Quantitation of Rh D antigen sites on weak D and D variant red cells by flow cytometry

Information on the number of D sites on weak D (D-u) and D variant cells is limited and incomplete. The aim of this study was to use a simple non-isotopic technique utilising a combination of flow cytometry and ELISA to quantitate the number of D sites on an extensive range of these cells. Five human monoclonal IgG anti-D (BRAD-7 (JAC10), BRAD-5, 2B6, BRAD-3, H27) and one affinity-purified polyclonal IgG anti-D were each used at a saturating concentration of 20 mu g/ml. In general, BRAD-3, BRAD-5 and 2B6 gave the highest number of D sites per cell (SPC), H27 and the polyclonal anti-D were slightly lower, while BRAD-7 gave the lowest SPC with all D-positive cells tested except DFR. Interestingly, BRAD-7 gave the highest SPC with DFR cells. Rh D antigen density for R(2)R(2) cells was approximately double that seen with either R(2)r Or R(o) (presumed R(o)r) cells. R(1)R(1) cells gave only moderately higher SPC than R(1)r cells. Higher SPC were obtained with the R(1) haplotype if the C-W antigen was present. Weak D, Va and VI cells of the R(1) haplotype had higher SPC than those of the R(0) or R(2) haplotypes. The majority of D variant cells were found to have lower SPC than normal cells, and for polyclonal anti-D, which was the only anti-D to react with all D variants, SPC decreased in the order IIIc>IIIa>HMii>IVa>Va>DFR>DBT>IVb>VII>II>HMi>VI. The number of molecules of IgG anti-D bound to D variant cells varied by up to 10 times with reactive monoclonal antibodies. The highest SPC on weak D and D variant cells were obtained with BRAD-7 (DFR, 9,500), BRAD-3 (Va, 12,500), BRAD-5 (II, 5,500; VII, 5,300; weak D, 1,300), H27 (III, 24,000; VI, 2,900; HMi, 3,000; HMii, 16,800) and polyclonal anti-D (IVa, 9,300; IVb, 4,000; DBT, 4,300).