Biospecimen Preservation

Most of the experiments performed in the OCEDP laboratories rely heavily upon having access to high-quality human biospecimens.  Ongoing studies for biomarker discovery and validation require human tissues (e.g. ovarian tumors, normal ovaries, and ovarian tissue with benign diseases), blood samples (sera, plasma, and white blood cells), ascites (i.e. the ascites fluid and the cell pellets), and Pap tests.  We have obtained ethical approval for all of our studies through the University of Minnesota Institutional Review Board.    

The OCEDP members are well aware of the detrimental effects that pre-analytical variables can have on research projects.  For example, if biospecimens are not handled properly when they are removed from the patient and then processed immediately, they will not be suitable for doing experiments and the data that is generated from these biospecimens will not be reliable.  The entire team of surgeons, clinicians, nurses, and clinical trial coordinators have played an important role in ensuring that the workflow of biospecimens in the clinics and operating rooms is optimized for patient care and biospecimen quality.

For this reason, Dr. Amy Skubitz established the Tissue Procurement Facility (TPF) in 1995 from the ground up as a core facility for the University of Minnesota’s Masonic Cancer Center.   She served as the Director or Assistant Director for almost two decades.  During this time, the Tissue Procurement Facility became part of the Academic Health Center’s BioNet, and it is currently a key component of the University of Minnesota’s Clinical and Translational Science Institute.  A main mission of the BioNet TPF is to consent patients prior to surgery and then procure and process tissue and blood samples for researchers, while maintaining patient confidentiality.

Dr. Skubitz has been a member of the International Society for Biological and Environmental Repositories (ISBER) since 1999.  She has served as a member of ISBER’s Education and Training Committee for 6 years; serving as an Associate Editor for three editions of the “Best Practices for Repositories”.  She is also a member of ISBER’s Biospecimen Science Working Group.  Dr. Skubitz and members of the OCEDP have contributed to the publication of manuscripts that emphasize the need for standard operating procedures, quality assurance, and quality controls in all aspects of biobanking.

Vitrification & Fluids

Isothermal vitrification methodology development for non-cryogenic storage of archival human sera.

Less RBoylan KLSkubitz APAksan A.

Biorepositories worldwide collect human serum samples and store them for future research. Currently, hundreds of biorepositories across the world store human serum samples in refrigerators, freezers, or liquid nitrogen without following any specific cryopreservation protocol. This method of storage is both expensive and potentially detrimental to the biospecimens.

View full article here.

State of the art in preservation of fluid biospecimens.

Hubel AAksan ASkubitz APWendt CZhong X.

Abstract: Fluid biospecimens (blood, serum, urine, saliva, cerebrospinal fluid and bronchial lavage fluid) contain not only cells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which are utilized as biomarkers. Availability of high-quality biospecimens is a requirement for biomarker discovery. The quality of the biospecimens depends upon preanalytical variables (ie, collection and processing techniques, freeze/thaw stability, and storage stability), which account for >60%-90% of the diagnostic errors. Currently, millions of fluid biospecimens are stored in hundreds of biorepositories across the nation, and tens of thousands of new biospecimens are added to the pool daily. Specimen stabilization is imperative because fluid biospecimens degrade quickly when kept untreated at room temperature. Achieving a high-quality fluid biospecimen requires understanding the effects of storage processing parameters (eg, freezing and thawing as well as cryo-/lyoprotectant additives) and storage conditions on biomarkers contained within the biospecimens. In this article, we will discuss the main issues related to the stabilization of specific biofluids by reviewing (a) the current preservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain biomarkers to current storage techniques.

View the article on PubMed.

Stabilization of tissue specimens for pathological examination and biomedical research

Unhale SA, Skubitz APN, Solomon R, Hubel A.

Abstract: Human tissue specimens are critical reagents in the diagnosis of disease and biomedical research. Tissues experience rapid degradation immediately after ligation from their blood supply. A variety of processing techniques are employed to prevent the degradation of tissue samples, principally chemical fixation and thermal processing. The success of processing techniques is measured by the preservation of tissue morphology, as well as the critical biomarkers. In preservation of tissue specimens, formaldehyde is the most widely used fixative that maintains tissue morphology. However, the cross-links resulting from chemical interactions between formaldehyde and biomolecules in the specimens introduce difficulties in detection and extraction of antigens for analysis. Alternative processing methods, such as chemical fixation (e.g., alcohol-based) or thermal processing (e.g., freezing) help avoid the loss of antigenicity due to cross-linking, but introduce morphological artifacts. In this article, we review methods of processing of fresh tissue samples, as well as the effects of these procedures on morphology and antigenicity of the preserved tissues as assessed by histology, immunohistochemistry, proteomics, and genomics.

View the article on PubMed.

Storage of biospecimens: Selection of the optimal storage temperature.

Hubel A, Spindler R, Skubitz APN.

Abstract: Millions of biological samples are currently kept at low tempertures in cryobanks/biorepositories for long-term storage. The quality of the biospecimen when thawed, however, is not only determined by processing of the biospecimen but the storage conditions as well. The overall objective of this article is to describe the scientific basis for selecting a storage temperature for a biospecimen based on current scientific understanding. To that end, this article reviews some physical basics of the temperature, nucleation, and ice crystal growth present in biological samples stored at low temperatures (-20°C to -196°C), and our current understanding of the role of temperature on the activity of degradative molecules present in biospecimens. The scientific literature relevant to the stability of specific biomarkers in human fluid, cell, and tissue biospecimens is also summarized for the range of temperatures between -20°C to -196°C. These studies demonstrate the importance of storage temperature on the stability of critical biomarkers for fluid, cell, and tissue biospecimens.
View the article on PubMed.

ISBER Best Practices

Best practices for repositories I: Collection, storage, and retrieval of human biological materials for research. (2005)

Pitt KE (Editor-in-Chief), Campbell LD, and Skubitz APN (Associate Editors), Aamodt RL, Anouna A, Baird P, Beck JC, Bledsoe MJ, De Souza Y, Grizzle WE, Gosh J, Holland NT, Hakimian R, Michels C, Pitt KE, Sexton KC, Shea K, Stark A, and Vaught J (Contributing Authors). Cell Preservation Technology 3(1):5-48. PMID: 24844904

View full publication here.

2008 best practices for repositories: Collection, storage, retrieval, and distribution of biological materials for research. (2008)

Pitt KE (Editor-in-Chief), Campbell LD, Skubitz APN, Somiari SB, Sexton KC, and Pugh RS (Associate Editors), 

Introduction: The availability of high quality biological and environmental specimens for research purposes requires the development of standardized methods for collection, long-term storage, retrieval and distribution of specimens that will enable their future use.

View full article here. 

2012 best practices for repositories collection, storage, retrieval, and distribution of biological materials for research international society for biological and environmental repositories.

Introduction: The availability of high quality biological and environmental specimens for research purposes requires the development of standardized methods for collection, long-term storage, retrieval and distribution of specimens that will enable their future use.

View full article here

Development of the ISBER Best Practices for Repositories: Collection, Storage, Retrieval and Distribution of Biological Materials for Research.

Campbell LD, Betsou F, Garcia DL, Giri JG, Pitt KE, Pugh RS, Sexton KC, Skubitz AP, Somiari SB.

View information here.

ISBER Working Group

Standard Preanalytical Coding for Biospecimens: Defining the Sample PREanalytical Code (SPREC).

Betsou F, Lehmann S, Ashton G, Barnes M, Benson EE, Coppola D, DeSouza Y, Eliason J, Glazer B, Guadagni F, Harding K, Horsfall DJ, Kleeberger C, Nanni U, Prasad A, Shea K, Skubitz A, Somiari S, Gunter E; [International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science]

View the full article here. 

Short-term stability study of RNA at room temperature.

Mathay C, Yan W, Chuaqui R, Skubitz A, Jeon J, Fall N, Betsou F, Barnes M.

View the full article here. 


Human biospecimen research: experimental protocol and quality control tools.

Betsou F, Barnes R, Burke T, Coppola D, Desouza Y, Eliason J, Glazer B, Horsfall D, Kleeberger C, Lehmann S, Prasad A, Skubitz A, Somiari S, Gunter E; [International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science].

View the full article here

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

Lehmann S, Guadagni F, Moore H, Ashton G, Barnes M, Benson E, Clements J, Koppandi I, Coppola D, Demiroglu SY, DeSouza Y, De Wilde A, Duker J, Eliason J, Glazer B, Harding K, Jeon JP, Kessler J, Kokkat T, Nanni U, Shea K, Skubitz A, Somiari S, Tybring G, Gunter E, Betsou F; International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science.

Abstract: 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.

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Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group.

Betsou F, Gunter E, Clements J, DeSouza Y, Goddard KA, Guadagni F, Yan W, Skubitz A, Somiari S, Yeadon T, Chuaqui R.

Abstract: Control of biospecimen quality that is linked to processing is one of the goals of biospecimen science. Consensus is lacking, however, regarding optimal sample quality-control (QC) tools (ie, markers and assays). The aim of this review was to identify QC tools, both for fluid and solid-tissue samples, based on a comprehensive and critical literature review. The most readily applicable tools are those with a known threshold for the preanalytical variation and a known reference range for the QC analyte. Only a few meaningful markers were identified that meet these criteria, such as CD40L for assessing serum exposure at high temperatures and VEGF for assessing serum freeze-thawing. To fully assess biospecimen quality, multiple QC markers are needed. Here we present the most promising biospecimen QC tools that were identified.

View the full article here.