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We report a method of in situ hybridization (ISH) of 10-year-old archival cervical smears with a cocktail of nick-translated human papillomavirus (HPV) DNA types 6, 11, 16, 18, and 31. The method, which does not require destaining, results in excellent preservation of morphological detail with only 2% cell loss. Methods of smear treatment and detection of the biotinylated probe with a multistep avidin-biotin-immunoperoxidase method are described. Biotinylated PBR 322 plasmid and biotinylated human DNA were used as negative and positive controls in each run. Twenty-nine of 50 smears (58%) showing changes consistent with CIN I-II were positive for HPV. Fourteen corresponding cervical biopsies were also studied by ISH, seven corresponding to HPV-positive smears and seven to HPV-negative smears. HPV DNA was demonstrated in six of seven biopsies (87%) from the positive group but none could be demonstrated in the negative group. We conclude that retrospective study can be performed on routine alcohol-fixed, Papanicolaou-stained cervical smears with biotinylated HPV probes with excellent cell preservation, minimal cell loss, and high degrees of specificity.

In situ hybridization with human papillomavirus using biotinylated DNA probes on archival cervical smears.