Alcohol based fixatives provide excellent tissue morphology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens

Milcheva, Rositsa; Janega, Pavol; Celec, Peter; Russev, Russy; Babál, Pavel

doi:10.1016/j.acthis.2012.08.002

Cited by 28 publications

(27 citation statements)

References 14 publications

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“…Such a fixative may also be available when longer fragment analysis was required for the pathology laboratory in the future. On the other hand, at present, it is thought that NeuBF is a superior fixative for both protein and nucleic acids than NonBF …”

Section: Discussionmentioning

confidence: 99%

Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry, DNA, and mRNA assays

Sato

Kojima

Nagatsuma

et al. 2014

Pathology International

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The purpose of this study was to set the optimal preanalytical fixation protocol to enhance analytical and postanalytical phase accuracy and consistency. Twenty-five normal colorectal tissues were fixed using various formalin concentrations, pHs, and fixation periods. All specimens were embedded in paraffin and 4 μm sections were used for immunohistochemistry of Ki-67, and extraction and amplification of DNA and RNA. The Ki-67 labeling index and the successful gene amplification rate for DNA and mRNA were evaluated and compared among variously fixed tissue samples. Ki-67 positivity was enhanced by low pH and short fixation time, and was influenced by the type of antibody, but not by the staining (with or without using an autostainer) method. DNA amplification by PCR was strongly influenced by pH of formalin. cDNA amplification could be accomplished only with the shortest PCR fragment of 142 bp, and longer fixation times impaired the amplification. These data suggest that multiple different factors influence immunohistochemical results and gene amplification using DNA and mRNA. We recommend, based on data from this comprehensive analysis, a 10% neutral buffered formalin and fixation times of no longer than 1 week to produce consistent immunohistochemical slides and DNA amplification within 500 bp in pathology laboratories. Key words: fixation, pathology, preanalytic phaseThe numerous components of laboratory test processes can be categorized into preanalytic, analytic, and postanalytic phases. Many laboratory tests using formalin-fixed and paraffin-embedded tissue samples in the preanalytic phase have been developed. Fixation is one of the most important steps in the preanalytic phase in pathological testing. Multiple factors including kinds of fixative, fixation duration, and acidity levels have been reported to have an effect on immunohistochemical results and gene amplification. For these reasons, a standardized and optimized fixation protocol for pathology laboratories would allow for more consistent and accurate data to be produced and enable better informed decisions based on the results. [1][2][3][4][5][6][7][8] However, the current recommendations for preanalytic phase testing vary depending on what organ, therapy, or procedure (such as protein, DNA, or RNA assay) is being used.9-18 Furthermore, most studies investigating the effects of fixation in laboratory testing have dealt with only one kind of laboratory test target. A standardized fixation protocol for pathology laboratories should be made available. We analyzed Ki-67 immunohistochemistry (IHC) testing and polymerase chain reaction (PCR)-based gene amplification using DNA and mRNA from one samples that was fixed by different protocols to clarify how variations in fixation influence postanalytic results in different assays. In regards to IHC testing, we also evaluated the effect of staining procedures, autostainer or manual staining, and different antibody clones, to search for the best way to reduce the effect of nonstandardized fixation protocols....

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Section: Discussionmentioning

confidence: 99%

Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry, DNA, and mRNA assays

Sato

Kojima

Nagatsuma

et al. 2014

Pathology International

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“…Neste trabalho, foi demonstrado que o Carnoy foi inferior ao Bouin e ao FT. Este resultado pode estar associado a dois principais fatores: (1) a fixação de tecido de pele apresenta baixa celularidade e abundância de matriz extracelular e (2) por ser tecido extraído de biópsia e devido a sua pequena espessura (aproximadamente 2cm), deveria ter sido reduzido o tempo de fixação para menos de 30 minutos. Em outros tecidos, como estômago, intestinos e tecidos extraídos de biópsias retais, já foram relatados associados a uma excelente fixação tanto em Carnoy quanto em Bouin (Puchtler et al, 1970;Mitchell et al, 1985;Milcheva et al, 2012;Rieger et al, 2013;Pereira et al, 2015).…”

Section: Resultsunclassified

“…Entretanto, estes resultados não se aplicaram quando tecidos fixados em Carnoy e incluídos tanto em glicol metacrilato quanto em parafina foram submetidos às diversas técnicas de imuno-histoquímica. Contudo, trabalhos mostram como resultados a utilização de técnicas de imuno-histoquímica para marcações de membrana e núcleos celulares em tecidos fixados em Carnoy e concluem que os fixadores à base de álcool são excelentes ferramentas para este tipo de estudo (Milcheva et al, 2012).…”

Section: Methodsunclassified

Avaliação de diferentes fixadores na preservação das características histológicas de pele de orelha de cães

Campos

Tafuri

Pinto

2016

Arq. Bras. Med. Vet. Zootec.

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RESUMOOs fixadores biológicos desempenham um papel importante na qualidade final da histologia. Na rotina veterinária, a biópsia de pele é um procedimento comum e a escolha do fixador é primordial para resultado final adequado. Os fixadores mais usados são à base de formalina, ainda que sejam tóxicos, cancerígenos, de baixa penetração e de fixação lenta. Mesmo assim, não existe um fixador ideal que substitua as suas qualidades. O objetivo deste trabalho foi avaliar qualitativamente a preservação das características histológicas de pele de cão utilizando diferentes fixadores de tecidos incluídos em parafina, cortados e corados pela hematoxilina-eosina. Utilizou-se uma caneta Punch de 4 milímetros para coletar amostras de pele de orelha em seis cadáveres de cães. Após coleta, os tecidos foram fixados em: (1) Bouin, durante seis horas; (2) Carnoy, durante quatro horas; (3) formaldeído tamponado 10% durante 24 horas, todos sob refrigeração (4ºC). Posteriormente, os tecidos foram processados, cortados e corados em hematoxilina e eosina. As lâminas foram avaliadas, às cegas, por quatro patologistas diferentes, que consideraram aspectos qualitativos a seguir: (1) qualidade da coloração; (2) preservação das características histológicas; e (3) preservação dos limites citoplasmáticos utilizando a escala de LIKERT de pontuação para cada lâmina. O fixador com a maior média de pontuação em todos os itens foi o formol tamponado com 3,76 pontos, seguido pelo Bouin (3,39) e pelo Carnoy (2,52). O formol pode trazer riscos à saúde do profissional que rotineiramente o manuseia, portanto se faz necessária a busca por fixadores com as mesmas qualidades, mas menos nocivos à saúde.Palavras-chave: cão, formol , Bouin, Carnoy, histologia ABSTRACT The biological fixatives have an important role in the final histology quality. In veterinary, routine skin biopsy is a common procedure and the choice of fixative is essential for the final result. The most common fixative is Formalin, even though it is toxic

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“…Ethanol and acetic acid were used as fixatives because they have been previously proved to be successfully employed in proteomic analysis (Ahram et al, 2003; De Souza et al, 2004; Milcheva et al, 2013) (Figure 1). Although the anther length is generally used as a criterion to determine the developmental stages of anthers in rice (Kerim et al, 2003; Itoh et al, 2005; Nonomura et al, 2011), an anther from each fixed flower was removed using fine forceps under a dissection microscope (Stemi 2000-C stereomicroscope, Carl Zeiss, Göttingen, Germany) equipped with a cold-light source to stage meiosis as much accurately as possible.…”

Section: Methodsmentioning

confidence: 99%

Unravelling the proteomic profile of rice meiocytes during early meiosis

2014

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Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

show abstract

Alcohol based fixatives provide excellent tissue morphology, protein immunoreactivity and RNA integrity in paraffin embedded tissue specimens

Cited by 28 publications

References 14 publications

Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry, DNA, and mRNA assays

Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry, DNA, and mRNA assays

Avaliação de diferentes fixadores na preservação das características histológicas de pele de orelha de cães

Unravelling the proteomic profile of rice meiocytes during early meiosis

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