Investigating Pathological Mechanisms in Non-alcoholic Fatty Liver Disease: Cross-sectional Comparative Study Between Patients and Healthy Controls
Conditions: Non-Alcoholic Fatty Liver Disease
Sponsor: Queen Mary University of London

 
 
 
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JRMO Research Protocol for  
Interventional Studies  
 
 
Full Title  
Investigating pathological mechanisms in non-alcoholic fatty liver 
disease: cross-sectional comparative study between patients and 
healthy controls 
 
Short Title Investigating mechanisms in non-alcoholic fatty liver disease 
 
Sponsor 
Queen Mary, University of London  
 
Contact person: 
 
Dr Mays Jawad 
Research & Development Governance 
Operations Manager 
Joint Research Management Office 
 
5 Walden Street 
 
London 
 
E1 2EF 
 
Phone: 020 7882 7275/6574 
 
Email: sponsorsrep@bartshealth.nhs.uk 
 
 
IRAS Number 
247449 
 
Sponsor (ReDA) Number 
012414 
 
REC Reference 
18/LO/1759  
 
Chief Investigator 
Dr William Alazawi 
 
 
Reader in Hepatology 
 
 
 
Barts Health NHS Trust 
Royal London Hospital 
Department of Hepatology 
Whitechapel Road 
London, E1 1BB 
 
Phone: 020 7882 2308 
 
Email: w.alazawi@qmul.ac.uk  
 
List of sites 
 
Homerton University Hospital 
Mr John Loy 
Consultant Bariatric Surgeon 
 
Bariatric Surgery Department 
Homerton University Hospital 

 
 
 
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Homerton Row 
London, E9 6SR 
020 8510 5555 
John.loy1@nhs.net 
 
 
King's College Hospital 
Professor Francesco Rubino 
Consultant Bariatric Surgeon 
 
Department for Bariatric Surgery 
Kings College Hospital 
Denmark Hill 
London 
SE5 9RS 
020 3299 9000 
Francesco.rubino@kcl.ac.uk 
 
 
Bromley by Bow Centre 
Claire Dunne 
Fit For Life Programme Manager 
 
Fit For Life 
Bromley by Bow Centre 
St Leonard’s Street 
London, E3 3BT 
020 8709 9705 
Claire.Dunne@bbbc.org.uk  
 
 
List of laboratories 
Blizard Institute 
Deborah Hampson 
 
Barts & the London School of Medicine & 
Dentistry 
Queen Mary University London 
4 Newark Street 
Whitechapel 
London, E1 2AT 
  
Tel: 020 7882 2289 
d.hampson@qmul.ac.uk  
 
 
 
 
 
 
 
 
 
 

 
 
 
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1. 
Contents 
 
1. 
Contents ......................................................................................................... 3 
2. 
Glossary ......................................................................................................... 5 
3. 
Signature page ............................................................................................... 6 
4. 
Summary and synopsis .................................................................................. 7 
5. 
Introduction ..................................................................................................... 8 
5.1 
Background and Rationale .......................................................................... 8 
5.2 
Risks / benefits .......................................................................................... 10 
6. 
Study objectives ........................................................................................... 12 
6.1 
Primary objective....................................................................................... 12 
6.2 
Secondary objective .................................................................................. 12 
6.3 
Primary endpoint ....................................................................................... 12 
6.4 
Secondary endpoint .................................................................................. 12 
7. 
Study population ........................................................................................... 12 
7.1 
Inclusion criteria ........................................................................................ 13 
7.2 
Exclusion criteria ....................................................................................... 13 
8. 
Study design ................................................................................................. 13 
9. 
Study procedures ......................................................................................... 14 
10. 
Assessment and management of risk ........................................................... 16 
11. 
Statistical considerations .............................................................................. 16 
12. 
Ethics ........................................................................................................... 17 
12.1 Annual Safety Reporting ........................................................................... 17 
13. 
Public Involvement........................................................................................ 17 
14. 
Data handling and record keeping ................................................................ 17 
14.1 Data management ..................................................................................... 17 
14.2 Source data ............................................................................................... 18 
14.3 Confidentiality ........................................................................................... 18 

 
 
 
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15. 
Laboratories ................................................................................................. 18 
15.1 Central laboratory...................................................................................... 18 
15.2 Sample collection and preparation ............................................................ 18 
15.3 Laboratory procedures .............................................................................. 19 
15.4 Sample storage and transfer ..................................................................... 19 
16. 
Interventions and tools .................................................................................. 20 
16.1 Devices ..................................................................................................... 20 
16.2 Techniques and interventions ................................................................... 20 
16.3 Tools ......................................................................................................... 20 
17. 
Safety reporting ............................................................................................ 21 
17.1 Adverse Events (AEs) ............................................................................... 21 
17.2 Adverse Reaction (ARs) ............................................................................ 21 
17.3 Notification and reporting of Adverse Events and Reactions ..................... 21 
17.4 Serious Adverse Events (SAEs) or reactions ............................................ 21 
17.5 Notification and reporting of Serious Adverse Events ................................ 21 
17.6 Urgent Safety Measures ............................................................................ 22 
17.7 Annual Safety Reporting ........................................................................... 22 
17.8 Overview of the Safety Reporting responsibilities ...................................... 22 
18. 
Monitoring and auditing ................................................................................ 22 
19. 
Trial committees ........................................................................................... 23 
20. 
Finance and funding ..................................................................................... 23 
21. 
Indemnity ...................................................................................................... 23 
22. 
Dissemination of research findings ............................................................... 23 
23. 
References ................................................................................................... 24 
 
 
 
 

 
 
 
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2. 
Glossary 
 
 
AE 
Adverse Event 
 
AR 
Adverse Reaction 
 
ASR 
Annual Safety Report 
 
BMI 
Body Mass Index 
 
CAP 
Controlled Attenuation Parameter 
 
CI 
Chief Investigator 
 
CRF 
Case Report Form 
 
CyTOF 
Mass Cytometry 
 
FNA 
Fine Needle Aspirate 
 
GLP-1 
Glucagon Like Peptide 1 
 
HOMA-IR 
Homeostatic Model Assessment of Insulin Resistance 
 
HRA 
Health Research Authority 
 
JRMO 
Joint Research Management Office 
 
NAFLD  
Non-alcoholic fatty liver disease 
 
NASH 
Non-alcoholic Steatohepatitis 
 
NIHR 
National Institute for Health Research 
 
Participant 
An individual who takes part in a clinical trial 
 
PI 
Principle Investigator 
 
PIS 
Patient Information Sheet 
 
REC 
Research Ethics Committee 
 
T2DM  
Type 2 Diabetes Mellitus 
 

 
 
 
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3. Signature page 
 
 
 
Chief Investigator Agreement 
 
The study as detailed within this research protocol will be conducted in accordance 
with the principles of Good Clinical Practice, the UK Policy Framework for Health and 
Social Care Research, and the Declaration of Helsinki and any other applicable 
regulations. I agree to take responsibility for the statistical analysis and oversight of 
this study. 
 
 
Chief Investigator Name:    Dr William Alazawi 
 
 
Signature: 
 
 
 
Date: 
04/09/2018 
 
 
 

 
 
 
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4. Summary and synopsis 
 
Short title
Investigating mechanisms in non-alcoholic fatty liver 
disease 
Methodology
Cross-sectional study 
 
Research sites
Barts Health NHS Trust,  
Homerton University Hospital NHS Foundation Trust 
Kings College Hospital, NHS Foundation Trust 
Bromley by Bow Centre 
Objectives / aims
To identify key characteristics of the tissue resident and 
peripherally circulating immune-phenotype in addition to 
blood markers, metabolic profile,  faecal and oral microbiota 
in non-alcoholic fatty liver disease within an ethnically 
diverse population 
Number of 
participants 
153 
Inclusion and 
exclusion criteria 
Over 18 years of age 
Confirmed Non-alcoholic Fatty Liver disease or Healthy 
Control 
 
Excluding participants with type 1 Diabetes Mellitus or 
immune-suppressant concomitant medication or auto-
immune disease or excessive alcohol intake 
Statistical 
methodology and 
analysis  
Comparative and descriptive 
Study duration
24 months 
 

 
 
 
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5. Introduction 
 
 
5.1 Background and Rationale 
 
 
Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality 
worldwide.  NAFLD is the hepatic manifestation of the metabolic syndrome and is 
strongly associated with type 2 diabetes mellitus, obesity, hypertension and 
hypercholesterolaemia. It affects 20-30% of the general population, 58% of 
overweight individuals, 70% of diabetics and almost 90% of obese individuals. 1.  
 
NAFLD reflects a spectrum of disease from simple steatosis without inflammation and 
12-20% of individuals with NAFLD who develop the aggressive form of disease, non-
alcoholic steatohepatitis (NASH).  This is characterised by liver cell death, 
inflammation and fibrosis which can lead to cirrhosis, liver failure and liver cancer2. It 
is recognised that NASH with fibrosis confers increased all-cause and liver-related 
mortality over simple steatosis or NASH without fibrosis.2–4 2 Cardiovascular disease 
dominates non-liver related mortality and this risk appears to be independent to the 
presence of metabolic syndrome. 
 
Classically NAFLD is associated with an elevated Body Mass Index (BMI), a well-
recognised measure of obesity. However, studies have identified that people of south 
Asian ethnicity develop NAFLD with a BMI outside of the ‘obese’ range. 5,6 Factors 
possibly involved in this observed phenomenon includes preponderance for insulin 
resistance 7 and a distinct genetic profile. 8 
 
Apart from performing a liver biopsy, there is no convenient or accurate way to 
distinguish patients who have NASH from those who have simple steatosis which 
does not progress. This is largely because the pathogenesis of NASH is not fully 
understood. Hepatic fat deposition, genetic abnormalities, oxidative stress, 
inflammatory cytokines and more recently bacterial dysbiosis have all been shown to 
be relevant, but, the mechanisms that link these phenomena to NASH have not been 
elucidated 9,10.For the same reasons, there are currently no drugs licensed for the 
treatment of NASH and prevention of its complications. Therefore the cornerstone of 
treatment is behaviour and lifestyle change, but adherence to such changes is limited 
and effect on long term outcomes has not been demonstrated. 11,12  However, public 
health measures such as education and legislation will take many years to impact on 
the burden of established liver disease in the population.  Therefore, there is a large 
unmet clinical need for drugs to target the pathogenic processes that drive 
inflammatory liver diseases. 
 
Therefore, what is needed is a better understanding of the mechanisms that are 
associated with NASH in order to develop effective biomarkers of disease stage and 
therapeutic targets.  In recent years, it has become clear that NASH is driven by a 
combination of metabolic dysfunction and an aberrant immuno-inflammatory 
response resulting in liver cell injury, and fibrosis. In a multisystem disease such as 
NASH, many tissues are affected and a comprehensive understanding of the disease 

 
 
 
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cannot take place without simultaneously studying the different tissues involved, 
including liver, fat tissue, muscle and blood.  Historically such co-ordinated tissue 
assessment has been very difficult, but the recent rise to prominence of bariatric 
surgery as a metabolic treatment intervention changes that. 
 
Bariatric surgery (increasingly referred to as ‘metabolic surgery’) has wide-ranging 
benefits on insulin sensitivity, body weight and gut hormone profile such as glucagon 
like peptide-1. 13,14 Bariatric surgery is safe with morbidity and mortality comparable to 
hip-replacement, cholecystectomy and appendicectomy. 15 Cohort studies and case 
series have shown benefits in both liver biochemistry and histology. 16–18 The Lille 
bariatric cohort demonstrated resolution of NASH in 85% of patients with significant 
reduction in steatosis fibrosis. 19 It is well recognised that there is marked 
improvement in insulin sensitivity within days post bariatric surgery driven particularly 
driven by reduced basal glucose production  and increased hepatic insulin 
sensitivity.20 Furthermore, immediate and dramatic changes in gut hormones such as 
glucagon-like peptide 1 (GLP-1) increases pancreatic insulin secretion in response to 
glucose and reduced glucagon production contributing to attenuation of the metabolic 
syndrome phenotype.21,22 Prior to bariatric procedures patient undergo two to three 
weeks of a low calorie diet which has been shown to reduce liver and intra-abdominal 
fat volume.23,24 It has been suggested this improves visibility intra-operatively, 
moreover, has been shown to decrease 30 day post-operative morbidity and 
complications.25 We aim to evaluate the impact of this intervention and bariatric 
surgery upon serological markers and the peripheral immunophenotype. 
 
Although only 1% of patients eligible for such operations are currently offered 
surgery26 the prominence of metabolic surgery in international guidelines for patients 
with obesity, diabetes and complications means that these numbers are rising and 
expected to rise further.  This provides an opportunity for mechanistic research such 
as ours. 
 
For patients with NAFLD whom bariatric procedures are not appropriate or not under 
the care of bariatric services the cornerstone of management is behaviour and 
lifestyle change, focussing on weight loss and exercise 12,27,28.  Weight loss reduces 
fatty acid supply to the liver, improves insulin sensitivity, and reduces circulating 
inflammatory cytokines which may be mediated by a change in the inflammatory and 
metabolic states of adipose tissue.29   
 
A previous meta-analysis has revealed weight loss improves steatosis and cardio-
metabolic risk factors including HOMA-IR, lipid profile and glycated haemoglobin 
(HbA1c).30 More recently, a cohort study of 293 NASH patients encouraged to adopt 
lifestyle changes over 52 weeks showed a dose-response between weight loss and 
all histological parameters of NASH with benefit seen with weight loss greater than 
5%.12  
 
In this study, we will collect tissue, blood, stool, urine and saliva samples at the same 
time in order to enable a comprehensive disease phenotyping and ability to compare 
immunological, microbiological and metabolic features in patients with varying stages 
of NAFLD and healthy controls. To date there are no published data evaluating 
simultaneously-obtained samples from adipose, gut and liver tissue and peripheral 
blood in NAFLD. The important regulatory role of adipose tissue in systemic 
inflammation has been highlighted previously and demonstrated a hyperactive state 

 
 
 
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in obesity. 31,32. Therefore, a significant proportion of the participants in this study will 
be recruited from the bariatric surgery services so that these multiple tissue samples 
can be collected at time of surgery with minimal additional risk to participants.  
 
The largest cohort of paired liver biopsies monitoring the effects of weight loss 
through lifestyle modification revealed that a significantly greater proportion of 
individuals had histological resolution of NASH if they lost greater than 5% of body 
weight.12 Insulin resistance is a key component to NAFLD, therefore, the dramatic 
impact upon hepatic insulin resistance and gut hormones seen post bariatric surgery 
may display and very different pathological phenotype compared the same amount of 
weight loss attained through lifestyle changes. This study will enable evaluation of 
these immunological, microbiological and metabolic features of NAFLD at baseline 
and in participants who have lost at least 5% of body weight by lifestyle interventions 
versus a bariatric surgery. This comparison may reveal unique insight into the 
pathological mechanisms in NAFLD altered by the rapid improvement of the 
metabolic syndrome seen after bariatric surgery independent of the amount of weight 
loss. 
 
The potential benefits of this study will include identifying components which could aid 
disease staging; reducing the need for invasive liver biopsy but also identify those 
individuals at risk of disease progression with associated elevated all-cause mortality. 
Collecting samples from the ethnically diverse population of East and South London 
will enable us to evaluate risk factors and pathological characteristics which may vary 
between ethnic groups. Furthermore, a greater understanding of the peripheral and 
tissue resident inflammatory responses could highlight potential therapeutic targets 
for future drug development. 
 
 
 
5.2 Risks / benefits  
 
We do not expect any significant additional risk to participants within this study. Blood 
samples would amount to no more than 55mls in total, substantially less than taken 
at time of a blood donation, which amounts to 400mls. Tissue biopsies will only be 
collected from participants undergoing a planned procedure such as a liver biopsy for 
existing clinical purposes or gastrointestinal surgery. A large review of the safety of 
liver 3806 biopsies without direct visualisation but guided by ultrasound revealed a 
low rate of haemorrhagic complication 0.32% or hypovolaemic shock 0.11%. 33  
Furthermore, the Lille bariatric cohort did not report any significant risks associated 
with the 1496 intra-operative liver biopsies performed within their study.19 Tissue 
samples taken at time of surgery have lower risk than percutaneous procedures as 
they are performed under direct vision of the operating surgeon with ability to ensure 
haemostasis post biopsy.  
 
Participants will have an optional fine needle aspiration (FNA) of their liver at 
baseline and at follow up, once attaining 5%-7% weight loss. This procedure has 
been in use for more than 20 years as a research and clinical tool with better safety 
profile than standard liver biopsy.34 A recent study revealed safe repeated fine needle 
aspiration from the same study participants with the only adverse event reported as 
pain post procedure requiring simple analgesia in two cases after 39 fine needle 
aspirates.35 Furthermore, another research group has reported no serious 
complications in a series of more than 500 liver FNA procedures.36  
 

 
 
 
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As part of this study if participants identified to have evidence of the more aggressive 
form of fatty liver disease, NASH, other liver abnormality, or signs of hepatic fibrosis 
they would be referred for ongoing monitoring and management and benefit from 
specialist advice from the Hepatology clinics at the Royal London Hospital or King’s 
College Hospital.  

 
 
 
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6. Study objectives 
 
6.1 Primary objective 
 
To determine the changes in tissue-specific and peripheral immuno-inflammatory 
and metabolic abnormalities in NALFD in people who achieve 5% to 7% weight 
loss by Roux-en Y gastric bypass surgery or lifestyle measures. 
 
 
6.2 Secondary objective 
 
1.  
Understand the association between variations in immune-profiling and existing 
markers of disease activity 
2.  
Identify which markers predict disease progression / regression after 5% to 7%  
weight loss 
 
 
6.3 Primary endpoint 
 
To determine differences between specific immune cell populations in peripheral 
blood, liver and adipose tissue between two methods of 5% to 7%  weight loss. 
 
 
6.4 Secondary endpoint 
 
1.  Expression of key immune system markers (genes and proteins) and immune cell 
population frequency and activation status 
2.  Number and type of bacterial species in stool and mouth samples. 
3.  Severity of NAFLD - based on histology and Fibroscan (transient elastography)  
4.   In a subset of patients, non-invasive assessment of liver function and metabolic 
control once 5% to 7%  weight loss is achieved 
5. Measure of quality of life at baseline and once achieved 5% to 7%  weight loss 
6. Healthcare utilization; populated from healthcare records 
 
 
7. Study population 
 
Participants will be identified from outpatient clinics of Bariatric, Gastroenterology, 
and Hepatology services at Homerton and King College Hospitals in addition to 
Gastroenterology outpatients from the Barts Health NHS Trust. An additional cohort 
will be recruited from the community weight loss program, Fit For Life, run in Tower 
Hamlets and Waltham Forest at the Bromley by Bow Centre.  
 
Healthy control participants will be identified from patients undergoing outpatient 
assessment, gastroscopy or surgery for other non-malignant gastrointestinal 
disorders at hospital sites. Participants will have the opportunity to discuss the study 
with a member of the research team when they receive a copy of the patient 
information sheet. Only adults 18 years of age and above and not classified as a 
vulnerable adult will be recruited. Most participants will only need to attend one or 
two study visits including consent. 

 
 
 
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7.1 Inclusion criteria 
 
 
Age ≥18 years 
 
 
Confirmed non-alcoholic Fatty liver disease (diagnosed clinically, 
radiologically or histologically) 
 
 
If diabetic, Diagnosed with Type 2 Diabetes Mellitus 
 
 
OR 
 
Healthy Control: no diagnosis of any liver condition including NAFLD 
o NAFLD excluded by Fibroscan Controlled Attenuation Parameter 
(CAP) score of <222 dB/m 
 
 
 
7.2 Exclusion criteria 
 
 
Unwilling or unable to give informed consent 
 
Type 1 Diabetes Mellitus 
 
Other form of liver disease (other than NAFLD) 
 
o Viral hepatitis, Auto-immune hepatitis, primary sclerosing cholangitis, 
primary biliary cholangitis, haemochromatosis, Sarcoidosis, cystic 
fibrosis, sickle cell disease  
 
Taking medication associated with liver dysfunction (except methotrexate) 
 
Auto-immune disease which in the investigator’s opinion may confound 
immune profiling 
 
Concomitant immunosuppressive medications (except methotrexate, short 
course oral steroids or inhaled corticosteroids) 
 
Currently pregnant 
 
Any major organ transplant (excluding corneal or hair transplant) 
 
Regular alcohol intake greater than 14 units a week for female participants 
and 21 units a week for male participants 
 
 
8. Study design 
 
This 
observational 
study 
will 
invite 
individuals 
from 
hospital 
outpatients 
(Gastroenterology and Bariatric services), community weight loss program or 
academic institutions associated with hospital sites to form the NAFLD and healthy 
control participants respectively. We plan to recruit 153 participants over an expected 
24 month period. Opening this study at two bariatric surgery services we would 
expect to meet recruitment targets. However, if opportunistic recruitment does not 
meet expectations we will collaborate with other centres with whom we have existing 
relations (Imperial College and University College, London).  
 
Participants will be approached at routine clinic appointments if pre-screened to meet 
study inclusion criteria. Community participants will have a short presentation at the 
beginning of their weight loss program where the study will be explained by one of 

 
 
 
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the research team with the opportunity to ask questions. All potential participants will 
be given a copy of the PIS at this visit. A combined screening/first study visit will then 
be arranged, usually by telephone or e-mail.  Following consent, bariatric surgery 
participants or healthy control participants undergoing elective abdominal surgery will 
have a second study visit defined as the day of surgery for tissue sample collection. 
Patients will be asked to monitor their weight and when they reach 95% of starting 
weight, they will be asked to contact the study team to arrange a visit.  All 
participants will have a final study visit once they have attained 5% to 7%  weight 
loss, this is expected to be between one to six months after baseline visit. 
Participants who do not achieve at least 5% weight loss 6 months after the baseline 
visit will also be asked to attend the visit. 
 
Healthy control patients will be identified from patients undergoing outpatient 
assessment, gastroscopy or elective surgery for other non-malignant gastrointestinal 
disorders at NHS organisations associated with the study; Kings College Hospital, 
Barts Health NHS Trust and Homerton University Hospital.  
 
In order to exclude significant hepatic steatosis in healthy control participants and 
ensure study eligibility we will utilise the Controlled Attenuation Parameter (CAP) 
function of the Fibroscan. This is particularly important due to the recognised poor 
negative predictive value of liver ultrasound. 37 This will be carried out at the 
participant’s screening visit after informed consent.  
 
There is range of published cut-off values to determine the presence of hepatic 
steatosis (215 dB/m- 268 dB/m).37–40 For the purposes of our study we have utilised 
the data from a recent publication comparing CAP score in 349 individuals with 
NAFLD and 437 healthy controls as determined by proton-magnetic resonance 
spectroscopy.37 At the cut-off of 222 dB/m, the sensitivity, specificity, positive 
predictive value and negative predictive value of CAP, for diagnosing NAFLD, were 
93.1%, 45.1%, 57.5% and 89.1%, respectively.37 This indicates it to be an 
appropriate cut-off for our study. 
 
 
 
9. Study procedures 
 
At the first study visit a researcher will obtain informed consent and determine that 
the participant meets all study inclusion and exclusion criteria to proceed. Consent 
will include agreeing to access NHS databases for medical information and access to 
existing tissue samples surplus to clinical requirements. The study researcher will 
guide the participant through the consent form and case report form (CRF).  This will 
also incorporate the Short Form (36) Health Survey (SF-36) as a measure of 
participant self-reported health status. This is in addition to medical and drug history 
in addition to smoking, alcohol, dietary and exercise data. The same assessment will 
take place at the final study visit, once they have achieved 5% to 7%  weight loss.  
 
 
 
 
 
 
 

 
 
 
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Data collection will include: 
1. History and examination: family history, smoking and drinking habits, past 
medical history, drug history, demographics (race, gender, age) and life-style 
(quality of life, dietary, exercise and sleeping habits). 
2. Anthropometric measurement including body weight, body height, waist 
circumference and Body Mass Index (BMI). 
3. Transient elastography (Fibroscan) 
4. Fasted clinical blood tests including liver function and metabolic profile.  
5. Fasted research blood tests - 55mls (3 tablespoons) extra.   
6. Urine, saliva and stool samples 
7. Optional fine needle aspiration of the liver 
 
Every effort will be made to arrange all study procedures at each time point in a 
single visit, however, if this is not possible, participants have the option to return at an 
alternative date for that component / those components.  
 
If a participant is undergoing bariatric or upper gastrointestinal surgery for a non-
malignant indication, a study investigator or research nurse will attend the day of their 
surgery and collect the tissue samples: liver and adipose tissue and gut tissue (if 
their operation includes bowel resection) having consented previously. All 
participants with NAFLD will be offered a follow up visit once they have achieved 5% 
to 7%  weight loss with repeat assessment of immune-profile and liver specific tests. 
Additional consent pertaining to fine needle liver aspirate will be obtained 
immediately prior to this procedure. The standard NHS consent form will be used for 
this. 
 
Study procedures by visit 
 
Study intervention  
Visit 1 
Visit 2 
(Surgical & biopsy 
participants only) 
Visit 3 
(5%-7% weight 
loss achieved) 
Informed Consent 
X 
 
 
Screening 
X 
 
 
Medical history 
X
X
X
Questionnaires 
X
 
X
Blood sample 
X
X 
X
Urine, stool, saliva samples 
X 
X 
X 
Liver Fibroscan 
X 
 
X 
Liver Fine Needle Aspiration 
(Optional) 
X 
 
X 
Liver Biopsy sample 
(If already clinically indicated) 
 
X 
 
Intra-abdominal tissue sampling  
(At time of planned surgery) 
 
X 
 
 
If the study procedures identify a significant liver abnormality, elevated liver function 
tests or elevated Fibroscan score. We will ensure the participant is informed by one 
of the study investigators and referred for further investigation and management 
within a liver clinic at either Barts Health or Kings College Hospital. 
 
If a participant wishes to fully, or partially, withdraw from the study, they will be asked 
if they wish to have existing data and samples to be destroyed or whether they can 
be still used for the purposes of the study. Withdrawal of consent will be documented 
in the participants CRF. 

 
 
 
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10. Assessment and management of risk 
 
We do not expect any significant additional risk to participants within this study. Blood 
samples would amount to no more than 55mls in total, substantially less than taken 
at time of a blood donation, approximately 400mls. Fresh tissue samples will only be 
collected from participants undergoing a planned procedure such as a liver biopsy for 
existing clinical purposes or bariatric surgery. A large review of the safety of liver 
3806 biopsies without direct visualisation but guided by ultrasound revealed an 
exceptionally low rate of haemorrhagic complication 0.32% or hypovolaemic shock 
0.11%. 33  Furthermore, the Lille bariatric cohort did not report any significant risks 
associated with the 1496 intra-operative liver biopsies performed within their study. 19 
Tissue samples taken at time of surgery have minimal risk as they are performed 
under direct vision of the operating surgeon with ability to ensure haemostasis post 
biopsy.  
 
Participants will have an optional fine needle aspiration of their liver at baseline and 
at follow up, once attaining 5% to 7%  weight loss. This procedure has been in use 
for more than 20 years as a research and clinical tool with better safety profile than 
standard liver biopsy.34 A recent study revealed safe repeated fine needle aspiration 
from the same study participants with the only adverse event reported as pain post 
procedure requiring simple analgesia in two cases after 39 fine needle aspirates.35 
Furthermore, another research group has reported no serious complications in a 
series of more than 500 fine needle liver aspirates.36 In this study, liver FNA will be 
only undertaken by individuals with adequate prior experience to minimise risk to 
participants.  
 
We expect small risk to study researchers undertaking analysis of biological samples 
from this study. Standard mitigating precautions including personal protective 
equipment, sealed containers opened only in fume hoods within containment level 2 
laboratory facilities will be adhered to. Further information can be found in section 15 
regarding laboratories. All research staff dealing with samples will have to 
demonstrate up to date safety training either from their employing NHS trust or 
academic institution. 
 
11. Statistical considerations 
 
Patients undergoing assessment for bariatric surgery will predominantly be classified 
as obese, BMI >30Kg/m2 (>27.5Kg/m2 for South Asian patients) or more likely 
severely obese, BMI greater than 35 Kg/m2. It is recognised that up to 90% of obese 
individuals will have concurrent NAFLD.1 A recent meta-analysis reports the 
incidence of NASH among NAFLD patients to be 59.1%41 although in a bariatric 
cohort with more risk factors, a higher proportion is expected. A previous NAFLD 
study in 109 predominantly Caucasian bariatric patients revealed that almost 90% of 
patients undergoing bariatric surgery had evidence of NASH with at least stage 1 (out 
of 4) fibrosis 19 . 
 
In this exploratory, basic science observational project, formal power calculations are 
not possible.  However similar work by our group suggests that 40 patients in each of 
the following groups: NASH with mild fibrosis (stages 0-1) and significant fibrosis 
(stages 2-4) will be informative.  Given that we cannot determine the stage of disease 
before surgery, and assuming a conservative NASH rate of 75% of all patients and a 

 
 
 
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relatively even split of patients into the two categories and 20 healthy control 
participants we estimate that a total sample size of 153 will enable us to recruit with 
20% contingency for dropout.  
 
Techniques involved in analysis of biological samples will include flow cytometry, 
quantitative polymerase chain reaction, RNA sequencing of immune cell populations, 
16S 
ribosomal 
RNA 
sequencing 
of 
the 
microbiome 
and 
enzyme-linked 
immunosorbent assays on serum or plasma samples. 
 
The data will be analysed using widely used statistical packages Graphpad Prism 
and Statistical Package for the Social Sciences (SPSS) utilising descriptive statistical 
tests. Specific techniques may require additional software such as CytoBank for 
CyTOF data and HistoCAT for imaging mass cytometry.  
 
 
12. Ethics 
 
The Principal Investigator must ensure that the study will be carried out in 
accordance with the ethical principles in the Research Governance Framework for 
Health and Social Care, Second Edition, 2005 and its subsequent amendments as 
applicable and applicable legal and regulatory requirements.  
 
None of the Investigators declare any conflicts of interest with regards to this study. 
 
 
12.1 
Annual Safety Reporting  
 
The CI will send an Annual Progress Report to the REC and the sponsor using the 
HRA template on the anniversary of the REC “favourable opinion”. 
 
13. Public Involvement 
 
The work of the group has been discussed with the North thames Diabetes lay Panel 
and our results will be reported back to them at their quarterly meetings.   
 
 
14. Data handling and record keeping 
 
14.1 Data management 
 
Data pertaining to study participants will be recorded directly recorded into CRF 
which in turn would be transcribed into an anonymised electronic excel database 
stored as per the conditions below. 
 
- Confidentiality 
Personal data will be stored in password-protected formats on hospital 
networks.  We will follow local Trust guidelines on the storage and transfer of 

 
 
 
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data and all databases / spreadsheets will be compliant with the Data 
Protection Act, NHS Caldecott Principles, The Research Governance 
Framework for Health and Social Care, and the conditions of Research Ethics 
Committee Approval. 
 
- Record Retention and Archiving 
Data will be stored in codified, password-protected formats. We will follow 
local Trust guidelines on the storage and transfer of data and databases / 
spreadsheets will be Caldicott compliant. The UK Policy Framework for 
Health and Social Care Research requires that research records are kept for 
20 years after the project has completed and will be archived in the Trust 
Corporate Records Centre.  
 
 
14.2 Source data 
 
Source data will include the CRF incorporating the questionnaires completed at time 
of first visit in addition to participant’s hospital medical records, NHS databases and 
General Practitioner records (if consent given to access these). The total 
accumulated data will be collated in an anonymised electronic database. 
 
 
14.3 Confidentiality 
 
Information related to participants should be kept confidential and managed in 
accordance with the Data Protection Act, the General Data Protection Regulation 
(GDPR), NHS Caldecott Principles, the UK Policy Framework for Health and Social 
Care Research, and the conditions of Research Ethics Committee favourable 
opinion. Only data outlined in the protocol will be collected from study participants 
and anonymised within the study database. Only members of the research team will 
have access to healthcare records and source documents. 
 
 
15. Laboratories  
 
15.1 Central laboratory 
Recipient Scientist:  Dr William Alazawi, Queen Mary University of London 
 
- 
 
 
 
15.2 Sample collection and preparation 
 
Research samples will be collected at all study sites pseudo-anonymised and 
labelled with the patient study number. Material transfer agreements will be arranged 
with each study site with regards to the transfer of participant samples. All sample 
processing will occur at the primary site run by the chief investigator. 
 
 

 
 
 
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15.3 Laboratory procedures  
 
These may include flow cytometry, mass cytometry (CyTOF), imaging mass 
cytometry, tissue culture, nucleic acid and protein detection, mass spectrometry, 
bacterial sequencing and identification, metabolic profiling. 
 
 
Working with human tissue products (blood, tumour tissue, urine, stool, saliva, 
plasma, serum) has inherent risks; tissue, whether screened or unscreened, can 
carry infectious agents and so will require careful handling.  
 
All researchers planning to work with human tissue must ensure that they: 
 
Are trained in all necessary procedures.  
 
Are vaccinated against Hepatitis B. 
 
PPE (gloves, lab coat and eye protection) is worn. 
 
Study personnel must: 
 
 
Avoid the use of sharps, but if unavoidable, follow safe sharps practice; If 
possible, no sharps should be used when handling blood samples that have 
been collected elsewhere.  
 
Where possible, process samples in a Safety Cabinet.  
 
Only work in designated areas  
 
All other work with samples should be carried out in at Containment Level 2  
 
 
 
 
15.4 Sample storage and transfer 
 
All human tissue/product samples must be separately transferred or stored in 
watertight plastic containers and labelled appropriately (contents, date collected, 
name of researcher and a biohazard label). The laboratory is equipped with a spill 
kits for infectious material. 
 
The triple packaging system described by the WHO must be used for all infectious 
substances (Category A and B), exempt substances and non-infectious GM 
substances.  
 
This consists of three layers as follows: 
 
Primary receptacle: water-tight and leak proof with enough absorbent material to 
absorb all fluid in the event of a breakage. 
 
Secondary receptacle: a durable, water-tight and leak-proof packaging to enclose 
and protect the primary receptacle. Several cushioned primary receptacles may be 
placed in one secondary package but sufficient absorbent material should be used to 
absorb all fluid in a breakage. Goods of an unrelated type must not be packed 
together. 
 
Outer packaging: secondary receptacles are placed in a durable outer packaging 
with suitable cushioning material to protect from outside influences. 

 
 
 
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Samples will be stored in either minus 80 freeze or liquid nitrogen storage to 
minimise degradation of participant samples. Only laboratory personnel trained in the 
safe use of these facilities will be permitted to undertake the storage of samples 
received. 
 
All biological fluids or cells/tissue suspended in fluid which requires disposal will be 
treated with 10% distel for 24 hours before disposal down laboratory drain. All 
laboratory equipment used will undergo de-sterilisation by autoclaving or, if 
disposable, placed in clinical waste bags for appropriate disposal. 
 
We do not expect to have sample left over, but if there is, this will be transferred to 
the Digestive Diseases Tissue Bank 
 
 
16.  Interventions and tools  
 
16.1 Devices 
 
Fibroscan – transient elastography device. Manufactured by Echosens 
 
Operated by individuals who have completed the certified training provided by 
manufacturer. 
 
Owned by Site: who has responsibility for appropriate device maintenance under 
contract with Echosens. 
 
 
 
16.2 Techniques and interventions 
 
Percutaneous liver biopsy – performed under ultrasound guidance as per Barts 
Health standard operating procedures 
 
Intra-operative tissue sample collection. Under direct vision of operating surgeon 
with biopsy equipment with ability to achieve post biopsy haemostasis 
utilising diathermy electrocoagulation. 
 
 
16.3 Tools 
 
Patient Questionnaires 
Short Form (36) Health Survey (SF-36 ), measure of self-reported health status  
 
 
 
 

 
 
 
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17. Safety reporting  
 
17.1 Adverse Events (AEs) 
 
An AE is any untoward medical occurrence in a participant to whom an intervention 
has been administered, including occurrences which are not necessarily caused by 
or related to that intervention. An AE can therefore be any unfavourable or 
unintended sign (including an abnormal laboratory finding), symptom or disease 
temporally associated with study activities. 
 
 
17.2 Adverse Reaction (ARs)  
 
An AR is any untoward and unintended response in a participant to an intervention.  
All adverse events judged by either the reporting investigator or the sponsor as 
having a reasonable causal relationship to the intervention qualify as adverse 
reactions. The expression ‘reasonable causal relationship’ means in general that 
there is evidence or an argument to suggest a causal relationship. 
 
 
17.3 Notification and reporting of Adverse Events and Reactions 
 
If the AE is not defined as serious, the AE will be recorded in the study documents 
and the participant followed up by the research team. The AE will be documented in 
the participants’ source documents, the Case Report Form (CRF), and, where 
appropriate, medical records. 
 
 
17.4 Serious Adverse Events (SAEs) or reactions 
 
A serious adverse event (SAE) is defined as an untoward occurrence that: 
 
Results in death, 
 
Is life-threatening, 
 
Requires hospitalisation or prolongation of existing hospitalisation, 
 
Results in persistent or significant disability or incapacity, 
 
Consists of a congenital anomaly or birth defect, or 
 
Is otherwise considered medically significant by the investigator. 
 
SARs will be reported to the REC where in the opinion of the Chief Investigator the 
event was serious and: 
 
Related (it may have resulted from administration of any of the research 
interventions), and 
 
Unexpected (the type of event is not listed in the protocol or other Reference 
Safety Information as an expected occurrence). 
 
 
17.5 Notification and reporting of Serious Adverse Events  
 

 
 
 
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Serious Adverse Events (SAEs) that are considered to be ‘related’ and ‘unexpected’ 
will be reported to the sponsor within 24 hours of learning of the event, and to the 
REC within 15 days in line with the required timeframe. Host sites will reports SAEs 
to the CI via NHS email or direct telephone contact to ensure data security. The SAE 
maybe reported to the PI at local sites who will directly notify the CI within the 24 
hour timeframe. 
 
17.6 Urgent Safety Measures 
 
The CI will take urgent safety measures if necessary to ensure the safety and 
protection of the clinical study participant from immediate hazards to their health and 
safety. The measures will be taken immediately. The approval of the REC prior to 
implementing urgent safety measures is not required. However the CI will inform the 
sponsor and Research Ethics Committee (via telephone) of this event immediately.  
 
The CI will inform the REC in writing within 3 days, in the form of a substantial 
amendment. The sponsor (Joint Research Management Office (JRMO)) will be sent 
a copy of the correspondence with regards to this matter.  
 
 
17.7 Annual Safety Reporting 
 
The CI will send the Annual Progress Report to the REC using the HRA template (the 
anniversary date is the date on the REC “favourable opinion” letter) and to the 
sponsor.  
 
17.8 Overview of the Safety Reporting responsibilities 
 
The CI is the medical assessor on behalf on the sponsor and will review all events 
reported. The CI will ensure that safety monitoring and reporting is conducted in 
accordance with the sponsor’s requirements.  
 
 
 
18. Monitoring and auditing 
 
The sponsor or delegate retains the right to audit any study, study site, or central 
facility. Any part of the study may be audited by the funders, if requested. As there is 
no investigative intervention in this study no regular monitoring schedule will be 
devised. Reports of SAE or SAR will trigger discussion with the study sponsor 
whether a trial monitoring should be commenced. Any concerns regarding delivery of 
the study highlighted at the trial committees can also instigate formal monitoring 
procedures by the JRMO. 
 
 
 
 
 
 
 

 
 
 
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19. Trial committees 
 
The study management group will include: 
Dr William Alazawi 
Dr James Brindley 
Ms Johanna Preston 
Mr John Loy 
Professor Francesco Rubino 
Ms Claire Dunne 
Patient representative – to be confirmed 
 
The Trial Committee (quorum is 3 members of the above list) will meet prior to 
recruitment, at 3 months and 6 months into recruitment and 1 year after initiation of 
the study.  If recruitment is below target, additional meetings will be convened.  
 
 
20. Finance and funding 
 
The Association of Physicians of Great Britain & Ireland 
Provided funding for laboratory experiments 
 
c/o BCD M&E 
3 Jubilee Way,  
Faversham Kent 
ME13 8GD 
01227378874 
 
 
21. Indemnity 
 
The insurance that Queen Mary University of London has in place provides cover for 
the design and management of the study as well as "No Fault Compensation" for 
participants, which provides an indemnity to participants for negligent and non-
negligent harm. 
 
 
22. Dissemination of research findings 
 
Results of this study will be published in peer-reviewed scientific journals, conference 
presentations, internal reports and presented locally at meetings within Barts Health 
NHS trust Queen Mary, University of London. If a study participant wishes to learn 
more about the research findings they can discuss this in more detail with the 
research team and given copies of published data. 
 
 
 
 
 
 
 

 
 
 
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