Controlled attenuation parameter (CAP) for the diagnosis of steatosis: A prospective study of 5323 examinations

Accepted Manuscript Controlled Attenuation parameter (CAP) for the diagnosis of steatosis: a prospective study of 5,323 examinations Victor de Lédinghen, Julien Vergniol, Maylis Capdepont, Faiza Chermak, JeanBaptiste Hiriart, Christophe Cassinotto, Wassil Merrouche, Juliette Foucher, Brigitte Le Bail PII: DOI: Reference:

S0168-8278(13)00886-6 http://dx.doi.org/10.1016/j.jhep.2013.12.018 JHEPAT 4980

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

22 July 2013 15 December 2013 16 December 2013

Please cite this article as: Lédinghen, V.d., Vergniol, J., Capdepont, M., Chermak, F., Hiriart, J-B., Cassinotto, C., Merrouche, W., Foucher, J., Bail, B.L., Controlled Attenuation parameter (CAP) for the diagnosis of steatosis: a prospective study of 5,323 examinations, Journal of Hepatology (2013), doi: http://dx.doi.org/10.1016/j.jhep. 2013.12.018

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Controlled Attenuation parameter (CAP) for the diagnosis of steatosis: a prospective study of 5,323 examinations. Victor de Lédinghen

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, Julien Vergniol , Maylis Capdepont , Faiza Chermak , Jean-Baptiste Hiriart , Christophe 1

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Cassinotto, Wassil Merrouche , Juliette Foucher , Brigitte Le Bail .

1. Centre d‟Investigation de la Fibrose hépatique, Hôpital Haut-Lévêque, Centre Hospitalo-Universitaire de Bordeaux, Pessac, France 2. INSERM U1053, Université Bordeaux Segalen, Bordeaux, France 3. Service de Chirurgie Viscérale, Hôpital Saint-André, Centre Hospitalo-Universitaire de Bordeaux, Bordeaux, France 4. Service d‟Imagerie Médicale, Hôpital Haut-Lévêque, Centre Hospitalo-Universitaire de Bordeaux, Pessac, France 5. Laboratoire de Pathologie, Hôpital Pellegrin, Centre Hospitalo-Universitaire de Bordeaux, Bordeaux, France

Running title: Controlled Attenuation Parameter (CAP) in clinical practice Key words: CAP, liver stiffness; transient elastography; FibroScan; obesity; NAFLD; metabolic syndrome, steatosis.

Word count: 5178 Correspondence to : Victor de Lédinghen, MD PhD Centre d'Investigation de la Fibrose hépatique Service d'Hépato-Gastroentérologie Hôpital Haut-Lévêque 33604 Pessac France E-mail: [email protected] Tel: + 33 5 57 65 64 39 Fax: + 33 5 57 65 64 45

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Victor de Lédinghen: Echosens consultant

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Julien Vergniol: No conflicts of interest exist Maylis Capdepont: No conflicts of interest exist Faiza Chermak: No conflicts of interest exist Jean-Baptiste Hiriart: No conflicts of interest exist Christophe Cassinotto: No conflicts of interest exist Wassil Merrouche: No conflicts of interest exist Juliette Foucher: No conflicts of interest exist Brigitte Le Bail: No conflicts of interest exist

We disclosed to study participants potential investigator conflicts of interest.

No financial support.

ABBREVIATIONS CAP: Controlled attenuation parameter BMI: body mass index AUROC: Area under the ROC curve FLI: Fatty liver index

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ABSTRACT

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Background & Aims: Controlled attenuation parameter (CAP) evaluated with transient elastography (FibroScan) is a recent method for non invasive assessment of steatosis. Its usefulness in clinical practice is unknown. We prospectively investigated the determinants of CAP failure and the relationships between CAP and clinical or biological parameters in a large cohort of consecutive patients. Methods: All CAP examinations performed in adult patients with suspected chronic liver disease were included. CAP failure was defined as zero valid shot. The following factors were analyzed for their influence on CAP value and the relationships between CAP and clinico-biological parameters: age, gender, body mass index, waist circumference, hypertension, diabetes, metabolic syndrome, alcohol use, liver stiffness measurement, indication, and different biological parameters. Results. CAP failure occurred in 7.7% of 5,323 examinations. By multivariate analysis, factors independently associated with CAP measurement failure were female gender, BMI, and metabolic syndrome. By multivariate analysis, factors significantly associated with elevated CAP were BMI ]25-30] kg/m², BMI > 30 kg/m², metabolic syndrome, alcohol > 14 drink/week and liver stiffness > 6 kPa. CAP increased with the number of parameters of metabolic syndrome, BMI, waist circumference, the presence of diabetes or hypertension, and the cause of the disease. In the 440 patients with liver biopsy, for the diagnosis of steatosis > 10%, steatosis > 33%, and steatosis > 66%, AUROCs of CAP were 0.79 (95%CI 0.74-0.84, p<0.001), 0.84 (95%CI 0.80-0.88, p<0.001), 0.84 (95%CI 0.80-0.88, p<0.001), respectively Conclusion. CAP provides an immediate assessment of steatosis simultaneously with liver stiffness measurement. The strong association of CAP with the metabolic syndrome and alcohol use could be of interest for the follow-up of NAFLD or alcoholic patients.

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INTRODUCTION 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Recently, a novel physical parameter based on the properties of ultrasonic signals acquired by the FibroScan has been developed using the postulate that fat affects ultrasound propagation. This novel parameter, named Controlled Attenuation Parameter (CAP), measures the ultrasound attenuation at the center frequency of the FibroScan® M probe (3.5 MHz) [1]. Values range from 100 to 400 dB/m. CAP can be used for steatosis detection and quantification and present several advantages: it is noninvasive, easy to perform, provide immediate results and is inexpensive in comparison with other measurement modalities. It is less influenced by sampling error than liver biopsy since it explores a liver volume approximately 100 times larger. Furthermore, CAP was designed to target specifically the liver. Therefore, CAP can be performed, simultaneously to liver stiffness measurement and in the same liver volume, making possible the simultaneous evaluation of both fibrosis and steatosis and consequently enhancing the spectrum of non-invasive methods for the exploration and follow-up of patients with chronic liver disease. Recent studies shown that CAP is significantly correlated with the percentage of steatosis and steatosis grade, and that median CAP is higher among patients with significant steatosis [2-4]. In a prospective study of 153 patients, the AUROCs of CAP for ≥ 5%, >33% and >66% steatosis were 0.79, 0.76 and 0.70, respectively [3]. In another prospective study of 112 patients, the AUROCs of CAP for 11 ~ 33%, 34 ~ 66%, and > 66% steatosis were 0.84, 0.86, and 0.93, respectively [4]. However, transient elastography has limitations: liver stiffness measurement results may be influenced by acute liver injury (as reflected by ALT flares), with a risk of overestimating liver stiffness, and also by extrahepatic cholestasis [5-7]. The transient elastography interpretation software (for liver stiffness and CAP measurements) indicates whether or not each measurement (or “shot”) is successful. When a shot is considered unsuccessful, the machine provides no result. The entire procedure is considered to have failed when no value is obtained after ten shots or more. Recommandations for successful measurements for CAP measurement are unknown. The aim of this prospective study was to investigate the frequency and determinants of CAP failure in clinical practice, and to investigate the relationships between CAP and different clinical and biological parameters in a large cohort of consecutive patients.

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PATIENTS AND METHODS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Study population Between April 2009 and November 2012, all consecutive patients who presented with chronic liver disease in our centre had CAP performed and were included. These patients were referred to our centre for a noninvasive assessment of liver fibrosis. The following clinical parameters were recorded at the time of measurement: age, gender, body mass index (BMI), waist circumference, hypertension, diabetes, metabolic syndrome, alcohol and coffee use, and indication for measurement. Alcohol abuse was defined as more than 14 drink/week in females, and more than 21 drink/week in males. The study protocol conformed to the ethical guidelines of the 1975 Helsinski declaration and patients were enrolled after giving their written informed consent.

Liver stiffness and CAP measurements and definition of failure ®

Liver stiffness and CAP measurements were performed by FibroScan (Echosens, Paris, France) by experienced operators. All patients were measured using the 3.5 MHz standard M probe. The final liver stiffness result was expressed in kPa and was the median value of 10 measurements performed between 25 and 65 mm depth. Only results with no shot or with 10 valid shots and IQR/median liver stiffness ratio < 30% were included. CAP has been designed to measure liver ultrasonic attenuation (go and return path) at 3.5 MHz on the signals acquired by the FibroScan®. Principle of CAP measurement has been described elsewhere [1]. CAP was computed only when the associated liver stiffness measurement was valid and using the same signals as the one used to measure liver stiffness, Therefore both stiffness and CAP were obtained simultaneously and in the same volume of liver parenchyma (namely between 25 and 65 mm). The final CAP value was the median of individual CAP values and was expressed in dB/m. Liver stiffness and CAP measurement failures were recorded when no value was obtained after at least 10 shots (valid shots =0).

Biological parameters The same day as liver stiffness and CAP measurements, the following biological parameters were recorded: aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), alkaline phosphatise (AP), total bilirubin, platelet count, prothrombin time, total cholesterol, HDL cholesterol, triglycerides, fasting glucose, and ferritin level. Blood sample analysis was centralised in the laboratory of our hospital.

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Metabolic syndrome was defined as follow: waist circumference > 94 cm for male and 80 cm for female,

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antihypertensive treatment or blood pressure ≥ 130/85 mmHg, type 1 or type 2 diabetes, triglycerides > 1.6 mmol/L and HDL cholesterol < 1.04 mmol/L for male and < 1.29 mmol/L for female [8]. The Fatty Liver Index (FLI) was calculated according to the formula: FLI = (e 0.953*loge (triglycerides in mg/dL) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference - 15.745) / (1 + e 0.953*loge (triglycerides) + 0.139*BMI + 0.718*loge (GGT) + 0.053*waist circumference - 15.745) * 100). [9].

Liver histology In some cases, when needed, a liver biopsy was performed by senior operators according to the Menghini ®

technique using a 1.6 mm diameter needle (Hepafix , Braun, Melsungen, Germany). Liver Biopsy specimens were fixed in formalin and paraffin embedded. Four-micrometer-thick sections were stained with hematoxylin-eosinsafran, Masson‟s trichromic stain for collagen, Perl‟s stain for iron, and Gordon Sweets reticulin stain. For the purpose of the present study, all liver biopsies were analysed by the same experienced hepatopathologist who was blinded to the results of CAP and liver stiffness measurements. Liver biopsy specimens that contained fewer than 10 portal tracts (except for cirrhosis) were excluded. The aggregated length of each liver biopsy specimen was measured in mm. Liver fibrosis stage was evaluated according to the Metavir or Brunt scoring system according to the cause of disease [10, 11]. Steatosis was categorized by visual assessment as S0: steatosis less than 10% of hepatocytes, S1: 11% to 33%, S2: 34% to 66% and S3: 67% to 100% of hepatocytes.

Statistical analysis In descriptive analyses, continuous variables were expressed as mean ± standard deviation (SD) or median [interquartile range], and categorical variables as absolute figures and percentages. In patients with reliable measurement, the relationship between each factor and CAP was investigated by using univariate logistic regression, p values <0.05 being considered to denote significant associations. Continuous variables such as age were dichotomized around the median, unless a cut-off was considered clinically relevant (for example BMI > 30 kg/m²). Factors significantly associated with CAP in univariate analyses were entered in a multivariate logistic model. In order to be clinically relevant, only parameters available in outpatients were analysed in multivariate analysis (clinical parameters and liver stiffness measurement). Two different cut-offs were evaluated: 250 and 300 dB/m. These two cut-offs were chosen because they include most of previously published cut-offs for the diagnosis of steatosis > 33% [3, 4]. Odds ratios were estimated from the model and are given with their 95% confidence intervals (CI). Areas under the ROC curves (AUROC) were used for the diagnostic analyses. Comparisons between diagnostic AUROCs were performed using the Obuchowski method to take into account 6

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the risk of multiple testing and the spectrum effect [12-15]. The Obuchowski measure allows to compare 2

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biomarkers with a single test, avoiding appropriate correction for the type I error when comparing 2 biomarkers for different stages or grades [14]. This measure is a multinomial version of the AUROC. Statistical analyses were performed with SPSS software (Statistical Systems, Kayville, UT).

RESULTS Characteristics of the study population A total of 5,323 examinations were performed in 4,451 patients. Characteristics of patients were as follow: males 54.3%, mean age 54.9 ± 13.4 years, mean BMI 26.6 ± 5.9 kg/m², 54.1% of patients overweight (BMI ]25-30] kg/m²), 22.5% of obese patients (BMI > 30 kg/m²), waist circumference 92.7± 15.8 cm, diabetes 18.6%, hyertension 36.4%, metabolic syndrome 27.3%, alcohol use 7.7 ± 25.9 drink/week. Causes of chonic liver diseases were as follow: HCV infection 32.8%, Non-Alcoholic Fatty Liver Disease (NAFLD) 24.2%, follow-up of methotrexate treatment 9.1%, HBV infection 7.5%, alcohol abuse 7.3%, HIV-HCV coinfection 4.2%, chronic unexplained cytolysis 2.9%, auto-immune hepatitis 1.3%, primary biliary cirrhosis 1.1%, and miscellaneous 9.6%. Clinical and biological characteristics of the 5,323 CAP examinations are indicated in Table1. The mean liver stiffness measurement value was 9.1 ± 10.5 kPa (range: 2.2-75 kPa), and the mean CAP value was 246.5 ± 67.1 dB/m (range: 100-400 dB/m).

Frequency and determinants of CAP measurement failure Overall, CAP measurement failed in 410 cases (7.7%). CAP measurement failure was independently associated with the following factors: female gender, age > 55 years, BMI ]25-30] kg/m², BMI > 30 kg/m², waist circumference, hypertension, diabetes, and metabolic syndrome (Table 2). By multivariate analysis, factors independently associated with CAP measurement failure were female gender, BMI, and metabolic syndrome (Table 2). CAP measurement failed in respectively 1.0%, 5.6%, 19.4% and 58.4% of patients with BMI ≤ 25 kg/m², 25 to 29.9 kg/m², 30 to 40 kg/m², and >40 kg/m².

Factors associated with CAP values CAP measurement was significantly correlated with many clinical and biological factors (Supplementary file 1).

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In order to be clinically relevant, by regression analysis, only clinical factors (including liver stiffness)

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were evaluated. By univariate analysis, factors associated with CAP were male gender, age > 55 years, BMI, elevated waist circumference, diabetes, hypertension, metabolic syndrome, and liver stiffness > 6 kPa (Table 3). By multivariate analysis, factors associated with CAP were mal gender, age > 55 years, BMI, metabolic syndrome, alcohol abuse and liver stiffness > 6 kPa (Table 3). In Supplementary file 2, 3 and 4 are indicated factors associated with elevated CAP values. As indicated on Figures 1 and 2, CAP significantly increased with the number of parameters of metabolic syndrome, BMI, waist circumference, the presence of diabetes or hypertension, and the cause of the disease (p<0.001). A significant relationship was observed between mean CAP and increasing alcohol use: 243 ± 67 dB/m in patients with ≤ 7 drinks/week, 253 ± 55 dB/m in patient with 8 to 14 drinks/week, and 261 ± 65 dB/m in patients with ≥ 14 drinks/week (p<0.001).

Relationships between CAP and steatosis A liver biopsy was available in 440 cases. Mean length of liver biopsy was 2.51 ± 0.5 cm. Liver fibrosis according to liver biopsy was a follow: F0 11.1%, F1 35.5%, F2 22.3%, F3 15.5%, and F4 15.7%. Steatosis grade was available in 423 cases. Mean steatosis was 25.1 ± 1.3% (extremes: 0-90%). Steatosis was ≤ 10% in 205 cases (48.5%), between 11 and 33% in 85 cases (20.1%), between 34 and 66% in 71 cases (16.8%), and > 66% in 62 cases (14.7%). CAP was strongly correlated with steatosis (r=0,456, p<0.0001) but slighlty with fibrosis (r=0,095, p=0,01). For the diagnosis of steatosis > 10%, steatosis > 33%, and steatosis > 66%, AUROCs of CAP were 0.79 (95%CI 0.75-0.84), 0.84 (95%CI 0.80-0.88), 0.84 (95%CI 0.80-0.88), respectively. For the diagnosis of steatosis > 10%, steatosis > 33%, and steatosis > 66%, AUROCs of FLI score were 0.74 (95%CI 0.69-0.79), 0.79 (95%CI 0.750.84), 0.76 (95%CI 0.70-0.82), respectively. For the diagnosis of steatosis, according to Obuchowski test, CAP measurement (AUC 0.914, 95% confidence interval 0.90-0.93) had significantly better performance than FLI score (AUC 0.885, 95% confidence interval 0.86-0.91).

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DISCUSSION 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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In this prospective study based on more than 5,300 examinations – the largest to date - we found that CAP values were significantly associated with all parameters of metabolic syndrome. Our findings may have important implications for current and future applications in patients with metabolic syndrome, especially with NAFLD. Indeed, the increasing prevalence of obesity, insulin resistance and the metabolic syndrome is changing the face of chronic liver disease; in particular, nearly one-third of American adults now have NAFLD [16-18]. With CAP, a quantitative method, we will able to follow patients with metabolic syndrome or NAFLD. The evolution of CAP values could be related to the evolution of metabolic syndrome. Another interesting result is that CAP values were significantly associated with alcohol abuse. This result could be very relevant for the diagnosis of steatosis associated with chronic alcohol use but also for the follow-up of alcoholic patients with or without persistent alcohol use. Of course, this result needs to be confirmed by other groups and prospective studies. CAP measurement failure was observed in 7.7% of cases. CAP was always valid when liver stiffness was measurable, because measurement is obtain with the same shot . Among the characteristics of patients, age, obesity

and other signs of the metabolic syndrome (diabetes and hypertension) influenced CAP failure. The influence of age, metabolic syndrome and BMI has already been observed in several studies with liver stiffness measurement [19-22]. We found that the frequency of CAP failure ranged from 0.5% in young male with no sign of the metabolic syndrome to 33% in elderly female with diabetes and hypertension. CAP measurement failure was significantly more frequent in female, as previously published for liver stiffness measurement [22]. This rate could be related to fat mass repartition. CAP accuracy and failure rates need to be studied more closely in western adults with NAFLD, but it is already clear that probe refinements will be needed for CAP measurement in this population. CAP values were significantly associated with all parameters of metabolic syndrome. This result emphasizes the fact that CAP has a relationship to steatosis. But more interesting is that CAP is a quantitative value. Therefore, with all 5 parameters of the metabolic syndrome, we could add CAP value for the evaluation of the risk of NAFLD. The accuracy of liver biopsy for assessing steatosis has also been questioned, because of sampling errors and intra- and interobserver variability that may lead to over- or under-staging of steatosis. There is thus a need for accurate non-invasive methods of measuring the degree of steatosis. However, we need to compare CAP values with the histological diagnosis of NASH to evaluate its performance for the prediction of NASH. Moreover, we need to evaluate CAP as a prognostic factor in NAFLD, but also in chronic liver diseases associated with steatosis (for example, HCV infection). Our study has several limitations. Our sample size was limited in part because of the difficulty of obtaining valid CAP measurements in obese patients using the FibroScan® M probe. Future studies are necessary to develop a 9

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CAP algorithm for the FibroScan® XL probe, which was designed for use in this population [23, 24]. Our study was

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performed in a tertiary centre, specialized in chronic liver diseases. Therefore, the generalizability of our findings to other patient populations (e.g. a „screening cohort‟ seen in primary care) requires confirmation. We did not compare our results to ultrasonography, an operator-dependent method. The sensitivity of ultrasonography for the diagnosis of fatty liver ranges between 49% and 94%, and is lower when the steatosis degree is mild or the BMI is high [25]. Saadeh et al. [25] reported a poor sensitivity of ultrasonography for the identification of steatosis extent under 30%. In contrast, a more recent study has shown that ultrasonography is sensitive for steatosis extent as low as 20% [26]. Only 440 liver biopsies were performed. Therefore, the results of CAP performances for the diagnosis of steatosis should be interpreted with cautious. At last, our study was not an intent-to-diagnose study. The first aim of this study was a clinical approach and interpretation of CAP, and the second aim was the evaluation of performance of CAP for the diagnosis of steatosis. Therefore, to be relevant with the first aim, we decided to evaluate only patients with no shots, and patients with reliable resulats of liver stiffness measurement. The prognostic value of CAP needs to be evaluated and compared to FLI. Indeed, FLI is independently associated with hepatic-related mortality. It is also associated with all-cause, cardio-vascular disease, and cancer mortality rates, but these associations appear to be tightly interconnected with the risk conferred by the correlated insulin-resistant state [27]. In conclusion, the accurate assessment of liver steatosis is crucial in clinical practice for the management of patients with chronic liver disease, and in clinical research for epidemiological and therapeutic studies. The ideal method for liver steatosis evaluation must be widely available, non-invasive, safe, sensitive, accurate for quantification, reproducible and inexpensive. In this perspective, CAP is a promising tool for the noninvasive detection of hepatic steatosis, and maybe for the evaluation of metabolic syndrome. Advantages of CAP include its simplicity, operator-independence and sensitivity to lesser degrees of steatosis than are detectable using other widely available imaging modalities. Moreover, CAP provides an immediate assessment of steatosis simultaneously with liver stiffness measurement used to stage hepatic fibrosis. The strong association of CAP with the metabolic syndrome and alcohol use could be of interest for the follow-up of NAFLD or alcoholic patients. Future studies are necessary to develop a CAP algorithm for the FibroScan XL probe that will facilitate measurement in a greater proportion of obese patients.

Contributions: Study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript and critical revision of the manuscript: Victor de Lédinghen; acquisition of data and critical revision of the manuscript: Julien Vergniol, Juliette Foucher, Faiza Chermak, Brigitte Le Bail, Wassil Merrouche, JeanBaptiste Hiriart, Christophe Cassinotto.

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Financial Support: None

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Competing Interests: Victor de Lédinghen received lecture fees from Echosens.

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1 Table 1. Characteristics of the 5,323 CAP examinations. 2 3 4 Parameters Whole Failure CAP CAP measurement p 5 examinations N=410 N=4,913 6 7 N=5,323 8 Gender (males) 9 2,893 (54.3) 201 (49.0) 2692 (54.8) 0.026 10 Mean age (years) 54.9 ± 13.4 59.2 ± 12.3 54.7 ± 13.4 <0.0001 11 12 BMI (kg/m²) 26.6 ± 5.9 34.8 ± 7.7 26.1 ± 5.4 <0.0001 13 Waist circumference (cm) 92.7 ± 15.8 113.8 ± 15.2 90.9 ± 14.5 <0.0001 14 15 Diabetes (%) 989 (18.6) 168 (41.0) 821 (16.7) <0.0001 16 Hypertension (%) 1,939 (36.4) 268 (65.4) 1,671 (34.0) <0.0001 17 18 Metabolic syndrome (%) 1,453 (27.3) 241 (58.8) 1,212 (24.7) <0.0001 19 Alcohol use (glass/week) 20 7.7 ± 25.9 7.4 ± 24.8 7.7 ± 26.0 NS 21 Tobacco (pack years) 12.2 ± 18.2 12.7 ± 21.8 12.2 ± 17.9 NS 22 Coffee (cup/day) 23 2.0 ± 2.6 1.9 ± 3.1 2.1 ± 2.5 NS 24 Platelet count (G/L) 212 ± 73 214 ± 72 212 ± 73 NS 25 26 Prothrombin Time (%) 99.2 ± 18.6 93.5 ± 22.1 99.7 ± 18.2 <0.0001 27 AST (IU/L) 53.1 ± 81.7 45.5 ± 40.8 53.8 ± 84.2 0.047 28 29 ALT (IU/L) 65.8 ± 106.2 51.7 ± 64.8 66.9 ± 108.9 0.005 30 GGT (IU/L) 116.2 ± 212.1 113.5 ± 186.9 116.4 ± 214.1 NS 31 32 Total bilirubin (µmol/L) 14.9 ± 23.6 16.3 ± 23.2 14.8 ± 23.6 NS 33 Albumin (g/L) 34 42.8 ± 4.2 41.4 ± 5.3 42.9 ± 4.1 <0.0001 35 Creatinin (µmol/L) 74.3 ± 53.2 75.3 ± 43.0 74.2 ± 54.0 NS 36 Fasting glucose (mmol/L) 37 5.44 ± 2.1 6.2 ± 2.6 5.4 ± 2.1 <0.0001 38 Triglycerides (mmol/L) 1.43 ± 1.1 1.6 ± 0.9 1.4 ± 1.1 <0.0001 39 40 HDL cholesterol (mmol/L) 1.40 ± 0.4 1.3 ± 0.4 1.4 ± 0.4 <0.0001 41 Ferritin (ng/mL) 227 ± 363 192 ± 291 230 ± 368 0.042 42 43 FLI score 53.3 ± 31.8 85 ± 19 51 ± 31 <0.0001 44 Median liver stiffness (kPa) 5.7 [4.4 - 8.7] 45 46 Median CAP (dB/m) 239 [200-287] 47 p: Failure CAP versus CAP measurement; AST: aspartate aminotrasnferase; ALT: alanine aminotrasnferase, 48 49 GGT: gamma glutamyltranspeptidase; NS: not significant.

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Table 2. Clinical factors associated with CAP failure in univariate and multivariate analyses. Univariate Parameters

OR

95% CI

Female gender

1.26

1.03-1.54

Age > 55 years

2.00

]25–30] versus < 25 kg/m²

Multivariate P

OR

95% CI

P

0.026

1.31

1.05 – 1.63

0.015

1.62-2.47

<0.001

1.56

1.24 - 1.97

<0.001

6.03

3.84 – 9.48

<0.001

5.38

3.40 - 8.52

<0.001

> 30 versus ]25–30] kg/m²

5.36

4.19 - 6.85

<0.001

4.75

3.68 - 6.12

<0.001

> 30 versus < 25 kg/m²

32.34

2.18 – 49.37

<0.001

25.55

16.44- 39.70

<0.001

Elevated waist circumference

50.80

24.01 - 107.47

<0.001

Diabetes

3.46

2.80 - 4.27

<0.001

Hypertension

3.66

2.96 - 4.53

<0.001

Metabolic syndrome

4.36

3.54 - 5.36

<0.001

1.48

1.17 - 1.87

0.001

BMI

OR: odds ratio - CI: confidence interval - Since hypertension, diabetes and elevated waist circumference are parameters included in "metabolic syndrome", these parameters were not included in the multivariate analysis.

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Table 3. Factors associated with CAP = ]250-300] dB/m and CAP > 300 dB/m versus CAP < 250 dB/m (logistic regression).

Univariate

CAP = ]250-300] dB/m

Parameters

OR

95% CI

P

OR

95% CI

P

Male gender

1.57

1.36-1.81

<0.001

1.56

1.35-1.81

<0.001

Age > 55 years

1.66

1.44-1.90

<0.001

1.83

1.58-2.12

<0.001

BMI ]25–30] versus ≤ 25 kg/m²

3.58

3.05-4.20

<0.001

7.54

6.08-9.34

<0.001

> 30 versus ]25–30] kg/m²

1.87

1.50-2.34

<0.001

4.94

4.00-6.10

<0.001

> 30 versus ≤ 25 kg/m²

6.71

5.38-8.40

<0.001

37.04

29.41-47.62

<0.001

Elevated waist circumference

4.64

3.99-5.39

<0.001

16.11

13.00-19.97

<0.001

Diabetes

1.99

1.64-2.43

<0.001

4.69

3.92-5.62

<0.001

Hypertension

1.91

1.65-2.21

<0.001

3.35

2.88-3.89

<0.001

Metabolic syndrome

2.89

2.44-3.43

<0.001

7.94

6.72-9.38

<0.001

Alcohol abuse

1.67

1.35-2.07

<0.001

1.96

1.59-2.43

<0.001

Coffee consumption > 1 cup/day

CAP > 300 dB/m

NS

Liver stiffness > 6 kPa

1.74

1.51-2.01

Multivariate

CAP = ]250-300] dB/m

Parameters

OR

95% CI

<0.001

NS 3.67

3.15-4.28

<0.001

CAP > 300 dB/m P

OR

95% CI

P

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Male gender

1.38

1.18-1.62

<0.001

1.37

1.14-1.64

0.001

Age > 55 years

1.46

1.25-1.71

<0.001

1.32

1.10-1.59

0.003

BMI ]25–30] versus ≤ 25 kg/m²

3.21

2.70-3.81

<0.001

5.86

4.64-7.39

<0.001

> 30 versus ]25–30] kg/m²

1.71

1.34-2.17

<0.001

3.69

2.93-4.65

<0.001

> 30 versus ≤ 25 kg/m²

5.47

4.28-7.00

<0.001

21.61

16.47-28.36

<0.001

Metabolic syndrome

1.46

1.20-1.78

<0.001

2.73

2.23-3.34

<0.001

Alcohol abuse

1.72

1.37-2.16

<0.001

2.22

1.72-2.88

<0.001

Liver stiffness > 6 kPa

1.32

1.12-1.54

0.001

2.00

1.67-2.41

<0.001

15

Figure legends

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

ACCEPTED MANUSCRIPT

Figure 1. Values of CAP according to different parameters. A: number of parameters of metabolic syndrome, B: metabolic syndrome, C: body mass index, D: presence of diabetes, E: waist circumference, F: causes of chronic liver disease (NAFLD: nonalcoholic fatty liver disease - HCV: Hepatitis C virus - HBV: Hepatitis B virus).

Figure 2. Categories of CAP values according to different parameters. A: waist circumference, B: body mass index, C: FLI score, D: liver stiffness.

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