Enhanced Formalin Fixation to Improve Tests on Solid Tissues

描述(由申请人提供)：标准的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处理，随后是在过去的一个世纪中为显微可视化而优化的染色方案。在过去的二十年里，分子分析越来越多地应用于福尔马林固定的石蜡包埋组织，尽管与从新鲜或冷冻组织中提取的核酸相比，这一努力受到了数量较少和质量较差的阻碍。有待检验的假设：我们建议，为了改进将被病理学界接受的固定技术，标准福尔马林固定的关键步骤不能改变。另一方面，在标准试剂中添加化学稳定剂，以及改变福尔马林固定的初始阶段的温度，都是现实的变化，可以改善下游分子分析，而不会对形态和免疫染色结果产生不利影响。在综合各种文献的基础上，我们提出了一个两部分假说来推动加强福尔马林固定方案的发展：a)。福尔马林固定过程中发生的核酸不可逆性损伤主要是生化损伤，通过抑制福尔马林注射过程中的内源性核酸酶活性可以在很大程度上防止这种损伤。为了解决这个问题，将确定足够小的广谱核酸酶抑制剂与福尔马林共同扩散到组织空间，并将在其他标准的自动组织处理方案中进行冷藏或不冷藏测试。b)。固定后，核酸损伤增加，主要是由于组织块内捕获的大气O2产生的活性氧物种(ROS)缓慢、持久的氧化。为了解决这一问题，我们将确定在第一个“后福尔马林”脱水步骤中，ROS清除剂是水溶性的，价格低廉，并且足够小，足以迅速扩散到组织空间中，但在酒精或二甲苯中很难溶解，因此，当组织转移到无水溶剂中时，清除剂被嵌入到脱水的组织块基质中，在那里它们准备在原位储存期间淬灭新形成的ROS。与后续R33的关系：当R21完成时，程序将得到改进，在普通的10%福尔马林缓冲固定和随后作为石蜡包埋组织存储期间保存DNA和RNA。在R33的工作中，这些化合物将作为测试盒进行中试生产，并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.