COAGULATION AND TRANSFUSION MEDICINE Original Article

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1 COAGULATION AND TRANSFUSION MEDICINE Heparin-induced Platelet Aggregation Vs Platelet Factor 4 Enzyme-Linked Immunosorbent Assay in the Diagnosis of Heparin-induced Thrombocytopenia-Thrombosis KELLY A. LOOK, MD, MERVYN SAHUD, MD, 2 SHEILA FLAHERTY, MT (ASCP), 2 AND JAMES L. ZEHNDER, MD 3 Thrombosis occurs in an unpredictable subset of patients with heparin-induced thrombocytopenia (HIT). The diagnosis of HIT requires clinical suspicion and laboratory confirmation. Although the "gold-standard" diagnostic test is considered to be the serotonin release assay (SRA), most laboratories use heparininduced platelet aggregation (HIPA), which is highly specific but reported to be less sensitive than the SRA. Recently, the heparinplatelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) has been reported to have comparable sensitivity to the SRA. We compared the HIPA and PF4 ELISA in serum samples from 46 patients examined for HIT and assessed whether either test predicted thrombotic risk. Results for 8 patients were positive for HIPA, PF4 ELISA, or both. Of these, 9% were HIPA-positive, while only 60% were PF4 ELISA-positive. Clinical information was available on 63 patients, 7 of whom had thrombotic events (0 venous, 6 arterial, and both). Neither the HIPA nor the PF4 ELISA predicted thrombotic risk, but the HIPA proved to be a more sensitive test for laboratory confirmation. (Key words: Heparin-induced thrombocytopenia; Heparin-induced thrombocytopenia-thrombosis; Thrombosis; Heparin-induced platelet aggregation; Platelet factor 4 ELISA) Am J Clin Pathol 997;08: Heparin-induced thrombocytopenia (HIT) occurs in 5% to 7% of persons requiring heparin therapy or prophylaxis. -5 The degree of thrombocytopenia is variable, but usually is less than 60 x 0 9 /L and occurs 5 to 7 days after heparin exposure. 6 Despite the thrombocytopenia, bleeding complications are uncommon. However, heparin-induced thrombocytopenia-thrombosis (HITT), arterial or venous, develops in an unpredictable subset of persons. The thrombotic complications are associated with a high mortality rate and risk of amputation. There is no explanation for what places a person at risk for HITT and, therefore, no way to predict in which patients the thrombotic complications will develop. The thrombocytopenia in HIT is due to an IgG directed at a heparin-platelet factor 4 (PF4) complex, ' 7-0 From the Department of Pediatric Hematology/Oncology, Stanford University Medical Center, Stanford, California; 2 The Coagulation Center, Oakland, California; and the ^Department of Pathology, Stanford University Medical Center, Stanford, California. Manuscript received November, 996; revision accepted January 6,997. Address reprint requests to Dr Zehnder: Department of Pathology, L235, Stanford University Medical Center, Stanford, CA which binds to platelet Fc receptors resulting in platelet activation, aggregation, and further release of PF4, potentiating the process. Occupation of the Fc receptors has been reported to generate microparticles with procoagulant activity, providing one pathophysiologic explanation for thrombosis in HIT. Alternatively, antibodies binding to PF4-heparan sulfate complexes on vascular endothelium also may induce endothelial injury and thrombosis. The laboratory tests most commonly used for confirming HIT include the serotonin release assay (SRA), the heparin-induced platelet aggregation (HIPA), and a newer PF4 enzyme-linked immunosorbent assay (ELISA). The SRA is considered the "gold standard," however, few laboratories in the United States perform this assay. The most commonly used test is the HIPA, which is highly specific, but reported to have variable sensitivity (30%-80%) The ELISA has been reported to have sensitivity and specificity comparable with that of the SRA, 5 but other investigators have found that in approximately 0% of patients with clinically evident HIT, the assay will show false-negative results. 6 Data comparing the HIPA and PF4 ELISA are lacking. Although the HIPA and PF4 ELISA have proved useful in confirming the diagnosis of HIT, their utility 78

2 LOOK ET AL 79 Diagnosis of Heparin-Induced Thromboa/topenia-Thrombosis in predicting or determining persons at risk for thrombotic complications has not been studied. We compared the sensitivity of the HIPA with that of the PF4 ELISA in the laboratory confirmation of HIT and determined whether either test predicted thrombotic risk. Patients PATIENTS, MATERIALS, AND METHODS We studied the serum samples of 46 patients with possible HIT. All patients had received 5 or more days of heparin therapy and had decreases in platelet counts of at least 50% from baseline. All serum samples were studied with the HIPA and PF4 ELISA in the coagulation laboratory at Stanford University Hospital (Stanford, Calif) or at The Coagulation Center (Oakland, Calif). Serum samples were obtained and studied on consecutive patients from January 995, through August 996. Samples were stored at -30 C until tested. Heparin-Induced Platelet Aggregation Healthy laboratory personnel who were not receiving medications or consuming alcohol were used as the source of platelet-rich plasma (PRP). Blood was drawn into citrated tubes and cenrrifuged at a relative centrifugal force of 00 for 8 minutes. The PRP was removed; the remaining blood was cenrrifuged at a relative centrifugal force of,000 for 5 minutes to obtain platelet-poor plasma (PPP). If the platelet count on the PRP was less than 350 x 0 9 /L, another donor was obtained. If the platelet count was more than 500 x 0 9 /L, PPP was added in a volume calculated to bring the final platelet count to 350 x 0 9 /L. Patient serum was inactivated by warming in a 56 C water bath for 30 minutes. Patient serum (300 ul) was added to 200 ul of normal platelets and incubated in the BioData Pap-4 aggregometer cuvette (BioData, Horsham, Pa) for 3 minutes. The patient's therapeutic heparin type was added (50 ul) in three different concentrations (0. U/mL, 0.4 U/mL, and U/mL). The Coagulation Center used one standard heparin concentration of U/mL. Aggregation was observed for 5 minutes. Each patient specimen was incubated with PRP from two donors known to respond to HIT-positive plasma. Known positive serum was incubated as a control of platelet reactivity, and control serum was tested for platelet overaggregation. Incubation of patient specimens with normal saline was used as a control for heparin-independent aggregation. A positive aggregation study was defined as a difference of 20% or more in aggregation response between patient and normal PPP in the presence of heparin. 7 The total percentage aggregation was determined from the difference between the baseline and maximal aggregation seen. Heparin-Platelet Factor 4 ELISA A research kit (Asserachrom HPIA, Diagnostica Stago, France) was used. All reagents were included in the kit except 3 mol/l sulfuric acid. Serum samples were diluted :00 with the supplied dilution buffer; 200 ul of each sample were run in duplicate on the precoated (with heparin-pf4 complex) wells and incubated for hour at room temperature. Plates were washed five times with solution containing trisodium citrate buffer and goat serum. Then 200 ul of peroxidase-conjugated goat antihuman IgG, IgA, and IgM was added to each well and incubated for additional hour at room temperature. After five washes, ortho-phenylenediamine dihydrochloride and urea peroxide tablets ( each) were dissolved in distilled water, and 200 ul was added to each well to develop the enzymatic activity. After 5 minutes, the color reaction was stopped with 50 ul of 3 mol/l H 2 S0 4. After 0 minutes, the optical density was measured at 492 nm. Each assay was run with negative and positive controls in duplicate. As defined by the manufacturer, a positive test was an optical density of 0.5 or more or 23% or more of the positive control. RESULTS We studied the serum samples from 46 patients with a clinical diagnosis of HIT. Results in 8 patients (55%) were HIPA-positive, PF4 ELISA-positive, or both. Of 8 patients, results for 74 (9%) were HIPApositive, while results for 49 (60%) were PF4 ELISA-positive. Results of both tests were positive in 42 (52%) of 8 patients (Table ). Of the 8 patients with HIPA-positive and PF4 ELISA-positive results, clinical information was available on 63 (78%). Clinical information was obtained by review of the medical records or direct discussion with the attending physician. Of 63 patients, 7 (27%) had thrombotic complications 0 experienced venous thrombosis; 6, arterial thrombosis; and, venous and arterial thrombosis. In two patients, deep vein thrombosis in a limb and pulmonary embolus developed (Table 2). Of the patients with thrombotic complications, primary diagnoses included coronary artery disease, valvular heart disease, pancreatitis, hepatic failure, myelofibrosis, aortic aneurysm, pancreatic cancer, arterial thrombosis, and chronic obstructive pulmonary disease Vol. 0 No.

3 80 COAGULATION AND TRANSFUSION MEDICINE (Table 3). Five (29%) of the 7 patients with thrombotic complications had underlying cardiac disease and had undergone an invasive cardiac procedure (eg, coronary artery bypass, angioplasty, or valvuloplasty). DISCUSSION Heparin-induced thrombocytopenia occurs in up to 5% of patients receiving heparin therapy. The clinical characteristics of the isolated thrombocytopenia have been well documented because of the relative frequency. The characteristics of patients who suffer thrombotic complications as a result of this syndrome are less clearly defined and, as yet, there is no laboratory test to determine which patients will have the more serious outcome. The laboratory diagnosis of HIT remains problematic. The gold-standard test is the SRA, but this is not TABLE. COMPARISON OF HIPA AND PF4 ELISA IN 8 OF 46 PATIENTS WITH POSITIVE ASSAY RESULTS Diagnosis HIPA-Positive HIPA-Negative Total HIT ELISA-positive ELlSA-negative Total Thrombosis ELISA-positive ELlSA-negative Total HIPA = heparin-induced platelet aggregation; PF4 = platelet factor 4; ELISA = enzymelinked immunosorbent assay; HIT = heparin-induced thrombocytopenia. TABLE 2. THROMBOSIS OBSERVED IN 63 PATIENTS WITH HITT Characteristic Type None Arterial Venous Both Description Arterial Cerebrovascular accident Limb ischemia Coronary bypass occlusion Venous Pulmonary embolus HITT = heparin-induced thrombocytopenia-thrombosis Number widely used. Platelet aggregometry relies on in vitro aggregation of normal donor platelets by the HIT antibody present in patient serum in the presence of heparin. Platelet aggregation has been criticized for its lack of sensitivity. 5,2 ' 4 This lack of sensitivity has been attributed to donor platelet variation in response to the HIT antibody. -3 In a recent report, Brandt et al 2 showed that this donor-specific platelet variability is influenced by an allelic polymorphism of the platelet Fc receptor (FcyRII). Specifically, platelets with histidine-histidine homozygosity at position 3 of the FcyRII were unresponsive to HIT antibody in vitro, but the arginine-arginine 3 phenotype responded well. This supports the recommendation to use platelets from at least two donors that have exhibited reactivity to known HIT-positive serum when performing the HIPA. This approach has been reported to increase the sensitivity of the HIPA. 7-3 Another factor potentially contributing to the reported lack of sensitivity of the HIPA is that the optimum concentration of heparin is not well defined. The usual recommendation is 0.5 U/mL heparin, in addition to a high concentration (00 U/mL) because reports indicate that high heparin concentrations inhibit platelet aggregation induced by HIT-positive TABLE 3. UNDERLYING DISORDERS IN 7 PATIENTS WITH HITT Thrombotic Event Primary Disorder Limb ischemia Pulmonary embolus, cerebral vascular event, pulmonary embolus, pulmonary embolus Coronary bypass occlusion HITT = heparin-induced thrombocytopenia thrombosis. Hepatic failure Arterial thrombosis Peripheral vascular disease Information not available Abdominal aortic aneurysm after repair Postcardiac transplantation Myelofibrosis Chronic obstructive pulmonary disease Abdominal aortic aneurysm after repair Pancreatic cancer Valvular heart disease Pancreatitis Information not available AJCP July 997

4 LOOK ET AL 8 Diagnosis ofheparin-i Thrombocytopenia-Thrombosis Aggregation Test Results Minutes Aggregation test results. Heparin-induced platelet aggregation using () saline, (2) 0. U/mL heparin, (3) 0.4 U/mL heparin, and (4).0 U/mL heparin. serum samples, but not that induced by serum samples that are thrombocytopenic because of other causes. 3 We have evaluated the HIPA and PF4 ELISA in 46 patients with clinically suspected HIT. In contrast to previous reports, we found the HIPA to be more sensitive than the PF4 ELISA (9% vs 60%). The reason for this increased sensitivity of the HIPA assay is likely the use of three heparin concentrations, 0. U/mL, 0.4 U/mL, and U/mL, as well as donor platelets known to respond to HIT-positive plasma. Of the 29 patients from Stanford with HIPApositive results, 6 (20%) had negative results on an aggregation study at a heparin concentration of 0. U/mL, but clearly positive aggregation at heparin concentrations of 0.4 or.0 U/mL. The Figure shows an example from one such patient. Previous reports have shown the PF4 ELISA to have a high sensitivity, comparable with that of the SRA. ' 5 In our study, we found agreement between the HIPA and PF4 ELISA to be only 52% (42/8). In a smaller study, Nguyen et al 8 reported a comparison between the HIPA (using a heparin concentration of 0.8 U/mL) and PF4 ELISA in 49 patients and found agreement to be 84%. Of patients with HIPA-positive results, they reported two PF4 ELISA-negative test results. In contrast, we found 32 PF4 ELISA-negative results in 74 patients with HIPA-positive results. This difference in correlation between the two tests likely represents variations in technical procedure, which is much more problematic when performing the HIPA than the PF4 ELISA. Our results suggest that the HIPA, under the conditions used in our study, is a more sensitive test than the PF4 ELISA in diagnosing HIT. At the outset of this study, we were interested in whether the PF4 ELISA would be useful in predicting thrombotic risk, because no information was available on this aspect of the assay. However, neither the HIPA nor the PF4 ELISA proved to be predictive of a HITT diagnosis or thrombotic risk. Of the 7 patients with thrombosis, two had HIPA-negative results, and four had PF4 ELISA-negative results. The majority (65%) had HIPA-positive and PF4 ELISA-positive results, so that neither test is more predictive. More efficient methods for the laboratory diagnosis of HIT continue to be pursued. As more is learned about the optimization of the tests, such as the polymorphism of the FcyRII influencing donor platelets, the sensitivities will continue to improve. Because no commonly used test is absolute, clinical suspicion and judgment remain invaluable. For laboratory confirmation of the diagnosis of HIT, we propose the following strategy. Because of its higher sensitivity, the initial test for HLT should be the HIPA. If results of the HIPA are negative, then a PF4 ELISA can be performed to detect the 0% of patients with HIT with HIPA-negative results. REFERENCES. Amiral J, Bridey F, Wolf M, et al. Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases. Throtnb Haemost. 995;73: Warkentin TE, Kelton JG. Heparin-induced thrombocytopenia. Annu Rev Med. 989;40: Boshkov LK, Warkentin TE, Hayward CPM, Andrew M, Kelton JG. Heparin-induced thrombocytopenia and thrombosis: clinical and laboratory studies. Br ] Haematol. 993;84: Greinacher A, Michels, Kiefel V, Mueller-Eckhardt C. A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia. Thromb Haemost. 99;66: Sheridan D, Carter C, Kelton JG. A diagnostic test for heparininduced thrombocytopenia. Blood. 986;67: Chong BH. Heparin-induced thrombocytopenia. Br ) Haematol. 995;89: Amiral J, Bridey F, Dreyfus M, et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparininduced thrombocytopenia. Thromb Haemost. 992;68: Visentin GP, Ford SE, Scott JP, Aster RH. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells. / Clin Invest. 994;83: Greinacher A, Potzsch B, Amiral J, et al. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen. Thromb Haemost. 994;7: Kelton JG, Smith JW, Warkentin TE, et al. Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4. Blood. 994;83: Vol. 08 No.

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