The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: i) Short-term storage (<8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; ii) Neutrophils from long-term storage (>24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.