Keywords
dabigatran - point-of-care testing - ecarin clotting time - thrombolysis - anticoagulation
Introduction
In patients treated with anticoagulants, the coagulation status must be assessed in
the emergency situation when relevant alterations of hemostasis would represent a
contraindication for intravenous thrombolysis in the case of ischemic stroke or an
indication for reversal therapy in the case of major/intracranial hemorrhage or prior
to urgent surgery.
For patients treated with vitamin K antagonists (VKAs), commercially available point-of-care
(POC) testing systems that measure prothrombin time/international normalized ratio
(INR) have proven their reliability in doing so significantly faster as compared with
laboratory-based assays, thus shortening the interval between admission and effective
therapy.[1]
[2]
In a rising number of patients treated with direct oral anticoagulants (DOACs), rapid
and accurate determination of suitable coagulation parameters remains a challenge:
laboratory-based DOAC-specific assays—such as anti-Xa activity for Xa inhibitors and
antithrombin (IIa) activity (i.e., diluted thrombin time, ecarin clotting time [ECT],
or chromogenic assays) for dabigatran—have long turnaround times and are still widely
unavailable.[3] Commercially available POC tests may be able to provide information about moderately
elevated DOAC concentrations; their diagnostic performance at low DOAC levels, however,
is limited.[4]
[5]
[6]
In this study, we evaluated the direct thrombin monitoring (DTM) test card on a Cascade
Abrazo POC device (both Helena Laboratories, Beaumont, Texas, United States). Neither
U.S. Food and Drug Administration approval nor the European CE mark has been obtained
for the DTM test cards so far. The DTM assay measures ECT and was originally devised
to monitor the anti-IIa activity of bivalirudin in patients undergoing catheter-based
coronary intervention. We hypothesized that POC-ECT may be suitable to accurately
determine dabigatran plasma concentrations in a blood sample. To our knowledge, this
study represents the first clinical evaluation of a DOAC-specific POC assay.
Methods
Anonymized source data and study protocol will be made available to other researchers
on request to the last author.
Standard Protocol Approvals
Independent review board approval was obtained prior to all study-related activity
from the Ethics Committee of Tübingen University (protocol no. 270/2015BO1). The trial
was registered at Clinicaltrials.gov under the unique identifier number NCT02825394.
Setting and Eligibility
The study was single-center prospective diagnostic study with blinded outcome assessment.
The study was conducted at the Department of Neurology and Department of Cardiology
at Tübingen University Hospital, a tertiary care facility in Germany. We planned to
include 40 patients either receiving first doses of dabigatran or being on continuous
dabigatran treatment (20 in each group). Patients being treated with other anticoagulants
(VKA within 14 days, other DOAC within 7 days, low-molecular-weight heparins [LMWHs]
within 24 hours, or unfractionated heparin [UFH] within 12 hours prior to enrolment),
spontaneously altered coagulation (INR >1.2, activated partial thromboplastin time
>37 seconds), or known coagulopathy were excluded.
Sample Collection and Measurements
Venous blood samples were taken from every patient at six prespecified time points:
before intake of the first or a regular dabigatran dose, 30 minutes, 1, 2, 8, and
12 hours after intake.
Whole blood (WB) was drawn directly into a noncitrated, nonheparinized syringe (Injekt,
B. Braun, Melsungen, Germany) and used to conduct POC-ECT within 15 seconds of sampling;
additional blood was drawn into a standard blood sampling tube for coagulation assays
(S-Monovette Citrate 3.2%, Sarstedt, Nümbrecht, Germany) to yield citrated blood (CB).
POC-ECT was conducted with CB and—after centrifugation (at 2,500 g for 15 minutes)—
with the supernatant citrated plasma (CP).
Chromogenic Biophen Direct Thrombin Inhibitor (BDTI, Hyphen BioMed, Neuville-sur-Oise,
France) assay, calibrated to yield estimated dabigatran plasma concentrations in the
standard range of 0 to 500 ng/mL, was performed on a Siemens Sysmex CS5100 instrument
(Siemens Healthcare Diagnostics, Eschborn, Germany).
Global coagulation tests as well as full blood count, electrolytes, inflammatory markers,
protein/albumin, liver, and kidney function tests were performed at baseline.
CP samples from each time point were stored at −80°C at our center, and later shipped
to the Institute for Laboratory and Transfusion Medicine at the Heart and Diabetes
Centre of the Ruhr University (Bad Oeynhausen, Germany) for ultra-performance liquid
chromatography/tandem mass spectrometry (UPLC-MS/MS), which was performed in a manner
previously described as the gold-standard method for exact determination of dabigatran
plasma concentrations.[7] Shortly, mass spectrometric measurements of dabigatran were performed by monitoring
the fragmentation of the single-charged molecular ion (dabigatran + H+) with a transition of m/z 472.2 → 289.2 for quantification of the drug. The corresponding [13C6]-isotope ([13C6-dabigatran + H+, transition of m/z 478.2 → 295.2) was used as internal standard. Furthermore, a second transition of
the single-charged dabigatran molecular ion (m/z 472.2 → 306.2) and a second transition of the single-charged internal standard ion
(m/z 478.2 → 312.2) were monitored for qualification to detect interferences, which may
be present in the complex biological matrix and which could be interfered with measurement
accuracy. In addition to UPLC-MS/MS, batch analysis of calibrated ECT was performed
using the STA-ECA II assay (Diagnostica Stago, Asnieres-sur-Seine, France) on a Siemens
BCS system (Siemens Healthcare Diagnostics, Eschborn, Germany).
All POC and laboratory-based tests were performed according to manufacturers' instructions
by thoroughly trained investigators and technicians.
Determination of POC-ECT
For POC-ECT measurements we used DTM test cards, provided by Helena Laboratories,
on a Cascade Abrazo analyzer. Underneath its transparent cover, the test card's reaction
chamber contains ecarin, calcium, buffers, and (paramagnetic) iron oxide particles.
When put into contact with a blood sample of approximately 30 µL, ecarin triggers
a coagulation reaction by conversion of prothrombin into meizothrombin, which subsequently
polymerizes fibrinogen into fibrin eventually leading to the formation of a stable
clot.[8] An oscillating magnetic field under the card produced by the analyzer causes the
iron particles to move constantly. The increasing restriction of movement caused by
the forming fibrin clot is detected by an infrared optical system and reported as
a clotting time. The presence of a thrombin inhibitor extends the clotting time proportionately
to its concentration in the blood sample.
Blinding
All POC device operators were blinded to the results of all other coagulation assays
as well as those of UPLC-MS/MS. External technicians conducting UPLC-MS/MS were blinded
to the results of all other coagulation assays (including POC-ECT) as well as patient
number and sampling time point. External technicians conducting laboratory-based ECT
were blinded to the results of all other coagulation assays and those of UPLC-MS/MS
as well as patient number and sampling time point. Fully automated laboratory-based
measurements (including BDTI) were conducted during routine operation at our central
laboratory where technicians were blinded to the results of POC-ECT as well as UPLC-MS/MS,
and laboratory-based ECT.
Calibration and Limit of Detection
To assess the feasibility and accuracy of calibrated POC-ECT measurements of dabigatran
plasma concentrations, we retrospectively analyzed 138 rethawed CP samples from two
previous studies.[4]
[5] POC-ECT values were correlated with actual dabigatran concentrations determined
by UPLC-MS/MS to yield a regression line, which was used for calculation of calibrated
POC-ECT from CP in the prospective study.
The limit of detection (LOD) was defined using POC-ECT values gained from the baseline
measurements of those patients who had not taken any dabigatran prior to any blood
samples. The estimated plasma concentration of these samples was determined using
the calibration function. Mean and standard deviation of the results were calculated.
The LOD was determined by adding three standard deviations to the mean value. For
further analyses, calibrated values below the LOD were defined as 0 ng/mL.
Interassay Variability, Interaction with Heparins and Direct Oral Xa-Inhibitors, and
Monitoring of Dabigatran Reversal Using Idarucizumab
In addition to the abovementioned experiments, we performed in vitro experiments to
determine interassay variability, interaction of UFH, LMWH, and direct oral Xa-inhibitors
with POC-ECT, and feasibility of drug reversal monitoring (for details please see
[Supplementary Methods] in the [Supplementary Material] [available in the online version]).
Statistical Analyses
Pearson's correlation coefficient was used to quantify the strength of correlation
between coagulation assay test results and UPLC-MS/MS. Diagnostic accuracy of coagulation
assays regarding detection of clinically relevant dabigatran plasma concentrations
(i.e., >30 and >50 ng/mL)[9]
[10]
[11]
[12] is expressed in terms of sensitivity, specificity, positive and negative predictive
value as well as likelihood ratio including respective 95% confidence intervals. Contrary
to prior publications from our group,[4]
[5] sensitivity is defined as the percentage of samples containing clinically relevant
dabigatran plasma concentrations that were correctly identified by the respective
coagulation assay and thus as theoretically belonging to a patient ineligible for
thrombolysis/surgery or requiring reversal therapy. Specificity is defined as the
percentage of samples containing dabigatran concentrations ≤30 ng/mL that were correctly
identified by the respective coagulation assay. Positive predictive value is defined
as the percentage of samples with dabigatran concentrations >30 ng/mL of all samples
identified as containing relevant drug levels by the respective coagulation assay
and negative predictive value is defined as the percentage of samples with dabigatran
concentrations ≤30 ng/mL of all samples identified as containing no relevant drug
levels by the respective coagulation assays. Receiver operating characteristic (ROC)
curves were drawn and the area under the ROC curve (AUROC) was calculated for each
coagulation assay. The ideal cut-off point was defined for each coagulation assay
to yield a target sensitivity of at least 95%—predefined as sufficiently safe for
clinical application—and the highest possible specificity to avoid false-negative
results but simultaneously to identify the largest number of patients eligible for
emergency treatment such as thrombolysis or emergency surgery.
The 95% confidence intervals for all proportions were calculated according to the
efficient-score method as described by Newcombe[13] using the free online VassarStats Clinical Calculator 1.[14] AUROCs were compared using the VassarStats Clinical Calculator according to suggestions
made by Hanley and McNeil.[15]
[16] SPSS version 24 (IBM, Armonk, New York, United States) was used for all other statistical
analyses as well as visualization of scatter and Bland–Altman plots and ROC curves.
Bland–Altman plots were modified following suggestions made by Krouwer.[17]
Using a two-tailed approach, an α-level of <0.05 was considered statistically significant.
This study was performed in accordance with the STARD (Standards for Reporting Diagnostic
Accuracy) guidelines for studies on diagnostic tests.[18]
Results
Clinical Study
Between September 2015 and April 2016, 42 patients gave written informed consent to
study participation. A total of 40 were enrolled in the study; 20 receiving first
dose of dabigatran and 20 already on dabigatran treatment. Baseline demographics are
summarized in [Table 1] and baseline laboratory values are summarized in [Supplementary Table S1] (available in the online version). Two patients were screened but did not meet eligibility
criteria.
Table 1
Patient baseline characteristics (N = 40)
Age
|
67 ± 14 y
|
Female sex
|
19 (47.5%)
|
Dabigatran dose
|
150 mg BD: 26 (65%)
110 mg BD: 14 (35%)
|
Body weight
|
78 ± 16.5 kg
|
Body mass index
|
27 ± 4.3 kg/m2
|
Risk factors
|
Arterial hypertension
|
27 (68%)
|
Hyperlipidemia
|
20 (50%)
|
Diabetes mellitus
|
10 (25%)
|
History of stroke
|
26 (65%)
|
Congestive heart failure
|
8 (20%)
|
Coronary heart disease or history of myocardial infarction
|
4 (10%)
|
Smoking
|
2 (5%)
|
Alcohol abuse
|
2 (5%)
|
Indication for dabigatran therapy
|
Atrial fibrillation
|
19 (48%)
|
AF ablation
|
6 (15%)
|
Stroke associated with patent foramen ovale
|
13 (33%)
|
Repeated embolic stroke of undetermined source
|
1 (3%)
|
Deep vein thrombosis
|
1 (3%)
|
Concomitant antiplatelet therapy
|
Acetylsalicylic acid
|
22 (55%)
|
Clopidogrel
|
1 (3%)
|
Abbreviations: AF, atrial fibrillation; BD, twice a day.
Note: Continuous variables are displayed as mean ± standard deviation. Nominal variables
are displayed as absolute quantity (percentage).
A total of 240 blood samples were collected and analyzed as described above. According
to UPLC-MS/MS, they contained 0 to 275 ng/mL of dabigatran. Using WB/CB/CP, POC-ECT
ranged from 20 to 186/184/316 seconds. Calibrated laboratory-based ECT yielded estimated
dabigatran concentrations of 0 to 265 ng/mL and calibrated laboratory-based BDTI assay
concentrations of 0 to 298 ng/mL. No samples were lost to analysis.
Correlation and Receiver Operating Characteristics
POC-ECT values showed a strong linear correlation with dabigatran plasma concentrations
as determined by UPLC-MS/MS (see [Fig. 1] for illustration of linear regression).
Fig. 1 Diagnostic accuracy of point-of-care ecarin clotting time (POC-ECT), laboratory-based
ECT, and laboratory-based Biophen Direct Thrombin Inhibitor (BDTI) assay. Scatter
plots illustrate the correlation between dabigatran plasma concentrations determined
by ultra-performance liquid chromatography/tandem mass spectrometry and (A) POC-ECT with whole blood (WB), (B) citrated blood (CB), (C) citrated plasma (CP) as well as (D) calibrated laboratory-based (lab.) ECT, and (E) calibrated laboratory-based (lab) BDTI. Green shaded bars indicate the treatment-relevant dabigatran plasma concentration thresholds of 30
(darker) and 50 ng/mL (lighter). Horizontal gray lines indicate suggested optimal cut-offs providing >95% sensitivity for detection of samples
containing >30 (solid) and >50 ng/mL (dashed) of dabigatran. Diagonal black lines indicate regression lines with their respective equations and the squared Pearson's
correlation coefficient (R2
) to be found in the upper left corner of the diagrams.
Correlation was highest for POC-ECT with CP (R2
= 0.92, p < 0.001; [Fig. 1C]) followed by CB (R2
= 0.90, p < 0.001; [Fig. 1B]) and WB (R2
= 0.78, p < 0.001; [Fig. 1A]). Full results of ROC analyses are summarized in [Tables 2] and [3] and [Supplementary Fig. S1] (available in the online version).
Table 2
Sensitivity and specificity regarding detection of dabigatran concentrations >30 and
>50 ng/mL
Test
|
Threshold (ng/mL)
|
(Ideal) cut-off
|
Sensitivity (%)
|
Specificity (%)
|
LR
|
PPV (%)
|
NPV (%)
|
POC-ECT WB
|
>30
|
36 s
|
95.1 [89.8–97.8]
|
81.4 [72.0–88.3]
|
5.13 [3.37–7.89]
|
88.3 [81.9–92.7]
|
91.9 [83.4–96.4]
|
>50
|
43 s
|
95.8 [89.1–98.7]
|
73.6 [65.5–80.4]
|
3.63 [2.76–4.79]
|
70.8 [62.0–78.2]
|
96.4 [90.4–98.8]
|
POC-ECT CB
|
>30
|
35 s
|
97.2 [92.5–99.1]
|
86.6 [77.8–92.4]
|
7.25 [4.37–12.04]
|
91.4 [85.5–95.2]
|
95.5 [88.1–98.5]
|
>50
|
45 s
|
97.9 [92.0–99.6]
|
60.4 [51.9–68.4]
|
2.47 [2.02–3.03]
|
62.3 [54.0–69.9]
|
97.8 [91.4–99.6]
|
POC-ECT CP
|
>30
|
45 s
|
96.5 [91.6–98.7]
|
93.8 [86.5–97.5]
|
15.60 [7.18–33.89]
|
95.8 [90.8–98.3]
|
94.8 [87.7–98.1]
|
>50
|
59 s
|
96.9 [90.5–99.2]
|
91.0 [84.8–94.9]
|
10.73 [6.38–18.04]
|
87.7 [79.6–93.0]
|
97.8 [93.1–99.4]
|
Calibrated POC-ECT
|
>30
|
28 ng/mL
|
96.5 [91.6–98.7]
|
93.8 [86.5–97.5]
|
15.60 [7.18–33.89]
|
95.8 [90.8–98.3]
|
94.8 [87.7–98.1]
|
30 ng/mL[a]
|
92.3 [86.3–95.9]
|
95.9 [89.2–98.7]
|
22.38 [8.56–58.50]
|
97.1 [92.2–99.1]
|
89.4 [81.5–94.3]
|
>50
|
50 ng/mL
|
95.8 [89.1–98.7]
|
90.1 [84.8–94.9]
|
10.62 [6.31–17.86]
|
87.6 [79.4–93.0]
|
97.0 [92.1–99.0]
|
Calibrated lab. ECT
|
>30
|
28 ng/mL
|
95.1 [89.8–97.8]
|
96.9 [90.6–99.2]
|
30.75 [10.09–93.74]
|
97.8 [93.3–99.4]
|
93.1 [85.8–96.9]
|
30 ng/mL[a]
|
94.4 [88.9–97.3]
|
96.9 [90.6–99.2]
|
30.52 [10.01–93.05]
|
97.8 [93.3–99.4]
|
92.2 [84.7–96.3]
|
>50
|
50 ng/mL
|
96.9 [90.5–99.2]
|
70.1 [61.9–77.3]
|
3.24 [2.52–4.18]
|
68.4 [59.8–75.9]
|
97.1 [91.2–99.3]
|
Calibrated lab. BDTI
|
>30
|
0 ng/mL
|
95.1 [89.8–97.8]
|
82.5 [73.1–89.2]
|
5.43 [3.52–8.37]
|
88.9 [82.5–93.2]
|
92.0 [83.6–96.4]
|
30 ng/mL[a]
|
74.1 [66.0–80.9]
|
100 [95.3–100]
|
–
|
100 [95.6–100]
|
72.4 [63.9–79.6]
|
>50
|
16 ng/mL
|
96.9 [90.5–99.2]
|
75.7 [67.7–82.3]
|
3.99 [2.98–5.33]
|
72.7 [63.9–80.0]
|
97.3 [91.8–99.3]
|
50 ng/mL[a]
|
69.8 [59.4–78.5]
|
97.2 [92.6–99.1]
|
25.13 [9.48–66.62]
|
94.4 [85.5–98.2]
|
82.8 [76.1–88.0]
|
Abbreviations: BDTI, Biophen Direct Thrombin Inhibitor assay; CB, citrated blood;
CP, citrated plasma; ECT, ecarin clotting time; lab., laboratory-based; LR, likelihood
ratio; NPV, negative predictive value; POC-ECT, point-of-care ecarin clotting time;
PPV, positive predictive value; WB, whole blood.
Note: The ideal cut-off point (in bold letters) was defined for each coagulation assay as the lowest test result yielding a target
sensitivity of at least 95% regarding detection of dabigatran concentrations >30 and
>50 ng/mL.
a For calibrated assays, all values were calculated for test results of “30 ng/mL”
and “50 ng/mL” in addition to the ideal cut-off. Sensitivity, specificity, PPV, and
NPV are displayed in % with 95% confidence intervals in square brackets. LR is displayed
with 95% confidence intervals in square brackets.
Table 3
Comparison of areas under the ROC curves for detection of dabigatran plasma levels
>30 and >50 ng/mL
Method
|
Threshold (ng/mL)
|
AUROC
|
Two-tailed p-Value
|
Lab. ECT
|
>30
|
0.99 [0.99–1.00]
|
Reference
|
>50
|
0.99 [0.99–1.00]
|
Reference
|
Lab. BDTI
|
>30
|
0.96 [0.94–0.98]
|
0.011[a]
|
>50
|
0.97 [0.95–0.99]
|
0.052
|
POC-ECT with WB
|
>30
|
0.97 [0.95–0.99]
|
0.028[a]
|
>50
|
0.95 [0.92–0.98]
|
0.011[a]
|
POC-ECT with CB
|
>30
|
0.98 [0.97–1.00]
|
0.215
|
>50
|
0.97 [0.95–0.99]
|
0.058
|
POC-ECT with CP
|
>30
|
0.99 [0.98–1.00]
|
0.700
|
>50
|
0.98 [0.97–1.00]
|
0.351
|
Abbreviations: AUROC, area under the receiver operating curve; BDTI, Biophen Direct
Thrombin Inhibitor assay; CB, citrated blood; CP, citrated plasma; ECT, ecarin clotting
time; POC-ECT, point-of-care ecarin clotting time; ROC, receiver operating curve;
WB, whole blood.
Note: This table lists the AUROCs found in [Supplementary Fig. S1] (available in the online version). We compared AUROCs of all test modalities, using
calibrated laboratory-based ECT (lab. ECT), which was performed in batch under controlled
conditions, as the reference. At the 30 ng/mL threshold, we determined the AUROC of
POC-ECT with WB and that of the calibrated laboratory-based BDTI (lab. BDTI) to be
significantly smaller while AUROC of POC-ECT with CB as well as CP did not differ
significantly from the reference. For the 50 ng/mL threshold, only POC-ECT with WB
performed significantly worse than lab. ECT. AUROC is displayed with 95% confidence
intervals in square brackets.
a Statistically significant.
Calibration and Limit of Detection
Frozen/rethawed CP samples used for retrospective POC-ECT measurements contained 0
to 371 ng/mL of dabigatran. POC-ECT values ranged from 20 to 219 seconds. Linear regression
analysis revealed a strong correlation of POC-ECT with dabigatran plasma concentrations
(R2
= 0.91). The calibration function was determined as: estimated dabigatran plasma
concentration (ng/mL) = 1.521 * POC-ECT from CP (s) – 40.369. Applying the calibration
function to dabigatran-free baseline measurements from the prospective study, the
LOD was determined to be 9 ng/mL.
Agreement of Calibrated POC-ECT and UPLC-MS/MS
Agreement of calibrated POC-ECT with CP and calibrated laboratory-based ECT and BDTI
with UPLC-MS/MS is visualized using Bland–Altman plots and mean, as well as standard
deviation of the differences between calibrated and mass-spectrometric dabigatran
concentrations was calculated both for the whole dataset and for a low concentration
range of 0 to 100 ng/mL ([Fig. 2]).
Fig. 2 Comparison of agreement with ultra-performance liquid chromatography/tandem mass
spectrometry (UPLC-MS/MS) between calibrated point-of-care ecarin clotting time (POC-ECT)
and two calibrated laboratory-based anti-IIa assays. Bland–Altman plots are used to
display the agreement level between UPLC-MS/MS and calibrated POC-ECT: (A) all data and (B) 0–100 ng/mL; calibrated laboratory-based (lab.) ECT: (C) all data and (D) 0–100 ng/mL; as well as calibrated laboratory-based (lab.) Biophen Direct Thrombin
Inhibitor (BDTI) assay: (E) all data and (F) 0–100 ng/mL; all measurements were performed using citrated plasma. Gray horizontal lines indicate a distance of 1.96 standard deviations (short-dashed) from the mean (long-dashed). Green shaded bars indicate the dabigatran plasma concentration treatment-relevant thresholds of 30
and 50 ng/mL. Diagonal red lines indicate regression curves with their respective equations and the squared Pearson's
correlation coefficient (R2
) to be found in the upper left corner of the diagrams.
Bias of calibrated laboratory-based ECT was negligible overall and in the low concentration
range. Bias of calibrated POC-ECT with CP was negligible in the low-concentration
range and slightly positive (8.5 ng/mL) overall with a trend toward overestimation
of true dabigatran plasma concentrations at higher levels. Calibrated laboratory-based
BDTI showed a negative bias overall (−11.4 ng/mL) and, more importantly, in the low
range (−12.1 ng/mL). The standard deviation of calibrated POC-ECT values from the
true dabigatran plasma concentration range was found to be higher than that of laboratory-based
ECT and comparable to that of laboratory-based BDTI.
Interassay Variability, Interaction with Heparins, Direct Oral Xa-Inhibitors and,
Idarucizumab
Assessment of interassay variability yielded a coefficient of variance of 12.3% for
normal plasma containing no dabigatran, 8.7% at 30 ng/mL, and 11.5% at 255 ng/mL for
calibrator plasma, and 8.1% at 100.6 ng/mL and 14.7% at 198.1 ng/mL for rethawed patient
CP samples. Artificial addition of therapeutic and supratherapeutic doses of UFH,
LMWH, or direct oral Xa-inhibitors did not alter the POC-ECT results (see [Supplementary Tables S2] and [S3], available in the online version). POC-ECT results accurately reflected idarucizumab-induced
reversal of anticoagulation. Plasma samples containing dabigatran bound to idarucizumab
yielded normal POC-ECT values (see [Supplementary Table S4], available in the online version).
Discussion
This study represents the first clinical study to qualitatively and quantitatively
evaluate a DOAC-specific POC coagulation assay.
Overall, POC-ECT using WB, CB, and CP is feasible and testing with all three different
sample types yielded POC-ECT values that strongly correlate with actual dabigatran
plasma concentrations as determined by UPLC-MS/MS (gold standard for dabigatran concentration
measurement) with little scattering and thus high accuracy in the clinically most
relevant concentration range around the current thresholds for thrombolysis, reversal
therapy, and emergency surgery.[9]
[10]
[11]
[12]
POC-ECT using WB and CB was performed under routine conditions at the bedside and
without relevant loss of time between sampling and testing. Regarding detection of
dabigatran plasma concentrations >30 and >50 ng/mL, accuracy of POC-ECT with CB is
comparable to that of laboratory-based ECT, which in our case was measured in batch
under stable conditions. Laboratory-based BDTI, on the other hand, did not reach this
level of performance.
For technical reasons, POC-ECT with WB is performed minimally faster than with CB.
Due to the limited stability of noncitrated, nonheparinized WB in a plastic syringe,
on the other hand, test performance may suffer (as reflected in poorer ROCs).
Accuracy of POC-ECT using CP may be comparable or even superior to POC-ECT with CB.
Due to acquisition and handling, however, use of CP is complicated and comes with
significant loss of valuable time. CB thus seems to represent the most suitable sample
type for emergency POC-ECT.
The agreement between calibrated POC-ECT and actual dabigatran plasma concentrations
shows that quantitative estimation of drug levels is possible. However, this is currently
only true for CP.
Due to the delay associated with the creation of plasma, we would encourage the use
of raw POC-ECT performed ideally with CB (or WB), and a sufficiently low cut-off ([Table 2]) to rule out the presence of clinically relevant dabigatran plasma concentrations
in patients who have not recently received any other anticoagulants influencing IIa
activity (except UFH), e.g., VKA, hirudins.
If calibrated assays are used for (emergency) decision making, test results given
in “ng/mL” cannot necessarily be taken literally and may involve significant imprecision,
as shown for the laboratory-based BDTI: despite its high correlation with UPLC-MS/MS,
we found safe cut-off values (i.e., detection of dabigatran concentrations >30 or
>50 ng/mL with >95% sensitivity) only below the test's LOD (0 ng/mL) or at “16 ng/mL,”
respectively. In contrast, for calibrated POC-ECT with CP as well as calibrated laboratory-based
ECT, the ideal cut-off values were either close (28 ng/mL) or equal (50 ng/mL) to
the real thresholds ([Table 2]).
Strengths and Limitations
POC measurements were performed at the bedside under real-life conditions. Patients
are well characterized and no artificial spiking of samples was performed for the
main analyses. Within our dataset, low to moderate dabigatran concentrations are well
represented, supporting the validity of our ROC analyses around the treatment-relevant
thresholds of 30 and 50 ng/mL. The sampling time points were chosen on the basis of
positive experience in earlier studies of our work group, where we were able to collect
samples containing a wide range of dabigatran plasma concentrations.[4]
[5]
When comparing our results to data from unspecific global coagulation POC published
by our group,[4]
[5] diagnostic performance in emergency decision making is significantly improved through
the use of POC-ECT.
A limitation of our study is that patients using other anticoagulants were excluded.
In real-life emergency situations, however, a detailed medical history of the patient
might be unavailable. To address this issue and assess the anti-IIa specificity of
the assay, we conducted measurements with samples containing dabigatran as well as
UFH, LMWH, and the currently approved direct oral Xa-inhibitors (apixaban, edoxaban,
and rivaroxaban) in high concentrations, which revealed no significant interaction
of the substances with POC-ECT.
The high anti-IIa specificity of POC-ECT assures accurate detection of dabigatran
in blood samples of a dabigatran-treated patient. Intake of other anticoagulants,
however, will still have to be excluded by the patient's history or other coagulation
assays. Unfortunately, there is currently no anti-Xa-specific POC assay available.
Despite the potential use of POC-ECT in the emergency department, we did not include
any patients in the emergency setting. The nonemergency setting was chosen to ensure
fast patient recruitment and feasibility of multiple POC measurements per patient
allowing for the collection of baseline blood samples as well as samples containing
a wide spectrum of dabigatran plasma concentrations.
The ideal cut-offs found in our dataset were defined retrospectively and do not necessarily
translate to other batches of POC-ECT test cards. To enable the safe use of POC-ECT
(with CB or WB) as an emergency diagnostic tool in the future, the manufacturer will
either have to provide respective POC-ECT cut-offs for the (current) treatment-relevant
dabigatran plasma concentration thresholds or offer a CB- or WB-based calibrated POC-ECT.
POC-ECT in the Context of Acute Stroke Care
Given the high diagnostic accuracy of the DTM test card-based POC-ECT in detecting
relevant plasma concentrations of dabigatran, we deem it suitable to be integrated
in the acute stroke care work flow, where POC-INR has already become the diagnostic
standard in VKA-treated patients. Pivotal features of the device and assay are the
portability of the battery powered Cascade Abrazo POC device, the possible use of
WB (noncitrated or citrated) without the need for centrifugation, and the fast availability
of test results as longer intervals would affect admission-to-treatment times.
At this moment in time, other POC systems are less suited for this task: rotational
thromboelastometry (ROTEM) and thromboelastography (TEG) have also been evaluated
for POC coagulation assessment in DOAC-treated patients as well as monitoring of dabigatran
reversal with idarucizumab (in vitro and in a porcine polytrauma model).[19]
[20]
[21]
[22] The size of respective devices, however, limits transportability and the duration
of measurements is much longer compared with the POC-ECT method. Furthermore, sensitivity
to low plasma concentrations, which are relevant for guiding emergency decision making,
has generally been reported as being low, at least if the reagents used are not DOAC-specific.
Akman and colleagues have suggested that dabigatran reversal using idarucizumab can
be monitored using ROTEM and TEG,[22] while a study by Takeshita and colleagues indicated that incomplete reversal of
dabigatran might not be accurately reflected by ROTEM.[21] One recent evaluation of a ROTEM method using a thrombin trigger showed promising
results regarding speed of measurements and correlation between clotting times and
dabigatran plasma concentrations in an in vitro study. The data acquired in a small
clinical substudy, however, were only analyzed qualitatively and for a threshold,
which is not regarded as relevant for clinical decision making.[23] A thorough prospective quantitative and quantitative clinical evaluation of ROTEM/TEG-based
POC coagulation testing in DOAC-treated patients, including clinically relevant safe-for-treatment
thresholds, is—to the best of our knowledge—still lacking.
A completely different approach is taken by a recently developed urine test, which
is designed to provide information about the presence or absence of a urine concentration
of dabigatran or FXa inhibitor above 95 ng/mL. The advantage of this assay is that
it will detect both substance classes simultaneously. The duration of this merely
qualitative measurement, however, is also more than 10 minutes and it naturally does
not allow for determination of drug plasma concentration and consequently the level
of anticoagulation.[24]
Previous publications of our group have shown that determination of DOAC plasma concentrations
(including dabigatran) is possible within certain limits. As a linear correlation
between POC test results and actual DOAC plasma concentrations is not sufficient for
qualitative estimation, POC testing using global coagulation assays is only able to
provide qualitative information about the presence of DOAC, however, with high accuracy
for predefined clinically relevant thresholds.[4]
[5]
[6] Using the suggested decision model, a relevant percentage of patients who may receive
intravenous thrombolysis (or in which reversal therapy is unnecessary) is safely identified.
The Cascade Abrazo currently lacks an anti-FXa specific coagulation assay. The so-called
ENOX test card is currently under evaluation (NCT02825394).
Conclusion
This study represents the first qualitative and quantitative evaluation of DOAC-specific
POC coagulation testing in dabigatran-treated patients. Correlation of POC with UPLC-MS/MS
results is excellent and—through calibration of plasma-based measurements—allows for
quantitative estimation of actual dabigatran plasma concentrations.
POC-ECT is suitable for distinguishing between dabigatran concentrations of above
and below 30 and 50 ng/mL, potentially enabling clinicians to reliably detect or exclude
the presence of relevant drug levels in the emergency setting without unnecessary
loss of time. POC-ECT is not influenced by UFH, LMWH, or direct oral Xa-inhibitors
and accurately reflects normalization of coagulation following dabigatran reversal
with idarucizumab.
What is known about this topic?
-
In stroke patients or prior emergency surgery, rapid assessment of coagulation status
is necessary to guide thrombolysis or reversal of anticoagulation.
-
Available point-of-care global coagulation assays do only provide (limited) qualitative
assessment, but do not allow for quantitative coagulation testing in patients treated
with direct oral anticoagulants including the thrombin inhibitor dabigatran.
What does this paper add?
-
This is the first study evaluating point-of-care ecarin clotting time (POC-ECT).
-
POC-ECT accurately reflected dabigatran plasma concentrations and is not influenced
by other anticoagulants.
-
POC-ECT could improve clinical care of dabigatran-treated patients by making fast
and precise assessment of coagulation available to emergency physician.