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Abstract Background This study aimed to compare sublingual microcirculatory parameters between anesthetized pigs and conscious adult humans using sidestream darkfield videomicroscopy. The overarching aim of the work was to validate the pig as an experimental model of changes in microcirculatory function following traumatic haemorrhagic shock and resuscitation. Methods Fourteen large white pigs and 14 humans were recruited for the study. Sublingual sidestream darkfield videomicroscopy clips were captured in anesthetized pigs and conscious humans. Clips underwent manual analysis in Automated Vascular Analysis 3.2 software. The total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPVs) and microvascular flow index (MFI) were quantified. An independent samples t test was used for between species comparison of microcirculatory parameters. Results and Conclusions Conscious humans had a significantly lower TVD, PVD and MFI than anesthetized pigs. No significant difference in PPVs was observed between the species. Perfusion of the microcirculation is a critical determinant of tissue metabolic function and viability. Whilst it may not be surprising that some interspecies differences in the sublingual microcirculatory anatomy were identified between pig and human subjects, it is interesting to report the insignificant difference in PPVs. This direct microcirculatory measure represents a relative change which should hold translatable value across species. We therefore conclude the pig is a suitable model for microcirculatory research and may be a suitable species to investigate changes in microcirculatory perfusion following perturbations in cardiovascular homeostasis, for example during traumatic haemorrhagic shock and resuscitation.

Background The treatment of traumatic haemorrhagic shock has been transformed through better haemorrhage control, use of tranexamic acid and use of blood products. The improved survival seen from these strategies has stimulated an interest in pre-hospital transfusion. Objectives To determine if the clinical effectiveness of resuscitation with red blood cells and lyophilised plasma was superior to 0.9% saline for improving tissue perfusion and reducing mortality in adults with haemorrhagic shock following major trauma. Design A multi-centre, allocation concealed, open-label, parallel group, randomised controlled trial (with internal pilot). Setting The trial was conducted in four civilian pre-hospital critical care services who operated within the National Health Service (NHS) England Major Trauma Networks. Participants Adults (aged ≥16 years) who had sustained traumatic injuries, were attended by a pre-hospital emergency medical team and were hypotensive (systolic blood pressure 1.5]; coagulation measured viscoelastically by rotational thromboelastometry (ROTEM); platelet function using multiple electrode impedance aggregometry (MultiPlate); total blood product receipt; acute respiratory distress syndrome; transfusion-related complications; organ failure-free day. Sample size and statistical analysis The trial set out to detect a 10% absolute difference between groups in the proportion of participants experiencing the primary outcome assuming an event rate of 20% in the control group and 10% in the intervention. For 80% power and type 1 error rate of 0.05, 438 participants (219 per group) were required. Allowing for 10% attrition, the sample size was set at 490 participants. During May 2018, the Data Monitoring Committee (DMC) reported a much higher than anticipated pooled event rate for the primary outcome (65%) to the Trial Steering Committee (TSC). The TSC advised to continue the trial with the original sample size unchanged, noting that the trial retained 80% power to detect a relative risk of 0.82 (71.7% control, 58.3% intervention). All primary analyses of the primary and secondary outcomes followed the intention-to-treat principle. The analyses used a model-based approach with pre-hospital critical care service included as a fixed effect covariate in the model. Treatment effects are presented with two-sided 95% confidence intervals (CIs). No adjustment for multiple comparisons was made. Binary outcomes were analysed using log-binomial regression models to obtain adjusted relative risks along with 95% CIs. A relative risk 0% or >10% was 48.2% and 1.3% (non-informative priors), 44.1% and 0.3% (sceptical priors) and 53.4% and 1.6% (informative priors) respectively. Secondary outcomes The event rates for the individual components of the primary outcome comprised of: Episode mortality 88/203 (43%) in the RBC/LyoPlas group compared to 99/218 (45%) in the 0.9% saline group [adjusted average differences −3% (−12% to 7%), p = 0.57]. Failure to clear lactate 98/196 (50%) compared to 113/206 (55%) [adjusted average difference −5% (−14% to 5%), p = 0.33]. Vital signs on arrival at hospital and through to 24 hours were similar between groups. The haemoglobin concentration on arrival to hospital was 133 (19) g/L in the RBC/LyoPlas group compared to 118 (23) g/L in the 0.9% saline group, an adjusted average difference of 15 g/L (95% CI 10 to 19), P < 0.0001. There were no between group differences in tests of coagulation, and platelet function was similar. Blood product use was similar following hospital admission through to 24 hours. A post-hoc analysis found that total (pre-hospital and hospital) blood and plasma use was higher in the RBC/LyoPlas group [mean difference 1.80 units (95% CI 0.58 to 3.01) and 1.54 units (95% CI 0.57 to 2.50)] respectively. Death within 3 hours occurred in 32/197 (16%) compared to 46/208 (22%), adjusted average difference −7% (−15% to 1%); P = 0.08, and within 30 days it occurred in 86/204 (42%) compared to 99/219 (45%), adjusted average difference −4% (−13% to 6%); P = 0.44. Adverse events Rates of complications and adverse events were similar across groups, and only two serious adverse events were recorded. Complications relating to transfusion in the first 24 hours were similar for the RBC/LyoPlas group 11/148 (7%); compared to 0.9% saline 9/137 (7%). Acute respiratory distress syndrome developed amongst 9/142 (6%) in the RBC/LyoPlas group and 3/129 (2%) in the 0.9% saline group [adjusted relative risk 2.71 (0.75 to 9.81)]. The number of organ failure-free days were also similar across groups [12.9 (SD 13.0) RBC/LyoPlas vs. 12.1 (13.1) 0.9% saline]. No patients required dose reductions or had treatment discontinued for drug-related toxicity. There were no treatment-related deaths. Conclusion In adults with severe injuries and haemorrhagic shock secondary to major trauma in a civilian setting, the RePHILL trial did not demonstrate that pre-hospital RBC/LyoPlas resuscitation was superior to 0.9% sodium chloride. Future research should seek to identify if specific groups of patients may benefit and explore the effects of alternative transfusion strategies. Trial registration This trial is registered as ISRCTN62326938. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation Programme (NIHR award ref: 14/152/14) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 2. See the NIHR Funding and Awards website for further award information.

Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects.37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented 400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells.EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes.