J Clin Pathol 1991;44:676-680

Detection of Epstein-Barr virus genomes in AIDSrelated lymphomas: Sensitivity and specificity of insitu hybridisation compared with Southernblotting

S J Hamilton-Dutoit, H J Delecluse, M Raphael, G Lenoir, G Pallesen

AbstractEighteen cases of AIDS related, non-Hodgkin's lymphomas were examinedfor the presence of Epstein-Barr virus(EBV) genomes using in situ hybridisa-tion with a "S-labelled probe. Theresults were compared with those ob-tained independently by Southern blotanalysis with a 32P-labelled probe offrozen tissue from the same tumours.Technically satisfactory results wereobtained with both methods in 15 lym-phomas. EBV DNA was detected in sevenof 15 (47%) cases by in situ hybridisationand in eight of 15 (53%) cases by South-ern blotting (including all the casespositive by in situ hybridisation). Theresults ofEBV DNA detection by the twotechniques were identical in 14 of 15(93%) cases. In situ hybridisation gaveno false positive results.This study shows that the sensitivity

and specificity ofin situ hybridisation forthe detection of EBV genomes in AIDSrelated lymphomas approaches that ofSouthern blotting, even when usingroutinely processed archival, paraffin waxembedded material.

Laboratory ofImmunohistology,University Institute ofPathology, Aarhus,DenmarkS J Hamilton-DutoitG PallesenInternational Agencyfor Research onCancer, Lyon, FranceH J DelecluseG LeniorDepartment ofHaematology, PitikSalpetriare Hospital,Paris, FranceM RaphaelCorrespondence to:Dr Stephen Hamilton-Dutoit, Laboratory ofImmunohistology,University Institute ofPathology, Finsensgade 12,DK-8000 Aarhus C,Denmark

Accepted for publication20 March 1991

Epstein-Barr virus (EBV) has been implicatedin the pathogenesis of a range of benign andmalignant lymphoproliferative and epitheliallesions.'-" Traditionally, the virus has beenidentified in tissues either by the use ofimmunocytochemistry to show the presence ofEBV specific antigens in cytological prepara-tions, or by the detection of EBV genomes inextracts of frozen tumour by filter-basednucleic acid hybridisation. Both these tech-niques require fresh or frozen tissue and thishas limited the possibilities available for thestudy of EBV associated lesions in tissue sec-

tions.Recently, methods have been described for

showing the presence of EBV genomes inroutinely processed paraffin wax embeddedmaterial by in situ hybridisation.7"'- Thistechnique is potentially of great value becauseit allows retrospective studies to be carried outon archival pathological material, and itprovides information on the nature and dis-tribution of cells infected with EBV which isnot available with other methods. We havepreviously shown the value of this techniquefor studying both epithelial and lympho-

proliferative EBV associated lesions." 12 16 Inparticular, we have shown that about a half ofAIDS related lymphomas contain EBVgenomes, as detected by paraffin wax sectionin situ hybridisation with radiolabelledprobes. 17-1"While previous investigations have shown

the potential of paraffin wax section in situhybridisation, there have been no systematicstudies to assess the specificity and sensitivityof this technique for showing the presence ofEBV genomes compared with establishedmethods using filter hybridisation. Previousstudies of EBV genome positive, AIDS re-lated lymphomas have found a range of viralcopy numbers comparable with that found inother types of EBV associated lympho-proliferation. AIDS related lymphomasprovide, therefore, a good model for testingthe sensitivity of paraffin wax section in situhybridisation when applied to lympho-proliferative disorders induced by EBV.

MethodsThe tumours were selected from a series ofover100 cases of non-Hodgkin's lymphomas frompatients seropositive for human immuno-deficiency virus (HIV), collected by theFrench-Danish Study Group on the pathologyof AIDS related lymphomas. Criteria for in-clusion were the availability of both routinelyprocessed, paraffin wax embedded material andsnap frozen tumour tissue. These criteria weremet in 18 cases. All were surgical biopsyspecimens of non-Hodgkin's lymphomas,selected from the archives of several differentinstitutions participating in the study. Tissuewas routinely fixed and processed before paraf-fin wax embedding, although the procedureswere not standardised. The fixative used in eachcase is shown in the table, although precisedetails of the protocol used were not available.Paraffin wax blocks had been stored for up tothree years. Cases were classified according tothe updated Kiel classification,20 with modifica-tions as reported previously.'9The lymphomas were examined in separate

laboratories for the presence of EBV DNAusing both in situ hybridisation on paraffin waxsections and Southem blotting of snap frozentissues. All studies were performed blind with-out knowledge of the results obtained in theother laboratory, and the findings were com-pared once all investigations had been com-pleted.

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Detection of EBVgenome in AIDS related lymphomas

Histological type and results ofEBVDNA detection by Southern blotting and paraffin wax section in situhybridisation

Epstein-Barr virus DNA

Case Site of Lymphoma Fiaxtive Southern In situNo lymphoma histology used blotting hybridisation

1 Lymph node Burkitt NBF + +2 Lymph node Burkitt NBF + +3 Lymph node Immunoblastic (PC) NBF + +4 Lung Immunoblastic (PB) AAF + +5 Palate Immunoblastic (PB) AAF + +6 Lymph node Immunoblastic (PB) NBF + +7 Gum Immunoblastic (PB) AAF + +8 Lymph node Burkitt NBF +9 Lymph node Burkitt NBF10 Bone marrow Burkitt NBF11 Lymph node Burkitt NBF12 Muscle Burkitt NBF NS13 Lymph node Centroblastic NBF -

14 Lymph node Centroblastic AAF -

15 Lymph node Centroblastic NBF -

16 Lymph node Immunoblastic (PB) NBF -

17 Gum Immunoblastic (PB) AAF + NS18 Gum Immunoblastic (PB) AAF + NS

The results ofEBV DNA detection using in situ hybridisation and Southern blotting were in agreement in 14 of the 15 lymphomaswhich gave technically satisfactory analyses.AAF = acetic acid, alcohol, formaldehyde (Lillie's AAF).NBF = neutral buffered formalin.NS = not satisfactory.PB = plasmablastic differentiation.PC = plasmacytic differentiation.

PROBESEBV DNA was detected using EBV BamHIWprobes specific for the viral internal repeats. Forin situ hybridisation total plasmid DNA was

labelled with "S-dCTP by nick translation2' toa specific activity of at least 1 x 109 dpm/pg.For Southern blot analysis, probes werelabelled with 32P-dCTP by the random primermethod.22For in situ hybridisation, probes for CMV

(EcoRI J fragment of human cytomegalovirusand plasmid pBR322 (without the EBV insert)were used as negative controls.

IN-SITU HYBRIDISATIONISH was performed on routinely fixed andprocessed, paraffin wax embedded tumour tis-sue as described previously. 12 9Briefly, sectionswere cut onto slides coated with 3-amino-propyltriethoxysilane (Merck; Darmstadt,Germany) to prevent section loss,23 baked over-

night at 65°C, and dewaxed in xylene. Slideswere incubated sequentially at room tem-perature in 0-2 N HCI (10 minutes) and 0 01%Triton X-100 (90 seconds) with interveningwashes in 2 x SSC (0 3 M sodium chloride/0 03 M sodium citrate, pH 7 6). Sections were

digested in pronase (1 mg/ml; BoehringerMannheim, Germany) for 10 minutes at 37°C,washed in 2 x SSC, acetylated with 0.1 Mtriethanolamine/0-25% acetic anhydride for 10minutes, and dehydrated. Hybridisation mix-ture (40 ng/ml "S-labelled probe, 0-25 mg/mlsalmon sperm DNA, 50% deionised forma-mide, 2 x SSC, 10% dextran sulphate, and50 mM dithiothreitol) was applied under sili-conised coverslips, and probe and tissue DNAwere denatured simultaneously on a 90°C heat-ing block for six minutes. After overnighthybridisation at 37°C coverslips were removedin 50% formamide/0-1 x SSC at 370C andthen washed, first in 50% formamide/0-1 x

SSC, for four hours at 37°C, and then in 2 x

SSC and 01 x SSC, each for 30 minutes atroom temperature. After dehydration in

graded alcohols containing 0 3 M ammoniumacetate slides were dipped in Ilford G5 emul-sion (with 0-3 M ammonium acetate), exposedat 4°C for between two and eight days,developed in Kodak D19, fixed with Kodakrapid fixer, and counterstained with haema-toxylin and eosin.

Cells were scored positive for viral DNA ifthey showed deposition of grains clearly inexcess of the background level seen over ran-dom cells, and if hybridisation with the controlprobes was negative.Controls: In all in situ hybridisationexperiments the "S-labelled CMV andpBR322 probes were used on adjacent sectionsas negative probe controls. Sections of cells ortissues known to contain EBV-that is, oralhairy leucoplakia, acute infectious mono-nucleosis in tonsils, post transplantation lym-phoma, or pellets of the EBV replicativelyinfected cell line P3HR1-were included ineach batch of experiments as positive tissuecontrols. Non-hybridised sections and plainglass objective slides were included in eachautoradiography batch as controls of back-ground levels of signal. All slides wereevaluated independently by two observers(SHD and GP). In cases of discrepancy, thecase was re-examined by both observers and aconsensus diagnosis reached.

SOUTHERN BLOT ANALYSISTotal cellular and viral DNA was extractedfrom tumour samples (snap frozen after sur-gical removal) using standard procedures.24Briefly, cell lysis and proteinaseK digestion wasfollowed by phenol/chloroform extraction andethanol precipitation. Ten pg of DNA wasdigested with the restriction endonuclease BamHI (Bethesda Research Laboratories; Gothers-burg, MD, USA) according to the supplier'srecommendations. Digested DNA was size-fractionated by electrophoresis on 0-7%agarose gels in TRI S-borate buffer, hydrolysedfor 15--minutes in 0-25 N HCI, denatured for 30

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Hamilton-Dutoit, Delecluse, Raphael, Lenoir, Pallesen

Figure I Partialinvolvement of a lymphnode by Burkitt's typelymphoma in a patientwith AIDS (case 1).Paraffin wax section in situhybridisation with a 3S-labelled EBVBamHI Wprobe. (A) Infiltratinglymphoma contains EBVDNA in every cell asshown by heavy depositionof silver grains. Residualstroma (lower part offield) shows onlybackground signal. (B) Athigh power all identifiabletumour cells are heavilylabelled (haematoxylinand eosin).

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minutes before being transferred to nylonmembrane (Hybond N, Amersham) accordingto the method of Southern.25 Filters werehybridised with "2P-labelled probe, as recom-mended by the manufacturer, and then washedat 60'C for 30 minutes each in 0-1% SDS/2 xSSC and 0-1% SDS/0-2 x SSC. Hybridisedprobe was detected by autoradiography for oneto three days at - 70'C using intensifyingscreens (Quanta III, Dupont; Wilmington,DE, USA).Controls: Positive and negative controls forSouthern blotting consisted ofDNA extractedfrom Raji and BJAB cell lines, respectively.*X4' *

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Figure 2 A polymorphic immunoblastic lymphoma with plasmablastic differentiation inan AIDS patient (case 4). Paraffin wax section in situ hybridisation with a 'S-labelledEBV BanHI W probe shows the presence ofEBVgenome. The hybridisation pattern ismuch more heterogenous than that seen in fig 1, with scattered tumour blasts showingheavy autoradiographic labelling (haematoxylin and eosin).

ResultsIn two out of the 18 cases of AIDS relatedlymphomas in situ hybridisation was tech-nically unsatisfactory because of high back-ground signal in both test and control slides. Ina third case Southern blot analysis could not beperformed because of the poor yield of DNAextracted from the frozen block. Thus tech-nically satisfactory results were obtained withboth techniques in 15 cases (table).

In situ hybridisation identified EBVgenomesin tumour cells of seven of 15 (47%) cases. Aspreviously reported,'9 there was considerablevariation in the number and distribution ofEBV positive cells both among cases (figs 1and 2), and from field to field within thesame tumour. Positive control tissues showed ahybridisation pattern identical with that repor-ted previously.'2 16 No signal over backgroundwas detected in any of the EBV positive caseswhen the CMV and pBR322 probes were sub-

3-2 KB-

Figure 3 Southern blot analysis of nine cases ofAIDSrelated lymphoma (lanes 3-11). The DNA in each lanewas digested with BamHI and hybridised with theBamHI Wprobe. Five tumours (lanes 4,8-11) show apositive 3-2 kilobase pair band corresponding to theBamHI Wfragment ofEBV. Lanes I (Raji) and 2

(BJAB) contain positive and negative control DNA,respectively.

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Detection ofEBVgenome in AIDS related lymphomas

stituted as negative controls for the EBV probe.EBVDNA was detected in eight of 15 (53%)

tumours by Southern blotting, including allcases positive by in situ hybridisation. Inpositive cases a 3-2 kilobase pair DNA frag-ment could be shown by hybridisation of theBamHI W probes to BamHI digests (fig 3).This band was also found in control DNAknown to contain EBV genomes, and corres-ponds to the reported size of the BamHI Wfragment of isolated EBV DNA. EBV genomeswere identified in one lymphoma (case 8) bySouthern blot analysis, but not by in situhybridisation. Thus the two techniques gaveidentical results in 14 out of the 15 (93%)technically satisfactory cases.

DiscussionTraditionally, Southern blot analysis of DNAextracted from frozen tissue has been used asthe "gold standard" when attempting to showthe presence of viral genomes in tumourssupposedly associated with EBV. The relativerarity of EBV associated lesions, however,combined with the need for frozen tissue foranalysis, have made it difficult to collect largeseries of such cases. Paraffin wax section in situhybridisation is, therefore, an attractive tech-nique as it permits retrospective studies onarchival material. Tissue structure is also main-tained during in situ hybridisation and this,combined with the superior morphologicaldetail of paraffin wax sections, allows viralsequences to be localised precisely within atissue. Several recent studies have confirmedthe value of paraffin wax section in situ hy-bridisation for EBV genome detection in bothepithelial and lymphoproliferative lesions.7 11-19Optimal conditions for performing EBV in

situ hybridisation have been studied in variousmodel experimental systems,2"29 but there havebeen no previous systematic investigations tocompare the sensitivity of paraffin wax sectionEBV in situ hybridisation with Southern blotanalysis in the same tumours. In our studythese two techniques have identical results forEBV detection in 14 of 15 (93%) cases ofAIDSrelated lymphomas. The EBV carriage ratesdetected by in situ hybridisation and Southernblotting (47% and 53%, respectively), how-ever, are similar to those found in a larger seriesof AIDS related lymphomas."9 This suggeststhat the tumours in the present study can betaken to be representative of AIDS relatedlymphomas in general.

Discrepancies between the results of in situhybridisation and Southern blot analysis mighthave been predicted in this study for severalreasons. In general, the minimum average copynumber per cell of a gene detectable by paraffinwax section in situ hybridisation will not be aslow as that which can be shown by Southernblotting of fresh tissue. This is attributable toseveral factors, including the possible degrada-tion of nucleic acids during fixation, thereduced access of probe to target DNA in fixedtissues, the restrictions placed on prehybridisa-tion treatments by the need to retain goodtissue morphology, and the introduction of

increased background noise by the use ofsensitive detection systems. This means thatoccasional lymphomas may exist in whichtumour cells contain so few EBV copies thatthey fall below the limit of detection by in situhybridisation, but they may still be identified bySouthern blot analysis. Case 8 in our study maybe an example of such a lymphoma.On the other hand, the pattern of viral

distribution in some tumours may mean thatEBV can be detected by in situ hybridisation,but not by Southern blotting. This might occurif a tumour contains only very few EBVpositive cells. The individual tumour cells maycontain sufficient viral copies to be identified insections by in situ hybridisation. WhenDNA isextracted for Southern blot analysis, however,these viral sequences may be so diluted byDNA from EBV negative cells as to be un-detectable.Another possible source of false negative

results when using in situ hybridisation isrelated to fixation of the tissues. It has beenshown that the sensitivity of in situ hybridisa-tion is strongly affected by both initial delay infixation and by the overall length offixation.3"32Furthermore, certain fixatives such as Bouin'sseem to be unsuitable if paraffin wax in situhybridisation is subsequently used, possiblybecause they cause high levels of DNA de-gradation.33 In our study fixation delay andoverall fixation times for the different cases werenot known. Standard prehybridisation proce-dures had, therefore, to be used, particularlywith regard to the conditions of protease treat-ment used to expose target DNA. The closeagreement of results obtained in this study byboth in situ hybridisation and Southern blot-ting indicates that in most cases this did notaffect the overall sensitivity of EBV detection.One lymphoma, which was negative by in situhybridisation, was found to contain EBV bySouthern blotting. Whether this false negativeresult was due to the tumour having a viral copynumber below the detection limits by in situhybridisation, or whether it reflected the use ofan unusual fixation protocol is not clear.We used 35S-labelled probes in this study as

in our experience these are more sensitive thannon-isotopic probes for the detection of lowabundance target sequences.'6 Other groupshave described the use of non-isotopic probesfor EBV genome detection in lymphomas byparaffin wax section in situ hybridisation.'415Separate studies will be required to assess .thesensitivity and specificity of these techniques.We have only examined the sensitivity of

paraffin wax section EBV in situ hybridisationwhen applied to AIDS related non-Hodgkin'slymphomas, but we believe these can be used asa model for lymphoproliferative disordersinduced by EBV in general. Our results showthat the sensitivity and specificity of paraffinwax section EBV in situ hybridisation using35S-labelled probes in these lesions approachesthat of Southern blot analysis of frozen tissueextracts. This technique can, therefore, be usednot only to provide information on the dis-tribution and nature of cells infected with EBVin tissues, but also to identify EBV genome

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positive tumours when fresh or frozen tissue isnot available to perform more traditionalanalyses.

This work was supported by the DanishMedical Research Council, the EEC(European Federation ofAIDS Research), andthe Lions Club of Denmark. Viral probes usedin this study were provided by Dr G WBornkamm, Freiburg, Germany (EBV) and DrB Fleckenstein, Erlangen, Germany (CMV).

AddendumAfter this paper had been accepted, we were

able to re-examine DNA from case 8 withSouthern blotting to estimate the EBV copy

number. The tumour contained an average ofless than two copies of EBV per cell comparedwith control cell lines, confirming that thefailure of in situ hybridisation to detect EBVDNA in this case could be attributed to the lowviral copy number present.

1 Epstein MA, Achong BG, Barr YM. Virus particles incultured lymphoblasts from Burkitt's lymphoma. Lancet1964;i:702-3.

2 Henle G, Henle W, Diehl V. Relation of Burkitt's tumor-associated herpes-type virus to infectious mononucleosis.Proc Natl Acad Sci USA 1968;59:94-101.

3 Saemundsen AK, Purtilo DT, Sakamoto K, et al. Documen-tation of Epstein-Barr virus infection in immunodeficientpatients with life-threatening lymphoproliferativediseases by Epstein-Barr virus complementary RNA/DNA and viral DNA/DNA hybridization. Cancer Res1981;41:4237-42.

4 Ho M, Miller G, Atchinson RW, et al. Epstein-Barr virusinfections and DNA hybridization studies in posttrans-plantation lymphoma and lymphoproliferative lesions: therole of primary infection. J Infect Dis 1985;152:876-86.

5 Ziegler J, Beckstead J, Volberding P, et al. Non-Hodgkin'slymphoma in 90 homosexual men. Relation to generalizedlymphadenopathy and the acquired immunodeficiencysyndrome. N Engl J Med 1984;311:565-70.

6 Weiss LM, Strickler JG, Warnke RA, Purtilo DT, Sklar J.Epstein-Barr viral DNA in tissues of Hodgkin's disease.Am J Pathol 1987;129:86-91.

7 Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection ofEpstein-Barr viral genomes in Reed-Sternberg cells ofHodgkin's disease. N Engl J Med 1989;320:502-6.

8 Harabuchi Y, Yamanaka N, Kataura A, et al. Epstein-Barrvirus in nasal T-cell lymphomas in patients with lethalmidline granuloma. Lancet 1990;335:128-30.

9 Greenspan JS, Greenspan D, Lennette ET, et al. Replica-tion ofEpstein-Barr virus within the epithelial cells oforal"hairy" leukoplakia, an AIDS-associated lesion. N Engl JMed 1985;313:1564-71.

10 zur Hausen H, Schulte-Holthausen H, Klein G, et al. EBVDNA in biopsies of Burkitt tumours and anaplasticcarcinomas of the nasopharynx. Nature 1970;228:1056-8.

11 Hamilton-Dutoit SJ. Therkildsen M, Nielsen NH, JensenH, Hansen JPH, Pallesen G. Undifferentiated carcinomaof the salivary gland in Greenlandic Eskimos: demonstra-tion of Epstein-Barr virus DNA by in situ hybridization.Hum Pathol (in press).

12 Niedobitek G, Hamilton-Dutoit S, Herbst H, et al. Iden-tification of Epstein-Barr virus infected cells in tonsils of

acute infectious mononucleosis by in-situ hybridization.Hum Pathol 1989;20:796-9.

13 Weiss LM, Movahed LA. In situ demonstration of Epstein-Barr viral genomes in viral-associated B cell lympho-proliferations. Am J Pathol 1989;134:651-9.

14 Bashir R, Harris N, Hochberg F, Singer R. Detection ofEBvirus in four CNS lymphomas by in situ hybridisation.Neurology 1989;39:813-17.

15 Murphy JK, Young LS, Bevan IS, et al. Demonstration ofEpstein-Barr virus in primary brain lymphoma by in situhybridisation in paraffin wax embedded tissue. J ClinPathol 1990;43:220-3.

16 Hamilton-Dutoit SJ, Pallesen G. Detection of Epstein-Barrvirus genomes in lymphoid and epithelial lesions using insitu nucleic acid hybridisation. In: Fenoglio-Preiser CM,Wolff M, Rilke F, eds. Progress in Pathology XII.Philadelphia: Field and Wood 1991:97-128.

17 Hamilton-Dutoit SJ, Pallesen G, Karkov J, Skinhoj P,Franzmann M, Pedersen C. Identification of EBV-DNAin tumour cells of AIDS-related lymphomas by in-situhybridisation. Lancet 1989;i:554-5.

18 Hamilton-Dutoit SJ, Karkov J, Franzmann MB, Pallesen G.AIDS-related central nervous system lymphoma.Demonstration of Epstein-Barr virus DNA by in situhybridisation. In: Racz P, Haase AT, Gluckman JC, eds.Modern pathology of AIDS and other retroviral infections,Basel: Karger, 1990:110-29.

19 Hamilton-Dutoit SJ, Pallesen G, Franzmann MB, et al.AIDS-related lymphoma. Histopathology, immuno-phenotype, and association with Epstein-Barr virus asdemonstrated by in situ hybridization. Am J Pathol1991;138:149-63.

20 Stansfeld AG, Diebold J, Kapanci Y, et al. Updated KielClassification for lymphomas. Lancet 1988;i:292.

21 Rigby P, Dieckmann M, Rhodes C, Berg P. Labellingdeoxyribonucleic acid to high specific activity in vitro bynick translation with DNA polymerase I. J Mol Biol1977;113:237-51.

22 Feinberg AP, Vogelstein B. A technique for radiolabellingDNA restriction endonuclease fragments to a high specificactivity. Analyt Biochem 1983;132:6-13.

23 Rentrop M, Knapp B, Winter H, Schweizer J. Aminoalkyl-silane-treated glass slides as support for in situ hybridiza-tion of keratin cDNAs to frozen tissue sections undervarying fixation and pretreatment conditions. Histochem J1986;18:271-6.

24 Sambrook J, Fritsch E, Maniatis T. Molecular cloning. Alaboratory manual. 2nd ed. New York: Cold SpringHarbor Press, 1989.

25 Southern EM. Detection of specific sequences among DNAfragments separated by gel electrophoresis. J Mol Biol1975;98:503-17.

26 Moar MH, Klein G. Detection of Epstein-Barr virus (EBV)DNA sequences using in situ hybridization. BiochimBiophys Acta 1978;519:49-64.

27 Lawrence JB, Villnave CA, Singer R. Sensitive, high-resolution chromatin and chromosome mapping in situ:Presence and orientation of two closely integrated copiesof EBV in a lymphoma line. Cell 1988;52:51-61.

28 Bashir R, Hochberg F, Singer RH. Detection of Epstein-Barr virus by in situ hybridization. Progress towardsdevelopment of a nonisotopic diagnostic test. Am J Pathol1989;135: 1035-44.

29 Teo CG, Griffin BE. Visualization of single copies of theEpstein-Barr virus genome by in situ hybridization.Analyt Biochem 1990;186:78-85.

30 Brigati DJ, Myerson D, Leary JJ, et al. Detection of viralgenomes in cultured cells and paraffin-embedded tissuesections using biotin-labeled hybridization probes.Virology 1983;126:32-50.

31 Haase A, Brahic M, Stowring L, Blum H. Detection of viralnucleic acids by in situ hybridization. IN: MaramoroschK, Koprowski H, eds. Methods in Virology, VII. NewYork: Academic Press, 1984:189-226.

32 Pringle JH, Homer CE, Warford A, Kendall CH, Lauder I.In situ hybridization: alkaline phosphatase visualization ofbiotinylated probes in cryostat and paraffin sections.Histochem J 1987;19:488-96.

33 Nuovo GJ, Silverstein SJ. Comparison of formalin, bufferedformalin, and Bouin's fixation on the detection of humanpapillomavirus deoxyribonucleic acid from genitallesions. Lab Invest 1988;59:720-4.

34 Nuovo GJ, Richart RM. Buffered formalin is the superiorfixative for the detection of HPV DNA by in situhybridization analysis. Am J Pathol 1989;134:837-42.

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