- Email: [email protected]
heparin antibody induction in patients undergoing percutaneous coronary revascularization
Incidence of Antiplatelet Factor 4/Heparin Antibody Induction in Patients Undergoing Percutaneous Coronary Revascularization Tyler J. Gluckman, MD, Jodi B. Segal, MD, MPH, Natalie L. Fredde, BSN, Kenneth E. Saland, MD, Jayesh T. Jani, MS, MT, Jeanine M. Walenga, PhD, M. Margaret Prechel, PhD, Kathleen M. Citro, BSN, David A. Zidar, MD, Emily Fox, Steven P. Schulman, MD, Thomas S. Kickler, MD, and Jeffrey J. Rade, MD The incidence of antiplatelet factor-4/heparin antibody formation in patients who receive contemporary doses of unfractionated heparin in the setting of percutaneous coronary revascularization is unknown. Also unknown is the ability of these antibodies to activate platelets or adversely affect clinical outcome in the absence of clinically recognized heparin-induced thrombocytopenia. To address these questions, we serially measured antiplatelet factor-4/ heparin antibody levels and performed serotonin release assays in patients who underwent percutaneous coronary intervention. Correlations were then made across antibody induction, heparin exposure, and clinical outcome at 6 months. 䊚2005 by Excerpta Medica Inc. (Am J Cardiol 2005;95:744 –747)
nfractionated heparin (UFH) is the most widely used anticoagulant in patients who undergo perU cutaneous coronary revascularization. Administration of UFH can induce antibodies directed against complexes of heparin and platelet factor-4 (PF4). AntiPF4/heparin antibodies have been implicated in the pathogenesis of heparin-induced thrombocytopenia type II (HIT), a syndrome characterized by immune complex–mediated platelet activation and a high incidence of arterial and venous thromboses.1 Even in the absence of clinically apparent HIT, anti-PF4/heparin antibodies may increase the risk of thrombosis by directly triggering tissue factor expression by endothelial cells and peripheral blood monocytes.2– 4 This concept is supported by 2 recent studies that found a correlation between the presence of anti-PF4/heparin antibodies and adverse clinical outcomes among patients who had acute coronary syndromes without thrombocytopenia.5,6 The goal of the present study was to determine the incidence and potential consequences of anti-PF4/heparin antibody induction in paFrom the Departments of Medicine and Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland; and the Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois. This work was supported in part by a research grant from The Medicines Company, Parsippany, New Jersey. Dr. Rade’s address is: Division of Cardiology, Johns Hopkins School of Medicine, Carnegie 568, 600 N. Wolfe Street, Baltimore, Maryland 21287. E-mail: jjrade@ jhmi.edu. Manuscript received August 6, 2004; revised manuscript received and accepted November 12, 2004.
744
©2005 by Excerpta Medica Inc. All rights reserved. The American Journal of Cardiology Vol. 95 March 15, 2005
BS,
tients who underwent percutaneous coronary revascularization. •••
Patients who underwent elective or urgent percutaneous coronary revascularization at Johns Hopkins Hospital (Baltimore, Maryland) between March 2001 and July 2003 were eligible for inclusion, provided that they received UFH during their procedure. Patients who received UFH for ⬎5 days before percutaneous coronary revascularization or low-molecularweight heparin during the index hospitalization were excluded, as were patients who had cardiogenic shock, respiratory failure, sepsis, or concurrent illness associated with a life expectancy of ⬍6 months. Patients received standard therapy during and after percutaneous coronary revascularization as directed by their physicians. The first platelet count obtained upon admission to a Johns Hopkins Medical Institution was considered the baseline value. Serum and plasma, separated from blood samples collected at the time of percutaneous coronary revascularization and ⬃2 weeks later, were stored in aliquots at ⫺70°C until analyzed. The amount of UFH each patient received was calculated from medical and pharmacy records and included the amount administered before transfer from outside medical facilities. The adverse clinical outcomes of death, myocardial infarction, need for revascularization, and noncardiac thromboembolic events were determined 6 months after percutaneous coronary revascularization by telephone interview and review of the medical records. Any intentionally staged percutaneous coronary revascularization was considered part of the index procedure. Antibodies to PF4/heparin were detected in citrated plasma with a commercially available sandwich-type enzyme-linked immunosorbent assay (Asserachrom HPIA, Diagnostica Stago, Asnieres, France) that recognizes immunoglobulin-G,-A, and -M isotypes. Duplicates of all samples were performed in a blinded fashion, and coefficients of variance routinely averaged ⬍10%. Samples were considered positive if the mean absorbance value exceeded that of a predefined percentage of a lot-specific reference sample (positive threshold range 0.465 to 0.710 absorbance units) supplied with each kit.7 Serotonin release assay was performed on serum samples as described by Sheridan et al.8 Briefly, normal donor platelet-rich plasma collected in acid-citrate-dextrose anticoagulant (pH 4.5, 1 part ⫹ 5 parts 0002-9149/05/$–see front matter doi:10.1016/j.amjcard.2004.11.026
TABLE 1 Patient Demographics
Characteristic Age (yrs)* Body mass index (kg/m2)* White Unstable angina or non–ST-elevation myocardial infraction at presentation ST-elevation myocardial infarction at presentation Previous heparin exposure ⱕ3 mo Previous heparin exposure ⱕ6 mo Platelet count before PCT (K/mm3)* Previous myocardial infarction Previous PCI Previous coronary bypass Previous noncardiac thromboembolic event Diabetes mellitus Hypercholesterolemia Hypertension Tobacco use
Study Cohort (n ⫽ 154) 60 30 123 112
(55–70) (26–33) (80%) (73%)
20 (13%) 12 27 209 52 64 46
(8%) (18%) (174–243) (34%) (42%) (30%) 0
58 135 116 95
(38%) (88%) (75%) (62%)
Antibody Positive at Baseline (n ⫽ 7) 73 31 6 5
(56–75) (26–36) (86%) (71%)
2 (29%) 2 4 196 2 3 1
5 7 6 2
(29%) (57%)† (123–265) (29%) (43%) (14%) 0 (71%) (100%) (86%) (29%)
Antibody Converters (n ⫽ 16) 60 30 14 11
(46–68) (27–31) (88%) (69%)
3 (19%)
3 191 4 4 4
3 13 10 9
0 (18%)† (164–218) (25%) (25%) (25%) 0 (19%) (81%) (63%) (56%)
Antibody Nonconverters (n ⫽ 78) 61 30 62 51
(55–69) (26–35) (79%) (65%)
11 (14%) 6 13 200 28 38 25
35 66 63 50
(8%) (17%) (165–238) (36%) (49%) (32%) 0 (45%) (85%) (81%) (64%)
*Medians (interquartile ranges). † p ⬍0.05 relative to nonconverters. PCI ⫽ percutaneous coronary intervention.
blood) was labeled with 0.1 Ci/ml of carbon-14 serotonin for 30 minutes. Washed, radiolabeled platelets (70 l) were incubated with 20 l of heat-treated (56°C for 45 minutes) patient serum and 10 l of heparin (final concentrations 0.1 and 100 U/ml) for 60 minutes at room temperature with gentle shaking. Ethylenediaminetetraacetic acid was added to stop the release reaction. Platelets were pelleted and radioactivity in the supernatant was quantified by scintillation counting. Samples were considered positive only if there was ⱖ20% release observed at 0.1 U/ml of heparin and ⬍20% release observed at 100 U/ml. Duplicates of all samples were run in blinded fashion, as were appropriate positive and negative controls. Baseline characteristics of patients who had antibodies were compared with those of patients who did not, and significance of differences was tested with Wilcoxon’s signed-rank test and Fisher’s exact test. Techniques of survival analysis, including the logrank test, were used to compare occurrence-free survival of patients who had antibodies with that of patients who did not for the outcomes of thrombosis, myocardial infarction, revascularization, and death. Analyses were performed with STATA 7.0 (STATA Corp., College Station, Texas). The relevant clinical characteristics of the 154 patients who were enrolled in the study are presented in Table 1. Seven of these patients (4.5%, 95% confidence interval 1.8 to 9.4) were positive for anti-PF4/ heparin antibodies at baseline. Of these patients, 4 had received UFH within the previous 6 months and 2 had received UFH 3 days before baseline antibody determination and thus may have been early converters. Two patients who had anti-PF4/heparin antibodies at baseline developed thrombocytopenia (nadir platelet
counts 28,000 and 84,000/mm3) acutely after receiving heparin and eptifibatide during percutaneous coronary revascularization. Although possibly due to early-onset HIT,9 the serotonin release assay in the 2 patients was persistently negative and the rapid recovery of the platelet count was more consistent with eptifibatide-induced thrombocytopenia.10 Of the 94 patients who were negative for antibody at baseline and in whom a follow-up blood sample was obtained, 16 (17.0%, 95% confidence interval 9.7 to 27.6) became positive for the antibody. The median UFH exposure in these 94 patients was 7,400 U. Fifteen of 47 patients (32%, 95% confidence interval 19 to 47) who received ⬎7,400 U of UFH became positive for the antibody compared with only 1 of 47 patients (2%, 95% confidence interval 0.05 to 11) who received ⬍7,400 U. The median total UFH exposure during the index hospitalization in antibody converters was sixfold higher than in patients who remained negative for the antibody (Table 2). Serotonin release assay was performed in parallel on all samples to determine the ability of anti-PF4/ heparin antibodies to stimulate heparin-dependent platelet activation. Only 1 patient was positive for the serotonin release assay at baseline. This patient was negative for the anti-PF4/heparin antibody and had a platelet count of 210,000/mm3 that remained largely unchanged after receiving UFH during percutaneous coronary revascularization. Two patients became positive for serotonin release assay at follow-up. The first was negative for anti-PF4/heparin antibody at baseline and follow-up. The second had high levels of antiPF4/heparin antibodies at baseline and follow-up (mean absorbance values 2.081 and 4.111, respectively). In these 2 patients, platelet counts at the time BRIEF REPORTS
745
TABLE 2 Heparin Exposure Antibody Positive at Baseline (n ⫽ 7)
Antibody Converters (n ⫽ 16)
Antibody Nonconverters (n ⫽ 78)
3,000 (3,000–4,000) 4,000 (3,500–13,250)
2,000 (3,000–3,500) 31,676 (21,732–47,639)†
3,750 (3,000–5000) 5,000 (4,000–22,480)
Variable* Heparin exposure during PCI (U)* Total heparin exposure during hospitalization, units§ Days between index heparin exposure and baseline blood draw Days between index heparin exposure and follow-up blood draw
0 (0–2) 17 (17–18) (n⫽5)
1 (0.5–1)
0 (0–1)
20 (12–36)‡
13 (9–21)
*Medians (interquartile ranges). † p ⬍0.001 relative to antibody nonconverters. ‡ p ⬍0.05 relative to antibody nonconverters. § Includes intravenous and subcutaneous dosing. Abbreviation as in Table 1.
TABLE 3 Procedural Characteristics
Outcome 3
Nadir platelet count after PCI (K/mm )* Percent change in platelet count from baseline to nadir (K/mm3)* Glycoprotein IIb/IIIa inhibitor used Aspirin used ADP-receptor antagonist used Average duration of ADP-receptor antagonist use (mo) Stent deployed Multivessel PCI Target coronary artery Left anterior descending Left circumflex Right Bypass graft
Antibody Positive (n ⫽ 23)
Antibody Negative (n ⫽ 78)
p Value
181 (134–214) 13 (⫺2–19) 23 (100%) 23 (100%) 23 (100%) 3.1 ⫾ 2.3 20 (87%) 2 (9%)
182 (148–216) 10 (2–21) 77 (98%) 78 (100%) 77 (98%) 2.7 ⫾ 2.3 68 (87%) 17 (22%)
0.59 1.0 1.0 1.0 1.0 0.47 1.0 0.23
7 8 7 2
(30%) (35%) (30%) (9%)
23 32 29 12
(29%) (41%) (37%) (15%)
1.0 0.64 0.63 0.51
*Medians (interquartile ranges). ADP ⫽ adenosine diphosphate. Other abbreviation as in Table 1.
TABLE 4 Six-month Clinical Outcomes
Outcome Death Myocardial infarction Repeat revascularization Noncardiac thromboembolic event Combined outcome (death, myocardial infarction, revascularization, or noncardiac thromboembolic event)
Antibody Positive (n ⫽ 23)
Antibody Negative (n ⫽ 78)
p Value
0 0 3 (13%) 0 3 (13%)
0 3(4%) 10 (13%) 0 11 (14%)
1.0 0.34 0.98 1.0 0.87
Values are numbers of patients (percentages).
of the positive serotonin release assays were 335,000 and 307,000/mm3, respectively, and neither was clinically suspected of having HIT. Clinical outcomes of the 78 patients who remained negative for the antibody were compared with those of the 23 patients who were positive for the antibody at baseline or through seroconversion. The groups were similar with regard to procedural characteristics and to the nadir platelet count during the index hospitalization and the percent change from baseline (Table 3). 746 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 95
The incidence of the composite adverse clinical outcome, which was driven primarily by the need for revascularization, did not differ between groups (Table 4). There was also no correlation between absolute antibody levels and outcome (data not shown). •••
There were 3 major findings of this study. (1) The incidence of antiPF4/heparin antibody induction in patients who received UFH in the setting of percutaneous coronary revascularization was substantial and depended on dose. (2) Antibody conversion was infrequently associated with in vitro evidence of heparin-dependent platelet activation or with clinical HIT. (3) The presence of anti-PF4/heparin antibodies did not appear to dramatically affect the combined incidence of death, myocardial infarction, repeat revascularization, or noncardiac thromboembolic events after percutaneous coronary revascularization. Previous studies have documented anti-PF4/heparin antibody conversion rates of 8% to 34% in patients MARCH 15, 2005
who were administered UFH for 5 to 14 days.5,11,12 In contrast, patient exposure to UFH in the present study was relatively limited: ⬃50% of patients received only a single intraprocedural bolus of UFH, and the longest periprocedural infusion of UFH was 5 days in duration. Despite the limited exposure to UFH, the anti-PF4/heparin antibody conversion rate in our population was substantial and was ⬎30% among patients who received ⬎7,400 U. Several factors may have even contributed to an underestimation of the frequency of heparin-dependent antibody induction. First, the enzyme-linked immunosorbent assay used in this study only detects antibodies against complexes of heparin and PF4.7 In ⱕ10% of patients who have suspected HIT, antibodies are formed against complexes of heparin and interleukin-8 or neutrophil-activating peptide-2, which are related chemokines with significant homology to PF4.13 Second, the enzymelinked immunosorbent assay uses a predefined optical density threshold for positivity based on studies of patients who have clinically defined HIT.7 Subthreshold antibody titers could have physiologic consequences in patients who do not have overt HIT. Third, follow-up blood samples were obtained sooner in antibody nonconverters than in converters (Table 2). Although anti-PF4/heparin antibody titers typically begin to increase 5 to 8 days after heparin exposure, a longer lag period may be observed in some patients.14 Some patients whose antibody titer did not reach the positivity threshold might have become positive if tested at a later time point. The clinical significance of anti-PF4/heparin antibodies lies mainly in their ability to cause thrombocytopenia and thrombosis by heparin-dependent platelet activation. In this study, serotonin release assay was used to determine the ability of anti-PF4/heparin antibodies to stimulate heparin-dependent platelet activation. It is considered the gold standard to confirm the diagnosis in patients who have thrombocytopenia and are clinically suspected of having HIT, with a reported specificity of ⬎90% and a sensitivity ⬎80%.8,15 The assay is independent of platelet aggregation and therefore not affected by the presence of glycoprotein IIb/IIIa inhibitors.16,17 Concurrent patient use of an adenosine diphosphate receptor antagonist is also unlikely to affect the assay because diluted serum is tested against normal donor platelets.18 Of the 23 patients who were positive for anti-PF4/ heparin antibodies, only 1 was positive for the serotonin release assay. This patient had very high levels of anti-PF4/heparin antibodies but did not have thrombocytopenia and was not clinically suspected of having HIT. Although a false-positive result cannot be excluded, this patient may have had a subclinical form of HIT. Although HIT is typically characterized by a nadir platelet count ⬍150,000/mm3 and/or a 50% decrease from baseline, a small percentage of patients develop thrombotic events in the absence of thrombocytopenia.19 The presence of anti-PF4/heparin antibodies was also found not to dramatically affect the combined clinical end points assessed 6 months after percutane-
ous coronary revascularization. This is not surprising because repeat revascularization for in-stent restenosis, which was the most frequently observed adverse outcome in this study, is not likely to be significantly influenced by prothrombotic risk factors. The incidence of thrombotically related adverse outcomes, including myocardial infarction, was very low in our study population. This was likely due to aggressive early revascularization with the use of stents in ⬎85% of patients, nearly universal use of glycoprotein IIb/ IIIa inhibitors during percutaneous coronary revascularization, and prolonged use of adenosine diphosphate receptor antagonists for 1 to 6 months thereafter. In addition to suppressing thrombosis, these 2 antiplatelet agents have been shown to inhibit heparininduced platelet aggregation by sera from patients who had HIT.18,20
1. Warkentin T, Kelton JG. A 14-year study of heparin-induced thrombocytope-
nia. Am J Med 1996;101:502–507. 2. Cines DB, Tomaski A, Tannenbaum S. Immune endothelial-cell injury in
heparin-associated thrombocytopenia. N Engl J Med 1987;316:581–589. 3. Pouplard C, Iochmann S, Renard B, Herault O, Colombat P, Amiral J, Gruel Y. Induction of monocyte tissue factor expression by antibodies to heparinplatelet factor 4 complexes developed in heparin-induced thrombocytopenia. Blood 2001;97:3300 –3302. 4. Arepally GM, Mayer IM. Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8. Blood 2001;98:1252–1254. 5. Mattioli AV, Bonetti L, Sternieri S, Mattioli G. Heparin-induced thrombocytopenia in patients treated with unfractionated heparin: prevalence of thrombosis in a 1 year follow-up. Ital Heart J 2000;1:39 – 42. 6. Williams RT, Damaraju LV, Mascelli MA, Barnathan ES, Califf RM, Simoons ML, Deliargyris EN, Sane DC. Anti-platelet factor 4/heparin antibodies: an independent predictor of 30-day myocardial infarction after acute coronary ischemic syndromes. Circulation 2003;107:2307–2312. 7. Woodhams BJ, Grimaux M. Detection of PF4-heparin auto-antibodies by ELISA. Thromb Haemost 1999;82:157–158. 8. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986;67:27–30. 9. Warkentin TE, Kelton JG. Temporal aspects of heparin-induced thrombocytopenia. N Engl J Med 2001;344:1286 –1292. 10. Hongo RH, Brent BN. Association of eptifibatide and acute profound thrombocytopenia. Am J Cardiol 2001;88:428 – 431. 11. Warkentin TE, Levine MN, Hirsh J, Horsewood P, Roberts RS, Gent M, Kelton JG. Heparin-induced thrombocytopenia in patients treated with lowmolecular-weight heparin or unfractionated heparin. N Engl J Med 1995;332: 1330 –1335. 12. Lindhoff-Last E, Nakov R, Misselwitz F, Breddin HK, Bauersachs R. Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated or low-molecular-weight heparin. Br J Haematol 2002;118:1137–1142. 13. Amiral J, Marfaing-Koka A, Wolf M, Alessi MC, Tardy B, Boyer-Neumann C, Vissac AM, Fressinaud E, Poncz M, Meyer D. Presence of autoantibodies to interleukin-8 or neutrophil-activating peptide-2 in patients with heparin-associated thrombocytopenia. Blood 1996;88:410 – 416. 14. Amiral J. Antigens involved in heparin-induced thrombocytopenia. Semin Hematol 1999;36:7–11. 15. Chong BH, Burgess J, Ismail F. The clinical usefulness of the platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost 1993;69:344 –350. 16. Kelton JG, Sheridan D, Santos A, Smith J, Steeves K, Smith C, Brown C, Murphy WG. Heparin-induced thrombocytopenia: laboratory studies. Blood 1988;72:925–930. 17. Jeske WP, Walenga JM, Szatkowski E, Ero M, Herbert JM, Haas S, Bakhos M. Effect of glycoprotein IIb/IIIa antagonists on the HIT serum induced activation of platelets. Thromb Res 1997;88:271–281. 18. Haas S, Walenga JM, Jeske WP, Fareed J. Heparin-induced thrombocytopenia: the role of platelet activation and therapeutic implications. Semin Thromb Hemost 1999;25(suppl 1):67–75. 19. Warkentin T. Clinical presentation of heparin-induced thrombocytopenia. Semin Hematol 1998;35:9 –16. 20. Polgar J, Eichler P, Greinacher A, Clemetson KJ. Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients. Blood 1998; 91:549 –554.
BRIEF REPORTS
747
nfractionated heparin (UFH) is the most widely used anticoagulant in patients who undergo perU cutaneous coronary revascularization. Administration of UFH can induce antibodies directed against complexes of heparin and platelet factor-4 (PF4). AntiPF4/heparin antibodies have been implicated in the pathogenesis of heparin-induced thrombocytopenia type II (HIT), a syndrome characterized by immune complex–mediated platelet activation and a high incidence of arterial and venous thromboses.1 Even in the absence of clinically apparent HIT, anti-PF4/heparin antibodies may increase the risk of thrombosis by directly triggering tissue factor expression by endothelial cells and peripheral blood monocytes.2– 4 This concept is supported by 2 recent studies that found a correlation between the presence of anti-PF4/heparin antibodies and adverse clinical outcomes among patients who had acute coronary syndromes without thrombocytopenia.5,6 The goal of the present study was to determine the incidence and potential consequences of anti-PF4/heparin antibody induction in paFrom the Departments of Medicine and Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland; and the Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois. This work was supported in part by a research grant from The Medicines Company, Parsippany, New Jersey. Dr. Rade’s address is: Division of Cardiology, Johns Hopkins School of Medicine, Carnegie 568, 600 N. Wolfe Street, Baltimore, Maryland 21287. E-mail: jjrade@ jhmi.edu. Manuscript received August 6, 2004; revised manuscript received and accepted November 12, 2004.
744
©2005 by Excerpta Medica Inc. All rights reserved. The American Journal of Cardiology Vol. 95 March 15, 2005
BS,
tients who underwent percutaneous coronary revascularization. •••
Patients who underwent elective or urgent percutaneous coronary revascularization at Johns Hopkins Hospital (Baltimore, Maryland) between March 2001 and July 2003 were eligible for inclusion, provided that they received UFH during their procedure. Patients who received UFH for ⬎5 days before percutaneous coronary revascularization or low-molecularweight heparin during the index hospitalization were excluded, as were patients who had cardiogenic shock, respiratory failure, sepsis, or concurrent illness associated with a life expectancy of ⬍6 months. Patients received standard therapy during and after percutaneous coronary revascularization as directed by their physicians. The first platelet count obtained upon admission to a Johns Hopkins Medical Institution was considered the baseline value. Serum and plasma, separated from blood samples collected at the time of percutaneous coronary revascularization and ⬃2 weeks later, were stored in aliquots at ⫺70°C until analyzed. The amount of UFH each patient received was calculated from medical and pharmacy records and included the amount administered before transfer from outside medical facilities. The adverse clinical outcomes of death, myocardial infarction, need for revascularization, and noncardiac thromboembolic events were determined 6 months after percutaneous coronary revascularization by telephone interview and review of the medical records. Any intentionally staged percutaneous coronary revascularization was considered part of the index procedure. Antibodies to PF4/heparin were detected in citrated plasma with a commercially available sandwich-type enzyme-linked immunosorbent assay (Asserachrom HPIA, Diagnostica Stago, Asnieres, France) that recognizes immunoglobulin-G,-A, and -M isotypes. Duplicates of all samples were performed in a blinded fashion, and coefficients of variance routinely averaged ⬍10%. Samples were considered positive if the mean absorbance value exceeded that of a predefined percentage of a lot-specific reference sample (positive threshold range 0.465 to 0.710 absorbance units) supplied with each kit.7 Serotonin release assay was performed on serum samples as described by Sheridan et al.8 Briefly, normal donor platelet-rich plasma collected in acid-citrate-dextrose anticoagulant (pH 4.5, 1 part ⫹ 5 parts 0002-9149/05/$–see front matter doi:10.1016/j.amjcard.2004.11.026
TABLE 1 Patient Demographics
Characteristic Age (yrs)* Body mass index (kg/m2)* White Unstable angina or non–ST-elevation myocardial infraction at presentation ST-elevation myocardial infarction at presentation Previous heparin exposure ⱕ3 mo Previous heparin exposure ⱕ6 mo Platelet count before PCT (K/mm3)* Previous myocardial infarction Previous PCI Previous coronary bypass Previous noncardiac thromboembolic event Diabetes mellitus Hypercholesterolemia Hypertension Tobacco use
Study Cohort (n ⫽ 154) 60 30 123 112
(55–70) (26–33) (80%) (73%)
20 (13%) 12 27 209 52 64 46
(8%) (18%) (174–243) (34%) (42%) (30%) 0
58 135 116 95
(38%) (88%) (75%) (62%)
Antibody Positive at Baseline (n ⫽ 7) 73 31 6 5
(56–75) (26–36) (86%) (71%)
2 (29%) 2 4 196 2 3 1
5 7 6 2
(29%) (57%)† (123–265) (29%) (43%) (14%) 0 (71%) (100%) (86%) (29%)
Antibody Converters (n ⫽ 16) 60 30 14 11
(46–68) (27–31) (88%) (69%)
3 (19%)
3 191 4 4 4
3 13 10 9
0 (18%)† (164–218) (25%) (25%) (25%) 0 (19%) (81%) (63%) (56%)
Antibody Nonconverters (n ⫽ 78) 61 30 62 51
(55–69) (26–35) (79%) (65%)
11 (14%) 6 13 200 28 38 25
35 66 63 50
(8%) (17%) (165–238) (36%) (49%) (32%) 0 (45%) (85%) (81%) (64%)
*Medians (interquartile ranges). † p ⬍0.05 relative to nonconverters. PCI ⫽ percutaneous coronary intervention.
blood) was labeled with 0.1 Ci/ml of carbon-14 serotonin for 30 minutes. Washed, radiolabeled platelets (70 l) were incubated with 20 l of heat-treated (56°C for 45 minutes) patient serum and 10 l of heparin (final concentrations 0.1 and 100 U/ml) for 60 minutes at room temperature with gentle shaking. Ethylenediaminetetraacetic acid was added to stop the release reaction. Platelets were pelleted and radioactivity in the supernatant was quantified by scintillation counting. Samples were considered positive only if there was ⱖ20% release observed at 0.1 U/ml of heparin and ⬍20% release observed at 100 U/ml. Duplicates of all samples were run in blinded fashion, as were appropriate positive and negative controls. Baseline characteristics of patients who had antibodies were compared with those of patients who did not, and significance of differences was tested with Wilcoxon’s signed-rank test and Fisher’s exact test. Techniques of survival analysis, including the logrank test, were used to compare occurrence-free survival of patients who had antibodies with that of patients who did not for the outcomes of thrombosis, myocardial infarction, revascularization, and death. Analyses were performed with STATA 7.0 (STATA Corp., College Station, Texas). The relevant clinical characteristics of the 154 patients who were enrolled in the study are presented in Table 1. Seven of these patients (4.5%, 95% confidence interval 1.8 to 9.4) were positive for anti-PF4/ heparin antibodies at baseline. Of these patients, 4 had received UFH within the previous 6 months and 2 had received UFH 3 days before baseline antibody determination and thus may have been early converters. Two patients who had anti-PF4/heparin antibodies at baseline developed thrombocytopenia (nadir platelet
counts 28,000 and 84,000/mm3) acutely after receiving heparin and eptifibatide during percutaneous coronary revascularization. Although possibly due to early-onset HIT,9 the serotonin release assay in the 2 patients was persistently negative and the rapid recovery of the platelet count was more consistent with eptifibatide-induced thrombocytopenia.10 Of the 94 patients who were negative for antibody at baseline and in whom a follow-up blood sample was obtained, 16 (17.0%, 95% confidence interval 9.7 to 27.6) became positive for the antibody. The median UFH exposure in these 94 patients was 7,400 U. Fifteen of 47 patients (32%, 95% confidence interval 19 to 47) who received ⬎7,400 U of UFH became positive for the antibody compared with only 1 of 47 patients (2%, 95% confidence interval 0.05 to 11) who received ⬍7,400 U. The median total UFH exposure during the index hospitalization in antibody converters was sixfold higher than in patients who remained negative for the antibody (Table 2). Serotonin release assay was performed in parallel on all samples to determine the ability of anti-PF4/ heparin antibodies to stimulate heparin-dependent platelet activation. Only 1 patient was positive for the serotonin release assay at baseline. This patient was negative for the anti-PF4/heparin antibody and had a platelet count of 210,000/mm3 that remained largely unchanged after receiving UFH during percutaneous coronary revascularization. Two patients became positive for serotonin release assay at follow-up. The first was negative for anti-PF4/heparin antibody at baseline and follow-up. The second had high levels of antiPF4/heparin antibodies at baseline and follow-up (mean absorbance values 2.081 and 4.111, respectively). In these 2 patients, platelet counts at the time BRIEF REPORTS
745
TABLE 2 Heparin Exposure Antibody Positive at Baseline (n ⫽ 7)
Antibody Converters (n ⫽ 16)
Antibody Nonconverters (n ⫽ 78)
3,000 (3,000–4,000) 4,000 (3,500–13,250)
2,000 (3,000–3,500) 31,676 (21,732–47,639)†
3,750 (3,000–5000) 5,000 (4,000–22,480)
Variable* Heparin exposure during PCI (U)* Total heparin exposure during hospitalization, units§ Days between index heparin exposure and baseline blood draw Days between index heparin exposure and follow-up blood draw
0 (0–2) 17 (17–18) (n⫽5)
1 (0.5–1)
0 (0–1)
20 (12–36)‡
13 (9–21)
*Medians (interquartile ranges). † p ⬍0.001 relative to antibody nonconverters. ‡ p ⬍0.05 relative to antibody nonconverters. § Includes intravenous and subcutaneous dosing. Abbreviation as in Table 1.
TABLE 3 Procedural Characteristics
Outcome 3
Nadir platelet count after PCI (K/mm )* Percent change in platelet count from baseline to nadir (K/mm3)* Glycoprotein IIb/IIIa inhibitor used Aspirin used ADP-receptor antagonist used Average duration of ADP-receptor antagonist use (mo) Stent deployed Multivessel PCI Target coronary artery Left anterior descending Left circumflex Right Bypass graft
Antibody Positive (n ⫽ 23)
Antibody Negative (n ⫽ 78)
p Value
181 (134–214) 13 (⫺2–19) 23 (100%) 23 (100%) 23 (100%) 3.1 ⫾ 2.3 20 (87%) 2 (9%)
182 (148–216) 10 (2–21) 77 (98%) 78 (100%) 77 (98%) 2.7 ⫾ 2.3 68 (87%) 17 (22%)
0.59 1.0 1.0 1.0 1.0 0.47 1.0 0.23
7 8 7 2
(30%) (35%) (30%) (9%)
23 32 29 12
(29%) (41%) (37%) (15%)
1.0 0.64 0.63 0.51
*Medians (interquartile ranges). ADP ⫽ adenosine diphosphate. Other abbreviation as in Table 1.
TABLE 4 Six-month Clinical Outcomes
Outcome Death Myocardial infarction Repeat revascularization Noncardiac thromboembolic event Combined outcome (death, myocardial infarction, revascularization, or noncardiac thromboembolic event)
Antibody Positive (n ⫽ 23)
Antibody Negative (n ⫽ 78)
p Value
0 0 3 (13%) 0 3 (13%)
0 3(4%) 10 (13%) 0 11 (14%)
1.0 0.34 0.98 1.0 0.87
Values are numbers of patients (percentages).
of the positive serotonin release assays were 335,000 and 307,000/mm3, respectively, and neither was clinically suspected of having HIT. Clinical outcomes of the 78 patients who remained negative for the antibody were compared with those of the 23 patients who were positive for the antibody at baseline or through seroconversion. The groups were similar with regard to procedural characteristics and to the nadir platelet count during the index hospitalization and the percent change from baseline (Table 3). 746 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 95
The incidence of the composite adverse clinical outcome, which was driven primarily by the need for revascularization, did not differ between groups (Table 4). There was also no correlation between absolute antibody levels and outcome (data not shown). •••
There were 3 major findings of this study. (1) The incidence of antiPF4/heparin antibody induction in patients who received UFH in the setting of percutaneous coronary revascularization was substantial and depended on dose. (2) Antibody conversion was infrequently associated with in vitro evidence of heparin-dependent platelet activation or with clinical HIT. (3) The presence of anti-PF4/heparin antibodies did not appear to dramatically affect the combined incidence of death, myocardial infarction, repeat revascularization, or noncardiac thromboembolic events after percutaneous coronary revascularization. Previous studies have documented anti-PF4/heparin antibody conversion rates of 8% to 34% in patients MARCH 15, 2005
who were administered UFH for 5 to 14 days.5,11,12 In contrast, patient exposure to UFH in the present study was relatively limited: ⬃50% of patients received only a single intraprocedural bolus of UFH, and the longest periprocedural infusion of UFH was 5 days in duration. Despite the limited exposure to UFH, the anti-PF4/heparin antibody conversion rate in our population was substantial and was ⬎30% among patients who received ⬎7,400 U. Several factors may have even contributed to an underestimation of the frequency of heparin-dependent antibody induction. First, the enzyme-linked immunosorbent assay used in this study only detects antibodies against complexes of heparin and PF4.7 In ⱕ10% of patients who have suspected HIT, antibodies are formed against complexes of heparin and interleukin-8 or neutrophil-activating peptide-2, which are related chemokines with significant homology to PF4.13 Second, the enzymelinked immunosorbent assay uses a predefined optical density threshold for positivity based on studies of patients who have clinically defined HIT.7 Subthreshold antibody titers could have physiologic consequences in patients who do not have overt HIT. Third, follow-up blood samples were obtained sooner in antibody nonconverters than in converters (Table 2). Although anti-PF4/heparin antibody titers typically begin to increase 5 to 8 days after heparin exposure, a longer lag period may be observed in some patients.14 Some patients whose antibody titer did not reach the positivity threshold might have become positive if tested at a later time point. The clinical significance of anti-PF4/heparin antibodies lies mainly in their ability to cause thrombocytopenia and thrombosis by heparin-dependent platelet activation. In this study, serotonin release assay was used to determine the ability of anti-PF4/heparin antibodies to stimulate heparin-dependent platelet activation. It is considered the gold standard to confirm the diagnosis in patients who have thrombocytopenia and are clinically suspected of having HIT, with a reported specificity of ⬎90% and a sensitivity ⬎80%.8,15 The assay is independent of platelet aggregation and therefore not affected by the presence of glycoprotein IIb/IIIa inhibitors.16,17 Concurrent patient use of an adenosine diphosphate receptor antagonist is also unlikely to affect the assay because diluted serum is tested against normal donor platelets.18 Of the 23 patients who were positive for anti-PF4/ heparin antibodies, only 1 was positive for the serotonin release assay. This patient had very high levels of anti-PF4/heparin antibodies but did not have thrombocytopenia and was not clinically suspected of having HIT. Although a false-positive result cannot be excluded, this patient may have had a subclinical form of HIT. Although HIT is typically characterized by a nadir platelet count ⬍150,000/mm3 and/or a 50% decrease from baseline, a small percentage of patients develop thrombotic events in the absence of thrombocytopenia.19 The presence of anti-PF4/heparin antibodies was also found not to dramatically affect the combined clinical end points assessed 6 months after percutane-
ous coronary revascularization. This is not surprising because repeat revascularization for in-stent restenosis, which was the most frequently observed adverse outcome in this study, is not likely to be significantly influenced by prothrombotic risk factors. The incidence of thrombotically related adverse outcomes, including myocardial infarction, was very low in our study population. This was likely due to aggressive early revascularization with the use of stents in ⬎85% of patients, nearly universal use of glycoprotein IIb/ IIIa inhibitors during percutaneous coronary revascularization, and prolonged use of adenosine diphosphate receptor antagonists for 1 to 6 months thereafter. In addition to suppressing thrombosis, these 2 antiplatelet agents have been shown to inhibit heparininduced platelet aggregation by sera from patients who had HIT.18,20
1. Warkentin T, Kelton JG. A 14-year study of heparin-induced thrombocytope-
nia. Am J Med 1996;101:502–507. 2. Cines DB, Tomaski A, Tannenbaum S. Immune endothelial-cell injury in
heparin-associated thrombocytopenia. N Engl J Med 1987;316:581–589. 3. Pouplard C, Iochmann S, Renard B, Herault O, Colombat P, Amiral J, Gruel Y. Induction of monocyte tissue factor expression by antibodies to heparinplatelet factor 4 complexes developed in heparin-induced thrombocytopenia. Blood 2001;97:3300 –3302. 4. Arepally GM, Mayer IM. Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8. Blood 2001;98:1252–1254. 5. Mattioli AV, Bonetti L, Sternieri S, Mattioli G. Heparin-induced thrombocytopenia in patients treated with unfractionated heparin: prevalence of thrombosis in a 1 year follow-up. Ital Heart J 2000;1:39 – 42. 6. Williams RT, Damaraju LV, Mascelli MA, Barnathan ES, Califf RM, Simoons ML, Deliargyris EN, Sane DC. Anti-platelet factor 4/heparin antibodies: an independent predictor of 30-day myocardial infarction after acute coronary ischemic syndromes. Circulation 2003;107:2307–2312. 7. Woodhams BJ, Grimaux M. Detection of PF4-heparin auto-antibodies by ELISA. Thromb Haemost 1999;82:157–158. 8. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986;67:27–30. 9. Warkentin TE, Kelton JG. Temporal aspects of heparin-induced thrombocytopenia. N Engl J Med 2001;344:1286 –1292. 10. Hongo RH, Brent BN. Association of eptifibatide and acute profound thrombocytopenia. Am J Cardiol 2001;88:428 – 431. 11. Warkentin TE, Levine MN, Hirsh J, Horsewood P, Roberts RS, Gent M, Kelton JG. Heparin-induced thrombocytopenia in patients treated with lowmolecular-weight heparin or unfractionated heparin. N Engl J Med 1995;332: 1330 –1335. 12. Lindhoff-Last E, Nakov R, Misselwitz F, Breddin HK, Bauersachs R. Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated or low-molecular-weight heparin. Br J Haematol 2002;118:1137–1142. 13. Amiral J, Marfaing-Koka A, Wolf M, Alessi MC, Tardy B, Boyer-Neumann C, Vissac AM, Fressinaud E, Poncz M, Meyer D. Presence of autoantibodies to interleukin-8 or neutrophil-activating peptide-2 in patients with heparin-associated thrombocytopenia. Blood 1996;88:410 – 416. 14. Amiral J. Antigens involved in heparin-induced thrombocytopenia. Semin Hematol 1999;36:7–11. 15. Chong BH, Burgess J, Ismail F. The clinical usefulness of the platelet aggregation test for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost 1993;69:344 –350. 16. Kelton JG, Sheridan D, Santos A, Smith J, Steeves K, Smith C, Brown C, Murphy WG. Heparin-induced thrombocytopenia: laboratory studies. Blood 1988;72:925–930. 17. Jeske WP, Walenga JM, Szatkowski E, Ero M, Herbert JM, Haas S, Bakhos M. Effect of glycoprotein IIb/IIIa antagonists on the HIT serum induced activation of platelets. Thromb Res 1997;88:271–281. 18. Haas S, Walenga JM, Jeske WP, Fareed J. Heparin-induced thrombocytopenia: the role of platelet activation and therapeutic implications. Semin Thromb Hemost 1999;25(suppl 1):67–75. 19. Warkentin T. Clinical presentation of heparin-induced thrombocytopenia. Semin Hematol 1998;35:9 –16. 20. Polgar J, Eichler P, Greinacher A, Clemetson KJ. Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients. Blood 1998; 91:549 –554.
BRIEF REPORTS
747