A Novel Assay for Neutrophil Extracellular Traps (NETs) Formation Independently Predicts Disseminated Intravascular Coagulation and Mortality in Critically Ill Patients

Rational: Neutrophil extracellular traps (NETs) are important in the host defence against infection, but also promote intravascular coagulation and multi-organ failure (MOF) in animal models. Its clinical significance remains unclear and available assays for patient care lack specificity and reliability. Objectives: To establish a novel assay and test its clinical significance Methods: A prospective cohort of 341 consecutive adult ICU patients was recruited. The NETs-forming capacity of ICU admission blood samples was semi-quantified by directly incubating patient plasma with isolated neutrophils ex vivo. The association of NETs–forming capacity with sequential organ failure assessment (SOFA) scores, disseminated intravascular coagulation (DIC) and 28-day mortality were analysed and compared with available NETs assays. Measurements and Main Results: Using the novel assay, we could stratify ICU patients into 4 groups with absent (22.0%), mild (49.9%), moderate (14.4%) and strong (13.8%) NETs formation, respectively. Strong NETs formation was predominantly found in sepsis (P <0.0001). Adjusted by APACHE II, multivariate regression showed that the degree of NETs formation could independently predict DIC and mortality whereas other NETs assays, e.g. cell-free DNA, myeloperoxidase and myeloperoxidase-DNA complexes, could not. IL-8 levels were found to be strongly associated with NETs formation and inhibiting IL-8 significantly attenuated NETosis. MAPK activation by IL-8 has been identified as a major pathway of NETs formation in patients. Conclusions: This assay directly measures the NETs-forming capacity in patient plasma. This could guide clinical management and enable identification of NETs-inducing factors in individual patients for targeted treatment and personalised ICU medicine.