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Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Accurately Measures Receptor Number
Kristina A. Matkowskyja,b, Randal Coxc, Robert T. Jensend, and Richard V. Benyaa,ba Department of Medicine, University of Illinois at Chicago, Chicago, Illinois
b Chicago VA Medical Center, Chicago, Illinois
c Bio-Informatics Group, Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois
d Digestive Diseases Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Correspondence to: Richard V. Benya, Dept. of Medicine, University of Illinois at Chicago, 840 South Wood Street (M/C 716), Chicago, IL 60612. E-mail: rvbenya@uic.edu
We previously demonstrated that quantitative immunohistochemistry (Q-IHC) performed by measuring the cumulative signal strength of the digital file encoding an image can be used to determine the absolute amount of chromogen present per pixel. We now show that Q-IHC so performed can be used to accurately determine the amount of peptide hormone receptor of interest in archived tissues. To do this we transfected Balb 3T3 fibroblasts with the cDNA encoding the human receptor for gastrin-releasing peptide (GRP), and selected six cell lines stably expressing between 102 and 106 receptors/cell. These cell lines were fixed in formalin, embedded in paraffin, and treated with antipeptide antibodies against the GRP receptor, followed by DAB chromogen to identify bound antibody. Images were acquired using a 4.9 million pixel digital scanning 24-bit RGB camera, saved in TIFF format, and used for subsequent analysis. Q-IHC was performed after digitally dissecting out the relevant portion of the image for analysis, and processing using a program written in C (available at http://www.uic.edu/com/dom/gastro/Freedownloads.html). Under the conditions defined here, chromogen quantity as determined by Q-IHC tightly correlated with GRP receptor number (r2=0.867) in these cell lines. Using the conversion factor identified as a result of these studies, we then determined GRP receptor number on eight randomly selected, archived human colon cancers. Overall GRP receptor expression in colon cancer depended on the degree to which cells within any particular tumor were differentiated, with well-differentiated cells expressing the greatest numbers of receptors (
55,000 ± 10,000 sites/cell). These studies indicate that Q-IHC can be used to determine receptor quantity in archived tissues and other samples of limited quantity.
(J Histochem Cytochem 51:205–214, 2003)
Key Words: bombesin, gastrin-releasing peptide, archived tissues, receptor number
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