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.

项目摘要/摘要 每年，血浆/血清的分析前处理和存储中的不正当和不一致 （p/s）标本在生物医学研究中产生了不可接受的大量昂贵的假铅。专家 该领域要求立即停止或至少最小化此问题。这个问题尤其 在NIH赞助的研究范围中很重要：最近的一项研究发现，在455个NCI赞助中 依赖生物测量的壁外赠款，有63％的人使用先前存在的生物素材 - 其中107 涉及预先存在的p/s。令人惊讶的是，尚未设置建立先前存在的样本完整性的标准 在NCI（纳税人）资金用于涉及的研究之前，没有任何质量阈值必须满足 预先存在的p/s。暴露于解冻状态（包括-20°C的临时存储）代表其中之一 p/s遇到的最常见的分析前变量（PAV）。考虑一生的研究 标本，没有其他PAV更难控制和跟踪，并且如研究策略所证明的那样， 即使是最受尊敬的手中最好的标准操作程序（SOP）也无法保证 理想地处理标本（即按照SOP）。我们的初步数据认为这一目标， 基于分子损伤的测量的分子级证据 - 放弃和超越唯一的缓解 SOP-对于明确建立样本完整性至关重要。但是，迄今为止，没有黄金标准标记 p/s的完整性却存在。该项目的目的是验证一个简单，廉价，快速测试，需要10 µL P/S的P/S提供了对P/S蛋白到期的氧化损伤的代表性评估 暴露于解冻状态。该测试是基于以下事实 （氧化）白蛋白（S-CYS-ALB）随着时间的推移大大增加（但最大值）当P/S为 在其冰点上方处理/存储-30°C。通过在故意之前和之后测量S-Cys-Alb 导致S-Cys-Alb达到其最大值的孵育周期，这些值之间的差异ΔS- 然后，cys-alb很容易解释为与发生的离体氧化程度成反比 在首次测量S-Cys-Alb之前。例如，ΔS-cys-alb值为零，将表明 严重虐待的样本。在此，我们将通过四个特定 目的：特定目标1：实验验证可以预期的δs-cys-alb的预测范围 新鲜收集的癌症患者血浆和血清样品。特定目标2：系统地绘制ΔS-的方式 Cys-Alb在“现实”的虐待条件下的行为以及已知的不稳定癌症标志物与ΔS-cys- Alb。特定目标3：进行盲目挑战以量化ΔS-cys-alb鉴定生物仿生的能力 癌症患者血浆和血清样品中的虐待。和特定的目标4：使用δs-cys-alb到 量化从代表性的大型癌症研究中收集的血浆样品的完整性，其中样品 在单个SOP下的多个位点收集，并最终转移到单个存储库中。