Validating controlled attenuation parameter in the assessment of hepatic steatosis in living liver donors.

INTRODUCTION: Hepatic steatosis (HS) negatively impacts transplant outcomes in living liver donors. To date, liver biopsy is preferred for HS evaluation. This study aims to evaluate the measurement of controlled attenuation parameter (CAP) as a diagnostic tool of HS in living liver donors.
METHODS: Candidates recruited to this study, conducted from April 2016 to February 2020, were potential donors who had undergone transient elastography using Fibroscan® and CAP measurements at liver segments VI and VII, followed by liver biopsy. The HS grades from liver biopsy were classified as S0 (<5%), S1 (5-33%), S2 (33-66%), and S3 (>66%). For CAP, they were S0 (≤218dB/m), S1 (218-249dB/m)), S2 (250-305dB/m)), and S3 (>305dB/m)). The CAP measurements were compared with the liver biopsy results.
RESULTS: Of the 150 potential donors [male, 73.3%; mean age, 30.0±7.0 years; body mass index (BMI), 24.7±3.5kg/m2], 92 (61.3%) had no or mild HS, while 58 (38.7%) and 10% had moderate to severe HS based on CAP and liver biopsy, respectively. Subjects with moderate to severe HS per CAP were mostly males (0.014), and had higher BMI (p = .006), alanine aminotransferase (ALT) (.001), gamma-glutamyl transferase (.026), and high-density lipoprotein (.008). On multivariate analysis, high ALT (OR, 1.051; 95% CI, 1.016-1.087; p = .004) was a predictor of significant HS. The sensitivity, specificity, positive and negative predictive values of CAP to detect significant HS were 93.3%, 67.4, 24.1%, and 98.9%, respectively.
CONCLUSION: The high sensitivity and negative predictive values of CAP make it a good screening test to exclude significant HS in potential living liver donors which, in turn, can help avoid unnecessary liver biopsies.

Introduction

For the appropriate selection of living liver donors, the evaluation of hepatic steatosis (HS) is essential because of its considerable negative impact on perioperative outcomes on donor and recipient [1]. Indeed, living donors with significant HS are at risk of increased blood loss during transection and impaired regeneration, which are potentially life-threatening complications, particularly when associated with a reduced volume of the remnant [2, 3]. Moreover, steatotic grafts in recipients are associated with higher rates of primary graft dysfunction, several postoperative complications, and poor overall graft survival [4]. Thus, accurate assessment of the presence and severity of HS in the living liver donor is fundamental to ensure primarily, the donor’s safety, as well as successful liver transplantation [4, 5]. Though liver biopsy is the current gold standard for diagnosis and severity assessment of HS, it carries several limitations [6]. Liver biopsy is an invasive procedure associated with several complications, such as bleeding and infection [7]. Further, the sampling technique is variable and carries a high rate of error, while there is the possibility of having non-univocal interpretations among pathologists [7]. Given the limitations of liver biopsy, several non-invasive imaging techniques have been proposed for assessment of HS in living liver donors, such as ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI) [8]. However, these techniques have their own limitations, and none of them have been proven to possess a higher sensitivity in diagnosing HS than liver biopsy [4].

Measurement of controlled attenuation parameter (CAP) is a promising, noninvasive tool for evaluation of HS [9, 10]. It is a qualitative and quantitative technique that measures the degree of ultrasonic attenuation by the hepatic fat at a frequency of 3.5 MHz. CAP is integrated with transient elastography (TE) in the Fibroscan® device (Echosens, Paris, France) [9]. In a recent meta-analysis of 1297 patients with nonalcoholic fatty liver disease (NAFLD) in 2019, the sensitivity of the CAP was 87%, 85%, and 58% in mild, moderate, and severe cases of HS [11]. CAP also has several other advantages, such as being non-invasive, non-ionizing, quantitative, inexpensive, and easy to perform [9]. The only significant limitation of CAP is its reduced ability to discriminate higher HS scores in individuals with a high body mass index (BMI) [12, 13].

Several reports have shown the role of CAP in the assessment of HS in patients with chronic liver disease (CLD) of various etiologies, and these reports suggest threshold CAP values for determining the presence of HS in CLD patients (e.g., > 5% or > 10% of hepatic fat) [14-17]. There is uncertainty, however, on the value of such a scale in those with perceived healthy liver tissue due to the scarcity of data about the role of CAP in the assessment of HS in potential living liver donors. This study aims to evaluate CAP as a diagnostic tool for the presence and severity assessment of HS in potential living liver donors by correlating it to the standard liver biopsy approach.

Materials and methods

Sample and setting

We conducted a prospective, single-center, cohort study in the Liver Transplant Unit at King Faisal Specialist Hospital and Research Centre (KFSH&RC) from April 2016 to February 2020. The study was conducted per the latest version of the Declaration of Helsinki and Good Clinical Practice, the policies and procedures of the Office of Research Affairs (ORA) of the KFSH&RC, and the laws of Saudi Arabia. All of the potential transplant donors included in this study provided written informed consent. Potential donors from vulnerable populations (such as minors, the incarcerated, or subjects with compromised mental capacity) were exempt from this study.

The Institutional Review Board within the ORA of KFSH&RC reviewed and approved the study protocol. All subjects signed informed consent before participation.

All potential living liver donors who fulfilled the donor selection criteria and were sent for assessment of HS were eligible for the study. The subjects were selected in a four-step approach according to the center’s policy on donor selection for living liver donation (Table 1). Subjects who refused to undergo a liver biopsy, had severe HS on any radiological modality (US, CT or MRI), planned for left lateral hepatectomy, had BMI above 30 kg/m2, and those who had unsatisfactory CAP assessment (interquartile range > 30%) were excluded from the study.

Table 1

Workup and steps for selection of living liver donors.

Step 0 TELEPHONE INTERVIEW (QUESTIONNAIRE)

Age <50

Compatible Blood group

Gender

Body mass index <30

Previous surgery

Medications

Relation to recipient

STEP 1

Bloodwork & urinalysis

Psychological/ psychiatric consultation

Electrocardiography

Chest X-ray

Pregnancy test for all females

STEP 2

CT abdominal angiography

Pulmonary function testing

Echocardiography

Mammogram (Females aged >40 years)

Vaccinations

Hepatology consultation

Magnetic resonance cholangiopancreatography

STEP 3

preoperative Anesthesiology clinic

Carotid Doppler (age >40 years)

Colonoscopy and Gastroscopy (age >50 years)

Surgical informed consent

STEP4

Liver biopsy

All donors included in the study were not previously registered as organ donors and they donated for their first or second degree relatives. Recruitment of living donors in our center require that potential donors are adults (18 years and older), come voluntarily for donation, fulfill the criteria mentioned in Table 1 and pass the medical and surgical clearance. The remaining live size should be at least 32% of their liver and should sign an informed consent that explain all the potential complications of the procedure. Donors who have abnormalities in their pre-transplant work up investigations should be cleared from the respective services. All donors who passed the evaluation process during the study period were approached for participation and those who agreed to participate were recruited if they fulfill inclusion and exclusion criteria.

Data collection

All patients recruited to this study had TE followed by liver biopsy. The TE was performed with Fibroscan® Touch 502 device (Echosens, Paris, France), and CAP measurements were taken at a frequency of 3.5 MHz and a depth of 25–65 mm at liver segments VI and VII [16]. At least ten valid measurements were taken, and the median values were considered for the analysis. Liver biopsies were done under ultrasound guidance from the same liver segments with a full-core biopsy needle (length 33 mm, diameter 18G). The specimens collected were fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin and chromotrope aniline blue stains, and evaluated by an experienced pathologist who was blinded to the result of CAP. The scores used to grade HS on liver biopsy were S0 (<5%), S1 (5%–33%), S2 (33%–66%), and S3 >66%, and the HS evaluation scores for CAP were graded as S0 (≤218 dB/m), S1 (218–249), S2 (250–305), and S3 (>305) [14].

Although the range in S1 is quite wide, however it remains the well-known grading system for reporting severity of steatosis. The degree of steatosis might affect the decision on donation and in most transplant centers subject will be accepted for right lobe donation if they have minimal steatosis (<5% or S0) while steatosis up to 30% (S0 or S1) can be accepted for left lobe donation. Considering subdivision of S1 however will require a separate study to validate such score.

Patient demographics, BMI, and lab investigations [glycated hemoglobin (HbA1c), liver function tests, and lipid profile] at the time of recruitment were collected.

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software version 22.0 (SPSS, Inc., Chicago, IL). Youden index was used to choose the cut-off value for CAP. Quantitative data were expressed as mean and standard deviation. Differences in the groups were analyzed with the unpaired student’s t-test for comparison of the means of numerical variables and the chi-squared test for comparison of categorical variables. Multiple logistic regression analysis was used to assess the predictors of HS. P-value < .05 was considered statistically significant. Odds ratios (OR), sensitivity, specificity, positive and negative predictive values (PPV and NPV), positive and negative likelihood ratios, and area under the receiver operator characteristic (AUROC) curve are reported.

Results

In total, 150 potential living liver donors were included in the analysis. The mean age of the cohort was 30.0±7.0 years, and 73.3% were males, giving a male to female ratio of 3:1. The baseline characteristics of the study subjects are shown in Table 2. Ninety-two subjects (61.3%) had no or mild HS based on CAP measurement, while moderate to severe HS was detected in 58 subjects (38.7%) compared to 10% on liver biopsy (Fig 1).

Table 2

Baseline characteristics of the study population (n = 150).

Variable (units)	Values*
Age (years)*	30.0 ± 7.0
Gender (Male/Female)	110/40 (73.3%/26.7%)
BMI (kg/m2)*	24.7 ± 3.5
HbA1c (%)*	5.2± 0.5
TB (μmol/L)*	10.4 ± 6.7
Alb (g/L)*	47.1± 3.0
ALT (IU/L)*	24.3 ± 13.1
AST (IU/L)*	21.3 ± 9.0
ALP (IU/L)*	72.8 ± 23.1
GGT (IU/L)*	24.8 ± 0.9
TC (mmol/L)*	4.4 ± 0.9
TG (mmol/L)*	1.0 ± 0.9
LDL (mmol/L)*	3.0 ± 0.8
HDL (mmol/L)*	1.3 ± 0.3

* means ± SD

BMI = body mass index; HbA1C = hemoglobin A1C; TB = total bilirubin; Alb = albumin, ALT = alanine aminotransferase; AST = aspartate aminotransferase; ALP = Alkaline phosphatase; GGT = gamma glutamyl transferase; TC = total cholesterol; TG = triglycerides; LDL = low-density lipoprotein’ HDL = high-density lipoprotein

Fig 1

Steatosis as detected by Fibroscan and liver biopsy.

On univariate analysis, subjects with moderate to severe HS based on CAP were predominantly males (p = 0.014) and had higher BMI (p = .006), higher HbA1C percentage (p = .04), alanine aminotransferase (ALT) (p = .001), gamma-glutamyl transferase (p = .026) and high-density lipoprotein (p = .008) levels. There was no significant difference in age, aspartate aminotransferase, triglycerides, or low-density lipoprotein levels between groups (Table 3). Multiple logistic regression showed that higher ALT [OR, 1.051; 95% confidence interval (CI), 1.016–1.087; p = .004) was a predictor for moderate to severe HS (Table 4).

Table 3

Comparison of patients based on the grade of steatosis (ST) by CAP.

Variable	No to Mild HS N = 92	Moderate to Severe HS N = 58	P-Value
Age	29.8 ± 6.8	30.3 ± 7.3	0.671
Gender	61/31	48/9	0.014
BMI	24.1 ± 3.6	25.7 ± 3.1	0.006
HbA1c	5.1 ± 0.5	5.3 ± 0.4	0.04
ALT	21.0 ± 10.1	29.5 ± 15.6	0.001
AST	20.3 ± 8.5	22.9 ± 9.6	0.089
GGT	22.5 ± 14.6	28.4 ± 17.2	0.026
TG	0.98 ± 1.01	1.13 ± 0.67	0.324
LDL	2.9 ± 0.8	3.0 ± 1.0	0.559
HDL	1.4 ± 0.4	1.2 ± 0.3	0.008

Results are given as means ± SD

Table 4

Multiple logistic regression analysis of variables predicting moderate to severe steatosis on CAP.

Variable	OR	P value	95% CI
Ageact	0.976	0.423	0.919–1.036
Gender	0.541	0.296	0.171–1.711
BMI	1.134	0.058	0.996–1.292
ALT	1.051	0.004	1.016–1.087
GGT	0.987	0.374	0.959–1.016
HDL	0.522	0.369	0.126–2.158
HbA1c	1.764	0.203	0.736–4.229

The sensitivity, specificity, PPV, and NPV of Fibroscan® to detect significant HS were 93.3%, 67.4%, 24.1%, and 98.9%, respectively. The positive likelihood ratio of CAP was 2.86, and the negative likelihood ratio was 0.1. Assuming that the pre-test probability of having HS (as indicated from biopsy results) was 10%, using the Fagan nomogram, the post-test probability for a positive test was 30%, and 1% for a negative test.

The AUROC curve for CAP was 0.841 (95% CI, 0.736–0.946) when the cut-off for HS used was greater than or equal to a Fibroscan® score of 2 (Fig 2). When the absolute CAP score was used, the AUROC curve was 0.883 (95% CI, 0.725–0.951). Using a cut-off of 276 dB/m, the sensitivity for CAP to detect HS was 86.7%, and specificity was 83.7% (Fig 3).

Fig 2

Receiver operator characteristic (ROC) curve for Fibroscan detection of steatosis for stage >2.

Fig 3

ROC curve for steatosis using a cutoff of 276 dB/m.

Discussion

There is no current consensus regarding how to assess HS in potential living liver donors. Noninvasive scoring methods have been investigated as potential alternatives to liver biopsy, the current gold standard for HS assessment. Due to its potential complications (bleeding, hematomas, or infections), liver biopsy is not universally utilized for assessing the donor liver in living donor liver transplantation (LDLT) [18]. However, having reliable preoperative information on the degree of HS would help to avoid marginal donor grafts, which potentially harm the donors and put recipients at risk of graft dysfunction.

Ayvazoglu et al. evaluated the importance of preoperative liver biopsy in selecting donor candidates, and they reported that 32% of liver biopsies had pathologic findings. Out of those findings, 44% were fatty changes, with 12% having portal inflammation. In this case, the high rate of pathologic findings in liver biopsy of healthy-appearing donor candidates appeared to indicate the importance of liver biopsy in the preoperative evaluation of donors [19].

To date, only a limited number of studies have reported the potential role of CAP in assessing HS in potential living liver donors [20-22]. This study has shown that CAP has high sensitivity (over 93%) to detect moderate to severe HS and a very high NPV of almost 99%, which can accurately identify patients without significant HS. A score of ≥2 was selected as the best cut-off value using the Youden index. The PPV, however, was low at 24.1%, indicating the need for liver biopsy in donors with positive CAP results to confirm the findings.

Several studies support the use of CAP instead of liver biopsy for HS assessment in various liver diseases. One systematic review evaluated CAP for the diagnosis of HS in patients with various liver conditions; in that study, CAP had good diagnostic accuracy in the evaluation of patients with HS, with similar results regardless of stage [23]. Shi et al. compared the accuracy of CAP versus biopsy, and determined that CAP was accurate for all HS stages [24].

In a study of 224 patients, Baumeler et al. evaluated CAP as a diagnostic tool for identifying the presence and degree of HS in consecutive patients in an outpatient liver unit of a tertiary center [10]. They found that irrespective of the underlying liver disease, CAP values strongly correlated with the degree of HS. These findings support integrating CAP measurements in the standard workup of CLD to identify patients with NAFLD, whether alone or with other causes.

Another systematic review, involving 1297 patients, evaluated the performance of CAP in the diagnosis and staging of HS and biopsy-proven NAFLD [11]. The pooled sensitivity and specificity of CAP in detecting mild HS was 87% and 91%, 85% and 74% for moderate HS, and 76% and 58% for severe HS, respectively. The mean AUROC value for CAP in the diagnosis of mild, moderate, and severe HS was 0.96, 0.82, and 0.70, respectively. Subgroup analysis indicated that variation in the geographic regions, cut-offs, age, and BMI could be the potential sources of heterogeneity in the diagnosis of moderate to severe HS. The study’s conclusion notes that CAP should be viewed, albeit with caution, as a potentially sound, noninvasive alternative to liver biopsy.

The findings in this study were well aligned with the results of previous smaller studies using CAP to assess and quantify HS in LDLT. A study of 54 donors by Yen et al., who underwent both CAP and biopsy, also showed high AUROC using a cut-off value of 257 dB/m, 100% sensitivity, and 100% NPV for CAP in evaluating HS. The study found a specific correlation between BMI and CAP values in subjects who did not have HS. They concluded that CAP could prove to be a useful method in identifying HS in living liver donors in East Asia [25].

Hong et al. evaluated the accuracy of CAP for detecting HS in potential liver donors with similar results of high AUROC, albeit with somewhat lower sensitivity and NPV. The CAP value again correlated positively with BMI, along with waist circumference, hip circumference, magnetic resonance fat signal fraction, and histologic HS grade. This study suggests that CAP, as a simple and noninvasive preoperative assessment for HS, may be sufficient for identifying and thus excluding significant HS in potential living liver donors [26].

Factor affecting the concordance between histology and CAP in assessment of HS like the sampling technique of the biopsy and the adequacy of the tissue are important to consider together with the presence of other liver pathology that might increase the stiffness like liver congestion and infiltration and these are less likely here as we are dealing with healthy subjects.

The findings of our study should be viewed in light of both their strengths and limitations. The cohort was prospectively recruited, and participants underwent a standardized, validated liver disease assessment. The presence and degree of HS were assessed by CAP against the accepted gold standard test (liver biopsy). The study was conducted at a single site, which may limit the generalizability of the results and conclusions. However, the consistency with other studies’ findings may imply more ubiquity to these results.

Conclusion

The high sensitivity and NPV of CAP make it a good screening test for assessment of HS in potential living liver donors to avoid unnecessary liver biopsies in those with a negative result. A positive CAP, however, has poor PPV, and liver biopsy is required to confirm the presence of significant steatosis and exclude other liver pathology.

24 Nov 2020

PONE-D-20-31215

VALIDATING CONTROLED ATTENUATION PARAMETER IN THE ASSESSMENT OF HEPATIC STEATOSIS IN LIVING LIVER DONORS

PLOS ONE

Dear Dr. Bzeizi,

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.

This is an important study for those in the field of living liver donor work up; it describes an innovative way (CAP) to assess liver steatosis in potential donors, which could replace the liver biopsy as gold standard, which would reduce the risk for potential donors, which is something we should always aspire to. The authors should be applauded for this work, and expert reviewers have highlighted its value for the literature. They have recommended minor revisions to be made, and I would encourage the authors to revise the MS accordingly and respond in a point-by-point fashion to the comments. Looking forward to receive the revised MS.

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Reviewer #1: Dear authors,

congrats for your paper describing a non invasive tool to investigate hepatic steatosis in the assessment of living liver donors.

I have two comments:

You're using the following scores to grade hepatic steatosis on liver biopsy - S0 (<5%), S1 (5%–33%), S2 (33%–66%), and S3 >66%. Decision making whether or not a living donor can be accepted and if yes, whether to use a right or left lobe, depends amongst other things on the liver remnant in % and the liver quality, e.g. hepatic steatosis (HS). In this regards score S1 includes a wide range of hepatic steatosis between 5 and 33 %. This range includes also a wide range of possible decisions whether or not a living donor is suitable or not at all, respectively if suitable for a right and left or only a left lobe. It seems that a subdivision of S1 would be valid, but should be at least discussed.

On page 15 is written "In the 54 donors of the study by xx, ...". I guess "xx" is just a spacer for the planned citation.

Reviewer #2: This is a very interesting paper about the potential use of the controlled attenuation parameter (CAP) in the assessment of hepatic steatosis in living liver donors.

The current gold standard is the liver biopsy but CAP may be a useful tool to avoid unnecessary biopsies, although it is still necessary due to the reported low diagnostic power of CAP in presence of severe steatosis,

This is a well-conducted prospective study, abstract, introduction, and discussion are clear and concise, aim and conclusion are coherent. Figures and tables are appropriate for the manuscript.

To-date, data on this specific topic (evaluation of steatosis in perceived healthy liver) are scarce and from relatively small case-series.

Evaluation of the necessary statistical power to use the term "validating" is required.

Other issues include:

- an evaluation of the parameters affecting the concordance between CAP and biopsy could add some value to this paper

- it is reported that an expert pathologist evaluates the biopsy blindly, but there is no specific indication about who performs the CAP, which is an operator-dependent test.

- why exclude left lateral hepatectomies from inclusion criteria?

Minor revisions:

- please revise the language used in the Methods section of the abstract

- the use of the Youden index to choose the cut-off should be mentioned first in the Method section

- "Another systematic review of the performance of CAP ... 1297 patients" is maybe linked to the ref 11 instead of 23

- "In the 54 donors of the study by xx" should be revised

- I would put the paragraph with the reference about the paper of Ayvazoglu at the beginning of the discussion since they stress the importance of the biopsy.

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Reviewer #1: Yes: Markus U. Boehnert

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9 Feb 2021

January 11, 2021

Frank JMF Dor, M.D., Ph.D., FEBS, FRCS

Academic Editor

PLOS ONE

Dear Professor Dor,

Re: “Validating controlled attenuation parameter in the assessment of hepatic steatosis in living liver donors”

Many thanks for giving us the opportunity to resubmit the manuscript after the revision as per your valuable suggestions and those of the reviewers. The manuscript has been proofread by a native English speaker with expertise in the field.

I hope that we covered most of the points raised and once again, many thanks for considering our study for publication in PLOS ONE

Best wishes,

Khalid Bzeizi

Point-By- Point Response to The Comments Raised by The Reviewers:

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.

3). Answer:

The preliminary and essential requirement for accepting potential donors to proceed with the liver transplant workup include:

1). Signing an informed consent for donation, (2). Being a direct relative to the patient, (3) Age between 18-50 years, (4). Body mass index (BMI) <30, (5). No previous abdominal surgery, (6). No history of alcohol or substance abuse, (7). No documented chronic medical history or on regular medication and (8). All donors were cleared by psychologist/psychiatrist from any psychiatric illness or of reduced mental capacity

b). Given the nature of donor process selection, the sample is not representative of the general population.

c). All donors were direct relatives of the patients.

4. We note that your study involved studying potential tissue/organ transplantation donors. Please provide the following information:

a) Please state in your response letter and ethics statement whether the potential donors involved any vulnerable populations; for example, prisoners, subjects with reduced mental capacity due to illness or age, or minors.

Answer: The following statement was added in page 6, para 2:

“None of the potential transplant donors was from a vulnerable population (e.g, prisoners, subjects with reduced mental capacity due to illness or age, or minors) and all potential donors provided written informed consent that was freely given.”

- If a vulnerable population was used, please describe the population, justify the decision to use them as potential organ donors, and clearly describe what measures were taken in the informed consent procedure to assure protection of the vulnerable group and avoid coercion.

Answer: NA

- If a vulnerable population was not used, please state in your ethics statement, “None of the potential transplant donors was from a vulnerable population and all potential donors provided written informed consent that was freely given.”

Answer: Done (Page 6, Para2)

b) Please provide a blank example of the form used to obtain consent from donors, and an English translation if the original is in a different language.

Answer: The English consent form has been uploaded

c) Please indicate whether the donors were previously registered as organ donors.

Answer: No. This is unfortunately not the practice in KSA

d) Please discuss whether medical costs were covered or other cash payments were provided to the potential donor. If so, please specify the value of this support (in local currency and equivalent to U.S. dollars).

Answer: None

5. Please include additional information regarding the questionnaire used for selecting living donors in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

6. Thank you for stating the following financial disclosure:

"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:

1. 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.

2. 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.”

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

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

Answer: No funding was provided for this study. All workup assessment measures were done as part of the transplant protocol requirements

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

7. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, on some of which you may be an author.

https://www.sciencedirect.com/science/article/pii/S0041134518308984?via%3Dihub

https://smw.ch/article/doi/smw.2019.20077

https://bmcgastroenterol.biomedcentral.com/articles/10.1186/s12876-019-0961-9

https://journals.lww.com/eurojgh/Abstract/2017/07000/Clinical_usefulness_of_controlled_attenuation.10.aspx

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

Answer: Many thanks for the valuable comment. The manuscript has been revised taking into consideration the overlaps mentioned. This has now been rectified

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors,

congrats for your paper describing a non invasive tool to investigate hepatic steatosis in the assessment of living liver donors.

I have two comments:

You're using the following scores to grade hepatic steatosis on liver biopsy - S0 (<5%), S1 (5%–33%), S2 (33%–66%), and S3 >66%. Decision making whether or not a living donor can be accepted and if yes, whether to use a right or left lobe, depends amongst other things on the liver remnant in % and the liver quality, e.g. hepatic steatosis (HS). In this regards score S1 includes a wide range of hepatic steatosis between 5 and 33 %. This range includes also a wide range of possible decisions whether or not a living donor is suitable or not at all, respectively if suitable for a right and left or only a left lobe. It seems that a subdivision of S1 would be valid, but should be at least discussed.

Answer: We have addressed this point in the methods section (page 7, para: 2):

Although the range in S1 is quite wide, however it remains the well-known grading system for reporting severity of steatosis. The degree of steatosis might affect the decision on donation and in most transplant centers subject will be accepted for right lobe donation if they have minimal steatosis (<5% or S0) while steatosis up to 30% (S0 or S1) can be accepted for left lobe donation. Considering subdivision of S1 however will require a separate study to validate such score.

On page 15 is written "In the 54 donors of the study by xx, ...". I guess "xx" is just a spacer for the planned citation.

Answer: Thank you. This has been rectified (page 10, para 2):

“In the 54 donors of the study by Yen et al, who underwent both CAP and biopsy, also showed high AUROC using a cut-off value of 257 dB/m, 100% sensitivity, and 100% NPV for CAP in evaluating HS.”

Reviewer #2: This is a very interesting paper about the potential use of the controlled attenuation parameter (CAP) in the assessment of hepatic steatosis in living liver donors.

The current gold standard is the liver biopsy but CAP may be a useful tool to avoid unnecessary biopsies, although it is still necessary due to the reported low diagnostic power of CAP in presence of severe steatosis,

This is a well-conducted prospective study, abstract, introduction, and discussion are clear and concise, aim and conclusion are coherent. Figures and tables are appropriate for the manuscript.

To-date, data on this specific topic (evaluation of steatosis in perceived healthy liver) are scarce and from relatively small case-series.

Evaluation of the necessary statistical power to use the term "validating" is required.

Other issues include:

- an evaluation of the parameters affecting the concordance between CAP and biopsy could add some value to this paper

Answer: Many thanks for the suggestion. This has been added in the discussion (page 11, para:8):

“Factor affecting the concordance between histology and CAP in assessment of HS like the sampling technique of the biopsy and the adequacy of the tissue are important to consider together with the presence of other liver pathology that might increase the stiffness like liver congestion and infiltration and these are less likely here as we are dealing with healthy subjects.”

- it is reported that an expert pathologist evaluates the biopsy blindly, but there is no specific indication about who performs the CAP, which is an operator-dependent test.

Answer: One operator (Dr M. Shawkat) performed the CAP

- why exclude left lateral hepatectomies from inclusion criteria?

Answer: As part of our center protocol. none of the donors selected for left lateral hepatectomies needed to undergo liver biopsy as a requirement for donation.

Minor revisions:

- please revise the language used in the Methods section of the abstract.

Answer: Done.

- the use of the Youden index to choose the cut-off should be mentioned first in the Method section

Answer: Many thanks for this suggestion. This has been added to the manuscript (page:7, para: 4)

“Youden index was used to choose the cut-off value for CAP.”

- "Another systematic review of the performance of CAP ... 1297 patients" is maybe linked to the ref 11 instead of 23.

Answer: Many thanks for highlighting this reference mislabeling. This has now been rectified in the manuscript (page: 9, para: 2).

- "In the 54 donors of the study by xx" should be revised

Answer: Thank you. This has been rectified (page 10, para 2):

“In the 54 donors of the study by Yen et al, who underwent both CAP and biopsy, also showed high AUROC using a cut-off value of 257 dB/m, 100% sensitivity, and 100% NPV for CAP in evaluating HS.”

- I would put the paragraph with the reference about the paper of Ayvazoglu at the beginning of the discussion since they stress the importance of the biopsy.

Answer: Many thanks for the suggestion. The authors have felt that the paragraph describing the findings of Ayvazoglu et al study is reasonably positioned in the discussion section and we prefer to keep it unchanged.

Submitted filename: Response to reviewers. CAP study.docx

Click here for additional data file.

10 Mar 2021

PONE-D-20-31215R1

VALIDATING CONTROLED ATTENUATION PARAMETER IN THE ASSESSMENT OF HEPATIC STEATOSIS IN LIVING LIVER DONORS

PLOS ONE

Dear Dr. Bzeizi,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Frank JMF Dor, M.D., Ph.D., FEBS, FRCS

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):

Congratulations on the revised MS. Reviewer 2 suggests a few further edits to be made, and i am confident you can address any remaining issues in the revisions.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: This is a very interesting paper about the potential use of the CAP in the assessment of hepatic steatosis in living liver donors.

Liver biopsy remains the current gold standard and, but CAP, despite the low diagnostic power of CAP with severe steatosis, may be a useful tool to avoid unnecessary biopsies.

This is a well-conducted prospective study. Abstract , introduction, and discussion are clear and concise and aim and conclusion are coherent. Figures and tables are adequate and coherent to the test.

To-date, data on this specific topic (assessment of steatosis in perceived healthy livers) are scarce and from relatively small case-series.

Evaluation of the necessary statistical power to use the term "validating" is required.

Further issues with this manuscript:

- an evaluation of the parameters affecting the concordance between CAP and biopsy could add value to this paper

- it is reported that an expert pathologist assessed the biopsies in a blind way. However it is not clear who performs CAP that is an operator-dependent exam

- why was left lateral hepatectomy chosen among the exclusion criteria?

Minor comments:

- please revise the language used in the methods section of the abstract

- the use of the Youden index to identify the cut-off should be mentioned with priority in the Methods section

- "Another systematic review of the performace of CAP ... 1297 patients" is possibly linked to ref 11 instead of 23 ?

- "In the 54 donors of the study by xx" should be revised

- The paragraph with the reference regarding the paper of Ayvazoglu should find a place earlier in the Discussion given that they clarify the importance of the biopsy.

**********

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Reviewer #1: Yes: Markus U. Boehnert

Reviewer #2: No

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11 Apr 2021

Dear Professor Dor,

Ref: PONE-D-20-31215R1

VALIDATING CONTROLED ATTENUATION PARAMETER IN THE ASSESSMENT OF HEPATIC STEATOSIS IN LIVING LIVER DONORS

PLOS ONE

Many thanks for the opportunity to resubmit a revised manuscript and we appreciate the valuable comments of the reviewers.

The following are the responses to the points raised by Reviewer #2:

1) An evaluation of the parameters affecting the concordance between CAP and biopsy could add value to this paper.

Response: In the earlier submission, we alluded to the factors and parameters that might impact on the concordance between CAP and biopsy. In paragraph-1 (page 11), we highlighted the fact that the donors are healthy cohorts and this might well be a factor in minimizing the potential factors that adversely impact on the concordance.

“Factor affecting the concordance between histology and CAP in assessment of HS like the sampling technique of the biopsy and the adequacy of the tissue are important to consider together with the presence of other liver pathology that might increase the stiffness like liver congestion and infiltration and these are less likely here as we are dealing with healthy subjects.”

2) It is reported that an expert pathologist assessed the biopsies in a blind way. However, it is not clear who performs CAP that is an operator-dependent exam.

Response: The Fibroscan & CAP were performed by a certified technician under the supervision of one hepatologist. The technician did more than 1000 scan on potential donors prior to his participation in this study.

3) Why was left lateral hepatectomy chosen among the exclusion criteria?

Response: As per our department transplant protocol, liver biopsy is not a requirement from donors of left lateral segment. The liver biopsies were only for right lobe donors.

4) Please revise the language used in the methods section of the abstract.

Response: Many thanks for the suggestion. These parts have been proofread.

5) The use of the Youden index to identify the cut-off should be mentioned with priority in the Methods section.

Response: This point is now highlighted in the last paragraph (page 7).

6) "Another systematic review of the performace of CAP ... 1297 patients" is possibly linked to ref 11 instead of 23 ?.

Response: Many thanks for the suggestions. This has now been corrected (para: 3, page 10):

Another systematic review, involving 1297 patients, evaluated the performance of CAP in the diagnosis and staging of HS and biopsy-proven NAFLD.(11)

7) "In the 54 donors of the study by xx" should be revised.

Response: Thank you. This has now been revised (para 4: page 10):

“ A study of 54 donors by Yen et al, who underwent both CAP and biopsy, also showed high AUROC using a cut-off value of 257 dB/m, 100% sensitivity, and 100% NPV for CAP in evaluating HS”.

8) The paragraph with the reference regarding the paper of Ayvazoglu should find a place earlier in the Discussion given that they clarify the importance of the biopsy.

Response: Many thanks for the suggestion. This paragraph has now moved up to page 9 (para: 2). The order of the references has now changed upon the transfer of this paragraph

We hope that the points raised by the reviewers have been addressed and once again, many thanks for the opportunity to resubmit the manuscript.

Best wishes

Khalid Bzeizi

Submitted filename: CAP study. Response to reviewers. April 2021.docx

Click here for additional data file.

28 Apr 2021

VALIDATING CONTROLED ATTENUATION PARAMETER IN THE ASSESSMENT OF HEPATIC STEATOSIS IN LIVING LIVER DONORS

PONE-D-20-31215R2

Dear Dr. Bzeizi,

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,

Frank JMF Dor, M.D., Ph.D., FEBS, FRCS

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors correctly and completely addressed all the issues raised. In my opinion, the topic is of interest for the readership

**********

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Reviewer #2: Yes: Paolo Muiesan

4 May 2021

PONE-D-20-31215R2

Validating controlled attenuation parameter in the assessment of hepatic steatosis in living liver donors

Dear Dr. Bzeizi:

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.

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

Kind regards,

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on behalf of

Dr. Frank JMF Dor

Academic Editor

PLOS ONE