By sensitizing human red blood cells with antisera specific for corresponding blood group antigens and subsequent rosette formation, we were able to distinguish homozygosity of the antigens from heterozygosity. Three types of rosette assays were utilized: a protein A rosette, an immunoglobulin-coated bead rosette, and an erythrocyte-antibody rosette with human lymphocytes. In the Rh blood group system, we were able to demonstrate the dosage effect of the Rho(D) antigen by rosette assay in addition to determining that the deletion of the C and/or E alleles increased the rosette forming cell count when red blood cells with different genotypes were sensitized with Rh immune globulin (anti-D). The assay proved to be reproducible in distinguishing between homozygous and heterozygous Rh red blood cell antigens, and may be adaptable to study many antigenic markers on cell surfaces.

Several methods were compared for quantitating the B lymphocytes in human peripheral blood. RBC rosette assays including sheep and bovine red blood cells, and immunobead rosette assays including beads coated with anti-kappalambda and trivalent anti-GAM antibodies respectively were investigated. The anti-kappalambda bead assay proved to be simpler, faster, higher in reproducibility, and appeared to be more accurate than any of the other assays.

Utilizing an EA rosette assay, we were able to distinguish genotypic differences of the Rho (D) antigen on human red blood cells (RBC) based on the number of rosettes formed between human peripheral B lymphocytes and RBC optimally sensitized with anti-Rho (D) antibody. In this study, RBC with possible Rh genotypes R1R1 (CDe/CDe), R1r (CDe/cde), R2R2 (cDE/cDE), R2r (cDE/cde) and R1R2 (cDe/cDE) were sensitized with either RhoGAM (anti-D) or Ripley serum (anti-G). When RBC were optimally sensitized with RhoGAM, R1r formed 60% as many rosettes as R1R1 and R2r formed 64% as many rosettes as did R2R2. Using Ripley serum for sensitization and comparing the number of rosettes formed, R1r exhibited 70% of R1R1 and R2r showed 76% R2R2. Furthermore, R1R1 cells sensitized with Ripley serum formed a higher percentage of rosettes than R1R2 or R2R2.

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