Ventana ish iview blue plus detection kit

For the detection of HPV-DNA in FFPE-tissue-slides by in situ hybridization (ISH), the Ventana INFORM^® HPV III Family 16 probe (Roche Diagnostics GmbH, Mannheim, Germany) was used according to the manufacturer’s instructions targeting the common high-risk HPV genotypes found to be associated with human malignancies including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. For the visualization of labeled DNA, the Ventana ISH iView Blue Plus detection kit (Roche Diagnostics GmbH, Mannheim, Germany) was used according to the manufacturer’s instructions. HPV-DNA-ISH was performed for all 153 HNSCC patients. HPV-DNA-ISH was evaluated by an experienced pathologist and rates as positive when at least 50% of the lesional cells showed distinct nuclear and/or cytoplasmic signals.

Detection of EBER1 by ISH was used to determine the presence of EBV allowing sample selection. Briefly, the formalin-fixed paraffin-embedded (FFPE) tissue sections were deparaffinized, rehydrated in a graded solution of xylene and alcohol, then deproteinized with proteinase K before incubation with the Ventana EBER 1 DNP Probe^® (Roche Diagnostics, Meylan, France; catalog number 760-1209) on the Benchmark XT automaton^TM (Roche Diagnostics). This was followed by staining with Ventana ISH iVIEW blue plus detection kit^® (Roche Diagnostics; catalog number 760-097) on the same apparatus.
For EBV positive samples, further work was performed on frozen material collected at the same time as the FFPE tissue.