A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears

Br J Cancer. 2000 Apr;82(8):1421-6. doi: 10.1054/bjoc.1999.1128.


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.

MeSH terms

  • Cervix Uteri / cytology
  • Cervix Uteri / pathology*
  • Cervix Uteri / virology*
  • DNA, Viral / analysis
  • Disinfectants
  • Female
  • Genome, Viral
  • Guanidines
  • Humans
  • Pancreatitis-Associated Proteins
  • Papanicolaou Test*
  • Papillomaviridae / genetics
  • Papillomaviridae / isolation & purification*
  • Polymerase Chain Reaction / methods
  • Retrospective Studies
  • Specimen Handling
  • Thiocyanates
  • Time Factors
  • Uterine Cervical Neoplasms / pathology
  • Uterine Cervical Neoplasms / virology*
  • Vaginal Smears*


  • DNA, Viral
  • Disinfectants
  • Guanidines
  • Pancreatitis-Associated Proteins
  • REG3A protein, human
  • Thiocyanates
  • guanidine thiocyanate