Plate reader assays to forensically assess exposure of plasma and serum to thawed conditions

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Project Summary/Abstract High quality (pre)clinical research relies on the use of high quality biospecimens; without the latter it is impossible to have the former. Unfortunately, cancer-relevant clinical analytes from all major biomolecular classes exhibit instability when the plasma or serum (P/S) in which they reside is temporarily exposed to thawed conditions (i.e., temperatures > -30 °C). Though the percentage of biomolecules that exhibit instability under commonly encountered thawed conditions is generally low (e.g. 2-20%), the number of genuine biomarkers waiting to be discovered is orders of magnitude lower—meaning that in mistreated samples the number of false leads (i.e., potential false discoveries) is orders of magnitude greater than the number of bona fide biomarkers waiting to be found. For this reason, every year, poor 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. Under NCI (IMAT) support (R33CA217702) we recently developed a simple, inexpensive, rapid assay requiring 10 µL of P/S known as ΔS-Cys-Albumin that provides an estimate of the amount of time that archived plasma or serum samples have spent at the equivalent of room temperature. This assay works very well but it requires an expensive liquid chromatograph-mass spectrometer (LC-MS) instrument and user expertise to run it. The need for LC-MS limits its availability to most biomedical research labs which further dis-incentivizes its use beyond the limited incentives that most investigators already have to conduct quality control (QC) analysis on their P/S samples. Fortuitously, we have discovered that the ex vivo biochemistry that governs the ΔS-Cys- Albumin marker can be accessed via plate reader-based absorbance or fluorescence measurements. Converting the ΔS-Cys-Albumin assay to a plate reader-based format would put P/S QC at the fingertips of nearly all biomedical research scientists. As such, the goal of this project is to develop and analytically validate plate reader-based absorbance and fluorescence assays that effectively substitute for the ΔS-Cys-Albumin assay. This will be done via two Specific Aims: Specific Aim 1: Develop plate reader-based absorbance and fluorescence assays that, by quantifying low molecular weight thiol and disulfide-bearing molecules in P/S, will be able to detect exposure of plasma and serum specimens to thawed conditions (i.e., temperatures > -30 °C). Specific Aim 2: A) Analytically characterize the assays and perform an initial analytical validation of them. B) Conduct a small population survey of fresh P/S samples and time course aliquots thereof from GI cancer patients and cancer-free age/gender matched controls to begin to link these assays to P/S exposure time to thawed conditions at -20 °C, 4 °C, and 23 °C.