Janossy George, Barry Simon M, Breen Ronan A M, Hardy Gareth A D, Lipman Marc, Kern Florian
Department of Immunology, Royal Free and University College Medical School, London, United Kingdom.
Cytometry B Clin Cytom. 2008;74 Suppl 1:S141-51. doi: 10.1002/cyto.b.20381.
TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.
结核病仍然未得到有效控制。在资源丰富的国家，只有约60%的诊断通过培养得到确认。在资源匮乏的环境中，这一比例更低。因此，替代诊断策略存在巨大的发展空间。通过结核抗原刺激8 - 16小时后根据细胞因子产生情况来计数抗原特异性淋巴细胞，是目前的首选策略。有几种方法，包括酶联免疫吸附测定（ELISA）、酶联免疫斑点法（ELISpots）和流式细胞术。虽然很明显，受早期分泌性抗原靶6（ESAT - 6）和培养滤液蛋白10（CFP - 10）抗原刺激的血样能够区分结核感染和卡介苗接种，但似乎只有流式细胞术能够区分活动性结核病与单纯的结核暴露。在各种流式检测方案中，包括四色检测（CD45 - CD3 - CD4 - IFNγ）、三色检测（CD3 - CD4 - IFNγ）和双色检测（CD4 - IFNγ），即使是最简单的方法也表现良好，前提是结果以每CD4 +淋巴细胞中IFN - γ +细胞的百分比（%IFNγ/CD4 +）来表示。使用支气管肺泡灌洗（BAL）和诱导痰（ISp）的研究表明，在肺结核和肺外结核中，结核特异性CD4 + IFN - γ + T细胞在肺部的积聚频率比在血液中高5 - 20倍以上。卡介苗免疫后不存在这种肺归巢现象。在活动性结核病中，使用结核菌素纯蛋白衍生物（PPD）刺激肺部的CD4 + IFN - γ +细胞所产生的反应比使用CFP - 10或ESAT - 6时大3 - 12倍，并且不存在卡介苗接种的任何干扰。这种方法不受人类免疫缺陷病毒（HIV）合并感染的影响，而HIV合并感染一直是其他基于免疫的诊断方法所面临的问题。此外，基于肺部的样本为IFN - γ检测和细菌学的快速检测提供了材料，重要的是，这些检测适合未来使用自动荧光图像细胞仪进行简化。流式细胞术多参数分析能力的另一个发展是使用血液中CD4 + T细胞“归巢至肺”群体的标志物，从而无需进行肺部采样。在活动性结核病患者中，而非卡介苗接种者中，可以在血液中发现结核特异性记忆CD4 + T细胞，这些细胞主要为CD27阴性，可能归巢至肺，并且可能具有诊断价值。
