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. 2021 Jun 23;11:13190. doi: 10.1038/s41598-021-92596-z

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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(A) TAM-based assay procedure overview from blood collection to delivery of results within 24 h. Briefly, a single millilitre of fresh blood was subjected to red blood cell lysis (steps 1 & 2) and white blood cells evenly split between 4 tubes before overnight stimulation (step 3). The following morning, cells were fixed and stained with fluorescent antibodies as detailed in the methods section (step 4). Samples were immediately acquired on a FACSCalibur delivering final results within 24 h following phlebotomy (steps 5 & 6). This figure was created using objects from Servier Medical Art, free to share and adapt, licensed under a Creative Commons Attribution 3.0 Unported License (CC BY 3.0) (http://smart.servier.com/). (B) Representative gating strategy of the TAM-based assay adapted for a 2-laser flow cytometer FACSCalibur apparatus. First, a gate based on Forward- (FSC) and Side-Scattered (SSC) signals is drawn to isolate lymphocytes from other blood cell types. Second, a CD4 T cell gate is obtained by tearing apart non-T cells in the y-axis from CD8 T cells in the x-axis. This gating strategy is applied to compare cytokine production and expression of CD27 or CD38 by CD4 T cells in samples stimulated or not with a Mycobacterium tuberculosis peptide pool. The index test result is obtained by dividing the frequency of CD4 T cells producing IFN-γ or TNF-α that express the investigated biomarker (Quadrant 2, Q2) by the frequency of those that did not express the biomarker (Q3).