Frequency analysis of B lymphocytes specific for Rh antigens in naturally immunized Rh-negative women

Background and Objectives Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rhalloimmunized individuals at different time intervals after parturition. Materials and Methods The donors' B lymphocytes were transformed with Epstein-Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19(+) B lymphocytes in peripheral blood wets determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). Results The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27 850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rhspecific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. Conclusions The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals.