Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.
Author(s): Servais MD, Galtier F, Nouvel A, Rebuffat S, Laget J, Géan A, Provost N, Lorcy F, Rigau V, Couderc G, Géraud P, Nocca D, Builles N, De Préville N, Lajoix AD
Publication: PLoS One, 2022, Vol. 17, Page e0278780
PubMed ID: 36584180 PubMed Review Paper? No
Purpose of Paper
This paper examined whether cold ischemia time (5-25 min on wet ice), and -80°C storage of snap-frozen tissue (≤4 y) or extracted RNA (≤4 y) affects the integrity of RNA (RNA integrity number, RIN; the percentage of fragments >200 nt, DV200) isolated from snap-frozen biopsy tissues (muscle; liver; subcutaneous adipose tissue, SCAT; visceral adipose tissue, VAT) collected during bariatric surgery from obese patients with type 2 diabetes, or insulin sensitivity or resistance. Effects of storing PAXgene blood specimens at -80°C for 4-65 months on DNA concentration and quality were also investigated.
Conclusion of Paper
Of the 57 tissue specimens that experienced different cold ischemia times (5-25 min) on wet ice, 36 had an A260/280 ratio that exceeded the authors’ purity threshold of ≥2.0 (range: 1.88-2.09), and 50 exceeded the authors’ RIN threshold of ≥7.0. The mean RIN of liver biopsies (6.82) was significantly lower than that of the other tissue biopsies examined (muscle, RIN = 7.76, p<0.01; SCAT, RIN=7.75, p<0.01; VAT, RIN=7.83, p<0.001). While the authors state that RIN did not differ significantly among the tissue types and cold ischemia times examined (≤15 min, ≤20 min, ≤25 min (adipose tissue only)), details on statistical analysis and p-values were not provided for these comparisons. No differences in intra- or inter-patient cycle threshold (Ct) values of six housekeeping genes were observed among the cold ischemia times for the VAT, liver, and muscle specimens with a RIN >6.7 (37 samples in total) evaluated, although details on statistical analysis were not provided.
RINs of RNA that was freshly extracted from liver, muscle, and VAT specimens stored at -80°C for 3 and 4 y were higher than RINs obtained from RNA extracted before storage of the same tissue specimens (SCAT, RIN=8.56 and 8.68 versus 7.75 and 8.57, respectively, p<0.01; VAT, RIN=8.73 and 8.50 versus 7.89 and 8.39, respectively, P<0.001; muscle, RIN=7.52 and 7.94 versus 7.76 and 7.28, respectively, P<0.05; liver, RIN=7.98 and 8.35 versus 6.83 and 7.70, respectively, P<0.001). When RNA extracted before frozen storage (3-4 years prior) was reanalyzed, RINs were comparable to the original data for SCAT and muscle and significantly higher for VAT (8.34 versus 7.89-8.39, P<0.05) and liver (8.35 versus 6.83-7.70, P<0.001). DV200 values of SCAT, VAT, liver, and muscle extracted after tissues were stored at -80°C for 1 year (88-94%) were comparable to values obtained from frozen, non-case-matched tissue controls that were stored for less than 1 year before extraction and analysis (89-93%). The pathologist concluded that all SCAT, muscle, and liver specimens (from three patients) that were stored for 7, 29, and 43 months at -80°C prior to staining and evaluation were of satisfactory quality, with the exception of one SCAT specimen that lacked adequate tissue; the authors concluded that the duration of frozen storage did not affect tissue quality when determined by histological evaluation.
The mean concentration of DNA isolated from PAXgene Blood DNA tubes, was not significantly different when analyzed immediately or following storage of extracted DNA at -80°C for 4-65 months (330 ± 125 µg/ml versus 308 ± 122 µg/ml). No evidence of DNA fragmentation was observed for any of the samples when assessed by gel electrophoresis, regardless of whether DNA samples were stored and for how long.
Studies
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Study Purpose
This study investigated the potential effects of cold ischemia time (5-25 min) on tissue quality and RNA integrity markers (RIN, DV200, generation of qRT-PCR amplicons) in tissue biopsy specimens from five patients diagnosed as obese (BMI ≥35 kg/m2) with type 2 diabetes, insulin sensitivity, or insulin resistance for the Collection of Metabolic Tissues (COLLECT) Biobank. Biopsies were sampled from liver, muscle, and subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) during bariatric surgery. Biopsies were dissected in a sterile Petri dish on wet ice with a sterile scalpel into smaller segments: 50 mg of adipose tissue (11 VAT samples, 18 SCAT samples), 20 mg liver (11 samples), and 20 mg muscle (12 samples). Tissue samples experienced the following cold ischemia times in the Petri dish on wet ice: ≤15 min (all tissue types), ≤20 min (all tissue types), ≤25 min (adipose tissue only). Tissues were transferred to a cryotube and snap frozen in a cryogenic tank containing liquid nitrogen, were transported in a dry shipper containing liquid nitrogen to the biobank, then stored in a -80°C freezer. Tissue (20 mg skeletal muscle, 20 mg liver, 50 mg adipose) was then homogenized in QIAzol Lysis Reagent with a gentleMACS Dissociator, and total RNA was extracted with the RNeasy (Lipid Tissue) Mini Kit and treated with DNase. RNA concentration was determined with a DropSense 96 machine. A Bioanalyzer 2100 machine was used to determine RIN and DV200 value. Total RNA (1.0 µg for liver and 0.5 µg for all other tissues) was reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor. Six housekeeping genes (beta-actin, ACTB; beta-2 microglobulin, B2M; glyceraldehyde 3-phosphate dehydrogenase, GAPDH; hypoxanthine phosphoribosyltransferase, HPRT; TATA-box binding protein, TBP, 18S rRNA) were amplified by qPCR using cDNA (20 ng for liver and 10 ng for all other tissues), a QuantiFast SYBR Green PCR Kit, and a 7500 Fast Real-time PCR System.
Summary of Findings:
Of the 57 tissue specimens that experienced different cold ischemia times (5-25 min) on wet ice, 36 had an A260/280 ratio that exceeded the authors’ purity threshold of 2.0 (range: 1.88-2.09), and 50 exceeded the authors’ RIN threshold of ≥7.0. The mean RIN of RNA from liver specimens (6.82) was significantly lower than that of the other tissue types examined (muscle, RIN = 7.76, p<0.01; SCAT, RIN=7.75, p<0.01; VAT, RIN=7.83, p<0.001). While the authors state that RIN did not differ significantly among the tissue types and cold ischemia times examined (≤15 min, ≤20 min, ≤25 min (adipose tissue only)), details on statistical analysis were not provided for these comparisons. No differences in intra- or inter-patient cycle threshold (Ct) values of six housekeeping genes were observed among the cold ischemia times for the VAT, liver, and muscle specimens with a RIN >6.7 (37 samples in total) evaluated, although details on statistical analysis were not provided.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Diabetes Type 2
- Obesity
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Muscle
Liver
Subcutaneous adipose tissue (SCAT)
Visceral adipose tissue (VAT)
Real-time qRT-PCR Specific Targeted nucleic acid ACTB
B2M
GAPDH
HPRT
TBP
18S
Biospecimen Acquisition Cold ischemia time ≤15 min
≤20 min
≤25 min (adipose tissue only)
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Study Purpose
This study compared RNA integrity markers (RIN, DV200) in a non-specified number of case-matched tissue biopsy specimens that were analyzed immediately or after storage at -80°C for 1, 3, or 4 years and RIN between RNA immediately after extraction and at re-analysis after 3-4 years of storage at -80°C. Tissue quality was assessed by a pathologist using hematoxylin and eosin-stained, OCT-embedded frozen sections of subcutaneous adipose tissue (SCAT), visceral adipose tissue (VAT), muscle, and liver specimens from three patients after 7, 29, or 43 months of storage at -80°C. Biopsies were sampled from the liver, muscle, SCAT, and VAT of patients diagnosed as obese (BMI ≥35 kg/m2) with type 2 diabetes, insulin sensitivity, or insulin resistance during bariatric surgery. Biopsies were dissected in a sterile Petri dish on wet ice with a sterile scalpel into small segments (50 mg of adipose tissue, 20 mg liver, and 20 mg muscle), transferred to a cryotube, snap frozen in a cryogenic tank containing liquid nitrogen within 5-25 min of excision, and transported in a dry shipper containing liquid nitrogen to the biobank. Tissues (unspecified number) underwent RNA extraction immediately or were stored in a -80°C freezer for 1, 3, or 4 years. For RNA extraction, tissue (20 mg skeletal muscle, 20 mg liver, 50 mg adipose) was homogenized in QIAzol Lysis Reagent with a gentleMACS Dissociator, and total RNA was extracted with the RNeasy (Lipid Tissue) Mini Kit and treated with DNase. RNA concentration was determined with a DropSense 96 machine. A Bioanalyzer 2100 machine was used to determine RIN and DV200 values. Tissue quality of SCAT, muscle, and liver specimens from three patients was analyzed by histological evaluation after specimens from three patients were stored for 7, 29, and 43 months at -80°C before being OCT-embedded, sectioned on a cryostat (3 µm), and hematoxylin and eosin stained.
Summary of Findings:
RINs of RNA that was freshly extracted from liver, muscle, SCAT, and VAT specimens stored at -80°C for 3 and 4 y were higher than RINs of RNA extracted before storage of the same tissue specimens (SCAT, RIN=8.56 and 8.68 versus 7.75 and 8.57, respectively, p<0.01; VAT, RIN=8.73 and 8.50 versus 7.89 and 8.39, respectively, P<0.001; muscle, RIN=7.52 and 7.94 versus 7.76 and 7.28, respectively, P<0.05; liver, RIN=7.98 and 8.35 versus 6.83 and 7.70, respectively, P<0.001). When RNA samples that were extracted 3-4 years prior were reanalyzed, RINs were comparable to the original data for SCAT and muscle and significantly higher for VAT (8.34 versus 7.89-8.39, P<0.05) and liver (8.35 versus 6.83-7.70, P<0.001). DV200 values of RNA from SCAT, VAT, liver, and muscle specimens that were analyzed after 1 year of storage at -80°C (88-94%) did not differ from frozen, non-case-matched tissue that were stored for < 1 year (89-93%). The pathologist concluded that all SCAT, muscle, and liver specimens (from three patients) that were stored for 7, 29, and 43 months at -80°C prior to staining and evaluation had satisfactory tissue quality, with the exception of one SCAT specimen that lacked an adequate amount of tissue; the authors concluded that the duration of frozen storage did not affect tissue quality when assessed by histological evaluation.
Biospecimens
Preservative Types
- Frozen
- OCT
Diagnoses:
- Diabetes Type 2
- Obesity
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration Fresh
1 y
3-4 y
Biospecimen Acquisition Biospecimen location Muscle
Liver
Subcutaneous adipose tissue (SCAT)
Visceral adipose tissue (VAT)
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Study Purpose
This study assessed whether prolonged frozen storage of extracted DNA from PAXgene-preserved blood specimens affects DNA concentration or quality. Blood from 12 obese patients (Body mass index, BMI ≥35 kg/m2) with type 2 diabetes, or insulin sensitivity or resistance were collected into PAXgene Blood DNA tubes approximately 1 month prior to bariatric surgery; blood was inverted 8-10 times, transported at room temperature, and stored at 4°C for <28 days before DNA extraction with the PAXgene Blood DNA Kit. The concentration and purity (A260/280) of the DNA sample were assessed immediately with a NanoDrop One spectrophotometer, and DNA degradation was assessed by gel electrophoresis. DNA samples were aliquoted into cryotubes (to minimize freeze-thaw cycling), stored at -80°C for 4-65 months (storage duration varied among patients), and re-analyzed.
Summary of Findings:
The mean concentration of DNA isolated from PAXgene Blood DNA tubes, was not significantly different when analyzed immediately or following storage of extracted DNA at -80°C for 4-65 months (330 ± 125 µg/ml versus 308 ± 122 µg/ml). No clear trend was observed when individual DNA samples stored for the longest durations (40-45 months) at -80°C were compared to case-matched controls that were analyzed immediately after extraction (108-153 versus 88-224 µg/ml, respectively). No evidence of DNA fragmentation was observed for any of the samples when assessed by gel electrophoresis, regardless of whether DNA samples were stored and for how long.
Biospecimens
Preservative Types
- PAXgene
Diagnoses:
- Diabetes Type 2
- Obesity
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration Fresh
4-65 months