Laboratory measurement of DOACs in specific clinical settings ISTH 2015 Congress Technical innovations in hemostasis Pr François Mullier Dr Sarah Lessire Ph Anne-Laure Sennesael Ph Jonathan Douxfils Laboratory measurement of DOACs in specific clinical settings 1

Monitoring of DOACs anticoagulant effect Gold standard for the accurate quantification of low levels of DOACs = LC-MS/MS method Limited in routine use and available in highly specialized centers Barrett YC and coll. Thromb Haemost 2010 Douxfils J and coll. Thromb Haemost 2013 Samama MM Thromb Haemost 2012 Gouin-Thibault I and coll. Thromb Haemost 2014 2

Evidence regarding laboratory measurement: a systematic review Cuker and coll. J Am Coll Cardiol 2014 ;64:1128-39

Dabigatran – Thrombin Time Several advantages over aPTT: no influence of factor deficiencies (except for fibrinogen deficiencies), of lupus anticoagulants and of elevated FVIII in inflammatory settings. Lack of standardisation and specificity Several biological and analytical variables: Thrombin origin Concentration of thrombin Lot to lot reagent differences Instrumentation/Storage 4

Dabigatran – Thrombin Time Using TT needs to consider the clinical context of the patient (heparin bridging, inflammatory syndrome, fibrin degradation products…) the laboratory to optimize its TT (thrombin concentration that can allow a measure from 0 to 120 s for dabigatran concentration from 0 to 50 ng/ml with an acceptable CV% = 10-12%). to be cautious with the storage condition of plasma (TT increased at room temperature with time)    Lessire S and coll. Tromb Res 2015 Submitted after revision

Dabigatran - dedicated coagulation test Hemoclot® Thrombin Inhibitors (HTI) 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. HTI: agreement with free dabigatran ECA: agreement with total dabigatran 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 - dedicated coagulation test HTI, HTI LOW and ECA II versus LCMS/MS: HTI LOW and ECA II performed better than HTI for low dabigatran plasma concentration ECA II useful for patient bridged with LMWH Meizothrombin is insensitive to heparins 7 Douxfils J, Lessire S. and coll. Thromb Haemost 2015

Which coagulation test to choose? Rivaroxaban and Apixaban: lSTH recommends PT to evaluate the intensity of their anticoagulant effect BUT: Different sensitivity for rivaroxaban and apixaban (different kon) Variation between reagents Do not allow quantification of plasmatic concentrations Bad correlation with the LC-MS/MS Other factors influencing PT ISSUE apixaban: PT can be normal with therapeutic concentrations Baglin T and coll. J Thromb Haemost 2013 Douxfils J and coll. Thromb Res 2012 Douxfils J and coll. Thromb Haemost 2013 Jourdi G and coll. Thromb Haemost 2015 8

Rivaroxaban – Apixaban: dedicated coagulation tests Specific anti-Xa chromogenic assays: Rivaroxaban: good correlation with LC-MS/MS for concentrations > 30 ng/mL Apixaban: good correlation with LC-MS/MS for concentrations > 15 ng/mL. Currently: assessment of dedicated coagulation assay for low concentrations of Riva and Api (<50 ng/ml) Exception apixaban: Biophen heparin ® used with ACL TOP: 40ng/ml Barrett YC, and coll. Thromb Haemost 2010 Douxfils J, and coll. Thromb Haemost 2013 Samama MM, Thromb Haemost 2012 Gouin-Thibault I. and coll. Thromb Haemost 2014 9

Rivaroxaban – Apixaban: dedicated coagulation tests Assessment of dedicated coagulation assay for low concentrations of rivaroxaban and apixaban (<50 ng/ml) Proof-of-concept study performed on 90 plasma samples BUT results from LC-MS/MS (external) were unreliable (interrun variation) Adaptation of previous validated methodology Important variation of the signal of the internal standard Incoherent results between the two methodologies (control: n=7) Bland-altman: mean difference: -17ng/mL 95%CI: -45 to 10 ng/mL R-square: 0.63 R-Spearman: 0.64 Exception apixaban: Biophen heparin ® used with ACL TOP: 40ng/ml First method (in ng/ml) Second method (in ng/ml) 96 84 124 93 99 74 98 66 80 59 94 89 123 128 10

Coagulation tests and NOACs HTI LOW ECA II Anti-Xa LOW? Horizontal bars and vertical hatching correspond to the approximate range of detectability (i.e., sensitivity) and linearity, respectively, of each assay to below, within, and above typical on-therapy concentrations of dabigatran, rivaroxaban, and apixaban. Ranges are approximations and may vary on the basi Anti-Xa LOW? Cuker and coll. J. Am. Coll. Cardiol. 2014

Proposition d’algorithme Lippi G. CCLM 2014

Influence of NOAC on DRVVT Intérêt du DRVVT? Lippi G. CCLM 2014 see poster PO125-WED Sennesael AL and coll. (new DRVVT method)

Unsolved technical issues (I) Sensitivity of HTI/HTI LOW to LMWH HTI LOW requires a new calibration curve and a new set of controls Sensitivity of Liquid anti Xa to LMWH Xabans: no accurate assay (or data) in the low range (<50ng/ml)

Unsolved technical issues (II) How to calibrate the assays? (home-made calibrators vs manufacturers calibrators) When to measure the drug level at Cmax (2-3-4h?) No data about sample stability No POCT devices (or few data Dias Arch Pathol Lab Med. 2015) 

How to calibrate the assay? ISTH recommends to assess the sensitivity of its own PT (for Riva) or aPTT (for dabi) reagents using commercially available calibrators Difference of response depending on the calibrators aPTT: e.g. STA® - C.K. Prest®

How to calibrate the assay? Calibration of aPTT (dabigatran) or PT (rivaroxaban and apixaban) requires specific calibrators dedicated and validated in this context Possible matrix effect Calibrators dedicated for chomogenic assays performed poorly in clotting assays Calibrators dedicated for specific clotting assays (e.g. for HTI) can be used for clotting assays* * Initially rivaroxaban calibrators from Stago were developed for STA®-Neoplastin CI+, this may explain the good performance in clotting assays even if they are now used for chromogenic assays No data available for apixaban calibrators but the explanation can be similar.

How to calibrate the assay? « Use of drug specific calibrators for assessing the relative sensitivity of NOAC to PT and APTT assays may not be optimal, as an overestimation of sensitivity occurred with drug specific calibrators compared with patient samples (exception: dabigatran with SynthASil and PTT-A APTT reagents) Clinically this may result in considerable underestimation of in vivo drug concentration in a given patient ». explanations;: 1) Frozen samples 2) Presence of antibiotics used as a preservative An antibiotic could possibly induce an artifactual prolongation of the clotting test, thereby overestimating the reagent sensitivity to lower levels of drug, as seen in our data 3) This discrepancy likely reflects variation in the citrate concentration of the manufactured calibrators compared to that used for APTT and PT assays 4) or may be due to the lyophilization process applied to the calibrator material. Adcock TR 2015 Gosselin RC. Thromb Haemost 2015

CONCLUSION EMA position

Thank you for your attention Aknowledgement Ph. Jonathan Douxfils Dr Sarah Lessire Ph. Anne-Laure Sennesael Prof. Jean-Michel Dogné Dr. Anne-Sophie Dincq Prof. Maximilien Gourdin Prof. Bernard Chatelain Prof. Edith Collard Ph. Anne-Sophie Larock Ph. Anne Spinewine Laboratory Technicians: Justine Baudar Maïté Guldenpfennig Françoise Biot Nicolas Bailly Thank you for your attention