RhD变异型的血清学及分子生物学研究  

Serological and Molecular Biological Detection of RhD Variants

作　　者：任道菊 陈春月 李小薇 肖军 张晓娟 李翠莹 REN Dao-Ju;CHEN Chun-Yue;LI Xiao-Wei;XIAO Jun;ZHANG Xiao-Juan;LI Cui-Ying(Air Force Clinical College,The Fifth Clinical Medical College,Anhui Medical University,Hefei 230032,Anhui Province,China;Department of Blood Transfusion,Air Force Specialty Medical Center,Air Force Medical University,Beijing 100142,China)

机构地区：[1]安徽医科大学第五临床医学院空军临床学院,安徽合肥230032 [2]空军军医大学空军特色医学中心输血科,北京100142

出　　处：《中国实验血液学杂志》2025年第2期498-503,共6页Journal of Experimental Hematology

基　　金：空军特色医学中心青年人才项目(2022YXQN028);空军军医大学临床研究项目(2024LC2406)。

摘　　要：目的:分析临床上RhD血清学初筛阴性样本的RHD基因分型及测序结果,进一步探讨RhD检测的实验室方法,为临床精准输血提供依据。方法:采用血清学微柱凝胶法对27200例全血样本进行RhD血型抗原初筛检测。对RhD血型抗原初筛结果为阴性的样本,使用3种不同克隆株IgG抗-D试剂进行血清学RhD阴性确认试验;同时,通过聚合酶链反应-序列特异性引物(PCR-SSP)法分析1号染色体上RHD基因的10个外显子,以确定RHD基因分型。当PCR-SSP法无法得出确切结果时,对其RHD基因进行三代测序分析。结果:27200例样本中RhD初筛阴性136例(0.50%);其中86例样本进行了RhD阴性确认试验和RHD基因分型检测,RhD阴性确认试验中3种抗-D试剂检测结果均为阴性的占88.37%(76/86例);RHD基因分型结果显示,58例(67.44%)为10个外显子全缺失,余28例分别是RHD*711delC(1例)、RHD*D-CE(1-9)-D(1例)、RHD*D-CE(2-9)-D(2例)、RHD*D-CE(3-9)-D(4例)、RHD*DEL1(c.1227G>A)(16例)、RHD*weak partial 15(845G>A)杂合突变(3例),1例样本通过三代测序发现c.165C>T碱基突变。结论:对RhD血清学检测为阴性的样本进行RHD基因型检测发现部分样本存在RHD基因变异,并非所有阴性样本均为RHD全缺失,表明血清学方法在RhD检测上存在一定局限性。由于RHD基因结构的多态性,不同的RhD变异型呈现出不同的血清学特征,需结合分子生物学检测进一步确认,尤其是对亚洲型DEL的鉴定,对临床精准输血具有重要意义。Objective:To analyze the RHD genotyping and sequencing results of RhD serology negative samples in the clinic,and to further explore the laboratory methods for RhD detection,in order to provide a basis for clinical precision blood transfusion.Methods:A total of 27200 whole blood samples were screened for RhD blood group antigen using microcolumn gel card method.Serologic RhD-negative confirmation tests were performed on blood samples that were negative for RhD on initial screening using three different clonal strains of IgG anti-D reagents.The 10 exons of the RHD gene on chromosome 1 were also analyzed by PCR-SSP to determine RHD genotyping.When the PCR-SSP method did not yield definitive results,the RHD gene of the sample was analyzed by the third-generation sequencing.Results:The results of the initial screening test by the microcolumn gel card method showed that 136 of the 27200 samples were RhD-negative,of which 86 underwent RhD-negative confirmation testing and RHD genotyping,88.37%(76/86 cases)of the RhD-negative confirmation test results were negative for the three anti-D reagents,and the results of RHD genotyping showed that 67.44%(58/86 cases)of the cases had a complete deletion of 10 exons,and the remaining 28 cases were RHD711delC(1 case),RHDD-CE(1-9)-D(1 case),RHDD-CE(2-9)-D(2 cases),RHDD-CE(3-9)-D(4 cases),RHDDEL1(c.1227G>A)mutation(16 cases),RHDweak partial 15(845G>A)mutation(3 cases),and a mutation of c.165C>T base was found in 1 sample by three-generation sequencing.Conclusion:RHD genotype testing of samples that are serologically negative for RhD antigen shows that some of the samples have RHD gene variants,not all of which are total deletions of RHD,suggesting that there are some limitations of the serologic method for RhD detection.Due to the polymorphism of the RHD gene structure,different RhD variants present different serologic features,which need to be further detected in combination with molecular biology testing,especially for the identification of Asian-type DELs,which is important for c

关 键 词：RHD抗原 RhD初筛 RHD基因分型 

分 类 号：R457.1[医药卫生—治疗学]