Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time

Abstract

<b><i>Introduction:</i></b> Optimization of pre-analytic procedures and tissue processing is a basic requirement for reliable and reproducible data to be obtained. Tissue fixation in formalin represents the extensively favored method for surgical tissue specimen processing in diagnostic pathology; however, formalin fixation exerts a blasting effect on DNA and RNA. <b><i>Methods:</i></b> A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2–7.4. Human (<i>n</i> = 27) and mouse (<i>n</i> = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed. <b><i>Results:</i></b> Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (&#x3e;5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (<i>p</i> &#x3c; 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues. <b><i>Conclusion:</i></b> Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.