Keith Harding

Standard Preanalytical Coding for Biospecimens: Defining the Sample PREanalytical Code

Background: Management and traceability of biospecimen preanalytical variations are necessary to provide effective and efficient interconnectivity and interoperability between Biobanks. Methods: Therefore, the International Society for Biological and Environmental Repositories Biospecimen Science Working Group developed a “Standard PREanalytical Code” (SPREC) that identifies the main preanalytical factors of clinical fluid and solid biospecimens and their simple derivatives. Results: The SPREC is easy to implement and can be integrated into Biobank quality management systems and databases. It can also be extended to nonhuman biorepository areas. Its flexibility allows integration of new novel technological developments in future versions. SPREC version 01 is presented in this article. Conclusions and Impact: Implementation of the SPREC is expected to facilitate and consolidate international multicenter biomarker identification research and biospecimen research in the clinical Biobank environment. Cancer Epidemiol Biomarkers Prev; 19(4); 1004–11. ©2010 AACR.

Standard preanalytical coding for biospecimens: review and implementation of the Sample PREanalytical Code (SPREC).

The first version of the Standard PREanalytical Code (SPREC) was developed in 2009 by the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group to facilitate documentation and communication of the most important preanalytical quality parameters of different types of biospecimens used for research. This same Working Group has now updated the SPREC to version 2.0, presented here, so that it contains more options to allow for recent technological developments. Existing elements have been fine tuned. An interface to the Biospecimen Reporting for Improved Study Quality (BRISQ) has been defined, and informatics solutions for SPREC implementation have been developed. A glossary with SPREC-related definitions has also been added.

Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm.

The physiological and molecular mechanisms associated with acclimation and survival have been examined in four Ribes genotypes displaying differential cryotolerance. Changes in DNA methylation, nucleic acid and nucleoside composition were determined during acclimation and recovery of in vitro shoot-meristems from cryopreservation. DNA methylation was induced in the tolerant genotype, while demethylation was evident in sensitive genotypes. This response initially occurred during sucrose simulated acclimation, with progressive changes as shoots recovered from successive stages of the encapsulation-dehydration protocol. These methylation patterns existed in the initial vegetative cycle but regressed to control values following subculture, indicating the changes in DNA methylation to be a reversible epigenetic mechanism. RNA levels indicating transcriptional activity during the acclimation of nodal tissue are inversely linked to methylation changes, where activity appears to be up-regulated in the cryosensitive genotypes. Conversely, cryopreserved shoots show increased levels of both RNA and DNA methylation in the cryotolerant genotypes. Other nucleosides show post-transcriptional activity corresponds with tolerance during acclimation and cryopreservation. These observations connect physiological attributes to differential molecular changes in Ribes, the implications of which are discussed in relation to cryopreservation-induced apoptosis and genetic stability.

Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP)

Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.

Exploring the physiological basis of cryopreservation success and failure in clonally propagated in vitro crop plant germplasm

An appraisal of potato and Ribes shoot meristem cryopreservation shows physiological factors influence survival and development, sometimes independently of protocol and genotype. Markers for oxidative damage incurred by cryostorage reveal two responses: (1) oxidative stress with an eventual decline in regrowth and (2) an oxidative burst associated with higher survival. Differential responses to cryoinjury are discussed in relation to in vitro ageing and genetic stability within the conceptual framework of cryobionomics. The possibility that cryopreservation-induced cell death and apoptosis occurs in plants is considered with respect to current concepts of animal cell cryoinjury. It is proposed that a more holistic approach is now required to understand the basis for success or failure of cryopreserved plant germplasm.;

Cryoconservation of South African plant genetic diversity

South Africa has a rich flora which exhibits among the highest species density in the world, distributed across nine biomes that support an impressive diversity of animal life. However, a variety of human actions, invasion by alien species, natural disturbances and climate change collectively impact negatively on the great diversity of both plant and animal species. In situ conservation has long been practised, primarily in nature reserves, complemented by ex situ conservation in national botanic gardens, but in vitro plant conservation is not common. In the context of animal biodiversity conservation, the Wildlife Biological Resource Centre of the National Zoological Gardens utilises cryobanking as one of its major focuses and is now poised to expand as the repository for the cryoconservation of plant germplasm, particularly for indigenous recalcitrant-seeded and poor-seeding species. However, there are particular problems associated with successful germplasm cryostorage of such tropical and subtropical plants. As we see the science and application of cryobiology and cryoconservation as cross-cutting and transdisciplinary, we have entrained formal networking among scientists offering a range of specialisations aimed at a deeper understanding of common problems and practical outcomes to facilitate both plant and animal biobanking. The endeavours are aimed at elucidating the basis of both successes and failures in our efforts to attain optimal outcomes. With focus on best practices, standard operating procedures, validation and risk management for cryopreserved and cold-stored plant and animal material, our ultimate aim is to facilitate restoration by the safe reintroduction of indigenous species.

Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections.

The Standard PREanalytical Code (SPREC) was developed by the medical/clinical biobanking sector motivated by the need to harmonize biospecimen traceability in preanalytical processes and enable interconnectivity and interoperability between different biobanks, research consortia, and infrastructures. The clinical SPREC (01) consists of standard preanalytical variable options (7-code elements), which comprise published and (ideally) validated methodologies. Although the SPREC has been designed to facilitate clinical research, the concept could have utility in biorepositories and culture collections that service environmental and biodiversity communities. The SPREC paradigm can be applied to different storage regimes across all types of biorepository. The objective of this article is to investigate adapting the code in nonclinical biobanks using algal culture collections and their cryostorage as a case study. The SPREC (01) is recalibrated as a putative code that might be adopted for biobanks holding different types of biodiversity; it is extended to include optional coding from the point of sample collection to postcryostorage manipulations, with the caveat that the processes are undertaken by biorepository personnel.

Can Biospecimen Science Expedite the Ex Situ Conservation of Plants in Megadiverse Countries? A Focus on the Flora of Brazil

Increasing the number of species conserved ex situ in Megadiverse countries is a major task exacerbated by many intricate factors including: biome complexity, wide range of biodiversity and an incomplete knowledge of life cycles, reproductive strategies, adaptations and species interactions. Although, establishing safe reserves is a crucial conservation measure their security and effective maintenance can be unfavourably compromised by climate change and the risks incurred by socioeconomic instability and changes in land use. Anthropogenic impacts, non-sustainable practices and habitat erosion have motivated current international efforts which focused on Brazil as host of ‘Rio+20’ the United Nations twentieth anniversary conference on sustainable development. The revised targets of the Global Strategy for Plant Conservation (GSPC) are responses to species decline and realizing Target 8, which concerns ex situ conservation, places the heaviest burdens on countries that are custodians of the highest levels of global biodiversity. At the scientific level, ex situ conservation of endemic species in genebanks is often hindered by a lack of information about molecular genetics and problematic (recalcitrant) storage behaviors that restrict the preservation of flora native to Megadiverse countries. The potential for applying the ‘Biospecimen Science’ paradigm in expediting conservation in biodiversity-rich biomes is considered using Brazil as an exemplar of a Megadiverse country. The impacts of process chains on the quality of preserved plant germplasm and using evidence-based research to improve conservation outcomes, risk and quality management systems are appraised. The Biospecimen Science approach is not intended to displace conventional conservation practices but rather, to enhance their effectiveness in terms of the scale and efficiency of their scientific and technical operations.

Cryopreservation of Shoot Tips and Meristems: An Overview of Contemporary Methodologies

Cryopreservation is the storage of viable bioresources at ultra-low temperatures in liquid nitrogen (LN). This chapter provides an overview of those protocols most commonly used to cryopreserve in vitro derived shoot tips and meristems; they are described generically, as sequential technical steps, including preparative and cryogenic treatments and the morphogenetic assessment of recovery. The importance of translating research-generated methods into formal Standard Operating Procedures (SOPs) is considered.

Biomarkers from Molecules to Ecosystems and Biobanks to Genebanks

The in vitro conservation of economically important tropical crops has made considerable progress, however there is a substantial proportion of tropical diversity that remains difficult to conserve. This chapter considers how molecular technologies including the Barcode of Life initiative and biomarkers may be applied to characterize and enable the conservation of tropical plant species. It explores the use of biomarkers, as biospecimen science research tools to expedite the ex situ conservation of tropical plant diversity and the utility of the Standard Preanalytical Code (SPREC) for the conservation of tropical plant genetic resources.