Looking for Biomarkers in Human Joint Fluid Using Metabonomic, Microbiomic and Tribological Analysis
Conditions: Osteoarthritis, Septic Arthritis, Rheumatoid Arthritis
Sponsor: Imperial College London

Version 1.3 
1 
08/01/2015 
 
 
PROPOSED PROTOCOL 
LOOKING FOR BIOMARKERS IN HUMAN JOINT FLUID 
SPONSOR: IMPERIAL COLLEGE LONDON 
STUDY CENTRE: IMPERIAL COLLEGE NHS TRUST 
 
Principle Investigator: 
Mr Chinmay Gupte 
St Mary’s Hospital 
Praed Street 
Paddington 
London 
 
Telephone +44203 311 1234 
 
Chief investigator: 
 
Matt Jaggard (study co-ordinator),  
St Mary’s Hospital 
Praed Street 
Paddington 
London 
 
 
Telephone +447904 066695 

Version 1.3 
2 
08/01/2015 
Aims:   
1. To utilise metabonomic analysis to analyse human joint fluid in tandem with other body fluids 
2. To analyse joint fluid from osteoarthritic joints to identify potential biomarkers of the disease and 
further understanding of the cartliage destruction pathways 
3. To attempt to further understand population variances in joint fluid composition and if this 
composition correlates to arthropathy of joints 
4. To study the lubrication and wear properties of the joint fluid in tandem with metabonomic 
analysis to better understand these properties; with a particular view to the impact of wear upon 
prosthetic joints 
5. To study joint fluid for biomarkers of bacterial colonisation and analyse in tandem with 
microbiological analysis in an attempt to correlate results 
 
Background: 
Joint fluid represents a source of possible disease detection largely uninvestigated by analytical 
chemistry to date.  We aim to chemically investigate the composition of joint fluid.  Joint fluid has 
many key functions.   
1. It is involved in lubrication of cartliage.  In osteoarthritis the cartliage is lost leading to bone on 
bone wear.  The mechanisms of the loss of cartliage and how the composition of joint fluid affects 
this are poorly understood. 
2. In the case of joint replacement corrosion and wear of the components is an important concern.  
It has a direct effect upon the lifetime of the any prosthesis.  It also has a bearing upon destructive 
process seen in some joints as a result of biological handling of corrosion and particulate wear. 
3. The presence of infection in any joint, replaced or not is a destructive and aggressive disease 
process.  It is difficult to treat due to the joint fluid being a happy medium for microbial growth with 
poor antibiotic penetration. 
 
To analyse the joint fluid composition, a metabonomic approach will be used.  Metabonomics is 
relatively in its infancy.  It is the study of profiling biological biproducts combined with powerful 
computer analysis.  Previous strategies for identifying metabolites have fallen foul of an inability to 
detect at low levels in complex samples with too many compounds to distinguish satisfactorily.  This 
necessitated complex and lengthy chemical purification techniques.  Utilising modern NMR analysis 
overcomes these challenges.  Body fluids can be analysed directly.    Once targetted compounds 
have been found, further study using alternative analysis techniques like mass spectroscopy can be 
used to help identification. 
 

Version 1.3 
3 
08/01/2015 
Microbiological assays will attempt to amplify any organisms present in the joint fluid by growth.  
These organisms will then by subjected to a series of selected stresses to identify them.  
Furthermore, polymerase chain reaction assays may be used to identify the genetic component of 
any organisms found within the joint fluid. 
 
Tribology testing will be carried out to determine lubrication and wear properties of synovial fluid 
samples.  A laboratory test device will be used to rub implant materials under simulated artificial 
joint conditions.  Only a small amount of fluid is required for the tests.  At the end of the test wear of 
the implant material surface will be measured.  The synovial fluid test sample will be recovered and 
wear products (particles, metal ions in solution) analysed. 
 
Once the human joint fluid has been characterised for composition, lubrication and microbiological 
content, the results will be correlated statistically in an attempt to identify which compounds in the 
fluid are key to these properties. 
 
Any results will be published in a peer review journal and components of the research may be used 
for the submission of a research thesis.  Joint fluid represents a source of possible disease detection 
largely uninvestigated by biochemistry to date.  As such there may be many potential biomarkers to 
joint disease. 
 
Collaborations: 
The study will be coordinated by the Department of Musculoskeletal Research, Imperial College 
London.  The research centre will be the Imperial College NHS Trust.  Analysis will be conducted 
within the department of microbiology at Imperial College NHS Trust and with the collaboration of 
the Department of Surgery and Cancer, Imperial College London.  
 
Duration: 
We aim to achieve ethical approval and begin collecting samples from patients at the beginning of 
2015.  We would hope to complete joint fluid analysis before the close of 2017.  Identification of 
relevant target markers would be completed before 2018.  Any proposal of clinical application and 
use would require extensive validation, testing and replication.  As a result we would not expect 
clinical application within 10 years of detection. 
 
Method: 

Version 1.3 
4 
08/01/2015 
Blood, urine and joint fluid will be collected from suitable and consenting patients.  The collection 
will be focused from the accident and emergency, rheumatology and orthopaedic departments.  In 
addition control subjects may be recruited to allow potential background matrix subtraction. 
Samples will be analysed using NMR and computer modelling analysis to identify key metabolites 
Samples will be stored at 4oC for no more than 48 hours before analysis.  Samples will then be stored 
at -80OC, should any further analysis be required. 
All details of subject background will be collected.  These include diet, medical history, drug history, 
exercise history and joint symptoms 
 
Power Calculation 
The previous evidence has demonstrated that power calculations in metabonomic methods of 
analysis has proved difficult.  This is due to the large and varied amount of data that is generated1  
This is due to the methodology and subsequent reliance upon powerful, computer generated 
statistical calculations to provide significance.  However, from previous evidence and experience we 
would expect the numbers to be relatively low and thus are hoping to sample 40 patients for each 
arm of the study. 
 
Subjects: 
Subjects will be recruited in the clinical setting to donate joint fluid, blood and urine.  These patients 
will be recruited in several scenarios. 
1. Anyone receiving a diagnostic aspirate of joint.   
2. Patients undergoing joint arthroscopy 
3. Patients undergoing joint replacement 
4. Patients undergoing prosthetic joint revision surgery 
No patient under 16 years or age or older than 80 years will be entered for the study.  Any female 
patient who is pregnant will be excluded.  Any patient medicated with anti-coagulants will be 
excluded. 
 
Analysis: 
Body fluids will be analysed using unextracted direct NMR analysis.  Statistical multi-variate analysis 
methods will then be employed to determine co-variates and significant metabolites. 
Parallel analysis of the lubrication properties will be performed using a thin film optical 
interferometric test device.   

Version 1.3 
5 
08/01/2015 
Joint fluid will be analysed using standard microbiological assays and polymerase chain reaction for 
any organisms present and their genetic make-up.  These results will be correlated using the multi-
variate analysis to identify how the composition of the joint fluid corresponds.   
 
Complications:   
No adverse events are expected arising from the sampling of body fluids.  A low risk of both intra-
articular infection and haemoarthrosis exists.  It is possible that subjects may experience minor 
bruising from blood sampling.   Should any complications occur they will be immediately reported to 
the study coordinator who will then inform the study centre and sponsor.  Any adverse events will 
be dealt with at Imperial College or the treating hospital.   
 
Reporting: 
The authors will attempt to publish those results in a peer review journal of relevance.   
 
Regulatory Issues: 
  
Ethics Approval 
The investigators are seeking approval from the North West London REC2. 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. 
Consent 
Samples will be taken from patients fulfilling the inclusion criteria on arrival.  Consent to enter the 
study must be sought from each participant only after a full explanation has been given and time 
allowed for consideration.  In all situations samples will be collected and not analysed until consent 
is gained.  The right of the participant to refuse to participate without giving reason is to be 
respected.  Irrespective of whether consent is gained, the treatment of the patient shall not be 
affected either in process or decision making.   Patients will be followed up for a period of six 
months after discharge from hospital. All participants are free to withdraw at any time from the 
study without prejudicing further treatment. 
Confidentiality 
The Chief Investigator will preserve the confidentiality of participants taking part in the study and is 
registered under the Data Protection Act. 
Indemnity  

Version 1.3 
6 
08/01/2015 
Imperial College London holds negligent harm and non-negligent harm insurance policies which 
apply to this study.  Imperial College London Healthcare NHS Trust's research is covered by the 
standard NHS Hospital Indemnity for negligent or wrongful harm and this is provided under clinical 
negligence scheme for Trusts clinical risk management NHS Litigation Authority (NHSLA) for NHS 
Trusts in England. 
Sponsor 
Imperial College Healthcare NHS Trust will act as the main Sponsor for this study.  Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study.  
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).