Evaluation of the Image-Pro Plus 4.5 software for automatic counting of labeled nuclei by PCNA immunohistochemistry

Abstract

The objective of this study was to create and evaluate a routine (macro) using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, USA) for automatic counting of labeled nuclei by proliferating cell nuclear antigen (PCNA) immunohistochemistry. A total of 154 digital color images were obtained from eleven sections of reticular oral lichen planus stained by PCNA immunohistochemistry. Mean density (gray-level), red density, green density, blue density, area, minor axis, perimeter rate and roundness were parameters used for PCNA labeled nuclei discrimination, followed by their outlined presentation and counting in each image by the macro. Mean density and area thresholds were automatically defined based, respectively, on mean density and mean area of PCNA labeled nuclei in the assessed image. The reference method consisted in visual counting of manually outlined labeled nuclei. Statistical analysis of macro results versus reference countings showed a very significant correlation (rs = 0.964, p < 0.001) for general results and a high level (89.8 +/- 3.8%) of correctly counted labeled nuclei. We conclude that the main parameters associated with a high correlation between macro and reference results were mean density (gray-level) and area thresholds based on image profiles; and that Image-Pro Plus 4.5 using a routine with automatic definition of mean density and area thresholds can be considered a valid alternative to visual counting of PCNA labeled nuclei.

Dynamics of immune granuloma formation in a Leishmania braziliensis‐induced self‐limiting cutaneous infection in the primate Macaca mulatta

“…Automatic counting of immunostained cells was performed using Image‐Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA) 24. Briefly, the sections were examined under a Nikon E400 light microscope and digital images were captured by an attached colour video camera (Sony DXC‐151A CCD, Tokyo, Japan) connected to a 500 MHz Pentium‐III (Intel Corp, Austin, TX, USA) personal computer by a 4 MB PCI All‐in‐Wonder Pro Image card (ATI Technologies, Santa Clara, CA, USA).…”

Morpho-functional response of Nile tilapia (Oreochromis niloticus) to a homeopathic complex

“…These measurements were analysed by Image Pro Plus Ò 4.5 Media Cybernetics software. 19,20 Branchiae: Branchial samples were stained with H&E 17 to determine the morphology and to verify any possible branchial changes, 12 which were adapted to a rising scale of mean modification values depending on the lesions' severity. 21 The following scale was used: level 0 = no histological alteration; level 1 = mild alterations; level 2 = fair and specific alterations; level 3 = severe and extensive alterations.…”

Heterogeneity of PTEN and PPAR‑γ in cancer and their prognostic application to bladder cancer

“…In the grey level analysis, the level is negatively correlated with the expression level of proteins. The detailed methods used are as reported previously (12). In this method, low expression is reported as a high grey level, and high protein expression is reported as a low grey level.…”