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

SUMMARY Cell death is of broad physiological and pathological importance, makingquantification of biochemical events associated with cell demise a high priority for experimentalpathology. Fibrosis is a common consequence of tissue injury involving necrotic celldeath. 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 immunohistochemicaland 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 terminaldeoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay to detect DNAfragmentation; and (c) evaluated with Masson’s trichrome staining. We developed novelalgorithm-based scoringmethods and validated themusingTMAs as a high-throughput format.The proposed computer-assisted scoring methods for digital images by brightfield microscopypermit linear quantification of immunohistochemical and histochemical stainings. Examples areprovided of digital image analysis performed in automated or semiautomated fashion for successfulquantification of molecular events associated with cell death in tissue sections.