Molecular Pathways Involved in Knee Pain: an Observational Study
Conditions: Knee Osteoarthritis, Knee Pain Chronic
Sponsor: University of Nottingham

 
 
 
Page 1 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
 
 
 
 
 
 
 
Molecular pathways involved in knee pain:  
an observational study 
 
Final Version 4.2 June 2022 
 
 
 
Short title:  
 
Molecular pathways involved in knee pain  
 
 
Ethics Reference: 
20/EM/0065 
 
 
Trial Sponsor: 
 
University of Nottingham 
 
 
Sponsor reference: 
19098 
 
 
Funding Source: 
Pain Centre Versus Arthritis and the Kennedy Trust via 
their Step-Up OA Consortium Fund 
 

 
 
 
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Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
STUDY PERSONNEL AND CONTACT DETAILS 
 
        Sponsor: 
University of Nottingham 
 
Ms Angela Shone 
Research and Graduate Services  
King's Meadow Campus  
Lenton Lane 
Nottingham  
NG7 2NR 
Email: sponsor@nottingham.ac.uk  
 
Chief investigator:  
Dr Ana M Valdes MA PhD 
 
 
 
 
Professor in Genetic and Molecular Epidemiology 
NIHR Nottingham Biomedical Research Centre 
School of Medicine 
University of Nottingham 
Phone: +44 (0) 115 82 31954  
Email: ana.valdes@nottingham.ac.uk 
 
 
Co-investigators:  
Professor David Walsh 
Director of Versus Arthritis (VA) Pain Centre 
FRCP, PhD 
A19 Academic Rheumatology 
Clinical Sciences Building 
City Hospital 
Nottingham 
NG5 1PB 
Phone: +44 (0) 115 823 1751 
 
Email: david.walsh@nottingham.ac.uk 
 
Dr Tony Kelly 
Research Metrologist, Faculty of Medicine & Health Sciences 
A27 Academic Rheumatologist 
Clinical Sciences Building 
Nottingham City Hospital 
Hucknall Road 
Nottingham 
NG5 1PB 
Phone: +44 (0) 115 823 1696 
 
Email: tony.kelly@nottingham.ac.uk 
 
 
Dr Daniel McWilliams 
Post-doctoral research fellow  
Faculty of Medicine & Health Sciences 
A25 Academic Rheumatologist, Clinical Sciences Building 
Nottingham City Hospital, Hucknall Road 
Nottingham, NG5 1PB 
Phone: +44 (0) 115 823 1942 
 
Email: daniel.mcwilliams@nottingham.ac.uk 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
Dr Bonnie Millar  
Musculoskeletal Project Manager, Faculty of Medicine & Health 
Sciences 
 
A27 Academic Rheumatology  
Clinical Sciences Building 
Nottingham City Hospital 
Hucknall Road 
Nottingham 
NG5 1PB 
Phone: +44 (0) 1158231754 
Email: Bonnie.Millar@nottingham.ac.uk 
 
Vasileios Georgopoulos  
 
 
 
 
Post-doctoral research fellow  
University of Nottingham,  
A25 Academic Rheumatology,  
Clinical Sciences Building,  
City Hospital Nottingham, NG5 1PB 
Phone: +44 (0) 115 823 1942 
 
Email: vasileios.georgopoulos@nottingham.ac.uk 
 
Professor Benjamin Ollivere 
Professor of Orthopaedic Trauma 
NIHR Nottingham Biomedical Research Centre 
School of Medicine 
University of Nottingham 
Phone: +44 (0)115 924 9924 (#81811) 
Email: Benjamin.Ollivere@nottingham.ac.uk 
 
Mr Ben Bloch 
Consultant Orthopaedic Surgeon 
Nottingham University Hospitals 
Nottingham City Campus 
C Suite 
NG5 1PB 
Email: Ben.bloch@nuh.nhs.uk 
 
Dr Jessica Nightingale 
Orthopaedic Clinical Research Manager 
Queens Medical Centre (WC1285) 
Derby Road 
Nottingham 
NG7 2UH  
Phone: +44 (0)115 924 9924 (#81811) 
Email: Jessica.nightingale@nuh.nhs.uk 
 
Study Statistician:  
Dr Ana M Valdes MA PhD 
Associate Professor and Reader in Musculoskeletal Genetics 
NIHR Nottingham Biomedical Research Centre 
School of Medicine 
University of Nottingham 
Phone: +44 (0) 115 82 31954  

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
Email: ana.valdes@nottingham.ac.uk 
 
Study-Coordinator:  
Dr Bonnie Millar  
Musculoskeletal Project Manager, Faculty of Medicine & Health 
Sciences 
 
A27 Academic Rheumatology  
Clinical Sciences Building 
Nottingham City Hospital 
Hucknall Road 
Nottingham 
NG5 1PB 
Phone: +44 (0) 1158231754 
Email: Bonnie.Millar@nottingham.ac.uk 
 
 
 
 
Study Coordinating  
Centre:  
Division of Academic Rheumatology 
 
Clinical Sciences Building 
 
Nottingham City Hospital  
 
Nottingham 
 
NG5 1PB 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
SYNOPSIS 
 
Title 
Molecular pathways involved in knee pain: an observational study  
Short title 
Molecular pathways involved in knee pain 
Chief Investigator 
Dr Ana M Valdes 
Objectives 
 
Observational Study: 
[1] To test whether there is an association between proteomic 
concentrations in the synovial fluid and pain hypersensitivity. 
 
[2] To identify proteomic signatures in synovial fluid of centrally vs 
peripherally driven knee pain. 
  
[3] To investigate the relationship of pain hypersensitivity and different 
proteomic profiles with radiologically-established joint structural changes 
and muscle strength. 
   
[4] To explore the correlation between knee inflammation (effusion, 
synovial hypertrophy or/and synovial hyper vascularity) and biomarkers 
of insulin resistance, stress, DNA markers, poor metabolism and knee 
pain. 
 
 
Study Configuration 
Observational cross-sectional study  
Setting 
University and secondary care 
Sample size estimate 
Observational Study: We propose to compare proteomic signatures in 
140 knee pain cases. To achieve a Bonferroni p < 1.25 x 10[-5] (adjusting 
for 4000 tests, i.e. the number of proteins in the Somalogic platform) 
effect sizes of beta=0.64 in units of standard deviations are needed to 
achieve 80% statistical power.  Based on the effects seen on 
radiographic progression (standardized beta =0.62) these effects appear 
reasonable and achievable with the proposed sample size.  
Reliability Sub-study: Considering that the study will comprise two 
different sessions (n=2) featuring one measurement within each and with 
minimally accepted reliability of ρ0=0.5 and expected reliability of ρ=0.8, 
the minimum sample size has been calculated to be 22 participants. 
High variability is not expected however, to account for such an 
occurrence, data will be collected from 25 individuals. 
Number of participants 
Observational Study: 140 
Sub-study: 25 
Eligibility criteria 
Inclusion criteria:  
• have the ability to give informed consent 
• be 45 years or over 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
• have complaints of knee pain for 3-6 months with or without 
radiographically established OA (K/L scale score ≥ 1) 
• have complaints of knee pain for 3-6 months with or without satisfying 
the non-radiographic American College of Rheumatology criteria for 
knee OA 
• are willing to undertake knee synovial fluid aspiration 
• be able to speak, read, and write in English as all instructions and 
questionnaires are designed in the English language 
Exclusion criteria:  
• Inability to give informed consent due to cognitive impairment or 
otherwise – (capacity levels are already established under GP care) 
• Inability to understand key aspects of the study due to cognitive 
impairment or otherwise 
• Giving history of additional co-morbidities such as cancer, neurological 
conditions, inflammatory joint diseases including rheumatoid arthritis, 
diabetic neuropathies, fractures or other conditions causing greater 
disability than their knee pain 
• Acute soft tissue injury to the knee within last 3 months before 
potential recruitment 
Same criteria apply for the sub-study 
Description of 
interventions 
Clinical assessment for this study will include self-report questionnaires, 
and ultra-sound scanning, synovial fluid aspiration, blood samples. and 
faecal extraction as well as balance and muscle strength assessment 
through a sensitive platform and an isokinetic dynamometer respectively. 
The duration of the clinical assessment will be approximately 1.5 hours. 
 
 
Duration of study 
18 months 
Planned recruitment date: January 2020 
Per participant appointment duration (Observational and Sub-study):   
1 x 2 hours 
Outcome measures 
Primary: Pain measured by patient reported pain scoring. 
 
Secondary: Knee joint morphology on X-ray, Central aspects of pain in 
knee (CAP-Knee), Synovial fluid protein concentration, Cognitive 
function (CFQ), Frailty (FRAIL), Inflammatory markers on ultrasound 
(Synovial fluid, synovial hypertrophy and hypervascularity), Muscle 
Strength, , Biomarkers of insulin resistance, DNA markers, compromised 
metabolism, and stress generators (cortisol, gut microbiota), Physical 
functioning (Time up and go test, 30-second sit to stand test), Western 
Ontario and McMaster Universities Osteoarthritis Index (WOMAC), 
General health questionnaire (EQ5D-5L) 
Statistical methods 
Standard parametric regression methods will be used to assess the 
levels of association between proteomic profiles, pain and inflammatory 
markers on ultrasound.  

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
ABBREVIATIONS  
 
VA 
 
Versus Arthritis 
CAP-Knee 
Central Aspects of Pain in the Knee 
CFCF  
Consent for Contact Form 
CFQ 
 
Cognitive Failures Questionnaire  
 
CI 
 
Chief Investigator overall 
CNS 
 
Central Nervous System 
CPA 
 
Computerised Pressure Algometer 
CS 
 
Central Sensitisation 
CSB 
 
Clinical Sciences Building 
cmRCT 
Cohort Multiple Randomised Controlled Trial 
CPM   
Conditioned Pain Modulation  
DAP 
 
Data Analysis Plan 
DCU 
 
Data Collection Unit 
FFQ 
 
Food Frequency Questionnaire 
FRAIL  
Simple Frailty Questionnaire 
GCP 
 
Good Clinical Practice 
GP 
 
General Practitioner 
HADS  
Hospital Anxiety and Depression Scale 
ICC 
 
Intraclass Correlation Coefficient 
 
ICF 
 
Informed Consent Form 
ICOAP  
Intermittent and Constant Osteoarthritis Pain 
JA 
 
Joint Academy  
K/L  
 
Kellgren and Lawrence system for classification of osteoarthritis of knee 
KPIC  
Knee Pain in the Community 
MLR 
 
Multiple Linear Regression 
MSK 
 
Musculoskeletal 
MSK-HQ  
The Versus Arthritis Musculoskeletal Health Questionnaire  
MSK-USS  
Musculoskeletal Ultrasound Scan 
MVC   
Maximum Voluntary Contraction 
NHS 
 
National Health Service 
 
NRS   
Numerical Rating Scale 
NUH 
 
Nottingham University Hospital Trust  
OA 
 
Osteoarthritis  
PA 
 
Posterior-Anterior                           
PIS 
 
Participant Information Sheet 
PPI 
 
Patient and Public Involvement  
PPT 
 
Pressure Pain Threshold 
PRU 
 
Patient Response Unit  
QST   
Quantitative Sensory Testing 
REC 
 
Research Ethics Committee 
R&D 
 
Research and Development department 
SAE 
 
Serious Adverse Event 
SF  
 
Synovial fluid  
TS 
 
Temporal Summation 
TUG 
 
Time Up and Go Test  
USGA  
Ultrasound-guided Aspiration  
WOMAC  
Western Ontario and McMaster Universities Osteoarthritis Index  
30CST  
30-second Sit to Stand Test   
 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
TABLE OF CONTENTS 
 
 
STUDY PERSONNEL AND CONTACT DETAILS ............................................................ 2 
SYNOPSIS ................................................................................................................................. 5 
ABBREVIATIONS ................................................................................................................... 7 
STUDY BACKGROUND INFORMATION AND RATIONALE ..................................... 10 
STUDY OBJECTIVES AND PURPOSE ............................................................................. 11 
PURPOSE ............................................................................................................................... 11 
HYPOTHESIS ........................................................................................................................ 11 
PRIMARY OBJECTIVE ....................................................................................................... 11 
SECONDARY OBJECTIVES .............................................................................................. 11 
DETAILS OF PRODUCTS ................................................................................................... 11 
KNOWN SIDE EFFECTS ..................................................................................................... 12 
STUDY DESIGN .................................................................................................................... 13 
STUDY CONFIGURATION ................................................................................................. 13 
PRIMARY ENDPOINT ......................................................................................................... 13 
SECONDARY ENDPOINT ................................................................................................... 13 
STOPPING RULES AND DISCONTINUATION .............................................................. 13 
STUDY MANAGEMENT ..................................................................................................... 13 
DURATION OF THE STUDY AND PARTICIPANT INVOLVEMENT ........................ 14 
END OF THE TRIAL ............................................................................................................ 14 
SELECTION AND WITHDRAWAL OF PARTICIPANTS ............................................. 14 
RECRUITMENT .................................................................................................................... 14 
INCLUSION CRITERIA ...................................................................................................... 15 
EXCLUSION CRITERIA ..................................................................................................... 16 
PARTICIPANT WITHDRAWAL ........................................................................................ 16 
INFORMED CONSENT ........................................................................................................ 16 
STUDY REGIMEN ................................................................................................................ 17 
MUSCLE, STRENGTH, AND BALANCE MEASUREMENTS ...................................... 17 
ULTRASOUND & ULTRASOUND-GUIDED ASPIRATION ......................................... 18 
COMPLIANCE ...................................................................................................................... 19 
CRITERIA FOR TERMINATING THE STUDY .............................................................. 19 
TRANSPORT AND STORAGE OF THE TISSUES .......................................................... 19 
LABORATORY ANALYSIS ................................................................................................ 20 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
MISSED APPOINTMENTS .................................................................................................. 20 
STATISTICS ........................................................................................................................... 20 
METHODS .............................................................................................................................. 20 
SAMPLE SIZE AND JUSTIFICATION ............................................................................. 21 
ADVERSE EVENTS .............................................................................................................. 21 
ETHICAL AND REGULATORY ASPECTS ..................................................................... 21 
ETHICS COMMITTEE AND REGULATORY APPROVALS ....................................... 21 
INFORMED CONSENT AND PARTICIPANT INFORMATION .................................. 22 
RECORDS ............................................................................................................................... 22 
STUDY FORMS ..................................................................................................................... 22 
SAMPLE LABELLING ......................................................................................................... 23 
SOURCE DOCUMENTS ...................................................................................................... 23 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS .................................................. 23 
DATA PROTECTION ........................................................................................................... 23 
QUALITY ASSURANCE & AUDIT .................................................................................... 23 
INSURANCE AND INDEMNITY ........................................................................................ 23 
STUDY CONDUCT ............................................................................................................... 24 
STUDY DATA ........................................................................................................................ 24 
RECORD RETENTION AND ARCHIVING ..................................................................... 24 
DISCONTINUATION OF THE TRIAL BY THE SPONSOR .......................................... 24 
STATEMENT OF CONFIDENTIALITY ........................................................................... 24 
PUBLICATION AND DISSEMINATION POLICY .......................................................... 25 
USER AND PUBLIC INVOLVEMENT .............................................................................. 25 
STUDY FINANCES ............................................................................................................... 25 
SIGNATURE PAGES ............................................................................................................ 26 
REFERENCES ....................................................................................................................... 27 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
STUDY BACKGROUND INFORMATION AND RATIONALE 
 
Osteoarthritis (OA) is the most common cause of disability in the elderly population and most 
individuals suffering from osteoarthritis are managed in the primary care setting (1). Knee OA 
is the most common form of arthritis and the most common cause of knee pain in the world 
(2). The rate of knee arthritis is as high as that of cardiac disease and is the most common 
problem in individuals over the age of 65 (3). In the United Kingdom, 10% of 65 to 74-year-old 
individuals consult their general practitioners about OA per year (4).  Out of the entire 
population, 4% attend their general practitioners as a result of knee OA, and half of them (2%) 
consult their general practitioner for the first time or with the acute flare of knee arthritis (1).  
 
We will recruit 140 individuals with OA-related knee pain, and we will record their pain levels 
on the Numerical rating scale (NRS) at a single time-point. The validity and sensitivity of NRS 
have been established numerous times (14-18).  
 
 
Metabolic evidence from body secretions can provide significant insight about the 
course of OA, as the presence of glucosamine and chondroitin sulfate in faecal samples 
has shown to improve the symptoms of the condition and delay its progression (56). 
Nutritional information and metabolic evidence from faecal samples can be used to 
explore associations of gut microbiota with pain sensitivity and stress levels (57, 58).   
 
The widely utilised Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) 
will be also used for the evaluation of knee OA. It is a self-administered questionnaire made 
of 24 items and consists of three subscales covering pain, stiffness and physical function.  
It has been used extensively and is considered a valid and reliable tool (59-63). Frailty has 
been also found to be a predictor of disability (66) and a determinant of treatment 
outcomes (67-69) and the Simple Frailty Questionnaire (FRAIL) is considered a valid and 
reliable tool for that purpose (70).  
 
Both the 30-second sit to stand test (30CST) and the ‘time up and go’ (TUG) test will be 
used to see if patients have improved their lower limb fitness levels. 30CST has shown 
excellent reliability and validity (63, 71, 72).  TUG has been widely used in clinical setups 
and is a valid tool to assess necessary functional mobility (73-79).  
 
 
 
Musculoskeletal Ultrasound scan (MSK-USS) will be also conducted on the knees of 
participants to establish if they have inflammation of the synovial membrane. There is enough 
evidence that inflammation is present in all stages of OA (108-111). Synovitis or inflammation 
of synovial fluid is associated with pain, disease severity and, OA progression (108, 112). 
Synovitis manifests as synovial membrane thickening, increased vascularity, and/or joint 
effusion (108, 113-115). During MSK-USS, the presence of synovial fluid, synovium 
hypertrophy, and Power Doppler will be also assessed. Synovial hypertrophy, synovitis and 
knee effusion are linked with arthritis in the knee and associated with knee pain in osteoarthritis 
(116-121). Power Doppler provides a reliable and accurate method for visualising blood flow 
in the synovial tissue, and histological findings support the value of this technique (119, 122, 
123). An ultrasound scan is considered to be a valid and reliable instrument for the assessment 
of synovial disease (113, 119, 120, 124) and synovitis is strongly associated with osteoarthritis 
as mentioned earlier. The synovial fluid will be aspirated (subject to participant consent), in 
order to establish a phenotype which is strongly associated with OA. Studying synovial fluid 
biomarkers alongside clinical, radiographic and ultra-sonographic characteristics is one 
strategy to improve resolution and stratification into targetable OA phenotypes (125). Synovial 
fluid aspiration will be ultrasound guided as it increases the accuracy of needle placement 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
compared to blind needling (95.8% versus 77.8%, p < 0.001) (126) reduces procedural pain 
by 43%, improves effusion detection by 200%, and volume of synovial fluid aspirated by 337% 
compared with blind synovial fluid aspiration (127). Ultrasound guidance also reduces 
procedural pain (43% reduction) in knees with no palpable effusion and increases the 
responder rate and therapeutic duration by 107% and 36% respectively (128-132).  
 
Optionally, isometric quadriceps strength will be also assessed to establish current strength 
levels of vastus medialis muscle and see whether muscle strength associates with centrally 
driven pain or proteomic synovial concentrations. Quadriceps muscles strength deficits are 
associated with knee osteoarthritis (133). Isometric testing will be done at 30 and 60 degrees 
of flexion as per the protocol of a previous study (134). Hand grip strength will also be 
measured as an option 
 
Blood samples will be also extracted to assess the biomarkers (including serum levels and 
gene expression levels of various molecules) and to establish insulin resistance (135, 136).  
 
Collection and study of these parameters can provide more insight into the traits of knee pain, 
allow the examination of possible correlations to each other, and highlight potential detrimental 
effects of them on knee joint health.  
 
STUDY OBJECTIVES AND PURPOSE 
 
PURPOSE 
The primary purpose of this study is to explore the potential molecular links between pain 
and structure on knee pain using synovial fluid proteomics. A secondary purpose is to 
explore the association of knee pain with biomarkers of stress and metabolism  
 
HYPOTHESIS 
Among individuals with moderate to severe knee pain different proteomic signatures will be 
seen in synovial fluid of the affected knees. 
 
PRIMARY OBJECTIVE 
1. To identify proteomic signatures in synovial fluid of the knee joint in individuals with 
knee pain.  
SECONDARY OBJECTIVES 
1. To test whether there is an association between proteomic concentrations in the 
synovial fluid and pain.  
2. To link the changes in pain  and physical activity to biomarkers of insulin resistance, 
stress, poor metabolism, inflammation and pain. 
3. To investigate the relationship of pain and different proteomic profiles with 
radiologically-established joint structural changes and muscle strength. 
4. To explore the correlation between  knee inflammation (effusion, synovial hypertrophy 
or/and synovial hyper vascularity), biomarkers of insulin resistance, DNA markers, 
stress, poor metabolism, gene expression and knee pain. 
DETAILS OF PRODUCTS  
 
We will be using ultrasound to assess participant knee joints.  
 
Ultrasound: 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
An ultrasound will be used to image both knee joints using a Toshiba Aplio SSA-770A 
machine with a multi-frequency (7 – 12 Hz). This equipment belongs to the university and is 
CE marked.  
 
Known Side Effects 
There are not any known side effects from ultrasound. There can be a small amount of pain 
from the aspiration site.  
 
 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
STUDY DESIGN 
 
Study Configuration 
 
A single-centre observational cross-sectional study in a university and hospital setting with 
participants suffering from knee pain.  
 
Primary endpoint 
The primary outcome measure is pain measured by patient reported scores.   
 
Secondary endpoint 
• 
Synovial fluid protein concentration (synovial fluid) 
• 
Knee joint morphology on X-ray 
• 
Central aspects of knee pain (CAP-Knee) 
• 
Inflammatory markers on ultrasound (Synovial fluid, synovial hypertrophy and 
hypervascularity) 
• 
Muscle Strength (Dynamometer) 
• 
Balance (Balance platform) 
• 
Biomarkers of insulin resistance (blood, urine) and metabolic rate (faeces) 
• 
Gene expression levels of molecules in pain 
• 
Physical functioning (Time up and go test, 30-second sit to stand test) 
• 
Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) 
• 
General health questionnaire (EQ5D-5L) 
 
All these endpoints will be measured at a single time-point (baseline) and at a follow up time 
point 120 days for those who subsequently go on to have surgery  
 
Stopping rules and discontinuation 
As the study involves only a single clinical assessment and does not involve investigational 
medicinal products or medical devices it is not envisaged that circumstances will arise that 
require termination or discontinuation of this study. 
 
Study Management 
The study will be managed in Academic Rheumatology, Clinical Sciences Building by the 
Research Manager under the supervision of the Chief Investigator who has overall 
responsibility for the study and shall oversee all study management. 
Other members of the team are Professor David Walsh (Director of Pain Centre Versus 
Arthritis (VA)), Dr Tony Kelly (Research Metrologist), Dr Daniel McWilliams (Post-doctorate 
Research Fellow), Vasileios Georgopoulos (Post-doctorate Research Fellow) and Dr Bonnie Millar 
(Musculoskeletal Project Manager). Professor Ollivere (Professor of Orthopaedic Surgery) 
will provide oversight of research procedures conducted within NHS facilities. Mr Ben Bloch 
(Consultant Orthopaedic Surgeon) will facilitate recruitment from NHS waiting lists. Dr 
Jessica Nightingale (NUH Orthopaedic Clinical Research Manager) will support recruitment 
and research delivery from NHS areas.  
The study management group will meet regularly and have documented quarterly meetings 
from the start of the study. The group will also monitor and supervise the progress of the study 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
and advice on recruitment rate and unforeseen logistical issues to ensure that its objectives 
are achieved.  
The data custodian will be the CI who will also have overall responsibility for the study and 
shall oversee all study management. 
 
Duration of the Study and Participant Involvement 
 
Study Duration and Schedule of Events:  
The trial is expected to start in April 2020, and enrolment will end in in the late summer of 
2022. After about two months from the end of recruitment, all data will be put together for 
coding and further processing. Data analysis will be performed by a co-investigator 
supervised by the CI.  
 
Participant Duration:  
Those individuals who agree to participate will be asked to attend one session, at a time of 
their convenience in order to receive all relevant examination and samples extraction 
 
Therefore, in total, individuals will be asked to attend a single session for no more than 2 
hours. 
 
End of the Trial 
The end of the study will be on the last participant visit. The end of the study overall will be 
when the results will be written up and publication submissions will be attempted. 
 
Selection and Withdrawal of Participants 
 
Recruitment 
 
There are several potential recruitment pathways for this study:  
 
1) A selection of eligible people for the study will be invited from existing databases 
(Knee Pain in the Community (KPIC) and Investigating MSK Health and Well-being 
(IMH&W) Databases) held at Academic Rheumatology of participants with knee pain 
who have agreed to be contacted for future studies. The databases can be accessed 
by a member of the research team who will also contact the potential participants to 
ask if they are interested. We aim to recruit 140 participants and out of those we also 
aim to recruit 25 to participate in the sub-study. 
2) Participants will also be recruited from consultant waiting lists and clinics for Total 
Knee Arthroplasty (TKR) at Nottingham University Hospitals NHS Trust. An invitation 
pack will be posted to eligible participants who are on the waiting list for TKA, these 
pack will be sent by the participants treating orthopaedic surgeons or a member of 
their administrative team. Interested individuals will be invited to declare their interest 
by mailing their reply slip via a prepaid envelope directly to the study team.  

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
The study team in Academic Rheumatology will phone interested participants to further 
establish eligibility and to screen them about x-ray scanning. From those who had an x-ray in 
the previous 12 months and their x-ray was undertaken within the Nottingham University 
Hospital (NUH) Trust, verbal consent will be requested overt the phone for a member of the 
study team with an honorary contract with NUH to access the relevant-only information in order 
to establish whether the existing scan is relevant to this research.  
Study visits will take place either in the Clinical Sciences Building in Academic Rheumatology, 
or in an NHS clinic room at the Queens Medical Centre site. Participants who satisfy the 
eligibility criteria upon the telephone screening will be invited to attend a study visit, at a day 
of their convenience. That visit will be considered as their single study session. The purpose 
of the visit will be to further establish suitability and gain full consent about knee aspiration and 
all other proceedings (samples, ultrasound, functional assessment) before any study activities 
are undertaken. Participants will be eligible for inclusion to the study if they are willing to 
undergo knee synovial fluid aspiration and demonstrate radiographic evidence of osteoarthritis 
(K/L= 1 or above) or if they satisfy the American College of Rheumatology clinical criteria for 
knee OA (139) even in the absence of radiographic findings (e.g. knee pain and three or more 
of the following: over the age of 50, less than 30 minutes of stiffness in the morning, no palpable 
warmth on synovium, bony tenderness, bony enlargement, crepitus on active motion).  
Participants will be free to leave the study at any time without giving any reason. Participants 
who indicate in their questionnaire that they are willing to receive information about future knee 
pain studies may withdraw this indication at any time, and this will be altered appropriately on 
the study database. Participants will be made aware via the questionnaire that all data will 
remain confidential and will be anonymised before use in further analysis.    
It will be explained to the potential participant that entry into the study is entirely voluntary and 
that their treatment and care will not be affected by their decision. It will also be explained that 
they can withdraw at any time, but attempts will be made to avoid this occurrence. In the event 
of their withdrawal, it will be explained that any data collected so far cannot be erased, and we 
will seek consent to use the data in the final analyses where appropriate (via the information 
sheet and consent form). Those who withdraw from the study will be made aware that this will 
not in any way affect their future care. 
Participants who have agreed to participate in the study will be asked directly by a member of 
the research team whether they would like to participate also in the sub-study. Those who will 
agree to participate will be provided with a separate Participant Information Sheet (PIS) 
composed specifically for the sub-study and they will have to sign an additional informed 
consent form (ICF). 
Inclusion criteria  
For individuals to be included in the study they must:   
• 
have the ability to give informed consent 
• 
be 45 years or over 
• 
have complaints of knee pain for 3-6 months with or without radiographically 
established OA (K/L scale score ≥ 1) 
• 
have complaints of knee pain for 3-6 months with or without satisfying the non-
radiographic American College of Rheumatology criteria for knee OA 
• 
are willing to undertake knee synovial fluid aspiration 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
• 
be able to speak, read, and write in English as all instructions and questionnaires are 
designed in the English language 
 
Exclusion criteria 
Individuals who meet one or more of the following criteria will be excluded: 
• 
Inability to give informed consent due to cognitive impairment or otherwise – (capacity 
levels are already established under GP care) 
• 
Inability to understand key aspects of the study due to cognitive impairment or 
otherwise 
• 
Giving history of additional co-morbidities such as cancer, neurological conditions, 
inflammatory joint diseases including rheumatoid arthritis, diabetic neuropathies, 
fractures or other conditions causing greater disability than their knee pain 
• 
Acute soft tissue injury to the knee within last 3 months before potential recruitment 
 
Participant Withdrawal 
Participants may be withdrawn from the study either at their own request or at the discretion 
of the investigator. The participants will be made aware that this will not affect their future care. 
Participants will be made aware (including via the information sheet and consent form) that 
should they withdraw the data collected to date cannot be erased and may still be used in the 
final analysis.  
Informed consent 
All participants will provide written informed consent. The ICF will be signed and dated by the 
participant before they enter the trial. The Investigator will explain the details of the trial and 
provide a Participant Information Sheet, ensuring that the participant has sufficient time to 
consider participating or not. The Investigator will answer any questions that the participant 
has concerning study participation.  
 
Informed consent will be collected from each participant before they undergo any interventions 
(including physical examination and history taking) related to the study. One copy of this will 
be kept by the participant, one will be kept by the investigator, and a third will be retained in 
the participant’s hospital records. 
 
The same process will be reiterated with a separate study specific information sheet and 
consent form for those individuals who will agree to participate in the sub-study and undergo 
the additional assessment. 
 
Should there be any subsequent amendment to the final protocol, which might affect a 
participant’s participation in the study at any part, continuing consent will be obtained using an 
amended Consent form which will be signed by the participant. 
 
Future contact 
The consent form includes an optional clause asking participants to confirm whether they are 
happy for their personal details to be retained in order to be informed about relevant future 
studies within the Pain Centre for which they might be suitable. If they consent, their data will 
be stored on a secure database on a University of Nottingham server and will be kept up-to-
date in accordance with Data Protection Act, 2018. If not, their contact details will be destroyed 
at the end of this study after it is no longer necessary to contact them.  

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
 
Study Regimen  
Participants seeking care for their knee pain will be informed of the study both orally and in 
writing. Those who qualify for the study will be asked to come to Academic Rheumatology 
for a single session. All research activities will be undertaken on the same session which is 
expected to last no longer than 2 hours. During the single session, participants will sign the 
informed consent form (ICF) and will be asked to go through the study procedure. All 
participants will have blood samples extracted. All participants will have a knee ultrasound 
scan by a trained member of the study team to assess their knee and locate the best needle 
insertion point and go through the ultrasound-guided aspiration (USGA) procedure. 
Participants will then have their pain sensitivity, strength, balance and function measured by 
special tests (sit to stand, time up and go). Outcome measures about pain mechanisms 
(CAP-Knee), OA (WOMAC), cognitive function (CFQ), frailty (FRAIL), and health (EQ5d-5L) 
will be completed by participants at home online or on paper copies. Participants will be also 
asked if they wish to donate a faecal sample and if they agree they will be given a collection 
kit to take home along which will be shipped back by post.  
 
To summarise, the total study duration, including recruitment and the assessment visit, will 
be 18 months. The key stages are detailed below:  
 
• 
Individuals who agree to attend for a clinical assessment will participate for a single 
appointment. This appointment will last approximately 2 hours.  
• 
They will be seen at the Academic Rheumatology Unit, Clinical Sciences Building, 
Nottingham City Hospital or in an NHS clinic room at Queens Medical Centre. 
• 
Transport (taxi) will be arranged if required and any travel expense will be 
reimbursed.   
• 
Our Research Nurses will get a 15ml blood sample from the participant  
• 
Once blood samples have been collected participants may be offered a light 
breakfast and drink refreshment before a brief assessment to confirm medical history 
and current medications.  
• 
Questionnaires such as WOMAC, EQ5D-5L , CFQ, CAP-Knee, FRAIL and Numeric 
VAS will be collected.  
• 
Physiological measurements such as 30 seconds sit to stand (30CST), time up and 
go (TUG), balance and muscle strength assessments will be taken at this point.  
• 
Diagnostic ultrasound of the most painful knee will be conducted and ultrasound 
guided synovial fluid aspiration will follow.  
• 
Follow up questionnaire will be posted or conducted through the telephone in line 
with the questionnaires  at the initial visit.  
 
Muscle, Strength, and Balance Measurements  
A sequence of four physiological measures will be undertaken:  
 
Time up and Go:  The participant will start in a seated position. The participant will stand 
up upon therapist’s command, walk 3 meters, turn around, walk back to the chair and sit 
down. The time will stop when the participant is seated. The subject can use an assistive 
device. If the assistive device is used, it will be documented. 
NOTE: A practice trial will be completed before the timed trial 
 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
30 seconds sit to stand.  The 30-Second Chair Test is administered using a folding chair 
without arms, with a seat height of 17 inches (43.2 cm). The chair, with rubber tips on the 
legs, is placed against a wall to prevent it from moving. The participant is seated in the 
middle of the chair, back straight; feet approximately shoulder width apart and placed on 
the floor at an angle slightly back from the knees, with one foot slightly in front of the other 
to help maintain balance. Arms are crossed at the wrists and held against the chest. 
Demonstrate the task both slowly and quickly. Have the participant practice a repetition 
or 2 before completing the test. If a participant must use their arms to complete the test, 
they are scored 0. The participant is encouraged to complete as many full stands as 
possible within 30 seconds. The participant is instructed to fully sit between each stand. 
While monitoring the participant’s performance to ensure proper form, the tester silently 
counts the completion of each correct stand.  The score is the total number of stands 
within 30 seconds (more than halfway up at the end of 30 seconds counts as a full stand). 
Incorrectly executed stands are not counted. The 30-second chair stand involves 
recording the number of stands a person can complete in 30 seconds rather than the 
amount of time it takes to complete a pre-determined number of repetitions.  
 
Muscle strength Assessment: Isometric testing will be done at 30 and 60 degrees of 
flexion as done in the previous study and the participant will be in sitting position with hips 
and knees strapped to keep the position standardised.  
 
Balance: Static balance and postural sway either in the medial-lateral or antero-posterior 
direction will be assessed using the RS Scan force plate. The participant will be asked to 
stand on the plate looking straight forward for 30 seconds in two conditions: first with their 
eyes open and then with eyes closed. Medial-lateral, antero-posterior and total sway will 
be recorded. 
 
Ultrasound & Ultrasound-guided Aspiration 
During the ultrasound scan, the supra-patellar pouch, medial and lateral recess of the knees 
will be assessed for synovial thickening, synovial fluid/effusion and for positive power Doppler. 
An ultrasonic probe will be used to direct ultrasonic waves onto the knee joint during 
sonography, and a computer converts the signals received so that they can be presented on 
the screen. During skin application, a sterile probe cover and sterile acoustic gel will be used. 
There is no radiation exposure to ultrasound due to the lack of radioactive rays and no 
detrimental side effects.  
 
Once the best aspiration location is identified and the location of the needle insertion marked, 
the skin will be prepared with a cleansing agent such as iodine or chlorhexidine. The needle 
may be introduced into the skin either parallel to the probe or perpendicular to the probe 
(Figure 1). When the needle runs parallel to the probe it will be seen in full length. If it is 
advanced perpendicular to the probe the needle is only represented by a hyperechoic spot in 
the ultrasound image. 
 
 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
 
Figure 1. Approaches of ultrasound guided injections with possible positions of needles and probes (141). 
 
Once the aspiration is completed, the needle will be removed, the skin cleansed, and a 
bandage will be applied. A woven elastic bandage or knee immobilizer may be applied to 
reduce post-procedural swelling and discomfort (140). 
 
 
 
Compliance 
Compliance will be judged by attendance for study-related appointments and during the study 
visit based on completion of key study procedures. Participants will be replaced only if they 
refuse to undertake ultrasound-guided synovial fluid extraction and quantitative sensory 
testing. Individuals will be given a contact number to contact us if they have any query or wish 
to reschedule an appointment.   
 
Criteria for terminating the study 
As the study involves only a single assessment and does not involve investigational medicinal 
products or medical devices it is not envisaged that circumstances will arise that require 
termination of this study.  
 
 
Transport and Storage of the Tissues 
The samples will be stored at University of Nottingham premises. Whole blood (5ml) samples 
will be stored in a refrigerator (at -20°C) within the Clinical Sciences Building. Remaining blood 
(5ml) will be centrifuged as soon after venesection as possible and the serum stored, with the 
urine, at -80C within freezers in University of Nottingham premises at the Clinical Sciences 
Building (CSB), City Hospital for future biochemical and molecular studies.  Excess samples 
(DNA, serum, saliva, urine and faeces) that remain after the study analyses have been 
undertaken will remain stored within the CSB for possible future research studies related to 
OA and/or pain provided that participants are agreeable and sign the optional clause on the 
consent form. These CSB facilities come within the remit of the Research Tissue Bank (DI Dr 
William Dunn- Licence Number 12265). Where participants do not agree to the future use of 
the samples, any samples remaining after the analysis will be destroyed in accordance with 
the Human Tissue Act, 2004 and GDPR. 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
 
Samples will be stored in the linked anonymised format in the CSB and labelled using a 
randomly generated unique participant identifier to permit accurate linkage to clinical data 
and the consent form.  The master database will be held by the CI in an encrypted password 
file. After 7 years all personal data linked to the samples will be destroyed. 
 
Laboratory Analysis  
Whole blood may be sent to a specialist company for total genomic DNA extraction and 
processing using standard protocols. The DNA will be used to identify genes that identify with 
pain progression and severity. Bacterial DNA will be extracted from faecal samples to identify 
bacterial lineages associated with pain and inflammation. Cortisol will be extracted from saliva 
samples. Protein and inflammatory markers will be extracted from urine samples. Faecal 
samples will be stored until all samples are collected (as this is optional) and then they will be 
sent to the laboratory. Similarly, biochemical/biomarker analyses may be undertaken by other 
academic or commercial groups. Such investigations will be undertaken on anonymised 
samples under usual Material Transfer Agreement arrangements. The study will be registered 
with the Human Tissue Management Group of the UoN. Storage of these tissues will be 
managed, transported and handled according to Standard Operating Procedure (SOP) HTMG 
004. All shipments will contain a complete inventory of all samples, along with the name of the 
person responsible for sending the samples. Companies which will be used for the analysis 
should process the samples within seven days upon receipt or companies with HTA licence 
will be considered. It is anticipated that samples will be destroyed following analysis. There is 
no plan for long-term storage of such samples by those companies and no samples are 
expected to be transported back to UoN. 
 
Missed Appointments 
Missed appointments are an avoidable cost and resource inefficiency which impacts upon 
research outcomes. Health care service colleagues are increasingly utilising reminder systems 
to manage these negative effects. A systematic review (145) found consistent evidence that 
all types of reminder systems are effective at improving appointment attendance across a 
range of care settings and patient populations. Therefore, a reminder mechanism (a reminder 
telephone call, a polite reminder e-mail and where the participant has provided their mobile 
telephone number, a reminder text message) will be considered. 
 
STATISTICS 
 
Methods  
The chief investigator, co-investigators and the study statistician will evaluate the findings 
and analysis will be carried out by the co-investigator. The data will be entered into MS 
Access and analysed using with STATA, SPSS (Statistical Packages for Social Scientists, 
version 19) and R software. Data will allow exploration of potential relationships between 
objective sensory measures and molecular findings and/or pain. Several factors that 
putatively mediate the primary or secondary outcomes are inter-correlated so investigation 
of their interaction must be ascertained. Simple univariate unadjusted correlations 
(Pearson/Spearman) and linear regression modelling will be used to examine associations 
between pain and molecular findings in the synovial fluids at baseline. Multiple linear 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
regression (MLR) modelling (backward stepwise regression) will be conducted to assess the 
impact of the significant independent variables and their interactions with the dependent 
variable. 
 
The results of the study will be available to the study management group. All data will be stored 
in Academic Rheumatology and will be backed up on the University of Nottingham servers.  
 
Sample size and justification 
We propose to compare proteomic signatures in 140 knee pain cases. To achieve a 
Bonferroni p < 1.25 x 10[-5] (adjusting for 4000 tests, i.e. the number of proteins in the 
Somalogic platform) effect sizes of beta = 0.64 in units of standard deviations are needed 
to achieve 80% statistical power.  Based on the effects seen on radiographic progression 
(standardized beta = 0.62) these effects appear reasonable and achievable with the 
proposed sample size.   
 
ADVERSE EVENTS 
As all procedures are being carried out following standard methods, the occurrence of an 
adverse event as a result of participation within this study is not expected however, participants 
will be asked to contact the study site immediately in the event of any serious adverse event 
which is not expected. Any adverse event will be recorded and closely monitored until 
resolution, stabilisation or until it has been shown that the study treatment/intervention is not 
the cause. The CI shall be informed immediately of any serious adverse events and shall 
determine seriousness and causality in conjunction with any treating medical practitioners.  
 
Any serious adverse event (s) related to this study will be recorded and reported to the REC 
as part of annual reports. Unexpected serious adverse events will be reported within 
timeframes to the REC and CI will be responsible for all adverse event reporting.  
  
ETHICAL AND REGULATORY ASPECTS 
 
Involvement of Participant’s General Practitioner  
Participant’s GPs will not be informed that their patient has agreed to participate in the study.  
 
Ethics Committee and Regulatory Approvals 
The study will not be initiated before the protocol, consent forms and participant information 
sheets have received approval / favourable opinion from the Research Ethics Committee 
(REC), and the respective National Health Service (NHS) Research & Development (R&D) 
department. Should a protocol amendment be made, that requires REC approval, the changes 
in the protocol will not be instituted until the amendment and revised informed consent forms, 
and participant information sheets (if appropriate) have been reviewed and received approval 
/ favourable opinion from the REC and R&D departments. A protocol amendment intended to 
eliminate an apparent immediate hazard to participants may be implemented immediately 
notifying the REC as soon as possible and approval is requested. Minor protocol amendments 
only for logistical or administrative changes may be implemented immediately, and the REC 
will be informed. 
 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
The study will be conducted in accordance with the ethical principles that have their origin in 
the Declaration of Helsinki, 1996; the principles of Good Clinical Practice, and the UK 
Department of Health Policy Framework for Health and Social Care, 2017. 
 
Informed Consent and Participant Information 
The process for obtaining participant informed consent will be in accordance with the REC 
guidance, and Good Clinical Practice (GCP) and any other regulatory requirements that might 
be introduced. The investigator or their nominee and the participant shall both sign and date 
the Consent Form before the person can participate in the study. 
 
The participant will receive a copy of the signed, and dated forms and the original will be 
retained in the Study records.  
 
The decision regarding participation in the study is entirely voluntary. The investigator or their 
nominee shall emphasize to them that consent regarding study participation may be withdrawn 
at any time without penalty or affecting the quality or quantity of their future medical care, or 
loss of benefits to which the participant is otherwise entitled. No study-specific interventions 
will be done before informed consent has been obtained. 
 
The investigator will inform the participant of any relevant information that becomes available 
during the course of the study and will discuss with them, whether they wish to continue with 
the study. If applicable, they will be asked to sign revised consent forms. 
 
If the Consent Form is amended during the study, the investigator shall follow all applicable 
regulatory requirements pertaining to approval of the amended Consent Form by the REC and 
use of the amended form (including for ongoing participants). 
 
Records 
  
Study Forms  
Each participant will be assigned a study identity code number, and this will be used on the 
study forms as well as other trial documents and the electronic database. This will be a 
randomly generated unique participant identifier.  
 
Study forms will be treated as confidential documents and held securely in accordance with 
regulations. The investigator will make a separate confidential record of the participant’s name, 
date of birth, local hospital number or NHS number, and Participant Study Number, to permit 
identification of all participants enrolled in the trial, in accordance with regulatory requirements 
and for follow-up as required. 
 
Study forms shall be restricted to those personnel approved by the Chief or local Principal 
Investigator and recorded in a Study Personnel Log.  All paper forms shall be filled in using a 
black ballpoint pen. Errors shall be lined out but not obliterated by using correction fluid and 
the correction inserted, initialled and dated. 
 

 
 
 
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transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
The Chief or local Principal Investigator shall sign a declaration ensuring the accuracy of data 
recorded in the study form. 
 
Sample Labelling  
Each participant will be assigned a unique study identification code number for use on the 
samples, consent forms and other study documents and the electronic database.  
 
Source documents  
Source documents shall be filed at the investigator’s site and may include but are not limited 
to, consent forms, study records, and. A study form may also completely serve as its own 
source data. 
 
Direct access to source data/documents 
The study form and all source documents, including progress notes and copies of laboratory 
and medical test results, shall be made be available at all times for review by the Chief 
Investigator, Sponsor’s designee and inspection by relevant regulatory authorities.  
 
Data Protection  
All trial staff and investigators will endeavour to protect the rights of the trial’s participants to 
privacy and informed consent and will adhere to the Data Protection Act, 2018. The study 
forms will only collect the minimum required information for the purposes of the trial. Study 
forms will be held securely, in a locked room, or locked cupboard or cabinet. Access to the 
information will be limited to the trial staff and investigators and relevant regulatory authorities 
(see above). The computer held data including the trial database will be held securely and 
password protected. All data will be stored on a secure dedicated web server. Access will be 
restricted by user identifiers and passwords (encrypted using a one-way encryption method). 
Information about the trial in the participant’s medical records / hospital notes will be treated 
confidentially in the same way as all other confidential medical information.  
 
Electronic data will be backed up every 24 hours to both local and remote media in an 
encrypted format 
 
 
QUALITY ASSURANCE & AUDIT  
 
Insurance and Indemnity 
Insurance and indemnity for clinical study participants and study staff are covered within the 
NHS Indemnity Arrangements for clinical negligence claims in the NHS, issued under cover of 
HSG (96)48. There are no special compensation arrangements, but study participants may 
have recourse through the NHS complaints procedures. 
 
The University of Nottingham as research sponsor indemnifies its staff, research participants 
and research protocols with both public liability insurance and clinical trials insurance. These 
policies include provision for indemnity in the event of a successful litigious claim for proven 
non-negligent harm.  
 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
Study Conduct 
Study conduct will be subject to systems audit for inclusion of essential documents; 
permissions to conduct the study; CVs of study staff and training received; local document 
control procedures; consent procedures and recruitment logs; adherence to procedures 
defined in the protocol (e.g. inclusion / exclusion criteria, random selection of suitable 
participants for Phase II and Phase III, timeliness of visits); accountability of study materials 
and equipment calibration logs. 
 
Study Data  
Monitoring of study data shall include confirmation of informed consent; source data 
verification; data storage and data transfer procedures; local quality control checks and 
procedures, back-up and disaster recovery of any local databases and validation of data 
manipulation. This will be managed by the direct study team.  
 
Entries on study forms will be verified by inspection against the source data. A sample of the 
forms (10%) will be checked on a regular basis for verification of all entries made. In addition, 
the subsequent capture of the data on the study database will be checked. Where corrections 
are required these will carry a full audit trail and justification. 
 
Study data and evidence of monitoring and systems audits will be made available for inspection 
by the REC as required. 
 
Record Retention and Archiving 
In compliance with the ICH/GCP guidelines, regulations and in accordance with the University 
of Nottingham Code of Research Conduct and Research Ethics, the Chief or local Principal 
Investigator will maintain all records and documents regarding the conduct of the study. These 
will be retained for at least seven years or for longer if required. If the responsible investigator 
is no longer able to maintain the study records, a second person will be nominated to take over 
this responsibility.  
 
The study master file held by the Chief Investigator on behalf of the Sponsor shall be finally 
archived at secure archive facilities at the University of Nottingham.  This archive shall include 
all anonymised study databases and associated meta-data encryption codes. 
 
Discontinuation of the Trial by the Sponsor  
The Sponsor reserves the right to discontinue this study at any time for failure to meet expected 
enrolment goals, for safety or any other administrative reasons.  The Sponsor shall take advice 
as appropriate in making this decision. 
 
Statement of Confidentiality  
Individual participant medical or personal information obtained as a result of this study are 
considered confidential, and disclosure to third parties is prohibited with the exceptions noted 
above. 
 
Participant confidentiality will be further ensured by utilising identification code numbers to 
correspond to treatment data in the computer files. 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
 
Such medical information may be given to the participant’s medical team and all appropriate 
medical personnel responsible for the participant’s welfare. 
 
Data generated as a result of this study will be available for inspection on request by the 
participating physicians, the University of Nottingham representatives, local R&D Departments 
and the regulatory authorities. 
 
PUBLICATION AND DISSEMINATION POLICY 
The study results will be submitted to Versus Arthritis, regulatory authorities, and peer-
reviewed journals for publication.  In addition, the results will be presented at national and 
international conferences.  Study participants’ identity will not be disclosed when publishing 
the results.  Study participants will also be informed of the results if requested.  
 
USER AND PUBLIC INVOLVEMENT 
On 17/05/2017 6 representatives from the Patient and Public Involvement (PPI) MSK group 
were asked about their views on this project. They all agreed on the need to identify molecular 
markers to distinguish patient subgroups, not only if this can be conducive to drug 
development, but more generally they were very appreciative of a stratified medicine approach, 
and to them it was mostly an issue related to the discrepancies between pain and joint damage 
seen in their own X-rays. All group members (6/6) indicated their willingness to participate in 
studies that are conducive to understanding molecular subtypes of OA.  Almost all (5/6) would 
be happy to have synovial aspirations for such a scientific project and would prefer US guided 
aspiration. They also expressed the view that having diagnostics that can better direct 
treatment based on molecular test from synovial aspirations or blood should be part of standard 
practice within the NHS. 
 
The latest versions of the participant facing documentation (PIS, consent form, invitation letter 
and flyer) has all been forwarded to three PPI representatives who have reviewed and 
commented on them 
 
STUDY FINANCES 
Funding source  
This study is part-funded by Versus Arthritis and the Kennedy Trust via their Step-Up OA 
consortium fund. 
 
Participant stipends and payments 
Participants will not be paid to participate in the study. However, all travel expenses to and 
from Academic Rheumatology, Clinical Sciences Building, Nottingham City Hospital or Queens 
Medical Centre will be covered.  
 
 

 
 
 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
SIGNATURE PAGES 
 
Signatories to Protocol: 
 
Chief Investigator: (name)__________________________________ 
 
 
Signature:__________________________________ 
 
 
Date: ___________ 
 
 
 
 
Co- investigator: 
(name) __________________________________ 
 
 
Signature:__________________________________ 
 
 
Date: ___________ 
 
 
 
Trial Statistician:  (name)__________________________________ 
 
 
Signature:__________________________________ 
 
 
Date: ___________ 
 
 
 
 
 
 
 

 
 
 
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Molecular pathways involved in knee pain 
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This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
REFERENCES 
 
 
1. 
Peat G, McCarney R, Croft P. Knee pain and osteoarthritis in older adults: a review of 
community burden and current use of primary health care. Ann Rheum Dis. 2001;60(2):91-7. 
2. 
Felson DT. Weight and osteoarthritis. The American journal of clinical nutrition. 
1996;63(3 Suppl):430s-2s. 
3. 
Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, et al. The 
effects of specific medical conditions on the functional limitations of elders in the 
Framingham Study. American Journal of Public Health. 1994;84(3):351-8. 
4. 
Royal College of General P, McCormick A, Fleming D, Charlton J, Great B, Office of 
Population C, et al. Morbidity statistics from general practice : fourth national study 1991-
1992. London: H.M.S.O.; 1995. 
5. 
Finan PH, Buenaver LF, Bounds SC, Hussain S, Park RJ, Haque UJ, et al. 
Discordance between pain and radiographic severity in knee osteoarthritis: findings from 
quantitative sensory testing of central sensitization. Arthritis and rheumatism. 
2013;65(2):363-72. 
6. 
Malfait A-M, Schnitzer TJ. Towards a mechanism-based approach to pain 
management in osteoarthritis. Nature Reviews Rheumatology. 2013;9(11):654. 
7. 
Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity 
by central neural plasticity. The Journal of Pain. 2009;10(9):895-926. 
8. 
Nijs J, Van Oosterwijck J, De Hertogh W. Rehabilitation of chronic whiplash: 
treatment of cervical dysfunctions or chronic pain syndrome? Clinical rheumatology. 
2009;28(3):243-51. 
9. 
Jull G, Sterling M, Kenardy J, Beller E. Does the presence of sensory hypersensitivity 
influence outcomes of physical rehabilitation for chronic whiplash?--A preliminary RCT. Pain 
[Internet]. 2007; 129(1-2):[28-34 pp.]. Available from: 
http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/676/CN-00577676/frame.html. 
10. 
Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. 
Pain. 2011;152(3):S2-S15. 
11. 
Murphy SL, Lyden AK, Phillips K, Clauw DJ, Williams DA. Subgroups of older 
adults with osteoarthritis based upon differing comorbid symptom presentations and potential 
underlying pain mechanisms. Arthritis research & therapy. 2011;13(4):1. 
12. 
French HP, Smart KM, Doyle F, editors. Prevalence of neuropathic pain in knee or hip 
osteoarthritis: a systematic review and meta-analysis. Seminars in arthritis and rheumatism; 
2017: Elsevier. 
13. 
Saunders J, Ding C, Cicuttini F, Jones G. Radiographic osteoarthritis and pain are 
independent predictors of knee cartilage loss: a prospective study. Internal medicine journal. 
2012;42(3):274-80. 
14. 
Ferreira-Valente MA, Pais-Ribeiro JL, Jensen MP. Validity of four pain intensity 
rating scales. PAIN®. 2011;152(10):2399-404. 
15. 
Hjermstad MJ, Fayers PM, Haugen DF, Caraceni A, Hanks GW, Loge JH, et al. 
Studies Comparing Numerical Rating Scales, Verbal Rating Scales, and Visual Analogue 
Scales for Assessment of Pain Intensity in Adults: A Systematic Literature Review. Journal of 
Pain and Symptom Management. 2011;41(6):1073-93. 
16. 
Karcioglu O, Topacoglu H, Dikme O, Dikme O. A systematic review of the pain 
scales in adults: Which to use? The American Journal of Emergency Medicine. 2018. 
17. 
Williamson A, Hoggart B. Pain: a review of three commonly used pain rating scales. 
Journal of Clinical Nursing. 2005;14(7):798-804. 

 
 
 
Page 28 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
18. 
Mawdsley RH, Moran KA, Conniff LA. Reliability of Two Commonly Used Pain 
Scales With Elderly Patients. Journal of Geriatric Physical Therapy. 2002;25(3):16-20. 
19. 
Geber C, Klein T, Azad S, Birklein F, Gierthmühlen J, Huge V, et al. Test–retest and 
interobserver reliability of quantitative sensory testing according to the protocol of the 
German Research Network on Neuropathic Pain (DFNS): a multi-centre study. PAIN®. 
2011;152(3):548-56. 
20. 
Pfau DB, Krumova EK, Treede R-D, Baron R, Toelle T, Birklein F, et al. Quantitative 
sensory testing in the German Research Network on Neuropathic Pain (DFNS): reference data 
for the trunk and application in patients with chronic postherpetic neuralgia. PAIN®. 
2014;155(5):1002-15. 
21. 
Sterling M, Jull G, Vicenzino B, Kenardy J, Darnell R. Physical and psychological 
factors predict outcome following whiplash injury. Pain. 2005;114(1-2):141-8. 
22. 
Backonja MM, Attal N, Baron R, Bouhassira D, Drangholt M, Dyck PJ, et al. Value of 
quantitative sensory testing in neurological and pain disorders: NeuPSIG consensus. Pain. 
2013;154(9):1807-19. 
23. 
Uddin Z, MacDermid JC, Galea V, Gross AR, Pierrynowski MR. The current 
perception threshold test differentiates categories of mechanical neck disorder. Journal of 
Orthopaedic & Sports Physical Therapy. 2014;44(7):532-C1. 
24. 
Cruccu G, Anand P, Attal N, Garcia‐Larrea L, Haanpää M, Jørum E, et al. EFNS 
guidelines on neuropathic pain assessment. European Journal of Neurology. 2004;11(3):153-
62 
25. 
Arendt-Nielsen L, Nie H, Laursen MB, Laursen BS, Madeleine P, Simonsen OH, et al. 
Sensitization in patients with painful knee osteoarthritis. Pain. 2010;149(3):573-81. 
26. 
Imamura M, Imamura ST, Kaziyama HH, Targino RA, Hsing WT, de Souza LP, et al. 
Impact of nervous system hyperalgesia on pain, disability, and quality of life in patients with 
knee osteoarthritis: a controlled analysis. Arthritis and rheumatism. 2008;59(10):1424-31. 
27. 
Lee YC, Lu B, Bathon JM, Haythornthwaite JA, Smith MT, Page GG, et al. Pain 
sensitivity and pain reactivity in osteoarthritis. Arthritis care & research. 2011;63(3):320-7. 
28. 
Neogi T, Frey-Law L, Scholz J, Niu J, Arendt-Nielsen L, Woolf C, et al. Sensitivity 
and sensitisation in relation to pain severity in knee osteoarthritis: trait or state? Ann Rheum 
Dis. 2015;74(4):682-8. 
29. 
Neogi T, Guermazi A, Roemer F, Nevitt MC, Scholz J, Arendt-Nielsen L, et al. 
Association of Joint Inflammation With Pain Sensitization in Knee Osteoarthritis: The 
Multicenter Osteoarthritis Study. Arthritis & rheumatology (Hoboken, NJ). 2016;68(3):654-
61. 
30. 
Wessel J. The reliability and validity of pain threshold measurements in osteoarthritis 
of the knee. Scandinavian journal of rheumatology. 1995;24(4):238-42. 
31. 
Pud D, Granovsky Y, Yarnitsky D. The methodology of experimentally induced 
diffuse noxious inhibitory control (DNIC)-like effect in humans. PAIN®. 2009;144(1):16-9. 
32. 
Yarnitsky D, Arendt-Nielsen L, Bouhassira D, Edwards RR, Fillingim RB, Granot M, 
et al. Recommendations on terminology and practice of psychophysical DNIC testing. 
European Journal of Pain. 2010;14(4):339. 
33. 
Lewis GN, Rice DA, McNair PJ. Conditioned Pain Modulation in Populations With 
Chronic Pain: A Systematic Review and Meta-Analysis. The Journal of Pain. 
2012;13(10):936-44. 
34. 
Chang L. Brain Responses to Visceral and Somatic Stimuli in Irritable Bowel 
Syndrome: a Central Nervous System Disorder? Gastroenterology Clinics of North America. 
2005;34(2):271-9. 

 
 
 
Page 29 of 33 
 
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Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
35. 
Julien N, Goffaux P, Arsenault P, Marchand S. Widespread pain in fibromyalgia is 
related to a deficit of endogenous pain inhibition. Pain. 2005;114(1):295-302. 
36. 
Kosek E, Hansson P. Modulatory influence on somatosensory perception from 
vibration and heterotopic noxious conditioning stimulation (HNCS) in fibromyalgia patients 
and healthy subjects. Pain. 1997;70(1):41-51. 
37. 
Lautenbacher S, Rollman GB. Possible deficiencies of pain modulation in 
fibromyalgia. Clinical Journal of Pain. 1997;13(3):189-96. 
38. 
Maixner W, Fillingim R, Booker D, Sigurdsson A. Sensitivity of patients with painful 
temporomandibular disorders to experimentally evoked pain. Pain. 1995;63(3):341-51. 
39. 
Pielsticker A, Haag G, Zaudig M, Lautenbacher S. Impairment of pain inhibition in 
chronic tension-type headache. Pain. 2005;118(1):215-23. 
40. 
Rossi P, Serrao M, Perrotta A, Pierelli F, Sandrini G, Nappi G. Neurophysiological 
approach to central pain modulation in primary headaches. Journal of Headache and Pain. 
2005;6(4):191-4. 
41. 
Sandrini G, Rossi P, Milanov I, Serrao M, Cecchini AP, Nappi G. Abnormal 
modulatory influence of diffuse noxious inhibitory controls in migraine and chronic tension-
type headache patients. Cephalalgia. 2006;26(7):782-9. 
42. 
Song GH, Venkatraman V, Ho KY, Chee MWL, Yeoh KG, Wilder-Smith CH. 
Cortical effects of anticipation and endogenous modulation of visceral pain assessed by 
functional brain MRI in irritable bowel syndrome patients and healthy controls. Pain. 
2006;126(1):79-90. 
43. 
Edwards RR, Ness TJ, Weigent DA, Fillingim RB. Individual differences in diffuse 
noxious inhibitory controls (DNIC): association with clinical variables. Pain. 
2003;106(3):427-37. 
44. 
Yarnitsky D, Crispel Y, Eisenberg E, Granovsky Y, Ben-Nun A, Sprecher E, et al. 
Prediction of chronic post-operative pain: pre-operative DNIC testing identifies patients at 
risk. Pain. 2008;138(1):22-8. 
45. 
Manresa JAB, Jensen MB, Andersen OK. Introducing the reflex probability maps in 
the quantification of nociceptive withdrawal reflex receptive fields in humans. Journal of 
electromyography and kinesiology : official journal of the International Society of 
Electrophysiological Kinesiology. 2011;21(1):67-76. 
46. 
Soni A, Kiran A, Hart D, Leyland K, Goulston L, Cooper C, et al. Prevalence of 
reported knee pain over twelve years in a community‐based cohort. Arthritis & Rheumatism. 
2012;64(4):1145-52. 
47. 
Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta psychiatrica 
scandinavica. 1983;67(6):361-70. 
48. 
Julian LJ. Measures of anxiety: state‐trait anxiety inventory (STAI), Beck anxiety 
inventory (BAI), and Hospital anxiety and Depression scale‐anxiety (HADS‐A). Arthritis care 
& research. 2011;63(S11):S467-S72. 
49. 
Spinhoven P, Ormel J, Sloekers P, Kempen G, Speckens A, Van Hemert A. A 
validation study of the Hospital Anxiety and Depression Scale (HADS) in different groups of 
Dutch subjects. Psychological medicine. 1997;27(2):363-70. 
50. 
Biondi M, Picardi A. Psychological stress and neuroendocrine function in humans: the  
58. 
Dinan TG, Cryan JF. Regulation of the stress response by the gut microbiota: 
implications for psychoneuroendocrinology. Psychoneuroendocrinology. 2012;37(9):1369-78. 
59. 
Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. Validation study of 
WOMAC: a health status instrument for measuring clinically important patient relevant 

 
 
 
Page 30 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. J 
Rheumatol. 1988;15(12):1833-40. 
60. 
Bombardier C, Melfi CA, Paul J, Green R, Hawker G, Wright J, et al. Comparison of 
a generic and a disease-specific measure of pain and physical function after knee replacement 
surgery. Medical care. 1995;33(4 Suppl):As131-44. 
61. 
Hawker G, Melfi C, Paul J, Green R, Bombardier C. Comparison of a generic (SF-36) 
and a disease specific (WOMAC) (Western Ontario and McMaster Universities Osteoarthritis 
Index) instrument in the measurement of outcomes after knee replacement surgery. J 
Rheumatol. 1995;22(6):1193-6. 
62. 
McConnell S, Kolopack P, Davis AM. The Western Ontario and McMaster 
Universities Osteoarthritis Index (WOMAC): a review of its utility and measurement 
properties. Arthritis care & research. 2001;45(5):453-61. 
63. 
Gill SD, de Morton NA, Mc Burney H. An investigation of the validity of six 
measures of physical function in people awaiting joint replacement surgery of the hip or knee. 
Clinical rehabilitation. 2012;26(10):945-51. 
66. 
Morley JE. Developing novel therapeutic approaches to frailty. Current 
pharmaceutical design. 2009;15(29):3384-95. 
67. 
Theou O, Stathokostas L, Roland KP, Jakobi JM, Patterson C, Vandervoort AA, et al. 
The effectiveness of exercise interventions for the management of frailty: a systematic review. 
Journal of aging research. 2011;2011. 
68. 
Morley JE, Argiles JM, Evans WJ, Bhasin S, Cella D, Deutz NE, et al. Nutritional 
recommendations for the management of sarcopenia. Journal of the american Medical 
Directors association. 2010;11(6):391-6. 
69. 
Srinivas-Shankar U, Roberts SA, Connolly MJ, O'Connell MD, Adams JE, Oldham 
JA, et al. Effects of testosterone on muscle strength, physical function, body composition, and 
quality of life in intermediate-frail and frail elderly men: a randomized, double-blind, 
placebo-controlled study. The Journal of Clinical Endocrinology & Metabolism. 
2010;95(2):639-50. 
70. 
Morley JE, Malmstrom T, Miller D. A simple frailty questionnaire (FRAIL) predicts 
outcomes in middle aged African Americans. The journal of nutrition, health & aging. 
2012;16(7):601-8. 
71. 
Gill S, McBurney H. Reliability of performance-based measures in people awaiting 
joint replacement surgery of the hip or knee. Physiotherapy research international : the journal 
for researchers and clinicians in physical therapy. 2008;13(3):141-52. 
72. 
Jones CJ, Rikli RE, Beam WC. A 30-s chair-stand test as a measure of lower body 
strength in community-residing older adults. Research quarterly for exercise and sport. 
1999;70(2):113-9. 
73. 
Bennell K, Dobson F, Hinman R. Measures of physical performance assessments: 
Self-Paced Walk Test (SPWT), Stair Climb Test (SCT), Six-Minute Walk Test (6MWT), 
Chair Stand Test (CST), Timed Up & Go (TUG), Sock Test, Lift and Carry Test (LCT), and 
Car Task. Arthritis care & research. 2011;63(S11):S350-S70. 
74. 
French HP, Fitzpatrick M, FitzGerald O. Responsiveness of physical function 
outcomes following physiotherapy intervention for osteoarthritis of the knee: an outcome 
comparison study. Physiotherapy. 2011;97(4):302-8. 
75. 
Halket A, Stratford PW, Kennedy DM, Woodhouse LJ. Using hierarchical linear 
modeling to explore predictors of pain after total hip and knee arthroplasty as a consequence 
of osteoarthritis. The Journal of arthroplasty. 2010;25(2):254-62. 

 
 
 
Page 31 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
76. 
Kennedy DM, Hanna SE, Stratford PW, Wessel J, Gollish JD. Preoperative function 
and gender predict pattern of functional recovery after hip and knee arthroplasty. The Journal 
of arthroplasty. 2006;21(4):559-66. 
77. 
Mizner RL, Petterson SC, Clements KE, Zeni JA, Jr., Irrgang JJ, Snyder-Mackler L. 
Measuring functional improvement after total knee arthroplasty requires both performance-
based and patient-report assessments: a longitudinal analysis of outcomes. The Journal of 
arthroplasty. 2011;26(5):728-37. 
78. 
Podsiadlo D, Richardson S. The Timed “Up & Go”: A Test of Basic Functional 
Mobility for Frail Elderly Persons. Journal of the American Geriatrics Society. 
1991;39(2):142-8. 
79. 
Zeni JA, Jr., Axe MJ, Snyder-Mackler L. Clinical predictors of elective total joint 
replacement in persons with end-stage knee osteoarthritis. BMC musculoskeletal disorders. 
2010;11:86. 
108. 
Atukorala I, Kwoh CK, Guermazi A, Roemer FW, Boudreau RM, Hannon MJ, et al. 
Synovitis in knee osteoarthritis: a precursor of disease? Annals of the Rheumatic Diseases. 
2014. 
109. 
de Lange-Brokaar BJ, Ioan-Facsinay A, van Osch GJ, Zuurmond AM, Schoones J, 
Toes RE, et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a 
review. Osteoarthritis Cartilage. 2012;20(12):1484-99. 
110. 
Englund M. The role of the meniscus in osteoarthritis genesis. Rheumatic diseases 
clinics of North America. 2008;34(3):573-9. 
111. 
Loeuille D, Chary-Valckenaere I, Champigneulle J, Rat AC, Toussaint F, Pinzano-
Watrin A, et al. Macroscopic and microscopic features of synovial membrane inflammation in 
the osteoarthritic knee: correlating magnetic resonance imaging findings with disease severity. 
Arthritis and rheumatism. 2005;52(11):3492-501. 
112. 
Attur M, Samuels J, Krasnokutsky S, Abramson SB. Targeting the synovial tissue for 
treating osteoarthritis (OA): where is the evidence? Best practice & research Clinical 
rheumatology. 2010;24(1):71-9. 
113. 
Hayashi D, Roemer FW, Katur A, Felson DT, Yang S-O, Alomran F, et al. Imaging of 
Synovitis in Osteoarthritis: Current Status and Outlook. Seminars in Arthritis and 
Rheumatism. 2011;41(2):116-30. 
114. 
Keen HI, Conaghan PG. Ultrasonography in Osteoarthritis. Radiologic Clinics of 
North America. 2009;47(4):581-94. 
115. 
Guermazi A, Eckstein F, Hellio Le Graverand-Gastineau M-P, Conaghan PG, Burstein 
D, Keen H, et al. Osteoarthritis: Current Role of Imaging. Medical Clinics of North America. 
2009;93(1):101-26. 
116. 
Fernandez-Madrid F, Karvonen RL, Teitge RA, Miller PR, An T, Negendank WG. 
Synovial thickening detected by MR imaging in osteoarthritis of the knee confirmed by 
biopsy as synovitis. Magnetic Resonance Imaging. 1995;13(2):177-83. 
117. 
Hill CL, Gale DG, Chaisson CE, Skinner K, Kazis L, Gale ME, et al. Knee effusions, 
popliteal cysts, and synovial thickening: association with knee pain in osteoarthritis. J 
Rheumatol. 2001;28(6):1330-7. 
118. 
Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: 
latest findings and interpretations. Therapeutic advances in musculoskeletal disease. 
2013;5(2):77-94. 
119. 
D’Agostino MA, Conaghan P, Le Bars M, Baron G, Grassi W, Martin-Mola E, et al. 
EULAR report on the use of ultrasonography in painful knee osteoarthritis. Part 1: Prevalence 
of inflammation in osteoarthritis. Annals of the Rheumatic Diseases. 2005;64(12):1703-9. 

 
 
 
Page 32 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
120. 
Sarmanova A, Hall M, Fernandes GS, Bhattacharya A, Valdes AM, Walsh DA, et al. 
Association between ultrasound-detected synovitis and knee pain: a population-based case-
control study with both cross-sectional and follow-up data. Arthritis research & therapy. 
2017;19(1):281. 
121. 
Sarmanova A, Hall M, Moses J, Doherty M, Zhang W. Synovial changes detected by 
ultrasound in people with knee osteoarthritis – a meta-analysis of observational studies. 
Osteoarthritis and Cartilage. 2016;24(8):1376-83. 
122. 
Walther M, Harms H, Krenn V, Radke S, Faehndrich TP, Gohlke F. Correlation of 
power Doppler sonography with vascularity of the synovial tissue of the knee joint in patients 
with osteoarthritis and rheumatoid arthritis. Arthritis and rheumatism. 2001;44(2):331-8. 
123. 
Labanauskaite G, Sarauskas V. [Correlation of power Doppler sonography with 
vascularity of the synovial tissue]. Medicina (Kaunas, Lithuania). 2003;39(5):480-3. 
124. 
Karim Z, Wakefield RJ, Quinn M, Conaghan PG, Brown AK, Veale DJ, et al. 
Validation and reproducibility of ultrasonography in the detection of synovitis in the knee: a 
comparison with arthroscopy and clinical examination. Arthritis and rheumatism. 
2004;50(2):387-94. 
125. 
Snelling SJB, Bas S, Puskas GJ, Dakin SG, Suva D, Finckh A, et al. Presence of IL-17 
in synovial fluid identifies a potential inflammatory osteoarthritic phenotype. PLOS ONE. 
2017;12(4):e0175109. 
126. 
Berkoff DJ, Miller LE, Block JE. Clinical utility of ultrasound guidance for intra-
articular knee injections: a review. Clinical interventions in aging. 2012;7:89. 
127. 
Haavardsholm EA, Aga A-B, Olsen IC, Lillegraven S, Hammer HB, Uhlig T, et al. 
Ultrasound in management of rheumatoid arthritis: ARCTIC randomised controlled strategy 
trial. Bmj. 2016;354:i4205. 
128. 
Balint PV, Kane D, Hunter J, McInnes IB, Field M, Sturrock RD. Ultrasound guided 
versus conventional joint and soft tissue fluid aspiration in rheumatology practice: a pilot 
study. J Rheumatol. 2002;29(10):2209-13. 
129. 
Sibbitt WL, Kettwich LG, Band PA, Chavez-Chiang NR, DeLea SL, Haseler LJ, et al. 
Does ultrasound guidance improve the outcomes of arthrocentesis and corticosteroid injection 
of the knee? Scandinavian journal of rheumatology. 2012;41(1):66-72. 
130. 
Nazarian LN. The Top 10 Reasons Musculoskeletal Sonography Is an Important 
Complementary or Alternative Technique to MRI. American Journal of Roentgenology. 
2008;190(6):1621-6. 
131. 
Fessell DP, Jacobson JA, Craig J, Habra G, Prasad A, Radliff A, et al. Using 
Sonography to Reveal and Aspirate Joint Effusions. American Journal of Roentgenology. 
2000;174(5):1353-62. 
132. 
Hassan F, Trebinjac S, Murrell WD, Maffulli N. The effectiveness of prolotherapy in 
treating knee osteoarthritis in adults: a systematic review. British medical bulletin. 
2017;122(1):91-108. 
133. 
Palmieri-Smith RM, Thomas AC, Karvonen-Gutierrez C, Sowers MF. Isometric 
Quadriceps Strength in Women with Mild, Moderate, and Severe Knee Osteoarthritis. 
American journal of physical medicine & rehabilitation / Association of Academic 
Physiatrists. 2010;89(7):541-8. 
134. 
Tan J, Balci N, Sepici V, Gener FA. Isokinetic and isometric strength in osteoarthrosis 
of the knee. A comparative study with healthy women. Am J Phys Med Rehabil. 
1995;74(5):364-9. 
135. 
Berry PA, Jones SW, Cicuttini FM, Wluka AE, Maciewicz RA. Temporal relationship 
between serum adipokines, biomarkers of bone and cartilage turnover, and cartilage volume 

 
 
 
Page 33 of 33 
 
Molecular pathways involved in knee pain 
Final Version 4.2 / June 2022/ IRAS: 275727 
 
This protocol is confidential and the property of the University of Nottingham. No part of it may be 
transmitted, reproduced, published, or used by others persons without prior written authorisation 
from the University of Nottingham 
loss in a population with clinical knee osteoarthritis. Arthritis & Rheumatism. 
2011;63(3):700-7. 
136. 
Richette P, Poitou C, Garnero P, Vicaut E, Bouillot J-L, Lacorte J-M, et al. Benefits of 
massive weight loss on symptoms, systemic inflammation and cartilage turnover in obese 
patients with knee osteoarthritis. Annals of the Rheumatic Diseases. 2011;70(1):139-44. 
139. 
Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of 
criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of 
the knee. Arthritis & Rheumatism: Official Journal of the American College of 
Rheumatology. 1986;29(8):1039-49. 
140. 
Thomsen TW, Shen S, Shaffer RW, Setnik GS. Arthrocentesis of the Knee. N Engl J 
Med. 2006;354(19):e19. 
141. 
D'Agostino M-A, Schmidt WA. Ultrasound-guided injections in rheumatology: actual 
knowledge on efficacy and procedures. Best Practice & Research Clinical Rheumatology. 
2013;27(2):283-94. 
142. 
Rosenberg TD, Paulos LE, Parker RD, Coward DB, Scott SM. The forty-five-degree 
posteroanterior flexion weight-bearing radiograph of the knee. The Journal of bone and joint 
surgery American volume. 1988;70(10):1479-83. 
143. 
Kellgren JH, Lawrence JS. Radiological Assessment of Osteo-Arthrosis. Annals of the 
Rheumatic Diseases. 1957;16(4):494-502. 
144. 
Kohn MD, Sassoon AA, Fernando ND. Classifications in Brief: Kellgren-Lawrence 
Classification of Osteoarthritis. Clinical Orthopaedics and Related Research. 
2016;474(8):1886-93. 
145. 
McLean SM, Booth A, Gee M, Salway S, Cobb M, Bhanbhro S, et al. Appointment 
reminder systems are effective but not optimal: results of a systematic review and evidence 
synthesis employing realist principles. Patient preference and adherence. 2016;10:479.