1. No category

Use of Thromboelastography (TEG) for Detection
of New Oral Anticoagulants
Joao
˜ D. Dias, PhD; Katherine Norem, BA; Derek D. Doorneweerd, PhD; Robert L. Thurer, MD; Mark A. Popovsky, MD;
Laurel A. Omert, MD
Context.—The clinical introduction of new oral anticoagulants (NOACs) has stimulated the development of tests
to quantify the effects of these drugs and manage
complications associated with their use. Until recently,
the only treatment choices for the prevention of venous
thromboembolism in orthopedic surgical patients, as well
as for stroke and systemic embolism in patients with atrial
fibrillation, were vitamin K antagonists, antiplatelet drugs,
and unfractionated and low-molecular-weight heparins.
With the approval of NOACs, treatment options and
consequent diagnostic challenges have expanded.
Objective.—To study the utility of thromboelastography
(TEG) in monitoring and differentiating between 2 currently approved classes of NOACs, direct thrombin
inhibitors (dabigatran) and factor Xa inhibitors (rivaroxaban and apixaban).
Design.—Blood samples from healthy volunteers were
spiked with each NOAC in both the presence and absence
of ecarin, and the effects on TEG were evaluated.
Results.—Both the kaolin test reaction time (R time) and
the time to maximum rate of thrombus generation were
prolonged versus control samples and demonstrated a dose
response for apixaban (R time within the normal range)
and dabigatran. The RapidTEG activated clotting time test
allowed the creation of a dose-response curve for all 3
NOACs. In the presence of anti-Xa inhibitors, the ecarin
test promoted significant shortening of kaolin R times to
the hypercoagulable range, while in the presence of the
direct thrombin inhibitor only small and dose-proportional
R time shortening was observed.
Conclusions.—The RapidTEG activated clotting time test
and the kaolin test appear to be capable of detecting and
monitoring NOACs. The ecarin test may be used to
differentiate between Xa inhibitors and direct thrombin
inhibitors. Therefore, TEG may be a valuable tool to
investigate hemostasis and the effectiveness of reversal
strategies for patients receiving NOACs.
(Arch Pathol Lab Med. 2015;139:665–673; doi: 10.5858/
arpa.2014-0170-OA)
T
agents, including unfractionated heparin and low-molecular-weight heparin.2
Currently licensed NOACs are dabigatran (Pradaxa;
Boehringer Ingelheim International GmbH, Ingelheim,
Germany), rivaroxaban (Xarelto; Bayer Pharma AG, Leverkusen, Germany, and Janssen Pharmaceuticals, Inc,
Titusville, New Jersey), and apixaban (Eliquis; Bristol-Myers
Squibb, New York, New York, and Pfizer EEIG, Sandwich,
United Kingdom). All of these agents, and others in
development, are under investigation for the management
of multiple thromboembolic disorders.3–10 Dabigatran is an
oral direct inhibitor of thrombin (factor IIa) that is ‘‘not
permanent,’’ selective, and competitive. Rivaroxaban and
apixaban are oral direct inhibitors of factor Xa and are also
competitive, selective, and not permanent.11 Dabigatran is
the only NOAC administered as a prodrug and metabolized
to the active form. All 3 agents are licensed in the European
Union and the United States to reduce the risk of venous
thromboembolism in orthopedic surgical patients, as well as
for stroke and systemic embolism in patients with nonvalvular atrial fibrillation. Furthermore, rivaroxaban is
approved in the European Union for the secondary
prevention of acute coronary syndrome. Rivaroxaban can
be administered in combination with acetylsalicylic acid or
with acetylsalicylic acid plus clopidogrel or ticlopidine for
he introduction of new oral anticoagulants (NOACs) has
changed the management of patients with venous and
arterial thromboembolic diseases. Unlike traditional oral
vitamin K antagonists, NOACs are given at fixed doses and
have a lower potential for drug and food interactions,
eliminating the requirement for routine laboratory monitoring.1,2 These novel agents show similar or improved
efficacy and safety profiles compared with vitamin K
antagonists such as warfarin and established parenteral
Accepted for publication July 11, 2014.
From Clinical Marketing, Haemonetics SA, Signy, Switzerland (Dr
Dias); Operations Department (Ms Norem) and Scientific Research &
Biomedical (Dr Doorneweerd), Haemonetics Corporation, Rosemont, and Medical Affairs, Haemonetics Corporation, Chicago (Dr
Omert), Illinois; Hospital Division (Dr Thurer) and Medical Affairs
(Dr Popovsky), Haemonetics Corporation, Braintree, Massachusetts;
and Department of Surgery, Einstein Medical Center, Philadelphia,
Pennsylvania (Dr Omert). Dr Omert is now with Acquired Bleeding,
CSL Behring, King of Prussia, Pennsylvania.
This work was supported by Haemonetics Corporation, Rosemont,
Illinois.
Drs Dias, Doorneweerd, Thurer, and Popovsky and Ms Norem are
employees of Haemonetics Corporation. Dr Omert was an employee
of Haemonetics Corporation at the time of the study.
Reprints: Joa˜ o Dias, PhD, Haemonetics SA, PO Box 262, 1274
Signy-Centre, Switzerland (e-mail: [email protected]).
Arch Pathol Lab Med—Vol 139, May 2015
Use of TEG for Detection of NOACs—Dias et al 665
Figure 1. Illustration of a thromboelastography tracing and accompanying parameters. A, Depiction of a thromboelastography tracing and
parameters measured throughout the life span of a clot. B, Thrombus generation curve (V-curve in green) overlaying a thromboelastography tracing.
A V-curve is plotted from the first derivative of changes in clot resistance, expressed as a change in clot strength per unit of time (dynes/cm2/s),
representing the maximum velocity of clot formation. Abbreviations: ACT, activated clotting time; a, a angle; K, coagulation time; Ly30, percentage
lysis 30 minutes after maximum amplitude; MA, maximum amplitude; MRTG, maximum rate of thrombus generation; R, reaction time; TMRTG, time
to maximum rate of thrombus generation.
the prevention of thrombotic events in adult patients with
elevated cardiac biomarkers after a coronary event.12
The NOACs present management challenges to both
clinicians and laboratory personnel when patients develop
bleeding diatheses. In some cases, there are no useful
methods to detect and monitor these agents, and no
‘‘antidotes’’ are available to reverse their effects. Miyares
and Davis13 recently reviewed the usefulness and sensitivity
of current coagulation assays for dabigatran, rivaroxaban,
and apixaban. The most easily available tests for emergency
situations, prothrombin time and partial thromboplastin
time, were described as ‘‘not ideal’’ with the exception of the
partial thromboplastin time for dabigatran.13 Less widely
available tests, including thrombin time, ecarin clotting
time, Heptest (American Diagnostica, Stamford, Connecticut), prothrombinase-induced clotting time, and chromogenic factor IIa, either do not correlate well with drug levels
or are cumbersome and lengthy to perform. The difficulty of
managing trauma patients receiving dabigatran was highlighted by Cotton et al in an editorial in which the authors
emphasized that ‘‘. . .there is no readily available means for
assessing the degree of anticoagulation with dabigatran,
there is no readily available reversal strategy, and lifethreatening bleeding complications can occur after an injury
in patients taking this drug.’’(pp2039–2040)
Viscoelastic measurements of coagulation provided by
tests such as thromboelastography (TEG) are increasingly
being used to assess trauma patients who arrive in shock
secondary to massive bleeding, as well as for acute care of
surgical patients with bleeding diatheses. Thromboelastography is widely used as a management tool for cardiac
surgery and transplant patients and provides information
to guide the administration of blood products.15 Thromboelastography is able to detect both low-molecularweight and unfractionated heparins and, with the use of
a heparinase cup, can illustrate whether the effects of these
agents have been completely reversed. Furthermore, the
666 Arch Pathol Lab Med—Vol 139, May 2015
TEG PlateletMapping assay (Haemonetics Corporation,
Braintree, Massachusetts) is used to quantify the response
to antiplatelet therapies, including clopidogrel and aspirin,
that can be used in combination with NOACs. Thromboelastography assays using ecarin have been used to monitor
recombinant hirudin and bivalirudin during cardiac surgery.16,17
In this study, we investigated whether TEG could detect
dabigatran, rivaroxaban, and apixaban in low, normal, and
high doses using spiked blood samples from healthy
volunteers. In addition, we tested an assay to differentiate
these agents from each other.
MATERIALS AND METHODS
We studied 3 NOACs, dabigatran, rivaroxaban, and apixaban,
using blood from 14 healthy volunteer donors. For each NOAC
tested, citrated blood from 3 donors was spiked with 3 different
concentrations of the active drug. The spiked blood samples and
control samples spiked with diluent were tested with the TEG 5000
Thrombelastograph hemostasis analyzer (Haemonetics Corporation) using kaolin and RapidTEG (rTEG) (Haemonetics Corporation) reagents. Each sample was run in triplicate. All samples were
tested with and without ecarin (Enzyme Research Laboratories,
South Bend, Indiana). The work in this study was institutional
review board approved, and all donors were 18 years or older and
signed informed consent forms.
Sample Preparation
Blood was drawn using standard venipuncture technique and a
Vacutainer push-button collection set (Becton, Dickinson and
Company, Franklin Lakes, New Jersey) with a 21-gauge needle.
Blood was spiked and tested within 2 hours of being drawn.
Dabigatran stock was prepared from the active dabigatran
moiety (Alsachim, Illkirch Graffenstaden, France) by dissolution
in 0.1M hydrogen chloride and further dilution in 1:1 dimethyl
sulfoxide and water. The final stock used to spike the blood had a
concentration of 20 ng/lL in 0.1M hydrogen chloride, dimethyl
sulfoxide, and water. Tubes of citrated blood were spiked with this
Use of TEG for Detection of NOACs—Dias et al
Arch Pathol Lab Med—Vol 139, May 2015
Use of TEG for Detection of NOACs—Dias et al 667
14.4
8.9
8.4
6.8
17
13.9
11.1
5.7
Rivaroxaban, ng/mL
500
89
22
Control
Dabigatran, ng/mL
500
200
50
Control
6
6
6
6
6
6
6
6
6
6
6
6
0.8¥¥,¤¤¤,***
0.5^^^,***
0.2***
0.4
1.1¥¥¥,¤¤¤,***
0.5**
0.7
0.4
0.8***
0.4***
0.3***
0.3
No Ecarin
13.5
10.6
6.7
3.3
2.7
2.5
2.3
2.3
1.6
1.2
1.6
1.2
6
6
6
6
6
6
6
6
6
6
6
6
0.4¥¥,¤¤¤,***
0.6^^^,***
0.3***
0.1
0.1¤¤,**
0.1
0.1
0.1
0.2¤,**
0.0
0.2
0.0
With Ecarin
.001§
.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
P Value
3.0
2.6
2.1
1.5
2.7
2.1
2.1
1.7
2.9
2.4
2.3
1.6
6
6
6
6
6
6
6
6
6
6
6
6
0.15¤,***
0.15^,***
0.13**
0.05
0.21¥,**
0.18
0.16
0.19
0.23***
0.18**
0.18*
0.18
No Ecarin
2.2
1.5
1.3
1.2
1.6
1.3
1.3
1.0
1.1
1.0
1.1
1.0
6
6
6
6
6
6
6
6
6
6
6
6
0.20¥¥,¤¤¤,***
0.06^^,**
0.03
0.08
0.13**
0.12*
0.08*
0.05
0.08
0.08
0.08
0.09
With Ecarin
K Time, min
.004§
,.001§
,.001§
.004§
.001§
.003§
.001§
.004§
,.001§
,.001§
,.001§
.01§
P Value
6
6
6
6
6
6
6
6
6
6
6
6
53.4
58.2
59.2
67.5
55.4
61.8
61.7
66.5
52.9
57.4
61.7
68.4
1.65¥,¤¤,***
1.32^,***
1.35***
0.71
2.31**
2.24
2.52
2.26
2.60**
1.93**
2.10*
2.13
No Ecarin
61.6
68.9
71.8
73.0
67.3
70.7
71.4
73.6
74.8
75.3
75.3
75.3
6
6
6
6
6
6
6
6
6
6
6
6
2.27¥¥,¤¤¤,***
0.50^^^,**
0.34
0.94
1.90*
1.85
1.30
1.02
1.34
0.97
1.13
1.08
With Ecarin
a Angle, 8
.007§
,.001§
,.001§
.001§
.001§
.008§
.004§
.01§
,.001§
,.001§
,.001§
.005§
P Value
7.34
8.32
10.53
12.72
8.82
11.19
10.69
13.67
7.81
9.17
9.57
14.21
6
6
6
6
6
6
6
6
6
6
6
6
0.37¤¤,***
0.55^,***
0.72*
0.64
0.67**
1.05
1.03
1.24
0.73**
0.81*
0.94*
1.92
No Ecarin
Extended
10.54
13.91
15.31
17.13
16.01
17.62
18.5
20.62
20.25
19.67
20.23
19.64
6
6
6
6
6
6
6
6
6
6
6
6
0.98¥¥,¤¤¤,**
0.32^^,*
0.31
1.34
1.74
1.75
1.46
1.76
1.70
1.45
1.68
1.51
With Ecarin
MRTG, mm/min
Table 1.
.008§
,.001§
,.001§
.009§
.001§
.006§
,.001§
.003§
,.001§
,.001§
,.001§
.04§
P Value
19.51
15.81
12.77
7.52
16.72
10.69
10.09
8.06
13.64
11.2
9.86
6.68
6
6
6
6
6
6
6
6
6
6
6
6
0.83¥¥,¤¤¤,***
0.70^^,***
0.31***
0.46
1.16¥¥¥,¤¤¤,***
0.48*
0.67*
0.45
0.69¥¥,¤¤¤,***
0.53***
0.52***
0.38
No Ecarin
15.13
11.87
7.76
4.11
4.08
3.72
3.54
3.30
2.25
2.20
2.18
2.05
6
6
6
6
6
6
6
6
6
6
6
6
0.44¥¥,¤¤¤,***
0.72^^^,***
0.31***
0.10
0.14¥,¤¤,***
0.08***
0.04**
0.05
0.04**
0.05
0.05
0.05
With Ecarin
TMRTG, min
,.001§
.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
P Value
Abbreviations: K Time, coagulation time; MRTG, maximum rate to thrombus generation; R Time, reaction time; TMRTG, time to maximum rate of thrombus generation.
a
Statistically significant between ¥ (higher dose and medium dose), ¤ (higher dose and lower dose), ^ (medium dose and lower dose), and * (control). Standard error of 3 independent experiments
measured in triplicate. Single symbol ¤, ^, and * indicates P , .05; 2 symbols ¥¥, ¤¤, ^^, and ** indicate P , .01; and 3 symbols ¥¥¥, ¤¤¤, ^^^, and *** indicate P , .001.
§ Statistically significant between paired sample with or without ecarin.
11.0
9.2
9.3
5.5
Apixaban, ng/mL
1000
500
250
Control
R Time, min
Thromboelastography Kaolin Test Coagulation Parameters’ Sensitivity in Healthy Donor Spiked Samples With Different Doses of Apixaban, Rivaroxaban,
and Dabigatran in the Presence or Absence of Ecarina
Variable
Table 1.
dabigatran stock to create final concentrations of 500, 200, and 50
ng/mL of citrated whole blood. Dabigatran is approved for the
prevention of venous thromboembolism following elective knee or
hip replacement (220 mg/d for patients without renal impairment
and 150 mg/d for patients with moderate renal impairment) and for
the prevention of stroke in patients with renal impairment and
atrial fibrillation in the United States (at a reduced dose of 75 mg/
d).18,19 A 150-mg oral dose of dabigatran has a maximum plasma
concentration of 110 ng/mL.20,21
Rivaroxaban stock was prepared by agitation of a 20-mg
rivaroxaban tablet in a 1:1 dimethyl sulfoxide and water solution,
which was diluted to a final concentration of 20 ng/lL of rivaroxaban
in 1:1 dimethyl sulfoxide and water. Tubes of citrated blood were
spiked with this rivaroxaban stock to create final concentrations of
500, 89, and 22 ng/mL in citrated whole blood. Rivaroxaban is
approved for the prevention of stroke and systemic embolism in
adults with nonvalvular atrial fibrillation (20 mg/d in the European
Union and the United States), as well as for the treatment of deep
venous thrombosis and pulmonary embolism and for the
prevention of recurrent deep venous thrombosis and pulmonary
embolism in adult patients (15 mg twice daily for 3 weeks,
followed by 20 mg/d, in the European Union and the United
States).12,22 An oral dose of 10 mg of rivaroxaban has a maximum
plasma concentration of 141 ng/mL.21,23 Apixaban stock was
prepared in a similar manner from a 2.5-mg apixaban tablet, with
final concentrations of 1000, 500, and 250 ng/mL in whole blood.
Apixaban is approved for the prevention of venous thromboembolism in elective hip or knee replacement surgery (2.5 mg twice
daily) and for the prevention of stroke and systemic embolism in
patients with nonvalvular atrial fibrillation (5 mg twice daily).24 An
oral dose of 20 mg of apixaban has a maximum plasma
concentration of 460 ng/mL.21,25
Control samples were prepared for each tested drug. These
included a solvent control containing only citrated blood and the
diluent used to dilute the drug stock, as well as an unadulterated
citrate blood tube.
Thromboelastography
Testing was performed on TEG 5000 analyzers (Haemonetics
Corporation) using kaolin tubes, 0.2M calcium chloride, rTEG vials,
diluent water, and disposable clear cups and pins provided by the
manufacturer (Haemonetics Corporation), as well as ecarin. All
testing was performed in triplicate at each dose and allowed to
continue until the maximum amplitude (MA) parameter had defined.
The various components of the TEG tracing are shown in Figure
1, A. The kaolin test generates a reaction time (R time) parameter,
which is measured in minutes, and is the time elapsed from the
initiation of the test until the point where the onset of clotting
provides enough resistance to produce a 2-mm amplitude reading
on the TEG tracing. This parameter represents the initiation phase
of coagulation related to the function of enzymatic clotting factors.
The R time parameter has a normal range of 5 to 10 minutes. A
prolonged R time indicates slower clot formation. K is a
measurement of the interval from the split point to the point
where fibrin cross-linking provides enough clot resistance to
produce a 20-mm amplitude reading measured in minutes. The
a angle is the angle formed by the slope of a tangent line traced
from the R time to the coagulation time (K time) and a central line
measured in degrees. The K time and the a angle denote the rate at
which the clot strengthens and are representative of thrombin’s
cleaving of the available fibrinogen into fibrin. The MA indicates
the point at which clot strength reaches its MA, measured in
millimeters on the TEG tracing, and reflects the end result of
maximum platelet-fibrin interaction via the glycoprotein IIb-IIIa
receptors.26
The rTEG test incorporates both tissue factor and kaolin to
generate the conventional kaolin parameters, as well as the TEG
activated clotting time (ACT) parameter, which is measured in
seconds. The TEG ACT is equivalent to the activated clotting time27
and has a normal range of 86 to 118 seconds. A prolonged TEG
ACT time indicates slower clot formation. In addition, velocity
668 Arch Pathol Lab Med—Vol 139, May 2015
curves derived from the above-mentioned kaolin and rTEG tests
were plotted using TEG software (Haemonetics Corporation).
These curves represent the speed of clot propagation (maximum
rate of thrombus generation [MRTG] and the time to MRTG
[TMRTG]) (Figure 1, B).
In the kaolin test, 1 mL of citrated blood sample was mixed with
kaolin, and a 340-lL aliquot of this blood was added to a TEG cup
containing 20 lL of 0.2M calcium chloride for recalcification. The
kaolin with ecarin test was performed in a similar fashion using 20
lL of an ecarin and calcium chloride solution (0.16M calcium
chloride and 19 Endotoxin Units/mL [EU/mL] of ecarin). In the
rTEG test, the reagent was reconstituted with 20 lL of diluent
water and allowed to stand for 5 minutes per the manufacturer’s
instructions. Ten microliters of this reconstituted reagent was
added to the TEG cup with 20 lL of 0.2M calcium chloride for
recalcification. A 360-lL aliquot of the citrated blood sample was
added to the cup with these 2 reagents, and the contents of the cup
were mixed 3 times by drawing the contents of the cup up into the
pipette and redispensing it into the cup. The test was started
immediately after mixing and allowed to run until the MA
parameter had defined. The rTEG with ecarin test was performed
as the rTEG test above using 20 lL of an ecarin and calcium
chloride solution (0.16M calcium chloride and 19 EU/mL of ecarin).
Statistical Analysis
Statistical analyses were performed using a 2-tailed Student t
test. For all analyses, P , .05 was deemed statistically significant.
RESULTS
Kaolin Test
The R time, K time, a angle, and MRTG parameters in the
kaolin test achieved statistical significance only for the
higher concentrations of rivaroxaban (Table 1 and Figure 2,
A) but were able to detect the presence of all tested
concentrations of apixaban (P .04) (Table 1 and Figure 2,
B) and dabigatran (P .04) (Table 1 and Figure 2, C). In
addition, for all drugs the TMRTG parameter was statistically different between the control group and all tested
concentrations. Furthermore, the R time, a angle, and
TMRTG parameters for the dabigatran samples were
significantly different between all concentrations, indicating
an appropriate dose response (Table 1 and Figure 2, C).
Finally, the MA values from the kaolin test for rivaroxaban
and dabigatran did not change with the addition of the
studied NOACs compared with the control, illustrating the
lack of effect of these agents on platelet-fibrin contribution
to clot strength. However, apixaban tracing at a concentration of 250 ng/mL demonstrated that the MA was
significantly different from the control group (P ,
.001) but was still within the normal range (data on
file).
rTEG Test
The TEG ACT parameter for all tested drugs in the rTEG
test was significantly different between the control group
and all tested concentrations of rivaroxaban, apixaban, and
dabigatran (Table 2 and Figure 2, D through F) with the
exception of the rivaroxaban concentration of 22 ng/mL (P
¼ .58) (Table 2 and Figure 2, D). Furthermore, the TEG
ACT parameter was able to distinguish between concentrations of rivaroxaban (Table 2 and Figure 2, D) and
dabigatran (Table 2 and Figure 2, F), indicating a good
dose-response curve. The K time, a angle, and MRTG
parameters for both apixaban and rivaroxaban from the
rTEG test did not show any statistical difference between
the control or between studied concentrations (Table 2).
Use of TEG for Detection of NOACs—Dias et al
Figure 2. The thromboelastography kaolin test reaction time (R Time) sensitivity as a function of different concentrations of rivaroxaban (A),
apixaban (B), and dabigatran (C) and RapidTEG activated clotting time (ACT) test time sensitivity as a function of different concentrations of
rivaroxaban (D), apixaban (E), and dabigatran (F). The R times for all doses of dabigatran, as well as the highest concentrations of apixaban and
rivaroxaban, tested significantly higher than the expected normal range. The ACT times for all concentrations of dabigatran, as well as the normal and
higher concentrations of apixaban and rivaroxaban, tested significantly higher than the normal range for ACT. The dotted parallel bars show the
normal ranges of R times. Statistically significant between ¥ (higher dose and medium dose),¤ (higher dose and lower dose), ^ (medium dose and
lower dose), and * (control). ¥¥, ^^, ** P , .01 and ¥¥¥, ¤¤¤, ^^^, *** P , .001. Error bars represent the standard error of 3 independent
experiments measured in triplicate.
However, the K time for the dabigatran group was
statistically different from the control for the lower tested
concentrations (P ¼ .003 for 200 ng/mL and P ¼ .003 for 350
ng/mL) but not for the concentration of 500 mg/mL (P ¼
.44), and the a angle from the dabigatran group was
statistically different from the control for the concentrations of 500 ng/mL (P , .001) and 50 ng/mL (P , .001) but
not for the concentration of 200 ng/mL (P ¼ .38) (Table 2).
In addition, both the K time and a angle were able to
differentiate between the highest dabigatran concentration
Arch Pathol Lab Med—Vol 139, May 2015
and the other concentrations (P ¼ .002 for 500 versus 200
ng/mL and P , .001 for 500 versus 50 ng/mL) (Table 2).
Furthermore, the MRTG parameter was sensitive to the 2
lowest concentrations of dabigatran (P ¼ .06 for 500 ng/mL,
P ¼ .002 for 200 ng/mL, and P , .001 for 50 ng/mL). The
TMRTG parameter from the rTEG test is sensitive to the
presence of both rivaroxaban and dabigatran but not
apixaban (Table 2). Furthermore, the TMRTG parameter is
able to differentiate between concentrations of dabigatran
(P , .001 for 500 versus 200 ng/mL and P , .001 for 200
Use of TEG for Detection of NOACs—Dias et al 669
670 Arch Pathol Lab Med—Vol 139, May 2015
Use of TEG for Detection of NOACs—Dias et al
154.4
119.8
110.2
108.6
342.4
237.6
146.6
108.6
Rivaroxaban, ng/mL
500
89
22
Control
Dabigatran, ng/mL
500
200
50
Control
6
6
6
6
6
6
6
6
6
6
6
6
17.6¥¥¥,¤¤¤,***
10.8^^^,***
4.5***
2.3
3.4¥¥¥,¤¤¤,***
2.4^^,**
2.3
1.9
4.7¥¥,¤¤¤,***
3.4***
2.3***
2.2
No Ecarin
612.7
400.4
275.7
170.1
215.9
194.3
202.9
175.2
171.8
162.2
147.5
148.4
6
6
6
6
6
6
6
6
6
6
6
6
37.3¥¥¥,¤¤¤,***
20.3^^,***
25.0**
16.4
7.8**
12.8
8.3*
5.8
4.1¤¤,***
4.9*
5.8
2.9
With Ecarin
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
,.001§
P Value
6
6
6
6
2.1
0.9
0.9
1.7
6
6
6
6
1.26
1.1 6
1.2 6
1.5 6
1.8
1.4
1.6
1.9
0.32¥¥,¤¤
0.02**
0.02**
0.25
0.08
0.05
0.11
0.19
0.20
0.18
0.21
0.29
No Ecarin
1.5
1.0
0.9
2.2
1.0
0.9
1.0
1.1
1.3
1.2
1.0
1.1
6
6
6
6
6
6
6
6
6
6
6
6
0.08¥¥¥,¤¤¤
0.05^^
0.04
0.65
0.07
0.07
0.07
0.15
0.04¤¤,**
0.05*
0.06
0.04
With Ecarin
K Time, min
.12
.004§
.62
.51
.21
.04§
.12
.11
.02§
.25
.02§
.008§
P Value
6
6
6
6
6
6
6
6
6
6
6
6
68.9
72.0
70.9
68.8
74.8
75.5
75.3
73.3
65.1
75.7
78.4
74.2
2.04¥¥,¤¤¤,**
1.44
0.29***
0.78
0.76
0.67
1.03
1.40
1.91
2.18
2.29
2.87
No Ecarin
65.0
71.1
75.8
73.4
75.1
76.0
75.3
73.8
66.9
67.9
66.9
67.9
6
6
6
6
6
6
6
6
6
6
6
6
2.56¤¤,*
1.56^
0.91
1.81
0.84
0.63
0.78
1.37
1.82
2.00
1.79
2.28
With Ecarin
a Angle, 8
.97
.04§
.02§
.68
.80
.59
.99
.81
.45
.19
.19
.80
P Value
11.99
21.17
20.84
14.63
16.3
16.61
16.69
14.13
12.68
15.1
13.90
12.43
6
6
6
6
6
6
6
6
6
6
6
6
1.08¥¥¥,¤¤¤
1.54**
0.48***
0.74
0.94
0.59
1.02
1.13
1.60
1.36
1.23
1.77
No Ecarin
Extended
14.72
19.13
22.24
15.23
18.48
18.81
20.95
19.38
18.64
19.78
16.64
17.76
6
6
6
6
6
6
6
6
6
6
6
6
0.45¥¥¥,¤¤¤
0.62^,*
0.88***
1.45
1.11
1.66
1.32
1.60
1.83
1.83
1.51
2.09
With Ecarin
MRTG, mm/min
Table 2.
.03§
.24
.18
.72
.15
.23
.02§
.02§
.03 §
.06
.18
.07
P Value
4.15
2.78
1.68
1.27
1.90
1.69
1.55
1.15
2.07
1.90
2.00
1.50
6
6
6
6
6
6
6
6
6
6
6
6
0.24¥¥¥,¤¤¤,***
0.11^^^,***
0.06**
0.11
0.08¤,***
0.11***
0.14**
0.02
0.21
0.16
0.20
0.07
No Ecarin
7.59
4.63
3.27
1.70
2.71
2.46
2.47
2.04
2.46
2.22
2.32
1.99
6
6
6
6
6
6
6
6
6
6
6
6
0.45¥¥¥,¤¤¤,***
0.24^^,***
0.27***
0.18
0.10***
0.12**
0.12**
0.08
0.10**
0.09
0.09*
0.07
With Ecarin
TMRTG, min
,.001§
,.001§
,.001§
.05
,.001§
,.001§
,.001§
,.001§
.12
.10
.16
.07
P Value
Abbreviations: ACT, activated clotting time; K Time, coagulation time; MRTG, maximum rate to thrombus generation; TMRTG, time to maximum rate of thrombus generation.
a
Statistically significant between: ¥ (higher dose and medium dose), ¤ (higher dose and lower dose), ^ (medium dose and lower dose), and * (control). Standard error of 3 independent experiments
measured in triplicate. Single symbol ¤, ^, and * indicates P , .05; 2 symbols ¥¥, ¤¤, ^^, and ** indicate P , .01; and 3 symbols ¥¥¥, ¤¤¤, ^^^, and *** indicate P , .001.
§ Statistically significant between paired sample with or without ecarin.
144.0
122.4
118.0
103.3
Apixaban, ng/mL
1000
500
250
Control
ACT, s
Rapid Thromboelastography Test Coagulation Parameters’ Sensitivity in Healthy Donor Spiked Samples With Different Doses of Apixaban, Rivaroxaban,
and Dabigatran in the Presence or Absence of Ecarina
Variable
Table 2.
versus 50 ng/mL) (Table 2). Finally, the MA values of the
rTEG test for rivaroxaban and apixaban did not change
with the addition of the studied drug concentrations
compared with the control, and only the MA values of
the dabigatran 500-ng/mL concentration were significantly
different from the control group (P , .001) but were still
within the normal range (data on file).
Ecarin Test
The addition of ecarin to the kaolin test caused a
significant decrease in the R time, K time, and TMRTG
values for both the treated and control groups (P .003 for
rivaroxaban, P .01 for apixaban, and P .004 for
dabigatran) (Table 1 and Figure 3, A through C) and a
significant increase in the a angle and MRTG values for both
the treated and control groups (P .01 for rivaroxaban, P .04 for apixaban, and P .009 for dabigatran) (Table 1).
Furthermore, the addition of ecarin to the kaolin test in the
presence of anti-Xa drugs severely decreases the R times to
the hypercoagulable range (, 5 minutes), with no statistical
difference from the control with the exception of the higher
studied dosages: these values for rivaroxaban were P ¼ .002
for 500 ng/mL, P ¼ .08 for 89 ng/mL, and P ¼ .90 for 22 ng/
mL and for apixaban were P ¼ .03 for 1000 ng/mL, P ¼ .76
for 500 ng/mL, and P ¼ .05 for 250 ng/mL (Table 1 and
Figure 3, A and B), while in the presence of dabigatran there
is only a dose-related decrease in the R time (Table 1 and
Figure 3, C). The addition of ecarin to the kaolin test did not
change the MA values of the samples for dabigatran or
rivaroxaban relative to samples run without ecarin, but in
the presence of apixaban the MA values were statistically
different (P ¼ .02 for 1000 ng/mL, P ¼ .009 for 500 ng/mL,
and P , .001 for 250 ng/mL) from samples run without
ecarin but were still within the normal range (data on file).
The addition of ecarin to the rTEG test significantly
increases the TEG ACT times for both anti-Xa and direct
thrombin inhibitor (DTI) drugs (P , .001 for apixaban, P
.001 for rivaroxaban, and P , .001 for dabigatran), as
well as the TMRTG times in the presence of both
rivaroxaban and dabigatran (P , .001 for rivaroxaban
and P , .001 for dabigatran), increasing the hypercoagulable status (Table 2). On the other hand, the rTEG a
angle did not change for any of the studied concentrations when ecarin was added in the presence of
rivaroxaban or apixaban. However, for the lowest
concentrations of dabigatran, there was a decrease in
the a angle (P ¼ .047 for 200 ng/mL and P ¼ .02 for 50 ng/
mL) (Table 2). The rTEG K times significantly decreased
for the highest and lowest concentrations of apixaban (P
¼ .02 for 1000 ng/mL and P ¼ .17 for 250 ng/mL) and the
middle concentration of rivaroxaban (P ¼ .04 for 89 ng/
mL) but increased in the middle concentration of
dabigatran (P ¼ .004 for 200 ng/mL) when ecarin was
added. Finally, the addition of ecarin to the rTEG test
significantly decreased the MA value of only the 200-ng/
mL concentration of dabigatran (P ¼ .02) but was still
within the normal range (data on file).
COMMENT
The lack of a readily available method to determine the
degree of anticoagulation creates a major challenge to
clinicians treating bleeding patients who are potentially
receiving NOACs. Moreover, the potentially irreversible
coagulopathy associated with their use is of great concern
Arch Pathol Lab Med—Vol 139, May 2015
to trauma and emergency physicians. Therefore, we
studied the effect of the currently approved NOAC drugs
(dabigatran, rivaroxaban, and apixaban) on TEG and the
ability of the ecarin test to distinguish a DTI from anti-Xa
drugs using TEG. We observed that both R time and
TMRTG parameters in the kaolin test were sensitive to
apixaban and dabigatran. Furthermore, it was found that
the rTEG test ACT parameter is sensitive to all 3 NOACs
tested. Finally, in the presence of anti-Xa inhibitors, the
ecarin test promoted significant shortening of kaolin R
times to the hypercoagulable range, while in the presence
of DTI only a small and dose-proportional R time
shortening was observed, allowing to distinguish the
presence of DTI from anti-Xa inhibitors.
Although the drugs investigated in this study are clinically
effective, they present challenges in monitoring and
difficulties in reversal should bleeding occur. Dabigatran
has a half-life of 12 to 17 hours, which is lengthened in
patients with renal dysfunction.18 The high renal clearance
of dabigatran (80%) precludes its use in the European Union
for patients with severe renal insufficiency, although it is
indicated for the prevention of stroke in patients with severe
renal impairment experiencing atrial fibrillation in the
United States (at a reduced 75-mg daily dose).18,19 Apixaban
and rivaroxaban have shorter half-lives than dabigatran.
However, apixaban also has an increased half-life of up to
44% in patients with severe renal impairment compared
with healthy volunteers.28 Furthermore, the presence of
moderate hepatic and renal impairment may lead to
significant changes in the pharmacokinetics of rivaroxaban
(mean, 2.3-fold increase in the area under the curve
compared with healthy volunteers), and there are no data
in patients with severe hepatic impairment.12 Rivaroxaban
and apixaban, which are eliminated in greater proportions
via other nonrenal routes, may be used with caution in such
patients.12,24
For patients, part of the attractiveness of NOACs is that
they do not require regular blood testing. Unlike warfarin,
however, there are currently no antidotes for NOAC
reversal. Dabigatran can only be partially removed from
circulation by dialysis.18,19 There is no established way to
reverse the anticoagulant effect of apixaban or rivaroxaban,
which can be expected to persist for at least 10 to 30 hours
after the last dose (ie, for about 2 half-lives). Hemodialysis
does not have a substantial impact on anti-Xa drug
levels.12,22,24 Activated charcoal reduces apixaban and
rivaroxaban absorption, reducing the plasma half-life;
however, the reductions are limited.12,22,24 Therefore, bleeding can be extremely resistant to known therapies. The
Randomized Evaluation of Long-term Anticoagulant Therapy (RE-LY trial) reported a 1.45% per year incidence of lifethreatening bleeding or death related to bleeding complications from treatment with dabigatran, and this led to
safety advisories being issued in several countries.6,13
Agents that may be used to reverse the effects of
dabigatran such as the activated prothrombin complex
concentrate (FEIBA; Baxter, Deerfield, Illinois) and the
recently approved 4-component prothrombin complex
concentrate (Kcentra; CSL Behring, King of Prussia,
Pennsylvania) may be effective, but without laboratory
guidance the dosing is difficult, and thrombosis may
result.28,29
The most commonly used coagulation tests, prothrombin
time and partial thromboplastin time, can detect the
presence of dabigatran but are lacking in sensitivity,
Use of TEG for Detection of NOACs—Dias et al 671
Figure 3. The thromboelastography kaolin test reaction time (R Time)
as a function of drug concentrations in the presence or absence of
ecarin for rivaroxaban (A), apixaban (B), and dabigatran (C).
Rivaroxaban and apixaban both show an equivalent and significant
quickening of the R time to a hypercoagulable status, despite the
concentration of drug. Dabigatran has a concentration-dependent
decrease in the R time. The dotted parallel bars show the normal ranges
of the R times for normal donors. Statistically significant between ¥
(higher dose and medium dose), ¤ (higher dose and lower dose), ^
(medium dose and lower dose), and * (control). § Statistically
significant between paired sample with or without ecarin (P .001).
Error bars represent the standard error of 3 independent experiments
measured in triplicate. ¤ P ¼ .02; ¤¤, ¥¥, ** P , .01; and ¤¤¤, ^^^,
*** P , .001.
especially the prothrombin time, which is only increased at
concentrations that are higher than therapeutic. Other
assays such as ecarin clotting time and chromogenic anti–
factor IIa are not readily available and/or have not been
specifically studied with dabigatran. For the anti-Xa
inhibitors rivaroxaban and apixaban, chromogenic assays
672 Arch Pathol Lab Med—Vol 139, May 2015
against human factor Xa are preferred.30,31 Although anti–
factor Xa chromogenic assays can provide accurate results
over a wide range of rivaroxaban concentrations, the
addition of exogenous antithrombin results in falsely
elevated results, suggesting unsuitability for use with
rivaroxaban.32 Furthermore, a recent trial investigated
interlaboratory variability of the measurement of rivaroxaban plasma concentrations with anti–factor Xa chromogenic assays and demonstrated an interlaboratory variation
greatest at lower concentrations.33 Although the prothrombin time results are prolonged in the presence of
rivaroxaban or apixaban, the results are reagent dependent,
making the test unreliable for use in actively bleeding
patients.30,31
Thromboelastography can provide a quick determination
of the effects of dabigatran, rivaroxaban, and apixaban
using either the rTEG or citrated kaolin assays. Both
illustrate prolongation of the enzymatic phase of coagulation with prolonged TEG ACT in the rTEG assay and a long
R time in the kaolin assay. In trauma patients who may
present with hemorrhagic shock and are either suspected
or known to have taken NOACs, the persistence of the
long R time/ACT following resuscitation and correction of
surgical bleeding can demonstrate persistent NOAC
effects. Furthermore, a universal reversal agent for antiXa inhibitors is currently in development (Andexanet Alfa,
PRT4445; Portola Pharmaceuticals, Inc, San Francisco,
California). The dosing of this drug could potentially be
monitored by TEG to avoid potential adverse effects.
Furthermore, patients receiving NOACs who require
emergent procedures are not well served by conventional
coagulation tests and can be successfully treated when TEG
is used to guide therapy.34
Some initial studies14,34,35 have identified the TEG kaolin
test as useful in monitoring the effects of dabigatran.
However, in the case of rivaroxaban, both the TEG kaolin
test and the ROTEM (Tem International GmbH, Munich,
Germany) INTEM and EXTEM tests lacked sensitivity.35,36
Our findings support this because we observed that a
reagent that tests both intrinsic and extrinsic pathways
(rTEG) is more sensitive to the presence of anti-Xa
inhibitors than single-pathway reagents. If the specific
oral anticoagulant is not known, an ecarin assay can be
performed to differentiate between dabigatran and the
anti-Xa inhibitors rivaroxaban or apixaban. The patients on
dabigatran will only have a dose-proportional shortening
of the R time, while the patients on anti-Xas will have a
dramatic shorting of the R times back to the control level
independently of the dose used. Thus, our findings support
the need to develop a new reference range in healthy
volunteers for the kaolin plus ecarin test to facilitate the
distinction when it is not known whether the patient is
receiving a DTI or an anti-Xa.
In conclusion, we conducted an in vitro characterization of
the effects of the currently approved NOACs on clot
formation using the TEG 5000. Thromboelastography is
sensitive to the effects of NOACs and can potentially be
used to guide NOAC reversal therapies. While the role of
TEG requires additional clinical validation, it may be a
valuable tool to investigate both the hemostatic derangements and the effects of reversal strategies in patients
treated with NOACs.
We thank Daniel P. Herbstman, BA, for his service to
Haemonetics Corporation, Rosemont, Illinois.
Use of TEG for Detection of NOACs—Dias et al
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