Abstract
Introduction
Hemorrhage control in austere environments is challenging, particularly for wounds that are not amenable to tourniquets. Hemostatic gauzes are crucial in such settings, but their efficacy may be compromised by suboptimal storage conditions, including extreme temperatures, where discoloration has been observed. This study evaluated the impact of extreme temperature exposure on the efficacy of hemostatic gauze using thromboelastography.
Methods
Blood from 30 healthy adults was diluted by 30% with hetastarch to mimic trauma-induced coagulopathy. Kerlix and QuikClot Combat Gauze stored for 3 weeks in cold (−10°C), hot (70°C), and room-temperature (22°C) environments were compared in the thromboelastography parameters of R (time to initiation of clot formation), K (clot amplification), α angle (clot formation rate), and MA (maximum amplitude of clot).
Results
Compared with whole blood, diluted blood had weaker clots with slower clot-formation kinetics (MA=58 vs 43 mm, P<0.0001; K=2.6 vs 4.0 min, P<0.0001; α angle=55 vs 47 degrees, P<0.0003) but faster clot initiation times (R=8.7 vs 7.1 min, P<0.0001). Addition of either gauze shortened clot initiation times (Kerlix: 7.1 vs 5.0 min, P<0.0001; QuikClot Combat Gauze: 7.1 vs 2.7 min, P<0.0001), with QuikClot Combat Gauze significantly shortening R compared with Kerlix. Reductions in R values were consistent across temperature extremes (P<0.05). The other parameters were consistently unaffected (P>0.05).
Conclusions
This in vitro laboratory study demonstrated that hemostatic gauze retained its ability to initiate clotting in vitro even after prolonged exposure to temperature extremes.
Introduction
Hemorrhage is the leading cause of preventable death in trauma, 1 and on arrival at an emergency facility, 28 to 38% of patients with severe traumatic injuries present with coagulopathy.2,3 Wound packing with hemostatic gauze is often the default initial treatment for injuries not amenable to tourniquets. 1 In both military and wilderness environments, evacuation of critically injured casualties can be delayed significantly. Therefore, first responders need to have a hemorrhage-control product that has a long shelf life, is lightweight and durable, and is able to endure wide variation in temperature storage and harsh environmental conditions. Although hemostatic gauzes seem to fit these criteria, they have been observed to discolor after prolonged storage in extreme heat. The significance of this discoloration and effect on functionality are unclear.
Thromboelastography (TEG) quantitatively measures the properties of clotting in whole blood, from the beginning of coagulation to fibrinolysis, and has been used in trauma resuscitation to assess for coagulopathy and guide administration of component blood products. 4 Two studies have established an in vitro model of the effect of hemostatic gauze on coagulation using TEG and human blood—one demonstrating that QuikClot Combat Gauze (Z-Medica, Wallingford, CT) significantly reduced time to clot formation and increased clot strength and overall clotting parameters and a second showing that these results held in a maritime environment.5,6
Future civilian and military operations are likely to occur increasingly in varying environmental settings, especially considering the accelerating rate of climate change. Therefore, there is a need to assess the functionality of medical products under extreme thermal conditions to ensure the fidelity of hemostatic mechanisms. The objective of this study was to evaluate the hemostatic efficacy of plain gauze and QuikClot Combat Gauze following prolonged exposure to extreme temperature environments in an in vitro dilutional coagulopathy model.
Whole blood vs diluted blood (mean±SD). TEG parameters compared between naive whole blood of each subject and blood diluted with 30% hetastarch. Whole blood performs better in all parameters except, paradoxically, R.
Diluted blood vs blood exposed to room-temperature gauzes (mean±SD). TEG parameters compared between diluted blood and blood exposed to gauzes stored at room temperature—only R is significantly different.
Blood exposed to gauzes at 3 temperatures (mean±SD). R remains statistically unchanged despite variation in temperature storage when comparing the same gauze, but R does vary significantly between gauzes.
Comprehensive results (mean).
Note: Whole blood performs better in all parameters except R. Diluted blood exposed to room-temperature gauzes shows a shorter R, whereas other parameters are not significantly different. Blood exposed to gauzes stored at each extreme temperature shows no significant difference in R when compared with the same gauze stored at room temperature. The other parameters have not been included because there was no significant difference appreciated in the control group (diluted blood vs blood exposed to room-temperature gauze).
Methods
This study was conducted under the provisions of Department of Defense (DOD) Instruction 3216.2, SECNAVINST 3900.39D, 32 CFR Part 219, NAVMEDCENPTSVAINST 6710.10E, and the Belmont Report and approved by the institutional review board at Navy Medicine Readiness and Training Command Portsmouth.
Procedures
Blood samples were obtained from 30 healthy active-duty volunteers between 18 and 45 years of age. Participants were screened by the study investigators for the presence of known coagulopathies, recent major trauma or surgery, personal or family history of thromboembolic disease, pregnancy, and exposure to any medications that may affect normal clotting processes. After written informed consent was obtained, blood specimens were collected via venipuncture into sodium citrate vacutainers according to standard clinical operating procedures. Samples from 3 subjects failed to coagulate on testing with a TEG analyzer and were excluded from analysis, leaving a total of 27 blood samples. Coagulation failure was likely due to a combination of donor-specific variability, the sensitivity of TEG to detect very weak or very delayed clot formation, and interactions with test materials (specifically a dilution agent known to variably impair clotting factor and platelet activity).
The naive citrated blood sample of each subject was analyzed first as a control for each subject. Any abnormal coagulation pattern identified by a blind investigator resulted in that subject being excluded from the data. To prevent any premature coagulation, blood samples were gently agitated while awaiting processing. All blood was processed within 4 h of collection because previous research has demonstrated that reliable TEG results can be obtained within 4 h using recalcified citrated blood. 7 The remaining citrated blood of each subject was then diluted with 30% hetastarch to induce a dilutional coagulopathy intended to mimic trauma-induced coagulopathy and provide a stringent test for the gauze, even at moderate dilution.
Two Different Gauzes
Kerlix (Covidien, Medline Industries, Inc, Northfield, IL), a 6-ply rolled-cotton gauze that absorbs blood and provides a scaffold for platelet aggregation, and QuikClot Combat Gauze (Z-Medica, Wallingford, CT), a z-folded nonwoven rayon-polyester gauze impregnated with kaolin (hydrated aluminum silicate) were evaluated. Each gauze, in its original packaging, was exposed to 1 of the following conditions for a minimum of 3 wk prior to use: room temperature at 22°C, a commercial freezer at −10°C, and an incubator at 70°C.
A 10-mg piece of each gauze was added to the diluted blood samples and gently inverted 8 times.5,6 Then 340 µL of blood was placed in TEG cups and allowed to clot spontaneously. Then 20 µL of calcium chloride (0.2 mol/mL) was added to the samples to overcome the anticoagulant effect of citrate.
A TEG 5000 Thromboelastograph Hemostasis Analyzer System (Haemonetics Corp, Braintree, MA) was calibrated in accordance with the manufacturer's protocol. TEG analyses were performed at 37°C to simulate the goal temperature for traumatically injured patients during resuscitation. For each TEG cycle, all gauzes were tested in the same chamber for standardization. The TEG parameters of time to initiation of clot formation (R, in minutes), clot amplification time (K, in minutes), the rate of clot formation (α, angle in degrees), and maximum amplitude (MA, in millimeters) were determined for each sample by a blinded investigator and recorded in an Excel spreadsheet (Microsoft Corp, Redmond, WA).
Statistics
Blood samples from each subject were analyzed under 8 conditions: whole blood, untreated diluted blood, and diluted blood treated with each gauze product at each of the 3 test storage temperatures. Data were analyzed in GraphPad Prism version 9.0.0 for Windows (GraphPad Software, San Diego, CA). First, to assess model validity, each subject's blood was compared before and after dilution with Hextend (Pfizer, Inc, New York, NY) using a paired t test. Second, analysis of variance was used to assess the effect of each gauze on the continuous TEG parameters (ie, R, K, MA, and α), comparing each gauze treatment group with the diluted-blood control using Dunnet's multiple-comparison test. Finally, a mixed-effects model fit using restricted maximum likelihood followed by Bonferroni's multiple-comparison test was used to assess statistical difference in TEG parameter R of gauze-treated diluted blood within 2 families of gauzes (Kerlix and QuikClot Combat Gauze) stored under each of 3 temperatures (22, −10, and 70°C). Data were matched within subjects across gauze type and storage conditions. Comparisons with gauze stored at room temperature were made within each family of gauze, with reported P values corrected for multiple comparisons.
Results
Model Validation: Mimicking Dilutional Coagulopathy
As expected, whole blood showed a significantly higher rate of clot formation (mean α=55 vs 47 degrees, P<0.0003), greater maximum amplitude (mean MA=58 vs 43 mm, P<0.0001), and lower clot amplification time (mean K=2.6 vs 4.0 min, P < .0001) when compared with diluted blood. However, as with our previous study, 6 this model showed a longer time to initiation of clot formation in whole blood when compared with diluted blood (mean R=8.7 vs 7.1 min, P<0.0001) (Figure 1).
Establishing Control Group (Diluted Blood Vs Room-Temperature Gauzes)
Diluted blood exposed to both gauzes showed a significantly shorter time to initiation of clot formation when compared with untreated diluted blood (Kerlix: mean R=7.1 vs 5.0 min, P<0.0001; QuikClot Combat Gauze: mean R=7.1 vs 2.7 min, P<0.0001). QuikClot Combat Gauze further reduced R when compared with plain Kerlix (mean R=2.7 vs 5.0 min, P<0.0001).
There was no significant difference between diluted blood and diluted blood exposed to either gauze with regard to the other measured parameters (Kerlix: mean α=47 vs 44 degrees, P=0.73; QuikClot Combat Gauze: mean, α=47 vs 48 degrees, P>0.99; Kerlix: mean MA=43 vs 45 mm, P=0.99; QuikClot Combat Gauze: mean MA=43 vs 46 mm, P=0.73; and Kerlix: mean K=4.0 vs 3.8 min, P=.99; QuikClot Combat Gauze: mean K= 4.0 vs 4.4 min, P=0.83) (Figure 2).
Gauze at Extreme Temperatures
Although QuikClot Combat Gauze was observed to take on a yellowish orange color after hot storage, there was no significant difference with regard to R between agents stored at room vs hot temperature (Kerlex: mean R=5.0 vs 4.6 min, P=0.25; QuikClot Combat Gauze: mean R=2.7 vs 2.5 min, P=0.58) or between room versus cold temperature (Kerlix: mean R=5.0 vs 4.9 min, P>1.0; QuikClot Combat Gauze: mean R=2.7 vs 2.6 min, P=0.73) (Figure 3 and Table 1).
Discussion
The results of this study indicate that hemostatic gauze remains functional despite exposure to temperature extremes and despite visual changes, supporting its potentially lifesaving use in hemorrhage control in austere environments and suboptimal storage conditions. While each gauze studied decreased the time to initiation of clot formation, this reduction was noted to be greatest for QuikClot Combat Gauze, which has previously demonstrated superior efficacy. 8 In general, hemostatic gauze's retained efficacy when frozen, heated, or immersed in saltwater, 6 as well as its ease of application and transportation, makes it suitable for nearly any austere or wilderness setting.
Although colloid and large-volume crystalloid solutions are no longer recommended in trauma resuscitation, coagulopathy is still a prominent and problematic complication of traumatic hemorrhage,2,3 incited by factors such as clotting factor consumption, metabolic derangements, cytokine production, hypoxia, hypothermia, blood components given in an unbalanced ratio, high levels of citrate found in stored blood products, and inappropriately given non-blood-based resuscitation products. Our model successfully mimicked trauma-induced coagulopathy, with diluted blood demonstrating a decreased rate of clot formation, decreased maximum amplitude of clots, and increased clot amplification time. Interestingly, R was actually reduced when compared with whole blood, which also was demonstrated in our previous study and appears to be a consistent effect across in vitro studies. 6 Nevertheless, both gauzes effectively further reduced R, demonstrating their persistent utility in potentially coagulopathic patients.
The established decrease in R values with the addition of gauze, and especially with QuikClot Combat Gauze, remained consistent across temperature extremes. This was not entirely unexpected because the melting point of the active ingredient in QuikClot Combat Gauze, kaolin, is >740°C, making disruption due to thermal stress unlikely. However, other mechanisms including degradation of the rayon-polyester base, degradation of packaging, exposure to cold, and compromise of sterility all theoretically could have impaired the function of the gauzes. Visual changes were observed in the QuikClot Combat Gauze exposed to heat stress, with the gauze taking on a yellowish orange color. This mirrored anecdotal reports of the same visual changes after prolonged high-temperature storage during military deployments that originally inspired this study. However, the color transformation did not alter the hemostatic function of the gauze, implying that visual inspection does not correlate with efficacy.
Limitations
One limitation of our study is the relatively short time of extreme temperature exposure; proposed mechanisms of impaired function would only compound with longer exposures and may be grounds for future studies. Our results also must be viewed in light of the limitations of the in vitro dilution model. Although fresh whole blood was used, the in vitro environment by nature does not replicate certain complex interactions found in vivo, such as the role of epithelial cells, inflammatory cytokines, cortisol, vasospasm, and so on that occur during tissue injury, hemorrhage, and subsequent coagulation. Furthermore, although our model aimed to mimic coagulopathy, we did so by administering a colloid solution that is no longer recommended in trauma resuscitation. Although the other factors noted previously still do contribute to coagulopathy, they do so in variable ways that may not be reproduced effectively. The paradoxically decreased R times observed here and across similar in vitro studies emphasize these limitations. Furthermore, most of our other diluted TEG values, although significantly different from whole-blood values, also were still within the TEG reference range. This suggests that even though the diluted blood was more coagulopathic than baseline, the model may not have gone far enough to mimic true coagulopathy. Future studies in vivo, using different or increased diluents or just using baseline whole blood, may better model the desired effects.
Conclusions
This in vitro laboratory study demonstrated that hemostatic gauzes retained their ability to initiate clotting in vitro even after prolonged exposure to temperature extremes. Gauze, especially QuikClot Combat Gauze, shortened time to initiation of clot formation in a coagulopathic in vitro model even when exposed to hot and cold stressors.9,10
Footnotes
Acknowledgments
The authors thank Emily Friedrich for assistance with statistical analysis and editing, Megan Bohan and Michael Boboc for assistance with data collection and analysis, and Eric Sulava for assistance with critical revision of the manuscript.