Non Invasive Evaluation OF Liver Fibrosis IN Patients With Chronic Hepatitis B and C

NON INVASIVE EVALUATION
OF
LIVER FIBROSIS
IN
PATIENTS WITH CHRONIC
HEPATITIS B AND C
(Ph.D. Thesis)
SHAHZAD ASHRAF
MBBS
Diplomate American Board of Internal Medicine
Dept of Medicine
Baqai Medical University
2006
Supervisor
Professor Lt Gen (R) Syed Azhar Ahmed

Hilal-e-Imtiaz (M), Sitara-e-Basalat
MBBS, Ph.D. (London), FRCPath (London), FCPS
Vice Chancellor
Co Supervisor
Professor Jameel Ahmed
MBBS, MRCP
Professor of Medicine
ii
This work is dedicated to
My parents & wife

for their constant encouragement and support
and to
Prof Lt Gen (R) S Azhar Ahmed

for his invaluable help
and
without whom this work could not have been possible
iii
Acknowledgements
Prof Thierry Poynard, Paris, France, for imparting great insight in to the
subject and providing guidance to an unknown research worker
Pakistan Medical Research Council for approving special grant for the
project
Lt Col Nadir Ali, Classified Pathologist and Hematologist, for providing
assistance at performing the biochemical assays, hematological
investigations; and also for his helpful suggestions in writing the
manuscript
Mr. Iqbal Mujtaba, for his assistance in statistical analysis, and patient
listening to scientific details
Dr Faisal, Dr Yasmin Wahid and Dr Bushra Ayaz for liver biopsy
interpretation
Dr Hina, for her help in the sensitive assays for hyaluronic acid, alpha 2
macroglobulin, and graphic analysis
Hafiz Zulfiqar for his help in assays, and supervising the laboratory staff to
carry out investigations
Paramedical staff of the Medical Dept. and laboratory for their support in
specially attending to the chronic hepatitis B and C patients taking time
out from their busy schedule
Library staff of the College of Physicians and Surgeons and Aga Khan
University Hospital for their courteous help in providing access to the
journals and textbooks by print and electronic media
Researches who have contributed in the past to the “Non invasive evaluation
of liver fibrosis”
iv
CONTENTS
Page
ABSTRACT 1
LIST OF TABLES 3
LIST OF GRAPHS 5
LIST OF FIGURES 6
INTRODUCTION 10
Chap 1 INTRODUCTION AND OVERVIEW 11
LITERATURE REVIEW 53
Chap 2 EPIDEMIOLOGY AND NATURAL HISTORY 54
OF CHRONIC HEPATITIS B AND C VIRUSES
Chap 3 HEPATIC INJURY, FIBROSIS, AND FIBROGENESIS 104
(THE CELL AND MOLECULAR BIOLOGY)
v
Chap 4 LIVER BIOPSY AND HEPATIC 185
HISTOPATHOLOGY
Chap 5 NON INVASIVE EVALUATION OF LIVER 279
FIBROSIS
PATIENTS AND METHODS 349
RESULTS 358
DISCUSSION 385
SUMMARY 415
CONCLUSION 420
vi
ABSTRACT
ABSTRACT
Chronic viral hepatitis is an important cause of morbidity and mortality in the present day
world [Goldstein et al, 2005; Baldo et al, 2008]1,2. The situation is particularly precarious
in the developing countries [Jafri et al, 2006]3. It is estimated that by the year 2020-5,
there will be three fold rise in cirrhosis, and hepatocellular carcinoma from HBV and
HCV [Nguyen et al, 2008; Law et al, 2003]4,5. In chronic viral hepatitis the prognosis and
management are highly dependent on the extent of liver fibrosis [Sebastiani et al, 2006]6.
Though classically considered the “gold standard”; the liver biopsy is far from perfect,
and has significant limitations [Poynard et al, 2004]7. This has led researchers to look for
other methods to assess the stage of liver fibrosis [Afdhal et al, 2004]8.The noninvasive
markers are the most widely used alternative to liver biopsy [ Manning et al, 2008;
Castera et al, 2007; Morra et al, 2007]9,10,11.
In the study presented, the association between serum markers, platelet parameters and
liver fibrosis was investigated taking liver biopsy as the reference standard. A set of 5
serum markers, Fibroscore, consisting of: bilirubin, gamma glutamyl transferase (GGT),
hyaluronic acid (HA), alpha 2 macroglobulin (A2M), and platelets; has shown very high
diagnostic accuracy for the near absence of fibrosis, and cirrhosis. The area under the
ROC for F2 (stage 2) fibrosis was 0.808, for F3 the ROC was 0.938, and for F4 the ROC
was 0.959. A central cut off point of > 0.5, in the model, predicted clinically significant
fibrosis, (F2, F3 and F4) with a sensitivity of 82%, specificity of 92%, and overall
diagnostic accuracy of 89%. By increasing the cut off to 0.65, for stages F2-F4, the PPV
was 95%. Lowering the cut off to < 0.08 for the exclusion of stages F2-F4 provided 98%
NPV, thus almost certainly ruling out stages F2-F4. The PDW index consists of platelet
distribution width (PDW), mean platelet volume (MPV), and platelet count. The area
under the ROC for advanced fibrosis (F3-F4) for PDW index was 0.840, compares with
the well known AST to Platelet Ratio Index (APRI) with area under ROC of 0.888.
It is concluded that, Fibroscore has a high diagnostic accuracy for stages F2-F4, and
PDW Index reliably predicts advanced fibrosis. The noninvasive markers will be helpful
in the screening and management of fibrotic liver disease [Morra et al, 2007]11, and will
replace liver biopsy in most patients with chronic liver disease from viral related causes
[Castera et al, 2007; Morra et al, 2007]10,11.
2
TABLES
Literature Review Page No.
Table 1 Prevalence of hepatitis B in various areas of the world 59
Table 2 Host and viral factors in HBV disease progression 66
Table 3 Factors associated with advanced fibrosis among patients with 79
chronic HCV infection
Table 4 Published studies of paired liver biopsy assessment of fibrosis 82
progression in patients with chronic HCV infection
Table 5 Causes of fibrosis and cirrhosis 108
Table 6 Indications for liver biopsy 192
Table 7 Indications for transjugular liver biopsy 199
Table 8 Indications for and contraindications to laparoscopic liver biopsy 201
Table 9 Complications of percutaneous liver biopsy 207
Table 10 Contraindications to percutaneous liver biopsy 209
Table 11 Knodell Histological Activity Index 241
Table 12 Modified Knodell Histological Activity Index – The Ishak Score 243
Table 13 METAVIR Score 247
Table 14 Scheuer scoring system 251
Table 15 Batts and Ludwig system 252
Table 16 Laennec scoring system for fibrosis 253
Table 17 Direct markers of liver fibrosis 296
3
Results Page No.
Table 18 Diagnostic indices of FIBROSCORE > 0.5 for clinically 364
significant fibrosis
Table 19 Clinical and demographic data of patients 365
Table 20 Virological profile of patients 366
Table 21 Liver biopsy and needle characteristics 367
Table 22 Histopathological characteristics 368
Table 23 Viral aetiology and histopathology 369
Table 24 Lipid profile and HCV 369
Table 25 Clinical/ultrasonographic/biopsy parameters vs significant fibrosis 370
Table 26 Biomarkers vs fibrosis categories (statistically significant) 371
Table 27 Biomarkers vs fibrosis categories (other) 372
Table 28 Platelet parameters vs advanced fibrosis 373
Table 29 Logistic regression analysis 374
4
GRAPHS Page No.
Graph 1 Receiver Operating Characteristic (ROC) curve Fibroscore vs 375
F3 Fibrosis
Graph 2 ROC curve, Fibroscore vs F2 Fibrosis 376
Graph 3 Boxplot of predicted probability of Fibrosis (Fibroscore) vs various 377
stages of Fibrosis
Graph 4 Boxplot of Hyaluronic Acid values vs different Fibrosis stages 378
Graph 5 Boxplot of Alpha 2 Macroglobulin values vs different Fibrosis stages 379
Graph 6 ROC curve of PDW Index vs F3 Fibrosis 380
Graph 7 APRI and PDW Index ROC vs Stage F3-F4 Fibrosis 381
Graph 8 Boxplot of Platelets vs various stages of Fibrosis 382
Graph 9 Boxplot PDW Index vs various stages of Fibrosis 383
5
FIGURES Page No.
Fig. 1 Gross image of a normal and cirrhotic liver 150
Fig. 2 Diagnosis of cirrhosis – CAT Scan 151
Fig. 3 Pathogenesis of liver fibrosis – Stellate Cell 152
Fig. 4 Scanning electron micrograph of the sinusoidal endothelium from rat 153
liver showing the fenestrated wall
Fig. 5 Fibrogenic cascade 154
Fig. 6 Stellate Cell Activation 155
Fig. 7 Pathogenesis of intrahepatic portal hypertension 156
Fig. 8 Chronic hepatitis with mild activity and no fibrosis, x 10 H & E 254
Fig. 9 Chronic hepatitis with fatty change (steatosis), moderate activity and 255
portal fibrosis, x 10 H & E
Fig. 10 Chronic hepatitis with fatty change (steatosis), moderate activity and 256
portal fibrosis, x 20 H & E
Fig. 11 Chronic hepatitis with moderate activity and portal fibrosis, x 10 H & E 257
Fig. 12 Chronic hepatitis with moderate activity and portal fibrosis, x 20 H & E 258
Fig. 13 Chronic hepatitis with moderate activity and bridging fibrosis, 259
x 10 H & E
6
Fig. 14 Chronic hepatitis with moderate activity and bridging fibrosis, 260
x 20 H & E
Fig. 15 Chronic hepatitis with moderate to marked activity and early cirrhosis, 261
x 10 H & E
Fig. 16 Chronic hepatitis with moderate to marked activity and early cirrhosis, 262
x 20 H & E
Fig. 17 Fibroscore assessment for clinically significant fibrosis 363
7
REFERENCES
1. Goldstein ST, Zhou F, Hadler SC, et al. A mathematical model to estimate

global hepatitis B disease burden and vaccination impact. Int J Epidemiol
2005;34(6):1329-39.
2. Baldo V, Baldovin T, Trivello R, et al. Epidemiology of HCV infection. Curr
Pharm Des 2008;14(17):1646-54.
3. Jafri W, Jafri N, Yakoob J, et al. Hepatitis B and C: prevalence and risk factors
associated with seropositivity among children in Karachi, Pakistan. BMC
Infectious Diseases 2006, 6: 101.
4. Nguyen VT, Law MG, Dore GJ. An enormous hepatitis B virus-related liver
disease burden projected in Vietnam by 2025. Liver Int 2008 ;28(4):525-31.
5. Law MG, Dore GJ, Bath N, et al. Modelling hepatitis C virus incidence,
prevalence, and long term sequelae in Australia, 2001. Int J Epidemiol 2003; 32:
717-724.
6. Sebastiani G, Alberti A. Noninvasive fibrosis biomarkers reduce but not
substitute the need for liver biopsy. World J Gastroenterol 2006;12(23):3682-
3694.
7. Poynard T, Munteanu M, Imbert-Bismut F, et al. Prospective analysis of
discordant results between biochemical markers and biopsy in patients with
chronic hepatitis C. Clin Chem 2004;50(8):1344-1355.
8. Afdhal NH, Nunes D. Evaluation of liver fibrosis: a concise review. Am J
Gastro 2004; 99; 1160-1174.
9. Manning DS, Afdhal NH. Diagnosis and quantitation of fibrosis.
Gastroenterology. 2008 ;134(6):1670-81.
8
10. Castera L, Denis J, Babany G, et al. Evolving practices of noninvasive markers
of liver fibrosis in patients with chronic hepatitis C in France: Time for new
guidelines? J Hepatol 2007; 46: 528-529.
11. Morra R, Munteanu M, Imbert-Bismut F, et al. FibroMAX™: towards a new
universal biomarker of liver disease? Expert Rev Mol Diagn 2007; 7(5): 481-
490.
9
Chapter 1
INTRODUCTION
&
OVERVIEW
Contents
Introduction 12
Burden of chronic hepatitis 12
Liver fibrosis 13
Anti fibrotic treatment 15
Evaluation of liver fibrosis 17
Liver biopsy 18
Noninvasive markers of liver fibrosis 21
Indirect markers of liver fibrosis 22
Direct markers of liver fibrosis 26
Gene Markers and Liver Fibrosis 28
Fibrosis Markers to Assess the Effect of Treatment and 29
Disease Progression
Fibroscan & Caffeine Breath Test 31
Meta-analysis of Studies of Fibrosis Markers 33
11
1. Introduction
Chronic liver diseases, CLD, are a major cause of morbidity and mortality in the
1-6
present day world . Chronic infections with hepatitis B and C viruses, alcoholic and
3,7-13
non-alcoholic fatty liver diseases are important causes of CLD . The major
determinant in their prognosis is the progressive accumulation of fibrosis, with distortion
of the hepatic architecture, and ultimate progression to cirrhosis and its allied
3,8,9,14-21
complications . In the recent years, with the great advancement in therapeutic
modalities to treat chronic liver diseases, accurate assessment of fibrosis has become of
paramount importance; to guide management decisions, predict outcome (prognosis), and
monitor disease activity in individual patients 21-26.
1.1 Burden of chronic hepatitis

HBV is the most common cause of cirrhosis and hepatocellular carcinoma in the
world today. Of approximately 2 billion people who have been infected with HBV
worldwide, more than 350 million, or about 5% of the world’s population are chronic
1,27
carriers, and with an annual incidence of more than 50 million . HBV accounts for
500,000 to 1.2 million deaths per year 4.
Similarly more than 3 % of the world population, or about 170-5 million people
may be infected with HCV 2,3,28,29. The prevalence of HCV is increasing and estimates of
the future burden of chronic hepatitis C predict at least a 3 fold rise in chronic liver
disease and cirrhosis by the year 20203,30-32.
Nonalcoholic fatty liver disease (NAFLD) affects 10-30% of the general
population in various countries. 10% of these people, or ~ 2-3% of the general
population, satisfy the criteria for nonalcoholic steatohepatitis, (NASH). The prevalence
of NAFLD is also expected to rise in developed countries given the epidemic of its major
determinant, obesity 6,11,12,20,25,33-36.
Studies indicate that advanced fibrosis and cirrhosis will develop in 20-40% of
patients with chronic hepatitis B or C and in a similar proportion of patients with
nonalcoholic fatty liver disease 3,4,5,7,9,10,11,12,37,38.
12
1.2 Liver fibrosis
Fibrogenesis is a ubiquitous process in chronic inflammatory diseases in human
beings, in response to injury. Progressive fibrosis with the development of cirrhosis, is a
complication of all chronic liver diseases whatever their etiology, whether viral,
autoimmune, biliary, toxic, or metabolic disease 13,17,18,19,24,39.
Fibrogenesis is a non-specific mechanism, which lasts as long as injury persists
and is believed to help limit the extension of inflammatory reaction. Fibrosis, therefore, is
a physiologic mechanism, which is at first beneficial, but becomes pathological if viral
infection and chronic hepatocellular injury persist 13,16,40.
Fibrosis is the deposition of extracellular matrix, ECM, components within the
liver parenchyma. It is the consequence of a dynamic mechanism of gene transcription
and protein synthesis of ECM compounds termed fibrogenesis 17,18,40,41.
In recent years, substantial progress has been made in our understanding of the
pathophysiology of extracellular matrix, ECM, deposition and metabolism. Extracellular
matrix, is a complex mixture of glycoproteins (collagen, elastin, fibronectin, laminin) and
proteoglycans organized into complex polymers, which are insoluble and arranged in a
13,16,40-44
tridimensional network that induces loss of liver architecture . Basement
membranes are usually absent along the sinusoid but are deposited during the process of
cirrhosis, a process termed ‘sinusoid capillarisation’ 45.
The collagens are the most important molecular targets, since 1) they represent
the major matrix proteins, 2) they form important mechanical scaffolds, and 3) their
proteolysis by specific proteases appears to be the rate limiting for ECM removal. In
cirrhosis the collagen content increases 10 fold. In normal liver, ECM comprises less than
3% of area on a liver cut section. In cirrhosis a quantitative increase of up to 30%-40%
and composition modification is observed. Other major compounds of liver ECM are
glycoproteins such as laminin and fibronectin, elastic fibers, and proteoglycans
13,18,42,44,46
.
It is now clear that ECM metabolism is a very dynamic process and that
deposition of ECM is much more reversible than was previously thought. Hepatic stellate
cells are the major source of extracellular matrix deposition, although other cell types
19,24,40,47,48
such as portal fibroblasts may play a role . Hepatic stellate cells are located
13
within the perisinusoidal space (or space of Disse) between the endothelial wall of the
sinusoid and the vascular face of the hepatocytes 49.
The mechanism of hepatic stellate cell activation has been divided into two steps:
initiation and perpetuation, both are closely integrated 50. Factors that trigger the initial
step of the process are initiators. After HSC’s activation is initiated, they engage in the
process and gain an activated phenotype, this phenotype is maintained because of the
perpetuation mechanism involving other growth factors 13,51.
The HSC’s can be activated by several cytokines (e.g. tumor growth factor beta
(TGF-b), tumor necrosis factor alpha (TNF-a), platelet derived growth factor (PDGF),
which are secreted in response to liver injury. On the other hand, other signals, e.g.
interleukin-10 (IL-10) promote ECM degradation. Once activated, HSC’s secrete
cytokines such as metalloproteinases, TGF-b1, PDGF, monocyte chemotactic protein 1
(MCP-1), and endothelin (ET-1). Some of these are directly involved in the fibrogenesis
(TGF-b 1, connective tissue growth factor), others in chemotaxis (MCP-1) and
proliferaton of HSC’s (PDGF, ET-1), and others in matrix degradation
(metalloproteinases) 13,19,24,40, 41.
A variety of adverse stimuli such as toxins, viruses, bile stasis, and hypoxia can
18
trigger fibrogenesis , and its pathogenesis is somehow related to the etiology of the
underlying CLD 24.
The mechanisms by which HBV and HCV induce liver fibrosis are only partially
understood. In chronic hepatitis B infection the pathogenesis of hepatic fibrosis has been
associated with cytokines, particularly TGF-b 152. HCV on the other hand induces
oxidative stress and recruitment of inflammatory cells, with HSC’s activation and
collagen deposition. In addition several HCV proteins directly stimulate the fibrogenic
pathways of HSC’s 53. By interaction with mitochondria, core protein induces reactive
54,55
oxygen intermediates and thus oxidative stress which can induce steatohepatitis .
Hepatitis C virus nonstructural genes (NS3 and NS5B) are able to induce increased
expression of TFG-b1 and of other profibrogenic factors in infected hepatocytes 56. These
cellular events directly mediated by HCV in infected hepatocytes could explain the
occurrence of progressive liver fibrosis with minimal inflammation 24.
14
In all forms of chronic liver disease, including chronic hepatitis C, the
development of fibrosis is a step by step process starting from minimal fibrosis limited to
in and around the portal tracts (periportal or zone 1 fibrosis), followed by more extensive
fibrosis extending in to the lobules in the liver parenchyma towards the central veins
(zone 3), which can form bridges between two portal tracts or portal tracts and central
veins eventually ending in cirrhosis 57,58,59.
In some patients, the rate of fibrosis is rapid so that cirrhosis eventually develops,
and, with it the major complications of chronic viral hepatitis; end stage liver disease,
portal hypertension, and hepatocellular carcinoma. In other patients fibrosis does not
develop or progresses so slowly that after decades of infection, little or no fibrosis is
found on liver biopsy. Such patients are unlikely to suffer the long term complications of
chronic viral hepatitis 16.
For e.g., in a retrospective study for the natural evolution of chronic hepatitis C;
1/3rd of the patients progressed to cirrhosis in approximately 20 years, 1/3rd within
approximately 50 years, and 1/3rd didn’t show any sign of progression 60.
In a mathematical model developed in a large number of patients with single liver
biopsy; the suggested average (median) rate of progression of fibrosis in chronic hepatitis
61
C was 0.13 Metavir points per year . Based on this rate, the average patient would
develop cirrhosis in 30 years. The rate of progression was higher in men, age > 40 years,
and heavy alcohol drinkers >50g/day. Moreover, the time required to progress from stage
1 to 2 may be far longer than the time required to progress from stage 3 to 4 ( or vice
versa) 16.
For these reasons, clinicians and patients require accurate information about the
degree of fibrosis to guide management decisions, monitor disease activity, and make an
assessment of prognosis 23.
1.3 Anti fibrotic treatment

The best way not to have fibrosis, is not to have CLD altogether; the old dictum,
‘prevention is better than cure.’ The next best option is the treatment of the underlying
cause of CLD, for e.g., chronic HBV and HCV. While immunosuppressive cytokines are
usually profibrogenic, the antiviral interferons are antifibrogenic. Interferon-a blocks
15
activation, proliferation and collagen synthesis of hepatic stellate cells and
myofibroblastic cells 62. Retrospective analyses and also prospective studies in patients
with chronic hepatitis C suggest that Interferon-a therapy can prevent fibrosis
progression, even in nonresponders to antiviral therapy 18,60,63,64.
Recently, new targets for therapies aimed at preventing or inhibiting liver fibrosis
19
independent of the underlying disease aetiology are being explored . Animal studies
have identified a number of potentially important therapeutic targets, which include
65 66 67
glucose regulation, leptin , endothelin , angiotensin II , and a possible role for
68
PPARg (peroxisome proliferator-activated receptors). All of these can be modulated
using a variety of currently available therapies 19.
New understanding and insights into the pathogenesis of hepatocellular damage,
hepatic fibrogenesis, and the development of molecular techniques; such as new viral or
non-viral vector systems, RNAi, ribozyme and antisense technologies have made it
possible to modulate the expression of specific genes involved in the key pathways of
liver injury and fibrosis. For instance by blocking the Fas-ligand activity by siRNA or
antisense against Fas receptor almost completely prevented the Fas ligand-induced
fulminant liver failure and animal mortality. It has been an important and noteworthy
observation. It is also striking that silencing of a key enzyme, caspase 3 or 7, in an
apoptotic cascade by siRNA blocks the cell damage 40.
Alternatively, when the overexpression of fibrogenic cytokines, such as TGF-b
and PDGF and their receptors are inhibited at the mRNA level with antisense, ribozymes
or siRNA, or interrupted by specific antibodies or protein molecules; the interventions
result in marked amelioration of hepatic fibrosis. Moreover, delivery of collagenases
genes by adenoviral vectors promotes the resolution of excessive deposition of ECM
components and reverses hepatic fibrosis 69,70,71.
The transition of these promising molecular therapies from animal models to
clinical trials for the validation of their clinical efficacy and adverse effects in the target
patient population usually will take a long period of time 40.
Human studies are urgently needed to evaluate the effectiveness of therapies
aimed at each of the molecular and genetic targets. Furthermore, it is vital that
noninvasive methods for the assessment of liver fibrosis be developed and validated, so
16
that the impact of any new therapies can be readily assessed in a simple way and
subsequently followed up easily19.
1.4 Evaluation of liver fibrosis

According to Afdhal NH 22, one of the major problems faced by the hepatologists
and gastroenterologists today is: how best to evaluate and manage the increasing burden
of patients with chronic viral and likely non-alcoholic fatty liver hepatitis.
In the last decade, advances in serologic and virologic testing and improvements
in therapy have led more patients to be identified and to seek treatment. Unfortunately,
despite our improved understanding of the mechanisms involved in ECM deposition, the
complex nature of the process continues to be elusive. Little progress has been made in
improving either our ability to determine the degree of hepatic injury, particularly
fibrosis, or to predict the risk of disease progression for the individual patient 22.
Understanding the pathogenesis of hepatic fibrosis on a molecular level has led to
the identification of several putative serum markers of hepatic fibrosis. Either
individually or in combination, these serum markers appear capable of determining early
and advanced hepatic fibrosis. It is vital that new noninvasive methods for the assessment
of liver fibrosis be developed and validated, so that the impact of any new therapies can
be rapidly assessed in a simple and meaningful way. This information is still provided by
the old fashioned liver biopsy. The clinicians rely on the liver biopsy for both prognostic
and therapeutic decision making, which can have a major impact on patient’s life 22.
A review of the accuracy of liver biopsy in the assessment of liver fibrosis is
necessary, as this has been used as the ‘gold standard’ for almost all studies of
noninvasive markers of liver fibrosis 19,22; but this ‘gold’ appears to have tarnished, in
certain respects.
17
1.5 Liver biopsy
There is no ideal reference for the assessment of liver histology 72. The diagnosis
of liver fibrosis has traditionally relied on liver biopsy. But for both the physician and the
patient, the decision to proceed with liver biopsy is not a trivial one. Patients are reluctant
to submit to repeated biopsies, which limits our ability to monitor disease progression and
effects of treatment. Recent studies have suggested that there can be up to a 33% error in
the diagnosis of cirrhosis 19. Liver biopsy, which is considered the ultimate standard, has
three major limitations 72: 1) a risk of adverse events73, 2) sampling error 74,75,76, and 3)
intra and inter observer variability 77. Over view of the current literature summarizes the
risk of liver biopsy as pain (~ 30%), severe adverse events (3 in 1000), prolonged
hospital stay 1-5%, and death (3 in 10,000) 73,78-81. Most, but not all, studies have shown
that the incidence of complications is related to both the prevalence of relative
contraindications as well as the number of biopsies taken 78,82.
Sampling variability is the major source of variation. Chronic hepatitis does not
affect the liver uniformly. The extent of fibrosis may vary from one part of the liver to
another; this is especially true of macronodular cirrhosis. The fibrosis starts in and
around the portal tracts, and then extends into the liver parenchyma towards the central
57,58,59
vein . Liver biopsy samples only 1/50,000th of the mass of liver, and carries a
substantial sampling error 19.
Both autopsy and laparoscopic studies have evaluated the accuracy of liver biopsy
for staging fibrosis and diagnosing cirrhosis. These studies have clearly shown that
76,83-87
cirrhosis can be missed on liver biopsy in 10-30% of the cases . The majority of
this error is due to the understaging of the disease and is more common in macronodular
cirrhosis 19.
Cirrhosis is macronodular if nearly all of the nodules size is greater than 3 mm,
and micronodular if nearly all of the nodules size is less than 3 mm. Nodularity and septa
may be readily evident in specimens from micronodular cirrhosis, whereas more subtle
criteria have to be relied on in macronodular cirrhosis 88.
Since the diameter of the biopsy needle is less than 3 mm – the size of a nodule,
there is always a chance of missing mild to moderate fibrosis. For instance, the biopsy
sample may be obtained along the line of portal-portal or portal-central fibrosis; or may
18
be tangential to it. Thus in the latter case, apart from the extensive fibrosis, other forms of
portal/periportal, or even bridging fibrosis between portal tracts or central veins may be
under represented. Thus it is recommended that the most adequate biopsy sample should
have 11 complete portal tracts, apart from the adequate length 75.
In addition to the underlying disease severity, the ease with which cirrhosis is
diagnosed further depends on the type of specimen (surgical vs needle), type of needle
(Menghini or Trucut), approach (intercostal or transjugular), and the quality of applied
reticulin stains. A further complicating factor is that biopsy is just a snap shot in time of
the dynamic nature of the process, and progression and regression cannot be determined
by a single biopsy 88.
Both the size of the biopsy sample and number of biopsies taken has a major
impact on accuracy 89. Earlier, it was suggested that an adequate biopsy should be at least
15mm in length and contain greater than 5 portal tracts 89. Recent studies have concluded
that an adequate specimen should be at least 20 mm in length with at least 11 complete
portal tracts 75; while others are recommending biopsy samples up to 25 mm in length 90,
and it is suggested that; the bigger is better 91. The need for obtaining a larger liver biopsy
sample of adequate size, contrasts with the patients’ requirement of a procedure causing
limited pain and hemorrhagic risks 24.
It has been reported that the correct diagnosis of cirrhosis increased from 80%
with a single biopsy to 100% when three specimens were analyzed 92. To the contrary, it
has also been reported that when biopsies are taken from more than one site the rate of
discordance between biopsies may be substantial. For instance, biopsies taken at
laparoscopy from both the right and left lobes were compared. It was reported that there
was a difference in the Scheuer stage of at least one grade in 33% of the patients.
Furthermore, in 14.5% of cases, the diagnosis of cirrhosis was made in one lobe but not
in the other 74.
Similarly intra- and inter-observer variability can lead to over- or under-staging.
Even when an experienced physician performs the liver biopsy, and an expert
pathologist reads and interprets the findings, up to a 20% error rate in disease staging
has been reported 22. It has also been observed that the experience of the pathologist, as
indicated by the longer duration of practice or belonging to an academic setting, may
19
have an outstanding impact on the diagnostic interpretation of liver biopsy, even higher
than determined by the sample size 93.
The type of needle used to perform the biopsy is also important. Data indicates
that cutting type needle (Trucut) is superior to other type (Menghini) in obtaining a
94, 95
better representation of liver fibrosis particularly those in the advanced disease .
Similarly, as expected, a thick needle is superior to the fine needle in assessing the
presence of advanced fibrosis and cirrhosis 96.
The criteria used to evaluate liver biopsy are equally important. The old
classification of chronic hepatitis made a rough grading distinction between milder and
more severe forms of liver disease. More recently, new insights in the aetiology and
therapy of CLD’s, particularly viral hepatitis, has led to a revised classification, aimed
to describe and quantify in more details the necroinflammation and fibrosis. Several
semiquantitative scoring systems have been proposed to measure the activity grade of
inflammation and to stage the amount and type of fibrosis in the liver 24. The use of
97
more standardized grading systems of hepatic fibrosis including the Knodell , later
98
modified by Ishak , METAVIR 77, Scheuer 99
, and the Batts and Ludwig 100
scores
amongst others have improved the reproducibility of these criteria. Many studies have
shown good to excellent inter- and intra-observer reproducibility for the staging of
fibrosis, but that the reproducibility of inflammatory scores is significantly worse101-105.
Although the METAVIR scoring system for liver fibrosis, has been frequently used in
recent times particularly for chronic hepatitis C 24, lack of a uniform scoring system of
fibrosis adds to the observer bias and difficulties in comparison between studies.
Whichever score is used, the histological staging of liver fibrosis on biopsy is
artificially represented as a quantitative categorical variable with a linear quantum
progression in severity from 0 to 4 or 6. This does not accurately reflect the dynamic
biological process of fibrosis. Fibrosis progression is likely to be nonlinear and there is
not equal temporal progression between sequential stages 23. So all the scoring systems
have some limits: being semiquantitative, not linear, prone to intra- and inter-observer
variation, to sampling variability, and numbers with linear progression not reflecting
the active and dynamic process of fibrosis 23,106.
20
Although the cost of liver biopsy is not a major factor in our country; yet it can be
elsewhere. A cost benefit analysis showed that in US the cost of a liver biopsy is $ 1032
and it could rise to $ 2745 when complications occur 107. So the procedure is invasive,
costly, and difficult to standardize24.
As we certainly are daunted at the prospect of performing 3 million liver biopsies
with the associated cost, manpower issues, and risks for the patient, we need to have a
reliable alternative, which, if not better can just as effectively guide our decision 22.
1.6 Noninvasive markers of liver fibrosis

In the recent years, there has been an increasing interest in identifying and
describing liver fibrosis through noninvasive surrogate markers measurable in the
peripheral blood. Serum markers of liver fibrosis offer an attractive alternative. They are
less invasive than biopsy, with no risk of complications, eliminate sampling and observer
variability which is in the case of liver biopsy (but there is inter-laboratory variability in
the measurement of noninvasive markers), may allow dynamic calibration of fibrosis, can
be performed repeatedly, and may be more cost effective in certain parts of the world 23.
Most noninvasive markers of liver fibrosis were developed with the aim of
discriminating between ‘insignificant”, (F0-F1) by METAVIR and clinically
“significant” fibrosis (> F2) by METAVIR or of identifying or excluding established
cirrhosis in patients with well compensated CLD. Both these aims are clinically the most
relevant.
The major clinical utility of the index biopsy in HCV is to enable the clinician to
determine the need for treatment. Because of the complexity and side effects of
interferon-based therapies, the liver biopsy has taken an increasing role in the clinician’s
decision whether to treat a patient. Presence of significant fibrosis in the liver is indeed
considered as the hallmark of a progressive liver disease and a clear indication for
immediate initiation of antiviral therapy, in agreement with International Guidelines and
22,108,109
recommendations for the management of these conditions . On the other hand,
patients with F0-F1 fibrosis usually do not progress or progress much slowly 110,111 and
are often not as aggressively offered treatment 22.
21
However as therapy for HCV improves, the clinical need for a biopsy may be less
apparent. For example, genotype 2 and 3 patients have a greater than 70% sustained
virologic response with pegylated interferons and ribavirin, and in uncomplicated cases a
rationale can be made to treat all patients without liver biopsy and only to biopsy those
who fail treatment 22,112,113. As newer, better tolerated, and more efficacious therapies are
being developed, the need for biopsying all HCV patients to grade and stage the disease
may become redundant. Therefore the development of noninvasive tests that can
differentiate between patients with mild disease (METAVIR F0 or F1) versus those with
more significant fibrosis (METAVIR F2-F4) could have a widespread clinical utility in
managing HCV patients in the future 22.
The search for the noninvasive marker(s) of liver fibrosis has evolved along two
main but quite different approaches: the direct and indirect markers of fibrosis. There are
several proposed serum markers; the ideal features of which are 114:
· Liver specific
· Independent of metabolic alterations
· Easy to perform
· Minimally influenced by urinary and biliary excretion
· Reflective of fibrosis irrespective of cause
· Sensitive enough to discriminate between stages of fibrosis
· Correlate between dynamic changes in fibrogenesis or fibrous resolution
1.7 Indirect markers of liver fibrosis

The indirect markers of fibrosis reflect alterations in the hepatic function, but do
not directly reflect ECM metabolism. Some of the test panels use general measures, such
as age and gender. The first indirect marker of liver fibrosis were transaminases, later
associated in the aspartate to alanine aminotransferase ratio (AAR) to detect cirrhosis 115-
118
. The strength of such a marker is its simplicity and availability to every hepatologist
and clinician 23. On the other side, studies showed that its accuracy is highly variable 119.
Other simpler parameters included platelet count 118, and prothrombin index 120.
22
A further evolution was later introduced by Wai, et al, who combined aspartate
amiotransferase (AST) with platelet count 121. The AST to Platelet Ratio Index (APRI)
was then assessed in several studies conducted with a cohort of patients with hepatitis C
and showed a rather good diagnostic performance and reproducibility, particularly for
cirrhosis (AUC range from 0.77 to 0.94) 121-124. The real strength of such an index is that
it is based on blood tests that are routinely performed in patients with liver disease; and
with no need for additional blood collection or costs. More recently, APRI has been
modified by adding alanine aminotransferase (ALT), and international normalized ratio
(INR), with further improvement of the diagnostic accuracy, particularly for cirrhosis 125.
Another index, the Göteborg University Cirrhosis Index (GUCI), using AST,
Prothrombin –INR, and platelet count proved slightly superior for sensitivity, specificity,
negative predictive value (NPV), positive predictive value (PPV), and the area under the
receiver operating chraracteristic (AUROC) curve for prediction of cirrhosis and
126
bridging fibrosis compared with APRI . Although, APRI test is very simple, it is
subject to issues related to the reproducibility of AST measurement and platelet count 127.
Forns and colleagues reported a fibrosis index (Forns’ index) based on platelet
count, γ-glutamyl transferase (GGT), and cholesterol levels. It has a rather good NPV of
96% for excluding significant fibrosis, but only 66% PPV for diagnosing significant
fibrosis 128. A study tested the role of APRI and Forns’ index as noninvasive markers in
patients co-infected with human immunodeficiency virus (HIV) and HCV. Overall the
markers showed lower diagnostic accuracy than in HCV mono-infected patients 129.
Important concerns about the Forns’ index are: the impact of the serum lipid
abnormalities, cholesterol altering medicines, as well as the reproducibility of platelet
estimations 19,21. However, the most important limit of both APRI and Forns’ index is that
they leave almost half of the patients unclassified 24.
The most widely investigated combination set of noninvasive markers of liver
fibrosis is the Fibrotest; a combination of five blood tests based on a mathematical
formula: gGT, bilirubin, haptoglobin, apolipoprotein A1, a2 macroglobulin adjusted for
gender and age 130. According to the investigators, for the exclusion of significant fibrosis
(METAVIR > F2), it has 100% negative predictive value, and more than 90% positive
predictive value, using liver biopsy as a reference. Over all liver biopsy could have been
23
avoided in 46% of patients on the basis of these findings 21,130. On the other hand, it uses
two rather uncommon parameters, apolipoprotein A1 and a2 macroglobulin, which
require precise standardization of laboratory procedures 131.
To date, Fibrotest is by far the most investigated and validated noninvasive
marker of liver fibrosis with around twenty studies reported in the literature 24. The areas
under the receiver operating characteristic curve, ROC, for the first year (0.836), and
second year (0.870) did not differ (p=0.44). With the best index, a high negative
predictive value (100% certainty of absence of F2, F3, or F4 fibrosis) was obtained in
scores ranging from zero to 0.10 (12% of all patients), and high positive predictive value
(>90% certainty of presence of F2, F3, or F4 fibrosis) for scores ranging from 0.60 to
130
1.00 (34% of all patients) . The detection of significant fibrosis F2 or greater had a
130
75% sensitivity and 85% specificity . The assay performed somewhat better for the
assessment of more advanced liver disease (METAVIR stages 3 and 4).
It performed slightly worse when validated by independent workers in chronic
19
HCV (NPV85%, PPV 78%) , and when validated by the same group, performed
similarly less well than in HCV, in patients with chronic HBV with an AUROC of 0.78
132
. In a study performed in HIV/HCV co-infected patients, Fibrotest performed well,
particularly for cirrhosis (AUROC 0.87) that could be excluded with 100% negative
predictive value133.
134
In the Fibropaca study , an independent prospective multicenter study
confirmed the diagnostic value of Fibrotest and Actitest found in the principal study and
suggested that both the tests could be an alternative to liver biopsy in most patients with
chronic HCV. The Actitest is a modification of the Fibrotest that incorporates ALT along
with other biomarkers, and is a measure of the degree of inflammation. The area under
the ROC for the diagnosis of activity (A2-A3) was 0.73 (0.69-0.77), for significant
fibrosis ( F2-F4) was 0.79 (0.75-0.82),and for severe fibrosis (F3-F4) was 0.80 (0.76-
0.83). Nevertheless, both the Fibrotest and Acitest bring new insights in the diagnosis of
fibrosis in patients with chronic hepatitis C.
A Fibrometer test has been proposed combining platelets, prothrombin index,
AST, a2 macroglobulin, hyaluronate, urea, and age. The area under the receiving
operating characteristic curve (AUROC) for stages F2-F4 was 0.883 for the Fibrometer,
24
0.808 for the Fibrotest, 0.820 for the Forns’ test, and 0.794 for the APRI 135. The authors
concluding that the Fibrometer has a high diagnostic accuracy for clinically significant
fibrosis (F2-F4) 135.
In the same study, the area of fibrosis (AOF) was estimated in viral hepatitis by
testing for hyaluronate, γ-glutamyl transferase, bilirubin, platelets, and apolipoprotein A1.
Why use the area of fibrosis? It stems from difficulties in transforming histological stages
in to a binary variable when staging is evaluated by noninvasive tools. The histological
staging usually includes five stages from F0 to F4, the cut off at F2 usually defines
clinically significant fibrosis as F2 or higher. Whereas it is well known that the amount of
fibrosis varies considerably between stages. Another limitation is the restriction of
cirrhosis to one stage (F4), whereas the amount of fibrosis in cirrhosis is four times that
of the other four stages.
Because cirrhosis corresponds to only to only one or two stages (depending on the
histopathological system used), the range of blood test results for advanced fibrosis in
patients with cirrhosis is very limited. For instance in the virus related CLD, the ranges in
the Fibrometer test for stages F0-F3 was 0.03-0.99, and for F4 was 0.99-1.00. The
authors concluding that the AOF estimation via the blood tests is the only statistically
validated quantitative test for the noninvasive diagnosis of fibrosis. However the aim of
AOF evaluation is not to distinguish mild F stages due to overlap of AOF values in these
stages 135.
Adams et al, proposed a model (Hepascore) with similar efficacy. It consists of
bilirubin, g GT, hyaluronic acid, a2 macroglobulin, age and sex and produced AUROC
of 0.85, 0.96, and 0.94 for significant fibrosis, advanced fibrosis, and cirrhosis 136.
Other markers are PGA index 137
; combines prothrombin index, g GT, and
apolipoprotein A1, and modified to PGAA index with the addition of a2 macroglobulin
which resulted in some improvement in its performance 138.
The Sud score includes age, past alcohol intake, AST, cholesterol, and HOMA-
139;
IR, (insulin resistance by the homeostatic model assessment) and performs well with
21
an area under the ROC of 0.77 .
25
1.8 Direct markers of liver fibrosis
The direct markers of liver fibrosis reflect the process of fibrogenesis, the ECM
24
deposition or removal . Hyaluronic acid is the most widely studied of the direct
markers. It has been studied in hepatitis C, hepatitis B, alcoholic fatty liver disease
AFLD, and nonalcoholic fatty liver disease NAFLD. In hepatitis C the AUROC’s have
24,140
ranged from 0.82-0.92 . In hepatitis B the AUROC is 0.98 but requires further
141
validation .
140
A study by Halfon P, et al, , focused on the diagnostic accuracy of HA alone,
(instead of combination of markers) in predicting fibrosis and cirrhosis in HCV infected
patients. In all 405 patients were studied. Absence of significant fibrosis, severe fibrosis,
and cirrhosis can be predicted by HA levels of 16, 25, and 50 µg/l respectively (with
NPV of 82%, 89%, and 100% in the same order). Presence of significant fibrosis, severe
fibrosis, and cirrhosis can be predicted by HA levels of 121, 160, and 237 µg/l
respectively (with PPV of 94%, 100%, and 57% in the same order). Thus serum HA is a
clinically useful as a noninvasive marker of liver fibrosis and cirrhosis. It suffers from the
need to limit, as much as possible, potential confounding variables such as the effects of
exercise and eating.
Another combination panel of matrix markers, FibroSpect (hyaluronic acid,
TIMP-1, and a2-macroglobulin) has been tested in a cohort of hepatitis C patients,
obtaining an AUROC of 0.83 with an accuracy of 75% 142.
143
In a recently reported retrospective study , hyaluronic acid, YKL-40, and
FibroSpect II (comprising hyaluronic acid, TIMP-1 (tissue inhibitor of metalloproteinase
1), and alpha 2 macroglobulin) were assessed with Ishak stages and digital quantification
of fibrosis (DQF). Among the serum markers, hyaluronic acid was effective in
discriminating Ishak stages 0-1 and Ishak stages 2-3 compared with FS-II, with area
under the ROC curve of 0.76 versus 0.66 respectively. All three serum markers predicted
advanced fibrosis and cirrhosis. YKL-40 had the highest false positive rates in all
categories of fibrosis.
144
The SHASTA index developed by Afdhal N, et al , consists of serum
hyaluronic acid, AST, and albumin. A cutoff of less than 0.30 was associated with a
26
sensitivity of more than 88% and a negative predictive value of more than 94%. The
SHASTA index in HIV/HCV has similar accuracy to the Fibrotest and in this study
performed significantly better than the APRI test.
Among the glycoproteins, laminin has been assessed as a noninvasive marker
mainly for significant liver fibrosis. It showed an overall accuracy of 81% in patients
with CLD 101. Laminin showed good performance, particularly when combined with type
IV collagen, with 87% accuracy, 100% specificity and positive predictive value 145.
YKL-40 is a recently described glycoprotein that belongs to the chitinase family.
It is strongly expressed in the human cartilage and human liver. It is a relatively new
24
marker of hepatic fibrosis and it has only been preliminary evaluated in CLD’s . In
patients with chronic hepatitis C, it has an AUROC of 0.81, with 78% sensitivity, and
81% specificity 146.
Among the collagens, type IV collagen has been extensively investigated as a
noninvasive marker of liver fibrosis. Its diagnostic performance for significant fibrosis in
patients with hepatitis C, has been with an AUROC of 0.83 147. Several studies evaluated
a possible role of procollagen III in patients with hepatitis C and AFLD. In comparative
studies conducted in hepatitis C, procollagen III performed less well than type IV
collagen and hyaluronic acid 146-148.
Collagenases and their inhibitors have also been proposed as surrogate markers
of liver fibrosis. As noninvasive markers of liver fibrosis, the performance of
metalloproteinase 2 (MMP-2) and tissue inhibitor metalloproteinase 1 (TIMP-1) has been
high; especially MMP-2 (AUROC 0.97) 149. Unfortunately, it has been difficult to obtain
24
good standardization of the method for routine clinical use . In another study,
metalloproteinase 3 (MMP-3) and TIMP-1 had an AUROC of 0.82 for significant fibrosis
(METAVIR F2-F4) with 85% specificity 150.
Measurements of serum cytokines (TGF-b, TNF-b) involved in fibrogenesis have
been assessed in a limited number of studies in which the cytokines have shown less
value in predicting liver fibrosis compared to the ECM tests 19,119.
Several authors have tried to combine different direct markers of liver fibrosis.
The Europeon Liver Fibrosis study 151, was an international multi-center cohort of 1021
patients with hepatitis C, nonalcoholic fatty liver disease, and alcoholic liver disease.
27
Discriminant analysis of a test set of samples was used to identify an algorithm
combining age, hyaluronic acid, amino-terminal propeptide of type III collagen (PIIINP),
and tissue inhibitor of matrix metalloproteinase 1. The sensitivity for the detection of
Scheuer stage 3 or 4 fibrosis was 90% at a threshold score of 0.102, and accurately
detected the absence of fibrosis (negative predictive value for significant fibrosis, 92%;
area under the curve of a receiver operating characteristic plot was 0.804. The algorithm
performed equally well in comparison with each of the pathologists 151. The AUROC was
discreet for hepatitis C (0.77), good in NAFLD (0.87), and excellent in AFLD (0.94) 151.
Some of the other techniques are DNA sequence-based protein glycomics, the
Glycocirrhotest. The combination of Fibrotest and Glycocirrhotest allows identification
of cirrhosis with 100% specificity and 75% sensitivity 152.
It is thought that increased vascular adhesion molecule-1 (VCAM-1) expression
occurs in association with capillarization of the sinusoidal spaces and fibrous septa 19. In a
study of 52 patients with hepatitis with C, soluble VCAM-1 was found to have excellent
discriminator power for the detection of advanced fibrosis, sensitivity 100%, and
specificity 85%. In this small study measurement of soluble VCAM was of greater
diagnostic value than PIIINP 153.
1.9 Gene Markers and Liver Fibrosis

The complexity of the fibrogenetic process and the high number of
factors/cytokines/molecules involved imply that several genetic polymorphisms could
influence progression of liver fibrosis. The genetic polymorphisms linked to hepatic
fibrogenesis have been investigated mainly in chronic hepatitis C and in alcoholic fatty
liver disease. There are many studies that report gene polymorphisms to either favour or
reduce fibrogenesis in patients with different forms of chronic liver disease 24.
154
One such study , consists of a set of seven marker genes, the cirrhosis risk
score (CRS), was found to be a better predictor of high risk versus low risk for cirrhosis
in Caucasian patients than clinical factors. The clinical factors such as age, gender,
155,156
alcohol use, and age at infection, obesity and hepatic steatosis , influence the
progression to cirrhosis, but cannot accurately predict the risk of developing cirrhosis in
patients with chronic hepatitis C 16.
28
The authors validated all significant markers from a genome scan in the training
cohort (n= 420), and selected 361 markers for the signature building. Subsequently, a
signature consisting of 7 markers most predictive for cirrhosis risk in Caucasian patients
was developed. The Cirrhosis Risk Score (CRS) was calculated to estimate the risk of
developing cirrhosis for each patient.
The area under the receiver operating characteristic (ROC) curve was 0.75 in the
training cohort. In the validation cohort, the ROC was only 0.53 for clinical factors,
increased to 0.73 for CRS, and 0.76 when CRS and clinical factors were combined. A
low CRS cutoff of < 0.50 to identify low risk patients would misclassify only 10.3% of
high risk patients, while a high cut off of > 0.70 to identify high risk patients would
misclassify 22.3% of low risk patients. Thus more importantly fewer of the high risk
patients would be misclassified. In conclusion, CRS is a better predictor than clinical
factors in differentiating high risk versus low risk for cirrhosis in Caucasian patients.
According to the authors, for the first time, one can estimate the risk of cirrhosis
rather than using findings of liver biopsy to project the future course of disease.
1.10 Fibrosis Markers to Assess the Effect of Treatment

and Disease Progression
According to Afdhal N 19, an attractive use of fibrosis markers would be to assess
the effects of treatment, and if they can be validated, they could be valuable tools in the
search of new antifibrotic treatments. However, none of the currently available markers
have yet been validated for use in this way, but several studies have shown that levels of
these markers are altered by treatment and they have prognostic value.
19
According to Afdhal N , Poynard reported on two studies describing the
relationship between response to interferon therapy and the results of Fibrotest performed
before and after therapy 157,158.
158
In one of these studies (n = 352) the authors studied the effect of interferon
plus ribavirin on both the Fibrotest and Actitest scores. On follow up, patients who had a
sustained virological response to interferon had a substantial reduction in Fibrotest and
Acitest scores, as compared to those who were either primary nonresponders or relapsed
29
following therapy. There was a significant decrease of Fibrotest among the 184 sustained
virological responders, from (0.39 + 0.02 to 0.28 + 0.02) at 72 weeks; in comparison with
126 nonresponders and with 42 relapsers.
Furthermore, there was a significant concordance between Fibrotest and fibrosis
stage variations. At baseline 32 patients had cirrhosis. Fibrotest scores fell substantially in
17 patients with cirrhosis at baseline and a post treatment reduction of 1 or more stages;
from 0.68 to 0.44 for 3 stages improvement, from 0.60 to 0.47 for 2 stages improvement
and from 0.61 to 0.56 for 1 stage improvement 158.
These data support the concept that these assays may be useful not only in the
initial staging of the live disease, but may also be of value in following the histological
response to therapy.
19
According to Afdhal N , many other studies have evaluated the effect of
interferon therapy on levels of fibrosis markers and have compared them to histological
159,160
findings . Most of these studies have shown that serum levels of several of these
markers including HA, PIIINP, YKL-40, and TIMP-1 fall in patients who achieve a
sustained virological and biochemical response. In these patients, levels continue to fall
following treatment and often return to normal levels. In patients who relapse following
treatment, the levels frequently fall during therapy but most often return to pretreatment
levels with virological and biochemical relapse.
Additionally, treatment with interferon has been associated with a fall in serum
161
markers of fibrosis, independent of a biochemical or virological response . This has
been used as evidence that interferon has a beneficial direct antifibrotic effect,
presumably through direct inhibition of TGF-β expression162.
If noninvasive markers of fibrosis truly reflect fibrogenic activity and the rate of
underlying disease progression; then they should have prognostic value: both for
predicting clinical outcomes and progression of fibrosis. In this regard the available data
are more limited 19. However several studies have looked at the prognostic value of these
fibrosis markers.
Guechot in year 2000 evaluated the predictive value of hylauronic acid in a cohort
of 91 patients with hepatitis C associated cirrhosis followed for a median of 38 months.
During this time, complications including death, liver transplantation, and severe
30
complications of cirrhosis occurred in 24 patients. Of all the laboratory tests evaluated,
hyaluronic acid was found to have the greatest predictive value and was equivalent to
Child-Pugh score163.
In a cohort of 97 patients with primary biliary cirrhosis, Poupon demonstrated that
by multivariate analysis HA, PIIINP, bilirubin, and prothrombin time were independently
predictive of disease progression 164.
19
According to Afdhal N , serum TGF-β levels have also been used to assess
165,166
disease progression . In a 12 month study of 39 patients with hepatitis C infection,
who underwent paired liver biopsies, Kanzler showed that those patients who had
progressive liver fibrosis had higher levels of serum and tissue expression of TGF-β than
those who did not progress 165.
Similarly, prognostic value of the Fibrotest was compared with biopsy staging for
predicting cirrhosis decompensation and survival in patients with chronic HCV infection.
The investigators concluded that the Fibrotest measurement of HCV biomarkers has a 5-
year prognostic value similar to that of liver biopsy 26. According to the authors, Fibrotest
was a better predictor than biopsy staging for HCV complications, with the area under the
ROC, 0.96 vs 0.91, respectively; it was also a better predictor for HCV related deaths,
AUROC 0.96 vs 0.87 respectively. The prognostic value of Fibrotest was also significant
in multivariate analyses after taking into account histology, treatment, alcohol
consumption, and HIV coinfection 26.
1.11 Fibroscan & Caffeine Breath Test

In the noninvasive evaluation of liver fibrosis, a new technology (Fibroscan) that
measures liver stiffness has been developed. The rationale has been based on correlation
between liver fibrosis and stiffness.
Using a probe (Fibroscan, Echosens, Paris) that includes an ultrasonic
transducer, a vibration of low frequency (50 MHz) and amplitude is transmitted into the
liver. The vibration wave induces an elastic shear wave that propagates through the
organ. The velocity of this wave as it passes through the liver correlates directly with
tissue stiffness and by simultaneously using a pulse-echo ultrasound by means of the
31
same probe, the velocity of the wave is measured. The harder or stiffer the tissue, the
faster the shear wave propagates. Results are expressed in kilopascals (kPa). Fibroscan
measures liver stiffness of a volume that is approximately a cylinder of 1 cm diameter,
and 2 cm long, 100 times greater in size than a standard liver biopsy. Thus it is more
representative of the entire hepatic parenchyma 167.
In a large multicenter study of 327 patients, to investigate the use of liver stiffness
measurement in the evaluation of liver fibrosis in patients with chronic hepatitis C, the
areas under the receiver operating characteristic curve (ROC); for F > 2 was 0.79, for F >
3 was 0.91, and for F > 4 was 0.97.
The authors concluded that they found significant positive correlation
between liver stiffness measurement and fibrosis stages in patients with chronic hepatitis
C 167.
In a prospective study with more than seven hundred patients with CLD’s of
different aetiologies, the Fibroscan obtained an AUROC ranging between 0.8 for
detecting significant fibrosis to 0.96 for diagnosing cirrhosis168. The limitation is that it
24
requires a costly device to measure liver stiffness . Similarly, many of the direct
markers of noninvasive evaluation of fibrosis and few of the indirect markers are only
available in highly specialized research laboratories and are thus not routinely available.
Combining a Fibroscan with biomarkers may increase the accuracy of both
tests169.
Caffeine has high oral bioavailability and has almost exclusive hepatic
metabolism, principally via demethylation by cytochrome P450 1A2 (CYP1A2). This
property renders it an ideal substrate for a quantitative assessment of liver function. In the
study, cirrhotic patients were characterized by significantly reduced caffeine breath test
(CBT) values (1.15), compared with controls (2.23), and hepatitic patients (1.83). There
was significant inverse relationship between the CBT and Child-Pugh score (r = - 0.74, p
13
= .002). The authors concluding that C-CBT represents a valid indicator of plasma
caffeine clearance and also correlates reproducibly with hepatic dysfunction 170.
32
1.12 Meta-analysis of Studies of Fibrosis Markers
A meta-analysis of studies of fibrosis markers was published in 2006. It included

14 studies up to the year 2004 with 10 different panels. The discussion includes some of
the important differences between studies and future research recommendations23:
Although the quality of the included studies in the meta-analysis was assessed
using the quality assessment of diagnostic accuracy studies (QUADAS) tool171; yet it
goes without mentioning that there were important differences:
(1) Whilst all studies included patients from specialist clinics, there was variation
between studies in population characteristics, prevalence of severe fibrosis,
and methods of test validation and type of markers used, some studies used
indirect markers, some direct markers or a combination thereof, some were
prospective some retrospective, patient characteristics varied widely between
studies; the quality of liver biopsy varied depending on length of the liver
biopsy and the number of portal tracts.
(2) Few tests are excellent and perform better than others 23,130. Since 2004, there
are other works published where the performance of noninvasive markers is
even better 135,136.
(3) In the well known studies, the number of individual patients correctly
121,130
classified is higher, more than 40%-50% . In the meta-analysis the
reported figure is 35% but at a PPV of 90% and NPV of 95%. Relaxing the
probability of making correct assignment can increase this figure 23.
(4) The prevalence of significant fibrosis varied between studies from 17 to 80%
(median 40%). Predictive values of tests are affected by disease prevalence,
leading to a lack of generalizability. It is possible some tests might perform
better in low or high prevalence populations. For example those with a high
sensitivity across lower test scores will perform best in low prevalence
populations as the NPV will be higher and the test is applicable to a
significant part of study population; the converse would apply in high
prevalence populations.
33
(5) As already discussed, the most important fundamental methodological
limitation in assessing noninvasive markers is the use of liver biopsy as a
reference standard, and this may contribute to the moderate performance of
these tests. The quality of the reference standard is impaired due to the
sampling error 74,75,76, length of the biopsy sample 75,90, fragmentation, number
75 22,77,93
of portal tracts , and observer variability . Data on the discordant
results between histology and one panel of markers has been explored with
attribution of discordance to biopsy failure in 18% cases, failure of markers in
72
2.4% and non-attributable in 8.2% cases . The authors concluding that in
many cases of differences it is the shortcomings of the biopsy that are
responsible and this leads to an underestimation of the diagnostic performance
of the serum markers23,72.
(6) The majority of the proposed biomarker tests-and in particular, those available
for use in clinical practice-have an AUC of between 0.80-0.85, not for staging
the disease, but for differentiating mild form (F0-F1) from significant fibrosis
(F2-F4). Since the biomarker is validated against the biopsy, and the accuracy
of the biopsy is only 80%, it is probably statistically impossible for a
biomarker to perform any better for staging fibrosis. Interestingly, the
performance of biomarkers is superior at the extremes of the disease; the
indeterminate results occur when patients have F1-F2 disease. This is not
surprising, since these stages are relatively artificial separations of spectrum
of a dynamic disease process.
34
The review of the literature is presented in subsequent chapters on noninvasive
evaluation of liver fibrosis under the following headings:
2. Epidemiology of and natural history of hepatitis B and C viruses
3. Hepatic injury, fibrosis, and fibrogenesis (The cell and molecular biology)
4. Liver biopsy and hepatic histopathology
5. Noninvasive evaluation of liver fibrosis
35
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1,2,9 and their inhibitors TIMP-1 and TIMP-2 as serum markers of liver
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hyaluronic acid. Am J Gastroenterol 2004; 99: 271-279.
151. Rosenberg WM, Voelker M, Thiel R, et al. Serum markers detect the
presence of liver fibrosis; a cohort study. Gastroenterology 2004; 127;
1704-1713.
152. Callewaert N, Van Vlierberghe H, Van Hecke A, Laroy W, Delanghe J,
Contreras R. Noninvasive diagnosis of liver cirrhosis using DNA
sequencer-based total serum protein glycomics. Nat Med 2004;10: 429-
434.
153. Lo Iacono O, Garcia-Monzon C, Almasio P, et al. Soluble adhesion
molecules correlate with liver inflammation and fibrosis in chronic
hepatitis C treated with interferon-alpha. Aliment Pharmacol Ther
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154. Huang H, Shiffman ML, Friedman SL, et al. A 7 gene signature identifies
the risk of developing cirrhosis in patients with chronic hepatitis C.
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METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997; 349: 825-
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156. Wright M, Goldin R, Fabre A, et al. Measurement and determinants of the
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157. Poynard T, Imbert-Bismut F, Ratziu V, et al. Biochemical markers of liver
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158. Poynard T, McHutchison J, Manns M, et al. Biochemical surrogate
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163. Guechot J, Serfaty l, Bonnand AM, et al. Prognostic value of serum
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164. Poupon RE, Balkau B, Guechot J, et al. Predictive factors in
ursodeoxycholic acid treated patients with primary biliary cirrhosis: role
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165. Kanzler S, Lohse AW, Keil A, et al. TGF-beta 1 in liver fibrosis: An
inducible transgenic mouse model to study liver fibrogenesis. Am J
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166. Neuman MG, Banhamou JP, Malkiewicz IM, et al. Kinetics of serum
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167. Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of
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168. Foucher J, Chanteloup E, Vergniol J, et al. Diagnosis of cirrhosis by
transient elastography (Fibroscan): a prospective study. Gut 2006;55:403-
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assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128:
343-350.
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171. Whiting P, Rutjes AW, Dinnes J, Reitsma J, Bossuyt PM, Kleijnen J.
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52
LITERATURE
REVIEW
Chapter 2
EPIDEMIOLOGY
&
NATURAL HISTORY
OF
HEPATITIS B AND C VIRUSES
54
Epidemiology and Natural History of HBV
History 56
Introduction 56
HBV Disease Burden 57
The Worldwide Prevalence of HBV 57
Transmission of HBV 58
Vertical Transmission 59
Horizontal Transmission 60
Natural History of Chronic Hepatitis B Infection 61
Perinatal or Childhood Acquired Infection 61
Adult Acquired Infection 63
HBeAg Negative Chronic Hepatitis B ( Precore/Core Promotor Mutants) 64
Vaccination 65
Risk Factors for the Progression of Disease 65
Epidemiology and Natural History of HCV
Introduction 67
History 67
The Epidemiology of HCV 69
The Prevalence of HBV and HCV in Pakistan 70
Epidemiological Studies in Pakistan 70
Modes of HCV Transmission 73
Natural History of Acute HCV Infection 75
Factors Associated with Development of Chronic hepatitis C 76
Virus Infection
Natural History of Chronic Hepatitis C Virus Infection 77
Factors Linked with Progression of Disease 78
55
EPIDEMIOLOGY AND NATURAL HISTORY OF HBV
2.0 History
Hepatitis B infection, although recognized nearly 40 years ago with the
identification of Australia antigen, remains a global health problem 1. Clinical and
epidemiological studies began to differentiate among various types of acute hepatitis in
the decades after World War II 2. The ground breaking studies of Krugman and
colleagues in 1967 firmly established the existence of at least two types of hepatitis 3, one
of which (then called serum hepatitis, and now called hepatitis B) was parenterally
transmitted. Serological studies were conducted independently by Prince and
colleagues4,5,6, and by Blumberg et al 1. Searching for serum protein polymorphisms
linked to diseases, Blumberg and colleagues identified an antigen (termed Au) in serum
from patients with leukemia, leprosy and hepatitis 1.
By systematically studying patients with transfusion associated hepatitis, Prince
and coworkers independently identified an antigen, termed SH, that appeared in the blood
of these patients during the incubation period of the disease, and further work established
that Au and SH were identical7,8. The antigen represented the HBsAg 9,10
. These
preliminary studies made possible the serologic diagnosis of hepatitis B virus and opened
up a whole new world of investigation and research in the field of Hepatology 2.
2.1 Introduction
It is estimated that 2 billion people worldwide have been infected with HBV, 350
11,12,13
million chronically infected, and 50 million new cases are diagnosed annually .
Implementation of HBV vaccination programs has led to significant reductions in the
HBV infection rate among children and health care workers 14. The clinical spectrum of
HBV infection is broad, ranging from asymptomatic chronic carriers to fulminant
hepatitis with many factors influencing the natural history of the disease: host (including
age at the time of infection, gender, immune status); viral characteristics (viral load,
genotype, mutation), and exogenous factors such as (viral co-infection, alcohol
15,16
consumption and chemotherapy) . The Hepatitis B e Antigen (HBeAg) negative
56
chronic hepatitis is increasingly recognized worldwide, which has important implications
for therapeutic strategies 15.
2.2 HBV Disease Burden

HBV is one of the most common infectious diseases in the world. Of the 2 billion
people who are infected with HBV, over 350 million are chronically infected, and over 1
million individuals die annually of HBV related chronic liver disease 17, 93% of which
are related to cirrhosis and HCC and 7% from fulminant acute HBV 18. Over all cirrhosis,
liver failure, or hepatocellular carcinoma develop in 15-40% of individuals with chronic
HBV infection 19. In the US, a fourfold increase in age-adjusted death rate due to HBV
was noted between 1979 and 1998, most notably in men and non whites, despite the
availability of HBV vaccine for the last 20 years 20. Hospitalization for HBV related liver
disease increased to 4.9 fold along with increases in hospitalizations for HCC and in-
hospital deaths. Hospital charges related to HBV also doubled over the 10 year period 15.
A decision analysis performed by the National Immunization program estimates that if
routine infant HBV vaccination with three dose coverage in 90% and no birth dose, could
be achieved; 68% of HBV related deaths could be prevented 18.
2.3 The Worldwide Prevalence of HBV

The prevalence of HBV infection varies markedly in different geographic areas of
the world, as well as in different population subgroups. The world can be roughly divided
into three distinct areas based on Epidemiology. The developed countries of the world
fall in low endemic areas, while the developing countries of the world have the high level
of endemicity, and other countries falling in between 16,17. Broadly speaking, the level of
endemicity depends on the socioeconomic development, and access to health care
facilities, barring few special population subgroups as exceptions 16.
The global distribution of various genotypes is: Genotype D (Pakistan,
Afghanistan, Iran and middle east), Genotype B and C (China, south east Asia),
Genotype A and D (Europe), Genotype A, B, C, and D (North America), Genotype F
57
(Central America), Genotype H and F (South America) and Genotype A, E and G
(Africa) 16.
The HBsAg prevalence is subdivided as low <2%, intermediate 2-8%, and high
>8% 16.
The regional prevalence of HBsAg varies from 0.1% -0.5%(>2%) in low
prevalence areas (US, Canada, Northern, Western and Central Europe, Australia & New
Zealand), to 2-5% in intermediate prevalence areas (Mediterranean region, Eastern
Europe, Russian Federation, Japan, Middle East, Central and South America) to 8-20% in
high endemic areas (sub-Saharan Africa, south east Asia: southern China, Taiwan & the
Pacific rim)21,22.
Chronic hepatitis B infection remains highly endemic in most Asian countries,
with prevalence rates as high as 16% 23, and is a major cause of morbidity and mortality
due to complications of cirrhosis and hepatocellular carcinoma (HCC). In the areas of
high endemicity, 70-90% of the general population has serologic evidence of current or
resolved HBV infection 22. In these areas, most HBV infection is transmitted perinatally
or in early childhood (< 5 years). It is estimated that the perinatal route accounts for 50%
of the cases. In the intermediate and low endemicity areas, transmission is more likely to
occur in later childhood or adulthood 15.
In the Asian countries, the prevalence is: 0.8% in Japan 23, 1.5% in Saudi Arabia
24 25
, 3.1% Vietnam , 4.6% in China, 2.5%-5% in Indonesia, 7.3% in Korea, >8% in
Thailand, 5-16% in Philippines, and >10% in Taiwan 23.
2.4 Transmission of HBV

HBV is ubiquitous in body fluids, including blood, saliva, sweat, breast milk,
tears, urine, vaginal secretions, semen, and menstrual blood 26. Because HBV is resistant
to breakdown outside of body, it is easily transmitted through contact with infected body
fluids 17.Viral transmission can be mother to child (vertical or perinatal transmission) or
by percutaneous or mucosal exposure to infectious body fluids (horizontal transmission).
After infection, the incubation period varies ranges from 45 to 160 days (mean 120) 15.
58
% of population pos Infection
Area for: Neonatal Childhood
HBsAg anti-HBS
North America (USA, Canada),
Northern, Western, and Central 0.2-0.5 4-6 rare infrequent
Europe, Australia, New Zealand
Eastern Europe, the Mediterranean,
Russia, Middle East, Central and 2-7 20-55 frequent frequent
South America
Parts of China, Taiwan, Southeast
Asia, Sub-saharan Africa 8-20 70-95 very freq. very freq.
Table 1: Prevalence of hepatitis B in various areas of the world

From Zuckerman AJ. Hepatitis viruses. In: Baron S, editor. Medical Microbiology, 4th
edition. Galveston TX: The University of Texas Medical Branch at Galveston; 1996. p.
849-63; and 15.
2.5 Vertical Transmission

Perinatal vertical transmission is the most common route of transmission
worldwide 16. The presence of HBeAg in the mother’s serum is associated with greater
infectivity 27,28. The risk of perinatal HBV infection among infants born to HBV-infected
mothers ranges from 10-40% in HBeAg negative mothers to 70-90% in HBeAg positive
29,30
mothers . If a pregnant woman contracts acute HBV infection during the first or
second trimester, HBV rarely infects the foetus, whereas infection occurring in the third
trimester or in the postpartum period will more likely lead to infection in the infant. This
observation suggests that infection of infants born to HBV positive mothers most likely
occurs in the perinatal period as opposed to in utero, and the risk is associated with
29,31 32
maternal replicative status . Wang et al, reported that in 33 HBeAg positive
mothers, 70% of their infants had HBeAg positivity at the time of delivery as compared
to 0% positivity in 21 infants born to HBeAg negative mothers; suggesting transplacental
33
HBeAg acquisition. Milich et al, had suggested that transplacental HBeAg movement
59
may suppress the development of cellular immune response to nucleocapsid proteins that
are a major target during immune clearance of infected hepatocytes. However more
recent animal data doesn’t support this hypothesis, showing that small amounts of
HBeAg transferred via transplacental, lactogenic, or renal routes did not induce tolerance
34
.
The concept of perinatal transmission has important practical application.
Immediate post natal vaccination with both passive and active immunoprophylaxis for at
risk infants born to HBsAg positive mothers significantly reduces the risk of
transmission. And HBV vaccination has been introduced in the EPI Programme. But
despite this 5%-10% of infants born to HBeAg positive mothers subsequently become
HBsAg positive in follow up 35. This may be related to high level of maternal viremia 36,
intrauterine infection 37, or HBV mutation in the surface protein 15,38. Anti-viral therapy to
suppress HBV vaccination in late pregnancy may reduce vertical transmission 16,39.
Nevertheless, universal infant vaccination has been a success story. For instance,
in the United States, vaccination coverage among children aged 19-35 months increased
from 16% in 1992 to 90% in 2000 40. From 1990-2002, the incidence of acute hepatitis B
decreased by 67% across all age groups, while in children under 20 years, the incidence
40
decreased by 89% . The vaccination is recommended for all adults at increased risk of
infection (e.g., immunocompromised individuals, health professionals, IV drug users); in
cases of chronic infection, vaccination of sexual partners and household contacts is also
27
recommended . It can also be offered to adult population at large, to desirous
29
individuals. In the US, vaccination is also recommended for all adolescents . Other
preventive measures include needle exchange programmes, screening of blood products,
and educational approaches 16,39.
2.6 Horizontal Transmission

In areas with a low prevalence of HBV, there is horizontal transmission via sexual
11
contact, injection drug use, or occupational exposure to blood and blood products .
41,42
Percutaneous routes of exposure include transmission of blood or blood products ,
43,44,45 42,46
contaminated health related equipment and needles , and injection drug use .
Less commonly, tattooing and acupuncture have also been implicated in HBV
60
transmission47,48. Blood product screening has virtually eliminated this source of HBV
transmission; however, in underdeveloped countries, re-use of medical instruments,
contaminations of multiple dose medicine vials, and re-use of disposable needles remains
15
as risks for infection . Contamination of dialysis equipment is also a source of
transmission if isolation of infected patients and strict adherence to infection control
measures are not practiced. Reflecting these lapses, clusters of acute HBV continue to be
reported in hemodialysis units 15. Person to person spread of HBV between household
49
contacts can occur, as HBV can survive in the environment for 7 days or more .
Contamination of surfaces with blood or other secretions is the most common source of
transmission. Reports among Southeast Asian refugee children showed that 6% to 11% of
children born to HBsAg negative mothers were HBsAg positive, indicating probable
child-to-child or household transmission 15,50,51.
2.7 Natural History of Chronic Hepatitis B Infection

One of the major determinants of acute HBV infection is age and immune
competence at the time of infection. In infants and children, the initial HBV infection is
typically subclinical and a large percentage of acute cases proceed to chronic infection. If
acquired in adulthood, as is often the case in areas of low endemicity, progression to
chronicity is uncommon and symptomatic acute HBV infection is more common 15.
2.8 Perinatal or Childhood Acquired Infection

With the development of more sensitive molecular assays, a better understanding
of the natural history of perinatally acquired HBV infection has been possible. Broadly,
four sequential phases of HBV infection can be defined 15:
1. Immune Tolerance
2. Immune Active/Clearance
3. Nonreplicative
4. Reactivation
In the immune tolerance phase, there is minimal activity against the
61
virus, viral replication is high, as evidenced by the high serum DNA levels, serum
aminotransferase levels are normal, and patients are generally asymptomatic. Histology
in this phase shows minimal inflammatory activity. This phase can last for the first two
decades of life in the perinatally infected, with a low rate of spontaneous HBsAg
clearance 15,52.
In the second phase of immune activity/clearance, previously inactive HBV
carriers have recurrent episodes of clinical reactivation as immune mediated destruction
of infected hepatocytes occurs, leading to elevated liver enzymes and decreased HBV
DNA levels. Vertically infected patients often become symptomatic for the first time in
this immune active phase, presenting with elevated liver enzymes and HBeAg positivity.
They may also seroconvert to anti-HBe positivity sooner. The duration of this second
phase is variable from months to years. Reactivation can be severe enough to mimic
fulminant acute infection 15.
In the third and nonreplicative phase, HBV DNA levels have fallen to lower
levels, seroconversion from HBeAg positive to HBeAg negative/anti-HBe positive
occurs, aminotransferase levels normalize, and histology is again quiescent. This third
phase is commonly referred to as “inactive carrier” state. Progression from the active
hepatitis seen in the second phase to the seroconversion of HBeAg in the third phase is
seen as favourable because it is associated with cessation of viral replication and reduced
necro-inflammatory activity with a diminished risk of disease progression 53,54. Around
55
50% of patients clear HBeAg within 5 years of diagnosis, 70% within 10 years .
15,56
Regression of fibrosis may occur months to years after seroconversion .
The outcome after HBeAg seroconversion depends on the degree of pre-existing
liver damage; patients without liver damage may suffer only slight fibrosis or mild
chronic hepatitis. Those with pre-existing cirrhosis may experience further complications
23
. Patients remain HBsAg positive with integration of viral DNA in to the host’s
11
hepatocyte genome , and with detectable HBV DNA in serum measured by sensitive
polymerase chain reaction (PCR) based assays 2,55.
With time, some of these patients become HBsAg negative. The annual rate of
delayed clearance of HBsAg among patients with chronic HBV infection has been
estimated to be 0.5% to 2% 57,58. HBsAg clearance is more likely to be delayed in female
62
patients, those who are older, and those with histological evidence of chronic hepatitis
and cirrhosis 59. Despite HBsAg clearance, some patients have residual liver disease, and
hepatocellular carcinoma, HCC, may develop 59. Crytogenic cirrhosis or HBsAg negative
HCC may be misdiagnosed in these patients if they are not previously known to be
HBsAg positive 60,61.
Some infected persons may have the fourth phase. Reactivation can occur
spontaneously or under circumstances of immunosuppression and is usually associated
with elevated ALT and HBV DNA levels 15. Reactivation of hepatitis B may be mild and
asymptomatic, or it may be severe and lead to fulminant hepatic failure, especially in
patients with underlying cirrhosis62,63,64. In rare instances, reactivation of HBV has been
reported after HBsAg seroconversion 65.
2.9 Adult Acquired Infections

In contrast to childhood acquired infection, only 1% to 5% of immune
competent adults become chronically infected after acute HBV infection. A much higher
66
percentage (30% to 50%) present with an icteric illness at the time of infection .
Fulminant hepatitis occurs in 0.1% to 0.5% of acute HBV cases 15.
Adult acquired HBV infection in those who are immune compromised due to HIV
co-infection can have altered disease progression. In HIV co-infected individuals,
spontaneous clearance rates for HBsAg and HBeAg are decreased compared with non
HIV infected patients. When followed in a long term study, co-infected individuals had
12% loss of HBeAg at 5 years versus 49% for non HIV infected patients 67. HIV/HBV
co-infection is also associated with lower ALT levels and higher HBV DNA serum
levels; indicating more active viral replication in the setting of a depressed immune
system unable to mount as vigorous a cytopathic response 68.
63
2.10 HBeAg Negative Chronic Hepatitis B Virus
( The Precore and Core Promotor Mutants)
During the second phase of HBV infection (the immune clearance phase),
mutations in the core promoter and precore regions may occur that decrease or prevent
the synthesis of HBeAg but do not impair viral replication. The host immune response
may select these precore and core promotor variants. The most common of the mutants
are in which there is a G-to-A change at nucleotide 1896 15.
The preC-C (precore-core) region encodes hepatitis B core antigen (HBcAg) and
hepatitis B e antigen (HBeAg). These two proteins are also derived by alternative
initiation of translation at two in-frame AUG codons69,70. The internal AUG encodes the
21-kD C protein, the structural polypeptide of the viral capsid, whereas the upstream
AUG directs production of the 24-kD preC protein. The preC region encodes a signal
sequence, which directs the chain into secretory pathway. As the chains traverse the
Golgi complex, cleavage by cellular proteases generates HBeAg, a 16-kD fragment that
is secreted into the blood 71. HBeAg plays no role in viral assembly, and its function is
not clear. It is not required for viral replication; mutants bearing chain–terminating
lesions within the preC region replicate well in culture, and in fact, arise frequently
during natural infection 2,72.
Patients who are HBsAg positive (>6 months), anti-HBe positive, HBeAg
negative with detectable levels of serum HBV DNA and who have evidence of disease
activity (elevated aminotransferases or histological inflammation) have HBeAg-negative
chronic hepatitis B 15. Funk et al 73, has suggested that HBeAg chronic hepatitis B is more
common worldwide than previously reported with a prevalence of 33% in the
Mediterranean, 15% in the Asia Pacific, and 14% in the United States and Europe. Chu 74
and the HBV Epidemiologic Study Group have described the presence of the precore
variant in 27% and the core promoter in 445 of 694 patients in the United States. Several
others also suggest an increasing prevalence of HBeAg–negative chronic hepatitis B 75,76.
The reason for this changing epidemiology is still not clear, but may be related to
several factors including more widespread treatment of HBeAg positive disease,
vaccination, and environmental changes, but further population based studies are
64
warranted to determine true prevalence rates. Vaccination, by diminishing the acute HBV
and subsequent chronic HBV in which HBeAg is typically found during the early phase
of infection, may have reduced the prevalence HBeAg positive HBV infection 15.
HBeAg negative chronic HBV and its increasing recognition have implications
for treatment and disease outcomes. Treatment with interferon and other drugs is more
difficult in this group due to a higher rate of relapse after cessation of treatment 77,78,79,80.
2.11 Vaccination
The first plasma derived vaccine for HBV was introduced in 1981; but
it wasn’t until 1986 when a recombinant vaccine was available, and vaccination became
widely accepted 15,81. As of 2003, 79% of the 192 WHO member states had adopted the
policies of universal childhood immunization against HBV 82,83. Thus, despite a safe and
efficacious vaccine that has been available for more than 20 years, more global institution
of immunization recommendations is needed. Nevertheless the vaccine has dramatically
reduced the occurrence of chronic HBV infection and hepatocellular carcinoma; and can
thus be considered the first anticancer vaccine 84.
The success of vaccination programs has been clearly demonstrated in Taiwan.
There the program was established in 1984, first for the high risk neonates, then
extending to cover the children and adults. As a result over all acute infection rates have
decreased by 90% (mostly among children and adolescents). The prevalence of chronic
HBV infection in children < 15 years old was reduced from 10% to 0.7%, and rates of
HCC among children also declined by 50% 14,81,85.
In the United States, due to immunization of all persons up to 18 years of age; the
overall incidence of acute HBV infection has reduced from 8.5 cases per 100,000 in
1990, to 2.8 per 100,000 in 2002, a decrease of 67% 86.
2.12 Risk Factors for the Progression of Disease

Longitudinal studies in patients chronically infected with HBV indicate that the
incidence of cirrhosis ranges from 2-5 per 100 person-years and the 5-year cumulative
incidence ranges between 8% and 20% 87. Fattovich et al 88
, has reported that HBeAg
65
negative chronic hepatitis had a higher progression to cirrhosis (8-10 per 100 person-
years) than HBeAg positive chronic hepatitis. This may reflect the duration of infection,
and a late phase in the natural history of the disease, as opposed to de novo infection with
a variant not producing HBeAg. The host and viral factors implicated in disease
progression are shown in table 2 15.
Host factors Viral factors

Co-infection: HCV, HDV, HIV Ongoing viral replication, HBV DNA
Alcohol use Recurrent episodes of reactivation
Older age Possible genotype
Table 2: Host and viral factors in HBV disease progression
66
EPIDEMIOLOGY AND NATURAL HISTORY OF HCV
2.13 Introduction
We are also facing a pandemic of HCV infection. More than 3 % of the world
population, or about 170 million people may be infected with HCV 89. The prevalence of
HCV is increasing and according to Centers for Disease Control (CDC) estimates of the
future burden of chronic hepatitis C predict at least a 4 fold rise in the number of persons
with long-standing (20 years or more) infection between 1990 and 2015 90.
2.14 History
Our awareness and understanding of viral hepatitis have increased dramatically
during the past three decades, but viral hepatitis is not a new problem 91. The descriptions
of jaundice exist in the literature as far back as several centuries before Christ and are
referenced in the Babylonian Talmud and the writings of Hippocrates 92. The infectious
93
nature of the disease was first recognized in the eight century by Pope Zacharis .
However, most of the reports of large population epidemics during the past several
centuries probably represent the enteral transmission of what is now known as Hepatitis
A. It was not until the practice of inoculation for small pox vaccination was introduced
in the 1880’s that the percutaneous route of transmission of the disease was recognized 94.
Numerous reports of jaundice in patients receiving vaccines or injections for diabetes or
95,96,97
syphilis followed during the early 20th century . The first association of blood
transfusion with the development of hepatitis was reported in 1943 98.
The landmark studies of Krugman and colleagues at the Willowbrook State
School in New York documented the transmissibility of hepatitis by human plasma 99;
and confirmed the long standing clinical observations that both parenteral (serum
hepatitis) and enteric (infectious hepatitis) transmission could occur 3. Frustrating and
largely unsuccessful efforts to identify the specific agents responsible for hepatitis
continued for several decades 100.
A serologic marker for hepatitis B was first identified by Blumberg et al in 1965
1
; although its association with the parenterally transmitted entity known as serum
67
hepatitis was not recognized until 2 years later 101. The specific viral agents responsible
for hepatitis B and A became recognized during next few years 102,103.
These discoveries were major breakthroughs, but it quickly became apparent that
either hepatitis A or B virus could not explain most cases of hepatitis 91.
The entity of non-A, non-B hepatitis was formally christened by Prince et al, in
1974 104. An infectious agent was suspected based on the observations that the disease can
be transmitted parenterally to the chimpanzees and humans by blood transfusion 105.
106,107
A series of experiments by Bradley and colleagues at the Centers for
Disease Control and Prevention (CDC) characterized the nature of the infectious agent.
Filtration studies suggested that the agent was between 30 and 50 nm in size. Its
infectivity was abolished by chloroform, which suggested the presence of lipid envelop
106
, and also by formalin, heat (100o C for 5 minutes or 60oC for 10 hours), and b
propiolactone ultraviolet light 107. However, the conventional virologic and immunologic
techniques of the time failed to isolate the responsible agent 91.
Scientists at the Chiron Corporation and in Japan used a different tact based on
what was then recently described molecular biologic techniques108,109. This was based on
Bradley’s work, which suggested that the non-A, non-B agent was a virus. However,
because the genomic nature of this putative virus was not known (although a flavivirus-
like RNA viral agent was suspected), both DNA and RNA were extracted for cloning
from a large volume of infected serum 108. Following extensive ultracentrifugation, which
was sufficient to pellet down the smallest of known infectious agents, total nucleic acid
was extracted, and both RNA and DNA were converted to complementary DNA (cDNA).
Restriction fragments were cloned into a recombinant bacteriophage vector to form a
cDNA library. These phages were then inserted in Escherichia coli capable of
transcribing and expressing the encoded peptide, and the resulting products were
screened against sera from patients with non-A, non-B hepatitis. The assumption was that
sera from infected patients should contain antibody against the agent. After more than
1,000,000 (1 million) clones were screened, 5 clones were found to be reactive. Of these,
one clone (5-1-1) was shown to bind not only antibodies in the serum of non-A, non-B
hepatitis patients but also those in experimentally infected chimpanzees who appeared to
110
undergo seroconversion several weeks after exposure . Identification of other clones
68
with overlapping regions of the viral cDNA allowed these investigators to establish the
entire viral genome 91.
This breakthrough in 1989 led to an explosion of research on the viral agent, now
designated as, hepatitis C virus, and its disease, now called, hepatitis C. With the
development of antibody based detection systems, HCV was found to be the major cause
of non-A, non-B viral hepatitis 111,112,113.
2.15 The Epidemiology of HCV

The prevalence of HCV infection throughout the world is ~3%, which
corresponds to about 170 million people 89. In comparison, Diabetes Mellitus, which is a
common disorder affecting individuals of all ages, has a worldwide prevalence is about
114
180 million ; and the prevalence of impaired glucose tolerance is estimated at 197
115
million .
These estimates of HCV in the population are often based on prevalence rates on
volunteer blood donors and may therefore underestimate the true population prevalence
116
. For example, in the United States, the prevalence of ant-HCV in the blood donor
117
population is 0.6%, whereas the population prevalence is 1.8% . Nonetheless, these
91
estimates provide some idea of the worldwide pattern of infection . Wasley and Alter
suggest that these variations represent variations in the predominant risk factors 116. For
example, in the United States and Europe, the prevalence is low and is concentrated in
young males who predominantly acquire infection in early adulthood during injection
drug use. By contrast, the infection is most common in older persons in Japan and Italy,
which indicates a risk in the distant past 116. The high prevalence across all age groups in
Egypt indicates a common and ongoing risk factor 118.
Extremely low anti HCV prevalence (0.01%-0.1%) has been reported among
blood donors in United Kingdom and Scandinavia; a slightly higher prevalence (0.2%-
1%) has been reported from other European countries, North America and Australia, &
New Zealand 116,119,120.
121
The prevalence is 0.01% in blood donors in Northern Ireland , in German
122
population 0.63% , 1.1% in France (randomly selected subjects)123, Spain 1.2%
69
voluntary blood donors 124, Italy, healthy young population in the north 3.2% to 16.2% in
the south 125.
In USA it is 1.8% in National Health and Nutrition Examination Survey 1988-94
117 126
, to 1.6% in National Health and Nutrition Examination Survey 1999-2002 , and
Australia 1.1% (pregnant women) 127.
Intermediate prevalence (1.1%-5%) is reported from Eastern Europe,
Mediterranean region, South America and Middle East 119, 120. It is for e.g., in Russia 0.93
% (voluntary blood donors), and 7.5% (paid blood donors) 128.
Very high prevalence >10% is reported from Taiwan, south east Asia, countries
of sub Saharan Africa and Egypt 120, for e.g. in Egypt 24.8% in a study 129 and 17% to
26% in other study 118.
Like HBV, the prevalence of HCV varies widely in the Asian countries. It is 1.1%
23 130
in Japan , 0.9%-5.3% in Saudi Arabia , 2.3% Indonesia 23, 5.52% Korea 131
, 5.6%
130 23 130
Thailand , 5.2% Philippines , 5.6-16.9% Taiwan , and 20.6% Vietnam (Ho Chi
Minh city area) 132.
2.16 The Prevalence of HBV and HCV in Pakistan

Pakistan falls in the intermediate endemicity area for HBV 133 and HCV 130. There
has been paucity of community based epidemiologic work in Pakistan in the general
population, other than healthy blood donors. Also there has been lack of representation
134
from across the country . Some of the representative work from different areas of the
country is submitted.
2.17 Epidemiological Studies in Pakistan

The earlier studies and studies on high risk patients noted a high prevalence of
hepatotropic viruses. For e.g., in elective surgical patients in Lahore, Shirazi et al,
reported 8.75% hepatitis BsAg positive 135.
A large general population based study of 47,538 individuals noted 2.56%
prevalence for HBsAg. Although it was a good study, two aspects require special
mention: (i) In the study, the predominant proportion were males, 94%, as compared to
70
136
females 6%, and (ii) a relatively younger population was studied . In another study,
137
Qasmi et al noted 3% seropositivity for HBsAg in normal individuals of Karachi .
Zakariya et al reported a prevalence of 3.2% for HBsAg 138 in healthy male naval recruits.
In a recent study of 15,550 young adults seeking recruitment in the Armed Forces, 504
(3.24%) were positive for HBsAg 139. The presence of HBsAg in young healthy Pakistani
population has ranged from 3.0% to 3.5% 140,141.
Most of the earlier studies were on healthy blood donors; Zuberi et al 142, Yousuf
143 144
et al , Rehman et al reported HBsAg prevalence of 3.1%, 0.99%, and 5%
respectively in healthy voluntary blood donors. Important to note that the figure quoted
by Zuberi et al corroborates with the present day studies. In a study of 1,03,858 blood
donors from northern Pakistan, 3.3% were HBsAg positive 145. In another study of blood
donors from northern part of the country, of 1885 patients, 6.4% were HBsAg positive
146
. In a study from the Multan region, 6000 blood donors were screened. 3.37% tested
positive for HBsAg 147. In Lahore, Bukhari, et al showed 25% prevalence of hepatitis B
virus in patients, overall prevalence 5% (1456/29,131). The carrier state in healthy donors
was 3.8% (925/27,057) 148.
There are not many studies in Pakistan on the prevalence of pre-core mutant
strains. In one of the study from northern Pakistan, HBV DNA was found in 19%
(19/100) patients of HBeAg negative and anti-HBe positive carriers suggestive of a
prevalence rate of 19% for precore mutants. Raised serum ALT (surrogate marker of liver
damage) was found in 42.1% (8/19) carriers with detectable HBV DNA and HBeAg
negative (pre-core mutants) as compared to only 11% patients infected with wild virus;
indicative of a more detrimental effect on the liver for pre-core mutants during the course
of chronic HBV infection 149.
Similarly, for HCV earlier studies and studies in high risk populations noted a
high prevalence. Bhopal et al, have reported anti HCV prevalence of 16.31% in general
surgical patients of Rawalpindi, but probably high risk population was studied 150. Shirazi
et al, in a study on elective surgical patients in Lahore, reported 9.24% hepatitis C
positive 135. The results show high seroprevalence of hepatitis B & C in elective surgical
patients 135,148,150.
71
Studies in the general population: a very widely cited study both locally, and in
the international literature, on randomly selected subjects in Hafizabad noted anti HCV
130,151
prevalence of 6.5% . In the large population based study, cited before of 47,538
individuals, the anti HCV prevalence was 5.31% 136. Zakariya et al noted a prevalence of
2.2% anti HCV in healthy male naval recruits 138. In the recent study on 15,550 healthy
subjects, 574 (3.69%) were positive for anti HCV 139.
In the study on 1,03,858 blood donors, the prevalence of anti HCV was 4.0% 145.
In another study on 1885 blood donors, the anti HCV prevalence was 4.7% 146. In the
study from Multan with 6000 screened blood donors anti HCV was 0.27% 147. Similarly
in other studies the prevalence of anti HCV in blood donors was reported around 5% 152.
Aziz et al, noted amongst health workers of Civil Hospital Karachi, 2.4%
prevalence for HBsAg, and 5.6% for anti HCV antibodies. Their results show that the
prevalence of antibodies to HCV in health workers was 20 folds higher than health
workers in developed countries 153. Zuberi et al 142
and Rehman et al 144
reported that in
health care personnel the prevalence of HBsAg was 2.8% and 5% respectively. In the
dental practice the prevalence was 1.66% for HBsAg and 1.26% for HCV antibody 154.
Zaidi, et al reported a decreasing trend in the Seroprevalence of viral hepatitis (B
& C), in the blood donor population of northern Pakistan. Blood donors screened at
H.M.C. Hospital Peshawar showed a seropositivity of 1.40% and 1.34% for hepatitis B &
C respectively from 1999-2003. Screening of blood donors from CMH Peshawar during
the same period detected 1.75% HBsAg positive and 2.60% anti HCV positive 155.
Although some studies have shown a decreasing trend, by far the diseases are
epidemic in our country.
Previous studies done in Pakistan have shown that the small pox eradication
programs conducted in Pakistan from 1964 to 1982 had given rise to an increased
positive serology for anti-HCV. These were noted as 15.9% in Lahore, and 23.8% in
Gujranwala 156. This could also be attributed to the increased number of injections used in
139
many healthy individuals for minor problems . A study cited before from Pakistan
revealed that more than 10 injections per year in the previous 10 years were far more
151
likely to be associated with increased occurrence of HCV antibodies . Studies from
Southern Italy had shown that individuals who had received Salk Polio vaccine between
72
1956 and 1965 by the multiple uses of unsafe glass syringes may have contracted HCV
157
. Like HCV, iatrogenic factors have also been found in the transmission of hepatitis B,
which in addition to transfusion of blood and blood products, has been associated with
parenteral medications, use of needles, and diagnostic procedures 158.
Because of lack of health facilities in the city slums and rural areas, the risk
factors are not addressed to. Unscreened blood transfusions, reuse of syringes and other
equipment is rampant. Similarly social customs like ear piercing, tattooing, circumcision
and shaving by barbers through use/reuse of unsterilised instruments require attention. As
the treatment for these diseases is not affordable by vast majority of people and in case of
HCV, a vaccine is not available; mass education of the general population is of
paramount importance 130.
2.18 Modes of HCV Transmission
Transfusion of Blood and Blood Products

Historically, transfusion of blood and blood products played an important role in
the epidemiology of HCV infection. Although transfusions never accounted for the
159
majority of infections among adults . As far back as 1981, two separate American
studies showed that there was increased risk of acquiring post-transfusion non-A, non-B
hepatitis following blood transfusion, if the donors have elevated ALT levels as
160,161
compared to normal ALT levels . In the 1980’s transfusion associated hepatitis
accounted for 20% of the cases 113. For e.g., by 1986 the incidence of post transfusion
HCV ranged from 5% to 13%, which declined to between 1.5% to 9% from 1986-1990
130
. Since 1990, when anti-HCV screening of blood donors became mandatory, the
incidence of post transfusion HCV hepatitis declined to <1%, but not completely
eliminated. This is because some of the earlier generation ELISA tests are not sensitive
enough to pick up early anti-HCV detection 162. Furthermore, seronegative HCV carriers
163
were responsible for 10%-15% of HCV transmission . Nucleic acid testing of all
donors for HCV RNA has been started in some countries, such as USA, and has reduced
this risk even further 163.
73
Plasma derived products such as clotting factors concentrates frequently were
contaminated with HCV until 1987, when manufacturers incorporated effective viral
164
inactivation procedures . Thus haemophiliacs have a very high incidence of HCV
infection, 46%-90% 165,166. Although viricidal procedures for blood products such as heat
treatment, pasteurization, and solvent –detergent treatment have nearly totally eliminated
the risk of HCV transmission, they nonetheless do not guarantee complete security 166,167.
This is because there have been a number of incidences of HCV transmission via
intravenous immunoglobulin preparations 167.
Transmission in the Health Care Setting

Unsafe therapeutic injections, contaminated needles and syringes, contaminated
surgical instruments are may be the most important source of HCV transmission
worldwide 151,168. This is why the developing countries have the higher incidence of HCV
than the developed countries 159. A notable example is Egypt, where the HCV prevalence
among all age groups is at least fivefold higher than in developed countries. These high
rates of infection have been attributed partially to mass schistosomiasis treatment
campaigns in the 1960’s and 1970’s, in which medications were administered with reused
syringes that had not been adequately disinfected169.
Transmission between patients is uncommon in developed countries and generally
is associated with inadequate infection control practices, such as breaks in aseptic
170
techniques, contamination of multi-dose vials , or inadequate cleaning of equipment
171,172
. With current infection control practices, digestive endoscopy is not associated with
HCV transmission 173. Transmission from HCV infected patients to health care workers
can occur, primarily through needle stick injuries, which carry a risk of infection of
approximately 1.8% 174,175.
Perinatal Transmission
Transmission to infants born to HCV RNA positive mothers ranges from 4.6% to
10% 176,177,178, but a threshold RNA concentration has not been identified 177. Most infants
do not test HCV RNA positive during the first month of birth, suggesting that infection
74
178
occurs at the time of delivery rather than in utero . Because maternal antibody may
remain detectable in the uninfected infant for more than 1 year 176,178, anti-HCV testing is
not recommended before 18 months of age 178.
Intrafamilial Transmission
The prevalence of HCV among sexual and household contacts of chronic hepatitis
C patients though varies between 0%-27%; with majority of studies reporting between
130
0%-11% . However, no conclusive data exists as to the threshold concentration of
HCV required to transmit infection 179. One study showed 180
direct correlation with the
duration of marriage (>20 years vs <20 years) and duration of actual exposure to the
index patients but not with serum HCV RNA titers. The nonsexual household
transmission of HCV infection is speculative, and includes sharing of toothbrushes,
dental appliances, razors and nail grooming equipment 130.
Injection Drug Use

It is currently the most common mode of HCV transmission in the developed
countries. For instance in the USA; the proportion of acute cases who reported injection
181
drug use increased from 31% in 1994 to 38% in 1999 and to 45% in 2003 .
2.19 Natural History of Acute HCV Infection

According to Bialek SR159, acute infection with HCV is asymptomatic in the
majority of individuals182,183. Recent studies have examined early virologic events in
acute infection 182,183. Four phases of infection are recognized 159:
1. Pre-ramp Phase
During this phase low level viremia is detected.
2. Ramp-up Phase
It occurs between 1 and 2 weeks after exposure. There is exponentially increasing
levels of HCV RNA. The estimated doubling time of HCV RNA is 11 to 17
hours 184,185.
75
3. High Titer Plateau Phase
It occurs between ramp up and seroconversion, typically lasts 40–60
184
days .
4. Seroconversion
Antibody development occurs 9 weeks after exposure on average, but
cases are described in which antibody development occurred more than 12
182
months after first detection of viremia . In persons who achieve long term
clearance of HCV following acute infection, HCV RNA becomes undetectable as
early as 3 months but can persist for up to 2 years after exposure 182,186.
2.20 Factors Associated with Development of

Chronic hepatitis C Virus Infection
The majority of patients who have acute HCV infection develop chronic infection.
Earlier studies in cohorts of blood donors, transfusion recipients, and adults acquiring
187,188,189
infection drug use reported chronicity rates of 76% to 86% . In contrast, in
cohorts of children infected through contaminated blood only 55% to 71% were
persistently infected 15 to 20 years later 190,191. In cohorts of young women infected with
HCV through contaminated anti-D immunoglobulin in Germany and Ireland, the
chronicity rate 15 to 20 years after exposure was 55% 192,193. Neither size of the inoculum
nor mode of HCV acquisition have been linked consistently with likelihood of achieving
viral clearance after exposure or severity of chronic disease. The factors most
consistently associated with development of chronicity are: age at the time of infection,
and immune status at the time of exposure. Whether females are more likely to clear
spontaneously than males is unclear 159.
Racial differences in rate of spontaneous clearance of HCV are suggested by
cohort studies showing higher rates of HCV RNA positivity in anti-HCV positive African
Americans and Asian Americans than in White Americans 117,189,194. Persons who have
compromised immune responses are at higher risk of developing persistent infection
following exposure, as shown in studies of HIV infected persons 189,194, and transplant
recipients acquiring HCV from infected organs 195,196.
76
2.21 Natural History of Chronic Hepatitis C Virus
Infection
According to Bialek, SR 159, in persons who have chronic HCV infection, disease
progression is variable; and only a proportion of infected patients develop the serious
complications, including cirrhosis and hepatocellular carcinoma (HCC). Available studies
indicate that cirrhosis develops in 4% to 24% of persons after 20 years of infection. These
197
estimates of cirrhosis risk are strongly influenced by the population studied , and
cohort recruitment methods 198. Estimates based upon referred patients from liver clinics
had a 20 year rate of cirrhosis of 22% (95% confidence interval [CI] 18% –26%),
whereas studies in blood donors and community cohorts were lower, with cirrhosis
estimated to occur in 4% (95% CI, 1%-7%), and 7% (95% CI, 4%-10%), respectively
197
after 20 years of disease . Community cohorts probably provide the most accurate
estimates of HCV disease progression in the general population of HCV infected
individuals 159.
Most estimates of disease progression use cross sectional data in persons who
have HCV associated liver disease. Prospective evaluation of sequential biopsies in
persons who have chronic infection is a more accurate method to assess progression and
159
risk of complications . In paired biopsy studies, fibrosis progression of at least one
fibrosis stage was shown in 27% to 41% with average follow up periods of 2.2 to 6.5
years (table 4, given at the end). These data support the practice of monitoring for fibrosis
159
progression with repeat biopsies every 3 to 5 years . And here comes the role of
noninvasive evaluation of liver fibrosis; to avoid repeat biopsies, and for more
frequent monitoring of hepatic fibrosis.
Among European cohorts of patients who have HCV associated cirrhosis, the rate
199,200
of developing decompensated liver disease is approximately 4% per year . North
American cohorts of patients who have chronic HCV infection and cirrhosis have not
been studied. In those who have advanced fibrosis, in particular, cirrhosis, hepatocellular
carcinoma (HCC) develops at the rate of 1% to 7% (median 3%) per year199-202. HCV
infection has emerged as one of the most common causes of liver cancer in the United
States. A study has shown that among persons aged 65 years or older who had HCC the
77
proportion who had HCV increased from 11% in the period from 1993 to 1996 to 21%
from 1996 to 1999 203.
The majority of natural history studies published have follow up periods of up to
2 decades, but rarely beyond. Outcomes beyond 20 years of infection are largely
unknown and are based primarily on modeling. This lack of information is a significant
limitation in counseling patients who may have been infected for longer periods; and it
also points to the importance of on-going follow up of established cohorts to allow
revised estimates of disease progression 159.
2.22 Factors Linked with Progression of Disease
The host, viral, and environmental factors most consistently associated with
progressive fibrosis and the development of cirrhosis include: age at infection, duration
of infection, heavy alcohol use, and HIV infection (table 3) 159.
Co-existent liver diseases, such as chronic hepatitis B infection 204,205, and most
recently, schistosomiasis 206, seem to be associated with more severe fibrosis than seen in
patients who have HCV mono-infection. An important emerging risk factor for fibrosis
progression and cirrhosis is steatosis and factors related to metabolic syndrome (body
mass index or obesity) 159.
Factors linked with fibrosis progression in some but not all studies include HCV
207
viral load , HCV genotype and quasispecies, 159 and daily cannabis use 208. There is an
increasing focus on the effect of host genetics on fibrosis progression. Specific HLA class
209,210,211
I alleles have not been consistently linked with fibrosis progression . HLA class
II allelic variation has been linked with severity of disease in studies from Asia and
Europe 212,213,214, but further confirmatory studies are needed. Studies of the relationship
between fibrosis progression and the genes encoding inflammatory mediators and
cytokines, as well as the enzymes involved in oxidative stress, lipid metabolism, and the
mutation in the hemachromatosis gene, have been examined, but consistent associations
have not emerged 215.
78
Type of factor Well established Emerging risk factors More controversial risk
factors factors
Host Age at infection Insulin resistance Race
Duration of infection Steatosis Baseline necroinflammation
Male Gender HLA Class II Other genetic polymorphisms
Elevated ALT polymorphisms
Baseline fibrosis
Viral HBV infection HCV viral load HCV genotype
HIV infection Viral quasispecies
External Heavy alcohol use Schistosomiasis Smoking
Cannabis use
Table 3: Factors associated with advanced fibrosis among patients with chronic HCV
infection 159.
Age at Infection
Both age and age at the time of infection are associated with risk of cirrhosis
development 216,217. Individuals exposed to HCV infection at a younger age (<40 years)
progress less rapidly than persons exposed at an older age. Additionally, ageing is likely
to be an important factor in disease progression. The capacity of an ageing liver to endure
ongoing chronic injury may be less than that of a young liver. Thus fibrosis progression is
less likely to be linear, and some models suggest that risk of fibrosis progression
218
increases with age . However, the effect of age on risk of progression may be
overestimated by a failure to account for the competing risks related to deaths from
natural causes 219.
Gender
Male gender is consistently associated with higher risk of fibrosis
progression(~2.5 fold) , and higher rates of cirrhosis and HCC than seen in females. The
effect is independent of alcohol consumption, higher body mass index, and iron overload
79
159
. Recent retrospective studies suggest a protective effect of estrogens. In one study of
157 women who had chronic HCV infection, the estimated rate of fibrosis progression
was higher and histologic activity was higher in postmenopausal and nulliparous women
than in premenopausal women who had had prior pregnancies 220. Additionally, among
postmenopausal women, the estimated rate of fibrosis progression was lower in women
who received hormone replacement therapy 221. Other studies of women who had chronic
HCV infection report that estrogen exposure, defined by duration of menses, number of
pregnancies, and use of hormone replacement therapy, is associated with a reduced risk
of HCC 221. The exact mechanisms by which estrogens may slow fibrosis progression and
modify the risk of liver-related complications have not been defined 159.
Alcohol Consumption
Heavy alcohol consumption is clearly associated with progression of fibrosis and
cirrhosis in patients with chronic HCV infection 159. A recent meta-analysis of 20 studies
including 15,000 persons chronically infected with HCV, reported a pooled relative risk
of cirrhosis associated with heavy alcohol intake of, 2.33 (95% CI 1.67-3.26) 222. Even
among HCV infected patients with persistently normal serum transaminases, heavy
223
alcohol use is predictive of more severe fibrosis . Studies indicate that at levels of
50g/day or higher, the effects of HCV and alcohol are additive, but at very high levels
(125g/day or more), the effects are synergistic 224.
HIV Co-infection
Among persons who have chronic HCV infection, HIV co-infection is associated
with more advanced stages of fibrosis and higher risk of cirrhosis225,226,227. In a French
study, the median time to cirrhosis was estimated to be 26 years(95% CI, 22-34) in HIV-
HCV co-infected patients; versus 38 years (95% CI, 32-47) in HCV mono-infected
patients 225. Very similar results were obtained in a study of injection drug users in the
United Kingdom (73% male); the median rate of fibrosis progression was 0.17 units/yr in
HIV-HCV co-infected patients and 0.13 units/yr in HCV mono-infected patients (p =
80
0.01). This equated to an estimated time from HCV infection to cirrhosis of 23 and 32
years respectively 226.
Steatosis
Steatosis is a common histologic finding in patients who have chronic HCV
disease (40%-86%), with the majority of patients having mild steatosis (< 30% of
228,229
hepatocytes affected) . The underlying mechanism for steatosis differs by the
genotype. Steatosis in persons, who have genotype 3 HCV infection is viral associated. In
these individuals, steatosis is best correlated with HCV RNA titers; and steatosis is
improved by attainment of sustained virologic response with antiviral therapy 230-234. In
vitro studies and transgenic mouse models suggest HCV core protein may play a role in
lipid accumulation within hepatocytes 235,236.
In contrast, steatosis in persons infected with HCV genotype 1 (and probably all
non-3 genotypes) is associated with the same risk factors as seen in non-HCV infected
persons who have fatty liver. Coexistent obesity, diabetes, and hyperlipidemia, as well as
excessive alcohol use, have been linked with the presence of steatosis in HCV infected
228,229,233,237
individuals . In studies assessing insulin resistance, steatosis and insulin
resistance are significantly correlated in persons who have non-genotype 3 HCV
infection233,237.
Of importance is: whether steatosis causes worsening of fibrosis. Several studies
232,234,238,239
have identified a strong association between steatosis and fibrosis . In the
largest study, a multinational study of 3068 persons who had chronic HCV infection,
independent predictors of fibrosis were inflammatory activity, steatosis, male sex, and
older age 234. It remains to be proven whether this association is caused by steatosis per se
or by the metabolic factor promoting steatosis or the associated necroinflammation. If
steatosis is the ‘cause’ of liver fibrosis, then potential interventions to reduce steatosis
may positively influence fibrosis progression. In paired biopsy studies, more progressive
fibrosis is seen in patients who have steatosis at baseline or worsening of steatosis during
follow up 240,241. Whether steatosis is an independent risk factor for HCC is less clear 159.
81
Published Studies of paired liver biopsy assessment of fibrosis progression in
persons with chronic HCV infection 159
Author, N Baseline Interval % with progression Predictors of

year, ref. characteristics between progression
biopsies
Collier, 105 68% male, avg age 3.4 year mean 26.7% progressed > Current
2005 40 yr; 70% IDU; (range 0.4- 1 fibrosis unit alcohol use
JDC’05 [242]
86% increased 15.3) (scale to 5); 5%
ALT progressed > 2
fibrosis units
Ryder, 214 59% male; median 2.5 median 32.7% progressed > Age at biopsy;
2004 age 1st biopsy 36 (range, 1.9-9.4) 1 fibrosis unit fibrosis score
SR’04 [243]
yr; 52% IDU as 86% F0 or F1 (scale to 6) at baseline
risk factor at baseline
Wilson, 121 82% male; median 4.2 yr median 21% progressed > 2 HCV viral
2006 age 42 yr; 92% (range 2.8-6.0); fibrosis unit; rate = load; ALT >
LEW’06 [207]
African Americans; 63% F0 or F1 0.04 fibrosis unit 60 IU/mL
100% IDU; 27% at baseline /yr (scale to 6)
HIV infected
Wali, 46 Mean age 37 yr 2.2 yr mean; 41% progressed > 1 ALT elevation
1999 median at fibrosis score; rate
MW’99 [244]
baseline F0 = 0.15 fibrosis
(range, 0-2) units/yr (scale to 4)
Colleta, 40 45% male; median 6.5 yr median; 35.7% progressed > High HCV
2005 age 43 yr; ALT 98% F0 or F1 1 fibrosis score viral load; past
CC’05 [245]
persistently < 1.2 at baseline alcohol intake
ULN > 20 g/d
Table 4
Abbreviations: ALT, alanine aminotransferase; F0, fibrosis stage 0, F1, fibrosis stage 1;
HCV, hepatitis C virus; IDU, injection drug user; ULN, upper limit of normal
82
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103
Chapter 3
HEPATIC INJURY
FIBROSIS
AND
FIBROGENESIS
THE CELL AND MOLECULAR

BIOLOGY
Contents
Introduction 106
Biological Principals of Hepatic Fibrosis 109
Morphological Basis of Liver Fibrosis 110
Initiation of Hepatic Fibrogenesis – The Cellular Apoptosis 111
Cellular Sources of Extracellular Matrix - Normal and Fibrotic Liver 112
Stellate Cell Activation – The Central Event in Hepatic Fibrogenesis 114
Initiation of Stellate Cell Activation 115
Perpetuation of Stellate Cell Activation 117
Cytokines & Small Peptides involved in Hepatic Fibrogenesis & 123
Stellate Cell Activation
Tyrosine Kinase Receptor Ligands 125
Immunomodulatory Cytokines 127
INTERFERONS – (The Immunomodulatory Cytokines) 129
Chemotactic Cytokines / Chemokines 130
Peptides 130
Reactive Oxygen Species and Free Radical Scavengers 132
Composition of Extracellular Matrix in Normal Liver & Hepatic Scar 132
Biological Activity of Extracellular Matrix in Liver 134
Remodeling/Degradation of Extracellular Matrix 137
Specificity of Liver Fibrosis in Chronic Hepatitis C 141
Are There Fibrogenic HCV Proteins? 142
HCV Core Protein & Non-structural Protein NS5A 142
HBV and Liver Injury 145
HDV and Liver Injury 148
105
3.1 Introduction
Fibrosis is a reversible wound healing process that occurs in almost all patients
1,2
with chronic liver injury . It is the response to various insults, such as viral agents,
alcohol, ischemia, medications and hepatotoxins 3. Fibrosis and cirrhosis may ensue after
any of the multiple types of liver injury (Fig. 1&2). The major etiologies of cirrhosis
include: chronic viral hepatitis (B or C), alcohol abuse, autoimmune hepatitis,
hemochromatosis, Wilson’s disease (copper overload), α-1-antitrypsin disease, recurrent
injury to the bile ducts (primary biliary cirrhosis, and primary sclerosing cholangitis), and
perhaps congenital lesions 2. Broadly speaking, the causes of cirrhosis are multiple, and
include many congenital, metabolic, inflammatory, and toxic liver diseases (table 1)1.
Regardless of the etiologic basis, with chronic injury and fibrosis, the clinical
outcome is similar, liver architecture and metabolism is disrupted, eventually manifesting
2
as cirrhosis and its complications . Which are: portal hypertension, ascites,
encephalopathy, varices, synthetic dysfunction, and impaired metabolic capacity 1. Thus
fibrosis is deleterious both by its direct effects on cellular function and by its mechanical
contribution to increased portal resistance 1,4.
Although the wound healing response often begins with injury to the hepatocyte,
the overall response extends far beyond this event 2. The injury to the hepatocytes results
in a cascade of events 5,6. These events include activation and mobilization of a variety of
inflammatory cells that release cytokines that not only lead to amplification of the overall
response, but also contribute directly or indirectly to the “activation” of the effector cells,
usually of the mesenchymal lineage 7,8. The typical effector cells are hepatic stellate cells
(HSC’s; Fig. 3) and Kupffer cells 1,2,3.
A brief outline is: the hepatocyte damage will cause release of cytokines and other
soluble factors by the Kupffer cells and other cell types in the liver. These factors lead to
the activation of hepatic stellate cells, HSC, which synthesize large amounts of
7,8
extracellular matrix components . The activation of hepatic stellate cells involve the
transdifferentiation from a quiescent state into myofibroblast-like cells with the
appearance of smooth muscle α-actin and loss of vitamin A storage 9. Once the effector
cells (HSC’s) are activated, cytokines (and biologically active peptides) are further
106
released from the effector cells themselves, creating an autocrine loop, which further
amplifies the response (Fig. 5). An important step in this response is the release of matrix
degrading proteases and their regulation by specific inhibitors and plasma proteins 2.
According to Rockey DC 2, no matter what the cause of liver injury, increased
production of extracellular matrix constituents is the key in all forms of hepatic
fibrogenesis. The most prominent and abundant extracellular matrix types include
10,11
interstitial collagens, types I, and III . Quantitative and qualitative changes in many
12,13
other matrix components have been described, including proteoglycans , and matrix
14,15
glycoproteins, such as laminin , fibronectin 16, and tenascin 17. Specific changes in
matrix composition are similar in all forms of liver injury and hepatic fibrogenesis; which
suggests that the general mechanisms of fibrosis are similar 2.
Discoveries during the past 2 decades have begun to clarify mechanisms of
fibrogenesis and have thereby indicated areas of potential therapeutic intervention. Thus
new therapies for hepatic fibrogenesis will be based on fundamental understanding of the
basic mechanisms involved rather than on empiric observations 2. In addition to treating
the underlying etiology, such as specific antiviral therapy; of lately, it has been
encouraging that novel strategies are being developed to directly address hepatic injury
and fibrosis at the subcellular and molecular levels 2.
With new understanding and insights into the pathogenesis of hepatocellular
damage and hepatic fibrogenesis, key steps such as signaling, activation and gene
expression in specific cell types of liver have been targeted by molecular modalities 18,19.
The state of the art techniques such as: new viral or non-viral vector systems, RNAi,
ribosome, and antisense technologies have made it possible to modulate the expression of
specific genes involved in the key pathways of liver injury and fibrosis. Gene therapy is
still in its infancy, and ideal gene delivery systems for selective gene transfer with high
and prolonged gene expression, as well as, less cytotoxicity or immuogenicity remain to
be developed. Nevertheless, molecular therapeutics has already proven to be a powerful
research tool and explorative experimental medicine. They have demonstrated some
promise as novel modalities in reducing liver injury, inhibiting HSC activation, and
promoting the resolution of fibrosis 2.
107
Although these progresses in treatment of liver injury and fibrogenesis are
encouraging, one issue that requires additional attention remains that of targeting these
molecular therapeutics to specific cell types (hepatocytes, Kupffer cells or HSC), is
critical in avoiding undesired effects on other organs or cell types 2,20-23.
This chapter intends to review the exciting new developments that have been
made towards unraveling the cellular and molecular basis of hepatic injury and fibrosis,
and partly deals with how the newly acquired insights are leading towards advances in
the diagnosis and treatment of chronic liver disease.
1. Presinusoidal fibrosis E. Metabolic/Genetic diseases

Wilson’s Disease
Schistosomiasis
Genetic hemochromatosis
Idiopathic portal fibrosis
a1-Antitrypsin deficiency
Carbohydrate metabolism disorders
2. Parenchymal fibrosis
Lipid metabolism disorders
A. Infections Urea cycle disorders
Chronic hepatitis B,C,D Porphyria
Brucellosis Amino acid metabolism disorders
Bile acid disorders
Echinococcosis
F. Biliary Obstruction
Congenital or Tertiary Syphilis
Primary biliary cirrhosis
B. Drugs and Toxins
Secondary biliary cirrhosis
Alcohol
Cystic fibrosis
Paracetamol
Biliary atresia/neonatal hepatitis
INH, Rifampicin, PZA
Congenital biliary cysts
Amiodarone
G. Idiopathic/Miscellaneous
Methotrexate
Nonalcoholic steatohepatitis
Vit A
Indian childhood cirrhosis
a-Methyldopa
Granulomatous liver disease
C. Autoimmune Disease
Polycystic liver disease
Autoimmune hepatitis
D. Vascular Abnormalities
3. Postsinusoidal fibrosis
Chronic passive congestion
Hereditary hemorrhagic telengiectasia
Venoocclusive disease
Table 5. Causes of Fibrosis and Cirrhosis 1
108
3.2 Biological Principals of Hepatic Fibrosis
According to Friedman SL 1, and Rockey DC 2 several discoveries have played a

critical role in understanding the pathophysiologic basis of hepatic fibrogenesis and
cirrhosis. These are discussed below:
i) Hepatic fibrosis initially is a wound healing response in which damaged regions
are encapsulated by extracellular matrix, (ECM) or scar. In all circumstances, the
characterization of the fibrotic scar is similar; specifically the extracellular matrix
proteins that make it up. The cells and soluble factors participating in this response in
liver are similar to those seen in parenchymal injury to the kidney 24, lung, and skin. This
understanding has helped to identify underlying mechanisms and may likely lead to new
therapies for fibrotic diseases of many organs, including liver 1.
ii) The role of cellular elements in extracellular matrix production. The discoveries
have led to the identification of cellular elements that are responsible for extracellular
matrix production and in defining how they respond to injury. One of the most important
cellular elements has been the hepatic stellate cells 2.
iii) The same cell type produces hepatic fibrosis regardless of the underlying cause.
The activated hepatic stellate cell has emerged as the key cellular source of hepatic scar.
How stellate cells become activated in response to a large variety of hepatic insults –
from inborn metabolic defects to chronic viral hepatitis – remains largely unknown 1,2.
iv) Soluble factors (cytokines / peptides) and cell-matrix interactions are important
mediators in wound response; certain soluble factors such as cytokines and peptides, as
well as extracellular matrix (including interactions with integrins as important receptors
mediating cell-matrix interactions) play a critical role in wound response 2.
v) The fibrotic scar, and ECM are biologically dynamic milieu, not inert ground
substances. It has been recognized that the fibrogenic process, ECM and the fibrotic scar
109
can be dynamically modulated (i.e., reversible) by way of the effect of matrix degrading
proteases and apoptosis of the effector cells 2.
iii) Hepatic fibrosis follows chronic but not self-limited injury. Scarring does not
develop in patients who survive fulminant hepatitis, despite an abundance of fibrogenic
stimuli, unless chronic injury follows. The reason for this observation is not understood,
but identifying the factors that make fibrosis reversible may provide important clues to
the pathogenesis of fibrosis and cirrhosis 1.
iv) Fibrosis occurs earliest in regions where injury is most severe, especially in
chronic inflammatory liver disease resulting from alcohol abuse or viral infection 1.
3.3 Morphological Basis of Liver Fibrosis
Fibrogenesis is a ubiquitous biological process observed in every human chronic

inflammatory disease; such as inflammatory diseases of the lung, kidney, skin, or
gastrointestinal tract 25.
Fibrosis is the deposition of extracellular matrix (ECM) components within the
liver parenchyma.
Fibrogenesis is the consequence of a dynamic mechanism of gene transcription
and protein synthesis of ECM compounds 25.
For instance, in chronic hepatitis C, liver fibrogenesis develops initially around
the portal tract leading to periportal stellate fibrous deposits and expansion (periportal or
26
zone 1 fibrosis) . When this process progresses, fibrous tissue extends to the
neighboring mesenchymal structures with the formation of fibrous septa. It gradually
extends out into the lobules towards the central veins (zone 3) with septa formation.
Finally, when most of the portal tracts or central veins are interconnected, bridging
fibrosis develops, and annular fibrosis surrounds nodules of the liver cells, and cirrhosis
develops 27,28.
110
The fibrous tissue deposition within the liver has several deleterious direct
consequences. It reduces the mass of functional parenchyma (liver cells) and disturbs
liver architecture and vascularisation 25.
3.4 Initiation of Hepatic Fibrogenesis – The Cellular

Apoptosis
According to Prosser CC 2, chronic hepatocellular death via necrosis and/or
apoptosis initiates inflammatory responses and hepatic fibrogenesis. Ideally, hepatocytes
regenerate and replace dying cells. However, the hepatocellular regeneration in chronic
liver injury is often inhibited due to the imbalance of growth factors and the distortion of
liver architecture and circulation. Cellular debris and apoptotic bodies accumulate and
initiate inflammatory responses, which may form a self-amplifying loop that further
compromises recovery from injury, and facilitates the fibrogenic process 29.
HSC may engulf the apoptic bodies, which has been demonstrated in vitro, and
phagocytosis of apoptic bodies by quiescent HSC facilitates the phenotypic
30
transdifferentiation to myofibroblasts . Activated HSC with engulfed apoptic bodies
were identified in a rat model of bile-duct ligation and liver biopsy specimens from HCV
infection 2,30,31.
There are two pathways of cellular apoptosis:
i) Extrinsic Pathway of Cellular Apoptosis

According to Prosser CC 3, this extrinsic pathway is signaled through cell surface
32
death receptors , including Fas (also known CD95), TNF-α-receptor-1, and TNF-α-
related-apoptosis-inducing-ligand receptors 1 & 2 (TRAIL-R1 and R2). Tumor necrosis
32,33
factor-α (TNF-α) via TNF-receptor-1 can initiate apoptosis . Examples of this
extrinsic pathway of programmed cell death include autoimmune hepatitis, viral hepatitis
34,35
, chronic alcohol consumption, D-galactosamine (GaIN) plus lipopolysaccaride (LPS)
induced liver injury, and ischemia/reperfusion associated liver injury. In these forms of
111
injury, cytotoxic cytokines or chemokines play a crucial role in the mediation of the
injury process 2,36-42.
ii) Intrinsic Pathway of Cellular Apoptosis

In contrast to the extrinsic apoptotic pathway, the intrinsic pathway is based on
damage or dysfunction of intracellular organelles, such as lysosomes, endoplasmic
reticulum, nucleus, and mitochondria 43. This mechanism involves changes in membrane
permeability and integrity of the subcellular organelles. Examples are: damage to nuclear
or mitochondrial DNA can initiate apoptosis, and the release of cytochrome C from
mitochondria triggers apoptotic cascades. The intrinsic pathway of programmed cell
death is often seen in drug toxicity or hepatotoxin-induced liver injury, such as
acetaminophen overdose, alcohol toxicity, etc 42,44.
Inhibiting apoptosis will alleviate liver injury, and in turn delay or stop the
progression of hepatic fibrosis 2.
3.5 Cellular Sources of Extracellular Matrix - Normal

and Fibrotic Liver
The clear identification of the cellular sources of extracellular matrix in hepatic
fibrosis is a significant advance. These are: 1) hepatic stellate cells, (HSC), 2)
myofibroblasts (MF) or portal fibroblasts, and 3) sinusoidal endothelial cells 1.
i) Hepatic Stellate Cells

Extensive investigation during the past 15 years has helped establish the critical
role of hepatic stellate cells in hepatic fibrogenesis 2. The identification and isolation of
hepatic stellate cells was a major advance in the understanding of the pathogenesis of
hepatic fibrogenesis because it has allowed careful characterization of their biology 45.
The hepatic stellate cell (previously called lipocyte, Ito, fat-storing, or perisinusoidal cell)
is the primary source of ECM in normal and fibrotic liver (Fig. 3). Minor contributions
by portal fibroblasts are likely 1,2.
112
Hepatic stellate cells are resident perisinusoidal cells in the subendothelial space
between the hepatocytes and sinusoidal endothelial cells 46,47. They are the primary site
48
for storing retinoids (vitamin A metabolites) within the body . Stellate cells can be
recognized by their Vit A autoflourescence, perisinusoidal orientation, and expression of
the cytoskeletal proteins, desmin, and glial acidic fibrillary protein. In strict terms,
“stellate cells” may represent a heterogenous population of mesenchymal cells with
respect to cytoskeletal phenotype, vitamin A content, and localization 49. But collectively
they are the key fibrogenic cell type in the liver. Stellate cells with fibrogenic potential
are not confined to liver; for example they have been identified in pancreas also 50.
Studies in situ in both animals and humans with progressive injury have defined a
series of changes within stellate cells that collectively are termed activation (see below)
1,2
. In this event, a quiescent resting vitamin A-rich cell is transformed into a
proliferative, highly fibrogenic and contractile cell characterized morphologically by
enlargement of the rough endoplasmic reticulum, diminution of the vitamin A droplets,
ruffling of the nuclear membrane, the appearance of the contractile filaments, and
proliferation 1,2. Cells with features of both quiescent and activated cells are often called
transitional cells 1.
Although simple in concept, the activation process is remarkably complex and
involves several important cellular changes 2. Although it is appreciated that several
factors which are prominent in the injured liver are important in stimulation activation
(cytokines, chemokines, extracellular matrix), recent data suggest that a much broader
range of factors contribute to activation. For example, apoptotic fragments that are
51
derived from hepatocytes seem to stimulate fibrogenesis in cultured stellate cells .
Further, hepatitis C virus (HCV) core and nonstructural (NS3-NS5) proteins directly
interact with stellate cells and facilitate many features of stellate cell activation (e.g.,
proliferation, secretion of biologically active transforming growth factor, TGF-β1 and
expression of procollagen α1 2,52.
Perhaps the most prominent feature of activation is the enhanced production of
15,53
extracellular matrix . Further, the available evidence now indicates that the overall
increase in extracellular matrix deposition that is typical of cirrhosis largely can be
15,54
ascribed to excess production by stellate cells . Activation is also associated with
113
55,56 57
other important events, including proliferation , contractility , release of
58,59
proinflammatory cytokines and release of matrix-degrading enzymes and their
inhibitors 60,61.
As noted above, the proliferation of stellate cells occurs in the region of greatest
injury. It is typically preceded by an influx of inflammatory cells and associated with the
subsequent accumulation of extracellular matrix 1.
Stellate cells have now been characterized in many human liver diseases.
Alcoholic liver disease is the best studied example 62. Activation may even occur in the
63
presence of steatosis alone without inflammation . Activated stellate cells have also
been observed in viral hepatitis and massive hepatic necrosis 64. Stellate cells have been
62
characterized in a number of other human conditions , including vascular disease 65,
hematologic malignancy, biliary disease, mucopolysaccharidosis, acetaminophen
overdose, leishmaniasis, allograft rejection, and in drug users1. In hepatocellular
carcinoma, activated stellate cells contribute to the deposition of tumor stroma 66.
ii) Sinusoidal Endothelial Cell

Extracellular matrix production by sinusoidal endothelial cells, although less than
that by the stellate cells, is nonetheless an important component of early fibrosis. Like
stellate cells, this cell type demonstrates considerable heterogeneity in normal and
49 15
fibrotic liver . Endothelial cells from normal liver produce type III , and type IV
collagens 67, laminin 15, syndecans 15, and fibronectin 16. Following acute liver injury, an
increased expression of cellular isoforms of fibronectin by these cells is a key early event
because their appearance creates a microenvironment that activates stellate cells 1.
3.6 Stellate Cell Activation – The Central Event in

Hepatic Fibrogenesis
According to Friedman SL 1, hepatic stellate cells are the major source of ECM,
and are the key players in liver fibrogenesis and remodeling. Activation consists of two
major phases; 1) initiation (also called the pre-inflammatory stage), and perpetuation 9,
although both are associated tightly 68. These are finely tuned mechanisms regulated by
114
several molecules including chemokines and growth factors organized into a complex
network (Fig. 6)69.
Initiation represents early stages in gene expression and phenotype that renders
the cells responsive to other cytokines and stimuli. Perpetuation represents the effects of
these stimuli to maintain the activated phenotype and generate fibrosis. Initiation is
largely a paracrine function, whereas perpetuation involves both autocrine and paracrine
pathways 1.
Upon activation, HSC’s change their phenotype and function into a myofibroblast
like cell; a specialized cell type involved in ECM production. In parallel to these
morphological changes, HSC gains new functions such as proliferation, migration,
contractility, and protein synthesis 15,54-59,68. Activated HSC produce specialized enzymes
such as matrix metalloproteases (MMP) that can destroy the normal pericellular ECM
before producing fibrous tissue remodeling. Therefore, HSC’s have a key role in the
control of liver fibrosis synthesis and destruction 60,61.
3.7 Initiation of Stellate Cell Activation

The earliest changes in stellate cells are likely to result from paracrine stimulation
by all the neighboring cell types; including sinusoidal endothelium, Kupffer cells,
hepatocytes, and platelets. Factors that trigger the initial step are called initiators 1.
Early injury to the endothelial cells stimulates the production of cellular
fibronectin, which has an activating effect on stellate cells 16. Endothelial cells are also
likely to participate in the conversion of TGF-β from a latent to very potent active
fibrogenic form 1.
Kupffer cell infiltration and activation also play a prominent role. The influx of
Kupffer cells coincides with the appearance of markers of stellate cell activation. Kupffer
cells can stimulate matrix synthesis, cell proliferation, and release of retinoids by stellate
cells through the actions of cytokines (especially TGF-β1) and reactive oxygen
intermediaries/lipid peroxides 1.
Due to the critical role of the Kupffer cells in the mediation of liver injury and
fibrogenesis, inhibiting Kupffer cell activity has been proposed as a means to reduce liver
115
injury 2. In the study as quoted in Prosser CC 2, Ogushi et al 70, modulated Kupffer cell
function by administering double stranded antisense ODN targeting NF-χB, the p65
subunit. The mice were first sensitized with intraperitoneal injection of heat killed
Propionibacterium acnes. Phosphorothionate modified antisense ODN was delivered with
hemagglutinating virus of Japan (HVJ)-liposomal complexes via portal vein injection on
day 4 and 7 after P acnes priming challenge. The HVJ-liposome-mediated antisense ODN
against NF-χB significantly improved animal survival, and the mortality was decreased to
15% in mice treated with NF-χB decoy ODN as compared to 90% in control mice that
died within 24 hours. The treatment with decoy antisense ODN against NF-χB also
suppressed the production of proinflammatory cytokines (IL-1β, TNF-α, IL-18, and IL-
12) by Kupffer cells and decreased mRNA expression of IFN-γ and Fas-L. Less
infiltration of inflammatory cells and lesser destruction of sinusoidal architecture were
documented by histopathology in the animals treated with antisense ODN against NF-χB
70
.
As mentioned by Friedman SL 1, in the initiation of stellate cell activation; the
early stimulation of stellate cells by lipid peroxides in vivo may be important in many
forms of liver fibrosis; particularly hepatitis C, nonalcoholic steatohepatitis (NASH), and
iron overload. Because antioxidant levels are typically depleted in cirrhotic liver 71, as
fibrosis advances, their loss may further amplify the injurious effects of lipid peroxides 1.
Hepatocytes are the most abundant cells in the liver, are a potent source of
fibrogenic lipid peroxides. A correlation has been noted in situ between the presence of
72
aldehyde adducts and collagen gene expression by stellate cells , and peroxides
73,74
stimulate collagen synthesis by cultured stellate cells . Steatosis in NASH and
hepatitis C correlates with increased stellate cell activation and fibrogenesis 75,76, possibly
because fat represents an enhanced source of lipid peroxides. In culture, activation of
stellate cells is provoked by the generation of free radicles and is blocked by antioxidants
77
. This activation may involve the transcription factors c-Myb and nuclear factor κB
because antibodies to these factors disrupt activation. These and other key transcriptional
events are an important early signs in the activation cascade 1,78.
Recently, peroxisome proliferator-activated receptors (PPAR’s) particularly
PPAR-γ have been identified in stellate cells, and their expression increases with
116
activation 79,80. Ligands for this newly identified nuclear receptor family down-regulate
stellate cell activation 1,80.
Platelets are an important paracrine stimulus, are present within the injured liver,
and are a potent source of growth factors. Potentially important platelet mediators include
PDGF, TGF-β1, and epidermal growth factor 1.
3.8 Perpetuation of Stellate Cell Activation

According to Friedman SL 1, the perpetuation of stellate cell activation involves at
least seven changes in cell behaviour: proliferation, chemotaxis, fibrogenesis,
contractility, matrix degradation, retinoid loss, white blood cell chemoattractant and
cytokine release. Either directly or indirectly, the net effect of these changes is to increase
the accumulation of extracellular matrix. For example, proliferation and chemotaxis lead
to increased numbers of collagen producing cells, but matrix production per cell is also
increased. Cytokine release by the stellate cells can amplify the inflammatory and
fibrogenic tissue responses. Matrix proteases may hasten the replacement of normal
matrix with matrix typical of the wound “scar” 1.
i) Proliferation
An increase in stellate cell number has been well documented after both
81,82,83
experimental and human liver injury . Indeed, proliferation is an important
component of the activation cascade because it amplifies the stellate cell–mediated
response to injury. A number of mitogens appear to be important in stimulating stellate
cell proliferation. These include PDGF 56, epidermal growth factor 84, fibroblast growth
85 86 87 88
factor , endothelin-1 , insulin-like growth factor , thrombin , and transforming
85,89
growth factor alpha (TGF-α) .
PDGF is the most potent stellate cell mitogen identified. The induction of PDGF
75,90
receptors early in stellate cell activation increases responsiveness to this mitogen .
Downstream pathways of PDGF signaling have been carefully characterized in stellate
cells 91. In addition to proliferation, PDGF stimulates Na+-H+ exchange, thereby providing
a potential site for therapeutic intervention through the blockage of ion transport 92,93.
117
Thus, neutralizing PDGF activity, by either ligand antagonists or receptor
blockade, is a potentially important therapeutic approach 2.
In animal models of liver fibrosis, stellate cell proliferation is important in liver
fibrosis, and it has been demonstrated that during spontaneous recovery of experimental
94
liver fibrosis, stellate cell apoptosis (programmed cell death) was prominent . These
data suggest that apoptosis of activated stellate cells may play a role in the resolution of
fibrosis, and that a balance between cell proliferation and death is important in
determining the dynamics of the total overall stellate-cell population in the liver 2.
ii) Chemotaxis
95,96
Stellate cells migrate towards cytokine chemoattractants . Such migration is
also characteristic of wound infiltrating mesenchymal cells in other tissues. Chemotaxis
of stellate cells explains in part why stellate cells align within inflammatory septa in
vivo1.
iii) Fibrogenesis
One of the earliest described and most prominent effects of stellate cell activation
is increased extracellular matrix production 8,15. The available evidence now indicates that
the overall increase in extracellular matrix deposition typical of cirrhosis can be largely
ascribed to excess production by stellate cells 15,54.
Increased matrix production is the most direct way in which activated stellate
cells generate hepatic fibrosis. Among the components of the hepatic scar, collagen type I
is the best studied; and numerous reports describe the regulation of the collagen I gene in
stellate cells 1.
The most potent stimulus to the collagen I production is TGF-β, which is derived
from both paracrine and autocrine sources. TGF-β also stimulates the production of other
97,98,99
matrix components, including cellular fibronectin, and proteoglycans .
Transforming growth factor beta-1 seems to act by directly (and, to a lesser extent,
100,101
indirectly) stimulating extracellular matrix production in stellate cells .
Transforming growth factor beta-1 can be produced in a paracrine manner from Kupffer's
102
cells or by stellate cells themselves, and thus can be an important autocrine factor
118
103,104
. When expression of TGF-β1 is blocked during experimental liver injury, fibrosis is
reduced 105.
According to Friedman SL 1, TGF-β1 stimulates collagen in stellate cells through
hydrogen peroxide- and C/EBP-β-dependent mechanism 106. Stellate cell responsiveness
to TGF-β1 is increased during activation by enhanced ligand binding to its cognate
receptors 107. Signals downstream of TGF-β include a family of bifunctional molecules
known as SMAD’s, on which many extracellular and intracellular signals converge to fine
tune and further enhance the effects of TGF-β during fibrogenesis downstream of its
receptors 91. The response of SMAD’s in stellate cells differs between acute and chronic
injury to favour matrix production in chronic injury 108. The increased expression of TGF-
β in patients with chronic hepatitis C underscores the potential importance of this
cytokine in chronic liver disease109.
Lipid peroxidation products are emerging as important stimuli to the production
of extracellular matrix 6. Their effects may be amplified by loss of the antioxidant
110
capacity of stellate cells as they become activated . These important insights have
stimulated efforts to use antioxidants as therapy for hepatic fibrosis 1.
Other cytokines and growth factors, many of which stimulate stellate cell
proliferation, are important in the fibrogenic cascade. Platelet-derived growth factor
56,111 59 112
(PDGF), monocyte chemotactic factor (MCP-1), hepatocyte growth factor,
insulin-like growth factors (IGF-1 and 2), 87 and interleukin-6 113 are produced by stellate
cells and seem to serve as autocrine stimulators of stellate-cell proliferation, in turn
indirectly augmenting fibrogenesis.
Recent evidence also indicates that peptides such as endothelin-1, a vasoactive
peptide that has pleotropic biologic effects, are important in fibrogenesis. Abundant
evidence in other wounding models indicates that this peptide stimulates extracellular
matrix synthesis 114,115,116. Indeed, endothelin-1 is overproduced in the cirrhotic liver 117,86
118
and stimulates hepatic fibrogenesis . Other biologically active peptides that are also
important in fibrogensis include angiotensin II, and adrenomedullin 119,120. It is likely that
other, as yet unidentified, biologically active peptides are important in mediating the
hepatic fibrogenic cascade; some examples are other compounds, which are not
119
catalogued easily as peptides or cytokines (e.g., adiponectin, leptin, and prostanoids)
121,122,123
.
Finally, although cytokines are important components of the fibrogenic response
to injury, it is clear that stimuli underlying stellate cell fibrogenesis extend beyond
cytokines such as TGF-β and include the matrix itself, which clearly modulates the
activation state and thus the production of extracellular matrix by stellate cells. For
example, culture of stellate cells on a basement membrane mimicking the normal
124
basement membrane inhibits stellate cell activation and matrix synthesis, whereas
culture of stellate cells on the EDA isoform of fibronectin leads to increased activation of
stellate cells synthesis of matrix and smooth muscle proteins 16. A further example of the
importance of the extracellular matrix comes from work that demonstrated that stellate
cells which are exposed to an abnormal basement membrane (type I collagen) exhibited
marked activation of MMP-2, which in turn would be predicted to degrade normal
125
basement membrane extracellular matrix further . Finally, type I collagen promoted
activation of hepatic stellate cells through discoidin domain tyrosine kinase receptor 2
(DDR2) signaling, which increased the expression of active MMP-2 and led to enhanced
proliferation and invasion 126. Thus abnormal matrix in the injured hepatic environment
seems to have critical consequences for cellular function and implies that the interruption
of certain cell-matrix interactions could be therapeutically beneficial 2.
iv) Contractility
The contractility of stellate cells may be a major determinant of early and late
increases in portal resistance during liver fibrosis. And this may be important in the
pathophysiology of portal hypertension 2. Activated stellate cells impede blood flow both
by constricting individual sinusoids and by contracting the cirrhotic liver. The collagen
127
bands typical of end stage cirrhosis contain large numbers of activated stellate cell .
The acquisition of a contractile phenotype during stellate cell activation has been
documented both in culture and in vivo and is mediated in part by integrin receptors that
interact with the extracellular matrix 91.
120
As stellate cells become contractile, their expression of the cytoskeletal protein α
128,129
smooth muscle actin and smooth muscle myosin is increased . This observation led
130,131,132
to work demonstrating that stellate cells are contractile, a feature common to
myofibroblasts in general24,133,134.
Stellate cell contraction seems to be induced by a number of compounds,
including endothelin-1 132, prostanoids 130, substance P 135, angiotensin II 131, and arginine
vasopressin 119.
The major contractile stimulus to stellate cells is endothelin 1; its receptors are
expressed on both quiescent and activated stellate cells, but their subunit composition
136
may vary . It is also postulated that; with activation, receptor expression doesn’t
increase, unlike that of PDGF receptors, but a shift in the type of endothelin receptor that
127
predominates is combined with increased sensitivity to autocrine endothelin 1 .
Increased endothelin levels result from increased endothelin-converting enzyme activity
that in turn is a result of stabilization of the endothelin-converting enzyme mRNA 127.
The contractility of stellate cells in response to endothelin1 has also been observed in
vivo 137. Other, less potent contractile stimuli have been identified 127.
Locally produced vasodilator substances may counteract the constrictive effects
of endothelin1 127. Nitric oxide, which is also produced by the stellate cells 138, or from
139
the sinusoidal endothelium , is a well characterized endogenous antagonist to
endothelin (Fig. 7). During acute endotoxemia, stellate cell production of nitric oxide is
increased. Studies in vivo suggest that carbon monoxide 140 and adrenomedullin 141also
mediate sinusoidal relaxation through their effects on stellate cells 1,2.
According to Rockey DC 2, the clinical significance of increased stellate cell
contractility during injury in vivo is a subject active investigation. Available data suggest
that stellate cells controls sinusoidal blood flow by perisinusoidal constriction, analogous
142
to the way that tissue pericytes control blood flow in systemic capillary structures .
Because stellate cell contractility is greatest after stellate cell activation, and because
endothelin-1 is overproduced in the injured liver, enhanced contractility after activation
seems to contribute to elevated intrahepatic resistance to blood flow 143,144. Contraction of
stellate cells residing within bands of extracellular matrix is likely to lead to the whole-
organ contraction characteristic of end-stage liver disease 145.
121
If smooth muscle actin is required for contraction, then its inactivation may
represent a target for treating portal hypertension 1.
v) Matrix Degradation
Quantitative and qualitative changes in matrix protease activity play an important
role in ECM remodeling that accompany fibrosing liver injury. Stellate cells express
virtually all the key components required for pathologic matrix degradation and therefore
play a key role not only in matrix production but also in matrix degradation 1. Discussed
in detail later.
vi) Retinoid Loss

As stellate cells become activated, they lose their characteristic perinuclear
retinoid (Vitamin A) droplets and acquire a more fibroblastic appearance. In culture,
retinoid is stored as retinyl esters; however as stellate cells become activated, the retinoid
release outside the cell is retinol, and intracellular hydrolysis of estrers probably takes
62
place before export . However, it is generally not known whether retinoid loss is
required for stellate cells to become activated, and which retinoids may accelerate or
prevent activation in vivo. For instance, one retinoic acid analogue, 9-cis-retinoic acid,
stimulates hepatic fibrosis in rats by increasing the activation of latent TGF-β1 146.
Several nuclear retinoid receptors have been identified in stellate cells147,
molecules that bind intracellular retinoid ligands and regulate gene expression, but it is
uncertain whether they play a regulatory role in fibrogenesis. The question has important
clinical implications because efforts are being made to use retinoids therapeutically.
vii) White Blood Cell Chemoattractant and Cytokine Release

Increased production or activity of cytokines may be critical for both autocrine
and paracrine perpetuation of stellate cell activation. Direct effects on stellate cell matrix
production and contractility have been attributed to autocrine TGF-β 59 and endothelin1,
respectively 1. Another important example is PDGF, which is produced by stellate cells
122
and binds to stellate cell PDGF receptors 111, it has prominent effects on the stimulation
of stellate cell proliferation 2. Discussed in detail later.
In addition to TGF-β1 59, and PDGF 111, stellate cells are the source of various
other cytokines and small peptides. For example, stellate cells secrete macrophage
colony-stimulating factor (M-CSF), which may contribute to recruitment and activation
of resident or infiltrating cells in the injured liver 58, and monocyte chemotactic protein-1
59
. In addition to this mononuclear cell chemoattractant, stellate cells produce neutrophil
chemoattractants, which may contribute to the neutrophil accumulation characteristic of
alcoholic liver disease 1,95.
Recent studies have further demonstrated that stellate cells produce connective
tissue growth factor (CTGF) 148 and vascular endothelial growth factor (VEGF) 149, the
latter of which may prove to be mitogenic 150; and many others 91,151
, which often have
effects on stellate cells themselves 2.
Finally, stellate cells also seem to produce cytokines that dampen the fibrogenic
response, suggesting that stellate cells themselves could play a role in inhibiting
fibrogenesis. For example stellate cells produce potent immunomodulatory cytokine
interleukin (IL)-10 152,153. This cytokine has profound inhibitory actions on macrophages,
cells that produce several cytokines themselves that modify the wound response. Stellate
cells also produce hepatocyte growth factor (HGF) 112, which has recently been shown to
inhibit hepatic fibrogenesis 154, and has the potential to be mitogenic 155.
3.9 Cytokines & Small Peptides involved in Hepatic

Fibrogenesis & Stellate Cell Activation
In addition to the hepatic stellate cells, Kupffer cells and sinusoidal endothelial
cells; various cytokines play an important role in the hepatic fibrogenesis. Several general
points merit emphasis:
i) Multiple cytokines are involved in the fibrogenic process, many of which are
unknown 2.
123
ii) The effects of cytokines are diverse and complex; and the functions of even
known cytokines are not fully defined yet 2.
1) For example, PDGF stimulates stellate cell proliferation and also

stimulates stellate cell motility, a function that is probably important in the
fibrogenic response 156.
2) TGF-β1, although a potent profibrogenic cytokine, is also
antiproliferative. Thus, the net effect of TGF-β1 on fibrogenesis may be
mixed 2, but more fibrogenic.
3) Interferon-γ itself appears to have direct antifibrotic effects on stellate
157 158
cells and may also inhibit interleukin-4 (IL-4) an apparently
profibrotic cytokine 159. In turn, IL-4 may also induce TGF-β 160; which is
a potent profibrogenic cytokine. Thus, it can be readily understood that
interferon-γ may have multiple downstream effects 2.
iii) Although the effects of certain cytokines are well characterized in controlled
experimental conditions in vitro, their effects in the complex extracellular
environment in vivo; that includes interactions with other cytokines and
extracellular matrix, may be more difficult to predict 2.
iv) This complexity raises questions about which cytokines represent the most
reasonable therapeutic targets 2.
v) Hepatic fibrogenesis is a dynamic phenomenon. Both the cellular source and the
target of cytokines in the liver fibrogenic response varies. This variability has
important implications for therapeutic measures directed at specific cellular
sources 2.
There are various cytokines involved in hepatic fibrogenesis. Stellate cells

themselves are an important source of several cytokines and biologically active peptides
which have effects not only on themselves (autocrine effect), (e.g., TGF-β1 in
fibrogenesis, PDGF and others in proliferation, endothelin-1 in contractility), but also on
other cells (paracrine effect) in the hepatic-wounding environment (for e.g., Interleukin-
10, colony stimulating factor 58, and monocyte chemotactic peptide-1 2,59
.
124
The multiple cytokines that are produced during the hepatic fibrogenic response
have diverse effects which include; but are not limited to: stimulation of inflammation,
cell growth, and fibrogenesis (as well as their inhibition). Cytokines can be divided into
those that are primarily fibrogenic, that stimulate growth, that are immunomodulatory,
and those that are chemotactic 2.
3.10 Tyrosine Kinase Receptor Ligands

Two of the most prominent cytokines produced in the injured liver are the
tyrosine kinase receptor ligands TGF-β and PDGF. As described previously, these
cytokines directly mediate stellate cell fibrogenesis and proliferation, respectively.
i) Transforming Growth Factor-β (TGF-β)

TGF-β is the most potent cytokine for enhancing hepatic fibrogenesis; and has
received the most attention. It is a tyrosine kinase receptor ligand. It suppresses
hepatocyte proliferation, stimulates the activation of HSC, promotes ECM production,
and mediates hepatocyte apoptosis 101,161,162.
Among the most convincing pieces of evidence indicating the involvement of
TGF-β1 in hepatic fibrogenesis is the profibrotic effect of transgenically expressed TGF-
99
β1 in hepatocytes ; further work which blocked TGF-β production in the liver
confirmed its critical role 105. The effect of TGF-β1 seems to be largely through direct and
indirect stimulation of extracellular matrix production in stellate cells 101,162. Sources of
102 103,104
TGF-β1 are paracrine from Kupffer cells and autocrine from stellate cells.
Increased binding of TGF-β1 has also been documented during stellate cell activation in
vivo and in culture,107 and increased activation of latent TGF-β1 may also contribute to a
net increase in TGF-β activity 163.
Soluble TGF-β decoy receptors or adenoviral constructs that block TGF-β
signaling have been developed that show antifibrotic efficacy in vitro and in vivo
105,161,164,165
.
As mentioned by Prosser CC, 3 Smad3 (a transcriptional factor in TGF-β receptor
166
downstream signal transduction) knock out mice did not develop hepatic fibrosis ;
125
while transgenic mice overexpressing TGF-β1 developed hepatic fibrosis faster than wild
type mice, and the fibrosis regressed slower after the withdrawal of the fibrogenic agent
99,167,168
.
For example, Qi et al105, evaluated adenoviral expression of truncated TGF-β
receptor type II to abolish TGF-β signaling in liver fibrosis mediated by
dimethylnitrosamine. Sprague-Dawley rats were given a single infusion of adenoviral
vectors (AdCATβ-TR) encoding the TGF-β receptor II gene (TGF-βRII) via portal vein
injection. A greater than 20 fold increase in truncated receptor mRNA expression with
the adenoviral gene delivery was detected in animals after the TGF-β receptor type II
gene delivery with adenoviral vectors. The gene delivery improved the animal mortality,
decreased liver hydroxyproline content by 3.4 fold, hyaluronate levels by nearly 20 fold,
AST by 100 fold, and ALT by 63 fold. Less hepatocyte injury and fibrosis with a
decreased infiltration of monocytes/macrophages and reduced semiquantitative score of
fibrosis in histopathology were noted; which was consistent with decreased gene
expression of collagen type I, fibronectin, smooth muscle α-actin (SMA), and TGF-β1 in
the treated animals 3.
With the importance of TGF-β1 for extracellular matrix production firmly
established, efforts are under way to develop therapies which neutralize this cytokine. Its
9,161,169
inhibition, therefore appears attractive . It appears that an approach targeting
activated HSC and MF is necessary, since TGF-β receptors are expressed on most cell
types and systemic inhibition that reaches sufficient levels to block hepatic fibrogenesis
may trigger autoimmune diseases and cellular de-differentiation 155.
ii) Platelet Derived Growth Factor (PDGF)

Another important cytokine of the tyrosine kinase receptor ligand type is the
PDGF. It is the most potent mitogen for HSC with effects in growth stimulation,
chemotaxis, and intracellular signaling. PDGF expression is upregulated in hepatic
injury, as are PDGF receptors in activated HSC 3,170.
Stellate cells express PDGF A- and B-chain mRNA and release bioactive PDGF,
111
indicating that PDGF serves as an important autocrine factor during liver injury .
Although regulation of PDGF production in stellate cells is complex (and intricately
126
171,172
related to other events in the wounding milieu) , this regulation is critical in the
fibrogenic cascade because PDGF seems to be the most potent stimulus of stellate cell
proliferation 85.
According to Prosser CC, 3 the tyrosine kinase activity of PDGF receptors signals
through PI-3K, Ras, Raf-1, and ERK, leading to nuclear translocation and activation of
173
nuclear transcription factors . Interrupting PDGF signal effects with specific agents
inhibiting tyrosine phosphorylation, or interrupting down stream signal transduction
pathways attenuates hepatic fibrosis by preventing proliferation and chemotaxis of HSC
in vitro and in vivo 173. Selectively targeting the PDGF receptor by specific antibodies or
agents has been considered a valuable strategy to block the progression of hepatic
fibrogenesis 22,174.
3.11 Immunomodulatory Cytokines

Immune mediated damage is linked to fibrogenesis, but the character of the
immune response determines fibrosis progression. Contrary to the general thinking,
fibrosis appears not to be the logical consequence of significant macroscopic
inflammation as reflected by the mononuclear infiltrate; but rather by the associated
intrinsic and extrinsic immunosuppression. Here the cytokines play an important role 155.
The immunomodulatory cytokines include tumor necrosis factor-alpha (TNF-α),
the interleukins, and the interferons (-α, -β, and -γ). Immunomodulatory cytokines are
produced by lymphocytes, natural-killer (NK) cells and macrophages and typically can be
divided into proinflammatory Th1 cytokines (interferon-γ, interleukins 2, 3, and 12, and
TNF-α) and anti-inflammatory, profibrogenic Th2 cytokines (interleukins 4, 5, 9, 10, and
13) 2.
They can be subdivided into fibrogenic, antifibrogenic, and neutral group. The
later has no effect on fibrogenic effector cells, i.e., the HSC and the myofibroblasts (MF)
155
.
As a general rule, certain Th1 cytokines that stimulate cellular immune responses,
(proinflammatory) rather trigger matrix dissolution, i.e., fibrolysis. Whereas Th2
127
cytokines, which stimulate the humoral immune response and can suppress Th1 T cells
promote fibrogenesis 155.
Evidence comes from patients coinfected with both HIV(Th2) and HCV (Th1 in
acute, Th2 in chronic infection) who progress more quickly to cirrhosis than patients
infected with HCV alone 175. Similarly accelerated fibrosis progression is found in HCV
patients coinfected with Schistosoma mansoni which triggers a Th2 cytokine profile 176.
i) Interleukin-10
IL-10 seems to have effects similar to those of interferon-γ in the wounding
milieu. Activated stellate cells produce this cytokine, which has potent inhibitory actions
152,153
on macrophages . Macrophages are cells that also produce a several cytokines that
modify the wounding response. In culture, IL-10 was found to be antifibrogenic toward
stellate cells. The potential importance of this cytokine is underlined by the finding that
IL-10 knock-out mice exhibited significantly more fibrosis and higher hepatic TNF levels
177
than wild-type controls . This finding suggests that IL-10 synthesized during
fibrogenesis may modulate Kupffer's cells and serve as an antifibrotic cytokine, much
like interferon-γ 178.
ii) Tumor Necrosis Factor-α (TNF-α)

According to Prosser CC, 3 TNF-α is a key cytokine involved in many forms of
liver injury, and may play a crucial role in HSC activation and hepatocyte regeneration
151,179
. The major cell type for TNF-α production in the liver is Kupffer cells which
release TNF-α when activated by factors released by damaged heptocytes , and by
Reactive Oxygen Species , ROS. TNF-α participates in the second phase of
hepatocellular damage via apoptosis and TRAIL receptor activation in alcoholic or other
hepatotoxic induced liver injury 180. It acts on HSC, and may contribute to the activation
181
process . Accordingly, reducing TNF-α production, or blocking its action will
significantly minimize liver injury caused by alcohol toxicity, acetaminophen overdose,
or ischemia/reperfusion-associated liver injury in animal models 180,182.
128
3.12 INTERFERONS (The Immunomodulatory Cytokines)
The interferons are unique biological compounds with major immunomodulatory,
anti-viral and anti-fibrogenic effects. Three major isoforms of interferon exist (α, β, and
γ) 183. Each of these isoforms is unique in protein structure and in biologic actions 178.
There are multiple interferon-α subtypes but only single interferon-β and
interferon γ-species. Interferon-α and interferon-β are more closely related to each other,
structurally (indeed, they each bind to the same receptor) and functionally, than they are
to interferon-γ. Interferon-α is stimulated by viral infection, whereas interferon-γ is
stimulated by mitogenic or antigenic stimulation of T lymphocytes or NK cells 178.
The major interferon isoforms, α and γ, each bind to unique receptors. Interferon-
α has much more potent antiviral effects than does interferon-γ. On the other hand
interferon-γ is 100 to 10,000 times more potent as an immunomodulator than interferon-α
184
. This observation has led to the belief that whereas interferon-α and -β are primarily
antiviral agents that have some immunomodulatory effects, interferon-γ is primarily an
immunomodulatory agent with some antiviral effects 178.
i) Interferon-α
It is well established that interferon-α is capable of eradicating hepatitis virus B
and hepatitis virus C from the liver. Evidence clearly indicates that for hepatitis C this
109,185-190
eradication is associated with a reduction in fibrosis . Studies in patients with
chronic hepatitis C suggest that IFN-α therapy can prevent fibrosis progression, even in
191-194
nonresponders to antiviral therapy . The effect was dependent on IFN-α dose
duration and most pronounced in sustained responders. A study has found reversion of
cirrhosis in 75 out of 153 patients 155,193.
Although some experimental evidence suggests that interferon-α has primary
antifibrogenic effects 195,196 (which may play a role in reducing fibrogenesis in patients,
independent of its effect on hepatitis C), these data are not been nearly as convincing as
those for interferon-γ 178. This effect is linked at least, in part, to the activation of stat-1
signalling pathways. In cell culture, interferon-α (IFN-α) blocks activation, proliferation,
and collagen synthesis of HSC and MF 197.
129
ii) Interferon-γ
178
According to Rockey DC , interferon-γ is ineffective at eradicating hepatitis
virus B or C, but extensive experimental evidence indicates that interferon-γ effectively
inhibits fibrogenesis in the liver 198-201 and in other organs 202,203. The mechanism for the
effect in the liver seems to be a global inhibition of stellate cell activation, 157,159,198,200
although the mechanism by which activation is inhibited remains unclear. It may also
inhibit interleukin-4 (IL-4) 204, an apparently profibrotic cytokine 159.
There has been little clinical interest in the use of interferon-γ in patients with
hepatic fibrogenesis because the overexpression of interferon-γ in the liver leads to
205
chronic hepatitis and because long-term side effects may result from its profound
178
immunomodulatory effects . A report in patients with pulmonary fibrogenesis,
however, suggests that it may be feasible to use interferon-γ to treat patients with hepatic
fibrogenesis 206.
3.13 Chemotactic Cytokines / Chemokines

According to Rockey DC 178, chemotactic cytokines or chemokines of the C-X-C
family (interleukin-8, platelet factor [PF]-4) are neutrophil chemoattractants, whereas
those of the C-C family (MCP-1, macrophage inflammatory protein [MIP]-1, and
regulated on activation of normal T cell expressed and secreted [RANTES]) are
chemotactic for mononuclear cells. Several of these cytokines, including colony-
158
stimulating factor and monocyte chemotactic peptide-1159, are secreted by stellate
cells and probably contribute to recruitment of inflammatory cells after injury.
3.14 Peptides
i) Endothelins
As mentioned by Rockey DC 178, the peptide that has received the most attention
207 208
is endothelin . This family of potent vasoconstrictors comprises three unique
endothelin peptides, each consisting of 21 amino acids, which have been termed
endothelin-1, endothelin-2 and endothelin-3209. They bind to at least two heptahelical (G-
protein–coupled) receptors, termed endothelin A (ETA) and endothelin B (ETB ) receptors
130
210,211
. Endothelins are usually produced by endothelial cells and exert paracrine effects
on adjacent smooth muscle cells. In this capacity, it is believed that their major role is the
212
local control of vascular tone, including regulation of basal blood pressure . The
endothelins induce vasoconstriction by stimulating ETA receptors on smooth muscle cells
and to induce vasodilation by stimulating ETB receptors on endothelial cells 213. Although
the vasoregulatory functions of the endothelins have been emphasized here, reports also
emphasize regulation growth and other effects 214-219.
220
In liver, endothelins are produced by sinusoidal endothelial cells . There is
substantial evidence that endothelin-1 is involved in regulating sinusoidal blood flow
137,221
. Accumulating evidence indicates that endothelin biology extends far beyond
vasoregulation. Indeed, a large body of work now indicates that the endothelins are
involved in the wound-healing response. Available data further indicate that the source of
endothelin is the injured tissue itself. In some systems, the cellular compartments
responsible for increased production of endothelin have been identified. For example, in
liver, the cellular source of endothelin in the liver shifts from the sinusoidal endothelial
cell to the hepatic stellate cell after injury 222. Also, the total amount of endothelin in the
liver is overproduced, and because stellate cells have many ETA and ETB receptors 136,
endothelin-1 has multiple effects on stellate cells, apparently in an autocrine fashion.
Endothelin-1 has potent effects on stellate cell contractility; as described earlier, this
143,144,223
effect is probably involved in elevated intrahepatic resistance to blood flow .
Finally, as in other systems, overproduced endothelin in the injured liver appears to have
major effect on the fibrogenic response to injury 118.
The mechanism underlying increased endothelin-1 synthesis seems to be based on
the regulation of endothelin converting enzyme-1, which converts precursor endothelin-1
to mature endothelin-1. Further, TGF-β seems to control endothelin-1 production, at least
in part, through modulation of endothelin converting enzyme-1224. This latter finding
emphasizes the complex inter-relationships by which cytokines control fibrogenesis 178.
131
3.15 Reactive Oxygen Species and Free Radical
Scavengers
Oxidative stress is an important mechanism in liver injury. ROS include
superoxide anions, hydroxyl radicals, hydrogen peroxide, and hydroxyethyl radicals
(HER). These are generated from a variety of insults, such as drugs/ toxin metabolites,
ischemia/ reperfusion, and alcohol metabolism. ROS are involved in necrosis and
apoptosis of hepatocytes, and contribute to HSC activation 18,225. Several major classes of
free radical scavengers, such as superoxide dismutase (SOD), catalase, and glutathione
peroxidase(GSH-P), as well as SOD mimics, were investigated in various forms of liver
injury, and they afforded effective protection against the oxidative insults to the tissue
226,227
.
3.16 Composition of Extracellular Matrix in Normal Liver

& Hepatic Scar
The extracellular matrix refers to the array of macromolecules comprising the
normal and fibrotic liver. Its components include several families of structural and
nonstructural molecules: collagens, noncollagen glycoproteins, matrix-bound growth
factors, glycosaminoglycans, proteoglycans, and matricellular proteins 1.
As described in detail by Freidman SL 1, remarkable progress has been made in
identifying new members of these families, and in understanding how these molecules
interact. Matrix composition within different tissue regions is markedly heterogenous in
respect to the various isoforms within each class of molecules, their stoichiometry, and
their intermolecular interactions. Moreover, hybrid molecules have been identified that
may contain, for example, both collagenous and proteoglycan domains. Additionally, a
variety of new roles of matrix molecules are now recognized, including their role as
transmembrane transducers of extracellular signals1,228.
The most abundant proteins in ECM are collagens, a family of proteins containing
25
19 isotypes with a common trihelicoidal structure . Of the 20 types of collagens
characterized thus far, 10 have been identified in liver 1. On a morphological basis,
collagens can be divided into those forming fibrils (interstitial collagens) and those
132
included within the basement membranes 25. In the normal liver, so-called fibril-forming
collagens (type I, III, V, and XI) are largely confined to the capsule, around large vessels,
and in the portal triad. Only scattered fibrils containing types I and III collagen are found
in the subendothelial space. Additionally, smaller amounts of other collagens, including
types VI, XIV, and XVIII are found. Glycoproteins and matricellular proteins are also
present, including subendothelial deposits of fibronectin, laminin, tenascin, and von
Willebrand factor. The proteoglycans, which are primarily of heparan sulfate
proteoglycans, include perlecan, and small amounts of decorin, biglycan, fibromodulin,
aggrecan, glypican, syndecan, and lumican 1,228.
The matrix composition of fibrotic liver differs both quantitatively and
qualitatively from that of normal liver; and these changes are similar regardless of the
229
type of liver injury. The total collagen increases 3 to 10 fold . For instance, in the
normal liver, ECM comprises less than 3% of area on a liver cut section. In fibrosis,
quantitative increase (30%-40% in cirrhosis) and composition modification of ECM are
observed, leading to liver architecture distortion and impairing of the exchanges between
blood and liver cells 25. Important to note is: basement membranes are usually absent
along the sinusoids but are deposited during the process of cirrhosis, termed “sinusoid
capillarisation” 230.
Although, in the fibrotic liver, there is ECM modification, but the collagen is not
“abnormal” in sequence or structure. Overall, the “interstitial matrix” typical of a healing
wound is markedly increased and includes fibril forming collagens (types I, III, V), some
non-fibrill forming collagens (types IV, and VI), several glycoproteins (cellular
fibronectin, laminin, osteonectin, tenascin, and von Willebrand factor), and a large
number of proteoglycans and glycosaminoglycans (perlecan, decorin, aggrecan, lumican,
fibromodulin). In particular, a shift occurs from heparin sulfate-containing proteoglycans
to those containing chondroitin and dermatan sulfates 1,231.
133
3.17 Biological Activity of Extracellular Matrix in Liver
i) Accumulation of Subendothelial Matrix

Although a prominent and highly visible effect of liver injury is the dramatic
accumulation of extracellular matrix, it is important to understand that the extracellular
matrix itself is highly dynamic and has profound effects on hepatic cellular function
16,124,232
.
Normally the subendothelial space contains the components of a basement
membrane, that, unlike true basement membrane, is not electron dense. These
components include the following: laminin, type IV collagen, some types I, III, V, and VI
collagen, the FACIT (fibril associated collagens with interrupted triple helices), collagen
233
type XIV; and several heparin sulphate proteoglycans, including decorin, perlecan ,
syndecans 1 through 4, and glypican 234.
The basement membrane constituents within the subendothelial space may be
essential to preserving the differentiated functions of hepatocytes, hepatic stellate cells,
and subendothelial cells.
For example, studies in cultured cells have demonstrated that the maintenance of
differentiated hepatocyte function requires contact with a matrix substratum similar to
that found in the subendothelial space of normal liver 232. A hallmark of early liver injury
is the replacement of this normal subendothelial matrix, which contains laminin, type IV
collagen, and fibronectin, with one enriched in interstitial collagens types I and III 235,236.
124
The high density matrix also activates stellate cells , and leads to a decrease in the
237
fenestrations of sinusoidal epithelial cells , which may impair the transport of solutes
1
from the sinusoid to the hepatocytes . Early accumulation of subendothelial matrix that
leads to “capillarisation” of the subendothelial space of Disse is a key event, that may be
more important than the overall increases in the matrix constituents 1,230.
This replacement may lead to deterioration of hepatocellular function and may
alter the biology of other cells in the subendothelial space of Disse. Indeed, clinical
hallmarks of chronic liver disease, such as impaired albumin and clotting factor synthesis,
134
almost certainly are in part the result of altered hepatocyte function caused by an altered
microenvironment 1.
Recent studies have begun to elucidate the mechanisms underlying changes in the
basement membrane extracellular matrix, and this understanding may ultimately lead to
new therapies. An early component of the altered subendothelial matrix is the fibronectin
isoform known as EDA (or cellular) fibronectin. This matrix protein is produced by
sinusoidal endothelial cells very early in the injury response 16. Once synthesized, it is, in
turn, capable of directly activating stellate cells, leading to enhanced stellate cell
synthesis of matrix and smooth muscle proteins 16. A further example of the interaction
between matrix and cells has been recently described in that stellate cells exposed to an
abnormal basement membrane (type I collagen) exhibited marked activation of MMP-2,
which, in turn, would be expected to cause further degradation of the normal basement
membrane extracellular matrix further 125. Further type I collagen promoted activation of
hepatic stellate cells through discoidin domain tyrosine kinase receptor 2 (DDR2)
signaling, which increased the expression of active MMP-2 and led to enhanced
proliferation and invasion 126.
Thus, abnormal matrix in the injured environment seems to have critical
consequences for cellular function. Interrupting certain cell-matrix interactions could be
therapeutically beneficial 2.
ii) Integrins
As discussed by Rockey DC 2, a major class of membrane receptors that mediate
cell-matrix interactions are the family of proteins known as integrins. Altered cellular
behavior induced by matrix alterations is typically mediated by these cell membrane
receptors 2.
Integrins, are the best characterized type of extracellular matrix receptors. These
are a large family of homologous membrane linker proteins. Integrins are noncovalent αβ
heterodimers (made up of an alpha chain and a beta chain) 2, that consist of a large
extracellular domain, a membrane-spanning domain, and a cytoplasmic tail 238.
Integrins control many cellular functions, including gene expression, growth, and
differentiation. A growing number of α and β subunits have been identified, with each
135
combination having a different cellular and ligand specificity 1. Integrins recognize a
number of motifs on extracellular proteins, including the amino acids Arg-Gly-Asp
(RGD) which mediate several important cellular functions 239. Integrin signaling across
the plasma membrane allows communication between the extracellular matrix and
cytoskeleton, in association with phosphorylation of several intracellular substrates. In
addition to this signaling “outside in”, typical of most transmembrane receptors, integrins
also signal “inside out”, meaning that integrin mediated cytoskeleton changes can lead to
an altered conformation of extracellular matrix molecules-for example, fibronectin 1.
Several integrin and nonintegrin receptors have been identified in situ on
hepatocytes and nonparenchymal cells 240,241,242. In liver, hepatic stellate cells express the
integrin α1 β1, a collagen-binding integrin that mediates stellate cell adhesion to type I
collagen and also stellate cell contraction of collagen I lattices 242. Stellate cells express a
multitude of other integrins that are important in a variety of responses in the wound
milieu 243.
Up-regulation of α6β1 and α2β1 receptors, 244 both of which bind laminin, has been
reported in experimental fibrosis. Studies have also reported the integrin phenotypes of
isolated cell types from liver. In particular, stellate cells express integrin receptors for
collagen and laminin240,242, which may contribute to their activation in response to
deposition of these matrix components during injury 1.
Antagonists directed against RGD sites are being widely investigated in many
processes in which integrin binding is important (e.g., platelet adhesion and T-cell
activation). Such antagonists may be involved in decreasing the severity of hepatic
fibrosis through RGD-mediated or other pathways. For example, the RGD mimetic, SF-
245
6,5 markedly inhibited the progression of thioacetamide-induced liver cirrhosis .
Treatment of stellate cells with soluble RGD peptides was also found to reduce the
accumulation of type I collagen, whereas a control peptide had no such effect 246. Finally,
it has been shown that the αvβ6 integrin binds and activates latent TGF-β1247, indicating
that integrins are involved in mediating cell matrix interactions and also in controlling the
activity of other important components within the wounding environment 178.
136
ii) Other Cell Matrix Receptors
In addition to integrins, a growing number of other adhesion proteins and cell
matrix receptors have been characterized, including cadherins and selectins, which
248-250
mediate interactions between inflammatory cells and the endothelial wall . Up-
regulation of a tyrosine kinase receptor, discoidin domain receptor 2 (DDR-2), has been
identified during stellate cell activation; its signaling in response to fibrillar collagens
leads to enhanced matrix metalloproteinase (MMP) expression and cell growth 126. DDR-
2 is the only receptor tyrosine kinase with a ligand that is an extracellular matrix
molecule rather than a peptide ligand, and its regulation may be a critical requirement for
perpetuating liver fibrosis 1.
3.18 Remodeling/Degradation of Extracellular Matrix
The degradation of extracellular matrix represents a very important component of

hepatic fibrosis due the following two reasons:
i) Early disruption of normal hepatic matrix by matrix proteases, hastens
replacement of the normal matrix by scar matrix, which in turn has deleterious effect on
cell function.
ii) In patients with chronic liver disease, and established fibrosis, the resorption of
excess wound matrix, “therapeutic matrix degradation”, is urgently needed to arrest or
reverse hepatic dysfunction and portal hypertension. Because fibrosis reflects a balance
between matrix production and degradation, this balance must be shifted in favour of
degradation for any antifibrotic therapy to succeed 1.
Although the net result of fibrogenesis is typically a progressive accumulation of
excess extracellular matrix, fibrogenesis clearly involves a dynamic interplay between
matrix synthesis and degradation. Under normal circumstances, a temporal and spatial
balance between matrix synthesis and degradation exists, and restoring this balance leads
to physical and functional restitution of the organ. In contrast, under abnormal
circumstances, the disrupted balance leads to scar formation. The molecular processes
137
that control the balance between normal and abnormal synthesis and the degradation of
extracellular matrix are only now becoming understood 2.
Significant progress has been made in elucidating the fundamental mechanisms of
matrix remodeling and how these apply to hepatic fibrosis.
An enlarging family of Matrix Metalloproteinases (MMP’s), also known as
(Matrixins) has been identified. These are calcium dependent enzymes that specifically
degrade collagens and non-collagen substrates 2,251. They can also be inhibited by binding
to specific inhibitors known as Tissue Inhibitors of Metalloproteinases (TIMP’s) 1,2.
As suggested previously, the remodeling process is mediated by key cellular
elements, among which are stellate cells. Stellate cells seem to accelerate the replacement
of the normal subendothelial matrix by one rich in abnormal matrix constituents through
secretion MMPs, such as MMP-1, type IV collagenase (MMP-2), or gelatinase, and
through modulation of TIMPs 252-256.
i) Matrix Metalloproteinases (MMP’s)

Broadly, they fall into five categories based on substrate specificity 1:
i) Interstitial Collagenases
(MMP’s-1, -8,and -13)
The collagenases (interstitial collagenases that include MMP-1, -8, and -
13 and the collagenase/gelatinase family) degrade a wide variety of
collagens 2.
ii) Gelatinases
Gelatinase A(MMP-2), Gelatinase B (MMP-9), and fibroblast activation
protein) 257.
These degrade denatured collagens or gelatins and also digest native type
IV collagen, an important component of the normal basement membrane 2.
iii) Stromelysins (MMP’s -3, -7, -10, and -11)
The stromelysins (-1, -2, and -3) degrade fibronectin, proteoglycans,
laminin, and a variety of other matrix proteins 2.
iv) Membrane type (MMP’s -14, -15, -16, -17, -24, and -25), and
v) Metalloelastase (MMP-12) 1.
138
MMP’s are regulated at many levels to restrict their activity to discreet regions
within the pericellular milieu. The extracellular activity of the MMPs is tightly regulated
by the interaction of activation pathways and specific inhibitors. The most prominent
activation systems are the uroplasminogen activator (uPA) and tissue plasminogen
activator (tPA) systems 2.
Inactive MMP’s can be activated through proteolytic cleavage by either
membrane-type matrix metalloproteinase-1 (MT1-MMP), or plasmin1.
They can also be inhibited by binding to specific inhibitors known as Tissue
Inhibitors of Metalloproteinases (TIMP’s) 1,2.
The stoichiometry and molecular basis of these interactions has been greatly
clarified. For example, membrane type matrix MMP-1and TIMP-2 form a ternary
complex with MMP-2, possibly including ανβ3-integrin, which is essential for optimal
251
MMP-2 activity . Plasmin activity is controlled by its activating enzyme
uroplasminogen activator (u-PA) and a specific inhibitor, plasminogen activator inhibitor
1 (PAI-1), and can be stimulated by active transforming growth factor β1 (TGF-β1). Thus
net collagenase activity reflects the relative amounts of activated MMP’s and their
inhibitors, especially TIMP’s 1.
In addition to TIMP’s other protease inhibitors may effect net degradative
activity, including α2-macroglobulin 1.
ii) Disruption of Normal Subendothelial Matrix - Initiation of

fibrogenesis
Several matrix-degrading enzymes are produced by stellate cells during
fibrogenesis 252,258 . In the liver, “pathologic” matrix degradation is the early disruption of
the normal subendothelial matrix, which occurs through the actions of at least four
enzymes: MMP-2 (gelatinase A or 72-kd type IV collagenase) and MMP-9 (gelatinase B
or 92-kd type IV collagenase), which both degrade type IV collagen; MT1-MMP, which
activates latent MMP-2; and stromelysin 1, which degrades proteoglycans and
glycoproteins and also activates latent collagenases. Stellate cells are a key source of
MMP-2 252,259 and stromelysin 260.
139
Stellate cells express the mRNA for the 72 kda type IV collagenase/gelatinase
(MMP-2) and secrete this enzyme, particularly after activation 60,252. Activation of latent
MMP-2 may require interaction with heaptocytes 261,262. Markedly increased expression
of MMP-2 is characteristic of cirrhosis 253.
HCV envelope E2 glycoprotein binds to stellate cells, and induces an increased
expression of MMP-2. This leads to increased degradation of the normal hepatic
extracellular matrix, which in turn, facilitates stellate cell activation and fibrogenesis 263.
Additionally, activation of DDR2 in stellate cells leads to increased expression of active
MMP-2 239.
251
MMP-9 is secreted locally by Kupffer cells . Stimulation with IL-1α causes
robust induction of pro-MMP-9 (the precursor of MMP-9) in stellate cells and induces
conversion of pro-MMP-9 to the active form when the cells are exposed to type I
collagen 264.
Disruption of the normal liver matrix is also a requirement for tumor invasion and
desmoplasia 1,265.
Because the enzyme/s exhibits degradative activity against basement-membrane
collagen, their release by activated hepatic stellate cells in the space of Disse disrupts the
normal subendothelial liver matrix. Enhanced production of abnormal interstitial
collagens (i.e., types I and III) subsequently leads to an abnormal basement membrane,
which, in turn, disrupts hepatocellular function and may lead to further stellate cell
activation. The net effect of this activity seems to be to accelerate the replacement of the
normal subendothelial matrix by one rich in abnormal scar constituents 178..
Urokinase plasminogen activator generates plasmin and is inhibited by
plasminogen-activator inhibitor (PAI)-1; uPA is localized to the cell surface by being
bound to a specific receptor. Plasmin degrades extracellular matrix both directly and by
activation of MMPs. After stellate cell activation, net uPA activity was increased,
elucidating a mechanism for increased MMP activation 266.
140
ii) Progression of fibrogenesis & fibrosis
More importantly, progressive fibrosis is associated with marked increases in
94,267 268
TIMP-1 , and TIMP-2 , which lead to a net decrease in protease activity and
therefore an increase in unopposed matrix accumulation. Stellate cells are the major
source of these inhibitors 269. Sustained TIMP-1 expression is a key factor in progressive
fibrosis, and its diminution is an important prerequisite for the reversal of fibrosis 1.
Moreover, TIMP-1 activity is increased in the fibrotic liver and with stellate cell
activation, leading to an imbalanced expression of TIMPs relative to interstitial
collagenase. This imbalance may promote the deposition of interstitial collagens in liver
254
fibrosis . An enhanced ability to convert precursor forms of these enzymes to active
forms after activation may help accelerate fibrosis 253.
From a practical standpoint, failure to degrade the increased interstitial or scar
matrix is a major determinant of progressive fibrosis. MMP-1 is the main protease that
degrades type I collagen, the principal collagen in the fibrotic liver; however, sources of
this enzyme are not clearly established as those of the type IV collagenases. Stellate cells
express MMP-1 messenger RNA (mRNA), but little enzyme can be detected 259.
270
Unique mechanisms of TIMP-1 regulation in stellate cells , offer the potential
for the selective inhibition of TIMP-1 expression to accelerate the resorption of scar
matrix 1.
3.19 Specificity of Liver Fibrosis in Chronic Hepatitis C
Because of the lack of HCV experimental models, specificities regarding cellular

and molecular mechanisms involved in relation between HCV and fibrosis have not been
explored in great detail at the molecular level, and the role of viral proteins in hepatic
fibrosis deserve further study. Although several co-factors related to the host have been
associated with a faster rate of fibrosis progression in hepatitis C, understanding of viral
specific factors and their role in the evolution of fibrosis require further understanding 193;
nevertheless, the representative work is submitted:
141
3.20 Are There Fibrogenic HCV Proteins?
A major impediment to the development of specific antiviral drugs has been the
lack of a highly replicative and low cost in vitro or in vivo model of HCV infection.
Several studies with HCV-transfected, preferably hepatocytic cell lines and HCV-
transgenic mouse models have emerged 271, that enable some conclusions to be drawn as
to possible direct profibrogenic and procarcinogenic mechanisms of certain HCV-
proteins, irrespective of the host’s immune response. However, only some transgenic
mouse strains develop liver damage, pointing to additional genetic predispositions.
Furthermore, numerous studies used a highly expressed single gene, or an incomplete set
of HCV-genes, a setting that does not mimic human infection, which is usually
characterized by moderate viral replication in liver. Finally, expression of a limited set of
genes ignores the interactive potential of HCV proteins with each other 155,272.
3.21 HCV Core Protein & Non-structural Protein NS5A
HCV core protein represents multifunctional activity. Earlier studies have shown
that HCV core protein regulate cellular events and induce hepatic carcinogenesis 273,274. It
has also been observed that the HCV core protein play a key role in signal transduction of
apoptosis 275; and signal transduction and induction of steatosis and lipid peroxidation 276.
Shin JY et al, 274 has shown that HCV core protein significantly produced fibrosis
related proteins in an in vitro co-culture system. TGF-β1 being recognized as the
strongest inducer of fibrogenesis, underwent pronounced expression in media of HSC co-
cultured with stable HepG2-core cells compared to that of HSC co-cultured with HepG2
cells. These results suggested that HCV core protein might regulate TGF-β1 expression.
In addition, collagen I, which was produced during fibrogenesis, was increased from
media of HSC co-cultured with stable HepG2 core cells, again strongly suggesting HCV
core protein is closely correlated to fibrogenesis. Taken together it is believed that HCV
core protein regulates TGF-β1 and TGFβRII expression to direction of progressive
fibrosis.
142
Connective Tissue Growth Factor (CTGF) is known to be a multifunctional
matricellular protein, which has been implicated in wound healing, and several fibrotic
diseases including atherosclerosis, pulmonary fibrosis, renal fibrosis, and scleroderma
274,277
. Although some data showed that CTGF was mainly derived from HSC during liver
148,278
fibrogenesis , it has been shown that all major cell types in the liver have the
potential to produce CTGF according to initial hepatic injury and type of damage 279.
274
Shin JY et al, immunocytochemically examined CTGF in HSC co-cultured
with either stable HepG2-core cells or with HepG2 cells. In their results, CTGF was
predominantly expressed in co-culture of HSC with stable HepG2-core cells than in HSC
with HepG2 cells. They noted that CTGF was expressed in both HSC and HepG2-core
cells. They also observed that CTGF mRNA and proteins were significantly expressed in
the stable HepG2-core cells compared to in the HepG2 cells alone and in normal human
liver. All this suggested that HCV core protein up-regulates CTGF in the liver with HCV
infection.
So the same authors concluded that: the expressions of α-SMA, TGF-β1, collagen
I, TGF-βRII, and MMP-2 were significantly increased in the co-culture of stable HepG2-
HCV core with HSC 274.
280
In cell culture the core protein has transforming potential . It can induce
apoptosis. It can interact with the intracytoplasmic TNF-α type 1 and the lymphotoxin β
281
receptors, enhancing their pro-apoptotic signal transduction , and activate the tumor
suppressor p53 282. In addition, HCV core can repress the cell cycle regulator p21283, and
via inhibition of the p38 mitogen activated kinase pathway promote Fas-induced
284
apoptosis . Taken together, these data rather suggest a pro-apoptotic effect of core
protein in hepatocytes, favouring the elimination of infected and potentially transformed
cells. On the other hand, anti-apoptotic Bcl-xl can be upregulated 285.
While the debate is open as to the predominantly anti- or proapoptotic effect of
core protein, the data regarding lipid accumulation in core transfected hepatocytes, one of
the second hits that favour hepatic fibrogenesis, are more homogenous. Thus it interacts
with apolipoprotein II and reduces microsomal triglyceride transfer protein in vivo,
286
inhibiting assembly and secretion of regular VLDL particles . As mentioned by
Schuppan D 155, overexpression of the cytoplasmic located HCV core has been analyzed
143
in detail. It has been associated with steatosis and hepatocellular carcinoma (HCC).
Sensitive C57BL/6 mice with the core transgene driven by the albumin promoter develop
hepatic lipid accumulation (steatosis) after 6 months and hepatocellular carcinoma (HCC)
after 16 months 287,288.
In transfected hepatoma cells core activates the retinoid X receptor alpha which
dimerises with the peroxisome proliferator activated receptor alpha to upregulate genes of
lipid metabolism like cellular retinol binding protein II and acetyl CoA-reductase 289.
Notably, by interaction with mitochondria, core protein induces reactive oxygen
intermediates and thus oxidative stress which can induce steatohepatitis 290.
The non-structural protein NS5A has also been implicated in HCV pathogenicity.
It can compromise the antiviral and hypothetically antifibrotic effect of interferon,
291
obviously, either by repression of protein kinase B or alternate pathways . NS5A
enhances the acute phase response via activation of NF-kappa B and STAT-3 and as the
core, causes oxidative stress 292. In addition NS5A has been implicated in favouring cell
cycle progression to the G2/M by binding to the cyclin dependent kinase 1 (cdk1)/cdk2-
complex 293.
Nunez et al 294, has shown that, intrahepatic COX-2, MMP-2, and MMP-9 over
expression is associated with progressive liver disease in chronic HCV infection. They
demonstrated that HCV core and NS5A proteins, alone and with the synergistic effect of
endotoxin and proinflammatory cytokines (including TNF-α and IL-1β), were able to
upregulate COX-2 and MMP-9 gene expression in cultured human hepatic cells. Their
study further showed an inducing effect on MMP-9 synthesis in human liver cells is
exerted by core protein, but not by NS5A, this induction being partially abrogated by a
specific COX-2 inhibitor.
These HCV proteins are known to function as transcriptional transactivators for a
wide number of cellular genes 295. It has been reported that core and NS5A proteins are
able to activate different transcription factors, such as nuclear factor κB, AP-1, SRE, and
STAT-3, suggesting their potential role in upregulation of COX-2 gene expression 292.
Alternative candidates transactivated by HCV proteins are PPAR-α ligands,
which are able to induce COX-2 expression in hepatocytes 296. Transcriptional regulation
by PPAR’s is achieved through PPAR-retinoid X receptor heterodimers and interaction
144
of core protein with retinoid X receptor α modulates transcriptional activity 289. The role
of C/EBP-α transcription factor in the regulation of COX-2 expression in hepatocytes has
also been described 297.
Transgenic mice that express very low levels of the complete reading frame of the
HCV polyprotein developed significantly more spontaneous HCC’s after 13 months
compared to their nontransgenic controls (5/38 vs 0/16), while transgenic mice
expressing significant levels of the HCV structural proteins (Core E1 and E2) only
298
developed HCC in a single case (1/43 vs 0/35) . While steatosis developed in all
animals, inflammation, apoptosis, and histochemical fibrosis were not enhanced in both
models, and increased hepatocellular proliferation was only found in the transgenic mice
harbouring the complete HCV genome. Matrix gene expression remains to be
investigated in these models which should also be challenged, to test how far a second hit
can promote both fibrosis and hepatocarcinogenesis 155.
3.22 HBV and Liver Injury

The mechanisms of HBV induced liver disease have also been delineated, and
some of the representative work is submitted.
Iloeje UH et al,299 has shown that progression to cirrhosis in hepatitis B–
infected persons is correlated strongly with the level of circulating virus. The risk for
cirrhosis increases significantly with increasing HBV-DNA levels and is independent of
hepatitis B e-antigen status and serum alanine transaminase level. During a mean follow-
up time of 11 years, the 3582 patients contributed 40,038 person-years of follow-up
evaluation and 365 patients were newly diagnosed with cirrhosis. The cumulative
incidence of cirrhosis increased with the HBV-DNA level and ranged from 4.5% to
36.2% for patients with a hepatitis B viral load of less than 300 copies/mL and 106
copies/mL or more, respectively (P < .001). In a Cox proportional hazards model
adjusting for hepatitis B e-antigen status and serum alanine transaminase level among
other variables, hepatitis B viral load was the strongest predictor of progression to
cirrhosis relative risk [95% confidence interval] was 2.5 [1.6–3.8]; 5.6 [3.7–8.5]; and 6.5
145
[4.1–10.2] for HBV-DNA levels ≥104 − <105; ≥105 − <106; ≥106 copies/mL,
respectively.
i) Apoptosis
As mentioned by Baumert300 , the induction of apoptosis is a hallmark of many
301
viruses infecting humans. Although HBV is considered as a non-cytopathic virus
hepadnavirus induced apoptosis and cytopathic effects have been described in several
experimental model systems: 1) a duck hepatitis B variant containing a single amino acid
change in the large surface antigen resulting in accumulation of cccDNA resulted in a
strong cytopathic effect in hepatocytes in vitro and in vivo 302,303.
In this system the level of viral replication and cccDNA formation correlated with
cytopathic effects in infected hepatocytes 302. 2) Intracellular retention of the HBV large
304
surface protein has been shown to induce apoptosis in cell lines . In this model
overexpression of the large surface antigen resulted in cellular vacuolization and
apoptosis of transfected hepatoma cells 304. 3) The HBX protein has been suggested to
induce apoptosis in both p-53-dependent and p53-independent manner 305. A viral variant
containing two core promoter mutations associate with fulminant hepatitis has been
shown to induce apoptosis in primary Tupaia hepatocytes306.
Interestingly in the latter model induction of apoptosis was independent of viral
replication, suggesting that viral protein synthesis was sufficient for the virus-induced
hepatocyte cell death. Since the two core promoter mutations resulted in two amino acid
changes of the HBX protein, the HBX protein may be a potential candidate mediating
this effect 306.
ii) Other mechanisms of HBV induced cellular injury:

According to experimental models, IFN-gamma counteracts several TGF-beta
effects. IFN-gamma displays antifibrotic effects in liver cells via STAT-1
phosphorylation, upregulation of Smad7 expression and impaired TGF-beta signaling. A
benefit of 9-month IFN-gamma treatment resulting in decreased fibrosis scores and a
reduced number of alpha-smooth muscle actin-positive hepatic stellate cells (HSCs) has
146
been identified. Approaches opposing profibrogenic activities of TGF-beta may be
amenable in chronic liver disease 307.
Antiproliferative, pro-apoptotic and immunosuppressive activity effects suggest
crucial role of transforming growth factor (TGF)-beta1, metalloproteinase (MMP)-1 and
its tissue inhibitor (TIMP)-1 in the pathogenesis of acute liver injury that in some patients
precede development of chronic liver diseases and fibrogenesis. Flisiak R et al, 308 has
shown significant correlation between both TGF-beta1 and ALT or AST as well as
between TIMP-1 and ALT, AST or bilirubin in acute HBV infection. Elevated baseline
levels of both TGF-beta1 and TIMP-1 decreased gradually in consecutive weeks of the
disease. TGF-beta1 but not TIMP-1 plasma concentrations were significantly lower in
3rd and 4th week than baseline values. MMP-1 concentration remained on baseline level
in the 2nd week of the disease. However in the 3rd week its values increased suddenly
but the significant difference in comparison to baseline was observed only in 4th week.
Thus there is an important role of TGF-beta1, TIMP-1 and MMP-1 in acute viral
hepatitis, that seems to be connected first of all with hepatocytes damage. Their role in
extracellular matrix metabolism during acute liver injury needs further evaluation.
Hepatic steatosis occurs frequently in patients with chronic hepatitis B virus
(HBV) or chronic hepatitis C virus (HCV) infection. Studies have suggested that steatosis
plays an important role as a cofactor in other liver diseases such as hepatic fibrosis,
hepatitis, and liver cancer. In contrast to HCV, however, the molecular mechanism by
which HBV mediates hepatic steatosis has not been clearly studied. Kim KH et al, 309has
shown the molecular mechanism by which hepatitis B virus X protein (HBx) induces
hepatic steatosis. The increased HBx expression causes lipid accumulation in hepatic
cells mediated by sterol regulatory element binding protein 1 and PPARγ, which could be
a putative molecular mechanism mediating the pathophysiology of HBV infection.
Overexpression of HBx induced hepatic lipid accumulation in HepG2-HBx stable cells
and HBx-transgenic mice. It also up-regulated the messenger RNA and protein levels of
sterol regulatory element binding protein 1, but not peroxisome proliferator-activated
receptor alpha (PPARα). Moreover, the expression of HBx increases PPARγ gene
expression as well as its transcriptional activity in hepatic cells, mediated by CCAAT
147
enhancer binding protein α activation. Finally, HBx expression is able to up-regulate the
gene expressions of various lipogenic and adipogenic enzymes in hepatic cells 309.
What is the role of host genetic factors in chronic HBV infection? Miyazoe S et
310
al, has shown in their study, in HBV carriers, the TNF-α gene promoter
polymorphisms were not linked to disease progression. In contrast, allelic frequencies of
T and A at positions −819 and −592, respectively, in the IL-10 gene promoter, as well as
the frequencies of ATA haplotype at positions −1082/ −819/ −592 (which is
characterized with low capacity for IL-10 production), were significantly higher in
asymptomatic carriers than in patients with chronic progressive liver disease. Even after
adjusting for individuals positive for anti-HBe, such a relationship could be found
between the two groups.
3.23 HDV and Liver Injury

Transforming growth factor-β (TGF-β) has been implicated in the pathogenesis of
liver disease. TGF-β is involved in liver regeneration and in the fibrotic and cirrhotic
transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes
fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV
pathogenesis, Choi SH et al, 311 examined the effects of HDV-encoded–only protein, the
small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on
TGF-β– and c-Jun–induced signaling cascades. They found out that: the LHDAg, but not
the SHDAg, potentiated TGF-β– and c-Jun–induced signal activation, and the
isoprenylation of LHDAg played a major role in signaling cascades. LHDAg
synergistically activated hepatitis B virus X protein–mediated TGF-β and AP-1 signaling
cascades. In addition, LHDAg enhanced the protein expression level of TGF-β–induced
plasminogen activator inhibitor-1. They concluded that: LHDAg may induce liver
fibrosis through the regulation of TGF-β–induced signal transductions. This regulation of
TGF-β–mediated signaling is accomplished by the isoprenylation of LHDAg, which is a
novel mechanism involved in HDV pathogenesis 311.
Farci P et al, 312 has shown that: high doses of interferon α-2a significantly
improved the long-term clinical outcome and survival of patients with chronic hepatitis
148
D, even though the majority had active cirrhosis before the onset of therapy. Thirty-six
patients with chronic hepatitis D who participated in a randomized controlled trial of a
48-week course of high (9 million units) or low (3 million units) doses of interferon α or
no treatment were followed for an additional 2 to 14 years.
Long-term survival was significantly longer in the high-dose group than in
untreated controls (P = 0.003) or in the low-dose group (P = 0.019) but did not differ
between patients treated with 3 million units and controls. Among surviving patients at
12 years of follow-up, a biochemical response was present in 7 of 12 treated with 9
million units, in 2 of 4 who received 3 million units, and in none of 3 controls. Long-term
alanine aminotransferase (ALT) normalization correlated with improved hepatic function
and loss of IgM antibody to hepatitis delta antigen (anti-HD). Patients in the high-dose
group had a sustained decrease in HDV replication (P = 0.008), leading to clearance of
HDV RNA and, eventually, hepatitis B virus (HBV) in some patients, as well as a
dramatic improvement in liver histology with respect to activity grade (P = 0.0004) and
fibrosis stage (P = 0.007). They documented an absence of fibrosis in the final biopsy of
4 patients with a long-term biochemical response and an initial diagnosis of active
cirrhosis 312.
149
Fig. 1 GROSS IMAGE OF A NORMAL AND A CIRRHOTIC LIVER
Gross images of two livers. On the left a normal liver with a smooth surface and
homogeneous appearance. On the right a cirrhotic liver with an irregular surface and
nodules that give it a heterogeneous appearance.
From: Garcia-Tsao G, Wongcharatrawee S, Kamath PS, et al. Cirrhosis and Portal

Hypertension. Power Point Presentation. Gastroenterology Teaching Project, American
Gastroenterological Association. CD-ROM: colour, 4 ¾ in.
150
Fig 2 DIAGNOSIS OF CIRRHOSIS – CT SCAN
Computed tomography findings in compensated cirrhosis. The contour of the liver is

irregular, there is obvious splenomegaly and the presence of collaterals indicates portal
hypertension and secures the diagnosis of cirrhosis.

151
Fig. 3 PATHOGENESIS OF LIVER FIBROSIS – STELLATE CELL
The key pathogenic feature underlying liver fibrosis and cirrhosis is hepatic stellate cell
activation. Hepatic stellate cells (also known as Ito cells or perisinusoidal cells) are
located in the space of Disse between hepatocytes and sinusoidal endothelial cells (that
normally are fenestrated). Normally, hepatic stellate cells are quiescent and serve as the
main storage site for retinoids (vitamin A).

152
Fig. 4 Low magnification scanning electron micrograph of the sinusoidal
endothelium from rat liver showing the fenestrated wall. Notice the clustering of
fenestrae in sieve plates. Scale bar, 1 µm.
From: Braet F, Wisse E. Structural and functional aspects of liver sinusoidal
endothelial cell fenestrae: a review. Comparative Hepatology 2002, 1:1doi:10.1186/1476-
5926-1-1
153
Fig. 5 Fibrogenic Cascade
Most forms of liver damage result in hepatocyte injury followed by recruitment of cells
and cytokines of inflammation, which, in turn, leads to activation of hepatic stellate cells.
Rockey DC, Bissell DM. Noninvasive measures of liver fibrosis. Hepatology 2006; 43(2)
(Suppl 1): S 114.
Rockey DC. Hepatic Fibrosis, Stellate Cells, and Portal Hypertension. Clin Liver Dis
2006; 10: 461.
154
Fig. 6 Stellate Cell Activation
Stellate cell activation is a key pathogenic feature that signifies liver fibrosis and
cirrhosis. It includes initiation and perpetuation phases.
Key phenotypic features of perpetuation include: proliferation, production of extracellular
matrix, contractility, loss of retinoids, and secretions of cytokines and peptides.
ET, endothelin; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase;

PDGF, platelet derived growth factor; TGF, transforming growth factor.
From; Rockey DC, Friedman SL. In: Boyer TD, Wright TL, Manns MP. Zakim and
Boyer’s Hepatology: a text book of liver disease. 5th edition. Philadelphia: Saunders;
2006. p. 93. And
2006; 10: 464.
155
Fig. 7 Pathogenesis of intrahepatic portal hypertension
In the normal sinusoid (left), quiescent stellate cells produce little or no endothelin-1 (ET-
1), whereas nitric oxide (NO) production by the endothelium is normal.
After liver injury (right), stellate cells become activated and produce increased quantities
of ET-1; moreover NO production by sinusoidal endothelial cells is reduced.
In addition, stellate cell activation, leads to greater expression of smooth muscle proteins.
The net effect is enhanced stellate cell contractility and resultant sinusoidal constriction
that leads to an increased resistant in sinusoidal blood flow and portal hypertension.
Rockey DC. Vascular mediators in the injured liver. Hepatology 2003; 37:9.
2006; 10: 473.
156
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184
185
Chapter 4
LIVER BIOPSY
&
HEPATIC
HISTOPATHOLOGY
Liver Biopsy
History 189
Introduction 189
Indications of liver biopsy 190
Types of liver biopsy needles 191
Techniques of liver biopsy 194
Percutaneous liver biopsy 194
Transthoracic and subcostal liver biopsy 195
Blind and guided liver biopsy 195
Ultrasound guided liver biopsy 195
Plugged liver biopsy 197
Transjugular, transvenous liver biopsy 197
Laparoscopic liver biopsy 200
Fine needle aspiration biopsy 200
Complications of percutaneous liver biopsy 202
Mortality 202
Causes of mortality 203
Morbidity 204
Intraperitoneal haemorrhage 204
Intrahepatic and subcapsular haematomas 205
Pain 205
Haemobilia 206
Miscellaneous 206
Contraindications 206
The uncooperative patient 208
186
Abnormal coagulation indices 208
Prothrombin time 210
Thrombocytopenia 210
Platelet function/bleeding time 211
Ascites 212
Cystic lesions 212
Quality of liver biopsy specimens
Sampling variability 213
Specimen size and histological evaluation of grading 216
and staging
Intra-observer and Inter-observer variation in the 217
histopathological assessment of chronic viral hepatitis
Length and the number of complete portal tracts 221
Type of needle 222
Needle size 222
Thin needle vs large needle for assessment of diffuse 223
liver disease
Center experience 225
Transjugular liver biopsy 225
The adequate liver biopsy sample 226
187
Histopathology of chronic hepatitis B and C
Elementary lesions of chronic hepatitis 229
Histopathology of hepatitis B virus 234
Histopathology of hepatitis C virus 235
Grading and staging of chronic hepatitis 236
History 236
Knodell score 238
Limitations of the Knodell score 239
Modified Histological Activity Index-The Ishak score 243
METAVIR score 246
Guidelines by International Association for the Study 250
of the Liver
Scheuer Scoring System 251
Batts and Ludwig system 252
Laennec scoring system for fibrosis 253
188
LIVER BIOPSY
4.1 History
Paul Ehlrich in Germany is credited with the first liver biopsy in 1883 1, and
subsequently liver biopsy for diagnostic purposes was reported in 1923 2. In the 1930’s
two series were published on the role of liver biopsy in evaluating acute and chronic liver
disease 3,4. The procedure became more popular and practical after Menghini reported a
quick, “ one-second needle biopsy of the liver” in 1958 5,6.
Before Menghini, the previous technique was cumbersome and required on
average 6 to 15 minutes of intrahepatic phase making it difficult and probably unsafe 5,7.
With this fundamental modification in technique, the liver biopsy for histologic
examination has been central to the investigation of hepatic disease for the last 50 years 8-
10
. Despite being an invasive procedures, the liver biopsy has become widely used 11,12 in
clinical practice, because of the low mortality (0.01-0.17%), and the relatively low
morbidity of the procedure12.
4.2 Introduction
The liver biopsy is an important diagnostic tool and helps make important
therapeutic decisions in acute and chronic liver disease 13,14. It is usually the most specific
test to assess the nature and severity of liver diseases 7; and has been considered the “gold
standard” 13,14. According to Cholongitas et al,13 in chronic hepatitis C, histopathologic
examination is considered mandatory for grading (necroinflammatory activity) and
16,17
staging (fibrosis) in most patients ; including patients with persistently normal
18,19
aminotransferase levels , and for evaluating steatosis, all histologic features that affect
20-22
the natural history, prognosis, and therapeutic outcome . In chronic hepatitis B the
13,23
same applies .
In addition to chronic hepatitis B & C, some of the other indications of liver
biopsy include diagnosis and evaluation of: nonalcoholic steatohepatitis, alcoholic liver
disease, autoimmune hepatitis, hemochromatosis, Wilson’s disease, primary biliary
189
cirrhosis, cholestatic liver disease, fever of unknown origin, liver mass, status of liver
post transplantation and of donor pre-transplantation 7.
The status of liver biopsy is being challenged by noninvasive tests for the
evaluation of fibrosis and its value called into a question owing to variable specimen size
and other factors 13.
The size of the liver biopsy specimen which varies between 1 and 3 cm in length
and between 1.2 and 2 mm in diameter, represents 1/50,000 of the total mass of the liver
7,24
. Usually for the evaluation of diffuse liver disease, a specimen of 1.5 cm in length is
adequate for a diagnosis to be made, but some authors claim that a specimen of 2.5 cm
represents the best optimal length 25. The number of portal triads present in the specimen
is important; most hepatopathologists are satisfied with a biopsy specimen containing at
least six to eight portal triads7; but in cases of chronic liver disease in which the extent of
injury may vary among portal triads, and due to this reason, one review recommends 11
13
complete portal tracts . All the needles currently used for liver biopsy usually provide
an adequate specimen. Specimens obtained with standard thin-bore or spring-loaded
needles measure between 1.4 and 1.8 mm in diameter, and those obtained with Menghini
or Trucut needles measure up to 2 mm in diameter 7,26,27.
Advances in medical technology at the molecular level such as markers of liver
fibrosis, improvements in imaging modalities, together with advances in drug therapy and
refinements in surgical techniques, such as, liver transplantation, have greatly influenced
the diagnosis and management of hepatic disease and as a consequence the role of liver
biopsy is also evolving 7,10,12.
4.3 Indications of liver biopsy

The indications of liver biopsy are outlined in the table 6. Even for patients in
whom serologic tests point to a specific liver disease, a liver biopsy can give valuable
information regarding staging, prognosis, and management 6,7.
According to Bravo AA 7, in patients with chronic hepatitis C infection,
not only is there a poor correlation between symptoms or levels of serum alanine
aminotransferase and histologic features of the liver, but also patients with completely
190
normal levels of liver enzymes may also be found to have clinically significant fibrosis or
cirrhosis on biopsy 18,19,28.
If the patient has mild disease and is infected with genotype 1a or 1b of hepatitis
C virus, a decision may be made to defer treatment, or the treatment may be stopped if
there are significant side effects. Conversely, if the patient has moderate to advanced
disease, treatment will most likely be offered. The finding of cirrhosis on liver biopsy
will determine the need for further examinations, such as upper GI endoscopy to rule out
esophageal varices and screening for hepatocelluar carcinoma 7.
In alcoholic liver disease, the severity of the clinical symptoms and the degree of liver
enzyme elevation correlate poorly7,29.
In patients with alcoholic liver disease, as well as nonalcoholic steatohepatitis,
liver biopsy may reveal fatty infiltration of the liver, balloon degeneration, Mallory’s
bodies, and hepatocyte necrosis, with or without clinically significant fibrosis or
cirrhosis7,29.
In primary biliary cirrhosis, serial liver biopsies help one to study the natural
history, monitor the effects of therapy, or identify a recurrence of the disease after liver
transplantation 7,30,31.
The elucidation of various processes that occur in a transplanted liver-
including immune rejection, systemic or infectious complications, drug toxicity, and the
recurrence of primary disease-requires a liver biopsy 32.
It has been claimed that liver biopsy is able to provide an accurate diagnosis in
approximately 90% of patients with unexplained abnormalities revealed on liver function
tests 7,33.
4.4 Types of liver biopsy needles
There are three general categories of needles used to obtain a percutaneous liver
biopsy (the most common of the liver biopsy techniques)34:
· Suction needles (Menghini needle, Sure-Cut [modified Menghini needle],
Klatskin needle, Jamshidi needle)
191
· Cutting needles (Trucut needle,Vim-Silverman needle)
· Spring loaded cutting needles that have a triggering mechanisms
Table 6 Indications for liver biopsy
Indications for liver biopsy

Grading and staging of chronic hepatitis B and C
Evaluation of abnormal results of biochemical tests of the liver in association with a
Serologic work up that is negative or inconclusive
Evaluation of fever of unknown origin, with a culture of tissue
Diagnosis, grading, and staging of nonalcoholic steatohepatitis, alcoholic liver disease, or
autoimmune hepatitis
Diagnosis of hemochromatosis in index patient and relatives, with quantitative estimation
of iron levels
Diagnosis of Wilson’s disease, with quantitative estimation of copper levels
Evaluation of cholestatic liver diseases, primary biliary cirrhosis and primary sclerosing
cholangitis
Evaluation of the efficacy or the adverse effects of treatment regimens (e.g., methotrexate
Therapy for psoriasis, effectiveness for therapies for chronic viral hepatitis)
Diagnosis of a liver mass/intrahepatic neoplasms
Evaluation of the status of the liver post transplantation, or of the donor liver before
transplantation
Diagnosis and evaluation of systemic disorders, such as, sarcoidosis, lymphoma, acquired
immunodeficiency syndrome, and amyloidosis
Evaluation of unexplained jaundice, acute hepatitis of uncertain cause, and hepatomegaly
Adapted from:
1. Bravo A, et al. Liver Biopsy. N Eng J Med 2001; 344(7): 495-500.
2. Reddy KR, et al. Evaluation of the Liver: Liver biopsy and laparoscopy. In:
Schiff ER, eds. Schiff’s Diseases of the Liver. 9th edition. Lippincott Williams
& Wilkins Philadelphia 2003; 257-280.
192
The two main types of needle currently being used are the Trucut and the Menghini
needles. These two needles use different methods for sampling hepatic tissue. The
former, as the name describes, is a cutting needle, whereas the latter uses a suction
technique 34.
These needles come in varying diameters, and the type and gauge of the needle
that is optimal for percutaneous liver biopsy has been the subject of several studies 12,13.
Specimens from the Trucut needles are larger and give more information about
12
liver architecture and may thereby increase the diagnostic yield , however this has
13
undergone re-evaluation . If cirrhosis is clinically suspected, a cutting needle is
preferred over a suction-type needle, as fibrotic tissue tends to be fragmented with the
latter 10,35,36.
12
According to Grant A , a large series to look at needle type in relation to
complications describes a complication rate of 3.5/1000 for the Trucut needle and 1/1000
for the Menghini needle. Death, serious haemorrhagic complications, pneumothorax, and
biliary peritonitis all occurred more frequently with the Trucut needle than with the
Menghini needle, whereas puncture of other viscera and sepsis were more frequent with
the Menghini needle 36. Others have reported conflicting results about their relative safety
13,37
.
Cutting needles, with the exception of the spring-loaded variety, require a
relatively longer time in the liver during the biopsy, a factor that may influence the risk of
34,38
bleeding . Some groups have compared the older Jamshidi suction needle with the
Trucut/Vim Silverman cutting needles and found no difference in complication rates 38,39.
The theoretical advantages of the Menghini suction technique have been
described 40, the main advantage being that the needle is only in the liver parenchyma for
a “second”. This allows less time for the patient to move, thereby minimizing the
potential for tearing the capsule 12.
Although not consistent, a greater risk of bleeding following a biopsy has been
observed with larger diameter needles 34,36. The needles are considered large when the
external diameter is 1.0 mm or more (14-19 gauge), and thin when it is less than 1.0 mm
(> 20 gauge) 13.
193
According to Grant A12, one group showed that larger needles produced more
bleeding after liver biopsy in anaesthetized pigs. This was statistically significant when
comparing 2.1 mm (14 gauge) with 1.6 mm (16 gauge) needles, and also when
comparing 1.6 mm with 1.2 mm (18 gauge) and smaller needles 41. Human studies on the
effect of biopsy needle diameter on complications are rare; however, Forssell and
colleagues42 could not show any difference in the incidence of intrahepatic hematoma
formation when they compared the 1.6 mm modified Menghini needle with a 1.9 mm
Jamshidi needle12.
4.5 Techniques of liver biopsy
There are currently several techniques available for obtaining the liver tissue 7,12:
1. Percutaneous biopsy
2. Transjugular biopsy
3. Laproscopic biopsy
4. Biopsy or fine needle aspiration under ultrasonography or computed
tomography
5. Peroperative biopsy
Each of these techniques has its own merits and demerits 7. The decision to select
a particular technique is based upon available expertise and the particular clinical
situation.
4.6 Percutaneous liver biopsy
The percutaneous technique for the liver biopsy is the most commonly used
procedure and lasts just a few seconds.
Percutaneous liver biopsy may be classified according to the site of entry of the
biopsy needle, whether the biopsy is performed in a blind or guided manner, or according
to Grant A 12 whether the biopsy track is plugged after the procedure 12.
194
4.6.1 Transthoracic and subcostal liver biopsy
According to one source Grant A12, the patient lies supine for both of the
approaches of the procedure. The borders of the liver are usually defined by percussion or
visualized by ultrasound. In most instances the intercostal space in the midaxillary line
just cephalad to the costal margin is then infiltrated with the local anaesthetic, the biopsy
needle is then advanced into the intercostal space. The patient then holds his/her breath in
expiration. The subsequent procedure for taking the liver biopsy then varies according to
whether the biopsy needle is of aspiration or cutting type.
If the patient has an enlarged liver extending below the costal margin, then the
12
site of entry of the biopsy needle may be subcostal . One view claimed that the
complications are slightly more frequent with the transthoracic (4.1%) than the subcostal
39
route (2.7%) , but usually this is not the preferred route because of risk of puncturing
the gall bladder.
4.6.2 Blind and guided liver biopsy

A blind biopsy is one which is done without imaging of the liver immediately
prior to taking the biopsy sample. A guided biopsy is one that is undertaken during
imaging of the liver, whether the imaging modality is ultrasound, CT, or MRI scan. Thus
guided biopsies should provide bigger specimens, should avoid the puncture of adjacent
organs, and should allow the accurate biopsy of focal hepatic lesions, where appropriate
12
. Apart from focal lesions, for diffuse disease, it is the experience of the operator that
counts more than the imaging modality, as discussed subsequently.
4.6.3 Ultrasound guided liver biopsy

Ultrasound guided percutaneous liver biopsy is used extensively in the
investigation of focal liver lesions, however its use in diffuse liver disease is more
controversial.
34
According to another source , ultrasonography prior to a liver biopsy identifies
silent mass lesions (such as haemangioma) and defines the anatomy of the liver and the
relative positions of the gallbladder, lung, and kidney. Many practitioners routinely
195
recommend an ultrasound of the liver prior to performing a percutaneous biopsy. Others
perform an ultrasound only in selected patients such as those who have a history of prior
upper abdominal surgery, those in whom the point of maximal hepatic dullness cannot be
elicited, and those who are suspected of having advanced cirrhosis with possible atrophy
of the right lobe of the liver based upon clinical or laboratory grounds 34.
In a study that evaluated 222 consecutive liver biopsies by a single operator,
ultrasonography was helpful in only 3.6 percent of the patients in whom the biopsy site
43
had to be moved after it had been marked by the percussion technique . A British
survey concluded that complications were not avoided by the use of ultrasonography
prior to liver biopsy 44.
Different conclusions were reached in a study of 165 consecutive outpatient liver
biopsies, in which ultrasound changed the biopsy site in 21 of 165 patients (13 percent)
45
and led to abortion of the procedure in four patients . Similarly, two large studies
demonstrated a lower complication rate and a higher diagnostic yield using
ultrasonography guidance 34,46,47.
13
In one of the reviews Cholongitas E et al , a total of 5,392 specimens from
ultrasound guided procedures and 1,369 specimens from blind biopsy procedures were
analyzed. Specimens from ultrasound-guided biopsies were longer than specimens from
blind biopsy procedures (20.5 vs 14.4 mm; p=0.021). However ultrasound guided biopsy
specimens did not contain significantly more Complete Portal Tracts (CPT’s) than
specimens from blind biopsy procedures (8.3 vs 5.3; p=0.13). Specimens obtained using
the Menghini needle and ultrasound guidance were significantly longer than specimens
obtained with the Trucut and ultrasound guidance (24.4 vs 13.6 mm; p=0.017), and
specimens obtained blindly using the Menghini (15.8 mm; p=0.017). There was no
significant difference in the length of specimens obtained with the Trucut needle with
ultrasound guidance vs blindly 13.
It has been postulated that ultrasound guided biopsy should reduce complications.
As the commonest cause of mortality is bleeding, it follows that the incidence of bleeding
should be proportional to the incidence of hematoma formation. The rate of hematoma
formation however is unaffected by the use of ultrasound guidance 48.
196
One way ultrasound can reduce the incidence of bleeding is potentially it can
49
reduce the number of passes made into the liver . This may be especially important in
the context of a shrunken liver where ultrasound may be used to perform the procedure
accurately the first time 12. The increased risk of bleeding associated with multiple biopsy
38
passes has been documented in patients with and without malignancy , and has led to
the suggestion that all hepatic tumors should be biopsied by ultrasound or CT guided fine
needle aspiration 12.
Additionally, it is also useful to have a pre-biopsy ultrasound/chest X-ray to rule
out any anatomical abnormalities such as Chilaiditi syndrome, where bowel lies between
the liver and the abdominal wall, thereby avoiding inadvertent puncture of an adjacent
50
viscus ; or a haemangioma in the liver. It is also helpful in patients in whom the liver
cannot be easily identified for reasons such as obesity 12.
4.6.4 Plugged liver biopsy

According to the source Grant A12, plugged liver biopsy is a modification of the
51,52
percutaneous approach first described in 1984 . It has been advocated as an
alternative method for obtaining liver tissue in patients with impaired coagulation where
transjugular biopsy is not available.
In this technique a biopsy sample is taken using a Trucut needle in the
conventional manner, but only the obturator containing the specimen is removed, leaving
the outer cutting sheath within the liver parenchyma. A plastic cannula is then inserted
down the sheath and while the breath is still held in expiration, gelatin or gelfoam is
injected as the sheath is withdrawn12.
4.7 Transjugular, transvenous liver biopsy

Disorders of coagulation commonly occur in patients with liver disease and the
conventional practice in circumstances where there is significant disturbance of clotting
is to avoid perutaneous liver biopsy because of risk of bleeding 12.
197
53
Transvenous liver biopsy was first described in 1964 . Then in 1967, the
transjugular catheterization of hepatic veins in human subjects was first described, as an
access to the biliary tree for cholangiography54. In the transjugular liver biopsy the liver
tissue is obtained from within the vascular system, it minimizes the risk of bleeding7.
This is usually done through the transjugular approach; but rarely also done via a femoral
approach. It is performed in the vascular catheterisation laboratory with videoflouroscopy
equipment and cardiac monitoring because of risk of cardiac arrythmias as the catheter
passes through the right atrium 55.
According to Bravo A, et al 7, the internal jugular vein is (usually) cannulated on
the right side and a sheath inserted via a Seldinger technique. A 45 cm long catheter is
then guided under fluoroscopic control through the right side of heart to the inferior vena
cava. The catheter is then loaded with the transvenous biopsy needle and advanced into
the hepatic veins and the position checked by injection of contrast medium. The needle is
then advanced rapidly 1-2 cm post the tip of the catheter with the patient holding his/her
breath and the liver tissue is retained in the needle by aspiration/suction by a syringe
attached to the other end of the needle while it is still inside the liver 12. The duration of
the procedure is between 30 to 60 minutes 7.
According Bravo A7, adequate tissue for histologic diagnosis can be obtained in
80-97% of the patients in centers where a large number of transjugular biopsies are
56
performed . The tissue specimen is usually 0.3 to 2 cm long, and the procedure
generally requires multiple passes 7. The samples obtained are small and fragmented, a
disadvantage of the technique that may be obviated with newer generation technology 57.
However, in centers where transjugular biopsies are performed regularly, larger samples
with fewer passes are obtained13. A transjugular liver biopsy may be performed at the
same time as the placement of a transjugular intrahepatic portosystemic shunt (TIPS) 7.
The rate of complication associated with transjugular liver biopsy ranges from
1.3% to 20.2%; and the mortality ranges from 0.1% to 0.55%7,55,56.
Complications of transjugular liver biopsy include: neck haematoma, transient
Horner’s syndrome, transient dysphonia, cardiac arrythmias, pneumothorax, abdominal
pain, fistula formation between hepatic artery or portal vein or the biliary tree, and the
perforation of the liver capsule (especially in small cirrhotic livers) 7.
198
Heterogeneity of the liver disease and interobserver or intraobserver variation has
not been evaluated in transjugular liver biopsy13.
One review 13, analyzed 15 studies of transjugular liver biopsy with 1,389 TJLB
specimens; (mean = 2.5 passes per patient), the mean + SD length was 13.5 + 4.5 mm (13
of 15 studies), and the mean + SD number of CPT’s was 6.8 + 2.3 (6 studies). According
to the review13, only the author’s own large series of TJLB (n=326) 58
detailed the
number of passes (n=3), needle size (Trucut 19 gauge), length (mean 22.5 mm), number
of CPT’s (mean 8.7), and fragmentation (median=5) 58, most studies on the other hand
were small series, and were deficient in one or estimates.
One study 59 compared Trucut (18 gauge) and Menghini type (16 gauge) needles
and found that using the Trucut resulted in significantly larger specimens (12 vs 7 mm, p
= <0.05) 58.
The authors13 concluded that the quality of TJLB, as well as interobserver and
intraobserver variation, requires further study because this method allows multiple passes
(to obtain adequate samples) with far less likelihood of increasing complications 13,54,60,61.
Indications for Transjugular Liver Biopsy

Severe coagulopathy
Massive ascites
Massive obesity
Suspected vascular tumor or peliosis hepatis
Need for ancillary vascular procedure (e.g. TIPS or venography)
Failure of percutaneous liver biopsy
Table 7. Indications for transjugular liver biopsy

Adapted from: Bravo A, et al. Liver Biopsy. N Eng J Med 2001; 344(7): 495-500.
How does TJLB compare with PLB? In this study58, 326 consecutive TJLB
specimens, using three passes (19 gauge Trucut) were compared with 40 consecutive
199
PLB specimens using (15 gauge Menghini) needle. The study concluded that TJLB with
3 passes is adequate for histologic diagnosis, with 89% of specimens being either > 15
mm or having > 6 portal tracts. Although like PLB, adequate sampling remains a
limitation for staging and grading of chronic hepatitis with TJLB, nonetheless, the
feasibility of TJLB is comparable to PLB in this respect 58.
4.8 Laparoscopic liver biopsy

Laparoscopic liver biopsy is well established, however its use varies between
centers. For instance, it is ideal in patients who have a combination of a focal liver lesion
and a coagulopathy, where a histologic diagnosis is essential in the management of that
patient. Some centers in USA perform laparoscopic liver biopsy on an outpatient basis 62
and in some Japanese centers more than 50% of liver biopsies are performed
laparoscopically 12,63.
However, generally, the use of laparoscopic liver biopsy by
gastroenterologists has declined in favour of: less invasive radiologic procedures, and
usually the procedure is performed by surgeons. The indications for and contraindications
to the laparoscopic liver biopsy are outlined in the table 8 7.
According to Bravo A7, the complications include perforation of a viscus,
bleeding, hemobilia, laceration of the spleen, leakage of ascitic fluid, hematoma in the
abdominal wall, vasovagal reaction, prolonged abdominal pain and seizures7,64.
4.9 Fine Needle Aspiration Biopsy

According to one source 7, Lundquist in 1971 demonstrated that fine needle
aspiration compared favourably with the final histologic diagnosis based on surgical
65
specimens . Patients with focal hepatic lesions are good candidates for fine needle
aspiration biopsy, especially if they have a history of cancer.
24
The diagnostic accuracy ranges from 80 to 95 percent , and is substantially
affected by the expertise of the cytopathologist. Cytologic findings that are negative for
cancer, however, do not rule it out.
200
Indications for and Contraindications to Laparoscopic
Liver Biopsy
Indications
Staging of cancer
Ascites of unclear cause
Peritoneal infections
Evaluation of an abdominal mass
Unexplained hepatosplenomegaly
Contraindications
Absolute
Severe cardiopulmonary failure
Intestinal obstruction
Bacterial peritonitis
Relative
Uncooperative patient
Severe coagulopathy
Morbid obesity
Large ventral hernia
Table 8. Indications for and contraindications to laparoscopic liver biopsy

Adapted from: Bravo A, et al. Liver Biopsy. N Eng J Med 2001; 344(7): 495-500.
Fine needle aspiration biopsy of the liver is performed under ultrasonographic or

CT guidance. Although it is usually reserved for focal hepatic lesions; limited data
suggest that diagnostically useful material can be obtained with automatic spring-loaded
201
66
biopsy needles guided by ultrasound in over 95% of patients , including those with
diffuse liver disease.
Fine needle aspiration biopsy is associated with a low risk of seeding of the
needle track with malignant cells, and is generally a very safe procedure, even in patients
with haemangiomas, and echinococcal cysts7,67,68.
4.10 Complications of percutaneous liver biopsy

The indications for, and methods of liver biopsy have changed over the last few
69
years , with the advent of new imaging techniques, better facilities at patient
monitoring, and new indications for liver biopsy such as liver transplantation70. All
invasive procedures have a mortality rate associated with them, and consequently the
benefits of obtaining liver sample for histology should always be weighed against the
possible morbidity and mortality of the procedure 12.
4.10.1 Mortality
According to Grant A, the reported mortality from liver biopsy varies
considerably. This is partly because most of the larger series reporting liver biopsy
36,55
complications have been retrospective . The overall mortality rate also varies
according to the center in which the liver biopsies were performed-for example, an audit
of liver biopsies performed in the UK district general hospitals the death rate was
10,12
between 0.13% to 0.33% . And in the Mayo Clinic the mortality from fatal
haemorrhage after percutaneous biopsy was 0.11% 38.
A generally accepted mortality rate in standard textbooks is between 0.1% and
0.01% 11,12.
According to Reddy KR, et al, 6 although the liver is a rich vascular organ with an
intricate meshwork of supply and drainage, the complications associated with
percutaneous liver biopsy are fortunately rare. This may be related to the elasticity of the
liver parenchyma itself or the elasticity of the biopsy track collapsing down after core has
been taken, or to the presence of high concentration of clotting factors within the liver
substance 71. It should, however, be borne in mind that during a blind percutaneous liver
202
biopsy, the liver is not the only structure to be punctured and the skin and subcutaneous
tissues (and occasionally other organs) can bleed. Thus, peripheral indexes of clotting
must still be taken into consideration 12.
The most dreaded of all the complications is haemorrhage, which can manifest as
free intraperitoneal bleed, intrahepatic or subcapsular hematoma, or hemobilia. Sixty
percent of complications occur within two hours after the procedure, and 90% within 24
36,72
hours . Fatal complications typically occur within 6 hours after a liver biopsy. A
hospitalization rate of 1.4% to 3.2% for the management of complications following a
liver biopsy has been reported, with pain or hypotension as the predominant cause 6,27,73.
4.10.2 Causes of mortality

According to Grant A, et al, 12 the main cause of mortality after percutaneous liver
biopsy is intraperitoneal haemorrhage as shown in a retrospective Italian study of 68,000
percutaneous liver biopsies in which all six patients who died did so from intraperitoneal
haemorrhage 36. Three of these patients had had a laparotomy, and all had either cirrhosis
38,71
or malignant disease, both of which are risk factors for bleeding . Other serious
complications responded to treatment; puncture of viscera was never followed by serious
complications. Other series have shown, however, that puncture of the gall bladder
followed by biliary peritonitis is a recognized cause of death 10.
As the main source of mortality after percutaneous liver biopsy is haemorrhage, it
is reasonable to assume that improvements in mortality rates can be made if the clinician
understands the risk factors for bleeding, recognizes bleeding promptly and aggressively
resuscitates the patient. It has also been suggested that patients who bleed significantly
(i.e., patients whose haemoglobin falls to >20g/l or who become haemodynamically
unstable) should be considered for either laparotomy or therapeutic angiography if the
10,12
bleeding does not stop with transfusion alone ; as well as those patients who are
suspected to have biliary peritonitis should also have an early laparotomy12.
4.10.3 Morbidity
According to Grant A, 12 like mortality, the overall morbidity from percutaneous
203
liver biopsy varies, and is difficult to ascertain as most studies are retrospective and
therefore symptoms such as post-biopsy pain requiring simple analgesia, or transient
vasovagal drop in blood pressure, are not recorded. Similarly, there is no agreement about
the division into major and minor complications, and whether complications such as
asymptomatic post-biopsy intrahepatic/subcapsular haematoma should be included in the
figures.
A morbidity rate of 5.9% for patients suffering from minor complications after
liver biopsy has been reported 39.
4.10.4 Intraperitoneal haemorrhage

According to Reddy KR, et al,6 significant intraperitoneal haemorhage is the most
serious of all complications and often becomes apparent within the first 2 to 3 hours after
36,38,73,74
the procedure . In a large study of the complications of 68,276 liver biopsies,
36
haemoperitoneum occurred in 0.32% of patients . Significant haemorrhage (indicated
by a drop in haemoglobin of >20 g/l) occured in 0.35 to 0.5% of all procedures 38,75. Free
intraperitoneal haemorrhage may be related to a laceration sustained during deep
inspiration as the biopsy is performed, or to a penetrating injury of a branch of the hepatic
artery or portal vein 6. However, the overall reported incidence ranged from 0.03% to
0.7% 36,38,39,73,74,76.
Delayed free intraperitoneal haemorrhage after a 24 hour period is infrequent and
unpredictable 77. The interval before delayed haemorrhage after liver biopsy has ranged
from 36 hours to 18 days, and delayed haemorrhage is associated with a high mortality
rate 77. Haemorrhage after liver biopsy is more common in patients who are elderly, have
cirrhosis or a malignant lesion, or undergo multiple passes 38,71.
Hypotension or tachycardia following a biopsy, particularly when associated with
abdominal pain, is usually the consequence of haemorrhage. Ultrasonography, preferably
performed at the bedside, or CT may readily demonstrate a free intraperitoneal collection.
Measures to improve haemodynamic status may be sufficient, blood or blood products
should be administered. Qualitative platelet abnormalities must be kept in mind. Success
with a conservative approach is apparent within a short period of time. If haemodynamic
204
instability persists despite aggressive resuscitative measures during a couple of hours,
alternative measures should be pursued 6.
According to Reddy KR6, angiography is the preferred procedure, and
experienced angiographers are able to embolize a peripheral bleeding vessel and any
associated arteriovenous fistula. Some of the agents used for embolization include gelatin
sponges, autologous blood clot, bucrylate, polyvinyl alcohol sponges, detachable
balloons, steel coils, and platinum microcoils. The major complications of transarterial
embolization include ischemia or infarction of the liver, gall bladder, or pancreas; rates
range from 4% to 15%. Minor complications include fever, bacteremia, pain and
78-80
elevation of transaminases . Surgical exploration is preferred, if haemodynamic
instability persists despite aggressive measures, for a relatively rapid access to the
bleeding site/vessel 6.
4.10.5 Intrahepatic and subcapsular haematomas
According to Reddy KR6, intrahepatic and subcapsular haematomas are noted

frequently when ultrasonography is performed after a liver biopsy 6. The incidence of
these haematomas following liver biopsy ranges from 17% to 23% 81,82. These are often
asymtomatic and the frequency is similar in both laparoscopically guided and “blind”
liver biopsies 81. The duration of bed rest after a liver biopsy, ranging 6 to 24 hours, does
82
not appear to influence the incidence of haematoma . A rapid enlargement of the liver
associated with tenderness may suggest subcapsular or intrahepatic haematoma 83. Fever
and leukocytosis may be present, and a rare occurrence is biliary obstruction caused by a
84
large haematoma impinging on the biliary tree . Conservative treatment is generally
sufficient, and angiography may rarely be required to embolize an arteriovenous fistula 6.
4.10.6 Pain
Pain is probably the commonest complication of liver biopsy occurring in up to
30% 10,85, with moderate and severe pain occurring in 3% and 1.5% respectively 12. After
a liver biopsy, approximately one third of patients experience pain in the right upper
quadrant or right shoulder 10. The pain is usually dull and mild, but occasionally may be
205
sharp and stabbing, and responds to analgesics. It is also of short duration, occasionally
lasting few hours. Generally, the long procedure or multiple passes are associated with
increased severity of pain. Ongoing severe abdominal pain should alert the physician to
the possibility of bleeding or peritonitis 6.
Transient hypotension and vasovagal episodes are common accompaniments to
39
pain, occurring in about 3% of liver biopsies , and vasovagal episodes occasionally
require the administration of atropine 12, and fluid boluses.
4.10.7 Haemobilia
Hemobilia is a well recognized but infrequent complication of liver biopsy,
presenting with a classic triad of gastrointestinal bleeding, biliary colic, and jaundice 86,87.
12 36
It has an incidence of 0.05% . Piccinino et al, reported 4 cases in 68,276 biopsies.
88 89
Cholecystitis , and pancreatitis are infrequent complications of hemobilia. Hemobilia
may resolve with conservative treatment, but ongoing or intermittent bleeding requires
intervention. Successful treatment is achieved angiographically in 95% of patients 6,86,87.
4.10.8 Miscellaneous complications

Puncture of other viscera occurs infrequently, with an incidence between 0.01%
36
and 0.1% . The puncture of lung, colon, kidney, and gall bladder, together with
pneumothorax, pleural effusion, and subcutaneous emphysema are well recognized
complications, which rarely require intervention 90.
Other complications include sepsis, reaction to anaesthetic, and breakage of the
biopsy needle 12,91.
4.11 Contraindications
Some of the contraindications of liver biopsy have changed overtime due to
patient selection, the availability of newer imaging modalities, choice of the technique
(transjugular/laparoscopic/ percutaneous), and modern patient care facilities.
206
Table 9. Complications of percutaneous liver biopsy 6
Pain (0.056% - 22%)
Pleuritic
Peritoneal
Diaphragmatic
Haemorrhage
Intraperitoneal (0.03 - 0.7%)
Intrahepatic and/or subcapsular (0.59% - 23%)
Haemobilia (0.059% - 0.2%)
Bile peritonitis (0.03% - 0.2%)
Bacteremia
Sepsis (0.088%) and abscess formation
Pneumothorax and/or pleural effusion (0.08% - 0.28%)
Haemothorax (0.18% - 0.49%)
Arteriovenous fistula (5.4%)
Subcutaneous emphysema (0.014%)
Reaction to anaesthetic (0.029%)
Breakage of the needle (0.02% - 0.059%)
Biopsy of other organs
Lung (0.001% - 0.014%)
Gall Bladder (0.034% - 0.117%)
Kidney (0.029% - 0.096%)
Colon (0.0038% - 0.044%)
Mortality (0.0088% - 0.3%)
Sources; [6,36,39,73,74,76,81,82].
Adapted from: Reddy KR, et al. Evaluation of the Liver. Liver biopsy and laparoscopy.
In: Schiff ER, eds. Schiff’s Diseases of the Liver. 9th ed. Lippincott Williams & Wilkins,
Philadelphia, 2003; 257-280.
207
For a patient with suspected chronic liver disease/cirrhosis, who has one or more
of the features of obesity, coagulopathy, and ascites, a transjugular approach is preferred
in most institutions 6. And, laparoscopy is preferred in patients with ascites of unknown
cause or in whom a mass lesion is suspected6.Liver biopsy is a safe procedure when
performed by experienced operators. Froelich et al, 92 noted a lower complication rate for
physicians who performed more than 50 biopsies a year 7.
4.11.1 The uncooperative patient

In percutaneous liver biopsy it is essential that the patient is cooperative as an
untoward movement when the biopsy needle is in the hepatic parenchyma can lead to a
tear of the parenchyma and/or the capsule and subsequent torrential bleeding. If the
patient is anxious, then the use of midazolam as sedation and/or meperidine6 can be
considered with no increased risk 93. If the patient remains uncooperative and the benefit
of obtaining liver histology outweighs the risk to the patient, then liver biopsy under
general anaesthesia should be considered 12.
4.11.2 Abnormal coagulation indices

According to Grant A, 12 there is no consensus about the values at which abnormal
coagulation indices become contraindications to percutaneous liver biopsy. A number of
investigators have shown that the degree of bleeding from the liver puncture site
(observed at laparoscopy) bears no correlation to peripheral blood coagulation
parameters, when these parameters are modestly increased 94,95.
It has been postulated that this discrepancy in liver bleeding time may be due to
the inherent elasticity of the biopsy track collapsing down after the core has been taken,
together with the high local concentrations of clotting factors within the hepatic
71
parenchyma . It should, however, be borne in mind that during a blind percutaneous
liver biopsy, the liver is not the only structure to be punctured and the skin and
subcutaneous tissues (and occasionally other organs) can bleed. Thus, peripheral indexes
of clotting must still be taken into consideration 12.
208
Table 10. Contraindications to percutaneous liver
biopsy
Absolute
Uncooperative patient
History of unexplained bleeding
Bleeding tendency
Prothrombin time > 3-5 sec more than control
Platelet count < 50,000
Prolonged bleeding time > 10 min
Unavailability of blood transfusion support
Suspected haemangioma or other vascular tumor
Inability to identify an appropriate biopsy site by percussion or ultrasonography
Suspected echinococcal cysts in the liver
Relative
Morbid obesity
Ascites
Infection in the right pleural cavity or below the right hemidiaphragm
Adapted from:
1. Bravo A, et al. Liver Biopsy. N Eng J Med 2001; 344(7):495-500.
2. Reddy KR, et al. Evaluation of the Liver: Liver biopsy and laparoscopy. In:
Schiff ER, et al. Schiff’s Diseases of the Liver. 9th edition. Lippincott
Williams & Wilkins. Philadelphia 2003; 257-280.
Liver biopsy may be helpful in determining the extent of liver damage in patients
with haemophilia and the benefits of treatment in those infected with hepatitis C virus. In
the absence of factor concentrate inhibitors, liver biopsy is safe if the clotting
abnormalities are corrected before and for 24 hours after biopsy 96.
209
4.11.3 Prothrombin time
12
According to Grant A, several large studies have not shown a greater risk of
bleeding associated with increased prothrombin time of four seconds above control values
38,71,94
, as well as a large retrospective study of percutaneous liver biopsy to date in which
the prothrombin time was prolonged by seven seconds36.
By contrast, a number of other studies, however, support the widely held belief
that a coagulopathy predisposes the patient to bleeding diathesis after percutaneous liver
biopsy 97. The 1991 BSG audit of the biopsy practice in 189 health districts in the United
Kingdom showed that bleeding occurred more commonly if the international normalised
ratio (INR) was raised, with 3.3% of the bleeds occurring when the INR was between
1.3 and 1.5, and 7.1% occurring when the INR was >1.5 10. This suggests that about 90%
of the bleeds occurred in patients with an INR<1.3 and reinforces the fact that having a
normal INR or prothrombin time is no reassurance that the patient will not bleed after the
procedure 12. However, it must be borne in mind that patients with prolonged INR ≥ 1.5
might not have been subjected to liver biopsy in the first place.
4.11.4 Thrombocytopenia
According to Grant A,12 there is no general agreement at the level at which
thrombocytopaenia becomes a contraindication to liver biopsy. In the UK the recognized
texts recommend that platelet count should be above 80 000/mm3,11. The Mayo Clinic
regards counts as low as 56 000/mm3 to be safe 38, whereas a survey of mostly US centers
showed a preference for platelet counts above 50 000/mm3,63. The evidence for a cut off
value remains scanty and takes no account of the function of the platelets 12.
The absolute value of the platelet count may not be crucial in determining the risk
of bleeding as it is well recognized that even those patients with normal prothrombin
times and platelet counts can have haemorrhage from severely deranged bleeding times.
Nevertheless, according to Grant A 12, for a percutaneous liver biopsy the minimum safe
limit for a platelet count is 60 000/mm3 12.
4.11.5 Platelet function/Bleeding time
210
Some authorities recommend that a bleeding time should be routinely obtained
27,98
, and an alternative approach considered if it is prolonged beyond 10 minutes 27. One
12
group suggests that BT is seldom if ever measured in UK centers prior to liver biopsy
even though the ingestion of aspirin and other non-steroidal anti-inflammatory drugs in
the week prior to invasive intervention is a recognized contraindication by several
authorities. And to their knowledge, however, there are no convincing data to support this
as a contraindication to percutaneous liver biopsy 12.
Patients with renal impairment usually have abnormalities of platelet function.

According to one small study, patients with end stage renal failure on haemodialysis are
at high risk (up to 50%) of haemorrhagic complications after percutaneous liver biopsy,
independent of the BT 99. This same study suggested that liver transplant recipients with a
BT above 10 minutes (upper limit of normal) had a higher incidence of bleeding
complications compared with those with a BT below 10 minutes. The sample size,
however, was too small to allow any firm conclusions to be drawn12. Several other
factors are likely to affect platelet function with or without affecting the BT. A bleeding
time may be elevated in patients with cirrhosis despite what appears to be an adequate
platelet count and prothrombin time.
In a study conducted at The Royal Free Hospital, within a group of cirrhotic

patients, those with abnormal BTs (42%) were more likely to have significantly lower
platelet counts, longer prothrombin times and higher blood urea and serum bilirubin than
those with normal BTs (58%). The investigators also demonstrated that the bilirubin
concentration and the platelet count were independently associated with the BT (although
the correlation for the latter was weak, and the raised serum bilirubin may well be just a
12,100
surrogate marker for the severity of liver disease) . However, this practice has not
been uniformly adopted 101.
One source recommends, 34 to check a bleeding time in patients with renal failure,
those who are HIV positive, or patients with a history of excessive bleeding despite a
normal prothrombin time, partial thromboplastin time, and platelet count; and to this list
patients with advanced cirrhosis may be added.
211
4.11.6 Ascites
12
According to Grant A, percutaneous liver biopsy in the presence of significant
ascites is considered a contraindication in many texts. The reasons are; from the high
likelihood of not obtaining a biopsy specimen because of the distance between the
abdominal wall and the liver, to the potential severity of underlying liver disease, and
increased risk of complications, such as uncontrollable bleeding into the ascites. Although
these reasons seem to be sensible, they are not substantiated in randomized, controlled
clinical trials. There is evidence, however, to support the fact that CT or ultrasound
guided liver biopsy in the presence of ascites does not affect the complication rate 102,103.
Notwithstanding these studies, generally, liver biopsy is avoided in the presence of tense
ascites. However, if strongly indicated, the options include; total paracentesis prior to
liver biopsy, image guided biopsy, transjugular liver biopsy, or laparoscopic biopsy 12.
4.11.7 Cystic lesions
12
According to Grant A , modern imaging techniques can identify benign cystic
lesions of the liver, thereby eliminate the need for biopsy in many cases. Cystic lesions
within the liver may communicate with several structures including the biliary tree and
therefore pose a risk of biliary peritonitis after biopsy 12.
Echinococcal cysts have been considered a contraindication to percutaneous liver

biopsy because of the risk of anaphylaxis from the dissemination of the hydatid cysts
throughout the abdomen. New develoments104, such as aspiration of hydatid cysts with
19-22 gauge needles under ultrasound guidance has been shown to be safe for both
diagnostic 105 and therapeutic 106 purposes12.
Quality of Liver Biopsy Specimens
212
The liver biopsy has an important diagnostic role that helps make important
therapeutic and prognostic decisions in acute and chronic liver disease13,14. The
histopathological examination has been considered the “gold standard”13. Though the
biopsy specimen samples approximately 1/50,000 of the mass of the liver, but is
considered reasonably representative of the whole liver pathology7. There have been
questions regarding the quality of liver biopsy specimens, and according to Cholongitas
et al13, studies have evaluated the following13:
1. What is the optimal size of the liver biopsy specimen required for the
accurate description of liver disease?
2. When there is heterogeneity of diffuse liver disease, does the small size of
constitute a problem in diagnosis?
3. Does interobserver and intraobserver variation significantly affect the liver
biopsy interpretation?
4. Which histopathological scoring system is the most reliable and
reproducible?
5. What is an adequate liver biopsy sample?
With the development of noninvasive tests of liver fibrosis, the quality of liver
biopsy specimens is further under scrutiny due to the above mentioned factors. Studies
25,107
have evaluated optimal length and number of portal tracts 107 for accurate grading
and staging in chronic viral hepatitis. Thus, there has been further emphasis on the quality
of liver biopsy leading to an accurate interpretation 13.
4.12 Sampling Variability

Even before the advent of the semiquantitative scoring systems for chronic
hepatitis, several studies already have pointed out sampling variability as a major
108-110
potential limitation of liver biopsy . These studies were published before the
development of semiquantitative scoring systems for chronic hepatitis, and underlined the
difficulty caused by sampling errors, especially when using biopsy specimens to diagnose
cirrhosis 25.
213
Maharaj et al,111 by performing 3 transcutaneous biopsies in the same patients
using different entry points, reported that in proven cirrhotic patients, a histologic feature
of cirrhosis was present in all 3 biopsy specimens of only 50% of the patients. Similarly,
110
Abdi et al, performed several postmortem biopsies and showed that the diagnosis of
cirrhosis could be obtained from one biopsy specimen in only 16 out of 20 cases, but that
the performance increased to 100% with 3 biopsy specimens 25.
For the diagnosis of chronic active hepatitis, several investigators raised the same
108
problems, although to a lesser extent. Soloway et al, found major discrepancies in
diagnosis in cirrhotic patients with sequential biopsies, they found a 90% degree of
consistency in grading individual histologic features of chronic hepatitis including
112
fibrosis. Less favourable results were published by Baunsgaard et al, who showed a
concordance in only 36 of 50 patients when evaluating the fibrosis amount with 2
biopsies in same patients 25.
113
According to Regev et al, therefore sampling error has been shown to exist in
needle liver biopsy, in respect to severity of inflammation,110,114,115 degree of fibrosis,
110,115
and the presence of cirrhosis 108,110,115-119.
113
Lately, Regev et al, studied 124 patients with chronic hepatitis C who
underwent simultaneous laparoscopy-guided biopsies of the right and left hepatic lobes.
The slides were randomly divided among two hepatopathologists after coding.
Inflammation and fibrosis were scored according to the modified Scheuer system (Batts
and Ludwig).
Thirty of the 124 patients (24.2%) had a difference of at least one grade, and 41 of
124 patients (33.1%) had a difference of at least one stage between the right and left
lobes. In 18 (14.5%) patients, interpretation of cirrhosis was given in one lobe, whereas
stage 3 fibrosis was given in the other. A difference of two stages or two grades was
found in only three (2.4%) and two (1.6%) patients, respectively. Of the 50 samples that
were examined twice, the grading by each pathologist on the second examination differed
from the first examination in 0% and 4% and the staging differed in 6% and 10%,
respectively. All observed variations were of one grade or one stage.
The authors concluded that: liver biopsy samples taken from the right and left
hepatic lobes differed in histological grading and staging in a large proportion of chronic
214
hepatitis C virus patients; however, differences of more than one grade or stage were
uncommon. A sampling error may have led to underdiagnosis of cirrhosis in 14.5% of the
patients 113.
One review13, compared 5 studies 113,120-123
regarding potential heterogeneity of
liver disease. The scoring systems used were Knodell, Ishak, and Scheuer. Only 1 study
had biopsy specimens of adequate length. In 50 patients with chronic HCV ultrasound-
guided PLB of the right lobe (28 ± 11 mm) and left lobe (25 ± 9 mm) showed no
difference between grading and staging in the paired biopsy specimens 123. According to
the review13, the studies had variations in length and designs. In the other studies, 1 did
not document length122, 1 had a mean length of only 12.3 mm 120
and 2 evaluated
113,121
biopsy specimens selected as 15 mm or longer . These 4 studies, however,
showed difference in agreement between grading and staging. But in the overall context,
the variations of studies documented by the review13, appear smaller than the variation in
grading and staging of liver disease.
These studies have pointed out one of the major limitations of fibrosis assessment:
sampling variation. As shown by these studies, the problem of heterogeneity could be
resolved partially by taking several biopsy specimens from the same patient; a procedure
that raises major ethical concerns due to an increase in the risk for morbidity and
mortality 25.
In the context of sampling variability, the size of the biopsy specimen must be
considered because it is evident that the longer the biopsy specimen, the lower the risk for
an erroneous evaluation due to sampling error 25.
4.13 Specimen size and histological evaluation of

grading and staging
124
According to the review 13, Holund et al , first studied diagnostic
reproducibility relative to specimen size in 100 selected liver biopsy specimens that were
215
25 mm or longer, and 1 mm or wider in patients with acute or chronic hepatitis or
cirrhosis. They concluded that an LB specimen 5 mm long or longer was adequate for
diagnosing acute hepatitis but inadequate for chronic hepatitis or cirrhosis. Subsequently,
the same group 114 focused on chronic hepatitis, using the same selection criteria (16-
gauge Menghini needles with an external diameter of 1.65 mm). Specimens of 15 mm
or more were necessary for an accurate diagnosis of chronic aggressive (active) hepatitis.
According to Cholongitas et al13, another study, in patients with chronic HCV,
using same methods evaluated 100 liver biopsy specimens. The specimens were 20 mm
or longer. The Metavir scoring system was assessed in different lengths: 5, 10, 15,
and 20 mm or longer, with the latter as the reference standard for concordance 126. A 10
mm length was adequate for reliable assessment of necroinflammatory activity and
fibrosis (weighted k, 0.81 and 0.85, respectively) 13.
Colloredo et al107, studied 161 liver biopsy specimens in patients with chronic
hepatitis B and C, using the Ishak scoring system. The biopsy length was ≥ 3cm and
width 1.4 mm. Subsequently the scoring was blindly repeated by reducing the length to
1.5 cm and 1 cm, and width to 1 mm.
Reducing the length resulted in increased diagnosis of mild grade and small stage
of fibrosis. Mild grade was diagnosed; 49.7% in > 3 cm, 60.2% in 1.5 cm, and 86.6% in 1
cm long specimens (p<0.001). Similarly, cases staged as having mild fibrosis
significantly increased in the shorter specimens: 59% in > 3 cm, 68.3% in 1.5 cm, and
80.1% in 1 cm long specimens (p<0.001)107.
According to the review13, severe grade was diagnosed in 11.8% of liver biopsy
specimens that were 3 cm or longer but only 0.6% of liver biopsy specimens that were
1.5 cm long (both were 1.4 mm wide), and severe stage was diagnosed in 11.2% of
liver biopsy specimens 3 cm or longer but in only 3.1% of liver biopsy specimens 3 cm or
longer but 1 mm wide. A specimen 20 mm or longer and/or containing 11 or more
complete portal tracts was necessary for reliable assessment of grading and staging in
chronic viral hepatitis. These criteria have been adopted rapidly as optimal standards 13.
The authors concluded that: liver biopsy size strongly influences the Grading and
Staging of chronic viral hepatitis. The use of fine needles should be discouraged in this
setting 107.
216
However, according to Cholongitas E13, whether a 16-gauge needle (external
diameter, 1.65 mm) results in a constant width of 1.4 mm needs to be questioned because
other studies using larger needles (14 gauge; external diameter, 2.1 mm) describe a
mean ± SD width of 0.9 ± 0.3mm 127. In practice, biopsy width is not uniform because
of variable tissue shrinkage and because the plane of section cannot always be through
the maximum diameter of the biopsy cylinder 13.
In an important study, Bedossa et al25, evaluated the adequacy of liver biopsy
samples obtained at least 3 cm from the tumor by using image analysis of 17 surgical
specimens following resection, in patients with chronic hepatitis C. They obtained
10,659 virtual liver samples varying from 2.5 to 200 mm in length and a constant 1.22
mm width.
The image analysis of fibrosis was converted to the METAVIR scoring system
(the reference METAVIR stage was based on the whole sample, at least 2 x 3 cm), and
compared with histopathological system. The coefficient of variation of fibrosis with 15
mm biopsy was 55%, and for 25 mm was 45%. Accurate evaluation of fibrosis was
achieved in only 65% of 15-mm-long and 75% of 25-mm virtual samples, with no
significant improvement with longer samples. The conclusion was that a specimen 25
mm was the minimum length for reliable staging25.
4.14 Intra-observer and Inter-observer variation in

the histopathological assessment of chronic
viral hepatitis
The way in which liver biopsies showing chronic hepatitis are reported is
undergoing re-evaluation. A related question is: how the inflammatory and fibrotic
changes in these liver biopsies can be semi-quantitatively assessed so that comparisons
can be made between groups of patients and the effect of treatment on disease
128
progression studied . In an attempt to standardize the histological assessment several
semi-quantitative scoring systems have been proposed and evaluated.
217
In one of the studies by Goldin et al128, a blinded trial was done in which 20 cases
of chronic viral hepatitis were assessed by five histopathologists, using the Knodell and
Scheuer scoring systems (two of the commonly used systems) on two separate occasions.
While both systems produced good inter- and intraobserver variation when fibrosis was
assessed; the Scheuer system produced slightly higher kappa values.
According to the authors, the Scheuer system also produced considerably better
agreement when the severity of inflammtion was examined. A multirater kappa analysis
confirmed that both systems showed better agreement for fibrosis scores than
inflammatory scores. The authors concluded that, while both systems produced
reasonable agreement, this was greater using the Scheuer system 128.
Westin J et al129, evaluated the interobserver reliability of Ishak scoring system in
95 liver biopsies from patients with chronic hepatitis C virus infection. For three
independent observers, the agreement for periportal hepatitis, focal necrosis, and portal
inflammation was found in 95%-96%. Kappa scores ranged from 0.11 to 0.41 and
weighted kappa scores from 0.18 to 0.53.
For staging the authors noted 84% agreement, kappa scores of 0.26-0.47, and
weighted kappa scores of 0.57-0.69.
The authors concluded that: the Ishak system is associated with good
interobserver reliability if a deviance of one categorical level in each variable of the
system is accepted as agreement. Compared to the Knodell system it provides more
detailed information but is less reliable regarding fibrosis 129.
In the study by Grønbæk et al130, five specialist histopathologists evaluated 46
liver biopsies from 20 patients treated with interferon-α. Knodell’s and Ishak’s scoring
systems, De Groote’s classification and a four level general necro-inflammatory activity
score (GNAS) were applied. In addition to kappa statistics, slide by slide analysis was
performed. An acceptable slide agreement was defined as eight of ten observer pairs
agreed on 80% of the slides.
The best agreement was seen for Knodell’s and Ishak’s fibrosis scores, De
Groote’s classification and GNAS (mean weighted kappa (κw) = 0.49, 0.51, 0.50, and
0.44, respectively). The concordance was substantial regarding cirrhosis (mean κ =0.69,
218
and 0.72, respectively), but only moderate in piecemeal necrosis (mean κ =0.40, and
0.39, respectively).
Slide by slide analysis showed the highest agreement on Knodell’s fibrosis score
and GNAS; only one point of difference in score was to be accepted to obtain ‘eight of
ten’ agreement. In contrast, five points of difference were necessary to accept in order to
reach the same agreement for Knodell’s total activity score130.
Regev et al113, studied 124 patients with chronic hepatitis C for sampling error
and intraobserver variation in liver biopsy. The patients underwent simultaneous
laparoscopic – guided biopsies of the right and left hepatic lobes. The sampling variation
part has been described earlier.
In the study, formalin fixed paraffin embedded sections of liver biopsies were
stained with hematoxylin and eosin and with trichrome. The slides were blindly coded
and randomly divided among two hepatopathologists. Inflammation and fibrosis were
scored according to the grading (inflammation) and staging (fibrosis) method based on
modified Scheuer system. Fifty of the samples were blindly resubmitted to each of the
pathologists to determine the intraobserver variation.
Of the 50 samples that were examined twice, the grading by each pathologist on
the second examination differed from the first examination in 0% and 4%, and the
staging differed in 6% and 10%, respectively. All observed variations were of one grade
or stage.
The authors concluded that the intraobserver variation appeared to be low 113.
One of the reviews by Cholongitas et al13, already cited to earlier, commented on
these and other studies 128-133. According to them the studies had variations in design, and
were not uniform. One study128, used samples of longer length ( >40 mm). The Scheuer
system had excellent results for intraobserver and interobserver agreement, as did the
Knodell system for fibrosis but not for inflammatory score. In the other studies, 1 did not
129 130,131,133
document length , 3 used samples 10 mm or longer , and 1 used samples 15
132 13
mm or longer . According to the review , the study that included histopathologists
133
with different levels of expertise, duration, and location of practice and had an
excellent design only used specimens 10 mm or longer, and, thus, its results may not be
applicable to optimal liver biopsy specimens. But the question remained that how many
219
of the liver biopsy samples obtained in routine practice are ≥ 40 mm. In clinical practice
the majority of the samples are between 10-20 mm, and one has to realistically assess the
interobserver variation and intraobserver variation in these biopsy samples with the
different histopathological scoring systems. Thus, it is ideal to have a longer liver biopsy
sample, but in how many patients this ideal is achieved. According to the review13, in
fact, agreement increased in relation to length and number of portal tracts.
In one of the excellent studies by Bedossa et al25, the authors investigated the
increase in the METAVIR score and the increase in the amount of fibrosis via image
analysis. They reported that the increase in the METAVIR stage is associated with a
progressive increase in the fibrosis area. Interestingly, this increase of fibrous tissue
accumulation is not linear; because for example, F2 has 3-fold more fibrosis than an F0
stage (normal liver), whereas F3 and F4 are respectively, 7- and 12-fold F0. They
concluded that if the METAVIR score of fibrosis appears to increase linearly with time,
this is in fact corresponds to an exponential accumulation of fibrous tissue within liver 25.
Therefore a difference of just one stage variation in liver biopsies may in fact
translate into a much more difference in the actual amount of fibrosis within the liver
parenchyma, and results in liver biopsy shown being less than accurate interpretation of
liver fibrosis.
In the studies described above, “agreement” was defined as variation in one grade
or stage. Due to the facts illustrated above, an agreement of one stage may be misleading,
and may amount to speculation, as potentially there could be significant difference in the
amount of fibrosis between different stages. The semi-quantitative grading systems
should also in some way incorporate this difference to over come this fallacy in reporting
different stages, as the total accumulation of fibrous tissue within the liver with
subsequent stages increases to a great extent, which is not reflected by the 4-5/6 stages of
fibrosis.
4.15 Length and the number of complete portal tracts
220
How the length of the biopsy specimen and the no. of CPT does correlate? Do we
always or most of the times get the required no. of CPT’s with adequate liver biopsy, or
is there any optimal length in this regard. Thus according to Cholongitas et al13, and
based on preceding discussion, the minimum standards for an optimal percutaneous liver
biopsy (PLB)25,107 for assessing chronic viral hepatitis require longer specimens than
before (≥ 20-25 mm vs ≥ 15 mm)7, and more CPT’s than before (≥11 vs ≥ 6-8)7.
Additionally, in a study107, 11 CPT’s were not obtained while evaluating biopsy

specimens 1 mm wide, suggesting that 1 mm is an insufficient width13.
A systematic review by Cholongitas et al13, evaluated these characteristics in 32

studies with a collective pool of 10,027 PLB specimens from 8,746 patients. All 32
studies reported length, but only 12 reported the number of CPT’s. Fragmentation was
described in 8 studies, but in only 4 the mean no. of fragments given. Five studies did not
report the mean length of the biopsy and the range, but just divided them into categories,
i.e., longer or longer than a certain length. The mean + SD length and the number of
13
CPT’s were 17.7 + 5.8 mm and 7.5 + 3.4 mm, respectively. According to the review ,
importantly the correlation between length and CPT’s was poor (Spearman r = 0.45, p =
0.04).
The review13 further goes on to analyze the length of the biopsy sample and the
period in which these were obtained. The percutaneous liver biopsy specimens obtained
during the 1996-2005 period compared with those obtained before 1996 were
significantly longer (19.8 vs 15.7 mm, p = 0.033); and were more frequently obtained
with ultrasound guidance (9 vs 2 studies, p = 0.001); and using smaller needles (18 or 19
gauge, 6 vs 2 studies, p = 0.023)13.
The Menghini needle yielded significantly longer samples (19.9 + 6.6 mm)
compared with the Trucut needle (14.3 + 3.2 mm, p = 0.016), but without a significant
difference in the number of CPT’s (7.3 vs 6.9, p = 0.8)13.
Few studies reported the number of complete portal tracts, and length. Even in
those studies, which reported both characteristics, the correlation between length and
number of complete portal tracts was poor. In addition, comparing the type of needle and
221
complete portal tracts; although, longer samples were obtained with the Menghini needle
as compared to the Trucut needle, again there was no significant difference in the number
of complete portal tracts.
4.16 Type of needle

13
According to the same review , in their analysis, there were a total of 4,481
Menghini needle biopsies, 4,134 Trucut needle biopsies, and 1,412 of unknown type. The
needle size varied from 14 to 19 gauge (median, 16 gauge).
As discussed in the previous section, the Menghini needle yielded significantly

longer samples (19.9 ± 6.6 mm) compared with the Trucut needle (14.3 ± 3.2 mm; P =
.016), but without a significant difference in the number of CPTs (7.3 vs 6.9; p = 0.8).
What are the elements behind a longer sample obtained by the Menghini needle.
The investigators13 commented that; the Trucut needle provides a maximum length of
sample determined by the notch in the needle shaft (usually 20-25 mm; compared with
the Menghini needle in which the length depends on the force of aspiration and operator
experience), and also that the Menghini needle is less traumatic as compared to the
Trucut needle, this could explain the longer samples obtained with the Menghini needle.
Another potential reason could be that more passes were performed with the Menghini
needle. However, the data does not support this notion. In 16 of 32 studies that gave such
information showed that more than 1 pass was performed in 108 (3.1%) of 3,535
biopsies using the Menghini compared with 199 (12.1%) of 1,646 biopsies using
the Trucut 13. Thus more passes were performed with the Trucut needle.
4.17 Needle size

Cholngitas et al13, reviewed the impact of needle diameter and gauge on the
length and no. of CPT’s. There was no significant difference in length (range,16.3- 20.7
mm) or number of complete portal tracts (range, 4.6-9.7) according to the needle
diameter.
222
Longer biopsy specimens (mean, 20.7 mm) containing a larger number of
complete portal tracts (mean, 9.7) were obtained by using 17-gauge needles, but these
results were derived from only 3 studies134,135.
PLB specimens obtained by 18- or 19-gauge needles compared with smaller ones
had similar mean length (18.4 vs 18.6 mm) but contained more CPTs (8.0 vs 6.0);
however, this difference was not significant.
The Menghini and Trucut needles were compared only in studies with 14-, 15-
, and 18- gauge needles. Liver biopsy specimens were longer with the Menghini needle
than the Trucut needle across all 3 gauges mentioned, the 14-gauge Menghini (23 vs
15.5 mm; P = .18), and 15-gauge Menghini (21 vs 14 mm; P = .47), and significantly
longer by using 18-gauge Menghini needle as compared to 18-gauge Trucut needle (26
vs 12.8 mm; P = .012) 13.
4.18 Thin needle vs large needle for assessment of

diffuse liver disease
According to the review13, one study136, compared the Menghini thin needle,
20 and 21 gauge, with the conventional Menghini large needle, 17 gauge, in patients with
similar indications for biopsy for histological diagnosis. From 258 patients, 343 biopsy
samples were obtained, excluding the Ishak stages 5 and 6. A 17-gauge needle used by
surgeons using several passes for 28 biopsies (17Gs); single-pass percutaneous for 79
biopsies using a 17-gauge needle (17Gp); and ultrasound guidance with a 20-gauge
needle in 88 biopsies (20Gp) and a 21-gauge needle in 80 biopsies (21Gp).
Astonishingly, the authors found that specimens in the 20Gp group, compared with
specimens in the 17Gp group, were longer (29.8 vs 25.3 ; P < .05) but contained fewer
portal tracts (6.7 vs 9.7). An insufficient sample was obtained in 4 cases in the 20Gp
group, and in only 1 in the 17Gp group. The authors concluded that 20-gauge needle
(20Gp) could be a reliable alternative for patients with diffuse liver disease and
contraindications for large-needle, 17 gauge needle (e.g., 17Gp) percutaneous biopsy.
223
137
Another study , examined the reliability of thin-needle biopsy for grading and
staging in chronic viral hepatitis: 59 patients underwent thin-needle biopsy (20-gauge,
0.9-mm needle) and 41 underwent large-needle biopsy (17-gauge, 1.4-mm needle). Two
independent pathologists using the Ishak scoring system read all samples first separately
and then together.
According to the review13, in thin-needle specimens, severe fibrosis (stage 5) and

cirrhosis (stage 6) tended to be underestimated, otherwise no significant difference was
found for grading and staging between thin-needle and large-needle specimens.
However, the limitations of the study were that the thin-needle and large-needle
samples were not paired, there was no randomization, and there may have been a bias
because there was a significantly lower platelet count in patients who had undergone
thin-needle biopsy that likely represented more advanced liver disease and/or cirrhosis,
with a greater prevalence of higher fibrosis stages in patients, but still in thin needle
biopsy specimens severe fibrosis and cirrhosis were underestimated 13.
According to Cholongitas et al13, these limitations were overcome in a study

in which biopsy specimens were obtained through the same puncture site from 149
consecutive patients with chronic HCV134. Two histopathologists evaluated the paired
thin-needle (0.8 mm) and large-needle (1.2 mm) samples using the Ishak scoring system.
Liver biopsy samples were considered adequate if they were 10 mm or longer, contained
4 or more portal tracts, and were not too fragmented.
Large-needle specimens were significantly longer than thin-needle specimens
(21.2 vs 12.2 mm; P < .001) and less fragmented (11% vs 42%; P < .001) and considered
adequate more frequently (94% vs 55.7%; P < .001). Comparison of the 83
paired and adequate specimens showed that in thin-needle specimens, fibrosis and all 4
categories of necroinflammatory activity were underscored. Finally, thin-needle biopsy
resulted in understaging of cirrhosis (2 of 3 biopsy specimens with stage 5/6). Similar
results were obtained with the METAVIR and Scheuer scoring systems. The authors
concluded that thin-needle biopsy should be avoided for grading and staging in patients
with chronic HCV13.
224
4.19 Center experience
According to the review,13 liver biopsy samples were longer in studies
with 100 or more percutaneous liver biopsies (PLB’s) than those with fewer than 100
PLBs (20.4 mm vs 16 mm; P = .026). However, this difference was not significant for the
number of CPTs (8 vs 7.3; P = .7). In these circumstances, even ultrasound guidance did
not help to obtain longer specimens (ultrasound-guided vs non–ultrasound guided,
17.9 vs 13.6 mm; P = .19).
Specimens obtained with Menghini needles were significantly longer in studies
with 100 or more PLBs than those with fewer than 100 PLBs (24 vs 16.1 mm; P = .005),
in contrast with studies in which Trucut needles were used13.
4.20 Transjugular liver biopsy
TJLB has been considered a second grade biopsy owing to the small specimen
size and increased fragmentation compared with PLB. TJLB, nevertheless, offers
certain advantages; it is being used in circumstances where PLB is avoided or
contraindicated, and offers the possibility of using multiple passes without increasing
complications 12,38 .
In the author’s review13, overall, 1,389 TJLB specimens (15 studies) were
evaluated (mean, 2.5 passes per patient); the mean ± SD length was 13.5 ± 4.5 mm (13 of
15 studies), and the mean ± SD number of CPTs was 6.8 ± 2.3 (6 studies)13. Most studies
described small series.
According to the authors13, only their series of TJLB (n = 326) detailed the
number of passes (n = 3), needle size (Trucut 19 gauge), length (mean, 22.5 mm), number
of CPTs (mean, 8.7) and fragmentation (median, 5)58. Only 1 study compared Trucut (18
gauge) and Menghini- type (16 gauge) needles and found that using the Trucut
resulted in significantly longer specimens (12 vs 7 mm; P < .05) 59.
The authors13 were of the view that with a mean of 2.5 passes, the biopsy
specimens were on average only 4.2 mm shorter compared with PLB (13.5 mm vs 17.7
mm, respectively), and, it is important to note, contain almost the same number of CPTs
225
(6.8 vs 7.5, respectively), which is similar to the difference between Trucut and Menghini
needles. In their study of TJLB58, 89% of specimens were either ≥ 15 mm long or had ≥ 6
CPT’s. The authors58, concluding that although adequate sampling remains a limitation
with TJLB for staging and grading of chronic hepatitis, TJLB is comparable to PLB in
this respect58.
According to the review 13, heterogeneity of liver disease and interobserver
or intraobserver variation have not been evaluated in TJLB.
Quality of TJLB requires study, for all the estimates of an adequate liver biopsy
sample, because this method allows multiple passes (to obtain adequate samples) with
far less likelihood of increasing complication rates 60,61.
The authors commented that, TJLB could be an alternative and safe approach to
obtain samples of adequate size and a reliable assessment of liver histologic features 13.
4.21 The adequate liver biopsy sample

So what is the ideal liver biopsy sample in texts and in practice, and how many
times one gets the standard PLB sample, and if not always, then what is the trade off.
According to the review cited extensively 13, the minimum standards for an optimal
25,107
percutaneous liver biopsy for assessing chronic viral hepatitis require longer
7
specimens than before (>20-25 vs >15 mm) and more CPTs than before (>11 vs
>6-8) 7. Additionally, in 1 study 107
, 11 CPTs were not obtained when evaluating
masked biopsy specimens 1 mm wide, suggesting that 1 mm is an insufficient width.
These new high standards would result in liver biopsy being less delusive.
128
Thus as evidenced in a study by Goldin et al , there was less interobserver and
intraobserver variation with biopsy specimens that were 40 mm or longer, i.e., a large
specimen. In addition, if the new standards are met, potential heterogeneity of liver
disease is not significant 138.
However, according to the review13, a study136, showed that all methods of LB
resulted in an insufficient sample size in a significant proportion of patients: 42% of
PLBs with a large 17-gauge needle contained 10 or more portal tracts. Only the surgically
obtained LB specimens with multiple passes provided adequate liver samples in a very
226
high proportion of cases. Using a thin needle allows multiple passes without increasing
complications, this advantage is overcome by its low diagnostic performance 134.
Although specimens obtained with Menghini needles are significantly longer
than those obtained with Trucut needles (19.5 vs 14.3 mm; P = .01) the number of CPTs
was no different. A new Trucut needle with a larger notch (at least 30 mm) may
overcome this restraint but could result in more complications13.
In addition to length, for a fully representative liver biopsy sample, the number of
complete portal tracts are also important. There is no uniform consensus on the definition
of a complete portal tract. The study by Rocken et al136, for PLBs, similar to the cited
author’s study for TJLBs 58, used the definition of Crawford et al127, for CPTs: complete
circumference with at least 2 portal structures within them. Colloredo et al107, on the
other hand, considered CPTs as only the portal triads with complete circumference, and
partial portal tracts were those incompletely represented (usually at the margin of the
specimens). Moreover, it is recognized that biopsy specimens obtained at the periphery of
the liver more frequently contain only a hepatic arterial branch and bile duct, missing the
portal vein 127. These portal dyads with a complete circumference are CPTs in the normal
liver. In addition, fragmentation will reduce number of CPTs if the break occurs through
them 13. This may explain in part why poor correlation was documented between length
and CPTs (although still statistically significant, r = 0.45; P = .04)13.
However, more than 1 pass is likely to be needed to obtain a PLB specimen of
adequate (ideal) size, which has the potential to increase the complication rate, which
12,38,139
increases with needle size and number of passes . For this reason, the clinical
applicability of the histopathologic requirement for larger liver biopsy specimens has
to be explored critically.
In this systematic review 13, comprising all documented series of PLB in the
literature, the liver biopsy specimens had an average length and number of portal
25,107
tracts well below the published minimum sample size requirements in more
13
than half the cases. Thus according to the review , it is surprising that only 2 of
147 studies of antiviral therapy for CHC and none for CHB had details on both the
length of the LB specimen and the number of CPTs.
227
How can adequate biopsy samples be obtained for reliable grading and staging
of chronic liver disease25?
A related question is whether LB can be regarded as the gold standard for the
140
staging and grading of diffuse liver diseases when risks of biopsy, inadequate
sampling, and intraobserver and interobserver errors are taken into account, not to
mention the different methods of reporting. If the currently proposed minimal criteria for
a liver biopsy specimen (>20-25 mm long and >11 CPTs) are to be used as a gold
standard, then more than 1 pass using a standard percutaneous liver biopsy will be
required, with more risk of complications13.
13
Thus according to Cholongitas E, et al , inadequate liver biopsy specimens (as
compared to a perfect sample with the most optimal sample length and CPT criteria),
have been used in studies of noninvasive markers of fibrosis.
These limitations of the liver biopsy lead to an elusive specimen which is not the
“gold standard”, does accurately reflect the underlying histopathology, may not be that
reliable to guide the clinicians, and finally impair the validity of nonhistological markers.
Thus, to achieve an ideal sample in routine clinical care; with a minimum
requirement for a liver biopsy specimen to always be > 2.5 cm longer in length, and to
have 11 or more complete portal tracts could be unrealistic and dangerous for the patient
13
on one hand, and practically very difficult on the other.
228
Histopathology of chronic hepatitis B and C
141
According to Goodman ZD, chronic necroinflammatory disease (chronic
hepatitis) is a morphologic pattern seem most often in chronic viral hepatitis, but also in
autoimmune hepatitis, drug reactions, and some metabolic diseases. Although the
histopathologic features are similar, some notable features are more characteristic of one
type than of other 141.
Parenterally transmitted viral hepatitis accounts for 90% of cases. Approximately

5% to 10% of cases of chronic hepatitis are autoimmune. Drug induced liver disease is a
142
rare but well documented cause of chronic hepatitis . Metabolic diseases, such as
Wilson’s disease, alpha 1 antitrypsin deficiency, and haemochromatosis, are also causes
of chronic hepatitis, but they are distinguished from the usual causes of chronic hepatitis
by clinical features, liver biopsy and laboratory tests 141.
As in acute necroinflammatory disease, hepatocellular injury and inflammation

are present, but in the chronic diseases, the brunt of the injury tends to be portal and
periportal rather than panacinar. And the injury is accompanied by fibrosis that can
141
progress to cirrhosis .
Chronic hepatitis regardless of the cause, is characterized by several pathologic

changes that are present to a variable extent in each case. These include the following:
Hepatic necrosis, portal inflammation and periportal injury and inflammation,

piecemeal necrosis, and fibrosis which may involve only the portal and periportal areas or
may form septa 141.
4.22 Elementary lesions of chronic hepatitis

143
According to Desmet V, the elementary lesions that constitute the
histopathologic picture of chronic hepatitis consist of: Spotty necrosis, confluent lytic
necrosis, portal inflammation, interface hepatitis, fibrosis, and cirrhosis 144.
229
Thus according to Desmet V143, Spotty necrosis is an older term, loosely applied
to apoptosis and necrosis of isolated hepatocytes. A more appropriate morphologic term
would be, “focal necroinflammation”, recognizable as a small cluster of mononuclear
cells (lymphocytes, possibly with histiocytes). Spotty necrosis in chronic hepatitis looks
the same as in acute hepatitis 143.
Confluent lytic necrosis, comprises of confluent necrosis of the lytic type of the
hepatic parenchyma, which results in dropout of adjacent groups of hepatocytes with
denudation of the reticulin framework. . As in acute hepatitis, the extent of confluent
necrosis may range from focal and zonal confluent necrosis to “bridging” necrosis, and to
the more wide spread involvement of the single or multiple lobes143. It occurs in more
severe forms of chronic hepatitis, and thus usually coincides with clinical episodes of
disease exacerbation 145.
Portal inflammation in chronic hepatitis may be mild, moderate, or dense. It is
composed of mononuclear cells, comprising mainly lymphocytes, variable numbers of
143
plasma cells, and other mononuclear cells (histiocytes and immature lymphocytes) .
Lymphoid aggregates or follicles with germinal centers may be present and are now
considered typical, although not pathognomonic, of chronic hepatitis.
Immunohistochemical studies have shown that even when the germinal centers are not
apparent by light microscopy, these are true functional lymphoid follicles 146.
141
Hepatitis associated bile duct lesions, according to Goodman ZD, were first
described in chronic hepatitis 147, but the lesions may also be found in specimens of acute
hepatitis. The lesion is characterized by swelling, vacuolization, nuclear irregularity, and
sometimes pseudostratification of the biliary epithelial cells. The basement membrane
appears to be ruptured, and lymphocytes, occasional plasma cells, and sometimes
neutrophils infiltrate the duct. The lesion looks like the “florid duct lesions” of the
primary biliary cirrhosis. However, the ducts are not destroyed as they are in PBC, and
features of cholestasis do not develop. Reconstructions of serial sections has
demonstrated that the most frequently observed lesions are actually blind diverticula
148
arising from injured ducts rather than ducts themselves . The ductal lesions have been
141,149
seen in all forms of hepatitis, but most often in hepatitis C .
230
Interface hepatitis, according to Goodman ZD141, is now the preferred term for
150,151
the lesion formerly known as piecemeal necrosis . The original term was defined
by an international group as “destruction of liver cells at an interface between
parenchyma and connective tissue, together with a predominantly lymphocytic or plasma
cell infiltrate” 152. It is now apparent that the destruction of liver cells occurs primarily
through apoptosis, and because the dead hepatocytes quickly disappear from the tissue,
and the mode of cell death is apoptosis not necrosis 153. In addition it is the location of the
inflammatory component that permits recognition of the lesion, so that interface hepatitis
is the more accurate term 141.
Interface hepatitis has traditionally been considered a key lesion in the
progression and pathogenesis of chronic hepatitis, and hence the distinction between
chronic persistent hepatitis and chronic active hepatitis was based on this finding. The
basis for this distinction is no longer considered valid, but the degree of periportal injury
(mild, moderate, or severe) is still used to grade the degree of activity 141.
Interface hepatitis may be mild (one or few foci around the portal perimeter
without noticeable fibrous expansion), moderate (with periportal destruction of groups of
liver cells along with fibrous septa) or severe (with wedge shaped extension of the
necroinflammatory lesion and obvious fibrosis deep into the lobule) 143.
141
According to Goodman ZD , Interface hepatitis can most easily be recognized
as irregularity of the limiting plate, and later its disappearance, caused by the extension of
portal inflammation through the plate into the periportal parenchyma. Inflammatory cells
surround and destroy injured hepatocytes (“emperipolesis”). Evidence of hepatocelluar
degeneration and death is characterized by either acidophilic or ballooning degeneration.
As in acute hepatitis, cell death occurs principally by the process of apoptosis, which
results in the formation of apoptotic or acidophilic bodies that rapidly disappear from the
liver plates or sinusoids. As chronic hepatitis progresses, continuous erosion of the
hepatic parenchyma takes place, with closer approximation of expanded portal areas, and
small groups of hepatocytes (“hepatocyte islets”) become trapped in expanded portal
zones. The necroinflammatory changes are gradually succeeded by fibrosis, often best
appreciated with a Masson or collagen stain. Initially, delicate collagen fibers are laid
231
down in areas of periportal liver cell loss, which eventually condense into scars and thick
fibrous tissue 141.
Even after cirrhosis has developed, interface hepatitis can continue at full pace
along the fibrous septa, causing further loss of hepatocytes and eventually clinical
decompensation of cirrhosis 141.
141
Fibrosis, according to Goodman ZD, accompanies chronic hepatitis, although
the degree of fibrous tissue deposition is quite variable from patient to patient. Fibrosis is
the progressive component of the disease 143, which ultimately leads to the architectural
distortion and cirrhosis. The long term prognosis of chronic hepatitis depends on the
fibrous tissue accumulation. It is thought that at least two pathways may lead to fibrosis
in chronic hepatitis 141.
Probably the most important in chronic viral hepatitis is the collagen deposition
that accompanies the periportal injury of interface hepatitis, causing fibrous expansion of
the portal tracts.
Intralobular fibrosis apparently results from continuous ongoing lobular
necroinflammatory damage 143.
As the disease progresses, portal-to-portal fibrous bridges are formed, filling zone
1 between adjacent acini. Central-to-portal and sometimes central-to central fibrous
bridges may also form, developing from superimposed episodes of portal-central
(bridging) confluent necrosis involving zone 3 141,143.
In the evaluation of needle biopsy specimens, it is important to distinguish
tangential cuts through enlarged portal areas, which contain preexisting bile ducts and
portal vessels, from true bridging fibrosis, in which septa form through parenchyma
141
where fibrous tissue previously did not exist . Thus it reflects on the importance of
reporting a certain number of complete portal tracts in liver biopsy specimens, as an
attribute of an adequate sample, in addition to biopsy size.
The scars of bridging fibrosis contain elastic fibers in addition to collagen. Like
scars in any tissue, these tend to contract. Contraction of the fibrous septa together with
nodular regeneration of the surviving parenchyma produces architectural distortion, and
when complete nodules have formed, surrounded by fibrous septa, the result is cirrhosis
141
.
232
143
Cirrhosis, according to Desmet V, is the end result of many chronic liver
diseases. It corresponds to diffuse hepatic fibrosis with replacement of normal lobular
architecture by parenchymal nodules separated by fibrous issue 155. Architectural changes
are best appreciated on a reticulin stain 156.
Portal-central septa linking portal tracts and central veins are an important
component of cirrhosis. Cirrhosis may be strongly suspected on clinical, laboratory, and
imaging parameters, but the final diagnosis requires morphological confirmation by liver
biopsy 143.
155
The morphologic classification of cirrhosis is based on the size of nodules .
Cirrhosis is micronodular if nearly all of the nodules are less than 3 mm in diameter, and
macronodular if greater than 3 mm in diameter. A mixed micro-macronodular
cirrhosis comprises approximately equal numbers of nodules greater than and less than 3
mm in diameter. The size of the nodules has some use in defining the aetiology of the
process, but is relative since micronodualr cirrhosis may convert into a macronodular
type under circumstances more favorable for parenchymal regeneration 143,157.
Nodular size is more important in determining the ease with which the diagnosis
of cirrhosis can be made in needle biopsy specimens. Nodularity and septa may be
readily evident in specimens from micronodular cirrhosis, whereas more subtle criteria
have to be relied on in macronodular cirrhosis.
According to Desmet V, helpful criteria in this respect include: fragmentation of
the biopsy specimen (especially with thin tissue cylinders provided by small aspiration
type needles such as Menghini needle); thin layers of connective tissue adhering to
rounded edges of nodular biopsy fragments; abnormal orientation of reticulin fibers
resulting from different rates of parenchymal growth in different areas; abnormal spacing
of portal tracts and central veins, and excess numbers of draining veins in relation to the
number of portal tracts; presence of minute and poorly formed portal tracts (mini portal
tracts); hepatocellular features of regeneration – double plates over widespread areas;
different appearance of hepatocytes in adjacent areas; liver cell dysplasia of the large cell
and small cell type 143.
Besides, confirming the presence or absence of distorted architecture, and stage of
development, the liver biopsy can help diagnose the aetiology of the liver disease, in
233
many instances with the use of some additional stains and attention to a number of
features 143.
Before the architecture is entirely obliterated, parts of the tissue are nodular while
adjacent areas maintain an acinar and viable structure, a state that can be regarded as
“incomplete cirrhosis” 141. It is the most difficult anatomic type to recognize 143.
This is best characterized by irregular nodularity, slender septa, some of which
end blindly, mini-portal tracts, excess efferent veins, and sinusoidal dilatation. There is
evidence of parenchymal hyperplasia with corresponding compression of reticulin fibers
158
in adjacent areas. Inflammation and necrosis are generally absent or minimal . The
diagnosis is more easy in surgical than needle specimens 143.
The ease with which cirrhosis is diagnosed further depends on multiple factors;
the type of biopsy procedure (surgical vs needle), the rout of the procedure (surgical,
percutaneous, and transjugular), the type of needle used (Menghini, Trucut, or other), the
adequacy of the biopsy specimen, and the quality of applied stains, and the
histopathological reporting. These attributes have been discussed at length previously. In
some instances, the histopathologist can only hint at the possibility of cirrhosis. A further
complicated factor is the process of change of the fibrous tissue deposition. Cirrhosis
143
does not appear overnight, but may take months or years to develop , a one time liver
biopsy specimen may not capture the dynamic nature of the process.
4.23 Histopathology of hepatitis B virus

141
According to Goodman ZD, hepatitis B virus can be diagnosed histologically
and distinguished from chronic hepatitis of other causes by demonstration of the virus in
tissue. Histochemical stains such as orcein or Victoria blue, or the more sensitive
immunostains can demonstrate HBsAg in 80% or more of cases of chronic hepatitis B 141.
Cells containing large quantities of HBsAg have cytoplasm with a uniform, finely
granular appearance. These ground-glass cells are scattered randomly throughout the
liver, often in clusters. The number of ground glass cells tends to be inversely related to
the activity of hepatitis. The greatest number of cells is found in livers with the least
234
active disease, whereas livers with the most activity tend to have the fewest ground glass
cells 141.
In acute hepatitis B, the immune response eliminates antigen containing cells, and
the results of immunostaining are entirely negative. Conversely, the presence of staining
surface antigen proves a chronic rather an acute infection, even when severe
hepatocellular injury is present 141.
Hepatitis B core antigen (HBcAg) can usually also be demonstrated by
imunohistochemistry in nuclei and sometimes cytoplasm in cases of chronic disease. The
presence of core antigen reflects active viral replication, so the amount of core is
generally directly proportional to the activity of hepatitis. Patients with recent acute
exacerbations have most core, with HBcAg often present in hepatocyte cytoplasm and
numerous nuclei. Increased cytoplasmic core antigen has also been found in strains of
virus with precore mutations associated with more severe disease 141,159.
4.24 Histopathology of hepatitis C virus

141
According to Goodman ZD, hepatitis C virus cannot reliably be demonstrated
in routinely processed tissue at the present time. However, certain histologic features are
characteristic, although not pathognomonic of hepatitis C 149, and there presence should
prompt a serologic evaluation if it has already not been carried out 141. The overall pattern
is such distinctive that entire scoring systems have been developed on the histopathologic
changes associated with HCV, such as the Metavir system.
Chronic hepatitis C tends to be associated with more intense chronic portal
inflammation than other types of chronic hepatitis, often with lymphoid aggregates and
sometimes follicles with germinal centers. The tendency to steatosis is also greater than
in other types. Approximately 50% of biopsy specimens have some fat, and in about
10%, the amount may be considerable. Patients infected with genotype 3 tend to have
even more steatosis, and it has been suggested that the increased fat may have a
cytopathic and fibrogenic effect in such cases 160. Hepatitis associated bile duct lesions
may be present in acute or chronic hepatitis of any cause, but they are most frequent in
hepatitis C. Severe degrees of bile duct injury can be seen in about 10% to 15% of
235
specimens from patients with chronic hepatitis C lesser degrees of duct irregularity and
lymphocytic infiltration are found more often 141.
4.25 Grading and staging of chronic hepatitis

The stage of any disease is how far it has progressed in terms of its natural
history, with the end stage being organ failure or death. The grade of a disease is meant to
reflect how quickly it is progressing to the end stage 141. Between these, there are caveats,
as discussed below:
The end stage of chronic hepatitis is cirrhosis with clinical decompensation;
fibrosis or cirrhosis is less severe in earlier stages. The grade is considered to be the
degree of inflammation and hepatocellular injury, which are thought to lead to the
fibrosis. Of lately, it has been shown that it is the degree of development of fibrosis,
which is more important of a measure of how quickly it is progressing to advanced stages
than the degree of inflammation 25.
4.26 History
143
According to Desmet V et al, the introduction of needle biopsy of liver has
greatly contributed to the recognition of non cirrhotic forms of chronic inflammatory
liver disease. After World War II, chronic sequelae of epidemic hepatitis were described
in several parts of the world 161.
By the 1960’s, chronic hepatitis was presumed to be a sequel of viral hepatitis,
although a causative agent had not been identified. A simple classification of chronic
hepatitis based on histology, was proposed by a group of hepatologists and pathologists
in 1968 162.
According to Desmet V143, at that time immunosuppression was the standard
treatment, to be reserved for the more “active” forms of the disease, the classification
distinguished between a milder form (chronic persistent hepatitis - CPH) with a low
degree of necroinflammatory activity, and a more severe variant (chronic aggressive or
active hepatitis – CAH) featuring higher degrees of necroinflammatory lesions; a term
implying disease highly likely to progress to cirrhosis; was a form of grading.
236
This classification of chronic hepatitis became widely accepted and was used
worldwide for nearly 40 years. Although it was emphasized that CPH and CAH were not
to be considered as distinct disease entities, but as variations in disease severity of a
single morphological pattern, this notion was not adhered to 143.
Since 1968, remarkable progress has been achieved in the elucidation of the
multiple etiologies of chronic hepatitis, with the discoveries of HBV, HCV and HDV 163.
Piecemeal necrosis, a very important diagnostic criterion in CAH, but
(incorrectly) thought by some to be pathognomonic of and synonymous with chronic
hepatitis, was also observed in chronic biliary diseases such as primary biliary cirrhosis
and primary sclerosing cholangitis 143.
Investigations into the natural course of the disease in chronic viral hepatitis B
revealed successive phases of viral replication, elimination, and integration, associated
successively with lesser, higher, and again decreasing degrees of necroinflammatory
disease activity 164,165. This implied successive occurrence of CPH and CAH variants in
individual patients, confusing those who considered CPH and CAH to be distinct disease
166
entities. A change of view on chronic hepatitis was indicated . Finally, and most
importantly, the development of more efficient treatment for viral types of chronic
hepatitis (interferon and other antiviral agents) necessitated a revision of chronic hepatitis
classification 167-169.
Thus advances in our understanding into the aetiologies of the diseases, the
discovery of some hepatotropic viruses, and their natural history, and refinements at
histopathological reporting, novel drugs of treatment, have introduced a myriad new
ideas, which have fundamentally changed our concepts, and rendered this classification
obsolete 141.
According to Desmet V143, there appears to be general agreement to restrict the
term “chronic hepatitis” to its original meaning: a clinical and pathologic syndrome with
several causes and characterized by varying degrees of hepatocellular necrosis and
inflammation. However, for lack of a better definition of chronicity, chronic hepatitis is
still defined (as in 1968) as a continuous disease process without improvement for at least
6 months 143,170.
237
Several methods of classification are currently in use to express the grade and
stage of chronic hepatitis. These can be categorized as: simple verbal descriptions,
relatively simple numeric grades and stages that correspond to the verbal descriptions,
and more complicated systems for numerically scoring the histologic features that
correspond to the grade and stage.
Many histologic classification systems have been proposed to provide a uniform
standard that can be used to compare histologic findings in clinical trials. Although some
of these classifications are qualitative, quantitative systems are most frequently used in
clinical trials since they are amenable to statistical analysis 171. In 1994 two proposals for
a new classification of chronic hepatitis were published 170,172 .
However, the most common quantitative system that has been used since 1981, in
the assessment of chronic viral hepatitis is the Knodell score173, and its modification by
151
Ishak . In addition, another semiquantitative system, the Metavir score, has been
126,131
increasingly used for chronic hepatitis C . Other scoring systems also widely used
169
are: a simple scoring system proposed by Scheuer , and its modification by Batts and
Ludwig 174.
Each method has its advantages and disadvantages, and the system used should be
appropriate for the task at hand. In general, more complex systems can provide more
information than simple ones, but they are less reproducible 141.
The numeric scores generated by these systems are very useful in investigational
studies that involve large number of patients and require statistical analysis. They are a
very good way to show differences in the histologic response between cohorts of patients
receiving different forms of therapy, and they have been used successfully in many large
clinical trials. However studies have shown fairly poor reproducibility of these scores
when applied to individual biopsy specimens, both between different pathologists and for
the same pathologist at different times 113,128,129,130,141.
4.27 Knodell score

175
According to Bonis PA, the Knodell score, also known as the histologic
activity index (HAI), is composed of the summation of four individual scores
representing periportal and/or bridging necrosis, intralobular degeneration and focal
238
necrosis, portal inflammation, and fibrosis; the score ranges from 0 to 22. Several
modifications of the HAI have also appeared, which were designed, in part, to address
histologic features specific to the disease under study 176-178. One modification (referred to
as the Ishak score) has six stages of fibrosis, permitting more detailed evaluation of
changes in fibrosis compared with the standard Knodell fibrosis score, which has only
three stages 151,175.
4.28 Limitations of the Knodell score
According to Bonis PA,175 the changes in the HAI are sometimes interpreted
inappropriately: The standard deviation in HAI scoring among six individual observers in
the original description of the Knodell score was 2.4. Thus, variation in the HAI score by
less than 2.4 does not necessarily represent a true difference in the histologic pattern. This
observation is not always adhered to in clinical trials and studies, in which changes of one
point or more have sometimes been considered to be clinically or statistically significant.
A difference of one grade has one meaning, but a difference of one stage has vastly great
diagnostic and prognostic importance25.
In the original description of the Knodell score, the inter- and intra-observer
reliability was relatively good. However, the Knodell score was originally validated on
only five patients (with a total of 14 biopsies), one of whom had hepatitis B and four of
whom were presumed to have non-A, non-B hepatitis, which is most likely to have been
hepatitis C 173,175.
The Knodell score is frequently used in drug trials in chronic hepatitis,
particularly hepatitis C, as well as natural history studies. A decrease in the Knodell score
is considered to represent histologic amelioration, or less progression to advanced
fibrosis. No histologic feature represented in the Knodell score can predict the response
to interferon 175,179.
The reliability of the HAI score has not been well-established in specific forms of
liver disease. Reliability refers to the extent to which repeated measurements of a
relatively stable phenomenon fall closely to each other 175.
239
A subsequent study evaluated the Knodell score among ten pathologists using a
131
cohort of 30 liver biopsy specimens from patients who had documented hepatitis C .
Interobserver correlation for three inflammatory components of the Knodell score was
relatively poor (with kappa coefficients ranging from 0.25 to 0.46 [a perfect coefficient
being 1.00]). The interobserver correlation for the total HAI was also relatively poor
ranging from 0.48 to 0.57. Only the fibrosis score had good reliability with a kappa
coefficient of approximately 0.80. Similar results were found for intraobserver reliability.
The HAI is weighted toward periportal necrosis and bridging necrosis. However,
this weighting may have limitations. First, the Knodell score is relatively insensitive to
changes in fibrosis, which is more important because it is fibrosis, and not inflammation
per se, which leads to many of the sequelae of chronic liver disease. Furthermore,
patients may have the same Knodell score despite having markedly different degrees of
fibrosis 175.
Second, the individual components of the Knodell score have not been well-
validated in the context of the natural history of viral hepatitis following treatment. Prior
to treatment, specific components of liver histology may have different impact on disease
prognosis 180-183. Thus, assessment of improvement following treatment should probably
also focus on this inflammatory component of liver histology, and fibrosis (rather than
lobular or portal inflammation only). This is particularly relevant in studies assessing the
efficacy of long-term interferon therapy in patients with hepatitis C who did not respond
to interferon or interferon in combination with ribavirin 175.
Histologic changes on serial liver biopsies are being used as a surrogate endpoint
for determining the efficacy of treatment. Although the Knodell Score can be used as a
categorical variable in statistical analysis, it is unclear what degree of change constitutes
a clinically meaningful histologic response. Changes in the mean fibrosis scores in
studies are used as end points, but it is not that clearly discernable in individual patients.
The Knodell score does not account for features specific to different types of viral
hepatitis. As an example, the HAI does not account for lymphoid aggregates, bile duct
injury, and macrovesicular fat that are often present in chronic hepatitis C 175,184.
240
Table 11. KNODELL Histological Activity Indexa
I. Periportal ± bridging necrosis Score

None 0
Mild piecemeal necrosis 1
Moderate piecemeal necrosis (involves less than 3
50 percent of the circumference of most portal tracts)
Marked piecemeal necrosis (involves more than 50 4
of most portal tracts)
Moderate piecemeal necrosis plus bridging necrosisb 5
Marked piecemeal necrosis plus bridging necrosisb 6
Multilobular necrosisc 10
II. Intralobular degeneration and focal necrosisd

None 0
Mild (acidophilic bodies, ballooning degeneration and/or 1
scattered foci of hepatocellular necrosis in < 1/3 of lobules
or nodules
Moderate (involvement of 1/3 to 2/3 of lobules or nodules) 3
Marked (involvement of >2/3 of lobules or nodules) 4
III. Portal inflammation

No portal inflammation 0
Mild (sprinkling of inflammatory cells in <1/3 of 1
portal tracts)
Moderate (increased inflammatory cells in 1/3 to 2/3 3
of portal tracts
241
Marked (dense packing of inflammatory cells in >2/3 4
of portal tracts)
IV. Fibrosis
No fibrosis 0
Fibrous portal expansion 1
Bridging fibrosis (portal-portal or portal- central linkage) 3
Cirrhosise 4
a
HAI score is the combined scores for necrosis, inflammation, and
fibrosis.
b
Bridging is defined as 2 bridges in the liver biopsy specimen; no
distinction is made between portal-portal and portal-central linkage.
c
Two or more contiguous lobules with panlobular necrosis.
d
Degeneration-acidophilic bodies, ballooning; focal necrosis-
scattered foci of hepatocellular necrosis.
e
Loss of normal hepatic lobular architecture with fibrous septae
separating and surrounding nodules.
Adapted from173: Knodell, RG, et al. Hepatology 1981; 1:431.
242
Table 12. Modified Histological Activity Index-
The Ishak score
Grading: Necroinflammatory scores
A. Periportal or periseptal interface hepatitis Score

(piecemeal necrosis)
Absent 0
Mild (focal, few portal areas) 1
Mild/moderate (focal, most portal areas) 2
Moderate (continuous around <50% of tracts or 3
septa)
Severe (continuous around >50% of tracts or 4
septa)
B. Confluent necrosis
Absent 0
Focal confluent necrosis 1
Zone 3 necrosis in some areas 2
Zone 3 necrosis in most areas 3
Zone 3 necrosis + occasional portal-central (P-C) 4
Bridging
Zone 3 necrosis + multiple P-C bridging 5
Panacinar or multiacinar necrosis 6
C. Focal (spotty) lytic necrosis, apoptosis
and focal inflammation*
Absent 0
243
One focus or less per 10 x objective 1
One to four foci per 10 x objective 2
Five to ten foci per 10 x objective 3
More than ten foci per 10 x objective 4
D. Portal inflammation
None 0
Mild, some or all portal areas 1
Moderate, some or all portal areas 2
Moderate/marked, all portal areas 3
Marked, all portal areas 4
Maximum possible score for grading 18
Modified Histological Activity Index Staging:

Architectural changes, fibrosis and cirrhosis
Change
No fibrosis 0
Fibrous expansion of some portal areas, with 1
or without short fibrous septa
Fibrous expansion of most portal areas, with 2
or without short fibrous septa
Fibrous expansion of most portal areas with 3
occasional portal to portal (P-P) bridging
Fibrous expansion of portal areas with marked 4
Bridging, portal-portal (P-P) as well as
portal-central (P-C)
244
Marked bridging (P-P and/or P-C) with occasional 5
nodules (incomplete cirrhosis)
Cirrhosis, probable or definite 6
________________________________________________
Maximum possible score 6
For grading:
* Does not include diffuse sinusoidal infiltration by
inflammatory cells
Additional features which should be noted but not
scored
Bile duct inflammation and damage
Lymphoid follicles
Steatosis, mild, moderate, marked
Hepatocellular dysplasia, large or small
Adenomatous hyperplasia
Iron or copper overload
Intracellular inclusions (e.g. PAS-positive globules,
Mallory bodies)
Immunohistochemical findings:
Information on viral antigens, lymphoyte subsets or
other features, when available, should be recorded and may be
semi-quantitatively expressed.
245
For staging:
Additional features which should be noted but not
scored
Intra-acinar fibrosis, perivenular (“chicken wire” fibrosis)
Phlebosclerosis of terminal hepatic venules.
From Ishak KG, et al, 151 J Hepatol 1995; 22: 696-699.
4.29 METAVIR score
175
According to Bonis PA, the Metavir score was developed in an attempt to
126,131
address some of the problems with the Knodell score . In contrast to the Knodell
score, which was designed as a generic scoring system for chronic hepatitis, the Metavir
score was specifically designed and validated for patients with hepatitis C.
The Metavir score is a semiquantitative classifications system consisting of an
activity and a fibrosis score: The fibrosis score is assessed on a five point scale (0 = no
fibrosis, 1 = portal fibrosis without septa, 2 = few septa, 3 = numerous septa without
cirrhosis, 4 = cirrhosis). Compared to the Knodell fibrosis score (which has only four
levels), the Metavir score permits recognition of subtler variation in the degree of
fibrosis. The activity score was graded according to the intensity of necroinflammatory
lesions (A0 = no activity, A1 = mild activity, A2 = moderate activity, A3 = severe
activity).
246
Table 13. METAVIR SCORE
1. Focal lobular necrosis

Less than one necroinflammatory foci per lobule 0
At least one necroinflammatory foci per lobule 1
Several necroinflammatory foci per lobule or confluent 2
or bridging necrosis
2. Portal Inflammation
Absent 0
Presence of mononuclear aggregates in some portal tracts 1
Mononuclear aggregates in all portal tracts 2
Large and dense mononuclear aggregates in all portal tracts 3
3. Piecemeal necrosis
Absent 0
Focal alteration of the periportal plate in some portal tracts 1
Diffuse alteration of the periportal plate in some portal tracts 2
or focal lesions around all portal tracts
Diffuse alteration of the periportal plate in all portal tracts 3
4. Bridging necrosis
Absent 0
Present 1
LN=0 A=0
PMN=0 LN=1 A=1
LN=2 A=2
247
PMN=1 LN=0,1 A=1
LN=2 A=2
PMN=2 LN=0,1 A=2
PMN=2 LN=2 A=3

PMN=3 LN=0,1,2 A=3
Algorithm for the evaluation of histological activity.

PMN, Piecemeal necrosis: 0, none; 1, mild; 2, moderate; 3, severe
LN, Lobular necrosis; 0, no or mild; 1, moderate; 2, severe
A, Histological activity; 0, none; 1, mild; 2, moderate; 3, severe
F 0= No fibrosis
F 1= Portal fibrosis without septa
F 2= Portal fibrosis with rare septa
F 3= Numerous septa without cirrhosis
F 4= Cirrhosis
The proposed algorithm provides an easy means of scoring the

activity.
Adapted from 126:
Pierre Bedossa and Thierry Poynard for the METAVIR Cooperative
Study Group. An algorithm for the grading of activity in chronic
hepatitis C. Hepatology. 1996;24:289-293.
248
The inter-and intraobserver reliability of the activity and fibrosis score of the
Metavir system are similar to the Knodell score. In one study, the kappa coefficients of
the Metavir activity score and the HAI, and the Metavir fibrosis score and the Knodell
fibrosis score were found to be similar (approximately 0.5 and 0.8, respectively) 131. A
subsequent study found that the interobserver agreement of the Metavir score depends
highly upon the experience of the hepatopathologist 185. Agreement was influenced more
heavily by the interpreter's experience compared with features of the specimen itself such
as its length 175.
On the other hand, a separate study evaluating the fibrosis and activity scores in
specimens of various length suggested that the length of the biopsy was also important
170
. The kappa coefficients for fibrosis were 0.75, 0.85, and 0.92 comparing specimens of
5, 10, and 15 mm respectively (considering the fibrosis score of a 20 mm specimen as the
reference standard). The corresponding figures for the activity scores were 0.73, 0.81,
and 0.77, respectively. The authors concluded that a specimen length of at least 10 mm
usually reflects the fibrosis and activity scores reliably. Another study, however,
suggested that a length of at least 25 mm is necessary to evaluate fibrosis accurately with
a semiquantitative score. Sampling variability becomes a major limitation when using
more accurate methods such as automated image analysis 25.
Thus, the main advantage of the Metavir score for hepatitis C is its relative
simplicity, its focus on necroinflammatory lesions, and its increased sensitivity in the
fibrosis score due to the addition of one extra fibrosis level. However, many of the
limitations of the Knodell score discussed above also apply to the Metavir score. In
particular, the fibrosis stages of the Metavir score have not been well-correlated to the
175
natural history of hepatitis C . Thus, earlier on, it was unclear whether individuals
progress from early to late stages at a constant, linear rate, although this hypothesis has
169
been proposed . But in subsequent studies it has been shown that the rates of
progression of fibrosis were not normally distributed and greatly different estimated rates
of progression were found in different patient groups 186.
249
4.30 Guidelines by International Association for the
Study of the Liver
141
For routine diagnosis and patient management, one authority , has
recommended a simple system of grading and staging as proposed by a panel of experts
convened by the International Association for the Study of Liver IASL in 1994 170.
Chronic hepatitis is graded according to whether the degree of activity is mild,
moderate, or marked. Although this concept seems rather simple, it is essentially
subjective and even this is not highly reproducible between pathologists or even the same
pathologist 131.
The principal features used to determine the grade are the degree of periportal
interface hepatitis (piecemeal necrosis) and spotty parenchymal injury. The staging of
chronic hepatitis is based on an assessment of the degree of fibrosis and a Masson
trichrome stain is required for a proper evaluation. The stage of disease progresses as an
absence of fibrosis incomplete cirrhosis, and finally to established cirrhosis 141.
170
Following the recommendations of IASL panel , the diagnostic line of
pathology report should indicate the cause of chronic hepatitis if known, the grade, and
the stage. Thus the report may read, “chronic hepatitis C with mild activity and portal
fibrosis” or “ chronic hepatitis B with moderate activity and extensive bridging fibrosis”
or “chronic autoimmune hepatitis with marked activity and established cirrhosis” 141.
141
According to the source cited above , the follow up biopsy specimen of a
patient who is being treated for chronic hepatitis should be evaluated in the context of
previous specimens. Biopsies are performed to determine whether the activity of the
patient’s liver disease has improved or the fibrosis has progressed. The only meaningful
evaluation is one in which the initial specimen is compared with a follow up specimen. A
comparison of pathology reports or numeric scores generated at different times,
according to the source, can only lead to confusion and incorrect conclusions about the
course of a patient’s disease 141.
250
Table 14. Scheuer Scoring System for
necroinflammatory activity and fibrosis
Grade Portal/periportal Lobular Fibrosis

Activity activity
0 Normal or minimal None None

1 Portal Inflammation (CPH) Inflammation but Enlarged fibrotic
no necrosis portal tracts
2 Mild piecemeal necrosis Focal necrosis or Periportal or portal
(mild CAH) acidophil bodies -portal septa but
intact architecture
3 Moderate piecemeal Severe focal cell Fibrosis with
necrosis (moderate CAH) damage architectural
distortion but no
cirrhosis
4 Severe piecemeal necrosis Damage includes Probable or
(severe CAH) bridging necrosis definite cirrhosis
Adapted from169:
Scheuer PJ. Classification of chronic viral hepatitis: a need for
reassessment. J Hepatol 1991; 13: 372-374.
251
Table 15. Batts and Ludwig system for Grading
and staging chronic hepatitis
Grade
Chronic hepatitis, minimal Grade 1
Chronic hepatitis, mild Grade 2
Chronic hepatitis, moderate Grade 3
Chronic hepatitis, marked Grade 4
Stage
No fibrosis Stage 0
Portal fibrosis Stage 1
Few bridges Stage 2
Many bridges Stage 3
Cirrhosis Stage 4
Adapted from 174:

Batts KP, Ludwig J. Chronic hepatitis: an update on terminology and
reporting. Am J Surg Pathol 1995; 19: 1409-1417.
252
Table 16. Laennec scoring system for fibrosis
Criteria:
Septa
(thickness
Grade Name & number) Descriptive examples
0 No definite fibrosis
1 Minimal fibrosis +/- No septa or rare thin septum, may have
portal expansion or mild sinusoidal fibrosis
2 Mild fibrosis + Occasional thin septa. May have portal
expansion or mild sinusoidal fibrosis
3 Moderate ++ Moderate thin septa, up to incomplete
fibrosis cirrhosis
4A Cirrhosis, mild +++ Marked septation with rounded contours or
definite or visible nodules. Most septa are thin (one
probable broad septum allowed)
4B Moderate ++++ At least two broad septa, but no very broad
cirrhosis septa and less than half of biopsy length
composed of minute nodules
4C Severe +++++ At least one very broad septum or more than
Cirrhosis half of biopsy length composed of minute
nodules (micronodular cirrhosis)
Adapted from 187:
Wanless IR. Cirrhosis. In: Johnson LR, ed. Encyclopedia of
Gastroenterology. New York: Academic Press; 2003: 356-62.
253
Fig 8 Chronic hepatitis with mild activity and no fibrosis, x 10 H& E
254
Fig. 9 Chronic hepatitis with fatty change (steatosis), moderate activity and portal
fibrosis, x 10 H & E
255
Fig. 10 Chronic hepatitis with fatty change (steatosis), moderate activity and portal
fibrosis, x 20 H & E
256
Fig. 11 Chronic hepatitis with moderate activity and portal fibrosis, x 10 H & E.
257
Fig. 12 Chronic hepatitis with moderate activity and portal fibrosis, x 20 H & E.
258
Fig. 13 Chronic hepatitis with moderate activity and bridging fibrosis, x 10 H & E
259
Fig. 14 Chronic hepatitis with moderate activity and bridging fibrosis, x 20 H & E
260
Fig. 15 Chronic hepatitis with moderate to marked activity and early cirrhosis,
x 10 H & E
261
Fig. 16 Chronic hepatitis with moderate to marked activity and early cirrhosis,
x 20 H & E
262
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278
Chapter 5
NON INVASIVE
EVALUATION
OF
LIVER FIBROSIS
Contents
Introduction 282
Serum Markers 283
Indirect Markers 285
AST/ALT Ratio 285
PGA Index 286
Fibrotest 287
Acti Test 290
Steato Test 291
Forns index 291
APRI (AST to Platelet Ratio Index) 292
Göteborg University Cirrhosis Index (GUCI) 294
Direct Markers of Liver Fibrogenesis 294
Hyaluronic Acid 297
YKL-40 (Chondrex) 299
Collagen 300
Matrix metalloproteinases and inhibitors 301
Cytokines 302
FibroSpect 303
FibroSpect II 303
European Liver Fibrosis Group assay 304
Fibrometer 304
Hepascore 306
SHASTA Index 307
APRICOT Clinical Investigators Assay 307

13
C- Caffeine Breath Test 308
280
Fibrosis Markers to Assess Effect of Treatment 309
Fibrosis Markers to Predict Disease Progression 312
Gene Markers and Liver Fibrosis 313
Radiology in the Noninvasive Assessment of Liver Fibrosis 316
Measurement of Hepatic Stiffness (Elastography) 317
Platelets in Chronic Liver Disease – The role of Spleen 322
Platelet Auto antibodies and Thrombocytopenia 322
Thrombopoietin and Chronic Liver disease 323
Platelet Parameters and Chronic Liver Disease 326
5.1 Introduction
Chronic liver diseases, CLD, are a major cause of morbidity and mortality in the
1-6
present day world . Chronic infections with hepatitis B and C viruses, alcoholic and
281
7-13
non-alcoholic fatty liver diseases are important causes of CLD . The major
determinant in their prognosis is the progressive accumulation of fibrosis, with distortion
of the hepatic architecture, and ultimate progression to cirrhosis and its allied
3,8,9-21
complications . In the recent years, with the great advancement in therapeutic
modalities to treat chronic liver diseases, accurate assessment of fibrosis has become of
paramount importance; to guide management decisions, predict outcome (prognosis), and
monitor disease activity in individual patients 21-26.
Chronic hepatitis from HBV and HCV are the most common causes of cirrhosis
and hepatocellular carcinoma in the world today. Of approximately 2 billion people who
have been infected with HBV worldwide, more than 350 million, or about 5% of the
world’s population are chronic carriers, and with an annual incidence of more than 50
million 1,27. HBV accounts for 500,000 to 1.2 million deaths per year 4.
Similarly more than 3 % of the world population or about 170-5 million people
may be infected with HCV 2,3,28,29. The prevalence of HCV is increasing and estimates of
the future burden of chronic hepatitis C predict at least a 3 fold rise in chronic liver
disease and cirrhosis by the year 2020 3,30-32.
Nonalcoholic fatty liver disease (NAFLD) affects 10-30% of the general
population in various countries. 10% of these people, or ~ 2-3% of the general
population, satisfy the criteria for nonalcoholic steatohepatitis, (NASH). The prevalence
of NAFLD is also expected to rise in developed countries given the epidemic of its major
determinant, obesity 6,11,12,20,25,33-36.
Studies indicate that advanced fibrosis and cirrhosis will develop in 20-40% of
patients with chronic hepatitis B or C and in a similar proportion of patients with
nonalcoholic fatty liver disease 3,4,5,7, 9,10,11,12,37,38.
Therefore it is imperative for both clinicians and patients to acquire accurate
information about the degree of fibrosis to guide management decisions predict outcome
and monitor disease activity 23.
Hepatic fibrosis is the final common pathway for a multitude of liver injuries.
Viral, immune, toxin mediated liver injuries all result in expansion of the extracellular
matrix, with the deposition of fibrous tissue, distortion of hepatic architecture and
ultimately the development of cirrhosis 39.
282
Percutaneous liver biopsy is considered the gold standard for grading and staging
the liver disease. But it has its limitations, which include: small but significant morbidity
and mortality rates, sampling error, inter- and intra-observer variability, limits of the
histopathological scoring systems, and the provision of a static picture of liver
architecture in a dynamic disease process 19,23.
With the expanding knowledge of fibrosis, more accurate, reproducible, and
noninvasive methods of determining liver fibrosis are required. Understanding the
pathogenesis of hepatic fibrosis at molecular level has led to the identification of several
potential serum markers of hepatic fibrosis. Either individually or in combination, these
serum markers appear capable of determining early and advanced fibrosis 39.
Serum markers of hepatic fibrosis offer an attractive alternative to liver biopsy; as
they are less invasive than biopsy, may allow dynamic calibration of fibrosis and may be
more cost effective 23.
Additionally, radiological means can also noninvasively assess liver fibrosis.
Ultrasonography and cross-sectional imaging with CT, MRI though enables detailed
images of the liver and surrounding structures, but resolution of hepatic parenchyma is
insufficient to determine any of the earlier stages of fibrosis before the establishment of
cirrhosis and portal hypertension 40. Transient hepatic elastography is a novel technology
demonstrating promise as a noninvasive means of fibrosis determination 41.
5.2 Serum Markers

According to Afdhal N 39, serum markers of hepatic fibrosis refer to the
measurement of one or more molecules within blood or serum sample as markers of
42
fibrosis in the liver . There are several proposed biomarkers or combinations of
biomarkers. Ideal features of serum markers are 39:
· Liver specific
· Independent of metabolic alterations
· Easy to perform
· Minimally influenced by urinary and biliary excretion
283
· Reflective of fibrosis irrespective of cause
· Sensitive enough to discriminate between stages of fibrosis
· Correlate with dynamic changes in fibrogenesis or fibrosis resolution
According to Afdhal N 19, none of the available markers fulfill, these ideal
criteria. None are liver specific, so there may be significant contribution from non-hepatic
sources, including bone, joints, skin, and lungs. Levels of these markers are altered by
changes in their clearance, metabolism, and excretion. For instance, both the liver (80%)
and the kidneys (20%) clear hyaluronan and the removal from circulation is also
43
dependent upon binding to specific receptors by hepatic endothelial cells . Increased
hyaluronan levels occur in the post prandial state, presumably as a result of competition
for these receptors 44.
Their relationship to the total matrix content of the liver and to the activity of
fibrogenesis or degradation is usually mixed. Indeed, in the absence of a golden standard
of matrix turnover, it is not possible to assess the relationship of the levels of these
markers to ongoing matrix remodeling and new matrix deposition or removal. Thus from
a practical and research standpoint, it would be ideal to have marker(s) that can (a)
noninvasively stage the degree of liver fibrosis, (b) reflect the rate of matrix deposition or
removal, both to monitor the impact of therapies and to give prognostic information. It is
unlikely that any single marker can meet the acid test, and can fulfill both of these ideal
criteria 19.
Proposed serum markers of hepatic fibrosis can be categorized broadly as either
direct or indirect. Indirect markers reflect alterations in hepatic function but do not
directly reflect extracellular matrix (ECM) metabolism, for instance platelet count,
coagulation studies, and assessment of liver transaminases. Direct markers of fibrosis
reflect ECM turnover. Their discovery has been linked directly to advances in
understanding of the molecular mechanisms of hepatic fibrosis. Serum assays for
products of matrix synthesis or degradation and the enzymes involved in these processes
45-53
have been investigated as markers of liver fibrosis in several studies . Combinations
of these markers, both direct and indirect, are emerging as a promising alternative to
routine liver biopsy for many patients with liver disease 54-57.
284
5.3 Indirect Markers
According to Afdhal N 39, various indirect markers of liver fibrosis have been
used in clinical practice for many years, such as on physical examination, hepatomegaly
and splenomegaly. Patients who have elevated AST, ALT, coagulopathy, hypersplenism
with thrombocytopenia invariably have cirrhosis and portal hypertension. Initial efforts
39
attempted to correlate these routine investigations with stage of hepatic fibrosis . For
example the AST/ALT ratio 58, platelet count 59, and the prothrombin index 60. It should
be reiterated that the indirect markers reflect the disturbance of hepatic function or
structure, rather than the deposition or the removal of ECM 19.
One of the main limitations to the clinical use of direct markers of liver fibrosis is
that they are not routinely available in all hospital settings. While direct markers of liver
fibrosis have shown promise in detecting liver fibrosis, the indirect markers satisfy the
request for a simple and easy to perform marker with diagnostic accuracy for detecting
liver fibrosis that is equal or better than direct markers.
5.4 AST/ALT Ratio

According to Afdhal N 39, Park and colleagues compared the AST/ALT ratio
between 30 known cirrhotics and 123 controls without cirrhosis and reported a ratio
greater than or equal to 1 had a 95.9% specificity and 73.7% positive predictive value in
distinguishing patients who had cirrhosis from those who did not have cirrhosis, with
46.7% sensitivity and 88.1% negative predictive value. It appeared that though relatively
insensitive, an AST/ALT greater than or equal to 1 is highly specific but not diagnostic
for the presence of cirrhosis in patients who have chronic HCV infection 61.
In a cohort of patients who had hepatitis C, Assy reported a moderate correlation
62
between AST levels and the presence of advanced fibrosis . A study of 252 patients
who had hepatitis C evaluated the diagnostic and prognostic value of the AST/ALT ratio
63
. Diagnostically, the ratio performed well; an AST/ALT ratio of greater than 1 was
strongly suggestive of cirrhosis with sensitivity of 78% and specificity 97%. Combined
use of the AST/ALT ratio with a platelet count of less than 130 x 109/l increased the
285
diagnostic accuracy of the test with positive and negative predictive values of 97% and
86% respectively 39. Furthermore, a progressive increase in AST/ALT ratio was observed
in patients with more advanced liver disease as determined by Child Pugh and MELD
scores as well as by lidocaine metabolism (MEGX test) 19.
The relative increase in AST is probably related to both reduced clearance of AST
by hepatic sinusoidal cells as well as to mitochondrial dysfunction 64-66.
The value of this ratio is greatest for the noninvasive diagnosis of cirrhosis, where
61
a ratio of >1.0 is suggestive of this diagnosis . This has been shown for both viral
67 68
disease and nonalcoholic fatty liver disease . The efficacy of this ratio, is however,
not relevant to all forms of the liver disease, in particular alcoholic liver disease
(associated with elevated AST) or conditions associated with predominantly with hepatic
inflammation (autoimmne hepatitis) 39.
The prognostic value of AST/ALT ratio was assessed in subset of 63 cirrhotic
patients who were followed for at least one year. In this subgroup, an AST/ALT ratio
greater than 1.16 was found to predict mortality and performed similarly to the Child
Pugh and MELD scores. It should be noted that this study included a significant
proportion of patients with clinically diagnosed cirrhosis and hence it would be expected
that the diagnostic accuracy of this ratio for the detection of asymptomatic cirrhosis and
earlier stages of fibrosis would be lower 19.
5.5 PGA Index

According to Afdhal N 39, PGA index was the original index of hepatic fibrosis
described in 1991. It was devised as a simple biological index for detecting alcoholic
69
liver disease in drinkers . It combines the measurement of the prothrombin index,
gamma glutamyl transferase (γGT) and apolipoprotein A1 (PGA). It has been validated in
patients with various chronic liver diseases, in particular alcohol (hence the use of γGT)
70,71
. The diagnostic accuracy of the PGA score for detecting cirrhosis is reported between
69, 70
66% and 72% . This was subsequently modified to the PGAA index by the addition
of α2- macroglobulin, which resulted in some improvement in its performance 72.
Oberti, in a study of 243 patients with chronic viral or alcoholic liver disease,
assessed the utility of a large number of clinical and ultrasonographic parameters as well
286
70
as indirect and direct markers of liver fibrosis to diagnose cirrhosis . Of the clinical
features of cirrhosis, a firm liver with a thin lower edge had the highest diagnostic
accuracy of 83%. Of the indirect markers of fibrosis, the prothrombin index had the
highest diagnostic accuracy 86%, while the performance of the PGA and PGAA scores
were somewhat less good at 78.5% and 80%, respectively.
70
In this study , of all the specific markers of fibrosis being assessed, including
laminin, procollagen III N-peptide (PIIINP), hyaluronan, and TGF-β 1, only hylauronan
performed as well as the prothrombin index with a diagnostic accuracy of 86%. Of
importance, it was noted that the aetiology of the liver disease affected the performance
of some of these tests. For example analysis of the AST/ALT ratio had a diagnostic
accuracy of 79% in viral liver disease, but as expected performed poorly in alcoholic
liver disease, 65%. The PGA and PGAA scores performed better in alcoholic than viral
liver disease, whereas, the prothrombin index and hyaluronan levels performed similarly
in both disease groups 19.
5.6 Fibrotest
Fibrotest 54, is the most widely known, and the most validated of the tests of the
noninvasive evaluation of liver fibrosis, with over 20 studies reported in the literature 24.
Developed by Poynard and colleagues, several biochemical markers of liver fibrosis were
assessed in 339 patients with hepatitis C. The aim of this study was to find out whether a
panel of biochemical markers could reliably identify patients with clinically significant
fibrosis (METAVIR F2 or greater) and thus be used to limit the need for liver biopsy in
the selection of patients for treatment of hepatitis C.
Of the assessed markers, α2 macroglobulin, α2 globulin (or haptoglobin), γ
globulin, apolipoprotein A 1, γ glutamyl transferase, and total bilirubin were the most
informative, and were used to derive a calculated index; now known as the Fibrotest. In
the study, a total of 11 markers were assessed: α2 macroglobulin, AST, ALT, γ glutamyl
transferase, total bilirubin, albumin, α1 globulin, α 2 globulin, β globulin, γ globulin, and
apolipoprotein A1. The α2 globulins mainly consisted of α2 macroglobulin and
haptoglobin. In the second period, Interleukin 10 (IL-10), tumor (transforming) growth
287
factor β 1(TGF β 1), hepatocyte growth factor, apolipoprotein A2, and apolipoprotein B
were also assessed.
However, the most informative markers were, in the decreasing rank: α2
macroglobulin, haptoglobin, GGT, γ globulin, total bilirubin, and apolipoprotein A1 54.
The areas under the receiver operating characteristic curve, ROC, for the first year
(0.836), and second year (0.870) did not differ (p=0.44). By selecting the upper and lower
cut off values, the authors were able to categorize their patients into three groups: (1)
those in which there was a high certainty of mild liver disease (METAVIR F0-1), (2)
those with high certainty of significant fibrosis (METAVIR F2-4), (3) and a group of
patients who could not be adequately characterized and in whom liver biopsy would be
necessary, the indeterminate group 39.
With the best index, a high negative predictive value (100% certainty of absence
of F2, F3, or F4 fibrosis) was obtained in scores ranging from zero to 0.10 ( 12% of all
patients), and high positive predictive value ( >90% certainty of presence of F2, F3, or F4
fibrosis) for scores ranging from 0.60 to 1.00 (34% of all patients). The detection of
significant fibrosis F2 or greater had a 75% sensitivity and 85% specificity. The assay
performed somewhat better for the assessment of more advanced liver disease
(METAVIR stages 3 and 4). Thus the correct identification of the disease as either mild
or severe was made in 46% of the patients overall, hence obviating the need for liver
biopsy in these patients. This included 100% negative predictive value for the exclusion
of METAVIR stages F2-4 fibrosis and a greater than 90% positive predictive value 54.
The same authors have validated this score in other hepatitis C positive cohorts
73-75
including those with HIVinfection , but an independent study did not achieve the
76
same results .
76
Rossi et al , studied 125 hepatitis C positive patients who had undergone liver
biopsy. Using local assays and the original authors’ computer program for the calculation
of Fibrotest score, the performance of the assay was somewhat less impressive.
The area under the receiver operating characteristic curve, ROC for significant
fibrosis staging (> F2 on Metavir Index) was 0.739; which was smaller than the area
under the ROC in the principal study. The negative and positive predictive values were
85% and 78%, respectively. Using these cut off values only 33 of the 125 patients would
288
have been saved liver biopsy. Six of these patients would have been misclassified as
having mild fibrosis, while the results of the liver biopsy demonstrated more significant
disease requiring treatment 76.
The reason for this discrepancy in results is unclear, since the original authors
have shown that the assay and score appear to be reproducible when performed in
different laboratories 77,78.
79
Commenting on the Rossi et al study, in another study the Fibropaca study, the
authors had the opinion that the smaller area under the ROC may be partially explained
by non respecting the analytical recommendations for performing the Fibrotest, the low
number of patients, and the lack of information concerning biopsy sample. More
importantly, Rossi et al, did not discuss the causes of failures for the Fibrotest and biopsy.
Various studies have highlighted the importance of the pre-analytical and analytical steps
in the validation of the values of biochemical markers for carrying out the Fibrotest 77,80.
The Fibropaca Study 79, an independent prospective multicenter study confirmed
the diagnostic value of Fibrotest and Actitest found in the principal study and suggested
that both the tests could be an alternative to liver biopsy in most patients with chronic
HCV. In this study 504 patients were followed, 46% were classified as F2-F4 fibrosis and
39% as A2-A3 activity. The area under the ROC for the diagnosis of activity (A2-A3)
was 0.73 (0.69-0.77), for significant fibrosis (F2-F4) was 0.79 (0.75-0.82), and for severe
fibrosis (F3-F4) was 0.80 (0.76-0.83). Among the 92 patients (18%) with 2 fibrosis stages
of discordance between Fibrotest and biopsy, the discordance was attributable to
Fibrotest in 5.4% of patients, to biopsy in 3.8%, and undetermined in 9.1%.
In analyzing the discordance between liver biopsy and biomarkers, the main
difficulty is the absence of a true gold standard of liver injury. The most frequent failures
attributable to the markers were false positives due to Gilbert’s disease and inflammation
and false negatives due to inflammation. The most frequent failures due to biopsy were
false negatives due to small biopsy size. Among the eight cirrhotic patients well defined
by radiological, endoscopic, or biochemical criteria with discordant Fibrotest and liver
biopsy, five were not diagnosed by liver biopsy and three were not diagnosed by
Fibrotest, underlining the absence of a gold standard in determination of liver fibrosis.
289
Nevertheless, both the Fibrotest and Acitest bring new insights in the diagnosis of
fibrosis in patients with chronic hepatitis C. However, the identification of risk factors of
Fibrotest and Actitest failures such as Gilbert’s syndrome, inflammation, and haemolysis
must be considered before and after the Fibrotest-Actitest interpretation.
The combined use of Fibrotest and Fibroscan (an instrument used to detect
transient hepatic elastography) among 183 patients with chronic hepatitis C demonstrated
an area under the ROC curve of 0.88 for F > 2, 0.95 for F > 3, and 0.95 for F > 4. When
the Fibrotest and Fibroscan results agreed, liver biopsy examination confirmed them in
81
84% cases for F > 2, in 95% of cases for F > 3, and in 94% of cases for F > 4 .
Therefore it is likely that a combination of serum markers and Fibroscan will
complement each other and enhance accuracy of fibrosis detection 39.
5.7 Acti Test

Acti Test is a modification of the Fibrotest that incorporates ALT and reflects
both necroinflammatory activity and liver fibrosis. According to the original study 54, the
best logistic regression for diagnosis of clinically significant fibrosis (F2, F3, or F4) or
substantial activity (A2 or A3) combined the same six markers as for fibrosis alone plus
ALT ( R2 =0.297, p<0.0001).
Acti Test appears to demonstrate improved identification of more advanced
77
fibrosis associated with greater histologic inflammation . Both Acti Test and Fibrotest
have shown reductions among those with sustained virological response to interferon and
ribavirin therapies, supporting a role in following response to therapy 82.
83
A meta-analysis of 16 publications and 1570 patients, by the same group
concluded that: at a cut off of 0.31, the Fibrotest negative predictive value for excluding
significant fibrosis was 91%. At a cut off of 0.36, the Acti Test negative predictive value
for excluding significant necrosis was 85%. Additionally, there was no difference
between the area under the ROC’s of Fibrotest/Acti Test according to the genotype or
viral load. Thus the use of biochemical markers of liver fibrosis (Fibrotest), and necrosis
(Acti Test) can be recommended as an alternative to liver biopsy for the assessment of
liver injury in patients with chronic hepatitis C 83.
290
5.8 Steato Test
Developed by Poynard and colleagues 84, Steato Test incorporates the five
components of Fibrotest (α2 macroglobulin, haptoglobin, apolipoprotein A1, γ glutamyl
transferase, total bilirubin) and Acti Test (ALT) plus body mass index, serum cholesterol,
triglycerides, and glucose adjusted for age and gender. A cut off of 0.30 had a 90%
sensitivity and a cut off of 0.72 had a 90% specificity permitting to achieve a useful
predictive value, 93% NPV and 63% PPV for a steatosis prevalence of 30%. Although
the PPV of the Steato test is not that high, but still is significantly higher than those of
previous markers to evaluate steatosis, such as, GGT, ALT, and ultrasonography 84.
5.9 Forns index

55
Forns et al , reported an index based on four readily available variables; age,
platelet count, γ glutamyl transferase, and cholesterol levels. The study was confined to
patients with hepatitis C and included both test and validation cohorts. The authors
constructed a simple score system applying a constant to the obtained formula:
7.811- 3.131.In (platelet count) + 0.781.In (GGT) + 3.467.In (age) – 0.014. (cholesterol)
Using the test cohort of 125 patients, in a manner similar to the Imbert-Bismut
study (Fibrotest) 54, the authors set thresholds for defining individuals with a high or low
probability of significant fibrosis (METAVIR F2-4). The area under the ROC curve was
55
0.86 for the estimation group and 0.81 for the validation group .When these criteria
were applied to the validation cohort, 51% of the population could be classified using
these upper and lower cut off values. The lower cut of value had a 96% negative
predictive value, whereas the upper cut off value had a positive predictive value of 66%.
Hence this test was useful at excluding patients with minimal fibrosis, but was of limited
19
value for the identification of patients with more advanced liver disease . In summary
half of the patients with chronic hepatitis C without significant liver fibrosis can be
identified with high accuracy with this panel; and liver biopsy could have been avoided in
more than one third of the patients 55.
One important aspect of the index is the use of very basic clinical parameters, and
the fact that the accuracy of this model compares with models based on more
291
sophisticated variables (such as Fibrotest). In comparing with the study by Imbert-Bismut
54
(Fibrotest) some of the variables used do provide a high level of certainty of the
presence or absence of significant fibrosis, but the usefulness may be curtailed by the fact
that some of the predictive markers (α2 macroglobulin, haptoglobin, and apolipoprotein
A1) are research tools not readily available in routine clinical practice in most centers.
In addition, the patient population analyzed in the Imbert-Bismut study included a
high proportion of patients with advanced hepatitis C infection, as indicated by the
presence of significant fibrosis in up to 40% of the subjects. In the authors’ series,
significant fibrosis was present in only 25% of the subjects a figure possibly closer to the
spectrum of the disease in the community at large 55.
Additionally, the performance of this index was compared to the Fibrotest by the
57
authors of the later test . They found that the results of the Forns index were
reproducible but performed slightly less well than Fibrotest.
Major caveats of the Forns index include concerns about the impact of lipid
57
abnormalities in patients with hepatitis C , cholesterol altering medicines, and the
reproducibility of platelet estimations 19.
5.10 APRI (AST to Platelet Ratio Index)

Reported by Wai and colleagues 56, APRI refers to the AST to platelet ratio index.
In this retrospective study, a total of 270 patients were evaluated, which included training
(n=192), and validation cohorts (n=78). The authors looked at a number of simple
laboratory studies and their relationship to two end points, significant liver fibrosis and
cirrhosis. Based on their analysis of simple laboratory measurements, they found that the
APRI was the simplest and the most accurate test for the evaluation of two end points 19.
It is calculated as follows:
APRI = AST level / ULN x 100

Platelet count (109/l)
292
Platelet counts and AST levels are the most important predictors of significant
fibrosis and cirrhosis. This novel index was developed to amplify the opposing effects of
fibrosis on AST and platelet count.
The area under the ROC curve of APRI for predicting significant fibrosis and
cirrhosis in the training set were 0.80 and 0.89, respectively. In the validation cohort the
same values were 0.88 and 0.94, respectively. While the upper and lower cut off levels
were selected with the aim of correctly characterizing this cohort into those with
significant fibrosis and cirrhosis, the index performed reasonably well. Thus, using the
optimized cut off values, significant fibrosis could be predicted accurately in 51% and
cirrhosis in 81% of patients 56.
APRI test successfully and simply can exclude those with and without significant
fibrosis and cirrhosis with negative predictive values of 86% and 98%, respectively; and
corresponding positive predictive values of 88% and 57% 56.
Its most important aspect is the use objective and readily available laboratory
values. Both platelet count and AST levels are routine tests performed in chronic hepatitis
C patients in clinical practice, so no additional tests are required. The finding of
decreased platelet count and increased AST level with progression of liver fibrosis has
been reported in many studies.
With increasing fibrosis and worsening portal hypertension, there is increased
sequestration and destruction of platelets in the enlarging spleen 85. In addition, studies in
liver transplant patients showed that the progression of liver fibrosis is associated with
decreased production of thrombopoietin by hepatocytes, and hence reduced platelet
production 86,87. Progression of liver fibrosis may reduce the clearance of AST, 65 leading
to increased serum AST levels. In addition, advanced liver disease may be associated
with mitochondrial injury, resulting in more marked release of AST, which is present in
mitochondria and cytoplasm, relative to ALT 64,66.
One caveat of the study is that: it included a sufficient proportion of patients with
significant fibrosis (47%) and cirrhosis (15%) from a tertiary care center; so the results
may not be totally generalizeable to community based practice.
293
Though slightly inferior, nevertheless, its simplicity (APRI can be determined by
the bedside with the help of a calculator), matched with performance to the more
sophisticated Fibrotest and Forn’s index is a great advantage 39.
5.11 Göteborg University Cirrhosis Index (GUCI)

In this study 88, samples from 179 patients with chronic hepatitis C were analyzed
using routinely available biochemical markers of liver disease, and compared with liver
biopsy using the Ishak protocol. By multivariate logistic regression analysis the authors
found strong association between AST, platelet count and prothrombin-INR. They
developed the Göteborg University Cirrhosis Index (GUCI) according to the formula:
Normalized AST x prothrombin-INR x 100

Platelet count (x 109/l)
Using a cut-off value of 1.0, the sensitivity was 80%, and the specificity 78% for
the diagnosis of cirrhosis, and the negative predictive value and the positive predictive
values were 97% and 31% respectively. The authors concluded that the GUCI score
proved slightly superior for sensitivity, specificity, NPV, PPV, and the area under the
receiver operating characteristic curve (ROC) for the prediction of cirrhosis and bridging
fibrosis compared with AST to platelet ratio index (APRI) 88.
5.12 Direct Markers of Liver Fibrogenesis

39
According to Afdhal N , the extracellular matrix refers to a group of
macromolecules (both soluble and insoluble) that comprise the supporting framework of
the normal and fibrotic liver. The liver fibrosis results in qualitative and quantitative
changes in ECM markers. Some ECM markers reflect fibrogenesis, and others reflect
fibrosis regression, a dynamic evaluation of liver fibrosis reflecting ECM activity is
possible.
The direct markers of liver fibrosis include a number of serum or urinary markers
which have been shown to be, or are thought to be directly involved in the deposition or
294
removal of ECM (see table 17) 19. The potential markers of fibrosis include products of
collagen synthesis or degradation, enzymes involved in matrix biosynthesis or
degradation, extracellular matrix glycoproteins, proteoglycans and glycosaminoglycans
39
.
None of the currently available direct markers completely fulfill the ideal criteria.
In particular, none is liver specific, and all are affected by changes in their clearance,
metabolism, and excretion. The serum half lives of these molecules is short lived, so
levels probably reflect the activity of ECM metabolism. Since the ECM turnover is
related to both new ECM deposition and removal, as well as remodeling of established
ECM, serum levels probably reflect both the activity of the process as well as the total
mass of ECM undergoing metabolism 19.
This is supported by at least three features. First levels of these markers are most
often elevated in conditions with rapidly progressing fibrosis (e.g., severe alcoholic
89,90
hepatitis or more active hepatitis) and may be high prior to the deposition of ECM .
Second, levels tend to fall in response to treatment of the underlying disease process,
91,92
often before to any discernible reduction in the stage of fibrosis . Third in chronic
liver diseases, elevations of several, but not all of these markers correlate independently
with the stage of fibrosis, rather than with either biochemical or histological features of
inflammation 53, 93-95.
However, in some studies serum levels of these markers correlated more strongly
with the degree of histological inflammation or transaminases96,97. The observation that
markers of ECM metabolism are increased in parallel with markers of liver inflammation
and necrosis may reflect the importance of these processes in upregulating fibrogenesis
19
.
According to Afdhal N 19, the direct markers of liver fibrosis can be conveniently
subdivided into three groups: Those reflecting (a) matrix deposition, (b) matrix removal,
or (c) where the relationship to the matrix deposition or removal is unclear.
One approach to the evaluation of matrix deposition is to simultaneously
measure markers of both matrix deposition and removal, with a view to define the net
effect upon the fibrotic process. However, using combination of these markers has added
295
little diagnostic accuracy, while increasing both the expense and the complexity of
interpreting results19.
Table 17. Direct markers of liver fibrosis19
Markers of matrix removal

Procollagen IV C peptide
Procollagen IV N peptide (7-S collagen)
Collagen IV
Metalloproteinase MMP
Undulin
Urinary desmosine and hydroxylysylpyridinoline
Markers of matrix deposition

Procollagen I carboxy terminal peptide (PICP)
Procollagen III amino terminal peptide (PIIINP)
Tissue inhibitor of metalloproteinase (TIMP)
Transforming growth factor beta (TGF-β)
Tenascin
Uncertain
Hyaluronic acid
YKL-40 (Chondrex)
Laminin
296
19
Fibrosis markers can also be classified according to their molecular structure .
These include: (a) the collagens; procollagen I and III, propeptides released into the
serum during matrix deposition and remodeling. Type IV collagen, which is released
during interstitial filament degradation, reflects matrix degradation and remodeling.
Several assays for type IV collagen are available but their performance tends to be
similar; (b) The glycoproteins and polysaccharides including hyaluronan, laminin,
tenascin, and YKL-40, which are found in association with the basement membranes and
in regions of matrix deposition. The relationship between these markers and matrix
metabolism has not been clearly defined; (c) Collagenases and their inhibitors, include
the metalloproteinases (MMP’s) and the tissue inhibitors of metalloproteinases (TIMP’s);
(d) Cytokines involved in liver fibrosis, the best studied of these is TGF-β. Others
including platelet derived growth factor and the antifibrotic cytokine IL-10, have been
less well evaluated 19.
5.13 Hyaluronic Acid

39
According to Afdhal N , in evaluating single marker assays that reflect ECM
concentration, the best individual test appears to be hyaluronic acid (HA), which has been
validated in many clinical trials. Studies have demonstrated that serum hyaluronic acid
levels correlate with the degree of hepatic fibrosis in patients who have chronic hepatitis
C 53,98.
HA is a high molecular weight glycosaminoglycan, which is an essential
component of extracellular matrix in virtually every tissue in the body 99. In the liver, HA
is mostly synthesized by the hepatic stellate cells and degraded by the sinusoidal
94
endothelial cells . HA levels are increased in chronic liver diseases 94. In patients with
chronic hepatitis C virus, HA levels increase with the development of liver fibrosis.
Moreover, in patients with cirrhosis, HA levels correlate with clinical severity 100,101,102.
Serum hyaluronic acid can identify those without cirrhosis accurately. A level of
60 µg/l had a 99% negative predictive value for the absence of cirrhosis. It had low
accuracy for diagnosing cirrhosis (30% positive predictive value) 53.
297
103
A study by Halfon et al, focused on the diagnostic accuracy of HA alone,
(instead of combination of markers) in predicting fibrosis and cirrhosis in HCV infected
patients. In all 405 patients were studied. Absence of significant fibrosis, severe fibrosis,
and cirrhosis can be predicted by HA levels of 16, 25, and 50 µg/l respectively (with
NPV of 82%, 89%, and 100% in the same order). Presence of significant fibrosis, severe
fibrosis, and cirrhosis can be predicted by HA levels of 121, 160, and 237 µg/l
respectively (with PPV of 94%, 100%, and 57% in the same order). Thus serum HA is a
clinically useful as a noninvasive marker of liver fibrosis and cirrhosis. It suffers from the
need to limit, as much as possible, potential confounding variables such as the effects of
exercise and eating.
70
According to Afdhal N , Oberti evaluated four specific markers of fibrosis
(PIINP, HA, Laminin, TGF-β). These specific markers of fibrosis were studied in 243
patients with viral and alcoholic liver disease 70. In this prospective study HA performed
best with a diagnostic accuracy of 86%, whereas the performance of other specific
markers of fibrosis was less impressive; PIIINP 74%, laminin 81%, and TGF-β 67%.
Overall, the results of this study did not demonstrate any diagnostic advantage of the
specific over the nonspecific markers of fibrosis19.
Other comparative studies have also supported the superiority of HA over PIIINP
for the diagnosis of cirrhosis 98,104,105. For example in a comparative study of 326 patients
with hepatitis C, areas under the receiver operating curve (ROC) for the diagnosis of
fibrosis and cirrhosis were 0.86 and 0.92 for HA, while they were 0.69 and 0.73 for
PIIINP respectively 98. One explanation for the difference in performance may be related
to the observation that PIIINP levels correlate more closely with histological and serum
markers of hepatic inflammation than HA 104.
The greatest clinical utility of HA may be its ability to exclude patients with
53, 103
significant fibrosis and cirrhosis . In a study of 486 patients who entered the
hepatitis C Consensus Interferon Study, an HA level of <60 mg/l was found to exclude
patients with cirrhosis and significant hepatic fibrosis with predictive values of 99% and
93% respectively. Hence a low HA level may have a special role in identifying patients
with early fibrosis and hence reduce the need for biopsy in this subgroup of patients 19,103.
298
5.14 YKL-40 (Chondrex)
YKL-40 is a novel marker of liver fibrosis. YKL-40 (chondrex, human cartilage
106
glycoprotein-39), according to Saitou et al , is a recently described glycoprotein that
107
belongs to the chitinase family . YKL-40 m RNA was strongly expressed in human
liver and arthritic articular cartilage107,108 and was elevated in the synovial fluid 109
,
110 106
alcoholic liver disease , recurrent breast cancer, and colorectal cancer .
Although its physiologic function is not known in detail, YKL-40 is thought to
contribute to tissue remodeling or degradation of the extracellular matrix in liver fibrosis
111
. YKL-40 is produced by a wide variety of cells, including: chondrocytes, synovial
109
cells , activated macrophages, neutrophils, and in particular from cells located in
tissues with increased remodeling/degradation or inflammation of the extracellular
111 112
matrix, such as hepatic stellate cells . It is growth factor for fibroblasts ,
113
chondrocytes and synovial cells. It is a chemo-attractant for endothelial cells ,
modulates vascular endothelial cell morphology by promoting the formation of branching
tubules, indicating that YKL-40 may have a role in angiogenesis 106.
106
The authors studied Type IV collagen, amino-terminal peptide of type III
procollagen (PIIIP), hyaluronic acid (HA), YKL-40 and biochemical parameters. The
authors concluded that HA and YKL-40 were more useful than other markers for
assessing the fibrosis stage. In particular, YKL-40 was the most useful for monitoring the
fibrosis of liver disease and for distinguishing extensive liver fibrosis from mild stage of
liver fibrosis, enabling to predict severe stage of fibrosis at 80% positive predictive value.
It appears that serum levels of YKL-40 represented ongoing fibrosis like HA, in addition
to fibrogenesis similar to type IV collagen and PIIIP of the liver disease.
Serum YKL-40 levels is valuable for diagnosing mild stage of fibrosis (value <
186.4), severe stage of fibrosis (186.4 < value < 284.8), and F4 (284.8 < value). HA
appeared to be slightly better for prediction of cirrhosis (F4) from chronic hepatitis (F0-3)
than YKL-40. Additionally, after Interferon therapy, only YKL-40 values significantly
decreased not only in the responder group, but also in the non-responder group 106.
Further studies have also shown that YKL-40 appears to be reduced in patients
with chronic hepatitis C undergoing treatment, in conjunction with several other direct
114
markers of hepatic fibrosis, including PIIINP, MMP-2, and TIMP-1 . In a study of
299
YKL-40 in alcoholic liver disease suggested that it could function as an independent
marker of clinical outcomes 115.
5.15 Collagen
39
According to Afdhal N , collagen, the major component of hepatic fibrosis, is
synthesized in a precursor form known as procollagen. Enzymatic cleavage by two
distinct enzymes liberates carboxy and amino terminal procollagen peptides that have
proposed correlation with fibrogenesis. Procollagen type III amino terminal peptide
(PIIINP) is known to be elevated in acute hepatitis, and levels reflect stage of fibrosis in
71,97,116
chronic liver disease . PIIINP does not perform as well as HA for predicting
fibrosis but does reflect inflammatory score better 98.
Type IV collagen has been immunolocalized to the periportal interstitium and
117
large fibrotic bands in alcoholic liver disease . Serum type IV collagen is increased in
118
chronic liver diseases relative to controls . Some studies have demonstrated equivalent
efficacy in fibrosis determination to that seen with HA, but results have been
inconsistent48,105,119.
According to Afdhal N 19, Murawaki et al, assessed the diagnostic performance of
two forms of type IV collagen (7s domain and central triple helix domain) in 151 patients
with viral hepatitis 119. While the two assays performed similarly, the 7s domain achieved
slightly greater diagnostic accuracy for identification of patients with cirrhosis, with
positive and negative predictive values of 75% and 92% respectively. In another study
from the same group, the correlation between two assays for type IV collagen and
fibrosis scores were similar to HA but better than PIIINP. However, the correlation to
histological inflammatory scores and ALT values were greater, suggesting that type IV
collagen may be a marker of both inflammation and fibrosis 104.
At this time there appears to be no clear advantage to the use of assays of type IV
collagen over HA for fibrosis staging 19.
300
5.16 Matrix metalloproteinases and inhibitors
39
According to Afdhal N , the matrix metalloproteinases (MMP’s) and their
inhibitors are a group of proteins involved in controlling matrix degradation. MMP’s are
enzymes that are produced intracellularly and secreted in a proenzyme form that requires
cleavage by cell surface mechanisms for functional activity. The action of MMP’s is
counteracted by tissue inhibitors of metalloproteinases (TIMP’s) 39.
As such these proteins act to both degrade ECM and to permit new matrix
deposition. However, the relationship is complex and in addition to the actions cited
above; these molecules have multiple other activities including: activation of growth
factors, effects on cell proliferation, and inhibition of apoptosis. Thus the association
between peripheral levels and liver fibrosis is not clear19.
The hypothesis that hepatic fibrogenesis is associated with disrupted matrix
degradation stems from hepatic stellate cell research. MMP-13 is decreased in activated
stellate cell culture, while TIMP activity increases, promoting accumulation of
extracellular matrix. This corresponds to findings in human cirrhotic livers explants that
120,121
demonstrate increased expression of TIMP’s . Therefore, it seems that imbalance
between MMP’s and TIMP’s affects rate of fibrosis progression, and their estimation
correlates with stage of fibrosis. Results from studies of these markers, however, have
been variable and dependent upon the MMP/TIMP being assessed 51,115,122,123.
19 122
According to NA , Boeker et al , reported on the diagnostic accuracy of
TIMP-1 and pro-MMP-2, the free precursor molecule of MMP-2, and their relationship to
histological inflammatory scores. In this study of 78 patients with hepatitis C infection,
both of these assays performed as well or better than HA for the diagnosis of cirrhosis,
but only TIMP-1 showed diagnostic value for the identification of patients with earlier
stages of fibrosis. MMP-2 levels become elevated only once cirrhosis has developed.
Disturbing characteristics of these assays include the observation that TIMP-1
levels correlate strongly with histological inflammatory scores and that in the
noncirrhotic liver MMP-2 levels demonstrated no relationship to the stage of fibrosis.
These results are similar to other studies and significantly limit their value for staging
123,124
liver disease . Serum MMP-1 and MMP-3 levels have not been shown to be of
significant diagnostic value51,125.
301
5.17 Cytokines
Cytokines are thought to mediate hepatic fibrogenesis have also been evaluated in
determining hepatic fibrogenesis in a limited number of studies with mixed results19.
TGF-β is the dominant stimulus for producing extracellular matrix by hepatic stellate
cells. In a study of 88 patients who had chronic hepatitis C, there was a correlation
between total TGF-β 1 and the degree of hepatic fibrosis 126.
In another study of 39 patients with two liver biopsies, a close correlation was
noted between TGF-β serum levels and the rate of fibrosis progression, which may
suggest the use of TGF-β to determine those with progressive disease suitable for
127
antiviral therapy . But the performance of TGF-β appears to be less than the other
already discussed markers 54,70,126.
According to Afdhal N 19, a few studies have evaluated the relationship between
128,129,130
the levels of soluble adhesion molecules and liver inflammation and fibrosis .
Increased expression of intracellular adhesion molecule-1 (ICAM-1) occurs in virus
infected hepatocytes; whereas expression of vascular adhesion molecule-1 (VCAM-1)
128-130
may be seen in association with progressive hepatic fibrosis . It is thought that
increased VCAM-1 expression occurs in association with capillarization of the sinusoidal
spaces and fibrous septa.
In a study of 52 patients with hepatitis C virus, soluble VCAM-1 was found to
have excellent discriminant power for the detection of advanced fibrosis, sensitivity
100%, and specificity 85%. In this small study, measurement of soluble VCAM was of
greater diagnostic value than PIIINP128. Further studies are needed to confirm these
results, in larger patient populations and in other disease cohorts 19.
Panels with Indirect & Direct markers of liver fibrosis

Combinations of direct markers have been tried with indirect markers to enhance
the accuracy of liver fibrosis detection. Some of the assays are:
302
5.18 FibroSpect
McHutchison and colleagues in a retrospective cohort study evaluated the
131
FibroSpect assay . The aim of the study was to evaluate the diagnostic accuracy of a
panel of markers in chronic hepatitis C patients, develop a predictive algorithm that
differentiates no/mild (METAVIR F0-F1) from moderate/severe (F2-F4) fibrosis and
validate the model in external cohorts. The assay involves three parameters: hyaluronic
acid, TIMP-1, and α2 macroglobulin. These three markers were selected as having the
best predictive accuracy for F2-F4 fibrosis (combined AUROC = 0.831) 131.
At an index cut off of >0.36 and prevalence for F2-F4 of 52%, results in all 696
patients indicated positive and negative predictive values of 74.3% and 75.8% with an
accuracy of 75%. All patients were evaluable by the FibroSpect with no indeterminate
values, and this is an advantage of the assay 39. Maximum sensitivity and specificity were
39
seen at the two extreme spectrums of disease, stage 0 and stage 4 . The authors
concluded that the three marker panel may reliably differentiate chronic hepatitis C
patients with moderate/ severe fibrosis from those with no/mild fibrosis; although
131
accurate delineation between stages was not possible . In clinical use, the test gives a
likelihood of prediction as to whether the patient has mild or advanced disease based on
the score 39.
5.19 FibroSpect II
In a recently reported retrospective study 132, hyaluronic acid, YKL 40, and
FibroSpect II (comprising hyaluronic acid, TIMP-1 (tissue inhibitor of metalloproteinase
1), and alpha 2 macroglobulin) were assessed with Ishak stages and digital quantification
of fibrosis (DQF). Among the serum markers, hyaluronic acid was effective in
discriminating Ishak stages 0-1 and Ishak stages 2-3 compared with FS-II, with area
under the ROC curve of 0.76 versus 0.66 respectively. All three serum markers predicted
advanced fibrosis and cirrhosis. YKL-40 had the highest false positive rates in all
categories of fibrosis.
303
5.20 European Liver Fibrosis Group assay
133
The ELF group reported a novel assay in an international multi-center cohort
of 1021 patients with hepatitis C, nonalcoholic fatty liver disease, and alcoholic liver
disease. The authors aimed to develop a panel of sensitive automated immunoassays to
detect matrix constituents and mediators of matrix remodeling in serum to evaluate their
performance in the detection of liver fibrosis.
Serum levels of 9 surrogate markers of liver fibrosis were compared with fibrosis
stage in liver biopsy specimens obtained from all 1021 subjects with chronic liver
disease. Discriminant analysis of a test set of samples was used to identify an algorithm
combining age, hyaluronic acid, amino-terminal propeptide of type III collagen (PIIINP),
and tissue inhibitor of matrix metalloproteinase 1.
The sensitivity for the detection of Scheuer stage 3 or 4 fibrosis was 90% at a
threshold score of 0.102, and accurately detected the absence of fibrosis (negative
predictive value for significant fibrosis, 92%; area under the curve of a receiver operating
characteristic plot, 0.804; standard error, 0.02; p < 0.0001; 95% confidence interval,
0.758-0.851). The algorithm performed equally well in comparison with each of the
pathologists. In contrast, the pathologists’ agreement over histological scores ranged from
very good to moderate (kappa = 0.97-0.46). Thus the algorithm achieved a similar level
of sensitivity and specificity when compared with the scoring of three different
pathologists, providing evidence that it could be used with similar accuracy in different
settings.
The authors concluding that assessment of liver fibrosis with multiple serum
markers used in combination is sensitive, specific, and reproducible, suggesting they may
be used in conjunction with liver biopsy to assess a range of chronic liver diseases 133.
5.21 Fibrometer
In this landmark study 134, the authors studied 383 patients with viral hepatitis, 95
patients with alcoholic liver disease in the exploratory step, and the validating population
consisted of 120 patients with chronic liver disease due to HCV. The objective was to
develop tests to characterize different fibrosis parameters (Fibrometer) in viral and
304
alcoholic liver diseases. Measurements included 51 blood markers, and Fibrotest,
Fibrospect, ELFG, APRI, and Forns scores. The clinically significant fibrosis was
evaluated via Metavir staging (F2-F4), and image analysis was used to determine the area
of fibrosis (AOF).
In patients with chronic viral hepatitis, the area under the receiving operator
characteristic (AUROC) curve for stages F2-F4 in a test termed the “Fibrometer” test,
combining platelets, prothrombin index, AST, α2 macroglobulin (A2M), hyaluronate,
urea, and age was 0.883 compared with 0.808 for the Fibrotest (p = 0.01), 0.820 for the
Forns test (p = 0.005), and 0.794 for the APRI test (p < 10-4). The Fibrometer AUROC
curve was 0.892 in the validating step in 120 patients. In patients with alcoholic liver
disease the AUROC curve for stages F2-F4 in a test combining prothrombin index, A2M,
hyaluronate, and age was 0.962.
The area of fibrosis was estimated in viral hepatitis by testing for hyaluronate, γ
2
glutamyl transferase, bilirubin, platelets, and apolipoprotein A1, the adjusted R
coefficient in linear regression (aR2 = 0.645), and in alcoholic liver diseases by testing for
hyaluronate, prothrombin index, A2M, and platelets (aR2 = 0.836).
The 95 patients with alcoholic CLD were significantly older and had more
marked fibrosis than patients with viral CLD, something that could have influenced the
statistical results.
The authors claimed that their study was original because it measures the area of
fibrosis, AOF, in addition to histological staging, takes into account the cause of CLD,
and includes a large number of blood variables (n = 51).
The results of blood tests for clinically significant fibrosis were better in the
largest specimens, thus test results could be even better with new criteria for specimen
135,136
length, > 20 mm . It has been suggested that the variability is greater for AOF
135
evaluation than it is for histological staging . AOF is the only quantitative
morphological method of determining the amount of liver fibrosis, and it has been
suggested that it is superior to histological staging 95. For instance, there is restriction of
cirrhosis to one stage (F4), whereas the amount of fibrosis in cirrhosis is four times that
of the other four stages 95.
305
Although there was less observer variability for histological staging when a
137
consensual reading was performed by two experts , blood test results were not perfect
(e.g., the diagnostic accuracy, DA, for the Fibrometer test was 83.3% in the validation
population). This might be due to preanalytical or analytical variability in blood variables
or variability in the pathological reference. Studies with the Fibrotest have suggested that
most errors are due to histological staging itself 80,138.
The authors commented that the plots of the blood test results and of interobserver
137
agreement in relation to F stages , all had the same V-shape with a nadir at the F2
stage, suggesting that the difficulty in distinguishing F2 from F1 or F3 stages via
histological staging was the main cause of misclassification by the blood tests 134.
The use of blood tests to estimate the area of fibrosis is new. Measurement of area
of fibrosis via image analysis is limited to clinical research. Unlike histological staging,
the AOF provides precise quantification of the extensive variations in fibrosis during
cirrhosis. AOF estimation via blood test is the only statistically validated quantitative test
for the noninvasive diagnosis of fibrosis. However, the aim of AOF evaluation is not to
distinguish mild stages due to overlap of AOF values in these stages 134.
The authors concluded that: the pathological staging and the area of liver fibrosis
can be estimated using different combinations of blood markers in viral and alcoholic
liver diseases. The Fibrometer has a high diagnostic accuracy for clinically significant
fibrosis; blood tests for the area of liver fibrosis provide a quantitative estimation of the
amount of fibrosis, which is especially useful in cirrhosis 134.
5.22 Hepascore
139
In this study , the authors evaluated 10 biochemical biomarkers at the time of
liver biopsy in 117 untreated hepatitis C patients, and further validated in 104 patients.
Multivariate logistic regression and ROC curve analyses were used to create a predictive
model for significant fibrosis (METAVIR F2, F3, and F4), advanced fibrosis (F3 and F4),
and cirrhosis (F4).
A model, Hepascore, consisting of bilirubin, γ glutamyl transferase, hyaluronic
acid, α2 macroglobulin, age and sex produced areas under the ROC curves of 0.85, 0.96
and 0.94 for significant fibrosis, advanced fibrosis, and cirrhosis, respectively. In the
306
training set, the model was 92% specific and 67% sensitive for significant fibrosis, 81%
specific and 95% sensitive for advanced fibrosis, and 84% specific and 71% sensitive for
cirrhosis.
Thus a model consisting of four serum markers plus age and sex provides
clinically useful information regarding different fibrosis stages among hepatitis C
patients.
5.23 SHASTA Index

Developed by Afdhal N and colleagues 140, the SHASTA index consists of serum
hyaluronic acid, AST, and albumin. It was evaluated in a cohort of 95 patients with
HIV/HCV coinfection. As with other biomarker assays, optimal results were noted in the
extreme categories. Using a cut off of 0.8 resulted in a specificity of 100% and a positive
predictive value of 100%, but this applied to less than 5% of patients. At the other end of
the spectrum, a cutoff of less than 0.30 was associated with a sensitivity of more than
88% and a negative predictive value of more than 94%. Overall 42% of patients could be
correctly classified at either extreme, but 58% would not be classifiable with scores
between 0.3 and 0.8. The SHASTA index in HIV/HCV has similar accuracy to the
Fibrotest and in this study performed significantly better than the APRI test.
5.24 APRICOT Clinical Investigators Assay

A retrospective analysis of liver biopsies was performed in 832 patients with
HIV/HCV co-infection, who were randomly assigned to training (n = 555), and validation
(n = 277) sets.
The authors derived a simple index (FIB-4) 141:
Age ([yr] x AST [U/L]) / ((Plt [109/L]) x (ALT [U/L]) (1/2))
The AUROC of the index was 0.765 for differentiation between Ishak stage 0-3
and 4-6. At a cut off of < 1.45 in the validation set, the negative predictive value to
exclude advanced fibrosis (stage 4-6) was 90% with a sensitivity of 70%. A cut off of
307
>3.25 had a positive predictive value of 65% and a specificity of 97%. Using these
cutoffs, 87% of the 198 patients with FIB-4 values outside 1.45-3.25 would be correctly
classified, and liver biopsy could be avoided in 71% of the validation group.
13
5.25 C- Caffeine Breath Test
142
According to Park et al , the properties of caffeine render it an ideal substrate
for a quantitative test of liver function. It has high oral bioavailability, and undergoes
almost exclusive hepatic metabolism, principally via demethylation by cytochrome P450
1A2 (CYP1A2) to CO2143. It has a low extraction ratio and low plasma binding 144, and at
the test doses used may be considered innocuous. These characteristics render caffeine an
ideal substrate for a breath test of hepatic function 142.
The authors studied that whether the caffeine breath test (CBT) using orally
13
administered C-caffeine correlates reliably with plasma caffeine clearance and reflects
varying degrees of liver dysfunction. The CBT was performed in 25 healthy controls; 20
subjects with non-cirrhotic, chronic hepatitis B or C; and 20 subjects with cirrhosis.
Cirrhotic patients were characterized by significantly reduced CBT values (1.15 +
0.75 Δ o/oo mg-1) compared with controls (2.23 + 0.76; p = 0.01), and hepatic patients
(1.83 + 1.05; p = 0.04). There was a significant inverse relationship between the CBT and
Child-Pugh score (r = -0.74, p = 0.002). The intraclass correlation between repeated
CBT’s in 20 subjects with normal and cirrhotic livers was 0.89. Multivariate analysis
revealed that only smoking (p< 0.001) and disease state (p = 0.001) were significant
predictors of CBT.
The authors concluded that the results from their study support the application of
CBT as a test of quantitative liver assessment. 1) The elimination of caffeine is impaired
in cirrhosis, characterized by a reduction in plasma clearance and a prolongation in
13
caffeine half-life; 2) the oral C-CBT using a single 1-hour measure correlates very
closely with plasma caffeine clearance; 3) cirrhotic subjects show significantly reduced
CBT results compared with control and hepatitis subjects; 4) the CBT values are
increased in smokers but still distinguish smokers of varying hepatic functional
impairment; 5) the CBT is administered easily and safely in subjects with liver disease; 6)
the CBT appears to be a reproducible assay142.
308
The CBT exhibited significant correlations with serum albumin and platelet count
and correlated modestly with INR, bilirubin, γ glutamyl transferase, and AST/ALT ratio,
markers traditionally associated with hepatic dysfunction. The CBT was associated
inversely with the Child-Pugh score and differentiated the grades of cirrhosis. In addition,
it was able to predict the cirrhotic state in two patients with biopsy proven cirrhosis who
had completely normal physical and laboratory findings but significantly reduced CBT
values. It thus may be viewed as a complementary test in the overall assessment of liver
status, to be taken in context with laboratory, radiologic, and histologic data where
available 142.
5.26 Fibrosis Markers to Assess Effect of Treatment

According to Afdhal N 19, an attractive use of fibrosis markers would be to assess
the effects of treatment, and if they can be validated, they could be valuable tools in the
search of new antifibrotic treatments. However, none of the currently available markers
have yet been validated for use in this way, but several studies have shown that levels of
these markers are altered by treatment and they have prognostic value.
Poynard and colleagues reported on two studies describing the relationship
between response to interferon therapy and the results of Fibrotest performed before and
after therapy 78, 82.
82
In the larger of these studies (n = 352) , the authors studied the effect of
interferon plus ribavirin on both the Fibrotest and Actitest scores. Actitest is a
modification of Fibrotest that incorporates ALT and reflects both liver fibrosis and
necroinflammatory activity. All patients had two interpretable liver biopsies and stored
serum sample before and after treatment. Of the patients, 208 patients received
peginterferon alfa-2b, 1.5 mcg per kg and ribavirin. The remaining 144 patients received
interferon alfa-2b, 3 MU and ribavirin. All patients received treatment for 48 weeks 82.
At baseline, Fibrotest performed fairly well for the diagnosis of significant hepatic
fibrosis, defined as METAVIR score of F2-4. The area under the receiver operating curve
was 0.733 + 0.03, but was lower than that which had been reported in the original study
by this group (0.827) 54. It increased to 0.766 + 0.03 at the end of follow up. For bridging
309
fibrosis and/or moderate necroinflammatory activity, the area under the ROC was 0.76 +
0.03 at baseline and 0.82 + 0.02 at the end of follow up. The index had 90% sensitivity
and 88% for the diagnosis of bridging fibrosis and moderate necroinflammatory activity
82
.
Actitest showed slightly greater diagnostic value for the identification of
individuals with more advanced fibrosis and with greater histological activity as
determined by Knodell score, and may thus be of value in guiding treatment decisions. Of
interest, the performance of Actitest improved with longer liver biopsies, those greater
than 15 mm or which contained six or more portal tracts, suggesting that these assays
truly reflect the more global liver histology 82.
On follow up, patients who had a sustained virological response to interferon had
a substantial reduction in Fibrotest and Acitest scores, as compared to those who were
either primary nonresponders or relapsed following therapy. There was a significant
decrease of Fibrotest among the 184 sustained virological responders, from (0.39 + 0.02
to 0.28 + 0.02) at 72 weeks; in comparison with 126 nonresponders from (0.59 + 0.03 to
0.55 + 0.02) at 72 weeks, p<0.001; and with 42 relapsers, from (0.49 + 0.04 to 0.45 +
0.02) at 72 weeks, p<0.001 82.
There was also a significant decrease of Actitest among the 184 sustained
virological responders, from (0.55 + 0.02 to 0.08 + 0.020) at 72 weeks, in comparison
with 126 nonresponders from 0.58 + 0.02 to 0.50 + 0.03 at 72 weeks, and in comparison
with 42 relapsers, from (0.58 + 0.03 to 0.43 + 0.03) at 72 weeks 82.
Furthermore, there was a significant concordance between Fibrotest and fibrosis
stage variations. At baseline 32 patients had cirrhosis. Fibrotest scores fell substantially in
17 patients with cirrhosis at baseline and a post treatment reduction of 1 or more stages;
from 0.68 to 0.44 for 3 stages improvement, from 0.60 to 0.47 for 2 stages improvement
and from 0.61 to 0.56 for 1 stage improvement. For 15 patients, the fibrosis stage
remained F4 and Fibrotest did not significantly change (0.67 + 0.05 vs 0.64 + 0.05).
These data support the concept that these assays may be useful not only in the
initial staging of the liver disease, but may also be of value in following the histological
response to therapy 82.
310
19
According to Afdhal N , many other studies have evaluated the effect of
interferon therapy on levels of fibrosis markers and have compared them to histological
93,105,114,145-153
findings . Most of these studies have shown that serum levels of several of
these markers including HA, PIIINP, YKL-40, and TIMP-1 fall in patients who achieve a
sustained virological and biochemical response. In these patients, levels continue to fall
following treatment and often return to normal levels. In patients who relapse following
treatment, the levels frequently fall during therapy but most often return to pretreatment
145
levels with virological and biochemical relapse . Where follow up biopsies have been
performed, levels of HA and PIIINP have often been found to correlate better with
improvement in fibrosis stage, than with biochemical or histological markers of
94,105,151,152
inflammation . Furthermore, levels of these markers often become normal
despite evidence of fibrosis on liver biopsy, suggesting that these markers do reflect the
underlying fibrogenic activity within the liver.
According to Afdhal N 19, treatment with interferon has been associated with a
fall in serum markers of fibrosis, independent of a biochemical or virological response
145,148,149,153
, while other studies have not confirmed these findings 93. And this reduction
in the levels of these markers tends to be less than which is observed in patients who
achieve a biochemical and virological response. This has been used as evidence that
interferon has a beneficial direct antifibrotic effect, presumably through direct inhibition
of TGF-β expression 154,155.
Serum TGF-β levels and tissue expression has been shown to decline in response
to successful therapy of hepatitis C and following treatment of autoimmune hepatitis
156,157,158
. Based on the available data it is not possible to discern whether interferon has a
clinically important antifibrotic effect and hence ongoing long-term low dose interferon
studies and studies of interferon –γ and interferon with more potent antifibrotic activity
and less antiviral effect are awaited with interest 19.
311
5.27 Fibrosis Markers to Predict Disease
Progression
19
According to Afdhal N , if noninvasive markers of fibrosis truly reflect
fibrogenic activity and the rate of underlying disease progression then they should have
prognostic value, both for predicting progression of fibrosis, and clinical outcomes. In
this regard, the available data are more limited. However, several studies looked at the
prognostic value of these fibrosis markers in patients with more advanced liver disease
101,102,159
.
Guechot evaluated the predictive value of HA in a cohort of 91 patients with
hepatitis C associated cirrhosis followed for a median of 38 months 159. During this time,
severe complications including death, liver transplantation, and severe complications of
cirrhosis occurred in 24 patients. Of all the laboratory tests evaluated, HA was found to
have the greatest predictive value and was equivalent to Child-Pugh score. Similarly, in a
cohort of 97 patients with primary biliary cirrhosis, Poupon demonstrated that by
multivariate analysis HA, PIIINP, bilirubin, and prothrombin time were independently
101
predictive of disease progression . Interestingly, in the subgroup of 49 patients who
were treated with ursodeoxycholic acid therapy, only HA was predictive of a poor
outcome.
Nojgaard reported on the ability of PIIINP and YKL-40 to predict survival in a
115
cohort of 370 patients with alcoholic liver disease . In this study, elevated levels of
PIIINP and YKL-40 were predictive of shorter survival and carried an increased relative
risk of dying of 3.32 (95% CI 1.05-10.5), and 4.24 (95% CI 2.18-8.26), respectively. As
prognostic assays, it is not possible to discern whether these markers simply reflect more
advanced liver disease or whether they identify individuals whose fibrotic disease is
progressing more rapidly and hence have a worse prognosis 19,115.
19
According to Afdhal N , serum TGF-β levels have also been used to assess
160,161
disease progression . In a 12 month study of 39 patients with hepatitis C infection,
who underwent paired liver biopsies, Kanzler showed that those patients who had
progressive liver fibrosis had higher levels of serum and tissue expression of TGF-β than
312
160
those who did not progress . No such correlation could be found for PIIINP, serum
transaminases, or viral load.
161
Neuman and colleagues , using a slightly different study design, obtained
similar results. They assessed two patient cohorts, a group of 56 patients with minimal or
no fibrosis and no inflammation and a second group of 103 patients with no fibrosis but
mild histological disease activity. When followed over 3 years, those patients who
showed progressive hepatic fibrosis had a parallel increase in TGF-β levels. Hence, TGF-
β levels were found to identify the subgroup of patients with mild liver disease that
subsequently progressed 19,161.
Similarly, prognostic value of the Fibrotest was compared with biopsy staging for
predicting cirrhosis decompensation and survival in patients with chronic HCV infection.
The investigators concluded that the Fibrotest measurement of HCV biomarkers has a 5-
year prognostic value similar to that of liver biopsy 26.
According to the authors, Fibrotest was a better predictor than biopsy staging for
HCV complications, with the area under the ROC, 0.96 vs 0.91, respectively; it was also
a better predictor for HCV related deaths, AUROC 0.96 vs 0.87 respectively. The
prognostic value of Fibrotest was also significant in multivariate analyses after taking
into account histology, treatment, alcohol consumption, and HIV coinfection 26.
5.28 Gene Markers and Liver Fibrosis

The hepatic fibrogenesis is a complex process requiring an intricate balance
between extracellular matrix (ECM) deposition and removal. Indeed, the ECM
metabolism is a very dynamic process, influenced by factors/cytokines/molecules that
contribute to its deposition and by others that mediate its degradation 24.
influence progression of liver fibrosis. The genetic polymorphisms linked to hepatic
fibrogenesis have been investigated mainly in chronic hepatitis C and in alcoholic fatty
313
liver disease. There are many studies that report gene polymorphisms to either favor or
reduce fibrogenesis in patients with different forms of chronic liver disease 24.
While these studies clearly indicate that many genetic factors have a definitive
influence on the risk of developing a more or less active and progressive fibrogenesis,
very few of them have found an application as a diagnostic/prognostic marker in clinical
practice, due to their complexity, difficulty to test and variable behavior in different
patient populations 24.
One such study, consists of a set of seven marker genes, the cirrhosis risk score
(CRS), was found to be a better predictor of high risk versus low risk for cirrhosis in
162
Caucasian patients than clinical factors . The clinical factors such as age, gender,
163-165
alcohol use, and age at infection, obesity and hepatic steatosis, influence the
progression to cirrhosis, but cannot accurately predict the risk of developing cirrhosis in
patients with chronic hepatitis C 16.
The authors hypothesized that host genetic factors, such as single nucleotide
polymorphisms (SNP’s) could play a primary role in determining fibrosis risk. The
preliminary results from their previous study suggested that replicated SNP’s identified
by large association studies had several advantages over clinical risk factors, such as
higher odds ratios, consistent percentages of risk population across different study
166
cohorts, and objective genotyping calls that are available for all patients . The key
question remained as to how these markers could be utilized in clinical settings.
The aim of the study was to develop and validate a predictive signature (of genes)
alone or in combination with standard clinical factors to assess the risk of cirrhosis in
Caucasian patients with chronic hepatitis C virus infection 162.
In the training set (n = 420), all patients had well characterized liver histology
and clinical factors. DNA was extracted from the whole blood for genotyping. The
authors validated all significant markers from a genome scan in the training cohort, and
selected 361 markers for the signature building. Subsequently, a signature consisting of 7
markers most predictive for cirrhosis risk in Caucasian patients was developed. The
Cirrhosis Risk Score (CRS) was calculated to estimate the risk of developing cirrhosis for
each patient. The CRS performance was then tested in an independently enrolled
validation cohort of 154 Caucasian patients.
314
increased to 0.73 for CRS, and 0.76 when CRS and clinical factors were combined. A
low CRS cutoff of < 0.50 to identify low risk patients would misclassify only 10.3% of
high risk patients, while a high cut off of > 0.70 to identify high risk patients would
misclassify 22.3% of low risk patients. Thus more importantly fewer of the high risk
patients would be misclassified. In conclusion, CRS is a better predictor than clinical
factors in differentiating high risk versus low risk for cirrhosis in Caucasian patients 162.
According to the authors, for the first time, one can estimate the risk of cirrhosis
rather than using findings of liver biopsy to project the future course of disease. Liver
biopsy represents only one time point in the long natural history of hepatitis C, whereas
the genetic markers are intrinsic and life long. Potentially the CRS could be used to
stratify patients’ cirrhosis risk prior to liver biopsy 162.
Unlike rare Mendelian disorders, and similar to other complex human diseases,
liver cirrhosis is caused by the interactions among multiple genetic and environmental
factors. Yang et al, estimated that for genes with very common genotype frequencies
(>30%) and moderate OR’s (1.2-1.5), 10-15 markers are needed to achieve appreciable
167
population attributable fraction (PAF) for disease occurrence . Consistent with this
finding, the CRS signature is comprised of seven markers with high frequencies (18.5%-
87.3%) and significant OR’s (1.86-3.23). In contrast, only two clinical factors, sex and
age, had significant associations with cirrhosis risk. Moreover, the CRS measurements
are objective 162.
In the CRS, each of the 7 most predictive markers only provide moderate
predictability; whereas the combination of these 7 was more robust and predictive. This
finding is consistent with multiple biological pathways known to be involved in hepatic
fibrogenesis 168.
Of the 7 genes, antizyme-Inhibitor-1 (AZIN1) and Toll-like receptor 4 (TLR4)
have an identified role in hepatic fibrosis. AZIN1 binds to ornithine decarboxylase
(ODC) antizyme and stabilizes ODC, thus inhibiting antizyme-mediated ODC
degradation 169. Regarding TLR4, it is expressed in all hepatic cell types in response to its
170
ligand lipopolysaccharide (LPS) . In patients with chronic hepatitis C, NS5A induces
315
171
the elevated expression of TLR4 in B cells 3- to 7- fold . Moreover, LPS-TLR4
172
pathway plays an important role in the hepatic fibrogenesis . The authors were in the
process of determining the functional mechanisms of the other 5 genes in fibrognesis, and
the applicability of the CRS in other liver diseases162.
Nevertheless, improved understanding of the genetic influence on liver
fibrogenesis is of paramount importance as it may lead in the near future to identification
of patients for high risk for fibrosis/cirrhosis, diagnosis of liver fibrosis, and new
therapeutic targets and strategies for development of effective antifibrotic treatments 24.
5.29 Radiology in the Noninvasive Assessment of

Liver Fibrosis
19
According to Afdhal N , radiological evaluation of liver to assess fibrosis has
been limited to the identification of individuals with cirrhosis and its complications. The
advent of cross sectional imaging with CT, MRI, and ultrasound enables detailed images
of the liver and surrounding structures to be made. Resolution of the hepatic parenchyma
with any of the available modalities, however, is insufficient to determine any of the
earlier stages of fibrosis before the establishment of cirrhosis and portal hypertension.
Established cirrhosis with portal hypertension can be determined with a high
specificity by identifying splenomegaly, an enlarged caudate lobe, or the presence of
39
large varices . Studies using CT, ultrasound, and MRI, have identified reduction in the
size of the right lobe of the liver with relative enlargement of the left and caudate lobes as
reliable markers of cirrhosis 40,173,174.
Harbin’s original description of this technique measured the ratio of transverse
width of the caudate lobe, to the transverse width of the right lobe of the liver. Using this
technique, cirrhosis could be correctly diagnosed with sensitivity 84%, a specificity of
40
100%, and diagnostic accuracy of 94% . These studies report a high specificity but
limited sensitivity because these morphological changes are only present in more
advanced disease 19.
Further studies using up to 11 sonographic and Doppler parameters, including:
measurements of liver morphology, assessment of portal venous blood flow, spleen size,
316
and liver surface nodularity; reported accuracy of cirrhosis detection between 82% and
175,176
88% , however anatomical limitations and interobserver variability remained
limiting factors 39. In addition, it should also be noted that a large number of patients had
decompensated liver disease, and the diagnosis of cirrhosis could have been made using
simple clinical criteria, including palpation of a nodular liver, or the presence of a hard
and enlarged left lobe 175.
When Oberti compared the ability of ultrasound to diagnose cirrhosis to clinical
examination and biochemical studies, he found ultrasonography to be of less diagnostic
value 70.
It should be noted that ultrasound also identified cirrhosis in a substantial number
of patients in which a definite diagnosis of cirrhosis was not made on liver biopsy, in the
175
study cited above . It can therefore, be argued that ultrasound examination has
substantial complementary value, i.e., a properly performed examination can identify
patients with cirrhosis where the biopsy findings are equivocal, or at variance with
19
clinical impression . Furthermore, progression in the ultrasonographic changes,
including an enlarging spleen has been associated with an increased risk of
complications177.
5.30 Measurement of Hepatic Stiffness

(Elastography)
Accepting the limitations of standard radiological techniques to accurately
determine fibrosis, transient elastography is an emerging technology that is more
sensitive for staging hepatic fibrosis. This technique rapidly and noninvasively measures
mean hepatic tissue stiffness 178,179.
Hepatic stiffness is related to the degree of fibrosis; palpation of a firm liver edge
has been used for centuries as a marker of hepatic injury. Using a probe (Fibroscan,
Echosens, Paris) that includes an ultrasonic transducer, a vibration of low frequency (50
MHz) and amplitude is transmitted into the liver. The vibration wave induces an elastic
shear wave that propagates through the organ. The velocity of this wave as it passes
through the liver correlates directly with tissue stiffness and by simultaneously using a
317
pulse-echo ultrasound by means of the same probe, the velocity of the wave is measured.
The harder or stiffer the tissue, the faster the shear wave propagates. Results are
expressed in kilopascals (kPa). Fibroscan measures liver stiffness of a volume that is
approximately a cylinder of 1 cm diameter, and 2 cm long, 100 times greater in size than
a standard liver biopsy. Thus it is more representative of the entire hepatic parenchyma
39,41
.
19
According to Afdhal N , initial studies of hepatic elastography in 19
180
hepatectomy specimens correlated with histologic analysis . In vivo study of a cohort
of 15 patients with hepatitis C virus (HCV) showed excellent intra- and inter-operator
reproducibility.
Furthermore, in 91 patients who had HCV reported by the same authors, the
receiver operating characteristic (ROC) curve, which estimates the diagnostic
performance of elastography, were 0.90, 0.88, 0.91, and 0.99, respectively, for the hepatic
fibrosis grade superior and equal to F1, F2, F3, and F4, respectively. Of the studied
patients with a score of less than 5.1 kPa, 93% were METAVIR F0 or F1, whereas for
those with a score of 7.6 kPa or higher, 94% were F2 or more 178.
In a large multicenter study 41, the authors enrolled 327 patients. The aim of the
study was to investigate the use of liver stiffness measurement in the evaluation of liver
fibrosis in patients with chronic hepatitis C. Patients underwent both liver biopsy and
liver stiffness measurement. METAVIR liver fibrosis stages were assessed on biopsy
specimens.
The areas under the receiver operating characteristic curve (ROC); for F > 2 was
0.79 (95% CI, 0.73-0.84), for F > 3 was 0.91 (0.87-0.96), and for F > 4 was 0.97 (0.93-
1.0). For larger biopsy specimens (longer than the median value in each category), these
values were, 0.81, 0.95, and 0.99 respectively. Optimal cut of values of 8.7 and 14.5 kPa
showed F > 2, and F = 4, respectively 41.
The authors concluded that they found significant positive correlation between
liver stiffness measurement and fibrosis stages in patients with chronic hepatitis C.
Although there was overlap in scores for those with early stages of fibrosis; yet there was
significant areas under the ROC curves for F > 3, and F = 4 fibrosis, and with high total
sensitivity, specificity, and high likelihood ratios suggest that liver elastometry is a
318
reliable method for the diagnosis of extensive fibrosis (F > 3), and cirrhosis (F = 4). In
the study 30% of patients were F > 3, or F = 4. However, in the community practice,
where the proportion of patients with F4 may be lower than in referral centers, liver
stiffness measurement accuracy in predicting patients with F2 or more METAVIR
fibrosis stage might be lower 41.
The study also confirmed that in chronic hepatitis C patients, the correlation
between liver stiffness and fibrosis stage is not affected by steatosis or activity grade.
Indeed, activity was not expected to modify the liver stiffness, whereas steatosis could
have been expected to soften the liver because it consists of fat deposits in the liver
parenchyma. But in the study the multivariate analysis showed that the potential effect of
steatosis on liver stiffness was hidden by the strong effect of fibrosis. These findings
support a study on elastic modulus measurements of ex vivo human liver samples that
reported a correlation between liver stiffness and fibrosis but did not show any obvious
correlation between steatosis and elastic modulus 180.
Results show that the diagnostic performances of liver stiffness measurement
were better in the larger specimens than in the smaller specimens. This suggests that the
real diagnostic performance of liver elastometry may be underestimated because of the
sampling error of the biopsy 41.
According to the authors41, the diagnostic performance of liver elastometry
appears to be equivalent to that of the best biochemical scores for patients with
significant fibrosis (F > 2), and is better than these tests for the diagnosis of extensive
fibrosis (F > 3) and cirrhosis (F = 4), (there are studies which report better performance
of serum markers in earlier stages). The main advantage of liver elastometry over the
fibrosis markers is that it measures a quantitative physical parameter directly on the liver
and there is no interference from extrahepatic disorders. It, therefore, is complementary
to the fibrosis markers to better assess liver fibrosis without resorting to liver biopsy 41.
There are certain limitations to the procedure. Elastometry cannot be applied to
patients with ascites, even if clinically undetected. Ascites is a physical limitation to the
technique because elastic waves do not propagate through the liquids. In addition,
elastometry is unsuccessful in patients with narrow intercostals spaces and in patients
with morbid obesity. In obese patients the fatty thoracic belt attenuates both elastic waves
319
and ultrasound, rendering liver stiffness measurement more difficult or even impossible.
Probes with smaller size and elongated shaped transducer tips are currently available for
these patients 41.
In another study, a total of 711 patients with chronic liver disease were evaluated.
In cirrhotic patients, liver stiffness measurements range from 12.5 to 75.5 kPa. However,
the clinical relevance of these values is unknown.
The aim of the prospective study was to evaluate the accuracy of liver stiffness
181
measurement for the detection of cirrhosis in patients with chronic liver disease .
Etiologies of chronic liver diseases were hepatitis C virus or hepatitis B virus infection,
alcohol, non-alcoholic steatohepatitis, other, or a combination of the above etiologies.
Liver fibrosis was evaluated according to the METAVIR score.
Areas under the receiver operating characteristic curve were 0.80 for patients with
significant fibrosis (F>2), 0.90 for patients with severe fibrosis (F3), and 0.96 for patients
with cirrhosis. Using a cut off value of 17.6 kPa, patients with cirrhosis were detected
with a positive predictive value and a negative predictive value (NPV) of 90%. Liver
stiffness was significantly correlated with complications of liver disease. With an NPV
>90%, the cut off values for the presence of esophageal varices stage 2/3, cirrhosis Child-
Pugh B or C, past history of ascites, hepatocellular carcinoma, and esophageal bleeding
were 27.5, 37.5, 49.1, 53.7, and 62.7 kPa, respectively181.
The authors concluding that transient elastography is a promising non-invasive
method for detection of cirrhosis in patients with chronic liver disease. Its use for the
follow up and management of these patients could be of great interest and should be
evaluated further.
24
The limitation is that it requires a costly device to measure liver stiffness .
Similarly, some of the direct markers of noninvasive evaluation of fibrosis are only
available in highly specialized research laboratories and are thus not routinely available.
Thus in conclusion, this simple noninvasive technique has proved beneficial in
detecting patients with advanced fibrosis or cirrhosis and more generally in assessing
fibrosis in patients with chronic hepatitis C.
Studies underway will evaluate the applicability of hepatic elastography across a
range of liver diseases. Further research also will determine its use for assessing hepatic
320
fibrosis over time and its responsiveness to detecting changes in fibrosis associated with
specific therapies, in particular the response to antiviral therapies for chronic viral
hepatitis. For now, the preliminary results and its ease of use predict a promising future in
the reliable staging of fibrosis 39.
321
5.31 Platelets in Chronic Liver Disease – The role of
Spleen
Thrombocytopenia is a commonly encountered hematological condition in

182
chronic liver disease and cirrhosis . It is a risk factor for gastrointestinal bleeding and
other life threatening hemorrhagic events 182.
Historically it has been attributed to the sequestration and destruction of platelets
in the enlarged spleen with an impaired ability of bone marrow to compensate by
85,183,184
increasing platelet production . Hypersplenism occurs in a varying percentage of
patients with advanced liver disease and is a common complication of portal
hypertension. Radiolabeled platelet studies have shown that human spleen contains a
sizeable fraction (about one third) of the total body platelet mass in the form of
85
exchangeable pool . The spleen can increase its pool by 30-90% when pathologically
enlarged. This redistribution of cells from the peripheral circulation to the spleen appears
sufficient to produce thrombocytopenia despite the normal platelet life span, normal total
body mass and unimpaired platelet production, as might be expected form the number of
megakaryocytes in the marrow 85,87.
However attempts to correct the low platelet levels by splenectomy and portal
decompression procedures have failed to consistently improve the platelet count in the
long term 85,185.
5.32 Platelet Autoantibodies and Thrombocytopenia
It has been postulated that auto-antibody mediated platelet destruction, as seen in

patients with idiopathic thrombocytopenia purpura (ITP) may also contribute to cirrhotic
thrombocytopenia186. In this situation, the thrombocytopenia is principally mediated by
enhanced platelet clearance in the periphery, resulting in accelerated platelet turnover 182.
Autoimmune mechanism mediated by platelet associated Ig may play an important role in
thrombocytopenia associated with viral hepatitis187.
322
In another study, antiplatelet autoantibodies were determined in blood serum with
the use of ELISA method in 15 patients with cirrhosis and thrombocytopenia (mean
platelet count 67.9 +/- 24.9 x 103/µl). Three patients (20%) presented with anti-GPIIb/IIIa
antibodies and 2 patients with anti-GPIa/IIa. These patients had liver failure (stage C
according to Child-Pugh classification) and splenomegaly. Platelet morphological
parameters were also evaluated. The significant decrease of plateletcrit as well as the
decrease of mean platelet volume (MPV) was observed in liver cirrhosis with
thrombocytopenia. The increase of megathrombocyte population (MPV > 20fl) up to
5.5% of all platelets was also observed. Megathrombocytes in healthy individuals were
2.25% of platelet population. Examinations confirmed that autoimmunological factors
play an important role in the development of thrombocytopenia in liver cirrhosis188.
The authors postulating that immunological disorders in patients with liver
cirrhosis, loss of tolerance to own antigens, and the change of platelet antigenicity enable
antiplatelet antibody formation under the influence of continuous activation188.
Another study has shown that serum thrombopoietin levels may not be directly
associated with thrombocytopenia in patients with chronic hepatitis and liver cirrhosis. In
contrast spleen volume and platelet associated immunoglobulin (PAIgG) are associated
with thrombocytopenia in such patients, suggesting that hypersplenism and immune
mediated processes are predominant thrombocytopenic mechanisms189.
5.33 Thrombopoietin and Chronic Liver disease
Impaired platelet production due to thrombopoiten (TPO) deficiency has also

been proposed as another cause of thrombocytopenia in cirrhosis patients.
Thrombopoietin, a principal regulator of megakaryopoiesis is predominantly produced by
190,191 182
the liver . Its levels are insufficient in advanced liver failure . This is further
supported by the observation that reduced circulating level of TPO in cirrhosis patients is
restored in conjunction with an increase in platelet count after orthotopic liver
transplantation 192.
323
Adinolfi et al, has shown that in patients without splenomegaly, the
thrombocytopenia was associated with the stage of fibrosis; platelets counts were the
highest in patients with fibrosis stage 0-2 (Knodell HAI), lower in those with stage 3 (p<
0.008) and lowest in those with stage 4 (p< 0.05). These findings were independent of
demographic, biochemical, hepatic necroinflammatory activity, portal hypertension and
splenomegaly. Patients with normal platelet counts showed higher thrombopoietin levels
than those with low platelet counts (p< 0.0001). An inverse correlation between
thrombopoietin levels and fibrosis grade was observed (r= -0.50; p, 0.0001). The authors
postulated that advanced hepatic fibrosis causing an altered production of thrombopoietin
and portal hypertension, plays a central role in the pathogenesis of thrombocytopenia in
chronic viral hepatitis 87.
Evaluating the association between the degree of fibrosis and the platelet count,
another study included seven hundred eighty-four patients (265 chronic viral hepatitis C
and 519 chronic viral hepatitis B). In an effort to avoid the effects of hypersplenism,
patients with splenomegaly and/or bi- or pancytopenia were excluded. In multivariate
analysis, the peripheral platelet count had a negative correlation with the fibrosis score
and age, but not with necroinflammatory activity, in both groups. The authors concluding
that, a decrease in peripheral platelet count may be a sign of an increase in the degree of
fibrosis during the course of chronic viral hepatitis B and C and factors other than
hypersplenism may play a role in this decrease in the peripheral platelet count 193.
However, several other studies have found that the circulating TPO level is
86,194
maintained or even increased in patients with cirrhosis including the study by
182
Kajihara et al .
In their study when compared with healthy controls, cirrhosis patients presented
with, (i) normal or slightly increased plasma TPO, (ii) accelerated platelet turnover based
on elevated %RP (reticulated platelets) and GCI (glycocalicin index), and (iii) reduced
platelet production based on decreased absolute RP count and plasma GC 182.
Reticulated platelets (RP) are young platelets that contain higher levels of nucleic
acid components than mature platelets. The absolute RP count is a reliable indicator of
the thrombopoiesis rate, analogous to the reticulocyte count to evaluate erythropoiesis.
Glycocalicin is a proteolytic fragment of the α-chain of glycoprotein (GP) Ib, which is
324
cleaved from the surface of megakaryocytes and platelets. The plasma glycocalicin
concentration is decreased in patients with aplastic anemia and greatly increased in
patients with essential thrombocythemia, indicating that it is a marker of platelet
production195.
In contrast, the proportion of reticulated platelets in total platelets (%RP) and the
plasma glycocalicin level normalized to the individual platelet count (GC Index; GCI)
have been shown to reflect platelet turnover196.
These markers in ITP and cirrhosis patients were comparable, but significantly
different from those in aplastic anemia patients. The bone marrow megakaryocyte density
in cirrhosis and ITP patients was similar, and significantly higher than in aplastic anemia
patients182.
The authors concluded that cirrhotic thrombocytopenia is a multifactorial
condition involving accelerated platelet turnover and moderately impaired
thrombopoiesis. Thrombopoietin deficiency is unlikely to be the primary contributor to
cirrhotic thrombocytopenia182.
It has also been shown in one of the studies that in acute liver failure (ALF) the
inverse relationship between platelet count and TPO levels was not observed. Despite
severe hepatic dysfunction, serum TPO levels were initially normal and increased during
hospitalization in acetaminophen-induced ALF, but did not prevent the development of
thrombocytopenia197.
TPO is synthesized primarily in the liver as a single 353-amino acid precursor
protein. Following removal of the 21 amino acid signal peptide, the remaining 332 amino
acids undergo glycosylation to produce a 60-70 kDa protein. Thrombopoietin is produced
at a constant rate by the liver and enters the circulation where most of it is removed by
avid thrombopoietin (c-mpl) receptors on normal platelets. The residual amount of
thrombopoietin (50-150 pg/ml) provides basal stimulation of megakaryocytes and a basal
rate of platelet production198.
It is the circulating platelet mass, not the platelet count, which is regulated by the
body. A practical demonstration of this principle is the effect of changes in the size of the
spleen, (which normally sequesters one third of the platelet mass), on the platelet count.
With increasing splenomegaly, thrombocytopenia becomes progressively more severe,
325
but the total body mass of platelets (circulating + splenic pools) remains constant. How
the body maintains this constant circulating platelet mass has been the subject of
considerable investigation198.
Thrombopoietin is released into the circulation at a constant rate. In the absence
of platelets, there is little clearance of thrombopoietin by platelets, levels rise, bone
marrow megakaryocytes are stimulated and platelet production increases. In contrast, in
the presence of platelets, thrombopoietin clearance increases, levels are low,
megakaryocytes are not stimulated and basal platelet production ensues. Unlike the
mechanism for red blood cells, there is no "sensor" of the platelet mass; instead, as occurs
in the regulation of neutrophils and monocytes where the regulated cells bind and clear
their regulatory cytokine, the circulating platelet mass directly determines the circulating
level of thrombopoietin198,199.
Since it is the total number of circulating platelet c-mpl receptors that determines
the clearance of thrombopoietin, the constancy of normal circulating platelet mass, and
not the platelet count can now be explained. Which is the body defends the total mass of
platelets, and not the platelet count. The platelet count decreases proportionally to the
increase in the size of spleen, but the total mass of platelets remains normal and
unchanged. This could be one explanation for low platelets and not having increased
thrombopoietin levels.
5.34 Platelet Parameters and Chronic Liver Disease
The quantitation of platelets in peripheral blood is a well-recognized tool.

Recently, new indices related to platelet counts have been provided by hematologic
analyzers. Concerning the platelet parameters, the important parameters are mean platelet
200
volume (MPV), platelet distribution width (PDW) . The evaluation of these two
parameters does constitute part of the routine evaluation of complete blood count (CBC)
in modern automated analyzers, but clinical inferences are usually not drawn. However,
there are interesting observations.
326
200
One observer , found a significant correlation between PDW and RDW. And
the anisocytosis of red blood cells and platelets might co-occur. However, these data are
basic observations; further in-depth evaluation of the platelet parameters is
recommended.
The mean platelet volume is the geometric mean of the transformed lognormal
platelet volume data in impedence technology systems. In some, optical systems, (e.g.,
Bayer), MPV is the mode of the measured platelet volume 201.
Under normal circumstances, there is an inverse relationship between platelet size
and number. Therefore, the total platelet mass, the product of MPV and the platelet count
(plateletcrit) is closely regulated. When platelets decrease in number, bone marrow
megakaryocytes are stimulated by thrombopoietin and produce larger platelets. Thus
platelets with a larger volume are expected to be seen in destructive thrombocytopenia
when megakaryocytic stimulation is present. Conversely, platelets with low MPV are
expected to be seen in thrombocytopenic states seen in marrow hypoplasia or aplasia 200.
A very important exception occurs in splenic sequestration, in which a low MPV
is seen, because spleen sequesters large platelets.
A decrease of mean platelet volume (MPV) was observed in liver cirrhosis with
thrombocytopenia 188. This fact has been known from before, for instance in a study from
1984 202, MPV in patients with liver disease (9.25 + 1.14 fl) was significantly lower than
that of controls (10.52 + 0.74 fl, p< 0.001). In control subjects, the MPV and platelet
counts were inversely correlated 202.
It has also been shown that, platelet distribution width is a better indicator of
203
altered platelet homeostasis than MPV in liver cirrhosis . In the study, platelet count,
mean platelet volume, giant platelet percentage, expressed as megathrombocytic index
(MTI), and platelet distribution width (PDW) were evaluated in 32 controls, and in 27
patients with cirrhosis and thrombocytopenia. MTI and PDW were linearly and inversely
correlated to platelet count both in controls and patients. MTI and PDW were markedly
increased in cirrhosis as compared to controls, while MPV was not significantly different.
The authors concluded that: MTI and PDW were good indicators of thrombopoietic
stimulus both in controls and in cirrhosis and are better indicators of altered platelet
homeostasis than MPV in cirrhosis203.
327
These platelet parameters are altered in similar direction in immune
thrombocytopenia, as in cirrhotic thrombocytopenia. Likewise, it has been postulated that
auto-antibody mediated platelet destruction, as seen in patients with idiopathic
thrombocytopenia purpura (ITP) may also contribute to cirrhotic thrombocytopenia 186, as
already discussed.
In a study the authors, investigated the significance of the platelet indices, mean
platelet volume (MPV), platelet size deviation width (PDW), and platelet-large cell ratio
(P-LCR), in the diagnosis of thrombocytopenia by comparing these levels in 40 patients
with hypo-productive thrombocytopenia (aplastic anaemia; AA) and 39 patients with
hyper-destructive thrombocytopenia (immune thrombocytopenia; ITP). The sensitivity
and specificity of platelet indices to make a diagnosis of ITP were also compared.
All platelet indices were significantly higher in ITP than in AA, and platelet
indices showed sufficient sensitivity and specificity. The area under the curve (AUC) of
the receiver operating characteristics curve of platelet indices was large enough to enable
the diagnosis of ITP. P-LCR and PDW had the largest AUCs, which indicated that these
values were very reliable for immune thrombocytopenia. The authors concluded that,
these indices provide clinical information about the underlying conditions of
thrombocytopenia. More attention should be paid to these indices in the diagnosis of
thrombocytopenia 204.
328
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PATIENTS
and
METHODS
PATIENTS
Inclusion criteria
One hundred patients, aged between 20 and 50 years, with chronic liver disease
who were tested positive with HCV RNA PCR and/or HBsAg, and had liver biopsy done
for evaluation purposes were consecutively enrolled from the medical outpatient
department in this cross sectional study. The patients had increased ALT for more than 6
months. Patients whose PCR remained positive after antiviral therapy were also enrolled,
provided they had liver biopsy done within the last 06 months. All patients gave informed
consent to enroll in the study and the study protocol was approved by the Board of
Advanced Studies and Research (BASR) of the Baqai Medical University, which
included the Ethics committee.
Exclusion criteria
Patients with following characteristics were excluded from the study: chronic
liver disease other than HBV and HCV, decompensated cirrhosis (modified Child-Pugh
class C), prior antiviral therapy and PCR negative afterwards. Patients with insufficient
liver biopsy specimen (adequate specimen was taken as containing ≥ 8 portal tracts or if
significant pathology was visible otherwise as assessed by two histopathologists), and
incomplete data on noninvasive markers were also excluded from the final analysis. No
patient had history of alcoholism, HIV or had liver transplantation.
METHODS
Clinical Data
The clinical data recorded were: age, gender, ethnicity, possible mode of
transmission, previous antiviral treatment and result, symptoms of decompensated liver
disease, body mass index (BMI), hepatomegaly, splenomegaly, and stigmata of chronic
liver disease.
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Laboratory Investigations
The lab investigations included: total bilirubin, Aspartate aminotransferase (AST),
Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Gamma glutamyl
transferase (GGT), albumin, total cholesterol, high density lipoprotein (HDL), low
density lipoprotein (LDL), triglycerides, urea, creatinine and blood sugar random. The
analyses for these variables were performed on microlab 200 using commercial reagents.
High control, normal control and low control sera were run before and during the analysis
of each batch. Globulins and Immunoglobulin G levels were measured by protein
electrophoresis. Prothrombin time (PT), international normalized ratio (INR), and partial
thromboplastin time with kaolin (PTTK) were performed using commercial tissue
thromboplastin reagent and results were interpreted visually. Hyaluronic acid and alpha 2
macroglobulin assays were performed on sera stored at - 20o C.
Total leucocyte count, haemoglobin concentration, mean corpuscular volume
(MCV), platelet count, mean platelet volume (MPV), and platelet distribution width
(PDW) were measured on automated haematology analyzer, Sysmex KX-21. Control
cells were run at the start of each day. Osmotic fragility test as an indirect marker of
erythrocyte rigidity, was performed in varying dilutions of isotonic saline and was
interpreted visually. Haemolysis was ascertained by haemoglobin estimation of
supernatant fluid. Percentage of giant platelets was assessed by slide examination of
peripheral blood smear stained with Leishman’s stain.
Hyaluronic Acid
The hyaluronic acid (HA) assay was performed on the 96 well microplate
(Corgenix Inc., CO, USA). It is an enzyme-linked binding protein assay that uses a
capture molecule known as hyaluronic acid binding protein (HABP). Properly diluted
serum or plasma and HA reference solutions were incubated in HABP-coated microwells,
allowing HA present in samples to react with the immobilized binding protein (HABP).
After the removal of unbound serum molecules by washing, HABP conjugated with
horseradish peroxidase (HRP) solution was added to the microwells to form complexes
351
with bound HA. Following another washing step, a chromogenic substrate of
tetramethylbenzidine and hydrogen peroxide was added to develop a colored reaction.
The intensity of the color was measured in optical density (O.D.) units with a
spectrophotometer at 450 nm. HA levels in patients and control samples were determined
against a reference curve prepared from the reagent blank (0 ng/ml), and the HA
reference solutions, both provided with the kit.
Alpha 2 Macroglobulin
Alpha 2 macroglobulin assay was performed on the 96 well polystyrene
microplate (Assaypro, MO, USA). The assay employed a quantitative sandwich enzyme
immunoassay technique which measured alpha 2 macroglobulin in 4 hours. A polyclonal
antibody specific for alpha 2 macroglobulin was pre-coated onto a microplate. Alpha 2
macroglobulin in standards and samples was sandwiched by the immobilized antibody
and a biotinylated polyclonal antibody specific for alpha 2 macroglobulin, which was
recognized by a streptavidin-peroxidase conjugate. All unbound material was then
washed away and a peroxidase enzyme substrate added. The color development stopped
and the intensity of the color measured.
HBsAg
In this assay, samples were added to microplate wells pre-coated with monoclonal
antibodies specific for HBsAg. During the course of the assay, the positive controls,
negative controls were also added. The microtiter plate wells were thoroughly washed to
remove unbound other components of the sample. A standardized preparation of
horseradish peroxidase (HRP) conjugated antibody specific for HBsAg was added to
each well to “sandwich” the antibodies immobilized during the first incubation.
Following a wash to remove any unbound HRP conjugate, a TMB (3, 3’, 5, 5’
tetramethylbenzidine) substrate solution was added to each well. The enzyme (HRP) and
substrate were allowed to react over a 10-minute incubation period. The enzyme-
substrate reaction was terminated by the addition of sulfuric acid solution and the color
change was measured spectrophotemetrically at a wavelength of 450nm ± 2nm. Only
352
those wells containing HBsAg and HRP conjugate exhibited a change in color. The
intensity of this color change was proportional to the concentration of HBsAg in the
sample.
Antibody to Hepatitis B e Antigen

The test was based on Microparticle Enzyme Immunoassay (MEIA) technology.
It utilized the principle of competitive binding between anti-HBe in the sample and anti-
HBe coated on the microparticles and in the Anti-HBe: Alkaline Phosphatase Conjugate
for a standardized amount of Neutralizing Reagent [recombinant DNA derived HBeAg
(rHBeAg)].
HBeAg
The test was also based on Microparticle Enzyme Immunoassay (MEIA)
technology, and utilized the principal of direct binding of the HBeAg in the sample to the
anti-HBe coated on the microplates, followed by the detection of the bound HBeAg by
the anti-HBe: Alkaline Phosphatase Conjugate.
Antibody to Hepatitis C Virus

The recombinant HCV antigen was coated on the multiple wells. The serum
sample and Anti-human IgG labeled with HRP (conjugated) were added to the coated
wells, and a complex of HCVAg-Anti-HCV-Anti-human IgG labeled with HRP was
formed. This enzyme reaction produced a color change, and the intensity of the
absorbance at 450 nm indicated the presence or absence of Anti-HCV in the sample.
RT RNA PCR
Serum separated within 6 hours and frozen at -70o C was used to perform RT
RNA PCR with Amplicor Roche reagent kit. The test utilized reverse transcription of
target RNA to gene rate complementary DNA (cDNA). Amplification of target c DNA
by polymerase chain reaction and nucleic acid hybridization for detection of HCV RNA
in serum was performed.
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HBV DNA PCR
HBV viral genome was determined by using PCR-restriction fragment length
polymorphism of the surface genome of HBV. Minimum of 10 copies of HBV DNA per
specimen by testing serial 10-fold dilutions of HBV transcripts with known amounts
(108copies/ml) was considered positive. The DNA was extracted from sera by using a
Qiagen (Hilden, Germany) viral DNA kit or equivalent. Amplification was performed in
18 µL of Light Cycler DNA Master SYBER Green or equivalent. 1 mix contained 3.5
mM of Mg Cl2 by using 2 µL of c DNA and appropriate primers. PCR was performed in
45 cycles of 1 s at 95o C (denaturation), 3 s at 55o (annealing), and 8 s at 72oC (extension).
Histopathological evaluation
Liver biopsy was performed by using the 18 gauge Surecut liver biopsy needle
(modified Menghini liver aspiration needle) or as otherwise mentioned, using the
subcutaneous intercostal approach. Prior to liver biopsy informed consent was obtained,
and blood CP, PT/INR and PTTK was done. Ultrasound abdomen was carried out to
mark the biopsy site, and to rule out any anatomical abnormality of the gall bladder, or
silent lesion (such as haemangioma) within the liver parenchyma. Similarly chest X-ray
was done to rule out Chiladiti Syndrome (the interposition of bowel between the inner
intercostal wall and liver parenchyma).
All biopsy material slides were stained with haematoxylin and eosin and reticulin
stains. The biopsies were interpreted by qualified histopathologists (B.A. and Y.W.; with
12 and 10 years experience in liver histopathological reporting respectively). The liver
biopsy was scored according to the Knodell HAI classification (mainly), and in few
circumstances by modified Knodell HAI system (Ishak scoring system). The
histopathologists were blinded to the patient’s clinical and laboratory profile. The biopsy
material was considered adequate if it contained > 8 portal tracts or if significant
pathology was visible otherwise.
Fibrosis was scored according to Knodell HAI: stage 0, no fibrosis; stage 1,
fibrous portal expansion; stage 3, bridging fibrosis (portal-portal or portal- central
354
linkage); and stage 4, cirrhosis. In Ishak scoring system, stage 2 was also recognized
corresponding to fibrous expansion of most portal areas with or without short fibrous
septa. Minimal fibrosis was defined as stage 0 or 1, significant fibrosis was defined as
stages 2, 3, and 4. Advanced fibrosis was considered for stages 3, and 4. The
necroinflammatory activity was graded according to the respective scoring system.
Following the recommendations of International Association for the Study of
Liver (IASL) panel, the diagnostic line of pathology report also read the pathologist’s
visual impression in terms of mild/moderate/severe (extensive) activity and fibrosis.
Ultarsonographic parameters
Ultrasound examination of abdomen was done using the Toshiba ECO-CEE
machine, evaluating liver size/echotexture/nodularity, presence or absence of any focal
lesion, portal vein diameter and splenomegaly.
Statistical Analysis
Statistical analysis was done by logistic regression, receiver operating
characteristic (ROC) curves, and by descriptive statistics; sensitivities, specificities,
positive and negative predictive values, and diagnostic accuracy. The main end point was
the identification of clinically significant fibrosis (F2-F3-F4) from insignificant fibrosis
(F0-F1). In the secondary analysis, recognition of moderate to severe necroinflammatory
activity from no to mild activity was also included. In addition, different platelet
parameters were separately studied for their association with advanced fibrosis (F3-F4).
Firstly, the association between different biochemical markers for the presence or
absence of clinically significant fibrosis (CSF; F2-F3-F4), was assessed in univariate
analysis. Continuous variables were compared with the student t-test, while categorical
variables were compared by the Chi-square test. A 2-sided p value of less than 0.05 was
considered statistically significant. The quantitative variables were expressed as mean +
SD, or median in certain circumstances, and as interquartile range in box plots. The
categorical variables were expressed as percentages.
355
In addition, the strength of association of individual biochemical markers with
CSF was also assessed by the ROC curve analysis.
The biochemical markers with strong association in univariate analysis, and high
area under the ROC curve were then subjected to multivariate analysis. The independent
discriminative value of markers for the diagnosis of fibrosis was then assessed by logistic
regression analysis. The best index for discrimination was the logistic regression function
that combined the most discriminatory independent factors. These markers were then
combined with age and sex and entered into a forward step wise logistic regression
analysis to determine a probability index ranging from 0 to 1. The dependent variable
was the presence or absence of CSF.
A central cut off of 0.5 was chosen for the prediction of dependent variable. The
scores presented were those of the study population where β coefficients of score
probability were provided by running multivariate analysis of composite variables. The
biochemical markers with the best discriminatory value were combined in various
logistic regression analyses to develop different models to predict CSF. These models
were then compared amongst each other by the ROC curve analysis. The model,
Fibroscore, with fewer variables and best area under the ROC was then selected. The
regression function was:
y = exp [ - 4.795 + (0.189 x bilirubin) + (0.120 x GGT) + (0.080 x hyaluronic

acid) + (0.518 x alpha 2 macroglobulin) – (0.040 x platelets)]
with bilirubin expressed in µmol/L; GGT in U/L; hyaluronic acid in µg/L; alpha 2
macroglobulin in g/L; and platelets in 109 /L.
The model predictability was presented as area under the ROC in logistic
regression. In addition to the central cut off point of 0.5, various other cut off points were
chosen on either side to predict the presence or absence of different stages of fibrosis.
The diagnostic values of the model were also presented by the sensitivities, specificities,
positive and negative predictive values, and diagnostic accuracy along these cut off
points between 0 and 1.
356
The Fibroscore model was also used to predict moderate to severe
necroinflammatory activity from no to mild activity.
The platelet parameters were also assessed in univariate analysis (student t-test)
for their association with advanced fibrosis (F3-F4). They were then combined into a
simple arithmetic index, PDW Index, to predict significant fibrosis.
PDW Index: ( PDW/√MPV)² x 100

Platelet count
It has shown better correlation with significant fibrosis. The PDW Index
diagnostic accuracy was then compared with the well known APRI, AST to Platelet Ratio
Index, via ROC curve analysis.
The statistical software used was SPSS version 16.0 (SPSS Inc., Chicago, IL,
USA).
357
ANALYSIS
DISCUSSION
385
Introduction
The present work is an original study for the evaluation of noninvasive markers of
liver fibrosis in our country. There has been few local studies describing the role of
1-3
noninvasive markers, but those have been limited in scope . These studies evaluated
only one or two markers. This study is comprehensive, in all 25 markers were studied,
includes direct and indirect markers, and evaluates these markers both individually and
collectively for the noninvasive evaluation of liver fibrosis. In addition some of the
platelet parameters were also assessed and related to the noninvasive evaluation of liver
fibrosis.
Due to the limitations4-8, and risks of biopsy9, as well as the improvements of the
diagnostic accuracy of new noninvasive markers, numerous studies strongly suggest that
liver biopsy should no longer be considered mandatory as a first line mode of evaluation
in some chronic liver diseases10-12.
For example, for the diagnosis of liver fibrosis one of the panels of biomarkers,
the Fibrotest13 has been extensively studied and validated both alone and in combination
with elastography14,15 or other indices in various algorithms. Between 2001 and 2006,
Fibrotest has been assessed in 51 publications; most studies have been performed in
hepatitis C (> 5000 patients), in hepatitis B (> 2000 patients), and also in nonalcoholic
fatty liver disease, NAFLD, (> 1000 patients)10.
The practices have been evolving rapidly and a nation wide survey in
France found that among 546 hepatologists; 81% used a noninvasive marker (Fibrotest-
Actitest), and 32% used elastography with a dramatic decrease in the use of liver biopsy
for more than 50% of patients with chronic hepatitis C16. A recent overview by the
French health authorities officially approved noninvasive biomarkers, Fibrotest, and
elastography (Fibroscan) as first line estimates of fibrosis in patients with chronic
hepatitis C; recommended reimbursement by social security and approved liver biopsy
only as a second line estimate in cases of discordance or non-interpretability of
noninvasive biomarkers10.
386
Fibroscore
In this study 25 markers were assessed for their association with liver fibrosis.
Those markers showing the best association were then entered into stepwise regression
analyses to create a predictive model, the (Fibroscore). It consists of 5 markers:
bilirubin, gamma glutamyl transferase, hyaluronic acid, alpha 2 macroglobulin, and
platelets.
The Fibroscore was quite useful in predicting different stages of fibrosis in all
patients with chronic viral hepatitis. The Fibroscore values (range 0.00-1.00) increased
as the fibrosis stage increased. The maximum score close to 1.00 was for stage 4 (F4,
Cirrhosis).
A central cut off of > 0.5 predicted clinically significant fibrosis, CSF, (F2-F3-F4)
with a sensitivity of 82% and specificity of 92%. It provided a positive predictive value
(PPV) of 79%, and overall diagnostic accuracy of 89%. The same cut off for advanced
fibrosis (F3-F4) increased the sensitivity to 85%.
Increasing the cut off value to 0.65 increased the specificity to more than 95%
(98%) with a PPV of 95%.
The patients with cirrhosis (F4) had values close to 1.00.
Similarly, the Fibroscore value of < 0.5 provided more than 90% negative
predictive value, NPV, for the exclusion of clinically significant fibrosis. The same cut
off value has 95% NPV for the exclusion of advanced fibrosis.
Lowering the cut off to < 0.08 for the exclusion of stages F2-F4 provided 98%
NPV, thus almost certainly ruling out clinically significant fibrosis.
Thus it can be seen that the Fibroscore is very useful in classifying patients in to
various fibrosis categories. This may guide decision to avoid liver biopsy in individual
patients, for treatment decisions in patients with chronic HCV (for e.g., genotype 1), and
chronic HBV (together with DNA and ALT levels), to continue or otherwise treatment in
patients with significant side effects from treatment, for re-treatment of non responder
chronic hepatitis patients. A lower Fibroscore values may provide prognostic information
in patients who are unlikely to develop liver related morbidity and mortality in the
absence of advanced fibrosis. Finally, it may be useful to avoid liver biopsy in patients in
387
whom occult cirrhosis is suspected and to guide decisions about screening these patients
for related complications such as varices.
Clinical application
The Fibroscore results can be applied to clinical practice in three ways, analogous
to similar panels reported previously17:
1. First, it can be applied as a qualitative variable (i.e., indicating the absence [< 0.5]
or presence [> 0.5] of CSF). Although this appears a rather simplistic
interpretation, the underlying statistical principle (binary logistic regression
analysis) between two categorical dependent variables (absence or presence of
CSF) has been designed towards this. The Fibroscore values range from 0.00-
1.00; a central cut off of 0.5 thus classifies patients on either side, presence or
absence of CSF.
2. Second, the probability score can be used as a semiquantitative variable by giving

the correspondence between the score and the five fibrosis stages, which is
interesting from a clinical point of view, as has been provided by the Fibrotest
investigators10.
3. Third, the probability score can be used as it is (i.e., as a quantitative variable,

such as fibrosis score ranging from 0 [lack of fibrosis] to 1 [cirrhosis]). It is more
reliable to use the probability score as a crude measure. Thus for example, a value
of 0.48 means no CSF, whereas the fibrosis stage is probably F1. However this
probability score is close to the probability threshold of 0.5, thus a value of 0.52
(which is close to 0.48) means that CSF and stage F2 are probable. This example
shows that a crude measure (0.48) is a better indicator of the patient’s results than
a qualitative interpretation such as no CSF or F1.
However it must be mentioned that a CSF probability score is a fibrosis meter

with no unit of reference and with a nonlinear relationship to the F stages17.
The noninvasive markers work best at the extremes of fibrosis stages. There are
few misclassified patients. What are factors behind misclassification of patients?
388
Important factors are liver biopsy related, sampling error and interobserver variability in
histopathological interpretation.
Another practical difficulty is that of transforming staging into a binary variable
when staging is evaluated by the noninvasive tools (the presence or absence of CSF).
Whereas staging usually includes five stages from F0 to F4, the cut off is fixed at F2 to
define clinically significant fibrosis, CSF, as F2 or higher17.
But more importantly the misclassification relates to the liver histopathology
itself. There is considerable overlap between fibrosis scores of F1 and F2 stages. This has
been described in the studies before. This may in part reflect the variability in
pathologists’ interpretation, with poor inter-observer agreement in the scoring of these
stages18.
Furthermore, it has been observed by previous studies that plots of blood tests’
results in relation to F stages, all had same V-shape with a nadir at the F2 stage,
suggesting that the difficulty in distinguishing F2 from F1 or F3 stages via histology was
the main cause of misclassification17.
Whichever scoring system is used, the histological staging of liver fibrosis on
biopsy is artificially represented as a quantitative categorical variable with a progression
in severity from 0-4 or 619. The staging system itself may not reflect a linear increase in
fibrosis. In particular, the increase in the degree of fibrosis between F1 (enlarged portal
tract) and F2 (enlarged portal tract with rare septae) may not be as great as the increase
between F2 and F3 (enlarged portal tract with many septae/bridging fibrosis)20. This does
not accurately reflect the dynamic biologic process of fibrosis and constrains the serum
marker test performances that are capable of generating continuous variables19.
Indeed, in the early stage disease, there is poor correlation between degree of liver
21
fibrosis as detected by digital image analysis and staging by a pathologist . Because
serum markers are likely to reflect the quantity of fibrotic matrix/tissue, they may
correlate better with fibrosis as detected by image analysis than fibrosis stages as
determined by the pathologist20. Thus the biochemical markers might represent a more
accurate description of fibrogenic events that occur across the liver13.
The quality of this study conformed to the “quality assessment of diagnostic
accuracy studies”, (QUADAS), tool 22. It is explicit about patient selection and outcomes,
389
and used an accepted reference standard (rather used the “gold standard” [liver
histopathololgy] for comparison). All patients included in the study received verification
using a reference standard (liver biopsy) of diagnosis which was regardless and
independent of the index test (noninvasive markers), i.e., the reference standard did not
form part of the index test.
This study was conducted in a primary care facility, which is in contrast to other
studies carried out in tertiary care centers where relatively sick patients were evaluated
(with over representation of advanced disease). Thus the spectrum of patients would be
the same who will receive the test in practice (i.e., a community based practice); where
the prevalence of the disease in the population being studied is closer to the general
population at large. All of these factors have been cited as the most important criteria that
impact on study quality 19.
Additionally, in the context of this particular study, the pros and cons of reference
standard are described in detail, the composition of panel of serum markers reported and
the formula derived for the panel of serum markers 19.
Importance of Direct and Indirect Markers

Broadly there are two types of noninvasive markers, indirect and direct markers.
Each particular type has its merits. For instance, the indirect markers are easily accessible
13,17,20,23-25
, but on the other hand, the direct markers are known for their putative
specificity 26-29. It has been proved by various studies that the combination of both types
of markers may increase certain advantages and limit other disadvantages 30-32.
The direct markers are more specific but are readily altered by other phenomenon
17
such as connective tissue damage . Indeed, their relationship with fibrosis is complex.
One explanation is that, the blood markers interact both with the dynamics and the cause
33,34
of fibrosis . For example, hyaluronic acid (HA) is a marker of fibrogenesis35, and its
blood level also depends on the persistence of the cause of chronic liver disease (CLD) 36.
Thus the exact meaning of these tests requires further investigation17.
Indeed, some of the previous studies have limited the inclusion of markers to one
of the two categories. Thus according to one source 17, Rosenberg et al 26, stated that the
390
inclusion of Prothrombin index (PI, the prothrombin time is expressed as percentage of
the standard value) and the platelet count didn’t increase the performance of direct
markers17.
Isolated markers have smaller diagnostic value than panels of noninvasive
markers11,27. Individually the markers are useful in the assessment of liver fibrosis, but
collectively they add more information to the diagnostic ability. The combination of
markers slightly increases sensitivity, specificity, negative and positive predictive values
presumably due to isolated variations in biomarkers for diseases other than liver
fibrosis17. Also in statistical analysis, by combining these markers (independent
variables) into a regression model, their collective contribution to the model (correlation
with the dependent variable [fibrosis on liver histopathology]) may be more than their
individual association (correlation) with the dependent variable 37.
Noninvasive Markers in the Study

In this study, age and different markers that have shown association with fibrosis
include: bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST),
gamma glutamyl transferase (GGT), cholesterol, prothrombin time, albumin,
immunoglobulin G (IgG), urea, hyaluronin acid (HA), alpha 2 macroglobulin (A2M),
platelet count, mean platelet volume (MPV), and platelet distribution width (PDW). All
these factors either reflect liver function and/or are fibrosis markers.
In the present study, age has not come up as a major factor influencing fibrosis, as
has been in some of the other studies, presumably due to certain demographic
circumstances, a relatively younger population was studied. Similarly the relative
percentage of patients in different stages of fibrosis is also variable.
Age at infection has been shown to influence the outcome of hepatitis C and
38-40
patients infected after the fourth decade have a higher risk of disease progression . It
is evident that the duration of the hepatitis infection would be a more precise indicator of
fibrosis than age, as also evidenced by our study, in which some of the relatively younger
population has evidence of advanced fibrosis. It has been previously mentioned in the
literature that the precise timing and the mechanism of acquisition of hepatitis infection
391
remains largely unknown in most of the patients, therefore the duration of hepatitis
infection is difficult to establish 23.
As fibrosis progresses the concentration of bilirubin increases as a result of
reduced hepatic excretion and less enterohepatic circulation attributable to portal
systemic shunting 41.
ALT is the most commonly investigated biomarker, but with a low sensitivity of
61-71%. This diagnostic value is lower than the combination of markers in all direct
comparisons. However, it is an invaluable component of the grade of necroinflammatory
activity as evidenced by its inclusion in the Actitest (a modification of the Fibrotest that
incorporates ALT in addition to other 5 parameters) 10.
AST is another commonly investigated marker with many studies either in
isolation or more commonly with other parameters 24. It is thought that the progression of
liver fibrosis may reduce clearance of AST 42, leading to increased serum AST levels. In
addition advanced liver disease may be associated with mitochondrial injury, resulting in
more marked increase of AST, which is present in mitochondria and cytoplasm, relative
24,43
to ALT . In the present study, although AST has shown correlation with fibrosis,
there were other markers which had shown stronger association. It may be that AST
levels significantly increase in advanced liver disease.
GGT has been strongly correlated with liver fibrosis among patients with hepatitis
13,20,23,44
B and C . Early steatosis or an increase in epidermal growth factor could be one
explanation for the GGT increase with the severity of fibrosis 45. In one of the studies it
was shown that in patients with chronic hepatitis C bile duct damage was present in more
than one third of patients with substantial fibrosis (stages 2-4) in comparison with less
than 10% in patients with fibrosis stage 0-1. In this study GGT was an independent
predictor of bile duct damage 46.
Albumin had no independent diagnostic value, most likely because patients with
advanced cirrhosis were excluded13. Urea synthesis is also decreased in patients with
cirrhosis 47.
IgG (γ-globulin) concentration is associated with cirrhosis and portosystemic
48
shunts . Serum immunoglobulins are produced by B lymphocytes and thus
measurement of these substances is not a direct test of liver function. Earlier on it has
392
been believed that, elevation of serum immunoglobulin levels in many patients with
chronic liver disease is believed to indicate impaired function of reticuloendothelial cells
in hepatic sinusoids or shunting of portal venous blood around the liver 49. Data indicate
that antibodies directed against antigens of the normal colonic flora account for much of
49
the increased serum immunoglobulin levels in patients with cirrhosis . In cirrhosis,
these antigens are not taken up and degraded by the hepatic reticuloendothelial cells as
they are normally, and reach the lymphoid tissue outside the liver, where they elicit an
inflammatory response50.
Studies indicate a strong association between serum immunoglobulin levels (IgA,
IgG and total) and hepatic fibrosis in patients with HCV infection51; and between levels
of serum globulin and IgG and extent of hepatic fibrosis in patients with chronic HBV
infection. For each increase of 0.33 mg/dL in serum globulin, there was a 0.5 point
increase in the stage of hepatic fibrosis in chronic HBV infection 52.
It has been postulated that the removal of immunoglobulins by the liver may be
impaired in patients with severe liver dysfunction because the liver is a major catabolic
site for immunoglobulins; possibly by a deficient receptor-mediated mechanism53.
In addition, in recent years several studies have reported that platelet associated
immunoglobulin G levels are elevated in patients with chronic hepatitis and cirrhosis and
have a role in immune mediated thrombocytopenia in these patients 54-56.
The relationship between cholesterol and fibrosis has been investigated before 23.
The decrease in cholesterol levels seen in patients with advanced cirrhosis is caused by a
reduction in cholesterol synthesis, and, therefore it is unlikely that the synthetic capacity
of hepatocytes can be altered before liver cirrhosis is established 23.
HCV infection is associated with clinically significant lower cholesterol levels
(TC, LDL and HDL) when compared with those of normal subjects. This finding is more
pronounced in patients infected with HCV genotype 3a57. In our study HDL, and TG
were significantly decreased in patients with HCV, as compared to HBV, while total
cholesterol and LDL levels had minimal difference.
Hepatocellular steatosis is common in patients with chronic hepatitis C. Steatosis
can be considered as a true cytopathic lesion induced by hepatitis C virus (HCV)
genotype 3. The authors in one study found that HCV core protein-lipid droplet
393
interaction could play a role in virus-induced steatosis. But importantly, they found no
genetic or functional differences between genotype 3a core proteins from patients with
and without HCV-induced steatosis. Thus the investigators concluding that other viral
proteins and/or host factors are involved in the development of hepatocellular steatosis in
patients infected by HCV genotype 3a58. In our study a significant proportion of patients
with HCV had hepatic steatosis as compared to HBV.
Hyaluronic Acid is a high molecular weight glycosaminoglycan, which is an
essential component of extracellular matrix in virtually every tissue in the body 59.
In patients with chronic hepatitis C virus, HA levels increase with the
development of liver fibrosis. Moreover, in patients with cirrhosis, HA levels correlate
60-62
with clinical severity . Liver injury increases hyaluronic acid (a glycosaminoglycan)
production by the hepatic stellate cells and decreases its clearance by the sinusoidal
63
endothelial cells . These cells degrade hyaluronic acid in a specific receptor mediated
process and its elevated levels in cirrhosis are presumed because of sinusoidal
capillarisation 50.
In one of the recent studies, direct quantitative association was observed between
HA level and hepatic hydroxyproline content in a cirrhotic rat model 64. Of all the direct
markers, HA has shown the strongest association with liver fibrosis 17,20,26-32,35.
Alpha 2 macroglobulin is another very important marker and has shown strong
association with liver fibrosis. Hardly known outside the research realms65, it became
widely known after the noninvasive test panels became popular.
Initially a significant diagnostic value of increased alpha 2 macroglobulin was
noted for fibrosis staging in patients with alcohol liver disease 65. It is a protease inhibitor
whose concentration increases with stellate cell activation and liver fibrosis. It is also an
acute phase protein and is produced at sites of inflammation and liver fibrosis by
hepatocytes, stellate cells and granuloma cells 66-68.
Alpha 2 macroglobulin is related to fibrosis since it is a feature of stellate cell
activation69. Being a proteinase inhibitor, its increased synthesis can inhibit catabolism of
66,69
matrix proteins and enhance fibrotic processes in the liver . As seen in experimental
fibrosis, an increase in hepatocyte growth factor could account for the unexpected fall in
reduction in transforming growth factor-β1 (TGF-β1), rise of A2M, and decrease of
394
haptoglobin. Transduction with hepatocyte growth factor gene suppresses increase of
TGF-β170 and the factor stimulates synthesis of A2M 13,71.
Unlike hyaluronic acid, which has stronger correlation with higher grades of liver
fibrosis, alpha 2 macroglobulin shows association with liver fibrosis across all stages. In
the original study by Imbert-Bismut et al, (Fibrotest), of all the markers, alpha 2
13
macroglobulin was the most informative . Alpha 2 macroglobulin has been the
component of all the major test panels of noninvasive markers of liver fibrosis 13,17,20,27,28.
Platelet Parameters, PDW Index, and Liver Fibrosis

The platelet parameters that have shown association with liver fibrosis include:
platelet count, Platelet distribution Width (PDW); and these together with Mean Platelet
Volume (MPV) were combined into an index, the PDW index.
For the prediction of advanced fibrosis (F3, F4), at a cut off of 7.00, the PDW
index had a sensitivity of 75%, specificity of 91%, and overall diagnostic accuracy of
87%. And for the prediction of advanced fibrosis, the PDW ROC was 0.840, while that of
APRI was 0.888. The ROC’s for APRI are similar to the original study by Wai, et al.
Thus it can be seen that the ROC of PDW Index is comparable to APRI for the prediction
of advanced fibrosis.
Thrombocytopenia is a commonly encountered hematological condition in
54
chronic liver disease and cirrhosis . Historically, it has been attributed to the
sequestration and destruction of platelets in the enlarged spleen with an impaired ability
72,73
of bone marrow to compensate by increasing platelet production . However attempts
to correct the low platelet levels by splenectomy and portal decompression procedures
have failed to consistently improve the platelet count in the long term 72,74.
A study has shown that in patients without splenomegaly, the thrombocytopenia
was associated with the stage of fibrosis; platelets counts were the highest in patients with
fibrosis stage 0-2 (Knodell HAI), lower in those with stage 3 (p< 0.008) and lowest in
those with stage 4 (p< 0.05)75.
It has been postulated that auto-antibody mediated platelet destruction, as seen in
patients with idiopathic thrombocytopenia purpura (ITP) may also contribute to cirrhotic
395
55
thrombocytopenia . In this situation, the thrombocytopenia is principally mediated by
enhanced platelet clearance in the periphery, resulting in accelerated platelet turnover 54.
Additionally, impaired platelet production due to thrombopoiten (TPO) deficiency has
also been proposed as another cause of thrombocytopenia in cirrhosis patients75.
However, several other studies have found that the circulating TPO level is maintained or
even increased in patients with cirrhosis 76.
Thus according to one review, in patients with chronic liver disease (CLD) from
HCV, and also in patients with CLD in general, both prevalence and severity of
thrombocytopenia increase in parallel with the extent of disease, usually becoming
clinically relevant when patients develop extensive fibrosis and/or cirrhosis77.
Pathogenetic mechanisms for thrombocytopenia in advanced liver disease include
hypersplenism secondary to portal hypertension, bone marrow suppression (resulting in
suppression of megakaryocytes) likely by the disease itself, and the use of drugs, and
aberrations of the immune system resulting in the formation of anti-platelet antibodies
and/or immune-complexes that bind to platelets and facilitate their premature clearance.
In chronic liver disease, the natural inverse relationship between TPO and platelet levels
is not maintained; therefore, blood TPO levels fail to have clinical relevance or predictive
value in assessing the thrombocytopenic status of a given patient 54,77.
The quantitation of platelets in peripheral blood is a well-recognized tool.
Recently, new indices related to platelet counts have been provided by hematologic
analyzers. The important parameters are mean platelet volume (MPV), and platelet
78
distribution width (PDW) . Though routinely evaluated, the clinical inferences are
usually not drawn.
A decrease of mean platelet volume (MPV) has been observed in liver cirrhosis
79
with thrombocytopenia . It has also been shown that, platelet distribution width is a
80
better indicator of altered platelet homeostasis than MPV in liver cirrhosis . PDW is
markedly increased in cirrhosis as compared to controls, while MPV was not
significantly different80.
One study reported that, in the diagnosis of thrombocytopenia in aplastic anaemia
(AA) and immune thrombocytopenia (ITP); platelet-large cell ratio (P-LCR), and platelet
size deviation width (PDW), had the larger area under the ROC, than mean platelet
396
volume (MPV). This indicated that these values were very reliable for immune
thrombocytopenia. Thus there is increase in PDW in auto-antibody mediated platelet
destruction in patients with idiopathic thrombocytopenia purpura (ITP).
In cirrhotic thrombocytopenia, one of the contributory factors is the auto-antibody
mediated platelet destruction, like ITP , as already discussed54. Thus this mechanism may
also contribute to the increase in PDW seen in patients with advanced liver disease.
Important studies of Noninvasive Evaluation of Liver

Fibrosis
In the past few years the noninvasive evaluation of liver fibrosis has been an
active area of research and investigation.
Of the noninvasive marker panels, Fibrotest13 is the most well known and
validated of all with more than 50 studies to its credit10. The 5 indices of the panel are:
total bilirubin, GGT, haptoglobin, alpha 2 macroglobulin and apolipoprotein A1; these
are entered with the patient’s age and gender in a patented artificial intelligence algorithm
(USPTO 6631330) to generate a measure of fibrosis stage10.
Actitest is a modification of Fibrotest that scores necroinflammtory activity
(Grade in liver histology), and incorporates ALT in addition to the five markers. In
addition to the Actitest, the other panels are: Steatotest, for the quantitative assessment of
steatosis; Nashtest, for the categorical assessment of nonalcoholic steatohepatitis; and
Ashtest, for the quantitative assessment of alcoholic steatohepatitis. The last three panels
combine all the six components of Fibrotest-Actitest and, AST, fasting glucose, total
cholesterol, triglycerides, height, weight (BMI), age and sex of the patient 10.
All the above mentioned panels are combined into a supercombination,
FibroMAX, in patients at risk of chronic liver diseases 10. The Fibrotest-Actitest provide
a continuous linear estimate ranging from 0.00 to 1.00 corresponding to the well
established Metavir scoring system of stages from F 0-4 and Grade A 0-3 10.
For Fibrotest, the area under the receiver operating characteristic curve, ROC, was
0.836 to 0.87 in the principal study13. In subsequent studies the ROC curves have varied
from 0.65 to 0.89. This could be attributed to the variations in the study protocols,
397
analytical variability of the tested markers, and variability of fibrosis stage prevalence
defining advanced and non-advanced fibrosis 10,81.
With the best index, a high negative predictive value (100% certainty of absence
of F2, F3, or F4 fibrosis) was obtained in scores ranging from zero to 0.10 ( 12% of all
patients), and high positive predictive value ( >90% certainty of presence of F2, F3, or F4
fibrosis) for scores ranging from 0.60 to 1.00 (34% of all patients). The detection of
significant fibrosis F2 or greater had a 75% sensitivity and 85% specificity. The assay
performed somewhat better for the assessment of more advanced liver disease
(METAVIR stages 3 and 4)13.
The discordances between the Fibrotest and biopsy are similar. According to one
study 18% of the discordances were attributable to biopsy failure (mostly due to short
82
length) and 2% to Fibrotest . In another prospective multicenter study, the Fibropaca
study, the discordance was attributable to biopsy in 3.8%, to Fibrotest in 5.4%, and
undetermined in 9.1% 31.
The Fibrotest scores fall in response to therapy. Patients who had a sustained
virological response to interferon had a substantial reduction in Fibrotest and Acitest
scores, as compared to those who were either primary nonresponders or relapsed
following therapy 83.
The 5 year prognostic value of Fibrotest is at least equal to liver biopsy 84.
In addition to analytic variability (discussed below), there are issues related to the
false positive/negative values of Fibrotest components, that could artificially
increase/decrease the suspicion of liver fibrosis. The most frequent abnormal value of
Fibrotest markers noted in the general population, since its inception is low haptoglobin
10
. Extremely low haptoglobin, especially when other exams are hardly modified could
have hemolysis. False positives due to increase in bilirubin, most likely due to Gilbert’s
syndrome have also been observed, although hemolysis is a second thought. Acute
sepsis/inflammation can also raise alpha 2 macroglobulin and haptoglobin levels. If these
conditions are suspected, the Fibrotest evaluation should be postponed 10.
398
There has been concern about the analytic variability of proteins of Fibrotest
components.
In this regard, a study compared the variability of Fibrotest proteins (including

alpha 2 macroglobulin) using two different nephelometric analyzers: the BN Prospec ®
(Dade-Berhing) i.e., the technique used by the original reference laboratory, and the
Immage ® (Beckman-Coulter) another widely distributed nephelometric instrument. For
both analyzers the reagents were standardized against the International Certified material
470 for alpha 2 macroglobulin 85.
Levels of alpha 2 macroglobulin measured with Immage ® analyzer were

significantly higher than those with BN Prospec ® analyzer. The median difference
between the results obtained with the two analyzers was 20.5% (range 4.8-32.3) 86.
Subsequently, the same group, however, in another study, for analytic variability
for Fibrotest components, used new reagents for alpha 2 macroglobulin and found
excellent transferability between the Immage ® and the BN Prospec ® analyzers, with
fewer than 10% of the values outside the accepted boundaries.
The reason for this dramatic improvement was that in the subsequent study, a new
antibody was used in the Beckman-Coulter reagent (the analyzer different from the
Fibrotest reference laboratory analyzer) 87.
The authors suggesting that in addition to the antibody, other factors could cause
variations, such as: the calibrator and the type of calibration, antigen excess, matrix
differences, molecular weight and/or the functional conformation of the proteins, length
of the immunological reaction, and finally individual genetic heterogeneity 87.
Another very widely reported index, APRI refers to the AST to Platelet ratio
index. It has been assessed in several studies and has shown rather good diagnostic
performance and reproducibility, particularly for cirrhosis ( ROC between 0.77-0.94). In
the original study by Wai et al, the model predicted bridging fibrosis with a ROC of 0.80-
0.88 24. APRI has lower accuracy (ROC of 0.74) for detecting lower grades of fibrosis 88.
Its most important aspect is the use objective and readily available
laboratory values. Both platelet count and AST levels are routine tests performed in
399
chronic hepatitis C patients in clinical practice, so no additional tests are required Though
slightly inferior, nevertheless, its simplicity (APRI can be determined by the bedside with
the help of a calculator), matched with performance to the more sophisticated Fibrotest
and Forn’s index is a great advantage 89.
The Forn’s index is based on four readily available variables; age, platelet count,
γ glutamyl transferase, and cholesterol levels. The area under the ROC curve was
between 0.81- 0.86 for the estimation group and the validation groups 23. The lower cut of
value had a 96% negative predictive value, whereas the upper cut off value had a positive
predictive value of 66%. Hence this test was useful at excluding patients with minimal
fibrosis, but was of limited value for the identification of patients with more advanced
liver disease 90.
One important aspect of the index is the use of very basic clinical parameters, and
the fact that the accuracy of this model compares with models based on more
sophisticated variables (such as Fibrotest). Major caveats of the Forns index include
91
concerns about the impact of lipid abnormalities in patients with hepatitis C ,
cholesterol altering medicines, and the reproducibility of platelet estimations 90.
The Sud score incorporated age, past alcohol intake, AST, cholesterol, and insulin
resistance had a ROC of 0.77 25.
The Europeon Liver Fibrosis Group study (ELFG) by Rosenberg et al, evaluated
an algorithm consisting of age, hyaluronic acid, tissue inhibitor of metalloproteinase 1
(TIMP-1), and N-terminal propeptide of type III collagen (PIIINP). It had a ROC of 0.78
for significant fibrosis from all etiologies, and a ROC of 0.77 when limited to patients
with hepatitis C 26.
In a recent study, in which hyaluronic acid (HA) was compared with Fibrospect II
(consisting of HA, TIMP-1, and alpha 2 macroglobulin), and YKL-40, concluded that:
HA was effective in discriminating between significant fibrosis as compared to FS II with
a ROC of 0.76 vs 0.66 28.
Hepascore another model consisting of bilirubin, GGT, hyaluronic acid, alpha 2

macroglobulin, age and sex had area under the ROC of 0.85 for significant fibrosis,
400
which increased to 0.96 and 0.94 for advanced fibrosis and cirrhosis respectively. The
Hepascore increased significantly (p<0.001) as the fibrosis stage increased. A central cut
off point of 0.5 predicted significant fibrosis with a sensitivity of 67% and a specificity of
92%. When the same cut off point of 0.5 was applied for the prediction of advanced
fibrosis (F3-F4) sensitivity was 95% and specificity was 81%. A cut off point of 0.84,
when applied for the detection of cirrhosis (F4) provided a 71%sensitivity and 84%
specificity20.
The Fibrometer test, developed by Calès et al, combined platelets, prothrombin

index, AST, alpha 2 macroglobulin, hyaluronic acid, urea and age. Its area under ROC in
patients with viral hepatitis for significant fibrosis (stage F2-4) was 0.883 compared with
0.808 for the Fibrotest (p = 0.01), 0.820 for the Forns test (p = 0.005), and 0.794 for the
APRI test (p < 10-4). The Fibrometer area under ROC curve increased to 0.892 in the
validating population17.
The authors also estimated the area of fibrosis in viral hepatitis by testing for
hyaluronate, γ glutamyl transferase, bilirubin, platelets, and apolipoprotein A1. The use
of blood tests to estimate the area of fibrosis (AOF) is new. Measurement of area of
fibrosis via image analysis is limited to clinical research. Unlike histological stages, the
AOF provides precise quantification of the extensive variations in fibrosis during
cirrhosis. Because cirrhosis corresponds to only one 92 or two 93 stages the range of blood
test results for clinically significant fibrosis (CSF) in patients with cirrhosis is very
limited 17.
Thus in viral related chronic liver disease, the interquartile range of blood tests for
AOF was 7.2% to 10.4% in F0-F3 versus 11.2% to 19.0% in F4; whereas the
corresponding ranges for the Fibrometer test for CSF were 0.03 to 0.99 and 0.99 to 1.00,
respectively. Moreover, the AOF estimation via blood test is the only statistically
validated quantitative test for the noninvasive diagnosis of fibrosis17.
401
Genetics and Proteomics
In addition to the indirect and direct noninvasive markers, other novel and
exciting new indices have been developed.

influence progression of liver fibrosis. There are many studies that report gene
polymorphisms to either favor or reduce fibrogenesis in patients with different forms of
chronic liver disease 11.
While these studies clearly indicate that many genetic factors have a definitive
influence on the risk of developing a more or less active and progressive fibrogenesis,
very few of them have found an application as a diagnostic/prognostic marker in clinical
practice, due to their complexity, difficulty to test and variable behavior in different
patient populations 11.
The clinical factors such as age, gender, alcohol use, and age at infection, obesity
and hepatic steatosis,38,94 influence the progression to cirrhosis, but cannot accurately
predict the risk of developing cirrhosis in patients with chronic hepatitis C 40.
One such study, consists of a set of seven marker genes, the cirrhosis risk score
(CRS), was found to be a better predictor of high risk versus low risk for cirrhosis in
95
Caucasian patients than clinical factors . The authors validated all significant markers
from a genome scan in the training cohort, and selected 361 markers for the signature
building. Subsequently, a signature consisting of 7 markers most predictive for cirrhosis
risk in Caucasian patients was developed.
increased to 0.73 for CRS, and 0.76 when CRS and clinical factors were combined. The
authors concluding that, CRS is a better predictor than clinical factors in differentiating
high risk versus low risk for cirrhosis in Caucasian patients 95.
A study using profiles of serum protein N-glycans found that a profile has a
similar area under ROC to Fibrotest for the diagnosis of compensated cirrhosis. When
402
compared with Fibrotest, this marker had 100% specificity and 75% sensitivity for the
diagnosis of compensated cirrhosis, which is not greatly different from the 92%
specificity and 67% sensitivity of the Fibrotest alone 96.
Proteomics studies are ongoing, and the components of the Fibrotest have been
97,98
identified in several proteomic studies . A combination of proteomic peaks has been
identified with a 7% increase in area under the ROC in comparison with the Fibrotest 10.
Even if the diagnostic value of these panels is significantly greater than the reference
panels, the cost utility and the availability to the population at large are important
concerns.
Elastography
Another noninvasive method for the assessment of liver fibrosis if elastography
(FibroScan). In a study by Castera et al, the area under the ROC by FibroScan was 0.83
for F< 2, 0.90 for F< 3, and 0.95 for F=4. The same authors demonstrated that, when
combined with Fibrotest, FibroScan was more precise than liver biopsy, the area under
the ROC reaching 0.88, 0.95 and 0.95 for fibrosis stages F< 2, F< 3 or F4 respectively 14.
In the study performed by Ziol et al, the area under the ROC value of FibroScan
as compared to liver biopsy was 0.79 for F< 2, 0.91 for F< 3, and 0.97 for F= 4. The
authors concluding that elastography appears reliable to detect significant fibrosis and
cirrhosis in patients with chronic hepatitis C99.
In one of the metanalysis, for significant fibrosis, the area under the ROC for
Fibrotest and FibroScan were 0.81 (95% CI 0.78-84) and 0.83 (0.03-1.00), respectively.
At a threshold of approximately 0.60, the sensitivity and specificity of the Fibrotest were
47% (35-59%) and 90% (87-92%). For FibroScan (threshold approximately 8 kPa),
corresponding values were 64% (50-76%) and 87% (80-91%), respectively. For cirrhosis,
the summary area under the ROC for Fibrotest and FibroScan were 0.90 (95% CI not
calculable) and 0.95 (0.87-0.99), respectively. The diagnostic accuracy of both measures
was associated with the prevalence of significant fibrosis and cirrhosis in the study
populations 100.
403
In one of the studies, in patients with HCV infection, the sensitivity of Fibrotest
appears higher for early stage fibrosis, and the combination of FibroScan and Fibrotest
improves the overall diagnostic value. Elastography is complementary because of its
specificity for severe fibrosis, and also in case of Fibrotest, some patients with high risk
profile of false positives and negatives15.
It is probable that in not too distant future, the combination of

Fibrotest (or other combination of noninvasive biomarkers) with FibroScan
could replace liver biopsy in most patients with chronic hepatitis14,101.
404
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414
RESULTS
Patient Characteristics
The clinical and laboratory characteristics of patients are presented in the
following Tables. A total of 100 patients were enrolled in the study, of which 88 were
included in the final analysis. Their mean age was 32.43 years (range 20-53 yrs). There
were 41 (46.6%) females. There were 74 (84%) patients with chronic liver disease from
HCV, 13 (15%) with HBV, and 1(1%) patient had both HCV RNA PCR and HBsAg
positive. The clinically significant fibrosis (CSF) as defined as stages 2, 3 and 4 ( F2, F3,
F4) was present in 23 (26%) patients; and advanced fibrosis, stages 3 & 4 (F3-F4) was
present in 20 (23 %) of patients, which generally is regarded as the prevalence in the
community. The mean length of the biopsy specimen was 1.15 cm (median 1.00 cm), and
the interobserver agreement (Kappa, κ coefficient) between the histopathologists was
moderate (κ = 0.60) for grading and good for staging (κ = 0.74).
Predictive Model (FIBROSCORE)

Various markers were assessed for their association with clinically significant
fibrosis Table. The most useful variables were then entered into step wise logistic
regression function to create different probability models. The best model with the largest
area under the receiver operating characteristic curve (ROC) with the fewer variables was
selected.
This model Fibroscore consisted of 5 markers: bilirubin, gamma glutamyl
transferase (GGT), hyaluronic acid (HA), alpha 2 macroglobulin (A2M), and platelets.
The area under the ROC for F2 (stage 2) fibrosis was 0.808 (95% CI; .613-1.00),
for F3 the ROC was 0.938 (95% CI; .888-.988), and for F4 the ROC was 0.959 (.893-
1.00) and for combined stages 2 and 3 (F2, F3) the ROC was 0.948 (95% CI .902-.994).
The overall diagnostic accuracy of the model for predicting clinically significant fibrosis
was 89%.
The Fibroscore values (range 0.00-1.00) increased as the fibrosis stage increased
(graph 3). The maximum score was for stage 4 (F4). The small box plot for F4 indicates
that the corresponding range for the Fibroscore for cirrhosis (F4) is small i.e., 0.99-1.00.
359
A central cut off of 0.5 was chosen to observe the significance for clinically
significant fibrosis. A score of > 0.5 was seen in 24 of 88 (27%) of patients. This central
cut off point in the model predicted clinically significant fibrosis (F2, F3 and F4) with a
sensitivity of 82% (95% CI; 63-93), specificity of 92% (95% CI; 83-96), positive
predictive value (PPV) of 79 % (95% CI; 59-90), negative predictive value (NPV) of
93% (95% CI; 85-97) and overall diagnostic accuracy of 89% (95% CI; 81-94).
Thus 19 of 24 patients (79%) had clinically significant fibrosis (The PPV). Of the
5 misclassified patients with values > 0.5, four patients had moderate activity (ALT range
58-139), and portal fibrosis (necroinflammatory grade in two patients, 8/18; and in two
patients, 7/18; fibrosis stage in all four patients 1/4). Only one patient had minimal
activity and no fibrosis (1/18, 1/22). Of the 5 misclassified patients with values < 0.5 only
two had fibrosis stage 3/4, one had stage 3/6, the other 2/6.
By applying the same cut off point for advanced fibrosis (F3, F4), the sensitivity
was 85% (95% CI; 64-94), specificity was 89% (95% CI; 80-94) and overall diagnostic
accuracy was about the same, 88% (95% CI; 80-93).
Increasing the cut off point to > 0.65 for clinically significant fibrosis increased
the specificity to 98% with a positive predictive value of 95%. Thus 19 of 20 (95%),
patients who had Fibroscore value more than 0.65, had clinically significant fibrosis (The
PPV). The one misclassified patient had high ALT 139 U/L, moderate activity (8/18), and
fibrosis stage 1 on liver biopsy.
Further increasing the cut off to 0.80 increased the positive predictive value to
100%, but only 15 patients out of 88 could be classified (17%).
A score of < 0.5 was observed in 64 (73%) patients, which excluded advanced
fibrosis (F3, F4) with a sensitivity of 89%, and specificity of 85%.
The Fibroscore reliably detects the absence of fibrosis. Lowering the cut off to
0.20 reliably predicts the absence of clinically significant fibrosis with a NPV of 96%.
Only 2 patients had score less than 0.20, none of these had established bridging fibrosis;
one had stage 3/6, other stage 2/6.
Almost 100% certainty of the absence of clinically significant fibrosis (actual
NPV 98% of the absence of F2,F3,F4) was predicted by scores between 0.00 to 0.08. The
360
only one misclassified patient had mild activity and a lower stage of fibrosis (grade 6/18,
and stage 2/6).
The Fibroscore was also very useful in differentiating moderate to severe from
minimal to mild necroinflammatory activity. The area under the ROC curve was 0.790
(95% CI; .696-.884), with the sensitivity of 63% and specificity of 77%.
Platelet Variables
The different platelet variables evaluated in the study for advanced fibrosis stages
3 and 4 (F3-F4) were: platelet count, mean platelet volume (MPV), platelet distribution
width (PDW), and giant platelet percentage. Their association with the advanced fibrosis
is shown in the Table 28. The covariates that showed statistical significance included:
platelet count (F0-F2, mean 266 x 109/l, F3-F4 mean 193 x 109/l, p = 0.000); PDW (F0-
F2, mean 12.37, F3-F4 mean 13.66, p = 0.004; and the PDW index described below (F0-
F2 mean 5.7, F3-F4 mean 9.5 p = 0.000).
Of the other parameters, MPV had shown some association (F0-F2 mean 10.66 fl,
F3-F4 mean 11.26 fl, p = 0.76), though not statistically significant. In the study giant
platelet percentage did not show any association with advanced levels of fibrosis. Apart
from the later, the other parameters were combined into an index that had shown good
predictability with advanced fibrosis.
PDW Index
The platelet count, mean platelet volume (MPV), and platelet distribution width
(PDW) were combined into different indices for the prediction of advanced fibrosis
(F3,F4); the one with the best association and the best ROC, was derived arithmetically
as PDW index.
The area under the ROC for advanced fibrosis (F3-F4) for PDW index was 0.840
(95% CI; .721-.958). The ROC for the prediction of stage 3 (F3) fibrosis was 0.801 (95%
CI; .671-.930). For the prediction of advanced fibrosis (F3, F4), at a cut off of 7.00, the
index had a sensitivity of 75%, specificity of 91%, PPV of 71%, NPV of 92%, and
overall diagnostic accuracy of 87%.
361
Out of 21 patients with scores more than 7.00, six patients were misclassified.
Out of these 6 patients, 4 patients had evidence of moderate activity, the other two had
mild activity; all patients had stage 1 fibrosis.
PDW Index performance for the lower grades of fibrosis falls, as for F2, the PDW
ROC was 0.635 (95% CI; .521-.749).
In this study, the PDW ROC for the prediction of F3 fibrosis was 0.801, while the
corresponding ROC for AST to Platelet ratio index (APRI) for F3, also evaluated in this
study, was 0.854. And for the prediction of F3-F4 fibrosis, the PDW ROC was 0.840,
while that of APRI was 0.888. The ROC’s for APRI are comparable to the original study
by Wai, et al. Thus, it can be seen that the ROC of PDW index has only slight difference
at the second decimal point as compared to the ROC of APRI.
Thus it is concluded that the PDW index compares with the renowned
noninvasive index, APRI, for the prediction of significant fibrosis. It’s most important
aspect is that, it does not use any biochemical markers, and is only derived from platelet
parameters, which are generally considered not of great clinical significance.
Additionally, in the context of our country with limited resources, it is
very helpful to have an index being derived from blood complete picture only, and unlike
the well known index, APRI, by not even using a single biochemical test. Thus as a
screening test it may guide towards the use of more expensive tests.
Though somewhat similar in performance (the difference is manifest at the second
decimal point), its simplicity (like APRI, PDW index can be determined by the bedside
with the help of a simple calculator), matched with performance to the more sophisticated
Fibrotest; and the liver fibrosis index, (Fibroscore), evaluated in this study, is a great
advantage.
362
Fibroscore
0.00-1.00
Fibroscore Fibroscore
< 0.5 > 0.5
64 patients 24 patients
F0-F1 F2-F4
F2-F4 F0-F1
Fig 17. The Fibroscore assessment for clinically significant fibrosis
363
Table 18. Diagnostic indices of FIBROSCORE > 0.5 for clinically
significant fibrosis
Parameter Estimate Lower – Upper 95 % CIs
Sensitivity 82.61% (62.86 – 93.02)
Specificity 92.31% (83.22 – 96.67)
PPV 79.17% (59.53 – 90.76)
NPV 93.75% (85.00 – 97.54)
Diagnostic Accuracy 89.77% (81.69 – 94.53)
364
____________________________________________________
Table 19. Clinical and demographic data of patients
____________________________________________________
Characteristic Mean (SD)(Range) /Percentage
Age (yrs) 32.43 + 6.1 (20-53) Median 32
Gender
Female 41 (46.6%)
Male 47 (52.4%)
Mode of Transmission
Unknown 54 (61.4%)
Injections (excluding procedure) 5 (5.7%)
Transfusions 3 (3.4%)
Minor Surgery 15 (17%)
Major Surgery 11 (12.5%)
BMI 23.9 + 3.64 (17-35) Median 24
Hepatomegaly 22 (25%)
Splenomegaly 6 (6.8%)
HCV RNA 74 (84%)
HBsAg 13 (14.7%)
Both 1 (1.13%)
Knodell HAI 82 (93%)
Modified Knodell (Ishak) 6 (7%)
Fibrosis score 1.21 + 1.14 (0-4), Median 1.00
Significant Fibrosis (F2-F4) 23 (26%)
Advanced Fibrosis (F3-F4) 20 (23%)
Advanced Activity (Mod-Severe) 44 (50%)
________________________________________________________________________
365
Table 20. Virological Profile of Patients
HCV RNA PCR 74 (84%)

HBsAg 13 (14.7%)
HBV DNA PCR 10 (11.36%)
HBeAg 9 (10.22%)
HBeAg (+) DNA PCR (-) 1 (1.13%)
HBeAg (-) DNA PCR (+) 2 (2.27%)
HCV RNA PCR & HBsAg 1 (1.13%)
366
_______________________________________________________
Table 21. Liver Biopsy & Needle Characteristics
_______________________________________________________
Length of biopsy (cm) Mean1.15 + 0.64, Median 1.00
No of fragments
Single fragment 44 (50%)
Two fragments 20 (22.7%)
More than two fragments 24 (27.3%)
Needle Type
Surecut Needle (Modified Menghini) 79 (88.8%)
Trucut Needle 1 (1.1%)
Unknown 8 (9.1%)
Needle Size
Surecut 18 Gauge 76 (86.4%)
Surecut 16 Gauge 3 (3.4%)
Unknown 9 (10.2%)
Scoring System
Knodell HAI 82 (93%)
Modified Knodell (Ishak) 6 (7%)
367
_______________________________________________________
Table 22. Histopathological Characteristics
Stage of Fibrosis, Knodell HAI (Metavir)

Stage 0 (F0) No Fibrosis 26 (29.5%)
Stage 1 (F1) Portal Fibrosis 39 (44.3%)
Stage 2 (Ishak) (F2) Portal fibrosis + septa 3 (3.4%)
Stage 3 (F3) Bridging Fibrosis 18 (20.5%)
Stage 4 (F4) Cirrhosis 2 (2.3%)
Activity (Knodell Grade)
Minimal activity (0-3) 11 (12.5%)
Mild activity (3-5) 33 (37.5%)
Moderate activity (6-11) 39 (44.3%)
Severe activity (> 11) 5 (5.7%)
Category of Fibrosis
Significant Fibrosis (F2-F4) 23 (26.1%)
Advanced Fibrosis (F3-F4) 20 (22.72%)
Insignificant Fibrosis (F0-F1) 65 (73.9%)
Category of Activity
Significant Activity (Moderate-Severe) 44 (50%)
Steatosis 42 (47.7%)
____________________________________________________
368
_______________________________________________________
Table 23. Viral Aetiology and Histopathology
HCV HBV
Significant Fibrosis (F3-F4) 18/75 (24%) 6/14 (43%)

Significant Activity (Mod-Severe) 36/75 (48%) 9/14 (64%)
Steatosis 39/75 (52%) 3/14 (21%)
____________________________________________________
Table 24. Lipid profile and HCV
________________________________________________
Anti HCV Negative Positive p value

Cholesterol (mmol/L) 4.46 +.39 4.56 +.45 0.43
Triglycerides (mmol/L) 2.03 +.80 1.67 +.29 .004
HDL (mmol/L) 1.01 +.19 0.94 +.07 .21
LDL (mmol/L) 2.70 +.50 2.92 +.39 .79
369
Table 25. Clinical/Ultrasonographic/Biopsy Parameters vs
Significant Fibrosis
Variable Stage F0-F1 Stage F2-F4 p value

___________________________________________________________
Age , years 31.2 + 5.46 35.9 + 6.88 0.001
Gender , Female, % 32 (49.2%) 9 (39.1%) 0.404
Ultrasound variables
Liver echogenicity (increased) 12 (18.5%) 10(43.5%) 0.017
Hepatomegaly 5 (7.7%) 8 (34.8%) 0.002
Portal vein diameter 2 (3.1%) 1 (4.3%) 0.773
(12-15 mm)
Splenomegaly 2 (3.1%) 3 (13%) 0.076
Biopsy variables
No to Mild Activity 42 (64.6%) 2 (8.7%) 0.000
(necroinflammatory)
Mod to Severe Activity 23 (34.5%) 21 (91.3%) 0.000
Steatosis 27(41.5%) 15 (65.2%) 0.051
370
________________________________________________
Table 26. Biomarkers vs Fibrosis categories
(statistically significant)

Bilirubin (µmol/L) 11.2 + 4.55 13.7 + 4.71 0.026
AST (U/L) 40.4 + 12.68 51.6 + 24.89 0.49
GGT (U/L) 33.7 + 12.1 53.4 + 22.89 0.001
PT (sec) 14.3 + 0.73 15.4 + 0.99 0.000
HDL (mmol/L) 0.94 + 0.06 1.00 + 0.15 0.017
IgG (g/L) 13.8 + 2.61 15.1 + 2.48 0.040
Hyaluronic Acid (µg/L) 31.2 + 16.63 73.9 + 32.73 0.000
Alpha 2 Macroglobulin (g/L) 2.3 + 2.17 3.9 + 2.36 0.004
Platelets (x 109/L) 267 + 45.98 199 + 45.88 0.000
___________________________________________________________
371
Table 27. Biomarkers vs Fibrosis Categories (other)

ALT (U/L) 88.2 + 37.22 93.6 + 50.60 0.58
Albumin (g/L) 43.3 + 5.45 43.2 + 4.08 0.95
Urea (mmol/L) 4.12 + 0.73 4.14 + 0.95 0.89
Cholesterol (mmol/L) 4.54 + 0.45 4.58 + 0.43 0.68
Triglycerides (mmol/L) 1.6 + 0.29 1.8 + 0.66 0.049
LDL (mmol/L) 2.8 + 0.42 2.9 + 0.37 0.15
Globulins (g/L) 22.5 + 7.46 23.1 + 5.02 0.69
___________________________________________________________
372
Table 28. Platelet Parameters vs Advanced Fibrosis
Platelet count (x109/L) 266 + 45.47 193 + 46.87 0.000
MPV (f/L) 10.6 + 1.35 11.2 + 1.08 0.076
PDW (f/L) 12.3 + 1.62 13.6 + 1.96 0.004
PDW Index 5.7 + 1.53 9.5 + 3.67 0.000
___________________________________________________________
373
________________________________________________
Table 29. Logistic Regression Analysis
Variable B Wald Test p value Odds Ratio 95% CI
Bilirubin 0.189 3.824 .051 1.208 1.000-1.459

GGT 0.120 5.768 .016 1.128 1.022-1.244
Hyaluronic 0.080 8.796 .003 1.084 1.028-1.143
Acid
Alpha 2 0.518 6.032 .014 1.678 1.110-2.537
Macroglobulin
Platelets -.040 5.694 .017 .960 .929-.993
Constant -4.795 1.289
___________________________________________________________
374
Graph. 1 Receiver Operating Characteristic (ROC) curve Fibroscore vs F3
Fibrosis
Area under the ROC .938 (95% CI; .888-.988)
375
Graph. 2 ROC curve, Fibroscore vs F2 Fibrosis
Area under the ROC .808 (95% CI; .613-1.00)
376
Graph 3. Boxplot of predicted probability of Fibrosis (Fibroscore) vs
various Stages of Fibrosis.
The Fibroscore range is from 0.00-1.00
The line through the box is the Median; the top and bottom edges of each
box represent 25th and 75th percentile, giving the interquartile range; the
vertical lines on either side of box represent distribution from the quartile to
the farthest observation.
377
Graph 4. Boxplot of Hyaluronic Acid values vs different fibrosis stages.
378
Graph 5. Boxplot of Alpha 2 Macroglobulin values vs different Fibrosis
stages
379
Graph 6. ROC curve of PDW Index vs F3 Fibrosis
Area under the ROC .801 (95% CI; .671-.930)
380
Graph 7. APRI and PDW Index ROC vs Stage F3-F4 Fibrosis
APRI area under the ROC .888(95% CI; .797-.980)
PDW Index area under the ROC .840 (95% CI; .721-.958)
381
Graph 8. Boxplot of Platelets vs various Stages of Fibrosis
382
Graph 9. Boxplot PDW Index vs various stages of Fibrosis
383
SUMMARY
&
CONCLUSION
SUMMARY
1. With the increasing number of patients suffering from chronic liver disease from
HBV and HCV, which may progress to cirrhosis with all its complications;
clinicians and patients require accurate information about the degree of liver
fibrosis, to guide management decisions, monitor disease activity, and predict
outcome.
2. Many treatment algorithms recommend treatment for patients with septal fibrosis
or greater ( > stage F2, Metavir/Ishak), this recommendation also depends on viral
cause, and particular viral subtype. However more efficacious treatments or
improvements in tolerability of therapy could easily alter this treatment paradigm;
increasing the physician and patient acceptability of therapy, and reducing the
need for liver biopsy.
3. There is no ideal reference for the assessment of liver histology.
4. Liver biopsy, which is considered the ultimate standard, has three major
limitations: a risk of adverse events, sampling error, and intra and inter observer
variability. Liver biopsy is only 80% accurate in assessing stage of liver fibrosis,
and could miss advanced fibrosis and cirrhosis in as high as 30% of patients. The
factors associated with this inaccuracy of liver biopsy include: the heterogeneous
nature of chronic liver disease; the relatively small size of liver biopsy sample
compared to the size of liver (1/50,000), or even the size of a nodule as in
macronodular cirrhosis, which is > 3mm; and the experience of the
histopathologist.
5. These limitations of biopsy have led clinical investigators to study alternative
methods to investigate liver disease.
6. It is vital that new methods for the assessment of liver fibrosis be developed and
validated, so that the liver fibrosis is diagnosed and impact of any new therapies
can be rapidly assessed in a simple and meaningful way.
7. Noninvasive markers are the most widely used alternative to liver biopsy to stage
chronic liver disease.
416
8. Serum markers of liver fibrosis offer an attractive alternative. They are less
invasive than biopsy, with no risk of complications, eliminate sampling and
observer variability, may allow dynamic calibration of fibrosis (liver biopsy is just
a snap shot in time), and can be performed repeatedly.
9. There are two kinds of noninvasive markers: direct and indirect. There are
advocates of both types of tests, but they are complementary to each other.
10. The ideal features of these serum markers are: noninvasive, liver specific,
correlates well with the severity of liver disease especially fibrosis, reflective of
fibrosis irrespective of cause, not confounded by other co-morbidities, minimally
influenced by urinary and biliary excretion, easy to perform, and be commonly
used in all patients.
11. There are three commercially available serum marker panels, which have been
most extensively validated (in thousands of patients); Fibrotest, FibroSpect, and
the European Liver Fibrosis Study Group Assay. Additionally, there have been
many studies of noninvasive markers of liver fibrosis.
12. These tests are extremely accurate > 95% in determining the near absence (F0-F1)
of fibrosis in CLD, and the presence of cirrhosis. They are also very accurate for
advanced fibrosis (F3-F4); the predictability is more than 85%-90%, and the area
under the ROC more than 0.90; but not for intermediate stages. Overall, the
majority of noninvasive marker panels have an area under the ROC of 0.80-0.85
not for staging the disease, but differentiating mild (F0-F1) from clinically
significant (F2-F3-F4) fibrosis; which is a significant advancement.
13. The relatively lower diagnostic performance in intermediate stages is related to
liver biopsy itself. Since the noninvasive marker is validated against the biopsy,
and the overall accuracy of biopsy is only 80%, it is probably statistically
impossible for a marker to perform any better than biopsy.
14. This is not surprising, since these stages are relatively artificial separations of a
spectrum of a disease process with progression in severity expressed as a
categorical variable from 0-4 to 6. This does not accurately reflect the dynamic
biologic process of fibrosis, and constrains the serum marker test performances
that are capable of generating continuous variables.
417
15. The noninvasive markers also correlate fairly well with the necroinflammatory
activity.
16. In addition to diagnosing hepatic fibrosis, these tests have proved valuable in
predicting changes in fibrosis content over time in individual patients. Those
patients who favorably respond to therapy have changes in mean serum values of
fibrosis markers. Thus, these assays may be useful not only in the initial staging
of the liver disease, but may also be of value in following the histological
response to therapy.
17. The long term (5 year) prognostic value of one of the test panels (Fibrotest) is at
least equal to that of liver biopsy.
18. Thus with the use of serum markers: the severity of fibrosis is diagnosed; the
stage of fibrosis helps predict the likelihood of response to therapy in CLD from
HBV and HCV, since advanced stages of fibrosis generally have a lower
response; treatment may be delayed or deferred if little fibrosis progression has
occurred over time, in certain HCV genotypes such as genotype 1; and
approximate time to the development of cirrhosis can be estimated.
19. Besides, the noninvasive markers allow a quantitative estimation of fibrosis,
which is a major breakthrough.
20. Although remarkable progress has been achieved in the technique of noninvasive
markers, there are some caveats.
21. These tests must be interpreted in the overall clinical and biological context. The
serum markers, especially the direct markers typically reflect the rate of matrix
turnover, not deposition and thus tend to be more elevated when there is high
inflammatory activity. None of the markers are liver specific, so their serum
levels may be affected by concurrent inflammation, fibrosis elsewhere, or
unrelated phenomenon such as haemolysis. Their serum levels may be affected by
clearance rates, such as in impaired renal or biliary excretion. Further refinements
in technique and even better noninvasive marker algorithms will decrease the
risks of false positives and false negatives.
22. An alternative method to stage chronic liver disease is elastography, or
measurement of hepatic elasticity or stiffness.
418
23. The sensitivity of noninvasive markers is higher for early stage fibrosis, but
elastography is helpful because of its specificity for severe fibrosis.
24. This technique, however, has its limitations: it uses expensive equipment, and has
decreased accuracy in obese patients and those who have ascites.
25. Nevertheless, elastography is complementary, as the combination of noninvasive
markers and elastography improves the overall accuracy.
26. The development of noninvasive markers derived from proteomics and genomics
is likely to be important in the years to come.
27. Several published studies and overviews have demonstrated the predictive value
and better benefit-to-risk ratio than liver biopsy of these combinations of simple
serum noninvasive markers in patients with chronic liver disease. They allow a
quantitative assessment of fibrosis according to the cause of liver disease, as well
as an assessment of the necroinflammatory activity. These tests which are now
available worldwide, can facilitate not only the screening and management of
fibrotic liver disease, but also provide useful prognostic value, and will be
extremely helpful to the clinicians.
28. It is probable that in not too distant future, the combination of noninvasive
marker panels and/or noninvasive markers + elastography could replace liver
biopsy in most patients with chronic hepatitis.
419
CONCLUSION
Chronic viral hepatitis is a common disease in the general population of which the
world is facing a pandemic. In the last decade, advances in serological and virological
testing, and improvements in therapy have led more patients to be identified and to seek
treatment. In chronic viral hepatitis the prognosis is highly dependent on the extent and
progression of hepatic fibrosis. Thus accurate information about the degree of fibrosis is
required to guide management decisions, monitor disease activity, and predict outcome.
The clinicians rely on liver biopsy for both prognostic and therapeutic decision
making, which can have a major impact on patient’s life. Though classically considered
the “gold standard”; the liver biopsy is far from perfect, and has significant limitations.
This has led researchers to look for other methods to assess the stage of liver fibrosis. The
noninvasive markers are the most widely used alternative to liver biopsy; and tremendous
progress has been made in this regard.
In the study presented, the Fibroscore results are extremely accurate (> 95%) in
determining the near absence of fibrosis in viral related chronic liver disease, and
cirrhosis. Fibroscore also correlates highly with clinically significant fibrosis, and
provides useful assessment of the necroinflammatory activity. The association between
platelet parameters and fibrosis was also looked into; the PDW Index reliably predicts
advanced fibrosis.
Thus the Fibroscore results have high diagnostic value to stage fibrosis; for either
the presence or exclusion of very early stage, clinically significant, or advanced stages of
fibrosis. These tests will assist in the screening and management of fibrotic liver disease;
and represent a valid alternative to liver biopsy
It is concluded that, the noninvasive markers will replace liver biopsy in most
patients with chronic liver disease from hepatitis B and C viruses.
420

Non Invasive Evaluation OF Liver Fibrosis IN Patients With Chronic Hepatitis B and C

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Non Invasive Evaluation OF Liver Fibrosis IN Patients With Chronic Hepatitis B and C

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NON INVASIVE EVALUATION

Professor Lt Gen (R) Syed Azhar Ahmed

My parents & wife

Prof Lt Gen (R) S Azhar Ahmed

Chap 1 INTRODUCTION AND OVERVIEW 11

Chap 2 EPIDEMIOLOGY AND NATURAL HISTORY 54

OF CHRONIC HEPATITIS B AND C VIRUSES

Chap 3 HEPATIC INJURY, FIBROSIS, AND FIBROGENESIS 104

(THE CELL AND MOLECULAR BIOLOGY)

Chap 5 NON INVASIVE EVALUATION OF LIVER 279

PATIENTS AND METHODS 349

Literature Review Page No.

Table 1 Prevalence of hepatitis B in various areas of the world 59

Table 2 Host and viral factors in HBV disease progression 66

Table 3 Factors associated with advanced fibrosis among patients with 79

chronic HCV infection

Table 4 Published studies of paired liver biopsy assessment of fibrosis 82

progression in patients with chronic HCV infection

Table 5 Causes of fibrosis and cirrhosis 108

Table 6 Indications for liver biopsy 192

Table 7 Indications for transjugular liver biopsy 199

Table 8 Indications for and contraindications to laparoscopic liver biopsy 201

Table 9 Complications of percutaneous liver biopsy 207

Table 10 Contraindications to percutaneous liver biopsy 209

Table 11 Knodell Histological Activity Index 241

Table 13 METAVIR Score 247

Table 14 Scheuer scoring system 251

Table 15 Batts and Ludwig system 252

Table 16 Laennec scoring system for fibrosis 253

Table 17 Direct markers of liver fibrosis 296

Table 18 Diagnostic indices of FIBROSCORE > 0.5 for clinically 364

Table 19 Clinical and demographic data of patients 365

Table 20 Virological profile of patients 366

Table 21 Liver biopsy and needle characteristics 367

Table 22 Histopathological characteristics 368

Table 23 Viral aetiology and histopathology 369

Table 24 Lipid profile and HCV 369

Table 25 Clinical/ultrasonographic/biopsy parameters vs significant fibrosis 370

Table 26 Biomarkers vs fibrosis categories (statistically significant) 371

Table 27 Biomarkers vs fibrosis categories (other) 372

Table 28 Platelet parameters vs advanced fibrosis 373

Table 29 Logistic regression analysis 374

Graph 1 Receiver Operating Characteristic (ROC) curve Fibroscore vs 375

Graph 2 ROC curve, Fibroscore vs F2 Fibrosis 376

Graph 3 Boxplot of predicted probability of Fibrosis (Fibroscore) vs various 377

Graph 4 Boxplot of Hyaluronic Acid values vs different Fibrosis stages 378

Graph 5 Boxplot of Alpha 2 Macroglobulin values vs different Fibrosis stages 379

Graph 6 ROC curve of PDW Index vs F3 Fibrosis 380

Graph 8 Boxplot of Platelets vs various stages of Fibrosis 382

Graph 9 Boxplot PDW Index vs various stages of Fibrosis 383

Fig. 1 Gross image of a normal and cirrhotic liver 150

Fig. 2 Diagnosis of cirrhosis – CAT Scan 151

Fig. 3 Pathogenesis of liver fibrosis – Stellate Cell 152

liver showing the fenestrated wall

Fig. 5 Fibrogenic cascade 154

Fig. 6 Stellate Cell Activation 155

Fig. 7 Pathogenesis of intrahepatic portal hypertension 156

portal fibrosis, x 10 H & E

portal fibrosis, x 20 H & E

Fig. 17 Fibroscore assessment for clinically significant fibrosis 363

1. Goldstein ST, Zhou F, Hadler SC, et al. A mathematical model to estimate

1.1 Burden of chronic hepatitis