1. 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

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

3. Why to measure DOACs? 3

4. 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
Weitz J, Eikelboom J. Circulation 2016 4 4

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

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

12. 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

13. 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

14. 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

15. Dabigatran and dedicated coagulation assays : External UKNEQAS survey

16. 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%).

17. 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

18. 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%

19. 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

20. 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!

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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: ~ ng/mL (sometimes lower Mani H, Thromb Haemost 2012 , Lessire S. CATH 2016 Accepted)
Upper limit of quantification: ~ 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

27. 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

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

29. Are the tests limited to specialized laboratories?
NO!

30. 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

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

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

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

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

35. 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 % in comparison to the baseline
Mc Grail. et al. Thromb Res 2016

36. 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 Sep 17;361(12):
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 Aug 6;373(6): 36

37. 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

38. 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