Introduction: Lung transplantation is a lifesaving therapy for patients with end-stage lung disease. However, obstacles to successful lung transplantation, such as a scarcity of donor lungs and development of primary graft dysfunction (PGD) in recipients, are prevalent. Normothermic ex vivo lung perfusion (EVLP), which preserves lungs in a physiological state until transplantation, offers a unique platform to examine cellular processes occurring within donor lungs. Neutrophil extracellular traps (NETs) represent a neutrophilic response to inflammation and have been associated in a variety of lung injury states, including PGD. This study aimed to assess for the presence of NETs in donor lung EVLP perfusate, and the utility of NETs in donor lung perfusate as a biomarker for recipient outcomes.

Methods: Clinical perfusate samples from consecutive EVLP cases performed at our center between 2009 and 2017 at the 4^th hour of perfusion were snap frozen. Neutrophil elastase (NE) bound to DNA in complexes, representative of NETs, were measured using ELISA. Optical densities of NE-DNA complexes from lungs that were transplanted were compared to recipient outcomes, including days on ventilator and days in the ICU (excluding patients who were in the ICU prior to transplantation), using the clinically relevant threshold of 4 days.

Results: From the 200 samples included, 111 were from donation after brain death and 89 were from donation after cardiac death.  NETs were significantly higher in donor lung perfusate associated with recipients who were on the ventilator for >4 days (p=0.04). This was especially pronounced in patients who received female donor lungs (p=0.0383). Results for recipient length of stay in ICU were similar. Regression analyses supported these associations, with increasing levels of NETs in perfusate significantly associated with more recipient days on ventilator (R2=0.1101, p=0.0027). NETs were most significantly associated with recipient days on ventilator in female donor lungs when stratified by donor sex (R2=0.2306, p=0.0099), and in DCD female donor lungs when stratified by donor sex and type (R2=0.5361, p=0.0009).

Conclusions: This study adds to the field because it demonstrates for the first time that NETs can be detected in donor lung EVLP perfusate. Additionally, donor lungs with higher levels of NETs in the perfusate were associated with recipients who had longer days on the ventilator. This suggests that NETs are a potential predictor of recipient outcomes post-lung transplantation. NETs are relatively stable and easily detectable with ELISA, which is a common laboratory technique. Therefore, this study suggests that detection of NETs in donor lung perfusate may be a feasible means by which to predict recipient outcomes. In addition, NETs seem to correlate well with lungs donated after cardiac death in female donors. This suggests that the biological processes underlying donor lung injury may differ, and further investigation into these variations may yield better predictors for recipient outcomes.