Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squamous cell carcinomas of the cervix

Abstract

The sensitivity of human papilloma virus type 16 (HPV-16) DNA detection by DNA in situ hybridization using biotinylated probes (bio-DISH) was estimated by performing this technique on snap-frozen tissue sections of 10 cervical squamous cell carcinomas containing increasing amounts of HPV-16 as determined by Southern blot hybridization. A protocol using serial sections for bio-DISH and DNA extraction was used. The number of positively stained cells and the detection limit were strongly dependent on the treatment of the sections with proteinase K prior to hybridization. At low proteinase K concentration (0.1 micrograms/ml), the detection limit appeared to be 30-40 HPV-16 DNA copies per carcinoma cell, whereas morphology was preserved. A high proteinase K concentration (1-5 micrograms/ml) often resulted in an increase in the number of positively stained cells but also in a poor morphology. The detection limit was improved to at least 20 HPV-16 DNA copies per carcinoma cell.