Scientific Advisory Committee Meeting, Werfen Benelux, June 8th, 2017 Update on DOAC's in a lab setting. Measurement when short TAT is needed. Prof. François Mullier Université catholique de Louvain, CHU UCL Namur Associate head of laboratory of clinical biology

Some figures in Belgium Already 107582 patients on DOACs in 2014 Pharmanet, INAMI 2

Why to measure DOACs? 3

When do we need to measure DOACs in urgent situations? Bleeding or recurrence of thrombosis (to assessing the potential contribution of DOACs) Before an invasive procedure (elective or urgent surgery at risk of bleeding, thrombolysis) to make decision about timing of these procedures or to determine whether patients with acute ischemic stroke can safely be given fibrinolytic therapy. To select patients who will benefit from antidote administration (RE-VERSE AD study; at baseline, 25 patients had a normal aPTT  more sensitive tests than the aPTT are needed to best identify patients who will benefit from idarucizumab and to monitor their response to treatment). To monitor the extent of reversal achieved when reversal agents are given Douxfils et al. Exp. Op. Drug. Saf. 2015. Weitz J, Eikelboom J. Circulation 2016 4 4

When do we need to measure DOACs in urgent situations? For andexanet administration: The dose is determined by which oral FXa inhibitor the patient is taking and the time from the last dose This can lead to unnecessary administration or underdosing if the clinical information is incorrect Ready access to rapidly available, calibrated tests is needed to ensure reversal agents are given appropriately The cost of andexanet is estimated to be between 3000 and 4000 EUR per dose. Therefore, ready access to rapidly available, calibrated tests is needed to ensure that reversal agents are given appropriately. Weitz J, Eikelboom J. Circulation 2016 5 5

Guidance of the SSC of the ISTH (2/2) For the administration of reversal agents: In patients with serious bleeding, a drug concentration, > 50 ng/mL is likely sufficiently high to warrant antidote administration whereas in those requiring an urgent intervention associated with a high risk of bleeding, antidote administration should be considered if the drug concentration exceeds 30 ng/mL Note: This algorithm needs to be validated prospectively Levy et al. J Thromb Hemost 2016

How to interpret DOACs assays in urgent situations? Use the appropriate assay! Limit of quantification, linearity and Interferences! Safety threshold for surgery with high risk of bleeding Drug reversal in case of serious bleeding Risk of bleeding Ischemic stroke: no consensus, IV thrombolysis in patients with 50 to 100 ng/mL < 30 ng/mL > 50 ng/mL Trough measurement 200ng/ml Levy JH, J Thromb Haemost 2016 Baglin T, J Thromb Haemost 2013 Pernod G, Arch Cardiovasc Dis 2013 Seiffge DJ, Circulation 2015 7 7

How to measure DOACs? Requirements Readily available 24h/day and 7 days/week Easily performed short turnaround time Reliable for measurement of “low” and “high “ concentrations < 30 to > 500ng/mL Since measurement of the conc is most of the time require in emergency situation, the assay must be readily available and have a short…. Gold standard: liquid chromatography with tandem mass spectrometry (LC-MS/MS) Baglin T, J Thromb Haemost 2014 Dale BJ, Br J Haematol 2015 Cuker A, J Thromb Thrombolysis 2016 8

How to measure DOACs: summary of systematic reviews and recommendations Focaliser sur dabigatran avec un cadre? Cuker A., et al. J Am Coll Cardiol. 2014 Lippi G. Clin Chem Lab Med. 2015 Samuelson BT, Cuker A. Blood Rev 2016

Mass spectrometry : advantages and limitations of the gold standard method specificity labour-intensive preparation sensitivity LOD around 1 ng/mL LOQ around 3 ng/mL complexity of the technique selectivity compared to coagulation-based assay cost >< availability reproducibility intra-assay precision < 6% inter-assay precision < 10% Lack of standardization: in-house validation matrix effect interference with plasma phospholipids presence of other compounds removal of proteins interference by drug metabolites (either active or not) accuracy external quality control turn-around time Douxfils J., et al. Trends in Analytical Chemistry. 2016

Standard laboratory assays (PT/APTT) for DOACs Poor comparability of APTT/PT with specific assays 11 Testa S. et al, J Thromb Haemost 2016

Dabigatran: aPTT APTT is not reliable: not sensitive enough to dabigatran not possible to estimate dabigatran concentration normal aPTT cannot exclude the presence of dabigatran multiple other biological variable interfering with aPTT Do not calibrate with commercial standards Douxfils J,, Mullier F et al. Thromb Haemost. 2012 Douxfils J,, et al. Thromb Haemost. 2013 Hawes E. M., et al. JTH. 2013 Baglin T., et al. JTH. 2013 Van Blerk M., et al. Thromb Haemost. 2015 Samuelson BT, et al. Chest 2016

TT may be useful for low concentrations Often used in clinical practice even if it is not recommended Normal TT excludes clinical relevant dabigatran level A prolonged TT is not always associated with a concentration higher than 30 ng/ml  some anesthesiologists/surgeons may uselessly delay an invasive procedure Lack of standardisation and specificity (heparin bridging, inflammatory syndrome, fibrin degradation products…) Several biological and analytical variables: Thrombin origin Concentration of thrombin Lot to lot reagent differences Instrumentation/Storage Lessire S, Douxfils J et al. Thromb Res 2015; Douxfils J, Lessire S et al. Thromb Haemost. 2015; Chin et al. Br J Clin Pharmacol 2014. 13

Dabigatran: Dilute Thrombin Time and Ecarin Chromogenic Assay Hemoclot® Thrombin Inhibitors (HTI) and Ecarin Chromogenic assay (ECA) and STA®-ECA II (ECA II) useful for normal and high dabigatran concentrations but lower limits of quantitation between 30 and 50 ng/mL. Stangier J and coll. Blood Coagul Fibrin 2012 Lange U and coll. Pathophysiology of Haemostasis and Thrombosis 2003 Gosselin RC and coll. Ann Pharmacother 2013 Douxfils J and coll. Thromb Haemost 2013 Hawes EM and coll. J Thromb Haemost 2013 Antovic JP and coll. Eur J Clin Pharmacol 2013 Douxfils J, Mullier F and coll. Thromb Haemost 2012 van Ryn J and coll. Thromb Haemost 2010 Skeppholm M. and coll. Thromb Res 2014

Dabigatran and dedicated coagulation assays : External UKNEQAS survey

Dabigatran and dedicated coagulation assays : External UKNEQAS survey High CVs were observed for the two samples with lower levels of dabigatran. Between center agreement was better for sample S14:13 (median concentration 158.5ng/ml, CV 19.1%).

Dabigatran and dedicated coagulation assays : External UKNEQAS survey Only 3 CE marked assays Most of the assays are useful for normal and high concentrations but have lower limits of quantitation between 30 and 50 ng/mL Stangier J et al. Blood Coagul Fibrin 2012; Lange U et al. Pathophysiology of Haemostasis and Thrombosis 2003; Gosselin RC et al. Ann Pharmacother 2013; Douxfils J et al. Thromb Haemost 2013, Hawes EM et al. J Thromb Haemost 2013; Antovic JP et al. Eur J Clin Pharmacol 2013; Douxfils J, Mullier F et al. Thromb Haemost 2012; van Ryn J et al. Thromb Haemost 2010; Skeppholm M. et al. Thromb Res 2014

Dabigatran and dedicated coagulation assays : Other international surveys UKNEQAS 2015 Sample 15:01(n=56) : median 92ng/ml: overall CV: 15.1% Sample 15:02 (n=55): median 154 ng/ml, overall CV: 16.7% ECAT 2015 Sample 15:88 (n=111): median 131 ng/ml, overall CV: 15.4% Sample 15:89 (n=112): median 235 ng/ml, overall CV: 11.6%

New specific tests are available for low dabigatran concentrations Good accuracy in the low concentration range HemosIL Direct Thrombin Inhibitor also accurate (UKNEQAS 2014) Douxfils J,, et al. Thromb Haemost. 2015.

Assays sensitive to low plasma concentrations of dabigatran are required to assess the reversal by idarucizumab RE-VERSE AD interim results: primary endpoint by dTT reversal Assay upper limit of normal Idarucizumab 2x 2.5 g dTT (s) 130 110 70 60 50 40 30 20 120 100 90 80 1h 2h 4h 12h 24h Baseline Between vials 10–30 min Time post idarucizumab Uncontrolled bleeding Emergency surgery or procedure Idarucizumab 2x 2.5 g dTT (s) 130 110 70 60 50 40 30 20 120 100 90 80 1h 2h 4h 12h 24h Baseline Between vials 10–30 min Time post idarucizumab Interim analysis includes data for the first 90 patients. Pollack CV et al. N Engl J Med 2015:373:511–20 Lower limit of quantitation: 30-50ng/ml!

Idarucizumab for dabigatran reversal- Does one dose fit all? The 5 g idarucizumab decreased dabigatran concentration from 3000ng/ml to 500 ng/mL. However, the dabigatran concentration returned to 1126 ng/mL at 7 h after idarucizumab administration, = rebound of drug levels This case highlights the need for continued monitoring of dabigatran concentrations and coagulation parameters following idarucizumab administration. Rottenstreich A et al. Thromb Res 2016 Miller L et al. J Emerg Med 2016 Tripodi A. J Thromb Haemost. 2017  Simon A. J Thromb Haemost 2017

Heparin-calibrated chromogenic anti-Xa assays Correlation only between low anti-Xa inhibitors conc. and heparin anti-Xa activity High inter-assay variability: no relationship between anti-Xa inhibitor concentration and heparin anti-Xa U/mL Given the availability of heparin calibrated anti-xa assays, the performance of these assays to mesure direct anti-Xa inhibitors have been tested. Direct anti-Xa inhibitors yield high heparin anti-Xa activity Correlation between drug conc. and heparin anti-Xa U/mL only over calibration ranges: low drug concentrations Variabiliy between commercial kits and calibrators: no relationship between drug conc. and heparin anti-Xa U/mL Suitable for ruling out presence of direct anti-Xa Should not be used to quantify direct anti-Xa Gosselin R, Ann Pharmacother 2015 Gosselin R, J Thromb Haemost 2016 Samama MM, Thromb Haemost 2010 22

Heparin-calibrated chromogenic anti-Xa assays: interassay variability Given the availability of heparin calibrated anti-xa assays, the performance of these assays to mesure direct anti-Xa inhibitors have been tested. Direct anti-Xa inhibitors yield high heparin anti-Xa activity Correlation between drug conc. and heparin anti-Xa U/mL only over calibration ranges: low drug concentrations Variabiliy between commercial kits and calibrators: no relationship between drug conc. and heparin anti-Xa U/mL High inter-assay variability Anti-Xa IU/ml <=0.1 may be associated with concentration <30ng/ml Sabor L et al Thromb Res 2017 23

Modification of Heparin-calibrated chromogenic anti-Xa assays Modification of sample dilution for rivaroxaban(standard curve): 1/10 instead of 1/2 in heparin application (Biophen LRT): No interference of heparin Correlation only between anti-Xa inhibitors conc. and heparin anti-Xa activity Rivaroxaban (low curve): dilution is still ½ (heparin interference) Apixaban (standard curve) 1/20- Apixaban (low curve) 1/3 Edoxaban (standard curve) 1/15 - Edoxaban (low curve) 1/3 Given the availability of heparin calibrated anti-xa assays, the performance of these assays to mesure direct anti-Xa inhibitors have been tested. Direct anti-Xa inhibitors yield high heparin anti-Xa activity Correlation between drug conc. and heparin anti-Xa U/mL only over calibration ranges: low drug concentrations Variabiliy between commercial kits and calibrators: no relationship between drug conc. and heparin anti-Xa U/mL Samama MM, Amiral J ISTH 2012 24

Specific tests for direct anti-Xa concentration measurement: chromogenic anti-Xa assays Advantages Drawbacks Same reagents for measurement of UFH, LMWH anti-Xa activity and direct anti-Xa inhibitors Ready-to-use reagents Not affected by pre-analytical variables and alterations of clotting factor levels Measurement in plasma/serum High correlation with LCMSMS Specific calibrators and controls for each anti-Xa inhibitor: Rivaroxaban: available Apixaban: available Edoxaban: available Lack of standardization: a reference international calibrator is required Each laboratory should establish, riva, api and edoxaban-specific standard curves Harenberg J, Clin Chem Lab Med 2016 Cuker A, J Thromb Thrombolysis 2016 Douxfils J, Bio Med Res Int 2015 Dale BJ, Br J Haematol 2015 Gosselin RC, Arch Pathol Lab Med 2014 Cuker A, JACC 2014 25

Intra-assay CV %: < 5% Inter-assay CV%: < 10% Specific chromogenic anti-Xa assays for rivaroxaban and apixaban measurement Reagents: one- or two-stage assay, different buffers, dilution factors, concentrations of Xa, types of chromogenic substrate Methods with exogenous antithrombin: over estimation of rivaroxaban Mani H, Thromb Haemost 2012 ; Helin TA, Clin Chem 2013 ; Asmis IM, Thromb Res 2012, Gosselin R Arch Pathol Lab Med 2014 Calibrators: 4 to 5 levels from 0 to 500 ng/mL (Stago, Hyphen, Technoclone) Lower limit of detection/quantification: ~ 20-30 ng/mL (sometimes lower Mani H, Thromb Haemost 2012 , Lessire S. CATH 2016 Accepted) Upper limit of quantification: ~ 400-600 ng/mL (if higher: dilution in buffer/plasma) Chromogenic anti-Xa assays are available from many commercial diagnostic companies. They differ in the type of methods, Biophen DiXa: specific for direct anti-Xa; Insensitive to the presence of heparin-like anti-Xa Berichrom contains AT Intra-assay CV %: < 5% Inter-assay CV%: < 10% Samama MM, Thromb Haemost 2010 ; Becker RC, J thromb Thrombolysis 2011 ; Douxfils J, Thromb Res 2012 ; Mani H, Thromb Haemost 2012 Douxfils J, Thromb Haemost 2013 ; Barrett YC, Thromb Haemost 2013 ; Francard S, Thromb Haemost 2014 Package inserts from diagnostic companies 26

Specific assays for low Xabans concentrations Prospective multicentre study Agreement between laboratories was high at peak level but limited at trough level (<50ng/ml) Studt JD,, et al. J Thromb Haemost. 2017.

Interference of heparin on DOAC specific assays Lessire S. et al. Clin App Thromb Hemost 2016.

Are the tests limited to specialized laboratories? NO!

Turn around time: specific assays 38 consecutive patients with acute ischemic or hemorrhagic stroke under Rivaroxaban (last intake <48 h) in which RivLev determined by Biophen assay TAT:34 min (IQR 29–65 min). similar for on-hours and off-hours-TATs as well as for early and late patient arrival Feasible for use in emergency treatment Further research to evaluate the role of rivaroxaban concentration to guide acute treatment decisions is warranted. Seiffge DJ, et al. J Thromb Thrombolysis 2016

Turn around time: POCT assays Iapichino, et al. Semin Thromb Hemost 2017

Turn around time: POCT assays Iapichino, et al. Semin Thromb Hemost 2017

DRVV-DOAC assay (X9211) (Haematex Research) Sennesael AL et al. Submitted 33

DRVV-DOAC assay (X9211) (Haematex Research) Sennesael AL et al. Submitted 34

Stability In whole blood, all DOACs were stable for up to 2 hours In plasma, rivaroxaban and apixaban were stable for up to 8 hours at 20-25°C In plasma, rivaroxaban and apixaban were stable for up to 48 hours at 5°C Rivaroxaban was the only freeze-thaw stable DOAC Laboratories have to consider these differences before analysis of DOACs Stability if 80-120% in comparison to the baseline Mc Grail. et al. Thromb Res 2016

Take home messages (1/2) 2 key opinion leaders says: Registration by regulatory agencies and their widespread implementation in clinical laboratories are “urgently” needed Dabigatran versus warfarin in patients with atrial fibrillation. Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, Pogue J, Reilly PA, Themeles E, Varrone J, Wang S, Alings M, Xavier D, Zhu J, Diaz R, Lewis BS, Darius H, Diener HC, Joyner CD, Wallentin L; RE-LY Steering Committee and Investigators. N Engl J Med. 2009 Sep 17;361(12):1139-51. Idarucizumab for Dabigatran Reversal. Pollack CV Jr, Reilly PA, Eikelboom J, Glund S, Verhamme P, Bernstein RA, Dubiel R, Huisman MV, Hylek EM, Kamphuisen PW, Kreuzer J, Levy JH, Sellke FW, Stangier J, Steiner T, Wang B, Kam CW, Weitz JI. N Engl J Med. 2015 Aug 6;373(6):511-20. 36

Take home messages (2/2) Specific assays should be preferred for DOAC quantification Adaptations of the specific assays are required for low concentrations (perioperative management, reversal by antidotes) 3 cut-offs in urgent situations: 30 – 50 – 200 ng/mL 37

Perspectives Development of point of care devices Development of a reference international calibrator Development of assays to remove the interference of DOACs Collect data about sample stability are lacking To validate low methodologies for apixaban and edoxaban in a perioperative setting To optimize the specificity of chromogenic assays for low DOACs plasma concentrations To reduce the turn-around time (TAT) of urgent DOAC level estimation To analyze the value of DOAC measurements in patients in emergency setting