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Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by the susceptibility of blood cells to attack by the complement system, inducing extracellular vesicle (EV) production. Thromboembolism is the leading cause of death in this condition. Eculizumab, a humanized monoclonal antibody which inhibits the C5 protein of the complement, reduces the thrombotic risk in PNH., Materials and Method: We conducted a pilot, prospective, open-label, longitudinal clinical study with six PNH patients treated with eculizumab. The aim was to measure, by flow cytometry, the EVs' production in the patients' platelet-free plasma (PFP) before and during the treatment. We also assessed the procoagulant activity in PFP using STA®-Procoag-PPL and thrombin generation assays (TGA). A high-sensitive version of TGA was also used to study the procoagulant profile induced by the EVs using EVs pelleted from PFP., Results: We observed a decrease in platelet EV count with eculizumab treatment (p<0.05). STA®-Procoag-PPL assay showed a decrease of the procoagulant profile induced by procoagulant phospholipids (PL) during treatment. These results were not confirmed by TGA on PFP, due to a lack of sensitivity. Thus, we used a high-sensitive version of TGA that enabled us to observe variation in the procoagulant profile induced by the EVs with eculizumab (p<0.05)., Conclusions: Eculizumab has an impact on the extent of EV production and on the procoagulant profile induced by the procoagulant PL and the EVs. One factor in the antithrombotic action of eculizumab is its ability to decrease EV production and the procoagulant profile induced by PL and EVs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Dilute Russell Viper Venom Time (DRVV-T) might be useful in urgent settings for screening patients on Non-VKA Oral Anticoagulants (NOACs)., Aim: To compare the accuracy of DRVV-T with gold standard assays for the assessment of pharmacodynamics of dabigatran, rivaroxaban and vitamin K antagonist (VKA) in plasma samples from patients., Methods: Sixty rivaroxaban, 48 dabigatran and 50 VKA samples from patients were included. DRVV-T was performed in all groups using STA®-Staclot®DRVV-Screen and -Confirm. For NOACs, PT and aPTT were performed using different reagents while plasma drug concentrations were measured by liquid mass-spectrometry (LC-MS/MS). For VKA, INR was performed using RecombiPlasTin 2G®., Results: For NOACs, correlations between calibrated STA®-Staclot®DRVV-Confirm and LC-MS/MS (rs=0.88 and 0.97 for rivaroxaban and dabigatran, respectively) were higher than the ones obtained with STA®-Staclot®DRVV-Screen (rs=0.87 and 0.91), PT (rs=0.83 to 0.86) or aPTT (rs=0.84 to 0.89). Bland Altman analyses showed that calibrated DRVV-T methods tend to overestimate plasma concentrations of NOACs. ROC curves revealed that cut-off to exclude supra-therapeutic levels at Ctrough (i.e. 200ng/mL) are different for dabigatran and rivaroxaban. Neither STA®-Staclot®DRVV-Screen nor -Confirm correlated sufficiently with the intensity of VKA therapy (rs=0.35 and 0.52)., Conclusions: STA®-Staclot®DRVV-Confirm provides a rapid estimation of the intensity of anticoagulation with rivaroxaban or dabigatran without specific calibrators. At Ctrough, thresholds for rivaroxaban and dabigatran can be used to identify supra-therapeutic plasma level. However, this test cannot differentiate the nature of the NOACs. The development of a point-of-care device optimising this method would be of particular interest in emergency situations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL). The aim of this study was to assess the capacity of untreated (APL) cells to shed procoagulant MVs. APL cells (NB4 and HL-60 cell lines) and MVs were separated by filtration (0.1-0.22-0.45-0.65 μm). The procoagulant activity (PCA) was assessed by thrombin generation assay (TGA). Alternatively, MVs were incubated with anti-Tissue Factor (TF) antibodies, with annexin V to assess the contribution of TF and phospholipids (PL) to the PCA, respectively. NB4 cells had a high PCA mainly triggered by MVs of size under 0.45 μm. The PCA of MVs was related to the expression of active TF and PL. HL-60 cells had a weaker PCA since TF is mostly present in its inactive form. Moreover, HL-60 do not produce MVs<0.65 μm associated with PCA. MVs could have a predicting value for VTE and DIC in patients with acute promyelocytic leukemia and could inform physicians about the optimal use of a thromboprophylaxis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)