Antigenic Responses to Bovine Thrombin Exposure During Surgery: A Prospective Study of 309 Patients*

Neil Winterbottom, PhD

Jacqueline M. Kuo, BS

Khanh Nguyen, BS

Cary J. Reich, PhD

Kristen J. Trent, BA

Joseph F. Rondinone, PhD

Narinder S. Shargill, PhD

Fusion Medical Technologies, Inc.

34175 Ardenwood Blvd.

Fremont, CA 94555

*This study was sponsored by Fusion Medical Technologies, Inc.

 

KEY WORDS: Bovine thrombin, hemostasis, coagulopathy, Factor V_a, antibodies

 

ABSTRACT

Three hundred and nine patients undergoing either cardiac, vascular, or spinal surgery were exposed to bovine thrombin intraoperatively, either in conjunction with FloSeal®, a bovine gelatin-based hemostatic agent, or with Gelfoam® sponge. Patient sera were collected before surgery and at 6 to 8 weeks after surgery and assayed for antibodies to bovine and human thrombin and Factor V_a. Prothrombin time was measured for the same samples to determine any clinically significant coagulopathy.

Nineteen percent of the serum samples were positive for bovine thrombin antibodies, and 30% were positive for bovine Factor V_a antibodies. None of the patients that tested positive for either one or both bovine antibodies was positive for human Factor V_a antibodies, while only 8 samples tested positive for human thrombin antibodies.

There was no evidence of antibody-induced coagulopathy in any patient testing positive for one or both antibodies. The presence of antibodies to bovine thrombin and/or bovine Factor V_a preoperatively or postoperatively did not result in an increased incidence of adverse clinical outcomes (P 5 .54 and P 5 .57, respectively). The use of bovine thrombin for hemostasis during surgery does not present an undue risk of bleeding resulting from an antibody-induced coagulopathy.

INTRODUCTION

Bovine thrombin preparations are frequently applied topically during surgery to achieve hemostasis. The use of commercial “fibrin glue” preparations with a human or bovine thrombin component in surgical procedures, especially during cardiac surgery, provide yet another source of exposure to exogenous thrombin.1 As with exposure to any foreign proteins, bovine thrombin preparations represent potential antigenic stimuli, and an immunologic response may occur.

Acquired antibodies to specific blood coagulation factors have been reported in association with a variety of conditions, including infection, malignancy, pregnancy, and autoimmune disorders.2 Despite the frequent use of bovine thrombin since the early 1940s, the development of bleeding diastheses resulting from the presence of antibodies to bovine thrombin or contaminant coagulation factors (chiefly Factor V) in the bovine thrombin preparations have been reported in only isolated cases,3,4 and have generally been associated with the presence of induced Factor V deficiency.4–8 Reports in the scientific literature on the immunologic response following exposure to bovine thrombin have been for either individual or a small number of case studies.4,6–13 Results from a prospective study on the immunologic impact after surgical exposure to bovine thrombin during cardiac surgical procedures have recently been reported by Ortel et al.14

We now report the results of a prospective study involving 309 patients exposed to topical bovine thrombin during surgery in whom the immunologic response to bovine thrombin was measured. Prothrombin time (PT) and antibodies against bovine and human thrombin and Factor V_a were measured pre-surgery as well as 6 to 8 weeks postexposure to a commercial preparation of bovine thrombin.

MATERIALS AND METHODS

Study Sample

Between March 1998 and November 1998, a total of 309 patients undergoing cardiac, vascular, or spinal surgery were enrolled in a multicenter, randomized study to evaluate the safety and effectiveness of FloSeal^® Matrix Hemostatic Sealant (FloSeal, Fusion Medical Technologies, Inc., Fremont, CA), a new device for controlling intraoperative bleeding. FloSeal consists of 2 components: a Gelatin-based Matrix and topical thrombin. FloSeal was compared to a control hemostatic agent, Gelatin Sponge (Gelfoam^®, Pharmacia & Upjohn Co., Kalamazoo, MI) 1 thrombin. In both cases, the thrombin used was of bovine origin (Thrombin-JMI^®, Jones Pharma, Inc., St. Louis, MO) and diluted to deliver a concentration of 800 to 1000 NIH units/mL. The Institutional Review Boards (IRBs) at all 10 sites participating in the study had approved the study protocol, and no patients were enrolled into the study prior to IRB approval. The study was conducted in accord with the ethical standards of the IRB for each institution and with the ethical standards of the Helsinki Declaration of 1975.

The inclusion criteria for the study were as follows: patients of either gender; aged 21 years or older; patients undergoing cardiovascular, vascular, or orthopedic surgery; patients willing and able to complete all follow-up visits; patients in whom surgical techniques to control bleeding (sutures and/or cautery) were inappropriate or failed to control bleeding; and patients who had read, understood, and signed an informed consent.

The exclusion criteria were as follows: patients with known sensitivity to any of the components of bovine thrombin preparations and/or material of bovine origin; patients who were pregnant; patients with an active infection at the operative site; and patients in whom the use of a topical hemostat was contraindicated.

Study Design

Patients undergoing cardiac, vascular, or spinal/orthopedic surgery were screened for potential enrollment into the study. A baseline blood sample was drawn within 24 hours before surgery. Patients were enrolled into the study intraoperatively when a bleeding site requiring the use of a topical hemostatic agent was identified. The patient was randomized into the Control (Gelatin Sponge 1 thrombin) or Treatment (FloSeal) group, and the appropriate hemostatic agent used.

Following surgery, patients were evaluated at 12 to 36 hours and at 6 to 8 weeks postsurgery, with follow-up throughout the period for any adverse events. Blood was drawn at each of the follow-up periods. Blood collected at all 3 time points was used to assay hematocrit and hemoglobin levels and red blood cell and white blood cell counts with differentials, PT, activated partial thromboplastin time (aPTT), sodium, potassium, chloride, bicarbonate, BUN, creatinine, total protein, albumin, alkaline phosphatase, AST, ALT, and total and direct bilirubin. In addition, a serum sample from each of the baseline and the 6 to 8 week follow-up period was stored in a 220ºC freezer for later assay of bovine thrombin antibody, bovine Factor V_a antibody, human thrombin antibody, and human Factor V_a antibody. At the baseline and each of the follow-up periods, any anticoagulant or antiplatelet medications that the patient was on were recorded.

Antibody Assays

Bovine thrombin, bovine Factor V_a, human thrombin, and human Factor V_a antibodies were assayed by the ELISA method of Tarantino et al.15

Bovine thrombin and bovine Factor V_a solutions were prepared in Tris Buffered Saline (TBS) at concentrations of 1 mg/mL and 2 mg/mL, respectively. Pre-labeled microtiter plate wells were coated with 100 mL of the antigen solutions by storing at 2ºC to 8ºC for 1 to 14 days. After warming to room temperature, the microtiter plate wells were washed 3 times with 300 mL of wash buffer (0.5% Tween-20 in TBS). Plates were blot dried with a paper towel, 150 mL of Blocking solution (1% BSA in TBS) was dispensed into each well, and the plates were covered with sealing tape and incubated either overnight at 2ºC to 8ºC or at room temperature for 2 hours.

Test samples and the negative control sample were diluted to 1:100 using the assay buffer (0.1% BSA and 0.005% Tween-20 in TBS). Positive control was diluted to 1:1000 using the assay buffer. The microtiter plates were washed and dried as before and 100 mL of the diluted test and control samples pipetted in duplicate into appropriately labeled wells. The covered plates were incubated for 2 hours at room temperature. Following the incubation, the plates were worked and dried and 100 mL of Goata–Human IgG-HRP conjugate was added to each well. The covered plates were incubated for 2 hours. Following incubation, the plates were washed and dried and 100 mL of a proprietary peroxidase substrate containing 3, 3’, 5, 5’ – tetra-methyl-benzidine (TMB substrate) was added to each well. Substrate reaction was monitored using a plate reader at 630 nm. When the O.D. value for the positive control samples read between 0.2 and 0.6, the reaction was stopped by the addition of 100 mL of TMB Stop Solution. The plates were carefully agitated, left to sit for 5 minutes, and read on the plate reader at 450 nm.

Any sample with an average O.D. greater than twice the average O.D. of the pooled negative control was read as positive for presence of antibodies. All positive samples were assayed again at dilutions ranging from 102 to 107, as needed.

Human thrombin and human Factor V_a antibodies were measured in the 6 to 8 week follow-up blood samples of only those patients that tested positive for either one or both of the bovine protein antibodies.

Reagents and Supplies

Tris Buffered Saline tablets, Bovine Serum Albumin (BSA), Tween-20, and ELISA Sealing tapes were purchased from Sigma Chemical Co. (St. Louis, MO); Bovine thrombin and bovine Factor V_a were purchased from Haematologic Technologies, Inc. (Essex Junction, VT); Goata–Human IgG-HRP Conjugate was purchased from Pierce (Rockford, IL); TMB 1-Component Substrate and TMB 1-Component Stop Solution were purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD); and blood sample used as positive control for bovine thrombin and bovine Factor V_a was kindly provided by Dr. T.L. Ortel (Div. Hematology, Duke University Medical Center, Raleigh, NC). The negative control was pooled serum from individuals who had not previously undergone surgical procedures that may have exposed them to bovine thrombin.

RESULTS

Ten institutions enrolled a total of 309 patients into the study over a period of 7 months. Of these, 156 patients were randomized to the Treatment (FloSeal) group and 153 patients were randomized into the Control (Gelatin Sponge 1 thrombin) group. Patient demographics as well as the distribution between the three surgical specialties are shown in Table 1.

Blood was drawn for laboratory testing, hematology, coagulation studies, and metabolic tests at Baseline (within 24 hours prior to surgery), 12 to 36 hours after surgery, and 6 to 8 weeks after surgery. Each test result was compared to the reference range for that test at the laboratory in which it was measured. None of the blood test results that were outside of the normal reference values at Baseline or at the 2 follow-up time points were judged by the surgeons to be clinically significant.

Pairs of Baseline and 6- to 8-week postoperative sera were available for 264 patients. There were 6 additional patients for whom the Baseline serum sample was not available to measure antibodies. The total of 270 patients on whom antibody data were collected represent 91% (270/296) of patients who were enrolled (309) and still living at follow-up (296). Results of the bovine thrombin and bovine Factor V_a antibody measurements for the 2 groups are summarized in Table 2.

In the FloSeal group, 25/139 (18%) of the patients presented with an increased titer over baseline for bovine thrombin antibodies. In the Control group, 26/131 (20%) of the patients presented with an increased titer over baseline for bovine thrombin. The corresponding numbers for bovine Factor V_a were 39/139 (28%) and 43/131 (33%) for the FloSeal and Control groups, respectively. The differences in the frequency of developing either bovine thrombin or bovine Factor V_a antibodies between the 2 groups were not statistically significant.

At the 6 to 8 week follow-up, bovine thrombin and/or bovine Factor V_a antibodies were detected in a total of 100 patients. Seventeen (17) of these patients had PT greater than 15 seconds (range 16.2 to 61.2 sec). All except one of these patients were taking some type of anticoagulant medication at the time. The one patient who tested positive for bovine Factor V_a antibody, and was not on any anticoagulant medication, presented with a PT of 18.3 sec. Eighty five (85) patients who tested positive for bovine thrombin and/or bovine Factor V_a antibodies had normal PT. There were 172 patients who had no measurable antibodies to either bovine thrombin or bovine Factor V_a and for whom PT was available. Twenty-one (21) of these patients had PT greater than 15 sec (range 15.1 to 45.2 sec). All except 4 of these patients were taking anticoagulant medication at the time.

Antibodies to human thrombin and human Factor V_a were measured in the 6- to 8-week follow-up samples of all the patients who had tested positive for either one or both of the bovine thrombin and bovine Factor V_a antibodies. Five (5/25) patients in the FloSeal group and three (3/29) patients in the Control group tested positive for human thrombin antibodies. There was no statistically significant difference between the 2 groups (P 5 .455, Fisher’s Exact Test). None of the patients in either group tested positive for human Factor V_a antibodies. Data for these 8 patients who tested positive for human thrombin antibody are presented in Table 3 along with their prothrombin times, which were all within the normal range.

Of the 309 patients enrolled in the study, only 5 patient records showed knowledge of previous exposure to bovine thrombin. One hundred and nineteen (119) patients reported no previous exposure to bovine thrombin, and 185 patients did not know whether they had previously been exposed to bovine thrombin. Table 4 shows details of the 5 patients with known previous exposure to bovine thrombin. Prothrombin times at Baseline and at the 6- to 8-week follow-up were within the normal range.

Twelve (12) patients in the FloSeal group and 13 patients in the Control group had sera that were positive for antibodies to bovine thrombin, bovine Factor V_a, or both at Baseline, suggesting previous exposure to these proteins. Of these, one patient had an elevated PT at Baseline (70.8 sec), and 3 patients had elevated PT at the 6- to 8-week follow-up (46 sec, 22 sec, and 26.7 sec). All these patients were taking anticoagulant medication (Coumadin) at the time.

A total of 384 complications were seen in 144 patients. Of these, 75 postoperative complications were blood-related and are listed in Table 5.

There was no correlation between all complications and the presence or absence of antibodies to bovine thrombin and/or bovine Factor V_a either at baseline (prior to surgery; P 5 .84; Chi-squared) or at the 6- to 8-week follow-up period (P 5 .86; Chi-squared). The data for the blood-related complications and the presence or absence of antibodies to the bovine proteins are given in Table 6. There was no correlation between the blood-related complications and the presence or absence of antibodies to bovine thrombin and/or bovine Factor V_a either at Baseline (prior to surgery; P 5 .54; Chi-squared) or at the 6- to 8-week follow-up period (P 5 .57; Chi-squared). Furthermore, although there was a trend to a correlation between these blood-related complications and the patients’ history of prior surgery, it did not reach statistical significance (P 5 .08; Chi-squared; Table 6).

 

DISCUSSION

A number of reports have shown that exposure to commercial bovine thrombin preparations can result in the development of antibodies against bovine thrombin and other coagulation factors.8–13,16 However, the clinical evidence of compromised hemostasis in treated patients is rare.16 The available scientific literature on this subject consists mainly of a series of brief reports that describe either individual or a small number of case studies.8–13,16 A prospective clinical trial to demonstrate the safety and effectiveness of a new gelatin-based hemostatic agent, which includes bovine thrombin as one of its components, has provided a unique opportunity to study the frequency of bovine thrombin antibody formation and its correlation, if any, to the development of coagulopathy or other complications that may be clinically relevant.

Patients in both the FloSeal treated group and Control group in this trial were exposed to similar levels of bovine thrombin during the intraoperative application of either hemostatic agent. This provided a study sample of 309 patients in whom bovine thrombin was applied topically either with gelatin matrix of FloSeal or the gelatin formulated as Gelfoam sponge. In agreement with the findings of others,16–18 intraoperative exposure to bovine thrombin during surgery resulted in the development of antibodies to bovine thrombin as well as bovine Factor V_a, a contaminant present in commercial bovine thrombin preparations, in some patients. Other coagulation factor antibodies were not measured in this study. There was no difference between the study groups in the frequency of antibody development. In the present study, bovine thrombin antibodies were measurable in the sera of 19% (pooled data from both FloSeal and Control groups) of the patients at 6 to 8 weeks following intraoperative exposure to bovine thrombin. A larger proportion of patients (30%) tested positive for bovine Factor V_a antibodies. These rates of antibody formation are comparable to the rate of 65% previously reported by Nichols et al18 and 38% reported by Banninger et al.10 A recently published Summary of Safety and Effectiveness of another topical hemostatic agent using a bovine thrombin preparation from the same supplier (Thrombin-JMI) also showed an antibody formation rate of 29% for bovine thrombin.19 In contrast, 2 recent studies have reported seropositive responses to bovine coagulation proteins in as many as 95% to 100% of patients exposed to bovine thrombin during cardiovascular surgery procedures.14,17 The first report of 6 patients exposed to fibrin sealant containing bovine thrombin (THROMBOGEN, Johnson & Johnson Medical, Inc., Arlington, TX) during cardiac surgery for left ventricular assist system (LVAS) placement reported the development of antibodies in all 6 patients.17 Because these patients had coagulopathies related to anticoagulant medications and cardiopulmonary bypass surgery, the role of the bovine thrombin antibodies in the coagulopathy is difficult to determine. The second report of a prospective study of 151 patients exposed to bovine thrombin (THROMBOGEN) during cardiac surgical procedures reported seropositive responses to bovine coagulation proteins in more than 95% of patients and seropositive responses to the corresponding human coagulation proteins in 51% of patients. The vast discrepancy in the rate of antibody formation between these 2 reports and the present study is probably due to the purity of the thrombin that was used, since Thrombin-JMI undergoes an additional purification step to reduce Factor V contaminant compared to Thrombogen.20

In the present study, of the 5 patients with knowledge of previous exposure to bovine thrombin, only 1 tested positive for bovine thrombin and bovine Factor V_a antibodies at Baseline (prior to surgery), and 4 tested positive for either one or both antibodies at the 6- to 8-week follow-up. This is consistent with knowledge that prior exposure to bovine thrombin triggers an anamnestic antibody response.

Despite the presence of thrombin antibodies in sera from 51 patients and Factor V_a antibodies in sera from 82 patients in the present study, none of these patients exhibited any unexplained clinical coagulopathy as assessed by an increase in PT. All but one of the 17 patients who tested positive for either the bovine thrombin or the bovine Factor V_a or both antibodies, and had elevated PT (.15 sec), were taking anticoagulant medications such as Coumadin. The one patient who was an exception had a PT of 18.3 sec—a PT value that was judged by the surgeon to be “not clinically significant.” Although a role of the inhibitory antibodies in the elevated PT of these patients could not be conclusively ruled out, it is likely that the elevated PTs were a result of the anticoagulant medications that the patients were prescribed and taking. The development of coagulopathy resulting from the presence of coagulation cascade protein antibody(s) is relevant when these antibodies are human protein antibodies that can cross react the corresponding human proteins. In the present study, the presence of cross-reactive human thrombin antibodies in 8 patients that had either bovine thrombin and/or bovine Factor V_a antibodies did not result in the development of coagulopathy since the PT for all 8 patients was within the normal range. To date, no reports of coagulopathy following the use of bovine thrombin in FloSeal have been reported, despite the use of FloSeal for intraoperative hemostasis in an estimated 35,000 patient procedures.

Postoperative clinical complications were not associated with the presence of preoperative antibodies to bovine thrombin and/or bovine Factor V_a or the development of antibodies to either bovine protein in the postoperative period. The absence of adverse clinical complications in spite of the presence of bovine and human coagulation protein antibodies in the present study appears to contrast with the findings reported by Ortel et al.14 However, a careful analysis of the data reported by Ortel et al reveals that the results are not dissimilar. Although Ortel et al showed a strong correlation between “preoperative elevated antibody levels to two or more bovine proteins” and adverse events (P 5 .0042), there was also a statistically significant correlation between “postoperative elevated antibody levels to two or more bovine proteins” and a lack of adverse events (P 5 .0229).14 In the present study, repeated exposure to bovine thrombin in patients who either had been previously exposed to bovine thrombin during surgery or had preexisting antibodies to bovine thrombin and/or bovine Factor V_a did not result in any untoward immune-mediated adverse reactions.

The safety profile of bovine thrombin demonstrated in the present study is further supported by a review of Medical Device Reports (MDRs) to the United States’ Food and Drug Administration (FDA). During the period from 1986 to 1994, before the approval of Thrombin-JMI, a total of 27 patients were reported via the MDRs as having a bovine thrombin-related adverse event. Sixteen of these patients presented with clinical signs or symptoms of systemic anaphylaxis (usually hypotension with/without shock, bradycardia, tachycardia, asthma, urticaria, angioedema), 8 had coagulopathies (4 with bleeding), 1 suffered cardiac arrest and died, and 1 died (but no etiology was given). One patient was reported as being a treatment failure. Three of the patients with anaphylaxis and 2 with coagulopathy also died. A total of six patients in this group of MDRs had prolonged bleeding. Of the 16 MDRs of thrombin-related adverse events reported to the FDA between 1995 and 1997, only 2 involved thrombin from the same supplier as that for the thrombin that is supplied in the FloSeal kit. The first of these reported that thrombin was ineffective at producing hemostasis, and the second reported an accidental intravascular injection of thrombin. Neither report involved a coagulopathy or anaphylaxis induced by the proper use of the thrombin.

Based on reported sales figures, it is estimated that during 1994, for example, approximately 370,000 patients may have been exposed to bovine thrombin preparations. During a 12-year period from 1986 through 1997, MDRs listed only 43 patients who had a serious untoward reaction to topical bovine thrombin, giving an incidence rate of about 4 cases per year. This yields an adverse event incidence rate of approximately 0.001%.

CONCLUSION

In summary, our results show that up to 30% of patients exposed to bovine thrombin preparations during surgery are likely to develop antibodies to bovine thrombin and/or contaminant proteins in the preparation, such as Factor V. There was no evidence of antibody-induced coagulopathy in any patient testing positive for one or both antibodies. Furthermore, there was no correlation between the presence of bovine protein antibodies and adverse reactions. The use of bovine thrombin for topical applications to achieve hemostasis during surgery (for example, when using FloSeal) is safe and does not present an undue risk of bleeding resulting from an antibody-induced coagulopathy.

ACKNOWLEDGEMENTS

The authors are grateful to the following Principal Investigators who recruited patients for the study and obtained blood samples that are the subject of this manuscript: Mehmet C. Oz, MD (Columbia Presbyterian Medical Center, New York, NY), Delos M. Cosgrove, MD (The Cleveland Clinic Foundation, Cleveland, OH), J. Donald Hill, MD (California Pacific Medical Center, San Francisco, CA), Brian R. Badduke, MD (Washington Hospital, Fremont, CA), Fred A. Weaver, MD (University of Southern California School of Medicine, Los Angeles, CA), Louis M. Messina, MD (UCSF School of Medicine, San Francisco, CA), Michael A. Zatina, MD (St. Agnes Healthcare, Baltimore, MD), Troy D. Payner, MD (Indianapolis Neurosurgical Group, Indianapolis, IN), Gunnar B. J. Andersson, MD (Rush Presbyterian-St. Luke’s Medical Center, Chicago, IL), and Kevin J. Lawson, MD (Mercy Medical Center & Redding Medical Center, Redding, CA).

The authors are also grateful to T L Ortel, MD (Div. Hematology, Duke University Medical Center, Raleigh, NC) for providing the samples positive for antibodies against bovine thrombin and bovine Factor V_a.

REFERENCES

 1. Jackson MR, MacPhee MJ, Drohan WN, Alving BM: Fibrin sealant: Current and potential clinical applications. Blood Coagulation Fibrinolysis 7:737–746, 1996.

 2. Shapiro SS, Hultin M: Acquired inhibitors to blood coagulation factors. Semin Thromb Hemost 1:336–384, 1975.

 3. Cronkite EP, Lozner EL, Deaver JM: Use of thrombin and fibrinogen in skin grafting. JAMA 124:976–978, 1944.

 4. Cmolik BL, Spero JA, Magovern GJ, Clark MD: Redo cardiac surgery: Late bleeding complications from topical thrombin-induced Factor V deficiency. J Thorac Cardiovasc Surg 105:222–228, 1993.

 5. Ortel TL, Charles LA, Keller FG, et al: Topical thrombin and acquired coagulation factor inhibitors: Clinical spectrum and laboratory diagnosis. Am J Hematol 45:128–135, 1994.

 6. Israels SJ, Israels ED: Development of antibodies to bovine Factor V in two children after exposure to topical bovine thrombin. Am J Pediatr Hematol Oncol 16:249–254, 1994.

 7. Zehnder JL, Leung LLK: Development of antibodies to bovine thrombin and Factor V with recurrent bleeding in a patient exposed to topical bovine thrombin. Blood 76:2011–2016, 1990.

 8. Rapaport SI, Zivelin A, Minow RA, et al: Clinical significance of antibodies to bovine and human thrombin and Factor V after surgical use of bovine thrombin. Am J Clin Pathol 97:84–91, 1992.

 9. Christie RJ, Carrington L, Alving B: Postoperative bleeding induced by topical bovine thrombin: Report of two cases. Surgery 121:708–710, 1997.

10. Banninger H, Hardegger T, Tobler A, et al: Fibrin glue in surgery: Frequent development of inhibitors of bovine thrombin and human Factor V. Br J Haematol 85:528–532, 1993.

11. Muntean W, Zenz W, Finding K, et al: Inhibitor to Factor V after exposure to fibrin sealant during cardiac surgery in a two-year old child. Acta Paedtiatr 83:84–87, 1994.

12. La Spada AR, Skalhegg BS, Henderson R, et al: Fatal hemorrhage in a patient with an aquired inhibitor of human thrombin. New Engl J Med 333:494–497, 1995.

13. Spero JA: Bovine thrombin-induced inhibitor of Factor V and bleeding risk in postoperative neurosurgical patients. Report of three cases. J Neurosurg 78:817–820, 1993.

14. Ortel TL, Mercer MC, Thames EH, et al: Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin. Ann Surg 33:88–96, 2001.

15. Tarantino MD, Ross MP, Daniels TM, et al: Modulation of an acquired coagulation Factor V inhibitor with intravenous immune globulin. J Pediatr Hematol Oncol 19:226–231, 1997.

16. Carroll JF, Moskowitz KA, Edwards NM, et al: Immunologic assessment of patients treated with bovine fibrin as a hemostatic agent. Thrombosis and Haemostasis 76(6):925–931, 1996.

17. Fastenau DR, McIntyre JA: Immunochemical analysis of polyspecific antibodies in patients exposed to bovine fibrin sealant. Ann Thorac Surg 69:1867–1872, 2000.

18. Nichols WL, O’Marcaigh AS, Daniels TM, et al: Incidence of immune-mediated coagulopathies associated with surgical use of fibrin sealant (“Glue”) or bovine topical thrombin. Thrombosis Haemostasis 78(Suppl 1):177, 1997.

19. Summary of Safety and Effectiveness Data: Absorbable Hemostatic Agent (PMA P990030). www.fda.gov/cdrh/pdf/p990030b/pdf.

20. Thrombin, Topical U.S.P. (Bovine Origin): THROMBIN-JMI^® Instructions for Use – Rev. 6/98.

 

 

Table 1. Patient Demographics and Distribution Among Specialties

Number of Patients

All Surgical Specialties     156     153

Cardiac Surgery   48 (31%)       45 (29%)

Vascular Surgery   43 (28%)       46 (30%)

Spinal/Orthopedic Surgery   65 (41%)       62 (41%)

Gender

Men       91 (58%)       88 (58%)   .840^a

Women  65 (42%)       65 (42%)

Age

Mean 6 SD (years)  60.67 6 14.13  59.48 6 14.48   .377^b

Range 19.6–88.7 17.3–89.3

^aP value from Cochran-Mantel-Haenszel Test for row mean scores, adjusted for investigational site.

^bP value for treatment comparisons from a Two-Way Analysis of Variance with factors of treatment and investigator.

 

 

Table 2. Antibody Data: Bovine Thrombin and Bovine Factor V_a

                                                             P Value^†

                  Variable                   FloSeal                   Control             FloSeal vs.                  Pooled

Number of Patients 156               153                                    309

Thrombin Antibody Titer

Increase 25 (18%)       26 (20%)  0.757       51 (19%)

No Increase     114 (82%)     105 (80%)               219 (81%)

Not Reported*       17       22                 39

Factor V_a Antibody Titer

Increase 39 (28%)       43 (33%)  0.428       82 (30%)

No Increase     100 (72%)       88 (67%)               188 (70%)

Not Reported*       17       22                 39

*Includes patients whose blood was not drawn or who expired.

^†P values obtained from Fischer’s Exact Test.

• Increase in titer was defined as Baseline (2) to Follow-up (1).

• No Increase in titer was Baseline (2) to Follow-up (2).

• For patients with a Baseline (1) to Follow-up (1), semi-quantitative titers were used to determine Increase or No Increase in titer.

• No patients were seen with a decrease in titer between Baseline and Follow-up.

 

 

Table 3. Table of Patients with Cross Reactivity to Human Thrombin and PT Times

  6–8 Week Follow-up

                                      Bovine   Human      Bovine   Human

      Patient   Treatment   Thrombin   Thrombin  Factor V_a Factor V_a        PT

        No.       Group    Antibody    Antibody    Antibody    Antibody    (sec.)

     011-013  Control        1 (4)         1 (2)        1 (4)               –          #

     091-047  Control        1 (2)         1 (2)        1 (2)               –       13.0*

     093-056  Control        1 (2)         1 (2)          –                  –       10.1*

     046-059  FloSeal          –            1 (2)        1 (4)               –       11.0*

     061-024  FloSeal        1 (2)         1 (2)        1 (3)               –       14.0*

     061-026  FloSeal        1 (2)         1 (2)          –                  –       14.1*

     081-013  FloSeal        1 (2)         1 (2)        1 (2)               –       11.4*

     101-027  FloSeal        1 (2)         1 (2)        1 (4)               –       12.0*

Values in parentheses are titers at Log base 10.

^#Prothrombin Time not measured.

*Prothrombin Times within Normal Range.

 

 

 

Table 4. Antibody Status and PT of Patients with Known Previous Exposure to Bovine Thrombin

                   Baseline  6–8 Week Follow-up

    Patient Treatment    Bovine     Bovine       PT        Bovine    Bovine            PT

       No.         Group  Thrombin    Factor V_a  (sec)  Thrombin  Factor V_a       (sec)

                                Antibody  Antibody              Antibody  Antibody

      21-6       FloSeal         –                  –     12.4*          1 (2)      1 (2)         11.7*

     21-16      FloSeal         –                  –     11.9*          1 (3)      1 (2)         11.4*

     21-20     Control      1 (2)           1 (3)     10.9*          1 (4)      1 (4)         11.7*

     101-7     Control         –                  –     13.1*              –         –            13.7*

    101-31    Control         –                  –     11.8*              –      1 (2)         11.8*

Values in parentheses are titers at Log base 10.

*Prothrombin Times within Normal Range.

 

Table 5. Blood-Related Postoperative Complications

Complication N

Hemorrhagic Complications

Excessive postoperative bleeding   9

Anemia      15

Transfusion Reaction      1

Decreased Platelets       2

Thromboembolic Complications   

Arterial Thrombosis 7

Deep Venous Thrombosis 1

Hypercoagulation             1

Congestive Heart Failure/Stroke                    5

Wound Complications

Abscess      1

Seroma         1

Other Serious Complications

Infection    11

Fever            4

Renal Failure                      1

Death         16

TOTAL        75

 

 

Table 6. Blood-Related Complications Versus Laboratory Variables

                     Complications

Variable Complications  Without    P Value*

           With Variable    Variable

Presence of Preoperative

Antibodies to Bovine Thrombin   8/25 (32%)         73/277 (26%)    .54

and/or Bovine Factor V_a

Presence of Postoperative

Antibodies to Bovine Thrombin 20/99 (20%)         39/168 (23%)    .57

and/or Bovine Factor V_a

Prior Surgical Procedure 28/85 (33%)         52/224 (23%)    .08

*Chi-Squared test.