Targeted Metabolic Profiling in Deep Vein Thrombosis
Conditions: Deep Vein Thrombosis
Sponsor: Imperial College London

1 
 
 
 
 
 
 
 
Targeted Metabolic Profiling in Deep Vein Thrombosis 
 
 
Version 4.0 14/05/19 
 
 
 
 
MAIN SPONSOR: Imperial College London 
FUNDING: GRAHAM DIXON CHARITY GRANT  
STUDY COORDINATION CENTRE: Academic Section of Vascular Surgery 
Department of Surgery & Cancer 
Imperial College London 
Room 4E04, 4 East 
Charing Cross Hospital 
Fulham Palace Road 
London, W6 8RF 
 
NRES reference: 245270

2 
 
Study Management Group 
 
Principal Investigator: 
 
 
Professor Alun H Davies 
Professor of Vascular Surgery 
Academic Section of Vascular Surgery,  Department of Surgery & Cancer 
Imperial College London 
Room 4E04, 4 East  
Charing Cross Hospital, Fulham Palace Road  
London, W6 8RF Telephone: 020 3311 7320 
Email: a.h.davies@imperial.ac.uk 
 
Co-investigators: 
 
Mr Kemal I Kemal 
Clinical Research Fellow 
Academic Section of Vascular Surgery,  Department of Surgery & Cancer 
Imperial College London 
Room 4N18, 4th Floor, North Wing 
Charing Cross Hospital, Fulham Palace Road 
London W6 8RF 
Telephone: 020 3311 1234 (extension 17250) 
 
Miss Sarah Onida 
NIHR Clinical Lecturer in Vascular Surgery and Honorary Vascular Specialty Registrar 
Academic Section of Vascular Surgery, Department of Surgery & Cancer  
Imperial College London 
Room 4N12, 4th Floor North Wing, 
Charing Cross Hospital, Fulham Palace Road 
London, W6 8RF 
Telephone 02033117317 
E-mail s.onida@imperial.ac.uk 
 
 
 
Professor Elaine Holmes, 
Professor of Chemical Biology 
Section of Computational and Systems Medicine,  Department of Surgery and Cancer 
Imperial College London 
Room 661, Sir Alexander Fleming Building 
South Kensington Campus 

3 
 
E-mail: elaine.holmes@imperial.ac.uk 
 
Professor Mike Laffan 
Professor of Haemostasis & Thrombosis  
Faculty o f Med icine,  Department of Medicine 
Imperial College London 
  
 
  
 
 E-mail: m.laffan@imperial.ac.uk 
 
Clinical Queries 
 
Clinical queries should be directed to Professor Alun H. Davies or Mr Kemal I Kemal who 
will direct the query to the appropriate person. 
 
 
Sponsor 
 
Imperial College London is the main research Sponsor for this study. For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at: 
 
Joint Research Compliance Office 
Imperial College London & Imperial College Healthcare NHS Trust Room 215, 
Level 2, Medical School Building, Norfolk Palace London 
W2 1PG 
Email: r.nicholson@imperial.ac.uk 
 
 
Funder 
 
 
There will be no participants or investigator payments.  The Graham- Dixon Charitable Trust has 
awarded £14 491 towards the consumables cost of this research for the duration of the project. Further 
grant applications will be submitted to fund the experimental analysis. 
 
This protocol describes the “Targeted Metabolic Profiling in Deep Venous Thrombosis” study and 
provides information about procedures for entering participants. Every care was taken in its drafting, 
but corrections or amendments may be necessary. These will be circulated to investigators in the 
study. Problems relating to this study should be referred, in the first instance, to the Chief 
Investigator. 
 
This study will adhere to the principles outlined in the NHS Research Governance Framework for Health 
and Social Care (2nd edition). It will be conducted in compliance with the protocol, the Data Protection 
Act and other regulatory requirements as appropriate. 
Room 5S5b, Hammersmith Hospital 
Hammersmith Campus 

4 
 
Table of Contents 
1. 
INTRODUCTION 
11 
1.1 BACKGROUND AND RATIONALE FOR CURRENT STUDY 
11 
2. STUDY OBJECTIVES 
12 
3. STUDY DESIGN 
12 
3.1 
INCLUSION CRITERIA 
13 
3.2 
EXCLUSION CRITERIA 
3.3 
WITHDRAWAL CRITERIA 
3.4 
STUDY PROCEDURES, SAMPLE COLLECTION AND PREPARATION 
14 
4. 
ASSESSMENT AND FOLLOW-UP 
15 
5. 
STATISTICS AND DATA ANALYSIS 
16 
6. 
ADVERSE EVENTS 
16 
6.1 
DEFINITIONS 
17 
6.2 
REPORTING PROCEDURES 
17 
7.1 
       REGULATORY ISSUES 
18 
7.2 
ETHICS APPROVAL 
18 
7.3 
CONSENT 
18 
7.4 
CONFIDENTIALITY 
18 
7.5 
INDEMNITY 
18 
7.6 
SPONSOR 
18 
7.7 
FUNDING 
18 
7.8 
AUDITS 
18 
8. 
STUDY MANAGEMENT 
   19 
9. 
PUBLICATION POLICY 
19 
10. 
REFERENCES 
19 
APPENDIX 1.  SUMMARY OF INVESTIGATIONS AND ASSESSMENTS 
20 

5 
 
GLOSSARY OF ABBREVIATIONS 
 
DUS 
Duplex ultrasound 
DVT 
Deep vein thrombosis 
LMWH 
Low molecular weight heparin 
MS 
Mass spectrometry 
NMR 
Nuclear magnetic resonance 
DOAC 
Direct oral anti-coagulant 
VTE 
Venous thromboembolism 
DNA 
Deoxyribonucleic acid 
RNA 
Ribonucleic acid 
RCF 
Relative centrifugal force 
PLS 
Partial least squares 
PCA 
Principal component analysis 
SAE 
Serious adverse event 
NRES 
National Research Ethics Service 
CSM 
Section of Computational and Systems Medicine, 
Department of Surgery and Cancer, Imperial 
College London 
OPLS 
Orthogonal partial least squares 
OPLS-DA 
Orthogonal partial least squares - discriminant 
analysis 
 
 
KEYWORDS 
Deep vein thrombosis, metabonomics, biomarkers 

6 
 
STUDY SUMMARY 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   TITLE 
Targeted Metabolic Profiling in Deep Venous Thrombosis 
                                 DESIGN 
Prospective observational case-control study 
    AIMS 
Identification of novel biomarkers for Deep Vein Thrombosis using 
biometabolomic platforms 
OUTCOME MEASURES 
Deep Vein Thrombosis biomarkers 
POPULATION 
40 patients diagnosed with a confirmed DVT and 40 healthy non-DVT controls 
ELIGIBILITY 
Disease - Adults, willing and able to consent, presenting with confirmed DVT.  
Controls - Adults, able and willing to consent, without a DVT diagnosis. 
Exclusion criteria - pregnancy, Hepatitis B, C or HIV positive, active cancer, 
steroid treatment 
DURATION 
      28 months 

7 
 
FLOWCHART-REFERENCE DIAGRAM 
 
 
 
 
 
 
     
 
 
 
 
Exclusion Criteria: 
Pregnancy (pregnancy excluded 
based upon patient history, 
documented last menstrual period and 
urinary pregnancy test if patient 
unsure) 
Patients with blood borne disorders 
(HIV, Hep B, C) 
Patients with an active malignancy 
currently undergoing chemo or 
radiotherapy 
Patients taking systemic steroids or 
immunomodulating drugs 
Patients involved in a different 
venous research project 
Patients aged >18 years with 
a suspected DVT 
Informed Consent 
Medical History 
Clinical examination 
Blood and urine sample 
Ultrasound not available 
during the first visit 
Ultrasound available 
during the first visit 
Anticoagulation 
commenced 
Blood and urine sample 
(if not already taken day 
before ) 
Ultrasound scan result 
Patients recruited 
N=40 
Return the next day 
(as per standard practice) 
Ultrasound scan 
negative for 
DVT 
Ultrasound scan positive 
for DVT N=40 
Excluded from 
study 
Second visit, 3 weeks   after 
the end of anticoagulation 
treatment 
Medical history 
Clinical examination 
Blood and urine sample 
Ultrasound scan 

8 
 
Healthy volunteers, aged >18 years 
 
 
Exclusion 
i
i
 
 
 
 
 
 
 
 
Pregnancy (pregnancy 
excluded based upon patient 
history, documented last 
menstrual period and urinary 
pregnancy test if patient 
unsure) 
Patients with blood borne 
disorders (HIV, Hep B, C) 
Patients taking systemic 
steroids or immunomodulating 
drugs 
Active cancer  
Participating in a 
different venous research 
project 
Consent Medical History 
Ultrasound Scan 
Clinical examination  
Blood and urine sample 
Volunteers recruited 
N=40 

9 
 
1. 
INTRODUCTION 
 
1.1 
BACKGROUND AND RATIONALE FOR CURRENT STUDY 
 
 
Deep venous thrombosis (DVT) is a blood clot, usually affecting the legs, causing pain, 
swelling, and redness. The clot damages the veins, which can result in chronic pain, swelling 
and ulceration. This is called the post-thrombotic syndrome, which impacts heavily on 
patients’ life and work. If the clot dislodges and travels to the lungs, it becomes a pulmonary 
embolus (PE), which can be life threatening. Together, DVT and PE affect 500,000 people in 
Europe every year, representing the most common preventable cause of hospital acquired 
death [1,2]. They are expensive conditions not only due to their treatment costs, but also 
secondary to the resulting loss of work days and productivity. 
 
DVT is diagnosed by clinical examination, risk scoring, usually via the Wells Score, and a 
blood test called D dimer, a fibrin degradation product. D-dimer is specific but not very 
sensitive. In other words, when the test is negative, it is unlikely that a DVT is present. 
However, many conditions can raise D-dimer levels, making it less useful when positive [3]. 
Duplex ultrasound can confirm the presence of clot but this can be difficult to visualize or is 
often not seen at all. The clot can take time to form and patients may not experience 
symptoms immediately. This is a problem for treatment, as new, clot-busting medication 
works best in the first 2 weeks after a DVT and it is currently impossible to tell when the 
thrombus formed [4]. 
 
Because of the importance of DVT, and ongoing issues regarding its diagnosis, ageing and 
prognosis, numerous research groups have been exploring novel candidate biomarkers to help 
develop a more accurate diagnostic and prognostic marker for DVT. The Section of Vascular 
Surgery and of Computational and Systems Medicine (CSM) at Imperial College, have had a 
longstanding collaboration working towards this end using metabonomics. 
 
Metabonomics is defined as: ‘the quantitative measurement of the multiparametric metabolic 
response of living systems to pathophysiological stimuli or genetic modification’. In other 
words, it examines the end products of cellular metabolism via platforms such as Nuclear 
Magnetic Resonance Spectroscopy (NMR) and Mass Spectrometry (MS), which are both 
high throughput analytical platforms. These enable the qualification (untargeted) and 
quantification (targeted) of different molecules in a given sample, to a degree of detail 
superior to that offered by other omic technologies (e.g. proteomics). Metabonomics has 
demonstrated clear applications in vascular disease, including chronic venous disease [5], 
atherosclerosis [6], venous leg ulceration [7], aneurysmal disease and stroke [8]. 
 
Untargeted preliminary work on deep venous thrombosis has revealed a metabolic signature 
for DVT using a murine animal model [9], and promising pilot data in a human study. The 
aim of the proposed study is to validate previous findings in a separate patient cohort and 
perform quantification of the metabolites identified in the pilot studies via targeted NMR and 
MS experiments, continuing the exploration of novel biomarkers for DVT. 

10 
 
2. STUDY OBJECTIVES 
 
Primary outcome: 
• Quantification of metabolites of interest and comparison of their concentrations 
in DVT and control participants 
Secondary outcomes: 
• Comparison of metabolite presence and concentration with other endogenous 
biomarkers associated with DVT, including D-dimer and P selectin. 
• Validation of previously identified metabolites in the human DVT pilot study 
• Assessment of the response to DVT treatment following anticoagulation 
• Identification of candidate biomarkers to explore thrombus ageing in future studies 
 
 
2.1 
STUDY OUTCOME MEASURES 
 
Primary Outcome: 
 Identification of diagnostic deep vein thrombosis metabolites  
 
Secondary Outcome: 
 Validation of previously identified molecules/chemicals in a separate population 
 Quantification of identified molecules in DVT patients and controls  
 Comparison of identified metabolite concentrations to known DVT biomarkers (e.g. D 
dimer) 
 Identification of potential molecules to study in future work with respect to thrombus 
ageing 
 Longer term, genetic testing will be performed to compare the results to the 
metabonomic analysis 
 
 
3. STUDY DESIGN 
 
Type of study 
The study is a prospective observational case-control study, recruiting 40 DVT patients and 
40 healthy subjects. Patients diagnosed with a DVT will be recruited at the sites of Imperial 
College Healthcare NHS Trust. 
 
Summary 
Forty patients diagnosed with a DVT will be recruited.  Participation in the study will not 
interfere with the decision making and diagnostic investigations by the direct care team. A 
second control group of 40 healthy volunteers will be also examined. Healthy subject 
recruitment will take place at the same sites. A patient information leaflet will be provided to 
participants for informed consent purposes. 
 
A member of the research team will explain the study to prospective participants. Both 
patients and volunteers will be given sufficient time to read the information sheet and make 
an informed decision regarding participation in the study; this will be confirmed by the 
participant signing a consent form. The study involves a comprehensive history and clinical 
examination, recording medication taken and collection of blood and urine samples. The 
samples will be labeled anonymously with a study number and transferred to the Imperial 

11 
 
College laboratories in South Kensington. Sample transfer will be performed securely 
according to established departmental standard operating procedures (SOPs). A blood sample 
will be stored for future DNA analysis to complement the findings from the metabonomic 
assays. No DNA analysis will be performed for this particular project. 
 
The DVT group will have a repeat ultrasound study, performed by a vascular research fellow 
on a dedicated (non-NHS) machine 3 weeks after anticoagulation treatment has ended. They 
will also have repeat urine and blood samples taken. None of the research interventions will 
have an impact on the clinical care the patients will receive. 
A letter explaining the purpose of the study will be sent to the General Practitioner if the 
participant wishes. All clinical and research information will remain confidential. Consent 
forms will be stored in a locked room accessible only to the researchers involved in the 
study. Any clinical data recorded electronically will be stored in an anonymised fashion 
using study numbers and saved in encrypted, password protected files. 
 
Patient Groups: 
 
• Group 1: Patients with DVT confirmed by lower limb venous duplex ultrasound 
(DUS) (n =40) 
• Group 2: Healthy volunteers with no DVT confirmed on DUS (n = 40) 
 
 
PARTICIPANT ENTRY: 
 
3.1 INCLUSION CRITERIA 
 
- 
>18 years of age 
- 
Willing/able to give written informed consent 
- 
Patients with DVT and age and sex matched controls confirmed on duplex ultrasound 
 
 
3.2 EXCLUSION CRITERIA 
 
- 
Pregnancy (excluded based upon patient history, documented last menstrual 
period and urinary pregnancy test if patient unsure) 
- 
Patients with blood borne disorders (HIV, Hepatitis B, C) 
- 
Patients on systemic steroids and immunomodulating drugs 
- 
Patients involved in a different venous research project or who have recently been 
involved with a venous research project 
 
3.3 WITHDRAWAL CRITERIA 
Participants can withdraw from the study at any point without giving any reason. 

12 
 
3.4 Study procedures, sample collection and preparation 
 
 
Clinical data: 
First Visit (Group 1 – DVT): Patient history (including medications, dietary habits and last 
meal) and clinical examination will be performed. A Wells’ score will be recorded (to test 
probability of DVT) and the presence of varicose veins noted. DUS will be performed on the 
affected limb by a vascular scientist, and by the clinical research fellow (CRF) to the 
asymptomatic limb. Blood and urine sampling will be performed. A pregnancy test will also 
be performed in all females with child bearing potential. The CRF will be trained by Mary 
Ellis, Lead Vascular Scientist at Imperial College Healthcare NHS Trust, in performing DUS 
examinations. The anatomical extent of the acute thrombus will be assessed using LET 
(Lower Extremity Thrombus) scoring. 
 
Occasionally it is not possible to carry out the DUS on the day of presentation; in this case, 
the patient will be recruited and samples taken before they return home. This may be before 
or after administration of parenteral anticoagulation. The patient will then return to hospital 
the next day to have the scan – participation in the study will be confirmed following the scan 
results. If the patient is excluded from this study we will retain the blood and urine samples 
taken for future DNA analysis on an established biobank. 
 
First Visit (Group 2 – controls): History (including medications, dietary habits and last 
meal) and clinical examination will be performed. DUS to both legs will be performed by a 
CRF or vascular scientist to ensure no DVT is present.  Blood and urine sampling will be 
performed and stored as per departmental SOPs. 
 
Sample preparation and storage: 
All sample collection will be performed according to departmental SOPs. 
Blood: 
Blood tests will be performed via aseptic technique by a trained healthcare professional. 
Samples collected at clinical sites will be placed in containers and labelled anonymously 
using a study participant number. Blood will be transferred into two 13 x 100 mm (5mL) 
plastic serum tubes (Becton, Dickinson and Company, New Jersey, USA), allowed to stand 
for 30 minutes at room temperature, centrifuged at 4500RCF for 10 minutes; this will cause 
serum separation. The serum fraction will generate a minimum of three 0.4mL aliquots, 
which will be transferred to 1.5 ml Eppendorf® tubes, which will be immediately placed on 
dry ice and transferred to a -80°C freezer. 
 
Urine: 
Patients will be asked to provide a mid-stream urine sample into a 50mL Falcon tube 
(Becton, Dickinson and Company, New Jersey, USA). Four 1mL aliquots of urine will be 
transferred to 1.5 ml Eppendorf® tubes, which will be immediately placed on dry ice and 
then transferred to a -80°C freezer. 
 
Sample separation: 
Sample separation and analysis will take place at the Section of Computational and Systems 
Medicine, Department of Surgery and Cancer, Imperial College London. We will isolate 
components from the blood and store these (e.g. DNA, RNA, plasma, serum, blood precursor 
cells, etc.) for use in future research (for a maximum of 10 years) for the patient group. 
 
For the control group, blood and urine samples will be discarded at the end of this project.  
For any participant in the patient group, whose duplex scan demonstrates that they do not 

13 
 
have a DVT then they will be withdrawn from the study and all blood/urine samples 
collected for the study will be discarded. 
   
Metabolite Profiling: 
 
Targeted NMR and ultra performance liquid chromatography (UPLC-MS) platforms will be 
employed for the metabolic profiling of urine and serum. UPLC-MS analysis will be 
performed on a Waters ACQUITY UPLCTM system (Waters, Milford, MA), coupled with a 
Waters XEVO G2 Mass Spectrometer (Waters MS Technologies, Manchester, UK). Internal 
standards available at CSM will be used for quantification purposes. All samples will be 
analysed in both positive and negative electrospray ionisation (ESI) MS modes. 1H NMR 
spectra will be acquired on a Bruker DRX-600 spectrometer (Bruker Biospin, Germany) 
operating at 600.13 MHz, with a probe temperature of 300 K. 
 
Metabolites to study: 
Detection and quantification of metabolites in human blood serum and urine will be 
completed using targeted MS and NMR metabolic profiling approaches. MS and NMR are 
the two main analytical techniques employed for metabolic profiling, providing 
complementary information. All endogenous metabolic profiling studies result in complex 
multivariate data sets that require pre-processing, use of visualization software and 
chemometric methods for interpretation. The MS and NMR spectra from the samples will be 
analysed in order to identify metabolite features whose intensities and quantity is 
significantly different between the disease and control groups. These important metabolites 
will be classified and ultimately identified. The identification of molecules is a challenging 
procedure. Consultation with in-house metabolite databases and online databases is the first 
step in metabolite structural elucidation. These databases contain information regarding 
thousands of endogenous and drug metabolites, including MS spectra, in some cases MS/MS 
spectra and experimental and predicted metabolite 1H- and 13C-NMR data. Well- 
characterized metabolites may be identified through database searches but for unambiguous 
metabolite identification, co-chromatography and comparison of MS/MS data with the 
authentic compound are necessary. 
 
DNA: extraction and storage 
Blood sample for DNA will be preferentially taken during the first visit. A 5ml EDTA sample 
will be obtained for DNA extraction and storage.  Extraction will be by standard DNA 
extraction columns (Qiagen, Germany). DNA will be aliquoted and stored at -80C. If 
indicated by the metabonomic analysis then the samples may be used for sequencing of 
specific genes. However, it is intended that if differences between patients and controls are 
identified, a submission for whole genomic sequencing will also be submitted. The rapidly 
falling costs of sequencing and increasing resource allocated to this analysis, make this a 
practical and important subsequent step. 
 
4. 
ASSESSMENT AND FOLLOW-UP 
The patients in Group 1 with confirmed DVT, receiving anticoagulation treatment 
according to standard care, will be reviewed 3 weeks after the end of their treatment. They 
will then have repeat urine and serum samples taken. Both limbs will be scanned again by 
the clinical research fellow to assess the deep venous system and check for clot resolution . 
The clinical study will be complete when all patients have been recruited, followed up and 
the last sample has been taken. The laboratory portion of the study will be complete once all 
samples have been analysed. 

14 
 
5. 
STATISTICS AND DATA ANALYSIS 
 
Sample size 
The sample size of 40 patients in each group has been determined following consultation 
with Professor Elaine Holmes, Head of the Section of Computational and Systems Medicine, 
and based upon the results of the previous preliminary study, which revealed a statistically 
significant difference between the DVT and control groups in a sample size of 40 patients in 
each group. This will suffice to validate the previous results and quantify metabolite(s) of 
interest.  
 
Data Analysis and Statistics 
Following NMR and MS experiments, raw data is generated in the form of spectra. Prior to 
the application of multivariate approaches, both NMR and MS datasets will undergo a 
number of key pre-processing steps including binning, peak picking, alignment and 
normalization. 
 
Spectral data is impossible to assess to the naked eye; therefore, multivariate statistical 
techniques must be employed to determine group differences (e.g. between disease and 
controls) and identify spectral areas responsible for driving the difference (representing 
metabolites of interest). This is performed by using software that presents data points and 
groupings in a visual manner. This permits identification of clusters, outliers, trends and 
batch effects. Multivariate statistics include unsupervised (i.e. principal component analysis 
[PCA]) and supervised methods (i.e. partial least squares [PLS], orthogonal-PLS [OPLS] and 
OPLS discriminant analysis [OPLS-DA]). Following multivariate analysis, metabolites will 
be identified and pathway analysis performed using in house and external tools (Chemspider, 
KEGG). 
 
 
 
Fig 1. Workflow in metabolic phenotyping. Metabolite analysis includes digitalisation of 
findings into spectra, which will be compared to known metabolite lists and will be correlated 
to biological pathways in order to identify possible biomarkers. 
 
 
Data and all appropriate documentation will be stored for a minimum of 10 years after the 
completion of the study, including the follow-up period. 
 
6. 
ADVERSE EVENTS 
 
6.1 
DEFINITIONS 
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject. 
 
Serious Adverse Event (SAE): any untoward and unexpected medical occurrence or effect that: 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused 
death if it were more severe 
• 
Requires hospitalisation, or prolongation of existing inpatients’ hospitalisation 
Spectra 
 
Pathways 
 

15 
 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other situations. 
Important AEs that are not immediately life-threatening or do not result in death or hospitalisation but 
may jeopardise the subject or may require intervention to prevent one of the other outcomes listed in 
the definition above, should also be considered serious. 
 
6.2 
REPORTING PROCEDURES 
All adverse events should be reported. Depending on the nature of the event the reporting procedures 
below should be followed. Any questions concerning adverse event reporting should be directed to the 
Chief Investigator in the first instance. 
 
6.2.1 Non serious AEs 
All such events, whether expected or not, should be recorded. 
 
6.2.2 Serious AEs 
An SAE form should be completed and faxed to the Chief Investigator within 24 hours. However, 
hospitalisations for elective treatment of a pre-existing condition do not need reporting as 
SAEs. 
 
All SAEs should be reported to the ………………..REC where in the opinion of the Chief Investigator, 
the event was: 
• 
‘related’, ie resulted from the administration of any of the research procedures; and 
• 
‘unexpected’, ie an event that is not listed in the protocol as an expected occurrence 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief Investigator 
becoming aware of the event, using the NRES SAE form for non-IMP studies. The Chief Investigator 
must also notify the Sponsor of all SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics Committee, 
Sponsor and/or Research & Development Office. 
 
 
 
 
 
 
 
Contact details for reporting SAEs : 
Please send SAE forms to: 
Professor Alun H. Davies 
Professor of Vascular Surgery 
Academic Section of Vascular Surgery 
Department of Surgery & Cancer 
Imperial College London 
Room 4E04, 4 East 
Charing Cross Hospital 
Fulham Palace Road 
London, W6 8RF 
Tel: 020 3311 7320 (Mon to Fri 09.00 – 17.00) 

16 
 
7. 
REGULATORY ISSUES 
 
7.1 
ETHICAL APPROVAL 
 
 
 
 
 
The Chief Investigator has obtained approval from the London Surrey NHS Research Ethics 
Committee). The study must also receive confirmation of capacity and capability from each 
participating NHS Trust before accepting participants into the study or any research activity is 
carried out. The study will be conducted in accordance with the recommendations for physicians 
involved in research on human subjects adopted by the 18th World Medical Assembly, Helsinki 
1964 and later revisions. 
 
 
 
7.2 
CONSENT 
Consent to enter the study must be sought from each participant only after a full explanation 
has been given, an information leaflet offered and time allowed for consideration. Signed 
participant consent should be obtained. The right of the participant to refuse to participate 
without giving reasons must be respected. After the participant has entered the study the 
clinician remains free to give alternative treatment to that specified in the protocol at any stage 
if he/she feels it is in the participant’s best interest, but the reasons for doing so should be 
recorded. In these cases, the participants remain within the study for the purposes of follow- 
up and data analysis. All participants are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudicing further treatment. 
 
7.3 
CONFIDENTIALITY 
The Chief Investigator will preserve the confidentiality of participants taking part in the study 
and is registered under the Data Protection Act. 
 
7.4 
INDEMNITY 
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study. 
 
7.5 
SPONSOR 
Imperial  College  London  will  act  as  the  main  Sponsor  for  this  study.  Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study. 
 
7.6 
FUNDING 
The funding will be internal. There will be no participants or investigator payments.  The Graham- 
Dixon Charitable Trust has awarded £14 491 towards the consumables cost of this research. Further 
grant applications will be submitted. 
 
7.7 
AUDITS 
The study may be subject to inspection and audit by Imperial College London under their remit 
as sponsor and other regulatory bodies to ensure adherence to GCP and the NHS Research 
Governance Framework for Health and Social Care (2nd edition).

17 
 
 
8. 
STUDY MANAGEMENT 
 
The day-to-day management of the study will be co-ordinated through the Clinical Research 
Fellow Mr Kemal Kemal, under the supervision of the Chief Investigator Professor Alun H. 
Davies. 
 
9. 
PUBLICATION POLICY 
 
The aim of this study is to generate results for dissemination at international meetings and peer 
reviewed articles in scientific journals. Authorship shall be determined by contribution to the 
various aspects of the study. All authors shall have the ability to review and comment on the 
manuscript of any work prior to submission. The study shall also form the basis of the higher 
degree submission of Mr Kemal Kemal at Imperial College London. 
 
 
10. 
REFERENCES 
 
1. Silverstein, M.D., et al., Trends in the incidence of deep vein thrombosis and pulmonary embolism a 25-year 
population-based study. Arch Intern Med, 1998. 158(6): p. 585-93. 
 
2. Cohen AT, Agnelli G, Anderson FA, et al. Venous thromboembolism (VTE) in Europe. The number of VTE 
events and associated morbidity and mortality. Thromb Haemost. 2007;98(4):756–764. 
 
 
3.'Angelo A, D'Alessandro G, Tomassini L, Pittet JL, Dupuy G, Crippa L. Evaluation of a new rapid 
quantitative D-dimer assay in patients with clinically suspected deep vein thrombosis. Thromb Haemost. 1996 
Mar;75(3) 412-416. PMID: 8701399. 
 
4.Venous thromboembolic diseases: diagnosis, management and thrombophilia testing | Guidance and 
guidelines | NICE Nov 2015 
 
5. Anwar, Muzaffar A. et al. “A Comprehensive Characterisation of the Metabolic Profile of Varicose Veins; 
Implications in Elaborating Plausible Cellular Pathways for Disease Pathogenesis.” Scientific Reports 7 (2017): 
2989. PMC. Web. 15 Aug. 2018. 
 
 
6. Shalhoub J, Sikkel MB, Davies KJ, Vorkas PA, Want EJ, Davies AH. Systems biology of human 
atherosclerosis. Vasc Endovascular Surg. 2014;48: 5–17. pmid:24212404 

18 
 
7. Metabolic Profiling Reveals Changes in Biofluids of Patients With Chronic Venous Ulceration 
Bergner, Richmond et al. Journal of Vascular Surgery: Venous and Lymphatic Disorders , Volume 6 , 
Issue 2 , 297 
 
 
8. Qureshi, Mahim & Vorkas, Panagiotis & Coupland, Alex & Harri Jenkins, I & Holmes, Elaine & Davies, 
Alun.(2016). Lessons from Metabonomics on the Neurobiology of Stroke. The Neuroscientist. 23. 
10.1177/1073858416673327. 
 
9. Deep Vein Thrombosis Exhibits Characteristic Serum and Vein Wall Metabolic Phenotypes in the 
Inferior Vena Cava Ligation Mouse Model Sung, Yeji et al. European Journal of Vascular and Endovascular 
Surgery , Volume 55 , Issue 5 , 703 - 713 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix 1. Summary of investigations and assessments 
 
Exam 
1st visit 
2rd visit (if applicable) 
Informed consent 
X 
History, physical exam 
X 
X 
Blood and urine sample 
X 
X 
Venous duplex ultrasound 
X 
X 
 
Group1: 
1st visit: medical history, examination blood and urine sample 
Patients will also undergo a venous duplex ultrasound examination either on the same day or 
the day after. 
All patients of group 1 will be asked to return 3 weeks after the end of their treatment During 
this 2nd visit the medical history will be revised, leg inspection and leg ultrasound will be 
performed and a blood and urine sample will be repeated. 
 
Group 2 (volunteers): 
Only 1st visit.