ABSTRACT
Hemorrhage is responsible for 91% of preventable prehospital deaths in combat. Bleeding from anatomic junctions such as the groin, neck, and axillae make up 19% of these deaths, and reports estimate that effective control of junctional hemorrhage could have prevented 5% of fatalities in Afghanistan. Hemostatic dressings are effective but are time-consuming to apply and are limited when proper packing and manual pressure are not feasible, such as during care under fire. CounterFlow-Gauze is a hemostatic dressing that is effective without compression and delivers thrombin and tranexamic acid into wounds. Here, an advanced prototype of CounterFlow-Gauze, containing a range of low thrombin doses, was tested in a lethal swine model of junctional hemorrhage. Outcomes were compared with those of Combat Gauze, the current dressing recommended by Tactical Combat Casualty Care.
CounterFlow-Gauze containing thrombin doses of 0, 20, 200, and 500 IU was prepared. Swine received femoral arteriotomies, and CounterFlow-Gauze was packed into wounds without additional manual compression. In a separate study using a similar model of junctional hemorrhage without additional compression, CounterFlow-Gauze containing 500 IU thrombin was tested and compared with Combat Gauze. In both studies, the primary outcomes were survival to 3 h and volume of blood loss.
CounterFlow-Gauze with 200 and 500 IU had the highest 3-h survival, achieving 70 and 75% survival, respectively. CounterFlow-Gauze resulted in mean peak plasma tranexamic acid concentrations of 9.6 ± 1.0 µg/mL (mean ± SEM) within 3 h. In a separate study with smaller injury, CounterFlow-Gauze with 500 IU achieved 100% survival to 3 h compared with 92% in Combat Gauze animals.
An advanced preclinical prototype of CounterFlow-Gauze formulated with a minimized thrombin dose is highly effective at managing junctional hemorrhage without compression. These results demonstrate that CounterFlow-Gauze could be developed into a feasible alternative to Combat Gauze for hemorrhage control on the battlefield.
INTRODUCTION
Hemorrhage is a leading cause of death on the battlefield, accounting for 91% of potentially preventable deaths in the prehospital environment.1 Rapid hemorrhage control is critical, as mortality increases by 0.35% for every minute that definitive hemostasis is not achieved.2 Continued bleeding rapidly increases the risk of developing deadly sequelae such as coagulopathy, acidosis, hypothermia, and hypocalcemia, which increase mortality four-fold.3–7 Currently, QuikClot Combat Gauze (Z-Medica) is the first-line hemostatic dressing recommended by Tactical Combat Casualty Care (TCCC) and is carried in the packs of U.S. Army soldiers.8 Combat Gauze is effective for severe bleeding when applied with at least 3 min of sustained manual compression.9,10
Hemostatic dressings that are effective without compression would be highly advantageous in difficult tactical scenarios, particularly in future conflicts forecasted to have mass casualties without air superiority.11,12 During care under fire, capacity to tightly pack and manually compress wounds is compromised, especially for lesser trained medics or in instances of buddy care or self-care.13 Without proper compression, high blood flow flushes topical hemostats out of wounds14,15 and decreases the efficacy of many hemostatic dressings.13 Current hemostatic dressings such as Combat Gauze, which contains kaolin, do not deliver pharmacologically active hemostatic agents. A dressing that can deliver pharmacologic agents deep into wounds and stops bleeding without compression could address multiple limitations of hemorrhage control in future conflicts.
CounterFlow-Gauze is a novel hemostatic dressing that can manage severe junctional bleeding without adjunct compression.15–21 It is composed of calcium carbonate (CaCO3), thrombin, and tranexamic acid (TXA), which are materials with many years of safe clinical use.22,23 Upon contact with blood, the calcium carbonate reacts with TXA and dissolves while releasing Ca2+ ions (coagulation factor IV) and effervescing to generate a small amount of carbon dioxide gas (CO2). CO2 propels thrombin, TXA, and Ca2+ deep into wounds even against the brisk outward flow of blood, thus increasing their local delivery and effectiveness. Through this unique composition and mode of action, CounterFlow-Gauze presents an important opportunity to deliver TXA and Ca2+ via alternate routes of administration in the far-forward environment.7,24 In a swine model of junctional hemorrhage similar to those used by the United States Department of Defense to evaluate new topical hemostatic agents, an early iteration of CounterFlow-Gauze containing 2000 IU thrombin had superior efficacy to Combat Gauze and demonstrating a higher survival rate.16
We have developed CounterFlow-Gauze to an advanced preclinical prototype that is manufactured via automated methods and has been reformulated with excipients, which improves handling, ruggedness, thrombin activity during storage, and overall shelf-life. This process has additionally decreased batch variability of thrombin and TXA doses, increased intuitiveness of use, and reduced the number of raw material inputs used during production.
Here, a dose-finding study was performed to determine the minimum thrombin dose in CounterFlow-Gauze, which significantly reduces mortality and blood loss in a lethal swine model of femoral artery injury.25 This gauze was then compared to Combat Gauze in a similar model of junctional hemorrhage without compression. We hypothesized that CounterFlow-Gauze with a minimized dose of thrombin between 0 and 500 IU could achieve high efficacy and comparable efficacy to Combat Gauze.
METHODS
Preparation of CounterFlow-Gauze
CounterFlow-Gauze was prepared by coating CaCO3 (3 µm, American Elements, Los Angeles, CA, USA), protonated TXA (TXA-NH3+), and recombinant human thrombin at 0, 20, 200, or 500 IU (Baxter, Deerfield, IL, USA) on a synthetic fabric gauze substrate using a slot-die automated coating system (Fig. 1A). TXA (Chem-Impex International, Wood Dale, IL, USA) was protonated using organic synthesis. The final size of CounterFlow-Gauze was 42 cm × 7 cm in the dose-finding study and 120 cm × 7 cm in the efficacy study compared to Combat Gauze. The thrombin activity was confirmed by a fluorogenic assay. Gauzes were labeled with radiopaque strips, Z-folded and vacuum sealed in aluminum packs, and stored at room temperature until the experiments were performed.
Ruggedized, shelf-stable CounterFlow-Gauze formulated with various doses of thrombin achieves high survival in junctional hemorrhage and can deliver tranexamic acid systemically. (A) The image of CounterFlow-Gauze coated with protonated tranexamic acid, calcium carbonate, and thrombin using an automated slot-die coating method. (B) Survival curves of animals receiving CounterFlow-Gauze with 0, 20, 200, and 500 IU thrombin in the swine model of junctional hemorrhage. (C) Total blood loss volumes of animals receiving different doses of thrombin with CounterFlow-Gauze. (D) Systemic tranexamic acid concentrations at different time points after groin packing in animals receiving 200 and 500 IU thrombin versions of CounterFlow-Gauze (n = 5).
Swine Hemorrhage Protocol for Dose-Finding Study
The study was approved by the University of Washington’s Institutional Animal Care and Use Committee (protocol #4329-02). Male Yorkshire swine weighing 25-30 kg was transported to the animal facility and acclimatized for 1 week with free access to food and water. Animals were sedated with ketamine (30 mg/kg IM), and anesthetically induced with isoflurane (5%) via nasal cone. When a surgical plane of anesthesia was reached, animals were intubated and the isoflurane concentration was reduced to 1-2% with a dose of buprenorphine (0.01 mg/kg IM). Fraction of inspired oxygen (FiO2) was titrated for arterial O2 saturation >95%. End-tidal carbon dioxide was maintained at 35-40 Torr via ventilator adjustment as necessary. Body temperature was monitored via a pulmonary artery catheter and maintained at 37 ± 1°C. The left femoral artery and venous catheters were placed for blood sampling and fluid/drug administration. A right carotid artery introducer catheter was placed for continuous blood pressure monitoring and blood sampling. A pulmonary artery thermodilution catheter was inserted into the right external jugular vein and advanced into the pulmonary artery for mixed venous blood sampling and for monitoring central venous pressure, mean pulmonary artery pressure, cardiac output (CO), and core temperature. A 4-cm incision was made in the right femoral area, and the femoral artery was exposed and bathed in lidocaine to dilate the artery and prevent vascular contraction in response to injury. Wound pockets were not standardized to have an equivalent volume. The artery was then clamped, and using a vascular punch, a 6-mm arteriotomy was made on the anterior portion of the vessel 2 to 3 cm above the bottom of the groin. Hemorrhage was initiated by removal of the femoral artery clamps, and 30 s of free bleeding was allowed. To measure blood loss, blood was collected on preweighed sponges. After free bleeding, animals were randomized to receive one unit of CounterFlow-Gauze containing a variable dose of thrombin, which was 0, 20, 200, or 500 IU. The gauze was finger-packed into the wound cavity by a single operator.
All surgical staff and data collectors on-site of the experiment were blinded to the study group at the time of the experiment. Gauzes were individually packaged and labeled off-site, with the labeling key kept by a single person. Once packed, the wound was not compressed. After 3 min, Hextend (15 mL/kg) was infused over 10 min. Then, lactated Ringer’s solution was infused as needed over the 3-h monitoring period to maintain a mean arterial pressure (MAP) of 60 mmHg. No additional fluid resuscitation was given. The criteria for euthanasia were cardiovascular collapse—as indicated by MAP <20 mmHg or pCO2 <15 mmHg, which lasted more than 2 min—and loss of pulsatile arterial waveform—or when 180 min had elapsed—whichever occurred first. Primary outcomes included survivability and blood loss. Secondary outcomes included rebleeding frequency, systemic TXA concentrations, volumes of fluid infused, CO, lactate levels, hemoglobin, prothrombin time measurements, and calcium ion concentrations.
TXA Quantification
Whole blood from swine at different timepoints was collected into standard tubes containing ethylenediaminetetraacetic acid (EDTA) and was centrifuged to isolate plasma. High-performance liquid chromatography was used to construct a standard curve of known TXA concentrations in plasma. This was used to determine TXA concentrations in experimental samples.
CounterFlow-Gauze Stability Test
To measure the stability of thrombin on CounterFlow-Gauze, units of gauze were stored in vacuum-sealed foil packaging for different lengths of time at room temperature. Throughout the study, thrombin activity was determined using a spectrophotometric fluorogenic assay. CounterFlow-Gauze was submerged in 20 mL of buffer (20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 90 mM NaCl, 2 mM CaCl2, pH 7.4) to generate CO2 and dissolve TXA and CaCO3. After allowing CO2 generation for 2 min, the mixture was centrifuged and the supernatant was isolated. The supernatant was diluted 1/100 added into a clear 96-well plate. The fluorogenic peptide substrate Boc-Asp(OBzl)-Pro-Arg-MCA (Peptide Institute Inc, Osaka, Japan) was reconstituted in dimethyl sulfoxide and mixed at a 1:1 volumetric ratio with the supernatant to achieve a final concentration of 16 µM, just before starting the reading. Fluorescence at 465 nm was measured every 15 s for 1 h. A fresh thrombin solution of known activity served as the standard, representing 100% target thrombin activity.
Swine Hemorrhage Protocol for Efficacy Study
This protocol is similar to the protocol for the dose-finding study with differences in anesthesia, fluid resuscitation, and the treatment groups. The study was approved by the St. Michael’s Hospital Animal Care Committee. To simulate the difficulty with giving isoflurane in the field, total intravenous anesthesia was used, which is more relevant to combat casualty care. Female pigs weighing 30-40 kg were given ketamine (20-30 mg/kg) and midazolam (0.1-1 mg/kg) by intramuscular injection. Animals were induced with isoflurane 3-5% and endotracheally intubated. Once anesthetized, the pigs were maintained on a ventilator (10 mL/kg) and they were given the following: a loading dose of propofol (2 mg/kg) followed by a constant rate infusion (CRI) (8 mg/kg/h), a loading dose of ketamine (11 mg/kg) followed by a CRI (11 mg/kg/hr), a loading dose of midazolam (0.5 mg/kg) followed by a CRI (0.4-0.7 mg/kg/hr), and isoflurane as needed to maintain anesthesia. Propofol is generally avoided in human trauma patients, but in this animal study, no negative effects of propofol use such as hypotension were observed. The injury creation and other instrumentation were identical to the dose-finding study described earlier although the size of the aortic punch instrument was changed from 6 to 4 mm. In this study, wound pockets were standardized by pouring 40 mL of saline into the pocket before bathing the vessel with lidocaine. Following the initiation of hemorrhage and free bleed, the wound was finger-packed and filled with one pack of Combat Gauze or CounterFlow-Gauze with additional plain gauze to reach an equivalent surface area of Combat Gauze. Packing was performed by a highly trained, military trauma surgeon with extensive experience. Manual compression was not applied. Five minutes after the onset of bleeding, all animals received one 350 mL bolus of lactated Ringer’s and were resuscitated to a target pressure of 60 mmHg (max. 100 mL/kg). Hextend was not used. Animals were observed for 3 h following initiation of hemorrhage or to the time of death. Death was defined as earlier. Primary outcomes were the same as earlier. Secondary outcomes included MAP levels, lactate levels, blood calcium concentrations, blood pH, pCO2, rotational thromboelastometry (ROTEM) clotting time, clot formation time, and maximum clot firmness.
ROTEM Analysis
Blood samples were taken from animals at baseline, 30 min and 1, 2, and 3 h postinjury, or death and collected into vacutainers containing EDTA. ROTEM’s EXTEM assay was performed on whole blood samples at each time point according to the manufacturer’s instructions to measure systemic blood coagulation and the incidence of coagulopathy in all animals that received CounterFlow-Gauze and Combat Gauze.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0.1. Baseline physiological characteristics were compared using a one-way analysis of variance (ANOVA) or a t-test. Survival curves in pigs were analyzed with a Kaplan–Meier log-rank test. Blood loss was analyzed using one-way ANOVA or a t-test. Lactate and calcium concentrations were analyzed using a repeated measures ANOVA. Other secondary outcomes including blood parameters, MAP, volume fluids infused, and ROTEM were compared using one-way ANOVA with Dunnett’s test for multiple comparisons or a t-test. All values were considered statistically significant at P < 0.05.
RESULTS
Low Doses of Thrombin Delivered by CounterFlow-Gauze Achieve High Survival and Display Reduced Blood Loss
The refined CounterFlow-Gauze was tested at four different thrombin concentrations in the swine model of junctional hemorrhage. The survival at 3 h in animals treated with 0, 20, 200, and 500 IU was 4/12 (33%), 1/5 (20%), 7/10 (70%), and 9/12 (75%), respectively (Fig. 1B). Survival between the pigs receiving 0 and 500 IU was statistically significant (P < 0.05). No other differences were significant. Total blood loss normalized to body weight at 0, 20, 200, and 500 IU was 63 ± 7.7 (mean ± SEM), 58 ± 13, 44 ± 12, and 42 ± 8.6 mL/kg, respectively (Fig. 1C). There was a trend to decreased blood loss as thrombin dose was increased, but the differences between groups were not significant.
TXA Delivered into Wounds Is Systemically Absorbed
Plasma TXA concentrations were serially measured in a subset of animals following packing with CounterFlow-Gauze, reflecting systemic absorption from the wound site. There was no TXA detectable at baseline. Plasma TXA concentrations following administration of 200 IU (n = 5) and 500 IU (n = 5) reached peak values of 10.0 ± 1.8 (mean ± SEM) and 9.2 ± 1.0 µg/mL (Fig. 1D) within 3 h of packing. Mean TXA levels in plasma at the end of the experiment were 9.6 ± 1.8 µg/mL in the 200 IU group and 7.4 ± 1.3 hours µg/mL in the 500 IU group.
CounterFlow-Gauze Thrombin Activity Is Stable Long Term
CounterFlow-Gauze with 200 IU thrombin was stored for up to 5 months at 4°C. Thrombin activity of the gauze immediately following manufacturing was 200 IU, which was considered the 100% activity reference mark to which all subsequent time points were compared. The relative thrombin activities at months 2, 3, and 5 were 85 ± 1.5%, 80 ± 2.7%, and 80 ± 3.7%, respectively.
Summary of Secondary Outcomes in Swine Dose-Finding Study
Secondary outcomes of the dose-finding study are summarized in Table I. The rebleeding rate decreased as thrombin dose was increased, suggesting that the increasing thrombin may increase the durability of hemostasis. MAP in surviving animals across all groups was similar at around 60 mmHg, which is comparable to permissive hypotension resuscitation strategies. There was a trend to reduced fluid requirements as the thrombin concentration increased. CO in survivors was similar across all groups and was similar among nonsurvivors as well. Nonsurvivors had reduced CO compared to survivors. There were no differences in lactate concentrations between survivors. Lactate concentrations in nonsurvivors were significantly increased in the 200 IU group compared to the 20 IU group (P < 0.01). Survivors across all groups had low lactate compared to nonsurvivors, indicating that nonsurvivors had a high degree of shock. Blood calcium concentrations were similar across all groups in survivors and nonsurvivors. Hemoglobin concentrations among survivors were similar across groups. However, there were significant differences in hemoglobin concentrations between groups in nonsurvivors. Overall, nonsurvivors had approximately two-fold less hemoglobin compared to survivors. Prothrombin time in survivors was significantly lower in the 500 IU group compared to the 0 IU group (P < 0.01). Nonsurvivors displayed significant differences in prothrombin time between groups. In all thrombin doses, nonsurvivors displayed a slightly prolonged prothrombin time compared to survivors, indicating mild coagulopathy.
Secondary Outcomes of Thrombin Dose-Finding Study Testing Different CounterFlow-Gauze Formulations
| Parameter | 0 IU | 20 IU | 200 IU | 500 IU |
|---|---|---|---|---|
| Number survived for 3 h/total in group (%) | 4/12 (33%) | 1/5 (20%) | 7/10 (70%) | 9/12 (75%) |
| Rebleeding observed (%) | 5/12 (42%) | 3/5 (60%) | 1/10 (10%) | 2/12 (17%) |
| Mean arterial pressure (mmHg) in surviving animals | 62 ± 2 | 63* | 62 ± 5 | 64 ± 1 |
| Volume fluid infused (mL/kg) | 75 ± 11 | 82 ± 16 | 46 ± 15 | 35 ± 12 |
| Cardiac output in survivors (L/min) | 3.7 ± 0.2 | 4.3* | 3.2 ± 0.4 | 3.6 ± 0.1 |
| Cardiac output in nonsurvivors (L/min) | 2.4 ± 0.8 | 1.1 ± 0.2 | 0.6 ± 0.2 | 1.3 ± 0.6 |
| Lactate in survivors (mM) | 1.2 ±0.2 | 0.1* | 1.9 ± 0.6 | 1.4 ± 0.4 |
| Lactate in nonsurvivors (mM) | 11.1 ± 0.4 | 5.8 ± 0.8a | 14.4 ± 2.0a | 12.6 ± 2.8 |
| Ca2+ (mM) in survivors | 1.11 ± 0.03 | 1.19* | 1.15 ± 0.01 | 1.28 ± 0.06 |
| Ca2+ (mM) in nonsurvivors | 1.02 ± 0.03 | 1.08 ± 0.02 | 1.07 ± 0.04 | 1.26 ± 0.02 |
| Hemoglobin in survivors (g/dL) | 7.5 ± 0.8 | 7.0* | 7.8 ± 0.7 | 7.7 ± 0.3 |
| Hemoglobin in nonsurvivors (g/dL) | 3.9 ± 0.3a | 5.3 ± 0.5b | 1.9 ± 0.1a,b | 3.3 ± 0.5 |
| Prothrombin time (s) in survivors | 14.5 ± 0.7a | 12.1* | 13.5 ± 0.3 | 12.6 ± 0.1a |
| Prothrombin time (s) in nonsurvivors | 18.1 ± 0.8a | 14.2 ± 0.3 a,c | 24.5 ± 2.2a,c | 19.9 ± 0.8a |
| Parameter | 0 IU | 20 IU | 200 IU | 500 IU |
|---|---|---|---|---|
| Number survived for 3 h/total in group (%) | 4/12 (33%) | 1/5 (20%) | 7/10 (70%) | 9/12 (75%) |
| Rebleeding observed (%) | 5/12 (42%) | 3/5 (60%) | 1/10 (10%) | 2/12 (17%) |
| Mean arterial pressure (mmHg) in surviving animals | 62 ± 2 | 63* | 62 ± 5 | 64 ± 1 |
| Volume fluid infused (mL/kg) | 75 ± 11 | 82 ± 16 | 46 ± 15 | 35 ± 12 |
| Cardiac output in survivors (L/min) | 3.7 ± 0.2 | 4.3* | 3.2 ± 0.4 | 3.6 ± 0.1 |
| Cardiac output in nonsurvivors (L/min) | 2.4 ± 0.8 | 1.1 ± 0.2 | 0.6 ± 0.2 | 1.3 ± 0.6 |
| Lactate in survivors (mM) | 1.2 ±0.2 | 0.1* | 1.9 ± 0.6 | 1.4 ± 0.4 |
| Lactate in nonsurvivors (mM) | 11.1 ± 0.4 | 5.8 ± 0.8a | 14.4 ± 2.0a | 12.6 ± 2.8 |
| Ca2+ (mM) in survivors | 1.11 ± 0.03 | 1.19* | 1.15 ± 0.01 | 1.28 ± 0.06 |
| Ca2+ (mM) in nonsurvivors | 1.02 ± 0.03 | 1.08 ± 0.02 | 1.07 ± 0.04 | 1.26 ± 0.02 |
| Hemoglobin in survivors (g/dL) | 7.5 ± 0.8 | 7.0* | 7.8 ± 0.7 | 7.7 ± 0.3 |
| Hemoglobin in nonsurvivors (g/dL) | 3.9 ± 0.3a | 5.3 ± 0.5b | 1.9 ± 0.1a,b | 3.3 ± 0.5 |
| Prothrombin time (s) in survivors | 14.5 ± 0.7a | 12.1* | 13.5 ± 0.3 | 12.6 ± 0.1a |
| Prothrombin time (s) in nonsurvivors | 18.1 ± 0.8a | 14.2 ± 0.3 a,c | 24.5 ± 2.2a,c | 19.9 ± 0.8a |
Significantly different (P < 0.05).
Significantly different (P < 0.01).
Significantly different (P < 0.001).
Groups where n < 3 and SEM could not be calculated.
Secondary Outcomes of Thrombin Dose-Finding Study Testing Different CounterFlow-Gauze Formulations
| Parameter | 0 IU | 20 IU | 200 IU | 500 IU |
|---|---|---|---|---|
| Number survived for 3 h/total in group (%) | 4/12 (33%) | 1/5 (20%) | 7/10 (70%) | 9/12 (75%) |
| Rebleeding observed (%) | 5/12 (42%) | 3/5 (60%) | 1/10 (10%) | 2/12 (17%) |
| Mean arterial pressure (mmHg) in surviving animals | 62 ± 2 | 63* | 62 ± 5 | 64 ± 1 |
| Volume fluid infused (mL/kg) | 75 ± 11 | 82 ± 16 | 46 ± 15 | 35 ± 12 |
| Cardiac output in survivors (L/min) | 3.7 ± 0.2 | 4.3* | 3.2 ± 0.4 | 3.6 ± 0.1 |
| Cardiac output in nonsurvivors (L/min) | 2.4 ± 0.8 | 1.1 ± 0.2 | 0.6 ± 0.2 | 1.3 ± 0.6 |
| Lactate in survivors (mM) | 1.2 ±0.2 | 0.1* | 1.9 ± 0.6 | 1.4 ± 0.4 |
| Lactate in nonsurvivors (mM) | 11.1 ± 0.4 | 5.8 ± 0.8a | 14.4 ± 2.0a | 12.6 ± 2.8 |
| Ca2+ (mM) in survivors | 1.11 ± 0.03 | 1.19* | 1.15 ± 0.01 | 1.28 ± 0.06 |
| Ca2+ (mM) in nonsurvivors | 1.02 ± 0.03 | 1.08 ± 0.02 | 1.07 ± 0.04 | 1.26 ± 0.02 |
| Hemoglobin in survivors (g/dL) | 7.5 ± 0.8 | 7.0* | 7.8 ± 0.7 | 7.7 ± 0.3 |
| Hemoglobin in nonsurvivors (g/dL) | 3.9 ± 0.3a | 5.3 ± 0.5b | 1.9 ± 0.1a,b | 3.3 ± 0.5 |
| Prothrombin time (s) in survivors | 14.5 ± 0.7a | 12.1* | 13.5 ± 0.3 | 12.6 ± 0.1a |
| Prothrombin time (s) in nonsurvivors | 18.1 ± 0.8a | 14.2 ± 0.3 a,c | 24.5 ± 2.2a,c | 19.9 ± 0.8a |
| Parameter | 0 IU | 20 IU | 200 IU | 500 IU |
|---|---|---|---|---|
| Number survived for 3 h/total in group (%) | 4/12 (33%) | 1/5 (20%) | 7/10 (70%) | 9/12 (75%) |
| Rebleeding observed (%) | 5/12 (42%) | 3/5 (60%) | 1/10 (10%) | 2/12 (17%) |
| Mean arterial pressure (mmHg) in surviving animals | 62 ± 2 | 63* | 62 ± 5 | 64 ± 1 |
| Volume fluid infused (mL/kg) | 75 ± 11 | 82 ± 16 | 46 ± 15 | 35 ± 12 |
| Cardiac output in survivors (L/min) | 3.7 ± 0.2 | 4.3* | 3.2 ± 0.4 | 3.6 ± 0.1 |
| Cardiac output in nonsurvivors (L/min) | 2.4 ± 0.8 | 1.1 ± 0.2 | 0.6 ± 0.2 | 1.3 ± 0.6 |
| Lactate in survivors (mM) | 1.2 ±0.2 | 0.1* | 1.9 ± 0.6 | 1.4 ± 0.4 |
| Lactate in nonsurvivors (mM) | 11.1 ± 0.4 | 5.8 ± 0.8a | 14.4 ± 2.0a | 12.6 ± 2.8 |
| Ca2+ (mM) in survivors | 1.11 ± 0.03 | 1.19* | 1.15 ± 0.01 | 1.28 ± 0.06 |
| Ca2+ (mM) in nonsurvivors | 1.02 ± 0.03 | 1.08 ± 0.02 | 1.07 ± 0.04 | 1.26 ± 0.02 |
| Hemoglobin in survivors (g/dL) | 7.5 ± 0.8 | 7.0* | 7.8 ± 0.7 | 7.7 ± 0.3 |
| Hemoglobin in nonsurvivors (g/dL) | 3.9 ± 0.3a | 5.3 ± 0.5b | 1.9 ± 0.1a,b | 3.3 ± 0.5 |
| Prothrombin time (s) in survivors | 14.5 ± 0.7a | 12.1* | 13.5 ± 0.3 | 12.6 ± 0.1a |
| Prothrombin time (s) in nonsurvivors | 18.1 ± 0.8a | 14.2 ± 0.3 a,c | 24.5 ± 2.2a,c | 19.9 ± 0.8a |
Significantly different (P < 0.05).
Significantly different (P < 0.01).
Significantly different (P < 0.001).
Groups where n < 3 and SEM could not be calculated.
CounterFlow-Gauze Is as Effective as Combat Gauze in a Swine Model of Femoral Artery Injury
In a separate study, performed at a separate institution, survival was compared between pigs that received Combat Gauze or that received CounterFlow-Gauze with 500 IU thrombin following junctional hemorrhage created using a smaller, 4-mm arteriotomy. The proportional 3-h survival was 11/12 (92%) in the Combat Gauze group and 12/12 (100%) in the CounterFlow-Gauze group (Fig. 2A). Blood loss volumes were 11.5 ± 3.6 and 10.8 ± 1.5 ml/kg in the Combat Gauze and CounterFlow-Gauze groups, respectively (Fig. 2B).
Optimized CounterFlow-Gauze has similar efficacy to Combat Gauze in a swine model of junctional hemorrhage. (A) Survival curves of treatment groups (n = 12). (B) Total hemorrhage volume during survival time, normalized to body weight. (C) Arterial blood lactate concentrations. (D) Arterial blood Ca2+ concentrations over 3 h.
Summary of Secondary Outcomes for Comparative Survival Study
Secondary outcomes of the study are tabulated in Table II. All secondary outcomes are reported for survivors only, as only one animal died in this study. There were no significant differences among any of the secondary outcomes across surviving animals between groups. MAP in survivors was approximately 60 mmHg, which was the target for permissive hypotension. Blood pH and pCO2 measurements to detect any increased CO2 in the blood were not different between groups. Calcium and potassium ion concentrations were not different between groups. Lactate levels remained low, which is consistent with the low blood loss in this study. ROTEM EXTEM analysis was performed at multiple time points, but there were no differences in any parameter. These are likely due to excellent packing of wounds by a highly skilled operator, a trauma surgeon, which prevented major blood loss and any subsequent coagulopathy.
Summary of Secondary Outcomes in Surviving Animals in Head-to-Head Survival Study Comparing Two Gauze Treatments
| Parameter | Combat Gauze (n = 12) | CounterFlow-Gauze (n = 12) |
|---|---|---|
| Mean arterial pressure (mmHg) | 68 ± 4 | 68 ± 2 |
| Blood pH | 7.41 ± 0.03 | 7.41 ± 0.02 |
| pCO2 (mmHg) | 38.7 ± 2.0 | 38.8 ± 1.7 |
| ROTEM EXTEM clotting time (s) (n = 4) | 47.5 ± 2.2 | 49.5 ± 3.4 |
| ROTEM EXTEM clot formation time (s) (n = 4) | 47.5 ± 1.3 | 49.0 ± 1.8 |
| ROTEM EXTEM maximum clot firmness (mm) (n = 4) | 73.2 ± 1.0 | 74.0 ± 1.3 |
| Parameter | Combat Gauze (n = 12) | CounterFlow-Gauze (n = 12) |
|---|---|---|
| Mean arterial pressure (mmHg) | 68 ± 4 | 68 ± 2 |
| Blood pH | 7.41 ± 0.03 | 7.41 ± 0.02 |
| pCO2 (mmHg) | 38.7 ± 2.0 | 38.8 ± 1.7 |
| ROTEM EXTEM clotting time (s) (n = 4) | 47.5 ± 2.2 | 49.5 ± 3.4 |
| ROTEM EXTEM clot formation time (s) (n = 4) | 47.5 ± 1.3 | 49.0 ± 1.8 |
| ROTEM EXTEM maximum clot firmness (mm) (n = 4) | 73.2 ± 1.0 | 74.0 ± 1.3 |
Abbreviations: ROTEM: Rotational Thromboelastometry.
Summary of Secondary Outcomes in Surviving Animals in Head-to-Head Survival Study Comparing Two Gauze Treatments
| Parameter | Combat Gauze (n = 12) | CounterFlow-Gauze (n = 12) |
|---|---|---|
| Mean arterial pressure (mmHg) | 68 ± 4 | 68 ± 2 |
| Blood pH | 7.41 ± 0.03 | 7.41 ± 0.02 |
| pCO2 (mmHg) | 38.7 ± 2.0 | 38.8 ± 1.7 |
| ROTEM EXTEM clotting time (s) (n = 4) | 47.5 ± 2.2 | 49.5 ± 3.4 |
| ROTEM EXTEM clot formation time (s) (n = 4) | 47.5 ± 1.3 | 49.0 ± 1.8 |
| ROTEM EXTEM maximum clot firmness (mm) (n = 4) | 73.2 ± 1.0 | 74.0 ± 1.3 |
| Parameter | Combat Gauze (n = 12) | CounterFlow-Gauze (n = 12) |
|---|---|---|
| Mean arterial pressure (mmHg) | 68 ± 4 | 68 ± 2 |
| Blood pH | 7.41 ± 0.03 | 7.41 ± 0.02 |
| pCO2 (mmHg) | 38.7 ± 2.0 | 38.8 ± 1.7 |
| ROTEM EXTEM clotting time (s) (n = 4) | 47.5 ± 2.2 | 49.5 ± 3.4 |
| ROTEM EXTEM clot formation time (s) (n = 4) | 47.5 ± 1.3 | 49.0 ± 1.8 |
| ROTEM EXTEM maximum clot firmness (mm) (n = 4) | 73.2 ± 1.0 | 74.0 ± 1.3 |
Abbreviations: ROTEM: Rotational Thromboelastometry.
DISCUSSION
The survival studies here demonstrated the efficacy of a refined CounterFlow-Gauze, which delivers thrombin, TXA, and calcium and meets many of the requirements set out by Kheirabadi et al.25 for hemostatic dressings for tactical applications. These studies both determined the optimal dose of thrombin and validated a new design of CounterFlow-Gauze that maintains its efficacy as previously reported. These design changes streamline manufacturing and ruggedize CounterFlow-Gauze to prepare it for fielding and advanced studies. The new manufacturing process allows for large-scale production and rapid scale-up, if required, and reduces risks caused by potential supply chain disruptions for raw material inputs. We also validated lightweight and low cube packaging, which minimizes moisture ingress and maintains product quality. CounterFlow-Gauze is positioned for scale-up for future conflicts where its high efficacy without the need for compression could enable it to save many lives.
In the dose-finding study, CounterFlow-Gauze with 500 IU thrombin was selected as the lead prototype since it significantly increased survival compared to animals that received 0 IU. Although our stability study showed that CounterFlow-Gauze’s thrombin activity remains stable over months, packaging CounterFlow-Gauze, which initially contains 500 IU, may help maximize survival when applying units that have been stored under extreme conditions; reduction of thrombin activity to 200 IU could still yield a high survival of 70%. In the dose-finding study, survival rates appear lower than that in the study comparing with Combat Gauze since wound pocket volumes were not rigorously normalized to ensure tightest possible packing. This enabled us to observe a higher difference in survival when comparing thrombin doses, which was essential to definitively choosing a thrombin dose for CounterFlow-Gauze. Within studies, operators were blinded to which gauzes were being applied to minimize variations because of unconscious differences in the packing technique. This dose-finding study also showed that the refined CounterFlow-Gauze with 500 IU thrombin, with other modifications that increased its usability and ruggedness, is as effective as previous iterations of CounterFlow-Gauze, which contained more thrombin.
Powerful procoagulants such as thrombin carry risks of thrombosis.14 The self-propelling action of CounterFlow-Gauze enables high hemostatic efficacy using doses of thrombin that are lower than clinically approved thrombin products.26,27 This is likely due to the increased transport of thrombin in the wound sites, which increases the local concentration at the site of damage, as previous data in a similar swine model showed that gauze, which delivered the same dose of thrombin but without effervescence, greatly reduced survival from 100% to 25%.16 Delivering lower doses of thrombin would likely further reduce risks of adverse events, although even when using CounterFlow-Gauze containing higher doses of thrombin, no thrombosis or other adverse effects have been seen through multiple bleeding types and methods of application.15–21
CounterFlow-Gauze also delivers TXA, which is a pharmacological therapy for managing bleeding. TXA acts to stabilize clots against fibrinolysis and is especially beneficial for addressing weak clots, which form in coagulopathic trauma patients. Delivering TXA locally can prevent rebleeding and maintain hemostasis during delayed evacuations and through Prolonged Field Care.24 Here, we showed that CounterFlow-Gauze delivers TXA systemically and that plasma concentrations are similar to 10 µg/mL which is known to inhibit fibrinolysis in vitro.28 We do not expect any increase in the risk of thromboembolic events from administering additional systemic TXA beyond the intravenous administration of 2 g TXA recommended by TCCC.29 Thromboembolic events have never been observed in multiple studies testing the absorption of high doses of CounterFlow from the intraperitoneal space in swine models of hemorrhage.20,21 TXA is highly researched and safely administered in trauma and many surgical procedures at intravenous doses as high as 4 g.30 Systemic TXA delivered with CounterFlow-Gauze could help to reduce hyperfibrinolysis, which increases mortality.31 CounterFlow-Gauze also delivers calcium ions into wounds, which is an important cofactor for coagulation enzymes. The absorption of Ca2+ could contribute to mitigating hypocalcemia, a common finding in patients with trauma-related shock.7 In future conflicts predicted to take place in austere battlespaces including extreme cold environments, mortality from hemorrhage will be exacerbated.32–34 Extreme cold environments will impact large-scale operational logistics, delay evacuations—particularly during mass-casualty events—and challenge the current paradigms of TCCC for halting hemorrhage and administering emergency first aid at the point of injury. CounterFlow-Gauze could therefore become an important additional source of TXA and Ca2+ in freezing Arctic environments where administering therapies intravenously is difficult.34 Future studies will verify the suitability of CounterFlow-Gauze for managing hemorrhage without compression at cold temperatures.
In the comparative study versus Combat Gauze, CounterFlow-Gauze with 500 IU thrombin achieved 100% survival at 3 h postinjury, although both gauze groups had high survival. Survival in this model appeared higher than that in the dose-finding study, although a smaller injury was created. However, the higher survival is consistent with the lower blood loss volumes and increased attention to tightness of packing. This study shows that both CounterFlow-Gauze and Combat Gauze can be highly effective at managing junctional hemorrhage in a highly controlled setting, even without wound compression, when applied by a highly experienced military trauma surgeon. These high survival rates may not reflect actual use cases in the prehospital environment that might be more severe. Using more severe models of junctional hemorrhage with a larger arteriotomy similar to the dose-finding study performed here, Combat Gauze achieved 33-67% survival even with additional manual compression.25 While no direct comparisons can be made, this is less than the 75% survival demonstrated by CounterFlow-Gauze with 500 IU without compression in the dose-finding study.
This study had several limitations. In both studies, wound packing was applied by a single blinded operator, which would not show differences in efficacy related to differences in the packing technique. To determine if CounterFlow-Gauze and Combat Gauze have similar usability, and if usability and user training affect survival, future experiments are required wherein personnel of less and varied experience levels are recruited to pack the wound.35 Differences in survival can also be highlighted by changing the severity of hemorrhage in the model or by inducing dilutional coagulopathy or hypothermia. As lactated Ringer’s solution and Hextend are no longer recommended for fluid resuscitation and have been replaced by whole blood when possible, administering these fluids to animals may have affected outcomes of the study. However, this resuscitation protocol is similar to previously published standardized models of junctional hemorrhage used for evaluating new topical hemostatic agents.25 It is likely that whole blood resuscitation in this model would have increased survivability, and using lactated Ringers and Hextend to resuscitate presented a greater challenge to the efficacy of CounterFlow-Gauze. Furthermore, specific studies designed to detect changes in arterial Ca2+ concentration would elucidate how CounterFlow-Gauze’s ability to deliver calcium into wounds and calcium’s subsequence distribution and clearance contribute to CounterFlow-Gauze’s hemostatic efficacy. This could be achieved by minimizing the volume of Hextend or lactated Ringer’s administered or replacing fluid resuscitation with calcium-depleted saline. Lastly, the longer-term hemostasis of CounterFlow-Gauze could not be evaluated in this study as it was a 3-h study. Continued testing of CounterFlow-Gauze will utilize longer-term studies to test the capability of CounterFlow-Gauze to provide prolonged hemorrhage control and systemic TXA.
CONCLUSION
CounterFlow-Gauze is a novel ruggedized hemostatic dressing that is effective without compression and is nearing field readiness for hemorrhage control in the prehospital environment, including potentially care under fire scenarios. Packing of CounterFlow-Gauze with a low thrombin dose of 500 IU achieves 75-100% short-term survival in two swine models of junctional hemorrhage and delivers therapeutically relevant TXA levels systemically.
SUPPLEMENT SPONSORSHIP
This article appears as part of the supplement “Proceedings of the 2022 Military Health System Research Symposium,” sponsored by the Assistant Secretary of Defense for Health Affairs.
ACKNOWLEDGMENTS
We thank Danielle Bince and the animal laboratory at the Li Ka Shing Knowledge Institute and Sandy Trpcic for their support with conducting experiments. We also thank the animals used in this study.
FUNDING
This work was supported by the U.S. Department of Defense (W81XWH2020006 and W81XWH-21-1-0969), a grant from the Surgeon General’s Health Research Program, Defence Research and Development Canada, and the Canadian Institute for Military and Veteran Health Research (Task 60/ W7714-145967/001/SV), along with support from the Center for Blood Research.
CONFLICT OF INTEREST STATEMENT
N.A., M.F.C., J.R.B., and C.J.K. are involved in commercialization activities for self-propelling hemostatic powder. A.B. is an active serving member of the Canadian Armed Forces. The rest of the authors have no conflicts of interest to declare for this work.
IACUC APPROVALS
Obtained, detailed in the Methods section.
REFERENCES
Author notes
Co-First Authors Contact Information
Nabil Ali-Mohamad, [email protected]
Massimo F. Cau, [email protected]
Christian J. Kastrup, [email protected]
Presented as a poster at the 2022 Military Health System Research Symposium, Kissimmee, FL; MHSRS-22-07404.
