Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction.
The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp.
de Roda Husman
A M
AM
Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.
Snijders
P J
PJ
Stel
H V
HV
van den Brule
A J
AJ
Meijer
C J
CJ
Walboomers
J M
JM
D001483:Base Sequence; D001609:Beta-Globulins; D001706:Biopsy; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D019286:Endopeptidase K; D005069:Evaluation Studies as Topic; D005240:Feasibility Studies; D005260:Female; D005615:Freezing; D005838:Genotype; D006146:Guanidines; D006801:Humans; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D065006:Papanicolaou Test; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011136:Polysorbates; D012697:Serine Endopeptidases; D012822:Silicon Dioxide; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D016707:Tissue Fixation; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology; D014626:Vaginal Smears
D001609:Beta-Globulins; D004279:DNA, Viral; D006146:Guanidines; D017879:Isothiocyanates; D011136:Polysorbates; C054435:guanidine isothiocyanate; D012822:Silicon Dioxide; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K