Studies of in vitro red cell autoantibody production in normal donors and in patients with autoimmune hemolytic anemia


3Director of Transfusion Medicine, UCLA Medical Center, 10833 Le Conte Avenue, Los Angeles, CA 90024–1713.


The in vitro production of red cell autoantibodies (RBC AuAbs) has been investigated for better understanding of the pathogenesis of autoimmune hemolytic anemia (AIHA). Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured for 14 days with or without added pokeweed mitogen (PWM), autologous RBCs, methyldopa, procainamide, and alpha-methylnorepinephrine. Also, isolated B cells were infected with Epstein-Barr virus (EBV) to produce polyclonal B-cell lines. Supernatants were tested for IgG and IgM RBC AuAbs by use of 125I-staphylococcal protein A (SPA). RBC AuAbs were detected in PBMNC cultures without additives to the culture medium of four of eight patients who had warm-antibody AIHA or a positive direct antiglobulin test (DAT) without hemolytic anemia. In two of these patients, RBC AuAb production was augmented by the addition of PWM, and in two additional patients, RBC AuAbs were detected only after the addition of PWM. Supernatants from PBMNC cultures from three of four normal donors produced RBC AuAbs independent of the presence of PWM; in two of these subjects, PWM augmented production of RBC AuAbs. PBMNC cultures from three DAT-negative patients with systemic lupus erythematosus produced RBC AuAbs, one in the presence of PWM and two in its absence. With one exception, there was no augmentation of AuAb production by the addition to the culture system of autologous RBCs or drugs. EBV infection of B cells from four patients with AIHA and four normal persons yielded B-cell lines secreting RBC AuAbs. The quantity of RBC AuAb after a 24-hour culture of EBV-transformed B cells was significantly greater in cultures from four patients who had AIHA than in cultures from four normal persons. It is concluded that in vitro cultures of lymphocytes can be used to study the production of RBC AuAbs. The presence of RBC AuAbs in supernatants of normal lymphocyte cultures and their frequent enhancement by a polyclonal activator suggest that the development of pathogenic concentrations of RBC AuAbs may depend on polyclonal stimulation of lymphocytes that are programmed to produce RBC AuAbs.