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.
The European Society for Reproduction and Embryology (ESHRE) will be holding their annual conference in the beautiful city of Geneva, Switzerland this year from the 2nd to the 5th July.
On Sunday 2nd July, there will be fifteen special interest group pre-congress presentations, before the main programme itself starts on Monday 3rd July.
Planer will be exhibiting once again at ESHRE, with some exciting new products. Don't forget to come and see us on stand D16 to find out more.
The AVA Clinic in Latvia is part of a European network of infertility treatment clinics founded in 1993 and the Riga clinic, shown here, opened in 2005. The experienced Riga team get high success rates, and as they adhere strictly to ISO 9001:2008 the quality of the equipment they buy is very important. So we were honoured that they chose Planer equipment - incubators, monitoring and low oxygen alarms when they recently upgraded their lab.
Shown here is the DATAcentre monitoring system on two incubators. This affordable system can monitor temperature, humidity, CO2, liquid nitrogen level, Oxygen (O2), door status and more. It can connect to up to 120 transmitters, including a mixture up to 120 wireless sensors or 14 hard wired sensors. Real time and historical logged data can be viewed in graphical or text format and any alarms can be easily managed. Optionally the system may be connected directly to a PC or to a LAN port in the building.
Andris Grunskis is both an embryologist with AVA and the Quality Systems Manager (for ISO9001:2008 & EUTCD) and has worked at AVA since 2009. After our engineers had finished installing the system, Andris commented " ... the engineers were great! They fixed all our issues and now the DATAcentre seems to be performing flawlessly".
Researchers at Kings College London have used Planer freezers for many years. Now Doctors Sharon Lehec and Ragai Mitry are among the authors of a new paper aiming to develop an optimised protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions in their Kryo 10.
Transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of these hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. So the establishment of banks for such cryopreserved microbeads would be an important step in emergency use. The aim of the study was to develop an optimised protocol for this cryopreservation for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated using human and rat hepatocytes. Cryopreservation was performed using a standard King’s College Hospital freezing protocol in a Planer controlled-rate freezer, the Kryo10.
The two freezing solutions which gave better results were studied with human and rat hepatocyte microbeads. Similar effects on cryopreserved microbeads morphology, viability and hepatocyte-functions post thawing were observed over seven days in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; ZVAD), an antioxidant (desferoxamine; DFO), and buffering and mechanical protection (human serum albumin; HSA) on RMBs. ZVAD (60µM) had beneficial effect on cell viability, greater than with DFO (1mM), HSA (2%) and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes, and a lower degree of cell apoptosis were observed with all three cytoprotectants, ZVAD tending to provide the greatest effect.
They concluded that cryopreservation of hepatocyte microbeads using UW solution containing 10% DMSO, 5% glucose and a cytoprotectant such as ZVAD had beneficial effects regarding cellular and physical damage leading to maintenance of cell viability and function post thawing. This initial optimised protocol for cryopreservation of hepatocyte microbeads supports the hypothesis that the development of freezing solutions containing cryoprotective agents and compounds targeting apoptosis during the cryopreservation process could improve the outcome of cryopreservation.
The hope is that, eventually, large amounts of hepatocyte microbeads may be cryopreserved and banked for immediate emergency clinical treatment in conditions such as acute liver failure. The work in the Dhawan lab at Kings College continues and a new PhD starts in April using clinical/GMP grade materials for future banking of these cryopreserved microbeads.
The Culture Collection of Freshwater Microalgae, the Coleção de Culturas de Microalgas de Água-Doce, plays an important role in Brazilian micro algal research. It is based at the Universidade Federal de São Carlos (www.ufscar.br) and was founded in 1977. The collection provides biological materials, substrates and education for a large proportion of past, and indeed current, projects throughout the Brazilian territory. Microalgae are found in most aquatic and many terrestrial ecosystems; their diversity of lifestyles has resulted in a large group of organisms with differing metabolic and functional characteristics; as a result they possess considerable biotechnological potential, particularly in the bioenergy, cosmeceutical and nutraceutical markets.
The establishment of a cryopreserved biobank linked to the culture collection was recently the focus of a PhD project, aiming to reduce the costs associated with the maintenance regime of cultures and the secure maintenance of the collection catalogue. The optimised cryopreservation protocol developed as part of the project has been tested with around 100 strains of microalgae from CCMA-UFSCar, including exemplar taxa across the different taxonomic groups in the collection catalogue, with elevated levels of success, particularly for the smaller taxa, such as the small green algae.
Letícia Piton Tessarolli (pictured above) recently successfully defended this PhD in algal cryobiology under the supervision of Prof Armando Vieira (Universidade Federal de São Carlos UFSCar) and Prof John Day (Scottish Association for Marine Science, University of the Highlands and Islands). Her work was mostly undertaken at the Coleção de Culturas de Microalgas de Água-Doce. Letícia used one of our freezers for some of the work: cryopreservation is regarded as the preferred method for the long-term, genetically and phenotypically stable, storage of stock cultures. The Planer controlled rate freezer used provides adjustable protocols which can be optimised to ensure maximum post-thaw viability
If Lucy Ing, Lab Manager at Primary IVF’s first clinic in Sydney Australia had to pick her favourite piece of equipment, it would have to be the ‘mini bench top incubators'. Why?
“Because they are home to the little embryos that grow in the lab. Being placed in the centre of the lab means that we walk past these incubators hundreds of times a day, keeping a close watchful eye on the embryos. They provide a safe home for the embryos and work to make an environment similar to how it would be in the female uterus. These incubators constantly glow a warm soothing green (what I like to always see) to indicate that all conditions are ideal and optimal for embryo growth. It also has in-built alarms to quickly alert us if something is slightly out of range, which is an important quality control measure.”
When setting up the Primary IVF labs the Scientific Director, Rosemarie Cullinan, thought carefully about the choice of incubators. “Having had experience with the Planer controlled rate freezers in the 1990s, a reliable workhorse, I had confidence that the BT37s would be built to the same high standard. The in-built battery was also a big plus for me. Along with the sleek design that suits our state-of-the-art laboratories!”
Primary IVF offers Australians IVF treatment through the Australian government Medicare system making IVF accessible and affordable for all Australians.
Since Primary IVF began operations in August 2014, an additional two labs have been established in Melbourne and Brisbane and over 600 babies have been born who started life in the BT37 incubators.
See a profile of Lucy Ing at https://primaryivf.com.au/2016/05/23/getting-know-team-profile-senior-scientist-lucy-ing/
At the second recent gathering of the Upper Egypt Assisted Reproduction Conferences in February 2107, 163 lectures were presented by 46 international and Egyptian ART scientists - and attendance was close to 1900 attendees. The main theme was “translational research in ART: basic science to improve clinical practice” and the conference included four workshops. The program covered, from a new perspective, laboratory procedures including for culture media, incubator management, oocytes and embryo manipulation, pH impact and adjustment, quality control, vitrification, embryo selection and other topics.
Participants also had a look at the emerging technologies that may have future applications in ART, including gene editing, stem cell researches, and mitochondrial transfer. For the first time in this area, the meeting conducted two consensus meetings that focused on controversial issues in the field; the first was to give some recommendations on laboratory air quality and environment and the resulting work will could be published September.
The next UEARC 2018 conference will be held in Cairo on February 21-23, 2018.
Back in 2008 we reported that a child had been born with semen stored for 22 years. We thought we did well - but our record seems to have been well and truly broken.
We wrote then that Mike Kuzminski, a rock musician from Canada with Hodgkin‘s Lymphoma, banked his sperm and forgot about it till 22 years later when he wanted to start a family and learned he was infertile. In 2003 he tracked down the clinic and got the news from them “We totally have you here -- and you owe us $2,000 for storage." And on the 1st of November 2007 a baby boy was born. The sperm they used had been frozen down in a Planer controlled rate freezer, and kept at liquid nitrogen temperatures.
But Andreas Szell and others report two live births from semen around 40 years old! In 1971, a man aged about 52, banked semen for frozen for long-term storage. In 2012 a patient using the donor sperm became pregnant after a second FET and had two healthy girls delivered. The authors conclusions are that low temperature storage in liquid nitrogen should maintain the viability of cells almost indefinitely because reactions that require molecular motion and/or activation do not occur at −196 °C. The adverse effects of background radiation during storage appear to be negligible since exposure of mouse embryos to the equivalent to 2,000 years of background radiation did not have a detectable effect on their viability.