QUANTITATIVE STUDIES OF THE EFFECT OF RED-BLOOD- CELL SENSITIZATION ON IN VIVO HEMOLYSIS M. Constantoulakis, … , R. S. Schwartz, W. Dameshek J Clin Invest. 1963;42(11):1790-1801. https://doi.org/10.1172/JCI104864. Research Article Find the latest version: https://jci.me/104864/pdf Journal of Clinical Investigation Vol. 42, No. 11, 1963 QUANTITATIVE STUDIES OF THE EFFECT OF RED-BLOOD-CELL SENSITIZATION ON IN VIVO HEMOLYSIS * By M. CONSTANTOULAKIS,t N. COSTEA,4 R. S. SCHWARTZ, AND W. DAMESHEK (From the Blood Research Laboratory, Pratt Clinic-New England Center Hospital, and the Department of Medicine, Tufts University School of Medicine, Boston, Mass.) (Submitted for publication January 22, 1963; accepted July 29, 1963) Although a positive antiglobulin reaction is a butes, both isoantibodies and autoantibodies had characteristic and distinctive feature of autoim- qualitatively distinctive effects on red-cell survival. mune hemolytic anemia (AIHA), the precise role of the erythrocyte-sensitizing globulin in the MATERIALS AND METHODS pathogenesis of this disease is poorly understood. Patients. Seventeen patients with AIHA of the Earlier studies have suggested that the severity "warm" antibody variety were studied; 11 were "idio- of hemolysis in AIHA is a function of the quan- pathic," and the rest had systemic lupus erythematosus tity of globulin adherent to the red blood cells (2), chronic lymphocytic leukemia (3), or Hodgkin's (1); direct estimation of the amount of disease (1). Characteristics of the individual cases are however, shown in Table I. autoantibody was possible in only a few cases. Isoantibodies. The anti-D and anti-Kell sera were of There is, in addition, a lack of quantitative data the incomplete, noncomplement-binding variety.' concerning the effects of isoantibodies on erythro- Eluates were prepared by Fudenberg, Barry, and cyte survival. In the experiments described in Dameshek's modification (1) of the Kidd (3) method this paper, the relationships between red-cell sen- and were stored at - 200 C until used. Each of the eluates was tested serologically and physicochemically sitization, by either auto- or isoantibodies, and in as follows. a) Serology. Eluates were titrated by the vivo hemolysis were examined through the tech- standard indirect antiglobulin technique (4), in which a niques of Cr51-labeled red-cell survival and the constant pool of red cells 2 (Hemantigen) and commer- radioactive antiglobulin (RAG) test (2). In cial antiglobulin serum 3 were used. The tests were car- this test, rabbit antihuman globulin labeled with ried out at 40, 220, and 370 C. In some instances, fresh human serum was added as a source of complement. radioactive iodine is used. After appropriate in- The serologic specificity of the eluates was determined cubation and washing, the amount of rabbit anti- by the indirect antiglobulin reaction using a panel of red human globulin reacting with the sensitized red cells of known antigenic compositions (Panocell). The cells may be ascertained from the specific radio- antigens present in this panel were: C, CW, D, E, V, c, e, activity of these cells. The amount of human f, K, k, Fya, Fyb, JOa, Le', Leb, Jka, Jkb, M, N, S s, P. and Lua. Normal red cells (Hemantigen) incubated antibody sensitizing the red blood cells is calcu- with the eluates were also tested for sensitization by the lated from the combining ratio between the rab- standard antiglobulin technique with specific goat anti- bit antibody and the erythrocyte-bound human human 7S and antihuman 19S sera.5 b) Physicochemical gamma globulin. When the human antibody was studies. Electrophoresis by standard techniques on pa- per and cellulose-acetate supporting media were car- of the gamma2 globulin variety, this ratio was ried out on a Durrum-type cell. Protein concentrations found to be 7 to 1, i.e., 7 molecules of rabbit anti- were determined by the method of Lowry, Rosebrough, globulin reacted with 1 molecule of red-cell-bound Farr, and Randall (5). A standard curve, utilizing a antibody. The results of these experiments indi- 1 Kindly provided by the Ortho-Research Foundation, cated that, irrespective of their serologic attri- Raritan, N. J., and the Blood Grouping Laboratory, Bos- ton, Mass. * Aided by American Cancer Society grant T-243 and 2 Hemantigen, Knickerbocker, New York, N. Y. U. S. Public Health Service grant E-3091. 3 Broad spectrum, Ortho-Research Foundation, Raritan, t Present address: 2 Ersis Street, Athens 7, Greece. N. J. t Present address: West Side VA Hospital, South 4Panocell, Knickerbocker, New York, N. Y. Damen Avenue, Chicago 12, Ill. 5 Hyland Laboratories, Los Angeles, Cal. 1790 RED-CELL SENSITIZATION AND IN VIVO HEMOLYSIS 1791 TABLE I Some clinical and laboratory features in 17 patients with AIHA * Antibody Cr5' Hemo- Reticui- Coombs N/RBCj survival Clinical Patient Diagnosis globin locytes Bilirubin ESRt test (RAG) ti Treatment assessment mg/ g/100 ml % 100 ml mm/hour jg/rnl days V.A. CLL§ 8.4 12.1 1.1 4 + 5.0 16.5 S11 + + R.P. Idiopathic 9.6 7.8 1.9 65 4+ 1.6 6.4 A + E.L. CLL 6.4 0.2 1.5 122 4+ 6.8 11.8 S ++++ M.H. Idiopathic 12.4 0.6 0.5 2 + 0.23 25.0 0 0 G.B. Idiopathic 9.1 7.0 0.9 110 4+ 1.0 5.7 S + + C.B. Idiopathic 14.9 3.2 1.8 2 4+ 0.45 6.0 A + Y.N. Idiopathic 6.7 10.4 3.4 45 4+ 3.46 4.3 A +++ A.P. Idiopathic 13.4 1.9 10 3 + 0.73 19.5 A + D.E. SLE** 10.7 4.0 10 4+ 1.1 9.5 A + D.C. Hodgkin's 10.9 6.4 45 2 + 0.25 14.5 HN2 + L.B. Idiopathic 12.6 14.0 32 1 + 0.53 7.5 S 0 D.S. Idiopathic 10.6 12.6 2.3 3 4+ 0.8 12.0 A/S +++ J.S. Idiopathic 13.5 5.2 0.5 28 4 + 2.9 10.2 5S E.S. Idiopathic 10.6 4 + 2.54 12.0 A -4 13.0 0.6 0.8 8 3 + 0.58 23.5 S Splenectomy 0 M.B. CLL 6.3 21.5 3.6 4 + 1.43 4.5 S +++ H.C. Idiopathic 9.4 29.0 1.8 75 3 + 0.4 3.5 S +++ C.C. SLE 14.0 3.4 16 4+ 1.7 14.5 S + * The clinical assessment was arbitrarily judged to range from complete remission (0) to severe illness (+ + + +). AIHA = autoimmune hemolytic anemia. t ESR = erythrocyte sedimentation rate. $ N = nitrogen; RBC = red blood cells; RAG = radioactive antiglobulin. CLL = chronic lymphocytic leukemia. llS = steroids. ¶ A = antimetabolites. ** SLE = systemic lupus erythematosus. solution in which the protein-nitrogen concentration Determiniation of the combbining ratio of RAG with was determined by the micro-Kjeldahl technique, was the red-cell antibodies. The globulin fractions of po- prepared for each new batch of Lowry reagents. Im- tent anti-D and anti-K sera obtained from individual munodiffusion in agar plates was performed with a donors were dissolved in borate buffer, pH 8. Immuno- modification of the Ouchterlony method (6). Specific electrophoretic analysis of these fractions showed only antihuman 7S and antihuman 19S globulins sera of goat one component, gamma2 globulin. These preparations origin were employed. Since some eluates were slightly were labeled with I"'5 by the method of Helmkamp and contaminated with free hemoglobin, each gel-diffusion his associates (9). In some experiments, the anti-D sera test was controlled for the possible precipitation of were treated by absorption on thoroughly washed Rh- methemalbumin in the agar medium by allowing the positive red cells and subsequent elution by Fudenberg eluate to react against normal human serum in a sepa- and his co-workers' (1) modification of the Kidd (3) rate agar plate. In no instance was a precipitin line technique. Other experiments showed that this pre- observed in these control tests. Eluates were next in- liminary isolation of the antibody did not affect the re- cubated with equal volumes of 0.2 M mercaptoethanol sults, and later studies dealing with the combining ratio (7, 8) at room temperature for 48 hours in tightly were performed directly with the labeled globulin frac- sealed tubes, then dialyzed in the cold against 20 L of tions. The radioactive antibody solutions containing 6% phosphate buffer (pH 7.2) containing 0.02 M iodoacetate. bovine or human serum albumin, added to minimize utake Controls in which saline was substituted for 2-mercap- of nonspecific proteins (2, 10), were incubated with 0.1- toethanol were run simultaneously. As an additional ml portions of packed red cells. After 15 minutes of control, a serum containing a high titer of cold ag- incubation at 370 C, the cells were washed in chilled 0.15 glutinin was treated in a similar manner and then tested MA saline until the radioactivity of the red-cell button for cold agglutinin activity. Indirect antiglobulin tests remained constant. All washing procedures were car- were carried out, as described above, on the eluates ried out in a refrigerated centrifuge. The amount of treated in this manner. antibody protein attached to the red-cell surface was RAG tests (2) were performed on red cells from pa- then determined from the SA of the antibody solution. tients with AIHA immediately before the red-cell sur- After this, the cells were incubated with RAG labeled vivals were started and on the artificially sensitized nor- with I131 for 30 minutes at 37° C. The cells were next mal erythrocytes just before the Cr" labeling procedure.