Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from regular and fine-needle biopsies.

Abstract

The aim of the study was to investigate the use of flow cytometry, as an alternative for immunohistochemistry, for the detection of viral antigens in the liver of patients with chronic hepatitis B virus (HBV) infection. Hepatocytes were obtained from regular- and fine-needle biopsy from HBV positive (n=17) and negative (n=7) patients and quantified by flow cytometry for intracellular hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg). Number of HBsAg positive hepatocytes ranged from 0 to 83%. A significant correlation was found between the percentage of infected hepatocytes and the intracellular expression level of HBsAg (R=0.841, p<0.001). The specificity and sensitivity of flow cytometry was similar to immunohistochemistry. Of the patients on anti-viral treatment with undetectable serum HBV DNA (<400 copies/ml), two had high HBsAg expression in the liver. HBcAg staining was found in 3 out of 15 patients, with 2-3% positive hepatocytes. The results obtained with fine-needle aspiration biopsy (n=12) were comparable to regular biopsy. In conclusion, flowcytometric quantitation of HBV antigens is sensitive and provides relevant information on the course of infection. The minimally invasive fine-needle biopsy provides a useful alternative for regular-needle biopsy for monitoring intrahepatic antiviral responses during therapy.

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Please cite this article in press as: van der Laan, L.J.W. et al., Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from
regular and fine-needle biopsies, J. Virol. Methods (2007), doi:10.1016/j.jviromet.2007.01.027
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Flowcytometric quantitation of hepatitis B viral antigens in
hepatocytes from regular and fine-needle biopsies
Luc J.W. van der Laana,∗, Pavel Taimrb, Alice Koka, Dave Sprengersb,
Pieter E. Zondervanc, Hugo W. Tilanusa, Harry L.A. Janssenb
aDepartment of Surgery, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
bDepartments of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
cDepartment of Pathology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands
Received 30 October 2006; received in revised form 17 January 2007; accepted 25 January 2007
Abstract
The aim of the study was to investigate the use of flow cytometry, as an alternative for immunohistochemistry, for the detection of viral antigens
in the liver of patients with chronic hepatitis B virus (HBV) infection. Hepatocytes were obtained from regular- and fine-needle biopsy from
HBV positive (n=17) and negative (n=7) patients and quantified by flow cytometry for intracellular hepatitis B surface antigen (HBsAg) and
hepatitis B core antigen (HBcAg). Number of HBsAg positive hepatocytes ranged from 0 to 83%. A significant correlation was found between the
percentage of infected hepatocytes and the intracellular expression level of HBsAg (R=0.841, p<0.001). The specificity and sensitivity of flow
cytometry was similar to immunohistochemistry. Of the patients on anti-viral treatment with undetectable serum HBV DNA (<400copies/ml), two
had high HBsAg expression in the liver. HBcAg staining was found in 3 out of 15 patients, with 2–3% positive hepatocytes. The results obtained
with fine-needle aspiration biopsy (n=12) were comparable to regular biopsy. In conclusion, flowcytometric quantitation of HBV antigens is
sensitive and provides relevant information on the course of infection. The minimally invasive fine-needle biopsy provides a useful alternative for
regular-needle biopsy for monitoring intrahepatic antiviral responses during therapy.
© 2007 Published by Elsevier B.V.
Keywords: HBV; Flow cytometry; Aspiration biopsy; FNAB; HBsAg; HBcAg; Intrahepatic
1. Introduction
Infection by HBV affects millions of people worldwide
(McMahon, 2005). Replication of HBV virus occurs almost
exclusively in hepatocytes, although other cells types may be
infected (Umeda et al., 2005). The liver is, therefore, the most
relevant site for monitoring the course of HBV infection and
antiviralresponses.Incurrentclinicalpractice,HBVinfectionis
monitored primarily by examining serum. In serum, viral DNA,
Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B surface anti-
gen; HBcAg, hepatitis B core antigen; geoMFI, geometric mean fluorescence
intensity
∗Correspondingauthorat:DepartmentsofSurgeryandGastroenterologyand
Hepatology, Erasmus MC-University Medical Center, Room L458, Molewater-
plein 40, NL-3015 GD, Rotterdam, The Netherlands. Tel.: +31 10 463 2759;
fax: +31 10 463 2793.
E-mail address: l.vanderlaan@erasmusmc.nl (L.J.W. van der Laan).
soluble hepatitis B e antigen (HBeAg) or virion-associated viral
proteins(e.g.HBsAgandHBcAg)canbedetectedreadily.How-
ever, it is still controversial whether the serum viral load always
reflects accurately the level and progression of infection in the
liver(Iloejeetal.,2006;vanderEijketal.,2006).Agoodexam-
ple is recurrence of HBV infection after apparent clearance of
the virus as a result of antiviral therapy (Ito et al., 2004). Indeed,
recentstudiesfoundthatdetectionofHBVDNAandreplication-
intermediate cccDNA in the liver, rather than in serum, are
better surrogate markers of a true sustained virological response
(Alberti,2003;Werle-Lapostolleetal.,2004;Sungetal.,2005).
Clearly, this indicates that when HBV DNA is not detectable in
serum, virus can still persist in the liver and can restart replicat-
ingonceantiviraltherapyisstopped.Determinationofpredictive
factor(s)ofresponsetotherapyisneeded(Zoulim,2006).There-
fore, to be able to assess successfully a sustained virological
response in patients on antiviral therapy, the liver is the most
relevant site for monitoring the clearance of the virus.
0166-0934/$ – see front matter © 2007 Published by Elsevier B.V.
doi:10.1016/j.jviromet.2007.01.027

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Please cite this article in press as: van der Laan, L.J.W. et al., Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from
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Intrahepatic monitoring of HBV infection can be undertaken
using liver tissue biopsies. Detection of intrahepatic viral DNA
or RNA has been reported. Although PCR techniques are very
sensitive, these do not provide information on the number
of infected hepatocytes or the intrahepatic immune response
against these cells, as can be observed by immunohistochem-
istry. Detection of viral antigens, in particular HBsAg and
HBcAg,byimmunohistochemistryiscommonpractice(Bianchi
and Gudat, 1979). However, standardization of these assays is
difficult.Furthermore,theuseofregulartissuebiopsyneedlesis
invasiveandcouldleadtoseriouscomplications(Reuben,2003).
As a less traumatic alternative, fine-needle aspiration biopsy
has been introduced successfully for the detection of allograft
rejection (Hayry et al., 1981; Hayry and Lautenschlager, 1992).
The diameter of this needle is significantly smaller (25-Gauge)
than the core-needle and therefore fine-needle aspiration
biopsy does not require anesthesia and has minimal risk of
complication due to damage of intrahepatic blood vessels or
biliarytracts.Aspirationbiopsywasshowntobeaseffectivefor
the cytological diagnosis of liver graft rejection as core-needle
biopsy (Lautenschlager et al., 1988; Kwekkeboom et al., 2003).
Though clearly useful for cytological, immunocytochemical
(Kuijf et al., 2002) and flowcytometric analysis (Sprengers et
al., 2005), to date the utility of aspiration biopsy for detection
of intrahepatic viral antigens has not been reported. As shown
previously, aspiration biopsy was shown to be suitable for
intrahepatic monitoring of virus-specific T-cell response in
patients with chronic HBV (Sprengers et al., 2005).
In the present study, we have investigated the use of flow
cytometry for quantitation of viral antigens in regular and aspi-
ration liver biopsy samples from chronic HBV patients. The
feasibility of intracellular detection of HBsAg and HBcAg by
flow cytometry was demonstrated. Using this method, both the
percentages of infected hepatocytes and the relative expression
level of viral antigens within the infected cells could be deter-
mined.
2. Material and methods
2.1. Patients
PatientswithchronicHBVundersurveillanceattheErasmus
MC outpatient clinic were included. Patients underwent a liver
biopsy as part of their diagnostic evaluation. Control liver biop-
sieswereobtainedfrompatientswithchronicHCVortransplant
donor livers. The characteristics of patients are summarized in
Table 1.
2.2. Regular- and fine-needle liver biopsy
Regular biopsy was performed after an ultrasound exam-
ination with tru-cut automate core-needle (14Ga, MD
Table 1
Patient characteristics
Patient #AgeSexHepatitisRegular-needle
biopsy
Fine-needle
biopsy
Serum viral loada
(×104GEQ/ml)
ND
23.0
4.3
<0.1
0.4
0.4
51.3
33,100
<0.1
ND
<0.1
<0.1
0.4
171.0
2.0
110,000
ND
NA
NA
NA
NA
NA
NA
NA
HBe AgAnti-HBeALTb
AVT at time
of biopsy
1
2
3
4
5
6
7
8
9
50
23
62
57
29
23
42
22
57
47
36
31
37
20
31
42
45
57
40
34
36
30
43
27
F
M
M
M
M
F
F
M
M
M
M
F
M
M
M
M
M
M
M
F
M
M
M
F
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
No
C
C
No
No
No
No
×
×
×
×
×
×
×
×
×
×
×
×
−
−
−
−
−
−
×
×
−
−
−
−
−
−
−
−
−
−
−
−
−
−
×
×
×
×
×
×
×
×
−
−
×
×
×
×
−
−c
+
−
−
−
−c
+
−
−
−
−
−
+
−c
+
−
NA
NA
NA
NA
NA
NA
NA
+
+
−
+
+
+
+
−
−
ND
+
+
+
−
+
−
ND
NA
NA
NA
NA
NA
NA
NA
29
50
197
48
61
55
30
40
23
80
38
33
52
29
93
ND
ND
38
ND
ND
290
81
ND
ND
No
No
Yesd
No
No
No
No
No
Yese
No
Yesd
Yese
No
Yesd
Yese
No
No
No
No
No
No
No
No
No
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
NA, not applicable; ND, not determined.
aWithin 4 weeks of biopsy time point.
bWithin 2 weeks of biopsy time point.
cPossible pre-core mutant (HBe Ag negative, anti-HBe positive with medium and high serum viral load).
dPeg-IFN-? and Lamividine combination therapy.
eLamividine monotherapy.

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Technologies, Inc., Gainsville, FL, Ref. 763114160X) under
standard conditions. All biopsies were examined by an
experienced pathologist. Fine-needle aspiration biopsies were
obtained under ultrasound guidance as described previously
(Lautenschlager et al., 1990, 1991). For each patient, two
aspiration biopsies were taken and evaluated macro- and micro-
scopically. When the aspirate contained predominantly blood
at macroscopic evaluation, an additional aspiration biopsy was
taken.Cytospinswerepreparedofindividualaspirationsamples
and stained with May-Gr¨ unwald-Giemsa (Merck, Darmstadt,
Germany) to determine the hepatocyte and leukocyte con-
tent. Only representative aspiration specimens, i.e. containing
at least seven hepatocytes per 100 leukocytes, were included
(Lautenschlager et al., 1988; Kwekkeboom et al., 2003). In a
seriesof48aspirationbiopsysamplesobtainedinthisperiod,the
average content of nucleated cells was 1.9×105±0.3 S.E.M.
2.3. Tissue processing for flow cytometry
Biopsy specimens were directly processed in the labo-
ratory. For regular liver biopsy samples, hepatocytes were
isolated by collagenase P (Roche, Mannheim, Germany) diges-
tion for 45min at 37◦C in the presence of 0.4U/ml DNAse
(Boehringer, Mannheim, Germany). For aspiration biopsy,
representative samples were pooled and hepatocytes were
enriched by low-speed centrifugation (50×g, 3min without
brake). In some cases, peripheral blood mononuclear cells
(PBMCs) were obtained at time of biopsy and used as negative
control for immunostaining. PBMCs were obtained from hep-
arinizedperipheralbloodbydensitygradientcentrifugationover
Ficoll–Paque plus (Amersham Biosciences, Buckinghamshire,
UK).
2.4. HBV replicating cell line (HepG2215)
HepatomacelllinecontainingduplicateHBVrepliconswere
used (Sells et al., 1988; Galle et al., 1994). HepG2215 replicon
cells were cultured in Williams E medium supplemented with
10% FCS and antibiotics. Parental HepG2 cells without HBV
were used as a control. Sub-confluent cell monolayers were har-
vested by trypsinization using trypsin/EDTA solution (Gibco
Invitrogen Corp., UK) directly prior to flowcytometric analysis
of HBsAg and HBcAg.
2.5. Flowcytometric analysis of HBsAg and HBcAg
Single cell suspension of liver samples and PBMC were
washed,fixed2%paraformaldehyde(ICNBiomedicals,Aurora,
OH)for15minatRT,washedwithPBSandpermeabilizedwith
PBScontaining0.5%Saponin(Sigma–Aldrich,St.Louis,USA)
to allow intracellular staining. Mouse anti-HBsAg Ab-1 (Neo-
Markers, Frement, CA) was used at 5?g/ml and anti-HBcAg
rabbitantibodies(DAKO,Carpintera,CA)wasusedat10?g/ml.
Primary antibodies were diluted in PBS containing 0.3% BSA
(Sigma–Aldrich, St. Louis, USA) and 0.5% Saponin (PBS-S)
and incubated for 30min at 4◦C. After washing the cells with
PBS-S, cells were stained with fluorescent secondary antibod-
ies rat anti-mouse PE 1:10 (BD, Qume Drive, San Jose) or goat
anti-rabbit FITC 1:50 (Beckman Coulter, Marseille, France) in
PBS-S and incubated for 30min at 4◦C. With each sample, a
negative control with isotype-matched control antibodies was
used. Labelled cells were analyzed by using FACScalibur flow
cytometry and CellQuest software (BD Biosciences, Mountain
View, CA) by gating for hepatocytes.
2.6. Immunohistochemistry for HBsAg and HBcAg
Parafin sections of liver biopsy were stained for HBsAg and
HBcAg using standard immunohistochemistry. Two indepen-
dentobserversassessedthepercentageofpositivecells(ranging
from 0 to 100%) in the complete tissue section at ×200- and
×400-times magnifications.
2.7. Statistical analysis
Statistical analysis was performed using software package
SPSSVersion11.5(SPSS,Chicago,IL).Significancewastested
using the Wilcoxon paired test or the Mann–Whitney U-test.
p-Values less than 0.05 were considered significant. Linear
regression was performed using Graphpad Prism software.
3. Results
3.1. Validation of the flowcytometric HBsAg and HBcAg
detection
In order to determine the specificity and sensitivity of intra-
cellular staining of HBV antigens by flow cytometry the HBV
positive hepatoma cell line, HepG2215, and the parental HBV-
free HepG2 cells were used. Cells were fixed and permeabilized
andstainedwithantibodiesdirectedagainstHBsAgandHBcAg.
AsshowninFig.1,specificstainingofbothHBsAgandHBcAg
wasseenintheHepG2215celllinebutnotintheHepG2parental
cells. Optimal antibody concentrations for anti-HBsAg were
5?g/mlandforanti-HBcAg10?g/ml,andwereusedforfurther
analysis.
3.2. Flowcytometric analysis of HBsAg and HBcAg in
regular liver biopsies
Using flowcytometric analysis, it was determined whether
intracellular expression of HBsAg and HBcAg in hepatocytes
could be detected in liver biopsies. Twelve consecutive biopsy
specimens of patients with chronic HBV (see Table 1) were
processed to obtain a single-cell suspension. These cells were
stained for HBsAg and HBcAg and analyzed by flow cytome-
try. Histograms of two patients are shown in Fig. 2A. The mean
number of hepatocytes that could be analyzed by flow cytom-
etry from the biopsy samples was 15,000±1400 S.E.M. For
the HBsAg staining, on average 3700±760 S.E.M. hepatocyte
eventswereexamined,rangingfrom130to15,000.Resultsofall
biopsy samples are summarized in Fig. 2B. HBsAg expression
was observed in 10/12 patients, ranging from less than 1–83%
positive cells. No statistically significant correlation was found

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Fig. 1. Intracellular detection of HBsAg and HBcAg in HepG2215 cells. To
validate the intracellular staining of HBV antigens by flow cytometry, the HBV
replicating hepatoma cell line HepG2215 and the parental HBV-free HepG2
cells were tested. Cells were fixed and permeabilized and stained with antibod-
ies for (A) HBsAg or (B) HBcAg. Isotype-matched antibodies were used as
control (gray line). Specific staining was observed for both HBsAg and HBcAg
in HepG2215 cells (gray filled histogram) but not HepG2 control cells (black
line). Shown is one of the three experiments.
between the serum viral load and the percentage of HBsAg pos-
itive hepatocytes (R=0.632, p=0.09). For HBcAg, only three
of the biopsies were positive and the percentage of positive cells
in these samples ranged from 2 to 3% (Fig. 2). The lymphocyte
population in liver samples showed little (<1%) or no specific
reactivityforHBsAgorHBcAg(Fig.2A).AlsonoclearHBsAg
or HBcAg staining was detected in lymphocytes in the periph-
eral blood (data not shown). Liver samples of HBV-negative
liver transplant donors or patients with chronic HBV showed no
staining for HBsAg or HBcAg (Fig. 2B).
Five patients on antiviral therapy had low or undetectable
HBV viral load in the serum at the time of biopsy. Two of
these patients (#9 and #12) with undetectable serum HBV-
DNA still had detectable HBsAg expression in the liver with
28 and 77% positive hepatocytes, respectively. Unfortunately,
no reliable information about virological response and disease
of these patients was available in time following biopsy. The
other patients with low or undetectable viral load (#3, #11 and
#15) had no or less than 1% HBsAg positivity in the liver.
3.3. Comparison of immunohistochemistry and flow
cytometry
Results from the flowcytometric analysis of HBsAg and
HBcAg were compared to the standard immunohistochemistry
of liver biopsies in order to determine the relative sensitiv-
ity. In Fig. 3, a representative immunohistochemical staining
is shown. In general, staining for HBsAg showed a patchy
distribution, with large areas without staining. The cellular dis-
tribution of HBsAg in infected hepatocytes was diffuse with
predominant staining in the cytoplasm (Fig. 3A). HBcAg was
predominantlylocatedinthenucleusinasubsetofinfectedhep-
atocytes (Fig. 3B). Some cytoplasmatic HBcAg staining was
seen near lobular lymphocyte infiltrates (not shown). Table 2
shows the side-by-side comparison of immunohistochemical
and flowcytometric results of 10 liver biopsies. Both methods
had a similar sensitivity. As shown in Fig. 3C, a significant cor-
relation was found between the percentage of HBsAg positive
hepatocytes determined by flow cytometry and immunohisto-
chemistry (R=0.886, p<0.001). The correlation for HBcAg
staining was less clear. Of the three patients who were posi-
tive for HBcAg by flow cytometry, only one was positive by
immunohistochemistry. Conversely, of the three samples pos-
itive by immunohistochemistry only one was positive by flow
cytometry (Table 2).
3.4. Flow cytometry detection of HBsAg and HBcAg in
liver aspiration biopsies
Liver aspiration biopsies from HBV patients (n=7) and con-
trols (n=5) were analyzed by flow cytometry. Representative
histograms are shown in Fig. 4A. Cells in the lymphocyte-
gate showed little positivity for HBsAg whereas cells in the
hepatocyte-gateclearlystainedpositiveforHBsAg.Sixofseven
aspirationbiopsiesthatwereexaminedstainedpositiveforintra-
cellular HBsAg. In two cases (Patient #11 and #12) paired
regular biopsy and fine-needle aspiration biopsy were analyzed
yielding comparable results (0% versus 0% HBsAg and 77%
versus 56% HBsAg positive cells, respectively). The aspiration
biopsyresultsaresummarizedinFig.4B.Thenumberofhepato-
cytes that could be analyzed from the aspiration biopsies ranged
Table 2
Flow cytometry vs. immunohistochemistry
Patient #%HBsAg positive hepatocytes% HBcAg positive hepatocytes
Flow cytometryImmunohistochemistryFlow cytometryImmunohistochemistry
1
2
3
5
6
7
8
9
36
70
20
30
0
2
0
0
2
ND
3
ND
0
0
<10
0
0
0
0
0
1
0
0
5
55
38
83
28
70
60
80
20
65
0
11
12
05<10
77800
ND, not determined.

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Fig. 2. Flowcytometric analysis of liver biopsies for HBsAg and HBcAg. Liver biopsy samples from 12 chronic HBV patients and 2 chronic HCV controls were
analyzedforintracellularHBsAgandHBcAg.(A)Forward/sidewardscatterprofileandhistogramsfromtwoHBVpatients(#2and#6).CellsintheLymphocyte-gate
(left panels) showed little reactivity, whereas cells in the Hepatocyte-gate clearly stained positive for HBsAg and HBcAg (gray histograms). The histogram marker
that defined the percentage of positive cells was based on the isotype-matched control staining (black lines). (B) Summary of the flowcytometric results of HBV and
control biopsies. The geometric mean fluorescence intensity (geoMFI) for HBsAg, corrected for the background geoMFI of the isotype-matched control staining,
was used as measure for the level of HBsAg expression in hepatocytes. The number of cells measured in the Hepatocyte-gate ranged from 130 to 15382. Three of
the 12 HBV samples stained positive for HBcAg (2–3% positive cells). None of the HBV negative samples stained positive for either HBsAg or HBcAg.
from 160 to 1560 cells. Five of seven HBV aspiration biop-
sies were analyzed for HBcAg, but none were positive (data not
shown). None of the HBV-free control aspiration biopsies was
positive for HBsAg (Fig. 4B) or HBcAg (not shown). Taken
together, these results demonstrate that the detection of HBV
viral antigens is feasible with aspiration biopsy despite the rel-
ative low number of cells that are obtained.
3.5. Correlation between intracellular HBsAg levels and
percentage of positive hepatocytes
As shown in Figs. 2 and 4, flowcytometric analysis is very
useful for determining the percentage of HBsAg positive cells,
andalsoprovidesinformationabouttherelativeexpressionlevel
of viral antigen. The assumption is that the mean fluorescence

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Fig. 3. Immunohistochemistry of liver biopsies from chronic HBV patients. Shown is a representative immunostaining for (A) HBsAg and (B) HBcAg. The staining
for HBsAg showed a patchy distribution of positive cells surrounded by areas without staining. The HBsAg localization within infected hepatocytes was diffuse with
predominant staining in the cytoplasm and with distinct nuclear exclusion in some of the cells. HBcAg is predominantly located in the nucleus in a subset of infected
hepatocytes. Isotype controls were negative (not shown). Original magnification ×400. (C) A significant correlation was found between the percentage of HBsAg
positive hepatocytes detected by flow cytometry (FACS) and by immunohistochemistry (IHC). Data are summarized in Table 2.
staining for HBsAg is proportional to the expression level of
HBsAg in infected cells. The specific geometric mean fluo-
rescence intensity (geoMFI) of HBsAg varied greatly between
patients, ranging from 0 to 688 (Fig. 2B). The relationship
between the percentage of HBsAg positive hepatocytes and the
relative HBsAg expression in regular and aspiration biopsies
(n=18) is shown in Fig. 5. A significant positive correlation
was found between the percentage of HBsAg positive hepato-
cytes and the level of HBsAg expression (i.e. geoMFI). Biopsy
samples that were positive for HBcAg showed highest HBsAg
expression (geoMFI 416±163 S.E.M., n=3) as compared to
the HBcAg negative samples (geoMFI 66±41 S.E.M.). Also
the percentage of HBsAg positive cells was high (>55%) in the
HBcAg positive samples (Fig. 2B). This finding suggests a link
between the number of infected hepatocytes and the extent of
HBsAg expression within the infected population of cells.
4. Discussion
Intrahepatic monitoring of viral infection and replication can
providerelevantinformationregardingthecourseofdiseaseand
the response to viral therapy. In the present study, the feasibil-
ity and value of flow cytometry analysis for the quantitation of
viral antigens in liver biopsy samples of patients with chronic
HBV was demonstrated. The percentages HBsAg and HBcAg
positive hepatocytes obtained by flow cytometry correlated sig-
nificantly with the results obtained by immunohistochemistry
(Fig. 3). In addition, the flow cytometry provided information
on the relative expression levels of HBsAg within the infected
cells, which is difficult to quantify by immunohistochemistry.
Interestingly, using this quantitation, a correlation was found
between intracellular expression of HBsAg and the percentage
ofinfectedhepatocytes.Furthermore,thisstudyshowedthatnot
onlyregulartissuebiopsiesbutalsoaspirationbiopsiescouldbe
used for flowcytometric analyses. The less invasive fine-needle
aspiration biopsy may be particularly useful to determine sus-
tained virological response and immune reactivity in the liver
during therapy.
It is known from immunohistochemistry that HBsAg anti-
gen is highly expressed in infected hepatocytes and is located
mainlyinthecytoplasm.Accordingly,HBsAgcouldbedetected
readilybyflowcytometry.Incontrast,HBcAgisonlyexpressed
in a subset of infected hepatocytes and is limited to cells with
active viral replication (Hadziyannis et al., 1983; Ballare et al.,
1989). HBcAg is located predominantly in the nucleus. Cyto-
plasmic HBcAg expression is associated with destruction of

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Fig. 4. Flowcytometric analysis of liver aspiration biopsies. Aspiration biopsies from seven chronic HBV patients and five non-HBV controls were analyzed for
intracellular HBsAg. (A) Representative forward/sideward scatter profile and histograms from aspiration biopsies of two HBV patients (#14 and #17). Cells in
the Lymphocyte-gate showed little positivity for HBsAg, whereas cells in the Hepatocyte-gate from these same samples clearly stained positive for HBsAg. The
histogram marker that defined the percentage of positive cells was based on the isotype-matched control staining (not shown). (B) Summary of flowcytometric results
of HBV and control aspiration biopsies. The percentage of HBsAg positive cells ranged from 0 to 71% and the relative HBsAg expression level within hepatocytes
is shown by the specific geoMFI for HBsAg. The number of cells measured in the Hepatocyte-gate ranged from 160 to 1560. None of the HBV-negative samples
showed specific staining for HBsAg.
hepatocytes in response to immune mediated cytotoxicity (Chu
and Liaw, 1987; Jazayeri et al., 2004; Tang et al., 2004). With
flowcytometric analysis only three of fifteen biopsies staining
positive for HBcAg with only 2–3% of positive cells (Fig. 2B).
Alsowithimmunohistochemistryonlythreebiopsieswereposi-
tive,however,thepercentageofpositivehepatocyteswashigher
(up to 65% positivity). This discrepancy could suggest that the
intracellular staining for flow cytometry is less sensitive or only
detects cytoplasmic and not nuclear HBcAg. HBcAg positivity
byflowcytometrywasfoundonlyincellswithveryhighHBsAg
expression, confirming a link with active viral replication.
To obtain tissue from the liver for diagnostic purposes,
Menghini type or core-needle biopsy is generally used. The
core-needle biopsy is undertaken under local anesthetics and
can cause serious complications, including bleeding, pain and
a mortality rate around 0.1% (Perrault et al., 1978; Lindor et
al., 1996; Thampanitchawong and Piratvisuth, 1999; Terjung
et al., 2003). As a less traumatic alternative, the fine-needle
aspiration biopsy has been introduced successfully for detec-
tion of allograft rejection (Hayry et al., 1981; Pitman, 1998).
The diameter of this needle is significantly smaller than the
core-needle and therefore does not require anaesthesia and has
a minimal risk of serious complication due to damage of the
intercostal space, intrahepatic blood vessels or biliary tracts.
Using aspiration biopsy, inflammatory leukocytes (including T
lymphoblasts and B plasmablasts), plasma cells, lymphocytes

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Please cite this article in press as: van der Laan, L.J.W. et al., Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from
regular and fine-needle biopsies, J. Virol. Methods (2007), doi:10.1016/j.jviromet.2007.01.027
ARTICLE IN PRESS
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L.J.W. van der Laan et al. / Journal of Virological Methods xxx (2007) xxx–xxx
Fig. 5. Flowcytometric data from regular and aspiration biopsy samples com-
bined (n=18) were analyzed for the relation between the percentage of HBsAg
positive hepatocytes and the level of HBsAg expression of these cells. The spe-
cific geoMFI is representative for the expression level of HBsAg. The geoMFI
is plotted on a logarithmic scale. By linear regression, a significant positive cor-
relation was found between the percentage of HBsAg positive hepatocytes and
the expression level of HBsAg in these cells.
and monocytes can be detected in the liver (Lautenschlager et
al., 1988). Cytological evaluation of aspiration biopsies was
shown to be as effective for the diagnosis of rejection of the
liver graft as regular biopsy (Lautenschlager et al., 1988; Kuijf
et al., 2002; Kwekkeboom et al., 2003). Furthermore, aspiration
biopsyhasbeendemonstratedtoimprovediagnosticaccuracyof
focal lesions of liver malignancy (Franca et al., 2003). Recently,
the use of aspiration biopsy for the flowcytometric detection of
hepatic T cells in HBV patients was demonstrated (Sprengers
et al., 2005). To our knowledge, the current study is the first
report on the use of flow cytometry of aspiration biopsy for
the detection of viral antigens. Combing flowcytometric anal-
ysis of infected hepatocytes with an intrahepatic detection of
HBV-specific immune response could provide comprehensive
view of the disease status. This combined analysis using aspi-
ration biopsy possibly provides more reliable information than
single serological or intrahepatic HBV DNA detection by any
method.
Of interest is the observation that two patients on antiviral
therapywithnodetectableviralloadintheserumshowedaclear
presenceofHBsAgintheliver.Thesepatientscouldbeatriskof
recurrenceafterantiviraltreatmentisstopped.Oneotherpatient
with undetectable serum HBV-DNA was negative for HBsAg
in the liver (Fig. 2B) suggesting complete virus eradication and
no risk of recurrence after discontinuation of therapy. These
findings emphasize the importance of evaluating the liver and
not only the blood for the accurate determination of a sustained
virologicalresponse.Itiswellknownthatmanychronicpatients
on antiviral therapy that became negative for the virus in the
serum, can still develop relapse of the virus as soon as therapy
is discontinued. One study looking at HBV DNA replication
intermediates(i.e.cccDNA)confirmedthateveninpatientswith
serological evidence of viral clearance cccDNA persists in the
liver (Werle-Lapostolle et al., 2004). Presumably only when the
virushasbeeneliminatedfromtheliverthesustainedvirological
response is complete and treatment can then be stopped safely.
Inconclusion,thefeasibilityofflowcytometryforthequanti-
tationofviralantigensinliverbiopsyfrompatientswithchronic
HBV was demonstrated. Intrahepatic monitoring of viral infec-
tion could provide relevant information regarding the course of
infection.
Acknowledgements
The authors like to thank Katja Seggeren, Anthonie
GroothuisminkandAngelaHeijensforexcellenttechnicalassis-
tance, Dr. Renate van der Molen, Dr. Jaap Kwekkeboom and
Dr. Hanneke van Vuuren for helpful discussions and Danielle
Wolvers for critically reading the manuscript.
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