Neutrophil extracellular traps (NETs) in aortic stenosis: Comparison of methods for assessment of NETs formation

Introduction. We previously showed increased neutrophil extracellular traps (NETs) formation (NETosis) in patients with severe aortic stenosis (AS). Our aim was to comprehensively assess NETosis in AS using a relatively simple and appropriate technique. We investigated circulating NETosis markers by ELISA and assessed the potential of blood neutrophils to release NETs by flow cytometry.Materials and Methods. We enrolled 13 patients aged 66 [Q1-Q3, 60-70] years with severe isolated AS without diabetes, chronic kidney disease, and atrial fibrillation. Nine apparently healthy volunteers of similar sex and age served as controls. Serum concentrations of citrullinated histone H3 (citH3), circulating nucleosomes, myeloperoxidase (MPO), and deoxyribonuclease-1 (DNASE1) were measured using ELISAs. Peripheral blood NET-releasing neutrophils were detected by flow cytometry as MPO/citH3-positive cells.Results. AS patients compared to controls presented 174% higher concentrations of citH3 (p<0.001), 456% higher nucleosomes (p<0.001), 136% higher MPO (p=0.021) and 19% higher DNASE1 levels (p=0.039), together with 101% elevated percentage of NET-releasing neutrophils assessed by flow cytometry (p=0.003). In AS patients, the proportion of blood NET-releasing neutrophils positively correlated with citH3 (r=0.86, p<0.001) and nucleosome (r=0.58, p=0.041) concentrations but not with MPO or DNASE1. Moreover, we observed a strong association between AS severity, measured as aortic valve area (AVA), and serum citH3 concentrations (r=-0.75, p=0.003), but not the number of NET-releasing neutrophils.Conclusions. Our study showed increased blood neutrophil potential to release NETs together with increased levels of serum markers of NETos is in severe AS patients. However, we recommend ELISA to assess NETosis in vivo as simpler technique giving more unequivocal results compared to flow cytometry.