基于γ干扰素释放试验的流式荧光免疫分析法检测结核分枝杆菌的方法学研究
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摘要
目的基于γ干扰素释放试验(IGRA),利用流式荧光免疫分析法建立一种检测结核分枝杆菌(TB感染的新方法。方法分别用植物凝集素(PHA)、TB特异性混合多肽[早期分泌靶向抗原(ESAT-6)和培养滤液蛋白10(CFP-10)]刺激全血标本产生γ干扰素(IFN-γ),用双抗体夹心法结合流式荧光技术检测培养后血浆中的IFN-γ浓度,使用受试者工作特征(ROC)曲线评价其对TB感染的诊断效能。对该方法的线性范围、最低检测限、重复性、抗干扰性能及其与市售同类产品检测的一致性进行评价。结果流式荧光免疫分析法检测IFN-γ的线性范围为2～1 000 pg/mL,最低检测限为0.3 pg/mL;检测100 pg/mL和500 pg/mL 2个浓度标本的重复性,2个浓度下变异系数分别为4.58%和2.46%;回收试验平均回收率为98.0%;三酰甘油≤50 mg/mL、胆红素≤0.6 mg/mL、血红蛋白≤10 mg/mL时对流式荧光免疫分析法检测结果无干扰。通过ROC曲线确定该方法诊断TB感染的最佳截断值为10 pg/mL,此时灵敏度为82.46%,特异度为87.30%。该方法与市售TB感染T淋巴细胞斑点试验(T-SPOT)试剂盒、QuantiFERON-TB Gold(QFT)试剂盒和万泰TB-IGRA试剂盒诊断TB感染的总符合率分别为92.5%、83.0%和85.4%,Kappa系数分别为0.822、0.622和0.630。结论本研究建立的TB感染检测方法性能良好,准确性达到市售同类产品的水平,且在重复性、检测流程等方面具有明显优势。
Objective To establish a new method for detecting Mycobacterium tuberculosis(TB) infection based on flow cytometry fluorescence immunoassay and interferon γ release assay(IGRA). Methods The whole blood samples were stimulated to produce interferon γ(IFN-γ) with phytagglutinin(PHA) and TB specific mixed peptides(early secretory antigenic target [ESAT-6] and culture filtrate protein 10 [CFP-10]), and the plasma was analyzed by double antibody sandwich method combined with flow cytometry fluorescence immunoassay. The IFN-γ concentration was evaluated by receiver operating characteristic(ROC) curve for the diagnostic efficacy of TB. The linear range, minimum detection limit,repeatability, anti-interference performance of the established method were observed, and the consistency of detection with similar products on the market was evaluated. Results The linearity of the flow cytometry fluorescence immunoassay ranged from 2 pg/mL to 1 000 pg/mL. The lowest detection limit was 0.3 pg/mL; the repeatability parameters(coefficient of variation)of the samples at 100 pg/mL and 500 pg/mL were 4.58% and 2.46%, respectively. The average recovery rate of recovery assay was 98.0%. There was no interference with flow cytometry fluorescence immunoassay when the highest concentrations of triglyceride, bilirubin and hemoglobin were 50 mg/mL, 0.6 mg/mL and 10 mg/mL, respectively. As the optimum cut-off value of the IFN-γ concontration was 10 pg/mL, the sensitivity of IFN-γ in diagnosis of TB infection was 82.46% and the specificity was 87.30%. The total coincidence rates with T-SPOT, QFT, and Wantai TB-IGRA reagent were 97.2%, 83.0%, and 85.4%,respectively; and the Kappa coefficients were 0.822, 0.622 and 0.630, respectively. Conclusion The method for diagnosis of TB infection established in this study has a good performance, with the accuracy reaching the level of similar products on the market, and our method has obvious advantages in terms of repeatability and detection process.
Objective To establish a new method for detecting Mycobacterium tuberculosis(TB) infection based on flow cytometry fluorescence immunoassay and interferon γ release assay(IGRA). Methods The whole blood samples were stimulated to produce interferon γ(IFN-γ) with phytagglutinin(PHA) and TB specific mixed peptides(early secretory antigenic target [ESAT-6] and culture filtrate protein 10 [CFP-10]), and the plasma was analyzed by double antibody sandwich method combined with flow cytometry fluorescence immunoassay. The IFN-γ concentration was evaluated by receiver operating characteristic(ROC) curve for the diagnostic efficacy of TB. The linear range, minimum detection limit,repeatability, anti-interference performance of the established method were observed, and the consistency of detection with similar products on the market was evaluated. Results The linearity of the flow cytometry fluorescence immunoassay ranged from 2 pg/mL to 1 000 pg/mL. The lowest detection limit was 0.3 pg/mL; the repeatability parameters(coefficient of variation)of the samples at 100 pg/mL and 500 pg/mL were 4.58% and 2.46%, respectively. The average recovery rate of recovery assay was 98.0%. There was no interference with flow cytometry fluorescence immunoassay when the highest concentrations of triglyceride, bilirubin and hemoglobin were 50 mg/mL, 0.6 mg/mL and 10 mg/mL, respectively. As the optimum cut-off value of the IFN-γ concontration was 10 pg/mL, the sensitivity of IFN-γ in diagnosis of TB infection was 82.46% and the specificity was 87.30%. The total coincidence rates with T-SPOT, QFT, and Wantai TB-IGRA reagent were 97.2%, 83.0%, and 85.4%,respectively; and the Kappa coefficients were 0.822, 0.622 and 0.630, respectively. Conclusion The method for diagnosis of TB infection established in this study has a good performance, with the accuracy reaching the level of similar products on the market, and our method has obvious advantages in terms of repeatability and detection process.
引文
[1]World Health Organization.Global tuberculosis report2017[R].Geneva:WHO Press,2017:1-2.
[2]韦海旭,席薇莲,陆利欢,陈健.γ-干扰素释放分析T-SPOT.TB在结核性疾病中的诊断价值[J].临床肺科杂志,2012,17:1439-1440.
[3]丰玫玫,苏文琴.结核分枝杆菌诊断技术的发展及目前临床应用的新型诊断技术产品分析[J].中国医药科学,2012,2:26-28.
[4]WHITWORTH H S,BADHAN A,BOAKYE A A,TAKWOINGI Y,REES-ROBERTS M,PARTLETT C,et al.Clinical utility of existing and second-generation interferon-γrelease assays for diagnostic evaluation of tuberculosis:an observational cohort study[J].Lancet Infect Dis,2019,19:193-202.
[5]姚见儿.Luminex高通量检测技术的应用和挑战[J].临床检验杂志,2010,28:250-251.
[6]Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative measurement procedures:a statistical approach;approved guideline.CLSI document EP6-A[S].Wayne:CLSI,2003.
[7]National Committee for Clinical Laboratory Standards.Interference testing in clinical chemistry;proposed guideline.Proposed Guideline,NCCLS document 1986;EP7-P[S].Wayne:NCCLS,1986.
[8]鲍磊,李涛,卢水华.全血γ干扰素释放试验在儿童和成人中的结核病诊断价值[J].微生物与感染,2014,9:21-27.
[9]WANG Y,YANG Y,LI H,LIANG Y,LIU J,YU T,et al.Evaluation of a whole blood chemiluminescent immunoassay of interferon-gamma inducible protein 10(IP-10)for diagnosis of tuberculosis patients[J].Clin Lab,2016,62(1/2):165-172.
[10]JEONG Y H,HUR Y G,LEE H,KIM S,CHO J E,CHANG J,et al.Discrimination between active and latent tuberculosis based on ratio of antigen-specific to mitogen-induced IP-10 production[J].J Clin Microbiol,2015,53:504-510.
[11]QIU X,TANG Y,YUE Y,ZENG Y,LI W,QU Y,et al.Accuracy of interferon-γ-induced protein 10 for diagnosing latent tuberculosis infection:a systematic review and meta-analysis[J].Clin Microbiol Infect,2019,25:667-672.
[2]韦海旭,席薇莲,陆利欢,陈健.γ-干扰素释放分析T-SPOT.TB在结核性疾病中的诊断价值[J].临床肺科杂志,2012,17:1439-1440.
[3]丰玫玫,苏文琴.结核分枝杆菌诊断技术的发展及目前临床应用的新型诊断技术产品分析[J].中国医药科学,2012,2:26-28.
[4]WHITWORTH H S,BADHAN A,BOAKYE A A,TAKWOINGI Y,REES-ROBERTS M,PARTLETT C,et al.Clinical utility of existing and second-generation interferon-γrelease assays for diagnostic evaluation of tuberculosis:an observational cohort study[J].Lancet Infect Dis,2019,19:193-202.
[5]姚见儿.Luminex高通量检测技术的应用和挑战[J].临床检验杂志,2010,28:250-251.
[6]Clinical and Laboratory Standards Institute.Evaluation of the linearity of quantitative measurement procedures:a statistical approach;approved guideline.CLSI document EP6-A[S].Wayne:CLSI,2003.
[7]National Committee for Clinical Laboratory Standards.Interference testing in clinical chemistry;proposed guideline.Proposed Guideline,NCCLS document 1986;EP7-P[S].Wayne:NCCLS,1986.
[8]鲍磊,李涛,卢水华.全血γ干扰素释放试验在儿童和成人中的结核病诊断价值[J].微生物与感染,2014,9:21-27.
[9]WANG Y,YANG Y,LI H,LIANG Y,LIU J,YU T,et al.Evaluation of a whole blood chemiluminescent immunoassay of interferon-gamma inducible protein 10(IP-10)for diagnosis of tuberculosis patients[J].Clin Lab,2016,62(1/2):165-172.
[10]JEONG Y H,HUR Y G,LEE H,KIM S,CHO J E,CHANG J,et al.Discrimination between active and latent tuberculosis based on ratio of antigen-specific to mitogen-induced IP-10 production[J].J Clin Microbiol,2015,53:504-510.
[11]QIU X,TANG Y,YUE Y,ZENG Y,LI W,QU Y,et al.Accuracy of interferon-γ-induced protein 10 for diagnosing latent tuberculosis infection:a systematic review and meta-analysis[J].Clin Microbiol Infect,2019,25:667-672.
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