Flow Cytometric Analysis of Erythrocytic D Antigen Density Profile

Red blood cells (RBC) were studied with flow cytometry (FCM) after staining with a directly fluoresceinated anti-D antiserum. The immunofluorescence distribution profiles were slightly asymmetrical, due to a significant, yet small skewness. This skewness was not associated specifically with D antigen positivity, making a homogeneous, normal D antigen distribution on the RBC very likely. With the exception of Du, D-positive RBC could always be distinguished from D-negative ones. FCM was successful in bringing the following phenomena to light: difference in mean fluorescence intensity between the different rhesus phenotypes, the D gene dosage effect, the depressing effect of C antigen on D antigenicity, the nonspecific attachment of IgG on (D-negative) RBC and rhesus mosaicism. The absolute number of D antigen sites could be calculated, using an absolute fluorescence standard.