Rh blood group phenotypes and distribution of RHD gene polymorphism among Han nationality voluntary blood donors with RhD negative of primary screening in Weifang City, Shandong Province

Objective To explore characteristics of Rh blood group phenotypes and RHD gene polymorphism among Han nationality voluntary blood donors with RhD negative of primary screening in Weifang City, Shandong Province. Methods From March 2015 to July 2017, a total of 1 307 Han nationality blood donors who were negative for RhD primary screening when participated in the voluntary blood donation at Weifang Blood Center, were selected as subjects. Among them, there were 921 male donors and 386 females, aged 18-55 years. Serological testing method was used to confirm RhD negative of 1 307 blood donors with RhD negative of primary screening. Anti-D blood grouping reagents from 3 different manufacturers and direct antiglobulin test were performed for serological tests. Blood donors who were confirmed RhD negative by serological test were classified for Rh blood group phenotype, and the phenotype frequency of each RhCcEe and their haplotype frequencies were calculated. From September 2015 to March 2016, a total of 116 blood donors with RhD negative of primary screening were selected for RHD gene detection. PCR-sequence-specific primer (SSP) method and human erythrocyte RHD genotyping kit were used for RHD gene detection. And the gene polymorphism of RHD gene was analyzed. The frequencies of Rh blood group phenotypes and haplotypes were calculated by square root method. The test for Hardy-Weinberg equilibrium of the phenotype frequency distribution of Rh blood group phenotypes were conducted by chi-square test. The procedures followed in this study were accordance with the requirements of the World Medical Association Declaration of Helsinki revised in 2013, and all the blood donors signed the informed consents. Results ① Among 1 307 blood donors with RhD negative of primary screening from Weifang in this study, there were 1 244 donors (95.2%) with RhD negative and 63 donors (4.8%) with RhD variants. ② Among the 1 244 RhD negative blood donors, there were 773 donors (62.14%) with ccee phenotypes, 329 cases (26.45%) with Ccee, 83 cases (6.66%) with ccEe, 40 cases (3.22%) with CCee, 18 cases (1.45%) with CcEe and 1 case (0.08%) with CCEe. The haplotype frequencies of cde, CdE, Cde and cdE were 78.83%, 0.06%, 16.99% and 4.12%, respectively. The result of test for Hardy-Weinberg equilibrium showed that there was no significant difference between the observed and expected values of Rh blood group phenotype frequencies in 1 244 RhD negative blood donors (χ2=2.17, P>0.05). ③ Among 116 blood donors with RhD negative of primary screening, there were 77 cases (66.4%) with whole RHD exon deletion genotypes, 13 cases (11.2%) with RHD-CE(2-9)-D, 3 cases (2.6%) with weak D15, 19 cases (16.4%) with homozygous DEL RHD1227A, and 4 cases (3.5%) with heterozygous DEL RHD1227A. Conclusions This study preliminarily investigate characteristics of Rh blood type phenotypes and RHD gene distribution among donors with RhD negative in Weifang City, which ensure the blood transfusion safety of RhD negative patients and provide scientific basis for the establishment of RhD negative rare blood group library. Key words: Rh-Hr blood-group system; Phenotype; RhD negative; RHD gene; RHD exons; Gene polymorphism