Study Protocol – MalMCSM2BPGi Version 3.
0 Date: 25 September 2024
Study Protocol Project 1
[Malaysia Study Sites Specific Protocol]
Title: Liver Fibrosis Assessment in Both Viral and Non-viral Etiologist Using
Sysmex High-Sensitivity Chemiluminescent Immunoassay (HISCL) Mac-2
Binding Protein Glycosylation Isomer (M2BPGi) biomarker.
Protocol number, version number and date:
Protocol number MalMCSM2BPGi Version 3.0, 2024
Name and Institution of Principal investigator:
Datuk Dr Hjh Rosaida binti Hj Md Said
Department of Medicine, Sultan Idris Shah Hospital, Serdang, Selangor
Name and Institution of Co-Investigators:
Prof. Rosmawati Mohamed
Department of Medicine, Hospital Canselor Tengku Mukhriz (UKM), Kuala Lumpur
Dr Haniza bt Omar
Medical Department, Hospital Selayang, Selangor
Dr Mohd Azri bin Mohd Suan
Clinical Research Center, Hospital Sultanah Bahiyah, Alor Setar, Kedah
Dr Siti Maisarah bin Md Ali
Clinical Research Center, Hospital Sultanah Bahiyah, Alor Setar, Kedah
Shahrul Aiman bin Soelar
Clinical Research Center, Hospital Sultanah Bahiyah, Alor Setar, Kedah
Faizah binti Ahmad
Clinical Research Center, Hospital Sultanah Bahiyah, Alor Setar, Kedah
Name and address of Sponsor:
1. Kementerian Kesihatan Malaysia
2. Sysmex Asia Pacific Pte Ltd
Study site/s:
1. Hospital Sultan Idris Shah, Serdang, Selangor
2. Hospital Sultanah Bahiyah, Kedah
3. Hospital Selayang, Selangor
4. Hospital Canselor Tunku Mukhriz, UKM, Kuala Lumpur
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
Research Synopsis
Study Title Liver Fibrosis Assessment in Both Viral and Non-viral
Etiologies Using HISCL M2BPGi Biomarker
Study Population Study subjects associated with various etiologies of liver
disease will be recruited to assess different stages of fibrosis
across different etiologies of liver disease.
Study Design An observational cross-sectional study
General Study 1. To evaluate the effectiveness of M2BPGi as a non-invasive
Objectives: biomarker for the assessment of liver fibrosis across
various etiologies, and to determine its clinical utility in the
early detection and management of liver disease.
Specific Study 1. To establish optimal cut-off values for M2BPGi to
Objectives: accurately classify different stages of liver fibrosis.
2. To compare the performance of M2BPGi with non-
invasive methods (FibroScan) for the assessment of liver
fibrosis.
3. To evaluate the correlation between M2BPGi levels and
other commonly used biomarkers for liver disease (e.g.,
APRI, FIB-4).
4. To assess the sensitivity and specificity of M2BPGi in
detecting liver fibrosis across different etiologies (viral and
non-viral).
5. To determine the clinical utility of M2BPGi in the early
detection and monitoring of liver disease progression.
Study Primary Outcome Measure
endpoints/outcomes 1 Establish optimal cut-off values for M2BPGi to accurately
classify different stages of liver fibrosis across various
etiologies.
2 Evaluate the sensitivity and specificity of the M2BPGi
assay in detecting liver fibrosis in a targeted population,
using both liver biopsy and FibroScan as reference
standards.
3 Correlate M2BPGi levels with other commonly used
biomarkers for liver disease (e.g. AFP level, APRI score,
FIB-4 index) and assess its association with hepatic
inflammatory response (ALT levels).
Secondary Outcome Measure
1 Conduct sub-group analyses comparing the performance
of liver biopsy, FibroScan, and M2BPGi in a limited cohort.
2 Evaluate the performance of M2BPGi in treatment-naïve
and treated cohorts.
3 Assess serial changes in M2BPGi levels to evaluate its
potential for monitoring disease progression or response to
treatment.
Sample Size The recruitment of 82 samples from Malaysia is planned.
Study Duration 1 October 2024 - 31 December 2026
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
1. Background and Significance
Liver disease is a significant health concern in the Asia Pacific region(1, 2), with several
risk factors contributing to its high incidence. The most common types of liver disease
in this region are viral hepatitis and non-alcoholic fatty liver disease (NAFLD)(3, 4).
According to recent studies, chronic hepatitis B and C infections are prevalent in Asia
Pacific, with an estimated 120 million individuals affected. These infections can lead to
cirrhosis and HCC, which are the leading causes of liver-related deaths in the region(5).
NAFLD is also on the rise in Asia Pacific(1, 6), particularly in countries with a high
prevalence of obesity and type 2 diabetes. NAFLD is characterized by the
accumulation of fat in the liver, which can progress to more severe conditions such as
non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Liver disease is a major
health issue in the Asia Pacific region, and efforts to prevent and treat these conditions
are essential to reduce their burden on public health.
For liver disease, early detection is crucial for effective treatment and improved
outcomes (7). However, there are several challenges in detecting liver disease in this
region, including limited access to screening and diagnostic tools, low awareness of
the disease among the population, and the high cost of testing. One approach to
overcoming these challenges is through the use of serum biomarker testing. Serum
biomarkers, which can be liver-specific or indirect markers in blood, can be measured
to indicate the presence of liver disease (8). Unlike traditional diagnostic methods such
as liver biopsy, serum biomarker testing is non-invasive and can provide results quickly
and at a lower cost.
The current study aims to evaluate the potential and feasibility of Sysmex HISCL Mac-
2 Binding Protein Glycosylation Isomer (M2BPGi) either as a single marker or
combined with other testing modalities for the assessment of liver fibrosis. M2BPGi
has been mentioned in the APASL recommendations for liver disease management (9)
and was extensively discussed in the recent APASL Oncology 2022 STC. This marker
is also reimbursable fully or partially in Japan and Korea. The main concern is that
extensive data were generated in Japan, but little exists in other Asia Pacific countries.
Prior art from renowned institutions out of Japan has shown positive outcomes, and
more needs to be done to generate sufficient clinical evidence to support its use. This
study aims to build credible evidence for M2BPGi testing in hopes of increasing
diagnostic tools available for liver disease assessment and improving early detection
rates.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
2. Study Objectives
1.1 Hypothesis: M2BPGi use in liver disease detection can serve as a reliable and
non-invasive biomarker for the assessment of liver fibrosis. M2BPGi levels will
correlate directly with the severity of liver fibrosis, as determined by histological
assessment, across various etiologies of liver disease, providing a reliable and
non-invasive biomarker for disease assessment and monitoring.
1.2 General Objective:
To evaluate the effectiveness of M2BPGi as a non-invasive biomarker for the
assessment of liver fibrosis across various etiologies, and to determine its clinical
utility in the early detection and management of liver disease.
1.3 Specific Objectives:
a. To establish optimal cut-off values for M2BPGi to accurately classify different
stages of liver fibrosis.
b. To compare the performance of M2BPGi with non-invasive methods
(FibroScan) for the assessment of liver fibrosis.
c. To evaluate the correlation between M2BPGi levels and other commonly used
biomarkers for liver disease (e.g. AFP level, APRI score, FIB-4 index).
d. To assess the sensitivity and specificity of M2BPGi in detecting liver fibrosis
across different etiologies (viral and non-viral).
e. To determine the clinical utility of M2BPGi in the early detection and monitoring
of liver disease progression.
3. Methods
3.1 Study Design
This study involves 17 centers within Asia including 4 sites in Malaysia. This is a cross-
sectional study. Eligible subjects will be recruited and enrolled in the study after
obtaining informed consent. Baseline data, including demographic information,
medical history, and laboratory results, will be collected. Blood samples will be drawn
for the measurement of M2BPGi and other biomarkers. Liver fibrosis assessment will
be conducted using FibroScan.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
The study endpoints are as follows:
1. Primary Outcome Measures
▪ Establish optimal cut-off values for M2BPGi to accurately classify different
stages of liver fibrosis across various etiologies.
▪ Evaluate the sensitivity and specificity of the M2BPGi assay in detecting liver
fibrosis in a targeted population, using both liver biopsy and FibroScan as
reference standards.
▪ Correlate M2BPGi levels with other commonly used biomarkers for liver
disease (e.g. AFP level, APRI score, FIB-4 index) and assess its association
with hepatic inflammatory response (ALT levels).
2. Secondary Outcome Measures
▪ Conduct sub-group analyses comparing the performance of liver biopsy,
FibroScan, and M2BPGi in a limited cohort.
▪ Evaluate the performance of M2BPGi in treatment-naïve and treated cohorts.
▪ Assess serial changes in M2BPGi levels to evaluate its potential for monitoring
disease progression or response to treatment.
3.2 Study Population
Potential study participants will be identified from patient registries (chronic HBV
infection and chronic HCV infection) and those actively followed up at the
gastroenterology clinic for HBV, HCV, non-alcoholic fatty liver disease (NAFLD) or
alcohol-related liver disease.
3.3 Inclusion Criteria
▪ Patients aged 18 years and older.
▪ Both Treatment-naïve and Treated patients with hepatitis B virus (HBV), hepatitis
C virus (HCV), non-alcoholic fatty liver disease (NAFLD) or alcohol-related liver
disease.
▪ NAFLD patients with diabetes will be included if they have well-controlled glycemic
levels (HbA1c < 8%) and have received lifestyle interventions for at least 6 months
duration.
▪ HCV or HBV infection with steatosis (CAP value of less than 240) will be included
if the patient does not meet the criteria for metabolic syndrome, as defined by
having at least three of the following conditions:
o Abdominal obesity (waist circumference more than 120cm for men and
more than 88cm for women
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
o High blood pressure
o Abnormal blood sugar
o High triglycerides
o Low HDL cholesterol
3.4 Exclusion Criteria
▪ Patients with HBV and HCV will be excluded if their FibroScan scores exceed a
CAP value of 240 or higher.
▪ Active intravenous drug-users. Patients who had not used intravenous drugs for at
least 6 months can be included.
▪ Patients with chronic lung diseases, except for asthma.
▪ Patients diagnosed with cardiomyopathy, ischemic heart disease, previous acute
myocardial infarction, percutaneous coronary intervention, New York Heart
Association Class II-IV congestive cardiac failure and severe tricuspid
incompetence.
▪ Patient with any significant medical condition at the discretion/judgment of
investigators.
▪ Patients with stage IV and V chronic kidney disease.
▪ Post organ transplantation.
▪ Pregnancy.
▪ Patients with a history of recent cancer within the past 5 years.
3.5 Study Withdrawal
Happens when participant voluntarily withdraws their consent to participate in the study.
Withdrawn participants will be replaced.
3.6 Sample Size
Following a power calculation using the parameters of a one-tailed test with a power
of 80% and a significance level (alpha) of 0.05, the proposed sample size, factoring in
a 10% patient ineligibility and lost to follow-up, the minimum sample size for all
Malaysia sites will be 82 patients.
3.7 Study Duration and Timeline
This study is planned to take place between 1st Oct 2024 and 31st December 2026.
• Stage 1, NMRR registration and MREC approval – 2 months (September 2024 -
October 2024)
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
• Stage 2, Study subject’s recruitment, data collection, data entry – 21 months
(October 2024 – June 2026)
• Stage 3, Data analysis – 4 months (June 2026 – September 2026)
• Stage 4, Write-up – 1 month (December 2026)
3.8 Study Visits and Procedures
• Potential participants will be approached by the investigators when they present to
the clinic.
• They will be screened for eligibility and briefed about the study.
• They will be given enough time to read and understand the patient information
sheet.
• They will be enrolled in the study only after providing informed consent.
• Their baseline characteristics will be gathered.
• Each of them will undergo transient elastography (FibroScan) at the point of
recruitment. However, using finding of transient elastography conducted within
30 days before or after recruitment is acceptable considering the chronic
nature of disease. While transient elastography is available in all the study sites
and has been routinely used to assess liver scarring as a standard practice, the
cost incurred by the scan, along with all the types of scans available in the study
sites, will be borne by the Ministry of Health.
• Blood will also be collected on the day of recruitment for: a) M2BPGi and AFP
testing; b) liver function test (to include AST, ALT, ALP, GGT, total bilirubin, direct
bilirubin, albumin, serum creatinine), full blood count (to include platelet count), c)
rapid test kit (HIV, HbsAg/ HbeAg, Anti-HCV/ HCV-cAg) and viral load test (HBV
DNA and HCV RNA), d) HbA1C test and e) lipid profile and fasting blood sugar
a. Measurement of biomarker levels (M2BPGi and AFP)
✓ One tube of 5mL blood sample will be collected (in SST II Advance –
yellow tube).
✓ Samples will be centrifuged. Serum will be immediately aliquoted into 2 plain
tubes (red cap, without additive) and frozen under conditions -20˚C or lower
until measurement.
✓ Analysis will be performed in batches within 3 months (90 days) of sample
collection using Sysmex HISCL (up to 30 tests at a time).
✓ Samples that are highly hemolyzed, present with high turbidity and/or with
microbial contamination (fungus) will not be used.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
b. Full blood count, platelet and liver function test
✓ Two tubes of blood samples will be collected: 1 tube of 3mL blood
samples for full blood count and platelet (in K2EDTA – purple tube) and 1
tube of 5mL blood samples for liver function test (in SST II Advance -
yellow tube).
✓ Blood samples will be sent to the hospital laboratory for analysis on the day
of recruitment.
✓ Past result is acceptable only if the test is performed within 30 days from
the day of recruitment.
c. Rapid test kit (RTK) for HIV, HbsAg/ HbeAg, anti-HCV/ HCV-cAg and Viral
Load Test (HBV DNA (for hepatitis B) or HCV RNA (for hepatitis C))
✓ Five tubes of blood samples will be collected: 1 tube of 5 ml blood
sample for RTK (in SST II Advance - yellow tube), and 4 tubes of blood
sample for viral load test (3 mL each in K2EDTA – purple tube).
✓ Blood samples will be sent to the hospital laboratory for analysis on the day
of recruitment.
✓ Past result is acceptable only if the test is performed within 90 days from
the day of recruitment.
d. Lipid profile and fasting blood sugar
✓ Two tubes of blood samples will be collected: 1 tube of 2 ml blood
sample for fasting blood sugar (in Sodium Fluoride Potassium Oxalate –
grey tube, and 1 tube of 5ml blood sample for lipid profile (in SST II
Advance - yellow tube)
e. HbA1C test
✓ One tube of 3 ml blood sample (in K2EDTA – purple tube) will be
collected.
✓ Blood samples will be sent to the hospital laboratory for analysis on the day
of recruitment.
✓ Past result is acceptable only if the test is performed within 6 months from
the day of recruitment.
• The blood taking, transient elastography and data collection are all one-off
procedures and will not be repeated.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
• The case report form (Appendix 1) will be completed right after all the imaging
and laboratory findings are available.
• The excess blood specimens will be discarded at the end of the study.
3.9 Statistical Analysis Plan
The statistical analysis will be performed using the SPSS version 24.0 (IBM, New
York). The types of analysis planned for this study include:
▪ Descriptive Statistics: Descriptive statistics will be used to summarize the
characteristics of the study population.
▪ Correlational Analysis: Correlation analysis will be used to assess the
relationship between M2BPGi levels and liver fibrosis severity.
▪ Regression Analysis: Regression analysis will be used to evaluate the
association between M2BPGi levels and other relevant factors (e.g., age, sex,
etiology of liver disease).
▪ Sensitivity and Specificity: Sensitivity and specificity of M2BPGi will be
calculated to assess its diagnostic accuracy.
▪ Subgroup Analyses: Subgroup analyses will be conducted to explore the
performance of M2BPGi in different patient populations (e.g., treatment-naïve
vs. treated, different etiologies of liver disease).
3.10 Risk and benefit to study participants
The study participants who were found to have any unaddressed medical conditions
of this study will be subjected to the standard treatment as provided by public health
institutions under the Ministry of Health, Malaysia. The study procedures, including
venous blood sample collection and imaging procedures, are all routine procedures for
the viral hepatitis management and will be performed by qualified health practitioners
(doctors and nurses). There is thus minimal risk for participants. This study is expected
to benefit HCV-infected patients by accelerating the treatment initiation.
If new information raises concern about the safety of the procedures involved in the
study, the consent form should be reviewed and updated if necessary. Participants will
be informed of the new information, given a copy of the revised form, and will need to
give their consent in order to continue in the study.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
3.11 Risk Benefit Assessment
As stated above, there is minimal risk from the study procedures. Study findings shall
potentially greatly improve the management of viral hepatitis and its complications in
the hospital. The expected benefit outweighs the minimal risk to participants and thus
this study should be supported. If any injuries, including blood taking-related swelling,
bruising and pain, do occur as a direct result of participating in the study, treatment for
such injuries shall be managed by the investigators at the study sites.
3.12 Ethics of Study
The study will be conducted in compliance with ethical principles outlined in the
Declaration of Helsinki and Malaysian Good Clinical Practice Guideline. The protocol
will be registered with the National Medical Research Register (NMRR), and ethics
approval will be sought from the Medical Research and Ethics Committee (MREC).
3.13 Informed Consent/ Assent Process
Patients shall be informed of the study when they present to the clinic. The patient
information sheet will be provided and explained to them. If they are willing to
participate, the consent forms will be signed and dated. If necessary, they are allowed
to take the information sheet home to discuss with their family members before
consenting to the study on another day.
3.14 Privacy and Confidentiality
The names of participants will be kept on a password-protected database and linked
only with a study identification number for this research. The identification number
instead of patient identifiers will be used on CRF. All data will be entered into a
computer that is password protected. On completion of study, data in the computer will
be copied to pendrives and the data in the computer erased. Pendrives and any
hardcopy data will be stored in a locked office of the investigators and maintained for
a minimum of three years after the completion of the study. The pendrives and data
will be destroyed after that period of storage. Participants can write to the investigators
to request access to personal information or study findings during or after the conduct
of the study.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
3.15 Conflict of Interest
The investigators declare they have no conflict of interest.
3.16 Publication Policy
Permission will be sought from the Director General of Health, Malaysia prior to the
publication of the study findings. No personal information will be disclosed, and
participants will not be identified when the findings of the study are published.
3.17 Termination of Study
The study team reserves the right to terminate the study at any time prior to inclusion
of the intended number of subjects for valid scientific or administrative reasons. Should
this be necessary, the study team will arrange the procedures on an individual basis
after review and consultation. In terminating the study, the investigator will assure that
adequate consideration is given to the protection of the interest of participants.
Reasons for termination may include but not be limited to:
• Too low enrolment rate.
• Protocol violations.
• Inaccurate or incomplete data.
• Unsafe or unethical practices.
• Questionable safety of the test products or treatment given.
• Suspected lack of efficacy of the test products or treatment given.
• Following the recommendation of the MREC.
• Administrative decision.
• Insufficient time or resource to conduct the study.
• Lack of eligible patients.
In the event that a study is terminated, the investigator has to:
• Complete all CRFs to the greatest extent possible.
• Return all test products to the sponsor who provides them.
• Answer all questions related to data of subjects enrolled prior to study
termination.
• Ensure that subjects are followed up with the necessary medical care.
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Study Protocol – MalMCSM2BPGi Version 3.0 Date: 25 September 2024
3.18 References
1. Wong MC, Huang JL, George J, Huang J, Leung C, Eslam M, et al. The
changing epidemiology of liver diseases in the Asia–Pacific region. 2019;16(1):57-73.
2. Sarin SK, Kumar M, Eslam M, George J, Al Mahtab M, Akbar SMF, et al. Liver
diseases in the Asia-Pacific region: a lancet gastroenterology & hepatology
commission. 2020;5(2):167-228.
3. Golabi P, Paik JM, Eberly K, de Avila L, Alqahtani SA, Younossi ZMJAoh.
Causes of death in patients with Non-alcoholic Fatty Liver Disease (NAFLD), alcoholic
liver disease and chronic viral Hepatitis B and C. 2022;27(1):100556.
4. Wong GL-H, Wong VW-SJTLG, Hepatology. How many deaths are caused by
non-alcoholic fatty liver disease in the Asia-Pacific region? 2020;5(2):103-5.
5. Bosch FX, Ribes J, Borràs J, editors. Epidemiology of primary liver cancer.
Semin Liver Dis; 1999: © 1999 by Thieme Medical Publishers, Inc.
6. Amarapurkar DN, Hashimoto E, Lesmana LA, Sollano JD, Chen PJ, Goh KL,
et al. How common is non‐alcoholic fatty liver disease in the Asia–Pacific region and
are there local differences? 2007;22(6):788-93.
7. Ginès P, Castera L, Lammert F, Graupera I, Serra‐Burriel M, Allen AM, et al.
Population screening for liver fibrosis: Toward early diagnosis and intervention for
chronic liver diseases. 2022;75(1):219-28.
8. Shirabe K, Bekki Y, Gantumur D, Araki K, Ishii N, Kuno A, et al. Mac-2 binding
protein glycan isomer (M2BPGi) is a new serum biomarker for assessing liver fibrosis:
more than a biomarker of liver fibrosis. 2018;53:819-26.
9. Yoshiji H, Nagoshi S, Akahane T, Asaoka Y, Ueno Y, Ogawa K, et al. Evidence-
based clinical practice guidelines for liver cirrhosis 2020. 2021;56(7):593-619.
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