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Introduction: Thrombin generation (TG) documents hypercoagulability. TG in platelet-poor plasma is exquisitely sensitive to heparins, which thus must be neutralized before testing. Heparinase and hexadimethrine bromide (polybrene) have been used for that purpose, but their effects per se on TG have been poorly studied so far., Methods: (i) TG was studied in commercial normal pooled plasma (NPP; CryoCheck ® , Cryopep) in absence or presence of neutralizing agents. (ii) NPP was spiked with increasing concentrations of unfractionated heparin (UFH; up to 1.0 IU/mL) or low-molecular-weight heparin (LMWH; enoxaparin up to 1.2 IU/mL) and TG studied after incubation of heparinase (Hepzyme ® ; 15 minutes) or polybrene (0.025 mg/mL; 10 minutes)., Results: (i) With ThromboScreen reagent to initiate TG, addition of heparinase was associated with increased peak, whereas polybrene caused lengthening of lag time and time to peak, compared with nonsupplemented NPP. (ii) With polybrene, TG was completely restored over the whole range of UFH and LMWH studied. By contrast, heparinase failed to fully restore TG in presence of UFH concentrations ≥0.8 IU/mL or LMWH concentrations ≥1.0 IU/mL. Those effects were matched with detectable tiny residual amounts of non-neutralized heparin (as assessed with an anti-Xa assay) and were less pronounced with a higher picomolar concentration of tissue factor (DrugScreen reagent)., Conclusion: Polybrene fully restored TG of heparinized plasma at the expense of an alteration of TG, pointing to the need to use adapted reference ranges. Heparinase failed to do so in presence of high concentrations of both heparins., (© 2021 John Wiley & Sons Ltd.)

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., (© 2021 John Wiley & Sons Ltd.)

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., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Calibrated automated thrombinography (CAT) is a sensitive method to assess coagulation. Dabigatran inhibits both free thrombin and the α 2 macroglobulin (α 2 M)-thrombin complex, which results in an erroneously increased peak and endogenous thrombin potential (ETP) without affecting lag time and time-to-peak. The aim of this study was to elucidate the artefacts in CAT when dabigatran is present., Materials and Methods: Thrombin generation (TG) was measured in vitro by using CAT in the presence or absence of 6 μM idarucizumab in plasma spiked with dabigatran. Additionally, ex vivo measurements were performed in plasmas of 63 patients using dabigatran in the presence and absence of idarucizumab., Results: The in vitro experiments confirmed that the ETP, peak and velocity index were artificially increased. This was mainly due to the inhibition of the calibrator by dabigatran and partly due to CAT algorithms. The calibration artefact could be resolved by adding idarucizumab to the calibrator well. However, the second, mathematical artefact remains when dabigatran is present in the TG well. These findings were corroborated by ex vivo experiments i.e. the lag time and time-to-peak were significantly reduced in patients upon addition of idarucizumab, but the ETP and peak were not significantly affected. The velocity index did change significantly, since this is a combination of time-dependent factors and the peak., Conclusions: The peak, ETP and velocity index do not represent the anticoagulant effect of dabigatran on TG measured with CAT. The lag time and time-to-peak, however, do reflect the effect of dabigatran., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction:  Betrixaban is a novel direct oral factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in adult patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anti-coagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced., Objective:  The aim of this study is to determine which coagulation assay(s) should be used to assess the impact of betrixaban on haemostasis and provide laboratory guidance for their interpretation., Materials and Methods:  Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/mL in platelet-poor plasma. Different reagents from several manufacturers were tested and the impact of betrixaban on pro-thrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russel viper venom time (dRVV-T), chromogenic anti-Xa assays, thrombin generation assay (TGA), and a large panel of haemostasis diagnostic tests has been assessed., Results:  A concentration-dependent prolongation of aPTT, PT and dRVV-T is observed. The sensitivity mainly depends on the reagent. Chromogenic anti-Xa assays show high sensitivity depending on the reagent and/or the methodology. These assays applicable for other direct factor Xa inhibitors have to be adapted to obtain a relevant range of measurement. TGA may also be attractive to assess the anti-coagulant activity of betrixaban., Conclusion:  Adapted chromogenic anti-Xa assays are the most appropriate assays to estimate the concentration of betrixaban. Betrixaban significantly affects several haemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests., Competing Interests: Among the authors, J. Douxfils is CEO and founder of QUALIblood s.a. and reports personal fees from Stago and Daiichi-Sankyo, outside the submitted work. F. Mullier reports institutional fees from Stago, Werfen, Nodia, Sysmex and Bayer. He also reports speaker fees from Boehringer Ingelheim, Bayer Healthcare and Bristol-Myers Squibb-Pfizer, all outside the submitted work. Other authors have no conflicts of interest to disclose., (Georg Thieme Verlag KG Stuttgart · New York.)

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., (© 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA ® -Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component., Materials and Methods: Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA ® -Procoag-PPL assays) were assessed., Results: The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA ® -Procoag-PPL were well correlated (partial r=-0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed., Discussion: The STA ® -Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate.

Background: The dilute Russell's viper venom time (dRVVT) has been suggested for the assessment of the intensity of anticoagulation of all direct oral anticoagulants (DOACs). This study aimed to compare the performance of an optimized liquid-stable dRVVT-based DOAC assay (DRVV-DOAC) on clinical samples before and after mixing these with normal pooled plasma (NPP)., Methods: Forty-one apixaban, 25 dabigatran, 56 rivaroxaban and 49 vitamin K antagonists (VKAs) plasma samples were included for retrospective analysis. Plasma DOAC concentrations were determined by liquid chromatography coupled with tandem mass-spectrometry. INR was determined for all VKA samples. DRVV-DOAC was performed with an original ready-to-use reagent (Haematex Research ™ ) where plasma samples were tested neat and in a 1:1 mix with NPP., Results: Plasma concentrations ranged from 1 to 406ng/ml for apixaban, 0 to 386ng/ml for dabigatran and 0 to 719ng/ml for rivaroxaban. INR ranged from 2.2 to 6.1. DRVV-DOAC correlated well with plasma concentrations (r 2 =0.70, 0.94, 0.63 (non-mixed procedure) and 0.77, 0.97, 0.86 (mixed procedure) for apixaban, dabigatran and rivaroxaban, respectively). DRVV-DOAC measurements in the normal range ruled out dabigatran and rivaroxaban concentrations above 30 and 50ng/ml, but performance was lower for apixaban. DRVV-DOAC was sensitive to VKA samples but poorly reflected INR values. When VKA samples were mixed with NPP, DRVV-DOAC measurements decreased to values close to baseline clotting time., Conclusions: DRVV-DOAC is a quick method which showed increased sensitivity compared with other phospholipid-rich dRVVT reagents already investigated. Mixing samples with NPP improved the specificity but reduced sensitivity, especially for apixaban., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© 2017 John Wiley & Sons Ltd.)

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). Eight patients had increased EVs with tissue factor activity (>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.

Assessment of plasma concentration/effect of edoxaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. It was our aim to determine coagulation tests useful for the assessment of edoxaban's pharmacodynamics and provide recommendations for the interpretation of haemostasis diagnostic tests. Edoxaban was spiked at concentrations ranging from 0 to 1,000 ng/ml in platelet-poor plasma which covers the on-therapy range (from ± 25 ng/ml at Ctrough to ± 170 ng/ml at Cmax). aPTT, PT, dRVVT, chromogenic anti-Xa assays, TGA and a large panel of haemostasis diagnostic tests were performed using several reagents. A concentration-dependent prolongation of aPTT, PT and dRVVT was observed. The effect was dependent on the reagents. FXa chromogenic assays showed high sensitivity and a linear correlation depending on the methodology. TGA may be useful to assess the pharmacodynamics of edoxaban but its turnaround time and the lack of standardisation are limitations. Edoxaban impairs the assessment of lupus anticoagulant, protein S (clotting method), APC-R, antithrombin (FXa-based assay) and measurement of clotting factor activity. Immunological assays and assays acting below the FXa are not influenced by edoxaban. In conclusion, some PT reagents could be used to estimate edoxaban activity. Chromogenic anti-Xa assays are required to assess the plasma concentration. TGA may be useful but requires standardisation. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake and the sampling is mandatory to provide a proper assessment of the result.

Non-VKA oral anticoagulants (NOACs) have now widely reached the lucrative market of anticoagulation. While the marketing authorization holders claimed that no routine monitoring is required and that these compounds can be given at fixed doses, several evidences arisen from the literature tend to demonstrate the opposite. New data suggests that an assessment of the response at the individual level could improve the benefit-risk ratio of at least dabigatran. Information regarding the association of rivaroxaban and apixaban exposure and the bleeding risk is available in the drug approval package on the FDA website. These reviews suggest that accumulation of these compounds increases the risk of experiencing a bleeding complication. Therefore, in certain patient populations such as patients with acute or chronic renal impairment or with multiple drug interactions, measurement of drug exposure may be useful to ensure an optimal treatment response. More specific circumstances such as patients experiencing a haemorrhagic or thromboembolic event during the treatment duration, patients who require urgent surgery or an invasive procedure, or patient with a suspected overdose could benefit from such a measurement. This paper aims at providing guidance on how to best estimate the intensity of anticoagulation using laboratory assays in daily practice.

The Belgian national External Quality Assessment Scheme performed a nationwide survey using lyophilised plasma samples spiked with dabigatran or rivaroxaban to demonstrate to the Belgian clinical laboratories how these drugs affect their routine coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin. Virtually all Belgian laboratories performing routine coagulation testing (189/192) participated in the survey. Both, dabigatran and rivaroxaban significantly prolonged the PT and aPTT in a concentration- and reagent-dependent manner. PT reagents were more influenced by rivaroxaban than by dabigatran and aPTT reagents more influenced by dabigatran than by rivaroxaban. Among PT reagents, Neoplastin R® was the most sensitive to rivaroxaban and Innovin® and Thromborel S® the least sensitive. Converting PT results to INR only increased the variability between reagents. Among aPTT reagents, Actin FSL® was the least sensitive to dabigatran while the other aPTT reagents showed slightly higher sensitivities. The presence of dabigatran led to falsely reduced fibrinogen concentrations when measured with a low thrombin concentration reagent. The presence of dabigatran caused an overestimation of the antithrombin level when measured with a thrombin-based activity assay and the presence of rivaroxaban an overestimation of the antithrombin level when measured with a FXa-based assay. Instrument-related differences were found for all tested parameters. In conclusion, this paper provides detailed information on the effect of dabigatran and rivaroxaban on routine coagulation assays as performed with a large number of reagent/instrument combinations.

Apixaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose for the individual patient may be useful in some situations. Moreover, there is a need for clinicians to know whether coagulation assays are influenced by apixaban use. The aim of this study was to determine which coagulation assay could be used to assess the impact of apixaban on haemostasis and provide good laboratory recommendations for the accurate interpretation of haemostasis assays. Apixaban is spiked at concentrations ranging from 5 to 500 ng/mlin platelet-poor plasma. Routinely used or more specific coagulation assays are tested. Results show a concentration dependent prolongation of aPTT, PT and dilute PT. The sensitivity mainly depends on the reagent, but none of these tests is sensitive enough to ensure an accurate estimation of the pharmacodynamic effect of apixaban. FXa chromogenic assays show high sensitivity and a linear correlation depending on the reagent and/or the methodology. Immunological assays and assays acting below the FXa are not influenced by apixaban. In conclusion, PT and/or dilute PT cannot be used to assess apixaban pharmacodynamic properties. More specific and sensitive assays such as chromogenic FXa assays using specific calibrators are required. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake of apixaban and the sampling is mandatory.

Introduction: Rivaroxaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations., Aim: To determine which coagulation assay could be used to assess the impact of rivaroxaban on haemostasis and provide guidelines for the interpretation of routine lab tests., Materials: Rivaroxaban was spiked at concentrations ranging from 11 to 1,090 ng/mL in plateletpoor plasma. A large panel of coagulation assays was tested., Results: A concentration dependent prolongation of aPTT, PT, dPT, PiCT was observed. PT and dPT were the most sensitive chronometric assays but results varied depending on the reagent (Triniclot PT Excel S>Recombiplastin 2G>Neoplastin R>Neoplastin CI+>Triniclot PT Excel>Triniclot PT HTF>Innovin). FXa chromogenic assays showed the highest sensitivity. In TGA, Cmax was the most sensitive parameter with the tissue factor induced pathway. Rivaroxaban interferes on haemostasis diagnostic tests such the measurement of clotting factors, fibrinogen, antithrombin, proteins C and S, activated protein-C resistance and Xa-based chomogenic assays., Conclusions: PT may be used as screening test to assess the risk of bleedings. A more specific and sensitive assay such as Biophen DiXaI using calibrators should be used to confirm the concentration of rivaroxaban. We also propose cut-off associated with a bleeding or thrombosis risk based on pharmacokinetic studies. Standardization of the time between the last intake of rivaroxaban and the sampling is mandatory., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Due to low bioavailability and high inter-individual variability, monitoring of dabigatran may be required in specific situations to prevent the risk of bleedings or thrombosis. The aim of the study was to determine which coagulation assay(s) could be used to assess the impact of dabigatran on secondary haemostasis. Dabigatran was spiked at concentrations ranging from 4.7 ng/ml to 943.0 ng/ml in pooled citrated human platelet-poor plasma. The following clotting assays were performed: prothrombin time (PT); activated partial thromboplastin time (aPTT); thrombin time (TT); ecarin clotting time (ECT); ecarin chromogenic assay (ECA); prothrombinase-induced clotting time (PiCT); activated clotting time (ACT); Hemoclot Thrombin Inhibitor (HTI) and thrombin generation assay (TGA). A concentration-dependent prolongation of PT, dPT, and aPTT was observed with aPTT being the more sensitive test. The results varied mostly due to the clotting reagent. HTI, ECT and TGA were the most sensitive tests but are not available 24 hours a day. In addition, HTI showed a linear correlation with a good reproducibility. Dabigatran induced a concentration-dependent delay and inhibition of tissue factor-induced TGA. Cut-offs related with higher risk of bleedings or thrombosis were defined for each reagent of aPTT and HTI. In conclusion, aPTT could be used for the monitoring of dabigatran and as screening test for the risk of overdose. However, because of its higher sensitivity, good reproducibility, excellent linear correlation at all doses, its simplicity of use, and possibilities of automation, HTI should be considered as the gold-standard.

We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO₂ ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Coagulation activation on medical devices remains a significant problem as it can lead to dramatic thromboembolic complications. Understanding its poorly described mechanisms and finding optimal pharmacological prevention means is crucial to improve patient safety. We developed an in vitro model to study thrombin generation (TG) initiated by the contact of plasma with the surface of catheters. Interventional cardiology catheters were cut into segments and inserted in the bottom of multi-well plates; TG was then measured with the calibrated automated thrombogram (CAT). Model performance (analytical, intra- and inter-individual variability) was investigated and compared with activation of thrombin generation by tissue factor (TF) or contact pathway activator (ellagic acid), in the presence (PRP) and absence (PPP) of platelets. Model response to unfractionated heparin (UFH) was also assessed. TG was greater when measured in presence of catheter segments, compared to conditions without activators. The analytical variability of the model was good (CV ≤ 5 %), both with PPP and PRP. Intra-individual variability was between 15 and 30 % with PPP and between 10 and 15 % with PRP. Inter-individual variability was between 15 and 30 % with both kinds of plasma samples. The analytical performance of the catheter-initiated TG model was equivalent to that observed when TG was initiated with TF or ellagic acid. Catheter-initiated TG was measurable until 0.1 IU/mL UFH with PPP and until 1.0 IU/mL UFH with PRP, highlighting the crucial requirement of platelets. Our model is suitable for studying TG initiated with catheters. Inhibition of TG by UFH is overestimated in the absence of platelets. • Foreign material, including catheters, strongly activates hemostasis. • Platelets are essential to assess the effect of heparin on thrombin generation (TG). • We developed a model to study TG at the surface of catheters in presence of platelets. • The model showed low analytical, intra- and inter-individual variability. • This model will enable to study pharmacological inhibition of TG at foreign surfaces. [ABSTRACT FROM AUTHOR]