Transcript Document

Quantifying and Estimating
Assessing DOACs
NOAC in
routine clinical Laboratory
Robert C Gosselin, CLS
UC Davis Health System
Department of Pathology and Laboratory Medicine
Sacramento, CA
[email protected]
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:
FXII
FXI
PT
FVII
APTT
FIX
FVIII
Direct Anti-Xa
FX
FXa
FII
FV
Fibrinogen
Thrombin
Fibrin
TT
Direct Anti-IIa
Expected DOAC levels
In healthy adults:
Dose
Median Cmax ng/mL [SD]
150mg
110 [24]
10mg
141 [17]
1Apixaban
2mg
460 [23]
2Edoxaban
60mg
300 [34]
1Dabigatran
1Rivaroxaban
1Mueck
et al Thromb J 2013:11:10
2Ogata,
et al J Clin Pharmacology 2010:50:743-53.
In treated patients
1Dabigatran
(n=35)
2Rivaroxaban
(n=29)
Dose
Mean Trough
level, ng/mL
(SD)
Mean Peak
level, ng/mL
(SD)
150mg bid
85 (48)
179 (104)
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.
3Becker
, et al. Thromb Haemost 2010;104:976-983
2Francart
4Weitz,
S, et al. Thromb Haemost 2014 doi 10.116-0/TH13-10-0871
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.
2.
3.
4.
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
5. 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:
Baglin T, Hillarp A, Tripodi A, et al, J Thromb Haemost 2013;11:756-60.
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)
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…
90.0
80.0
APTT, s
70.0
60.0
50.0
40.0
Actin FS
Actin
Actin FSL
SynthasIL
APTT-A
30.0
20.0
0
100
200
300
Dabigatran, ng/mL
Modified from Hawes E, et al. J Thromb Haemost 2013;11:1493-1502
400
500
600
40.0
Stago Neoplastin CI+
35.0
Recombiplastin 2G
Innovin
25.0
20.0
15.0
10.0
5.0
0.0
0
100
200
300
400
500
600
700
2.0
Rivaroxaban by LC-MS/MS, ng/mL
1.9
Modified from Francart S, et al. Thromb Haemost 2014; 111(5) Epub
Neoplastin CI+: Riva > Apix
R2G: Apix > Riva
Innovin Riva ~ Apix
Prothrombin time ratio
Prothrombin times, s
30.0
Neoplastin CI+
1.8
Recombiplastin 2G
1.7
Innovin
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0
100
200
300
400
500
Apixaban, ng/mL
Gouin-Thibault, et al Thromb Haemost 2014: 240-248
600
700
40.0
Rivaroxaban
R2 G Prothrombin time, s
35.0
30.0
25.0
Edoxaban
20.0
Apixaban
15.0
10.0
5.0
0.0
0
100
200
300
400
500
600
700
800
Anti-Xa DOAC, ng/mL
25.0
Rivaroxaban
Innovin Prothrombin time, s
Previous slide with clinicial
samples indicated:
R2G: Apix > Riva
Innovin Riva ~ Apix
20.0
Edoxaban
15.0
Apixaban
10.0
5.0
0.0
0
200
400
Anti-Xa DOAC, ng/mL
RG Unpublished data using drug enriched plasma
600
800
Concepts #2 and #3:
2. The APTT is more sensitive to Dabigatran
3. Then PT is more sensitive to anti-Xa DOACs
Dabigatran v Rivaroxaban: APTT
80.0
Dabigatran = 0.0632x + 32.788
R2 = 0.6745
A
c 70.0
t 60.0
i
50.0
n
40.0
F
30.0
S
L 20.0
Rivaroxaban = 0.026x + 27.914
R2 = 0.6977
0
100 200 300 400 500 600 700 800 900
Drug concentration, ng/ml
Dabigatran v Rivaroxaban: PT
20.0
19.0
18.0
I
n
n
o
v
i
n
Dabigatran = 0.0099x + 11.102
R2 = 0.7022
17.0
Similar
16.0
15.0
14.0
13.0
Rivaroxaban = 0.0079x + 10.962
R2 = 0.6788
12.0
11.0
10.0
0
100
200
300 400 500 600
Drug concentration, ng/ml
700
800
900
Dabigatran v Rivaroxaban: PT
R
2
G
30.0
28.0
26.0
24.0
22.0
20.0
18.0
16.0
14.0
12.0
10.0
Rivaroxaban = 0.024x + 10.91
R2 = 0.872
Dabigatran = 0.011x + 11.577
R2 = 0.5346
0
100
200
300
400
500
600
Drug concentration, ng/ml
700
800
900
DOACs concepts not always true…
Rivaroxaban: Actin APTT > Innovin PT
3.0
2.5
Actin
Ratio
2.0
Innovin
1.5
1.0
0.5
0.0
0
100
200
300
400
500
Rivaroxaban, ng/mL
600
700
Concept #4:
Thrombin time useful for excluding presence of
dabigatran
Thrombin time, s
300.0
275.0
250.0
225.0
200.0
175.0
150.0
125.0
100.0
75.0
50.0
25.0
0.0
3 reagent manufacturers
for 11 results at 10 sites
25ng/mL
0
50 100 150 200 250 300 350 400 450 500 550
Dabigatran level, ng/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
Assay
NOAC Anti-IIa
NOAC Anti-Xa
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
Falsely present
Not tested
APTT- based factor assays,
one stage
Possibly Falsely 
Possibly Falsely 
PT- based factor assays,
one stage#
Possibly Falsely 
Possibly Falsely 
No effect
Possibly Falsely 
a. FXa based
a. No effect
a. Falsely 
b. FIIa based
b. Falsely 
b. No effect
a. Clot based
a. Falsely 
a. Falsely 
b. Chromogenic
b. No effect
b. No effect
a. Markedly 
a. Falsely 
b. No effect
b. No effect
Possible to misclassify as LA
Possible to misclassify as LA
Falsely  ratio
Falsely  ratio
TCT
Bethesda assay
Chromogenic FVIII activity
AT Activity
PC Activity
PS Activity
a. Clot based
b. ELISA or LIA based
LA Tests
APCR
DRVVT formulations and DOAC interference
1.60
Siemens LA Ratio
1.40
LA1/LA2 ratio
1.20
1.00
Hematex LA ratio
0.80
0.60
0.40
0.20
0.00
0
100
200
300
400
Apixaban, ng/mL
500
600
700
So what about the SSC
recommendations for using drug
calibrators for determining reagent
sensitivity to DOACs?
25
Hyphen calibrators = 0.0184x + 10.139
R² = 0.974
PT Doubling time:
Calibrators – 395ng/mL
Patient samples – 636ng/mL
15
Patient samples = 0.0099x + 11.102
R² = 0.7022
10
5
0
0
100
200
300
400
Dabigatran, ng/mL
500
90
600
80
Hyphen calibrators = 0.0857x + 37.707
R² = 0.9962
70
APTT Doubling time:
Calibrators – 268ng/mL
Patient samples – 431ng/mL
Actin FSL APTT, s
Innovin PT, s
20
60
50
40
Patient samples = 0.0632x + 32.788
R² = 0.6745
30
20
10
Gosselin, et al Thromb Haemost 2015; 113(1):77-84
0
0
100
200
300
Dabigatran, ng/mL
400
500
600
25.0
Hyphen calibrators = 0.0173x + 10.752
R² = 0.9956
PT Doubling time:
Calibrators – 384ng/mL
Patient samples – 1007ng/mL
15.0
10.0
Patient samples = 0.006x + 11.349
R² = 0.3929
5.0
0.0
0
100
200
300
400
500
600
Rivaroxaban, ng/mL
700
800
900
80.0
Hyphen calibrators = 0.0743x + 34.093
R² = 0.9964
70.0
60.0
APTT Doubling time:
Calibrators – 349ng/mL
Patient samples – 1512ng/mL
Actin FSL APTT, s
Innovin PT, s
20.0
50.0
40.0
30.0
Patient samples = 0.0205x + 29.001
R² = 0.439
20.0
10.0
Gosselin, et al Thromb Haemost 2015; 113(1):77-84
0.0
0
100
200
300
400
500
600
Rivaroxaban, ng/mL
700
800
900
Is in-vitro drug enrichment providing truth?
Dabigatran: Comparison between sample types
120.0
Enriched NPP
100.0
Actin FS APTT, s
Hyphen calibrators
80.0
Patient samples
60.0
40.0
20.0
0.0
0
100
200
300
400
Dabigatran, ng/mL
500
600
• 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
Easy, no FDA approved kits
LAB A = 0.8453x + 11.77
R2 = 0.9475
450
Dabigatran, ng/ml
400
500
LAB B = 0.9238x - 2.4958
R2 = 0.9842
350
LAB C = 0.7845x - 4.1644
R2 = 0.9794
300
250
200
150
100
50
0
0
50
100
ECA
400
X
150
200
250
300
dTT
450
Lab D Dabigatran, ng/ml
500
350
400
450
350
300
250
200
Lab D = 0.8106x - 5.5632
R2 = 0.9367
150
100
50
0
500
0
50
100
150
BI-MS Dabigatran, ng/ml
250
300
350
400
450
500
450
500
BI-MS Dabigatran, ng/ml
200
500
LAB E = 1.0633x + 8.2818
R2 = 0.9771
450
180
400
160
350
140
Clotting time, s
Dabigatran, ng/ml
200
300
250
200
LC-MS/MS
150
120
100
80
ECT
60
100
40
50
20
0
LAB A ECT = 0.3112x + 27.999
R2 = 0.9702
0
0
50
100
150
200
250
300
350
BI-MS Dabigatran, ng/ml
Gosselin, et al Am J Clin Pathol 2014;141(2):262-7
400
450
500
0
50
100
150
200
250
300
BI-MS Dabigatran, ng/ml
350
400
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
Anti-Xa
methods
LC-MS/MS
COAMATIC
Hyphen
COAMATIC
Technoview
Berichrome
Rotachrome
BIOPHEN
R2: 0.988
m: 0.89
p <0.001
R2: 0.948
m: 0.85
p <0.001
R2: 0.975
m: 0.78
p <0.001
R2: 0.978
m: 0.86
p<0.001
R2: 0.985
m: 0.76
0.003
BIOPHEN calibration difference, ng/ml
LA Confirmatory reagent, s
120 40
110
Precision Biologics method (x) = 0.083x + 48.832
R2 = 0.8011
100 20
90
0
80
70
0
100
200
300
60 -40
50
40
400
500
600
700
800
-20
Siemens LA2 method (∆) = 0.0734x + 36.966
R2 = 0.8736
-60
30 -80
20
10
-100
0 -120
0
Anti-Xa method bias
100
200
300 [LC-MS/MS
400 + Anti-Xa
500 Method/2],
600 ng/ml
700
Mean
Rivaroxaban by LC-MS/MS, ng/ml
Gosselin, et al. Arch Pathol Lab Med 2014 Dec;138(12):1680-4
800
900
So what happens if we run
a patient on DOAC using
routine Anti-Xa methods??
Anti-Xa activity
plasma [heparin] + Excess FXa
[Antithrombin]
DXa -inhibitors
[AT]
heparin-Xa
complex + residual fXa
Chromogenic substrate
yellow color
Peptide
cleavage
Berichrom UFH
1.20
STA Liquid Heparin UFH
COAMATIC UFH
0.80
0.60
0.40
0.20
0.00
0
25
50
75
100
125 150 175 200
Rivaroxaban, ng/ml
225
250
275
300
Berichrom UFH
1.20
STA Liquid Heparin UFH
1.00
Anti-Xa level
Anti-Xa level
1.00
COAMATIC UFH
0.80
0.60
0.40
0.20
0.00
Gosselin, Moll, Adcock Ann Pharmacotherapy 2015
0
10
20
30
40
50
60
Rivaroxaban, ng/ml
70
80
90
100
1.40
UFH Anti-Xa U/mL
1.20
1.00
0.80
0.60
Apixaban
0.40
Rivaroxaban
0.20
0.00
0
100
200
300
400
500
600
700
DOAC, ng/mL
2.00
1.80
Edoxaban will be different!
LMWH Anti-Xa U/mL
1.60
1.40
1.20
1.00
0.80
Apixaban
0.60
Rivaroxaban
0.40
0.20
0.00
0
100
200
300
400
DOAC level, ng/mL
Gosselin, Adcock unpublished data
500
600
700
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
present or absent
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
????