document: hepatitis c virus ( hcv ) is a single - stranded rna virus , a member of the flaviviridae family . since the first identification of the hcv genome by choo et al . 
   , hcv study has progressed mainly in the field of hcv functional analysis and therapeutic implications . 
   because of low viral levels in the serum , the diagnosis of hcv infection has been made with a branched chain dna signal amplification assay and reverse - transcription polymerase chain reaction ( rt - pcr ) [ 24 ] . 
   pathologists are often requested to detect viral pathogens within the routinely prepared biopsy tissue samples , and therefore simple and reliable histochemical techniques are needed . 
   reports have described the detection of hcv in human liver tissue by using immunohistochemistry and in situ hybridization ( ish ) [ 414 ] , as well as by in situ rt - pcr [ 4 , 6 , 1520 ] . at the moment , hcv detection with immunohistochemistry and ish is not yet reproducible enough , we believe . in situ rt - pcr 
   reliable histochemical techniques for detecting hcv in formalin - fixed , paraffin - embedded ( ffpe ) liver tissues are needed , particularly for routine diagnostic purpose . in the present study 
   , we established an ish technique using a locked nucleic acid ( lna ) probe and biotin - free tyramide amplification system ( csaii ) for detecting hcv - rna in ffpe tissue sections of the liver . 
   as positive controls , we sampled the humanized liver of hcv - infected chimeric mice [ 21 , 22 ] , and ffpe tissue sections were prepared . 
   the chimeric mice were maintained by m. kohara , department of microbiology and cell biology , tokyo metropolitan institute of medical science , tokyo . 
   a total of 13 humanized liver specimens from hcv - infected chimeric mice were used ( noninfected 3 , genotype 1a - infected 3 , genotype 1b - infected 4 , and genotype 2a - infected 3 ) . 
   a total of 11 needle biopsy liver specimens of chronic hepatitis c were culled from the computer file at the diagnostic pathology division of fujita health university hospital , toyoake , japan , and ffpe sections were cut . 
   needle biopsy specimens of hepatitis - b - virus- ( hbv-)infected chronic hepatitis , virus - negative fatty liver and autoimmune hepatitis ( n = 1 , resp . ) , as well as two surgically removed hcc lesions caused by hcv or hbv infection , were also examined . 
   the nonneoplastic part of the liver tissue ( chronic hepatitis b ) in the hbv - related hcc case was also evaluated . 
   non - neoplastic liver tissue was scarcely included in the hcv - related hcc case . 
   the activity of inflammation and the degree of fibrosis were evaluated according to the new inuyama classification ( 1996 ) . 
   serum hcv - rna levels in the patients were measured using an rt - pcr assay , the amplicor hcv monitor assay ( roche diagnostics ) , and serum hcv subtypes were determined with nested rt - pcr ( roche diagnostics ) . the sequence of an hcv - common probe was designed according to the previous description . 
   the target was within the hcv 5-untranslated region ( hcv-5utr ) , which is a highly conserved portion in the hcv genome . 
   an lna - modified oligonucleotide probe labeled with digoxygenin at the 3-end was prepared in gene design inc . , 
   the nucleotide sequence of the 45-mer probe was 5-altttlggglcgtlgcclccclgcglagalctglctalgcclgagltagltgtltgglgt-3 , in which la , lt , lc , and lg represent lna monomers corresponding to the bases a , t , c , and g , respectively . 
   endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 60 minutes at room temperature ( rt ) . 
   after rinsing thrice in diethyl - pyrocarbonate- ( depc-)treated water , sections were digested with 40  g / ml proteinase k ( roche diagnostics , tokyo , japan ) at 37c for 15 minutes , washed thrice in depc - treated water , submerged in 95% ethanol for 1 minute , and air - dried completely . 
   then , sections were heat - treated for 5 minutes at 95c on a hot plate and hybridized with 0.01 
   mol / l lna - modified oligonucleotide probe diluted with in situ hybridization buffer ( enzo life science , bulter pike , pa , usa ) in an incubation chamber overnight at 37c . after rinsing in 1x saline sodium citrate ( ssc ) for 30 minutes at 50c 
   , sections were rinsed twice in 50  mm tris - hcl - buffered saline ( tbs ) , ph 7.6 , at rt . a horseradish - peroxidase - labeled antidigoxygenin antibody ( roche diagnostics ) diluted at 1  :  100 with 1% bovine serum albumin in tbs was applied for 60 minutes at rt . 
   for amplifying signals , fluorescein - isothiocyanate- ( fitc-)conjugated tyramide enclosed in csaii ( dako , glostrup , denmark ) was applied to the sections for 15 minutes at rt . 
   finally , a horseradish - peroxidase - labeled anti - fitc antibody equipped in the csaii kit was reacted for 30 minutes at rt . 
   reaction products were visualized in 50  mm tris - hcl buffer , ph 7.6 , containing 20  mg / dl diaminobenzidine tetrahydrochloride and 0.006% hydrogen peroxidase . 
   the nuclei were lightly counterstained with mayer 's hematoxylin . in order to verify the specificity of the ish technique for hcv - rna detection 
   , we carried out the treatment with dnase and rnase before the hybridization with the lna probe on consecutive sections . 
   namely , either 10  iu / ml dnase i ( wako , osaka , japan ) or 100  g / ml rnase ( sigma - aldrich , st . louis , mo , usa ) was incubated for 30 minutes at 37c . in order to distinguish mouse hepatocytes from chimeric human hepatocytes in hcv - infected chimeric mice 
   , we utilized a human - specific ck8/18 monoclonal antibody ( clone : ncl 5d3 , mp biochemicals , santa ana , ca , usa ) . after blocking endogenous peroxidase activity , 
   hydrated heat - assisted epitope retrieval was employed using a pressure pan cooker ( delicio 6l , t - fal , clichy , france ) for 10 minutes . 
   preliminary study revealed that as a soaking solution , 1  mm ethylenediamine tetraacetic acid solution , ph 8.0 , was optimal to retrieve the antigenicity . 
   after pressure cooking , sections were left for 30 minutes at rt for cooling . in order for avoiding nonspecific signals in mouse hepatocytes , a mouse stain kit ( nichirei bioscience , tokyo ) 
   was applied before and after the primary antibody incubation . the human - specific ck8/18 monoclonal antibody at a 1  :  100 dilution was incubated overnight at rt . 
   after rinsing in 10  mm phosphate - buffered saline ( pbs ) , ph 7.2 , the sections were reacted with a secondary polymer reagent , novolink ( novocastra , newcastle , uk ) . 
   total rna was extracted from dewaxed sections using recoverall total nucleic acid isolation kit ( applied biosytems , austin , tx , usa ) , according to the manufacturer 's protocol , and stored at 80c until use . 
   the present study was approved by the institutional ethical review board for clinical and epidemiological investigations at fujita health university , toyoake . 
   in he - stained sections , mouse hepatocytes showed densely eosinophilic cytoplasm , while chimeric human hepatocytes were weakly eosinophilic with frequent deposition of fat droplets . 
   immunoreactivity of human ck8/18 was seen only in human hepatocytes . in all the 10 hcv - infected livers but not the livers of three hcv noninfected mice , ish demonstrated diffuse cytoplasmic signals in the human hepatocytes . in figure 1 employing consecutive sections , 
   hcv - rna was visualized with the ish technique in human ck8/18-positive chimeric hepatocytes from chimeric mice infected hcv genotypes 1a , 1b , and 2a ( in figure 1 , dotted lines encircle the mouse hepatocyte area ) . 
   it is of note that hcv - rna was demonstrated in the cytoplasm of almost all chimeric human hepatocytes . 
   the positive signals remained after dnase treatment but were completely abolished after rnase treatment . 
   figure 2 illustrates hcv genotype 1b - infected chimeric mouse liver , showing he histology and positive cytoplasmic signals , which were resistant to dnase but sensitive to rnase . 
   most of the hcv - positive patients showed high levels of hcv - rna in the serum . 
   exceptionally , a surgical case of hcv - related hcc showed a low level of hcv - rna in the serum . 
   nested rt - pcr genotyping for hcv using total rna extracted from ffpe sections was successful in 8 ( 73% ) of 11 biopsied chronic hepatitis c lesions , and in all the 8 cases , the genotypes were comparable with the serum analysis . 
   lna - based ish detected signals in 9 ( 82% ) of 11 chronic hepatitis c lesions , including three showing diffuse cytoplasmic positivity , and six showing focal or partial positivity . in three nested rt - pcr negative lesions , ish revealed negativity ( 1 lesion ) or focal / partial positivity ( 2 lesions ) . 
   no hcv - rna was detected in hbv - related lesions , fatty liver , autoimmune hepatitis , and in hcc lesions . 
   nonspecific binding of the csaii reagent to kupffer cells or lymphoid cells was occasionally observed in both the hcv - infected and hcv - unrelated lesions , as arrows indicate . 
   we showed herein that lna - based ish for hcv - rna yielded clear cytoplasmic reactivity in infected hepatocytes in ffpe sections . 
   the lna - modified oligonucleotide probe we employed represents one of the most sensitive probes for ish [ 2529 ] . 
   lna - based ish has been applied to detecting cellular microrna in ffpe tumor tissues [ 3032 ] . 
   the 45-mer lna probe was targeted at the hcv-5utr containing a sequence common to the hcv subtypes . 
   positive signals for hcv - rna were clearly demonstrated in hcv 1a- , 1b- , and 2a - infected humanized livers of chimeric mice . 
   hcv - infected chimeric mice with humanized liver thus functioned as a useful model for histochemically demonstrating the hcv genome , particularly when immunostaining for human - specific ck8/18 was combined . in needle - biopsied human liver specimens , 
   hcv - rna was detected in 8 of 11 hcv - infected samples with the nested rt - pcr analysis and in 9 of 11 with the ish technique . in nested rt - pcr negative cases , 
   ish was negative or partially positive , suggesting that the detection threshold may depend on the virus load . 
   hcv - rna was undetectable in the hepatocytes in hbv - infected livers , nonviral liver disorders ( fatty liver and autoimmune hepatitis ) , and in hcv - related hcc . 
   with ish using a digoxygenin - labeled cdna probe , tang et al . reported positivity in 8 ( 67% ) of 12 hcv - related hcc . 
   it has been documented with an in situ rt - pcr technique that hcv - rna decreases along with the progression from liver cirrhosis to hcc . 
   more cases of hcc need to be analyzed , in relation to the distribution of the hcv genome in cancerous lesions . 
   revie and salahuddin reviewed that hcv replicated in macrophages , b and t lymphocytes , and other nonhepatocellular components . 
   however , we judged the nonhepatocellular bindings as nonspecific reactions , primarily because of the staining in non - hcv cases . under our present condition , nonspecific binding of the reagents , particularly csaii , to kupffer cells and lymphoid cells 
   further technical improvement is requested for reliable and reproducible hcv - rna detection in ffpe specimens . in conclusion , 
   the ish technique using the lna - modified oligonucleotide probe and csaii can be applied to detecting hcv - rna in liver biopsy specimens , the sensitivity probably being comparable to in situ rt - pcr . for detecting the hcv genome in routinely prepared liver biopsy specimens , 
   our ish sequence is relatively simple and requires no special equipment to perform . at the moment 
   , this method seems to be suitable for demonstrating hcv genome in ffpe sections and will become a valuable tool for routine histopathological diagnosis of hcv infection using ffpe liver biopsy specimens .
summary: background . in situ hybridization ( ish ) with high sensitivity has been requested to demonstrate hepatitis c virus ( hcv ) rna in formalin - fixed , paraffin - embedded ( ffpe ) sections of the liver . 
   methods . 
   ish employing a locked - nucleic - acid- ( lna-)modified oligonucleotide probe and biotin - free catalyzed signal amplification system ( csaii ) was applied to hcv - rna detection in the liver tissue . 
   nested reverse - transcription polymerase chain reaction ( rt - pcr ) was performed for hcv genotyping using total rna extracted from ffpe sections . 
   the target tissues included ffpe tissue sections of humanized livers in hcv - infected chimeric mice ( hcv genotypes 1a , 1b , and 2a and noninfected ) and of needle - biopsied livers from hcv - infected patients . results . 
   hcv - rna was demonstrated with the ish technique in hcv - infected liver tissues from both chimeric mice and 9 ( 82% ) of 11 patients with hcv infection . 
   the hcv signals were sensitive to rnase . 
   nested rt - pcr confirmed the genotype in 8 ( 73% ) of 11 livers ( type 1b : 6 lesions and type 2a : 2 lesions ) . 
   hcv - rna was not identified in chronic hepatitis b lesions , fatty liver , autoimmune hepatitis , and hepatocellular carcinoma . 
   conclusion . 
   ish using the lna - modified oligonucleotide probe and csaii was applicable to detecting hcv - rna in routinely prepared ffpe liver specimens .