widespread presence of hbcag in by methods · pare direct andindirect immunofluorescence in car-bon...

J Clin Pathol 1985;38:393-398

Widespread presence of cytoplasmic HBcAg inhepatitis B infected liver detected by improvedimmunochemical methodsEJ GOWANS, CJ BURRELL

From the Division of Medical Virology, Institute of Medical and Veterinary Science, Adelaide, SouthAustralia 5000

SUMMARY Cytoplasmic and cell membrane associated hepatitis B core antigen (HBcAg) were

found to be more widespread within infected liver using indirect immunofluorescence on frozensections than with the widely used direct immunofluorescence method. Fixation of frozen sectionswith carbon tetrachloride improved tissue histology without reducing the sensitivity of antigendetection. In tissue blocks fixed with formalin or ethanol-acetic acid, detection of HBcAg wasreduced in comparison with frozen sections, and many cells containing low concentrations of(usually cytoplasmic and membranous) HBcAg could not be identified even using indirectimmunofluorescence or peroxidase-antiperoxidase reactions. In contrast, intracellular hepatitis Bsurface antigen (HBsAg) was well detected in fixed sections, but membrane associated HBsAgwas not detectable after fixation.

Among long term carriers of hepatitis B surfaceantigen, the additional presence of hepatitis B coreantigen (HBcAg) in infected liver tissue is usuallyassumed to identify those patients in whom appreci-able virus replication is occurring.' - HBcAg is clas-sically seen within hepatocyte nuclei. CytoplasmicHBcAg has also been reported, however, althoughthe significance of different cellular locationsremains unknown.56 Many carriers with this andother markers of virus replication show a greatertendency for ongoing hepatocyte injury than thosecarriers without such markers, and HBcAg has beenproposed as a possible immunological target for Tcell mediated hepatocyte damage.' This correlationis not absolute, however, and no universal mechan-ism for hepatocyte injury in such patients has beenestablished. In some livers hepatocytes thought tobe supporting hepatitis B virus (HBV) DNA replica-tion often show evidence of cell injury, but cellinjury may also be seen in other livers without evi-dence of viral DNA replication!9 Further under-standing of the significance of the presence and loca-tion of cellular HBcAg, and its relation to virusreplication and hepatocyte injury, has been ham-pered by the insensitivity of the assays used.

Accepted for publication 21 December 1984

HBcAg in infected liver sections has usually beendetected by direct immunofluorescence usingfluorescein conjugated human anti-HBc'-4; occa-sionally indirect immunofluorescence orimmunoperoxidase assays using human anti-HBchave been used.56 The use of the direct test wasusually necessary because (a) high titre anti-HBcfrom an animal species was unavailable and (b)many HBV infected livers contain bound autologousIgG which interferes with the interpretation ofresults obtained with human anti-HBc.

Recently, HBcAg has been expressed inEscherichia coli transfected with HBV DNA'° I andused to prepare high titre anti-HBc in rabbits. Thisreagent now permits the ready use of indirectimmunofluorescence or peroxidase-antiperoxidase(PAP) reactions for HBcAg detection. A recentlyavailable commercial kit for HBcAg detection usesthis reagent in a peroxidase-antiperoxidase reactionon formalin fixed liver sections. Full comparisonsbetween direct and indirect immunofluprescenceand between frozen and fixed tissue sections havenot been published.

This report addresses the conflicting demandsbetween, on the one hand, the need for good his-tological preservation and, on the other hand, thecare required to obtain maximum sensitivity of viralantigen detection.

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Patients and methods

Livers obtained at necropsy from five welldocumented HBcAg positive patients with chronicactive hepatitis and cirrhosis were included in thestudy. Samples were removed as soon as possibleafter death, cut into 2-3 cm slices, and stored at-700C.Frozen sections (6 gtm) were air dried for 15-20

min, fixed if required, washed in phosphate bufferedsaline, and stained in a moist chamber at 37°C withappropriately diluted reagents. Frozen sections werefixed either by immersion in ethanol-acetic acid(3:1) for 10 min at room temperature followed by abrief rinse in ethanol and air drying, or by immer-sion in carbon tetrachloride at 4°C for 10 min andair drying.'2From areas of necropsy tissue adjacent to those

examined above, tissue blocks, measuring 0 5 cm3,were cut from each liver and fixed either in 10%buffered formalin for 24 h or in freshly preparedethanol-acetic acid (3:1) for 20 min at room tem-perature before dehydration and processing intoparaffin wax blocks. Sections (6 ,um) were dried at37°C overnight before use. Before immunostainingthe fixed sections were dewaxed in xylene andhydrated. Sections were then stained in a moistchamber at 37°C using appropriately diluted re-agents.HBcAg was detected by (a) a high titre HBeAg

positive, anti-HBc positive human serum directlyconjugated to fluorescein isothiocyanate (FITC),diluted 1/5 for use (titre = 320 by immunofluores-cence); or (b) an indirect assay using rabbit anti-HBc serum (Dako Corporation) diluted 1/100 fol-lowed by FITC conjugated sheep antirabbit (Well-come) diluted 1/8; or (c) by the Dako PAP kit sys-tem (Dako Corporation) using the manufacturer'srecommended protocol. HBsAg was detected using(a) an indirect immunofluorescence reaction withrabbit anti-HBs (Behringwerke) diluted 1/30 fol-lowed by sheep antirabbit FITC (as above) or (b) byPAP performed exactly as for HBcAg except thatthe anti-HBs (diluted 1/40) was substituted foranti-HBc in the primary antibody step. All dilutionswere in phosphate buffered saline. All reagents usedin immunofluorescence reactions were previouslyabsorbed against normal human liver, and the sheepantirabbit FITC was also adsorbed against normalhuman IgG to remove non-specific cross reactions.The specificity of each reaction was established

using several different uninfected livers as negativecontrols and, in the case of the indirect assays, bysubstitution of the first antiserum with a non-immune serum at the same dilution on infected liversections.

Gowans, Burrell

Our original observations on the five livers weremade using the Dako PAP kit to detect HBcAg asrecommended by the manufacturer. In our hands,however, the substrate supplied in the kit,aminoethylcarbazole, produced a reaction productwhich faded with time, and for this reason subse-quent results were obtained using diaminoben-zidine'3 as the substrate. Although the aminoethyl-carbazole produced a more aesthetically pleasingand readily recognisable reaction product, wedecided that a permanent record of reactions bydiaminobenzidine was more desirable.FITC stained sections were examined in a Ziess

microscope equipped with FITC specific Ploemillumination. PAP stained sections were reactedwith the aminoethylcarbazole supplied by themanufacturer or with diaminobenzidine andmounted in glycerine jelly or DPX respectively.

Results

FIXATION OF FROZEN SECTIONSThe detection of HBsAg and HBcAg by indirectimmunofluorescence in unfixed frozen sections oflivers 1 and 2 was compared with results obtainedwhen sequential frozen sections cut from the sameblock were fixed in ethanol-acetic acid or carbontetrachloride. Ethanol-acetic acid completely dena-tured both HBcAg and HBsAg so that only a fewnuclei showed a weak staining reaction for HBcAgwhile HBsAg was completely negative. Carbon tet-rachloride fixed sections,'2 on the other hand, gavesimilar staining for HBsAg and HBcAg to unfixedsections and resulted in improved histologicalappearance and better retention of sections on slides(data not shown). Carbon tetrachloride was there-fore chosen as a routine fixative for frozen sections.Frozen sections fixed in this manner and stained byindirect immunofluorescence to detect HBsAg andHBcAg were then used as the standard to which allsubsequent fixation and antigen detection was com-pared.

DETECTION OF HBcAg BY DIRECT AND INDIRECTIMMUNOFLUORESCENCEWe then examined all five livers for HBcAg to com-pare direct and indirect immunofluorescence in car-bon tetrachloride fixed, frozen sections. The directand indirect assays detected nuclear HBcAg equallywell, but cytoplasmic HBcAg was stained moreintensely and was present in a higher number of cellsusing the indirect method (Fig. 1 and Table 1). Twolivers (nos 4 and 5) which showed low concentra-tions of cytoplasmic HBcAg in a few cells by directimmunofluorescence now showed low concentra-tions of cytoplasmic HBcAg in virtually every



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Immunochemical methods for detecting cytoplasmic HBcAg in hepatitis B infected liver

Fig. 1 Comparison of the samefield in two sequential frozensections from liver no 4 stained forHBcAg by directimmunofluorescence (a) or indirectimmunopluorescence (b). (b) wasphotographed using the cameraautomatic exposure meter and (a)was subsequently photographedusing manual exposure for thesame exposure time as (b). x630.

Table 1 Comparison between direct and indirectimmunofluorescence for HBcAg on frozen sectionssubsequently fixed with carbon tetrachloride

Liver no HBcAg

Nuclear Cytoplasmic*

Direct Indirect Direct Indirect

++ ++ ++ +++2 ++ ++ ++ +++3 + + ++++ ++++4 + + ++++5 ++++ ++++ ++++

*Including membranous HBcAg.Samples were scored on a 0++++ scale corresponding to 0,1-25%, 25-50%, 50-75%, and ¢75% of total hepatocytesexamined; in this instance the +++++ scale does not representimmunofluorescence intensity.

hepatocyte when examined by the indirectimmunofluorescence reaction. This was in contrastto nuclear HBcAg, which was detected using bothmethods in a few foci of cells in liver no 4 and inevery hepatocyte in liver no 5. Cytoplasmic HBcAgdetected by indirect immunofluorescence was pres-ent as discrete dots throughout the infected cell,occasionally as a large cytoplasmic inclusion, orlocalised to the cell membrane (Fig. lb).

DETECTION OF HBsAg IN FROZEN AND FIXEDTISSUE BLOCKSWe then prepared fixed tissue blocks from adjacentareas of tissue to those examined above, using eitherethanol-acetic acid or formalin. The results of indi-rect immunofluorescence or PAP staining for

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396 Gowans, BurrellTable 2 Detection ofHBsAg and HBcAg in (carbon tetrachloride fixed) frozen sections detected by indirectimmunofluorescence, compared with peroxidase-antiperoxidase staining in sections from formalin fixed tissue blocks

Liver no HBsAg HBcAg

Cytoplasmic Membrane Nuclear Cytoplasmic*

Frozen Formalin Frozen Formalin Frozen Formalin Frozen Formalinfixed fixed fixed fixed

1+ + +++ +++++2 +++ +++ ++ - ++ ++ +++ +++3 + + +++ + ++++4 + - +++ + +++5 + + +++ - ++++ ++++

*Includes membranous HBcAg.Samples were scored as in Table 1-that is, 0 - ++++ represents cell numbers and not staining intensity.

HBsAg on sections from these fixed blocks werethen compared with the results obtained by indirectimmunofluorescence on carbon tetrachloride fixed,frozen sections.

Indirect immunofluorescence and PAP detectedHBsAg in the fixed blocks to a similar extent,although PAP appeared to produce a relativelymore intense reaction. Both detection systemsshowed similar cytoplasmic HBsAg staining in fixed

blocks to that seen in carbon tetrachloride fixed, fro-zen sections (described above), but HBsAg detectedat the hepatocyte membrane in fixed frozen sectionswas undetectable in sections from fixed blocks byeither immunofluorescence or PAP (Table 2). Thisdiffers from some previous reports that detectedmembranous HBsAg in formalin fixed tissue5 145but is consistent with other reports,'6I7 where mem-branous HBsAg was undetected or greatly reduced

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Immunochemical methods for detecting cytoplasmic HBcAg in hepatitis B infected liver

in formalin fixed tissue. Blocks fixed in formalinwere slightly superior for HBsAg detection to thosefixed in ethanol-acetic acid.

DETECTION OF HBcAg IN FROZEN AND FIXEDTISSUE BLOCKSIn contrast to the above HBsAg results, more cellscontaining HBcAg were detected in fixed tissue withPAP than with indirect immunofluorescence, andethanol-acetic acid was slightly superior to formalin(data not shown). But tissue fixed by either methodshowed poor detection of HBcAg present in lowconcentrations compared with the above results byindirect immunofluorescence on frozen sections.Such low concentrations of HBcAg were particu-larly seen in livers nos 3-5, in which a majority ofhepatocytes were HBcAg positive by indirectimmunofluorescence on frozen sections but negativewhen fixed tissue was examined. IntracytoplasmicHBcAg detected by PAP in fixed tissue in livers nos1 and 2 was in general uniformly distributedthroughout the cytoplasm and on the cytoplasmicmembrane, although a proportion of cells containeda discrete, intensely stained inclusion (Fig. 2) similarto those seen by immunofluorescence.We attempted to increase the sensitivity of the

PAP reaction to detect HBcAg in formalin fixed liv-ers 3-5 by substituting the primary anti-HBc reag-ent (supplied already diluted for use in the kit) witha more concentrated preparation of anti-HBc pur-chased separately from Dako. No appreciableincrease in HBcAg detection was achieved using arange of higher concentrations of primary anti-HBc.Non-specific reactions were encountered unless theanti-HBc was first absorbed with normal liverhomogenate. We concluded that the sensitivity ofHBcAg detection by PAP in formalin fixed sectionscould not be increased by increasing the primaryanti-HBc concentration over that supplied in thecommercial kit.

Discussion

FIXATION OF FROZEN SECTIONSThe detection of HBsAg and HBcAg in infectedliver tissue is most reliably accomplished in'frozensections. It is advisable, however, to fix these frozensections before immunostaining in order to preservethe histological integrity of the tissue. This studyshowed that carbon tetrachloride retained the anti-genicity of HBsAg and HBcAg and also preservedthe histological appearance. The distribution andconcentrations of HBsAg and HBcAg in infectedcells of carbon tetrachloride fixed, frozen sectionswere then used as the standard to which otherfixation methods were compared.

DETECTION OF HBcAg IN FROZEN SECTIONSComparison of direct with indirect immunofluores-cence to detect HBcAg showed quite clearly that theindirect method was more sensitive. The indirecr,reaction detected HBcAg in the cytoplasm of apopulation of cells previously thought to be negativefor HBcAg and in some cells which were thought tocontain nuclear HBcAg only. Indeed, in the liversamples studied most HBcAg positive cells ex-pressed the antigen in the cytoplasm, while nuclearHBcAg was seen in only a varying proportion ofthese cytoplasmic HBcAg positive cells. We do notbelieve these results are due to leakage of nuclearHBcAg in these necropsy samples, as we have seensimilar HBcAg distribution patterns in biopsy sam-ples snap frozen within a few minutes of sampling(data not shown).The significance of this finding is as yet unclear. It

is possible that nuclear HBcAg is not directlyinvolved in virus replication, since nuclei containingHBcAg detected by immunofluorescence containlittle if any virus DNA as shown by us previouslyusing in situ hybridisation.9 In any case, futurestudies of the significance of hepatic HBcAg as anindicator of virus replication and as a possible targetfor immune cell damage should clearly distinguishnuclear from cytoplasmic HBcAg and should use amethod capable of detecting low concentration,widespread cytoplasmic HBcAg, if present.

DETECTION OF HBsAg AND HBcAg IN FIXEDTISSUEThis part of the study was performed as there werefew reports which compared the detection ofHBsAg and HBcAg in fixed tissue with resultsobtained in frozen sections. Examination of tissuefixed by either of the two methods described indi-cated that PAP was more sensitive than indirectimmunofluorescence. Despite the use of PAP, lowconcentrations of HBsAg and HBcAg (shown to bepresent, usually at the cell membrane, by indirectimmunofluorescence examination of frozen tissue)were undetected in fixed tissue. Other workers havecommented on similar results with HBsAg'819 orHBV-unrelated membrane antigens fixed in forma-lin.20 Of the two fixatives studied, formalin wasslightly better for HBsAg detection, and ethanol-acetic acid was slightly better for HBcAg detection.The extent of antigen loss, however, may depend onthe time and degree of fixation as well as other lesswell defined factors, and satisfactory detection ofHBsAg or HBcAg should not be assumed after anytype of fixation without proper evaluation.

CONCLUSIONSThree main points have emerged from this study:

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1 Cytoplasmic HBcAg is much more abundant inHBV infected liver tissue than was previouslydemonstrable.2 HBV infected liver tissue fixed and processed intoparaffin wax may fail to show the same pattern ofHBV antigen expression seen in frozen sections ofthe same liver. This is particularly true of viral anti-gens expressed on the cytoplasmic membrane ofinfected hepatocytes.3 Studes relating HBcAg expression with virusreplication or hepatocyte injury should allow for theabove and should clearly distinguish nuclear andcytoplasmic HBcAg localisation in view of theirpossibly differing significance.

This work was supported by a grant from theNational Health and Medical Research Council ofAustralia and carried out in a category 1 laboratorywith approval from the Institute of Medical andVeterinary Science Biohazard Committee. Wethank Allison Jilbert and Tom MacNaughton forhelpful discussion, Judy Findlay for skilled technicalassistance, Nancy Mitchell and Deloraine Mauvielfor typing, and the staff of the gastroenterologylaboratory, IMVS, for a constant supply of sections.

References

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2 Ray MB, Desmet VJ, Bradburne AF, Desmyter J, Fevery J, DeGroote J. Differential distribution of hepatitis B surface anti-gen and hepatitis B core antigen in the liver of hepatitis Bpatients. Gastroenterology 1976;71:462-7.

3Hadziyannis Si, Lieberman HM, Karvountzis GB, Shafritz DA.Analysis of liver disease, nuclear HBcAg, viral replication, andhepatitis B virus DNA in liver and serum of HBcAg vs. anti-HBe positive carriers of hepatitis B virus. Hepatology1983;3:656-62.

Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin J.Hepatitis B virus DNA in the sera of HBsAg carriers: amarker of active hepatitis B virus replication in the liver.Hepatology 1981;1:386-91.

Huang SN, Neurath AR. Immunohistologic demonstration ofhepatitis B viral antigens in liver with reference to itssignificance in liver injury. Lab Invest 1979;40: 1-17.

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Gowans, Burrell

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Requests for reprints to: Dr CJ Burrell, Division of Medi-cal Virology, Institute of Medical and Veterinary Science,PO Box 14 Rundle Mall PO, Adelaide SA 5000, Australia.



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