CONTEXT AND OBJECTIVE 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. DESIGN 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. RESULTS 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 (R=0.8094), even under the less-standardized condition, but this correlation was better under the improved standardized condition using corrected γ=1 (R=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. CONCLUSIONS 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.

中文翻译：

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组织厚度干扰免疫组织化学强度的估计

背景和目的 在不考虑组织厚度的情况下，将免疫组织化学 (IHC) 结果从 3 维组织转换为 2 维视觉图像，这会带来相当大的误导 IHC 强度的风险。本研究旨在澄清组织厚度是否会干扰 IHC 染色强度的估计，并引入控制系统来管理它。设计 我们制备了用作人表皮生长因子受体 2 (HER2) IHC（MDA-MB-231、MDA-MB-175VII、MDA-MV-453 和 SK-BR-3）（一种多克隆抗体）对照的细胞系。 HER2 抗体、测量福尔马林固定石蜡包埋切片组织厚度的干涉仪、带卤素或 LED 光源的显微镜、互补金属氧化物半导体相机（其中输出信号可校正为 γ=1） ，以及估计颜色元素（色调、饱和度和亮度）的程序。检查组织厚度是否干扰常规 HER2 评分系统的实验评分系统和实际分类。结果 在管理组织厚度方面，非细胞对照被证明比细胞对照更好。即使在标准化程度较低的条件下，HER2 的 IHC 强度也与组织厚度相关 (R=0.8094)，但在使用校正 γ=1 的改进标准化条件下，这种相关性更好 (R=0.9282)。厚度 <2 和 >5 μm 的切片中实际 HER2 评分的差异更大。引入了管理组织厚度的控制系统。结论 在实验和实际评分系统中，组织厚度都会干扰 HER2 IHC 强度的估计。用于管理组织厚度的控制系统对于提高 IHC 作为临床应用标准化检测的优势至关重要。