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Comparative efficacy of topical hemostatic agents in a rat kidney model
Comparative
Efficacy of Topical Hemostatic in a Rat Kidney Model
Agents
Joseph S. Raccuia, MD, Gregory Simonian, BS, Michael Dardik, BA, David Hallac, Salvatore V. Raccuia, BS, Rosalyn Stahl, MD, Herbert Dardik, MD, FACS, Englewood. New Jersey
The efficacies of four topical hemostatic agents were compared in a rat model employing a standardkd renal injury. The materials used to effect hemostasis were oxidized cellulose, microfibrillar collagen powder, positively charged modified collagen, and single donor heterologous fibrin glue. Animals that were treated only with surgical gauze served as controls. Hemostasis was achieved by application of one of the topical hemostatic agents plus moderate digital pressure. The time necessary to achieve complete hemostasis was recorded for each animal. Control animals bled profusely and suffered an increased postoperative mortality rate compared with the experimental animals. Microscopic studies demonstrated progressive healing of the injuries with varying degrees of inflammation and scar formation. Fibrin glue was by far the most effective agent in controlling hemostasis. The collagen materials, though effective, required a longer time to control bleeding and did not differ statistically in their activity from one another.
From the Surgical Research Laboratory, Department of Surgery, Englewood Hospital, En&wood, New Jersey. Requests for reprints should be addressed to Herbert Dardik, MD, 375 Engle Street, Englewood, New Jersey 0763 1. Manuscripts submitted April 10, 1990, and accepted in revised form September 13,199O. 234
THE AMERICAN JOURNAL OF SURGERY
he attainment of adequate hemostasis for surgical T procedures is often complicated by the presence of friable tissues and coagulopathy. Attempts to control such bleeding by the placement of additional sutures may in fact further aggravate the situation. An alternative method to control bleeding is the application of topical hemostatic agents. In order to assess the comparative efficacies of the various hemostatic agents currently available, we undertook the following set of experiments. Three commercially available hemostatic materials, as well as heterologous fibrin, were compared in their ability to achieve effective hemostasis upon the cut surface of the rat kidney. MATERIALS AND METHODS Experimental preparation and protocol: SpragueDawley rats weighing between 400 and 500 g were used to assess the efficacy of the intraoperative application of topical hemostatic agents. Anesthesia was induced with 50 mg intramuscular ketamine hydrochloride and 1.25 mg of midazolam hydrochloride. In randomly chosen animals, an incision was made in the left neck over the carotid pulse. The sternocleidomastoid muscle and vagus nerve were dissected, and a 20-gauge angiocatheter (Terumo Medical Corp., Elkton, MD) inserted into the carotid artery. The catheter was attached to a blood pressure transducer, and blood pressure was monitored throughout the procedure. The peritoneum was entered, and a segment of each kidney representing approximately 10% of renal volume was excised with a standardized premeasured template, leaving an actively bleeding area measuring approximately 1 by 5 cm. Profuse bleeding was thereby produced, and a topical hemostatic agent was then applied with gentle pressure. Fibrin glue was applied with a dual syringe injector, and no pressure was applied. The time required to achieve total hemostasis was recorded, and the results compared for statistical significance by the Student’s t-test and the chi-square test. The control of bleeding was evaluated by releasing the pressure on the hemostatic agent at intervals of 30 seconds. This was not required in the case of fibrin glue. Compression of renal hilar vessels to augment hemostasis was not employed in this study. The kidneys were then returned to the peritoneal cavity. Specimens were removed every 24 hours for 4 weeks after the application of the hemostatic agents. Microscopic examination was performed after formalin fixation and staining with hematoxylin and eosin. Hemostatic agents: The following materials were used in this study: (1) oxidized cellulose (Surgicel, Johnson and Johnson Inc., New Brunswick, NJ), (2) microtibrillar collagen powder (Avitene, Medical Chemical Products, Inc., Woburn, MA), (3) positively charged modified collagen (Superstat, Edward Week Inc., Prince-
VOLUME 163 FEBRUARY 1992
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ton, NJ), and (4) single donor heterologous fibrin glue. Oxidized cellulose, microfibrillar collagen powder, positively charged modified collagen, and fibrin glue were applied directly to the wound sites. Oxidized cellulose is formed by passing surgical gauze over fuming nitric acid and thus oxiding the cellulose and creating cellulosic acid [ 1). Microfibrillar collagen powder is a specifically crosslinked collagen prepared from bovine dermis [2]. Positively charged collagen powder is a modified collagen sponge (no cross-linkage), treated to provide a net positive charge. Fibrin glue was made from single donor fresh-frozen plasma with modification of the method described by Dresdale et al [3]. A unit of fresh-frozen plasma from a single donor, screened for hepatitis B, syphilis, and human immunodeficiency virus, was obtained from the blood bank and apportioned into SO-mL polypropylene centrifuge tubes. The tubes are kept in a freezer at -8OOC for at least 12 hours. The fibrinogen is prepared for use by thawing the tubes for at least 6 hours at 4OC, followed by centrifugation at 1,500g for 15 minutes. The supematant is decanted, leaving a yellowish precipitate of fibrinogen (cryoprecipitate). The precipitate is resuspended in a small amount of supematant. This concentrated fibrinogen suspension is aspirated into a syringe with a large bore needle. Fibrin sealant is formed by mixing the fibrinogen suspension with thrombin, which in turn is prepared by mixing 500 IU/mL with 5 mL sterile saline. The two solutions (fibrinogen and thrombin), in separate syringes, are applied simultaneously to the wound with a dual syringe holder. Upon application, the components form a clear viscous solution that adheres firmly to the wound surface. Application of hemostatic agents Fifty rats were divided into five groups of ten animals each. In the control group, only digital pressure was applied with a standard 4 by 4-inch sterile gauze sponge. In the oxidized cellulose group, microfibrillar collagen powder group, and positively charged modified collagen group, direct pressure was also applied with gauze but with the hemostatic material directly on the bleeding surface of the kidney. THE AMERICAN
The wounds were examined at intervals of 30 seconds until bleeding ceased. In the fibrin glue group, fibrinogen and thrombin were applied simultaneously to the bleeding kidney surface in a drop by drop fashion until hemostasis was achieved. Digital pressure was not applied in this group. RESULTS
Hemostasis was achieved in all animals prior to closure of the peritoneum. Seven of the animals in the control group died within 48 hours of surgery. Four of these animals died from delayed hemorrhage evidenced at autopsy by gross hemoperitoneum, and the other three died of complications of anesthesia. No animals died in the fibrin glue group, 2 in the positively charged modified collagen group, 3 in the oxidized cellulose group, and 3 in the microfibrillar collagen powder group. Deaths in the positively charged modified collagen group, oxidized cellulose group, and microfibrillar collagen powder group were directly attributed to complications from anesthesia, since no hemorrhage was evident at autopsy. Average blood pressure was monitored, and there was no signilicant hypotension during the procedure (134.6 f 4.6 mm Hg) (p <0.5). There was a significant difference in the mortality from late hemorrhage between the control and the experimental animals by Student’s t-test (p
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Figure 2. Photomicrograph at 4 weeks of traumatizedrat kidney followed by appHcatkn of podtlvely chargedmodifiedcollagen.Note mlnimalscatdngonsurfaceandamild intedtid infiltrateof chronic lnflan+ mdoryfAlS.Thl!JhlMOgkplctue exempliflea the appearance at 4 weeksofallvaWnatizedareasfoc lowedby applkatkn of oxklizeclcells kWposltlWchergedmodlRedcollagen,or flbrlnglue. (Hematoxylinand eosln, orlglnal magnification X100, reducedby35%.)
F~S.Photomlcrogmphat 1 week oftraumatizedareaofratkldneyfoL kwed by applkatkn of mkroflbrlllar collagen powder. Note exuberant ~karre8ctkntothe fore@~ mat&al. (liematoxylin and eosln, original magllfkatkn x100, reducedby35%.)
sured in seconds. There was no significant difference by Student’s t-test (p <0.05) between the fibrin glue group and all other groups. Cross findings: Animals surviving after 72 hours were killed at intervals up to 4 weeks. None of the animals exhibited evidence of hematoma formation, recurrent bleeding, or disruption of renal parenchyma. There were also no abscesses or signs of peritonitis. Gross tissue reactions were limited to thin omental adhesions to the injured surfaces. Microscopic findings: Histologic evaluation demonstrated a series of progressive changes from acute infarc tion with varying degrees of hemorrhage at 24 hours to healing with varying degrees of scar formation. By 4 236
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weeks, all agents, except microfibrillar collagen powder, left minimal scarring and complete healing with little evidence of past injury (FIgure 2). Microfibrillar collagen powder elicited the most exuberant inflammatory re sponse with granuloma formation surrounclmg the fibrillary collagen at one week (Hgure 3), which progressed to dense scarring and chronic inflammation with foreign body reaction that was histologically visible at 4 weeks (WWe 4). COMMENTS The need for effective hemostasis with the use of topical hemostatic agents to control bothersome and sometimes lethal bleeding has been established. By the turn of
VOLUME 163 FEBRUARY 1992
COMPARATIVE EFFICACIES OF TOPICAL HEMOSTATIC AGENTS
Flgure 4. Photomicrograph at 4 weaksofbawMkdwaaofratk# iiziE!--&sL= pt3mwu3dvisiMe~matarL al. BuIwndbd W m bodv siant cebanddelnmpopullwarofchronic innMmataycelk.ThlsiBinsmking anWasttoF~2.~xylinand mE@btbn x100. -,orighlel t=ducedby35%.)
the century, it was generally appreciated that gauze was the most effective hemostatic agent, which it did by trap ping blood elements within its fibers and thereby facilitating a chemical response in addition to its physical characteristics. Fibrin patches and foams in varied cross-linkages were also introduced but had unpredictable efficacy. The hemostatic effect of cellulosic acid, first noted by Olwin and Wahl [I], is based on its physical matrix, which collects blood into its interstices, entrapping and concentrating the coagulation factors. The pH changes in the tissues caused by this material are also a factor in procoagulation. Although the fabric is easy to work with, it provides only a moderate degree of hemostasis [4-7). Correll et al [8] introduced a new absorbable sponge in 1945 that consisted of an insoluble, organic polymer de rived from formal&tanned gelatin. This material (Gelfoam, Upjohn Co., Washington, DC) effects coagulation by physically absorbing blood within its interstices, but it is cumbersome to use and crumples easily. Even if this material is presoaked with thrombin, it is difficult to maintain in position and disintegrates under pressure while effecting only a moderate degree of hemostasis [ 93. In 1962, Beck, Duckert, and Ernst [ZO] reported on the in uiuo function and morphologic characteristics of fibrin glue. The use of thin metallic and gold foils was subsequently described for topical hemostasiq however, to date, these methods have never been applied in clinical practice [ 1I ,I21 . Microfibrillar collagen powder was introduced in 1967 [2] and consists of partial acid salt material of specifically cross-linked collagen prepared from bovine dermis. It is water insoluble, biodegradable, nonallergenic, and can be sterilized. Hemostatic action occurs after contact with blood, forming an insoluble gelfibrin mesh that adheres to the wound surface. The material is very difficult to handle due to its high electrostatic THE AMERICAN
charge in the powder form. Fibrillar forms of collagen can also be fabricated into fleece or felt, which are easier to handle than other forms [5,7,9,13]. Positively charged modified collagen differs from other collagen products in that the material is water soluble and is not cross-linked. Hemostasis is achieved by its special charge characteristics, interacting with blood to convert fibrinogen to fibrin. As various collagen agents continue to gain favor for hemostasis, the use of commercial fibrin glue has also begun to be appreciated, although problems pertaining to the use of pooled donor plasma persist [Z&20]. Preparation of fibrin glue from singledonor autologous and homologous whole blood or freshfrozen plasma is cumbersome and requires anticipation of the need for it prior to surgery. This method requires 1 or 2 days of preparation for a small finite volume of glue, so that autologous glue may not be available for emergency cases. Cryoprecipitate as a source for fibrin glue has also been described, but it is expensive and variable in the amount of end-product. Comparison of autologous fibrin glue with the lyophilized, commercial European product (Tissucol, Immuno AG, Vienna, Austria) indicates that the tear coefficient in the latter is higher [Z5], but it has not been adequately determined if this product has an increased risk for serologically transmitted infections. The risk of transmission of hepatitis B and AIDS by the use of nonautologous blood products is of paramount concern, and the use of sensitive screening tests has significantly reduced the risk of transferring these and other serologically transmitted illnesses. The use of fibrin glue derived from homologous sources must be weighed against the risk of transfusing multiple units of blood products in the event that hemorrhage is significant. In the future, as blood screening tests become more sensitive, the possibility for transmitting these diseases from homologous donors might be negligible.
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Our study evaluates the effectiveness of the most pop ular topical hemostatic agents now currently available. In this experiment, the injured kidney surface bled profusely in the control group and was associated with a higher mortality rate from delayed hemorrhage when compared with the other groups. On the other hand, although time for achieving hemostasis was basically unchanged from the control time in the oxidized cellulose, microlibrillar collagen powder, and positively charged modified collagen groups, the decreased postoperative mortality in these groups might reflect improved long-term control of bleeding. This is further supported by the smaller amount of hematoma found in the experimental group compared with the amount found in the control group. The most effective agent was clearly fibrin glue in that bleeding was controlled almost immediately. Histopathologic findings were most dramatic with the microfibrillar collagen powder group. Microscopic evaluation of microfibrillar collagen powder revealed dense scars with inflammation. At 4 weeks, dense scarring extended into the fat with adhesions and infiltration of lymphocytes. Foreign body reactive cells and palisading histiocytes with granuloma formation were also present. These findings have been confirmed by other investigators [ 7,21,22]. On a practical basis, oxidized cellulose was no more effective in producing complete hemostasis than surgical gauze alone despite prior reports in which controls were not employed [ 5,141. The other coilagen groups, microiibrillar collagen powder and positively charged modified collagen, also averaged about the same amount of time for complete hemostasis but were clearly superior to oxidized cellulose and the control group, findings which have been observed by others [4,5,9,14,21-241. However, our findings suggest a trend of the collagen products being superior in controlling bleeding from parenchymal organs, but this is not statistically significant. Fibrin glue was the most effective agent studied. A clear clot was formed almost immediately upon contact with the bleeding surface, and, within five seconds, a fibrin clot formed and the bleeding completely ceased. Furthermore, fibrin glue was the easiest of the agents to apply, although care must be taken to direct the contents of the syringe over only the bleeding surface. Extrinsic pressure is not necessary. We conclude that fibrin glue is the most effective hemostatic agent when compared with oxidized cellulose and collagen materials. Histopathologic examination demonstrated that the microfibrillar collagen powder group had the most obvious and prolonged tissue reaction, which resulted in granuloma formation. A decreased mortality rate from late hemorrhage in the experimental groups compared with the control animals also indicates that hemostatic agents are important in controlling prolonged and/or delayed oozing or bleeding and thereby preventing hematomas, abscesses, and even death. Magical hemostatic agents abound: this is an especially careful study with a well-defined model, which may describe a way out of a tight surgical corner. 238
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REFERENCES 1. Olwin JH, Wahl FJ. The effect of absorbable sponge materials on the activity of thrombin. Surg Gynecol Obstet 1948; 86: 203. 2. Bat&a OA, Erdi NZ, Ferraso CF, et al. Colloidal macromolecular phenomena. II. Novel microcrystals of polymer. J Appl Polymer Sci 1967; 11: 481. 3. Dresdale A, Bowman FO, Ma1 Jr, et al. Preparation of fibrin glue derived from single donor fresh frozen plasma. Ann Thorac Surg 1985; 40: 385-7. 4. Hait MR, Robb CA, Baxter CR, Borgman AR, Tippett LO. Comparative evaluation of Avitene microcrystalline collagen he mostat in experimental animal wounds. Am J Surg 1973; 125: 284-7. 5. Abbott W, Austen WG. The effectiveness and mechanism of collagen-induced topical hemostasis. Surg 1975; 78: 723-9. 6. Dineen, P. Antibacterial activity of oxidized regenerated cellulose. Surg Gynecol Obstet 1976; 142: 481-7. 7. Voorman JHC, Ringers J, Bats GTAM, van der Heide A, Hermans J. Hemostatic agents: brain tissue reaction and effectiveness: a comparative animal study using collagen fleece and oxidized cellulose. Neurosurgery 1987; 20: 702-9. 8. Correll JT, Prentice HR, Wise FC. Biologic investigations of a new absorbable sponge. Surg Gynecol Obstet 1945; 81: 585. 9. Silverstein ME, Chvapil M. Experimental and clinical experiences with collagen fleece as a hemostatic agent. J Trauma 1981; 21: 388-93. 10. Beck E, Duckert F, Ernst M. Der einfluB des librinstabilisierenden faktors (FSF) auf funktion und morphologie von fibroblastin in-vivo. Z Zellforsch 1962; 57’: 327. 11. Dardik H, Altman A, Podalsky S, Dardik I. Evaluation of hemostasis with goId foil. Am J Surg 1968; 116: 419-22. 12. Porter JM, Acinapura AJ, Silver D. The effectiveness of thin metallic films in overcoming hemostasis. J Surg Res 1966,6: 205. 13. Jakob H, Campbell CD, Stemberger A, Wreidt-Lubbe I, Blume1 G, Replogle RL. Combined application of heterologous collagen and fibrin sealant for liver injuries. J Surg Res 1984; 36: 57 l-7. 14. Coln D, Horton J, Ogden ME, Buga LM. Evaluation of hem* static agents in experimental splenic lacerations. Am J Surg 1983; 145: 256-9. 15. Dresdale A, Rose EA, Jecvanandam V, et al. Preparation of Bbrin glue from single donor fresh frozen plasma. Surgery 1985; 97: 750-5. 16. Ikossi-O’Connor MG, Ambrus JL, Rae U. The role of fibrin adhesive in vascular surgery. J Surg Oncol 1983; 23: 151-2. 17. Kram HB, Reuben BI, Fleming AW, Shoemaker WC. Use of fibrin glue in hepatic trauma. J Trauma 1988; 28: 1195-201. 18. Spotnitz WD, Dalton MS, Baker JW, Nolan SP. Reduction of perioperative hemorrhage by anterior mediistinal spray application of fibrin glue during cardiac operations. Ann Thorac Surg 1987; 44: 529-31. 19. Stuart JD, Kenney JG, Lettieri J, Spotnitz W, Baker J. Application of single-donor fibrin glue to burns. J Bum Care Rehab 1988; 9: 619-22. 20. Rousou JA, EngIeman RM, Breyer RH. Fibrin glue: an effective hemostatic agent for nonsuturable intraoperative bleeding. Ann Thorac Surg 1984; 38: 409-10. 21. Rybock JD, Long DM. Use of microtibrillar collagen as a topical hemostatic agent in brain tissue. J Neurosurg 1977; 46: 501-5. 22. Silverstein ME, Keown K, Gwen JA, Chavapil M. Collagen fibers as a fleece hemostatic agent. J Trauma 1980; 29: 688-94. 23. Chvapil M, Owens JA, DeYoung D. A standardized animal model for evaluating hemostatic effectiveness of various materials. J Trauma 1983; 23: 1042-7. 24. Zoucas, E, Groansson G, Benmark S. Comparative evaluation of local hemostatic agents in experimental liver trauma: a study in the rat. J Surg Res 1984; 37: 145-50.
VOLUME 163 FEBRUARY 1992
Efficacy of Topical Hemostatic in a Rat Kidney Model
Agents
Joseph S. Raccuia, MD, Gregory Simonian, BS, Michael Dardik, BA, David Hallac, Salvatore V. Raccuia, BS, Rosalyn Stahl, MD, Herbert Dardik, MD, FACS, Englewood. New Jersey
The efficacies of four topical hemostatic agents were compared in a rat model employing a standardkd renal injury. The materials used to effect hemostasis were oxidized cellulose, microfibrillar collagen powder, positively charged modified collagen, and single donor heterologous fibrin glue. Animals that were treated only with surgical gauze served as controls. Hemostasis was achieved by application of one of the topical hemostatic agents plus moderate digital pressure. The time necessary to achieve complete hemostasis was recorded for each animal. Control animals bled profusely and suffered an increased postoperative mortality rate compared with the experimental animals. Microscopic studies demonstrated progressive healing of the injuries with varying degrees of inflammation and scar formation. Fibrin glue was by far the most effective agent in controlling hemostasis. The collagen materials, though effective, required a longer time to control bleeding and did not differ statistically in their activity from one another.
From the Surgical Research Laboratory, Department of Surgery, Englewood Hospital, En&wood, New Jersey. Requests for reprints should be addressed to Herbert Dardik, MD, 375 Engle Street, Englewood, New Jersey 0763 1. Manuscripts submitted April 10, 1990, and accepted in revised form September 13,199O. 234
THE AMERICAN JOURNAL OF SURGERY
he attainment of adequate hemostasis for surgical T procedures is often complicated by the presence of friable tissues and coagulopathy. Attempts to control such bleeding by the placement of additional sutures may in fact further aggravate the situation. An alternative method to control bleeding is the application of topical hemostatic agents. In order to assess the comparative efficacies of the various hemostatic agents currently available, we undertook the following set of experiments. Three commercially available hemostatic materials, as well as heterologous fibrin, were compared in their ability to achieve effective hemostasis upon the cut surface of the rat kidney. MATERIALS AND METHODS Experimental preparation and protocol: SpragueDawley rats weighing between 400 and 500 g were used to assess the efficacy of the intraoperative application of topical hemostatic agents. Anesthesia was induced with 50 mg intramuscular ketamine hydrochloride and 1.25 mg of midazolam hydrochloride. In randomly chosen animals, an incision was made in the left neck over the carotid pulse. The sternocleidomastoid muscle and vagus nerve were dissected, and a 20-gauge angiocatheter (Terumo Medical Corp., Elkton, MD) inserted into the carotid artery. The catheter was attached to a blood pressure transducer, and blood pressure was monitored throughout the procedure. The peritoneum was entered, and a segment of each kidney representing approximately 10% of renal volume was excised with a standardized premeasured template, leaving an actively bleeding area measuring approximately 1 by 5 cm. Profuse bleeding was thereby produced, and a topical hemostatic agent was then applied with gentle pressure. Fibrin glue was applied with a dual syringe injector, and no pressure was applied. The time required to achieve total hemostasis was recorded, and the results compared for statistical significance by the Student’s t-test and the chi-square test. The control of bleeding was evaluated by releasing the pressure on the hemostatic agent at intervals of 30 seconds. This was not required in the case of fibrin glue. Compression of renal hilar vessels to augment hemostasis was not employed in this study. The kidneys were then returned to the peritoneal cavity. Specimens were removed every 24 hours for 4 weeks after the application of the hemostatic agents. Microscopic examination was performed after formalin fixation and staining with hematoxylin and eosin. Hemostatic agents: The following materials were used in this study: (1) oxidized cellulose (Surgicel, Johnson and Johnson Inc., New Brunswick, NJ), (2) microtibrillar collagen powder (Avitene, Medical Chemical Products, Inc., Woburn, MA), (3) positively charged modified collagen (Superstat, Edward Week Inc., Prince-
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ton, NJ), and (4) single donor heterologous fibrin glue. Oxidized cellulose, microfibrillar collagen powder, positively charged modified collagen, and fibrin glue were applied directly to the wound sites. Oxidized cellulose is formed by passing surgical gauze over fuming nitric acid and thus oxiding the cellulose and creating cellulosic acid [ 1). Microfibrillar collagen powder is a specifically crosslinked collagen prepared from bovine dermis [2]. Positively charged collagen powder is a modified collagen sponge (no cross-linkage), treated to provide a net positive charge. Fibrin glue was made from single donor fresh-frozen plasma with modification of the method described by Dresdale et al [3]. A unit of fresh-frozen plasma from a single donor, screened for hepatitis B, syphilis, and human immunodeficiency virus, was obtained from the blood bank and apportioned into SO-mL polypropylene centrifuge tubes. The tubes are kept in a freezer at -8OOC for at least 12 hours. The fibrinogen is prepared for use by thawing the tubes for at least 6 hours at 4OC, followed by centrifugation at 1,500g for 15 minutes. The supematant is decanted, leaving a yellowish precipitate of fibrinogen (cryoprecipitate). The precipitate is resuspended in a small amount of supematant. This concentrated fibrinogen suspension is aspirated into a syringe with a large bore needle. Fibrin sealant is formed by mixing the fibrinogen suspension with thrombin, which in turn is prepared by mixing 500 IU/mL with 5 mL sterile saline. The two solutions (fibrinogen and thrombin), in separate syringes, are applied simultaneously to the wound with a dual syringe holder. Upon application, the components form a clear viscous solution that adheres firmly to the wound surface. Application of hemostatic agents Fifty rats were divided into five groups of ten animals each. In the control group, only digital pressure was applied with a standard 4 by 4-inch sterile gauze sponge. In the oxidized cellulose group, microfibrillar collagen powder group, and positively charged modified collagen group, direct pressure was also applied with gauze but with the hemostatic material directly on the bleeding surface of the kidney. THE AMERICAN
The wounds were examined at intervals of 30 seconds until bleeding ceased. In the fibrin glue group, fibrinogen and thrombin were applied simultaneously to the bleeding kidney surface in a drop by drop fashion until hemostasis was achieved. Digital pressure was not applied in this group. RESULTS
Hemostasis was achieved in all animals prior to closure of the peritoneum. Seven of the animals in the control group died within 48 hours of surgery. Four of these animals died from delayed hemorrhage evidenced at autopsy by gross hemoperitoneum, and the other three died of complications of anesthesia. No animals died in the fibrin glue group, 2 in the positively charged modified collagen group, 3 in the oxidized cellulose group, and 3 in the microfibrillar collagen powder group. Deaths in the positively charged modified collagen group, oxidized cellulose group, and microfibrillar collagen powder group were directly attributed to complications from anesthesia, since no hemorrhage was evident at autopsy. Average blood pressure was monitored, and there was no signilicant hypotension during the procedure (134.6 f 4.6 mm Hg) (p <0.5). There was a significant difference in the mortality from late hemorrhage between the control and the experimental animals by Student’s t-test (p
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Figure 2. Photomicrograph at 4 weeks of traumatizedrat kidney followed by appHcatkn of podtlvely chargedmodifiedcollagen.Note mlnimalscatdngonsurfaceandamild intedtid infiltrateof chronic lnflan+ mdoryfAlS.Thl!JhlMOgkplctue exempliflea the appearance at 4 weeksofallvaWnatizedareasfoc lowedby applkatkn of oxklizeclcells kWposltlWchergedmodlRedcollagen,or flbrlnglue. (Hematoxylinand eosln, orlglnal magnification X100, reducedby35%.)
F~S.Photomlcrogmphat 1 week oftraumatizedareaofratkldneyfoL kwed by applkatkn of mkroflbrlllar collagen powder. Note exuberant ~karre8ctkntothe fore@~ mat&al. (liematoxylin and eosln, original magllfkatkn x100, reducedby35%.)
sured in seconds. There was no significant difference by Student’s t-test (p <0.05) between the fibrin glue group and all other groups. Cross findings: Animals surviving after 72 hours were killed at intervals up to 4 weeks. None of the animals exhibited evidence of hematoma formation, recurrent bleeding, or disruption of renal parenchyma. There were also no abscesses or signs of peritonitis. Gross tissue reactions were limited to thin omental adhesions to the injured surfaces. Microscopic findings: Histologic evaluation demonstrated a series of progressive changes from acute infarc tion with varying degrees of hemorrhage at 24 hours to healing with varying degrees of scar formation. By 4 236
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weeks, all agents, except microfibrillar collagen powder, left minimal scarring and complete healing with little evidence of past injury (FIgure 2). Microfibrillar collagen powder elicited the most exuberant inflammatory re sponse with granuloma formation surrounclmg the fibrillary collagen at one week (Hgure 3), which progressed to dense scarring and chronic inflammation with foreign body reaction that was histologically visible at 4 weeks (WWe 4). COMMENTS The need for effective hemostasis with the use of topical hemostatic agents to control bothersome and sometimes lethal bleeding has been established. By the turn of
VOLUME 163 FEBRUARY 1992
COMPARATIVE EFFICACIES OF TOPICAL HEMOSTATIC AGENTS
Flgure 4. Photomicrograph at 4 weaksofbawMkdwaaofratk# iiziE!--&sL= pt3mwu3dvisiMe~matarL al. BuIwndbd W m bodv siant cebanddelnmpopullwarofchronic innMmataycelk.ThlsiBinsmking anWasttoF~2.~xylinand mE@btbn x100. -,orighlel t=ducedby35%.)
the century, it was generally appreciated that gauze was the most effective hemostatic agent, which it did by trap ping blood elements within its fibers and thereby facilitating a chemical response in addition to its physical characteristics. Fibrin patches and foams in varied cross-linkages were also introduced but had unpredictable efficacy. The hemostatic effect of cellulosic acid, first noted by Olwin and Wahl [I], is based on its physical matrix, which collects blood into its interstices, entrapping and concentrating the coagulation factors. The pH changes in the tissues caused by this material are also a factor in procoagulation. Although the fabric is easy to work with, it provides only a moderate degree of hemostasis [4-7). Correll et al [8] introduced a new absorbable sponge in 1945 that consisted of an insoluble, organic polymer de rived from formal&tanned gelatin. This material (Gelfoam, Upjohn Co., Washington, DC) effects coagulation by physically absorbing blood within its interstices, but it is cumbersome to use and crumples easily. Even if this material is presoaked with thrombin, it is difficult to maintain in position and disintegrates under pressure while effecting only a moderate degree of hemostasis [ 93. In 1962, Beck, Duckert, and Ernst [ZO] reported on the in uiuo function and morphologic characteristics of fibrin glue. The use of thin metallic and gold foils was subsequently described for topical hemostasiq however, to date, these methods have never been applied in clinical practice [ 1I ,I21 . Microfibrillar collagen powder was introduced in 1967 [2] and consists of partial acid salt material of specifically cross-linked collagen prepared from bovine dermis. It is water insoluble, biodegradable, nonallergenic, and can be sterilized. Hemostatic action occurs after contact with blood, forming an insoluble gelfibrin mesh that adheres to the wound surface. The material is very difficult to handle due to its high electrostatic THE AMERICAN
charge in the powder form. Fibrillar forms of collagen can also be fabricated into fleece or felt, which are easier to handle than other forms [5,7,9,13]. Positively charged modified collagen differs from other collagen products in that the material is water soluble and is not cross-linked. Hemostasis is achieved by its special charge characteristics, interacting with blood to convert fibrinogen to fibrin. As various collagen agents continue to gain favor for hemostasis, the use of commercial fibrin glue has also begun to be appreciated, although problems pertaining to the use of pooled donor plasma persist [Z&20]. Preparation of fibrin glue from singledonor autologous and homologous whole blood or freshfrozen plasma is cumbersome and requires anticipation of the need for it prior to surgery. This method requires 1 or 2 days of preparation for a small finite volume of glue, so that autologous glue may not be available for emergency cases. Cryoprecipitate as a source for fibrin glue has also been described, but it is expensive and variable in the amount of end-product. Comparison of autologous fibrin glue with the lyophilized, commercial European product (Tissucol, Immuno AG, Vienna, Austria) indicates that the tear coefficient in the latter is higher [Z5], but it has not been adequately determined if this product has an increased risk for serologically transmitted infections. The risk of transmission of hepatitis B and AIDS by the use of nonautologous blood products is of paramount concern, and the use of sensitive screening tests has significantly reduced the risk of transferring these and other serologically transmitted illnesses. The use of fibrin glue derived from homologous sources must be weighed against the risk of transfusing multiple units of blood products in the event that hemorrhage is significant. In the future, as blood screening tests become more sensitive, the possibility for transmitting these diseases from homologous donors might be negligible.
JOURNAL
OF SURGERY
VOLUME 163
FEBRUARY
1992
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Our study evaluates the effectiveness of the most pop ular topical hemostatic agents now currently available. In this experiment, the injured kidney surface bled profusely in the control group and was associated with a higher mortality rate from delayed hemorrhage when compared with the other groups. On the other hand, although time for achieving hemostasis was basically unchanged from the control time in the oxidized cellulose, microlibrillar collagen powder, and positively charged modified collagen groups, the decreased postoperative mortality in these groups might reflect improved long-term control of bleeding. This is further supported by the smaller amount of hematoma found in the experimental group compared with the amount found in the control group. The most effective agent was clearly fibrin glue in that bleeding was controlled almost immediately. Histopathologic findings were most dramatic with the microfibrillar collagen powder group. Microscopic evaluation of microfibrillar collagen powder revealed dense scars with inflammation. At 4 weeks, dense scarring extended into the fat with adhesions and infiltration of lymphocytes. Foreign body reactive cells and palisading histiocytes with granuloma formation were also present. These findings have been confirmed by other investigators [ 7,21,22]. On a practical basis, oxidized cellulose was no more effective in producing complete hemostasis than surgical gauze alone despite prior reports in which controls were not employed [ 5,141. The other coilagen groups, microiibrillar collagen powder and positively charged modified collagen, also averaged about the same amount of time for complete hemostasis but were clearly superior to oxidized cellulose and the control group, findings which have been observed by others [4,5,9,14,21-241. However, our findings suggest a trend of the collagen products being superior in controlling bleeding from parenchymal organs, but this is not statistically significant. Fibrin glue was the most effective agent studied. A clear clot was formed almost immediately upon contact with the bleeding surface, and, within five seconds, a fibrin clot formed and the bleeding completely ceased. Furthermore, fibrin glue was the easiest of the agents to apply, although care must be taken to direct the contents of the syringe over only the bleeding surface. Extrinsic pressure is not necessary. We conclude that fibrin glue is the most effective hemostatic agent when compared with oxidized cellulose and collagen materials. Histopathologic examination demonstrated that the microfibrillar collagen powder group had the most obvious and prolonged tissue reaction, which resulted in granuloma formation. A decreased mortality rate from late hemorrhage in the experimental groups compared with the control animals also indicates that hemostatic agents are important in controlling prolonged and/or delayed oozing or bleeding and thereby preventing hematomas, abscesses, and even death. Magical hemostatic agents abound: this is an especially careful study with a well-defined model, which may describe a way out of a tight surgical corner. 238
THE AMERICAN JOURNAL OF SURGERY
REFERENCES 1. Olwin JH, Wahl FJ. The effect of absorbable sponge materials on the activity of thrombin. Surg Gynecol Obstet 1948; 86: 203. 2. Bat&a OA, Erdi NZ, Ferraso CF, et al. Colloidal macromolecular phenomena. II. Novel microcrystals of polymer. J Appl Polymer Sci 1967; 11: 481. 3. Dresdale A, Bowman FO, Ma1 Jr, et al. Preparation of fibrin glue derived from single donor fresh frozen plasma. Ann Thorac Surg 1985; 40: 385-7. 4. Hait MR, Robb CA, Baxter CR, Borgman AR, Tippett LO. Comparative evaluation of Avitene microcrystalline collagen he mostat in experimental animal wounds. Am J Surg 1973; 125: 284-7. 5. Abbott W, Austen WG. The effectiveness and mechanism of collagen-induced topical hemostasis. Surg 1975; 78: 723-9. 6. Dineen, P. Antibacterial activity of oxidized regenerated cellulose. Surg Gynecol Obstet 1976; 142: 481-7. 7. Voorman JHC, Ringers J, Bats GTAM, van der Heide A, Hermans J. Hemostatic agents: brain tissue reaction and effectiveness: a comparative animal study using collagen fleece and oxidized cellulose. Neurosurgery 1987; 20: 702-9. 8. Correll JT, Prentice HR, Wise FC. Biologic investigations of a new absorbable sponge. Surg Gynecol Obstet 1945; 81: 585. 9. Silverstein ME, Chvapil M. Experimental and clinical experiences with collagen fleece as a hemostatic agent. J Trauma 1981; 21: 388-93. 10. Beck E, Duckert F, Ernst M. Der einfluB des librinstabilisierenden faktors (FSF) auf funktion und morphologie von fibroblastin in-vivo. Z Zellforsch 1962; 57’: 327. 11. Dardik H, Altman A, Podalsky S, Dardik I. Evaluation of hemostasis with goId foil. Am J Surg 1968; 116: 419-22. 12. Porter JM, Acinapura AJ, Silver D. The effectiveness of thin metallic films in overcoming hemostasis. J Surg Res 1966,6: 205. 13. Jakob H, Campbell CD, Stemberger A, Wreidt-Lubbe I, Blume1 G, Replogle RL. Combined application of heterologous collagen and fibrin sealant for liver injuries. J Surg Res 1984; 36: 57 l-7. 14. Coln D, Horton J, Ogden ME, Buga LM. Evaluation of hem* static agents in experimental splenic lacerations. Am J Surg 1983; 145: 256-9. 15. Dresdale A, Rose EA, Jecvanandam V, et al. Preparation of Bbrin glue from single donor fresh frozen plasma. Surgery 1985; 97: 750-5. 16. Ikossi-O’Connor MG, Ambrus JL, Rae U. The role of fibrin adhesive in vascular surgery. J Surg Oncol 1983; 23: 151-2. 17. Kram HB, Reuben BI, Fleming AW, Shoemaker WC. Use of fibrin glue in hepatic trauma. J Trauma 1988; 28: 1195-201. 18. Spotnitz WD, Dalton MS, Baker JW, Nolan SP. Reduction of perioperative hemorrhage by anterior mediistinal spray application of fibrin glue during cardiac operations. Ann Thorac Surg 1987; 44: 529-31. 19. Stuart JD, Kenney JG, Lettieri J, Spotnitz W, Baker J. Application of single-donor fibrin glue to burns. J Bum Care Rehab 1988; 9: 619-22. 20. Rousou JA, EngIeman RM, Breyer RH. Fibrin glue: an effective hemostatic agent for nonsuturable intraoperative bleeding. Ann Thorac Surg 1984; 38: 409-10. 21. Rybock JD, Long DM. Use of microtibrillar collagen as a topical hemostatic agent in brain tissue. J Neurosurg 1977; 46: 501-5. 22. Silverstein ME, Keown K, Gwen JA, Chavapil M. Collagen fibers as a fleece hemostatic agent. J Trauma 1980; 29: 688-94. 23. Chvapil M, Owens JA, DeYoung D. A standardized animal model for evaluating hemostatic effectiveness of various materials. J Trauma 1983; 23: 1042-7. 24. Zoucas, E, Groansson G, Benmark S. Comparative evaluation of local hemostatic agents in experimental liver trauma: a study in the rat. J Surg Res 1984; 37: 145-50.
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