Quantitative liver parameters of HCV infection: relation to HCV genotypes, viremia and response to interferon treatment

Journal of Hepatology 1991; 26: 179-186 Printed in Denmark . AN rights reserved Munksgaard . Copenhagen

Copyright 0 EuropeanAssociation for the Studv of the Liver 1997

Journal of Hepatology ISSN 0168-8278

Quantitative liver parameters of HCV infection: relation to HCV genotypes, viremia and response to interferon treatment Giorgio Ballardini, Aldo Manzin i, Fabrizio Giostra, Raffaella Francesconi, Paolo Groff, Albert0 Grassi, Laura Solforosi’, Sabrina Ghettij Daniela Zauli, Massimo Clementi and Francesco B. Bianchi Semeiotica Medica II, Azienda Ospedaliera, University of Bologna, ‘Institute of Microbiology, University of Ancona, and ‘Dipartimento di Scienze Biomediche. University of Trieste, Italy

Background/Aims: This study aimed to evaluate the relation between the number of hepatocytes positive for HCV antigens and the amount of HCV RNA in the liver and to evaluate the relationship between the above parameters and viremia levels, HCV genotype and response to interferon treatment. Methods: This was a retrospective study on 31 consecutive patients with chronic HCV-related liver disease, selected on the basis of the availability of frozen liver tissue for both liver HCV antigens detection and liver HCV RNA quantitation. HCV antigens (immunohistochemistry), liver and plasma HCV RNA (competitive RT-PCR), and HCV genotype (commercial kit) were studied. Results: A significant correlation (p=O.OOOS) was found between the amount of liver HCV RNA (log 10 copylpg of extracted RNA) and the number of HCVinfected hepatocytes (scored from 0 to 3). These parameters were not significantly correlated with viremia levels. The highest liver HCV RNA levels and HCV

antigen scores were found in patients infected with genotype lh Liver HCV RNA (median 541~10~ vs 118~10~ copy number/pg, p=O.O31) and liver HCV antigens (mean score 2.3 vs 1.3, p=O.O18) but not plasma HCV RNA (median 14956X lo3 vs 2.885~10~ copy number/ml, ns) were significantly higher in patients not responding to interferon treatment compared to responders. Conclusions: The tissue parameters tested in this study were significantly correlated, shared the same clinical implications and predicted short-term response to interferon treatment better than tiemia levels. We suggest that these tests should be included in the study protocol of patients under evaluation for interferon treatment, basing the choice on local facilities.

rDENrrrrcArroN of virological parameters useful for correct clinical management of patients with hepatitis C virus (HCV)-related chronic liver disease is still a controversial issue. It has been suggested that qualitative parameters (such as HCV genotype) can help in the selection of patients to be treated with interferon (14). A great deal of information has also indicated that quantitative evaluation of viral activity might be of value. Most attention so far has been focused on serological parameters: response to interferon treatment seems to be more frequent in patients with

low viremia (5-7). The possibility that viremia and HCV genotype might be related to the clinical course of the disease is still under evaluation (8-10). Recently, more attention has been focused on the qualitative and quantitative determination of tissue parameters such as liver HCV RNA and the expression of hepatocellular HCV antigens (HCVAgs). The diffusion of the use of tissue parameters is hampered by the need for an invasive approach (liver biopsy), but their evaluation should provide more direct information on pathogenic mechanisms operating in viva In fact, while viral replication in the liver, the expression of HCV antigens on hepatocytes and their recognition by the immune system represent the leading pathogenic events of the disease, viremia represents the net balance of two major events: emission in the blood of viral particles due to

?”

Received II June: revised 21 October; accepted 6 November 1996

Correspondence: Dott. Giorgio Ballardini, Semeiotica Medica II, Pol. S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. Tel: 39 51/392524. Fax: 39 51/340877.

Key words: Antigens, viral; Chronic hepatitis; Liver

disease; Treatment outcome.

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viral replication (in the liver and possibly extrahepatic sites) and rate of viral elimination from the blood (11). The possibility of detecting HCVAgs in liver biopsies has been definitely established (12-21) and it has been shown that a high number of positive hepatocytes is associated with a low chance of response to interferon treatment (14,20). The absence of HCV RNA in posttreatment biopsy proved to be a good prognostic indicator of long-term response (22). Quantitation of liver HCV RNA has also been performed, using different techniques (competitive, commercial or “in house”, PCR assay or branched DNA signal amplification assay). Conflicting data have been reported on the correlation between liver and serum or plasma levels (2328). Even if a codistribution of HCV RNA and HCVAgs has been shown by combining in situ hybridization with immunohistochemistry (1 S), no quantitative information is at present available on the relationship between the number of HCVAgs containing hepatocytes (infected) and the amount of liver HCV RNA. The aim of this study was to investigate this aspect and to compare the utility of the two tissue parameters in clinical practice. In particular, the relationship of these parameters with viremia, HCV genotype and response to interferon was considered.

Patients and Methods Thirty-one consecutive IFN-treated patients were retrospectively selected on the basis of pure HCV etiology, the availability of a frozen liver biopsy and of liver samples properly stored for immunohistochemical evaluation of HCVAgs, and HCV RNA quantification in the liver. All patients had given informed consent to liver biopsy, which was part of the diagnostic routine. The coexistence of HBV infection was excluded by the use of appropriate commercial RIAs. Alcohol abuse was excluded on the basis of clinical history, Wilson’s disease, al-antitrypsin deficiency and hemochromatosis were ruled out by the quantification of serum ceruloplasmin, a 1-antitrypsin and ferritin and percent of saturation of transferrin, respectively. Autoi~une hepatitis was excluded by testing autoantibodies to actin, homogeneously distributed nuclear antigens and LKM 1, by indirect immunofluorescence on rodent substrates and HEp-2 cells. Anti-HCV positivity was assessed by a third-generation ELISA (Ortho Diagnostic System, Raritan, NJ, USA). The schedule of treatment included the use of either recombinant or lymphoblastoid a-interferon (IFN) (6 MU t.i.w for 3 months, 3 MU t.i.w for 9 months). Twelve subjects displayed a primary response, defined as normalization of ALT levels and disappearance of 780

serum HCV RNA within 3 months and persisting during treatment. Sixteen were considered non-responders, and the pattern of primary response was not defined in three (interferon treatment just started in two, suspended after 1 month in one). Liver biopsy studies Biopsy specimens were obtained percutaneously with a 1.6 mm Hepafix needle. Samples were divided into three fragments: one was fixed in formalin and embedded in paraffin for histological examination, the second (length 8-14 mm) was immediately embedded in TISSUE-TEK O.C.T. compound (Miles Inc. Elkhart, IN, USA), mounted on a piece of cork, snap frozen in N-methylbutane, pre-cooled in liquid nitrogen, and kept at -80°C until examined, and the third (length 35 mm, weight 3.1-7.3, median 5.0 mg) was obtained under sterile conditions and stored at -80°C for HCV RNA quantification. Serum HCV RNA Serum HCV RNA was tested by nested PCR using primers derived from the S’UTR as previously described (29). Blood samples were drawn under sterile conditions, the serum was separated within 2 h, divided into aliquots and stored at -70°C until tested. Serum RNA was extracted from 100 ~1 by the guanidinium thiocyanate-phenol-chloroform procedure (30). Kwok & Higuchi’s contamination avoidance measures (31) were strictly observed throughout the study. One negative control and three positive samples (dilutions lo-‘, lop2 and low3 of a positive serum in a negative one) were employed for every 10 sera tested. Positive and negative results were systematically confirmed. HCV genotype HCV genotype was determined by a commercially available kit (INNO-LIPA HCV, Innogenetics, Zwijndrecht, Belgium) (32) and classified according to Simmond’s classification (33). Liver and plasma HCV RNA quantitation RNA extraction and purification from liver and piasma samples Liver samples were weighed. Total RNA was extracted from biopsy-derived liver samples and from plasma samples using the guanidinium thiocyanate/phenol/ chloroform method. In particular, liver tissue was homogenized by 10 to 20 strokes in a Potter-Elveheim tissue grinder in 400 ~1 of denaturing buffer containing 4 mol/l guanidinium thyocianate, 25 mmolfl sodium citrate (pH 7.0), 0.5% sodium N-Lauroylsarcosinate, and 0.1 molil mercaptoethanol. Forty microliters of 2

Liver HCV-RNA and HCV antigens

mol/l sodium acetate (pH 4.0), 400 ~1 of water-saturated phenol and 80 ~1 of chloroform-isoamylalcohol (49: 1) were subsequently added, with thorough mixing of each new reagent. After cooling in ice for 20 min, the final suspension was centrifuged at 15 000 rpm for 20 min, and the aqueous phase precipitated with absolute ethanol. The samples were then pelleted by centrifuging at 15000 rpm for 45 min, washed with 70% ethanol and vacuum dried. Total RNA was measured by spectrophotometric analysis at 260 and 280 nm (550s UVNIS Spectrophotometer, Perkin Elmer Cetus, Norwalk, Connect., USA). The amount of extracted RNA ranged from 1.1 to 4.2 ,uglmg of liver tissue (median 2.05). Viremia was evaluated on plasma samples which, in our hands, are more reliable than serum samples for quantitation, as previously described (34). Plasma samples, obtained the same day as liver biopsy, were available in only 26 cases. EDTA-treated plasma were centrifuged at 3500 rpm for 10 min soon after collection, and RNA was directly extracted from 100 ~1 of the supernatant as described above. HCV RNA copy numbers quantitation by competitive RT-PCR The cRTPCR assay was performed as previously described (34). Briefly, a specific 86 bp fragment of the HCV genome (from position -91 to -6 of the 5’-UTR sequence) was specifically amplified using PCR. The competitor RNA was a similar 5’-UTR HCV fragment (with a 15-bp internal deletion from position -67 to -53) and was obtained by in vitro transcription of the pC71AN plasmid (30). CRT’-PCR was performed in two steps: constant aliquots (5 ~1) of purified RNA resuspended in diethyl pyrocarbonate (DEPC)-treated water were reverse transcribed at 42°C for 30 min, along with different concentrations (100 000, 20 000, 4 000 and 800 molecules) of the RNA competitor, in the presence of 10 pmol of antisense primer HCVS (position -6 to -29 in the 5’-UTR region), 100 U of Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD, USA), 200 mmol/l of each deoxynucleotide triphosphate and 20 U of ribonuclease inhibitor (Promega Corp., Madison, WI, USA) in a final volume of 20 ~1. After denaturation at 92°C for 10 min, the amplification reaction was carried out for 45 cycles using an automated thermal cycler (Mod 9600, Perkin Elmer Cetus, Norwalk, Connect., USA) in a mixture (final volume 100 ~1) containing 1 XPCR buffer ( 50 mmol/l NaCl, 10 mmol/l Tris-Hcl, pH 8.3, 1.5 mmol/l MgC12, 0.01% gelatin), 2.5 U Taq DNA polymerase (BRL, Gaithersburg, MD, USA), and primers HCV4 (positions -91 to -68 in the 5’-UTR region) and HCVS

(final concentration 50 pmol each). The amplification profile was as follows: denaturation at 94°C for 30 s; annealing at 55°C for 15 s; extension at 72°C for 45 s. Visualization of CRT-PCR amplicons and competition analysis Following PCR amplification, 5 ~1 aliquots of the reaction mixture were run on a 10% polyacrylamide gel at 180 V for 40 min in order to obtain complete separation of the 86 bp wild type from the deleted 71 bp fragment. After ethidium bromide staining the gels were scanned using a video densitometer (Ultra Violet Products Ltd., Cambridge, UK) by positive fluorescent emission on the UV transilluminator. Peak areas (Wa =wild-type area; Ca =competitor area) of both amplified products were calculated by the machine software for each lane. The Ca value was corrected (Cue) for its lower molar ethidium bromide incorporation according to: CacX = Ca wild-type length deleted length= CaX 1.2112. The Cac/Wa ratio was calculated for each sample and plotted on the Y-axis against the copy number of the deleted RNA competitor (C). A regression curve was fitted for each sample: the copy numbers of the wild-type template were calculated from the curve expression for Cac/Wa= 1. The results were expressed as HCV genome copy numbers per ~1 plasma and per pg of total RNA extracted from liver specimen, respectively. The sensitivity of the method (calculated by amplification of endpoint dilutions of the competitor RNA and by determining the Poisson distribution of positive results) has been established to be nearly 4 target molecules of synthetic RNA, and 10 to 100 HCVRNA molecules per ml of plasma samples, respectively. The ratio between liver and plasma HCVRNA, was calculated on a weight to volume basis (copy number/g of liver tissue, copy number/ml, respectively) Zmmunostaining of HCVAgs Five micrometer serial cryostat sections were air dried overnight, fixed with acetone for 5 min, wrapped in aluminium foil and stored at -20 C” until used. Before immunostaining, sections were further fixed with chloroform for 5 minutes. Tissue HCV antigens were searched for using fluorescein conjugated human IgG as previously described (19,20). In brief, IgG were obtained using protein G columns (Pharmacia, Uppsala, Sweden) from a high titer antiHCV positive sera of a patient affected by chronic active hepatitis negative for serum autoantibodies. IgG were conjugated at a protein concentration of 1.5-2 mg/ml with fluorescein isothiocyanate (FITC; isomer 781

G. Ballardini et al.

I, Sigma, St. Louis, MO, USA) by the dialysis method (35). FITC-conjugated IgG were adsorbed overnight with 10 mgiml normal human liver acetone powder; 2 volumes of commercial normal human Ig (Sandoz SA, Basel, Switzerland, protein concentration 33 mglml) were added. The reagent was further diluted 1:10 with phosphate buffered saline (PBS), sections were incubated overnight in a moist chamber at 4°C. After washing (PBS, 30 min), a monoclonal antibody to fluorescein (DAKFITC4, Dako A/S Glostrup Denmark, diluted 150 in PBS) was applied, followed by peroxidase conjugated rabbit anti-mouse Ig (Dako A/S Glostrup, Denmark, diluted 150 with PBS containing 5% normal group AB human serum) and peroxidase conjugated swine antirabbit Ig (Dako A/S Glostrup Denmark, diluted I : 100 with PBS containing 5% normal group AB human serum) (each of the 3 reagents was incubated for 30

min at room temperature, followed by a 15 min PBS wash). The reaction was developed with OS mg/ml diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO, USA) in 0.05 M Tris HCl buffer, pH 7.8, containing 0.003% H202, for 7 min in the dark. Sections were dehydrated in alcohol, cleared in xylol and mounted with Eukit (0. Kindler GmbH, Freiburg, Germany). Controls of specificity were previously reported and included the study of anti-HCV negative cases, blocking experiments with anti-HCV positive and antiHCV negative sera, and reproduction of the staining pattern by anti-HCV specific immunoglobulins eluted by immunoblot assay strips (Ortho Diagnostic System, Raritan, NJ, USA) (18,19). The slides were evaluated blindly by two independent observers who scored the following parameters: a. Histological activity, defined as mild (absent or occasional mild piecemeal necrosis), moderate (pres-

TABLE 1 Clinical, histological, biochemical and virological parameters of the study population Patient

Age

Sex

PP D.L. P.S. S.A. CA. R.G. T.S. B.H. B.G. M.E. VA. R.L. VC. C.E R.N. PD. VS. A.E EA. B.L. C.G. S.C. G.A. D.M M.E. R.G. GE M.D. CM. B.M. BE.

51 21 30 30 61 32 28 33 55 18 24 57 35 42 62 28 23 30 53 51 52 59 64 33 21 55 59 42 54 62 36

F M F M F M M M F M F F F M F F M M F M F M F M M F F M M F M

Histology

moderate mild mild cirrhosis mild mild mod cirrhosis mild mild moderate mild mild severe mild mild mild severe severe

moderate moderate cirrhosis moderate mild mild mild mild cirrhosis severe

ALT

Genotype

Response to IFN

83 140 95 64 69 32 456 302 167 282 194 109 100 46 114 98 49 54 91 214 291 109 69 230 34 69 157 85 334 124 44

2 la lb 4a 2 3a 3a 3a lb la 2 2 lb la lb 3a la lb 2 lb 2 2 lb 3a 2 lb lb lb lb lb la

R un R un R NR R R NR R R R NR NR NR R NR NR NR NR NR R NR NR R R NR NR NR NR un

HCVAgs score

3 3 3 3

Liver HCV RNA

Plasma HCV RNA

14 23 24 43 144 259 4 22 33 77 101 134 906 52 146 1534 1808 3312 62 224 380 404 431 651 691 1512 1518 1541 2586 3107 3805

12234 1894 2425 2340 nd 13483 2885 469 2183 102 29104 1719 nd 10158 nd 60256 332 10352 16690 20809 874 881 nd 51675 8761 11355 20663 16429 67430 nd 4565

M=male; F=Female; Mild =chronic hepatitis without or with occasional mild piecemeal necrosis; Moderate=presence of piecemeal but not bridging necrosis; Severe =presence of piecemeal and bridging necrosis. ALT=normal value<37 U/l; R=Primary Responder (normalization of ALT and disappearance of serum HCV RNA within 3 months and persisting during treatment); NR=Non Responder; un=protocol of treatment not completed. Liver HCV RNA: 1X103 copy number/pg of extracted RNA. Plasma HCV RNA: 1X lo3 copy number/ml of plasma. nd=not done.

Liver HCV-RNA

ence of piecemeal but not bridging necrosis) or severe (presence of piecemeal and bridging necrosis). Presence of cirrhosis was also recorded. b. The presence of HCVAgs was scored according to Krawczynski et al. (12), considering the number of hepatocytes positive for HCVAgs as: 0, no staining; 1,<5%; 2, 5-20%; 3, >20% positive hepatocytes. Statistical

and HCV antigens

tensity of positive hepatocytes, from a few faint cells (Fig. 1) to many strongly positive cells (Fig. 2). Weak and strong positive cells were usually contemporarily observed in the same biopsy, the distribution was often

analysis

Mean values of the different groups were compared by t-test. Fisher’s exact and Mann-Whitney’s tests, linear correlation and Spearman’s rank correlation were used when appropriate.

Results Clinical, biochemical, histological and virological data of the 31 patients at the time of liver biopsy are summarized in Table 1. No correlation was found between HCV antigens score, liver HCV RNA, plasma HCV RNA and age, sex, histology, or ALT levels. HC V genotype

HCV genotype was found to be type lb in 12 patients, type la in 5, type 2 in 8, type 3a in 5 and 4a in 1. Genotype 1b was more frequently found in non-responder than in responder patients (lo/16 vs 2/12, Fisher’s exact test, p=O.O19).

Fig. 2. Liver. Immunohistochemical detection of HCVAgs. Cytoplasmicpositivity of several (score 3) hepatocytes with different intensity of staining. Original magntfkation 50X.

liver HCV-RNA

log10 copy number lpg RNA

Tissue HC VAgs

Hepatocytes positive for HCVAgs were found in 25 of the 31 patients (80.6 %). Of these, seven scored 1, five scored 2 and 13 scored 3. Positive reactions were confined to the cytoplasm of hepatocytes. The staining pattern showed great variability in the number and in-

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Fig. 3. Liver HCV RNA, plasma HCV RNA and liver HCVAgs in patients with (R) and without (NR) ALT normalization and HCV RNA disappearance during interferon treatment. Different symbols correspond to different genotypes. Liver HCV RNA and HCVAgs score are signi@ cantly higher in NR patients. Plasma HCV RNA median value is higher in NR patients, but the difference is not statistically signiJcant. HCV genotype lb is more frequent in NR patients. M=mean value. m=median value.

783

G. Ballardini et al.

Correlation between liver HCV-RNA and HCVAgs 7,00

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Quantitation

of liver and plasma HCV

RNA

Liver HCV RNA ranged from 4 to 3805 (median 259)~ lo3 copy number/pg of extracted RNA. Plasma HCV RNA ranged from 102 to 67430 (median 9460)X lo3 copy number/ml (Table I).

p=O.O005

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The median liver (HCV RNA copy number/g) to plasma (HCV RNA copy ~urn~er~rnl~ ratio was 55

Liver HCV RNA was significantly correlated with HCVAgs score (Spearman’s rank correlation, r,=0.64, p=O.OOOS) (Fig. 4). No significant correlation was observed between plasma and liver HCV RNA (r=O.33, p=O.O9) or liver HCVAgs (t-,=0.32, p=O.lO). Liver HCV RNA was significantly higher (median 541 vs 118, Mann-Whitney test, p=O.O31) in patients not responding to interferon treatment than in responders (Fig. 3). Although the median value of plasma HCV RNA was higher in non-responders than in responders (median 14 956 vs 2885), the difference was not statistically significant (Fig. 3). Patients infected by genotype lb displayed the highest values of liver and plasma HCV RNA. The former was significantly higher with respect to other genotypes (Mann-Whitney, p=O.O47) (Table 2).

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b

1

2

3

HCVAgs Score Fig. 4. Even ifan partial overlap of liver HCV RNA values corresponding to different HCVAgs scores is observed, liver HCV RNA is sig~~~cant~y correlated with HCVAgs score (reflecting the number of infected ~epatocytes~. rs=Spearman rank correlation coefficient.

Discussion The present study was planned to answer some of the questions concerning the relationship between HCV load in different compartments (liver and plasma), HCV genotype and response to IFN therapy We have demonstrated that the number of HCVAgs-positive hepatocytes and the amount of liver HCV RNA are significantly correlated. This is in line with what is known about the mechanism of HCV replication. Moreover, a codistribution between HCV RNA and HCVAgs has been shown combining in situ hybridization with immunohistochemistry (1 S), and we have pre-

focal, with areas of several positive cells and areas with no stained hepatocytes. A preferential lobular distribution was not observed. The number of positive cells was clearly higher in non-responder than in responder patients (Fig. 3) (mean scores 2.3 and 1.3, respectively, t-test: p=O.O18). Higher (although not significantly) scores of positivity were observed in patients infected by genotype lb (Table 2).

TABLE 2 Quantitative parameters and HCV genotype Genotype No. of patients Liver HCV RNA (m) 1X lo3 copy number/pg Plasma HCV RNA (m) 1X lo3 copy number/ml HCVAgs score (M) m=median; M=mean; *=7 patients; +=8 patients.

784

2

la

3a

5

8

17

139

1894 1.6

8670 * 1.8

I b vs others

lb 5

12

259

1209

0.047 Mann-Whitney

13892

ns Mann-Whitney

13483 1.4

t

2.3

P

ns t test

Liver HCV-RNA

viously shown that structural and non-structural HCVAgs (including core and NS5 epitopes) are usually codistributed within the liver (19). Both tissue parameters displayed higher values in patients not responding to interferon treatment compared to responders and in patients infected by genotype lb. Even if the capacity of the method used to discriminate between genotypes lb and la is not absolute, these observations provide further explanation of the divergent sensitivity to interferon of these subgroups of patients. Although a positive trend was found, no significant correlation was observed between viral load in the liver (HCV RNA and HCVAgs) and plasma HCV RNA. The absence of correlation between HCVAgs score and viremia (calculated by serum dilutions) has been previously reported (14). A significant correlation between liver and serum HCV RNA levels has been reported (24,27,28) but poor correlation has been described by others (23,25,26). The median values and the liver-toplasma ratio found in this study were similar to those reported in the literature (23,24), and thus the above discrepancies might possibly be due to the different series of patients, to the low number of cases described in the various studies (including the present) or to the use of different techniques, which possibly shared diverging patterns of sensitivity and reliability when tested with different samples (plasma, serum or liver tissue). The possibility of sampling errors has to be taken into account for liver parameters. A strong correlation between the amount of liver HCV RNA in right and left liver lobes has, however, been described, suggesting an even distribution of the virus (23). We made similar observations for HCVAgs distribution in different samples from explanted livers (unpublished). It should be noted that high HCV viremia can result from both high HCV replication in the liver (and possibly in extrahepatic sites) and a low rate of viral elimination from the blood (11). For the above reasons it is possible that discrepancies between liver and plasma HCV RNA might reflect individual situations, which might influence the natural history of the disease and the long-term outcome of interferon treatment. From a clinical point of view, our study confirms and extends previous data suggesting that liver parameters are more closely related to the pattern of interferon response than viremia levels (26) and , since liver biopsy is included in the routine diagnostic protocol of patients with chronic hepatitis C, strongly support their implementation in clinical practice for prospective evaluation in controlled studies. HCV viremia, which is currently the most frequently evaluated virological parameter, cannot be considered as an alternative

and HCV antigens

to the quantitation of liver viral load. A more precise quantitative analysis of viral load in different compartments is needed for a better understanding of the dynamics of HCV infection and for a more rational approach to treatment. In conclusion, liver HCV RNA and liver HCVAgs are significantly correlated and share the same clinical implications. The choice between them should be based on the facilities and experience of individual laboratories. It has recently been shown possible to detect HCV RNA on liver samples routinely processed for immunohistochemistry (36). Provided that this material is adequate, the quantitation of liver RNA could easily be expanded. To date, only occasional reports suggest that HCVAgs can be detected, with loss of sensitivity, on routine liver sections (20); frozen sections are usually needed. If immunohistochemistry on frozen sections is feasible, it should be more cost effective than quantitation of liver HCV RNA.

Acknowledgement Supported by the: M.U.R.S.T. 1995.

“Progetto

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