Journal of Investigative Surgery, 27, 360–365, 2014 C 2014 Informa Healthcare USA, Inc. Copyright ISSN: 0894-1939 print / 1521-0553 online DOI: 10.3109/08941939.2014.941444
NEW METHODOLOGY
Hemostatic Efficacy of EVARRESTTM, Fibrin Sealant Patch R vs. TachoSil in a Heparinized Swine Spleen Incision Model John P. Matonick, PhD1 Jeffrey Hammond, MD, MPH2
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1
Preclinical Research, Ethicon, Inc., Somerville, New Jersey, USA, 2 Medical Affairs, Ethicon, Inc., Somerville, New Jersey, USA
ABSTRACT Background: First-generation single-component hemostats such as oxidized regenerated cellulose (ORC), fibrin, collagen, and gelatin have evolved into second and third generations of combination hemostats. Objective: This study compares two FDA approved products, EVARRESTTM , Fibrin Sealant Patch, a hemostat comprised of a maR trix of nonwoven polyglactin 910 embedded in ORC coated with human fibrinogen and thrombin to TachoSil medicated sponge, an equine collagen pad coated with human fibrinogen and thrombin. Materials and Methods: Swine were anticoagulated with heparin to 3X their baseline activated clotting time and a 15 mm long × 3 mm deep incision was made to create a consistent moderate bleeding pattern. Test material was then applied to the wound site and compressed manually for 3 min with just enough pressure to prevent continued bleeding. Hemostatic effectiveness was evaluated at 3 min and 10 min. Results: At 3 min, the hemostasis success rate was 86% in R group, p < .0001. The overall success rate at 10 min was 100% the EVARRESTTM group and 0% in the TachoSil R , p < .0001. Adhesive failure, in which the test material did not stick with EVARRESTTM and 4% with TachoSil R sites. In contrast, 100% of the EVARRESTTM applications adhered to to the tissue, occurred in 96% of TachoSil TM the test site. Conclusions: EVARREST , Fibrin Sealant Patch demonstrated greater wound adhesion and more R R . Adhesive failure was the primary failure mode for TachoSil in this model. effective hemostasis than TachoSil Keywords: EVARRESTTM , Fibrin Sealant Patch; oxidized regenerated cellulose; ORC; polyglactin 910; R ; hemostasis TachoSil
INTRODUCTION
polymers, and inorganic compounds [7]. Some of these rely on physical means for passive coagulation (ORC, gelatin, collagen) while others provide biologically active components of the clotting cascade (thrombin and fibrin sealants) [8]. The latest generation of topical hemostats combines both active and passive mechaR medicated sponge nisms of action, such as TachoSil TM and EVARREST , Fibrin Sealant Patch. The present study was initiated to compare R to that of the hemostatic efficacy of TachoSil TM in a uniform and reproducible model, EVARREST and thereby provide a benchmark comparison of the currently available collagen/thrombin/fibrinogen hemostasis pad and ORC/PG910/thrombin/fibrinogen hemostasis pad.
Prolonged or uncontrolled bleeding following trauma [1] or after surgery of the cardiovascular system [2], liver [3, 4], kidney [5], spleen [6], and other organs is a serious complication associated with significant morbidity, mortality, and cost of care. Meticulous surgical technique is essential for primary hemostasis, but when conventional techniques such as compression, ligation, clipping, and electrocautery are impractical or ineffective, topical hemostatic agents are relied upon to help control bleeding [2]. Many approved and investigational topical hemostats have been described in the literature, including oxidized regenerated cellulose (ORC), collagen, gelatin, fibrinogen, thrombin,
Received 2 March 2014; accepted 1 July 2014. Disclosure: This research project was supported by Ethicon, Inc., Somerville, NJ 08876. The authors are employees of Ethicon, Inc. Address correspondence to John P. Matonick, PhD, Ethicon, Inc., P.O. Box 151, Somerville, NJ 08876, USA. E-mail: [email protected]
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R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
MATERIALS AND METHODS Test Materials 4×4 inch EVARRESTTM is a recently FDA approved bio-absorbable hemostat comprised of a matrix of nonwoven PG910 fibers embedded in an ORC backing; the matrix is coated with a lyophilized layer of human fibrinogen and human thrombin, identical to the approved fibrin sealant product EvicelTM Fibrin Sealant (Human) (Omrix Biopharmaceuticals, Ethicon, Inc., Somerville, NJ). R medicated sponge (Nycomed, Linz, AusTachoSil tria) is an equine-derived collagen pad coated with a dry layer of human fibrinogen and human thrombin.
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5 to 10 ml/hr. Vital signs and oxygenation were monitored throughout. All surgical sites were clipped and the animal was placed in dorsal recumbency. No antibiotics were administered. No postoperative analgesics were administered because the animals were sacrificed while fully sedated under general anesthesia. A baseline activated clotting time (ACT) was determined and heparin was administered at an initial intravenous bolus dose of 220 U/kg. The ACT was checked again approximately 10 min after the initial heparin dose and periodically thereafter. Additional heparin was administered as needed to maintain an ACT of 3X baseline.
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Surgical Procedure Experimental Animals Crossbred Yorkshire swine, 12 females and 2 males, weight range 40–59 kg, were used in this study. Animals were individually housed in stainless-steel cages, fed once a day with a standard pig chow, provided water ad libitum, and kept on a 12-hr light/dark cycle at 22◦ C ± 4◦ C and 30% to 70% relative humidity. All test animals were handled and maintained under a protocol approved and monitored by the Ethicon Animal Care and Use Committee. The animals received care in compliance with the Ethicon Program of Care and Use of Laboratory Animals and the current Guide for the Care and Use of Laboratory Animals [9]. Each animal was humanely euthanized at the conclusion of R Solution at a dosage of 10 the trial utilizing Euthasol to 15 ml/45 kg body weight and documented in the clinical record (pentobarbital sodium and phenytoin sodium, Virbac Animal Health, Fort Worth, TX).
Preparation and Monitoring Anesthesia was induced with an intramuscular inR , Fort Dodge jection of 5 mg/kg tiletamine (Telazol Animal Health, Fort Dodge, IA), 5 mg/kg xylazine R HCl Injection, RXV, Westlake, TX), and (Xylazine R , Fort Dodge 0.011 mg/kg glycopyrrolate (Robinul-V Animal Health, Fort Dodge, IA). When a sufficient level of anesthesia was reached, an intravenous catheter was then placed in the marginal ear vein or medial auricular vein. An endotracheal tube was inserted and attached to a veterinary anesthesia machine; anesthesia was maintained by a semi-closed circuit inhalation of 0.5% to 4% isoflurane and oxygen at a flow rate of 1 to 2 l/min. Assisted ventilation was accomplished with a mechanical ventilator set at 8 to 12 respirations/min and a tidal volume of approximately 11 ml/kg body weight. Ophthalmic ointment was applied to both eyes of each anesthetized animal. Lactated Ringer’s solution was administered intravenously at approximately C
2014 Informa Healthcare USA, Inc.
A ventral midline incision, approximately 15 cm in length, was made from a point just below the xyphoid process in a caudal direction. The spleen was located, incrementally exteriorized as needed, and kept moist with saline-soaked gauze. A surgical marker was used to delineate the 15-mm-long incision site. The depth of the defect was approximately 3 mm, created with a #11 surgical blade and attached depth gauge to control the depth of the incision. The depth (but not the length) was increased as needed to produce a consistent moderate bleeding pattern among all test sites. Moderate bleeding was defined as flowing venous and/or nonspurting arterial bleeding. After the defect was created and a moderate bleeding pattern was achieved, the test article was given to the surgeon. The test articles were randomized within each animal (2 EVARRESTTM treatment sites R treatment sites per animal) through a and 2 TachoSil blinded drawing process following the creation of the defect. A total of 4 linear incisions and individual treatment applications were performed on the spleen of each animal proceeding from the tail to the head of the spleen, with the sites referred to as distal, mid-distal, midproximal, and proximal, respectively. Each test article was applied to 28 incisions. A schematic of the procedure is shown in Figure 1.
Hemostat Application Technique The instructions for use for each product were rigidly adhered to throughout the testing. The surgeon had more than 20 years’ clinical experience in general surgery and hemostasis testing. Extensive pre-study application trials were performed to ensure the technique was optimal prior to starting the study. Each hemostat was trimmed to 3.0 cm × 4.5 cm with scissors so it extended 1.5 cm beyond the edges of the wound. Excess blood or fluid was removed from the application site to improve visibility, and the hemostat
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Hemostasis Evaluation Hemostasis at the wound site was assessed at 3 and 10 min after application of the hemostat and rated as one of the following: (1) hemostasis with complete adhesion of the test article; (2) hemostasis with local delamination of the test article; (3) incomplete hemostasis with adhesive failure; and (4) incomplete hemostasis with cohesive failure. Adhesive failure was defined as free-flowing blood exiting the perimeter of the test article resulting from a delamination of the test article from the tissue surface. Cohesive failure was defined as free-flowing blood exiting through the test article.
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Statistics The application of test material to the wound sites was assigned by an “out of the hat” type randomization scheme. Fischer’s exact test was conducted on the 3 min data set and the overall data set. Groups were considered significantly different if p ≤ .05.
RESULTS
FIGURE 1 Flow chart of the surgical procedure and hemostasis evaluation. After 10 min, excessive bleeding was terminated by arterial cross clamp or application of EVARRESTTM .
was placed centrally over the wound. EVARRESTTM was placed with the fuzzy side toward the wound and the embossed side facing up; the active side of R was moistened in saline and placed with the TachoSil yellow side against the wound. Saline-moistened surgical gauzes were held over the hemostat to help ensure full contact with the tissue. Manual compression was applied over the entire surface of the hemostat for 3 min with a compression force that stopped all wound sites bleeding during tamponade. The saline moistened gauzes were removed gently from the site of application, taking care not to disrupt or dislodge the hemostat. When persistent bleeding occurred at a test site after the 10 min evaluation period, the bleeding was terminated by the surgeon either by occlusion of the splenic artery, through cross-clamping the spleen immediately proximal to the incision site, or by applying EVARRESTTM to the bleeding site before moving on to the next site.
At 3 min, hemostasis was achieved more frequently in R the EVARRESTTM group (86%) than in the TachoSil R group (0%), p < .0001 (Table 1). In the TachoSil group, all 28 incised defects were bleeding at the 3 min mark; there were 27 adhesive failures and 1 cohesive failure. In the EVARRESTTM group, the 4 sites with incomplete hemostasis were cohesive failures. Figure 2 shows a R pad with hemostatic failure at 3 min. typical TachoSil Figure 3 shows a typical EVARREST pad with hemostatic success at 3 min. At 10 min, hemostasis was achieved in 100% of the EVARRESTTM group (Table 1). The pad was fully adhered to the wound at all 28 sites, with full
FIGURE 2 Hemostatic failure. In this representative image taken R at 3 min, free-flowing blood exits the perimeter of the Tachosil patch. There was no adhesion of the patch to the wound site. Journal of Investigative Surgery
R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
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DISCUSSION The present study is among the relatively few published trials that directly compare the efficacy of marketed hemostatic products, as summarized in a review by Seyednejad and colleagues that showed comparative trials comprised only 12% of nearly 200 published journal articles [7]. The benefit of such models is that they provide direct comparison of efficacy under controlled and reproducible conditions. This study compared the hemostatic efficacy, time to hemostasis, and tissue adhesion of EVARRESTTM , Fibrin Sealant Patch R ) in a hepand a collagen-based product (TachoSil arinized splenic wound model. R is a third-generation hemostat in a famTachoSil ily of collagen patches coated with a mixture of fibrinogen and thrombin. The initial formulation, introduced in the 1990s, consisted of a sponge-like equine collagen patch coated on one side with a mixture of human fibrinogen, bovine thrombin, and bovine aprotinin. The formulation was later refined by replacing bovine thrombin with human thrombin. Bovine aprotinin was omitted in the final and present formulation, which contains equine collagen, human fibrinogen, and human thrombin. The coated side of the pad also contains riboflavin, which imparts a yellow color and indicates the side to be placed against the wound [10]. EVARRESTTM , Fibrin Sealant Patch has two major components: a fully absorbable composite matrix and human-derived active biologics, each with hemostatic properties. The absorbable matrix is a fabric of plant-derived ORC that is needle-punched with polyglactin 910 (PG910) filaments [8]. ORC has been used for decades in SurgicelTM Absorbable Hemostat (Ethicon Inc., Somerville, NJ) and its related hemostats. The PG910 component of the matrix has also been safely used in surgical settings for decades as the essential material in Coated VICRYLTM Suture and mesh products (Ethicon Inc., Somerville, NJ). Absorption of the ORC component occurs within 28 days
FIGURE 3 Hemostatic success. In this representative image taken at 3 min, there was no free-flowing blood either through or under the EVARRESTTM patch.
resolution of the 4 cohesive failures observed at 3 min. R group only 8 sites were evaluable In the TachoSil at 10 min; of these, hemostasis was achieved at 1 site and incomplete hemostasis due to adhesive failure occurred at 7 sites. Hemostasis evaluation was aborted at 20 sites due to persistent uncontrolled bleeding in an effort to preserve the physiologic conditions of the animal. The overall hemostasis success rate, defined as complete hemostasis within 10 min, was 100% in the R group, EVARRESTTM group and 4% in the TachoSil R p < .0001 (Table 1). In the TachoSil group, adhesive failure was evident in 27/28 (96%) of the nonhemostatic sites at 3 min, with cohesive failure evident in 1 failed site. Adhesive failure was evident in all 7 failed sites at 10 min; the 1 site with complete hemostasis displayed local delamination of the hemostat. In the EVARRESTTM group, cohesive failure was evident in 4/4 (100%) of nonhemostatic sites at 3 min, but all sites had complete adhesion of the test article to the wound site. By 10 min all cohesive failures resolved and hemostasis was achieved at all sites.
TABLE 1 Hemostasis outcomes Hemostasis with local delamination
Incomplete hemostasis with adhesive failure
Incomplete hemostasis with cohesive failure
Group
Test material
Hemostasis with complete adhesion
3 min 3 min
TS (n = 28) EP (n = 28)
0 86%b
0 0
96% 0
4% 14%
10 min 10 min
TS (n = 8)a EP (n = 28)
0 100%
13% 0
88% 0
0 0
Overall Overall
TS (n = 28) EP (n = 28)
0 100%b
4% 0
96% 0
0 0
R TS = TachoSil ; EP = EVARRESTTM , Fibrin Sealant Patch. a Only 8 of the 28 sites were evaluable at 10 min. Evaluation was aborted at 20 sites to preserve the animals with uncontrolled bleeding. b p < .0001 for EP vs. TS.
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and EVARRESTTM is essentially fully absorbed by 56 days [8]. The needle-punched PG910 filaments create a “fuzzy” microfibrillar surface that forms the application side of the patch. This fuzzy side is loaded with the human-derived active biologics thrombin and fibrinogen that are sourced from EvicelTM Fibrin Sealant (Human) (Ethicon Inc., Somerville, NJ), a commercially available fibrin sealant with hemostatic indications. The hemostatic efficacy of EVARRESTTM is due to the combined hemostatic properties of its components. ORC is a long-used passive hemostat that denatures proteins, mechanically activates the clotting cascade, and induces local vasoconstriction. The microfibrillar structure of the fuzzy PG910 application surface triggers both the intrinsic and extrinsic pathways of the coagulation cascade and provides a scaffold for platelet aggregation. Complementing the physical matrix are the active biologic components fibrinogen and thrombin, which form fibrin in the absence of patient coagulation factors or metabolic activation [8, 11]. The pig was selected for this model because it closely replicates human anatomy, physiology, blood volume, and tissue properties [12, 13]. It also provides an appropriate anatomic size in relation to product dimensions to facilitate testing. The linear incision spleen model used in the present study was found to be a reliable and reproducible method to evaluate the efficacy of absorbable hemostatic formulations by Hutchinson et al. [14]. The blood flow pattern from the wound site on the spleen was consistent, stable, and persistent; it was important that the bleeding pattern did not change throughout the evaluation period and that it remained consistent over each of the test sites. A heparinized model was selected because heparin abrogates endogenous fibrin clotting mechanisms and forces each product to induce hemostasis based on its own structure and formulation. Additionally, no vasoactive drugs were administered that could alter peripheral resistance and stimulate vasoconstriction during the procedure. The experimental animals were heparinized to create a “worst-case” bleeding scenario, to emulate human patients with severe coagulopathies. The model tests the ability of the hemostatic pad itself to induce clotting in the absence of an active host coagulation system. Both ORC-based (EVARRESTTM ) and R ) hemostatic products have collagen-based (TachoSil been evaluated in normal and coagulopathic models. The present study is one of the first to compare the two products head to head in a heparinized model. Both products use clotting factors (fibrinogen and thrombin) whose activity would be inhibited by heparin. The heparinized model shows that a support matrix of ORC positively contributes to hemostatic activity, whereas a collagen-based support matrix does not. We found the EVARRESTTM to be significantly more effective and to provide significantly faster R . hemostasis and better tissue adhesion than TachoSil TM had no adhesive failures, whereas EVARREST
R had a 96% adhesive failure rate. We believe TachoSil the adhesive performance of EVARRESTTM was due to the three-dimensional porous design of the ORC and polyglactin matrix, which allows migration of blood through the pad, reduces the blood–pad interface pressure levels, and provides a secondary hemostatic effect from the ORC component. The reduced stress at the blood–pad interface precluded the hemostasis pad from being “pushed off” the wound site, resulting in R matrix, on the other an adhesive failure. The TachoSil hand, is a closed-cell equine collagen-based product that creates a thin monolithic layer on the wound site. It is not especially porous and therefore does not allow blood to flow into the matrix, which results in increased stress at the interface. The increased stress level at the hemostat tissue interface, as the material peeled away, resulted in complete delamination from the wound site and re-bleeding. R group, evaluation was aborted at 20 In the TachoSil of the 28 sites because uncontrolled bleeding was a serious risk to the pig’s life. Bleeding was stopped at these sites by one or both of the following methods: (1) A soft tissue clamp was applied across the spleen to stop blood flow; in working from the tip of the spleen back, cross-clamping did not affect perfusion to the next site; R (2) EVARRESTTM was placed on the failed TachoSil sites, which stopped the bleeding in all instances. In the R sites, EVARRESTTM was majority of failed TachoSil used instead of cross-clamping because it was easier to continue the procedure and manipulate the spleen without clamps attached. An important finding of the present study is that R did not adhere to actively bleeding tissue TachoSil under heparinized conditions after 3 min of physical tamponade and was effectively pushed off the surface of the spleen by normal physiologic blood pressure. These results are quite different from Berdajs and colR adhesion and burst leagues, who studied TachoSil pressure in a silicone rubber surgical tubing model [15]. Although burst pressure and adhesion were acceptR able, the model involved application of the TachoSil pad to unpressurized tubing, holding it in place for 10 min to affect a seal, then finally pressurizing the tubing to determine burst strength. In our in vivo model, R pad did not adhere to livwe found that the TachoSil ing tissue with underlying physiologic blood flow after 3 min of tamponade pressure. It is important to note that 3 min of tamponade is within the range specified R instructions for use [16]. by the TachoSil TM EVARREST resulted in less adhesive failure R , which is believed to mode outcomes than TachoSil be, in part, attributed to the design of the matrix. The EVARRESTTM matrix is a three-dimensional porous structure that allows the migration of blood through the material, therefore, reducing the blood–tissue interface stress level, as well as providing a secondary hemostatic affect from the ORC component. The reduction of elevated stress levels overcomes an
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R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
adhesion-based lamination failure mode, which is difficult to resolve in a coagulopathic condition. R matrix is a closed cell equine The TachoSil collagen-based product, which when applied, creates a thin monolithic layer that does not allow for the blood to flow within the matrix, resulting in increasing blood applied stress level at the matrix to tissue interface. The increased stress level and blood flow result in delamiR . nation of TachoSil The EVARRESTTM , Fibrin Sealant Patch described in this study was approved by FDA in 2012 and reported as biocompatible and 100% hemostatically effective in a kidney swine model [8] and 100% in a kidney model [17]. Under conditions of the present study, we confirmed these previous efficacy levels R . and found EVARRESTTM to be superior to TachoSil TM EVARREST has been studied in several clinical trials involving different tissue types (i.e. soft tissue and parenchymal tissue) and increased bleeding severities; the development program is still ongoing [18–21]. Declaration of interest: The authors alone are responsible for the content and writing of the article.
REFERENCES [1] Holcomb JB. Optimal use of blood products in severely injured trauma patients. Hematol Am Soc Hematol Educ Program. 2010;2010:465–469. [2] Maisano F, Kjaerg˚ard HK, Bauernschmitt R, et al. TachoSil surgical patch versus conventional haemostatic fleece material for control of bleeding in cardiovascular surgery: a randomised controlled trial. Eur J Cardiothorac Surg. 2009;36(4):708–714. [3] Frilling A, Stavrou GA, Mischinger H-J, et al. Effectiveness of a new carrier-bound fibrin sealant versus argon beamer as haemostatic agent during liver resection: a randomized prospective trial. Langenbecks Arch Surg. 2005;390(2):114–120. [4] Fischer L, Seiler CM, Broelsch CE, et al. Hemostatic efficacy of TachoSil in liver resection compared with argon beam coagulator treatment: an open, randomized, prospective, multicenter, parallel-group trial. Surgery. 2011;149(1):48–55. [5] Siemer S, Lahme S, Altziebler S, et al. Efficacy and safety of TachoSil as haemostatic treatment versus standard suturing in kidney tumour resection: a randomised prospective study. Eur Urol. 2007;52(4):1156–1163. [6] Carbon RT, Baar S, Waldschmidt J, et al. Innovative minimally invasive pediatric surgery is of therapeutic value for splenic injury. J Pediatr Surg. 2002;37(8):1146–1150. [7] Seyednejad H, Imani M, Jamieson T, et al. Topical haemostatic agents. Br J Surg. 2008;95(10):1197–1225.
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[8] Muench TR, Kong W, Harmon AM. The performance of a hemostatic agent based on oxidized regenerated cellulose – polyglactin 910 composite in a liver defect model in immunocompetent and athymic rats. Biomaterials. 2010;31(13):3649–3656. [9] Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals, 8th ed.; The National Academies Press, Washington, DC, 2011. Available from: https://www.aaalac.org/resources/theguide.cfm [10] Rickenbacher A, Breitenstein S, Lesurtel M, et al. Efficacy of TachoSil a fibrin-based haemostat in different fields of surgery—a systematic review. Expert Opin Biol Ther. 2009;9(7):897–907. [11] Dalpiaz O, Neururer R, Bartsch G, et al. Haemostatic sealants in nephron-sparing surgery: what surgeons need to know. BJU Int. 2008;102(11):1502–1508. [12] Pusateri AE, Modrow HE, Harris RA, et al. Advanced hemostatic dressing development program: animal model selection criteria and results of a study of nine hemostatic dressings in a model of severe large venous hemorrhage and hepatic injury in swine. J Trauma. 2003;55(3):518–526. [13] Pusateri AE, Holcomb JB, Kheirabadi BS, et al. Making sense of the preclinical literature on advanced hemostatic products. J Trauma. 2006;60(3):674–682. [14] Hutchinson RW, George K, Johns D, et al. Hemostatic efficacy and tissue reaction of oxidized regenerated cellulose hemostats. Cellulose. 2013;20:537–545. ¨ [15] Berdajs D, Burki M, Michelis A, et al. Seal properties of TachoSil: in vitro hemodynamic measurements. Interact Cardiovasc Thorac Surg. 2010;10(6):910–913. [16] TachoSil Prescribing Information. Available from: http:// www.tachosil.com/howtoapply/illustrations. Cited Jan 1, 2014. [17] Hutchinson RW, Broughton D, Barbolt TA, et al. Hemostatic effectiveness of fibrin pad after partial nephrectomy in swine. J Surg Res. 2011;167(2):e291–e298. [18] Nativ O, Patel B, Shen, J, et al. Safety and hemostatic efficacy of fibrin pad in partial nephrectomy: results of an open-label phase I and a randomized, standard-of-carecontrolled phase I/II study BMC Nephrol. 2012 Nov8;13:147. doi: 10.1186/1471-2369-13-147. [19] Koea JB, Baldwin P, Hart J, Fischer CP, Garden OJ. A phase III, randomized, controlled, superiority study evaluating the fibrin pad versus standard of care treatment in controlling severe soft tissue bleeding during abdominal, retroperitoneal, pelvic, and thoracic surgery. Association of Surgeons of Great Britain and Ireland, International Surgical Congress, Expertise and Excellence, Liverpool, England, May 9–12, 2012, Abstract #0617. [20] Koea JB, Batiller J, Patel B, et al. A phase III, randomized, controlled, superiority trial evaluating the fibrin pad versus standard of care in controlling parenchymal bleeding during elective hepatic surgery. HPB. 2013;15:61–70. [21] Fischer CP, Bochicchio G, Batiller J, et al. A prospective, radomized, controlled trial of the efficacy and safety of fibrin pad as an adjunct to control soft tissue bleeding during abdominal, retroperitoneal, pelvic, and thoracic surgery. J Am Coll Surg. 2013;217(3):385–933.
NEW METHODOLOGY
Hemostatic Efficacy of EVARRESTTM, Fibrin Sealant Patch R vs. TachoSil in a Heparinized Swine Spleen Incision Model John P. Matonick, PhD1 Jeffrey Hammond, MD, MPH2
J Invest Surg Downloaded from informahealthcare.com by University of Laval on 12/01/14 For personal use only.
1
Preclinical Research, Ethicon, Inc., Somerville, New Jersey, USA, 2 Medical Affairs, Ethicon, Inc., Somerville, New Jersey, USA
ABSTRACT Background: First-generation single-component hemostats such as oxidized regenerated cellulose (ORC), fibrin, collagen, and gelatin have evolved into second and third generations of combination hemostats. Objective: This study compares two FDA approved products, EVARRESTTM , Fibrin Sealant Patch, a hemostat comprised of a maR trix of nonwoven polyglactin 910 embedded in ORC coated with human fibrinogen and thrombin to TachoSil medicated sponge, an equine collagen pad coated with human fibrinogen and thrombin. Materials and Methods: Swine were anticoagulated with heparin to 3X their baseline activated clotting time and a 15 mm long × 3 mm deep incision was made to create a consistent moderate bleeding pattern. Test material was then applied to the wound site and compressed manually for 3 min with just enough pressure to prevent continued bleeding. Hemostatic effectiveness was evaluated at 3 min and 10 min. Results: At 3 min, the hemostasis success rate was 86% in R group, p < .0001. The overall success rate at 10 min was 100% the EVARRESTTM group and 0% in the TachoSil R , p < .0001. Adhesive failure, in which the test material did not stick with EVARRESTTM and 4% with TachoSil R sites. In contrast, 100% of the EVARRESTTM applications adhered to to the tissue, occurred in 96% of TachoSil TM the test site. Conclusions: EVARREST , Fibrin Sealant Patch demonstrated greater wound adhesion and more R R . Adhesive failure was the primary failure mode for TachoSil in this model. effective hemostasis than TachoSil Keywords: EVARRESTTM , Fibrin Sealant Patch; oxidized regenerated cellulose; ORC; polyglactin 910; R ; hemostasis TachoSil
INTRODUCTION
polymers, and inorganic compounds [7]. Some of these rely on physical means for passive coagulation (ORC, gelatin, collagen) while others provide biologically active components of the clotting cascade (thrombin and fibrin sealants) [8]. The latest generation of topical hemostats combines both active and passive mechaR medicated sponge nisms of action, such as TachoSil TM and EVARREST , Fibrin Sealant Patch. The present study was initiated to compare R to that of the hemostatic efficacy of TachoSil TM in a uniform and reproducible model, EVARREST and thereby provide a benchmark comparison of the currently available collagen/thrombin/fibrinogen hemostasis pad and ORC/PG910/thrombin/fibrinogen hemostasis pad.
Prolonged or uncontrolled bleeding following trauma [1] or after surgery of the cardiovascular system [2], liver [3, 4], kidney [5], spleen [6], and other organs is a serious complication associated with significant morbidity, mortality, and cost of care. Meticulous surgical technique is essential for primary hemostasis, but when conventional techniques such as compression, ligation, clipping, and electrocautery are impractical or ineffective, topical hemostatic agents are relied upon to help control bleeding [2]. Many approved and investigational topical hemostats have been described in the literature, including oxidized regenerated cellulose (ORC), collagen, gelatin, fibrinogen, thrombin,
Received 2 March 2014; accepted 1 July 2014. Disclosure: This research project was supported by Ethicon, Inc., Somerville, NJ 08876. The authors are employees of Ethicon, Inc. Address correspondence to John P. Matonick, PhD, Ethicon, Inc., P.O. Box 151, Somerville, NJ 08876, USA. E-mail: [email protected]
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R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
MATERIALS AND METHODS Test Materials 4×4 inch EVARRESTTM is a recently FDA approved bio-absorbable hemostat comprised of a matrix of nonwoven PG910 fibers embedded in an ORC backing; the matrix is coated with a lyophilized layer of human fibrinogen and human thrombin, identical to the approved fibrin sealant product EvicelTM Fibrin Sealant (Human) (Omrix Biopharmaceuticals, Ethicon, Inc., Somerville, NJ). R medicated sponge (Nycomed, Linz, AusTachoSil tria) is an equine-derived collagen pad coated with a dry layer of human fibrinogen and human thrombin.
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5 to 10 ml/hr. Vital signs and oxygenation were monitored throughout. All surgical sites were clipped and the animal was placed in dorsal recumbency. No antibiotics were administered. No postoperative analgesics were administered because the animals were sacrificed while fully sedated under general anesthesia. A baseline activated clotting time (ACT) was determined and heparin was administered at an initial intravenous bolus dose of 220 U/kg. The ACT was checked again approximately 10 min after the initial heparin dose and periodically thereafter. Additional heparin was administered as needed to maintain an ACT of 3X baseline.
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Surgical Procedure Experimental Animals Crossbred Yorkshire swine, 12 females and 2 males, weight range 40–59 kg, were used in this study. Animals were individually housed in stainless-steel cages, fed once a day with a standard pig chow, provided water ad libitum, and kept on a 12-hr light/dark cycle at 22◦ C ± 4◦ C and 30% to 70% relative humidity. All test animals were handled and maintained under a protocol approved and monitored by the Ethicon Animal Care and Use Committee. The animals received care in compliance with the Ethicon Program of Care and Use of Laboratory Animals and the current Guide for the Care and Use of Laboratory Animals [9]. Each animal was humanely euthanized at the conclusion of R Solution at a dosage of 10 the trial utilizing Euthasol to 15 ml/45 kg body weight and documented in the clinical record (pentobarbital sodium and phenytoin sodium, Virbac Animal Health, Fort Worth, TX).
Preparation and Monitoring Anesthesia was induced with an intramuscular inR , Fort Dodge jection of 5 mg/kg tiletamine (Telazol Animal Health, Fort Dodge, IA), 5 mg/kg xylazine R HCl Injection, RXV, Westlake, TX), and (Xylazine R , Fort Dodge 0.011 mg/kg glycopyrrolate (Robinul-V Animal Health, Fort Dodge, IA). When a sufficient level of anesthesia was reached, an intravenous catheter was then placed in the marginal ear vein or medial auricular vein. An endotracheal tube was inserted and attached to a veterinary anesthesia machine; anesthesia was maintained by a semi-closed circuit inhalation of 0.5% to 4% isoflurane and oxygen at a flow rate of 1 to 2 l/min. Assisted ventilation was accomplished with a mechanical ventilator set at 8 to 12 respirations/min and a tidal volume of approximately 11 ml/kg body weight. Ophthalmic ointment was applied to both eyes of each anesthetized animal. Lactated Ringer’s solution was administered intravenously at approximately C
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A ventral midline incision, approximately 15 cm in length, was made from a point just below the xyphoid process in a caudal direction. The spleen was located, incrementally exteriorized as needed, and kept moist with saline-soaked gauze. A surgical marker was used to delineate the 15-mm-long incision site. The depth of the defect was approximately 3 mm, created with a #11 surgical blade and attached depth gauge to control the depth of the incision. The depth (but not the length) was increased as needed to produce a consistent moderate bleeding pattern among all test sites. Moderate bleeding was defined as flowing venous and/or nonspurting arterial bleeding. After the defect was created and a moderate bleeding pattern was achieved, the test article was given to the surgeon. The test articles were randomized within each animal (2 EVARRESTTM treatment sites R treatment sites per animal) through a and 2 TachoSil blinded drawing process following the creation of the defect. A total of 4 linear incisions and individual treatment applications were performed on the spleen of each animal proceeding from the tail to the head of the spleen, with the sites referred to as distal, mid-distal, midproximal, and proximal, respectively. Each test article was applied to 28 incisions. A schematic of the procedure is shown in Figure 1.
Hemostat Application Technique The instructions for use for each product were rigidly adhered to throughout the testing. The surgeon had more than 20 years’ clinical experience in general surgery and hemostasis testing. Extensive pre-study application trials were performed to ensure the technique was optimal prior to starting the study. Each hemostat was trimmed to 3.0 cm × 4.5 cm with scissors so it extended 1.5 cm beyond the edges of the wound. Excess blood or fluid was removed from the application site to improve visibility, and the hemostat
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Hemostasis Evaluation Hemostasis at the wound site was assessed at 3 and 10 min after application of the hemostat and rated as one of the following: (1) hemostasis with complete adhesion of the test article; (2) hemostasis with local delamination of the test article; (3) incomplete hemostasis with adhesive failure; and (4) incomplete hemostasis with cohesive failure. Adhesive failure was defined as free-flowing blood exiting the perimeter of the test article resulting from a delamination of the test article from the tissue surface. Cohesive failure was defined as free-flowing blood exiting through the test article.
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Statistics The application of test material to the wound sites was assigned by an “out of the hat” type randomization scheme. Fischer’s exact test was conducted on the 3 min data set and the overall data set. Groups were considered significantly different if p ≤ .05.
RESULTS
FIGURE 1 Flow chart of the surgical procedure and hemostasis evaluation. After 10 min, excessive bleeding was terminated by arterial cross clamp or application of EVARRESTTM .
was placed centrally over the wound. EVARRESTTM was placed with the fuzzy side toward the wound and the embossed side facing up; the active side of R was moistened in saline and placed with the TachoSil yellow side against the wound. Saline-moistened surgical gauzes were held over the hemostat to help ensure full contact with the tissue. Manual compression was applied over the entire surface of the hemostat for 3 min with a compression force that stopped all wound sites bleeding during tamponade. The saline moistened gauzes were removed gently from the site of application, taking care not to disrupt or dislodge the hemostat. When persistent bleeding occurred at a test site after the 10 min evaluation period, the bleeding was terminated by the surgeon either by occlusion of the splenic artery, through cross-clamping the spleen immediately proximal to the incision site, or by applying EVARRESTTM to the bleeding site before moving on to the next site.
At 3 min, hemostasis was achieved more frequently in R the EVARRESTTM group (86%) than in the TachoSil R group (0%), p < .0001 (Table 1). In the TachoSil group, all 28 incised defects were bleeding at the 3 min mark; there were 27 adhesive failures and 1 cohesive failure. In the EVARRESTTM group, the 4 sites with incomplete hemostasis were cohesive failures. Figure 2 shows a R pad with hemostatic failure at 3 min. typical TachoSil Figure 3 shows a typical EVARREST pad with hemostatic success at 3 min. At 10 min, hemostasis was achieved in 100% of the EVARRESTTM group (Table 1). The pad was fully adhered to the wound at all 28 sites, with full
FIGURE 2 Hemostatic failure. In this representative image taken R at 3 min, free-flowing blood exits the perimeter of the Tachosil patch. There was no adhesion of the patch to the wound site. Journal of Investigative Surgery
R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
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DISCUSSION The present study is among the relatively few published trials that directly compare the efficacy of marketed hemostatic products, as summarized in a review by Seyednejad and colleagues that showed comparative trials comprised only 12% of nearly 200 published journal articles [7]. The benefit of such models is that they provide direct comparison of efficacy under controlled and reproducible conditions. This study compared the hemostatic efficacy, time to hemostasis, and tissue adhesion of EVARRESTTM , Fibrin Sealant Patch R ) in a hepand a collagen-based product (TachoSil arinized splenic wound model. R is a third-generation hemostat in a famTachoSil ily of collagen patches coated with a mixture of fibrinogen and thrombin. The initial formulation, introduced in the 1990s, consisted of a sponge-like equine collagen patch coated on one side with a mixture of human fibrinogen, bovine thrombin, and bovine aprotinin. The formulation was later refined by replacing bovine thrombin with human thrombin. Bovine aprotinin was omitted in the final and present formulation, which contains equine collagen, human fibrinogen, and human thrombin. The coated side of the pad also contains riboflavin, which imparts a yellow color and indicates the side to be placed against the wound [10]. EVARRESTTM , Fibrin Sealant Patch has two major components: a fully absorbable composite matrix and human-derived active biologics, each with hemostatic properties. The absorbable matrix is a fabric of plant-derived ORC that is needle-punched with polyglactin 910 (PG910) filaments [8]. ORC has been used for decades in SurgicelTM Absorbable Hemostat (Ethicon Inc., Somerville, NJ) and its related hemostats. The PG910 component of the matrix has also been safely used in surgical settings for decades as the essential material in Coated VICRYLTM Suture and mesh products (Ethicon Inc., Somerville, NJ). Absorption of the ORC component occurs within 28 days
FIGURE 3 Hemostatic success. In this representative image taken at 3 min, there was no free-flowing blood either through or under the EVARRESTTM patch.
resolution of the 4 cohesive failures observed at 3 min. R group only 8 sites were evaluable In the TachoSil at 10 min; of these, hemostasis was achieved at 1 site and incomplete hemostasis due to adhesive failure occurred at 7 sites. Hemostasis evaluation was aborted at 20 sites due to persistent uncontrolled bleeding in an effort to preserve the physiologic conditions of the animal. The overall hemostasis success rate, defined as complete hemostasis within 10 min, was 100% in the R group, EVARRESTTM group and 4% in the TachoSil R p < .0001 (Table 1). In the TachoSil group, adhesive failure was evident in 27/28 (96%) of the nonhemostatic sites at 3 min, with cohesive failure evident in 1 failed site. Adhesive failure was evident in all 7 failed sites at 10 min; the 1 site with complete hemostasis displayed local delamination of the hemostat. In the EVARRESTTM group, cohesive failure was evident in 4/4 (100%) of nonhemostatic sites at 3 min, but all sites had complete adhesion of the test article to the wound site. By 10 min all cohesive failures resolved and hemostasis was achieved at all sites.
TABLE 1 Hemostasis outcomes Hemostasis with local delamination
Incomplete hemostasis with adhesive failure
Incomplete hemostasis with cohesive failure
Group
Test material
Hemostasis with complete adhesion
3 min 3 min
TS (n = 28) EP (n = 28)
0 86%b
0 0
96% 0
4% 14%
10 min 10 min
TS (n = 8)a EP (n = 28)
0 100%
13% 0
88% 0
0 0
Overall Overall
TS (n = 28) EP (n = 28)
0 100%b
4% 0
96% 0
0 0
R TS = TachoSil ; EP = EVARRESTTM , Fibrin Sealant Patch. a Only 8 of the 28 sites were evaluable at 10 min. Evaluation was aborted at 20 sites to preserve the animals with uncontrolled bleeding. b p < .0001 for EP vs. TS.
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and EVARRESTTM is essentially fully absorbed by 56 days [8]. The needle-punched PG910 filaments create a “fuzzy” microfibrillar surface that forms the application side of the patch. This fuzzy side is loaded with the human-derived active biologics thrombin and fibrinogen that are sourced from EvicelTM Fibrin Sealant (Human) (Ethicon Inc., Somerville, NJ), a commercially available fibrin sealant with hemostatic indications. The hemostatic efficacy of EVARRESTTM is due to the combined hemostatic properties of its components. ORC is a long-used passive hemostat that denatures proteins, mechanically activates the clotting cascade, and induces local vasoconstriction. The microfibrillar structure of the fuzzy PG910 application surface triggers both the intrinsic and extrinsic pathways of the coagulation cascade and provides a scaffold for platelet aggregation. Complementing the physical matrix are the active biologic components fibrinogen and thrombin, which form fibrin in the absence of patient coagulation factors or metabolic activation [8, 11]. The pig was selected for this model because it closely replicates human anatomy, physiology, blood volume, and tissue properties [12, 13]. It also provides an appropriate anatomic size in relation to product dimensions to facilitate testing. The linear incision spleen model used in the present study was found to be a reliable and reproducible method to evaluate the efficacy of absorbable hemostatic formulations by Hutchinson et al. [14]. The blood flow pattern from the wound site on the spleen was consistent, stable, and persistent; it was important that the bleeding pattern did not change throughout the evaluation period and that it remained consistent over each of the test sites. A heparinized model was selected because heparin abrogates endogenous fibrin clotting mechanisms and forces each product to induce hemostasis based on its own structure and formulation. Additionally, no vasoactive drugs were administered that could alter peripheral resistance and stimulate vasoconstriction during the procedure. The experimental animals were heparinized to create a “worst-case” bleeding scenario, to emulate human patients with severe coagulopathies. The model tests the ability of the hemostatic pad itself to induce clotting in the absence of an active host coagulation system. Both ORC-based (EVARRESTTM ) and R ) hemostatic products have collagen-based (TachoSil been evaluated in normal and coagulopathic models. The present study is one of the first to compare the two products head to head in a heparinized model. Both products use clotting factors (fibrinogen and thrombin) whose activity would be inhibited by heparin. The heparinized model shows that a support matrix of ORC positively contributes to hemostatic activity, whereas a collagen-based support matrix does not. We found the EVARRESTTM to be significantly more effective and to provide significantly faster R . hemostasis and better tissue adhesion than TachoSil TM had no adhesive failures, whereas EVARREST
R had a 96% adhesive failure rate. We believe TachoSil the adhesive performance of EVARRESTTM was due to the three-dimensional porous design of the ORC and polyglactin matrix, which allows migration of blood through the pad, reduces the blood–pad interface pressure levels, and provides a secondary hemostatic effect from the ORC component. The reduced stress at the blood–pad interface precluded the hemostasis pad from being “pushed off” the wound site, resulting in R matrix, on the other an adhesive failure. The TachoSil hand, is a closed-cell equine collagen-based product that creates a thin monolithic layer on the wound site. It is not especially porous and therefore does not allow blood to flow into the matrix, which results in increased stress at the interface. The increased stress level at the hemostat tissue interface, as the material peeled away, resulted in complete delamination from the wound site and re-bleeding. R group, evaluation was aborted at 20 In the TachoSil of the 28 sites because uncontrolled bleeding was a serious risk to the pig’s life. Bleeding was stopped at these sites by one or both of the following methods: (1) A soft tissue clamp was applied across the spleen to stop blood flow; in working from the tip of the spleen back, cross-clamping did not affect perfusion to the next site; R (2) EVARRESTTM was placed on the failed TachoSil sites, which stopped the bleeding in all instances. In the R sites, EVARRESTTM was majority of failed TachoSil used instead of cross-clamping because it was easier to continue the procedure and manipulate the spleen without clamps attached. An important finding of the present study is that R did not adhere to actively bleeding tissue TachoSil under heparinized conditions after 3 min of physical tamponade and was effectively pushed off the surface of the spleen by normal physiologic blood pressure. These results are quite different from Berdajs and colR adhesion and burst leagues, who studied TachoSil pressure in a silicone rubber surgical tubing model [15]. Although burst pressure and adhesion were acceptR able, the model involved application of the TachoSil pad to unpressurized tubing, holding it in place for 10 min to affect a seal, then finally pressurizing the tubing to determine burst strength. In our in vivo model, R pad did not adhere to livwe found that the TachoSil ing tissue with underlying physiologic blood flow after 3 min of tamponade pressure. It is important to note that 3 min of tamponade is within the range specified R instructions for use [16]. by the TachoSil TM EVARREST resulted in less adhesive failure R , which is believed to mode outcomes than TachoSil be, in part, attributed to the design of the matrix. The EVARRESTTM matrix is a three-dimensional porous structure that allows the migration of blood through the material, therefore, reducing the blood–tissue interface stress level, as well as providing a secondary hemostatic affect from the ORC component. The reduction of elevated stress levels overcomes an
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R EVARRESTTM vs. TachoSil Hemostasis in Splenic Wounds
adhesion-based lamination failure mode, which is difficult to resolve in a coagulopathic condition. R matrix is a closed cell equine The TachoSil collagen-based product, which when applied, creates a thin monolithic layer that does not allow for the blood to flow within the matrix, resulting in increasing blood applied stress level at the matrix to tissue interface. The increased stress level and blood flow result in delamiR . nation of TachoSil The EVARRESTTM , Fibrin Sealant Patch described in this study was approved by FDA in 2012 and reported as biocompatible and 100% hemostatically effective in a kidney swine model [8] and 100% in a kidney model [17]. Under conditions of the present study, we confirmed these previous efficacy levels R . and found EVARRESTTM to be superior to TachoSil TM EVARREST has been studied in several clinical trials involving different tissue types (i.e. soft tissue and parenchymal tissue) and increased bleeding severities; the development program is still ongoing [18–21]. Declaration of interest: The authors alone are responsible for the content and writing of the article.
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[8] Muench TR, Kong W, Harmon AM. The performance of a hemostatic agent based on oxidized regenerated cellulose – polyglactin 910 composite in a liver defect model in immunocompetent and athymic rats. Biomaterials. 2010;31(13):3649–3656. [9] Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals, 8th ed.; The National Academies Press, Washington, DC, 2011. Available from: https://www.aaalac.org/resources/theguide.cfm [10] Rickenbacher A, Breitenstein S, Lesurtel M, et al. Efficacy of TachoSil a fibrin-based haemostat in different fields of surgery—a systematic review. Expert Opin Biol Ther. 2009;9(7):897–907. [11] Dalpiaz O, Neururer R, Bartsch G, et al. Haemostatic sealants in nephron-sparing surgery: what surgeons need to know. BJU Int. 2008;102(11):1502–1508. [12] Pusateri AE, Modrow HE, Harris RA, et al. Advanced hemostatic dressing development program: animal model selection criteria and results of a study of nine hemostatic dressings in a model of severe large venous hemorrhage and hepatic injury in swine. J Trauma. 2003;55(3):518–526. [13] Pusateri AE, Holcomb JB, Kheirabadi BS, et al. Making sense of the preclinical literature on advanced hemostatic products. J Trauma. 2006;60(3):674–682. [14] Hutchinson RW, George K, Johns D, et al. Hemostatic efficacy and tissue reaction of oxidized regenerated cellulose hemostats. Cellulose. 2013;20:537–545. ¨ [15] Berdajs D, Burki M, Michelis A, et al. Seal properties of TachoSil: in vitro hemodynamic measurements. Interact Cardiovasc Thorac Surg. 2010;10(6):910–913. [16] TachoSil Prescribing Information. Available from: http:// www.tachosil.com/howtoapply/illustrations. Cited Jan 1, 2014. [17] Hutchinson RW, Broughton D, Barbolt TA, et al. Hemostatic effectiveness of fibrin pad after partial nephrectomy in swine. J Surg Res. 2011;167(2):e291–e298. [18] Nativ O, Patel B, Shen, J, et al. Safety and hemostatic efficacy of fibrin pad in partial nephrectomy: results of an open-label phase I and a randomized, standard-of-carecontrolled phase I/II study BMC Nephrol. 2012 Nov8;13:147. doi: 10.1186/1471-2369-13-147. [19] Koea JB, Baldwin P, Hart J, Fischer CP, Garden OJ. A phase III, randomized, controlled, superiority study evaluating the fibrin pad versus standard of care treatment in controlling severe soft tissue bleeding during abdominal, retroperitoneal, pelvic, and thoracic surgery. Association of Surgeons of Great Britain and Ireland, International Surgical Congress, Expertise and Excellence, Liverpool, England, May 9–12, 2012, Abstract #0617. [20] Koea JB, Batiller J, Patel B, et al. A phase III, randomized, controlled, superiority trial evaluating the fibrin pad versus standard of care in controlling parenchymal bleeding during elective hepatic surgery. HPB. 2013;15:61–70. [21] Fischer CP, Bochicchio G, Batiller J, et al. A prospective, radomized, controlled trial of the efficacy and safety of fibrin pad as an adjunct to control soft tissue bleeding during abdominal, retroperitoneal, pelvic, and thoracic surgery. J Am Coll Surg. 2013;217(3):385–933.
