Ex vivo cytokine induction in whole blood
HEAT
Ex vivo cytokine induction in blood was evaluated using the Rules-Based Medicine (RBM) TruCulture system containing either of the TLR stimulants R848 or LPS. An unstimulated null tube was also collected at each timepoint to evaluate the effect of stimulation in each patient for each analyte. One milliliter of whole blood was collected directly into each of the three TruCulture tubes. Once blood was collected, TruCulture tubes were inverted 3–5 times and immediately placed on a 37 °C heat block and incubated overnight and up to 24 h. Cells were manually separated from the supernatant using the provided filter system, and the entire sample was then frozen at −20 °C and batch shipped to RBM for analysis. Samples were run on the OptiMAP 13 analyte panel to measure changes in cytokine levels. The percent change from baseline for each analyte was calculated by subtracting the post-dose from the pre-dose and then dividing by the pre-dose. Our internal criteria for response of cytokines for each analyte was ≥2× the value of the unstimulated (null) baseline value. The TruCulture system with OptiMAP detection is a commercially available and clinically validated system that was developed by RBM.