Advancing wide implementation of precision oncology: A liquid nitrogen-free snap freezer preserves molecular profiles of biological samples.
Author(s): van der Wijngaart H, Jagga S, Dekker H, de Goeij R, Piersma SR, Pham TV, Knol JC, Zonderhuis BM, Holland HJ, Jiménez CR, Verheul HMW, Vanapalli S, Labots M
Publication: Cancer Med, 2023, Vol. 12, Page 10979-10989
PubMed ID: 36916528 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA integrity, RNA sequencing profiles, and mass spectrometry-based global phosphoproteomic profiles of case-matched core needle biopsies of normal adjacent liver specimens that were (1) preserved by snap-freezing in liquid nitrogen (LN2) within 5 min of resection, (2) preserved in LN2 after a cold ischemia time of 2 h, and (3) preserved using an electric snap freezer device (-73°C) with an adjustable cooling rate that operates independently of liquid nitrogen.
Conclusion of Paper
Analysis of the global phosphoproteome of twelve core needle biopsies from four patients resulted in the identification of 15,262 unique peptides, 68% of which were phosphorylated. Unsupervised clustering analysis clustered samples by patient (not by freezing method), although subclusters of specimens that experienced a cold ischemia time of 2 h formed apart from those with a cold ischemia time ≤5 min (frozen with LN2 or a snap freezer grouped together) in two of the four patients. RIN did not differ significantly between RNA from case-matched specimens snap-frozen in LN2 or with a snap-freezer device (p=0.89). RINs were comparable among RNA from specimens that experienced a cold ischemia time of 2 h or ≤5 min. Unsupervised cluster analysis of gene expression profiles clustered samples by patient and not freezing method or cold ischemia time.
When combinations of different types of vials with different thermal conductive properties and two different coolants were compared, polypropylene vials submersed in LN2 took one of the shortest times to achieve -80°C (2 s) while polypropylene tubes submersed in pre-cooled isopentane took the longest (25 s); unsupervised cluster analysis of the global phosphoproteome did not segregate the two sample types with different freezing rates. Correlations in the phosphoproteome between all samples frozen in different vials with different coolants were very strong (Pearson’s r = 0.93-0.99). Of all the phosphopeptides identified, 51% were present in all five vial/coolant variations and ≤1.6% were unique to only one sample.
Studies
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Study Purpose
This study compared RNA integrity, RNA sequencing profiles, and mass spectrometry-based global phosphoproteomic profiles of case-matched core needle biopsies of normal adjacent liver specimens that were preserved in LN2 after a cold ischemia time of 2 h or within 5 min of resection by snap-freezing in liquid nitrogen (LN2) or with an electric snap freezer device (-73°C) with an adjustable cooling rate that operates independently of liquid nitrogen. Six core needle biopsies (14-gauge needle) were collected from normal adjacent regions of surgically resected livers of three patients due to a cancer diagnosis. Two biopsies from each patient were snap-frozen within 5 min of resection by (1) immersion in LN2 or (2) placement in an aluminum vial and frozen using an electric snap freezer device (-73°C); the remaining two biopsies per patient experienced a cold ischemia time of 2 h at room temperature before being snap-frozen in LN2. All frozen samples were transported in LN2 and stored at -80°C. One biopsy from each patient and freezing method was analyzed by LC-MS/MS and the remaining biopsy underwent next-generation sequencing. Tissue lysates were isolated from 10 µm thick sections in the presence of phosphatase inhibitors and stored at -80°C until reduction, alkylation, and digestion; phosphopeptide enrichment was performed with titanium oxide beads and aliphatic hydroxy-acid modified metal oxide chromatography. LC-MS/MS spectra were searched against the UniProt human reference proteome. RNA was extracted from frozen sections (20 µm thick) using the RNeasy Plus Mini Kit. RNA was quantified using a NanoDrop Spectrophotometer. cDNA libraries were constructed using Illumina’s TruSeq Small RNA Sample Preparation protocol and were sequenced on a HiSeq 2000 machine. Cells from the colorectal cancer cell line (HCT116) were also used to investigate differences in freezing rate due to different thermal conductive properties of vial composition (polypropylene, aluminum, aluminum vials covered in paper tape) and different coolants (LN2, isopentane).
Summary of Findings:
The global phosphoproteome of twelve core needle biopsies from four patients were generated by LC-MS/MS, resulting in the identification of 15,262 unique peptides, 68% of which were phosphorylated. Unsupervised clustering analysis clustered samples by patient (not by freezing method), although subclusters of specimens that experienced a cold ischemia time of 2 h formed apart from those with a cold ischemia time ≤5 min (frozen with LN2 or a snap freezer grouped together) in two of the four patients. RIN did not differ significantly between RNA from case-matched specimens snap-frozen in LN2 or in a snap-freezer device (p=0.89). Specimens that experienced a cold ischemia time of 2 h also yielded RNA with RINs that were comparable to specimens with a cold ischemia time of ≤5 min that were snap-frozen in LN2 or with a snap-freezer device. Unsupervised cluster analysis of gene expression profiles clustered samples by patient and not freezing method or cold ischemia time.
When three types of vial compositions with different thermal conductive properties and two different coolants were compared, polypropylene vials submersed in LN2 took one of the shortest times to achieve -80°C (2 s) while polypropylene tubes submersed in pre-cooled isopentane took the longest (25 s); unsupervised cluster analysis of the global phosphoproteome did not segregate the two sample types with different freezing rates. Samples in aluminum vials covered in paper tape reached -80°C in < 2 s. Correlations in the phosphoproteome between all samples frozen in different vials with different coolants were very strong (Pearson’s r = 0.93-0.99). Of all the phosphopeptides identified, 51% were present in all five vial/coolant variations and ≤1.6% were unique to only one sample.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Protein LC-MS or LC-MS/MS RNA DNA sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Polypropylene
Aluminum
Aluminum vials covered in paper tape
Biospecimen Preservation Cooling or freezing method/ rate Liquid nitrogen
Pre-cooled isopentane
Snap freezer device