Increased plasma NETs correlate with increased COVID-19 severity. (A) We stained for neutrophils and NETs, using immunofluorescence, in lung tissue obtained at autopsy from COVID-19 patients (n = 3). Commercially available normal lung tissue was stained as a negative control. Neutrophils express MPO (red), and early-stage NET-forming neutrophils also express citrullinated histone H3 (green). DAPI serves as a nuclear DNA counterstain (blue). Cyan fluorescence represents the colocalization of citrullinated histone H3 with DNA. The yellow arrowheads point to neutrophils not making NETs, and the white arrows point to neutrophils making NETs. The dashed line highlights a thrombus in the microvasculature (top right panel). Scale bar, 100 μm. Higher-magnification images of the boxed areas in the top row (middle row) are shown along with single-channel images (bottom row); scale bars, 50 μm. (B-F) We used MPO-DNA ELISA to assess NETs in plasma and tracheal aspirate samples from patients in our COVID-19 prospective cohort and in age- and sex-matched healthy donors; each colored dot represents an individual participant. (B) We compared plasma NET levels across all groups: healthy adult donors (n = 17), adults hospitalized with COVID-19 but not intubated for ARDS (n = 22), adults intubated for COVID-19 ARDS (n = 6), and adults recovered from COVID-19 (n = 5). The y-axis depicts plasma NETs expressed as a percentage of healthy adult controls ± SD, arbitrarily set at 100%. (C) We compared plasma NET levels in 2 groups of COVID-19 hospitalized patients: survivors (n = 24) vs nonsurvivors (n = 4). The y-axis depicts plasma NETs expressed as a percentage of healthy adult donors ± SD, arbitrarily set at 100% (dashed line). (D) We correlated plasma NET levels with the Pao₂/FiO₂ ratio measure of respiratory failure for all hospitalized COVID-19 patients (n = 28). (E) We correlated plasma NET levels with the SOFA Illness Severity Scores for all hospitalized COVID-19 patients (n = 28). (F) We compared plasma NET levels in adult COVID-19 patients (n = 28) with NET levels quantified in the available tracheal aspirate samples of intubated COVID-19 patients (n = 3). The y-axis depicts NET levels expressed as a percentage of healthy adult donors ± SD, arbitrarily set at 100% (dashed line). One-way ANOVA with Tukey’s multiple-comparisons post hoc testing (B-C), Spearman’s rank-correlation test (D-E), Student t test (F).