Shock-Driven Endotheliopathy in Trauma Patients Is Associated with Leucocyte Derived Extracellular Vesicles.

Endotheliopathy following trauma is associated with poor outcome, but the underlying mechanisms are unknown. This study hypothesized that an increased extracellular vesicle (EV) concentration is associated with endotheliopathy after trauma and that red blood cell (RBC) transfusion could further enhance endotheliopathy. In this post hoc sub study of a multicentre observational trial, 75 trauma patients were stratified into three groups based on injury severity score or shock. In patient plasma obtained at hospital admission and after transfusion of four RBC transfusions, markers for endotheliopathy were measured and EVs were labelled with anti CD41 (platelet EVs), anti CD235a (red blood cell EVs), anti CD45 (leucocyte EVs), anti CD144 (endothelial EVs) or anti CD62e (activated endothelial EVs) and EV concentrations were measured with flow cytometry. Statistical analysis was performed by a Kruskall Wallis test with Bonferroni correction or Wilcoxon rank test for paired data. In patients with shock, syndecan-1 and von Willebrand Factor (vWF) were increased compared to patients without shock. Additionally, patients with shock had increased red blood cell EV and leucocyte EV concentrations compared to patients without shock. Endotheliopathy markers correlated with leucocyte EVs (ρ = 0.263, p = 0.023), but not with EVs derived from other cells. Injury severity score had no relation with EV release. RBC transfusion increased circulating red blood cell EVs but did not impact endotheliopathy. In conclusion, shock is (weakly) associated with EVs from leucocytes, suggesting an immune driven pathway mediated (at least in part) by shock.

Treatment with ddAVP improves platelet-based coagulation in a rat model of traumatic hemorrhagic shock.

Objectives: Trauma-induced hemorrhagic shock is characterized by increased endothelial permeability and coagulopathy. Vasopressin analog ddAVP (desmopressin) acts by reorganizing and redistributing adhesive and tight junction molecules, enhancing endothelial barrier function. Furthermore, ddAVP increases von Willebrand factor (vWF) plasma levels and thereby potentially enhances platelet-based coagulation. The objective of this study was to assess whether the use of ddAVP results in improvement of both endothelial barrier function and platelet-based coagulation, thereby improving shock reversal and reduce organ failure in a rat model of trauma and transfusion. Methods: Blood products were prepared from syngeneic rat blood according to blood bank standards. Polytrauma was induced in Sprague Dawley rats by a fractured femur and crush injury to the intestines and liver. The rats were hemorrhaged until a mean arterial pressure of 40 mm Hg and transfused with RBCs, fresh frozen plasmas and platelets in a 1:1:1 ratio, and randomized to receive a single dose of ddAVP (n=7 per group). Blood samples were taken up to 6 hours after trauma to assess biochemistry, markers of endothelial injury and coagulation status by rotational thromboelastometry (ROTEM). Organ damage was assessed by histopathology. Results: Rats receiving ddAVP showed significantly better shock reversal compared with controls. Also, coagulation parameters remained stable in the ddAVP treated group, whereas rats in the control group showed deterioration of coagulation parameters, including decreased clot strength and decreased platelet functioning (89% (IQR 82% to 92%) of baseline values). Platelet count and vWF antigen levels at exsanguination did not differ between groups. ddAVP did not reduce markers of endothelial dysfunction nor markers of organ injury. Conclusions: The use of ddAVP in a rat trauma-transfusion model improved shock parameters and ROTEM parameters of clot formation. However, this did not abrogate the amount of organ failure. Level of evidence: Level III.

Platelet-to-red blood cell ratio and mortality in bleeding trauma patients: A systematic review and meta-analysis.

BACKGROUND: In traumatic bleeding, transfusion practice has shifted toward higher doses of platelets and plasma transfusion. The aim of this systematic review was to investigate whether a higher platelet-to-red blood cell (RBC) transfusion ratio improves mortality without worsening organ failure when compared with a lower ratio of platelet-to-RBC. METHODS: Pubmed, Medline, and Embase were screened for randomized controlled trials (RCTs) in bleeding trauma patients (age ≥16 years) receiving platelet transfusion between 1946 until October 2020. High platelet:RBC ratio was defined as being the highest ratio within an included study. Primary outcome was 24 hour mortality. Secondary outcomes were 30-day mortality, thromboembolic events, organ failure, and correction of coagulopathy. RESULTS: In total five RCTs (n = 1757 patients) were included. A high platelet:RBC compared with a low platelet:RBC ratio significantly improved 24 hour mortality (odds ratio [OR] 0.69 [0.53-0.89]) and 30- day mortality (OR 0.78 [0.63-0.98]). There was no difference between platelet:RBC ratio groups in thromboembolic events and organ failure. Correction of coagulopathy was reported in five studies, in which platelet dose had no impact on trauma-induced coagulopathy. CONCLUSIONS: In traumatic bleeding, a high platelet:RBC improves mortality as compared to low platelet:RBC ratio. The high platelet:RBC ratio does not influence thromboembolic or organ failure event rates.

Therapeutic application of recombinant human ADAMTS-13 improves shock reversal and coagulation status in a trauma hemorrhage and transfusion rat model.

INTRODUCTION: In hemorrhaging trauma patients, the endothelium is activated, resulting in excessive endothelial synthesis of von Willebrand Factor (vWF), which may enhance micro-thrombi formation, resulting in obstruction of the microcirculation and endothelial injury, aggravating bleeding, as well as contributing to organ failure. Under normal conditions, vWF is cleaved by the metalloprotease ADAMTS-13. After trauma, ADAMTS-13 levels are reduced. OBJECTIVES: To assess whether recombinant human ADAMTS-13 inhibits endothelial injury and organ failure in a rat trauma-transfusion model. METHODS: Blood products were prepared from syngeneic rat blood according to blood bank standards. Polytrauma was induced in rats by crush injury to the intestines and liver and by fracture of the femur. The rats were hemorrhaged until a mean arterial pressure (MAP) of 40 mmHg was reached. Rats were randomized to receive transfusion of RBCs, FFPs, and platelets in a 1:1:1 ratio to achieve a MAP of 70 mmHg, with or without the addition of ADAMTS-13 (50 µg/kg). Blood samples were assessed for biochemistry and rotational thromboelastometry (ROTEM). Syndecan-1 and VE-cadherin levels were measured as a reflection of endothelial integrity. The amount of leakage of dextran-FITC from the vascular system to the parenchyma in lungs was quantified. To assess inflammation, IL-6 and IL-8 levels were determined. Organ damage was assessed by histopathology. RESULTS: All rats were severely shocked, with no significant differences in shock parameters between groups. Rats treated with ADAMTS-13 showed signs of a more effective shock reversal (higher blood pressure, lower lactate levels) compared to controls. Also, ROTEM parameters of clot formation in rats receiving ADAMTS-13 improved compared to controls, which was mainly platelet-dependent. Syndecan-1 levels relative to baseline trended to be lower in ADAMTS-13 treated rats compared to controls (107 vs 149%, p = 0.08). ADAMTS-13 reduced albuminuria (1.7 vs 4.4 g/L, p < 0.01) and organ-specific inflammation (pulmonary IL-6 243 vs 369 pg/mL, p = 0.08; splenic IL-6 253 vs 307, p = 0.03) compared to controls, but did not improve histopathological scores. CONCLUSIONS: The use of ADAMTS-13 in a rat trauma-transfusion model improves parameters of shock, platelet-driven coagulation, endothelial damage, and organ inflammation. These results suggest that ADAMTS-13 is important in mediating outcome of trauma. Whether ADAMTS-13 can be used as a therapeutic adjunct to treat bleeding trauma patients remains to be determined.

Autonomic nervous system activity and the risk of nosocomial infection in critically ill patients with brain injury.

PURPOSE: Nosocomial infection contributes to adverse outcome after brain injury. This study investigates whether autonomic nervous system activity is associated with a decreased host immune response in patients following stroke or traumatic brain injury (TBI). METHODS: A prospective study was performed in adult patients with TBI or stroke who were admitted to the Intensive Care Unit of our tertiary university hospital between 2013 and 2016. Heart rate variability (HRV) was recorded daily and assessed for autonomic nervous system activity. Outcomes were nosocomial infections and immunosuppression, which was assessed ex vivo using whole blood stimulations with plasma of patients with infections, matched non-infected patients and healthy controls. RESULTS: Out of 64 brain injured patients, 23 (36%) developed an infection during their hospital stay. The ability of brain injured patients to generate a host response to the bacterial endotoxin lipopolysaccharides (LPS) was diminished compared to healthy controls (p < 0.001). Patients who developed an infection yielded significantly lower TNF-α values (86 vs 192 pg/mL, p = 0.030) and a trend towards higher IL-10 values (122 vs 84 pg/mL, p = 0.071) following ex vivo whole blood stimulations when compared to patients not developing an infection. This decreased host immune response was associated with altered admission HRV values. Brain injured patients who developed an infection showed increased normalized high-frequency power compared to patients not developing an infection (0.54 vs 0.36, p = 0.033), whereas normalized low-frequency power was lower in infected patients (0.46 vs 0.64, p = 0.033). CONCLUSION: Brain injured patients developing a nosocomial infection show parasympathetic predominance in the acute phase following brain injury, reflected by alterations in HRV, which parallels a decreased ability to generate an immune response to stimulation with LPS.

Effectiveness of prothrombin complex concentrate for the treatment of bleeding: A systematic review and meta-analysis.

Prothrombin complex concentrate (PCC) is increasingly being used as a treatment for major bleeding in patients who are not taking anticoagulants. The aim of this systematic review and meta-analysis is to evaluate the effectiveness of PCC administration for the treatment of bleeding in patients not taking anticoagulants. Studies investigating the effectivity of PCC to treat bleeding in adult patients and providing data on either mortality or blood loss were eligible. Data were pooled using Mantel-Haenszel random effects meta-analysis or inverse variance random effects meta-analysis. From 4668 identified studies, 17 observational studies were included. In all patient groups combined, PCC administration was not associated with mortality (odds ratio = 0.83; 95% confidence interval [CI], 0.66-1.06; P = .13; I2  = 0%). However, in trauma patients, PCC administration, in addition to fresh frozen plasma, was associated with reduced mortality (odds ratio = 0.64; CI, 0.46-0.88; P = .007; I2  = 0%). PCC administration was associated with a reduction in blood loss in cardiac surgery patients (mean difference: -384; CI, -640 to -128, P = .003, I2  = 81%) and a decreased need for red blood cell transfusions when compared with standard care across a wide range of bleeding patients not taking anticoagulants (mean difference: -1.80; CI, -3.22 to -0.38; P = .01; I2  = 92%). In conclusion, PCC administration was not associated with reduced mortality in the whole cohort but did reduce mortality in trauma patients. In bleeding patients, PCC reduced the need for red blood cell transfusions when compared with treatment strategies not involving PCC. In bleeding cardiac surgery patients, PCC administration reduced blood loss.

The impact of blood product ratio and procoagulant therapy on the development of thromboembolic events in severely injured hemorrhaging trauma patients.

INTRODUCTION: Transfusion therapy in hemorrhaging trauma patients is associated with the development of thromboembolic events. It is unknown whether current resuscitation strategies, including large volumes of plasma and early administration of procoagulant therapy, increases this risk. METHODS: A systematic search was conducted in MEDLINE, PubMed, and Embase. Studies were screened by two independent reviewers and included if they reported on thromboembolic events in patients with severe trauma (injury severity score ≥16) who received transfusion of at least 1 unit of red blood cells. The ratio by which blood products were transfused, as well as use of procoagulant or antifibrinolytic medication, was recorded. RESULTS: A total of 40 studies with 11.074 bleeding trauma patients were included, in which 1.145 thromboembolic events were reported, yielding an incidence of 10% thromboembolic events. In studies performing routine screening for thromboembolic complications, the incidence ranged from 12% to 23%. The risk of thromboembolic events was increased after administration of tranexamic acid (TXA; odds ratio [OR], 2.6; 95% confidence interval [CI], 1.7-4.1; p < 0.001) and fibrinogen concentrate (OR, 2.1; 95% CI, 1.0-4.2; p = 0.04). Blood product ratio, the use of prothrombin complex concentrate or recombinant factor VIIa were not associated with thromboembolic events. CONCLUSION: This systematic review identified an incidence of thromboembolic events of 10% in severely injured bleeding trauma patients. The use of TXA and fibrinogen concentrate was associated with the development of thromboembolic complications.

Red-blood-cell manufacturing methods and storage solutions differentially induce pulmonary cell activation.

BACKGROUND AND OBJECTIVES: Red-blood-cell (RBC) transfusion is associated with lung injury, which is further exacerbated by mechanical ventilation. Manufacturing methods of blood products differ globally and may play a role in the induction of pulmonary cell activation through alteration of the immunomodulatory property of the products. Here, the effect of different manufacturing methods on pulmonary cell activation was investigated in an in vitro model of mechanical ventilation. MATERIALS AND METHODS: Pulmonary type II cells were incubated with supernatant from fresh and old RBC products obtained via whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD) or whole blood-derived (WBD) methods. Lung cells were subjected to 25% stretch for 24 h. Controls were non-stretched or non-incubated cells. RESULTS: Fresh but not old AD products and WBF products induce lung cell production of pro-inflammatory cytokines and chemokines, which was not observed with WBD or RCF products. Effects were associated with an increased amount of platelet-derived vesicles and an increased thrombin-generating capacity. Mechanical stretching of lung cells induced more severe cell injury compared to un-stretched controls, including alterations in the cytoskeleton, which was further augmented by incubation with AD products. In all read-out parameters, RCF products seemed to induce less injury compared to the other products. CONCLUSIONS: Our findings show that manufacturing methods of RBC products impact pulmonary cell activation, which may be mediated by the generation of vesicles in the product. We suggest RBC manufacturing method may be an important factor in understanding the association between RBC transfusion and lung injury.

Use of a high platelet-to-RBC ratio of 2:1 is more effective in correcting trauma-induced coagulopathy than a ratio of 1:1 in a rat multiple trauma transfusion model.

BACKGROUND: Platelet dysfunction importantly contributes to trauma-induced coagulopathy (TIC). Our aim was to examine the impact of transfusing platelets (PLTs) in a 2:1 PLT-to-red blood cell (RBC) ratio versus the standard 1:1 ratio on transfusion requirements, correction of TIC, and organ damage in a rat multiple trauma transfusion model. METHODS: Mechanically ventilated male Sprague Dawley rats were traumatized by crush injury to the small intestine and liver and a fracture of the femur, followed by exsanguination until a mean arterial pressure (MAP) of 40 mmHg. Animals were randomly assigned to receive resuscitation in a high PLT dose (PLT to plasma to RBC in a ratio of 2:1:1) or a standard PLT dose (ratio of 1:1:1) until a MAP of 60 mmHg was reached (n = 8 per group). Blood samples were taken for biochemical and thromboelastometry (ROTEM) assessment. Organs were harvested for histopathology.Outcome measures were transfusion requirements needed to reach a pretargeted MAP, as well as ROTEM correction and organ failure. RESULTS: Trauma resulted in coagulopathy as assessed by deranged ROTEM results. Mortality rate was 19%, with all deaths occurring in the standard dose group. The severity of hypovolemic shock as assessed by lactate and base excess was not different in both groups. The volume of transfusion needed to reach the MAP target was lower in the high PLT dose group compared to the standard dose, albeit not statistically significant (p = 0.054). Transfusion with a high PLT dose resulted in significant stronger clot firmness compared to the standard dose at all time points following trauma, while platelet counts were similar. Organ failure as assessed by biochemical analysis and histopathology was not different between groups, nor were there any thromboembolic events recorded. CONCLUSIONS: Resuscitation with a high (2:1) PLT-to-RBC ratio was more effective compared to standard (1:1) PLT-to-RBC ratio in treating TIC, with a trend towards reduced transfusion volumes. Also, high PLT dose did not aggravate organ damage. Transfusion strategies using higher PLT dose regiments might be a feasible treatment option in hemorrhaging trauma patients for the correction of TIC.

Towards patient-specific management of trauma hemorrhage: the effect of resuscitation therapy on parameters of thromboelastometry.

Essentials The response of thromboelastometry (ROTEM) parameters to therapy is unknown. We prospectively recruited hemorrhaging trauma patients in six level-1 trauma centres in Europe. Blood products and pro-coagulants prevent further derangement of ROTEM results. ROTEM algorithms can be used to treat and monitor trauma induced coagulopathy. SUMMARY: Background Rotational thromboelastometry (ROTEM) can detect trauma-induced coagulopathy (TIC) and is used in transfusion algorithms. The response of ROTEM to transfusion therapy is unknown. Objectives To determine the response of ROTEM profiles to therapy in bleeding trauma patients. Patients/Methods A prospective multicenter study in bleeding trauma patients (receiving ≥ 4 red blood cell [RBC] units) was performed. Blood was drawn in the emergency department, after administration of 4, 8 and 12 RBC units and 24 h post-injury. The response of ROTEM to plasma, platelets (PLTs), tranexamic acid (TXA) and fibrinogen products was evaluated in the whole cohort as well as in the subgroup of patients with ROTEM values indicative of TIC. Results Three hundred and nine bleeding and shocked patients were included. A mean dose of 3.8 g of fibrinogen increased FIBTEM CA5 by 5.2 mm (IQR: 4.1-6.3 mm). TXA administration decreased lysis by 5.4% (4.3-6.5%). PLT transfusion prevented further derangement of parameters of clot formation. The effect of PLTs on EXTEM ca5 values was more pronounced in patients with a ROTEM value indicative of TIC than in the whole cohort. Plasma transfusion decreased EXTEM clotting time by 3.1 s (- 10 s to 3.9 s) in the whole cohort and by 10.6 s (- 45 s to 24 s) in the subgroup of patients with a ROTEM value indicative of TIC. Conclusion The effects of therapy on ROTEM values were small, but prevented further derangement of test results. In patients with ROTEM values indicative of TIC, the efficacy of PLTs and plasma in correcting deranged ROTEM parameters is possibly more robust.

Washing or filtering of blood products does not improve outcome in a rat model of trauma and multiple transfusion.

BACKGROUND: Transfusion is associated with organ failure and nosocomial infection in trauma patients, which may be mediated by soluble bioactive substances in blood products, including extracellular vesicles (EVs). We hypothesize that removing EVs, by washing or filtering of blood products, reduces organ failure and improves host immune response. MATERIALS AND METHODS: Blood products were prepared from syngeneic rat blood. EVs were removed from RBCs and platelets by washing. Plasma was filtered through a 0.22-µm filter. Rats were traumatized by crush injury to the intestines and liver, and a femur was fractured. Rats were hemorrhaged until a mean arterial pressure of 40 mm Hg and randomized to receive resuscitation with standard or washed/filtered blood products, in a 1:1:1 ratio. Sham controls were not resuscitated. Ex vivo whole blood stimulation tests were performed and histopathology was done. RESULTS: Washing of blood products improved quality metrics compared to standard products. Also, EV levels reduced by 12% to 77%. The coagulation status, as assessed by thromboelastometry, was deranged in both groups and normalized during transfusion, without significant differences. Use of washed/filtered products did not reduce organ failure, as assessed by histopathologic score and biochemical measurements. Immune response ex vivo was decreased following transfusion compared to sham but did not differ between transfusion groups. CONCLUSION: Filtering or washing of blood products improved biochemical properties and reduced EV counts, while maintaining coagulation abilities. However, in this trauma and transfusion model, the use of optimized blood components did not attenuate organ injury or immune suppression.

Blood manufacturing methods affect red blood cell product characteristics and immunomodulatory activity.

Transfusion of red cell concentrates (RCCs) is associated with increased risk of adverse outcomes that may be affected by different blood manufacturing methods and the presence of extracellular vesicles (EVs). We investigated the effect of different manufacturing methods on hemolysis, residual cells, cell-derived EVs, and immunomodulatory effects on monocyte activity. Thirty-two RCC units produced using whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD), and whole blood-derived (WBD) methods were examined (n = 8 per method). Residual platelet and white blood cells (WBCs) and the concentration, cell of origin, and characterization of EVs in RCC supernatants were assessed in fresh and stored supernatants. Immunomodulatory activity of RCC supernatants was assessed by quantifying monocyte cytokine production capacity in an in vitro transfusion model. RCF units yielded the lowest number of platelet and WBC-derived EVs, whereas the highest number of platelet EVs was in AD (day 5) and in WBD (day 42). The number of small EVs (<200 nm) was greater than large EVs (≥200 nm) in all tested supernatants, and the highest level of small EVs were in AD units. Immunomodulatory activity was mixed, with evidence of both inflammatory and immunosuppressive effects. Monocytes produced more inflammatory interleukin-8 after exposure to fresh WBF or expired WBD supernatants. Exposure to supernatants from AD and WBD RCC suppressed monocyte lipopolysaccharide-induced cytokine production. Manufacturing methods significantly affect RCC unit EV characteristics and are associated with an immunomodulatory effect of RCC supernatants, which may affect the quality and safety of RCCs.

Viscoelastic Testing in Trauma.

Traumatic-induced coagulopathy (TIC) is a complex condition which develops both as a response to trauma as well as to clinical care interventions. Accurate and timely diagnostics are necessary to enable therapy aimed at correction of TIC. Viscoelastic hemostatic assays (VHA) are increasingly recognized for their potential to diagnose TIC as well as for guidance of treatment. This narrative review focuses on the evidence of the use of VHAs to diagnose TIC, to monitor efficacy of treatment of TIC during bleeding and to prognosticate outcome. Pitfalls of the use of VHA are also discussed.

Risk factors for trauma-induced coagulopathy- and transfusion-associated multiple organ failure in severely injured trauma patients.

BACKGROUND: Both trauma-induced coagulopathy (TIC) and transfusion strategies influence early outcome in hemorrhagic trauma patients. Their impact on late outcome is less well characterized. This study systematically reviews risk factors for TIC- and transfusion-associated multiple organ failure (MOF) in severely injured trauma patients. MATERIALS AND METHODS: A systematic search was conducted in PubMed and Embase. Studies published from 1986 to 2013 on adult trauma patients with an injury severity score ≥16, investigating TIC or transfusion strategies with MOF as primary or secondary outcome, were eligible for inclusion. Results of the included studies were evaluated with meta-analyses of pooled data. RESULTS: In total, 50 studies were included with a total sample size of 63,586 patients. Due to heterogeneity of the study populations and outcome measures, results from 7 studies allowed for pooling of data. Risk factors for TIC-associated MOF were hypocoagulopathy, hemorrhagic shock, activated protein C, increased histone levels, and increased levels of markers of fibrinolysis on admission. After at least 24 h after admission, the occurrence of thromboembolic events was associated with MOF. Risk factors for transfusion-associated MOF were the administration of fluids and red blood cell units within 24 h post-injury, the age of red blood cells (>14 days) and a ratio of FFP:RBC ≥ 1:1 (OR 1.11, 95% CI 1.04-1.19). CONCLUSION: Risk factors for TIC-associated MOF in severely injured trauma patients are early hypocoagulopathy and hemorrhagic shock, while a hypercoagulable state with the occurrence of thromboembolic events later in the course of trauma predisposes to MOF. Risk factors for transfusion-associated MOF include administration of crystalloids and red blood cells and a prolonged storage time of red blood cells. Future prospective studies investigating TIC- and transfusion-associated risk factors on late outcome are required.