Original Articles
The Pathology of Hepatitis C
PETERJ. SCHEUER,' PARVIN ASHRAFZADEH,~
SHEILASHERLOCK,' DAVIDBROWN3 AND
M.DUSHEIK03
GEOFFREY
Departments of Histopathology,' surge^ and M e d i ~ i n eRoyal
,~
United Kingdom
To determine the histologic pattern of hepatitis C, 54
liver biopsy specimens from 45 patients w i t h a clinico-
pathological diagnosis of hepatitis C were studied. All
patients were seropositive f o r antibody to hepatitis C
virus by second-generation testing. Both transfusion-
related and sporadic cases were included. More than
half the samples showed chronic hepatitis without
cirrhosis, whereas 44% showed developing or fully
established cirrhosis. A histological pattern of mild
chronic hepatitis w i t h portal lymphoid follicles and
varying degrees of lobular activity was found in many
of the patients. Lymphoid aggregates or follicles were
seen in 78% of biopsy specimens, but aggregates, less
prominent than in hepatitis C, w e r e also seen in 14 of
27 samples (52%) f r o m patients with hepatitis B. We
conclude that a characteristic histological pattern
exists in chronic hepatitis C, that this pattern is not
always found and that prominent lymphoid follicles,
though not unique to hepatitis C, provide a useful
diagnostic clue. (HEPATOLOGY 1992; 15:567-571.)
The histopathological changes in non-A, non-B
(NANB) hepatitis have been described in several papers
over the past decade (1-5). However, accurate as-
sessment of these changes has been hampered by lack of
specific serological tests to support the diagnosis, and it
is possible that in some instances the changes have
resulted from infection with a number of different
hepatitis viruses. Occasionally, epidemiological evidence
has suggested that the disease studied was caused by a
single agent; examples are the enterically transmitted
form of NANB hepatitis now often referred to as
hepatitis E (6, 7), and a group of patients with coagu-
lation disorders who contracted a short-incubation
hepatitis after administration of factor VIII (1).In most
studies, however, evidence of infection with a single
virus could not be conclusively established.
The development of a serological test for antibody to
a nonstructural antigen (C100-3) of a parenterally-
transmitted NANB hepatitis agent, hepatitis C virus
(HCV) (81, provided the opportunity to study the
Received February 22, 1991; accepted November 18, 1991.
Address reprint requests to: Professor P.J. Scheuer, Department of Histo-
pathology, Royal Free Hospital, Pond Street, London NW3 2QG, UK.
31/1/35182
Free Hospital and School of Medicine, London NW3 2QG,
pathology of at least one form of this disease. However,
first-generation tests gave both false-positive and false-
negative results (9-11).Second-generation tests detect
antibodies t o both structural and nonstructural compo-
nents of the virus, and they have demonstrated that
most patients with parenterally transmitted NANB
hepatitis are infected with HCV (12). A substantial
proportion of patients with sporadic NANB hepatitis can
also be shown t o be infected with this virus.
PATIENTS AND METHODS
Fifty-four liver biopsy specimens from 45 patients with
hepatitis C were studied. All patients were HBsAg seronegative
and were considered to have NANB hepatitis on the basis of
exclusion of other causes of hepatitis by clinical, serological,
immunological and biochemical criteria. All were positive for
antibody to hepatitis C (anti-HCV) by second-generation
testing. Thirty-three were men. The mean age of the cohort
was 47 yr (range = 25 to 74 yr). In addition to patients from
Britain and southern Europe, a substantial number originated
from the Middle East or the Indian subcontinent. In 26
patients, onset of hepatitis was related to definite parenteral
exposure by blood transfusion, intravenous drug abuse, occu-
pational needle-stick or human bite (13). The interval from
exposure to biopsy in these patients averaged 13 yr (range,
< I yr to 46 yr). In one multiple-transfusion patient with
thalassemia, the date of onset could not be accurately ascer-
tained. The patient who contracted hepatitis C after sustaining
a human bite underwent liver biopsy 6 mo after presumed
infection. The mean interval from first clinical diagnosis of
chronic hepatitis or cirrhosis to the diagnostic liver biopsy was
2.9 yr (range = 6 mo to 10 yr). Several patients had had
cirrhosis established clinically at the time of presentation with
symptoms or abnormal liver function test scores, suggesting
that the disease, although only recently diagnosed, was in fact
long-standing.
Selected patients had been enrolled in a controlled thera-
peutic trial of interferon-a,, treatment of NANB hepatitis.
This trial was approved by the ethical committee of the Royal
Free Hospital. Liver biopsy samples were taken with patients'
informed consent before treatment began (in some cases
during or after treatment). All specimens were obtained by the
percutaneous or transjugular route.
Hepatitis C markers were measured by second-generation
ELISA (Abbott Laboratories, North Chicago, IL; and Ortho
Laboratories, Raritan, NJ). Both assays used detect antibody
to nonstructural (NS3 and NS4) regions and t o the structural
core of the HCV. Test specimens were diluted in microtiter
wells to form antigen-antibody complexes to bound antigens.
567
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568 SCHEUER ET AL. HEPATOLOGY

TABLE1. Overall histological diagnoses and portal-tract features in hepatitis C

Lymphoid aggregates
and follicles" Bile duct damagem
Diagnosis n 0 1 2 3 0 1 2

Acute hepatitis 1 1 0 0 0 0 0 1
Mildimoderate chronic hepatitis (CPH, CLH, CAH) 29 4 5 15 5 23 6 0
Severe CAH and/or developing cirrhosis 5 0 2 3 0 3 0 2
Established cirrhosis 19 7 7 4 1 16 3 0
biopsy specimens 54 12 14 22 6 42 9 3
- TOTAL

CLH = Chronic lobular hepatitis.

"For explanation of scoring system, see text.

TABLE2. Pathological features in acini in hepatitis C

Lymphocytic infiltration
Acidophil bodies" of sinusoids- Fatty change"
Diagnosis n 0 1 2 0 1 2 0 1 2

Acute hepatitis 1 0 1 0 1 0 0 0 1 0
Mildimoderate chronic hepatitis (CPH, CAH, CLH) 29 23 6 0 16 10 3 15 11 3
Severe CAH and/or developing cirrhosis 5 3 2 0 4 1 0 1 2 2
Established cirrhosis 19 17 2 0 19 0 0 9 7 3
TWI'AI. biopsy specimens 54 43 11 0 40 11 3 25 21 8

CLH = Chronic lobular hepatitis.

"For explanation of scoring system, see text.

3. Overall histological diagnoses and lymphoid aggregates or follicles in hepatitis B

TABLE
Lymphoid aggregates and follicles"
Diagnosis n 0 1 2 3

Mildimoderate chronic hepatitis (CPH, CAH, CLH) 19 10 6 3 0

Severe CAH and/or developing cirrhosis 1 1 0 0 0
Established cirrhosis 7 2 3 2 0
TOTAL biopsy specimens 27 13 9 5 0
CLH = Chronic lobular hepatitis.
"For explanation of scoring system, see text.

Murine monoclonal antibody conjugate was then added. In the
final stage, o-phenylenediamine 2 hydrochloric acid and
hydrogen peroxide was added, and the optical density of the
end product was measured.
All anti-HCV-positive specimens were tested by second-
generation recombinant immunoblot assay (RIBA) (Chiron
Corp., Emeryville, CA). In this procedure, four HCV antigens
(5-1-l,ClO0-3,C33c,C22-3) and a control, superoxide dis-
mutase are immobilized as individual bands on nitrocellulose
strips. The strips are incubated with serum specimens.
Antibodies in serum, if present, bind to the antigens. After
washing, the strips are incubated with goat antibody to human
IgG conjugated with horseradish peroxidase. After incubation,
hydrogen peroxide and 4-chloro-1-naphthol are added to
develop the band patterns. If antibodies to two HCV antigens
are detected, the test is scored as positive; if antibody to one
antigen is present, the test is scored as indeterminate. If no
bands are present, the test is scored as nonreactive. For this
study, .ELISA-positivebut RIBA-indeterminate samples were
also considered positive because the RIBA test appears more
specific but less sensitive (14). Only RIBA-reactive or RIBA-
indeterminate patients were included.
Liver biopsy specimens were fixed in buffered formalin,
processed to paraffin and routinely stained by the following
methods: hematoxylin and eosin, diastaselperiodic acid-Schiff,
Perls' stain for iron and Gordon and Sweets' method for
reticulin. Overall histologic diagnoses were made using
standard criteria (15). Semiquantitative assessment of histo-
logic features was carried out by one author (PA), with
independent assessment of a proportion of the biopsies by
another (PJS). The following features were chosen for this
assessment on the basis that they had been previously
considered characteristic of or common in NANB hepatitis
(1-5): lymphoid aggregates or follicles in portal tracts, infil-
tration of small bile ducts by inflammatory cells or damaged
duct epithelium, acidophil body formation, steatosis and
infiltration of sinusoids by lymphoid cells.
Lymphoid aggregates and follicles were scored 0 to 3: 1
represented ill-defined condensations of lymphoid infiltrates, 2
represented definite aggregations or follicles without identi-
fiable germinal centers and 3 represented lymphoid follicles
with germinal centers. Other features were graded 0 to 2: 1
represented mild to moderate changes and 2 represented
severe changes.
A control group of 27 biopsy specimens from 24 patients
with chronic hepatitis B was studied to compare the prevalence
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Vol. 15, No. 4, 1992 PATHOLOGY OF HEPATITIS C 569

FIG.1. Photomicrograph of part of a liver biopsy specimen from a patient seropositive for anti-HCV showing a commonly found histological
pattern. The chronic hepatitis is characterized by mild piecemeal necrosis, a lymphoid follicle with germinal center in a portal tract (arrow) and
focal inflammatory infiltration in the acinus (H & E; original magnification x 175).

FIG.2. High-power view of part of a liver biopsy sample from patient with chronic NANB hepatitis who was seropositive for anti-HCV showing
lobular activity. Many lymphocytes are seen, and an acidophil body is present (arrow). Some hepatocytes contain fat vacuoles (H & E; original
magnification x 440).

of lymphoid aggregates and follicles with that in hepatitis C. patients varied from mild to severe. Nineteen patients
All patients were seronegative for anti-HCV by second- had established cirrhosis. No HCCs were seen in any of
generation testing as described above. the biopsy specimens.
Nine of the 45 patients underwent more than one
RESULTS biopsy. The interval between first and last biopsy ranged
Overall diagnoses are given in Table 1. One patient from 1 to 6 yr. In no patient did the diagnosis change
had histological features of acute hepatitis. Twenty-nine substantially from one biopsy to the next, although
had mild or occasionally moderately severe chronic there were minor differences in the pattern and extent
hepatitis without severe disturbance of liver archi- of histological activity.
tecture. The pattern included all the described histo- The frequency of individually assessed histological
logical categories of chronic hepatitis: chronic persistent features is shown in Tables 1 and 2. The most striking
hepatitis (CPH), CAH and chronic lobular hepatitis. In feature was the presence of lymphoid aggregates or
five patients, cirrhosis appeared to be developing or follicles in portal tracts, either alone or part of a general
nearly developed. Necroinflammatory activity in these inflammatory infiltration of the tracts. The structures

570
FIG.:3. Fatty cirrhosis in a patient seropositive for anti-HCV. Note the small lymphocytic aggregate in a septum (arrow) (H & E; original
magnification x 70)
varied from simple aggregations of lymphoid cells to
well-farmed follicles with germinal centers (Fig. 1).They
were more easily seen in specimens not showing cir-
rhosis (25 of 30 specimens) but could also be identified
in cirrlhotic or near-cirrhotic livers (17 of 24 specimens).
Overall, these features were detected in 42 of 54 samples
(78951, usually in the form of easily recognizable aggre-
gates or follicles with germinal centers. In the group of
27 biopsy specimens from patients with hepatitis B,
lymphoid aggregates were found in 14 of 27 samples
(52%) (Table 3). In 9 of the 14, the aggregates were
poorly defined (score 11, and in no patient were well-
defined follicles with germinal centers seen.
Other histological features were less striking. Signif-
icant bile duct damage was only found in 3 of 54
specimens, although minor epithelial irregularity or
infiltration of lymphocytes was found in another nine
samples (Table 1). In the acinar parenchyma (Table 21,
acidophil bodies were found in 11 specimens, but were
relatively scanty in all. Lymphocytic infiltration of
sinusoids was striking in three samples and present t o a
moderate degree in another 11.Fatty change was mild to
moderate in 2 1 and severe in eight specimens.
The presence of lymphoid aggregates or follicles,
together with an overall diagnosis of mild CAH or CPH,
fatty change and/or lobular activity, gave many of the
biopsy specimens a very characteristic and easily recog-
nizable appearance (Fig. 1).However, all four features
were found together in only a small minority of
specimens. Lobular activity took the form of diffuse or
focal infiltration by lymphocytes with or without liver
cell dropout, acidophilic change in hepatocytes and
acidophil bodies (Fig. 2). The latter were more often seen
than acidophilic change in intact liver-cell plates.
Cirrhosis was seen in a high proportion of patients and
was present in 15 at first biopsy. Apart from the common
finding of lymphoid follicles with fatty change - the
latter usually not severe -the cirrhosis did not differ
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SCHEUER ET AL. HEPATOLOGY
markedly in its histological characteristics from so-
called cryptogenic cirrhosis (Fig. 3). Minimal hepatocel-
lular dysplasia of large-cell type was seen in a minority
of samples.
DISCUSSION
The histological features observed correspond closely
to those given for NANB hepatitis in the literature (1-5).
They are consistent with the results of a semiquanti-
tative analysis of liver biopsy specimens from patients
with or without antibody to C100-3 in their serum (161,
but no histological details were provided in that study.
False-positive results were obtained with the first-
generation tests for anti-HCV (9, 101, particularly in
patients with autoimmune diseases, but the patients in
this study were all selected because they had established
NANB hepatitis as determined by clinical criteria and
serologic tests, including supplemental assays, so that it
was likely that the positive serum results were a true
indication of HCV infection. The histological features
found in our patients were therefore considered to
reflect accurately the pathology of hepatitis C.
A common histological pattern has emerged from this
study of a mild chronic hepatitis on the borderline of
CPH and CAH, with lymphoid aggregates or follicles in
portal tracts, lobular activity including acidophil body
formation, and fatty change. The most characteristic
single feature of this picture is the lymphoid follicle. The
lymphoid lesions seen ranged from loose aggregates of
lymphocytes to well-defined structures with germinal
centers. They were usually recognized easily in reticil-lin
preparations. Clearly follicles are not restricted to
hepatitis C; they are seen, for example, in PBC, and may
be found in biopsy specimens from patients with chronic
HBV infection, as confirmed in our control group.
However, in this study, aggregates were found less
frequently in hepatitis B than hepatitis C and were
generally less well formed. Follicles with germinal

Vol. 15, No. 4, 1992 PATHOLOGY OF HEPATITIS C
centers were seen in six samples from patients with
hepatitis C and in none with hepatitis B. The reason for
the formation of lymphoid follicles remains obscure and
deserves further study. Their presence supports the
participation of the patient’s immune system in the
pathogenesis of the liver lesion.
Many of the patients in this series exhibited cirrhosis;
in some it had developed over a period of many years or
even decades. The pathology of the cirrhosis is often not
characteristic, but lymphoid aggregates and follicles -
together with fatty change-were seen in some of the
patients. As in the case of the noncirrhotic livers, the
presence of lymphoid follicles in cirrhosis should alert
the pathologist to the possibility of HCV infection.
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