MSACL 2022 Abstract

Introduction: Biomolecular integrity can be compromised when specimens are exposed to improper storage or handling conditions. Measurement of compromised analytes can then lead directly to the generation of incorrect and potentially misleading results--without any indication that this has occurred. We recently introduced an LC/MS based marker of blood plasma/serum exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of plasma/serum to temperatures greater than their freezing point of -30 °C. To begin to empirically link ΔS-Cys-Albumin to clinical proteins that may be destabilized by common thawed-state exposures, we have simultaneously measured both ΔS-Cys-Albumin and 21 such proteins in plasma samples from 24 patients during thawed-state time courses conducted at 23 °C, 4 °C, and -20 °C.

Methods: ΔS-Cys-Albumin was measured at nine different time points per exposure temperature in K2EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and -20 °C using a dilute-and-shoot LC/MS method that requires only 10 µL of plasma. Twenty-one clinically relevant proteins were then measured at four different time points per sample and temperature using multiplexed ELISA on the Luminex platform. Protein stability was assessed by mixed effects models with correction for multiple comparisons. Coordinated shifts in stability between ΔS-Cys-Albumin and several of the 21 clinical proteins across all temperature and time exposures were documented by Pearson correlation.

Results: As expected, ΔS-Cys-Albumin dropped from a mean value of 20% to under 5% within 96 hrs at 23 °C, 28 days at 4 °C, and 65 days at -20 °C. On average, 5 of the 21 proteins significantly increased or decreased in apparent concentration at each exposure temperature (p < 0.0008 and relative concentration shift of > 10%). Considering the protein stability data from all temperatures together, a linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r > 0.9; p < 0.01)—regardless of the specific time/temperature of exposure.

Conclusions: ΔS-Cys-Albumin is useful for forensically tracking approximate times of exposure of archived plasma/serum samples to thawed conditions (> -30 °C). The data to be presented now enable quantitative estimation of resulting plasma specimen damage relevant to clinical proteins based on a single measurement of ΔS-Cys-Albumin.