Flow‐Cytometric Analysis of Erythrocytic Blood Group A Antigen Density Profile

Abstract

Blood group A antigen density on red blood cells (RBC) was studied using flow cytometry (FCM) and fluoresceinated polyclonal and monoclonal IgG anti-A antisera. Agglutination was a problem, which could only be solved by prefixation of the RBC with glutaraldehyde or formaldehyde. However, this treatment resulted in a significant reduction of the number of antigen sites as compared to the native (i.e. nonfixed) RBC. Two major new findings came out of this study: (1) A antigen density on native RBC seems to be higher than previously recognized, and (2) A antigen density distribution is probably non-Gaussian. The absolute number of A antigen sites was determined, using a human polyclonal IgG antiserum and commercially available absolute fluorescence standards. The site numbers on fixed RBC were comparable to those found by earlier radioimmunological studies (x 10(6)/RBC): A1, 1.07 +/- 0.28; A2, 0.21 +/- 0.09; A1B, 0.79 +/- 0.26 sites (mean +/- SD). The values found for native RBC were considerably higher (x 10(6)/RBC): A1, 2.86 +/- 0.95; A2, 0.47 +/- 0.29; A1B, 1.98 +/- 0.58 sites (mean +/- SD). With the 1 monoclonal and the 3 polyclonal antisera used in this study, and in contrast to Rh D, the erythrocytic A antigen density distribution of a given sample is highly asymmetrical. This non-Gaussian distribution profile does not seem to be affected by such factors as antibody heterogeneity, variability in antibody fluoresceination range, RBC density and reticulocyte content. This suggests that the asymmetrical A antigen distribution may be an intrinsic property of the RBC population.