Study On Weak D Phenotype Distribution And Gene Polymorphisms In Hebei Area

Objective:The Rh blood group system is the most complex in red cell blood group system, and second in importance to ABO blood group system in the clinical blood transfusion. Until now, the amoun of identified Rh system antigen are more than 40. RHD gene and RHCE gene located on human chromosome 1p34.3-36.1 are respectively encoded to be Rh blood group system antigens Rh D, Rh C/c, and Rh E/e, of which antigen D is the more essential in blood transfusion medicine and obstetrics. Generally, it is termed Rh- blood group when D antigen is negative, while the positive D antigen called Rh+ blood group. Weak D phenotype refers to ones whose D epitopes totally exist while the amounts of D epitopes were decreased, but their quality is not involved. Distributions of weak D phenotype and gene polymorphisms are not consistent all over the world, and most reports focused on Chinese Taiwanese. Few researches on weak D phenotype performed in southern areas of our country. The study on distribution and the molecular background of Rh weak D is not yet well known in Hebei region. Here, the serological identification was performed and the molecular mechanism was detected by sequence analysis to the blood samples from Hebei region.In Rh blood group system, there are five important antigens: C, c, D, E, e antigen, among which the immunogenicity of the antigen D is the strongest one. Weak D antigen of red blood cells may stimulate the Rh- recipients to produce antibodies. In fact, some recipients were sensitized to weak D antigen and occurred hemolytic reaction, which has lead to health workers and researchers pay more attention. Whether the donors’ specimens are weak D or not are required by << American Association of Blood Banks and Transfusion transfusion agency Standard >>. As long as there are anti-D in blood serum of the recipient, weak D’s red blood cells were refuse to the recipitent, because anti-D serum of the recipient will fastly reacted with the red blood cells with weak D antigen. Therefore, it is an important method to improve clinical transfusion safety by discovering the genetic background and gene polymorphism of weak D phenotype, designing appropriate PCR to detect accurately and efficiently weak D phenotype.This study will randomly select Rh-negative blood donors in Hebei Province to be serological screening. The analysis and experiments for molecular genetics background of weak D phenotype were performed in the typing laboratory of Hebei Province Blood Center. Through this project serological screening, we can know the distribution proportion of weak D in Rh-negative blood donors in Hebei Province. By analyzing the molecular background, we can discover the genetic polymorphism of weak D and try to find new new alleles. By this project, identification methods ofweak D gene will be used as weak D genotyping means in Hebei province, which will facilitate to use weak D blood more rationally and effectively in the future according to the molecular basis of weak D antigen.Methods:1 Screening Rh D antigens were performed to 78000 blood donors in Hebei Blood Center by standard serological methods; then the Rh D antigen of those Rh-negative donors will be identified by three different sources of anti-D sera.2 Preparation on genomic DNA and Rh phenotype: high-purity genomic DNA was extracted from 214 cases of Rh negative donors by micro-column method, and then the DNA concentration and purity was checked.3 The sequence-specific primers PCR(sequence specific primers, PCR-SSP) : compares to HGH(human growth hormone, HGH,as control), RHD 3,4,5,6,7,9- allele-specific gene exons were amplified by PCR.. According to the length of the gene fragment and the presence or absence of the PCR product, whether RHD gene epitope lose or not was determined after the PCR products were electrophoresed on 2.5% agarose gel.4 Genetic Test:the epitope deficiency samples were analyzed by "human blood erythrocytes RHD weak D, Part D gene detection kit"(Xiupeng Tianjin Biotechnology Development Co, Ltd.)Results:1 Serological test: among 78000 donors, 214 cases are Rh negative. The percentage of Rh-negative population was 0.275%, of which 35 cases are weak Rh D-type, so the percentage of D-type weak was 0.045%, compared with the normal population.2 The 214 cases of Rh-negative blood type were tested by serotyping techniqueindividually. The results showed that 88 cases are whole-small factors ccee phenotype, 67 cases are Ccee phenotype, 24 cases are cc Ee phenotype, 15 cases are Cc Ee phenotype, 12 cases are CCee phenotype and 8 cases are cc EE.3 The increasing results showed that the negative Rh D gene exons in Han nationality are polymorphism obviously in Hebei Area. In 35 samples of Weak D, there are 30 complete Rh D gene exon, while 5 lost them partially.4 It shows by genetic testing that 27 ones are Weak D15, 3 ones are Weak D24 in 30 complete Rh D gene exon cases; in the 5 partially D missing cases, 3 ones are Weak RHD gene 2- 9 exons fused, which termed as RHD-CE(2-9)-D; 1 sample was termed as D Cat.Va type 2; 1 sample was for D Cat.VI type 2.Conclusion:1 In Hebei area, among 78,000 blood donors, there were 214 Rh D negative cases by common serological identification, and the proportion was 0.275%, of which 35 cases were detected as weak D, and the ratio was 0.045 %.2 88 cases are whole-small factors ccee phenotype, 67 cases are Ccee phenotype, 24 cases are cc Ee phenotype, 15 cases are Cc Ee phenotype, 12 cases are CCee phenotype and 8 cases are cc EE among the 214 cases of Rh-negative blood type.3 30 cases complete Rh D gene exons of 35 cases were determined by SSP-PCR, among them, 27 cases were detected with weak type D15, and 3 cases were detected with D24 weak type respectively; Additionally, 5 cases with partial exon genes lost Rh D, among them, three ones were Weak RHD gene 2- 9 exons fused, which expressed as RHD-CE(2-9)-D, 1 sample was tested as D Cat.Va type2, and 1 sample was for D Cat.VI type 2.4 Weak D detection rates are 16.4% among Rh D negative cases on the basis of normal measure to the cases from Hebei area, and the weak D15 type is the main ones, Accounting for around 77.1 per cent of weak D types.