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

项目摘要/摘要 每年，在分析前处理和储存血浆/血清方面的不当和不一致 (P/S)样本在生物医学研究中产生了大量代价高昂的虚假线索，令人无法接受。专家们在 该领域呼吁立即停止这一问题，或者至少将其降至最低。这个问题特别突出 在NIH赞助的研究范围内很重要：最近的一项研究发现，在NCI赞助的455项研究中 依赖生物检疫剂的校外赠款，63%使用了预先存在的生物检疫剂--其中107 涉及预先存在的P/S。令人惊讶的是，没有为建立先前存在的样本完整性设定标准 在NCI(纳税人)资金用于研究之前，不存在必须达到的质量门槛 预先存在的P/S暴露在解冻状态(包括在-20°C下的临时存储)表示 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 除了标本，没有其他PAV比这更难控制和跟踪--而且，正如研究战略所表明的那样， 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 样品的处理是理想的(即，与SOP一致)。我们的初步数据表明， 基于分子损伤测量的分子水平证据-超过或超出单独依赖 标准操作规程--对于明确地建立样品完整性至关重要。然而，到目前为止，还没有金本位标志 P/S的诚信仍然存在。该项目的目标是验证一种简单、廉价、快速的检测方法，需要10微米L 对P/S蛋白的氧化损伤进行了具有代表性的评估 暴露在解冻状态下。这项测试是基于这样一个事实，即S-半胱氨酸化的相对丰度 (氧化)白蛋白(S-半胱氨酸-白蛋白)随时间显著增加(但达到最大值)，当P/S为 在冰点-30°C以上处理/储存。因此，通过在有意的 导致S-半胱氨酸-白蛋白达到最大值的潜伏期，这些值之间的差异，ΔS- Cys-Alb很容易被解释为与所发生的体外氧化程度成反比 首次测量前S-半胱氨酸-白蛋白。因此，例如，ΔS-半胱氨酸-白蛋白的值为零将指示 被严重虐待的样品。在这里，我们将验证ΔS-半胱氨酸-白蛋白作为P/S诚信的标志 目标：具体目标1：实验验证ΔS-半胱氨酸-白蛋白的预测范围 新鲜采集的癌症患者血浆和血清样本。具体目标二：系统规划ΔS如何-- Cys-Alb在“现实”虐待条件下的行为，并将已知的、不稳定的癌症标志物与ΔS-Cys- 阿尔布。具体目标3：进行盲法挑战，以量化ΔS-半胱氨酸-白蛋白识别生物胺的能力 癌症患者血浆和血清样本中的不当处理。和具体目标4：使用ΔS-半胱氨酸-白蛋白 量化从具有代表性的大型癌症研究中收集的血浆样本的完整性 在单一SOP下在多个地点收集，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.