Department of Pathology and Laboratory Medicine Quantifying and Estimating Assessing NOAC in routine clinical Laboratory DOACs Robert C Gosselin, CLS UC Davis Health System Department of Pathology and Laboratory Medicine Sacramento, CA robert.gosselin@ucdmc.ucdavis.edu Cell: 916-612-7086

Objectives Describe the effect of oral anticoagulants on laboratory screening tests Review the recommendations on assessing these drugs General concepts Specific recommendations

Disclosures North American Advisory Committee Instrumentation Laboratory Speaker honoraria Diagnostica Stago Siemens Healthcare Diagnostics

What’s in a name? New or Novel oral anticoagulants - NOACs Target specific oral anticoagulants – TSOACs Non-vitamin K oral anticoagulants - NOACs Direct oral anticoagulants - DOACs

Dabigatran (Boehringer Ingelheim) Apixaban (Karen Rossi Bristol-Myers Squibb)

“Waterfall” Coagulation Cascade: PT FXII APTT FXI FVII FIX FVIII Direct Anti-Xa FX FXa FV FII Thrombin Fibrinogen Fibrin Direct Anti-IIa TT

Mean Trough level, ng/mL Expected DOAC levels In healthy adults: Dose Median Cmax ng/mL [SD] 1Dabigatran 150mg 110 [24] 1Rivaroxaban 10mg 141 [17] 1Apixaban 2mg 460 [23] 2Edoxaban 60mg 300 [34] 1Mueck et al Thromb J 2013:11:10 2Ogata, et al J Clin Pharmacology 2010:50:743-53. In treated patients Dose Mean Trough level, ng/mL (SD) Mean Peak level, ng/mL 1Dabigatran (n=35) 150mg bid 85 (48) 179 (104) 2Rivaroxaban (n=29) 20mg qd 45 (38) 335 (174) 3Apixaban (n=26) 2.5mg bid 57 (37) 68 (32) 4Edoxaban (N=1,691) 60mg qd ~25 (15) ~175 (50) 1Hawes E, et al. J Thromb Haemost 2013;11:1493-1502. 2Francart S, et al. Thromb Haemost 2014 doi 10.116-0/TH13-10-0871 3Becker , et al. Thromb Haemost 2010;104:976-983 4Weitz, et al. Thromb Haemost 2010;104:633-641

DOACs and the clinical laboratory Questions “they” ask of us What do these drugs do to lab tests? What do these results mean? Question we should be asking “them” What do you want to know? Question we ask of us? How sensitivity is my stuff? What else can I use to assess drug effect?

General concepts Not all reagents respond the same APTT more sensitive to Dabigatran PT more sensitive to anti-Xa DOACs Thrombin time useful for excluding presence of dabigatran If the drug has effects on coagulation in-vivo, may likely affect on coagulation ex-vivo

Measuring Recommendations Scientific and Standardization Subcommittee on the Control of Anticoagulation of the International Society for Thrombosis and Haemostasis (April 2013) recommendations: In emergent situations the use of an easily performed assay(s) that to provide semiquantitative results but these assays are not to be used to determine drug concentration – PT, APTT, TT Assay(s) that provides quantitative results should be used to determine concentration of drug in serum or plasma – drug calibrated methods (ecarin, dTT, anti-Xa, LC-MS/MS) Baglin T, Hillarp A, Tripodi A, et al, J Thromb Haemost 2013;11:756-60.

ISTH Subcommittee: Control of Anticoagulation For Dabigatran, thrombin time can be used to exclude presence of drug For Rivaroxaban Use of calibrated anti-Xa measurements For Apixaban PT less sensitive than with rivaroxaban Use of spiked samples to estimate sensitivity Harenberg et al J Thromb Haemost 2012:10:1433-6 Harenberg et al J Thromb Haemost 2014: epub

Concept #1: Not all reagents respond the same…

Modified from Hawes E, et al. J Thromb Haemost 2013;11:1493-1502

Neoplastin CI+: Riva > Apix R2G: Apix > Riva Innovin Riva ~ Apix Modified from Francart S, et al. Thromb Haemost 2014; 111(5) Epub Neoplastin CI+: Riva > Apix R2G: Apix > Riva Innovin Riva ~ Apix Gouin-Thibault, et al Thromb Haemost 2014: 240-248

Previous slide with clinicial samples indicated: R2G: Apix > Riva Innovin Riva ~ Apix RG Unpublished data using drug enriched plasma

Concepts #2 and #3: The APTT is more sensitive to Dabigatran Then PT is more sensitive to anti-Xa DOACs

Dabigatran v Rivaroxaban: APTT

Dabigatran v Rivaroxaban: PT Similar

Dabigatran v Rivaroxaban: PT

DOACs concepts not always true… Rivaroxaban: Actin APTT > Innovin PT

Concept #4: Thrombin time useful for excluding presence of dabigatran

3 reagent manufacturers for 11 results at 10 sites 25ng/mL Dager, et al Ann Pharmacotherapy 2012; 46:1627-36

Concept #5: If it effects coagulation in-vivo, more than likely affects coagulation testing ex-vivo

Incomplete correction Possible to misclassify as LA Assay NOAC Anti-IIa NOAC Anti-Xa TCT Markedly  No effect Clauss Fibrinogen No effect or falsely  APTT Mixing Study Incomplete correction PT Mixing Study# Bethesda assay Falsely present Not tested APTT- based factor assays, one stage Possibly Falsely  PT- based factor assays, one stage# Chromogenic FVIII activity AT Activity   a. FXa based a. No effect a. Falsely  b. FIIa based b. Falsely  b. No effect PC Activity a. Clot based b. Chromogenic PS Activity a. Markedly  b. ELISA or LIA based LA Tests Possible to misclassify as LA APCR Falsely  ratio Falsely  ratio

DRVVT formulations and DOAC interference

So what about the SSC recommendations for using drug calibrators for determining reagent sensitivity to DOACs?

Patient samples – 636ng/mL PT Doubling time: Calibrators – 395ng/mL Patient samples – 636ng/mL APTT Doubling time: Calibrators – 268ng/mL Patient samples – 431ng/mL Gosselin, et al Thromb Haemost 2015; 113(1):77-84

Patient samples – 1007ng/mL PT Doubling time: Calibrators – 384ng/mL Patient samples – 1007ng/mL APTT Doubling time: Calibrators – 349ng/mL Patient samples – 1512ng/mL Gosselin, et al Thromb Haemost 2015; 113(1):77-84

Is in-vitro drug enrichment providing truth?

Dabigatran: Comparison between sample types

Plausible causes for calibrator discrepancy: Theory versus reality In theory, since DOACs have direct inhibition, plasma enrichment should be acceptable Reality is that not all patients have 100% factor levels so perhaps we are seeing that variation with patient samples Plausible causes for calibrator discrepancy: Higher citrate concentration Lyophilization process

Quantifying DOAC levels WHY? Suspicion of overdose Bleeding patient Emergency surgery or trauma Acute stroke – candidate for thrombolysis Renal insufficiency and possibly the elderly

Quantifying DOACs Direct IIa class Mass spectrophotometry Accurate, but not timely Dilute TT Easy, no FDA approved methods LDT possible using TT reagents Ecarin Clotting time Easy, no FDA approved kits Ecarin Chromogenic assay

X ECA dTT LC-MS/MS ECT Gosselin, et al Am J Clin Pathol 2014;141(2):262-7

No FDA approved calibrators or controls Quantifying DOACs Direct Xa class of drugs Mass spectrophotometry Accurate, but not timely Chromogenic anti-Xa Easy, fast, and cheap Same kits as UFH/LMWH No FDA approved calibrators or controls

Quantifying Rivaroxaban   COAMATIC Hyphen Technoview Berichrome Rotachrome BIOPHEN LC-MS/MS R2: 0.988 m: 0.89 p <0.001 R2: 0.948 m: 0.85 R2: 0.975 m: 0.78 R2: 0.978 m: 0.86 p<0.001 R2: 0.985 m: 0.76 0.003 Anti-Xa methods Anti-Xa method bias Gosselin, et al. Arch Pathol Lab Med 2014 Dec;138(12):1680-4

So what happens if we run a patient on DOAC using routine Anti-Xa methods??

[AT] - heparin-Xa complex + residual fXa Anti-Xa activity plasma [heparin] + Excess FXa [Antithrombin] [AT] - heparin-Xa complex + residual fXa DXa inhibitors - Chromogenic substrate Peptide cleavage yellow color

Gosselin, Moll, Adcock Ann Pharmacotherapy 2015

Edoxaban will be different! Gosselin, Adcock unpublished data

UCDHS Consideration for DOAC Creating an alternative screening panel for ED PT APTT Anti-IIa screen (TT) – qualitative present or absent Anti-Xa screen – qualitative

Summary Variability in PT and APTT response Reference literature to guesstimate sensitivity General rules may not apply PT more sensitive to anti-Xa DOAC APTT more sensitive to anti-IIa DOAC Determining presence of drug could be done at any laboratory using existing methods Quantifying drug levels can be done at any laboratory with clot or chromogenic based testing using LDTs Recommendations using calibrators to estimate reagent sensitivity does not appear to be viable for all drugs and reagents Perhaps drug enrichment studies should be used to suggest effects on tests in lieu of suggesting reagent sensitivity Challenges are those patients where medication history is unavailable

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