Enhanced Formalin Fixation to Improve Tests on Solid Tissues

描述（由申请人提供）：标准病理学实践依赖于在10%中性缓冲福尔马林中固定的组织的自动化处理，随后是在过去世纪中优化的用于显微镜可视化的染色方案。在过去的二十年中，分子检测越来越多地应用于福尔马林固定，石蜡包埋的组织，虽然这一努力受到阻碍的数量较少，质量较差的核酸相比，从新鲜或冷冻的组织。待检验的假设：我们建议，为了改善固定技术，将接受病理学界，标准福尔马林固定的关键步骤不能改变。另一方面，向标准试剂中添加化学稳定剂以及改变福尔马林固定初始阶段的温度是可以改善下游分子分析而不会对形态学和免疫染色结果产生不利影响的现实变化。基于对不同文献的综合，我们提出了两部分假设来推动增强福尔马林固定方案的发展：A）。在福尔马林固定过程中发生的对核酸的不可逆损伤主要是生物化学的，并且可以通过在福尔马林灌注过程中抑制内源性核酸酶活性来在很大程度上防止。为了解决这个问题，将鉴定出足够小以与福尔马林共扩散到组织空间中的广谱核酸酶抑制剂，并且将在其他标准的自动化组织处理方案中在有或没有冷藏的情况下对这些抑制剂进行测试。B）。固定后核酸损伤增加，主要是由于来自大气O2的活性氧（ROS）的缓慢、持续氧化，被困在组织块内。为了解决这个问题，将鉴定ROS清除剂，其是水溶性的、便宜的并且足够小以在第一“福尔马林后”脱水步骤期间快速扩散到组织空间中，但在醇或二甲苯中溶解性差，使得在组织转移到无水溶剂中时，清除剂包埋在脱水的组织块基质中，在那里它们随时准备在原位储存期间淬灭新形成的ROS。与后续R33的关系：当该R21完成时，将进行程序改进，在普通10%缓冲福尔马林固定和随后作为石蜡包埋组织储存期间保存DNA和RNA。在R33工作中，这些化合物将作为β测试试剂盒进行中试规模生产，在多个地点的不同人类癌症组织上进行验证。

DESCRIPTION (provided by applicant): Standard pathology practice relies on automated processing of tissues fixed in 10% neutral buffered formalin followed by staining protocols that were optimized over the past century for microscopic visualization. In the last two decades, molecular assays are increasingly applied to formalin fixed, paraffin embedded tissues although this effort is hampered by lesser quantity and poorer quality of nucleic acid compared with that recovered from fresh or frozen tissue. Hypothesis to be tested: We propose that, in order to improve fixation technology that will be embraced by the pathology community, key steps of standard formalin fixation cannot be altered. On the other hand, addition of chemical stabilizers to standard reagents, and altering the temperature of the initial phase of formalin fixation, are realistic changes that could improve downstream molecular analysis without adversely impacting morphology and immunostain outcomes. Based on synthesis of a diverse literature, we present a two-part hypothesis to drive development of enhanced formalin fixation protocols: A). The irreversible damage to nucleic acid occurring during formalin fixation is mainly biochemical and can be largely prevented by inhibiting endogenous nuclease activity during formalin infusion. To address this, broad-spectrum nuclease inhibitors will be identified that are small enough to co-diffuse with formalin into tissue spaces, and these will be tested with or without refrigeration in an otherwise-standard, automated tissue processing protocol. B). Nucleic acid damage accrues after fixation, due mainly to slow, persistent, oxidation by reactive oxygen species (ROS) derived from atmospheric O2, trapped inside the tissue block. To address this, ROS scavengers will be identified that are water-soluble, inexpensive, and small enough to diffuse rapidly into tissue spaces during the first "post-formalin" dehydration step, yet are poorly soluble in alcohol or xylene so that, upon tissue transfer into water-free solvents, the scavengers are embedded in the dehydrated tissue block matrix where they stand ready to quench newly-formed ROS during storage in situ. Relation to a follow-on R33: When this R21 is completed, procedural improvements will have been made which preserve DNA & RNA during ordinary 10% buffered formalin fixation and subsequent storage as paraffin embedded tissue. In R33 work, these compounds will be subjected to pilot scale manufacture as beta test kits, to be validated on diverse human cancer tissues at multiple sites.