Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.

Introduction: After worldwide closures due to the COVID-19 pandemic, schools have reopened in most European countries in late 2020. Consequently, for children with chronic diseases the risks of COVID-19 have to be weighed against the long-time risks of missing school.Methods: To evaluate the impact of chronic diseases on school attendance for children in Europe during the COVID-19 pandemic we conducted a survey among members of the European Society for Pediatric Nephrology (ESPN) between September and November 2020. We asked for current forms of schooling, the existence of national guidelines, parental concerns, and the pediatric nephrologists recommendations for school attendance for specific virtual patients with chronic kidney disease (CKD).Results: Recommendations varied widely among pediatric nephrologists. A minority stated that specific recommendations for COVID-19 risk in children with kidney diseases existed in their country from local health authorities (9 of 29 countries; 31%) and/or national pediatric nephrology societies (9 of 29 countries; 31%). Over 90% of physicians have experienced parents keeping their children out of school against medical advice of their health providers and about 50% have experienced their patients being refused by school authorities. Consequently, 25% of all pediatric nephrologists estimated that more than 10% of their patients will not attend school regularly.Conclusion: COVID-19 causes educational deficits in the already vulnerable population of children with CKD. As the evidence for the course of COVID-19 in children with chronic diseases grows, rapidly adapted recommendations from pediatric societies could help reduce uncertainty among doctors, patients, and parents.

ObjectiveWe developed a fiberoptic-assisted tracheoscopy (FAST) method to avoid direct laryngoscopy during surfactant replacement therapy and compared two training approaches on a very low birth weight (VLBW) infant simulator.DesignThis prospective randomized controlled study was conducted at the Department of Neonatology and Pediatric Intensive Care Medicine of the University Medical Center Hamburg-Eppendorf, Germany.ParticipantsWe recruited physicians, trainees, students, and nurses without prior experience in endoscopic techniques.InterventionsParticipants were assigned randomly to a group that received instructions according to Peyton’s Four-Step Approach and a control group that received standard bedside teaching only.Main outcome measuresPrimary endpoints were the total and the component times required to place the bronchoscope and the method success.ResultsWe recruited 186 participants. Compared with the control group, the Peyton group had a lower mean (±standard deviation) FAST completion time (33.2 ± 27.5 s vs. 79.5 ± 47.9 s, p < 0.001; d = 1.12) and a higher FAST success rate (95% vs. 84%, p = 0.036, V = 0.18).ConclusionAfter standardized training, the vast majority of novices completed FAST successfully. Peyton’s four-step approach resulted in faster and more successful performance than standardized training.