1. 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

2. 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

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

4. 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

5. 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 Submitted after revision

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. Proposition d’algorithme
Lippi G. CCLM 2014

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

14. 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)

15. 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) 

16. 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®

17. 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.

18. 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

19. CONCLUSION
EMA position

20. 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