Assessment of the effects of repeated freeze thawing and extended bench top processing of plasma samples using untargeted metabolomics.
Author(s): Goodman K, Mitchell M, Evans AM, Miller LAD, Ford L, Wittmann B, Kennedy AD, Toal D
Publication: Metabolomics, 2021, Vol. 17, Page 31
PubMed ID: 33704583 PubMed Review Paper? No
Purpose of Paper
The purpose of the paper was to compare levels of compounds by untargeted metabolomics in matched plasma subjected to 0-3 freeze/thaw cycles and in matched plasma subjected to thawing on ice for 0, 2, 4, and 6 h.
Conclusion of Paper
The number of compounds with a significant >15% change in level compared to non-freeze/thaw cycled specimens increased with increasing number of freeze/thaw cycles (7 after 1 cycle, 14 after 2 cycles, and 19 after 3 cycles). Five of the seven compounds that were altered after a single freeze/thaw cycle were also found to be significantly altered after 2 or 3 freeze/thaw cycles and an additional seven compounds were significantly affected by 2 and 3 freeze/thaw cycles. Further investigation of the timeline identified trends for three compounds classified as antioxidants, two compounds classified as central carbon metabolism, a single compound characterized as lipid metabolism, and three compounds classified as nucleotides.
Compared to specimens analyzed immediately after thawing, specimens thawed on ice for 2 h had lower levels of three compounds, specimens thawed on ice for 4 h had lower levels two of compounds, and specimens thawed on ice for 6 h had lower levels of four compounds and higher levels of one compound. Only thioproline was decreased at all three timepoints. While not significantly altered at any timepoint, a trend toward increasing maltose and dihydroorotate was found with increased thaw duration.
Studies
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Study Purpose
The purpose of the study was to compare levels of compounds by untargeted metabolomics in matched plasma subjected to 0-3 freeze/thaw cycles and in matched plasma subjected to thawing on ice for 0, 2, 4, and 6 h. EDTA blood from 24 donors (diagnosis not specified) was collected into Vacutainer tubes and centrifuged (speed and duration not specified) to obtain plasma. Plasma aliquots (four/patient) were stored at -80°C for <8 months. Aliquots from twelve patients were thawed on ice for 0, 2, 4, or 6 h. Aliquots of plasma from the remaining twelve patients was subjected to up to three cycles of thawing on ice, sampling, vortexing, centrifugation, and refreezing at -80°C before metabolomic analysis by ACQUITY ultra-performance liquid chromatography (UPLC) and Q-Exactive high resolution/accurate mass spectrometer interfaced with heated electrospray ionization (HESI-II). Instability was defined as >15% change relative to control (0 freeze/thaw cycle or 0 h thaw), q value ≤0.3, and P≤0.05.
Summary of Findings:
A total of 7, 14, and 19 compounds were found to be unstable after 1, 2, and 3 freeze/thaw cycles, respectively. Three (E,E isoform of bilirubin, sphingosine, and maltose) of the four compounds that increased after a single freeze/thaw cycle were also found to be significantly higher after 2 or 3 freeze/thaw cycles. Two (glycerol-3-phosphate and dihydroorotate) of the four compounds that decreased after a single freeze/thaw cycle were also found to be significantly lower after 2 or 3 freeze/thaw cycles. An additional seven compounds were significantly affected by 2 and 3 freeze/thaw cycles but considered stable after a single freeze/thaw cycle. More changes were apparent when the unstable compounds were characterized as antioxidants, central carbon metabolism, lipid metabolism, and nucleotides and analyzed as a timeline rather than as single timepoint. Freeze/thaw cycles consistently led to decreases in the antioxidant compounds threonate and cysteine-glutathione disulfide and to increases in bilirubin. Of the central carbon metabolism compounds, both maltose and 2-hydroxyglutarate increased with freeze/thaw cycles. Of the lipid metabolism compounds, sphingosine increased with increased cycles but the difference was not significant for any individual pair. Investigation of the biomarkers in the nucleotide family found freeze/thaw cycles increased AMP and ADP ribose but decreased dihydroorotate.
Compared to specimens analyzed immediately after thawing, specimens thawed on ice for 2 h had lower levels of three compounds, specimens thawed on ice for 4 h had lower levels two of compounds, and specimens thawed on ice for 6 h had lower levels of four compounds and higher levels of one compound. Only thioproline was decreased at all three timepoints but levels of cysteine-glutathione disulfide were decreased relative to control in specimens thawed for 4 or 6 h. Importantly, while not significantly altered at any timepoint, a trend toward increasing maltose and dihydroorotate was found with increased thaw duration.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Carbohydrate LC-MS or LC-MS/MS Lipid LC-MS or LC-MS/MS Small molecule LC-MS or LC-MS/MS Protein LC-MS or LC-MS/MS Peptide LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
Storage Thaw duration 0 h
2 h
4 h
6 h