Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.
Author(s): Jeffs JW, Jehanathan N, Thibert SMF, Ferdosi S, Pham L, Wilson ZT, Breburda C, Borges CR
Publication: Mol Cell Proteomics, 2019, Vol. 18, Page 2121-2137
PubMed ID: 31324658 PubMed Review Paper? No
Suggested by: Chad Borges, Arizona State University
Purpose of Paper
The purpose of this paper was to develop and validate a mass spectrometric assay (the change in S-cysteineylated form of albumin, ∆S-Cys-Alb) that can be used to quantify the cumulative exposure of frozen serum and plasma specimens to thaw temperatures (> -30°C).
Conclusion of Paper
Both initial S-Cys-Alb levels and the change in (𝛥)S-Cys-Alb levels in plasma from patients undergoing a nonacute cardiac event were modestly but significantly higher than in case-matched serum specimens (p<0001 for both). Using time course experiments the authors determined the rate law for S-Cys-Alb formation via oxidation for delays up to 28 h at 23°C. ∆S-Cys-Alb values of control plasma and serum samples stored at -80°C were significantly higher than case-matched specimens exposed to warmer temperatures (23°C to -20°C) for 2 to 48 h (p<0.1). When a subset of aliquots that were exposed to longer thaw durations (23°C for 24, 48, 72 h; 4°C for 7 or 14 days; -20°C for 60 or 90 days) were examined individually, all 60 samples were correctly assigned as a control (-80°C) or thawed specimens based whether ∆S-Cys-Alb values fell above or below 2.58 standard deviations of mean ∆S-Cys-Alb values for reference plasma and serum samples. . Serum specimens from healthy donors that displayed unexpectedly elevated glucose levels and significantly lower ∆S-Cys-Alb levels than observed among serum of lung cancer patients (p<1 x 10-20) were found to have been housed in a freezer that experienced a 3-4 day power outage. Based on the determined rate law for S-Cys-Alb formation the authors estimate that the control samples were exposed to ambient temperature (23°C) for 23 h.
Studies
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Study Purpose
The purpose of this study was to develop a mass spectrometric assay that can be used to quantify the cumulative exposure of frozen serum and plasma specimens to thaw temperatures (> -30°C). The assay developed entails quantification of the S-cysteineylated form of albumin (S-Cys-Alb) by mass spectrometry in plasma and serum specimens, initially and after sample incubation at 37°C for 18 h; thus, ∆S-Cys-Alb reflects the relative change in sample S-Cys-Alb. S-Cys-Alb levels were determined using blood collected into K2EDTA, sodium heparin, and serum tubes that were processed within 30 min of collection (plasma) or allowed to clot for 40 min (serum) prior to processing, aliquoting, and freezing at -80°C. The reference range of S-Cys-Alb and calculated ∆S-Cys-Alb were assessed using blood from 106 patients undergoing testing for nonacute cardiac events that was collected in to K2EDTA and BD vacutainer red top serum tubes. The time course of changes in S-Cys-Alb was assessed in plasma and serum from three of the patients that were incubated at 23°C for up to 4 days, at 4°C for up to 28 days, and at -20°C for up to 65 days. ∆S-Cys-Alb was determined by mass spectrometry in plasma and serum samples following dilution (1:1000, in 0.1% TFA) and injection into the LC-MS immediately (initial S-Cys-Alb) and after incubation in a 37°C oven for 18 h (maximum S-Cys-Alb).
Summary of Findings:
Both initial S-Cys-Alb levels and ∆S-Cys-Alb levels in plasma from patients undergoing a nonacute cardiac event (106 patients) were modestly but significantly higher than case-matched serum specimens (p<0001 for both). A timecourse experiment using plasma and serum from three patients revealed that ∆S-Cys-Alb levels were lower in both plasma and serum samples that experienced thaw conditions at 23°C for up to 4 days, at 4°C for up to 28 days, and at -20°C for up to 60 days compared to those that did not experience a thaw event. The authors inferred that S-cysteinylation occurs ex vivo in samples during exposure to temperatures <-30°C, resulting in a smaller difference between initial and maximum values of S-Cys-Alb (∆S-Cys-Alb) in thawed samples compared to those that were not thawed. The time course experiment allowed the authors to determine the rate law for S-Cys-Alb formation via oxidation for delays at 23°C, which facilitated prediction of thaw durations.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cardiovascular Disease
- Normal
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Thaw duration 0-4 d
0-28 d
0-65 d
Biospecimen Acquisition Biospecimen location Plasma
Serum
Storage Thaw temperature/condition 23°C
4°C
-20°C
-
Study Purpose
The purpose of this study was to validate a mass spectrometric assay that quantifies the cumulative exposure of frozen serum and plasma specimens to thaw temperature (23°C). The assay developed involves the mass spectrometry quantification S-Cys-Alb in plasma and serum specimens initially and after sample incubation at 37°C for 18 h; thus, ∆S-Cys-Alb reflects the relative change in sample S-Cys-Alb levels. S-Cys-Alb levels were measured in blood collected from a single donor on 3 different days (6 days between draws) into K2EDTA and serum tubes that were processed within 30 min of collection (plasma) or allowed to clot for 40 min (serum) prior to processing, aliquoting, and freezing at -80°C. Aliquots (50 µl) of plasma and serum samples were either stored at -80°C or subjected to 23°C for 2 or 4 h; 4°C for 8 or 16 h; -20°C for 24 to 48 h for group analysis or 23°C for 24, 48, 72 h; 4°C for 7 or 14 days; -20°C for 60 or 90 days for analysis of individual samples. The integrity assay was also applied to serum specimens collected from stage I lung cancer patients (n=36) and control healthy donors (n=40) collected as part of another study. Samples were analyzed for ∆S-Cys-Alb in a blinded fashion by mass spectrometry; plasma and serum samples were diluted (1:1000, in 0.1% TFA) and injected into the LC-MS immediately (initial S-Cys-Alb) and after incubation in a 37°C oven for 18 h (maximum S-Cys-Alb).
Summary of Findings:
Distributions of ∆S-Cys-Alb values for plasma and serum samples analyzed immediately after -80°C storage and those subjected to different thaw temperature and durations before analysis overlapped. When analyzed as groups, ∆S-Cys-Alb values of control plasma and serum samples stored at -80°C were significantly higher than all other groups that included exposure to warmer temperatures (23°C to -20°C) for 2 to 48 h (p<0.1). When aliquots that were exposed to longer thaw durations (23°C for 24, 48, 72 h; 4°C for 7 or 14 days; -20°C for 60 or 90 days) were examined individually, all 60 samples were correctly assigned as a control (-80°C) or thawed specimen based whether ∆S-Cys-Alb values fell above (control) or below (thawed) 2.58 standard deviations of mean ∆S-Cys-Alb values of reference plasma and serum samples.. Serum specimens from healthy donors that displayed unexpectedly elevated glucose levels also displayed significantly lower ∆S-Cys-Alb levels compared to serum from stage I lung cancer patients (p<1 x 10-20). Upon further inquiry, the authors confirmed that the freezer housing serum samples from control donors experienced a 3-4 day power outage. Based on the rate law for S-Cys-Alb formation ex vivo, the authors estimate that the control samples were exposed to ambient temperature (23°C) for 23 h.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Thaw duration 24 h
48 h
72 h
7 d
14 d
60 d
90 d
Storage Thaw temperature/condition 23°C
4°C
-20°C
-80°C