Aliquot-level visual indicators of biospecimen exposure to thawed conditions

项目摘要/摘要 临床肿瘤学家和癌症研究人员都感兴趣的许多生物分析物在以下情况下不稳定 它们所居住的未固定的生物群落暴露在解冻的条件下。例如，美国社会 临床肿瘤学(ASCO)/美国病理学家学会(CAP)指南指出，切除后- 收集用于乳腺癌临床HER2检测的组织的固定暴露时间间隔必须较短 不到1个小时。对于要冷冻的血浆/血清和许多类型的组织标本，适当的 冷藏温度远低于实验室常见的-20°C的冷藏温度。 许多其他原因，每年在分析前样品处理和 存储在生物医学研究中产生了大量代价高昂的虚假线索，这是不可接受的。不出所料， 该领域的专家一致认为，必须立即将这一问题降至最低。目前，几乎没有工具和 而不是一种被广泛接受的方法，通过这种方法来实施基于证据的生物胺跟踪 暴露在解冻的条件下。在实践中，生物医学研究人员实际上很少雇佣任何 基于证据的QA/QC工具-这表明易于使用的、个人等分级别的解冻状态 指标可对改进生物检疫系统质量跟踪产生重大影响，从而对实际质量产生重大影响。 在这里，我们将开发基于颜色变化的指示器，用于12种不同的生物示踪剂处理/存储 最终将通过放置到定制设计的双腔、柔性- 激活的(像发光棒一样)可安装在特制外壳(凹槽中)的透明设备 设计了样品储存瓶。一旦指示剂中高锰酸盐的亮粉色(Mn(Vii))变得透明 由于它还原为Mn(II)(从储存瓶的外部很容易看到)，这表明 小瓶内的样本暴露在超过允许的时间-温度范围内 存储/搬运参数。重要的是，这些指标的子集在-33.5°C或-76°C之前不会冻结， 促进了前所未有的跟踪暴露在非常寒冷但不适当的存储中的能力 温度--所有这些都是任何处理样本的人都很难忽视的。 这些指标的制定将通过两个具体目标来实现： 具体目标1：通过设计反应建立高锰酸盐/草酸盐反应体系的动力学控制 启动条件，从而开发出12个唯一有用的时间/温度指示解决方案。 具体目标2：验证12个指标的反应动力学和温度敏感性表现为 预期--即，A)准确、精确(可重现)，并且在25°C下运行时间显著增加， 4°C、0°C和-20°C-和B)在不同的原液、试剂批次、 分析师，或不同时期的冻结期都参与其中。

Project Summary/Abstract Many biological analytes of interest to both clinical oncologists and cancer researchers are unstable when the unfixed biospecimens in which they reside are exposed to thawed conditions. For example, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines state that the post-excision- up-to-fixation exposure time span for tissues collected for clinical HER2 testing in breast cancer must be less than 1 hour. For blood plasma/serum and many types of tissue specimens that are to be frozen, the proper cold storage temperature is well below the common laboratory freezer temperature of -20 °C. For this and many other reasons, every year improprieties and inconsistencies in pre-analytical sample handling and storage generate unacceptably large numbers of costly false leads in biomedical research. Unsurprisingly, experts in the field agree that this problem must be minimized immediately. Currently there are few tools and not so much as one widely accepted approach by which to implement evidence-based tracking of biospecimen exposure to thawed conditions. In practice, it is actually quite rare for biomedical researchers to employ any evidence-based QA/QC tools at all—which suggests that easy-to-use, individual aliquot-level thawed-state indicators could have a major impact on improving biospecimen quality tracking and therefore actual quality. Here, we will develop color change-based indicators designed for 12 different biospecimen handling/storage requirements that ultimately will be implemented by placement into custom-designed dual-chambered, flex- activated (like a glow-stick) see-through devices that can be housed on (notched into) the outside of a specially designed sample storage vial. Once the bright pink color of permanganate (Mn(VII)) in the indicator turns clear due to its reduction to Mn(II) (as easily seen from the outside of the storage vial), this indicates that the specimen inside the vial has been exposed to a time-temperature span that is outside of the allowed storage/handling parameters. Importantly, subsets of these indicators will not freeze until -33.5 °C or -76 °C, facilitating the unprecedented ability to track exposures to very cold, but nevertheless improper storage temperatures—all in a manner that will be difficult to ignore by anyone handling the samples. Development of these indicators will be accomplished via two specific aims: Specific Aim 1: Establish kinetic control of the permanganate/oxalate reaction system by designing reaction starting conditions so that 12 uniquely useful time/temperature indicating solutions are developed. Specific Aim 2: Verify that reaction kinetics and temperature sensitivities for the 12 indicators behave as expected—that is, A) accurately, precisely (reproducibly), and with substantially increasing run times at 25 °C, 4 °C, 0 °C, and -20 °C—and B) remain accurate and precise when different stock solutions, reagent lots, analysts, or varying periods of freezing are involved.