Inform hpv 3 family 16 probe b

Automated in situ hybridizations were carried out on TMAs on a Discovery XT (VMS) instrument using Research ISH UltraMap XT procedure and Ventana products (INFORM HPV III Family 16 Probe (B), #800‐4295). For details, see Appendix S2. Each TMA slide contained HeLa cells as positive staining controls. Additionally, sections of a known HPV positive OPSCC were also used as positive controls. A no probe control containing only RiboHybe and RiboWash served as a negative control.
Blinded for the clinicopathological data, the HPV DNA ISH results were scored by TMS and DS. The in situ results were interpreted following the manufacturer's guidelines (Interpretation Guide for Ventana INFORM HPV Probes ISH). Any blue nuclear dots in the tumor cells were regarded as positive staining and all of the samples were classified binary as either positive or negative.

HR- HPV DNA in-situ hybridisation (HR-HPV ISH) was carried out using proprietary reagents (Inform HPV III Family 16 Probe (B), Ventana Medical Systems Inc., USA) on a Benchmark Autostainer (Ventana Medical Systems Inc., USA). The Inform HPV III Family 16 Probe (B) detects high risk genotypes HPV-16, − 18, − 31, − 33, − 35, − 39, − 45, − 51, − 52, − 56, − 58 and − 66. Three control samples were used: FFPE CaSki cells (HPV-16 positive; 200–400 copies per cell), HeLa cells (HPV-18 positive; 10–50 copies per cell) and C-33A (HPV negative; Ventana Medical Systems Inc., USA). The HR- HPV ISH test was scored as positive if there was any blue reaction product that co-localised with the malignant cells [21 (link)].

The INFORM HPVIII Family 16 Probe(B) which is capable of detecting 12 oncogenic HPV genotypes (Ventana Medical Systems) was used to stain cervical tissues. Untransfected and HPV18 genome transfected human keratinocytes grown as organotypic rafts were used as negative and positive controls8 (link), in addition to the HPV positive and negative xenograft tissues provided by Ventana. A blue reaction product, either in the form of diffuse nuclear and cytoplasmic or punctate nuclear staining, was accepted as positive hybridisation signals. Three pathologists independently reviewed the ISH slides (MP, RG and GN) and agreed on the results for all cases.
Eleven cases were excluded from this analysis; 6 because of high background staining in non-epithelial and endocervical cells and in the cytoplasm of squamous epithelia; and 5 because of significant squamous epithelial disruption; 88 evaluable cases remained. Two other ISH methods were evaluated during this study, GenPoint DAKO HPV DNA ISH assay, Abbott Vysis Cervical FISH HPV DNA ISH assay and Bond^TM DNA ISH assay; these were optimised on organotypic raft sections of untransfected and HPV18 genome transfected keratinocytes but discarded because of high background staining and low sensitivity after testing on archival FFPE cervical tissue sections.