Fig. 1.

Variability of the WB-ICS assay for detecting cytokine-expressing CD4 and CD8 T cells. Establishing the lower limit of quantification of the WB-ICS assay (A–D). Unstimulated cells were spiked with a series of 2-fold diluted BCG-stimulated cells, then stained and analysed. Expected frequencies of cytokine positive cells were derived by dividing the frequencies of BCG-specific cytokine-expressing CD4 and CD8 T cells in the undiluted samples by the dilution factor. The plots show the expected and observed frequencies of BCG-specific IFN-γ (A), TNF-α (B), IL-2 (C) and IL-17 (D) CD4 T cells for each dilution factor. Establishing variability in detection of cytokine-expressing CD4 and CD8 T cells (E–H). Aliquots of fixed cells from a single BCG-stimulated (and unstimulated) whole blood sample were thawed and stained independently by two operators on 4 different days. For each operator on each day, 2–5 aliquots of cells were stained and acquired to calculate the coefficient of variation (CV) for frequencies of BCG-specific IFN-γ-, TNF-α-, IL-2- or IL-17-expressing CD8 T cells (E, F) and CD4 T cells (G, H) for one operator are shown. The vertical dashed lines are a 30% CV mark. Frequencies of cytokine-expressing CD8 T cells with CVs less than 30% (IFN-γ) and more than 30% (TNF-α-, IL-2- and IL-17) (panels E and F) are shown. For each repeat, frequencies detected in the unstimulated sample were subtracted from frequencies in the respective BCG-stimulated sample. (I) Frequencies of non-specific IFN-γ-, TNF-α-, IL-2- or IL-17-expressing CD4 T cells, detected in unstimulated cells, in 54 adults. Horizontal lines within the boxes represent medians, boxes represent the IQR and whiskers represent the range. The black horizontal lines with stars represent the established limit of reliable quantification of the flow cytometer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)