European Journal of Internal Medicine 122 (2024) 11–19

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European Journal of Internal Medicine

journal homepage: www.elsevier.com/locate/ejim

Review Article

Non-invasive testing and risk-stratification in patients with MASLD

Mirko Zoncapè a, b, c, 1, Antonio Liguori a, b, d, 1, Emmanuel A. Tsochatzis a, b, *
a
Sheila Sherlock Liver Unit, Royal Free Hospital, London, UK
b
UCL Institute for Liver and Digestive Health, University College London, UK
c
Liver Unit, Department of Medicine, University of Verona and Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy
d
Medical and Surgical Sciences Department, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Università Cattolica del Sacro Cuore, Rome 00168, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The development and validation of non-invasive fibrosis tests (NITs) has changed clinical practice in Hepatology
NAFLD over the last 15 years. Metabolic associated steatotic liver disease (MASLD), formerly known as non-alcoholic
Fibroscan fatty liver disease (NAFLD), is the most prevalent liver disease in western countries, with up to a third of the
FIB4
unselected adult population affected. In this article, we review the use of NITs in the diagnosis and staging of
ELF
Cirrhosis
MASLD. We discuss their use in the diagnosis of steatosis, steatohepatitis and fibrosis and critically evaluate
recently published data. These NITs include a variety of approaches, such as serum markers like FIB-4, pro-C3
and ELF, imaging techniques like Fibroscan® and MRE, and combined scores like Agile 3+ and Agile 4, offering a
range of options for healthcare providers. Furthermore, these non-invasive tests also serve as valuable prognostic
tools, allowing for better risk assessment and improved patient management, particularly in predicting liver-
related events and overall mortality.

1. Introduction (cACLD) according to the latest Baveno VII consensus [4] is an inde­
pendent risk factor for the development of both liver-related events and
Hepatic steatosis denotes the excessive accumulation of fat within non-liver related morbidity and mortality [5,6]. The presence of stea­
liver cells. In June 2023, an international expert consensus panel tosis in isolation is not associated with an increased risk of liver-related
introduced the term steatotic liver disease (SLD), serving as an inclusive events [7].
term encompassing all the different aetiologies of hepatic steatosis, such Currently, histological assessment through a liver biopsy, remains
as metabolic, alcohol-related, drug-induced and cryptogenic [1]. the gold standard for the diagnosis of MASH, which is characterized by a
Of particular interest is metabolic-dysfunction associated steatotic combination of steatosis, lobular or portal inflammation and hepato­
liver disease (MASLD) – formerly known as non-alcoholic fatty liver cellular ballooning, and for staging of liver fibrosis. However, liver bi­
disease (NAFLD) – that is currently the most prevalent liver disease opsy has its limitations, including invasiveness, risk of complications,
globally, affecting approximately 30% of the world population [2]. To sampling variability, and the potential for patient reluctance due to its
correctly define the MASLD, hepatic steatosis must be identified by invasive nature [8,9]. Furthermore, a biopsy length of at least 25 mm is
imaging or biopsy, and the patient should be affected by at least one recommended for accurate staging, which is not always attenable [10].
cardiometabolic risk factor [1]. The increasing prevalence of MASLD and the previously cited limits
MASLD can progress over time causing inflammation in the liver of liver biopsy led to the development of several non-invasive tests
tissue (metabolic-associated steatohepatitis, MASH), liver fibrosis, and (NITs) for accurate staging and risk stratification. These include NITs for
ultimately liver cirrhosis. Early detection and management of MASLD diagnosing steatosis, steatohepatitis and for staging liver fibrosis. NITs
can help prevent the progression to more severe liver fibrosis and that were initially developed for staging fibrosis, are also increasingly
improve overall liver health. Liver fibrosis is the most important prog­ used to determine liver-related prognosis. NITs can be broadly catego­
nostic factor for liver-related events in MASLD [3]. Advanced fibrosis rized in serum tests, imaging techniques and scores that combine the two
(≥F3), also termed compensated advanced chronic liver disease [11].

* Corresponding author at: UCL Institute for Liver and Digestive Health, Royal Free Hospital and UCL, London, UK.
E-mail address: [email protected] (E.A. Tsochatzis).
1
Both authors had equal contribution and are joint first authors.

https://doi.org/10.1016/j.ejim.2024.01.013
Received 9 November 2023; Received in revised form 12 January 2024; Accepted 14 January 2024
Available online 20 January 2024
0953-6205/© 2024 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

Table 1 summarizes the performances and cut-off values of the main fasting blood sugar, triglycerides, and cholesterol level; apolipoprotein
NITs for the diagnosis of steatosis, steatohepatitis, and hepatic fibrosis. A1; alpha-2-microglobulin, haptoglobin. Normal liver blood tests do not
exclude the presence of MASLD, MASH or fibrosis [20].
2. Non-invasive diagnosis of liver steatosis Some scores were created and validated using different blood tests
often associated with demographic characteristics, to diagnose steatosis.
Hepatic steatosis can be diagnosed and quantified using various These include:
methods. Usually, the medical history and physical examination rise the
suspicion of excessive liver fat accumulation and different risk factors • Fatty Liver Index (FLI): a simple algorithm based on BMI, waist
should be taken into account: obesity/overweight, T2DM, alcohol con­ circumference, triglycerides, and GGT, created using US as reference.
sumption, sedentary lifestyle and sub-optimal diet. FLI can detect the presence of steatosis with high accuracy (AUROC
Many radiological imaging techniques, such as abdominal ultra­ 0.85; 95 %CI 0.82–0.89) but not the severity of steatosis [8,21]. FLI
sounds (US), abdominal computed tomography (CT) scans, or magnetic has undergone external validation and correlates with cardiovascu­
resonance imaging (MRI), can be used to non-invasively identify liver lar, liver and cancer-related mortality, as well as reduced insulin
steatosis [12]. sensitivity, risk of T2DM, accelerated atherosclerosis, and cardio­
US is often the first non-invasive test performed, as it can assess the vascular risk [22].
size and structure of the liver, and detect the presence of fatty deposits, • NAFLD Liver Fat Score (LFS): it was developed using magnetic
by identifying hyper-echogenicity of the liver in comparison with the resonance spectroscopy as gold standard, but not directly compared
renal parenchyma. It is a simple, non-invasive technique, generally to hepatic histology. It is based on AST, ALT, fasting insulin level,
faster and less expensive than other radiological imaging methods, but it presence of T2DM and metabolic syndrome. LFS detect the presence
is operator-dependent. of steatosis (but not the severity grade) with high accuracy (AUROC
Liver US gives a subjective evaluation of hepatic steatosis, with good 0.80; 95 % CI 0.69–0.88). Like FLI, LFS has been externally vali­
sensitivity and specificity in detecting moderate to severe levels of dated, and also correlates with insulin resistance and T2DM [22].
steatosis (about 85 % and 94 %, respectively) [13]. However, US is • Hepatic Steatosis Index (HSI): it was created using ALT, AST, BMI,
subject to various limitations, including the inherent subjectivity of the T2DM, gender, and US as reference; the AUROC of HSI for detecting
criteria employed to distinguish between fatty and normal liver, as well steatosis is 0.81 (95 % CI 0.801–0.824) [23].
as the absence of well-defined sonographic parameters to categorize • SteatoTest-2: constructed using a combination of the 6 components
varying degrees of steatosis. Notably, the sensitivity and specificity of of FibroTest-ActiTest (GGT, total bilirubin, alpha-2-macroglobulin,
B-mode sonography decrease as body mass index (BMI) rises. Steatosis apolipoprotein A1, haptoglobin, ALT) plus BMI, serum cholesterol,
by US can be missed if it is less than 20 % [14]. triglycerides, and glucose adjusted for age and gender; the reference
Fibroscan® is a non-invasive medical device designed to perform standard was biopsy and the AUROC 0.78 for any grade of steatosis
liver stiffness measurement (LSM) by vibration-controlled transient [24].
elastography (VCTE). Fibroscan® can be equipped with a module called
controlled attenuation parameter (CAP), based on an algorithm which, The above tests are better suited for large epidemiological studies
during the execution of the exam, automatically provides a rather pre­ rather than for use in individual patients due to their sub-optimal
cise estimate of any excess fat identified in the liver tissue [15]. More in specificity.
detail, CAP was found to correctly identify patients with steatosis with:
3. Non-invasive diagnosis of MASH and fibrotic-MASH
• an AUROC of 0.87 (95 % CI 0.82–0.92) for steatosis ≥S1,
• an AUROC of 0.77 (95 % CI 0.71–0.82) for steatosis ≥S2, Distinguishing between MASLD and MASH poses significant clinical
• an AUROC of 0.70 (95 % CI 0.64–0.75) for steatosis S3 [16] and diagnostic challenges. The evolving understanding of these condi­
tions necessitates accurate diagnostic methods to guide appropriate in­
A CT or MRI scan can provide more detailed information about the terventions. Existing non-invasive markers do not have adequate
liver and help diagnose the presence of cirrhosis or portal hypertension. diagnostic accuracy, therefore accurate diagnosis still relies on a liver
In CT scans, steatosis reduces liver attenuation, measured by Hounsfield biopsy.
Units (HU), causing hypodense liver. Enhanced CT has a limited role in Among various biomarkers, cytokeratin-18 (CK-18) is the most
diagnosing steatosis, due to contrast injection rate and timing affecting widely studied candidate, however it remains inadequate for reliable
liver attenuation, while unenhanced CT could qualitatively assess stea­ clinical use, underscoring the need for further research for appropriate
tosis, using the spleen as a reference organ: spleen attenuation is about NITs [25,26].
8–10 HU lower than the liver in healthy individuals [17]. A retrospec­ The entity of “fibrotic MASH” which is defined as MASH with sig­
tive study revealed that hepatic venous phase-normalized liver attenu­ nificant liver fibrosis (stage 2 or higher) has emerged as a diagnosis of
ation with spleen could aid steatosis assessment in unacceptable liver clinical significance, as this defines the population that is currently
donation candidates with moderate to severe steatosis, potentially enrolled in clinical trials and is also associated with a higher risk of liver-
averting needless biopsies [18]. related events. Several scores have been developed for this which are
The MRI-estimated proton density fat fraction (MRI-PDFF) repre­ outlined below.
sents the gold standard for the assessment and quantification of liver Newsome et al. developed and validated a score known as FAST
steatosis. In fact, MRI-PDFF has proven to be highly accurate in quan­ (FibroScan-AST), which combines LSM, CAP, and AST levels. The score
tifying hepatic steatosis, but also very precise and reproducible, over uses two cut-offs, one for ruling out and one for diagnosing fibrotic
other invasive (liver biopsy) and non-invasive assessment techniques MASH, and has an AUROC of 0.85 (95 % CI 0.83–0.87) [27].
[19]. MAST is an MRI-serum-based score designed to identify patients with
Blood tests can help assess liver function and detect elevated liver fibrotic MASH, developed using data from MRI-PDFF, MR elastography
enzymes, which might indicate liver damage in the context of a steatotic (MRE), and a blood test for AST. MAST score demonstrated high accu­
liver. Common tests include: full blood count; liver blood tests, such as racy in identifying Fibrotic MASH patients, with an AUC of 0.93 (95 % CI
alanine aminotransferase (ALT) and aspartate aminotransferase (AST), 0.88–0.97) in the validation cohort. Two specific cut-off points were
released into the blood when liver cells are damaged, total bilirubin, and identified: at the 90 %-sensitivity cut-off of 0.165, the test showed a
gamma-glutamyl transferase (GGT), which may arise in certain liver specificity of 72.2 %, while at the 90 %-specificity threshold of 0.242,
diseases, including hepatic steatosis; gluco‑lipid profile, including the test exhibited a sensitivity of 75.0 %. About 17.6 % of the patients in

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M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

Table 1
Main Non-Invasive Tests (NITs) used for diagnosis and staging of steatosis, MASH, MASH with significant fibrosis and advanced fibrosis.
NIT Use Cut-off values AUROC (95 % CI) Sensitivity (%) Specificity (%)

HEPATIC STEATOSIS
Ultrasounds (US) [13] Diagnosis of liver steatosis NA 0.93 (0.91–0.95) 85 94
Fibroscan® controlled Diagnosis of liver steatosis grade 248 dB/m 0.82 (0.81–0.84) 68.8 82.2
attenuation parameter (CAP) ≥S1
[15,104] Diagnosis of liver steatosis grade 268 dB/m 0.87 (0.85–0.88) 77.3 81.2
≥S2
Diagnosis of liver steatosis grade 280 dB/m 0.88 (0.86–0.91) 88.2 77.6
S3
Liver-to-spleen attenuation ratio Diagnosis of steatosis presence 1.12 0.87 (0.77–0.96) 81 79
on non-enhanced phase of with single cut-off (best sensitivity
computed tomography CT scan and specificity)
[18] Rule-in steatosis 0.99 52 100
Detecting ≥20 % steatosis with a 0.97 0.88 (0.76–0.99) 75 92
single cut-off (best sensitivity and
specificity)
Rule-in ≥20 % steatosis 0.91 63 100
Liver to spleen attenuation ratio Diagnosis of steatosis presence 0.98§ 0.93 (0.87–0.99) 77 100
on hepatic venous phase of with single cut-off (best sensitivity
computed tomography (CT) and specificity)
scan [18] Detecting ≥20 % steatosis with a 0.94 0.95 (0.88–1.01) 94 90
single cut-off (best sensitivity and
specificity)
Rule-in ≥20 % steatosis 0.87 56 100
Magnetic Resonance Imaging Discriminating ≥S1 From 8.9 to 6.4 %** 0.99 96 100
with estimated proton density Discriminating ≥S2 From 15 to 17.4 %** From 0.83 to 0.95 From 52 to 93 From 81 to 85
fat fraction (MRI-PDFF)* [19, Discriminating ≥S3 From 22.1 to 25 %** From 0.89 to 0.95 From 74 to 93 From 81 to 85
105-107]
Fatty Liver Index (FLI) [21] Rule-out steatosis 30 0.85 (0.82–0.89) 87 64
Rule-in steatosis 60 61 86
NAFLD Liver Fat Score (LFS) [22] Diagnosis of steatosis ≥5 % 0.16 0.80 (0.69–0.88) 65 87
Hepatic Steatosis Index (HIS) Rule-out steatosis ≥S1 30 0.81 (0.80–0.82 92.5 40.0
[23] Rule-in steatosis ≥S1 36 46.0 92.4
SteatoTest-2 [24] Diagnosis of steatosis ≥5 % (single 0.40 0.77 (0.71–0.82); 0.79 (0.73–0.83) 79.0 50.0
cut-off, adjusted for a prevalence ***
of steatosis of 18 %)
MASH (NASH)
Cytokeratin-18 (CK-18) Diagnosis of NASH/MASH in patients 260 U/L 0.77 82.7 57.4
[108] with FIB-4 ≥ 2.67
Diagnosis of NASH/MASH in patients 260 U/L 0.77 82.4 56.9
with FIB-4 <2.67
FibroScan-AST (FAST) [27] Rule-out NASH (NASH/MASH + 0.35 0.85 (0.83–0.87) (pooled external 89 64
NAS≥4 + F ≥ 2) validation cohort).
Rule-in NASH (NASH/MASH + 0.67 92 49
NAS≥4 + F ≥ 2)
MRI-PDFF-AST (MAST) score Rule-out fibro-NASH 0.165 0.93 (0.88–0.97) 90.0 72.2
[28] Rule-in fibro-NASH 0.242 75.0 90.0
Magnetic Resonance Diagnosis of fibro-NASH F ≥ 2 MRE≥3.3 kPa + FIB-4 ≥ 0.90 (0.85–0.95) (CSD-NAFLD NA NA
Elastography (MRE) with 1.6 cohort); 0.84 (0.78–0.89) (Japan-
FIB-4 (MEFIB score) [84] NAFLD cohort)
Fibrotic NASH index (FNI) Rule-out fibro-NASH ≤0.10 0.78 (0.71–0.85) (derivation cohort). 89 (derivation 37 (derivation
[29] From 0.80 (0.75–0.83) to 0.95 cohort) cohort)
Rule-in fibro-NASH ≥0.33 (0.92.− 0.98) (validations cohorts). 52 (derivation 90 (derivation
cohort) cohort)
Liquid biopsy based on PLIN- Diagnosis of NASH (with NAS>3) NA (score derived from a 0.98 (0.95–1.00) (derivation cohort) 95 (derivation 90 (derivation
2 mean florescence neural network classifier cohort) cohort)
intensity [109] including PLIN2)
Liquid biopsy based on Diagnosis of liver fibrosis NA (score derived from a 0.96 (0.88–1.00) (derivation cohort) 100 95.8
RAB14 mean florescence neural network classifier (derivation (derivation
intensity [109] including RAB14) cohort) cohort)
NIS2+™ [30] Rule-out at-risk NASH 0.4564 0.81 (0.80–0.83) 85 61
Rule-in at-risk NASH 0.6815 62 85
MACK-3 [32] Rule-out of fibrotic-NASH 0.135 0.79 (0.77–0.81) 90.8 48.4
Rule-in of fibrotic-NASH 0.549 48.0 86.4
ADVANCED FIBROSIS (F ≥ 3)
Fibrosis-4 index (FIB-4) [38] Rule-out advanced fibrosis 1.30 0.80 (0.77–0.84) 77.8 71.2
Rule-in advanced fibrosis 3.25 37.3 95.8
NAFLD Fibrosis Score (NFS) [38] Rule-out advanced fibrosis − 1.455 0.78 (0.75–0.81) 72.9 73.8
Rule-in advanced fibrosis 0.676 43.1 88.4
Aspartate Aminotransferase to Rule-out advanced fibrosis 0.5 0.75 (0.72–0.77) 72.9 67.7
Platelet Ratio Index (APRI) Rule-in advanced fibrosis 1.5 32.9 90.5
[38]
BARD score [38] Diagnosis of advanced fibrosis 2 0.73 (0.71–0.75) 75.2 61.6
Enhanced Liver Fibrosis (ELF) Diagnosis of advanced fibrosis 9.8 0.948 (0.88–1.00) 86.7 92.5
[44]
(continued on next page)

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M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

Table 1 (continued )
NIT Use Cut-off values AUROC (95 % CI) Sensitivity (%) Specificity (%)

Pro-C3 [34] Diagnosis of advanced fibrosis NA 0⋅75 (0.70–0.80) NA NA

ADAPT [51] Diagnosis of advanced fibrosis 6.3287 0.87 (0.83–0.91) NA NA
Vibration-controlled transient Rule-out advanced fibrosis 7 kPa 0.87 (0.86–0.88) 95.6 48.6
elastography (VCTE) with Rule-in advanced fibrosis 12 kPa 61.1 88.3
Fibroscan® [64]
Point Shear Wave Elastography Diagnosis of advanced fibrosis 2.06 m/s 0.94 (0.91–0.96) 90 90
(pSWE) [69]
Two-dimensional shear [79] Diagnosis of advanced fibrosis 9.2 m/s 0.928 93.1 80.9
wave elastography (2D-SWE)
[71]
Magnetic Resonance Diagnosis of advanced fibrosis 4.11 kPa 0.94 (0.91–0.98) 89 0.84
Elastography (MRE) [76]
Agile 3+ [79] Rule-out advanced fibrosis 0.451 0.87 (0.85–0.89) 83 75
Rule-in advanced fibrosis 0.679 61 90
Agile 4 [79] Rule-out cirrhosis 0.251 0.89 (0.86–0.92) 88 71
Rule-in cirrhosis 0.565 44 97
§
represents the cut-off value that provided a balance between sensitivity and specificity, and yielded a 100 % specificity.
*
data presented as single value or range of values, when declared in the original studies included in the cited review.
**
percentage of estimate steatosis in the liver using a generate PDFF map with ranges from 0 % to 50 %, representative of the fat content in the liver.
***
values varying according to subsets and the prevalence of steatosis, as stated by Authors.
NA: Not-Available.

the validation cohort fell in the “grey-zone” [28]. It has been suggested that the low cut-offs should be higher in patients
The Fibrotic NASH Index (FNI) is a non-invasive scoring system, >65 years old at 2.0 for FIB-4 and 0.12 for NFS [37,38]. FIB-4 un­
proposed in 2023, and designed to identify fibrotic MASH in MASLD derestimates fibrosis in patients <35 years old and has decreased diag­
patients. FNI is based on AST, HDL, and HbA1c. It showed good per­ nostic accuracy, even after cut-off adjustments, in those >65 years.
formance in validation cohorts with AUROCs ranging from 0.80 to 0.95. Since the calculation of both these scores is based on simple pa­
Using a rule-out cut-off of 0.10, FNI demonstrated high sensitivity and rameters that can be easily obtained with a clinical evaluation and
NPV, meaning it accurately identified individuals without fibrotic routine blood tests, their use is encouraged in primary care and dia­
MASH, reducing the need for referral to liver specialists [29]. betology clinics to identify patients at higher risk of advanced fibrosis
NIS2+™ is a novel two-biomarker-test corrected for sex, consisting that should be referred to secondary care/hepatology clinics [9,39,40].
of miR-34a-5p and YKL-40. In the derivation cohort, NIS2+™ exhibited Other simple markers, such as the Aspartate Aminotransferase to
an AUROC of 0.81 for detecting at-risk MASH. Moreover, it consistently Platelet Ratio Index (APRI) [41] and the BARD score [42] are not used
maintained high and stable AUROC values (ranging from 0.780 to routinely in clinical practice.
0.807), when utilized for identifying at-risk F3, MASH and F>=2 [30]. Direct serum biomarkers related to the fibrogenic process and
Importantly, the diagnostic accuracy of NIS2+™ is maintained in people extracellular matrix turnover have been proposed and validated for
>65 years old [31]. staging of liver fibrosis. The Enhanced Liver Fibrosis (ELF) test is based
The MACK-3 score, based on 3 biomarkers (AST, homeostasis model on the measurement of three serum biomarkers involved in matrix
assessment [HOMA], and CK-18), was recently validated in an interna­ turnover: tissue inhibitor of metalloproteinase-1, hyaluronic acid and N-
tional multicentric study (AUROC=0.79), suggesting that it could be a terminal procollagen III peptide [43]. ELF has a good diagnostic per­
valuable tool in improving patient selection for MASH therapeutic trials formance for diagnosis of advanced fibrosis (AUROC ≥0.8) [34,44] with
[32]. a cut-off of >9.8 being highly sensitive for advanced fibrosis [45,46]
All the above scores are suboptimal if used on their own for the ac­ although the presence of extra-hepatic inflammatory or fibrotic diseases
curate diagnosis of fibrotic MASH. It is still uncertain if the presence of could lead to false positive results [47].
fibrotic MASH (rather than fibrosis alone) will be required for treatment ELF has become increasingly available in laboratories in recent
eligibility once pharmacotherapy becomes available. The concomitant years, especially in Western countries, but remains expensive when
use of two or more the above tests (with a liver biopsy if discordant) applied to the general population. A two-step approach of FIB-4 fol­
needs to be tested in future studies. At the moment, the utility of these lowed by ELF allows the test to be used only in the population with
scores is to better select patients for screening biopsies for participation indeterminate FIB-4 results. This approach reduces costs and improve
in clinical trials. the selection of patients for secondary care referral [48,49].
The recent EASL and AASLD guidelines recommend that patients at
4. NITs for fibrosis assessment risk of MASLD with high FIB-4 or NFS and patients with intermediate
FIB-4 or NFS and high Fibroscan® (>8 KPa) or ELF (>9.8 for EASL, >7.7
4.1. Serum markers for AASLD) should be referred to secondary care for further evaluation
[9,39].
Serum markers can be categorized as either indirect or direct. Indi­ Pro-C3 is a direct biomarker of type-III collagen formation and has
rect serum markers comprise combinations of routine laboratory pa­ been proposed and validated as NIT for liver fibrosis assessment either
rameters along with demographic factors such as BMI, age or the alone or in algorithms (ADAPT) [50,51]. They showed a good perfor­
presence of diabetes. Among indirect serum biomarkers the most mance for diagnosis of advanced fibrosis in patients with MASLD with an
extensively validated and commonly used are Fibrosis 4 (FIB-4) and AUROC of 0.75 (Pro-C3) and 0.81 (ADAPT) in a recent multicentric
NAFLD Fibrosis Score (NFS) for advanced fibrosis. Their ability to study [34]. Currently, its high cost and low availability render it sel­
discriminate advanced fibrosis is good with an AUROC among 0.7 and domly used in routine clinical practice.
0.8 according to different multicentric studies and meta-analysis
[33–36]. These biomarkers have dual cut-offs, whith a low cut-off to 4.2. Liver elastography
rule out and a high cut-off to diagnose advanced fibrosis. Their main use
is to rule out advanced fibrosis, at <− 1.455 for NFS and <1.3 for FIB-4. In addition to serum biomarkers and scoring systems, LSM is closely

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M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

associated with liver fibrosis and can be determined using various US 0.93) and cirrhosis (AUC: 0.92) and it is not affected by gender, BMI,
and magnetic resonance elastography (MRE) techniques [52]. US elas­ steatosis and inflammation grade. Proposed cut-offs are 3.66 kPa for
tography methods include Vibration Controlled Transient Elastography significant fibrosis (sensitivity: 79 %, specificity: 81 %), 4.11 kPa for
(VCTE or Fibroscan®), acoustic radiation force impulse (ARFI), point advanced fibrosis (sensitivity: 85 %, specificity: 85 %) and 4.71 kPa for
shear wave elastography (p-SWE), and two-dimensional shear wave liver cirrhosis (sensitivity: 91 %, specificity: 81 %) [76].
elastography (2D-SWE) [53]. However, it must always be kept in mind
that other characteristics of the liver tissue can significantly influence its 4.3. Elastography scores
rigidity and limit the diagnostic accuracy of elastography. Liver tissue
stiffness could be potentially influenced by steatosis, The gradual increase in the availability of elastography in secondary
necro-inflammation, postprandial hepatic hyperaemia, thickness of care settings, in particular the Fibroscan®, has highlighted some limi­
subcutaneous tissue, cholestasis and increased central venous pressure tations in the reliability of elastography methods. Many co-factors such
[54–57]. as age, obesity, liver congestion and significant elevation of liver en­
VCTE is the first-developed, most validated and extensively used US- zymes have proved to impact on elastography results [77,78].
based elastography method worldwide. It measures liver stiffness by In this scenario, scores combining clinical, laboratory and elastog­
assessing the speed of shear wave propagation through the hepatic tis­ raphy variables have been proposed to overcome those limitations and
sue. According to two recent meta-analysis, VCTE has an excellent to more accurately identify MASLD patients with advanced fibrosis or
diagnostic accuracy for diagnosis of advanced fibrosis and cirrhosis in cirrhosis.
MASLD patients with AUROCs close to 0.90 [58,59]. VCTE (and other The Agile 3+ proposed by Sanyal et al. takes into account LSM
US elastography methods) are less accurate in diagnosing significant or (Fibroscan®), platelets, AST, ALT, diagnosis of diabetes age and sex.
lesser degrees of fibrosis [60]. Although data have shown that the This score showed a very good diagnostic performance of advanced
presence of steatosis can increase liver stiffness values [61], this was not fibrosis in the European and US validation courts with AUROC>0.85,
confirmed in a prospective study of patients with suspected NAFLD [62]. significantly higher than LSM alone, FIB4 and NFS [79]. Interestingly,
A limitation of VCTE is that there are no established cut-offs for the Agile3+ superiority compared to LSM alone is not confirmed in diabetic
diagnosis of advanced fibrosis and cirrhosis. When a single cut-off is populations, arising questions about the net benefit of using the score in
used, VCTE is better in excluding that ruling in advanced fibrosis or such patients. The proposed cutoff for the exclusion of advanced fibrosis
cirrhosis, also due to the relatively low prevalence of these stages. To (0.451) showed a sensitivity >80 % in both European and US cohorts,
overcome this, a dual cut-off strategy has been proposed in the Baveno while the proposed cutoff for the diagnosis of cACLD (0.679) showed a
VI and VII consensus - a VCTE cut-off of <10 kPa to rule out and a cut-off specificity >85 % in both European and US validation cohorts. The
of >15 kPa to diagnose cACLD [4,63]. A recent real-world validation of percentage of patients falling into the grey area is small compared to
these thresholds revealed that the optimal cut-offs for diagnosing cACLD other NITs and is between 8 and 20 % in both European and American
in patients with MASLD are <8 kPa (with a sensitivity >90 %) and >12 validation cohorts [80–82].
kPa (with a specificity of 88.2 %) [64]. pSWE is a method that can assess Interestingly, similar results were not evident in Asian cohorts. In a
the elastic characteristics of the liver in real-time B mode US examina­ recent Asian multicentre study in a population of 641 patients with
tion using ARFI technology [65]. pSWE is based on the quantification of MASLD, the AUROC of Agile 3+ for diagnosis of advanced fibrosis was
shear wave travelling speed, which is directly reliant on viscoelastic 0.82, and was equal to that of LSM alone. Furthermore, the percentage of
properties of the parenchyma [52,66]. Compared with VCTE, ARFI patients with an indeterminate result was significantly high for both
showed similar accuracy to detect cirrhosis (AUROC 0.87 and 0.85 for LSM and Agile3+ (34 % and 39 %), respectively, compared to the
VCTE and ARFI, respectively) with a lower failure rate [67]. Recent multicentric US and European studies [83].
meta-analyses demonstrated a good accuracy of pSWE for detecting The Agile 4 aims to diagnose cirrhosis and consists of the following
significant fibrosis (≥2) with a pooled sensitivity of 80.2 % and a variables: LSM, platelets, ALT, AST, Gender and T2DM status [80]. It
specificity of 85.2 % [68]. Diagnostic performance of pSWE for showed a high diagnostic performance for cirrhosis both in European
advanced fibrosis (F ≥ 3) was excellent with an AUROC of 0.94 [69]. and Nord American validation cohorts with AUC of 0.89 and 0.85
2D-SWE uses real time conventional B-mode imaging to produce a respectively. A dual cutoff strategy uses 0.251 as a rule-out cutoff and
two-dimensional quantitative map of liver tissue stiffness. The operator 0.565 as a rule-in cutoff. In European validation cohorts this strategy led
can specify of the region of interest where liver stiffness is measured. to a low percentage of patients falling in the gray zone (11–16 %
This allows the measurement of stiffness to be oriented towards a spe­ depending on different cohorts) whereas in North American validation
cific area of the liver but on the other hand it has the disadvantage of cohort that percentage was higher (23 %) [80,81].
increasing intra- and inter-observer variability [70]. A recent Jung et al. assessed the diagnostic accuracy of combining Magnetic
meta-analysis showed that the diagnostic performance of 2D-SWE for Resonance Elastography (MRE) with FIB-4 (MEFIB score) in identifying
various fibrosis stages was good-to-optimal with AUROCs of 0.855, candidates with significant fibrosis. When MRE (≥3.3 kPa) was com­
0.928 and 0.917 for ≥F2, ≥F3 and cirrhosis, respectively. Proposed bined with FIB-4 (≥1.6), it produced a clinical-prediction-rule with a
cut-offs were 7.1 kPa for significant fibrosis (≥F2), 9.2 kPa for advanced PPV of 97.1 % for diagnosing significant fibrosis. These findings were
fibrosis (≥F3) and 13.0 kPa for cirrhosis (F4) [71]. validated in the “Japan-NAFLD” cohort, where the MEFIB score had an
Some studies have compared the diagnostic performance of fibrosis AUROC of 0.84 (95 % CI 0.78-0.89), demonstrating its potential as a
of different US elastography techniques. No significant differences were non-invasive tool for identifying MASH patients in need of treatment
highlighted in terms of performance although VCTE remains the most [84].
validated technique and therefore still the most recommended by in­
ternational guidelines [9,39,72,73]. 5. Sequential use of NITs for optimized management
MRE studies the propagation of shear waves in liver tissue converting
the wavelength information into tissue stiffness maps [74,75]. International guidelines recommend the use of a two-tier testing in
Compared with US elastography techniques, MRE offers a panoramic primary care, starting from the FIB4, which is an inexpensive widely
assessment of fibrosis and a superior spatial resolution, exhibiting su­ available serum test that can rule out with high sensitivity people who
perior performance in stratifying intermediate stages of fibrosis. How­ do not have advanced fibrosis, before moving to a second tier test [9,39].
ever, its high cost and low availability limits its practical application A proposed algorithm for testing patients at risk of MASLD in
primarily to research purposes [9,12]. MRE, is reliable for diagnosis of non-hepatology settings is shown in Fig. 1. In the primary care setting, it
significant fibrosis (F ≥ 2, AUC: 0.88), advanced fibrosis (F ≥ 3, AUC: is necessary to use a test that is easy and inexpensive to calculate or

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M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

Fig. 1. Testing algorithm for patients with diagnosed or suspected MASLD in primary care or endocrinology clinics. This consists of a two-tier sequential testing,
starting with a FIB-4 and followed by either Fibroscan or ELF, depending on what is locally available.

measure, and given the low prevalence of advanced fibrosis, the goal 6. NITs for assessment of prognosis in MASLD patients
should be to use a cut-off that can reliably exclude disease (high sensi­
tivity and NPV) and refer to secondary care patients at risk of disease Liver fibrosis represents the most relevant prognostic factor in pa­
[85]. In the secondary care setting, the size of the population to be tested tients with MASH, particularly regarding the risk of liver cancer, liver-
is smaller and the prevalence of cACLD is higher. This allows the use of related events, cardiovascular events, liver transplant and death [3,
NITs that have a better diagnostic accuracy but also a higher cost, with 90–93].
the aim of reliably identifying patients with the target condition (high The need of histopathological staging of fibrosis for the assessment of
specificity and high PPV) and undertaking the correct clinical and the prognosis of patients with MASH is a major limitation as liver biopsy
therapeutic management of cACLD. The main limitation of the dual is invasive, expensive and therefore can’t be performed on a large
cut-off strategy lies in the fact that a relevant percentage of patients, 40 population. The use of non-invasive tests as prognostic factors that guide
to 60 % depending on the setting and the NIT, have an indeterminate the clinical and therapeutic choices of patients with MASH represents an
result. This limitation can be partially overcome by using independent overcoming of this limit and is strongly encouraged as an area of
NITs in a sequential or simultaneous approach or by performing a liver research by the main scientific societies worldwide.
biopsy [6,57,86]. Ultimately, the widespread use of such testing algo­ NFS and FIB-4 showed a good performance for prediction of liver
rithms in populations at risk of liver disease will require a substantial related events (hazard ratio [HR], 2.77 and 1.68, respectively), HCC
increase in testing capacity, both in terms of funding and also facilities. (HR, 4.2 and 1.67, respectively) and overall mortality (HR, 2.86 and
The use of population-derived risk scores, such as the LiverRisk score, 2.00 respectively) whereas their association with risk of cardiovascular
can potentially refine the patient population that requires testing [87]. events (HR, 1.46 and 1.24, respectively) and extrahepatic cancers (HR,
The implementation of a two-tier approaches utilizing NITs is overall 1.66 and 1.24, respectively) is lower [94].
a promising strategy in the management of MASLD in primary care, According to a recent meta-analysis, NFS and FIB-4 have the best
offering cost-effective and clinically efficient pathways for identifying performance for prediction of all-cause mortality compared with other
patients at risk of advanced fibrosis and streamlining referrals to sec­ indirect NITs (APRI and BARD) with a HR>3. Interestingly, NFS appear
ondary care. to be a strong predictor also of cardiovascular-related mortality in this
In a modelling study, Crossan et al. tested a pathway for triaging study (HR 3.09), probably due to the fact that NFS includes known
patients with NAFLD in primary care using NITs to identify those at risk cardiovascular risk factors such as T2DM and BMI, not considered in the
of advanced fibrosis. The sequential use of NITs, starting with FIB-4 FIB-4 [95]. Changes over time of NFS and FIB-4 are significantly asso­
followed by ELF or Fibroscan® for indeterminate results, effectively ciated with fibrosis progression: for every unit change in FIB-4 or NFS a
reduced unnecessary secondary care referrals by 90 %, resulting in a 40 mean fibrosis stage progression of 0.26 and 0.19 was observed, respec­
% cost savings per patient. The proposed two-tier approach accurately tively [96].
identified patients at the highest risk of advanced fibrosis and disease A prognostic role in MASLD patients has also been demonstrated for
progression, offering an efficient strategy for managing NAFLD in pri­ ELF. Many authors showed that patients with ELF>11.3 have a higher
mary care settings [88]. risk (5-fold) of liver related events in patients with MASLD [97].
Srivastava et al. assessed the clinical and cost effectiveness of LSM has been proposed as a reliable prognostic factor in MASLD
implementing NITs in primary care for patients with NAFLD, using a patients [98,99]. Boursier et al. showed that a prognostic stratification
similar two-tier model. Their study revealed that the adoption of NITs could be achieved according to baseline LSM in terms of overall survival
led to a notable reduction in unnecessary referrals, resulting in sub­ and risk of liver related events [100]. Similar results have been
stantial cost savings (from 3 to 17 %). This underscores the significant confirmed by other studies and meta-analyses [101,102]. LSM values
benefits of integrating NITs into primary care for effective NAFLD pa­ above 10–12 kPa are associated with a higher risk of liver related events
tient management [89]. and a lower overall survival, confirming advanced fibrosis as a main
prognostic factor for MASLD. A recent individual patient meta-analysis

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M. Zoncapè et al. European Journal of Internal Medicine 122 (2024) 11–19

by Mozes et al. showed that the prognostic stratification based on LSM patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2019;49:
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