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Limited Resuscitation With Fresh or Stored Whole Blood Corrects Cardiovascular and Metabolic Function in a Rat Model of Polytrauma and Hemorrhage

Chen, Jacob; Wu, Xiaowu; Keesee, Jeffrey; Liu, Bin; Darlington, Daniel N.; Cap, Andrew P.

doi: 10.1097/SHK.0000000000000748
Basic Science Aspects
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Introduction: We have recently shown that human whole blood stored at 4°C maintains hemostatic and platelet function. In this study, we compared restoration of hemodynamic, metabolic and hemostatic function after limited resuscitation with rat fresh whole blood, rat stored whole blood, or Lactated Ringers in traumatized rats.

Methods: Rat whole blood was stored for 10 days at 4°C for evaluation of hemostatic function. Polytrauma was performed on isoflurane-anesthetized Sprague–Dawley rats (350–450 g) by damage to the intestines, liver, right leg skeletal muscle, and right femur fracture, followed by 40% hemorrhage. At 1 h, rats were resuscitated (20%) with either fresh whole blood (FWB), stored whole blood, 4°C for 7 days (SWB), Lactated Ringers (LR), or nothing. Blood samples were taken before and 2 h after trauma and hemorrhage to evaluate metabolic and hemostatic function.

Results: Whole blood stored for 10 days showed a significant prolongation in prothrombin time (PT) and activated partial thromboplastin time (aPTT), and fall in fibrinogen concentration, but no change in Maximum Clot Firmness or speed of clot formation. Platelet function was maintained until day 7 in storage, than fell significantly. Polytrauma and hemorrhage in rats led to a fall in arterial pressure, plasma bicarbonate, fibrinogen, and platelet function, and a rise in plasma lactate, PT, aPTT, and creatinine. Resuscitation with either FWB or 7 day SWB, but not LR, returned arterial pressure, plasma lactate and plasma bicarbonate to levels similar to control, but had no effect on the fall in fibrinogen or platelet function, or the rise in PT, aPTT, or creatinine.

Conclusion: Hemostatic and platelet function of rat whole blood stored at 4°C is preserved for at least 7 days in vitro. Low volume resuscitation with SWB or FWB, but not LR, restores hemodynamic and metabolic function, but not the coagulopathy after severe trauma and hemorrhage.

*Israeli Defense Forces, Israel

United States Army Institute of Surgical Research, Fort Sam Houston, Texas

Department of Surgery, University of Texas Health Science Center, San Antonio, Texas

Address reprint requests to Daniel N. Darlington, PhD, United States Army Institute of Surgical Research, 3698 Chambers Pass, Fort Sam Houston, TX 78234-6315. E-mail: daniel.n.darlington.civ@mail.mil

Received 8 July, 2016

Revised 26 July, 2016

Accepted 30 August, 2016

The opinions or assertions expressed herein are the private views of the authors and are not to be construed as official or as reflecting the views of the US Department of the Army or the US Department of Defense or the Israeli Defense Forces.

JC prepared the animal experiments, performed all assays, and helped edit the manuscript. XW prepared the animal experiments, performed all assays, assisted in statistical analysis, and helped edit the manuscript. DND conceived the experimental design, analyzed data, graphed data, performed statistics, wrote and edited manuscript. APC conceived the experimental design, wrote and edited manuscript. BL and JK assisted with animal preparation, ran all assays.

Source of Support: US Army Medical Research Material Command.

The authors report no conflicts of interest.

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INTRODUCTION

Human fresh whole blood (FWB) that is collected and transfused within 24 h is an efficient resuscitation fluid for restoration of hemodynamic and hemostatic function in patients with severe trauma and hemorrhage (1–3). Military experience suggests that FWB may achieve better outcomes when compared with component therapy (1–3). Whole blood can be stored at 4°C for up to 21 days in Citrate Phosphate Dextrose or 35 days in Citrate Phosphate Dextrose Adenine-1 anticoagulants. Room temperature storage of whole blood increases the risk of pathogen growth over time, and causes loss of labile coagulation factors, platelet metabolic exhaustion, as well as morphological and biochemical changes in erythrocytes (4, 5). Refrigeration can mitigate these problems, but partially activates platelets and reduces their circulating half-life after transfusion (6–8). Irrespective of this issue, cold stored whole blood has been shown to retain acceptable hemostatic function for up to 14 days (9–12). Whole blood, either fresh or stored, has been proposed for use in trauma patients to restore blood volume, oxygen delivery and mitigate the progression of acute traumatic coagulopathy (10–13). In particular, whole blood has been proposed for use for prehospital resuscitation because it provides all the components of blood in one product in a setting where resuscitation options are limited. Although whole blood is likely superior for use in a prehospital setting, little experimental data exist to test the efficacy of whole blood in trauma.

We have recently developed a rat model of polytrauma and hemorrhage that is coagulopathic and shows a prolongation of prothrombin time and platelet dysfunction that closely parallels clinical findings in human trauma patients (14). In this study, we hypothesize that rat whole blood stored at 4°C is similar to fresh whole blood, and that both would produce similar benefits after resuscitation in this trauma model. However, we first set out to determine whether rat blood stored at 4°C for 10 to 14 days maintains its hemostatic function in vitro as has been shown in human blood (9–11). Here, we found that rat blood retains its hemostatic function for 7 days. We then compared the efficacy of fresh whole blood and stored whole blood (4°C for 7 days) to each other, and to Lactated Ringers, in restoring hemodynamic, metabolic, and hemostatic function after severe trauma and hemorrhage.

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MATERIALS AND METHODS

This study was approved by the Institutional Animal Care and Use Committee of the US Army Institute of Surgical Research. This study was conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations, and the Principles of the Guide for the Care and Use of Laboratory Animals. All experiments were started between 0800 and 0900 h in a room separate from home caging. All rats (Sprague–Dawley from Charles Rivers, www.criver.com) were male, and were group housed. The light/dark cycle was 12 h light/12 h dark. The rats ate Laboratory Rodent Diet 5001 (www.LabDiet.com). Food and water were given ad libitum.

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Whole blood storage at 4°C

Ten rats (300–450 g) were anesthetized with isoflurane and had their left femoral arteries cannlulated. For each rat, 15 mL of whole blood was collected from the femoral cannula into syringes containing standard citrate phosphate dextran anticoagulant and aliquoted into 2.5 mL blood bags. The blood bags were made in our laboratory from 15 mL bags (Blood Cell Storage Inc, Seattle, Wash) by heat-pressing sections of the bag to create a smaller volume and to maintain sterility. Each rat had blood put in five 2.5 mL bags, and stored for 1, 3, 5, 7, and 10 days. Each bag was used for a complete analysis of coagulation function (as described below) per rat on days 1, 3, 5, 7, and 10. The blood was stored in these bags at 4°C in a cold room without agitation. Complete blood count, clotting function, platelet function, blood gas, and chemistries were assessed on whole blood taken fresh from each rat (day 0) and from bags stored for 1, 3, 5, 7, and 10 days. Clotting function was determined on whole blood by measuring prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration using ST-4 (Diagnostic Stago Inc, Parsippany, NJ). Clotting time (CT, min, the time that the initial fibrin formation is detected); α angle (degrees, the speed of clot development); mean clotting firmness (MCF, mm, or strength of the developed clot); and Lysis 60 (LI60, %, percent of lysis at 60 min) were measured by thromboelastometry (ROTEM, TEM International GmbH) as per manufacturer's instructions for the Extem Test (15). The Fibtem test was also performed on paired blood samples to remove the contribution of platelet (cytochalasin D) on ROTEM parameters. Platelet function was measured on whole blood at 37°C diluted in half with saline using multiple electrode aggregometry (Multiplate, Diapharma, Philadelphia, Pa) after stimulation with 1 mM ADP (Sigma-Aldrich Corp, St. Louis, Mo), 1 mM Thrombin mimetic (PAR4 agonist, peptide sequence GYPGKF, AnaSpec), 1 mg/mL rat tail collagen (Life Technology, Thermo Scientific, Waltham, Mass) or an equal volume of vehicle (as a control) as per manufacturer's instructions. Platelet function was expressed as the degree of aggregation (area under the curve [AUC]) or speed of aggregation (velocity). Complete blood counts were measured on a Coulter Ac Tdiff2 (Beckman Coulter, Brea, Calif). Blood gases and chemistries were measured on an I-STAT using Chem4 and Chem 8 cartridges (Abbott Point of Care, Princeton, NJ) as per manufacturer's instruction.

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Whole blood resuscitation in a polytrauma rat model

Four resuscitation groups were used in this study: resuscitation with fresh whole blood (FWB), resuscitation with whole blood stored at 4°C for 7 days (SWB), resuscitation with Lactated Ringer (LR), and no resuscitation (NR). A separate group of rats (donors) were used as a blood source for resuscitation. Polytrauma and hemorrhage was modeled as previously described (14). Rats (350–450 g, n = 10/group) were anesthetized with Isoflurane and had their femoral artery and vein cannulated. Polytrauma was induced after laparotomy by damaging the small intestines, the left and medial liver lobes, and by fracturing the right femur, and damaging the overlying leg skeletal muscle. Specifically, the liver was exposed, and three crush injuries were performed on the right and medial lobes each with a 3 inch hemostat covered with silastic tubing. Ten centimeters of intestine proximal to the cecum was exposed and run gently through a silastic covered clamp with a 2 mm separation between the blades. The intestines showed petechial hemorrhaging, and the liver showed suture lines with clots from the clamps, but no uncontrolled bleeding. The organs were returned to the abdominal cavity and the laparotomy sutured in two layers. The right leg was placed on two pedestals and 6 × 65 g balls were dropped through a tube to impact on a rounded blade placed mid femur. Post-mortem examination showed a shattered bone mid femur with a small (approximately 0.25 mL) hematoma. The skeletal muscle of the right leg was clamped ×10 with a 5 inch hemostat in various places. Damage to the femur or skeletal muscle caused no uncontrolled hemorrhaging. The rats were then bled to a mean arterial pressure of 40 mm Hg. Arterial pressure was held at 40 mm Hg by withdrawing blood while monitoring pressure. Bleeding was stopped when 40% of the estimated blood volume was removed. Total blood volume was estimated as 7% of body weight. Hemorrhage was completed between 30 and 45 min postinjury. Resuscitation was started at 1 h, and lasted up to 5 min. Whole blood used in resuscitation was derived from donor rats of the same strain and same vendor. Donor rats were anesthetized with isoflurane and cannulated as described above, but not traumatized. Whole blood was collected from the donors in standard citrate phosphate dextrose anticoagulant and used either immediately, or after 7 days 4°C static storage and warmed to room temperature before use. Donor rats were euthanized after blood collection. Both FWB and SWB were filtered (Y-Type Blood Plumset with 210 μm filter, Hospira Inc, Lake Forest, Ill) before transfusion. The resuscitation volume was 20% of blood volume (half of shed blood volume). This represents the approximate volume of resuscitation used in prehospital care of trauma patients in both civilian and military settings (16–18) assuming a 5 L blood volume in a 75 kg patient getting approximately two units of whole blood or 1 L of Lactated Ringer. The experiment lasted 2 h to model a military field care scenario where resuscitation would occur within 2 h of trauma. The rats were then euthanized. Resuscitation occurred 1 h after the end of trauma and the beginning of hemorrhage. Blood samples (venous) were taken in citrate phosphate dextrose anticoagulant before (time 0) and 2 h after trauma for measurement of complete blood count, hemostasis and platelet function as described above (for ROTEM analyses, both Extem and Fibtem were performed per manufacturer instructions).

Data Analysis: Comparisons between groups was analyzed by 1- or 2-way ANOVA, or 1- or 2-way ANOVA corrected for repeated measures followed by Holms–Sidac post hoc test. Friedman Repeated Measures Analysis of Variance on Ranks, followed by post hoc comparisons by the method of Dunn, or by Wilcoxon Signed Rank Test was used on data that was not normally distributed (Shapio–Wilk test). The comparison of two means was done by paired t test. All statistics were performed using SigmaPlot (Systat Software Inc, San Jose, Calif).

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RESULTS

Whole blood stored at 4°C

There were significant changes in both the extrinsic and intrinsic coagulation systems over time in whole blood stored at 4°C. PT and fibrinogen concentration fell significantly over time with the largest change in PT occurring on the first day (Fig. 1). Both PT and fibrinogen remained relatively stable over days 3 to 10. aPTT was significantly elevated throughout the storage period; the largest changes occurring over the first 3 days. Red blood cell and platelet counts did not change significantly over the 10 days.

Fig. 1

Fig. 1

Clotting function was also assessed using thromboelastometry and showed fewer significant changes. Storage of whole blood at 4°C had no significant effect on α angle or MCF over the 10 days (Fig. 1). CT decreased significantly on day 1 in parallel with PT, but did not show any greater change through day 10.

Platelet aggregation was assessed after stimulation with ADP, thrombin mimetic, or collagen. These agonists represent three natural stimuli that are involved in platelet aggregation during trauma. However, each agonist uses a different mechanism to initiate aggregation as can be seen from their aggregation profile. There was an initial rise in the ability of the thrombin mimetic, PAR4, and collagen to stimulate aggregation (days 1–5 for thrombin, days 1–7 collagen), followed by a drop below baseline after day 7 (Fig. 2). The ability of ADP to stimulate aggregation steadily decreased and was below 50% of baseline after day 7. The velocity of aggregation to the thrombin mimetic steadily fell after an initial elevation on day 1. The velocity steadily fell for ADP stimulation, and was below 50% of baseline by day 5. There was no significant change in velocity after stimulation with thrombin mimetic or collagen until day 10.

Fig. 2

Fig. 2

PCO2 fell over the 10 days suggesting adequate CO2 diffusion from the storage bag over time (Table 1). Lactate rose and HCO3 fell suggesting that cellular metabolism was maintained, producing lactate, and using HCO3 to buffer the accumulation of H+ ion. The rise in BUN indicates the maintenance of cellular nitrogen metabolism. The fall in creatinine indicates movement of creatinine into cells. The fall in Na+ and rise in K+ suggests a slow loss of cellular Na+/K+ ATPase activity, consistent with a fall in ATP over time.

Table 1

Table 1

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Resuscitation with fresh whole blood, stored whole blood, or LR

We chose 7 days as the maximum storage time for rat whole blood at 4°C because the greatest changes in platelet function occurred after 7 days (comparing day 10 to day 0). The ability of ADP, thrombin minetic, and collagen to stimulate platelet aggregation was significantly lower on day 10, when compared with either day 0 or day 7. Thromboelastometry (ROTEM) values were preserved through days 7 to 10.

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Effects of polytrauma and hemorrhage

Upon necropsy, we found no tissue lacerations or free bleeding into the abdominal cavity after crush of the liver and intestine. No noticeable bleeding occurred after crush in the leg skeletal muscle. However, a small hematoma was seen at the site of the femur break. A line of clot was seen at each site of liver crush. Petechial hemorrhaging was observed at the site of crush injury to the intestines. This suggests that clotting, and therefore thrombin formation, occurred soon after injury, and prevented uncontrolled bleeding later in the experiment, including the post-resuscitation stage.

Polytrauma and hemorrhage led to a fall in mean arterial blood pressure (Fig. 3). However, resuscitation with FWB or SWB returned mean pressure to levels that were similar to baseline (before trauma and hemorrhage) and no rebleeding occurred. In both the NR and LR groups, mean arterial pressure did not recover, and was significantly lower at 90 and 120 min when compared with either the FWB or the SWB groups. In both the NR and LR groups, polytrauma and hemorrhage led to a rise in lactate and a fall in HCO3 (Fig. 4, A and B). However, resuscitation with FWB or SWB attenuated the rise in lactate and completely abolished the fall in HCO3 (Fig. 4, A and B). Polytrauma and hemorrhage led to a significant rise in BUN and creatinine in all groups suggesting the development of acute kidney injury (Fig. 4, C and D).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Polytrauma and hemorrhage led to a rise in PT and aPTT, and a fall in plasma fibrinogen (NR group, Fig. 5, A, C, and E). In all groups, polytrauma and hemorrhage significantly reduced clotting time and mean clotting firmness (Table 2). This was true for both Extem and Fibtem tests and the changes were similar between groups regardless of resuscitation. There was no change in speed of clot formation (α angle) or lysis over 60 min (LI60). In this study, platelets made up approximately 70% of the clot strength as shown by the difference in mean clotting firmness between Extem and Fibtem (Table 2). Fibtem uses cytochalasin D to prevent platelet aggregation and represents the contribution of fibrin to the clot strength.

Fig. 5

Fig. 5

Table 2

Table 2

Polytrauma and hemorrhage led to a significant fall in the ability of collagen and thrombin mimetic to stimulate platelet aggregation (NR group, Fig. 5, B and D). Resuscitation with LR, FWB, or SWB did not restore the ability of collagen or thrombin mimetic to stimulate aggregation. The ability of ADP to stimulate aggregation (Fig. 5F) was not significantly affected by polytrauma and hemorrhage (NR group) or resuscitation at 2 h.

Polytrauma and hemorrhage led to a significant fall in red blood cell count, hemoglobin, and hematocrit (0 vs. 2 h) in all groups (Table 3) consistent with the expected movement of extravascular fluid into the vascular space (19, 20). Resuscitation with whole blood led to a significantly smaller drop in the RBC count for both the FWB or SWB groups, as RBCs were added to the vascular space (Table 3). Platelet count fell modestly, but significantly after polytrauma and hemorrhage (NR group) as has been reported previously (14). Resuscitation with FWB restored platelet count (Table 3) whereas SWB did not. Polytrauma and hemorrhage did not affect overall WBC count in any of the groups. However, there was a significant fall in lymphocytes, and a rise in monocytes and granulocytes at 2 h in all groups.

Table 3

Table 3

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DISCUSSION

This study shows that early resuscitation with a limited volume of whole blood stored at 4°C for 7 days was not different from FWB in terms of restoring hemodynamic and metabolic function after polytrauma in rats. Resuscitation with the same volume of LR did not restore cardiovascular, metabolic, or hemostatic function. The physiologic responses after LR resuscitation were not different from the NR group. Limited resuscitation with either FWB or SWB had little effect on normalization of hemostatic laboratory values, at least 1 h after transfusion in this model.

Guidelines for Tactical Combat Casualty Care has recently focused interest on the benefits of whole blood and blood product for resuscitation in the prehospital setting (13, 21, 22). Indeed, the Norwegian Naval Special Operation Commando protocol calls for collection and transfusion of fresh whole blood on the front lines (23) and recommendations and guidelines are being proposed for use of blood and blood products in austere environments (21, 23, 24). It would be beneficial to the trauma patient if whole blood could be stored, carried, and used at the site of injury. To that end, cold stored whole blood has been proposed (9). The present study shows that cold stored blood has the same beneficial effect as fresh blood in that limited volume that is commonly used prehospital.

This rat model has been shown to be coagulopathic, and the coagulopathy is associated with a rise in plasmin activity, tissue plasminogen activator, and d-dimers. This suggests that elevated fibrinolysis is at least partially involved in the development of the coagulopathy (25). The coagulopathy is also likely due to a decrease in platelet function, which may have multiple etiologies. In this study, the ability of thrombin and collagen to stimulate platelet aggregation was reduced (Fig. 5). Although this reduction was small, it was significant, and may be due in part to the fall in fibrinogen as fibrinogen has been shown to potentiate the effects of natural agonist like ADP and thrombin (26). Because platelets make up 70% to 80% of clot strength (14) (and Table 2), any resuscitation strategy that targets the reversal of this coagulopathy must take into account restoration of platelet function and fibrinogen, as well as the correction of the fibrinolysis.

The present experiments characterize the hemostatic function of rat stored blood. The results presented here are very similar to what was found in human blood stored at 4°C. As shown by Pidcoke et al. (9), human blood stored at 4°C for 21 days exhibited a prolongation in PT and aPTT, and a fall in fibrinogen, and no change in viscoelastic clotting properties (ROTEM) and platelet aggregation over 10 to 14 days (9). Although this is similar to the present study, we observed that rat blood acquires a storage lesion more quickly than human blood, reaching minimal platelet aggregation at 7 days instead of 10 to 14 (human). It should be noted that differences between the studies may be due to a number of factors, such as the blood bag composition and interspecies differences. Irrespective of these differences, both human and rat studies found very little change in clotting time, α angle, and clotting strength during storage, suggesting that rat blood is similar enough to human blood that it can be used in preclinical studies for resuscitation after polytrauma and hemorrhage.

In these experiments, we use a volume of resuscitation equivalent to 1 L (2–3 units of whole blood) for a 75 kg male (approximately 20% of blood volume). This volume has been shown to be feasible in prehospital resuscitation for both the military and civilian populations (16–18) and is relevant to the projected requirements of prolonged field care in future military settings (27, 28). Although this small volume is relevant for prehospital use, the results of this study are different from results obtained after massive transfusion (29).

A wealth of clinical studies demonstrates a deficit in clotting function in severely traumatized patients, and includes an elevation in PT, aPTT, and a decrease in clot strength (30–35). Because an elevation in PT or aPTT is associated with an increase in mortality (36, 37), the correct treatment for this condition is essential in terms of survival, morbidity, and treatment costs. Coagulopathy can be attributed to many factors, including inhibition of thrombin generation or an increase in fibrinolysis (38). Inhibitors of thrombin generation include antithrombin, α2-macroglubulin, Tissue Factor Pathway Inhibitor, and activated protein C. The former two bind and neutralize active thrombin, the latter two inhibit thrombin formation. In the present study, the coagulopathy was not corrected after resuscitation with either blood product. It was therefore possible that the amount of transfused FWB and SWB was not sufficient to correct the hemostatic deficits generated in this study or that correction of coagulopathy lags circulatory improvement due to a need to clear activated factors, platelets, etc. in the liver and spleen. It appears that the coagulopathy induced by trauma and hemorrhage may thus be sustained in the first hour after limited whole blood resuscitation despite hemodynamic and metabolic restoration.

Our data shows that limited resuscitation with FWB and SWB corrected the cardiovascular and metabolic changes after trauma and hemorrhage. This suggests that the hypoperfusion, which contributes to the development of coagulopathy, was significantly reduced. However, the correction of the cardiovascular and metabolic insult did not correct the changes in PT, aPTT, or platelet dysfunction. Therefore, optimal resuscitation may require more clotting factors than is provided by our limited FWB or SWB resuscitation. Fibrinolysis has been reported in many clinical studies of trauma as an elevation in tissue plasminogen activator, plasmin-anti-plasmin, and D-dimers (39, 40). It would follow that inhibition of fibrinolysis with tranexamic acid, or supplementation with fibrinogen, may be a beneficial additive to FWB or SWB for the early treatment of coagulopathy (41–43). Alternatively, higher relative doses of FWB or SWB may be sufficient to correct the coagulopathy and platelet dysfunction.

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REFERENCES

1. Nessen SC, Eastridge BJ, Cronk D, Craig RM, Berseus O, Ellison R, Remick K, Seery J, Shah A, Spinella PC. Fresh whole blood use by forward surgical teams in Afghanistan is associated with improved survival compared to component therapy without platelets. Transfusion 2013; 53 (suppl 1):107S–113S.
2. Perkins JG, Cap AP, Spinella PC, Shorr AF, Beekley AC, Grathwohl KW, Rentas FJ, Wade CE, Holcomb JB. G. st Combat Support Hospital Research. Comparison of platelet transfusion as fresh whole blood versus apheresis platelets for massively transfused combat trauma patients (CME). Transfusion 2011; 51 2:242–252.
3. Spinella PC, Perkins JG, Grathwohl KW, Beekley AC, Holcomb JB. Warm fresh whole blood is independently associated with improved survival for patients with combat-related traumatic injuries. J Trauma 2009; 66 (4 suppl):S69–S76.
4. Hoehn RS, Jernigan PL, Chang AL, Edwards MJ, Caldwell CC, Gulbins E, Pritts TA. Acid sphingomyelinase inhibition prevents hemolysis during erythrocyte storage. Cell Physiol Biochem 2016; 39 1:331–340.
5. Hoehn RS, Jernigan PL, Japtok L, Chang AL, Midura EF, Caldwell CC, Kleuser B, Lentsch AB, Edwards MJ, Gulbins E, et al. Acid sphingomyelinase inhibition in stored erythrocytes reduces transfusion-associated lung inflammation. Ann Surg 2016; [Epub ahead of print].
6. Grozovsky R, Giannini S, Falet H, Hoffmeister KM. Regulating billions of blood platelets: glycans and beyond. Blood 2015; 126 16:1877–1884.
7. Kaufman RM. Uncommon cold: could 4 degrees C storage improve platelet function? Transfusion 2005; 45 9:1407–1412.
8. Shrivastava M. The platelet storage lesion. Transfus Apher Sci 2009; 41 2:105–113.
9. Pidcoke HF, McFaul SJ, Ramasubramanian AK, Parida BK, Mora AG, Fedyk CG, Valdez-Delgado KK, Montgomery RK, Reddoch KM, Rodriguez AC, et al. Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time. Transfusion 2013; 53 (suppl 1):137S–149S.
10. Jobes D, Wolfe Y, O’Neill D, Calder J, Jones L, Sesok-Pizzini D, Zheng XL. Toward a definition of “fresh” whole blood: an in vitro characterization of coagulation properties in refrigerated whole blood for transfusion. Transfusion 2011; 51 1:43–51.
11. Strandenes G, Austlid I, Apelseth TO, Hervig TA, Sommerfelt-Pettersen J, Herzig MC, Cap AP, Pidcoke HF, Kristoffersen EK. Coagulation function of stored whole blood is preserved for 14 days in austere conditions: a ROTEM feasibility study during a Norwegian antipiracy mission and comparison to equal ratio reconstituted blood. J Trauma Acute Care Surg 2015; 78 (6 suppl 1):S31–S38.
12. Yazer MH, Glackin EM, Triulzi DJ, Alarcon LH, Murdock A, Sperry J. The effect of stationary versus rocked storage of whole blood on red blood cell damage and platelet function. Transfusion 2016; 56:596–604.
13. Cap AP. Platelet storage: a license to chill! Transfusion 2016; 56 1:13–16.
14. Darlington DN, Craig T, Gonzales MD, Schwacha MG, Cap AP, Dubick MA. Acute coagulopathy of trauma in the rat. Shock 2013; 39 5:440–446.
15. Lang T, Bauters A, Braun SL, Potzsch B, von Pape KW, Kolde HJ, Lakner M. Multi-centre investigation on reference ranges for ROTEM thromboelastometry. Blood Coagul Fibrinolysis 2005; 16 4:301–310.
16. Hervig T, Doughty H, Ness P, Badloe JF, Berseus O, Glassberg E, Heier HE. Prehospital use of plasma: the blood bankers’ perspective. Shock 2014; 41 (suppl 1):39–43.
17. O’Reilly DJ, Morrison JJ, Jansen JO, Apodaca AN, Rasmussen TE, Midwinter MJ. Prehospital blood transfusion in the en route management of severe combat trauma: a matched cohort study. J Trauma Acute Care Surg 2014; 77 (3 suppl 2):S114–S120.
18. Sixta SL, Hatch QM, Matijevic N, Wade CE, Holcomb JB, Cotton BA. Mechanistic determinates of the acute coagulopathy of trauma (ACoT) in patients requiring emergency surgery. Int J Burns Trauma 2012; 2 3:158–166.
19. Darlington DN, Jones RO, Magnuson TA, Gann DS. Role of intestinal fluid in restitution of blood volume and plasma protein after hemorrhage in awake rats. Am J Physiol 1995; 268 (3 Pt 2):R715–R722.
20. Darlington DN, Jones RO, Marzella L, Gann DS. Changes in regional vascular resistance and blood volume after hemorrhage in fed and fasted awake rats. J Appl Physiol 1995; 78 6:2025–2032.
21. Butler FK, Holcomb JB, Schreiber MA, Kotwal RS, Jenkins DA, Champion HR, Bowling F, Cap AP, Dubose JJ, Dorlac WC, et al. Fluid resuscitation for hemorrhagic shock in tactical combat casualty care: TCCC guidelines change 14-01-2 June 2014. J Spec Oper Med 2014; 14 3:13–38.
22. Fisher AD, Miles EA, Cap AP, Strandenes G, Kane SF. Tactical damage control resuscitation. Mil Med 2015; 180 8:869–875.
23. Strandenes G, De Pasquale M, Cap AP, Hervig TA, Kristoffersen EK, Hickey M, Cordova C, Berseus O, Eliassen HS, Fisher L, et al. Emergency whole-blood use in the field: a simplified protocol for collection and transfusion. Shock 2014; 41 (suppl 1):76–83.
24. Cap AP, Pidcoke HF, DePasquale M, Rappold JF, Glassberg E, Eliassen HS, Bjerkvig CK, Fosse TK, Kane S, Thompson P, et al. Blood far forward: time to get moving!. J Trauma Acute Care Surg 2015; 78 (6 suppl 1):S2–6.
25. Wu X, Darlington DN, Cap AP. Procoagulant and fibrinolytic activity after polytrauma in rat. Am J Physiol Regul Integr Comp Physiol 2016; 310:R323–R329.
26. Schneider DJ, Taatjes DJ, Howard DB, Sobel BE. Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein: a potential contributor to cardiovascular risk. J Am Coll Cardiol 1999; 33 1:261–266.
27. Gerhardt RT, Cap AP, Cestero R, Dubick MA, Heiner J, Koller AR, Lairet J, McClinton AR, Manifold C, Stewart R, et al. The Remote Trauma Outcomes Research Network: rationale and methodology for the study of prolonged out-of-hospital transport intervals on trauma patient outcome. J Trauma Acute Care Surg 2013; 75 (2 suppl 2):S137–S141.
28. Gerhardt RT, Strandenes G, Cap AP, Rentas FJ, Glassberg E, Mott J, Dubick MA, Spinella PC, Network T, Rem TSG. Remote damage control resuscitation and the Solstrand Conference: defining the need, the language, and a way forward. Transfusion 2013; 53 (suppl 1):9S–16S.
29. Repine TB, Perkins JG, Kauvar DS, Blackborne L. The use of fresh whole blood in massive transfusion. J Trauma 2006; 60 (6 suppl):S59–S69.
30. Cotton BA, Faz G, Hatch QM, Radwan ZA, Podbielski J, Wade C, Kozar RA, Holcomb JB. Rapid thrombelastography delivers real-time results that predict transfusion within 1 hour of admission. J Trauma 2011; 71 2:407–414.
31. Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson PI, Stanworth S, Thiemermann C, Brohi K. Definition and drivers of acute traumatic coagulopathy: clinical and experimental investigations. J Thromb Haemost 2010; 8 9:1919–1925.
32. Johansson PI, Sorensen AM, Perner A, Welling KL, Wanscher M, Larsen CF, Ostrowski SR. Disseminated intravascular coagulation or acute coagulopathy of trauma shock early after trauma? An observational study. Crit Care 2011; 15 6:R272.
33. Park MS, Martini WZ, Dubick MA, Salinas J, Butenas S, Kheirabadi BS, Pusateri AE, Vos JA, Guymon CH, Wolf SE, et al. Thromboelastography as a better indicator of hypercoagulable state after injury than prothrombin time or activated partial thromboplastin time. J Trauma 2009; 67 2:266–275.
34. Shaz BH, Winkler AM, James AB, Hillyer CD, MacLeod JB. Pathophysiology of early trauma-induced coagulopathy: emerging evidence for hemodilution and coagulation factor depletion. J Trauma 2011; 70 6:1401–1407.
35. Tauber H, Innerhofer P, Breitkopf R, Westermann I, Beer R, El Attal R, Strasak A, Mittermayr M. Prevalence and impact of abnormal ROTEM(R) assays in severe blunt trauma: results of the ’diagnosis and treatment of trauma-induced coagulopathy (DIA-TRE-TIC) study’. Br J Anaesth 2011; 107 3:378–387.
36. Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie RC, Pittet JF. Acute traumatic coagulopathy: initiated by hypoperfusion: modulated through the protein C pathway? Ann Surg 2007; 245 5:812–818.
37. Frith D, Davenport R, Brohi K. Acute traumatic coagulopathy. Curr Opin Anaesthesiol 2012; 25 2:229–234.
38. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, Pittet JF. Acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. J Trauma 2008; 64 5:1211–1217.
39. Raza I, Davenport R, Rourke C, Platton S, Manson J, Spoors C, Khan S, De’Ath HD, Allard S, Hart DP, et al. The incidence and magnitude of fibrinolytic activation in trauma patients. J Thromb Haemost 2013; 11 2:307–314.
40. Schochl H, Voelckel W, Maegele M, Solomon C. Trauma-associated hyperfibrinolysis. Hamostaseologie 2012; 32 1:22–27.
41. Grottke O. Coagulation management. Curr Opin Crit Care 2012; 18 6:641–646.
42. Lippi G, Favaloro EJ, Cervellin G. Massive posttraumatic bleeding: epidemiology, causes, clinical features, and therapeutic management. Semin Thromb Hemost 2013; 39 1:83–93.
43. Perel P, Prieto-Merino D, Shakur H, Roberts I. Development and validation of a prognostic model to predict death in patients with traumatic bleeding, and evaluation of the effect of tranexamic acid on mortality according to baseline risk: a secondary analysis of a randomised controlled trial. Health Technol Assess 2013; 17 24:1–45.
Keywords:

aPTT; clotting firmness; coagulation; cold storage; fibrinogen; hemorrhage; platelets; polytrauma; PT; resuscitation

© 2017 by the Shock Society