Heparin-platelet factor 4 antibodies are frequent after vascular surgery but are not a frequent cause of graft thrombosis or thrombocytopenia
Article Outline
Objective
Approximately 10% of infrainguinal bypass surgeries are complicated by early conduit failure. The cause is unclear in most cases. A prospective study was conducted to monitor the development and function of platelet factor 4 (PF4)/heparin antibodies after infrainguinal bypass procedures and to evaluate their clinical significance in early graft occlusion.
Methods
Blood samples were obtained before surgery and at the 7-, 14-, and 28-day postsurgical evaluation. Relevant demographic and laboratory data were collected, and plasma samples were assayed for the presence and function of PF4/heparin-antibody by enzyme-linked immunosorbent assay (ELISA) and a two-point platelet aggregation assay. All tests were performed in duplicate or triplicate.
Results
Of the 79 patients who were enrolled, 67 reported previous heparin exposure. Six patients (7.6%) tested positive for the presence of PF4/heparin antibodies before surgery with ELISA, and four of these (67%) also had a positive result on the aggregation assay. During the 28-day follow-up, 22 subjects (32%) converted to positive according to the ELISA results; and five (22.7%) of these also tested positive for platelet-activating antibodies. No participants presented with thrombocytopenia or a ≥50% decrease in platelet count during the study period. Early graft occlusion was detected in three patients, all with negative ELISA and functional assay results throughout the study.
Conclusion
Patients undergoing vascular surgery frequently develop PF4/heparin antibodies, with platelet-activating antibodies detected in up to 11% of these individuals. However, thrombocytopenia and vascular graft thrombosis both appear to be an uncommon consequence.
Approximately 10 million individuals have symptomatic peripheral artery disease in the United States, and almost 100,000 reconstructive procedures performed annually.1 Owing to the refinements in conduit selection and operative techniques, aortoiliac and femoropopliteal bypass surgeries can provide effective revascularization and a favorable immediate operative result for most patients, thus reducing the incidence and complications of peripheral ischemia.2
However, subsequent graft closure is not uncommon and poses a significant clinical problem. Early graft failure is observed in up to 10% of patients owing to the acute thrombotic occlusion of the conduit.3 The incidence of late graft stenosis is reported to reach 30% due to intimal hyperplasia at the proximal anastomosis or near the valve leaflets.4 Advanced atherosclerosis of the contiguous arteries predisposes these vessels to clamp injury during the bypass procedure, further increasing the risk of late graft failure.
The underlying cause of early conduit occlusion is less clearly understood, with multiple possible pathologic mechanisms implicated. These can involve anatomic or structural defects of the bypass graft promoting early thrombosis, postsurgical conditions leading to a prothrombotic state, inflow disease, or poor runoff.3, 5 We recently observed recurrent, early conduit thrombosis in two patients after lower extremity bypass procedures who were subsequently diagnosed with heparin-induced thrombocytopenia (HIT). Thus, we conducted this prospective study to evaluate the development of platelet factor 4 (PF4)/heparin antibodies after infrainguinal bypass surgery and to explore the pathophysiologic significance of HIT in early graft occlusion.
Methods
Consecutive adult patients undergoing infrainguinal bypass surgery for atherosclerotic occlusive vascular disease at the Los Angeles County-University of Southern California (USC) Medical Center or USC University Hospital were eligible for the study. All relevant demographic and clinical data were collected, and serial measurements of enzyme-linked immunosorbent assay (ELISA) were performed with platelet aggregometry to test for the titer and activity of PF4/heparin antibodies. No routine preoperative screening was used with ELISA or platelet aggregometry. Samples in the present study were batch tested so that assay results were often not available until days to weeks after surgery, with the exception of patients with suspected HIT, who immediately underwent laboratory testing.
Clinical HIT was defined by the development of thrombocytopenia (platelet count <100,000/μL) or a ≥50% drop in platelet count, with or without thrombosis. Enrolled patients received only a single dose of unfractionated heparin intraoperatively (average dose, 6088 ± 1246 U). No patients were re-exposed to heparin in the postoperative period. All participants provided informed consent, and the study was approved by the Human Subjects Committee of the Keck School of Medicine, USC.
Exclusion criteria included failure to sign informed consent, uncertain patient follow-up, individuals with known allergy to heparin, and any history of HIT. A 30-mL blood sample was obtained by sterile antecubital venipuncture into citrate (3.2%) from eligible subjects preoperatively on the day of surgery (before the administration of heparin) and at the 7-, 14-, and 28-day postsurgical evaluation. Plasma was isolated by centrifugation (2500g, 10 minutes) and stored at −80°C until assayed.
All samples were tested for the presence of PF4/heparin antibodies using a commercially available ELISA kit (Asserachrom HPIA, Diagnostica Stago, Parsippany, NJ). Absorbance was measured at 492 nm; the cutoff value for a positive test result was specific for each assay and was provided by the manufacturer. For the purposes of the study, positive ELISA results were analyzed in quartiles, with quartile 4 representing subjects with the highest antibody titer. Tests were performed in duplicate or triplicate.
All ELISA-positive and selected negative patient samples were further tested for their ability to promote platelet activation using a two-point aggregation assay. Initially, 12 healthy, drug-free volunteers were prescreened for platelet reactivity to PF4/heparin antibodies using plasma samples known to be positive for HIT, and four sensitive donors were identified. Approximately 40 mL of blood was collected from these volunteers by sterile antecubital venipuncture into anticoagulant citrate dextrose solution. Samples were centrifuged immediately at 150g for 10 minutes to separate platelet-rich plasma (PRP). Meanwhile, a batch of patient plasmas was defrosted in a 37°C waterbath, followed by heat inactivation (56°C for 45 minutes) and centrifugation (2500g, 10 minutes). Supernatants were removed and used as platelet-poor plasma (PPP) for the functional assay.
Aliquots of 145 μL of PRP and 145 μL of test PPP were combined and preincubated at 37°C for 3 minutes in plastic corvettes. Upon testing, 10 μL of unfractionated heparin (American Pharmaceuticals Partners, Inc, Schamburg, Ill) was added to the samples, yielding the final concentrations of 0.5 U/mL and 100 U/mL. An equal volume of physiologic saline was mixed to the control corvettes (negative control).
Platelet function was monitored and aggregation curves were recorded at 37°C for 15 minutes with a Carat TX-4 platelet aggregometer (Carat Ltd, Budapest, Hungary), using the turbidimetric method described by Born and Cross.6 Maximal aggregation was expressed as a percentage of 100% light transmission, calibrated for each specimen. The functional assay was considered positive if the change in optical density exceeded 20% upon the addition of 0.5 U/mL heparin, with no aggregation in the presence of 100 U/mL of heparin and in the control sample.
Platelet response was deemed nonspecific and thus negative if positive assay results were obtained with both heparin concentrations tested. Negative samples were retested with adenosine diphosphate (final concentration, 5μM), collagen (2 μg/mL), and epinephrine (10μM) to ensure the viability of donor platelets. All functional assays were performed in duplicate and were repeated on at least two sensitive donors to reduce false-positive and false-negative results. To be deemed a positive assay, positive aggregation assays had to be obtained from both sensitive platelet donors.
Results
The study enrolled 79 subjects (42 men, 37 women; mean age, 67 ± 12 years) undergoing infrainguinal bypass surgery. An autogenous conduit was used in 68 patients, a prosthetic graft in nine, and a biograft in two. No participants had significant liver function impairment or any family history for coagulation or bleeding disorders. Seventy patients (89%) reported previous heparin exposure. Detailed characteristics of the study population are summarized in Table I.
Table 1. Patient characteristics
| Variables | All patients | ELISA+ | ELISA− |
|---|---|---|---|
| Subjects, No. | 79 | 28 | 51 |
| Age, mean (SD) years | 67 (12) | 65(9) | 69(11) |
| Males/females | 0.54 | 0.64 | 0.45 |
| Average heparin dose, mean (SD) U | 6088(1246) | 6026(1241) | 6167(1291) |
| Previous heparin exposure, % | 84.8 | 85.7 | 84.3 |
| Previous warfarin exposure, % | 20.3 | 28.6 | 15.7 |
| Hypertension, % | 87.3 | 89.3 | 86.3 |
| Diabetes mellitus, % | 73.4 | 71.4 | 74.5 |
| History of thrombocytopenia, % | 5.1 | 3.6 | 5.9 |
| History of DVT, % | 10.1 | 10.7 | 9.8 |
| Positive functional assay, % | 11.4 | 32.1 | 0 |
Of the 79 enrolled subjects, 28 (35.4%) had at least one ELISA-positive blood sample, with nine (11%) positive in the aggregation assay. Six patients (7.6%) tested positive for PF4/heparin antibody by ELISA before the surgery, with four of the six specimens (67%) also positive in the functional assay. Antibody titers increased after surgery in four of the six patients. Seventy-three subjects (92%) provided all four blood samples described in the protocol, only the presurgical specimen was collected from six patients (8%). Of the 68 follow-up patients, 22 (32.3%) with a negative presurgical test result produced antibodies to the PF4/heparin complex during the postsurgical evaluations. Two of the nine patients with no previous heparin exposure seroconverted in the postoperative period. All participants developed a positive ELISA result by day 14 (Table II). Five subjects (22.7%) tested positive in the functional platelet assay.
Table II. Follow-up laboratory data
| Pre-op (n = 79) | Day 7 (n = 68) | Day 14 (n = 68) | Day 28 (n = 68) | |||||
|---|---|---|---|---|---|---|---|---|
| ELISA+ | ELISA− | ELISA+ | ELISA− | ELISA+ | ELISA− | ELISA+ | ELISA− | |
| Patients, No. (%) | 6(7.6) | 73(92.4) | 14(20.6) | 54(79.4) | 23(33.8) | 45(66.2) | 17(25.0) | 51(75.0) |
| New ELISA+ patients, No. | 6 | … | 10 | … | 12 | … | 0 | … |
| Positive functional assay (n) | 4 | 0 | 3 | 0 | 1 | 0 | 1 | 0 |
| Platelet count, mean (SD) ×109/L | 251.0 (124.5) | 276.9 (87.05) | 340.3 (150.0) | 339.5 (76.2) | 329.0 (141.0) | 410.0 (94.4) | 287.2 (124.4) | 286.7 (69.4) |
| Patients with HIT, No.a | None | None | None | None | None | None | None | None |
| Early graft occlusion, No. | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 |
aClinical HIT was defined as the development of thrombocytopenia (platelet count <100,000/μL) or a ≥50% drop in platelet count, with or without thrombosis. |
No patient in our series showed any laboratory assays results, clinical signs, or symptoms suggestive for HIT. Also, none of the ELISA-positive patients developed early graft closure, despite the presence of platelet-activating antibodies in nine subjects (Table II). In contrast, early conduit occlusion was detected in three participants, and all of them tested negative with ELISA and the functional assay.
Discussion
HIT is of special interest for vascular surgeons inasmuch as heparin is the predominant anticoagulant used to prevent or treat perioperative thrombotic complications. It represents a potentially serious acquired form of drug-induced autoimmune disorder characterized by antibody formation against the complex of heparin and PF4.7, 8 Antibodies of the immunoglobulin G subclass bind to platelets through the FcγRIIa receptor and induce their activation, microparticle release, and thrombin generation, thus promoting a hypercoagulable state.9, 10, 11, 12 The development of a new thrombotic complication, a reduction in platelet count >50%, or unexplained heparin resistance are possible clinical indicators for HIT, typically appearing 5 to 10 days after heparin exposure.13, 14 Once HIT is suspected, rapid laboratory confirmation is required; the most commonly used tests include ELISA to detect the titer and two functional assays (ie, serotonin release assay, two-point platelet aggregation test) to explore the functionality of PF4/heparin antibodies.15, 16
The incidence of HIT is dependent on the patient population studied and is reported to be 0.3% to 2.7%; HIT complications, such as HIT thrombosis (HITT), are detected in approximately 50% of these subjects.17, 18 Venous or arterial thrombi might develop days or even weeks after heparin exposure, suggesting that HIT might be implicated in early graft occlusion after lower extremity bypass procedures.19, 20 Despite the potentially serious clinical complications of HIT, only a limited number of prospective studies have focused on patients undergoing vascular surgery. Jackson et al21 used ELISA to evaluate the development of PF4/heparin antibodies in 54 patients. They found that only one subject converted to positive after surgery, and one thrombotic complication was recorded that was independent of the patient's ELISA status. No functional platelet tests were performed in these series, however.
Calaitges et al22 conducted a prospective study to investigate the incidence and clinical significance of heparin-associated antiplatelet antibodies in 106 subjects undergoing major vascular reconstruction. A two-point platelet aggregation assay was performed before surgery and at least 4 days after the intervention; 22 patients (21%) had at least one positive test result. Although statistically not significant (P = .21), the authors found that the presence of functionally active heparin-associated antiplatelet antibodies is associated with a 2.6-fold increased risk for perioperative thrombotic complications, with thrombocytopenia being an unreliable clinical marker.
Lindhoff-Last et al23 report their experience with 50 patients undergoing vascular surgery. All participants received intravenous unfractionated heparin for ≥5 days preoperatively. The therapy was continued for 7 days after surgery and was followed by the administration of subcutaneous low-molecular-weight heparin for 9 days.23 After ceasing the heparin treatment, 34% of patients were positive in the PF4/heparin antibody ELISA and 14% in the functional test, with no association between the assay results and the clinical manifestations of HIT.
The purpose of this prospective study was to determine the incidence of PF4/heparin antibodies by ELISA and test their functionality using a two-point platelet aggregation assay in patients undergoing infrainguinal reconstructive surgery. Assay results and short-term surgical outcome of incidence of early graft thrombosis ≤28 days postoperatively were also compared to evaluate the clinical significance of PF4/heparin antibodies.
Pre-existing PF4/heparin antibodies were detected in six subjects (7.6%) before the bypass procedure, despite the extensive history of heparin exposure in our study population. When present, however, these antibodies were found to be functionally active in the platelet aggregation assay in four patients (67%). After the single unfractionated heparin exposure used in the present study, ELISA-detected PF4/heparin antibodies developed in 22 patients (32%). The incidence of seroconversion increased from 14.4% on postoperative day 7 to 17.6% on day 14, with no new ELISA-positive patients detected on day 28. Of the nine patients with no previous heparin exposure, two were identified to have PF4/heparin antibodies present by postoperative day 14.
Five of the 22 subjects (23%) tested positive with platelet aggregometry; the antibody titer of these patients were all in the highest quartile, as determined by ELISA. However, not all subjects in quartile 4 tested positive in the functional assay; thus, we were unable to establish a close correlation between the results of the two laboratory tests.
Despite the frequent appearance of PF4/heparin antibodies after infrainguinal bypass surgery, none of our patients developed thrombocytopenia or any other clinical symptoms characteristic for HIT. This observation most likely is due to the single intraoperative heparin exposure and the absence at our institution of routine postoperative heparin infusions after lower extremity revascularization. However, the documentation of nine patients having PF4/heparin antibodies with positive platelet aggregation studies in the postoperative period suggests that prolonged heparin infusions may put patients at risk for HIT and HITT and, consequently, should be used sparingly. Also, the titer and biologic activity of PF4/heparin antibodies showed no correlation with the incidence of early conduit failure in our study population. Three patients presented with early graft thrombosis on postoperative day 7; however, samples from all these subjects tested negative with ELISA and did not promote platelet aggregation in the functional assay.
Conclusion
Patients undergoing vascular surgery frequently develop PF4/heparin antibodies, with platelet-activating antibodies detected in up to 11% of the population. However, both thrombocytopenia and early conduit thrombosis appear to be an uncommon consequence.
Author contributions
We would like to thank Leanne Rochanda for her excellent technical support.
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Competition of interest: none.
This study was partly supported by an unrestricted research grant from Glaxo Smith Kline and research support from Max Gondon.
PII: S0741-5214(08)00440-0
doi:10.1016/j.jvs.2008.03.028
© 2008 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
