Qualitative and Quantitative Analysis of Circulating NET-derived chromatin and nucleosomes in Severe Thermal and Traumatically-injured patients

Objectives: This study evaluates the dynamic changes in the quantity and quality of neutrophil-derived cell-free DNA (cfDNA) in whole blood and plasma samples from severe thermal and traumatically-injured patients. Background: Neutrophil-derived cfDNA, a component of neutrophil extracellular traps (NETs), is released during immune responses to injury and infection. Excessive NET formation mediates inflammation, tissue damage and coagulopathy. Increased cfDNA levels in burn and trauma patients are reported to be associated with poor clinical outcomes, indicating their potential as biomarkers for predicting sepsis and MODS. Methods: Blood samples were obtained from two cohorts of burns (N= 96) and trauma (N= 147) patients. Neutrophil-derived cfDNA was quantified in plasma by SYTOX Green fluorescence and the size of extracted DNA was measured by electrophoresis. A microfluidic assay was also performed to capture NET-derived chromatin from burn patients' fresh whole blood samples. Captured NETs were stained with SYTOX green and anti-citrullinated histone antibodies. Stimulated whole blood or isolated neutrophils from healthy controls (HC) were used to generate NETs as positive controls. Results: Circulating cfDNA levels were significantly elevated in plasma from both burns (days 1-14 post-injury) and trauma patients (<1-72 hours) compared to HC. Electrophoresis revealed a prominent nucleosome band (~150 bp) in both burns and trauma plasma, with nucleosome oligomers detected within ultra-early samples (<1-hour post-trauma). The density of nucleosome bands correlated with cfDNA concentrations. Microfluidic capture of circulating NET-derived chromatin demonstrated increased levels in whole blood from burns, particularly at day 1 post-injury. Conclusion: NET-derived chromatin, cfDNA and nucleosomes are elevated following severe burns and trauma. The dynamic changes in cDNA support their contribution to inflammation and as a key biomarker.