SENSITIVE CHEMILUMINESCENCE IN-SITU HYBRIDIZATION FOR THE DETECTION OF HUMAN PAPILLOMAVIRUS GENOMES IN BIOPSY SPECIMENS

We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and sem iquantitative evaluation of the results. The hybridization reaction wa s performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent sub strate. The light emitted from the hybridized probes was detected, ana lyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal compu ter for quantification of the photon fluxes and for image analysis. Th e system operated in consecutive steps: First, hybridized specimens we re recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transm itted light and by the luminescent signal allowed the spatial distribu tion of the target DNA to be localized, measured, and evaluated. Biops y specimens from different pathological conditions associated with HPV , which had previously been proved positive for HPV DNA with the polym erase chain reaction (PCR), were analysed. The chemiluminescence in si tu hybridization proved sensitive and specific with digoxigenin-, biot in-, or fluorescein-labeled probes, and provided an objective evaluati on of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ h ybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the po ssibility of detecting HPV DNA with great sensitivity in biopsy specim ens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.