Genetic testing analysis of samples with multiple RhD negative confirmations and inconsistent serological results from voluntary blood donors in Jinan region from 2012 to 2022
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摘要: 目的 借助分子生物学方法探讨常规血清学方法进行无偿献血者RhD阴性确认多次结果不一致情况的原因分析。方法 对2012年1月—2022年12月无偿献血员RhD初筛阴性的标本,采用微柱凝集间接抗人球蛋白(IAT)法进行血清学RhD阴性确认,同时进行RhC、c、E、e鉴定分型检测。对阴性确认试验中发现的多次结果不一致的标本采用荧光PCR-SSP方法进行RhD等位基因分型鉴定,对于基因分型无法确认的标本,特异性扩增RHD基因10个外显子,并进行正反向Sanger测序,明确其基因型。结果 RhD阴性确认试验中共发现12例多次结果不一致标本。标本均含有RhC或RhE抗原,经PCR-SSP法基因分型和Sanger测序共检出5种基因型,其中:8例RHD*01EL.01/RHD*01N.01、1例RHD*01N.03/RHD*01N.01、1例RHD*15/RHD*01N.01、1例RHD*01N.01/RHD*01N.01、1例RHD*01W.24/RHD*01N.01。结论 常规血清学方法进行RhD阴性确认存在多次结果不一致的情况,应联合应用血清学和分子生物学检测技术对无偿献血者RhD进行鉴定,保证血型鉴定的准确性,保障临床用血安全。Abstract: Objective To explore the reasons of repeatedly inconsistent RhD negative serological confirmation results in voluntary blood donors using molecular biology method.Methods From January 2012 to December 2022, the samples of voluntary blood donors who were initially screened negative for RhD were confirmed by microcolumn agglutination, and RhC, c, E and e were identified and subtyped. Repeatedly inconsistent RhD negative confirmation samples were selected for RhD alleles typing by fluorescent PCR-SSP method. For the specimens whose genotyping could not be confirmed, 10 exons of RHD gene were specifically amplified and their genotypes were identified by two directional Sanger sequencing.Results 12 samples in total with repeatedly inconsistent results of the negative confirmation were found to contain RhC or RhE antigens. Five gene types were detected through genetic testing of PCR-SSP and Sanger sequencing, including 8 cases of RHD*01EL.01/RHD*01N. 01, 1 case of RHD*01N. 03/RHD*01N. 01, 1 case of RHD*15/RHD*01N. 01, 1 case of RHD*01N. 01/RHD*01N. 01 and 1 case of RHD*01W. 24/RHD*01N. 01.Conclusion Repeatedly inconsistent RhD negative confirmation results were found using serological method. Serological and molecular biology method were combined to identify the RhD of voluntary blood donors to ensure the accuracy of blood type identification and ensure the safety of blood transfusion.
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Key words:
- RhD negative confirmation /
- serological method /
- molecular biology method
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表 1 血清学格局汇总
标本编号 直抗 RhD阴性确认试验血清学格局 RhCcEe分型 抗-D1 抗-D2 抗-D3 抗-D1 抗-D2 抗-D3 1a 0 0 0 0 0 0 w Ccee 2 0 0 0 0 w w w Ccee 3b 0 0 0 0 w 1+ 1+ Ccee 4 0 0 0 0 1+ 1+ 1+ ccEe 5 0 0 0 0 0 w w Ccee 6 0 0 0 0 0 0 w Ccee 7 0 0 0 0 0 0 w Ccee 8c 0 0 0 0 w 0 w Ccee 9 0 0 0 0 w w w Ccee 10 0 0 0 0 w 0 w CcEe 11d 0 0 0 0 w 1+ 2+ ccEe 12 0 0 0 0 0 0 w Ccee 注:a1号标本5次RhD确认2次为RhD阳性,3次为RhD阴性;b3号标本7次RhD确认3次为RhD阳性,4次为RhD阴性;c8号标本4次RhD确认2次为RhD阳性,2次为RhD阴性;d11号标本:8次RhD确认6次为RhD阳性,2次为RhD阴性。 表 2 RHD基因和合子基因分型结果
标本编号 检测结果 1 RHD*01N.03/RHD*01N.01[RHD-CE(2-9)-D合并D全缺失] 2 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 3 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 4 基因初筛阳性,sanger测序法进一步验证(合子阳性) 5 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 6 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 7 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 8 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 9 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) 10 RHD*01N.01/RHD*01N.01(D全缺失) 11 RHD*15/RHD*01N.01(弱D15合并D全缺失) 12 RHD*01EL.01/RHD*01N.01(DEL1227A合并D全缺失) -
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