Original Paper Vox Sang 1992;63:198-203

H . De Beenhouwer H. Verhaert H . Clacys C. Verrnylen Belgian Centre, Red Leuven, Cross Belgium Blood Transfusion -

Confirmation of Hepatitis C Virus Positive Blood Donors by lmmunoblotting and Polymerase Chain Reaction

................................................................................................. Abstract In a series of 385 sera obtained from volunteer blood donors positive for the first-generation hepatitis C virus assay (Ortho), the viral genome was detected by polymerase chain reaction (PCR) in 89 sera (23%). Most PCR-positive sera were found positive with the c100-3 neutralisation assay (Abbott) and by two second-generation enzyme immunoassays (Abbott, Ortho). However overall specificity of these assays was rather low. By immunoblotting (Innogenetics and Chiron/Ortho) the specificity could be considerably improved and the best correlation with carrier state was obtained when analysing the results for lane-specific reaction: all 89 viral carriers and only 9 other donors had antibodies against structural ‘core’ epitopes. From the present data we can conclude that in screening a volunteer blood donor population the confirmation of antibodies against ‘core’ epitopes by immunoblotting is strongly associated with viral carriage.

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Introduction

Following the discovery by Choo et al. [I] of a cDNA clone reactive in a high percentage of chronic non-A, non-B hepatitis (NANBH) patients the genome of an etiological agent was identified and is now called hepatitis C virus (HCV). Commercial enzyme immunoassays (EIAs) based on a recombinant, non-structural protein of the virus (c100-3) have allowed large-scale testing for HCV antibodies. However, low specificity and sensitivity are the major drawbacks [2-4].

Received: Nov. 13, 1991 Revised nianuscript received: January 20. 1992 Accepted: February 4, 1992

Since August 1990 HCV testing of blood and plasma donors has been compulsory in Belgium and positive units are withdrawn. This implies a loss of about 0.6% of the donor blood. For these donors as well as for the transfusion centres confirmation is needed urgently. The purpose of this study was to develop a confirmatory strategy for reactive sera and, because of the importance in transfusion medicine, to define a serological pattern correlated with the HCV carrier state. During 3 months 400 samples from 13 collaborating blood transfusion centres were received. We investigated the samples with the polymerase chain reaction (PCR) and with 5 dif-

Dr. Hans De Beenhouwer Institute of Tropical Medicine Virology Natioiialc btrnat 15s B-2NX) Antwerpen (Belgium) ~

I Y Y ? S . Karger AG. Basel ~10-12~00lJ7/‘~2/1J~h.13-(1 I9H

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ferent serological tests: a c100-3 neutralisation EIA (Abbott Diagnostics), two second-generation EIAs (Ortho Diagnostics and Abbott Diagostics), a four-antigen recombinant immunoblot assay (4-RIBA: Chiron/Ortlio Diagnostics), and a synthetic peptide immunoblot assay (INNO-LIA: Innogenetics).

Materials and Methods Sarnple.~ All sera used for this study were obtained from volunteer blood donors found positive in the screening with the Ortho c100-3 EIA (first generation). Aliquotsfrom the co-operating centres were mostly sent by post without special conservatory precautions. On arrival all samples were stored at -20°C until PCR and serological testing were performed. Serological Tests Neutralisation Assay (Ahhott). The neutralisation assay is an EIA basedon thec100-3 antigen, originatingfrom the fourthnon-structural region (NS4) of the viral polyprotein, executed with and without neutralisation diluent. Result of the plain EIA (without neutralisation) is expressed as S/CO ratio (optical density of the sample/optical density of the cut-off). When, after neutralisation, a signal reduction of at least 50% is observed the sample is interpreted positive. Second-Generation EIA (Ortlio and Abbott). The second-generation assays contain the c100-3antigen supplemented with recombinant HCV structural (c22-3) and non-structural (from NS3: c33) antigens. Results are expressed as S/CO ratios. 4-RIBA (ChironlOrtlio). A recombinant immunoblot assay (5 lanes) withHCVstructural (c22-3) and non-structural (c33: c100-3: 5-1-1)antigens and the fusion protein (SOD) control (5-1-1is a smaller part of c100-3). The intensity of the colour reaction is compared with IgG standards and scaled from I f to 4 + . A signal of at least I f in at least 2 HCV antigen lanes ist required for a specimen to be 'reactive' (R). Reaction in only one lane or in the SOD lane is interpreted 'indeterminate' (ID). INNO-LIA (Innogenerics). A synthetic peptide immunoblot (6 lanes) representingepitopes from non-structural(2 lanes with several peptides of the NS4 and NSS region, respectively) and structural (4 lanes with different 'core'epitopes) gene regions. The colour reaction is compared with IgG standards and scaled from I+ to 4f.A signal of at least 2+ in one lane or I + in two lanes is required for a specimen to be R. Lower reactivity is considered ID. All assays were performed according to the manufacturer's instructions. Polymerase C h i n Renction Samples were prepared for PCR essentially as described [S]. In short: 100-200 p1serum was diluted to 1 ml with sterile distilled water. 500 yl polyethylene glycol (30% polyethylene glycol 8000 in 0.1 MNaCI) was added, the tubes were gently mixed and cooled on ice for 30 min. Viral particles were pelleted by centrifugation (15,000 g ) for 12 min. The pellet was resuspended by vortexing in 400 1.11 TE (10 mMTris-HCI, pH 8.0, I mM EDTA); 10 1.11of 10% sodium dodecyl sulphate was added immediately followed by two extractions with phenol-chloroform andone with chloroform. The aqueousphase was

supplemented with 20 kg glycogen and 0.1 M Na acetate. RNA was precipitated with 1 ml ice-cold absolute ethanol for 12 h at -70"C, pelletedbycentrifugation (15,000g,4"C,15 min), washedwith500 p1 ethanol, air-dried and dissolved in 10 PI diethyl pyrocarbonate-treated water. Reverse transcription (RT) was performed on 2 1.11 RNA in 20 pl RTmix (SO mM Tris-HCI, pH 8.3, 75 mM KCI, 3 mM MgC12. 10 mM DTT) with 0.5 mM of each dNTP (Boehringer). SO p M random primers (Boehringer), 12 U RNAse inhibitor (Pharmacia) and 200 U Moloney murine leukaemia virus reverse transcriptase (BRL). This mixture was incubated for 60 min at 37"C, the cDNA heated for 1 min to 100°C and quickly chilled on ice. PCR was done on 5 pl cDNA in 45 PI PCRmix (10 mM Tris-HCI, pH8.3,50 mMKC1,2.0 mMMgC12,0.01w/v% gelatine)with150 pM of each dNTP and 0.75 U Taq-polymerase (Boehringer). In the first round 35 cycles were performed (95°C: 30 s/46"C: 60 s/72"C: 60 s) with 10 pmol primers PI3 and Pt6. The second round with 25 cycles (95°C:30 s/48"C:45 s/72"C: 60 s)wasdoneunderidenticalconditions but with 50 pmol primers PI4 and PI5 and 1 kl of the first round product. Primers were designed on the basis of the sequence of the 5' non-coding region of the virus [6]. The sequence of the outer primers (PI3 and P16) was CCATAGATCACTCCCCTGT and GTGCACGGTCTACGAGACCT, and of the inner primers (P14 and P15) CGAAGCTTCACGCAGAAAGCGT and CAAGCACCCTATCAGGCAGT (cloning site = underlined). First and second round products were analysed by electrophoresis on a 1.5% agarose, stained by ethidium bromide andvisualised by UV transillumination. A fragment of 310 bp for the first round and of 251 bp for the second round was expected. Every run included a weak (dilution 1/10,000) positive control plasma, a PCR reaction control and a negative control plasma. All precautions were taken to avoid contamination [7,8]. Samples were processed coded and the sera, only positive in the second round, were repeated.

Statistics Descriptive statistics will be reported for the EIA ratios using median, 25Ih and 75"' percentiles. Due to the upper limit for the optical density values the Wilcoxon test for censored values was used to compare the serological groups statistically. For all calculations the statistical package SAS (SAS/STAT, SAS Institute Inc.. N.C.) was used.

ResuIts

From a total of 400 first-generation R samples received between August 1 and October 31,1990,389 were repeatedly R and 385 had sufficient volume for further testing.

Polymerase Chain Reaction To evaluate the primers and the general set-up of the PCR procedure 18 stored sera from untreated chronic NANBH patients and 18 negative control samples were processed. In the 18 patient sera PCR was positive in 17 (of which 15 already showed up after the first round). All the 18 controls remained negative.

199

When investigating the 385 sera of the donor population, only 89 (23.1%) were positive in the PCR assay. Here 71 were already clearly positive after the first round. No correlation was found in the PCR-positive samples between the appearance in the first or second round and the EIA ratio. ‘Nested priming’ designed to enhance sensitivity was in most cases only required to confirm the specificity of the result.

Neutralisation Assay In a first attempt to confirm the positive EIA (Ortho) results the neutralisation assay (Abbott) was selected. 303 samples (78.7%) were positive in the plain Abbott EIA and of these 168 (55.4%) could be neutralised (fig. 1). This last group of 168 sera contained 87 of the 89 PCRpositive (97.7%) specimens. One PCR-positive sample was not neutralisable and one was negative for the Abbott EIA. Thus considering the ‘neutralisable’ samples 51.8% were found positive by PCR. Second-Generation E I A When second-generation assays became available we retested all 385 samples to compare first- and second-generation EIA. The results are presented in figure 2. In the Ortho assay 284 (73.8%) samples and in the Abbott assay 182 (47.3%) samples remained positive. Median ratio ortho: 3.455 (25 t” percentile: 1,877/75t” percentile: 4.184); median ratio Abbott: 4.950 (1.432/4.950). These results allow to discriminate three groups: *A: A group of 98 (25.5%), negative in both assays. *B: A group of 108 (28.1%), positive in only one assay. Median ratio 105 Ortho single positives: 1.850 (1.362/ 2.522); median ratio 3 Abbott single positives: 1.562 (1.052/2.782). *C: A group of 179 (46.5%), positive in both assays, median ratio Ortho: 3.781(3.290/4.385);median ratio Abbott: 4.950 (1.432/4.950). Within this group there was a difference between the 89 PCR-positive [median ratio Ortho: 4.184 (3.727/4.385) and Abbott: 4.950 (4.950/4.950)] and the 90 PCR-negative sera [median ratio Ortho: 3.410 (2.0727/4.010); median ratio Abbott: 1.437 (1.198/2.072)] which was statistically significant (pgo%) confirmed samples HCV genome is detectable.

Acknowledgements We want to thank Ortho Diagnostics, Abbott Diagnostics and Innogenetics for supplying test kits. Primers were kindly synthesised by Prof. F. Van Leuven (K.U. Leuven-Genetics). Statistical advice was given by Prof. M. Lesaffre (K.U. Leuven). We are grateful to Prof. J. Desmyter and Dr. P. Goubeau (K.U. Leuven -Virology) for critical reading, to Mrs. W. Mertens, E. Noe, A. Volckaerts and G. Wuyts for technical support and to Dr. K. Gautarna for data rnanagement.

De BeenhouwerNerhaertlClaeysNermylen

HCV Confirmation

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Confirmation of hepatitis C virus positive blood donors by immunoblotting and polymerase chain reaction.

In a series of 385 sera obtained from volunteer blood donors positive for the first-generation hepatitis C virus assay (Ortho), the viral genome was d...
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