Browsing by Author "Pote, Alexandra"
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- Carcinoma adenoescamoso: um estudo de caso em citologia ginecológicaPublication . Pote, Alexandra; Sousa, R.; Mendonça, PaulaO carcinoma adenoescamoso é um tumor raro que representa cerca de 4% dos tumores cervicais. Este carcinoma ocorre maioritariamente em mulheres com cerca de 50 anos, podendo ocorrer em mulheres mais jovens e está também associado ao estado de gravidez e à infeção pelo Papiloma Vírus Humano. No presente trabalho, relata-se o caso de uma mulher de 45 anos, com coitorragias e uma lesão dura, elevada e hemorrágica no colo do útero, que foi submetida a citologia esfoliativa. O exame microscópico permitiu visualizar achados citológicos compatíveis com coexistência de componente pavimentosa- células pleomórficas com citoplasma denso com aumento da relação núcleo citoplasma - e componente glandular- pseudoestratificação, feathering e núcleos alongados. O relatório citológico foi de carcinoma pavimento celular, não excluindo a possibilidade de carcinoma adenoescamoso. No seguimento da doente, foi realizada biópsia ao colo uterino, cujo diagnóstico histológico foi de adenocarcinoma invasor produtor de muco com uma componente do tipo pavimentoso. Esta neoplasia faz diagnóstico diferencial principalmente com “tumores de colisão” onde ocorrem duas neoplasias primárias, uma glandular e outra pavimentosa. Para este tipo de tumor, a interpretação citológica pode ser desafiante, pela dificuldade de interpretar nos aspetos citomorfológicos a componente pavimentosa e glandular na mesma amostra.
- Determinação da eficiência de obtenção de DNA a partir de secções histológicas submetidas a técnicas histoquímicasPublication . Pote, Alexandra; Freitas, Beatriz; Boghenco, Otília; Marques-Ramos, AnaIntrodução: Devido à deteção precoce de lesões, as amostras para análise molecular são cada vez mais reduzidas e a quantidade DNA pode não ser suficiente para obter resultados, tornando-se necessário extrair DNA diretamente de tecidos corados. Apesar da influência das colorações na obtenção de DNA já ter sido estudada, esta é uma área pouco explorada. Objetivo do estudo: Apresentar a recolha de dados referentes à influência de técnicas histoquímicas na integridade do DNA.
- Influence of H&E and Papanicolau stains on DNA integrity: systematic reviewPublication . Pote, Alexandra; Boghenco, Otília; Marques-Ramos, AnaThe success of the Human Genome Project along with the development of new molecular and bioinformatics methods has allowed a rapid evolution of personalized health care. In that context, molecular pathology plays a key role in providing patient-specific disease signatures allowing to develop targeted and patient-driven therapeutic strategies and to predict therapeutic response, particularly in oncology. For that, the presence or absence of mutations is assessed by molecular techniques such as polymerase chain reaction (PCR), Sanger sequencing and next-generation sequencing (NGS). This information complements the morphological analysis obtained by microscopic inspection of tissue sections, commonly from formalin-fixed paraffin-embedded (FFPE) samples, or of cytological specimens (CS). As cells and other tissue components are invisible, to render them visible under the microscope, it is necessary to perform a previous staining procedure. The routine stain in clinical pathology laboratories for FFPE samples is Hematoxylin and Eosin (H&E), as it allows the visualization of general tissue architecture assisting the evaluation of structural and morphological changes. For cytological specimens, the routine stain is Papanicolau (Pap). In some cases, FFPE or CS samples are scarce and it is necessary to extract DNA directly from FFPE or CS stained-samples. Furthermore, samples from tumors are usually very heterogeneous containing a variable mixture of neoplastic cells and a variety of normal cells. Molecular analysis of DNA extracted from these mixtures can lead to confusing or false negative results and erroneous conclusions. In order to diminish the interference of normal cells, the tumor area is usually selected and isolated before molecular analysis is performed. This enrichment of neoplastic cells can be performed through manual or automated-assisted microdissection of FFPE, CS or frozen samples. In these cases, it is mandatory to extract nucleic acids directly from stained samples. Although this is a common practice in molecular pathology laboratories, a survey of the literature demonstrates that the reagents used in H&E and Pap stains may induce chemical modifications on DNA. This review collects the available information about the influence of H&E and Pap stains in DNA integrity and explores the mechanisms by which the dyes might interfere with molecular analysis, particularly on PCR-based methods.
- Influence of histochemical stains on DNA obtained from FFPE samplesPublication . Boghenco, O.; Freitas, B.; Pote, Alexandra; Marques-Ramos, AnaIntroduction: Formalin-fixed, paraffin embedded (FFPE) samples are used for diagnostic and prognostic purposes. Histopathological analysis frequently includes not only histomorphological evaluation but also histochemical and molecular studies. In some cases FFPE samples are scarce and it is necessary to use the same histological section for histochemical analysis and DNA extraction. In molecular pathology labs this is a common practice, allowing the analysis of DNA specifically from altered cells. However, histochemical techniques use reagents that may induce chemical modifications on DNA. To perform a literature review about the influence of histochemical stains on DNA integrity. Materials and methods: PubMed and Research Gate were used to survey original articles published until December 2017. Results: For this review articles about the analysis of DNA extracted from stained FFPE sections were considered. The studies demonstrated that: DNA extracted from sections stained with Azure B, toluidine blue and methyl green (MG) was successfully amplified by Polymerase Chain Reaction (PCR) whereas Mayer’s hematoxylin stain inhibits the reaction. Another study demonstrated that DNA amplification by PCR had better results with eosin Y and MG stains comparatively to Mayer’s hematoxylin and May-Grunwald. Banaschak et al. showed that DNA analysis by PCR and capillary electrophoresis was successful with Hematoxylin-Eosin (HE), Periodic Acid Schiff (PAS), Azan and Perl's stains. Phosphotungstic acid hematoxylin (PTHA) and Gomori stains had negative results. Two different studies concluded that DNA is refractory to HE stain as capillary electrophoresis demonstrated similar degradation to that of unstained samples and it was successfully amplified by PCR. Conclusions: Histochemical analysis allows demonstration of cellular components whose alterations are typical from pathological conditions. These techniques encompass reagents that may alter biomolecules. Nevertheless, from the analyzed studies it is possible to conclude that DNA integrity is maintained in techniques such as Azure B, toluidine B, MG, eosin Y, HE, PAS, Azan and Perls. On the other hand, Mayer’s hematoxylin, May-Grunwald, PTHA and Gomori resulted in inhibition of DNA amplification. Since this analysis was not performed in common routine techniques such as Masson’s Trichrome and PAS-Alcian Blue, it is important to deepen the knowledge, performing new studies for future appliance.
- Molecular analysis of H&E - and Papanicolau-stained samples: systematic reviewPublication . Pote, Alexandra; Boghenco, Otília; Marques-Ramos, AnaMolecular pathology allows the identification of causative agents in infectious diseases and the detection of biomarkers important for prediction of disease susceptibility, diagnosis and personalized therapy. Accordingly, nucleic acid-based methods have gained a special role in clinical laboratories particularly to evaluate solid and hematological tumors. Extraction of nucleic acids is commonly performed in microdissected formalin-fixed paraffin-embedded (FFPE) or cytological samples that had been previously evaluated through the use of hematoxylin and eosin (H&E) or Papanicolau (Pap) stains, respectively. Although the effect of both stains on nucleic acids integrity has been explored by several authors, the results are not consistent and require further examination. Accordingly, the goal of this review was to assess the influence of H&E and Pap stains on DNA and RNA integrity and to address the mechanism by which each staining compromises molecular based-analysis. The analyzed studies demonstrate that H&E- and Pap-staining result in low DNA recovery and some degree of DNA fragmentation. Additionally, it is concluded that hemalum inhibits PCR by interfering with DNA extraction, preventing DNA polymerase attachment and possibly by rescuing divalent cations. Accordingly, proper sample purification and adjustment of PCR conditions are of key importance to achieve satisfactory results by PCR in H&E- and Pap-stained samples. Furthermore, although H&E results in RNA fragmentation, it is possible to perform expression analysis in H&E-stained frozen sections, using RNase-free conditions, low amounts of hematoxylin and a rapid protocol from sample collection to RNA analysis. It The effect of Pap-staining on RNA integrity remains to be determined.