Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions*[S]

Abstract

Exposure of blood plasma/serum (P/S) to thawed conditions (> −30 °C) can produce biomolecular changes that skew measurements of biomarkers within archived patient samples, potentially rendering them unfit for molecular analysis. Because freeze-thaw histories are often poorly documented, objective methods for assessing molecular fitness before analysis are needed. We report a 10-μl, dilute-and-shoot, intact-protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The relative abundance of S-cysteinylated (oxidized) albumin in P/S increases inexorably but to a maximum value under 100% when samples are exposed to temperatures > −30 °C. The difference in the relative abundance of S-cysteinylated albumin (S-Cys-Alb) before and after an intentional incubation period that drives this proteoform to its maximum level is denoted as ΔS-Cys-Albumin. ΔS-Cys-Albumin in fully expired samples is zero. The range (mean ± 95% CI) observed for ΔS-Cys-Albumin in fresh cardiac patient P/S (n = 97) was, for plasma 12–29% (20.9 ± 0.75%) and for serum 10–24% (15.5 ± 0.64%). The multireaction rate law that governs S-Cys-Alb formation in P/S was determined and shown to predict the rate of formation of S-Cys-Alb in plasma and serum samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. A blind challenge demonstrated that ΔS-Cys-Albumin can detect exposure of groups (n = 6 each) of P/S samples to 23 °C for 2 h, 4 °C for 16 h, or −20 °C for 24 h—and exposure of individual specimens for modestly increased times. An unplanned case study of nominally pristine serum samples collected under NIH-sponsorship demonstrated that empirical evidence is required to ensure accurate knowledge of archived P/S biospecimen storage history.

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data

“…The fact that blood as a biological fluid experiences a change in temperature from 37°C to 20°C during venous draw, may introduce additional and not fully understood physiological processes should not be overlooked. Apart from plasma storage conditions, the number of freeze-thaw cycles add further pre-analytical variables 41 ; the relative abundance of S-cysteinylated albumin can provide an estimate of the time for which specimens have been exposed to thawed conditions 42 . In particular, extensive delays of several hours prior to separating blood cells from fluids can alter plasma proteome composition due to erythrocyte and platelet degradation 43 .…”

Biochemically Tracked Variability of Blood Plasma Thawed-State Exposure Times in a Multisite Collection Study

“…For nearly all of these candidate markers, the indication of disrupted sample integrity is based on the apparent loss of target analytes beyond their associated reference rangegenerally without a known mechanism of apparent loss or associated rate law. As previously reported, 29,39,40 human serum albumin contains a single free cysteine residue that can undergo spontaneous disulfide bond exchange with free cystine, forming S-cysteinylated albumin (S-Cys-Albumin) to a limited degree in vivo and to a much greater extent when P/S samples are exposed to thawed conditions-that is, temperatures greater than -30°C. 41,42 Charge deconvoluted electrospray ionization (ESI)-mass spectra of albumin that illustrate the ex vivo formation of S-Cys-Albumin and the DS-Cys-Albumin concept as a whole are provided in Figure 1.…”

“…[20][21][22][23][24][25][26][27] In addition, efforts have been made toward the development of standard operating procedures (SOPs) for sample collection, processing, and storage. 1,28 However, the universal adoption of a single rigorous SOP, while in principle appealing as a solution to the development of potential biospecimen integrity problems, can, in practice, become impractical or impossible for laboratory personnel to implement on a rigorous basis for every single sample or in situations where infrastructure issues arise, such as unexpected power losses 29,30 or when other problems occur such as inspection/customs delays during shipment. These potential problems are amplified in studies with multiple collection sites involved-each of which must contend with different personnel, unique infrastructure constraints, and shipment routes.…”

“…31 Recently, using our newly developed DS-Cys-Albumin marker, we discovered a major integrity discrepancy in a set of serum samples collected under NCI sponsorship and slated for distribution by the U.S. National Institutes of Health (NIH). 29 It would be naive to assume that this integrity discrepancy was a single, isolated incident among all archived P/S samples slated for use in future research studies (or that may have already been used in published research studies). As such, there is a present and urgent need for accurate quality control/quality assurance (QC/QA) markers and tests to retrospectively and empirically assess the integrity of archived biospecimens-even those for which a nominally pristine paper trail may exist.…”

Testing the Stability of Plasma Protein and Whole Blood RNA in Archived Blood of Loggerhead Sea Turtles, Caretta caretta

“…Likewise, RNA is increasingly used for the evaluation of gene expression from blood transcriptomes. [9][10][11] Storage temperature can affect concentrations of plasma proteins, 12,13 and RNA degrades rapidly if not properly stored. 14 However, few studies have assessed stability of whole blood RNA and plasma proteins of reptiles.…”