Iscan coreo scanner

For visual point counting (VPC), photomicrographs of three randomly selected fields were taken at 40X with a background grid and color printed (more fields to achieve the minimal cell count). Each observer counted both the total number tumor cells and the number of Ki-67-positive cells that intersect with first grid line. This process is repeated on every third gridline. All the cells on the slide were counted if three fields could not be obtained however at least 200 total tumor cells were required. For Ki-67 image analysis of the CLIA clinical trial assay, slides were scanned with the iScan Coreo scanner (Ventana). The computer image was reviewed and “Areas of Interest” (AOI) were selected at 4X magnification using the following guidelines: (1) identify the largest AOI of representative clear invasive tumor; (2) exclude DCIS, vessels, lymphocytes; (3) avoid AOIs in peri-necrotic or necrotic areas; (4) identify at least 3 AOIs and a maximum of 10. The image analysis was performed using the FDA cleared VENTANA Companion Algorithm Ki-67 (30-9) and the VENTANA VIRTUOSO software (Roche).

Formalin-fixed and paraffin-embedded specimens were prepared for histological sectioning. Antigen recovery was performed on renal carcinoma specimens incubated with Tris–EDTA buffer (pH 8.4) at 99 °C for 60 min. Endogenous peroxidase activity was inactivated by incubating sections with a methanol and 3% H₂O₂ solution. Sections were incubated with the primary antibody for 60 min, secondary antibody for 8 min, and DAB developer for 8 min. All procedures were performed using the Ventana BenchMark XT automated stainer, and the sections were scanned using a Ventana iScanCoreo scanner. IHC results were quantified by experienced pathologists. The intensity was calculated according to the positive area and the degree of positive staining. The sections were stained with SIRT5 (1:100), PDHA1 (1:100), Succ-K351-PDHA1 (1:100) and Ki67 (1:100) antibodies using an ultraView Detection Kit.