Fatores que interferem na qualidade do DNA extraído de amostras biológicas armazenadas em blocos de parafina / Factors that affect the quality of DNA extracted from biological samples stored in paraffin blocks

Os avanços tecnológicos e metodológicos da biologia molecular criaram a possibilidade de obter DNA de amostras teciduais estocadas em blocos de parafina, alternativa esta que permite estudos retrospectivos de grandes bancos de tecidos contendo inúmeras doenças raras. O objetivo deste trabalho foi a realização de uma breve revisão de alguns fatores inerentes à qualidade do produto extraído proveniente de blocos de parafina armazenados. Os processos envolvidos nas fases de prefixação, fixação, pós-fixação apresentam aspectos que são causa de resultados de produtos finais insatisfatórios. Na fase de prefixação, alterações bioquímicas influenciam na preservação das macromoléculas. As mudanças moleculares causadas pelos fixadores para evitar autólise celular podem ser um limitante no momento da extração de DNA. A qualidade do DNA obtido nas duas primeiras fases é considerada importante para os procedimentos de pós-fixação (extração e purificação do DNA). Existem na literatura vários protocolos, com diferentes passos que podem ser modificados, para a obtenção de DNA de material parafinado. Assim, a compreensão das reações em cada fase é importante para solucionar ou minimizar problemas, que influenciam na qualidade das macromoléculas de DNA.

Technological and methodological advances in molecular biology have enabled the obtainment of DNA from paraffin embedded tissue, thus allowing the use of extensive pathological archive sources and samples of uncommon diseases in retrospective studies. The aim of this work was to carry out a brief review of some factors inherent in the quality of product from paraffin embedded material. The processes involved in the pre-fixation, fixation and post-fixation phases have several aspects that may result in unsatisfactory final products. In the pre-fixation phase, biochemical changes influence the preservation of macromolecules. The molecular changes caused by fixation, an attempt to prevent cell autolysis, may be a limiting factor at the time of DNA extraction. The quality of DNA obtained in the first two phases is regarded important for post-fixation procedures (DNA extraction and purification). In the literature there are several protocols whose steps can be modified to obtain DNA from paraffin embedded material. Therefore, the understanding of reactions at each stage is important in order to solve or minimize problems that interfere in the quality of DNA macromolecules.

HER-2/neu immunoreactivity in invasive mammary carcinomas: a comparative study using monoclonal and polyclonal antibodies including the HercepTestTM

INTRODUÇAO: A superexpressão de HER-2/neu tem sido testada por imunoistoquímica como um método seguro e de baixo custo para selecionar pacientes com carcinoma mamário invasivo (CMI) para tratamento com trastuzumab, entretanto não há consenso sobre qual o melhor anticorpo a ser utilizado. O objetivo deste trabalho foi testar cinco diferentes anticorpos para determinar a superexpressão do HER-2/neu em CMI. MATERIAL E MÉTODOS: Sessenta e seis casos de CMI fixados em formalina e incluídos em parafina foram submetidos a imunoistoquímica, utilizando-se dois anticorpos policlonais: HercepTestTM (Dako) e A0485 (Dako), e três anticorpos monoclonais: CB11 de Novocastra Laboratories e da Biogenex e 4D5 (Genentech). Todas as reações imunoistoquímicas foram interpretadas de acordo com as instruções do HercepTestTM. RESULTADOS: O A0485 foi positivo em 25/66 casos (37,9 por cento). O HercepTestTM foi positivo em 14/66 casos (21,2 por cento). Houve concordância integral nos casos corados com CB11 de ambos os fabricantes: 9/66 (13,7 por cento). O 4D5 foi positivo em somente 4/66 casos (6,1 por cento). Todos os casos positivos para CB11 e 4D5 foram positivos para o HercepTestTM. Todos os casos HercepTestTM foram positivos para o A0485. A maioria dos casos 2+ para o HercepTestTM e A0485 foi negativa com os outros anticorpos. DISCUSSAO: Houve maior incidência de superexpressão do HER-2/neu com o HercepTestTM e o A0485 do que com os anticorpos monoclonais, principalmente nos casos classificados como 2+. Não houve diferença na imunorreatividade usando-se o anticorpo CB11 dos dois diferentes fabricantes. CONCLUSAO: O uso clínico da imunoistoquímica para determinar a superexpressão do HER-2/neu necessita ainda de validações prospectivas.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates.

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Kl³ver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Kl³ver-Barrera stain. These procedures have the advantage of permitting Nissl and Kl³ver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.(AU)