The role of flow cytometry in the interferon-gamma-based diagnosis of active tuberculosis and its coinfection with HIV-1--A technically oriented review.

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.