New York Blood Center Enterprises, Community Blood Center of KC, United States
Background/Case Studies: RHD genotyping is often performed to detect variant alleles and in samples with altered or unexpected serology. Variant alleles may be suspected in cases of variable D typing or anti-D formation in D+ individuals. There are currently over 700 reported RHD alleles, many encoding partial, weak, or null phenotypes. Characterizing RHD alleles that encode partial D is important to prevent alloimmunization. Commercial genotyping assays are constrained by both the number of targets they interrogate and their allele-calling algorithms, which may lead to miscalls even when variants are detected. We investigated a sample in a D+ white female with apparent anti-D in her plasma. We report a novel RHD allele, encoding partial D phenotype, that had incomplete characterization by a commercial SNP-based genotyping assay.
Study
Design/Methods: Serologic testing was performed by standard hemagglutination in tube or by manual gel column agglutination testing (CAT, Ortho). RhD typing was performed in tube using Gamma-clone and Series 4 and 5 (Werfen), Bioclone (Ortho), Seraclone (Bio-Rad), and Albaclone (Alba). Antibody identification was performed by CAT or PEG IAT. Genomic DNA was isolated from leukocytes (Qiagen). Genotyping included RHD zygosity by the hybrid Rhesus box assay, RHD BeadChip (Werfen), Sanger sequencing of RHD exon 5, and next-generation sequencing (NGS) (Illumina) of RH genes.
Results/Findings: The patient was group A, D+ and DAT negative. The patient’s plasma contained anti-D by PEG IAT and CAT; autocontrol tests were negative. RBCs reacted strongly (4+) with all Anti-D reagents at immediate spin. Anti-LW was excluded based on reactivity with D+ and D– cord cells. Hybrid Rhesus box assay revealed the patient was RHD hemizygous. RHD Beadchip reported RHD*DUC2(variant c.733C); however, NGS revealedc.733C and c.712A variants. Sanger sequencing confirmed c.712A and c.733C with no other changes detected. See Table 1 for a summary of results. Conclusions: We report a novel allele RHD*712A, 733C, which likely encodes a partial D phenotype based on the apparent alloanti-D and the partial D status of numerous RHD alleles with c.712A and c.733C. This case displays the limitations of allele-calling algorithms in SNP-based arrays and the advantages of using sequencing-based RHD genotyping, while contributing to our evolving understanding of the diversity of the RH blood group system.
