Fixation and Immunohistochemistry

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, preparou-se um total de 90 lâminas, nas quais foram aplicados 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 feita com base num grelha que contempla itens como preservação, intensidade/proporção de marcação específica, marcação inespecífica e contraste, criando um resultado final entre 0 e 100. Para o tratamento estatístico foi utilizado o teste one-way ANOVA (alfa=0.05). Quando sujeitas a técnicas de pós-fixação em etanol, acetona e formaldeído obtiveram-se as médias/desvio-padrão de 85,76/5,14; 84,40/6,67 e 91,18/4,53, respetivamente. Verificou-se ainda que existe diferença estatística na globalidade dos resultados obtidos (p=.000). Apesar de todos os métodos de pós-fixação permitirem uma qualidade da imunomarcação aceitável, o melhor método de entre os que foram testados neste estudo, é o que utiliza formaldeído.

Texto/Resumo (Introdução, objectivos, metodologia, resultados, conclusão e bibliografia) Introdução – Os tecidos processados histologicamente são fixados, por acção de agentes químicos, para inibição de enzimas endógenas e eliminação de bactérias. Essa fixação permite também dar consistência aos tecidos, criando diferentes texturas e pormenores macroscópicos. Consequentemente, no caso das peças de esvaziamento axilar da mama (EAM), a fixação pode facilitar ou dificultar a identificação dos gânglios linfáticos (gl) cuja detecção é essencial para o prognóstico e tratamento, pois permitem avaliar a proliferação metastática da neoplasia. Objectivos – Identificar o fixador que permite evidenciar gl de menores dimensões e em maior número no EAM, mantendo a preservação morfológica. Metodologia – realizou-se um estudo retrospectivo, sendo escolhidos os fixadores: formol 10% neutro tamponado (FNT), carnoy e álcool/formol/ácido acético (AFA). A partir do arquivo seleccionou-se aleatoriamente e comparou-se 32 casos diferentes de EAM sujeitos a cada um dos fixadores (total 96 casos). Em cada caso foi avaliado o número, tamanho e preservação morfológica dos gl detectados. Resultados – No grupo de FNT foram identificados, em média, 9,38gl/caso com um tamanho médio de 7,87mm e um score de preservação de 24,75. No grupo de carnoy foram identificados 16,88gl/caso com tamanho médio de 6,18mm e um score de preservação de 18,75. No grupo de AFA foram identificados 20,41gl/caso com tamanho médio de 5,05mm e um score de preservação de 23,56. Conclusão - O AFA detectou mais gl do que carnoy e FNT (p=.000). O mesmo fixador conseguiu detectar gl de menores dimensões que carnoy e FNT (p=.000). Por último a preservação morfológica foi equivalente entre AFA e FNT, sendo estes superiores ao carnoy (p=.000).

Context: There are very few circumstances where the diagnosis of malignancy is made in the absence of histopathological confirmation. For prognosis and therapeutic guidance, it is also essential to support all decisions with knowledge of the oncobiology provided by immunohistochemistry and other molecular methodologies. Regarding this goal, it is essential to obtain/preserve adequate diagnostic material. In case of tissue samples, the most widely used fixative is 10% neutral buffered formalin. However, this concentration is not supported by consistent scientific evidence. Aim: To compare immunohistochemical results considering less than 10% formalin concentrations, in order to support the proper use of this reagent and also to explore the possibility of decreasing the formalin use, since its toxicity is known. Methods: Three formalin concentrations (10; 7,5; 2,5%) and 70% ethanol (representing non-formalin fixation) were used to fix, for 48 hours at room temperature, similar sized human placenta samples, with a cold ischemia time of 15 minutes. For each fixative, 30 paraffin blocks were prepared, which, after sectioning, were subjected to vimentin (NCL-L-VIM-V9) immunohistochemical detection (Ventana Benchmark GX - OptiView DAB), has this structural protein is commonly used as a fixation quality biomarker. Immunohistochemical results were assessed by two experts using the Global Immunohistochemistry Score (GIS) providing a 0–100 points score. The GIS results were analysed using descriptive statistics and Kruskal-Wallis test with pairwise comparison. Results: The results were (mean-standard deviation): 2,5% formalin (90,10–6,94); 7,5% formalin (86,77–7,22); 10% formalin (82,94–9,62); 70% ethanol (84,67–9,49). The Kruskal-Wallis test revealed statistical differences between 2,5% formalin and all the other samples (p < 0,05). The internal consistency between the two observers was considered satisfactory (α=0,72). Conclusions: Results sustain that 2.5% formalin provided best immunohistochemical results in this context. Even considering the exploratory type of this research, it is possible to consider the reduction of formalin concentration used in fixation.

There are several hazards in histopathology laboratories, and the laboratory staff members must ensure that their professional activity is set to the highest standards while complying with the best safety procedures. Formalin is one of the chemical hazards to which such professionals are routinely exposed. To decrease this contact, it is suggested that 10% neutral buffered liquid formalin (FL) is replaced by 10% formalin-gel (FG), given the latter reduces the likelihood of spills and splashes, and decreased fume levels are released during its handling, proving less harmful. However, it is mandatory to assess the effectiveness of FG as a fixative and ensure that the subsequent complementary techniques, such as immunohistochemistry (IHC), are not compromised. Two groups of 30 samples from human placenta were fixed with FG and FL fixatives for different periods of time (12, 24, and 48 hours) and, thereafter, processed, embedded, and sectioned. IHC for six different antibodies was performed and the results were scored (0–100) using an algorithm that took into account immunostaining intensity, percentage of staining structures, non-specific immunostaining, contrast, and morphological preservation. Parametric and non-parametric statistical tests were used (alpha50.05). All results were similar for both fixatives, with global score means of 95.36¡6.65 for FL and 96.06¡5.80 for FG, and without any statistical difference (P.0.05). The duration of the fixation had no statistical relevance also (P.0.05). So it is proved here FG could be an effective alternative to FL.

There are very few circumstances where the diagnosis of malignancy is made in the absence of histopathological confirmation 1. For prognosis and therapeutic guidance, it is also essential to support all decisions with knowledge of the oncobiology provided by immunohistochemistry and other molecular methodologies 2-3. Regarding this goal, it is essential to obtain/preserve adequate diagnostic material 4. In case of tissue samples, the most widely used fixative is 10% neutral buffered formalin 4. However, this concentration is not supported by consistent scientific evidence. The results were (mean-standard deviation): 2,5% formalin (90,10-0,89); 7,5% formalin (86,77-0,93); 10% formalin (82,94-1,24); 70% ethanol (84,66-1,22). The Kruskal-Wallis test revealed differences between 2,5% formalin and all the other samples (Kruskal-Wallis with multiple comparisons without bonferroni correction-Kruskal-Wallis H=19,54; p<0,05). The internal consistency between the two observers was considered satisfactory (α=0,72). To compare immunohistochemical results considering less than 10% formalin concentrations, in order to support the proper use of this reagent and also to explore the possibility of decreasing the formalin use, since its toxicity is known 5-6. Three formalin concentrations (10%; 7,5%; 2,5%) and 70% ethanol (representing non-formalin fixation) were used to fix, for 48 hours at room temperature, similar sized human placenta samples, with a cold ischemia time of 15 minutes. For each fixative, 30 paraffin blocks were prepared, which, after sectioning, were subjected to vimentin (NCL-L-VIM-V9) immunohistochemical detection (Ventana Benchmark GX-OptiView DAB), has this structural protein is commonly used as a fixation quality biomarker 7. Immunohistochemical results were assessed by two experts using the Global Immunohistochemistry Score (GIS) providing a 0-100 points score 8. The GIS results were analyzed using descriptive statistics and Kruskal-Wallis test with pairwise comparison. Results sustain that 2.5% formalin provided best immunohistochemical results in this context. Even considering the exploratory type of this research, it is possible to consider the reduction of formalin concentration used in fixation. Diagram 1: a) experimental procedure; b) immunohistochemistry results; c) statistical results. a) b) c)