UNIVERSITY OF MEDICINE AND PHARMACY IULIU HATIEGANU CLUJ -NAPOCA [606773]

UNIVERSITY OF MEDICINE AND PHARMACY “IULIU HATIEGANU” CLUJ -NAPOCA
FACULTY OF MEDICINE

LICEN CE THESIS

Scientific coordinato r:
Assist. Prof. Dr. Dan Gheban PhD
Graduate student: [anonimizat]

2011

UNIVERSITY OF MEDICINE AND PHARMACY “IULIU HATIEGANU” CLUJ -NAPOCA
FACULTY OF MEDICNE

LICEN CE THESIS

Immunohistochemistry in children’s chronic B
hepatitis. A role in prognosis and treatment

Scientific coordinato r:
Assist. Prof. Dr. Dan Gheban PhD
Graduate student: [anonimizat]

2011

CONTENT S
1st Part – Theory
Chapter 1
Role of histopathology in liver pathology pag.4
Chapter 2
Chronic hepatitis pag.16

2nd Part – Practical
1. Introduction pag. 49
2. Material and Methods pag. 50
3. Results and Discussions pag. 65
4. Conclusions pag. 74
5. References pag. 75

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1st Part
Theory

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Chapter 1
ROLE OF HISTOPATHOLOGY IN LIVER PATHOLOGY

Introduction
Examination of liver tissue from living patients is performed on surgical and needle
biopsies. It comprises mainly of histopathological study, and may serve diagnostic and/or
research purposes. Diagnostic investigations require close collaboration with the clinician to
assure optimal reliability. Of paramount importance are adequate biopsies (1) and impeccable
histopathological and histoc hemical techniques. (2)

I. DIAGNOSTIC INVESTIGATIONS
1. Histopathological study
Histopathology of the liver biopsy allows diagnosis of acute hepatitis, its degree of
severity, and in most cases differentiation from chronic hepatitis.
Since histopathologically acute viral hepatitis B appears essentially similar to other
forms of acute hepatitis, an etiological diagnosis is less reliable. Acute viral hepatitis B may
show different patterns; shortly described below. (3-5)
Acute hepatitis with s potty necrosis is the classical picture of self -limiting acute
hepatitis. Cell damage tends to predominate in the centrolobular areas. (6)
Distinction can be made between an early, fully developed, late, and residual stage. (7)
Acute hepatitis with bridging necrosis represents more severe hepatitis with confluent
areas of necrosis of the lytic type. The necrosis may link afferent and efferent vascular
landmarks (portal -central bridging necrosis). (8)

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Extensive confluent necrosis is often followed by collapse of the denuded reticulin
framework, resulting in scarring fibrous septa. Older scars can be identified by their elastin
content. (9, 10 )
Acute hepatitis with panlobular or multilobular necrosis is more severe, seen in
fulminant hepatitis.
Acute hepatitis with periportal necrosis features periportal interface hepatitis
(piecemeal necrosis). The occurrence of this lesion in acute hepatitis B was considered an
indicator of possible transition to chronicity (11); as was also bridging hepatic necrosis. (8) It
appears that in the later stage of acute hepatitis (after 2 months) both lesions are unfavorable
prognostic signs. (12, 13 ) The most reliable predictor of chronicity is the demonstrable
presence of HBV antigens in scattered hepatocytes. (14)
The histol ogical differentiation of acute hepatitis B from viral -like drug -induced and
auto-immune hepatitis may sometimes be difficult. Chronic hepatitis is recognizable by
predominance of portal and periportal changes, possible presence of elastin -containing septa ,
and detectable viral antigens (HBsAg, HBcAg) on immunostaining. Differentiation from bile
duct obstruction and acute alcoholic hepatitis is feasible .
The histopathology of chronic viral hepatitis B comprises the elementary lesions of
any form of chronic hepatitis; and some specific features. (15)
Common features in chronic hepatitis.
Portal infiltration. Most portal tracts are infiltrated by inflammatory cells,
predominantly lymphocytes. (16)
Interface hepatitis (previously termed piecemeal necrosis) is typical of more active
disease. It corresponds to lymphocytic infiltration at the interface between connective tissue
(portal tracts and septa) and parenchyma, associated with apoptotic death of local
hepatocytes. (15, 17 ) The lesion may be minimal, mild o r severe.
True interface hepatitis must be differentiated from periportal necro -inflammation in
hepatitis A, from “biliary piecemeal necrosis” in chronic biliary diseases, and from spill -over
of inflammatory cells unassociated with liver cell damage. (16)

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Intralobular focal necro -inflammation (spotty necrosis) varies in extent with the
severity of disease. Confluent lytic necrosis (bridging, panlobular, multilobular) characterizes
severe disease, and clinical exacerbations of chronic disease. (18)
Hepatitic rosettes correspond to clusters of surviving hepatocytes in areas of
extensive necro -inflammation. (5)
Most authors consider bridging necrosis an ominous prognostic finding (19, 20 )
although the lesion may also heal. (21) Apparently bridging necrosis carr ies a sinister long –
term prognosis when associated with interface hepatitis, whereas the cirrhogenic evolution of
chronic disease marked by interface hepatitis (“chronic active hepatitis”) is accelerated by
bridging hepatic necrosis. (7)
Hepatic fibrosis in chronic hepatitis B occurs mostly in a septal pattern, comprising of
so-called active and passive septa. (11) Active septa are rich in cells; they represent extensive
interface hepatitis eventually leading to portal -portal septal fibrosis. (7) Passive septa carry
few or no inflammatory cells, are sharply delineated, and derive from postnecrotic collapse
after confluent necrosis.
The mesenchymal cells responsible for fibrosis comprise portal/septal myofibroblasts,
interface myofibroblasts and activated i ntralobular hepatic stellate cells. (22)
Parenchymal regeneration is evidenced by thickening of liver cell plates (2 -3 cells
wide), and increased numbers of bi – and tri -nucleated hepatocytes. (15) Activation of liver
progenitor cells also participates in r egeneration in chronic hepatitis. (23, 24 )
Parenchymal regeneration in between a restructuring fibrous scaffold, leads to
progressive disturbance of the lobular architecture and results in cirrhosis, usually of the
macronodular type. (25)
Necro -inflammatio n may continue in the cirrhotic stage (active cirrhosis) or burn out
(inactive cirrhosis). (25)
The common elementary lesions of chronic hepatitis constitute the base for grading
and staging in chronic liver disease (see below).

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Specific features of chronic hepatitis B
The most distinctive histological feature for identifying HBV etiology is the “ground –
glass hepatocyte”. (26) It has a finely granular, and faintly eosinophilic cytoplasm due to
proliferation of the smooth endoplasmic reticulum containing accumulated Hepatitis B
surface Antigen (HBsAg). (27, 28 ) Ground -glass cells are highlighted by special stains,
including Shikata’s orcein or aldehyde fuchsin (29) and Victoria blue. (30)
Orcein staining needs to be critically performed. (31) Ground -glass hepatocytes can
be specifically stained by more sensitive immunohistochemistry (32) (see below).
Ground -glass cells must be differentiated from oncocytic change, due to densely
packed mitochondria (mitochondriosis) of unclear significance. (33, 34 ) The differential
diagnosis of HBsAg positive ground -glass cells further includes a similar appearance due to
drug-induction, to cyanamide toxicity, to Lafora’s disease, and to fibrinog en storage disease.
(35)
Ground -glass inclusions from several causes may co -exist in the same patient and in
the same hepatocyte. (36)
“Sanded nuclei” are another, though not obvious change in some cases of chronic
hepatitis B. These are pale finely granul ar inclusions in nuclei containing huge amounts of
HBcAg particles (37), staining reddish violet with chromotrope aniline blue. (38)
A study on the frequency of chracteristic features for chronic hepatitis B, C,
autoimmune and cryptogenic hepatitis conclud ed that the respective histological patterns
have low sensitivity, but high specificity and predictability. (39)
Chronic hepatitis B can be distinguished from acute hepatitis by older fibrosis
(positive elastin stain) and presence of orcein positive (HBsAg positive) ground -glass cells or
inclusions. (5) Bilirubinostasis is rare in chronic and frequent in acute hepatitis.
Chronic biliary diseases (e.g. PBC, PSC) are differentiated by their features of
ductopenia, cholate stasis, cholestatic liver cell rosett es and biliary type fibrosis. Chronic
alcoholic liver disease has its own characteristics of steatosis, pericellular fibrosis, Mallory
bodies, satellitosis and micronocular pattern of cirrhosis.

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2. Immunohistochemistry, in situ hybridization, electron and immuno -electron
microscopy
Immunohistochemical staining with specific antibodies for HBV antigens allows to
specify the HBV etiology of chronic hepatitis and the viral phase in the disease. (3, 40 )
In situ hybridization (ISH) of viral DNA is helpful in det ecting HBV infection and its
topography in the infected cells.
Various direct and indirect immunofluorescence and immunoperoxidase procedures
can be applied on fresh frozen and paraffin embedded tissue. The sensitivity of
immunoperoxidase methods can be en hanced by techniques of antigen retrieval and signal
amplification like the Immunomax (41) and the EnvisionTM+ system. (42)
In acute hepatitis B, very little or no viral antigens are demonstrable (4, 43, 44 ),
except in the very early phase (45, 46 ) and according to one study in patients infected with a
mutant HBV (“silent HBV”). (47)
In chronic hepatitis B, antigen localization patterns vary. The course of chronic
hepatitis B comprises three successive phases: virus tolerance (viral replication) ph ase, virus
clearance and residual integration phase. (48-50)
During the immunotolerant, viral replicative phase there is only mild hepatocellular
damage and inflammation. Immunostaining reveals predominant nuclear localization of
HBcAg. (51, 52 ) HBcAg and HBeAg generally have a coincident cellular expression (53-55),
but the ratio of HBcAg to HBeAg may differ in subcellular locations. Strong cytoplasmic
HBeAg is a marker of high viral replication. (52, 54 ) HBsAg is found in the cytoplasm of
some hepatocytes and in the membrane of numerous cells in a honeycomb -like pattern. (56,
57)
The viral clearance phase is characterized by immune elimination of virus -infected
hepatocytes, seroconversion from HBeAg to HBe antibody, and reduction of viral replication.
(58) Liver histopathology consists of more severe lesions, including confluent necrosis of
variable extent. (59) Immunostaining reveals nuclear, cytoplasmic and membranous HBcAg.
Cytoplasmic HBcAg correlates predominantly with liver damage (51, 52, 60 ) and

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proliferation61 HBsAg shows weak cytoplasmic positivity in some hepatocytes and a
membranous staining pattern. (49, 57, 62, 63 ) HBeAg has been found localized in nucleus
and cytoplasm of hepatocytes (52, 54, 64, 65 ) and in liver cell membranes (66).
Some viru s-infected hepatocytes apparently escape immune elimination, resulting in
persistence of viral infection and integration of viral DNA into the host genome (viral
integration phase). (67, 68 ) If damage during the virus clearance phase was not extensive, th e
liver may recover to only minimal abnormalities. (69) In case several exacerbations occurred
(70) the liver may have developed cirrhosis. (49)
Active replication of HBV has ceased, but HBsAg is produced by hepatocytes that
contain integrated HBV -DNA. (71) HBsAg accumulates in clusters of hepatocytes. HBcAg is
usually undetectable. (72) Mild inflammation may persist for some time, but substantial
necro -inflammation should alert for a possible superinfection with another virus. (73)
Some patients show ongoing active disease after HBeAg seroconversion, due to
persistence of a precore stop mutant HBV with deficient HBeAg synthesis (74, 75 ) or other
mutations. (76) Immunostaining in such cases reveals cytoplasmic HBcAg (75, 77, 78 ) and
precore peptide. (79)
HBxAg has been visualized in the nucleus and cytoplasm of hepatocytes, more widely
present than HBsAg or HBcAg, perhaps representing a more sensitive immunohistochemical
test for HBV infection. (80) Its presence in hepatocellular carci noma cells in HBV+ patients
supports its involvement in hepatocarcinogenesis. (81)
In situ hybridization (ISH) for detection of HBV sequences has been performed with
radioactively and with chemically labeled probes, the former yielding higher sensitivity, the
latter better resolution. ISH identified the hepatocyte cytoplasm as the site of HBV
replication. (82-86) Combination of ISH with immunostains for HBV antigens revealed that
HBV DNA is present in numerous antigen negative cells, but co -localizes mainly with
cytoplasmic and less with nuclear HBcAg. (85, 87, 88 ) Hepatocytes with cytoplasmic HBsAg
accumulation are not virus replicating. (89)

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HBV specific sequences were also found in bile duct epithelium, endothelial and
vascular smooth muscle cells. (84) In hepatitis B, immunohistochemistry is more sensistive
than ISH. (90)
Electron and immuno -electron microscopy allow to visualize and identify structural
components of HBV in liver cells.
HBc particles appear as spherical structures, 24 -27nm in diameter, in the
nucleoplasm, and in the hyaloplasm between hepatocellular organelles. (89, 91 ) The finding
that cells with nuclear HBcAg are mostly free from HBV -DNA by ISH87, (88) suggests an
accumula tion of empty or non -replicating core particles. (89)
Encapsulation of core particles within the endoplasmic reticulum (Dane particle
formation) has been documented. (92-94) Membrane localization of HBcAg in the
hepatocellular plasmalemma was confirmed by immuno -electron microscopy. (95)
HBsAg appears as filaments within the cisternae of the endoplasmic reticulum. (96-
98) Ground -glass hepatocytes exhibit a marked hyperplasia of the endoplasmic reticulum
which dislocates the cytoplasmic organelles to the cel l periphery. Within the cisternae, there
are typical filaments giving a positive reaction for HBsAg and pre -S by immuno -electron
microscopy. (27, 28, 89, 98 )

3. Grading and staging of chronic viral hepatitis B. Semiquantitative scoring
The old classification of chronic hepatitis (99) was essentially a rough grading
system, distinguishing milder from more severe variants of disease.
Progress in aetiological insight and treatment options necessitated a revised
classification.
The new classificatio n of chronic hepatitis is based on etiology (viral, autoimmune,
drug-induced and cryptogenic), also taking into account the histological grade of disease
activity and the stage of evolution in terms of fibrosis and architectural derangement
(cirrhosis). (100, 101 )

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Grading and staging is done in (preferably standardized) verbal descriptions, for instance:
mild, moderate and severe chronic hepatitis as grades; and mild, moderate, severe fibrosis
and cirrhosis as stages. (100)
Several semiquantitative scoring systems have been proposed, in which numerical
scores are assigned to different grades and stages of chronic hepatitis. These methods are
primarily indicated in the context of therapeutic trials or research projects; they are not
intended to replace verbal reports in routine diagnostic practice. (100, 102 -107)
The first successful scoring system specifically designed for chronic hepatitis is
widely known as the Knodell Histological Activity Index (HAI). (108) Scores for the
individual lesion categories are added to obtain an overall “histological activity index”. This
broadly used system is subject to a number of criticisms, the most important being that scores
for necro -inflammation (grading) are added to those for fibrosis (staging). (100, 109 )
Subsequent scoring systems have taken these deficiencies into account and comprise
more simple and more complex prescriptions. Several simple and easily applicable systems
are available. (110-112) The French METAVIR group devised a simple algorithm for
standardized g rading (113), and an assessment of fibrosis (staging) in five categories. (114)
Simpler scoring systems provide less information but tend to be more reproducible than
complex ones. (115)
An updated version of the Knodell HAI116 separates grading from stagi ng and is
more detailed to provide more information. It assures adequate interobserver reliability. (117)
Several problems are inherent to all scoring systems available: use of arbitrary scores
that are not mathematically valid, observer variation, samplin g variability and aetiological
diversity. (103)
Accuracy and consistency of scoring procedures can be improved by previous
planning and agreement between clinician(s) and pathologist(s), by excluding biopsy
specimens of inadequate size and quality, by agre ement on definition of histological criteria
at the start of the study, by preferable use of dual observers, by avoiding long time intervals
between scoring of biopsies, and by checking intra – and interobserver variation.116

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In general, staging of fibrosis and architectural changes has proved to be more reproducible
than grading of necro -inflammatory lesions. (115, 118 )
Semiquantitative scoring of liver biopsies has been extensively applied in clinical
trials for new treatments of chronic hepatitis B, C and D. (119)
Morphometry has been used as well for quantitation, mostly for assessment of
fibrosis. (120, 121 ) Image analysis based auto mated quantification of liver fibrosis was
claimed to be a sensitive, precise, objective and reproducible method for quantification, thus
supplementing scoring systems that are more based on distribution patterns of fibrosis. (122)
The potential usefulness of a mathematical scoring system based on fractal geometry
for quantifying irregular patterns of fibrosis as observed in chronic hepatitis was recently
addressed. (123)
4. Precancerous lesions
Both HBV and HCV infections may lead to chronic hepatitis, cir rhosis and
hepatocellular carcinoma (HCC). (124, 125 ) Some cellular changes and nodular lesions are
considered premalignant or precursors of HCC. Recognition and reporting of such lesions is
important for adequate patient surveillance. Liver cell dysplasia (LCD), which may be of the
large cell type (126) or of the small cell type (127) belongs to this type of change.
Large cell LCD in biopsies of patients with chronic hepatitis B (or C) is an
independent risk factor for the development of HCC. (128, 129 ) Small cell LCD might
originate from hepatic progenitor cells (130) and may represent an early step in
carcinogenesis. (129)
Clusters of LCD measuring less than 1mm in diameter are termed “dysplastic foci”.
Lesions measuring more than 1mm in diameter are desi gnated as nodules. (131)
A macroregenerative nodule (measuring 0,8 cm or more) is particularly common in
macronodular cirrhosis. Histologically it shows hyperplastic liver parenchyma but no cellular
atypia nor disorder in muralia arrangement. Dysplastic nodules do display atypical features,
but not severe enough to qualify for frank HCC. Dysplastic nodules are subclassified as low

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or high grade, and considered distinct stages in the multistep process of hepatocarcinogenesis.
(132) Foci and nodules should be carefully identified and reported. (5, 133)

5. Special cases of chronic viral hepatitis B
Short reference is made to those conditions in which liver tissue examination may be
contributory.

5.1. Coinfection with Hepatitis D vir us
Coinfection with Hepatitis D virus (HDV, delta agent) alters the course of acute
hepatitis B, favors chronic evolution and enhances severity of disease. (110, 134, 135 )
Microvesicular steatosis of hepatocytes (morula cells) was a notable feature in an outbreak of
HDV infection in Venezuelan Indians. (136-138)
Specific immunostaining allows to confirm HDV aetiology. HDAg is detected in
hepatocyte nuclei (139, 140 ) and in the cytoplasm and plasmalemma of hepatocytes. (141)
Large amounts of nuclear HDAg may cause a “sanded nuclei” appearance. (142, 143 )
Double immunostaining reveals separate expression of HDAg versus HBsAg and
HBcAg; co -expression in the same cell is found though rarely with HBcAg. (141, 144 )
In situ hybr idization for HDV RNA may be more sensitive than immunostaining for
HDAg. (84, 145, 146 )
HDV RNA localizes in nuclei, usually together with HDAg, but occasionally alone.
(146)

5.2. Recurrent HBV infection post transplantation
Recurrent HBV infection post transplantation may either show the typical features
observed in non -transplanted liver, or, more rarely, atypical patterns of liver damage.

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Atypical patterns include hepatocyte ballooning, fatty change and a distinctive
cholestatic syndrome, which also ma y occur in combination. (147)
Hepatocyte ballooning typically occurs without significant inflammation, but shows
high level of viral replication reflected in diffuse nuclear and cytoplasmic immunostaining for
HBcAg and HBeAg. (148) Ballooning associated wi th steatosis may explain the term
“steatoviral hepatitis”. (149)
The terms “fibrosing cholestatic hepatitis” (fibrosing cytolytic hepatitis, fibroviral
hepatitis) refer to a distinctive pattern of injury associated with rapidly progressive graft
failure. (147-153) The histological pattern comprises periportal fibrosis surrounding ductular
structures, prominent hepatocyte ballooning, bilirubinostasis, and mild or absent
inflammation. Immunostaining reveals markedly positive cytoplasmic HBcAg. This stage
may be followed by extensive postnecrotic collapse, fibrosis and ductular reaction.
This lesional pattern was also reported in other immunodeficiency states, including
HIV infection (154) and following renal transplantation. (155)

5.3. Association of HBV with other viral infection
Dual and triple infections with HBV, HCV and HDV occur. Histopathologically,
there are no specific findings to suggest the possibility of multiple infections. (156)
Immunohistochemical analysis may reveal suppression of HBsAg and HBc Ag by
simultaneous HCV infection. (157)
HCV infection is suspected on the base of characteristic histological features (158)
and positive immunostaining with specific antibody. (159)
Co-infection of HBV with cytomegalovirus (CMV) infection can be identifie d by
typical nuclear inclusions, abnormal basophilic granular cytoplasm, microabcesses, and
immunohistochemical staining for CMV antigen. (5)
Co-infection of HBV with HIV usually results in diminished histological activity
(160), although more severe activ ity was also reported. (161) Immunostaining revealed more

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diffuse staining for HBV and HDV antigens in tissue specimens of HIV coinfected patients.
(160)

5.4. Association of HBV with concomitant liver disease
Histopathological study often reveals clinically unsuspected concomitant liver
disease. Examples include: alpha -1-antitrypsin dificiency, alcoholic liver disease, primary
sclerosing cholangitis, haemochromatosis, amongst others. (2)

II. SPECIAL STUDIES (RESEARCH PURPOSE )
Inventive investigations in different ways of tissue examination are applied to broaden
the insight in pathophysiology. Examples include: semiquantitative evaluation of activated
hepatic stellate cells (162-164); immunohistochemical studies on lymphocyte s ubsets, antigen
presenting cells, adhesion molecules and apoptosis markers (165, 166 ); study of cytokines
(167); elution of liver infiltrating lymphocytes (168); microdissection of sublobular regions
of high and low degree of liver damage and their relationship to HBV variants (169); and
cDNA microarray based analysis of differences in gene expression between chronic hepatitis
B and C. (170)
To meet future requirements, pathology evaluates app ropriate techniques of tissue
preparation for high throughput molecular analysis. (171)

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Chapter 2
CHRONIC HEPATITIS

Chronic hepatitis indicates a persistent, and often progressive, inflammatory process
of the liver that is characterized histologic ally by lymphocytic infiltration of the portal tracts
together with varying degrees of parenchymal inflammation, hepatocellular injury, and
fibrosis.
Although this definition appears straightforward, there are certain difficulties in its
practical applica tion. The crux of the problem is in establishing that the process is indeed
chronic and thus potentially progressive. Chronicity can be judged in several ways – clinical
findings, laboratory abnormalities, or morphologic assessment – but, unfortunately, none of
these is an absolutely reliable guide in separating acute hepatitis, which is usually self –
limited, from chronic hepatitis. When all these evaluations are in agreement, the diagnosis
poses few problems, but sometimes the criteria are discordant or ambiguous. (172) In those
cases, the duration of the disease becomes an important consideration in designating
chronicity. Although the choice is somewhat arbitrary, a minimum of six months duration is
the commonly agreed -upon period. This temporal criteri on is particularly useful in deciding
when a patient with protracted acute viral hepatitis can be definitively said to have developed
chronic hepatitis.
Chronic hepatitis encompasses several distinct disorders with differing etiologies and
natural histories. The causes include several of the hepatitis viruses, presumed autoimmune
reactions, and drug -induced injury. Certain inherited metabolic disturbances such as Wilson's
disease and alpha -1-antitrypsin deficiency may also manifest with chronic hepatitis . As
defined above, chronic hepatitis could also embrace such conditions as the chronic biliary
disorders – primary biliary cirrhosis, primary sclerosing c holangitis, and chronic biliary
obstruction – but these are generally considered to be separate conditions.

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Chronic hepatitis has customarily been divided into two major nosologic groups,
chronic persistent hepatitis and chronic active hepatitis. An addit ional group, chronic lobular
hepatitis, was introduced later, but represents a minor and not uniformly accepted category.
(173) These three groups were distinguished primarily on morphologic grounds but their
discrimination was further justified by differences in prognosis. Thus, chronic persistent
hepatitis and chronic lobular hepatitis, characterized by uncomplicated portal inflammati on
and pronounced lobular inflammation respectively, were considered benign, nonprogressive
lesions, and chronic active hepatitis, with its aggressive hepatocellular necrosis and fibrosis,
was regarded as a serious, progressive process leading to cirrhosis .
This widely -employed categorization, which was originally formulated 25 years ago,
has contributed greatly to our understanding of chronic hepatitis and has provided a basis for
patient evaluation and management, but it has certain drawbacks. Our curren t understanding
of the biology and natural history of chronic hepatitis suggests that the three categories do not
represent distinct disease entities; they instead define portions of a broad histologic and
clinical spectrum. Some overlap among the groups i s therefore inevitable. Moreover,
although the prognostic differences continue to be generally accurate, they have become less
distinct as further knowledge about the causes and evolution of chronic hepatitis accumulates.
The classification nevertheless re mains a practical means of designating the extent and
severity of disease, expressing the range of histologic findings, and providing partial
prognostic information, despite its weaknesses.
2.1. CAUSES
The primary causes of chronic hepatitis are the hepat itis viruses and autoimmune
reactions, with drugs and metabolic disturbances being less frequent. Clinical information
and laboratory tests are the chief means of establishing an etiologic diagnosis, although
histologic examination can yield useful informa tion in some circumstances. (174)
Hepatitis B
Between 1% and 10% of adults with acute hepatitis B will develop a chronic
infection. The factors that control viral persistence are not well understood, but chronic

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infections are more apt to develop in infan ts and in individuals who are immunosuppressed,
receiving cancer chemotherapy or hemodialysis, or have Down's syndrome. (175)
The natural history of chronic hepatitis B infection, although incompletely defined, is
separated into two general phases. The ea rly phase is characterized by active, high -level viral
replication with production of intact infectious virions. Serologic markers of replication, such
as e antigen (HBeAg), HBV DNA, and DNA polymerase, are consequently detectable in the
serum. This stage of the infection tends to correlate most strongly with hepatic injury,
although there is a wide range in the severity of the damage: Severe chronic active hepatitis
develops in some patients, but milder disease is more commonly the result and is particular ly
frequent among children, hemodialysis patients, and immunosuppressed individuals. (176)
After an interval ranging from months to years, the infection evolves into a low -grade
replicative phase and viral production declines. Serologically, this is marke d by
disappearance of HBeAg, HBV DNA, and DNA polymerase from the serum and the
corresponding acquisition of antibody to HBe (anti -HBe). Seroconversion to anti -HBe occurs
spontaneously in about 10% to 20% of infected patients per year.
The transition from the high to the low replication phase may be preceded by an
episode of hepatocellular injury and inflammation, which may even result in massive hepatic
necrosis and death. However, once seroconversion to anti -HBe has occurred, the liver
damage tends to re solve and become inactive. The liver may completely return to normal, a
circumstance often referred to as the "healthy" viral carrier state, but any fibrosis that has
supplanted earlier hepatic necrosis may still be evident. Moreover, portions of the HBV
genome may later become integrated into the host DNA. This can enable continued synthesis
of HBsAg despite the lack of complete virus production. However, approximately 1% of
patients annually also lose detectable HBsAg. Incorporation of the viral genome ap pears to be
an important step in the development of hepatocellular carcinoma, a major long -term
complication of chronic hepatitis B infections.
Although the later stages of the infection are therefore generally accompanied by
quiescent disease, there are exceptions. Some patients experience an acute exacerbation with
a reactivation of active viral replication and hepatic injury. This reactivation, which may be

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transient, recurring, or persistent, can occur spontaneously or follow immunosuppressive or
antin eoplastic therapy, and it may lead to severe or progressive disease. Another exception is
noted in other patients who, despite being anti -HBe positive, have ongoing viral replication
and suffer continuing liver disease. In some instances, viral mutants app ear to be responsible
for these anomalous findings.
The serologic diagnosis of chronic hepatitis B is established by detecting HBsAg in
the serum for more than six months. Usually no (or only low -titer) IgM anti -HBc is present,
and, depending on the phase of the disease, HBeAg, HBV DNA, or DNA polymerase activity
may be noted.
Although many aspects remain unclear, the pathogenesis of chronic hepatitis B is
generally considered to involve cell -mediated immunologic mechanisms. The general idea is
that the i nfected hepatocyte, perhaps through the expression of HBcAg on its surface,
becomes a target for attack by cytotoxic T lymphocytes. Although this is a process similar to
that occurring in acute hepatitis, it differs because the immune response is deficient in some
way and fails to eliminate the virus; the destruction of liver cells therefore continues apace.
The reasons for this immunologic impotence are not known, but several possibilities with
variable experimental support have been advanced. For example, the virus itself may disrupt
the production or response to the endogenous anti -viral agent interferon, thereby curbing viral
eradication. Deficient interferon activity may also affect the expression of the class I
histocompatibility products that are requ ired for appropriate recognition of the viral antigens.
Another suggestion is that high -titers of anti -HBc, by binding up the HBcAg on the
hepatocyte surface, might inhibit the T lymphocyte response. Spontaneous mutation of the
viral genome during the cour se of the disease could potentially alter viral antigen expression,
disrupt the immune response, and modify the activity of the disease. All these mechanisms
reflect alterations in host -viral interactions, and further insights in their nature should be
forthcoming.

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Hepatitis C
Hepatitis C is a disease of world -wide importance. While there are areas where the
prevalence in the general population is extremely high, such as Egypt, Saudi Arabia, the
Phillippines, and Papua New Guinea, the prevalence of detect able antibody to hepatitis C in
volunteer blood donors seems to be quite similar, averaging 0.4% to 1.0%. In the United
States, most epidemiologic data have derived from programs administered by the Centers for
Disease Control, Atlanta, Georgia, especially , in recent years, from the elegant Sentinel
Counties Surveillance Program.
Disease Prevalence:
Earlier data from the Sentinel Counties studies indicated that approximately 180,000
new cases of acute hepatitis C developed each year and that hepatitis C accounted for about
21% of all cases of acute viral hepatitis. While that data have proven to be ex tremely useful,
it is not clear just how accurate they are. Unlike hepatitis A and B, diseases in which most
infected adults have overt clinical illness, hepatitis C uncommonly begins with symptoms
generally attributable to viral hepatitis. Therefore, a fu dge factor is required when data are
accumulated through surveillance of patients with symptomatic illness. If apparently a cause
for the minority of instances of acute viral hepatitis, HCV is associated with the majority of
instances of chronic hepatitis and liver disease. HCV appears to be present in slightly more
than one -half of all persons whose death certificate is coded as chronic liver disease, although
in about one -half of these, there is associated chronic alcoholism.
Recent data suggest that the annual incidence of acute hepatitis C has fallen from
180,000 to 35,000, that there are 3.9 million Americans who are currently infected, and that
hepatitis C now accounts for about 15% of all new cases of acute viral hepatitis.
Nevertheless, residual cas es from the period of peak incidence abound and are the subject of
current concern with respect to outcome (see later). There has probably been a background
prevalence of HCV infection of about 0.4%, the incidence presumably skyrocketting as the
U.S. enter ed the periods of expanded narcotic drug use in the late 1960's and early 1970's. A
recent survey seeking the nationwide prevalence of anti -HCV utilized sera obtained through
the NHANES surveillance program, finding seropositivity in 1.8% of instances. How ever

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there are pockets in which far higher prevalences have been identified, such as hemophiliacs,
parenteral narcotic drug addicts, and inner city populations (18% in a survey of persons
coming to the Johns Hopkins University Hospital emergency room; 21% of almost 1,000
consecutive admissions to the VA Medical Center, Washington, DC).
Routes of Transmission:
HCV infection is generally acquired through parenteral exposure (e.g. blood and
blood product transfusion in the past though not currently, parentera l narcotic drug abuse,
tattooing, body piercing, accidental needlesticks, hemodialysis centers, donated organs or
semen from an HCV carrier), snorting of drugs such as cocaine (causing damage to nasal
mucosa and transmitted by sharing of straws), rarely by sexual and maternal -fetal
transmission, and also by other mechanisms not fully defined (perhaps the sharing of razors
and toothbrushes, nicks by straight razors in the barber shop).
Natural History of Chronic HCV Infection
There are many uncertain issues regarding hepatitis C virus (HCV) infection, chief
among which is the question of its natural history. Outcome data are needed for two obvious
reasons: (1) the need to inform hepatitis C virus (HCV) carriers of what to anticipate in their
future and, if p ossible, to reassure them about potential sequelae, and (2) the need to make
rational decisions regarding treatment with drugs that currently are of only limited therapeutic
value. The problem is compounded by the conflicting views held with respect to the natural
history, some regarding chronic HCV infection as a universally progressive disease that will
inevitably be responsible for serious chronic liver disease or liver disease -related demise
provided other disease entities do not intervene first, while others regard it as a progressive
disease limited to a minority of infected individuals, its course dictated by as yet not fully
defined factors that might promote liver disease progression.
What is recognized is that persons who develop acute HCV infecti on rarely recover
completely, more than 80% of them remaining HCV -infected. The result is progression to
chronic hepatitis, generally defined by persistence of serum enzyme abnormalities for at least
six months, advancement in a proportion of instances to cirrhosis, and culmination among a

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few in the development of hepatocellular carcinoma (HCC). What is not well established is
the frequency of these changes, the tempo of advancement, and whether or not there are
existing factors that curb or promote diseas e progression.
Basis for Uncertainty Regarding Natural History: The difficulties inherent in
attempting to define the natural history of HCV infection are numerous. First, a natural
history study requires knowledge of onset of acute disease in order to es tablish frequency and
rate of progression. Unfortunately, onset of acute HCV infection is rarely recognized; in 80%
or more of instances, persons with chronic HCV infection do not recall ever having had an
illness resembling acute hepatitis. Second, full k nowledge of natural history must include
long-term evaluation of the entire spectrum of the acute disease, ranging from the mildest to
the most severe forms. Because of the paucity of symptoms in most persons infected with
HCV, those with the milder diseas e are generally overlooked, thus biasing outcome data in
the favor of those with the more severe illness. Third, a valid study requires equally intense
evaluation of a non -infected control group. Again, the lack of symptoms inhibits selection of
such a gro up thus foiling conduct of a case -control study. Fourth, the extraordinarily indolent
nature of the disease process in chronic hepatitis C coupled with its highly extended course
before sequelae are recognized makes study of the natural history a daunting task, difficult to
accomplish. Indeed, the natural history of the disease is generally more extended than the
natural history of the clinical investigator. Finally, the disease course is obviously altered if
treatment is instituted. Currently, it is almost routine to attempt treatment of most persons
with compensated chronic HCV infection. It is these items that have made study of the
natural history of hepatitis C so difficult.
Available data therefore have derived either from relatively short term prospe ctive
studies beginning from onset of acute disease, mostly transfusion -related, or from study of
persons with already established chronic liver disease.
Follow -Up Studies Beginning with Acute Hepatitis : Most such studies have not
exceeded 10 to 15 years in duration (with the exception of one which is a 17 -year follow -up
of an outbreak of HCV infection induced by contaminated immunoglobulin). This time
period is obviously insufficient since in two retrospective/prospective studies, one from Japan

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and one f rom the U.S.A., progression from apparent acute to chronic hepatitis was estimated
to average 10 years, to cirrhosis, 20 years, and to HCC, 30 years, some developing HCC 40 to
50 years after the presumed acute infection. Nevertheless, these studies have yi elded useful
and important information. In five prospective studies, clinical symptoms have been noted in
about 10% of cases, histologic cirrhosis in 15% to 20%, and HCC identified in 0.7% to 1.3%.
Mortality ranged from 1.6% to 6.0%. In the 17 -year follow -up study of imunoglobulin
recipients, outcome was reported to be benign in the majority of instances; 60% did have
moderate enzyme abnormalities, but only 1.8% had histologic evidence of severe fibrosis
(cirrhosis). These prospective studies thus revealed that during the first two decades
following acute infection, liver -related morbidity and death is modest in frequency.
Follow -Up Studies Beginning with Already Established Chronic Liver Disease : Not
surprisingly, in four such studies, two from Japan, one from the U.S., and one from Australia,
far more serious sequelae were noted. In the two studies from Japan, cirrhosis developed in
30% and 42% respectively, and HCC in 15% and 19%. In the U.S. study, similar devastating
outcomes were identified. Thus, in s tudies beginning with already identifiable chronic liver
disease, serious sequelae are common. The problem with such studies is that they are
"biased" in the direction of severe forms of the disease; persons infected with HCV who have
no symptoms – perhaps the bulk of cases – do not come to the attention of investigators in
tertiary care or liver transplantation centers.
Predictive Factors for Outcome of Chronic HCV Infection:
Since it seems likely that only a limited proportion of HCV -infected individuals
manifest progressive disease, there is speculation that there may be definable factors that
might be predictive of progressive disease. Viral -related factors that have been examined
include viral dose, viral genotype, and the presence of quasispecies. Hos t factors scrutinized
have included the age at the time of infection, race, gender, and geographic location.
Extraneous influences sought have included co -infection with other viruses, exposure to viral
contaminants, smoking, and concomitant chronic alcoho lism. Of these, the latter has proven
to play the most obviously important role, but further studies are warranted.

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Autoimmune -Associated
The cause of this important and classic type of chronic hepatitis is not known, but
many of its characteristic featu res point to an autoimmune origin. These include a female
predominance, the frequent presence of circulating autoantibodies, an association with other
presumed autoimmune disorders (for example, thyroiditis, vasculitis, uveitis, and arthritis),
and a drama tic response to corticosteroid therapy. Several names have been applied to this
entity since its original description in the 1950's – including lupoid hepatitis, plasma cell
hepatitis, and active juvenile cirrhosis – but the term autoimmune chronic hepat itis is
commonly employed today. Some patients with idiopathic, nonviral ("cryptogenic") chronic
hepatitis lack the typical immunoserologic abnormalities, but they are otherwise comparable
to those with autoimmune features.
The typical patient is a woman of either 15 to 35 years or perimenopausal age, but
examples are reported at all ages and in both sexes. A genetic predisposition is suggested by
the presence of serum autoantibodies and other autoimmune manifestations in family
members and by the increase d prevalence, in common with other autoimmune diseases, of
HLA haplotypes A1, B8, and DR3. Serum IgG levels are elevated, often to a striking degree,
and a variety of non -organ -specific autoantibodies can be detected. These variably include
antinuclear ant ibodies, antibodies to double -stranded DNA, positive tests for lupus
erythematosus cells, antismooth muscle antibodies directed against actin, and, uncommonly,
antimitochondrial antibodies at low titer. Some investigators have identified an anti –
liver/kidn ey microsomal antibody (anti -LKM -1) that reacts to a cytochrome P450 isozyme
and have further suggested that its presence defines a distinct subgroup. Although in most
respects this uncommon variant conforms to the usual picture of autoimmune hepatitis, it
tends to occur in younger patients, often in the absence of antinuclear and antismooth muscle
antibodies, and is associated with more serious disease and a worse prognosis.
The natural history of autoimmune chronic hepatitis is not completely characteriz ed,
and, in particular, the course of the less severe forms is poorly defined. In general, the disease
evolves with recurring bouts of hepatic injury that variably, and often insidiously, terminate
in cirrhosis. An acute onset has sometimes been recognized , but it is unclear whether this is a

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true inaugural phase of the disease or simply represents an episode of active exacerbation
complicating an ongoing chronic process. Progression can be swift in patients presenting with
markedly active disease, but the evolution in milder cases appears less aggressive and the
exact risk of cirrhosis unsettled. Immunosuppressive therapy is effective in repressing hepatic
damage, retarding disease progression, and prolonging survival in severe cases, but the
development of cirrhosis is not prevented.
The pathogenesis of this condition is a matter of considerable conjecture, but it is
generally thought to involve a immunologically -mediated attack on hepatocytes. There is
indirect and circumstantial support for this concept, but the initiation, target, and nature of the
attack remain unknown. Numerous experimental studies using in vitro assays have
demonstrated evidence of immune sensitization to the liver cell membrane and, in particular,
to an antigen complex referred to as liver specific protein. Almost all patients with
autoimmune chronic hepatitis have autoantibodies directed against liver specific protein;
recent evidence indicates that the asialoglycoprotein receptor is the major target. By binding
to the hepatocyte sur face, these antibodies presumably provoke an assault through antibody –
dependent cellular cytotoxicity. A decrease in suppressor T cell activity is also commonly
noted and may act to facilitate the synthesis of the autoantibodies. These possibilities are
intriguing, and their role in inducing or perpetuating chronic hepatitis is under intense
investigation.
Drug -Induced
Drug reactions are an unusual – but perhaps underrated – cause of chronic hepatitis.
Several agents have been implicated: the now -discontinued laxative oxyphenisatin,
methyldopa, and nitrofurantoin are most often cited, and sporadic reports incriminate s uch
drugs as isoniazid, dantrolene, halothane, ticrynafen, and aspirin. Prolonged exposure to these
drugs is usually a prerequisite for the development of chronic hepatitis; presumably the
persistence of an initial low -grade or unrecognized acute hepatitis is the responsible process.
The clinical and histologic features of drug -induced chronic hepatitis may be
indistinguishable from any of the other forms, and a careful drug history prompted by a
proper degree of suspicion is the sole means of establishing the correct diagnosis. The

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importance of recognizing a drug etiology is that, once the offending agent is discontinued,
the disease resolves.
Others
Two inherited metabolic disorders, Wilson's disease and alpha -1-antitrypsin
deficiency, can give rise to a clinical and pathologic syndrome indistinguishable from chronic
hepatitis. These uncommon causes, are important to recognize because of the implications for
treatment and family counseling. The clinical setting and histologic features may be
suggestive, b ut appropriate laboratory studies (such as hepatic copper determinations or
serum phenotyping) are needed to establish the diagnosis. With both conditions, advanced
disease and cirrhosis are commonly present at the time of presentation.
Chronic hepatitis i s also occasionally noted in conjunction with chronic alcohol abuse,
and an etiologic relationship between the two has been proposed. The issue is complicated,
however, because alcoholic patients have a greater exposure to hepatitis viruses, particularly
the hepatitis C virus, than the general population. The suspicion is therefore that a
superimposed chronic viral infection is actually responsible for the observed chronic hepatitis
in some patients. In other patients though, viral markers are absent and no other cause of
chronic hepatitis can be implicated: these may represent examples of alcohol -induced chronic
hepatitis, presumably arising on an immunologic basis, although such cases are rare.

2.2. CLINICAL FEATURES
As befits a heterogeneous disorder, c hronic hepatitis encompasses a wide range of
clinical manifestations. Many patients are either asymptomatic or have only minor complaints
while others exhibit features of chronic liver disease or hepatic failure. The disease may have
an insidious onset or may present abruptly; occasionally it follows an episode of acute
hepatitis that fails to remit. In many patients, the sum of the clinical, biochemical, and
histologic findings clearly indicate that the patient is suffering chronic disease, but this is not

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always the case, and documenting a six -month or longer duration of disease may then be
necessary.
The most common symptoms include fatigue or malaise, with mild abdominal
discomfort, jaundice, anorexia, fever, nausea, or arthralgias less often noted. Extr ahepatic
manifestations such as amenorrhea, acne, gynecomastia, or Cushinoid changes can be
prominent, particularly with autoimmune chronic hepatitis, and may represent the presenting
complaint. Physical examination findings are few: hepatomegaly, mild spl enomegaly, and,
occasionally, spider angiomas or palmar erythema may be discovered. In advanced disease,
hepatic decompensation may supervene, as manifest by ascites, varices, or encephalopathy.
Unfortunately, the clinical findings are not specific and cor relate poorly with the severity of
the disease.
The characteristic laboratory abnormalities include an increase, usually of two – to 10 –
fold, in serum transaminase levels. There is no good correlation between the degree of
elevation and the state of progre ssion, although, as a general rule, substantial increases
signify markedly active hepatocellular damage. Other liver tests are generally normal or only
mildly abnormal; increasing serum bilirubin, falling albumin levels, and a prolonged
prothrombin time si gnal advanced, progressive disease.
The etiology is typically established through serologic studies. Chronic viral hepatitis
is suggested by the appropriate antibodies and confirmed by markers of ongoing viral
replication. Autoimmune chronic hepatitis is largely a diagnosis of exclusion; the finding of
elevated serum immunoglobulins and antinuclear or antismooth muscle antibodies,
particularly at high titers, points to this condition, but these alterations are not specific and
may be missing in some cases.
The overall survival of chronic hepatitis is approximately 80% at five years, but the figure
varies from 40% to almost 100% depending on the cause, the severity of the disease, and
extent of progression. In general, patients who have severe parenchymal i njury, developed
advanced disease, or ongoing viral replication suffer the poorest outcome, but the variables
are many and confounding. The long -term outcome is also compromised by the development
of hepatocellular carcinoma, a clearly established complica tion of chronic hepatitis B and

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increasingly recognized with chronic hepatitis C as well. Autoimmune chronic hepatitis has
less often been associated with carcinoma, but the risk may be greater than usually
appreciated, particularly in patients with long -standing cirrhosis.
Immunosuppressive therapy represents the principal treatment for chronic hepatitis,
and the greatest benefit is found in symptomatic patients with severe chronic autoimmune
hepatitis. In these patients, corticosteroids (with or without added azathioprine) diminishes
disease activity, eases symptoms, and prolongs five -year survival from approximately 40% to
over 80%. However, most patients with autoimmune hepatitis have disease of lesser severity,
posing a therapeutic dilemma for the clin ician since corticosteroids are not of proven benefit
in this setting.
In viral -induced chronic hepatitis, immunosuppressive drugs have limited value,
except perhaps in patients in severe and rapidly progressing disease. Antiviral and
immunomodulating age nts would be logical therapeutic choices, but most studies have
yielded disappointing results. Recently, however, interferon alfa has shown promise: A
biochemical and histologic remission is induced in approximately 30% to 50% of patients
with chronic hepa titis B or C. Whether this alters the natural history of the disease is yet
uncertain, and further studies are necessary to define the optimal treatment regime and the
cost-benefit ratio of its use.
Liver transplantation remains a therapeutic option for d ecompensated, end -stage chronic
hepatitis. The results are generally favorable, but chronic hepatitis B is the disappointing
exception. The hepatitis B infection recurs in most patients, leading to a return of hepatic
injury with consequently greater risk of graft loss and death.
The indications for transplantation in this population thus remain unclear, although various
strategies to prevent reinfection are being actively investigated. In contrast, recurrence of
hepatitis C appears in preliminary studies to occur in only a minority of patient; the
reappearance of autoimmune chronic hepatitis is an exceptional event.

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2.3. PATHOLOGIC FEATURES
General Considerations
Chronic hepatitis encompasses a broad range of histologic appearances that center
around thr ee primary alterations: portal inflammation, lobular inflammation and damage, and
piecemeal necrosis. Fibrosis may also develop as a complication of these lesions, and, in
advanced cases, cirrhosis may result. The alterations vary considerably in degree an d are
often distributed irregularly across the liver; sampling error therefore becomes an important
consideration when assessing severity and progression in needle biopsy specimens.
The central feature of most cases of chronic hepatitis is a prominent inf lammatory
infiltration of the portal tracts.

Fig.1: Mononuclear portal inflammation in chronic hepatitis. The density and distribution of
the infiltrate can vary remarkably

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This infiltrate consists primarily of lymphocytes with variable plasma cells and
macrophages and occasionally a few neutrophils or eosinophils. The lymphocytes may be
organized into lymphoid follicles, sometimes with germinal centers.

Fig.2: The portal infiltrate take the shape of lymphoid aggregate, this aspect being suggestive
for HCV etiology.
Most portal tracts are involved to some extent, but the intensity of involvement is
ranges from a sparse collection to a dense crowding of the portal tract; som e portal tracts may
be spared. Bile ductular proliferation is occasionally noted but is generally of mild degree,
and interlobular bile ducts may be damaged, particularly in chronic hepatitis C, as manifest
by epithelial swelling, vacuolation, and inflamma tory infiltration.
Lobular inflammation and damage is an inconsistent feature of chronic hepatitis.

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Fig.3: Lobular inflammation in chronic HBV hepatitis. Focal hepatocyte necrosis with a
scattering of mononuclear inflammatory cells.

It is characterize d by changes similar to those seen in acute hepatitis, including
varying degrees of ballooning or acidophilic degeneration, focal hepatocellular necrosis,
acidophilic bodies, and an accompanying mononuclear inflammatory component. In severe
cases, confluen t necrosis including bridging or multilobular necrosis may be noted. The
magnitude of these lobular changes generally mirrors the clinical and biochemical activity of
the disease: minor alterations are noted in quiescent phases and more conspicuous finding s
seen with exacerbations of active hepatic injury or with superinfections by other hepatitis
viruses. In chronic hepatitis, this lobular reaction tends to be milder and less regularly
distributed than in acute hepatitis, but this is not always the case.

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Piecemeal necrosis designates the presence of periportal injury and inflammation. It is
characterized by extension of the portal inflammatory infiltrate into the adjacent parenchyma
together with destruction of individual hepatocytes alon g the edges of the portal tract .

Fig.4: Piecemeal necrosis (interface hepatitis) in chronic HBV hepatitis. The affected
periportal hepatocytes are engaged in apoptotic destruction

The interface between liver cells and connective tissue consequently becomes ragged
and irregular, sometimes with trapping of small clusters of liver cells within the infiltrate.
Periportal fibrosis is an almost universal accompaniment and imparts a stella te profile to the
edge of the portal tract best appreciated on connective tissue stains.

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Fig.5: Stellate shape periportal fibrosis. Trichrom Masson special stain for connective tissue.

The extent and distribution of piecemeal necrosis is variable and often uneven. In
mild disease, only occasional foci of periportal necrosis may be seen, whereas with more
vigorous involvement, the entire portal tract circumference may be affected.
The nature of piecemeal necrosis is not well understood, but, from the time the term
was introduced, it has been considered to be an immunologically -mediated phenomenon.
That concept derives indirect support from several investigative studies. The lymphocytes
that accumulate in piecemeal necrosis are largely suppressor/cytoto xic (CD8+) T
lymphocytes together with a variable number of CD4+ cells. They express various cellular
adhesion molecules and receptors, indicating that they are biologically active and presumably
engaged in liver cell injury. This is further implied by the intimate contact between the
membranes of the lymphocytes and damaged hepatocytes. Moreover, antigen -presenting cells

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such as interdigitating and dendritic reticulum cells are also found in the inflammatory
infiltrate. These observations suggest that piec emeal necrosis represents a liver -based
immune response fundamentally similar to that occurring in lymphoid organs. The reasons
for the periportal localization of such processes are unknown, but this may relate to the
differential distribution of the targe t antigens.
Based on the relative predominance of these alterations, the three customary types of
chronic hepatitis – chronic persistent hepatitis, chronic active hepatitis, and chronic lobular
hepatitis – can be defined. These should be considered not as discrete diagnostic entities but
rather as histologic patterns that denote the extent and severity of disease within a
morphologic continuum. Consequently, no absolute lines of demarcation among the three
types exist, and their distinction blurs at the borders of their definitions. In addition,
transitions from one type to another occur spontaneous and following treatment. Fortunately,
once clinical information is taken into account, these pathologic problems do not translate
into great differences in pr ognosis or treatment. As noted before, there are drawbacks to
classifying chronic hepatitis into the these traditional groups, but the scheme serves as a
practical strategy for characterizing the histologic spectrum of chronic hepatitis.
A full histologic description of a liver biopsy specimen of chronic hepatitis involves
the assessment of each of the independent morphologic features, including the degree of
portal inflammation and piecemeal necrosis, the severity of associated lobular activity, and
the e xtent and distribution of fibrosis. Chronic active hepatitis, because of its wide range of
appearance, has often been separated into mild, moderate, and severe grades, based usually
on the degree of piecemeal necrosis and the presence of bridging necrosis.

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Fig.6: Bridging necrosis in chronic HBV hepatitis.

Ishak’s Histologic Activity Index

Modified HAI Grading: Necroinflammatory Scores* Score
A. Periportal or periseptal interface heptatitis (piecemeal necrosis)
Absent 0
Mild (focal, few portal areas) 1
Mild/moderate (focal, most portal area s) 2
Moderate (continuous around <50% of tracts or septa) 3
Severe (continuous around >50% of tracts or septa) 4
B. Confluent necrosis
Absent 0
Focal confluent necrosis 1
Zone 3 necrosis in some areas 2
Zone 3 necrosis in most are as 3
Zone 3 necrosis + occasional portal -central (P -C) bridging 4
Zone 3 necrosis + multiple P -C bridging 5
Panacinar or multiacinar necrosis 6
C. Focal (spotty) lytic necrosis, apoptosis and focal inflammation†
Absent 0
One focus or less per 10× objective 1
Two to four foci per 10× objective 2
Five to ten foci per 10× objective 3
More than ten foci per 10× objective 4
D. Portal inflammation
None 0
Mild, some or all portal areas 1
Moderate, some or all portal a reas 2
Moderate/marked, all portal areas 3
Marked, all portal areas 4
Maximum possible score for grading 18
Modified Staging: Architectural Changes, Fibrosis, and Cirrhosis ‡
Change Score
No fibrosis 0
Fibrous expansion of some portal a reas, with or without short fibrous septa 1
Fibrous expansion of most portal areas, with or without short fibrous septa 2
Fibrous expansion of most portal areas with occasional portal to portal (P -P) bridging 3
Fibrous expansion of portal areas wit h marked bridging (P -P) as well as portal -central (P -C) 4
Marked bridging (P -P and/or P -C) with occasional nodules (incomplete cirrhosis) 5
Cirrhosis, probable or definite 6
Maximum possible score 6

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Etiologic Aspects
As a general rule, the various causes of chronic hepatitis cannot be conclusively
distinguished on histologic grounds.
The one exception is chronic hepatitis B, which can sometimes be identified by the
presence of "ground -glass" hepatocytes. The cytoplasm of these cells has a uniform, finely
granular, and faintly eosinophilic appearance that is likened to frosted glass. Th is change
derives from a marked proliferation of endoplasmic reticulum containing accumulated
particles of HBsAg. All or part of the cytoplasm may be affected, and an unstained halo
typically separates the involved from the uninvolved portion. Ground -glass cells are scattered
Modified HAI Grading: Necroinflammatory Scores* Score
A. Periportal or periseptal interface heptatitis (piecemeal necrosis)
Absent 0
Mild (focal, few portal areas) 1
Mild/moderate (focal, most portal area s) 2
Moderate (continuous around <50% of tracts or septa) 3
Severe (continuous around >50% of tracts or septa) 4
B. Confluent necrosis
Absent 0
Focal confluent necrosis 1
Zone 3 necrosis in some areas 2
Zone 3 necrosis in most are as 3
Zone 3 necrosis + occasional portal -central (P -C) bridging 4
Zone 3 necrosis + multiple P -C bridging 5
Panacinar or multiacinar necrosis 6
C. Focal (spotty) lytic necrosis, apoptosis and focal inflammation†
Absent 0
One focus or less per 10× objective 1
Two to four foci per 10× objective 2
Five to ten foci per 10× objective 3
More than ten foci per 10× objective 4
D. Portal inflammation
None 0
Mild, some or all portal areas 1
Moderate, some or all portal a reas 2
Moderate/marked, all portal areas 3
Marked, all portal areas 4
Maximum possible score for grading 18
Modified Staging: Architectural Changes, Fibrosis, and Cirrhosis ‡
Change Score
No fibrosis 0
Fibrous expansion of some portal a reas, with or without short fibrous septa 1
Fibrous expansion of most portal areas, with or without short fibrous septa 2
Fibrous expansion of most portal areas with occasional portal to portal (P -P) bridging 3
Fibrous expansion of portal areas wit h marked bridging (P -P) as well as portal -central (P -C) 4
Marked bridging (P -P and/or P -C) with occasional nodules (incomplete cirrhosis) 5
Cirrhosis, probable or definite 6
Maximum possible score 6

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throughout the lobule in variable numbers, but there is a rough inverse correlation with the
activity of the disease: Many ground -glass cells are seen in "healthy" viral carriers with
minimal hepatocellular injury, but they are sparse or absent in severe chronic active hepatitis.
Note that they are not seen in uncomplicated acute hepatitis B.

Fig.7: Typical aspect of chronic HBV hepatitis. 1. Portal inflammation; 2. Piecemeal
necrosis; 3. Lobular inflammation. The red arrows points to “ground -glass” modification of
hepatocytes who are considered to be highly suggestive for HBV etiology.

Similar ground -glass cells are seen in other settings, including Lafora's disease
(myoclonic epilepsy), type IV glycogenosis , therapy with cyanamide, and exposure to drugs
that induce proliferation of the endoplasmic reticulum, such as phenobarbital or phenytoin.

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Other cases of unknown significance reflect the intracellular accumulation of fibrinogen or
other proteins. The hepa titis B ground -glass cells are distinguished by their positive staining
with orcein, Victoria blue, or aldehyde fuschin stains, or by specific immunostaining for
HBsAg .
Fig.8: Immunostain for HBs antigen. Cytoplasmic diffuse pattern of stain.

In addition , hepatocyte nuclei in chronic hepatitis B may develop a finely granular
"sanded" look that reflects the accumulation of HBcAg. This feature can be difficult to
identify with certainty, and the presence of HBcAg is better secured by
immunohistochemistry.

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Fig.9: The sanded look of HBc antigen integrated in hepatocyte nuclear region. In HE stain is
a very rare encounter.

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Fig.10: Immunostain for HBc antigen. Nuclear pattern of distribution .

Chronic hepatitis C can demonstrate the same inconsistent and nonspecific histologic
changes seen in acute disease. Although not definitive, these features are frequently
suggestive and include fatty change, prominent lobular activity that is often characterized by
a sinusoidal arrangement of inflammatory cells, dens e portal inflammation with lymphoid
aggregates or germinal centers, and bile duct damage .

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Fig.11: Bile duct damage in chronic HCV hepatitis

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Fig.12: Fatty change and lobular inflammation in chronic HCV hepatitis

The conspicuous lobular activity often results in difficult in separating acute from
chronic hepatitis C on morphologic appearances alone.
Autoimmune chronic hepatitis is often characterized by severe hepatocellular injury
and broad areas of reticulin co llapse. Plasma cells may be abundant, as emphasized in early
reports, but this is neither an absolute nor required feature.
Wilson's disease is suggested by such features as copper accumulation (as detected
with copper stains), Mallory bodies, fatty chang e, and glycogenated nuclei (Chapter 9). These

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are not specific and, moreover, are not invariably present; appropriate suspicion when chronic
hepatitis is encountered in a patient less than 30 years is the key to considering the diagnosis.
The histologic m arker for alpha -1-antitrypsin deficiency is the PAS -positive, diastase
resistant cytoplasmic globule. Typically located in periportal (or periseptal) hepatocytes,
these globules can be specifically stained by immunohistochemistry.
Immunohistochemistry
Immunohistochemistry is a sensitive and specific method for detecting hepatitis B
antigens in liver tissue, but it seldom plays an important diagnostic role when serologic tests
are available. HBsAg is generally identified within the cytoplasm of the hepat ocyte (see
fig.8) – particularly in ground -glass cells – or along the cell membrane .
Fig. 13: Membrane pattern of HBs antigen immunostain in HBV chronic hepatitis

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HBcAg is typically located in the nucleus (see fig.10) but cytoplasmic and
membranous staini ng are also seen. Several patterns of viral antigen expression have been
described and correlations with the severity and course of disease have been proposed, but
the results have been variable. In particular, the presence of HBcAg signifies active viral
replication, and its cytoplasmic expression appears to be related to more active disease .
Hepatitis C viral antigens have been demonstrated in liver tissue by
immunoperoxidase techniques, but the procedure is not widely available and awaits further
confirmation of its utility.
The hepatitis D viral antigen is detected by immunohistochemistry in both acute and
chronic hepatitis D. The antigen is expressed primarily in nuclei an d occasionally in
cytoplasm, and its presence is noted in almost all patients with chronic hepatitis D. Positive
staining, which correlates with active viral replication, is therefore frequently useful in
confirming the diagnosis.

2.4. PROGNOSTIC FACTORS
The course of a patient with chronic hepatitis depends on many factors, but the issue
is clouded by differences among various studies in patient selection, etiology, histologic
criteria, and length and rigor of follow -up. Firm conclusions are therefore di fficult to reach.
Histologic features are likely important indicators of outcome, but these must be tempered by
the clinical circumstances, the exact cause, and, in viral -associated cases, the presence of
continuing viral replication.
The histologic patter ns continue to be useful overall guides to prognosis, despite their
weaknesses as nosologic categories. Chronic persistent hepatitis and chronic lobular hepatitis
generally tend to be benign conditions, typically with stationary courses. On the other hand,
chronic active hepatitis can be a serious and progressive disorder leading to cirrhosis and
death; in severe cases, the five -year survival may be as low as 40% to 50%.

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Nevertheless, the histologic pattern only denotes the disease severity at a particular
time, and the general prognostic precepts therefore have exceptions that limit their
unconditional applicability. Chronic persistent hepatitis B, for example, may progress if viral
replication is sustained. Immunosuppressive therapy of chronic active hepa titis can reduce the
periportal inflammation and piecemeal necrosis, thereby converting it into chronic persistent
hepatitis, but this does not necessarily imply a favorable long -term outcome.
Moreover, chronic active hepatitis is associated with a range of outcomes that, in part,
depend the severity of additional histologic changes. Most notably, the presence of bridging
(or multilobular) necrosis indicates a group with a particularly poor prognosis, and this
feature should be specifically evaluated in bi opsy specimens. Chronic active hepatitis that
displays only piecemeal necrosis and lacks bridging necrosis appears to have a better
prognosis, although the exact outcome is incompletely defined. At the minimal end of the
spectrum, the disease may be nonpro gressive and the course indistinguishable from that of
chronic persistent hepatitis.
The degree of fibrosis, as a measure of the severity of previous hepatic injury, can be
used to stage chronic hepatitis. Although no formal staging schemes have been prop osed,
fibrosis can be assessed in the same fashion as other types of chronic liver disease, and, based
on the low -power architectural appearance, three stages can be distinguished: portal and
periportal fibrosis, bridging fibrosis, and cirrhosis. No study has examined the prognostic
implications of fibrosis independently of other features, and, in most investigations, bridging
fibrosis has not been clearly distinguished from bridging necrosis. Nevertheless, more
advanced fibrosis and, in particular, cirrhos is portend greater mortality. A practical difficulty
in evaluating the stage is the uneven distribution of fibrosis in chronic hepatitis; although
therefore subject to sampling error, a rough estimation is generally possible.

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2.5. DIFFERENTIAL DIAGNOSI S
Although the diagnosis of chronic hepatitis can often be suggested by the
morphologic picture, none of the various histologic features is entirely specific, and a final
diagnosis therefore generally requires clinical correlation.
The mild portal and lob ular inflammation seen in the lesser forms of chronic hepatitis
can be noted in such disorders as resolving acute hepatitis and nonspecific reactive hepatitis.
The histologic findings overlap greatly, and the distinction depends largely on the duration of
the disease and the presence of appropriate laboratory abnormalities. Unless this ancillary
evidence indicates that a low -grade but nonetheless chronic inflammatory disease of the liver
is present, a diagnosis of chronic hepatitis should not be entertained .
The lobular activity of chronic hepatitis represents the primary feature of acute
hepatitis, and distinguishing the two can be difficult. In chronic hepatitis, the lobular
inflammation is typically distributed in a more focal manner than in acute diseas e, but this is
often difficult to appreciate. In addition, bridging necrosis can be seen in either acute or
chronic hepatitis, further complicating the distinction. The presence of underlying fibrosis is
the best clue to chronicity, but attention to the cl inical setting, serologic data, and time course
of the disease are often required. In equivocal cases, a six -month duration may be the only
indicator of chronic disease.
Portal inflammation – with or without accompanying periportal inflammation or
pieceme al necrosis – occurs in wide range of conditions. The principal considerations are
primary biliary cirrhosis and primary sclerosing cholangitis, both of which share
characteristics with chronic hepatitis. Their main distinguishing features are chronic
cholestasis and loss of the interlobular bile ducts (Chapter 5). Chronic cholestasis, although
accompanied by periportal inflammation and hepatocyte injury, is characterized by a
heterogeneous inflammatory population that includes neutrophils and macrophages, the
occasional presence of Mallory bodies, and the accumulation of copper. Stains for copper and
copper -associated protein are therefore useful: Typically positive in either of the biliary
conditions, they are negative in precirrhotic chronic hepatitis. B ile duct loss is identified by
finding hepatic arteries that are missing their accompanying bile duct; when more than half of

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the arteries lack ducts, bile duct loss is clearly present. In addition, granulomas may be seen
in primary biliary cirrhosis but n ot the usual case of chronic hepatitis. Before chronic
hepatitis is diagnosed in a woman over 40 years, serious consideration should be given to
primary biliary cirrhosis. Although patients with ulcerative colitis can certainly develop
chronic hepatitis, t he possibility of primary sclerosing cholangitis should be entertained when
a histologic pattern suggesting chronic hepatitis is encountered; the diagnosis of primary
sclerosing cholangitis can be established by cholangiography.
Chronic biliary obstruction , especially when intermittent or low -grade, can produce a
picture similar to that of chronic hepatitis. Chronic cholestasis may be present, but the clinical
and radiographic data generally permit the distinction to be made.
Malignant lymphomas, primarily the low -grade lymphocytic varieties, mimic chronic
hepatitis by filling the portal tracts and sometimes spilling out into the adjacent parenchyma.
The malignant infiltrates tend to be cytologically atypical and more dense and
monomorphous in character than inflammatory infiltrates; in questionable cases, lymphocyte
markers may be helpful in establishing the nature of the cells.

Second Part
Practical study

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INTRODUCERE

Immunohistochemical screening of HBV antigens is not routinely applied in
practice, being a technique somehow reserved for research purposes only. It is
admitted that in cases with cryptogenic hepatitis, such immunohistochemical
investigations are indicated, but otherwise the serological viral diagnosis is enough.
In research purposes, the immunohistostaining of HBs and HBc antigens can
be useful for the quantification of the viral replicative state of the patient1.
According to Desmet, the replicative phase of HBV is characterized by:
 Light to moderate necro -inflammatory activity
 Nuclear staining of HBc antigen
 Cytoplasmic staining for HBe antigen
 Membranar staining for HBs antigen

The clearance phase of HBV virus, triggered usually by interferon treatment, is
characterized by:
 Moderate to intense necro -inflammatory activity
 Nuclear, cytoplasmic and membranar positivity for HBcAg
 Focal cytoplasmic and membranar positivity for HBsAg

The integrative phase of the HBV virus infection is characterized by:
 Absent of light necro -inflammatory activity
 Negative HBc Ag stain
 Focal cytoplasmic stain for HBs

Our goal was to verify by immunohistochemical staining, the etiologic
diagnosis, established by serologic techniques and to add at diagnosis, the viral
replicative state of each studied patient, because the treatment opportunity’s and
treatment strategy (interfero n, antiviral, antifibrotic) seems to be dependent on this
replicative state.

1 Desmet V.J., Liver tissue examination, Journal of Hepatology, 39 (2003), 843 -849

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MATERIAL AND METHOD
Study group
The study is based on a group of 26 children’s with chronic HBV hepatitis ,
admitted in Children Hospital from Cluj -Napoca . From all this patients, we have a
liver biopsy, taken for the evaluation of the case before treatment indication. The
grading and staging of all this cases was established, the reference system in this study
being the Ludwig/Bat ts grading and staging system for liver biopsy’s2.
The study group age was between 4 and 18 yrs. (average 11 yrs.).
Sex distribution of the study group was 10 girls (38.46%) and 16 boys
(61.53%).
Al data about patients were extracted from the clinician referral note sanded
with the biopsy.

Fig. 1 4: Age distribution of the study group

Method
The paraffin embedded material was extracted from the Anatomic Pathology
Laboratory of the Children Hospital Cluj . From this material, new histologic sections
was performed , and immunohistochemical stains were performed. The mono clonal
antibodies against HBc and HBs antigens were paid from a Grant study of Dr. Lazar

2 Zachary D. Goodman , Grading and staging systems for inflammation and fibrosis in chronic liver
diseases , Journal of Hepatology 47 (2007) 598 –607 00,511,522,533,544,5
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Calin from Children Hospital Cluj. De manufacturer of this antibody’s was Sigma
group (clone MCA for HBs antigen and clone AHP for HBc antigen). The
monoclonal antibod y against HBe antigen was not available for us.
The entire staining process was performed in the Anatomic Pathology
department of the University, by technician Rodica Boros.

HISTOLOGIC TECHNIQUE
Analyzed histological slides were stained in HE for a dia gnostic purpose and
with Gömöry, Masson trichrom and Sirius Red for the connective tissue analysis.
Coloration method is detailed in the following.

Paraffin inclusion
It’s the method that permits the inclusion of the piece in a solid environment,
from which there can be selected thin sections, successive, in a ribbon, so the lesion
can be traced. It undergoes all the stains after all the fixations.

Material.
 Slightly changed paraffin by addition of 5 -10% of bee wax to make it homogeny
and fusible at 52 -56 degrees.
 Paraffin solvents: xilene, benzen or toluen.
 Paraffin oven, adjustable at 56 Celsius degrees.
 Carved plug bottles.
 Ethanol of different concentrations or dioxane.
 Borrel glass battery.

Technique
First step after fixation and proper wash of the piece is dehydration . This is
done with ethanol, methanol or dioxane.
With ethanol . The piece is passed in successive bathes of ethanol:
o bath I: alcohol 80 degrees

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o bath II: alcohol 90 degrees
o bath III: alcohol 95 degrees (or absolute)
o bath IV: absolute alcohol
o bath V: absolute alcohol

Each bathing takes 1 -6 hours, dependent on the nature and volume of the
piece. Dehydratio n must be perfect, on it depending the clarity and inclusion.
With methanol . Out of the 5 ethanol bathes (as above), it continues with
another 3 bathes of methanol. From here it passes straight into paraffin for inclusion.
Being toxic and irritative to th e respiratory system, there must be a lot of care at the
process.
With dioxane . Two or three bathes with pure dioxane of 8 hour duration, from
where it goes straight into paraffin for inclusion.

Clarification consists in the alcohol removal from the samp le (xilene, benzen or
toluen).
The sample being removed from the last alcohol, it will pass through 3 bathes
of xilen (benzen or toluen) of a duration of 1 -8 hours each, depending on the volume
of the piece. When the piece becomes semitransparent it is cl ear. The xilen bath time
must not be extended as it hardens then piece!
Paraffin -wax inclusion. Consists of the imbibing of the piece in paraffin.
From the last xilen bath, the piece passes through 3 successive 560C melted
paraffin bathes, with the objec tive consisting of the substitution of the reactive
clarification in the piece. The duration of the bathes varies between 1 and 8 hours, and
even more, until the complete liberation of the solvent, so the piece doesn’t retract.
This technique can also be done automatically, using a automated processor of
tissues for paraffin inclusion.

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Figure 15: Automatic paraffin -wax inclusion device

Specimen inclusion in paraffin block . After the Leckardt bars have been
placed to form the perimeter of the block, the melted paraffin is poured (from the 3rd
bath) until the top level of the perimeter, then with a 600C heated pence the piece is
removed from the last paraffin bath and introduced into the new formation. It is then
left to cool down.

Figure 16: Paraff in block production

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Paraffin sectioning
Preparation of the block is made by cutting it up with a blade, in the shape of a
parallelepiped, with lateral faces, to obtain a continuous and straight ribbon from
sectioning.
The portobject piece of the micro tome is heated up, over which the block is
placed with it’s base, producing a slight melt and then left to cool down. The block
remains attached to the portobject.

Figure 17: Circular vertical microtome

Sectioning is made using a circular or linear mic rotome.

Figure 18: Sectioning the paraffin block

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Posting the sections on the slide. The skimmed slides, kept in alcohol, are
dried and waxed, in the moment of usage, with Mayer albumin, in a very thin layer.
The waxed slide is placed on heating platinum and water is poured upon it,
over which the sections are applied with their white surface downwards. Through heat
the sections move and spread.
This technique can be made on a water bath as well, if available.

Figure 19: Water bath for posting sections on slides

The water is then poured carefully and the slide is placed tilted to dry at the
thermostat.

Figure 20: The paraffin -wax section posted on slide
Mayer albumin preparation . A egg white is mixed in the vessel, without
spuming, filtered and added upon an equally quantic glycerin substance (50 ml

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aprox.). It is then placed in a bottle and there are some timol crystals added or sodium
salicite.
Note. Absolute Ethanol is prepar ed from 95 degree ethanol, from which water
is removed.
Many methods are used. The most frequent one is the copper sulfate method.
By adding 95 degree alcohol with calcinated CuSO 4, this will absorb the water from
the alcohol. 1 Volume of CuSO 4 is added t o 2 volumes of 95 degree alcohol; it is then
passed through 3 jars with CuSO 4 in a 24 -48 hour interval. The jars will be well
sealed with a carved close. After 3 -5 days it will filter. An absolute alcohol is
obtained, with an acidic pH.
Calcination of the CuSO 4, which is added in the shape of blue -green crystals,
is done in a porcelain capsule through moderate heating until the crystals become a
white -shadowy powder.
How is absolute alcohol tested? In a well or tube a slight bit of xilene is
poured, over which is added a drop of the tested alcohol. The absolute alcohol doesn’t
have to bruise the xylene, it is an alcohol that contains under 3% water.
The antrachinon probe is more sensible. It shows the presence of traces of
water. One small quantity of tes t alcohol is added at 1mg of antrachinon and 4mg of
sodium amalgam. If the alcohol contains even traces of water, a red color is produced.
If it is anhydric, a green coloration is produced.

The following colorations have been practiced:
 H-E
 Masson Trich rome
o Green light version
o Anilinic blue version

General technique of section staining
A. Preparing and staining of the sections

Paraffin sections, whom are already spread and blended to the slide, must
first be treated to erase the paraffin with which they were dipped, this not being

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miscible neither with water nor alcohol. In this purpose, before staining, they must be
deparaffinated and then hydrat ed.
Deparaffination of the sections is made by passing the slides through bathes of
paraffine dissolvent (benzene, xylene or toluene). Usually being used a battery with 6
Borrel glasses, first 3 with benzene (xylene or toluene), and the last 3 bathes of
alcohol (I – 96 degrees; II – 80 degrees; III – 70 degrees).
Hydration . From the last alcohol, the sliders with sections are passed in 2 -3
glasses of distilled water.
It is recommended that when passing the slides from a bath to another, these
are to be ra pidly wiped from the excessed dissolvent, as the paraffin it includes would
deposit in the next bathes. The same thing will be done when passing from one
alcohol to another.

Fig. 21: Manual deparaffination of slides

Staining will be done by passing the slides in the Borell glasses with colorants,
whether by placing the slide horizontally on a staining tray and turning a sufficient
quantity of colorant over it, and the second case consuming large quantities of
colorants and reactive.

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Fig. 22: Manual staining kit for histological slides

Fig. 23: Manual staining procedure

To avoid drying the sections, becoming thus unusable, care must be taken in
all these stages (deparaffination operations, hydration and staining) sections being
covered in liquids from the glasses that form the respective baths.
The coloration technique c an be completely automatized in services with high
working volume and well equipped.

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Fig. 24: Automatic staining of histological slides

Mayer hematoxiline eozine staining
Acidulated Mayer hemalaun substance: 75 g of alaun is dissolved in heat in
1000ml of distilled water. 75ml hematoxiline is filtrated in heat (from the base
solution of 10%) and 0.30 g of Potassium iodate in water. Then it all accidulates in
100ml of solution, 0.5 ml of acetic acid.
Eozine. It is boiled into a jar:
 100ml alcohol 70 
 1g alcoholic eozine (Eosinblau siritus soluble)
 1g watery eozine (Eosingelb aqua soluble)
 0,25g orange G
 1ml officinalis HCl acid
After cooling 20 ml of saturated liquid substance of lithium carbonate is added . It is
filtered.
Lithium carbonate substance: 100ml of distilled water + 15g of lithium carbonate.
Technique :
– Mayer hemalaun stain, 10 minutes;

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– Wash in water;
– Clorhydric alcohol differentiation, 1 -2 seconds;
– Wash in water;
– Change in lithium carbonate, 2-4 minutes;
– Wash in water;
– Eozine coloration, 2 -3 minutes;
– Wash in water;
– Ethanol dropped on slide until no more eozine;
– Dehydration with absolute and 95  alcohol;
– Clarification in fenicated xylene, xylene.
– Mounting in Canadian fabric.
Results:
 nucleus: dark blue
 cytoplasm: pink -red
 red cells: bright red
 collagen: pale pink
 elastic fibers: bright pink

Masson Trichrome stain
It is applied after all the fixations, especially after Susa. It offers details on
contrast, highlighting very well the collagen. Sections are made only at paraffin.
Preparing the substance
1. The R. Ponceau Substance (red Ponceau) or Ponceau -xilidine. It
dissolves at heat in 300ml of distilled water: 2 g of R Ponceau and 1 g
of acid fuxine; after dissolution, 4ml of acetic ac id is added. It is

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filtered before staining. The background is stained (cytoplasm in violet
red; red cells in orange).
2. The fosfomolibdenic acid substance (mordant and differentiative). 1g
of fosfomolibdenic acid is dissolved in 100ml of distilled water. I t is
filtered before usage.
3. G orange -aniline blue substance. 0.5 g of hydro soluble blue aniline
and 2 g of orange G is dissolved in 100ml of distilled water. Then after
dissolution, 2.5ml of acetic acid is added. It is filtered before usage.
Blue aniline stains the connective fibers; the G orange stains the
background and blood.
4. 1/500 acidulated water, Weigert hematoxiline (in 100ml of 95  ethanol
1 g of crystalized hematoxiline), clorhidric alcohol, 15% lithium
carbonate substance.
Principles:
 time 1: the nucleus is stained with ferric hematoxiline, followed by
differentiation;
 time 2: R Ponceau coloration of the cytoplasm and the differentiation using
fosfomolibdic acid.
 time 3: staining the collagen with aniline blue and G orange.
Technique:
 deparaffination (xilene)
 hydration (absolute alcohol of 90, 80 )
 wash in water
 wash in distilled water
 Water sections are stained in the Wiegert ferric hematoxiline slide for 15 -20
minutes
 wash in distilled water

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 differentiation using hydrochloric alcoho l
 wash in water
 change in lithium carbonate water
 wash in water
 R Ponceau fucsine staining for 10 minutes
 Proper wash with pipe water
 Coverage of the slides with 1% fosfomolibdenic acid for 3 -4 minutes.
 No wash, passes through aniline blue G orange fo r 1-2 minutes (under
microscopic control)
 wash in water
 differentiation in 1/500 acidic water for 3 -5 minutes (under microscopic
control to not stain the nucleus, connective fibers appearing blue)
 wash in distilled water
 alcohol dehydration (960 and abso lute)
 xylene
 Mounting in Canadian fiber

Results:
Nucleus shadowy -red
Cytoplasm violet -pink
Red cells orange
Connective Tissue blue
Muscular Tissue red
Mucus pale-pink
Amyloid bright -violet

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Imunohistochemical reaction principle
Of all the imunohistochemical techniques, the imunoenzimatic techniques
have spread the most in current practice, thus they will be detailed the most. As basic
general principle, these methods use antibodies whom have one or more enzimatic
molecules attached which catalise the transformation of a non -coloured underlevel
through a product of coloured reaction. The most frequently used enzimes are
peroxidase and alcaline fosfatase, while other enzymes such as glucozoxidase and
galactozidase are used only exceptionally.
For peroxidase, the most used underlevel is diamonobenzidine (DAB) in a
distilled water substance. The resulted reaction from DAB is the brown colouring.
This product being intensely osmophilic, can be used even in the applied
imunohistochemistry for the electronic microscope.
Because DAB is a cancerigenic product, lately there is a tendency to give up
on it and to replace it with other less perilous substances such a s: 3-amino -9-
ethilecarbazole or AEC which results in a red reaction product, 4 -chrolo -1-nafthole
which gives a blue reaction product or the so called Hanker -Yates reactive which
gives a black reaction product.
For the alcaline phosphotase sistems the chro mogenic underlevels are used:
AS-Mx-naftole or the AS -Bi naftol also known as „fast red” and „fast blue”. (2).
The enzyme is coupled covalently to the Fc portion of the antibody. Based on
the nature of this antibody, coupled with the enzyme, we can distin guish the best
methods of work. For our purpose, according with Sigma indications, we used the
PAP method.

PAP method
PAP method (peroxidase -antiperoxidase) or the circulant immune method, has
been introduced in 1979 by Sternberg. This method does no longer use marked
covalent antibodies with an enzime, but uses a immune complex in which the enzime
(as antigen) is coupled with 2 anti -enzime antibodies.
Even in this method there are 3 steps necessary. In the first step, the
primary antibody is coupled with the antigen searched for on the slide. In the second
step, a 2nd antibody is introduced, unmarked anti antibody, also named bridge
antibody, but which is added in high concentration, thus this 2nd antibody binds with
the Fc portion of the primary antibody, only with one of the 2 Fab portions of his. In

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this moment the PAP reactive is being introduced (peroxidase -antipero xidase
complex) or the APAAP reactive (alcaline fosfatase – alcaline antifosfatase complex).
These commercially availible reactives (soluble complexes) bind with the second
available Fab arm of the secondary antibody.

Fig.25: PAP method (APAAP)

Through this method, at the place of the marking there is a large quantity of
enzymatic molecules, thus, the intensity of the chromogenic reaction will be very big.
Due to this, the method has a special sensitivity, being used especially for tracking a
small concentration of antigens such as the case for the fixed specimens in
formaldehyde and refined in paraffin. Then when the investigated tissue has a high
peroxidazic activity, capable to determine a heightened background reaction, the
APAAP method is pre ferred.

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4. RESULTS and DISCUTIONS
This study is a complementary study of my colleague Rafael Kazarel with the
purpose to compare de results obtained din adults with those in children’s.

Fig.26-27: HBsAg x100 immunostain . Focal -diffuse placed cytoplasmic staining
pattern for the infected hepatocytes .

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Fig.28: Detail. HBsAg x400. Focal -diffuse placed cytoplasmic staining pattern for the
infected hepatocytes .

Fig.29: HBsAg x100. Nodular placed cytoplasmic staining pattern for the infected
hepatocytes .

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Fig.30: HBsAg x200. Nodular placed cytoplasmic staining pattern for the infected
hepatocytes .

Fig.31: HBsAg x400. Membranar immunostaining pattern for infected hepatocytes .

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Fig.32-33: Immuno staining for HBcAg. Pure intranuclear immuno staining pattern,
characteristic to the HBV virus replication phase.

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Fig. 34: HBcAg immuno staining . Intra-cytoplasmic immuno staining pattern ,
characteristic to the viral clearance phase follow ing interpherone therapy.

Fig. 35: HBcAg immuno staining . Negative immuno staining pattern, characteristic for
the viral integration phase.

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As a result of adequate interpretation of this immunostaings, a Excel 2010
database was establi shed and exploited for obtaining the res ults.

The 26 cases were analyzed following the next correlations:
 Desmet classification of the hepatitis evolutionary phase based upon
the immunohistocoloration pattern.
 Desmet class correlation between the age and gender of the children’s .

Case repartition according to Desmet’s immunohistochemical classification
(Desmet V.J., Liver tissue examination, Journal of Hepatology, 39 (2003), 843 -849)

Fig. 36: Desmet’s class repartition

a. In replicative phase: 13 out of 26 cases ( 50%)
b. In clearance phase: 8 out of 26 cases ( 30.76 %)
c. In integration phase: 5 out of 26 cases ( 19.23 %)

The figure 36 indicates, that most children’s (50%) with chronic HBV
infection are diagnosed early in replication phase, only a minority of cases (~20%)
Replication
Clearance
Integration

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being already in integrative phase of the viral infection. Another ~30% of cases are
capable to enter in the clearance phase, spontaneously (the analyzed biopsies being in
all cases taken before initiation of any treatment). By comparison, in adults, the vast
majority of cases were in integrative phase, only a minority being in replicative or
clearance phase.

„Desmet class” correlation with age and sex of the children’s
The girls (10 examined cases) presented 5 cases in replicative phase ( 50%)
and 2 cases in clearance phase ( 20%) and 3 (30%) in integrative phase.
The boys (16 cases) presented 8 (50%) in replicative phase, 4 (25%) in
clearance phase and 4 (25%) in integrative phase.

Fig. 37: Desmet class repartition of the cases based on gender
Girls
Replicative
Clearance
Integrative
Boys
Replicative
Clearance
Integrative

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Fig. 38: Desmet class repartition of the cases based on gender

No gender significant differences were observed in our children study group,
comparing with adults were in the replicative phase, no women was present. Integrative
Girls
Boys
Replicative
Girls
Boys
Clearance
Girls
Boys

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Age correlation shows that all cases in clearance phase were older than 14 yrs.
This information correlates with the known idea that after puberty, the immune
system maturation of children’s is capable to induce spontaneous healing of such
chronic HBV hepatitis.

All this data reveals that 50% of c hildren’s, without any sex discrimination,
are in the replicative phase in the moment of diagnosis. This means that the HBV
antiviral specific treatment (lamivudine and interferon) is useful and the results are
expected to be good. In adults this group is insignificant and as a result the same
treatment in children’s and adults will have significantly better result in children
cases.
The clearance phase means that immunity became strong enough to clear the
viral infection. In adult’s, this phase is rarely seen, but in children’s they represent
30% of cases, all of them being after puberty age. This means that in children’s with
chronic HBV infection, older than 14 yrs. the treatment is not only even more
efficient than in the rest, but also is possible to b e based on only one therapeutic agent
(lamivudine or interferon) the combination being not necessary. Even more, is
possible that some teenagers to cure de chronic HBV infection even without any
treatment at all.
The integrative phase is the main phase for adults, bat less than 20% of
children cases are in this hopeless situation. In this phase, the only beneficial
treatment is the control of necro -inflammatory activity (by non -steroidal or even
steroidal anti -inflammatory drugs) and the advance of fibrosis (by antifibrotic drugs
like interferon gamma). In adults and children’s in this situation, interferon gamma
seems momentarily to be the best hope because it have slight antiviral effects and
strong antifibrotic effects. Unfortunately this treatment has no t yet approval to be used
in children’s.

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74 | P a g e
CONCLUSIONS

1. Immunohistochemical stains for HBV antigens seems to be a very
important tool in the effort of diagnose correctly a cryptogenic
hepatitis but even more useful in an adequate prescription of
treatment.
2. The efficacy of the treatment is expected to be better in children’s
than in adults.
3. In children, about 20% of cases will not be beneficial from the
actual standard treatment based on alpha2 -interferon and
lamivudine, other treatments like gamm a-interferon being more
adequate for their integrative phase of the evolution of viral
infaction.

FINAL CONCLUSION

Children’s are not small adults

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75 | P a g e
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