JG Marguerite Sylvain

À mes co internes de pédiatrie, Chloé, Pascaline et Julien ! Plus dans la même spé mais toujours dans mon coeur ! À ma tutrice préférée, Grâce THOMAS, qui m'a appris la recherche fondamentale et la vie en générale ! Merci pour ta gentillesse infinie et ton aide pour des faire de belles diapos ! À mes amis de Nice, Isabeau, Marie-Alice

10

REMERCIEMENTS

À Monsieur le Professeur Émile FERRARI, pour l'honneur que vous me faites de présider ce jury. À Monsieur le Docteur Pierre TOULON, je vous remercie de m'avoir proposé ce travail de thèse. Merci pour l'enseignement que vous m'avez transmis durant ces années au laboratoire de Nice.

À Madame le Docteur Laetitia MAUGE, pour avoir accepté de participer à ce jury et pour l'enseignement que tu m'as transmis durant ce stage à l'HEGP ! Merci pour ta gentillesse et pour avoir apporté à cette thèse tes riches connaissances sur l'hémostase et la thrombose.

À Monsieur le Docteur Nicolas GENDRON, pour avoir accepté de participer à ce jury et pour tout ce que tu m'as appris sur les sur-experts de la thrombose ! Ce fut un réel plaisir de participer à tes consultations d'hémostase spécialisée. Merci pour tous les cafés, les chocolats et blagues réconfortantes ! À madame le Professeur Pascale GAUSSEM, pour m'avoir accueillie dans son laboratoire à l'HEGP pour mon inter CHU qui m'a permis d'apprendre énormément de choses sur la thrombose! Merci pour votre bienveillance et vos précieuses remarques pour la rédaction de cette thèse! À Florence FISCHER, pour m'avoir fait découvrir la plus belle spécialité de l'hémostase! Merci pour ta gentillesse et ton enseignement ! À Corinne FERRERO, pour les moments sympas au microscope pour apprendre la cytologie ! À tous les Biologistes du CHU de Nice qui m'ont permis de découvrir tous les aspects de la biologie médicale, particulièrement à Nicolas DEGAND en bactério, Lilia HASSEINE avec qui c'était une joie de travailler, et Pascal DELAUNAY qui a été un fort soutien durant mon parcours ! À toute l'équipe de techniciennes et de techniciens du laboratoire de Nice qui ont récolté et congelé tous les plasmas, et particulièrement à Émilie, Mélanie, Stéphanie, Julie, Cécile, Sylvain, Julien, Gabriel, Yann, qui m'ont appris à dompter a ACL-Star ! À mes co internes (puis chefs) de Nice, Mathilde B et Mathilde V, Célia, Caroline, Alicia, Simona, Amaya, Greg, Jacques, Sami, Stéphane, Elamine, sans oublier les anapath Yuta, Florent, Pierre et Alex ! Que de bons moments ensemble autour de la piscine de l'Archet, le parvis de Pasteur et les bars de Nice ! À mes co internes de l'HEGP, Amna, Kahina et Aurélien. Je suis très heureuse d'être votre co interne, vaillants soldats, toujours prêt à aider dans la joie et à valider (bisou) ! Merci Carla d'avoir été un soutien pendant les derniers moments de Results: A total of 713 plasma samples were evaluated, obtained from patients treated with dabigatran (n=152), rivaroxaban (n=131), or apixaban (n=1134), and from 420 patients with various disease states and not on DOAC, including 35 patients on warfarin and 32 patients on heparins (UFH: n=18; LMWH: n=14). In untreated patients, the DOAC Remove® had no impact on test results and LA interpretation. In the plasma from patients treated on DOAC, the DOAC Remove® eliminated all DOACs, with residual levels below the quantification limit of the techniques, leading to a correction of the DOAC-induced prolongations of PT and APTT, as well as of their lowering effect on FV, FVIII and FIX activities. The same applied to the DOAC-induced elevation of PC and PS anticoagulant activities (clotting assays), and antithrombin activity in the plasma from patients on rivaroxaban and apixaban. DRVVT screen/confirm ratios were normalized and DOAC Remove® eliminated 86% and 79% of false positive on rivaroxaban and dabigatran samples. The impact of the DOACs on the SCT screen/confirm ratio was less noticeable with baseline positive test results in only one of the samples from patients on dabigatran (n=31), and in three patients on rivaroxaban (n=32).

Treatment with the carbon leaded to a correction in the positive sample from patient on dabigatran and in one of the three positive samples from patients on rivaroxaban. Only one patient on apixaban was positive in dRVVT and SCT test and no change was observed after DOAC Remove®.

Conclusion:

The DOAC Remove® had no effect on test results obtained in plasmas from untreated patients and from patients on traditional anticoagulants. It removed all three tested DOACs from the plasma of treated patients, allowing an accurate measurement of routine coagulation and thrombophilia tests in DOAC treated patients without necessity to withhold treatment.

INTRODUCTION

Direct oral anticoagulants (DOAC) are used for a number of indications [START_REF] Favaloro | Replacing warfarin therapy with the newer direct oral anticoagulants, or simply a growth in anticoagulation therapy? Implications for pathology testing[END_REF]. The most common includes stroke prevention in non-vascular atrial fibrillation, venous thrombosis prevention in major orthopedic surgery, and treatment and secondary prevention of venous thromboembolism (VTE) [START_REF] Khider | Inherited Thrombophilia in the Era of Direct Oral Anticoagulants[END_REF]. Three DOAC are available in France: dabigatran is a direct inhibitor of thrombin, rivaroxaban and apixaban are direct inhibitors of activated factor (FXa).

DOACs have many benefits, they are rapidly-acting, target specific and they do not require routine laboratory monitoring (3). However, DOAC have an impact on routine and specialized coagulation assays by a prolongation on clot-based screening test [START_REF] Douxfils | Update of the International Council for Standardization in Haematology Recommendations for Laboratory Measurement of Direct Oral Anticoagulants[END_REF][START_REF] Kitchen | Measurement of non-coumarin anticoagulants and their effects on tests of Haemostasis: Guidance from the British Committee for Standards in Haematology[END_REF][START_REF] Bonar | The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples[END_REF][START_REF] Bonar | The effect of dabigatran on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples[END_REF][START_REF] Adcock | Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review[END_REF][START_REF] Samuelson | Laboratory Assessment of the Anticoagulant Activity of Direct Oral Anticoagulants[END_REF][START_REF] Douxfils | Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians[END_REF][START_REF] Goodwin | Thrombophilia Testing and Venous Thrombosis[END_REF][START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays[END_REF][START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays[END_REF]. Routine coagulation screening assays, including the prothrombin time (PT) and activated partial thromboplastin time (aPTT), vary depending on the patients and the DOAC [START_REF] Adcock | Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review[END_REF]. PT and aPTT are false prolonged and Plasma levels of coagulation factors are under estimated when measured under DOAC therapy. Furthermore, clot-based protein C and protein S activity are significantly affected by DOAC, resulting in factitious overestimation, which could conclude to a false normal result in a patient with PC or PS deficiency [START_REF] Bonar | The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples[END_REF][START_REF] Bonar | The effect of dabigatran on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples[END_REF][START_REF] Adcock | Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review[END_REF][START_REF] Goodwin | Thrombophilia Testing and Venous Thrombosis[END_REF][START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays[END_REF][START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays[END_REF]. Also, the dilute Russel Viper Venom time (dRVVt) is the most influenced by DOAC with false-positive results and false positive lupus anticoagulation (LA) interpretation [START_REF] Hoxha | Detection of lupus anticoagulant in the era of direct oral anticoagulants[END_REF][START_REF] Flieder | Interference of DOACs in different DRVVT assays for diagnosis of lupus anticoagulants[END_REF]. Thrombophilia testing is not recommended during the acute phase of VTE [START_REF] Sanchez | Recommendations of good practice for the management of thromboembolic venous disease in adults. Short version][END_REF]. Moreover, thrombophilia testing is often prescribed when the patient is still treated by DOAC therapy 3 to 6 months after VTE for reevaluation of anticoagulant treatment. Because of DOAC interference, patients should not be tested for thrombophilia during the anticoagulation period [START_REF] Goodwin | Thrombophilia Testing and Venous Thrombosis[END_REF].

However, interruption of anticoagulation therapy, in particular in case of patients treated for unprovoked VTE, could be harmful for the patient given the high risk of VTE recurrence [START_REF] Sanchez | Recommendations of good practice for the management of thromboembolic venous disease in adults. Short version][END_REF][START_REF] Khan | Long-Term Risk for Major Bleeding During Extended Oral Anticoagulant Therapy for First Unprovoked Venous Thromboembolism : A Systematic Review and Meta-analysis[END_REF]. Owing the high inter-individual variability of DOAC plasma level, the temporary interruption of DOAC treatment may need to last more than 72h to achieve an undetectable concentration [START_REF] Adcock | Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review[END_REF][START_REF] Gong | Importance of pharmacokinetic profile and variability as determinants of dose and response to dabigatran, rivaroxaban, and apixaban[END_REF][START_REF] Gay | Lupus anticoagulant diagnosis in patients receiving direct oral FXa inhibitors at trough levels: A real-life study[END_REF]. Furthermore, switching from DOAC to low-molecular-weight heparin may increase the risk of major bleeding and the day-to-day practice can sometimes be complicated for the patient who must not forget to switch of anticoagulation [START_REF] Douxfils | Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians[END_REF]. It is therefore necessary to find a solution to counter the interferences of DOAC on tests, to avoid stopping anticoagulation for thrombophilia testing.

To address these issues, different methods have been tested to limit the DOAC interference. The dabigatran antidote, idarucizimab, has been tested to remove the dabigatran [START_REF] Jacquemin | The adsorption of dabigatran is as efficient as addition of idarucizumab to neutralize the drug in routine coagulation assays[END_REF] and andexanet alfa for rivaroxaban [START_REF] Favaloro | Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa[END_REF]. These methods were found efficient but are costexpensive and require to be sure of the treatment taken by the patient. The filtration device DOAC Filter (Diagnostica Stago) has been tested to remove DOAC on LA but filtration might induce a procoagulant effect [START_REF] Sevenet | Evaluation of DOAC Filter, a new device to remove direct oral anticoagulants from plasma samples[END_REF][START_REF] Farkh | A Diagnostic Solution for Lupus Anticoagulant Testing in Patients Taking Direct Oral FXa Inhibitors Using DOAC Filter[END_REF][START_REF] Linskens | Direct Oral Anticoagulant removal by a DOAC filter: Impact on lupus anticoagulant testing -Evaluation on spiked and patient samples[END_REF].

Several studies have tried in vitro drug adsorption using activated charcoal using DOAC Remove® [START_REF] Kopytek | DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients[END_REF][START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF][START_REF] Favre | Direct oral anticoagulant neutralization by activated charcoal DOAC-Remove for thrombophilia screening[END_REF][START_REF] Al-Qawzai | Effect of DOAC-Remove on coagulation screening assays in samples from patients receiving oral or parenteral anticoagulation[END_REF] and DOAC Stop™ [START_REF] Favaloro | Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa[END_REF][START_REF] Favaloro | Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects[END_REF][START_REF] Exner | Simple method for removing DOACs from plasma samples[END_REF][START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF][START_REF] Favresse | Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests[END_REF][START_REF] Slavik | Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban[END_REF][START_REF] Ząbczyk | The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants[END_REF][START_REF] Ząbczyk | The Effect of Direct Oral Anticoagulants on Antithrombin Activity Testing Is Abolished by DOAC-Stop in Venous Thromboembolism Patients[END_REF]. The adsorption power of activated charcoal was described in 1946 [START_REF] Andersen | Experimental studies on the pharmacology of activated charcoal; adsorption power of charcoal in aqueous solutions[END_REF]. Activated charcoal allowed the absorption of drugs and toxins through weak intermolecular forces [START_REF] Olson | Activated charcoal for acute poisoning: one toxicologist's journey[END_REF] and it is currently used as an antidote for patients having ingested toxic substances [START_REF] Olson | Activated charcoal for acute poisoning: one toxicologist's journey[END_REF][START_REF] Juurlink | Activated charcoal for acute overdose: a reappraisal[END_REF][START_REF] Skov | The effect of activated charcoal on drug exposure following intravenous administration: A meta-analysis[END_REF][START_REF] Hoegberg | Systematic review on the use of activated charcoal for gastrointestinal decontamination following acute oral overdose[END_REF][START_REF] Mégarbane | Management of pharmaceutical and recreational drug poisoning[END_REF]. In healthy volunteers, Activated Charcoal reduced the area under the rivaroxaban concentration-time curve when administered two at eight hours post dose [START_REF] Ollier | Effect of Activated Charcoal on Rivaroxaban Complex Absorption[END_REF]. DOAC Remove® [START_REF] Kopytek | DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients[END_REF][START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF][START_REF] Favre | Direct oral anticoagulant neutralization by activated charcoal DOAC-Remove for thrombophilia screening[END_REF][START_REF] Al-Qawzai | Effect of DOAC-Remove on coagulation screening assays in samples from patients receiving oral or parenteral anticoagulation[END_REF] and DOAC Stop™ [START_REF] Favaloro | Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa[END_REF][START_REF] Favaloro | Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects[END_REF][START_REF] Exner | Simple method for removing DOACs from plasma samples[END_REF][START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF][START_REF] Favresse | Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests[END_REF][START_REF] Slavik | Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban[END_REF][START_REF] Ząbczyk | The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants[END_REF][START_REF] Ząbczyk | The Effect of Direct Oral Anticoagulants on Antithrombin Activity Testing Is Abolished by DOAC-Stop in Venous Thromboembolism Patients[END_REF] can neutralize DOACs from human citrated plasma samples and eliminate their interference on routine or more specialized coagulation assays. DOAC Remove® (5-Diagnostics, Basel, Switzerland) is composed of 20 mg activated carbon, specially formulated with additives, and can absorb molecules up to MM 6000 Daltons.

The aim of this study was to evaluate the effect of DOAC Remove® on routine coagulation tests and thrombophilia testing in plasmas of patients treated by rivaroxaban, apixaban and dabigatran. We suggest a solution to allow routine assay and thrombophilia testing of patients receiving DOAC without withholding anticoagulant therapy.

MATERIAL AND METHODS: 1. Plasma samples

Patient's samples were collected routinely into citrated tubes (0,109 M trisodium citrate 9:1 v/v) at various clinics and sent for testing at laboratories of University Hospital of Nice and Monaco Princess Grace Hospital. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki. According to the GFHT guidelines, a double centrifugation at 2500g for 15 minutes at room temperature with plasma decantation in a second tube in between led to platelet-poor plasma (PPP) with was frozen at -80°C until analysis. Prior to experiments, the samples were thawed at 37°C during 4 minutes. Patients received apixaban, dabigatran or rivaroxaban. For control group, patients received Vitamine K antagonist (VKA) or heparin or no anticoagulant. First, non-DOAC patients were tested to ensure that activated charcoal did not affect results of routine and specialized coagulation assays (control group). There were two aliquots per patient from the same blood sample. One aliquot served as control without charcoal and the second was treated with activated charcoal to compare the results of coagulation assay. In cases where there was not enough plasma, the same aliquot was used for all steps of pre-and post-activated charcoal treatment testing.

Treatment with activated charcoal DOAC Remove®

According to the manufacturer instructions, one tablet of activated charcoal DOAC Remove® ™ (5-Diagnostics, Basel, Switzerland) was added to 800 µL of plasma sample.

Sample was gently mixed for 10 minutes at room temperature using a rotating shaker, then centrifuged for 2 minutes at 2000g at room temperature. Supernatant devoid of charcoal particles was used for coagulation assays.

DOAC measurement

Rivaroxaban and apixaban concentrations were measured using the Liquid Anti-Xa kit, a stage chromogenic assay based on a synthetic chromogenic substrate and on Factor Xa inactivation (Liquid Anti -Xa, HemosIL®). For dabigatran, the quantitative determination anti-IIa activity was performed using a dilute thrombin time test (DTI, HemosIL®). The lower limit of quantification (LLOQ) and of the detection (LLOD) was indicated by the manufacturers. The LLOQ was equal to 20 ng/ml for dabigatran and rivaroxaban, and 15ng/ml for apixaban. The LLOD was equal to 10 ng/ml for the three DOAC.

Heparin anti-Xa activity was measured with the Liquid Anti-Xa (HemosIL®).

Routine and specialized coagulation assays

Routine assay: The quantitative determination of PT was performed using a high sensitivity thromboplastin reagent (RecombiPlasTin 2G, HemosIL®). APTT was determined using a highquality synthetic phospholipid reagent (SynthASil, HemosIL®). Fibrinogen was measured by the Clauss method, with Q.F.A Thrombin (Bovine) (HemosIL®) and Fibrinogen C (HemosIL®). This reagent is composed of different quantity of thrombin (100 UI/ml Q.F.A Thrombin (R1) vs 35UI/ml fibrinogen C (R2)). Immunoturbidimetric assay was used to determine plasma D-Dimer (D-Dimer HS 500 HemosIL®). All reagents were from Werfen (Bedford, MA, USA) and all assays were performed on the ACL-TOP 700® analyzer (Werfen) except for the VWF:GP1bR assay on the AcuStar™ analyzer (Table 1).

Statistical analysis

Results were expressed as median [min-max]. Statistical analysis was performed using statistical sofware MedCalc. Differences in median values were assessed by Wilcoxon test. A p-value <0.05 was considered as statistically significant. The average biases were evaluated according Bland and Altman.

RESULTS

Overall, we collected 134, 134 and 152 plasmas from patients treated by apixaban, rivaroxaban and dabigatran, respectively. We collected 296 plasmas from patients without DOAC, among us 10 who received LMWH, 18 who received heparin, 35 who received Vitamin K antagonists (VKA).

Effect of DOAC Remove® on DOAC levels

Activated charcoal significantly reduced plasma levels of apixaban, rivaroxaban and dabigatran (p<0.0001) (Table 2). Indeed, after DOAC Remove® procedure, apixaban level was superior to the LLOD in only four plasmas. All DOAC levels were below LLOQ limits after treatment with DOAC Remove®.

Effect of DOAC Remove® on heparin anti-Xa

In patients receiving heparins (10 on LMWH and 18 on HNF), there was no significant difference between specific anti Xa activity before (median [Min-Max] = 0.23 [0.09-0,88]) and after (median [Min-Max] = 0.21 [0.04-0,82]) treatment with DOAC Remove® (p= 0.001) (Table 3). This difference was minimal and inferior to the acceptable limits of GHFT. We can conclude than activated charcoal has not effect on the anti-Xa activity.

Routine assay a. Without DOACs:

For PT, aPTT and fibrinogen testing, no significant changes were observed after sample treatment with DOAC Remove® in the control group. Only Factor V level significantly increased after treatment with DOAC Remove®. This difference (5%) was below the acceptable bias given by the GHFT (17.7%). Such as for D-dimer levels, the difference had no impact and was inferior to the uncertainly measurement. In conclusion, DOAC Remove® has not impact on PT, aPTT, fibrinogen, D-dimer and Factor V level in control group (table 3).

b. With DOAC:

After DOAC Remove®, PT was significantly reduced from a median of 15.3 sec to 12.9 sec (p<0.0001) in apixaban samples, from 21.0 sec to 12.5 sec (p<0.0001) in rivaroxaban samples and from 14.7 to 12.7 in dabigatran samples (p<0.0001) (Table 4). After DOAC Remove®, aPTT was significantly reduced from a median of 32.3 sec to 30.3 sec (p<0.0001) in apixaban samples, from 35.6 sec to 30.1 sec (p<0.0001) in rivaroxaban samples and from 44 sec to 28.4 sec in dabigatran samples (p<0.0001).After DOAC Remove®, factor V level was significantly increased from a median from 85% to 92% (p<0.0001) in apixaban sample, from 68% to 94% (p<0.0001) in rivaroxaban samples and from 78% to 85% in dabigatran samples (p<0.0001).

For fibrinogen measurement, we tested two reagents with different quantity of thrombin (100 UI/ml Q.F.A Thrombin (R1) vs 35UI/ml Fibrinogen C (R2)). With reagent R1, fibrinogen level was not modified by DOAC Remove® whereas with reagent R2, fibrinogen level increased after DOAC Remove® in dabigatran sample (p< 0.05). In apixaban and rivaroxaban samples, the difference in fibrinogen level with reagent R2 was minimal and inferior to the acceptable bias of GFHT (14.8%).

Concerning D-dimer levels, we observed a decreasing trend of D-dimer levels after treatment with DOAC Remove® in apixaban samples. In rivaroxaban and dabigatran samples, the difference of D-dimer levels after treatment with DOAC Remove® was inferior to the acceptable bias of uncertainly measurement (100ng/ml).

Only two patients of 97 would have a change in interpretation of D-Dimer level with the threshold at 500 ng/ml. For one dabigatran sample, D-dimer level increased from 491 to 511 ng/ml and for one apixaban sample, he decreased from 564 to 482 ng/ml. For these patients, with age-appropriate threshold (850ng/ml and 610 ng/ml), the interpretation was negative before and after DOC Remove procedure and there was no clinical impact. Therefore, DOAC Remove® significantly removed the effect of DOAC on PT, aPTT and factor V level.

Endogen factors level a. Without DOACs:

We collected 47 and 65 plasmas from patients without DOAC for measuring respectively factor VIII and factor IX level. Factor FVIII level was decreased after treatment with DOAC Remove® in the group control. This difference was minimal (5%) and inferior to the acceptable bias of GFHT (18.8%) and had no clinical impact. For the FIX level, no significant changes were observed after sample treatment with DOAC Remove® in the group control (Table 3).

b. With DOAC :

After the action of DOAC Remove®, factor VIII level significantly increased from a median value of 80% to 96% (p<0.0001) in apixaban samples, from 82% to 96% (p<0.0001) in rivaroxaban samples and from 104% to 147% in dabigatran samples (p<0.0001) (Table 4).

After DOAC Remove® factor IX level significantly increased from a median value of 80% to 102% (p<0.0001) in apixaban samples, from 81% to 107% (p<0.0001) in rivaroxaban samples and from 69% to 117% in dabigatran samples (p<0.0001).

In consequence, DOAC Remove® significantly removed the effect of DOAC on factor VIII and IX level.

Von Willebrand factor assay a. Without DOACs:

In 29 plasmas from patients without DOAC, no significant changes were observed in VWF:Ag level and VWF:RCo level after sample treatment with DOAC Remove® in group control (table 3).

b. With DOAC:

For VWF:Ag level and VWF:RCo level, after the action of DOAC Remove®, no significant changes were observed in apixaban, rivaroxaban and dabigatran samples (Table 4).

Thrombophilia testing a. Without DOACs:

For group control, we collected 49 plasmas from patients without DOAC, among them 10 were treated by VKA, 9 had an inflammatory syndrome and 7 had PT superior to 14.6 seconds. No significant changes were observed after sample treatment with DOAC Remove® in the group control, except for free PS antigen level from 58 % to 57% before and after treatment with DOAC Remove® (p<0.01) (Table 3). Nevertheless, this difference was minimal, inferior to the acceptable bias of GHFT and had no clinical impact.

b. With DOAC:

After the action of DOAC Remove®, AT co-factor activity level was significantly reduced from a median from 118% to 93% (p<0.0001) in apixaban samples, from 124% to 101% (p<0.0001) in rivaroxaban samples. In dabigatran samples, AT level were similar after DOAC Remove® procedure (from 87% to 86%) and the difference had no clinical impact (Table 5).

After DOAC Remove®, with clotting method, PC level was significantly reduced from a median value of 126% to 117% (p<0.0001) in apixaban samples, from 139% to 116% (p<0.0001) in rivaroxaban samples and from 162% to 91% in dabigatran samples (p<0.05).

With chromogenic method, no significant changes were observed on PC level after DOAC Remove® procedure. After DOAC Remove®, PS activity level was significantly reduced from a median value of 129% to 100% (p<0.0001) in apixaban samples, from 150% to 99% (p<0.0001) in rivaroxaban samples and from 150% to 81% in dabigatran samples (p<0.0001).

For free PS antigen level, no significant changes were observed after DOAC Remove® procedure in apixaban samples and the difference in rivaroxaban and dabigatran sample was minimal, inferior to the acceptable bias of GHFT, and had no clinical impact (from 81 to 79 % in rivaroxaban samples and from 75 to 73 % in dabigatran samples). Activated charcoal significantly removed the effect of DOAC on AT level for apixaban and rivaroxaban, PC level with clotting method and PS activity level for the three DOAC tested.

DOAC has no impact in PC level with chromogenic method and free PS antigen level.

LA testing a. Without DOACs:

We collected 49 plasmas from patients without DOAC, among us 18 were LA positive and 31 were LA negative, for SCT and dRVVT. The interpretation of LA results did not change after treatment with DOAC Remove® in the group control. Furthermore, the difference between dRVVT and SCT before and after DOAC Remove® procedure was not significant in control group (table 3).

b. With DOAC:

DRVVT screen/confirm and SCT screen/confirm was performed in 33, 35 and 31 plasma samples from patients who received respectively apixaban, rivaroxaban and dabigatran.

With dRVVT, after treatment with DOAC Remove®, in rivaroxaban and dabigatran sample respectively 24 and 11 patients initially positive were found negative. False positive were eliminated with dRVVT in 86% (24/28) and 79% (11/14) of respectively rivaroxaban and dabigatran positive samples (table 6). SCT screen/confirm ratio was less affected by DOAC than dRVVT. In rivaroxaban samples, one patient of three initially positive patients was found negative after DOAC Remove® procedure. In dabigatran samples, the unique positive patient was negative after DOAC Remove® procedure.

Nevertheless, SCT screen significantly reduced after DOAC Remove® procedure (p<0.005).

The impact of apixaban on LA testing was small: 97% (32/33) were negative with dRVVT and SCT. No significant changes were observed after sample treatment with DOAC Remove® in apixaban sample.

DISCUSSION

We have shown that activated charcoal DOAC Remove® had no impact on plasma without DOAC and on analyses initially not influenced by DOAC. The differences observed in the results before and after DOAC Remove® procedure in the control group were not significative or inferior to the acceptable bias of GFHT and had no clinical impact (GFHT 2014). This suggests that there is no adsorption of coagulation proteins by DOAC Remove®.

However, the level of D-dimer showed a decreasing trend after DOAC Remove® procedure.

Even if this decrease would have no clinical impact in our study, we can wonder if activated charcoal does not interfere with the quantification of D-Dimer.

On patients who received apixaban, rivaroxaban and dabigatran, our study has shown that pre-treatment of citrated plasma with DOAC Remove® reduced DOAC levels. In previous studies, activated charcoal proved his efficacy in eliminating DOAC from spiked plasma or from patients plasma with charcoal powder [START_REF] Frans | Resolving DOAC interference on aPTT, PT, and lupus anticoagulant testing by the use of activated carbon[END_REF][START_REF] Vercruyssen | Resolving DOAC interference on antithrombin activity testing on a FXa based method by the use of activated carbon[END_REF], DOAC Remove® [START_REF] Kopytek | DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients[END_REF][START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF] and DOAC Stop™® [START_REF] Favaloro | Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects[END_REF][START_REF] Exner | Simple method for removing DOACs from plasma samples[END_REF][START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF][START_REF] Favresse | Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests[END_REF]. Residual concentration of DOAC were measured by high-performance liquid chromatography-coupled tandem mass spectrometry (HPL MS/MS) evidenced a small number of plasma with a detectable level of DOAC after activated charcoal, suggesting incomplete neutralization [START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF][START_REF] Slavik | Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban[END_REF].

Concerning routine assay, in our study, PT and aPTT were significantly reduced after DOAC Remove® procedure. This was in agreement with previous studies who used activated charcoal: on plasma spiked with apixaban and rivaroxaban, DOAC Stop™ restoring aPTT results of the original value [START_REF] Exner | Simple method for removing DOACs from plasma samples[END_REF], and in patients who received apixaban and rivaroxaban, PT and aPTT were decreased after treatment by DOAC Stop™ [START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF] and DOAC Remove® [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF].

The impact of DOAC on fibrinogen levels depends on the reagent used [START_REF] Van Blerk | Influence of dabigatran and rivaroxaban on routine coagulation assays. A nationwide Belgian survey[END_REF][START_REF] Lindahl | Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays[END_REF]. In our study, we tested two reagents with different amounts of thrombin (100UI/ml Q.F.A Thrombin vs 35UI/ml fibrinogen C). There was no impact of dabigatran on the reagent containing enough excess thrombin. However, the reagent containing less thrombin may be affected by dabigatran.

On this reagent, the DOAC Remove® procedure may eliminate the interference of dabigatran.

Factor V, VIII and IX were significantly higher after DOAC Remove® procedure in our study. The study that used DOAC Stop™ showed an increase of factor VIII and FIX levels after treatment by DOAC Stop™ on plasma spiked with rivaroxaban (29) and on plasma from patients who received apixaban and rivaroxaban [START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF]. With DOAC Remove®, a study showed a significant increase of intrinsic factor levels in patients on DOAC anti-Xa therapy [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF]. With more patients and by testing the dabigatran, we confirm these results with DOAC Remove®.

Our study has shown that DOAC have no impact on VWF:AG and VWF: RCO level and DOAC Remove® procedure did not modify results. Our experiments confirmed that activated charcoal only neutralizes DOAC and does not adsorb VWF. To the best of our knowledge, this study is the first evaluating the effect of DOAC interference in von Willebrand's disease testing and the neutralization by DOAC Remove®.

Thrombophilia testing is necessary to detect and characterize the different types of PS and PC deficiency (quantitative and qualitative). Thrombophilia assessment can help to adapt an anticoagulant treatment in prevention and to perform family investigations [START_REF] Khider | Inherited Thrombophilia in the Era of Direct Oral Anticoagulants[END_REF]. Our study confirms the absence of impact of DOAC on free protein S antigen and protein C activity by chromogenic method [START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays[END_REF][START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays[END_REF]. Indeed, previous study with activated charcoal procedure showed no impact on these analyses on DOAC spiked plasma [START_REF] Exner | Simple method for removing DOACs from plasma samples[END_REF] and on plasma from patients on DOAC therapy [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Favresse | Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests[END_REF].

We have completed the thrombophilia testing with protein S activity level and protein C level by chronometric method. We have shown that after treatment with DOAC Remove®, protein S activity level and protein C level measured with chronometric method were decreased. With Werfen reagents, our results are similar to the tests performed with Stago reagents [START_REF] Favre | Direct oral anticoagulant neutralization by activated charcoal DOAC-Remove for thrombophilia screening[END_REF].

In Favre study, a decrease in PS activity after DOAC Remove® in one sample reveals an authentic protein S deficiency confirmed by genotype, showing the interest of DOAC Remove® [START_REF] Favre | Direct oral anticoagulant neutralization by activated charcoal DOAC-Remove for thrombophilia screening[END_REF].

AT co-factor activity assay is affected by DOAC but depend on the assay used and the nature of DOAC and the substrate used (FIIa or FXa chromogenic substrate). DOAC Remove® had non-impact on thrombin based anti-thrombin activity in apixaban and rivaroxaban sample [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF]. With FXa based (Innovance), DOAC Stop™ treatment induced a decrease of AT levels in patients taking rivaroxaban and apixaban. In our study, on patients taking rivaroxaban and apixaban, we also observed that antithrombin level were decreased with anti-Xa chromogenic substrate (Werfen) after DOAC Remove®.

The major impact of DOAC interference concerns the lupus anticoagulant interpretation. On spiking plasma sample with DOAC, initially negative LA assay became positive due to interference of rivaroxaban and became negative again after action of activated charcoal powder [START_REF] Frans | Resolving DOAC interference on aPTT, PT, and lupus anticoagulant testing by the use of activated carbon[END_REF]. Likewise, on spiking plasma sample with rivaroxaban, DOAC Stop™ procedure were effective in eliminating LA false-positive [START_REF] Favaloro | Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa[END_REF][START_REF] Kesel | Direct oral anticoagulant adsorption: Impact on lupus anticoagulant testing-Review of the literature and evaluation on spiked and patient samples[END_REF]. On sample from patients taking DOAC, DRVVT screen and confirm decreased and there were less positive-LA test after DOAC Remove® [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF] and DOAC Stop™ procedure [START_REF] Platton | Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban[END_REF][START_REF] Favresse | Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests[END_REF][START_REF] Ząbczyk | The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants[END_REF]. We have shown that DOAC Remove® allowed 86% neutralization of false positive on DRVVT testing in rivaroxaban samples and 79% in dabigatran samples. According to the other studies, we confirm the efficacy of pre-treatment of citrated plasma with DOAC Remove® to avoid the interference of DOAC on dRVVT testing. Previous study showed that there was less influence in total in aPTT based LA assay in spiking samples [START_REF] Hillarp | Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays[END_REF][START_REF] Flieder | Interference of DOACs in different DRVVT assays for diagnosis of lupus anticoagulants[END_REF] and in patients taking with rivaroxaban, apixaban and dabigatran [START_REF] Gay | Lupus anticoagulant diagnosis in patients receiving direct oral FXa inhibitors at trough levels: A real-life study[END_REF][START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF][START_REF] Martinuzzo | Frequent Falsepositive results of lupus anticoagulant tests in plasmas of patients receiving the new oral anticoagulants and enoxaparin[END_REF]. Our study showed a low interference of DOAC with SCT test (aPTT based test) in rivaroxaban, dabigatran and apixaban samples.

Spiking experiment showed that apixaban has less effect on LA assay [START_REF] Kesel | Direct oral anticoagulant adsorption: Impact on lupus anticoagulant testing-Review of the literature and evaluation on spiked and patient samples[END_REF] and on patient samples, apixaban has less pronounced effect on the dRVVT screen [START_REF] Jourdi | Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants[END_REF][START_REF] Favaloro | Laboratory testing for lupus anticoagulant (LA) in patients taking direct oral anticoagulants (DOACs): potential for false positives and false negatives[END_REF]. Likewise, in our study, we did not observe any impact of apixaban on LA testing. In the case of LA testing positive after DOAC Remove® procedure (one patient on apixaban, four patients on rivaroxaban, and three patients on dabigatran), it is difficult to differentiate a positive result from persistent interference. Even at plasma concentration below the limit of quantification, interference by DOAC is always possible [START_REF] Ratzinger | Lupusanticoagulant testing at NOAC trough levels[END_REF]. In practice, with DOAC Remove® procedure, we can reduce false-positive LA in patients who received DOAC without stopping anticoagulation and we can exclude LA with a negative result. However, for patients whose LA testing remains positive after DOAC Remove® procedure, we cannot eliminate the interference of very low non-detectable doses.

DOAC Remove® can also be used during the switch DOAC-heparin.For heparin monitoring, Anti-Xa activity of heparin is confounded by the persistent activity of DOAC, ever after stopping DOAC therapy [START_REF] Plum | The Relationship between the Initial Antifactor Xa Measurement and the Duration of Direct Oral Anticoagulant Influence in Patients Transitioning to Heparin[END_REF]. In our experiments, in patients on heparin, the anti-Xa level did not change after DOAC Remove® procedure. These data confirm that DOAC Remove® reduced DOAC concentration without affecting anti Xa level in plasma [START_REF] Cox-Morton | A diagnostic solution for haemostasis laboratories for patients taking direct oral anticoagulants using DOAC-Remove[END_REF][START_REF] Favre | Direct oral anticoagulant neutralization by activated charcoal DOAC-Remove for thrombophilia screening[END_REF] like DOAC Stop™ [START_REF] Exner | Effect of an activated charcoal product (DOAC Stop TM ) intended for extracting DOACs on various other APTT-prolonging anticoagulants[END_REF]. The DOAC Remove® procedure eliminated the anti-Xa activity of the AOD without affecting the heparin ones. This allows the adjustment of the patient's heparin treatment.

Further studies are needed to validate this application in patients with heparin after stopping DOAC.

Our study has some limitations. Interference from very low levels of undetectable circulating drug cannot be ruled out. We cannot conclude the presence of a LA if the test remains positive after DOAC Remove® procedure. In addition, this method should be validated in each laboratory with its own reagents and its own automated coagulometers. Furthermore, all plasmas collected were not tested for all the assays. We tested an average of 35 plasmas of each DOAC per assay.

To conclude, our study validated DOAC Remove® to eliminate the interference DOAC on chronometric test and thrombophilia testing. Our experiments have shown that after treatment with activated charcoal, DOAC concentration was below detection limits and the different assays were adjusted (the factor V, VIII and IX, AT level, PC level with clotting method and PS activity level, LA ratio). DOAC Remove® could be a solution to allow routine and thrombophilia testing for patient taking DOAC, without stopping anticoagulation.

TABLES

  stress ! Merci à Kahina et à Thomas pour tous nos fous rires ! INTRODUCTION .................................................................................................................... MATERIAL AND METHODS: ............................................................................................... 1. Plasma samples.............................................................................................................. 2. Treatment with activated charcoal DOAC Remove® ................................................... 3. DOAC measurement ..................................................................................................... 4. Routine and specialized coagulation assays .................................................................. 5. Statistical analysis ......................................................................................................... RESULTS ................................................................................................................................. 1. Effect of DOAC Remove® on DOAC levels................................................................ 2. Effect of DOAC Remove® on heparin anti-Xa ............................................................ 3. Routine assay................................................................................................................. 4. Endogen factors level .................................................................................................... 5. Von Willebrand factor assay .......................................................................................... 6. Thrombophilia testing ................................................................................................... 7. LA testing ...................................................................................................................... DISCUSSION .......................................................................................................................... TABLES ................................................................................................................................... REFERENCES ......................................................................................................................... SERMENT D'HIPPOCRATE .................................................................................................. RESUME Contexte : Les anticoagulants oraux directs sont de plus en plus prescrits en première intention dans la maladie thromboembolique veineuse. Cependant, ils ont un impact significatif sur les résultats des tests de coagulation de routine et le bilan de thrombophilie. Pour éviter une mauvaise interprétation de ces tests, il est nécessaire de lever les interférences des AOD sans interrompre le traitement du patient à risque de thrombose. Objectif : Le but de notre étude est d'évaluer l'efficacité du charbon activé DOAC Remove® (5-Diagnostics, Bâle, Suisse) pour neutraliser l'apixaban, le rivaroxaban et le dabigatran dans le plasma du patient afin de lever leurs interférences analytiques. Matériel et méthodes : Les plasmas de patients proviennent du laboratoire du Centre Hospitalier Universitaire de Nice et le Centre Hospitalier de Monaco. Les analyses ont été réalisées avant et après incubation avec le DOAC Remove®. Les concentrations des AOD ont été mesurées à l'aide d'un inhibiteur direct spécifique de la thrombine ou d'un test anti-Xa avec des calibrations spécifiques. Les tests de coagulation de routine (TP, TCA et facteur(F)V, FVIII, FIX, fibrinogène et D-dimères), les tests explorant le facteur von Willebrand (vWF :Ag et vWF :GP1bR), ainsi que ceux du bilan de thrombophilie [antithrombine (test basé sur l'inhibition du FXa), Protéine C (activité amidolytique et activité anticoagulante), Protéine S (antigène PS libre et activité anticoagulante)], et la recherche d'anticoagulant lupique (LA) [temps de coagulation de la silice (SCT), temps de coagulation du venin de Russell dilué (dRVVT)] ont été réalisés avec des réactifs de Werfen (Bedford, MA, USA). Résultats : Nous avons évalué un total de 713 échantillons de plasma, dont 152 traités par dabigatran, 131 sous rivaroxaban, 134 sous apixaban, comparés à 420 patients ne prenant pas d'AOD (dont 35 patients sous coumadine, 18 patients sous Héparine Non Fractionnée et 10 sous Héparine de Bas Poids Moléculaire). Chez les patients non traités par AOD, le DOAC Remove® n'a pas eu d'impact sur les résultats des tests d'hémostase et aucune différence d'interprétation des résultats n'a été observée pour la recherche de LA après DOAC Remove®. Dans les plasmas de patients traités par AOD, DOAC Remove® a neutralisé les trois AOD testés, avec des taux inférieurs à la limite de quantification après la procédure. Nous avons observé une correction significative des allongements du TP et du TCA induits par les AOD, ainsi que de la sous-estimation des activités des FV, FVIII, FIX. De même, nous avons observé la correction de la surestimation des activités anticoagulantes PC et PS et de l'activité cofacteur de l'antithrombine pour les plasmas sous rivaroxaban et apixaban. Pour la recherche de LA, DOAC Remove® a éliminé respectivement 86% et 79 % des patients faux positifs sous rivaroxaban et dabigatran. L'impact des AOD sur le SCT était moins fort. Un patient sur les trois positifs sous rivaroxaban et le patient positif sous dabigatran se sont négativés après DOAC Remove®. Sous apixaban, seulement un patient positif en dRVVT et SCT est resté identique après DOAC Remove®. Conclusion : Le DOAC Remove® n'a eu aucun effet sur les résultats des tests obtenus dans le plasma de patients non traités et de patients sous héparine ou AVK. Il a éliminé l'apixaban, le rivaroxaban et le dabigatran du plasma des patients traités, permettant une mesure précise des tests de coagulation de routine et du bilan de thrombophilie. Ces résultats confirment les études sur le DOAC Remove® avec d'autres réactifs. ABSTRACT Background: Direct oral anticoagulants (DOACs) have a significant impact on various coagulation test results. As the correct interpretation of these tests is mandatory to prevent misclassification and subsequent clinical consequences, withholding treatment could be necessary, with the associated risk of thrombosis. Aims: To evaluate the performance of the activated carbon DOAC Remove® (5-Diagnostics, Basel, Switzerland) in extracting DOACs from plasma samples and its effect on various routine coagulation test and thrombophilia results. Materials and Methods: Plasmas from patients were collected in the university hospital center of Nice and of hospital center of Monaco. Tests were performed before and after incubation with DOAC Remove®. DOACs were measured using either a specific direct thrombin inhibitor or an anti-Xa assay with specific calibrations. Routine coagulation tests (PT, aPTT, and factor(F)V, FVIII:C, FIX, fibrinogen, and D-dimer) and von Willebrand testing were performed, as well as thrombophilia panel [antithrombin, protein C (PC, chromogenic and clotting assays), PS (free PS antigen and clotting assays)], and lupus anticoagulant (LA) panel [silica clotting time (SCT), and dilute Russell venom clotting time (dRVVT)]. All assays used reagents from Werfen (Bedford, MA, USA).

  Russel viper venom time GFHT: Groupe Français d'étude sur l'Hemostase et la Thrombose LA: Lupus anticoagulant LLOD: Lower limit of detection LLOQ: Lower limit of quantification LMWH: Low molecular weight heparin PC

Factor assay :

 assay Factor V, VII, IX activities were measured using human plasmas immunodepleted of factors (Factor deficient plasma, HemosIL®), based on aPTT on IL coagulation System (SynthASil, HemosIL®). Thrombophilia testing: Determination of antithrombin (AT) used a chromogenic assay anti-Xa based (Liquid Antithrombin, HemosIL®). Proteins S and C were measured by different methods: Clot-based (ProClot, HemosIL®) and chromogenic (Protein C, HemosIL®). Free protein S antigen (Free Protein S, HemosIL®) used an immunologic method and Protein S activity (HemosIL®) a chronometric method. LA detection: Two reagents were used: dRVVT Screen/Confirm (HemosIL®) and Silica Cloting Time (SCT Screen and SCT confirm, HemosIL®). The normalized ratio Screen/Confirm was calculated only if Screen was positive (dRVVT Screen >1.20 and SCT Screen > 1.16). LA DRVVT positivity was defined as a ratio screen/confirm > 1.20 and LA SCT positivity is defined as a ratio screen/confirm > 1.16. Von Willebrand's factor assay: Von Willebrand Factor Antigen (VWF:Ag) was quantified by immunoassay (HemosIL®) and von Willebrand Factor Ristocetin Cofactor activity (VWF:GP1bR) by a chemiluminescent immunoassay (HemosIL®) on the ACL AcuStar™.

Jourdi,

  et al. have shown that DOAC Remove® corrected DOAC interference with DRVVT assays in 76, 85 and 95% of patients on apixaban, rivaroxaban and dabigatran respectively (26). Platton, et al. have shown that DOAC Stop™ corrected DOAC interference with DRVVT assays in 86% of patients on rivaroxaban (31). In our study, DOAC Remove® significantly reduced the number of false positive of LA interpretation in patients on DOAC.

Table 1 :

 1 Reagents used on the ACL TOP 700 analyzer (all were from Werfen) i.e. brand, assay principles, and units (for details, see Subjects, Materials and Methods section). All assays were performed on the ACL TOP 700 analyzer, except for the VWF:GP1bR assay on the AcuStar analyzer.

	Parameters

Table 2 :

 2 Plasma levels (in ng/mL) of dabigatran, rivaroxaban and apixaban measured before and after incubation with the DOAC Remove® activated carbon. Tests results are expressed as the median values with their ranges. N denotes the number of evaluated samples. The reported lower limits of quantitation (LLOQ) and of detection (LLOD) of the techniques are those indicated by the manufacturer of the reagents in the corresponding package inserts.

	Parameter	n	Before	After	p	Mean Bias (95% CI), % Desirable Bias* (%)
	PT (sec)	118	14.2 (10.3-72.7)	14.1 (10.1-70.5)	0.0067	-0.3 (-2.1 +1.5) ; -1.1%	2.0
	(percentage activity)		73.0 (11-17)	73.5 (11-121)	0.0057	+0.7
	INR		1.21 (0.89-5.72)	1.20 (0.88-5.55)	0.0057	-0.03
	aPTT (sec)	118	32.9 (24.8-124.9)	32.8 (25.3-149.3)	NS (0.10)	ND
	(ratio)		1.09 (0.82-4.14)	1.09 (0.84-4.94)	NS (0.13)	ND
	Fibrinogen (g/L) R1	118	3.91 (0.93-10.90)	3.91 (0.78-11.92)	NS (0.50)	ND	4.8
	R2	70	3.36 (1.01-8.43)	3.28 (1.09-9.06)	NS (0.24)	ND
	D-dimer (ng/mL)	116 1,096 (127-21915) 1,076 (108-21720)	<0.0001	-15 ; -9.4%	8.82
	Factor V (IU/dL)	116	112.1 (9.3-169.2)	107.2 (9.5-169.2)	<0.0001	-6.2 (-20.6 +6.9) ; -6.5%	ND
	Factor VIII:C (IU/dL)	47	88.7 (<1-148.7)	84.6 (<1-139.9)	<0.0001	-4.2	4.9
	Factor IX (IU/dL)	65	84.1 (17.5-140.4)	82.2 (15.6-136.0)	NS (0.19)	ND
	VWF:GP1bR (IU/dL)	29	70.0 (23.3-397.0)	69.8 (24.3-385.5)	NS (0.23)	ND
	VWF:Ag (IU/dL)	29	89.6 (29.1-234.9)	88.3 (28.8-233.5)	NS (0.20)	ND
	Antithrombin (IU/dL)	39	94.0 (44.0-124.0)	91.0 (46.0-124.0)	NS (0.27)	ND	4.0
	Protein C chromogenic (IU/dL)	45	88.0 (27.0-167.0)	91.0 (27.0-165.0)	NS (0.10)	ND
	Protein C clotting (IU/dL)	43	105.9 (4.9-236.0)	105.0 (4.6-256.0)	NS (0.75)	ND
	Protein S clotting (IU/dL)	39	74.1 (9.4-128.5)	76.1 (10.0-136.4)	NS (0.15)	ND
	Protein S free antigen (IU/dL)	39	58.4 (10.0-110.6)	57.3 (11.3-104.6)	0.0027	-1.5 (-6.9 +3.8) ; -2.4%	ND
	Anti-Xa UFH, LMWH (IU/mL)	28	0.23 (0.09-0.88)	0.21 (0.04-0.82)	0.001	-0.01 (0.08 +0.05); -4.6%	ND
	dRVVT Screen (NR)	49	1.05 (0.70-3.26)	1.05 (0.80-3.09)	NS (0.06)	ND
	n with NR>1.20		18/49	16/49
	dRVVT Confirm (NR)	49	0.93 (0.80-2.84)	0.97 (0.82-1.46)	0.0004	0.03	ND
	dRVVT Screen/Confirm (NR)*		1.45 (0.71-2.89)	1.55 (1.01-2.63)	NS (0.31)	ND
	n with NR>1.20		16/18	16/16
	SCT Screen (NR)	49	1.06 (0.68-4.65)	1.04 (0.67-5.15)	0.0018	0.02	ND
	n with NR>1.16		20/49	19/49
	SCT Confirm (NR)	49	1.05 (0.79-2.25)	1.06 (0.79-2.14)	NS (0.84)	ND
	SCT Screen/Confirm (NR)*		1.72 (0.64-3.56)	1.90 (0.66-3.87)	0.004	0.01	ND
	n with NR>1.16		17/20	17/19	NS
	LA+ profiles (n)		17	17	NS	N Appl

Table 3 :

 3 Test results of routine and specialist coagulation parameters measured in the plasma of patients not treated with any DOAC before and after incubation with the DOAC Remove® activated carbon. Tests results are expressed as the median values with their ranges. N indicate the number of samples evaluated for each parameter. For details, see patients, Materials, and Methods section. N Appl: not applicable; ND: not determined; NS: not significant dRVVT: dilute Russsell Viper Venom Time; SCT: silica clotting time; LA: lupus anticoagulant; IU: international unit; PT: prothrombin time; aPTT: activated partial thromboplastin time; VWF: von Willebrand factor; CI: confidence interval; NR: normalized ratio. *: only performed if dRVVT or SCT screen was positive i.e. above the cut-off values of 1.20 and 1.16 respectively. LA positive profile was defined by a dRVVT and/or a SCT screen-to-confirm NR above the cut-off values.

			Dabigatran			Rivaroxaban				Apixaban
		n	Before	After	p	n	Before	After	p	n	Before	After	p
	PT (sec)	33	14.7 (12.1-26.2)	12.7 (10.2-18.2)	<0.0001	32 21.0 (12.6-42.6)	12.5 (10.5-14.7)	<0.0001	32	15.3 (10.1-50.7)	12.9 (9.7-28.5)	<0.0001
	(%)		69 (34-91)	86 (52-119)	<0.0001		44 (19-86)	87 (69-114)	<0.0001		66 (16 -111)	84 (30-129)	<0.0001
	INR		1.25 (1.04-2.17)	1.09 (0.88-1.53)	<0.0001		1.76 (1.08-3.44)	1.08 (0.91-1.25)	<0.0001		1.30 (0.88-4.06)	1.10 (0.84-2.35)	<0.0001
	aPTT (sec)	33	44.0 (28.6-76.0)	28.4 (21.5-35.3)	<0.0001	32 35.6 (27.4-49.5)	30.1 (24.3-38.4)	<0.0001	32	32.2 (28.5-44.5)	30.3 (26.8-37.2)	<.00001
	(ratio)		1.46 (0.95-2.52)	0.94 (0.71-1.17)	<0.0001		1.18 (0.91-1.64)	1.00 (1.08-1.23)	<0.0001		1.07 (0.94-1.47)	1.00 (0.89-1.23)	<0.0001
	ratio >1.20		n=26 (76.5%)	n=0 (0%)	<0.0001		n=14 (43.8%)	n=2 (6.3%)	0.0006		n=6 (18.8%)	n=3 (9.4%)	NS (0.28)
	Fg (g/L) R1	33	3.35 (1.95-5.65)	3.40 (2.01-6.11)	NS	32	3.88 (2.47-9.63)	3.74 (2.30-9.63)	NS	32	3.98 (1.86-7.74)	3.78 (2.17-5.51)	NS
	R2	30	2.37 (1.38-3.94)	2.87 (1.86-4.16)	(0.82)	30	3.18 (1.86-6.08)	2.96 (1.80-6.09)	0.006	31	3.07 (1.83-7.37)	3.18 (2.14-7.87)	0.06
					0.0002
	D-dimer (ng/mL)	32	363 (92-12900)	349 (92-12900)	0.0001	32	604 (81-3776)	548 (81-3520)	<0.0001	32	773 (116-25443)	704 (102-26629)	<0.0001
	FV (IU/dL)	33	78 (33-128)	85 (55-144)	<0.0001	32	68 (26-144)	94 (55-177)	<0.0001	31	85 (21-117)	92 (33-122)	<0.0001
	FVIII:C (IU/dL)	37	104 (26-242)	147 (45-447)	<0.0001	38	82 (42-209)	96 (54-307)	<0.0001	35	80 (43-136)	96 (51-145)	<0.0001
	FIX (IU/dL)	37	69 (17-203)	117 (61-245)	<0.0001	38	81 (49-133)	107 (74-199)	<0.0001	35	80 (47-135)	102 (97-150)	<0.0001
	VWF:GP1bR	24	217 (122-436)	219 (109-443)	NS	23	218 (109-526)	209 (122-487)	NS	22	256 (74-420)	250 (91-408)	NS
	(IU/dL)				(0.84)
	VWF:Ag (IU/dL)	22	222 (134-470)	223 (131-440)	NS	23	228 (152-670)	226 (152-627)	NS	23	229 (129-524)	230 (123-520)	NS

Table 4 :

 4 Routine coagulation tests results and factor levels, measured in the plasma of patients treated with DOAC before and after incubation with the DOAC Remove® activated carbon. Tests results are expressed as the median values with their ranges. For details, see Patients, Materials, and Methods part. NS: not significant

	Dabigatran	Rivaroxaban	Apixaban

Table 5 :

 5 Test results of coagulation inhibitors i.e., antithrombin (AT), protein C (PC) and protein S (PS) expressed in IU/mL, measured in the plasma of patients treated with DOAC before and after incubation with the DOAC Remove® activated carbon. Tests results are expressed as the median values with their ranges. For details, see Patients, Materials, and Methods part. NS: not significant

Table 6 :

 6 LA test results, measured in the plasma of patients treated with DOAC before and after incubation with the DOAC Remove® activated carbon. Tests results are expressed as the median values with their ranges. For details, see Patients, Materials, and Methods part. NS: not significant * Calculated only if screen positive

		Dabigatran (n=31)		Rivaroxaban (n=35)		Apixaban (n=33)
		Before	After	P	Before	After	p	Before	After	p
	ratio n>1.20 (%)	1.55 (0.93-2.49) n=26 (83.9%)	1.00 (0.75-1.39) n=2 (6.5%)	<0.0001	1.15 (0.88-2.21) n=11 (31.4%)	0.99 (0.82-2.05) n=1 (2.9%)	<0.0001	1.07 (0.87-1.70) n=8 (24.2%)	1.00 (0.79-1.68) n=4 (12.1%)	<0.0001
	Screen (NR) n>1.20 (%)	2.37 (1.18-3.75) n=30 (96.8%)	1.11 (0.57-1.37) n=10 (32.3%)	<0.0001	2.01 (1.13-3.79) n=34 (97.1%)	0.98 (0.73-3.24) n=6 (17.2%)	<0.0001	1.32 (0.76-3.84) n=22 66.6%)	1.00 (0.65-1.78) n=3 (6.1%)	<0.0001
	Confirm (NR) n>1.20 (%)	2.00 (1.25-3.62) n=31 (100%)	1.06 (0.71-1.92) n=5 (16.1%)	<0.0001	1.39 (1.04-2.06) n=29 (82.9%)	0.96 (0.73-1.19) n=0 (0%)	<0.0001	1.46 (1.00-3.12) n=28 (84.8%)	0.95 (0.72-1.33) n=2 (6.1%)	<0.0001
	Screen/Confirm (NR)* n>1.20	1.14 (0.82-1.69) n=14/30	1.05 (0.62-1.26) n=3/10	<0.0001	1.41 (0.78-2.21) n=28/34	1.08 (0.94-1.87) n=4/6	<0.0001	0.89 (0.63-1.75) n=1/22	1.16 (0.88-1.75) n=1/3	<0.0001
	LA Positive Profile (n, %)	14/31 (45.2%)	3/31 (9.7%)	0.004	28/35 (80.0%)	4/35 (11.4%)	<0.0001	n=1/33 (3.0%)	n=1/33 (3.0%)	NS
	Screen (NR) n>1.16 (%)	1.60 (0.81-3.43) n=27 (87.1%)	0.96 (0.56-1.47) n=1 (3.2%)	<0.0001	1.15 (0.81-3.55) n=14 (40.0%)	0.97 (0.72-3.24) n=5 (14.3%)	<0.0001	1.05 (0.68-2.41) n=7 (21.2%)	0.97 (0.64-1.91) n=4 (12.1%)	<0.0005
	Confirm (NR) n>1.16 (%)	2.01 (1.30-4.25) n=31 (100%)	1.09 (0.81-2.11) n=14 (45.2%)	<0.0001	n=26 (74.3%) 1.23 (0.96-2.79)	n=9 (25.7%) 0.98 (0.76-1.85)	<0.0001	n=18 (54.5%) 1.18 (0.84-1.95)	n=8 (24.2%) 1.01 (0.72-1.49)	<0.0001
	Screen/Confirm (NR)* n>1.16	0.82 (0.39-1.46) n=1/27	0.98 n=0/1	0.0035	0.97 (0.62-2.55) n=3/14	0.97 (0.68-2.65) n=2/5	<0.0001	0.86 (0.60-2.05) n=1/7	0.90 (0.64-2.02) n=1/4	<0.0001
	LA Positive Profile (n, %)	1/31 (3.2%)	0/31 (0%)		3/35 (8.6%)	2/35 (5.7%)		1/33 (3.0%)	1/33 (3.0%)