Intracellular cytokine flow cytometry (ICCFC) for measurements of TB-specific T cells

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Abstract

Background
Tuberculosis (TB) is a communicable disease that is a major cause of ill health, one of the top 10 causes of death worldwide, and the leading cause of death from a single infectious agent (ranking above HIV/AIDS). Multifunctional T cells, defined by their ability to coexpress two or more cytokines, e.g., (TNF-α and IFN-γ), have shown a good diagnostic yield to detect TB infection.
The aim of the study
Assessment of the diagnostic sensitivity of intracellular cytokine flow cytometry analysis of multifunctional specific CD4+ T cells coexpressing TNF-α and IFN-γ after stimulation with phytohemagglutinin for the diagnosis of active pulmonary TB.
Patients and methods
This study was performed on 30 active pulmonary tuberculosis (APTB) patients who were admitted to Fayoum Chest Hospital and 30 healthy controls. CD4+ T cells were stimulated by phytohemagglutinin (PHA) and then were measured using the intracellular cytokine staining technique by flow cytometry to detect CD4+ T cells expressing TNF-α and IFN-γ on whole-blood samples.
Results
Receiver-operator characteristic (ROC) analysis was done to determine the optimum cutoff value for the studied diagnostic markers, which was the difference between the percentages of CD4 lymphocytes coexpressing TNF+α and IFN+γ before phytohemagglutinin (PHA) stimulation (inactivated CD4+) and after phytohemagglutinin (PHA) stimulation (activated CD4) in the cases and controls, which was 4.9 with sensitivity 90% and specificity 50%. Using this cutoff, it scored 27 (90%) as positive from 30 APTB of active TB patients and 15 (50%) as negative from 30 of healthy controls.
Conclusion
Multifunctional flow cytometry analysis of specific CD4+ T-cell response may represent a relatively simple and rapid immune-based approach to distinguish between MTB-infected and -uninfected patients.

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