Clinical Study to Identify Biological Markers to Predict Wound Healing in Patients With Chronic Venous Ulceration of the Lower Limb
Conditions: Varicose Ulcer
Sponsor: Imperial College London

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“MOJITO” 
Clinical Study to Identify Biological Markers to Predict Wound 
Healing in Patients with Chronic Venous Ulceration of the 
Lower Limb 
Date and Version Number: 07/06/2013 Version 5 
Main Sponsor: Imperial College London 
Funder: Laborotoires Urgo 
NRES reference: <13/EE/0137> 
Department Reference number: Mojito-Protocol1 
Amendment Number: N/A 
 
Date 
Signature 
Protocol Authorised by: 
 
 
 
Name and Role: 
 
 
 
 
 
 
 

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Study Management Group 
Chief Investigator: Professor Alun H. Davies 
Co-Investigators: Mr Rahul Velineni, Dr Konstantina Spagou, Mr Muzaffar A Anwar, Mr Manjit S 
Gohel, Mr George Geroulakos, and Professor Elaine Holmes 
Statistical Advice:  
Professor Alun H Davies 
Study Management:  
Professor Alun H Davies 
Professor of Vascular Surgery 
Academic Section of Vascular Surgery 
Imperial College London 
4E04 East Wing 
Charing Cross Hospital 
Fulham Palace Road 
London 
W6 8RF 
 
Telephone: 020 3311 7320 
 
a.h.davies@imperial.ac.uk  
Mr Rahul Velineni – Clinical Research Fellow 
Clinical Research Fellow in Vascular Surgery 
Imperial College London 
4N13C North Wing 
Charing Cross Hospital 
Fulham Palace Road 
London 
W6 8RF 
Telephone: 07525 480568 
r.velineni@imperial.ac.uk  
Clinical Queries 
Clinical queries should be directed to Professor Davies or Mr Velineni 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 and Imperial College Healthcare NHS Trust 
Room 5L10C, 5th Floor Lab Block 
Charing Cross Hospital 
Fulham Palace Road 
London W6 8RF 
 
Telephone: 
0203 311 0204 
Fax: 
 
0203 311 0203 

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Funder 
Laborotoires Urgo have provided an unrestricted academic grant to Imperial College London. 
Laborotoires Urgo 
42 Rue de Longvic,  
21300 Chenôve,  
France 
This Protocol will describe the MOJITO 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 outlines in the NHS Research Governance Framwork for 
Health and Social Care (2nd edition). It will be conducted in compliance with the protocl, the Data 
Protection Act and other regulatory requirements as appropriate. 
 
 

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Table	  of	  Contents	  
STUDY	  MANAGEMENT	  GROUP	  
2	  
CLINICAL	  QUERIES	  
2	  
SPONSOR	  
2	  
FUNDER	  
3	  
1.	  BACKGROUND	  AND	  OBJECTIVES	  
6	  
1.1	  OBJECTIVES	  
6	  
2.	  ELIGIBILITY	  AND	  INCLUSION	  
6	  
3.	  EXCLUSION	  CRITERIA	  
6	  
4.	  STUDY	  DESIGN	  AND	  METHODOLOGY	  
7	  
4.1	  PROCEDURES	  AND	  ASSESSMENTS	  
7	  
4.1.1	  INITIAL	  ASSESSMENT	  
7	  
4.1.2	  SUBSEQUENT	  ASSESSMENTS	  
7	  
4.1.3	  WEEK	  TWENTY	  –	  SPECIAL	  ARRANGEMENTS	  
7	  
4.1.4	  END	  OF	  THE	  STUDY	  
8	  
4.2	  STANDARD	  OPERATING	  PROCEDURE	  FOR	  SAMPLE	  COLLECTION	  AND	  PREPARATION	  
8	  
4.2.1	  DIGITAL	  AND	  MANUAL	  PLANIMETRY	  
8	  
4.2.1.1	  URINE	  
8	  
4.2.1.2	  BLOOD	  
8	  
4.2.1.3	  LEG	  ULCER	  FLUID	  
8	  
4.3	  PROPOSED	  ANALYSES	  
9	  
4.3.1	  METABONOMICS	  
9	  
4.3.2	  FUNCTIONAL	  ASSAYS	  OF	  ANGIOGENESIS	  
9	  
4.3.3	  OTHER	  FIRST	  LINE	  ASSAYS	  
9	  
4.3.4	  SECOND	  LINE	  ASSAYS	  
9	  
4.4	  DATA	  HANDLING	  AND	  RECORD	  KEEPING	  
10	  
4.4.1	  CLINICAL	  DATA	  
10	  
4.4.2	  SAMPLE	  LABELLING	  AND	  STORAGE	  
10	  
4.5	  STATISTICS	  
10	  
4.6	  SUMMARY	  OF	  STUDY	  PATIENT	  PROCEDURES	  
10	  
5.	  ADVERSE	  EVENTS	  
11	  
5.1	  DEFINITIONS	  
11	  
5.2	  REPORTING	  PROCEDURES	  
12	  
5.2.1	  NON	  SERIOUS	  ADVERSE	  EVENTS	  
12	  

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5.2.2	  SERIOUS	  ADVERSE	  EVENTS	  
12	  
6.	  REGULATORY	  ISSUES	  
12	  
6.1	  ETHICAL	  APPROVAL	  
12	  
6.2	  CONSENT	  
12	  
6.3	  CONFIDENTIALITY	  
12	  
6.4	  INDEMNITY	  
12	  
6.5	  SPONSOR	  
12	  
6.6	  FUNDING	  
13	  
6.7	  AUDITS	  AND	  INSPECTIONS	  
13	  
7.	  STUDY	  MANAGEMENT	  
13	  
8.	  PUBLICATION	  POLICY	  
13	  
9.	  REFERENCES	  
13	  
 
 
 
 
 

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1. Background and Objectives 
Chronic venous ulceration of the lower limb represents a significant problem to patients and providers 
of healthcare worldwide. It is estimated that 0.3% of the population suffer from an open venous ulcer 
and that 1% of the population have either an open ulcer or chronic venous ulcer (1). In the UK, the 
cost of treating an unhealed ulcer is approximately £1300 a year (2).  
The established treatment of chronic venous ulceration in the United Kingdom is to provide a course 
of compression bandaging <reference>. However, despite the use of this treatment recurrence at one 
year has been shown by one study to be 17% (3). In addition, some ulcers will not heal with 
compression (3). 
When dealing with chronic venous ulceration it may be useful to visualise the process of a failure of 
normal healing to progress through its well defined pathway. Multiple groups have attempted to 
unpick the mechanism of failed wound healing. However, our knowledge of this area is incomplete 
and requires further clarification. 
1.1 Objectives 
We seek to further examine the biology of chronic venous ulceration. Our rationale for this study is: 
1. Can we effectively predict from biological markers in whom standard therapy will not culminate in 
wound healing? 
2. Can potential therapeutic targets be identified? 
3. What changes occur in the biological profile of a chronic venous ulcer over various points in time? 
Our belief is that the potential benefits of this study to wider healthcare community may be in 
effectively stratifying patients with chronic venous ulceration by risk of treatment failure. This may 
allow resources to be concentrated on high risk patients. 
In addition, identifying therapeutic targets may in the future reduce the proportion of patients in whom 
standard treatment fails or ulcers recur.  
Study of the same patients over time will help us understand the process of healing in chronic venous 
ulceration and determine the differences in patients who ultimately heal versus those who do not. 
2. Eligibility and Inclusion 
• 
Forty patients to be recruited from Vascular Surgery Clinics at Imperial College NHS Trust, 
and Cambridge University Hospitals NHS Foundation Trust. 
• 
Male or female over the age of 18 years 
• 
Chronic venous ulceration – Defined as wound of greater than four weeks in duration 
between the foot and the ankle with an Ankle Brachial Pressure Index greater than 0.85. 
• 
Ulceration present for at least four weeks. 
• 
Colour venous duplex evidence of chronic venous insufficiency showing either reflux or 
obstruction. 
3. Exclusion Criteria 
• 
Acute infection in the studied lower limb within the last four weeks 
• 
History of malignancy in the lower limb to be studied  
• 
History of connective tissue disease 

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• 
Patients on medications that can cause immunosuppression – Corticosteroids, chemotherapy 
or radiotherapy for cancer and recombinant immunological medications. 
4. Study Design and Methodology 
Forty patients who fulfil the criteria for the study will be recruited from Vascular Surgery clinics in 
hospitals under the auspices of Imperial NHS Trust in London and Cambridge University Hospitals 
NHS Foundation Trust in Cambridge. 
In summary, this study is a prospective cohort study of patients with venous leg ulceration with 
samples of blood, urine and venous leg ulcer fluid collected over multiple time points over an initial 
twenty-week period with standard treatment for leg ulceration. In patients not healed fully at twenty 
weeks, they will be offered an Urgostart® dressing and further assessments carried out to the end of 
follow up at twenty-eight weeks. 
The study will take place between August 2013 and February 2015.  
4.1 Procedures and Assessments 
4.1.1 Initial Assessment 
• 
A full history and examination will be completed.  
• 
Venous severity scoring (4). 
• 
CEAP (Clinical-etiologic-anatomy-pathophysiology) scoring (5). 
• 
Colour venous duplex to establish integrity of the venous system. 
• 
Ankle brachial pressure index. 
• 
Routine blood tests: Full blood count, urea, electrolytes, glucose, glycosylated haemoglobin (if 
patient suffers from diabetes).  
• 
Assessment of wound healing: This will be performed using digital planimetry by which 
electronic representations of the ulcer are formed and the area calculated.  
• 
Wound ulcer fluid – Two techniques performed simultaneously. 
1. “Occlusive dressing technique” (6). This technique involves the placement of a clear 
adherent dressing over the ulcer. After ninety minutes in a seated position. The 
dressing is pierced with a 21 gauge needle and any accumulated fluid is sampled. 
2. “Filter disc technique” (7). This involves placement of filter discs on top of the ulcer 
bed separated by a permeable transparent dressing to filter out solid material. 
• 
Urine sampling: A mid-stream urinary sample shall be requested from the patient in a 
standard specimen pot without additives. 
• 
Special blood tests: A sample shall be taken from the antecubital fossa and placed in a 
standard bottle without additives. 
• 
The patient’s ulcer will be managed using compression bandaging. 
4.1.2 Subsequent Assessments 
• 
Reassessments of the patient will take place at weeks 1, 2, 4, 8, 12 and 20. 
• 
These will involve reviewing any significant changes in health. 
• 
The ulcer area will be measured using digital planimetry to chart progress of healing. 
• 
Compression bandaging will be continued as per the requirements of the patient. 
• 
Wound fluid, urine and special blood tests will be repeated as described above. 
4.1.3 Week Twenty – Special Arrangements 
If the ulcer has healed by week 20 then the patient will have completed the study. If the ulcer has not 
healed, the patient’s standard of therapy (compression bandaging) will be complemented by the use 
of an Urgostart® dressing. This product will be used within its licence. 

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These patients will be followed up to a total of 28 weeks. Assessments as described above will be 
repeated at weeks 21, 22, 23, 25 and 28. These assessments will be undertaken to detect changes in 
the microenvironment of the wound in response to a new treatment. 
4.1.4 End of the Study 
The study is complete once the last patient visit has taken place. 
4.2 Standard Operating Procedure for Sample Collection and Preparation 
4.2.1 Digital and Manual Planimetry 
4.2.1.2 Digital Planimetry 
1. Digital camera to take photograph of wound with calibration scale adjacent to wound 
2. Computer based calculation of wound size  
4.2.1.3 Manual Planimetry 
1. Transparent, flexible planimetry grid placed onto wound. Edge of wound traced with 
permanent marker pen.  
2. Calculate area of ulcer and log into clinical data sheet. 
4.2.1.1 Urine 
1. Mid stream urine sample  
2. Transfer to 50ml Falcon tube 
3. Aliquot 10 x 1ml samples into 2ml Eppendorf 
4. Transported to South Kensington Campus on dry ice 
5. Freeze at -80 degrees Celsius for storage 
4.2.1.2 Blood 
1. Sampled from antecubital fossa avoiding the use of alcohol based skin preparation. Collection 
into plain blood bottles.21g needle and syringe technique 
2. Transfer into 15ml Conical Dome Polypropylene Falcon Tube  
3. Stand for 30 minutes to clot on dry ice 
4. Centifugation at 2,500g  for 10 minutes to obtain serum from supernatant fluid  
5. Transfer serum into 10ml Falcon tube 
6. Aliquot 0.4ml into 1.5 Eppendorf x 2 (minimum, ideally 3) 
7. Freeze at -80 degrees Celsius for storage 
4.2.1.3 Leg Ulcer Fluid 
a. Occlusive Dressing Technique (6) 
1. Ulcerated area covered in Tegaderm dressing 
2. 90 minutes with the leg dependent in a seated position 
3. Fluid aspirated with hypodermic 21g needle and syringe 
4. Fluid transferred into 10ml 15ml Conical Dome Top Polypropylene Falcon tube and kept on 
dry ice 
5. Collected fluid kept on ice and centrifuged within 30 minutes at 13,000g 
6. Supernatant filtered using methylcellulose filter (8) 
7. Transfer 0.4ml into 1.5ml Eppendorfs  
8. Transport to South Kensington Campus on dry ice 
9. Flash frozen and stored at -80 degrees Celsius 
b. Filter Disc Technique (7) 
1. Preprepared 1cm2 absorptive filters from Whatman 54 paper, sterilised in ethanol and oven 
dried at 60 degrees Celsius. Preweighed in sterile 2ml tubes. 

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2. Sterile Tegapore mesh placed cut into 4cm2 segments and placed onto ulcer. Ten discs 
placed on top of mesh. 
3. After 90  minutes, disc removed from mesh and placed back into tube.  
4. Transferred to South Kensington Campus on dry ice 
5. Putative Imperial method – Analyse filter discs directly after separation into aqeous and 
organic extracts 
6. Aqueous extract – mass spectrometry and NMR 
7. Organic extract – Mass spec ± examination for eicosanoids / inflammatory molecules 
Tarlton method  
1. Filter discs reweighed to determine quantity of fluid absorbed and placed into extraction buffer. 
2. Four hours of agitated extraction and fluid removed for freezing and stored at -80 degrees. 
4.3 Proposed Analyses 
These assays would be performed on ulcer fluid, urine and blood samples collected at the initial and 
each subsequent assessment. 
4.3.1 Metabonomics 
This technique has been pioneered at Imperial College London and is defined as a technique to 
broadly measure the “global metabolic metabolic response of living biological systems to biological 
stimuli or genetic manipulation.”(9) 
We would seek to obtain a comprehensive metabolic profile of a chronic venous ulcer throughout its 
cycle of healing or failure to heal. This would be performed using established techniques of nuclear 
magnetic resonance spectroscopy and mass spectrometry. 
Biomarkers of potential significance will be identified from the comprehensive list of metabolites using 
chemometric and bioinformatics software.   
4.3.2 Functional Assays of Angiogenesis 
Commercial angiogenesis assays will also be performed to evaluate the angiogenesis stimulating 
ability of paired wound exudate specimens. 
4.3.3 Other First Line Assays 
• 
PAI-1, lactotransferrine, S100A9, Annexine 1, AGEs (advanced glycation end-products)  
• 
Markers of oxidative stress-alantoinuric acid ratio, protein carbonyl content  
• 
Plasminogen activators - uPA, tPA 
4.3.4 Second Line Assays 
These tests will be performed if the sample volume is adequate. 
• 
Proteolysis – MMP2, MMP9, TIMP1, TIMP2 
• 
Angiogenesis – VEGF, bFGF 
• 
Inflammation – IL1 (α And β), IL6, TNFα 
• 
Fibrosis – TGFβ1 
• 
SERPINB4, 
• 
SERPIN D1, 
• 
Lipocaline 2, 

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• 
IFN gamma 
 
Comprehensive metabolic profile will be attained in ulcer fluid at the initial assessment. Changes in 
this profile over the subsequent assessments will highlight the metabolic response of ulcer healing. 
Using the chemometric software and multivariate statistical methods, potential biomarkers of ulcer 
healing will be identified. The changes in metabolic picture will also be correlated with the changes 
other mediators (PAI-1, lactotransferrine, S100A9, Annexine 1, AGEs, markers of oxidative stress, 
MMPs, cytokines, growth factors) to elucidate the pathogenesis of chronic venous ulcer formation. 
Moreover, the effect of Urgostart® dressing on the potential biomarkers of non-healing ulcer will help 
us to predict the treatment response. 
4.4 Data Handling and Record Keeping 
4.4.1 Clinical Data 
The initial assessment comprising history, examination, routine blood investigations, colour venous 
duplex result, venous clinical severity score, CEAP classification and assessment of wound status 
shall be recorded onto a standard document <clinical data document v2>. Subsequent assessments 
shall be recorded onto <follow up clinical data document v1>.  
Clinical data shall be placed into a computerised database and each patient will be given a unique 
study identifying number. Clinical data documentation will be placed into the study master file. 
Unique identifying numbers will be held at Imperial College and at the relevant clinical sites.  
The study master file will be kept at the secure vascular clinical research office at Imperial College’s 
Charing Cross Campus. 
4.4.2 Sample Labelling and Storage 
Samples will be marked with the patient’s unique identifying number and a suffix to the sample will be 
added as per the specimen type. 
Blood serum 
B 
Urine 
U 
Wound fluid – Occlusive dressing 
fX 
Wound fluid – Filter disc 
fN 
Filter disc for analysis 
fW 
 
Prior to analysis, all samples will be stored in a secure freezer at the Section of Computational and 
Systems Medicine, South Kensington Campus Imperial College London at -80 degrees Centrigrade. 
4.5 Statistics 
Statistical analysis will be performed to analyse the patient recruitments in terms of baseline clinical 
characteristics. Survival analysis will be used to demonstrate ulcer healing. Biological assays will be 
analysed using univariate statistical analysis. Metabonomic assay data will be mathematically 
modelled and analysed using multivariate statistical models. 
4.6 Summary of Study Patient Procedures 
First Meeting 
Introductions, discussion and consent 
 

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Initial Assessment – Week 0 
Preliminary evaluation – Interview about 
medical problems, physical examination. 
Blood, urine and ulcer fluid sampling. Wound 
dressing.  
 
Week 1,2, 4, 8 and 12 
Clinical review 
Blood, urine and wound ulcer fluid sampling 
Week 20 
Clinical review 
Blood, urine and wound ulcer fluid sampling 
If wound has not healed, apply Urgostart® 
dressing to wound dressing.  
If wound has healed – end of study. 
Week 21, 22, 23 and 25 
Clinical review 
Blood, urine and wound ulcer fluid sampling 
Continue dressing with Urgostart® 
Week 28 
Clinical review 
Blood, urine and wound ulcer fluid sampling 
Completion of Urgostart® dressing 
End of study 
 
5. Adverse Events 
5.1 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  
- 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.  
 

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5.2 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.  
 
5.2.1 Non Serious Adverse Events 
All such events, whether expected or not, should be recorded.  
 
5.2.2 Serious Adverse Events 
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 <name of 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.  
6. Regulatory Issues 
6.1 Ethical Approval 
The Chief Investigator has obtained approval from the xxx Research Ethics Committee. The study 
must be submitted for Site Specific Assessment (SSA) at each participating NHS Trust. The Chief 
Investigator will require a copy of the Trust R&D approval letter before accepting participants into the 
study. 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.  
6.2 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.  
6.3 Confidentiality 
The Chief Investigator will preserve the confidentiality of participants taking part in the study and is 
registered under the Data Protection Act.  
6.4 Indemnity 
Imperial College London holds negligent harm and non-negligent harm insurance policies which apply 
to this study.  
6.5 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.  

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6.6 Funding 
Laborotoires Urgo are funding this study with an unrestricted academic grant. No specific payments 
are being made to participants or investigators. 
6.7 Audits and Inspections 
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).  
7. Study Management 
Day to day management of the study will be co-ordinated through Mr Rahul Velineni, Clinical 
Research Fellow in Vascular Surgery under the supervision of the Chief Investigator Professor Alun 
H. Davies. 
8. Publication Policy 
The aim of this study is to generate results for dissemination to 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 Velineni at Imperial College London. 
9. References 
1. 
Fowkes FG, Evans CJ, Lee AJ. Prevalence and risk factors of chronic venous insufficiency. 
Angiology. 2001 Aug;52 Suppl 1:S5-15. PubMed PMID: 11510598. Epub 2001/08/21. eng. 
2. 
Iglesias CP, Nelson EA, Cullum N, Torgerson DJ, Ven USIc. Economic analysis of VenUS I, a 
randomized trial of two bandages for treating venous leg ulcers. The British journal of surgery. 2004 
Oct;91(10):1300-6. PubMed PMID: 15382101. 
3. 
Gohel MS, Taylor M, Earnshaw JJ, Heather BP, Poskitt KR, Whyman MR. Risk factors for 
delayed healing and recurrence of chronic venous leg ulcers--an analysis of 1324 legs. European 
journal of vascular and endovascular surgery : the official journal of the European Society for Vascular 
Surgery. 2005 Jan;29(1):74-7. PubMed PMID: 15570275. Epub 2004/12/01. eng. 
4. 
Rutherford RB, Padberg FT, Jr., Comerota AJ, Kistner RL, Meissner MH, Moneta GL. Venous 
severity scoring: An adjunct to venous outcome assessment. J Vasc Surg. 2000 Jun;31(6):1307-12. 
PubMed PMID: 10842165. Epub 2000/06/08. eng. 
5. 
Eklöf B, Rutherford RB, Bergan JJ, Carpentier PH, Gloviczki P, Kistner RL, et al. Revision of 
the CEAP classification for chronic venous disorders: Consensus statement. Journal of Vascular 
Surgery. 2004 12//;40(6):1248-52. 
6. 
Gohel MS, Windhaber RAJ, Tarlton JF, Whyman MR, Poskitt KR. The relationship between 
cytokine concentrations and wound healing in chronic venous ulceration. Journal of Vascular Surgery. 
2008 11//;48(5):1272-7. 
7. 
Tarlton JF, Bailey AJ, Crawford E, Jones D, Moore K, Harding KD. Prognostic value of 
markers of collagen remodeling in venous ulcers. Wound repair and regeneration : official publication 
of the Wound Healing Society [and] the European Tissue Repair Society. 1999 Sep-Oct;7(5):347-55. 
PubMed PMID: 10564563. Epub 1999/11/17. eng. 
8. 
De Mattei M, Ongaro A, Magaldi S, Gemmati D, Legnaro A, Palazzo A, et al. Time- and dose-
dependent effects of chronic wound fluid on human adult dermal fibroblasts. Dermatologic surgery : 
official publication for American Society for Dermatologic Surgery [et al]. 2008 Mar;34(3):347-56. 
PubMed PMID: 18177397. Epub 2008/01/08. eng. 
9. 
Nicholson 
JK, 
Lindon 
JC. 
Systems 
biology: 
Metabonomics. 
Nature. 
2008 
10/23/print;455(7216):1054-6. 
 

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