Analysis of Del phenotype and genotype in RhD-negative blood donors in Guangzhou area

Abstract

Objective To investigate the distribution, phenotype and genotype of D-elute type(Del) in blood donors with RhD negative blood in Guangzhou, so as to understand the molecular biological background of DEL blood group in this area. Methods During the period from November 1, 2021 to June 30, 2022, the RhD-negative blood initially screened by saline method was confirmed by indirect anti-human globulin test(IAT) serology, and RhCE phenotype was determined by RhCE typing card. A total of 1 146 RhD-negative samples, including all RhD-negative samples with RhCE C+ (n=459) and a randomly selected subset of RhCE C-(n=175), were subjected to adsorption-elution(Del) screening(a total of 634 samples). DNA from Del-positive samples was extracted for real-time fluorescent PCR detection of the RHD gene c. 1227 locus using high-resolution melting curve analysis(HRM). For samples without mutations detected at the RHD*1227 locus by HRM, restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP) was performed to amplify the product which was subsequently digested with PstI enzyme and analyzed by electrophoresis to determine RHD gene haplotypes. Sanger sequencing was performed for exon sequencing(exon 1-10) of the RHD gene, and gene mutations were analyzed using SeqMan software. Suspected Del-positive samples were subjected to RHD whole gene analysis using third-generation single-molecule sequencing technology. Results Among the 634 confirmed RhD-negative samples, 229(36.1%) displayed Del phenotype, accounting for 20%(229/1 146) of the total confirmed RhD-negative samples. The RhCE phenotypes of the 229 DEL cases were as follows: Ccee in 181 cases, CCee in 40 cases, CcEe in 7 cases, and ccEe in 1 case. HRM combined with RHD haplotype analysis showed that there were 170 cases with RHD gene as RHD*1227A/01N.01, 32 cases with RHD gene as RHD*1227A/1227G, 26 cases with RHD gene as RHD*1227A/1227A, and 6 cases with RHD gene as RHD*1227G/1227G(sequencing results included 1 case of weak D type 12, 4 cases of D-, and 1 case of RHD*01EL.02). Conclusion The individual genotype of DEL blood donors in Guangzhou area is mainly characterized by RHD*1227A/01N.01, and their RhCE phenotypes are all C+. HRM can be used as a molecular biology method for routine screening of Asian-type DEL blood type genes.

Keywords

• voluntary blood donors
• rhd-negative
• asian-type del
• rhce phenotype
• high-resolution melting(hrm)