News 2017

The International Society for Biological and Environmental Repositories (ISBER) is an international forum that addresses the technical, legal, ethical, and managerial issues relevant to repositories of biological and environmental specimens. This year's Annual Meeting took place in Toronto Canada - here is a round of abstracts that we felt might be of interest:-

The Effect of Liquid Nitrogen Storage on ctDNA Extraction from Plasma
ISBER abstract reference BRS-8 
Kenney1, J. Sosa-Baez2, E. Lin1, E. Hernandez1, P. McNeil1, C. Mariano1, L. Villafania2, J. Padilla2, A. Samoila2, E. Peerschke2, M. H. Roehrl1
1Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, United States,
2Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States

The identification of a biomarker for cancer that is procured in a safe, non-invasive, and efficient manner for the detection of disease has been made possible in recent years via the discovery of cell-free circulating tumor DNA (ctDNA). The full scope of potential for the uses of ctDNA have not yet been realized; therefore, there is a call to preserve valuable samples in the hope that these resources can be utilized with forthcoming technologies. Traditionally, either due to cost or ease of use, tissue samples are stored at −80°C where they can be kept for many years, whereas in LN2, they can potentially be preserved indefinitely without degradation of the sample for future genetic and proteomic applications. Currently, plasma samples taken for ctDNA extraction are routinely stored at −80°C for extended periods of time. The trend toward banking samples in LN2, however, presents the question of whether the quality ctDNA in plasma samples stored in LN2 will be suitable for assays in the future. It has been shown that LN2 preserves many elements of the plasma, such as antibodies/proteins; however, the ability to preserve ctDNA has not yet been tested.

Methods and Results 
ctDNA from standard plasma samples stored at room temperature, −4°C, −80°C, and in vapor phase LN2 (−180°C) over various amounts of time (days to months) was extracted in a Hamilton easyBlood robot and ctDNA yields were compared.

Storing quality control plasma standards at −80°C produces a mean concentration of ctDNA of 0.95 ng/μl. We stored aliquots of a parallel standard in LN2 for 2 weeks and were able to extract a ctDNA concentration of 1.019 ng/μl. Additional data will be presented at the meeting. Initial results indicate that plasma storage in LN2 is at least not inferior, if not superior, to storage at −80°C. Storage at temperatures higher than −80°C is not recommended and further data will be acquired to study how time-sensitive cfDNA is with respect to “needle-to-freezer” speed and handling.

 We aim to provide evidence that ctDNA extracted from plasma stored in LN2 for various amounts of time will have equal to or better yields of ctDNA than plasma stored at other temperatures. Encouraged by the results, we plan to test more samples at varying temperatures, optimal and suboptimal, for extended periods of time in order to discover the ideal long term storage conditions for plasma used for ctDNA extraction.

Impact of Room Temperature on Tissue Quality as Assessed by RNA Integrity Number (RIN)
ISBER abstract reference HSR-12 
Bhanot1, C. Lai1, M. Santin1, C. Mariano1, P. McNeil1, M. R. Weiser2, M. H. Roehrl1
1Pathology, MSKCC, New York, New York, United States
2Surgery, MSKCC, New York, New York, United States

The practice of oncology is being propelled by new emerging technologies in genomics, transcriptomics, and proteomics to help better understand molecular events that result in tumor initiation, development, and progression. However, the results of genomic, transcriptomic, and proteomic analysis can reflect true alterations only when biospecimens used are of very high quality. Therefore, the impact of pre-analytic variables on tissue quality needs to be studied, as this will help identify areas for improvement in biobanking protocols.

Due to high volume and engagement of biobank personnel in various operations in a busy cancer center, it is not always possible to transfer the tissue in to liquid nitrogen immediately upon receipt. We are studying the impact of transport/storage at room temperature on fresh tissue kept at room temperature (RT) for various time points.

Tissues aliquots from the same patient (tumor and normal) were divided and frozen immediately in vapor phase liquid nitrogen or kept at room temperature (20°C) for various amounts of time (0-24 hours) before freezing. Total RNA was extracted from tissues using RNeasy Mini Kit (Qiagen) and RNA analysis was performed on the Agilent Bioanalyzer.

Initial data show that the quality of tissue as determined by RNA integrity numbers (RIN) is best maintained when the tissue is transferred to liquid nitrogen immediately upon arrival. When the tissue is transferred to liquid nitrogen immediately on arrival, tissues yielded RIN values close to 10 (optimal). When kept at RT for short time intervals, i.e., up to 1 hour, the RIN values are relatively unchanged. However, when the transfer of tissues into liquid nitrogen was delayed and the tissue is kept at RT for longer than 2 hours, a decline in the RIN scores was observed. Interestingly, we noticed that RIN values are typically higher in tumor vs. normal tissues from the same patient.

Any modern biorepository needs to closely monitor its protocols and implement an ongoing internal as well as external quality improvement plan that will help identify areas for improvement to better maintain the integrity of banked tissues.

Assessing the Quality of RNA from Fresh Frozen Human Tumour Tissues Stored Long-Term at Cryogenic Temperatures
ISBER Abstract Reference RS-15 
Kelly1, M. de Ladurantaye2, M. Albert1, M. Moore3, S. Dokun4, J. Bartlett1,5
1Ontario Tumour Bank, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
2Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montreal, Quebec, Canada,
3Ontario Health Study, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
4Health Services Research, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
5Transformative Pathology, Ontario Institute for Cancer Research, Toronto, Ontario, Canada

It is widely recognized that the integrity of tissue specimens preserved at temperatures below the glass state is stable long-term. Scientific studies in literature, however, do not generally extend beyond a few years. With biobanks reaching a degree of maturity where specimens may be stored for over a decade, this assumption should be tested to provide the data to support extended long-term storage and demonstrate continued fit-for-purpose. Since its inception in 2004, the Ontario Tumour Bank (OTB) has had an ongoing commitment to quality and, in accordance with biobanking best practices, has embedded stringent quality control (QC) and quality assurance (QA) measures into its routine procedures. One such measure, triggered twice annually, includes the random selection of cryopreserved tissues to undergo external quality assessment (EXTQA) by a third party to measure the integrity of the tissue's DNA and RNA. Here, as an extension of OTB's routine EXTQA, we analyzed second aliquots of previously evaluated tissues collected between 2005 and 2014 to determine if RNA integrity is affected by extended long-term storage in liquid nitrogen vapor phase.

RNA was extracted from duplicate aliquots of 70 cryopreserved tissue samples across 11 disease sites previously analyzed in prior years during routine EXTQA and having recorded RNA integrity number (RIN) scores of 7.5 or greater. Extractions and quality determinations were performed at the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) according to CTRNet SOPs. RNA quality was determined by the RIN assigned by the Agilent Bioanalyzer.

The results of external quality assurance 1 (EXTQA1) and external quality assurance 2 (EXTQA2) for RNA extracted from fresh frozen tissues were compared; 94% of EXTQA2 samples were Acceptable to Very Good in quality. There was no significant correlation (r = 0.10, p = 0.80) between the quality of RNA extracted in EXTQA2 and storage time. There was also no significant correlation (r = 0.04, p = 0.92) between the change in RIN score between EXTQA1 and EXTQA2 and storage time.

These data suggest that extended long-term storage of tumor tissue samples in vapor phase does not negatively affect the quality of RNA derivatives. From this, we conclude that OTB samples banked since inception continue to be viable for downstream applications and are fit to be used in high-impact cancer research studies.

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