Flow Cytometric Analysis of Erythrocytic D Antigen Density Profile

Abstract
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.