Background

Systemic and local tissue hypoxia are contributors to both morbidity and mortality in critical illness and necessitate timely intensive care-based vital organ support. There is a need for better biomarkers to allow early intervention to support recovery for intensive care cases. The aim of this thesis was to first assess factors related to poor outcome in critical care patients with a focus on a regional cohort in the early phase of critical illness, and with a focus on circulatory and respiratory impairment. Then, the aim was to assess early responses to hypoxic and inflammatory stress on a cellular level in lab-based studies, with a focus on extracellular vesicles (EVs) released by cells as potential biomarkers.

Methods

In papers I and II, intensive care cases transported by fixed-wing air ambulance between 2000-2016 were screened for inclusion. A total of 2146 cases were included and all-cause mortality at 90 days post transport was assessed using regression models with relevant co-factors. In Paper III mammalian cells were cultured and EVs were isolated from the conditioned culture media and studied with regard to histone content using an array of molecular biological and biochemical methods including western blotting, immunocytochemistry, transmission electron microscopy and mass spectrometry. In the exploratory study Paper IV, plasma from intensive care cases and healthy volunteers was collected, from which EVs were isolated. Various isolation methods were used and compared, and the EVs were studied with focus on associated histones.

Results

Paper I and II: An increased all-cause mortality risk was found for impaired oxygenation and hemodynamic instability. In addition, the risk of non-daytime transporting of patients was associated with higher mortality risk, not explained by oxygenation or hemodynamic instability. Total transport mission time was assessed for all-cause mortality risk and clinical deterioration during transport. Total mission time was not associated with increased mortality risk. Small negative changes in oxygenation SpO2/FiO2 ratio and mean arterial pressure during transport were observed for the cohort.

Paper III: It was found that histones associated with the outer membrane surface of EVs, and EV secretion increased following cell stress. Histone secretion could be targeted pharmacologically and genetically by targeting intracellular signaling/trafficking pathways and by altering histone posttranslational modifications.

Paper IV: Established methods proved inadequate for reliable EV purification and histone quantification in plasma EV samples. This was likely due to an abundance of plasma proteins co-purifying with EVs, including immunoglobulins. The presence of histones in EVs was confirmed using liquid chromatography mass-spectrometry.

Conclusion

The fixed-wing air ambulance system in Northern Sweden is safe and comparable to other international transport systems, even though the stressors hypoxia and hemodynamic instability were found to contribute to significant all-cause mortality. Histones are associated with EVs and their degree of association can be affected by cell stress, including hypoxia and inflammatory stress. In plasma, improved methods are needed to quantify histone levels in EVs in order to assess their potential use as biomarkers.v