酶联免疫斑点术和流式细胞术检测γ干扰素对结核性胸膜炎的辅助诊断价值

目的 探讨胸腔积液PEMC中CD+4T淋巴细胞分泌或产生IFN-γ的检测对结核性胸膜炎的辅助诊断价值.方法 分离40例结核性胸腔积液(结核组)和30例恶性胸腔积液患者(疾病对照组)PEMC,经克隆表达的早期分泌抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)融合蛋白(简称"E/C")刺激后,用ELISpot检测分泌IFN-γ细胞的斑点形成细胞(SFC)数量和流式细胞术(FCM)结合胞内细胞因子染色检测产生IFN-γ细胞比率,并比较这2项指标在结核组和疾病对照组间的差异,同时评价2种方法检测IFN-γ对结核性胸膜炎的辅助诊断效能.结果 PEMC经E/C刺激后用ELISpot检测结核组SFC数量为205(125 ～450)SFC/5×104PEMC,疾病对照组为5(2～18)SFC/5×104PEMC,两组间的差异有统计学意义(U=20.00,P<0.01);FCM检测结核组CD+4T淋巴细胞产生IFN-γ细胞比率为3.27%(1.81%～7.34%),疾病对照组为0.12%(0.06%～0.46%),两组间的差异有统计学意义(U=45.00,P<0.01).ELISpot检测PEMC经E/C刺激后分泌IFN-γ的敏感度为92.5%(37/40)、特异度为80.0%(24/30)、阳性预测值为0.86、阴性预测值为0.89、阳性似然比为4.63和阴性似然比为0.09,准确性为87.1%;FCM检测经E/C刺激后产生IFN-γ的敏感度为87.5%(35/40)、特异度为90.0%(27/30)、阳性预测值为0.92、阴性预测值为0.84、阳性似然比为8.75和阴性似然比为0.14,准确性为88.6%.结论 经E/C刺激后,用FCM和ELISpot检测CD+4T淋巴细胞产生和分泌IFN-γ细胞的方法对结核性胸膜炎诊断的敏感度和特异度高,且对结核性胸膜炎有辅助诊断价值.

Abstract：

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method.