Frequency determination of RhD specific B-lymphocytes in RhD antagonism

Immunoprophylaxis with Rh‐D immunoglobulin has been proven effective for the prevention of haemolytic disease of the newborn (HDN) in > 90% of cases. At present, anti RhD immunoglobulin is prepared from plasma of volunteer RhD‐donors who are immunized with RhD+ erythrocytes, while human monoclonal RhD antibodies (RhD‐HuMAbs) are in development. Selection of donors is entirely based on the progression of plasma RhD antibody titre during the course of repeated boosting. Donor selection would improve if a predictive test were available for estimating the number of B‐lymphocytes involved in RhD antibody secretion. We have developed a B cell precursor assay (BCPF) for specific antibody producing cells. Immunomagnetically isolated CD19+ B‐cells are cultured for 10 days in limiting dilution format and supernatants are tested for the presence of agglutinating antibodies. We validated this method using B‐cells from women immunized by pregnancy or deliberate immunization. RhD specific BCPF values were found to range from 42 to 16000 per 10 6 B‐cells in immunized women. We are currently correlating serum RhD antibody agglutination titres with RhD‐BCPF titres. This method will be useful for: (i) prediction of donors who will or will not develop high titreed RhD antibodies upon boosting; (ii) evaluation of the efficiency of immunoprophylaxis in the presence of passive antibodies; (iii) evaluation of the downregulating effects of immunosuppressive drugs or agents on B‐cells; (iv) assessing donor suitability for RhD‐HuMAb hybridoma development.