In vitro neutrophil functional assay in microliter whole blood for days-long extraction of donor-specific information.

For in vitro neutrophil functional assays, isolated neutrophils are typically used as the standard input. However, isolated neutrophils show a varying and limited lifespan as short as only a few hours ex vivo , significantly shorter than the lifespan of neutrophils in vivo up to 3-5 days. The limited assay time window leads to a significant loss of donor-specific information, which is attributed to non-specific activation (i.e., baseline cell death without operator-defined stimuli) of neutrophils after being pulled out of whole blood. In addition, due to neutrophils' inherently high sensitivity, removing neutrophils from whole blood generates operator-derived inconsistencies in the information extracted from the assays. Here we present a method - named "μ-Blood" - that supports long-term (days) multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control) using a microliter of unprocessed whole blood. In autologous whole blood, neutrophils show sustained migration and limited non-specific activation kinetics [<0.1% non-specific activation >3 days]. By contrast, isolated neutrophils show altered activation kinetics [10-70% non-specific activation in 3 h and diminished (>90%) recruitment]. Using unprocessed whole blood, μ-Blood captures a distinct neutrophil functional heterogeneity between healthy donors and cancer patients against microbial stimuli through days-long assays, showing the potential for in vitro immune assays with improved extraction of donor-specific information and assay consistency over time.