Background/Case Studies: Some individuals carry a very low expression of the D-antigen, called the Del phenotype. Red cell units from blood donors with DEL alleles are RhD protein-positive, despite being routinely labelled D-negative. These units have been shown to cause both primary and secondary anti-D alloimmunization when transfused to patients lacking the RHD gene. In the U.S. and Europe, D-antigen typing in blood donors is performed using automated systems designed to detect most weak D samples. In Europe, serologic D-negative first-time donors are confirmed using the indirect antiglobulin test (IAT). However, in some European countries, however, the IAT has been replaced by the more sensitive method of molecular RHD testing. In the U.S., an IAT is generally not used for confirming serologic D-negative donors. The objective of this study was to screen the serologic D-negative, multiethnic donor population at the NIH Clinical Center for the presence of the RHD gene.
Study
Design/Methods: The study included 16,589 routine blood donors at the NIH Blood Center between November 2009 and October 2024. D-antigen typing of donors was outsourced to Creative Testing Solutions (CTS). D-negative donor samples were screened individually using 3 real-time PCR assays targeting RHD specific DNA sequences in intron 4, exon 5, and exon 7. The real-time PCR assays were performed in separate reaction tubes (modular) and in triplicates. D-negative donors that had at least one positive real-time PCR result for the detection of the RHD gene underwent additional testing to identify RHD gene variants using commercial genotyping kits or nucleotide sequencing.
Results/Findings: Over 15 years, 2,254 D-negative donors were individually tested for the RHD gene. With a sensitivity of detecting 5 RHD positive gDNA copies per reaction, 42 donors tested positive (1.9%). Among them, 34 carried the common RHDΨ allele (80.9%), 7 harbored 5 known RHD alleles, and 1 had a novel RHD deletion. Additionally, we inadvertently detected 2 other donors with DVI, for a population frequency of 1 in 731. Conclusions: A modular approach for RHD screening is suitable for blood donors when sample pooling is not feasible among multiethnic donor populations. Since 2009, we have transitioned donors from serologic D-negative to molecularly RHD-negative status at the NIH Clinical Center. In December 2018, the U.S. Food and Drug Administration approved a supplement to our Biologics License Application to label red blood cells from DEL phenotype donors as Rh positive. Molecular RHD screening of serologic D-negative donors is an effective way to identify individuals harboring DEL alleles, which can cause alloimmunization in transfusion recipients.
