Comparative p16^INK4A Expression in Laryngeal Carcinoma and Cervical Cancer Precursors: A Real-time Grid-based Immunocytochemistry Analysis

p16 analysis on tissue and cytological specimens. (a) A tissue section of LSCC with diffuse over-expression of p16. (b) Scattered cells with LSIL cytomorphology with p16 diffuse over-expression. (c) Implementation of a grid-based coverslip on a liquid fixed cytological slide. Arrows show the thin grey lines of the corresponding grid. Inside the red circle, a p16 over-expressing abnormal cell has been detected. (d) Schematic illustration of a spatial grid featuring the segmented and indexed cells as microscopy observation windows. (e) Application of a digital image analysis protocol for estimating p16 protein expression levels. Digitized snapshot of a microscopic field is analyzed by a macro focused on staining intensity values (0-255 at RGB). Red areas show different expression of the marker. The final value is the result of a synthetic approach of all stained pixels (DAB chromogen for p16 ICC&IHC, original magnification 100× and 400×). LSCC, Laryngeal squamous cell carcinoma; LSIL, low-grade squamous intraepithelial lesions; RGB: red green blue; DAB, 3-3,diaminobenzidine tetrahydrocloride; ICC, immunocytochemistry; IHC, immunohistochemistry.