Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system

Abstract

Background: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation.

Materials and methods: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time.

Results: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times.

Conclusion: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

Fig 1. DNA integrity according time of storage in the biobank.

After extraction, DNA was separated on 0.8% agarose gel and visualized by Gel Red staining on an imager. (A) Electrophoresis of increasing amounts of DNA after extraction from PAXgene tube. (B) Electrophoresis of DNA after extraction and storage at –80°C. The year in which the extraction is carried out is indicated. MM: molecular markers.

Fig 2. Electropherograms of RNA samples obtained for the four tissue types.

Electropherograms were obtained on RNA samples using 2100 Bioanalyser and RNA 6000 Nano chips. Typical electrophoretic traces and RIN were shown for VAT, SCAT, liver, and muscle extracted in 2016. SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.

Fig 3. RIN according to handling time for tissue preparation for the first five patients.

RNA was extracted from frozen tissues. RIN was shown according to handling times of liver, muscle, SCAT and VAT samples resected from the first five patients, before snap–freezing. The handling times between tissue resection and snap–freezing were: ≤ 10, ≤ 15 and ≤ 20 minutes (all tissues) and ≤ 25 minutes (adipose tissues only). SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.

Fig 4. Cycle threshold (Ct) values of six housekeeping genes according to handling time for tissue preparation for the first five patients.

RNA was used to perform a RT–qPCR in order to amplify six housekeeping genes (ACTB, B2M, GAPDH, HPRT, TBP, 18S). Ct values were shown for the genes according the type of tissue samples resected from the first five patients and the handling time between tissue resection and snap–freezing. VAT, visceral adipose tissue.

Fig 5. RIN according to the time of sample storage in the biobank.

For each patient, RIN was shown for RNA samples 1) obtained after extraction from the four tissue types carried out in 2016 or 2017, 2) after their storage at –80°C during 3 to 4 years and re–evaluation in 2020, 3) obtained after destocking of frozen tissues from the biobank and extraction in 2019 and 2020. Results are shown for samples obtained from the first five patients included in 2016 and other patients included in 2017. SCAT, sub–cutaneous adipose tissue; VAT, visceral adipose tissue.

Fig 6. Histological integrity according to the time of sample storage in the biobank.

Sections of SCAT, liver and muscle stored since 2016 were performed and stained with hematoxylin–eosine in 2020. Images were shown according to the material preparation protocol: (A) direct evaluation on frozen tissue sections (B) evaluation after the tissue was thawed, fixed and embedded in paraffin. Magnification is x200. SCAT, sub–cutaneous adipose tissue.