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 ex traction from fixed and Papanicolaou-stained cells from the cervical c ancer cell line Siha was measured by beta-globin polymerase chain reac tion (PCR). The GTC/silica beads method, which appeared superior, reve aled a human papillomavirus (HPV) general primer-mediated PCR sensitiv ity 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 st orage time. The longer the storage time, the more repetitions of the w hole procedure, including the lysis step, were required to extract suf ficient amplifiable DNA. In this way, an overall beta-globin PCR posit ivity for 98% of the smears was reached. Further analysis revealed tha t a maximum size of 200 bp could be amplified from smears stored for u p to 9 years. The method was validated by demonstrating by PCR the sam e HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method a ppears 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 maxi mum of about 200 bp.