Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Quality Assessment of Cryopreserved Human Biological Samples from the Biobank of Barretos Cancer Hospital.

Author(s): Neuber AC, Komoto TT, da Silva ECA, Duval VDS, Scapulatempo-Neto C, Marques MMC

Publication: Biopreserv Biobank, 2023, Vol. 21, Page 74-80

PubMed ID: 35613409 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to assess the integrity of DNA (DNA integrity number, DIN) and RNA (RNA integrity number, RIN) isolated from tumor tissue, buffy coat, and peripheral blood mononuclear cells (PBMCs) after frozen storage at -80°C for up to 9 years; the DNA and RNA integrity of buffy coat and PBMC samples stored at -30°C and -80°C for 2 to 7 years were also compared.  In total, 317 retrospectively collected tumor specimens that had paired blood specimens (PBMCs or buffy coat) and met the study criteria of a tumor percentage ≥60% and ≤20% necrosis were included in analysis. Tumor specimens that had been stored at -80°C for up to 9 years  were embedded in OCT compound, sectioned (5 µm thick), mounted on glass slides, stained with hematoxylin and eosin, and microdissected.  DNA and RNA was extracted from 20-30 mg of tumor tissue using the QIAsymphony DNA and RNA Mini Kits, respectively, after tissue homogenization with ceramic beads.  A subset of buffy coat (n=171) and PBMC (n=37) specimens were stored at -30°C for 2 to 7 years before being transferred to -80°C (number of specimens not specified), while the remaining specimens were stored at -80°C for the entire 9 year period. Buffy coat specimens were thawed at 37°C and centrifuged (200 rpm at 15-25°C for an unspecified duration) before DNA extraction with the QIAsymphony DNA Midi Kit. DNA was extracted from PBMC specimens using the DNA Blood Mini Kit. DNA and RNA were quantified using a NanoDrop spectrophotometer and the Qubit fluorimeter with dsDNA B and RNA-BR Kits.  DNA and RNA integrity numbers (DIN and RIN, respectively) were determined with A TapeStation 4200 and the DNA Genomic Kit and RNA Screen Tape Assay, respectively. The authors applied a minimum acceptability DIN/RIN ratio threshold of ≥7.  Cut-offs for acceptable storage durations based on indicators of degradation were determined using "the area under the receiver operating characteristic (AUROC) curve."

   Summary of Findings:

   Based on AUROC curve analysis, thresholds of effect for the duration of -80°C storage on DIN and RIN ≥7 were determined for DNA from tumor tissue specimens (<8.1 y. P<0.001), RNA from tumor tissue specimens (<4.5 y, p=0.003), and DNA from buffy coat (<8 y, p<0.001) and PBMCs (7.2 y, p<0.018).  When cutoffs based on these thresholds were applied, 92% of tumor tissue specimens stored at -80°C for <8.1 y had DNA with a DIN ≥7 compared to just 68% of tumor tissue specimens stored for ≥8.1 y (p<0.001).  Similarly, the percentage of tumor tissue specimens yielding DNA with a RIN≥7 was higher among specimens stored at -80°C for <4.5 y versus those stored for ≥4.5 y (45% versus 17%, p<0.001). The percentage of buffy coat samples that had DNA with a DIN ≥7 was 95% versus 74% in specimens stored for <7.2 or ≥7.2 y, respectively (p<0.001), and the percentage of PBMC samples that yielded DNA with a DIN ≥7 was 96% versus 54% in specimens stored for <7.2 or ≥7.2 y, respectively (p=0.003).

   Buffy coat and PBMC samples (data was pooled) stored at -30°C for 2-7 years before being transferred to -80°C had a higher percentage of samples yielding DNA with a DIN <7 than those stored at -80°C for the entire study duration (26% versus 1%, P<0.001).

   Biospecimens

   • Tissue - Bone Marrow
   • Cell - White Cells
   • Tissue - Skin
   • Tissue - Esophagus
   • Tissue - Other
   • Tissue - Head and Neck
   • Tissue - Bladder
   • Tissue - Uterus
   • Tissue - Nerve
   • Tissue - Prostate
   • Tissue - Breast
   • Tissue - Lymph Node
   • Tissue - Stomach

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	Fluorometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Male genital tract Skin Breast Head and neck Gynecological tract Central nervous system Urinary tract Bone marrow Lymph node Thoracic Upper digestive Lower digestive Torso and limbs
   Storage	Storage duration	1-9 y
   Storage	Storage temperature	-30°C (buffy coat and PBMC specimens) -80°C (tumor, buffy coat, and PBMC specimens)

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