Low thrombin generation in postmenopausal women using estetrol.

OBJECTIVE: Estetrol (E4) represents a novel estrogen of interest to relieve vasomotor symptoms. E4 activates the nuclear estrogen receptor α (ERα) but antagonizes the estradiol ERα-dependent membrane-initiated steroid signaling pathway. The distinct pharmacological properties of E4 could explain its low impact on hemostasis. This study aimed to assess the effect of E4 on coagulation in postmenopausal women, using the thrombin generation assay (TGA). METHODS: Data were collected from a multicenter, randomized, placebo-controlled, dose-finding study in postmenopausal women (NCT02834312). Oral E4 (2.5 mg, n = 42; 5 mg, n = 29; 10 mg, n = 34; or 15 mg, n = 32) or placebo (n = 31) was administered daily for 12 weeks. Thrombograms and TGA parameters were extracted for each subject at baseline and after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, all treatment groups showed a mean thrombogram (±95% confidence interval [CI] of the mean) within the reference ranges, that is, the 2.5th-97.5th percentile of all baseline thrombograms (n = 168), as well as for TGA parameters. CONCLUSIONS: The intake of E4 15 mg for 12 weeks led to significant but not clinically relevant changes compared to baseline as the mean values (±95% CI of the mean) remained within reference ranges, demonstrating a neutral profile of this estrogen on hemostasis.

D-dimer testing: A narrative review.

D-dimer containing species are soluble fibrin degradation products derived from plasmin-mediated degradation of cross-linked fibrin, i.e., 'D-dimer'. D-dimer can hence be considered a biomarker of in vivo activation of both coagulation and fibrinolysis, the leading clinical application in daily practice of which is ruling out venous thromboembolism (VTE). D-dimer has been further evaluated for assessing the risk of VTE recurrence and helping define optimal duration of anticoagulation treatment in VTE, for diagnosing disseminated intravascular coagulation (DIC), and for screening those at enhanced risk of VTE. D-dimer assays should however be performed as intended by regulatory agencies, as their use outside these indications might make them a laboratory-developed test (LDT). This narrative review is aimed at: (1) reviewing the definition of D-dimer, (2) discussing preanalytical variables affecting D-dimer measurement, (3) reviewing and comparing the assays performance and some postanalytical variables (e.g., different units and age-adjusted cutoffs), and (4) discussing the interest of D-dimer measurement across different clinical settings, including pregnancy, cancer, and coronavirus disease 2019 (COVID-19).

Impact of estetrol (E4) on hemostasis, metabolism and bone turnover in postmenopausal women.

OBJECTIVE: This study aimed to determine the effects of estetrol (E4) on hemostasis, lipids, carbohydrate metabolism and bone turnover in postmenopausal women. METHODS: This study was a multicenter, randomized, double-blind placebo-controlled phase 2 trial. Participants (n = 180, age 43-64 years) received E4 2.5 mg, 5 mg, 10 mg and 15 mg or placebo once daily for 12 weeks. Changes from baseline at week 12 were evaluated versus placebo for hemostasis parameters, sex hormone binding globulin (SHBG), lipids, carbohydrate metabolism and bone markers. RESULTS: Changes for hemostasis parameters were minimal with a small increase only in the normalized activated protein C sensitivity ratio in the E4 15 mg group versus placebo. SHBG increased in the E4 5 mg, 10 mg and 15 mg groups versus placebo. High-density lipoprotein cholesterol increased in all E4 groups; changes were not consistent for other lipids. Significant decreases versus placebo were seen for insulin resistance (E4 10 mg group), hemoglobin A1c (E4 15 mg group) and type 1 collagen C-terminal telopeptide (E4 10 mg and 15 mg groups). Small decreases in osteocalcin in the E4 5 mg, 10 mg and 15 mg groups were significant versus the increase observed in placebo. CONCLUSION: E4 had limited impact on hemostasis and potentially beneficial effects on lipids, carbohydrate metabolism and bone turnover.

Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory.

Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.

Clotting test results correlate better with DOAC concentrations when expressed as a "Correction Ratio"; results before/after extraction with the DOAC Stop reagent.

INTRODUCTION: Clotting test results are currently not useful for estimating direct oral anti-coagulant (DOAC) concentrations because baseline results vary. DOAC Stop is a DOAC extracting agent with no effect on clotting factors. We investigated if aPTT (activated partial thromboplastin time) and dRVVT (dilute Russells viper venom time) results might correlate better with DOAC concentrations if results after DOAC extraction were used to estimate a "before/after" value (Correction Ratio). MATERIALS AND METHODS: We used activated partial thromboplastin time (aPTT, PTT-LA) and dilute Russells viper venom time clotting test (dRVVT) results previously recorded on DOAC patient plasmas (25 dabigatran, 15 apixaban, 19 rivaroxaban) without known thrombotic risk factors before and after DOAC extraction. DOAC concentrations had been determined by standard chromogenic assays. RESULTS: Correlations between aPTT and dabigatran, apixaban, and rivaroxaban concentrations were initially poor (0.64, 0.15 and 0.39 respectively). However, they improved significantly to 0.94, 0.89 and 0.80 when the ratios of initial aPTT to the aPTT obtained after DOAC extraction were plotted against DOAC concentration. Still better correlations (0.99, 0.97, 0.95) and much higher sensitivities to the DOACs were obtained when dRVVT (LA Confirm) tests were used following this procedure on the same samples. CONCLUSIONS: The correlations of aPTT and dRVVT tests with DOAC concentrations were significantly improved by using the ratio of result "before" to those "after" DOAC extraction. The results indicate that dRVVT (especially LA Confirm) and similar tests might be useful for determining DOAC concentrations more reliably and with better sensitivity than currently possible with clotting tests.

Andexanet alfa for the reversal of factor Xa inhibitors.

INTRODUCTION: Andexanet alfa is a recombinant modified factor Xa protein that has been developed to reverse factor Xa inhibitors. Since May 2018, the FDA has approved its utilization in patients treated with apixaban and rivaroxaban in case of life-threatening or uncontrolled bleeding. On 28 of February 2019, the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a conditional marketing authorization for andexanet alfa in Europe. Area covered: The authors provide an overview of andexanet alfa development and its pharmacokinetic and pharmacodynamic properties. The results of the clinical phase III trial ANNEXA as well as current limitations related to andexanet alfa are also discussed. Expert opinion: Although phase I and II studies have proven that andexanet alfa can be effective in reversing the effect of factor Xa inhibitors, its efficacy in major bleeding patients has only been shown for apixaban and rivaroxaban, without any comparator group. Well-designed studies comparing the efficacy and safety of andexanet alfa to other reversal strategies are required to confirm preliminary data. The benefit of andexanet alfa in specific settings needs to be investigated and its use in clinical practice needs to be facilitated by the implementation of international guidelines.

Atherothrombosis and Thromboembolism: Position Paper from the Second Maastricht Consensus Conference on Thrombosis.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians.

Click to hear Dr Baglin's perspective on the role of the laboratory in treatment with new oral anticoagulants SUMMARY: One of the key benefits of the direct oral anticoagulants (DOACs) is that they do not require routine laboratory monitoring. Nevertheless, assessment of DOAC exposure and anticoagulant effects may become useful in various clinical scenarios. The five approved DOACs (apixaban, betrixaban, dabigatran etexilate, edoxaban and rivaroxaban) have different characteristics impacting assay selection and the interpretation of results. This article provides an updated overview on (i) which test to use (and their advantages and limitations), (ii) when to assay DOAC levels, (iii) how to interpret the results relating to bleeding risk, emergency situations and perioperative management, and (iv) what is the impact of DOACs on routine and specialized coagulation assays. Assays for anti-Xa or anti-IIa activity are the preferred methods when quantitative information is useful, although the situations in which to test for DOAC levels are still debated. Different reagent sensitivities and variabilities in laboratory calibrations impact assay results. International calibration standards for all specific tests for each DOAC are needed to reduce the inter-laboratory variability and allow inter-study comparisons. The impact of the DOACs on hemostasis testing may cause false-positive or false-negative results; however, these can be minimized by using specific assays and collecting blood samples at trough concentrations. Finally, prospective clinical trials are needed to validate the safety and efficacy of proposed laboratory thresholds in relation to clinical decisions. We offer recommendations on the tests to use for measuring DOACs and practical guidance on laboratory testing to help patient management and avoid diagnostic errors.

Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

Inactivation of human coagulation factor X by a protease of the pathogen Capnocytophaga canimorsus.

Essentials Capnocytophaga canimorsus causes severe dog bite related blood stream infections. We investigated if C. canimorsus contributes to bleeding abnormalities during infection. The C. canimorsus protease CcDPP7 causes factor X dysfunction by N-terminal cleavage. CcDPP7 inhibits coagulation in vivo, which could promote immune evasion and trigger hemorrhage. SUMMARY: Background Capnocytophaga canimorsus is a Gram-negative bacterium that is present in the oral flora of dogs and causes fulminant sepsis in humans who have been bitten, licked, or scratched. In patients, bleeding abnormalities, such as petechiae, purpura fulminans, or disseminated intravascular coagulation (DIC), occur frequently. Objective To investigate whether C. canimorsus could actively contribute to these bleeding abnormalities. Methods Calibrated automated thrombogram and clotting time assays were performed to assess the anticoagulant activity of C. canimorsus 5 (Cc5), a strain isolated from a fatal human infection. Clotting factor activities were measured with factor-deficient plasma. Factor X cleavage was monitored with the radiolabeled zymogen and western blotting. Mutagenesis of Cc5 genes encoding putative serine proteases was performed to identify the protease that cleaves FX. Protein purification was performed with affinity chromatography. Edman degradation allowed the detection of N-terminal cleavage of FX. Tail bleeding times were measured in mice. Results We found that Cc5 inhibited thrombin generation and increased the prothrombin time and the activated partial thromboplastin time of human plasma via FX cleavage. A mutant that was unable to synthesize a type 7 dipeptidyl peptidase (DPP7) of the S46 serine protease family failed to proteolyse FX. The purified protease (CcDPP7) cleaved FX heavy and light chains from the N-terminus, and was active in vivo after intravenous injection. Conclusions This is, to our knowledge, the first study demonstrating a detailed mechanism for FX inactivation by a bacterial protease, and it is the first functional study associating DPP7 proteases with a potentially pathogenic outcome.

Effects of dietary linseed oil on innate immune system of Eurasian perch and disease resistance after exposure to Aeromonas salmonicida achromogen.

This study was designated to investigate the effects of dietary fish oil (FO diet) replacement by linseed oil (LO diet) on regulation of immune response and disease resistance in Eurasian perch (Perca fluviatilis). A control diet containing fish oil (FO = cod liver oil) and characterized by high levels of n-3 high LC-PUFA (6% EPA, 7.5% of total fatty acids (FAs)) was compared to linseed oil diet (LO diet) composed of low LC-PUFA contents (1% EPA, 2.3% DHA of total FAs) but high C18 fatty acids levels. The experiment was conducted in quadruplicate groups of 80 fish each. After 10 weeks of feeding, the innate immune status was evaluated in various organs (liver, spleen, and head-kidney) (feeding condition). Two days later, a bacterial challenge was performed on fish from 2 rearing conditions: fish infected with Aeromonas salmonicida (bacteria condition) and fish injected with sterile medium but maintained in the same flow system that fish challenged with bacteria (sentinel condition). Three days after injection of bacteria, a significant decrease of lymphocyte, thrombocyte and basophil populations was observed while neutrophils were not affected. In addition, plasma lysozyme activity and reactive oxygen species production in kidney significantly increased in fish challenged with A. salmonicida while the plasma alternative complement pathway activity was not affected. Increase of plasma lysozyme activity as well as reactive oxygen species production in spleen and kidney of sentinel fish suggest that these immune defenses can also be activated, but at lower bacteria concentration than infected fish. No differences in leucocyte populations, plasma lysozyme and alternative complement pathway activities were observed between dietary treatments. Similarly, expression of genes related to eicosanoid synthesis in liver were not affected by the dietary oil source but were strongly stimulated in fish challenged with A. salmonicida. These findings demonstrated that the use of linseed oil does not deplete the innate immune system of Eurasian perch juveniles.

Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis, L.).

Domestication might be a possible way to reduce the physiological response to long-term stressors and deleterious effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeated emersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) and long (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiological stress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cortisol and glucose elevated within 6h post-stress and splenosomatic index (SSI) decreased within 48h, indicating that the species was affected by emersion stressor. In the second experiment, F1 and F4 generations were submitted to repeated water emersions (3 times/week during 44days). On day 9, 18 and 44, samplings were performed 48h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lysozyme activity were evaluated and serum proteome was analyzed using 2D-DIGE. Any of the tested variables were affected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) were up-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2M isoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor exposure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch, probably explaining why domestication had no influence on fish responses. Changes associated with domestication are highly complex and certainly need further investigations.

Rapid exclusion of the diagnosis of immune HIT by AcuStar HIT and heparin-induced multiple electrode aggregometry.

BACKGROUND: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential but remains challenging. We have previously demonstrated, in a retrospective study, the usefulness of the combination of the 4Ts score, AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) with optimized thresholds. OBJECTIVES: We aimed at exploring prospectively the performances of our optimized diagnostic algorithm on suspected HIT patients. The secondary objective is to evaluate performances of AcuStar HIT-Ab (PF4-H) in comparison with the clinical outcome. METHODS: 116 inpatients with clinically suspected immune HIT were included. Our optimized diagnostic algorithm was applied to each patient. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) of the overall diagnostic strategy as well as AcuStar HIT-Ab (at manufacturer's thresholds and at our thresholds) were calculated using clinical diagnosis as the reference. RESULTS: Among 116 patients, 2 patients had clinically-diagnosed HIT. These 2 patients were positive on AcuStar HIT-Ab, AcuStar HIT-IgG and HIMEA. Using our optimized algorithm, all patients were correctly diagnosed. AcuStar HIT-Ab at our cut-off (>9.41 U/mL) and at manufacturer's cut-off (>1.00 U/mL) showed both a sensitivity of 100.0% and a specificity of 99.1% and 90.4%, respectively. CONCLUSION: The combination of the 4Ts score, the HemosIL® AcuStar HIT and HIMEA with optimized thresholds may be useful for the rapid and accurate exclusion of the diagnosis of immune HIT.

Platelet microparticle generation assay: a valuable test for immune heparin-induced thrombocytopenia diagnosis.

BACKGROUND: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance. OBJECTIVES: The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of (14)C-serotonin release assay (SRA) on the same series of patients. METHODS: Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1IU and 500IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient's medical records. Performances of PMPGA and SRA (n=53) were evaluated using ROC curve analysis with clinical outcome as reference. RESULTS: In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of (14)C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2). CONCLUSIONS: PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with (14)C-SRA performances and predominately with clinical outcome.

Comparison of calibrated dilute thrombin time and aPTT tests with LC-MS/MS for the therapeutic monitoring of patients treated with dabigatran etexilate.

Ways to monitor dabigatran etexilate (DE) therapy would be useful in certain situations. Functional assays such as aPTT or Hemoclot® Thrombin Inhibitor (HTI) have been proposed to evaluate dabigatran concentrations, but previous findings are based on in vitro studies and results must be confirmed in clinical samples. The aim of this study was to compare aPTT and HTI measurements with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of dabigatran in plasma samples from DE treated patients. Seventy-one plasma samples were included. aPTT was performed using STA-CKPrest® and SynthASil®. HTI was performed according to instructions from the manufacturer. The LC-MS/MS method utilised dabigatran-d3 as internal standard. The plasma concentration range was 0 to 645 ng/ml as measured by LC-MS/MS. Overall, the HTI and LC-MS/MS analyses correlated well (r²=0.97). The Bland-Altman analysis showed a mean difference of 9 ng/ml (SD: 20 ng/ml). However, the HTI performed poorly at concentrations <50 ng/ml. LC-MS/MS was sensitive (limit of quantification 1.1 ng/ml) and specific for dabigatran. The aPTT methods did not correlate well with plasma concentrations measured by LC-MS/MS (r² = 0.59 with SynthASil® and 0.50 with STA-CKPrest®). In conclusion, the poor sensitivity, the important inter-individual variability, and the poor correlation with LC-MS/MS preclude the use of aPTT to estimate dabigatran concentrations. Due to its small inter-individual variability and good agreement with LC-MS/MS measurements, we recommend the use of HTI assays to rather accurately estimate concentrations of dabigatran >50 ng/ml. Quantification of lower dabigatran levels in DE-treated patients requires the "reference" LC-MS/MS method.

Assessment of the performances of AcuStar HIT and the combination with heparin-induced multiple electrode aggregometry: a retrospective study.

BACKGROUND: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is challenging. HemosIL® AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) were recently proposed as rapid diagnostic methods. OBJECTIVES: We conducted a study to assess performances of AcuStar HIT-IgG (PF4-H) and AcuStar HIT-Ab (PF4-H). The secondary objective was to compare the performances of the combination of Acustar HIT and HIMEA with standardised clinical diagnosis. METHODS: Sera of 104 suspected HIT patients were retrospectively tested with AcuStar HIT. HIMEA was performed on available sera (n=81). The clinical diagnosis was established by analysing in a standardized manner the patient's medical records. These tests were also compared with PF4-Enhanced®, LTA, and SRA in subsets of patients. Thresholds were determined using ROC curve analysis with clinical outcome as reference. RESULTS: Using the recommended thresholds (1.00AU), the negative predictive value (NPV) of HIT-IgG and HIT-Ab were 100.0% (95% CI: 95.9%-100.0% and 95.7%-100.0%). The positive predictive value (PPV) were 64.3% (95% CI: 35.1%-87.2.2%) and 45.0% (95% CI: 23.2%-68.6%), respectively. Using our thresholds (HIT-IgG: 2.89AU, HIT-Ab: 9.41AU), NPV of HIT-IgG and HIT-Ab were 100.0% (95% CI: 96.0%-100.0% and 96.1%-100.0%). PPV were 75.0% (95% CI: 42.7%-94.5%) and 81.8% (95% CI: 48.3%-97.7%), respectively. Of the 79 patients with a medium-high pretest probability score, 67 were negative using HIT-IgG (PF4-H) test at our thresholds. HIMEA was performed on HIT-IgG positive patients. Using this combination, only one patient on 79 was incorrectly diagnosed. CONCLUSION: Acustar HIT showed good performances to exclude the diagnosis of HIT. Combination with HIMEA improves PPV.