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Advisor(s)
Abstract(s)
A imunocitoquímica possui diversas aplicações em citopatologia, nomeadamente no diagnóstico diferencial e identificação de neoplasias. No entanto apresenta, por vezes, uma reduzida precisão e reprodutibilidade, principalmente ao nível dos falsos-negativos e da uniformidade da marcação. Sabendo que a preservação estrutural e a qualidade da imunomarcação são fulcrais em citopatologia, definiu-se como objetivo deste trabalho avaliar o impacto da etapa de pós-fixação das lâminas de secreções brônquicas processadas em ThinPrep® na técnica de imunocitoquímica, no sentido de selecionar a melhor metodologia para aplicação prática. Com recurso a 26 amostras de secreções brônquicas prepararam-se 90 lâminas, nas quais foram aplicados três métodos de pós-fixação baseados em etanol, acetona e formaldeído. A avaliação da imunomarcação de Citoqueratinas (clones AE1/AE3), Citoqueratinas 8/18 e Vimentina, foi efetuada por 3 avaliadores independentes, com base numa grelha de avaliação que contempla os parâmetros: preservação da morfologia; intensidade da imunomarcação específica; quantidade relativa de estruturas marcadas; marcação inespecífica; fundo; contraste. O resultado final variou entre 0 e 100 pontos. Para o tratamento estatístico foi utilizado o teste one-way ANOVA (α= 0,05). Quando sujeitas a técnicas de pós-fixação baseadas em etanol, acetona e formaldeído obtiveram-se os seguintes valores de média e desvio-padrão (̅//σ): 85,76/4,25; 84,40/6,67 e 91,18/5,25, respetivamente. Verificou-se ainda que existe diferença estatisticamente
significativa na globalidade dos resultados obtidos (p < 0,001). Apesar de todos os métodos de pós-fixação permitirem uma qualidade aceitável da imunomarcação, o melhor método, de entre os que foram testados neste estudo, é o que utiliza formaldeído.
ABSTRACT - Immunocytochemistry has several applications in cytopathology, particularly in the differential diagnosis and identification of neoplasms. However, sometimes it has reduced accuracy and reproducibility, with false-negative results and uneven immunolabeling. Being aware that structural preservation and immunostaining quality are central in cytopathology, the aim of this study was to evaluate the impact of post- fixation step on immunocytochemistry of bronchial secretions processed in ThinPrep™ in order to select the best methodology for practical application. From 26 samples of bronchial secretions it was prepared 90 slides, in which were applied three post-fixation methods based in ethanol, acetone and formaldehyde. The evaluation of immunostained cytokeratin (clones AE1/AE3), cytokeratins 8/18 and vimentin was performed by three independent evaluators, using an assessment grid that includes the parameters: morphology preservation, intensity of specific immunostaining; relative amount of labeled structures; nonspecific labeling; background; contrast.. The final score could vary from 0 to 100 points. For statistical analysis it was used the one-way ANOVA test (α =0,05). The immunocytochemistry that used post-fixation techniques based on ethanol, acetone and formaldehyde obtained the average/standard deviation (/σ) of: 85,76/4,25, 84,40/6,67 and 91,18/5,25, respectively. It was also found that there is statistical difference in the overall results (p <0.001). Although all methods of post-fixation permit an acceptable quality of immunostaining, the best method is the one that uses formaldehyde.
ABSTRACT - Immunocytochemistry has several applications in cytopathology, particularly in the differential diagnosis and identification of neoplasms. However, sometimes it has reduced accuracy and reproducibility, with false-negative results and uneven immunolabeling. Being aware that structural preservation and immunostaining quality are central in cytopathology, the aim of this study was to evaluate the impact of post- fixation step on immunocytochemistry of bronchial secretions processed in ThinPrep™ in order to select the best methodology for practical application. From 26 samples of bronchial secretions it was prepared 90 slides, in which were applied three post-fixation methods based in ethanol, acetone and formaldehyde. The evaluation of immunostained cytokeratin (clones AE1/AE3), cytokeratins 8/18 and vimentin was performed by three independent evaluators, using an assessment grid that includes the parameters: morphology preservation, intensity of specific immunostaining; relative amount of labeled structures; nonspecific labeling; background; contrast.. The final score could vary from 0 to 100 points. For statistical analysis it was used the one-way ANOVA test (α =0,05). The immunocytochemistry that used post-fixation techniques based on ethanol, acetone and formaldehyde obtained the average/standard deviation (/σ) of: 85,76/4,25, 84,40/6,67 and 91,18/5,25, respectively. It was also found that there is statistical difference in the overall results (p <0.001). Although all methods of post-fixation permit an acceptable quality of immunostaining, the best method is the one that uses formaldehyde.
Description
Keywords
Anatomia patológica Imunocitoquímica Pós-fixação Secreções brônquicas Immunocytochemistry Post-fixation method Bronchial aspirations and washings ThinPrep
Citation
Barata C, Anágua M, Roque R, Ferro AB. Imunocitoquímica em amostras brônquicas processadas em ThinPrep™: comparação de três métodos de pós-fixação. NewsLab. 2017;(141):24-8.