#OA3-AM25-ST-21 - Protein Variance Affects the Highly Immunogenic Rh Complex in the Red Cell Membrane: A Flow Cytometry Analysis

Rh is a highly polymorphic blood group system critical to transfusion medicine. Within the Rh complex, there are antigen presenting proteins RhD, RhCE, and RhAG. However, the structural composition of these proteins and their antigens has not been fully studied in most Rh phenotypes. Quantification using flow cytometry has become an important tool to measure antigen densities (number of antigens on the red cell surface). In 2023, a previously published flow cytometry method had been revised to allow for both absolute and relative quantification of RhD antigen. Expanding on this method, we aimed to quantify both RhCE and RhAG antigens in common Rh phenotypes.

Study

Design/Methods:

The absolute number of RhAG antigens per red cell was measured using the allophycocyanin (APC)-labeled monoclonal antibody LA1818 and Quantum Simply Cellular beads. For the relative quantification of RhCE and RhAG antigens, we used 9 monoclonal antibodies targeting RhC (MS-24, MS-273), Rhc (MS-42, H48) RhE (DEM-1, C2), Rhe (MS-63/P3GD512, MS-16/MS-21/MS-63) and RhAG (LA1818). We tested samples representing the common Rh phenotypes R₁R₂, R₁R₁, R₂R₂, R₀r, and rr. Because RhCE antigens could not be quantified absolutely, we determined the expression in Rh phenotypes relative to an R₁R₂ cell as reference.

Results/Findings:

We tested 10 samples with distinct Rh phenotypes. The absolute RhAG antigen density of the reference R₁R₂ cell was determined to be 102,000 (Table 1). Antigen expression varied for RhAG, RhCE, and RhD antigens across different Rh phenotypes. Notably, the 2 R₁R₁ phenotypes showed considerable variation in RhAG antigen expression (87,636 and 140,621). The mean of the RhAG antigen densities was 100,234 ± 18,069 (standard deviation, n=10). Red cells with homozygous RhCE alleles expressed higher antigen densities than those with heterozygous RhCE alleles. In red cells with the RhD-negative rr phenotype, RhAG antigen expression remained 100%-105%, while RhCE antigen expression may have increased to 123%-149%.

Conclusions: The revised assay enabled quantification of RhAG and RhCE antigens on the red cell membrane. RhAG antigen densities corresponded with 5 published D+ and D- samples. Further, RhCE expression varied among the common red cells. RhCE and RhD proteins are influenced by genetic variation, such variation likely contributed to the observed differences in expression within the Rh complex. Applying this method to additional Rh variants will further clarify the structural organization of the Rh complex. Using quantitative flow cytometry to measure the absolute number of RhCE antigens on the red cell surface will determine the specific antigen densities of RhCE proteins in the Rh complex.