Robbie K. Montgomery

Hemostatic function of apheresis platelets stored at 4 °C and 22 °C

ABSTRACT Introduction: Platelet refrigeration decreases the risk of bacterial contamination and may preserve function better than standard-of-care room temperature (RT) storage. Benefits could include lower transfusion-related complications, decreased costs, improved hemostasis in acutely bleeding patients, and extended shelf life. In this study, we compared the effects of 22°C and 4°C storage on the functional and activation status of apheresis platelets. Methods: Apheresis platelets (n = 5 per group) were stored for 5 days at 22°C with agitation (RT) versus at 4°C with agitation (4°C + AG) and without (4°C). Measurements included platelet counts, mean platelet volume, blood gas analytes, aggregation response, thromboelastography, thromboxane B2 and soluble CD40 ligand release, activation markers, and microparticle formation. Results: Sample pH levels were within acceptable limits for storage products (pH 6.2–7.4). Platelet glucose metabolism (P < 0.05), aggregation response (adenosine diphosphate: RT 0; 4°C + AG 5.0 ± 0.8; 4°C 5.6 ± 0.9; P < 0.05), and clot strength (maximum amplitude: RT 58 ± 2; 4°C + AG 63 ± 2; 4°C 67 ± 2; P < 0.05) were better preserved at 4°C compared with RT storage. Refrigerated samples were more activated compared with RT (P < 0.05), although thromboxane B2 (P < 0.05) and soluble CD40 ligand release (P < 0.05) were higher at RT. Agitation did not improve the quality of 4°C-stored samples. Conclusions: Apheresis platelets stored at 4°C maintain more viable metabolic characteristics, are hemostatically more effective, and release fewer proinflammatory mediators than apheresis platelets stored at RT over 5 days. Given the superior bacteriologic safety of refrigerated products, these data suggest that cold-stored platelets may improve outcomes for acutely bleeding patients.

Storage of platelets at 4°C in platelet additive solutions prevents aggregate formation and preserves platelet functional responses

Platelet (PLT) storage has been limited to 5 days at room temperature due to metabolic decline and risk for bacterial contamination. Refrigeration preserves PLT metabolism and function as well as limits bacterial growth; however, cold storage of PLTs also leads to aggregate formation. We hypothesized that storage of PLT concentrates at 4°C leads to glycoprotein (GP)IIb‐IIIa activation and thus aggregate formation through fibrinogen binding and that this could be prevented by storing PLTs in PLT additive solution (PAS) without compromising PLT function.

Comprehensive evaluation of coagulation in swine subjected to isolated primary blast injury.

ABSTRACT Blast is one of the major causes of injury and death in recent armed conflicts. With increased use of improvised explosive devices in Iraq and Afghanistan, more than 71% of combat casualties are caused by explosions. Blast injuries can range from primary (caused by shock wave) to quaternary injuries (e.g., burns), and such injuries can result in an acute coagulopathy denoted by a hypocoagulable state. It is not clear if this coagulopathy observed in victims of explosion is caused by local or general effect of the primary blast injury itself. In this study, 13 pigs were subjected to severe isolated open-field blast injury and we measured indices of coagulation impairment during the first hour after injury: ROTEM, prothrombin time, activated partial thromboplastin time, coagulation factors, thrombin generation potential, platelet count, platelet activation, platelet function, and procoagulant microparticle formation. After 1 h, the mortality was 33%. No coagulation dysfunction was observed in the survivors in this period. This study presented a highly reproducible and consistent isolated blast injury in large mammals with comprehensive coagulation testing. The data suggest that isolated primary blast injury is not responsible for acute coagulopathy of trauma in victims of explosion but seems to lead to an early hypercoagulable state.

Severe Acinetobacter baumannii Sepsis Is Associated with Elevation of Pentraxin 3

ABSTRACT Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 105 CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.

Pro-coagulant activity of human mesenchymal stem cells

BACKGROUND Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and procoagulant activity to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis. METHODS Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography and calibrated automated thrombogram assays using platelet-poor plasma or whole blood. RESULTS Mesenchymal stem cells express tissue factor on their surfaces and are procoagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more procoagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. Bone marrow MSCs were identified that exhibited low procoagulant activity, whereas all Ad-MSCs examined exhibited high procoagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional procoagulant activity. Mesenchymal stem cell tissue factor expression and procoagulant activity change over time in culture. CONCLUSIONS All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs seem more consistently procoagulant than BM-MSCs, presenting a potential safety concern for systemic administration in coagulopathic patients. Donor variation exists between different cell populations, and culture handling conditions may also determine coagulation activity. Cells must be routinely monitored during preparation to ensure that they retain the desired characteristics before patient administration.

Platelets stored at 4°C contribute to superior clot properties compared to current standard‐of‐care through fibrin‐crosslinking

Currently, platelets for transfusion are stored at room temperature (RT) for 5–7 days with gentle agitation, but this is less than optimal because of loss of function and risk of bacterial contamination. We have previously demonstrated that cold (4°C) storage is an attractive alternative because it preserves platelet metabolic reserves, in vitro responses to agonists of activation, aggregation and physiological inhibitors, as well as adhesion to thrombogenic surfaces better than RT storage. Recently, the US Food and Drug Administration clarified that apheresis platelets stored at 4°C for up to 72 h may be used for treating active haemorrhage. In this work, we tested the hypothesis that cold‐stored platelets contribute to generating clots with superior mechanical properties compared to RT‐stored platelets. Rheological studies demonstrate that the clots formed from platelets stored at 4°C for 5 days are significantly stiffer (higher elastic modulus) and stronger (higher critical stress) than those formed from RT‐stored platelets. Morphological analysis shows that clot fibres from cold‐stored platelets were denser, thinner, straighter and with more branch points or crosslinks than those from RT‐stored platelets. Our results also show that the enhanced clot strength and packed structure is due to cold‐induced plasma factor XIII binding to platelet surfaces, and the consequent increase in crosslinking.

Low-dose heparin anticoagulation during extracorporeal life support for acute respiratory distress syndrome in conscious sheep

Background: Over 32% of burned battlefield causalities develop trauma-induced hypoxic respiratory failure, also known as acute respiratory distress syndrome (ARDS). Recently, 9 out of 10 US combat soldiers’ survived life-threatening trauma-induced ARDS supported with extracorporeal membrane oxygenation (ECMO), a portable form of cardiopulmonary bypass. Unfortunately, the size, incidence of coagulation complications, and the need for systematic anticoagulation for traditional ECMO devices have prevented widespread use of this lifesaving technology. Therefore, a compact, mobile, ECMO system using minimal anticoagulation may be the solution to reduce ARDS in critically ill military and civilian patients. Methods: We conducted a prospective cohort laboratory investigation to evaluate the coagulation function in an ovine model of oleic acid induced ARDS supported with veno-venous ECMO. The experimental design approximated the time needed to transport from a battlefield setting to an advanced facility and compared bolus versus standard heparin anticoagulation therapy. Results: Comprehensive coagulation and hemostasis assays did not show any difference because of ECMO support over 10 h between the two groups but did show changes because of injury. Platelet count and function did decrease with support on ECMO, but there was no significant bleeding or clot formation during the entire experiment. Conclusions: A bolus heparin injection is sufficient to maintain ECMO support for up to 10 h in an ovine model of ARDS. With a reduced need for systematic anticoagulation, ECMO use for battlefield trauma could reduce significant morbidity and mortality from ventilator-induced lung injury and ARDS. Future studies will investigate the mechanisms and therapies to support patients for longer periods on ECMO without coagulation complications. Level of Evidence: V—therapeutic animal experiment.

Platelet and coagulation function before and after burn and smoke inhalation injury in sheep

BACKGROUND Smoke inhalation and burn injury remain a major source of morbidity and mortality. There is known dysregulation of hemostasis in burn patients, but either hypercoagulation or hypocoagulation states are reported. Sheep are an established animal model for studying burn pathology and provide robust data on hemostatic function at baseline and after injury. METHODS After an IACUC-approved protocol, 15 sheep were anesthetized and subjected to a 40% full thickness burn with smoke inhalation. Blood was sampled at baseline, 1 day postinjury (early effects) and days 2, 3, and 4 (late effects) after injury. Assays at each timepoint assessed: hemostatic function by thromboelastography (TEG), platelet counts and function by flow cytometry and aggregometry, coagulation protein levels, and free hemoglobin. Data were analyzed by the Wilcoxon paired test (nonparametric) with significance set at less than 0.05. RESULTS By 24 hours postinjury, platelet counts had dropped, whereas the percent activated platelets increased. Absolute platelet functional response to the agonist adenosine diphosphate (ADP) decreased, whereas response to collagen showed no significant difference. On a per platelet basis, ADP response was unchanged, whereas the collagen response was elevated. Prothrombin time and activated partial thromboplastin time were prolonged. TEG parameters decreased significantly from baseline. Fibrinogen and factor V were trending up; coagulation proteins ATIII, factors IX and X were decreased. Late effects were followed in six animals. At day 4, platelet counts remained depressed compared with baseline with a nadir at day 2; responses to agonist on a per platelet basis remained the same for ADP and stayed elevated for collagen. Platelets continued to have elevated activation levels. Fibrinogen and factor V remained significantly elevated, whereas TEG parameters and prothrombin time, factors IX and X returned to near baseline levels. CONCLUSION Coagulation parameters and hemostasis are dysregulated in sheep after smoke inhalation and burn. By 24 hours, sheep were hypocoagulable and subsequently became hypercoagulable by day 4. These results suggest a three-stage coagulopathy in burn injuries with a known early consumptive hypercoagulable state which is followed by a relatively hypocoagulable state with increased bleeding risk and then a return to a relatively unknown hypercoagulability with increased susceptibility to thrombotic disorders.

Procoagulant activity of human mesenchymal stem cells

BACKGROUND Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and procoagulant activity to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis. METHODS Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography and calibrated automated thrombogram assays using platelet-poor plasma or whole blood. RESULTS Mesenchymal stem cells express tissue factor on their surfaces and are procoagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more procoagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. Bone marrow MSCs were identified that exhibited low procoagulant activity, whereas all Ad-MSCs examined exhibited high procoagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional procoagulant activity. Mesenchymal stem cell tissue factor expression and procoagulant activity change over time in culture. CONCLUSIONS All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs seem more consistently procoagulant than BM-MSCs, presenting a potential safety concern for systemic administration in coagulopathic patients. Donor variation exists between different cell populations, and culture handling conditions may also determine coagulation activity. Cells must be routinely monitored during preparation to ensure that they retain the desired characteristics before patient administration.

Cold storage of platelets in platelet additive solution: an in vitro comparison of two Food and Drug Administration-approved collection and storage systems: COLD STORAGE IN ISOPLATE VS. INTERSOL PAS

Refrigeration of platelets (PLTs) in a PLT additive solution (PAS) reduces PLT activation compared to storage in plasma and preserves function for at least 15 days. Currently only two PASs are licensed by the Food and Drug Administration, each for use with only one apheresis platform. In this study, we compared the metabolic, functional, and activation status of PLTs collected on a Trima apheresis collection system and stored refrigerated in Isoplate (ISO) PAS to PLTs collected on an Amicus collection system and stored refrigerated in Intersol (INT) PAS.