Microliter Whole Blood Neutrophil Assay Preserving Physiological Lifespan and Functional Heterogeneity

Abstract For in vitro neutrophil functional assays, neutrophils are typically isolated from whole blood, having the target cells exposed to an artificial microenvironment with altered kinetics. Isolated neutrophils exhibit limited lifespans of only a few hours ex vivo, significantly shorter than the 3–5 day lifespan of neutrophils in vivo. In addition, due to neutrophils’ inherently high sensitivity, neutrophils removed from whole blood exhibit stochastic non‐specific activation that contributes to assay variability. Here, a method – named “µ‐Blood” – is presented that enables functional neutrophil assays using a microliter of unprocessed whole blood. µ‐Blood allows multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control). In µ‐Blood, neutrophils show sustained migration and limited non‐specific activation kinetics (<0.1% non‐specific activation) over 3–6 days. In contrast, neutrophils isolated using traditional methods show increased and divergent activation kinetics (10–70% non‐specific activation) in only 3 h. Finally, µ‐Blood allows the capture and quantitative comparison of distinct neutrophil functional heterogeneity between healthy donors and cancer patients in response to microbial stimuli with the preserved physiological lifespan over 6 days.