Rhesus D Variants in Pregnant Women and Donor Blood: Implications for Anti-D Alloimmunization

Abstract

Background: Blood grouping systems with most diversity is the rhesus (Rh) system. About 56 rhesus antigens have been specified so far with five of them (D, C, E, c, and e) being the most clinically significant because of their involvement in haemolytic transfusion reactions (HTRs). Chromosome one houses a pair of closely linked genes, the Rh D gene, responsible for encoding the D antigen, and the Rh CE gene, which encodes the C/c and E/e antigens. The main cause of haemolytic reactions is the immunogenic D antigen. Certain variants, usually result from a single nucleotide polymorphism that affects antigen membrane integration resulting in reduced antigen expression (weak D) or alter the formation of the antigen (partial D). Molecular classification is therefore crucial to prevent discrepancies and inaccuracies from commercial reagents. Aim: The study objective was to identify RHD variants present in pregnant women and donors to evaluate the risk of haemolytic disease of the foetus and newborn and other haemolytic reactions due to alloimmunization. Method: A cross-sectional study was employed involving pregnant women and blood donors in St. Gregory Catholic Hospital. Serotyping was carried out for RHD and RHCE phenotypes using monoclonal and polyclonal antibodies using Coombs reagents, an indirect antiglobulin test verified weak D expression. In an initial multiplex PCR reaction, amplification was performed on two regions: exon 7, which shows homology to both RHCE and RHD, and exon 10, which is unique to the D sequence. This was followed by six sets of Rh D specific primers in a second multiplex PCR to amplify Rh D exons 3, 4, 5, 6, 7, and 9 to identify the variations for RHD genotyping. Results: The study analysed the Rh D and Rh CE phenotypes of 100 participants: 72 pregnant women and 28 blood donors. Serological testing revealed that 78% participants were Rh D positive, 20% Rh D negative, and 2% weak D. Molecular analysis confirmed the Rh D-positive genotype in 94.9% of serologically positive samples. However, discrepancies were noted, with some samples displaying amplification of RHCE but not RHD genes (false negatives). The weak D phenotypes detected in 2% of participants showed amplification of both exon 7 and exon 10 of the RHD gene. Conclusion: Although no evidence of alloimmunization was found among participants, the detection of weak D phenotypes highlighted the importance of precise Rh D typing to minimize alloimmunization risks. Discrepancies between serological and molecular methods, including those due to gene dosage effects, emphasize the need for optimized molecular assays to improve Rh D variant detection and prevent clinical misclassifications in transfusion and obstetric care.