Supplementary Material for: Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time

<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 (&gt;5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (<i>p</i> &lt; 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.

引言：优化分析前操作流程与组织处理方案，是获得可靠且可重复实验数据的基本前提。福尔马林固定是诊断病理学领域中外科组织标本处理的主流优选方法，但福尔马林固定会对DNA与RNA造成严重的裂解破坏作用。
方法：本研究通过离子交换碱性树脂去除酸性成分，制备得到无甲酸甲醛溶液；随后将浓缩的无酸甲醛（acid-deprived formaldehyde, ADF）溶液配制为0.1 M磷酸盐缓冲液（pH 7.2~7.4）中的4% ADF溶液。将27例人体组织与20例小鼠组织在相同条件下，分别采用ADF与中性缓冲福尔马林（neutral buffered formalin, NBF）进行平行固定。随后提取各组组织的DNA与RNA，并开展片段化分析。
结果：在提取产率与吸光度比值方面，ADF固定组与NBF固定组无显著差异；但ADF固定可降低DNA片段化程度，即从ADF固定组织中纯化得到的DNA内，大于5000 bp的大片段占比显著更高（两个队列均满足p < 0.001）。此外，相较于NBF固定的组织，ADF固定组织的DNA保存稳定性更优。
结论：尽管福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）组织的DNA片段化是多因素参与的过程，但本研究证实，去除甲酸可显著改善DNA的保存效果。