QUANTIFICATION OF IGG ANTI-D BOUND TO D-POSITIVE RED-CELLS INFUSED INTO D-NEGATIVE SUBJECTS AFTER INTRAMUSCULAR INJECTION OF MONOCLONAL ANTI-D

A flow-cytometric method was developed to determine the number of mole cules of IgG bound to D-positive red blood cells (RBC) when sensitized with low plasma concentrations of IgG anti-D in the presence of an ex cess of D-negative RBC. D-positive RBC were infused into 12 D-negative male volunteers 2 days after intramuscular injection of monoclonal an ti-D (BRAD-3, IgG3 or BRAD-5, IgG1). Blood samples were taken immediat ely before, 3 min and 3 h after injection of the RBC, incubated for 1 h at 37 degrees C, washed, then incubated sequentially with FITC-conju gated anti-IgG, IgM monoclonal anti-D, biotin-conjugated anti-IgM, and R-phycoerythrin-conjugated streptavidin. To prepare a standard curve, O R(1)R(2) RBC were incubated in duplicate with varying dilutions of BRAD-3 or BRAD-5, and RBC from one set were mixed with an excess of D- negative RBC (1:100) and labelled as above, while cell-bound IgG on th e other set was quantified by ELISA. The test samples and standards we re analysed by flow cytometry and the mean channel (FITC) fluorescence was plotted against molecules IgG/cell, from which the sensitization level of D-positive RBC in the test samples was determined. The use of IgM anti-D enhanced the discrimination between D-positive and D-negat ive RBC, especially when fewer than about 3000 molecules IgG/cell were bound. The assay was sensitive to about 1000 molecules IgG/cell. The sensitization levels of the D-positive RBC in samples taken 3 h after injection were found to be in the range 1500-10,000 molecules IgG anti -D per cell.