Enhanced Formalin Fixation to Improve Tests on Solid Tissues

描述（由申请人提供）：标准病理实践依赖于用10%中性缓冲福尔马林固定组织的自动处理，随后是染色方案，该方案在过去的一个世纪中被优化用于显微镜可视化。在过去的二十年中，分子分析越来越多地应用于福尔马林固定，石蜡包埋的组织，尽管与从新鲜或冷冻组织中回收的核酸相比，这种努力受到数量较少和质量较差的核酸的阻碍。有待检验的假设：我们建议，为了改进将被病理界接受的固定技术，标准福尔马林固定的关键步骤不能改变。另一方面，在标准试剂中加入化学稳定剂，改变福尔马林固定初始阶段的温度，是现实的改变，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。基于多种文献的综合，我们提出了一个由两部分组成的假设，以推动增强福尔马林固定方案的发展：a)。福尔马林固定过程中对核酸的不可逆损伤主要是生化损伤，在很大程度上可以通过抑制福尔马林灌注过程中内源性核酸酶活性来预防。为了解决这个问题，广谱核酸酶抑制剂将被确定为足够小，可以与福尔马林共同扩散到组织空间，这些抑制剂将在标准的自动化组织处理方案中进行冷藏或不冷藏的测试。B)。核酸在固定后发生损伤，主要是由于被组织块内来自大气O2的活性氧（ROS）缓慢、持续地氧化。为了解决这个问题，活性氧清除剂将被确定为水溶性、廉价且足够小，可以在第一个“后福尔马林”脱水步骤中迅速扩散到组织空间，但在酒精或二甲苯中难溶，因此，当组织转移到无水溶剂中时，清除剂被嵌入脱水的组织块基质中，在原位储存期间，它们随时准备淬灭新形成的活性氧。与后续R33的关系：当R21完成后，将对程序进行改进，在普通的10%缓冲福尔马林固定期间保存DNA和RNA，随后作为石蜡包埋组织保存。在R33工作中，这些化合物将作为beta测试试剂盒进行中试规模生产，在多个部位对不同的人类癌症组织进行验证。

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.