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

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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 (× 106/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 (× 106/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.

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