The definition and diagnostic criteria for MASLD have undergone a fundamental paradigm shift in recent years, transitioning from an exclusion-based diagnosis (NAFLD) to a positive, criteria-based diagnosis (MASLD) based on hepatic steatosis and metabolic risk factors^[2,21]. This shift not only simplifies the clinical diagnostic process but also lays a clearer foundation for the application of NITs. The term NAFLD has been widely used since 1980. Its diagnosis relied on an exclusion-based approach: ruling out excessive alcohol consumption (≤ 30 g/day for men, ≤ 20 g/day for women) and other specific liver disease etiologies, while confirming hepatic steatosis^[22]. This diagnostic framework did not explicitly acknowledge the central roles of obesity, insulin resistance, and metabolic syndrome in disease pathogenesis, and the term itself carried stigmatizing connotations^[21]. To address this limitation, an international panel of experts proposed in 2020 the term “metabolic associated fatty liver disease (MAFLD)”, advocating for a definition based on positive metabolic criteria - a pivotal shift from “what to exclude” to “what to include”^[22]. Following extensive consultation, the 2023 multi-society Delphi consensus - involving the American Association for the Study of Liver Diseases (AASLD), the European Association for the Study of the Liver (EASL), and other major societies - officially adopted the term “MASLD” to promote global uniformity in clinical practice and research^[2]. This change renders the diagnostic process more intuitive and practical, encourages clinicians to actively screen for metabolic risk factors, acknowledges that multiple liver diseases frequently coexist in clinical practice, and formally establishes metabolic dysfunction as the central disease paradigm^[21,22]. Beyond pathophysiological considerations, concerns over patient stigmatization associated with the term “fatty” also contributed to the nomenclature change^[21].

However, the evidence for stigma as a primary driver of nomenclature change is more nuanced than commonly assumed. A global survey across 21 countries (1,976 patients and 825 providers) found a marked discordance: only 8% of patients reported having experienced stigmatization due to NAFLD, whereas 38% of providers believed the term “fatty” was stigmatizing. Moreover, 26% of patients reported stigma related to overweight/obesity - more than three times the rate attributable to the disease name itself. Stigma perception varied substantially by region, ranging from 22% in the USA to 3% in the Middle East and North Africa for disease-name-related stigma^[23]. This regional variation may partly reflect cultural factors: in some settings, plumpness is regarded as a positive sign of prosperity, fertility, and health rather than a health risk. Notably, physicians’ own attitudes may contribute to internalized self-stigma: 46% of providers reported discomfort in caring for patients with NAFLD, viewing them as lacking willpower or self-control, while 33% of patients believed they were at least partly to blame for their liver disease^[24]. These findings suggest that the rationale for renaming is not a universally experienced patient-level stigma, but a more complex interplay of provider perception, cultural context, and internalized blame.

The diagnostic criteria for MASLD follow a positive-definition approach, comprising one mandatory criterion and at least one concomitant criterion^[2]:

Alcohol intake must be carefully quantified in all patients with steatotic liver disease, as it determines sub-classification: MASLD (≤ 20 g/day for women, ≤ 30 g/day for men), metabolic and alcohol-associated liver disease (MetALD) (> 20 to ≤ 50 g/day for women, > 30 to ≤ 60 g/day for men), and alcoholic liver disease (ALD) (> 50 g/day for women, > 60 g/day for men)^[2]. This distinction acknowledges that metabolic dysfunction and alcohol-related liver injury frequently coexist.

The diagnosis of MASLD is established by confirming hepatic steatosis and at least one cardiometabolic risk factor, together with the exclusion of other specific causes of steatotic liver disease^[21]. Exclusion of other aetiologies is therefore an integral component of the diagnostic process, rather than a secondary consideration. While the new nomenclature has shifted the diagnostic emphasis toward positive metabolic criteria, the exclusion of other causes remains essential to confirm the diagnosis^[2]. In contrast, the previous MAFLD definition allowed a diagnosis based on metabolic criteria alone without requiring exclusion of other aetiologies, which was a key point of divergence^[22].

The table below clearly compares the core differences between the NAFLD and MASLD diagnostic paradigms [Table 1]^[2,21,22,25]:

The new MASLD diagnostic criteria strengthen the central role of NITs in clinical practice. Because diagnosis no longer requires liver biopsy to exclude other etiologies, the assessment focus shifts to confirming hepatic steatosis ≥ 5% and evaluating both metabolic risk and fibrosis stage. Imaging techniques (e.g., ultrasound, CAP, MRI-PDFF) and serum biomarkers/scores (for assessing metabolic abnormalities and fibrosis risk) are well suited as non-invasive tools for these purposes. Thus, NITs have evolved from a supporting role to central components of MASLD diagnosis, risk stratification, and comprehensive management.

The FIB-4 index is among the most widely used serum scoring systems for liver fibrosis in clinical practice. Its formula integrates four routine clinical parameters: age, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and platelet count. Because all variables are obtained from routine tests at no additional cost, FIB-4 is a highly cost-effective and readily accessible initial screening tool^[2,29,30].

In clinical practice, the FIB-4 index guides decision-making through clearly defined risk stratification thresholds. The exclusion threshold is set at FIB-4 < 1.3, yielding a very high NPV for ruling out advanced fibrosis (F3-F4)^[31], rendering it particularly suitable for primary care. The referral threshold of FIB-4 > 2.67 indicates an increased likelihood of advanced fibrosis or cirrhosis in specialty care settings, although PPV vary substantially with disease prevalence and should not be interpreted as fixed test properties^[2]. Patients with scores above this threshold warrant referral for further evaluation^[2,32]. However, for patients with scores in the grey zone (1.3-2.67), the fibrosis stage remains highly uncertain, and second-line imaging assessment - such as VCTE - is often required for further risk stratification.

Despite its core advantages (low cost, simple calculation, extensive validation, and high NPV), recent studies have identified several limitations. First, the index exhibits age bias, with potentially limited accuracy in young (< 35 years) and elderly patients; particularly in those over 65 years of age, FIB-4 may yield false-positive results. To improve specificity in this age group, the EASL guidelines recommend using a higher lower threshold of 2.0 (instead of 1.3) for ruling out advanced fibrosis, while the upper threshold of 2.67 remains unchanged^[33]. Second, because thrombocytopenia and elevated transaminases are not liver-specific, FIB-4 has limited ability to distinguish hepatic inflammation from fibrosis, and its diagnostic accuracy may be compromised in patients with severe obesity or metabolic comorbidities^[34]. Third, the performance of FIB-4 is particularly suboptimal in higher-risk populations, especially in patients with T2D, where it has been shown to yield high false-positive rates and limited specificity, prompting some guidelines to question its routine use as a first-line screening tool in this population^[34,35]. Given these limitations, FIB-4 is often recommended as an initial screening tool to identify low-risk individuals and avoid unnecessary referrals, whereas patients with indeterminate results require sequential evaluation using further testing^[35]. In addition, FIB-4 may reflect systemic congestion rather than liver fibrosis alone, as supported by its prognostic value in heart failure^[36].

The NFS is another widely used serum-based scoring tool^[37]. It integrates routine clinical parameters - including age, BMI, fasting glucose or diabetes status, platelet count, albumin, and the AST/ALT ratio - for fibrosis risk assessment. In clinical stratification, NFS and FIB-4 are often recommended as first-line screening tools to identify the risk of advanced fibrosis, thereby optimizing referral pathways and reducing unnecessary liver biopsies^[2]. The main limitation of NFS is its susceptibility to BMI and age, which may lead to a high false-positive rate^[38].

The ELF test is a direct biomarker assay that quantifies three serum markers involved in extracellular matrix metabolism: hyaluronic acid (HA), procollagen III N-terminal peptide (PIIINP), and tissue inhibitor of metalloproteinases-1 (TIMP-1), thereby providing more direct pathophysiological information on fibrosis^[39]. Studies show that the ELF test has good diagnostic performance for detecting significant fibrosis, advanced fibrosis, and cirrhosis, with AUROC values of 0.811, 0.812, and 0.810, respectively^[40]. Despite its diagnostic accuracy advantages, the ELF test has limited availability and higher cost, and its specificity may decrease in low-prevalence populations. Therefore, guidelines suggest its use as an alternative to imaging for identifying advanced liver fibrosis or as a supplementary assessment for patients with indeterminate screening results.

PRO-C3 (procollagen III N-terminal peptide) is a direct serum biomarker that reflects active type III collagen formation during fibrogenesis, providing a real-time measure of fibroblast activity^[41]. In a cohort of 431 biopsy-proven NAFLD patients, PRO-C3 levels progressively increased with fibrosis stage (rho = 0.50, P < 0.0001) and remained independently associated with advanced fibrosis after adjustment for multiple confounders (OR = 1.05, P = 0.003)^[42].

Based on PRO-C3, the ADAPT score was developed to identify patients with advanced fibrosis (F ≥ 3). The score integrates four variables: Age, presence of DiAbetes, PRO-C3, and plaTelet count. In the derivation cohort (n = 150), ADAPT achieved an AUROC of 0.86 [95% confidence interval (CI): 0.79-0.91], which was superior to the AST to platelet ratio index (APRI) (0.73) and numerically higher than FIB-4 (0.78) and NFS (0.78). These findings were confirmed in an independent validation cohort (n = 281), with an AUROC of 0.87 (95%CI: 0.83-0.91), outperforming APRI (0.78, P = 0.0005) and NFS (0.79, P = 0.02), and comparable to FIB-4 (0.85, P = 0.32)^[42]. Notably, ADAPT maintained robust performance across various subpopulations, including diabetics vs. non-diabetics and different BMI categories.These data support PRO-C3 and ADAPT as promising tools for identifying patients with advanced fibrosis and for monitoring treatment response in MASH. ADAPT was derived and validated in biopsy-proven NAFLD cohorts from Australia, the UK, and Japan.

The LiverRisk score is a next-generation serum scoring system. Derived from an international prospective cohort, it integrates eight variables: age, sex, and six standard laboratory parameters - AST, ALT, gamma-glutamyl transferase (GGT), fasting glucose, total cholesterol, and platelet count - to quantify liver-related mortality risk. Using three cut-offs (6, 10, and 15 points), the score classifies individuals into four risk levels (very low, low, medium, and high), providing a dynamic basis for clinical decision-making. In terms of diagnostic performance, the LiverRisk score achieves an AUROC of 0.83 for predicting liver stiffness ≥ 10 kPa, markedly outperforming the traditional FIB-4 index (AUROC 0.68)^[43]. More importantly, the score demonstrates strong discriminative ability for long-term prognosis; the hazard ratio (HR) for liver-related mortality is 471 in the high-risk group compared with the very low-risk group^[43], underscoring its clinical value for risk stratification and prognostic assessment.

The SAFE score is a non-invasive assessment tool specifically designed for MASLD to optimize risk stratification in primary care settings^[44]. It integrates readily available clinical parameters, including age, BMI, diabetes status, platelet count, AST, ALT, and globulin levels. Compared with traditional blood biomarkers, the SAFE score demonstrates superior ability to identify low-risk patients, effectively distinguishing mild fibrosis (F0-F1) from significant fibrosis (≥ F2), thereby reducing reliance on invasive tests. In NAFLD populations, the SAFE score outperforms the FIB-4 index and NFS, particularly in patients with metabolic risk factors such as obesity or T2D; however, its broader applicability requires further validation across diverse populations. The SAFE score was developed using the NASH Clinical Research Network observational cohort as the training set, and validated in the FLINT trial cohort and in a Stanford cohort with MRE-based fibrosis assessment. The LiverRisk score was derived and validated in large European general population cohorts^[45-47].

The LiverPRO score is an innovative software tool designed for primary care settings, aiming to achieve efficient risk stratification using routine clinical data. The tool is highly flexible, automatically selecting 3-9 variables from a candidate pool of nine routine laboratory tests plus age, and generating 466 multivariable models to accommodate varying data availability across clinical settings. For diagnostic performance, the LiverPRO score achieves an AUROC of 0.86 for detecting LSM ≥ 8 kPa in the derivation cohort and 0.80 in the DECIDE validation cohort^[48]. Validation data indicate that its diagnostic performance is comparable to that of the ELF and LiverRisk scores and significantly superior to traditional FIB-4 and MAFLD scores.

The Agile scoring system is an innovative assessment tool specifically developed for the MASLD population to address the clinical need for accurate diagnosis of advanced fibrosis and cirrhosis. The system includes two sub-scores: Agile 3+ for assessing advanced fibrosis (F3) and Agile 4 for cirrhosis (F4)^[49]. A meta-analysis of seven studies (6,037 participants) showed that in MASLD patients, Agile 4 had a pooled sensitivity of 0.90 (95%CI: 0.80-0.95) for excluding cirrhosis (< 0.251) and a pooled specificity of 0.93 (95%CI: 0.86-0.97) for ruling in cirrhosis (≥ 0.565)^[50]. Another external validation study showed that Agile 3+ had an AUROC of 0.86 for identifying F3 fibrosis and Agile 4 had an AUROC of 0.89 for identifying F4 fibrosis, though these values were not significantly different from the diagnostic accuracy of LSM alone. Agile scores have been validated in MASLD and ALD populations, but their performance in viral hepatitis is inferior to LSM^[49].

The MAST score integrates MRI-PDFF, MRE-derived liver stiffness, and AST to identify patients with fibrotic NASH [NAFLD activity score (NAS) ≥ 4, F ≥ 2)]. In a validation cohort, it achieved an AUROC of 0.93 (95%CI: 0.88-0.97), with rule-out (< 0.165) and rule-in (> 0.242) cut-offs, outperforming FIB-4, NFS, and the FibroScan-AST (FAST) score. MAST was validated in tertiary care cohorts with a low prevalence of Fibro-NASH, reflecting real-world clinical settings^[51].

The MEFIB index combines MRE ≥ 3.3 kPa and FIB-4 ≥ 1.6 as a binary rule-in for significant fibrosis (≥ F2). It demonstrated a PPV of 97.1% in the training cohort and 91.0% in an independent Japanese validation cohort^[52].

Both scores are limited to specialized centers with MRE availability but offer high diagnostic accuracy for identifying candidates for pharmacotherapy and clinical trial enrichment.

FibroSure (also known as FibroTest) is a proprietary algorithm-based assay that integrates several serum markers - alpha-2-macroglobulin (α2-M), haptoglobin (Hp), apolipoprotein A1 (ApoA1), GGT, and total bilirubin (TBil) - to assess the degree of liver fibrosis and necroinflammatory activity. In patients with NAFLD, the FibroTest (FibroSure) has demonstrated good diagnostic accuracy for advanced fibrosis and cirrhosis, with AUROCs of 0.86 for F2-F4 and 0.92 for F3-F4 in a prospective validation study^[53]. However, it may be affected by acute inflammation or hemolysis. Therefore, guidelines recommend its combined use with other diagnostic modalities, such as VCTE, to provide a comprehensive assessment of liver pathology.

VCTE is the most widely used non-invasive imaging assessment tool. Its core technology generates shear waves in the liver via transient vibration, measures their propagation speed to calculate liver stiffness, and simultaneously uses CAP to quantify hepatic steatosis. VCTE has good diagnostic accuracy for advanced fibrosis (F3-F4), with reported AUROC values ranging from 0.82 to 0.88 for detecting F3-F4 fibrosis^[54]. However, the diagnostic accuracy of VCTE can be compromised by liver inflammation, cholestasis, and hepatic congestion - all of which can transiently elevate stiffness measurements independent of fibrosis stage, contributing to spectrum bias - as well as narrow intercostal spaces. In addition, the failure rate is significantly higher in patients with severe obesity, primarily due to increased skin-to-capsule distance and thicker subcutaneous adipose tissue, which attenuate shear wave propagation. Therefore, in clinical practice, VCTE is often recommended as a second-line assessment tool after serum scoring (e.g., FIB-4) for risk stratification and dynamic monitoring of disease progression^[2].

MRE is currently the most accurate non-invasive imaging technique for assessing liver fibrosis. It combines conventional magnetic resonance imaging (MRI) with elastography principles to generate visual maps that reflect liver stiffness distribution across the entire organ^[55]. MRE demonstrates high diagnostic accuracy for significant fibrosis (≥ F2), with an AUROC of 0.89, and is widely regarded as the most accurate non-invasive method for fibrosis staging, although liver biopsy remains the definitive histological reference standard^[14]. This technique is particularly advantageous in obese patients and for detecting early-stage fibrosis (F0-F2), because its diagnostic performance is less affected by abdominal wall thickness^[56]. Nevertheless, widespread use of MRE is limited by high cost, limited equipment availability, and high patient cooperation requirements. It is currently used mainly as a second- or third-line tool in specialized centers for patients with inconclusive serum or VCTE results, those requiring precise diagnosis, or those enrolled in clinical trials.

SWE is an ultrasound-based real-time technique for measuring liver stiffness, with diagnostic accuracy comparable to that of VCTE^[57]. However, its sensitivity for early-stage fibrosis remains relatively low^[58].

The core advantage of SWE lies in its ability to provide objective, quantitative, and organ-specific liver stiffness data, with diagnostic accuracy significantly higher than that of serum scores, especially for detecting at least moderate fibrosis^[57-59]. Some studies have suggested that SWE may be feasible in patients with obesity when using deep-penetration probes, although measurement reliability is still largely dependent on skin-to-capsule distance and adequate acoustic windows. Limitations include higher cost than serum tests, and measurement accuracy can be affected by operator skill, severe obesity (particularly with increased skin-to-capsule distance), ascites, and acute hepatitis^[57,60]. Furthermore, as with MRE, advanced imaging techniques such as SWE remain poorly accessible in primary care.

Conventional B-mode ultrasonography is the most widely available and affordable imaging modality for hepatic steatosis. It relies on qualitative features such as increased liver echogenicity (bright liver), hepatorenal echo contrast, blurring of intrahepatic vessel margins, and posterior beam attenuation. Its advantages include low cost, broad availability in primary care, lack of radiation, and feasibility for repeated assessments. However, its sensitivity for detecting mild steatosis is limited; it generally requires hepatic fat content > 20%-30% to be reliably detected, and its accuracy is reduced in obesity and by operator dependence^[61].

To improve standardization, semiquantitative ultrasonographic indices have been developed, including the hepatorenal index (HRI), the ultrasound fatty liver index (US-FLI), and graded scoring systems (mild, moderate, severe). These indices provide a more reproducible estimate of steatosis severity than qualitative B-mode alone and can detect mild steatosis in some patients, although they remain operator-dependent and are less accurate than MRI-PDFF or CAP for quantifying small changes in hepatic fat. Nevertheless, given their affordability and broad availability, conventional and semiquantitative ultrasonography remain useful first-line tools for identifying hepatic steatosis in primary care and for large-scale screening, particularly in resource-limited settings.

Quantitative ultrasound techniques, such as tissue attenuation imaging (TAI) and tissue scatter distribution imaging (TSI), represent major advances in assessing hepatic steatosis on ultrasound platforms. TAI quantifies hepatic fat content by measuring the rate of ultrasound energy attenuation in liver tissue, and its attenuation coefficient correlates highly with histological steatosis grade. TSI evaluates tissue microstructure by analyzing the statistical distribution of ultrasound backscatter signals. Notably, studies have reported that TSI parameters correlate with histological activity scores; however, it is important to emphasize that, like all current NITs, TSI cannot directly assess microscopic features such as lobular inflammation or hepatocyte ballooning, which require histological examination^[2]. These two techniques extract information from different physical dimensions (energy attenuation vs. structural scattering), enabling simultaneous quantitative assessment of the two core pathological changes in MASLD - steatosis and inflammatory activity.

In terms of diagnostic performance, TAI has good accuracy for assessing hepatic steatosis, whereas TSI shows strong performance in detecting inflammation. The combined model of TSI and SWE demonstrates excellent diagnostic performance for predicting MASH^[62]. The principal value of this technique is that it equips widely available conventional ultrasound devices to perform accurate staging and risk stratification, serving as a bridge between primary screening and specialized assessment. Ideal applications include immediate quantitative assessment of incidentally identified hepatic steatosis in primary care, or use as a complement to VCTE in specialized clinics to evaluate inflammatory activity risk.

Compared with existing technologies, TAI can cross-validate with CAP for fat quantification, but its key advantage lies in performing TAI, TSI, and conventional ultrasound on the same device during a single examination^[62,63]. Although its quantitative accuracy does not yet match that of MRI-PDFF, quantitative ultrasound offers substantial advantages in accessibility, cost, and convenience^[63]. It provides direct tissue information with greater organ specificity than indirect serum models. Notable advantages include high platform ubiquity, an integrated “conventional ultrasound + TAI + TSI + SWE” workflow, and no additional cost or risk. Current challenges include the need for further research to standardize scanning protocols, establish diagnostic thresholds, and overcome operator dependence. Future trends involve deep integration with AI algorithms to develop more powerful automated diagnostic and predictive models.

Composite scoring systems represent one approach. The FAST score, which combines VCTE-derived liver stiffness with serum AST levels, identifies individuals at high risk for fibrotic MASH (i.e., MASH with significant fibrosis). A meta-analysis of 12 studies comprising 5,835 patients reported a pooled sensitivity of 89%, specificity of 89%, and NPV of 92% at optimal cut-offs, demonstrating excellent exclusion efficacy^[64].