结核病患者外周血淋巴细胞亚群以及12项血浆细胞因子流式检测分析

Analysis of peripheral blood lymphocyte subsets and 12 plasma cytokines by flow cytometry in patients with tuberculosis

目的:分析结核病患者淋巴细胞亚群和12项血浆细胞因子的流式检测结果,探讨其在结核病中的诊断效能。方法:回顾性病例对照研究。选取2022年1月至2023年12月在解放军总医院第八医学中心初诊的有病原学证据或临床确诊的结核病患者128例为研究对象,根据结核分枝杆菌感染部位分为肺结核组(83例)与肺外结核组(45例)。另选择性别、年龄与结核病患者相匹配的健康体检者100例为对照组。采用流式细胞术检测各组外周血淋巴细胞亚群和12项血浆细胞因子[包括10项促炎因子:白细胞介素(IL)-5、干扰素(IFN)-α、IL-2、IL-6、IL-1β、IFN-γ、IL-8、IL-17、IL-12p70、肿瘤坏死因子(TNF)-α和2项抑炎因子:IL-4、IL-10]水平。比较各组淋巴细胞亚群和血浆细胞因子水平的差异,采用Spearman相关法分析淋巴细胞亚群与细胞因子的相关性,二元Logistic回归筛选结核病相关因素,受试者工作曲线(ROC)评价结核病相关因素的诊断效能。结果:与对照组比较,肺结核组和肺外结核组CD3+T淋巴细胞、CD3+CD8+T淋巴细胞、CD3+CD4+T淋巴细胞、NK细胞和B细胞绝对数较低(P均<0.05),肺结核组除IL-1β外的其他11项细胞因子水平均较高(P均<0.01),肺外结核组IL-6、IFN-γ、IL-17、TNF-α、IL-4和IL-10水平较高(P均<0.05)。与肺外结核组比较,肺结核组IL-8水平较高(P=0.026)。Spearman相关性分析显示,IL-6、IFN-γ、IL-8分别与CD3+T淋巴细胞、CD3+CD8+T淋巴细胞、CD3+CD4+T淋巴细胞、NK细胞和B细胞绝对数均呈负相关(IL-6:R^(2)=-0.30、-0.28、-0.32、-0.26、-0.28;IFN-γ:R^(2)=-0.36、-0.31、-0.37、-0.25、-0.36;IL-8:R^(2)=-0.14、-0.13、-0.16、-0.14、-0.22;P均<0.001),IL-10与CD3+CD4+T淋巴细胞、NK细胞和B细胞绝对数呈负相关(R^(2)=-0.14、-0.19、-0.21,P均<0.05);二元Logistic回归分析显示,IL-6、IFN-γ、IL-8、IL-10是结核病的相关因素(OR=1.809、1.136、0.910、2.218,P均<0.05);ROC曲线分析显示IL-6、IFN-γ、IL-8、IL-10联合诊断结核病的ROC曲线下面积为0.845,敏感度为0.766,特异度为0.820。结论:肺结核和肺外结核患者的淋巴细胞亚群绝对数和细胞因子水平显示其免疫功能处于低下状态,促炎因子(IL-6、IFN-γ、IL-8)和抑炎因子(IL-10)水平明显升高,4项细胞因子联合检测对结核病具有较高的诊断效能。

Objective To analyze the results of lymphocyte subsets and 12 plasma cytokines in patients with tuberculosis by flow cytometry and to evaluate their diagnostic efficacy in these patients.Methods This is a retrospective case-control study.A total of 128 patients with evidence of tuberculosis disease or clinically confirmed tuberculosis who were admitted to the 8th Medical Center of PLA General Hospital from January 2022 to December 2023 were included.According to the location of mycobacterium tuberculosis infection,the patients were divided into the pulmonary tuberculosis group(83 cases)and the extrapulmonary tuberculosis group(45 cases),and 100 healthy age-and sex matched people who underwent health check up during the study period were selected as the control group.Flow cytometry was used to detect peripheral blood lymphocyte subsets and 12 plasma cytokines[including 10 pro-inflammatory factors:interleukin(IL)-5,interferon(IFN)-α,IL-2,IL-6,IL-1β,IFN-γ,IL-8,IL-17,IL-12P70,Tumor necrosis factor(TNF)-α,and two anti-inflammatory factors:IL-4,IL-10]in participants of all groups.Spearman correlation method was used to analyze the correlation between lymphocyte subsets and cytokines,binary Logistic regression was used to screen the TB related factors,and receiver operating curve(ROC)was used to evaluate the diagnostic efficacy of TB related factors.Results Compared with the control group,the absolute number of CD3+T lymphocytes,CD3+CD8+T lymphocytes,CD3+CD4+T lymphocytes,NK cells and B cells were lower in pulmonary tuberculosis group and extrapulmonary tuberculosis group(all P<0.05).Except for IL-1β,the levels of other 11 cytokines are all significantly higher in the pulmonary tuberculosis group(all P<0.01),and the levels of IL-6,IFN-γ,IL-17,TNF-α,IL-4 and IL-10 were significantly higher in extrapulmonary tuberculosis group(all P<0.05).Compared with extrapulmonary tuberculosis group,the level of IL-8 was higher in pulmonary tuberculosis group(P=0.026).Spearman correlation analysis showed that IL-6,IFN-γand IL-8 were negatively correlated with the absolute numbers of CD3+T lymphocytes,CD3+CD8+T lymphocytes,CD3+CD4+T lymphocytes,NK cells and B cells(IL-6:R^(2)=-0.30,-0.28,-0.32,-0.26,-0.28;IFN-γ:R^(2)=-0.36,-0.31,-0.37,-0.25,-0.36;IL-8:R^(2)=-0.14,-0.13,-0.16,-0.14,-0.22;all P<0.05),IL-10 was negatively correlated with the absolute number of CD3+CD4+T lymphocytes,NK cells and B cells(R^(2)=-0.14,-0.19,-0.21,all P<0.05);Binary Logistic regression analysis showed that IL-6,IFN-γ,IL-8 and IL-10 were the related factors of tuberculosis(OR=1.809,1.136,0.910,2.218,all P<0.05),ROC curve analysis showed that the AUC of IL-6,IFN-γ,IL-8 and IL-10 in the joint diagnosis of tuberculosis was 0.845,the sensitivity was 0.766,and the specificity was 0.820.Conclusion The lower absolute number of lymphocyte subsets and cytokine levels in patients with pulmonary tuberculosis and extrapulmonary tuberculosis indicate that their immune function is in a low state,and the higher levels of pro-inflammatory factors(IL-6,IFN-γ,IL-8)and anti-inflammatory factor(IL-10)indicates the higher inflammatory status,and evaluation of these 4 cytokines has satisfactory diagnostic efficacy for tuberculosis.