AJCP / Original Article
Performance of Various Laboratory Assays in the
Measurement of Dabigatran in Patients Receiving
Therapeutic Doses
A Prospective Study Based on Peak and Trough Plasma Levels
Robert Gosselin, CLS,1 Emily Hawes, PharmD,2 Stephan Moll, MD,3 and Dorothy Adcock, MD4
From the 1Department of Medical Pathology and Laboratory Medicine, UC Davis Medical Center, Sacramento, CA; 2Department of Pharmacy, University
of North Carolina Hospitals and Clinics, Chapel Hill; 3Department of Medicine, University of North Carolina School of Medicine, Chapel Hill; and
4Esoterix, Englewood, CO.
Key Words: Dabigatran; Direct thrombin inhibitor; Mass spectrophotometry; Ecarin clotting time; Ecarin chromogenic assay; Dilute
thrombin time
DOI: 10.1309/AJCPRNUMI4PVSJ7Q
ABSTRACT
Objectives: To study dabigatran etexilate, a new oral
anticoagulant that functions as a direct thrombin inhibitor.
Methods: This study evaluates four methods, one of which
is performed in three different laboratories, and compares
results against dabigatran levels measured by BoehringerIngelheim (Ingelheim, Germany) using mass spectrometry.
Results: Although routine monitoring is not required,
measurement of plasma concentrations may be necessary in
certain clinical situations. Routine coagulation assays such
as the prothrombin time, activated partial thromboplastin
time, and thrombin time do not reliably determine levels
of dabigatran anticoagulation. Alternative assays, when
calibrated with a dabigatran standard, such as the modified
dilute thrombin time, ecarin clotting time, and ecarin
chromogenic assay, may be appropriate, although a
comparison of these methods using samples from patients
taking dabigatran has not been performed.
Conclusions: Although results using all methods in this study
demonstrate adequate correlation, measured dabigatran
levels varied in a statistically significant manner, even
when the same method was used by different laboratories.
The clinical significance of this variation in dabigatran
concentrations is uncertain.
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Dabigatran extilate (Pradaxa; Boehringer-Ingelheim,
Ingelheim, Germany) is a novel oral anticoagulant that belongs
to the direct thrombin inhibitor class of anticoagulants. Dabigatran’s mechanism of anticoagulant action is through inhibition of free, fibrin-bound, and clot-bound thrombin. It is
approved by the US Food and Drug Administration for stroke
reduction in individuals with atrial fibrillation not caused by
a heart valve problem. Unlike warfarin, dabigatran has predictable pharmacodynamic and pharmacokinetic profiles, has
fewer drug-drug interactions compared with warfarin, is not
affected by diet, requires no bridging therapy, and is distinct
from other direct thrombin inhibitors (eg, bivalirudin) because
it does not require routine monitoring. Patients receiving
dabigatran therapy may, however, require assessment of
plasma drug concentration due to unforeseen circumstances,
such as suspicion of overdose, the occurrence of bleeding,
or in situations of increasing renal insufficiency, emergency
surgery, or trauma. Published studies have illuminated the
limitations of routine coagulation tests, such as prothrombin
time (PT), activated partial thromboplastin time (APTT), and
thrombin time (TT), to determine the relative concentration
of plasma dabigatran.1-3 Alternative assays, when calibrated
with a dabigatran standard, such as the dilute TT (dTT), ecarin
clotting time (ECT), ecarin chromogenic assay (ECA), or the
prothrombinase-induced clotting time, have been suggested
methods to quantify the amount of dabigatran in plasma.1-5
It is not known, however, whether these methods provide
comparable results. In this study, we assessed four different means for measuring dabigatran levels compared with
a standard method in patients administered dabigatran for
thromboprophylaxis.
© American Society for Clinical Pathology
AJCP / Original Article
Materials and Methods
The study was approved by the University of North
Carolina Institutional Review Board for human subject
experimentation. Detailed study methods have been described
elsewhere, and the data presented here are a further analysis
of data from that study.3 In brief, a total of 35 participants,
taking 150 mg dabigatran twice daily at steady state, provided
written consent and were enrolled in the study. Blood was collected by standard phlebotomy techniques into 3.2% sodium
citrate tubes for trough (just before dose) and peak (2.5 hours
after dose) dabigatran levels. Once collected, the blood was
processed within 2 hours, centrifuged to obtain platelet-poor
plasma, aliquoted into cryovials, and stored at –70°C until
analysis. Each stored sample was labeled with a study ID
number with designation of “trough” or “peak” level. Frozen
samples were distributed to testing laboratories via express
delivery in Styrofoam containers with ample dry ice to ensure
maintenance of a frozen state. Frozen samples were sent to
a central laboratory for predicate dabigatran measurements
using mass spectrometry (courtesy of Boehringer-Ingelheim,
Ingelheim, Germany [designated BI-MS]) and to five expert
laboratories based in the United States.
Reagents, calibration materials, instrumentation used,
and trough/peak dabigatran results were provided by each
participating site. Of the five participating laboratories reporting dabigatran levels, three used the ECA method,6 one
applied a commercial dTT method,1 and one employed liquid
chromatography/tandem mass spectrometry (LC-MS/MS).
One site that reported the ECA also provided results using
the classic ECT method.7 Laboratory sites A, B, and C used
a commercially prepared ECA (ECA-T Kit; Diagnostica
Stago, Asnieres, France), and laboratory D used commercially
prepared dTT (HEMOCLOT; Hyphen-BioMed, NeuvilleSur-Oise, France). All ECA and dTT methods used the same
dabigatran calibrator source and control material (Dabigatran
Calibrator Plasma and Dabigatran Control Plasma; HyphenBioMed) but not necessarily the same lot of materials. Laboratory E used an internally validated LC/MS-MS measurement
for dabigatran levels. Laboratory A also performed a classic
noncalibrated ECT, and for this reason, results are reported as
clotting times and do not quantify dabigatran concentration.
All values were reported in nanograms/milliliters of drug,
except for ECT, which was reported in seconds. Laboratory
A also calibrated the ECT using aforementioned dabigatran
calibrators to determine whether the ECT quantification of
dabigatran would be equivalent to other methods.
Regression analysis was used to determine the correlation
between the predicate method (BI-MS) and other methods,
with an R2 greater than 0.95 indicating acceptable agreement.
Biases between the reporting laboratory and predicate method
were assessed using the Bland-Altman calculation (bias =
laboratory – [laboratory + BI-MS]/2). The paired t test P
value of less than .05 was used to determine whether statistically significant differences exist between methods. Comparison evaluation between laboratory and predicate dabigatran
measurement methods was determined for (1) all measured
dabigatran (trough and peak) values, (2) expected dabigatran
trough levels based on the RE-LY trial (95th percentile range,
~40-240 ng/mL),8 and (3) changes in drug concentrations
between trough and peak measures.
Results
Thirty-five pairs (trough and peak levels) of samples
were drawn for each enrolled participant. The laboratory
testing matrix included three ECAs on two different analyzers, one dTT assay, one ECT, and one LC/MS-MS method
❚Table 1❚.
❚Table 1❚
Testing Matrix Used for Determining Dabigatran Levels
Laboratory
SiteMethod
Reagent
Calibrator
Instrument
A
Chromogenic
ECA-T Kit (Diagnostica Stago, Dabigatran Calibrator Plasma STA Compact (Diagnostica Stago)
ecarin assay Asnieres, France) (Hyphen-BioMed, Neuville- Sur-Oise, France)
A
Ecarin clotting time
Ecarin Reagent None
STart 4 Hemostasis Analyzer
(Diagnostica Stago)
(Diagnostica Stago)
B
Chromogenic ECA-T Kit (Diagnostica Stago) Dabigatran Calibrator Plasma BCS XP System (Siemens Healthcare
ecarin assay (Hyphen-BioMed) Diagnostics, Marburg, Germany)
C
Chromogenic ECA-T Kit (Diagnostica Stago) Dabigatran Calibrator Plasma STA-R Evolution (Diagnostica Stago)
ecarin assay (Hyphen-BioMed)
D
Dilute thrombin time
HEMOCLOT (Hyphen-BioMed) Dabigatran Calibrator Plasma STA Compact (Diagnostica Stago)
(Hyphen-BioMed)
E
Liquid chromatography, In-house developed
mass spectrometry
© American Society for Clinical Pathology
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For the chromogenic ecarin assays, Pearson correlation
was 0.948, 0.984, and 0.979 for laboratories A, B, and C,
respectively ❚Figure 1A❚, but all sites varied significantly (P <
.001) from the predicate BI-MS method. Bland-Altman bias
plots demonstrate that most of these result differences are
directional, indicating that overall, the ECA method underestimates drug level compared with the BI-MS method ❚Figure
1B❚. There was no statistical improvement when comparing
the range of expected trough dabigatran levels ❚Table 2❚.
When comparing the change in drug concentration between
trough and peak drug concentration, only laboratory B demonstrated no significant differences (P = .10) (when compared
with changes between drug concentrations as measured by
BI-MS). When comparing among laboratories that report the
ECA, there were no significant differences between values
reported between laboratories A and B (P = .10).
500
450
400
350
300
250
200
150
100
50
0
B
Lab A
Lab B
Lab C
Lab D
Lab E
Lab A
Lab B
Lab C
60
40
ECA Dabigatran
Difference (ng/mL)
Dabigatran (ng/mL)
A
There was borderline correlation (R2 = 0.937) and
statistically significant differences (P < .001) between the
commercial dTT kit (laboratory D) and BI-MS–measured
dabigatran levels (Figure 1A). The bias plots indicate that
values determined using the dTT method are primarily
lower than BI-MS measurements, with increasing bias as
drug concentration increases ❚Figure 1C❚. There was no
observed improvement in correlation or drug concentration
differences between this dTT and predicate BI-MS when
evaluating those samples in the expected trough range of
results (~40-240 ng/mL) or the observed changes between
trough and peak drug levels (Table 2). Results obtained
with the dTT method demonstrated no significant differences (P = .54) between this method and the chromogenic
ecarin method for only one of the three laboratories that
performed the chromogenic method.
20
0
–20 0
50 100 150 200 250 300 350 400 450 500 550
–40
–60
–80
–100
–120
0
50
100 150 200 250 300 350 400 450 500
Mean (Lab + BI-MS)/2 (ng/mL)
BI-MS Dabigatran (ng/mL)
100
75
50
25
0
–25 0
–50
–75
–100
–125
–150
–175
D
50 100 150 200 250 300 350 400 450 500
Mean (Lab D + BI-MS)/2 (ng/mL)
Lab E LC/MS-MS Dabigatran
Difference (ng/mL)
Lab D dTT Dabigatran
Difference (ng/mL)
C
100
90
80
70
60
50
40
30
20
10
0
–10 0
50 100 150 200 250 300 350 400 450 500
BI-MS Dabigatran (ng/mL)
❚Figure 1❚ A, Regression analysis between laboratories A, B, and C performing the ecarin chromogenic assay (ECA); laboratory
D performing dilute thrombin times (dTT); and laboratory E performing liquid chromatography/tandem mass spectrometry
(LC-MS/MS) for determining dabigatran levels and predicate measurements using Boehringer-Ingelheim (Ingelheim, Germany)
mass spectrometry (BI-MS). Laboratory A = 0.85x + 11.8; R2 = 0.948. Laboratory B = 0.92x – 2.58; R2 = 0.984. Laboratory C
= 0.78x – 4.16; R2 = 0.979. Laboratory D = 0.81x – 5.56; R2 = 0.937. Laboratory E = 1.06x + 8.28; R2 = 0.977. B-D, An overlay
of Bland-Altman–style plots demonstrating the bias between (B) each respective laboratory site’s ECA-determined dabigatran
concentration and predicate mass spectrometry quantitation of dabigatran concentration, (C) dTT-determined dabigatran
concentration and predicate mass spectrometry quantitation of dabigatran concentration, and (D) LC/MS-MS–determined
dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration.
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© American Society for Clinical Pathology
AJCP / Original Article
❚Table 2❚
Statistical Analysis for Each Laboratory Testing Sitea
Laboratory
Characteristic
A BC D E
All samples (n = 70)
Pearson correlation
0.948
0.984
0.979
0.937
0.977
P value (paired t test) <.001
<.001
<.001
<.001
<.001
Samples 40-240 ng/mL (n = 57)
Pearson correlation
0.855
0.962
0.956
0.849
0.937
P value (paired t test)
<.001
<.001
<.001
<.001
<.001
Difference in drug concentration between trough and peak levels
Pearson correlation
0.933
0.958
0.966
0.836
0.937
P value (paired t test)
<.001
.10
<.001
.03
.57
Testing laboratories A, B, and C used the ecarin chromogenic assay, laboratory D used dilute thrombin times, and laboratory E used liquid chromatography/tandem mass
spectrometry for measuring dabigatran levels. Each site was compared with dabigatran levels obtained from the parent drug company using Boehringer-Ingelheim (Ingelheim,
Germany) mass spectrometry (BI-MS), with a P value of less than .05 indicating significant differences between methods. A subset analysis of samples between 40 and 240 ng/
mL dabigatran by BI-MS would reflect the 90th percentile of trough levels in patients receiving a 150-mg twice-daily regimen. The difference between trough and peak drug
concentration represents the absolute change in drug value between these time frames.
There was acceptable correlation (R2 = 0.977) for laboratory E using LC/MS-MS compared with the predicate BI-MS
values (Figure 1A). Bias plots indicate that the LC/MS-MS
method of laboratory E overestimates drug concentration (P
< .001) compared with BI-MS ❚Figure 1D❚. There was no statistical improvement between methods when evaluating drug
levels from 40 to 240 ng/mL, but no statistically significant
difference between methods (P = .57) was observed when
assessing the change between trough and peak drug concentrations (Table 2).
There was acceptable correlation (R2 = 0.970) between
BI-MS and the ECT, but a relatively flat slope ❚Figure 2❚
was evident. As expected, there were significant differences
between BI-MS, which measures drug concentration, and
ECT, which merely reports time in seconds. These differences did not improve when evaluating expected trough levels
or differences between trough and peak samples. There was
good correlation (R2 = 0.962) but significant differences (P <
.001) between dabigatran levels obtained from calibrated ECT
and BI-MS measurements. The ECT-dabigatran results correlated with other laboratory methods, with R2 values ranging
from 0.874 (laboratory A) to 0.934 (laboratory E). There were
no significant differences between ECT-dabigatran levels and
those levels reported by laboratory C using ECA (P = .43) and
laboratory D using LC-MS/MS (P = .23).
measure. While the use of a dTT for measuring dabigatran
has been advocated by some, these data suggest that the chromogenic ecarin method is also acceptable when both assay
methods are calibrated using a dabigatran standard.1,3,9,10
Significant differences in dabigatran concentrations were
demonstrated between some laboratories in this study, despite
the fact that they used the same methods and calibration
source. It is unclear why there were significant differences
between laboratory sites that used ECA methods, even though
the same calibrator source was used. The only two significant
Dabigatran (ng/mL) or ECT (s)
a
350
325
300
275
250
225
200
175
150
125
100
75
50
25
0
–25 0
–50
ECT-Dabigatran
ECT
50 100 150 200 250 300 350 400 450 500
BI-MS Dabigatran (ng/mL)
Discussion
This multisite study used different methods of determining dabigatran concentration in the plasma of patients treated
with 150 mg dabigatran twice daily. All methods demonstrate
a linear relationship with drug concentrations determined
by BI-MS, which in this study is considered the predicate
© American Society for Clinical Pathology
❚Figure 2❚ Regression analysis of a single site performing
both classic ecarin clotting time (ECT); y = 0.311x + 28.0; R2
= 0.970) and dabigatran levels obtained from calibrated ECT;
y = 0.920x – 25.48; R2 = 0.962) compared with dabigatran
levels measured by Boehringer-Ingelheim (Ingelheim,
Germany) mass spectrometry (BI-MS).
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differences between assays at the ECA testing sites were (1)
use of a 0-ng/mL calibrator, not included in the commercial
dabigatran calibrator kit, and (2) instrumentation. Although
the 0-ng/mL calibrator may enhance the sensitivity to lower
drug concentrations (<30 ng/mL), this would not affect results
greater than 30 ng/mL. These data suggest that subtle differences in processing, reading, calibration, accuracy, and result
extrapolation between instrumentation, possibly even within
the same instrument manufacturer, may account for differences seen in ECA measurements.
This study is distinct from other published findings in
several ways. Douxfils and colleagues6 used pooled normal
plasma spiked with different concentrations of dabigatran, but
the calibration of dTT and ECA curves is unclear. Another
study measuring dabigatran levels used local laboratory-prepared calibrators to compare levels obtained using either dTT
or ECT.11 In the study reported by Samama and colleagues,12
the observed dabigatran concentration in patients receiving
dabigatran after major orthopedic surgery was similar between
dTT and ECA when using the same instrumentation and
calibrator at a single testing center. Current recommendations
from the Scientific and Standardization Committee (SSC) of
the International Society for Thrombosis and Haemostasis
indicate that either the APTT or a dabigatran-calibrated dTT
can be used to estimate drug level.10 This SSC report also
notes that ECT is sensitive and linear to 300 ng/mL of dabigatran, but it takes no position on chromogenic ecarin methods
to determine dabigatran levels. We also demonstrated that the
classic ECT, when calibrated with dabigatran calibrators, correlates well with other dabigatran measurement methods and
therefore may also be considered a laboratory-developed (and
equivalent) method for determining dabigatran levels.
To our knowledge, this is the first multisite comparison of methods for measuring dabigatran concentration in
patients receiving the drug for thromboprophylaxis. Recently,
a European-based proficiency assessment survey program
(ECAT; Leiden, the Netherlands) incorporated plasma samples containing novel anticoagulants, including dabigatran, as
part of the program. For the 2012 ECAT proficiency dabigatran sample survey, 26 participating laboratories determined
dabigatran levels under the category “anti-IIa assay” (which
is a chromogenic prothrombin inactivation assay similar to
the anti-Xa used to measure heparin), one laboratory used
the ECT, and 36 used the dTT. No participating laboratories
used mass spectrometry. The coefficients of variation associated with anti-IIa and dTT methods were 8.3% to 14.3%
and 11.9% to 18.9%, respectively, with the acceptable result
range of 235 to 410 ng/mL and 85 to 200 ng/mL for samples
containing approximately 300 ng/mL and 100 ng/mL, respectively.13 This proficiency survey data would suggest that
variability for dabigatran measurements is not uncommonly
observed in coagulation testing.
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There are some limitations to our findings. First, the
chromogenic ecarin method was used by most laboratories.
This is in contrast to ECAT findings that suggest that dTT and
anti-IIa methods are more abundant. However, the ECAT proficiency assessment program does not offer ECA as a method
option, and perhaps those laboratories using ECA are recording under the chromogenic method (anti-IIa) in lieu of a clotbased method (dTT). In our study, only a single site used the
dTT method for reporting dabigatran levels, and none used a
chromogenic prothrombin inactivation assay. The calibrators
used by each ECA testing site are optimized for dTT methods
and not ECA per the manufacturer’s instructions. However,
the calibrator values reported for these calibrators are assessed
using BI-MS and thus could be used in different methods,
assuming that the lyophilization processing of this material
does not interfere with the testing method. Indeed, our data
would suggest that these dTT-optimized commercial calibrators are suitable for ECA methods and measuring dabigatran
concentrations in plasma.
In conclusion, we find good correlation between existing laboratory methods (ECT, ECA, and dTT) and predicate
BI-MS measurements for dabigatran, but significant differences in measured dabigatran concentrations exist between
laboratories and methods. It is unclear whether these differences have any clinical significance or impact, since this drug
is not routinely monitored For those clinical situations that
may require virtual absence of drug, employing a more sensitive measure for assessing dabigatran (eg, TT testing) should
be considered.
Address reprint requests to Dr Adcock: 8490 Upland Dr, Suite
100, Englewood, CO 80112-7116.
Acknowledgments: We thank Cheryl Jeanneret, University
of North Carolina at Chapel Hill, McAllister Heart Institute,
for patient enrollment; Herbert Whinna, University of North
Carolina at Chapel Hill, Department of Pathology and Laboratory
Medicine; Mike Taylor, Esoterix Coagulation, and Anne Winkler,
Emory University, Department of Pathology, for dabigatran
results provided from their respective institution; Joann van
Ryn, Boehringer-Ingelheim, for providing predicate dabigatran
measurements; and Abigail Cook, Loyola University Health
System, for assistance with study design.
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