The role of flow cytometry in the interferon‐γ‐based diagnosis of active tuberculosis and its coinfection with HIV‐1—A technically oriented review

• Published in: Cytometry. Part B, Clinical cytometry Clinical cytometry, 2008, Vol.74B (S1), p.S141-S151
• Main Authors: Janossy, George, Barry, Simon M., Breen, Ronan A. M., Hardy, Gareth A. D., Lipman, Marc, Kern, Florian
• Format: Article
• Language: English
• Subjects: Antigens, Bacterial; Bacterial Proteins; bronchoalveolar lavage; CD4; ELISpot; flow cytometry; Flow Cytometry - methods; HIV Infections - complications; HIV Infections - diagnosis; human immunodeficiency virus; Humans; immunological monitoring; Interferon-gamma; interferon‐γ; sputum; Tuberculin - blood; tuberculosis; Tuberculosis, Pulmonary - complications; Tuberculosis, Pulmonary - diagnosis; Tuberculosis, Pulmonary - pathology
• ISSN: 1552-4949; 1552-4957
• DOI: 10.1002/cyto.b.20381

TB remains uncontrolled. In resource‐rich countries, only ∼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‐IFNγ), three‐color tests (CD3‐CD4‐IFNγ) and two‐color tests (CD4‐IFNγ), even the simplest is performing well, provided that the results are expressed as percentage of IFN‐γ+ cells per CD4+ lymphocytes (%IFNγ/CD4+). Studies using broncho‐alveloar lavage (BAL) and Induced‐Sputum (ISp) show that TB‐specific CD4+IFN‐γ+ 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‐γ+ 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‐γ 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. © 2007 Clinical Cytometry Society