Detection of Human Papilloma Virus by means of Chromogen in situ hybridization (CISH)

Abstract

Introduction. The human papillomavirus (HPV) can cause infections in humans, although most women will be infected with HPV at some time in their life only a few cases will progress into invasive disease. Of all the HPV types only the ‘high-risk’ viruses are associated with cervical carcinomas. In order to prevent cervical cancer, multiple countries have implemented a cervical cancer screening programme. The detection mechanism in these screening programmes are commonly based on the Papanicolaou test, colposcopic analysis and PCR. However the sensitivity of such tests have several limitations. Another detection tool is HPV in situ hybridization either with fluorescence (FISH) or with an enzyme reaction (CISH). The use of FISH ensures high sensitivity with low endogenous background. Furthermore, this method will result in high resolution and the possibility to quantify the signal intensity. However, the fluorescent signal fades after light exposure, and samples might have auto-fluorescence. Thus in a setting where routine analysis has to be carried out the CISH procedure is more convenient, as only a standard bright-field microscope is needed for the detection procedure. The aim of this thesis is to determine if there are enzymatic substrates that have stronger reaction products in comparison to the control substrate DAB. Secondly this thesis aims to determine which conjugate detection system results in the best localisation and least background. Finally, the main aim is to investigate the possibilities of developing such an CISH that the end staining is comparable or better than the detection with FISH. Materials & Methods. Seven horseradish peroxidase substrates and six alkaline phosphatase substrates have been tested on CasKi cells with the centromere 1 and 1p36 probes as targets for the in situ hybridization reaction to determine the sensitivity and specificity of the substrates. The selected substrates were further tested with the HPV16 probe as target on SiHa cells and on formalin-fixed and paraffin-embedded (FFPE) tissue sections to determine the best conjugate detection system. The DAB DAKO substrate and the FISH with FITC were used as controls. The selected substrate in combination with the selected conjugate detection system was then tested on a series of eighteen clinical samples. Results. The horseradish peroxidase substrates Vina Green and Seramun Grün resulted in superior precipitates in comparison to the DAB DAKO control. These substrates show very high sensitivity as the spots visible for 1p36 were similar in size as the spots for 1C of the control staining. The substrates Vulcan Fast Red and Ferangi Blue are the selected substrates for the alkaline phosphatase enzyme reaction. Both these substrates have good localised sensitive reaction products with, respectively, clear red and blue precipitate products. The Av-PO system in combination with the Vina Green substrate lead to the best results in comparison to the fluorescent control. All clinical samples tested with the Av-PO system tested positive for HPV infection and had the same sensitivity and specificity as the fluorescent samples. Discussion and Conclussion. The Av-PO system results in the same specificity and sensitivity on clinical samples as the FISH. However, the ease of localizing the infected area when only the integrated pattern is present is much better with FISH. In conclusion we can say that the Vina Green substrate in combination with the Av-PO systems targeting HPV in clinical samples gives similar results as FISH. For this reason we developed a CISH method which could be used in the routine setting for histopathological diagnosis.