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Liver Steatosis Assessed by Controlled Attenuation Parameter (CAP) Measured with the XL Probe of the FibroScan: A Pilot Study Assessing Diagnostic Accuracy
Ultrasound in Med. & Biol., Vol. -, No. -, pp. 1–12, 2015 Copyright Ó 2015 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.08.008
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Original Contribution LIVER STEATOSIS ASSESSED BY CONTROLLED ATTENUATION PARAMETER (CAP) MEASURED WITH THE XL PROBE OF THE FIBROSCAN: A PILOT STUDY ASSESSING DIAGNOSTIC ACCURACY MAGALI SASSO,* STEPHANE AUDIERE,* ASTRID KEMGANG,y FARID GAOUAR,y CHRISTOPHE CORPECHOT,y OLIVIER CHAZOUILLERES,y CELINE FOURNIER,z OLIVIER GOLSZTEJN,x STEPHANE PRINCE,x YVES MENU,x LAURENT SANDRIN,* and VERONIQUE MIETTE* * R&D Department, Echosens, Paris, France; y Assistance Publique-H^ opitaux de Paris, H^ opital Saint-Antoine, Centre de Reference Maladies Rares des Maladies Inflammatoires des Voies Biliaires, et Service d’hepatologie, Centre de Recherche Saint-Antoin, Sorbonne Universite, Paris, France; z Medical Affairs Department, Echosens, Paris, France; and x Department of Radiology, H^opital Saint-Antoine, APHP, Paris, France (Received 6 May 2015; revised 9 July 2015; in final form 11 August 2015)
Abstract—To assess liver steatosis, the controlled attenuation parameter (CAP; giving an estimate of ultrasound attenuation 3.5 MHz) is available with the M probe of the FibroScan. We report on the adaptation of the CAP for the FibroScan XL probe (center frequency 2.5 MHz) without modifying the range of values (100–400 dB/m). CAP validation was successfully performed on Field II simulations and on tissue-mimicking phantoms. In vivo performance was assessed in a cohort of 59 patients spanning the range of steatosis. In vivo reproducibility was good and similar with both probes. The area under receiver operative characteristic curve was equal to 0.83/0.84 and 0.92/ 0.91 for the M/XL probes to detect .2% and .16% liver fat, respectively, as assessed by magnetic resonance imaging. Patients can now be assessed simultaneously for steatosis and fibrosis using the FibroScan, regardless of their morphology. (E-mail: [email protected]) Ó 2015 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound attenuation, Controlled attenuation parameter (CAP), Liver, Steatosis, Steato-hepatitis, Non-alcoholic fatty liver disease (NAFLD), Elastography, Vibration-controlled transient elastography (VCTE), FibroScan.
INTRODUCTION
fibrosis, which can further progress to cirrhosis, liver failure or hepatocellular carcinoma (Farrell et al. 2004). When the cause of steatosis is the result of metabolic dysfunction, the disease is called non-alcoholic fatty liver disease (NAFLD). In approximately 30% of NAFLD patients, steatosis is associated with liver inflammation that can lead to non-alcoholic steato-hepatitis (NASH; Farrell and Larter 2006), which has a more severe outcome. The prevalence of NAFLD in the general population is high, approximately 20%–30% in affluent countries (Chalasani et al. 2012). With the rising epidemic of obesity and metabolic disorders, NAFLD is rapidly becoming one of the most common liver diseases worldwide. Liver biopsy is the gold standard to assess steatosis but suffers from many drawbacks and contraindications (Bravo et al. 2001). Alternative non-invasive methods, mainly involving conventional imaging, have been proposed to detect steatosis. Ultrasonography (US) is the
Hepatic steatosis is the accumulation of excess lipids (mainly triglycerides) in hepatocytes (Farrell et al. 2004). Steatosis is considered abnormal when the hepatic-fat content exceeds 5% of liver weight and, more practically, when more than 5% of hepatocytes contain fatty droplets. Steatosis can be a result of several causes, such as alcohol consumption, viral hepatitis or metabolic dysfunction (obesity, type 2 diabetes, hyperglycemia, hypertriglyceridemia; Farrell et al. 2004). Isolated steatosis is considered a benign and reversible condition; however, when steatosis is associated with inflammation (steato-hepatitis) it can progress to liver
Address correspondence to: Magali Sasso, 30 place d’Italie, 75013 Paris, France. E-mail: [email protected] 1
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most common liver-imaging technique because of its ease of use, accessibility and low cost (Schwenzer et al. 2009). However, its diagnostic value is controversial (Schwenzer et al. 2009) as it is highly operator and machine dependent. Furthermore, it cannot quantify steatosis and can only detect steatosis when at least 30% of hepatocytes are affected (Schwenzer et al. 2009). A recent metaanalysis (Bohte et al. 2011) that compared the performances of US, computed tomography, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (1H-MRS) to evaluate hepatic steatosis revealed that MRI and 1H-MRS were the techniques of choice for accurate evaluation of hepatic steatosis. However, these methods are costly, are not readily available, are not standardized and cannot be used to screen for NAFLD in the general population. Recently, a novel non-invasive parameter called the controlled attenuation parameter (CAP), has been developed to assess liver steatosis. CAP is measured with the FibroScan (Echosens, Paris, France), which is based on vibration-controlled transient elastography (VCTE), a technique initially developed to assess liver stiffness (LS), which is highly correlated with liver fibrosis (Sandrin et al. 2003). CAP is derived from the properties of ultrasonic signals acquired by the FibroScan. It uses the postulate that fat affects ultrasound propagation and is a measure of ultrasound attenuation at 3.5 MHz (i.e., the ultrasound center frequency of the FibroScan M probe). CAP was introduced recently (Sasso et al. 2010) and has already been assessed in many clinical studies, most of them evaluating steatosis in liver biopsies as the gold standard. The performance of CAP for the detection of steatosis has been reported to be good to excellent in most studies in patients with chronic liver disease of various etiologies (Chon et al. 2014; de Ledinghen et al. 2012; Sasso et al. 2010), with viral hepatitis (Mi et al. 2015; Sasso et al. 2012) and with NAFLD (Karlas et al. 2014). In this last study, CAP performance was compared to 1H-MRS and was shown to have a comparable diagnostic value. However, in some studies dealing with overweight and obese patients (Chan et al. 2014; Myers et al. 2012a), it has been shown that CAP performance was impaired by an increased body mass index (BMI). This phenomenon is attributed to the fact that overweight or obese patients have an increased skin-to-capsula distance (SCD) resulting from a high subcutaneous fat thickness. In these cases, the region of interest for CAP on the M probe will contain not only liver parenchyma but also a portion of the subcutaneous fat layer, which causes an overestimation of CAP. Recently, a probe dedicated to overweight and obese patients was developed by Echosens (Myers et al. 2012b). Indeed, the M probe has been shown to
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be inaccurate or to fail to assess LS in some patients with an increased BMI and a thick layer of subcutaneous fat (Castera et al. 2010; Foucher et al. 2006). This new probe, named the XL probe (Echosens, Paris, France), makes measurements to a greater depth in the liver (i.e., 35–75 mm vs. 25–65 mm) using a lower ultrasound frequency transducer (2.5 vs. 3.5 MHz) to increase ultrasound penetration. As LS values change as a function of frequency, the shear-wave center frequency remains unchanged (i.e., 50 Hz) with the XL probe. The first objective of this study was to validate the development of the CAP with the XL probe on Field II simulations and on tissue-mimicking phantoms. The difficult part of the development was to get CAP values around 3.5 MHz using the XL probe, whose center frequency is at 2.5 MHz. This was necessary to obtain comparable CAP values between the M and XL probes and, thus, to enable physicians to have the same range of CAP values for interpretation and diagnostic purposes. This is particularly important from a clinical point of view to prevent any misdiagnosis. Indeed, attenuation values measured on the same medium using a 2.5 or 3.5 MHz center frequency do not differ greatly; therefore, if the physician had to read off CAP values from different scales using the M and the XL probes, he/she might easily confuse the CAP values. In addition, obtaining CAP values on the same scale with both probes will allow us to use for the XL probe all published cutoffs and results found in literature for the M probe. This is of great interest as it can be difficult, from an ethical standpoint, to set up clinical studies on steatosis diagnosis using liver biopsies as the gold standard. The second aim of the study was to validate the reproducibility and diagnostic performance of CAP measured using the XL probe in a cohort of patients undergoing steatosis quantification assessed by MRI and to compare these data with CAP measured using the M probe. The study was conducted and written according to the Standards for Reporting of Diagnostic Accuracy (Bossuyt et al. 2003). MATERIALS AND METHODS CAP measurement principle CAP (Sasso et al. 2010) is an estimate of the ultrasonic attenuation coefficient at 3.5 MHz measured with the FibroScan, a VCTE device that uses ultrasound signals to track shear-wave propagation within the liver to perform a liver stiffness measurement (LSM; Mueller and Sandrin 2010; Sandrin et al. 2003). CAP is evaluated using the same radio-frequency data and in the same region of interest as that used for LSM and is
CAP with the XL probe of the FibroScan d M. SASSO et al.
only appraised if the LSM is ‘‘valid,’’ ensuring the operator obtains an ultrasonic attenuation value of the liver. CAP is an estimate of the total ultrasonic attenuation (go-and-return path, which was initially developed from signals acquired with the FibroScan M probe to assess in vivo liver attenuation using a proprietary algorithm at the center frequency of the probe, i.e., 3.5 MHz). CAP is expressed in dB/m. The new development of CAP on signals acquired with the XL probe requires three steps (see Fig. 1): 1. Evaluation of the raw attenuation around 2.5 MHz, the center frequency of the XL probe, using the same proprietary algorithm used on signals acquired on the M probe. 2. Evaluation of the frequency dependence of attenuation using a method similar to that described in Kuc and Schwartz (1979) and Wear (2003). 3. Estimation of CAP around 3.5 MHz using frequencydependent attenuation as a correction factor. In this step, attenuation was assumed to be linearly dependent with frequency. Note that the correction procedure was also performed using a proprietary algorithm. The correction factor developed here was based on estimation of frequency-dependent attenuation, assuming that attenuation varies linearly with frequency, which is a common assumption made when dealing with in vivo liver ultrasound attenuation (see Kuc 1980; Maklad et al. 1984). The linear dependence of ultrasound attenuation with frequency within the 0.5–7 MHz range has been shown experimentally in mammalian livers in (Goss et al. 1979). The hypothesis made in this paper is that the frequency dependence of attenuation is almost linear, whatever the steatosis. One of the objectives of this paper is to validate that the corrective factor is still valid with varying fat contents.
Fig. 1. Measurement principle of the controlled attenuation parameter (CAP) with the XL probe.
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Justification of the necessity to develop CAP on the XL probe As suggested in previous studies (Chan et al. 2014; Myers et al. 2012a), the performance of CAP is downgraded in cases of a large SCD (i.e., greater than 25 mm) because the measurement window not only contains liver parenchyma but also a portion of the subcutaneous fat layer, which causes overestimation of CAP. This phenomenon was first verified on a custommade tissue-mimicking phantom, made by ATS (Bridgeport, CT, USA) to mimic homogeneous liver parenchyma (background attenuation at 3.5 MHz of 250 dB/m) and SCD zones (hyper-echogenic zones, contrast 5 115 dB). Successive CAP measurements were performed using the M and XL probes in the homogenous zone of the phantom and in the zone mimicking a SCD equal to 32 mm (see Fig. 2). Validation of CAP measured with the XL probe Field II simulations. The validity of CAP measured with the XL probe to estimate the attenuation coefficient around 3.5 MHz was first appraised with Field II simulations (Technical University of Denmark, Copenhagen, Denmark; Jensen 1996; Jensen et al. 1993). A-mode simulations were performed in the FibroScan configuration, using a single 7-mm diameter flat
Fig. 2. Experimental setup to test the influence of a large skinto-capsula distance (SCD). The plain line represents the homogenous zone of the phantom and the dotted line represents the zone mimicking a SCD equal to 32 mm.
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piston transducer (9-mm, respectively) and a 3.5 MHz (2.5 MHz, respectively), two-cycle sinusoidal excitation for the M probe configuration (XL probe configuration, respectively). Tissue scatterers were modeled as a 3-D spatial distribution of independent point scatterers with uniformly drawn random positions (Jensen and Nikolov 2000) and a Gaussian-distributed amplitude (Jensen and Nikolov 2000; Wagner et al. 1983). Phantom size was set to 10 3 10 3 80 mm3. Scatterers number was set to 100,000, which is the minimal amount of scatterer needed to reach the traditionally defined maximal speckle signal-to-noise ratio of 1.91 (Burckhardt 1978) for the considered phantom size. Simulations were made for different homogeneous media with different attenuations, ranging from 100 dB/ m at 3.5 MHz to 400 dB/m at 3.5 MHz, in 20 dB/m steps. The CAP with the M probe was assessed for each simulated data set. When using the XL probe configuration, raw attenuation was first estimated, the corrective factor was then calculated and CAP was finally assessed. Experiments on tissue-mimicking phantoms. The validity of CAP measured with the XL probe to estimate the attenuation coefficient at 3.5 MHz was assessed on tissue-mimicking phantoms using both the FibroScan M and XL probes. The phantoms used were custom-made ultrasound attenuation tissue-mimicking phantoms made of waterbased gel with powdered graphite and glass bead scatterers, manufactured by the University of Wisconsin (Madison, WI, USA; Madsen et al. 1991). In our case, to assess CAP through its entire range (100–400 dB/m), three kinds of phantoms were made: (i) a low attenuation phantom, with attenuation at 150 dB/m at 3.5 MHz; (ii) an intermediate attenuation phantom, with attenuation at 250 dB/m at 3.5 MHz; and (iii) a high attenuation phantom, with attenuation at 350 dB/m at 3.5 MHz. The phantom’s characteristics at 20 C, given by the manufacturer, are shown in Table 1. For phantom testing, a probe-holding system was used and phantoms were placed in a tank filled with demineralized, degassed water (see Fig. 3). Water temperature was controlled at 20 C 6 0.5 C using a cooling and warming system (TC5; TECO, Ravenna, Italy). Acquisitions were performed using a FibroScan probe (both M and XL type; Echosens, Paris, France). During all the tests, the probe was held by a support (see Fig. 3) and moved to the different measurement points by a motorized linear translation stage (Newport Corporation, Irvine, CA, USA) driven using Matlab software (Mathworks, Natick, MA, USA). The translation stages had an accuracy of 1 mm. Each measurement set consisted of a scan of the whole phantom surface (57.5 mm in the X and Y axes)
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Table 1. Characteristics of phantoms at 20 C, as supplied by the manufacturer Phantom name Ph150 Ph250 Ph350
Speed of sound (m/s)
Reference attenuation (dB/m @ 3.5 MHz)
Uncertainty of reference attenuation (dB/m)
1496.1 1492.6 1490.0
145 235 340
5 5 5
with a 2.5-mm step, and five consecutive measurements were performed to assess bias, linearity and repeatability. In vivo study population. To constitute the cohort for the in vivo evaluation of CAP using both probes, a cohort was constituted using medical records from the Liver Unit of Saint Antoine Hospital, Paris, France. The objective was to sample, in a small cohort (objective: 60 patients), the whole spectrum of steatosis grades from no to massive steatosis. Enrolled patients had undergone a biopsy within one year of the medical records being accessed. The objective was to find the same proportions of patients within the following steatosis groups: no steatosis (0%–10% of hepatocytes), mild steatosis (10%– 33% of hepatocytes), moderate steatosis (33%–67% of
Fig. 3. Experimental setup for phantom testing: the probe is held by a probe-holding system.
CAP with the XL probe of the FibroScan d M. SASSO et al.
hepatocytes) and massive steatosis (67%–100% of hepatocytes), as described in Sasso et al. (2012). Patients with a BMI .40 kg/m2 were not eligible for the study. A posteriori exclusion criterion was having a SCD .25 mm. When a patient corresponding to the inclusion criteria was identified, he or she was contacted to participate in the study. After their enrollment, patients underwent both an MRI and a FibroScan on the same day for steatosis quantification. Liver fat fraction was quantified using liver MRI (Signa HDTX 1.5 Tesla, General Electrics, Milwaukee, WI, USA) using the three-point Dixon method. To measure fat fraction, three regions of interest (ROIs) were drawn across the liver parenchyma on in-phase and outphase images. ROIs were copied and pasted from one image to the other to ensure complete mirror measurements. Fat fraction was calculated using eqn (1): Fat fraction 5
ðIn2OutÞ 2 In
(1)
where In was the mean intensity of the ROIs drawn across the in-phase images and Out was the mean intensity of the ROIs drawn across the out-phase images. In addition, to avoid confusing the results related to potential liver iron deposition, an evaluation of liver iron concentration was performed and the fat fraction was validated only if liver iron concentration was less than 36 mmol/g. Although this formula may be ambiguous for high values of fat deposition because it has a theoretical signal intensity peak at 50%, and the signal may decrease above, this is not a clinical problem as this would only correspond to situations of extreme severity, which are higher than histologic grade 3 as described by Kleiner et al. (2005) and were not encountered in this study. The histologic grade and the measured fat fraction, even if expressed as percentages, are dissimilar. A fat fraction of 30%– 40% assessed by MRI corresponds to a 66% histologic evaluation (Tang et al. 2013). FibroScan examination was performed using a Fibroscan 502 Touch (Echosens, Paris, France) three consecutive times with the M probe and three times with the XL probe by two trained operators blinded to the patients’ bio-clinical data and using the acquisition procedure described in de Ledinghen and Vergniol (2008). During the clinical examination, the raw ultrasonic radio-frequency signals were stored in the FibroScan examination file to enable computation of CAP with the XL probe off-line and retrospectively. The final CAP (LSM, respectively) results were expressed in dB/m (kPa, respectively) and corresponded to the median of 10 valid individual measurements. Bio-clinical parameters were prospectively collected at the time of inclusion. Recorded data included
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the following: gender, age, weight, height, BMI, waist circumference, diabetic status, hypertension status, alcohol use, platelet count, prothrombin time, aspartate aminotransferase, alanine aminotransferase (ALAT), gamma-glutamyl transpeptidase activity, total bilirubin, fasting glucose, triglycerides, high-density lipoprotein (HDL) cholesterol, ferritin and transferrin saturation. The protocol was performed in accordance with the Helsinki Declaration and was approved by an independent ethics committee. Patients were enrolled after giving their written informed consent. Statistical analyses For CAP validation on the Field II simulations, differences between CAP measured with the XL probe and the true attenuation set in the simulations was appraised using the root mean-square error (RMSE). For CAP validation on the tissue-mimicking phantoms, bias (B) and precision (P) were assessed using the definitions (ISO 2011) illustrated in eqns (2) and (3): B5
CAPi 2Aref Aref
(2)
P5
stdðCAPi Þ Aref
(3)
where CAPi is the average of the five replicate measurements (CAPi , i 5 1,.,5) and Aref is the reference attenuation value given by the manufacturer. Repeatability was assessed using repeatability coefficient (RC) and limits of agreements (LOA; Raunig et al. 2015). RC can be calculated from the within subject variance s2w obtained from one-way analysis of variance as follows in eqn (4): pffiffiffiffiffiffiffi RC 5 1:96 2s2w (4) and LOA is defined as the interval where the difference between two measurements under repeatability conditions for a randomly selected subject is expected to be 95% of the time and is expressed as eqn (5): LOA 5 ½2RC; RC
(5)
For repeatability results, the intra-class coefficient (ICC) of variation was also appraised, as suggested in Raunig et al. (2015). Agreement was classified as poor (ICC 5 0.00–0.20), fair to good (ICC 5 0.40–0.75) or excellent (ICC .0.75; Fleiss et al. 1981). Concerning the clinical data, except for evaluating reproducibility, only the first examination in the series of three measurements acquired using each probe type was considered. In the descriptive analyses, the continuous variables were expressed as their median
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[interquartile range] and the categorical variables as absolute figures and percentages. Reproducibility of CAP and LSM were assessed using ICC and the agreement scale described above. The relationship between CAP and the bio-clinical parameters was assessed using either Spearman’s rank correlation coefficient r or Student’s t-test (homoscedasticity was tested using the F-test). Parameters that were significantly related to CAP (or at the limit of significance p , 0.10) were entered in a multivariate model (Katz 2011). The normality of CAP was tested using Lilliefors’ test (Lilliefors 1967). Multiple linear regression (Katz 2011) was performed using a backward procedure based on minimization of the Akaike information criterion to select independent features significantly associated with CAP. The diagnostic performance of CAP was assessed using receiver operating characteristic curve analysis, using the MRI-based hepatic fat fraction dichotomized according to its distribution (first quartile, median and third quartile). The area under the receiver operating characteristic curve (AUROC) was computed as well as its 95% confidence interval. Cut-off values, specificities, sensitivities and accuracy were computed for the cut-off based on simultaneously maximizing sensitivity and specificity (Gallop et al. 2003). All statistical analyses were performed using R software (The R Foundation for Statistical Computing: Vienna, Austria). ICC was estimated using the psych package. A p value of less than 0.05 was considered statistically significant. RESULTS Justification for the development of CAP on the XL probe Results for the CAP measurements performed using the M and XL probes in the homogenous zone of the phantom and in the zone mimicking an SCD equal to 32 mm are given in Table 2. When measurements were performed in the homogeneous zone of the phantom, both probes gave similar results that matched the reference’s attenuation value given by the manufacturer. When measurements were performed in the zone that mimicked an SCD equal to 32 mm, measurements performed with the XL probe matched the reference’s attenuation value given by the manufacturer, whereas CAP performed using the M probe over-estimated the attenuation. Validation of the CAP as an estimate of ultrasonic attenuation Results on Field II simulations. Several simulations were performed on homogeneous attenuating media with attenuation values at 3.5 MHz set from 100 to 400 dB/m in 20 dB/m steps. Estimated CAP with the M probe, the XL probe and the raw attenuation around 2.5 MHz are
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Table 2. Controlled attenuation parameter (CAP) with both the M and XL probes on the homogeneous part of the phantom and in the part that mimicked a skin-tocapsula distance (SCD) of 32 mm
CAP with the M probe (dB/m) CAP with the XL probe (dB/m)
Homogeneous zone of the phantoms
Zone that mimicked a SCD of 32 mm
249 (32) 252 (50)
330 (35) 251 (58)
The results are given in term of median. The interquartile range is given in parentheses.
shown in Figure 4a, and the relative difference between the reference attenuation set in the simulation and estimated CAP with both the M and XL probes are given in Figure 4b. The RMSE for attenuation estimation at 3.5 MHz, using the M and XL probes, was 2.1 and 5.6 dB/m, respectively. Results on tissue-mimicking phantoms. CAP values for the five replicate measurements are given in Table 3, together with results in terms of bias and precision on each phantom and each probe type. The repeatability coefficient was equal to 0.7 and 3.6 dB/m for the M and XL probe, respectively. Corresponding LOA were [20.7; 0.7] dB/m and [23.6; 3.6] dB/m, respectively. ICC was equal to 1 [1; 1] for both M and XL probes, showing perfect agreement. In vivo results Fifty-nine patients who underwent an MRI and a FibroScan on the same day were enrolled between January 2013 and July 2013. One patient was excluded because of an SCD .25 mm. All patients were successfully measured by the Fibroscan. The patients’ characteristics are shown in Table 4. CAP measured with the M probe and the XL probe were very well correlated (r 5 0.82, p , 3.3 3 10215). A plot of individual CAP values measured with the M probe versus the XL probe is shown in Figure 5. CAP was correlated with the MRI-based hepatic fat fraction (r 5 0.73, p , 5.6 3 10211, and r 5 0.74, p , 1.6 3 10211 for measurements using the M and XL probes, respectively). In vivo reproducibility ICC for CAP was equal to 0.83 [0.76; 0.89] and 0.84 [0.77; 090] for the M and XL probes, respectively. For LSM, ICC was equal to 0.97 [0.96; 0.98] and 0.94 [0.91; 0.96] for the M and XL probes, respectively. Relationship between CAP and the bio-clinical parameters The parameters significantly related (or at the limit of significance) to CAP measured with the M
CAP with the XL probe of the FibroScan d M. SASSO et al.
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Fig. 4. (a) Controlled attenuation parameter (CAP) with the M and XL probe configurations as a function of the reference attenuation set in Field II; (b) relative difference (%) between the reference attenuation set in Field II and estimated CAP with the M and XL probes as a function of the reference attenuation set in Field II.
and XL probes are given in Table 5. In univariate analyses, CAP appeared to be related to BMI, waist circumference, hypertension status, diabetic status, ALAT, triglyceride, HDL cholesterol, ferritin and steatosis as assessed by MRI. CAP was independent of the other bio-clinical parameters given in Table 4, especially LSM. All the parameters significantly related to CAP or at the limit of significance were entered in a multivariate model, and only BMI and steatosis (as assessed by MRI) were related to CAP on both the M and XL probes. A plot of CAP values for both M and XL probes versus BMI and steatosis assessed by MRI is shown in Figure 6 (a, b), respectively.
CAP performance for steatosis diagnosis The CAP performance for steatosis diagnosis is given in Table 6. The CAP performance in terms of AUROC was 0.83 (0.71–0.95) for the M probe and 0.84 (0.73–0.95) for the XL probe for the diagnostic of MRIbased liver fat fraction .2%, 0.87 (0.78–0.97) for the M probe and 0.90 (0.82–0.99) for the XL probe for the diagnostic of liver fat fraction of .8%, and 0.92 (0.85–0.99) for the M probe and 0.91 (0.83–0.99) for the XL probe for the diagnostic of liver fat fraction of .16%. No statistical differences were found for the diagnostic performances in terms of AUROC between the two probes.
DISCUSSION
Table 3. Results for phantom testing using the probeholding system
Measurement 1, M probe Measurement 2, M probe Measurement 3, M probe Measurement 4, M probe Measurement 5, M probe Average, M probe (dB/m) Std, M probe (dB/m) Bias, M probe (%) Precision, M probe (%) Measurement 1, XL probe Measurement 2, XL probe Measurement 3, XL probe Measurement 4, XL probe Measurement 5, XL probe Average, XL probe (dB/m) Std, XL probe (dB/m) Bias, XL probe (%) Precision, XL probe (%)
Ph150
Ph250
Ph350
143 143 143 143 143 143.0 0 21.4 0 157 153 154 154 154 154.4 1.5 6.5 1.0
234 234 234 234 234 234.0 0 20.4 0 226 226 227 228 228 227.0 1.0 23.4 0.4
323 323 323 323 324 323.2 0.45 24.9 0.1 328 327 327 329 330 328.2 1.3 23.5 0.4
In the present study, the CAP was successfully implemented with the XL probe of the FibroScan. This implementation necessitated a scaling procedure to get CAP values to correspond to an attenuation around 3.5 MHz when using the XL probe of the FibroScan, whose center frequency is around 2.5 MHz. This scaling procedure was shown to be efficient in the simulations and tissue-mimicking phantoms, as well as in vivo on the whole attenuation range, from that of a normal liver (no steatosis) up to massive steatosis (100–400 dB/m at 3.5 MHz). This scaling procedure assumes that the frequency dependence of attenuation is linear with frequency. Even if this approximation is gross in the case of fatty infiltration, as suggested in Narayana and Ophir (1983), it was shown here that it did not impair the performance of the scaling procedure. This was also shown in the phantom testing because, when SCD was greater than 25 mm, measuring CAP with the M probe of the FibroScan led to overestimation (330 dB/m instead of 250 dB/m). These results are consistent with previous
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Table 4. Characteristics of the study population Characteristic Gender (female) Age (y) BMI (kg/m2) Waist circumference (cm) Diabetic status Hypertension status Alcohol use Platelet count (103/mm3) Prothrombin time (%) ASAT (IU/L) ALAT (IU/L) GGT (IU/L) Total bilirubin (mmol/L) Fasting glucose Triglycerides HDL cholesterol Ferritin (ng/mL) Ferritin saturation (%) Liver stiffness (kPa) CAP (dB/m) LB steatosis classification (retrospective selection) MRI-based liver fat fraction (%) MRI-based liver iron concentration (mmol/g)
Patient distribution* 23 (39.0%) 53.0 [11.5] 27.0 [5.2] 96.5 [15.3] (7y) 10 (16.9%) 19 (32%) 19 (32%) 197 [97] (3y) 94 [14] (15y) 38.5 [31.0] (1y) 48.0 [45.6] (1y) 71.5 [101.8] (3y) 11.5 [6.7] (7y) 6.1 [1.4] (22y) 1.3 [0.8] (2y) 1.1 [0.6] (8y) 226 [338] (10y) 30.9 [18.3] (13y) M probe (3 series): 7.4 [4.6]/7.3 [5.7]/7.2 [4.3] XL probe (3 series): 6.4 [3.7]/6.9 [3.9]/6.7 [3.7] M probe (3 series): 275 [101]/276 [82]/268 [73] XL probe (3 series): 279 [91]/272 [114]/275 [105] Absent/Minimal/Moderate/Massive 10 (17%)/23 (39%)/14 (24%)/12 (20%) 8 [15] 10 [15]
BMI 5 body mass index; ASAT 5 aspartate aminotransferase; ALAT 5 alanine aminotransferase; GGT 5 gamma-glutamyl transpeptidase; HDL 5 high-density lipoprotein; CAP 5 controlled attenuation parameter; LB 5 liver biopsy; MRI 5 magnetic resonance imaging. * Continuous variables were expressed as the median [interquartile range], and categorical variables as absolute figures (percentages). y Number of missing data.
in vivo studies (Chan et al. 2014; Myers et al. 2012a) in which it was observed that CAP performance was downgraded when SCD was greater than 25 mm. With the development of CAP for the XL probe of the FibroScan it will now be possible to properly assess
Fig. 5. Controlled attenuation parameter (CAP) measured with the M probe versus the CAP measured with the XL probe for the 59 patients.
steatosis and reduce the risk of misdiagnosis in overweight and obese patients, who are particularly vulnerable to steatosis. CAP measured with the XL probe of the FibroScan was validated first with Field II simulation. CAP estimated on simulated signals with the XL probe were very close to values set in the simulation as the RMSE was equal to 5.6 dB/m. It was also shown in the Field II simulation that the scaling procedure worked in a satisfactory way for each attenuation value between 100 and 400 dB/m. CAP measured with the XL probe of the FibroScan was then validated on ultrasound attenuation tissuemimicking phantoms. The repeatability was shown to be almost perfect as the LOA was (23.6; 3.6) dB/m and the ICC was 1. Bias and precision were also satisfactory, as bias in absolute value was always smaller than 7% and precision was less than or equal to 1%. Results on the tissue-mimicking phantoms indicated that performance of the CAP with the XL probe, even though satisfactory, was always slightly below that of the M probe. This was because (i) the transducer of the XL probe is not the same (lower center frequency, lower sensitivity) as the transducer of the M probe and, therefore, its performance is slightly different; and (ii) the scaling process, even though
CAP with the XL probe of the FibroScan d M. SASSO et al.
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Table 5. Factors associated with controlled attenuation parameter (CAP) on M and XL probes in univariate and multivariate analyses CAP with the M probe Univariate analysis BMI Waist circumference Hypertension status Diabetic status ALAT Triglycerides HDL cholesterol Ferritin MRI-based hepatic fat fraction
CAP with the XL probe
Multivariate analysis 26
r 5 0.55, p 5 6.5 3 10 r 5 0.50, p , 1.0 3 1023 p 5 0.03 p 5 0.06 r 5 0.27, p 5 0.04 r 5 0.30, p 5 0.02 r 5 20.31, p 5 0.03 r 5 0.29, p 5 0.04 r 5 0.75, p , 5.6 3 10211
p 5 0.03 x x x x x x p , 1.3 3 1026
Univariate analysis
Multivariate analysis 24
r 5 0.50, p 5 1.6 3 10 r 5 0.52, p , 1.3 3 1025 p 5 0.01 p 5 0.06 r 5 0.33, p 5 0.01 r 5 0.28, p 5 0.04 r 5 20.14, p 5 0.34 r 5 0.26, p 5 0.07 r 5 0.77, p , 1.6 3 10211
p 5 0.06 x x x x x x x p , 6.7 3 1028
BMI 5 body mass index; ALAT 5 alanine aminotransferase; HDL 5 high-density lipoprotein; MRI 5 magnetic resonance imaging.
satisfactory, is not completely perfect and therefore it may have introduced a certain amount of variability. Note that the effect of hand-holding the probe was tested on the same tissue-mimicking phantoms by two operators and led to a LOA of (212.5; 12.5) dB/m for the M probe and of (214.0; 14.0) dB/m for the XL probe (data not shown). Reproducibility of the CAP with the M and XL probes was assessed in vivo, yielding similar results (0.83 and 0.84 for the M and XL probes, respectively) and good performances using both probes. However, CAP reproducibility was lower than LSM reproducibility (0.97 and 0.94 for the M and XL probes, respectively). Similar results were found by Recio et al. (2013), who found an ICC of 0.83 (0.77–0.88) for the CAP measured with an M probe and 0.96 (0.94–0.97) for the LSM measured with an M probe in a cohort of 118 patients with human immunodeficiency virus and/or hepatitis C virus infection.
Although CAP has good reproducibility, it appears to be less reproducible than LSM. This phenomenon is because the CAP, and more generally ultrasound attenuation, is very sensitive to heterogeneities such as blood vessels, nodules, etc. (Audiere et al. 2013; Sasso et al. 2013). This is why it is important to perform CAP examinations in a very homogenous part of the parenchyma and why a liver-guidance tool has been developed on the interface of the FibroScan to help the operator accurately conduct a CAP examination (Audiere et al. 2013; Sasso et al. 2013). In the present study, CAP assessed on both the probes was shown to be mainly related, in univariate analyses, to steatosis (p , 10211), BMI (p , 1024) and waist circumference (p , 1024). CAP assessed on both probes was also related to hypertension status, diabetic status, ALAT triglycerides and ferritin. Note that CAP assessed with the M probe was related to HDL cholesterol, but not when measured with the XL probe. This was
Fig. 6. Controlled attenuation parameter (CAP) measured with the M and XL probes as a function of (a) body mass index (kg/m2) and (b) MRI-based liver fat fraction (%).
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Table 6. Diagnostic performance of the controlled attenuation parameter (CAP)
S $2% (Prevalence 5 69%)
S $8% (Prevalence 5 51%)
S $16% (Prevalence 5 22%)
CAP with the M probe
CAP with the XL probe
p Value (Delong test)
AUROC 5 0.83 [0.71–0.95] Cutoff 5 251 dB/m Se 5 0.78/Sp 5 0.78 Acc 5 0.80 AUROC 5 0.87 [0.78–0.97] Cutoff 5 267 dB/m Se 5 0.80/Sp 5 0.79 Acc 5 0.81 AUROC 5 0.92 [0.85–0.99] Cutoff 5 299 dB/m Se 5 0.92/Sp 5 0.88 Acc 5 0.90
AUROC 5 0.84 [0.73–0.95] Cutoff 5 254 dB/m Se 5 0.83/Sp 5 0.78 Acc 5 0.83 AUROC 5 0.90 [0.82–0.99] Cutoff 5 270 dB/m Se 5 0.88/Sp 5 0.79 Acc 5 0.85 AUROC 5 0.91 [0.83–0.99] Cutoff 5 301 dB/m Se 5 0.92/Sp 5 0.81 Acc 5 0.85
0.76
0.50
0.78
S 5 steatosis; AUROC 5 area under the receiver operating characteristic curve; Se 5 sensitivity; Sp 5 specificity; Acc 5 accuracy.
probably because of the small number of patients and the influence of outlier values. Except for that parameter, CAP measured with both the M and XL probes was linked to the same bio-clinical parameters. Note that CAP was independent of LSM for both probes (p . 0.10). In multivariate analyses, CAP was only related to BMI and steatosis for both the M and XL probes. This result is in agreement with published studies, in which the only two parameters linked to CAP were steatosis and BMI (Chan et al. 2014; Chon et al. 2014; Ferraioli et al. 2014; Jung et al. 2014; Kumar et al. 2013; Mi et al. 2015; Shen et al. 2014). CAP diagnostic performance has been assessed for the first time using the hepatic fat-fraction measured by MRI. A recent meta-analysis (Bohte et al. 2011) reported that MRI and 1H-MRS were accurate techniques for evaluating hepatic steatosis. Taking another non-invasive technique as a gold standard can be relevant in many clinical studies when a liver biopsy cannot be performed or is not planned, especially in NAFLD patients. Karlas et al. (2014) compared CAP and 1H-MRS performance, taking a liver biopsy as a gold standard for NAFLD patient and providing good to excellent and similar performances using both non-invasive techniques. The diagnostic performance of CAP to assess the MRI-based hepatic fat fraction was good to excellent using both probes. The performances were shown to be similar. The optimal cut-off values determined by simultaneous maximizing sensitivity and specificity (Gallop et al. 2003) studies have been found to be similar for both probes, showing that the scaling process used to develop CAP for the XL probe works quite well and that physicians will be able to use the same reading scale with the M and XL probe. However, as suggested by Karlas et al. (2014) and Recio et al. (2013), taking a single cut-off point to classify a patient might be risky. Indeed, reproducibility for CAP, even if good, is not perfect, with relative variability between measurements. Furthermore, cut-off points are very dependent on the study’s popula-
tion (limited number of patients, usually between 100 and 200) and on the prevalence of steatosis stages in each study population. The cut-offs determined in this study should be considered with caution. First, the number of patients was very small. Second, the gold standard here was the MRI-based fat fraction, which cannot be compared directly to the percentage of steatosis assessed in liver biopsies. In future studies, a different approach to classify patients should be used, such as for instance the use of intervals instead of a single value (Recio et al. 2013), the use of a ‘‘grey area’’ (the use of two cut-off points to determine if the patient is healthy [no steatosis] or sick [massive steatosis]) and careful examination for interfering factors if the patient is in between classification schemes (Karlas et al. 2014). One perspective of this work was to validate the diagnostic performance of CAP with the XL probe in patients that actually need to be measured by the XL probe. In our study, CAP was measured on patients that could be measured by both probes, as the objective of our study was to validate the development of the CAP with the XL probe and to show that its performance was similar to the performance of CAP with the M probe. CAP with the XL probe will, therefore, need to be also clinically validated in overweight and obese populations. CONCLUSION In summary, CAP was successfully adapted to the XL probe of the FibroScan with the same measurement range as the CAP with the M probe. CAP with the XL probe was validated in Field II simulations and in tissue-mimicking phantoms. Its in vivo diagnostic performance for steatosis, assessed by MRI, was good to excellent. Reproducibility in vivo was also shown to be good: both performance and reproducibility in vivo were shown to be similar. The diagnostic performance of CAP with an XL probe should be validated in larger studies.
CAP with the XL probe of the FibroScan d M. SASSO et al.
CAP can now be assessed properly with no overestimation when SCD is greater than 25 mm, which is especially useful for overweight and obese patients who are particularly exposed to steatosis. NASH patients will benefit from this new development as they can be assed simultaneously for steatosis and fibrosis, regardless of their morphology.
REFERENCES Audiere S, Clet M, Sasso M, Sandrin L, Miette V. Influence of heterogeneities on ultrasound attenuation for liver steatosis evaluation (CAP): Relevance of a liver guidance tool. Ultrasonics Symposium (IUS), 2013 IEEE International 2013;401–404. Bohte AE, van Werven JR, Bipat S, Stoker J. The diagnostic accuracy of US, CT, MRI and 1 H-MRS for the evaluation of hepatic steatosis compared with liver biopsy: A meta-analysis. Eur Radiol 2011;21: 87–97. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig LM, Lijmer JG, Moher D, Rennie D, de Vet HC, Standards for Reporting of Diagnostic A. Towards complete and accurate reporting of studies of diagnostic accuracy: The STARD Initiative. Ann Intern Med 2003;138:40–44. Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med 2001;344: 495–500. Burckhardt CB. Speckle in Ultrasound B-Mode Scans. IEEE Trans Sonics Ultrason 1978;25:1–6. Castera L, Foucher J, Bernard PH, Carvalho F, Allaix D, Merrouche W, Couzigou P, de Ledinghen V. Pitfalls of liver stiffness measurement: A 5-year prospective study of 13,369 examinations. Hepatology 2010;51:828–835. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ. The diagnosis and management of nonalcoholic fatty liver disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 2012;55:2005–2023. Chan WK, Nik Mustapha NR, Mahadeva S. Controlled attenuation parameter for the detection and quantification of hepatic steatosis in non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2014; 29:1470–1476. Chon YE, Jung KS, Kim SU, Park JY, Park YN, Kim do Y, Ahn SH, Chon CY, Lee HW, Park Y, Han KH. Controlled attenuation parameter (CAP) for detection of hepatic steatosis in patients with chronic liver diseases: A prospective study of a native Korean population. Liver Int 2014;34:102–109. de Ledinghen V, Vergniol J. Transient elastography (FibroScan). Gastroenterol Clin Biol 2008;32:58–67. de Ledinghen V, Vergniol J, Foucher J, Merrouche W, le Bail B. Noninvasive diagnosis of liver steatosis using controlled attenuation parameter (CAP) and transient elastography. Liver Int 2012;32: 911–918. Farrell GC, George G, de la M, Hall P, Mc Cullough AJ. Fatty liver disease: NASH and related disorders. Oxford, UK: Blackwell Publishing; 2004. Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: From steatosis to cirrhosis. Hepatology 2006;43:S99–S112. Ferraioli G, Tinelli C, Lissandrin R, Zicchetti M, Dal Bello B, Filice G, Filice C. Controlled attenuation parameter for evaluating liver steatosis in chronic viral hepatitis. World J Gastroenterol 2014;20: 6626–6631. Fleiss JL, Levin B, Paik MC. The measurement of interrater agreement. Stat Methods Rates Proportions 1981;2:212–236. Foucher J, Castera L, Bernard PH, Adhoute X, Laharie D, Bertet J, Couzigou P, de Ledinghen V. Prevalence and factors associated with failure of liver stiffness measurement using FibroScan in a prospective study of 2114 examinations. Eur J Gastroenterol Hepatol 2006;18:411–412.
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Gallop RJ, Crits-Christoph P, Muenz LR, Tu XM. Determination and interpretation of the optimal operating point for ROC curves derived through generalized linear models. Understanding Stat 2003;2: 219–242. Goss SA, Frizzell LA, Dunn F. Ultrasonic absorption and attenuation in mammalian tissues. Ultrasound Med Biol 1979;5:181–186. Jensen JA. Field: A program for simulating ultrasound systems. 10th Nordicbaltic conference on Biomedical Imaging 1996;4:351–353. Jensen JA, Nikolov SI. Fast simulation of ultrasound images. Ultrasonics Symposium, 2000 IEEE 2000;2:1721–1724. Jensen JA, Gandhi D, O’Brien WD Jr. Ultrasound fields in an attenuating medium. Ultrasonics Symposium, 1993 Proceedings, IEEE 1993; 1993:943–946. Jung KS, Kim BK, Kim SU, Chon YE, Cheon KH, Kim SB, Lee SH, Ahn SS, Park JY, Kim DY, Ahn SH, Park YN, Han KH. Factors affecting the accuracy of controlled attenuation parameter (CAP) in assessing hepatic steatosis in patients with chronic liver disease. PLoS One 2014;9:e98689. Karlas T, Petroff D, Garnov N, Bohm S, Tenckhoff H, Wittekind C, Wiese M, Schiefke I, Linder N, Schaudinn A, Busse H, Kahn T, Mossner J, Berg T, Troltzsch M, Keim V, Wiegand J. Non-invasive assessment of hepatic steatosis in patients with NAFLD using controlled attenuation parameter and 1 h-mr spectroscopy. PLoS One 2014;9:e91987. Katz MH. Multivariable analysis: A practical guide for clinicians and public health researchers. Cambridge, UK: Cambridge University Press; 2011. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ, Nonalcoholic Steatohepatitis Clinical Research N. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–1321. Kuc R, Schwartz M. Estimating the acoustic attenuation coefficient slope for liver from reflected ultrasound signals. IEEE Trans Son Ultrason 1979;26:353–361. Kuc R. Clinical application of an ultrasound attenuation coefficient estimation technique for liver pathology characterization. IEEE Trans Biomed Eng 1980;27:312–319. Kumar M, Rastogi A, Singh T, Behari C, Gupta E, Garg H, Kumar R, Bhatia V, Sarin SK. Controlled attenuation parameter for noninvasive assessment of hepatic steatosis: Does etiology affect performance? J Gastroenterol Hepatol 2013;28:1194–1201. Lilliefors HW. On the Kolmogorov-Smirnov test for normality with mean and variance unknown. J Am Stat Assoc 1967;62:399–402. Madsen EL, Zagzebski JA, Macdonald MC, Frank GR. Ultrasound focal lesion detectability phantoms. Med Phys 1991;18:1171–1180. Maklad NF, Ophir J, Balsara V. Attenuation of ultrasound in normal liver and diffuse liver disease in vivo. Ultrason Imaging 1984;6:117–125. Mi YQ, Shi QY, Xu L, Shi RF, Liu YG, Li P, Shen F, Lu W, Fan JG. Controlled attenuation parameter for noninvasive assessment of hepatic steatosis using Fibroscan: Validation in chronic hepatitis B. Dig Dis Sci 2015;60:243–251. Mueller S, Sandrin L. Liver stiffness: A novel parameter for the diagnosis of liver disease. Hepat Med 2010;2:49–67. Myers RP, Pollett A, Kirsch R, Pomier-Layrargues G, Beaton M, Levstik M, Duarte-Rojo A, Wong D, Crotty P, Elkashab M. Controlled Attenuation Parameter (CAP): A noninvasive method for the detection of hepatic steatosis based on transient elastography. Liver Int 2012a;32:902–910. Myers RP, Pomier-Layrargues G, Kirsch R, Pollett A, Duarte-Rojo A, Wong D, Beaton M, Levstik M, Crotty P, Elkashab M. Feasibility and diagnostic performance of the FibroScan XL probe for liver stiffness measurement in overweight and obese patients. Hepatology 2012b;55:199–208. Narayana PA, Ophir J. On the frequency dependence of attenuation in normal and fatty liver. IEEE Trans Son Ultrason 1983;30:379–382. Raunig DL, McShane LM, Pennello G, Gatsonis C, Carson PL, Voyvodic JT, Wahl RL, Kurland BF, Schwarz AJ, Gonen M, Zahlmann G, Kondratovich M, O’Donnell K, Petrick N, Cole PE, Garra B, Sullivan DC, Group QTPW. Quantitative imaging
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biomarkers: A review of statistical methods for technical performance assessment. Stat Methods Med Res 2015;24:27–67. Recio E, Cifuentes C, Macias J, Mira JA, Parra-Sanchez M, Rivero-Juarez A, Almeida C, Pineda JA, Neukam K. Interobserver concordance in controlled attenuation parameter measurement, a novel tool for the assessment of hepatic steatosis on the basis of transient elastography. Eur J Gastroenterol Heptaol 2013;25:905–911. Sandrin L, Fourquet B, Hasquenoph JM, Yon S, Fournier C, Mal F, Christidis C, Ziol M, Poulet B, Kazemi F, Beaugrand M, Palau R. Transient elastography: A new noninvasive method for assessment of hepatic fibrosis. Ultrasound Med Biol 2003;29:1705–1713. Sasso M, Beaugrand M, de Ledinghen V, Douvin C, Marcellin P, Poupon R, Sandrin L, Miette V. Controlled attenuation parameter (CAP): A novel VCTE guided ultrasonic attenuation measurement for the evaluation of hepatic steatosis: preliminary study and validation in a cohort of patients with chronic liver disease from various causes. Ultrasound Med Biol 2010;36:1825–1835. Sasso M, Tengher-Barna I, Ziol M, Miette V, Fournier C, Sandrin L, Poupon R, Cardoso AC, Marcellin P, Douvin C, de Ledinghen V, Trinchet JC, Beaugrand M. Novel controlled attenuation parameter for noninvasive assessment of steatosis using Fibroscan((R)): Validation in chronic hepatitis C. J Viral Hepat 2012;19:244–253.
Volume -, Number -, 2015 Sasso M, Audiere S, Sandrin L, Miette V. Influence of heterogeneities on liver steatosis evaluation by Fibroscan (CAP): Relevance of a liver guidance tool. Eur J Ultrasound 2013;34. PS7_11. Schwenzer NF, Springer F, Schraml C, Stefan N, Machann J, Schick F. Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance. J Hepatol 2009;51:433–445. Shen F, Zheng RD, Mi YQ, Wang XY, Pan Q, Chen GY, Cao HX, Chen ML, Xu L, Chen JN, Cao Y, Zhang RN, Xu LM, Fan JG. Controlled attenuation parameter for non-invasive assessment of hepatic steatosis in Chinese patients. World J Gastroenterol 2014;20: 4702–4711. Tang A, Tan J, Sun M, Hamilton G, Bydder M, Wolfson T, Gamst AC, Middleton M, Brunt EM, Loomba R, Lavine JE, Schwimmer JB, Sirlin CB. Nonalcoholic fatty liver disease: MR imaging of liver proton density fat fraction to assess hepatic steatosis. Radiology 2013; 267:422–431. Wagner RF, Smith SW, Sandrik JM, Lopez H. Statistics of speckle in ultrasound B-scans. IEEE Trans Son Ultrason 1983;30:156–163. Wear KA. The effect of trabecular material properties on the frequency dependence of backscatter from cancellous bone. J Acoust Soc Am 2003;114:62–65.
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.08.008
d
Original Contribution LIVER STEATOSIS ASSESSED BY CONTROLLED ATTENUATION PARAMETER (CAP) MEASURED WITH THE XL PROBE OF THE FIBROSCAN: A PILOT STUDY ASSESSING DIAGNOSTIC ACCURACY MAGALI SASSO,* STEPHANE AUDIERE,* ASTRID KEMGANG,y FARID GAOUAR,y CHRISTOPHE CORPECHOT,y OLIVIER CHAZOUILLERES,y CELINE FOURNIER,z OLIVIER GOLSZTEJN,x STEPHANE PRINCE,x YVES MENU,x LAURENT SANDRIN,* and VERONIQUE MIETTE* * R&D Department, Echosens, Paris, France; y Assistance Publique-H^ opitaux de Paris, H^ opital Saint-Antoine, Centre de Reference Maladies Rares des Maladies Inflammatoires des Voies Biliaires, et Service d’hepatologie, Centre de Recherche Saint-Antoin, Sorbonne Universite, Paris, France; z Medical Affairs Department, Echosens, Paris, France; and x Department of Radiology, H^opital Saint-Antoine, APHP, Paris, France (Received 6 May 2015; revised 9 July 2015; in final form 11 August 2015)
Abstract—To assess liver steatosis, the controlled attenuation parameter (CAP; giving an estimate of ultrasound attenuation 3.5 MHz) is available with the M probe of the FibroScan. We report on the adaptation of the CAP for the FibroScan XL probe (center frequency 2.5 MHz) without modifying the range of values (100–400 dB/m). CAP validation was successfully performed on Field II simulations and on tissue-mimicking phantoms. In vivo performance was assessed in a cohort of 59 patients spanning the range of steatosis. In vivo reproducibility was good and similar with both probes. The area under receiver operative characteristic curve was equal to 0.83/0.84 and 0.92/ 0.91 for the M/XL probes to detect .2% and .16% liver fat, respectively, as assessed by magnetic resonance imaging. Patients can now be assessed simultaneously for steatosis and fibrosis using the FibroScan, regardless of their morphology. (E-mail: [email protected]) Ó 2015 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound attenuation, Controlled attenuation parameter (CAP), Liver, Steatosis, Steato-hepatitis, Non-alcoholic fatty liver disease (NAFLD), Elastography, Vibration-controlled transient elastography (VCTE), FibroScan.
INTRODUCTION
fibrosis, which can further progress to cirrhosis, liver failure or hepatocellular carcinoma (Farrell et al. 2004). When the cause of steatosis is the result of metabolic dysfunction, the disease is called non-alcoholic fatty liver disease (NAFLD). In approximately 30% of NAFLD patients, steatosis is associated with liver inflammation that can lead to non-alcoholic steato-hepatitis (NASH; Farrell and Larter 2006), which has a more severe outcome. The prevalence of NAFLD in the general population is high, approximately 20%–30% in affluent countries (Chalasani et al. 2012). With the rising epidemic of obesity and metabolic disorders, NAFLD is rapidly becoming one of the most common liver diseases worldwide. Liver biopsy is the gold standard to assess steatosis but suffers from many drawbacks and contraindications (Bravo et al. 2001). Alternative non-invasive methods, mainly involving conventional imaging, have been proposed to detect steatosis. Ultrasonography (US) is the
Hepatic steatosis is the accumulation of excess lipids (mainly triglycerides) in hepatocytes (Farrell et al. 2004). Steatosis is considered abnormal when the hepatic-fat content exceeds 5% of liver weight and, more practically, when more than 5% of hepatocytes contain fatty droplets. Steatosis can be a result of several causes, such as alcohol consumption, viral hepatitis or metabolic dysfunction (obesity, type 2 diabetes, hyperglycemia, hypertriglyceridemia; Farrell et al. 2004). Isolated steatosis is considered a benign and reversible condition; however, when steatosis is associated with inflammation (steato-hepatitis) it can progress to liver
Address correspondence to: Magali Sasso, 30 place d’Italie, 75013 Paris, France. E-mail: [email protected] 1
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most common liver-imaging technique because of its ease of use, accessibility and low cost (Schwenzer et al. 2009). However, its diagnostic value is controversial (Schwenzer et al. 2009) as it is highly operator and machine dependent. Furthermore, it cannot quantify steatosis and can only detect steatosis when at least 30% of hepatocytes are affected (Schwenzer et al. 2009). A recent metaanalysis (Bohte et al. 2011) that compared the performances of US, computed tomography, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (1H-MRS) to evaluate hepatic steatosis revealed that MRI and 1H-MRS were the techniques of choice for accurate evaluation of hepatic steatosis. However, these methods are costly, are not readily available, are not standardized and cannot be used to screen for NAFLD in the general population. Recently, a novel non-invasive parameter called the controlled attenuation parameter (CAP), has been developed to assess liver steatosis. CAP is measured with the FibroScan (Echosens, Paris, France), which is based on vibration-controlled transient elastography (VCTE), a technique initially developed to assess liver stiffness (LS), which is highly correlated with liver fibrosis (Sandrin et al. 2003). CAP is derived from the properties of ultrasonic signals acquired by the FibroScan. It uses the postulate that fat affects ultrasound propagation and is a measure of ultrasound attenuation at 3.5 MHz (i.e., the ultrasound center frequency of the FibroScan M probe). CAP was introduced recently (Sasso et al. 2010) and has already been assessed in many clinical studies, most of them evaluating steatosis in liver biopsies as the gold standard. The performance of CAP for the detection of steatosis has been reported to be good to excellent in most studies in patients with chronic liver disease of various etiologies (Chon et al. 2014; de Ledinghen et al. 2012; Sasso et al. 2010), with viral hepatitis (Mi et al. 2015; Sasso et al. 2012) and with NAFLD (Karlas et al. 2014). In this last study, CAP performance was compared to 1H-MRS and was shown to have a comparable diagnostic value. However, in some studies dealing with overweight and obese patients (Chan et al. 2014; Myers et al. 2012a), it has been shown that CAP performance was impaired by an increased body mass index (BMI). This phenomenon is attributed to the fact that overweight or obese patients have an increased skin-to-capsula distance (SCD) resulting from a high subcutaneous fat thickness. In these cases, the region of interest for CAP on the M probe will contain not only liver parenchyma but also a portion of the subcutaneous fat layer, which causes an overestimation of CAP. Recently, a probe dedicated to overweight and obese patients was developed by Echosens (Myers et al. 2012b). Indeed, the M probe has been shown to
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be inaccurate or to fail to assess LS in some patients with an increased BMI and a thick layer of subcutaneous fat (Castera et al. 2010; Foucher et al. 2006). This new probe, named the XL probe (Echosens, Paris, France), makes measurements to a greater depth in the liver (i.e., 35–75 mm vs. 25–65 mm) using a lower ultrasound frequency transducer (2.5 vs. 3.5 MHz) to increase ultrasound penetration. As LS values change as a function of frequency, the shear-wave center frequency remains unchanged (i.e., 50 Hz) with the XL probe. The first objective of this study was to validate the development of the CAP with the XL probe on Field II simulations and on tissue-mimicking phantoms. The difficult part of the development was to get CAP values around 3.5 MHz using the XL probe, whose center frequency is at 2.5 MHz. This was necessary to obtain comparable CAP values between the M and XL probes and, thus, to enable physicians to have the same range of CAP values for interpretation and diagnostic purposes. This is particularly important from a clinical point of view to prevent any misdiagnosis. Indeed, attenuation values measured on the same medium using a 2.5 or 3.5 MHz center frequency do not differ greatly; therefore, if the physician had to read off CAP values from different scales using the M and the XL probes, he/she might easily confuse the CAP values. In addition, obtaining CAP values on the same scale with both probes will allow us to use for the XL probe all published cutoffs and results found in literature for the M probe. This is of great interest as it can be difficult, from an ethical standpoint, to set up clinical studies on steatosis diagnosis using liver biopsies as the gold standard. The second aim of the study was to validate the reproducibility and diagnostic performance of CAP measured using the XL probe in a cohort of patients undergoing steatosis quantification assessed by MRI and to compare these data with CAP measured using the M probe. The study was conducted and written according to the Standards for Reporting of Diagnostic Accuracy (Bossuyt et al. 2003). MATERIALS AND METHODS CAP measurement principle CAP (Sasso et al. 2010) is an estimate of the ultrasonic attenuation coefficient at 3.5 MHz measured with the FibroScan, a VCTE device that uses ultrasound signals to track shear-wave propagation within the liver to perform a liver stiffness measurement (LSM; Mueller and Sandrin 2010; Sandrin et al. 2003). CAP is evaluated using the same radio-frequency data and in the same region of interest as that used for LSM and is
CAP with the XL probe of the FibroScan d M. SASSO et al.
only appraised if the LSM is ‘‘valid,’’ ensuring the operator obtains an ultrasonic attenuation value of the liver. CAP is an estimate of the total ultrasonic attenuation (go-and-return path, which was initially developed from signals acquired with the FibroScan M probe to assess in vivo liver attenuation using a proprietary algorithm at the center frequency of the probe, i.e., 3.5 MHz). CAP is expressed in dB/m. The new development of CAP on signals acquired with the XL probe requires three steps (see Fig. 1): 1. Evaluation of the raw attenuation around 2.5 MHz, the center frequency of the XL probe, using the same proprietary algorithm used on signals acquired on the M probe. 2. Evaluation of the frequency dependence of attenuation using a method similar to that described in Kuc and Schwartz (1979) and Wear (2003). 3. Estimation of CAP around 3.5 MHz using frequencydependent attenuation as a correction factor. In this step, attenuation was assumed to be linearly dependent with frequency. Note that the correction procedure was also performed using a proprietary algorithm. The correction factor developed here was based on estimation of frequency-dependent attenuation, assuming that attenuation varies linearly with frequency, which is a common assumption made when dealing with in vivo liver ultrasound attenuation (see Kuc 1980; Maklad et al. 1984). The linear dependence of ultrasound attenuation with frequency within the 0.5–7 MHz range has been shown experimentally in mammalian livers in (Goss et al. 1979). The hypothesis made in this paper is that the frequency dependence of attenuation is almost linear, whatever the steatosis. One of the objectives of this paper is to validate that the corrective factor is still valid with varying fat contents.
Fig. 1. Measurement principle of the controlled attenuation parameter (CAP) with the XL probe.
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Justification of the necessity to develop CAP on the XL probe As suggested in previous studies (Chan et al. 2014; Myers et al. 2012a), the performance of CAP is downgraded in cases of a large SCD (i.e., greater than 25 mm) because the measurement window not only contains liver parenchyma but also a portion of the subcutaneous fat layer, which causes overestimation of CAP. This phenomenon was first verified on a custommade tissue-mimicking phantom, made by ATS (Bridgeport, CT, USA) to mimic homogeneous liver parenchyma (background attenuation at 3.5 MHz of 250 dB/m) and SCD zones (hyper-echogenic zones, contrast 5 115 dB). Successive CAP measurements were performed using the M and XL probes in the homogenous zone of the phantom and in the zone mimicking a SCD equal to 32 mm (see Fig. 2). Validation of CAP measured with the XL probe Field II simulations. The validity of CAP measured with the XL probe to estimate the attenuation coefficient around 3.5 MHz was first appraised with Field II simulations (Technical University of Denmark, Copenhagen, Denmark; Jensen 1996; Jensen et al. 1993). A-mode simulations were performed in the FibroScan configuration, using a single 7-mm diameter flat
Fig. 2. Experimental setup to test the influence of a large skinto-capsula distance (SCD). The plain line represents the homogenous zone of the phantom and the dotted line represents the zone mimicking a SCD equal to 32 mm.
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piston transducer (9-mm, respectively) and a 3.5 MHz (2.5 MHz, respectively), two-cycle sinusoidal excitation for the M probe configuration (XL probe configuration, respectively). Tissue scatterers were modeled as a 3-D spatial distribution of independent point scatterers with uniformly drawn random positions (Jensen and Nikolov 2000) and a Gaussian-distributed amplitude (Jensen and Nikolov 2000; Wagner et al. 1983). Phantom size was set to 10 3 10 3 80 mm3. Scatterers number was set to 100,000, which is the minimal amount of scatterer needed to reach the traditionally defined maximal speckle signal-to-noise ratio of 1.91 (Burckhardt 1978) for the considered phantom size. Simulations were made for different homogeneous media with different attenuations, ranging from 100 dB/ m at 3.5 MHz to 400 dB/m at 3.5 MHz, in 20 dB/m steps. The CAP with the M probe was assessed for each simulated data set. When using the XL probe configuration, raw attenuation was first estimated, the corrective factor was then calculated and CAP was finally assessed. Experiments on tissue-mimicking phantoms. The validity of CAP measured with the XL probe to estimate the attenuation coefficient at 3.5 MHz was assessed on tissue-mimicking phantoms using both the FibroScan M and XL probes. The phantoms used were custom-made ultrasound attenuation tissue-mimicking phantoms made of waterbased gel with powdered graphite and glass bead scatterers, manufactured by the University of Wisconsin (Madison, WI, USA; Madsen et al. 1991). In our case, to assess CAP through its entire range (100–400 dB/m), three kinds of phantoms were made: (i) a low attenuation phantom, with attenuation at 150 dB/m at 3.5 MHz; (ii) an intermediate attenuation phantom, with attenuation at 250 dB/m at 3.5 MHz; and (iii) a high attenuation phantom, with attenuation at 350 dB/m at 3.5 MHz. The phantom’s characteristics at 20 C, given by the manufacturer, are shown in Table 1. For phantom testing, a probe-holding system was used and phantoms were placed in a tank filled with demineralized, degassed water (see Fig. 3). Water temperature was controlled at 20 C 6 0.5 C using a cooling and warming system (TC5; TECO, Ravenna, Italy). Acquisitions were performed using a FibroScan probe (both M and XL type; Echosens, Paris, France). During all the tests, the probe was held by a support (see Fig. 3) and moved to the different measurement points by a motorized linear translation stage (Newport Corporation, Irvine, CA, USA) driven using Matlab software (Mathworks, Natick, MA, USA). The translation stages had an accuracy of 1 mm. Each measurement set consisted of a scan of the whole phantom surface (57.5 mm in the X and Y axes)
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Table 1. Characteristics of phantoms at 20 C, as supplied by the manufacturer Phantom name Ph150 Ph250 Ph350
Speed of sound (m/s)
Reference attenuation (dB/m @ 3.5 MHz)
Uncertainty of reference attenuation (dB/m)
1496.1 1492.6 1490.0
145 235 340
5 5 5
with a 2.5-mm step, and five consecutive measurements were performed to assess bias, linearity and repeatability. In vivo study population. To constitute the cohort for the in vivo evaluation of CAP using both probes, a cohort was constituted using medical records from the Liver Unit of Saint Antoine Hospital, Paris, France. The objective was to sample, in a small cohort (objective: 60 patients), the whole spectrum of steatosis grades from no to massive steatosis. Enrolled patients had undergone a biopsy within one year of the medical records being accessed. The objective was to find the same proportions of patients within the following steatosis groups: no steatosis (0%–10% of hepatocytes), mild steatosis (10%– 33% of hepatocytes), moderate steatosis (33%–67% of
Fig. 3. Experimental setup for phantom testing: the probe is held by a probe-holding system.
CAP with the XL probe of the FibroScan d M. SASSO et al.
hepatocytes) and massive steatosis (67%–100% of hepatocytes), as described in Sasso et al. (2012). Patients with a BMI .40 kg/m2 were not eligible for the study. A posteriori exclusion criterion was having a SCD .25 mm. When a patient corresponding to the inclusion criteria was identified, he or she was contacted to participate in the study. After their enrollment, patients underwent both an MRI and a FibroScan on the same day for steatosis quantification. Liver fat fraction was quantified using liver MRI (Signa HDTX 1.5 Tesla, General Electrics, Milwaukee, WI, USA) using the three-point Dixon method. To measure fat fraction, three regions of interest (ROIs) were drawn across the liver parenchyma on in-phase and outphase images. ROIs were copied and pasted from one image to the other to ensure complete mirror measurements. Fat fraction was calculated using eqn (1): Fat fraction 5
ðIn2OutÞ 2 In
(1)
where In was the mean intensity of the ROIs drawn across the in-phase images and Out was the mean intensity of the ROIs drawn across the out-phase images. In addition, to avoid confusing the results related to potential liver iron deposition, an evaluation of liver iron concentration was performed and the fat fraction was validated only if liver iron concentration was less than 36 mmol/g. Although this formula may be ambiguous for high values of fat deposition because it has a theoretical signal intensity peak at 50%, and the signal may decrease above, this is not a clinical problem as this would only correspond to situations of extreme severity, which are higher than histologic grade 3 as described by Kleiner et al. (2005) and were not encountered in this study. The histologic grade and the measured fat fraction, even if expressed as percentages, are dissimilar. A fat fraction of 30%– 40% assessed by MRI corresponds to a 66% histologic evaluation (Tang et al. 2013). FibroScan examination was performed using a Fibroscan 502 Touch (Echosens, Paris, France) three consecutive times with the M probe and three times with the XL probe by two trained operators blinded to the patients’ bio-clinical data and using the acquisition procedure described in de Ledinghen and Vergniol (2008). During the clinical examination, the raw ultrasonic radio-frequency signals were stored in the FibroScan examination file to enable computation of CAP with the XL probe off-line and retrospectively. The final CAP (LSM, respectively) results were expressed in dB/m (kPa, respectively) and corresponded to the median of 10 valid individual measurements. Bio-clinical parameters were prospectively collected at the time of inclusion. Recorded data included
5
the following: gender, age, weight, height, BMI, waist circumference, diabetic status, hypertension status, alcohol use, platelet count, prothrombin time, aspartate aminotransferase, alanine aminotransferase (ALAT), gamma-glutamyl transpeptidase activity, total bilirubin, fasting glucose, triglycerides, high-density lipoprotein (HDL) cholesterol, ferritin and transferrin saturation. The protocol was performed in accordance with the Helsinki Declaration and was approved by an independent ethics committee. Patients were enrolled after giving their written informed consent. Statistical analyses For CAP validation on the Field II simulations, differences between CAP measured with the XL probe and the true attenuation set in the simulations was appraised using the root mean-square error (RMSE). For CAP validation on the tissue-mimicking phantoms, bias (B) and precision (P) were assessed using the definitions (ISO 2011) illustrated in eqns (2) and (3): B5
CAPi 2Aref Aref
(2)
P5
stdðCAPi Þ Aref
(3)
where CAPi is the average of the five replicate measurements (CAPi , i 5 1,.,5) and Aref is the reference attenuation value given by the manufacturer. Repeatability was assessed using repeatability coefficient (RC) and limits of agreements (LOA; Raunig et al. 2015). RC can be calculated from the within subject variance s2w obtained from one-way analysis of variance as follows in eqn (4): pffiffiffiffiffiffiffi RC 5 1:96 2s2w (4) and LOA is defined as the interval where the difference between two measurements under repeatability conditions for a randomly selected subject is expected to be 95% of the time and is expressed as eqn (5): LOA 5 ½2RC; RC
(5)
For repeatability results, the intra-class coefficient (ICC) of variation was also appraised, as suggested in Raunig et al. (2015). Agreement was classified as poor (ICC 5 0.00–0.20), fair to good (ICC 5 0.40–0.75) or excellent (ICC .0.75; Fleiss et al. 1981). Concerning the clinical data, except for evaluating reproducibility, only the first examination in the series of three measurements acquired using each probe type was considered. In the descriptive analyses, the continuous variables were expressed as their median
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[interquartile range] and the categorical variables as absolute figures and percentages. Reproducibility of CAP and LSM were assessed using ICC and the agreement scale described above. The relationship between CAP and the bio-clinical parameters was assessed using either Spearman’s rank correlation coefficient r or Student’s t-test (homoscedasticity was tested using the F-test). Parameters that were significantly related to CAP (or at the limit of significance p , 0.10) were entered in a multivariate model (Katz 2011). The normality of CAP was tested using Lilliefors’ test (Lilliefors 1967). Multiple linear regression (Katz 2011) was performed using a backward procedure based on minimization of the Akaike information criterion to select independent features significantly associated with CAP. The diagnostic performance of CAP was assessed using receiver operating characteristic curve analysis, using the MRI-based hepatic fat fraction dichotomized according to its distribution (first quartile, median and third quartile). The area under the receiver operating characteristic curve (AUROC) was computed as well as its 95% confidence interval. Cut-off values, specificities, sensitivities and accuracy were computed for the cut-off based on simultaneously maximizing sensitivity and specificity (Gallop et al. 2003). All statistical analyses were performed using R software (The R Foundation for Statistical Computing: Vienna, Austria). ICC was estimated using the psych package. A p value of less than 0.05 was considered statistically significant. RESULTS Justification for the development of CAP on the XL probe Results for the CAP measurements performed using the M and XL probes in the homogenous zone of the phantom and in the zone mimicking an SCD equal to 32 mm are given in Table 2. When measurements were performed in the homogeneous zone of the phantom, both probes gave similar results that matched the reference’s attenuation value given by the manufacturer. When measurements were performed in the zone that mimicked an SCD equal to 32 mm, measurements performed with the XL probe matched the reference’s attenuation value given by the manufacturer, whereas CAP performed using the M probe over-estimated the attenuation. Validation of the CAP as an estimate of ultrasonic attenuation Results on Field II simulations. Several simulations were performed on homogeneous attenuating media with attenuation values at 3.5 MHz set from 100 to 400 dB/m in 20 dB/m steps. Estimated CAP with the M probe, the XL probe and the raw attenuation around 2.5 MHz are
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Table 2. Controlled attenuation parameter (CAP) with both the M and XL probes on the homogeneous part of the phantom and in the part that mimicked a skin-tocapsula distance (SCD) of 32 mm
CAP with the M probe (dB/m) CAP with the XL probe (dB/m)
Homogeneous zone of the phantoms
Zone that mimicked a SCD of 32 mm
249 (32) 252 (50)
330 (35) 251 (58)
The results are given in term of median. The interquartile range is given in parentheses.
shown in Figure 4a, and the relative difference between the reference attenuation set in the simulation and estimated CAP with both the M and XL probes are given in Figure 4b. The RMSE for attenuation estimation at 3.5 MHz, using the M and XL probes, was 2.1 and 5.6 dB/m, respectively. Results on tissue-mimicking phantoms. CAP values for the five replicate measurements are given in Table 3, together with results in terms of bias and precision on each phantom and each probe type. The repeatability coefficient was equal to 0.7 and 3.6 dB/m for the M and XL probe, respectively. Corresponding LOA were [20.7; 0.7] dB/m and [23.6; 3.6] dB/m, respectively. ICC was equal to 1 [1; 1] for both M and XL probes, showing perfect agreement. In vivo results Fifty-nine patients who underwent an MRI and a FibroScan on the same day were enrolled between January 2013 and July 2013. One patient was excluded because of an SCD .25 mm. All patients were successfully measured by the Fibroscan. The patients’ characteristics are shown in Table 4. CAP measured with the M probe and the XL probe were very well correlated (r 5 0.82, p , 3.3 3 10215). A plot of individual CAP values measured with the M probe versus the XL probe is shown in Figure 5. CAP was correlated with the MRI-based hepatic fat fraction (r 5 0.73, p , 5.6 3 10211, and r 5 0.74, p , 1.6 3 10211 for measurements using the M and XL probes, respectively). In vivo reproducibility ICC for CAP was equal to 0.83 [0.76; 0.89] and 0.84 [0.77; 090] for the M and XL probes, respectively. For LSM, ICC was equal to 0.97 [0.96; 0.98] and 0.94 [0.91; 0.96] for the M and XL probes, respectively. Relationship between CAP and the bio-clinical parameters The parameters significantly related (or at the limit of significance) to CAP measured with the M
CAP with the XL probe of the FibroScan d M. SASSO et al.
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Fig. 4. (a) Controlled attenuation parameter (CAP) with the M and XL probe configurations as a function of the reference attenuation set in Field II; (b) relative difference (%) between the reference attenuation set in Field II and estimated CAP with the M and XL probes as a function of the reference attenuation set in Field II.
and XL probes are given in Table 5. In univariate analyses, CAP appeared to be related to BMI, waist circumference, hypertension status, diabetic status, ALAT, triglyceride, HDL cholesterol, ferritin and steatosis as assessed by MRI. CAP was independent of the other bio-clinical parameters given in Table 4, especially LSM. All the parameters significantly related to CAP or at the limit of significance were entered in a multivariate model, and only BMI and steatosis (as assessed by MRI) were related to CAP on both the M and XL probes. A plot of CAP values for both M and XL probes versus BMI and steatosis assessed by MRI is shown in Figure 6 (a, b), respectively.
CAP performance for steatosis diagnosis The CAP performance for steatosis diagnosis is given in Table 6. The CAP performance in terms of AUROC was 0.83 (0.71–0.95) for the M probe and 0.84 (0.73–0.95) for the XL probe for the diagnostic of MRIbased liver fat fraction .2%, 0.87 (0.78–0.97) for the M probe and 0.90 (0.82–0.99) for the XL probe for the diagnostic of liver fat fraction of .8%, and 0.92 (0.85–0.99) for the M probe and 0.91 (0.83–0.99) for the XL probe for the diagnostic of liver fat fraction of .16%. No statistical differences were found for the diagnostic performances in terms of AUROC between the two probes.
DISCUSSION
Table 3. Results for phantom testing using the probeholding system
Measurement 1, M probe Measurement 2, M probe Measurement 3, M probe Measurement 4, M probe Measurement 5, M probe Average, M probe (dB/m) Std, M probe (dB/m) Bias, M probe (%) Precision, M probe (%) Measurement 1, XL probe Measurement 2, XL probe Measurement 3, XL probe Measurement 4, XL probe Measurement 5, XL probe Average, XL probe (dB/m) Std, XL probe (dB/m) Bias, XL probe (%) Precision, XL probe (%)
Ph150
Ph250
Ph350
143 143 143 143 143 143.0 0 21.4 0 157 153 154 154 154 154.4 1.5 6.5 1.0
234 234 234 234 234 234.0 0 20.4 0 226 226 227 228 228 227.0 1.0 23.4 0.4
323 323 323 323 324 323.2 0.45 24.9 0.1 328 327 327 329 330 328.2 1.3 23.5 0.4
In the present study, the CAP was successfully implemented with the XL probe of the FibroScan. This implementation necessitated a scaling procedure to get CAP values to correspond to an attenuation around 3.5 MHz when using the XL probe of the FibroScan, whose center frequency is around 2.5 MHz. This scaling procedure was shown to be efficient in the simulations and tissue-mimicking phantoms, as well as in vivo on the whole attenuation range, from that of a normal liver (no steatosis) up to massive steatosis (100–400 dB/m at 3.5 MHz). This scaling procedure assumes that the frequency dependence of attenuation is linear with frequency. Even if this approximation is gross in the case of fatty infiltration, as suggested in Narayana and Ophir (1983), it was shown here that it did not impair the performance of the scaling procedure. This was also shown in the phantom testing because, when SCD was greater than 25 mm, measuring CAP with the M probe of the FibroScan led to overestimation (330 dB/m instead of 250 dB/m). These results are consistent with previous
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Table 4. Characteristics of the study population Characteristic Gender (female) Age (y) BMI (kg/m2) Waist circumference (cm) Diabetic status Hypertension status Alcohol use Platelet count (103/mm3) Prothrombin time (%) ASAT (IU/L) ALAT (IU/L) GGT (IU/L) Total bilirubin (mmol/L) Fasting glucose Triglycerides HDL cholesterol Ferritin (ng/mL) Ferritin saturation (%) Liver stiffness (kPa) CAP (dB/m) LB steatosis classification (retrospective selection) MRI-based liver fat fraction (%) MRI-based liver iron concentration (mmol/g)
Patient distribution* 23 (39.0%) 53.0 [11.5] 27.0 [5.2] 96.5 [15.3] (7y) 10 (16.9%) 19 (32%) 19 (32%) 197 [97] (3y) 94 [14] (15y) 38.5 [31.0] (1y) 48.0 [45.6] (1y) 71.5 [101.8] (3y) 11.5 [6.7] (7y) 6.1 [1.4] (22y) 1.3 [0.8] (2y) 1.1 [0.6] (8y) 226 [338] (10y) 30.9 [18.3] (13y) M probe (3 series): 7.4 [4.6]/7.3 [5.7]/7.2 [4.3] XL probe (3 series): 6.4 [3.7]/6.9 [3.9]/6.7 [3.7] M probe (3 series): 275 [101]/276 [82]/268 [73] XL probe (3 series): 279 [91]/272 [114]/275 [105] Absent/Minimal/Moderate/Massive 10 (17%)/23 (39%)/14 (24%)/12 (20%) 8 [15] 10 [15]
BMI 5 body mass index; ASAT 5 aspartate aminotransferase; ALAT 5 alanine aminotransferase; GGT 5 gamma-glutamyl transpeptidase; HDL 5 high-density lipoprotein; CAP 5 controlled attenuation parameter; LB 5 liver biopsy; MRI 5 magnetic resonance imaging. * Continuous variables were expressed as the median [interquartile range], and categorical variables as absolute figures (percentages). y Number of missing data.
in vivo studies (Chan et al. 2014; Myers et al. 2012a) in which it was observed that CAP performance was downgraded when SCD was greater than 25 mm. With the development of CAP for the XL probe of the FibroScan it will now be possible to properly assess
Fig. 5. Controlled attenuation parameter (CAP) measured with the M probe versus the CAP measured with the XL probe for the 59 patients.
steatosis and reduce the risk of misdiagnosis in overweight and obese patients, who are particularly vulnerable to steatosis. CAP measured with the XL probe of the FibroScan was validated first with Field II simulation. CAP estimated on simulated signals with the XL probe were very close to values set in the simulation as the RMSE was equal to 5.6 dB/m. It was also shown in the Field II simulation that the scaling procedure worked in a satisfactory way for each attenuation value between 100 and 400 dB/m. CAP measured with the XL probe of the FibroScan was then validated on ultrasound attenuation tissuemimicking phantoms. The repeatability was shown to be almost perfect as the LOA was (23.6; 3.6) dB/m and the ICC was 1. Bias and precision were also satisfactory, as bias in absolute value was always smaller than 7% and precision was less than or equal to 1%. Results on the tissue-mimicking phantoms indicated that performance of the CAP with the XL probe, even though satisfactory, was always slightly below that of the M probe. This was because (i) the transducer of the XL probe is not the same (lower center frequency, lower sensitivity) as the transducer of the M probe and, therefore, its performance is slightly different; and (ii) the scaling process, even though
CAP with the XL probe of the FibroScan d M. SASSO et al.
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Table 5. Factors associated with controlled attenuation parameter (CAP) on M and XL probes in univariate and multivariate analyses CAP with the M probe Univariate analysis BMI Waist circumference Hypertension status Diabetic status ALAT Triglycerides HDL cholesterol Ferritin MRI-based hepatic fat fraction
CAP with the XL probe
Multivariate analysis 26
r 5 0.55, p 5 6.5 3 10 r 5 0.50, p , 1.0 3 1023 p 5 0.03 p 5 0.06 r 5 0.27, p 5 0.04 r 5 0.30, p 5 0.02 r 5 20.31, p 5 0.03 r 5 0.29, p 5 0.04 r 5 0.75, p , 5.6 3 10211
p 5 0.03 x x x x x x p , 1.3 3 1026
Univariate analysis
Multivariate analysis 24
r 5 0.50, p 5 1.6 3 10 r 5 0.52, p , 1.3 3 1025 p 5 0.01 p 5 0.06 r 5 0.33, p 5 0.01 r 5 0.28, p 5 0.04 r 5 20.14, p 5 0.34 r 5 0.26, p 5 0.07 r 5 0.77, p , 1.6 3 10211
p 5 0.06 x x x x x x x p , 6.7 3 1028
BMI 5 body mass index; ALAT 5 alanine aminotransferase; HDL 5 high-density lipoprotein; MRI 5 magnetic resonance imaging.
satisfactory, is not completely perfect and therefore it may have introduced a certain amount of variability. Note that the effect of hand-holding the probe was tested on the same tissue-mimicking phantoms by two operators and led to a LOA of (212.5; 12.5) dB/m for the M probe and of (214.0; 14.0) dB/m for the XL probe (data not shown). Reproducibility of the CAP with the M and XL probes was assessed in vivo, yielding similar results (0.83 and 0.84 for the M and XL probes, respectively) and good performances using both probes. However, CAP reproducibility was lower than LSM reproducibility (0.97 and 0.94 for the M and XL probes, respectively). Similar results were found by Recio et al. (2013), who found an ICC of 0.83 (0.77–0.88) for the CAP measured with an M probe and 0.96 (0.94–0.97) for the LSM measured with an M probe in a cohort of 118 patients with human immunodeficiency virus and/or hepatitis C virus infection.
Although CAP has good reproducibility, it appears to be less reproducible than LSM. This phenomenon is because the CAP, and more generally ultrasound attenuation, is very sensitive to heterogeneities such as blood vessels, nodules, etc. (Audiere et al. 2013; Sasso et al. 2013). This is why it is important to perform CAP examinations in a very homogenous part of the parenchyma and why a liver-guidance tool has been developed on the interface of the FibroScan to help the operator accurately conduct a CAP examination (Audiere et al. 2013; Sasso et al. 2013). In the present study, CAP assessed on both the probes was shown to be mainly related, in univariate analyses, to steatosis (p , 10211), BMI (p , 1024) and waist circumference (p , 1024). CAP assessed on both probes was also related to hypertension status, diabetic status, ALAT triglycerides and ferritin. Note that CAP assessed with the M probe was related to HDL cholesterol, but not when measured with the XL probe. This was
Fig. 6. Controlled attenuation parameter (CAP) measured with the M and XL probes as a function of (a) body mass index (kg/m2) and (b) MRI-based liver fat fraction (%).
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Table 6. Diagnostic performance of the controlled attenuation parameter (CAP)
S $2% (Prevalence 5 69%)
S $8% (Prevalence 5 51%)
S $16% (Prevalence 5 22%)
CAP with the M probe
CAP with the XL probe
p Value (Delong test)
AUROC 5 0.83 [0.71–0.95] Cutoff 5 251 dB/m Se 5 0.78/Sp 5 0.78 Acc 5 0.80 AUROC 5 0.87 [0.78–0.97] Cutoff 5 267 dB/m Se 5 0.80/Sp 5 0.79 Acc 5 0.81 AUROC 5 0.92 [0.85–0.99] Cutoff 5 299 dB/m Se 5 0.92/Sp 5 0.88 Acc 5 0.90
AUROC 5 0.84 [0.73–0.95] Cutoff 5 254 dB/m Se 5 0.83/Sp 5 0.78 Acc 5 0.83 AUROC 5 0.90 [0.82–0.99] Cutoff 5 270 dB/m Se 5 0.88/Sp 5 0.79 Acc 5 0.85 AUROC 5 0.91 [0.83–0.99] Cutoff 5 301 dB/m Se 5 0.92/Sp 5 0.81 Acc 5 0.85
0.76
0.50
0.78
S 5 steatosis; AUROC 5 area under the receiver operating characteristic curve; Se 5 sensitivity; Sp 5 specificity; Acc 5 accuracy.
probably because of the small number of patients and the influence of outlier values. Except for that parameter, CAP measured with both the M and XL probes was linked to the same bio-clinical parameters. Note that CAP was independent of LSM for both probes (p . 0.10). In multivariate analyses, CAP was only related to BMI and steatosis for both the M and XL probes. This result is in agreement with published studies, in which the only two parameters linked to CAP were steatosis and BMI (Chan et al. 2014; Chon et al. 2014; Ferraioli et al. 2014; Jung et al. 2014; Kumar et al. 2013; Mi et al. 2015; Shen et al. 2014). CAP diagnostic performance has been assessed for the first time using the hepatic fat-fraction measured by MRI. A recent meta-analysis (Bohte et al. 2011) reported that MRI and 1H-MRS were accurate techniques for evaluating hepatic steatosis. Taking another non-invasive technique as a gold standard can be relevant in many clinical studies when a liver biopsy cannot be performed or is not planned, especially in NAFLD patients. Karlas et al. (2014) compared CAP and 1H-MRS performance, taking a liver biopsy as a gold standard for NAFLD patient and providing good to excellent and similar performances using both non-invasive techniques. The diagnostic performance of CAP to assess the MRI-based hepatic fat fraction was good to excellent using both probes. The performances were shown to be similar. The optimal cut-off values determined by simultaneous maximizing sensitivity and specificity (Gallop et al. 2003) studies have been found to be similar for both probes, showing that the scaling process used to develop CAP for the XL probe works quite well and that physicians will be able to use the same reading scale with the M and XL probe. However, as suggested by Karlas et al. (2014) and Recio et al. (2013), taking a single cut-off point to classify a patient might be risky. Indeed, reproducibility for CAP, even if good, is not perfect, with relative variability between measurements. Furthermore, cut-off points are very dependent on the study’s popula-
tion (limited number of patients, usually between 100 and 200) and on the prevalence of steatosis stages in each study population. The cut-offs determined in this study should be considered with caution. First, the number of patients was very small. Second, the gold standard here was the MRI-based fat fraction, which cannot be compared directly to the percentage of steatosis assessed in liver biopsies. In future studies, a different approach to classify patients should be used, such as for instance the use of intervals instead of a single value (Recio et al. 2013), the use of a ‘‘grey area’’ (the use of two cut-off points to determine if the patient is healthy [no steatosis] or sick [massive steatosis]) and careful examination for interfering factors if the patient is in between classification schemes (Karlas et al. 2014). One perspective of this work was to validate the diagnostic performance of CAP with the XL probe in patients that actually need to be measured by the XL probe. In our study, CAP was measured on patients that could be measured by both probes, as the objective of our study was to validate the development of the CAP with the XL probe and to show that its performance was similar to the performance of CAP with the M probe. CAP with the XL probe will, therefore, need to be also clinically validated in overweight and obese populations. CONCLUSION In summary, CAP was successfully adapted to the XL probe of the FibroScan with the same measurement range as the CAP with the M probe. CAP with the XL probe was validated in Field II simulations and in tissue-mimicking phantoms. Its in vivo diagnostic performance for steatosis, assessed by MRI, was good to excellent. Reproducibility in vivo was also shown to be good: both performance and reproducibility in vivo were shown to be similar. The diagnostic performance of CAP with an XL probe should be validated in larger studies.
CAP with the XL probe of the FibroScan d M. SASSO et al.
CAP can now be assessed properly with no overestimation when SCD is greater than 25 mm, which is especially useful for overweight and obese patients who are particularly exposed to steatosis. NASH patients will benefit from this new development as they can be assed simultaneously for steatosis and fibrosis, regardless of their morphology.
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Volume -, Number -, 2015 Sasso M, Audiere S, Sandrin L, Miette V. Influence of heterogeneities on liver steatosis evaluation by Fibroscan (CAP): Relevance of a liver guidance tool. Eur J Ultrasound 2013;34. PS7_11. Schwenzer NF, Springer F, Schraml C, Stefan N, Machann J, Schick F. Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance. J Hepatol 2009;51:433–445. Shen F, Zheng RD, Mi YQ, Wang XY, Pan Q, Chen GY, Cao HX, Chen ML, Xu L, Chen JN, Cao Y, Zhang RN, Xu LM, Fan JG. Controlled attenuation parameter for non-invasive assessment of hepatic steatosis in Chinese patients. World J Gastroenterol 2014;20: 4702–4711. Tang A, Tan J, Sun M, Hamilton G, Bydder M, Wolfson T, Gamst AC, Middleton M, Brunt EM, Loomba R, Lavine JE, Schwimmer JB, Sirlin CB. Nonalcoholic fatty liver disease: MR imaging of liver proton density fat fraction to assess hepatic steatosis. Radiology 2013; 267:422–431. Wagner RF, Smith SW, Sandrik JM, Lopez H. Statistics of speckle in ultrasound B-scans. IEEE Trans Son Ultrason 1983;30:156–163. Wear KA. The effect of trabecular material properties on the frequency dependence of backscatter from cancellous bone. J Acoust Soc Am 2003;114:62–65.