Comparing the controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound as a novel measurement for liver steatosis.

BACKGROUND/AIMS: In a recent study, attenuation imaging (ATI) with ultrasound was used as a new approach for detecting liver steatosis. However, although there are many studies on ATI and controlled attenuation parameter (CAP) that prove their practicability, there are few studies comparing these two methods. As such, this study compared CAP and ATI for the detection and evaluation of liver steatosis.
METHODS: A prospective analysis of 28 chronic liver disease patients who underwent liver biopsy, FibroScan® imaging, and ATI with ultrasound was conducted. The presence and degree of steatosis, as measured with the FibroScan® device and ATI, were compared with the pathological results obtained using liver biopsy.
RESULTS: The areas under the receiver operating characteristic curve (AUROC) of ATI and CAP for differentiating between normal and hepatic steatosis were 0.97 (95% confidence interval [CI] 0.83-1.00) and 0.96 (95% CI 0.81-0.99), respectively. ATI has a higher AUROC than CAP does in liver steatosis, at 0.99 (95% CI, 0.86-1.00) versus 0.91 (95% CI, 0.74-0.98) in grade ≥ 2 and 0.97 (95% CI, 0.82-1.00) versus 0.88 (95% CI, 0.70-0.97) in grade = 3, respectively.
CONCLUSION: The ATI and CAP results showed good consistency and accuracy for the steatosis grading when compared with the liver biopsy results. Moreover, ATI is even better than CAP in patients with moderate or severe steatosis. Therefore, ATI represents a non-invasive and novel diagnostic tool with which to support the diagnosis of liver steatosis in clinical practice.

Introduction

The diagnosis of liver steatosis is important to facilitate the treatment of chronic liver disease in clinical medicine [1]. Liver steatosis is generally considered to be a reversible and benign disease, yet researchers are increasingly attempting to reveal its role in the pathogenesis of various liver diseases [2, 3]. The development of fatty liver is related to steatohepatitis, and it can progress to liver fibrosis, cirrhosis, and even end-stage liver disease. In patients with chronic hepatitis C, liver steatosis accelerates the process of fibrosis [4-6], thus adversely affecting the sustained viral response rate of antiviral therapy [7, 8], and can predict the development of hepatocellular carcinoma due to liver steatosis [8, 9]. The evaluation of liver steatosis is also very important in the prognosis of liver transplantation donors because follicular steatosis of the donor liver is correlated with a risk of transplant failure after liver transplantation [10, 11].

According to the Clinical Practice Guidelines from the European Association for the Study of the Liver (EASL), the gold standard for the diagnosis and evaluation of fatty liver is liver biopsy [12], as the presence or absence of fatty liver and non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH) can only be distinguished by liver biopsy [13, 14]. However, this procedure is invasive and prone to triggering complications, such as pain, bleeding, and infection [15], and its results may be delayed due to the need to generate pathological reports. In addition, due to the high incidence of steatosis, its benign course, and the lack of clear association with liver enzyme changes, liver biopsy can only be deployed for some patients in need, such as in cases of non-alcoholic steatohepatitis, and because it is an invasive examination, it cannot be repeated, resulting in the inability to continuously and repeatedly monitor changes in patients with steatosis over a period of time.

Some studies have shown that using an ultrasound scanner such as FibroScan® (Echosens, Paris, France) together with the controlled attenuation parameter (CAP) can quantitatively and accurately assess the severity of liver steatosis in concordance with liver biopsy [16]. CAP estimates the total ultrasonic attenuation at the central frequency of the M probe or XL probe with FibroScan by using a go-and-return path [17]. However, FibroScan® is not an imaging device and cannot perform B-mode ultrasound assessments at the same time [18, 19].

In recent years, Canon Medical Systems has introduced an attenuation imaging (ATI) mode to the market as a novel ultrasound technique for diagnosing steatosis with the advantages of being easy to use and involving built-in ultrasonic machines [20]. ATI is a mode used to estimate the ultrasonic attenuation coefficient in tissues and can adjust the region of interest to measure liver attenuation and quantitatively evaluate liver steatosis through ultrasound imaging [21]. Based on two-dimensional images obtained in a daily ultrasound examination within a runtime of less than 2 minutes, ATI showed its convenience in routine screening of liver steatosis [22]. Compared with CAP, ATI’s advantage is the existence of an ultrasonic inspection mode; therefore, there is no need for additional equipment for examinations [23].

Another non-invasive tool is magnetic resonance imaging proton density fat fraction (MRI-PDFF), which can accurately quantify liver steatosis and provide a value of imaging diagnosis [24]. However, the cost of the instrument and the inconvenient operation are disadvantages [25, 26].

Given the current tools available for detecting liver steatosis, only few accurate, non-invasive, and easy-to-use detection methods other than CAP and ATI exist; therefore, this study aimed to compare the accuracy of CAP and ATI in assessing liver steatosis to support the identification of convenient and reliable methods for its clinical screening and treatment.

Materials and methods

From January 1, 2019, to July 31, 2019, we prospectively included 48 patients with chronic hepatitis scheduled for liver biopsy at Changhua Christian Hospital who met the following study inclusion criteria: (1) age of 18 to 80 years; (2) body mass index of less than 35 but greater than 17 kg/m2; (3) signed the informed consent form. Meanwhile, the exclusion criteria were as follows: (1) malignancy, including hepatocellular carcinoma and cholangiocarcinoma; (2) chronic systematic disease, such as coronary artery disease, chronic kidney disease, and chronic respiratory disease; (3) alcohol consumption. The method of participant recruitment was the notification of liver biopsy from the hospital during this period. Once the patient provided signed consent, they were included in the study. Ultimately, 28 patients underwent liver biopsy, ATI, CAP assessment, and analysis. The demographic details of the participants show that they are all Asian and living in central Taiwan, mainly in the Changhua county. Moreover, all relevant data are within the paper. As stated, all participants signed an informed consent form for this study, which was reviewed and approved by the institutional review board of Changhua Christian Hospital (approval no. 210202).

Liver biopsy

Under ultrasound guidance, a 16-gauge needle was used to obtain liver biopsy specimens. To be considered sufficient to score, the liver biopsy specimen had to measure at least 10 mm and have six portal tracts. The entire biopsy process was performed through percutaneous puncture over the right-side intercostal area of segments V-VI, and specimens were fixed with formalin, transferred to the pathology department of Changhua Christian Hospital within one hour [27], and stained with Masson’s trichrome. Meanwhile, reticulin staining was used to facilitate histological evaluation. All specimens were analyzed by two independent pathologists who were blinded to the clinical and laboratory characteristics of the patient under review. The steatosis grade was determined by the percentage of fat-containing cells in the liver sample that could be seen on a glass slide as follows: less than 5%, S0; 5% to 33%, S1; 34% to 66%, S2; and greater than 66%, S3.

CAP measurement method

The CAP was measured by an experienced technician who did not know the patients’ clinical data, at the FibroScan® facility, in dB/m. A 3.5-MHz standard probe (≥ 2.5 cm skin distance, XL probe; <2.5 cm skin distance, M probe) was used to measure the right liver lobe of the intercostal space while the patient lay on their back. The data reported by FibroScan® had to meet the following conditions: (1) at least 10 effective shots, (2) a success rate of at least 60%, and (3) the interquartile range (IQR) was less than 30% of the median CAP.

ATI measurement method

ATI was determined using data obtained from the TOSHIBA® i800 ultrasonic instrument (Toshiba, Tokyo, Japan) operated by a technician who did not know the results of the other reports. ATI allows for quantifying and color-coding changes in liver attenuation coefficients, which may be triggered by changes in the liver composition (e.g., increased fat content). The signal difference from point A to point B divided by the distance indicates the value of ATI, offering comprehensive quantitative data on liver steatosis (Fig 1). It also provides the slope of the signal attenuation (slope = attenuation coefficient). The value of ATI was defined as dB/cm/MHz × 100. The measurement was considered valid if the following criteria were met: (1) at least five effective values were collected, (2) a success rate of at least 60% was achieved, (3) an R2 value of 0.9 or greater at every single data point was recorded, and (4) the interquartile range was less than 30% of the median ATI.

Fig 1

Mechanism of attenuation imaging with ultrasound.

The signal difference from point A to point B divided by the distance shows the value of attenuation imaging.

Statistical analysis

First, we evaluated the normal distribution of the quantitative variables, in which the data are reported as mean and standard deviation values or median and interquartile range. The Kruskal–Wallis test, followed by the Dunn–Bonferroni post-test, was used to analyze the difference in the degree of ultrasound fatty liver between the four groups (S0, S1, S2, and S3). A two-tailed p-value of less than 0.05 was considered statistically significant.

The receiver operating characteristic curve (ROC) was used to evaluate the diagnostic performance of each non-invasive model. We calculated the area under the ROC curve (AUROC) and the 95% confidential interval (CI) of the AUROC. Then, using the De Long method, we compared the same data to judge the AUC value of different diagnostic criteria. We used Pearson’s correlation coefficient for ATI and CAP to confirm whether they are significantly related.

To evaluate the feasibility of the two measurements, we recalculated their diagnostic values (i.e., sensitivity, specificity, positive predictive value, and negative predictive value). These values were determined by original research. We used MedCalc statistical software version 19.4.0 (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; 2020) for statistical analysis.

Results

From January 2019 to July 2019, we included 48 patients with chronic liver disease scheduled for liver biopsy. Thereafter, four patients were excluded for malignancy, including hepatocellular carcinoma (n = 2) and cholangiocarcinoma (n = 2); furthermore, 11 were excluded for chronic systematic disease, such as coronary artery disease, chronic kidney disease, and chronic respiratory disease, and 5 were excluded for current alcohol consumption. Finally, 28 patients met the eligibility criteria for the following analysis (Fig 2).

Fig 2

Study flowchart.

The characteristics of the study participants are presented in Table 1. The patients had a mean age of 50.8 (± 14.0) years and eight (28.5%) were male. The risk factors for liver steatosis are baseline mean body mass index (BMI) value of 27.2 kg/m2, mean waist circumference of 89.4 cm, and diabetes mellitus (seven patients; 25%). The lipid profiles were as follows: mean triglyceride level: 149.0 mg/dL; cholesterol level: 188.2 mg/dL; high-density lipoprotein level: 50.4 mg/dL; low-density lipoprotein level: 125.2 mg/dL. Patient distribution according to steatosis grade was as follows: 6 (21.4%) patients with S0, 5 (17.8%) patients with S1, 9 (32.1%) patients with S2, and 8 (28.5%) patients with S3. The etiology distribution included 7 (25.0%) patients with hepatitis B virus infection, 9 (32.1%) patients with hepatitis C virus infection, 7 (25.0%) patients with nonalcoholic fatty liver disease, and 5 (17.8%) patients with autoimmune hepatitis; see Table 1.

Table 1

Clinical characteristics of the patients (n = 28).

Characteristic	Mean (±SD) or absolute count or median (interquartile range)
Age, years	50.8 ± 14.0
Male sex, n (%)	8 (28.5)
BMI, kg/m2	27.2 ± 3.8
Waist circumference (cm)	89.4 ± 12.8
Diabetes mellitus, n (%)	7 (25.0)
Triglyceride, mg/dL	149.0 ± 60.5
Cholesterol, mg/dL	188.2 ± 59.5
HDL, mg/dL	50.4 ± 20.2
LDL, mg/dL	125.2 ± 48.6
ALT, U/L	77.5 (34.3–125.8)
AST, U/L	56.5 (32.0–79.4)
Biopsy length, mm	16.5 ± 3.4
Liver stiffness, kPa	8.1 ± 2.3
Histology of steatosis grade*, n (%)
S0	6 (21.4)
S1	5 (17.8)
S2	9 (32.1)
S3	8 (28.5)
Etiology of liver disease, n (%)
HBV	7 (25.0)
HCV	9 (32.1)
NAFLD	7 (25.0)
AIH	5 (17.8)
ATI, dB/cm/MHz × 100	81.5 ± 14.1
CAP, dB/m	262.3 ± 51.1

AIH: autoimmune hepatitis; ALT: alanine transaminase; AST: aspartate transaminase; ATI: attenuation imaging; BMI: body mass index; CAP: controlled attenuation parameter; HBV: hepatitis B virus; HCV: hepatitis C virus; HDL: high-density lipoprotein; LDL: low-density lipoprotein; NAFLD: non-alcoholic fatty liver disease; SD: standard deviation

*Steatosis grade S0: <5%; S1: 5%–33%; S2: 34%–66%; S3: >66%

This study mainly compared the differences between the ATI and CAP assessment methods for liver steatosis of varying stages in patients with general chronic liver disease. The median (interquartile range) values for ATI and CAP according to liver steatosis grade were 67.5 (54.0–69.0) and 198.5 (193.0–206.0) for S0, 72 (72.0–78.5) and 261 (227.5–268.7) for S1, 82.0 (81.5–85.7) and 275 (255.7–283.5) for S2, and 98 (89.5–102) and 324.0 (290.0–338.0) for S3, respectively, with trends correlated to liver steatosis grading (p < 0.001, Jonckheere–Terpstra trend test); see Fig 3. ATI showed a significant difference in liver steatosis grading between S0 and S2, S0 and S3, and S1 and S3, with p < 0.05 in the post hoc analysis (Fig 3A), and CAP assessment showed the same result between S0 and S2 and S0 and S3, with p < 0.05 (Fig 3B). In addition, based on these data, ATI and the CAP were positively correlated (r = 0.8111; p < 0.05) (Fig 4).

Fig 3

The distribution of CAP and ATI values according to histologic steatosis grade.

A: ATI was significantly different between S0 and S2, S0 and S3, and S1 and S3 (p < 0.05) with the Kruskal–Wallis test in the post hoc analysis. B: The CAP assessment results were significant between S0 and S2 and S0 and S3, with p < 0.05. The vertical axis is a logarithmic scale. The top and bottom of the boxes are the first and third quartiles, respectively. The length of each box represents the interquartile range, within which is located 50% of the values. The lines through the middle of the boxes represent median values. ATI: attenuation imaging; CAP: controlled attenuation parameter, *: p < 0.05.

Fig 4

The association between ATI and CAP.

ATI and CAP were significantly correlated (r = 0.8111; p < 0.05).

We then analyzed the cut-off values of ATI and CAP to correctly predict steatosis. For this reason, we performed a comparative AUROC plot analysis, including all study participants (n = 28) with different steatosis grades. The AUROC of ATI according to liver steatosis grade was higher than that of CAP at 0.97 (0.83–1.00) to 0.96 (0.81–0.99) in S ≥ 1; 0.99 (0.86–1.00) to 0.91 (0.74–0.98) in S ≥ 2; and 0.97 (0.82–1.00) to 0.88 (0.70–0.97) in S3; see Fig 5. The sensitivity value that distinguished normal and liver steatosis (≥ S1) was 100% for ATI, which was higher than that of CAP at 95%. The positive predictive value (PPV) of CAP of 100% is higher than that of ATI of 95%, but the negative predictive value (NPV) of ATI of 100% is higher than that of CAP of 85%; see Table 2.

Fig 5

Comparing AUROC for ATI and CAP for detecting different grades of liver steatosis.

A: Liver steatosis grade S1 or higher with AUROC values of 0.97 (95% CI: 0.83–1.00) for ATI (blue line) and 0.96 (95% CI: 0.81–0.99) for CAP (green line). B: Liver steatosis grade S2 or higher with AUROC values of 0.99 (95% CI: 0.86–1.00) for ATI and 0.91 (95% CI: 0.74–0.98) for CAP. C: Liver steatosis grade S3 with AUROC values of 0.97 (95% CI: 0.82–1.00) for ATI and 0.88 (95% CI: 0.70–0.97) for CAP. AUROC: area under the receiver operating characteristic curve; ATI: attenuation imaging; CAP: controlled attenuation parameter.

Table 2

CAP and ATI values for diagnosing liver steatosis.

Model	Steatosis stage	AUROC (95% CI)	Cutoff	Sen	Spe	PPV	NPV
CAP	S ≥ 1	0.96 (0.81–0.99)	220	0.95	1.00	1.00	0.85
	S ≥ 2	0.91 (0.74–0.98)	267	0.76	0.90	0.92	0.71
	S = 3	0.88 (0.70–0.97)	290	0.75	1.00	1.00	0.90
ATI	S ≥ 1	0.97 (0.83–1.00)	69	1.00	0.83	0.95	1.00
	S ≥ 2	0.99 (0.86–1.00)	78	1.00	0.90	0.94	1.00
	S = 3	0.97 (0.82–1.00)	82	1.00	0.85	0.72	1.00

ATI: attenuation imaging; AUROC: area under the receiver operating characteristic curve; CAP: controlled attenuation parameter; CI: confidence interval; NPV, negative predictive value; PPV, positive predictive value; Sen, sensitivity; Spe, specificity. Cut-off values were obtained from the original article.

Discussion

In this study, the ATI and CAP values of mild and severe steatosis grades were found to increase significantly with increasing histologically diagnosed steatosis grade (p < 0.001, Jonckheere–Terpstra trend test). The AUROC values of ATI for diagnosing hepatic steatosis of grades S1 or higher, S2 or higher, and S3 were greater than those of CAP, suggesting that ATI is a more reliable method than CAP assessment for diagnosing liver steatosis.

Recent studies have suggested that CAP assessment via transient elastography (TE) can quantify the diagnosis of liver steatosis [17]. The CAP mode of TE is a non-image-based ultrasound technology able to measure the stiffness of tissues in real time and accurately [28]. Simultaneously, this technology can measure liver steatosis in CAP mode using M and XL probes; here, it revealed the AUROC for hepatic steatosis grades S1 or higher, S2 or higher, and S3 or higher to be 0.82 (95% CI 0.77–0.88), 0.83 (95% CI 0.77–0.88), and 0.89 (95% CI 0.84–0.93) for the M probe and 0.88 (95% CI 0.82–0.93), 0.92 (95% CI 0.89–0.96), and 0.93 (95% CI 0.89–0.97) for the XL probe, respectively [19]. Another study of patients with steatosis assessed by CAP showed an AUROC of 0.87 (95% CI 0.82–0.92) for S≥S1, 0.77 (95% CI 0.71–0.82) for S≥S2, and 0.70 (95% CI 0.64–0.75) for S = S3, and the Youden cut-off values of CAP for S≥S1, S≥S2, and S≥S3 were 302, 331, and 337 dB/m, respectively [29].

Another report showed that using the CAP mode of TE to detect liver steatosis in patients with hepatitis C presented AUROC values of 0.80 (95% CI 0.75–0.84) for S1 or higher, 0.86 (0.81–0.92) for S2 or higher, and 0.88 (0.73–1) for S3. CAP also exhibited a good ability to differentiate steatosis grades in hepatitis C patients (Obuchowski measure = 0.92) [30].

Compared with our study’s results, CAP yielded higher AUROC values in liver steatosis grades S1 or higher, S2 or higher, and S3. This result showed that CAP assessment could distinguish different grades of liver steatosis with a significant difference.

Recent studies have shown that, when comparing the measurement results for the proton density fat fraction based on magnetic resonance imaging with CAP assessment results based on TE, the former is more effective than CAP is in evaluating liver steatosis. A study showed that MRI-PDFF AUROCs for classifying steatosis grades 0 vs. 1–3, 0–1 vs. 2–3, and 0–2 vs. 3 were 0.98, 0.91, and 0.90, respectively [31]. Notably, ATI has a similar diagnostic mechanism in this study [32].

We acknowledge that there are several limitations to this study, including the small sample size, relatively obese patients (in patients with a BMI of 28 kg/m2 or more, CAP has no correlation with actual liver fat percentage) [33], patients being unblinded, possible selection bias in screening of patients, and other easily negligent and unobserved biases due to not using a randomized controlled clinical trial. The advantages of this study are as follows: (1) it is the first comparative study of ATI and CAP, (2) the prospective nature of the study with operators blinded makes the reports more credible, and (3) this study demonstrates that ATI is more reliable than CAP for diagnosing liver steatosis.

Conclusions

ATI is a new method that is highly concordant with liver biopsy in detecting steatosis. ATI has a higher AUROC value in detecting different grades of liver steatosis than CAP, thus demonstrating ATI’s excellent and accurate diagnostic ability. In the future, ATI may be a promising technique for liver steatosis screening or diagnosis.

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10 Mar 2021

PONE-D-20-37505

A prospective comparative study of the controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound in evaluating liver steatosis

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Reviewer #1: The authors describe a comparative study of controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound in liver steatosis. The topic of the work is interesting and serves a good base to detect liver steatosis by noninvasive manner. For these reason, the manuscript is suitable for publication after revision of some sections, reported here:

Minor revision:

I missed the two different investigated techniques from the keywords.

The references in the main text from 10 to 19 are missing.

At row 82 there is a mistyping by the numbers of inclusion criteria.

At row 84 there is an extra ‘s’.

The number of excluded patients should move from the material and method section to the result part.

The Liver biopsy section in the material and methods is superficial. There is not enough information about the applied technique. Any other case, a reference article should be inserted which describes the details of biopsy and staining.

There is a mistyping at row 114 dB/cm/MHz.

At row 125 the authors write about three groups but there are 4 different ones in parenthesis.

The Fig 2 seems a little bit over-explained.

At row 155 ATI definition is missing.

Major revision:

The introduction part contains detailed description (18 lines long) about the steatosis and the gold standard liver biopsy. But there are only 2-2 sentences about the compared techniques. This part should be extended with much more information related to these techniques.

At the last paragraph of introduction the authors mention there are other detection methods than CAP and ATI for the diagnosis of steatosis but the relevant references are missing.

The image quality of Fig 4 and Fig 5 should be increased.

The Fig 4 legend is just a simple sentence of the result. It should contain a title and at least one descriptive sentence.

The cohesion power is missing from the discussion section. The consecutive paragraphs are independent from each other. The fourth paragraph would be more appropriate at the introduction part.

In the fifth paragraph of discussion, without any introduction or background information there is a comparison with proton-density fat fraction based MRI.

The conclusion is too short and not so demonstrative.

Other questions:

How could you define the success rate and effective shot by the US scans?

What does effective data point mean?

Reviewer #2: This is a concise paper with the advantage of easily reading the results and judging the significance. A merit of the paper is the accompanying rather detailed patient data as well as the rigorous statistics.

There are a number of important concerns, though, which are to be addressed. I do not list all of them one by one, some are issue types, and it is expected that the authors improve all instances of these types.

Data availability should be stated in the methods section.

Page 2 and Page 13 "No study to date has confirmed the clinical utility of...". This is clearly wrong. There are a number of studies recently published 2020 and 2021. Please include them. With these, the purpose of this study should be specified in more detail. Also your study should be compared to all these.

Page 4 "is invasive and prone to triggering complications such as pain..." etc. Reference citing required, and also to all similar statements, that refer to clinical "facts".

Page 7 "to make the statistic significant". Determining the unit and display multiplier has nothing to do with significance, please correct the sentence.

Page 14a "same from a previous study". Need to cite the previous study.

Page 14b "there are several limitations". The reader will need numerical estimation of liver steatosis diagnosis errors coming from obese patients. Cite a study with clinical data, CAP e.g., and/or repeat with a couple of non-obese patients with negative steatosis at least, so that a baseline can be shown: either as non-confounding, or establishing correction factors with subdermal fat amount.

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Reviewer #1: No

Reviewer #2: No

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10 Apr 2021

Dear Editor,

We appreciate your editorial comments, as well as those of the reviewers, concerning our manuscript. Based on these comments, we have made several revisions to our manuscript, which is resubmitted for your consideration. If there is anything needing to be further improved, please do not hesitate to inform us at your earliest convenience. Your assistance is highly appreciated. We look forward to your message.

The followings are point-by-point responses to the comments.

All authors thank the reviewer’s suggestions. Your assistance is highly appreciated.

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response:

All authors thank the reviewer’s suggestions. We have tried our best to confirm that the format complies with the regulations. Thank you for your advice if there are errors.

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2. Thank you for stating in the text of your manuscript "all participants signed an informed consent form for this study". Please also add this information to your ethics statement in the online submission form.

Response:

All authors thank the reviewer’s suggestions. We will add this information to my ethics statement in the online submission form.

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3. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants.

Please ensure you have provided sufficient details to replicate the analyses such as:

a) a statement as to whether your sample can be considered representative of a larger population, and

b) a description of how participants were recruited.

Response:

All authors thank the reviewer’s suggestions. Thank you for your comments. We have added the participant recruitment method to the article and adding a description of how participants were recruited . We added on “The method of participant recruitment is the notification of liver biopsy from the hospital during this period. After the patient's consent signed, this will be included in the study.” in Row 123- 125, page 7, for participant recruitment method, and added on “Demographic details of the participants show that they are all Asians, and living in central Taiwan mainly in the Changhua county.” in Row 127- 128, page 7for demographic details of participants.

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4. We noticed you have some minor occurrence of overlapping text with the following previous publications, which needs to be addressed:

- https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0182784

- https://researchportal.bath.ac.uk/en/publications/endothelin-receptor-aa-regula tes-proliferation-anddifferentiatio-2

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section.

Further consideration is dependent on these concerns being addressed.

Response:

All authors thank the reviewer’s suggestions. Thank you for your comments.

We have revised and strengthened related paragraphs, such as the following:

1. We changed the title of “Compared the Ccontrolled attenuation parameter using FibroScan with and attenuation imaging with ultrasound as an novel measurement for evaluating liver steatosis” in Row 2-3, page 1.

2. changed the duplicated text “reliable” to “novel” in Row 2, page 1.

3. changed the duplicated text and added on “According to the Clinical Practice Guidelines from European Association for the Study of the Liver (EASL), the golden standard for diagnosis and evaluation fatty liver is liver biopsy [7]. Currently, the gold-standard technique for diagnosing and evaluating liver steatosis is liver biopsy; In addition to distinguishing the presence or absence of fatty liver through liver biopsy, non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) can only be distinguished through liver biopsy. Hhowever, this procedureapproach is invasive and prone to triggering complications, such as pain, bleeding, and infection, and its results may be delayed due to the need to generate pathological reports.”, and we also cited the journal of “https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0182784

in Row 77- 82, page 5

4. changed the duplicated text and added on “Some studies have shown that using an ultrasound scanner such as FibroScan® (Echosens, Paris, France) together with the controlled attenuation parameter (CAP) to measure the amount of ultrasound attenuation can quantitatively and accurately assess the severity of the liver steatosis fatty liver in concordance with liver biopsy [8]. However, FibroScan® is not an imaging device and cannot perform B-mode ultrasound assessments at the same time” in Row 89-95, page 6.

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5. Thank you for stating the following financial disclosure:

"NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." At this time, please address the following queries:

a. Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c. If any authors received a salary from any of your funders, please state which authors and which funders.

d. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response:

We will add on the text ”The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript” within the cover letter.

Thanks for your suggestions.

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6. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation.

Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any intext citations to match accordingly. Please see our Supporting Information guidelines for more information:

http://journals.plos.org/plosone/s/supporting-information.

Response: We will add on the text and make it more perfect. Thanks for your suggestions.

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Reviewer: 1

The authors describe a comparative study of controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound in liver steatosis. The topic of the work is interesting and serves a good base to detect liver steatosis by noninvasive manner. For these reason, the manuscript is suitable for publication after revision of some sections, reported here:

Minor revision:

I missed the two different investigated techniques from the keywords.

Response:

All authors thank the reviewer’s suggestions. Thank you for your comments.

We added on the keywords as follow “controlled attenuation parameter; attenuation imaging” in Row 62-63, page 4.

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The references in the main text from 10 to 19 are missing.

Response:

We are very sorry, this is our typesetting negligence, and we will make up the relevant reference.

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At row 82 there is a mistyping by the numbers of inclusion criteria.

Response: Thank you for your advice, this is our major mistake and made the following revisions: (2) signed the informed consent form to (3) signed the informed consent form

in Row 125, page 7

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At row 84 there is an extra ‘s’.

Response: Thank you for your advice, this is our major mistake and made the correction

in Row 122, page 7

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The number of excluded patients should move from the material and method section to the result part.

Response: Thank you for your advice, this is our major mistake and made the correction.

We moved the excluded patient number to the result

in Row 188- 192, page 10 in Result section.

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The Liver biopsy section in the material and methods is superficial. There is not enough information about the applied technique. Any other case, a reference article should be inserted which describes the details of biopsy and staining.

Response: Thank you for your advice, this is our major negligence. We have added some discussion and cited related articles of liver biopsy

in Row 133- 138, page 8

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There is a mistyping at row 114 dB/cm/MHz.

Response: Thank you for your advice, this is our major mistake and made the correction

in Row 161, page 9

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At row 125 the authors write about three groups but there are 4 different ones in parenthesis.

Response: Thank you for your advice, this is our major mistake and made the correction from three groups to four groups

in Row 171, page 9

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The Fig 2 seems a little bit over-explained.

Response: Thank you for your advice, this is our major mistake and made the correction from “Fig 2. Study flowchart” without figure Legends, just entitled with the “Fig 2. Study flowchart”.

in Row 193, page 10

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At row 155 ATI definition is missing.

Response: Thank you for your advice, this is our major mistake and made the correction with adding “attenuation imaging” for ATI,

in Row 208, page 13 in Table 1 legends.

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Major revision:

The introduction part contains detailed description (18 lines long) about the steatosis and the gold standard liver biopsy. But there are only 2-2 sentences about the compared techniques. This part should be extended with much more information related to these techniques.

Response: Thanks for your suggestion, we will revise the proportion of these two checks in the article.

We added on the CAP at in Row 89-94, page 6, in introduction section.

We also added on the ATI in Row 96- 105, page 6 in introduction section.

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At the last paragraph of introduction the authors mention there are other detection methods than CAP and ATI for the diagnosis of steatosis but the relevant references are missing.

Response: Thank you for your advice, this is our major mistake and made the correction with added on the “reference 25 to 27” showed MRI for liver steatosis with “Another non-invasive tool is magnetic resonance imaging proton density fat fraction (MRI-PDFF), which can accurately quantify liver steatosis and provide the value of imaging diagnosis [24]. However, the cost of the instrument and the inconvenient operation are also disadvantages “

in Row 106- 109, page 6

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The image quality of Fig 4 and Fig 5 should be increased.

Response: Thank you for your advice, we will improved the quality of the Fig 4 and Fig 5.

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The Fig 4 legend is just a simple sentence of the result. It should contain a title and at least one descriptive sentence.

Response: Thank you for your advice, this is our major mistake and made the correction.

First, we added the “We used Pearson's correlation coefficient for ATI and CAP to confirm whether they are positively related”

in Row 177- 179, page 10. In Statistic section.

Second, We added on a descriptive sentence with “ATI and CAP were positively correlated (r = 0.8111; p < 0.05)” and entitled with “The association between ATI and CAP”

in Row 232- 233, page 13 in Fig 4.

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The cohesion power is missing from the discussion section. The consecutive paragraphs are independent from each other. The fourth paragraph would be more appropriate at the introduction part.

Response: Thank you for your advice, this is our major mistake and made the correction.

We will move the text of the fourth paragraph in discussion section to introduction part as ” Basing on two-dimensional images in daily ultrasound examination within less than 2 minutes performing time, ATI showed the convenience in routine screening of liver steatosis[23]. As compared with CAP, ATI's advantage is the existence of an ultrasonic inspection mode, so there is no need to arrange for additional equipment for examinations [24]”,

in Row 101- 105, page 6.

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In the fifth paragraph of discussion, without any introduction or background information there is a comparison with proton-density fat fraction based MRI.

Response: Thank you for your advice, this is our major mistake and added the MRI-PDFF in introduction with “ Another non-invasive tool is magnetic resonance imaging proton density fat fraction (MRI-PDFF), which can accurately quantify liver steatosis and provide the value of imaging diagnosis [25]. However, the cost of the instrument and the inconvenient operation are also disadvantages [26] [27].”and discussion section with “ A study showed that MRI-PDFF with AUROC for classifying steatosis grades 0 vs. 1–3, 0–1 vs. 2–3, and 0–2 vs. 3 were 0.98, 0.91, and 0.90, respectively.

in Row 293- 294, page 17

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The conclusion is too short and not so demonstrative.

Response: Thank you for your advice, this is our major mistake and made the correction with “ATI has a higher AUROC values in different grading of liver steatosisthan CAP demonstrating ATI’s excellent and accurate diagnostic ability. . In the future, ATI may be a promising technique for liver steatosis screening.”

in Row 305- 310, page 17

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Other questions:

How could you define the success rate and effective shot by the US scans?

Response: Thank you for your questions. We defined the “ a success rate of at least 60% was achieved” in ATI measurement method : Fully meet the following conditions (1) at least five effective values were collected, (2) R2 value of 0.9 or greater at every single data point was recorded, and (3) the interquartile range was less than 30% of the median ATI.

If the technician completes an inspection and fails to meet any of the three items, it is considered a failure.

If the success rate is not higher than 60%, the inspection case must be excluded.

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What does effective data point mean?

Response: Thank you for your questions. The text does not express the meaning very accurately, so we changed it from "at least five effective data points” to “ at least five effective values “ at

in Row 162, page 9.

Thank you again for your careful review.

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Reviewer #2: This is a concise paper with the advantage of easily reading the results and judging the significance. A merit of the paper is the accompanying rather detailed patient data as well as the rigorous statistics. There are a number of important concerns, though, which are to be addressed. I do not list all of them one by one, some are issue types, and it is expected that the authors improve all instances of these types.

Data availability should be stated in the methods section.

Response: Thank you for your advice. We added on Data availability stating in the methods section.

in Row 128, page 7 in method section.

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Page 2 and Page 13 "No study to date has confirmed the clinical utility of...". This is clearly wrong. There are a number of studies recently published 2020 and 2021. Please include them.

With these, the purpose of this study should be specified in more detail. Also your study should be compared to all these.

Response: Thank you for your advice. This is obviously our mistake. We made these corrections:

1. delete the “No study to date has confirmed the clinical utility of...".

in Row 39, page 3.

.

2. Adding the text “ However, there are many studies on ATI and controlled attenuation parameter (CAP) to prove their practicability, but there are few studies comparing these two methods.no study to date has confirmed the clinical utility of this technique. As such, this study compared the controlled attenuation parameter (CAP) gleaned using FibroScan® (Echosens, Paris, France) and ATI for the detection and evaluation of liver steatosis.”

in Row 39- 43, page 3.

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Page 4 "is invasive and prone to triggering complications such as pain..." etc. Reference citing required, and also to all similar statements, that refer to clinical "facts".

Response: Thank you for your advice. This is obviously our mistake.

We has changed text cited with No.16 reference.

in Row 82-83 , page 5.

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Page 7 "to make the statistic significant". Determining the unit and display multiplier has nothing to do with significance, please correct the sentence.

Response: Thank you for your advice. This is obviously our mistake. This is just a unit, we have deleted the redundant words that follow, thank you for your review,

in Row 162, page 9.

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Page 14a "same from a previous study". Need to cite the previous study.

Response: Thank you for your advice. This is obviously our mistake. The previous study means our current study data. So, we delete the text and change it to “ This result showed CAP assessment could distinguish different grades of liver steatosis with a significant difference.”

in Row 285, page 16.

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Page 14b "there are several limitations". The reader will need numerical estimation of liver steatosis diagnosis errors coming from obese patients. Cite a study with clinical data, CAP e.g., and/or repeat with a couple of nonobese patients with negative steatosis at least, so that a baseline can be shown: either as non-confounding, or establishing correction factors with subdermal fat amount.

Response: Thank you for your advice. This is obviously our mistake. We has mede the correction with “relatively obese patients (in the patients with BMI of 28 kg/m2 or more, CAP has no correlation with actual liver fat percentage) [34]” and cite No. 34 reference

in Row 298-299, page 17.

Submitted filename: Response to reviewer - 1-2021.docx

Click here for additional data file.

24 May 2021

PONE-D-20-37505R1

Comparing the controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound as a novel measurement for liver steatosis

PLOS ONE

Dear Dr. Hsu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically, the following author, James Cheng-Chung Wei Chung Shan Medical University, was added to the revised version. It did not appear to meet the authorship policies unless a specific contribution was made and mentioned by him in the original version, particularly the newly-added author abruptly served as the corresponding author of the manuscript. The Authors should have a clear declaration and signed agreement by all authors for the ethic concern. Otherwise, the authorship should only list the original authors.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

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Jee-Fu Huang, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The following author, James Cheng-Chung Wei Chung Shan Medical University, was added to the revised version. It did not appear to meet the authorship policies unless a specific contribution was made and mentioned in the original version, particularly the newly-added author abruptly served as the corresponding author of the manuscript. The Authors should have a clear declaration and signed agreement regarding it.

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

5 Jul 2021

Dear Editor,

We appreciate your editorial comments, as well as those of the reviewers, concerning our manuscript. Based on these comments, we have made several revisions to our manuscript, which is resubmitted for your consideration. If there is anything needing to be further improved, please do not hesitate to inform us at your earliest convenience. Your assistance is highly appreciated. We look forward to your message.

The followings are point-by-point responses to the comments.

All authors thank the reviewer’s suggestions. Your assistance is highly appreciated.

Journal Requirements:

Specifically, the following author, James Cheng-Chung Wei Chung Shan Medical University, was added to the revised version. It did not appear to meet the authorship policies unless a specific contribution was made and mentioned by him in the original version, particularly the newly-added author abruptly served as the corresponding author of the manuscript. The Authors should have a clear declaration and signed agreement by all authors for the ethic concern. Otherwise, the authorship should only list the original authors.

Response:

After discussing with various authors, the original author group accepted the editor’s kind suggestions.

Decided to remove the relevant information of James Cheng-Chung Wei Chung Shan Medical University in order to comply with the ethics of scientific research on Page 1 , Line 6.

We also fixed the Author Contributions on Page 18, Line 292.

This is our negligence. We are very sorry.

Updated with IRB No. 210202

Response:

Because this research is being updated and reviewed again this year(2021), the IRB number is updated at this time to more accurately comply with the research ethical basis. Thank you reviewer for the suggestion.

We also attach the latest IRB in the attachment to ensure the rigor of the research.

We revised the IRB no 210202. at Page 7, Line 118

Submitted filename: Response to reviewer 2.docx

Click here for additional data file.

7 Jul 2021

Comparing the controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound as a novel measurement for liver steatosis

PONE-D-20-37505R2

Dear Dr. Wu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Jee-Fu Huang, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The Authors have sufficiently responded to the comments raised.

Reviewers' comments:

8 Oct 2021

PONE-D-20-37505R2

Comparing the controlled attenuation parameter using FibroScan and attenuation imaging with ultrasound as a novel measurement for liver steatosis

Dear Dr. Wu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jee-Fu Huang

Academic Editor

PLOS ONE