Baseline DOAC Measurement in Non Valvular Atrial Fibrillation Patients and Incidence of Bleeding or Thromboembolic Complications During Follow-up: a Prospective, Multicenter, Observational Study. The MAS (Measure And See) Study
Conditions: Atrial Fibrillation, Anticoagulant-induced Bleeding
Sponsor: Arianna Anticoagulazione Foundation

 
Protocol MAS Study v2.0 dated 11.10.2018  
 
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PROTOCOL  
MAS STUDY 
Final version 2.0 date 11.10.2018 
 
Title of the Study 
 
 
BASELINE DOAC MEASUREMENT IN NVAF 
PATIENTS AND INCIDENCE OF BLEEDING OR 
THROMBOEMBOLIC COMPLICATIONS DURING 
FOLLOW-UP: A PROSPECTIVE, MULTICENTER, 
OBSERVATIONAL STUDY  
The MAS (Measure And See) Study 
 
Code of the Study 
FAA O3 12-2017 (MAS) 
 
Version of Protocol 
Final Version 2.0 
 
Date 
11th October 2018 
Promoter 
Arianna Anticoagulazione Foundation 
Via Paolo Fabbri 1/3  
40138 Bologna (BO) 
Italy 
Tel +39051341471 
Fax +39051343604 
Mail segreteria@fondazionearianna.org 
Coordinator site 
 
SC Laboratorio Analisi Aziendale, Dipartimento di 
Laboratorio, AO Istituti Ospitalieri, Viale 
Concordia, 26100 Cremona (CR) 
Italy 
Principal Investigator 
Dott.ssa Sophie Testa 
SC Laboratorio Analisi Aziendale, Dipartimento di 
Laboratorio, AO Istituti Ospitalieri, Viale 
Concordia, 26100 Cremona (CR) 
Italy 
Tel. +390372405456     
s.testa@asst-cremona.it  
Co-Investigators 
Dr. Oriana Paoletti, Dr. Rossella Morandini, Dr. 
Anke Zimmermann, Dr. Claudia Dellanoce 
 
 
 

 
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CONTACT INFORMATION 
 
Contact of Promoter 
Gualtiero Palareti 
Fondazione Arianna Anticoagulazione 
Via Paolo Fabbri 1/3  
40138 Bologna (BO) 
Italy 
Tel +39051341471 
Fax +39051343604 
gualtiero.palareti@unibo.it  
segreteria@fondazionearianna.org  
 
 
 
 
 

 
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INDEX 
List of abbreviations and acronyms ..................................................................................................... 4 
1. Background Information and rationale ............................................................................................ 5 
2. Study objectives ............................................................................................................................... 5 
3.Investigational plan ........................................................................................................................... 5 
3.1 Study design ............................................................................................................................... 5 
3.2 Setting ........................................................................................................................................ 6 
3.3 Study population ........................................................................................................................ 6 
3.3.1 Inclusion criteria .................................................................................................................. 6 
3.3.2 Exclusion criteria ................................................................................................................. 6 
4. Study procedures ............................................................................................................................. 6 
4.1 Enrolment procedure ................................................................................................................. 6 
4.2 Follow-up procedure .................................................................................................................. 7 
4.3 Definition of the conclusion of the study ................................................................................... 8 
5. Study evaluations and measurements ............................................................................................. 8 
5.1 Laboratory assessment .............................................................................................................. 8 
5.2 Outcomes during the follow-up ................................................................................................. 9 
5.2.1 Primary outcomes ............................................................................................................... 9 
5.2.2 Secondary outcomes ........................................................................................................... 9 
5.3 Safety control ............................................................................................................................. 9 
5.4 Results of the study .................................................................................................................... 9 
6. Statistical Considerations ................................................................................................................. 9 
6.1 Bias ............................................................................................................................................. 9 
6.2 Sample size ............................................................................................................................... 10 
6.3 Statistical analysis plan ............................................................................................................. 10 
7. Safety management ....................................................................................................................... 10 
8. Study administration ...................................................................................................................... 11 
8.1 Data collection and management ............................................................................................ 11 
8.2 Ethics ........................................................................................................................................ 11 
8.3 Informed consent forms ........................................................................................................... 11 
8.4 Administrative issues ................................................................................................................ 12 
8.4.1 Support of the study ......................................................................................................... 12 
9.1 Role of Promoter and Investigators .......................................................................................... 12 
9.2 Data handling and record keeping ........................................................................................... 12 
9.3 Final report and publication ..................................................................................................... 12 
10. References .................................................................................................................................... 13 
APPENDIX 1 ........................................................................................................................................ 15 
Labelling code ................................................................................................................................ 15 
APPENDIX 2 ........................................................................................................................................ 16 
OUTCOME DEFINITIONS ................................................................................................................. 16 
VENOUS THROMBOEMBOLISM (VTE) ........................................................................................ 16 
DEATH ......................................................................................................................................... 16 
BLEEDING EVENTS ...................................................................................................................... 17 
APPENDIX 3 ........................................................................................................................................ 18 
Sample collection protocol ............................................................................................................ 18 
 

 
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List of abbreviations and acronyms 
 
 
 
VKA  
Vitamin K Antagonists 
eCRF 
Electronic Case Report Form  
DOAC  
Direct Oral Anticoagulants 
SD 
Standard Deviation 
dTT 
Diluted Thrombin Time 
ECA 
Ecarin Clotting Assay 
FCSA 
Italian Federation of Thrombosis Centres (Federazione Centri per la 
diagnosi della trombosi e la Sorveglianza delle terapie Antitrombotiche) 
FDA 
Food and Drug Administration 
GCP 
 
Good Clinical Practice 
LC-MS/MS 
Liquid chromatography-tandem mass spectrometry 
NVAF 
Non Valvular Atrial Fibrillation  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1. Background Information and rationale 
 Oral anticoagulant therapy is recommended in many clinical conditions to treat and prevent 
venous and arterial thromboembolism. Until a few years ago, vitamin K antagonists (VKA) were the 
only available drugs that, owing to their pharmacological characteristics, require frequent 
laboratory monitoring and expert dose adjustment (1). The direct oral anticoagulants (DOAC) have 
been introduced in clinical practice for the prevention of stroke and systemic embolism in patients 
with non-valvular atrial fibrillation (NVAF) and for the prevention and treatment of venous 
thromboembolism (2-5). Contrary to VKA, DOACs have been proposed without the need for 
laboratory testing and dose-adjustment because phase III clinical trials showed efficacy and safety 
at fixed dose based only on clinical criteria (2-6). Nevertheless, DOAC inter-individual variability has 
been evaluated in previous studies, and a high inter-individual variability has been demonstrated 
with all DOAC at different dosages. Furthermore, post-hoc analysis and FDA reports of RE-LY, 
Rocket and Hokusai trials (7-13) showed that plasma concentrations of dabigatran, rivaroxaban, 
and edoxaban were associated with the relative risk of bleeding and thromboembolic 
complications, suggesting that DOAC measurements could be clinically relevant for increasing 
safety and efficacy of these treatments. As a consequence, an open clinical question is if, in real 
world patient population, measurements of DOAC levels are associated with subsequent bleeding 
and/or thromboembolic events. 
The guidelines of the Italian Federation of Thrombosis Centres (Federazione Centri per la diagnosi 
della trombosi e la Sorveglianza delle terapie Antitrombotiche, FCSA) (14) suggest to do follow-up 
visits after 1 month from the first therapy prescription, and later every 3-6 months, to assess the 
patient’s compliance with treatment (since a low adherence rate could diminish the benefit of 
treatment), to investigate regarding the occurrence of thromboembolic and bleeding complications 
potentially related to the prescribed DOAC, and any new co-medications with possible drug–drug 
interactions, and to perform a blood sampling for the necessary exams (15, 16).  The execution of 
specific laboratory tests after 15-30 days gives a DOAC level at the steady- state.   
2. Study objectives 
The MAS Study is an observational, multicentre, prospective cohort study in NVAF patients treated 
with one the DOACs available in Italy for NVAF patients.   
The general aim is to deepen the knowledge of DOAC treatment in NVAF patients, by measuring 
the plasma concentration of anticoagulant drugs and their correlation with any adverse events 
that may occur during treatment. 
The study has an exclusively observational purpose in order to be the starting point for 
collaborative clinical studies, enabling their planning and execution; it is not aimed to influence 
the treatment of the individual patients included in the study. 
The primary study objective is to evaluate the possible relationship between DOAC anticoagulant 
levels at the trough, measured at steady state (within the first 2-4 weeks of treatment) and 
occurrence of bleeding and thromboembolic events during the subsequent one year follow up. 
3.Investigational plan 
3.1 Study design   
The MAS Study is an observational, prospective cohort study, double blind, multicentre, 
international and no Profit.  

 
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3.2 Setting 
Anticoagulation clinics, affiliated or not to the Italian FCSA, will be asked to take an active part in 
the study, provided they have the facilities for blood sampling and processing. 
3.3 Study population 
4000 consecutive NVAF outpatients, 1000 for each single drug, aged >18 years and starting 
anticoagulation with one of the four DOAC (apixaban, dabigatran, edoxaban, rivaroxaban) will be 
enrolled at the moment of the first prescription. Patients will receive the type and dosage of DOAC 
on the base of clinical characteristics at the discretion of the attending physician, as the normal 
clinical practice, and the study will not influence the decision of the type and dosage of DOAC. 
3.3.1 Inclusion criteria  
- NVAF patients starting DOAC anticoagulation  
- age > 18 years  
- ability to give written informed consent  
- availability, as part of the normal withdrawals, to the blood sampling for the study purpose 
- availability for 12-months follow-up 
3.3.2 Exclusion criteria 
- age < 18 years  
- indication for electrical cardioversion at the moment of drug prescription  
- participation in Phase II or III clinical trials 
- indication for treatment different from NVAF 
- not suitable to give or not giving informed consent 
- not available for blood collection or follow-up 
4. Study procedures 
4.1 Enrolment procedure 
The active participants will propose to participate in the study to the NVAF subjects treated with 
DOAC that satisfy the inclusion and exclusion criteria 
4000 consecutive NVAF patients, 1000 for each single drug, aged>18years and starting 
anticoagulation with one of the four DOAC (apixaban, dabigatran, edoxaban, rivaroxaban) will be 
enrolled at the moment of the first prescription. The enrolment will end at the achievement of 
4000 patients, 1000 for each drug. 
The study requires a mandatory plasma collection for the measurement of the anticoagulant 
activity of the drug at the steady state, that is within the first 2-4 weeks of treatment, obtained at 
the time of trough, immediately before the subsequent intake of the drug. It is up to the 
participating centres, depending on their availability and organization, to perform an additional 
plasma collection on the same day 2 hours after the last intake, or to make further blood sampling 
after 3, 6 or 9 months from the time of the first prescription.  
The Italian FCSA guidelines recommend a follow-up visit approximately one month after the first 
prescription to confirm the necessary patient's adherence to the treatment and to complete the 
routine exams not available at the time of prescription. On that occasion, and together with the 
routine blood sampling, it will be collected the plasma needed to measure the DOAC concentration 
for the study, taking 10 ml more of blood. Samples will be treated and identified locally in order to 

 
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maintain patient's anonymity, later they will be centralized to the Biobank of Fondazione Arianna 
Anticoagulazione in Bologna (Italy). Afterwards, samples will be distributed to laboratories 
identified by the Promoter for the measurement of DOAC plasma concentration. Test results will be 
centrally stored and communicated to the patient's recruiting centre only at the end of the study. 
Each patient will have a 12-month follow-up period, that will start at the time of the plasma 
collection, established within 15-30 days from the beginning of DOAC treatment.  
The end of the study is at the conclusion of the 12-month follow-up of the last enrolled patient. 
In the 12 months following the end of the follow-up period of the last enrolled patient, all the data 
will be collected and the statistical analysis and the drafting of the final report will be carried out. 
All patients with NVAF who start DOAC treatment for the first time and meet the inclusion criteria 
are eligible for the study. 
The investigator, after collection of the patient's informed consent, will collect and record in a 
specific electronic database (electronic CRF), the essential information collected during the routine 
checks performed within the previous 60 days. 
The following data will be registered in the electronic CRF: patient identification number, date of 
birth, gender, blood cell count, creatinine, liver enzymes, diabetes, previous stroke/TIA, other 
comorbidities, concomitant medications (with a special attention to antiplatelet drugs), and past 
clinical history.  The CHADS2VASC and the HASBLED score will be calculated for all patients. 
A unique anonymous identifying code will be given to each patient to ensure anonymity, which will 
be used both to collect clinical information and to identify biological samples. The investigator of 
the participating centre has to keep the correspondence between the patient's personal details 
and the related identification code (Appendix 1). 
All events occurring during the 12-month follow-up will be recorded. 
Data entered in the eCRF will be stored in the database located at a server of the S. Orsola-
Malpighi University Hospital in Bologna, which, thanks to an agreement between Fondazione 
Arianna Anticoagulazione and the S. Orsola-Malpighi Hospital, guarantees the storage, backup and 
maintenance of the database. 
An Adjudication Committee will evaluate the adverse events occurring during the year of follow-
up, examining a possible correlation with the anticoagulant activity of the drug, measured in the 
sample taken at the entry of the study. 
4.2 Follow-up procedure 
The follow-up of patients will last 12 months after blood sampling, also in patients that will stop 
anticoagulant treatment. In that case, a telephone follow-up is scheduled, to assess the patient's 
health status. A structured follow-up, as defined by FCSA guidelines, including clinical evaluation at 
the moment of blood sampling and each 3 to 6 months thereafter is required, in order to assess 
the patient’s adherence to the treatment and compliance. During the follow-up, bleeding (major 
and clinically relevant non-major) and thromboembolic complications (Appendix 2) will be 
registered. Patients not referring at established dates are promptly looked for, to avoid as much as 
possible, lost at follow-up. 
Clinical follow up will be performed each 3 to 6 months, and the following data will be registered: 
weight, blood pressure, change in clinical and health status and co-medications, creatinine 
clearance, blood cell count, liver enzymes. The need for an adequate adherence to the treatment 
will be stressed to each patient on every contact occasion; on these occasions, adherence to the 
treatment will be evaluated by a questionnaire according to the "Morisky medication adherence 
scale" (25, 26), given to the patient and to be completed by the patient himself. The negative 

 
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answer to all four questions in the questionnaire indicates a good adherence to the treatment, 
while the presence of even one positive answer suggests criticality in the adherence to therapy 
(26). 
What is required to do during follow up should be in line with the indications for local and usual 
care management. In patients discontinuing anticoagulant treatment for whatever reason before 
the end of the follow-up period, a form detailing reason for discontinuation should be completed 
in the eCRF. Any surgical interventions or invasive procedures resulting in temporary therapy 
interruption (for at least 2 days) with no DOAC administration must be reported in the eCRF. 
4.3 Definition of the conclusion of the study 
The study will be concluded for each patient at the end of the 12-months follow-up and, globally, 
at the completion of the last follow-up of the last patient enrolled 
5. Study evaluations and measurements  
5.1 Laboratory assessment 
Plasma samples will be collected in NVAF patients, after collection of written informed consent, 
within the first 15-30 days of treatment. During routine periodic checks, clinical information 
(weight, blood pressure, changes in clinical status and co-medications, creatinine clearance, blood 
count), and adverse events defined by the protocol (Appendix 2) will be recorded. The drug dose 
intake is recommended in the morning and evening for dabigatran and apixaban, and in the 
morning for rivaroxaban (during breakfast) and for edoxaban.  
Blood sampling should be performed as follows:  
1. at trough level for each anticoagulant drug used, in fasting subject: 12 hours or 24 hours 
after the last dose intake of dabigatran and apixaban, or of rivaroxaban and edoxaban, 
respectively. This blood sample is mandatory for the study. 
It is up to the participating centres, depending on their availability and organization, to perform a 
blood sample at peak, after 2 hours of dose intake, or further blood samples at 3rd, 6th and 9th 
month of treatment. 
 
See Appendix 3 for technical aspects of blood sampling and processing (19). 
The frozen samples in anonymous form will be initially stored at the participating centre, then 
centralized and stored c/o the Biobank of Fondazione Arianna Anticoagulazione in Bologna for 5 
years (compatibly with their quantitative availability). The following specific tests for DOAC levels 
measurement will be performed: dTT (diluted thrombin time) and ECA (Ecarin chromogenic assay) 
calibrated for dabigatran; chromogenic anti-Xa assays, calibrated for apixaban, rivaroxaban, or 
edoxaban. These assays are indirect methods that do not measure quantitatively DOAC 
concentrations (as the gold standard liquid chromatography-tandem mass spectrometry LC-
MS/MS), but the biological activity of the drug. Nevertheless, many published studies have shown 
that the DOAC activity measured by coagulative and chromogenic methods is significantly 
correlated with the DOAC levels measured by LC-MS/MS (20-23), and their use is recommended by 
Italian (24) and international (8) guidelines.  
All the dedicated methods available (from all the companies) will be assessed in laboratories 
identified by the Promoter to measure the anticoagulation level of the four direct oral 
anticoagulant drugs. Each assay, for all collected samples, will be carried-out in the same 
laboratory for that assay (to avoid inter-laboratory variability). The tests will be carried out when 

 
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appropriate in relation to the optimal number of samples to be tested. Results of the tests will be 
kept blind both to attending clinicians and patients; they will be open to the participating centre 
only at the end of follow-up period. 
5.2 Outcomes during the follow-up 
5.2.1 Primary outcomes 
1. Bleeding complications (Appendix 2): a composite of: 
- Major bleeding (ISTH definition) (17) 
- Clinically relevant non-major bleeding (18) 
2. Thromboembolic complications (Appendix 2): 
- objectively documented cerebral vascular event 
- systemic embolism with angiographic demonstration of abrupt arterial occlusion 
3. Mortality (cardiovascular and total) 
5.2.2 Secondary outcomes 
- Drug discontinuation 
- Adverse drug reaction (determining change of treatment) 
- Thromboembolic complications (venous and/or arterial) 
- A composite of thromboembolic and total mortality 
- A composite of bleeding complications and total mortality 
- Incidence of thromboembolic/haemorrhagic complications by CHADS-Vasc subgroups 
5.3 Safety control 
Every 6 months data regarding the events occurred in the interval period and the possible 
relationship with the individual results of drug levels measured at inclusion will be communicated 
to the Safety Committee. The Safety Committee, nominated by the Promoter, may propose the 
interruption of the study in case of excessive outcomes in relation to the drug levels results. 
5.4 Results of the study 
The study will evaluate DOAC plasma concentrations. In particular, dabigatran levels will be 
measured by dTT (diluted thrombin time) or ECA (chromogenic test for anti-IIa activity), while 
apixaban, edoxaban and rivaroxaban levels will be measured by a chromogenic method for anti-Xa 
activity. The possible relationship between DOAC plasma concentrations and the occurrence of 
haemorrhagic or thrombotic events during follow-up will be examined. 
 
6. Statistical Considerations 
6.1 Bias 
To avoid patient's selection bias, the investigators are asked to consecutively include the observed 
patients or, if it is not possible, to declare the criterion adopted for enrolment (e.g. one day a 
week, one week a month, etc.). 

 
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6.2 Sample size  
We estimate that the global incidence of thromboembolic events and bleeding complications to be 
around 2% and 10% in NVAF patients per year. The range of drug levels at trough or peak, from the 
quantification level to the highest measured level, will be divided into classes. Comparisons will be 
made by contrasting the first vs the fourth class (for efficacy: increased incidence of 
thromboembolism in the first vs last class; for safety: increased incidence of bleeding in the last vs 
first class). We assume that the incidence of thromboembolism is 6% in the first class, and 1% in 
the fourth class, thus requiring 211 patients per class (total 844) to demonstrate differences in the 
efficacy end point, with an alpha and beta error equal to 0.05 and 0.80, respectively. We assume 
that the incidence of bleeding is 8% in the first class, and 16% in the fourth class, thus requiring 
252 patients per class (total 1004) to demonstrate differences in the safety end point, with an 
alpha and beta error equal to 0.05 and 0.80, respectively.   
We therefore aim at evaluating 1000 NVAF patients per drug subgroup (total 4000 patients). 
For the statistical analysis, follow-up will be interrupted and patients censored when: 
- a primary outcome is reached 
- therapy is definitively interrupted 
- end of study 
6.3 Statistical analysis plan  
Calculation of the mean (+ -SD) and median (interquartile distribution) of the drug blood levels at 
the trough and peak level. Investigation of the distribution of primary and secondary events in 
relation to drug blood levels, and study of correlations. 
Patients who will not be present at the controls on the established dates will be immediately 
contacted, to avoid as much as possible the number of subjects lost to follow-up. 
Patients with no frozen plasma aliquots to perform the tests, or for which test can not be 
performed for any reason, are excluded from the study. 
Patients with no available clinical follow-up are excluded from the study, but the performed 
laboratory tests are used to evaluate the overall variability of the anticoagulation levels. 
Patients with an available follow-up lower than the expected 1-year observation, are considered 
only for the available follow-up period. 
 
7. Safety management 
Since the study procedures are not greater than minimal risk, Serious Adverse Events (SAEs) are 
not expected. If any unanticipated problems related to the research involving risks to subjects or 
others happen during the course of this study (including SAEs) these will be reported to the IRB in 
accordance with CHOP IRB SOP 408: Unanticipated Problems Involving Risks to Subjects. Clinical 
adverse events (AEs) that are not serious but that are notable and could involve risks to subjects 
will be summarized in narrative or other format and submitted to the IRB at the time of continuing 
review. Adverse drug reactions should be reported according to the rules for spontaneous 
reporting (post-marketing).  

 
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8. Study administration 
8.1 Data collection and management 
Patient’s personal data will be collected (in anonymous form), date of birth, gender, weight, height, 
liver and hepatic enzymes, past clinical history, thrombotic/haemorrhagic risk factors, lifestyle 
habits, type of treatment, clinical events during the follow-up. 
The monitor of the study will check data quality and completeness by remote monitoring.  
Data will be anonymously entered by the investigator in the eCRF, by assigning a patient 
identification code. 
The adopted database is mySQL and it will be produced by Softime90 Snc company. 
Data anonymously entered in the eCRF will be recorded and stored in the server of the S. Orsola-
Malpighi University Hospital in Bologna.  
Each access to the eCRF will be managed by personal user name and a password. 
The security system requires that the password be changed at the first access; it requires a length 
of at least 8 characters and provides for a periodic 3-month expiry. 
It is obviously possible to disable the login, if it is deemed necessary by the system administrator. 
Investigators can have full access only to their patient’s data. 
Every night the data backup is automatically performed. 
The Study Monitor will send blind to the Safety Committee the data of the occurred events and all 
the necessary related information for their assessment. 
8.2 Ethics 
The study will be conducted in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki and in compliance with the protocol and ICH-GCP.  
This is an observational study and all drug prescriptions and clinical investigations are independent 
from the study. 
Patients will receive the type and dosage of DOAC on the base of clinical characteristics at the 
discretion of the attending physician, as the normal clinical practice, independently from the 
decision to enrol the patient in the study.  
The protocol and any amendments and the informed consent form (or information/non-opposition 
letter, as allowed by local regulations) will have to obtain Institutional Review Board or Ethics 
committee (IRB/EC) approval prior to initiation of the study.  
8.3 Informed consent forms 
Participation in this study is entirely voluntary. Informed consent will be obtained in accordance 
with the Declaration of Helsinki, ICH GCP and local regulations. A properly executed, written, 
informed consent will be obtained from each subject prior to entering the subject into the trial.  
Information should be given in both oral and written form and subjects (or their legal 
representatives) must be given ample opportunity to inquire about details of the study.  If 
appropriate and required by the local IRB/IEC, assent from the subject will also be obtained.  If a 
subject is unable to sign the informed consent form (ICF) a legal representative may sign for the 
subject. A copy of the signed consent form will be given to the subject or legal representative of 
the subject and the original will be maintained with the subject’s records. 
Once a patient has been enrolled in the study, he/she may withdraw his/her consent to participate 
in the study at any time without prejudice. Patients will not be entered in the database if the 
informed consent has not been obtained.  

 
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8.4 Administrative issues 
8.4.1 Support of the study 
Participation in the MAS study is entirely voluntary and no form of remuneration or refund is given 
to the investigators. 
The MAS study is supported by own funds of the Promoter, for the preparation, working and 
maintenance of all the necessary activities, i.e. informatic and monitoring activities, centralization, 
storage and distribution of plasma aliquots, reagents for laboratory activities, secretary needs, 
statistical analysis of results, production of reports and publication of scientific papers. 
Fondazione Arianna Anticoagulazione will free provide to the Italian centres the material for 
plasma collection, buying the cryogenic storage tubes. 
Becton Dickinson supports the study by the free supply of blood sampling tubes for Italian centres. 
The Promoter will also support the costs for the shipment of the material to Italian centres and for 
the centralization of Italian plasma samples and their further distribution to the centres chosen for 
the execution of laboratory tests. 
 
9. Publication  
9.1 Role of Promoter and Investigators 
The Promoter and the investigators may suggest specific analysis, regarding all or some data of the 
central database. The authorship of the article must provide who proposed the analysis, who 
performed the data analysis, who took part in writing the article. 
The role of the Promoter and investigators in the study (study design, collection, management, 
data analysis and interpretation, report writing, authorship) should be described. 
9.2 Data handling and record keeping 
All data and results and all intellectual property rights to the data and results derived from the 
study will be the property of the Promoter Fondazione Arianna Anticoagulazione according to D.M. 
17 Dicembre 2004. This study is a No Profit Study (D.M. 17 Dicembre 2004, Art. 1, comma 2, lettera 
c) 
9.3 Final report and publication   
Within 12 months from the completion of the trial and in accordance with ICH-GCP, the promoter 
provides the IRB/IEC with a summary of the trial’s outcome, and the regulatory authority(ies) with 
any reports required. All data and results and all intellectual property rights to the data and results 
derived from the study will be the property of Fondazione Arianna Anticoagulazione, who may 
utilize the data in various ways, such as for submission to government regulatory authorities or 
disclosure to other investigators.  All publication or communication (oral or written) will respect 
the international requirements: “Uniforms requirements for Manuscripts Submitted to Biomedical 
Journals”.   

 
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10. References 
 
1. 
Ageno W, Gallus AS, Wittkowsky A, Crowther M, Hylek EM, Palareti G. Oral Anticoagulant 
Therapy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest 
Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012;141(2 Suppl):e44S-88S. 
2. 
Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, et al. Dabigatran 
versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361(12):1139-51. 
3. 
Patel MR, Mahaffey KW, Garg J, Pan G, Singer DE, Hacke W, et al. Rivaroxaban versus 
warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011;365(10):883-91. 
4. 
Granger CB, Alexander JH, McMurray JJ, Lopes RD, Hylek EM, Hanna M, et al. Apixaban 
versus warfarin in patients with atrial fibrillation. N Engl J Med. 2011;365(11):981-92. 
5. 
Giugliano RP, Ruff CT, Braunwald E, Murphy SA, Wiviott SD, Halperin JL, et al. Edoxaban 
versus warfarin in patients with atrial fibrillation. N Engl J Med. 2013;369(22):2093-104. 
6. 
Pengo V, Crippa L, Falanga A, Finazzi G, Marongiu F, Palareti G, et al. Questions and answers 
on the use of dabigatran and perspectives on the use of other new oral anticoagulants in patients 
with atrial fibrillation. A consensus document of the Italian Federation of Thrombosis Centers 
(FCSA). Thromb Haemost. 2011;106(5):868-76. 
7. 
Testa S, Tripodi A, Legnani C, Pengo V, Abbate R, Dellanoce C, et al. Plasma levels of direct 
oral anticoagulants in real life patients with atrial fibrillation: Results observed in four 
anticoagulation clinics. Thromb Res. 2016;137:178-83. 
8. 
Kitchen S, Gray E, Mackie I, Baglin T, Makris M. Measurement of non-coumarin 
anticoagulants and their effects on tests of Haemostasis: Guidance from the British Committee for 
Standards in Haematology. Br J Haematol. 2014;166(6):830-41. 
9. 
Reilly PA, Lehr T, Haertter S, Connolly SJ, Yusuf S, Eikelboom JW, et al. The effect of 
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Thromb Haemost. 2015;13(11):2119-26. 

 
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APPENDIX 1 
Labelling code 
1. Alphanumeric code chosen by the centre 
2. Date of collection 
3. V (uppercase) for trough sample and P (lowercase) for peak sample 
 
Example of labelling: “Alphanumeric code/01/01/2018/V”, that identify the trough sample of a 
patient dated 01/01/2018. 
That code has to be inserted in the proper field in the eCRF (tube code) 
 
 
 
 

 
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APPENDIX 2 
 
OUTCOME DEFINITIONS 
VENOUS THROMBOEMBOLISM (VTE) 
 
Pulmonary embolism (PE) 
Symptoms of PE with one of the following findings. 
 A new intraluminal filling defect in (sub) segmental or more-proximal branches on spiral 
computed tomography (CT) of the chest. 
 A new intraluminal filling defect, or an extension of an existing defect, or a new sudden 
cutoff of vessels more than 2.5 mm in diameter on the pulmonary angiogram. 
 A new perfusion defect of at least 75% of a segment, with a local normal ventilation result 
(high probability) on ventilation/perfusion lung scintigraphy (VQ scan). 
 Inconclusive spiral CT, pulmonary angiography, or VQ scan evidence of a new or recurrent 
PE, with demonstration of a new or recurrent deep vein thrombosis (DVT) in the lower 
extremities by compression ultrasound (CUS) or venography. 
 
Proximal deep vein thrombosis (DVT) 
Symptoms of DVT with one of the following findings. 
(a) For a NEW DVT: abnormal CUS, or an intraluminal filling defect on venography. 
(b) For a RECURRENT DVT: 
 abnormal CUS where compression had been normal or, if non-compressible during 
screening, a substantial increase (4 mm or more) in diameter of the thrombus during full 
compression, or 
 an extension of an intraluminal filling defect, or a new intraluminal filling defect, or an 
extension of non-visualization of veins in the presence of a sudden cut-off on venography. 
 
DEATH 
For all patients who died during the study, the cause of death was adjudicated to one of the 
following categories. 
 VTE-related death 
 PE (based on objective diagnostic testing, autopsy) 
 Unexplained death (and VTE cannot be ruled out) 
 Sudden death (and VTE cannot be ruled out). 
 Cardiovascular (CV)-related death 
 Myocardial infarction (MI) 
 Stroke  
 Other CV event (to be specified). 
 Other 
 Cancer 
 Bleeding 

 
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 Infectious disease 
 Other known cause (to be specified). 
 
BLEEDING EVENTS 
Major bleeding event 
A major bleeding event was defined as a bleeding event (as per International Society on 
Thrombosis and Haemostasis guidelines 11), as follows. 
 Acute clinically overt bleeding accompanied by one or more of the following. 
 a decrease in hemoglobin of 2 g/dl or more 
 a transfusion of 2 or more units of packed red blood cells 
 bleeding that occurs in at least one of the following critical sites: intracranial, intraspinal, 
intraocular, pericardial, intra-articular, intramuscular with compartment syndrome, 
retroperitoneal 
 fatal bleeding 
 
Clinically relevant non-major bleeding event 
The definition of clinically relevant non-major bleeding was acute clinically overt bleeding that 
consists of: 
 any bleeding compromising hemodynamics 
 any bleeding leading to hospitalization 
 subcutaneous hematoma larger than 25 cm2, or 100 cm2 if there was a traumatic cause 
 intramuscular hematoma documented by ultrasonography 
 epistaxis that lasted for more than 5 minutes, was repetitive (i.e. two or more episodes of 
bleeding more extensive than spots on a handkerchief within 24 hours), or led to an 
intervention (e.g. packing or electrocoagulation) 
 gingival bleeding occurring spontaneously (i.e. unrelated to eating or tooth brushing) or 
lasting for more than 5 minutes 
 hematuria that was macroscopic and was spontaneous or lasted for more than 24 hours 
after instrumentation (e.g. catheter placement or surgery) of the urogenital tract 
macroscopic gastrointestinal hemorrhage, including at least one episode of rectal blood 
loss, if more than a few spots on toilet paper 
 hemoptysis, if more than a few speckles in the sputum and not occurring within the 
context of PE 
 any other bleeding type considered to have clinical consequences for a patient such as 
medical intervention, the need for unscheduled contact (visit or telephone call) with a 
physician, or temporary cessation of a study drug, or associated with pain or impairment of 
activities of daily life 
 
Minor bleeding events 
All acute clinically overt bleeding events not meeting the criteria for either major bleeding or 
clinically relevant non-major bleeding were classified as minor bleeding. 
 
Fatal bleeding event 
A fatal bleeding event was defined as a bleeding event that the adjudication 
committee determined was the primary cause of death or contributed directly to death. 

 
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APPENDIX 3 
Sample collection protocol 
 
1. Select the patient treated with one of the direct acting oral anticoagulants, which you want 
to include in the study. Besides other characteristics (see protocol for further details) the 
patient must express (after appropriate information) the availability to perform a trough 
and a peak (optional) withdrawal, to be carried out preferably in the morning, just before 
taking the daily dose (or one of two daily doses, for double-dose drugs). Trough withdrawal 
should be performed 12 hours after the last dose of dabigatran or apixaban and 24 hours 
after the last intake of rivaroxaban or edoxaban. 
2. For each sample it is required a total blood volume of 9-10 ml (e.g. 2 tubes of 4.5 ml or 4 
tubes of 2.7 ml), taken with a vacuum system, containing citrate at the concentration of 
0.109 M (3.2%). 
3. Centrifuge (within 2 hours from sampling) for 15 minutes at 2,000xg at controlled room 
temperature (20°C). 
4. Separate the plasma, without disturbing the cells, and mix it in a single plastic tube. 
Dispense the plasma into cryogenic storage tubes in aliquots of 0.5 mL, taking care to 
prepare as many as possible (with that blood volume - see above - the tubes will be at least 
7-8). Each tube will be labelled by a code (see Appendix 1) that identifies centre, patient, 
date of collection and indicates whether it is a trough or peak sample. 
5. Cap the tubes and move them into a freezer -70°C as soon as possible. If it is possible, it 
would be recommended to quickly freeze the plasma, before transfer it to the freezer, by 
immersion for a few seconds in liquid nitrogen or 10 minutes in an alcohol and dry-ice bath. 
If a freezer -70°C is not available, samples can be stored in a freezer -30°C for up to 3 
months. 
6. Move the samples to the Biobank of the Promoter, according to the planning and method 
indicated in the study protocol.