title: A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears.
abstract: The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome equivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3-6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.
authors.affiliation: Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: M V
authors.initials: MV
authors.lastname: Jacobs
authors.forename: D
authors.initials: D
authors.lastname: Zielinski
authors.forename: C J
authors.initials: CJ
authors.lastname: Meijer
authors.forename: R P
authors.initials: RP
authors.lastname: Pol
authors.forename: F J
authors.initials: FJ
authors.lastname: Voorhorst
authors.forename: F A
authors.initials: FA
authors.lastname: de Schipper
authors.forename: A P
authors.initials: AP
authors.lastname: Runsink
authors.forename: P J
authors.initials: PJ
authors.lastname: Snijders
authors.forename: J M
authors.initials: JM
authors.lastname: Walboomers
mesh_terms: D002584:Cervix Uteri / Q000166:cytology / Q000473:pathology* / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D004202:Disinfectants; D005260:Female; D016679:Genome, Viral; D006146:Guanidines; D006801:Humans; D000073718:Pancreatitis-Associated Proteins; D065006:Papanicolaou Test; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012189:Retrospective Studies; D013048:Specimen Handling; D013861:Thiocyanates; D013997:Time Factors; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D014626:Vaginal Smears
chemical_list: D004279:DNA, Viral; D004202:Disinfectants; D006146:Guanidines; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D013861:Thiocyanates; C054436:guanidine thiocyanate