Histological assessment of non-alcoholic fatty liver disease.pdf

REVIEW

Histological assessment of non-alcoholic fatty liver disease

S G HubscherDepartment of Pathology, University of Birmingham, Birmingham, UK

Hubscher S G

(2006) Histopathology 49, 450465

Histological assessment of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is an import-ant complication of the metabolic syndrome, which isbecoming an increasingly common cause of chronicliver disease. Histological changes typically mainlyaffect perivenular regions of the liver parenchyma andinclude an overlapping spectrum of steatosis, steato-hepatitis and persinusoidal or pericellular fibrosis, insome cases leading to cirrhosis. Once cirrhosis hasdeveloped, typical hepatocellular changes are often nolonger conspicuous, leading to such cases being mis-takenly diagnosed as cryptogenic. Portal inflamma-tion, ductular reaction and periportal fibrosis can alsobe seen as part of the morphological spectrum ofNAFLD, particularly in the paediatric population.

Hepatocellular carcinoma has also been described asa complication of NAFLD-associated cirrhosis. NAFLDis also an important cofactor in other chronic liverdiseases, especially hepatitis C. Histological assessmentshave an important role to play in the diagnosis andmanagement of NAFLD. These include making thepotentially important distinction between simple stea-tosis and steatohepatitis and providing pointers to theaetiology, including cases where a dual pathologyexists. A number of systems have been devised forgrading and staging the severity of fatty liver disease.These require further evaluation, but have a potentiallyimportant role to play in determining prognosis andmonitoring therapeutic responses.

Keywords: cirrhosis, fatty liver, grading and staging, insulin resistance, liver biopsy, metabolic syndrome,non-alcoholic, steatohepatitis, steatosis

Abbreviations: ALD, alcoholic liver disease; ASH, alcoholic steatohepatitis; HCC, hepatocellular carcinoma;HCV, hepatitis C virus; IR, insulin receptor; LFT, liver function test; NAFLD, non-alcoholic fatty liver disease;NASH, non-alcoholic steatohepatitis; ROS, reactive oxgen species; TNF, tumour necrosis factor

Introduction

Non-alcoholic fatty liver disease (NAFLD) comprises amorphological spectrum of liver lesions, closely resem-bling those seen in alcoholic liver disease (ALD), butdeveloping in individuals who do not consume exces-sive amounts of alcohol. The great majority of NAFLDoccurs in the setting of the metabolic syndrome inwhich insulin resistance plays a key role (primaryNAFLD).1 Other much less common causes of NAFLDare summarized in Table 1.2,3

The metabolic syndrome is an increasingly commonmetabolic disorder related to the increasing worldwideprevalence of obesity.4,5 Diagnostic criteria vary, butimportant components are central or visceral obesity,glucose intolerance (Type 2 diabetes), systemic hyper-tension and dyslipidaemia (hypertriglyceridaemia andlow high-density lipoprotein-cholesterol). Prevalencerates of the metabolic syndrome are as high as 50% insome western countries. Most of the adverse effects ofthe metabolic syndrome are associated with com-plications related to diabetes and cardiovascular dis-ease. However, there is an increasing recognition thatNAFLD is also a common component of the metabolicsyndrome, with estimated prevalence rates in theregion of 1030% in countries where the metabolicsyndrome is common.3,610

Address for correspondence: Prof. Stefan G Hubscher, Department of

Pathology, University of Birmingham, Birmingham B15 2TT, UK.

e-mail: [email protected]

2006 The Author. Journal compilation 2006 Blackwell Publishing Limited.

Histopathology 2006, 49, 450465. DOI: 10.1111/j.1365-2559.2006.02416.x

This article will review the histological changesoccurring in NAFLD, highlighting areas in which liverbiopsy is most important in the diagnosis andmanagement of patients with this disease. Briefconsideration will also be given to clinical aspects ofNAFLD and some of the proposed pathogenic mech-anisms.

Clinical impact of non-alcoholic fatty liverdisease

Although a non-alcoholic form of fatty liver diseasewas first recognized some 25 years ago,11 the clinicalsignificance of NAFLD has started to be properlyrecognized only during the last few years. Severalstudies have shown that NAFLD is the commonesthistological diagnosis in patients with otherwiseunexplained persistently abnormal liver function tests(LFTs), being present in 4070% of such cases.1215

Perhaps of greater concern is the recognition that thefull histological spectrum of fatty liver disease can alsobe seen in up to 70% of people with the metabolicsyndrome who have normal LFTs.16,17 Epidemiologicalstudies have suggested that NAFLD is the mostimportant cause of so-called cryptogenic cirrhosis18,19

and it has also been implicated in the pathogenesis ofhepatocellular carcinoma.2024 Based on these obser-vations, it has been suggested that, in countries whererisk factors for the metabolic syndrome are prevalent,NAFLD is likely to become the commonest cause ofchronic liver disease.8

Pathogenic mechanisms

The presence and severity of NAFLD are closely relatedto risk factors for insulin resistance and the metabolicsyndrome.6,25,26 The relationship between insulinresistance and NAFLD is complex and the pathogenicmechanisms involved in mediating liver damage in thissetting are still not fully understood.13,6,10,2733 Atwo hit process has been proposed.

The first hit involves accumulation of fat inhepatocytes. This is closely associated with metabolicderangements related to central obesity and insulinresistance. An increased delivery of free fatty acids tothe liver is combined with impaired fatty acid meta-bolism in hepatocytes, leading to a net accumulationof triglyceride within the liver. It has also beenrecognized more recently that the accumulation of fatin the liver exacerbates insulin resistance by inter-ferzing with phosphorylation of insulin receptor sub-strates.34 Increased hepatocellular expression of themicrosomal cytochrome P450 2E1 (CYP2E1) may beimportant in mediating this process,35 which leads toa potential vicious cycle where the metabolic syn-drome causes fatty change in the liver and viceversa.6

The factors involved in determining the progressionfrom steatosis to the more serious lesions of steato-hepatitis and fibrosis (discussed further below) are morecomplex and less clearly established. Oxidative stress is

Table 1. Classification and aetiology of fatty liver disease(from Reid 2001 and Ramesh 2005)2,3

Alcoholic

Non-alcoholicPrimary (insulin resistance)Obesity, diabetes, hyperlipidaemia

Secondary (other causes)Drugs

Amiodarone

Glucocorticoids

Perhexiline maleate

Synthetic oestrogens

Tamoxifen

4,4diethylaminoethoxyhexestrol

Isoniazid

Industrial exposure to petrochemicals

Surgical procedures

Jejunoileal bypass

Biliopancreatic diversion

Extensive small bowel resection

Gastroplasty for morbid obesity

Total parenteral nutrition

Inborn errors of metabolism

Wilson disease

Abetalipoproteinaemia

Tyrosinaemia

Hypobetalipoproteinaemia

Lipodystrophy

Congenital

Acquired (HIV-related)

Non-alcoholic fatty liver pathology 451

2006 The Author. Journal compilation 2006 Blackwell Publishing Ltd, Histopathology, 49, 450465.

thought to play a key role in the second hit, whichalso involves peroxidation of lipid accumulated withinsteatotic hepatocytes. Factors promoting lipid peroxi-dation and oxidative stress include induction of hepaticCYP2E128 and mitochondrial dysfunction leading tothe formation of reactive oxgen species (ROS).33

Immunohistochemical techniques have demonstratedincreased staining for lipid oxidation products in liverswith fatty change, with further increases in thoseshowing steatohepatitis.36,37 Immune responses tolipid peroxidation products may also be involved indisease progression.38 Other changes occurring in themetabolic syndrome that could mediate hepaticinflammation include increased levels of proinflam-matory cytokines such as tumour necrosis factor(TNF)-a, which may be released directly from adipo-cytes in visceral fat, and decreased levels of anti-inflammatory cytokines such as adiponectin, alsoproduced by adipocytes.1,5,26 These observations sug-gest that the metabolic syndrome itself may be involvedin mediating the second as well as the first hit inNAFLD.29 Adiponectin is also produced within theliver, mainly by endothelial cells, whilst its receptorsare expressed on hepatocytes.39 A significantly reducedintrahepatic expression of adiponectin and its recep-tors has been observed in biopsies showing non-alcoholic steatohepatitis (NASH) compared with simplesteatosis.39

Interactions between lipid-laden hepatocytes, Kupf-fer cells, inflammatory cells and hepatic stellate cellsare important in the subsequent development offibrosis and cirrhosis.40 Factors regulating this pro-cess include activation of profibrogenic cytokines,such as interleukin-10 and transforming growthfactor-b, which in turn are regulated by otherfactors including leptin and neurotransmitters suchas noradrenaline.1,26 Lipid released from damagedhepatocytes may also result in mechanical and orinflammatory cell-mediated occlusion of hepaticvenules, leading to parenchymal collapse andfibrosis.41

Liver lesions in non-alcoholic fatty liverdisease

Similar to the changes seen in ALD, the histologicalchanges occurring in NAFLD typically predominantlyaffect the liver parenchyma, where they are mainlypresent in perivenular regions (acinar zone 3). How-ever, there is an increasing recognition that portal andperiportal lesions can also be seen as part of thespectrum of fatty liver disease.

parenchymal les ions in non-alcoholic fattyliver disease

These can be divided into three main categories: non-alcoholic fatty liver (NAFL), NASH and cirrhosis(Figure 1). As with ALD, areas of overlap exist betweenthese three main patterns of liver injury and they areprobably best regarded as different parts of a broadhistological spectrum. Histological changes are oftenreversible, particularly during the early stages of thedisease. However, with progression to fibrosis andcirrhosis, the potential for reversibility diminishes(Figure 1).

Fatty change is predominantly macrovesicular intype (Figure 2). The severity of fatty change is gen-erally determined by estimating the proportion ofhepatocytes containing fat droplets: < 1 3 mild,1 32 3 moderate, > 2 3 severe.31,42 Two stud-ies have suggested lower thresholds for grading theseverity of steatosis (< 10% mild, 1030% mod-erate, > 30% severe), although these were bothrelated to steatosis occurring in the setting of chronichepatitis C virus (HCV) infection.43,44 It has beensuggested that very mild degrees of steatosis, involving< 5% of hepatocytes, may not actually represent a truepathological abnormality.6,31,44 However, in theabsence of definite evidence to the contrary, it is stillappropriate to document any degree of steatosis present

Normal liver

Fatty change

Steatohepatitis/fibrosis

Cirrhosis

Hepatocellular carcinoma

5090%

2030%

25%

Figure 1. Histological spectrum and estimated prevalence of liver

lesions in non-alcoholic fatty liver disease (NAFLD).The majority of

people with risk factors for NAFLD will develop fatty change, which is

reversible. A smaller proportion progress to more severe liver disease,

which is less readily reversible.

452 S G Hubscher


in the liver biopsy report. Although fatty change tendsmainly to involve perivenular regions, in severe cases itcan extend to a panacinar distribution.

Features of steatohepatitis include hepatocellularinjury (beyond simple fatty change), inflammationand fibrosis (Table 2) (Figure 3). These changes alsopredominantly involve acinar zone 3. Hepatocyteballooning is the most characteristic feature of steato-hepatitis and is typically associated with formation ofMallorys hyaline. Mallory bodies in NAFLD are oftensmall and poorly formed and may be difficult to detectin routinely stained sections.45 Immunohistochemi-cal techniques can be used to demonstrate antigensassociated with Mallorys hyaline. These includeubiquitin, p62 and cytokeratins 8 and 1831,46,47

(Figure 4). Death of hepatocytes may occur by apop-tosis or necrosis, probably mainly the former,4850

although apoptotic bodies are not usually conspicuoushistologically. Fatty liver disease is associated withincreased hepatocellular expression of the membranereceptor Fas, rendering these cells more susceptibleto apoptosis by Fas-ligand-expressing inflammatorycells.51 The severity of steatosis appears to be importantin determining the extent of apoptosis.52 Furthemore,formation of ROS is associated with Fas-ligand expres-sion on hepatocytes, thus producing a pathway for

H

Figure 2. Non-alcoholic fatty liver (NAFL). In this case of mild NAFL

steatosis is mainly macrovesicular and is present in a predominantly

perivenular (acinar zone 3) distribution. (Haematoxylin and

eosin. H, Hepatic vein.)

Table 2. Typical lobular changes occurring in steatohepatitis

Hepatocellular injury Ballooning

Apoptosis necrosis

Mallorys hyaline

Giant mitochondria

Inflammation Neutrophil polymorphs

Other cells (e.g. T lymphocytes,macrophages)

Fibrosis Perisinusoidal

Pericellular

H

Figure 3. Non-alcoholic steatohepatitis (NASH). In addition to fatty

change, many hepatocytes in acinar zone 3 show ballooning and

contain Mallorys hyaline. There is a mixed infiltrate of inflammatory

cells, including a prominent component of neutrophils. (Haemat-

oxylin and eosin. H, Hepatic vein.)

Figure 4. Mallorys hyaline in non-alcoholic steatohepatitis. In this

example there are numerous ballooned hepatocytes containing

Mallorys hyaline-like material immunoreactive for ubiquitin.

Immunohistochemical staining for ubiquitin may also help to

demonstrate small amounts of Mallorys hyaline, which are not

detectable in routinely stained sections. (Immunoperoxidase staining

for ubiquitin.)



fratricidal apoptosis.33 Megamitochondria, which aremore typically present in ALD, have also been recog-nized in non-alcoholic steatohepatitis.36,37,53 Onestudy showed that mitochondrial defects in the formof loss of cristae and paracrystalline inclusions werepresent in patients with NASH but not in those withfatty change alone,36 supporting the suggestion thatmitochondrial abnormalities play an important rolein the pathogenesis of progressive liver injury inNAFLD.33 Parenchymal inflammation is typicallymild and comprises a mixed population includingneutrophils, lymphocytes (mainly CD3+ T cells) andmacrophages Kupffer cells.54 Neutrophils typicallypredominate (Figure 3). Natural killer cells may alsobe involved.1

The parenchymal fibrosis that occurs in fatty liverdisease typically has a perisinusoidal and or pericellu-lar distribution (Figure 5) and, in a proportion of cases,eventually progresses to cirrhosis. This initially has amicronodular pattern, but larger nodules may subse-quently evolve. Typical features of steatohepatitis oftenbecome less conspicuous once cirrhosis has developedand may disappear altogether.18,55 This is the reasonwhy many cases of NAFLD-related cirrhosis werepreviously classified as cryptogenic.18,19 Vascularchanges occurring in cirrhosis have been postulatedas possible reasons for this phenomenontheseinclude portosystemic shunting, resulting in a lowerexposure of hepatocyes to insulin, and sinusoidalcapillarization, which may impair the trans-sinusoidalpassage of lipoproteins carried to the liver in the portalcirculation.56

Hepatocellular carcinoma (HCC) is recognized tooccur as a complication of cirrhosis related toNAFLD,2024 although the relative risk of HCC inNASH-associated cirrhosis compared with other forms

of chronic liver disease is uncertain. HCC has also beennoted as a rare complication of precirrhotic NAFLD.57

In addition to the carcinogenic factors associated withcirrhosis in general, there may also be risk factorsspecifically associated with fatty liver disease. Amongstpatients with cryptogenic (presumed NAFLD-related)cirrhosis, the presence of obesity and diabetes issignificantly associated with the development ofHCC.20,21 In another population-based casecontrolstudy, diabetes was found to be associated with anincreased risk of HCC, regardless of the presence ofother major HCC risk factors.58 The insulin resistancesyndrome is emerging as a risk factor for a wide varietyof other cancers, including colon, breast and endo-metrium.24 Hepatic steatosis is associated with replica-tive senescence and apoptosis of hepatocytes, stimulifor progenitor cell proliferation,59 which has been identi-fied as a potentially important step in hepatocellularcarcinogenesis.60,61 Furthermore, the ROS and lipidperoxidation products that are formed with progressionfrom steatosis to steatohepatitis cause DNA damageand gene mutations.33

portal/periportal les ions in non-alcoholicfatty liver disease

These include inflammatory changes similar to thoseseen in chronic hepatitis and biliary features resem-bling those occurring in low-grade biliary obstruction(Figure 6). Both of these patterns of damage provide amechanism for the development of progressive peripor-tal fibrosis (Figure 7).

Chronic hepatitis-like changesMinor degrees of portal inflammation associated withinterface hepatitis and periportal fibrosis are commonlypresent and can produce changes resembling thoseseen in various forms of chronic hepatitis.42,62 Theyusually occur in combination with typical parenchy-mal features of steatohepatitis, but can sometimesoccur as an isolated phenomenon (isolated portalfibrosis).6365 The latter particularly applies to fattyliver disease occurring in the paediatric population.Portal inflammation and fibrosis may become moremarked during the later stages of NASH.45 In someinstances these changes can be attributed to anotherconcurrent disease. This possibility should be consid-ered particularly if the severity of portal inflammationis disproportionate to the more typical lobular featuresof steatohepatitis or if there are atypical featuressuch as lymphoid follicles (suggestive of hepatitis C)or a prominent plasma cell component (suggestiveof autoimmune hepatitis). The relationship between

Figure 5. Pericellular fibrosis in non-alcoholic steatohepatitis.

Delicate strands of collagen surround individual hepatocytes in

acinar zone 3. (Haematoxylin van Gieson.)

454 S G Hubscher


hepatitis C and NAFLD will be considered further later.Autoantibodies have been found in up to 50% ofindividuals with NAFLD, although their pathogenicsignificance in this setting is uncertain.66,67 Autoanti-bodies are also commonly present in low titre in otherchronic liver diseases, where they are considered to bea non-specific response to hepatocellular injury.68,69

One study has shown that NAFLD patients who wereautoantibody positive had significantly higher inflam-matory grades and more advanced fibrosis than those

who were autoantibody negative, suggesting that hostimmune mechanisms may interact with other factorscausing liver injury in NAFLD.66 However, anotherstudy failed to identify any significant clinical orhistological differences in NASH patients who hadautoantibodies compared with those in whom theywere absent.67

Biliary featuresDuctular reaction is commonly seen and may producea picture resembling that seen in various types ofbiliary tract disease. It is usually present to a minordegree, but may become more severe, particularly inlate-stage disease.70 The ductular reaction whichoccurs in NAFLD is thought to be related to aprogenitor cell response occurring as a consequenceof steatosis-induced impaired hepatocellular replica-tion.59,71 The severity of steatosis in the setting ofHCV-associated NAFLD has been shown to correlatewith the extent of ductular reaction72 and this inturn provides a mechanism for the developmentof progressive periportal fibrosis.73 A cholestatic vari-ant of NAFLD with bile duct inflammation and ductloss has recently been described.74 However, bile ductdamage and duct loss are not typical features of NAFLDand the presence of these additional findings shouldraise the possibility of another cause of chronic biliarydisease such as primary biliary cirrhosis or sclerosingcholangitis. Likewise, the presence of unusually prom-inent ductular reaction or abundant periportal depositsof copper-associated protein should also prompt asearch for an additional biliary tract pathology.

Isolated portal fibrosisThe term isolated portal fibrosis has been used todescribe cases of fatty liver disease developing fibrousportal expansion without typical lobular features ofsteatohepatitis.6365 In the adult population, this pat-tern of damage is particularly seen amongst patientswith morbid obesity, where it occurs in 1833% ofcases.63,64 Fatty change is typically mild or moderate inseverity and inflammation is also predominantly portalin distribution. Portal fibrosis may persist followingtreatment, despite improvement in other histologicalfeatures,65 suggesting that different pathogenic mech-anisms are involved in mediating portal changes infatty liver disease.75

other histological f indings in non-alcoholicfatty liver disease

Minor degrees of siderosis are commonly present.2,76,77

The significance of iron overload in potentiating liver

Figure 6. Portal inflammation and ductular reaction in non-

alcoholic steatohepatitis. Portal tract contains a moderately dense

infiltrate of mononuclear inflammatory cells, associated with mild

interface hepatitis. These changes may resemble those occurring in

various forms of chronic hepatitis. There is also a moderate degree of

ductular reaction. (Haematoxylin and eosin.)

H

Figure 7. Periportal fibrosis in non-alcoholic steatohepatitis. In this

case fibrosis has a predominantly periportal distribution and is

associated with portalportal linkage. By contrast, only mild peri-

cellular fibosis is present in the perivenular region. (Haematoxylin

van Gieson. H, Hepatic vein.)



damage in NAFLD is uncertain, but the overallevidence suggests that it is unlikely to be of majorimportance.7880 The presence of more than mildsiderosis should raise the possibility of other causes ofhepatic iron overload.

Liver biopsy occasionally reveals previously un-suspected abnormalities (e.g. a1-antitrypsin globules)indicating an alternative or additional diagnosis to fattyliver disease.

non-alcoholic fatty liver disease in children

The incidence of paediatric NAFLD is rising as child-hood obesity becomes increasingly prevalent.8184

NAFLD has been implicated as the most commoncause of liver disease in children.85 Amongst obesechildren, male sex and Hispanic or Asian ethnicityhave been associated with an increased risk of develo-ping fatty liver disease.8486 Steatohepatitis in childrenappears to have different features to the typicalspectrum of NASH in the adult population.2,8791

Portal periportal changes in the form of inflammationand fibrosis tend to be more prominent, whereaslobular changes, including hepatocyte ballooning,Mallorys hyaline, inflammation and pericellular fibro-sis, are frequently less well developed. In a recent studyof 100 children with NAFLD, two different forms ofsteatohepatitis were identified.91 Type 1, characterizedby steatosis, ballooning degeneration and perisinusoi-dal fibrosis (similar to steatohepatitis in the adultpopulation) was present in 17% of cases; type 2,characterized by steatosis, portal inflammation andportal fibrosis, was present in 51%. Sixteen percentof biopsies had overlapping features of types 1 and2 disease and the remaining 16% showed simplesteatosis.

Role of liver biopsy in fatty liver disease

There are three main areas in which liver biopsy is usedin the diagnosis and management of fatty liver disease:1 Establishing a morphological diagnosis, includingthe distinction between simple steatosis and steato-hepatitis.2 Providing pointers to the aetiology, including casesin which a dual pathology appears to be present.3 Assessing disease severity using histological gradingand staging.

In addition to establishing an initial morphologicaldiagnosis, repeat liver biopsies may be useful inmonitoring responses to treatment, particularly in thecontext of clinical trials testing novel therapeuticapproaches.

Establishing a morphological diagnosis

Although fatty liver disease has been identified as themost common diagnosis in people with otherwiseunexplained persistently abnormal alanine amino-transferase values, in at least 2030% of such casesliver biopsy reveals an alternative diagnosis.3,12,92 Liverbiopsy may therefore be helpful to confirm the presenceof fatty liver disease, particularly in those cases whererisk factors for the metabolic syndrome are lacking.3

dist inction between steatosis andsteatohepatit is

Although fatty change can be reliably diagnosed bynon-invasive methods, the distinction between purefatty change (NAFL) and steatohepatitis (NASH) canonly be made histologically.2,93,94 Likewise, non-inva-sive methods are unreliable in diagnosing mild degreesof fibrosis. Because of the generally poor correlationbetween clinical, biochemical and histological findingsin NAFLD, it could be argued that liver biopsy shouldbe carried out on all people with a suspected diagnosisof fatty liver disease. However, for logistic and safetyreasons the use of liver biopsy is generally restricted tothose patients with risk factors for more severe formsof fatty liver disease in which there is a likelihood ofprogression to fibrosis. These risk factors include age,obesity, hypertension and the presence of diabetesmellitus.29,9597

What are the minimum criteria required to makea diagnosis of steatohepatitis?An important problem has been the lack of universallyaccepted criteria for making the histological diagnosisof steatohepatitis.31 Some studies have defined steato-hepatitis as fatty change and inflammation of any type.This approach lacks specificity and may result in otherunrelated diseases, including systemic illnesses associ-ated with non-specific reactive hepatitis, being mis-takenly classified as fatty liver disease.98 The use ofmore rigid diagnostic criteria including the presence ofhepatocyte ballooning with Mallorys hyaline and orpericellular perisinusoidal fibrosis improves diagnosticspecificity but may lack sensitivity in detecting caseswith mild disease or those where portal periportalchanges predominate. In an attempt to resolve thisissue a working party organized by the AmericanAssociation for the Study of Liver Diseases has pro-duced a consensus document in which a number ofessential and non-essential features of steatohepatitishave been identified.31 These are summarized inTable 3.

456 S G Hubscher


The most important diagnostic criterion for distin-guishing steatohepatitis from simple steatosis is thepresence of hepatocyte ballooning. The classical bal-looned hepatocyte in fatty liver disease is associatedwith a partly cleared cytoplasm in which residualcytoplasmic fragments condense to form Malloryshyaline. Ballooned hepatocytes are typically perivenu-lar in location and are closely associated with otherhistological components of fatty liver disease includinginflammation and pericellular fibrosis. However, lesssevere forms of hepatocyte ballooning frequently lack

typical Mallory bodies and may be more difficultto diagnose reliably. They may also be difficult todistinguish from the minor degrees of hepatocyteswelling, which can sometimes arise from processingartefacts. Not surprisingly, therefore, the histologicaldiagnosis of hepatocyte ballooning is associated withmore observer variability than other features ofNAFLD such as steatosis and fibrosis.99,100 Importantpointers to genuine hepatocyte ballooning are itsperivenular location and close association with stea-totic hepatocytes.62,101,102 In cases where there is

Table 3. Histologicalabnormalities which maybe present in non-alcoholicsteatohepatitis fibrosis(NASH) (based on AmericanAssociation for the Studyof Liver Diseases SingleTopic Conference on NASH,Atlanta, GA, September2002)31

1 Necessary componentsSteatosis, macro > micro, mainly zone 3

Mixed, mild lobular inflammationpolymorphs as well as mononuclear cells

Hepatocyte ballooning, most apparent near steatotic cells

2 Usually present, but not necessary for diagnosisPerisinusoidal fibrosis (zone 3)

Glycogenated nuclei (zone 1)

Lipogranulomas (usually small)

Occasional acidophil bodies or periodic acidSchiff-stained Kupffer cells

3 May be present, but not necessary for diagnosisMallorys hyaline in ballooned hepatocytes, usually zone 3 (typically poorly formedmayrequire immunostaining for ubiquitin, p62, cytokeratins 7, 8, 19 to confirm)

Mild siderosis (hepatocytes and or sinusoidal cells)

Megamitochondria

4 Unusual for NASH, consider other causes of liver diseaseMacrovesicular steatosis (< 30% of parenchyma involved or non-zonal distribution)

Pure or predominant microvesicular steatosis

Sclerosing hyaline necrosis, veno-occlusive lesions, perivenular fibrosis, phlebosclerosis

Portal inflammation > lobular inflammation, lymphoid aggregates, plasma cells

Significant eosinophils in portal or lobular inflammation, epithelioid granulomas

Portal periportal fibrosis in the absence of, or markedly greater than, zone 3perisinusoidal fibrosis

Lobular disarray, marked inflammation, confluent or bridging necrosis, endophlebitis

Acute cholestasis, bile plugs

Chronic cholestasis + florid bile duct lesions, bile duct loss, ductular proliferation,copper granules in periportal hepatocytes

Periodic acidSchiff diastase-resistant globules (a1-antitrypsin) in periportal hepatocytes

Significant granular iron in hepatocytes + zone 1 to zone 3 gradient



doubt about the presence of ballooning, immuno-histochemical staining may also help to demonstratesmall amounts of Mallorys hyaline-like material thatcannot be reliably identified in conventionally stainedsections.

Is the distinction between steatosis and steatohepatitisclinically important?A number of studies have indicated that pure fatty liveris a non-progressive reversible lesion, whereas thepresence of features of steatohepatitis indicates thepotential for progressive liver damage, in some casesleading to cirrhosis.9,103,104 In two studies with acombined population of 149 patients with a pure fattyliver, none developed clinical or histological featuresof advanced liver disease over median follow-up periodsof > 11 years.9,103 In a third study of 132 patientsundergoing liver biopsy for NAFLD who were followedup for a median period of 8.3 years, only two of 59(3%) with fatty change alone or fatty change andlobular inflammation progressed to cirrhosis, whereas18 of 73 patients (25%) whose biopsies showedadditional features of steatohepatitis (ballooning, Mal-lorys hyaline, fibrosis) became cirrhotic.104 This studyalso emphasizes the importance of using more rigidcriteria than fatty change and inflammation alone forthe diagnosis of steatohepatitis.

The suggestion that pure fatty change (NAFL) isassociated with a favourable outcome has recentlybeen challenged. Three studies have shown that up to10% of patients with pure fatty liver progress tosteatohepatitis with fibrosis or cirrhosis.105107 How-ever, in one of these studies the risk of progression tocirrhosis was significantly greater in patients whoseinitial diagnosis was NASH (20%) than in those withsteatosis alone (3%).107 Other recent studies havesuggested that other factors such as diabetes, bodymass index and obesity are more reliable predictorsof fibrosis progression than baseline biopsy find-ings.108,109 Furthermore, many of these cases developfibrosis progression despite improvement in otherhistological abnormalities including steatosis, hepato-cyte ballooning and Mallorys hyaline. Biochemicalabnormalities including serum transaminase levelsmay also improve despite progression of fibrosis.109

These observations suggest that repeat liver biopsiesmay have a role in monitoring disease progression evenin cases where the initial biopsy shows simple steatosisand in those where there appears to be an improve-ment in liver biochemistry. They also confirm previousobservations that features of fatty liver disease arefrequently lacking in cases that have progressed tocirrhosis.

Aetiological considerations

alcoholic versus non-alcoholicsteatohepatit is

A generally accepted diagnostic criterion for NAFLDis the exclusion of significant alcohol consumption(no more than 2040 g day).3 Moderate alcoholconsumption has been associated with a lower risk ofdeveloping NASH, possibly by reducing insulin resist-ance.63 However, in people with heavy alcohol con-sumption, obesity has been implicated as a risk factorfor the development of acute alcoholic hepatitis andcirrhosis, suggesting that there are common pathwaysof liver damage in alcoholic and non-alcoholic fattyliver disease.80,110

Faced with a liver biopsy showing features of fattyliver disease, are there any particular features thatsuggest a non-alcoholic rather than an alcoholic aeti-ology? In the great majority of such cases a distinctionbetween these two processes cannot be made onhistological grounds alone and the final diagnosis isbased on clinicopathological correlation. In general,NAFLD tends to be associated with relatively moresevere fatty change, whereas features of steatohepatitissuch as ballooning, Mallory body formation, neutrophi-lic infiltration and pericellular fibrosis are generally lesssevere than in ALD.31,45,111,112 Nuclear vacuolation ofhepatocytes is seen in 7080% of cases of NAFLDcompared with only 510% of cases of ALD and may be auseful pointer to glycogen accumulation occurring inthe setting of insulin resistance111,112 (Figure 8). Nuc-lear vacuolation of hepatocytes can also be seen inNAFLD-related cirrhosis, even when other features ofsteatohepatitis are no longer conspicuous.113 Florid

Figure 8. Nuclear vacuolation of hepatocytes in non-alcoholic fatty

liver disease. Many hepatocytes show nuclear vacuolation, a feature

much more commonly seen when fatty liver has a non-alcoholic

rather than alcoholic aetiology.

458 S G Hubscher


zone 3 changes falling into the spectrum of severealcoholic hepatitis or central sclerosing hyaline necrosisare rarely seen in NASH and point to an alcoholic causefor liver damage.31,114 These changes include abun-dant deposits of Mallorys hyaline, dense infiltration byneutrophils, severe bilirubinostasis, extensive zone 3fibrosis associated with sinusoidal obliteration andhepatic veno-occlusive lesions. However, occasionalcases of florid NASH with subacute liver failure havebeen documented.115 Other recent studies have sug-gested that NASH can be distinguished from alcoholicsteatohepatitis (ASH) on the basis of different patterns offibrosis (lattice pattern in NAFLD, solid in ALD)116 or bydifferent immunohistochemical staining patterns forthe protein tyrosine phosphatase 1B (PTP1B) andinsulin receptor on hepatocytes.117 PTP1B negativelyregulates the insulin receptor (IR) through dephospho-rylation and was found to be up-regulated in thecytoplasm of hepatocytes in biopsies obtained frompatients with NASH compared with those from cases ofASH. This was accompanied by loss of membranousstaining for IR in hepatocytes from NASH biopsies,compared with ASH biopsies where it was still preserved.

primary versus secondary causes of nafld

The great majority of NAFLD occurs in the setting ofthe metabolic syndrome (primary NAFLD) (Table 1). Incases where risk factors for primary NAFLD are lackingand a history of excess alcohol consumption can beconfidently excluded, a number of less common causesof fatty liver disease can be considered in the differentialdiagnosis. Although there are many potential causesof steatosis, too numerous to discuss in detail here,relatively few of these are associated with additionalfeatures of steatohepatitis (Table 1). In the adultpopulation, drug-induced NASH is the main differentialdiagnosis. In drug-related causes of NASH, Malloryshyaline is often present in large amounts out ofproportion to only mild fatty change and may have aperiportal rather than perivenular distribution.118 Inchildren, the diagnosis of NASH requires exclusion ofinherited metabolic causes of fatty liver disease.83 Thehepatocyte ballooning and Mallorys hyaline occurringin Wilsons disease tend to have a predominantlyperiportal (zone 1) rather than perivenular distributionand pericellular fibrosis is rarely seen.

nafld co-existing with other chronic liverdiseases

With the increasing prevalence of fatty liver dis-ease, histological features of NAFLD are now seen

with increasing frequency in association with othercauses of chronic liver disease, particularly HCVinfection.80,119121

The relationship between NAFLD and HCV infectionis complex. Fatty change is a common finding inchronic HCV infection. This may in part be a directcytopathic effect of the virus itself, possibly related toviral proteins interfering with fatty acid oxidation orother pathways of lipid metabolism.80 Chronic HCVinfection is a risk factor for the development of insulinresistance, thus also predisposing to the development ofNAFLD.122124 The development of insulin resistancein HCV-infected individuals may relate to viral-inducedproduction of TNF-a, which interferes with insulinsignalling.121,125 In cases infected with genotype 3 theseverity of steatosis correlates with levels of intra-hepatic viral replication.126128 Genotype 3 has alsobeen implicated as a risk factor for progression tosteatohepatitis.128 The severity of steatosis in HCVgenotype 3-infected patients correlates with fibrosisdevelopment.129,130 In contrast, in HCV+ individualsinfected with other genotypes, the severity of steatosisand the risk of progression to steatohepatitis andfibrosis are associated with risk factors for the metabolicsyndrome.80,127,128,131135 Overall, approximately 520% of patients with chronic HCV infection havesuperimposed features of steatohepatitis.120,128,132

Recognizing the presence and severity of fatty liverdisease in HCV-infected individuals is potentiallyimportant, both as a prognostic factor for the develop-ment of liver fibrosis43,44,124,127,131,132,136,137 and indetermining the likelihood of poor response to antiviraltherapy.134,135,138 Steatosis predisposes to the devel-opment of both the perisinusoidal and periportalpathways of fibrosis in chronic HCV infection,139

apparently independently of HCV-associated necro-inflammation.137 Portal fibrosis appears to be relatedto the activation and proliferation of portal myofibro-blasts, possibly via a paracrine pathway.139,140 Alter-natively, as discussed earlier, steatosis is associatedwith impaired hepatocyte regeneration, predisposing toprogenitor cell activation and bile ductular reactionand the extent of ductular reaction has been shown tocorrelate with fibrosis severity.72

Risk factors for NAFLD have been implicated in thepathogenesis of fatty change occurring in some indi-viduals with chronic hepatitis B virus infection.141

Fatty change is generally mild in severity and, incontrast to steatosis occurring in the setting of chronicHCV infection, is not associated with progression tosteatohepatitis or fibrosis. Other diseases in whichNAFLD has been implicated as a cofactor include ALD(discussed earlier), drug toxicity (e.g. methotrexate,



tamoxifen), a1-antitrypsin deficiency and iron-overloaddisorders.80,119,120,142,143

Assessing disease severity

The prevalence and natural history of the different liverlesions seen in NAFLD are difficult to determineaccurately as many of these patients are not biopsiedand few studies have investigated disease progressionin serial biopsies.144 It has been estimated that amongsta population of obese diabetic individuals approxi-mately 5090% will have fatty change, 2030% willprogress to steatohepatitis fibrosis and 25% willeventually become cirrhotic (Figure 1).2,31,63,141 Cross-sectional studies have suggested a higher preval-ence of cirrhosis (in the region of 1030%) amongstpatients undergoing liver biopsy for NAFLD, perhapsreflecting a degree of selection bias.8,16,29,95,145,146 In areview of four previously published series describing30 patients with NAFLD, who had paired liver biopsiesat intervals ranging from 1.0 to 9.0 years, 14 showedprogression in fibrosis, six cases resulting in cirrho-sis.146 Amongst the other 16 cases, one showedimprovement in liver histology, whereas the other 15showed no change. Other studies have suggested thatfibrosis may remain stable or regress in up to 6070%of cases.106,108,109

grading and staging of fatty liver disease

Histological grading and staging are widely used in theassessment of liver biopsies from patients with chro-nic viral hepatitis, particularly hepatitis C. A similarapproach has been applied to the assessment of diseaseseverity in fatty liver disease. Features which aregraded are fatty change, ballooning and inflammation(parenchymal and portal). Staging involves an assess-ment of the severity of fibrosis.

A number of systems have been proposed for asses-sing the severity of fatty liver disease.31,42,100,104,147,148

The main features of a scheme devised by Brunt andcolleagues, which has been most widely used forassessing disease severity in non-alcoholic steatohepa-titis, are summarized in Table 4.31,42 One criticism ofthis approach is the incorporation of fatty change,ballooning and inflammation into an overall grade.This implies that these three histological featuresincrease in parallel to each other, which is notnecessarily the case. For example, a biopsy may showsevere (grade 3) fatty change but only mild (grade1)ballooning and inflammation and it is not clear whatoverall grade should be applied to such a specimen.Other limitations of this system relate to the fact that it

applies only to NASH (and does not thus encompassthe entire spectrum of NAFLD) and to cases withclassical lobular features of steatohepatitis. An alter-native approach is thus required for cases which have apredominantly portal-based disease process; this par-ticularly applies to fatty liver disease occurring inchildren. In an attempt to address some of theseproblems a modified histological scoring system hasbeen devised by a group of North American patholo-gists working under the auspices of the Non-alcoholicSteatohepatitis Clinical Research Network (Table 5).100

This system evaluates the same three main features ofdisease activity that were used in the original systemdevised by Brunt et al.,42 but instead of being incor-porated into an overall grade they are scored separatelyand the individual scores then added to produce anoverall NAFLD Activity Score (NAS). There is alsorecognition of portal fibrosis as a separate pathway fordisease progression. The main purpose of the systemproposed by Kleiner et al. was to determine robustcriteria for establishing a histological diagnosis of

Table 4. A proposed scheme for histological grading andstaging of non-alcoholic steatohepatitis fibrosis (NASH)(based on Brunt 1999 and Neuschwander-Tetri and Caldwell2003)31,42

a. Grading of NASH

Overallgrade Steatosis

Ballooning(zone 3) Inflammation

Mild 12 Minimal Lobular 12Portal 01

Moderate 23 Present Lobular 2Portal 12

Severe 3 Marked Lobular 3Portal 12

Individual features graded semiquantitatively on a scale of0 absent, 1 mild, 2 moderate, 3 severe.Severity of steatosis based on proportion of hepatocytesinvolved: 1 < 33%, 2 3366%, 3 > 66%.Severity of lobular inflammation based on inflammatory fociper 200 field: 1 12, 2 up to 4, 3 >4.b. Staging of NASH

Stage 1 Zone 3 perivenular, persinusoidal orpericellular fibrosis; focal or extensive

Stage 2 As for stage 1 plus focal or extensiveportal fibrosis

Stage 3 Bridging fibrosis, focal or extensive

Stage 4 Cirrhosis

460 S G Hubscher


NASH (Table 5). However, they also have potentialapplications for determining disease severity andresponses to treatment, similar to the approach thathas been used for hepatitis activity scores in chronicviral hepatitis.

Problems inherent to all histological scoring systemsthat have been used in assessment of liver disease relateto observer reproducibility and sampling variation.Amongst the features which have been assessed in fattyliver disease, observer agreement is generally good forsteatosis, moderate for ballooning and less good forlobular inflammation and Mallorys hyaline.99,100

The use of image analysis methods has questioned theaccuracy of the traditional approach to assessing theseverity of steatosis according to the estimated percent-age of hepatocytes containing fat droplets.149,150 Stud-ies of paired liver biopsies have suggested that samplingvariability presents a greater problem.151154 Althoughfatty change has a reasonably uniform distribution,there is considerable variation in the severity of otherhistological features, particularly fibrosis. Heterogeneityof fibrosis scores is increased in small biopsies(< 16 mm long).155

Conclusion and future developments

It is likely that histological assessments will continue toplay an important role in the diagnosis and manage-ment of people with NAFLD. In those cases where thediagnosis of NAFLD has already been made clinically,liver biopsy is required to make the distinction betweensimple steatosis and steatohepatitis and, where appro-priate, to determine the severity of liver damage withinthe broad spectrum of steatohepatitis fibrosis. In somecases where NAFLD is suspected clinically, liver biopsymay reveal an additional or alternative cause for liverdamage. It is also becoming increasingly apparent thatNAFLD is an important cofactor in other chronic liverdiseases, particularly hepatitis C. In addition to estab-lishing that a dual pathology is present, liver biopsymay help to determine which of the two diseases is thepredominant cause of liver damage. Further studies arestill required to determine the natural history of NAFLDand the role of liver biopsy in monitoring therapeuticresponses. The utility of recently devised systems forgrading and staging NAFLD also requires furtherevaluation.

References

1. Diehl AM, Li ZP, Lin HZ, Yang SQ. Cytokines and the

pathogenesis of non-alcoholic steatohepatitis. Gut 2005; 54;

303306.

2. Reid AE. Nonalcoholic steatohepatitis. Gastroenterology 2001;

121; 710723.

3. Ramesh S, Sanyal AJ. Evaluation and management of non-

alcoholic steatohepatitis. J. Hepatol. 2005; 42 (Suppl.); S212.

4. Alberti KG, Zimmet P, Shaw J. The metabolic syndrome

a new worldwide definition. Lancet 2005; 366; 10591062.

Table 5. Histological scoring system for non-alcoholic fattyliver disease (NAFLD) (from Kleiner et al. 2005)100

NAFLD Activity Score (NAS) (08)Sum of scores for steatosis, lobular inflammation andhepatocellular ballooning

Steatosis (03)0 66% hepatocytes involved

Lobular Inflammation (03)0 none1 4 foci per 200 field

Hepatocyte ballooning (02)0 none1 few ballooned cells2 many cells prominent ballooning

Correlation between total NAFLD activity scores andan overall histological diagnosis of steatohepatitis

NAFLDactivity score

Histological diagnosisof steatohepatitis

5 Probable or definite NASH

34 Uncertain

2 Not NASH

Fibrosis stage

1 Perisinusoidal or periportal1A Mild, zone 3, perisinusoidal

1B Moderate, zone 3, perisinusoidal

1C Portal periportal fibrosis only

2 Perisinusoidal and portal periportal fibrosis

3 Bridging fibrosis

4 Cirrhosis



5. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome.

Lancet 2005; 365; 14151428.

6. Adams LA, Angulo P. Recent concepts in non-alcoholic fatty

liver disease. Diabet. Med. 2005; 22; 11291133.

7. Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G,

Bellentani S. Prevalence of and risk factors for nonalcoholic

fatty liver disease: the Dionysos nutrition and liver study.

Hepatology 2005; 42; 4452.

8. Clark JM, Brancati FL, Diehl AM. Nonalcoholic fatty liver

disease. Gastroenterology 2002; 122; 16491657.

9. Dam-Larsen S, Franzmann M, Andersen IB et al. Long term

prognosis of fatty liver: risk of chronic liver disease and death.

Gut 2004; 53; 750755.

10. Cortez-Pinto H, de Moura MC, Day CP. Non-alcoholic steato-

hepatitis: from cell biology to clinical practice. J. Hepatol. 2006;

44; 197208.

11. Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic

steatohepatitis: Mayo Clinic experiences with a hitherto

unnamed disease. Mayo Clin. Proc. 1980; 55; 434438.

12. Skelly MM, James PD, Ryder SD. Findings on liver biopsy to

investigate abnormal liver function tests in the absence of

diagnostic serology. J. Hepatol. 2001; 35; 195199.

13. de Ledinghen V, Combes M, Trouette H et al. Should a liver biopsy

be done in patients with subclinical chronically elevated trans-

aminases? Eur. J. Gastroenterol. Hepatol. 2004; 16; 879883.

14. Madan K, Batra Y, Panda SK et al. Role of polymerase chain

reaction and liver biopsy in the evaluation of patients with

asymptomatic transaminitis: implications in diagnostic

approach. J. Gastroenterol. Hepatol. 2004; 19; 12911299.

15. Torezan-Filho MA, Alves VA, Neto CA, Fernandes HS,

Strauss E. Clinical significance of elevated alanine amino-

transferase in blood donors: a follow-up study. Liver Int.

2004; 24; 575581.

16. Mofrad P, Contos MJ, Haque M et al. Clinical and histologic

spectrum of nonalcoholic fatty liver disease associated with

normal ALT values. Hepatology 2003; 37; 12861292.

17. Sorrentino P, Tarantino G, Conca P et al. Silent non-alcoholic

fatty liver diseasea clinicalhistological study. J. Hepatol.

2004; 41; 751757.

18. Caldwell SH, Oelsner DH, Iezzoni JC, Hespenheide EE, Battle EH,

Driscoll CJ. Cryptogenic cirrhosis: clinical characterization and

risk factors for underlying disease. Hepatology 1999; 29; 664

669.

19. Poonawala A, Nair SP, Thuluvath PJ. Prevalence of obesity

and diabetes in patients with cryptogenic cirrhosis: a case

control study. Hepatology 2000; 32; 689692.

20. Bugianesi E, Leone N, Vanni E et al. Expanding the natural

history of nonalcoholic steatohepatitis: from cryptogenic

cirrhosis to hepatocellular carcinoma. Gastroenterology 2002;

123; 134140.

21. Ratziu V, Bonyhay L, Di MV et al. Survival, liver failure,

and hepatocellular carcinoma in obesity-related cryptogenic

cirrhosis. Hepatology 2002; 35; 14851493.

22. Marrero JA, Fontana RJ, Su GL, Conjeevaram HS, Emick DM,

Lok AS. NAFLD may be a common underlying liver disease in

patients with hepatocellular carcinoma in the United States.

Hepatology 2002; 36; 13491354.

23. Shimada M, Hashimoto E, Taniai M et al. Hepatocellular

carcinoma in patients with non-alcoholic steatohepatitis.

J. Hepatol. 2002; 37; 154160.

24. Bugianesi E. Review article: steatosis, the metabolic syndrome

and cancer. Aliment. Pharmacol. Ther. 2005; 22 (Suppl. 2);

4043.

25. Spaulding L, Trainer T, Janiec D. Prevalence of non-alcoholic

steatohepatitis in morbidly obese subjects undergoing gastric

bypass. Obes. Surg. 2003; 13; 347349.

26. Bugianesi E, McCullough AJ, Marchesini G. Insulin resistance:

a metabolic pathway to chronic liver disease. Hepatology 2005;

42; 9871000.

27. Day CP, James OF. Steatohepatitis. a tale of two hits?

Gastroenterology 1998; 114; 842845.

28. Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steato-

hepatitis. Semin. Liver Dis. 2001; 21; 2741.

29. Day CP. Non-alcoholic steatohepatitis (NASH): where are we

now and where are we going? Gut 2002; 50; 585588.

30. Younossi ZM, Diehl AM, Ong JP. Nonalcoholic fatty liver

disease: an agenda for clinical research. Hepatology 2002; 35;

746752.

31. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steato-

hepatitis: summary of an AASLD Single Topic Conference.

Hepatology 2003; 37; 12021219.

32. Choudhury J, Sanyal AJ. Insulin resistance and the pathogen-

esis of nonalcoholic fatty liver disease. Clin. Liver Dis. 2004; 8;

575594.

33. Pessayre D, Fromenty B. NASH: a mitochondrial disease.

J. Hepatol. 2005; 42; 928940.

34. Samuel VT, Liu ZX, Qu X et al. Mechanism of hepatic insulin

resistance in non-alcoholic fatty liver disease. J. Biol. Chem.

2004; 279; 3234532353.

35. Schattenberg JM, Wang Y, Singh R, Rigoli RM, Czaja MJ.

Hepatocyte CYP2E1 overexpression and steatohepatitis lead to

impaired hepatic insulin signaling. J. Biol. Chem. 2005; 280;

98879894.

36. Sanyal AJ, Campbell-Sargent C, Mirshahi F et al. Nonalcoholic

steatohepatitis: association of insulin resistance and mito-

chondrial abnormalities. Gastroenterology 2001; 120; 1183

1192.

37. Le TH, Caldwell SH, Redick JA et al. The zonal distribution of

megamitochondria with crystalline inclusions in nonalcoholic

steatohepatitis. Hepatology 2004; 39; 14231429.

38. Albano E, Mottaran E, Vidali M et al. Immune response

towards lipid peroxidation products as a predictor of progres-

sion of non-alcoholic fatty liver disease to advanced fibrosis.

Gut 2005; 54; 987993.

39. Kaser S, Moschen A, Cayon A et al. Adiponectin and its

receptors in non-alcoholic steatohepatitis. Gut 2005; 54; 117

121.

40. Marra F, Aleffi S, Bertolani C, Petrai I, Vizzutti F. Review article:

the pathogenesis of fibrosis in non-alcoholic steatohepatitis.

Aliment. Pharmacol. Ther. 2005; 22 (Suppl. 2); 4447.

41. Wanless IR, Shiota K. The pathogenesis of nonalcoholic

steatohepatitis and other fatty liver diseases: a four-step model

including the role of lipid release and hepatic venular obstruc-

tion in the progression to cirrhosis. Semin. Liver Dis. 2004; 24;

99106.

42. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri

BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for

grading and staging the histological lesions. Am. J. Gastro-

enterol. 1999; 94; 24672474.

43. Castera L, Hezode C, Roudot-Thoraval F et al. Worsening of

steatosis is an independent factor of fibrosis progression in

untreated patients with chronic hepatitis C and paired liver

biopsies. Gut 2003; 52; 288292.

44. Fartoux L, Chazouilleres O, Wendum D, Poupon R, Serfaty L.

Impact of steatosis on progression of fibrosis in patients with

mild hepatitis C. Hepatology 2005; 41; 8287.

462 S G Hubscher


45. Nonomura A, Enomoto Y, Takeda M et al. Clinical and

pathological features of non-alcoholic steatohepatitis. Hepatol.

Res. 2005; 33; 116121.

46. Banner BF, Savas L, Zivny J, Tortorelli K, Bonkovsky HL.

Ubiquitin as a marker of cell injury in nonalcoholic steato-

hepatitis. Am. J. Clin. Pathol. 2000; 114; 860866.

47. Zatloukal K, Stumptner C, Fuchsbichler A et al. p62 Is a

common component of cytoplasmic inclusions in protein

aggregation diseases. Am. J. Pathol. 2002; 160; 255263.

48. Feldstein AE, Canbay A, Angulo P et al. Hepatocyte apoptosis

and fas expression are prominent features of human non-

alcoholic steatohepatitis. Gastroenterology 2003; 125; 437

443.

49. Ribeiro PS, Cortez-Pinto H, Sola S et al. Hepatocyte apoptosis,

expression of death receptors, and activation of NF-kappaB in

the liver of nonalcoholic and alcoholic steatohepatitis patients.

Am. J. Gastroenterol. 2004; 99; 17081717.

50. Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and

chronic liver injury. Gut 2005; 54; 10241033.

51. Feldstein AE, Canbay A, Guicciardi ME, Higuchi H, Bronk SF,

Gores GJ. Diet associated hepatic steatosis sensitizes to Fas

mediated liver injury in mice. J. Hepatol. 2003; 39; 978983.

52. Walsh MJ, Vanags DM, Clouston AD et al. Steatosis and liver

cell apoptosis in chronic hepatitis C: a mechanism for increased

liver injury. Hepatology 2004; 39; 12301238.

53. Caldwell SH, Swerdlow RH, Khan EM et al. Mitochondrial

abnormalities in non-alcoholic steatohepatitis. J. Hepatol.

1999; 31; 430434.

54. Lefkowitch JH, Haythe JH, Regent N. Kupffer cell aggregation

and perivenular distribution in steatohepatitis. Mod. Pathol.

2002; 15; 699704.

55. Powell EE, Cooksley WG, Hanson R, Searle J, Halliday JW,

Powell LW. The natural history of nonalcoholic steatohepatitis:

a follow-up study of forty-two patients for up to 21 years.

Hepatology 1990; 11; 7480.

56. Caldwell SH, Crespo DM. The spectrum expanded: cryptogenic

cirrhosis and the natural history of non-alcoholic fatty liver

disease. J. Hepatol. 2004; 40; 578584.

57. Bullock RE, Zaitoun AM, Aithal GP, Ryder SD, Beckingham IJ,

Lobo DN. Association of non-alcoholic steatohepatitis without

significant fibrosis with hepatocellular carcinoma. J. Hepatol.

2004; 41; 685686.

58. Davila JA, Morgan RO, Shaib Y, McGlynn KA, El Serag HB.

Diabetes increases the risk of hepatocellular carcinoma in the

United States: a population based case control study. Gut 2005;

54; 533539.

59. Yang S, Koteish A, Lin H et al. Oval cells compensate for

damage and replicative senescence of mature hepatocytes in

mice with fatty liver disease. Hepatology 2004; 39; 403411.

60. Libbrecht L, Desmet V, Van Damme B, Roskams T. The

immunohistochemical phenotype of dysplastic foci in human

liver: correlation with putative progenitor cells. J. Hepatol.

2000; 33; 7684.

61. Libbrecht L, Desmet V, Roskams T. Preneoplastic lesions in

human hepatocarcinogenesis. Liver Int. 2005; 25; 1627.

62. Brunt EM. Nonalcoholic steatohepatitis: definition and pathol-

ogy. Semin. Liver Dis. 2001; 21; 316.

63. Dixon JB, Bhathal PS, OBrien PE. Nonalcoholic fatty liver

disease: predictors of nonalcoholic steatohepatitis and liver

fibrosis in the severely obese. Gastroenterology 2001; 121; 91

100.

64. Abrams GA, Kunde SS, Lazenby AJ, Clements RH. Portal

fibrosis and hepatic steatosis in morbidly obese subjects: a

spectrum of nonalcoholic fatty liver disease. Hepatology 2004;

40; 475483.

65. Dixon JB, Bhathal PS, Hughes NR, OBrien PE. Nonalcoholic

fatty liver disease: improvement in liver histological analysis

with weight loss. Hepatology 2004; 39; 16471654.

66. Adams LA, Lindor KD, Angulo P. The prevalence of auto-

antibodies and autoimmune hepatitis in patients with nonal-

coholic fatty liver disease. Am. J. Gastroenterol. 2004; 99;

13161320.

67. Cotler SJ, Kanji K, Keshavarzian A, Jensen DM, Jakate S.

Prevalence and significance of autoantibodies in patients with

non-alcoholic steatohepatitis. J. Clin. Gastroenterol. 2004; 38;

801804.

68. Alvarez F, Berg PA, Bianchi FB et al. International Auto-

immune Hepatitis Group Report: review of criteria for diagnosis

of autoimmune hepatitis. J. Hepatol. 1999; 31; 929938.

69. Czaja AJ, Homburger HA. Autoantibodies in liver disease.


70. Ayata G, Gordon FD, Lewis WD et al. Cryptogenic cirrhosis:

clinicopathologic findings at and after liver transplantation.

Hum. Pathol. 2002; 33; 10981104.

71. Roskams T, Yang SQ, Koteish A et al. Oxidative stress and oval

cell accumulation in mice and humans with alcoholic and

nonalcoholic fatty liver disease. Am. J. Pathol. 2003; 163;

13011311.

72. Clouston AD, Powell EE, Walsh MJ, Richardson MM, Demetris

AJ, Jonsson JR. Fibrosis correlates with a ductular reaction in

hepatitis C: roles of impaired replication, progenitor cells and

steatosis. Hepatology 2005; 41; 809818.

73. Roskams T, Desmet V. Ductular reaction and its diagnostic

significance. Semin. Diagn. Pathol. 1998; 15; 259269.

74. Sorrentino P, Tarantino G, Perrella A, Micheli P, Perrella O,

Conca P. A clinical-morphological study on cholestatic pres-

entation of nonalcoholic fatty liver disease. Dig. Dis. Sci. 2005;

50; 11301135.

75. Gramlich T, Kleiner DE, McCullough AJ, Matteoni CA, Boparai

N, Younossi ZM. Pathologic features associated with fibrosis in

nonalcoholic fatty liver disease. Hum. Pathol. 2004; 35; 196

199.

76. Younossi ZM, Gramlich T, Bacon BR et al. Hepatic iron and

nonalcoholic fatty liver disease. Hepatology 1999; 30; 847

850.

77. Chitturi S, Weltman M, Farrell GC et al. HFE mutations, hepatic

iron, and fibrosis: ethnic-specific association of NASH with

C282Y but not with fibrotic severity. Hepatology 2002; 36;

142149.

78. Chitturi S, George J. Interaction of iron, insulin resistance, and

nonalcoholic steatohepatitis. Curr. Gastroenterol. Rep. 2003; 5;

1825.

79. Bugianesi E, Manzini P, DAntico S et al. Relative contribution

of iron burden, HFE mutations, and insulin resistance to

fibrosis in nonalcoholic fatty liver. Hepatology 2004; 39; 179

187.

80. Powell EE, Jonsson JR, Clouston AD. Steatosis: co-factor in

other liver diseases. Hepatology 2005; 42; 513.

81. Roberts EA. Steatohepatitis in children. Best. Pract. Res. Clin.

Gastroenterol. 2002; 16; 749765.

82. Roberts EA. Nonalcoholic steatohepatitis in children. Curr.

Gastroenterol. Rep. 2003; 5; 253259.

83. Marion AW, Baker AJ, Dhawan A. Fatty liver disease in

children. Arch. Dis. Child. 2004; 89; 648652.

84. Roberts EA. Non-alcoholic fatty liver disease (NAFLD) in

children. Front Biosci. 2005; 10; 23062318.



85. Lavine JE, Schwimmer JB. Nonalcoholic fatty liver disease in

the pediatric population. Clin. Liver Dis. 2004; 8; 549558.

86. Nanda K. Non-alcoholic steatohepatitis in children. Pediatr.

Transplant. 2004; 8; 613618.

87. Baldridge AD, Perez-Atayde AR, Graeme-Cook F, Higgins L,

Lavine JE. Idiopathic steatohepatitis in childhood: a multicenter

retrospective study. J. Pediatr. 1995; 127; 700704.

88. Manton ND, Lipsett J, Moore DJ, Davidson GP, Bourne AJ,

Couper RT. Non-alcoholic steatohepatitis in children and

adolescents. Med. J. Aust. 2000; 173; 476479.

89. Rashid M, Roberts EA. Nonalcoholic steatohepatitis in children.

J. Pediatr. Gastroenterol. Nutr. 2000; 30; 4853.

90. Molleston JP, White F, Teckman J, Fitzgerald JF. Obese children

with steatohepatitis can develop cirrhosis in childhood. Am. J.

Gastroenterol. 2002; 97; 24602462.

91. Schwimmer JB, Behling C, Newbury R et al. Histopathology of

pediatric nonalcoholic fatty liver disease. Hepatology 2005; 42;

641649.

92. Sorbi D, McGill DB, Thistle JL, Therneau TM, Henry J, Lindor

KD. An assessment of the role of liver biopsies in asymptomatic

patients with chronic liver test abnormalities. Am. J. Gastro-

enterol. 2000; 95; 32063210.

93. Saadeh S, Younossi ZM, Remer EM et al. The utility of

radiological imaging in nonalcoholic fatty liver disease.


94. Joy D, Thava VR, Scott BB. Diagnosis of fatty liver disease: is

biopsy necessary? Eur. J. Gastroenterol. Hepatol. 2003; 15; 539

543.

95. Angulo P, Keach JC, Batts KP, Lindor KD. Independent

predictors of liver fibrosis in patients with nonalcoholic

steatohepatitis. Hepatology 1999; 30; 13561362.

96. Chitturi S, Abeygunasekera S, Farrell GC et al. NASH and

insulin resistance: insulin hypersecretion and specific associ-

ation with the insulin resistance syndrome. Hepatology 2002;

35; 373379.

97. Pagano G, Pacini G, Musso G et al. Nonalcoholic steatohepatitis,

insulin resistance, and metabolic syndrome: further evidence

for an etiologic association. Hepatology 2002; 35; 367372.

98. Lee RG. Nonalcoholic steatohepatitis: tightening the morpho-

logical screws on a hepatic rambler. Hepatology 1995; 21;

17421743.

99. Younossi ZM, Gramlich T, Liu YC et al. Nonalcoholic fatty liver

disease: assessment of variability in pathologic interpretations.

Mod. Pathol. 1998; 11; 560565.

100. Kleiner DE, Brunt EM, Van Natta M et al. Design and validation

of a histological scoring system for nonalcoholic fatty liver

disease. Hepatology 2005; 41; 13131321.

101. Brunt EM, Neuschwander-Tetri BA, Oliver D, Wehmeier KR,

Bacon BR. Nonalcoholic steatohepatitis: histologic features and

clinical correlations with 30 blinded biopsy specimens. Hum.

Pathol. 2004; 35; 10701082.

102. Brunt EM. Pathology of nonalcoholic steatohepatitis. Hepatol.

Res. 2005; 33; 6871.

103. Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural

history of nonalcoholic fatty liver: a follow-up study. Hepatology

1995; 22; 17141719.

104. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC,

McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of

clinical and pathological severity. Gastroenterology 1999; 116;

14131419.

105. Saksena S. Natural history and determinants of disease

progression in non-alcoholic fatty liver disease: good and bad

news. Hepatology 2003; 38; 232A.

106. Harrison SA, Torgerson S, Hayashi PH. The natural history of

nonalcoholic fatty liver disease: a clinical histopathological

study. Am. J. Gastroenterol. 2003; 98; 20422047.

107. McCullough AJ. The clinical features, diagnosis and natural

history of nonalcoholic fatty liver disease. Clin. Liver Dis. 2004;

8; 521533.

108. Fassio E, Alvarez E, Dominguez N, Landeira G, Longo C.

Natural history of nonalcoholic steatohepatitis: a longitudinal

study of repeat liver biopsies. Hepatology 2004; 40; 820826.

109. Adams LA, Sanderson S, Lindor KD, Angulo P. The histological

course of nonalcoholic fatty liver disease: a longitudinal study

of 103 patients with sequential liver biopsies. J. Hepatol. 2005;

42; 132138.

110. Diehl AM. Obesity and alcoholic liver disease. Alcohol 2004;

34; 8187.

111. Diehl AM, Goodman Z, Ishak KG. Alcohollike liver disease in

nonalcoholics. A clinical and histologic comparison with

alcohol-induced liver injury. Gastroenterology 1988; 95;

10561062.

112. Itoh S, Yougel T, Kawagoe K. Comparison between nonalco-

holic steatohepatitis and alcoholic hepatitis. Am. J. Gastro-

enterol. 1987; 82; 650654.

113. Contos MJ, Cales W, Sterling RK et al. Development of nonalco-

holic fatty liver disease after orthotopic liver transplantation for

cryptogenic cirrhosis. Liver Transpl. 2001; 7; 363373.

114. Brunt EM. Alcoholic and nonalcoholic steatohepatitis. Clin.

Liver Dis. 2002; 6; 399420.

115. Kuwabara H, Yoshii Y, Mori H et al. Nonalcoholic steato-

hepatitis-related cirrhosis with subacute liver failure: an

autopsy case. Dig. Dis. Sci. 2003; 48; 16681670.

116. Nakano M, Fukusato T. Histological study on comparison

between NASH and ALD. Hepatol. Res. 2005; 33; 110115.

117. Sanderson SO, Smyrk TC. The use of protein tyrosine

phosphatase 1B and insulin receptor immunostains to differ-

entiate nonalcoholic from alcoholic steatohepatitis in liver

biopsy specimens. Am. J. Clin. Pathol. 2005; 123; 503509.

118. Farrell GC. Drug-induced steatohepatitis. In Drug-induced liver

disease. Edinburgh: Churchill Livingstone 1994; 431438.

119. Clouston AD, Powell EE. Interaction of non-alcoholic fatty liver

disease with other liver diseases. Best. Pract. Res. Clin.


120. Brunt EM, Ramrakhiani S, Cordes BG et al. Concurrence of

histologic features of steatohepatitis with other forms of

chronic liver disease. Mod. Pathol. 2003; 16; 4956.

121. Asselah T, Rubbia-Brandt L, Marcellin P, Negro F. Steatosis in

chronic hepatitis C: why does it really matter? Gut 2006; 55;

123130.

122. Hui JM, Sud A, Farrell GC et al. Insulin resistance is associated

with chronic hepatitis C virus infection and fibrosis progres-

sion. Gastroenterology 2003; 125; 16951704.

123. Narita R, Abe S, Kihara Y, Akiyama T, Tabaru A, Otsuki M.

Insulin resistance and insulin secretion in chronic hepatitis C

virus infection. J. Hepatol. 2004; 41; 132138.

124. Lonardo A, Adinolfi LE, Loria P, Carulli N, Ruggiero G, Day CP.

Steatosis and hepatitis C virus: mechanisms and significance

for hepatic and extrahepatic disease. Gastroenterology 2004;

126; 586597.

125. Zekry A, McHutchison JG, Diehl AM. Insulin resistance and

steatosis in hepatitis C virus infection. Gut 2005; 54; 903

906.

126. Rubbia-Brandt L, Quadri R, Abid K et al. Hepatocyte steatosis is

a cytopathic effect of hepatitis C virus genotype 3. J. Hepatol.

2000; 33; 106115.

464 S G Hubscher


127. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R.,

Ruggiero G. Steatosis accelerates the progression of liver

damage of chronic hepatitis C patients and correlates with

specific HCV genotype and visceral obesity. Hepatology 2001;

33; 13581364.

128. Younossi ZM, McCullough AJ, Ong JP et al. Obesity and non-

alcoholic fatty liver disease in chronic hepatitis C. J. Clin.


129. Rubbia-Brandt L, Fabris P, Paganin S et al. Steatosis affects

chronic hepatitis C progression in a genotype specific way. Gut

2004; 53; 406412.

130. Westin J, Nordlinder H, Lagging M, Norkrans G, Wejstal R.

Steatosis accelerates fibrosis development over time in hepatitis

C virus genotype 3 infected patients. J. Hepatol. 2002; 37; 837

842.

131. Ong JP, Younossi ZM, Speer C, Olano A, Gramlich T, Boparai N.

Chronic hepatitis C and superimposed nonalcoholic fatty liver

disease. Liver 2001; 21; 266271.

132. Monto A, Alonzo J, Watson JJ, Grunfeld C, Wright TL. Steatosis

in chronic hepatitis C: relative contributions of obesity, diabetes

mellitus, and alcohol. Hepatology 2002; 36; 729736.

133. Ramalho F. Hepatitis C virus infection and liver steatosis.

Antiviral Res. 2003; 60; 125127.

134. Poynard T, Ratziu V, McHutchison J et al. Effect of treatment

with peginterferon or interferon alfa-2b and ribavirin on

steatosis in patients infected with hepatitis C. Hepatology 2003;

38; 7585.

135. Patton HM, Patel K, Behling C et al. The impact of steatosis

on disease progression and early and sustained treatment

response in chronic hepatitis C patients. J. Hepatol. 2004; 40;

484490.

136. Ortiz V, Berenguer M, Rayon JM, Carrasco D, Berenguer J.

Contribution of obesity to hepatitis C-related fibrosis progres-

sion. Am. J. Gastroenterol. 2002; 97; 24082414.

137. Wyatt J, Baker H, Prasad P, Gong YY, Millson C. Steatosis and

fibrosis in patients with chronic hepatitis C. J. Clin. Pathol.

2004; 57; 402406.

138. Bressler BL, Guindi M, Tomlinson G, Heathcote J. High body

mass index is an independent risk factor for nonresponse to

antiviral treatment in chronic hepatitis C. Hepatology 2003; 38;

639644.

139. Clouston AD, Jonsson JR, Purdie DM et al. Steatosis and

chronic hepatitis C: analysis of fibrosis and stellate cell

activation. J. Hepatol. 2001; 34; 314320.

140. Hickman IJ, Clouston AD, Macdonald GA et al. Effect of weight

reduction on liver histology and biochemistry in patients with

chronic hepatitis C. Gut 2002; 51; 8994.

141. Gordon A, McLean CA, Pedersen JS, Bailey MJ, Roberts SK.

Hepatic steatosis in chronic hepatitis B and C: predictors,

distribution and effect on fibrosis. J. Hepatol. 2005; 43; 3844.

142. Langman G, Hall PM, Todd G. Role of non-alcoholic steato-

hepatitis in methotrexate-induced liver injury. J. Gastroenterol.

Hepatol. 2001; 16; 13951401.

143. Bruno S, Maisonneuve P, Castellana P et al. Incidence and risk

factors for non-alcoholic steatohepatitis: prospective study of

5408 women enrolled in Italian tamoxifen chemoprevention

trial. BMJ 2005; 330; 932.

144. Ratziu V, Poynard T. NASH: a hidden and silent fibroser finally

revealed? J. Hepatol. 2005; 42; 1214.

145. Ratziu V, Giral P, Charlotte F et al. Liver fibrosis in overweight

patients. Gastroenterology 2000; 118; 11171123.

146. Ong JP, Younossi ZM. Nonalcoholic fatty liver disease

(NAFLD)two decades later: are we smarter about its natural

history? Am. J. Gastroenterol. 2003; 98; 19151917.

147. Promrat K, Lutchman G, Uwaifo GI et al. A pilot study of

pioglitazone treatment for nonalcoholic steatohepatitis. Hepa-

tology 2004; 39; 188196.

148. Mendler MH, Kanel G, Govindarajan S. Proposal for a

histological scoring and grading system for non-alcoholic fatty

liver disease. Liver Int. 2005; 25; 294304.

149. Franzen LE, Ekstedt M, Kechagias S, Bodin L. Semiquantitative

evaluation overestimates the degree of steatosis in liver

biopsies: a comparison to stereological point counting. Mod.

Pathol. 2005; 18; 912916.

150. Marsman H, Matsushita T, Dierkhising R et al. Assessment

of donor liver steatosis: pathologist or automated software?

Hum. Pathol. 2004; 35; 430435.

151. Merriman RB, Ferrell LD, Patti MG et al. Histologic correlation

of paired right lobe and left lobe liver biopsies in morbidly obese

individuals with suspected non alcoholic fatty liver disease.

Hepatology 2003; 38; 232A.

152. Mendez P, Regev A, Molina E et al. Sampling error and

reliability of liver biopsy in patients with non-alcoholic fatty

liver disease (NAFLD). Hepatology 2003; 38; 673A.

153. Janiec DJ, Jacobson ER, Freeth A, Spaulding L, Blaszyk H.

Histologic variation of grade and stage of non-alcoholic fatty

liver disease in liver biopsies. Obes. Surg. 2005; 15; 497501.

154. Ratziu V, Charlotte F, Heurtier A et al. Sampling variability of

liver biopsy in nonalcoholic fatty liver disease. Gastroenterology

2005; 128; 18981906.

155. Goldstein NS, Hastah F, Galan MV, Gordon SC. Fibrosis

heterogeneity in nonalcoholic steatohepatitis and hepatitis C

virus needle core biopsy specimens. Am. J. Clin. Pathol. 2005;

123; 382387.



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