1b). Calculation of reproducibility of the cytokines induced by H3N2 or Con A resulted in check details CV values ranging between 5% and 32% and 2–45%, respectively (Table 2). These CV

values are considered to be acceptable bioassay limits [34]. Only for IL-17 detection, the CV value for repeated analysis of influenza induced culture supernatant was above 50%, which may be due to the fact that the CV increases at levels approaching the detection limit [34] and [35]. Indeed, the IL-17 CV was below 20% for Con A induced IL-17 responses that were well above the detection limit. As described above, the cytokine assay shows acceptable variability on standard samples of culture supernatant. For the ultimate application of the assay in large scale vaccine trials, we determined the overall robustness by using PBMC for validation. Each research group performed the standard stimulation procedure on four different days with the same batch of frozen PBMC isolated from two donors. Supernatants were collected and analyzed. After stimulation with H3N2, significant productions of IFN-γ, TNF-α, IL-2, IL-10 and in addition for donor 1 of IL-4, IL-13 and GM-CSF were detected (Fig. Perifosine price 3a). For these cytokines and the log IFN-γ/IL-10

ratio (Fig. 3b), the intra-laboratory robustness was 52% and the inter-laboratory robustness was 49% (Table 3). In addition, all laboratories determined similar cytokine productions and significant differences in mock or H3N2-specific responses (Supplementary Table 1). Influenza H3N2-specific production of IL-17 was absent (not shown). Importantly, Con A stimulation resulted in upregulation of all cytokines, indicating that the PBMC were viable and capable of producing all CYTH4 ten cytokines that were analyzed. Moreover, all laboratories found higher levels of IFN-γ, IL-10 and IFN-γ:IL-10 ratios in donor 1 as compared to donor 2. Collectively, these data indicate that the cytokine detection assay is robust and capable of generating similar responses between different laboratories. This study introduces two standardized and validated

assays for determining influenza vaccine efficacy based on PBMC responses. The cytokine and granzyme B assays allowed to distinguish between high and low responses of PBMC isolated from different donors. In addition, significant differences were observed between negative control (mock) and influenza-specific responses. Most importantly, the assays showed mean inter-laboratory robustness CV values of lower than 50%. Although specific guidelines setting minimal requirements for CV values of assays determining influenza immune responses in man are lacking, our validation results are within an acceptable range considering the European Pharmacopoeia Guidelines for vaccine studies in animals [37], [38] and [39]. The validated assays have distinctive strengths, since they were developed to reliably detect low or high PBMC responses.