- Email: [email protected]
Heparin-induced thrombocytopenia: bovine versus porcine heparin in cardiopulmonary bypass surgery
Heparin-Induced Thrombocytopenia: Bovine Versus Porcine Heparin in Cardiopulmonary Bypass Surgery Barbara A. Konkle, MD, Thomas L. Bauer, MD, Gowthami Arepally, MD, Douglas B. Cines, MD, Mortimer Poncz, MD, Stephen McNulty, DO, Richard N. Edie, MD, and John D. Mannion, MD Department of Medicine, Cardeza Foundation for Hematologic Research, Departments of Surgery and Anesthesiology, Jefferson Medical College of Thomas Jefferson University, Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania, and Division of Pediatric Hematology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania
Background. Studies have demonstrated a high incidence of antibodies to heparin/platelet factor 4 complexes, the antigen in heparin-induced thrombocytopenia, in patients after cardiopulmonary bypass surgery. In many hospitals, beef lung heparin has been used historically for cardiopulmonary bypass, and there has been reluctance to change to porcine heparin despite concerns of an increased incidence of heparin-induced thrombocytopenia in patients receiving bovine heparin. Methods. A prospective randomized trial comparing bovine and porcine heparin in cardiopulmonary bypass surgery was conducted. Presurgery and postsurgery heparin antibody formation was studied using the serotonin release assay and a heparin/platelet factor 4 enzymelinked immunosorbent assay.
Results. Data available on 98 patients, randomized to receive either bovine or porcine heparin, revealed no significant difference in patient positivity by serotonin release assay (12% in both groups) or by the heparin/ platelet factor 4 enzyme-linked immunosorbent assay (29% with porcine and 35% with bovine heparin) postoperatively. There were no significant differences between preoperative and postoperative platelet counts or thromboembolic complications. Conclusions. Our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to heparin/platelet factor 4. (Ann Thorac Surg 2001;71:1920 – 4) © 2001 by The Society of Thoracic Surgeons
D
sion, pulmonary embolism, phlegmasia cerulae dolens, and death [2]. A number of recent studies have demonstrated a very high prevalence of antibodies to heparin/ platelet factor 4 (PF4) complexes, the antigen in HIT, in patients post– cardiopulmonary bypass [3– 6]. The clinical significance of the antibody formation is uncertain, but it may predispose patients to the development of HIT with continued heparin exposure or reexposure at a later date. Heparin is prepared from either beef lung or porcine intestinal mucosa. Some case reports and retrospective studies have found a higher incidence of HIT in patients receiving bovine heparin, and some authorities recommend avoidance of beef lung preparations [7]. However, in many centers, beef lung heparin has historically been used for cardiopulmonary bypass, and there has been a reluctance to change preparations. The finding of a high prevalence of heparin-associated antibodies in this setting raised the possibility that a difference between the antigenicity of the two heparin preparations could be detected with a small clinical trial. For this reason, we conducted a prospective double-blind randomized trial comparing the development of heparin-associated antibodies in patients receiving either beef lung or porcine mucosal heparin preparations for anticoagulation during cardiopulmonary bypass.
espite a low but serious incidence of side effects, unfractionated heparin remains the anticoagulant of choice for cardiopulmonary bypass. Alternatives to unfractionated heparin, such as heparinoids, direct thrombin inhibitors, or defibrinogenating agents, have not proved practical for routine use. As a result, most cardiovascular patients are still exposed to potential adverse responses to heparin, the most serious of which is heparin-induced thrombocytopenia (HIT). From a review of retrospective studies, the frequency of HIT has been estimated to be ⬃3% and of HIT with thrombosis to be ⬃1% in patients receiving heparin for 5 days or more [1]. The incidence probably varies by the circumstance in which heparin is administered and the patient population being treated. Heparin-induced thrombocytopenia is reported frequently in patients undergoing cardiopulmonary bypass. One study of 1,500 consecutive patients who underwent cardiopulmonary bypass reported 11 patients (0.75%) with HIT with thrombosis resulting in limb amputations, stroke, graft occluAccepted for publication Feb 7, 2001. Address reprint requests to Dr Konkle, Penn Comprehensive Hemophilia and Thrombosis Program, Hematology-Oncology, University of Pennsylvania, PMC, MAB 103, 39th and Market St, Philadelphia, PA 19104; e-mail: [email protected].
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
0003-4975/01/$20.00 PII S0003-4975(01)02534-6
Ann Thorac Surg 2001;71:1920 – 4
Material and Methods
KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
Table 1. Patient Characteristics
Prospective Clinical Trial One hundred six consecutive patients undergoing cardiopulmonary bypass surgery were asked to participate in this trial; complete data were available on 98 patients. All patients had been exposed to heparin at some time preoperatively during cardiac catheterization, and some had been treated for unstable angina. Only porcine heparin is used outside of the operating room at Thomas Jefferson University Hospital, where this study was conducted. The protocol was approved by the Institutional Review Board of Thomas Jefferson University, and informed written consent was obtained from all participants. Blood samples for serotonin release assay, heparin/PF4 enzyme-linked immunosorbent assay (ELISA), and platelet count were obtained before the induction of anesthesia on the morning of surgery and again on day 5 postoperatively. Platelet counts were obtained as clinically indicated on additional postoperative days. Patients were randomized to receive either bovine or porcine heparin by the pharmacist, and other caregivers, including the surgeon, were blinded as to the type of heparin received. The heparin-coated Swan Ganz catheters were removed routinely on postoperative day 1, and no patients were exposed to heparin in flush solutions. Therapeutic heparin (porcine; Elkins-Sinn, Cherry Hill, NJ) was administered only in a small percentage of patients postoperatively and did not differ between the two groups.
Anesthetic and Surgical Management A heparin-coated Swan Ganz catheter and a radial arterial line were placed in each patient before induction of anesthesia. High-dose fentanyl (50 to 100 g/kg) was used to induce and maintain anesthesia. Either beef lung (Pharmacia and Upjohn, Peapack, NJ) or porcine mucosal (Elkins-Sinn) heparin was administered at a dose of 300 U/kg before bypass. Additional heparin was given by bolus as needed to achieve a whole-blood activated clotting time of more than 480 seconds. Anticoagulation was maintained at this level throughout bypass. The cardiopulmonary bypass circuit consisted of a membrane oxygenator (Baxter-Bentley, Irvine, CA), closed venous reservoir (Baxter-Bentley), and centrifugal pump (Medtronic-Biomedicus, Eden Prairie, MN). Moderate systemic hypothermia, hemodilution, and blood cardioplegia were used. All patients received ⑀-aminocaproic acid (150 g/kg) to minimize postoperative blood loss. At the end of bypass, heparin activity was reversed with protamine.
Laboratory Assays Heparin-dependent plateletreactive antibodies were detected with a two-point serotonin release assay as described [8], except that heatinactivated plasma was used. Because patient platelets are not used in the assay, aspirin therapy had no effect on the results. Each sample was run in triplicate, and the results were averaged. The test was considered positive when there was a more than 20% serotonin release at 0.1
SEROTONIN RELEASE ASSAY.
1921
Heparin Source Age (years) % male % white Bypass cross-clamp time (min) Intraoperative heparin dose (units)
Porcine (n ⫽ 49)
Bovine (n ⫽ 49)
62 ⫾ 12 76 90 84 ⫾ 37
63 ⫾ 11 76 90 80 ⫾ 36
30,000 ⫾ 13,543
32,452 ⫾ 11,991
U/mL and less than 20% release at 100 U/mL heparin, but no release when patient plasma was added to the platelets in the absence of heparin. HEPARIN/PF4 ELISA. Heat-treated plasma samples were tested using the Asserachrom H PF4 ELISA kit (Diagnostica STAGO, Parsippany, NJ) as previously described [3]. The protocol was followed, and results were interpreted according to the manufacturer’s directions. A negative result was defined as an A492 nm less than 0.25, an intermediate result was defined as an A492 nm greater than or equal to 0.25 and less than 0.5, and a positive result was defined as an A492 nm greater than or equal to 0.5. These cutoff values, recommended by the manufacturer, are based on studies of the heparin/PF4 ELISA in patients with established HIT and in various control populations [9]. An absorbance of 0.5 is more than 8 standard deviations above the values obtained from healthy normal subjects and is a value characteristically seen in patients with HIT. Samples that generate absorbances in the intermediate range of detection are less than 8 standard deviations (ie, less than necessary for a positive result) and more than 2 standard deviations above those of healthy normal subjects.
Statistical Analysis Results are expressed as mean ⫾ standard deviation. For paired data, such as comparison of two assays or preoperative to postoperative comparisons, McNemar’s 2 test was used. Quantitative comparisons of ELISA results were performed using Wilcoxon rank sum statistics.
Results Data from 49 patients who received porcine heparin and 49 patients who received bovine heparin for cardiopulmonary bypass were available. The general patient characteristics of the two groups were similar and are listed in Table 1. The majority of the patients underwent primary coronary revascularization (85%), with a smaller percentage undergoing valve operations (13%) or combined procedures (12%). The percentage of patients testing positive for anti– heparin/PF4 antibodies by ELISA or for heparindependent platelet-reactive antibodies by serotonin release assay preoperatively and postoperatively were
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KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
Ann Thorac Surg 2001;71:1920 – 4
Table 2. Heparin Antibody Test Results Preoperatively and Postoperatively Preoperative
Postoperative
ELISA ELISA ELISA SRA ELISA ELISA ELISA SRA N I P P N I P P Porcine Bovine a
42 38
6 8
1 3
3 3
7 8
28* 24*
14* 17*
6 6
p ⬍ 0.001 versus preoperative values.
ELISA ⫽ enzyme-linked immunosorbent assay; I ⫽ intermediate; N ⫽ negative; P ⫽ positive; SRA ⫽ serotonin release assay.
similar for the two groups, as shown in Table 2. Cardiopulmonary bypass was associated with a significant increase in the percentage of patients developing anti– heparin/PF4 antibodies. This is illustrated for the ELISA results in Figure 1. There was an overall increase in the ELISA optical density reading (bovine heparin: 0.13 ⫾ 0.01 preoperative, 0.55 ⫾ 0.54 postoperative; porcine heparin: 0.19 ⫾ 0.04 preoperative, 0.50 ⫾ 0.27 postoperative), but there was no statistically significant difference in results between those receiving bovine and those receiving porcine heparin (p ⫽ 0.12 for preoperative values; p ⫽ 0.74 for postoperative values). Platelet counts preoperatively and on postoperative days 1 to 5 are illustrated in Figure 2. There was no significant difference between the platelet counts on any of the days tested. The percentage of patients with thrombocytopenia, intraoperatively or on any postoperative day, did not differ between the groups. There also was no significant difference in postoperative thromboembolic complications (Table 3). In the group that received bovine heparin, 2 patients suffered cerebrovascu-
Fig 1. Number of patients testing negative (Neg), intermediate (Int), or positive (Pos), by enzyme-linked immunosorbent assay for anti– heparin/PF4 antibodies preoperatively (Pre-Op) and postoperatively (Post-Op).
Fig 2. Mean platelet count of patients receiving either bovine or porcine heparin preoperatively (Pre-Op) and on days 1 to 5 postoperatively (Post-Op).
lar accidents, and 1 patient experienced a postoperative myocardial infarction. In the group receiving porcine heparin, 3 patients suffered cerebrovascular accidents. These complications were not associated with thrombocytopenia or antibody results that differed from the study group as a whole.
Comment This study confirms previous findings concerning the development of anti– heparin/PF4 antibodies after cardiovascular surgery—an unexpected number of patients have developed antibodies before surgery is even undertaken, and the majority increase these antibodies during the perioperative period. The percentage of patients testing positive by ELISA postoperatively in this study (32%) was less than in our previous study (51%). However, both values are in the range of positivity (25% to 61%) that has been reported in the postbypass state [3– 6]. Additionally, almost all patients had an increase in absorbance reading on the ELISA, with 85% of patients having either intermediate or positive readings postoperatively compared to 18% preoperatively (p ⬍ 0.001). It is surprising that these antibodies do not often result in either thrombocytopenia or thrombosis. Although some patients in our series developed thrombotic complications, there was no correlation with antibody formation or platelet count at the time of the event, and thus no diagnosis of HIT or HIT with thrombosis was made. Nevertheless, the antigenicity of heparin preparations is of importance, because even a small percentage of patients developing HIT with thrombosis results in a substantial human and economic cost. In its most serious form, it can result in devastating generalized thrombosis, resulting in multiple limb losses, strokes, cerebral injury, or death. Although the incidence of HIT with thrombosis is low, the large number of patients undergoing cardiopulmonary bypass makes prevention of this condition an important therapeutic goal.
Ann Thorac Surg 2001;71:1920 – 4
KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
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Table 3. Thromboembolic Complications Heparin Type
Surgery Type
Complication
Preop SRA
Postop SRA
Preop ELISA
Postop ELISA
Preop Plateleta
Postop Plateleta
Bovine Bovine Bovine Porcine Porcine Porcine
CABG CABG CABG/CEA CABG CABG MVR
MI CVA CVA CVAb CVA, DVT CVA
Neg Neg Pos Neg Neg Neg
Neg Neg Neg Neg Neg Neg
Neg Neg Int Pos Neg Neg
Pos Int Pos Pos Pos Neg
203 277 211 248 176 142
NA 200 196 285 209 249
a
Platelet count ⫻ 103 per mL.
b
With atrial fibrillation.
CABG ⫽ coronary artery bypass grafting; CEA ⫽ carotid endarterectomy; CVA ⫽ cerebrovascular accident; DVT ⫽ deep vein thrombosis; ELISA ⫽ enzyme-linked immunosorbent assay; Int ⫽ intermediate; MI ⫽ myocardial infarction; MVR ⫽ mitral valve replacement; NA ⫽ not available; Neg ⫽ negative; Pos ⫽ positive; SRA ⫽ serotonin release assay.
Effective treatment of cardiovascular disease often requires anticoagulant therapy. Although antiplatelet and thrombolytic therapies are becoming increasingly important in the treatment of acute coronary syndromes, antithrombotic therapy with heparin remains of central importance. For cardiopulmonary bypass, there are no substitutes available for widespread use. The two most commonly used approaches in patients with HIT who require cardiopulmonary bypass is to use anticoagulation with lepirudin [10, 11] or with recombinant hirudin, a direct thrombin inhibitor, or to use danaparoid [12], a heparinoid compound. Neither is FDA approved for this indication, although lepirudin is FDA approved for anticoagulation in patients with HIT and associated thromboembolic disease. Neither of these agents is ideal for this use. Both are excreted renally [13] and require specialized laboratory monitoring. Danaparoid does not affect the activated clotting time or activated partial thromboplastin time (aPTT), requiring antifactor Xa determinations. It also has a very long half-life. Lepirudin has a much shorter half-life, but at the concentrations needed for cardiopulmonary bypass, neither the activated clotting time nor the aPTT reflect drug concentration or activity. The ecarin clotting time has been used successfully in this setting [14] but is not widely available. In addition, there is no antidote to reverse anticoagulation for either lepirudin or danaparoid. The two heparin preparations used in our study might have been expected to induce different antigenic responses. In 1992, Amiral and colleagues [15] reported that heparin-induced antiplatelet antibodies were not directed against heparin, but against heparin/PF4 complexes, a finding confirmed by other investigators [16 – 19]. The affinity of different heparin preparations for PF4 could result in varying degrees of antigenicity. Lowmolecular-weight heparins are thought to be less likely to induce an immunogenic response, presumably because of their low affinity for PF4. However, in laboratory assays, a variety of negatively charged substances can substitute for heparin in the antigen complex, suggesting that the specificity for antibody formation may be low [1]. Some of our patients who received bovine heparin on bypass had received porcine heparin earlier in their hospital course. However, if bovine heparin resulted in
higher antibody formation, one would not expect prior porcine heparin exposure to prevent a further immune stimulation. The differences in the incidence of HIT reported in the past in patients receiving either bovine or porcine heparin may have been caused by the specific composition of the older bovine heparin preparations, which are no longer available. Since those studies were performed, the major manufacturer of bovine heparin and the supplier for our current study changed their manufacturing process. In a small prospective study after that change, the authors did not find a difference in incidence of thrombocytopenia between those receiving bovine (1 of 22) or porcine (0 of 22) heparin anticoagulation for at least 5 days [20]. Despite these changes and findings it is still widely stated that bovine heparin is more likely to result in HIT, even though there are no recent studies to support that statement. It is also possible that the incidence of HIT in our population would have been higher in patients receiving bovine heparin if anticoagulation had been continued. In a study of the effect of different heparin preparations (unfractionated heparin versus low-molecular-weight heparins) used after cardiopulmonary bypass, there was a higher incidence of HIT in patients anticoagulated with unfractionated heparin (source not defined) than with low-molecular-weight heparins, although antibody formation did not differ. However, these groups differed in that almost all patients who received postoperative lowmolecular-weight heparins had undergone coronary artery bypass grafting, and almost all patients who received unfractionated heparin had undergone valve replacement surgery [3]. Better options for anticoagulation during cardiovascular procedures are clearly needed. Alternatives to heparin and heparin-like substances are promising in terms of decreasing the risk of HIT. Compounds that allow easy monitoring and reversibility are greatly needed. However, our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to PF4/heparin or to stimulate increased levels of preexisting antibodies, because unfractionated preparations from either species seem to be equally immunogenic.
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KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
We thank Bernadette Mestichelli, MT (ASCP), for excellent technical assistance. This study was supported, in part, by a Special Purpose Grant from Thomas Jefferson University Hospital and grants HL44956 (Barbara A. Konkle), HL54749 (Mortimer Poncz, Douglas B. Cines), HL40387 (Douglas B. Cines), HL50790 (Douglas B. Cines), HL49517 (Douglas B. Cines), HL37419 (Mortimer Poncz), HL56265 (Mortimer Poncz), and a fellowship award from the Southeastern Pennsylvania Chapter of the American Heart Association (Gowthami Arepally).
References 1. Warkentin T. Heparin-induced thrombocytopenia: a tenyear retrospective. Annu Rev Med 1999;50:129– 47. 2. Singer RL, Mannion JD, Bauer TL, Armenti FR, Edie RN. Complications from heparin-induced thrombocytopenia in patients undergoing cardiopulmonary bypass. Chest 1993; 104:1436– 40. 3. Bauer TL, Arepally G, Konkle BA, et al. Prevalence of heparin-associated antibodies without thrombosis in patients undergoing cardiopulmonary bypass surgery. Circulation 1997;95:1242– 6. 4. Visentin GP, Malik MI, Cyganiak KA, Aster RH. Patients treated with unfractionated heparin during open heart surgery are at high risk to form antibodies reactive with heparin:platelet factor 4 complexes. J Lab Clin Med 1996;128: 376– 83. 5. Trossaert M, Gaillard A, Commin PL, Amiral J, Vissac A, Fressinaud E. High incidence of anti– heparin/platelet factor 4 antibodies after cardiopulmonary bypass surgery. Br J Haematol 1998;101:653–5. 6. Pouplard C, May MA, Iochmann S, et al. Antibodies to platelet factor 4 – heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin. Circulation 1999;99:2530– 6. 7. King DJ, Kelton JG. Heparin-associated thrombocytopenia. Ann Intern Med 1984;100:535– 40. 8. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986;67:27–30. 9. Amiral J, Bridey F, Wolf M, et al. Antibodies to macromolecular platelet factor 4-heparin complexes in heparininduced thrombocytopenia: a study of 44 cases. Thromb Haemost 1995;73:21– 8.
Ann Thorac Surg 2001;71:1920 – 4
10. Greinacher A, Vo¨lpel H, Janssens U, et al. Recombinant Hirudin (lepirudin) provides safe, and effective anticoagulation in patients with heparin-induced thrombocytopenia. Circulation 1999;99:73– 80. 11. Latham P, Revelis AF, Joshi GP, DiMaio JM, Jessen ME. Use of recombinant Hirudin in patients with heparin-induced thrombocytopenia with thrombosis requiring cardiopulmonary bypass. Anesthesiology 2000;92:263– 6. 12. Gillis S, Merin G, Zahger D, et al. Danaparoid for cardiopulmonary bypass in patients with previous heparin-induced thrombocytopenia. Br J Haematol 1997;98:657–9. 13. Koster A, Pasic M, Bauer M, Kuppe H, Hetzer R. Hirudin as anticoagulant for cardiopulmonary bypass: importance of preoperative renal function. Ann Thorac Surg 2000;69:37– 41. 14. Po¨tzsch B, Madlener K, Seelig C, Riess CF, Greinacher A, Mu¨ller-Berghaus G. Monitoring of r-Hirudin anticoagulation during cardiopulmonary bypass—assessment of the whole blood ecarin clotting time. Thromb Haemost 1997;77: 920–5. 15. Amiral J, Bridey F, Dreyfus M, et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haemost 1992;68:95– 6. 16. Greinacher A, Po¨tzsch B, Amiral J, Dummel V, Eichner A, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen. Thromb Haemost 1994;71:247–51. 17. Suh JS, Aster RH, Visentin GP. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis recognize different epitopes on heparin: platelet factor 4. Blood 1998;91:916–22. 18. Kelton JG, Smith JW, Warkentin TE, Hayward CP, Denomme GA, Horsewood P. Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4. Blood 1994;83:3232–9. 19. Ziporen L, Li ZQ, Park KS, et al. Defining an antigenic epitope on platelet factor 4 associated with heparin-induced thrombocytopenia. Blood 1998;92:3250–9. 20. Bailey RT, Ursick JA, Heim KL, Hilleman DE, Reich JW. Heparin-associated thrombocytopenia: a prospective comparison of bovine lung heparin, manufactured by a new process, and porcine intestinal heparin. Drug Intell Clin Pharm 1986;20:374– 8.
Background. Studies have demonstrated a high incidence of antibodies to heparin/platelet factor 4 complexes, the antigen in heparin-induced thrombocytopenia, in patients after cardiopulmonary bypass surgery. In many hospitals, beef lung heparin has been used historically for cardiopulmonary bypass, and there has been reluctance to change to porcine heparin despite concerns of an increased incidence of heparin-induced thrombocytopenia in patients receiving bovine heparin. Methods. A prospective randomized trial comparing bovine and porcine heparin in cardiopulmonary bypass surgery was conducted. Presurgery and postsurgery heparin antibody formation was studied using the serotonin release assay and a heparin/platelet factor 4 enzymelinked immunosorbent assay.
Results. Data available on 98 patients, randomized to receive either bovine or porcine heparin, revealed no significant difference in patient positivity by serotonin release assay (12% in both groups) or by the heparin/ platelet factor 4 enzyme-linked immunosorbent assay (29% with porcine and 35% with bovine heparin) postoperatively. There were no significant differences between preoperative and postoperative platelet counts or thromboembolic complications. Conclusions. Our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to heparin/platelet factor 4. (Ann Thorac Surg 2001;71:1920 – 4) © 2001 by The Society of Thoracic Surgeons
D
sion, pulmonary embolism, phlegmasia cerulae dolens, and death [2]. A number of recent studies have demonstrated a very high prevalence of antibodies to heparin/ platelet factor 4 (PF4) complexes, the antigen in HIT, in patients post– cardiopulmonary bypass [3– 6]. The clinical significance of the antibody formation is uncertain, but it may predispose patients to the development of HIT with continued heparin exposure or reexposure at a later date. Heparin is prepared from either beef lung or porcine intestinal mucosa. Some case reports and retrospective studies have found a higher incidence of HIT in patients receiving bovine heparin, and some authorities recommend avoidance of beef lung preparations [7]. However, in many centers, beef lung heparin has historically been used for cardiopulmonary bypass, and there has been a reluctance to change preparations. The finding of a high prevalence of heparin-associated antibodies in this setting raised the possibility that a difference between the antigenicity of the two heparin preparations could be detected with a small clinical trial. For this reason, we conducted a prospective double-blind randomized trial comparing the development of heparin-associated antibodies in patients receiving either beef lung or porcine mucosal heparin preparations for anticoagulation during cardiopulmonary bypass.
espite a low but serious incidence of side effects, unfractionated heparin remains the anticoagulant of choice for cardiopulmonary bypass. Alternatives to unfractionated heparin, such as heparinoids, direct thrombin inhibitors, or defibrinogenating agents, have not proved practical for routine use. As a result, most cardiovascular patients are still exposed to potential adverse responses to heparin, the most serious of which is heparin-induced thrombocytopenia (HIT). From a review of retrospective studies, the frequency of HIT has been estimated to be ⬃3% and of HIT with thrombosis to be ⬃1% in patients receiving heparin for 5 days or more [1]. The incidence probably varies by the circumstance in which heparin is administered and the patient population being treated. Heparin-induced thrombocytopenia is reported frequently in patients undergoing cardiopulmonary bypass. One study of 1,500 consecutive patients who underwent cardiopulmonary bypass reported 11 patients (0.75%) with HIT with thrombosis resulting in limb amputations, stroke, graft occluAccepted for publication Feb 7, 2001. Address reprint requests to Dr Konkle, Penn Comprehensive Hemophilia and Thrombosis Program, Hematology-Oncology, University of Pennsylvania, PMC, MAB 103, 39th and Market St, Philadelphia, PA 19104; e-mail: [email protected].
© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
0003-4975/01/$20.00 PII S0003-4975(01)02534-6
Ann Thorac Surg 2001;71:1920 – 4
Material and Methods
KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
Table 1. Patient Characteristics
Prospective Clinical Trial One hundred six consecutive patients undergoing cardiopulmonary bypass surgery were asked to participate in this trial; complete data were available on 98 patients. All patients had been exposed to heparin at some time preoperatively during cardiac catheterization, and some had been treated for unstable angina. Only porcine heparin is used outside of the operating room at Thomas Jefferson University Hospital, where this study was conducted. The protocol was approved by the Institutional Review Board of Thomas Jefferson University, and informed written consent was obtained from all participants. Blood samples for serotonin release assay, heparin/PF4 enzyme-linked immunosorbent assay (ELISA), and platelet count were obtained before the induction of anesthesia on the morning of surgery and again on day 5 postoperatively. Platelet counts were obtained as clinically indicated on additional postoperative days. Patients were randomized to receive either bovine or porcine heparin by the pharmacist, and other caregivers, including the surgeon, were blinded as to the type of heparin received. The heparin-coated Swan Ganz catheters were removed routinely on postoperative day 1, and no patients were exposed to heparin in flush solutions. Therapeutic heparin (porcine; Elkins-Sinn, Cherry Hill, NJ) was administered only in a small percentage of patients postoperatively and did not differ between the two groups.
Anesthetic and Surgical Management A heparin-coated Swan Ganz catheter and a radial arterial line were placed in each patient before induction of anesthesia. High-dose fentanyl (50 to 100 g/kg) was used to induce and maintain anesthesia. Either beef lung (Pharmacia and Upjohn, Peapack, NJ) or porcine mucosal (Elkins-Sinn) heparin was administered at a dose of 300 U/kg before bypass. Additional heparin was given by bolus as needed to achieve a whole-blood activated clotting time of more than 480 seconds. Anticoagulation was maintained at this level throughout bypass. The cardiopulmonary bypass circuit consisted of a membrane oxygenator (Baxter-Bentley, Irvine, CA), closed venous reservoir (Baxter-Bentley), and centrifugal pump (Medtronic-Biomedicus, Eden Prairie, MN). Moderate systemic hypothermia, hemodilution, and blood cardioplegia were used. All patients received ⑀-aminocaproic acid (150 g/kg) to minimize postoperative blood loss. At the end of bypass, heparin activity was reversed with protamine.
Laboratory Assays Heparin-dependent plateletreactive antibodies were detected with a two-point serotonin release assay as described [8], except that heatinactivated plasma was used. Because patient platelets are not used in the assay, aspirin therapy had no effect on the results. Each sample was run in triplicate, and the results were averaged. The test was considered positive when there was a more than 20% serotonin release at 0.1
SEROTONIN RELEASE ASSAY.
1921
Heparin Source Age (years) % male % white Bypass cross-clamp time (min) Intraoperative heparin dose (units)
Porcine (n ⫽ 49)
Bovine (n ⫽ 49)
62 ⫾ 12 76 90 84 ⫾ 37
63 ⫾ 11 76 90 80 ⫾ 36
30,000 ⫾ 13,543
32,452 ⫾ 11,991
U/mL and less than 20% release at 100 U/mL heparin, but no release when patient plasma was added to the platelets in the absence of heparin. HEPARIN/PF4 ELISA. Heat-treated plasma samples were tested using the Asserachrom H PF4 ELISA kit (Diagnostica STAGO, Parsippany, NJ) as previously described [3]. The protocol was followed, and results were interpreted according to the manufacturer’s directions. A negative result was defined as an A492 nm less than 0.25, an intermediate result was defined as an A492 nm greater than or equal to 0.25 and less than 0.5, and a positive result was defined as an A492 nm greater than or equal to 0.5. These cutoff values, recommended by the manufacturer, are based on studies of the heparin/PF4 ELISA in patients with established HIT and in various control populations [9]. An absorbance of 0.5 is more than 8 standard deviations above the values obtained from healthy normal subjects and is a value characteristically seen in patients with HIT. Samples that generate absorbances in the intermediate range of detection are less than 8 standard deviations (ie, less than necessary for a positive result) and more than 2 standard deviations above those of healthy normal subjects.
Statistical Analysis Results are expressed as mean ⫾ standard deviation. For paired data, such as comparison of two assays or preoperative to postoperative comparisons, McNemar’s 2 test was used. Quantitative comparisons of ELISA results were performed using Wilcoxon rank sum statistics.
Results Data from 49 patients who received porcine heparin and 49 patients who received bovine heparin for cardiopulmonary bypass were available. The general patient characteristics of the two groups were similar and are listed in Table 1. The majority of the patients underwent primary coronary revascularization (85%), with a smaller percentage undergoing valve operations (13%) or combined procedures (12%). The percentage of patients testing positive for anti– heparin/PF4 antibodies by ELISA or for heparindependent platelet-reactive antibodies by serotonin release assay preoperatively and postoperatively were
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KONKLE ET AL BOVINE VERSUS PORCINE HEPARIN IN HIT
Ann Thorac Surg 2001;71:1920 – 4
Table 2. Heparin Antibody Test Results Preoperatively and Postoperatively Preoperative
Postoperative
ELISA ELISA ELISA SRA ELISA ELISA ELISA SRA N I P P N I P P Porcine Bovine a
42 38
6 8
1 3
3 3
7 8
28* 24*
14* 17*
6 6
p ⬍ 0.001 versus preoperative values.
ELISA ⫽ enzyme-linked immunosorbent assay; I ⫽ intermediate; N ⫽ negative; P ⫽ positive; SRA ⫽ serotonin release assay.
similar for the two groups, as shown in Table 2. Cardiopulmonary bypass was associated with a significant increase in the percentage of patients developing anti– heparin/PF4 antibodies. This is illustrated for the ELISA results in Figure 1. There was an overall increase in the ELISA optical density reading (bovine heparin: 0.13 ⫾ 0.01 preoperative, 0.55 ⫾ 0.54 postoperative; porcine heparin: 0.19 ⫾ 0.04 preoperative, 0.50 ⫾ 0.27 postoperative), but there was no statistically significant difference in results between those receiving bovine and those receiving porcine heparin (p ⫽ 0.12 for preoperative values; p ⫽ 0.74 for postoperative values). Platelet counts preoperatively and on postoperative days 1 to 5 are illustrated in Figure 2. There was no significant difference between the platelet counts on any of the days tested. The percentage of patients with thrombocytopenia, intraoperatively or on any postoperative day, did not differ between the groups. There also was no significant difference in postoperative thromboembolic complications (Table 3). In the group that received bovine heparin, 2 patients suffered cerebrovascu-
Fig 1. Number of patients testing negative (Neg), intermediate (Int), or positive (Pos), by enzyme-linked immunosorbent assay for anti– heparin/PF4 antibodies preoperatively (Pre-Op) and postoperatively (Post-Op).
Fig 2. Mean platelet count of patients receiving either bovine or porcine heparin preoperatively (Pre-Op) and on days 1 to 5 postoperatively (Post-Op).
lar accidents, and 1 patient experienced a postoperative myocardial infarction. In the group receiving porcine heparin, 3 patients suffered cerebrovascular accidents. These complications were not associated with thrombocytopenia or antibody results that differed from the study group as a whole.
Comment This study confirms previous findings concerning the development of anti– heparin/PF4 antibodies after cardiovascular surgery—an unexpected number of patients have developed antibodies before surgery is even undertaken, and the majority increase these antibodies during the perioperative period. The percentage of patients testing positive by ELISA postoperatively in this study (32%) was less than in our previous study (51%). However, both values are in the range of positivity (25% to 61%) that has been reported in the postbypass state [3– 6]. Additionally, almost all patients had an increase in absorbance reading on the ELISA, with 85% of patients having either intermediate or positive readings postoperatively compared to 18% preoperatively (p ⬍ 0.001). It is surprising that these antibodies do not often result in either thrombocytopenia or thrombosis. Although some patients in our series developed thrombotic complications, there was no correlation with antibody formation or platelet count at the time of the event, and thus no diagnosis of HIT or HIT with thrombosis was made. Nevertheless, the antigenicity of heparin preparations is of importance, because even a small percentage of patients developing HIT with thrombosis results in a substantial human and economic cost. In its most serious form, it can result in devastating generalized thrombosis, resulting in multiple limb losses, strokes, cerebral injury, or death. Although the incidence of HIT with thrombosis is low, the large number of patients undergoing cardiopulmonary bypass makes prevention of this condition an important therapeutic goal.
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Table 3. Thromboembolic Complications Heparin Type
Surgery Type
Complication
Preop SRA
Postop SRA
Preop ELISA
Postop ELISA
Preop Plateleta
Postop Plateleta
Bovine Bovine Bovine Porcine Porcine Porcine
CABG CABG CABG/CEA CABG CABG MVR
MI CVA CVA CVAb CVA, DVT CVA
Neg Neg Pos Neg Neg Neg
Neg Neg Neg Neg Neg Neg
Neg Neg Int Pos Neg Neg
Pos Int Pos Pos Pos Neg
203 277 211 248 176 142
NA 200 196 285 209 249
a
Platelet count ⫻ 103 per mL.
b
With atrial fibrillation.
CABG ⫽ coronary artery bypass grafting; CEA ⫽ carotid endarterectomy; CVA ⫽ cerebrovascular accident; DVT ⫽ deep vein thrombosis; ELISA ⫽ enzyme-linked immunosorbent assay; Int ⫽ intermediate; MI ⫽ myocardial infarction; MVR ⫽ mitral valve replacement; NA ⫽ not available; Neg ⫽ negative; Pos ⫽ positive; SRA ⫽ serotonin release assay.
Effective treatment of cardiovascular disease often requires anticoagulant therapy. Although antiplatelet and thrombolytic therapies are becoming increasingly important in the treatment of acute coronary syndromes, antithrombotic therapy with heparin remains of central importance. For cardiopulmonary bypass, there are no substitutes available for widespread use. The two most commonly used approaches in patients with HIT who require cardiopulmonary bypass is to use anticoagulation with lepirudin [10, 11] or with recombinant hirudin, a direct thrombin inhibitor, or to use danaparoid [12], a heparinoid compound. Neither is FDA approved for this indication, although lepirudin is FDA approved for anticoagulation in patients with HIT and associated thromboembolic disease. Neither of these agents is ideal for this use. Both are excreted renally [13] and require specialized laboratory monitoring. Danaparoid does not affect the activated clotting time or activated partial thromboplastin time (aPTT), requiring antifactor Xa determinations. It also has a very long half-life. Lepirudin has a much shorter half-life, but at the concentrations needed for cardiopulmonary bypass, neither the activated clotting time nor the aPTT reflect drug concentration or activity. The ecarin clotting time has been used successfully in this setting [14] but is not widely available. In addition, there is no antidote to reverse anticoagulation for either lepirudin or danaparoid. The two heparin preparations used in our study might have been expected to induce different antigenic responses. In 1992, Amiral and colleagues [15] reported that heparin-induced antiplatelet antibodies were not directed against heparin, but against heparin/PF4 complexes, a finding confirmed by other investigators [16 – 19]. The affinity of different heparin preparations for PF4 could result in varying degrees of antigenicity. Lowmolecular-weight heparins are thought to be less likely to induce an immunogenic response, presumably because of their low affinity for PF4. However, in laboratory assays, a variety of negatively charged substances can substitute for heparin in the antigen complex, suggesting that the specificity for antibody formation may be low [1]. Some of our patients who received bovine heparin on bypass had received porcine heparin earlier in their hospital course. However, if bovine heparin resulted in
higher antibody formation, one would not expect prior porcine heparin exposure to prevent a further immune stimulation. The differences in the incidence of HIT reported in the past in patients receiving either bovine or porcine heparin may have been caused by the specific composition of the older bovine heparin preparations, which are no longer available. Since those studies were performed, the major manufacturer of bovine heparin and the supplier for our current study changed their manufacturing process. In a small prospective study after that change, the authors did not find a difference in incidence of thrombocytopenia between those receiving bovine (1 of 22) or porcine (0 of 22) heparin anticoagulation for at least 5 days [20]. Despite these changes and findings it is still widely stated that bovine heparin is more likely to result in HIT, even though there are no recent studies to support that statement. It is also possible that the incidence of HIT in our population would have been higher in patients receiving bovine heparin if anticoagulation had been continued. In a study of the effect of different heparin preparations (unfractionated heparin versus low-molecular-weight heparins) used after cardiopulmonary bypass, there was a higher incidence of HIT in patients anticoagulated with unfractionated heparin (source not defined) than with low-molecular-weight heparins, although antibody formation did not differ. However, these groups differed in that almost all patients who received postoperative lowmolecular-weight heparins had undergone coronary artery bypass grafting, and almost all patients who received unfractionated heparin had undergone valve replacement surgery [3]. Better options for anticoagulation during cardiovascular procedures are clearly needed. Alternatives to heparin and heparin-like substances are promising in terms of decreasing the risk of HIT. Compounds that allow easy monitoring and reversibility are greatly needed. However, our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to PF4/heparin or to stimulate increased levels of preexisting antibodies, because unfractionated preparations from either species seem to be equally immunogenic.
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We thank Bernadette Mestichelli, MT (ASCP), for excellent technical assistance. This study was supported, in part, by a Special Purpose Grant from Thomas Jefferson University Hospital and grants HL44956 (Barbara A. Konkle), HL54749 (Mortimer Poncz, Douglas B. Cines), HL40387 (Douglas B. Cines), HL50790 (Douglas B. Cines), HL49517 (Douglas B. Cines), HL37419 (Mortimer Poncz), HL56265 (Mortimer Poncz), and a fellowship award from the Southeastern Pennsylvania Chapter of the American Heart Association (Gowthami Arepally).
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