NIH Common Fund HuBMAP / SCAP Mini Workshop …€¦ · ORIGINAL ARTICLES A Novel Approach to...

WEDNESDAY, JUNE 28, 2017

12:20 pm Introductory Remarks - Robert Star (Director Division of Kidney, Urologic, and Hematologic Diseases National Institute of Diabetes and Digestive and Kidney Diseases, HuBMAP Co-Chair)

12:30 – 2:30pm Challenges in collecting and pre-analytical processing of tissue

Moderator: Robert Star (NIDDK)

There are many tissue collection and processing factors that influence data quality, from length of ischemia time to storage conditions and collection method. These factors influence the distribution and degradation of biomolecules at different rates. Therefore, it is critical to match the choice of tissue source, collection method and preservation technique with the types of biomolecules being studied by different downstream assays.

The purpose of this session is to identify some of the challenges in collecting, preserving, and annotating high quality human tissue that will be used for downstream analytical techniques in the HuBMAP program. These techniques include single cell RNAseq, FISH, immuno-fluorescence as well as emerging techniques such as MERFISH, FISSEQ, seqFISH, MIBI-TOF, and 3-dimensional high-end imaging. Through the discussion, we hope to have a better understanding of the challenges HuBMAP might face in collecting and pre-analytical processing of tissue specimens and how this processing will impact the quality of data collected by different single cell, tissue, and imaging assays.

A number of components add to these challenges. One component is to record the spatial orientation of samples relative to anatomical landmarks (and build this into the sample management pipeline). A second component is the analysis, then integration and iteration of data from multiple imaging and omics assays to develop comprehensive molecular (and omic) profiles of the cells within the tissue, including location information. A third key component is to understand when sources of variability are biologically relevant (within tissue samples from same patient, across multiple tissues, and across multiple donors) or artifacts of the collection and processing of the samples.

Questions for the breakout session to consider include:

• Quality: What are practical quality measures for assessing the impact of tissue collection methods and the degree of degradation? How does the magnitude of ischemia signatures compare with collection, dissociation or storage signatures? Is there a common set of quality biomarkers that can be used across all tissues and that are compatible with downstream assays?

• Metadata: Beyond SPREC 2.0, are there common data elements describing collection and processing that are relevant to mapping DNA, RNA and proteins biomolecular distributions in tissues?

• Assay Workflow: What are best practices for assessing the impact of single cell (liberase) and tissue (LCM, super-resolution, imaging MS/MS) based tissue “dissociation” methods on assay measurements? Can tissue sections be used for multiple assays (RNA in situ, then protein, then routine stains)?

• Collection: For what assays and tissue types do tissues need to be collected from live donors? Rapid autopsy protocols?

• Staining: Do common stains (e.g. H&E, trichrome, toluidine) influence the sensitivity and specificity of downstream assays?

• Orientation: How do we preserve orientation of a tissue specimen through the processing chain?

NIH Common Fund HuBMAP / SCAP Mini Workshop Neuroscience Center, Bethesda, MD

• Fixing, clearing and embedding: Are there tissue stabilization techniques that can be used before or during collection? For current and emerging fixatives/preservatives of excised tissue, which biomolecular species do they preserve with good fidelity (not only nucleic acids and proteins, but how effective are these techniques at preserving metabolites or carbohydrates), what compatibility issues are there with different tissue types, cell types, dissociation techniques and assays? What are some of the challenges associated with clearing techniques?

• Sectioning: What are tissue-specific considerations in preparing tissue sections? How does the choice of tissue size and format influence ischemia and preservation timing and in term the quality of the tissue for different downstream assays?

• End-users: What format, quantity, and quality level is needed for: RNAseq, DropSeq, MERFISH / FISSEQ / seqFISH, immuno-florescence, MIBI-TOF and CyTOF approaches?

2:30 – 3:00pm Break

3:00 – 5:00 pm Data Analysis, Standards, and Benchmarks for Single Cell Analysis

Moderator: Junhyong Kim (University of Pennsylvania)

Because of the difficulty of obtaining measurements at the single cell scale, the field has been driven by technological advances, including various RNA/DNA sequencing technologies, high-resolution proteomics and metabolomics, multiplexing strategies, cell handling technologies, etc. Despite these technological advances, single cell measurements remain difficult and is fundamentally challenged by the fact that the units of measurement, each cell, has no replication. It has been extremely difficult to assess the efficiency of measurements, establish benchmarks or controls, agree on protocols for data analysis, and coherently define standards for reporting experiments and data analysis. An especially important challenge is placing single cell data in their organismal context, including spatial coordinates.

Questions for this breakout session to consider include:

• Is there benchmark data to compare new experimental or computational methods? • How do we establish material standards such as specific cells or spike-in RNA? • What metadata about calibration is important to know? • What information is important to collect about the sample and its preparation? • How can we work together with manufacturers to build standards into their methods? • Does an ontology need to be established for single cell analysis? • How can we associate single cells to tissue orientation information? More generally, how can data be organized

from the single cell scale to whole organism scale? • What are the common data elements between imaging and sequencing assays? Is there a common header we

can use for all data, similar to FITS or DICOM?

5:00 pm Closing Remarks

Suggested background reading for these breakouts:

• Unhale, S. A., Skubitz, A. P., Solomon, R., & Hubel, A. (2012). Stabilization of tissue specimens for pathological examination and biomedical research. Biopreservation and biobanking, 10(6), 493-500. [http://online.liebertpub.com/doi/abs/10.1089/bio.2012.0031]

• Hubel, A., Spindler, R., & Skubitz, A. P. (2014). Storage of human biospecimens: selection of the optimal storage temperature. Biopreservation and biobanking, 12(3), 165-175. [http://online.liebertpub.com/doi/abs/10.1089/bio.2013.0084]

• Hubel, A., Aksan, A., Skubitz, A. P., Wendt, C., & Zhong, X. (2011). State of the art in preservation of fluid biospecimens. Biopreservation and biobanking, 9(3), 237-244. [http://online.liebertpub.com/doi/abs/10.1089/bio.2010.0034]

• Chung, Cho H, Hewitt SM (2016). The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissue. Biotechniques. May 1;60(5):239-44 [http://www.biotechniques.com/BiotechniquesJournal/2016/May/The-paraffin-embedded-RNA-metric-PERM-for-RNA-isolated-from-formalin-fixed-paraffin-embedded-tissue/biotechniques-364401.html]

• Carithers, L. J., Ardlie, K., Barcus, M., Branton, P. A., Britton, A., Buia, S. A., ... & Guan, P. (2015). A novel approach to high-quality postmortem tissue procurement: the GTEx project. Biopreservation and biobanking, 13(5), 311-319. [http://online.liebertpub.com/doi/full/10.1089/bio.2015.0032]

ORIGINAL ARTICLES

A Novel Approach to High-QualityPostmortem Tissue Procurement:

The GTEx Project

Latarsha J. Carithers,1 Kristin Ardlie,2 Mary Barcus,3 Philip A. Branton,1 Angela Britton,3

Stephen A. Buia,3 Carolyn C. Compton,1 David S. DeLuca,2 Joanne Peter-Demchok,1

Ellen T. Gelfand,2 Ping Guan,1 Greg E. Korzeniewski,3 Nicole C. Lockhart,1

Chana A. Rabiner,1 Abhi K. Rao,1 Karna L. Robinson,3 Nancy V. Roche,3 Sherilyn J. Sawyer,1

Ayellet V. Segre,2 Charles E. Shive,3 Anna M. Smith,3 Leslie H. Sobin,3 Anita H. Undale,3

Kimberly M. Valentino,3 Jim Vaught,1 Taylor R. Young,2

Helen M. Moore,1 on behalf of the GTEx Consortium4

The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to studythe correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals.A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here wedescribe how a successful infrastructure for biospecimen procurement was developed and implemented bymultiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, andcell lines for the GTEx project. Other research projects can follow this model and form beneficial partnershipswith rapid autopsy and organ procurement organizations to collect high quality biospecimens and associatedclinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Proceduresguiding biospecimen collection for the GTEx project are available to the research community.

Introduction

The aim of the Genotype-Tissue Expression (GTEx)Project of the U.S. National Institutes of Health (NIH)

Common Fund (https://commonfund.nih.gov/GTEx) is todetermine how genetic variation affects normal gene ex-pression in human tissues, and ultimately to assess how thisrelationship correlates with the development of disease. Toachieve this goal, the project planned to collect multipledifferent human tissues from each of hundreds of donors,isolate nucleic acids from the tissues and perform geno-typing, gene expression profiling, whole genome sequenc-ing, and RNA sequencing, and analyze the data to identifyexpression quantitative trait loci (eQTL).1–3 The scientificgoals of the project required that the donors and theirbiospecimens present with no evidence of disease (hence-forth termed ‘‘normal tissues’’ or ‘‘normal biospecimens’’).

The project began as a 2.5-year pilot study to assess thefeasibility of collecting tissue from up to 40 different tissuetypes from women and 34 different tissue types from men,from hundreds of individual donors.1,4 The resulting biospe-cimens needed to yield RNA with an RNA Integrity Number(RIN)5 of at least 6 for optimal RNA sequencing results. TheNational Cancer Institute (NCI)’s Biorepositories and Bios-pecimen Research Branch (BBRB) worked with the NationalHuman Genome Research Institute (NHGRI), the NationalInstitute of Mental Health (NIMH), and other GTEx projectpartners to develop a plan for collecting the normal humanbiospecimens for GTEx.

A group of experts working in the ethical, scientific, andoperational aspects of biobanking was assembled to identifykey challenges of collecting normal biospecimens and tohelp develop a framework for the project. This planninggroup recognized that donations of normal biospecimens

1Biorepositories and Biospecimen Research Branch, Cancer Diagnosis Program, National Cancer Institute (NCI), National Institutes ofHealth (NIH), Bethesda, Maryland.

2The Broad Institute of MIT and Harvard, Cambridge, Massachusetts.3Biospecimen Research Group, Clinical Research Directorate, Leidos Biomedical Research, Inc., Frederick, Maryland.4Membership of the GTEx consortium is provided in the Acknowledgments.

ª The Author(s) 2015; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the CreativeCommons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use,distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

BIOPRESERVATION AND BIOBANKINGVolume 13, Number 5, 2015Mary Ann Liebert, Inc.DOI: 10.1089/bio.2015.0032

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from living donors were rare, typically only occurring as asecondary research donation when surgery is performed forcancer treatment, limb amputation, or other surgical diseasetreatments. Thus it was envisioned that the GTEx biospe-cimens would be collected from deceased donors, due to theproject’s need for multiple tissues per individual donor.

The GTEx project set out to partner with organ pro-curement organizations (OPOs) and institutions with rapidautopsy programs to obtain the high number of normalbiospecimens needed for this study.4 The expert planninggroup established a comprehensive set of recommendationsfor acquiring high-quality, normal biospecimens. A summaryof the recommendations has been published4 and the fullrecommendations are posted for public use (http://biospecimens.cancer.gov/global/pdfs/caHUB_ANTWG_Postmortem_BPs.pdf). GTEx management used these recommendations to de-velop the eligibility criteria for GTEx donors (Fig. 1).

Herein we provide an overview of how we developed atissue collection platform focused on meeting the ethical,scientific, informatics, and operational challenges of biospeci-men procurement for the GTEx project. The success of thisproject depended on tissue donations from families who have losta loved one. With sincere respect and gratitude, we thank theGTEx donors and their families for their generous contributions.

Materials and Methods

Consenting donors and addressing ethical,legal and social issues

The medical institutions and OPOs that collected thebiospecimens for the GTEx pilot study, Biospecimen SourceSites (BSS), chose to either submit a GTEx research pro-tocol and undergo full or expedited IRB review, or uponconsultation with their Office of Research Subject Protec-tion determined that the research does not constitute humansubjects research and did not require further review becausethe donors were deceased. The GTEx pilot study requiredexplicit next-of-kin or legally authorized representative au-thorization for participation in the project.

Under the law, deceased individuals are not considered tobe human subjects and do not require consent for research;however, GTEx management decided to require authoriza-

tion due to the large amount of sequencing data to be pro-duced and made publically available. With respect toconsent, the BSSs sometimes had different approaches as tothe specific manner in which authorization was obtained, butproject staff worked closely with the BSSs to ensure that thebasic requirements and essential consent elements (Table 1)for the GTEx project were met.

Consent was obtained in person as well as over the phone.The GTEx project included a sub-study that evaluated theattitudes and concerns of family decision-makers regardingthe consent process and other ethical issues concerning thedonation process.6 This sub-study provides information tohelp ensure that the project effectively addresses the concernsand expectations of the study participants. This sub-study alsocreated and provided novel training materials for consentingpersonnel, in order to improve interactions with and under-standing of donor families. The training can be found athttp://gtextraining.org/.

Privacy and confidentiality of donor information also re-ceived careful consideration. Although the project collected alimited data set from the BSSs, only de-identified data (http://

FIG. 1. GTEx EligibilityRequirements. A set of eli-gibility requirements weredeveloped to align with thescientific needs of the projectand to reduce the risk ofcollecting tissue that wasdiseased, autolyzed, or oth-erwise unsuitable for molec-ular analysis. The eligibilitycriteria were developedwithin a framework thatconsidered the limited dataimmediately available in thetimeframe surrounding a po-tential donor’s death and thefeasibility of the Biospeci-men Source Sites to obtainthe tissues and associateddata in a timely manner.

Table 1. Essential GTEx Consent Elements

A description that genetic and genomic research may beconducted on the donated biospecimens

The donated biospecimens may be shared with researcherswho are approved by an access committee, includinginternational researchers

The donated biospecimens may be used for broad future researchCommercial products may be developed using the donated

biospecimens however the donor families will notfinancially profit from these products

There may be a risk of loss of privacy and confidentialityThe biospecimens may be withdrawn; however, molecular

data may not be retrieved once it is generatedNo individual genetic information will be returned to the

next-of-kin or legal representative; however, results fromthe collective GTEx biospecimen set will be availableon the GTEx portal ((http://www.gtexportal.org/home)and the NIH’s National Center for BiotechnologyInformation’s database of Genotypes and Phenotypes(dbGAP) http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000424.v1.p1

312 CARITHERS ET AL.

www.hhs.gov/ocr/privacy/hipaa/administrative/privacyrule/index.html) will be distributed to collaborators in the GTExproject or to downstream secondary researchers. An electronicdata capture system, the Comprehensive Data Resource de-scribed later in this article, controlled the display and access todata based on user roles and entitlements. BSSs could only viewdata from their own sites, and only a limited number of approvedstaff members within the GTEx project could view protectedhealth information. In addition, the project established acombined material transfer agreement/data use agreementthat covered all parties receiving either biospecimens or data.

The template agreement is posted publicly at http://biospecimens.cancer.gov/global/pdfs/caHUB_Material_Transfer_and_Data_Use_Agreement_072512-508.pdf. While thedevelopment and negotiation of such an agreement requiredan initial time investment, the agreement clearly laid outresponsibilities and requirements for privacy protection forall parties and was written broadly enough to cover thecollection process throughout the course of the project.

Developing an infrastructure to supportthe collection of human biospecimens

To meet the challenging biospecimen requirements of theGTEx pilot study, NCI’s BBRB and its partners developed a

novel infrastructure for collecting normal human biospeci-mens for research purposes. BBRB, together with the Fre-derick National Laboratory for Cancer Research, developedan operational plan to contract medical centers and OPOs toscreen potential donors, consent next-of-kin, and collect andship biospecimens. Each BSS complied with their respectiveinstitution’s biospecimen handling policies to procure GTExbiospecimens that were sent to a central, separately con-tracted Comprehensive Biospecimen Resource (CBR).

The CBR inventoried and divided biospecimens forthree purposes: (1) shipment to the separately contractedGTEx Laboratory, Data Analysis, and Coordinating Center(LDACC) for molecular analysis, (2) histology for pathologyreview, and (3) local storage for future analysis. Whole brainswere sent directly from the BSSs to a separate Brain Bank forproper sectioning and were then subsequently sent to theLDACC for analysis. Also, blood and skin samples were sentdirectly to the LDACC to generate EBV-transformed lym-phoblastoid cell lines and fibroblasts, respectively.

The entire biospecimen collection, processing, storage,and transfer operation was coordinated through a centralquality control program and uses a custom web portal fordata entry. A Pathology Resource Center (PRC), comprisedof board-certified pathologists, evaluated the quality of thebiospecimens. Figure 2 outlines the biospecimen platform

FIG. 2. The GTEx Biospecimen Collection Infrastructure. The GTEx Biospecimen Source Sites were responsible fordonor recruitment, tissue procurement and processing, and data collection. Brain and hair samples were sent to the MiamiBrian Bank for quality control purposes, coronal sectioning of brain tissue, and storage of brain tissue. The ComprehensiveBiospecimen Resource handled biospecimen receipt, processing, distribution and storage, histology and imaging, and kitdevelopment and production. The Comprehensive Data Resource is a data repository that served as an honest broker to keeplimited data set information confidential and distribute de-identified data. The Pathology Resource Center performed casereview through tissue identification and quality assessments. The Laboratory, Data Analysis, and Coordinating Center con-ducted molecular and data analysis as well as served as a project management and data-coordinating center. Clinical, demo-graphic, handling, genetic and molecular data from GTEx biospecimens can be accessed through the National Center forBiotechnology Information’s database of Genotypes and Phenotypes. Color images available online at www.liebertpub.com/bio

THE GTEx PROJECT 313

and the flow of biospecimens and data to and from eachsite within the infrastructure. Detailed SOPs can be foundat http://biospecimens.cancer.gov/resources/sops/library.aspthat describe tissue procurement, shipping, kit utilization,data entry, pathology review, and other aspects of the GTExproject.

Pathology review

The PRC was created and implemented in the GTEx projectto validate tissue origin, content, and integrity, and to ensurethat the collected tissues meet prescribed quality standards. ThePRC reviewed the disease state of the tissue for evidence ofcancer, infectious disease, or inflammatory disease to confirmthat collected biospecimens were ‘‘normal’’ or non-diseased,and subsequently annotate tissue dimensions and compositionas well as determine acceptability of the final biospecimen forinventory.

After tissues were sectioned and stained at the CBR,tissue sections were scanned using a digital whole slideimaging system (Aperio).7 The PRC pathologists were ableto review these images remotely via an Internet web portal.The PRC assessed and generated a report on multiple pa-rameters, including verifying that the correct target tissuehas been obtained and is of the correct size; the degree ofautolysis; the presence of clinically unsuspected malignancyor infection; and the presence of significant ‘‘contaminant’’but normal adjacent tissue, such as excessive adherent fataround an aliquot.

When all quality control measures were completed, thereports were made available to the LDACC to assess whe-ther to go forward with genomic processing and to providecritical, real-time feedback in process improvement for theBSSs. Table 2 lists the criteria required for a biospecimen tobe included in the molecular analysis pipeline.

The pathology review added another layer of confidencewhen interpreting data from GTEx samples. Instances oc-curred when a biospecimen’s gene expression profile did notcorrelate with the gene expression profiles of other samplesfrom the same biospecimen type or same donor, for exam-ple, when a GTEx donor was the recipient of a donatedorgan. The pathology report helped to identify tissue typesnot suitable for the GTEx project due to tissue samplinginconsistencies, problems with poor preservation, and tissueheterogeneity issues (Fig. 3). Pathology review via rapiddigital pathologic assessment of biospecimens streamlinedthe targeted collection of appropriate tissue types withconsistent quality.

Developing a total quality program

The establishment and implementation of a robust qualitymanagement program was integral for obtaining GTExbiospecimens that were suitable for genomic analysis.Hallmarks of established quality management methodologyincluding data management, Standard Operating Procedure(SOP) development, and auditing have been adopted by theproject.

A major challenge for GTEx was in the management ofdata associated with the biospecimens; each case could in-clude more than 500 data elements. To help manage thedata, a system for data queries was implemented and thisprocess proved to be an effective method for producingwell-organized, easily accessible, and reliable data. Im-plementation of data management techniques can be timeconsuming and burdensome. However, such techniquesprovided real time feedback to the BSSs on their protocolconduct, which resulted in site improvements and improvedadherence to protocol.

Queries were also used to guide data collection bestpractices, resulting in an increase in data fidelity and a de-crease in the issuance of certain query types. The develop-ment of these standards allowed for harmonization across allBSSs and was an important contributor to the project’ssuccessful acquisition of high quality biospecimens.

One lesson learned from the GTEx pilot study is that thereis not a ‘‘one size fits all approach’’ to SOP development forbiospecimen procurement; this lesson was largely based oninstitutional differences, many of which were unavoidable.The BSSs were consulted when new SOPs were developed toensure that the SOPs were operationally feasible. Documentcontrol software was also utilized to ensure that current ver-sions of SOPs were being used and training was conducted toensure comprehension of new procedures. Approximately100 supporting quality documents were developed to provideconsistency and clarity to this complex project. Many of thesedocuments are available to the public, including SOPs,workflows and project-related tools (http://biospecimens.cancer.gov/resources/sops/library.asp).

Results

Biospecimen annotation

The utility of a biospecimen for research purposes de-pends a great deal on the degree of annotation associatedwith the biospecimen. The GTEx pilot study used theComprehensive Data Resource (CDR) to facilitate the inputand analysis of multiple data types related to informedconsent, the donor’s medical history, biospecimen collectionand handling, and pathology annotations. The CDR is adistributed, multi-tenant informatics platform that controlsdisplay and access to data based on user roles and entitle-ments. Personally identifiable information and protectedhealth information were restricted to a limited data set andto individuals with authorized access through dynamiccontent redaction.

Web service application program interfaces (APIs)connected remote Laboratory Information ManagementSystems, whole-slide imaging systems and molecular anal-ysis APIs for real-time, two-way data transfers. The CDRproved to be user-friendly, durable, and scalable. Its com-mon data model was easy to query and easy to integrate with

Table 2. Criteria Required for a Biospecimen

to Be Included in The Molecular Analysis Pipeline

Pathology review confirms that the correct target tissue hasbeen obtained and comprises at least 50% of the sample(with few exceptions)

Pathology review confirms that the correct size of the targettissue has been obtained

Pathology review confirms that there is no presenceof malignancy

RNA extracted from the biospecimen has at least500 ng of total RNA

RNA extracted from the biospecimen has a RINof 5.7 or higher


outside information technology systems for the benefit ofsharing and exchanging data. The CDR was accessed bymultiple users around the clock for entering and accessingdata necessary for the collection, shipment, processing, andanalysis of biospecimens.

The challenging biospecimen collection setting and dif-ferences among partner organizations complicated the pro-cess of uniform data collection for donor characteristics. Forexample, data related to the health of the donor could havebeen derived from multiple sources, including the donor’smedical record (if available) and information from thedonor’s next-of-kin. Accordingly, additional CDR datafields were included to annotate the source of various in-formation reported. Terminology related to data elements,such as medication name and medical condition or cause ofdeath, varied across the BSSs, most often due to the use ofsimilar but different terms or synonyms.

The CDR interfaced with a common vocabulary system,derived in part from the International Statistical Classifica-tion of Diseases (ICD-10-CM), Current Procedural Termi-nology (CPT4) and the National Drug Code, to standardize

the data collected about GTEx donors and to improvecomparability across information sets from different BSSs.These vocabulary elements were presented dynamically andin context to data entry personnel during data entry.

Data from the CDR was exported to the LDACC-developed GTEx web portal (http://www.gtexportal.org/home), where members of the public can request access toGTEx samples, and to dbGAP (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000424.v1.p1),where the research community can request access to geno-mic data and corresponding biospecimen data, such as clin-ical, demographic, and biospecimen handling data.

Number of biospecimens collected

The GTEx pilot biospecimen collection included up to41 different PAXgene preserved postmortem target tissuetypes (Fig. 4). The PAXgene Tissue preservation system9

developed by Qiagen was selected for GTEx because itfacilitates good histological analysis as well as extractionof high-quality RNA and DNA. Hair, whole brain (frozen),

FIG. 3. GTEx PathologyReview. Certain tissues werenot considered acceptable forGTEx analysis purposes. (a, b)Autolysis. (a) Well-preservedpancreas with distinguishableexocrine and endocrine ele-ments (RNA integrity number[RIN] 6.3). (b) Severely auto-lyzed pancreas (RIN 2.4). (c,d) Heterogeneous tissue sam-pling. (c) Well-preserved gas-tric mucosa with a RIN of 7.3.The higher RIN reflects mul-tiple cell types: the abundantwell-fixed gastric glands in themucosal layer combined withmuscularis mucosa. (d) Poorlypreserved colon mucosa with aRIN of 7.3. Although the mu-cosa was also the intendedtarget in this biospecimen, itwas badly autolyzed and theRIN reflects the residual colonmuscularis propria. (e, f) Ac-ceptable diseased tissues. (e)Normal thyroid. (f) Thyroidwith Hashimoto’s germinalcenter formation was identi-fied but was still considered tobe eligible for the GTEx study.Color images available onlineat www.liebertpub.com/bio


and sections from 11 brain sub-regions (frozen) were alsocollected. The tissue types collected for GTEx were chosenbased on their relevance to the scientific goals of the pro-ject, their clinical significance, logistical feasibility, andtheir relevance to the research community. Recoveringtarget tissues from donors in rapid autopsy programs,where the donors were not donating tissue for transplantpurposes, was frequently achievable. The OPO settingproved to be more challenging due to several factors, in-cluding the need to prioritize tissue and organ donations to

living recipients and delays due to cause-of-death investi-gations; however this obstacle did not hinder GTEx fromreaching its collection goal, ultimately procuring 10,152PAXgene preserved tissue aliquots from190 donors.

RNA quality

High quality RNA was extracted from the majority of thebiospecimens collected for the GTEx project. Fig. 5 showsthe average RIN values5 derived by the LDACC, on a per

FIG. 4. The number and type of GTEx tissues collected. 41 different PAXgene preserved tissues types were collected forthe GTEx project. Six tissues were female specific and two tissues were male specific. Only five tissues were mandatory foreach case collected, which partially explains the significant differences among the number of tissues collected per tissue type.

FIG. 5. GTEx RIN values. Here we show the average RNA integrity number (RIN) values by tissue type for 190 cases.Twenty-four tissue types had average RNA integrity values greater than 6 during the pilot study.


tissue basis for tissues collected from the first 190 GTExcases (see Supplementary Text S1 for the percentages ofindividual tissue types that have RIN values greater than 6;Supplementary material is available online at www.liebertpub.com/bio). Eleven tissue types had at least 70% of thesamples with RIN greater than 6, and more than 60% of allRNA from GTEx tissues had RIN values greater than 6,making them suitable for high dimensional genomic ana-lyses. A RIN value of 6 was set as a goal because, in gen-eral, tissues with a RIN of 6 and above yield high qualityRNA sequencing data, whereas tissues with a RIN valuebelow 6 are more likely to have samples with failed se-quencing reactions.8 At the outset of the project, we alsotested RNAs of different quality (RIN 2.0–9.0) acrossmultiple different library construction protocols for RNAsequencing. The most scalable and robust library construc-tion protocol was the Illumina TruSeq protocol with Poly-Aselection, which performed best with more intact RNA withRIN values of 6.0 or higher.

Because autolysis sets in immediately after death andmay adversely affect the quality of RNA from postmortemtissues, well-planned measures were taken to reduce thepostmortem interval (PMI) for GTEx biospecimens. PMI isdefined as the interval between the time of death or thecessation of blood flow and the time that the tissue is placedin preservative. The average RIN value was 8.6 for tissueswith a PMI less than 4 hours, and 6.7 for tissues with a PMIbetween 4 and 8 hours, while tissues with a PMI of >8 hoursresulted in average RIN values below 6 (Fig. 6). An hourlycorrelation between PMI and RIN is presented in Supple-mentary Text S2. Based on these findings, the GTEx projectmade an effort to collect all tissues from each GTEx casewithin 8 hours from time of death or cessation of blood flowto maximize the number of tissues that will present withRIN values ‡6.

Discussion

A novel biospecimen collection platform was needed toaddress the many unique aspects of the GTEx project. Most

established tissue networks do not contain a large number ofnormal, non-disease biospecimens, but instead have a col-lection of retrospective disease or condition-specific bios-pecimens, frequently with limited annotation available.BBRB met the GTEx pilot requirements by creating theinfrastructure described here for the acquisition of normalbiospecimens through contractual relationships with OPOsand rapid autopsy programs.

The biospecimen collection project of the GTEx pilotfaced many challenges. Collecting biospecimens for re-search purposes using a novel preservation method(PAXgene Tissue) presented a new challenge for OPOs dueto the fact that the staff needed to be trained in proper useof the preservative including the required step of switchingfrom fix to stabilizer solutions. Ongoing feedback fromproject partners was essential to synergize project needsand local operations. Throughout the initiation, start-up andsteady state phases of the pilot GTEx project, collaborationwith all partners was paramount to the success of GTExanalysis, and continues to be an essential factor in theproject.

The pilot phase GTEx biospecimens and data are alreadyvaluable resources for the scientific community. RNA se-quencing data and expression array data generated by theLDACC, as well as clinical annotation for the GTEx bios-pecimen collection, are publicly available on dbGAP10

(http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000424.v1.p1) and are being analyzed by aninternational team of bioinformatics experts.1

Multiple aliquots of GTEx tissues have been madeavailable to the scientific community through a Request forApplication funding mechanism (http://grants.nih.gov/grants/guide/rfa-files/RFA-RM-12-009.html) and a biospe-cimen access policy has been developed for already-fundedinvestigators (http://www.gtexportal.org/static/form/GTEx_sample_access_policy_to_the_public_v20131024.doc). Inaddition, 40 SOPs from the GTEx project are available forpublic use and are a major contribution to the researchbiobanking community (http://biospecimens.cancer.gov/resources/sops/default.asp).

A meeting entitled ‘‘The GTEx Symposium: All ThingsConsidered- Biospecimens, Omics and Data’’ was held onthe NIH campus on May 20 and 21, 2015, that includedpresentations on each of the topics discussed in this reportand some molecular analysis presentations. The meetingvideocast was archived and can be accessed at http://videocast.nih.gov/Summary.asp?File=19024&bhcp=1 (May20) and http://videocast.nih.gov/Summary.asp?File=19026&bhcp=1 (May 21). Project policy mandates that all datagenerated by researchers from the GTEx biospecimens mustbe made publicly available. Such availability will greatlyenhance the value of GTEx resources by complementingother initiatives aimed at identifying functional elements inthe human genome, such as the ENCyclopedia Of DNAElements (ENCODE) project (http://www.genome.gov/10005107) and the Library of Integrated Network-basedCellular Signatures (LINCS) program (http://commonfund.nih.gov/lincs), in translating genome-wide associationstudy findings to help prioritize the advancement of thera-peutic targets.

One goal of the GTEx pilot study was to assess the fea-sibility of collecting a large number of normal, high-qualitybiospecimens. This goal was met in 2.5 years with the

FIG. 6. The effect of ischemic time on RIN values. RINvalues greatly decreased when the postmortem interval(PMI) was ‡8 hours. PMI is based on interval between thetime of death or the cessation of blood flow and the time thatthe last tissue is placed in preservative.


collection of samples from 190 donors. The first set ofmolecular analysis data from the GTEx pilot, includingRNA sequencing data was published in May 2015.11–17 Dueto the overwhelming proof of feasibility during the pilot, theNIH Common Fund committed support to continue theGTEx project. Therefore the GTEx study is ongoing andwill continue through the end of 2015 and aims to collect atotal of 900 cases.

To meet the demands of such a large biospecimen col-lection beyond the pilot phase, the GTEx project developedrelationships with several additional BSSs and worked withall other partners to ensure that proper staffing and resourcesare in place to accommodate the influx of biospecimenscollected.

The project has added the collection of frozen tissue samplesfor a limited number of tissue types based on feedback from arequest for information (RFI) regarding the potential uses ofstored GTEx biospecimens (http://grants.nih.gov/grants/guide/notice-files/NOT-RM-12-028.html). Adding frozen biospeci-mens will increase the utility of the biospecimen set for theresearch community in terms of being able to compare theGTEx tissue directly to past and future studies with frozentissue since PAXgene preserved tissue is not yet widely uti-lized. The novel infrastructure put in place by BBRB andits partners is an essential part of the groundbreaking GTExscientific project, which has the potential to change our un-derstanding of gene regulation and how gene interactionscontribute to disease development.

Acknowledgments

Members of the GTEx consortium include Laura Barker,Margaret Basile, Alexis Battle, Joy Boyer, Debra Bradbury,Jason P. Bridge, Amanda Brown, Robin Burges, ChristopherChoi, Deborah Colantuoni, Nancy Cox, Emmanouil T.Dermitzakis, Leslie K. Derr, Michael J. Dinsmore, KenyonErickson, Johnelle Fleming, Timothee Flutre, Barbara A.Foster, Eric R. Gamazon, Gad Getz, Bryan M. Gillard,Roderic Guigo, Kenneth W. Hambright, Pushpa Hariharan,Rick Hasz, Hae K. Im, Scott Jewell, Ellen Karasik, ManolisKellis, Pouya Kheradpour, Susan Koester, Daphne Koller,Anuar Konkashbaev, Tuuli Lappalainen, Roger Little, JunLiu, Edmund Lo, John T. Lonsdale, Chunrong Lu, Daniel G.MacArthur, Harold Magazine, Julian B. Maller, YvonneMarcus, Deborah C. Mash, Mark I. McCarthy, JeffreyMcLean, Bernadette Mestichelli, Mark Miklos, Jean Mon-long, Magboeba Mosavel, Michael T. Moser, Sara Mosta-favi, Dan L. Nicolae, Jonathan Pritchard, Liqun Qi,Kimberly Ramsey, Manuel A. Rivas, Barnaby E. Robles,Daniel C. Rohrer, Mike Salvatore, Michael Sammeth, JohnSeleski, Saboor Shad, Laura A. Siminoff, Matthew Ste-phens, Jeff Struewing, Timothy Sullivan, Susan Sullivan,John Syron, David Tabor, Mehran Taherian, Jorge Tejada,Gary F. Temple, Jeffrey A. Thomas, Alexander W. Thom-son, Denee Tidwell, Heather M. Traino, Zhidong Tu, DanaR. Valley, Simona Volpi, Gary D. Walters, Lucas D. Ward,Xiaoquan Wen, Wendy Winckler, Shenpei Wu, and JunZhu. The authors would like to thank the donors and theirfamilies for their generous tissue donation to the GTExstudy.

The authors would also like to thank the following indi-viduals for their previous and ongoing efforts in the GTExstudy: Assya Abdallah, Anjene Addington, James M. An-

derson, Patrick K. Bender, Mark Cosentino, Norma Diaz-Mayoral, Theresa Engel, Fernando Garci, Allen Green,Tiffanie Hammond, Katherine Jaffe, Judy Keen, MaryKennedy, Peter Kigonya, Brent Lander, Sreenath Nampally,Cathy Ny, James Robb, Vikram Santhanum, NataliyaSharopova, Shilpi Singh, Conrado Soria, Anne Sturcke,Surendra Sukari, Elizabeth J. Thomson, Magda To-maszewski, Casandra Trowbridge, Ferdinand Udoye, DavidVanscoy, Negin Vatanian, Elizabeth L. Wilder, and Penel-ope Williams.

This work was supported by the National Institutes ofHealth (HHSN261200800001E (Leidos Prime contract withNCI); 10XS170 (NDRI), 10XS171 (Roswell Park CancerInstitute), 10X172 (Science Care Inc.), 12ST1039 (IDOX);10ST1035 (Van Andel Institute); HHSN268201000029C(Broad Institute); and R01 DA006227-17 (U Miami BrainBank).

Author Disclosure Statement

The authors disclosed no conflicting financial interests.J.V is a member of the Board of Directors of NDRI.

References

1. The GTEx Consortium. The Genotype-Tissue Expression(GTEx) project. Nature Genetics 2013;45:580–585.

2. Sun W, Hu Y. eQTL mapping using RNA-seq data. StatBiosci 2013;5:198–219.

3. The GTEx Consortium. The Genotype-Tissue Expression(GTEx) Pilot Analysis: Multi-tissue gene regulation in hu-mans. Science 2015;348:648–660.

4. Mucci NR, Moore H, Brigham LE, et al. Meeting researchneeds with postmortem biospecimen donation: Summaryof recommendations for postmortem recovery of normalhuman biospecimens for research. Biopreserv Biobank 2013;11:77–82.

5. Schroeder A, Mueller O, Stocker S, et al. The RIN: AnRNA integrity number for assigning integrity values toRNA measurements. BMC Mol Biol 2006;7:3.

6. Siminoff LA, Traino HM, Mosavel M, Barker L, Gudger G,Undale A. Family decision maker perspectives on thereturn of genetic results in biobanking research. Genet Med2015. Epub ahead of print 9 April 2015.

7. Staniszewski W. Virtual microscopy, data management andimage analysis in Aperio ScanScope system. Folia His-tochem Cytobiol 2009;47:699–701.

8. Gallego Romero I, Pai AA, Tung J, Gilad Y. RNA-seq:Impact of RNA degradation on transcript quantification.BMC Biol 2014;12:42.

9. Groelz D, Sobin L, Branton P, Compton C, Wyrich R,Rainen L. Non-formalin fixative versus formalin-fixedtissue: A comparison of histology and RNA quality. ExperMol Pathol 2013;94:188–194.

10. Mailman MD, Feolo M, Jin Y, et al. The NCBI dbGaPdatabase of genotypes and phenotypes. Nature Genetics2007;39:1181–1186.

11. The GTEx Consortium. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: Multitissue generegulation in humans. Science 2015;348:648–660.

12. Baran Y, Subramaniam M, Biton A, et al. The landscape ofgenomic imprinting across diverse adult human tissues.Genome Res 2015;25:927–936.

13. Gibson G. GTEx detects genetic effects. Science 2015;348:640–641.


14. Mele M, Ferreira PG, Reverter F, et al. The human tran-scriptome across tissues and individuals. Science 2015;348:660–665.

15. Pierson E, Consortium GT, Koller D, Battle A, MostafaviS. Sharing and specificity of co-expression networks across35 human tissues. PLoS Comput Biol 2015;11:e1004220.

16. Rivas MA, Pirinen M, Conrad DF, et al. Effect of predictedprotein-truncating genetic variants on the human tran-scriptome. Science 2015;348:666–669.

17. Roosing S, Hofree M, Kim S, et al. Functional genome-wide siRNA screen identifies as mutated in Joubert syn-drome. eLife. 2015;4.

Address correspondence to:Helen M. Moore, Ph.D.

Chief, Biorepositories and BiospecimenResearch Branch

Cancer Diagnosis ProgramDivision of Cancer Treatment and Diagnosis

National Cancer Institute, Rm 3W428, MSC 97289609 Medical Center Drive

Bethesda, MD 20892

E-mail: [email protected]


www.BioTechniques.com239Vol. 60 | No. 5 | 2016

The need to determine RNA quality is a result of advances in RNA-based assays, as well as the diversity of biospec-imens from which RNA can be isolated. The advent of RNA-based biomarker assays of disease that interrogate the expression and sequences of RNAs in tissue has increased the demand for accurate measures of RNA quality. It is critical that the metric for RNA quality be defined in terms of its suitability for specific assays. Efforts to define RNA quality have thus accelerated with the development of microarrays (1). The RNA integrity number (RIN) has been widely adopted as a measure of RNA quality for RNA isolated from fresh and frozen tissue (2,3). However, the RIN remains an imperfect measure of quality; it lacks strong corre-lation to gene-specific measurements and cannot be accurately applied to RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue (2).

The design of a robust metric of RNA quality requires the development of a model to explain the differences observed in RNA isolated from frozen and FFPE tissues (4). The nature of RNA degradation within FFPE tissue was unclear until our previous work demonstrated that the quality of the RNA obtained from FFPE tissue was affected by (i) tissue hypoxia during the fixation phase (5) and (ii) challenges in RNA isolation related to the impregnation protocols (6). These studies resulted in a new model of tissue preservation in which chemical fixation protocols result in prolonged tissue hypoxia/anoxia from the time the tissue is devitalized until the penetration and reaction of the fixative halts cellular processes. During this interval, cellular programming in response to hypoxia initiates mRNA degradation via the activation of RNases that randomly degrade mRNAs and also

target for degradation their poly-A tails and 5´-caps. These processes seek to conserve energy that would otherwise be diverted to protein translation. Concurrent with mRNA degradation, all other classes of RNA are subject to degradation by RNases. On electropherograms, frozen tissues show a broad distribution of RNA lengths; however, this signal is overwhelmed by the 18S and 28S peaks in this frequency distribution represen-tation. With formalin fixation and paraffin embedding, the 18S and 28S peaks are generally not observed, largely due to hypoxia related to immersion fixation, and the distribution of RNA fragments is left-biased, with a tail representing the longer RNA fragments extending to the right (6,7). This distribution has been described previously (4), but methods to define a metric of RNA quality that corre-lates with gene-specific measures have not been previously reported.

The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissueJoon-Yong Chung1, Hanbyoul Cho1,2, and Stephen M. Hewitt1

1Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD and 2Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea

BioTechniques 60:239-244 (May 2016) doi 10.2144/000114415 Keywords: formalin-fixed; paraffin-embedded; RNA integrity; molecular pathology

RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investi-gative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue.

Reports

METHOD SUMMARYUsing an electropherogram created by the Agilent 2100 Bioanalyzer, the paraffin-embedded RNA metric (PERM) can be determined by a simple calculation based on the number of fluorescent units at specific time points. The PERM can be used to assess the integrity of RNA obtained from FFPE tissue in an application-specific manner.

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In the present study, we prepared specimens with varying RNA quality based on various fixation and tissue processing conditions, as previously described (5). In addition, we measured mRNA expression levels using the QuantiGene assay, which is based on branched-chain DNA technology. Furthermore, we created the paraffin-embedded RNA metric (PERM) formula, which is a novel quality assessment tool for RNA extracted from FFPE tissue. We also evaluated the value of PERM as an RNA quality tool and demonstrated the reliability of the PERM for RNA quality assessment in FFPE tissue specimens.

Materials and methodsTissue specimensTo create different levels of RNA degra-dation, we prepared FFPE tissue blocks as previously reported (5). Brief ly, 6-week-old female Balb/c mouse kidneys were fixed and processed with varying fixation times, fixation buffers, and tissue processing times. A whole kidney was used as the standard tissue specimen for the experiment, and independent experiments were carried out in triplicate. Mice were acquired from the Small Animals Section, Veter-inary Resources Branch, National Insti-tutes of Health (NIH). The animals were housed and euthanized in accordance with NIH guidelines for care and use of laboratory animals (6).

RNA extractionTotal RNA was extracted as previously reported (5–7). Two 10 mm-thick tissue sections were trimmed of excess wax and deparaffinized by three rounds of incubation in PROTOCOL Tissue Clear ing Agent (Fisher Scienti f ic, Kalamazoo, MI) for 15 m at 95°C with shaking followed by centrifugation at room temperature for 2 m at 10,000 × g. After deparaffinization, the sections were resuspended and ground in a solution of 4 M guanidine isothiocy-anate, 20 mM sodium acetate, and 25 mM m-mercaptoethanol (pH 5.5), followed by incubation for 72 h at 65°C with mild shaking. Subsequently, total RNA was isolated by phenol–chloroform extraction. In order to eliminate possible genomic DNA contamination, the isolated RNA was treated with TURBO DNase (Invitrogen, Carlsbad, CA).

The quantity of RNA extracted from FFPE tissue specimens was measured using a NanoDrop ND-1000 UV spectro-photometer (NanoDrop Technologies, Wilmington, DE). In addition, RNA was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), using the RNA 6000 LabChip kit (Agilent Technologies) to assess RNA integrity and

the ratio of the ribosomal RNAs. Using the Agilent 2100 Expert Software, we measured the RIN.

Paraffin-embedded RNA metric (PERM)To assess the RNA integrity of the isolated FFPE tissue specimen, we created a simple formula called the PERM. This novel RNA integrity assessment tool involved the use of

Figure 1. Paraffin-embedded RNA metric (PERM) for RNA extracted from formalin-fixed, paraffin-em-bedded (FFPE) tissues. Total RNA was extracted from three different mouse kidney FFPE tissue speci-mens treated with different fixatives (not buffered, Tris-buffered, and phosphate-buffered formalin). (A) Calculation of the PERM. RNA quality was assessed using the Agilent 2100 Bioanalyzer combined with the RNA 6000 LabChip kit (Agilent Technologies), and the PERM was calculated using the formula shown underneath the electropherogram. FUn is the number of fluorescent units at time n (in seconds) in the electropherogram. (B) Comparison of the PERM and RNA integrity number (RIN). The RIN was calculated using the Agilent 2100 Expert Software. Black and gray bars represent the PERM and RIN, respectively. Fo, formalin; Tris/Fo, Tris-buffered formalin; Phosphate/Fo, phosphate-buffered formalin.

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an electropherogram created by the Agilent 2100 Bioanalyzer. The PERM is based on a formula that approximates a weighted area-under-the-curve approach (Figure 1A):

PERM = FU25 + (2 × FU30 ) + (3 × FU35 ) +

(4 × FU40 ) + (5 × FU45 ) +

(6 × FU50 ) + (7 × FU55 ) +

(8 × FU60 ) + (9 × FU65 )

The PERM is calculated as the fluores-cence units at 25 s, plus 2× the fluores-cence units at 30 s, plus 3× the fluorescence units at 35 s, continuing in a multiplicative progression until the signal returns to base line. This approach provides a metric that places progressively greater value on the length of RNA. The PERM value is only appli-cable to RNA extracted from tissue that is subjected to chemical fixation via immersion and impregnation. We have validated the PERM against a variety of chemical fixatives paired with paraffin impregnation.

mRNA expressionTranscriptional expression levels in FFPE tissue specimens were assessed using the QuantiGene 2.0 Reagent System (Panomics, Fremont, CA). The QuantiGene assay was performed as previously described (5,8–10). To measure mRNA expression signals in FFPE tissue specimens, we used probes against glyceraldehye 3-phosphate dehydro-genase (GAPDH), cyclin-dependent kinase 4 (CDK4), and actin (ACTB), all specific for mouse RNA. Briefly, we used 200 ng of total RNA extracted from FFPE tissue samples for both the GAPDH and ACTB genes, whereas we used 500 ng total RNA for CDK4. The total RNA was resuspended in 10 µL RNA and then mixed with 40 µL capture buffer, 40 µL lysis mixture, and 10 µL target gene probe set (capture extender, label extender, and blocker). These mixtures were incubated at 53°C for 16–20 h within a 96-well capture plate. Subsequently, the plates were incubated with a branched DNA amplifier at

46°C for 1 h and then the label probe working reagents were added. After incubation with the substrate dioxetane solution at 46°C for 30 m, luminescence was measured using a GloRunnerTM Microplate Luminometer (TunerBiosystems, Sunnyvale, CA). Data represent the mean of three independent experiments.

We have additionally evaluated a series of alcohol-based alternative fixatives (11). The effect of fixatives on RNA integrity was evaluated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) amplification of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene. To further evaluate the PERM as an FFPE RNA integrity tool, we examined the correlation between PERM values and Cq values of HPRT using a retrospective data set.

Statistical analysisStatistical analyses were performed using IBM SPSS version 21.0 (IBM Corp., Armonk,

Figure 2. Correlation of the paraffin-embedded RNA metric (PERM) with mRNA expression levels in formalin-fixed, paraffin-embedded (FFPE) tissues. We extracted total RNA from 20 different mouse kidney FFPE tissue specimens. mRNA expression was measured using the QuantiGene assay with mouse GAPDH, CDK4, and ACTB gene-specific probe sets using the QuantiGene reagent system (Panomics). The relative expression level of each gene was normalized to that of frozen kidney tissue. (A) Scatter plot of the PERM versus GAPDH gene expression level (r = 0.963, P < 0.001). (B) Scatter plot of the PERM versus CDK4 gene expression level (r = 0.869, P < 0.001). (C) Scatter plot of the PERM versus ACTB gene expression level (r = 0.974, P < 0.001). (D) Scatter diagram of the PERM versus the average expression levels of the three genes (r = 0.974, P < 0.001). (E) Scatter plot of the RIN versus the average expression levels of the three genes (r = 0.060, P = 0.801). Data represent the mean of three independent experiments.

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NY). Pearson’s correlation coefficient analysis was used to evaluate the associ-ation not only between RNA measurement and the PERM or RIN but also the correlation between the PERM and RT-qPCR data. A P value <0.05 was considered statistically significant.

Results and discussionThe reliability of the PERM for assessing RNA integrity was evaluated in experimental mouse kidney FFPE tissue blocks. Total RNA was isolated from 20 mouse kidney FFPE tissue specimens. Optimal A260/A280 ratios for RNA were obtained from all specimens (1.81–1.93, data not shown). All samples were run on the Agilent 2100 Bioanalyzer. For the initial assessment of the PERM, three RNA specimens were analyzed using the Agilent 2100 Expert Software, and the PERM was then calculated using electro-pherograms (Figure 1). As shown in Figure 1B, the PERM had a greater dynamic range (8.05–51.65) than the RIN (2.4–2.5). These data demonstrate that the RIN is not appre-ciably correlated with the quality of RNA extracted from FFPE tissue.

To evaluate the reliability of the PERM as an RNA integrity tool for FFPE tissue, we performed the QuantiGene assay using mouse GAPDH, CDK4, and ACTB probe sets on all of the specimens. There was an excellent correlation between mRNA expression measured by the QuantiGene assay and the PERM values for GAPDH (r = 0.963, P < 0.001) (Figure 2A), CDK4 (r = 0.869, P < 0.001) (Figure 2B), ACTB (r = 0.974, P < 0.001) (Figure 2C), and the average of the 3 genes (r = 0.974, P < 0.001) (Figure 2D). However, there was no corre-lation between the RIN and mRNA using the QuantiGene assay (r = 0.060, P = 0.801) (Figure 2E).

In a retrospective study, PERM values were also correlated with RT-qPCR data (11). An alternative fixative showed the highest PERM values (mean = 121.23), while neutral buffered formalin showed the lowest PERM values (mean = 32.02) among tested fixatives (11). RNA isolated from tissue fixed in the alternative fixative resulted in a mean Cq value of 34.46 for HPRT, whereas the Cq value was higher in samples generated from formalin-fixed tissues (mean Cq = 41.27). Notably, the PERM values of RNA isolated from samples treated with different fixatives were negatively correlated with the Cq values for the HPRT gene (r = -0.900, P < 0.001)

(Figure 3). Altogether, these data suggest that the PERM is a robust tool for RNA quality measurement in fixed tissues.

RNA obtained from FFPE tissue is subject to substantial degradation. Our previous studies suggest that degradation is a function of cellular hypoxia/anoxia during chemical fixation, as well as strand-breakage induced by the fixatives (5,6). We have utilized the PERM in as yet unpublished studies of alternative fixatives and demon-strated that it provides a simple and accurate measure of RNA quality when tested against PCR-based assays.

The quality of extracted RNA is a critical factor in downstream quantification assays such as microarray analysis and RT-qPCR (1,12). RNA integrity has conven-tionally been measured by the ratio of 28S to 18S rRNA, which is neither robust nor accurate. Furthermore, this methodology is unsuitable for highly degraded RNA from FFPE tissues. To overcome these problems, several publications have discussed RNA quality control (QC) tools, for example the RIN (2,3), RNA integrity score (RIS) (13), DV200 (2014. Expression Analysis of FFPE Samples. Illumina Technical Note Pub. No. 470-2013-002), RT-qPCR assay (14,15), and multiplex RT-qPCR (16). Although the PCR-based assays are the most reliable and sensitive assays among these RNA QC tools, challenges remain, such as the appropriate choice of target genes, primer design, optimization of PCR conditions, and long processing times. On the other hand, microcapillary electrophoresis–based methods have been recently developed and introduced as RNA QC tools. The RIN is an algorithm based on a combination of electro-phoretic features including the total rRNA ratio, the height of the 18S peak, the fast area

ratio, and the height of the lower marker. The RIN is a reliable RNA QC method for RNA prepared from fresh and frozen fresh tissue and used in gene expression experiments. However, prior studies have suggested that the RIN is not good predictor of the success of gene expression experiments, especially in cases where a small number of RIN values are calculated. In addition, Unger et al. recently demonstrated that the precision of the RIN as an RNA QC test depends on the biochemical or biophysical RNA degradation method used, such as heat, enzymatic, or UV light. Recently, RIS (13) and DV200 (Illumina Technical Note, Pub. No 470-2013-002) metrics were described in the latest versions of analysis software using QIAxcel Screen Gel software (Ver. 1.2.0) and Agilent 2100 Expert software (Ver. B. 02.07.S1532), respectively, for automated capillary electrophoresis of extracted RNA. The RIS uses a similar scoring system (ranging from 1 to 10) as the RIN, whereas DV200 calcu-lates the percentage of RNA fragments >200 nucleotides in electrophoretic measure-ments. Although these latest RNA QC tools potentially improve the assessment of the integrity of RNA extracted from archival FFPE tissue, both systems require a special kit and sophisticated instrumentation.

Much like the RIN, the PERM was origi-nally developed without comparison to gene-specific measures as an intermediate assay of RNA quality in investigations of tissue preservation. In our evaluation of the PERM, we demonstrated a correlation of PERM values with mRNA expression and RT-qPCR data. Notably, the PERM is applicable to FFPE tissue and indicates the suitability of isolated RNA for downstream applications. In practice, PERM values should be interpreted as PERM ± 2.5 units. We primarily applied

Figure 3. Correlation between the paraffin-embedded RNA metric (PERM) and RT-qPCR data. We applied the PERM to retrospective data for the eval-uation of its value as an RNA quality control tool. The mean quantitation cycle (Cq) value of the housekeeping gene HPRT was determined in kidney tissue treated with different fixatives (n = 19). The scat-ter plot shows that the PERM number is negatively corre-lated with the Cq values for HPRT (r = -0.900, P < 0.001).

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the PERM to the Agilent 2100 Bioanalyzer; however, instruments from other vendors can be used, and simple densitometry of gel images would also be sufficient. Although initially used with the Bioanalyzer, the PERM can be used as an RNA quality metric for any method of quantifying RNA fragment length. One weakness of the PERM is that it requires laboratory validation to correlate the efficiency and performance of RNA extraction protocols with downstream assays. However, a laboratory can validate the PERM for its specific protocols and then use it as a robust method of qualifying the integrity of RNA prepared from FFPE tissues.

Author contributionsJ.-Y. C. and H.C. contributed to experi-mental design, conducted experiments, and analyzed data. S.M.H. conceived the study and contributed to the experimental design and data analysis. All authors partic-ipated in the drafting of the manuscript.

AcknowledgmentsThis study was supported by the Intra-mural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The authors would like to thank Kris Ylaya and Candice Perry for excellent technical assistance. This paper is subject to the NIH Public Access Policy.

Competing interestsThe authors declare no competing interests.

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13. Unger, C., O. Kofanova, K. Sokolowska, D. Lehmann, and F. Betsou. 2015. Ultraviolet C radiation influences the robustness of RNA integrity measurement. Electrophoresis 36:2072-2081.

14. Nolan, T., R.E. Hands, and S.A. Bustin. 2006. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1:1559-1582.

15. Penland, S.K., T.O. Keku, C. Torrice, X. He, J. Krishnamurthy, K.A. Hoadley, J.T. Woosley, N.E. Thomas, et al. 2007. RNA expression analysis of formalin-fixed paraffin-embedded tumors. Lab. Invest. 87:383-391.

16. Takano, E.A., T. Mikeska, A. Dobrovic, D.J. Byrne, and S.B. Fox. 2010. A multiplex endpoint RT-PCR assay for quality assessment of RNA extracted from formalin-fixed paraffin-embedded tissues. BMC Biotechnol. 10:89.

Received 20 October 2015; accepted 28 January 2016.

Address correspondence to Stephen M. Hewitt, Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, MSC 1500, Bethesda, MD 20892. E-mail: [email protected]

To purchase reprints of this article, contact: [email protected]

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Stabilization of Tissue Specimens for PathologicalExamination and Biomedical Research

Sanket A. Unhale,1,2 Amy P.N. Skubitz,1,3 Robin Solomon,4 and Allison Hubel1,2

Human tissue specimens are critical reagents in the diagnosis of disease and biomedical research. Tissuesexperience rapid degradation immediately after ligation from their blood supply. A variety of processingtechniques are employed to prevent the degradation of tissue samples, principally chemical fixation and thermalprocessing. The success of processing techniques is measured by the preservation of tissue morphology, as wellas the critical biomarkers. In preservation of tissue specimens, formaldehyde is the most widely used fixativethat maintains tissue morphology. However, the cross-links resulting from chemical interactions betweenformaldehyde and biomolecules in the specimens introduce difficulties in detection and extraction of antigensfor analysis. Alternative processing methods, such as chemical fixation (e.g., alcohol-based) or thermal proces-sing (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 procedureson morphology and antigenicity of the preserved tissues as assessed by histology, immunohistochemistry,proteomics, and genomics.

Introduction

Tissue biopsies and biospecimens collected during sur-gery are valuable resources for diagnostic and research

purposes. These samples facilitate diagnosis, selection oftreatment, and monitoring of response. As soon as a tissue isdisconnected from its blood supply, it experiences hypoxicconditions. The enzymatic alteration of proteins and nucleicacids ensues, leading to loss of antigens.1–3 Chemical orthermal processing of tissue is used to arrest degradation.Each method of preservation has its advantages and limita-tions. This article will review the parameters that influencethe quality of critical biomarkers after preservation by ther-mal and chemical processing.

Tissue Ischemia

Ischemia is the state in which tissue has experiencedhypoxia due to lost or restricted blood supply. The hypoxicconditions and enzymatic alteration of proteins and nucleicacids cause the loss of antigens.1–3 Moreover, this process isselective, affecting certain molecules more than others.4,6 Inparticular, nucleic acids and signaling molecules exhibitsignificant degradation in tissues subjected to hypoxia.5–9

Chung et al.1 simulated warm ischemia in the laboratoryusing rat kidneys and found a decrease in the rRNA

quality (assayed as a ratio of 18S and 28S bands by elec-tropherogram) and RNA yield within 4 h at 25�C or 37�C,compared to 4�C. However, the use of RNAlater, a stabi-lizing storage buffer, maintained the RNA quality for upto 24 h at 25�C.1

Huang et al.6 investigated the effect of warm ischemia ongene expression by using cDNA microarrays. They demon-strated that, although the standard assay of mRNA stabilitybased on the 18S and 28S bands showed that the mRNA wasstable, the microarray technique revealed significant alter-ation in gene expression profiles within 20 min after excisionof human colon cancer specimens. In similar studies,Spruessel et al.9 investigated the effect of ischemia on geneand protein expression in normal and malignant colon tis-sues. They found that the expression of 10%–15% of thegenes differed significantly from baseline within 15 min oftissue resection. Also, analysis of protein profiles by massspectrometry (SELDI-TOF) showed that 30% of the peaks inprotein profiles changed more than 2-fold within 30 min,with most of the changes occurring within the first 15 min. Incontrast, investigation of gene expression patterns in radicalprostatectomy specimens by Dash et al.8 yielded very littleoverall variation after 5 h of ischemia. However, among thegenes associated with prostate cancer, EGR1 showed almosta 2-fold increase in expression, while hepsin exhibited arelatively steady expression throughout the 5 h of ischemia.

1Biopreservation Core Resource, Departments of 2Mechanical Engineering and 3Laboratory Medicine and Pathology, University ofMinnesota, Minneapolis, Minnesota.

4Department of Pathology, Veterans Administration Hospital, Minneapolis, Minnesota.

BIOPRESERVATION AND BIOBANKINGVolume 10, Number 6, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/bio.2012.0031

493

These results suggest that tissue ischemia is a significant factorin detection and interpretation of molecular biomarkers.

Ischemic time can also influence other methods of bio-marker detection, including immunohistochemistry. Khouryet al.10 investigated the effects of delay in fixation on the ex-pression of the estrogen receptor (ER), progesterone receptor(PR), and HER2. Immunohistochemical (IHC) staining ofbreast cancer tissues for ER and PR showed a decrease instaining when tissue fixation had been delayed for 1–2 h, witha significant loss of expression after a 4 to 8 h delay. HER2staining assayed by fluorescence in situ hybridization (FISH)showed a decrease in fluorescence with increasing delay infixation, starting within 1 h, which prompted the authors torecommend fixation of breast cancer biopsies within 1 h ofresection to avoid degradation of these markers and henceavoid inappropriate therapeutic decisions. Yildiz-Aktas et al.also noted reduced staining of ER, PR, and HER2 markers inbreast carcinoma samples.11 Pinhel and colleagues12 com-pared the expression of breast cancer biomarkers in resectedtumor specimens that were processed immediately or afterknown periods of delay in a clinical setting. The markers Ki-67, ER, PR, and HER2 did not vary significantly among thecore-cut biopsies taken immediately after tumor resection andafter 20 to 80 min delays in fixation.

Jones et al.13 measured variations in the levels of Src ty-rosine kinase markers, due to delays in processing, in breastand bladder cancer specimens. Delays in processing in breastcancer biopsy specimens for 60 min after ligation led to sta-tistically significant increases in the levels of phosphopax-illin, while the levels of total paxillin decreased in the sametime period. In contrast, the same markers did not varysignificantly in bladder cancer specimens under similarprocessing conditions. The stability of phosphoproteins wasexamined by Espina et al.14 who quantified 53 phospho-protein biomarkers in 6 different tissue types over a periodup to 2 h past tissue excision. Fluctuations were recorded inthe expression levels of different phosphoproteins in differ-ent tissue types and storing tissues at 4�C was ineffective atreducing changes in phosphoprotein levels. Phosphatase andkinase inhibitors in the fixative or storage solution stabilizedthe phosphoproteins.15 The authors recommended tissueharvesting and processing within 20 min post-excision inorder to preserve phosphoproteins.

Thermal Processing of Frozen Sections

Another strategy to reduce tissue degradation involvesquick freezing. This technique is commonly used when nu-cleic acids or proteins are to be extracted.4,6,14,16–20 Samplefixation by freezing is a rapid process (when compared tochemical fixation)21 and therefore is useful for interoperativediagnostics.22–24 Frozen tissue biospecimens are also com-monly analyzed using immunohistochemistry, proteomics,and genomics.4,7,18–20 The most commonly used freezingtechnique involves immersion of the sample in an ultra-coldbath of quenching fluid such as liquid nitrogen or iso-pentane.6,17,19,26–35 Briefly, a bath of isopentane in a metalcontainer is cooled using a liquid nitrogen bath to the stagewhere the isopentane starts to solidify around the edges,resulting in a temperature of about - 150�C. A tissue sampleis dissected into small pieces and transferred to standardcryovials and frozen by immersing the sealed cryovialinto the isopentane bath.29 Alternatively, liquid nitrogen

quenching ( - 196�C) or an isopentane bath cooled using dryice ( - 80�C) may be employed, depending upon the tissuetype, specimen size, and availability of resources. Theseprotocols for biospecimen freezing, termed ‘‘snap freezing’’or ‘‘flash freezing’’, are recommended by the National Can-cer Institute (NCI),29 European Organization for Researchand Treatment of Cancer,19 and International Society forBiological and Environmental Repositories (ISBER).35

Another method of processing tissue samples involvesfirst embedding the tissue into Tissue Freezing Medium(TFM) or Optimal Cutting Temperature (OCT) com-pound,20,29 then freezing the tissue. NCI and ISBER recom-mend placing the tissue sample into a plastic cryomold,which is then filled with the liquid embedding medium andthe entire system is frozen (e.g., in the vapor phase of liquidnitrogen).29,35 For small specimens, Loken and Demetrick20

proposed using a type ‘00’ VCap capsule that is partiallyfilled with OCT. By this technique, a small piece of tissue isplaced in the capsule and the rest of the capsule is filled withmore OCT, and then immediately frozen by immersion inliquid nitrogen. The capsules can be stored in cryovials andthe specimen can be harvested by slicing the capsule asneeded while maintaining the remaining sample in thecryovial for subsequent use.

The main disadvantage of allowing liquid nitrogen tocontact tissues directly (i.e., with or without a cryovial) as aquenching fluid, is that when a biospecimen at room tem-perature is introduced into the liquid, a vapor film formsaround the tissue which reduces the heat transfer efficiencydrastically.36–38 Moline and Glenner21 showed that coatingtissues with fine powders (e.g., confectioners’ sugar, talcumpowder, flour, or starch) can achieve very fast cooling rates.The coated samples were frozen at least twice as fast asuncoated tissues frozen with liquid nitrogen or isopentane.Damage to tissue morphology was dependent upon the sizeof the coating granule; the best and most consistent mor-phology preservation was achieved when tissues werecoated with talcum powder.

Tissue from the nervous system such as the brain is deli-cate and particularly susceptible to processing artifacts, re-quiring more extensive processing. For example, afterdissection, brain tissue blocks may be fixed chemically for12–24 h, followed by sucrose filtration.39 To reduce tissueshrinkage resulting from the osmotic effects of exposure tosucrose, these tissue blocks are transferred to 20% sucrose inphosphate buffer for 2–3 days, followed by infiltration with30% sucrose in phosphate buffer for another 2–3 days. Theseblocks can then be frozen by placing them into metal trayscovered by powdered dry ice.39,40 Though infiltration of 30%sucrose is often recommended in the freezing of brain tissue,Rosene et al.41 noted that this procedure might not provideoptimum protection against freezing artifacts, especially inlarge blocks of brain tissue. They suggested infiltrating largebrain tissue blocks (larger than 60 cc) with 10% glycerol plus2% dimethylsulfoxide (DMSO) in fixative or buffer for 1–3days, followed by 3–5 days of immersion in 20% glycerolplus 2% DMSO. The tissue blocks were then encased intoalbumin-gelatin blocks and frozen by putting the blocks ontoa metal plate surrounded by dry ice pellets or by using anisopentane bath cooled using alcohol-dry ice slush. Thismethod yielded tissue blocks with fewer freezing artifacts.Additionally they reported that the use of cryoprotectantsfacilitated sectioning on the cryostat.41

494 UNHALE ET AL.

Chemical Processing

Chemical fixation is an alternative method of preventingdegradation of tissue samples. Chemically fixed specimensare subsequently dehydrated and embedded in paraffin wax,which facilitates long-term storage as well as thin sectioningof the preserved specimen for analysis. The fixatives used inhistotechnology are broadly categorized into two types,cross-linking fixatives and coagulating fixatives.

Buffered formalin or formaldehyde is the most commoncross-linking fixative. Formalin, which is a solution offormaldehyde in water, mainly consists of monomericformaldehyde or methylene hydrate. The fixation and cross-linking process requires several hours, if not days, to becompleted. The cross-links and conformational changes in-duced by fixation may result in the masking of epitopes andhinder detection of antigens by antibodies.43–45 The cross-linking process is reversible in most cases and antigens canbe retrieved by breaking the bonds through the applicationof heat or chemical reactions.34,43,45 Heat-induced antigenretrieval reverses most of the cross-linking artifacts and hasbecome a standard procedure for tissue processing in diag-nostic pathology.34,43,45–47

The most common coagulating fixatives are alcohol- oracetone-based solutions. They function by removal and re-placement of water in the tissue and precipitating or coagu-lating the proteins, which results in preservation of tissuestructure.42–44 Furthermore, the displacement of water fromthe vicinity of the proteins causes a reduction in the repulsiveforces acting on hydrophobic moieties. This then causes adestabilization of hydrogen bonds in the hydrophilic areas ofthe protein, leading to the loss of tertiary structure. The irre-versible disruption of tertiary structure or denaturation makesthe proteins insoluble and also causes a loss of function.42,43

The chemical fixation process is preferred in diagnosticpathology.5,34,44 Formalin-fixed paraffin-embedded (FFPE)specimens exhibit superior tissue morphology and are mostsuitable for microscopic analysis and immunohistochemis-try. However, FFPE samples have disadvantages: it is diffi-cult to extract high quality DNA and RNA from FFPEsamples, there are health hazards related to the use offormaldehyde, and the fixation process is quite slow.48–50

Several reports have demonstrated that alcohol-based fixa-tives achieve comparable immunohistochemical staining andmorphology and achieve superior preservation of nucleicacids compared with formaldehyde fixation.48–52 Zinc-basedfixatives, which contain salts of zinc in buffered solution, arenon-cross-linking or noncoagulative. They show excellentpreservation of morphology, superior antigenicity and im-munohistochemical staining, as well as good quality RNAand DNA yield from fixed specimens.53,54

It is a common practice in both the clinical as well asresearch settings to store precut, unstained tissue sections onslides for a variety of reasons, including later use as positivecontrols, retrospective analysis, specimen conservation bylimiting the sectioning sessions, ease of storage of slides, andability to maintain FFPE blocks at a central repository whilekeeping slides in different locations.55–57 However, there isstrong evidence that the immunoreactivity of antigens inthese precut tissue sections decreases significantly withstorage time.55–62 ER, PR, Her-2, and p53 showed moderateto significant decreases in antigenicity with storage times of 3to 6 months.55,57,59 Cytokeratins, smooth muscle actin, PS-

100, CD45, CD20, and CD30 exhibited no significant changesin expression over 3 to 10 years, and a moderate increase inimmunostaining of vimentin was also observed.58 Themechanisms involved in loss of antigenicity in stored sectionsare not fully understood. Low temperature (4�C) storage doesnot prevent the loss of antigenicity.55,59 Exposure to air re-sulting in oxidation was thought to be one of the importantmechanisms affecting antigenicity in stored slides.60,62 Yet theaddition of paraffin layers or storage in a nitrogen environ-ment did not offer sufficient protection to precut tissue sec-tions, while the use of wax-soluble antioxidants diminishedthe protective effects of the paraffin coating.60 Recent studiesin which precut tissue sections were stored in a vacuumchamber with desiccant showed that endogenous or exoge-nous water in the tissues protected the antigens and therebysignificantly affected their immunoreactivity.61

Analysis Using Preserved Tissue Sections

Histology

Any method of preserving tissue biospecimens (chemicalor thermal) will result in processing artifacts. Trained pa-thologists are accustomed to seeing artifacts in the processedtissue sections and can make accurate diagnostic decisionsdespite the presence of alterations in morphology of thetissue. Using intra-operative frozen sections, trained pathol-ogists can make diagnoses with an accuracy level of 94%–99%.22–24 Although the tissue is expected to shrink due tofixation and subsequent processing due to dehydration54,63–65

and may have other fixation artifacts, FFPE sections arepreferred by most pathologists mainly because of their fa-miliarity with FFPE morphological patterns.64 Examinationof histomorphology of the tissue is critical to the making ofdiagnostic decisions by pathologists. For example, in pros-tatic carcinoma the epithelial lining of lumens is distorted,(specifically a loss of basal cells or alterations of lumen ap-pearance and spacing is observed); whereas high-gradeprostate intraepithelial neoplasia (HGPIN) is characterizedby an increase in nuclear size or loss of nuclear content.30,66

Chemical processing. Several investigators have studiedthe effects of fixatives and embedding media on the mor-phology of preserved specimens. Hoetelmans and col-leagues67 used several microscopic techniques to assess theefficacy of acetone, methanol, paraformaldehyde, and glu-taraldehyde in preserving the structure and morphology incultured MCF-7 cells. They demonstrated that fixation withacetone and methanol resulted into disruption of cellularstructures, plasma membranes, and nuclear integrity. Acet-one and methanol fixation resulted in suboptimal preserva-tion of subcellular organelles compared to aldehyde-basedfixatives. Though ethanol-fixed paraffin-embedded tissuesections showed comparable or superior morphology whencompared to formalin-fixed or snap frozen samples, the levelof nuclear details was not on par with a formalin-zinc fixa-tive.49 UMFIX, a fixative cocktail containing methanol andpropylene glycol, adequately preserved tissue architecture,cellular and nuclear morphology, and tinctorial reactioncompared to formalin-fixed tissue.68 Cox et al.63 did not findany significant differences in the morphology of rat liversamples preserved using various fixatives (e.g., formalde-hyde, ethanol, methacarn, as well as UMFIX). However,modified methacarn fixative resulted in the best

STABILIZATION OF TISSUE SPECIMENS 495

histomorphological features among the fixatives tested. Asignificant benefit of using zinc-based fixatives was exhibitedfor a variety of tissue types when histology was compared toformalin-fixation and snap freezing.53,54 Wester and col-leagues54 also showed that exposure to zinc-based fixativesprior to snap freezing yielded crisper, more well-preservedtissue morphology. Gillespie et al.49 also reported that ethanolfixation or formalin fixation of prostate cancer specimensyielded superior structural morphology and staining quality,though it did not affect the clinical diagnosis.

Thermal processing. When compared to FFPE tissuespecimens, frozen tissue sections exhibit a variety of mor-phological artifacts. The most prominent artifact seen infrozen sections is due to nucleation and growth of ice crystalsin the samples during freezing, subsequent storage, andthawing.17,41,69,70 Steu et al.17 demonstrated that tumorsamples frozen using carbon dioxide gas showed significantice crystal artifacts compared to tissue frozen using iso-pentane or liquid nitrogen. However, the complex and ir-regular morphological patterns seen in carcinomas of lung,endometrium, and kidney were preserved in tissue samplesfrozen using an isopentane bath pre-cooled to - 80�C. Fal-coneiri et al.70 noted that snap-frozen specimens exhibittechnical artifacts due to the crushing of tissues duringprocessing, nuclear smudges caused by tissue tears, ridges,or folds, and ice crystal artifacts that may preclude diagnosisor make the microscopic examination difficult. They com-mented that the frozen section technique may be used withconfidence in cases where tumor sizes are large, but it isproblematic to diagnose smaller cancers or assign Gleasonscores for prostatic cancers. Scott et al.30 also noted promi-nent artifacts in frozen sections from prostatectomy biopsies.They observed that in frozen sections, the nuclear enlarge-ment and size variations in cancerous cells appeared to beaccentuated. While histologic patterns that are usually seenin FFPE samples were maintained in frozen sections, nuclearchromatin textural features were lost and heterochromatinwas homogeneous. Despite these artifacts, they were able tomake accurate diagnoses and the frozen tissues could beused in immunohistochemistry and gene expression studies.

Immunohistochemistry

Immunohistochemistry (IHC) is used for the detection andlocalization of specific biomarkers within tissue sections.71

IHC is extremely sensitive to tissue fixation and processingprotocols. Conventional fixation techniques inherently alterthe biomarkers through cross-linking, dehydration, precipi-tation, and coagulation; each of these affects IHC analysis.For example, hepatocellular carcinoma is characterized by theexpression of CD34 in a unique sinusoidal pattern with col-lagen around tumor nests.72 Also, in breast cancer patients,IHC staining for ER and PR is the determinant for anti-hormonal therapy, while IHC staining for the HER2/neu re-ceptor determines the treatment with trastuzumab.73–75

Chemical processing. Formaldehyde fixation protocolsresult in masking of the epitopes due to cross-linking of theproteins and hinder detection of these antigens with specificantibodies.43–45 This necessitates the use of antigen retrievaltechniques, which reverse the cross-links and facilitate antigendetection.34,43,45–47 By using individual peptide spots as wellas cellular and tissue controls, Sompuram et al.46 demon-strated that IHC techniques are unable to detect antigens in

samples after formalin fixation, which can be reversed byapplying heat to the samples. Sample processing protocolsand antibodies have been developed for formalin-fixed sam-ples49 and as a result IHC staining can be achieved in mosttissue types for a variety of antigens.73–77

Several alternative fixatives have been evaluated for theirability to preserve antigenicity as assessed by IHC. By ana-lyzing the expression of epidermal growth factor receptor invarious cancerous tissues, Atkins et al.75 noted that the bestIHC staining was observed in samples fixed using 4% un-buffered formalin, acetic formalin alcohol, and a proprietaryfixative called Pen-Fix. In contrast, tissues fixed with 4%neutrally buffered formalin or Bouin’s fixative solution dis-played slightly weaker staining. Cerio et al.78 reported thatthe immunoreactivity of the antigens in preserved cutaneoustissue samples decreased rapidly when fixation lasted morethan 4 h in acetone, ethanol, or formalin, whereas fixationwith ammonium sulfate medium for 7 days did not affect theimmunoreactivity. When compared to formalin fixation,tissue samples fixed in acetone,52,78 ethanol,49,52,78 modifiedmethanol-Carnoy,52 and methanol-based UMFIX68 demon-strated adequate preservation of antigenicity. Furthermore,the zinc-based fixatives resulted in excellent antigen preser-vation and showed superior IHC staining results in absenceof any antigen retrieval processes compared to the FFPEsections.53,54

Thermal processing. Antibodies that recognize nativeepitopes frequently require the use of frozen tissue sectionsfor IHC.30 Shi et al.73 noted that frozen sections are consid-ered ‘‘gold standards’’ for the evaluation of new markers ornew reagents. They compared acetone or ethanol-fixed fro-zen sections to formaldehyde-fixed frozen sections, andshowed that the latter technique with antigen retrieval yiel-ded the best IHC detection for many antibodies. In anotherstudy, Scott et al.30 used frozen sections from prostatectomyspecimens for the investigation of integrin expression. Threeantibodies specific for a3, a6, and b4 integrins, each detectedtheir antigen in the frozen tissue sections. However, the in-tegrins were not detected in FFPE sections, even after antigenretrieval. Steu et al.,17 reported that Ki-67 was detected invarious frozen tumor tissues and was not affected by theprocessing methods they tested.

Proteomics

Advances in techniques for protein extraction, separation(e.g., electrophoresis and chromatography), and quantitation(e.g., mass spectrometry) have led to increasing interest andapplication of proteomics in diagnostic pathology.79 Tradi-tionally, fresh or frozen tissues are used commonly for pro-teomic analysis due to minimal alterations in the native stateof the proteins that is an inherent characteristic of the che-mical fixation methods.4,79–82 Proteomic analysis is utilizedlargely in the investigation of disease pathogenesis andidentification of important diagnostic, predictive, and prog-nostic biomarkers of disease progression.80, Detection ofphosphoproteins has become essential for the developmentof individualized cancer therapy.14 In the clinical setting,proteomic techniques enable the differential diagnosis oftumors that appear histologically identical.83

Chemical processing. One of the goals of chemical fixationis to preserve the proteome in the tissue sample. However, ithas been reported that soluble proteins are lost when

496 UNHALE ET AL.

alcohol-based fixatives are used, while cross-linking fixativeslike paraformaldehyde preserve the localization of thesesoluble proteins.81 Soluble proteins move with the water andare consequently lost to the aqueous fixative solutions, aswell as to the dehydrating agents such as ethanol duringtissue processing, even in presence of aldehyde-based fixa-tives.84 Although stabilization of tissue specimens with for-malin fixation achieves superior structural preservation, theinter-molecular and intra-molecular cross-links that areformed as a result of the fixation process actually hinder theeffective extraction of the proteins of interest and subsequentanalysis.80–82,85 O’Leary et al.47 showed that fixation withformalin led to the aggregation of proteins and the formationof heterogeneous protein-formaldehyde adducts. By SDS-PAGE, they observed spreading of the characteristic proteinspots and the presence of multiple uncharacteristic bands.The antigen retrieval process successfully reversed the cova-lent methylene bridge cross-links and restored immunoreac-tivity. However, they also showed that formalin-fixationfollowed by ethanol dehydration resulted in the loss of pro-tein tertiary structure in cross-linked proteins, which essen-tially protected the cross-links from reversing during heat-induced antigen retrieval. Recently, it has been demonstratedthat using a buffer solution containing SDS improved theefficacy of the heat-induced antigen retrieval techniques, al-lowing the extraction of full length, reactive proteins fromFFPE tissue sections.80 FFPE tissue blocks can be used forproteomic analysis even after prolonged storage.79 Coagula-tive fixatives such as ethanol49,82 and methanol-based UM-FIX68 are compatible with proteomic analysis, resulting in awell-preserved protein profile that is superior to the formalin-fixed samples in the absence of any antigen retrieval.

Thermal processing. Fresh frozen tissues are preferred forproteomic analysis since their proteome is typically well pre-served. The proteomic profile of frozen tissue sections hasbeen analyzed by a variety of assays and demonstrates onlyminimal alteration.4,17,49,68,82,86 Steu et al.17 showed that pro-tocols involving isopentane freezing and OCT embedding didnot negatively influence the preservation of key cancer proteinbiomarkers such as Ki-67 when compared to snap-frozenspecimens. Also, when compared to ethanol-fixed specimens,25%–50% more proteins were extracted from frozen sections;the proteins from frozen sections appeared as sharp bands by2D SDS-PAGE as opposed to the smeared appearance ofprotein bands extracted from ethanol-fixed samples.49,82

One of the advantages of using frozen sections for pro-teomic analysis is that phosphoproteins and proteins withother post-translational modifications (PTMs), which areotherwise difficult to preserve using standard fixation tech-niques, are found in higher abundance in frozen samples.PTMs are critical components of signaling pathways andinfluence biochemical interactions and cellular responses tovarious conditions. Many of the PTMs are dynamic and theyare sensitive to tissue processing techniques. Hence, theability to capture and measure the PTMs provides an im-portant tool for investigating cellular regulatory mecha-nisms, as well as irregularities and abnormalities that arecharacteristic of a disease and have the potential to assist inpersonalized molecular therapies.14,87 Recent reports indicatethat snap-freezing specimens soon after resection can capturethe PTM states of several biomarkers which would otherwisebe lost or further modified.13,14,87 Furthermore, Ahmedet al.87 showed that heat stabilization of resected brain tissue

to 95�C and the presence of a stabilizer buffer immediatelyafter resection improved the preservation of specific phos-phoproteins.

Nucleic acids and genomics

Advancement in the field of genomic sequencing andtranscriptional profiling stimulated the use of nucleic acidbased assays for molecular profiling of diseases in clinicalpractice. Microarray technology facilitates analysis of thedifferential expression of thousands of genes simultaneouslywhile real-time polymerase chain reaction (RT-PCR) allowsrapid analysis of the transcriptional factors in the samples.These techniques allow investigators to analyze biochemicaland regulatory pathways associated with diseasestates.1,5,7,8,49 Furthermore, genome-wide expression analysishas paved the way to identify subsets of several diseasesclinically, elucidate distinctions among highly related tumortypes, and stratify patients with differential responses totherapies.7 The widespread use of genomic profiling de-pends upon the successful isolation and recovery of nucleicacids from preserved tissues; conventionally frozen tissueshave been the most reliable source of nucleic acids.

Chemical processing. FFPE has been the preferred methodof tissue processing for clinical applications. Therefore, it isimportant to understand the effects of formalin-based tissuefixation and processing on the nucleic acids within tissues.The cross-linking process that is characteristic of formalde-hyde fixation is most detrimental to the nucleic acids, mak-ing it difficult to extract and isolate them. Althoughenzymatic digestion or heat-induced antigen retrieval helpsin recovering the antigenicity of proteins, these proceduresare ineffective or may cause disruptions in some noncovalentinteractions in the RNA, which affects its integrity for sub-sequent downstream analysis.7,88,89 Masuda et al.88 showedthat formalin fixation for 16 h at 4�C caused the addition ofmono-methylol groups to almost 40% of the adenine basesfrom extracted RNA; this increased to 62% when sampleswere fixed for 7 days. However, heat treatment in Tris-EDTAbuffer reversed most of the methylol additions from theoligo-RNA. RNA isolated from FFPE samples is mostlyfragmented and does not extend beyond 200 to 300 bases byRT-PCR.7,68,89 Furthermore, formalin-fixation results in arti-ficial mutations or sequence alterations in the DNA due tocross-links as well as promotion of jumping the templatesduring PCR amplification.89,90

Most of the alternative fixatives preserve the nucleic acidsbetter than FFPE. Due to the absence of cross-linking, ethanoland methanol preserve nucleic acids quite well. Most of theconformational changes brought about by these alcohol-basedfixatives can be reversed by rehydration.89 Though the qualityof the RNA extracted from ethanol-fixed paraffin-embeddedtissues was reduced, as indicated by the 18S and 28S bands,the RNA was sufficient for techniques like RT-PCR andcDNA microarrays. Also, the quality of the DNA isolatedfrom these samples was superior to formalin-fixed samples;resulting in longer fragments that could be successfully am-plified using PCR.49 However, several limitations were re-ported for the ethanol-fixed samples used in mRNA analysisdue to increased laser microdissection time and loss of mea-sureable transcripts.48 Kim et al.91 reported that methacarn-fixed paraffin-embedded tissues yielded superior qualityRNA with intact 18S and 28S bands; the extracted RNA could


be successfully used for subsequent RT-PCR based assays.UMFIX-based,50,68 as well as zinc-based fixation protocols,54

have also demonstrated excellent nucleic acid recovery that iscomparable to frozen specimens.

Thermal processing. Molecular profiling of clinical sam-ples has relied on freezing as the best method to preserveproteins and nucleic acids in a way that is free from anyfixation-related artifacts. Hence, frozen tissues are mostcommonly used for genomic analysis. Despite the issues of alack of morphological preservation during freezing, frozensamples have been successfully used for laser capture mi-crodissection, a technique that facilitates gene expressionanalysis by isolating specific regions or cells from tis-sues.48,91–93 The quality of the RNA isolated from frozenspecimens and the quality and yield of DNA is superior,compared to most chemically fixed samples.17,48–50,68,93

Summary

Preservation of fresh tissue samples resected from thehuman body for diagnostic pathology is extremely importantand is sensitive to processing methods. Tissues experienceischemia and intrinsic enzymatic degradation, which alterthe state of the biomarkers. Quick processing of the tissue,using either chemical fixation or freezing, arrests the degra-dation and facilitates the use of tissues for subsequent anal-ysis and long-term storage. Thermal processing is thefavored procedure for many applications including in-traoperative diagnostics, proteomics, and genomics. In con-trast, chemical fixation, using cross-linking fixatives such asformalin or coagulative fixatives such as alcohols, is thepreferred procedure for archiving, histology, and immuno-histochemical applications. No one processing technique isperfect for all applications; rather each technique has ad-vantages and shortcomings. Therefore, procedures should beselected to optimize the specific downstream application ofthe tissues.


No competing financial interests exist.

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sequence alterations is due to formalin fixation of archivalspecimens. Am J Pathol 1999;155:1467–1471.

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Address correspondence to:Allison Hubel, Ph.D.

Mechanical Engineering DepartmentUniversity of Minnesota

111 Church St SEMinneapolis, MN 55455


500 UNHALE ET AL.

State of the Art in Preservation of Fluid Biospecimens

Allison Hubel,1,2 Alptekin Aksan,1,2 Amy P.N. Skubitz,1,3 Chris Wendt,4 and Xiao Zhong5

Fluid biospecimens (blood, serum, urine, saliva, cerebrospinal fluid and bronchial lavage fluid) contain not onlycells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which areutilized as biomarkers. Availability of high-quality biospecimens is a requirement for biomarker discovery. Thequality 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 thousandsof new biospecimens are added to the pool daily. Specimen stabilization is imperative, because fluid biospeci-mens degrade quickly when kept untreated at room temperature. Achieving a high-quality fluid biospecimenrequires 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 currentpreservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain bio-markers to current storage techniques.

Introduction

Fluid biospecimens (blood, urine, saliva, cerebrospinalfluid (CSF) bronchial lavage fluid, tear fluid, seminal

fluid, and ascites fluid) contain not only cells and subcellularcomponents, but also proteins, enzymes, lipids, metabolites,and peptides, which are utilized as biomarkers. At theforefront of personalized medicine, proteomic, peptidomic,lipidomic (in general, ‘‘-omics’’) research is discovering in-creasingly more biomarkers for risk assessment, diagnosis,management, and treatment of various diseases. Newly de-veloped microfluidic technologies can detect, capture,1 andquantify circulating tumor cells2 and conduct single-cellmolecular analysis.3,4

Availability of high-quality biospecimens is a requirementfor biomarker discovery and for determining the specificityand sensitivity of the discovered biomarkers. The quality ofthe biospecimens depends upon preanalytical variables (ie,collection and processing techniques, freeze/thaw stability,and storage stability), which account for > 60%–90% of thediagnostic errors.5–7

Hypothetically speaking, a freeze/thaw stable fluid bios-pecimen is one that is not affected by the thermal, mechan-ical, and chemical stresses induced during freezing andthawing. For these biospecimens, post thaw cell viability andfunction would be high and the macromolecules in the so-lution would regain their native states in terms of structure

and function. Aggregation and precipitation of proteins andother macromolecules would not be observed in the thawedbiospecimen. Proper stabilization of a fluid biospecimenwould ensure that potential issues associated with cryo-/lyo-protectant toxicity, ice damage, repeated freeze/thaw damage,osmotic damage, and recrystallization damage are carefullymanaged and minimized and/or accurately predicted. Onthe other hand, storage stability implies that the biochemicaldegradation of the biospecimen is halted or minimized.

Currently, millions of fluid biospecimens are stored inhundreds of biorepositories across the nation (in freezer-farms), and tens of thousands of new biospecimens are ad-ded to the pool daily (*500 million biospecimens are storedin biobanks as of 2010). Most people assume that thesecryogenically stored biospecimens are stable with biomarkerintegrity retained. However, in most biorepositories, fluidbiospecimens are stored by freezing without following anypreservation protocol. In most cases, samples are directlyplaced into - 20�C to - 80�C freezers, in the absence of anycryoprotectant, where they experience very slow cooling( - 1�C–2�C/min). These conditions often destroy the cellsand also inflict serious damage on macromolecules,8–16 al-tering their characteristics (eg, structure and activity), oftenirreversibly.8,17–24 This directly affects the quality of thestored biospecimens, thus altering the biomarkers in thestored biospecimens. Proteomic studies can be biased bystorage conditions25 and preanalytical variables affect the

1Biopreservation Core Resource, University of Minnesota, Minneapolis, Minnesota.Departments of 2Mechanical Engineering, 3Laboratory Medicine and Pathology, 4Medicine, and 5Biomedical Engineering, University of

Minnesota, Minneapolis, Minnesota.

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results, even overwriting the real biological variations.5–7 Thelow quality of the existing biospecimens in biobanks hasbeen identified as one of the major issues inhibiting scientificprogress.26

Specimen stabilization is imperative, because fluid biospe-cimens degrade quickly when kept untreated at room tem-perature. Degradation rates and biomarker susceptibilitydiffer considerably and those susceptible cannot be easilypredicted based on whether the biomarker is a small molecule,a metabolite, or a large macromolecule or whether it serves aspecific function or belongs to a certain family. For example,urine hydrogen peroxide levels change by 3–4-fold within 24 hat room temperature,27 whereas the urinary levels of a similaroxidative stress marker, 8-hydroxy-2’-deoxyguanosine, areperfectly stable under identical conditions.28

The success of biomarker research depends not only uponthe availability of the tools (eg, proteomic, peptidomic, lipi-domic, and metabolomic technologies) to extract informationfrom biospecimens, but also upon the availability of ‘‘high-quality’’ biospecimens.29 Achieving a high-quality fluidbiospecimen requires understanding the effects of storageprocessing parameters (eg, freezing and thawing as well ascryo-/lyoprotectant additives) and storage conditions onbiomarkers contained within the biospecimens. Establishingstandard protocols for sample collection, handling, and sto-rage is the key to having high-quality biospecimens thatyield reproducible and reliable results. Without establishingthe manner by which storage conditions influence candidatebiomarker stability, meaningful methods of biomarker de-tection cannot be developed.

Most of the proteins present in fluid biospecimens are af-fected by freezing and storage at cryogenic temperatures, withsome of the proteins being more extensively affected thanothers.8,12,17–19,21–24 The UK Biobank has acknowledged thatdifferent serum biomarkers (eg, TNF, IFN-a, IFN-g, IL-la,IL-Ib, and IL-6) require different storage conditions for stabi-lization.30 Some of the most promising cancer proteomicbiomarkers are very susceptible to freeze/thaw and frozen-state storage.31,32 For example, in sera from cancer patients,the levels of albumin, fibrinogen, and C3a significantly de-crease in correlation with the length of time spent in storage.33

At one time, C3a was considered as a potential biomarker forbreast cancer,34 until it was discovered to be too sensitive tostorage conditions.33 Similarly, freezing lactate dehydro-genase (LDH)35 under any condition is detrimental. LDH is aclinical biomarker for patients with sickle cell disease36 and isalso being evaluated as a testicular cancer biomarker. In ad-dition, matrix metalloproteinase-9 (MMP-9) starts to degradeat - 80�C, dropping by 65% in activity within 2 years of sto-rage.37 More recent studies that utilize highly sensitive tech-niques such as LC-MALDI-TOF and MALDI-FT-ICR massspectrometry (MS) have reported significant effects of re-peated freeze/thaw on the proteome.38,39 Other proteins thatare very susceptible to freeze/thaw include the MMP family(MMP-1, MMP-7, MMP-9, MMP-13)37 and a related family,ADAMs (a disintegrin and metalloprotease), which are con-sidered to be diagnostic and prognostic biomarkers in allmajor cancers including breast, pancreas, lung, bladder, col-orectal, ovarian, prostate, and brain40; tissue inhibitors ofmetalloproteinases41; polymeric proteins such as transthyretin(that forms fibrils leading to amyloidosis)38 as well as glyco-proteins22; and even small molecules such as folate,21

d-dimer,42 and thyroid hormones.24

Most of the studies of biomarker storage stability con-ducted to date have been confined to MS analysis, which isadequate for the measurement of the relative amounts ofpeptides/proteins or their absence/presence. However, MSdoes not provide information on changes in the secondaryand tertiary structure, state of denaturation, aggregation, orfunctional activity of proteinaceous biomarkers.38,43 There-fore, MS may underestimate the changes in biomarkers thatcould be measured using alternative methods such as en-zyme linked-immunosorbent assay (ELISA).

In the remainder of this article, we will discuss the mainissues related to the stabilization of specific biofluids by re-viewing (a) the current preservation and storage practicesapplied in biobanks/biorepositories and (b) the sensitivity ofcertain biomarkers to current storage techniques.

Storage Stability of Plasma and Serum

Plasma and sera are easy to collect and abundant (con-stituting *7% of the total body water).44 More importantly,this fluid bathes all tissues and organs, picking up macro-molecules and solutes from all major organ systems, pro-viding a rich source for biomarkers. One of the mainchallenges in studying plasma/serum samples is the largedynamic concentration range of the macromolecules it con-tains. To date, over 10,000 proteins have been identified inplasma/serum, with concentrations spanning 10 orders ofmagnitude.45 Many of the studies examining the stability ofplasma and serum proteins have used MS and examinedtrends in large numbers of proteins (vs. studying the stabilityof specific biomarkers of interest).

Plasma and serum protein levels obtained from wholeblood can be influenced by relatively short periods of holdingtime (*2 h) prior to processing. Ayache et al. observed thatlevels of 37 different factors (principally cytokines) changedsignificantly within 2 h after venipuncture.46 Banks et al.47

determined that significant changes to low molecular weightserum protein profiles occurred between 30 and 60 min aftervenipuncture and then remained constant up to 4 h post-collection. Ostroff et al.48 determined that the majority of the498 proteins examined were stable if the sample was cen-trifuged within 2 h of venipuncture and frozen within 2 h aftercentrifugation. Holding temperatures also influence proteinstability. Significant protein losses were observed when sam-ples were kept at room temperature for > 4 h or at 24 h whenstored at 4�C.49

Studies have demonstrated the differential response ofbiomarkers to the temperature of storage. For example,Pieragostino et al. utilized MALDI-TOF MS spectra analysisto show that specimens stored at - 20�C demonstratedongoing biochemical processes resulting in protein mod-ifications such as oxidation, amino acid truncation, and car-bonylation, whereas none of these effects was observedduring storage at - 80�C.50 Proteins found to be specificallysusceptible to modification during storage at high tempera-tures include C3/C4, a1B glycoprotein, a2-macroglobulin,apolipoprotein, and hemopexin.22 Three breast cancerbiomarkers (C3a anaphylatoxin, albumin, and fibrinogen)were found to be significantly degraded when stored at- 30�C (C3a, months; albumin, 1.4 years; fibrinogen,6 years),51 whereas free and total prostate-specific antigenin the sera of 160 prostate cancer patients were shown to bestable during frozen-state storage at - 70�C for 5 years.52

238 HUBEL ET AL.

Endothelial microparticles obtained from platelet-poorplasma were affected by cryostorage at - 80�C for 1week.53 Coagulation proteins exhibit significant degrada-tion when stored for > 2 years at - 74�C.54 In contrast,West-Nielsen et al.49 reported no effect on protein stabilityduring storage at - 20�C or - 80�C.

To minimize protein degradation, a protease inhibitorcocktail may be added to the plasma samples. This can helpprotect the stability of the proteins during storage at both- 20�C and - 70�C.55 Moreover, the use of stabilizing agentssuch as glycerol combined with storage in liquid nitrogen isalso recommended for the stability of serum samples.56

Repeated freeze–thaw cycles can also affect protein stabi-lity. Rai56 and Mitchell et al.57 observed significant de-gradation in protein stability with 2 freeze/thaw cycles.

Storage Stability of Urine

Urine is a biospecimen that can be collected noninvasively.The large array of proteins present in urine not only reflectthe physiology of the kidneys and the urogenital tract butalso the blood (plasma proteins contribute *30% of theproteins found in the urine).58 Several biomarkers (such asNMP22, Calreticulin, Clusterin, CystatinB, Proepithelin,UHRF1, a-1B-Glycoprotein, PCA3, and Cathepsin D) forurinary tract carcinoma, prostate cancer, and bladder cancerhave been successfully identified in urine.59–62

An interesting phenomenon observed in frozen/thawedurine is the formation of a precipitant.63 The precipitates,which are usually discarded before analysis, are determined tobe calcium oxalate dihydrate and amorphous calcium crystals.However, these precipitates have been shown to deplete notonly the calcium ions but also urinary proteins during storageat - 20�C in as little as 12 h of storage.63 As the normal urinarycalcium levels are 1 order of magnitude higher than those inthe sera, the effect of this ion on proteins during freezing isamplified and thus becomes easily detectable in the urine.64

Creatinine is a clinically relevant measure of renal functionand its stability in storage is important. Multiple reports havefound that creatinine is stable for up to 2 years when storedin the frozen state at - 20�C or - 80�C.65,66 However, it isstable for only 30 days at 4�C and 2 days at 55�C.67

Albumin is another important urinary biomarker for renaland cardiovascular diseases68 and its increase in urinary se-cretion mirrors disease progression. However, as detailed byInnanen et al.,69 freeze/thaw and storage stability of albuminis controversial with the reports supporting mainly 2 oppositeconclusions: freeze/thaw does not have an effect on albumincontent70,71 vs. freeze/thaw causes a significant decrease inalbumin content.72,73 Moreover, Brinkman et al.74 studied thestability of urinary albumin up to 24 months using im-munonephelometry. They found that samples could be storedat - 20�C for 5 months without significant change in theaverage albumin concentration; however, extended storage atthis temperature resulted in decreased levels.75 Similarly,Collins et al.76 reported that urinary albumin concentrationremained stable for up to 6 months at - 20�C. Parekh et al.66

examined urine samples stored for 8 months at - 70�C. Usingan immunoturbidimetric method, they found minimal declinein urinary albumin concentration for up to 2.5 years, with adecrease of *0.25% every 30 days over the entire storageperiod. These contradictory reports on albumin stability inurine are partially explained by Innanen et al.,69 in which the

decrease in albumin content is minimized when samples arewell mixed after freeze/thaw. These studies demonstrate thatfreezing-induced albumin aggregation (which is reversible byextensive mixing) is to a degree responsible for the observeddifferences. Other factors that vary between samples and im-pact protein stability include relative protein concentrations,pH, and ionic strength.77 It has not yet been determinedwhether the aggregated albumin presents a ‘‘sink’’ for the lowabundance proteins in the solution, irreversibly decreasingtheir concentrations.

Immunoglobulin G (IgG) in urine is found to be verysensitive to storage conditions, showing marked decrease inconcentration at - 20�C, with relative stability at - 70�C.77

This behavior is also mirrored by a1-microglobulin andtransferrin.77

Many proteins are vulnerable to various storage techniques;urinary cystatin C, a biomarker for glomerular filtration rate,has been shown to be stable using multiple preservationtechniques.78 Herget-Rosenthal et al.79 examined the stabilityof cystatin C under different storage conditions using a par-ticle-enhanced nephelometric immunoassay. They found thatcystatin C remained stable for 7 days at both 4�C and - 20�Cand for 48 h at 20�C. Repeated rounds of freezing and thawingfor up to 3 cycles did not cause significant protein destabili-zation. Herget-Rosenthal et al.79 also studied the influence ofurine pH on protein stability, as urine pH can be highlyvariable. Cystatin C was shown to be stable at pH > 5, but itlost its stability at lower pH. Similarly, Kidney Injury Protein 1was stable when stored at pH 6.7–8; however, its levels de-creased when stored outside this range.80

Recently, neutrophil gelatinase-associated lipocalin hasemerged as a biomarker for acute kidney injury.81 Grenieret al.82 used a chemiluminescent assay to study the effect ofpreanalytical variables on protein stability. Storage at 4�C for7 days only brought a < 2% change in protein concentration.It was also reported that long-term freezing at - 75�C isbetter than storage at - 20�C, resulting in smaller sample-to-sample variation.

Additional studies have examined the influence of storagetemperature on disease-specific biomarkers using specificpopulations. Schultz et al.65 investigated the effects of sto-rage temperature on urine proteins from 262 children withtype I diabetes using an enzyme-linked assay (ELISA).Urinary disease biomarkers such as albumin, retinol-bindingprotein, and N-acetyl glucosaminidase were less stable whenstored for 6–8 months at - 20�C compared with - 70�C.

Urinary exosomes and exosome-associated proteins havethe potential to serve as biomarkers for early diagnosis andtreatment of kidney diseases. Zhou et al.83 reported thatexosomes in the urine could be recovered after storage if thestorage temperature was - 80�C and if protease inhibitorswere present. They noted that storage at - 20�C caused amajor loss of exosome-associated proteins (even when com-pared with urine stored at 4�C).

Gazzolo et al.84 studied the stability of S100B, an acidiccalcium-binding protein used in the clinics to monitor high-risk newborns. Using an immunoluminometric assay,S100B was found to be stable when left for up to 72 h atroom temperature. Using high-pressure liquid chromato-graphy analysis, it was shown that the oxidative stressmarker, 8-hydroxy-2’-deoxyguanosine, is stable when storedat room temperature for up to 24 h, and for over 2 yearswhen stored at - 80�C.28

STATE OF THE ART IN PRESERVATION OF FLUID BIOSPECIMENS 239

Most studies focused on individual proteins, whereas re-cent studies have examined the effect of storage on a largenumber of urinary proteins by MS. Traum et al.85 usedSELDI-TOF MS to characterize changes in protein profileswhen urine samples were held at 4�C prior to freezing. Forholding times up to 24 h at 4�C, no differences in proteinprofiles were found. However, when held at room tem-perature, Papale et al.86 observed (using SELDI-TOF MS)degradation of urine protein profiles. They reported thatstability could be increased to 2 h postcollection with the useof protease inhibitor compound (PIC). They also observedthat protein profiles were stable for up to 5 freeze/thawcycles. Fiedler et al.87 used magnetic beads to separateurine peptides from urine and then characterized the pepti-dome using MALDI-TOF. Differences in the peptide profilewere observed between the fresh samples and those thatwere frozen/thawed once. Additional freeze/thaw cycles(up to 3) did not result in any significant change in thepeptide profile. Schaub et al.88 found that urinary proteinswere stable for up to 4 freeze/thaw cycles; however, certainproteins were not detectable after the fifth freeze/thaw cycle.

Some biomarkers are highly susceptible to storage,whereas some molecules can remain relatively stable in urineover long periods of time. Phthalates and metabolites, suchas dimethyl phthalate and mono-methyl phthalate, wereshown to be stable for over 20 years when the urine sampleswere stored at - 20�C.89 This shows the differential stabilityof small molecules and metabolites with respect to largemacromolecules in the urine.

Storage Stability of Saliva

Proteomic studies of saliva have drawn increasing atten-tion because saliva is easily accessed and noninvasivelycollected.90 Saliva contains a rich source of proteins andpeptides, some of which are plasma proteins. The salivaryproteins are not only responsible for oral health, but they canalso serve as a useful investigational tool for detection andearly diagnosis of other diseases such as cancers91–94 andSjogren’s syndrome.95 Further, some salivary antibodieshave been found to be elevated in patients with infectiousdiseases such as hepatitis and HIV.96–98

Preanalytical variables such as the methods used for thecollection and processing of saliva influence protein stability.Hu et al.93 added PIC to saliva supernatant and stored it at- 80�C until further analysis for oral cancer biomarkers.Shpitzer et al.94 performed multiple rounds of centrifugationon saliva samples, then incubated the cell pellets with lysisbuffer, and stored them at room temperature. Other studies,including Myers’ research on hepatitis C biomarkers, fail tomention conditions used for sample collection, preparation,or storage.97 The lack of standardized sample handlingprotocols for saliva samples remains a significant challengein data analysis and comparisons.

Specific salivary antibodies have been used to detect in-fectious diseases. Gaudette et al.99 studied the stability of IgGand HIV-1 antibody in whole saliva and oral fluid underdifferent storage times and temperatures. Oral fluid wascollected on a salt-treated cotton-padded filter and immersedin an antimicrobial solution. After 7 days of storage at roomtemperature, 93% of IgG in the oral fluid was stable. How-ever, HIV-1 antibody remained stable at - 20�C to 37�C forup to 21 days in oral fluid.99

Salivary-reduced glutathione (GSH) and tissue factor (TF)have been used as measures of oxidative stress in patients.100

Emekli-Alturfan et al.101 found that GSH levels were highlyvariable during storage, with the levels increasing over the first2 months and then decreasing significantly after 6 months ofstorage at - 20�C. TF activity also decreased significantly over6 months when stored at - 20�C and had a positive correlationwith GSH levels. Long-term freezing at - 20�C reduced GSHintegrity and TF activity, and therefore, storage for < 30 dayswas proposed as optimum for these 2 markers.

Using sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis, Jiang et al.102 evaluated the stability of 2 salivaryprotein markers, b-actin and cystatin C, by immunoblotting.About 90% of b-actin and 70% of cystatin C remained stablein the presence of a commercial stabilizing agent of unknowncomposition for up to 6 days at room temperature. However,only 20% of b-actin remained stable under the same condi-tions in the whole saliva without the stabilizing agent.

Some groups have investigated global salivary proteinprofile stability by varying the preanalytical procedures.Chevalier et al.103 evaluated the effects of storage time,temperature, and the use of PIC on saliva protein stabilityusing 1- and 2-dimensional gel electrophoresis. They reportedthat salivary protein stability decreased during storage.Schipper et al.104 examined the stability of samples stored at- 20�C and - 80�C for up to 6 months. Protein degradationwas observed for samples stored at - 20�C, whereas proteinsstored at - 80�C were stable (with a couple of exceptions).Additionally, for samples that underwent up to 4 freeze–thaw cycles, they reported no change in the protein profiles asmeasured using SELDI-TOF MS.

Storage Stability of Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is in direct contact with tissuesin the brain and spinal cord. CSF proteomic studies aretherefore very helpful in the detection and study of disordersin the central nervous system. Schoonenboom et al.105 in-vestigated the effect of storage time and repeated freeze–thawcycles on amyloid b (I-42) and tau, 2 biomarkers for Alzheimerdisease, using ELISA. The tau protein was still stable follow-ing 6 freeze–thaw cycles, but the concentration of amyloid b(I-42) decreased by 20% after 3 freeze–thaw cycles, in con-tradiction to earlier studies.106 Changes in the levels of the2 proteins were observed at high-temperature storage (4�Cand 37�C), and therefore, the authors recommended - 80�Cstorage. The consensus protocol published by a group of sci-entists recommended - 80�C storage based on the idea that‘‘liquid nitrogen storage is not feasible’’ for CSF, albeit withoutoffering any data to support the rationale for the idea.107

However, previous studies indicated decreased samplequality during storage at - 20�C and - 80�C beyond 3months, especially when PIC was not added.108

Storage Stability of BronchoalveolarLavage Fluid

A thin layer of epithelium lining fluid (ELF) covers thehuman airways protecting them from the external environ-ment and helping to maintain normal gas exchange. ELFcontains cells and many soluble components of the lung,which have been sampled by a variety of techniques to studythe physiology of the respiratory tract. The most commonly

240 HUBEL ET AL.

used sampling procedure is bronchoscopy, with the collec-tion of bronchoalveolar lavage fluid (BALF). Biochemicalanalysis of BALF has revealed an abundance of proteins inthe airways of patients with pulmonary diseases, andtherefore, BALF proteomics is being increasingly studied inthe hope to identify biomarkers of disease.

In addition to the usual preanalytical variation of time instorage, temperature, and processing, there are several uniquechallenges for the biopreservation of BALF. As the sample iscollected via the installation of saline, dilution of ELF proteinsand the presence of high salt concentrations are inherentchallenges. Therefore, it is imperative to first standardize theprocessing, handling and storage conditions of the BALF. Inaddition, many plasma proteins, such as albumin and IgG, arefound in abundance in BALF and can interfere with the de-tection of less-abundant proteins of interest.

Mucus is abundant in the respiratory tract and is oftenremoved from the BALF by filtering through a single layer ofgauze followed by centrifugation to separate cells and debrisfrom the fluid. This process may result in the loss of manyproteins and potential biomarkers. Although many methodsto study BALF proteins have been reported to date, no stu-dies have been conducted to examine the effects of differentpreservation conditions on BALF protein stability.

In a recent study, interleukin-8 and neutrophil elastase le-vels in BALF collected from patients with cystic fibrosis wascompared with their initial values after storage at 4�C for 7days and at - 80�C for up to 6 years. It was observed thatinterleukin-8 levels were stable, independent of storage tem-perature and time. Neutrophil elastase levels were stable forup to 6 months at - 80�C but decreased after 7 days at 4�C.109

Conclusion

Hundreds of biomarkers found in biofluids such as plas-ma, serum, urine, and saliva are used to monitor health,disease, and response to treatment. Clearly, these fluidscontain as-yet to-be identified biomarkers that could be usedto inform patient care. The studies described in this reviewmake it apparent that different collection and storage con-ditions have profound effects on protein stability, bringingartifacts to experimental results and resulting in invalidconclusions. Note that the only ‘‘preservation stability’’ stu-dies conducted on fluid biospecimens focus on the tem-perature of storage and the duration of storage, and thus, allthe current information summarized in this review aretherefore limited. In addition, the vast majority of studies arelimited to studying relatively few molecules and there is apaucity of studies examining the global effects of storage onproteins, cells, and subcellular particles.

The optimal protocols for the collection, processing, andstorage of each different type of fluid biospecimen has yet tobe determined. However, even with the limited preanalyticaldata that are currently available, some trends have emerged,which lead us to propose the following advice when one iscontemplating the preservation of fluid biospecimens:

1. Biofluid specimens should be stored in the vapor phase ofliquid nitrogen or, if liquid nitrogen is not feasible, at aminimum of - 80�C.

2. Specimens should experience a constant cooling rateduring freezing, using a controlled rate freezer or com-mercially available containers for smaller samples.

3. Temperature fluctuations during storage (due to repeatedfreezer access) and repeated freeze/thaw should beminimized.

4. Fast thawing methods should be utilized (eg, immersingthe sample in a continuously stirred 37�C water bath).Thawing rates should be monitored and validated.

5. For biofluid samples that contain high concentrations ofproteins that are freeze susceptible (such as albumin orLDH) or high concentrations of specific ions (such ascalcium in urine), frozen-state storage should be opti-mized (potentially by using cryo-/lyoprotectants).

6. If specific biomarkers or family of biomarkers are of in-terest, their freeze/thaw response, characteristics, andsusceptibility should be characterized not only by tech-niques such as MS, which is based on the charge densityof the macromolecules, but also by techniques that de-termine the secondary and tertiary structural changes (eg,infrared, Raman, and intrinsic fluorescence spectro-scopies).

7. More research is required to develop alternative biopre-servation techniques that will offer significant advantagesover cryopreservation.



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86. Papale M, Pedicillo MC, Thatcher BJ, et al. Urine profiling bySELDI-TOF/MS: monitoring of the critical steps in sample col-lection, handling and analysis. J Chromatogr B 2007;856:205–213.

87. Fiedler GM, Baumann S, Leichtle A, et al. Standarized pepti-dome profiling of human urine by magnetic bead separationand matrix-assisted laser desorption/ionization time-of-flightmass spectrometry. Clin Chem 2007;53:421–428.

88. Schaub S, Wilkins J, Weiler T, et al. Urine protein profiling withsurface-enhanced laser-desorption/ionization time-of-flightmass spectrometry. Kidney Int 2004;65:323–332.

89. Baird DD, Saldana TM, Nepomnaschy PA, et al. Within-personvariability in urinary phthalate metabolite concentrations:measurements from specimens after long-term frozen storage.J Expo Sci Environ Epidemiol 2010;20:169–175.

90. Aps JKM, Martens LC. Review: The physiology of saliva andtransfer of drugs into saliva. Forensic Sci Int 2005;150:119–131.

91. Maring JG, Wachters FM, Maurer M, et al. Gemcitabine andEpirubicin plasma concentration-related excretion in saliva inpatients with non-small cell lung cancer. Ther Drug Monit2010;32:364–368.

92. Brooks MN, Wang JG, Yang L, et al. Salivary protein factors areelevated in breast cancer patients. Mol Med Rep 2008;1:375–378.

93. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics fororal cancer biomarker discovery. Clin Cancer Res 2008;14:6246–6252.

94. Shpitzer T, Hamzany Y, Bahar G, et al. Salivary analysis of oralcancer biomarkers. Br J Cancer 2009;101:1194–1198.

95. Ryu OH, Atkinson JC, Hoehn GT, et al. Identification of parotidsalivary biomarkers in Sjogren’s syndrome by surface-enhancedlaser desorption/ionization time-of-flight mass spectrometryand two-dimensional difference gel electrophoresis. Rheuma-tology 2006;45:1077–1086.

96. Pomarico L, de Souza IPR, Castro G, et al. Levels of salivaryIgA antibodies to Candida spp. in HIV-infected adult patients:a systematic review. J Dent 2010;38:10–15 (review).

97. Myers T, Allman D, Xu KY, et al. The prevalence and correlatesof hepatitis C virus (HCV) infection and HCV-HIV co-infection


in a community sample of gay and bisexual men. Int J Infect Dis2009;13:730–739.

98. Arrieta JJ, Rodriguez-Inigo E, Ortiz-Movilla N, et al. In SituDetection of Hepatitis C Virus RNA in Salivary Glands. Am JPathol 2001;158:259–264.

99. Gaudette D, Griffin K, North L, et al. Stability of clinically sig-nificant antibodies in saliva and oral fluid. J Clin Immunoassay1994;17:171–175.

100. Arana C, Cutando A, Ferrera MJ, et al. Parameters of oxidativestress in saliva from diabetic and parenteral drug addict pa-tients. J Oral Pathol Med 2006;35:554–559.

101. Emekli-Alturfan E, Kasikci E, Alturfan AA, et al. Effect ofsample storage on stability of salivary glutathione, lipid per-oxidation levels, and tissue factor activity. J Clin Lab Anal 2009;23:93–98.

102. Jiang J, Park NJ, Hu S, et al. A universal pre-analyticsolution for concurrent stabilization of salivary proteins, RNAand DNA at ambient temperature. Archiv Oral Biol 2009;54:268–273.

103. Chevalier F, Hirtz C, Chay S, et al. Proteomic Studies of saliva:a proposal for a standarized handling of clinical samples. ClinProteomics 2007;3:13–21.

104. Schipper R, Loof A, de Groot J, et al. SELDI-TOF-MS of saliva:methodology and pre-treatment effects. J Chromatogr B 2007;847:45–53.

105. Schoonenboom NSM, Mulder C, Vanderstichele H, et al. Effectsof processing and storage conditions on amyloid beta (I-42) and

tau concentrations in cerebrospinal fluid: implications for use inclinical practice. Clin Chem 2005;51:189–195.

106. Sjogren M, Vanderstichele H, Agren H, et al. Tau and A{beta}42in cerebrospinal fluid from healthy adults 21–93 years of age:establishment of reference values. Clin Chem 2001;47:1776–1781.

107. Teunissen CE, Petzold A, Bennett JL, et al. A consensus protocolfor the standardization of cerebrospinal fluid collection andbiobanking. Neurology 2009;73:1914–1922 (review).

108. Berven FS, Kroksveen AC, Berle M, et al. Pre-analytical influ-ence on the low molecular weight cerebrospinal fluid proteome.Proteomics Clin Appl 2007;1:699–711.

109. Berry LJ, Sheil B, Garratt L, et al. Stability of interleukin 8 andneutrophil elastase in bronchoalveolar lavage fluid followinglong-term storage. J Cystic Fibrosis 2010;9:346–350.

Address correspondence to:Dr. Allison Hubel

Department of Mechanical EngineeringUniversity of Minnesota

111 Church St. SEMinneapolis, MN 55455


Received 23 December, 2010/Accepted 4 February, 2011

244 HUBEL ET AL.

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REVIEW ARTICLE

Storage of Human Biospecimens:Selection of the Optimal Storage Temperature

Allison Hubel,1,2 Ralf Spindler,1,2 and Amy P.N. Skubitz1,3

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

Introduction

B iospecimens include tissues, cells, and bodily fluids(and their constituent macromolecules). It is common for

biospecimens to be collected for use at a later time and dif-ferent location. The critical biological properties of the bio-specimen must be preserved during processing, transport, andstorage. The most common method of preserving biospecimensis freezing and storage of samples at low temperatures in orderto inhibit degradation. Other stabilization methods includechemical fixation,1 plastination,2 drying,3 lyophilization,4 ionicliquids,5 dry state storage,6, 7 and confinement.8 The focus ofthis review will be on long-term, low temperature storage ofhuman biospecimens. Issues associated with ischemia or stor-age studies of non-human samples will not be included in thisreview. It is noteworthy that many of the biophysical and bi-ological events that occur during freezing will influence thestability of any biospecimen, regardless of origin.

Physics of Biospecimens at Low Temperatures

As water is necessary for most biochemical reactions(including those that degrade molecules), understanding themobility of water during freezing is important. The mobility(and therefore ability of the water to participate in reactions)differs between water that is liquid, solid (e.g., ice), andglass (highly viscous liquid). The use of low temperaturestorage to stabilize biological specimens reflects the desireto control, on some level, the behavior of water in the sys-tem (see Ref. 9 for review) and specifically to reduce mo-

bility of water by freezing or vitrification. In order tounderstand what happens to water during freezing, a brieftutorial of the physics at low temperature is useful.

Most fluid biospecimens are complex mixtures contain-ing at minimum water, salt, and proteins, and therefore theydo not solidify at a single temperature, but freeze over arange of temperatures. The thermodynamic states of mul-ticomponent solutions can be described by phase diagrams10

and by time-dependent temperature-time-transition (TTT)diagrams.11 Specifically, the fraction of water that has beensolidified and the corresponding concentration of the unfro-zen fraction of solution can be determined based on tem-perature, for a given initial concentration and pressure.12–16

Water and all other components (e.g., lipids within the bio-logical sample) undergo phase transition and can be describedby phase diagrams as well.17

Unlike most materials, water can undercool significantlybelow its equilibrium freezing temperature.18 Pure water canfreeze at temperatures ranging from 0�C to - 40�C.19 It isnot uncommon for biospecimens to first form ice at - 5�C to- 15�C. The onset of freezing is called nucleation and canresult from water molecules forming stable ice crystals aidedby irregularities in the sides of the container or particles inthe sample. The undercooling of the sample (where thesample nucleates at temperatures below the melting tem-perature) is an important parameter for post thaw recovery ofcellular biospecimens and is frequently controlled using avariety of methods.20 After nucleation of a sample occurs,water is removed from the sample in the form of ice. Solutes

1Biopreservation Core Resource, 2Mechanical Engineering Department, 3Department of Laboratory Medicine and Pathology, Universityof Minnesota, Minneapolis, Minnesota.

BIOPRESERVATION AND BIOBANKINGVolume 12, Number 3, 2014ª Mary Ann Liebert, Inc.DOI:10.1089/bio.2013.0084

1

(e.g., proteins and salts) are not incorporated into the solid icecrystal. This results in a partitioning of the sample into a solidcomponent and the unfrozen liquid component whose soluteconcentration increases as the temperature of the sample de-creases. Cells, proteins, lipids, and other components are se-questered in the gap of unfrozen liquid between adjacent icecrystals.21, 22 When much of the water has been removed in theform of ice, the remaining unfrozen liquid is highly concen-trated 23 and the molecules, cells, and tissues present aresubjected to solute concentrations of *10,000 mOsm.24 Thedistribution of molecules may not be uniform in these partiallyfrozen solutions. Microsegregation of protein solutions hasbeen imaged during freezing using Raman spectroscopy25 anddemonstrated that even in gaps as small as 1mm, the distri-bution of protein was not uniform and the protein exhibitedaggregation near the ice–water interface.

The binary phase diagram of NaCl-water is often used asa model of the freezing behavior of biological systems in theabsence of cryoprotective agents.10 For example, whenisotonic saline solutions [0.9% (w/w) NaCl] freeze slowly,the gap between adjacent ice crystals will decrease until thesample fully solidifies (at the eutectic temperature) or vi-trifies (the glass transition temperature, Tg). For an isotonicsaline solution, the eutectic concentration is 23.3% (w/w)NaCl and occurs at - 21.2�C.

For cellular biospecimens and proteins stabilized forpharmaceutical applications, it is common for stabilizingagents to be added (dimethlysulfoxide and glycerol forcellular biospecimens26 and sugars and sugar alcohols forproteins27), resulting in multicomponent solutions. Empiri-cal equations have been used to estimate the glass transitiontemperature for multicomponent mixtures:

Tg(mixtures)¼ Tg1 � (1� x)þ Tg2 � xþ k � x � (1� x)

< Eq:1 >

with the glass transition temperatures Tg,i (i = 1,2) of thecomponents, the weight fraction x of the cryoprotective agentand an interaction parameter k. The glass transition temperatureof a 10% (w/w) dimethylsulfoxide (DMSO) solution withTg1 = - 135.2�C for water, Tg2 = - 122.2�C for DMSO, x = 0.1,and k = 16 can be calculated to be - 132.58�C.28 Compared toaqueous DMSO solutions, the glass transition temperature of thecommonly used cryoprotective agent trehalose is comparativelyhigher (Tg = - 113.9�C).13 Below the glass transition tempera-ture, the mobility of molecules within the sample is reduced dueto an increased viscosity of 1013 Pa-s. Angell29 reported thatNaCl solutions cannot vitrify under ‘‘normal’’ cooling proce-dures. However, the crystallization of isotonic systems can beprevented when applying very fast cooling rates.30 The glasstransition temperature is dependent upon several parameters,including concentration, storage temperature, and storagetime.16,31 More information on the thermodynamic principals ofthe phenomena described above (nucleation, crystal growth, andsolidification) can be found in Ref. 32.

Solidification of water in the system is also influenced bythe presence of extracellular matrix present in tissue bios-pecimens. Water interacts strongly with collagen and othermatrix components and these interactions influence solidifi-cation in tissues.33 Specifically, ice crystals form preferen-tially in blood vessels (for vascularized tissues)34 andpropagate down the vessel as the sample cools. For avasculartissues (e.g., cartilage, cornea), ice crystals form at different

locations fed by water flow down very narrow channels thatform in the tissue.33 These large ice crystals (or ice lenses)can result in significant disruption/reorganization of extra-cellular matrix.35 Therefore, the presence of extracellularmatrix may also influence the solidification process.

As described above, biospecimens are complex solutionsthat freeze over a range of temperatures. During freezing,water is removed in the form of ice; solute, proteins, andeven cells are partitioned from the ice. The sample is notfully solidified until a eutectic or glass forms in the re-maining unfrozen fraction of liquid. The presence of ex-tracellular matrix also influences the solidification of water.Therefore, storage of samples at temperatures in whichliquid water is still present (versus frozen or vitrified water)will result in reduced stability.

Characterizing Samples at Low Temperatures

Various modalities exist that allow for the investigation ofspecimens at low temperatures. These methods permit re-searchers to obtain detailed knowledge about the effects oftemperatures on the biological sample during cooling andrewarming, and thereby contribute to improvements in thecooling and rewarming protocols. Several modalities havebeen adapted for low temperature measurements, including:spectroscopy, calorimetry, tomography, and microscopy.Fourier transform infrared (FTIR) and Raman spectroscopyare used to detect molecular vibrations of the sample, whichcan deliver information about water transport and ice crystalgrowth.36,37 Commonly used calorimetric methods includedifferential scanning calorimetry (DSC) and differential ther-mal analysis (DTA); these techniques are used to create phasediagrams, quantify water transport through the plasma mem-branes,38 and detect ice crystal growth within cells.39 Mag-netic resonance imaging (MRI)40 and computer tomography(CT)41,42 have been used to visualize three-dimensional icecrystal growth and permeation of cryoprotective agents withinspatially extended specimens. Microscopic methods includecryomicroscopy,43 directional solidification,44 cryo electronmicroscopy,45 and multiphoton microscopy.46 Some of thesedifferent modalities have been used in combinations, for ex-ample, cryomicroscopy with DSC47 or Raman spectroscopy.25

Biological Activity at Low Temperature

All biological specimens contain degradative molecules.Lipases, carbohydrases, proteases, and nucleases may bepresent in fluid and/or tissue biospecimens. Proteases suchas matrix metalloproteinases can act as both biomarkers aswell as to degrade biomarkers in a sample.48 Enzyme ac-tivity is a function of the dynamics of the specific protein.49

Temperature has a strong influence on protein dynamics;reduced temperatures result in reduced protein dynamics/activity. The reduction in protein activity with decreasingtemperature is one mechanism by which biospecimens arestabilized at low temperatures. The temperature dependenceof protein activity with temperature follows an Arrheniusrelationship,50 whereby the rate constant k of a chemicalreaction is dependent on the absolute temperature T:

k¼A � e�Ea=(R�T) < Eq:2 >

with the pre-exponential factor A, the activation energyEa, and the universal gas constant R. Therefore, low

2 HUBEL ET AL.

temperatures result in a low rate constant k of chemicalreactions.

Optimal storage temperatures should be selected below thethreshold temperature for activity of the protein. Distinctchanges have been observed in the dynamic properties ofdifferent proteins near - 53�C.51–54 The temperature depen-dent behavior of ribonuclease A (RNase A) was studied byRasmussen and colleagues.55 They determined that the sub-strate failed to bind the enzyme at 215 K ( - 58�C). Similarresults were observed by Tilton et al.56 in which RNase Awas observed to change behavior at 180–200 K ( - 93 to- 73�C). In contrast, More and colleagues observed activityof b-glucosidase at temperatures as low as - 70�C.22

These studies demonstrate that for the limited number ofproteins studied, protein activity may persist at very lowtemperatures ( < - 80�C). Therefore, storage at temperaturesat which water is still mobile and proteins are still activewill result in degradation of the biospecimen.

Identifying Subpotimal Storage Conditions

Measuring the mobility of water and activity of proteinsin actual biospecimens is not practical in most situations.Most biobanks will have to rely on strong and effectivequality control programs to identify suboptimal storageconditions.57 One approach would be to collect a smallnumber of biospecimens for quality control testing (notdistribution). These samples could be monitored prefreeze,postfreeze, and as a function of time for prevalence ofbiomarkers of interest. Degradation of biomarkers with timein storage suggests that the specimen is not being storedoptimally and should be stored at lower temperatures. Aproper quality control program will also permit determina-tion of biomarkers that respond poorly to cryopreservation(versus those that are not stable for a given storage condi-tion). For example, lactate dehydrogenase loses all func-tional activity upon freezing,58 so its stability with storagewill appear poor only because it has been damaged simplyby freezing. Therefore, a robust quality control program willfacilitate determination of biomarkers that respond poorly tocryopreservation versus those that are not stable in storage.Biospecimens containing biomarkers that respond poorly tocryopreservation must be stored using alternative modalities(e.g., dry state storage, liquid storage) and biospecimenscontaining biomarkers that are not stable with time instorage need to be stored at lower temperatures. It is note-worthy that for some biospecimens, specifically cells,damage to the biospecimen may be manifest in post thawapoptosis,59 which evolves over time.60–65 Therefore, postthaw assessment of cellular biospecimens must accommo-date the time to evolution of this damage mechanism.Monitoring biospecimens for damage resulting from freez-ing, stability in storage, and proper post thaw assessmentmethods will be critical for the continuous improvement inquality for biobanking.

Literature Review of Storage Studies

The effect of storage conditions on the stability of bio-markers has been studied for a variety of systems. As in-dicated previously, the following sections will describestudies to date on the stability of human protein, cell, andtissue biospecimens. Additional references outside of thescope of this review can be found in the ISBER Biospeci-

men Sciences Working Group literature compilation foundon the ISBER website at (http://c.ymcdn.com/sites/www.isber.org/resource/resmgr/Files/ISBER_BIOSPECIMEN_SCIENCE_LI.pdf ) and a working review outlining trans-lating cryobiology principles into biobanking practice.66

Storage of Proteins and Purified Nucleic Acids

Fluid biospecimens (e.g., serum, plasma, urine, cerebralspinal fluid, etc.) are common sources of protein biomarkersand the influence of storage temperature on the stability of arange of biomarkers is summarized in Table 1. Millions offluid biospecimens are currently stored in biobanks aroundthe world. The ability to use these biospecimens for down-stream applications depends strongly upon the processingand storage conditions for the biospecimens,67 leading touncertainty as to the interpretation of biomarker studies.68

For instance, a recent study followed the concentration oftwo serum cancer markers (cancer antigens CA125 andCA15-3) over a 10-year period and observed an increase of15% in prevalence of these biomarkers as a function of timein storage.69 In another study, Kisand et al.70 reported thatvascular endothelial growth factor (VEGF) levels decreaseddramatically after serum was repeatedly frozen and thawedwhile stored at either - 20�C or - 75�C. Another serumcomponent, matrix metalloproteinase-7 (MMP-7), was sta-ble after 5 years of storage at - 20�C, and would be stablefor 100 years at - 75�C as calculated using the Arrheniusequation.70 Overall, these investigators concluded thatstorage of serum at - 20�C is unsuitable for biomarkers.70

Matrix metalloproteinase-9 (MMP-9), a biomarker for car-diovasular risk detection in clinical studies, was found todegrade over time in storage at - 80�C. After 24 months,MMP-9 levels in plasma dropped to 65%, and after 43months the level decreased to only 1%.71

The stability of free prostate-specific antigen (PSA) inserum under different storage conditions was tested byWoodrum et al.,72 who found that free PSA levels decreasedover time. Storage at - 20�C led to a 0.9% loss, whilestorage at - 70�C led to a 0.4% loss for each month instorage.72 Schiele et al.73 measured the stability of apoli-poprotein concentration in human serum after up to 4 yearsat different temperatures ( - 20�C and - 80�C). They foundthat apolipoprotein concentrations were not significantlyaffected following storage for 3 months at - 20�C or- 80�C, or storage for 4 years at - 80�C. Qvist et al.74 studiedthe stability of C-telopeptides of type I collagen (CTX), abone resorption marker, in blood and serum samples. After 3years of storage at different temperatures ( - 20�C, - 80�C,and - 150�C), CTX was reported to be stable only whenfrozen (i.e., at - 20�C and lower temperatures).74

Karlsen et al. found that vitamin C (ascorbic acid anddehydroascorbic acid) degenerated within plasma after only 1day at - 20�C, leading the authors to recommend storage ofthese samples at - 70�C.75 Rai et al.76 studied pre-analyticalfactors by storing plasma at different temperatures ( - 80�C,- 196�C) and performing proteomic analysis (SDS-PAGEand SELDI-TOF). They concluded that plasma should bestored in liquid nitrogen.76

Interestingly, some analytes were found to increase inconcentration over time during low temperature storage. Forexample, Mannisto et al.77 evaluated thyrotropin (TPO),thyroid hormones (TSH, fT4, fT3), and thyroid autoantibody

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concentrations (TPO-Ab, TG-Ab) in human serum afterstorage at - 25�C for up to 23 years. TSH and fT3 con-centrations were not affected by - 25�C storage. However,fT4 showed significant concentration fluctuations over time.TPO-Ab and TG-Ab levels increased over time.77 In anotherstudy, Panesar et al.78 measured the levels of thyroid hor-mones (TSH, fT4, and fT3) after 8–11 years of storage at- 80�C and found a decrease in TSH and an increase in fT3and fT4 concentrations with storage times.

Kubasik et al.79 examined the effects of duration andtemperature of storage on 14 different analytes in serumwhich are often used for radioimmunoassay procedures.They found that the analytes cholylglycine, cortisol, di-goxin, ferritin, follitropin, immunoglobulin E, lutropin,prolactin, thyroxin, and triiodothyronine were stable for 2weeks at room temperature. However, insulin and gastrinwere less stable and needed to be refrigerated or stored at- 70�C, respectively.79

The stability of purified and nonpurified forms of nucleicacids has also been studied. Vindeløv et al.80 investigatedthe effects of storage for 1 year at - 80�C on DNA extractedfrom JB-1 tumor cells and fine-needle aspirates of solidtumor tissue. Cells and fine-needle aspirates in cryovialswere preserved by immersion in dry ice and 99% ethanol at- 80�C. Their DNA deconvolution results revealed no sig-nificant changes after one year of storage at - 80�C.80 Chanet al.81 studied pre-analytical factors including freeze-thaw of plasma on the integrity of circulating cell-free DNA.In their study, the plasma was stored at - 80�C for 24 h,followed by repetitive freeze-thaw cycles, which led toDNA fragmentation.81

The stability of purified mRNA is strongly dependentupon the method of storage. Riesgo et al.82 compared twopreservation methods: (a) a flash-freezing method usingliquid nitrogen and subsequent storage at - 80�C, and (b) afixation method that involves immersion in RNAlater� for1 hour at 4�C, overnight storage at - 20�C, and then storageat - 80�C. After 1 month of storage, the flash freezingmethod delivered better results (measuring the A260/230ratio).82 These examples show that many but not all sub-cellular components can be stored in a - 20�C freezer andsome of them degrade at storage temperatures of - 80�C.

Storage of Cryopreserved Cells

The influence of storage conditions on cryopreservedcells has been studied extensively since the function of cellsfollowing thawing is critical for many applications. Thescientific literature suggests that optimal storage conditionsare a function of both the cell type and the preservationmedium used. A summary of relevant studies related tostorage of cells can be found in Table 1.

Blood cells (e.g., red blood cells, hematopoietic pro-genitors, platelets) that have been used therapeutically fordecades have been studied most extensively.83 When frozenin 40% (w/v) glycerol, the stability of red blood cells storedat - 80�C for up to 37 years has been demonstrated byValeri and colleagues.84 Red blood cells have also beenfrozen in solutions of 24% (w/w) hydroxyethyl starch (HES)and then stored at temperatures ranging from - 10�C to- 75�C.85 The storage temperatures had a strong effect onhemolysis of the cells, and the authors proposed an expo-nential law describing the measured hemolysis over time for

a given storage temperature.85 Furthermore, they proposedthat devitrification and denaturation of subcellular compo-nents were the damaging factors.85

Human hematopoietic progenitor cells (HPCs) are com-monly preserved using two different solutions: 5% DMSOwith 6% (w/w) HES, or 10% DMSO.86 The stability ofHPCs frozen in these solutions has been determined asa function of storage times and DMSO concentrations at- 80�C.87 While there was no difference in viability afterthawing between the different DMSO concentrations, theviability changed dramatically over time. Specifically, theviability decreased steadily from about 80% after 1 monthto 32% after 31 months.87 Halle et al.88 cryopreservedperipheral blood progenitor cells (PBPCs) in 3.5% DMSOusing uncontrolled-rate freezing. After about 7 weeks instorage at - 80�C, the nucleated cells, CD34 + cells, colony-forming units granulocyte-macrophages (CFU-GM), andburst-forming units erythroids (BFU-E) had acceptable re-coveries of 60.8%, 79.6%, 35.6%, and 32.6%, respective-ly.88 In another freezing study 89, autologous PBPCs werecooled to - 90�C at 1.5�C/min and stored for 1–2 yearseither at - 80�C or below - 170�C. No significant differencein cell count was observed between the two storage tem-peratures, however, only the cells stored at - 80�C had asignificant reduction in membrane integrity and clonogenicpotential. The authors stated that it was unclear if this re-duction was solely because of the relatively high storagetemperature of - 80�C or was also caused by the formationof intracellular ice.89 Cilloni et al.90 cryopreserved PBPCswith 10% DMSO and 10% human serum albumin (HSA) to- 140�C using an uncontrolled-rate freezing technique. Thesamples were stored at - 140�C for 10–12 months. Uponthawing, the absolute number of nucleated cells was re-duced. Also, a multilineage colony-forming assay showed areduction in recoveries. The assay’s long-term culture-initiating cell units and BFU-E demonstrated a significantloss.90

Parker et al.91 loaded bone marrow cells with 10% DMSOand then stored them at - 85�C, - 140�C, or - 190�C for40–42 months. Cell number and granulocyte-monocytecolony-forming cell (CFU-c) assays were performed beforeand after cryopreservation. While dilution and washing stepswere associated with quality decrease, the CFU-c mea-surements showed that storage in the vapor phase of liquidnitrogen is adequate for the long-term storage of humanbone marrow cells.91 Also, bone marrow cells and periph-eral blood mononuclear cells (PBMCs) were frozen byAyello et al.92 to - 90�C using 5% DMSO and 6% HES.These investigators tested in vitro cell recovery, CFU-GM,clonogenic potential of autologous HPCs and cell viability.They found no significant change and concluded that it isfeasible to store these cells for more than three years at- 90�C.92

Katayama et al. 93 loaded human peripheral blood stemcells (PBSCs) with 10% DMSO and 8% HSA and then useda non rate-controlled freezing method for cooling to - 80�C.Samples were stored for 1–61 months to study the thawingprocess and the effect of long-term cryopreservation.93

CFU-GM and BFU-E assays were used as quality tests.Recovery rates of CFU-GM and BFU-E showed no signif-icant difference after 5 years of storage. The authors con-cluded that this cryopreservation procedure is useful to storePBSCs for clinical applications.93 McCullough et al. 94 froze

6 HUBEL ET AL.

human PBSCs under different combinations of 10% DMSO,5% DMSO, and 6% HES with subsequent storage temper-atures of - 80�C or - 135�C. After 5 years of storage,sample quality was determined by quantifying the totalnumber of nucleated cells, cell viability, CD34 + cell con-tent, and CFU-GM content. Most of the quality tests re-vealed no significant differences; only the total nucleatedcell count for samples stored for 24 hours at 1�C–6�C beforecryopreservation declined over time.94

Massie et al.95 investigated the effects of storage tem-perature on viability of alginate-encapsulated liver cellspheroids. Spheroids were stored at either - 80�C or*- 170�C for up to 12 months. The viable cell count and thefunction of the liver spheroids stored at - 80�C decreasedsubstantially after 1 month and continued to degrade duringthe 12-month storage period. In contrast, liver cell spheroidsstored for 12 months at - 170�C maintained viability.95

These examples for the storage of cellular samples showthat some cell types were sucessfully stored at - 80�C andretained their viability over time. However, other cell typeswere found to lose their viability even after short periods oftime at - 80�C and thus require lower storage temperatures(e.g., - 170�C).

Storage of Tissues

Tissue biospecimens are important resources for diagno-sis of disease, selection of therapy, and monitoring responseto treatment, and can be used for treatment as well (i.e.,transplantation of islets of Langerhans to treat diabetes).Studies have demonstrated that rapid changes in the ex-pression of biomarkers occur after ligation of the bloodsupply.96 Freezing of tissues is used to arrest the degradationof biomarkers. Long-term stability of tissue-based bio-markers depends upon long-term storage conditions. Asummary of scientific studies describing the stability oftissue-based biomarkers in storage can be found in Table 1.

Jewell et al.97 analyzed the stability of DNA and RNAisolated from human tissues (from breast, colon, liver, lung,ovary, endometrium, and cervix) which were frozen at - 80�Cfor 0 to ‡ 12 months. They used several methods for analysis,including electrophoresis, reverse-transcriptase polymerasechain reaction, and Northern blot. They found the DNA to beof good quality in 80% of the tissues, while the RNA was ofgood quality in only 60% of the tissues; suggesting that tissueDNA was more stable than RNA.

Mato et al.98 found that storing of postmortem humantissue at - 25�C strongly affected the receptor density andresponse of the G-protein [35S]GTPgS, a marker that is usedto analyze cannabimimetic drugs for the treatment of nu-merous nervous disorders. After 12–24 months of storage at- 25�C, a 50% reduction of [35S]GTPgS was measuredwithin the tissues.98

In another study,99 1-mm square pieces of foreskin tissuewere placed into a solution of 0.8% DMSO and serum, andthen these samples were cooled in the gas phase of liquidnitrogen and stored at - 196�C for 1, 3, 6, 9, and 12 months.After thawing, the tissues were minced into smaller frag-ments to allow cell culture for chromosomal, morphological,and enzymatic analyses. No chromosomal damage or re-arragement was measured in any of the cryopreserved tissuesamples. No significant morphological changes were re-vealed upon microscopic analysis of the cells. Furthermore,

the six lysosomal acid hydrolases that were tested all re-mained within the normal range. The authors recommendthis tissue cryopreservation procedure, ‘‘when it is impos-sible or impractical to initiate a cell culture immediately’’.99

Estrogen receptors and progesterone receptors are used asprotein biomarkers in the prognosis of gynecologic carcino-mas. Toppila et al.100 investigated the level of estrogen andprogesterone receptors in human female reproductive tracttissues after excision. Tissues were analyzed after 2, 4, 6, and8 weeks of storage at - 196�C and in the presence or absenceof sodium molybdate. The freezing process caused a 30%loss in the steroid receptors, but once the tissues were kept at- 196�C no further loss was found over the 2–8 week timeperiod.100

Crawford et al.101 compared the storage of human breasttumor tissues at - 196�C to storage in sucrose and glycerolat - 20�C for up to 100 days. They reported that proges-terone in these tissues was stable for 1 month at - 20�C.They performed Scatchard analysis to investigate time-dependent estrogen receptor levels, which are used to predictoutcome following therapy in breast cancer. They concludedthat estrogen receptors were stable under these conditions at- 20�C, which is sufficient for any routine purpose.101

A series of studies demonstrate that storage below- 135�C is necessary for long-term storage of tissues.Brockbank et al.102 studied human allograft heart valveleaflets to evaluate the effect of different storage tempera-tures ( - 80�C and below - 135�C) on viability of fibroblastsembedded in the valve. Samples stored at - 80�C revealed atime-dependent loss of fibroblast viability. After 6 and 24weeks, a significant decline in tissue function was observed;only 15% of the tissue valves survived with full function-ality. In contrast, short-term ( < 3 months) and long-term (1–2 years) storage of the tissue samples in liquid nitrogen(below - 135�C) sustained their cellular function (i.e., fi-broblast protein synthesis).102

These examples demonstrate that some tissue biomark-ers are stable at relatively high temperatures (e.g., even at- 20�C).101 However, storage temperatures below - 135�Cappear to be necessary in order to preserve a wider variety ofbiomarkers (including viability).

Conclusion

Biospecimens are an important reagent in biomedicalresearch and suboptimal preservation and long-term storageconditions strongly influence their quality. This reviewdescribes critical events during the freezing and storageprocesses. Any biospecimen will contain water plus a bio-logical system (e.g., proteins, cells, tissues). As water iscritical for many biological reactions, the physical state ofwater in the solution (solid, liquid, or glass) is important inunderstanding and controlling degradation of biologicalmolecules. Storing the solution at low enough temperaturesto reduce mobility of water by forming a solid or glass,reduces the potential for water acting to degrade the bio-logical molecules in solution.

In addition, biospecimens contain degradative molecules(e.g., proteases, lipases, nucleases) whose activities arestrongly influenced by temperature. Storage conditions needto be selected such that these molecules are inactive so thatbiomarkers will be preserved. Optimal storage temperaturesfor biological cells are a function of solution composition


and biospecimen type. Implementation of proper qualitycontrol systems for monitoring stability of biomarkers inlong-term storage will permit determination of suboptimalstorage conditions and continuous improvement in biospe-cimen quality.



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Address correspondence to:Allison Hubel, PhD

University of MinnesotaMechanical Engineering Department

111 Church Street SEMinneapolis, MN 55455





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http://online.liebertpub.com/action/showLinks?pmid=1779480&crossref=10.1265%2Fjjh.46.976

http://online.liebertpub.com/action/showLinks?pmid=11460005&crossref=10.1097%2F00001721-200106000-00002






Meeting Location

This meeting will be located in Conference Room D on the ground floor of 6001 Executive Blvd.

Visitor Parking

Located at 6001 Executive Blvd, NIH offers two convenient garages and several parking lots for visitor parking. While the NIH parking Office does not issue permits to visitors, a validation sticker will be provided upon request.

Restaurants

Executive Deli Located at 6011 Executive Blvd (adjacent to 6001 Executive Blvd), the Executive Deli offers a variety of food options to pick from. Hours of operation are 7:00am-3:30pm. Menu available at the registration table. Pike & Rose Located at 11580 Old Georgetown Rd., North Bethesda, MD 20852, the Pike and Rose offers a fine selection of dining choices. Pike and Rose offers two convenient garages, and free parking for the first two hours. Click Here for More Information

Webex Information

These breakout sessions will also be videocast through Webex:

https://nih.webex.com/nih/onstage/g.php?MTID=e8179a0e2db31cb7a2c7abc135a4a76b2

http://www.pikeandrose.com/dine/

https://nih.webex.com/nih/onstage/g.php?MTID=e8179a0e2db31cb7a2c7abc135a4a76b2

NIH Common Fund HuBMAP / SCAP Mini Workshop …€¦ · ORIGINAL ARTICLES A Novel Approach to...

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Transcript of NIH Common Fund HuBMAP / SCAP Mini Workshop …€¦ · ORIGINAL ARTICLES A Novel Approach to...