时间分辨荧光法检测乙肝表面抗原LOD及参考区间的验证和建立
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摘要
目的验证时间分辨荧光分析法检测乙肝表面抗原(HBs Ag)的检出限(LOD),评价参考区间的合适性。方法自2017年10月～2018年1月对HBs Ag二级标准物倍比稀释后采用时间分辨荧光分析法及酶联免疫吸附法(ELISA)进行配对检测,标准物浓度除以稀释后检测结果出现阴性的上一稀释度为LOD,验证厂商的声明,比较两种方法检测同一稀释度样品结果,评估厂商声明的参考区间的合适性;根据美国临床实验室标准委员会(CLSI)EP17-A2规定的方法建立空白限(LOB)及LOD,评价已建立的LOD作为参考区间上限的合适性。结果时间分辨荧光法的LOD验证结果为0.1 U/m L,与厂商声明相符。如使用时间分辨荧光检测系统厂商声明的参考区间,标准物稀释3倍后结果判为假阴性,与ELISA检测结果不符。重庆医科大学附属永川医院(以下简称"我院")检验科建立的HBs Ag的LOB及LOD分别为0.048 ng/m L及0.137 ng/m L,如以我院检验科建立的LOD制订参考区间,稀释3倍后两种方法结果一致,均为阳性。结论时间分辨免疫荧光分析系统声明的LOD可以接受,但厂商声明的参考区间不适宜,易导致假阴性。检验科应建立自己的LOD来制订参考区间。建议在对病毒标志物定量检测项目进行性能验证时增加参考区间的验证。
Objectives To verify the detection limit(LOD) of hepatitis B surface antigen(HBsAg) by time-resolved fluorimmunoassay, and to evaluate the suitability of the reference interval. Methods From October 2017 to January 2018,the HBsAg secondary standard substance was diluted and then paired with time-resolved fluorescence method and enzyme-linked immunosorbent assay(ELISA) for detection, the detection limit was the last dilution degree with negative test results after standard substance concentration divided by dilution, to verify the manufacturer′s statement, the results of samples with the same dilution degree detected by the two methods was compared, and the suitability of the reference interval declared by the manufacturer was evaluated. According to the American clinical laboratory standards board(CLSI) EP17-A2 method, create blank limits(LOB) and LOD, and the suitability of the established LOD as the upper limit of the reference interval was evaluated. Results The detection limit of time-resolved fluorimmunoassay method was 0.1 U/mL, which was consistent with the manufacturer′s statement. If the reference interval declared by the manufacturer of the time-resolved fluorimmunoassay detection system was used, the standard substance was diluted 3 times and the result was found to be false negative, which was inconsistent with the ELISA test result, the LOB and LOD of HBsAg established in Department Clinical Laboratory, Yongchuan Hospital of Chongqing Medical University(hereinafter heferred to as "our hospital"), were 0.048 ng/mL and 0.137 ng/mL, respectively. If the reference interval was established with the LOD established chinical laboratory of our hospieal, the results of the two methods were consistent after 3 times of dilution, and both were positive. Conclusion The detection limit declared by the time-resolved fluorimmunoassay analysis system is acceptable, but the reference interval declared by the manufacturer is not appropriate, which is likely to lead to false negative. The clinical laboratory should establish its own LOD to establish the reference interval. It is suggested to increase the validation of the reference interval in the performance verification of the quantitative detection items of virus markers.
Objectives To verify the detection limit(LOD) of hepatitis B surface antigen(HBsAg) by time-resolved fluorimmunoassay, and to evaluate the suitability of the reference interval. Methods From October 2017 to January 2018,the HBsAg secondary standard substance was diluted and then paired with time-resolved fluorescence method and enzyme-linked immunosorbent assay(ELISA) for detection, the detection limit was the last dilution degree with negative test results after standard substance concentration divided by dilution, to verify the manufacturer′s statement, the results of samples with the same dilution degree detected by the two methods was compared, and the suitability of the reference interval declared by the manufacturer was evaluated. According to the American clinical laboratory standards board(CLSI) EP17-A2 method, create blank limits(LOB) and LOD, and the suitability of the established LOD as the upper limit of the reference interval was evaluated. Results The detection limit of time-resolved fluorimmunoassay method was 0.1 U/mL, which was consistent with the manufacturer′s statement. If the reference interval declared by the manufacturer of the time-resolved fluorimmunoassay detection system was used, the standard substance was diluted 3 times and the result was found to be false negative, which was inconsistent with the ELISA test result, the LOB and LOD of HBsAg established in Department Clinical Laboratory, Yongchuan Hospital of Chongqing Medical University(hereinafter heferred to as "our hospital"), were 0.048 ng/mL and 0.137 ng/mL, respectively. If the reference interval was established with the LOD established chinical laboratory of our hospieal, the results of the two methods were consistent after 3 times of dilution, and both were positive. Conclusion The detection limit declared by the time-resolved fluorimmunoassay analysis system is acceptable, but the reference interval declared by the manufacturer is not appropriate, which is likely to lead to false negative. The clinical laboratory should establish its own LOD to establish the reference interval. It is suggested to increase the validation of the reference interval in the performance verification of the quantitative detection items of virus markers.
引文
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[12]赵薇.血清中乙型肝炎病毒转录的检测在母婴垂直传播中的临床意义[J].中国医药导报,2017,14(15):117-120.
[13]Trépo C,Chan HL,Lok A.Hepatitis B virus infection[J].The Lancet,2014,384(9959):2053-2063.
[14]Cai QC,Zhao SQ,Shi TD,et al.Relationship between hepatitis B virus infection and chronic kidney disease in Asian populations:a meta-analysis[J].Ren Fail,2016,38(10):1581-1588.
[15]张红岩,仲曙光.医院医师职业暴露危险因素监控与防护对策分析[J].中国病原生物学杂志,2017(9):851-854.
[16]姜新华,高国生,胡爱荣.国产PCR试剂和COBAS系统血清HBV-DNA检测结果不一致的原因分析[J].中国现代医生,2017,55(2):108-101.
[17]陈德娟.输血和手术前患者血清5项感染性指标调查结果分析[J].中国病原生物学杂志,2013(4):355-357.
[18]曾健强,聂冬梅,马兰,等.深圳无偿献血乙肝筛查阳性结果确证分析[J].实用预防医学,2006,13(2):258-259.
[19]莫展,曾强,冉季于,等.时间分辨荧光免疫技术测量乙肝标志物可疑结果分析与处理[J].中国卫生检验杂志,2014(15):2188-2190.
[20]李金明.定性测定的性能验证[C]//中华医学第九次全国检验医学学术会议暨中国医院协会临床检验管理专业委员会第六届全国临床检验实验室管理学术会议,北京,2011:45-46.
[21]陈洪生,陈佳微,张三明,等.ELISA“灰区”范围的设定对献血者血液检测安全和血源保护的意义[J].中国输血杂志,2015,28(9):1140-1141.
[22]张艳丽.HBsAg ELISA灰区、弱反应标本与FQ-PCR检测结果对比分析[J].临床血液学杂志(输血与检验),2016(5):794-796.
[23]陈静,李帮芬,先秀,等.酶联免疫四项检测结果灰区与窗口期感染的关系[J].检验医学与临床,2017,14(17):2554-2556.
[2]方婉仙,李志雄,董志宁,等.总前列腺特异性抗原定量测定试剂盒(化学发光免疫分析法)的性能验证[J].分子诊断与治疗杂志,2015,7(5):323-327.
[3]沙广群,王启龙,刘萍,等.游离前列腺特异性抗原性能指标的建立[J].检验医学与临床,2017,14(14):2097-2099
[4]张秀明,冯仁丰.正确理解和使用分析灵敏度及LOD[J]中华检验医学杂志,2014,37(9):669-672.
[5]CLSI.Evaluation of detection capability for clinical laboratory measurement procedures[M].PA:Clinical and Laboratory Standards Institute,2012.
[6]杨有业,张秀明.临床检验方法学评价[M].北京:北京人民卫生出版社,2008:142-167.
[7]CLSI.EP12-A2 User protocol for evaluation of qualitative test performance;approved guideline[S].PA,USA:Clinical and Laboratory Standards Institute,2009.
[8]黄永富,黄介飞,丛辉,等.时间分辨荧光免疫分析法检测乙型肝炎血清标志物的分析方法性能验证[J].国际检验医学杂志,2011,32(5):543-547.
[9]康凤凤,王薇,王治国.临床实验室检测方法空白限、LOD和定量限评价新方法[J].中国卫生统计,2014,31(5):901-904.
[10]Ji Z,Wang T,Shao Z,et al.A population-based study examining hepatitis B virus infection and immunization rates in Northwest China[J].PLoS One,2014,9(5):e97 474.
[11]何龙芳.抗病毒治疗停药后乙型肝炎复发与血清HBVDNA水平的相关性分析[J].中国医药科学,2017,7(21):251-254.
[12]赵薇.血清中乙型肝炎病毒转录的检测在母婴垂直传播中的临床意义[J].中国医药导报,2017,14(15):117-120.
[13]Trépo C,Chan HL,Lok A.Hepatitis B virus infection[J].The Lancet,2014,384(9959):2053-2063.
[14]Cai QC,Zhao SQ,Shi TD,et al.Relationship between hepatitis B virus infection and chronic kidney disease in Asian populations:a meta-analysis[J].Ren Fail,2016,38(10):1581-1588.
[15]张红岩,仲曙光.医院医师职业暴露危险因素监控与防护对策分析[J].中国病原生物学杂志,2017(9):851-854.
[16]姜新华,高国生,胡爱荣.国产PCR试剂和COBAS系统血清HBV-DNA检测结果不一致的原因分析[J].中国现代医生,2017,55(2):108-101.
[17]陈德娟.输血和手术前患者血清5项感染性指标调查结果分析[J].中国病原生物学杂志,2013(4):355-357.
[18]曾健强,聂冬梅,马兰,等.深圳无偿献血乙肝筛查阳性结果确证分析[J].实用预防医学,2006,13(2):258-259.
[19]莫展,曾强,冉季于,等.时间分辨荧光免疫技术测量乙肝标志物可疑结果分析与处理[J].中国卫生检验杂志,2014(15):2188-2190.
[20]李金明.定性测定的性能验证[C]//中华医学第九次全国检验医学学术会议暨中国医院协会临床检验管理专业委员会第六届全国临床检验实验室管理学术会议,北京,2011:45-46.
[21]陈洪生,陈佳微,张三明,等.ELISA“灰区”范围的设定对献血者血液检测安全和血源保护的意义[J].中国输血杂志,2015,28(9):1140-1141.
[22]张艳丽.HBsAg ELISA灰区、弱反应标本与FQ-PCR检测结果对比分析[J].临床血液学杂志(输血与检验),2016(5):794-796.
[23]陈静,李帮芬,先秀,等.酶联免疫四项检测结果灰区与窗口期感染的关系[J].检验医学与临床,2017,14(17):2554-2556.