Non-Invasive Assessment of Liver Fibrosis

Cirrhosis is the leading cause of morbidity & mortality amongst patients with Hepatitis C. The
gold standard test for detecting the varied features of cirrhosis is liver biopsy. This test however
is wrought with complications, leading observers to call it an "imperfect Gold Standard". 30%
patients have come with complains of pain, 3% have required hospitalization whereas death has
been reported in 0.01-0.3% [1-8].
Because of its complications, liver biopsy has been challenged as a sole modality for assessing
liver fibrosis. Doctors & researchers alike have sought for a noninvasive approach towards
assessment of liver fibrosis. This has paved way for development of non-invasive methods that
could rely on one of the 2 following principles
1.) Biological - based on serum biomarkers of fibrosis
2.)Physical - based on measurement of liver stiffness;
these technologies are complementary to one another but assess different parameters affected
by disease processes [9-11]
Modalities like FibroScan, , ,etc open new windows for pain free testing & provide an alternate
approach to diagnosis. Following is a brief overview of the disease process and various
technologies that utilize the same and have resulted in development of a non invasive approach
& intends to educate the reader & equip them with a sense of decision-making that involves a
pain free diagnosis.

Liver Fibrosis - The Disease Process

Liver fibrosis can be defined as the accumulation of Extracellular Matrix (ECM) in the liver
parenchyma, as a part of the normal healing response to injury. The normal response inculcates
recruitment of inflammatory molecules to the site of injury with resultant deposition of ECM,
remodelling and possible regeneration of the hepatic nodule. In chronic inflammation this
response could be excessive and lead to disruption of the normal architecture of the liver &
function.
Myofibroblast (MF) is an important cellular player in the formation of ECM. Hepatic Stellate Cells
(HSC; Ito Cells) are the predominant MF producing cells. Non MF cells also take part in the
process of fibrogenesis. For eg. hepatocytes can react to produce Reactive oxygen species (ROS)
or kupfer cells can recruit inflammatory mediators that release more pro inflammatory and pro
fibrotic mediators including cytokines such as TNF- and various interleukins. As a result of this
response the quiescent HSC are converted into activated myofibroblasts that inturn produce
chemokines that attract leukocytes to the site of injury that release more proinflammatory
mediators. HSC express platelet derived growth factor receptor and transforming growth factor
receptor. All this inturn results in the gradual replacement of the basement membrane like ECM

within the space of Disse by collagen rich fibers & production of fibrous bands. In advanced
stages of fibrosis liver can contain more than 8 times the normal ECM. It includes collagen,
fibronectin, undulin, elastin, hyaluronan, laminin & proteoglycans. A number of biomarkers can
thus be used to evaluate fibrosis. (R05) Liver fibrosis due to various causes differ in their
fibrogenic mechanism and therefore the markers involved in a particular disease are different
and have to be evaluated like wise in separate cohort studies.
Increased evidence shows that unlike cirrhosis, fibrosis can be treated and is reversible in the
early stages laying great scope and importance for its serial assessment[12].

Liver Biopsy : Shortcomings

Liver biopsy is still the recommended test for fibrosis evaluation and treatment indication in
patients with chronic viral hepatitis [13-18]. However, it is as stated earlier an invasive and
painful procedure, 1-5% of the patients have developed complications that have required
hospitalization & a mortality rate has been reported between 1:1000 & 1:10000 [1-8] .
As it assesses only 1/50,000th of the liver, there is considerable chance of a sampling error.
Moreover studies show that 10-30% of blind liver biopsies miss detecting cirhosis [8,19-24].
Several recent studies highlight these and more crucial drawbacks that also include variable
accessibility, high cost, sampling errors & inaccuracy due to inter- & intra- observer variability
of pathological interpretations[25-26]. Included in this is an important but small risk of biopsy
associated morbidity & mortality. Among them, pain (20% of patients 4, R10) & hypotension are
the most frequent whereas intraperitoneal haemorrhage and injury to the biliary system are the
most serious complications(0.57% of patients) [16, 26-30].
It is important to have an effective as well as a safe diagnostic tool for liver fibrosis. This is
because patients with liver fibrosis represent the group that is at greatest risk for developing
liver disease related morbidities and mortalities and as such the group that would inturn benefit
from diagnosis and treatment with appropriate antifibrotic agents and also liver fibrosis is silent
till its end stages [31].

The existing scoring systems for Liver Biopsy : Yardsticks for the non-invasive
model
Various scoring systems have been devised to grade/stage a liver biopsy and to evaluate its
prognostic significance.
1.) Ishak Score/Revised Knodell Systemwas one of the 1st scoring system applied for assessing chronic hepatitis B & C. This test
considers grading and staging to be 2 separate categories; liver fibrosis is classified as
0 = absent
1-2 = mild

3-4 = moderate
5-6 = severe/cirrhosis
Necroinflammatory grade of the disease is assessed by the first 3 axes of the Knodell HAI
(Histological Activity Index) whereas the 4th assesses the stage by evaluating the degree of
fibrous portal tract expansion, fibrous portal-portal linking, portal central fibrous bridges, & the
formation of fibrous septa & parenchymal nodules [26,119]. Interobserver variation and
nonlinearity are 2 drawbacks of this system and its modified versions [26,32-34]
2.) METAVIR Scoring system was designed keeping patients with chronic hepatits C in mind. The score includes a
category of necroinflammatory grade under A for activity and stage of fibrosis represented by F.
Grade is number based on the degree of inflammation ranging from 0-4 with A0 being minimum
inflammation and A4 being its severe form. Likewise Fibrosis Score is defined as :
F0 = no scarring
F1 = portal fibrosis without septa
F2 = portal fibrosis with rare septa
F3 = numerous septa without cirrhosis
F4 = cirrhosis or advanced scarring of the liver
the metavir scoring system also suffers from similiar drawbacks of the Knodell system however
it enjoys decreased errors related to inter- & intra-observer variability.
Due to the aforementioned complications many patients forego the option of liver biopsy that
makes the availibility of non-invasive tests extremely important. The tests available in the
market range from a study of biomarkers to using radiologic imaging to study the fibrogenic
process. The scoring systems discussed above are used as a yardstick to compare their efficacy
with biopsy. Every non-invasive test is evaluated using the Area Under the Receptor Operator
Curve (AUROC) that combines the sensitivity and specifity of a given quantitative marker for the
diagnosis of a specific definition of fibrosis. Below is a review of all the available resources and
the comparison of their results with the comparative standard - Liver Biopsy. [26]

RADIOLOGY - measuring liver stiffness

Reduction in the size of right lobe of the liver and relative enlargement of the left &
caudate lobes has been found to be a reliable marker of cirrhosis, on USG, CT & MRI. (8,
35-37) Harbin used this technique by measuring the ratio of the transverse width of the

caudate lobe with the transverse width of the right lobe. This way a sensitivity of 84% &
specificity of 100% was achieved. A diagnostic accuracy of 94% has been reported.

Transient Elastography
Transient Elastography (using Fibro Scan) is a noninvasive modality that uses pulse-echo
ultrasound acquisitions to measure liver-stiffness. This technology uses an ultrasound probe
mounted on the long axis of a vibrator, vibrations of mild amplitude & low frequency are
transmitted by a transducer that creates an elastic shear wave that then travels thru the
tissues[11,38] . Liver Stiffness is proportioinated to this shear velocity, using the formula E =
3V2 where is the mass density (constant for tissues) & V is the shear velocity. Stiffer the
tissue, faster the shear wave. TE measures liver stiffness in volume of a cylinder 1cm wide and
4cm long, this volume is 100 times superior to liver biopsy size and therefore very much
representative of the hepatic parenchyma [11]. This one dimensional method does not produce
images as data is collected on the axis of a single element transducer [12]. TE is a painless
procedure and can be performed rapidly on the bedside with results available immediately, they
are expressed in kPa and range between 2.5-75kPa, with normal values around
5.5kPa[11,39,40].TE may correlate with portal hypertension upto the point where liver fibrosis is
contributing to pathology, it cannot, however, predict disease change when the HVPG >
12mmHg. This is the point where other hemodynamic factors (such as hyperdynamic
circulation, the splanchnic vasodilatation, and the resistance opposed to portal blood flow by
the portosystemic collaterals) take over the complex pathophysiology, this also makes the
technology useless in monitoring drug therapy that targets these hemodynamic parameters.
Likewise, using the parameters of liver stiffness cannot be correlated with the detection of
varices, however, this does not affect the test's ability to predict risk of bleeding. [41,42].
Because of its non invasive nature TE could be used to assess severity of recurrent HepC in
patients with liver transplantation. Similiarly, In a research done by Carrion et al [41, 43] it is
found that values above the cut-off for liver stiffness directly correlated with severe fibrosis
(Metavir - F3) or cirrhosis(F4) or HVPG > 10mmHg, highlighting the value of TE in these cases,
especially. In recent research it is shown that patients with sustained viral eradication of Hep C
show significantly decreased values of liver stiffness, making TE a promising tool in monitoring
response to therapy [41, 44-46].
TE can be useful as an indicator for the risk of Heptocellular Carcinoma, as suggested by a
research conducted in Japan [16, 57]. TE could also be useful for assessing severity of recurrent
hepatitis C after liver transplantation which would then reduce the need for follow-up biopsies
[16, 43,48-50].
Diagnostic Performance of TE

The interequipment, intraobserver (96-98%) and interobserver agreement (89-98%) has been
shown to be excellent but success depends on a couple of factors that include observer
expertise, patient body mass index and intercostal space [12, 51-53]
Researches conducted to assess the diagnostic performance of TE show AUROC ranging from
0.75-0.99 for significant fibrosis and 0.90-0.99 for cirrhosis [11]. The mean AUROC was 0.84
(95% CI; 0.82 - 0.86) for significant fibrosis giving cut-off of 7.6 kPa as obtained from 3 metaanalysis [11, 54-56], these same analysis gave 0.94 (95% CI, 0.93 - 0.95) for diagnosis of cirrhosis
with optimal cut-off being placed at 13.0 kPa. These results show that TE can be employed for
diagnosing cirrhosis & therefore is better at excluding than predicting it.
While TE is promising, it has some limitations; principal reasons being obesity, particularly
increased waist circumference & limited operator experience, demanding operators be trained
adequately & pushing the companies to innovate the technology.
Also, as liver is enclosed in a distensible but non-elastic capsule, changes such as edema,
inflammation, cholestasis & congestion would effect measurement of liver stiffness (LSM). Most
importantly , its accuracy in predicting significant cirrhosis is affected by necroinflammatory
activity and steatosis which is common in patients with NAFLD [26, 57]
Consequently, it has been found that ALT flares in individuals with acute viral hepatitis or
chronic hepatitis B are prone to overestimating liver stiffness [11, 58-60] Similiar problems have
been found to confound results in cases of extrahepatic cholestatsis [96, R02] or congestive
heart failure [61].
{a paragraph on combination of approaches} it has been shown that combining TE & Fibrotest
gives better results than standalone [62].

Acoustic Radiation Force Impulse

ARFI joins the use of conventional ultrasound with assessment of local liver stiffness. ARFI allows
the operator avoidance of large obstacles as local areas for evaluation can be chosen [26, 63,
64]. A small study done on 91 patients shows comparative efficacy with TE [65]. An important
advantage of ARFI is that it can be performed with software integrated into a conventional
ultrasound instead of obtaining expensive equipments as is required for FibroScan.

MRI/MRE
MRI can be used to measure both liver stiffness and the water-diffusion abnormalities
associated with liver cirrhosis. A modification on this method Magnetic Resonance Elastography
quantitatively measures acoustic shear wave propogating through the liver tissue. It is basically
a combination of phase-contrast MRI sequence combined with various ways of wave generation
by resistive electromechanical drivers, piezoelectric devices, electromagnetic coils or pneumatic
drivers [12, 66-69]. MRE gives an accurate idea of the fibrous distribution of the liver and as such

acts as a pointer towards the nature of injury. It has been shown to give good reproducibility
for viscosity and elasticity [12, 70]. It has also been shown superior to APRI [71].
A study of 50 patients with biopsy-proven liver disease & 35 healthy patients reveals that
analysis of AUROC with a cutt-off of 2.94kPa MRE has a predicted sensitivity of 98% and a
specificity of 99% [26,72] The technical success rate is higher of MRE as compared to transient
elastography (98% vs 84%).
Drawbacks include its substantially higher cost that precludes its widespread use and limits it for
research purposes and a subset of patients that are claustrophobic &/or those with
hemochromatosis. Another limitation is that it would be difficult to perform on an obese patient
who may not fit into the magnetic bore when an additional transducer also has to be placed.

Methodological Considerations
Any non-invasive test that could assess fibrosis would have to be compared against the goal
standard that is liver biopsy. This is done by obtaining an AUROC (area under the receptor
operator curve) that plots sensitivity against 1-specifity. The AUROC shows the probability of the
test in identifying 2 random individuals, in whom liver biopsy shows that they are "normal" or
"diseased". AUROC is shown to vary with respect to biopsy length & fragmentation as well as
stratification of the stages of fibrosis among the studied population. For an imperfect gold
standard like liver biopsy , AUROC of a perfect test will never reach its maximal value of 1.0 & an
ideal value of >0.9 [11, 73]

BIOLOGICAL Approach : Serum Biomarkers

In the recent years there has been a great interest in quantification and studying of biomarkers
of liver fibrosis. As discussed above studying markers taking part in the fibrogenic process is non
invasive, pain free, easy to perform , not wrought with sampling errors and operator related
variability. The tests can be done on bed side and serial monitoring gives it both therapeutic and
prognostic advantage. Many low-throughput studies have been able to identify the biomarkers
while evaluating the pathogenic mechanisms of liver fibrosis but to prove statistical significance
of these markers in therapy and disease evaluation as it evolves remains a technical challenge.

The Ideal Biomarker

An ideal biomarker would have all of the qualities stated below
-highly sensitive & specific in identifying various stages of liver fibrosis
- safe, cheap and readily available
- useful in monitoring disease progression or regression as a part of the natural history of
disease

-low false positive results

Although no single ideal biomarker exists many have been studied and divided into 2 types,
those that assess liver fibrosis directly; that depend on deposition and removal of extracellular
matrix like hyaluronate & those that are indirect markers like prothrombin index, platelet count
and AST/ALT ratio which are released into the blood stream as a part of the inflammatory
process or are indicators of disruption of liver function. More sophisticated approaches have
sprung from the combination of these 'direct' and 'indirect' markers.
Direct Biomarkers
1.) Procollagen type III amino-terminal peptide (PIIINP) & Procollagen type 1 carboxy-terminal
peptide (PICP)
Liver fibrosis is associated with a 8-fold rise in type I collagen, as well as increase in normal ratio
of typeI:III from 1:1 to 1:2 [74]
PIIINP is an important constituent of the connective tissue. Elevated serum levels in the acute
state reflect aminotransferase levels whereas in the chronic state it reflects the stage of liver
fibrosis [26,75,76]. However, PIIINP is not specific for liver fibrosis as it is raised in acromegaly,
chronic pancreatitis, lung fibrosis & rheumatologic disease[77].
PICP levels are elevated in 50% of the patients with chronic hepatits. Different PIIIP essays
utilized collectively have shown significant correlation with periportal necrosis, fibrosis and
hepatic activity index [18, 78].
2.) Matrix Metalloproteinases (MMPs)
MMPs are a family of proteolytic enzymes that are responsible for degrading the extracellular
matrix & the basement membranes.
The most studied among them are MMP2 (gelatinase A), MMP3 (stromelysin) and MMP-9
(gelatinase B). MMP 2 levels have been found to be markedly elevated in various liver diseases,
there expression is upregulated and they are released by activated HSCs, contradictory data
released about these enzymes limits their practicality[26,79,80]. MMP 9 on the other hand have
been found to negatively correlate with histological severity in patients with chronic liver
disease due to hepatitis C [26, 81] and is also discussed below.
3. Tissue inhibitors of matrix metalloproteinases (TIMPs)
TIMPs are proteins secreted for modulating the activation & functioning of MMPs. TIMPs are
involved in inhibiting ECM degradation and hence promote fibrosis. As has been shown through
research TIMP levels are elevated in chronic Hepatits C as well as their correlation with fibrosis
progression has been established [26, 79]. A study comparing MMP 9, TIMP & fibrosis was
recently done on 50 patients with various forms of chronic liver disease and it was found that

levels of MMP 9 decreased (p < 0.05) whereas TIMP increased (r = 0.73, p < 0.001), respectively
as fibrosis progressed [81] making TIMP a good candidate for assessing liver fibrosis[26]. TIMP 1
and HA have also been found to show good significance in HCV-HIV coinfected patients [18, 82].
4. Transforming Growth Factor -1
TGF-1 is a cytokine responsible for growth & differentiation and ECM production. Its levels
have been found to correlate with liver fibrosis [83,84]
5. Hyaluronic Acid
Hyaluronan is a glycosaminoglycan component of the ECM and is synthesized by the HSC.
Lydatakis H et al in their research on NAFLD showed that HA was that best class I biomarker of
fibrosis with AUROC of 0.97 [85]
Other direct markers include a mammalian analogue of bacterial chitanases involved in the
remodelling of ECM, YKL-40 (chondrex) [86,87]- best to differentiate between F2-4 from F0-1
(AUROC 0.81)[ 18, 88]; Laminin a non collagenous glycoprotein in the basement membrane of
the liver , elevated levels of laminin and pepsin resistant laminin have been found to correlate
with degree of perisinusoidal fibrosis[87]; Connective Tissue Growth Factor; Paraxonase 1 (PON
1) an enzyme that hydrolyzes lipid peroxidases , its serum levels have been proposed as a
potential measure of liver function but has limited practicality due to toxicity of its substrate
paraxon; and Microfibril Associated Glycoprotein 4 (MFAP-4) serum levels have found to have
high diagnostic accuracy with AUROC 0.97 p <0.0001.
Direct biomarkers have some limitations. Because they represent matrix turnover and will only
be elevated when there are massive changes in ECM metabolism they are unable to predict
changes in minimal inflammation. Moreover, serum levels of biomarkers are influenced by their
clearance & excretion in turn influenced by abnormal functioning of endothelial cells, biliary
tract or kidneys.
Indirect Biomarkers
Indirect markers are mostly studied as part of a panel of collected entities. Pioneer among these
has been FibroTesttm . With an aim to assess whether a panel of biochemical markers could
identify patients with clinically significant fibrosis(METAVIR F2 or greater), Imbert-Bismuth &
MULTI VIRC group published their assessment of these biomarkers in 339 patients with Hepatitis
C . Of the assessed markers 2 macroglobulin, 2 globulin, globulin, apolipoprotein A1, GT
were found to be the most useful and used to derive a calculated index that is the FibroTesttm
[89]. With this test the authors were able to stratify their patients into those with mild liver
disease (METAVIR F0-1), those with high certainity of significant fibrosis (METAVIR F2-4) and
those who could not be classified and in whom biopsy was deemed necessary and with this
stratification they were able to rule out biopsy in 46% of their patients successfully [8]. Other
tests such as the patented Fibrosure, Fibrometers, FibroSpect II, ELF, Forn's Index & Hepascore

are similiar mathematical formulas that combine the aforementioned parameters and help as a
non invasive guide.
With regards to diagnostic performance of these markers, a recent article by L.Castera [11]
reports a meta-analysis[90], which pooled 6,378 subjects (with analysis of individual data in
3,282) with both FibroTest and biopsy (3,501 HCV, 1,457 HBV, 267 NAFLD, 429 ALD, and 724
mixed),the mean standardized AUROC for diagnosing significant fibrosis was 0.84 (95% CI, 0.83
0.86), without differences between causes of liver disease: HCV 0.85 (0.820.87), HBV 0.80
(0.770.84), NAFLD 0.84 (0.760.92), ALD 0.86 (0.800.92), mixed 0.85 (0.800.93). In another
meta-analysis[91], which pooled 4266 HCV patients from 22 studies, the mean AUROCs of APRI
for diagnosing significant fibrosis and cirrhosis were 0.76 (0.740.79) and 0.82 (0.790.86),
respectively [11]. It has also been shown through various studies [11,92-96] that different
patented scores have similiar performance in detecting significant fibrosis.
Similiar tests for the AST/ALT ratio > 1 showed strong diagnostic significance for cirrhosis,
sensitivity > 78% & specificity of 97% [8, 97]. In this same research platelet count < 130 x 109 /L
increased the diagnostic accuracy of the test with positive and negative predictive value as 97%
& 86% respectively.
AST to Platelet Ratio Index (APRI)is another index that was previously useful as a surrogate
marker for fibrosis in HIV/HCV coinfected patients. However, through a metanalysis, it has
recently been shown to identify Hepatitis C related fibrosis with a moderate degree of accuracy
[26, 98].
A number of attempts are being made to combine these biomarker panels to generate a
sequential approach towards assessment of liver fibrosis in order to rule out the use of liver
biopsy in a patient with CLD. Sebestiani et al have put this idea to work by combining APRI,
Forn's Index & FibroTest to reduce the need of biopsy in patients with chronic hepatitis by 5070% [99]. Later these same authors combined APRI with Fibrotest/Fibrosure generating a SAFE
biopsy (Sequential Algorithm for fibrosis evaluation) assessed in a very large cohort of hepatitis
C patients (N = 2035) [100]. The results showed that SAFE biopsy was accurate upto 92.5% (95%
Confidence Interval, 0.89 -0.94) alleviating the need for biopsy in 81.5% of the liver biopsies.
Although biomarkers are promising in the field of non invasive assessment , none of these are
liver-specific and their levels are influenced by excretion & clearance. For eg. , post prandially
hyauronate levels are found to be increased, they are also increased in the elderly with chronic
inflammatory diseases such as rheumatoid arthritis. Also reproducibility of indirect markers like
AST, platelet count has been observed to be questionable. On the other hand, a critical analysis
of the tests can help avoid false positives or negatives, in their interpretation [89, 101-103].
An excellent use of biomarkers would be to use them to asses disease progression. As they
provide information about the current ECM metabolism these markers can give therapeutic and
prognostic information regarding progression to fibrogenic state. Studies have been carried out
to evaluate the effect of interferon therapy on the level of biomarkers and correlate these with

histological findings. The result has shown significant reduction in certain markers(HA, PIIINP,
YKL-40,TIMP-1) when sustained virological and biochemical response has been achieved [104114]. Likewise, if these non invasive markers truly represent the ongoing metabolism of the
extracellular matrix than they would be able to give great prognostic information however,
limited studies are available to validate this point.

Discovery-Based Technologies
In contrast to the biomarker approach, discovery-based technologies have been proposed and
investigated upon. They use a hypothesis-generating approach towards diagnostics and include
genomics, proteomics, metabonomics and lipidomics [R10]. These techniques help discover
biomarkers that were not found before and reveal associations that were'nt considered
influential before. However these technologies do not show causal relationships and further
studies need to be done to determine the same.
The use of genomics has lead to the discovery of several single nucleotide polymorphisms (SNPs)
that can predict the progression to advanced liver fibrosis in Chronic Hepatitis C [16 R10].
Examples include an SNP in the dead box polypeptide 5 gene found to be significantly associated
with severe fibrosis and an SNP in carnitine palmitoyltransferase 1A that was found to be
associated with a decreased risk of fibrosis [31].
Similiarly, a proteomic index has been recently shown to be superior to Fibrotest (AUC of 0.88
v/s 0.81) in terms of determining severe fibrosis [31, 115]. A proteomic algorithm was found by
Poon and his colleagues for Chronic Hepatitis B that had an AUC of 0.91 for detecting significant
fibrosis in 46 patients [116]. Glyomics have also been used to assess liver fibrosis through
modifying DNA sequencer analyzers to measure serum protein N-glycans; AUC 0.87 [117]. On
these lines Mooney investigated the glycosylation of alpha-1-acid glycoprotein (AGP), and found
it to be higher in patients with significant fibrosiscirrhosis *18, 118].
Although these technique offer much practical application they are limited by their ease of
access and cost & because of high false positive discovery rate.
Other methods have been devised that include caspase activity, serum globulins, plasma amino
acids and insulin-like growth factor (IGF-1). Caspase activity has been studied as a marker of
tissue damage in chronic hep C because of its role as a proapoptotic agent [120 121]. Amino
acids have also been analysed for the similiar purpose using a validating index [122]. Maruyama
observerd that serum gamma (y)-globulin and immunoglobulin (IgG) co-related well with HAI
score (both; P< 0.0001), grading score (both P< 0.01) and staging score (bothP< 0.0001) [123].
Lorenzo-Zuniga derived that mean IGF-I values were significantly lower in patients with
advanced fibrosis (F4,65.917.9 ngmL) vs. F0, 145.247.1; F1-F2, 150.389.6; and F3, 121.435.2
ngmL;P< .05)*124].
Artificial Neural Networks (ANNs) have been devised from clinical variables and patient data sets
to predict significant fibrosis in patients with chronic hepatitis C [18]. Haydon assimilated 15

routine clinical and virological factors into a model correlated from 112 HCV patients, with
sensitivity,specificity, NPV and PPV values all greater than 92% for Ward-type ANNs for
prediction of cirrhosis [125]. A similiar analysis by Piscaglia in post-liver transplant patients gave
100% sensitivity and NPV [126]. Cucchetti meanwhile observed that ANNs measured the
mortality risk of patients with cirrhosis more accurately than the model for end-stage liver
disease (MELD) score [127].
A glycoprotein, galactose-deficient anti-Gal immunoglobulin G, identified recently has been
found to be altered in amount as well as in glycosylation as a function of liver fibrosis and
cirrhosis [128].

Monitoring Disease Progression

One of the greatest practicality & lucrative offering of non invasive modalities is their ability to
monitor progression of disease. In patients with chronic Hepatitis C attaining sustained viral
eradications TE & Biomarkers could be useful for evaluating regression [129-133]
As such Castera [11] in her article after comparing & contrasting the various non invasive
approaches concludes that TE, Fibrotest, etc need to be used in an integrated system with liver
biopsy & not as a standalone hallmark.
She also promotes the shift from cross-sectional diagnosis to using these methods in longitudnal
studies to look at disease progression, regression and clinical outcomes and maintains that
validation studies should be done on a large scale [11]

Conclusion
Significant progress is being made towards centralizing noninvasive tests in the
diagnosis and management of liver fibrosis. These tests have indeed taken centre stage
over the years but may not eventually relieve liver biopsy of its role. As more and more
researches are conducted in this novel field diagnostic criteria using these tests needs to
be developed and its significance needs to be studied over several cohorts. Many such
studies are already underway and promise significant breakthrough in the near future.
They can be used in monitoring disease response as well as picking up disease
progression as compared with liver biopsy.
Noninvasive tests can mostly only differentiate cirrhosis from minimal fibrosis. As such
liver biopsy is still needed to find significant fibrosis (F>2) therefore their utility is
restricted for prescreening which may allow the physician to narrow down the patient
population that could actually benfit from a liver biopsy.

References
1.) Janes; Lindor, KD (1993). "Outcome of patients hospitalized for complications after
outpatient liver biopsy.". Ann Intern Med 118 (2): 968. PMID 8416324
2.) Pash; Gabriel, Sherine; Therneau, Terry; Dickson, E. Rolland; Lindor, Keith D. (1998). "Costeffectiveness of ultrasound-guided liver biopsy.". Hepatology 27 (5): 1220
6. doi:10.1002/hep.510270506. PMID 9581674

3.) Perrault J, McGill DB, Ott BJ et al, Liver Biopsy : Complications in 1000 inpatients and
outpatients Gastroenterology 1978;74,103-6
4.) Thampanitchawong P, Pirathvisuth T. Liver Biopsy : Complications and risk factors. World J
Gastroenterol 1999;5,301-4
5.) Little AF, Ferris JV, Dodd GD, 3rd, et al. Image-guided percutaneous hepatic biopsy: Effect of
ascites on the complication rate. Radiology 1996;199,79-83.
6.) Lindor KD, Bru C, Jorgensen RA, et al. The role of ultrasonography and automatic needle
biopsy in outpatient percutaneous liver biopsy. Hepatology 1996;23: 1079-83.
7.) Froehlich F, Lamy O, Fried M, et al. Practice and complications of liver biopsy. Results of a
nationwide survey in Switzerland. Dig Dis Sci 1993;38:1480-4
8.) Afdhal NH, Nunes D et al. Evaluation of Liver Fibrosis : A Concise Review, American Journal of
Gastroenterology 2004 [R04]
9.) Castera L, Pinzani M. Non-invasive assessment of liver fibrosis: are we ready? Lancet
2010;375(9724):141920
10.) Bedossa P, Carrat F. Liver biopsy: the best, not the gold standard. J Hepatol 2009;50(1):13.
11.) Castera L, Invasive and non-invasive methods for the assessment of fibrosis and disease
progression in chronic liver disease, Best Practice & Research Clinical Gastroenterology
2011.[R02]
12.) S. Bonekamp, I. Kamel, S. Solga, J. Clarke et al. Can imaging modalities diagnose and stage
hepatic fibrosis and cirrhosis accurately? Journal of Hepatology 2009 [R07]
13.) Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and treatment of
hepatitis C.Hepatology 2004; 39(4): 11471171.
14.) Alberti A, Clumeck N, Collins Set al. Short statement of the first European Consensus
Conference on the treatment of chronic hepatitis B and C in HIV co-infected patients. J Hepatol
2005; 42(5): 615624.

15.) Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B: summary of a
clinical research workshop.Hepatology 2007; 45(4): 10561075.
16.) L. Castera, Transient elastography and other noninvasive tests to assess hepatic fibrosis in
patients with viral hepatitis. Journal of Viral Hepatitis, 2009,16,300314. [R08]
17.) Bravo AA, Sheth SG, Chopra S. Liver biopsy.N Engl J Med2001;344: 495500.
18.) J. O. Smith & R. K. Sterling, Systematic review: non-invasive methods of fibrosis analysis in
chronic hepatitis C, Aliment Pharmacol Ther 2009;30, 557576 [R13]
19.) Bruguera M, Bordas JM, Mas P, et al. A comparision of the accuracy of peritoneoscopy and
liver biopsy in the diagnosis of cirrhosis. Gut 1974;15,799-800.
20.) Poniachik J, Bernstein DR, Reddy KR, et al. The role of laparoscopy in the diagnosis of
cirrhosis. Gastrointest Endosc 1996;43,568-71.
21.) Pagliaro L, Rinaldi F, Craxi A, et al. Percuataneous blind biopsy versus laparoscopy with
guided biopsy in the diagnosis of cirrhosis. A prospective randomized trial Dig Dis Sci
1983;28,39-43.
22.) Olsson R, Hagerstrand I, Broome U et al. Sampling variability of percutaneous liver biopsy in
primary sclerosing cholangitis, J Clin Pathol 1995;48,933-5.
23.) Maharaj B, Maharaj RJ, Leary WP, et al. Sampling variability and its influence on the
diagnostic yield of percutaneous needle biopsy of the liver. Lancet 1986;1,523-5.
24.) Angelucci E, Barociani D, Lucarelli G, et al. Needle liver biopsy in thalassemia Analysis of
diagnostic accuracy and safety in 1184 consecutive biopsies. Br J Haematol 1995;89,757-61.
25.) Sebastiani G, Alberti A:Non invasive fibrosis biomarkers reduce but not substitute the need
for liver biopsy.World J Gastroenterol2006, 12(23):3682-3694.
26.) A Baranova, P Lal, A Birerdinc and ZM Younossi et al. Non-Invasive markers for hepatic
fibrosis BMC Gastroenterology 2011,11:91 [R05]
27.) Cadranel JF, Rufat P, Degos F. Practices of liver biopsy in France: results of a prospective
nationwide survey. For the Group of Epidemiology of the French Association for the Study of the
Liver (AFEF). Hepatology2000; 32(3):477481.
28.) Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following percutaneous liver
biopsy. A multicentre retro-spective study on 68,276 biopsies. J Hepatol 1986; 2(2):1651673.
29.) Bedossa P, Darge`re D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C.
Hepatology2003; 38:14491457.

30.) Regev A, Berho M, Jeffers LJet al.Sampling error and in-traobserver variation in liver biopsy
in patients with chronic HCV infection.Am J Gastroenterol2002; 97(10):26142618.
31.) IN. Guha, W.M. Rosenberg. Noninvasive Assessment of Liver Fibrosis : Serum Markers,
Imaging, and Other Modalities, Clin Liver Dis 12 (2008) 883900
doi:10.1016/j.cld.2008.07.0101089-3261/08/$ [R10]
32.) Scheuer PJ:Classification of chronic viral hepatitis: a need for reassessment. Journal of
Hepatology1991,13:372-4.
33.) Desmet V J, Gerber M, Hoofnagle JH,et al: Classification of chronic hepatitis: Diagnosis,
grading and staging.Hepatology1994,19:1513-20.
34.) Ratnakar KS, Sudha S:Image analysis in the evaluation of chronic hepatitis. Bahrain Medical
Bulletin 2001
35.) Harbin WP, Robert NJ, Ferucci JT, Jr. Diagnosis of cirrhosis based on regional changes in
hepatic morphology: A radiological and pathological analysis. Radiology 1980;135,273-83.
36.) Honda H, Onitsuka H, Masuda K, et al. Chronic Liver Disease: Value of volumetry of liver &
spleen with computed tomography. Radiat Med 1990;8,222-6.
37.) Hess CF, Schmiedl U, Koelbel G, et al. Diagnosis of liver cirrhosis with US: ReceiverOperating characteristic of analysis of multidimensional caudate lobe indexes. Radiology
1989;171:349-51.
38.) Sandrin L, Fourquet B, Hasquenoph JM, Yon S, Fournier C, Mal F, et al. Transient
elastography: a new noninvasive method for assessment of hepatic fibrosis. Ultrasound Med
Biol 2003;29(12):170513.
39.) Castera L, Forns X, Alberti A. Non-invasive evaluation of liverfibrosis using transient
elastography. J Hepatol 2008;48(5):83547.
40.) Roulot D, Czernichow S, Le Clesiau H, Costes JL, Vergnaud AC, Beaugrand M. Liver stiffness
values in apparently healthy subjects: influence of gender and metabolic syndrome. J Hepatol
2008;48(4):60613.
41.) L. Castera, X. Forns, A. Alberti et al. Non-invasive evaluation of liver fibrosis using transient
elastography, Journal of Hepatology 48 (2008) 835847. [R01]
42.) Lim JK, Groszmann RJ. Transient elastography for diagnosis of portal hypertension in liver
cirrhosis: is there still a role for hepatic venous pressure gradient measurement? Hepatology
2007;45:10871090.

43.) Carrion JA, Navasa M, Bosch J, Bruguera M, Gilabert R, Forns X. Transient elastography for
diagnosis of advanced fibrosis and portal hypertension in patients with hepatitis C recurrence
after liver transplantation. Liver Transpl 2006;12:17911798.
44.) deLedinghen V, Castera L, Foucher J, Tournan R, Bernard PH, Moisset G, et al. Evaluation of
fibrosis evolution using non-invasive methods according to sustained virological response in HCV
patients: a pilot prospective controlled study (abstract). Hepatology 2006;44:337A.
45.) He zode C, Castera L, Rosa I, Roulot D, Leroy V, Bouvier-Alias M, et al. Dynamics of liver
stiffness during peginterferon alpha-ribavirin treatment in patients with chronic hepatitis C
(abstract). Hepatology 2007;46:366A.
46.) Takeda T, Yasuda T, Nakayama Y, Nakaya M, Kimura M, Yamashita M, et al. Usefulness of
non-invasive transient elastog-raphy for assessment of liver fibrosis stage in chronic hepatitis C.
World J Gastroenterol 2006;12:77687773.
47.) Masuzaki R, Tateishi R, Yoshida Het al.Risk assessment of hepatocellular carcinoma in
chronic hepatitis C patients by transient elastography.J Clin Gastroenterol2008; 42(7):839843.
48.) Rigamonti C, Donato MF, Fraquelli M et al. Transient elastography predicts fibrosis
progression in patients with recurrent hepatitis c after liver transplantation.Gut2008; 57: 821
827.
49.) Corradi F, Piscaglia F, Flori Set al. Assessment of liver fibrosis in transplant recipients with
recurrent HCV infec-tion: usefulness of transient elastography. Dig Liver Dis 2009; 41(3): 217
225.
50.) Harada N, Soejima Y, Taketomi Aet al. Assessment of graft fibrosis by transient elastography
in patients with recurrent hepatitis C after living donor liver transplanta-tion. Transplantation
2008; 85(1): 6974.
51.) Sandrin L, Fourquet B, Hasquenoph JM, Yon S, Fournier C, Mal F, et al. Transient
elastography: A new non-invasive method for assessment of hepatic fibrosis. Ultrasound Med
Biol 2003;29:17051713.
52.) Fraquelli M, Rigamonti C, Conte D, Casazza G, Donato F, Ronchi G, et al. Reproducibility of
transient elastography (TE) in assessing hepatic fibrosis. Gut 2007;56:968973.
53.) Konate A, Boursier J, Reaud S, Quemener E, Fouchard-Hubert I, Oberti F. Liver stiffness
measurement by transient elastogra-phy: predictive factors of accuracy, success and
reproducibility. J Hepatol 2006;44:S195.
54.) Shaheen AA, Wan AF, Myers RP. FibroTest and FibroScan for the prediction of hepatitis Crelatedfibrosis: a systematic review of diagnostic test accuracy. Am J Gastroenterol
2007;102(11):2589600.

55.) Talwalkar JA, Kurtz DM, Schoenleber SJ, West CP, Montori VM. Ultrasound-Based transient
elastography for the detection of hepaticfibrosis: systematic review and meta-analysis. Clin
Gastroenterol Hepatol 2007;5(10):121420.
56.) Friedrich-Rust M, Ong MF, Martens S, Sarrazin C, Bojunga J, Zeuzem S, et al. Performance of
transient elastography for the staging of liverfibrosis: a meta-analysis. Gastroenterology
2008;134(4):96074.
57.) Fraquelli M, Rigamonti C, Casazza G, Donato MF, Ronchi G, Conte D,Rumi M, Lampertico P,
Colombo M:Etiology-related determinants of liver stiffness values in chronic viral hepatitis B or
C.J Hepatol 2011,54(4):621-8
58.) Coco B, Oliveri F, Maina AM, Ciccorossi P, Sacco R, Colombatto P, et al. Transient
elastography: a new surrogate marker of liver fibrosis influenced by major changes of
transaminases. J Viral Hepat 2007;14(5):3609.
59.) Sagir A, Erhardt A, Schmitt M, Haussinger D. Transient elastography is unreliable for
detection of cirrhosis in patients with acute liver damage. Hepatology 2007;47(2):5925.
60.) Arena U, Vizzutti F, Corti G, Ambu S, Stasi C, Bresci S, et al. Acute viral hepatitis increases
liver stiffness values measured by transient elastography. Hepatology 2008;47(2):3804.
61.) Millonig G, Reimann FM, Friedrich S, Fonouni H, Mehrabi A, Buchler MW, et al. Extrahepatic
cholestasis increases liver stiffness (FibroScan) irrespective of fibrosis. Hepatology
2008;48(5):171823
62.) Millonig G, Friedrich S, Adolf S, Fonouni H, Golriz M, Mehrabi A, et al. Liver stiffness is
directly influenced by central venous pressure. J Hepatol 2010;52(2):20610.
63.) Takahashi H, Ono N, Eguchi Y, Eguchi T, Kitajima Y, Kawaguchi Y, Nakashita S, Ozaki I, Mizuta
T, Toda S, Kudo S, Miyoshi A, Miyazaki K, Fujimoto K:Evaluation of acoustic radiation force
impulse elastography for fibrosis staging of chronic liver disease: a pilot study. Liver Int2010,
30(4):538-45.
64.) Yoneda M, Suzuki K, Kato S, Fujita K, Nozaki Y, Hosono K, Saito S, Nakajima A:Nonalcoholic
fatty liver disease: US-based acoustic radiation force impulse lastography.
Radiology2010,256(2):640-7
65.) Rifai K, Cornberg J, Mederacke I, Bahr MJ, Wedemeyer H, Malinski P, BantelH, Boozari B,
Potthoff A, Manns MP, Gebel M:Clinical feasibility of liver elastography by acoustic radiation
force impulse imaging (ARFI). Dig Liver Dis2011.
66.) Muthupillai R, Lomas DJ, Rossman PJ, Greenleaf JF, ManducaA, Ehman RL. Magnetic
resonance elastography by direct visualization of propagating acoustic strain waves. Science
1995;269:18541857.

67.) Muthupillai R, Rossman PJ, Lomas DJ, Greenleaf JF, Riederer SJ, Ehman RL. Magnetic
resonance imaging of transverse acoustic strain waves. Magn Reson Med 1996;36:266274.
68.) Rouviere O, Yin M, Dresner MA, Rossman PJ, Burgart LJ, Fidler JL, et al. MR elastography of
the liver: preliminary results. Radiology 2006;240:440448.
69.) Yin M, Woollard J, Wang X, Torres VE, Harris PC, Ward CJ, et al. Quantitative assessment of
hepatic fibrosis in an animal model with magnetic resonance elastography. Magn Reson Med
2007;58:346353.
70.) Huwart L, Peeters F, Sinkus R, Annet L, Salameh N, ter Beek LC, et al. Liver fibrosis: noninvasive assessment with MR elastography. NMR Biomed 2006;19:173179.
71.) Huwart L, Sempoux C, Salameh N, Jamart J, Annet L, Sinkus R, et al. Liver fibrosis: noninvasive assessment with MR elastography versus aspartate aminotransferase-to-platelet ratio
index. Radiology 2007;245:458466.
72.) Yin M, Talwalkar JA, Glaser KJ, Manduca A, Grimm RC, Rossman PJ, Fidler JL, Ehman RL, Yin,
M:Assessment of hepatic fibrosis with magnetic resonance elastography. Clin Gastroenterol
Hepatol2007,5:1207-1213.
73.) Bedossa P, Carrat F. Liver biopsy: the best, not the gold standard. J Hepatol 2009;50(1):13.
74.) Grigorescu M:Noninvasive Biochemical Markers of Liver Fibrosis.J Gastrointestin Liver
Dis2006,15(2):149-159.
75.) Veidal SS, Vassiliadis E, Bay-Jensen AC, Tougas G, Vainer B, Karsdal MA: Procollagen type I
N-terminal propeptide (PINP) is a marker for fibrogenesis in bile duct ligation-induced fibrosis in
rats. Fibrogenesis Tissue Repair2010,3(1):5.
76.) Lieber CS, Weiss DG, Paronetto F, Veterans Affairs Cooperative Study 391 Group:Value of
fibrosis markers for staging liver fibrosis in patients with precirrhotic alcoholic liver
disease.Alcohol Clin Exp Res2008,32(6):1031-9.
77.) Gressner OA, Weiskirchen R, Gressner AM:Biomarkers of liver fibrosis: clinical translation of
molecular pathogenesis or based on liver dependent malfunction tests.Clin Chim
Acta2007,381:107-113.
78.) Walsh KM, Fletcher A, MacSween RN, Morris AJ. Comparison of assays for N-amino terminal
propeptide of type III procollagen in chronic hepatitis C by using receiver operating
characteristic analysis. Eur J Gastroenterol Hepatol 1999;11: 82731.
79.) Walsh KM, Timms P, Campbell S, MacSween RN, Morris AJ:Plasma levels of matrix
metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and
TIMP-2) as non invasive markers of liver disease inchronic hepatitis C: comparison using ROC
analysis.Dig Dis Sci1999,44:624-630.

80.) Murawaki Y, Ikuta Y, Idobe Y, Kawasaki H:Serum matrix metalloproteinase-1 in patients with
chronic viral hepatitis.J Gastroenterol Hepatol 1999, 14:138-145.
81.) Badra G, Lotfy M, El-Refaie A, Obada M, Abdelmonem E, Kandeel S, Fathy A: Significance of
serum matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in chronic
hepatitis C patients. Acta Microbiol Immunol Hung 2010,57(1):29-42.
82.) Larrousse M, Laguno M, Segarra M, et al. Noninvasive diagnosis of hepatic fibrosis in HIV
HCV-coinfected patients. J Acquir Immune Defic Syndr2007;46:30411.
83.) Manning DS, Afdhal NH: Diagnosis and quantitation of fibrosis. Gastroenterology 2008,
134.Gastroenterology2008,134(6):1670-1681.
84.) Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, Dienes HP, Lohse
AW:Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of
transforming growth factor-beta.J Viral Hepat 2001,8(6):430-7.
85.) Lydatakis H, Hager IP, Kostadelou E, Mpousmpoulas S, Pappas S, Diamantis I:Non-invasive
markers to predict the liver fibrosis in non alcoholic fatty liver disease.Liver Int2006,26:864-871.
86.) Tran A, Benzaken S, Saint-Paul MC, Guzman-Granier E, Hastier P, Pradier C, Barjoan EM,
Demuth N, Longo F, Rampal P:Chondrex (YKL-40), a potential new serum fibrosis marker in
patients with alcoholic liver disease.Eur J Gastroenterol Hepatol2000,12(9):989-993.
87.) Berres ML, Papen S, Pauels K, Schmitz P, Zaldivar MM, Hellerbrand C, Mueller T, Berg T,
Weiskirchen R, Trautwein C, Wasmuth HE:A functional variation in CHI3L1 is associated with
severity of liver fibrosis and YKL-40 serum levels in chronic hepatitis C infection. J Hepatol 2009,
50(2):370-6.
88.) Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Polynard T; MULTIVIRC
Group. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a
prospective study. Lancet, The2001;357: 106975.
89.) Imbert-Bismut F, Messous D, Thibault V, Myers RB, Piton A, Thabut D, Devers L, Hainque B,
Mercadier A, Poynard T:Intra-laboratory analytical variability of biochemical markers of fibrosis
(Fibrotest) and activity(Actitest) and reference ranges in healthy blood donors. Clin Chem Lab
Med2004,42(3):323-333.
90.) Poynard T, Morra R, Halfon P, Castera L, Ratziu V, Imbert Bismut F, et al. Meta-analyses of
Fibrotest diagnostic value in chronic liver disease. BMC Gastroenterol 2007;7(1):40.
91.) Shaheen AA, Myers RP. Diagnostic accuracy of the aspartate aminotransferase-to-platelet
ratio index for the prediction of hepatitis C-relatedfibrosis: a systematic review. Hepatology
2007;46(3):91221.

92.) Parkes J, Guha IN, Roderick P, Rosenberg W. Performance of serum marker panels for
liverfibrosis in chronic hepatitis C. J Hepatol 2006;44(3):46274.
93.) Halfon P, Bacq Y, De Muret A, Penaranda G, Bourliere M, Ouzan D, et al. Comparison of test
performance profile for blood tests of liverfibrosis in chronic hepatitis C. J Hepatol
2007;46(3):395402.
94.) Leroy V, Hilleret MN, Sturm N, Trocme C, Renversez JC, Faure P, et al. Prospective
comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C. J
Hepatol 2007;46(5):77582.
95.) Cales P, de Ledinghen V, Halfon P, Bacq Y, Leroy V, Boursier J, et al. Evaluating the accuracy
and increasing the reliable diagnosis rate of blood tests for liverfibrosis in chronic hepatitis C.
Liver Int 2008;28(10):135262.
96.) Degos F, Perez P, Roche B, Mahmoudi A, Asselineau J, Voitot H, et al. Diagnostic accuracy of
FibroScan and comparison to liver fibrosis biomarkers in chronic viral hepatitis: a multicenter
prospective study (the FIBROSTIC study). J Hepatol 2010; 53(6):101321.
97.) Giannini E, Risso D, Botta F, et al. Validity and clinical utility of the aspartate
aminotransferase-alanine aminotransferase ratio in assessing disease severity and prognosis in
patients with hepatits C virus-related chronic liver disease. Arch Intern Med 2003;163,218-24.
98.) Lin ZH, Xin YN, Dong QJ, Wang Q, Jiang XJ, Zhan SH, Sun Y, Xuan SY: Performance of the
aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis C-related fibrosis:
an updated meta-analysis. Hepatology2011,53(3):726-36.
99.) Sebastiani G, Vario A, Guido M, Noventa F, Plebani M, Pistis R, Ferrari A, Alberti A: Stepwise
combination algorithms of non-invasive markers to diagnose significant fibrosis in chronic
hepatitis C.J Hepatol2006, 44(4):686-93.
100.) Sebastiani G, Halfon P, Castera L, Pol S, Thomas DL, Mangia A, Di Marco V, Pirisi M,
Voiculescu M, Guido M, Bourliere M, Noventa F, Alberti A: SAFE biopsy: a validated method for
large-scale staging of liver fibrosis in chronic hepatitis C.Hepatology2009,49(6):1821-7.
101.) Piton A, Poynard T, Imbert-Bismut F, Khalil L, Delattre J, Pelissier E, et al. Factors associated
with serum alanine trans-aminase activity in healthy subjects: consequences for the definition of
normal values, for selection of blood donors, and for patients with chronic hepatitis C.
MULTIVIRC Group. Hepatology 1998;27(5):12139.
102.) Cales P, Veillon P, Konate A, Mathieu E, Ternisien C, Chevailler A, et al. Reproducibility of
blood tests of liverfibrosis in clinical practice. Clin Biochem 2008;41(12):108.

103.) Poynard T, Munteanu M, Imbert-Bismut F, Charlotte F, Thabut D, Le Calvez S, et al.

Prospective analysis of discordant results between biochemical markers and biopsy in patients
with chronic hepatitis C. Clin Chem 2004;10:10.
104.) Leroy V, De Traversy C, Barnoud R, et al. Changes in histological lesions and serum
fibrogenesis markers in chronic hepatits C patients non-responders to interferon alpha. J
Hepatol 2001;35:120-6.
105.) Ninomiya T, Yoon S, Hayashi Y, et al. Clinical significance of serum hyaluronic acid as a
fibrosis marker in chronic hepatitis C patients treated with interferon alpha: Histological
evaluation by modified histological activity index scoring system. J Gastroenterol Hepatol
1998;13,68-74.
106.) Nojgaard C, Johansen JS, Krarup HB et al. Effect of antiviral therapy on the markers of
fibrogenesis in patients with chronic hepatitis C. Scand J Gastroenterol 2003;38:659-65.
107.) Mitsuda S, Suou T, Ikuta Y, et al. Changes in tissue inhibitor of matrix metalloproteinase-1
after interferon alpha treatment in chronic hepatitis C J. Hepatol 2000;32,666-72.
108.) Guechot J, Poupon RE, Poupon R. Serum Hyaluronan as a marker of liver fibrosis. J Hepatol
1995;22,103-6
109.) Ishibashi K, Kashiwaqi T, Ito A, et al. Changes in serum fibrogenesis markers during
interferon therapy for chronic hepatits C. Hepatology 1996;24,27-31.
110.) Hiramatsu N, Hayashi N, Kasahara A, et al. Improvement of liver fibrosis in chronic hepatits
C patients treated with natural interferon alpha. J Hepatol 1995;22:135-42.
111.) Mazzoran L, Tamaro G, Mangiarotti MA, et al. Effects of interferon therapy on fibrosis
serum markers in HCV positive chronic liver disease. Eur J Gastroenterol Hepatol 1998;10:12531.
112.) Abe S, Tabaru A, Ono M, et al. High dose interferon alpha therapy lowers the levels of
serum fibrogenesis markers over 5 years in chronic hepatits C. Hepatol Res 2003;25,22-31.
113.) Kojima H, Hongo Y, Harada H, et al. Long term histological prognosis and serum fibrosis
markers in chronic hepatits C. Hepatol Res 2003;25,22-31.
114.) Serejo F, Costa A, Oliviera AG, et al. Alpha-interferon improves liver fibrosis in chronic
hepatitis C: Clinical Significance of serum N-terminal propeptide of procollagen type III. Dig Dis
Sci 2001;46:1684-9.
115.) Morra R, Munteanu M, Bedossa P, et al. Diagnostic value of serum protein profiling by
SELDI-TOF ProteinChip compared with a biochemical marker, FibroTest, for the diagnosis of
advanced fibrosis in patients with chronic hepatitis C. Aliment Pharmacol Ther 2007;26(6):847
58.

116.) Poon TC, Hui AY, Chan HL, et al. Prediction of liver fibrosis and cirrhosis in chronic hepatitis
B infection by serum proteomic fingerprinting: a pilot study. Clin Chem 2005;51(2):32835.
117.) Callewaert N, Van Vlierberghe H, Van Hecke A, et al. Noninvasive diagnosis of liver
cirrhosis using DNA sequencer-based total serum protein glycomics. Nat Med 2004;10(4):429
34.
118.) Mooney P, Hayes P, Smith K. The puta-tive use of alpha-1-acid glycoprotein as a noninvasive marker of fibrosis. Bio-med Chromatogr2006;20: 13518.
119.) Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP:An appraisal of the
histopathological assessment of liver fibrosis.Gut2006,55:569-578.
120.) Bantel H, Lugering A, Poremba C,et al. Caspase activation correlates with thedegree of
inflammatory liver injury in chronic hepatitis C virus infection.Hepa-tology 2001;1(4 Pt 1): 758
67.
121.) Bantel H, Lugering A, Heidemann J,et al. Detection of apoptotic caspase activation in sera
from patients with chronic HCV infection is associated with fibrotic liver injury.
Hepatology2004;40: 107887.
122.) Zhang Q, Takahashi M, Noguchi Y,et al. Plasma amino acid profiles applied for diagnosis of
advanced liver fibrosis in patients with chronic hepatitis C infec-tion. Hepatology research 2006;
34: 1707.
123.) Maruyama S, Hirayama C, Horie Y,et al. Serum immunoglobulins in patients with chronic
hepatitis C: a surrogate marker of disease severity and treatment out-come.
Hepatogastroenterology2007;54: 4938.
124.) Lorenzo-Zuniga V, Bartol R, Masnou H, Montoliu S, Morillas RM, Planas R. Serum
concentrations of insulin-like growth factor-I (igf-I) as a marker of liver fibrosis in patients with
chronic hepatitis C.Dig Dis Sci2007;52: 324550.
125.) Haydon GH, Jalan R, Ala-Korpela M, et al. Prediction of cirrhosis in patients with chronic
hepatitis C infection by artificial neural network analysis of virus and clinical factors.J Viral
Hepat1998;5: 25564.
126.) Piscaglia F, Cucchetti A, Benlloch S, et al.Prediction of significant fibrosis in hepatitis C virus
infected liver transplant recipients by artificial neural network analysis of clinical factors. Eur J
Gastro-enterol Hepatol2006;18: 125561.
127.) Cucchetti A, Vivarelli M, Heaton ND, et al. Artificial neural network is supe-rior to MELD in
predicting mortality of patients with end-stage liver disease. Gut 2007;56: 2538.

128.) Mehta AS, Long RE, Comunale MA, et al. Increased levels of galactose-defi-cient anti-Gal
immunoglobulin G in the sera of hepatitis C virus-infected individ-uals with fibrosis and cirrhosis.
J Virol 2008;82: 125970.
129.) Hzode C, Castra L, Roudot-Thoraval F, Rosa I, Roulot D, Leroy V, et al. Prospective
evaluation of liver stiffness dynamics during and after peginterferon alpha-ribavirin treatment in
patients with chronic hepatitis C (abstract). J Hepatol 2009; 50(Suppl.1):S226.
130.) Ogawa E, Furusyo N, Toyoda K, Takeoka H, Maeda S, Hayashi J. The longitudinal
quantitative assessment by transient elastography of chronic hepatitis C patients treated with
pegylated interferon alpha-2b and ribavirin. Antiviral Res 2009;83(2):12734.
131.) Vergniol J, Foucher J, Castera L, Bernard PH, Tournan R, Terrebonne E, et al. Changes of
non-invasive markers and FibroScan values during HCV treatment. J Viral Hepat 2009;16(2):132
40.
132.) Poynard T, McHutchison J, Manns M, Myers RP, Albrecht J. Biochemical surrogate markers
of liver fibrosis and activity in a randomized trial of peginterferon alfa-2b and ribavirin.
Hepatology 2003;38(2):48192.
133.) Poynard T, Munteanu M, Colombo M, Bruix J, Schiff E, Terg R, et al. FibroTest is an
independent predictor of virologic response in chronic hepatitis C patients retreated with
pegylated interferon alfa-2b and ribavirin in the EPIC(3) program. J Hepatol 2011;54(2):22735