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Impact of preanalytical freezing delay time on the stability of metabolites in oral squamous cell carcinoma tissue samples.

Author(s): Wang S, Sun Y, Zeng T, Wu Y, Ding L, Zhang X, Zhang L, Huang X, Li H, Yang X, Ni Y, Hu Q

Publication: Metabolomics, 2022, Vol. 18, Page 82

PubMed ID: 36282338 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the metabolome of oral squamous cell carcinoma and normal adjacent specimens subjected to 30-120 min delays to freezing by untargeted liquid chromatography–mass spectrometry/mass spectrometry.  Paired tumor and normal adjacent specimens were collected from five patients with oral squamous cell carcinoma during surgical resection. After the blood flow was surgically isolated from the tissue specimens (0 min), the tumor was fully resected (5 min) and 11 segments were generated. Segments were stored on wet ice for 30, 40, 50, 60, 70, 80, 90 and 120 min before snap-freezing in liquid nitrogen. All specimens were stored at -80°C until homogenization in liquid nitrogen and metabolome extraction with methanol. The metabolome was analyzed by untargeted liquid chromatography–mass spectrometry/mass spectrometry using ACQUITY UPLC HSS T3 columns.

   Summary of Findings:

   A total of 190 metabolites were identified in snap-frozen oral squamous cell carcinoma and normal adjacent tissue specimens. Unsupervised clustering grouped both tumor and normal adjacent specimens based on patient, not on the duration of the freezing delay. Thus, the authors conclude that individual variability has a greater effect on the metabolome than a delay to freezing. However, after removal of inter-specimen differences using the limma software package, some metabolites were identified that consistently increased or decreased with progressive delays to freezing. Metabolites formed 10 clusters based on similar trends in change during delays to preservation. Levels of 39 and 38 metabolites were significantly correlated (P<0.05) with time to freezing in normal adjacent and tumor tissues, respectively. In normal adjacent specimens, 6 metabolites decreased and 33 increased with delayed freezing; in tumor specimens, 12 metabolites decreased and 26 increased with delayed freezing. In normal tissue, the metabolites affected by time to freezing included those involved in fatty acid metabolism, glutamate metabolism, and the TCA cycle. In tumor tissues, metabolites in the TCA cycle, purine metabolism, and alanine, aspartate and glutamate metabolism were enriched, with the TCA cycle and purine metabolism pathways significantly affected. The authors suggest that  account the stability of the metabolites analyzed should be taken into account when establishing maximal acceptable delays to preservation.

   Biospecimens

   • Tissue - Oral Cavity

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	30 min 40 min 50 min 60 min 70 min 80 min 90 min 120 min

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