Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin

OBJECTIVE: To determine prospectively the immunologic response and adverse clinical events in surgical patients exposed to bovine thrombin during cardiac surgical procedures. SUMMARY BACKGROUND DATA: Topical bovine thrombin is used extensively as a hemostatic agent during cardiovascular surgery. Antibodies developing after exposure to bovine thrombin have been anecdotally associated with hemorrhagic complications. METHODS: One hundred fifty-one patients undergoing cardiac surgical procedures were prospectively recruited for this study before surgical exposure with topical bovine thrombin. Immunoassays were used to determine antibody levels against both bovine and human coagulation proteins before and after exposure to bovine thrombin. Alterations in coagulation assay parameters and adverse clinical events were followed in all patients enrolled in the study. RESULTS: Baseline elevated antibody levels to one or more bovine coagulation proteins were observed most frequently in patients with a prior history of a surgical procedure during which bovine thrombin is frequently used. More than 95% of patients developed a seropositive response to bovine coagulation proteins, and 51% manifested elevated antibody levels to the corresponding human coagulation proteins after bovine thrombin exposure. Postoperative coagulation abnormalities were more common in patients with antibodies to human coagulation proteins. Patients with multiple elevated antibody levels to bovine proteins before surgery were more likely to sustain an adverse clinical outcome after surgery. Using a logistic regression model, the adjusted odds ratio for sustaining an adverse event with multiple elevated antibody levels to bovine proteins before surgery was 5.40. CONCLUSIONS: Bovine thrombin preparations are highly immunogenic and appear to be associated with an increased risk for adverse clinical outcomes during subsequent surgical procedures. The clinical safety of these commonly used preparations needs to be reassessed, and reexposure to these agents should likely be avoided.     

A phase 3, randomized, double-blind comparative study of the efficacy and safety of topical recombinant human thrombin and bovine thrombin in surgical hemostasis

BACKGROUND: Plasma-derived bovine thrombin is used as a topical agent to improve surgical hemostasis, but development of antibodies to bovine hemostatic proteins has been associated with increased bleeding and thrombotic complications. Recombinant human thrombin could reduce the risk of these complications. STUDY DESIGN: The objective of this randomized, double-blind, comparative trial was to compare the efficacy, safety, and antigenicity of recombinant human thrombin (rhThrombin) and bovine thrombin as adjuncts to hemostasis in liver resection, spine, peripheral arterial bypass, and dialysis access surgery. Blinded study drug was applied topically to bleeding sites with an absorbable gelatin sponge. The primary efficacy end point was time to hemostasis, summarized as the incidence of hemostasis within 10 minutes. Safety analyses were conducted for 1 month after operation, and the development of antibodies to rhThrombin or to the bovine product was evaluated. RESULTS: Four hundred one patients completed this trial. Hemostasis was achieved at the time-to-hemostasis evaluation site within 10 minutes in 95% of patients in each treatment group. Overall complications, including operative mortality, adverse events, and laboratory abnormalities, were similar between groups. Forty-three (21.5%) patients receiving bovine thrombin developed antibodies to the product; three patients (1.5%; p < 0.0001) in the rhThrombin group developed antibodies to rhThrombin. None of the three patients who developed antirhThrombin antibodies had abnormal coagulation laboratory results or bleeding, thromboembolic, or hypersensitivity events. CONCLUSIONS: Results of this trial suggest that rhThrombin has comparable efficacy, a similar safety profile, and is considerably less immunogenic than bovine thrombin when used for surgical hemostasis.     

Quality of thrombin produced from the patient's own plasma using the TPD, a new Thrombin-processing Device

Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.     

Relative purity of thrombin-based hemostatic agents used in surgery

BACKGROUND: Hemostatic agents used in surgery contain thrombin isolated from either a bovine or human source. The use of thrombin derived from a bovine source has been associated with the development of an abnormal immune response, but a study of the immunoreactivity of the various commercially available thrombin preparations has not been conducted. This study determined the relative purity of commercially available thrombin preparations, if humans have natural antibodies that recognize these preparations, and if elicited antibodies against bovine thrombin cross-react with other bovine or human hemostatic agents. STUDY DESIGN: The purity of hemostatic agents was determined by protein and substrate assays, electrophoresis, and immunoblotting. The natural antigenicity and cross-reactivity of elicited antibodies were measured by ELISA using serum samples from 82 donors from the Red Cross and serum collected from patients exposed to bovine thrombin, respectively. RESULTS: All of the bovine thrombin preparations were found to contain the xenogeneic carbohydrate galactosealpha1-3galactose. The natural antigenicity of the bovine thrombin preparations was greater than that of a human thrombin preparation and similar to that of porcine aortic endothelial cells. Antibodies elicited against bovine thrombin were found to cross-react with other bovine preparations and other xenoantigens but not with human hemostatic preparations. CONCLUSIONS: All patients have antibovine thrombin antibodies, even before exposure to bovine thrombin-containing hemostatic agents. The cross-reactivity of elicited antibovine thrombin antibodies indicates that if a patient has been sensitized to a bovine product, it is likely safer to use a human-derived product in lieu of a bovine product.     

Perioperative topical bovine thrombin exposure is not associated with hemodialysis graft thrombosis

Arteriovenous (AV) graft use as hemodialysis access remains highly prevalent, with a consequent high thrombosis rate. The magnitude of this problem requires that all potentially modifiable risk factors for graft thrombosis be thoroughly investigated. During graft surgery, topical bovine thrombin is often administered, which can lead to the development of antibovine thrombin antibodies and subsequent hemostatic changes. A recent study correlated the presence of plasma antibovine thrombin antibodies with graft thrombosis in hemodialysis patients. We therefore hypothesized that perioperative topical bovine thrombin exposure would lead to the development of antibovine thrombin antibodies and graft thrombosis. We screened 314 hemodialysis patients and identified 73 patients who had 74 grafts placed for whom complete data on perioperative topical bovine thrombin exposure and subsequent graft outcomes was available. Sixty-one of these patients were available for retrospective measurement of antibovine thrombin antibodies, antihuman thrombin antibodies, and the thrombin activation markers thrombin activatible fibrinolysis inhibitor (TAFI) and thrombin precursor protein (TpP). In these grafts, there was no significant association between topical bovine thrombin exposure and primary assisted patency (P = 0.37). The presence of antibovine thrombin antibodies (P = 0.13), antihuman thrombin antibodies (P = 0.10), and increased TAFI (P = 0.18) were associated with trends toward reduced primary assisted patency which did not reach significance. There was a correlation between antibovine thrombin antibodies and antihuman thrombin antibodies (r = 0.30, P < 0.0001) and between TAFI and TpP trade mark (r = 0.30, P < 0.0001), but no significant correlation between topical bovine thrombin exposure and elevated levels of antibovine thrombin antibodies, antihuman thrombin antibodies, TAFI or TpP trade mark. We conclude that perioperative topical bovine thrombin exposure is not associated with subsequent graft thrombosis.     

A comparison of recombinant thrombin to bovine thrombin as a hemostatic ancillary in patients undergoing peripheral arterial bypass and arteriovenous graft procedures

OBJECTIVES: Recombinant thrombin (rThrombin) is a potential hemostatic alternative to bovine and human plasma-derived thrombin. This report examines the clinical results for the vascular surgery subgroup of patients enrolled in a larger double-blind, randomized, multicenter trial, which evaluated the comparative safety and efficacy of rThrombin and bovine plasma-derived thrombin (bThrombin) when used as adjuncts to surgical hemostasis. METHODS: Data from the 164 vascular patients who underwent either a peripheral arterial bypass (PAB) or arteriovenous graft (AV) procedure are included in this analysis. Time to hemostasis at proximal and distal anastomotic sites at 1.5-, 3-, 6-, and 10-minute intervals was determined by procedure (PAB or AV) and overall (PAB + AV). Baseline and day 29 immunologic sera were analyzed. The incidences of postoperative adverse events were compared between treatment groups. Categorical adverse events were evaluated in relation to thrombin product antibody formation. RESULTS: Patients were randomized to either bThrombin (n = 82) or rThrombin (n = 82). Procedures included PAB (n = 88) and AV (n = 76). The bThrombin and rThrombin groups were well matched for demographics and baseline characteristics. A comparable incidence of anastomotic hemostasis was observed in both treatment groups at 10 minutes (94% bThrombin, 91% rThrombin). The incidence of hemostasis was lower at all time points for PAB procedures compared with AV procedures. In the PAB group, a significantly greater proportion of patients receiving rThrombin (55%) achieved hemostasis at 3 minutes compared with bThrombin (39%; P < .05). Adverse event profiles and laboratory findings were similar between groups. No patients in the rThrombin group developed anti-rThrombin product antibodies at day 29, whereas 27% of patients in the bThrombin group developed antibodies to bThrombin product (P < .0001). CONCLUSIONS: rThrombin or bThrombin used as a hemostatic ancillary for anastomotic bleeding was equally effective at 10 minutes; however, rThrombin compared with bThrombin may provide a more rapid onset of hemostasis at 3 minutes in PAB procedures. Adverse events were similar between the two thrombins. In patients undergoing vascular surgery, both treatments were similarly well tolerated, although rThrombin demonstrated a superior immunogenicity profile.     

Antihuman factor V antibodies after use of relatively pure bovine thrombin

Although bovine thrombin is commonly used in the operating room, there is evidence that exposure to bovine thrombin can result in the development of autoimmune antibodies, usually against factor V, which can lead to a profound coagulopathy. It is thought that impurities in bovine thrombin preparations are responsible for the adverse reactions in patients. Here we describe a case in which exposure to a relatively pure bovine thrombin preparation resulted in the development of an antihuman factor V antibody-associated coagulopathy. This report calls into question the safety of even relatively pure bovine thrombin.     

Coagulopathy as a result of factor V inhibitor after exposure to bovine topical thrombin

We describe a case of severe coagulopathy after mesenteric revascularization. Laboratory investigation results revealed the presence of plasma inhibitors of factor V believed to result from exposure to bovine thrombin used for intraoperative hemostasis. Vascular and cardiothoracic surgeons commonly use topical thrombin for surgical hemostasis, and many patients undergo multiple exposure. More patients likely have factor V inhibitors develop than has previously been realized, and this may account for some otherwise unexplained postoperative coagulation disorders. This report may alert surgeons to coagulation disturbances that can result from exposure to bovine thrombin and provide guidelines for diagnosis and management.     

Ultrasound Guided Human Thrombin Injection. A New Modality in the Management of Femoral Artery Pseudo-aneurysms

OBJECTIVES: We report our initial results of a prospective study of duplex ultrasound-guided injection (UGTI ) of thrombin in the management of femoral artery pseudo-aneurysms. We used human thrombin to avoid the increase in the human antibodies directed against fibrinogen, with the use of bovine thrombus, that preclude further utilisation of the bovine fibrin glue during cardio-thoracic surgery. METHODS: From 1999 to 2001, 19 patients, aged 69 (range 52-85) years presented with 21 femoral pseudo-aneurysms were treated. The mean pseudoaneurysm diameter was 30 (15-55) mm. All but two were secondary to cardiac procedures and the common femoral artery was the injured vessel in all instances. Patients were referred within 2-21 days following their iatrogenic injury. RESULTS: Immediate thrombosis of the sac occurred in 19 (90%) of the 21 pseudo-aneurysms. After a second injection, complete occlusion occurred in the remaining two patients. Two patients (CI 95%; 1-19) with three femoral pseudo-aneurysms developed leg pain. Duplex ultrasound follow-up showed two recurrences (9.5% - CI 95%; 1-19) and both were treated by repeat UGTI. There was no conversion to surgical repair. CONCLUSION: This percutaneous minimally invasive technique is safe and effective in the management of iatrogenic pseudo-aneurysms in this high-risk group of patients. Human thrombin has significant advantages over bovine thrombin.     

Update in hematology: heparin-induced thrombocytopenia and bivalirudin

Heparin-induced thrombocytopenia (HIT) is important because it is common, and it significantly increases mortality after cardiac surgery. Although thrombocytopenia after cardiac surgery is common, it predicts serious adverse outcome when it is severe. Despite the high prevalence of heparin/platelet factor 4 antibodies in cardiac surgical patients, they typically do not indicate a higher perioperative risk. Recent evidence suggests, however, that when these antibodies are in the immunoglobulin M class, there is an increased risk of nonthrombotic adverse outcomes after cardiac surgery. According to the guidelines from the American College of Chest Physicians, patients with HIT require parenteral anticoagulation with a direct thrombin inhibitor such as lepirudin, argatroban, or bivalirudin. The transition to oral anticoagulation must be undertaken cautiously and only after the platelet count has recovered. Patients with a remote history of HIT can have cardiac surgery safely with unfractionated heparin. Patients with clinically active HIT who require cardiac surgery before the resolution of the HIT preferably should be anticoagulated with bivalirudin, dosed according to body weight and the goal-activated coagulation time. Given that bivalirudin is an established alternative to heparin as a thrombin inhibitor for cardiac surgery, it is likely that future trials will investigate which anticoagulant confers better outcomes after cardiac surgery, as is the case in percutaneous coronary intervention.     

Use of Bovine Thrombin to Reduce Blood Loss in Primary Total Knee Arthroplasty : A Controlled Randomized Trial

Reducing blood loss during primary total knee arthroplasty (TKA) can improve outcomes by reducing transfusion requirements and wound complications. We examined the use of bovine thrombin to augment hemostasis during primary TKA. A double-blinded randomized trial was performed with 80 primary TKA patients. Half received intraarticular bovine thrombin at the time of wound closure, and half did not. Hemoglobin levels in the study group did decline less than the control group, but no statistically significant difference was found in rates of transfusion, drain outputs, length of stay, or Knee Society scores. This agent does appear to slightly reduce blood loss, but routine use is not cost effective. Thrombin may be considered for patients who would benefit more from greater blood conservation.     

Immunochemical analysis of polyspecific antibodies in patients exposed to bovine fibrin sealant

Background. Patients exposed to bovine thrombin preparations in fibrin sealant often develop antibodies to bovine coagulation proteins, which cause significant bleeding by cross-reacting with human homologues. Recipients of our left ventricular assist system (LVAS) routinely are exposed to fibrin sealant; therefore, we determined whether they developed antibodies.

Methods. We compared sera from 6 LVAS recipients exposed to fibrin sealant (THROMBOGEN, Johnson & Johnson, Arlington, TX ) during LVAS placement to that of 5 nonexposed LVAS recipients. Pre-LVAS and weekly post-LVAS sera were tested for immunoglobulin (Ig)G, IgA, and IgM reactivity to THROMBOGEN by enzyme-linked immunosorbent assay. Peak IgG and IgA reactive sera were characterized by immunoblotting.

Results. All patients exposed to THROMBOGEN developed antibodies: 5 developed IgG, 4 IgA, and 3 IgM. In contrast, nonexposed patients did not develop antibodies. Only some antibody reactivity was contributed by antithrombin or antifactor V antibodies. Silver stain sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of THROMBOGEN showed more than 18 bands, many of which were recognized in Western blot by positive patient sera.

Conclusions. We found both IgG and IgA polyspecific antibody responses in patients exposed to bovine thrombin preparations.     

Intraocular human thrombin infusion in diabetic vitrectomies

The effectiveness of intravitreal thrombin infusion during vitrectomy was prospectively evaluated in eight diabetic patients with persistent vitreous hemorrhages and severe proliferative vitreoretinopathy. A concentration of 80 units/ml of human thrombin diluted in BSS-plus infusion fluid was used. Bleeding time ranged between 2 and 15 seconds (mean, 5 seconds). The reduction of bleeding facilitated surgery. In five of the eight cases the vitreous was clear one day postoperatively. One month postoperatively six eyes had clear media. No additional postoperative intraocular inflammation developed. These results suggest that human thrombin causes less inflammation than bovine thrombin.     

Different hypotensive responses to intravenous bovine and human thrombin preparations in swine

BACKGROUND: Accidental intravenous introduction of commercial bovine thrombin (BT) during use of fibrin glue may result in profound hypotension. Commercial human thrombin (HT) is now available. This study compared the effects of intravenous BT versus HT in swine. METHODS: Swine received 30 U/kg BT, 60 U/kg BT, 30 U/kg HT, or 60 U/kg HT intravenously. Mean arterial pressure (MAP) and survival were monitored for 30 minutes. Thrombin purities and in vitro activities were examined. RESULTS: MAP nadir was lower (p < 0.05) after BT, 27.7 +/- 3.3% (mean +/- SEM) of pretreatment MAP, compared with 41.1 +/- 3.7% after HT. Five of six animals died after 60 U/kg BT, whereas all others survived (p < 0.05). Histology suggested more severe disseminated intravascular coagulation after BT. HT was purer than BT. In vitro activities were similar. CONCLUSION: Both BT and HT produced hypotension. HT appeared safer, because of higher purity. Regardless of source and purity, thrombin must be used with caution.     

Thrombography reveals thrombin generation potential continues to deteriorate following cardiopulmonary bypass surgery despite adequate hemostasis

The intrinsic and extrinsic activation pathways of the hemostatic system converge when prothrombin is converted to thrombin. The ability to generate an adequate thrombin burst is the most central aspect of the coagulation cascade. The thrombin-generating potential in patients following cardiopulmonary bypass (CPB) may be indicative of their hemostatic status. In this report, thrombography, a unique technique for directly measuring the potential of patients' blood samples to generate adequate thrombin bursts, is used to characterize the coagulopathic profile in post-CPB patients. Post-CPB hemostasis is typically achieved with protamine reversal of heparin anticoagulation and occasionally supplemented with blood product component transfusions. In this pilot study, platelet poor plasma samples were derived from 11 primary cardiac surgery patients at five time points: prior to CPB, immediately post-protamine, upon arrival to the intensive care unit (ICU), 3 hours post-ICU admission, and 24 hours after ICU arrival. Thrombography revealed that the Endogenous Thrombin Potential (ETP) was not different between [Baseline] and [PostProtamine] but proceeded to deteriorate in the immediate postoperative period. At the [3HourPostICU] time point, the ETP was significantly lower than the [Baseline] values, 1233 +/- 591 versus 595 +/- 379 nM.min (mean +/- SD; n=9, p < .005), despite continued adequacy of hemostasis. ETPs returned to baseline values the day after surgery. Transfusions received, conventional blood coagulation testing results, and blood loss volumes are also presented. Despite adequate hemostasis, thrombography reveals an underlying coagulopathic process that could put some cardiac surgical patients at risk for postoperative bleeding. Thrombography is a novel technique that could be developed into a useful tool for perfusionists and physicians to identify coagulopathies and optimize blood management following CPB.     

The effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic response after cardiac surgery

Background: Thrombin formation during cardiac surgery could result in disordered hemostasis and thrombosis. The aim of the study was to examine the effects of aprotinin and tranexamic acid on thrombin generation and fibrinolytic activity in patients undergoing cardiac surgery. Methods: Data were collected prospectively from 60 patients undergoing coronary artery bypass grafting using cardiopulmonary bypass (CPB). In a randomized sequence, 20 patients received aprotinin, 20 patients received tranexamic acid, and in 20 patients placebo was used. Results: Significant thrombin activity was found in all the studied patients. Thrombin generation was less in the aprotinin group than in the tranexamic acid and the placebo group (thrombin/anti‐thrombin III complexes 33.7 ± 3.6, 53.6 ± 7.0 and 44.2 ± 5.3 µg/l 2 h after CPB and F1 + 2 fragment 1.50 ± 0.10, 2.37 ± 0.37 and 2.04 ± 0.20 nmol/l 6 h after surgery, respectively). The inhibition of fibrinolysis was significant with both anti‐fibrinolytic drugs (d ‐dimers 0.427 ± 0.032, 0.394 ± 0.039 and 2.808 ± 0.037 mg/l 2 h after CPB, respectively). The generation of d ‐dimers was inhibited until 16 h after CPB in the aprotinin group. The plasminogen activation was significantly less in the aprotinin group (plasmin/anti‐plasmin complexes 0.884 ± 0.095, 2.764 ± 0.254 and 1.574 ± 0.185 mg/l 2 h after CPB, respectively). Conclusion: Thrombin formation is inevitable in coronary artery bypass surgery when CPB is used. The suppression of fibrinolytic activity, either with aprotinin or with tranexamic acid interferes with the hemostatic balance as evaluated by biochemical markers. Further investigations are needed to define the role of hemostatic activation in ischemic complications associated with cardiac surgery.     

Use of a lyophilized bovine collagen matrix in postoperative wound healing.

BACKGROUND: Immediate reconstruction is the preferred approach to the management of defects following Mohs micrographic surgery. In a minority of patients, however, reconstruction is contraindicated, and a long-term biological dressing that stimulates wound healing and minimizes wound care is desirable. OBJECTIVE: We wanted to assess the utility of a lyophilized, type I bovine collagen matrix (SkinTemp) in wound care and wound healing following Mohs micrographic surgery. METHODS: Fifteen patients were treated with a bovine collagen matrix following Mohs micrographic surgery. Study wounds were evaluated for time to complete granulation, time to complete epithelialization, and adverse reactions including infection and allergy. The time to complete healing (granulation and epithelialization) for this group was compared to 15 size- and site-matched surgical defects. RESULTS: The use of bovine collagen matrix provided more rapid wound healing than traditional second intention healing at all anatomic sites studied. The time to complete healing averaged 6.1 weeks with bovine collagen matrix versus 9.4 weeks for the control group. Use of bovine collagen matrix required an average of 3.0 dressing changes weekly compared to 7.0 changes weekly in the control group. There were no wound infections or allergic reactions to it. CONCLUSIONS: A Type I bovine collagen matrix provided a safe, readily available alternative to traditional methods of second intention healing. It minimized wound care while reducing the time for complete healing. A larger study should be performed to confirm the results of this pilot study.     

血液凝固:止血与凝血酶调节

围手术期出血是一个棘手的问题,尤其是伴随着强效抗凝药物临床应用的日趋增多,这一问题变得更具挑战性.理解和掌握当今的凝血概念对于手术前确定患者围手术期的出血风险并在围手术期给予有效的止血治疗十分重要.具有丝氨酸蛋白酶活性的凝血酶在激活其他丝氨酸蛋白酶原(无活性的酶前体)、辅助因子和细胞表面受体的过程中发挥着关键的作用.机...     

Successful use of fibrin glue during 2 years of surgery at a university medical center

A method for producing concentrated fibrinogen, an essential component of fibrin glue, from individually stored, single-donor units of human plasma is reported. The plasma is screened for hepatitis B antigen and HIV-1 virus to reduce the risk of transmission of hepatitis and acquired immunodeficiency syndrome (AIDS). This material is routinely stocked in some operating rooms. It is thus readily available when requested by a surgeon for use in combination with topical bovine thrombin to produce fibrin glue. From April 1985 to March 1987 this material was used by surgeons from eight different surgical specialties on 413 patients with a 91 per cent success rate (376/413). Uses have included sealing vascular suture lines, reinforcing pulmonary and esophageal staple lines, closing dural cerebrospinal fluid leaks, fixing split-thickness skin grafts, reducing lymphatic leakage, and controlling bone bleeding. Additional uses include closure of bronchopleural fistulas by means of the flexible bronchoscope, reduction of perioperative hemorrhage by spraying fibrin glue on the anterior mediastinum during cardiac surgery, and reduction of bleeding during debridement of burn eschars. Careful monitoring and patient follow-up detected no cases of transmission of blood-borne diseases. Only one complication, a local wound infection, has been documented. This material has been an important adjunct for the surgical services and may be safely used at hospitals with local blood bank facilities.