Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165

Abstract

AbstractBackgroundAn original methodology for determining the D antigen density on red cells was published in 2000 and has been applied in many publications since. This flow cytometry‐based assay remained largely unrevised utilizing monoclonal anti‐Ds that are not readily available anymore. We updated the methodology to quantify erythrocyte D antigen sites using microspheres and monoclonal anti‐Ds that are commercially available today.MethodsThe absolute D antigen density of a frozen standard CcDEe cell, drawn in 2003, a fresh blood donation from the same individual, drawn in 2022, and an internal control CcDEe cell, was quantified by flow cytometry using fluorescence‐labeled microspheres. The internal control CcDEe cell was used in conjunction with 9 commercial anti‐Ds to determine D antigen densities of 7 normal D, 4 partial D, and 11 weak D type samples, including 2 novel alleles.ResultsThe reproducibility of the updated assay was evaluated with red cells of published D antigen densities. The current results matched the known ones closely. The new weak D types 164 and 165 carried 4500 and 1505 D antigens/red cell, respectively. The absolute D antigen density decreased from 27,231 to 26,037 in an individual over 19 years.DiscussionThe updated assay gave highly reproducible results for the D antigen densities of Rh phenotypes. Readily available anti‐Ds allowed for the determination of the D antigen densities of 7 weak D types. The assay is suitable to evaluate the effects of distinct amino acid substitutions on the RhD phenotype.