Review

Non-invasive assessment of non-alcoholic fatty liver disease:

Clinical prediction rules and blood-based biomarkers
Eduardo Vilar-Gomez⇑, Naga Chalasani⇑

Summary Keywords: NAFLD; NASH;

The correct identification of patients at increased risk of non-alcoholic steatohepatitis (NASH) and FIB-4; APRI.
advanced fibrosis is a critical step in the assessment of non-alcoholic fatty liver disease (NAFLD). Since
Received 1 November 2017;
liver biopsy is invasive, expensive and prone to sampling error, several clinical prediction rules and
received in revised form 3
blood-based biomarkers have been developed as attractive and affordable alternatives for identification November 2017; accepted 9
of patients at high risk of NASH and advanced fibrosis. Current biomarkers constitute predictive models November 2017
(e.g. NAFLD fibrosis score, FIB-4 index and BARD score) or direct measures of inflammation (e.g. circu-
lating keratin 18 fragments), or fibrosis (e.g. FibroTestÒ, ELFTM or Pro-C3 tests). In the clinical setting,
biomarkers may discriminate between patients with NASH or advanced fibrosis, predict dynamic
changes in NASH/fibrosis over time, and provide long-term prognostic information. Although clinically
useful, current biomarker predictions may be influenced by hepatic and extrahepatic conditions (e.g.
age, patient comorbidities, and fibrosis or NASH prevalence), which may lead to inaccurate estimates
in small subsamples of patients. No highly sensitive and specific tests are available to differentiate NASH
from simple steatosis. However, diagnostic accuracy can be improved by combining blood biomarkers.
NAFLD fibrosis score and FIB-4 index are both cost-effective and highly sensitive tools to exclude
patients with advanced fibrosis. Moreover, their higher scores may identify patients at higher risk of
non-liver- and liver-related morbidity and mortality. More expensive tests such as FibroTest or ELF
are more specific for detection of patients with significant and advanced fibrosis. Recent efforts have
concentrated on ‘‘omics” approaches for developing and validating novel biomarkers. Herein, we
describe currently available clinical prediction rules and blood-based biomarkers for identifying NASH
and advanced fibrosis in patients with NAFLD, discussing their advantages and disadvantages, as well
as their potential clinical utility for predicting dynamic changes over time and identifying patients at
increased risk of adverse outcomes.
Ó 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Prediction of NASH

Non-alcoholic fatty liver disease (NAFLD) is a Many biomarkers have been investigated to pre- Division of Gastroenterology and
Hepatology, Indiana University
leading cause of end-stage liver disease, hepatocel- dict NASH in patients with NAFLD. These range School of Medicine, IN, USA
lular carcinoma, and liver transplantation world- from clinical (age, gender, diabetes, body mass
wide.1–4 Non-alcoholic steatohepatitis (NASH) and index [BMI]), biochemical (aminotransferases,
most importantly fibrosis severity have been bilirubin and ferritin), metabolic (glycated hae- Key point
strongly implicated in the long-term prognosis of moglobin [HbA1c], insulin and HOMA-IR) and
Understanding the advan-
NAFLD patients.5–8 Thus, it is critical to identify lipid (triglycerides, cholesterol) parameters to tages and limitations of
patients at higher risk of NASH and advanced fibro- markers that reflect specific and complex molec- current biomarkers is
sis in order to optimise their management. Liver ular mechanisms underlying the pathogenesis imperative for selecting the
biopsy is still required to identify patients with and progression of NAFLD, including inflamma- appropriate biomarker in
the evaluation and
NASH and early fibrosis. However, its invasive nat- tion, oxidative stress, apoptosis, and glucose
management of NAFLD
ure, high cost and inter- and intra-observer vari- and lipid metabolism. As the pathogenesis of patients.
ability make it less suitable for diagnosis and NASH is complex and likely involves multiple
disease monitoring in clinical practice, and unsuit- biological aberrations, it is unlikely that a single
able for screening at a population level.9 During the biomarker could discriminate between simple
last decade, several non-invasive biomarkers have steatosis and NASH. Thus, most of current mod- Key point
been developed and validated to rule out patients els include at least two or more variables to add Because NASH and most
with NASH or advanced fibrosis. Thorough under- robustness to the non-invasive prediction importantly fibrosis sever-
standing of the advantages and limitations of cur- models. ity are critical determi-
rently available biomarkers is imperative for nants of long-term
prognosis of NAFLD, non-
selecting the appropriate biomarker for the evalua- Data from cross-sectional studies invasive biomarkers may
tion and management of patients with NAFLD. This Clinical prediction rules be useful to accurately
review article focusses on clinical prediction rules Several clinical and biochemical models have been distinguish patients at
and the blood-based biomarkers for non- evaluated to identify NASH in patients with higher risk of NASH and
invasively identifying NASH and advanced fibrosis advanced fibrosis.
NAFLD. Routinely tested blood parameters, such
in patients with NAFLD. as alanine aminotransferase (ALT) or aspartate

Journal of Hepatology 2017 vol. xxx j xxx–xxx

Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
Review
Key point
None of the currently
available tools differentiate
NASH from simple steato-
sis with high sensitivity
and specificity, however,
their diagnostic accuracy
can be improved by com-
bining blood biomarkers.
Key point
More recent efforts con-
centrating on ‘‘omics”
approaches have shown
promising results but are
not yet available for rou-
tine clinical practice.
⇑ Corresponding authors.
Address: 702 Rotary Circle,
Suite 225, Indiana University
School of Medicine,
Indianapolis, IN 46202, USA.
E-mail addresses: evilar@iu.edu
(E. Vilar-Gomez), nchalasa@iu.
edu (N. Chalasani).
2 Journal of Hepatology 2017 vol. xxx j xxx–xxx
Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
aminotransferase (AST), are associated with some
histological parameters such as inflammation
and steatosis. However, correlations appear to be
influenced by severity of disease.10 Aminotrans-
ferase levels do not correlate with the degree of
fibrosis.11 The new normal ALT cut-off values
(men, 30 U/L and women, 19 U/L) were found to
be associated with NASH, suggesting that their
adoption in clinical practice may identify patients
at risk of NASH.12,13 Many clinical features such as
diabetes, hypertension, BMI, age and gender have
been consistently associated with NASH. However,
they are inaccurate when used alone. Several clin-
ical prediction models, which combine clinical and
laboratory variables, have been published in the
literature.14–17 These models perform reasonably
well in predicting NASH, with an area under the
receiver operating characteristic curve (AUROC)
of 0.76–0.80, and some of them yield acceptable
negative predictive values (NPV) of 80–93%, when
using their lower cut-offs to exclude patients with
NASH.14–17
Blood-based models
Keratin 18 (K18) is a major intermediate filament
protein in hepatocytes and a prominent substrate
of caspase 3 cleavage that can be measured in
serum by immunoassay.18 Higher serum concen-
trations of K18 fragments have been detected in
patients with NASH than those with simple steato-
sis.19 Data from two recent meta-analyses have
reported a pooled AUROC of 0.82 (0.76–0.88) for
discriminating NASH patients with a median sen-
sitivity and specificity of 66%–78% and 82%–87%,
respectively.18,20 For detecting NASH, a wide range
of K18 cut-offs with their respective diagnostic
accuracy for M30 ELISA assays have been reported,
making it difficult to choose appropriate thresh-
olds for use in clinical practice. The M30 ELISA
assay measures the caspase-cleaved K18 frag-
ments and detects apoptosis, which is a hallmark
of steatohepatitis. The M65 ELISA assays detect
total cell death and interestingly have shown sim-
ilar diagnostic accuracy for detecting NASH
(pooled AUROC = 0.82). In one study, changes in
M65 were able to identify patients with NAFLD
and early fibrosis stages (stages 1 and 2), as well
as fibrosis progression.21 Other combinatorial
models which add K18 to soluble Fas,22 adipocy-
tokines,23 clinical (diabetes, gender, BMI) and rou-
tine blood-based parameters (ALT, platelets and
triglycerides)24,25 showed better NASH prediction
in patients with NAFLD. More recently, a model
combining K18 fragments with C-terminal cleav-
age site of procollagen type-III N-terminal peptide
(Pro-C3), acetyl-high mobility group box 1 and
patatin-like phospholipase domain-containing
protein 3 (PNPLA3) rs738409 has significantly
improved the accuracy of NASH diagnosis (AURO
C = 0.87, sensitivity: 71% and specificity: 87%) in
patients with NAFLD.26 However, this needs to
be validated and made commercially available
before it can be incorporated into clinical practice
(Fig. 1).
Adipocytokines are involved in the pathogene-
sis of NAFLD and have been strongly related to its
severity. Tight links between circulating levels of
adiponectin, leptin, ghrelin, interleukin-6 and
tumour necrosis factor-a, and insulin resistance,
diabetes, obesity and dyslipidaemia may explain
their potential role in the progression of NAFLD.
Machado et al. found that a panel including adipo-
nectin, leptin and ghrelin could differentiate
patients with NASH and simple steatosis with an
AUROC = 0.79.27
One of the more novel strategies to detect
patients at risk of NASH is to identify molecules
by OMICS approaches (genomic, epigenomic,
metabolomic, transcriptomic, proteomic) using
high-throughput technologies. OMIC approaches
result in the identification of a set of molecules
highly related to the disease. The discovery of
new molecules via OMICS provides a very useful
framework for designing and validating highly
accurate predictive models. Genomics, epige-
nomics, metabolomics, lipidomics and proteomics
are some of the new methods used to identify new
biomarkers able to distinguish between different
patterns of NAFLD severity. Using a metabolomic
approach in patients with NAFLD, a model com-
bining glucose, glutamate, taurine and lactate
levels identified different patterns of severity of
NAFLD.28 Loomba et al. reported that levels of
polyunsaturated fatty acid metabolites were dif-
ferent between patients with biopsy-proven sim-
ple steatosis vs. NASH, and that combining some
metabolites such as 11,12-diHETrE, dhk PGD2,
and 20-COOH, enabled excellent NASH prediction
(AUROC = 1), although this was a proof-of-
concept study and results require further valida-
tion.29 Puri et al. also reported a significant reduc-
tion in the lipogenic activity (docosahexanoic acid
[22:6 n3] to docosapentenoic acid [22:5n3] ratio
was significantly decreased within phosphatidyl-
choline, and phosphati-dylethanolamine pools),
while the lipoxygenase metabolites were mark-
edly increased in subjects with NASH.30 A recent
model including 28 serum lipid molecules (OWLi-
verÒ test) yielded high predictive capability for
detection of subjects with NASH (AUC = 0.87 and
0.85 in derivation and validation sets).31 Finally,
Zhou and colleagues have recently reported that
a model ‘‘NASH ClinLipMet Score” based on lipids,
metabolites, clinical markers and PNPLA genotype
identified patients with NASH, with an AUROC =
0.86.32
Data from longitudinal and interventional
studies
As a standalone measure, improvement in amino-
transferases is associated with improvement in
steatosis and inflammation over time.33 However,
it fails to accurately detect patients with overall
improvement in NAFLD activity score (NAS) or
 JOURNAL OF HEPATOLOGY

Exclude
Secondary causes of steatosis
Significant alcohol consumption
Obesity
Imaging evidence of
Type 2 diabetes Risk factors ALT/GGT abnormalities
fat accumulation
Metabolic syndrome

NAFLD

Rule out advanced fibrosis (F ≥3)

NAFLD fibrosis score or FIB-4 index

NFS : < -1.455 NFS : -1.455 –0.672 NFS : >0.672 or 0.12 if >65 y
FIB -4: <1.30 FIB -4: 1.30 –3.25 FIB -4: >3.25 or 2.0 if >65 y
NPV: 88 -95% PPV: 75 -90%

55-58%* 30%* 12-15%*

Low risk Intermediate risk High risk

Exclude F ≥3 Diagnose F ≥3
- +
Consider repeating FibroTest: 0.3 FibroTest: 0.7 Consider liver biopsy
NFS/FIB-4 every 2 years FibroMeter: 0.61 FibroMeter: 0.71
Hepascore: 0.37 Hepascore: 0.7
NPV >90% PPV : 60-80%

ELF test <10.35 ELF test >10.35

Imaging methods (VCTE, MRE)

Fig. 1. Algorithm to non-invasively assess patients with NAFLD and advanced fibrosis using prediction rules and blood-based biomarkers. *Estimated
prevalence for low, intermediate and high risks groups. ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; MRE, magnetic resonance
elastography; NAFLD, non-alcoholic fatty liver disease; NFS, NAFLD fibrosis score; NPV, negative predictive value; PPV, positive predictive value; VCTE,
vibration controlled transient elastography.

resolution of NASH after therapeutic interven-
tions.13,34 Dynamic changes in serum K18 frag-
ment levels over time and their association with
histological improvement have recently been
studied in 231 adults with NASH and 152 children
with NAFLD, who participated in two separate
prospective randomised clinical trials.34 Although
changes in serum K18 levels over time were clo-
sely linked to improved liver histology, these
changes did not perform better than changes in
ALT for detecting patients with overall histological
NAS improvement or resolution of NASH. More-
over, diagnostic accuracy was not improved by
adding changes in ALT to K18 levels.34 In the set-
ting of NASH and lifestyle interventions, a model
including age, diabetes, baseline NAS ≥5, weight
loss and normalisation of ALT (19 U/L and 30 U/L
for women and men, respectively) at end-of-
intervention accurately identified patients with
resolution of NASH (AUROC = 0.96 and 0.95 in
training and validation cohorts, respectively) after
52 weeks of diet and exercise. By using two cut-
offs to identify patients with a low and high prob-
ability of NASH resolution, 91% of patients were
correctly classified.13 Although clinically useful,
patients require a liver biopsy at baseline to com-
pute NAS, limiting its use in real-life clinical
practice.
Prediction of advanced fibrosis
Data from cross-sectional studies
Clinical prediction rules
Since fibrosis stage is a major determinant of all-
cause and liver-related mortality,5–8,35,36 non-
invasive assessment of fibrosis severity is crucial
in the management of patients with NAFLD. Sev-
eral non-invasive algorithms based on clinical
and biochemical variables have been developed
to detect individuals with NAFLD and advanced
fibrosis. However, major drawbacks include
reduced accuracy for detection of earlier fibrosis
stages and the high proportion of patients with
undetermined results (30%). These non-invasive
scoring systems, including NAFLD fibrosis score
(NFS),37 FIB-4 index,38 AST/platelet ratio (APRI)
index,39 and BARD score40 yield high NPV but poor
positive predictive values (PPV), suggesting that
they are best applied to exclude subjects without
advanced fibrosis, thereby avoiding unnecessary
liver biopsies. A recent meta-analysis compared
the diagnostic efficacy of NFS, FIB-4 index and

Journal of Hepatology 2017 vol. xxx j xxx–xxx 3

Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
Review
Key point
Although more expensive,
ELF test, FibroTest and
FibroMeter are better at
identifying patients with
significant and advanced
fibrosis. High scores of
non-invasive scores (NFS,
APRI, FIB-4) can be imple-
mented to predict long-
term adverse outcomes in
population-based
screening.
Key point
Although certain biomark-
ers appear to predict pro-
gression, improvement or
even development of clini-
cal outcomes in NAFLD
patients, future studies
should evaluate the diag-
nostic accuracy of direct
biomarkers of liver inflam
mation/fibrogenesis/fibri
nolysis as indicators of
efficacy of therapeutic
interventions.
4 Journal of Hepatology 2017 vol. xxx j xxx–xxx
Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
BARD score for detecting advanced fibrosis. By
using their low cut-offs to rule out advanced fibro-
sis, pooled AUROC were 0.84 and 0.85 for NFS
(cut-off = 1.455) and FIB-4 (cut-off = 1.30),
respectively. Whereas, using high thresholds to
diagnose advanced fibrosis, pooled AUROC were
0.65 and 0.84 for NFS (cut-off = 0.676) and FIB-4
(cut-off = 3.25). A BARD score of two showed
lower diagnostic accuracy than NFS and FIB-4
score (AUROC = 0.76).41 These results indicate that
both FIB-4 thresholds (1.30 and 3.25) have good
diagnostic accuracy to discriminate patients with
advanced fibrosis, whereas a 1.455 cut-off in
NFS may accurately exclude patients with
advanced fibrosis. Despite NFS and FIB-4 display-
ing good diagnostic efficacy, many patients
(30%) fall in-between the lower and upper
threshold values (indeterminate results), and
many factors such as age, diabetes, and prevalence
of fibrosis, among others, may influence their diag-
nostic performance. Recently, new age-adjusted
cut-offs have been proposed to improve the
diagnostic efficacy of NFS and FIB-4 for advanced
fibrosis.42 McPherson et al. found lower diagnostic
specificity for NFS (20%) and FIB-4 (35%) in
patients ≥65 years old and new thresholds (FIB-4
= 2.0 and NFS = 0.12) for those patients were able
to improve specificity (70%) without affecting
diagnostic sensitivity.
FibroTestÒ is a commercial algorithm that has
shown good predictive values for diagnosing
advanced fibrosis (AUROC = 0.88) in patients with
NAFLD. However, its diagnostic performance may
be reduced in the presence of some disease-
related interferences, such as acute inflammation,
sepsis and extrahepatic cholestasis.43
Hepascore is a widely used algorithm to detect
significant fibrosis in many chronic liver diseases.
It combines clinical variables of age and gender
with blood-based parameters including bilirubin,
gamma-glutamyl transferase, hyaluronic acid,
and a2-macroglobulin.44 In patients with NAFLD,
a threshold of 0.37 helps to identify individuals
with advanced fibrosis, with an AUROC of 0.81,
and NPV and PPV of 97% and 60%, respectively.45
Blood-based models
The enhanced liver fibrosis (ELFTM) test is another
algorithm that includes HA, procollagen type III
N-terminal peptide (PIIINP), and tissue inhibitor
of metalloproteinase 1 (TIMP-1). This has consis-
tently demonstrated good predictive values for
identifying patients with advanced fibrosis, with
an AUROC of 0.90, sensitivity of 80% and specificity
of 90%, using a cut-off of 10.35.46 However, its per-
formance can be notably influenced by age and
gender in patients with chronic hepatitis C,
although this caveat would require confirmation
in patients with NAFLD.47
Ò
Fibrometer has been recognised as another
blood test of fibrosis with very good diagnostic
accuracy for detecting patients with advanced
fibrosis.44,48 In patients with NAFLD, Fibrometer
showed higher diagnostic robustness in detecting
significant (AUROC = 0.94) or advanced fibrosis
(AUROC = 0.93).
PIIINP is a serum marker of collagen turnover
and increased levels occur as a consequence of tis-
sue repair and fibrosis. PIIINP as a single biomar-
ker has been associated with advanced fibrosis in
patients with NAFLD. For diagnosing advanced
fibrosis, a cut-off of 6.6 ng/ml and 11 ng/ml gave
an NPV and PPV of 95% and 100%, respectively.49
Pro-C3 is a new serum marker derived exclusively
from collagen III synthesis and deposition that has
recently been validated as a predictor of fibrosis
progression in patients with chronic hepatitis
C.50 A recent study in 150 biopsy-proven patients
with NAFLD reported that Pro-C3 was able to iden-
tify patients with advanced fibrosis yielding an
AUROC of 0.91, and an NPV and PPV of 97% and
56%, respectively.51 Pro-C3 correctly classified
82% of patients using a previously published cut-
off (>1.6738).51 Most recently, serum Pro-C3 com-
bined with clinical variables (age, BMI, diabetes
and platelets) was superior to established serolog-
ical fibrosis tests at identifying individuals with
NAFLD and advanced fibrosis, yielding AUROC of
0.87 and 0.85 in training and validation cohorts.52
Another model incorporating serum HA, CK-18
and TIMP-1 yielded an AUROC of 0.90, with a sen-
sitivity of 88% and specificity of 84% for predicting
advanced fibrosis in patients with NAFLD.53
FIBROSpectÒ is a test marketed to detect fibro-
sis in patients with chronic liver disease.54 This
panel incorporates a2-macroglobulin, HA and
TIMP-1. Data from a recent study including a large
cohort of histologically confirmed patients with
NAFLD showed that this panel accurately detects
patients with advanced fibrosis displaying AUROC
of 0.87 and 0.85 in training and validation sets.55
Data from longitudinal and interventional
studies
Since fibrosis severity is strongly related to non-
liver and liver-related morbidity and mortality,
biomarkers of fibrosis may help to identify those
patients at high risk of developing major clinical
outcomes and death. In population-based studies,
high NFS, FIB-4, APRI and FORNs56 scores have
consistently been associated with increased risk
of cardiovascular- and liver-related mortality.57–59
In the same context, the ELF test has been
strongly related to liver-related complications
and death in a wide range of liver-related diseases,
including NAFLD, reinforcing the importance of
these models in predicting long-term adverse clin-
ical outcomes.60,61 In terms of disease progression,
baseline and dynamic changes in ELF scores were
evaluated in a cohort of patients with NASH and
advanced fibrosis who were enrolled in two phase
IIb trials with simtuzumab, a humanised mono-
clonal antibody designed for the treatment of
fibrosis. Both trials were stopped at 96 weeks
 JOURNAL OF HEPATOLOGY

Table 1. Clinical predictors and blood-based biomarkers and their accuracy in the prediction of NASH. Data from cross-sectional studies.
Markers n NAFLD phenotype AUC* Diagnostic accuracy
Serum based variables Training Validation
set set
NAFIC score:75 619 All patients 0.85 0.78 NR
Ferritin ≥200 ng/ml (female) – ≥300ng/ml (male),
fasting insulin ≥10 lU/ml and type IV collagen 7 S
≥5.0ng/ml
Exhaled breath test:76 65 Morbidly obese 0.77 NR Sensitivity: 90%, specificity: 69%
N-tridecane, 3-methyl-butanonitrile, 1-propanol PPV: 81% and NPV: 82%
y
Resistin, cleaved CK-18, adiponectin23 101 All patients 0.91 0.73 cut-off = 0.4320
Sensitivity: 72%, specificity: 91%
Adiponectin, leptin, ghrelin27 82 Morbidly obese 0.79 NR Sensitivity: 82%, specificity: 76%
PPV: 35%, NPV: 96%
Terminal peptide of procollagen III49 136 All patients 0.83 0.78 Cut-off = 6.6, NPV: 85–100%
Cut-off = 11.0, PPV: 80–100%,
CK-18, soluble Fas22 177 All patients 0.93 0.79 Cut-off = probability 36%
Sensitivity: 88%, specificity: 89%
(13) C-methionine77 118 All patients 0.87 NR Cut-off <4.20%
Sensitivity: 81%, specificity: 76%
CK-18 fragments19,78 139 All patients 0.83 NR Wide range of cut-offs provided
201 (adults) 0.93 NR Wide range of cut-offs provided
All patients
(children)
CK-18 fragments (CK-18-M30, M65, M65ED) pooled 2,415 All patients 0.82à – CK-18-M30
analysis20 Sensitivity: 68%, specificity: 74%
CK-18-M65Sensitivity: 76%, specificity: 74%
CK-18-M65EDSensitivity: 91%, specificity:
64%
NASH ClinLipMet Score: glutamate, isoleucine, 318 All patients 0.86 NR Cut-off 0.134
glycine, lysophosphatidylcholine 16:0, Sensitivity: 86%, specificity: 72% NPV: 95%,
phosphoethanolamine 40:6, AST, fasting insulin, and PPV: 45%
PNPLA3 genotype32
OWLiver test: twenty-eight serum triglycerides31 467 All patients 0.87 0.85 Cut-off 0.54
Sensitivity: 71%, specificity: 92%
NPV: 82%, PPV: 84%
Combinations with serum based and clinical variables
OxNASH: BMI, age, AST, 13-HODE79 122 All patients 0.83 0.74 Cut-off <55, sensitivity: 81–84%
Cut-off >73, specificity: 97–63%
CK-18, ALT, platelets, triglycerides24 95 All patients 0.92 NR Cut-off value = 0.361
Sensitivity: 89%, specificity: 86%
PPV: 89%, NPV: 89%
Age ≥50, female, AST ≥45 IU/L, BMI ≥30, AST/ALT 80 All patients 0.76 NR Sensitivity: 74%, specificity: 66%
ratio ≥0.80 and serum HA ≥55 mcg/l15 PPV: 68%, NPV: 71%
diabetes, gender, BMI, triglycerides, M30 (apoptosis), 79 All patients 0.81 NR Cut-off = 0.2188, NPV: 85%
and M65–M30 (necrosis)25 Cut-off = 0.5689, PPV: 81%
Age, sex, height, weight, and serum levels of 257 All patients 0.79 0.79 No cut-off provided
triglycerides, cholesterol, A2M, apolipoprotein A1, PPV: 74%, NPV: 72%
haptoglobin, GGT, aminotransferases ALT, AST, and
total bilirubin16
Hypertension, diabetes, AST ≥27 IU/L, ALT ≥27 IU/L, 200 Morbidly obese 0.80 0.75 ≥6 – PPV: 87–93%
obstructive sleep apnoea, and non-black race14 ≤2 – NPV: 93–80%
Diabetes, triglycerides >150 mg/dl, ALT, and 253 Morbidly obese 0.76 NR ≤1, NPV: 90%
obstructive sleep apnoea17 ≥4, PPV: 60%
Novel combinations
Collagen Pro-C3, CK-18 (M30) fragments, AST, ALT, 374 All patients 0.87 NR Sensitivity: 71%, specificity: 87%
procollagen III N-terminal peptide, acetyl-HMGB-1,
and PNPLA3 rs73840926
MiR34a, YKL-40, HBA1C, A2M80 238 All patients 0.82 NR Sensitivity: 73%, specificity: 78% NPV: 79%
and PPV: 72%
A2M, alpha-2-macroglobulin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the receiver operating characteristic curve; BMI, body mass
index; CK, cytokeratin; GGT, gamma-glutamyl transferase; HA, hyaluronic acid; HBA1C, glycated haemoglobin; HMBG, high mobility group box protein; NAFLD, non-
alcoholic fatty liver disease; NR, not reported; PNPLA, patatin-like phospholipase domain containing protein A; PPV, positive predictive value; NPV, negative predictive
value; NASH, non-alcoholic steatohepatitis; YKL-40, chitinase-3-like protein 1.
*
Studies reporting AUC over 0.75 were included.
y
This cut-off was calculated in overall cohort to improve the model performance.
à
Pooled data from two meta-analysis.

Journal of Hepatology 2017 vol. xxx j xxx–xxx 5

Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
Review

Table 2. Clinical predictors and blood-based biomarkers and their accuracy in the prediction of advanced fibrosis (F ≥3). Data from cross-sectional studies.
Markers n NAFLD phenotype AUC* Diagnostic accuracy
Serum based variables Training set Validation set
AST/ALT81–83 174 All patients 0.83 0.83–0.90 Cut-off = 0.8, NPV: 93%, PPV: 44%
Terminal peptide of procollagen III49 136 All patients 0.82 0.84 Cut-off = 6.6 ng/ml, NPV: 95%
Cut-off = 11 ng/ml, PPV = 100%
Pro-C3 levels51 150 All patients 0.91 NR Cut-off >1.6738:
82% correctly classified
FibroTest: haptoglobin, a2-macroglobulin, 1,202 All patients 0.86 0.85 AUC: 0.88 for F ≥3 (METAVIR)
apolipoprotein-A, bilirubin, and GGT84–86 Cut-off = 0.3, NPV: 98%
Cut-off = 0.7, PPV: 60%
Cut-off = 0.47, PPV:51%, NPV: 89% [45]
APRI score: AST, Platelets82,87 175 All patients 080 0.56–0.67 Cut-off = 1, NPV: 84%, PPV: 37%
ELF test: hyaluronic acid, PIIINP, and 1,329 All patients 0.87 0.90 Cut-off = 8.5–10.18, AUC: 0.82 for ≥F2
TIMP-146,48,88 Cut-off = 10.35, AUC: 0.90 for ≥F3
Combinations with serum based and clinical variables
NAFLD fibrosis score: age, BMI, albumin, AST/ 733 All patients 0.88 0.77–0.84 Cut-off = 81.45, NPV: 78–93%
ALT ratio, hyperglycaemia, and Cut-off = 0.67, PPV: 82–90%
platelets82,89,90,42 Cut-off = 0.12, >65 yrs, NPV:81–98%
Fibrometer: age, weight, glucose, AST, ALT, 1,021 All patients 0.94 0.94 F ≥2, AUC: 0.94
ferritin, and platelets48 F ≥3 AUC: 0.93
FIB-4 index: age, AST, ALT, and 686 All patients 0.80 0.86 Cut-off = 1.30, NPV: 90–95%
platelets38,82,90,42 Cut-off = 2.67, PPV: 80%
Cut-off = 3.25, PPV: 75%
Cut-off = 2.0, >65 yrs, NPV = 82–98%
BARD score: BMI, diabetes, and AST/ALT 1,513 All patients 0.81 0.77–0.78 Cut-off = 2, PPV: 27%, NPV: 95–97%
ratio40,82,90
BARDI score: BMI, diabetes, AST/ALT ratio, and 107 All patients 0.88 NR Cut-off = 3, PPV: 51%, NPV: 96%
INR91
Hepascore: age, sex, bilirubin, GGT, 242 All patients – 0.81 Cut-off = 0.37, PPV: 57%, NPV:92%
a2-macroglobulin, and hyaluronic acid45
Novel combinations
HA, CK-18 and TIMP-153 180 All patients 0.90 NR Sensitivity: 88.2%, specificity: 84.1%
NPV: 97%, PPV: 60%
FIB-C3: Pro-C3, age, BMI, diabetes, and 433 All patients 0.86 0.85
platelets52
FIBROSpect test: a2-macroglubulin, 792 All patients 0.87 0.85 Sensitivity: 84–81%, specificity: 72–74%.
hyaluronic acid, and TIMP-155
ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the receiver operating characteristic curve; BMI, body mass index; CK, cytokeratin; INR,
international normalized ratio; GGT, gamma-glutamyl transferase; HA, hyaluronic Acid; NAFLD, non-alcoholic fatty liver disease; PPV, positive predictive value; NPV,
negative predictive value; Pro-C3, C-terminal cleavage site of N-terminal type III collagen propeptide; PIIINP, N-terminal type III collagen propeptide; TIMP-1, tissue
inhibitor of metalloproteinase-1.
*
Studies reporting AUROC over 0.75 were included.

owing to a lack of efficacy and patients were com-
bined for analysis of disease progression. Baseline
ELF values were able to predict progression to cir-
rhosis in patients with bridging fibrosis (c-
statistic: 0.80) or development of clinical events
(c-statistic: 0.69). However, changes in ELF values
over time did not improve predictions when
added to baseline ELF values.62
In another therapeutic trial performed in dia-
betic patients, Pro-C3 was able to identify patients
at high risk of advanced fibrosis at baseline, as
well as those who progressed over time and
responded to a potential anti-fibrotic therapy, con-
firmed by increased or attenuated liver fibrosis on
paired biopsies.63
Since lifestyle interventions have demonstrated
a significant impact on liver histology improve-
ment,64 identifying biomarkers that may detect
those patients with fibrosis improvement is a
priority. A recent study has demonstrated that a
simple panel that combines changes from baseline
in platelets and HbA1c alongside normalisation of
ALT (19 U/L and 30 U/L for women and men) is
able to accurately identify patients (AUROC = 0.9
6) with one-point improvements in fibrosis after
one year of diet and exercise. Changes from base-
line in other biomarkers such as NFS, APRI, FIB-4
and AST/ALT ratio yielded low diagnostic accuracy
for changes in fibrosis at end-of-intervention.65
We summarise the studies reporting models
for NASH and advanced fibrosis predictions and
their diagnostic accuracy (Tables 1–3). Selected
clinical prediction rules for non-invasive assess-
ment of advanced fibrosis are also described
(Table 4).
Proteomic investigations and NAFLD
biomarkers
There has been significant interest in the develop-
ment of protein-based biomarkers that utilise
mass spectrometry to identify NASH and advanced
fibrosis in patients with NAFLD. In a systematic
review, Ladaru and colleagues summarised 22

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Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
 JOURNAL OF HEPATOLOGY

Table 3. Biomarkers and their potential role in predicting dynamic changes in NASH or fibrosis and providing long-term prognostic information. Data
from longitudinal and interventional studies.
Markers n Study design Outcome prediction Risk AUC Diagnostic accuracy
Longitudinal or cohort studies
FIB-4, APRI, NAFLD 14,841 Population-based Liver-related APRI (high/intermediate), HR: 9.44 – –
fibrosis score and prospective survey in mortality FIB-4 (intermediate), HR: 3.15
FORNs score59 viral hepatitis (high), HR: 25.14
negative adult from NFS (high), HR: 6.52
NHANES III. FORNs score (intermediate), HR:
3.6 (high), HR: 63.1
APRI, NAFLD fibrosis 320 Cohort study in Liver-related APRI (intermediate), HR: 8.8 – –
score, and BARD biopsy-proven NAFLD mortality (high), HR: 20.9
score58 patients BARD score (intermediate), HR: 6.2
(high), HR: 6.6
NFS (intermediate), HR: 7.7 (high),
HR: 34.2
Interventional studies
ELF test62 477 Phase IIb trials of Histological Baseline ELF test Progression to –
simtuzumab. The progression to Progression from bridging fibrosis cirrhosis: 0.80
trials were stopped at cirrhosis to cirrhosis, HR: 3.13 Development of
96 wks due to lack of Development of Development of clinical events, clinical events:
efficacy. clinical events HR: 2.37 0.69
Changes in ELF over time does not
improve prediction to baseline
values
Pro-C363 297 Interventional trial Fibrosis attenuation Robust reduction in Pro-C3 as a – –
Glitazone therapy in in high-risk groups function of therapy. This
diabetes. biomarker at baseline is useful for
identification of responders to
glitazone therapy.
CK-18 fragments34 384 Interventional trial Overall histological Relative odds for overall Overall
(PIVENS and TONIC) improvement at 96 histological improvement (1.28) histological
wks* and NASH resolution (1.37) improvement:
0.71–0.72
NASH resolution:
0.64–0.69
NASH resolution score: 261 Interventional trial Histological – 0.96 and 0.95 in Cut-off ≤46.15,
weight loss, diabetes, (lifestyle changes for resolution of NASH training and NPV: 92%
NAS ≥5 and ALT 52 wks) at 52 wks validation sets Cut-off ≥69.72,
normalisation13 PPV: 89–92%
Fibrosis improvement 261 Interventional trial 1-point fibrosis – 0.96 in training Cut-off ≥0.497,
score: changes from (lifestyle changes for improvement at 52 set PPV: 94%,
baseline in A1C and 52 wks) wks Change in NFS: NPV: 91%
platelets, and ALT 0.77, FIB-4 index:
normalisation65 0.63, AST/ALT
ratio: 0.60, and
APRI: 0.50
ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, cytokeratin: ELF, enhanced liver fibrosis; HR, hazard ratio; NAS, NAFLD activity score; NASH, non-
alcoholic steatohepatitis; NFS, NAFLD fibrosis score; NPV, negative predictive value; Pro-C3; C-terminal cleavage site of N-terminal type III collagen propeptide; PPV,
positive predictive value.
*
Histological improvement as one point or greater improvement in the hepatocellular ballooning score, no increase in the fibrosis score, and either a decrease in the NAFLD
activity score (NAS) to a score of three or less or a decrease in the NAS of two points or fewer, with at least a 1-point decrease in either the lobular inflammation or steatosis
score.

studies which reported 21 confirmed serum pro-
tein biomarkers.66 Unfortunately, none of these
candidate proteins have been translated into com-
mercially available diagnostic tests.
Bell et al. have conducted a serum proteomics
and biomarker discovery study in 69 patients with
well characterised NAFLD (simple steatosis: 24,
steatohepatitis without advanced fibrosis: 23,
and steatohepatitis with stage 3/4 fibrosis [F3/4]:
22) and 16 controls without NAFLD.67 Using a
label-free mass spectrometry-based approach,
these investigators identified over 1,700 serum
proteins with a peptide ID confidence level of
>75%, 605 of which changed significantly between
any two patient groups (false discovery rate <5%).
Importantly, expression levels of 55 and 15 pro-
teins changed significantly between the simple
steatosis and NASH F3/F4 group, and the NASH
and NASH F3/F4 group, respectively. The priority
1 proteins with >30% change between any two
groups were subsequently considered for develop-
ing biomarker candidates. A panel consisting of six
priority 1 proteins (fibrinogen b chain, retinol
binding protein 4, serum amyloid P component,
lumican, transgelin 2, and CD5 antigen-like) differ-
entiated all four patient groups with an overall
success rate of 76% (AUROC for control group =
1.0, simple steatosis = 0.83, NASH = 0.86, and
NASH with F3/F4 = 0.91). A group of three priority
1 proteins (complement component C7, insulin-
like growth factor acid labile subunit, and trans-
gelin 2) correctly categorised 90% of patients as

Journal of Hepatology 2017 vol. xxx j xxx–xxx 7

Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013
Review

Table 4. Selected clinical prediction rule for non-invasive assessment of advanced fibrosis in patients with NAFLD.
Model Components and regression equation Cut-offs for advanced fibrosis Clinical benefits and limitations
NAFLD fibrosis score 1.675 + 0.037  Age (yrs) + 0.094  <1.45 (low) – NPV: 88–93% Cheap and reproducible Yes
BMI (kg/m2) + 1.13  IFG/diabetes >0.67 (high) – PPV: 78–90% Sensitivity to exclude F ≥3 High
(yes = 1, no = 0) + 0.99  AST/ALT ratio Age >65 yrs Specificity to diagnose F ≥3 Modest
 0.013  Platelet (109/L) 0.66  <0.12 (low) – 81–98% Influenced by age Yes
Albumin (g/dl) Allows predict clinical outcomes Yes
FIB-4 index Age (yrs)  AST [U/L]/(Platelet [109/L] <1.30 (low) – NPV: 90–95% Cheap and reproducible Yes
 (ALT [U/L])1/2) >3.25 (high) – PPV: 75% Sensitivity to exclude F ≥3 High
Age >65 yrs Specificity to diagnose F ≥3 Modest
<2.0 (low) – 82–98% Influenced by age Yes
Allows predict clinical outcomes Yes
BARD score AST/ALT ratio ≥0.8 = 2 points BMI ≥28 Single cut-off = 2 Cheap and reproducible Yes
= 1 point NPV: 95–97%, PPV: 27% Sensitivity to exclude F ≥3 High
Presence of diabetes = 1 point Specificity to diagnose F ≥3 Low
Score ranges from 0 to 4 points Influenced by age Unknown
Allows predict clinical outcomes Yes
APRI (AST [IU/L])/(AST upper limit of normal Single cut-off = 1 Cheap and reproducible Yes
[IU/L])/(Platelet [109/L]) NPV: 84%, PPV: 37% Sensitivity to exclude F ≥3 High
Specificity to diagnose F ≥3 Low
Influenced by age Unknown
Allows predict clinical outcomes Yes
Hepascore Y = exp [4.185818  (0.0249  Age) Single cut-off = 0.37 Cheap and reproducible Yes
+ (0.7464  Sex) + (1.0039  a2- NPV: 92%, PPV: 57% Sensitivity to exclude F ≥3 High
macroglobulin) + (0.0302  Specificity to diagnose F ≥3 Modest
Hyaluronic acid) + (0.0691  Influenced by age Unknown
Bilirubin)  (0.0012  GGT)] Allows predict clinical outcomes Yes
Hepascore = Y/(1 + Y)
ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; IFG, impaired fasting glucose; F ≥3, advanced fibrosis (bridging fibrosis and
cirrhosis); NAFLD, non-alcoholic fatty liver disease; NPV, negative predictive value; PPV, positive predictive value.
Variables included in the prediction models are in bold.
Individual patient scores for each model can be easily obtained using free online calculator: http://gihep.com/calculators/hepatology/.

having NAFLD (simple steatosis and NASH) or
NASH F3/F4 (AUROC = 0.91). Interestingly, two
proteins (prothrombin fragment and paraoxonase
1) were able to totally differentiate control sub-
jects from those with all forms of NAFLD com-
bined with an AUROC = 1.0. This group has also
reported the results of serum proteomic analysis
from their Ossabaw NASH model.68 This study
identified seven priority 1 proteins which were
different between pigs which developed NASH
and pigs without NASH. Importantly, these inves-
tigators found seven priority 1 proteins
(apolipoprotein C-III, apolipoprotein B, serum
amyloid P component, transthyretin, paraoxonase,
protein similar to a-2-macroglobulin precursor,
and orosomucoid I) to be common between their
human and Ossabaw swine proteomic
investigations.67,68
A promising new sensitive and quantitative
proteomic technology (SOMAscan assay, SomaLo-
gic; Boulder, CO), that is worth watching closely,
has recently emerged. It uses Slow Off-rate Modi-
fied Aptamers (SOMAmers), single-stranded apta-
mers with modified nucleotides that have a high
affinity for specific protein targets in the serum,
which are subsequently quantified as DNA. In a
preliminary study, a proteomic classifier based
on eight proteins had an AUROC of 0.932 for iden-
tifying steatosis in obese individuals undergoing
bariatric surgery.69 Interestingly, a proteomic clas-
sifier has performed as well as a multicomponent
classifier based on PNPLA3 genotype, proteomics,
and phenomics at identifying steatosis in this
cohort. This approach is being applied to differen-
tiate various histological stages in patients with
biopsy-proven NASH.
Other promising non-invasive approaches for
detecting patients with advanced NAFLD have
recently been developed and these biologically
based strategies could improve or complement
the success of other non-invasive markers.
Other promising non-invasive approaches
In a recently published paper, Decaris et al.
described a novel method to non-invasively
describe the hepatic fibrogenesis flux rates in the
liver tissue and in the blood. They have shown that
hepatic fibrogenesis flux rates correlate signifi-
cantly with the degree of liver fibrosis in 24 sub-
jects who underwent diagnostic liver biopsy for
suspected NAFLD.70 Liver collagen fractional syn-
thesis rate (FSR) and plasma lumican (liver extra-
cellular matrix protein) FSR were measured
based on 2H labelling using tandem mass spec-
trometry. In this method, patients undergoing
clinically indicated liver biopsy drank heavy water
(2H20, 50 ml two to three times daily) for three to
five weeks prior to their biopsy. Key observations
from this study were (a) there was active remod-
elling of hepatic extracellular matrix even in
patients with advanced fibrosis; (b) hepatic colla-
gen FSR correlated significantly with fibrosis stage
as well as non-invasive fibrosis indices, such as
FIB-4 and liver stiffness by magnetic resonance
elastography (MRE); (c) plasma lumican FSR

8 Journal of Hepatology 2017 vol. xxx j xxx–xxx

Please cite this article in press as: Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: Clinical prediction rules and blood-based biomarkers. J Hepa-
tol (2017), https://doi.org/10.1016/j.jhep.2017.11.013

correlated significantly with hepatic collagen FSR,
liver fibrosis by histology, and non-invasive fibro-
sis markers such as liver stiffness by MRE and FIB-
4. If this method is reproduced in follow-up publi-
cations, this may serve as a novel approach to test
anti-fibrotic compounds in short term proof-of-
concept studies. It is disappointing that this study
did not compare the hepatic collagen FSR and
lumican FSR with the ELF score.
Loomba et al. recently reported the utility of a
metagenomics signature based on the gut micro-
biome for non-invasively detecting advanced
fibrosis in 86 patients with NAFLD (14 had stage
3 or 4 fibrosis).71 Their gut microbiome composi-
tion was characterised using whole-genome shot-
gun sequencing of the stool DNA. A random forest
classifier, consisting of 40 variables including 37
bacterial species, was able to identify patients
with F3/4, with an AUROC of 0.936. This study
lacked a validation cohort.
There are several ongoing investigations to
examine the utility of blood-based molecular
markers (signatures derived from circulating
microRNA, cell-free RNA, DNA methylation, and
cell-free DNA methylation).72–74 These approaches
hold promise but they need to be validated exter-
nally using independent cohorts.
Conclusion
In summary, no highly sensitive and specific tests
are available to differentiate NASH from simple
steatosis. However, diagnostic accuracy can be
improved by combining blood biomarkers. More
investigations are needed before prediction mod-
els and blood-based biomarkers become available
for routine clinical care. Combinatorial and com-
plex models including clinical, routine blood-
based variables and markers that reflect the
dynamic nature of the fibrogenic process appear
to have higher diagnostic accuracy and predictive
value in identifying advanced fibrosis, in patients
with NAFLD. NFS and FIB-4 are useful screening
tools to be routinely applied in clinical practice,
since they can accurately exclude patients with
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Conflict of interest non-expert physician and
Eduardo Vilar-Gomez: Nothing to disclose. Naga patients.
Chalasani has ongoing consulting activities (or
had in preceding 12 months) with NuSirt, Abbvie,
Eli Lilly, Afimmune (DS Biopharma), Tobira (Aller-
gan), Madrigal, Immuron, Shire, Ardelyx, and Axo-
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research grant support from Intercept, Lilly,
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where his institution receives the funding. Over
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