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

项目总结/摘要 每年，血浆/血清分析前处理和储存中的不当和不一致 （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错误线索。专家 该领域呼吁立即停止或至少尽量减少这一问题。这个问题尤其 在NIH赞助的研究范围内很重要：最近的一项研究发现，在455个NIH赞助的研究中， 依赖于生物标本的校外赠款，63%采用了预先存在的生物标本-其中107个， 涉及预先存在的P/S。令人惊讶的是，没有建立预先存在的样品完整性的标准 在NCI（纳税人）资金用于研究之前，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（包括在-20 °C下暂时储存）代表以下情况之一： P/S遇到的最常见的分析前变量（PAV）。考虑到研究的整个生命周期 标本，没有其他PAV更难以控制和跟踪，正如研究策略中所证明的那样， 即使是最受尊敬的人手中的最好的标准操作程序（SOP）也不能保证所有 理想地处理样本（即，符合SOP的雅阁）。我们的初步数据表明，客观， 基于分子损伤测量的分子水平证据-超越了对 SOP-是明确建立样品完整性的关键。然而，迄今为止，没有金本位制 的P/S完整性还存在。该项目的目标是验证一种简单、廉价、快速的检测方法， 的P/S，提供了一个代表性的评估的氧化损伤，P/S蛋白已招致由于 暴露在解冻状态下该测试基于以下事实：S-半胱氨酸化的相对丰度 （氧化）白蛋白（S-Cys-Alb）随着时间的推移而显著增加（但达到最大值）， 在-30 °C的冰点以上处理/储存。因此，通过测量S-Cys-Alb之前和之后的故意 导致S-Cys-Alb达到其最大值的潜伏期，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值为零将指示 被严重虐待的样本在此，我们将通过四个特异性的方法来验证ΔS-Cys-Alb作为P/S完整性的标志物。 目的：具体目的1：通过实验验证ΔS-Cys-Alb的预测范围， 新鲜收集的癌症患者血浆和血清样品。具体目标2：系统地绘制ΔS- Cys-Alb在“现实”治疗条件下表现出行为，并将已知的不稳定癌症标志物与ΔS-Cys- 阿尔布具体目标3：进行盲态挑战，以定量ΔS-Cys-Alb鉴别生物标本的能力 在癌症患者血浆和血清样品中的治疗。具体目标4：使用ΔS-Cys-Alb， 量化从代表性大型癌症研究中收集的血浆样品的完整性， 根据单一SOP在多个地点收集，并最终转移到单一储存库。

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.