Quantifying and Estimating Assessing NOAC in

routine clinical Laboratory

Robert C Gosselin, CLSUC Davis Health System

Department of Pathology and Laboratory MedicineSacramento, CA

robert.gosselin@ucdmc.ucdavis.eduCell: 916-612-7086

DOACs

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 LaboratorySpeaker honoraria Diagnostica Stago Siemens Healthcare Diagnostics

What’s in a name?

New or Novel oral anticoagulants - NOACsTarget specific oral anticoagulants – TSOACsNon-vitamin K oral anticoagulants - NOACs

Direct oral anticoagulants - DOACs

Dabigatran (Boehringer Ingelheim)

Apixaban (Karen Rossi Bristol-Myers Squibb)

FXI

FIX

FXII

FX

FVII

FII Thrombin

Fibrinogen Fibrin

FVIII

FV

APTTPT

TT

“Waterfall” Coagulation Cascade:

Direct Anti-Xa

FXa

Direct Anti-IIa

Expected DOAC levels

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

Dose Median Cmax ng/mL [SD]1Dabigatran 150mg 110 [24]

1Rivaroxaban 10mg 141 [17]

1Apixaban 2mg 460 [23]2Edoxaban 60mg 300 [34]

In healthy adults:

1Mueck et al Thromb J 2013:11:10 2Ogata, et al J Clin Pharmacology 2010:50:743-53.

Dose Mean Trough level, ng/mL

(SD)

Mean Peak level, ng/mL

(SD)

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)

3Becker , et al. Thromb Haemost 2010;104:976-983

In treated patients

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

1. Not all reagents respond the same2. APTT more sensitive to Dabigatran3. PT more sensitive to anti-Xa DOACs4. Thrombin time useful for excluding presence

of dabigatran5. 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 calibrated anti-Xa measurements

• 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

0 100 200 300 400 500 60020.0

30.0

40.0

50.0

60.0

70.0

80.0

Actin FS Linear (Actin FS) Actin Linear ( Actin )Actin FSL Linear (Actin FSL)

Dabigatran, ng/mL

APTT

, s

Modified from Francart S, et al. Thromb Haemost 2014; 111(5) Epub

0 100 200 300 400 500 600 7000.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0Stago Neoplastin CI+Linear (Stago Neoplastin CI+)Recombiplastin 2GLinear (Recombiplastin 2G)

Rivaroxaban by LC-MS/MS, ng/mL

Prot

hrom

bin

times

, s

0 100 200 300 400 500 600 7001.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

Neoplastin CI+Polynomial (Neoplastin CI+)Recombiplastin 2GPolynomial (Recombiplastin 2G)InnovinLinear (Innovin)

Apixaban, ng/mL

Prot

hrom

bin

time

ratio

Gouin-Thibault, et al Thromb Haemost 2014: 240-248

Neoplastin CI+: Riva > ApixR2G: Apix > RivaInnovin Riva ~ Apix

0 100 200 300 400 500 600 700 8000.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

f(x) = 0.0143634408602151 x + 10.9732795698925R² = 0.997533081868792

f(x) = 0.0373514486291931 x + 11.2917735371845R² = 0.998587894716841

f(x) = 0.0140208837930796 x + 10.1433430816155R² = 0.992306993572355

Anti-Xa DOAC, ng/mL

R2 G

Pro

thro

mbi

n tim

e, s

0 100 200 300 400 500 600 700 8000.0

5.0

10.0

15.0

20.0

25.0

f(x) = 0.0103204301075269 x + 10.4281182795699R² = 0.998489545307138

f(x) = 0.00147838770251062 x + 10.1185170434444R² = 0.709036248121753

f(x) = 0.0148701637391704 x + 10.8793913135633R² = 0.997291519152309

Anti-Xa DOAC, ng/mL

Inno

vin

Prot

hrom

bin

time,

s

Previous slide with clinicial samples indicated:

R2G: Apix > RivaInnovin Riva ~ Apix

RG Unpublished data using drug enriched plasma

Concepts #2 and #3:

2. The APTT is more sensitive to Dabigatran3. Then PT is more sensitive to anti-Xa DOACs

Rivaroxaban = 0.026x + 27.914R2 = 0.6977

Dabigatran = 0.0632x + 32.788R2 = 0.6745

20.0

30.0

40.0

50.0

60.0

70.0

80.0

0 100 200 300 400 500 600 700 800 900

Actin

FSL

Drug concentration, ng/ml

Dabigatran v Rivaroxaban: APTT

Dabigatran = 0.0099x + 11.102R2 = 0.7022

Rivaroxaban = 0.0079x + 10.962R2 = 0.6788

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

20.0

0 100 200 300 400 500 600 700 800 900

Innovin

Drug concentration, ng/ml

Dabigatran v Rivaroxaban: PT

Similar

Dabigatran = 0.011x + 11.577R2 = 0.5346

Rivaroxaban = 0.024x + 10.91R2 = 0.872

10.012.014.016.018.020.022.024.026.028.030.0

0 100 200 300 400 500 600 700 800 900

R2G

Drug concentration, ng/ml

Dabigatran v Rivaroxaban: PT

0 100 200 300 400 500 600 7000.0

0.5

1.0

1.5

2.0

2.5

3.0

f(x) = 0.00168704711355155 x + 1.10603243585401R² = 0.610356175864619f(x) = 0.00118316085841073 x + 1.07401415612445R² = 0.656188747820327

Rivaroxaban, ng/mL

Ratio

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

Concept #4:

Thrombin time useful for excluding presence of dabigatran

0.025.050.075.0

100.0125.0150.0175.0200.0225.0250.0275.0300.0

0 50 100 150 200 250 300 350 400 450 500 550

Dabigatran level, ng/ml

Thro

mbi

n tim

e, s

Dager, et al Ann Pharmacotherapy 2012; 46:1627-36

3 reagent manufacturersfor 11 results at 10 sites

25ng/mL

Concept #5:

If it effects coagulation in-vivo, more than likely affects coagulation testing ex-vivo

Assay NOAC Anti-IIa NOAC Anti-Xa

TCT Markedly No effect

Clauss Fibrinogen No effect or falsely No effect

APTT Mixing Study Incomplete correction Incomplete correction

PT Mixing Study# Incomplete correction Incomplete correction

Bethesda assay Falsely present Not tested

APTT- based factor assays, one stage Possibly Falsely Possibly Falsely

PT- based factor assays, one stage# Possibly Falsely Possibly Falsely

Chromogenic FVIII activity No effect Possibly Falsely

AT Activity

a. FXa based a. No effect a. Falsely

b. FIIa based b. Falsely b. No effect

PC Activity

a. Clot based a. Falsely a. Falsely

b. Chromogenic b. No effect b. No effect

PS Activity

a. Clot based a. Markedly a. Falsely

b. ELISA or LIA based b. No effect b. No effect

LA Tests Possible to misclassify as LA Possible to misclassify as LA

APCR Falsely ratio Falsely ratio

0 100 200 300 400 500 600 7000.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

f(x) = 0.00013393973973595 x + 0.957375009522393

f(x) = 0.000566827218325118 x + 1.11945263097683

Apixaban, ng/mL

LA1/

LA2

ratio

DRVVT formulations and DOAC interference

So what about the SSC recommendations for using drug

calibrators for determining reagent sensitivity to DOACs?

0 100 200 300 400 500 6000

5

10

15

20

25

f(x) = 0.0184433549053029 x + 10.1390200259407R² = 0.973969079429466

f(x) = 0.00986439115702643 x + 11.1015012389874R² = 0.702223448282168

Dabigatran, ng/mL

Inno

vin

PT

, s

0 100 200 300 400 500 6000

10

20

30

40

50

60

70

80

90

f(x) = 0.0856698062597193 x + 37.706639662092R² = 0.996230231585745

f(x) = 0.0632364021746181 x + 32.7884003285407R² = 0.674514388010614

Dabigatran, ng/mL

Act

in F

SL

AP

TT

, s

PT Doubling time:Calibrators – 395ng/mLPatient samples – 636ng/mL

APTT Doubling time:Calibrators – 268ng/mLPatient samples – 431ng/mL

Gosselin, et al Thromb Haemost 2015; 113(1):77-84

0 100 200 300 400 500 600 700 800 9000.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

f(x) = 0.0742741469057258 x + 34.0932909196067R² = 0.996394570620228

f(x) = 0.0205080099322491 x + 29.0011196001673R² = 0.438992676051146

Rivaroxaban, ng/mL

Act

in F

SL

AP

TT

, s

0 100 200 300 400 500 600 700 800 9000.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

f(x) = 0.0173192596876807 x + 10.7523134759977R² = 0.995633003162987

f(x) = 0.00597102629741967 x + 11.3485333226763R² = 0.39286747602895

Rivaroxaban, ng/mL

Inno

vin

PT

, s

Gosselin, et al Thromb Haemost 2015; 113(1):77-84

PT Doubling time:Calibrators – 384ng/mLPatient samples – 1007ng/mL

APTT Doubling time:Calibrators – 349ng/mLPatient samples – 1512ng/mL

Is in-vitro drug enrichment providing truth?

0 100 200 300 400 500 6000.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

f(x) = 0.110632558565371 x + 34.1558906158074R² = 0.98228370880693

f(x) = 0.0762825256247874 x + 32.902319783104R² = 0.6998822138277

f(x) = NaN x + NaNR² = 0

Dabigatran, ng/mL

Acti

n FS

APT

T, s

Dabigatran: Comparison between sample types

• 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 overdoseBleeding patient Emergency surgery or traumaAcute stroke – candidate for thrombolysisRenal insufficiency and possibly the elderly

Quantifying DOACs

Direct IIa classMass spectrophotometry

Accurate, but not timelyDilute TT

Easy, no FDA approved methodsLDT possible using TT reagents

Ecarin Clotting timeEasy, no FDA approved kits

Ecarin Chromogenic assay Easy, no FDA approved kits

LAB B = 0.9238x - 2.4958

R2 = 0.9842

LAB A = 0.8453x + 11.77

R2 = 0.9475

LAB C = 0.7845x - 4.1644

R2 = 0.9794

0

50

100

150

200

250

300

350

400

450

500

0 50 100 150 200 250 300 350 400 450 500

BI-MS Dabigatran, ng/ml

Dab

igat

ran

, n

g/m

l

Lab D = 0.8106x - 5.5632R2 = 0.9367

0

50

100

150

200

250

300

350

400

450

500

0 50 100 150 200 250 300 350 400 450 500

BI-MS Dabigatran, ng/ml

Lab

D D

ab

igatr

an

, n

g/m

l

LAB E = 1.0633x + 8.2818R2 = 0.9771

0

50

100

150

200

250

300

350

400

450

500

0 50 100 150 200 250 300 350 400 450 500

BI-MS Dabigatran, ng/ml

Dab

igatr

an

, n

g/m

l

LAB A ECT = 0.3112x + 27.999R2 = 0.9702

0

20

40

60

80

100

120

140

160

180

200

0 50 100 150 200 250 300 350 400 450 500

BI-MS Dabigatran, ng/ml

Clo

ttin

g t

ime,

s

ECA dTT

LC-MS/MS ECT

Gosselin, et al Am J Clin Pathol 2014;141(2):262-7

X

Quantifying DOACs

Direct Xa class of drugsMass spectrophotometry

Accurate, but not timelyChromogenic anti-Xa

Easy, fast, and cheapSame kits as UFH/LMWH

No FDA approved calibrators or controls

COAMATICHyphen

COAMATICTechnoview Berichrome Rotachrome

BIOPHEN

LC-MS/MS

R2: 0.988m: 0.89p <0.001

R2: 0.948m: 0.85p <0.001

R2: 0.975m: 0.78p <0.001

R2: 0.978m: 0.86p<0.001

R2: 0.985m: 0.760.003

-120

-100

-80

-60

-40

-20

0

20

40

0 100 200 300 400 500 600 700 800

Mean [LC-MS/MS + Anti-Xa Method/2], ng/ml

BIO

PH

EN

cal

ibra

tion

diffe

renc

e, n

g/m

l

Quantifying Rivaroxaban

Anti-Xa methods

Anti-Xa method bias

Gosselin, et al. Arch Pathol Lab Med 2014 Dec;138(12):1680-4

Precision Biologics method (x) = 0.083x + 48.832R2 = 0.8011

Siemens LA2 method (∆) = 0.0734x + 36.966R2 = 0.8736

0

10

20

30

40

50

60

70

80

90

100

110

120

0 100 200 300 400 500 600 700 800 900

Rivaroxaban by LC-MS/MS, ng/ml

LA C

onfir

mat

ory

reag

ent,

s

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

Anti-Xa activity

Chromogenic substrate

plasma [heparin] + Excess FXa

[AT] - heparin-Xa complex + residual fXa

yellow color

[Antithrombin]

Peptide cleavage

DXa inhibitors -

0 25 50 75 100 125 150 175 200 225 250 275 300

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20 Berichrom UFH

STA Liquid Heparin UFH

COAMATIC UFH

Rivaroxaban, ng/ml

Ant

i-X

a le

vel

0 10 20 30 40 50 60 70 80 90 100

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20 Berichrom UFH

STA Liquid Heparin UFH

COAMATIC UFH

Rivaroxaban, ng/ml

Ant

i-X

a le

vel

Gosselin, Moll, Adcock Ann Pharmacotherapy 2015

0 50 100 150 200 250 3000.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Apixaban Rivaroxaban

DOAC level, ng/mL

LMW

H An

ti-Xa

U/m

L

0 50 100 150 200 250 3000.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Apixaban

Rivaroxaban

DOAC, ng/mL

UFH

Anti-

Xa U

/mL

Gosselin, Adcock unpublished data

Edoxaban will be different!

UCDHS Consideration for DOAC

Creating an alternative screening panel for EDPTAPTTAnti-IIa screen (TT) – qualitative

present or absentAnti-Xa screen – qualitative

present or absent

SummaryVariability in PT and APTT response

Reference literature to guesstimate sensitivityGeneral rules may not apply

PT more sensitive to anti-Xa DOACAPTT more sensitive to anti-IIa DOAC

Determining presence of drug could be done at any laboratory using existing methodsQuantifying drug levels can be done at any laboratory with clot or chromogenic based testing using LDTsRecommendations 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 sensitivityChallenges are those patients where medication history is unavailable

????