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Ann Thorac Surg 2001;71:1920-1924
© 2001 The Society of Thoracic Surgeons

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Original article: cardiovascular

Heparin-induced thrombocytopenia: bovine versus porcine heparin in cardiopulmonary bypass surgery

^a Department of Medicine, Cardeza Foundation for Hematologic Research, Philadelphia, Pennsylania, USA
^b Department of Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylania, USA
^c Department of Anesthesiology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylania, USA
^d Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylania, USA
^e Division of Pediatric Hematology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Accepted for publication February 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: konkleb{at}mail.med.upenn.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
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 enzyme-linked 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.

	Introduction

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Despite 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 occlusion, 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.

	Material and methods

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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 µg/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 µg/kg) to minimize postoperative blood loss. At the end of bypass, heparin activity was reversed with protamine.

Laboratory assays
Serotonin release assay
Heparin-dependent platelet-reactive antibodies were detected with a two-point serotonin release assay as described [8], except that heat-inactivated 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 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 A_{492 nm} less than 0.25, an intermediate result was defined as an A_{492 nm} greater than or equal to 0.25 and less than 0.5, and a positive result was defined as an A_{492 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

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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%).

View larger version (51K): [in this window] [in a new window]   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).

View larger version (24K): [in this window] [in a new window]   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).

	Comment

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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. Low-molecular-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 low-molecular-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.

	Acknowledgments

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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).

	References

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