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Image Analysis Algorithms for Immunohistochemical Assessment of Cell Death Events and Fibrosis in Tissue Sections

Maryla Krajewska¹, Layton H. Smith¹, Juan Rong, Xianshu Huang, Marc L. Hyer, Nikolajs Zeps, Barry Iacopetta, Steven P. Linke, Allen H. Olson, John C. Reed and Stan Krajewski

Burnham Institute for Medical Research, La Jolla, California (MK,JR,XH,MLH,JCR,SK) and Lake Nona (Orlando), Florida (LHS); St. John of God Pathology, Bendat Family Comprehensive Cancer Centre, Subiaco, Western Australia (NZ); University of Western Australia, Nedlands, Western Australia (BI); Prediction Sciences, La Jolla, California (SPL); and Aperio Technologies, Inc., Vista, California (AHO)

Correspondence to: Stan Krajewski, Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037. E-mail: stan{at}burnham.org

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections. (J Histochem Cytochem 57:649–663, 2009)

Key Words: cell death • automated image analysis • algorithm • autoscore • virtual pathology

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