Performance qualification of a 13-color intracellular cytokine staining assay for the assessment of T cell responses in human PBMCs (TECH3P.932)

Abstract One challenge to tuberculosis vaccine research is the lack of a correlate of immunity, which researchers are attempting to address through use of more high-throughput, high-data assays for assessing trial specimens. Multiparameter intracellular cytokine staining (ICS) assays have proven to be one of the most cost-effective tools available. Previously, we reported the development of a 13-color ICS panel for use in assessing T cell responses from human PBMCs. In an effort to better understand the metrics of the assay performance, a qualification study under GCLP conditions was executed. The parameters assessed in this performance qualification included specificity, sensitivity, selectivity, linearity, and intermediate precision. The lower limits of quantification for cytokine detection were shown to be under 0.03%. Using Fisher’s exact test to determine positivity, the false positive and false negative rates were shown to be 0% in this performance qualification. Furthermore, assessment of intermediate precision suggested that the CD3, CD4, CD8, CCR7, and CD45RO markers each showed a percent coefficient of variation (%CV) of 25% or under, while intracellular and functional markers were generally under a 30% CV as measured via responses to Staphylococcal enterotoxin B. The results of this performance qualification study illustrate the capabilities of this panel for assessing clinical trial responses and suggest it is a reliable assay for the assessment of T cell responses.