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

Wound healing: comparative study in hypertensive rats untreated and treated with an angiotensin converting enzime inhibitor

BACKGROUND: We evaluated the influence of captopril on the skin wound healing process of hypertensive rats. METHODS: 111 rats were placed in 4 groups: normotensive control (N=30); hypertensive control (N=30), which received an oral daily dose of saline solution 0,9%; group experiment (N=31) was treated with 7.5mg/kg/day of captopril; and an aferition group (N=20) with 10 hypertensive and 10 normotensive animals in which arterial blood pressure was mesured in the aorta in the last day of the experiment. After 15 days of treatment, an skin incision of 4 cm was made in the animals. Samples of the dorsal wall scar were taken 4, 7 and 14 days after the last procedure. The wounds were excised and divided in 2 pieces. They were sent to tensiontrial and histological analysis. RESULTS: The aferition group showed mean arterial blood pressure of 82.5±7.55 mmHg in the normotensive animals and 150.5± 10.66 mmHg in the hypertensive ones. The resistance analysis showed that the scars of treated and untreated hypertensives were less resistant than those of normotensives rats in the initial days (p<0.05) and that on the 14th day the resistances became similar. There were no diferences among treated and untreated groups. Total collagen had higher density in normotensives rats throughout the study (p<0.05) and there were no diferences among treated and untreated hypertensive rats. Epitelization, inflammatory response and granulation tissue formation were similar in all groups. CONCLUSION: Captopril, doesn't modify the wound healing process in rats, being the differences due to hypertension.OBJETIVO: Reconhecer a interferência do captopril na cicatrização de feridas cutâneas de ratos hipertensos. MÉTODOS: Distribuíram-se 111 ratos em quatro grupos: controle normotenso (N=30); controle hipertenso (N=30), os quais receberam 1 ml/dia de solução de cloreto de sódio a 0.9% por via oral; grupo experimento (N=31), hipertensos que receberam 7,5mg/kg/dia de captopril e um grupo aferição (N=20), 10 hipertensos e 10 normotensos, nos quais aferiu-se a pressão na aorta abdominal, no último dia de experimento. Após 15 dias de medicação, fez-se uma incisão da pele e da tela subcutânea, na região médio-dorsal dos grupos I, II e III, seguida de síntese. Ressecaram-se as cicatrizes de 10 animais de cada grupo, no 4.º, 7.º e 14.º dias após a operação, que divididas em duas partes foram enviadas para a tensiometria e para análise histológica. RESULTADOS: A pressão arterial média de 83,18 ± 7,51 mmHg nos normotensos e 151,36 ± 10,51 mmHg nos hipertensos. As cicatrizes dos hipertensos tratados e não tratados eram menos resistentes que as dos normotensos, nos tempos iniciais (p<0,05) e que ao 14.º dia as resistências se igualaram. Não houve diferença entre o grupo tratado e o não tratado. A densidade de colágeno total foi maior nos normotensos em todos os tempos (p<0,05) e não houve diferença entre hipertensos tratados e não tratados. A epitelização, a reação inflamatória e a formação do tecido de granulação foi semelhante nos três grupos. CONCLUSÕES: O captopril, em ratos, não modifica a cicatrização, ficando as diferenças relacionadas à hipertensão.UNIFESP-EPMPUCPRUniversidade Tuiuti do ParanáThe National Hospital for Nervous Diseases Institute of Neurology Department of NeuropathologyUFSCUNIFESP, EPMSciEL

Increased Incidence of Choroid Plexus Carcinoma Due to the Germline TP53 R337H Mutation in Southern Brazil

International audienceBACKGROUND: Choroid plexus carcinomas (CPC) are rare tumors predominantly found in children. Given the high frequency of the germline R337H mutation in the TP53 gene in southern Brazil, we have evaluated the frequency of the R337H mutation in families with CPC in children. METHODOLOGY/PRINCIPAL FINDINGS: The present series included 29 patients that were admitted to the same institution from 1992 to 2010, including 22 children with CPC (0.08-13.6 years of age at diagnosis) and 7 children with papilloma of the choroid plexus (Pp; 0.5-9.8 years of age). Surgical resection was possible in 28 children. Blood and/or tumor DNA was extracted and analyzed using PCR-RFLP and results were confirmed by sequencing 240 bp of the TP53 exon 10. The patients, all parents, and some relatives submitted samples for blood DNA analysis. In addition, we have also examined the presence of the mutation in DNA from paraffin-embedded tumor samples to evaluate loss of heterozygosity. We found 63.3% (14/22) of the CPC patients positive for the germline R337H mutation; CPC samples were either heterozygous (n = 7), lost only the wild-type (n = 4), or only the R337H copy (n = 2). One CPC sample was not available. All Pp cases (7/7, 100%) were negative for R337H. Cure (>5 years survival free of disease) was observed in 18.1% of the CPC cases with the R337H mutation (2/11), 71.4% of the Pp (5/7), and 25% of CPC cases negative for the R337H mutation (2/8). Family history of cancer (with 2 or more cancer cases) was exclusively identified on the parental side segregating the R337H mutation, and 50% (7/14) of them were compatible with Li-Fraumeni-like syndrome. SIGNIFICANCE: Our results show for the first time that the R337H TP53 mutation is responsible for 63% of the CPC cases in children, suggesting a higher incidence of CPC in southern Brazil