COVID-19 PMNs demonstrate increased activation and NETs at baseline and fail to respond to PMA with increased NET formation. (A) We assessed granularity using flow cytometry in PMNs isolated from healthy donors (n = 6) and COVID-19 patients (n = 4) as a measure of baseline PMN activation. (B-C) We assessed NETs in PMNs isolated from healthy donors (n = 5) and COVID-19 patients (n = 4), with and without PMA stimulation (20 nM; 2 hours), using confocal microscopy and cell-free DNA quantitation. (B) Representative images show NETs (magenta; white arrows) and nuclear DNA (green). Scale bars, 20 μm. (C) We quantified NETs using a fluorescence-based cell-free DNA assay as described in Patients and methods. The y-axis depicts NETs shown as relative cell-free DNA fluorescence ± SEM. Student t test (A), 1-way ANOVA with Tukey’s multiple-comparisons post hoc test (C). n.s., not significant.