Fibrin Glue
M. B r e n n a n S U M M A R Y. Fibrin glue is a topical biological adhesive, the effect of which imitates the final stages of coagulation. The glue consists of a solution of concentrated human fibrinogen which is activated by the addition of bovine thrombin and calcium chloride. The resultant clot aids haemostasis and tissue sealing and is completely absorbed during wound healing without foreign body reaction or extensive fibrosis. The fibrinogen component of fibrin glue can be produced from fresh frozen plasma obtained from single unit donations thereby reducing the risks of transfusion transmitted infections encountered by exposure to pools from large numbers of donors. Methods involving precipitation of fibrinogen by cryoprecipitation, polyethylene glycol or ammonium sulphate have been described and evaluated. The risk of transmission of infection can be further reduced by using plasma from 'accredited donors' who are plasma donors regularly tested for ALT and markers of viral infection or by use of fibrinogen prepared in advance of surgery from autologous blood. The second component, a mixture of thrombin and CaC! 2, is quantitatively and qualitatively well defined and commercially available (Armour Pharmaceutical Co., Thrombinar (bovine thrombin)). Thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. In the UK a commercial heat treated fibrin glue prepared from pooled plasma is available on a doctor[named patient basis (Tisseel, Immuno, Vienna). The haemostatic and adhesive properties of fibrin glue can be employed in virtually every surgical speciality. 1 The usefulness of the glue is particularly well documented in the fields of cardiovascular surgery, ENT and neurosurgery. Although widely used, fibrin glue is not a haemostatic panacea and its effectiveness relies on careful surgical technique.
The action of fibrin glue resembles the last phase of blood clotting, i.e., the conversion of fibrinogen to fibrin. The glue has two components which are drawn up in separate syringes. The first consists predominantly of fibrinogen with factor XIII and other plasma proteins i.e. fibronectin and plasminogen. The second is a combination of bovine thrombin and calcium chloride. When the two solutions are mixed, thrombin converts fibrinogen to fibrin monomers and M. Brennan,MRCP, MRCPath, Research Fellow, North London Blood TransfusionCentre, ColindaleAvenue, London NW9 5BG, UK. Blood Revtews (1991) 5, 240-244
© 1991 Longman Group UK Ltd
the mixture gels due to hydrogen bond formation. Simultaneous activation of factor XIII by thrombin (in the presence of ionized calcium) stabilises cross linkage of fibrin monomers within 3-5 min thereby increasing the tensile strength of the clot. The rate of fibrin clot formation depends on the concentration of thrombin activating solution. A thrombin concentration of 4 iu/ml produces clot formation in 1 min and is appropriate where the surfaces to be glued require subsequent adjustment e.g. skin grafts, whereas a concentration of 500 iu/ml produces fibrin sealing within a few seconds and is indicated where
BLOOD REVIEWS 241 haemostasis is of primary importance. Solidification of the glue can be visualised as its colour becomes milky white when it begins to gel. The adhesive strength of fibrin glue is directly proportional to the concentration of fibrinogen available, 2 reaching optimal strength in 3-5 min. Apart from adhesive and haemostatic properties, the glue has been found to enhance wound healing possibly due to the presence of fibronectin. 1 Wound healing is initiated when fibroblasts from the surrounding tissue migrate into the clot which serves as a matrix. Collagen synthesis by fibroblasts marks the beginning of connective tissue formation. As wound healing progresses, gradual resorption of the sealant is required and is determined by the amount of glue applied, the fibrinolytic activity in the wound area and phagocytosis by macrophages and granulocytes. Tissue plasminogen activators convert plasminogen to plasmin which lyses crosslinked fibrin into soluble fibrin degradation products. An antifibrinolytic agent, such as aprotinin, a naturally occuring protease inhibitor, can be added to either component of fibrin glue to delay clot lysis in areas with high local fibrinolytic activity, e.g. lung, prostate, uterus and highly vascularised tissues.
Historical Review
In World War I, fibrin patches were used to control bleeding from parenchymatous organs. The combination of autologous fibrinogen and thrombin solution was first used in the 1940's to fix human skin grafts;3 however, the adhesive effect was poor presumably due to an inadequate fibrinogen concentration. Two developments have led to a revival of the technique. Firstly, the ability to produce highly concentrated fibrinogen and secondly improvement in microsurgical techniques. In the 1970's, animal experiments demonstrated that fibrin glue reduced the number of sutures required to repair anastomoses and fibrin glue was used successfully to seal experimental dural lesions in dogs. In 1974, fibrin glue prepared from autologous cryoprecipitate and a thrombin solution was used for repair of peripheral nerves in humans. These encouraging reports prompted the use of fibrin glue in wound closure, skin grafting and the bony union of osteotomies. Current Uses
Cardiovascular Surgery Cardiovascular surgery is often associated with severe bleeding problems which can be aggravated by heparinisation, platelet dysfunction, hypothermia and adhesions due to previous surgery. Fibrin glue is a useful adjuvant haemostatic agent which is especially useful when low pressure bleeding is not readily controlled by conventional methods; for instance diffuse bleeding from friable tissues,
bleeding needle holes, and oozing suture lines. 4 Fibrin glue is effective in controlling bleeding on the raw surface of the heart at re-operation which may not be controlled by surgical techniques. 5 Since fibrin glue does not require an intact coagulation system and remains active at body temperatures of 26-37°C it is effective in heparinised patients on extracorporeal circulation. No adverse effects have been described although it is a reasonable precaution to avoid fibrinogen or thrombin entering the intravascular space or the suction port of the cardiopulmonary bypass pump. 6 Good results were obtained by Stark and de Leval7 using fibrin glue in operations for congenital heart defects. They recommend its use for presealing Dacron conduits, complex and multiple suture lines, and controlling bleeding near major coronary artery branches.
ENT Surgery In 1979 Staindl described the use of fibrin glue in ENT surgery) The amount of adhesive required ranges from very small amounts (microdrops) that will fit on the head of the stapes to large amounts for sealing dural defects. Fibrin glue is valuable in middle ear reconstructive surgery enabling accurate ossiculoplasty by fixing small pieces of cartilage, bone or prosthetic implants. Improved closure of the airbone gap has led to better post operative results in sites where access is difficult. Facial nerve grafting, closure of dural leaks with fascial grafts, nasal septal surgery and treatment of post tonsillectomy haemorrhage, even in patients with known bleeding tendencies, are all facilitated by fibrin glue.
Neurosurgery Biological glue has been applied to anchor dural patches and to prevent CSF leakage through dural defects, for instance, following removal of pituitary tumors via the trans-ethmoidal route, thereby reducing the risk of meningitis (personal communication, Mr David Thomas). Further uses include peripheral nerve repair following trauma or tumour excision. However, correct nerve fibre alignment and the amount of glue applied influence the outcome; too much glue will inhibit healing. 9
Plastic Surgery Split and full thickness skin grafts can be fixed with fibrin glue as can the donor areas? ° Graft fixation without sutures reduces the duration of surgery which is advantageous to patients especially those with an increased operative risk. Other advantages of the technique are that local haemostasis prevents haemorrhage underneath the transplant and immediate immobilisation of the graft at the recipient site aids its early vascularisation and wound healing.
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FIBRINGLUE
General Surgery Using a combination of collagen fleece soaked with fibrin glue, bleeding from parenchymatous organs following accidental trauma or resection can be controlled, ix In one study biological glue achieved haemostasis in 16 splenic injuries in 14 patients, lz Fibrin glue can seal liver biopsy needle tracks in patients at high risk of bleeding 13 and achieve local haemostasis in nephrostomy tracts following percutaneous nephrolithotomy.
Thoracic Surgery Initial optimism about the success of fibrin glue in management of recurrent spontaneous pneumothorax was unfounded with failure of treatment attributed to lack of direct vision of the target site. 14 It was also unsuccessful in reducing pulmonary air leaks after lobectomy. 25 The use of fibrin glue has been favourably described in other surgical specialties: in ophthalmology during repair of retinal tears and treatment of corneal perforations, 16 orthopaedic surgery for repair of chondral and osteochondral injuries, gynaecological surgery to control intraoperative haemorrhage and in dental surgery.
Methods of Application Fibrin glue can be applied in different ways depending on the specific indication. Sequential application of the components is not recommended as poor mixing reduces the tensile strength of the glue. This can be overcome by premixing (suitable for low thrombin concentration only) or by using a double syringe applicator, e.g. the Duploject (Figure). This system can be operated singlehandedly and consists of a clip for two syringes enabling a single plunger to dispense equal volumes of the solutions into a common joining piece to which a single application needle is attached. Good mixing of constituents occurs in the delivery needle which can be changed if the application is interrupted. A microdrop delivery system has been described iv which is suitable for middle ear microsurgery. Spray applications are useful for covering
Figure Duploject system for application of fibrin glue.
large surfaces: a multi-channel catheter can be fixed to a conventional pressurised gas source. The two components are injected simultaneously into separate, additional channels and are dispersed by the continuous gas jet at the tip of the catheter producing a thin film of glue at the wound surface. Fibrin glue can also be applied by a carrier such as collagen fleece, dura or grafts. Bonding power of biological adhesives is affected by the dryness of the tissue surface; the drier the surface, the greater the bonding, which seems to increase when both components are kept at 37oc. 18 The possibility of combining antibiotics with fibrin glue is being explored. Drug release occurs by simple diffusion across a concentration gradient. In vivo studies have demonstrated that adequate local drug levels persist for 3 days, which is longer than when antibiotics are injected directly into a body cavity but shorter than when they are mixed with cement.
Methods for Preparing Fibrinogen A variety of methods have been described for the preparation of fibrinogen from human plasma 19 including normal centrifugation, cryoprecipitation and precipitation using various agents, e.g. ether, ethanol, glycine, polyethylene glycol (PEG) or ammonium sulphate. Some techniques are time consuming and complex requiring cold processing and may produce degradation or contamination. For elective surgical procedures it is possible to make fibrin glue from autologous plasma avoiding the risk of transfusion transmitted diseases associated with pooled plasma. The risk of disease transmission can be minimised by using plasma from 'accredited' donors who have donated plasma at least 12 times in the past year and who have had blood samples tested at intervals of not greater than 3 weeks. All samples should be negative for hepatitis B virus markers: surface antigen, surface antibody and core antibody and negative for antibodies to human immunodeficency viruses (HIV) 1 and 2. Accredited donors are also tested for HIV antigen at the end of the accreditation period. All samples during and after accreditation should have normal alanine aminotransferase and ~-glutamyl transpeptidase activity. Methods suitable for producing fibrinogen from a small volume of single donor plasma or autologous blood are given in Appendix 1. Procedures are carried out under sterile conditions and fibrinogen produced may be stored frozen for up to 1 year at -80°C without adversely effecting the adhesive properties of the preparation. Cryoprecipitation is a simple, economical and reproducible procedure which has the advantage that it can be performed in a closed system but the disadvantages of requiring freezer capacity and of taking several hours to perform. Cryoprecipitation produces a fibr!nogen concentrate of approximately 21 g/l, a 10-fold increase compared with
BLOOD REVIEWS
plasma. P E G precipitation is performed at room temperature and yields a fibrinogen concentrate o f approximately 60 g/1. Varying the molecular weight of P E G (200-8000) selectively precipitates various plasma proteins. Increasing the amount of P E G from 10 15 % w/v increases the concentration of fibrinogen but also decreases the purity of the final product in which a decreased percentage of the precipitate is made up of fibrinogen. 23 Ammonium sulphate precipitation is a rapid and simple method performed at room temperature which produces fibrinogen of similar concentration to that produced by P E G precipitation. Higher concentrations of ammonium sulphate produce a less pure product.
Commercially Available Fibrin Glue Products Tisseel (Immuno, Vienna) is a 'lyophylised Sealer Protein Concentrate', which undergoes a viral inactivation process of heat treatment at 60°C for 10 h under conditions of increased pressure and moisture content. This product consists of two components: one solution containing fibrinogen, Factor XIII and aprotinin, and the second containing bovine thrombin and CaC12. Fibrinogen and Factor XIII are derived from pooled human plasma by eryoprecipitation. All plasma units are obtained from licenced plasmapheresis centres and all units used are non reactive in tests for HBsAg and HIV antibodies and have ALT values determined. Reconstitution of fibrinogen takes about 15 min and requires a constant temperature of 37°C. A comparison of the adhesive strengths of a commercial preparation (Beriplast, Behringwerke A.G., West Germany) with two autologous glues prepared by ammonium sulphate precipitation and by P E G precipitation indicated that the adhesive strength of fibrin glue is directly proportional to the concentration of fibrinogen. 2 All three preparations were serviceable, although the highest concentration of fibrinogen (as measured by clot weight), was achieved with the commercial product. A similar study found that fibrinogen precipitation by ammonium sulphate yielded a higher concentration of fibrinogen than cryoprecipitation or precipitation by P E G and hence had the greatest bonding strength. The bonding power of autologous glue, made by ammonium sulphate precipitation of fibrinogen, was comparable to that of a commercially manufactured adhesive.
Conclusion Fibrin glue has been shown to be an effective topical haemostatic agent in numerous surgical procedures. Advantages of a biological glue include excellent tissue tolerance, improved healing and complete reabsorption of the material; in contrast cyanoacrylate glues can cause inflammatory reactions leading to
243
tissue necrosis and poor wound healing. The use of fibrin glue can be effective when conventional techniques have failed 6 or access is difficult. Its use may reduce tissue ischaemia caused by sutures placed too close together and may avoid sutures cutting into parenchymatous organs. Disadvantages of fibrin glue are that its use is limited to low pressure bleeding and that it will fail if used inappropriately. There is a small risk of anaphylactic reaction because the thrombin component is bovine in origin. In theory there is a risk of dense adhesions forming after use of fibrin glue but in practice this does not appear to happen. 24 A questionnaire distributed in 1990 to all Consultant Surgeons in the North West Thames Region (UK) revealed that 10% of surgeons are currently using fibrin glue although only one surgeon works with autologous glue. The bulk of usage is in three specialties: E N T surgery, neurosurgery and orthopaedics. 25% of surgeons stated that they would use a biological glue if available and those already using commercial glue would use more were they not limited by availability and cost. Although there are concerns about the transmission of viral diseases by biological adhesives these can be minimised by screening donors and by heat treatment of commercial products. The risk of transfusion transmitted disease incurred by using fibrin glue is perhaps lower than the risk of multiple transfusions required in the event of unremitting haemorrhage. Current evidence demonstrates a favourable risk to benefit ratio of using fibrin glue.
Acknowledgements I wish to thank Mr R. Nichotson, Director Immuno Ltd for kind permission to reproduce the photograph and assistance in obtaining referencesand also Mrs V. Sproule for excellentsecretarial services.
Appendix 1 Methods for Preparation of Fibrinogen A. Cryoprecipitation 2° Method The procedure is performed in a laminar flow hood under sterile conditions. A unit of fresh frozen plasma (FFP) is divided into 40 ml aliquots in 50 ml polypropylene centrifuge tubes which are placed in the freezer at -80°C for 12 h and subsequently thawed at 4°C for several hours. The tubes containing thawed fibrinogen are centrifuged at 1000 g for 15 rain and the supernatant is decanted leaving a precipitate of fibrinogen which is resuspended in the small amount of residual supernatant. Since the final concentration of fibrinogen is partly determined by the volume of residual supernatant used to resuspend the pellet, care should be taken to remove as much liquid as possible. The fibrinogen suspension is aspirated with a wide bore needle into a syringe. Each 40 ml of FFP yield approximately 2 ml of concentrated fibrinogen. The measured fibrinogen is 21 g/1 in this concentrate compared with 2.6 g/1 in unprocessed FFP. Factor XIII is also present. Fibrinogen remains active for up to 1 year at - 80°C. It retains activity for 4 h at room temperature.
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FIBRIN GLUE
B. Precipitation Using Polyethylene Glycol (PEG) 21 Method The isolation procedure is performed at r o o m temperature under sterile conditions. 45 ml of autologous blood is drawn into a syringe containing 5 ml of 3.8% sodium citrate (pH 7.4). The blood is centrifuged at 2000 rpm for 15 min and the supernatant plasma is saved. To absorb out prothrombin, BaSO4 (2.25 g) and MgSO4 (0.01235 g) are incubated with 20 ml of plasma for 1 h. The prothrombin free plasma is decanted and sufficient 30% P E G (M.W. 1000) solution (citrate buffer, p H 7.4) is added to make a 10% wt/vol P E G mixture. This mixture is centrifuged at 104g for 20 min to precipitate fibrinogen and factor XIII. The precipitate pellet is resuspended in sodium citrate buffer to a final volume of 1 ml and divided into 0.1 ml aliquots which can be frozen for later use. The preparation contains fibrinogen at a concentration of approximately 50-60 g/1 and sufficient factor X I I I for normal clot polymerisation. The isolation process requires approximately 2 h to perform and the concentrate retains full activity up to 3 weeks when stored at - 2 0 ° C .
C. Precipitation Using Ammonium Sulphate 22 Me~od 18 ml of autologous blood is drawn into a syringe containing 2 ml of sterile 10% sodium citrate. The blood is centrifuged for 10 rain at 3000 rpm. The supernatant plasma ( ~ 11 ml) is put into a sterile vial and 2.0-2.5 ml of sterile saturated, purified a m m o n i u m sulphate is added until the solution becomes opaque. It is centrifuged for 5 rain at 3000 rpm and the supernatant is discarded. A button of fibrinogen is left in the vial to which is added a maximum of 2 ml of CaCI 2 (40 mmol/l). If the fibrinogen remains insoluble, gentle beating will ensure complete dissolution. The preparation of fibrinogen takes less than 30 rain and 2-3 ml are produced from 18 ml of blood.
References 1. Matras H 1985 Fibrin seal: The state of the art Journal of Oral Maxillofacial Surgery 13:605-611 2. Laitakari K, Luotonen J 1989 Autologous and homologous fibrinogen sealants: adhesive strength. Laryngoscope 99: 974-976 3. Tidrick R T, Warner E D 1944 Fibrin fixation of skin transplants. Surgery 15:90-95 4. Koveker G 1982 Application of fibrin glue in cardiovascular surgery. The Thoracic and Cardiovascular Surgeon 30: 228-229
5. Rousou J A, Engelman R M, Breyer R H 1984 Fibrin glue: an effective haemostatic agent for nonsuturable intraoperative bleeding. The Annals of Thoracic Surgery 38: 409-410 6. Lupinetti F M, Alford W C, Glassford D M, Thomas C S 1985 Cryoprecipitate--topical thrombin glue. Journal of Thoracic Cardiovascular Surgery 90:502-505 7. Stark J, de Leval M 1984 Experience with fibrin seal (Tisseel) in operations for congenital heart defects. Annals of Thoracic Surgery 38:411-413 8. Staindl O 1979 Tissue adhesion with highly concentrated human fibrinogen in otolaryngology. Annals of Otology 88. 413-418 9. Narkas A 1988 The use of fibrin glue in repair of peripheral nerves. Orthopaedic Clinics of North America 19:187-199 10. Staindl O 1982 The fibrin-adhesive system in plastic surgery of head and neck. Journal of Head and Neck Pathology 3: 78-85 11. Chung S W, Nagy A G 1988 Preservation of the spleen using human fibrin seal. Canadian Journal of Surgery 31:195-197 12. Kram H B, del Junco T, Clark S R, Ocampo H P, Shoemaker W C 1990 Techniques of splenic preservation using fibrin glue. Journal of Trauma 30:97-101 13. Chisholm R A, Jones S N, Lees W R 1989 Fibrin sealant as a plug for the post liver biopsy. Clinical Radiology 6: 627-628 14. Jessen C, Sharma P 1985 Use of fibrin glue in thoracic surgery. The Annals of Thoracxc Surgery 39:521-524 15. Fleisher A G, Evans K G, Nelems B, Finley R J 1990 Effects of routine fibrin glue use on the duration of air leaks after lobectomy. Annals of Thoracic Surgery 49:133-134 16. La goutte F M, Fauthier L, Comte P R M 1989 A fibrin Sealant for perforated and preperforated corneal ulcers. British Journal of Ophthalmology 73:757-761 17. Arenberg I K, Altshuler J H 1989 Autologons fibrin glue (AFG) and sealant: standard and microdrop delivery systems. Otolaryngology--Head and Neck Surgery 101: 709-712 18. Siedentop K H, Harris D M, Sanchez B 1986 Extended experimental and preliminary surgical findings with autologous fibrin tissue adhesive made from the patient's own blood. Larynogoscope 96:1062-1064 19. Gibble J W, Ness P M 1990 Fibrin glue: the perfect operative sealant? Transfusion 30:741-747 20. Dresdale A, Rose E, Jeevanandam V, Reemtsma K, Bowman O, Malta J R 1985 Preparation of fibrin glue from single-donor fresh frozen plasma. Surgery 97" 750-755 21. Epstein G H, Weisman R A, Zwillenberg S, Schrelber A D 1986 A new autologous fibrinogen based adhesive for otologic surgery Annals of Otorhinolaryngology 95:40-45 22. Durham L H, Willat D J, Yung M W, Jones I, Stevens P A, Ramadan M F 1987 A method of preparation of fibrin glue. The Journals of Laryngology and Otology 101: 1182-I 186 23. Weisman R A, Torsiglieri A J, Schreiber A D, Epstein G H 1987 Biochemical characterisation of autologous fibrinogen adhesive. Laryngoscope 97:1186-I 190 24. Jakob H, Cambell C D, Qiu Z K, Pick R, Replogle R L 1984 Evaluation of fibrin sealing for cardiovascular surgery. Circulation 70:138-146
M. B r e n n a n S U M M A R Y. Fibrin glue is a topical biological adhesive, the effect of which imitates the final stages of coagulation. The glue consists of a solution of concentrated human fibrinogen which is activated by the addition of bovine thrombin and calcium chloride. The resultant clot aids haemostasis and tissue sealing and is completely absorbed during wound healing without foreign body reaction or extensive fibrosis. The fibrinogen component of fibrin glue can be produced from fresh frozen plasma obtained from single unit donations thereby reducing the risks of transfusion transmitted infections encountered by exposure to pools from large numbers of donors. Methods involving precipitation of fibrinogen by cryoprecipitation, polyethylene glycol or ammonium sulphate have been described and evaluated. The risk of transmission of infection can be further reduced by using plasma from 'accredited donors' who are plasma donors regularly tested for ALT and markers of viral infection or by use of fibrinogen prepared in advance of surgery from autologous blood. The second component, a mixture of thrombin and CaC! 2, is quantitatively and qualitatively well defined and commercially available (Armour Pharmaceutical Co., Thrombinar (bovine thrombin)). Thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. In the UK a commercial heat treated fibrin glue prepared from pooled plasma is available on a doctor[named patient basis (Tisseel, Immuno, Vienna). The haemostatic and adhesive properties of fibrin glue can be employed in virtually every surgical speciality. 1 The usefulness of the glue is particularly well documented in the fields of cardiovascular surgery, ENT and neurosurgery. Although widely used, fibrin glue is not a haemostatic panacea and its effectiveness relies on careful surgical technique.
The action of fibrin glue resembles the last phase of blood clotting, i.e., the conversion of fibrinogen to fibrin. The glue has two components which are drawn up in separate syringes. The first consists predominantly of fibrinogen with factor XIII and other plasma proteins i.e. fibronectin and plasminogen. The second is a combination of bovine thrombin and calcium chloride. When the two solutions are mixed, thrombin converts fibrinogen to fibrin monomers and M. Brennan,MRCP, MRCPath, Research Fellow, North London Blood TransfusionCentre, ColindaleAvenue, London NW9 5BG, UK. Blood Revtews (1991) 5, 240-244
© 1991 Longman Group UK Ltd
the mixture gels due to hydrogen bond formation. Simultaneous activation of factor XIII by thrombin (in the presence of ionized calcium) stabilises cross linkage of fibrin monomers within 3-5 min thereby increasing the tensile strength of the clot. The rate of fibrin clot formation depends on the concentration of thrombin activating solution. A thrombin concentration of 4 iu/ml produces clot formation in 1 min and is appropriate where the surfaces to be glued require subsequent adjustment e.g. skin grafts, whereas a concentration of 500 iu/ml produces fibrin sealing within a few seconds and is indicated where
BLOOD REVIEWS 241 haemostasis is of primary importance. Solidification of the glue can be visualised as its colour becomes milky white when it begins to gel. The adhesive strength of fibrin glue is directly proportional to the concentration of fibrinogen available, 2 reaching optimal strength in 3-5 min. Apart from adhesive and haemostatic properties, the glue has been found to enhance wound healing possibly due to the presence of fibronectin. 1 Wound healing is initiated when fibroblasts from the surrounding tissue migrate into the clot which serves as a matrix. Collagen synthesis by fibroblasts marks the beginning of connective tissue formation. As wound healing progresses, gradual resorption of the sealant is required and is determined by the amount of glue applied, the fibrinolytic activity in the wound area and phagocytosis by macrophages and granulocytes. Tissue plasminogen activators convert plasminogen to plasmin which lyses crosslinked fibrin into soluble fibrin degradation products. An antifibrinolytic agent, such as aprotinin, a naturally occuring protease inhibitor, can be added to either component of fibrin glue to delay clot lysis in areas with high local fibrinolytic activity, e.g. lung, prostate, uterus and highly vascularised tissues.
Historical Review
In World War I, fibrin patches were used to control bleeding from parenchymatous organs. The combination of autologous fibrinogen and thrombin solution was first used in the 1940's to fix human skin grafts;3 however, the adhesive effect was poor presumably due to an inadequate fibrinogen concentration. Two developments have led to a revival of the technique. Firstly, the ability to produce highly concentrated fibrinogen and secondly improvement in microsurgical techniques. In the 1970's, animal experiments demonstrated that fibrin glue reduced the number of sutures required to repair anastomoses and fibrin glue was used successfully to seal experimental dural lesions in dogs. In 1974, fibrin glue prepared from autologous cryoprecipitate and a thrombin solution was used for repair of peripheral nerves in humans. These encouraging reports prompted the use of fibrin glue in wound closure, skin grafting and the bony union of osteotomies. Current Uses
Cardiovascular Surgery Cardiovascular surgery is often associated with severe bleeding problems which can be aggravated by heparinisation, platelet dysfunction, hypothermia and adhesions due to previous surgery. Fibrin glue is a useful adjuvant haemostatic agent which is especially useful when low pressure bleeding is not readily controlled by conventional methods; for instance diffuse bleeding from friable tissues,
bleeding needle holes, and oozing suture lines. 4 Fibrin glue is effective in controlling bleeding on the raw surface of the heart at re-operation which may not be controlled by surgical techniques. 5 Since fibrin glue does not require an intact coagulation system and remains active at body temperatures of 26-37°C it is effective in heparinised patients on extracorporeal circulation. No adverse effects have been described although it is a reasonable precaution to avoid fibrinogen or thrombin entering the intravascular space or the suction port of the cardiopulmonary bypass pump. 6 Good results were obtained by Stark and de Leval7 using fibrin glue in operations for congenital heart defects. They recommend its use for presealing Dacron conduits, complex and multiple suture lines, and controlling bleeding near major coronary artery branches.
ENT Surgery In 1979 Staindl described the use of fibrin glue in ENT surgery) The amount of adhesive required ranges from very small amounts (microdrops) that will fit on the head of the stapes to large amounts for sealing dural defects. Fibrin glue is valuable in middle ear reconstructive surgery enabling accurate ossiculoplasty by fixing small pieces of cartilage, bone or prosthetic implants. Improved closure of the airbone gap has led to better post operative results in sites where access is difficult. Facial nerve grafting, closure of dural leaks with fascial grafts, nasal septal surgery and treatment of post tonsillectomy haemorrhage, even in patients with known bleeding tendencies, are all facilitated by fibrin glue.
Neurosurgery Biological glue has been applied to anchor dural patches and to prevent CSF leakage through dural defects, for instance, following removal of pituitary tumors via the trans-ethmoidal route, thereby reducing the risk of meningitis (personal communication, Mr David Thomas). Further uses include peripheral nerve repair following trauma or tumour excision. However, correct nerve fibre alignment and the amount of glue applied influence the outcome; too much glue will inhibit healing. 9
Plastic Surgery Split and full thickness skin grafts can be fixed with fibrin glue as can the donor areas? ° Graft fixation without sutures reduces the duration of surgery which is advantageous to patients especially those with an increased operative risk. Other advantages of the technique are that local haemostasis prevents haemorrhage underneath the transplant and immediate immobilisation of the graft at the recipient site aids its early vascularisation and wound healing.
242
FIBRINGLUE
General Surgery Using a combination of collagen fleece soaked with fibrin glue, bleeding from parenchymatous organs following accidental trauma or resection can be controlled, ix In one study biological glue achieved haemostasis in 16 splenic injuries in 14 patients, lz Fibrin glue can seal liver biopsy needle tracks in patients at high risk of bleeding 13 and achieve local haemostasis in nephrostomy tracts following percutaneous nephrolithotomy.
Thoracic Surgery Initial optimism about the success of fibrin glue in management of recurrent spontaneous pneumothorax was unfounded with failure of treatment attributed to lack of direct vision of the target site. 14 It was also unsuccessful in reducing pulmonary air leaks after lobectomy. 25 The use of fibrin glue has been favourably described in other surgical specialties: in ophthalmology during repair of retinal tears and treatment of corneal perforations, 16 orthopaedic surgery for repair of chondral and osteochondral injuries, gynaecological surgery to control intraoperative haemorrhage and in dental surgery.
Methods of Application Fibrin glue can be applied in different ways depending on the specific indication. Sequential application of the components is not recommended as poor mixing reduces the tensile strength of the glue. This can be overcome by premixing (suitable for low thrombin concentration only) or by using a double syringe applicator, e.g. the Duploject (Figure). This system can be operated singlehandedly and consists of a clip for two syringes enabling a single plunger to dispense equal volumes of the solutions into a common joining piece to which a single application needle is attached. Good mixing of constituents occurs in the delivery needle which can be changed if the application is interrupted. A microdrop delivery system has been described iv which is suitable for middle ear microsurgery. Spray applications are useful for covering
Figure Duploject system for application of fibrin glue.
large surfaces: a multi-channel catheter can be fixed to a conventional pressurised gas source. The two components are injected simultaneously into separate, additional channels and are dispersed by the continuous gas jet at the tip of the catheter producing a thin film of glue at the wound surface. Fibrin glue can also be applied by a carrier such as collagen fleece, dura or grafts. Bonding power of biological adhesives is affected by the dryness of the tissue surface; the drier the surface, the greater the bonding, which seems to increase when both components are kept at 37oc. 18 The possibility of combining antibiotics with fibrin glue is being explored. Drug release occurs by simple diffusion across a concentration gradient. In vivo studies have demonstrated that adequate local drug levels persist for 3 days, which is longer than when antibiotics are injected directly into a body cavity but shorter than when they are mixed with cement.
Methods for Preparing Fibrinogen A variety of methods have been described for the preparation of fibrinogen from human plasma 19 including normal centrifugation, cryoprecipitation and precipitation using various agents, e.g. ether, ethanol, glycine, polyethylene glycol (PEG) or ammonium sulphate. Some techniques are time consuming and complex requiring cold processing and may produce degradation or contamination. For elective surgical procedures it is possible to make fibrin glue from autologous plasma avoiding the risk of transfusion transmitted diseases associated with pooled plasma. The risk of disease transmission can be minimised by using plasma from 'accredited' donors who have donated plasma at least 12 times in the past year and who have had blood samples tested at intervals of not greater than 3 weeks. All samples should be negative for hepatitis B virus markers: surface antigen, surface antibody and core antibody and negative for antibodies to human immunodeficency viruses (HIV) 1 and 2. Accredited donors are also tested for HIV antigen at the end of the accreditation period. All samples during and after accreditation should have normal alanine aminotransferase and ~-glutamyl transpeptidase activity. Methods suitable for producing fibrinogen from a small volume of single donor plasma or autologous blood are given in Appendix 1. Procedures are carried out under sterile conditions and fibrinogen produced may be stored frozen for up to 1 year at -80°C without adversely effecting the adhesive properties of the preparation. Cryoprecipitation is a simple, economical and reproducible procedure which has the advantage that it can be performed in a closed system but the disadvantages of requiring freezer capacity and of taking several hours to perform. Cryoprecipitation produces a fibr!nogen concentrate of approximately 21 g/l, a 10-fold increase compared with
BLOOD REVIEWS
plasma. P E G precipitation is performed at room temperature and yields a fibrinogen concentrate o f approximately 60 g/1. Varying the molecular weight of P E G (200-8000) selectively precipitates various plasma proteins. Increasing the amount of P E G from 10 15 % w/v increases the concentration of fibrinogen but also decreases the purity of the final product in which a decreased percentage of the precipitate is made up of fibrinogen. 23 Ammonium sulphate precipitation is a rapid and simple method performed at room temperature which produces fibrinogen of similar concentration to that produced by P E G precipitation. Higher concentrations of ammonium sulphate produce a less pure product.
Commercially Available Fibrin Glue Products Tisseel (Immuno, Vienna) is a 'lyophylised Sealer Protein Concentrate', which undergoes a viral inactivation process of heat treatment at 60°C for 10 h under conditions of increased pressure and moisture content. This product consists of two components: one solution containing fibrinogen, Factor XIII and aprotinin, and the second containing bovine thrombin and CaC12. Fibrinogen and Factor XIII are derived from pooled human plasma by eryoprecipitation. All plasma units are obtained from licenced plasmapheresis centres and all units used are non reactive in tests for HBsAg and HIV antibodies and have ALT values determined. Reconstitution of fibrinogen takes about 15 min and requires a constant temperature of 37°C. A comparison of the adhesive strengths of a commercial preparation (Beriplast, Behringwerke A.G., West Germany) with two autologous glues prepared by ammonium sulphate precipitation and by P E G precipitation indicated that the adhesive strength of fibrin glue is directly proportional to the concentration of fibrinogen. 2 All three preparations were serviceable, although the highest concentration of fibrinogen (as measured by clot weight), was achieved with the commercial product. A similar study found that fibrinogen precipitation by ammonium sulphate yielded a higher concentration of fibrinogen than cryoprecipitation or precipitation by P E G and hence had the greatest bonding strength. The bonding power of autologous glue, made by ammonium sulphate precipitation of fibrinogen, was comparable to that of a commercially manufactured adhesive.
Conclusion Fibrin glue has been shown to be an effective topical haemostatic agent in numerous surgical procedures. Advantages of a biological glue include excellent tissue tolerance, improved healing and complete reabsorption of the material; in contrast cyanoacrylate glues can cause inflammatory reactions leading to
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tissue necrosis and poor wound healing. The use of fibrin glue can be effective when conventional techniques have failed 6 or access is difficult. Its use may reduce tissue ischaemia caused by sutures placed too close together and may avoid sutures cutting into parenchymatous organs. Disadvantages of fibrin glue are that its use is limited to low pressure bleeding and that it will fail if used inappropriately. There is a small risk of anaphylactic reaction because the thrombin component is bovine in origin. In theory there is a risk of dense adhesions forming after use of fibrin glue but in practice this does not appear to happen. 24 A questionnaire distributed in 1990 to all Consultant Surgeons in the North West Thames Region (UK) revealed that 10% of surgeons are currently using fibrin glue although only one surgeon works with autologous glue. The bulk of usage is in three specialties: E N T surgery, neurosurgery and orthopaedics. 25% of surgeons stated that they would use a biological glue if available and those already using commercial glue would use more were they not limited by availability and cost. Although there are concerns about the transmission of viral diseases by biological adhesives these can be minimised by screening donors and by heat treatment of commercial products. The risk of transfusion transmitted disease incurred by using fibrin glue is perhaps lower than the risk of multiple transfusions required in the event of unremitting haemorrhage. Current evidence demonstrates a favourable risk to benefit ratio of using fibrin glue.
Acknowledgements I wish to thank Mr R. Nichotson, Director Immuno Ltd for kind permission to reproduce the photograph and assistance in obtaining referencesand also Mrs V. Sproule for excellentsecretarial services.
Appendix 1 Methods for Preparation of Fibrinogen A. Cryoprecipitation 2° Method The procedure is performed in a laminar flow hood under sterile conditions. A unit of fresh frozen plasma (FFP) is divided into 40 ml aliquots in 50 ml polypropylene centrifuge tubes which are placed in the freezer at -80°C for 12 h and subsequently thawed at 4°C for several hours. The tubes containing thawed fibrinogen are centrifuged at 1000 g for 15 rain and the supernatant is decanted leaving a precipitate of fibrinogen which is resuspended in the small amount of residual supernatant. Since the final concentration of fibrinogen is partly determined by the volume of residual supernatant used to resuspend the pellet, care should be taken to remove as much liquid as possible. The fibrinogen suspension is aspirated with a wide bore needle into a syringe. Each 40 ml of FFP yield approximately 2 ml of concentrated fibrinogen. The measured fibrinogen is 21 g/1 in this concentrate compared with 2.6 g/1 in unprocessed FFP. Factor XIII is also present. Fibrinogen remains active for up to 1 year at - 80°C. It retains activity for 4 h at room temperature.
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FIBRIN GLUE
B. Precipitation Using Polyethylene Glycol (PEG) 21 Method The isolation procedure is performed at r o o m temperature under sterile conditions. 45 ml of autologous blood is drawn into a syringe containing 5 ml of 3.8% sodium citrate (pH 7.4). The blood is centrifuged at 2000 rpm for 15 min and the supernatant plasma is saved. To absorb out prothrombin, BaSO4 (2.25 g) and MgSO4 (0.01235 g) are incubated with 20 ml of plasma for 1 h. The prothrombin free plasma is decanted and sufficient 30% P E G (M.W. 1000) solution (citrate buffer, p H 7.4) is added to make a 10% wt/vol P E G mixture. This mixture is centrifuged at 104g for 20 min to precipitate fibrinogen and factor XIII. The precipitate pellet is resuspended in sodium citrate buffer to a final volume of 1 ml and divided into 0.1 ml aliquots which can be frozen for later use. The preparation contains fibrinogen at a concentration of approximately 50-60 g/1 and sufficient factor X I I I for normal clot polymerisation. The isolation process requires approximately 2 h to perform and the concentrate retains full activity up to 3 weeks when stored at - 2 0 ° C .
C. Precipitation Using Ammonium Sulphate 22 Me~od 18 ml of autologous blood is drawn into a syringe containing 2 ml of sterile 10% sodium citrate. The blood is centrifuged for 10 rain at 3000 rpm. The supernatant plasma ( ~ 11 ml) is put into a sterile vial and 2.0-2.5 ml of sterile saturated, purified a m m o n i u m sulphate is added until the solution becomes opaque. It is centrifuged for 5 rain at 3000 rpm and the supernatant is discarded. A button of fibrinogen is left in the vial to which is added a maximum of 2 ml of CaCI 2 (40 mmol/l). If the fibrinogen remains insoluble, gentle beating will ensure complete dissolution. The preparation of fibrinogen takes less than 30 rain and 2-3 ml are produced from 18 ml of blood.
References 1. Matras H 1985 Fibrin seal: The state of the art Journal of Oral Maxillofacial Surgery 13:605-611 2. Laitakari K, Luotonen J 1989 Autologous and homologous fibrinogen sealants: adhesive strength. Laryngoscope 99: 974-976 3. Tidrick R T, Warner E D 1944 Fibrin fixation of skin transplants. Surgery 15:90-95 4. Koveker G 1982 Application of fibrin glue in cardiovascular surgery. The Thoracic and Cardiovascular Surgeon 30: 228-229
5. Rousou J A, Engelman R M, Breyer R H 1984 Fibrin glue: an effective haemostatic agent for nonsuturable intraoperative bleeding. The Annals of Thoracic Surgery 38: 409-410 6. Lupinetti F M, Alford W C, Glassford D M, Thomas C S 1985 Cryoprecipitate--topical thrombin glue. Journal of Thoracic Cardiovascular Surgery 90:502-505 7. Stark J, de Leval M 1984 Experience with fibrin seal (Tisseel) in operations for congenital heart defects. Annals of Thoracic Surgery 38:411-413 8. Staindl O 1979 Tissue adhesion with highly concentrated human fibrinogen in otolaryngology. Annals of Otology 88. 413-418 9. Narkas A 1988 The use of fibrin glue in repair of peripheral nerves. Orthopaedic Clinics of North America 19:187-199 10. Staindl O 1982 The fibrin-adhesive system in plastic surgery of head and neck. Journal of Head and Neck Pathology 3: 78-85 11. Chung S W, Nagy A G 1988 Preservation of the spleen using human fibrin seal. Canadian Journal of Surgery 31:195-197 12. Kram H B, del Junco T, Clark S R, Ocampo H P, Shoemaker W C 1990 Techniques of splenic preservation using fibrin glue. Journal of Trauma 30:97-101 13. Chisholm R A, Jones S N, Lees W R 1989 Fibrin sealant as a plug for the post liver biopsy. Clinical Radiology 6: 627-628 14. Jessen C, Sharma P 1985 Use of fibrin glue in thoracic surgery. The Annals of Thoracxc Surgery 39:521-524 15. Fleisher A G, Evans K G, Nelems B, Finley R J 1990 Effects of routine fibrin glue use on the duration of air leaks after lobectomy. Annals of Thoracic Surgery 49:133-134 16. La goutte F M, Fauthier L, Comte P R M 1989 A fibrin Sealant for perforated and preperforated corneal ulcers. British Journal of Ophthalmology 73:757-761 17. Arenberg I K, Altshuler J H 1989 Autologons fibrin glue (AFG) and sealant: standard and microdrop delivery systems. Otolaryngology--Head and Neck Surgery 101: 709-712 18. Siedentop K H, Harris D M, Sanchez B 1986 Extended experimental and preliminary surgical findings with autologous fibrin tissue adhesive made from the patient's own blood. Larynogoscope 96:1062-1064 19. Gibble J W, Ness P M 1990 Fibrin glue: the perfect operative sealant? Transfusion 30:741-747 20. Dresdale A, Rose E, Jeevanandam V, Reemtsma K, Bowman O, Malta J R 1985 Preparation of fibrin glue from single-donor fresh frozen plasma. Surgery 97" 750-755 21. Epstein G H, Weisman R A, Zwillenberg S, Schrelber A D 1986 A new autologous fibrinogen based adhesive for otologic surgery Annals of Otorhinolaryngology 95:40-45 22. Durham L H, Willat D J, Yung M W, Jones I, Stevens P A, Ramadan M F 1987 A method of preparation of fibrin glue. The Journals of Laryngology and Otology 101: 1182-I 186 23. Weisman R A, Torsiglieri A J, Schreiber A D, Epstein G H 1987 Biochemical characterisation of autologous fibrinogen adhesive. Laryngoscope 97:1186-I 190 24. Jakob H, Cambell C D, Qiu Z K, Pick R, Replogle R L 1984 Evaluation of fibrin sealing for cardiovascular surgery. Circulation 70:138-146
