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

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.