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）：标准病理实践依赖于固定在10％中性缓冲福尔马林中的组织的自动处理，然后是在过去一个世纪中对显微镜可视化进行了优化的染色方案。在过去的二十年中，尽管与从新鲜或冷冻组织中回收的核酸的数量较小，核酸的质量较小，核酸质量较差，但分子测定越来越多地应用于福尔马林固定的石蜡嵌入组织。要测试的假设：我们建议，为了改善病理学界将接受的固定技术，标准福尔马林固定的关键步骤无法改变。另一方面，将化学稳定剂添加到标准试剂中，并改变福尔马林固定的初始阶段的温度，是现实的变化，可以改善下游分子分析而不会不利影响形态和免疫剂结果。基于多种文献的综合，我们提出了两部分假设，以推动增强的福尔马林固定方案的发展：a）。福尔马林固定期间发生的核酸的不可逆转损害主要是生化的，可以在很大程度上通过抑制福尔马林输注过程中的内源性核酸酶活性来预防。为了解决这个问题，将确定宽光谱核酸酶抑制剂足够小，可以与福尔马林共进组织空间，并且在其他标准的自动化组织加工方案中，这些都将在有或没有冷藏的情况下进行测试。 b）。固定后的核酸损伤主要是由于源自大气O2的活性氧（ROS）的缓慢，持续的氧化，被困在组织块内。为了解决这个问题，将确定ROS清除剂是水溶性，廉价且足够小的，可以在第一个“型号后正象”脱水步骤中迅速扩散到组织空间中，但在酒精或二甲苯中却很差，因此，在无液体中，散布的液体在无处在原位存储期间。与后续R33有关：当完成此R21时，将进行程序改进，以在普通10％缓冲福尔马林固定期间保留DNA和RNA，并随后作为石蜡嵌入式组织存储。在R33的工作中，这些化合物将作为Beta测试试剂盒进行试验量表制造，并在多个地点的各种人类癌组织中进行验证。