10.4081/btvb.2022.37
Direct oral anticoagulants (DOAC) measurement is recommended in specific conditions. A point-of-care testing should be used in emergency to qualitatively rule out relevant DOAC concentrations. The DOAC-CHECK Study aims to evaluate whether the use of CoaguChek® Pro II (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) coagulation testing device can provide reliable information in patients treated with DOAC. The study was carried out in two FCSA (Italian Federation of Thrombosis Centers) centers. We choose 3 different concentration thresholds for our analysis (30, 50 and 100 ng/mL) and by ROC curves the ideal cut-off point was selected to be the one that yielded a sensitivity of at least 95% associated with the highest possible specificity. 512 patients were enrolled. For Edoxaban and Rivaroxaban, both CoaguChek® Pro II prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests showed a sensitivity &gt;95% corresponding to satisfying specificity values; negative predictive values resulted in the range 90-100%. At variance, CoaguChek® Pro II PT and aPTT tests did not seem to be useful for identifying Apixaban and Dabigatran concentrations higher than the pre-defined thresholds. Our results suggest that CoaguChek® Pro II coagulation testing device can be used to qualitatively identify relevant concentrations of Edoxaban or Rivaroxaban, but not of Apixaban or Dabigatran.
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Gualtiero Palareti
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Bleeding, Thrombosis and Vascular Biology
POCT
Evaluation of Coaguchek®Pro II coagulation testing device performance to assess direct oral anticoagulant action. The DOAC-CHECK study
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Original Articles
2022-12-01
2022-07-08
2022-10-17
2022-12-01
Bleed Thromb Vascul Biol
Gualtiero Palareti
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Evaluation of Coaguchek®Pro II coagulation testing device performance to assess direct oral anticoagulant action. The DOAC-CHECK study
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1
                                                                                                           ORIGINAL ARTICLE

Evaluation of Coaguchek®Pro II coagulation testing device performance
to assess direct oral anticoagulant action. The DOAC-CHECK study
                                                                            Cristina Legnani,1 Michela Cini,1 Sophie Testa,2
                                                                            Alberto Tosetto,3 Claudia Dellanoce,2
                                                                            Stefania Bellesso,3 Giuseppe Carli,3 Ilaria Nichele,3
 Correspondence: Cristina Legnani, Fondazione Arianna Anticoa-
                                                                            Laura Lissandrini,3 Serena Zorzi,1 Emilia Antonucci,1
 gulazione, Via Paolo Fabbri, 1/3, 40138 Bologna, Italy.                    Gualtiero Palareti1
 Tel.: +39.051.2812339 - Fax +39.051.343604.
 E-mail c.legnani@fondazionearianna.org                                     Arianna Anticoagulazione Foundation, Bologna;
                                                                            1

                                                                            Haemostasis and Thrombosis Center, Laboratory Medicine
                                                                            2
 Key words: CoaguChek, direct oral anticoagulant, DOAC, Emer-               Department, Cremona Hospital, Cremona; 3Department of
 gency, Point-of-care testing, POCT.
                                                                            Hematology, San Bortolo Hospital, Vicenza, Italy
 Funding: Roche Diagnostics contributed to the present study by
 providing instruments, controls/strips, and an unrestricted research
 grant to carry out the work, most of which was used to create the
 central database and electronic case report form and to monitor the        ABSTRACT
 study. An unrestricted grant was also received by Fondazione Cassa             Direct oral anticoagulants (DOAC) measurement is recom-




                                                                                              ly
 di Risparmio di Bologna.                                                   mended in specific conditions. A point-of-care testing should be




                                                                                      on
                                                                            used in emergency to qualitatively rule out relevant DOAC con-
 Acknowledgment: to Stephen Jewkes for English editing.
                                                                            centrations. The DOAC-CHECK Study aims to evaluate whether
 Contributions: CL: performed statistical analysis and wrote the ini-       the use of CoaguChek® Pro II (Roche Diagnostics International
                                                                            Ltd, Rotkreuz, Switzerland) coagulation testing device can pro-




                                                                                e
 tial draft of the manuscript. MC: contributed to statistical analysis
 and to the data collection. ST, AT and EA: contributed to the study        vide reliable information in patients treated with DOAC. The
 conception. CD, SC, IN, and LL: contributed to material prepara-
 tion and data collection. SZ: obtained authorization for the study
 from the National Health Authority and local Ethical Committees.
                                                                           us
                                                                            study was carried out in two FCSA (Italian Federation of Throm-
                                                                            bosis Centers) centers. We choose 3 different concentration
                                                                            thresholds for our analysis (30, 50 and 100 ng/mL) and by ROC
                                                                           al
 GP: contributed to the study conception and design and critically          curves the ideal cut-off point was selected to be the one that
 reviewed the draft. All the authors read and commented on the
                                                        ci

                                                                            yielded a sensitivity of at least 95% associated with the highest
 paper and approved submission.
                                                                            possible specificity. 512 patients were enrolled. For Edoxaban and
                                                      er


 Conflict of interest: The Authors declare no potential conflict of         Rivaroxaban, both CoaguChek® Pro II prothrombin time (PT)
 interest.                                                                  and activated partial thromboplastin time (aPTT) tests showed a
                                                     m



                                                                            sensitivity >95% corresponding to satisfying specificity values;
 Availability of data and material: Data from the case report forms         negative predictive values resulted in the range 90-100%. At vari-
                                         om




 (CRF) included in the study are available for consultation, accord-        ance, CoaguChek® Pro II PT and aPTT tests did not seem to be
 ing to the Italian law on the personal data protection.                    useful for identifying Apixaban and Dabigatran concentrations
                                                                            higher than the pre-defined thresholds. Our results suggest that
                                   -c




 Ethics approval: Approval was obtained by the Ethics Committee
 of Cremona and Vicenza hospitals.                                          CoaguChek® Pro II coagulation testing device can be used to
                                                                            qualitatively identify relevant concentrations of Edoxaban or Ri-
                          on




 Consent to participate: Written informed consent was obtained              varoxaban, but not of Apixaban or Dabigatran.
 from each patient before enrollment. The promoter of the study
                    N




 provided the measures to safeguard the subject’s privacy and the
 protection of personal data according to the EU GDPR 2016/679
 and Italian law.                                                           INTRODUCTION
 Received for publication: 9 May 2022.                                           Direct oral anticoagulants (DOACs), including Dabiga-
 Accepted for publication: 3 November 2022.                                 tran (anti-IIa agent), and Apixaban, Edoxaban, Rivaroxaban
 Publisher’s note: All claims expressed in this article are solely those    (all anti-Xa agents), have been licensed in many countries
 of the authors and do not necessarily represent those of their affili-     for prevention and treatment of cardioembolic complica-
 ated organizations, or those of the publisher, the editors and the re-     tions in patients with non-valvular atrial fibrillation (NVAF),
 viewers. Any product that may be evaluated in this article or claim
                                                                            and for prophylaxis, acute-long-term, and extended treat-
 that may be made by its manufacturer is not guaranteed or endorsed
 by the publisher.                                                          ment of venous thromboembolism (VTE). One of the most
                                                                            valued characteristics of these agents, that has been an im-
 ©
  Copyright: the Author(s), 2022                                            portant factor for their fast-increasing use worldwide, is that
 Licensee PAGEPress, Italy
 Bleeding, Thrombosis and Vascular Biology 2022; 1:37                       they are administered at fixed and unmonitored doses, dif-
 doi:10.4081/btvb.2022.37                                                   ferent in relation to the clinical indications of patients. Ran-
                                                                            domized controlled trials included almost one hundred
 This work is licensed under a Creative Commons Attribution-
 NonCommercial 4.0 International License (CC BY-NC 4.0).
                                                                            thousand NVAF or VTE patients and proved that the use of
                                                                            these agents at fixed-dose was safer (especially for reduction

                                             Bleeding, Thrombosis and Vascular Biology 2022; 1:37
52                                                          C. Legnani et al.




of intracranial hemorrhage occurrence) and as effective as              Helsinki and in compliance with the protocol. Independ-
dose-adjusted vitamin K antagonists (VKAs). Although                    ent review board approval was obtained prior to all study-
DOACs do not need routine monitoring, some situations or                related activity from the Ethics Committee of both
specific patient conditions may require an assessment of an-            Cremona and Vicenza hospitals. Written informed consent
ticoagulation levels, which is currently possible by using              was obtained from each patient before enrollment. The
laboratory tests.1,2 Unfortunately, for several reasons includ-         promoter of the study provided the measures to safeguard
ing the need of expert and dedicated laboratory staff, these            the subject’s privacy and the protection of personal data
tests have insufficiently been implemented in clinical labo-            according to the EU GDPR 2016/679 and Italian law.
ratories, resulting in a generally lower than required meas-
urement of DOAC activity levels. The lack of promptly                   Setting and eligibility
available DOAC assessment is particularly important in the
                                                                            The study was conducted in two tertiary care facilities
setting of urgent/emergent conditions, in which clinicians
                                                                        affiliated to FCSA (Italian Federation of Thrombosis Cen-
should face not only the scarcity of laboratories able to per-
                                                                        ters): the Haemostasis and Thrombosis Center of Cre-
form the specific tests but also, what is even more important,
                                                                        mona hospital and the Department of Hematology of
the time needed to receive results timely useful for the nec-
                                                                        Vicenza hospital. Between October 2020 and May 2021,
essary clinical decisions. Point-of-care testing (POCT), per-




                                                                                                  ly
                                                                        512 outpatients treated with a DOAC (Apixaban, Edoxa-
formed where clinical care is delivered, can provide
                                                                        ban, Rivaroxaban or Dabigatran) [either for non-valvular




                                                                                         on
significant benefits regarding improvement of time to test
                                                                        atrial fibrillation (NVAF) or venous thromboembolism
results and would be particularly helpful in case of urgently
                                                                        (VTE)], naïve or shifted from AVK or heparin, were con-
needed surgery, operative procedures, or treatments (throm-
                                                                        secutively enrolled in the participating centers at the mo-



                                                                                    e
bolysis after stroke). Unfortunately, POCT specifically de-
                                                                        ment of a routine follow-up visit. The inclusion criteria
signed to measure DOAC activity are not available at
present.3 In contrast, POCT designed to measure VKAs or
                                                                                us
                                                                        were: age ≥ 18 years, ability to give written informed con-
                                                                        sent to the blood sampling for the study purpose during a
heparin activity by performing prothrombin time (PT) and
                                                                      al
                                                                        routine visit. Patients already enrolled in clinical trials
activated partial thromboplastin time (aPTT) are available.4
                                                                        from phase 1 to 3 were excluded. Most of the patients
                                                         ci

However, these tests do not accurately assess the levels of
                                                                        were enrolled at Cremona hospital (78.9%).
DOAC-induced anticoagulation.5 Since the clinical deci-
                                                       er


                                                                            Patients were excluded from the study if: they were
sions in some conditions do not require a precise measure-
                                                                        treated with other anticoagulants (VKAs within 14 days,
                                                      m



ment of DOAC activity but only to verify that the
                                                                        low molecular weight heparin within 24 hours, or unfrac-
anticoagulant activity is below prespecified cutoff levels to
                                                                        tionated heparin within 12 hours prior to enrollment), had
                                             om




allow safe clinical procedures, it is possible that the currently
                                                                        changed the DOAC within 7 days, had spontaneous al-
available POCT may be useful to this scope.
                                                                        tered coagulation (PT INR > 1.2, aPTT >1.20 ratio), had
    The present study aimed to evaluate whether Co-
                                       -c




                                                                        known coagulopathies, or presented a lupus anticoagulant
aguChek® [Trademark of Roche] Pro II coagulation test-
                                                                        positivity and/or high antiphospholipid antibody levels.
ing device (Roche Diagnostics International, Rotkreuz,
                               on




                                                                        Use of antiplatelet agents was permitted.
Switzerland) use can provide reliable information in
DOAC treated patients; in particular, we aimed to deter-
                          N




                                                                        Sample collection and measurements
mine the diagnostic accuracy of this POCT to qualita-
tively rule out relevant DOAC concentrations                                Blood samples (venous and capillary) were taken dur-
(concentrations that are considered safe for surgery and                ing a routine clinical and laboratory control. Most of the
thrombolysis) in real-life patients. The study promoted by              samples (90.5%) were collected at trough level (12 hours
Fondazione Arianna Anticoagulazione, was a national                     after Apixaban or Dabigatran and 24 hours after Edoxaban
multi-center, prospective, observational and no-profit trial            or Rivaroxaban); the remaining samples were collected
with blinded end-point assessment.                                      at pick level (2-4 hours after drug intake).
                                                                            Baseline characteristics (age, sex, type of drug, clini-
                                                                        cal indication for anticoagulation, weight, body mass
MATERIALS AND METHODS                                                   index, kidney and liver function, concomitant medications
                                                                        and diseases) were recorded in a structured database. The
   Anonymized source data and study protocol will be made
                                                                        REDCap (Research electronic data capture) software was
available to other researchers on request to the first Author.
                                                                        used for data entry. Results of CoaguChek® PRO II tests
                                                                        and of DOAC level measurements were kept blind to the
Standard protocol approval
                                                                        patients and not used to take clinical decisions. Results
    The study was conducted in accordance with the eth-                 were recorded in the patient’s medical records and tran-
ical principles that have their origin in the Declaration of            scribed into the REDCap electronic case report form

                                        Bleeding, Thrombosis and Vascular Biology 2022; 1:37
                                                  The DOAC-CHECK study                                                    53



(eCRF). Various procedures in clinical data management            are currently limited we choose 3 different concentration
including eCRF designing, eCRF annotation, database de-           thresholds for our analysis (30, 50 and 100 ng/mL).6-10
signing, data-entry, data validation, discrepancy manage-             Sensitivity is defined as the percentage of samples
ment, medical coding, data extraction, and database               containing clinically relevant DOAC plasma concentra-
locking were assessed for quality at regular intervals dur-       tions that tested positive by the CoaguChek® Pro II PT
ing the study period. This was an observational study, and        or aPTT tests and thus correctly identified as patient in-
the analysis was performed on the total monitored patient         eligible for thrombolysis/surgery or requiring reversal
sample. Patients were excluded from analysis if they had          therapy. Specificity is defined as the percentage of sam-
no exact birth date so that the legal age was questionable,       ples containing no clinically relevant DOAC plasma con-
or no exact date of consent.                                      centrations that tested negative and were correctly
    PT and aPTT tests on CoaguChek® Pro II coagula-               identified by the CoaguChek® Pro II PT or aPTT tests.
tion testing device were performed using capillary blood              Positive predictive value (PPV) is defined as the per-
obtained through a direct finger puncture using a lancing         centage of samples with clinically relevant DOAC con-
device. Two POCT instruments for each participating cen-          centrations out of all samples identified as containing
ter were provided, completed with controls and strips.            clinically relevant DOAC levels by the CoaguChek® Pro
    Venous samples for specific DOAC measurements                 II PT or aPTT tests; negative predictive value (NPV) is




                                                                                   ly
were collected concomitantly with use of POCT, in                 defined as the percentage of samples with no clinically




                                                                            on
0.109M sodium citrate (9:1); plasma aliquots obtained             relevant DOAC plasma concentrations out of all samples
after centrifugation at 2000Xg for 15 min were initially          identified as containing no clinically relevant drug levels
stored at -80°C at the participating centers, and later           by CoaguChek® Pro II PT or aPTT tests.




                                                                       e
shipped to the Haemostasis and Thrombosis Center of                   Receiver operating characteristic (ROC) curves were
Cremona Hospital, where the specific drug measurements
were performed. DOAC levels expressed as drug concen-
                                                                usdrawn and the area under the ROC curves (AUROCs)
                                                                  were calculated for CoaguChek® Pro II PT and aPTT
                                                                  tests at the three different thresholds of DOAC plasma
tration-equivalent (ng/mL) were measured with STA-ECA
                                                          al
II (Diagnostica Stago, Asnieres-sur-Seine, France) for            concentrations (>30, >50 and > 100 ng/mL) (Figures 1-
                                                                  3); AUROCs were considered as a measure of overall test
                                              ci

Dabigatran, and STA-Liquid anti-Xa (Diagnostica Stago)
for Apixaban, Edoxaban, and Rivaroxaban. All these tests          performance and are given with the two-sided 95% CI.
                                            er


were calibrated using commercial plasmas from the same            Using the ROC curves, for each test/DOAC threshold, as
supplier and performed on STA-R instrument (Diagnos-              the ideal cut-off point was selected the one that yielded a
                                           m




tica Stago).                                                      sensitivity of at least 95% (misprediction percentage <
                                  om




    CoaguChek® Pro II PT and aPTT tests and specific              5%, considered sufficiently safe for clinical application)
drug measurements were performed according to manu-               associated to the highest possible specificity, to avoid
facturers’ instructions by thoroughly trained investigators       false-negative results but simultaneously to identify the
                            -c




and technicians.                                                  largest number of patients eligible for emergency treat-
                                                                  ment such as thrombolysis or emergency surgery.
                       on




Blinding                                                              This study was performed in accordance with the
                                                                  STARD (Standards for Reporting Diagnostic Accuracy)
                 N




    CoaguChek® Pro II coagulation testing device oper-            guidelines for studies on diagnostic tests.11
ators were blinded of the results of the specific drug meas-          All statistical analyses were performed using Graph-
urements as well as technicians performing specific drug          Pad Prism for MacOS version 9.3.1 (GraphPad Software,
measurements were blinded of the CoaguChek® Pro                   San Diego, CA, USA) and online MedCalc version
II coagulation testing device results.                            20.027 Ltd (MedCalc Software, Ostend, Belgium).

Statistical analysis
    Categorical variables are presented with absolute and         RESULTS
relative frequency; continuous variables with median and             The characteristics of patient population and the
range (min-max).                                                  DOAC treatments are described in Table 1. The median
    Diagnostic accuracy of CoaguChek® Pro II PT and               age of patients was 74 years (range 21-94) and 59.2%
aPTT tests (reported in seconds) regarding detection of           were males. Apixaban was used in most of the subjects
clinically relevant DOAC plasma concentrations was                (n=222, 43.4%), Edoxaban and Rivaroxaban in 23.4%
evaluated in terms of sensitivity, specificity, positive and      (n=120) and 21.7% (n=111) of cases, respectively, while
negative predictive values as well as likelihood ratios in-       few enrolled patients were treated with Dabigatran
cluding respective two-sided 95% confidence intervals             (n=59, 11.5%). This drug distribution reflects the current
(CI). Since data to define relevant DOAC concentrations           real-life situation of DOAC prescription in Italy.

                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:37
54                                                      C. Legnani et al.




                                                                                            ly
                                                                                     on
                                                                                e
                                                                            us
                                                                  al
                                                      ci
                                                    er
                                                   m
                                          om
                                     -c
                             on
                         N




Figure 1. Receiver operating characteristic (ROC) curves for        Figure 2. Receiver operating characteristic (ROC) curves for
CoaguChek®Pro II PT and aPTT tests when testing for detection       CoaguChek®Pro II PT and aPTT tests when testing for detection
of samples containing 30 ng/mL DOAC concentrations.                 of samples containing 50 ng/mL DOAC concentrations.


                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:37
                                                  The DOAC-CHECK study                                                            55



                                                                  Low doses of DOACs were used in about half of the pa-
                                                                  tients [41.9% Apixaban (2.5 mg/BID); 55.0% Edoxaban
                                                                  (30 mg); 37.8% Rivaroxaban (10 or 15 mg); 42.3%
                                                                  Dabigatran (110 mg/BID)]. Approximately half patients
                                                                  were treated for NVAF and less than half for a previous
                                                                  VTE event (50.4% and 47.7%, respectively), whereas
                                                                  only 1.9% of cases were treated for both VTE and
                                                                  NVAF. Concerning the associated treatments, no signif-
                                                                  icant differences between groups treated with the differ-
                                                                  ent DOACs were found regarding antiplatelet,
                                                                  antiarrhythmic, antidiabetic, antihypertensive, thyroid
                                                                  dysfunction and lipid lowering drugs; no patients were
                                                                  using antiviral or antiepileptic drugs. The only signifi-
                                                                  cant difference in co-medications was a higher preva-
                                                                  lence (p=0.030) of gastroprotective use in patients
                                                                  treated with Dabigatran and Edoxaban (42.4 and 47.5%,




                                                                                      ly
                                                                  respectively) vs those treated with Apixaban and Ri-




                                                                              on
                                                                  varoxaban (34.2 and 30.6%, respectively).
                                                                      Edoxaban and Rivaroxaban median levels (25 and 31
                                                                  ng/mL, respectively) resulted lower than that of Apixaban




                                                                       e
                                                                  and Dabigatran (90 and 83 ng/mL, respectively); this
                                                                usprobably because, differently for Apixaban and Dabiga-
                                                                  tran, Edoxaban and Rivaroxaban are taken once daily and
                                                          al
                                                                  blood sampling was drawn in the morning, at the trough
                                                                  level, in most patients.
                                              ci

                                                                      Using the ROC analysis, we calculated optimized
                                            er


                                                                  “ideal” cut-off values for CoaguChek® Pro II PT and
                                                                  aPTT tests (Table 2) which provide the > 95% sensitivity
                                           m



                                                                  and the highest specificity for different threshold DOAC
                                                                  concentrations: 30 ng/mL (Fig. 1), 50 ng/mL (Fig. 2) and
                                  om




                                                                  100 ng/mL (Fig. 3). Based on AUROC values,12 both Co-
                                                                  aguChek® Pro II PT and aPTT tests resulted from mod-
                            -c




                                                                  erately (0.7<AUROC≤0.9) to highly informative
                     on




                                                                  Table 1. Characteristics of the 512 enrolled patients
                 N




                                                                   Males, n (%)                                      303 (59.2)
                                                                   Age, median (min-max), y                          74 (21-94)
                                                                   BMI, median (min-max), kg/m2                    26.9 (17.0-46.1)
                                                                   eGRF, median (min-max), mL/min/1.73m2          71.4 (18.7-150.5)
                                                                   ALT, median (min-max), U/L                      19.5 (4.0-85.0)
                                                                   AST, median (min-max), U/L                      21.0 (9.0-72.0)
                                                                   DOAC treatment, n (%)
                                                                    Apixaban                                         222 (43.4)
                                                                    Edoxaban                                         120 (23.4)
                                                                    Rivaroxaban                                      111 (21.7)
                                                                    Dabigatran                                        59 (11.5)
                                                                   Indication for treatment, n (%)
                                                                     VTE                                             244 (47.7)
                                                                     NVAF                                            258 (50.4)
Figure 3. Receiver operating characteristic (ROC) curves for         VTE+NVAF                                         10 (1.9)
CoaguChek®Pro II PT and aPTT tests when testing or detection      eGRF, Glomerular Filtration Rate (estimated by the Chronic Kidney
of samples containing 100 ng/mL DOAC concentrations.              Disease-Epidemiology Collaboration equation); NVAF, non-valvular
                                                                  atrial fibrillation; VTE, venous thromboembolism.


                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:37
56                                                               C. Legnani et al.




(0.9<AUROC<1.0) for Edoxaban and Rivaroxaban for all                           threshold DOAC concentrations; the NPVs resulted in the
threshold DOAC concentrations. AUROC values were                               range 90-100% both for CoaguChek® Pro II PT and
lower for Apixaban compared to those obtained for Edox-                        aPTT tests at all DOAC thresholds. Finally, probably due
aban and Rivaroxaban, while both CoaguChek® Pro II PT                          to the low number of enrolled patients, CoaguChek® Pro
and aPTT tests seem not to be informative for Dabigatran                       II PT and aPTT tests did not seem to be useful to identify
at all DOAC threshold concentrations.                                          patients with Dabigatran levels above the pre-defined
    Table 2 reports the CoaguChek® Pro II PT and aPTT                          threshold Dabigatran concentrations; indeed, the speci-
diagnostic test evaluation regarding detection of DOAC                         ficity values were very low thus generating a large num-
concentrations at 30, 50 and 100 ng/mL. Regarding Apix-                        ber of false positive cases; only at 100 ng/mL Dabigatran
aban, for both CoaguChek® Pro II PT and aPTT tests, a                          threshold the NPV was acceptable for CoaguChek® Pro
sensitivity > 95% corresponded to low specificity values                       II aPTT test. Finally, only for Edoxaban and Rivaroxaban
at all Apixaban thresholds, that reached the highest values                    the negative likelihood ratio values resulted closer to zero,
at the 100 ng/mL Apixaban threshold (28.3 and 21.3% for                        indicating a markedly low probability of having DOAC
CoaguChek® Pro II PT and aPTT tests, respectively); the                        concentrations above the pre-defined thresholds, if the
NPVs were acceptable only at the 100 ng/mL Apixaban                            CoaguChek® Pro II PT or aPTT tests resulted negative.




                                                                                                           ly
threshold (94.7% and 87.1% for CoaguChek® Pro II PT
and aPTT tests, respectively). Conversely, for Edoxaban




                                                                                                   on
                                                                               DISCUSSION
and Rivaroxaban both for CoaguChek® Pro II PT and
aPTT tests a sensitivity > 95% corresponded to satisfying                          DOACs are administered at fixed doses, without rou-




                                                                                              e
specificity values, especially at the 50 and 100 ng/mL                         tine frequent laboratory monitoring. However, DOAC

                                                                                        us
Table 2. CoaguChek® Pro II PT and aPTT diagnostic test evaluation regarding detection of DOAC concentration at 30, 50 and 100
                                                                               al
ng/mL (95% confidence interval in brackets)
                                                              ci

Test       Threshold       (Ideal)         Sensitivity        Specificity            NPV               PPV           Negative LR     Positive LR
            (ng/mL)     Cut-off (sec)         (%)                 (%)                   (%)            (%)
                                                            er


                                                                      Apixaban
                                                           m



PT           >30            11.5        96.1 (92.4-98.3)     5.6 (0.1-27.3)     11.1 (1.6-48.6)   92.0 (91.1-92.8)   0.7 (0.1-5.3)   1.0 (0.9-1.1)
             >50            11.3        98.3 (95.1-99.6)     2.1 (0.0-11.3)     25.0 (3.4-75.8)   78.9 (78.1-79.7)   0.8 (0.1-7.6)   1.0 (0.9-1.1)
                                                 om




             >100           12.1        97.9 (92.6-99.7)    28.3 (20.7-37.0)   94.7 (81.6-98.6)   50.5 (47.7-53.4)   0.1 (0.0-0.3)   1.4 (1.2-1.5)
aPTT         >30            28.8        95.1 (91.2-97.6)    16.7 (3.6-41.4)     23.1 (8.3-49.8)   92.8 (91.3-94.1)   0.3 (0.1-1.0)   1.1 (0.9-1-4)
             >50            30.0        95.4 (91.2-98.0)    19.1 (9.1-33.3)    52.9 (31.5-73.4)   81.5 (79.2-83.5)   0.2 (0.1-0.6)   1.2 (1.1-1.4)
                                          -c




             >100           31.5        97.8 (89.6-98.8)    21.3 (14.5-29.4)   87.1 (71.0-94.9)   47.6 (45.2-50.1)   0.2 (0.1-0.6)   1.2 (1.1 (1.3)
                                                                     Edoxaban
                                   on




PT           >30            11.9        97.9 (88.7-99.9)    21.9 (13.1-33.1)   94.1 (68.7-99.1)   44.7 (41.5-47.9)   0.1 (0.0-0.7)   1.2 (1.1-1.4)
             >50            12.2        96.1 (80.4-99.9)    30.8 (21.7-41.2)   96.7 (80.6-99.5)   27.8 (24.8-31.0)   0.1 (0.0-0.9)   1.4 (1.2-1.6)
                             N




             >100           13.8        100 (78.2-100)      72.4 (62.8-80.7)         100          34.1 (27.5-41.3)         0         3.6 (2.7-4.9)
aPTT         >30            31.2        95.7 (85.5-99.5)    23.3 (14.2-34.7)   89.5 (67.3-97.2)   44.5 (41.1-48.0)   0.2 (0.1-0.7)   1.2 (1.1-1.4)
             >50            33.4        96.1 (80.4-99.9)    39.4 (29.4-50.0)   97.4 (84.2-99.6)   30.5 (26.8-34.4)   0.1 (0.0-0.7)   1.6 (1.3-1.9)
             >100           37.3        100 (78.2-100)      60.9 (50.9-70.3)         100          26.8 (22-4-31.7)         0         2.6 (2.0-3.2)
                                                                    Rivaroxaban
PT           >30            12.2        98.2 (90.6-99.7)    31.5 (19.5-45.6)   94.4 (70.1-99.2)   60.2 (55.7-64.3)   0.1 (0.0-0.4)   1.4 (1.2-1.7)
             >50            12.2         100 (89.4-100)     23.1 (14.3-34.0)         100          35.5 (32.7-38.3)         0         1.3 (1.1-1-5)
             >100           13.9        95.0 (75.1-99.9)    85.7 (76.8-92.2)   98.7 (92.0-99.8)   59.4 (46.7-70.9)   0.1 (0.0-0.4)   6.6 (4.0-11.1)
aPTT         >30            32.6        96.4 (87.7-99.6)   33.3 (21.1-47.5)    90.0 (68.7-97.4)   60.0 (55.2-64.6)   0.1 (0.0-0.4)   1.4 (1.2-1.8)
             >50            38.7        96.9 (83.8-99.9)   74.4 (63.2-83.6)    98.3 (89.3-99.7)   60.8 (51.4-69.5)   0.0 (0.0-0.3)   3.8 (2.6-5.5)
             >100           42.6        95.0 (75.1-99.9)   84.4 (75.3 (91.2)   98.7 (91.8-99.8)   57.6 (45.3-68.9)   0.1 (0.0-0.4)   6.1 (3.7-10.0)
                                                                     Dabigatran
PT           >30            14.5        95.2 (83.8-99.4)     5.9 (0.1-28.7)     33.3 (4.6-83.8)   71.4 (68.6-74.1)   0.8 (0.1-8.3)   1.0 (0.9-1.2)
             >50            11.5        95.2 (83.8-99.4)     5.9 (0.1-28.7)     33.3 (4.6-83.8)   71.4 (68.6-74.1)   0.8 (0.1-8.3)   1.0 (0.9-1.2)
             >100           11.5        95.4 (77.1-99.9)     5.4 (0.7-18.2)    66.7 (16.2-95.4)   37.5 (34.7-40.3)   0.8 (0.1-8.7)   1.0 (0.9-1.1)
aPTT         >30            30.2        96.1 (86.8-99.5)    14.3 (0.3-57.9)    33.3 (4.9-82.8)    89.3 (86.0-91.9)   0.3 (0.0-2.6)   1.1 (0.8-1.5)
             >50            32.8        95.2 (83.8-99.4)     23.5 (6.8-49.9)   66.7 (28.7-90.8)   75.5 (70.1-80.2)   0.2 (0.0-1.0)   1.2 (0.9-1.6)
             >100           34.1        95.4 (77.1-99.9)    27.0 (13.8-44.1)   90.9 (57.8-98.6)   43.7 (38.5-49.1)   0.2 (0.0-1.2)   1.3 (1.0-1.6)
LR, Likelihood ratio; NPV, Negative predictive value; PPV, Positive predictive value.


                                           Bleeding, Thrombosis and Vascular Biology 2022; 1:37
                                                    The DOAC-CHECK study                                                   57



measurement may be useful in some clinical conditions                  For Edoxaban and Rivaroxaban we found that a sensi-
such as emergencies.2,13,14 Acute clinical scenarios, includ-      tivity higher than 95% was associated to an
ing major bleeding, need for emergency surgery, or deci-           acceptable/good specificity for both CoaguChek® Pro II®
sion making for DOAC reversal, would utilize a one-time            PT and aPTT tests at all selected threshold concentra-
measurement to determine if a DOAC is present. In these            tions; overall, negative predictive values resulted between
situations, DOAC testing should consist of simple sample           90-100%. This means that CoaguChek® Pro II PT and
processing, rapid turnaround times, and considerable sen-          aPTT tests could identify patients with Edoxaban and Ri-
sitivity to be useful. Specific coagulation tests including        varoxaban concentrations above the pre-defined thresh-
diluted thrombin time, ecarin chromogenic assay, and               olds with lower number of false-positive cases, thus
drug-specific anti-Xa assays are available, but, unfortu-          allowing a higher number of patients eligible for throm-
nately, their applicability is still limited in the setting of     bolysis or emergency surgery. Surprisingly, our study
urgent/emergent conditions; furthermore, these tests are           showed that for Edoxaban- and Rivaroxaban-treated pa-
not implemented in many laboratories. In most cases, the           tients similar results were obtained either with PT or
only coagulation tests always available are PT and aPTT,           aPTT CoaguChek® Pro II coagulation testing device. Re-
for this reason several authors have argued these unspe-           sults of DOAC-specific tests, when available, are com-
cific global tests might suffice to rule out relevant DOAC         monly slower to be obtained in comparison with POCT;




                                                                                    ly
concentrations.7,15,16 However, both PT and aPTT are per-          POCT and DOAC-specific coagulation tests can therefore




                                                                             on
formed in the central laboratory and therefore results are         be used sequentially to optimize speed and patient man-
inevitably delayed by sample transportation and subse-             agement: if POCT yield results above pre-specified cutoff
quent sample handling, which under ideal conditions                values, emergency procedure may not be initiated, with-




                                                                        e
takes approximately 35 minutes.17 Moreover, standard PT            out the need of further coagulation testing. Calibrated
and aPTT are not adequate when used to monitor DOACs
due to significant variability in methodology and sensi-
                                                                 usDOAC-specific tests are only required if the results of
                                                                   POCT are below pre-specified cutoff values. Such an ap-
tivity of reagents used.13,14,18-20                                proach has been recently proposed.21
                                                            al
    Therefore, a coagulation test that can be processed                Conversely, CoaguChek® Pro II PT and aPTT tests
                                               ci

shortly and at the patient’s bed by a POCT, provides a bi-         could identify patients with Apixaban concentrations
nary yes or no answer for the presence of DOAC antico-             above the pre-defined thresholds, but only at the expense
                                             er


agulant effect, exhibits improved reliability and sensitivity      of a high number of false-positive cases, thus excluding
over standard coagulation tests, with a favorable cost-ef-         from thrombolysis/surgery a too large number of patients.
                                            m




fectiveness would be extremely useful in critical condi-           Finally, CoaguChek® Pro II PT and aPTT tests did not
                                   om




tions and/or in emergency situations.                              seem to be useful to identify Dabigatran concentrations
    Our study aimed to evaluate the diagnostic accuracy            higher than the pre-defined thresholds; acceptable results
of the CoaguChek® Pro II® coagulation testing device,              were only obtained for CoaguChek® Pro II aPTT test at
                             -c




a POCT coagulometer, to qualitative rule out relevant con-         the threshold of 100 ng/mL Dabigatran concentration.
centrations of Apixaban, Dabigatran, Edoxaban and Ri-                  Our results agree with those of previously published
                      on




varoxaban in real-life patients. Data on what constitutes          data. Indeed, all previous papers evaluating a similar
a relevant DOAC concentration are scarce, and concen-              POCT, found that the results of PT/INR obtained with this
                 N




trations that leads to clinically significant coagulation im-      device can accurately rule out relevant concentrations of
pairment have not been established in prospective clinical         Edoxaban22 or Rivaroxaban23-25 but not of Apixaban23,25 or
trials. Different thresholds have been proposed for each           Dabigatran.23 Our study confirms that the three factor Xa
DOAC, which differed between thrombolysis for acute                inhibitors, Edoxaban, Rivaroxaban and Apixaban, differed
stroke and emergency surgery.6-10 Based on these recom-            in their effects on different POCT devices.23,25,26 Other
mendations, we investigated 3 different concentration              studies have recorded less-pronounced effects of Apixa-
thresholds (30, 50 and 100 ng/mL) that, at present time,           ban on PT test performed in laboratory.27,28 As expected,
may be considered relevant for a safe urgent/elective sur-         CoaguChek® Pro II PT test resulted not accurate to iden-
gery and may permit thrombolysis.                                  tify samples with no relevant Dabigatran concentrations;
    Using the ROC curves, for each test/DOAC threshold,            surprisingly, this was the case also for CoaguChek® Pro
we selected as ideal cut-off point the one that yielded a          II aPTT test.
sensitivity of at least 95% (false-negative results <5%,               Prompt availability and instantaneous results make
considered sufficiently safe for clinical application) asso-       CoaguChek® Pro II coagulation testing device adoption
ciated to the highest possible specificity, to avoid false-        an attractive option in emergency situations, since it can
negative results but also to identify the largest number of        be used at patient’s bedside and without loss of time be-
patients eligible for emergency treatment such as throm-           tween sampling, testing and results. Indeed, the lag time
bolysis or emergency surgery.                                      between blood sampling and laboratory test execution

                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:37
58                                                       C. Legnani et al.




should particularly be considered, because the DOAC                  aguChek® Pro II coagulation testing device may be a fast
plasma concentration increases rapidly after intake until            and reliable alternative for guiding emergency
reaching a peak after 2 to 4 hours.                                  decision/treatment in patients on Edoxaban or Rivaroxaban
     Our study has strengths and limitations. Strengths in-          therapy. However, as the suggested cut-offs were deter-
clude the large sample size and the acquisition of all sam-          mined retrospectively, further evaluation in a prospective
ples from real-life patients. The use of real-life patients          clinical trial, ideally in emergency situations, is warranted
instead of spiked plasma has increased the inter-sample              to investigate the clinical safety of this approach.
variability, thus DOAC plasma concentrations around 30-
50-100 ng/mL thresholds are well represented in the
dataset. Furthermore, collecting samples during treatment,           REFERENCES
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                                                                       us
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                                                m
                                      om
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                        on
                   N




                                          Bleeding, Thrombosis and Vascular Biology 2022; 1:37
