Extracellular vesicles as biomarkers for metabolic dysfunction-associated steatotic liver disease staging: Methodological innovations and future perspectives

Table 1 Overview of current non-invasive diagnostic approaches for metabolic dysfunction-associated steatotic liver disease

Category	Method/model	Principle	Key variables	Advantages	Limitations
Imaging-based	MRI-PDFF/MAST	Quantitative imaging of liver fat & fibrosis	Liver fat fraction, stiffness	High accuracy, quantitative	Expensive, limited availability
	MEFIB	MRI elastography + FIB-4	Liver stiffness + clinical data	High PPV for fibrosis	Requires MRI access
	FibroScan (FAST, Agile)	Transient elastography	CAP, liver stiffness, AST	Non-invasive, widely used	Intermediate zone exists
	Ultrasound (ATI, 2D-SWE)	Acoustic attenuation/elasticity	Echo attenuation, stiffness	Accessible, bedside use	Lower accuracy vs MRI
Serum biomarkers	FIB-4	Routine lab-based index	Age, AST, ALT, platelets	Simple, widely available	Limited specificity
	ELF test	Fibrosis marker panel	HA, PIIINP, TIMP-1	Good for advanced fibrosis	Cost, lab dependence
	NIS4/NIS2+	Multi-marker panel	miRNA, proteins, HbA1c	Detects at-risk MASH	Limited accessibility
	ADAPT/PRO-C3	ECM remodeling biomarkers	PRO-C3, platelets, diabetes	Mechanism-based	Needs validation
	CK-18/FGF21	Cell death markers	Apoptosis-related proteins	Reflects inflammation	Moderate performance
Metabolic indices	TyG index	Insulin resistance proxy	TG, glucose	Simple, scalable	Indirect marker
Machine learning	Clinical ML models	Data-driven prediction	Anthropometric + lab data	High scalability	Data dependency
Microbiome-based	Gut microbiome signature	Metagenomic profiling	Bacterial composition	Novel mechanism insight	Not standardized
Digital pathology/AI	qFIBS	ML-assisted histology	Fibrosis, inflammation	High reproducibility	Requires biopsy
EV-based (emerging)	EV size + ML	Nanoparticle + AI	EV size, concentration, clinical data	Non-invasive, innovative	Needs validation

ADAPT: Algorithm for Disease Assessment in Progressive Fibrosis Testing; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ATI: Attenuation imaging; CAP: Controlled attenuation parameter; CK-18: Cytokeratin-18; ELF: Enhanced liver fibrosis test; EV: Extracellular vesicle; FAST: FibroScan-AST; FGF21: Fibroblast growth factor 21; FIB-4: Fibrosis-4 index; HA: Hyaluronic acid; MASH: Metabolic dysfunction-associated steatohepatitis; MAST: MRI-based assessment of steatosis and fibrosis; MEFIB: Magnetic resonance elastography and FIB-4 index; MRI-PDFF: Magnetic resonance imaging-proton density fat fraction; NIS2⁺: Non-Invasive Scoring system version 2⁺; NIS4: Non-invasive scoring system version 4; PIIINP: Procollagen III N-terminal propeptide; PRO-C3: N-terminal propeptide of type III collagen; qFIBS: Quantitative fibrosis, inflammation, ballooning, steatosis; TG: Triglyceride; TIMP-1: Tissue inhibitor of metalloproteinases-1; TyG index: Triglyceride-glucose index; 2D-SWE: 2-dimensional shear wave elastography; AI: Artificial intelligence; ML: Machine learning; HbA1c: Hemoglobin A1c.