Objectives
Increased plasma levels of cell free DNA (cf-DNA) are observed in patients after severe thermal and traumatic injuries. The majority of cf-DNA is generated from neutrophil extracellular traps (NETs) and not only implicated in the pathophysiology of multiple organ failure (MOF) but may provide an early biomarker for sepsis (Hampson et al, Annals of Surgery, 2017). DNAse degrades NETs into nucleosomes with ~150 base pairs (bp) of DNA wrapped around a histone core. Recently, large circulating chromatin fibres have also been observed in burns (Otawara et al, Scientific Reports, 2018). Here, we measured the size of c-fDNA in burns and trauma patients to understand the post-injury dynamics of NET-cf-DNA generation and breakdown.
Methods
Cf-DNA concentrations were measured in plasma samples obtained from severe burns (>15% TBSA) and traumatically-injured (injury severity score >8) patients across a timescale of days 1-28 and ≤1-72 hours respectively. Concentrations were compared to those measured in plasma samples from healthy controls. DNA was also extracted from plasma and analysed by the high sensitivity Agilent 2100 Bioanalyzer to determine DNA size (bp). As an in vitro control, DNA was also measured from purified NETs generated from neutrophils stimulated with 50 nM PMA for 4 hours and digested with DNase, MNase or DNase 1L3.
Results
Unresolved high molecular weight DNA was detected in the acute post-burn phase (days 1-4), which was digested down to nucleosome bands at ~150 (bp) at later time-points (days 6-14) that persisted up to 28 days. The density of nucleosomes correlated with cf-DNA levels. Healthy donor plasma, with low cf-DNA levels contains no nucleosome bands. A ~150 bp nucleosome band was detected in trauma patient plasma at all sampling time-points. In samples acquired 1-hour post-injury, higher molecular weight nucleosome oligomers (i.e., ~ 300 and 450 bp) were also detected. DNase 1L3 and MNase digestion of NETs generated nucleosome bands in vitro,, although DNAse-1 completely digests away remnant nucleosomes.
Conclusions
This study demonstrates the nature of circulating NET derived DNA fragments in post-injury burn and trauma samples with a predominant ~150 (bp) nucleosome band apparent at most time points which correlates with cf-DNA levels. Ultra-early trauma samples (<1h after injury) also contain nucleosome oligomers. In vitro digestion of NETs confirms the role of DNases in breaking down NET derived chromatin into nucleosomes. These studies will help elucidate the role of NET derived chromatin and nucleosomes in the pathophysiology of DIC, MODS and sepsis.

Objectives
Increased plasma levels of cell free DNA (cf-DNA) are observed in patients after severe thermal and traumatic injuries. The majority of cf-DNA is generated from neutrophil extracellular traps (NETs) and not only implicated in the pathophysiology of multiple organ failure (MOF) but may provide an early biomarker for sepsis (Hampson et al, Annals of Surgery, 2017). DNAse degrades NETs into nucleosomes with ~150 base pairs (bp) of DNA wrapped around a histone core. Recently, large circulating chromatin fibres have also been observed in burns (Otawara et al, Scientific Reports, 2018). Here, we measured the size of c-fDNA in burns and trauma patients to understand the post-injury dynamics of NET-cf-DNA generation and breakdown.
Methods
Cf-DNA concentrations were measured in plasma samples obtained from severe burns (>15% TBSA) and traumatically-injured (injury severity score >8) patients across a timescale of days 1-28 and ≤1-72 hours respectively. Concentrations were compared to those measured in plasma samples from healthy controls. DNA was also extracted from plasma and analysed by the high sensitivity Agilent 2100 Bioanalyzer to determine DNA size (bp). As an _in vitro_ control, DNA was also measured from purified NETs generated from neutrophils stimulated with 50 nM PMA for 4 hours and digested with DNase, MNase or DNase 1L3.
Results
Unresolved high molecular weight DNA was detected in the acute post-burn phase (days 1-4), which was digested down to nucleosome bands at \~150 (bp) at later time-points (days 6-14) that persisted up to 28 days. The density of nucleosomes correlated with cf-DNA levels. Healthy donor plasma, with low cf-DNA levels contains no nucleosome bands. A ~150 bp nucleosome band was detected in trauma patient plasma at all sampling time-points. In samples acquired 1-hour post-injury, higher molecular weight nucleosome oligomers (i.e., \~ 300 and 450 bp) were also detected. DNase 1L3 and MNase digestion of NETs generated nucleosome bands _in vitro_,, although DNAse-1 completely digests away remnant nucleosomes.
Conclusions
This study demonstrates the nature of circulating NET derived DNA fragments in post-injury burn and trauma samples with a predominant ~150 (bp) nucleosome band apparent at most time points which correlates with cf-DNA levels. Ultra-early trauma samples (<1h after injury) also contain nucleosome oligomers. _In vitro_ digestion of NETs confirms the role of DNases in breaking down NET derived chromatin into nucleosomes. These studies will help elucidate the role of NET derived chromatin and nucleosomes in the pathophysiology of DIC, MODS and sepsis.

P.012 Characterisation of Neutrophil-derived Cell Free DNA (cf-DNA) In Burns and Trauma

Dear friends and colleagues,

We will be pleased to welcome you in Turin (Italy) for the 19th European Burns Association´s Congress in 2022. Italy is honored to hold the meeting of the European Burns Association for the second time, after Verona in 1995. Turin is easily reachable from everywhere in Europe, either travelling by car, train or choosing flight connections. During the meeting, held in the renewed industrial center of the main Italian historical automotive factory (i.e. Lingotto-FIAT), we will meet international burn specialists, focusing on standards of care, progresses in burn treatment, strategies to face disaster situations, with a special attention to education of younger colleagues. However, in your spare time, you might enjoy the sweet days of Italian late summer, simply wandering in the green parks and gardens along the two rivers which cross the city, or you might decide to discover the numerous art galleries, churches, palaces, libraries, walking across elegant squares and large boulevards, passing through centuries of art and history; finally, you may choose to immerse yourself in some of the most exciting museum experiences, such as the Cinema Museum and the Turin’s Egyptian museum.

Looking forward to seeing you all in Turin for the 19th European Association´s Congress in 2022!
Maurizio Stella, Congress President, Turin Burn Centre