Quantitative flow cytometric analysis of ABO red cell antigens

Abstract

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluoroscein‐conjugated anti‐H lectin and the A and B antigens by indirect staining first with monoclonal anti‐A or anti‐B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti‐mouse immunoglobulin (Ig) antibodies. More than a ten‐fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of >200 and >30, respectively, while heterozygotes (AO or BO) had ratios of <5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti‐A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population. These results suggest that in addition to a specific gene dosage effect, other factors (e.g., aging of the RBC) may affect the expression of all these antigens in a coordinate way. Copyright © 1991 Wiley‐Liss, Inc.