Comparative observations of the hemostatic effects of bio-zeolite gauze and QuickClot Combat Gauze on massive hemorrhage with coagulopathy

doi:10.21203/rs.3.rs-2434504/v1

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Comparative observations of the hemostatic effects of bio-zeolite gauze and QuickClot Combat Gauze on massive hemorrhage with coagulopathy

https://doi.org/10.21203/rs.3.rs-2434504/v1

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BACKGROUND

The presence of coagulopathy after bleeding negatively affects the hemostatic effects of gauze. In this study, we compared the hemostatic effects of bio-zeolite gauze (BZG) and QuickClot Combat Gauze (QCG) on massive hemorrhage complicated by coagulopathy.

Methods

In 30 rabbits, diluted coagulopathy was prepared by extraction of blood at a dose of 15 mL/kg body weight following infusion with saline three times the volume of blood loss. Then, simulated blast-induced pelvic injury was initiated to produce massive hemorrhage using a custom-made machine, as previously reported. The rabbits were then randomly allocated to three groups, namely A, B, and C, in which BZG, QCG, and ordinary gauze were used to prevent bleeding, respectively. The primary outcomes, including the immediate hemostasis rate, blood loss, and survival rate, were recorded at designated time points. Additionally, blood samples were taken for laboratory examinations, including conventional coagulation, thromboelastography, full blood count, blood biochemistry, and enzyme-linked immunosorbent assay.

Results

Animal models of diluted coagulopathy and major bleeding were successfully developed with decreased concentrations of coagulation factors XII and X. The overall effects of BZG were better than those of QCG and ordinary gauze, with a higher immediate hemostatic rate, less blood loss at 10 min and 30 min after gauze application, and higher red blood cell and platelet counts, higher maximum amplitude, lower R values, lower levels of creatinine, and lower levels of alanine aminotransferase at 30 min after gauze application. Furthermore, a higher survival rate was found in group A rabbits than in group B and group C rabbits at 90 min after gauze application.

Conclusions

BZG demonstrated better hemostatic performance than QCG and normal gauze for massive hemorrhage complicated by coagulopathy. BZG is a suitable hemostatic gauze for control of massive hemorrhage complicated by coagulopathy.

hemostatic gauze

bio-zeolite gauze

QuickClot Combat Gauze

major bleeding

coagulopathy

Massive hemorrhage is the leading cause of death in trauma, and effective on-site hemostasis measures can prevent many deaths due to massive hemorrhage [1, 2]. Hemostatic gauze is one of the effective hemostatic measures, particularly for bleeding in junctional zones and torsos [3, 4]. Various types of hemostatic gauzes have been developed, among which QuikClot Combat Gauze (QCG) is the most commonly used and tested in both military and civilian settings. Indeed, QCG is recommended by the Committee on Tactical Combat Casualty Care to control bleeding in combat first aid, and has shown promising hemostatic effects [4–8].

Trauma induced coagulopathy (TIC) is a common and severe complication after major bleeding, which can occur rapidly after major bleeding, particularly following infusion of a large amount of crystalloid fluid. The presence of coagulopathy after bleeding will negatively affect the hemostatic effect of gauze [9, 10]. Multiple factors might contribute to the reduce hemostatic effects of gauze in the presence of coagulopathy, among which a decreased level of coagulation factors is one of the most important reasons. Animal experiments have shown that QCG can effectively stop bleeding complicated by coagulopathy, although most experiments use a negative control without a positive control [11–14]. More recently, a new type of hemostatic gauze, bio-zeolite gauze (BZG) has been introduced, which exhibits superior hemostatic performance to QCG [15–17]. Additionally, in vitro experiments have demonstrated that BZG has excellent procoagulant performance even in plasma that is deficient in coagulation factor X [15–17]. Thus, we hypothesized that BZG may have good hemostatic performance for massive hemorrhage complicated by TIC. In the current study, we comparatively observed the hemostatic effects of QCG, BZG, and ordinary gauze on major bleeding in rabbits complicated by TIC, with the aim to establish a suitable hemostatic gauze with promising hemostatic performance for massive hemorrhage complicated by TIC.

All procedures involving animals were approved by the Ethics Committee of the Army Medical University of China PLA and were performed in accordance with relevant regulations of the Ethics Committee of the Army Medical University of China PLA P. R. China.

Animal groups, animal model, instrumentation and monitoring, sample collection, and animal welfare

Thirty rabbits of both sexes (weight: 2.45 ± 0.31 kg, age: 8.8 ± 0.37 months) were randomly and evenly divided into three groups (n = 10 per group) and housed at a temperature of 19–22°C. All rabbits were subjected to diluted coagulopathy and then blast- and fragment-induced inguinal major bleeding as described below, and BZG (Hangzhou Zeolite Innovation Medical Technology Co., Ltd., Hang Zhou city, China), QCG (Z-Medica, Wallingford, CT), and ordinary gauze (Chongqing Medical Inc., Chongqing City, China) were used to control the massive hemorrhage in rabbits of groups A, B, and C, respectively.

Rabbits were anesthetized with 1% pentobarbital sodium via the ear rim vein at a dose of 5 mL per kg body weight. Then the right common carotid artery was cannulated for continuous monitoring of heart rate (HR) and mean arterial pressure (MAP), and the left internal jugular vein was cannulated for blood removal, saline infusion, and collection of blood samples for lab examinations [9,18].

After the instrumentation, diluted coagulopathy was induced by the removal of blood at a dose of 15 mL/body weight (kg) and at a fixed speed of 100 mL/min, followed by infusion of saline at a volume of three times the blood loss [11,13,14]. The overall infusion time was controlled at 10 min. Blood samples were immediately after anesthesia and at 10 min after saline infusion for enzyme-linked immunosorbent assay (ELISA) to determine the levels of coagulation factors X and XII, and conventional coagulation tests to determine whether coagulopathy was successfully introduced. The criterion for coagulopathy is that the international ratio (INR) is more than 1.2 times greater than the basic value [9–11].

After the induction of TIC, simulated blast-induced pelvic injury was produced using a custom-made machine as previously reported [18]. Briefly, rabbits were fixed in a supine position on a plate with the inguinal region exposed to the tube, and a small irregular copper chip was placed on the inner side of the aluminum membrane, before applying a pipe outlet pressure of 5 mPa. After pressing the “Fire” button, simulated blast- and fragment were produced and then applied onto the inguinal region of the rabbits. Free bleeding was allowed for 30 s, followed by wound packing with different types of gauze in different groups as described above. Manual pressure was then applied for 3 min, followed by gentle release. The blood loss after free bleeding for 30 s, and at 10 min and 30 min after wound packing, was collected by vacuum suction and by absorbent pads for weighing to calculate the total blood loss throughout the experiment [3,18,19]. Hemostasis is defined as a lack of visible blood pooling outside the injury site [3,18,19]. Immediate hemostasis was defined as hemostasis occurring within 3 min after the end of compression [3,18,19].

During the experimental process, all animals were kept warm with a towel covering and were maintained under anesthesia before sacrifice. Following completion of the experimental procedures, the animals were humanely sacrificed by overdose injection of potassium chloride intravenously.

Recording, lab examinations, and ELISA

HR and MAP were monitored continuously and were recorded immediately after anesthesia and at 10 min and 30 min after gauze packing. Immediately after anesthesia and at 30 min after gauze packing, blood samples were taken for ELISA, thromboelastography (TEG), full blood count (FBC), and blood biochemistry. Additionally, the survival rates at 10 min, 30 min, and 90 min after gauze application were recorded.

Laboratory examinations were conducted as previously reported [18,20]. Briefly, FBC was examined using an automatic full blood analyzer in accordance with the manufacturer’s instructions (BC-5180 CRP, Mindray Inc., Shengzhen, China), and red blood cell (RBC) and platelet (Plt) counts were recorded. TEG was performed using a TEG analyzer (CFMS LEPU-8800, Le Pu Medical Science Technology Inc., Beijing, China), and the R value (R) and maximum amplitude (MA) were recorded. Blood biochemistry was analyzed using an automatic biochemistry analyzer (HB-P01, Guang Bao Medical Instrument Inc., Changzhou, China). The levels of creatine and alanine aminotransferase (ALT) were recorded. ELISA kits for coagulation factors X and XII were purchased from Signalway Antibody LLC (Baltimore, Maryland, USA), and were conducted according to the manufacturer’s manual and as previously reported [18,20].

Statistical analysis

All data are expressed as the mean ± standard deviation. Chi-squared tests were used to determine significance among groups in tests with binary outcomes including survival rates and immediate hemostatic rates. The Kolmogorov–Smirnov test was used to examine the normality of the data distribution of other parameters including the MAP, HR, values of lab examinations, and volume of blood loss. Then, multi-group comparisons were conducted by one-way ANOVA. Statistical analysis was conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The confidence interval was set at 95% (95% CI). A P-value ≤ 0.05 was considered significant.

Successful development of animal models of TIC

The values of all examined parameters in the present study followed a normal distribution as examined by Kolmogorov–Smirnov test. One-way ANOVA test revealed no statistical differences both in the values of INR immediately after anesthesia and 10 min after saline infusion among the rabbits in the three groups. Furthermore, the values of INR at 10 min after saline infusion were 1.40 ± 0.07, 1.44 ± 0.08, and 1.46 ± 0.07 in groups A, B, and C, respectively, which were 1.2 times more than those immediately after anesthesia in each group (i.e., 1.04 ± 0.09, 1.06 ± 0.09, 1.03 ± 0.11 in groups A, B, and C, respectively, Figure 1A).

ELISA showed that the concentrations of coagulation factor XII 10 min after saline infusion were 219.2.8 ± 19.7 ng/mL, 215.9 ± 17.5 ng/mL, and 208.6 ± 15.8 ng/mL in groups A, B, and C, respectively, which were significantly lower than those immediately after anesthesia in the same group (Figure 1B). The change patterns of coagulation factor X were similar to those of coagulation factor XII (Figure 1C). These data indicated that animal models of TIC were successfully developed and the induced TIC was accompanied by a decreased concentration of coagulation factors XII and X.

BZG demonstrated better hemostatic performance than QCG for massive hemorrhage complicated by TIC

The amount of blood loss after free bleeding in groups A, B, and C showed no significant difference, however, the amount of blood loss in group A rabbits at 10 min and 30 min after wound packing was significantly lower than that in groups B and C rabbits, and the amount of blood loss in group B rabbits at 10 min and 30 min after wound packing was significantly lower than that in group C rabbits (Figure 2). After using gauze for wound packing and compression, the rate of immediate hemostasis in the animals of group A was 90%, which was significantly higher than that in group B (60%) and in group C (40%).

The survival rates were 100%, 100%, and 80% at 10 min after wound packing in groups A, B, and C rabbits, respectively, which showed no significant difference. At 30 min after wound packing, the survival rate of group A rabbits (90%) showed no significant difference compared to that of group B rabbits (80%), but was significantly higher than that of group C rabbits (60%). At 90 min after wound packing, the survival rate of group A rabbits (70%) was significantly higher than those of group B rabbits (40%) and group C rabbits (10%).

These data indicated that BZG demonstrated better hemostatic performance than QCG and ordinary gauze for massive hemorrhage complicated by TIC, and in turn improved the survival rate at 90 min after wound packing.

BZG improved parameters of hemodynamic monitoring, FBC, biochemistry, and TEG compared to QCG and ordinary gauze

There was no significant difference among groups A, B, and C in MAP and HR after anesthesia, after the introduction of TIC, and 30 s after free bleeding (Figure 3). However, the MAP of group A was significantly higher than that in groups B and C at 10 min and 30 min after wound packing, while the HR of group A was significantly lower than that of groups B and C at 10 min and 30 min after wound packing (Figure 3).

There was no significant difference in the RBC and platelet counts among all three groups immediately after anesthesia (Figure 4). At 30 min after wound packing, the RBC counts in group A rabbits were significantly higher than those in group B and group C rabbits, and the RBC counts in group B rabbits were significantly higher than those in group C rabbits (Figure 4A). The same tendency was found for the platelet counts (Figure 4B).

There was no significant difference in R and MA values among groups A, B, and C rabbits immediately after anesthesia (Figure 5). However, 30 min after wound packing, the R values in group A rabbits were significantly shorter than those in group B rabbits and group C rabbits, and the R values in group B rabbits were significantly shorter than those in group C rabbits (Figure 5A). In contrast, the MA values in group A rabbits were significantly higher than those in group B rabbits and group C rabbits, and the R values in group B rabbits were significantly higher than those in group C rabbits (Figure 5B).

There was no significant difference in the concentrations of creatinine and ALT levels among groups A, B, and C rabbits immediately after anesthesia (Figure 6). At 30 min after wound packing, the concentrations of creatinine and ALT in group A rabbits were significantly lower than those in group B and group C rabbits (Figure 6). Additionally, the concentrations of creatinine and ALT in group B rabbits were significantly lower than those in group C rabbits (Figure 6).

In this study, we found that BZG demonstrated better hemostatic performance than QCG and ordinary gauze for massive hemorrhage complicated by TIC, and in turn, improved the parameters of hemodynamic monitoring and laboratory examinations and eventually the survival rate 90 min after wound packing.

After tissue injuries and massive hemorrhage, three types of physiological activities are initiated to stop bleeding at the injured site. First, small blood vessels at the injured sites contract immediately after injury, in an attempt to close blood vessels. Second, damage to the vascular intima leads to exposure of the subintimal tissue, which in turn activates and attracts platelets to migrate and adhere onto the injured intima to form a platelet cluster. Third, damage to the vascular intima simultaneously activates coagulation [16]. The basic process of coagulation is generally divided into three phases: the formation of the prothrombinase complex, thrombin formation, and fibrin polymer formation [16]. According to the different initiating pathways and participating factors of prothrombin activator formation, coagulation can be divided into intrinsic coagulation and extrinsic coagulation pathways. Coagulation factor XII is necessary for the intrinsic coagulation pathway, and tissue factor (also called coagulation factor III) is necessary for the extrinsic coagulation pathway. Tissue factor is released after tissue damage to activate the extrinsic coagulation pathway. After the activation of intrinsic and extrinsic coagulation pathways, the prothrombinase complex, which comprises the serine protein, factor X, and the protein cofactor, factor V, is formed by activating different types of enzymes, and then coagulation factors II and I are sequentially activated to form fibrin polymers. As coagulation factor III is release during injury, sufficient amounts of coagulation factors X and XII are important for coagulation activation. Our results showed that after the induction of TIC, the concentrations of coagulation factors X and XII decreased significantly compared to their baseline values. This is one of the reasons for the presence of coagulopathy after bleeding negatively affecting the hemostatic effect of gauze. BZG is a zeolite-based bio-inorganic hybrid hemostatic gauze with a surface Ca ion exchange rate of 87%, which can promote the conversion of prothrombin from normal plasma and factor X deficient plasma to thrombin [16, 17]. In contrast, kaolin is the main hemostatic component of QCG and exerts its hemostatic effect through activating the endogenous coagulation pathway [12, 21]. When TIC occurs, the level of coagulation factor XII and the activation efficiency of the endogenous activation pathway decrease, resulting in a decrease in the hemostatic effect of QCG; while the decreased level of coagulation factor X will further decrease the hemostatic effect of QCG. This explains our finding that BZG demonstrated better hemostatic performance than QCG and ordinary gauze for massive hemorrhage complicated by TIC with decreased coagulation factors X and XII. The clinical relevance of these findings is that BZG is a suitable hemostatic gauze to control massive hemorrhage complicated by TIC.

The current study has several limitations. First, TIC was induced by a previously reported method, i.e., by blood removal plus infusion of saline, which might be different from real clinical scenarios. In clinical practice, multiple factors (e.g., combined trauma and hemorrhagic) other than blood loss induce TIC [9, 22, 23]. Moreover, the pathophysiology of TIC caused by different factors might be different, which may have different influences on the hemostatic effects of gauzes. Future work should focus on establishing the hemostatic effects of gauzes on massive hemorrhage companied by TIC induced by different factors. Second, the animal model used in the current study is less common than that used by other researchers, i.e., the femoral artery and femoral vein transection massive hemorrhage model [17, 19, 24]. Instead, we used a simulated blast-induced inguinal major bleeding [18]. This might lead to weak comparability between our data and other data reported in the literature; however, we believe that our model is closer to the injuries observed in combat.

In summary, we found that BZG demonstrated better hemostatic performance than QCG and ordinary gauze for massive hemorrhage complicated by TIC. Therefore, BZG is a suitable hemostatic gauze for controlling massive hemorrhage

ALT

alanine aminotransferase

BZG

bio-zeolite gauze

ELISA

enzyme-linked immunosorbent assay

FBC

full blood count

heart rate

INR

international ratio

maximum amplitude

MAP

mean arterial pressure

Plt

platelet

QCG

QuikClot Combat Gauze

RBC

red blood cell

TEG

thromboelastography

TIC

trauma induced coagulopathy

Ethics approval

All of the procedures involving animals were approved by the Ethics Committee of the Army Medical University of China PLA (AMUWEC2019471) and were performed in accordance with the relevant regulations of the Ethics Committee of the Army Medical University of China PLA, P. R. China.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Key logistics scientific project of the Special biosafety project (19WAQ28), the “Thirteenth Five Year Plan” of Medical Research of PLA (ALJ19J001), Key Clinical Innovation Project of Army Medical University and Xin Qiao Hospital (CX2019JS107 /2018JSLC0023). The sponsors played no role in study design, collection, analysis and interpretation of data, the writing of the manuscript, and in the decision to submit the manuscript for publication.

Authors' contributions

HY: participation of study design, development of animal model and data acquisition, YJ: TEG, NX: data analysis, XZ: full blood count, RJ: blood biochemistry, ZY: conventional coagulation test, WD: ELISA, TL: data interpretation, ZZ: conceptualization, original draft, and funding acquisition. All authors read and approved the final manuscript.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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No competing interests reported.

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Comparative observations of the hemostatic effects of bio-zeolite gauze and QuickClot Combat Gauze on massive hemorrhage with coagulopathy

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Abstract

BACKGROUND

Methods

Results

Conclusions

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Background

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

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