Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

HPV RNA CISH allows the detection of an active human papilloma virus infection. Since the in-situ hybridization targets HPV RNA this technique allows the visualization of an active transcription of the virus. It has precise spatial resolution and good diagnosis performances.

The detection of HPV may support the diagnosis of several HPV-related lesions, such as oropharyngeal squamous cell carcinoma or cervical neoplasia. It may also has an influence on the prognosis. After preparing buffers and counter-staining reagents, prepare one x target retrieval reagent in a large beaker by adding 70 milliliters of 10x target retrieval reagent to 630 milliliters of distilled water.

Place the beaker on a heating plate with magnetic stirrer and cover it with aluminum foil. Boil its contents at 100 degrees Celsius for 10 to 15 minutes making sure not to boil it for longer than 30 minutes. To block peroxidase activity on slides with tissue sections previously deparaffinized and placed in the slide rack, add four to six drops, just enough to cover the sample, of hydrogen peroxide to each slide, and incubate them for 10 minutes at room temperature.

Wash the slides two times for two minutes in distilled water at room temperature. To break RNA tissue bounds in the RNA tissue sections, first remove the aluminum foil from the boiling one x TRR using a claw, and stop stirring. Immerse the slide rack slowly and very carefully.

Cover the beaker again with the aluminum foil, and incubate for 15 minutes. Use the claw to immediately transfer the hot slide rack to a distilled water bath and wash it for two minutes. Wash the slides then in fresh 100%ethanol for two minutes, and let them dry at room temperature for two minutes.

Next, use a hydrophobic barrier pen to draw a barrier around each sample, and let it dry out for at least five minutes. For protease digestion, place the slides in the humidity controlled tray, and add approximately four drops of Protease Plus per sample. Cover the tray with a lid, and insert it into the hybridization oven for 30 minutes at 40 degrees Celsius.

Remove the tray from the oven, and remove the slide rack. Working one slide at a time, quickly remove any excess liquid, and place the slide in the slide rack submerged in a staining dish filled with distilled water. Wash the slides two times for two minutes in distilled water at room temperature with constant agitation.

To start hybridization, tap and flick the slides to remove any excess liquid and place them back in the slide rack. Remove pre-warmed HPV probe from the oven, and add approximately four drops to entirely cover each section. Cover the tray with the lid, and insert it into the oven for two hours at 40 degrees Celsius.

After completing incubation, remove the tray from the oven and remove the slide rack. One slide at a time, quickly remove any excess liquid, and place the slide in the slide rack submerged in a staining dish filled with one x wash buffer, washing the slides for two minutes at room temperature with constant agitation, and repeat with fresh one x wash buffer. Tap and flick to remove any excess liquid from the slides, and place them in the slide rack again.

Add approximately four drops of room temperature AMP1 per section to entirely cover each section. Cover the tray with the lid, and insert it into the oven for 30 minutes at 40 degrees Celsius. After removing the tray from the oven, remove the slide rack.

Working one slide at a time, quickly remove any excess liquid, and place the slide in the slide rack submerged in a staining dish filled with one x wash buffer, washing for two minutes at room temperature with constant agitation, and repeat with fresh one x wash buffer. Dispense the same number of drops of each DAB-A and DAB-B solution in an appropriately sized tube to make approximately 120 microliters of DAB substrate per section, and vortex. Take each slide, one at a time, from the slide rack, and tap and flick to remove excess liquid, and place it back in the slide rack.

Pipette approximately 120 microliters of DAB mixture onto each tissue section, making sure that the sections are covered. Incubate for 10 minutes at room temperature, and proceed with counterstaining. Place one drop of mounting medium per glass slat, and then place the slats on the slides and let them air-dry.

After a few hours, proceed with evaluation using an optical microscope as described in the manuscript. As described here in head and neck squamous cell carcinoma, presence of brown punctiform staining in the cytoplasm or in the nuclei of tumor cells defined a positive case. The results were divided into two scores, RNA CISH high and low score as observed with a 20x objective.

RNA CISH score staining is considered low when observed in under 50%of the tumor cells and covers less than 80%of the cell surface. On the other hand, RNA CISH score is high when observed in more than 50%of the stained cancer cells, or with the staining surface exceeding 80%in more than 30%of the tumor cells. One should never let the slides dry out for hybridization.

Do not forget to preheat the probe. For each sample, perform positive and negative control in order to assess the quality of the RNA.