Analysis of RHD variant alleles in serologically D-negative Thai patients: Prevalence and novel discoveries

D-negative blood donors are rare (0.3%) in Thailand. Patients with Asian DEL are D-seronegative but can receive D-positive blood without anti-D alloimmunization. To improve blood management, this study aimed to determine screening methods for detecting RHD variant alleles in serologic D-negative Thai patients.

Serologic D-negative blood samples were subjected to adsorption/elution for the DEL phenotype. The Hybrid Rhesus box, RHD exon 4, and RHD1227A were analyzed using polymerase chain reaction (PCR) and Sanger sequencing for RHD1227A. For inconclusive results, whole genome sequencing (WGS) was conducted. Genetic variants on RHD and RHCE genes were confirmed using deletion-spanning PCR and Sanger sequencing.

Among 80 patients, 57 (71.3%) cases of total RHD gene deletion, 20 (25.0%) of Asian DEL hemizygosity, two (2.5%) of novel genetic variants, and one (1.2%) with an inconclusive result were identified. Two patients had a novel RHD exon 3 frameshift variant, c.441delG p.V147fs, producing a truncated protein. Serology of all patients with novel variants showed D-negative. The adsorption/elution testing showed 34.8% false positive and 9.4% false negative rates for Asian DEL.

Our study suggests that PCR and Sanger sequencing for Asian DEL is helpful for serologic D-negative Thai patients, while adsorption/elution is unreliable. WGS, if available, is useful to identify rare and new variants, whereas its cost is not worth for routine testing in the Asian population.