Processamento de amostras fixadas em formalina e preservadas em parafina para uso diagnóstico e caracterização de doenças através de biologia molecular

Abstract

Scientific advances aimed at clinical diagnosis and the emergence of the Polymerase Chain Reaction (PCR) technique have made biological samples fixed in formalin and preserved in paraffin, the so-called FFPE (formalin-fixed paraffin-embedded), become valuable resources of genetic material for molecular analysis to complement clinical diagnoses. The great challenge from then on is faced with the use of protocols that are effective in the recovery and amplification of DNA. The aim of the study was to standardize the use of material (FFPE) from different pathological processes, representative of human diseases, for use in molecular investigation. This is a retrospective cross-sectional study, in which samples with different preservation times in paraffin were included, from the Pathology Laboratory of Hospital São Vicente de Paulo, Passo Fundo, RS and the Pathology Laboratory of UNESP – Botucatu, SP. Samples from different tissues (tumor and non-tumor), divided into two groups according to the fixative, Group 1 (G1 - unbuffered formalin, n=90) and Group 2 (G2 - buffered formalin, n=68). Were submitted to mcrotome sections (3 and 6 sections of 10 micrometers (μm) – G1 and 6 sections of 10 micrometers (μm) – G2), and the preservation period of the samples was 2, 5 and 10 years. Subsequently, the sectioned samples were submitted to deparaffinization and total DNA extraction and analyzed by spectrophotometry to verify the quality and quantity of DNA recovered from each tissue. The DNA recovery efficiency was confirmed by endogenous beta globin gene PCR amplification , using specific primers. Data were analyzed following statistical assumptions and the significance level adopted was 5%. It was observed that the concentration of Total DNA isolated from samples of periods of 2, 5 or 10 years was not different. Total DNA concentration was lower in biopsy samples (G1 and G2) compared to surgical specimens (G1 and G2) (p<0.0001), however this result was inverse when considering the concentration of DNA/ fragment area (G1, p=0.003 and G2, p=0.0005). Regarding the DNA quality, a better result was observed in the biopsy samples (G1 and G2) in relation to the surgical specimens. Considering the ratio >1.8, the number of Biopsies and surgical specimens in G2 were higher than in G1. As for PCR positivity, the success rate of endogenous beta globin gene amplification was about 70% in G1 and 100% in G2. For Biopsy samples (G1) 6 cuts, greater positivity was observed compared to 3 cuts (p=0.04). It is concluded that the storage time of FFPE samples does not interfere with the amount of DNA recovered. The use of buffered formalin is preferable in front of tissue fixation. FFPE biopsies for obtaining DNA have a greater amount of DNA/area and better DNA quality, its use being preferable in relation to the pieces, however a greater number of cuts (material) was more effective in the face of recovery and positivity in the face of biopsies preserved in common formalin. The easy and economical DNA extraction protocol is feasible for molecular analysis of a large number of FFPE samples, but it is necessary to know all the variables that can interfere in the recovery of genetic material.