Serotonin Release Assay Results Research Articles

Rethinking the Gold Standard: Real-World Performance of the Serotonin Release Assay (SRA) and PF4-Dependent P-Selectin Expression Assay (PEA) in the Diagnosis of Heparin-Induced Thrombocytopenia (HIT)

Background Heparin-induced thrombocytopenia (HIT) is a complication of heparin exposure with potential for significant morbidity and mortality. The serotonin release assay (SRA) has been considered the gold standard for HIT diagnosis. However, the SRA is technically challenging and results can be influenced by specimen handling and preparation. Therefore, the performance of the real-world assays may differ from published standards but reports of real-world performance are not widely available. A new assay, the PF4-dependent P-selectin expression assay (PEA), has demonstrated comparable accuracy in HIT diagnosis employing a simpler laboratory technique. Herein, we report the real-world performance of a reference laboratory SRA and PEA and describe how incorporation of the PEA into the testing algorithm positively influenced the HIT diagnostic process at a large academic medical center. Methods At the University of Alabama at Birmingham, we incorporated the PEA into HIT testing at provider discretion in patients with inconclusive SRA results starting January 1, 2020. Since July 15, 2020, we solidified the use of PEA in our algorithm (Figure 1) in which all patients with an inconclusive SRA had a PEA on the same specimen. A PEA could also be ordered ad hoc if the SRA result did not correlate with clinical suspicion for HIT. We retrospectively compared patients tested for HIT for 2 years before PEA use (January 1, 2018 to December 31, 2019; pre-PEA period) to the >2 years since using the PEA (January 1, 2020 to March 1, 2022; PEA period). All patients tested with PEA at our institution are included in Table 1. Laboratory testing: Enzyme immunoassay (EIA) was performed at our institution using Asserachrom® HPIA, Diagnostica Stago (France). The SRA and PEA assays were performed at Versiti (Milwaukee, WI). SRA was positive if there was >20% release of serotonin with low dose heparin (0.1 U/mL) and <20% release in with high dose heparin (100 U/mL). An inconclusive result was defined as a percentage of serotonin release not meeting criteria for a positive result (e.g., >20% release of serotonin with both low and high dose heparin, or some release but <20% with low dose along with inhibition with high dose). Statistical analysis Descriptive statistics were employed to examine the sample of patients with an inconclusive SRA result, comparing those with and without PEA testing. Bivariate analyses were conducted to compare testing patterns in the pre-PEA and PEA periods, and T-tests of significance were conducted to analyze assay results. All models were estimated using Stata 17 (StataCorp. 2019. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC). ResultsSRA results: In the pre-PEA and PEA periods, there were 1,252 and 1,403 EIAs performed, respectively. The rate of EIA positivity was similar in both groups (14% vs 15%, p=0.813). A total of 379 SRAs were sent in both periods, with 24 inconclusive results (6%, 15 pre-PEA and 9 PEA). PEA performance: In the 24 patients with inconclusive SRA results, HIT was diagnosed in 7 of 15 patients (47%) pre-PEA, compared to 3 of 9 patients (33%) in the PEA period. Table 1 presents the details of each case where PEA was used due to diagnostic uncertainty. Of the 9 patients with an inconclusive SRA result in the PEA period, PEA provided a definitive result in 8 cases (89%). In 2 cases, the PEA confirmed the SRA result, 1 positive and 1 negative. In 1 case with a positive SRA and low clinical suspicion, the PEA was negative. Conclusion Evaluation of the real-world performance of the SRA reveals that although inconclusive results are rare (6%), they can present a significant diagnostic challenge in centers with high testing volume. The PEA demonstrated reliable performance in patients with an inconclusive SRA, producing a definitive result in 89% of cases. The percentage of patients diagnosed with HIT after an inconclusive SRA result decreased with incorporation of the PEA (47% to 33%), suggesting that PEA use helped refute a HIT diagnosis when there was clinical uncertainty. Further studies are needed to determine the optimal testing algorithm to produce rapid, reliable HIT diagnosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical care pathway for the evaluation of patients with suspected VITT after ChAdOx1 nCoV-19 vaccination

AbstractVaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccination. In British Columbia (BC), Canada, a provincial clinical care pathway was developed to guide clinicians in evaluating for VITT among patients who present with thrombocytopenia or thrombosis symptoms within 4 to 28 days after adenoviral vector vaccine exposure. All patients had enzyme-linked immunosorbent assay (ELISA) testing for platelet factor 4 (PF4) antibodies, and all cases with positive PF4-ELISA or d-dimer levels ≥2.0 mg/L fibrinogen equivalent units (FEU) had further testing for platelet-activating PF4 antibodies using a modified serotonin release assay (SRA). Between 1 May and 30 June 2021, 37% of 68 patients investigated for VITT had thrombosis, but only 3 had VITT confirmed by PF4-ELISA and SRA. Platelet counts, d-dimer levels, and ELISA optical density values were significantly different between those with and without VITT. Three patients had thrombocytopenia and thrombosis with d-dimer levels >4.0 mg/L FEU but had negative PF4-ELISA and SRA results. Patients with VITT were treated successfully with IV immunoglobulin, nonheparin anticoagulants, and corticosteroids. Our pathway demonstrated that thrombosis is common among patients investigated for VITT and that PF4-ELISA testing is necessary to confirm VITT in those presenting with thrombosis and thrombocytopenia.

A Road Paved with Good Intentions: A Platelet Count-Based Alert to Facilitate Diagnosis of Heparin-Induced Thrombocytopenia

Background: Heparin-induced thrombocytopenia (HIT) is a rare disorder with potential to cause significant morbidity and mortality. Early identification and initiation of non-heparin anticoagulation can prevent devastating thrombotic events. However, over-testing is common and can lead to result misinterpretation, unnecessary heparin avoidance, and increased cost. When there is concern for HIT, guidelines from the American Society of Hematology recommend calculation of the 4Ts score to determine the need for laboratory testing. The Choosing Wisely® initiative recommends against laboratory testing in patients with a low probability score of ≤3. In patients with an intermediate or high probability score (≥4), screening with enzyme-linked immunosorbent assay (ELISA) is performed first. If positive, the diagnosis of HIT is confirmed with a functional assay, commonly the serotonin release assay (SRA).Methods: In an effort to increase recognition of HIT, providers at a large academic medical center received a non-interruptive alert in the electronic medical record (EMR) on all patients in whom the platelet count declined by ≥50% starting in Aug 2017. We performed a retrospective evaluation of 1) the number of alerts and 2) all ELISA results obtained with or without an alert, over a 90-day period (Dec 2019 to March 2020). A 4Ts score was calculated by chart review by the first author in real-time as the alert was sent (blinded to ELISA and SRA results). Among those patients with multiple alerts or test orders, the first instance was used for analysis. Demographic and clinical characteristics were reported using frequencies and percentages, means (standard deviation, SD), and medians (interquartile range, IQR). Patients with alerts and ELISA testing ordered were compared with 2 groups: 1) patients with alerts but no ELISA ordered; 2) patients with no alerts but ELISA ordered. Comparisons were performed using chi squared tests, Fisher's exact tests, t-tests and Wilcoxon rank-sum tests as appropriate.Results: In the 90-day observation period, 302 alerts were fired in 270 patients (Figure 1). Thirty alerts (28 patients, 10%) were generated for patients admitted for organ donation or post-stem cell transplantation, for whom platelet count decline was expected. Excluding these patients, there were 272 alerts in 242 patients (approximately 3 alerts per day in a 1,157-bed hospital). Of patients with alerts, 22 (8%) had a platelet count inaccuracy (i.e. platelets clump or another reason) and 18 (7%) did not receive heparin prior to platelet decline, for a cumulative total of 40 (15%) inappropriate alerts.In patients with an alert, the ELISA was ordered more frequently for those with a lower platelet nadir (77x10 9/L vs. 115x10 9/L, p<0.0001) or in those with a thrombotic event (11 patients (17%) vs. 6 patients (4%), p=0.0021) (Table 1). Those without an ELISA ordered were more likely to have a low 4Ts score (23 patients (36%) vs. 81 patients (58%), p<0.0001).In addition to 71 patients with an alert, an ELISA was also ordered for 67 patients without an alert (n=138) (Figure 1). Close to half of ELISA-tested patients had a low 4Ts score (n=51, 46%) (Figure 2). In patients with an alert and ELISA not ordered, 18 (27%) had an intermediate or high 4Ts score.Seven patients were diagnosed with HIT based on a positive SRA, 6 with an alert and 1 without. The alert demonstrated a sensitivity of 86% (95% CI, 59.8-100%) and specificity of 50% (95% CI, 41.8-58.9%) with a positive predictive value of 0.0845 (95% CI, 0.0198-0.1492) and negative predictive value of 0.9851 (95% CI, 0.9560-1.0000).Conclusion: An EMR alert based on platelet count decline had multiple shortcomings including frequent inappropriate firings and a lack of guidance on appropriate indications for testing. This evaluation of institutional testing practices indicates frequent use and misinterpretation of ELISA discordant with evidence-based guidelines. Although prompt diagnosis of HIT is important, alternative strategies for identification of at-risk patients and communication of recommended actions to providers should be considered. Because the 4Ts score includes variables difficult to automate in the EMR, our institution is exploring electronic consultation and real-time expert provider access to overcome these limitations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Heparin‐induced thrombocytopenia: Construction of a pretest diagnostic score derived from the analysis of a prospective multinational database, with internal validation

BackgroundDiagnosis of heparin‐induced thrombocytopenia (HIT) requires pretest probability assessment and dedicated laboratory assays. ObjectiveTo develop a pretest score for HIT. DesignObservational; analysis of prospectively collected data of hospitalized patients suspected with HIT (ClinicalTrials.gov NCT00748839). SettingThirty‐one tertiary hospitals in France, Switzerland, and Belgium. PatientsPatients tested for HIT antibodies (2280 evaluable), randomly allocated to derivation and validation cohorts. MeasurementsIndependent adjudicators diagnosed HIT based on the prospectively collected data and serotonin release assay results. ResultsHeparin‐induced thrombocytopenia was diagnosed in 234 (14.7%) and 99 (14.5%) patients in the two cohorts. Eight features were associated with HIT (in brackets, points assigned for score calculation of the score): unfractionated heparin (1); therapeutic‐dose heparin (1); cardiopulmonary bypass (cardiac surgery) (2); major trauma (3); 5‐ to 21‐day interval from anticoagulation initiation to suspicion of HIT (4); ≥40% decrease in platelet count over ≤6 days (3); thrombotic event, arterial (3) or venous (3). The C‐statistic was 0.79 (95% CI, 0.76–0.82). In the validation cohort, the area under the receiver operating characteristic curve was 0.77 (95% CI, 0.74–0.80). Three groups of scores were defined; HIT prevalence reached almost 30% in the high‐probability group. LimitationThe performance of the score may depend on settings and practices. ConclusionThe objective, easy‐to‐collect, clinical features of HIT we evidenced were incorporated into a pretest score, which may guide clinical decisions regarding diagnostic testing and anticoagulation.

Platelet‐activating immune complexes identified in critically ill COVID‐19 patients suspected of heparin‐induced thrombocytopenia

BackgroundThrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID‐19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID‐19 patients may be suspected of heparin‐induced thrombocytopenia (HIT), given similar clinical features. ObjectivesWe investigated the presence of platelet‐activating anti‐platelet‐factor 4 (PF4)/heparin antibodies in critically ill COVID‐19 patients suspected of HIT. Patients/MethodsWe tested 10 critically ill COVID‐19 patients suspected of HIT for anti‐PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti‐human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS‐CoV‐2 antibodies were measured using an in‐house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin‐13 Domain (ADAMTS13) activity and the presence of anti‐ADAMTS13 antibodies. ResultsHeparin‐induced thrombocytopenia was excluded in all samples based on anti‐PF4/heparin antibody and SRA results. Notably, six COVID‐19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)‐mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS‐CoV‐2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID‐19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy. ConclusionsOur study identifies platelet‐activating ICs as a novel mechanism that contributes to critically ill COVID‐19.

Indeterminate Serotonin Release Assays Are Associated with a High Mortality Rate

INTRODUCTION: The serotonin release assay (SRA) is considered the gold standard for diagnosis of heparin-induced thrombocytopenia (HIT). While the SRA holds high sensitivity and specificity when results are definitive, up to 10% of samples from patients with suspected HIT yield "indeterminate" results. In classic HIT, the SRA typically reveals high levels of serotonin release when serum is mixed with low concentrations of heparin and low levels of serotonin release when serum is mixed with high concentrations of heparin. Results are considered "indeterminate" when high levels of serotonin release are seen at both low and high concentrations of heparin, indicating a failure of high-dose heparin to saturate the heparin binding sites of PF4 molecules and inhibit platelet activation. Explanations for indeterminate assays include the presence of heparin-binding proteins that interfere with the assay, high titers of HLA class I alloantibodies, or immune complexes. Since the diagnosis of HIT carries such significance and has so many ramifications, an indeterminate SRA may leave therapeutic indecision. The etiologies, platelet trends, clinical course and outcomes of patients that receive indeterminate SRA results are not well-understood. We conducted a retrospective review of 2,056 patients that underwent SRA testing as part of their evaluation for HIT. METHODS: Using the electronic medical record data extraction software Clinical Looking Glass, we identified patients that underwent SRA testing between 1/1/2014 and 12/31/2018. SRA results were considered "indeterminate" when serotonin release exceeded 19% at all heparin dilutions (0.1 U/mL, 0.5 U/mL and 10 U/mL). We conducted a retrospective chart review to study the clinical course among patients who had "indeterminate" SRA results, the trends in platelet count, the timing of platelet drops and the physician response to this result. Statistical analysis was performed using chi-square testing for categorical variables. RESULTS: We identified 2,056 patients that underwent SRA testing between 2014 and 2019. Of these, 90 patients (4.4%) had "positive" SRA results and 1,814 patients (88.2%) had "negative" SRA results. Among the 152 patients (7.4%) with "indeterminate" SRA results, the mean 4T score was just 2.9, corresponding to a HIT probability of &amp;lt;5%. One-hundred and twenty of these 152 patients (78.9%) had heparin-PF4 antibody testing with optical densities below 0.60 OD, while only 4 of 152 patients (2.6%) had optical densities above 2.00 OD. Seventy eight of 152 patients (51.3%) either continued or were re-exposed to heparin after the indeterminate SRA result, and in 71 of 78 cases (91.0%), the platelet count stabilized or improved despite heparin exposure. In the remaining 7 cases, no acute VTE were found and no diagnosis of HIT was made. Four of the 71 patients (5.6%) that continued or were re-exposed to heparin were noted to have an acute VTE. A significantly higher portion of patients with an indeterminate SRA died during admission compared to those with a positive or negative SRA (49.3% vs. 21.1% and 27.2%, p &amp;lt;2.4 x10-10). The prevalence of thrombocytopenia &amp;lt;50,000 was substantially higher in patients with an indeterminate or positive SRA, compared to those with a negative SRA (39.5% and 40.0% vs. 27.5%, p &amp;lt;4.0 x 10-4). Re-exposure or continuation of heparin did not affect mortality; patients that were given heparin after an indeterminate SRA had a 47.4% mortality, compared to 51.4% in those given no more heparin (p = 0.63). Table 1 summarizes these findings. CONCLUSIONS: This is the largest study to date looking at patients with indeterminate SRA testing results, in which platelets exhibit high levels of serotonin release at both low and high heparin concentrations. Our data suggest 1) the majority of patients with indeterminate SRAs likely did not have HIT, 2) as evidenced by the low 4T scores and heparin-PF4 antibody levels, an indeterminate SRA suggests thrombocytopenia related to in-vivo platelet activation, and 3) an indeterminate SRA is associated with increased mortality. SRA results warrant a case-by-case assessment of the clinical picture in order to avoid unnecessary cessation of heparin products. Further studies exploring the mechanism leading to high serotonin release at both low and high heparin concentrations, causing an "indeterminate" SRA result, are warranted. Disclosures No relevant conflicts of interest to declare.

AORTIC THROMBOSIS AFTER CARDIAC SURGERY FROM HEPARIN-INDUCED THROMBOCYTOPENIA

SESSION TITLE: Fellows Critical Care Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a complication caused by exposure to heparin. Although incidence after cardiac surgery is of 0.3%, there is a 50% increase in mortality when HIT occurs [1]. CASE PRESENTATION: A 65-year-old male with history of diabetes mellitus presented with chest pain. Two days prior to coronary artery bypass grafting (CABG), coronary angiography revealed triple vessel disease. He received 3000 units of heparin for the procedure. He underwent on-pump CABG, where 19,500 units of heparin were given. After surgery, platelet count decreased with nadir of 105×103 /μL (40% decrease) on postoperative day (POD) 4. He complained of abdominal and lower extremities’ pain on POD 5. Computed tomography (CT) showed a thrombosed infrarenal aorta (Fig 1). Vascular team performed axillary and femoral artery bypass surgery. He complained of abdominal pain and CT showed dilated loops of bowel with pneumatosis intestinalis on POD 7. Laparotomy was performed and necrosed small bowel was resected. HIT was suspected due to thromboembolic events (4Ts score was 6; high risk of HIT) [2]. Particle Immuno-Filtration Assay (PIFA®) and Serotonin Release Assay (SRA) were ordered and results came negative. Assays were done for second time on POD 10 and PIFA® resulted negative. Echocardiogram revealed small thrombus in left ventricle apex on POD 11. Although SRA result was pending, heparin was discontinued and argatroban was administered. SRA test came positive on POD 12. Platelet count began recovering and there was no more thromboembolic event. Platelet count returned to normal at 5-month follow-up (Fig 2). DISCUSSION: There are two requirements to determine presence of HIT: a) clinical diagnosis and b) detection of heparin antibodies activating platelet (through positive SRA). Clinical diagnosis can be completed by use of 4Ts score [2] (Fig 3). If 4Ts score is more than 4 (intermediate risk), the serological tests should be performed. There are two types of tests for HIT: one is the antigen assay PIFA® and the other is the functional assay SRA. Both have high sensitivity of 90%. PIFA® is used for screening, has a specificity ranging from 50% to 89% and its result can be available within 15 minutes. SRA has a higher specificity of more than 90%. To confirm the diagnosis, SRA result should be positive, although several days may take to obtain result. CONCLUSIONS: If the serological test shows negative and HIT is still suspected clinically, the test should be repeated. High clinical suspicion should lead to termination of heparin and initiation of argatroban. Reference #1: Seigerman M., Cavallaro P., Itagaki S., Chung I., Chikwe J. Incidence and outcomes of heparin-induced thrombocytopenia in patients undergoing cardiac surgery in North America: an analysis of the nationwide inpatient sample. J Cardiothorac Vasc Anesth. 2014;28:98-102. Reference #2: East M.J., Cserti-Gazdewich M.C., Granton T.J. Heparin-induced thrombocytopenia in the critically ill patient. Chest. 2018; 154:678-690. Reference #3: Ortel T.L. Heparin-induced thrombocytopenia: when a low platelet count is a mandate for anticoagulation. Hematology Am Soc Hematol Educ Program. 2009;225-232. Fig 1; Contrast computed tomography (CT) showed total occlusion of the infrarenal abdominal artery and bilateral iliac arteries. DISCLOSURES: No relevant relationships by Youdelman Benjamin, source=Admin input No relevant relationships by Shinichiro Ikeda, source=Web Response No relevant relationships by Ronald Kaleya, source=Web Response No relevant relationships by Robert Rhee, source=Web Response

Effective Implementation of a Structured Protocol for Facilitation of the Judicious Use of Antibody Test for Diagnosis of Heparin Induced Thrombocytopenia

Introduction: Heparin induced thrombocytopenia (HIT) is a serious prothrombotic condition, usually triggered by exposure to heparin products with formation of antibodies to platelet factor 4/ heparin polyanion complexes. Diagnostic algorithm for HIT combines clinical scoring (4T score) with time sensitive screening for HIT antibodies (HIT-ab), while serotonin release assay (SRA) is remains the gold standard for confirmatory diagnosis. The rate of utilization of 4T score was low in our institution, resulting in inappropriate orders for HIT-Ab test and subsequent administration of unnecessary alternative anticoagulation (AC) in patients with false positive results. In this project, we designed a structured HIT diagnostic workflow incorporating 4T score calculation in our electronic medical record (EMR) and replaced particle immunofiltration assay (PIFA) with latex immunoturbidometric assay (LIA) in our laboratory for HIT-Ab screening, with an aim to improve the rate of 4T utilization and accuracy of HIT diagnosis in a cost-efficient manner. Methods: In phase I, we performed a retrospective chart review of all patients with PIFA ordered between March 2017-March 2018. Two investigators independently calculated 4T, collected data on results of HIT-Ab, confirmatory SRA tests, and the duration of alternative AC from each record. Any variations in 4T score were resolved by a senior investigator. In phase II, we implemented a new workflow in the EMR incorporating mandatory calculation of 4T score with every order for HIT-Ab test. Our lab started using LIA in place of PIFA. Charts were reviewed on patients with HIT-Ab orders (LIA) from January-June of 2019. Results: On review of data from phase I, we noted that 4T score was documented in only 5 (0.02%) of 170 patients in whom a PIFA test was ordered. Per investigators assessment, 113 (66.4%) patients had low probability (4T ≤ 3), 47 (27.6%) had intermediate probability (4T 4 or 5), and 10 (5.8%) had a high probability (4T ≥ 6) for a diagnosis of HIT. SRA was ordered in 32 patients, although 17 of them had low probability per investigator assessment. PIFA test came back positive in 26 patients, of whom 16 had corresponding SRA results, and three samples were positive for SRA. PIFA was negative in two patients with confirmed HIT (SRA positive). A total of 19 patients received alternate AC in the first phase, 7 of them had low 4T score per our assessment. In phase II, 69 records were found with available LIA results, showing a relative decrease in HIT-Ab orders compared to earlier phase at the six months mark. Documentation of 4T score has been 100% by ordering physicians, a certain improvement from phase I. Investigator calculated 4T score showed low probability in 33 (47.8%) patients, intermediate probability in 31 (44.9%) patients, high probability in 5 (0.07%) patients. LIA was positive in 7 of the 69 ordered tests, 6 of whom scored high/intermediate in the 4T score. HIT diagnosis was confirmed in 3 of these 7 patients with a positive SRA result. During this period, all the 7 of the eight patients who received alternate AC had a high or intermediate probability for HIT as per 4T. Conclusion: Our study demonstrated that the successful implementation of a structured protocol for HIT diagnosis ensured 100% adherence to the calculation and documentation of 4T score by clinicians, and significantly reduced the number of inappropriate HIT-Ab test orders in our institution. Use of alternate AC was also more consistent with the level of probability for HIT. Table Disclosures No relevant conflicts of interest to declare.

Performance of the New Automated Latex Immunoturbidometric Assay in the Diagnosis of Heparin Induced Thrombocytopenia: A Single Institution Experience

Introduction: Heparin induced thrombocytopenia (HIT) is a severe prothrombotic condition, usually triggered by exposure to heparin products. It is characterized by platelet activation induced by the formation of antibodies to the platelet factor 4 (PF4)/ heparin polyanion complexes. Diagnostic algorithm includes clinical scoring (4T score) alongside serological test for detection of these antibodies (HIT-Ab), while serotonin release assay (SRA) remains the gold- standard for confirmation. The automated latex immunoturbidometric assay (LIA) has recently been FDA approved as a screening tool for HIT and is a potential alternative to the conventional particle immunofiltration assay (PIFA) for time-sensitive detection of HIT-Ab to guide treatment considerations. We recently introduced LIA in our institution. In this study, we present our experience with LIA in comparison to PIFA in the diagnosis of HIT. Methods: We retrospectively reviewed the charts of all the patients on whom a PIFA was ordered between March 2017 and March 2018 in our hospital. We collected information on the results of the PIFA and SRA (if available). We replaced PIFA with LIA for HIT screening. Then, we introduced a structured protocol for diagnosis of HIT in our institution by incorporating 4T scoring alongside LIA order in the electronic medical record (EMR), in December 2018. We reviewed the EMR of all the patients on whom HIT-Ab test (LIA) was ordered between January and June of 2019, and collected similar information as before. All the data were compiled in a single master excel sheet for calculation of performance characteristics (sensitivity, specificity, positive and negative predictive values) for both PIFA and LIA. A patient was considered to have the diagnosis of HIT if the result of SRA was available and positive. Results: In the first phase, a total of 31 orders for SRA was noted against 170 PIFA orders. Five patients had a positive SRA, of whom two were PIFA negative. Half the patients with a negative SRA result were positive for PIFA. Hence, the sensitivity and specificity of PIFA test for our study population were noted to be 60% and 50%, respectively. PIFA had a positive predictive value (PPV) of mere 18.75% for the diagnosis of HIT, whereas the negative predictive value (NPV) was found to be 86.66%. Introduction of structured protocol for HIT diagnosis substantially reduced the number of inappropriate SRA orders in the second phase. On review of data for six months with the new HIT-Ab test LIA, SRA was ordered in only eight patients, to go with 69 orders for the LIA. The result of LIA was positive in all three patients with a positive SRA, whereas it was false positive in four instances. Only one patient was negative for both LIA and SRA during this period. LIA was found to be 100% sensitive and 20% specific for the diagnosis of HIT in our sample. PPV and NPV for LIA were 42.85% and 100%, respectively. Conclusion: The sensitivity and specificity of LIA were found to be 100% and 20%, respectively, in our study population, which is different from the earlier report (Warkentin et al. 2017). The small sample size is a limitation of our study. Higher PPV and NPV for LIA, with its quick turnaround time, make it a useful alternative for the time-sensitive determination of post-test probability for HIT in patients. [HIT- Ab- Heparin Induced Thrombocytopenia Antibody, PIFA- Particle Immunofiltration Assay, LIA- Latex Immunoturbidometric Assay, SRA- Serotonin Release Assay, +ve- Positive, -ve - Negative, PPV- Positive Predictive Value, NPV- Negative Predictive Value] Disclosures No relevant conflicts of interest to declare.

PS1495 FUNCTIONAL ASSAYS USING WASHED PLATELETS FOR HEPARIN‐INDUCED THROMBOCYTOPENIA DIAGNOSIS: A RETROSPECTIVE STUDY COMPARING HEPARIN‐ INDUCED PLATELET ACTIVATION TEST VERSUS 14C‐SEROTONIN RELEASE ASSAY

Background:Early recognition of heparin‐induced thrombocytopenia (HIT) is crucial because of increased risk of life threatening venous and/or arterial thrombosis. In case of positive immunoassay, HIT diagnosis has to be confirmed by functional assays detecting heparin‐dependent platelet‐activating antibodies (anti‐PF4/H). Heparin‐induced platelet activation test (HIPA) and serotonin release assay (SRA) are two functional assays that use washed platelets. SRA is considered the gold standard, but HIPA offers several advantages over SRA: it does not require radioactivity or specialized equipment and is less time consuming. HIPA closely correlates to SRA but 20 years after its first description, there is no additional published data regarding its diagnostic performances.Aims:To evaluate HIPA performances compared to SRA in a retrospective cohort of patients suspected for HIT. Secondary objectives were to evaluate negative or positive cut‐off criteria for HIPA and to identify clinical and/or biological factors associated with positive HIPA.Methods:We retrospectively analyzed the medical records of 81 consecutive patients who were suspected for HIT in our center between October 2010 and October 2015. Clinical suspicion of HIT was based on otherwise unexplained fall of platelet count (&gt;40%) 5 to 21 days after the initiation of heparin and/or the occurrence of any thrombotic event during heparin treatment. Patients with positive anti‐PF4/H IgG immunoassay (Zymutest HIA IgG, Hyphen BioMed, France) who were tested by SRA and HIPA were included in this study. Anti‐PF4/H IgG was positive when optical density was greater than 0.5 OD units. Patients with indeterminate SRA or HIPA results or who were tested with less than two and more than four platelet donors for the HIPA were excluded. SRA was considered positive when serotonin release was greater than 20% at 0.1 U/mL and/ or 0.5 IU/mL unfractionated heparin (UFH) and decreased by at least 50% at high UFH concentration (100 IU/mL). HIPA was considered positive when the suspension became transparent due to platelet aggregation with UFH 0.2 IU/ml and/or 0.5 IU/ml but not with UFH 48 IU/ml and when positive results were obtained with at least two platelet donors. HIPA was considered negative if negative results were obtained with four platelets donors. Positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA) and Cohen's kappa (κ) and their 95% confidence intervals (CI) were calculated.Results:HIPA and SRA results were compared in 47 patients fulfilling eligibility criteria. PPA and NPA were 83.8% (95% CI 71.9–95.7%) and 75.0% (95% CI 53.8–96.2%), respectively. The OPA was 81.1% (95% CI 78.4–83.8%; κ = 0.58, CI 0.33–0.80). Defining HIPA positive when positive results were obtained with two donors was associated with a higher NPA (80.0% versus 75.0%) with a minimal decrease of the PPA (81.3% versus 83.8%) compared to positive result with only one patient. No association was found between surgery, extracorporeal circulation, anti‐PF4/H IgG titer, platelet count nadir or type of heparin and HIPA results.Summary/Conclusion:HIPA showed a good performance against SRA when testing suspected HIT patients. A good concordance was found between the two assays. HIPA performance can be enhanced considering two positive results to establish HIT diagnosis and four negative results to rule out HIT diagnosis. HIPA appears to be an optional sensitive and specific functional assay for HIT diagnosis easy to handle without radioactivity and safety issues.

Multi-Year Study of Unfractionated Heparin Serotonin Release Testing at a Large U.S. Reference Laboratory

Introduction: The serotonin release assay (SRA) is a functional assay for the detection of heparin-induced thrombocytopenia (HIT). Laboratory practice suggests that it is important to test several low doses of unfractionated heparin (UFH), followed by suppression at a high dose (100 U/mL) of UFH to demonstrate the specificity for HIT. However, little is known of the demographic associations with UFH SRA testing at a large reference laboratory.Purpose: To characterize the population characteristics of UFH SRA testing at a large US reference laboratoryMethods: We performed a multi-year retrospective review of positivity rates among adults (n=63,651) and children (<18 yrs, n=233) who underwent SRA testing; we examined positivity rates at 0.1 and/or 0.5 U/mL of UFH. A positive SRA test was defined by ≥20% release at either low dose (0.1 or 0.5 U/mL) of UFH and >50% suppression of release at high dose (100 U/mL) of UFH. Indeterminant results were defined by ≥20% release at either low dose of UFH, but without >50% suppression at the high dose of UFH. A negative result was defined as <20% release at both low doses of UFH. All testing was performed at Quest Diagnostics, Nichols Institute in Chantilly, VA. Demographic and laboratory information collected included age, gender, date of collection, and SRA results at 0.1 and 0.5 U/mL UFH. Descriptive statistics were performed as well as non-parametric statistical analyses using the Mann-Whitney U test (two-tailed) with significance defined at p <0.05.Results: Over the study period, SRA testing included 63,051 adults and 233 children who encompassed 99.5% and 0.5% of the total population tested, respectively. Over the years studied, the positivity rate increased steadily from 4.08% to 7.50%, which was seen in adults but not in children. For positive SRA results, the ratio of male to female was nearly identical to the overall male to female ratio (55:45) in the tested population. In additional, no seasonal variation was noted. Yearly median percent positive, indeterminant, and negative SRA results were 5.42%, 3.32%, and 89.73%, respectively, for adults, and 1.96%, 6.52%, and 93.48%, respectively, for children, with children having significantly lower SRA positivity than adults (p<0.037).Among positive tests in adults, we examined the results by level of low dose UFH: the median positivity rates were 3.46% for 0.1 U/mL only, 1.03%, for 0.5 U/mL only, and 95.89% for both 0.1 and 0.5 U/mL UFH. In contrast, children demonstrated positive tests only when both 0.1 and 0.5 U/mL UFH results were positive. These children (n=6, median age 14 yrs with range 3 to 15 yrs) demonstrated median serotonin release of 81% and 77% at 0.1 and 0.5 U/mL of UFH, respectively, which was not significantly different from each other and similar to the corresponding adult positive (both 0.1 and 0.5 U/mL UFH) SRA population.Conclusions: Adults have a higher prevalence of SRA testing compared to children and have median positivity rates that are significantly higher (2-3 fold).The consistent increase in yearly percentage of positive tests, suggests improved clinical screening and/or diagnostic pathways for HIT. Testing was not influenced by gender or seasonal variation. To our knowledge, these findings provide, for the first time, a detailed picture of SRA testing from a large US reference laboratory. DisclosuresWong:Quest Diagnostics: Employment, Other: stock . Sahud:Quest Diagnostics: Consultancy. Noh:Quest Diagnostics: Employment. Aboueyada:Quest Diagnostics: Employment. Frock:Quest Diagnostics: Employment. Haley:Quest Diagnostics: Employment. Wu:Quest Diagnostics: Employment. Dlott:Quest Diagnostics: Employment, Other: stock.

Evaluation of a Rapid and Automated Heparin-Induced Thrombocytopenia Immunoassay

Introduction:Heparin-Induced thrombocytopenia (HIT) is a potentially life-threatening, immune complex mediated adverse reaction of heparin therapy, which may be suspected in patients who develop thrombocytopenia and/or thrombosis during, or following exposure to, unfractionated or low molecular weight heparin. Laboratory demonstration of heparin-platelet factor 4 (HPF4) antibodies supports more timely cessation of heparin therapy and initiation of alternative anticoagulation, and is an essential component of the diagnosis of HIT. The serotonin release assay (SRA) is considered the gold standard, but is technically difficult, requires normal donor platelets and radioactive isotopes. As a result, the SRA is not widely available in routine clinical laboratories. Enzyme immunoassays (EIA) are commonly used to support an HIT diagnosis; however, currently available EIAs typically require sample batching, which usually delays HIT diagnosis, and may be unavailable in laboratories with low test volumes. We evaluated the HemosIL HIT-Ab(PF4-H) (Instrumentation Laboratory, Bedford, MA) assay, a rapid, on-demand, automated method that can be performed 24/7 and provides results more quickly than existing tests.Methods:The assay cut-off determination was performed by a method comparison with SRA on HIT-suspected plasma samples (n=63; 31 SRA-positive and 32 SRA-negative). The reference interval was calculated using citrated plasma samples from normal donors (n=131) and HIT-suspected patients that were negative by EIA (n=122). A multicenter study of HemosIL HIT-Ab(PF4-H) assay on ACL TOP Family systems was conducted between March 2013 and June 2015. Method comparisons were conducted between HemosIL HIT-Ab(PF4-H), Asserachrom HPIA assay (Diagnostica Stago, Asnieres, France) (n=632), and SRA (n=537). HemosIL HIT-Ab(PF4-H) was also compared with clinical probability using the 2013 American Society of Hematology (ASH) guideline, a diagnostic algorithm that classifies samples as (HIT Likely) or (HIT Unlikely) based on the 4T score, EIA, and SRA results.Results:The optimal cut-off determined by Receiver Operator Curve (ROC) analysis was 1.0 U/mL (95.2% agreement, 95% confidence interval (CI) 86.7-99.0). The 95% reference interval was 0.0-0.7 and 0.0-0.9 U/mL for healthy donors and HIT negative suspected patients, respectively. Thus, based on these results, a value of equal or greater than 1.0 U/mL was considered positive for HIT antibodies. The overall agreement of HemosIL HIT-Ab(PF4-H) and EIA and SRA results were 90% (95% CI 88-92%), and 79% (95% CI 75-82%), respectively (Table 1). The overall agreement of the clinical probability compared to HemosIL HIT-Ab(PF4-H) results was 86% (95% CI 83-89%) with a Negative Predictive Value (NPV) of 99.6% (95% CI 98-100%) (Table 1).Discussion:Timing is critical in HIT diagnosis and affects clinical decisions in HIT-suspected patients. The advantage of an on-demand, automated assay is that test results can be provided in a more timely fashion, even in a routine clinical laboratory. Our data show that the HemosIL HIT-Ab(PF4-H) results in HIT-suspected patients are comparable to EIA and SRA. In addition, substantial correlation is noted between HemosIL HIT-Ab(PF4-H) results and the clinical probability of HIT with a NPV approaching 100%, which demonstrates the clinical utility of this assay in excluding a diagnosis of HIT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel PF4-Dependent P-Selectin Expression Assay (PEA) Facilitates Early Detection of Pathogenic HIT Antibodies: Implications for Diagnosis and Treatment of HIT

The PF4-dependent P-Selectin Expression Assay (PEA) is a novel assay for diagnosis of heparin-induced thrombocytopenia (HIT) which, in preliminary testing, demonstrates higher diagnostic accuracy than the "gold standard" serotonin release assay (SRA) (Padmanabhan et al. Chest, 2016 epub). Here, we describe two cases of severe thrombotic HIT in which the PEA detected pathogenic HIT antibodies (abs) before the SRA tested positive, and in one case, earlier than the highly sensitive PF4 ELISA. These findings have implications for diagnosis and monitoring of the response to therapy in HIT.Patient 1, a 67 yr old woman was admitted for treatment of a Bochdalek hernia and was started on unfractionated heparin (UFH). Her course was complicated by a declining platelet count and on the eighth day of admission PF4 ELISA and SRA tests were strongly positive (Fig 1A). Vascular imaging demonstrated venous thrombosis in both upper and lower extremities. Pre- and post-diagnosis patient samples were tested in the PF4 ELISA, SRA and PEA (Fig 1A). The PEA was positive at 152 hrs (~6 days) post-heparin exposure (69% expression), but the PF4 ELISA was only weakly positive (OD 0.67) and the SRA was negative (0%) at a time when the platelet count was 52% of baseline (Fig 1A). The SRA was still negative (18%) 16 hours later (168 hrs). Thirty hours after the first positive PEA test result, the SRA became positive (100% release). The patient's condition was then further complicated by sepsis-associated multisystem organ failure (MSOF) and therapeutic plasma exchange (TPE) was instituted. TPE treatment led to a rapid reduction in the SRA but only a gradual decline in PEA and ELISA test results (Fig 1B), in line with gradual improvement in platelet counts. Dissociation between SRA and PF4 ELISA test results following TPE has been described previously (Blood. 2015;125:195-8). In our patient, a PEA-SRA dissociation was also observed. Early in this patient's course, PEA-positive/SRA-negative abs were clearly "pathogenic," causing a significant decrease in platelet counts. Thus, it is plausible that PEA-positive/SRA-negative abs detected during the course of TPE were also "pathogenic". PEA results during the course of TPE appeared to correlate better with clinical status in light of the gradual improvement in platelet counts. Unfortunately, the patient expired after the 4th TPE secondary to complications of MSOF.Patient 2, a 57 yr old man was admitted for a creation of a thigh flap in preparation for knee replacement. Postoperatively, the flap became pale with poor pulses and UFH was started. On day 5 platelets dropped 25% from baseline and PF4 ELISA and SRA were both negative (Fig 1C). A strong suspicion of HIT prompted repeat testing on day 6, and yielded positive SRA (79%) and PF4 ELISA (2.8 OD) test results. Notably, the PEA was positive (74%) on Day 5 when both PF4 ELISA and SRA were negative. All tests remained strongly positive through day 12. Thrombocytopenia persisted (Fig 1C) and extensive venous thrombosis of the right upper extremity developed on day 15. The patient was transitioned to Coumadin after platelet recovery and discharged on Day 30.Previous studies indicate that the PEA is at least as accurate as the SRA for detection of pathogenic abs (Padmanabhan et al Thromb Haemost. 2015;114:1322-3, Padmanabhan et al. Chest, 2016 epub). Experience with these two cases suggests that the PEA may in some cases detect pathogenic antibodies before the SRA becomes positive and may be superior to the SRA for monitoring response to treatment. Because of its technical simplicity, the PEA has the potential to be performed on demand, rather than through a referral laboratory, facilitating early diagnosis and treatment. [Display omitted] DisclosuresPadmanabhan:Fenwal (Fresenius Kabi): Research Funding; Schlesinger & Associates: Consultancy, Honoraria; LEK Consulting: Consultancy, Honoraria; Mallinckrodt Pharmaceuticals: Consultancy, Honoraria; Terumo BCT: Consultancy, Honoraria; Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Jones:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Bougie:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Aster:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591.

Are We Doing Too Many HIT Tests? II

Back Ground: Heparin Induced Thrombocytopenia is a complex immune disorder related to exposure to heparin that results in arterial and venous thrombosis and moderate to severe thrombocytopenia. Incidence is 0.8-1% in hospitalized patients exposed to heparin. However there is a problem with over testing for HIT syndrome that it is not only financially burdensome but that treating false positive patients with anticoagulation can increase their risk for bleeding.Objective: To determine the actual incidence of Type II-HIT compared to the frequency and necessity of testing in a single urban, teaching, community hospital in Chicago.Methods: A retrospective review of lab records for patients who were tested for HIT syndrome by H-PF4 -ELISA through 2 years Jan 2013-Dec 2014. Then each patient's chart was reviewed. Patients were categorized according to requesting department (i.e. ICU, ED, Medical and surgical floors) then screened for date of admission, date of onset of platelet drop, degree of platelet drop, incidence of DVT, PE, arterial thrombosis and bleeding and finally for possible reasons of platelet count drop other than HIT for calculation of Pretest probability by the 4T score. According to the 4T criteria patients were categorized as low (score 0-3), intermediate (score 4, 5) and high probability (score 6-8). All charts with positive ELISA were reviewed for SRA (Serotonin Release Assay) results. Also contacted lab and pharmacy for cost of HIT testing, confirmatory test with SRA and the cost of Argatroban to be able to determine the economic burden of over testing for HIT syndrome.Results: an average of about 20,000 admissions per year. Over the year 2013, 110 patients were screened for HIT. Over the year 2014, 87 patients were tested for HIT. Of total of 197 tests ordered over the 2 years period 19 Patients did not have enough data in the charts for determination of 4T score and were so excluded. Of the remaining 178, 85(45%) were sent from ICU, 61(32.6%) from Medical floors, 28(15%) from CCU, 9(4.8%) from Surgical floors, 1(0.5%) from Rehab unit, 1(0.5%) from ED and 1(0.5%) from Observation Unit. 17(9.5%) Patients had proven DVT, PE or arterial thrombosis. 2(1%) Patients had evidence of bleeding. According to 4T score139 (74.3%) of 178 were low, 34(18%) intermediate and 5(2.6%) were high probability. ELISA test was positive for only 34(19%) patients of the 178 of which only 3(1.6%) were proven true positive by SRA, 12(6%) had no SRA result in their chart and 19(10.6%) had negative SRA results. ELISA was positive in 22 cases with Low Probability score, 9 Intermediate probability and 3 high probability. Of the 22 low probability ELISA positive cases only 1 (4%) was SRA positive, 15(68%) were SRA negative and 6(27%) did not have SRA in the chart. ELISA test costs 233$, SRA test costs 50$ and one day of Argatroban costs average of 663.44$.Thus the calculated cost of testing for low probability patients reached 32,387 $ for ELISA and 800$ for SRA. Cost of treating false positive patients with Argatroban for an average of 3 days until SRA results are available is 43,758$.Conclusion: From this study we concluded that in our facility we continue to do too many HIT studies without appropriate prescreening with added cost of testing and treatment for low probability patients of almost 77,000$ over the period of two years. In planning to avoid unnecessary testing and treatment for false positive patients we plan to build a 4T score calculator into our Electronic Medical Record System that is started once a HIT test is ordered to improve the screening process. We will also continue work with the Internal Medicine residency program and medical staff to improve teaching on HIT syndrome and other conditions with similar presentations.Table 1Number of Tests per DepartmentDepartmentNumber of tests sentICU86(45%)CCU28(15%)Medical Floors61(32.6%)Surgical Floors9(4.8%)Rehab Unit1(0.5%)ED1(0.5%)Observation Unit1(0.5%)Total187Table 2Tests Categorized into Low, Intermediate and High Probability according to 4T scoreLow ProbabilityIntermediate ProbabilityHigh Probability139(74.3%)34(18%)5(2.6%)Table 3SRA Results for ELISA positive patients per Each Probability CategoryLow ProbabilityIntermediate ProbabilityHigh Probability22(64.7%)9(26.4%)3(8%)SRA PositiveSRA NegativeSRA UnavailableSRA PositiveSRA NegativeSRA UnavailableSRA PositiveSRA NegativeSRA Unavailable1(4%)15(68%)6(27%)2(22%)4(44%)3(33%)01(33%)2(66.6%) DisclosuresNo relevant conflicts of interest to declare.

Efficiency of Diagnosing HIT at a Large Academic Medical Center

Background: Thrombocytopenia during heparin administration is common in hospitalized patients, leading to frequent testing for heparin-induced thrombocytopenia (HIT). The 4T score, which is based on degree and timing of thrombocytopenia, and whether there are other explanations for thrombocytopenia or the presence of thrombosis, is a commonly used tool to assess the clinical probability of HIT prior to performing laboratory testing. Published guidelines recommend further testing with an anti-PF4/heparin enzyme immunoassay (EIA) and confirmation with the serotonin-release assay (SRA) when the pretest probability of HIT is intermediate or high. The diagnosis of HIT confers high morbidity and mortality; therefore it is crucial to establish a definitive diagnosis in patients with suspected HIT. However, empiric treatment with alternative non-heparin anticoagulants increases the cost of hospitalization and may cause unnecessary risks in patients with a false positive EIA. The purpose of our study was to evaluate the utilization of EIA and SRA at a large tertiary care center and to assess the agreement between the 4T score, the EIA, and the SRA.Methods: We retrospectively reviewed all EIAs performed in our special hematology laboratory and SRAs sent to a reference laboratory at our institution in patients with suspected HIT between July 2012 and June 2014. First we evaluated the 4T score in all patients in whom laboratory testing was requested and subsequently assessed the correlation between the 4T score and the EIA and SRA results. A 4T score of 6 to 8 was high and a 4T score of 4 to 5 was intermediate. A strong positive EIA was defined as an EIA with an optical density (OD) of ≥ 1.00 and a weak/indeterminate EIA had an OD of 0.40-0.99.Results: A total of 583 unique PF4 EIAs were requested during the study period (Figure 1). Of these, 472 (81%) and 77 (13%) were performed in patients with intermediate/ high probability and with low-probability of HIT, respectively and in 34 patients (6%) the EIA was requested without documentation of the 4T score. Of the 472 patients with an intermediate or high 4T score, 36 (8%) had an EIA with an OD ≥ 1.0. Of these 36 patients, 27 (75%) had SRA performed and were positive in 14 patients (52%). Twenty-three (5%) of the 472 patients had a weakly positive EIA and in 18 (78%) of them the SRA was sent and were all negative. Five of the 472 patients (1%) had a positive SRA in the setting of an intermediate or high 4T score and a negative or indeterminate/weakly positive EIA. The SRA was sent in 24 patients with low probability 4T score (0-3) and in 9 patients with no documented 4T score. Two of these 33 (6%) patients had a positive SRA. Overall, the incidence of HIT based on a 4T score of 4 to 8, a positive EIA and positive SRA was found in 16 (3.4%) of the 472 patients; 2 of these 16 patients (12.5%) had a indeterminate/weakly positive EIA. Of the 216 SRAs sent, 21 were positive (9.7%). Patients with a lower pre-test probability based on a low or intermediate 4T score and negative/weakly positive EIA accounted for 33% of HIT cases observed. Patients with an intermediate/high 4T and strong or weakly positive EIA accounted for the remaining 67%.Conclusions: The incidence of definitive HIT in our study was low. There was a poor correlation between the 4T score and SRA positivity and between the EIA and SRA results. The combination of an intermediate/high 4T score and a strong EIA confirmed the diagnosis of HIT in only half of the patients. These findings underscore the need for improved screening tools for HIT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rivaroxaban for Treatment of Suspected or Confirmed Heparin-Induced Thrombocytopenia Study

▪Rivaroxaban is an ideal potential alternative for treatment of heparin-induced thrombocytopenia because it is administered orally by fixed dosing, requires no routine coagulation monitoring and has been proven effective in the treatment of venous and arterial thromboembolism in other settings. The Rivaroxaban for HIT Study prospectively evaluated the efficacy and safety of patients with suspected or confirmed HIT who were treated with rivaroxaban [NCT01598168 - investigator sponsored study funded by Bayer]Methods: Canadian multicenter, single-arm, prospective cohort study of patients with confirmed or suspected HIT (4Ts score ≥4) treated with rivaroxaban 15 mg bid until the diagnosis was supported or refuted using the local HIT assay. Participants with HIT (positive local assay result) received rivaroxaban 15 mg bid until platelet recovery (or until Day 21 if the patient had acute thrombosis; HITT) then stepped down to rivaroxaban 20 mg daily until Day 30. Central testing with the serotonin-release assay (SRA) was performed (not in real-time at all centres). HIT positive was defined as a 4Ts score ≥4 plus serotonin release ≥50%.The primary outcome measure was the incidence of new symptomatic, objectively-confirmed venous and arterial thromboembolism in the combined cohort of patients with suspected and confirmed HIT at 30 days. Secondary objectives included incidence of symptomatic thromboembolism while on treatment with rivaroxaban (combined cohort) and the following outcomes among SRA-positive participants while on treatment with rivaroxaban: incidence of venous and arterial thromboembolism, incidence of major bleeding, and time to platelet recovery.Sample size of 200 participants (10 to 30 with SRA-confirmed HIT) was based on feasibility and an anticipated thrombotic event rate in the study population (combined cohort) of 6.5% at 30 days (5% in HIT negative; 11% in HIT positive while on rivaroxaban).Results: 22 participants (12 HIT positive) were enrolled between January 2013 and July 2015. The study was terminated early due to poor recruitment, but after enrolling the minimum expected number of HIT positive participants. Of the 12 HIT positive participants (SRA, mean release 95%), 3(25%) had HITT at time of study entry and 6 had received at least one dose of fondaparinux prior to study enrolment. Half of the HIT positive participants were enrolled in study after the SRA result had already been reported as positive.After 371 days of exposure to rivaroxaban (combined cohort), 1 HIT positive participant had possible symptomatic recurrent VTE (4.5%, 95% CI: 0 to 23.5%), 1 HIT positive participant had major bleeding (9 days after rivaroxaban was held) and there were 4 deaths (cancer 2, sepsis 1, end-stage COPD 1).The single episode of possible recurrent VTE was extension of previously documented apheresis catheter-related arm DVT in a HIT positive participant who presented on Day 7 with worsening arm pain. A repeat ultrasound showed extension of DVT; however a baseline scan had not been performed at time of study entry. Interestingly, the same participant failed treatment with fondaparinux prior to study enrolment (development of erythematous plaques at injection sites and failure of platelets to rise). His apheresis catheter was removed on Day 8, rivaroxaban was continued and complete resolution of his symptoms as well as platelet recovery was achieved.One HIT positive participant presented with evidence of bilateral lower limb arterial ischemia (HIT-related acute arterial thrombosis on documented chronic peripheral vascular disease) at the time of study enrolment. Despite achieving platelet recovery, he underwent bilateral below knee amputation on Day 16.Out of the 12 HIT positive participants, 11 achieved platelet recovery with mean time to recovery 9 days. The single participant who did not achieve platelet recovery received only 2 doses of rivaroxaban before it was held due to a transient rise in liver enzymes. Rivaroxaban was never restarted because he bled while receiving fondaparinux as an alternative.Conclusions: Rivaroxaban appears to be effective for treating patients with confirmed HIT, although lack of a comparator and a small sample size are limitations of our findings. The advantages of rivaroxaban over other agents currently used to treat HIT such as ease of administration, lack of routine coagulation monitoring and low cost make it an attractive option. DisclosuresLinkins:Pfizer: Honoraria; Bayer: Honoraria, Research Funding. Off Label Use: rivaroxaban has not been approved for treatment of heparin-induced thrombocytopenia. Warkentin:W.L. Gore: Consultancy, Research Funding; Instrumentation Laboratory: Consultancy, Honoraria; Pfizer: Consultancy. Pai:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS-Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Shivakumar:Bayer: Honoraria. Wells:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS-Pfizer: Research Funding. Wu:Pfizer Canada: Membership on an entity's Board of Directors or advisory committees; Leopharma: Membership on an entity's Board of Directors or advisory committees.

Abstract 13476: Association of HLA-DRB3*01:01 With Heparin-Induced Thrombocytopenia

Introduction: Variation in the human leukocyte antigen (HLA) region is now used clinically to prevent immune-mediated adverse drug reactions. Heparin-induced thrombocytopenia (HIT) is an unpredictable, life-threatening, immune-mediated reaction to heparin treatment. Previous studies have implicated HLA-DR variants in HIT, but HLA alleles which predispose patients to HIT have not been identified. Hypothesis: Our hypothesis is that sequenced HLA alleles are associated with HIT. Methods: We identified HIT cases and heparin-exposed controls in an electronic medical record coupled to a DNA biobank. Cases were defined based on HIT antibody results, HIT risk scoring (4Ts score), and serotonin release assay results. Controls were matched to cases based on age, gender, and type of heparin exposure (unfractionated versus low molecular weight heparin). We performed high resolution, four-digit HLA sequencing for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ using long-read 454 FLX sequencing. We tested association of sequenced HLA alleles using conditional univariate logistic regression in a dominant model. Analysis was restricted to individuals with European ancestry and HLA alleles with a frequency greater than 0.01 (n=101). P values were corrected using a Bonferroni correction for 101 sequenced HLA alleles (alpha=4.95x10 -4 ). Results: We identified 77 HIT cases and 345 matched controls. No statistical differences were observed between cases and controls for baseline characteristics. The HLA-DRB3*01:01 allele was significantly associated with HIT (odds ratio 3.55, p=1.09x10 -4 ). No other alleles showed a significant association with HIT. Conclusions: We implicate the HLA-DRB3*01:01 allele as a risk factor for HIT. This allele represents a biologically plausible candidate for HIT, considering the previous literature implicating HLA-DR variation in HIT, the central role of HLA-DR in T cell-dependent antibody production, and the importance of T cells in HIT pathogenesis. Validation of HLA-DRB3*01:01 as a predictor of HIT pathogenesis would lead to preventive genotyping to reduce the risk of HIT.

Evaluation of clinical and laboratory data for early diagnosis of heparin-induced thrombocytopenia.

Results of a study to determine the utility of combining laboratory values and clinical probability scores to improve the detection of heparin-induced thrombocytopenia (HIT) are reported. In a retrospective, single-site, chart review-based investigation, 156 cases in which patients with suspected HIT had positive results on a widely used diagnostic test (the anti-heparin/platelet factor 4, or anti-PF4, assay) were identified; in all cases, the blood specimens had been sent to a reference laboratory for confirmation of HIT via serotonin release assay (SRA). After investigator scoring of the clinical probability of HIT in each case by the 4T's method, a multiple logistic regression model was used to evaluate the combined effect of 4T's scores and anti-PF4 assay values in predicting SRA results. 4T's scores indicating an intermediate or high probability of HIT combined with high anti-PF4 test values (i.e., optical density [OD] value of ≥1.4) were strongly predictive of a positive SRA result, as were high-probability 4T's scores alone. Low-probability 4T's scores alone or in combination with anti-PF4 OD values of <1.4 were highly correlated with negative SRA results. Controlling for potential confounding factors, logistic regression analysis indicated that the 4T's score was a better predictor of SRA results than the anti-PF4 test value. The combination of anti-PF4 OD values and 4T's scores accurately predicted SRA results, suggesting that the SRA may not be necessary to confirm HIT in patients with a relatively low 4T's score and a low anti-PF4 OD value.

Combination of 4Ts score and PF4/H-PaGIA for diagnosis and management of heparin-induced thrombocytopenia: prospective cohort study

AbstractRapid exclusion of heparin-induced thrombocytopenia (HIT) is needed to determine which patients can continue to receive heparin. In this prospective management study, 526 participants had a 4Ts score, rapid particle gel immunoassay (platelet factor 4/heparin [PF4/H]-PaGIA), and serotonin-release assay (SRA) performed. While awaiting SRA results, participants with a low 4Ts score (irrespective of PF4/H-PaGIA result) or intermediate 4Ts score plus a negative PF4/H-PaGIA result received prophylactic doses of danaparoid or fondaparinux; all others received therapeutic doses of nonheparin anticoagulants. The primary outcome was the frequency of management failures defined as HIT-positive participants with a low 4Ts score (irrespective of PF4/H-PaGIA result) or intermediate 4Ts score plus negative PF4/H-PaGIA result. Six participants (1.1–; 95– confidence interval [CI], 0.2-2.1–) were management failures. A negative PF4/H-PaGIA result reduced the pretest probability of HIT from 1.9– to 0– (95– CI, 0-1.3–), 6.7– to 0– (95– CI, 0-2.7–), and 36.6– to 0– (95– CI, 0-14.3–) in the low, intermediate, and high score groups, respectively. A positive PF4/H-PaGIA result increased the probability of HIT in the low score group to 15.4– (95– CI, 5.9-30.5). Thus, a low or intermediate 4Ts score plus negative PaGIA result excluded HIT, whereas any other combination of results justified the use of alternative anticoagulants until HIT could be excluded. This trial was registered at www.clinicaltrials.gov as #NCT00489437.

Efficacy and safety of argatroban and bivalirudine in patients with suspected heparin-induced thrombocytopenia.

Argatroban is the only commercially available Food and Drug Administration (FDA)-approved anticoagulant for managing heparin-induced thrombocytopenia (HIT). However, bivalirudin may be an attractive alternative. To assess the efficacy and safety of argatroban and bivalirudin in patients with suspected HIT. This single-center, retrospective analysis included patients who received argatroban or bivalirudin for at least 24 hours between January 1, 2000, and June 30, 2012. The primary end point assessed anticoagulation goals, specifically time to therapeutic activated partial thromboplastin time (aPTT) goal and percentage of aPTT values within therapeutic range. Secondary end points included new thromboembolic events, bleeding, and mortality. Of the 68 patients who met the inclusion criteria, 48 received argatroban and 20 received bivalirudin. Baseline characteristics were similar between the 2 groups except for age, percentage of patients with liver dysfunction, aPTT immediately prior to drug initiation, and the serotonin release assay results. The mean ± SD times to reach therapeutic aPTT goal for argatroban and bivalirudin were 14 ± 15 and 7 ± 8 hours, respectively (P = 0.024). The mean ± SD percentage of aPTT values within therapeutic aPTT goal was 69% ± 23% for argatroban and 84% ± 18% for bivalirudin (P = 0.005). Rates of thromboembolic events were similar between the 2 groups, as were the rates of bleeding and all-cause mortality. Bivalirudin appears to reach therapeutic aPTT goal faster with more aPTT values within therapeutic aPTT goal while achieving similar clinical outcomes. Although not approved by the FDA for managing HIT, bivalirudin may be an attractive alternative anticoagulant.