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Background: Tissue injury and hemorrhagic shock induce significant systemic metabolic reprogramming in animal models and critically injured patients. Recent expansions of the classic concepts of metabolomic aberrations in tissue injury and hemorrhage opened the way for novel resuscitative interventions based on the observed abnormal metabolic demands. We hypothesize that metabolic demands and resulting metabolic signatures in pig plasma will vary in response to isolated or combined tissue injury and hemorrhagic shock., Methods: A total of 20 pigs underwent either isolated tissue injury, hemorrhagic shock, or combined tissue injury and hemorrhagic shock referenced to a sham protocol (n = 5/group). Plasma samples were analyzed by UHPLC-MS., Results: Hemorrhagic shock promoted a hypermetabolic state. Tissue injury alone dampened metabolic responses in comparison to sham and hemorrhagic shock, and attenuated the hypermetabolic state triggered by shock with respect to energy metabolism (glycolysis, glutaminolysis, and Krebs cycle). Tissue injury and hemorrhagic shock had a more pronounced effect on nitrogen metabolism (arginine, polyamines, and purine metabolism) than hemorrhagic shock alone., Conclusion: Isolated or combined tissue injury and hemorrhagic shock result in distinct plasma metabolic signatures. These findings indicate that optimized resuscitative interventions in critically ill patients are possible based on identifying the severity of tissue injury and hemorrhage.

Background: Trauma is the leading cause of mortality under the age of 40 years. Recent observations on metabolic reprogramming during hypoxia and ischemia indicate that hypoxic mitochondrial uncoupling promotes the generation of succinate, which in turn mediates reperfusion injury and inflammatory sequelae upon reoxygenation. Plasma levels of succinate significantly increase in response to trauma and hemorrhage in experimental models and clinical samples, suggesting that succinate may represent a candidate marker of systemic perfusion in trauma., Methods: Quantitative mass spectrometry-based metabolomics was used to quantify succinate and lactate in 595 plasma samples from severely injured patients enrolled at the Denver Health Medical Center, a Level I trauma center in Denver, Colorado., Results: A total of 95 severely injured patients were sampled for up to 10 time points (595 total samples), from field blood to 7 days postinjury. Results indicate that plasma levels of succinate increased up to 25.9-fold in deceased patients versus the median of the surviving patients (p = 2.75e-100; receiver operating characteristic area under the curve, 0.911). On the other hand, only 2.4-fold changes increases in lactate were observed (p = 5.8e-21; area under the curve, 0.874)., Conclusion: Succinate represents a uniquely sensitive biomarker of postshock metabolic derangement and may be an important mediator of sequelae., Level of Evidence: Prognostic study, level III.

Background: Thrombelastography (TEG) has been used to characterize the coagulation changes associated with injury and shock. Animal models developed to investigate trauma-induced coagulopathy (TIC) have failed to produce excessive bleeding. We hypothesize that a native TEG will demonstrate marked differences in humans compared with these experimental models, which explains the difficulties in reproducing a clinically relevant coagulopathy in animal models., Methods: Whole blood was collected from 138 healthy human volunteers, 25 swine and 66 Sprague-Dawley rats before experimentation. Citrated native TEGs were conducted on each whole blood sample within 2 h of collection. The clot initiation (R-time, minutes), angle (degrees), maximum amplitude (MA; millimeter), and lysis 30 min after MA (LY30; percentage) were analyzed and contrasted between species with data represented as the median and 25 th to 75 th quartile range. Difference between species was conducted with a Kruskal-Wallis test with alpha adjusted with a Bonferroni correction for multiple comparisons (alpha = 0.016)., Results: Median R-time (clot initiation) was 14.65 min (IQR: 13.2-16.3 min) for humans, 5.7 min (4.9-8.8) for pigs, and 5.2 min (4.4-6) for rodents. Humans had longer R-times than both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.4439). Angle (fibrin cross-linking) was 42.3° (interquartile range [IQR]: 37.5-50.2) for humans, 71.7° (64.3-75.6) for pigs, and 61.8° (56.8-66.7) for rats. Humans had reduced angle compared with both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.6052). MA (clot strength) was 55.5 mm (IQR: 52.0-59.5) for humans, 72.5 mm (70.4-75.5) for pigs, and 66.5 mm (56.5-68.6) for rats. Humans had reduced MA compared with both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.0161). LY30 (fibrinolysis) was 1.5% (IQR: 0.975-2.5) for humans, 3.3% (1.9-4.3) for pigs, and 0.5% (0.1-1.2) for rats. Humans had a lesser LY30 than pigs (P = 0.0062) and a greater LY30 than rats (P < 0.0001), and pigs had a greater LY30 than rats (P < 0.0001)., Conclusions: Humans, swine, and rodents have distinctly different coagulation systems, when evaluated by citrated native TEG. Animals are hypercoagulable with rapid clotting times and clots strengths nearly 50% stronger than humans. These coagulation differences indicate the limitations of previous models of trauma-induced coagulopathy in producing coagulation abnormalities associated with increased bleeding. The inherent hypercoagulable baseline tendencies of these animals may result in subclinical biochemical changes that are not detected by conventional TEG and should be taken into consideration when extrapolated to clinical medicine., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: The transfusion-related respiratory complications, transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO), are leading causes of transfusion-related morbidity and mortality. At present, there are no effective preventive strategies with red blood cell (RBC) transfusion. Although mechanisms remain incompletely defined, soluble biological response modifiers (BRMs) within the RBC storage solution may play an important role. Point-of-care (POC) washing of allogeneic RBCs may remove these BRMs, thereby mitigating their impact on post-transfusion respiratory complications., Methods and Analysis: This is a multicenter randomised clinical trial of standard allogeneic versus washed allogeneic RBC transfusion for adult patients undergoing cardiac surgery testing the hypothesis that POC RBC washing is feasible, safe, and efficacious and will reduce recipient immune and physiologic responses associated with transfusion-related respiratory complications. Relevant clinical outcomes will also be assessed. This investigation will enrol 170 patients at two hospitals in the USA. Simon's two-stage design will be used to assess the feasibility of POC RBC washing. The primary safety outcomes will be assessed using Wilcoxon Rank-Sum tests for continuous variables and Pearson chi-square test for categorical variables. Standard mixed modelling practices will be employed to test for changes in biomarkers of lung injury following transfusion. Linear regression will assess relationships between randomised group and post-transfusion physiologic measures., Ethics and Dissemination: Safety oversight will be conducted under the direction of an independent Data and Safety Monitoring Board (DSMB). Approval of the protocol was obtained by the DSMB as well as the institutional review boards at each institution prior to enrolling the first study participant. This study aims to provide important information regarding the feasibility of POC washing of allogeneic RBCs and its potential impact on ameliorating post-transfusion respiratory complications. Additionally, it will inform the feasibility and scientific merit of pursuing a more definitive phase II/III clinical trial., Registration: ClinicalTrials.gov registration number is NCT02094118 (Pre-results)., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Systemic hyperfibrinolysis is an integral part of trauma-induced coagulopathy associated with uncontrolled bleeding. Recent data suggest that plasma-first resuscitation attenuates hyperfibrinolysis; however, the availability, transport, storage, and administration of plasma in austere environments remain challenging and have limited its use. Freeze-dried plasma (FDP) is a potential alternative due to ease of storage, longer shelf life, and efficient reconstitution. FDP potentially enhances clot formation and resists breakdown better than normal saline (NS) and albumin and similar to liquid plasma., Study Design and Methods: Healthy volunteers underwent citrated blood draw followed by 50% dilution with NS, albumin, pooled plasma (PP), or pooled freeze-dried plasma (pFDP). Citrated native and tissue plasminogen activator (t-PA)-challenge (75 ng/mL) thrombelastography were done. Proteins in PP, pFDP, and albumin were analyzed by mass spectroscopy., Results: pFDP and PP had superior clot-formation rates (angle) and clot strength (maximum amplitude) compared with NS and albumin in t-PA-challenge thrombelastographies (angle: pFDP, 67.9 degrees; PP, 67.8 degrees; NS, 40.6 degrees; albumin, 35.8 degrees; maximum amplitude: pFDP, 62.4 mm; PP, 63.5 mm; NS, 44.8 mm; albumin, 41.1 mm). NS and albumin dilution increased susceptibility to t-PA-induced hyperfibrinolysis compared with pFDP and PP (NS, 62.4%; albumin, 62.6%; PP, 8.5%; pFDP, 6.7%). pFDP was similar to PP in the attenuation of t-PA-induced fibrinolysis. Most proteins (97%) were conserved during the freeze-dry process, with higher levels in 12% of pFDP proteins compared with PP., Conclusion: pFDP enhances clot formation and attenuates hyperfibrinolysis better than NS and albumin and is a potential alternative to plasma resuscitation in the treatment of hemorrhagic shock., (© 2017 AABB.)

Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography-mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo-injected 13 C 15 N-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Background: Coagulopathy is associated with massive transfusion in trauma, yet most clinical scores to predict this end point do not incorporate coagulation assays. Previous work has identified that shock increases circulating tissue plasminogen activator (tPA). When tPA levels saturate endogenous inhibitors, systemic hyperfibrinolysis can occur. Therefore, the addition of tPA to a patient's blood sample could stratify a patients underlying degree of shock and early coagulation changes to predict progression to massive transfusion. We hypothesized that a modified thrombelastography (TEG) assay with exogenous tPA would unmask patients' impending risk for massive transfusion., Study Design: Trauma activations were analyzed using rapid TEG and a modified TEG assay with a low and high dose of tPA. Clinical scores (shock index, assessment of blood consumption, and trauma-associated severe hemorrhage) were compared with TEG measurements to predict the need for massive transfusion using areas under the receiver operating characteristic curves., Results: Three hundred and twenty-four patients were analyzed, 17% required massive transfusion. Massive transfusion patients had a median shock index of 1.2, assessment of blood consumption score of 1, and trauma-associated severe hemorrhage score of 12. Rapid TEG and tPA TEG parameters were significantly different in all massive transfusion patients compared with non-massive transfusion patients (all p < 0.02). The low-dose tPA lysis at 30 minutes had the largest the area under the receiver operating characteristic curve (0.86; 95% CI 0.79 to 0.93) for prediction of massive transfusion, similar to international normalized ratio of prothrombin time of 0.86 (95% CI 0.81 to 0.91), followed by trauma-associated severe hemorrhage score (0.83; 95% CI 0.77 to 0.89). Combing trauma-associated severe hemorrhage and tPA-TEG variables results in a positive prediction of massive transfusion in 49% of patients with a 98% negative predictive value., Conclusions: The tPA-TEG identifies trauma patients who require massive transfusion efficiently in a single assay that can be completed in a shorter time than other scoring systems, which has improved performance when combined with international normalized ratio. This new method is consistent with our understanding of the molecular events responsible for trauma-induced coagulopathy., (Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Pre-storage leucoreduction has been universally adopted in most developed countries in Asia, Europe and the Americas. It decreases febrile transfusion reactions, alloimmunisation to HLA antigens, cytomegalovirus exposure, the accumulation of a number of pro-inflammatory mediators in the supernatant, including the accumulation of platelet-and leucocyte-derived proteins and metabolites during routine storage. This review will highlight the lipids and proteins, biological response modifiers (BRMs) that accumulate, their clinical effects in transfused hosts, and methods of mitigation.

Background: Recent research focused on understanding stored red blood cell (RBC) quality has demonstrated high variability in measures of RBC function and health across units. Studies have historically linked this high variability to variations in processing, storage method, and age. More recently, a large number of studies have focused on differences in donor demographics, donor iron sufficiency, and genetic predisposition of the donor to poor storage, particularly through mechanisms of accelerated oxidative damage. A study was undertaken to evaluate a potential additional source of unit to unit variation in stored RBC: the role of variable percent oxygen saturation (%SO 2 ) levels on blood quality parameters during storage., Materials and Methods: %SO 2 data from 492 LR-RBC/AS-3 units used for internal and external collaborative research was included in the analysis. Whole blood units were processed into red blood cells, AS-3 added, leucocyte reduced, in compliance with American Association of Blood Banks guidelines. LR-RBC/AS-3 products were subsequently analysed for %SO 2 levels within 3-24 hours of phlebotomy using a co-oximeter. Separately, to evaluate the impact of pre-storage as well as increasing levels of %SO 2 during storage, a pool-and-split study was performed. Four units of LR-RBC/AS-3 were split 6 ways; "as is" (control), hyperoxygenated to more than 90%, and four levels of pre-storage %SO 2 . The units were periodically sampled up to 42 days and analysed for %SO 2 , pCO 2 , methaemoglobin, ATP, 2,3-BPG as well as with the metabolomics workflow., Results: The measured mean %SO 2 in LR-RBC/AS-3 within 24 hours of collection was 45.9±17.5% with (32.7-61.0 IQR). %SO 2 in all products increased to approximately 95-100% in three weeks. Measured blood quality parameters including ATP, % haemolysis, methaemoglobin, oxidised lipids, and GSH/GSSG indicated suppressed cellular metabolism and increased red cell degradation in response to higher %SO 2 levels., Discussion: The surprisingly high variability in starting %SO 2 levels, coupled with negative impacts of high oxygen saturation on red blood cell quality indicates that oxygen levels may be an important and under-appreciated source of unit-to-unit variability in RBC quality.

Background: Uncontrolled hemorrhage is a leading cause of mortality after trauma accounting for up to 40% of deaths. Massive transfusion protocols offer a proven benefit in resuscitation of these patients. Recently, the superiority of thrombelastography (TEG)-guided resuscitation over strategies guided by conventional clotting assays has been established. We seek to determine optimal thresholds for rapid (r)-TEG driven resuscitation., Methods: The r-TEG data were reviewed for 190 patients presenting to our level 1 trauma center from 2010 to 2015. Criteria for inclusion were highest level trauma activation in patients 18 years or older with hypotension presumed due to acute blood loss. Exclusion criteria included isolated gunshot wound to the head, pregnancy, and chronic liver disease. Receiver operating characteristic (ROC) analysis was performed to test the predictive performance of r-TEG for massive transfusion requirement defined by need for (1) >10 units of RBCs total or death in the first 6 hours or (2) >4 units of RBCs in any hour within the first 6 hours. Cutpoint analysis was then performed to determine optimal thresholds for TEG-based resuscitation., Results: The ROC analysis of r-TEG yielded areas under the curve (AUC) greater than 70% for all outputs with respect to both transfusion thresholds considered, with exception of activated clotting time and lysis at 30 minutes for greater than 4 U RBC in any hour in the first 6 hours. Optimal cutpoint analysis of the resultant ROC curves was performed and for each value, the most sensitive cutpoint was identified, respectively activated clotting time of 128 seconds or longer, angle (α) of 65 degrees or less, maximum amplitude of 55 mm or less, and lysis at 30 minutes of 5% or greater., Conclusions: Through ROC analysis of prospective TEG data, we have identified optimal thresholds to guide hemostatic resuscitation. These thresholds should be validated in a prospective multicenter trial., Level of Evidence: Therapeutic study, level V.

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca 2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47 phox ). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET) + interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47 phox Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47 phox In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET + interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47 phox ., (© Society for Leukocyte Biology.)

Background: Transfusion-related acute lung injury (TRALI) is a significant cause of mortality, especially after transfusions containing antibodies to major histocompatibility complex (MHC) class II antigens. We hypothesize that a first event induces both 1) polymorphonuclear neutrophils (PMNs) to express MHC class II antigens, and 2) activation of the pulmonary endothelium, leading to PMN sequestration, so that the infusion of specific MHC class II antibodies to these antigens causes PMN-mediated acute lung injury (ALI)., Study Design and Methods: Rats were treated with saline (NS), endotoxin (lipopolysaccharide [LPS]), or cytokines (interferon-γ [IFNγ], macrophage colony-stimulating factor [MCSF], tumor necrosis factor-α [TNFα]); the PMNs were isolated; and the surface expression of the MHC class II antigen OX6 and priming by OX6 antibodies were measured by flow cytometry or priming assays., Results: A two-event model of ALI was completed with NS, LPS, or IFNγ/MCSF/TNFα (first events) and the infusion of OX6 (second event). Compared with NS incubation, rats treated with either LPS or IFNγ/MCSF/TNFα exhibited OX6 PMN surface expression, OX6 antibodies primed the formyl-methionyl-leucyl phenylalanine (fMLF)-activated respiratory burst, and PMN sequestration was increased. OX6 antibody infusion into LPS-incubated or IFNγ/MCSF/TNFα-incubated rats elicited ALI, the OX6 antibody was present on the PMNs, and PMN depletion abrogated ALI., Conclusion: Proinflammatory first events induce PMN MHC class II surface expression, activation of the pulmonary endothelium, and PMN sequestration such that the infusion of cognate antibodies precipitates TRALI., (© 2016 AABB.)

Background: Metabolomic investigations have consistently reported succinate accumulation in plasma after critical injury. Succinate receptors have been identified on numerous tissues, and succinate has been directly implicated in postischemic inflammation, organ dysfunction, platelet activation, and the generation of reactive oxygen species, which may potentiate morbidity and mortality risk to patients. Metabolic flux (heavy-isotope labeling) studies demonstrate that glycolysis is not the primary source of increased plasma succinate during protracted shock. Glutamine is an alternative parent substrate for ATP generation during anaerobic conditions, a biochemical mechanism that ultimately supports cellular survival but produces succinate as a catabolite. We hypothesize that succinate accumulation during hemorrhagic shock is driven by glutaminolysis., Methods: Sprague-Dawley rats were subjected to hemorrhagic shock for 45 minutes (shock, n = 8) and compared with normotensive shams (sham, n = 8). At 15 minutes, animals received intravenous injection of C5-N2-glutamine solution (iLG). Blood, brain, heart, lung, and liver tissues were harvested at defined time points. Labeling distribution in samples was determined by ultrahigh-pressure liquid chromatography-mass spectrometry metabolomic analysis. Repeated-measures analysis of variance with Tukey comparison determined significance of relative fold change in metabolite level from baseline., Results: Hemorrhagic shock instigated succinate accumulation in plasma and lungs tissues (8.5- vs. 1.1-fold increase plasma succinate level from baseline, shock vs. sham, p = 0.001; 3.2-fold higher succinate level in lung tissue, shock vs. sham, p = 0.006). Metabolomic analysis identified labeled glutamine and labeled succinate in plasma (p = 0.002) and lung tissue (p = 0.013), confirming glutamine as the parent substrate. Kinetic analyses in shams showed constant total levels of all metabolites without significant change due to iLG., Conclusion: Glutamine metabolism contributes to increased succinate concentration in plasma during hemorrhagic shock. The glutaminolytic pathway is implicated as a therapeutic target to prevent the contribution of succinate accumulation in plasma and the lung-to-postshock pathogenesis., Competing Interests: The authors disclose no conflicts of interest.

The use of aggressive crystalloid resuscitation to treat hypoxemia, hypovolemia, and nutrient deprivation promoted by massive blood loss may lead to the development of the blood vicious cycle of acidosis, hypothermia, and coagulopathy and, utterly, death. Metabolic acidosis is one of the many metabolic derangements triggered by severe trauma/hemorrhagic shock, also including enhanced proteolysis, lipid mobilization, as well as traumatic diabetes. Appreciation of the metabolic benefit of plasma first resuscitation is an important concept. Plasma resuscitation has been shown to correct hyperfibrinolysis secondary to severe hemorrhage better than normal saline. Here, we hypothesize that plasma first resuscitation corrects metabolic derangements promoted by severe hemorrhage better than resuscitation with normal saline. Ultra-high-performance liquid chromatography-mass spectrometry-based metabolomics analyses were performed to screen plasma metabolic profiles upon shock and resuscitation with either platelet-free plasma or normal saline in a rat model of severe hemorrhage. Of the 251 metabolites that were monitored, 101 were significantly different in plasma versus normal saline resuscitated rats. Plasma resuscitation corrected lactate acidosis by promoting glutamine/amino acid catabolism and purine salvage reactions. Plasma first resuscitation may benefit critically injured trauma patients by relieving the lactate burden and promoting other non-clinically measured metabolic changes. In the light of our results, we propose that plasma resuscitation may promote fueling of mitochondrial metabolism, through the enhancement of glutaminolysis/amino acid catabolism and purine salvage reactions. The treatment of trauma patients in hemorrhagic shock with plasma first resuscitation is likely not only to improve coagulation, but also to promote substrate-specific metabolic corrections.

HSCT is a lifesaving procedure for children with malignant and non-malignant conditions. The conditioning regimen renders the patient severely immunocompromised and recovery starts with neutrophil (PMN) engraftment. We hypothesize that children demonstrate minimal PMN dysfunction at engraftment and beyond, which is influenced by the stem cell source and the conditioning regimen. Peripheral blood was serially collected from children at 1 to 12 months following allogeneic HSCT. PMN superoxide (O2-) production, degranulation (elastase), CD11b surface expression, and phagocytosis were assessed. Twenty-five patients, mean age of 10.5 yr with 65% males, comprised the study and transplant types included: 14 unrelated cord blood stem cells (cords), seven matched related bone marrow donors, three matched unrelated bone marrow donors, and one peripheral blood progenitor cells. Engraftment occurred at 24 days. There were no significant differences between controls and patients in PMN O2- production, phagocytosis, CD11b surface expression, and total PMN elastase. Elastase release was significantly decreased <6 months vs. controls (p < 0.05) and showed normalization by six months for cords only. The conditioning regimen did not affect PMN function. PMN function returns with engraftment, save elastase release, which occurs later related to the graft source utilized, and its clinical significance is unknown., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Massive transfusion protocols (MTPs) have become standard of care in the management of bleeding injured patients, yet strategies to guide them vary widely. We conducted a pragmatic, randomized clinical trial (RCT) to test the hypothesis that an MTP goal directed by the viscoelastic assay thrombelastography (TEG) improves survival compared with an MTP guided by conventional coagulation assays (CCA)., Methods: This RCT enrolled injured patients from an academic level-1 trauma center meeting criteria for MTP activation. Upon MTP activation, patients were randomized to be managed either by an MTP goal directed by TEG or by CCA (ie, international normalized ratio, fibrinogen, platelet count). Primary outcome was 28-day survival., Results: One hundred eleven patients were included in an intent-to-treat analysis (TEG = 56, CCA = 55). Survival in the TEG group was significantly higher than the CCA group (log-rank P = 0.032, Wilcoxon P = 0.027); 20 deaths in the CCA group (36.4%) compared with 11 in the TEG group (19.6%) (P = 0.049). Most deaths occurred within the first 6 hours from arrival (21.8% CCA group vs 7.1% TEG group) (P = 0.032). CCA patients required similar number of red blood cell units as the TEG patients [CCA: 5.0 (2-11), TEG: 4.5 (2-8)] (P = 0.317), but more plasma units [CCA: 2.0 (0-4), TEG: 0.0 (0-3)] (P = 0.022), and more platelets units [CCA: 0.0 (0-1), TEG: 0.0 (0-0)] (P = 0.041) in the first 2 hours of resuscitation., Conclusions: Utilization of a goal-directed, TEG-guided MTP to resuscitate severely injured patients improves survival compared with an MTP guided by CCA and utilizes less plasma and platelet transfusions during the early phase of resuscitation.

Background: Water-soluble components in mesenteric lymph have been implicated in the pathophysiology of acute lung injury and distal organ failure following trauma and haemorrhagic shock. Proteomics analyses have recently shown similarities and specificities of post-trauma/haemorrhagic shock lymph and plasma. We hypothesise that the metabolic phenotype of post-trauma/haemorrhagic shock mesenteric lymph and plasma share common metabolites, but are also characterised by unique features that differentiate these two fluids., Materials and Methods: Matched samples were collected from 5 brain-dead organ donors who had suffered extreme trauma/haemorrhagic shock. Metabolomics analyses were performed through ultra-high performance liquid chromatography mass spectrometry., Results: Overall, 269 metabolites were identified in either fluid. Despite significant overlapping, metabolic phenotypes of matched lymph or plasma from the same patients could be used to discriminate sample fluid or biological patient/traumatic-injury origin. Metabolites showing relatively high levels in both fluids included markers of haemolysis and cell lysis secondary to tissue injury., Discussion: High positive correlations were observed between the quantitative levels of markers of systemic metabolic derangement following traumatic/haemorrhagic hypoxaemia, such as succinate, oxoproline, urate and fatty acids. These metabolites might contribute to coagulopathies of trauma and neutrophil priming driving acute lung injury. Future studies will investigate whether the observed compositional specificities mirror functional or pathological adaptations after trauma and haemorrhage.

Postinjury fibrinolysis can manifest as three distinguishable phenotypes: 1) hyperfibrinolysis, 2) physiologic, and 3) hypofibrinolysis (shutdown). Hyperfibrinolysis is associated with uncontrolled bleeding due to clot dissolution; whereas, fibrinolysis shutdown is associated with organ dysfunction due to microvascular occlusion. The incidence of fibrinolysis phenotypes at hospital arrival in severely injured patients is: 1) hyperfibrinolysis 18%, physiologic 18%, and shutdown 64%. The mechanisms responsible for dysregulated fibrinolysis following injury remain uncertain. Animal work suggests hypoperfusion promotes fibrinolysis, while tissue injury inhibits fibrinolysis. Clinical experience is consistent with these observations. The predominant mediator of postinjury hyperfibrinolysis appears to be tissue plasminogen activator (tPA) released from ischemic endothelium. The effects of tPA are accentuated by impaired hepatic clearance. Fibrinolysis shutdown, on the other hand, may occur from inhibition of circulating tPA, enhanced clot strength impairing the binding of tPA and plasminogen to fibrin, or the inhibition of plasmin. Plasminogen activator inhibitor -1 (PAI-1) binding of circulating tPA appears to be a major mechanism for postinjury shutdown. The sources of PAI-1 include endothelium, platelets, and organ parenchyma. The laboratory identification of fibrinolysis phenotype, at this moment, is best determined with viscoelastic hemostatic assays (TEG, ROTEM). While D-dimer and plasmin antiplasmin (PAP) levels corroborate fibrinolysis, they do not provide real-time assessment of the circulating blood capacity. Our clinical studies indicate that fibrinolysis is a very dynamic process and our experimental work suggests plasma first resuscitation reverses hyperfibrinolysis. Collectively, we believe recent clinical and experimental work suggest antifibrinolytic therapy should be employed selectively in the acutely injured patient, and optimally guided by TEG or ROTEM., (© 2016 AABB.)

Background: Trauma-induced coagulopathy (TIC) is associated with a fourfold increased risk of mortality. Hyperfibrinolysis is a component of TIC, but its mechanism is poorly understood. Plasminogen activation inhibitor (PAI-1) degradation by activated protein C has been proposed as a mechanism for deregulation of the plasmin system in hemorrhagic shock, but in other settings of ischemia, tissue plasminogen activator (tPA) has been shown to be elevated. We hypothesized that the hyperfibrinolysis in TIC is not the result of PAI-1 degradation but is driven by an increase in tPA, with resultant loss of PAI-1 activity through complexation with tPA., Methods: Eighty-six consecutive trauma activation patients had blood collected at the earliest time after injury and were screened for hyperfibrinolysis using thrombelastography (TEG). Twenty-five hyperfibrinolytic patients were compared with 14 healthy controls using enzyme-linked immunosorbent assays for active tPA, active PAI-1, and PAI-1/tPA complex. Blood was also subjected to TEG with exogenous tPA challenge as a functional assay for PAI-1 reserve., Results: Total levels of PAI-1 (the sum of the active PAI-1 species and its covalent complex with tPA) are not significantly different between hyperfibrinolytic trauma patients and healthy controls: median, 104 pM (interquartile range [IQR], 48-201 pM) versus 115 pM (IQR, 54-202 pM). The ratio of active to complexed PAI-1, however, was two orders of magnitude lower in hyperfibrinolytic patients than in controls. Conversely, total tPA levels (active + complex) were significantly higher in hyperfibrinolytic patients than in controls: 139 pM (IQR, 68-237 pM) versus 32 pM (IQR, 16-37 pM). Hyperfibrinolytic trauma patients displayed increased sensitivity to exogenous challenge with tPA (median LY30 of 66.8% compared with 9.6% for controls)., Conclusion: Depletion of PAI-1 in TIC is driven by an increase in tPA, not PAI-1 degradation. The tPA-challenged TEG, based on this principle, is a functional test for PAI-1 reserves. Exploration of the mechanism of up-regulation of tPA is critical to an understanding of hyperfibrinolysis in trauma., Level of Evidence: Prognostic and epidemiologic study, level II.

Background: We developed a hemorrhagic shock animal model to replicate an urban prehospital setting where resuscitation fluids are limited to assess the effect of saline versus plasma in coagulopathic patients. An in vitro model of whole blood dilution with saline exacerbated tissue plasminogen activator (tPA)-mediated fibrinolysis, while plasma dilution did not change fibrinolysis. We hypothesize that shock-induced hyperfibrinolysis can be attenuated by resuscitation with plasma while exacerbated by saline., Methods: Sprague-Dawley rats were hemorrhaged to a mean arterial pressure of 25 mm Hg and maintained in shock for 30 minutes. Animals were resuscitated with either normal saline (NS) or platelet-free plasma (PFP) with a 10% total blood volume bolus, followed by an additional 5 minutes of resuscitation with NS to increase blood pressure to a mean arterial pressure of 30 mm Hg. Animals were observed for 15 minutes for the assessment of hemodynamic response and survival. Blood samples were analyzed with thrombelastography paired with protein analysis., Results: The median percentage of total blood volume shed per group were similar (NS, 52.5% vs. PFP, 55.7; p = 0.065). Survival was 50% in NS compared with 100% in PFP. The change in LY30 and tPA levels from baseline to shock was similar between groups (LY30 PFP, 10; interquartile range [IQR], 4.3-11.2; NS, 4.5; IQR, 4.1-14.2; p = 1.00; tPA PFP, 16.6 ng/mL; IQR, 13.7-27.8; NS, 22.4; IQR, 20.1-25.5; p = 0.240). After resuscitation, the median change in LY30 was greater in the NS group (13.5; IQR, 3.5-19.9) compared with PFP (-4.9%; IQR, -9.22 to 0.25 p = 0.004), but tPA levels did not significantly change (NS, 1.4; IQR, -6.2 to 7.1 vs. PFP, 1.7; IQR, -5.2 to 6.8; p = 0.699)., Conclusion: Systemic hyperfibrinolysis is driven by hypoperfusion and associated with increased levels of tPA. Plasma is a superior resuscitation fluid to NS in a prehospital model of severe hemorrhagic shock as it attenuates hyperfibrinolysis and improves systemic perfusion.

Background: Postinjury hyperfibrinolysis (HF), defined as LY30 of 3% or greater on rapid thrombelastography (rTEG), is associated with high mortality and large use of blood products. We observed that some cases of HF are reversible and are associated with patients who respond to hemostatic resuscitation; however, other cases of severe HF seem to be associated with these patients' inevitable demise. We therefore sought to define this unsurvivable subtype of HF as a recognizable rTEG tracing pattern., Methods: We queried our trauma registry for patients who either died or spent at least 1 day in the intensive care unit, received at least 1 U of packed red blood cells, and had an admission rTEG. Within this group of 572 patients, we identified 42 pairs of nonsurvivors and survivors who matched on age, sex, injury mechanism, and New Injury Severity Score (NISS). We inspected the rTEG tracings to ascertain if any pattern was found exclusively within the nonsurviving group and applied these findings to the cohort of 572 patients to assess the predictive value for mortality., Results: Within the matched group, 17% of the patients developed HF. Within the HF subgroup, a unique rTEG pattern was present in 14 HF patients who died and in none of the survivors. This pattern was a "diamond-shaped" tracing with a short time to maximum amplitude of 14 minutes or shorter and complete lysis before the LY30 point. When these criteria are applied to the 572 unmatched patients, this pattern had a 100% positive predictive value for mortality., Conclusion: Patients displaying the "death diamond" pattern on their admission rTEG are at higher risk for mortality. Given the volume of blood products and other resources that these patients consume, this thrombelastography pattern may represent an objective criterion to discontinue efforts at hemostatic resuscitation., Level of Evidence: Prognostic/epidemiologic study, level III.

Background: Systemic hyperfibrinolysis is a lethal phenotype of trauma-induced coagulopathy. Its pathogenesis is poorly understood. Recent studies have support a central role of platelets in hemostasis and in fibrinolysis regulation, implying that platelet impairment is integral to the development of postinjury systemic hyperfibrinolysis., Objective: The objective of this study was to identify if platelet function is associated with blood clot sensitivity to fibrinolysis. We hypothesize that platelet impairment of the ADP pathway correlates with fibrinolysis sensitivity in trauma patients., Methods: A prospective observational study of patients meeting the criteria for the highest level of activation at an urban trauma center was performed. Viscoelastic parameters associated with platelet function (maximum amplitude [MA]) were measured with native thrombelastography (TEG), and TEG platelet mapping of the ADP pathway (ADP-MA). The contribution of fibrinogen to clotting was measured with TEG (angle) and the TEG functional fibrinogen (FF) assay (FF-MA). Another TEG assay containing tissue-type plasminogen activator (t-PA) (75 ng mL(-1) ) was used to assess clot sensitivity to an exogenous fibrinolytic stimulus by use of the TEG lysis at 30 min (LY30) variable. Multivariate linear regression was used to identify which TEG variable correlated with t-PA-LY30 (quantification of fibrinolysis sensitivity)., Results: Fifty-eight trauma patients were included in the analysis, with a median injury severity score of 17 and a base deficit of 6 mEq L(-1) . TEG parameters that significantly predicted t-PA-LY30 were related to platelet function (ADP-MA, P = 0.001; MA, P < 0.001) but not to fibrinogen (FF-MA, P = 0.773; angle, P = 0.083). Clinical predictors of platelet ADP impairment included calcium level (P = 0.001), base deficit (P = 0.001), and injury severity (P = 0.001)., Results and Conclusions: Platelet impairment of the ADP pathway is associated with increased sensitivity to t-PA. ADP pathway inhibition in platelets may be an early step in the pathogenesis of systemic hyperfibrinolysis., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Metabolic derangement is a key hallmark of major traumatic injury. The recent introduction of mass spectrometry-based metabolomics technologies in the field of trauma shed new light on metabolic aberrations in plasma that are triggered by trauma and hemorrhagic shock. Alteration in metabolites associated with catabolism, acidosis and hyperglycemia have been identified. However, the mechanisms underlying fluxes driving such metabolic adaptations remain elusive., Methods: A bolus of U-(13)C-glucose was injected in Sprague-Dawley rats at different time points. Plasma extracts were analyzed via ultra-high performance liquid chromatography-mass spectrometry to detect quantitative fluctuations in metabolite levels as well as to trace the distribution of heavy labeled carbon isotopologues., Results: Rats experiencing trauma did not show major plasma metabolic aberrations. However, trauma/hemorrhagic shock triggered severe metabolic derangement, resulting in increased glucose levels, lactate and carboxylic acid accumulation. Isotopologue distributions in late Krebs cycle metabolites (especially succinate) suggested a blockade at complex I and II of the electron transport chain, likely due to mitochondrial uncoupling. Urate increased after trauma and hemorrhage. Increased levels of unlabeled mannitol and citramalate, metabolites of potential bacterial origin, were also observed in trauma/hemorrhagic shock rats, but not trauma alone or controls., Conclusions: These preliminary results are consistent with observations we have recently obtained in humans, and expand upon our early results on rodent models of trauma and hemorrhagic shock by providing the kinetics of glucose fluxes after trauma and hemorrhage. Despite the preliminary nature of this study, owing to the limited number of biological replicates, results highlight a role for shock, rather than trauma alone, in eliciting systemic metabolic aberrations. This study provides the foundation for tracing experiments in rat models of trauma. The goal is to improve our understanding of substrate specific metabolic derangements in trauma/hemorrhagic shock, so as to design resuscitative strategies tailored toward metabolic alterations and the severity of trauma.

Unlabelled: Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity., Methods: Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin., Results: α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required., Conclusions: α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.

The existing evidence shows great promise for plasma as the first resuscitation fluid in both civilian and military trauma. We embarked on the Control of Major Bleeding After Trauma (COMBAT) trial with the support of the Department of Defense to determine if plasma-first resuscitation yields hemostatic and survival benefits. The methodology of the COMBAT study represents not only 3 years of development work but also the integration of nearly two decades of technical experience with the design and implementation of other clinical trials and studies. Herein, we describe the key features of the study design, critical personnel and infrastructural elements, and key innovations. We will also briefly outline the systems engineering challenges entailed by this study. The COMBAT trial is a randomized, placebo-controlled, semiblinded, prospective, phase IIB clinical trial conducted in a ground ambulance fleet based at a level I trauma center and part of a multicenter collaboration. The primary objective of the COMBAT trial is to determine the efficacy of field resuscitation with plasma first compared with standard of care (normal saline). To date, we have enrolled 30 subjects in the COMBAT study. The ability to achieve intervention with a hemostatic resuscitation agent in the closest possible temporal proximity to injury is critical and represents an opportunity to forestall the evolution of the "bloody vicious cycle." Thus, the COMBAT model for deploying plasma in first-response units should serve as a model for randomized clinical trials of other hemostatic resuscitative agents.

Introduction: Systemic hyperfibrinolysis (accelerated clot degradation) and fibrinolysis shutdown (impaired clot degradation) are associated with increased mortality compared with physiologic fibrinolysis after trauma. Animal models have not reproduced these changes. We hypothesize rodents have a shutdown phenotype that require an exogenous profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis., Methods: Fibrinolysis resistance was assessed by thrombelastography (TEG) using exogenous tissue plasminogen activator (tPA) titrations in whole blood. There were 3 experimental groups: (1) tissue injury (laparotomy/bowel crush), (2) shock (hemorrhage to mean arterial pressure of 20 mmHg), and (3) control (arterial cannulation and tracheostomy). Baseline and 30-minute postintervention blood samples were collected, and assayed with TEG challenged with taurocholic acid (TUCA)., Results: Rats were resistant to exogenous tPA; the percent clot remaining 30 minutes after maximum amplitude (CL30) at 150 ng/mL (P = .511) and 300 ng/mL (P = .931) was similar to baseline, whereas 600 ng/mL (P = .046) provoked fibrinolysis. Using the TUCA challenge, the percent change in CL30 from baseline was increased in tissue injury compared with control (P = .048.), whereas CL30 decreased in shock versus control (P = .048). tPA increased in the shock group compared with tissue injury (P = .009) and control (P = .012)., Conclusion: Rats have an innate fibrinolysis shutdown phenotype. The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing different procedures. Tissue injury inhibits fibrinolysis, whereas shock promotes tPA-mediated fibrinolysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Metabolic staging after trauma/hemorrhagic shock is a key driver of acidosis and directly relates to hypothermia and coagulopathy. Metabolic responses to trauma/hemorrhagic shock have been assayed through classic biochemical approaches or NMR, thereby lacking a comprehensive overview of the dynamic metabolic changes occurring after shock. Sprague-Dawley rats underwent progressive hemorrhage and shock. Baseline and postshock blood was collected, and late hyperfibrinolysis was assessed (LY30 >3%) in all of the tested rats. Extreme and intermediate time points were collected to assay the dynamic changes of the plasma metabolome via ultra-high performance liquid chromatography-mass spectrometry. Sham controls were used to determine whether metabolic changes could be primarily attributable to anesthesia and supine positioning. Early hemorrhage-triggered metabolic changes that built up progressively and became significant during sustained hemorrhagic shock. Metabolic phenotypes either resulted in immediate hypercatabolism, or late hypercatabolism, preceded by metabolic deregulation during early hemorrhage in a subset of rats. Hemorrhagic shock consistently promoted hyperglycemia, glycolysis, Krebs cycle, fatty acid, amino acid, and nitrogen metabolism (urate and polyamines), and impaired redox homeostasis. Early dynamic changes of the plasma metabolome are triggered by hemorrhage in rats. Future studies will determine whether metabolic subphenotypes observed in rats might be consistently observed in humans and pave the way for tailored resuscitative strategies., (Copyright © 2015 the American Physiological Society.)

Background: In most countries, red blood cells (RBCs) can be stored up to 42 days before transfusion. However, observational studies have suggested that storage duration might be associated with increased morbidity and mortality. While clinical trials are under way, impaired metabolism has been documented in RBCs stored in several additive solutions (ASs). Here we hypothesize that, despite reported beneficial effects, storage in AS-3 results in metabolic impairment weeks before the end of the unit shelf life., Study Design and Methods: Five leukofiltered AS-3 RBC units were sampled before, during, and after leukoreduction Day 0 and then assayed on a weekly basis from storage Day 1 through Day 42. RBC extracts and supernatants were assayed using a ultra-high-performance liquid chromatography separations coupled online with mass spectrometry detection metabolomics workflow., Results: Blood bank storage significantly affects metabolic profiles of RBC extracts and supernatants by Day 14. In addition to energy and redox metabolism impairment, intra- and extracellular accumulation of amino acids was observed proportionally to storage duration, suggesting a role for glutamine and serine metabolism in aging RBCs., Conclusion: Metabolomics of stored RBCs could drive the introduction of alternative ASs to address some of the storage-dependent metabolic lesions herein reported, thereby increasing the quality of transfused RBCs and minimizing potential links to patient morbidity., (© 2014 AABB.)

Background: Severe trauma is associated with massive alterations in metabolism. Thus far, investigations have relied on traditional bioanalytic approaches including calorimetry or nuclear magnetic resonance. However, recent strides in mass spectrometry (MS)-based metabolomics present enhanced analytic opportunities to characterize a wide range of metabolites in the critical care setting., Methods: MS-based metabolomics analyses were performed on plasma samples from severely injured patients' trauma activation field blood and plasma samples obtained during emergency department thoracotomy. These were compared against the metabolic profiles of healthy controls., Results: Few significant alterations were observed between trauma activation field blood and emergency department thoracotomy patients. In contrast, we identified trauma-dependent metabolic signatures, which support a state of hypercatabolism, driven by sugar consumption, lipolysis and fatty acid use, accumulation of ketone bodies, proteolysis and nucleoside breakdown, which provides carbon and nitrogen sources to compensate for trauma-induced energy consumption and negative nitrogen balance. Unexpectedly, metabolites of bacterial origin (including tricarballylate and citramalate) were detected in plasma from trauma patients., Conclusion: In the future, the correlation between metabolomics adaptation and recovery outcomes could be studied by MS-based approaches, and this work can provide a method for assessing the efficacy of alternative resuscitation strategies.

Purpose: Viscoelastic hemostatic assays are emerging as the standard-of-care in the early detection of post-injury coagulopathy. TEG and ROTEM are most commonly used. Although similar in technique, each uses different reagents, which may affect their sensitivity to detect fibrinolysis. Therefore, the purpose of this study is to determine the ability of each device to detect fibrinolysis., Methods: TEG (Rapid, Kaolin, Functional Fibrinogen) and ROTEM (EXTEM, INTEM, FIBTEM) were run simultaneously on normal blood as well as blood containing tPA from healthy volunteers (n = 10). A two-tailed, paired t-test and ANOVA were used to determine the significance between parameters obtained from normal blood and blood with tPA, and individual TEG and ROTEM assays, respectively., Results: TEG detected significant changes in clot strength and 30-min lysis after the addition of tPA (p < 0.0001). All ROTEM assays detected changes in the 30-min lysis (p < 0.0001), but only INTEM detected changes in clot strength (p < 0.05). Kaolin and Rapid TEG assays detected greater changes in clot strength and lysis, but INTEM and EXTEM had decreased lysis onset times compared to TEG (p < 0.001). Functional Fibrinogen and FIBTEM assays detected lysis sooner than other TEG/ROTEM assays, and were comparable., Conclusions: TEG assays detect greater changes in clot strength compared to ROTEM. Despite this, Functional Fibrinogen and FIBTEM assays detect fibrinolysis sooner than their corresponding intrinsic and extrinsic assays. Therefore, fibrinogen assays should be employed in actively bleeding trauma patients in order to provide timely antifibrinolytic therapy.

Background and Objectives: The safety and efficacy of stored red blood cells (RBCs) transfusion has been long debated due to retrospective clinical evidence and laboratory results, indicating a potential correlation between increased morbidity and mortality following transfusion of RBC units stored longer than 14 days. We hypothesize that storage in Optisol additive solution-5 leads to a unique metabolomics profile in the supernatant of stored RBCs., Materials and Methods: Whole blood was drawn from five healthy donors, RBC units were manufactured, and prestorage leucoreduced by filtration. Samples were taken on days 1 and 42, the cells removed, and mass spectrometry-based metabolomics was performed., Results: The results confirmed the progressive impairment of RBC energy metabolism by day 42 with indirect markers of a parallel alteration of glutathione and NADPH homeostasis. Moreover, oxidized pro-inflammatory lipids accumulated by the end of storage., Conclusion: The supernatants from stored RBCs may represent a burden to the transfused recipients from a metabolomics standpoint., (© 2014 International Society of Blood Transfusion.)

Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender., Biological Significance: The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet releasates from apheresis platelets have not been hitherto addressed by means of extensive proteomics technologies. Indeed, apheresis platelets are resuspended in donors' plasma, which hampers detection of less abundant proteins, owing to the overwhelming abundance of albumin (and a handful of other proteins), and the dynamic range of protein concentrations of plasma proteins. In order to cope with these issues, we hereby performed an immuno-affinity column-based depletion of the 14 most abundant plasma proteins. Samples were thus assayed via GeLC-MS, a workflow that allowed us to cover an unprecedented portion of the platelet supernatant proteome, in comparison to previous transfusion medicine-oriented studies in the literature. Finally, we hereby address the issue of biological variability, by considering the donor gender as a key factor influencing the composition of apheresis platelet supernatants. As a result, we could conclude that platelet supernatants from male and female donors are not only different in the first place, but they also store differently. This conclusion has been so far only suggested by classic transfusion medicine studies, but has been hitherto unsupported by actual biochemistry/proteomics investigations. In our opinion, the main strengths of this study are related to the analytical workflow (immunodepletion and GeLC-MS) and proteome coverage, the translational validity of the results (from a transfusion medicine standpoint) and the biological conclusion about the intrinsic (and storage-dependent) gender-related differences of platelet supernatants., Competing Interests: The authors disclose no conflict of interest., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: We have recently identified a spectrum of fibrinolysis in response to injury, in which there is increased mortality in patients who have either excessive fibrinolysis (hyperfibrinolysis [HF]) or impaired fibrinolysis (shutdown). The regulation of the fibrinolytic system after trauma remains poorly understood. Our group's previous proteomic and metabolomic work identified elevated red blood cell (RBC) degradation products in trauma patients manifesting HF. We therefore hypothesized that hemolysis was contributory to the pathogenesis of HF. Given the central role of platelets in the cell-based model of coagulation, we further investigated the potential role of platelet lysis in mediation of the fibrinolytic system., Methods: Red blood cells from healthy donors were frozen in liquid nitrogen and vortexed to create mechanical membrane disruption. Platelets were prepared in a similar fashion. Assays were performed with citrated whole blood mixed ex vivo with either RBC or platelet lysates. Tissue plasminogen activator (tPA) was then added to promote fibrinolysis, mimicking the tPA release from ischemic endothelium during hemorrhagic shock. The degree of fibrinolysis was evaluated with thromboelastography. To identify the mediators of the fibrinolysis system present in RBC and platelet lysates, these lysates were passed over immobilized tPA and plasminogen affinity columns to capture protein-binding partners from RBC or platelet lysates., Results: The addition of 75 ng/mL of tPA to whole blood increased fibrinolysis from median 30-min lysis of 1.4% (interquartile range [IQR], 0.9%-2.0%) to 8.9% (IQR, 6.5%-11.5%). Red blood cell lysate with tPA increased fibrinolysis to 20.1% (IQR, 12.5%-33.7%), which was nearly three times as much lysis as tPA alone (P < 0.001). Conversely, the addition of platelet lysate decreased tPA-mediated fibrinolysis to 0.35% (IQR, 0.2%-0.8%; P < 0.001). Affinity chromatography coupled with tandem mass spectrometry identified a number of proteins not previously associated with regulation of fibrinolysis and trauma., Conclusion: Red blood cell lysate is a potent enhancer of fibrinolysis, whereas platelet lysate inhibits fibrinolysis. Intracellular proteins from circulating blood cells contain proteins that interact with the two key proteins of tPA-mediated fibrinolysis. Understanding the effect of tissue injury and shock on the lysis of circulating cells may provide insight to comprehending the spectrum of fibrinolysis in response to trauma.

Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.

Studies on animal models have documented a role for the water-soluble protein fraction of mesenteric lymph as a conduit from hemorrhagic shock to acute lung injury and postinjury multiple organ failure. We hypothesize that mesenteric lymph is not an ultrafiltrate of plasma and contains specific protein mediators that may predispose patients to acute lung injury/multiple organ failure. Mesenteric lymph and plasma were collected from critically ill or injured patients and from nine patients with lymphatic injuries, during semielective spine reconstruction, or immediately before organ donation. Proteomic analyses were performed through immunoaffinity depletion of the 14 most abundant plasma proteins and 1D gel electrophoresis followed by liquid chromatography coupled online with mass spectrometry analyses. Overall, 548 proteins were identified in the patients undergoing semielective surgery, of which 155 were uniquely present in the lymph. In addition, the postshock plasma proteome was characterized by peculiar features, suggesting that only a partial overlap exists between the plasma and mesenteric lymph from trauma patients. Differential proteins between the matched plasma and mesenteric lymph from trauma patients could be related to coagulopathy and hypercoagulability, cell lysis, proinflammatory responses and immune system activation, extracellular matrix remodeling, lymph-specific immunomodulation and vascular hypoactivity/neoangiogenesis, and energy/redox metabolic adaptation to trauma. In conclusion, the proteome of mesenteric lymph is biologically different (in qualitative and quantitative terms) than that of a mere plasma ultrafiltrate.

Early events triggered by posttrauma/hemorrhagic shock currently represent a leading cause of morbidity and mortality in these patients. The causative agents of these events have been associated with increased neutrophil priming secondary to shock-dependent alterations of mesenteric lymph. Previous studies have suggested that unknown soluble components of the postshock mesenteric lymph are main drivers of these events. In the present study, we applied a label-free proteomics approach to further delve into the early proteome changes of the mesenteric lymph in response to hemorrhagic shock. Time-course analyses were performed by sampling the lymph every 30 min after shock up until 3 h (the time window within which a climax in neutrophil priming was observed). There are novel, transient early post-hemorrhagic shock alterations to the proteome and previously undocumented postshock protein alterations. These results underlie the triggering of coagulation and proinflammatory responses secondary to trauma/hemorrhagic shock, metabolic deregulation and apoptosis, and alterations to proteases/antiproteases homeostasis, which are suggestive of the potential implication of extracellular matrix proteases in priming neutrophil activation. Finally, there is a likely correlation between early postshock mesenteric lymph-mediated neutrophil priming and proteomics changes, above all protease/antiproteases impaired homeostasis (especially of serine proteases and metalloproteases).

Background: Fibrinolysis is a physiologic process maintaining patency of the microvasculature. Maladaptive overactivation of this essential function (hyperfibrinolysis) is proposed as a pathologic mechanism of trauma-induced coagulopathy. Conversely, the shutdown of fibrinolysis has also been observed as a pathologic phenomenon. We hypothesize that there is a level of fibrinolysis between these two extremes that have a survival benefit for the severely injured patients., Methods: Thrombelastography and clinical data were prospectively collected on trauma patients admitted to our Level I trauma center from 2010 to 2013. Patients with an Injury Severity Score (ISS) of 15 or greater were evaluated. The percentage of fibrinolysis at 30 minutes by thrombelastography was used to stratify three groups as follows: hyperfibrinolysis (≥3%), physiologic (0.081-2.9%), and shutdown (0-0.08%). The threshold for hyperfibrinolysis was based on existing literature. The remaining groups were established on a cutoff of 0.8%, determined by the highest point of specificity and sensitivity for mortality on a receiver operating characteristic curve., Results: One hundred eighty patients were included in the study. The median age was 42 years (interquartile range [IQR], 28-55 years), 70% were male, and 21% had penetrating injuries. The median ISS was 29 (IQR, 22-36), and the median base deficit was 9 mEq/L (IQR, 6-13 mEq/L). Distribution of fibrinolysis was as follows: shutdown, 64% (115 of 180); physiologic, 18% (32 of 180); and hyperfibrinolysis, 18% (33 of 180). Mortality rates were lower for the physiologic group (3%) compared with the hyperfibrinolysis (44%) and shutdown (17%) groups (p = 0.001)., Conclusion: We have identified a U-shaped distribution of death related to the fibrinolysis system in response to major trauma, with a nadir in mortality, with level of fibrinolysis after 30 minutes between 0.81% and 2.9%. Exogenous inhibition of the fibrinolysis system in severely injured patients requires careful selection, as it may have an adverse affect on survival., Level of Evidence: Prognostic study, level III.

Many receptors involved with innate immunity activate the inhibitor kappa B kinase signalosome (IKK). The active complex appears to be assembled from the two kinase units, IKKα and IKKβ with the regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNFα-induced signaling and phosphorylation of inhibitor kappa B (IκB), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKKα and NEMO interactomes. Analysis of the affinity interactomes by mass spectrometry detected clathrin with both baits with high confidence. Using the same antibodies for indirect digital immunofluorescence microscopy and FRET, the CHC-IKK complexes were visualized together with NEMO or HSP90. The natural variability of protein amounts in unsynchronized A549 cells was used to obtain statistical correlation for several complexes, at natural levels and without invasive labeling. Analyses of voxel numbers indicated that: (i) CHC-IKK complexes are not part of the IKK signalosome itself but, likely, precursors of IKK-NEMO complexes. (ii) CHC-IKKβ complexes may arise from IKKβ-HSP90 complexes., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Background: Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality and has been linked to the infusion of donor antibodies directed against recipient HLA class I antigens. We hypothesize that antibodies against HLA class I antigens bind to the antigens on the neutrophil (PMN) surface and induce priming and PMN cytotoxicity as the second event in a two-event in vitro model of PMN-mediated cytotoxicity., Methods: Isolated PMNs from HLA-A2 homozygotes, heterozygotes and null donors were incubated with a monoclonal antibody to HLA-A2 and a human polyclonal IgG to HLA-A2 and priming of the oxidase was measured. The monoclonal antibodies and PMNs from these three groups were then used in a two-event model of PMN cytotoxicity., Results: The antibodies to HLA-A2 both primed PMNs from HLA-A2 homozygotes but not from heterozygotes or nulls. Antibodies to HLA-A2 also served as the second event in a two-event model to induce PMN cytotoxicity of HLA-A2 homozygous PMNs., Conclusion: Antibodies to HLA class I antigens may directly prime/activate PMNs through the ligation of the antigen on the cell surface, and the antigen density appears to be important for these changes in PMN physiology., (© 2013 International Society of Blood Transfusion.)

Background: Microparticles (MPs) are submicron size cell fragments that are released from cells., Objectives: We hypothesise that MPs increase during red blood cell (RBC) storage and are part of the pro-inflammatory activity, which accumulates in the RBC supernatant., Methods/materials: RBC units were separated from whole blood of eight healthy donors: 5 U were split, with 50% undergoing leucoreduction (LR) and the remaining left as unmodified controls. The remaining 3 U were leucoreduced. Samples were obtained at days (D) 1 and 42 and cell-free supernatants separated and stored. The supernatants were centrifuged at 17 000 × g (60 min) or 100 000 × g (120 min) into microparticle-rich (MPR) and microparticle-poor (MPP) portions, resuspended in albumin, incubated with antibodies to CD235 (RBCs), CD45 [white blood cells (WBCs)] and CD41a [platelets (Plts)], and analysed by flow cytometry. Isolated neutrophils were incubated with these samples, and priming activity measured., Results: Total MPs increased during storage; however, MPs that marked for precursor cell types did not. Significant priming accumulated in the MPP fraction during storage with some activity present in the MPR fraction from D1 and D42 LR-RBCs., Conclusion: Most of the pro-inflammatory priming activity from stored RBCs resides in the MPP supernatant, although the MPR fraction from D42 LR-RBCs does contain some priming activity., (© 2014 The Authors. Transfusion Medicine © 2014 British Blood Transfusion Society.)

Introduction: Injury is the second leading cause of death worldwide, and as much as 40% of injury-related mortality is attributed to uncontrollable hemorrhage. This persists despite establishment of regionalized trauma systems and advances in the management of severely injured patients. Trauma-induced coagulopathy has been identified as the most common preventable cause of postinjury mortality., Methods: A review of the current literature was performed by collecting PUBMED references related to trauma-induced coagulopathy. Data were then critically analyzed and summarized based on the authors' clinical and research perspective, as well as that reported by other institutions and researchers interested in trauma-induced coagulopathy. A particular focus was placed on those aspects of coagulopathy in which agreement among clinical and basic scientists is currently lacking; these include, pathophysiology, the role of blood components and factor therapy, and goal-directed assessment and management., Results: Trauma-induced coagulopathy has been recognized in approximately one-third of trauma patients. There is a vast range of severity, and the emergence of viscoelastic assays, such as thrombelastography and rotational thromboelastogram, has refined its diagnosis and management, particularly through the establishment of goal-directed massive transfusion protocols. Despite advancements in the diagnosis and management of trauma-induced coagulopathy, much remains to be understood regarding its pathophysiology. The cell-based model of hemostasis has allowed for characterization of endothelial dysfunction, impaired thrombin generation, platelet dysfunction, fibrinolysis, endogenous anticoagulants such as protein-C, and antifibrinolytic proteins. These concepts collectively compose the contemporary, but still partial, understanding of trauma-induced coagulopathy., Conclusion: Trauma-induced coagulopathy is a complex pathophysiological condition, of which some mechanisms have been characterized, but much remains to be understood in order to translate this knowledge into improved outcomes for the injured patient., Competing Interests: DECLARATION OF CONFLICTING INTERESTS None., (© The Finnish Surgical Society 2014.)

Transfusion-related acute lung injury (TRALI) remains a significant cause of transfusion-related mortality with red cell transfusion. We hypothesize that prestorage filtration may reduce proinflammatory activity in the red blood cell (RBC) supernatant and prevent TRALI. Filters were manufactured for both small volumes and RBC units. Plasma containing antibodies to human lymphocyte antigen (HLA)-A2 or human neutrophil antigen (HNA)-3a was filtered, and immunoglobulins and specific HNA-3a and HLA-2a neutrophil (PMN) priming activity were measured. Antibodies to OX27 were added to plasma, and filtration was evaluated in a 2-event animal model of TRALI. RBC units from 31 donors known to have antibodies against HLA antigens and from 16 antibody-negative controls were filtered. Furthermore, 4 RBC units were drawn and underwent standard leukoreduction. Immunoglobulins, HLA antibodies, PMN priming activity, and the ability to induce TRALI in an animal model were measured. Small-volume filtration of plasma removed >96% of IgG, antibodies to HLA-A2 and HNA-3a, and their respective priming activity, as well as mitigating antibody-mediated in vivo TRALI. In RBC units, experimental filtration removed antibodies to HLA antigens and inhibited the accumulation of lipid priming activity and lipid-mediated TRALI. We conclude that filtration removes proinflammatory activity and the ability to induce TRALI from RBCs and may represent a TRALI mitigation step., (© 2014 by The American Society of Hematology.)

Hemorrhage is the most preventable cause of death in civilian and military trauma, and despite tremendous advances in patient transport in the field, survival within the first hour has changed little over the past 40 years. The pathogenesis of trauma-induced coagulopathy is multifactorial, but most authorities believe there is an early depletion of clotting factors. While fresh frozen plasma delivered early in the emergency department has been shown to be beneficial, the rapid onset of trauma-induced coagulopathy suggests advancing this concept to the scene may improve patient outcome. The purpose of this report was to describe the rationale and design of a randomized trial to test the hypothesis that prehospital "plasma-first" resuscitation will benefit the critically injured patient. The rationale includes the possibility that plasma-first resuscitation may be advantageous beyond direct effects on clotting capacity. The study design is based on a ground ambulance system that allows rapid prehospital thawing of frozen plasma.

Background: Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO-activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO-mediated AKI., Methods: Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine., Results: AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine., Conclusion: 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury.

Background: Venous thromboembolism (VTE) prophylaxis remains debated following trauma, and recommendations have not been established. Although hyperfibrinogenemia is a marker of proinflammatory states, it also contributes to thrombus formation. Postinjury hyperfibrinogenemia is common, but the effect of hyperfibrinogenemia on VTE prophylaxis has not been fully elucidated. Therefore, we hypothesized that heparin is less effective for VTE prophylaxis following severe injury due to hyperfibrinogenemia., Methods: In vitro studies evaluated thromboelastography (TEG) parameters in 10 healthy volunteers after the addition of fibrinogen concentrate and heparin. Data from a recent randomized controlled trial, conducted at an academic level I trauma center surgical intensive care unit, were reviewed. Critically injured patients were randomized to standard VTE prophylaxis (5,000 U low-molecular-weight heparin daily) or TEG-guided prophylaxis (up to 10,000 U low-molecular-weight heparin daily) and were followed up for 5 days. Analysis was performed to evaluate the relationship between fibrinogen levels, measures of anticoagulation, and TEG parameters., Results: In vitro studies revealed increased fibrinogen reversed the effects of heparin as measured by TEG. Fifty patients were enrolled in the clinical study with 25 in each arm. Thromboelastography parameters, fibrinogen, platelet count, and anti-Xa levels did not differ between groups despite treatment provided. Fibrinogen levels increased over the 5-day study period (597 ± 24.0 to 689.3 ± 25.0), as well as clot strength (9.8 ± 0.4 to 14.5 ± 0.6), which had a significant correlation coefficient (P < 0.01). Moreover, there was a moderate inverse correlation between fibrinogen level and the effect of heparin (RF), which was significant on study days 1 and 3, implicating hyperfibrinogenemia in heparin resistance., Conclusions: Hypercoagulability and heparin resistance are common following trauma. The preclinical and clinical relationships between fibrinogen levels and hypercoagulability implicate hyperfibrinogenemia as a potential factor in heparin resistance.

Objective: The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors., Methods: Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak., Results: There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p<0.01, n=8) and a contribution by contaminating leukocytes was excluded. Exogenous 125I-VEGF bound avidly and specifically to the lung vasculature, and unlabeled VEGF in the lung perfusate caused vascular leak., Conclusion: Rising concentrations of VEGF occur during storage of single donor platelet concentrates due to platelet secretion or disintegration, but not due to leukocyte contamination. Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels. At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs. These data provide further evidence that VEGF may contribute to the increased lung permeability seen in TRALI associated with platelet products.

Hyperunstable hemoglobinopathy (HUH) [dominantly inherited β-thalassemia (β-thal)] is a relatively rare form of congenital hemolytic anemia in which mutations occur in the genes encoding for α and β chains, or both chains of the hemoglobin (Hb) molecule. We describe two Hispanic adolescents with a new unstable Hb variant (HBB: c.348&#95;349delinsG; p.His117IlefsX42), resulting from a frameshift mutation at codons 115/116 of the β-globin gene. Both patients also have a 3.7 kb deletion on one α gene, leading to a decreased imbalance between α and β chain formation, and subsequently a milder phenotype than that seen in other hyperunstable Hb variants.

Background: The acute coagulopathy of trauma is present in up to one third of patients by the time of admission, and the recent CRASH-2 and MATTERs trials have focused worldwide attention on hyperfibrinolysis as a component of acute coagulopathy of trauma. Thromboelastography (TEG) is a powerful tool for analyzing fibrinolyis, but a clinically relevant threshold for defining hyperfibrinolysis has yet to be determined. Recent data suggest that the accepted normal upper bound of 7.5% for 30-minute fibrinolysis (LY30) by TEG is inappropriate in severe trauma, as the risk of death rises at much lower levels of clot lysis. We wished to determine the validity of this hypothesis and establish a threshold value to treat fibrinolysis, based on prediction of massive transfusion requirement and risk of mortality., Methods: Patients with uncontrolled hemorrhage, meeting the massive transfusion protocol (MTP) criteria at admission (n = 73), represent the most severely injured trauma population at our center (median Injury Severity Score [ISS], 30; interquartile range, 20-38). Citrated kaolin TEG was performed at admission blood samples from this population, stratified by LY30, and evaluated for transfusion requirement and 28-day mortality. The same analysis was conducted on available field blood samples from all non-MTP trauma patients (n = 216) in the same period. These represent the general trauma population., Results: Within the MTP-activating population, the cohort of patients with LY30 of 3% or greater was shown to be at much higher risk for requiring a massive transfusion (90.9% vs. 30.5%, p = 0.0008) and dying of hemorrhage (45.5% vs. 4.8%, p = 0.0014) than those with LY30 less than 3%. Similar trends were seen in the general trauma population., Conclusion: LY30 of 3% or greater defines clinically relevant hyperfibrinolysis and strongly predicts the requirement for massive transfusion and an increased risk of mortality in trauma patients presenting with uncontrolled hemorrhage. This threshold value for LY30 represents a critical indication for the treatment of fibrinolysis., Level of Evidence: Prognostic study, level III.