HBsAg酶联免疫吸附试验灰区设置研究

目的评价与验证该实验室乙型肝炎表面抗原(HBsAg)试验设置0.9倍临界值(CO值)的合理性。方法参照美国临床和实验室标准协会(CLSI)发布的EP12-A2指南,通过试验确定HBsAg试验C5~C95区间即灰区。对HBsAg灰区标本进行抗体确认试验。绘制受试者工作特征曲线(ROC曲线)确定HBsAg试验最佳CO值。通过实验室既往数据,分析乙型肝炎病毒DNA(HBV-DNA)单阳性标本酶联免疫吸附试验(ELISA)结果分布(S/CO值)与灰区的关系。结果 (C50±20%)水平检测结果阴性数和阳性数均大于或等于95%,(C50-20%)~(C50+20%)水平范围包含C5~C95区间,灰区范围应在0.712~1.103倍CO值区间内。对44例HBsAg灰区标本(S/CO值0.900~0.990)进行中和试验,结果均为无反应性。绘制HBsAg的ROC曲线,曲线下面积(AUC)为0.981,最适CO值为0.063(现用CO值在0.055~0.060)。2010年11月2日至2013年12月31日检测标本研究886 291例患者,HBsAg阳性标本包括135例灰区标本,其中7例核酸检测法(NAT)检测结果均为反应性;共检出HBV-DNA单阳性标本421例,其HBsAg检测结果(S/CO值)分布区间为0.200~0.400,与阴性标本分布区间重叠、距0.9倍CO值较远。结论该实验室现阶段HBsAg试验设置0.900的CO灰区过于严苛,试验结果支持取消灰区设置。报道所提供的4种灰区评价方法为其他实验室在设置ELISA试验灰区方面提供了1种思路。

The discussion of method for grey area of HBsAg enzyme linked immunosorbent assay

Objective To evaluate and verify the rationality of grey area of HBsAg enzyme linked immunosorbent assay(ELISA) in the laboratory .Methods According to CLIS EP12‐A2 guide ,HBsAg concentration of the range between C5 and C95 concentra‐tion were determined as grey area by ELISA test .Samples of HBsAg in grey area were identified by ELISA .Using ROC curve anal‐ysis ,the optimal CO level of HBsAg test was determined .Combined with previous data ,the relationship between disposition of HBV‐DNA single positive simple of ELISA results(S/CO value) and grey area were explored .Results Positive and negative re‐sults of (C50 ± 20% ) concentration were all higher than 95% .(C50 - 20% ) to (C50 + 20% ) concentration range included C5 to C95 ,gray area of S/CO value was 0 .712 to 1 .103 times of CO .Forty four cases of HBsAg ELISA grey area samples(S/CO value was 0 .900 to 0 .990) were confirmed negatively by neutralization confirmatory test .According to ROC curve analysis ,AUC was 0 .981 ,the optimal CO level was 0 .063(CO level in use was 0 .055 to 0 .060) .And 886 291 samples were tested since November 2 , 2010 to December 31 ,2013 in our lab ,including 135 grey area samples which were tested out in HBsAg positive samples .Seven samples were reactive by NAT test .A total of 421 cases were HBV‐DNA single positive simples ,and their distribution interval of ELISA results(S/CO value) were between 0 .200 and 0 .400 ,which overlapped the distribution interval of ELISA negative samples and far from 0 .9 times of CO level .Conclusion The setting of grey area(0 .9 times of CO level) is too strict for the lab ,the grey ar‐ea could be cancelled based on experimental data .In addition ,the four evaluation methods of grey area we offered will give other labs idea for setting ELISA grey area .