Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Biospecimen Qualification in a Clinical Biobank of Urological Diseases.

Author(s): Kofanova O, Paul S, Pexaras A, Bellora C, Petersons A, Schmitt M, Baker Berjaoui M, Qaoud Y, Kenk M, Wagner H, Fleshner N, Betsou F

Publication: Biopreserv Biobank, 2024, Vol. 22, Page 257-267

PubMed ID: 37878356 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study explored potential differences in analyte levels in biobanked (non-case-matched) serum, plasma, buffy coat, urine supernatants, and urine pellets that were stored in liquid nitrogen vapor for 1, 5, or 10 years; specimens were collected from patients with bladder or prostate cancer. Specifically, levels of  sCD40L, IL-8, VCAM1, hemoglobin, and platelets were compared following storage serum specimens; levels of IL-16, complement C5, cfDNA, hemoglobin, and platelets were compared following storage of plasma specimens; PSA levels and ALP activity were compared following storage of urine supernatants; yield, purity, and integrity of DNA were compared following storage of urine pellets; yields, purity, and integrity of DNA and RNA were compared following storage of buffy coat specimens. Blood was collected in serum separator tubes (serum) and K2EDTA tubes (plasma and buffy coat) and urine was collected into sterile specimen containers from 72 patients with either prostate or bladder cancer using protocols of the McCain GU Biobank (MGB) in Toronto, Canada. Serum, plasma, and buffy coat were separated by centrifugation at 1500 g for 15 min at 4°C. Urine was stored on ice until separation of the pellet and supernatant by centrifugation at 400 g for 10 min at 4°C at a slow deceleration rate. Plasma, serum, buffy coats, urine supernatant, and urine pellets (resuspended in ~2mL supernatant) were snap-frozen and stored in liquid nitrogen vapor in 2 mL cryovials. A total of 24 specimens each were retrieved after 1, 5, or 10 years of storage.  In plasma and serum specimens, hemoglobin levels were measured in serum and plasma specimens using the DetectX Hemoglobin Detection Kit and platelets were enumerated using a CASY cell counter.  In serum specimens, sCD40L, IL-8 and VCAM1 were quantified by ELISA. In plasma specimens, IL-16, Complement C5a, and ICAM1 were quantified by ELISA; cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit and analyzed using a Cell-free DNA ScreenTapes on a 4200 TapeStation instrument. From buffy coat specimens, RNA was extracted using the miRNeasy Mini Kit and analyzed by spectrometry, bioanalyzer, and with an in-house assay based on real-time PCR amplification of GAPDH. DNA was isolated from buffy coat specimens using the automated Chemagic DNA Blood 4k Kit special H24 kit, quantified by spectrophotometer, and the Quant-iT PicoGreen dsDNA Assay Kit and analyzed using a Genomic DNA ScreenTapes on the 4200 TapeStation instrument. In urine supernatant, ALP activity and PSA levels were quantified by ELISA. From urine pellets, DNA was extracted using the QIAamp MinElute Virus Spin Kit, quantified by spectrophotometry and fluorometry, and analyzed using a Genomic DNA ScreenTapes on the 4200 TapeStation.

   Summary of Findings:

   Average platelet counts and hemoglobin, IL-8, and IL-16 concentrations were comparable in blood-based specimens stored in liquid nitrogen vapor for all storage durations investigated (1, 5 and 10 years), which the authors state confirms consistent processing over the study period. Further, the authors state all specimens had IL-8 and IL-16 levels below the quality thresholds, indicating that none experienced a delay to processing at 20C of >= 24 h..  Plasma specimens had a higher average hemoglobin concentration (0.37-39.3 mg/mL versus 0.03-14.5 mg/mL) and a lower percentage of specimens classified as platelet poor (87% versus 100%) than serum. Serum levels of VCAM1 and IL-8 were comparable across the frozen storage durations examined, but levels of scD40L increased significantly with frozen storage (One way ANOVA P=0.038); significant differences were observed in serum specimens stored for 1 or 5 years versus 10 years (3.6 ng/mL and 3.5 ng/mL versus 4.3 ng/mL; p<0.05, both).  In plasma specimens, level of ICAM-1 and complement C5 (but not IL-16) were affected by storage (one-way analysis of variance on ranks, P=0.028), but in pairwise comparisons, significant differences were limited to lower levels of each in plasma specimens stored for 5 years than 10 years (P<0.05, both). The mean percentage of cfDNA fragments in plasma specimens was approximately 72%, regardless of the storage duration, but the variation in the percentage of DNA was significantly lower among specimens stored for 5 years than 1 year (P<0.05). ALP activity and PSA levels were comparable among urine supernatants stored for 1, 5, or 10 years.  The cfDNA yield from urine pellets was highly variable (16.7 ng-1.1 mg, 8.8 ng-2.2 mg, and 7.6 ng-1.5 mg among specimens stored 1, 5, and 10 years, respectively), but no effect of storage duration on yield or DIN was observed. The authors noted that visible blood contamination in some urine pellet specimens affected DNA quantification and that the DNA had a A260/A280 greater than 2, indicating the presence of an RNA carrier. The DNA yield was higher from buffy coat specimens stored for 5 or 10 years than 1 year (P<0.05, both), but the percentage of dsDNA did not differ, and the average DIN was non-significantly lower in specimens stored for 5 or 10 years than 1 year. While A260/A280 values were between 1.7–1.90 for most DNA from buffy coat, the average A260/A280 was higher and less variable among DNA extracted from specimens stored for 5 years than 1 or 10 years (P<0.05). RNA yields were higher from buffy coats stored for 5 years relative to those stored for 1 or 10 years (P<0.05, both), but A260/A280 ratios were not significantly different among the storage durations evaluated. RNA integrity was lower in RNA extracted from buffy coats that were stored for 5 or 10 years than 1 year, with lower average RNA integrity numbers (RIN) observed among specimens stored for 5 or 10 years compared to those stored for 1 year (P<0.05, both) and a decreased percentage of fragments >200 nt (DV200) among specimens stored for 10 years than 1 year (P<0.05). The mRNA quality index (an in-house assay based in real-time RT-PCR amplification of GAPDH) was comparable among the storage durations evaluated, which the authors state indicated a low degree of mRNA degradation. 

   Biospecimens

   • Bodily Fluid - Buffy Coat
   • Bodily Fluid - Plasma
   • Cell - Urine
   • Bodily Fluid - Urine
   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Hematology/ auto analyzer
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   DNA	Spectrophotometry
   Protein	Colorimetric assay
   RNA	Fluorometry
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   Protein	ELISA
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 year 5 years 10 years

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