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Objectives: Metabolic dysfunction‐associated steatotic liver disease (MASLD) is the most prevalent liver disorder in Western countries, with approximately 20%–30% of the MASLD patients progressing to severe stages. There is an urgent need for noninvasive, cost‐effective, widely accessible, and precise biomarkers to evaluate liver steatosis. This study aims to assess and compare the diagnostic performance of a novel reference frequency method‐based ultrasound attenuation coefficient estimation (ACE) in both fundamental (RFM‐ACE‐FI) and harmonic (RFM‐ACE‐HI) imaging for detecting and grading liver steatosis. Methods: An Institutional Review Board‐approved prospective study was carried out between December 2018 and October 2022. A total number of 130 subjects were enrolled in the study. The correlation between RFM‐ACE‐HI values and magnetic resonance imaging proton density fat fraction (MRI‐PDFF), as well as between RFM‐ACE‐FI values and MRI‐PDFF were calculated. The diagnostic performance of RFM‐ACE‐FI and RFM‐ACE‐HI was evaluated using receiver operating characteristic (ROC) curve analysis, as compared to MRI‐PDFF. The reproducibility of RFM‐ACE‐HI was assessed by interobserver agreement between two sonographers. Results: A strong correlation was observed between RFM‐ACE‐HI and MRI‐PDFF, with R = 0.88 (95% confidence interval [CI]: 0.83–0.92; P <.001), while the correlation between RFM‐ACE‐FI and MRI‐PDFF was R = 0.65 (95% CI: 0.50–0.76; P <.001). The area under the ROC (AUROC) curve for RFM‐ACE‐HI in staging liver steatosis grades of S ≥ 1 and S ≥ 2 was 0.97 (95% CI: 0.91–0.99; P <.001) and 0.98 (95% CI: 0.93–1.00; P <.001), respectively, and 0.76 (95% CI: 0.65–0.85) and 0.80 (95% CI: 0.70–0.88) for RFM‐ACE‐FI, respectively. Great reproducibility was achieved for RFM‐ACE‐HI, with an interobserver agreement of R = 0.97 (95% CI: 0.94–0.99; P <.001). Conclusions: The novel RFM‐ACE‐HI method offered high liver steatosis diagnostic accuracy and reproducibility, which has important clinical implications for early disease intervention and treatment evaluation. [ABSTRACT FROM AUTHOR]

Ultrasound localization microscopy (ULM) enables microvascular imaging at spatial resolutions beyond the acoustic diffraction limit, offering significant clinical potentials. However, ULM performance relies heavily on microbubble (MB) signal sparsity, the number of detected MBs, and signal-to-noise ratio (SNR), all of which vary in clinical scenarios involving bolus MB injections. These sources of variations underscore the need to optimize MB dosage, data acquisition timing, and imaging settings in order to standardize and optimize ULM of microvasculature. This pilot study investigated temporal changes in MB signals during bolus injections in both pig and human models to optimize data acquisition for clinical ULM. Quantitative indices were developed to evaluate MB signal quality, guiding selection of acquisition timing that balances the MB localization quality and adequate MB counts. The effects of transmitted voltage and dosage were also explored. In the pig model, a relatively short window (approximately 10 seconds) for optimal acquisition was identified during the rapid wash-out phase, highlighting the need for real-time MB signal monitoring during data acquisition. The slower wash-out phase in humans allowed for a more flexible imaging window of 1-2 minutes, while trade-offs were observed between localization quality and MB density (or acquisition length) at different wash-out phase timings. Guided by these findings, robust ULM imaging was achieved in both pig and human kidneys using a short period of data acquisition, demonstrating its feasibility in clinical practice. This study provides insights into optimizing data acquisition for consistent and reproducible ULM, paving the way for its standardization and broader clinical applications., Competing Interests: Competing Interests: The Mayo Clinic and some of the authors (C.H., JZ and S.C.) have a potential financial interest related to the technology referenced in the research.