291

Noninvasive Evaluation of Nonalcoholic Fatty

Liver Disease
Laurent Castera, MD, PhD1

1 Department of Hepatology, Hôpital Beaujon, Assistance Publique- Address for correspondence Laurent Castera, MD, PhD, Service
Hôpitaux de Paris, Université Paris-VII, Clichy, France d’Hépatologie, Hôpital Beaujon, 100 Boulevard du Général Leclerc,
92110 Clichy, France (e-mail: laurent.castera@bjn.aphp.fr).
Semin Liver Dis 2015;35:291–303.

Abstract Key issues in patients with nonalcoholic fatty liver disease (NAFLD) are the differentia-
tion of nonalcoholic steatohepatitis (NASH) from simple steatosis and staging of liver
fibrosis, as patients with NASH/advanced fibrosis are at greatest risk of developing
complications of end-stage liver disease. The controlled attenuation parameter is the
most promising noninvasive technique for detecting and quantifying hepatic steatosis,
Keywords but needs to be implemented with the XL probe and compared with ultrasound that,
► nonalcoholic fatty despite its limitations, remains the most widely used method. Cytokeratin-18 is
liver disease currently the most extensively validated serum marker of NASH as a stand-alone test
► noninvasive

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or as part of prediction models. However, it is not widely available and thus has not been
► steatosis introduced yet into practice. Transient elastography, as well as FIB-4 and NAFLD fibrosis
► nonalcoholic scores are the best methods to rule out severe fibrosis and cirrhosis. However, the high
steatohepatitis rate of unreliable results with transient elastography remains a challenge, which is not
► fibrosis completely addressed by the use of the XL probe. Given the high prevalence of NAFLD in
► serum markers the general population, these noninvasive methods could be used in clinical practice as
► transient first-line tools to screen patients with NAFLD to help determine those who may still
elastography require a liver biopsy.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause
of liver disease worldwide, affecting around one third of the
population in the Western world.1 However, the vast majority
will not progress; only a minority of patients, namely those
with nonalcoholic steatohepatitis (NASH), are at greatest risk
of developing complications of chronic liver disease, such as
cirrhosis, liver failure, and hepatocellular carcinoma. Thus,
key issues in patients with NAFLD are the differentiation of
NASH from simple steatosis and staging of hepatic fibrosis.
The diagnosis of NASH and the staging of fibrosis are essen-
tially based on histological examination of a liver specimen
obtained by liver biopsy.2 However, liver biopsy has well
known limitations (invasiveness and sampling variability)
and cannot be proposed to all patients, given the high
prevalence of NAFLD worldwide.3 Over the past decade, there
has been a growing interest for alternative novel noninvasive
strategies, relying on two different, but complementary
approaches: the assessment of serum biomarkers and the
measurement of liver stiffness using ultrasound- (US-) based
elastography techniques.4
The advantages and limitations of noninvasive methods for
the management of patients with NAFLD, including the
diagnosis and quantification of steatosis, the diagnosis of
NASH, and the staging of hepatic fibrosis are discussed in
this review.
Diagnosis and Quantification of Steatosis
Serum Biomarkers
Five indices have been developed for the diagnosis and
quantification of steatosis. They include the SteatoTest (Bio-
predictive, Paris, France), a proprietary formula based on the

Issue Theme Nonalcoholic Fatty Liver
Disease (NAFLD); Guest Editors,
Christopher P. Day, MD, PhD, FMedSci,
and Quentin M. Anstee, BSc, MBBS, PhD,
MRCP(UK)
Copyright © 2015 by Thieme Medical DOI http://dx.doi.org/
Publishers, Inc., 333 Seventh Avenue, 10.1055/s-0035-1562948.
New York, NY 10001, USA. ISSN 0272-8087.
Tel: +1(212) 584-4662.
292 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease
six variables included in the FibroTest-ActiTest (α2-macro-
globulin, haptoglobin, apolipoprotein A1, gamma-glutamyl
transpeptidase [GGT], total bilirubin, alanine transaminase
[ALT]; discussed in detail below) plus body mass index (BMI),
cholesterol, triglycerides, and glucose adjusted by age and
gender.5 A cutoff of 0.3 has a sensitivity of 85% or more to
make the diagnosis of fatty liver, and a cutoff of 0.7 has a
specificity of 80%.5 In a meta-analysis by the developer in 494
patients undergoing bariatric surgery, the SteatoTest had a
0.80 area under the receiver operating characteristic (AUROC)
for diagnosing steatosis > 30%.6 The fatty liver index (FLI)
includes four variables: BMI, waist circumference, triglycer-
ides, and GGT.7 The FLI can be calculated using a specific
formula with a score of 30 or less having a sensitivity of 87%,
and a score of 60 or more having a specificity of 86% in the
diagnosis of steatosis.7 The NAFLD liver fat score is based on
five variables: metabolic syndrome, type 2 diabetes, fasting
insulin, fasting aspartate aminotransferase (AST), and AST/
ALT ratio.8 The AUROC curve was 0.87 in the estimation group
and 0.86 in the validation group. A cutoff point of -0.640
predicted increased liver fat content with a sensititivity of
86% and specificity of 71%. Using the same variables, a liver fat
equation was created for prediction of the percentage of liver
fat. The lipid accumulation product or LAP includes three
variables, waist circumference, triglycerides, and gender.9
Another score is the hepatic steatosis index (HSI), which
includes three variables: AST/ALT ratio, BMI, and diabetes.
The HSI had an AUROC curve of 0.812.10 A cutoff point less
than 30 had sensitivity for steatosis diagnosis of 93% and a
cutoff point greater than 36 ruled out steatosis with a
specificity of 92%.
Although SteatoTest and FLI have been independently
validated,6,11–13 they cannot be compared for their diagnostic
performance as they have been validated against different
standards: liver biopsy, ultrasonography, or 1H-magnetic
resonance spectroscopy. Finally, these indices have not gained
much popularity and they may not add much to the informa-
tion provided by clinical, laboratory, and imaging studies
done routinely in patients with suspected NAFLD.
Controlled Attenuation Parameter
Recently, using transient elastography (TE; FibroScan, Echos-
ens, Paris, France), a novel parameter, the controlled attenu-
ation parameter (CAP) has been proposed for noninvasive
grading of hepatic steatosis. The CAP measures the degree of
US attenuation by hepatic fat at the center frequency of the
FibroScan M probe (3.5 MHz). Results are expressed as
decibels per meter (dB/m) and range from 100 to 400 dB/
m. In a preliminary study14 in 115 patients with various
chronic liver diseases using liver biopsy as reference, the CAP
was able to accurately detect steatosis
11%,
33%, and
66%
with AUROCs of 0.91, 0.95, and 0.89, respectively. Many
studies have confirmed these results since15–29 (►Table 1).
A recent meta-analysis, based on nine studies (n ¼ 1,771
patients), reported AUROCs of 0.85 (95% confidence interval
[CI] ¼ 0.81–88), 0.88 (95% CI ¼ 0.85–0.91), and 0.87 (95%
CI ¼ 0.84–0.90) for steatosis
11%,
33%, and
66%, respec-
tively.30 It should be stressed, however, that although CAP
Seminars in Liver Disease Vol. 35 No. 3/2015
Castera
was well correlated with pathological grades of steatosis in
these studies, the results overlapped between grades and CAP
showed poor accuracy for the differentiation of adjacent
grades of steatosis. Cutoffs were variable from one study to
another, but the cutoff associated with significant steatosis
(>33% of hepatocytes) was almost always > 250 dB. Also the
number of patients with NAFLD included in these studies was
too small (< 150) to draw any firm conclusions. Further
studies including large cohorts of patients with NAFLD are
now required to validate these results. Interestingly, in the
largest study to date, based on 5,323 examinations performed
on 4,451 patients with chronic liver disease,22 failure rate was
observed in 7.7% of cases, ranging from 0.5% in young men
with no sign of metabolic syndrome to 33% in elderly women
with diabetes and hypertension. Failure was significantly
associated with older age, BMI, presence of metabolic syn-
drome, and with female gender. These results are not sur-
prising considering that technical failures of liver stiffness
measurement with the standard M FibroScan probe that is
used for CAP measurement, occur more often in such pa-
tients.31 Very recently, a study from Hong Kong screening
1,918 diabetic patients for NAFLD using the CAP reported a
failure rate of 6.2%.32 Clearly, a CAP algorithm for the Fibro-
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Scan XL probe, specifically designed for the obese population,
would be welcome.33
When CAP performances were compared with serum
indices of steatosis (SteatoTest, FLI, and HSI), conflicting
results were observed.15,16,22,34 Also studies comparing
CAP with ultrasonography are inconclusive: Although in
one study the CAP was able to detect steatosis in patients
with normal US, suggesting that it might be more accurate,35
in another study both methods had a similar, good accuracy
for the diagnosis of steatosis.34 Even though its sensitivity is
reduced in the morbidly obese and when < 30% of the liver
parenchyma is infiltrated by fat, and its performance is
operator-dependent, ultrasonography is simple and remains
the most widely used method for first-line screening of
steatosis in clinical practice.36
Another important issue is the generalizability of the
findings of these studies, conducted in tertiary referral cen-
ters, to primary care populations, in which it is important to
detect NAFLD because it is an independent predictor of
cancer, cardiovascular disease, and mortality from any
cause.36 Studies targeting the general population are still
scarce, but recent data confirm that the CAP correlates with
the features of metabolic syndrome in this setting.34,35 Final-
ly, future studies should also focus on assessing whether the
CAP is able to accurately mirror changes in steatosis over time.
Diagnosis of Nonalcoholic Steatohepatitis
Serum Biomarkers
Cytokeratin- (CK-) 18 (commercially available assay), a mark-
er of apoptosis, is to date the serum marker of NASH that has
been the most widely investigated as a stand-alone test or as
part of prediction models. Cytokeratin-18 fragments come
from apoptosis of hepatocytes accomplished by the enzyme
caspase 3. In the original study,37 39 patients with suspected
 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease Castera 293

Table 1 Performance of controlled attenuation parameter (CAP) for grading steatosis

Study Patients Etiologies Steatosis Steatosis Cutoff AUC Se Sp

Total/NAFLD (n) grading (%) (dB/m) (%) (%)
Sasso et al, 201014 115/17 CLD
11% 58 238 0.91 91 81

33% 39 259 0.95 89 86

66% 8 292 0.89 100 78
de Ledinghen et al, 201215 112/28 CLD
11% 51 266 0.84 69 85

33% 31 311 0.86 57 94

66% 15 318 0.93 87 91
Myers et al, 201216 153/72 CLD
11% 75 283 0.81 76 79

33% 35 288 0.75 85 62

66% 10 283 0.70 94 47
Sasso et al, 201217 615/0 HCV
11% 31 222 0.80 76 71

33% 13 233 0.86 87 74

66% 1 290 0.88 78 93
Friedrich-Rust et al, 201218 46/46 NAFLD
11% 98    

33% 74 245 0.78 97 67

66% 46 301 0.72 76 68
Masaki et al, 201319 155/40 CLD
5% 35 232 0.88 87 77
Kumar et al, 201324 317/63 CLD
11%     

33% 70 258 0.79 78 76

66% 11 263 0.76 71 68

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De Ledinghen et al, 201422 440/ CLD
11% 51  0.79  

33% 32  0.84  

66% 15  0.84  
Chan et al, 201420 161/101 CLD
5% 97 263 0.97 92 94

33% 64 281 0.86 97 68

66% 14 283 0.75 100 53
Chon et al, 201428 135/56 CLD
5% 69 250 0.88 73 95

33% 25 299 0.89 82 86

66% 7 327 0.80 78 84
Wang et al, 201427 88/0 HBV
11% 54 219 0.71 70 72

33% 28 230 0.87 83 78

66% 9 283 0.97 100 97
Ferraoili et al, 201423 115/0 HCV/HBV
5% 53 219 0.76 91 52

33% 14 296 0.82 60 91

66% 4    
Jung et al, 201429 161/72 CLD
5% 74 250 0.86 69 93

33% 24 301 0.90 82 88

66% 4 325 0.74 50 81
Shen et al, 201426 151/51 CLD
5% 59 253 0.92 89 82

33% 30 285 0.92 93 83

66% 9 310 0.88 92 79

Abbreviations: AUC, area under the curve; CLD, chronic liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; NAFLD, nonalcoholic fatty liver
disease; Se, sensitivity; Sp, specificity.

NAFLD were included, and CK-18 plasma values of 395 U/L
had a pretty high AUROC with high sensitivity and specificity
to differentiate between patients with NASH and non-NASH.
Subsequently, a validation study was performed including
139 patients with liver biopsy-confirmed NAFLD.38 The
AUROC was 0.83 with a sensitivity of 75% and a specificity
of 81% for a CK-18 plasma value of approximately 250 U/L.
Many studies in small populations37,39–47 have shown similar
results. However, in a recent study in a large population of
NAFLD patients (318 and 199 with NASH), the CK-18 AUROC
to predict NASH was 0.65 with overall sensitivity and speci-
ficity of 58% and 68%.48 A meta-analysis, including 11 studies
(n ¼ 822 patients, 389 with NASH) suggested a modest
diagnostic accuracy of CK-18.49 In the pooled estimates of
diagnostic accuracy, the seven studies that used a single
“best” overall cutoff level showed 66% sensitivity and 82%
specificity. In the six studies using separate high sensitivity
and high specificity cutoffs, the pooled estimates were 82%
sensitivity, 65% specificity, and 58% sensitivity and 98%
specificity, respectively. There was considerable variability
in the suggested cutoffs and their respective diagnostic
accuracy among studies. In clinical practice, this makes
choosing which threshold to use very difficult. In summary,
this meta-analysis suggests plasma CK-18 levels may help in

Seminars in Liver Disease Vol. 35 No. 3/2015

294 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease Castera

distinguishing between simple steatosis from NASH, but the
test is far from perfect as indicated by the lower 95% CI in the
0.60 range for sensitivity and specificity.
Other biomarkers or predictive models combining clini-
cal and laboratory parameters have been proposed to make
the diagnosis of NASH. One is the NashTest (Biopredictive,
Paris, France), a proprietary formula that includes 12
variables and has an AUROC of 0.79.50 In a recent meta-
analysis from the developer, in 494 obese patients with a
prevalence of NASH of 17.2%, the weighted AUROC of the
NashTest was 0.84.6 Other predictive models include scores
combining other parameters with CK-18 such as the the
NASH diagnostics (CK18, adiponectin, and resistin),46,51
and the Nice model (ALT, CK-18, and the presence of
metabolic syndrome),52 or without CK-18 such as the
HAIR (hypertension, increased ALT, and IR), 53 the oxNASH
(13-hydroxyl-octadecadienoic acid/linoleic acid ratio, age,
BMI, and AST),54 the NASH score (PNPLA3 genotype, AST,
and fasting insulin) 55 and the Palekar’ score. 56 However,
most of these scores rely on small and highly selected
populations (morbidly obese patients) and have not been
externally validated.57
Imaging Techniques
Although there are numerous experimental studies that have
explored different imaging modalities to differentiate simple
steatosis from NASH (diffusion-weighted magnetic resonance
imaging [MRI], MR elastography, TE, computed tomography
[CT] perfusion), there are too few studies in humans to draw
any conclusions.58
Diagnosis and Staging of Hepatic Fibrosis
Serum Biomarkers
Among the available serum biomarkers, some are specific
such as the BARD score and the NAFLD fibrosis score (NFS),
whereas others are not specific and mostly come from the
hepatitis C virus (HCV) field such as AST/ALT ratio, APRI, FIB-4,
FibroTest, Fibrometer NAFLD, Hepacore, and enhanced liver
fibrosis panel (ELF) score.
As shown in ►Table 2, the NFS was created using six
variables that were significantly associated in multivariate
analysis with severe fibrosis-cirrhosis in an international
multicenter study including 733 patients (estimation group
480; validation group 253) with liver-biopsy confirmed

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Table 2 Performance of serum biomarkers of fibrosis for severe fibrosis-cirrhosis

Predictive score Patients (n) Variables/formula Cutoff AUC Se Sp

(%) (%)
Specific for NAFLD 733 1.675 þ 0.037  age [y] þ 0.094  1.455 0.88 82 77
NAFLD fibrosis score59  BMI [kg/m2] þ 1.13  IFG/
0.676 51 98
diabetes [yes ¼ 1, no ¼ 0] þ 0.99
 AST/ALT ratio  0.013  platelet
count [109/L]  0.66  albumin
[g/dL]
BARD score62 827 BMI
28 ¼ 1 < 2 points  62 66
AST/ALT ratio
0.8 ¼ 2
Diabetes ¼ 1
Score
2, odds ratio for adv.
fibrosis ¼ 17
Not specific 541 Age [y]  AST [U/L] / platelet  1.30 0.80 74 71
FIB-4 score66 [109/L]  ALT [U/L]
2.67 33 98
FibroMeter NAFLD69 235 Patented formula using 0.94  
0.4184 glucose (mmol/L) þ 0.0701
AST (IU/L) þ 0.0008 ferritin (μg/L)
0.0102 platelet (G/L)  0.0260 ALT
(IU/L) þ 0.0459 body weight (kg)
þ 0.0842 age (y) þ 11.6226
Hepascore71 242 Exp[4.185818 – (0.0249 age) 0.37 0.81 75 84
þ (0.7464 sex) þ (1.0039 α2-mac-
roglobulin) þ (0.0302 hyaluronic
acid) þ (0.069 bilirubin) – (0.0012
GGT)
FibroTest68 267 Patented formula 0.3 0.88 92 71
0.7 25 97
ELF74 192 ¼ 7.412 þ (ln(HA)  0.681) þ 0.1068 0.93 90 75
(ln(P3NP)  0.775) þ (ln(TIMP1) 
0.3576 80 90
0.494)

Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; AUC, area under the curve; BMI, body mass index; ELF, enhanced liver
fibrosis panel; GGT, gamma-glutamyl transpeptidase; IFG, impaired fasting glucose; NAFLD, nonalcoholic fatty liver disease; Se, sensitivity; Sp,
specificity.

Seminars in Liver Disease Vol. 35 No. 3/2015

 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease
NAFLD.59 The AUROC curve for this score to distinguish
between patients with and without advanced fibrosis was
high, 0.88 in the estimation group and 0.82 in the validation
group. A score less than 1.455 had high accuracy in exclud-
ing advanced fibrosis with a negative predictive value (NPV)
of 93 and 88% in the training and validation groups, respec-
tively; whereas a score greater than 0.676 had high accuracy
in identifying advanced fibrosis with a positive predictive
value (PPV) of 90 and 92%, respectively, in the training and
validation groups. If the NFS had been applied to the entire
cohort of 733 patients, the liver biopsy for fibrosis staging
could have been avoided in 75% of patients—of those correctly
identified—and performed in only the 25% of patients that fell
in the indeterminate range. Several studies of independent
populations have since reproduced the high accuracy of the
NFS in distinguishing patients with and without advanced
fibrosis.60,61 The BARD score was created by analyzing data
collected retrospectively from a group of 827 patients with
NAFLD.62 Based on logistic regression analysis, the BARD
score included a combination of three variables (►Table 2).
They reported a score of 2 to 4 associated with an odds ratio
(OR) of 17 for advanced fibrosis. A BARD score of 2 to 4 was
associated with an OR for advanced fibrosis of 17.3 and a NPV
of 97%. The BARD score has since been cross-validated in a
Polish population of 104 patients with NAFLD.63
Among the nonspecific tests, simple laboratory ratios
such as the AST/ALT ratio and the APRI have been proposed.
The AST/ALT ratio is typically < 1 in patients who have
NAFLD without advanced fibrosis, but tends to reverse as
the degree of fibrosis progresses to bridging fibrosis or
cirrhosis.64 The APRI has been validated in a cohort of 111
patients with NAFLD65 with an AUROC of 0.85 at an optimal
cutoff of 0.98, leading to a sensitivity and specificity of 75%
and 86%, respectively. The PPV of the APRI was only 54%, with
a NPV of 93%. The APRI may therefore be useful in identifying
patients unlikely to have advanced fibrosis, but is less useful
in predicting the presence of advanced fibrosis. The FIB-4
score was originally developed to predict advanced fibrosis
in patients coinfected with HCV and human immunodefi-
ciency virus (HIV). The FIB-4 score was validated in 541
patients with NAFLD with AUROC of 0.80.66 Using a cutoff
of
2.67, the PPV was 80% and the NPV was 83%. Using a
cutoff of less that 1.30, the PPV was only 43%, but the NPV
was 90%, suggesting that the FIB-4 index may be useful in
excluding patients without advanced fibrosis. The FIB-4
score has been cross-validated in a cohort of 576 Japanese
patients with biopsy proven NAFLD,67 although the cutoff
values used were different from those used in the original
study.66 In the Japanese study, the lower cutoff used was
< 1.45. Only 6 of 308 patients with a FIB4 index below the
proposed low cutoff point (< 1.45) were understaged, giving
a high NPV of 98%. Twenty-eight of 59 patients with a FIB4
index above the high cutoff point (> 3.25) were overstaged,
giving a low PPV of 53%. Using these cutoffs, 91% of the 395
patients with FIB-4 values outside 1.45 to 3.25 would be
correctly classified, and implementation of the FIB-4 index
in the Japanese population would be estimated to avoid 58%
of liver biopsies.
Castera 295
Among the patented tests, the FibroTest is one algorithm,
consisting of a combination of age, gender, bilirubin, g-
glutamyltransferase, apolipoprotein A1, haptoglobin, and
a2-macroglobulin. It has been validated in a variety of chronic
liver conditions and was examined in a cohort of 267 patients,
85% of whom had NAFLD.68 A score of < 0.3 (range ¼ 0.0–
1.0) provided a NPV of 98% for the presence of bridging
fibrosis or cirrhosis, whereas a score of > 0.7 provided a
60% PPV for bridging fibrosis or cirrhosis. However, 33% of
individuals had a score between 0.3 and 0.7, indicating that
the FibroTest cannot predict severity of liver fibrosis in one-
third of patients with NAFLD. The FibroMeter NAFLD (Echos-
ens, Paris, France) is a proprietary panel that was created
using a population of 235 patients with NAFLD.69 The AUROC
curve to predict advanced (stage 3–4) fibrosis was 0.94, which
was similar to an AUROC of 0.93 for the NFS, but significantly
better than the AUROC curve of 0.86 for the APRI.70 The
FibroMeter NAFLD was equally accurate as the NFS for the
prediction of cirrhotic stage, but better than the NFS for the
prediction of significant (stage 2–4) fibrosis. However, the
NFS was created to predict advanced (stage 3–4) fibrosis, not
significant (stage 2–4) fibrosis. Both the FibroTest and the
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FibroMeter NAFLD need appropriate validation by investiga-
tors others than those that created them. The Hepascore
includes the six variables described in ►Table 2.71 The AUROC
curve for prediction of advanced (stage 3–4) fibrosis was 0.81,
and it seemed to be more accurate than the BARD and APRI,
but similar to the FIB-4 score and FibroTest.72 The hepascore
seemed more accurate in the prediction of cirrhotic stage
disease than the other four scores.72 Finally, the European
Liver Fibrosis group assessed the combination of age and
serum levels of hyaluronic acid, aminoterminal propeptide of
type III collagen, and tissue inhibitor of matrix metallopro-
teinase 1 in predicting advanced fibrosis in patients who had
a wide range of liver diseases.73 The proposed algorithm had
an acceptable accuracy overall, but only 61 out of the 912
patients studied had NAFLD—a number too small to derive
meaningful conclusions about the NAFLD population. The
same group therefore evaluated the same three serum
markers: hyaluronic acid, aminoterminal propeptide of
type III collagen, and tissue inhibitor of matrix metallopro-
teinase 1 (named enhanced liver fibrosis panel [ELF]) in
predicting fibrosis in 192 patients with NAFLD.74 An ELF
score of 0.3576 had an AUROC curve of 0.93, and a sensitivity
of 80% for detecting advanced fibrosis and a specificity of 90%
in ruling out advanced fibrosis. The ELF was also evaluated in
112 children with NAFLD75; the AUROC curve to distinguish
among the stages of fibrosis varied from 0.90 to 0.99. In that
study, values of ELF from 9.28 to 10.51 had a sensitivity of 88
to 100% and a specificity of 76 to 98% to distinguish among the
stages.
In summary, among the different serum biomarkers stud-
ied in NAFLD, only NFS and FIB-4 have been externally
validated more than once, in different NAFLD populations
and with consistent results.58 These tests perform best at
excluding severe fibrosis-cirrhosis (with negative predictive
values > 90%) and could therefore be used as a first-line
triage to identify patients at low risk of severe fibrosis.76
Seminars in Liver Disease Vol. 35 No. 3/2015
296 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease Castera

Measurement of Liver Stiffness Using Transient
Elastography
Liver fibrosis can be staged using one-dimensional ultra-
sound TE (FibroScan), which measures the velocity of a
low-frequency (50 Hz) elastic shear wave propagating
through the liver.77 This velocity is directly related to tissue
stiffness, called the elastic modulus (expressed as E ¼ 3ρv2,
where v is the shear velocity, and ρ is the density of tissue,
assumed to be constant). The stiffer the tissue, the faster the
shear wave propagates. Transient elastography measures
liver stiffness in a volume that approximates a cylinder that
is 1 cm wide and 4 cm long, 25 to 65 mm below skin surface.
The results are expressed in kilopascals (kPa), corresponding
to the median value of 10 validated measurements and range
from 2.5 to 75 kPa, with normal values around 5.5 kPa.78
Advantages to TE include a short procedure time (< 5 min),
immediate results, and the ability to perform the test at the
bedside or in an outpatient clinic: It is not a difficult proce-
dure to learn. However, accurate results require careful
interpretation of data, based on at least 10 validated measure-
ments, a success rate (the ratio of valid measurements to the
total number of measurement) above 60%, and an interquar-
tile range (IQR; reflects variations among measurements)
of < 30% of the median value (IQR/M, < 30%).79
Transient elastography has been shown to be reliable in
the assessment of liver fibrosis initially in patients with
chronic hepatitis C with a strong correlation of liver stiffness
values with Metavir fibrosis stages and AUROCs ranging from
0.79 to 0.83 for the diagnosis of significant fibrosis and from
0.95 to 0.97 for cirrhosis.80 Transient elastography has also
been investigated in NAFLD patients, but in a smaller number
of studies (►Table 3).81–90 A recent meta-analysis, based on
nine studies including a total pool of 1,047 NAFLD patients
from different ethnic backgrounds, suggests that TE is excel-
lent in diagnosing severe fibrosis (85% sensitivity, 82% speci-
ficity) and cirrhosis (92% sensitivity, 92% specificity) and has
moderate accuracy for significant fibrosis (79% sensitivity,
75% specificity).49 However, these results deserve several
comments. First, some of these studies have been conducted
in heterogeneous or peculiar populations such as Asian

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Table 3 Performance of transient elastography for diagnosis of severe fibrosis-cirrhosis

Study Patientsa BMI (kg/m2) Histological Endpoints Cutoff AUC Se Sp Failure

(n) score (kPa) or unreliable
(%)
Yoneda et al, 200881 97 26.6  4.2 Brunt F
2 6.6 0.86 88 74 4.9
F
3 9.8 0.90 85 81
F4 17.5 0.99 100 97
Nobili et al, 200882 50 25.7  4.9 Brunt F
2 7.4 0.99 100 92 3.8
F
3 10.2 1.00 100 100
F4    
Wong et al, 201083 246 28.0  4.5 Kleiner F
2 7.0 0.84 79 76 10.2
F
3 8.7 0.93 84 83
F4 10.3 0.95 92 88
Lupsor et al, 201084 65 28.7a (21.0–41.5) Brunt F
2 6.8 0.79 67 84 9.7
F
3 10.4 0.98 100 97
F4    
Petta et al, 201185 146 29.1  4.1 Kleiner F
2 7.2 0.79 69 70 14
F
3 8.2 0.8- 76 78
F4   
Gaia et al, 201186 72 27.5a (21.1–40.4) Brunt F
2 7.0 0.80 76 80 8
F
3 8.0 0.75 65 80
F4 10.5 0.94 78 96
Myers et al, 201287 75 30.0a Kleiner F
2 7.8 0.86 84 79 50
(28.0–53.0) F
3  0.87  
F4 22.3 0.88 80 91
Wong et al, 201288 193 28.9  4.8 Kleiner F
2 7.0 0.83 79 64 33
F
3 8.7 0.87 83 78
F4 10.3 0.89 81 83
Kumar et al, 201390 120 26.1  3.6 Kleiner F
2 7.0 0.85 77 78 10
F
3 9.0 0.94 85 88
F4 11.8 0.96 90 88
Naveau et al, 201489 100 42.3  0.5 Kleiner F
2 7.6 0.81 73 78 21
F
3 7.6 0.85 100 74
F4    

Abbreviations: AUC, area under the curve; BMI, body mass index; Se, sensitivity; Sp, specificity.
a
Patients with reliable results.

Seminars in Liver Disease Vol. 35 No. 3/2015

 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease
patients or children with low BMI (< 28 kg/m2); second, most
of them are underpowered with small sample size (< 100
patients) and very few patients with cirrhosis; third, the
scoring systems (Brunt or Kleiner) and endpoints (significant
fibrosis or severe fibrosis) are heterogeneous with uneven
distribution of fibrosis stages, that is a likely explanation for
the observed differences between proposed cutoffs for a given
endpoint, known as the spectrum bias.91,92 Finally, it should
be stressed that all these studies have been conducted in
tertiary referral centers where the proportion of patients
with severe fibrosis is higher that in the general population,
thus making it difficult to extrapolate the performance of TE if
used to detect cirrhosis in large populations. Nevertheless, TE
could be of interest to exclude confidently severe fibrosis and
cirrhosis with high NPV (around 90%) in these patients.76
Such strategy has been used in a large Chinese diabetic
population (n ¼ 1918 patients) with increased liver stiffness
(> 9.5 kPa) suggestive of severe fibrosis-cirrhosis in around
18% of patients of whom a subset accepted to undergo liver
biopsy.32
The major challenge for the use of TE in NAFLD patients in
clinical practice is the high rate of failure (no valid shot) or
unreliable results (not meeting manufacturer’s recommen-
dations, i.e., valid shots < 10, success rate < 60%, or IQR/
LSM > 30%) in these patients, ranging from 3.8 to 50%
(►Table 3). Such a wide range may be explained by the
differences in the definitions used (failure vs. unreliable
results) as well as in the BMI of the studied populations
(higher rates in the populations with higher BMI). In the
largest series to date, on more than 13,000 examinations in
7,261 European patients seen over a 5-year period, failure to
obtain any measurement was observed in 4% of examinations
and unreliable results in 17%.31 Failure was independently
associated with BMI > 30 kg/m2 (OR ¼ 7.5; 95% CI ¼ 5.6–
10.2; P < 0.0001), operator experience fewer than 500 ex-
aminations (OR ¼ 2.5 [1.6–4.0]; P < 0.0001); age greater
than 52 years (OR ¼ 2.3 [1.6–3.2]; P < 0.0001), and type 2
diabetes (OR ¼ 1.6 [1.1–2.2]; P < 0.009). Unreliable results
were independently associated with BMI > 30 kg/m2 (OR
¼ 3.3 [2.8–4.0]; P < 0.0001), operator experience fewer than
500 examinations (OR ¼ 3.1 [2.4–3.9]; P < 0.0001), age
greater than 52 years (OR ¼ 1.8 [1.6–2.1]; P < 0.0001), fe-
male sex (OR ¼ 1.4 [1.2–1.6], P < 0.0001), hypertension (OR
¼ 1.3 [1.1–1.5]; P < 0.003), and type 2 diabetes (OR ¼ 1.2
[1.0–1.5]; P < 0.05). Similar results were reported in a large
series of Asian patients (n ¼ 3205) with failure and unreliable
results rates of 2.7% and 11.6%, respectively.93 These results
emphasize the need for adequate operator training and for
technological improvements in NAFLD patients.
A new probe (XL) has been proposed to overcome these
limitations for overweight and obese patients.87,88,94–96
Myers et al87 have shown in 276 patients with chronic liver
disease (42% viral hepatitis, 46% NAFLD) and a BMI >28 kg/
m2, that failure was significantly less frequent with the XL
probe than the M probe (1.1 vs. 16%; p < 0.00005). However,
unreliable results were still observed with the XL probe in
50% of cases instead of 73% with the M probe (p < 0.00005). In
a subgroup of 75 NAFLD patients with reliable results using
Castera 297
both probes and liver biopsy, diagnostic performances were
similar (AUROC for significant fibrosis, 0.90 vs. 0.86, and
cirrhosis, 0.95 vs. 0.88), for XL and M probes, respectively).
In a larger series of 193 NAFLD patients,88 the failure rate of XL
probe was significantly lower than that of the M probe (2% vs.
10%, respectively; p ¼ 0.002), but the unreliable result rate,
although lower, did not differ significantly (25% vs. 33%,
respectively; p ¼ 0.093). The overall diagnostic accuracy of
the M probe and the XL probe was similar with the AUROC of
the M and the XL probes of 0.83 and 0.80 for F2 or higher, 0.87
and 0.85 for F3 or higher, and 0.89 and 0.91 for F4 disease,
respectively. However, by intention-to-diagnose the perfor-
mance of the M probe was unsatisfactory due to the high rate
of unreliable results with the AUROCS of the M and the XL
probes of 0.56 and 0.74 for F2 or higher, 0.57 and 0.78 for F3 or
higher, and 0.53 and 0.86 for F4 disease, respectively. Among
155 patients with 10 valid measurements by both the M
probe and the XL probe, 124 (80%) patients had lower liver
stiffness values by the XL probe than the M probe. Pairwise
examination showed that liver stiffness measurement (LSM)
by the XL probe was 1.7  2.3 kPa lower than that by M probe
(95% CI ¼ 4–2.1 kPa; P < 0.001). Because of this phenome-
Downloaded by: NYU. Copyrighted material.
non, a separate set of diagnostic cutoff values for the XL probe
was required. The following cutoffs were proposed for XL and
M probes for F2 and greater (6.2 vs. 7.2 kPa), F3 and greater
(7.2 vs. 8.7 kPa) and F4 (7.9 vs. 10.3 kPa), respectively. When
compared with histology, overestimation of fibrosis staging
by the XL probe measurement was observed in patients with
BMI > 35 kg/m2. The authors recommend using the M probe
as the first line and to use the XL probe as salvage in the 30% of
patients in whom the M probe is not reliable.
The risk of overestimating liver stiffness values has been
reported with other confounding factors including ALT
flares,97–99 extrahepatic cholestasis,100 congestive heart fail-
ure,101 and more recently, food intake,102–104 suggesting that
TE should be performed in fasting patients and after having
excluded these factors.76 The influence of steatosis is still a
matter of debate with conflicting results: Some studies
suggest that steatosis is associated with an increase in liver
stiffness,86,105,106 whereas others do not.83,107
Altogether these results, suggest that TE could be of
interest to exclude confidently severe fibrosis and cirrhosis
in NAFLD patients, but the high rate of unreliable results
remains the main challenge for the use of TE. The XL probe
could be used as second line in the subset of patients (around
30%) in whom the regular probe (M) fails. However, appro-
priate cutoffs remain to be defined.
Other Imaging Techniques for the Measurement of
Liver Stiffness
Several other liver elasticity-based imaging techniques are
being developed, including (two-dimesional) acoustic radia-
tion force impulse imaging (ARFI) and three-dimensional MR
elastography. Acoustic radiation force impulse imaging in-
volves mechanical excitation of tissue using short-duration
(262 µs) acoustic pulses that propagate shear waves and
generate localized, microscale displacements in tissue.108 The
shear-wave velocity (expressed in m/s) is measured in a
Seminars in Liver Disease Vol. 35 No. 3/2015
298 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease
smaller region than in TE (10 mm long and 6 mm wide), but
can be chosen by the operator. The major advantage of ARFI is
that it can be easily implemented on a modified commercial
US machine (Acuson 2000 Virtual Touch Tissue Quantifica-
tion, Siemens Healthcare, Erlangen, Germany). Acoustic radi-
ation force impulse imaging has been investigated in NAFLD
in few studies,18,109–111 with better performances for severe
fibrosis and cirrhosis than for significant fibrosis (AUROCs
ranging from 0.91 to 0.98 and from 0.66 to 0.86, respectively).
In the largest study to date (n ¼ 172 patients),110 ARFI
showed good performance in differentiating between pa-
tients with severe fibrosis-cirrhosis and those with mild-
significant fibrosis. However, this study was performed on a
noncommercial prototype machine with results expressed
in kPa after reconstruction in 135 patients, thus making it
difficult to extrapolate its results to those obtained with a
commercial machine. Interestingly, 80% of patients with BMI
between 30 to 40 kg/m2 and 58% of patients with BMI > 40
kg/m2 could be successfully evaluated using ARFI.110
In the studies18,111 that compared ARFI and TE (n < 100
patients), the diagnostic performances of ARFI were similar to
those of TE. However, the feasibility of ARFI in obese patients
was better than that of TE with the M probe and similar to that
of TE with the XL probe. It should be stressed that quality
criteria for ARFI imaging have yet to be established; ARFI
values, in contrast to TE values, have a narrow range (0.5–4.4
m/s) and this may limit definitions of cutoff values for patient
management decisions.112 Therefore, larger prospective trials
Fig. 1. Algorithm for diagnosing noninvasively severe fibrosis-cirrhosis in patients with NAFLD. NAFLD, nonalcoholic fatty liver disease; NPV,
negative predictive value; PPV, positive predictive value.
Seminars in Liver Disease Vol. 35 No. 3/2015
Castera
are necessary to develop cutoff values for the staging of liver
fibrosis using these new methods.
Magnetic resonance elastography uses a modified phase-
contrast method to image the propagation characteristics of
the shear wave in the liver.113 Elasticity is quantified by MR
elastography (expressed in kPa) using a formula that deter-
mines the shear modulus, which is equivalent to one-third
the Young’s modulus used with TE.114 The theoretical advan-
tages of MR elastography include its ability to analyze almost
the entire liver and its applicability to patients with obesity or
ascites. Preliminary results suggest that MR elastography
could be of interest to detect advanced fibrosis in patients
with NAFLD.115,116 In a study in 117 American patients with
biopsy-proven NAFLD, MR elastography had an AUROC of 0.92
for diagnosing severe fibrosis-cirrhosis with a sensitivity of
0.86 and a specificity of 0.91, at a cutoff of 3.63 kPa.116 In
another study in 102 patients, MR elastography outper-
formed serum biomarkers such as NFS, BARD score, FIB-4,
and APRI.117 Further validation is required before any firm
conclusion can be drawn. Also it should be stressed that MR
elastography cannot be performed in livers of patients with
iron overload because of signal-to-noise limitations, and it is
Downloaded by: NYU. Copyrighted material.
too costly and time-consuming to use in routine practice.
Comparison and Combination of Approaches
Data comparing TE and serum biomarkers still remain limit-
ed. When compared with serum biomarkers (AST/ALT ratio,
 Noninvasive Evaluation of Nonalcoholic Fatty Liver Disease Castera 299

APRI, FIB-4, BARD score, and NFS), TE performed better for CAP controlled attenuation parameter
diagnosing severe fibrosis and cirrhosis.83,90,118 Strategies CI confidence interval
combining TE and serum biomarkers have been shown to CK-18 cytokeratin-18
increase diagnostic accuracy in patients with chronic hepati- ELF enhanced liver fibrosis panel
tis C.119,120 Very recently, a retrospective study in 321 Italian FIB-4 Fibrosis 4
patients (training cohort 179; validation cohort 142) sug- FLI fatty liver index
gested that the combination of TE and NFS, two complemen- GGT gamma-glutamyl transpeptidase
tary, easy to perform, and widely available tools, was able to HAIR hypertension increased ALT and insulin resistance
accurately diagnose or exclude the presence of severe liver HCV hepatitis C virus
fibrosis, also reducing by approximately 50 to 60% the num- HIS hepatic steatosis index
ber of needed diagnostic liver biopsies.118 Although such HIV human immunodeficiency virus
strategy is attractive, more data are needed before any IQR interquartile range
conclusions can be drawn. LSM liver stiffness measurement
NASH nonalcoholic steatohepatitis
NFALD nonalcoholic fatty liver disease
Conclusion
NFS NAFLD fibrosis score
Significant progress has been made over the past decade NPV negative predictive value
regarding the noninvasive evaluation of NAFLD. Regarding OR odds ratio
grading of steatosis, the CAP is the most promising technique, PPV positive predictive value
but needs to be to be implemented with the XL probe and TE transient elastography
compared with ultrasound that, despite its limitations, re-
mains the most widely used tool for steatosis assessment.

Downloaded by: NYU. Copyrighted material.

Regarding the diagnosis of NASH, in the absence of any
imaging technique, CK-18 is the most extensively validated
marker, but it has not gained much use in practice. Finally, TE,
as well as FIB-4 and NFSs are the best methods to rule out
severe fibrosis and cirrhosis. However, the high rate of
unreliable results with TE remains a challenge that is not
completely addressed by the use of the XL probe. The search
for the ideal noninvasive test has not been accomplished yet,
but we believe that noninvasive methods and liver biopsy
should be employed as an integrated system to allow a more
efficient and convenient management of patients with
NAFLD.121 Given the high prevalence of NAFLD in the general
population, these noninvasive methods could be used in
clinical practice as the first line tools to screen patients
with NAFLD and select those that may still require a liver
biopsy according to the proposed algorithm (►Fig. 1).
There is also increasing evidence for the prognostic value
of serum biomarkers and liver stiffness in patients with
chronic liver disease such as viral hepatitis and alcoholic liver
disease.122–127 Similarly, recent data indicate that noninva-
sive scoring systems including the NFS, the APRI score, and
the FIB-4 score accurately identify the subgroup of patients
with NAFLD at a higher risk to reach the outcome of liver-
related complications and death/liver transplantation.128,129
However, the role of transient elastography as a noninvasive
method to predict long-term outcomes has not been yet
determined in NALFD.
Abbreviations
ALT alanine transaminase
ARFI acoustic radiation force impulse imaging
AST aspartate aminotransferase
AUC area under the curve
AUROC area under the receiver operating characteristic
BMI body mass index
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