Subjects
A prospective study was conducted between May 2021 and July 2021 in the Second Affiliated Hospital of Zhejiang University School of Medicine. The study population comprised 63 consecutive subjects of different BMI grades who were approved by our institutional review board. Informed consent was obtained from all participants. All methods were performed in accordance with the Declaration of Helsinki.
The inclusion criteria were the ability to provide informed consent, age older than 18 years, and no history of liver surgery. Exclusion criteria were an inability to hold their breath for more than 3 seconds during the US examination, a possible other cause of chronic liver disease (i.e., excessive alcohol consumption, viral hepatitis, hepatoxic drugs, autoimmune hepatitis, etc.) and known liver lesions. Finally, 63 adults with lean weight (BMI< 24 kg/m2) (n=13), overweight (24 kg/m2≤ BMI< 28 kg/m2) (n=27) and obesity (BMI>28 kg/m2) (n=23) constituted the study population.
Clinical Data
Patient age, sex, height, weight and distance from the skin to the liver capsule were recorded. In this study, we defined BMI (weight in kilograms divided by the square of height in meters) of <24 kg/m2 as normal/lean weigh, 24 kg/m2≤ BMI< 28 kg/m2 as overweight, and BMI ≥28 kg/m2 as a diagnosis of obesity on the basis of the Chinese criteria[15].
UGAP measurements
The patient was in the supine position with the right upper extremity extended above the head to stretch the intercostal muscles and obtain the proper scanning window during the examination. First, B-mode US images were scanned to detect if any focal liver lesion existed. Second, the UGAP mode was activated, and examinations were performed on liver segment V, inferior to the right anterior lobe, through the intercostal space, with the transducer perpendicular to the skin surface while the participant held his or her breath for 3–5 seconds. Third, a fixed region of interest (ROI) of 4 cm×1 cm was placed in the liver parenchyma at least 20 mm from the liver capsule to avoid subcapsular areas, bile ducts, hepatic vessels, and artifacts (Figure 2B). Twelve consecutive measurements on the different frames were recorded, and median and interquartile range (IQR)/median values were displayed. We defined IQR/median<15% of twelve measurements as effective and successful measurements. The AC was calculated using the method from a prior report by Yao et al.[16].
Group 1: Evaluation of UGAP reproducibility in healthy subjects
Before the study, all 18 healthy adults with BMI less than 25 kg/m2 fasted for at least 8 hours. UGAP measurements were performed in the supine position on the hepatic V segment during breath holding, free breathing (SP, S5, 1), deep inspiration (SP, EI), and expiration (SP, EE), and then in the lateral decubitus position during breath holding and free breathing (LP) by radiologist A. Then, radiologist B performed the same protocol in the supine position for hepatic V segment during breath holding and free breathing (SP, S5, another radiologist). After 15-minute intervals and one week later, the participants were measured again by radiologist A (SP, S5, 2) (SP, S5, 3). Finally, the measurements were repeated 2 hours after a meal (Figure 1).
Group 2: Evaluation of UGAP in overweight and obese participants
We consecutively enrolled 45 subjects with a BMI greater than 25 kg/m2 for further evaluation. Most of the procedures were similar to those in Group 1 (Figure 1), except that two UGAP measurements were added to different liver segments (SP, non-S5), and the ROI position was fixed perpendicular to the liver capsule (SP, S5, ROI PLC).
Comparison of the UGAP results with the visual grades of hepatic steatosis
The visual grade of hepatic steatosis in all 63 objects was graded on a 4-point scale through B-mode US by two independent double-blind reviewers with more than ten years of experience in abdominal ultrasonography. The diagnostic criteria were based on the following characteristics[17-19]: Grade 0, echogenicity of the liver parenchyma slightly greater than or equal to that of the renal cortex, with visible periportal and diaphragmatic echogenicity, Grade 1, increased hepatic echogenicity with visible periportal and diaphragmatic echogenicity, and Grade 2, increased hepatic echogenicity with impaired visualization of periportal echogenicity, without obscuration of the diaphragm (Figure 2A). Grade 3, increased hepatic echogenicity with impaired visualization of periportal echogenicity and obscuration of the diaphragm.
Statistical analysis
Statistical analysis was performed using SPSS software version 17 (IBM Corp., Armonk, NY, USA) and MedCalc software version 12.1.00 (MedCalc Software, Mariakerke, Belgium). Continuous data are expressed as the mean ± standard deviation, and count data are presented as absolute numbers or percentages. The normality of the continuous data was tested by the one-sample Kolmogorov–Smirnov test. UGAP value differences in different BMI groups were analyzed by one-way ANOVA and LSD tests for pairwise comparisons (the data satisfied the normal distribution and homogeneity of variance) and the Kruskal–Wallis H tests and Mann–Whitney U tests for pairwise comparisons (p value less than 0.017) (the data did not satisfy a normal distribution or homogeneity of variance). Intraobserver reproducibility and interobserver reproducibility of the UGAP values were assessed using intraclass correlation coefficients (ICCs). Visual grades of hepatic steatosis between the two reviewers were tested by the quadratic weighted kappa test. A paired sample t-test was used to evaluate the differences in UGAP values under different conditions. The Spearman rank correlation coefficient was used to evaluate the correlation between the UGAP values and the visual grade of hepatic steatosis. All significance tests were two-sided, and p values less than 0.05 were considered statistically significant.