干血斑样本用于梅毒酶联免疫吸附试验检测方法的初步评价及应用研究

Evaluation and preliminary application of ELISA method based on dried blood spots sample(DBS) for detection of syphilis

目的验证本实验室已建立的干血斑样本用于梅毒酶联免疫吸附试验(ELISA)检测方法的实验室性能指标并进行初步应用评价,为干血斑样本用于梅毒ELISA检测提供具有参考价值的实验数据。方法通过构建实验室线性盘、基础盘、干扰盘、精密度盘,分析所建立方法的线性范围、敏感性、特异性及精密度等性能并评估干血斑样本稳定性。2019年1-7月共采集329份血液样本,平行检测血浆与干血斑样本中梅毒抗体。以血浆检测结果作为参考,比较血浆与干血斑样本检测结果,分析其临床敏感性、临床特异性并评估二者间一致性及相关性。结果该方法的线性范围23~27(R^2=0.98,P <0.05);阳性符合率与阴性符合率均为15/15。检测不受其他非特异性干扰物质及合并感染影响;精密度CV天间、CV批内及CV斑间分别为0.49%~6.21%、0.49%~10.31%、0.51%~7.09%。与血浆结果相比,临床敏感性及临床特异性分别为86.5%[95%可信区间(CI):0.70~0.95]、100.0%(95%CI:0.98~1.00);阳性预测值、阴性预测值分别为100%(95%CI:0.87~1.00)、98.3%(95%CI:0.96~0.99)。Spearman相关性系数r=0.204(P <0.01),一致性检验Kappa值为0.92(95%CI:0.85~0.99),S/CO比值结果分布比较表明,二者分布特征相似,无明显差异,比值分布集中。结论已建立干血斑样本用于梅毒ELISA的检测方法精密度良好、抗干扰能力强,干血斑样本稳定性良好;与血浆检测结果一致性高但发现在弱阳性样本中存在漏检。初步认为干血斑样本可用于进行梅毒抗体检测,为干血斑样本用于梅毒抗体检测应用具有重要参考价值,但仍需改进并进行基于大量临床样本验证。

Objective To validate the performance of ELISA method based on dried blood spots sample(DBS) for anti-TP test(Treponema pallidum, TP) and to evaluate the preliminary application, so as to provide experimental data for DBS application in anti-TP test. Methods By building the lab linear panel, the lab basic panel, lab analytical specificity panel and lab precision panel, the performance of the established method and stability of DBS samples was assessed. A total of 329 blood samples were collected and anti-TP test in plasma and DBS was performed by ELISA. Compared to plasma as a standard reference, clinical sensitivity and clinical specificity of DBS anti-TP test were evaluated. The agreement and correlation were performed between DBS versus plasma. Results The established method had a good linear range of 23-27(R^2=0.98, P < 0.05);the positive/negative coincident rate were 15/15;DBS anti-TP test was not influenced by other non-specific interfering substances or co-infection. Precision analysis presented that the CV of inter-days were 0.49%-6.21%;the CV of intra-assay 0.49%-10.31%;and the CV of inter-spots 0.51%-7.09%. Compared to plasma, DBS anti-TP test had a sensitivity of 86.5%(95%CI: 0.70-0.95) and a specificity of 100.0%(95%CI: 0.98-1.00). The PPV and NPV were100%(95%CI: 0.87-1.00), and 98.3 %(95%CI: 0.96-0.99). The Spearman correlation coefficient(r) was 0.204(P < 0.01), and Kappa was 0.92(95%CI: 0.85-0.99).The distributions comparison of S/CO value showed that they had similar characteristics. Conclusion DBS anti-TP by ELISA has a good precision and anti-interference, and DBS sample has a stable preservation. There is a good concordance for anti-TP test between plasma and DBS, with undetectable anti-TP found in the weak positive plasma DBS samples. The study indicated that DBS samples can be used to anti-TP test. However, it still needs improvement and further verification based on large number of clinical samples.