Specimen quality evaluation in Canadian biobanks participating in the COEUR repository.
Author(s): Le Page C, Köbel M, de Ladurantaye M, Rahimi K, Madore J, Babinszky S, Bachvarov DR, Bachvarova M, Beauchamp MC, Cass CE, Chadwick D, Colleen C, Damaraju S, Dufour J, Gotlieb WH, Kalloger SE, Portelance L, McAlpine JN, Matte I, Piché A, Shaw P, Roehrl MH, Vanderhyden BC, Watson PH, Huntsman DG, Provencher DM, Mes-Masson AM
Publication: Biopreserv Biobank, 2013, Vol. 11, Page 83-93
PubMed ID: 24845429 PubMed Review Paper? No
Purpose of Paper
The paper assessed RNA and DNA quality in fresh frozen specimens and protein expression levels of matched formalin-fixed, paraffin-embedded (FFPE) specimens from high-grade serous ovarian tumors preserved and stored using different methods at nine independent Canadian biobanks. The paper also examined the effects method of preservation as well as storage temperature and duration on specimen quality.
Conclusion of Paper
Ovarian tumor specimens from nine different biobanks were comparable in DNA integrity, RNA integrity, and immunohistochemical staining for WT1, p53, Ki67, E-cadherin, and cytokeratin 7 (CK-7) despite differences in delay, preservation, and frozen storage temperature and duration. The time interval between surgical resection and storage (≤ 30 min vs. > 30 min) and method of preservation (OCT, RNAlater, or snap freezing) did not significantly affect DNA or RNA integrity. However, specimen storage at -80°C (as opposed to -150°C or LN2) adversely affected DNA integrity while an inverse relationship between the duration of frozen storage and rRNA 28S/18S ratio was observed.
Studies
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Study Purpose
The study compared nucleic acid quality in snap-frozen specimens and protein levels among FFPE specimens. Specimens were procured from high-grade serous ovarian tumors at nine independent Canadian biobanks that differed in processing, preservation, and storage practices. Matched FFPE (≥ 4 mm2) and frozen (≥ 9 mm3) specimens from fifteen cases of high-grade serous ovarian carcinomas per biobank were randomly chosen. Tissues were flash frozen in LN2 (109 specimens), frozen in OCT medium (15 specimens), or preserved in RNAlater (11 specimens) and stored at -80°C, -150°C, or in LN2. RNA quality was assessed with a bioanalyzer and RNA Integrity Number (RIN) was calculated. DNA quality was determined by PCR amplification of four different sized ß-globin amplicons (268, 536, 989, 1327 bp). TMAs created from FFPE blocks were sectioned at 4 mm; stained with H&E and reviewed by a pathologist to confirm tumor content; and immunohistochemically stained for WT1, p53, Ki67, E-cadherin, and cytokeratin 7 (CK-7).
Summary of Findings:
There was no statistically significant difference in the 260/280 nm ratios of DNA from snap-frozen ovarian tumor specimens from different biobanks (P = 0.593) and 85.8% (115 specimens) were of good quality with at least three amplified ß-globin bands. Ribosomal 28S/18S RNA ratios were ≥ 1 in 82% of specimens (112 out of 134 specimens) with 63% of those (85/134) with a ratio between 1.5 and 2. The average RIN value of the 135 samples was 7 (range 2.4–9.5) with 60% (80/135) assigned a RIN value of 7 or more, 31% (41/135) between 4 and 7, and only 10% (13/135) below 4. A significant difference in RNA quality was observed among specimens from a single site (P < 0.001) due to a higher number of samples with low RIN values (RIN = 4.71 – 0.6). The processing time interval between surgical resection and storage (≤ 30 min vs. > 30 min) did not significantly affect DNA integrity as determined by PCR amplification of at least 3 ß-globin fragments (100% vs. 92% for specimens processed within or in excess of 30 min; P = 0.07) or RNA integrity as determined by 28S/18S rRNA ratios or RIN values (P = 0.67). Specimen storage in OCT or RNAlater at -80°C or in LN2 did not affect the quality of extracted DNA or RNA in comparison to specimens stored at the same temperatures in the absence of media, although sample size was low (15 OCT specimens, 11 RNAlater). Storage temperature affected DNA integrity, as the number of specimens that only generated one ß-globin amplicon was significantly higher among specimens stored at -80°C (14 of 69 specimens) compared to those stored at -150°C (1 of 19 specimens) or in LN2 (1 of 46 specimens) (P = 0.001); however, the temperature of frozen storage did not affect RNA quality (P = 0.238). Storage duration (< 6 months vs. ≥ 6 months) did not affect DNA integrity (P = 0.16) or RIN value (P < 0.001) but a trend toward a higher 28S/18S ratio was observed for specimens stored for ≥ 6 months (P = 0.057). The percentage of immunopositive cells did not differ among sites for most of the clinical biomarkers investigated: WT1 (P=0.57), p53 (P=0.07), Ki67 (P=0.80), or E-cadherin (P=0.011) and only CK7 displayed a weak decline in immunostaining with block age (r = - 0.183, P = 0.065).
Biospecimens
Preservative Types
- Formalin
- RNAlater
- OCT
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR Protein Tissue microarray Protein Immunohistochemistry RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time ≤30 min
>30 min
Immunohistochemistry Specific Targeted peptide/protein WT1
p53
Ki67
E-cadherin
CK-7
Storage Storage duration <6 months
≥ 6 months
Biospecimen Preservation Type of fixation/preservation OCT
RNAlater
Snap frozen
Formalin (buffered)