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In clinical routine pathology today, detection of protein in intact formalin-fixed, paraffin-embedded tissue is limited to immunohistochemistry, which is semi-quantitative. This study presents a new and reliable quantitative immunohistochemistry method, qIHC, based on a novel amplification system that enables quantification of protein directly in formalin-fixed, paraffin-embedded tissue by counting of dots. The qIHC technology can be combined with standard immunohistochemistry, and assessed using standard bright-field microscopy or image analysis. The objective was to study analytical performance of the qIHC method. qIHC was tested under requirements for an analytical quantitative test, and compared with ELISA and flow cytometry for quantitative protein measurements. Human epidermal growth factor receptor 2 (HER2) protein expression was measured in five different cell lines with HER2 expression from undetectable with immunohistochemistry to strong positive staining (IHC 3+). Repeatability, reproducibility, robustness, linearity, dynamic range, sensitivity, and quantification limits were evaluated. Reproducibility and robustness were assessed in a setup to resemble daily work in a laboratory using a commercial immunohistochemistry platform. In addition, qIHC was correlated to standard HER2 immunohistochemistry in 44 breast cancer specimens. For all evaluated parameters, qIHC performance was either comparable or better than the reference methods. Furthermore, qIHC has a lower limit of detection than both immunohistochemistry and the ELISA reference method, and demonstrated ability to measure HER2 accurately and precise within a large dynamic range. In conclusion, the results show that qIHC provides a sensitive, quantitative, accurate, and robust assay for measurement of protein expression in formalin-fixed, paraffin-embedded cell lines, and tissue.

modern pathology

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2