A definição dos limites de corte para densidade média e área foi realizada automaticamente em função, respectivamente, da média da densidade e da média da área dos núcleos imunopositivos presentes em cada imagem. Os parâmetros densidade média (nível de cinza), densidades de vermelho, de verde e de azul, área, eixo menor, taxa de perímetro e redondeza foram usados para a discriminação dos núcleos imunopositivos pela macro, que, no final do processo, apresentava estes núcleos delineados e contados na imagem estudada. Utilizamos 154 imagens microscópicas digitalizadas coloridas obtidas de onze cortes histológicos de líquen plano oral reticular processados por imuno-histoquímica para PCNA. O objetivo deste trabalho foi desenvolver e avaliar uma macro (rotina informatizada) usando o programa Image-Pro Plus 4.5 (Media Cybernetics, Silver Spring, EUA) para a contagem automática de núcleos imunopositivos para o antígeno nuclear em célula proliferante (PCNA). Image processing, computer-assisted Lichen planus Proliferating cell nuclear antigen 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. Statistical analysis of macro results versus reference countings showed a very significant correlation (r s = 0.964, p < 0.001) for general results and a high level (89.8 ± 3.8%) of correctly counted labeled nuclei. The reference method consisted in visual counting of manually outlined labeled nuclei. Mean density and area thresholds were automatically defined based, respectively, on mean density and mean area of PCNA labeled nuclei in the assessed image. 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. A total of 154 digital color images were obtained from eleven sections of reticular oral lichen planus stained by PCNA immunohistochemistry. 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.
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