ALBERT

Description: Background: Platelets are required for effective treatment of severe hemorrhage. Standard-of-care storage is at room temperature (RT), but leads to a storage lesion characterized by loss of hemostatic function and increased risk of bacterial contamination. Refrigeration (4C) mitigates adverse effects; however, it results in pre-activation or priming, which may be suggestive of prothrombotic tendencies. We previously showed that 4C-stored platelets (4C-PLT) retain responses to physiological inhibitors comparable to those of fresh platelets (FR-PLT) in two static aggregation assays. To more closely mimic in-vivo conditions, we tested adhesive response in a microfluidic environment under physiologic high-shear flow. We hypothesized that 4C-PLT display superior adhesion compared to standard-of-care (RT-PLT) and that platelet hemostatic inhibition due to prostacyclin and nitric oxide (NO) would be similar to fresh. Methods: Apheresis platelets (AP) collected from 4 healthy donors were stored for 5 days at RT (22-24°C) or 4C (1-6°C). Additional whole blood was collected to obtain red blood cells (RBCs). Platelet samples were assayed on Day 1 (fresh) and Day 5 (RT-PLT and 4C-PLT) in the presence or absence of prostacyclin (10 nM Prostaglandin I2, PGI2) or an NO donor (50 uM S-Nitrosoglutathione, GSNO). Bioflux plates (Fluxion) were coated with 100 µg/ml type-1 collagen. Prior to perfusion, platelets were stained with calcein-AM (300x103PLT/ul) and RBCs were added to a hematocrit of 40%. Samples were perfused through the collagen-coated wells at an arterial shear rate of 720s-1, and compared to bovine serum album (BSA)-coated channels as a control to assess nonspecific binding. A fluorescence microscope acquired images every 30 sec for 6 min. Data were reported as fluorescence intensity units (FIU) and surface coverage (SC%) measured with Bioflux Montage (MetaMorph) software. Data were analyzed using two-way ANOVA and a post hoc Tukey test for multiple comparisons. Significance was p

Description: 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

Description: 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

Description: Introduction As of June 2015 the FDA has approved an alternative procedure under 21 CFR 640.120 that allows for storage of apheresis platelets at refrigerator temperature (1-6 C; 4°C) without agitation for up to 3 days for use in the resuscitation of actively bleeding patients. Understanding underlying mechanisms responsible for enhanced hemostatic function at 4°C will be critical for such improvements in platelet transfusion. We hypothesized that 4°C platelets display better mitochondrial respiratory function for up to 7 days compared to standard 5-day RT platelets and that mitochondrial gene expression differences between RT and 4°C -stored platelets will correlate with mitochondrial function. Methods Platelets were collected from healthy donors by apheresis according to an IRB-approved protocol. Apheresis platelets (AP) were rested for 1 h before allocation into platelet minibags (Blood Cell Storage, Seattle, WA) and stored for 4 storage durations (Baseline (BL), Day 3, 5, and 7). Mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration were assessed with high-resolution respirometry (O2k, Oroboros). Mitochondrial ROS generation in response to storage condition or stimulation (to assess oxidative burst capacity as a measure of function) was visualized with fluorescent imaging and assayed with flow cytometry using a superoxide stain (Life Technologies). Total RNA was extracted both immediately following apheresis (BL) and on Day 5 from RT and 4°C-stored platelets using Trizol (Molecular Research Center, Cincinnati, OH) after centrifuging the platelets at 900 x g for 10 min. Platelet RNA was quantified using the NanoDrop 2000. RNA quality was examined using gel electrophoresis with the Reliant Gel System (Cambrex, Rockland, ME). Platelet mitochondrial gene expression analysis was evaluated using the 96-well RT2 Profiler PCR Array (Qiagen, Valencia, CA) which profiled 84 mitochondria-focused targets and 12 control genes per sample. Gene expression data analysis was based on the ΔΔCt method with normalization of the raw data to housekeeping genes located on each 96-well plate. Results Mitochondrial respiration was lower in platelets stored at 4°C compared to RT on Days 3, 5, and 7 (Day 5= -57%±0.3; P 〈 0.05), demonstrating that refrigeration slows metabolism. Additionally, maximal mitochondrial oxygen utilization (electron transport system capacity) was better preserved in platelets stored at 4°C (Figure 1). Fluorescent imaging and flow cytometry demonstrated that mROS generation was higher in RT-stored platelets compared to 4°C, reflecting mitochondrial damage. Mitochondrial burst during de novo mROS generation due to stimulation was also preserved at 4°C. Mitochondrial gene expression studies revealed distinct differences in expression profiles for 4°C versus RT-stored platelets after 5 days of storage when normalized to BL measures. Storage at 4°C resulted in significantly greater preservation of 15 gene products at Day 5 (P

Description: Hemorrhage remains the leading cause of preventable morality, resulting in the death of over a third of all trauma patients. Additionally, twenty-five percent of trauma patients present on admission with acute traumatic coagulopathy (ATC) which portends a mortality approaching fifty percent. ATC has been defined by multiple parameters including international normalized ratio (INR) 〉1.2, rotational thromboelastometry (ROTEM) clot amplitude at 5 minutes (CA5) ≤ 35 mm and lysis at 60 minutes (LI60) ≤ 85%. Damage control resuscitation (DCR), the practice of the Joint Theater Trauma System in Iraq and Afghanistan, is based on rapid hemorrhage control, permissive hypotension and transfusion of blood products in a ratio that aims to deliver the functionality of whole blood (1:1:1, red cells:plasma:platelets), in addition to limiting crystalloid resuscitation. ROTEM defined ATC has not been observed over time among DCR eligible combat casualties. The goal of this study was to identify ATC and the effects of DCR in trauma patients treated at level III trauma hospitals in Afghanistan. In this prospective observational study, 88 trauma patients were treated at Craig Air Force Theatre Hospital – Bagram, or Kandahar NATO Hospital in the Afghanistan Theatre. We included only patients from coalition forces identified as having injury that would result in the loss of life or total disability resulting in activation of DCR. Blood was obtained for analysis upon admission and at 6 and 24 hours after admission by a designated research team. Blood was analyzed by ROTEM with multiple assays (EXTEM, FIBTEM, APTEM); however, data was not available to the treatment team. Complete blood counts and INR were also obtained and Injury Severity Scores (ISS) were determined. Transfusion requirements of red blood cells (RBCs), platelets (PLT), fresh frozen plasma (FFP) and cryoprecipitate were recorded for the first 24 hours following admission. ROTEM changes over time were analyzed using Wilcoxon signed-rank test. Forty patients in the cohort had ROTEM (EXTEM) data obtained for evaluation as equipment was unavailable during a portion of the study. The median ISS was 21.5 (IQR 14-27). Four of the patients in the cohort died. The median admission hemoglobin and hematocrit were 11.1 g/dL (IQR 10.1-12) and 32.3% (IQR 29-34.5) respectively. The median INR was 1.3 (IQR 1.2-1.4). The median patient RBC to FFP to PLT ratio was 1:1:0.8. The median clot time (CT) and maximum clot firmness (MCF) were 58.5 sec (IQR 51-65.5) and 56 mm (IQR 51-60.5) respectively. Median CA5 was 37.5 mm (IQR 31-45). ATC as identified by CA5 ≤ 35 mm was present in 15 of 40 patients (38%) upon admission. The median CA5 of patients who met criteria for ATC on admission was 26 mm (IQR 15-34) which improved to 38 mm (IQR 33-44) at 24 hours (p 〈 0.01). Median LI45 was 98% (IQR 96-99). Hyperfibrinolysis as defined by LI45 ≤ 85% was observed in 4 of 40 patients (10%) upon admission which did not change significantly at 24 hours. The incidence of acute traumatic coagulopathy as defined by ROTEM parameters in this high risk military cohort appears to be higher compared to that reported for civilian populations. These data suggest that current DCR practices including a 1:1:1 RBC:FFP:PLT ratio appropriately target high risk trauma patients with ATC and that this strategy appears to be associated with a reduction in the burden of coagulopathy by 24 hours. Disclosures No relevant conflicts of interest to declare.

Description: 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 (p

Description: 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