Liver injury is common following blunt abdominal trauma. It is often managed conservatively and does not require operative intervention. However, a subset of higher-grade injuries requires surgery (grades V and VI), with an associated mortality rate of 50 to 100%. 1,2,3 Exsanguination is the primary cause of death in these patients. Hemostasis is often hindered by coagulopathy, which results from hypothermia, dilution of clotting factors, thrombocytopenia and acidosis. Topical hemostatic agents are frequently used as adjuncts in the control of intraoperative bleeding. While a number of different materials have been used with limited success, each has potential drawbacks making a more effective topical hemostatic agent desirable.
A new polysaccharide product, fully acetylated poly-N-acetyl glucosamine (p-GlcNAc; Marine Polymer Technologies, Danvers, MA) has been recently developed. 4 P-GlcNAc is a polysaccharide polymer produced by a fermentation process and isolated from controlled, aseptic microalgal cultures grown on a defined culture medium. 4 Gel formulations of this substance have been evaluated as hemostatic agents in rabbit and canine models of variceal bleeding, and they have been found to be effective. 5,6 The membrane formulation of p-GlcNAc applied to one surface of a standard gauze bandage has been termed the Rapid Deployment Hemostat (RDH) bandage. This product is especially versatile because it can be cut or shaped into many different forms.
The mechanism of action of p-GlcNAc has not been fully defined, but is probably multifactorial. At least one mechanism is thought to be unrelated to the coagulation cascade, most likely by endothelial-dependent contraction of the microvasculature. For this reason, the RDH bandage should be efficacious even in the setting of coagulopathy. Patients with severe liver injuries rapidly develop hypothermia and coagulopathy, complicating hemostasis. In pilot studies, we took advantage of the versatility of this material and redesigned the RDH bandage for internal use. The result was termed the “Miami-modified Rapid Deployment Hemostat” (MRDH). We hypothesized that the addition of the MRDH to standard gauze packing for hemorrhage control would result in reduced blood loss compared with standard treatment with gauze packing alone. This study was performed to evaluate the effectiveness of this MRDH bandage for hemorrhage control following severe liver injury in the presence of both hypothermia and a dilutional coagulopathy.
MATERIALS AND METHODS
These experimental protocols were approved by the University of Miami School of Medicine, Animal Care and Use Committee and met National Institutes of Health guidelines for animal use.
Two protocols were performed. Each involved a traumatic liver injury in the setting of a hypothermic, hemodilutional coagulopathy. Both protocols were similar up until the time of the liver injury; details of each are provided below. Briefly, in series 1, the packing was removed after 1 hour and the observations were recorded. In series 2, the packing was removed at 1 hour, but then 1 unit of blood + an unlimited volume of lactated Ringers was administered to restore mean arterial pressure > 70 mm Hg and there was 2 more hours of observation.
Crossbred pigs (n = 14, 33.4 ± 1.8 kg) were fasted overnight then sedated with intramuscular ketamine (30 mg/kg) + xylazine (3.5 mg/kg). General anesthesia was then maintained by continuous intravenous infusion of ketamine (10 mg/hr) + xylazine (0.3 mg/hr) plus fentanyl (20 μg/kg/hr). Animals were orotracheally intubated and mechanically ventilated (Bear MA-2, FiO2=0.21 PEEP = 0) in the supine position. Bilateral external jugular vein and left internal carotid artery introducers were placed via cervical incisions. A pulmonary artery catheter was placed via the right jugular vein.
Cardiopulmonary variables included heart rate, mean arterial pressure (MAP), arterial hemoglobin oxygen saturation (Sao2), end-tidal CO2, central venous, pulmonary artery and wedge pressures, mixed venous hemoglobin oxygen saturation (SvO2), cardiac index, and core temperatures. Arterial blood samples were drawn for analysis of blood gases, lactate, electrolytes (Nova Stat Profile Ultra, Waltham, Ma) and complete blood counts (Abbott Cell-Dyn 1600, Abbott Park, IL). Animals were allowed a 15-minute equilibration period after catheter placement.
Over the following 20 minutes, three sets of baseline labs were drawn. Baseline inclusion parameters were MAP ≥ 70 mm Hg and a normal coagulation profile. Coagulation status was determined with a hemostasis analyzer (Thromboelastograph TEG; Hemoscope Corp, Niles, IL).
Creation of Coagulopathy
A hypothermic, hemodilutional coagulopathic state was induced by isovolemic, hypothermic exchange transfusion of 45% of the estimated blood volume with 6% hetastarch (Hextend) at 4°C. The exchange transfusion was accomplished over three, 10-minute intervals. Fifty percent of the hemorrhage proceeded during the first 10 minutes, followed by 25% of the total hemorrhage volume over each of the following 10-minute periods. Blood was removed from the arterial line, and simultaneously replaced with refrigerated Hextend through the jugular venous line. While the exchange transfusion was taking place, a laparotomy was performed and ice packs were placed in the paracolic gutters to facilitate the reduction in core temperature. The ice packs were removed when a desired core temperature of 33–34°C was achieved. Arterial blood samples were then drawn and a coagulopathic state documented by TEG.
A reproducible, severe liver injury was induced by the crushing and avulsing of the left, lateral hepatic lobe and its major vessels. The same individual produced the injury in each animal. Immediately following the injury, a Pringle maneuver was performed and pressure placed against the injury surface with a laparotomy pad. After 5 minutes, pressure was released and the presence of brisk bleeding was ensured. An envelope was then opened, revealing either a MRDH bandage or standard gauze bandage. While all participants were blinded up until this point, differences in texture and appearance of bandages were apparent once the envelopes were opened. Pringle was then reinstituted and pressure held against the injury surface with the bandage for 10 minutes. After 10 minutes, the Pringle was removed and the abdomen was packed with laparotomy pads, with care taken to ensure the study bandage remained against the injury surface. The abdomen closed with towel clips. No resuscitation fluids were given. Animals were then observed for 1 hour, with blood samples drawn every 10 minutes. After 1 hour, the abdomen was opened and all packs removed. A small amount (10 mL or less) of lactated Ringers (LR) was used to dampen the bandage lying against the injury surface, which was then removed, taking care not to disrupt any formed clot.
Crossbred pigs (n = 10, 40.6 ± 2.1 Kg) were fasted overnight. The protocol was followed as described above up until the point at which the abdomen was closed, then each animal was resuscitated with one unit of shed blood and as much LR as required to maintain a MAP ≥ 70 mm Hg. Arterial blood samples were drawn every ten minutes for the first hour following the liver injury.
After 1 hour, the abdomen was reopened and all laparotomy pads were removed. A small amount (10 mL or less) of LR was used to dampen the bandage lying against the liver surface, which was then removed, again taking care not to disrupt any formed clot. The abdomen was then closed and the animal followed for 2 more hours or until time of death. The animals continued to receive resuscitation fluids as needed to maintain a MAP ≥ 70 mm Hg. Arterial blood samples were drawn every 20 minutes for the remainder of the experiment. After 2 hours or at time of death, the abdomen was opened. The presence of bleeding at the injury surface was noted (Fig. 1) Pringle was performed, ensuring cessation of bleeding, and the abdomen was wiped clean of blood using pre-weighed laparotomy pads. Blood loss was calculated by weighing these laparotomy pads. Animals were then killed by infusion of a concentrated potassium solution.
Statistical Analysis; SAS Institute, Cary, NC
Using commercially-available software (Statview), continuous variables were compared by analysis of variance while discrete variables were compared with χ2. Differences were considered statistically significant with p ≤ 0.05.
Documentation of Coagulopathy
At baseline the coagulation index averaged 4.4 ± 1.2, corresponding to a normal coagulation state. Following isovolemic hemodilution and hypothermia, the coagulation index was -2.8 ± 1.48, corresponding to a coagulopathic state (p < 0.001). These changes are similar to those reported earlier in animals 7 and trauma patients. 8
The study population was comprised of 14 animals (n = 7 in each group). There were no statistically significant differences in physiologic parameters between the groups at baseline (control vs. MRDH: body weight, 31.4 ± 1.4 vs. 35.3 ± 3.3 kg; MAP, 87 ± 5 vs. 87 ± 3 mm Hg; lactate, 1.1 ± 0.3 vs. 1.2+0.2 mmol/L; hemoglobin, 9.1 ± 0.6 vs. 9.6 ± 0.8 g/dL; platelets, 391 ± 45 vs. 417 ± 80 × 103/μL). There were no significant differences between groups at the time of injury (control vs. MRDH: hemorrhage volume, 933 ± 42 vs. 1049 ± 98 mL; avulsed liver weight, 82 ± 8 vs. 96 ± 15 g; MAP, 84 ± 6 vs. 81 ± 3 mm Hg; lactate, 3.2 ± 1.1 vs. 3.6 ± 0.6 mmol/L; hemoglobin, 4.5 ± 0.4 vs. 4.9 ± 0.2 g/dL; platelets, 158 ± 28 vs. 187 ± 38 × 103/μL). Hemoglobin and hematocrit dropped by approximately 50% in both groups. Induction of the coagulopathic state did not cause any significant change in MAP, but did cause a rise in lactate that was similar in both groups. There were no significant differences between groups at 1 hour post-injury (control vs. MRDH: MAP, 53 ± 6 vs. 44 ± 5 mm Hg; lactate, 6.2 ± 2.0 vs. 8.0 ± 1.4 mmol/L, hemoglobin, 4.7 ± 1.3 vs. 5.2 ± 0.4 g/dL; platelets, 253 ± 96 vs. 166 ± 25 × 103/μL). At the time that packing was removed at 1 hour post injury, 6/7 in the control group had active bleeding vs. 1/7 with MRDH (p = 0.0291).
The study population was comprised of 10 animals (n = 5 in each group). There were no statistically significant differences in baseline physiologic parameters between the groups (Table 1). There were no significant differences in avulsed liver weight (90 ± 10 vs. 97 ± 14 g) or hemorrhage volume (1294 ± 103 vs. 1115 ± 47 mL) at the time of injury. There were no significant differences in physiologic parameters at the time of injury (Table 1). Induction of the coagulopathic state did not cause any significant change in MAP, but did cause a rise in lactate (similar in both groups). Hemoglobin and hematocrit dropped by approximately 50% in both groups. At 1 hour post-injury, there were significant differences in MAP and lactate between groups (both p < 0.05, Table 1).
No animals in the control group survived from the time of the liver injury to the end of the experiment, i.e., 0% survival rate at 3 hours. The mean survival time was 76 ± 20 minutes. In the control group, 3/5 died before unpacking the abdomen. With MRDH, 4/5 survived for the length of the experiment, i.e., 80% survival rate at three hours (p = 0.048). The mean survival time was 174 ± 11 minutes (p < 0.01).
Total blood loss in the control group was 3740 ± 1004 mL. Taking into account weight and survival time, this amounted to 1.19 ± 0.13 mL/kg/min. Total blood loss in the MRDH group amounted to 1514 ± 547 mL, or 0.26 ± 0.13 mL/kg/min (p = 0.001).
Total resuscitation fluid in the control group was 5420 ± 1860 mL. Taking into account weight and survival, this corresponds to 1.57 ± 0.28 mL/kg/min. Total fluid requirement in the MRDH group was 3500 ± 1400 mL, or 0.58 ± 0.27 mL/kg/min (p = 0.026) (Fig. 2).
The results from this study clearly demonstrate the hemostatic effectiveness of the MRDH bandage in a porcine model of severe liver injury with coagulopathy. Two series of experiments were conducted in a total of 24 animals. The addition of the MRDH bandage to gauze packing was compared with gauze packing alone in each series.
The first series judged the ability of the bandage to achieve complete hemostasis at 1 hour following a severe liver injury (with no resuscitation). Hemostasis was defined as the total absence of any arterial or venous bleeding. Animals did not receive fluid resuscitation and blood loss was not quantified. The data showed abdominal packing plus MRDH bandage more effectively achieved hemostasis than gauze packing alone.
The second series judged the ability of the bandage to achieve complete hemostasis at 3 hours following a severe liver injury (with resuscitation). We found that, when compared with gauze packing alone, the addition of RDH decreased blood loss, decreased fluid requirements, and improved survival.
Rapid and effective achievement of hemostasis is vital to the survival after hepatic injury. Hoyt et al., documented that 82% of all early civilian trauma deaths were due to uncontrolled hemorrhage, and 50% of these deaths were from severe liver injuries. 9 Achieving hemostasis is made more difficult due to the coagulopathy that commonly occurs in these patients. Certainly, more effective methods for obtaining rapid hemostasis would be a significant addition to the surgeon’s armamentarium.
The RDH bandage is comprised of a membrane formulation of a new polysaccharide product, fully acetylated poly-N-acetyl glucosamine (p-GlcNAc), placed on a gauze backing. It has recently been approved by the U.S. Food and Drug Administration for the treatment of extremity trauma. P-GlcNAc is a pure compound, free of proteinaceous debris and contaminants. 4 It is a polymer and can be formulated into membranes, fibers, sponges, or gels that can be directly applied to wound surfaces. The membrane formulation is easy to handle and coapts to the wound surface. It is fully biodegradable and can be left in place on a bleeding surface to provide continued hemostasis after wound closure; it is approved for internal use for up to 30 days. This product is easy to use, does not require special storage conditions, and does not require pre-mixing of reagents or the use of donated blood products.
The proposed mechanism of action of pGlcNAc is based on in vitro and in vivo studies and suggests that its functions are unrelated to the clotting cascade. When treating patients with coagulopathies, this gives the RDH bandage a potential advantage over other hemostatic agents that rely on activation of the clotting cascade. Immediately upon contact with blood, p-GlcNAc activates the formation of a red cell aggregates at the surface of the wound dressing that is in contact with blood. These aggregates may act as nitric oxide (NO) sinks, decreasing the local concentration of NO, causing a localized vasoconstriction. In addition, the decreased concentration of NO may induce the exposed endothelium of injured arteries to secrete endothelin-1, which causes further vasoconstriction. When in contact with blood, p-GlcNAc stimulates platelet aggregation and activation, leading to secretion of thromboxane. Thromboxane adds a further stimulus for local vasoconstriction. The vasoconstriction induced by the above mechanisms causes cessation of bleeding, leading to wound closure and subsequent clot formation. 10
P-GlcNAc has previously been tested in animal models of hemorrhage and compared with existing hemostatic products. Cole et al., used a swine splenic incision and splenic capsular stripping hemorrhage model to compare the hemostatic effectiveness of p-GlcNAc, oxidized cellulose, and absorbable collagen. 11 They concluded that p-GlcNAc was the most effective hemostatic agent. The authors note that this model produced small wounds that are not representative of injuries faced in actual operating room settings. In addition, the animals were not coagulopathic. Chan et al., used a similar model to compare p-GlcNAc to fibrin sealant;12 in a second series of experiments, the animals were heparinized and p-GlcNAc was compared with absorbable collagen. Again, p-GlcNAc was found to be the most effective hemostatic agent in this study.
We worked with the design team of Marine Polymer Technology to modify the existing RDH dressing in an effort to make a clinically effective preparation for internal use in the setting of liver injury. The earlier prototype RDH dressing was quite thin and was difficult to apply to the irregular surface of the injured viscera. We selected the final version of the bandage from a group of samples with different surfaces, thicknesses, and textures, each adherent to a 4” × 4” gauze pad. The final MRDH version was easy to apply and appeared to be effective at inducing hemostasis in major liver, spleen, and kidney injuries.
In the current study, a severe liver injury was induced in the setting of a hemodilutional, hypothermic coagulopathy. We compared the addition of the MRDH bandage to standard gauze packing as measures to control hemorrhage. No other measures such as suture ligation, electrocautery, or other topical hemostatic agents were used. In series one, 6/7 in the control group had active bleeding present at 1 hour post-injury, while only 1/7 had any evidence of bleeding in the MRDH bandage group (p < 0.05). The addition of the MRDH bandage was more effective than gauze packing alone in this model of uncontrolled hemorrhage. However, it is important to note that animals were not resuscitated in this series, and both groups were hypotensive at the experimental endpoint. While it is significant that the group receiving routine gauze packing continued to bleed despite this hypotension, underscoring the severity of the injury and coagulopathy, resuscitation and improvement of the MAP may have led to rebleeding in the MRDH treatment group.
The question of rebleeding was addressed in series two. The addition of the MRDH bandage led to decreased total blood loss, decreased fluid requirements, and improved survival. We feel this is due to the rapid achievement of hemostasis, which is sustained despite resuscitation to pre-injury MAP. The continued bleeding in the group receiving standard gauze packing led to further deterioration of MAP and rise in lactate and, ultimately, a 100% mortality at three hours.
This animal study compares the use of a new hemostatic bandage plus gauze packing to gauze packing alone in a reproducible model of severe liver injury combined with a hemodilutional, hypothermic coagulopathy. 7 This injury model closely resembles what is actually seen in the operating room. However, the results must be viewed within the context of the limitations of the study. The study was performed in anesthetized, mechanically-ventilated swine, and their compensatory physiologic responses may have been altered. There could be species-related differences with human beings. In addition, the sample size was relatively small. The MRDH bandage was tested only against gauze packing alone. There was no electrocautery, or surgical ligation and no comparison to other topical hemostatic agents. Most important, however, was the inability to blind the investigators to the type of bandage being applied to the injury surface. This may have introduced bias when holding pressure against the injury surface and when packing the abdomen. The fact that three of five animals in the control group of series two died during the first hour after injury, despite abdominal packing, was a surprise to us. However, we feel this is due to the severity of the injury and coagulopathy, rather than a bias in favor of the MRDH bandage, and lends further support to its efficacy for achieving hemostasis.
In summary, the addition of the MRDH bandage to gauze packing more rapidly achieved hemostasis (Fig. 3), leading to decreased fluid requirements and increased survival as compared with standard gauze packing alone in this porcine model of severe liver injury with coagulopathy.
We are grateful to Paul Segall, PhD, of BioTime, Inc. (Berkeley, CA) for providing the Hextend; Terry Shirey, PhD, of Nova Biomedical (Waltham, MA) for providing the Stat Ultra Blood Gas and Electrolyte Analyzer; Eli Cohen, PhD, of Hemoscope (Niles, IL) for providing the Thromboelastograph Hemostasis Analyzer.