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目的分析广州地区人群中RHD*960A突变型等位基因个体的血型血清学与分子生物学特征。方法收集RhD变异型标本,采用2种不同的单克隆抗-D试剂鉴定RhD血型,抗球蛋白凝胶卡法进行直接抗球蛋白试验,并用RhD抗原表位检测试剂盒(D-Screen)检测RhD抗原表位;此外,采用Rh血型分型卡检测Rh CE抗原;采用多重连接酶依赖的探针扩增(MLPA)方法检测RHD及RHCE基因型;对MLPA方法无法给出检测结论的标本,进行RHD基因全部10个外显子的PCR扩增及产物直接测序。结果发现3例RhD变异型标本,直抗阴性,初步血清学结果显示RhD抗原弱阳性表达,RhD抗原表位检测结果显示其红细胞与RhD抗原epD6.6、epD8.2和epD9.1表位特异性单克隆抗体无凝集反应,与其余表位抗体呈弱阳性凝集反应;RHCE抗原分型1例为CCee,其余2例为Ccee;RHD基因的MLPA基因分型显示其基因型为正常的RHD/d(即未鉴定出其携带的突变型RHD等位基因),后续RHD基因外显子直接测序发现其第7外显子携带c.960G>A(p.Leu302Leu)同义突变,RHCE基因MLPA基因分型结果与血清学表型一致。结论首次在广州地区人群中发现了RHD*960A突变型等位基因个体,并进行血清学抗原表位和基因型检测,其血清学表现为部分D。
Objective To analyze the serological and molecular biological characteristics of RHD * 960A mutant alleles in Guangzhou population. Methods RhD variants were collected and RhD blood samples were identified by two different monoclonal anti-D reagents. The anti-globulin gel assay was used to detect the direct antiglobulin activity. RhD antigen epitope detection kit (D-Screen) In addition, the Rh CE antigen was detected by Rh-type genotyping card; RHD and RHCE genotypes were detected by multiplex ligase-dependent probe amplification (MLPA); the MLPA method could not give the conclusion of the detection, All 10 exons of RHD gene were amplified by PCR and sequenced directly. The results showed that three RhD variant specimens were directly negative and negative, and the preliminary serological results showed weak positive expression of RhD antigen. The results of RhD antigen epitope showed that the erythrocytes and epidermal growth factor receptor epitopes epD6.6, epD8.2 and epD9.1 were specific The results showed that there was no agglutination reaction with the monoclonal antibody and weakly positive agglutination reaction with the other epitope antibodies. One case of RHCE antigen typing was CCee and the other two cases was Ccee. MLPA genotyping of RHD gene showed that its genotype was normal RHD / (ie, the mutant RHD allele carried by it was not identified). The direct sequencing of the exon of RHD gene revealed that the seventh exon carries synonymous mutations of c.960G> A (p.Leu302Leu), RHCE gene MLPA Genotyping results were consistent with serological phenotypes. Conclusions The RHD * 960A mutant allele was found in Guangzhou population for the first time, and serological epitopes and genotypes were detected.