Flow cytometric measurements of TB-specific T cells comparing with QuantiFERON-TB gold.
BACKGROUND
Interferon-gamma (IFN-gamma) release assays and the detection of IFN-gamma synthesis in the cytoplasm of activated CD4+ 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-gamma 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 CD4+ T cell response to TB antigens was measured using the intracellular cytokine staining technique and four color FC (CD3, CD4, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha)) 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-alpha+ IFN-gamma+ 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-alpha+ IFN-gamma+ 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.
Won
Dong Il
DI
Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Republic of Korea. wondi@knu.ac.kr
Park
Jung Ran
JR
D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*
D000942:Antigens, Bacterial; D007371:Interferon-gamma