Nuclear morphometric analysis: description of the methodology and the role of image-editing softwares

The morphometric analysis of images, which comprises measuring and counting, is used in pathology to obtain accurate data of cellular and tissue components that are important for diagnosis and prognosis of several tumors. Although it is a useful and low-cost tool, it is restricted to a few diagnosis and research centers. The objective of this study is to present and describe the method of nuclear morphometry with supplementary use of image-editing softwares, making detailed analysis of the critical stages of the process. The correction of problems found during the capture of the image, such as erroneously adjusted color temperature and color shade and unevenly illuminated images from an improperly adjusted microscope, is fundamental for the accurate analysis. Through the color mask resource present in countless image-editing softwares, it is possible to select and to separate objects of similar colors, resulting in a final image that shows only the objects of interest for analysis. Morphometry is accomplished through a specific software, such as ImageTool (version 3.00), that permits the accurate automatic measuring of one or more parameters in a short interval of time. The interface of ImageTool allows one to work simultaneously with the original and the processed images, in which the similar colors were selected. Therefore, it is possible to confront the selected object with the same object in its original image, what ensures its exact selection. This system allows interobserver comparison of 100% of the objects, enabling the rejection of those that should not have been selected and measured.A análise morfométrica de imagens é utilizada em patologia para se obter dados precisos de componentes celulares e teciduais, por meio da mensuração e contagem, que são importantes para o diagnóstico e prognóstico de vários tumores. Essa técnica, mesmo sendo ferramenta útil e de baixo custo, mantém-se restrita a poucos centros de diagnóstico ou pesquisa. O objetivo deste trabalho é apresentar e descrever o método de morfometria nuclear com a utilização complementar de softwares de edição de imagens, fazendo a análise detalhada das etapas críticas do processo. A correção de problemas encontrados durante a captura das imagens, como temperatura e matiz da cor erroneamente ajustados, e de imagens com iluminação desigual provenientes de microscópio mal regulado, é fundamental para a correta análise. Assim, utilizando o recurso máscara de cor disponível em vários softwares de editoração de imagens, é possível selecionar e separar objetos de cores semelhantes, resultando numa imagem final que apresenta somente os objetos de interesse para análise. A morfometria é realizada por meio de softwares específicos como o ImageTool (versão 3.0), que possibilitam a mensuração automática de um ou mais parâmetros, com precisão e em curto intervalo de tempo. A interface do ImageTool permite ainda trabalhar simultaneamente com a imagem original e a imagem processada em que se fez a seleção das cores semelhantes. Dessa forma, pode-se confrontar o objeto que está sendo selecionado com o mesmo objeto na sua imagem original, garantindo assim correta seleção. Esse sistema possibilita conferência interobservador de 100% dos objetos, o que permite desprezar aqueles que posteriormente constatou-se que não deveriam ter sido selecionados e mensurados.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de PatologiaUniversidade Federal do ParanáUNIFESP, EPM, Depto. de PatologiaSciEL

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds