Lipid Microparticle-Based Phantoms Modeling Hepatic Steatosis for the Validation of Quantitative Imaging Techniques

Metabolic dysfunction-associated steatotic liver disease (MASLD) typically presents as "macrovesicular steatosis", where each hepatocyte contains a large fat vacuole (30-50 µm), indicating a more indolent form. In about 20% of cases, "microvesicular steatosis" occurs, with smaller vacuoles (1-15 µm) linked to steatohepatitis, cirrhosis progression, and increased risk of liver cancer. Emerging quantitative ultrasound (QUS) liver fat quantification (QUS-LFQ) tools measure various acoustic properties, but few methods compare techniques and imaging modalities, and the impact of fat vacuole size remains unclear. This study introduces a methodology to create ultrasound (US) phantoms that replicate fat vesicle size in MASLD. While imaging phantoms validate quantitative tools, no model currently links QUS-LFQ measurements to steatosis severity. Existing homogeneous phantoms assessing properties like attenuation, backscatter, and speed of sound overlook the microstructure of steatosis, despite the known effect of particle size on acoustic interactions. Here, agar-based phantoms simulate fat accumulation in steatotic hepatocytes using stable peanut oil droplets as analogs for lipid vacuoles. Microscopy and sizing confirm stability at 4 °C, 23 °C, and 50 °C. Both microscopy and US imaging confirm uniform distribution, with QUS-LFQ measurements reflecting fat content. These phantoms hold promise for validating quantitative imaging methods, particularly for US-based MASLD screening tools.