Noninvasive Biomarkers of Metabolic Liver Disease 1.1
Phase: NA
Conditions: Nonalcoholic Steatohepatitis, Nonalcoholic Fatty Liver
Sponsor: Massachusetts General Hospital

NIMBLE Study 1.1 Clinical Study Protocol 
Protocol Version number: 4.0        
 
     
 
 
 
 
 
 
     11 August, 2021 
 
 
 
NON-INVASIVE BIOMARKERS OF METABOLIC LIVER DISEASE 
(NIMBLE) 
 
AN FNIH BIOMARKERS CONSORTIUM STUDY 
 
 
Protocol Number: NIMBLE Study 1.1 
Sponsor: FNIH 
Funded by: FNIH 
Version Number: 4.0 
11 August 2021 
 
 
 
 
 
 
 
 
 
 
 
 
 

NIMBLE Study 1.1 Clinical Study Protocol 
Protocol Version number: 4.0        
 
     
 
 
 
 
 
 
     11 August, 2021 
Table of Contents 
STATEMENT OF COMPLIANCE....................................................................................................................... 1 
1 
PROTOCOL SUMMARY ........................................................................................................................ 1 
1.1 
Synopsis ........................................................................................................................................ 1 
1.2 
Schema .......................................................................................................................................... 3 
1.3 
Schedule of Activities (SoA) ......................................................................................................... 3 
2 
INTRODUCTION ..................................................................................................................................... 4 
2.1 
Study RationalE............................................................................................................................. 4 
2.2 
Background.................................................................................................................................... 4 
2.3 
Risk/Benefit Assessment .............................................................................................................. 5 
2.3.1 
Known Potential Risks ............................................................................................... 5 
2.3.2 
Known Potential Benefits ........................................................................................... 5 
2.3.3 
Assessment of Potential Risks and Benefits ............................................................ 5 
3 
OBJECTIVES AND ENDPOINTS .......................................................................................................... 6 
4 
STUDY DESIGN ...................................................................................................................................... 7 
4.1 
Overall Design ............................................................................................................................... 7 
4.2 
Scientific Rationale for Study Design .......................................................................................... 9 
4.3 
Justification for Dose..................................................................................................................... 9 
4.4 
End of Study Definition ................................................................................................................. 9 
5 
STUDY POPULATION ............................................................................................................................ 9 
5.1 
Inclusion Criteria............................................................................................................................ 9 
5.2 
Exclusion Criteria ........................................................................................................................ 10 
5.3 
Lifestyle Considerations.............................................................................................................. 11 
5.4 
Screen Failures ........................................................................................................................... 11 
5.5 
Strategies for Recruitment and Retention ................................................................................. 11 
6 
STUDY INTERVENTION ...................................................................................................................... 12 
6.1 
Study Intervention(s) Administration .......................................................................................... 12 
6.1.1 
Study Intervention Description ................................................................................ 12 
6.1.2 
Dosing and Administration ....................................................................................... 12 
6.2 
Preparation/Handling/Storage/Accountability ........................................................................... 12 
6.2.1 
Acquisition and accountability ................................................................................. 12 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ............................................. 12 
6.2.3 
Product Storage and Stability .................................................................................. 12 
6.2.4 
Preparation ............................................................................................................... 13 
6.3 
Measures to Minimize Bias: Randomization and Blinding ....................................................... 13 
6.4 
Study Intervention Compliance .................................................................................................. 13 
6.5 
Concomitant Therapy.................................................................................................................. 13 
6.5.1 
Rescue Medicine ...................................................................................................... 13 
7 
STUDY INTERVENTION ...................................................................................................................... 13 
7.1 
Discontinuation of Study Intervention ........................................................................................ 13 
7.2 
Participant Discontinuation/Withdrawal from the Study ........................................................... 14 
7.3 
Lost to Follow-Up ........................................................................................................................ 14 
8 
STUDY ASSESSMENTS AND PROCEDURES ................................................................................. 14 
8.1 
SCREENING AND EFFICACY ASSESSMENTS ..................................................................... 14 
8.2 
Safety and Other Assessments.................................................................................................. 16 
8.3 
Adverse Events and Serious Adverse Events .......................................................................... 16 
8.3.1 
Definition of Adverse Events (AE) ........................................................................... 16 
8.3.2 
Definition of Serious Adverse Events (SAE) .......................................................... 17 
8.3.3 
Classification of an Adverse Event.......................................................................... 17 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ..................... 17 
8.3.5 
Adverse Event Reporting ......................................................................................... 18 

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8.3.6 
Serious Adverse Event Reporting ........................................................................... 18 
8.3.7 
Reporting Events to Participants ............................................................................. 18 
8.3.8 
Events of Special Interest ........................................................................................ 18 
8.3.9 
Reporting of Pregnancy ........................................................................................... 18 
8.4 
Unanticipated Problems.............................................................................................................. 18 
8.4.1 
Definition of Unanticipated Problems (UP) ............................................................. 19 
8.4.2 
Unanticipated Problem Reporting ........................................................................... 19 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................ 20 
9 
STATISTICAL CONSIDERATIONS ..................................................................................................... 20 
9.1 
Statistical Hypotheses ................................................................................................................ 20 
9.2 
Sample Size Determination ........................................................................................................ 20 
9.3 
Populations for Analyses ............................................................................................................ 21 
9.4 
Statistical Analyses ..................................................................................................................... 21 
9.4.1 
General Approach .................................................................................................... 21 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s).......................................................... 21 
9.4.3 
Analysis of the Secondary Endpoint(s) ................................................................... 22 
9.4.4 
Safety Analyses ........................................................................................................ 23 
9.4.5 
Baseline Descriptive Statistics ................................................................................ 23 
9.4.6 
Planned Interim Analyses ........................................................................................ 23 
9.4.7 
Sub-Group Analyses ................................................................................................ 23 
9.4.8 
Tabulation of Individual participant Data ................................................................ 23 
9.4.9 
Exploratory Analyses ............................................................................................... 23 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ............................ 23 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ....................................................... 23 
10.1.1 
Informed Consent Process ...................................................................................... 23 
10.1.2 
Study Discontinuation and Closure ......................................................................... 24 
10.1.3 
Confidentiality and Privacy ...................................................................................... 25 
10.1.4 
Future Use of Stored Specimens and Data ............................................................ 25 
10.1.5 
Key Roles and Study Governance .......................................................................... 25 
10.1.6 
Safety Oversight ....................................................................................................... 25 
10.1.7 
Clinical Monitoring .................................................................................................... 26 
10.1.8 
Quality Assurance and Quality Control ................................................................... 26 
10.1.9 
Data Handling and Record Keeping ....................................................................... 26 
10.1.10 
Protocol Deviations .................................................................................................. 26 
10.1.11 
Publication and Data Sharing Policy ....................................................................... 26 
10.1.12 
Conflict of Interest Policy ......................................................................................... 27 
10.2 
Additional Considerations ........................................................................................................... 27 
10.3 
Abbreviations ............................................................................................................................... 27 
10.4 
Protocol Amendment History...................................................................................................... 28 
11 
REFERENCES ...................................................................................................................................... 28 
 

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STATEMENT OF COMPLIANCE 
 
The trial will be conducted in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP) and applicable United States Code of Federal Regulations (CFR). The Principal 
Investigator at each site will assure that no deviation from, or changes to the protocol will take place 
without prior agreement from the Institutional Review Board (IRB), except where necessary to eliminate 
an immediate hazard(s) to the trial participant(s). All personnel involved in the conduct of this study 
have completed Human Subjects Protection and CITI GCP Training. 
 
The protocol and informed consent form(s) will be submitted to the IRB for review and approval. 
Approval of both the protocol and the consent form must be obtained before any participant is enrolled. 
Any amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study. All changes to the consent form will be IRB approved; a determination will be 
made regarding whether a new consent needs to be obtained from participants who provided consent, 
using a previously approved consent form. 
1 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS 
Title 
NON-INVASIVE BIOMARKERS OF METABOLIC LIVER DISEASE (NIMBLE); AN 
FNIH BIOMARKERS CONSORTIUM STUDY: Study 1.1 
Study Description 
(NIMBLE) 
NIMBLE is a comprehensive, five-year collaborative effort to standardize, 
compare, validate, and advance the regulatory qualification of imaging and 
circulating biomarkers to diagnose and stage nonalcoholic steatohepatitis 
(NASH), and to predict and assess response to therapeutic intervention 
(https://fnih.org/what-we-do/biomarkers-consortium/programs/nimble) 
Study Description 
(Study 1.1) 
This study, Study 1.1, is a prospective, observational, two-center, short-
term cross-sectional study to assess the reproducibility and repeatability 
of a set of specified ultrasound-based quantitative imaging biomarkers. 
The primary focus will be on imaging biomarkers of the liver fibrosis 
component of nonalcoholic fatty liver disease (NAFLD), rather than the 
steatosis or inflammation component. The rationale is that the fibrosis 
component is linked most closely to survival and other clinical outcomes. 
Study 1.1 will also collect data to explore vendor- or device-specific 
investigational biomarkers on other components of NAFLD such as 
steatosis and possibly inflammation. The data collected will be used to 
inform a decision of which of these biomarkers have sufficient precision to 
be advanced to NIMBLE Stage 2. 
Objectives (NIMBLE) 
The primary objectives of NIMBLE are to: 
• 
Standardize, compare, validate, and advance the regulatory 
qualification of a set of non-invasive imaging biomarker(s) for the 
diagnosis and staging of NASH. 
• 
Standardize, compare, validate, and advance the regulatory 
qualification of a set of non-invasive imaging biomarker(s) to predict 
and assess response to therapeutic intervention in patients with NASH. 
Objectives (Study 1.1) 
The primary objectives of Study 1.1 are: 

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• 
To evaluate the pooled different-day, different-operator 
reproducibility coefficient of ultrasound measurements of shear wave 
speed (SWS). 
• 
To evaluate the different-day, different-operator reproducibility 
coefficient of vibration controlled transient elastography (VCTE) on the 
measurement of SWS.  
 
Endpoints (Study 1.1) 
Primary endpoints: 
• 
Evaluation of pooled different-day, different-operator 
reproducibility coefficient of ultrasound measurements of SWS. 
• 
Evaluation of different-day, different-operator reproducibility 
coefficient of VCTE on the measurement of SWS.  
Secondary endpoints: 
• 
Evaluation of pooled same-day, same-operator repeatability 
coefficient of ultrasound-based measurements of SWS. 
• 
Evaluation of pooled different-scanner, same-day reproducibility 
coefficient of ultrasound-based measurements of SWS. 
• 
Evaluation of same-day, same-operator repeatability coefficient of 
VCTE on the measurement of SWS and Young’s modulus. 
• 
Evaluation of different-day, different-operator reproducibility 
coefficient of VCTE on the measurement of Young’s modulus. 
Exploratory endpoints 
• 
Precision and other reliability metrics of vendor- or device-specific 
investigational measurements of other components of NAFLD such 
as steatosis and possibly inflammation. 
 
Study Population 
This study will enroll patients with suspected or confirmed diagnosis of 
NAFLD. Based on protocol-specified FIB-4 values, about one-third are 
expected to have low, one-third to have intermediate, and one-third to 
have high likelihood of advanced fibrosis. The sample size of 40 
participants was determined to provide sufficient sample to provide >80% 
power to qualify SWS (or equivalent) as an ultrasound (US)-based 
biomarker for advancement to Stage 2.  The threshold for automatic 
qualification for advancement to Stage 2 will be an upper bound of the 
95% confidence interval (CI) of the reproducibility coefficient less than 
35%. Dropouts and technical failures were included in this sample size 
determination. 
Sex: ~ 50:50 – Note- no stratification will be done based on sex 
Age: ≥ 18 yrs 
Demographic group: Patients with a high probability of NAFLD based on 
the eligibility criteria 
General health status: Patients with suspected or confirmed diagnosis of 
NAFLD 
Geographic location: San Diego, CA and Boston, MA (greater metropolitan 
areas) 
Phase 
N/A 

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Description of 
Sites/Facilities Enrolling 
Participants 
Planned facilities/participating sites enrolling participants: University of 
California- San Diego (UCSD) and Massachusetts General Hospital (MGH) 
Number of sites: 2 
Description of Study 
Intervention 
Study will not have any therapeutic intervention. The goal of the study will 
be evaluating the variability of ultrasound-based measures for the 
diagnosis and monitoring of NAFLD/NASH. 
Study Duration 
12 months 
Participant Duration 
< 1 month 
 
 
1.2 
SCHEMA 
  
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
Procedures 
Screening 
(Day -7 to 1a) 
Visit 1 
(Day 1) 
Study Visit 2 
(Day 2 to 7)  
Informed consent b 
X 
 
 
Demographics 
X 
 
 
Medical history 
X 
 
 
Interim medical history 
 
X 
X 
Medication review 
X 
 
 
Change in medication review 
 
X 
X 
Physical activity review 
X 
 
 
Change in physical activity review 
 
X 
X 
AUDIT questionnaire 
X 
 
 
Vital signs 
X 
X 
X 
Height 
X 
 
 
Weight 
X 
X 
X 
Fasting blood collection c 
X 
 
 
Ultrasound Imaging 
 
X 
X 
Complete Case Report Forms 
(CRFs) 
X 
X 
X 

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a. Screening visit can occur on the same day as Visit 1. If visits occur on the same day, repeat tests (i.e. vital 
signs) and nonapplicable tests (e.g., change in medication review) do not need to be collected. 
b. Informed consent to be collected first at Screening Visit before all other procedures.  Informed consent 
can be collected up to 45 days prior to Visit 1.   
c. 
CBC, platelet count, complete metabolic panel (albumin, alkaline phosphatase, gamma glutamyl 
transferase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, 
potassium, total protein, AST, ALT, sodium), lipids (triglycerides, total cholesterol, HDL, LDL).  If the labs 
have been collected within the last 3 months the PI may opt to not repeat. Blood collected at MGH will 
be analyzed by the MGH clinical laboratory. Blood collected at UCSD will be analyzed by the UCSD clinical 
laboratory 
 
2 
INTRODUCTION 
 
2.1 
STUDY RATIONALE 
 
Nonalcoholic fatty liver disease (NAFLD) is a common liver problem which affects 30% of the United 
States population. Liver biopsy is accepted as the most accurate technique to detect patients at risk of 
developing serious liver conditions. However, liver biopsy is an invasive test with risk for complications 
and risk of mortality. Alternative imaging and blood-based biomarkers are needed to replace liver biopsy 
for screening and longitudinal assessment, to assist drug development process and to detect patients at 
high risk of developing liver-related complications. 
 
2.2 
BACKGROUND 
 
NAFLD is defined as fat accumulation in liver, when other fat-accumulating factors like alcohol 
consumption and steatogenic medications are excluded. NAFLD can be classified into two sub-types; 1) 
Nonalcoholic Fatty Liver (NAFL), and 2) Nonalcoholic Steatohepatitis (NASH). Both NAFL and NASH can 
progress to cirrhosis, however, NASH patients progress more frequently and more rapidly when 
compared to NAFL. NASH-related cirrhosis is currently the second most common indication for liver 
transplantation in the United States and is projected to become the leading cause in the near future 1,2,3. 
NASH is associated with a vast economic burden, estimated at $103 billion in medical costs each year in 
the United States alone4. 
 
Currently, liver biopsy is the reference standard for diagnosis and risk stratification of NASH, as it can 
evaluate both the inflammatory and fibrosis components of NAFLD. However, biopsy is limited by 
sampling error, and high intra- and inter-observer variability5. Moreover, liver biopsy is invasive, painful, 
costly, and associated with morbidity and even mortality6. Considering the large disease burden with 
high prevalence, it is not feasible to screen, diagnose, or monitor all suspected patients with non-focal 
liver biopsy. As a result, only a small minority of NASH patients typically undergo liver biopsy and an 
estimated 95% of patients remain undiagnosed7, 8. Non-invasive and inexpensive methods are needed to 
diagnose, risk-stratify, and monitor NASH. 
 
Noninvasive tests including imaging techniques and blood biomarkers might be useful in management of 
high-risk patients. Liver-specific ultrasound-based measurements including shear wave speed, or 
equivalently Young’s modulus, and sonographic fat assessment methods have the potential to provide 
complementary information, improving diagnostic confidence and guiding clinical and research practice 
9, 10. 

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2.3 
RISK/BENEFIT ASSESSMENT 
 
2.3.1 KNOWN POTENTIAL RISKS 
 
There are no known physical risks to study participants from the imaging procedures, as shear wave 
elastography (SWE), vibration controlled transient elastography (VCTE), and any exploratory imaging 
procedures are non-invasive ultrasound-based imaging tests. These techniques are performed using 
FDA-cleared devices that comply with United States Food and Drug administration (FDA) safety 
requirements, have no known bioeffects, and cause no patient discomfort. Images will be obtained on 
multiple ultrasound devices over 1.5 to 3 hours. If the patient becomes fatigued during this period, 
breaks will be provided. Inadvertent disclosure of protected health information is a potential risk that 
will be mitigated by storage of paper records in a locked filing cabinet and de-identification of clinical 
data.  There may be mild discomfort or pain from blood collection during screening.  The study is 
therefore considered a minimal risk study. 
 
There is a small but non-negligible risk of incidental findings.  An incidental finding is one unknown to 
the subject that has potential health or reproductive importance, which is discovered unexpectedly in 
the course of a research study but is unrelated to the purpose and beyond the aims of the study. This is 
a potential risk since the discovery of incidental findings may cause anxiety and lead to additional 
workup and even treatment, with attendant costs and risks, possibly without health benefit. 
Management of incidental findings is discussed in Section 8.2. 
  
 
2.3.2 KNOWN POTENTIAL BENEFITS 
 
There are no known direct benefits to study participants. The benefit of participation in this study is the 
contribution to the advancement of non-invasive biomarkers for NAFL and NASH, a contribution which 
could benefit the participant in their own future clinical surveillance or screening exams. 
 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS 
 
Non-invasive biomarkers could be valuable tools for advancing the care of patients with NAFL and NASH. 
Participation in NIMBLE could advance the knowledge of imaging biomarkers to allow for more routine 
use in clinical trials and routine clinical care. The broader benefits of biomarker development are as 
follows: 
 
Drug and Biomarker Development 
 
Difficulty in finding high-risk patients and risks of repetitive liver biopsy as a tool to assess 
treatment response, are major limitations in NASH drug development. By advancing the 
qualification of candidate ultrasound biomarkers and selecting reliable techniques, this 
limitation will be addressed. 
 
Regulatory agency approval process 
 

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Acquiring ultrasound data, analysis and research results will be helpful to obtain clearance and 
permission from regulatory agencies. 
 
Clinical practice 
 
Qualified ultrasound-based biomarkers might replace or reduce the need for liver biopsy, which 
is an invasive technique with limitations. 
 
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
The primary objectives of Study 1.1 
are: 
• 
To evaluate the pooled 
different-day, different-
operator reproducibility 
coefficient of ultrasound 
measurements of shear wave 
speed (SWS). 
• 
To evaluate the different-
day, different-operator 
reproducibility coefficient of 
VCTE on the measurement of 
SWS.  
 
• Evaluation of pooled different-day, 
different-operator reproducibility 
coefficient of ultrasound 
measurements of SWS. 
• Evaluation of different-day, 
different-operator reproducibility 
coefficient of VCTE on the 
measurement of SWS. 
 
Pooled diff-day, diff-operator 
reproducibility coefficient 
(pooled RDCdiff-day, diff-operator) 
Different-day and 
different-operator 
reproducibility are 
needed to inform the 
context of use of 
ultrasound and VCTE  
measurements for 
future clinical trials and 
clinical care. 
Secondary 
Secondary objectives of Study 1.1 
build on the understanding of 
repeatability and reproducibility of 
shear wave elastography and VCTE 
ultrasound-based measurements of 
shear wave speed (SWS) or, 
equivalently, Young modulus. 
 
The secondary objectives of Study 
1.1 are: 
• 
To evaluate the pooled same-
day, same-operator 
repeatability coefficient of 
ultrasound-based 
measurements of SWS. 
• 
To evaluate the pooled 
different-scanner, same-day 
reproducibility coefficient of 
• Evaluation of pooled same-day, 
same-operator repeatability 
coefficient of ultrasound-based 
measurements of shear wave 
speed (SWS). 
• Evaluation of same-day, same-
operator repeatability coefficient 
of VCTE on the measurement of 
SWS. 
 
Pooled same-day, same-operator 
repeatability coefficient 
(pooled RCsame-day, same-operator) 
 
• Evaluation of pooled different-
scanner, same-day reproducibility 
coefficient of ultrasound-based 
measurements of shear wave 
These endpoints will 
help refine the context 
of use of ultrasound 
and VCTE 
measurements for 
future clinical trials and 
clinical care. 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
ultrasound-based 
measurements of SWS. 
• 
To evaluate the same-day, 
same-operator repeatability 
coefficient of VCTE on the 
measurement of SWS. 
 
speed (SWS) and Young’s 
modulus. 
 
Pooled Diff-scanner, same-day 
reproducibility coefficient 
(pooled RDCdiff-scanner, same-operator) 
 
• Evaluation of different-day, 
different-operator reproducibility 
coefficient of VCTE on the 
measurement of Young’s modulus.  
 
Exploratory 
Exploratory objectives may include 
the evaluation of vendor- or 
device-specific ultrasound-based 
measurements of other tissue 
properties. 
Precision and other reliability metrics 
of vendor- or device-specific 
investigational measurements of 
other components of NAFLD such as 
steatosis and possibly inflammation.  
 
Exploratory analyses 
may contribute to the 
development of new 
ultrasound-based 
biomarkers. 
 
4 
STUDY DESIGN 
 
4.1 
OVERALL DESIGN 
 
Hypothesis. The upper bound of the 95% Confidence Interval (CI) of the pooled different-day, different-
operator reproducibility coefficient (true RDCdiff-operator, diff-day) of shear-wave speed values for the five 
participating ultrasound vendors (Canon, GE, Philips, Siemens, and Supersonic) will be less than 35%. 
The conservative assumption underlying this hypothesis is that the true RDCdiff-operator, diff-day values for the 
five ultrasound vendors will be 23, 25, 25, 27, and 30%, with no assumption made about how those true 
values correspond to the five vendors. 
 
Design. This is a prospective, low-interventional, two-center, short-term cross-sectional precision study. 
 
Study interventions. Ultrasound exams, blood collection, physical measurements, clinical history and 
medication reviews. 
 
Methods. At each of the two participating clinical sites (MGH and UCSD), approximately equal numbers 
of eligible participants will be enrolled, for a projected total enrollment of 40 participants across both 
sites (about 20 at each site). To ensure adequate representation of the disease severity spectrum, 
participants will be enrolled into three categories according to the likelihood for advanced fibrosis based 
on Fibrosis-4 (FIB-4) values: 
 
• 
low likelihood of advanced fibrosis (FIB-4 ≤ 1.3) 
• 
intermediate likelihood of advanced fibrosis (1.3 < FIB-4 < 2.67) 
• 
high likelihood of advanced fibrosis (FIB-4 ≥ 2.67) 

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About one-third of total participants (about 13 participants total -minimum 8, maximum 18), about 6 at 
each site (minimum 3, maximum 10) will be enrolled in each category. 
 
The rationale for using FIB-4 is that it is independent of the ultrasound-based measurements being 
tested in this Study. Although developed for assessment of liver fibrosis in Hepatitis C viral infection, it 
has been shown to stratify patients with NAFLD into the three likelihood levels with reasonable accuracy 
11 12. Perfect accuracy is not needed for this study because the aim of the stratification is to ensure a 
reasonable distribution of fibrosis severity. The FIB-4 levels will not be used as reference standard for 
fibrosis stage, and the accuracy of the ultrasound-based measurements to diagnose advanced fibrosis 
will not be tested in Study 1.1. The components of FIB-4 are age (years), AST level (U/L), platelet count 
(109/L), and ALT (U/L). The FIB-4 value is derived from a simple algebraic formula of these four 
components. 
 
Participants will be consecutively assigned to their corresponding category until the category is closed 
(reaches maximum of 11). Once a category is closed at a given site, participants will no longer be 
enrolled into that category at that site. 
 
Each participant will undergo ultrasound imaging at Visit 1 (by operator 1) and at Visit 2 (by operator 2), 
separated by 1 to 7 days. The 7-day window for Visit 2 is selected to allow flexibility in scheduling while 
helping to ensure that there is no substantial biological change in the interim. Ultrasound exams at 
Visits 1 and 2 will be performed at about the same time of day (target is ± 2 hours) and with identical 
fasting and other preparation instructions. For each visit, each participant will be assigned to three of 
the five ultrasound scanners plus VCTE. As shown in the Schema (Section 1.2), for Visit 1, two of the 
three ultrasound exams will be repeated; the third ultrasound exam and the VCTE exam will be 
performed once.  For Visit 2, the third ultrasound exam and the VCTE exam will be repeated; the other 
two ultrasound exams will be performed once.  The selection of exams to be repeated will be based on a 
block assignment strategy to ensure an approximately equal number of scans are performed by each 
ultrasound system, in addition to VCTE. Thus, each subject will undergo a total of six exams at each of 
their two visits, for a total of 12 exams across both visits. For the repeated exams, subjects will get off 
the table with a short break (target about 5 minutes) before getting back on the table. Subjects will not 
be required to leave the room. The machine will not be turned off and powered up again to save time 
 
Ultrasound operators will follow the acquisition procedures outlined in the imaging manual. The imaging 
manual will address patient positioning, image acquisition and quality control with scanning protocol 
input from the relevant ultrasound vendors. Additional investigational ultrasound data may be acquired, 
including with use of non-FDA-approved vendor proprietary software. 
 
As outlined in the Schedule of Activities (SOA), screening data will include demographic, medical history, 
medication review, physical activity review, vital signs, height, and weight. Body mass index (BMI) will be 
calculated. Fasting blood samples will be collected if needed at PI’s discretion based on availability of 
previous blood samples. The Alcohol Use Disorders Identification Test (AUDIT) questionnaire will be 
administered to assess alcohol consumption, drinking behaviors, and alcohol-related problems, 
information useful for assessing eligibility.  
 
On the subsequent visit, interim medical history, and changes in medications, physical activity, vital 
signs, and weight will be collected.  
 

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Methods that will be used to minimize bias. The following six methods will be used to minimize bias. 
 
1. Enrollment into fibrosis likelihood categories to ensure adequate representation of disease severity. 
The determination of fibrosis likelihood will be made by FIB-4, which is independent of any 
investigational ultrasound biomarker. 
 
2. None of the ultrasound operators will be involved in participant screening, or in the assignment of 
enrolled participants into the above three enrollment categories. 
 
3. The Visit 2 operator will be instructed to not review the Visit 1 exam results. 
 
4. The ultrasound operators will be blinded to clinical and laboratory data, and to enrollment category. 
 
5. On-site quality control, central quality control, and central reading.  Analysis will be performed by 
analysts and investigators who are not employed by vendor companies, and who have completed 
conflict of interest documentation for review and management by the Project Team. 
  
6.   Central analysts will be blinded to clinical and laboratory data. 
 
Interim analysis. None planned. 
 
Name(s) of sub-studies. None planned. 
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
All study participants will be scanned on a subset of ultrasound-based scanners.  All study participants 
will contribute data to the primary and secondary outcomes while minimizing burden on the patient.   
 
4.3 
JUSTIFICATION FOR DOSE 
 
Not applicable; this is not a clinical trial and there is no drug administration. 
 
4.4 
END OF STUDY DEFINITION 
 
A participant will be considered to have completed this study if: 
• 
all scheduled Visit 1 and Visit 2 ultrasound exams have been performed, and 
• 
all requested demographic, history, vital signs, and anthropometric information have been 
collected. 
• 
blood has been collected to measure the needed laboratory values 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
 
1. 
Adult (age ≥ 18 years) 

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2. 
Known or suspected NAFLD based on  
• 
Prior biopsy ≤ 36 months before enrollment consistent with NAFLD  
OR 
• 
Clinical and laboratory data ≤ 3 months before enrollment consistent with NAFLD: abnormal ALT 
(>30 U/L for men, > 19 U/L for women) without other common causes such as HCV, HBV AND 
meets criteriaf or ATP III criteria (2005 revision) for metabolic syndrome with any 3 of the 5: 
i. Waist circumference (WC) > 102 cm (M) or > 88 cm (F) 
ii. Fasting glucose ≥ 100 mg/dL or Rx 
iii. TG ≥ 150 mg/dL or Rx 
iv. Elevated blood pressure (SBP ≥ 130 mmHg or DBP ≥ 85 mmHg) 
v. Reduced HDL-C < 40 mg/dL (M) or < 50 gm/dL (W) 
 
3. 
Able and willing to participate, including maintaining steady-state: physical activity, alcohol use, 
medications 
4. 
Classifiable into one of the following enrollment categories by FIB-4 (ALT, AST, platelets, date of 
birth) collected at screening visit if not available already within 3 months prior: 
 
• Low likelihood of advanced fibrosis: FIB-4 ≤ 1.3 (about one-third of enrolled participants, 
minimum 8, maximum 18) 
• Intermediate likelihood of advanced fibrosis: 1.3 < FIB-4 < 2.67 (about one-third of enrolled 
participants, minimum 8, maximum 18) 
• High likelihood of advanced fibrosis: FIB-4 ≥ 2.67: (about one-third of enrolled participants, 
minimum 8, maximum 18) 
 
5.2 
EXCLUSION CRITERIA 
 
1. Liver disease other than NAFLD 
 
2. Excess alcohol consumption (≥ 2 units/day for women and ≥ 3 units/day for men) 
 
3. Current diagnosis of drug induced liver injury 
 
4. Receiving drug or placebo in treatment trial now or within 30 days 
 
5. Weight loss or gain of ≥ 5 kg in prior 3 months 
 
6. Other factors that in the judgment of the principal investigator might preclude study completion 
 
7. Women who state they are pregnant. Women who state they are pregnant will be excluded in an 
abundance of caution, since pregnancy might increase intra-abdominal pressure which in turn might 
affect the assessment of the different-day reproducibility coefficient of ultrasound and VCTE 
measurements.  
 

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8. Patients with active implants such as pacemakers or defibrillators or any other contraindication to 
ultrasound or VCTE scanning.  
 
5.3 
LIFESTYLE CONSIDERATIONS 
 
Participants will be asked to maintain their lifestyle over the course of their involvement in this study 
(i.e., no changes in medication; levels or types of exercise; amounts, types, or frequency of alcohol 
intake, etc.). 
 
5.4 
SCREEN FAILURES 
 
Screen failures will only be allowed in exceptional circumstances.  Potential participants who screen-
failed based on the Inclusion Criteria will not be allowed to re-screen.  Only potential participants who 
screen-failed based on the Exclusion Criteria 4, 5 or 6 will be allowed to re-screen if they no longer meet 
the Exclusion Criteria.   
 
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
Achieving target enrollment. It is not anticipated that there will be difficulty screening enough 
participants to meet the enrollment target of a total of 40 participants in this study. The goal will be to 
enroll approximately equal numbers of males and females, and to enroll a participant population 
representative of the sex, racial and ethnic mix of patients with NAFLD at the two clinical sites 
participating in this study. No special stratification will be done based on sex or race. Our effective 
screen-fail rate is expected to be up to about 25%, taking into account failure to satisfy inclusion and 
exclusion criteria, loss of participants due to possible uneven filling of our three enrollment categories, 
dropout, and technical failure. Thus, we expect to need to screen 50 participants to achieve our target 
enrollment of 40 participants. 
 
Anticipated accrual rate. We anticipate being able to enroll one participant at each site every 2 weeks, 
and therefore expect to be able to complete enrollment in about 40 weeks. 
 
Number of sites. There will be two clinical sites in this study (MGH and UCSD), and both have agreed to 
participate. 
 
Source of participants. Participants at MGH will be recruited from EMR review and the Fatty Liver 
Disease Clinic. Participants at UCSD will be recruited from the UCSD NAFLD Research Center. 
 
Methods of identification and approach for potential participants. At both sites, recruitment may be by 
informational flyers placed at the recruitment venues and/or by physicians at those venues discussing 
possible participation in this study with potential participants.  Patients identified through EMR review 
may be approached via opt-out mailing sent on behalf of their primary physician.  If subjects do not opt-
out they will be contacted by study staff.   
 
Recruitment strategies. It is not anticipated that additional recruitment methods or strategies will be 
needed to achieve our planned target enrollment within one year. 
 

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Participant retention. Given the short participation period for each participant (i.e., about a week or less 
to complete both imaging visits), it is not anticipated that participant retention will be a problem in this 
study. 
 
Recruitment and retention of participants from historically under-represented populations. Given the 
expected strong ability to recruit fairly from the outpatient clinic populations at both sites, the 
disproportionate prevalence of NAFLD in Latino minorities, and the short duration of participant 
involvement in this study, we do not expect that recruitment or retention of participants from 
historically under-represented populations will be a problem for this study. 
 
Vulnerable populations. No subjects from vulnerable populations will be recruited for this study.  
 
Participant compensation. Participants will be given up to $50 per hour for their participation in this 
study to compensate them for their time, any inconvenience, and parking. Compensation will be made 
upon completion of all study procedures or after completion of screening if participant screen fails. 
 
6 
STUDY INTERVENTION  
 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
 
Study interventions will include questionnaires, anthropometric measurements, blood collection, and 
non-invasive ultrasound-based imaging exams. All ultrasound-based devices use energies below FDA 
limits for diagnostic ultrasound.   
6.1.2 DOSING AND ADMINISTRATION 
 
There will be no dosing or administration of investigational products as part of this study. 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
 
There is no study intervention other than ultrasound-based exams. All ultrasound-based exams will be 
acquired by trained operators. 
 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
 
This section is not applicable as there is no study interventional product. 
 
6.2.3 PRODUCT STORAGE AND STABILITY 
 
This section is not applicable as there is no study interventional product. 
 

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6.2.4 PREPARATION 
 
This section is not applicable as there is no study interventional product. 
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 
Study participants will not be randomized to individual treatment groups or be blinded. In order to 
ensure a uniform distribution of scanner combinations, participants will follow a block-randomization 
pattern with different scanner combinations for different participants, however, patients and operators 
will not be blinded to ultrasound scanners being used. 
 
All efforts will be made to keep ultrasound operators blinded to clinical and laboratory data, however, it 
is not believed that this will significantly affect the ultrasound acquisition.  The Visit 2 operator will be 
asked to not review the Visit 1 exam results.   
 
Central analysts will be blinded to key clinical and laboratory findings to minimize potential bias.  
Blinding of the central analysts will be outlined in a separate Image Review Charter.  
 
6.4 
STUDY INTERVENTION COMPLIANCE 
 
Acquisition and analysis of ultrasound exams will be captured in a separate Acquisition Manual and 
Image Review Charter. These documents will outline the acquisition procedures and ultrasound analysis 
procedures. 
 
Participant weight will be measured and interim medical history, medication use, alcohol use, physical 
activity will be ascertained at each visit to assess compliance by participants with instructions to 
maintain steady-state weight, exercise, alcohol use, and medications. 
 
6.5 
CONCOMITANT THERAPY 
 
Current medications will be collected as part of a participant's medical history. 
 
6.5.1 RESCUE MEDICINE 
 
As there is no study intervention, no rescue medication will be used. 
 
7 
STUDY INTERVENTION 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
 
If participants become intolerant to ultrasound scanning they may discontinue. If they have not 
completed Visit 2 they will be withdrawn from the study. 
 

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7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
 
Participants are free to withdraw from participation in the study at any time upon request. 
 
An investigator may discontinue or withdraw a participant from the study if, in the opinion of the 
investigator, there have been significant lifestyle changes or other factors that could affect study results. 
 
7.3 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if he or she fails to return for Visit 1 and/or Visit 2, and 
is unable to be contacted by the study site staff. 
 
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit within the 
specified time frame and counsel the participant on the importance of maintaining the assigned 
visit schedule and ascertain if the participant wishes to and/or should continue in the study. 
• 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
If a participant is lost to follow-up and has only completed one study visit, they may or may not be 
replaced as part of the targeted enrollment. This decision will be based on the number of subjects who 
have completed the study to date. 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
SCREENING AND EFFICACY ASSESSMENTS 
 
Several assessments will be completed across Screening, Visit 1 and Visit 2. These are listed below and 
specific ultrasound image acquisition will be outlined in the ‘NIMBLE 1.1 Ultrasound Imaging Manual’. 
 
• 
Demographics, anthropometrics and medical history will be collected at the Screening Visit 
• 
Physical examination 
o Height will be recorded at Screening. 
o Weight and vital signs will be recorded at each visit. 
o Body mass index will be calculated at each visit. 
o All measurements will be made by trained coordinators using standard and calibrated 
instruments. 
 
• 
Imaging assessments will be collected at Visit 1 and Visit 2 
 
Ultrasound based imaging parameters will be collected from all patients in two visits. These will 
include but may not be limited to: 
o Shear wave elastography results in m/s and/or kPa 
o Quantitative ultrasound parameters, where available, including: 

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 
Attenuation Coefficient 
 
Backscatter Coefficient 
 
Shear Wave Dispersion 
 
Speed of Sound 
 
Ultrasound derived fat fraction 
o Conventional B mode (gray-scale) and Doppler ultrasound images, including: 
 
An image of the liver and right kidney on the same image for hepato-renal index 
calculation 
 
Right liver lobe for skin to liver capsule distance calculation 
 
Portal vein Doppler for portal vein pulsatility index measurement 
o VCTE and Controlled Attenuation Parameter measurements with the Fibroscan system  
 
Measurements will be collected using the following systems: 
o Ultrasound devices: 
o GE LOGIQ E10 
o Siemens Sequoia 
o Canon Aplio i800 
o Philips EPIQ 7 
o SuperSonic Aixplorer Mach 30 
o VCTE 
o Fibroscan 530 Compact 
 
For detailed ultrasound image acquisition protocol, please refer to the 'NIMBLE 1.1 Ultrasound 
Imaging Manual'.  Note:  Ultrasound-based exams will be reviewed centrally and results will not 
be shared with study participants.  
 
• 
Biological specimen collection and laboratory evaluations will be collected at the Screening 
Visit. 
o Blood will be collected by trained phlebotomists at each site using routine methods and 
standard collection tubes.   
o Total volume is expected to be about 10 mL or less 
o Blood collected at MGH will be analyzed by the MGH clinical laboratory.  
o Blood collected at UCSD will be analyzed by the UCSD clinical laboratory.  
o Blood results obtained within the 3-month interval prior to the screening visit at the 
MGH or UCSD laboratories will be considered acceptable for study analyses and may be 
used at PI discretion. 
o For each laboratory, the normal ranges for each blood test will be recorded and filed 
 
• 
Special assays or procedures required 
o None 
 
• 
Administration of questionnaires or other instruments 
o The following questionnaires will be administered at Screening: 
 
Medical history questionnaire 
 
Medication use questionnaire 
 
Alcohol consumption questionnaire (AUDIT questionnaire) 
 
Physical activity questionnaire 
o The following questionnaires will be administered at Visit 1 and Visit 2: 

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 
Interim medical history questionnaire 
 
Change in medication use questionnaire 
 
Change in physical activity questionnaire 
 
8.2 
SAFETY AND OTHER ASSESSMENTS 
 
Laboratory values will be reviewed by the PI at each site. The PI will report clinically important incidental 
findings to the referring physician or, if there is no referring physician, to the participant. 
 
If a lesion is identified by the operator during real-time scanning the site PI will be alerted by study staff 
and will review the images for safety.   
 
Additionally, ultrasound images will be reviewed by a central reviewer.  The purpose of this review is to 
evaluate images for liver stiffness and other exploratory biomarkers relevant to NAFLD and 
NASH.  Central image review is not a complete medical review of the subject.  If during the central 
review process, an unexpected observation is identified and this finding could, in the opinion of the 
central reviewer, have a significant health or reproductive consequence, this finding may be shared with 
the principal investigator.   
 
All follow-up testing and final diagnosis will be left to the discretion of the medical professionals at the 
site or those with an existing physician-patient relationship.  The principal investigator will be 
responsible for reporting any adverse events identified from incidental findings as described in the 
Adverse Event Reporting section.  Identification of such incidental findings during the central review 
process should not be expected, and the site maintains responsibility for performing a general safety 
review of all images as per site protocols.  
 
There are no dedicated Safety Assessments as part of this study as this study does not involve study 
medication. 
 
 
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
An AE is any untoward or unfavorable medical occurrence in a human subject, including any abnormal 
physical exam or certain unexpected abnormal laboratory finding, symptom, or disease, temporally 
associated with a subject's participation in the research. 
 
In this clinical study, we anticipate only minimal risk of adverse events because the investigational 
procedure is limited to noninvasive diagnostic ultrasound imaging of the abdomen using the vendor 
specific investigational liver fat quantification and shear wave elastography/VCTE software. All 
ultrasound systems that will be used in this clinical trial use ultrasound energies below FDA limits for 
diagnostic ultrasound. At these energies, these ultrasound systems and software have no known 
bioeffects and are not expected to cause risk to participants.  
 
Anticipated nonserious AEs may include; 
• 
Potential discomfort during the ultrasound scans due to body positioning, and 

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• 
Potential discomfort during the ultrasound scans due to ultrasound transducer pressure. 
• 
       Potential discomfort related to IV stick following blood draw at screening 
 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
A SAE is any AE that: 
1. 
Results in death, or 
2. 
Is life-threatening, or 
3. 
Results in hospitalization or prolongation of existing hospitalization, or 
4. 
Results in a persistent or significant disability/incapacitation, or 
5. 
Results in a congenital anomaly/birth defect, or 
6. 
May jeopardize the participant's health and may require medical or surgical intervention 
to prevent one of the other outcomes listed above. 
 
No SAEs are anticipated in this clinical trial.   
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
 
8.3.3.1 SEVERITY OF EVENT 
 
For adverse events (AEs), the following guidelines are used to describe severity. 
Mild - Events require minimal or no treatment and do not interfere with the participant's daily 
activities. 
Moderate - Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
Severe - Events interrupt a participant's usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating. Of note, the term 'severe' does not necessarily equate to 'serious'. 
 
AEs will be assessed by site PIs using the criteria mentioned above. Site PIs are responsible for reporting 
and managing AEs. 
 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
 
All AEs will have their relationship to study procedures assessed by the site principal investigator. The 
following criteria will be used to identify causality; (0) Not related, (1) Unlikely to be related, (2) 
Potentially related, (3) Potentially related, (4) Probably related, (5) Definitely related. 
 
8.3.3.3 EXPECTEDNESS 
 
Site PIs will be responsible for determining whether an adverse event (AE) is expected or unexpected in 
this NIMBLE clinical trial. An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with known ultrasound risks and complications, which are very limited. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 

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The occurrence of an AE or SAE may come to the attention of study personnel during ultrasound scans 
or screening visits. Any medical condition that is present at the time that the participant is screened will 
be considered as baseline and not reported as an AE. However, if the study participant's condition 
deteriorates at any time during the study, it will be recorded as an AE. 
 
8.3.5 ADVERSE EVENT REPORTING 
 
The site investigators will record adverse events and report them to the NIMBLE leadership, and to MGH 
and UCSD IRBs according to the timetable for reporting specified in the protocol. Any AE regarding 
research participants' health will be reported in 10 working days after the AE becomes known. 
 
Abnormal liver function tests and elevated blood lipids can be accepted as Disease-Related Events (DRE). 
These DREs are common in the target study population, patients with NAFLD. High liver function test 
and blood lipids are not associated with ultrasound exam. These factors will not be reported as adverse 
events. 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
 
In case of a serious adverse event, the site investigator will inform NIMBLE leadership and the MGH or 
UCSD IRBs as soon as possible, but in no event later than 10 working days after the site investigator first 
learns of the effect. NIMBLE leadership is responsible for conducting an evaluation of an unanticipated 
adverse device effect and shall report the results of such evaluation to the FNIH and MGH or UCSD IRB 
and participating investigators within 10 working days after NIMBLE first receives notice of the effect. 
Thereafter, the sponsor or designee (i.e. NIMBLE Leadership) shall submit such additional reports 
concerning the effect as FDA requests. 
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
 
Any AE regarding research participants' health will be reported to participant’s physician, or in the 
absence of a physician directly to the participant, via phone call or secure e-mail in 72 hours or less after 
the PI becomes aware of the AE. AE management strategies and next steps will be handled by the 
participant’s physician.  
 
8.3.8 EVENTS OF SPECIAL INTEREST  
 
This section is not applicable to this protocol.  
 
8.3.9 REPORTING OF PREGNANCY  
 
Women who state they know they are pregnant will be excluded from the study. Pregnancy might 
increase the intra-abdominal pressure which in turn might affect the reproducibility of elastographic 
measurements. 
 
8.4 
UNANTICIPATED PROBLEMS 
 

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8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 
In this clinical study, UP will be accepted as any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) ultrasound scans that 
are described in this protocol and NIMBLE 1.1 Ultrasound Imaging Manual; and (b) the 
characteristics of the NAFLD population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means 
there is a reasonable possibility that the incident, experience, or outcome may have 
been caused by the ultrasound scan); and 
• 
Suggests that the research places participants or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously known 
or recognized. 
 
In this clinical study, unanticipated adverse device effects will be accepted as, any serious adverse effect 
on health or safety or any life-threatening problem or death caused by, or associated with, ultrasound 
systems, if that effect, problem, or death was not previously identified in nature, severity, or degree of 
incidence in this protocol or any other unanticipated serious problem associated with ultrasound that 
relates to the rights, safety, or welfare of participants. 
 
8.4.2  UNANTICIPATED PROBLEM REPORTING  
 
The site primary investigators will report unanticipated problems (UPs) to the reviewing MGH and UCSD 
IRBs and to NIMBLE leadership. The UP report will include the following information: 
 
• 
Protocol identifying information: protocol title and number, PI’s name, and the site IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline: 
 
• 
UPs that are serious adverse events (SAEs) will be reported to MGH and UCSD IRBs and to the 
NIMBLE leadership within 24 hours of the investigator becoming aware of the event. 
 
• 
Any other UP will be reported to MGH and UCSD IRBs and to the NIMBLE leadership within 72 
hours of the investigator becoming aware of the problem. 
 
An investigator shall submit to the sponsor and NIMBLE Leadership and to the site IRB a report of any 
unanticipated adverse device effect occurring during ultrasound scans as soon as possible, but in no 
event later than 10 working days after the investigator first learns of the effect (21 CFR 812.150(a)(1)), A 
sponsor who conducts an evaluation of an unanticipated adverse device effect under 812.46(b) shall 
report the results of such evaluation to the Food and Drug Administration (FDA) and to all reviewing 

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IRBs and participating investigators within 10 working days after NIMBLE consortium first receives notice 
of the effect. Thereafter the sponsor or designee (i.e. NIMBLE Leadership) shall submit such additional 
reports concerning the effect as FDA requests (21 CFR 812.150(b)(1)). 
 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
 
Any UP regarding research participants’ health (i.e. SAE) will be reported to participant in 24 hours. UP 
management strategies and next steps will be explained by site PI. 
 
9 
STATISTICAL CONSIDERATIONS 
 
9.1 
STATISTICAL HYPOTHESES 
 
• 
Primary Efficacy Endpoint(s): 
 
Different-day, different-operator reproducibility coefficient of ultrasound measures 
 
• 
Secondary Efficacy Endpoint(s): 
 
Same-day, same-operator repeatability coefficient of ultrasound measures; 
Different-scanner, same-day reproducibility coefficient of ultrasound measures; 
Precision and other metrics measured by investigational analyses of ultrasound measures; and 
Vendor- or device-specific metrics of ultrasound measures 
 
9.2 
SAMPLE SIZE DETERMINATION 
 
The following assumptions were made in determining sample size for the primary study objective: 
 
1. Repeat measurements on the same participant follow a normal distribution. 
2. RDCdiff-day, diff-operator is in the range of 20-30% (wCV of 7.2-10.8%). 
3. A balanced design is planned (i.e. equal number of participants per scanner) where the following 
scenarios describe the possible RDCs across the five scanners: 
• 
25, 25, 25, 25, 25%  
• 
24, 25, 25, 26, 27% 
• 
23, 25, 25, 27, 29% 
• 
23, 25, 25, 27, 30% 
4. A study with 80% power is desired. 
 
The null hypothesis for the primary study objective is Ho: RDCdiff-day, diff-operator >35%, versus the alternative 
hypothesis that RDCdiff-day, diff-operator <35%, where RDC is the reproducibility coefficient for different day 
and different operator. The upper 95% confidence bound for the RDC will be constructed. 

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A Monte Carlo simulation study was conducted to determine the sample size needed. For each 
simulated participant, two observations were generated from a normal distribution with the specified 
variance. Samples of varying size were simulated. From each sample RDCdiff-day, diff-operator was estimated, 
along with its 95% CI. This process was repeated 10,000 times. 
In the table below the sample size is reported such that 80% of the samples of that size met the 
precision qualification (i.e. upper 95% confidence bound for RDC <35%). The results suggest that by 
pooling across the five scanners, up to 55 independent observations are needed.  With each subject 
scanned on three different scanners at the two time points and assuming moderate correlation between 
observations from the same subject (r=0.25), we consider the design effect to account for the clustering 
of the data (i.e. 3 observations/subject).  The design effect is 1+(s-1)r, where r=correlation and s=# 
of observation/subject, i.e. 1+(3-1)0.25=1.5. Thus, (55 x 1.5)/3=27.5 so a study with 28 total participants 
would provide 80% power.  
For estimating the RDCdiff-day, diff-operator of VCTE, assuming an RDCdiff-day, diff-operator of 25%, 32 subjects 
provides 80% power to show that the RDC<35%.  Taking into consideration the possibility of a drop-out 
or technical failure rate of no more than 25%, a study with 40 total participants (20 per site) is proposed. 
 
Number of Independent Observations needed for >80% power as a function of the five scanners’ 
RDCdiff-day, diff-operator 
RDCs across 5 scanners: 
25, 25, 25, 25, 
25%  
 
24, 25, 25, 
26, 27%  
 
23, 25, 25, 27, 
29%  
 
23, 25, 25, 27, 
30%  
 
Total # independent 
observations 
40 
45 
50 
55 
 
9.3 
POPULATIONS FOR ANALYSES 
 
All participants completing Visit 1 and Visit 2 with evaluable ultrasound images as defined during central 
review will be included in the analysis. 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
 
Means, medians, standard deviations (SDs), coefficients of variation (CVs), and relative frequencies will 
be used to describe the study sample’s characteristics and to report the observed imaging 
measurements. 95% CIs will be constructed for measures of precision. Log transformations of imaging 
measurements will be performed, as appropriate. 
 
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
 
RDCdiff-day, diff-operator will be estimated as follows: 

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𝑅𝑅𝑅𝑅𝑅𝑅
෣𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑−𝑑𝑑𝑑𝑑𝑑𝑑,𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑−𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜= 2.77 × ඨ෍
෍
(𝑌𝑌1𝑖𝑖𝑖𝑖−𝑌𝑌2𝑖𝑖𝑖𝑖)2/2𝑌𝑌ത𝑖𝑖𝑖𝑖
2𝑁𝑁
𝑁𝑁𝑗𝑗
𝑖𝑖=1
5
𝑗𝑗=1
 
where Y1ij is the biomarker measurement at timepoint 1 and Y2ij is the biomarker measurement on the 
same scanner j by a different operator at timepoint 2 for the ith participant. 𝑌𝑌ത𝑖𝑖𝑖𝑖 is the mean of Y1 and Y2 
for participant i for scanner j. Nj is the total number of participants per scanner, and N is the total 
number of observations. The 95% upper bound for RDCdiff-day, diff-operator will be constructed as follows: 
𝑅𝑅𝑅𝑅𝑅𝑅
෣ඨ
𝑁𝑁
𝜒𝜒𝑁𝑁,𝛼𝛼
2
 
where 𝜒𝜒𝑁𝑁,𝛼𝛼
2
 is the 𝛼𝛼th percentile of the chi square distribution with N degrees of freedom and α is 0.95. 
A bootstrap 95% CI will also be constructed for RDC, where sampling with replacement will be 
performed at the subject level. If the upper 95% confidence bound is < 0.35 (< 35%), then it will be 
concluded that the biomarker meets the prespecified precision criterion. 
 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
 
For each biomarker and each scanner, RCsame-day,same operator will be estimated as follows: 
𝑅𝑅𝑅𝑅
෢𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝑑𝑑𝑑𝑑𝑑𝑑,𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜= 2.77 × ඨ෍
(𝑌𝑌1𝑖𝑖−𝑌𝑌2𝑖𝑖)2/2𝑌𝑌ത𝑖𝑖
2𝑁𝑁𝑗𝑗
𝑁𝑁
𝑖𝑖=1
 
where Y1i is the biomarker measurement at timepoint 1 and Y2i is the replicate biomarker measurement 
on the same scanner by the same operator at timepoint 1 for the ith participant. 𝑌𝑌ത𝑖𝑖 is the mean of Y1 and 
Y2 for participant i. Nj is the total number of participants per scanner. 
For each biomarker and each scanner, RCdiff-day,diff-operator will be estimated as follows: 
𝑅𝑅𝑅𝑅
෢𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑−𝑑𝑑𝑑𝑑𝑑𝑑,𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑−𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜= 2.77 × ඨ෍
(𝑌𝑌1𝑖𝑖−𝑌𝑌2𝑖𝑖)2/2𝑌𝑌ത𝑖𝑖
2𝑁𝑁𝑗𝑗
𝑁𝑁
𝑖𝑖=1
 
where Y1i is the biomarker measurement at timepoint 1 and Y2i is the biomarker measurement on the 
same scanner by the same operator at timepoint 2 for the ith participant. 𝑌𝑌ത𝑖𝑖 is the mean of Y1 and Y2 for 
participant i. Nj is the total number of participants per scanner. 
For each biomarker and a pair of scanners, RDCscanner will be estimated as follows: 
𝑅𝑅𝑅𝑅𝑅𝑅
෣𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠= 2.77 × ඨ෍
(𝑌𝑌1𝑖𝑖−𝑌𝑌2𝑖𝑖)2/2𝑌𝑌ത𝑖𝑖
2𝑁𝑁𝑗𝑗,𝑗𝑗′
𝑁𝑁𝑗𝑗,𝑗𝑗′
𝑖𝑖=1
 
where Y1i is the biomarker measurement at timepoint 1 and Y2i is the biomarker measurement on a 
different scanner for the ith participant. This RDC will be estimated both with the same and different 
operators, and on the same and different days. 𝑌𝑌ത𝑖𝑖 is the mean of Y1 and Y2 for participant i. Nj,j’ is the 
number of participants scanned on the scanners j and j’. 

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For each biomarker and each scanner, 95% upper bounds for RCsame-day,same-operator, RCdiff-day,diff-operator, and 
RDCscanner will be constructed as follows: 
𝑅𝑅𝑅𝑅
෢ඨ
𝑁𝑁
𝜒𝜒𝑁𝑁,𝛼𝛼
2  
where 𝜒𝜒𝑁𝑁,𝛼𝛼
2
 is the 𝛼𝛼th percentile of the chi square distribution with N degrees of freedom and α is 0.95.  
9.4.4 SAFETY ANALYSES 
 
There are no safety analyses planned for this study.  All AEs, including their severity and relatedness to 
study interventions, will be recorded, however, there are no further planned analyses. 
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
Means, medians, standard deviations (SDs), coefficients of variation (CVs), and relative frequencies will 
be used to describe the study sample's characteristics. There are no intervention groups for comparison. 
 
9.4.6 PLANNED INTERIM ANALYSES 
 
No interim analysis is planned for this study. 
 
9.4.7 SUB-GROUP ANALYSES 
 
There are no sub-groups planned for this study. Any additional analyses will be outlined in the Statistical 
Analysis Plan. 
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
Individual participant data may be listed by measure and time points. 
 
9.4.9 EXPLORATORY ANALYSES 
 
There are no exploratory analyses that are planned as part of this study protocol other than exploratory 
endpoints.   
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 

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Consent forms describing in detail the ultrasound scans, blood sampling, and risks will be given to the 
participant and written documentation of informed consent will be required prior to starting ultrasound 
scans.  
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
 
Informed consent forms will be MGH and UCSD IRB approved for each institution, and the participant 
will be asked to read and review the document at the start of the Screening visit. The investigators, or 
their designees (e.g., coordinators), will explain the research study to the participant and answer any 
questions that may arise. This explanation will be provided in terms suited to the participant’s 
comprehension of the purposes, procedures, and potential risks of the study and of their rights as 
research participants. Participants will have the opportunity to carefully review the ICF and ask 
questions prior to signing.  The participants will have the opportunity to discuss the study with their 
family or surrogates or think about it prior to agreeing to participate. 
 
The participant will sign the ICF prior to any procedures being done specifically for the NIMBLE 1.1 
study. Participants will be informed that participation is voluntary and that they may withdraw from the 
study at any time, without prejudice. Both investigator and study participant will sign the informed 
consent form (ICF). A copy of the signed informed consent form will be given to participants for their 
records. The informed consent process will be conducted and documented in the source document 
(including the date), and the form signed, before the participant undergoes any study-specific 
procedures. 
 
The rights and welfare of the participants will be protected by emphasizing to them that the quality of 
their medical care will not be adversely affected if they decline to participate in this study. Appropriate 
efforts will be made at an institutional level to provide adequate interpretative services for participants 
who are not English speaking. Children or other vulnerable population will not be included in the study. 
 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
 
NIMBLE 1.1 study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or termination, 
will be provided to study participants, investigators, site IRBs, the NIMBLE consortium, and FNIH. Study 
participants will be contacted, as applicable, and be informed of changes to ultrasound scan visit 
schedule. 
 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping 
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the FNIH, NIMBLE and the IRBs. 
 

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10.1.3 CONFIDENTIALITY AND PRIVACY 
 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, MGH and 
UCSD staff, NIMBLE consortium and FNIH. Therefore, the study protocol, documentation, data, and all 
other information generated will be held in strict confidence. No information concerning the NIMBLE 1.1 
study or the data will be released to any unauthorized third party without prior written approval of the 
sponsor and/or NIMBLE Leadership.  Data may be included in the participant’s EMR as determined by 
the site PI.  
 
All research activities will be conducted in as private a setting as possible. Study participant’s contact 
information and PHI will be securely stored at each clinical site for internal use during the study. At the 
end of the study, all records, including signed consent form copies, will continue to be kept in a secure 
location. 
 
Study participant de-identified ultrasound imaging data, lab and clinical background information, will be 
transmitted to and stored in secure servers. Copy of the data will be stored at NIMBLE 1.1 Ultrasound 
Image Analysis Center (MGH). Investigators at MGH will be responsible for storing this data copy in 
secure server. Participants and their research data will be identified by a unique study identification 
number. The data entry and management in an image management system will be secured and 
password protected. Access will be limited to authorized personnel. 
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA 
 
Data collected for this study will be analyzed and stored at UCSD and MGH. After the study is 
completed, the de-identified, archived ultrasound images, ultrasound data and clinical data will be 
transmitted to and stored at a secure location. Permission to transmit data to the data storage center 
will be included in the informed consent. 
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
ultrasound images and clinical data stored for future research. 
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
 
 
Principal Investigator (MGH) 
Principal Investigator (UCSD) 
Anthony Edward Samir, MD MPH, 
Service Chief, Body Ultrasound, 
Director, Center for Ultrasound 
Research & Translation 
Kathryn Fowler, MD 
Massachusetts General Hospital  
UC San Diego  
55 Fruit Street, Boston, MA 02114 
9500 Gilman Drive 
+1-617-852-3241 
858-246-2196 
asamir@mgh.harvard.edu 
k1fowler@health.ucsd.edu 
 
 
10.1.6 SAFETY OVERSIGHT 
 

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There will be no Safety Oversight committee for this study as there is no intervention and all analyses 
will be conducted using FDA approved non-invasive imaging devices. 
 
10.1.7 CLINICAL MONITORING 
 
There will be no external clinical monitoring as part of this study.  Clinical monitoring will be the 
responsibility of the PI and appropriate team members at MGH and UCSD respectively. 
 
 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
 
MGH and UCSD will perform internal quality management of study conduct, data collection, 
documentation and management. An external auditor as a representative of the NIMBLE 1.1 team may 
perform independent assessments on an as-needed basis. 
 
10.1.9 DATA HANDLING AND RECORD KEEPING 
 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES 
 
Data collection is the responsibility of MGH and UCSD staff under the oversight of the PI at each site. 
Coordinators under PI oversight will ensure accurate, complete, legible, and contemporaneous data 
entry in source documents. 
 
10.1.9.2 STUDY RECORDS RETENTION 
 
All study records, de-identified data and research findings will be retained for 7 years after the study 
completion. No records will be destroyed without the written consent of FNIH and NIMBLE consortium. 
It is the responsibility of the FNIH and NIMBLE consortium to inform the investigators when these 
documents no longer need to be retained. 
 
10.1.10 PROTOCOL DEVIATIONS 
 
It is the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations within 10 working days of identification of the protocol deviation. All deviations will be 
reported to the NIMBLE leadership team. Protocol deviations will be sent to MGH and UCSD IRBs per 
their policies. The site investigator is responsible for knowing and adhering to the reviewing IRB 
requirements. 
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
 
As per National Institutes of Health (NIH) Public Access Policy, final peer-reviewed journal manuscripts 
will be submitted to the digital archive PubMed Central upon acceptance for publication. 
 
This study will comply with the FNIH Data Sharing and result dissemination policy. As such, this trial will 
be registered at ClinicalTrials.gov, and results information from this trial will be submitted to 
ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-reviewed journals. 

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NIMBLE consortium leadership will be responsible for developing publication procedures and resolving 
authorship issues 
 
10.1.12 
CONFLICT OF INTEREST POLICY 
Any potential conflicts of interest will be managed by FNIH. 
10.2 ADDITIONAL CONSIDERATIONS 
 
None. 
 
10.3 ABBREVIATIONS 
 
AE 
Adverse Event 
AUDIT 
Alcohol Use Disorders Identification Test 
BMI 
Body Mass Index 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CV 
Coefficient of Variation 
CRF 
Case Report Form 
DRE 
Disease-Related Event 
eCRF 
Electronic Case Report Forms 
EMR 
Electronic Medical Record 
FDA 
United States Food and Drug Administration 
FIB-4 
Fibrosis-4 
FNIH 
Foundation for the National Institute of Health 
GCP 
Good Clinical Practice 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation  
IRB 
Institutional Review Board 
MGH 
Massachusetts General Hospital 
NAFL 
Nonalcoholic fatty liver 
NAFLD 
Nonalcoholic fatty liver disease 
NASH 
Nonalcoholic steatohepatitis 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
NIMBLE 
Non-Invasive Biomarkers of Metabolic Liver Disease  
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RC 
Repeatability Coefficient 
RDC 
Reproducibility Coefficient 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
SWE 
Shear Wave Elastography 

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SWS 
Shear wave speed 
UCSD 
University of California,San Diego 
U/L 
Units per Liter 
UP 
Unanticipated Problem 
US 
Ultrasound 
VCTE 
Vibration Controlled Transient Elastography 
WC 
Waist Circumference 
 
10.4 PROTOCOL AMENDMENT HISTORY 
 
The table below is intended to capture changes of IRB-approved versions of the protocol, including a 
description of the change and rationale. A Summary of Changes table for the current amendment is 
located in the Protocol Title Page. 
 
Version 
Date 
Description of Change 
Brief Rationale 
Version 1.0 
24-Jan-2020 
Original document 
Original Version 
Version 2.0 
03-Nov-
2020 
Removed ‘Alcohol follow-back’ 
questionnaire.  
Due to the short time between 
Visit 1 and Visit 2 this 
questionnaire did not seem 
necessary.  
Version 3.0 
23 June 
2021 
Clarified metabolic syndrome criteria 
and minor site operation updates 
Inconsistency between protocol 
language and published criteria. 
Version 4.0 
11 August 
2021 
Remove pre-screening to allow sites 
to manage this individually, removed 
urine pregnancy test requirement at 
screening and extended the time 
before scanning that sites can collect 
ICDs.  
Updated to align with study site 
operations.  
 
 
 
 
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