Enhanced Formalin Fixation to Improve Tests on Solid Tissues

增强福尔马林固定以改进固体组织测试

基本信息

批准号：
8326059
负责人：
Margaret L Gulley
金额：
$ 13.84万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8326059
关键词：
Academia Address Alcohols Antibodies Applied Genetics Automation Biochemical Biological Assay Blinded Buffers Chemicals Communities DNA Dehydration Deoxyribonucleases Development Diffuse Dyes Epitopes Ethanol Fixatives Formaldehyde Formalin Freezing Genomics Glass Goals Gold Hematoxylin and Eosin Staining Method Histocytochemistry Hospitals Human Imagery Immunohistochemistry In Situ In Vitro Infusion procedures Laboratories Length Literature Liver Malignant Neoplasms Measures Medical Methods Microscopic Microscopy Modification Molecular Molecular Analysis Morphology Nucleic Acids Organic solvent product Outcome Paraffin Paraffin Embedding Pathologist Pathology Phase Phosphate Buffer Procedures Process Proteins Protocols documentation RNA Rattus Reaction Reactive Oxygen Species Refrigeration Ribonucleases Screening procedure Site Slide Solid Solvents Staging Staining method Stains Techniques Technology Temperature Testing Tissue Sample Tissues Validation Water Work Xylene aqueous base chemical addition clinically significant comparative genetic technology human tissue improved inhibitor/antagonist nuclease oxidation prevent programs reagent standard sample fixation small molecule solute tissue fixing tissue processing

项目摘要

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.

描述（由申请人提供）：标准病理学实践依赖于对固定在 10% 中性缓冲福尔马林中的组织进行自动处理，然后进行上个世纪针对显微镜可视化而优化的染色方案。在过去的二十年中，分子测定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，这种努力受到核酸数量较少和质量较差的阻碍。待检验的假设：我们建议，为了改进病理学界所接受的固定技术，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初始阶段的温度，都是现实的改变，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。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来推动增强福尔马林固定方案的开发：A)。福尔马林固定过程中发生的核酸不可逆损伤主要是生化性的，可以通过在福尔马林输注过程中抑制内源性核酸酶活性来很大程度上预防。为了解决这个问题，将鉴定出足够小的广谱核酸酶抑制剂，以与福尔马林共同扩散到组织空间中，并且将在有或没有冷藏的情况下，在其他标准的自动化组织处理方案中对这些抑制剂进行测试。 B）。固定后会产生核酸损伤，这主要是由于来自大气 O2 的活性氧 (ROS) 被困在组织块内而造成的缓慢、持续的氧化。为了解决这个问题，我们将鉴定出活性氧清除剂，它们是水溶性的、廉价的，并且足够小，可以在第一个“福尔马林后”脱水步骤中快速扩散到组织间隙中，但在酒精或二甲苯中溶解度很差，因此，在组织中转移到无水溶剂中后，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猝灭新形成的ROS。与后续 R33 的关系：当 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留 DNA 和 RNA。在 R33 工作中，这些化合物将作为 beta 测试套件进行中试规模生产，并在多个部位的不同人类癌症组织上进行验证。