Flow cytometric measurements of TB-specific T cells comparing with quantiFERON-TB gold

Background: Interferon-γ (IFN-γ) release assays and the detection of IFN-γ synthesis in the cytoplasm of activated CD41 T cells by flow cytometry have recently been used for tuberculosis (TB) diagnosis. The aim of this study was to compare the performance of IFN-γ assay between ELISA (QuantiFERON-TB Gold, QFT) and intracellular cytokine flow cytometry (ICCFC), and to investigate the significance of an optimal gating strategy in ICCFC. Methods: The CD41 T cell response to TB antigens was measured using the intracellular cytokine staining technique and four color FC (CD3, CD4, IFN-γ, and tumor necrosis factor-α (TNF-α)) on whole blood samples. The results were compared with those of QFT. Results: Regarding sensitivity in the TB group, patients in the QFT positive TB group (N = 22) were all ICCFC positive and 9 patients (64%) in the QFT negative TB group (N = 14) were ICCFC positive. In all test tubes (N = 72), sensitivity of "targeted" gating for TNF-α+ IFN-γ+ CD4+ T cells was significantly higher than customary gating (72%, 54%, respectively, P = 0.001). Conclusions: The diagnostic sensitivity of ICCFC was further confirmed to be much higher than that of QFT. In the ICCFC analysis, TNF-α+ IFN-yγ+ CD4+ T cells should be sequentially gated through appropriately defined regions, minimizing interferents and reflecting characteristics of light scatter and marker expressions of CD4+ T cells activated by TB antigens.