Journal of Medical Virology 37310-314 (1992)

Detection of Hepatitis “C” Virus in Formalin-Fixed Liver Tissue byNested Polymerase Chain Reaction Richard Sallie, Anne Rayner, Bernard Portmann, A.L. W.F. Eddleston, and Roger Williams Institute of Liver Studies, King$ College School of Medicine and Dentistry, King’s College Hospital, London, England Interpretation of antibody t o hepatitis C virus (HCV) in patients with liver disease is difficult due to false-positive reactivity in some conditions. To evaluate the feasibility of HCV in archival material, HCV was sought in formalinfixed, paraffin-embedded liver biopsy specimens. Nested polymerase chain reaction was used t o detect hepatitis C virus in formalin-fixed, paraffin-embedded liver biopsy specimens after total RNA was extracted from tissue by proteinase K digestion and phenol/chloroform purification. The relative efficiency of amplification of HCV RNA from formalin-fixed material was estimated semiquantitatively by serial dilution of cDNA synthesised from RNA extracted from fresh and formalin-fixed sections from the same liver. Although HCV RNA could be detected in formalinfixed liver tissue by nested PCR in 5/5 cases in which HCV was detected in serum, amplification was -5-fold less efficient than when HCV was amplified from fresh tissue. Nevertheless, nested PCR of HCV from formalin-fixed liver tissue represents a useful technique i n addressing some important questions related t o the pathogenesis of liver disease. (C 1992 Wiley-Liss, Inc.

body to HCV in patients with liver disease is presently uncertain [McFarlane et al., 19901, and serological tests to define the HCV carrier state are urgently required. Recently, the technique of nested polymerase chain reaction (nPCR) has been successfully applied to define HCV viraemia in blood donors thought on epidemiological grounds to have transmitted HCV [Garson, et al., 19901. Although polymerase chain reaction (PCR) detection of HCV circulating in serum is clearly a very important and useful technique, it does little to further the understanding of the effects of HCV on the liver, the presumed reservoir and primary site of viral replication. In particular, the putative contribution of HCV to cryptogenic cirrhosis, autoimmune chronic active hepatitis, and hepatoma, all suggested by serum antibody studies [Esteban et al., 1990; Sbolli et al., 19901, requires positive identification of HCV within the liver. In many patients in whom a diagnosis of HCV might be sought using the previously described method of tissue PCR for HCV [Weiner et al., 19901, fresh liver tissue is less likely to be available than formalin-fixed tissue. We report the detection of hepatitis C virus in formalin-fixed, paraffin-embedded tissue using the nested polymerase chain reaction in 5 patients coming to orthotopic liver transplantation.

MATERIALS AND METHODS Five patients thought likely to have chronic liver sue, polymerase chain reaction, disease consequent upon hepatitis C both on clinical RNA grounds and as a result of a positive HCV polymerase chain reaction performed on serum were initially studied. The demographic data of these patients are deINTRODUCTION scribed in Table I. Normal liver tissue was obtained as The predominant infective agent responsible for post- negative control material from five normal “cut-down” transfusion hepatitis [Alter et al., 19891, a positive livers used in organ transplantation. Three to four secstranded RNA virus, has recently been isolated, cloned, tions 10 pm thick were cut from a formalin-fixed, parafand sequenced [Choo et al., 1989; Kubo et al., 19891, fin-embedded pathological block obtained either a t and has been designated hepatitis C (HCV). Develop- Trucut liver biopsy obtained prior to OLT or from ment of various serological antibody tests based on recombinant viral proteins [Kuo e t al., 19891derived from the nucleic acid sequence of the HCV has resulted in widespread use of the tests for diagnostic purposes and Accepted for publication February 6,1992 for screening of blood and other transfusion products. Address reprint requests to Dr. Roger Williams, Institute of Although the finding of a positive HCV antibody test in Liver Studies, King’s College School of Medicine and Dentistry, the screening of transfusion blood is sufficient reason King’s College Hospital, Denmark Hill, London, England SE5 for rejection of the donation, the significance of anti- 8RX. KEY WORDS: hepatitis C, formalin-fixed tis-

0 1992 WILEY-LISS, INC.

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TABLE I. Demographic, Serological, Histological, and Risk Factors on the

5 Patients Positive for HCV by nPCR in Serum and Formalin-Fixed Liver Tissue

Patient S.P A.D S.E A.B K.P

Age 54 23 30 46 45

Sex M F F M F

Risk factora HRA transfusions nil known IVDA HRA

C-100 test positive positive negative Not done Not done

Diagnosisb C HCC aCAH + HCC SAH C SAH

+

aHRA = patient from a high risk area; IVDA = intravenous drug user. bC = cirrhosis; HCC = hepatocellular carcinoma; aCAH = autoimmune chronic active hepatitis; SAH = subacute hepatic necrosis. The patient with aCAH had been diagnosed i n childhood on the basis of compatible high titre autoantibodies, immunoglobulins, and biopsy, but HCV seroconverted following a 22-unit blood transfusion for bleeding varices.

Primera HCV JR12 HCV JR19 HCV JR 13 HCV JR 14 Probe Albumin Albumin

TABLE 11. Primer Sequences Used in Study Position Sequences sense 5’-GGCGACACTC CACCATAGAT-3’ nt 1-20 antisense nt 5’-CGCCCAAATC TCCAGGCATT-3’ 197-2 16 sense 5’GAACTACTGT CTTCACGCA-3’ nt 35-53 antisense nt 5’-GGCAATTCCG GTGTACTCAC C-3’ 140-161 115-135 5’-GAGAGCCATA GTGGTCTGCG-3’ sense 5’-TGAAATGGCT GACTGCTGTG-3’ 5’-GCAGC‘I“ITAT CAGCAGCTTG-3‘ antisense

Product size 216bp

126bp

“HCV primers as described by Ulrich et al. [1990]and primers directed against albumin mRNA (flanking a n intron of -250 base pairs), thus allowing for differentiation of RNA/DNA amplification.

wedge biopsy if the resected liver was available. To compare the efficiency of extraction of HCV RNA from formalin-fixed or fresh tissue, RNA was extracted from a 0.5 cm3 cube of liver tissue, known to be HCV RNA positive by PCR, that had been divided in two and the identical halves either formalin fixed or processed as a frozen section. The cDNA synthesised from these specimens was titred (analogous to the method described by Simmonds et al. [1990] prior to PCR to allow relative quantitation of the efficiency of extraction from these different methods of processing. This comparison was performed in triplicate on the same specimen, extracting RNA from a n identical number of sections from either the formalin fixed or the frozen block.

Nucleic Acid Extraction Formalin-fixed tissue. The biopsy shavings were placed into a 1.5 ml screw-capped Eppendorf tube and nucleic acid extracted in a modification of the methods described by Shibata et al. [1988] and by Jackson et al. [1990]. The specimen was de-waxed in xylene a t 50°C for 5-10 minutes, centrifuged a t 12,000 g for 5 minutes, and the xylene carefully aspirated from the resulting pellet. Addition of 50 pl of dithylpyrocarbonate (DEPC)-treated water prior to centrifugation of the xylene facilitated removal. Most of the xylene was then removed by careful aspiration. The remaining xylene was washed from the tissue pellet with a single 70% alcohol wash, followed by repelleting by centrifugation

for 5 minutes at 12,000 g. The pellet was resuspended in 200 p1 digestion buffer containing proteinase K a t 100 p/ml, 0.5% SDS, 0.005 M EDTA, 0.01 Molar Tris, pH 7.8, and vanadyl-ribunucleoside complexes (VRCs, Sigma) at 40 mM, before vortexing vigorously for 20 seconds, brief centrifugation, and addition of three drops of mineral oil (Sigma) to prevent evaporation. Digestion was carried out in a water bath a t 42” for 2-3 days. Following digestion, the nucleic acids were extracted with sodium acetate-buffered phenol (pH 5.2), phenol/chloroform, and finally chlorofordisoamyl alcohol before addition of 0.1 volumes of 3 M sodium acetate (pH 5.21, 2.5 volumes absolute alcohol, and 20 pg glycogen. RNA was then precipitated overnight at -70°C. The resulting pellet was washed once in 70% alcohol, dried, and resuspended in 20 pl sterile diethylpyrocarbonate (DEPCMreated water by gentle pipetting. Fresh tissue. For comparative purposes, RNA was also extracted from fresh tissue using the single step RNA extraction method described by Chomczynski et al. [1987]. In this method, the 10 pm frozen sections were solubilized in 4 M guanadinium isothiocyanate, 0.5% N-Lauryl sarcosyl, 0.2 M 2-mercaptoethanol before phenol extraction, and overnight absolute ethanol precipitation of nucleic acid at -70°C. RNA pellets were washed with 70% alcohol and resuspended in 20 p.1 of DEPC-treated water immediately before reverse transcription.

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Fig. 1. Agarose gel (3% Nusieve, 1%agarose) and corresponding Southern blot obtained from formalin-fixed tissue. Lane 1: molecular weight marker, 123 base pair ladder; lane 2 negative (-ve) control (albumin primers); lane 3 positive (+ve) control for albumin; lane 4: -ve control for HCV (normal human liver); lane 5 +ve control for HCV; lanes 6 and 7: patient -ve for HCV, lane 6 showing albumin

band, lane 7 -ve for HCV; lanes 8-12- HCV +ve liver, fresh and formalin processed: lane 8 +ve albumin band, lane 9 “outer” primer +ve HCV band, formalin fixed, lane 10 “inner” band, formalin fixed; lane 11 “outer” band, fresh tissue, lane 12 “inner” band, fresh tissue; lane 1 3 M.W. marker.

cDNA Synthesis Four pl of the resulting RNA/DNA suspension was then added to a reverse transcription mixture containing 5 units AMV reverse transcriptase (RT, Promega, U.K), 2 pl of 1OX reverse transcription buffer, 1 mM dithiothrietol (DTT), 2.5 mM dNTPs (Pharmacia, UK), 10 mM random hexamers, and 20 units of RNAse inhibitor (Promega, UK) in a total volume of 20 pl overlaid with oil. The mixture was chilled on ice for 5 minutes prior to incubation at 42” for 60 minutes. Following reverse transcription, the mixture was boiled for 5 minutes to inactivate the RT.

PCR mix, which contained 60 ng of each the “outer” primers, 0.2 mM dNTPs, 2.5 pl of 1OX Taq polymerase buffer, 0.5 units of Taq polymerase (Biotech, Bentley, Western Australia) in a total reaction volume of 25 pl, which was overlaid with 2 drops of light oil (Sigma) before being subject to 35 rounds of amplification using a Perkin-Elmer-Cetus thermal cycler set for the following cycling parameters: 94” for 1minute 30 seconds, 55” for 1 minute 30 seconds, and 72” for 1 minute before a final extension step of 72” for 7 minutes. Four pl of first round product was then taken into a second round mixture, which was identical to the first except that 120 ng of the inner primers were used. The annealing temperature during second round amplification was reduced to 50”. To avoid contamination, the general measure suggested by Kwok and Higuchi [19891 were adhered to except that separate sets of pipettes for different phases of the work were substituted for positive displacement pipettes. In addition, to avoid contamination from the microtome blade, sections were cut using a fresh blade for each sample and separate duplicate samples were

Polymerase Chain Reaction The primers used for HCV amplification were those described by Ulrich et al. [1990] and are described fully in Table I. As a n internal control for the reverse transcription step, albumin mRNA was co-amplified using primers based on the nucleotide sequence described by Minghetti et al. [1986] (Table I). Four p1 of the resulting DNAicDNA solution was then added to a first round

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Fig. 2. Comparative slot blot of PCR amplified, titred cDNA obtained from fresh and formalin-fixed tissue. Lane A Titred cDNA prepared from fresh tissue. Lane B: Titred cDNA prepared from formalinfixed tissue, showing a -5X increased sensitivity of amplification from fresh tissue when compared to formalin-fixedtissue.

19891. Recently, the specificity of nested polymerase chain reaction for HCV detection has been questioned on the basis that nonspecific bands of incorrect size are produced as a consequence of primer self-annealing and extension when the primer quarter of Kaneko et al. was employed [Clewley, 19901, a criticism that might apply Analysis to any PCR method. To negate this criticism, we conTen pl of the PCR product obtained after second firmed product specificity by Southern blotting with a round amplification was then either run on a n ethid- probe internal to the PCR primers. Although difficult to ium bromide-stained agarose gel and Southern blotted quantify, PCR amplification of HCV extracted from for(Fig. 1)to confirm product specificity or, in the case of malin-fixed tissue has tended, in our hands, to produce the comparison between fresh and formalin fixed tis- more “smeared” bands that amplified from fresh tissue. sue, applied to nylon membranes (Hybond N+ , Amer- Furthermore, despite using several different pairs of sham, UK) via a dot-blot apparatus (Fig. 2). In either primers to different “housekeeper” or structural genes case, membranes were probed using a P32 labelled syn- (notably the growth hormone receptor and actin), we thetic oligonucleotide probe internal to and not inclu- have found PCR amplification of RNA fragments greater than 500 base pairs from formalin-fixed, parafsive of the “inner” PCR primers (Table I). fin-embedded tissue to be inconsistent, a finding reRESULTS ported by others [Greer et al., 19911. It would therefore We were able to amplify albumin RNA extracted seem logical to limit the size of the HCV fragment to be from formalin-fixed liver tissue in every biopsy studied. amplified (including the “outer” primer product) to as HCV RNA was only amplifiable from tissue obtained small a fragment as can be conveniently visualized on from patients in whom HCV RNA was found in serum. a n ethidium-stained gel. The addition of VRCs to the The sensitivity of PCR amplification from either fresh digestion buffer is probably unnecessary, as RNA vior formalin-fixed tissue was compared in triplicate by ruses have been successfully amplified from formalinserial endpoint dilution of cDNA. Amplification of fresh preserved tissue following simple proteinase K digestissue was always more efficient when compared to for- tion [Godec et al., 1990; Jackson et al., 19901. However, malin-fixed tissue (Fig. 21, and typically it was possible VRCs do not seem to interfere with subsequent reverse to amplify HCV from fresh tissue at 2-5-fold greater transcription or polymerase chain reaction and may dilutions of the resultant cDNA than that seen with confer stability on the HCV RNA during prolonged digestions. formalin-processed material. Although extraction from formalin-fixed material is tedious and time-consuming when compared to extracDISCUSSION tions from serum or fresh material, application of the We have shown the feasibility of amplifying HCV technique to the vast amount of formalin-fixed archival RNA from formalin-fixed liver tissue obtained a t Tru- tissue available makes it possible to address important cut needle liver biopsy, using a PCR technique that has questions concerning the relationship of HCV to the been applied to the detection other viral agents in tis- development of cryptogenic cirrhosis, hepatocellular sue previously [Sakamoto et al., 1988; Shibata et al., carcinoma, and, possibly, chronic active hepatitis.

cut on different days. Both cDNA preparation and HCV amplification were carried out in duplicate and during different days. Screw-capped Eppendorf tube were used to reduce the likelihood of cross contamination through aerosol formation.

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