Liver International ISSN 1478-3223
NAFLD/NASH
Clinical value of liver ultrasound for the diagnosis of nonalcoholic fatty liver disease in overweight and obese patients Fernando Bril1,2, Carolina Ortiz-Lopez3, Romina Lomonaco1,2, Beverly Orsak3, Michael Freckleton4, Kedar Chintapalli4, Jean Hardies4, Song Lai5, Felipe Solano6, Fermin Tio6 and Kenneth Cusi1,2,3,7 1 2 3 4 5 6 7
Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL, USA Malcom Randall Veterans Administration Medical Center, Gainesville, FL, USA Division of Diabetes, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Radiology Department, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Clinical Translational Science Institute Human Imaging Core, McKnight Brain Institute, University of Florida, Gainesville, FL, USA Pathology Department, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Audie L. Murphy Veterans Administration Medical Center, San Antonio, TX, USA
Keywords hepatic steatosis – NAFLD – NASH – Obesity – steatohepatitis Abbreviations 1 H-MRS, magnetic resonance imaging and proton spectroscopy; ALT, alanine aminotransferase; AUROC, area under the ROC curve; BMI, body mass index; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OGTT, oral glucose tolerance test; ROC, receiver operating characteristic; T2DM, type 2 diabetes mellitus; US, ultrasound. Correspondence Kenneth Cusi, MD, FACP, FACE, Endocrinology, Diabetes and Metabolism Division University of Florida, 1600 SW Archer Road, room H-2. Gainesville, FL 32610, USA Tel: 352 273 8662 Fax: 352 273 7441 e-mail: [email protected] Received 17 January 2015 Accepted 23 March 2015
Abstract Background & Aims: Liver ultrasound (US) is usually used in the clinical setting for the diagnosis and follow-up of patients with nonalcoholic fatty liver disease (NAFLD). However, no large study has carefully assessed its performance using a semiquantitative ultrasonographic scoring system in overweight/obese patients, in comparison to magnetic resonance spectroscopy (1H-MRS) and histology. Methods: We recruited 146 patients and performed: a liver US using a 5-parameter scoring system, a liver 1H-MRS to quantify liver fat content, and a liver biopsy to assess histology. All measurements were repeated in a subgroup of patients (n = 62) after 18 months of follow-up. Results: The performance of liver US (parenchymal echo alone) was rather modest, and significantly worse than 1H-MRS (AUROC: 0.82 [0.69–0.94] vs. 0.96 [0.90–1.00]; P = 0.04). However, the AUROC improved when different echographic parameters were taken into account (AUROC: 0.89 [0.83–0.96], P = 0.15 against 1H-MRS). Optimum sensitivity for liver US was achieved at a liver fat content ≥12.5%, suggesting that below this threshold, liver US is less sensitive. Liver 1H-MRS showed a high accuracy for the diagnosis of NAFLD, and correlated strongly with histological steatosis (r = 0.73, P < 0.0001). None of the imaging tests was adequate enough to predict changes over time in histology. Conclusions: Despite its widespread use, liver US has several important limitations that healthcare providers should recognize, particularly because of its low sensitivity. Using a combination of echographic parameters, liver US showed a significant improvement in its diagnostic performance, but still was of limited value for monitoring treatment over time.
Handling Editor: Luca Valenti DOI:10.1111/liv.12840 Liver Int. 2015; 35: 2139–2146
As a consequence of the obesity epidemic, nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the USA (1, 2) and worldwide (3, 4). This condition has a broad spectrum of severity that ranges from benign liver fat accumulation (simple steatosis) to severe necroinflammation (nonalcoholic steatohepatitis, NASH), and eventually cirrhosis (5). It is currently estimated that ~60% of obese patients Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
have NAFLD by magnetic resonance imaging and proton spectroscopy (1H-MRS). However, the prevalence of NAFLD varies according to the diagnostic tool being used. Liver ultrasound (US) is widely used in the clinical setting to diagnose a fatty liver because of its low cost, simplicity and safety profile. However, its value for the diagnosis of hepatic steatosis has been rather conflicting
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Key points
Use of an ultrasonographic scoring system combining five echographic parameters improved the diagnostic performance of liver ultrasound for NAFLD, and its ability to assess changes over time. The optimum sensitivity of liver US for the diagnosis of NAFLD was reached with a liver fat content ≥12.5%. This ‘detection’ threshold is much better than commonly believed. Liver fat measured by 1H-MRS, and its changes over time, were strongly correlated with liver steatosis assessed by histology. Changes over time in steatosis did not predict changes in other histological parameters. Therefore, liver biopsy remains the gold standard to follow patients with NASH.
providing there was no evidence of any chronic liver disease other than NAFLD/NASH (i.e. hepatitis B or C, autoimmune hepatitis, hemochromatosis, Wilson’s disease, drug-induced, others). Patients were also excluded if they had a history of alcohol abuse (>30 g per day in men or >20 g per day in women) (9), type 1 diabetes mellitus, or a history of clinically significant renal, pulmonary or heart disease. A complete medical history, physical exam, routine blood and urine chemistries and electrocardiography were performed in all patients to confirm eligibility. This study was approved by the University IRB, and a written informed consent was obtained from each patient prior to participation. Some patients were included in previous reports on the role of ethnicity (10), plasma vitamin D (11) and insulin resistance (12–14) in the pathogenesis of NAFLD. Study design
with studies reporting a broad spectrum of sensitivities that ranged from 60 to 94% (6). In addition, the sensitivity of liver US is considered to be reduced in patients with liver fat 5.5% by 1H-MRS) and elevated plasma aminotransferases, or with normal aminotransferases but at high risk of NASH because of severe insulin resistance, obesity or with T2DM, were offered a percutaneous liver biopsy to establish the diagnosis of NASH. A subset of patients with biopsy-proven NASH (n = 62) were included in a randomized controlled trial assessing the effects of pioglitazone vs. placebo for 18 months (15). After therapy, these patients underwent a second liver US, 1H-MRS and a second liver biopsy. This information was used to assess the ability of liver ultrasound and 1H-MRS to detect changes in steatosis over time. Liver US examination
All liver ultrasounds (US) were performed the day of the liver biopsy after an overnight fast using a Phillips IU22 unit (Cleveland, OH, USA). Grey-scale images of the liver were obtained using a 2–5 MHz multifrequency transducer. Transverse and longitudinal images showing the inferior vena cava with hepatic vein confluence, porta hepatis and gallbladder were obtained. The field of view was adjusted to include the diaphragm on the longitudinal images. The gain settings, focal zones and field of view were adjusted as needed for individual studies. Two board certified radiologists were provided the ultrasound images for grading of hepatic steatosis. Images were reviewed by each radiologist independently from each other and in a blinded fashion without any clinical data available. Ultrasound parameters used to semiquantitatively assess fat scores were: parenchymal echogenicity of the liver, far gain attenuation in right intercostal view at posterior axilla line, gallbladder wall blurring in right sagittal subcostal view, portal vein blurring in right intercostal view at anterior axilla line, and hepatic vein blurring in right intercostal view at middle axilla line Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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(see Table S1 for details). Each parameter was scored with 0, 1 or 2 and the US fatty liver score was calculated as the sum of the scores of these five parameters (8). Measurement of liver fat content by 1H-MRS
For the measurement of hepatic fat content, localized proton nuclear magnetic resonance spectra of the liver were acquired using methodology previously described (10, 12, 14). Briefly, a 30 mm3 voxel was localized in three areas of the liver avoiding vessels and bile ducts. Liver fat content was calculated as fat fraction (area under the curve [AUC] fat peak/[AUC fat peak + water peak]) using commercial software (NUTS, Acorn NMR). Measurements were corrected for T1 and T2 relaxation using methods previously described (16). A liver fat content of >5.5% was considered diagnostic of NAFLD (1). Liver biopsy
An ultrasound-guided liver biopsy was performed in patients with a positive 1H-MRS when liver transaminases were elevated, and all other causes of liver disease were ruled out, and also in patients with normal liver transaminases if they had significant risk factors for the development of NASH such as T2DM, metabolic syndrome and/or severe insulin resistance. All biopsies were evaluated by the same pathologist that was unaware of the subjects’ identity or clinical information. Histological characteristics for the diagnosis of NASH were determined using standard criteria (17). Histological quantification of lipid droplets by Aperio ImageScope
The slides provided for this analysis were scanned using the Aperio ScanScope slide scanner & Aperio ScanScope Console v.10.2.0.0 at 209 magnification. The scans were maintained in Aperio’s Spectrum v.11.0.0.725, which is a database for the scans. They were viewed and prepared for analysis using Aperio ImageScope v.11.0.2.725. A total of 118 baseline images were collected plus 62 images after 18 months of follow-up. The algorithm that was used to determine the area and per cent area of the fat was the Microvessel Analysis v1, with the only changing variable being that of the Light Staining Threshold with values ranging from 125 to 215 (dark–light). Statistical analysis
Data were summarized in percentages for categorical variables and as mean ± standard error or median (interquartile range) for numeric variables. Categorical variables were compared performing Chi-square or Fisher’s Exact test. For comparisons between two groups, we performed Kruskal–Wallis or Student’s t-test for numeric variables depending on variables’ distribution. Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Diagnostic role of imaging in NAFLD
Comparisons among three or more groups were performed with ANOVA (Bonferroni method for post hoc testing) or Kruskal–Wallis test. Comparisons between AUROCs were performed using methodology described by DeLong et al. (18). Optimum cut-off points were calculated using Youden’s index, and inter-observer variations were compared with kappa coefficient. A twotailed P-value of less than 0.05 was considered to indicate statistical significance. Analyses were performed with Stata 11.1 (StataCorp LP, College Station, TX, USA). Results Patients’ characteristics
In Table 1, we have summarized patients’ demographical and clinical characteristics. As can be observed, this cohort consisted of predominantly obese patients (BMI: 34.1 ± 0.4 kg/m2), with a high prevalence of T2DM (54%), and elevated plasma alanine aminotransferase (64% had abnormal plasma alanine aminotransferase [ALT] defined as ≥40 IU/L). A total of 66% of the patients were diagnosed with NAFLD according to the ultrasonographic fatty liver score. Liver US performance for the diagnosis of NAFLD
In Fig. 1, we have plotted the ROC curves showing the performances of plasma ALT, liver ultrasound (based on parenchymal echo alone and on the ultrasonographic fatty liver score), and liver 1H-MRS for the diagnosis of hepatic steatosis when considering liver histology as the Table 1. Clinical and laboratory characteristics of patients All patients (n = 146) Age, years Gender (male), % Body mass index, kg/m2 Prevalence of obesity, % Total body fat, % Type 2 diabetes mellitus, % Fasting plasma glucose, mg/dL HbA1c, % Fasting plasma insulin, lIU/mL Fasting FFA, mM ALT, IU/mL AST, IU/mL Liver fat by 1H-MRS, % NAFLD activity score (NAS) Steatosis grade Inflammation grade Ballooning grade Fibrosis stage Patients with fibrosis stage 3–4, % Total cholesterol, mg/dL LDL-C, mg/dL Triglycerides, mg/dL HDL-C, mg/dL
50 ± 1 72% 34.1 ± 0.4 81% 33 ± 1 54% 121 ± 2 6.3 ± 0.1 14 ± 1 0.56 ± 0.02 61 ± 3 44 ± 2 16 ± 1 4.0 ± 0.1 1.7 ± 0.1 1.5 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 10% 178 ± 4 106 ± 3 152 (101–200) 37 ± 1
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gold standard (i.e. hepatic steatosis was defined as steatosis grade ≥1). As can be observed, liver ultrasound based on parenchymal echo alone had a modest performance for the diagnosis of NAFLD (AUROC: 0.82 [0.69–0.94]) and was considerably worse than liver 1HMRS (AUROC: 0.96 [0.90–1.00], P = 0.04). Of note, although it was better than plasma ALT (AUROC: 0.65 [0.41–0.89]), this difference did not reach statistical significance. Using the US fatty liver score (combination of five parameters), the performance of the test improved (shaded area in Fig. 1A) and became statistically indistinguishable from liver 1H-MRS (AUROC: 0.89 [0.83–0.96] vs. 0.96 [0.90–1.00], P = 0.15). The optimum cut-off point of the US fatty liver score for the diagnosis of hepatic steatosis in this population was 6 of 10. Applying this cut-off point, sensitivity was relatively low: 70% (61–77%) and specificity was 100% (63– 100%) to detect hepatic steatosis in the biopsy. However, this was still better than the performance of parenchymal echo alone: sensitivity of 63% (54–71%) and specificity of 88% (47–100%). In Fig. 2, we have summarized the sensitivity of the liver US scoring system (using its best cut-off point of 6) to detect different amounts of liver fat quantified by 1 H-MRS. As can be observed, the maximum sensitivity (plateau) was reached at ~12.5% of liver triglyceride content, showing worse performance below this threshold. Interestingly, when only parenchymal echogenicity was used as a criterion to diagnose hepatic steatosis, the plateau was reached at approximately the same amount of liver fat accumulation, but with overall lower sensitivity, especially at low thresholds of liver fat (P ≤ 0.02 for all comparisons using cut-off points of liver fat ≤10%). This suggests that using the scoring system improves sensitivity of liver US in patients with small elevations of liver fat content (from 5.5 to 10%). In Table 2, we report on general correlations between each echographic parameter and results of liver fat content measured by liver histology or 1H-MRS. As can be
Sensitivity (%)
Ultrasound fatty liver score
* *
80
* Parenchymal echogenicity alone
60 40 20
Plateau reached
0 0
5
10
15
20
25
30
Liver fat (%) Fig. 2. Sensitivity of the ultrasound fatty liver score and parenchymal echogenicity for the diagnosis of NAFLD using different cut-off points of liver fat content by 1H-MRS. *P ≤ 0.02 for comparisons of sensitivities between the ultrasound fatty liver score and parenchymal echogenicity alone.
observed, the echographic total score (sum of the five parameters) correlated well with the steatosis grade (rs = 0.49, P < 0.001), and with the amount of liver fat measured by digital fat quantification (DFQ) of liver tissue (rs = 0.57, P < 0.001) or by 1H-MRS (rs = 0.50, P < 0.001). No correlations were observed between echographic characteristics and histological parameters (inflammation, ballooning and fibrosis) after adjustment for steatosis grade, suggesting that liver US cannot differentiate between simple steatosis and NASH. As operator-dependency remains an important concern regarding liver ultrasound, inter-observer agreement was assessed between two independent examiners, for each of the 5 parameters used to score the ultrasounds (0, 1 or 2), as well as for the overall diagnosis of hepatic steatosis (from 0 to 10). The best inter-rater agreements were found for portal vein blurring (agreement: 79%, j = 0.45, P < 0.001), far field attenuation (agreement: 70%, j = 0.43, P < 0.001) and hepatic vein blurring (agreement: 65%, j = 0.36, P < 0.001). Parenchymal echo (agreement: 51%, j = 0.13, P = 0.04) and gallbladder wall blurring (agreement: 51%, j = 0.11, P = 0.07) P = 0.02
(B)
1
H-MRS
1.00
US
80
ALT
60
AUROCs
Sensitivity (%)
(A) 100
100
ECHO
40
0.50
20 0
0.00 0
20
40
60
80
100
ALT
ECHO
US
MRS
100 - Specificity (%) Fig. 1. (A) Receiver operating characteristic (ROC) curves describing the performance of plasma alanine aminotransferase (ALT), liver ultrasound by means of parenchymal echogenicity alone (ECHO), 5-parameter ultrasound fatty liver score (US), and liver 1H-MRS for the diagnosis of hepatic steatosis in predominantly obese patients with high risk of NAFLD. Liver histology was considered the gold standard. Shaded area represents the difference between using parenchymal echogenicity alone and the ultrasonographic scoring system. (B) AUROCs of different imaging techniques. P-value represents overall comparison of AUROCs.
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Table 2. Correlation analyses between ultrasound parameters and histological features
Severity of liver fat accumulation Steatosis grade % fat accumulation by DFQ % fat accumulation by 1H-MRS Severity of histology Inflammation grade‡ Ballooning grade‡ Fibrosis grade‡
Parenchymal echogenicity
Far gain attenuation
GB wall blurring
Portal vein blurring
Hepatic vein blurring
Steatosis score
0.44† 0.52† 0.43†
0.40† 0.43† 0.39†
0.41† 0.36* 0.43†
0.49† 0.50† 0.48†
0.52† 0.48† 0.45†
0.49† 0.57† 0.50†
0.10 0.08 0.11
0.17* 0.11 0.16
0.06 0.07 0.10
0.02 0.06 0.00
0.05 0.10 0.10
0.02 0.05 0.05
DFQ, digital fat quantification; GB, gallbladder. *P < 0.05 and †P < 0.0001. No significant differences were observed, when coefficients of parenchymal echogenicity were compared with the ones of the total score. ‡Adjusted for steatosis grade.
showed the worst agreement. Overall, the agreement for the diagnosis of NAFLD (US fatty liver score ≥6) was adequate (agreement: 74%, j = 0.34, P < 0.001), and significantly better than for parenchymal echo alone. Role of liver ultrasound in patients with NASH
In Table 2, we have also summarized the correlations between changes in histological grades/stages over time and changes in the scores of the echographic parameters. As can be appreciated, after adjustment for steatosis grade, none of the key histological characteristics of NASH (inflammation, ballooning and fibrosis) were correlated with any of the echographic parameters, or the overall echographic fatty liver score. To test whether the presence of advanced fibrosis could affect the sensitivity of the ultrasound in the diagnosis of NAFLD, patients were divided according to their fibrosis stage into those with no or minimal fibrosis (stages 0 and 1) and those with advanced fibrosis (stages ≥3). No significant difference was observed in the sensitivity of the US fatty score in patients with advanced fibrosis when compared to those without advanced fibrosis (71% vs. 60%, P = 0.39), or if patients with fibrosis stages 0–1 were compared to patients with fibrosis stages 2–4 (71% vs. 64%, P = 0.50). Most importantly, changes in the echographic fatty liver score failed to predict changes in other histological parameters, such as inflammation, ballooning and fibrosis. Prospective use of liver US for the follow-up of patients with NAFLD
Sixty-two patients in this report completed 18 months of follow-up, and underwent a second liver US, 1HMRS and biopsy. This was performed to assess if liver US could eventually be used to predict changes over time in hepatic steatosis. As can be observed in Table 3, changes in parenchymal echogenicity alone were not correlated with changes in liver fat by 1H-MRS or DFQ, and only modestly correlated with changes in steatosis Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
grade. On the contrary, the use of changes in the echographic fatty liver score after 18 months allowed to predict changes in liver fat accumulation measured either by steatosis grade (rs = 0.54, P < 0.0001), digital fat quantification (rs = 0.45, P = 0.003) or 1H-MRS (rs = 0.48, P = 0.002). These correlations were somewhat stronger than when parenchymal echogenicity was taken into account individually, but still failed to provide a robust test for monitoring changes during therapy. In the best scenario, changes in US fatty liver score had a sensitivity of 70% and a specificity of 79% to detect any improvement in steatosis grade by histology. Role of 1H-MRS in the diagnosis of NAFLD
To validate liver fat quantification by 1H-MRS, we compared liver fat content measured by 1H-MRS, and by digital fat quantification of the biopsy. Correlation between these two quantification tools was very robust (r = 0.73, P < 0.0001). Moreover, this was also the case when changes in liver triglyceride content by 1H-MRS were compared with changes assessed by digital fat quantification after 18 months of follow-up (r = 0.63, P < 0.0001). However, changes in liver fat by 1H-MRS failed to predict changes in histology (other than changes in steatosis grade). Discussion
To our knowledge, this is the largest study performing a three-way comparison between liver US, 1H-MRS and histology for the diagnosis of hepatic steatosis in overweight and obese patients with high risk of NAFLD. Our results suggest that the combination of different echographic parameters can improve the diagnostic performance of liver US for the diagnosis of NAFLD in overweight/obese patients. Of note, this improvement was more pronounced at lower levels of liver fat accumulation (Fig. 2). Optimum US sensitivity was achieved with a liver fat content ≥12.5%, suggesting that under this threshold liver US underperforms. Moreover,
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Table 3. Correlation analyses between changes in ultrasound parameters, and histological features.
ΔSteatosis grade ΔLiver fat by DFQ ΔLiver fat by 1H-MRS ΔPlasma ALT
ΔParenchymal echogenicity
ΔFar gain attenuation
ΔGB wall blurring
ΔPortal vein blurring
ΔHepatic vein blurring
ΔSteatosis score
0.42† NS NS 0.28*
NS NS NS NS
0.52† 0.50* NS NS
0.33* 0.38* NS NS
0.51† 0.47† 0.58† NS
0.54† 0.45† 0.48† NS
DFQ, digital fat quantification; GB, gallbladder; Δ: change after 18 months of follow-up for each parameter. *P < 0.05; †P < 0.01.
relying on different echographic parameters, liver US also improved its ability to detect changes in steatosis over time. Several methodological characteristics make this study unique. Although several investigators have assessed the performance of liver ultrasound using liver histology as the gold standard (6), limited data exist regarding the performance of different individual ultrasound parameters, as most studies have relied only on parenchymal echogenicity for the diagnosis of steatosis. In addition, no previous study has assessed if semiquantitative ultrasonographic scoring systems could be used to follow changes over time in patients with NAFLD. Also, only few small studies have simultaneously compared liver US and 1H-MRS for the diagnosis of NAFLD against the gold standard liver histology (19, 20). A relatively larger study by Lee et al. (21) was performed, but it mainly included young (mean 32.2 years) and lean (mean BMI: 23.4 kg/m2) subjects from the general population who were donor candidates for living liver transplantation. Finally, there is limited information on the use of digital fat quantification in patients with NAFLD (22). Taken together, our results highlight the limitations of liver US when relying only on parenchymal echogenicity, while suggesting that the combination of different echographic parameters may somewhat improve the performance of the test. Also, we have provided strong validation for the use of 1H-MRS for the diagnosis of NAFLD in overweight and obese patients. The main shortcoming of the liver US was its low sensitivity to detect patients with NAFLD (only 70% [61–77%]), particularly if liver fat accumulation was mild. In a meta-analysis by Hernaez et al. (6) including a total of 4720 patients, overall sensitivity of liver US was significantly higher: 85% (80–89%). However, this was the sensitivity to distinguish moderate-to-severe fatty liver from the absence of steatosis, not taking into account mild-to-moderate cases of NAFLD. Unfortunately, a significant proportion of patients with NAFLD usually have only mild to moderate liver fat, emphasizing the importance of diagnostic tools that can indeed detect these patients. Whether the US can detect small amounts of liver fat has been a topic of extensive discussion. Ryan et al. (7) showed that liver US sensitivity was as low as ~5% in patients with 5–9% of liver fat by histology and that it improved to ~80% in patients with ≥30% of liver fat. In
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a similar way, when we calculated sensitivities for different cut-off points of liver fat (by 1H-MRS), we found that sensitivity improved with higher cut-off points of liver fat until it reached a plateau with liver fat ≥12.5% (sensitivities of ~80–85%; Fig. 2). Technological improvements in ultrasound equipment may explain this relatively lower threshold for liver fat (when compared with 30% as reported by Ryan et al.), and bring hope for further improvements in the near future. In a recent study by Lin et al. (23) assessing a new quantitative ultrasound technique, results were encouraging. However, magnetic resonance imaging-estimated proton density fat fraction was used as reference as biopsies were not available. When the performance of parenchymal echogenicity alone vs. the combination of the five parameters was assessed, we found that sensitivities were somewhat lower for parenchymal echogenicity alone (P ≤ 0.02 for all comparisons when liver fat content was ≤10%). This finding suggests that the use of a combined echographic scoring system may in fact improve the performance of the liver US for the diagnosis of hepatic steatosis in patients with mild elevations in liver fat content. Perhaps the most significant advantage of using a semiquantitative US scoring system, instead of relying only in parenchymal echogenicity, consists in the possibility of better predicting changes in steatosis over time, as can be appreciated in Table 3, where gallbladder wall blurring, portal vein wall blurring and hepatic vein blurring performed better. However, the overall performance of liver US to follow steatosis changes over time, as a stand-alone test, was rather weak with a sensitivity of 70% (49.8–86.2%) and a specificity of 79% (57.8– 92.9%). Our results confirm and extend prior work (24, 25): although liver US is widely used in the clinical setting because of simplicity, low cost and wide availability, one must keep in mind that its overall performance is rather suboptimal. Several studies have compared liver 1H-MRS to liver histology (20, 26–28). However, most of these studies were relatively small (n = 33–52) and only compared 1 H-MRS to liver fat assessed by manual (histological) reading, which has been questioned because of interobserver variability (29). Recently, magnetic resonance imaging-estimated proton density fat fraction (MRIPDFF) has been suggested as a very promising technique for the diagnosis of NAFLD (30). Other novel techLiver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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niques based on magnetic resonance imaging, such as magnetic resonance elastography, also hold promise to help physicians identify those patients with more advanced disease (31). Future studies will likely validate their use for the diagnosis and monitoring of patients with NAFLD. It has been suggested that liver US could potentially help in the diagnosis of NASH, as there might be some echographic changes secondary to the development of NASH (32, 33). In our hands, after adjusting for liver steatosis, no significant association was found between ultrasonographic parameters and histological components of NASH (Table 2), and therefore liver US should not be recommended to this end. Moreover, the presence of fibrosis did not affect the performance of liver US to diagnose hepatic steatosis. Finally, our study was performed largely in a high-risk population for NAFLD (mainly overweight or obese patients, and with a high prevalence of T2DM and of elevated plasma ALT). Although we did not find any differences in the sensitivity of the ultrasonographic score to diagnose NAFLD in obese vs. overweight patients (P = 0.72), it is possible that its performance could be different in lean subjects. Therefore, findings should not be directly extrapolated to other populations with a lower risk of NAFLD, or to screening studies for populations at large. In summary, liver US has significant limitations for the diagnosis and monitoring of changes over time of hepatic steatosis in patients with NAFLD, and they should be kept in mind in clinical practice. The use of a semiquantitative ultrasonographic score assessing five different parameters can, in fact, improve the performance of the liver US for the diagnosis and follow-up of patients with NAFLD. However, it is more cumbersome and time-consuming for clinical practice. Liver 1H-MRS has shown to be strongly correlated with hepatic steatosis measured on liver biopsies, and it is the technique of choice to follow changes in steatosis over time. However, none of the imaging techniques could predict changes over time in inflammation, ballooning or fibrosis, implying that liver biopsy remains the gold standard for following patients with NASH. Acknowledgements
Financial support: Supported by the Burroughs Wellcome Fund (K. C.), the American Diabetes Association (1-08-CR-08 [K. C.]) and a VA Merit Award (1 I01 CX000167-01 [K. C.]). Conflicts of interest: The authors do not have any disclosures to report. References 1. Browning JD, Szczepaniak LS, Dobbins R, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004; 40: 1387–95.
Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Diagnostic role of imaging in NAFLD
2. Szczepaniak LS, Nurenberg P, Leonard D, et al. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population. Am J Physiol Endocrinol Metab 2005; 288: E462–8. 3. Loomba R, Sanyal AJ. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol 2013; 10: 686–90. 4. Lopez-Velazquez JA, Silva-Vidal KV, Ponciano-Rodriguez G, et al. The prevalence of nonalcoholic fatty liver disease in the Americas. Ann Hepatol 2014; 13: 166–78. 5. Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology 2012; 142: 711–25. e716. 6. Hernaez R, Lazo M, Bonekamp S, et al. Diagnostic accuracy and reliability of ultrasonography for the detection of fatty liver: a meta-analysis. Hepatology 2011; 54: 1082–90. 7. Ryan CK, Johnson LA, Germin BI, Marcos A. One hundred consecutive hepatic biopsies in the workup of living donors for right lobe liver transplantation. Liver Transpl 2002; 8: 1114–22. 8. Liang RJ, Wang HH, Lee WJ, et al. Diagnostic value of ultrasonographic examination for nonalcoholic steatohepatitis in morbidly obese patients undergoing laparoscopic bariatric surgery. Obes Surg 2007; 17: 45–56. 9. Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 2012; 142: 1592–609. 10. Lomonaco R, Ortiz-Lopez C, Orsak B, et al. Role of ethnicity in overweight and obese patients with nonalcoholic steatohepatitis. Hepatology 2011; 54: 837–45. 11. Bril F, Maximos M, Portillo-Sanchez P, et al. Relationship of vitamin D with insulin resistance and disease severity in non-alcoholic steatohepatitis. J Hepatol 2015; 62: 405–11. 12. Lomonaco R, Ortiz-Lopez C, Orsak B, et al. Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease. Hepatology 2012; 55: 1389–97. 13. Ortiz-Lopez C, Lomonaco R, Orsak B, et al. Prevalence of prediabetes and diabetes and metabolic profile of patients with nonalcoholic fatty liver disease (NAFLD). Diabetes Care 2012; 35: 873–8. 14. Bril F, Lomonaco R, Orsak B, et al. Relationship between disease severity, hyperinsulinemia, and impaired insulin clearance in patients with nonalcoholic steatohepatitis. Hepatology 2014; 59: 2178–87. 15. Cusi K, Orsak B, Lomonaco R, et al. Extended treatment with pioglitazone improves liver histology in patients with prediabetes or type 2 diabetes mellitus and NASH. Hepatology 2013; 58: 248a. 16. Perman WH, Balci NC, Akduman I. Review of magnetic resonance spectroscopy in the liver and the pancreas. Top Magn Reson Imaging 2009; 20: 89–97. 17. Sanyal AJ, Brunt EM, Kleiner DE, et al. Endpoints and clinical trial design for nonalcoholic steatohepatitis. Hepatology 2011; 54: 344–53. 18. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 1988; 44: 837–45.
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19. Edens MA, van Ooijen PM, Post WJ, et al. Ultrasonography to quantify hepatic fat content: validation by 1H magnetic resonance spectroscopy. Obesity (Silver Spring) 2009; 17: 2239–44. 20. van Werven JR, Marsman HA, Nederveen AJ, et al. Assessment of hepatic steatosis in patients undergoing liver resection: comparison of US, CT, T1-weighted dual-echo MR imaging, and point-resolved 1H MR spectroscopy. Radiology 2010; 256: 159–68. 21. Lee SS, Park SH, Kim HJ, et al. Non-invasive assessment of hepatic steatosis: prospective comparison of the accuracy of imaging examinations. J Hepatol 2010; 52: 579–85. 22. Turlin B, Ramm GA, Purdie DM, et al. Assessment of hepatic steatosis: comparison of quantitative and semiquantitative methods in 108 liver biopsies. Liver Int 2009; 29: 530–5. 23. Lin SC, Heba E, Wolfson T, et al. Noninvasive Diagnosis of Nonalcoholic Fatty Liver Disease and Quantification of Liver Fat Using a New Quantitative Ultrasound Technique. Clin Gastroenterol Hepatol 2015; 13: 1337–45. 24. Dasarathy S, Dasarathy J, Khiyami A, et al. Validity of real time ultrasound in the diagnosis of hepatic steatosis: a prospective study. J Hepatol 2009; 51: 1061–7. 25. Hepburn MJ, Vos JA, Fillman EP, Lawitz EJ. The accuracy of the report of hepatic steatosis on ultrasonography in patients infected with hepatitis C in a clinical setting: a retrospective observational study. BMC Gastroenterol 2005; 5: 14. 26. Noworolski SM, Lam MM, Merriman RB, Ferrell L, Qayyum A. Liver steatosis: concordance of MR imaging and MR spectroscopic data with histologic grade. Radiology 2012; 264: 88–96.
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27. Georgoff P, Thomasson D, Louie A, et al. Hydrogen-1 MR spectroscopy for measurement and diagnosis of hepatic steatosis. AJR Am J Roentgenol 2012; 199: 2–7. 28. Urdzik J, Bjerner T, Wanders A, et al. The value of preoperative magnetic resonance spectroscopy in the assessment of steatohepatitis in patients with colorectal liver metastasis. J Hepatol 2012; 56: 640–6. 29. Ratziu V, Charlotte F, Heurtier A, et al. Sampling variability of liver biopsy in nonalcoholic fatty liver disease. Gastroenterology 2005; 128: 1898–906. 30. Noureddin M, Lam J, Peterson MR, et al. Utility of magnetic resonance imaging versus histology for quantifying changes in liver fat in nonalcoholic fatty liver disease trials. Hepatology 2013; 58: 1930–40. 31. Loomba R, Lam J, Wolfson T, et al. Magnetic resonance elastography accurately predicts advanced fibrosis in NAFLD: a pilot study with paired-liver biopsy. Gastroenterology 2013; 144: S-1012. 32. Zardi EM, De Sio I, Ghittoni G, et al. Which clinical and sonographic parameters may be useful to discriminate NASH from steatosis? J Clin Gastroenterol 2011; 45: 59–63. 33. Ballestri S, Lonardo A, Romagnoli D, et al. Ultrasonographic fatty liver indicator, a novel score which rules out NASH and is correlated with metabolic parameters in NAFLD. Liver Int 2012; 32: 1242–52.
Supporting information
Additional Supporting Information may be found in the online version of this article: Table S1. Ultrasonographic fatty score description.
Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NAFLD/NASH
Clinical value of liver ultrasound for the diagnosis of nonalcoholic fatty liver disease in overweight and obese patients Fernando Bril1,2, Carolina Ortiz-Lopez3, Romina Lomonaco1,2, Beverly Orsak3, Michael Freckleton4, Kedar Chintapalli4, Jean Hardies4, Song Lai5, Felipe Solano6, Fermin Tio6 and Kenneth Cusi1,2,3,7 1 2 3 4 5 6 7
Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL, USA Malcom Randall Veterans Administration Medical Center, Gainesville, FL, USA Division of Diabetes, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Radiology Department, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Clinical Translational Science Institute Human Imaging Core, McKnight Brain Institute, University of Florida, Gainesville, FL, USA Pathology Department, University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, USA Audie L. Murphy Veterans Administration Medical Center, San Antonio, TX, USA
Keywords hepatic steatosis – NAFLD – NASH – Obesity – steatohepatitis Abbreviations 1 H-MRS, magnetic resonance imaging and proton spectroscopy; ALT, alanine aminotransferase; AUROC, area under the ROC curve; BMI, body mass index; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OGTT, oral glucose tolerance test; ROC, receiver operating characteristic; T2DM, type 2 diabetes mellitus; US, ultrasound. Correspondence Kenneth Cusi, MD, FACP, FACE, Endocrinology, Diabetes and Metabolism Division University of Florida, 1600 SW Archer Road, room H-2. Gainesville, FL 32610, USA Tel: 352 273 8662 Fax: 352 273 7441 e-mail: [email protected] Received 17 January 2015 Accepted 23 March 2015
Abstract Background & Aims: Liver ultrasound (US) is usually used in the clinical setting for the diagnosis and follow-up of patients with nonalcoholic fatty liver disease (NAFLD). However, no large study has carefully assessed its performance using a semiquantitative ultrasonographic scoring system in overweight/obese patients, in comparison to magnetic resonance spectroscopy (1H-MRS) and histology. Methods: We recruited 146 patients and performed: a liver US using a 5-parameter scoring system, a liver 1H-MRS to quantify liver fat content, and a liver biopsy to assess histology. All measurements were repeated in a subgroup of patients (n = 62) after 18 months of follow-up. Results: The performance of liver US (parenchymal echo alone) was rather modest, and significantly worse than 1H-MRS (AUROC: 0.82 [0.69–0.94] vs. 0.96 [0.90–1.00]; P = 0.04). However, the AUROC improved when different echographic parameters were taken into account (AUROC: 0.89 [0.83–0.96], P = 0.15 against 1H-MRS). Optimum sensitivity for liver US was achieved at a liver fat content ≥12.5%, suggesting that below this threshold, liver US is less sensitive. Liver 1H-MRS showed a high accuracy for the diagnosis of NAFLD, and correlated strongly with histological steatosis (r = 0.73, P < 0.0001). None of the imaging tests was adequate enough to predict changes over time in histology. Conclusions: Despite its widespread use, liver US has several important limitations that healthcare providers should recognize, particularly because of its low sensitivity. Using a combination of echographic parameters, liver US showed a significant improvement in its diagnostic performance, but still was of limited value for monitoring treatment over time.
Handling Editor: Luca Valenti DOI:10.1111/liv.12840 Liver Int. 2015; 35: 2139–2146
As a consequence of the obesity epidemic, nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the USA (1, 2) and worldwide (3, 4). This condition has a broad spectrum of severity that ranges from benign liver fat accumulation (simple steatosis) to severe necroinflammation (nonalcoholic steatohepatitis, NASH), and eventually cirrhosis (5). It is currently estimated that ~60% of obese patients Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
have NAFLD by magnetic resonance imaging and proton spectroscopy (1H-MRS). However, the prevalence of NAFLD varies according to the diagnostic tool being used. Liver ultrasound (US) is widely used in the clinical setting to diagnose a fatty liver because of its low cost, simplicity and safety profile. However, its value for the diagnosis of hepatic steatosis has been rather conflicting
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Key points
Use of an ultrasonographic scoring system combining five echographic parameters improved the diagnostic performance of liver ultrasound for NAFLD, and its ability to assess changes over time. The optimum sensitivity of liver US for the diagnosis of NAFLD was reached with a liver fat content ≥12.5%. This ‘detection’ threshold is much better than commonly believed. Liver fat measured by 1H-MRS, and its changes over time, were strongly correlated with liver steatosis assessed by histology. Changes over time in steatosis did not predict changes in other histological parameters. Therefore, liver biopsy remains the gold standard to follow patients with NASH.
providing there was no evidence of any chronic liver disease other than NAFLD/NASH (i.e. hepatitis B or C, autoimmune hepatitis, hemochromatosis, Wilson’s disease, drug-induced, others). Patients were also excluded if they had a history of alcohol abuse (>30 g per day in men or >20 g per day in women) (9), type 1 diabetes mellitus, or a history of clinically significant renal, pulmonary or heart disease. A complete medical history, physical exam, routine blood and urine chemistries and electrocardiography were performed in all patients to confirm eligibility. This study was approved by the University IRB, and a written informed consent was obtained from each patient prior to participation. Some patients were included in previous reports on the role of ethnicity (10), plasma vitamin D (11) and insulin resistance (12–14) in the pathogenesis of NAFLD. Study design
with studies reporting a broad spectrum of sensitivities that ranged from 60 to 94% (6). In addition, the sensitivity of liver US is considered to be reduced in patients with liver fat 5.5% by 1H-MRS) and elevated plasma aminotransferases, or with normal aminotransferases but at high risk of NASH because of severe insulin resistance, obesity or with T2DM, were offered a percutaneous liver biopsy to establish the diagnosis of NASH. A subset of patients with biopsy-proven NASH (n = 62) were included in a randomized controlled trial assessing the effects of pioglitazone vs. placebo for 18 months (15). After therapy, these patients underwent a second liver US, 1H-MRS and a second liver biopsy. This information was used to assess the ability of liver ultrasound and 1H-MRS to detect changes in steatosis over time. Liver US examination
All liver ultrasounds (US) were performed the day of the liver biopsy after an overnight fast using a Phillips IU22 unit (Cleveland, OH, USA). Grey-scale images of the liver were obtained using a 2–5 MHz multifrequency transducer. Transverse and longitudinal images showing the inferior vena cava with hepatic vein confluence, porta hepatis and gallbladder were obtained. The field of view was adjusted to include the diaphragm on the longitudinal images. The gain settings, focal zones and field of view were adjusted as needed for individual studies. Two board certified radiologists were provided the ultrasound images for grading of hepatic steatosis. Images were reviewed by each radiologist independently from each other and in a blinded fashion without any clinical data available. Ultrasound parameters used to semiquantitatively assess fat scores were: parenchymal echogenicity of the liver, far gain attenuation in right intercostal view at posterior axilla line, gallbladder wall blurring in right sagittal subcostal view, portal vein blurring in right intercostal view at anterior axilla line, and hepatic vein blurring in right intercostal view at middle axilla line Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Bril et al.
(see Table S1 for details). Each parameter was scored with 0, 1 or 2 and the US fatty liver score was calculated as the sum of the scores of these five parameters (8). Measurement of liver fat content by 1H-MRS
For the measurement of hepatic fat content, localized proton nuclear magnetic resonance spectra of the liver were acquired using methodology previously described (10, 12, 14). Briefly, a 30 mm3 voxel was localized in three areas of the liver avoiding vessels and bile ducts. Liver fat content was calculated as fat fraction (area under the curve [AUC] fat peak/[AUC fat peak + water peak]) using commercial software (NUTS, Acorn NMR). Measurements were corrected for T1 and T2 relaxation using methods previously described (16). A liver fat content of >5.5% was considered diagnostic of NAFLD (1). Liver biopsy
An ultrasound-guided liver biopsy was performed in patients with a positive 1H-MRS when liver transaminases were elevated, and all other causes of liver disease were ruled out, and also in patients with normal liver transaminases if they had significant risk factors for the development of NASH such as T2DM, metabolic syndrome and/or severe insulin resistance. All biopsies were evaluated by the same pathologist that was unaware of the subjects’ identity or clinical information. Histological characteristics for the diagnosis of NASH were determined using standard criteria (17). Histological quantification of lipid droplets by Aperio ImageScope
The slides provided for this analysis were scanned using the Aperio ScanScope slide scanner & Aperio ScanScope Console v.10.2.0.0 at 209 magnification. The scans were maintained in Aperio’s Spectrum v.11.0.0.725, which is a database for the scans. They were viewed and prepared for analysis using Aperio ImageScope v.11.0.2.725. A total of 118 baseline images were collected plus 62 images after 18 months of follow-up. The algorithm that was used to determine the area and per cent area of the fat was the Microvessel Analysis v1, with the only changing variable being that of the Light Staining Threshold with values ranging from 125 to 215 (dark–light). Statistical analysis
Data were summarized in percentages for categorical variables and as mean ± standard error or median (interquartile range) for numeric variables. Categorical variables were compared performing Chi-square or Fisher’s Exact test. For comparisons between two groups, we performed Kruskal–Wallis or Student’s t-test for numeric variables depending on variables’ distribution. Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Diagnostic role of imaging in NAFLD
Comparisons among three or more groups were performed with ANOVA (Bonferroni method for post hoc testing) or Kruskal–Wallis test. Comparisons between AUROCs were performed using methodology described by DeLong et al. (18). Optimum cut-off points were calculated using Youden’s index, and inter-observer variations were compared with kappa coefficient. A twotailed P-value of less than 0.05 was considered to indicate statistical significance. Analyses were performed with Stata 11.1 (StataCorp LP, College Station, TX, USA). Results Patients’ characteristics
In Table 1, we have summarized patients’ demographical and clinical characteristics. As can be observed, this cohort consisted of predominantly obese patients (BMI: 34.1 ± 0.4 kg/m2), with a high prevalence of T2DM (54%), and elevated plasma alanine aminotransferase (64% had abnormal plasma alanine aminotransferase [ALT] defined as ≥40 IU/L). A total of 66% of the patients were diagnosed with NAFLD according to the ultrasonographic fatty liver score. Liver US performance for the diagnosis of NAFLD
In Fig. 1, we have plotted the ROC curves showing the performances of plasma ALT, liver ultrasound (based on parenchymal echo alone and on the ultrasonographic fatty liver score), and liver 1H-MRS for the diagnosis of hepatic steatosis when considering liver histology as the Table 1. Clinical and laboratory characteristics of patients All patients (n = 146) Age, years Gender (male), % Body mass index, kg/m2 Prevalence of obesity, % Total body fat, % Type 2 diabetes mellitus, % Fasting plasma glucose, mg/dL HbA1c, % Fasting plasma insulin, lIU/mL Fasting FFA, mM ALT, IU/mL AST, IU/mL Liver fat by 1H-MRS, % NAFLD activity score (NAS) Steatosis grade Inflammation grade Ballooning grade Fibrosis stage Patients with fibrosis stage 3–4, % Total cholesterol, mg/dL LDL-C, mg/dL Triglycerides, mg/dL HDL-C, mg/dL
50 ± 1 72% 34.1 ± 0.4 81% 33 ± 1 54% 121 ± 2 6.3 ± 0.1 14 ± 1 0.56 ± 0.02 61 ± 3 44 ± 2 16 ± 1 4.0 ± 0.1 1.7 ± 0.1 1.5 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 10% 178 ± 4 106 ± 3 152 (101–200) 37 ± 1
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gold standard (i.e. hepatic steatosis was defined as steatosis grade ≥1). As can be observed, liver ultrasound based on parenchymal echo alone had a modest performance for the diagnosis of NAFLD (AUROC: 0.82 [0.69–0.94]) and was considerably worse than liver 1HMRS (AUROC: 0.96 [0.90–1.00], P = 0.04). Of note, although it was better than plasma ALT (AUROC: 0.65 [0.41–0.89]), this difference did not reach statistical significance. Using the US fatty liver score (combination of five parameters), the performance of the test improved (shaded area in Fig. 1A) and became statistically indistinguishable from liver 1H-MRS (AUROC: 0.89 [0.83–0.96] vs. 0.96 [0.90–1.00], P = 0.15). The optimum cut-off point of the US fatty liver score for the diagnosis of hepatic steatosis in this population was 6 of 10. Applying this cut-off point, sensitivity was relatively low: 70% (61–77%) and specificity was 100% (63– 100%) to detect hepatic steatosis in the biopsy. However, this was still better than the performance of parenchymal echo alone: sensitivity of 63% (54–71%) and specificity of 88% (47–100%). In Fig. 2, we have summarized the sensitivity of the liver US scoring system (using its best cut-off point of 6) to detect different amounts of liver fat quantified by 1 H-MRS. As can be observed, the maximum sensitivity (plateau) was reached at ~12.5% of liver triglyceride content, showing worse performance below this threshold. Interestingly, when only parenchymal echogenicity was used as a criterion to diagnose hepatic steatosis, the plateau was reached at approximately the same amount of liver fat accumulation, but with overall lower sensitivity, especially at low thresholds of liver fat (P ≤ 0.02 for all comparisons using cut-off points of liver fat ≤10%). This suggests that using the scoring system improves sensitivity of liver US in patients with small elevations of liver fat content (from 5.5 to 10%). In Table 2, we report on general correlations between each echographic parameter and results of liver fat content measured by liver histology or 1H-MRS. As can be
Sensitivity (%)
Ultrasound fatty liver score
* *
80
* Parenchymal echogenicity alone
60 40 20
Plateau reached
0 0
5
10
15
20
25
30
Liver fat (%) Fig. 2. Sensitivity of the ultrasound fatty liver score and parenchymal echogenicity for the diagnosis of NAFLD using different cut-off points of liver fat content by 1H-MRS. *P ≤ 0.02 for comparisons of sensitivities between the ultrasound fatty liver score and parenchymal echogenicity alone.
observed, the echographic total score (sum of the five parameters) correlated well with the steatosis grade (rs = 0.49, P < 0.001), and with the amount of liver fat measured by digital fat quantification (DFQ) of liver tissue (rs = 0.57, P < 0.001) or by 1H-MRS (rs = 0.50, P < 0.001). No correlations were observed between echographic characteristics and histological parameters (inflammation, ballooning and fibrosis) after adjustment for steatosis grade, suggesting that liver US cannot differentiate between simple steatosis and NASH. As operator-dependency remains an important concern regarding liver ultrasound, inter-observer agreement was assessed between two independent examiners, for each of the 5 parameters used to score the ultrasounds (0, 1 or 2), as well as for the overall diagnosis of hepatic steatosis (from 0 to 10). The best inter-rater agreements were found for portal vein blurring (agreement: 79%, j = 0.45, P < 0.001), far field attenuation (agreement: 70%, j = 0.43, P < 0.001) and hepatic vein blurring (agreement: 65%, j = 0.36, P < 0.001). Parenchymal echo (agreement: 51%, j = 0.13, P = 0.04) and gallbladder wall blurring (agreement: 51%, j = 0.11, P = 0.07) P = 0.02
(B)
1
H-MRS
1.00
US
80
ALT
60
AUROCs
Sensitivity (%)
(A) 100
100
ECHO
40
0.50
20 0
0.00 0
20
40
60
80
100
ALT
ECHO
US
MRS
100 - Specificity (%) Fig. 1. (A) Receiver operating characteristic (ROC) curves describing the performance of plasma alanine aminotransferase (ALT), liver ultrasound by means of parenchymal echogenicity alone (ECHO), 5-parameter ultrasound fatty liver score (US), and liver 1H-MRS for the diagnosis of hepatic steatosis in predominantly obese patients with high risk of NAFLD. Liver histology was considered the gold standard. Shaded area represents the difference between using parenchymal echogenicity alone and the ultrasonographic scoring system. (B) AUROCs of different imaging techniques. P-value represents overall comparison of AUROCs.
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Table 2. Correlation analyses between ultrasound parameters and histological features
Severity of liver fat accumulation Steatosis grade % fat accumulation by DFQ % fat accumulation by 1H-MRS Severity of histology Inflammation grade‡ Ballooning grade‡ Fibrosis grade‡
Parenchymal echogenicity
Far gain attenuation
GB wall blurring
Portal vein blurring
Hepatic vein blurring
Steatosis score
0.44† 0.52† 0.43†
0.40† 0.43† 0.39†
0.41† 0.36* 0.43†
0.49† 0.50† 0.48†
0.52† 0.48† 0.45†
0.49† 0.57† 0.50†
0.10 0.08 0.11
0.17* 0.11 0.16
0.06 0.07 0.10
0.02 0.06 0.00
0.05 0.10 0.10
0.02 0.05 0.05
DFQ, digital fat quantification; GB, gallbladder. *P < 0.05 and †P < 0.0001. No significant differences were observed, when coefficients of parenchymal echogenicity were compared with the ones of the total score. ‡Adjusted for steatosis grade.
showed the worst agreement. Overall, the agreement for the diagnosis of NAFLD (US fatty liver score ≥6) was adequate (agreement: 74%, j = 0.34, P < 0.001), and significantly better than for parenchymal echo alone. Role of liver ultrasound in patients with NASH
In Table 2, we have also summarized the correlations between changes in histological grades/stages over time and changes in the scores of the echographic parameters. As can be appreciated, after adjustment for steatosis grade, none of the key histological characteristics of NASH (inflammation, ballooning and fibrosis) were correlated with any of the echographic parameters, or the overall echographic fatty liver score. To test whether the presence of advanced fibrosis could affect the sensitivity of the ultrasound in the diagnosis of NAFLD, patients were divided according to their fibrosis stage into those with no or minimal fibrosis (stages 0 and 1) and those with advanced fibrosis (stages ≥3). No significant difference was observed in the sensitivity of the US fatty score in patients with advanced fibrosis when compared to those without advanced fibrosis (71% vs. 60%, P = 0.39), or if patients with fibrosis stages 0–1 were compared to patients with fibrosis stages 2–4 (71% vs. 64%, P = 0.50). Most importantly, changes in the echographic fatty liver score failed to predict changes in other histological parameters, such as inflammation, ballooning and fibrosis. Prospective use of liver US for the follow-up of patients with NAFLD
Sixty-two patients in this report completed 18 months of follow-up, and underwent a second liver US, 1HMRS and biopsy. This was performed to assess if liver US could eventually be used to predict changes over time in hepatic steatosis. As can be observed in Table 3, changes in parenchymal echogenicity alone were not correlated with changes in liver fat by 1H-MRS or DFQ, and only modestly correlated with changes in steatosis Liver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
grade. On the contrary, the use of changes in the echographic fatty liver score after 18 months allowed to predict changes in liver fat accumulation measured either by steatosis grade (rs = 0.54, P < 0.0001), digital fat quantification (rs = 0.45, P = 0.003) or 1H-MRS (rs = 0.48, P = 0.002). These correlations were somewhat stronger than when parenchymal echogenicity was taken into account individually, but still failed to provide a robust test for monitoring changes during therapy. In the best scenario, changes in US fatty liver score had a sensitivity of 70% and a specificity of 79% to detect any improvement in steatosis grade by histology. Role of 1H-MRS in the diagnosis of NAFLD
To validate liver fat quantification by 1H-MRS, we compared liver fat content measured by 1H-MRS, and by digital fat quantification of the biopsy. Correlation between these two quantification tools was very robust (r = 0.73, P < 0.0001). Moreover, this was also the case when changes in liver triglyceride content by 1H-MRS were compared with changes assessed by digital fat quantification after 18 months of follow-up (r = 0.63, P < 0.0001). However, changes in liver fat by 1H-MRS failed to predict changes in histology (other than changes in steatosis grade). Discussion
To our knowledge, this is the largest study performing a three-way comparison between liver US, 1H-MRS and histology for the diagnosis of hepatic steatosis in overweight and obese patients with high risk of NAFLD. Our results suggest that the combination of different echographic parameters can improve the diagnostic performance of liver US for the diagnosis of NAFLD in overweight/obese patients. Of note, this improvement was more pronounced at lower levels of liver fat accumulation (Fig. 2). Optimum US sensitivity was achieved with a liver fat content ≥12.5%, suggesting that under this threshold liver US underperforms. Moreover,
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Table 3. Correlation analyses between changes in ultrasound parameters, and histological features.
ΔSteatosis grade ΔLiver fat by DFQ ΔLiver fat by 1H-MRS ΔPlasma ALT
ΔParenchymal echogenicity
ΔFar gain attenuation
ΔGB wall blurring
ΔPortal vein blurring
ΔHepatic vein blurring
ΔSteatosis score
0.42† NS NS 0.28*
NS NS NS NS
0.52† 0.50* NS NS
0.33* 0.38* NS NS
0.51† 0.47† 0.58† NS
0.54† 0.45† 0.48† NS
DFQ, digital fat quantification; GB, gallbladder; Δ: change after 18 months of follow-up for each parameter. *P < 0.05; †P < 0.01.
relying on different echographic parameters, liver US also improved its ability to detect changes in steatosis over time. Several methodological characteristics make this study unique. Although several investigators have assessed the performance of liver ultrasound using liver histology as the gold standard (6), limited data exist regarding the performance of different individual ultrasound parameters, as most studies have relied only on parenchymal echogenicity for the diagnosis of steatosis. In addition, no previous study has assessed if semiquantitative ultrasonographic scoring systems could be used to follow changes over time in patients with NAFLD. Also, only few small studies have simultaneously compared liver US and 1H-MRS for the diagnosis of NAFLD against the gold standard liver histology (19, 20). A relatively larger study by Lee et al. (21) was performed, but it mainly included young (mean 32.2 years) and lean (mean BMI: 23.4 kg/m2) subjects from the general population who were donor candidates for living liver transplantation. Finally, there is limited information on the use of digital fat quantification in patients with NAFLD (22). Taken together, our results highlight the limitations of liver US when relying only on parenchymal echogenicity, while suggesting that the combination of different echographic parameters may somewhat improve the performance of the test. Also, we have provided strong validation for the use of 1H-MRS for the diagnosis of NAFLD in overweight and obese patients. The main shortcoming of the liver US was its low sensitivity to detect patients with NAFLD (only 70% [61–77%]), particularly if liver fat accumulation was mild. In a meta-analysis by Hernaez et al. (6) including a total of 4720 patients, overall sensitivity of liver US was significantly higher: 85% (80–89%). However, this was the sensitivity to distinguish moderate-to-severe fatty liver from the absence of steatosis, not taking into account mild-to-moderate cases of NAFLD. Unfortunately, a significant proportion of patients with NAFLD usually have only mild to moderate liver fat, emphasizing the importance of diagnostic tools that can indeed detect these patients. Whether the US can detect small amounts of liver fat has been a topic of extensive discussion. Ryan et al. (7) showed that liver US sensitivity was as low as ~5% in patients with 5–9% of liver fat by histology and that it improved to ~80% in patients with ≥30% of liver fat. In
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a similar way, when we calculated sensitivities for different cut-off points of liver fat (by 1H-MRS), we found that sensitivity improved with higher cut-off points of liver fat until it reached a plateau with liver fat ≥12.5% (sensitivities of ~80–85%; Fig. 2). Technological improvements in ultrasound equipment may explain this relatively lower threshold for liver fat (when compared with 30% as reported by Ryan et al.), and bring hope for further improvements in the near future. In a recent study by Lin et al. (23) assessing a new quantitative ultrasound technique, results were encouraging. However, magnetic resonance imaging-estimated proton density fat fraction was used as reference as biopsies were not available. When the performance of parenchymal echogenicity alone vs. the combination of the five parameters was assessed, we found that sensitivities were somewhat lower for parenchymal echogenicity alone (P ≤ 0.02 for all comparisons when liver fat content was ≤10%). This finding suggests that the use of a combined echographic scoring system may in fact improve the performance of the liver US for the diagnosis of hepatic steatosis in patients with mild elevations in liver fat content. Perhaps the most significant advantage of using a semiquantitative US scoring system, instead of relying only in parenchymal echogenicity, consists in the possibility of better predicting changes in steatosis over time, as can be appreciated in Table 3, where gallbladder wall blurring, portal vein wall blurring and hepatic vein blurring performed better. However, the overall performance of liver US to follow steatosis changes over time, as a stand-alone test, was rather weak with a sensitivity of 70% (49.8–86.2%) and a specificity of 79% (57.8– 92.9%). Our results confirm and extend prior work (24, 25): although liver US is widely used in the clinical setting because of simplicity, low cost and wide availability, one must keep in mind that its overall performance is rather suboptimal. Several studies have compared liver 1H-MRS to liver histology (20, 26–28). However, most of these studies were relatively small (n = 33–52) and only compared 1 H-MRS to liver fat assessed by manual (histological) reading, which has been questioned because of interobserver variability (29). Recently, magnetic resonance imaging-estimated proton density fat fraction (MRIPDFF) has been suggested as a very promising technique for the diagnosis of NAFLD (30). Other novel techLiver International (2015) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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niques based on magnetic resonance imaging, such as magnetic resonance elastography, also hold promise to help physicians identify those patients with more advanced disease (31). Future studies will likely validate their use for the diagnosis and monitoring of patients with NAFLD. It has been suggested that liver US could potentially help in the diagnosis of NASH, as there might be some echographic changes secondary to the development of NASH (32, 33). In our hands, after adjusting for liver steatosis, no significant association was found between ultrasonographic parameters and histological components of NASH (Table 2), and therefore liver US should not be recommended to this end. Moreover, the presence of fibrosis did not affect the performance of liver US to diagnose hepatic steatosis. Finally, our study was performed largely in a high-risk population for NAFLD (mainly overweight or obese patients, and with a high prevalence of T2DM and of elevated plasma ALT). Although we did not find any differences in the sensitivity of the ultrasonographic score to diagnose NAFLD in obese vs. overweight patients (P = 0.72), it is possible that its performance could be different in lean subjects. Therefore, findings should not be directly extrapolated to other populations with a lower risk of NAFLD, or to screening studies for populations at large. In summary, liver US has significant limitations for the diagnosis and monitoring of changes over time of hepatic steatosis in patients with NAFLD, and they should be kept in mind in clinical practice. The use of a semiquantitative ultrasonographic score assessing five different parameters can, in fact, improve the performance of the liver US for the diagnosis and follow-up of patients with NAFLD. However, it is more cumbersome and time-consuming for clinical practice. Liver 1H-MRS has shown to be strongly correlated with hepatic steatosis measured on liver biopsies, and it is the technique of choice to follow changes in steatosis over time. However, none of the imaging techniques could predict changes over time in inflammation, ballooning or fibrosis, implying that liver biopsy remains the gold standard for following patients with NASH. Acknowledgements
Financial support: Supported by the Burroughs Wellcome Fund (K. C.), the American Diabetes Association (1-08-CR-08 [K. C.]) and a VA Merit Award (1 I01 CX000167-01 [K. C.]). Conflicts of interest: The authors do not have any disclosures to report. References 1. Browning JD, Szczepaniak LS, Dobbins R, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004; 40: 1387–95.
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Supporting information
Additional Supporting Information may be found in the online version of this article: Table S1. Ultrasonographic fatty score description.
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