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

ii

Supervisor

Professor Lt Gen (R) Syed Azhar Ahmed

Hilal-e-Imtiaz (M), Sitara-e-Basalat

MBBS, Ph.D. (London), FRCPath (London), FCPS

Vice Chancellor

Baqai Medical University

Co Supervisor

Professor Jameel Ahmed

MBBS, MRCP

Professor of Medicine

Baqai Medical University

iii

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

iv

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”

v

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)

vi

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

ABSTRACT

2

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.

3

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

4

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

5

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

6

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

7

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

8

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.

9

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.

Chapter 1

INTRODUCTION

&

OVERVIEW

11

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

12

1. Introduction

Chronic liver diseases, CLD, are a major cause of morbidity and mortality in the

present day world 1-6. Chronic infections with hepatitis B and C viruses, alcoholic and

non-alcoholic fatty liver diseases are important causes of CLD 3,7-13. 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

complications 3,8,9,14-21. 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

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 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.

13

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

tridimensional network that induces loss of liver architecture 13,16,40-44. 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

such as portal fibroblasts may play a role 19,24,40,47,48. Hepatic stellate cells are located

14

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

trigger fibrogenesis 18, 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

oxygen intermediates and thus oxidative stress which can induce steatohepatitis 54,55.

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.

15

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

C was 0.13 Metavir points per year 61. 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

16

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

independent of the underlying disease aetiology are being explored 19. Animal studies

have identified a number of potentially important therapeutic targets, which include

glucose regulation, leptin 65, endothelin 66, angiotensin II 67, and a possible role for

PPARg 68 (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

17

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.

18

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

vein 57,58,59. 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

cirrhosis can be missed on liver biopsy in 10-30% of the cases 76,83-87. 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

19

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

20

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

better representation of liver fibrosis particularly those in the advanced disease 94, 95.

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

more standardized grading systems of hepatic fibrosis including the Knodell 97, later

modified by Ishak 98, 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.

21

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

recommendations for the management of these conditions 22,108,109. 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.

22

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.

23

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

bridging fibrosis compared with APRI 126. 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

24

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

1.00 (34% of all patients) 130. The detection of significant fibrosis F2 or greater had a

75% sensitivity and 85% specificity 130. 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

HCV (NPV85%, PPV 78%) 19, 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.

In the Fibropaca study 134, 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,

25

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-

IR, (insulin resistance by the homeostatic model assessment) 139; and performs well with

an area under the ROC of 0.77 21.

26

1.8 Direct markers of liver fibrosis

The direct markers of liver fibrosis reflect the process of fibrogenesis, the ECM

deposition or removal 24. 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

ranged from 0.82-0.92 24,140. In hepatitis B the AUROC is 0.98 but requires further

validation 141.

A study by Halfon P, et al, 140, 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.

In a recently reported retrospective study 143, 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.

The SHASTA index developed by Afdhal N, et al 144, consists of serum

hyaluronic acid, AST, and albumin. A cutoff of less than 0.30 was associated with a

27

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

marker of hepatic fibrosis and it has only been preliminary evaluated in CLD’s 24. 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

good standardization of the method for routine clinical use 24. 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.

28

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.

One such study 154, 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,

alcohol use, and age at infection, obesity and hepatic steatosis 155,156, influence the

progression to cirrhosis, but cannot accurately predict the risk of developing cirrhosis in

patients with chronic hepatitis C 16.

29

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.

According to Afdhal N 19, 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.

In one of these studies (n = 352) 158 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

30

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.

According to Afdhal N 19, many other studies have evaluated the effect of

interferon therapy on levels of fibrosis markers and have compared them to histological

findings 159,160. 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

markers of fibrosis, independent of a biochemical or virological response 161. 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

31

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.

According to Afdhal N 19, serum TGF-β levels have also been used to assess

disease progression 165,166. 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

32

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

requires a costly device to measure liver stiffness 24. 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

= .002). The authors concluding that 13C-CBT represents a valid indicator of plasma

caffeine clearance and also correlates reproducibly with hepatic dysfunction 170.

33

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

classified is higher, more than 40%-50% 121,130. 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.

34

(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

of portal tracts 75, and observer variability 22,77,93. 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

2.4% and non-attributable in 8.2% cases 72. 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.

35

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

36

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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

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153. Lo Iacono O, Garcia-Monzon C, Almasio P, et al. Soluble adhesion

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154. Huang H, Shiffman ML, Friedman SL, et al. A 7 gene signature identifies

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Hepatology 2007; 46: 297-306.

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Fibrosis progression in patients with chronic hepatitis C. the OBSVIRC,

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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161. Yagura M, Murai S, Kojima H, et al. Changes of liver fibrosis in chronic

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165. Kanzler S, Lohse AW, Keil A, et al. TGF-beta 1 in liver fibrosis: An

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166. Neuman MG, Banhamou JP, Malkiewicz IM, et al. Kinetics of serum

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LITERATURE

REVIEW

54

Chapter 2

EPIDEMIOLOGY

&

NATURAL HISTORY

OF

HEPATITIS B AND C VIRUSES

55

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

56

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

million chronically infected, and 50 million new cases are diagnosed annually 11,12,13.

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

consumption and chemotherapy) 15,16. The Hepatitis B e Antigen (HBeAg) negative

57

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

58

(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, 3.1% Vietnam 25, 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.

59

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

mothers 29,30. 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

maternal replicative status 29,31. Wang et al, 32 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

HBeAg acquisition. Milich et al, 33 had suggested that transplacental HBeAg movement

Area

% of population pos for: HBsAg anti-HBS

Infection Neonatal Childhood

North America (USA, Canada),

Northern, Western, and Central

Europe, Australia, New Zealand

0.2-0.5 4-6

rare infrequent

Eastern Europe, the Mediterranean,

Russia, Middle East, Central and

South America

2-7 20-55

frequent frequent

Parts of China, Taiwan, Southeast

Asia, Sub-saharan Africa

8-20 70-95

very freq. very freq.

60

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

decreased by 89% 40. 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

recommended 27. It can also be offered to adult population at large, to desirous

individuals. In the US, vaccination is also recommended for all adolescents 29. 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

contact, injection drug use, or occupational exposure to blood and blood products 11.

Percutaneous routes of exposure include transmission of blood or blood products 41,42,

contaminated health related equipment and needles 43,44,45, and injection drug use 42,46.

Less commonly, tattooing and acupuncture have also been implicated in HBV

61

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

as risks for infection 15. 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

contacts can occur, as HBV can survive in the environment for 7 days or more 49.

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

62

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

50% of patients clear HBeAg within 5 years of diagnosis, 70% within 10 years 55.

Regression of fibrosis may occur months to years after seroconversion 15,56.

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

hepatocyte genome 11, 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

63

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

percentage (30% to 50%) present with an icteric illness at the time of infection 66.

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.

64

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

65

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

66

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.

Table 2: Host and viral factors in HBV disease progression

Host factors Viral factors

Co-infection: HCV, HDV, HIV Ongoing viral replication, HBV DNA

Alcohol use Recurrent episodes of reactivation

Older age Possible genotype

67

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

nature of the disease was first recognized in the eight century by Pope Zacharis 93.

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

syphilis followed during the early 20th century 95,96,97. 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

68

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.

A series of experiments by Bradley and colleagues 106,107 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

undergo seroconversion several weeks after exposure 110. Identification of other clones

69

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

180 million 114; and the prevalence of impaired glucose tolerance is estimated at 197

million 115.

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

population is 0.6%, whereas the population prevalence is 1.8% 117. Nonetheless, these

estimates provide some idea of the worldwide pattern of infection 91. 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.

The prevalence is 0.01% in blood donors in Northern Ireland 121, in German

population 0.63% 122, 1.1% in France (randomly selected subjects)123, Spain 1.2%

70

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, to 1.6% in National Health and Nutrition Examination Survey 1999-2002 126, 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%

in Japan 23, 0.9%-5.3% in Saudi Arabia 130, 2.3% Indonesia 23, 5.52% Korea 131, 5.6%

Thailand 130, 5.2% Philippines 23, 5.6-16.9% Taiwan 130, 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

from across the country 134. 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

71

females 6%, and (ii) a relatively younger population was studied 136. In another study,

Qasmi et al noted 3% seropositivity for HBsAg in normal individuals of Karachi 137.

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

et al 143, Rehman et al 144 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.

72

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

prevalence of 6.5% 130,151. 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

many healthy individuals for minor problems 139. A study cited before from Pakistan

revealed that more than 10 injections per year in the previous 10 years were far more

likely to be associated with increased occurrence of HCV antibodies 151. Studies from

Southern Italy had shown that individuals who had received Salk Polio vaccine between

73

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

majority of infections among adults 159. 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

compared to normal ALT levels 160,161. 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

were responsible for 10%-15% of HCV transmission 163. 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.

74

Plasma derived products such as clotting factors concentrates frequently were

contaminated with HCV until 1987, when manufacturers incorporated effective viral

inactivation procedures 164. 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

techniques, contamination of multi-dose vials 170, 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

75

occurs at the time of delivery rather than in utero 178. 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

0%-11% 130 . 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

drug use increased from 31% in 1994 to 38% in 1999 and to 45% in 2003 181.

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.

76

3. High Titer Plateau Phase

It occurs between ramp up and seroconversion, typically lasts 40–60

days184.

4. Seroconversion

Antibody development occurs 9 weeks after exposure on average, but

cases are described in which antibody development occurred more than 12

months after first detection of viremia 182. 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

infection drug use reported chronicity rates of 76% to 86% 187,188,189. 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.

77

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

estimates of cirrhosis risk are strongly influenced by the population studied 197, 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

after 20 years of disease 197. 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

risk of complications 159. 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

progression with repeat biopsies every 3 to 5 years 159. 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

of developing decompensated liver disease is approximately 4% per year 199,200. 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

78

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

viral load 207, 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

I alleles have not been consistently linked with fibrosis progression 209,210,211. 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.

79

Type of factor Well established

factors

Emerging risk factors More controversial risk

factors

Host Age at infection

Duration of infection

Male Gender

Elevated ALT

Baseline fibrosis

Insulin resistance

Steatosis

HLA Class II

polymorphisms

Race

Baseline necroinflammation

Other genetic polymorphisms

Viral HBV infection

HIV infection

HCV viral load HCV genotype

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

increases with age 218. 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

80

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

alcohol use is predictive of more severe fibrosis 223. 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 =

81

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

hepatocytes affected) 228,229. 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

individuals 228,229,233,237. 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

have identified a strong association between steatosis and fibrosis 232,234,238,239. 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.

82

Published Studies of paired liver biopsy assessment of fibrosis progression in

persons with chronic HCV infection 159

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

Author,

year, ref.

N Baseline

characteristics

Interval

between

biopsies

% with progression Predictors of

progression

Collier,

2005 JDC’05 [242]

105 68% male, avg age

40 yr; 70% IDU;

86% increased

ALT

3.4 year mean

(range 0.4-

15.3)

26.7% progressed >

1 fibrosis unit

(scale to 5); 5%

progressed > 2

fibrosis units

Current

alcohol use

Ryder,

2004 SR’04 [243]

214 59% male; median

age 1st biopsy 36

yr; 52% IDU as

risk factor

2.5 median

(range, 1.9-9.4)

86% F0 or F1

at baseline

32.7% progressed >

1 fibrosis unit

(scale to 6)

Age at biopsy;

fibrosis score

at baseline

Wilson,

2006 LEW’06 [207]

121 82% male; median

age 42 yr; 92%

African Americans;

100% IDU; 27%

HIV infected

4.2 yr median

(range 2.8-6.0);

63% F0 or F1

at baseline

21% progressed > 2

fibrosis unit; rate =

0.04 fibrosis unit

/yr (scale to 6)

HCV viral

load; ALT >

60 IU/mL

Wali,

1999 MW’99 [244]

46 Mean age 37 yr 2.2 yr mean;

median at

baseline F0

(range, 0-2)

41% progressed > 1

fibrosis score; rate

= 0.15 fibrosis

units/yr (scale to 4)

ALT elevation

Colleta,

2005 CC’05 [245]

40 45% male; median

age 43 yr; ALT

persistently < 1.2

ULN

6.5 yr median;

98% F0 or F1

at baseline

35.7% progressed >

1 fibrosis score

High HCV

viral load; past

alcohol intake

> 20 g/d

83

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Hepatology 2005;42(4):838-45.

Chapter 3

HEPATIC INJURY

FIBROSIS

AND

FIBROGENESIS

THE CELL AND MOLECULAR BIOLOGY

105

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

106

3.1 Introduction

Fibrosis is a reversible wound healing process that occurs in almost all patients

with chronic liver injury 1,2. 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

as cirrhosis and its complications 2. 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

extracellular matrix components 7,8. 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

107

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

interstitial collagens, types I, and III 10,11. Quantitative and qualitative changes in many

other matrix components have been described, including proteoglycans 12,13, and matrix

glycoproteins, such as laminin 14,15, 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.

108

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.

Table 5. Causes of Fibrosis and Cirrhosis 1

1. Presinusoidal fibrosis

Schistosomiasis Idiopathic portal fibrosis 2. Parenchymal fibrosis A. Infections Chronic hepatitis B,C,D Brucellosis Echinococcosis Congenital or Tertiary Syphilis B. Drugs and Toxins Alcohol Paracetamol INH, Rifampicin, PZA Amiodarone Methotrexate Vit A a-Methyldopa C. Autoimmune Disease Autoimmune hepatitis D. Vascular Abnormalities Chronic passive congestion Hereditary hemorrhagic telengiectasia

E. Metabolic/Genetic diseases Wilson’s Disease Genetic hemochromatosis a1-Antitrypsin deficiency Carbohydrate metabolism disorders Lipid metabolism disorders Urea cycle disorders Porphyria Amino acid metabolism disorders Bile acid disorders F. Biliary Obstruction Primary biliary cirrhosis Secondary biliary cirrhosis Cystic fibrosis Biliary atresia/neonatal hepatitis Congenital biliary cysts G. Idiopathic/Miscellaneous Nonalcoholic steatohepatitis Indian childhood cirrhosis Granulomatous liver disease Polycystic liver disease 3. Postsinusoidal fibrosis Venoocclusive disease

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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

110

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

zone 1 fibrosis) 26. 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.

111

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

transdifferentiation to myofibroblasts 30. 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

death receptors 32, 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

factor-α (TNF-α) via TNF-receptor-1 can initiate apoptosis 32,33. 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

112

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.

113

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

for storing retinoids (vitamin A metabolites) within the body 48. 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

derived from hepatocytes seem to stimulate fibrogenesis in cultured stellate cells 51.

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

extracellular matrix 15,53. Further, the available evidence now indicates that the overall

increase in extracellular matrix deposition that is typical of cirrhosis largely can be

ascribed to excess production by stellate cells 15,54. Activation is also associated with

114

other important events, including proliferation 55,56, contractility 57, release of

proinflammatory cytokines 58,59 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

presence of steatosis alone without inflammation 63. Activated stellate cells have also

been observed in viral hepatitis and massive hepatic necrosis 64. Stellate cells have been

characterized in a number of other human conditions 62, 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

fibrotic liver 49. Endothelial cells from normal liver produce type III 15, 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

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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

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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

aldehyde adducts and collagen gene expression by stellate cells 72, and peroxides

stimulate collagen synthesis by cultured stellate cells 73,74. 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

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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

experimental and human liver injury 81,82,83. 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

factor 85, endothelin-1 86, insulin-like growth factor 87, thrombin 88, and transforming

growth factor alpha (TGF-α) 85,89.

PDGF is the most potent stellate cell mitogen identified. The induction of PDGF

receptors early in stellate cell activation increases responsiveness to this mitogen 75,90.

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.

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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

liver fibrosis, stellate cell apoptosis (programmed cell death) was prominent 94. 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

Stellate cells migrate towards cytokine chemoattractants 95,96. 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

matrix components, including cellular fibronectin, and proteoglycans 97,98,99.

Transforming growth factor beta-1 seems to act by directly (and, to a lesser extent,

indirectly) stimulating extracellular matrix production in stellate cells 100,101.

Transforming growth factor beta-1 can be produced in a paracrine manner from Kupffer's

cells 102 or by stellate cells themselves, and thus can be an important autocrine factor

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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

capacity of stellate cells as they become activated 110. 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

(PDGF), 56,111 monocyte chemotactic factor (MCP-1), 59 hepatocyte growth factor, 112

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

and stimulates hepatic fibrogenesis 118. 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

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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

basement membrane inhibits stellate cell activation and matrix synthesis, 124 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

basement membrane extracellular matrix further 125. 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

bands typical of end stage cirrhosis contain large numbers of activated stellate cell 127.

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.

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As stellate cells become contractile, their expression of the cytoskeletal protein α

smooth muscle actin and smooth muscle myosin is increased 128,129. This observation led

to work demonstrating that stellate cells are contractile, 130,131,132 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

may vary 136. 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

predominates is combined with increased sensitivity to autocrine endothelin 1 127.

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

the sinusoidal endothelium 139, 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

to the way that tissue pericytes control blood flow in systemic capillary structures 142.

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.

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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

place before export 62. 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

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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.

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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

cells 157 and may also inhibit interleukin-4 (IL-4) 158 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.

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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-

β1 in hepatocytes 99; 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

TGF-β1 are paracrine from Kupffer cells 102 and autocrine 103,104 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

downstream signal transduction) knock out mice did not develop hepatic fibrosis 166;

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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

inhibition, therefore appears attractive 9,161,169. 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,

indicating that PDGF serves as an important autocrine factor during liver injury 111.

Although regulation of PDGF production in stellate cells is complex (and intricately

127

related to other events in the wounding milieu) 171,172, 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

nuclear transcription factors 173. 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

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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

on macrophages 152,153. 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

than wild-type controls 177. 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

process 181. 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.

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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

eradication is associated with a reduction in fibrosis 109,185-190. Studies in patients with

chronic hepatitis C suggest that IFN-α therapy can prevent fibrosis progression, even in

nonresponders to antiviral therapy 191-194. 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.

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ii) Interferon-γ

According to Rockey DC 178, 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

chronic hepatitis 205 and because long-term side effects may result from its profound

immunomodulatory effects 178. 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-

stimulating factor 158 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

is endothelin 207. This family of potent vasoconstrictors 208 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

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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

local control of vascular tone, including regulation of basal blood pressure 212. 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.

In liver, endothelins are produced by sinusoidal endothelial cells 220. 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

effect is probably involved in elevated intrahepatic resistance to blood flow 143,144,223.

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.

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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

19 isotypes with a common trihelicoidal structure 25. 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

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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

type of liver injury. The total collagen increases 3 to 10 fold 229. 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.

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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

type XIV; and several heparin sulphate proteoglycans, including decorin, perlecan 233,

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.

The high density matrix also activates stellate cells 124, and leads to a decrease in the

fenestrations of sinusoidal epithelial cells 237, which may impair the transport of solutes

from the sinusoid to the hepatocytes 1. 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,

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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

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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-

6,5 markedly inhibited the progression of thioacetamide-induced liver cirrhosis 245.

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.

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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

mediate interactions between inflammatory cells and the endothelial wall 248-250. 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

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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.

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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

MMP-2 activity 251. 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.

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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.

MMP-9 is secreted locally by Kupffer cells 251. Stimulation with IL-1α causes

robust induction of pro-MMP-9 (the pre- cursor 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.

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ii) Progression of fibrogenesis & fibrosis

More importantly, progressive fibrosis is associated with marked increases in

TIMP-194,267, and TIMP-2 268, 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

fibrosis 254. 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.

Unique mechanisms of TIMP-1 regulation in stellate cells 270, 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:

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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.

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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

fibrogenesis 148,278, 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.

Shin JY et al, 274 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.

In cell culture the core protein has transforming potential 280. It can induce

apoptosis. It can interact with the intracytoplasmic TNF-α type 1 and the lymphotoxin β

receptors, enhancing their pro-apoptotic signal transduction 281, 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

apoptosis 284. 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,

inhibiting assembly and secretion of regular VLDL particles 286. As mentioned by

Schuppan D 155, overexpression of the cytoplasmic located HCV core has been analyzed

144

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,

obviously, either by repression of protein kinase B or alternate pathways 291. 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

145

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

developed HCC in a single case (1/43 vs 0/35) 298. 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

146

[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

viruses infecting humans. Although HBV is considered as a non-cytopathic virus 301

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

surface protein has been shown to induce apoptosis in cell lines 304. 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

147

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

148

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

al, 310 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

149

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.

150

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.

151

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.

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.

152

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).

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.

153

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

154

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.

155

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 Rockey DC. Hepatic Fibrosis, Stellate Cells, and Portal Hypertension. Clin Liver Dis 2006; 10: 464.

156

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. Rockey DC. Hepatic Fibrosis, Stellate Cells, and Portal Hypertension. Clin Liver Dis 2006; 10: 473.

157

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185

Chapter 4

LIVER BIOPSY

&

HEPATIC

HISTOPATHOLOGY

186

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

187

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

188

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

189

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

staging (fibrosis) in most patients 16,17; including patients with persistently normal

aminotransferase levels 18,19, and for evaluating steatosis, all histologic features that affect

the natural history, prognosis, and therapeutic outcome 20-22. In chronic hepatitis B the

same applies 13,23.

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

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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

complete portal tracts 13. 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

191

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)

192

· Cutting needles (Trucut needle,Vim-Silverman needle)

· Spring loaded cutting needles that have a triggering mechanisms

Table 6 Indications for liver biopsy

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.

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

193

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

liver architecture and may thereby increase the diagnostic yield 12, however this has

undergone re-evaluation 13. 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.

According to Grant A 12, 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

bleeding 34,38. 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.

194

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.

195

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

site of entry of the biopsy needle may be subcostal 12. One view claimed that the

complications are slightly more frequent with the transthoracic (4.1%) than the subcostal

route (2.7%) 39, 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. According to another source 34, 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

196

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

had to be moved after it had been marked by the percussion technique 43. 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)

and led to abortion of the procedure in four patients 45. Similarly, two large studies

demonstrated a lower complication rate and a higher diagnostic yield using

ultrasonography guidance 34,46,47.

In one of the reviews Cholongitas E et al 13, 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.

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One way ultrasound can reduce the incidence of bleeding is potentially it can

reduce the number of passes made into the liver 49. 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

passes has been documented in patients with and without malignancy 38, 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

viscus 50; 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

percutaneous approach first described in 1984 51,52. 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.

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Transvenous liver biopsy was first described in 1964 53. 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

performed 56. 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.

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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

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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

specimens 65. Patients with focal hepatic lesions are good candidates for fine needle

aspiration biopsy, especially if they have a history of cancer.

The diagnostic accuracy ranges from 80 to 95 percent 24, and is substantially

affected by the expertise of the cytopathologist. Cytologic findings that are negative for

cancer, however, do not rule it out.

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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

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biopsy needles guided by ultrasound in over 95% of patients 66, 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

years 69, 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

complications have been retrospective 36,55. 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

between 0.13% to 0.33% 10,12. 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

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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

hours 36,72. 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

or malignant disease, both of which are risk factors for bleeding 38,71. 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

bleeding does not stop with transfusion alone 10,12; 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

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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

the procedure 36,38,73,74. In a large study of the complications of 68,276 liver biopsies,

haemoperitoneum occurred in 0.32% of patients 36. 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

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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

elevation of transaminases 78-80. 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

not appear to influence the incidence of haematoma 82. 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

large haematoma impinging on the biliary tree 84. 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

206

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

pain, occurring in about 3% of liver biopsies 39, 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.

It has an incidence of 0.05% 12. Piccinino et al, 36 reported 4 cases in 68,276 biopsies.

Cholecystitis 88, and pancreatitis 89 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%

and 0.1% 36. 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.

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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.

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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

parenchyma 71. 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.

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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.

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4.11.3 Prothrombin time

According to Grant A, 12 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

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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

group 12 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

surrogate marker for the severity of liver disease) 12,100. 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.

212

4.11.6 Ascites

According to Grant A, 12 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

According to Grant A 12, 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

213

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

have evaluated optimal length 25,107 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

potential limitation of liver biopsy 108-110. 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.

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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,

Abdi et al, 110 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

problems, although to a lesser extent. Soloway et al, 108 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

fibrosis. Less favourable results were published by Baunsgaard et al, 112 who showed a

concordance in only 36 of 50 patients when evaluating the fibrosis amount with 2

biopsies in same patients 25.

According to Regev et al, 113 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.

Lately, Regev et al, 113 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

215

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

biopsy specimens selected as 15 mm or longer 113,121. 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

According to the review 13, Holund et al 124, first studied diagnostic

reproducibility relative to specimen size in 100 selected liver biopsy specimens that were

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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.

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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

progression studied 128. In an attempt to standardize the histological assessment several

semi-quantitative scoring systems have been proposed and evaluated.

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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 intra- observer 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,

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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

document length 129, 3 used samples 10 mm or longer 130,131,133, and 1 used samples 15

mm or longer 132. According to the review13, the study that included histopathologists

with different levels of expertise, duration, and location of practice 133 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

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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

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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

CPT’s were 17.7 + 5.8 mm and 7.5 + 3.4 mm, respectively. According to the review 13,

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

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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

According to the same review 13, 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.

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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.

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Another study 137, 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 under- staging 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.

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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

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(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

percutaneous liver biopsy 25,107 for assessing chronic viral hepatitis require longer

specimens than before (>20-25 vs >15 mm) 7 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.

Thus as evidenced in a study by Goldin et al 128, 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

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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

increases with needle size and number of passes 12,38,139. 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

tracts well below the published minimum sample size requirements 25,107 in more

than half the cases. Thus according to the review 13, 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.

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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

staging and grading 140 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.

Thus according to Cholongitas E, et al 13, 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.

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Histopathology of chronic hepatitis B and C According to Goodman ZD, 141 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

rare but well documented cause of chronic hepatitis 142. 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

progress to cirrhosis 141.

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 According to Desmet V, 143 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.

230

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

plasma cells, and other mononuclear cells (histiocytes and immature lymphocytes) 143.

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.

Hepatitis associated bile duct lesions, according to Goodman ZD, 141 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

arising from injured ducts rather than ducts themselves 148. The ductal lesions have been

seen in all forms of hepatitis, but most often in hepatitis C 141,149.

231

Interface hepatitis, according to Goodman ZD141, is now the preferred term for

the lesion formerly known as piecemeal necrosis 150,151. 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.

According to Goodman ZD 141, 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

232

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.

Fibrosis, according to Goodman ZD, 141 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

where fibrous tissue previously did not exist 141. 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.

233

Cirrhosis, according to Desmet V, 143 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.

The morphologic classification of cirrhosis is based on the size of nodules 155.

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

234

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

in adjacent areas. Inflammation and necrosis are generally absent or minimal 158. 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

does not appear overnight, but may take months or years to develop 143, a one time liver

biopsy specimen may not capture the dynamic nature of the process.

4.23 Histopathology of hepatitis B virus

According to Goodman ZD, 141 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

235

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

According to Goodman ZD, 141 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

236

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

According to Desmet V et al, 143 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.

237

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

entities. A change of view on chronic hepatitis was indicated 166. 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.

238

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

Ishak 151. In addition, another semiquantitative system, the Metavir score, has been

increasingly used for chronic hepatitis C 126,131. Other scoring systems also widely used

are: a simple scoring system proposed by Scheuer 169, 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

According to Bonis PA, 175 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

239

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.

240

A subsequent study evaluated the Knodell score among ten pathologists using a

cohort of 30 liver biopsy specimens from patients who had documented hepatitis C 131.

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.

241

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

242

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.

243

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

244

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)

245

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.

246

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

According to Bonis PA, 175 the Metavir score was developed in an attempt to

address some of the problems with the Knodell score 126,131. 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).

247

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

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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.

249

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

natural history of hepatitis C 175. Thus, earlier on, it was unclear whether individuals

progress from early to late stages at a constant, linear rate, although this hypothesis has

been proposed 169. 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.

250

4.30 Guidelines by International Association for the

Study of the Liver

For routine diagnosis and patient management, one authority 141, 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.

Following the recommendations of IASL panel 170, 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.

According to the source cited above 141, 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.

251

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.

252

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.

253

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.

254

Fig 8 Chronic hepatitis with mild activity and no fibrosis, x 10 H& E

255

Fig. 9 Chronic hepatitis with fatty change (steatosis), moderate activity and portal

fibrosis, x 10 H & E

256

Fig. 10 Chronic hepatitis with fatty change (steatosis), moderate activity and portal

fibrosis, x 20 H & E

257

Fig. 11 Chronic hepatitis with moderate activity and portal fibrosis, x 10 H & E.

258

Fig. 12 Chronic hepatitis with moderate activity and portal fibrosis, x 20 H & E.

259

Fig. 13 Chronic hepatitis with moderate activity and bridging fibrosis, x 10 H & E

260

Fig. 14 Chronic hepatitis with moderate activity and bridging fibrosis, x 20 H & E

261

Fig. 15 Chronic hepatitis with moderate to marked activity and early cirrhosis,

x 10 H & E

262

Fig. 16 Chronic hepatitis with moderate to marked activity and early cirrhosis,

x 20 H & E

263

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Chapter 5

NON INVASIVE

EVALUATION

OF

LIVER FIBROSIS

Contents

280

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 13C- Caffeine Breath Test 308

281

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

present day world 1-6. Chronic infections with hepatitis B and C viruses, alcoholic and

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non-alcoholic fatty liver diseases are important causes of CLD 7-13. 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

complications 3,8,9-21. 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.

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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

fibrosis in the liver 42. 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

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· 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

dependent upon binding to specific receptors by hepatic endothelial cells 43. 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

have been investigated as markers of liver fibrosis in several studies 45-53. 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.

285

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

attempted to correlate these routine investigations with stage of hepatic fibrosis 39. 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

between AST levels and the presence of advanced fibrosis 62. 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

286

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

a ratio of >1.0 is suggestive of this diagnosis 61. This has been shown for both viral

disease 67 and nonalcoholic fatty liver disease 68. 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

liver disease in drinkers 69. 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

66% and 72% 69, 70. 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

287

as indirect and direct markers of liver fibrosis to diagnose cirrhosis 70. 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.

In this study 70, 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

288

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

including those with HIVinfection 73-75, but an independent study did not achieve the

same results 76.

Rossi et al 76, 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

289

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.

Commenting on the Rossi et al study, in another study the Fibropaca study, 79 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.

290

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

84% cases for F > 2, in 95% of cases for F > 3, and in 94% of cases for F > 4 81.

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

fibrosis associated with greater histologic inflammation 77. 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.

A meta-analysis of 16 publications and 1570 patients, by the same group 83

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.

291

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

Forns et al 55, 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

0.86 for the estimation group and 0.81 for the validation group 55.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

value for the identification of patients with more advanced liver disease 19. 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

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sophisticated variables (such as Fibrotest). In comparing with the study by Imbert-Bismut

(Fibrotest) 54 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

authors of the later test 57. 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

abnormalities in patients with hepatitis C 57, 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)

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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.

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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

According to Afdhal N 39, 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

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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

hepatitis or more active hepatitis) and may be high prior to the deposition of ECM 89,90.

Second, levels tend to fall in response to treatment of the underlying disease process,

often before to any discernible reduction in the stage of fibrosis 91,92. 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

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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

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Fibrosis markers can also be classified according to their molecular structure 19.

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

According to Afdhal N 39, 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

endothelial cells 94. 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.

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A study by Halfon et al, 103 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.

According to Afdhal N 70, 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

significant fibrosis and cirrhosis 53, 103. 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.

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5.14 YKL-40 (Chondrex)

YKL-40 is a novel marker of liver fibrosis. YKL-40 (chondrex, human cartilage

glycoprotein-39), according to Saitou et al 106, is a recently described glycoprotein that

belongs to the chitinase family 107. YKL-40 m RNA was strongly expressed in human

liver and arthritic articular cartilage107,108 and was elevated in the synovial fluid 109,

alcoholic liver disease 110, recurrent breast cancer, and colorectal cancer106.

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

cells 109, activated macrophages, neutrophils, and in particular from cells located in

tissues with increased remodeling/degradation or inflammation of the extracellular

matrix, such as hepatic stellate cells 111. It is growth factor for fibroblasts 112,

chondrocytes and synovial cells. It is a chemo-attractant for endothelial cells 113,

modulates vascular endothelial cell morphology by promoting the formation of branching

tubules, indicating that YKL-40 may have a role in angiogenesis 106.

The authors 106 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

markers of hepatic fibrosis, including PIIINP, MMP-2, and TIMP-1 114. In a study of

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YKL-40 in alcoholic liver disease suggested that it could function as an independent

marker of clinical outcomes 115.

5.15 Collagen

According to Afdhal N 39, 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

chronic liver disease 71,97,116. 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

large fibrotic bands in alcoholic liver disease 117. Serum type IV collagen is increased in

chronic liver diseases relative to controls 118. 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.

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5.16 Matrix metalloproteinases and inhibitors

According to Afdhal N 39, 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

demonstrate increased expression of TIMP’s 120,121. 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.

According to NA 19, Boeker et al 122, 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

liver disease 123,124. Serum MMP-1 and MMP-3 levels have not been shown to be of

significant diagnostic value51,125.

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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

antiviral therapy 127. 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

the levels of soluble adhesion molecules and liver inflammation and fibrosis 128,129,130.

Increased expression of intracellular adhesion molecule-1 (ICAM-1) occurs in virus

infected hepatocytes; whereas expression of vascular adhesion molecule-1 (VCAM-1)

may be seen in association with progressive hepatic fibrosis 128-130. 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:

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5.18 FibroSpect

McHutchison and colleagues in a retrospective cohort study evaluated the

FibroSpect assay 131. 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

seen at the two extreme spectrums of disease, stage 0 and stage 4 39. 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

accurate delineation between stages was not possible 131. 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.

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5.20 European Liver Fibrosis Group assay

The ELF group 133 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

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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

glutamyl transferase, bilirubin, platelets, and apolipoprotein A1, the adjusted R 2

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

length, > 20 mm 135,136. It has been suggested that the variability is greater for AOF

evaluation than it is for histological staging 135. 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.

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Although there was less observer variability for histological staging when a

consensual reading was performed by two experts 137, 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

agreement in relation to F stages 137, 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

In this study 139, 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

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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

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>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.

5.25 13C- Caffeine Breath Test

According to Park et al 142, 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

administered 13C-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

caffeine half-life; 2) the oral 13C-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.

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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.

In the larger of these studies (n = 352) 82, 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

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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.

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According to Afdhal N 19, many other studies have evaluated the effect of

interferon therapy on levels of fibrosis markers and have compared them to histological

findings 93,105,114,145-153. 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 145. 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

inflammation 94,105,151,152. 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.

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5.27 Fibrosis Markers to Predict Disease

Progression

According to Afdhal N 19, 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

predictive of disease progression 101. 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

cohort of 370 patients with alcoholic liver disease 115. 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.

According to Afdhal N 19, serum TGF-β levels have also been used to assess

disease progression 160,161. 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

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those who did not progress 160. No such correlation could be found for PIIINP, serum

transaminases, or viral load.

Neuman and colleagues 161, 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.

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

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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

Caucasian patients than clinical factors 162. The clinical factors such as age, gender,

alcohol use, and age at infection, obesity and hepatic steatosis, 163-165 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

cohorts, and objective genotyping calls that are available for all patients 166. 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.

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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 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

population attributable fraction (PAF) for disease occurrence 167. 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

ligand lipopolysaccharide (LPS) 170. In patients with chronic hepatitis C, NS5A induces

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the elevated expression of TLR4 in B cells 3- to 7- fold 171. Moreover, LPS-TLR4

pathway plays an important role in the hepatic fibrogenesis 172. 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

According to Afdhal N 19, 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

large varices 39. 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

100%, and diagnostic accuracy of 94% 40. 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,

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and liver surface nodularity; reported accuracy of cirrhosis detection between 82% and

88% 175,176, 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

study cited above 175. 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

clinical impression 19. 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

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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.

According to Afdhal N 19, initial studies of hepatic elastography in 19

hepatectomy specimens correlated with histologic analysis 180. 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

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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

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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

measurement for the detection of cirrhosis in patients with chronic liver disease 181.

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.

The limitation is that it requires a costly device to measure liver stiffness 24.

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

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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.

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5.31 Platelets in Chronic Liver Disease – The role of

Spleen

Thrombocytopenia is a commonly encountered hematological condition in

chronic liver disease and cirrhosis 182. 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

increasing platelet production 85,183,184. 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

exchangeable pool 85. 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.

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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

the liver 190,191. Its levels are insufficient in advanced liver failure 182. 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.

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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

maintained or even increased in patients with cirrhosis 86,194 including the study by

Kajihara et al182.

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

325

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,

326

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

volume (MPV), platelet distribution width (PDW) 200. 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.

327

One observer 200, 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

altered platelet homeostasis than MPV in liver cirrhosis 203. 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.

328

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.

329

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PATIENTS

and

METHODS

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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

352

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

353

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.

354

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

355

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.

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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.

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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).

ANALYSIS

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DISCUSSION

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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

noninvasive markers, but those have been limited in scope 1-3. 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.

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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

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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?

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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

fibrosis as detected by digital image analysis and staging by a pathologist 21. 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,

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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

such as connective tissue damage 17. Indeed, their relationship with fibrosis is complex.

One explanation is that, the blood markers interact both with the dynamics and the cause

of fibrosis 33,34. 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

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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

patients infected after the fourth decade have a higher risk of disease progression 38-40. 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

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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

to ALT 24,43. 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

B and C 13,20,23,44. 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

shunts 48. 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

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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

the increased serum immunoglobulin levels in patients with cirrhosis 49. 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

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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

with clinical severity 60-62. Liver injury increases hyaluronic acid (a glycosaminoglycan)

production by the hepatic stellate cells and decreases its clearance by the sinusoidal

endothelial cells 63. 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

matrix proteins and enhance fibrotic processes in the liver 66,69. 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

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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

macroglobulin was the most informative 13. 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

chronic liver disease and cirrhosis 54. 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 increasing platelet production 72,73. 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

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thrombocytopenia 55. 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

distribution width (PDW) 78. Though routinely evaluated, the clinical inferences are

usually not drawn.

A decrease of mean platelet volume (MPV) has been observed in liver cirrhosis

with thrombocytopenia 79. It has also been shown that, platelet distribution width is a

better indicator of altered platelet homeostasis than MPV in liver cirrhosis 80. 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

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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,

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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

length) and 2% to Fibrotest 82. 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.

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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

400

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

concerns about the impact of lipid abnormalities in patients with hepatitis C 91,

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,

401

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.

402

Genetics and Proteomics

In addition to the indirect and direct noninvasive markers, other novel and

exciting new indices have been developed.

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. 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

Caucasian patients than clinical factors 95. 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 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. 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

403

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

identified in several proteomic studies 97,98. 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.

404

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.

405

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99. Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of liver

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hepatitis C-related fibrosis: a systematic review of diagnostic test accuracy. Am J

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chronic liver disease. World J Gastroenterol 2008; 14(21): 3396-3402.

RESULTS

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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.

360

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

361

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%.

362

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.

363

Fig 17. The Fibroscore assessment for clinically significant fibrosis

Fibroscore 0.00-1.00

Fibroscore < 0.5

64 patients

Fibroscore > 0.5

24 patients

F2-F4 19 patients

F0-F1 5 patients

F0-F1 60 patients

F2-F4 4 patients

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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)

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____________________________________________________

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%)

________________________________________________________________________

366

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%)

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_______________________________________________________

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%)

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_______________________________________________________

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%)

____________________________________________________

369

_______________________________________________________

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

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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

(12-15 mm)

2 (3.1%) 1 (4.3%) 0.773

Splenomegaly 2 (3.1%) 3 (13%) 0.076

Biopsy variables

No to Mild Activity

(necroinflammatory)

42 (64.6%) 2 (8.7%) 0.000

Mod to Severe Activity 23 (34.5%) 21 (91.3%) 0.000

Steatosis 27(41.5%) 15 (65.2%) 0.051

371

________________________________________________

Table 26. Biomarkers vs Fibrosis categories

(statistically significant)

Variable Stage F0-F1 Stage F2-F4 p value

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

___________________________________________________________

372

Table 27. Biomarkers vs Fibrosis Categories (other)

Variable Stage F0-F1 Stage F2-F4 p value

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

___________________________________________________________

373

Table 28. Platelet Parameters vs Advanced Fibrosis

Variable Stage F0-F2 Stage F3-F4 p value

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

___________________________________________________________

374

________________________________________________

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

Acid

0.080 8.796 .003 1.084 1.028-1.143

Alpha 2

Macroglobulin

0.518 6.032 .014 1.678 1.110-2.537

Platelets -.040 5.694 .017 .960 .929-.993

Constant -4.795 1.289

___________________________________________________________

375

Graph. 1 Receiver Operating Characteristic (ROC) curve Fibroscore vs F3

Fibrosis

Area under the ROC .938 (95% CI; .888-.988)

376

Graph. 2 ROC curve, Fibroscore vs F2 Fibrosis

Area under the ROC .808 (95% CI; .613-1.00)

377

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.

378

Graph 4. Boxplot of Hyaluronic Acid values vs different fibrosis stages.

379

Graph 5. Boxplot of Alpha 2 Macroglobulin values vs different Fibrosis

stages

380

Graph 6. ROC curve of PDW Index vs F3 Fibrosis

Area under the ROC .801 (95% CI; .671-.930)

381

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)

382

Graph 8. Boxplot of Platelets vs various Stages of Fibrosis

383

Graph 9. Boxplot PDW Index vs various stages of Fibrosis

SUMMARY

&

CONCLUSION

416

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.

417

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.

418

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.

419

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.

420

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.