Aliquot-level visual indicators of biospecimen exposure to thawed conditions

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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.