A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed,
Kristian Jensen1, Rikke Krusenstjerna-Hafstrøm1, Jesper Lohse1
1Dako Denmark A/S, an Agilent Technologies Company, Produktionsvej 42, Glostrup, Denmark.
Insights
A new quantitative immunohistochemistry (qIHC) method offers reliable protein quantification in formalin-fixed, paraffin-embedded tissues. This sensitive and robust assay surpasses traditional methods for accurate protein expression analysis.
Area of Science:
- Biopathology
- Molecular Pathology
- Biotechnology
Background:
- Current protein detection in formalin-fixed, paraffin-embedded (FFPE) tissues relies on semi-quantitative immunohistochemistry (IHC).
- There is a need for reliable quantitative methods to measure protein expression directly in FFPE samples.
Purpose of the Study:
- To evaluate the analytical performance of a novel quantitative immunohistochemistry (qIHC) method.
- To compare qIHC with established methods like ELISA and flow cytometry for protein quantification.
Main Methods:
- Developed a qIHC method utilizing a novel amplification system for direct protein quantification in FFPE tissues via dot counting.
- Assessed analytical parameters including repeatability, reproducibility, robustness, linearity, dynamic range, sensitivity, and quantification limits.
- Correlated qIHC results with standard HER2 IHC in 44 breast cancer specimens.
Main Results:
- qIHC demonstrated performance comparable or superior to reference methods (ELISA, flow cytometry) across all evaluated analytical parameters.
- The qIHC method exhibited a lower limit of detection than standard IHC and ELISA.
- Accurate and precise quantification of HER2 protein expression was achieved over a wide dynamic range.
Conclusions:
- The qIHC method provides a sensitive, quantitative, accurate, and robust assay for protein expression measurement in FFPE cell lines and tissues.
- qIHC is a valuable tool for clinical pathology, offering improved protein quantification capabilities.
- This technology can be integrated with standard IHC workflows and assessed using bright-field microscopy or image analysis.
Abstract:
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.
