Development of Room-Temperature Storage Technique for Plasma/Serum Biospecimens

描述(申请人提供)：目前，数以百万计的血清生物样本被储存在全国各地的生物仓库中，而每天都有数以万计的新生物样本被添加到池中。这些生物标记物被储存起来以供未来研究，主要用于蛋白质类生物标记物的发现和验证(例如，用于诊断、治疗和流行病学结果)。生物标记物研究的成功不仅取决于从生物样本中提取信息的工具(蛋白质组、多肽、脂组和代谢组学技术)的可用性，而且还取决于“高质量”生物样本的可用性。然而，在大多数生物仓库中，血清是通过冷冻的方式储存的，不遵循任何保存协议；在没有任何冷冻保护剂的情况下，样品被直接放入-20、-40或-80oC的冰柜中，在那里它们经历非常缓慢的冷却(1-20oC/分钟)。众所周知，这些条件对大分子施加了非常苛刻的化学和物理压力，改变了它们的特征(结构和活性)，往往是不可逆转的。近期 有证据表明，一些最有希望的蛋白质类癌症生物标记物确实非常容易受到冷冻/解冻和冷冻状态储存的影响。因此，冰冻状态储存可能会导致储存的血清生物标本中的大部分潜在生物标志物信息永远丢失。我们的长期目标是消除冷冻保存的需求，并开发使用等温玻璃化技术在室温下保存血清生物标本的技术。等温玻璃化是液体 掺入糖的物质被干燥成“玻璃”(一种非常粘稠的液体)。在这种状态下，生化反应停止，样品的降解停止，大分子稳定在它们的自然状态。等温玻璃化将消除蛋白质类生物标志物对冻结/融化应力和冷冻储存损伤的暴露，从而大大提高储存的生物样品的质量。这也将提供一种比冷冻更经济的选择，因为不再需要在冷冻场储存标本。我们将通过实现以下四个目标来实现我们的目标：目标1.开发一组用于恒温玻璃化冷冻的冷冻保护剂：证明冷冻保护剂鸡尾酒能够在稳定血清蛋白的同时实现快速而均匀的玻璃化冷冻。目的#2.测定血清蛋白的保留和洗脱效率 使用研制的冷冻保护剂鸡尾酒，在标准滤纸中通过吸附进行玻璃化。目的#3.确定特定的血清蛋白质是否在优化的玻璃化冷冻和洗脱条件下从最佳基质中改变；记录蛋白质的耗尽、聚集和降解。目的#4.开发和验证一种电纺海绵，用于血清采集点的血清样本的简化玻璃化冷冻。 公共卫生相关性：用于个性化医疗的基因组学和蛋白质组学的出现将生物谱学研究置于NIH优先事项的前列，因为研究项目取决于所使用的生物谱系药物的好坏。我们正在开发在室温下稳定存储液体生物标本(如血清)的方法。这项技术将消除在冷冻场长期储存血清样本的需要，这是非常昂贵的操作。

DESCRIPTION (provided by applicant): Currently, millions of serum biospecimens are being stored in biorepositories across the nation, while tens of thousands of new biospecimens are added to the pool daily. These biospecimens are stored for future research, mainly for proteinaceous biomarker discovery and verification (e.g. for diagnostic, therapeutic, and epidemiologic outcomes). The success of biomarker research not only depends upon the availability of the tools (proteomic, peptidomic, lipidomic and metabolomic technologies) to extract information from biospecimens, but also on the availability of "high quality" biospecimens. However, in most biorepositories, serum is stored by freezing without following any preservation protocol; the samples are directly placed in -20, -40 or -80oC freezers, in the absence of any cryoprotectant, where they experience very slow cooling (1-2o C/min). It is well known that these conditions impose very harsh chemical and physical stresses on macromolecules, altering their characteristics (structure and activity), often irreversibly. Recent evidence has shown that some of the most promising proteinaceous cancer biomarkers are indeed very susceptible to freeze/thaw and frozen state storage. Therefore, it is plausible that frozen state storage may cause most of the potential biomarker information in the stored sera biospecimens to be lost forever. Our long-term goal is to eliminate the requirement for frozen state storage and develop the techniques to store serum biospecimens at room temperature using isothermal vitrification technology. Isothermal vitrification is the process by which liquids doped with sugars are desiccated to a "glass" (a very viscous fluid). In this state, biochemical reactions are halted, degradation of the specimen is stopped, and macromolecules are stabilized in their native states. Isothermal vitrification will eliminate the exposure of the proteinaceous biomarkers to freeze/thaw stresses and to frozen state storage damage and thus will substantially increase the quality of the stored biospecimens. It will also present a more economical alternative to freezing, since storage of specimens in freezer-farms will no longer be needed. We will accomplish our goal by achieving the following four Aims: Aim #1. Develop a panel of lyoprotectant chemicals to be added to serum samples for isothermal vitrification: Demonstrate that the lyoprotectant cocktail enables rapid and uniform vitrification while stabilizing sera proteins. Aim #2. Determine the retention and elution efficiency of sera proteins following vitrification by adsorption in standard filter paper using the developed lyoprotectant cocktail. Aim #3. Determine whether specific sera proteins are altered following optimized vitrification and elution conditions from the optimal matrix; document protein depletion, aggregation and degradation. Aim #4. Develop and validate an electrospun sponge for streamlined vitrification of serum samples at sera collection sites. PUBLIC HEALTH RELEVANCE: The advent of genomics and proteomics for personalized medicine has placed biospecimen research on the forefront of NIH priorities, since research projects are only as good as the biospecimens used. We are developing methodology to stably store liquid biospecimens, such as sera, at room temperature. This technology will eliminate the need for long-term storage of serum samples in freezer farms, which are very costly to operate.