Validation and Advanced Development of Albumin Oxidizability as a Marker of Plasma/Serum Integrity

白蛋白氧化性作为血浆/血清完整性标志的验证和高级开发

• 批准号: 9759884
• 负责人: CHAD R BORGES
• 金额: $ 37.19万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-08 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9759884
• 关键词: Advanced Development; Albumins; Aliquot; Apolipoprotein A-I; Awareness; Biological Markers; Biomedical Research; Cancer Patient; Chemicals; Clinical; Clinical Research; Collection; Cysteine; Data; Dry Ice; Enzyme-Linked Immunosorbent Assay; Event; Exposure to; Extramural Activities; Forensic Medicine; Freezing; Funding; Goals; Gold; Grant; Hand; Incubated; Individual; Level of Evidence; Life; Link; Literature; Malignant Neoplasms; Maps; Measurement; Measures; Methionine; Minor; Molecular; Oxides; Plasma; Population; Procedures; Proteins; Publishing; Reference Values; Research; Sampling; Serum; Serum Proteins; Ships; Site; Specimen; Specimen Handling; Sulfoxide; Sulfur; Technology; Temperature; Testing; Time; United States National Institutes of Health; Validation; base; blind; cancer biomarkers; candidate marker; cost; evidence base; experience; genetic pedigree; improved; in vivo; maltreatment; oxidation; oxidative damage; repository; sound; success; theories; tool

Project Summary/Abstract Every year, improprieties and inconsistencies in pre-analytical handling and storage of blood plasma/serum (P/S) specimens generate unacceptably large numbers of costly false leads in biomedical research. Experts in the field are calling for this problem to be immediately stopped or at least minimized. The issue is particularly important in the scope of NIH-sponsored research: A recent study found that out of 455 NCI-sponsored extramural grants that relied on biospecimens, 63% employed pre-existing biospecimens—107 of which involved pre-existing P/S. Surprisingly, no criteria for establishing pre-existing sample integrity have been set and no quality thresholds exist that must be met before NCI (taxpayer) funds are spent on studies involving pre-existing P/S. Exposure to the thawed state (which includes temporary storage at -20 °C) represents one of the most common pre-analytical variables (PAVs) encountered by P/S. Considering the entire life of a research specimen, no other PAV is more difficult to control and track—and, as demonstrated in the Research Strategy, even the best standard operating procedures (SOPs) in the most respected hands cannot guarantee that all specimens are handled ideally (i.e., in accord with the SOP). Our preliminary data argue that objective, molecular-level evidence based on measurements of molecular damage—above and beyond sole reliance on SOPs—is crucial to unambiguously establishing sample integrity. To date, however, no gold standard marker of P/S integrity yet exists. The goal of this project is to validate a simple, inexpensive, rapid test requiring 10 µL of P/S that provides a representative assessment of the oxidative damage that P/S proteins have incurred due to exposure to the thawed state. The test is based on the fact that the relative abundance of S-cysteinylated (oxidized) albumin (S-Cys-Alb) increases substantially over time (but to a maximum value) when P/S is handled/stored above its freezing point of -30 °C. Thus by measuring S-Cys-Alb before and after an intentional incubation period that causes S-Cys-Alb to hit its maximum value, the difference between these values, ΔS- Cys-Alb, is then readily interpreted as inversely proportional to the degree of ex vivo oxidation that occurred prior to the first measurement of S-Cys-Alb. Thus, for example, a ΔS-Cys-Alb value of zero would indicate a badly mistreated sample. Herein we will validate ΔS-Cys-Alb as a marker of P/S integrity via four Specific Aims: Specific Aim 1: Experimentally validate the predicted range of ΔS-Cys-Alb that can be expected from freshly collected cancer patient plasma and serum samples. Specific Aim 2: Systematically map out how ΔS- Cys-Alb behaves under “realistic” mistreatment conditions and link known, unstable cancer markers to ΔS-Cys- Alb. Specific Aim 3: Conduct a blind challenge to quantify the ability of ΔS-Cys-Alb to identify biospecimen mistreatment within cancer patient plasma and serum samples. And Specific Aim 4: Use ΔS-Cys-Alb to quantify the integrity of plasma samples collected from a representative large cancer study in which samples were collected at multiple sites under a single SOP and eventually transferred to a single repository.

项目概要/摘要 每年，血浆/血清的分析前处理和储存都存在不当和不一致的情况 (P/S) 样本在生物医学研究专家中产生了令人无法接受的大量昂贵的错误线索。 该领域呼吁立即停止或至少最小化这个问题。 在 NIH 资助的研究范围内很重要：最近的一项研究发现，在 455 项 NCI 资助的研究中， 依赖生物样本的校外资助，63% 使用了已有的生物样本——其中 107 令人惊讶的是，没有设定建立现有样本完整性的标准。 在将 NCI（纳税人）资金用于涉及以下方面的研究之前，不存在必须满足的质量阈值： 预先存在的 P/S 暴露于解冻状态（包括 -20 °C 下的临时储存）代表以下之一： 考虑到研究的整个生命周期，P/S 遇到的最常见的分析前变量 (PAV)。 样本，没有其他 PAV 比它更难控制和跟踪——而且，正如研究策略所证明的那样， 即使是最受尊敬的人手中最好的标准操作程序（SOP）也不能保证所有 我们的初步数据表明，样本得到了理想的处理（即符合 SOP）。 基于分子损伤测量的分子水平证据——超越了单纯依赖 SOP——对于明确建立样品完整性至关重要，但是迄今为止，还没有黄金标准标记。 该项目的目标是验证一种需要 10 µL 的简单、廉价、快速的测试。 P/S 的分析提供了对 P/S 蛋白因氧化损伤而造成的氧化损伤的代表性评估 该测试基于S-半胱氨酸的相对丰度这一事实。 当 P/S 为 在 -30 °C 的冰点以上进行处理/储存，因此通过在有意的前后测量 S-Cys-Alb。 导致 S-Cys-Alb 达到最大值的潜伏期，这些值之间的差值，ΔS- Cys-Alb，然后很容易解释为与发生的离体氧化程度成反比 因此，例如，ΔS-Cys-Alb 值为零表示 在此，我们将通过四种特定方法验证 ΔS-Cys-Alb 作为 P/S 完整性的标志。 目标： 具体目标 1：通过实验验证可预期的 ΔS-Cys-Alb 的预测范围 新鲜采集的癌症患者血浆和血清样本 具体目标 2：系统地绘制出 ΔS- 的变化情况。 Cys-Alb 在“现实”的虐待条件下表现良好，并将已知的不稳定癌症标记物与 ΔS-Cys-联系起来 Alb. 具体目标 3：进行盲目挑战以量化 ΔS-Cys-Alb 识别生物样本的能力 癌症患者血浆和血清样本中的虐待和具体目标 4：使用 ΔS-Cys-Alb 来 量化从代表性大型癌症研究中收集的血浆样本的完整性，其中样本 在单个 SOP 下在多个站点收集并最终转移到单个存储库。