Quantitation of Rh D Antigen Sites on Weak D and D Variant Red Cells by Flow Cytometry

Abstract

Information on the number of D sites on weak D (Du) 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 micrograms/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 R2R2 cells was approximately double that seen with either R2r or Rzero (presumed R0r) cells. R1R1 cells gave only moderately higher SPC than R1r cells.Higher SPC were obtained with the R1 haplotype if the CW antigen was present. Weak D, Va and VI cells of the R1 haplotype had higher SPC than those of the Rzero or R2 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).

PCR screening for common weak D types shows different distributions in three Central European populations

“…Its epitope distribution lacking epitopes 4, 11, and 31 did not resemble D HR, which lacks epitopes 1, 2, 12, and 20. The low antigen density of both D HO (1300 D antigens/cell) and D HR (3800 D antigens/cell 12 ) might have confounded epitope determination. However, it should be noted that the nonconservative amino acid substitution in D HO (lysine eliminated) differs from the conservative one in D HR.…”

Molecular biology of Rh proteins and relevance to molecular medicine

“…45), and therefore it is important to distinguish weak D from partial D. The working definition of a weak D versus a partial D phenotype is that individuals with the former do not produce antibodies when exposed to D-positive erythrocytes by either transfusion or pregnancy, unlike partial D individuals. 45), and therefore it is important to distinguish weak D from partial D. The working definition of a weak D versus a partial D phenotype is that individuals with the former do not produce antibodies when exposed to D-positive erythrocytes by either transfusion or pregnancy, unlike partial D individuals.…”

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry

“…(1999) suggested that the routine administration in the UK of a 500 IU dose of prophylactic anti‐D postpartum was probably sufficient to prevent anti‐D immunization when the FMH is much greater than 4 ml of weak D fetal red cells. As there are at least nine times more RhD sites on RhD‐positive red cells compared with weak D red cells (Jones et al. , 1996; Wagner et al.…”

“…, 1995). With the introduction of high avidity monoclonal anti‐D reagents, such as RUM‐1, many individuals given D u status may now simply be regarded as RhD positive because of complete reactivity (Jones et al. , 1996).…”