An accessible, absorbance-based plate reader assay to assess cumulative exposure of blood plasma & serum to thawed conditions.

The integrity of human plasma and serum (P&S) is crucial for reliable biomarker discovery and validation, but it is affected by preanalytical exposure to thawed conditions (temperatures > -30 °C). The limited number of existing quality control methods for assessing plasma or serum (P/S) integrity often require resource-intensive techniques like mass spectrometry. We have developed and validated an accessible, cost-effective absorbance-based plate reader assay based on the oxidative ex vivo consumption of most small molecule thiols & disulfides (SMT&D) by albumin. The assay separates SMT&D from proteins, then uses tris(2-carboxyethyl)phosphine to reduce SMT&D, with subsequent quenching by 4-azidobenzoic acid, followed by reaction of thiols with Ellman's reagent. The method was optimized for yield, including steps to remove trace metals and minimize thiol re-oxidation. Due to inter-individual matrix effects, it was determined that measurements of each specimen before and after intentional expiration (which produce a single Δ-absorbance value, calibrated to Δ-SMT&D) are required to forensically evaluate prior exposure to thawed conditions. The assay demonstrated robust linearity, precision, and accuracy across diverse inter-individual matrices. Stability assessments at 23 °C, 4 °C and -20 °C revealed consistent exponential decay patterns in SMT&D, similar to those observed in the established LC/MS-based ΔS-Cys-Albumin assay. Estimated population reference ranges for P&S demonstrated sufficient dynamic range for the analysis of single samples with unknown histories. This potential was verified with blinded challenges. The assay provides a practical, readily accessible tool for quantifying P&S exposure to thawed conditions, facilitating improved quality control for investigators working with P&S for clinical or research purposes.