Impact of acquisition geometry on ultrasound-derived fat fraction measurements: implications for standardized acquisition and measurement interchangeability

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is among the most prevalent chronic liver diseases worldwide, affecting approximately 30–40% of the adult population and representing a substantial and growing public health burden (1,2). Reliable quantification of hepatic steatosis is essential for diagnosis, risk stratification, longitudinal monitoring, and quantitative imaging research. Although liver biopsy remains the reference standard, its invasiveness, sampling variability, and limited suitability for repeated assessment limit its practicality as a quantitative imaging biomarker (3). Consequently, noninvasive imaging-based approaches for hepatic fat quantification have received increasing attention (4).

Magnetic resonance imaging proton density fat fraction (MRI-PDFF) enables accurate whole-liver fat quantification and has been widely adopted as a noninvasive reference technique in research settings (5,6). However, its broader clinical implementation is limited by cost, restricted accessibility, breath-holding requirements, and susceptibility to confounding factors such as iron overload (7). These constraints underscore the need for a practical, repeatable, and widely accessible quantitative imaging biomarker for hepatic steatosis assessment in routine clinical practice and large-scale or longitudinal studies.

Ultrasound is the most widely available modality for liver imaging. Although conventional B-mode assessment of steatosis remains qualitative and operator dependent, recent quantitative ultrasound techniques based on radiofrequency data, including the attenuation coefficient and backscatter coefficient, enable objective characterization of hepatic tissue composition (8). Building on these approaches, ultrasound-derived fat fraction (UDFF) integrates attenuation- and backscatter-related information to provide a composite quantitative estimate of hepatic steatosis and has demonstrated linear correlation with MRI-PDFF, along with favorable intra-observer repeatability and inter-observer reproducibility (9).

As a composite parameter derived from acoustic backscatter- and attenuation-related processes, UDFF is inherently sensitive to acquisition geometry and beam-path characteristics (10). Preliminary studies have explored UDFF measurements obtained at different depths and anatomical acquisition sites (11). However, acquisition-related variability and its impact on the interchangeability of UDFF measurements remain insufficiently characterized. In routine clinical practice, preferred acquisition conditions may not always be achievable because of rib shadowing, body habitus, or limited acoustic access, potentially resulting in variation in acquisition window selection and region of interest (ROI) positioning. The contribution of skin-to-liver capsule distance (SCD) to this variability is also poorly understood. Importantly, high repeatability within a given acquisition configuration does not necessarily imply interchangeability between measurements obtained under different acquisition configurations. Unaccounted geometry-related variability may introduce systematic bias, be misinterpreted as biological variation, and compromise longitudinal comparability of UDFF measurements, particularly in follow-up and multicenter settings. At present, acquisition protocols for UDFF are not fully standardized, and existing recommendations remain largely vendor-specific.

Accordingly, this prospective study was designed to systematically characterize the impact of anatomical measurement window, subcapsular sampling depth, and SCD on UDFF measurements, with specific emphasis on geometry-related systematic variability, individual-level variability, and measurement interchangeability. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0219/rc).

Methods

Study design and participants

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This prospective study was conducted at the Department of Medical Ultrasound, between September and December 2024, and was approved by the Institutional Biomedical Ethics Committee of West China Hospital, Sichuan University (approval No. 1397). Written informed consent was provided by all participants. Adult volunteers undergoing conventional liver ultrasound and UDFF examinations were consecutively recruited. Of 258 initially enrolled participants, 238 were included in the final analysis after application of the predefined inclusion and exclusion criteria (Figure 1). The inclusion criteria were as follows: (I) age ≥18 years; (II) ability to comply with and complete standardized ultrasound and UDFF examinations; (III) normal liver appearance on conventional B-mode ultrasound, with no sonographic evidence of diffuse or focal liver disease; and (IV) provision of written informed consent. The exclusion criteria were as follows: (I) history of significant alcohol consumption (men >30 g/day, women >20 g/day) or use of steatogenic or hepatotoxic medications; (II) known chronic liver disease or sonographic evidence of hepatic steatosis, focal liver lesions, chronic hepatitis, cirrhosis, or biliary abnormalities; (III) prior hepatic surgery, trauma, or interventional procedures; and (IV) incomplete or technically inadequate UDFF acquisitions. Demographic and anthropometric data, including age, sex, height, weight, and body mass index (BMI), were recorded.

Figure 1 Study inclusion and exclusion flowchart. UDFF, ultrasound-derived fat fraction.

Conventional ultrasound examination

All conventional liver ultrasound examinations were performed by a single radiologist with more than 10 years of experience (D1), who completed dedicated training for the study protocol. Examinations were performed using an ACUSON Sequoia system (Siemens Healthineers, Erlangen, Germany). Participants were examined in a supine or slight left lateral position (≤30°) with the right forearm held behind the head and the arm in maximum abduction (180° from the resting position) to widen the intercostal space. Standardized upper abdominal scanning planes were obtained to exclude structural liver abnormalities. SCD was measured on the right anterior intercostal window of the liver.

UDFF acquisition

Following conventional ultrasound, UDFF measurements were acquired using the DAX transducer (1.0–3.5 MHz). A rectangular ROI (3 cm × 3 cm) was positioned parallel to the liver capsule within homogeneous hepatic parenchyma, while avoiding large vessels, biliary structures, rib shadowing, and focal echogenic artifacts. Eight predefined acquisition sites (sites 1–8) were selected to disentangle window- and depth-dependent effects (Figure 2). Subcapsular sampling depth, defined as the distance from the liver capsule to the upper edge of the ROI, was set at 1.0, 1.5, 2.0, and 3.0 cm, depending on the site. According to the manufacturer’s recommendations, the right anterior intercostal window with a subcapsular sampling depth of 1.5 cm (site 5) was prespecified as an internal reference configuration for within-case comparisons. Detailed definitions of all acquisition sites are provided in Table 1. At each site, participants were instructed to perform a brief end-expiratory breath-hold during image acquisition. All acquisitions were performed according to a predefined standardized protocol across participants. Five consecutive acquisitions were performed, and the median UDFF value was used for analysis to reduce the influence of transient acquisition instability. For each acquisition, both the predefined subcapsular sampling depth and the skin-to-ROI center distance were documented.

Figure 2 Representative ultrasound images corresponding to the eight predefined acquisition sites used for UDFF measurements. (A-H) Sites 1–8, respectively, as defined in Table 1. UDFF, ultrasound-derived fat fraction.

Repeatability assessment

To evaluate measurement reliability, 80 participants were randomly selected. Two trained radiologists (D1 and D2, each with more than 5 years of experience) independently performed UDFF measurements at the reference configuration (site 5) during end-expiratory breath-hold to assess inter-observer agreement. For intra-observer assessment, D1 repeated the measurements in the same participants after a minimum interval of 2 hours under identical acquisition conditions.

Statistical analysis

Statistical analyses were performed using R software (version 4.4.2; R Foundation for Statistical Computing, Vienna, Austria). Continuous variables were summarized as mean ± standard deviation or median with interquartile range, as appropriate. Categorical variables were summarized as frequencies and percentages. Global differences were assessed using the Friedman test, followed by Bonferroni-adjusted Wilcoxon signed-rank tests for pairwise comparisons. A prespecified 5% UDFF threshold was used as a pragmatic benchmark for potentially meaningful within-case disagreement, intended to represent disagreement beyond expected measurement variability and potentially relevant to longitudinal assessment and threshold-based steatosis interpretation. Associations between UDFF and skin-to-ROI center distance were evaluated using linear regression. To account for repeated measurements and assess the independent effects of acquisition-related factors on systematic differences and absolute deviations, linear mixed-effects models were fitted with participant identity specified as a random intercept. Intra-observer repeatability and inter-observer reproducibility were evaluated using two-way random-effects intraclass correlation coefficients (ICCs). Bland-Altman analysis was used to assess agreement and interchangeability between UDFF measurements obtained under different acquisition configurations, as well as intra- and inter-observer repeatability. Effect estimates are presented with corresponding 95% confidence intervals (CIs), where appropriate. A two-sided P value <0.05 was considered statistically significant.

Results

Participant characteristics

A total of 238 participants were included in the final analysis. The mean age was 42.5±13.9 years (range, 21–83 years), and the mean BMI was 22.5±2.4 kg/m² (range, 16.5–29.1 kg/m²). The mean SCD was 1.64±0.30 cm (range, 1.1–3.2 cm). Overall, 93 participants (39.1%) were male. Detailed baseline characteristics are summarized in Table 2.

Impact of anatomical window selection on UDFF

With subcapsular sampling standardized at a depth of 1.5 cm, UDFF differed significantly across the five predefined anatomical acquisition windows (P<0.001; Figure 3). UDFF values obtained from left lobe windows (sites 1 and 2) were significantly higher than those obtained from right lobe windows (sites 3–5) (all adjusted P<0.001). No significant difference was observed between lateral and medial left lobe measurements (adjusted P=0.293). Within the right hepatic lobe, UDFF also differed significantly across acquisition windows (all adjusted P<0.001). Measurements obtained at the right subcostal window yielded higher UDFF values than those obtained at intercostal windows, and measurements at the posterior intercostal window were higher than those at the anterior intercostal window.

Figure 3 UDFF across anatomical acquisition windows at a subcapsular sampling depth of 1.5 cm. ****, P<0.0001. UDFF, ultrasound-derived fat fraction.

Using the right anterior intercostal window as the within-case reference, left lobe measurements demonstrated positive biases, with mean reference-based differences of +4.96% (95% CI: 4.36–5.56) at the lateral site and +6.05% (95% CI: 5.30–6.81) at the medial site (both P<0.001). Within the right lobe, measurements obtained at the right subcostal window showed a moderate positive bias of +2.63% (95% CI: 2.22–3.05), whereas the posterior intercostal window demonstrated a smaller but still statistically significant positive bias of +1.07% (95% CI: 0.70–1.44).

Interchangeability of UDFF across anatomical windows

In addition to systematic mean differences, substantial individual-level variability was observed across acquisition windows. Bland-Altman analysis demonstrated wide limits of agreement between non-reference windows and the reference window, with the greatest dispersion observed for left-lobe windows. The medial left lobe window exhibited the widest limits of agreement, ranging from −5.53% to +17.64% (Figure 4).

Figure 4 Bland-Altman analyses comparing UDFF measurements obtained at different anatomical acquisition windows, using the right anterior intercostal window as the reference. Mean UDFF values were concentrated in the lower range of the x-axis. SD, standard deviation; UDFF, ultrasound-derived fat fraction.

Using a prespecified deviation threshold of 5% UDFF relative to the reference window, exceedance rates were highest for left lobe windows, occurring in 45.0% of measurements at the medial site and 37.8% at the lateral site. Among right lobe windows, exceedance rates were lower, at 16.8% for the right subcostal window and 6.7% for the right posterior intercostal window.

Effect of subcapsular sampling depth on UDFF

UDFF differed significantly across standardized subcapsular sampling depths (1.0, 1.5, 2.0, and 3.0 cm; P<0.001; Figure 5). Mean UDFF values were highest at a depth of 1.0 cm, decreased at 1.5 cm, and remained relatively stable at deeper sampling depths (2.0 and 3.0 cm).

Figure 5 UDFF across subcapsular sampling depths at the right anterior intercostal window. ***, P<0.001; ****, P<0.0001. UDFF, ultrasound-derived fat fraction.

Using 1.5 cm as the reference depth within the right anterior intercostal window, measurements obtained at 1.0 cm demonstrated a positive bias of +2.03% (95% CI: 1.68–2.39) with wide limits of agreement (−3.46% to +7.52%). In contrast, measurements obtained at depths of 2.0 cm and 3.0 cm showed small but statistically significant negative biases of −0.42% (95% CI: −0.60 to −0.24) and −0.38% (95% CI: −0.63 to −0.13), respectively, relative to the reference depth (Figure 6).

Figure 6 Bland-Altman analyses comparing UDFF measurements obtained at different subcapsular sampling depths, with 1.5 cm serving as the reference depth. Mean UDFF values were concentrated in the lower range of the x-axis. SD, standard deviation; UDFF, ultrasound-derived fat fraction.

Factors associated with systematic bias and absolute deviations

In univariable analysis, UDFF decreased by a mean of 0.73% per 1-cm increase in skin-to-ROI center distance (P<0.001). In multivariable mixed-effects analyses, both anatomical acquisition window and subcapsular sampling depth remained independently associated with systematic bias and absolute deviations after adjustment for SCD, BMI, age, and sex (all P<0.001). When anatomical windows were varied at a standardized depth of 1.5 cm, left lobe windows demonstrated the largest independent positive biases and greatest absolute deviations, followed by the right subcostal window, whereas right intercostal windows exhibited the smallest independent effects. Higher BMI was independently associated with small increases in systematic bias (β=+0.15% per kg/m², P=0.021) and absolute deviations (β=+0.13% per kg/m², P=0.028). When subcapsular sampling depth was varied within the reference right anterior intercostal window, greater SCD was independently associated with larger absolute deviations (β=+1.05% per cm, P<0.001), whereas BMI, age, and sex were not independently associated.

Repeatability at a standardized reference configuration

At the reference right anterior intercostal window with a subcapsular sampling depth of 1.5 cm, UDFF demonstrated good repeatability. The intra-operator ICC was 0.83 (95% CI: 0.73–0.89), and the inter-operator ICC was 0.83 (95% CI: 0.73–0.89). Bland-Altman analysis demonstrated minimal systematic differences, with a mean difference of 0.06% (limits of agreement, −1.34% to 1.47%) for intra-operator measurements and 0.01% (−1.36% to 1.39%) for inter-operator measurements (Figure 7).

Figure 7 Intra- and inter-observer agreement of UDFF measurements at the reference acquisition configuration. SD, standard deviation; UDFF, ultrasound-derived fat fraction.

Discussion

UDFF is increasingly used for the noninvasive assessment and longitudinal monitoring of hepatic steatosis. In this prospective study, we demonstrate that acquisition geometry—particularly anatomical window selection and subcapsular sampling depth—is associated with systematic differences and individual-level variability in UDFF measurements. These findings identify acquisition geometry as an important determinant of quantitative UDFF assessment.

Anatomical window selection emerged as a dominant source of systematic UDFF variability. Even under rigorously standardized subcapsular depth conditions, UDFF values differed substantially across hepatic acquisition windows. Left lobe acquisition windows yielded consistently higher UDFF values than right-lobe windows, with mean positive biases of approximately 5–6% relative to the right anterior intercostal window. This systematic left-right discrepancy differs from that reported in MRI-PDFF-based studies, in which left lobe fat content is typically lower than that of the right lobe, suggesting that the inter-window differences observed with UDFF may not be fully explained by underlying regional heterogeneity of hepatic fat distribution alone (12). Rather, these differences are more plausibly attributable to acquisition-related factors inherent to ultrasound-based measurements. Left lobe windows are known to be more susceptible to cardiac motion, gastrointestinal gas, limited acoustic access, and unstable probe contact, and higher technical failure rates for left lobe ultrasound measurements have been reported previously (13). Consistent with prior work supporting right lobe acquisition as the preferred approach for quantitative ultrasound assessment of hepatic steatosis, our findings further demonstrate that even within the right lobe, acquisition window selection introduces non-negligible systematic variability, likely reflecting differences in beam-path characteristics between intercostal and subcostal approaches (14,15). The magnitude of these reference-relative differences may be clinically relevant, as measurements obtained under different acquisition conditions could plausibly cross steatosis classification thresholds in individuals near diagnostic cutoffs. Bland-Altman analysis further demonstrated substantial discordance across acquisition windows, indicating limited measurement interchangeability. These observations emphasize the importance of maintaining consistent acquisition conditions when serial UDFF measurements are compared.

Beyond anatomical window selection, our findings indicate that UDFF measurements are strongly influenced by beam path-dependent acoustic factors inherent to clinical ultrasound acquisition. The detected UDFF signal reflects the combined effects of tissue backscatter and acoustic attenuation along the transmit–receive path, both of which vary systematically with sampling depth and total propagation distance (16). Prior quantitative ultrasound studies have demonstrated that such depth dependence is intrinsic to both attenuation and backscatter estimation, and that combining these parameters into composite metrics such as UDFF does not eliminate depth-related variability (17,18). Within this established framework, our data provide in vivo confirmation that subcapsular sampling depth is a major determinant of UDFF under routine clinical acquisition conditions. Specifically, very shallow acquisitions yielded higher UDFF values with greater variability, whereas intermediate-depth measurements demonstrated lower variability. These findings are consistent with known acoustic field and propagation effects that influence quantitative ultrasound measurements at different sampling depths (19).

In addition to intrahepatic sampling depth, total acoustic propagation distance, quantified as the skin-to-ROI center distance, showed a significant inverse association with UDFF. This finding is consistent with prior quantitative ultrasound studies demonstrating that increasing beam path length introduces cumulative attenuation- and propagation-related effects that influence quantitative parameter estimation (20). In contrast, inter-individual variation in SCD predominantly increased absolute deviations rather than inducing systematic directional bias. This distinction suggests that prehepatic tissues may contribute predominantly to measurement variability rather than consistent directional shifts in estimated fat fraction.

After accounting for acquisition geometry, participant-related factors exerted comparatively modest effects on UDFF. Higher BMI was associated with small but statistically significant increases in systematic differences and absolute deviations when anatomical windows were varied, but this association disappeared when acquisition was constrained to the reference right anterior intercostal window. Under these controlled conditions, SCD emerged as the dominant contributor to absolute deviations. Together, these findings suggest that a substantial proportion of the observed BMI-related variability may be mediated through its influence on beam path characteristics.

Consistent with prior reports, UDFF demonstrated high intra- and inter-operator repeatability (21). This finding supports the technical reliability of UDFF under standardized acquisition conditions.

Several limitations should be acknowledged. First, participants were recruited on the basis of normal conventional ultrasound findings, and the magnitude of acquisition-dependent differences may differ in populations with a broader spectrum of hepatic steatosis severity. Further validation in populations with obesity or MASLD is warranted. Second, UDFF measurements were not directly compared with histologic or MRI-based reference standards. Future studies incorporating external reference standards will be important for further validation. Third, the present study was performed using a single vendor-specific UDFF platform. The numerical magnitude of the observed differences should not be directly extrapolated to other vendor implementations without independent validation.

Conclusions

Acquisition geometry is a key technical determinant of UDFF measurements. Anatomical window selection, subcapsular sampling depth, and beam path characteristics were associated with systematic differences and individual-level variability in UDFF measurements. As a result, UDFF measurements obtained under different acquisition geometries are not interchangeable at the individual level, with direct implications for longitudinal follow-up and inter-study comparability. Our findings therefore support the need for standardized UDFF acquisition protocols, particularly with respect to anatomical window selection and subcapsular ROI depth.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0219/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0219/dss

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0219/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Biomedical Ethics Committee of West China Hospital, Sichuan University (approval No. 1397), and informed consent was obtained from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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