A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression

Abstract

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 of RHD zygosity 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. RHD zygosity 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 with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (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.

Figure 1

Identification of RHD gene deletion. Panel A (adapted from Ref. 27) illustrates the RHD gene locus on chromosome 1, its proximity to the RHCE gene, and the presence of flanking upstream and downstream rhesus boxes. Most D-negative haplotypes (particularly among Caucasians) are due to a deletion of the RHD gene, leading to the formation of the “hybrid rhesus box [22].” Panel B illustrates the distinct banding pattern obtained with PCR amplification of the upstream and hybrid boxes (using primers rez7 and rnb31) and subsequent digestion with the PstI restriction enzyme. Amplification of the downstream Rhesus box alone (indicating DD homozygosity) results in three DNA fragments of 1,888, 746, and 397 bp. The hybrid Rhesus box (present in D-negative individuals) has an additional PstI site and results in fragments of 1,888, 567, 397, and 179 bp. Heterozygous Dd individuals have one allele expressing the RHD gene and one allele lacking the RHD gene (and hence expressing the hybrid Rhesus box) and thereby has fragments of 1,888, 746, 567, 397, and 179 bp.

Figure 2

Quantitation of erythrocyte D antigen expression by flow cytometry. Panel A demonstrates clustering of RBC, allowing for easy gating. The subsequent panels demonstrate clearly distinguishable fluorescence intensities of typical dd (panel B), Dd (Panel C), and DD (Panel D) individuals. FL-1 geometric mean fluorescence was used in conjunction with the calibration curve to calculate the number of D antigen sites per RBC, as described in METHODS.

Figure 3

Erythrocyte D antigen expression and RHD Zygosity. The Y-axis represents number of D antigen sites per RBC as determined by quantitative flow cytometry, as described in METHODS. On average, homozygous DD individuals expressed nearly double the number of D antigen sites than heterozygous Dd individuals (DD mean antigen sites 5 33,560 ± 8,222, median 5 32,720; Dd mean antigen sites 5 17,720 ± 4,471, median 5 16,970).