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Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia.

BACKGROUND The evaluation of the activated protein C resistance (APCr) based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives. Results are usually expressed as "normalized APC sensitivity ratio" (nAPCsr) using a reference plasma that should achieve an ETP ratio of 0.1 in presence of exogenous APC. Because of the interassay variability, achieving exactly an ETP ratio of 0.1 in each run is almost impossible, which significantly affects the theoretical 0-10 scale of nAPCsr. OBJECTIVES To compare the nAPCsr to the nAPCsr10 , a newly proposed method to express the degree of APC resistance. METHODS Individual plasma samples (n = 854) were analyzed to compare nAPCsr and nAPCsr10 . These values were obtained using the validated ETP-based APCr assay. RESULTS The Spearman correlation between nAPCsr and nAPCsr10 had a coefficient of 0.99. Linear regression showed the following equation y = 0.9315*x + 0.03942 (r2 = .97). When differences (nAPCsr10 - nAPCsr) were plotted against nAPCsr10 , the mean difference equaled 0.16% or 4.95%. The correction obtained with the use of the nAPCsr10 showed that the results of the nAPCsr were statistically different (P < .0001). CONCLUSIONS This new scale provides a harmonization and normalization of the nAPCsr. Results show a better reproducibility with the nAPCsr10 . It avoids the additional variability and the unharmonized scale introduced by the use of a reference plasma. This adapted method for the calculation of the APC resistance could provide the regulatory and scientific bodies with more reproducible and harmonized evaluations.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Edoxaban is the only anti-Xa inhibitor metabolized in pharmacologically active moiety that could interfere with chromogenic anti-Xa assays, especially in case of drug-drug interactions or physiological disorders. We evaluated the contribution of the main metabolite of edoxaban, edoxaban-M4 (M4), in 79 plasma samples from patients taking edoxaban. The total anti-Xa activity was evaluated on three different chromogenic factor Xa-based assays. Results were compared with a validated ultra-high-performance liquid chromatography coupled with a tandem mass spectrometry measurement. Edoxaban and its active M4 metabolite have also been spiked separately in normal pooled plasma to assess the sensitivity of chromogenic anti-Xa assays to both molecules individually. Spiked edoxaban or M4 provided different slopes of linear regression models between chromogenic and chromatographic measurement (from 0.97 for STA Liquid Anti-Xa to 1.10 for Biophen Heparin LRT Low with edoxaban and from 0.70 for Biophen DiXaI High to 0.83 for Biophen Heparin LRT High, respectively). A positive correlation is observed between the increase of the ratio M4/edoxaban with the difference between chromogenic and chromatographic measurements. Edoxaban and M4 do not similarly impact chromogenic assays, leading to biased chromogenic estimations of ponderal concentrations. In patient samples, this impact is even more important at low concentrations or in the case of an increase in the M4/edoxaban ratio because of hepatic or renal impairments or in case of drug interactions. This study highlights the limitations and risks of error of expressing results in ponderal concentrations instead of global activity anti-Xa.

Background Regulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay. Methods The validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool. Results Repeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R2 > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Background: Thrombin generation testing has been used to provide information on the coagulation phenotype of patients. The most used technique is the calibrated automated thrombogram (CAT) but it suffers from a lack of standardization, preventing its implementation in routine. The ST Genesia is a new analyzer designed to assess thrombin generation based on the same principle as the CAT. Unlike the CAT system, the ST Genesia is a benchtop, fully automated analyzer, able to perform the analyses individually and not by batch, with strict control of variables such as temperature and volumes, ensuring, theoretically, maximal reproducibility. Objectives: This study aimed at assessing the performance of the STG-DrugScreen application on the ST Genesia analyzer. We also aimed at exploring stability of plasma samples after freezing and defining a reference normal range. Results: Results demonstrated the excellent interexperiment precision of the ST Genesia and confirmed that the use of a reference plasma helps reducing the inter-experiments variability. Stability revealed that plasma samples are stable for at least 11 months at −70°C or lower, except for those containing low molecular weight heparins which have to be tested within 6 months. Freezing had no effect on the majority of thrombin generation parameters except on time to peak. Conclusions: Our results suggest an easy implementation of thrombin generation with the use of ST Genesia in the routine laboratory. This will facilitate the design of multicentric studies and enable the establishment of reliable and evidence-based thresholds, which may improve the management of patients treated with anticoagulants.

INTRODUCTION: Double centrifugation before freezing is recommended before thrombin generation assays (TGA). However, this procedure is not mandatory for routine hemostasis tests, precluding the use of these samples for TGA. The aim of this study is to assess the impact of single and double centrifugation on TGA performed on frozen samples from healthy volunteers (HVs) and patients receiving direct oral anticoagulants (DOACs).METHODS: Forty HVs and 57 patients receiving a DOAC (dabigatran, rivaroxaban, apixaban, or edoxaban) were included in this prospective double-center observational study. Blood was collected into 109 mmol/L citrated tubes and frozen at -70°C before TGA using ST Genesia with STG-DrugScreen reagent. Four pre-analytical conditions were studied: (A) single centrifugation (2000 g, 15 minutes) before freezing; (B) one centrifugation before freezing and another after thawing (2000 g, 15 minutes for both); (C) one centrifugation before freezing(2000 g, 15 minutes) and another after thawing (2000 g, 10 minutes); (D) double centrifugation (2000 g, 15 minutes) before freezing (reference). Centrifugation conditions (A), (B), and (C) were compared with the reference condition (D). Acceptable relative differences were defined at 6%, 8%, and 10% for normalized lag time, endogenous thrombin potential, and peak height, respectively.RESULTS: Centrifugation conditions had a small but acceptable impact on HVs samples, but single centrifugation always resulted in unacceptable reductions in normalized lag times for DOAC samples. A second centrifugation after thawing permitted the recovery of acceptable differences for the three TGA parameters for edoxaban but not for apixaban, rivaroxaban, nor dabigatran.CONCLUSION: Double centrifugation before freezing should remain the recommended pre-analytical condition before TGA.

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

Introduction: Clot waveform analysis (CWA), a new methodology to assess coagulation process, can be usefully applied in various clinical settings. However, its clinical use is limited mainly because of the absence of standardization. No consensus exists regarding the wavelengths at which CWA has to be performed what is crucial for the sensitivity of the CWA. Objectives: The primary aim of this study is to determine which wavelength is the most sensitive and specific for CWA. Interindividual baseline absorbance will also be assessed as the impact of reagents from the intrinsic, extrinsic, and common coagulation pathway will be determined. Methods: Plasma samples were screened at wavelengths from 280 to 700 nm to provide absorbance spectra in clotted and nonclotted plasma. The interindividual variability of baseline absorbance was obtained by screening plasma from 50 healthy individuals at 340, 635, and 671 nm. The inner-filter effect of reagents was assessed in plasma or serum when appropriate at the same wavelengths. The reagents were those commonly used for activated partial thromboplastin time, prothrombin time, thrombin time, and dilute Russell's viper venom time. Results: Clotted plasma has higher absorbance value than nonclotted plasma (P 600 nm (P

Introduction: Chromogenic anti-Xa assays are the most appropriate tests to estimate the amount of betrixaban in plasma but the sensitivity of available tests is limited and improvements are needed to encompass the on-therapy range. Methods: Betrixaban was spiked at concentrations ranging from 0 to 500 ng/mL in plasma from healthy donors. Three commercial tests were used (Biophen®DiXaI®, STA®Liquid Anti-Xa, and HemosIL®Liquid Anti-Xa), and adaptation of their sample dilution scheme was performed. These new methodologies were also tested on plasma spiked with amounts of unfractionated heparin (UFH), low molecular weight heparins (LMWH), or fondaparinux covering the on-therapy ranges to evaluate their sensitivity to indirect factor Xa inhibitors. Results: Results showed concentration-dependent decreases in OD/min inversely proportional to the dilutions. While modifications improve the sensitivity of these tests to betrixaban (eg, ½*OD/min of 502 ng/mL [95% CI: 495-508 ng/mL] for Biophen®DiXaI® [1:50] is reduced to 51 ng/mL [95% CI: 50-52 ng/mL] for improved Biophen®DiXaI® [1:5]), results also showed an increased sensitivity to indirect factor Xa inhibitors, except for Biophen®DiXaI® which remains insensitive to UFH and LMWH. Conclusions: Results showed that the improvement of current chromogenic anti-Xa methodologies enhances the sensitivity of these assays to betrixaban but also to indirect factor Xa inhibitors. This lack of specificity may lead to overestimation of betrixaban concentrations in patients bridged with heparins. To avoid this cross-interference, the use of the Biophen®DiXaI® may be a solution except for fondaparinux which remains active even in the presence of the Biophen®DiXaI®’s specific buffer. For the other chromogenic assays, the conception and validation of specific buffer is required.

Objectives: Diffuse large B-cell lymphoma (DLBCL) is highly heterogeneous in terms of phenotype and treatment response in patients. These characteristics make the prognosis difficult to establish and hinder the use of new personalized treatments in clinical practice. In this context, there is currently a need to define new biomarkers enabling a better definition of DLBCL subtypes, prognosis evaluation, and an overview of the resistance to chemotherapeutics. The aim of this study was to evaluate the use of microRNAs found in plasma from patients with DLBCL as biomarkers of tumor evolution in these patients. Method: For this purpose, a plasma biobank was created with samples from patients with DLBCL. The evolution of the level of selected microRNAs during treatment has been studied. A total of 19 patients with DLBCL were included in this pilot mono-centered study and a total of 68 samples were analyzed. Results: The first step of this study was the selection of the microRNAs to be quantified in all the samples of the biobank and that could potentially be used as biomarkers. To this end, quantification of 377 microRNAs was performed on the plasma samples of 2 selected patients with DLBCL and 1 healthy donor with no history of cancer. Among the 377 microRNAs evaluated, 7 were selected and analyzed in the entire biobank. Conclusions: This study highlighted 5 circulating microRNAs whose plasma levels would be worth further investigating for the characterization of DLBCL evolution in patients. MiR-21 and miR-197 had a significant higher plasmatic level in patients with tumors unresponsive to treatment. With a higher plasma level in patients with complete remission, miR-19b, miR-20a, and miR-451 could enable to differentiate, at the remission review, patients with residual tumor, from patients with complete remission.

Haemostatic complication is common for patients with hematologic malignancies. Recent studies suggest that the procoagulant activity (PCA) of extracellular vesicles (EV) may play a major role in venous thromboembolism and disseminated intravascular coagulation (DIC) in acute leukaemia. To study the impact of EVs from leukaemic patients on thrombin generation and to assess EV-PCA as a potential biomarker for thrombotic complications in patients with acute leukaemia. Blood samples from a cohort of patients with newly diagnosed acute leukaemia were obtained before treatment (D-0), 3 and 7 days after treatment (D-3 and D-7). Extracellular vesicles were isolated and concentrated by ultracentrifugation. EV-PCA was assessed by thrombin generation assay, and EV-associated tissue factor activity was measured using a commercial bio-immunoassay (Zymuphen MP-TF®). Of the 53 patients, 6 had increased EV-PCA at D-0 and 4 had a thrombotic event. Patients without thrombotic events (n = 47) had no elevated EV-PCA. One patient had increased EVs with procoagulant activity at D-3 and developed a DIC at D-5. This patient had no increased EVs-related tissue factor activity from D-0 to D-7 (2 pg/ml), of these, four had a thrombosis and two had haemorrhages. Procoagulant activity of extracellular vesicles could have a predictive value in excluding the risk of thrombotic events. Our findings also suggest a possible association between thrombotic events and EV-PCA.

Resistance of cancer cells to chemotherapy is a major issue in acute myeloid leukemia (AML) and is usually due to a clonal selection of resistant leukemic cells. Recently, a horizontal transfer of chemoresistance among tumor cells has been reported via extracellular vesicles (EVs). EVs are vesicles ranging from 0.03 to 1µm generated by almost all cell types. They carry cellular components such as nucleic acids, membrane and cytosolic proteins. By interacting with cells and by transferring their content, these vesicles could modify target cells' phenotype. Because of their presence in biological fluids, a lot of studies have tried to use EVs as biomarkers. The aim of this research is to investigate the role of EVs in chemoresistance in AML. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was compared to its multiresistant strain: HL60/AR. The latter overexpresses multidrug resistance-associated protein 1 (MRP1), an efflux pump conferring resistance to anthracyclines. The transfer of this resistance between the two strains is obtained by treating HL60 with EVs generated by HL60/AR. The production of EVs by HL60/AR was confirmed by transmission electron microscopy. EVs from around 100 to 800 nm were highlighted. EVs from HL60/AR were then isolated by ultracentrifugation and their interaction with HL60 was investigated by labeling EVs with a fluorescent lipid membrane linker. The transmission of this fluorescence to HL60 was monitored by flow cytometry and demonstrated a binding or an incorporation of EVs. In order to check if this interaction has an impact on HL60 resistance to daunorubicin, HL60 were treated with EVs, once for 20 hours or four consecutive times for 48 hours each. The resistance to daunorubicin of EVs-treated HL60 was then assessed by a MTT cytotoxicity assay and revealed that EVs increase survival of cells to daunorubicin. The resistant profile between the cells treated once or four times with EVs were somewhat different and highlighted a potential implication of nucleic acids cargo in this resistance transfer. Indeed, nucleic acids transferred by EVs would need more than 20 hours to impact target cells. Afterwards, we investigated the changes in phenotype that could be responsible for this increased resistance of EVs-treated cells. First, the expression of MRP1 was evaluated by a flow cytometry drug retention assay based on the fluorescence exhibited by daunorubicin and consisting in analysis by flow cytometry of the daunorubicin retention of cells with or without the presence of a specific MRP1 inhibitor. This experiment emphasized an expression of MRP1 after EVs treatment. Oxidative stress also plays an important role in daunorubicin cytotoxicity. Thus reactive oxygen species (ROS) generation assays were performed on EVs-treated cells. These cells showed a decreased production of daunorubicin-induced ROS. We finally analyzed the microRNAs content of isolated EVs. This was performed by TaqMan® Human MicroRNA Array on EVs from HL60/AR but also on EVs generated by HL60. This experiment aimed at highlighting the microRNAs cargo potentially involved in this chemoresistance transfer but also at identifying microRNAs differentially expressed between the two strains to serve as biomarker of chemoresistance in vivo. Out of the 380 microRNAs tested, 32 presented a differential expression between HL60 and HL60/AR. Among them, 5 microRNAs presented a more than 8-fold change between the two strains: miR-19b, miR-20a, miR-488, miR-519a and miR-523. miR-519a was the only microRNA more expressed in EVs produced by HL60, compared to EVs from HL60/AR. In conclusion, this study demonstrates the ability of resistant leukemic cells to transfer, at least partially, their chemoresistance via EVs. This could be due to a direct transfer of MRP1 transported by EVs but also to a nucleic acid transfer. In this context, some microRNAs have been highlighted for their high differential expression in EVs between sensitive and chemoresistant cells. These results bring out a potential clinical use of these circulating microRNAs as chemoresistance biomarkers in AML. Disclosures No relevant conflicts of interest to declare.