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Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

THE ANTIGLOBULIN TEST IN

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AUTOIMMUNE HEMOLYTIC ANEMIA Wendell F. Rosse, MD. Hematology Division, Department of Medicine, Duke University Medical Center and the Durham Veterans Administration Hospital, Durham, North Carolina 27710

Autoimmune hemolytic anemia (AIHA) is a process wherein red cells are destroyed because of their interaction with an antibody made by the immune system of the patient himself. In this sense, immunological tolerance has been broken since the antigens against which the antibody is directed are those of the organism making the antibody (1). In a few instances these antigens have been either chemically or serologically defined, but for the most part they remain unknown. The antibodies involved in autoimmune hemolytic anemia are usually either IgG or IgM (2). The distribution of the subclasses among the IgG antibodies does not correspond to the distribution of these subclasses in the IgG of serum. Antibodies of the IgGI and IgG3 subclasses seem to predominate although IgG2 and IgG4 antibodies have been identified (3, 4). IgA antibodies have been identified (5, 6) but to date no IgD or IgE antibodies have been implicated in the causation of autoim­ mune hemolytic anemia. In almost all instances the interaction between antigen and antibody results in the fixation to the membrane of immunoproteins, either antibody or the components of complement (7). The immunoglobulins may in some instances be detected by their ability to effect agglutination; this is usually not true of the immunoglobulins of the IgG class (7). Antibody and the components of complement fixed to the red cell membrane may be detected by the antiglobulin reaction first proposed by Coombs (8). In this reaction antibodies produced in other animals (hetero-antibodies) against human immunoproteins are reacted with washed red cells. If immunoprotein molecules are fixed to the red cell membrane, agglutination will occur. This reaction may be used to detect free antibody in the serum if normal red cells are incubated with the serum, washed, and then reacted with the hetero-antibody. By use of specific antisera containing antibodies to a single immunoprotein, it is possible to discriminate which of the several immunoproteins is fixed to the mem­ brane. Using such antisera, one can distinguish four types of immunoprotein coating on the red cells of patients with autoimmune hemolytic anemia: 331

Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

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Type I: immunoglobulin alone is present on the membrane. Type II: both immunoglobulin and complement are present on the red cell mem­ brane. Type I11:only complement components are present on the membrane. Type IV: neither complement nor immunoglobulins is detectable on the red cell membrane. In this review, I shall analyze each of the types of reactions as they relate to the pathogenetic mechanisms and to disease processes. TYPE I-IMMUNOGLOBULIN ALONE IS FIXED TO THE RED ' CELL MEMBRANE

In order for this type of direct antiglobulin test to occur, two conditions must be met: (a) The immunoglobulin must be firmly fixed to the antigen at body tempera­ ture. If it is not, it will be washed away during the preparation of the cells for the antiglobulin test. (b) The first component of complement (CI) is not fixed by the antibody. This may occur either because the antibody is intrinsically unable to fix CI, or the antigen sites are too distant, so that two such molecules or molecular subunits cannot form the "doublet" necessary for the fixation of C1 (9). The former occurs in the case of IgG4, IgA, and perhaps some IgM molecules (10), but the vast majority of autoimmune antibodies are IgM, IgG1, and IgG3, which are intrinsically capable of fixing C1 (11). The latter condition, placement of antigen sites, explains the majority of the instances in which complement is not fixed. Most autoimmune antibodies which do not fix complement have some specificity for the Rh system (12), and Rh antigens appear to be so dispersed that doublets cannot form (13). Red cells coated with antibody which does not fix complement do not hemolyze in vitro but may be destroyed in vivo. This is thought to be due to activation of a part of the heavy chain portion of the antibody molecule to enable it to interact with specific sites on phagocytic cells (14, 15). This activation occurs only with IgG1, IgG2, and IgG3, molecules upon interaction with antigen and does not occur in IgG4, IgM, or IgA molecules (14, 16). Because of this interaction, the red cell is affixed to phagocytic cells, in particular to fixed macrophages in the spleen. It is either completely phagocytosed or a portion of its membrane is removed (17). In the latter case, the cell membrane is able to reseal and a spherocyte results; this abnormally shaped cell may then be trapped in subsequent circulation through the spleen (18). Thus, hemolysis of this type is characterized by relatively little evidence of intravascular lysis (increased plasma hemoglobin, hemoglobinuria) and often by the presence of spherocytes and splenomegaly. With recent techniques which allow estimation of antibody concentration on red cells, it is found that the degree of hemolysis depends in part upon the concentration of antibody, but there is considerable variation in the amount of IgG attached for a given amount of hemolysis (19, 20). In about one third to one fourth of all patients with AIHA in which IgG antibody is detectable on the cell membrane, no complement is fixed (2). This occurs in patients with chronic lymphocytic leukemia (CLL) as well as in patients with

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Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

"idiopathic" immune disease. The antibodies of patients with systemic lupus ery­ thematosus (SLE) and other "collagen vascular" diseases usually fix complement (12). Of the syndromes of drug-induced hemolysis, both that initiated by alpha­ methyldopa and penicillin are usually characterized by the fixation of antibody without the fixation of complement (21). TYPE II-BOTH ANTIBODY AND COMPLEMENT ARE DETECTED ON THE RED CELL MEMBRANE

When the conditions for continued antibody attachment as well as those for fixation of CI are met, both IgG and complement components (mostly C4 and C3) may be found on the red cell surface by the use of specific antisera (22). Thus, warm-reacting IgG1, IgG2, or IgG3 antibodies of reasonably high affinity which react with plentiful antigens will give this reaction. These antibodies are encountered in"idiopathic" AIHA, and AIHA secondary to CLL and SLE, as well as other syndromes of AIHA. However, with some exceptions (23), the antibodies involved in drug­ induced immune hemolysis do not fix both antibody and complement. Hemolysis in vivo may occur both because of the activation of antibody (see above) or the activation of complement. Two mechanisms appear to be important in effecting hemolysis. 1. When the third component is affixed in its active form, a site on the molecule is able to fix to receptors on phagocytic cells, thus allowing phagocytosis (24, 25); this mechanism is "defused" by an enzyme which cleaves the C3 molecule, thus detaching its red cell binding site from its phagocyte binding site (26-28). 2. The fixation of the last component, C9, appears to breach the lipid bilayer, causing rupture of the cell (29, 30). When this occurs in vivo, intravascular hemolysis results. For unknown reasons, the normal red cell is difficult to breach by this mechanism; hence, large amounts of defused C3 and other components of complement may accumulate on the red cell surface (28). If both antibody and complement were fixed, one would expect greater hemolysis for a given amount of antibody than if complement were not fixed. Careful studies in animals seem to confirm this (31) and studies are currently under way to deter­ mine, if possible, the relative roles of antibody and complement in effecting lysis in AIHA. TYPE III-ONLY COMPLEMENT IS FIXED TO THE RED CELL SURFACE

In many instances of AIHA only the components of complement are found fixed to the red cell surface by the direct antiglobulin test using specific antisera. This may mean that the antibody has been there but was removed either in vivo or during the washing of the cells on preparation for the test. There are several reasons for this. 1. The antibody is "cold-reacting" and is thus eluted at the temperatures at which the test is performed, such as IgM cold agglutinins and the IgG Donath-Landsteiner antibody.

Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

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2. The antibody, while capable of reacting at 37° C, has a low affinity and is easily eluted during the washing of the cells. Since treatment of the red cells with proteo­ lytic enzymes usually increases the affinity of antibody and antigen (32), such antibodies can often be detected in the serum using enzyme-treated cells in the indirect antiglobuiln test. 3. The antigen is not firmly fixed to the red cell surface. In this instance, both antigen and antibody may be removed during the washing. If complement has been fixed, it is not removed and is detected. The best examples of unfixed antigens occur in certain instances of drug-induced immune hemolysis (the "innocent bystander" reactions) (21). This reaction may occur with antigens other than drugs, but this has not been proven. In some instances of Type III reactions, antibody may be present but not detected by the antiglobulin serum for several reasons: 1. The antibody is not IgG. Most autoimmune warm-reacting antibodies are IgG but IgM and IgA antibodies are found, usually in conjunction with IgG antibodies (5, 6). These will not be detected by anti-IgG but will with specific antisera. 2. The antibody is present in too low concentration to be detected by the anti­ globulin serum. The limits of the agglutination test for detection of antibody are discussed below. 3. The antibody is detected only at reduced temperature. Certain IgG antibodies are fixed to the red cell at 37° C but are not able to react with antiglobulin molecules unless the temperature is reduced (33). These antibodies frequently fix complement which is detectable on the red cell surface at any temperature. The hemolysis which ensues in patients exhibiting Type III direct antiglobulin test is probably predominantly due to the presence of complement components on the surface. The amount of hemolysis may vary from relatively little to consider­ able. TYPE IV-NEITHER ANTIBODY NOR COMPLEMENT IS DETECTED ON THE RED CELL MEMBRANE

In a few patients with what clinically appears to be AIHA, the direct antiglobulin test appears to be negative with both anti-IgG and anti-C3 (2). This may be due to the presence of amounts of antibody too small to be detected in the agglutination reaction with antiglobulin sera. Probably somewhere between 200 and 1000 mole­ cules of antibody per red cell are required before the agglutination reaction is positive as usually performed (34), but amounts less than this can be detected by special techniques (35, 36). It should be remembered that the amount of antibody on the cell surface may be very much larger in vivo before the cells are washed in preparation for the antiglobulin test. Some patients with AIHA and a completely negative Coombs' test may have considerable hemolysis. SUMMARY

The foregoing summarizes what can be learned from a carefully performed anti­ globulin test using specific antisera. Clinical syndromes can be more easily consid­ ered, and mechanisms of destruction can be more certainly discerned.

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Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

Literature Cited

I. Pirofsky, B. 1969. Autoimmunization and the Autoimmune Hemolytic Anemias, Baltimore: Williams & Wil­ kins 2. Dacie, 1. V., Worlledge, S. M. 1969. Auto-immune hemolytic anemias. Progr. Hematol. 6:82-120 3. Ishizaka, T., Ishizaka, K., Borsos, T., Rapp, H. 1966. C'I fixation by human isoagglutinins: fixation of C'1 by AG and AM but not by AA antibody. J. Im­ munol. 97:716-26 4. Yount, W. J., Dorner, M. M., Kunkel, H. G., Kabat, E. A. 1968. Studies on human antibodies. VI. Selective varia­ tions in subgroup composition and ge­ netic markers. J. Exp. Med. 127:63346. 5. Hsu, T. C. S., Rosenfield, R. E., Bur­ kart, D., Wong, K. Y., Kochwa, S. 1974. Vox Sang. 26:305 6. Engelfriet, C. P., Von dem Borne, A. E., Van der Geissen, M., Beckers, Do, Van Loghem, J. J. 1968. Autoimmune ha­ emolytic anaemias. I. Serological stud­ ies with pure anti-immunoglobulin rea­ gents. Clin. Exp. Immunol. 3:605-14 7. Dacie, J. V. 1962. The Haemolytic Ana­ emias, Part II, The Auto-Immune Ha­ emolytic Anaemias. New York: Grune & Stratton 8. Coombs, R. R. A., Mourant, A. E., Race, R. R. 1945. A new test for the detection of weak and "incomplete" Rh agglutinins. Brit. J. Exp. Pathol. 26:255-66 9. Rosse, W. F. 1968. Fixation of the first component of complement C'la) by hu­ man antibodies. J. Clin. Invest. 47:2430--45 10. Ishizaka, T., Ishizaka, K., Borsos, T., Rapp, H. 1966. C'I fixation by human isoagglutinins: fixation of C'1 by AG and AM but not by AA antibody. J. Im­ munol. 97:716-26 11. Borsos, T., Rapp, H. J. 1965. Comple­ ment fixation on cell surfaces by 19S and 7S antibodies. Science 150:505-6 12. Leddy, J. P., Peterson, P., Yeaw, M. A., Bakemeier, R. F. 1970. Patterns of sero­ logic specificity of human AG ery­ throcyte autoantibodies. Correlation of antibody specificity with complement­ fixing behavior. J. Immunol. 105:677-8 13. Nicholson, G. L., Masouredis, S. P., Singer, S. J. 1971. Quantitative two­ dimensional ultrastructural distribution of Rho (D) antigenic sites on human

erythrocyte membranes. Proc. Nat. Acad. Sci. USA 68:1416-20 14. Abramson, N., Gelfand, Jandl, J. H., Rosen, F. S. 1970. The interaction be­ tween human monocytes and red cells. Specificity for IgG subclasses and IgG fragments. J. Exp. Med. 132:1207-15 15. Abramson, N. Lo Buglio, A. F., Jand!, J. H., Cotran, R. S. 1970. The interac­ tion between human monocytes and red cells. Binding characteristics. J. Exp. Med. 32:1191-1206 16. Abramson, N., Schur, P. H. 1972. The IgG subclasses of red cell antibodies and relationship to monocyte binding. Blood 40:500--8 17. Lo Buglio, A. F., Cotran, R. S., Jandl, J. H. 1967. Red cells coated with im­ munoglobulin G: Binding and sphering by mononuclear cells in man. Science 158:1582-85 18. Weed, R. I., Reed, C. 1966. Membrane alteration leading to red cell destruc­ tion. Am. J. Med. 41:681-98 19. Rosse, W. F. 1971. Quantitative immunology of immune hemolytic anemia. II. The relationship of cell­ bound antibody to hemolysis and the effect of treatment. J. Clin. Invest. 50:734-43 20. Constantoulakis, M., Costea, N., Schwartz, R. S., Dameshek, W. 1963. Quantitative studies of the effect of red­ blood-cell sensitization on in vivo hemolysis. J. Clin Invest. 42:1790-1801 21. Worlledge, S. M. 1973. Immune drug­ induced hemolytic anemias. Semin. Hematol. 10:327 22. Harboe, M., Muller-Eberhard, H. J., Fudenberg, H., Polley, M. J., Mollison, P. L. 1963. Identification of the compo­ nents of complement participating in the antiglobulin reaction. Immunology 6:412-20 23. Kerr, R. 0., Cardamone, J., Dalmasso, A. P., Kaplan, M. E. 1972. Two mecha­ nisms of erythrocyte destruction in penicillin-induced hemolytic anemia. N Engl. J. Med. 287:1322-25 24. Lay, W. H., Nussenzweig, V. 1968. Receptors for complement on leuko­ cytes. J. Exp. Med. 128:991-1009 25. Gigli, I., Nelson, R. A. Jr. 1968. Com­ plement dependent immune phagocyto­ sis. I Requirements for C' l , C'4, C'2, C'3. Exp. Cell Res. 51:45-67 26. Ruddy, S., Austen, K. F. 1971. C3b in­ activator of man. II. Fragments pro­ duced by C3b inactivator cleavage of

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Annu. Rev. Med. 1975.26:331-336. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 02/02/15. For personal use only.

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ROSSE cell-bound or fluid phase C3b. J. Im­ munol. 107:742-50 Ruddy, S., Austen, K. F. 1969. C3 inac­ tivator of man. I. Hemolytic measure­ ment by the inactivation of cell-bound C3. J. Immunol. 102:533-43 Logue, G. L., Rosse, W. F., Adams, I. P. 1973. Complement-dependent im­ mune adherence measured with human granulocytes: Changes in the antigenic nature of red cell bound C3 produced by incubation in human serum. Clin. Im­ munol. Immunopathol 1:398-407 Mayer, M. M. 1972. Mechanism of cytolysis by complement. Proc. Nat. Acad. Sci. USA 69:2954-58 Kolb, W. P., Haxby, J. A., Arroyave, C. M., Miiller-Eberhard, H. J. 1972. Molecular analysis of the membrane at­ tack mechanism of complement. J. Exp. Med. 135:549-66 Schreiber, A. D., Frank, M. M. 1972.

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The autinglobulin test in autoimmune hemolytic anemia.

The foregoing summarizes what can be learned from a carefully performed antiglobulin test using specific antisera. Clinical syndromes can be more easi...
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