic diseases, Hosp. Pract.. 24, 87, 1985. 23. Bernstein, M. L., Schneider, B. K., and Naiman, J. L., Plasma exchange in refractory acute autoimmune hemolytic anemia, J. Pediatr., 98, 774, 1981. 24. Imbach, P., Barandun, S., d'Apuzzo, V. et al., High-dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood, Lancet. I, 1228, 1981.

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25. Bussell, J. B., Schulman, I., Hilgartner, M. W. et al., Intravenous use of agammaglobulin in the treatment of chronic immune thrombocytopenic purpura as a means to defer splenectomy, J. Pediatr., 103, 651, 1983. 26. Fehr, J., Hofman, V., and Kappeler, V., Transient reversal of thrombocytopenia in idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin, N. Engl. J. Med.. 306, 1254, 1982. 27. MacIntyre, E. A., Linch, D. C., Macey, M. G. et al., Successful response to intravenous immunoglobulin in autoimmune hemolytic anemia, Br. J. Haematol., 60, 387, 1985. 28. Oda, H., Honda, A., Sugita, K. et al., High-dose intravenous intact IgG infusion in refractory autoimmune hemolytic anemia (Evans syndrome), J. Pediatr., I07,744, 1985. 29. Schreiber, A. D., Herskovitz, B. S., and Goldwein, M., Low-titre cold hemagglutinin disease. Mechanism of hemolysis and response to corticosteroids, N. Engl. J. Med., 296, 1490, 1977. 30. Weiler, J. M. and Packard, B. D., Methylprednisolone inhibits the alternative and amplification pathways of complement, Infect. Immun., 38, 122, 1982.