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D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context ofRHDzygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens.RHDzygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density withRHDzygosity and expression of Rh CcEe antigens. Subjects homozygous forRHDexpressed nearly twice the number of D antigen sites compared withRHDhemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd,P< 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.