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BACKGROUND Hemorrhage is the leading cause of preventable trauma-related mortality and is frequently aggravated by acute traumatic coagulopathy (ATC). Viscoelastic tests such as rotational thromboelastometry (ROTEM) may improve identification and management of ATC. This study aimed to prospectively evaluate changes in ROTEM among combat casualties during the first 24 hours and compare the capabilities of our conventional clotting assay (international normalized ratio [INR], >1.2) to a proposed integrated ROTEM model (INR >1.2 with the addition of tissue factor pathway activation thromboelastometry [EXTEM] A5 ≤35 mm and/or EXTEM LI30

Background This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. Study design and methods PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. Results Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. Conclusion Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.

BackgroundTo study central hypovolemia in humans, lower body negative pressure (LBNP) is a recognized alternative to blood removal (HEM). While LBNP mimics the cardiovascular responses of HEM in baboons, similarities in hemostatic responses to LBNP and HEM remain unknown in this species.MethodsThirteen anesthetized baboons were exposed to progressive hypovolemia by HEM and, four weeks later, by LBNP. Hemostatic activity was evaluated by plasma markers, thromboelastography (TEG), flow cytometry, and platelet aggregometry at baseline (BL), during and after hypovolemia.ResultsBL values were indistinguishable for most parameters although platelet count, maximal clot strength (MA), protein C, thrombin anti-thrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI) activity significantly differed between HEM and LBNP. Central hypovolemia induced by either method activated coagulation; TEG R-time decreased and MA increased during and after hypovolemia compared to BL. Platelets displayed activation by flow cytometry; platelet count and functional aggregometry were unchanged. TAFI activity and protein, Factors V and VIII, vWF, Proteins C and S all demonstrated hemodilution during HEM and hemoconcentration during LBNP, whereas tissue plasminogen activator (tPA), plasmin/anti-plasmin complex, and plasminogen activator inhibitor-1 did not. Fibrinolysis (TEG LY30) was unchanged by either method; however, at BL, fibrinolysis varied greatly. Post-hoc analysis separated baboons into low-lysis (LY30 2%) whose fibrinolytic state matched at both HEM and LBNP BL. In high-lysis, BL tPA and LY30 correlated strongly (r = 0.95; PConclusionsCentral hypovolemia induced by either LBNP or HEM resulted in activation of coagulation; thus, LBNP is an adjunct to study hemorrhage-induced pro-coagulation in baboons. Furthermore, this study revealed a subset of baboons with baseline hyperfibrinolysis, which was strongly coupled to tPA and uncoupled from TAFI activity.

Background: Lower body negative pressure (LBNP) has been established in human studies as an alternative method to blood removal (HEM) to study central hypovolemia. In a baboon model, LBNP also mimics the cardiovascular response of HEM. Hemostatic responses, including platelet activation, coagulation and fibrinolysis in response to LBNP and HEM in this baboon model are reported here. A previous meeting report from this study focused on hemostatic interactions; this report expands the assay of fibrinolytic proteins and includes detailed post-hoc analysis of interactions. Methods: Thirteen anesthetized baboons were exposed to progressive central hypovolemia by HEM and, four weeks later, by LBNP. Whole blood cell counts were determined; pro-coagulant and fibrinolytic activity was evaluated by plasma markers, thrombelastography, flow cytometry and platelet aggregometry at baseline (BL), intermediate hypovolemia (MID), presyncope or maximal hypovolemia (MAX) and after recovery (REC). Results: Baseline values for HEM (day 0) and LBNP (day 28) were indistinguishable for most analyzed parameters; however platelet number, maximal clotting (MA), Protein C, TAT, TAFI-activity, plasma renin, angiotensin and epinephrine were all significantly different (p2) whose fibrinolytic state matched at both HEM and LBNP BL. High lysis samples (n=8) showed a strong correlation (r=0.95; p Conclusions: Central hypovolemia resulted in activation of coagulation by either LBNP or HEM; thus LBNP is a powerful model for studying hemorrhage. This study also revealed a subset of baboons with baseline activation of fibrinolysis which was strongly coupled to tPA and uncoupled from TAFI activity. The methods demonstrated hemodilution with blood removal and hemoconcentration with LBNP, as has been observed in humans, but these modest changes had no significant effect on coagulation parameters. Unexpectedly, this study demonstrated that the effects of blood removal were sustained 28 days later on baseline parameters. Overall, these results in large primates suggest that baseline differences between apparently healthy and superficially homogeneous individuals may determine outcomes in studies of hemostasis. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage. STUDY DESIGN AND METHODS: WB units were stored for 21 days either at 4 C or 22 C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4 C); CON 22 (WB at 22 C); PRT 4 (PRT WB at 4 C); and PRT 22 (PRT WB at 22 C). Testing was at baseline, Days 1-7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG). RESULTS: Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p /= 0.009). Aggregation decreased over time (p /= 0.001); losses were attenuated by refrigeration (p /= 0.001). Refrigeration preserved TEG parameters (p /= 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p /= 0.001) and microparticle formation (p /= 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p /= 0.032), as well as GPIb-expressing platelets (p /= 0.009). CONCLUSION: The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.

Central hypovolemia elevates hemostatic activity which is essential for preventing exsanguination after trauma, but platelet activation to central hypovolemia has not been described. We hypothesized that central hypovolemia induced by lower body negative pressure (LBNP) activates platelets. Eight healthy subjects were exposed to progressive central hypovolemia by LBNP until presyncope. At baseline and 5 min after presyncope, hemostatic activity of venous blood was evaluated by flow cytometry, thrombelastography, and plasma markers of coagulation and fibrinolysis. Cell counts were also determined. Flow cytometry revealed that LBNP increased mean fluorescence intensity of PAC-1 by 1959±455 units (P

Introduction Hemostatic resuscitation in combat casualty care presents unique challenges not typically encountered in metropolitan areas. Fresh whole blood and blood components are considered preferable to crystalloid or colloid solutions, but availability may be limited in austere wartime environments. Autologous shed blood from a traumatic hemothorax or hemoperitoneum, in theory, is an attractive alternative. This position is supported by the American Academy of Blood Banking, Eastern Association for the Surgery of Trauma, and Society of Critical Care Medicine, despite the fact that the safety and efficacy of shed blood has never been evaluated in randomized trials. Recent reports suggest that shed blood has poor hemostatic properties. This study was performed to evaluate the effects of shed hemothorax plasma and microparticles on platelet function and global tests of coagulation. Methods Shed traumatic hemothorax blood was obtained from 17 adult trauma patients requiring tube thoracostomy, every hour up to 4 hours after placement, according to IRB approved protocol. The hemothorax plasma was then isolated and frozen for later analysis. The effects of shed frozen plasma at 1 and 4 hours (HFP1; HFP4) was examined by performing mixing studies with fresh platelet rich plasma from normal donors at pre-specified serial dilutions of 1:8, 1:4, and 1:2 to mimic intravascular resuscitation . This was then analyzed by several assays. Thromboelastometry (ROTEM) of HFP was done to evaluate clotting time (CT), clot formation time (CFT), alpha angle, and lysis index at 45 minutes. Platelet aggregometry was done to assess the effect of shed HFP on platelet function in response to adenosine diphosphate (ADP), arachidonic acid (ASPI) and collagen. Fibrin degradation products (FDP), von Willebrand factor (vWF) and tissue factor (TF) levels in HFP were determined by enzyme-linked immunosorbent assays (ELISA ). vWF multimers in HFP were further separated by size using gel electrophoresis and detected by immunostaining. Microparticle analysis was performed using isolated microparticles from ultra-centrifuged HFP1 and HFP4 (HMP1 and HMP4) and compared to control isolated microparticles from fresh frozen plasma (FMP). HMP and FMP were then diluted with platelet rich plasma to a predetermined platelet count after which, ROTEM and platelet aggregometry were performed as described above. Non-diluted HFP and resuspended HMP1, HMP4 and FMP were then analyzed by flow cytometry for a variety of microparticle (MP) derived antigens including APC-H7-CD41a (GPIIb/IIIa derived MP), and APC-CD235a (Glycophorin A from red cell derived MP), and FITC-Lactadherin phosphatidylserine (platelet derived MP). Results ROTEM of HFP demonstrated a CT of 55.8% and 60.6% at the 1:8 dilution and 69.3% and 71.4% at the 1:2 dilution, for HFP1 and HFP4 respectively, compared to platelet rich plasma (PRP) and FFP (p HMP1 and HMP4 inhibited platelet aggregation in response to ADP, TXA2, and collagen with data expressed as area under the curve (U), when compared to FMP and PRP, all statistically significant (p700ng/mL vs 0ng/mL p500 pg/mL vs Flow cytometry showed increased microparticle concentrations in HFP1, HFP4, HMP1, and HMP4 samples with greater than 10 fold increase (MP/uL) in several markers (p Conclusion These results demonstrate that autologous shed hemothorax blood promotes a prothrombotic state due to increased tissue factor and microparticle content. Furthermore, clot localization may be impaired through inhibition of platelet aggregation by microparticles and degradation of vWF. These data suggest that randomized trials are indicated to establish the safety and efficacy of autologous shed traumatic hemothorax blood for trauma resuscitation. Disclosures No relevant conflicts of interest to declare.

Up to 50% of preventable trauma deaths are due to truncal hemorrhage resulting in cardiovascular collapse. Lower body negative pressure (LBNP) causes central hypovolemia in humans, resulting in coagulation changes, platelet activation, and fibrinolysis. The model allows investigators to study impending hemorrhagic shock in a minimally invasive human model, but correlations to changes in clotting and lysis due to hemorrhage are not fully described. Published human data to date has not discriminated between altered coagulation due to the LBNP method versus changes attributable to incipient hemorrhagic shock. We measured multiple coagulation parameters in 16 baboons undergoing hemorrhage followed by LBNP and hypothesized that this model elicits early changes associated with acute coagulopathy due to hemorrhage. Baboons underwent incremental hemorrhage (HEM) under an ACURO-approved protocol, followed by LBNP 2-4 weeks later. Blood was sampled at baseline (T0) and after approximately 18.75% (T1), and 25% (T2) blood loss (or until presyncope), and at recovery (T3). LBNP levels were determined by matching the pulse and central venous pressures elicited by hemorrhage. Measurements included: complete blood count; blood chemistries; and coagulations tests. Parameters measured by thromboelastography (TEG) included: R-time, K-time, angle, maximum amplitude (MA), and lysis at 30 and 60 minutes (LY30, LY60). Coagulation factors were quantified over time during both experiments (STA-R Evolution, STAGO).Platelet count was measured and response to agonists (ADP, collagen, TRAP) assessed with impedance aggregometry (Multiplate, Roche Diagnostics). Markers of platelet activation were evaluated under flow cytometry (P-selectin, PAC-1, and leukocyte-platelet aggregates (CD45:CD41)). LBNP and hemorrhage caused activation of the coagulation system over time as measured by TEG R-time (HEM: T0=5.8±0.3 s; T2=4.7±0.3 s; LBNP: T0=6.5±0.3 s; T2=5.1±0.3 s; p LBNP-induced central hypovolemia and hemorrhage in the absence of tissue damage caused activation of the coagulation system in baboons, as well as increased clot formation velocity and strength. TEG and and Protein C results matched most closely between experiments and can be used with LBNP to study pre-hospital changes due blood loss. Lysis data, in contrast to human data, was contradictory. Central hypovolemia due to severe hemorrhage and LBNP caused global activation of coagulation function, indicating that this is the initiating event in acute coagulopathy of trauma. Disclosures No relevant conflicts of interest to declare.

A 3rd generation perfluorocarbon (PFC) is under development for the treatment of traumatic brain injury (TBI). PFCs have high gas solubility and can deliver oxygen to support ischemic brain tissue. This drug increased brain tissue oxygen consumption, decreased infarct size, and improved functional deficits in animal models of brain injury, but it also caused transient thrombocytopenia of unknown etiology. Acute inflammation also causes platelet deficits and is common in the setting of severe TBI. The interaction between the two could exacerbate thrombocytopenia. Here we studied PFC effects on platelet function, platelet morphology and global hemostasis in a lipopolysaccharide (LPS) baboon model of inflammation. Perfluorocarbon (Oxycyte”, Oxygen Biotherapeutics Inc, Morrisville, NC) infusion was performed with and without administration of LPS. The groups studied included: 1. Saline control (20 ml saline + 12 ml/kg saline: SALINE) 2. Saline and LPS control (0.3 mg/kg LPS in 20 ml saline + 12 ml/kg saline: SAL-LPS) 3. Medium dose PFC with saline vehicle (20 ml SALINE + 3 ml/kg PFC: SAL-PFC3) 4. High dose PFC with saline vehicle (20 ml SALINE + 12 ml/kg PFC: SAL-PFC12) 5. Medium dose PFC with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 3 ml/kg PFC: LPS -PFC3) 6. High dose PFC with with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 12 ml/kg PFC: LPS –PFC12) Platelet count, transmission electron microscopy, and flow cytometry were performed at baseline (BL) and at 2, 24, 48, 72, and 96 hours after perfluorocarbon infusion. Microparticles were quantified and characterized by flow cytometry and morphology examined via transmission electron microscopy. Impedance aggregometry and thromboelastography (TEG) were quantified at baseline (BL) and at T2, T72, and T96 (hours). Statistical analysis was by ANOVA, followed by pairwise comparisons and a Bonferroni adjustment. PFCs caused delayed thrombocytopenia which began 2 days post-infusion and persisted beyond the 5-day period of observation (nadirs≤50% of baseline at day 5, p PFC infusion caused clinically significant thrombocytopenia, particularly after LPS administration, and exacerbated LPS-induced platelet activation. The interaction between the two resulted in decreased hemostatic capacity, diffuse bleeding, and shock. Disclosures: Shade: Oxygen Biotherapeutics, Inc.: Research Funding. Anderson:Oxygen Biotherapeutics, Inc.: Employment.

Abstract 2272 Introduction: Fresh whole blood (WB) collected from a “walking blood bank” is used by the U.S. military to supplement component therapy when blood component supplies are exhausted. Currently, WB is used for emergency transfusion within 24 hours of collection, before results of pathogen testing are available. A pathogen reduction technology (PRT), which uses riboflavin and ultraviolet light to damage nucleic acids in pathogens, is being considered as a transfusion-transmitted disease (TTD) risk mitigation measure. The effect of this technology on the hemostatic properties of whole blood, particularly on clotting capacity and clot lysis, are poorly understood, and optimal storage conditions are not defined. We previously reported that activated partial thrombin time (aPTT) and prothrombin time (PT) are prolonged by treatment; however, these tests do not always correlate with clinical findings. Thromboelastography is a more robust measure of clot formation and stability over time; we previously found that maximum amplitude (MA), which represents clot strength, did not decrease with PRT treatment and was preserved by storage at 4°C. Here we explore the effects of PRT on other parameters important to clotting capacity and clot lysis, and present the effects of WB storage at 4°C compared to 22°C. Hypothesis: WB treated with PRT demonstrates similar hemostatic function to non-treated WB, and storage at 4°C reduces degradation of blood components essential to clotting capacity and clot lysis compared to 22°C. Methods: Under an IRB-approved protocol, 8 units per treatment group of WB were collected in CPD anticoagulant from healthy donors of normal hemostatic status according to standard blood donor guidelines. Pathogen reduction was performed using riboflavin and ultraviolet light (265–400nm phosphor; Mirasol® System, CaridianBCT) dosed at 80 J/mLRBC. Treatment groups included: control WB stored at 4° C (CON-04); control WB stored at 22° C (CON-22); PRT-treated WB stored at 4° C (PRT-04); and PRT-treated WB stored at 22° C (PRT-22). The hemostatic function of the blood was assessed at baseline, days 1–7, 10, 14, and 21. Factor VIII and fibrinogen were measured from assayed samples (BCS® XP system, Siemens). Thromboelastography (TEG®, Haemoscope Corp.) estimated total thrombin generation by calculating the first derivative of the TEG tracing, the Total Thrombus Generation variable (TTG). TEG was also used to measured lysis (LY30). Data were analyzed as repeated measures, followed by analysis of variance to assess interactions. Significance was set at p Results: Treatment with PRT caused an initial drop in fibrinogen (baseline: 244 ± 77.5 mg/dL versus post-treatment: 185 ± 63.2 mg/dL, p≤0.04) and factor VIII (baseline: 96 ± 39% versus day post-treatment: 46 ± 23%, p≤0.001); however, levels stabilized thereafter (p≥0.987 and p≥0.871, respectively; see Fig. 1–2). Baseline fibrinogen levels were similar between groups p≥0.386). PRT-04 was the only group in which both fibrinogen and Factor VIII levels fell below clinical reference ranges (fibrinogen: p'0.039; factor VIII: p≤0.016). TTG was unaffected by PRT and was preserved through day 14 by storage at 4° C (p≥0.979, see Fig. 3), but only through day 10 when stored at 22°C (p≤0.290 at day 10). PRT treatment inhibited clot lysis (LY30) compared to storage at 22°C (p≤0.001), and variability was the lowest in the PRT-04 group (p≤0.001, see Fig. 4). Conclusions: Our data demonstrate that pathogen reduction inhibited clot lysis. Decreased clot formation could conceivably account for the smaller degree of lysis; however, we previously found that MA is unaffected, and now demonstrated that thrombin generation was preserved despite a treatment-related decrease in factor VIII levels. While fibrinogen levels were diminished in the PRT-04 group, they were preserved in the PRT-22 group, which also demonstrated the diminished lysis. Cold storage preserved WB clotting capacity compared to storage at room temperature. The clinical significance of these findings has yet to be established; a coagulopathic animal hemorrhage model could determine whether the effects of PRT-induced lysis inhibition and cold storage are beneficial. Disclosures: Mora: CaridianBCT: Research Funding. Pidcoke:CaridianBCT: Research Funding. Valdez-Delgado:CaridianBCT: Research Funding. Fedyk:CaridianBCT: Research Funding. Reddy:CaridianBCT: Employment, Research Funding. Goodrich:CaridianBCT: Employment, Research Funding. Cap:CaridianBCT: Research Funding.