Tissue Thickness Interferes With the Estimation of the Immunohistochemical Intensity: Introduction of a Control System for Managing Tissue Thickness.

Abstract

The conversion of immunohistochemical (IHC) results from 3-dimensional tissue to a 2-dimensional visual image without considering tissue thickness poses a considerable risk of misleading IHC intensities. The present study aimed to clarify whether tissue thickness interferes with the estimation of IHC staining intensity and to introduce a control system to manage it. We prepared cell lines that are used as controls for human epidermal growth factor receptor 2 (HER2) IHC (MDA-MB-231, MDA-MB-175VII, MDA-MV-453, and SK-BR-3), a polyclonal antibody for HER2, an interferometry to measure the tissue thickness of formalin-fixed paraffin-embedded sections, a microscope with a Halogen or an LED light source, a complementary metal-oxide semiconductor camera in which the output signal can be corrected to γ=1, and a program to estimate color elements (hue, saturation, and luminance). It was examined whether tissue thickness interferes with the experimental scoring systems and practical classification of the routine HER2 scoring system. A noncellular control was shown to be better than a cellular control for managing tissue thickness. The IHC intensity for HER2 was correlated with tissue thickness (R2=0.8094), even under the less-standardized condition, but this correlation was better under the improved standardized condition using corrected γ=1 (R2=0.9282). Discrepancies in practical HER2 scores were increased in sections with thicknesses <2 and >5 μm. A control system to manage tissue thickness was introduced. Tissue thickness interferes with the estimation of the IHC intensity of HER2 in both experimental and practical scoring systems. A control system for managing tissue thickness is essential to increase the benefits of IHC as a standardized assay for clinical applications.