Quantification of Protein Expression on an Immunofluorescence Section by Using the MetaMorph Image Analysis System

In recent years, cancer treatment response and disease progression increasingly rely on immunofluorescence to monitor the changes in targeted antigens. Estimation of abundances of antigens has so far relied primarily on visual scoring and to a lesser extent on computer-assisted image processing techniques. The quantification of immunostaining by means of digital image analysis by using Leica MetaMorph AF 1.4 allows the characterization of protein expression revealed by immunohistochemistry on normal or pathological tissue slices. Our study focuses on the practical aspects of image acquisition, segmentation of staining and counterstaining areas, and extraction of quantitative features. We illustrate our findings by using a Red-Green-Blue (RGB) system to quantify different immunofluorescence markers targeting proteins with different expression patterns (cytoplasmic and membranous) in human breast cancer. Our investigations led us to identify several steps that we consider essential for standardizing computer-assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to compare measurements between different samples. In conclusion, we recommend certain critical prerequisites that commercial or in-house systems should satisfy to permit valid immunostaining quantification. In conclusion, image analysis-based quantitative immunofluorescence has the potential to accurately improve biomarker evaluation in research, clinical, and therapeutic applications for the study of cancer and other diseases.