Heparin-induced Thrombocytopenia ELISA Research Articles

Comparison of Interobserver Agreement between the 4T and the HIT Expert Probability (HEP) Scores: We Agreed Not to Agree

Background: Diagnosis of heparin induced thrombocytopenia (HIT) is challenging. The 4T score is the principal scoring system for HIT. The HIT Expert Probability (HEP) score, based on expert opinion, was proposed in 2010. Both scores have a very high sensitivity, being capable of ruling out HIT in low-risk individuals. However inter-observer agreement (IOA) of HEP score in comparison to the 4T score remains uncertain. Aim: This study aimed to compare IOA between the 4T and the HEP score among hospitalized patients. Specific objectives: Comparison of IOA between the 4T and the HEP score in the 1-whole cohort 2-non-ICU 3- ICU Identify which item of the score have the lowest IOA (magnitude of thrombocytopenia, timing of thrombocytopenia, existence of other causes of thrombocytopenia, presence of thrombosis) Methods: Patients: This is a retrospective study at a single tertiary hospital. All inpatients 16 years or older who have been tested for HIT between June 2019 to end of June 2021 were included. Patients were excluded if the reason for sending HIT was a repeat test. Data were collected retrospectively by manual chart review. Assessment of HEP score and 4Ts score: Two hematologists rated the HEP score and 4Ts score independently for every patient. The score's calculation was blinded between observers and, they were blinded to the HIT Eliza result. Low 4Ts score is defined as score less than 4 and for HEP score if less than 3. HIT test was done using Stago Asserachrom HPIA-IgGAM ELISA with level of < 0.4 considered negative. Statistical analysis: Continuous variables are reported as mean ± standard deviation (SD) or median (interquartile range; IQR), as appropriate. Categorical variables are reported as frequency and percentage. The Kappa coefficient (к) with 95 % confidence interval (CI) was used to assess the IOA of 4T score and HEP score, it was calculated for the individual risk categories - low vs high score in: 1-the whole patients 2-ICU 3- non-ICU We also calculated the IOA for each item of the score, magnitude of thrombocytopenia, timing of thrombocytopenia, existence of other causes of thrombocytopenia, presence of thrombosis (as total score). A P-value less than 0.05 was considered significant. Statistical analyses were performed using SAS version 9.4 (SAS institute, CARY, NC, USA). The study was approved by the local ethical board. Informed consent was waived. Results: After exclusion of the repeat test (13), 302 patients were included in this study. Median age 66 years old. Most common reason for request was thrombocytopenia 288 (95%). HIT ELISA ≥ 0.4 in 34 (11%). Inter-observer agreement: All patients: We found similar IOA between the 4T score and the HEP score к (95 % CI) of 4T score was .14(-0.13-0.41, P < 0.01), compared 0.17(-0.16-0.5, P < 0.01) in the HEP score. Non-ICU patients: The IOA was similar between the 4T score and the HEP score, к of 4T score was 0.24(0.11-0.38, P < 0.01) compared 0.17(0.03-0.31 P < 0.01) in the HEP score. ICU patients: The IOA of 4Tscore was lower than HEP score к of 4T score was -020(-0.20.16, P = 0.83) compared 0.16(-0.037-0.37, P = 0.86) in the HEP score. Among the four individual items of 4T score, “existence of other causes of thrombocytopenia” achieved the lowest к with 0.03 and 0.1 in 4Tscore and HEP score respectively. Conclusion: In this retrospective study, we found a lower interobserver agreement of the 4T score compared to the HEP score in ICU patients. The poor IOA of the 4T score in ICU patients is attributed to worse agreement in (existence of other causes of thrombocytopenia item). Ascertaining the cause of thrombocytopenia in ICU patients is challenging and the 4T score seems to depend on physician gestalt rather than objective points. Our data suggests that the HEP score is the preferred score in in patients with critical illness.

A Thrombospondin-1 Release Assay (TRA) Coupled to PF4-Treated Cryopreserved Platelets for the Detection of Pathogenic HIT and VITT Antibodies

Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis.Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry.Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation.Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D).Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis.Figure Legend:Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. [Display omitted] DisclosuresPruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Vaccine-induced immune thrombotic thrombocytopenia: current evidence, potential mechanisms, clinical implications, and future directions.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) (also termed thrombosis with thrombocytopenia syndrome or vaccine-induced thrombotic thrombocytopenia or vaccine-induced immune thrombocytopenia) is characterized by (i) venous or arterial thrombosis; (ii) mild-to-severe thrombocytopenia; (iii) positive antiplatelet factor 4 (PF4)–polyanion antibodies or anti-PF4–heparin antibodies detected by the HIT (heparin-induced thrombocytopenia) ELISA; (iv) occurring 5–30 days after ChAdOx1 nCoV-19 (AstraZeneca) or Ad26.COV2.S (Johnson & Johnson/Janssen) vaccination. VITT’s incidence is 1 per 100 000 vaccinated people irrespective of age and up to 1 in 50 000 for people <50 years of age with the AstraZeneca COVID-19 vaccine. The exact mechanism by which adenovirus-vectored COVID-19 vaccines trigger this syndrome is still unclear, as for the increased risk for acute cerebral sinus venous thrombosis and splanchnic vein thrombosis as compared to other locations of venous thrombotic events. VITT is associated with the detection of anti-PF4 antibodies, unrelated to previous use of heparin therapy. PF4 antibodies are thought to activate platelets via the platelet FcγRIIA receptors leading to further platelet activation that causes thrombosis and thrombocytopenia.

Vaccine‐induced Immune Thrombocytopenia and Thrombosis (VITT)

Vaccine‐induced Immune Thrombocytopenia and Thrombosis (VITT)

HIT: Anti-Platelet Factor 4-Herapin (PF4-H) Assay Validation and Comparison Study

<h3>ABSTRACT</h3> <h3>INTRODUCTION:</h3> Heparin is a common anticoagulant prescribed to many heart and surgical patients. These patients may develop antibodies to platelet factor 4 (PF4) and heparin complexes, which may lead to severe thrombocytopenia and deep vein thrombosis. The implementation of a rapid and automated laboratory test that can detect platelet factor 4-herapin (PF4-H) complexes qualitatively, can assist physicians in screening for heparin-induced thrombocytopenia (HIT) and decide whether to halt the heparin dosage. The purpose of the study is to validate the HemosIL HIT-Ab (PF4-H) assay on the ACL TOP 750, determine the sensitivities and specificities of 3 HIT assay methods (ACL TOP, particle immunofiltration assay [PIFA], and enzyme-linked immunosorbent assay [ELISA]), and compare them to sensitivity and specificity of the confirmatory serotonin-release assay (SRA), which is considered the gold standard. <h3>OBJECTIVES:</h3> The aims of the study were to perform an instrument validation and calibration of the HemosIL HIT-Ab (PF4-H) assay in the ACL TOP 750 analyzer and to compare the sensitivities and specificities of the 3 HIT tests (HemosIL HIT-Ab [PF4-H] assay, PIFA Heparin/PF4 assay, and Immucor PF4 ELISA) versus the gold standard SRA performed in Associated Regional and University Pathologists (ARUP) Laboratories and versus each other. <h3>METHODS:</h3> The HemosIL HIT-Ab (PF4-H) assay was validated on the ACL TOP 750 using calibrators, controls, and samples from University of Florida (UF) Pathology Laboratories and ARUP Laboratories that were previously tested for the HIT-Ab immunoglobin-G via an ELISA method. Results were entered in a data collection sheet. A 2 × 2 data conversion table in Microsoft Excel was used to calculate the sensitivities and specificities for each assay and EP Evaluator was used to calculate mean, SD, coefficient of variation, sensitivity, specificity, positive predictive value, and negative predictive value. <h3>RESULTS:</h3> A total of 116 samples were assayed using the HemosIL HIT-Ab (PF4-H) assay on the ACL TOP 750 and the PIFA Heparin/PF4 Rapid assay. Overall, there were 125 positive test results and 255 negative test results for anti-PF4-H (ACL TOP: 39 positive, 77 negative; PIFA: 11 positive, 105 negative; and ELISA: 60 positive, 56 negative). There were 15 positive and 17 negative test results for the SRA. The comparison between the ACL TOP and SRA had a sensitivity of 80.0% and specificity of 35.3%. ELISA versus SRA had a sensitivity of 100.0% but 0.0% specificity. The PIFA versus SRA correlation had a sensitivity of 0.0% and specificity of 88.2%. The ACL TOP versus UF Health’s current HIT ELISA method had a good comparison as well with a sensitivity of 61.7% and a specificity of 96.4%. <h3>CONCLUSION:</h3> The 3 HIT methods (ACL TOP, ELISA, and PIFA), compared to the SRA, showed that the ACL TOP had a good positive agreement but poor negative agreement; however, the ACL TOP had a very good negative agreement with the ELISA method. The implementation of the new HIT-antibody assay will benefit the institution’s standard practice of care because it proved to be a reliable test. The high negative predictive value in the HemosIL HIT-Ab test may assist physicians in the rule out of HIT amongst HIT-suspected patients. The PIFA cartridge test had an overall poor performance with no positive agreement with the SRA and poor sensitivity versus the ACL TOP and ELISA methods.

Risk of Subsequent Thrombosis in Patients with Isolated Heparin-Induced Thrombocytopenia

Background: Heparin-Induced Thrombocytopenia (HIT) is one of the most important and commonly encountered immune-mediated drug reactions which can lead to significant morbidity and mortality. In up to 25% of patients, thrombosis can be the presenting finding of HIT. Isolated HIT occurs in patients without clinically evident thrombosis at the time of diagnosis. The management of HIT focuses on reducing the risk of thrombotic complications. In addition to prompt discontinuation of heparin products, anticoagulation is recommended. The risk of thrombosis is thought to remain high for up to 4 weeks due to persistent circulating PF4-heparin antibodies. Prior studies have shown that in patients with isolated HIT, the subsequent 30-day risk of thrombosis is up to 53%.Guidelines recommend 3 months of anticoagulation for patients with HIT-associated thrombosis. The optimal duration of anticoagulation in isolated HIT is unknown. Many experts recommend prophylactic anticoagulation for up to 4 weeks after diagnosis. However in the era of direct thrombin inhibitors, the need for prophylactic anticoagulation and risk of thrombosis beyond normalization of platelet count remains to be determined. The aim of this study was to determine the incidence of subsequent thrombosis in patients with isolated HIT.Methods: In this retrospective review, we identified patients with a documented positive HIT (heparin-PF4 antibody) enzyme immunoassay (ELISA) at our affiliated hospitals between January 2006 and December 2016. Laboratory data from these patients were reviewed. Patients who met criteria for HIT, defined as HIT ELISA optical density ≥ 2 or positive serotonin release assay were included in the analysis. From this cohort of patients with confirmed HIT, clinical data was extracted from the electronic medical record, including anticoagulation management and documented thrombotic events within 3 months of HIT diagnosis. Patients with evidence of thrombosis at the time of diagnosis were excluded in the final analysis as our focus was on patients with isolated HIT. The incidence of subsequent thrombosis was compared in patients who received anticoagulation versus those who did not receive anticoagulation.Results: A total of 403 charts were reviewed from patients with a documented positive HIT ELISA, defined as an optical density of > 0.4. Of the 403 patients, 49.5% (n = 107) met criteria for HIT, with an optical density > 2 or positive serotonin release assay. In patients with HIT, 48.6% (n = 52) did not have evidence of thrombosis at the time of diagnosis. Of these patients with isolated HIT, 14 patients (26.9%) received no prophylactic anticoagulation, 8 patients (15.4%) received prophylactic anticoagulation for at least 4 weeks but less than 3 months, and 30 patients (57.7%) were anticoagulated for 3 months or longer. The total incidence of subsequent thrombosis in isolated HIT was 7.7%. In the cohort of patients that did not receive prophylactic anticoagulation, two patients (14.3%) developed a thrombotic event in the 3 months following the HIT diagnosis. No documented thrombotic complications occurred in the eight patients that received prophylactic anticoagulation for at least 4 weeks but less than 3 months. Three patients (10%) who received long-term anticoagulation (≥3 months) developed a subsequent thrombotic event. There was no significant difference in the incidence of subsequent thrombosis in patients who did not receive prophylactic anticoagulation versus patients who received short-term prophylactic anticoagulation (p=0.141). In addition, there was no significant difference in the incidence of subsequent thrombosis in patients who did not receive prophylactic anticoagulation versus those who received long-term anticoagulation (p=0.701).Conclusions: The optimal duration of anticoagulation in patients with isolated HIT is unknown. Our incidence of thrombosis following the diagnosis of HIT was lower than previously described. Patients who did not receive prophylactic anticoagulation did not have a significantly higher incidence of thrombosis compared to patients who received short-term prophylactic or long-term anticoagulation. The study was limited by the retrospective nature and small sample size. Further studies are needed to better understand the ideal length of anticoagulation to prevent thrombotic complications without leading to unnecessary increased risk of bleeding. DisclosuresNo relevant conflicts of interest to declare.

Platelet Trends Are Not Predictive of Heparin Induced Thrombocytopenia in Patients on Extra Corporeal Membrane Oxygenation

Introduction: Extracorporeal membrane oxygenation (ECMO) is a form of a cardiopulmonary bypass circuit that is utilized for patients in dire need of cardiac or pulmonary support. The circuit exposes blood to artificial surfaces which leads to platelet activation, clotting and thrombocytopenia. The addition of heparin is required in order to prevent clots from forming along the circuit. Heparin-induced thrombocytopenia (HIT) and heparin induced thrombocytopenia and thrombosis (HITT) are serious adverse effects of heparin treatment characterized by formation of antibodies to heparin platelet factor 4 (PF4) complexed with heparin which leads to thrombocytopenia and a greatly increased risk of arterial and venous thrombosis. The well-established incidence of HIT ranges from 0.5-5% in patients exposed to heparin for more than four days. The reported incidence of HIT in ECMO patients ranges from less than 1% to 8.3%. However this data comes from only a few studies with small sample sizes. Our institution has a large lung center and as a result ECMO is utilized frequently. When patients are suspected to have HIT, they are taken off heparin and switched to a different anticoagulant. For patients without HIT, the switch to a different anticoagulant is unnecessary and exposes the patient to additional risks such as a higher risk of bleeding. Our aim was to calculate the incidence of HIT and HITT at our institution. Furthermore we wanted to examine the platelet trends of all patients on ECMO specifically comparing and contrasting the patients who are deemed true HIT positive vs. all other patients on ECMO.Methods: We conducted a retrospective analysis of all patients on ECMO at Temple University Hospital from July 1, 2012 to December 31, 2017. The data for this study was drawn from the T'PAU (Temple Population Access Utility) research data warehouse that has successfully merged data from the Epic EMR and the McKesson administrative system that houses important summary data on inpatient admissions going back through July 2012. Our inclusion criteria consisted of all patients on ECMO who had a clinical suspicion of HIT and thus had a screening test sent for HIT, the ELISA polyclonal IgG testing against PF4. The diagnosis of HIT or HITT was confirmed by PF4 antibody positivity with an optical density (OD) >1.0, serotonin release assay (SRA) positivity or a positive PF4 antibody test >0.4 and the presence of thrombosis. Linear mixed-effects model was used to estimate and compare platelet count change over time between true HIT patients and patients who tested negative for the HIT ELISA.Results: There were 164 patients on ECMO between July 1st, 2012 and December 31, 2017. A total of 77 patients had the HIT ELISA sent while on ECMO. Of those, 15 patients had a positive test result (OD > 0.4) and 62 had a negative test result. Of the patients with positive results, 5 patients had an OD > 1.0. Three of the 10 patients with an OD < 1.0 were found to have thrombosis. Therefore 8 patients had true HIT out of a total of 164 patients on ECMO, for an incidence of 4.88%. There was no difference of the change of platelet count over time between the HIT (n=8) and non-HIT (n=62) groups (P=0.98).Conclusion: The incidence of HIT in our ECMO patient population is similar to the incidence of HIT in all patients who have been exposed to heparin for four or more days. Patients requiring ECMO are very ill and with the accompanying thrombocytopenia that develops with the initiation of ECMO, the clinical suspicion for HIT is high. Unlike the majority of hospitalized patients, patients on ECMO are a unique population and platelet count trends are not necessarily a clinically reliable indicator for HIT. Our research supports that the full clinical picture should be taken in account when suspecting HIT in patients on ECMO, especially when considering a change in anticoagulation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Appropriateness of Heparin Antibody Testing. A Single Institution Retrospective Analysis

Background: Heparin-induced thrombocytopenia (HIT) is a drug-induced, immune-mediated prothrombotic disorder associated with thrombocytopenia and venous and/or arterial thrombosis.Up to 5% of patients exposed to heparin for at least one week develop HIT, and approximately 50% of them will have thrombosis. Diagnosis of HIT is suspected from the clinical picture based on the ''4 T's'' (Thrombocytopenia, Timing, Thrombosis, no other cause of platelet fall) or the HIT Expert Probability (HEP) scoring system. However, often these "4 T's" are ignored and testing is inappropriately ordered in low risk patients. This could lead to possible morbidity and increase length of hospital stay. We opted to look back at the appropriateness of testing done within our academic center.Methods/ Design:All hospitalized patients with thrombocytopenia who underwent HIT antibody testing (HIT ELISA) during February 2013 were screened using our internal electronic medical record.Patients were subdivided to low, intermediate or high pretest probability group according to the 4Ts scoring system.Appropriateness of testing was determined according ASH 2013 Clinical Practice Guideline on the Evaluation and Management of Adults with Suspected Heparin-Induced Thrombocytopenia (HIT). HIT Ab test is recommended to be tested when there is intermittent to high probability of HIT (4T's score above or equal to 4).The percentage of appropriate/inappropriate testing was calculated.The results were then subdivided by ordering physician group.Results:120 HIT ab tests were performed during February 2013 in our institution. Only 13 patients tested had a positive HIT Ab. Internal medicine ordered the majority of these tests. Of the 120 patients tested, 7 patients had high risk and another 50 had intermediate risk as per 4T's scoring. 61 patients were low risk and should not have been tested. (Table 1)Table 1HIT antibody test ordered per Service and probability of HIT.Service ordering testLow probability of HITIntermediate/high probability of HITTOTALInternal Medicine282856Cardiothoracic Surgery131124Hematology Oncology7512Cardiology314Surgery145Emergency medicine011Infectious disease404Pulmonary011Critical care167Nephrology123Ob Gyn101Family Medicine202TOTAL6159120Conclusions:HIT testing has been overused within our institution. Low platelets, without other signs or symptoms of typical HIT were used as a trigger for testing the antibody. This has potential to cause harm due to increased bleeding risk, increased length of hospital stay, and the potential to utilize extended anticoagulation when not necessary. Better education on the appropriateness of the test can limit the potential harm of its use. DisclosuresNo relevant conflicts of interest to declare.

Clinical Features of Heparin-Induced Thrombocytopenia Diagnosed By Lumi-Aggregometry

Background: Heparin-induced thrombocytopenia (HIT) is associated with anti-platelet factor-4 (PF4)/heparin antibodies that activate platelets, resulting in thrombocytopenia and a pro-thrombotic state. At our institution antibody-mediated platelet activation is demonstrated by lumi-aggregometry, which is a method previously validated against the gold standard serotonin-release assay (SRA). Lumi-aggregometry does not involve radioactive isotopes, which is its major advantage over the SRA. The clinical course of HIT diagnosed via SRA and ELISA has been previously described, and clinical prediction tools such as the 4-T score were validated using these diagnostic tests. However, the clinical picture of HIT diagnosed by lumi-aggregometry has not been previously described.Aims: The objective of this study is to describe the clinical and laboratory presentation of patients diagnosed with HIT by lumi-aggregometry.Methods: Patients with clinically suspected HIT and quantitative anti-PF4 IgG-specific ELISA OD ≥0.400 (Gen-Probe, San Diego) received confirmatory HIT testing by lumi-aggregometry. Briefly, HIT antibody-induced activation of washed healthy donor platelets was tested at therapeutic (0.1U/mL and 0.5U/mL) and high (100U/mL) porcine heparin concentration. The degree of platelet activation was quantitated luminographically based on the light flash reaction of ATP (released from platelet dense-granules) with luciferin luciferase reagent. A ratio of therapeutic to high heparin luminescence amplitude of >5.0 and platelet aggregation at therapeutic, but not high, concentrations was considered a positive result. The results of assays performed by our regional HIT testing referral laboratory from June 2009 to July 2012 were reviewed to identify patients with positive HIT testing by lumi-aggregometry. Patient records were retrospectively reviewed to obtain predefined data on baseline patient characteristics, heparin exposure, platelet counts, and thrombotic events occurring in the 5 days preceding or the 30 days following the date of positive HIT testing.Results: We identified 43 patients diagnosed with HIT by lumi-aggregometry (median age 68.0, 49% male) while under the care of local academic (46%) or urban community hospitals (37.2% medical; 53.5% surgical; 9.3% intensive care). Median baseline platelet count was 187 (14-349). Median date of platelet drop post-heparin exposure was 6 days (range 3-14) in patients without prior heparin exposure or platelet transfusions (Figure 1). Platelet drop >50% and platelet nadir ≥20x109/L were present in the majority of patients (Table 1). Thrombocytopenia occurred prior to (70.5%) or the same day (23.5%) as thrombosis in 16/17 patients with serial platelet counts who developed HIT-associated thromboembolism.Conclusion: Patients diagnosed with HIT by lumi-aggregometry present with similar findings to those described in SRA-confirmed HIT. These findings lend support to the use of lumi-aggregometry as an accurate diagnostic assay for the clinico-pathologic syndrome of HIT. [Display omitted] Table 1Percentage platelet drop from baseline and platelet nadirPercent platelet dropPlatelet nadir>50%30-50%<30%≥20 x 109/L283210-19 x 109/L510<10 x 109/L110 DisclosuresSzkotak:Alexion Pharmaceuticals: Research Funding. Sandhu:Celgene: Honoraria; Jansen: Honoraria; Novartis: Honoraria.

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Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin treatment characterized by formation of antibodies to heparin–platelet factor 4 (H–PF4) complexes. Clinically, patients with HIT demonstrate a fall in platelet count of more than fifty percent, occurring 5–10 days after the start of heparin treatment; some also develop HIT related thrombosis (HITT). Cardiopulmonary bypass (CPB) is associated with platelet activation, thrombocytopenia and the release into the plasma of large amounts of PF4, as well as exposure to high doses of unfractionated heparin (UFH). Up to 50% of patients develop antibodies to H–PF4 complexes following CPB. However, less than 3% of those patients develop actual HITT, characterized by a second episode of thrombocytopenia 5-10 days after CPB ("P1 pattern" reported by Pouplard et al.). ECMO is a modified CPB circuit that critically ill patients may be exposed to for days and weeks. The incidence of HIT in patients undergoing ECMO is unknown. Furthermore, clinical scores used to diagnose HIT may not be useful in ECMO patients given the high prevalence and persistence of thrombocytopenia caused by artificial circuit and underlying disease. We sought to determine the incidence of HIT/HITT and platelet patterns potentially suggestive of HIT in a cohort of ECMO patients at our institution Methods: After IRB approval, a prospectively collected data set encompassing all ECMO patients from 2009 to 2012 at our institution was reviewed. To compare platelet trends, we only included patients who survived longer than 48 hours after ECMO initiation. Patients were tested for HIT based on presence of thrombocytopenia and clinical suspicion. Institutional criteria defines patients as "HIT positive" if they meet any of the following serological criteria: HIT ELISA Ab(+)/SRA(+); HIT ELISA Ab(-)/SRA(+); HIT ELISA(+) with optical density (OD)>1. "HIT positive" patients with evidence of thrombosis were considered as having HITT. Patients who didn't meet these criteria, or were not tested due to lack of clinical suspicion, were considered "HIT negative". Daily platelet counts were recorded for 10 days inclusive of day of ECMO initiation. Statistical analysis was done using the Wilcoxon rank sum test and Fisher exact test as appropriate for the data sets. Results: 85 patients underwent ECMO from 2009 to 2012. 68 (80%) patients survived longer than 48 hours after ECMO initiation. Of those, 50 patients(73.5%) were tested for HIT ELISA, SRA or both. 4 patients were deemed "HIT positive" and 3 of those patients had documented thrombo-embolic events (HITT) based on predefined criteria (HIT and HITT incidence in the study group of 5.9% and 4.4% respectively). HIT positive patients were significantly younger (53.1 vs. 37.5 years, p=0.048) and had a trend toward higher average HIT ELISA OD (1.246 vs. 0.258, p=0.056). Only significantly different average platelet count between "HIT negative" and "HIT positive group" was at day 10 (163 vs. 53.7, p=0.024).This difference remained significant when we excluded patients from "HIT negative" group who were not tested for HIT ELISA, SRA or both (127.9 vs. 53.7, p=0.033). No other platelet parameter, such as average highest platelet count, platelet nadir, days to nadir and percentage platelet drop, differed significantly between the groups. However, plots of average daily platelet counts suggests a potentially revealing pattern in "HIT positive" group, where a gradual drop to platelet nadir (post ECMO day 5-6) is followed by a period of persistently depressed platelet counts (days 6-10). Conclusions: Incidence of both HIT and HITT is relatively high in patients who survive ECMO for longer than 48 hours at our institution. Traditional clinical scores for diagnosis of HIT such as "4 Ts", or presence of "P 1" platelet pattern (typical of HIT in cardiac surgery patients) are not useful in ECMO patients. Pattern of depressed platelet counts after nadir may be suggestive of HIT in these patients.

Correlation Of Heparin Confirmatory Test (HCT) With Optical Density (OD) and Serotonin Release Assay (SRA) In The Diagnosis Of Heparin Induced Thrombocytopenia (HIT)

Introduction Heparin-induced thrombocytopenia (HIT) is a potentially fatal, thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin. Accurate and rapid diagnosis with prompt commencement of therapy are imperative as delays in treatment are associated with an increasing risk of thrombosis, amputation, or death. On the flip side, initiation of therapy with direct thrombin inhibitors without laboratory confirmation carries a significant risk of bleeding. Two types of laboratory tests are available for detection of these antibodies: a widely available immunoassay (ELISA), which is very sensitive to the presence of anti-heparin/PF4 antibodies, but is less specific to the clinical syndrome of HIT because of detection of non-pathological antibodies. The Serotonin Release Assay (SRA) is a functional assay that is now considered the gold standard for confirmatory diagnosis of HIT due to its high specificity. However, the downside of SRA is the cost involved, limited availability and a turnaround time of 5-7 days. As such, a heparin confirmatory test (HCT) with excess heparin has been in use since mid 2011 on positive ELISA samples in our laboratory to improve test specificity. This test is more cost and time efficient, with a turnover time of no more than 48 hours. As noted in prior studies, inhibition of a positive ELISA result by 50% or more in the presence of excess heparin is considered confirmatory of heparin-dependent antibodies. Likewise a negative confirmatory test is defined as a decrease of 50% or less in antibody binding in the presence of heparin. Aim a) Correlation of Heparin Confirmatory test (HCT) with strength of HIT ELISA, vis-à-vis optical density (OD) of 0.4 - 0.99 and OD of &gt;/= 1.0. b) Correlation of HCT results with SRA, to see if the latter can be replaced by the heparin confirmatory test. Patients and Methods A retrospective chart review of adult patients hospitalized at our institution with suspected HIT from July 2011 until January 2013 was done. There were 101 such patients. All patients who had a positive HIT ELISA, then had HCT as per our standard lab practice, with an SRA test done for diagnosis/confirmation of HIT, as per standard clinical practice. Historically, the major strength of SRA assay is its specificity. The optical density on HIT ELISA and SRA results were then compared with the Heparin Confirmatory test to establish clinical significance. Results Of the 101 patients tested for HIT ELISA, 49 were positive. HCT and SRA were performed on all 49 samples, 1 out of which was reported as indeterminate. Hence 48 samples were used for primary analysis, comparing HCT to the OD as well as the SRA results. Out of 48 patients, 6 had positive SRA with Heparin inhibition of &gt;50% (sensitivity 6/6 = 100%). Remaining 42 patients had negative SRA, 7 out of which had Heparin inhibition of &lt;50% (specificity 7/42 = 16.6%). All 7 patients with a negative HCT had a negative SRA, making the negative predictive value of the HCT 100%; however positive predictive value was only 14.6% (6/41). There was no correlation between the OD and Heparin Confirmation test. Conclusions Although there is data suggesting that there might be some value to the Heparin Confirmation test, we were unable to show a significant correlation between HCT and OD or between HCT and SRA. The prospect of having a cost effective and rapid assay for laboratory confirmation of HIT will always be a relevant need. We feel that a larger, prospective study should be conducted to definitively assess the relationship between HCT and SRA. Disclosures: No relevant conflicts of interest to declare.

External Validation Of The HIT Expert Probability (HEP) Score, a Novel Pre-Test Probability Model For Heparin-Induced Thrombocytopenia Based On Broad Expert Opinion

IntroductionThe diagnosis of heparin-induced thrombocytopenia (HIT) is challenging due to the overlap of its clinical features with other entities, variable specificity of ELISA-based tests, poor availability of functional tests such as the serotonin release assay (SRA) and suboptimal positive predictive values of pre-test probability clinical scoring models, such as the 4T and Lillo-Le Louėt models. Improved diagnostic tools are desirable to guide early therapeutic decisions in patients with suspected HIT and to reduce inappropriate use of alternative anticoagulants. The HIT Expert Probability (HEP) Score, a pre-test probability clinical scoring model for HIT based on broad expert opinion, has recently been proposed to improve the diagnosis of HIT compared to the 4T score. We sought to externally and prospectively validate the HEP score. MethodsPre-test probability of HIT was prospectively assessed for 51 consecutive patients referred to the Cleveland Clinic Vascular Medicine Consultative Service for evaluation of possible HIT between August 1, 2012 and February 1, 2013. Electronic medical records were reviewed to obtain clinical, biochemical (including IgG specific HIT ELISA and SRA tests) and imaging data, and two Vascular Medicine fellows independently applied the 4T and HEP scores for each patient. Two independent HIT expert adjudicators rendered a diagnosis of HIT likely or unlikely. Both scorers and adjudicators were blinded to the Results of HIT laboratory testing, consultative service inputs and scores generated by other raters. ResultsThe median 4T and HEP scores were 4.5 [range 3.0, 6.0] and 5 [range 3.0, 8.5], respectively. Interobserver agreement was lower for the HEP Score compared to 4T score, intraclass correlation coefficient: 0.50 (95% CI 0.16–0.85) vs. 0.69 (0.54–0.86). There were no significant differences between area under receiver-operating characteristics curves of 4T and HEP score against HIT ELISA testing (0.62 vs. 0.58, P = 0.62), SRA (0.74 vs. 0.73, P = 0.97), expert panel diagnosis (0.71 vs. 0.79, P = 0.20), concordant HIT ELISA and SRA (0.76 vs. 0.72, P = 0.65), and positive SRA or positive HIT ELSA with HIT likely expert panel diagnosis (0.73 vs. 0.75, P = 0.80). The HEP score model was 100% sensitive and 17% specific for determining the presence of HIT as defined by positive SRA or positive HIT ELISA with HIT likely expert panel diagnosis compared to 4T score (sensitivity, 94%; specificity 38%) (Figure 1, Table 1). [Display omitted] ConclusionThe HEP and 4T scores are excellent screening pre-test probability models for HIT, however, in this prospective validation study, test characteristics for the diagnosis of HIT based on confirmatory laboratory testing and expert opinion are similar. Given the complexity of the HEP scoring model compared to that of the 4T model, further validation of the HEP score is warranted prior to widespread acceptance. Disclosures:Kim:Philips US: Consultancy; GE: Research Funding.

Evaluation of a Pretest Scoring System (4Ts) for the Diagnosis of Heparin-Induced Thrombocytopenia in a University Hospital Setting

The initial diagnosis of heparin–induced thrombocytopenia (HIT) is made on clinical grounds because the assays with the highest sensitivity (eg, heparin–platelet factor 4 antibody enzyme–linked immunosorbent assay [ELISA]) and specificity (eg, serotonin release assay) may not be readily available. The clinical utility of the pretest scoring system, the 4Ts, was developed and validated by Lo et al in the Journal of Thrombosis and Haemostasis in 2006. The pretest scoring system looks at the degree and timing of thrombocytopenia, thrombosis, and the possibility of other etiologies. Based on the 4T score, patients can be categorized as having a high, intermediate, or low probability of having HIT We conducted a retrospective study of 100 consecutive patients who were tested for HIT during their hospitalization at Hahnemann University Hospital (Philadelphia, PA) in 2009. Of the 100 patients analyzed, 72, 23, and 5 patients had 4T pretest probability scores of low, intermediate, and high, respectively. A positive HIT ELISA (optical density > 1.0 unit) was detected in 0 of 72 patients (0%) in the low probability group, in 5 of 23 patients (22%) in the intermediate probability group, and in 2 of 5 patients (40%) in the high probability group. The average turnaround time for the HIT ELISA was 4 to 5 days. Fourteen (19%) of the 72 patients with a low pretest probability of HIT were treated with a direct thrombin inhibitor. Ten (71 %) of the 14 patients in the low probability group treated with a direct thrombin inhibitor had a major complication of bleeding requiring blood transfusion support. In this retrospective study, a low 4T score showed 100% correlation with a negative HIT antibody assay. We recommend incorporating the 4T scoring system into institutional core measures when assessing a patient with suspected HIT, selecting only patients with intermediate to high probability for therapeutic intervention, which may translate into reduced morbidity and lower health care costs.

687 Aortic Surgery Patients With a Positive Hit Elisa Are at Increased Risk of Thrombosis

Heparin induced thrombocytopenia (HIT) and thrombosis (HITT) is an important cause of morbidity/mortality/cost after cardiac surgery (CS). Thrombotic complications include deep venous thrombosis (DVT), pulmonary emboli (PE) and arterial occlusion (AO). The rapidly available ELISA (positive if the optical density is ≥ 0.4) is sensitive but non-specific. The serotonin release assay (SRA) is specific but generally not immediately available. If HITT is clinically suspected (positive ELISA), a non-heparin based anticoagulant should be started which is associated with increased cost and risk of bleeding. If there were known pre- and intraoperative factors associated with thrombosis, this would assist in clinical decision making with regard to starting a non-heparin anticoagulant. We sought to determine pre- and intraoperative factors associated with increased risk of DVT,PE and AO post CS in patients suspected of HIT. The CS patients having a HIT ELISA between 2008 - 2010 were divided into 2 groups: the high risk thrombosis (HRT) group had a positive ELISA with a positive SRA, or positive ELISA with evidence of DVT/PE/AO (but negative SRA); the low risk thrombosis (LRT) group had a negative ELISA, a negative SRA, or a positive ELISA with negative SRA. Regression analysis was performed to determine factors associated with thrombotic risk. Of the 269 patients having ELISA assays, 47 (17.4%) were positive. Of these 47, 11 had a positive SRA, and 6 had negative SRA but evidence of thrombosis - i.e. 17 patients in HRT group. The remaining 252 were the LRT group. (See table for univariate analysis). Multivariate analysis revealed aortic surgery to be associated with HRT (OR 4.9, 95% CI 1.3 - 17.8, p=0.017). ICU LOS was longer in HRT group - 4.8 (3.8 - 8.0) vs. 3.8 (2.2 - 6.8) days in LRT group, p=0.05.Tabled 1 Our data suggest that there appears to be an increased risk of thrombosis in patients having aortic surgery and who have a positive HIT ELISA.

111 Pre-operative, intra-operative and post-operative factors associated with heparin induced thrombocytopenia post cardiac surgery

If heparin induced thrombocytopenia (HIT) is strongly suspected, a non-heparin anticoagulant should be started as risk of thrombosis is high; however, this needs to be balanced by unnecessary and potentially fatal bleeding (eg. cardiac tamponade) if HIT is misdiagnosed. A high (eg. optical density OD ≥1.4) ELISA reading is used at our institution to suggest the presence of “true HIT” with a high concentration of platelet activating antibodies. Many institutions do not have access to rapid ELISA results, thus knowing which pre-, intra-, and post-operative factors are associated with HIT post cardiac surgery may assist in patient management prior to obtaining HIT ELISA results.

The HIT Expert Probability (HEP) Score: a novel pre-test probability model for heparin-induced thrombocytopenia based on broad expert opinion.

The diagnosis of heparin-induced thrombocytopenia (HIT) is challenging. Over-diagnosis and over-treatment are common. To develop a pre-test clinical scoring model for HIT based on broad expert opinion that may be useful in guiding clinical decisions regarding therapy. A pre-test model, the HIT Expert Probability (HEP) Score, was constructed based on the opinions of 26 HIT experts. Fifty patients referred to a reference laboratory for HIT testing comprised the validation cohort. Two hematology trainees scored each patient using the HEP Score and a previously published clinical scoring system (4 T's). A panel of three independent experts adjudicated the 50 patients and rendered a diagnosis of HIT likely or unlikely. All subjects underwent HIT laboratory testing with a polyspecific HIT ELISA and serotonin release assay (SRA). The HEP Score exhibited significantly greater interobserver agreement [intraclass correlation coefficient: 0.88 (95% CI 0.80-0.93) vs. 0.71 (0.54-0.83)], correlation with the results of HIT laboratory testing and concordance with the diagnosis of the expert panel (area under receiver-operating curve: 0.91 vs. 0.74, P = 0.017) than the 4 T's. The model was 100% sensitive and 60% specific for determining the presence of HIT as defined by the expert panel and would have allowed for a 41% reduction in the number of patients receiving a direct thrombin inhibitor (DTI). The HEP Score is the first pre-test clinical scoring model for HIT based on broad expert opinion, exhibited favorable operating characteristics and may permit clinicians to confidently reduce use of alternative anticoagulants. Prospective multicenter validation is warranted.

Evaluation of the Pre-Test Scoring System (4Ts) for the Evaluation and Management of Heparin Induced Thrombocytopenia

AbstractAbstract 2551The initial diagnosis of Heparin Induced Thrombocytopenia (HIT) is made on clinical grounds, because the assays with the highest sensitivity (Heparin-PF4 Ab ELISA) and specificity (Serotonin Release Assay) may not be readily available and may have a slow turn-around time. The clinical utility of the pretest scoring system, the 4Ts, was developed and validated by Lo, GK et al in the Journal of Thrombosis and Haemostasis in 2006. The pretest scoring system looks at the degree of thrombocytopenia, timing of platelet fall, thrombosis or other sequelae, and the possibility of other etiologies for thrombocytopenia. Based on the 4T score, patients can be divided into high, intermediate, and low probability of having HIT. The American Society of Hematology developed a clinical practice guideline in 2009 incorporating the 4T scoring system in the evaluation and management of patients with suspected HIT. It is recommended that patients with intermediate to high probability for HIT start direct thrombin inhibitor (DTI) therapy without delay. We conducted a retrospective study on 100 consecutive patients who were tested for the HIT assay during their hospitalization at Hahnemann University Hospital in 2009. In the 100 patients analyzed, the distribution of the 4T score of low, intermediate, and high pretest probability were seen in 73, 23 and 4 patients, respectively. A positive HIT ELISA (optical density > 1.0) was detected in 0/73 (0%) of the low probability group, 5/23 (22%) of the intermediate probability group and 2/4 (50%) of the high probability group. The average turn-around time for the HIT ELISA was 4 to 5 days. Fourteen (19%) out of the 73 patients with a low pre-test probability for HIT were treated with a direct thrombin inhibitor (DTI). Ten out of the 14 (71%) patients in the low probability group treated with a DTI had a major complication of bleeding requiring blood transfusion support. Overall, twenty five patients received a DTI. Argatroban was the DTI used in 24 of 25 patients. Fourteen patients started on argatroban had a contraindication for the use of lepirudin. In this retrospective study, a low 4T score correlated 100% with a negative HIT antibody assay. The 4T score also predicted the patients likely to test positive for HIT into the intermediate to high probability group. There was significant patient morbidity in the group with low probability when treated with a direct thrombin inhibitor. Based on previous data and this retrospective study, empiric direct thrombin inhibitor therapy should not be initiated in patients with low probability of having HIT. Moreover, many of these patients were started on the more expensive direct thrombin inhibitor, argatroban. For our institution, if no contraindication exists, we recommend lepirudin for the treatment of HIT because it is easier to monitor and less costly. Furthermore, we recommend incorporating the 4T scoring system into institutional core measures when assessing a patient with suspected HIT, selecting only patients with intermediate to high probability for further therapeutic intervention, which may translate into reduced morbidity and lower health care costs. Disclosures:No relevant conflicts of interest to declare.

Pitfalls of Clinical Diagnosis Strategies for Heparin-Induced Thrombocytopenia (HIT) After Cardiac Surgery (CS).

Abstract 3516Poster Board III-453 BackgroundHIT is an infrequent but potentially serious complication of heparin therapy. Its diagnosis is complex and depends on a combination of clinical suspicion and laboratory confirmation through ELISA and functional tests such as the serotonin release assay (SRA). The 4Ts score comprises 4 clinical parameters (severity and timing of onset of thrombocytopenia, development of thrombosis, and clinician's appraisal of the likelihood of alternate causes for thrombocytopenia) and has been proposed to predict the probability of HIT in patients deemed to be at risk. However, the validity of the 4Ts score in patients undergoing cardiac surgery (CS) is questionable considering the numerous other factors that predispose such patients to thrombocytopenia and thrombosis. In addition, in CS patients the HIT ELISA assay has been reported to have 25 - 50% false positive results making it less useful. ObjectivesTo determine the usefulness of the 4T score in the post cardiac surgical population and the value of the HIT ELISA optical density for predicting HIT. MethodsRetrospective case-control study of patients admitted for cardiac surgery to the London Health Sciences Centre between January 2006 and December 2008 and for whom a HIT ELISA assay was requested. Patients with an equivocal or positive ELISA test were tested by SRA which considered the gold standard. Information collected included clinical variables related to the surgery and post-operative period, calculated 4T scores, ELISA optical density (OD) and SRA results. Categorical variables were compared using chi2 or Fisher's exact tests as appropriate. Continuous variables were compared using a Mann-Whitney U test. Covariates achieving a p value ≤0.1 in univariate analysis and the components of the 4Ts score were incorporated in logistic regression models using stepwise forward selection. Finally, we constructed a Receiver Operating Characteristic (ROC) curve for the ELISA OD. Results73 patients were included in the analysis. Results of the univariate analysis are shown in the table. On regression analysis only the ELISA optical density (per each OD arbitrary unit increase) was correlated with a diagnosis of HIT (OR 37.266; 95% CI 2.342-593.013; p=0.010). For the ELISA OD the area under the ROC curve was 0.990 (SE 0.013) (Figure). A cutoff value for the OD of 0.475 had a Sensitivity of 1, a specificity of 0.9, a positive likelihood ratio (LR) of 10 and a negative LR of 0.00. Assuming a prevalence proportion of 0.082 the posterior probability of HIT if the ELISA has an OD <0.475 is 0 (95% CI 0 – 9). On the other hand, an OD >0.92 resulted in a LR+ of 20 with a posterior probability of 64% (95% CI 35 – 80). ConclusionsIn this study, we found that the 4T score does not accurately predict HIT in post CS patients. Limitations of this study include a reduced sample size and its retrospective nature. Our findings suggest that in post CS patients developing thrombocytopenia between 10 and 100 × 109 or a platelet drop of 50% or more (100% of our population) a HIT ELISA with an OD < 0.475 could be used to rule out HIT. Our findings need to be confirmed in prospective studies.ControlsCasesPAge (years) [Median]74.00066.0000.175Gender1.000Male [%]58.20066.700Cardiac surgery type [%]0.556CABG22.433.3Valve replacement28.40CABG + Valve replacement43.366.7Other30Transplant30Use of intra-aortic balloon pump [%]26.933.30.663Clamp time (minutes) [Median]121.000112.0000.651Pump time (minutes) [Median]174.000167.0000.507Heparin used in surgery (units) [Median]55000.00085500.0000.007Baseline platelet count (x 10∧9) [Median]211.000250.0000.315Nadir platelet count [Median]39.00038.5000.912Percent platelet fall [Median]81.41080.7970.659Time from cardiac Sx to HIT ELISA request OR Plt Nadir (days) [Median]4.0007.0000.032Time to HIT ELISA /Plt nadir >=5 days [%]29.983.30.016Heparin exposure within 100 days [%]49.333.30.676Confirmed new thrombosis [%]033.30.006Suspected new thrombosis [%]1.501Skin necrosis [%]016.70.082Alternate cause for thrombocytopenia[%]0.221No34.30Possible20.933.3Definite44.866.74T Score Calculated [Median]4.0005.0000.0594Ts Score pretest probability category [%]0.125Low37.30Intermediate43.383.3High19.416.7HIT ELISA Assay<0.001Negative80.60Equivocal11.90Positive7.5100ELISA optical density (arbitrary units) [Median]0.1702.306<0.001 Disclosures:No relevant conflicts of interest to declare.

Heparin Induced Thrombocytopenia; A Diagnostic Dilemma.

Abstract 4468 IntroductionThe diagnosis of HIT can be challenging. Short of the serotonin release assay, which can be difficult to get in a timely manner, the 4T method developed by Lo et al., in conjunction with the HIT elisa assay (EIA), is the best established criteria for the rapid diagnosis of Heparin Induced Thrombocytopenia (HIT). We sought to determine how often the 4Ts were being applied at a large academic institution. In addition, we sought to understand the outcome of patients started on direct thrombin inhibitors (DTIs) who did not meet the 4T/EIA criteria for HIT. MethodsA retrospective case series identified patients placed on DTI for HIT treatment from 10/2006 to 5/2008. The 4T pretest probability was then applied retrospectively. HIT EIA Ab testing, optical densities, duration of DTI treatment, and bleeding incidence were recorded. Liberal and modified conservative criteria from Lo et al.'s study were used to define patients with HIT. Overdiagnosis of HIT was then recorded based on the above criteria. Results76 patients were included for analysis. The average age was 61.4 years. Based on the 4T pretest probability, 30 patients were classified as low risk, 37 intermediate risk, and 9 high risk for HIT. The average HIT OD for low, intermediate, and high pretest probability was 0.49, 1.17, and 2.05.76% and 36% of patients treated with DTIs for HIT met the liberal and modified conservative HIT definitions, respectively. 18 patients (24%) did not meet liberal HIT criteria but remained on DTI treatment for 1 to 60 days. Cost of DTI overuse in this subset was $84,960. Cost of DTI use in patients who did not meet modified conservative criteria was $462,246. All 16 patients who developed bleeding complications fulfilled a liberal diagnosis of HIT while only 6 of these patients fulfilled the modified conservative definition of HIT. Conclusions1) The 4T method for diagnosing HIT is not being applied to a large percentage of patients in a tertiary academic center. 2) There is an overdiagnosis of HIT regardless of definition criteria with 24% of patients treated for HIT not meeting the liberal definition while 64% of patients did not meet the modified conservative definition. This overdiagnosis has both a significant monetary and morbidity cost. Disclosures:No relevant conflicts of interest to declare.

Cross Reactivity of Heparin-Induced PF4 Antibodies with Heparin- Bound IL-8.

Background: Heparin-Induced Thrombocytopenia (HIT) is caused by polyclonal antibodies, formed to conformational neoepitopes exposed in platelet factor 4 (PF4) bound to heparin (H). HIT antibodies are commonly measured by one of several commercially available ELISAs. The literature reports cases of clinical HIT and/or positive HIT antibody-mediated platelet activation tests in patients without antibodies detectable in PF4 based ELISAs, and suggests that antibodies to other chemokines, such as interleukin-8 (IL-8), may be involved. The current study was undertaken to characterize the frequency of such antibodies in a population of consecutive serum specimens submitted for HIT diagnosis by 14C-serotonin release assay (SRA), and also in a group of retrospectively selected SRA-positive specimens.Methods: Specimens (n=90) analyzed by SRA between March and July 2008 were subsequently tested by PF4 Enhanced ELISA (GTI, Waukesha, WI), and the Zymutest HIA ELISA (Hyphen BioMed, Neuville sur Oise, France). An additional ELISA was carried out on the specimens using the Zymutest heparin-bonded plate coated with purified, recombinant human interleukin-8 (IL-8) (72 aa form, Prospec, Rehovot, Israel). The second group consisted of archived specimens (n=32) that tested positive by SRA but negative by GTI HAT ELISA; these specimens were subsequently tested by the Zymutest HIA and the H:IL-8 ELISA.Results: Recombinant IL-8 (1μg/well) added to heparin-bound ELISA plates was recognized by anti-IL-8 (BD Biosciences), but not anti-PF4 (Leinco Technologies, Inc.) antibodies and was not displaced from the plate by addition of PF4 from normal serum at the dilution (1:100) used in the ELISA. Of the 90 consecutive specimens, 21 (23.3%) tested positive in both the GTI and the Zymutest HIA tests; 15 of the 90 (16.6%) tested positive by one but not both of the PF4-based ELISAs (Table 1). In this group of 90 specimens, 17 (18.9%) had OD > 0.500 on the heparin-bound IL-8 plate. Only 3 of the specimens bound to the H:IL-8 exclusively, while 14 were also positive in the GTI and/or Zymutest HIA ELISA. Thus, of the 36 specimens that tested positive in one or both of the PF-4-based ELISAs, 14 (38.9%) had antibodies that also targeted heparin-bound IL-8. Ten of the 90 specimens tested positive in the SRA. Six of SRA-positives had PF4-specific antibodies that did not cross react with H:IL-8, 3 had H:IL-8 reactive antibodies in addition to PF4 reactive antibodies, and 1 SRA positive specimen tested negative in all ELISAs.Table 1. Comparison of GTI HAT vs Zymutest HIA results: n = 90 consecutive patientsNumbers in parentheses indicate how many specimens in each subgroup contained antibodies that bound to H:IL-8.Zymutest HIA+−Total+21 (11)9 (1)30GTI HAT−6 (2)54 (3)60Total276390Among the set of 32 specimens that tested positive by SRA and negative by GTI ELISA, 8 tested positive in the Zymutest ELISA, including 4 that had OD > 0.500 on the H:IL-8 plate. The 24 specimens that tested negative by both HIT ELISAs ranged in percent serotonin release (tested with 0.1 U/ml H) between 21–84% with a median of 45.6%, and did not contain antibodies that bound the H:IL-8 plate.Conclusions: The data agree with other studies that show differences between results of commercially available HIT ELISA kits. The study also shows that over one third of specimens that test positive in the HIT ELISAs contain antibodies that are not specific for PF4, but also recognize heparin-bound IL-8. These results do not prove that the antibodies that bind PF4 targets are the same ones that bind IL-8. However, the significant degree of homology (40%) between the peptides suggests that the same antibodies may cross react with either target. The relevance of these antibodies is as yet unclear. Only 3% of specimens bound H:IL-8 plates but not PF4 plates, and these were not associated with SRA activity. Also HIT ELISA negative specimens with SRA activity, which showed only moderate percent serotonin release, did not contain H:IL-8-binding antibodies. The study did not find evidence of a role for H:IL-8 antibodies in HIT-related platelet activation tests.