Variability in TLR function influences susceptibility to infectious as well as immune-mediated diseases. Given the outbred nature of humans, identifying functional Toll-like receptor variability and its role in clinical disease requires such analysis to be conducted in large, often multi-centered cohorts. Yet the technically complex measurements involved in innate immune analysis benefit from centralized processing of samples. Centralization requires shipping of samples or prolonged storage, possibly even cryopreservation. Deviation from standard operating procedures (SOP) for sample procurement, storage and processing may alter the final innate immune read out. We here set out to define the impact of variables most likely to be encountered during large, multi-site studies: (i) the source of the sample, (ii) time between sample procurement to processing, and (iii) processing of fresh vs. cryopreserved samples. We found that all of these variables exert a profound impact on the final innate response to TLR stimulation. Specific innate responses appeared to be affected in response to specific TLR stimuli by a particular variable under study, proving it impossible to provide global generalizations. Based on our studies and other published work on this topic, we propose a minimal list of variables that have to be met for samples to be comparable within and across studies: a) timing between procurement and processing cannot vary by more than 10%; b) all samples have to be stored the same; and c) the source of samples needs to be the same. In summary, for innate immune analysis scrupulous adherence to standard operating procedures is paramount.
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