Introduction Directs oral anticoagulants (DOACs) can interfere with coagulation assays, especially in thrombophilia workup. To avoid these interferences, a new device, DOAC Filter, allows the removal of DOACs from citrated plasma. This study aims to confirm that DOAC Filter efficiently removes DOACs and to ascertain that coagulation assays are not impacted by filtration. Methods Directs oral anticoagulants Filter (Diagnostica Stago, France) is a filtration cartridge in which DOAC molecules are trapped by noncovalent binding, while plasma is filtered through a solid phase. Normal pool plasma (NPP) spiked with DOACs up to 300 ng/mL, with dabigatran etexilate (n = 27), rivaroxaban (n = 35), apixaban (n = 33), and edoxaban (n = 27) or 120 ng/mL for betrixaban (n = 4), and 18 plasma's samples from DOAC‐treated patients were used to assess efficacy. The potential impact of DOAC Filter on coagulation assays was evaluated with NPP and plasma's samples from positive and negative lupus anticoagulant (LA) patients. Results Directs oral anticoagulants concentrations measured after filtration were below the limit of detection (LoD) of DOAC‐specific assays for all plasmas tested, except for one apixaban plasma sample, with postfiltration concentration slightly higher than anti‐Xa assay LoD (25.1 ng/mL). Coagulation assays results varied between −4 and +8% after filtration and between −6 and +8% for LA plasmas. Such limited variations are not expected to have any clinical impact. Conclusion Directs oral anticoagulants Filter efficiently removes DOACs from plasma and achieves concentrations below DOAC‐specific assays LoD, except in the case of one apixaban sample. The integrity of plasma is respected, and the cartridge seems not to impact LA diagnosis.

Introduction Andexanet alfa (AnXa) was developed for anticoagulant effect reversal of direct factor Xa inhibitors (DXaI) (apixaban, rivaroxaban, edoxaban) in emergency situations. Regular anti‐Xa assays are not suitable to evaluate anti‐Xa activity after AnXa administration because of the high sample dilution resulting in the AnXa‐DXaI dissociation which gives inaccurately high DXaI measured concentrations. This study aimed at developing dedicated STA‐Liquid anti‐Xa test set‐ups for accurately measuring DXaI after reversal with AnXa. Methods Modified anti‐Xa test set‐ups, with reduced sample dilution, were developed to overcome regular assays limitations and to improve measured accuracy with results comparable to Portola microplate reference method used in clinical studies. Both regular and optimized assays were used to measure DXaI concentration in AnXa‐containing samples. Quality controls, normal pooled plasma spiked with five DXaI and three AnXa concentrations, samples from DXaI‐treated patients spiked with AnXa and ex vivo healthy volunteers having received both DXaI and AnXa were used. Results The lower limit of quantitation of optimized anti‐Xa assays was <10 ng/mL with CVs ≤10%. DXaI samples containing 300 ng/mL and 1 µmol/L AnXa resulted in DXaI residual concentrations of 29‐72 ng/mL depending on the DXaI (76%‐90% reversal), compared to 20‐28 ng/mL with reference method (92%‐94% reversal) and 135‐165 ng/mL with regular assays (about 50% reversal). Conclusion Modified test set‐ups are automated alternative to reference method with improved precision and reproducibility. They can be run in all laboratories where regular anti‐Xa assays are performed using commercially available reagents.

Edoxaban is a reversible orally active factor Xa inhibitor approved in Japan for Venous ThromboEmbolism (VTE) prophylaxis in major orthopedic surgery and submitted for approval in multiple markets for Stroke Prevention in non-valvular Atrial Fibrillation (SPAF) and VTE treatment and recurrence prevention. Although routine monitoring is not required, determination of anti-Xa activity with results expressed in edoxaban plasma concentration may be helpful in some special clinical settings such as urgent invasive procedures or in cases of bleeding. We have developed a specific, automated, user-friendly assay for measuring plasma edoxaban-related anti-Xa activity using the STA® Liquid Anti-Xa with an edoxaban dedicated test set-up, along with specific edoxaban calibrator and control sets, namely STA® Edoxaban Calibrator and STA® Edoxaban Control, on the STA® line analyzers. These calibrator and control sets are freeze-dried in vitro edoxaban spiked citrated plasmas. Test results are expressed in ng/mL of edoxaban. Assay performances including Limit of Blank (LOB), Lower Limit of Detection (LLOD) both according to CLSI EP-17-A guideline, Lower Limit of Quantification (LLOQ), Upper Limit of Quantification (ULOQ) according to CLSI EP6-A guideline, with and without automated re-dilution of plasma sample, and within and between-run reproducibility have been determined. Anti-Xa assay results were compared to those obtained with Mass Spectrometry Liquid Chromatography (LC-MS) reference method to evaluate assay recovery. All study assays were performed using freeze-dried of frozen in vitro edoxaban-spiked citrated plasma samples. Main potential interferences, i.e., hemoglobin, non-conjugated bilirubin, and lipemia, have been assessed. Assay performance results are summarized in Table I. Table I: Main Edoxaban assay performances as determined during test development Parameter Results obtained with prototype reagent batch and test set-up LOB 10 ng/mL LLOD 15 ng/mL LLOQ (preliminary estimation) 20 ng/mL ULOQ Without sample re-dilution 150 ng/mL With sample re-dilution 450 ng/mL Reproducibility Within run (n = 21) Freeze-dried controls 40 ng/mL ≤ 4.5% 120 ng/mL ≤ 6.0% Frozen spiked samples 50 ng/mL ≤ 7.1% 100 ng/mL ≤ 4.9% 200 ng/mL ≤ 4.3% 350 ng/mL ≤ 4.0% Between run (n = 10) Freeze-dried controls 40 ng/mL ≤ 7.0% 120 ng/mL ≤ 3.6% Frozen spiked samples 50 ng/mL ≤ 5.0% 100 ng/mL ≤ 4.0% 350 ng/mL ≤ 4.6% Recovery(freeze-dried samples) 40 ng/mL 87.4% 120 ng/mL 101.9% Edoxaban calibrator and control stability Calibrators Onboard 4 hours Controls Onboard 24 hours +2 – 8°C 7 days Interferences Hemoglobin None up to 1 g/L Non-conjugated bilirubin None up to 200 µM Lipemia None up to 2.5 g/L (as Intralipid® concentration) In conclusion, the proposed edoxaban assay developed using STA® Liquid Anti-Xa reagent with a dedicated test set-up and specific STA® Edoxaban Calibrator and STA® Edoxaban Control sets allows an accurate, reproducible, automated, and user-friendly, edoxaban plasma concentration determination. Further studies are required to confirm assay performance in ex vivo samples. Disclosures Herve: Diagnostica Stago: Employment. Beaufils:Diagnostica Stago: Employment. Kochan:Daiichi Sankyo: Employment. He:Daiichi Sankyo: Employment. Depasse:Diagnostica Stago: Employment.

Introduction: The anticoagulants rivaroxaban and apixaban inhibit Factor Xa (FXa) activity and are effective therapeutic agents for the prevention and treatment of thromboembolism. Andexanet alfa (andexanet) is the only specific reversal agent approved for anticoagulation reversal in patients presenting with life-threatening bleeding treated with rivaroxaban or apixaban. Anti-FXa assays are functional assays used for measuring direct and indirect FXa inhibitor levels in plasma. Current commercially available anti-FXa assays are not suitable for accurately measuring anti-FXa activity in patient samples in the presence of andexanet due to the high sample dilutions (e.g.,1:44 with STA-Liquid Anti-Xa), which causes dissociation of the inhibitor from andexanet (due to reversible binding). This results in substantial underestimation of the reversal activity of andexanet, and in some cases with erroneously elevated anti-FXa levels in patient samples following andexanet treatment. Therefore, we modified the current anti-FXa assays to measure apixaban and rivaroxaban anti-FXa levels in the presence of andexanet. Methods: The modified anti-FXa assays were performed on STA®-Compact analyzer using reagents from STA-Liquid Anti-Xa assay kits (Stago). Andexanet was provided as a 10 mg/mL frozen stock. Plasma samples with FXa inhibitor in the presence or absence of andexanet were prepared using Stago Calibrator 1 and pooled normal human plasma (Precision Biologic). Previous anti-FXa assays (calibrator range: 0-500 ng/mL) employed 1:4 dilution of test plasma in Owren-Koller buffer followed by addition of FXa substrate and bovine FXa with an overall 1:44 sample dilution. In the modified assays, calibration curves for the anti-FXa assay were generated using apixaban or rivaroxaban calibrators in the range of 0 to 80 ng/mL; test samples were analyzed neat with an overall 1:2.6 sample dilution. Rivaroxaban and apixaban concentrations in the test samples were interpolated from the respective assay specific calibration curves. Validation of the modified anti-FXa assays was performed by assessing a) linearity and precision of the calibration curve and controls, b) lower limit of quantitation (LLOQ), c) inter-assay precision and d) sample stability. Results: The calibration curves for both apixaban and rivaroxaban assays demonstrated good correlation with a mean "r" value of 0.997 and 0.998, respectively (n=6). The percent recovery and precision of the modified anti-FXa assay are summarized in Table 1. The LLOQ for apixaban and rivaroxaban using the modified assays were &lt;18.8 and &lt;20.6 ng/mL, respectively, based on the preliminary results. Both apixaban and rivaroxaban assays demonstrated potent reversal of anti-FXa activity in presence of andexanet. Samples containing 232 ng/mL apixaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 77.18 ng/mL of quantifiable apixaban (~66.7% reversal). Similarly, samples containing 242 ng/mL rivaroxaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 29.44 ng/mL quantifiable rivaroxaban (~87.8% reversal). The short-term stability study with the modified anti-FXa assay included storage of plasma samples at 2-8oC up to 24 hours and under frozen conditions at -20oC up to 2 weeks. Conclusions: The modified anti-FXa assays intended for measuring FXa inhibitor levels in the presence of andexanet produced acceptable analytical performance characteristics. Application of the modified anti-FXa assays in plasma samples from andexanet-treated human subjects has yet to be studied. Table 1 Disclosures Cardenas: CirQuest Labs/MLM Medical Labs: Current Employment. Kotha:CirQuest Labs/MLM Medical Labs: Current Employment. Lu:Portola Pharmaceuticals, Inc.: Current Employment. Conley:Portola Pharmaceuticals, Inc.: Current Employment. Bourdin:Diagnostica Stago: Current Employment. Herve:Diagnostica Stago: Current Employment. Jennings:CirQuest Labs/MLM Medical Labs: Current Employment, Current equity holder in private company.