unit 2. biomarkers: practical aspects

chapter 3. Biological sample collection, processing, storage

and information management Unit 2 Chapter 3 Chapter Jimmie B. Vaught and Marianne K. Henderson

Summary

The collection, processing and study that will involve the collection to assure that biological samples storage of biological samples occur of biological samples, many are of consistent quality and right in the larger context of organizations decisions need to be made that will for the intended analyses and study known as biological resource affect the quality of the samples goals. centres or biospecimen resources. and the outcome of the study. The Biological resource centres are (1,2) appropriate sample type(s) needs to Introduction service providers and repositories be chosen. The processing protocol of living cells, as well as genomes that will result in samples of suitable Although biological specimens have of organisms, archived cells and quality for the intended laboratory been collected for use in a variety tissues, and information relating to analyses must be selected from of molecular epidemiology, clinical these materials. The US National among various possible protocols. trial and basic research studies for Cancer Institute (3) defines a Consideration must be given to many years, it has only recently biospecimen resource as a “… the proper storage conditions been recognized that the protocols collection of human specimens and to maintain sample quality until and practices involved in collecting, associated data for research analyses are completed. All of these processing and storing specimens purposes, the physical entity activities must be monitored and actually comprise “biospecimen where the collection is stored, controlled by appropriate sample science.” As a result, many and all relevant processes and tracking and laboratory informatics organizations (Appendix 3.1) have policies.” The complexities involved systems. A comprehensive quality engaged in producing guidelines and in proper sample management management system, with standard best practices for these endeavours, policies and procedures are often operating procedures and other now known as biological resource underestimated. Prior to initiating a appropriate controls, is necessary centres or biospecimen resources.

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 23 http://www.bioethics-singapore.org/uploadfile/52533%20PMHT%20AppendixB-Dr%20Kon.pdf http://www.abrn.net/ http://www.oecd.org/dataoecd/55/48/2487422.pdf http://www.tubafrost.org/ http://www.mrc.ac.uk/Utilities/Documentrecord/index.htm?d=MRC002420 http://biospecimens.cancer.gov/bestpractices http://www.unece.org/trans/danger/publi/unrec/rev13/13files_e.html http://www.labcorp.com/datasets/labcorp/html/frontm_group/frontm/section/speccol.htm Cell Preserv Technol 2008;6:3-58 Reference/Link Australian BiospecimenAustralian Network, ABN/ Australia OECD/International NetherlandsTUBAFROST/The MRC/UK International Society Biological for & Repositories/USA Environmental Europe, for Commission Economic UN UNECE/International LabCorp/International NCI/USA Authors/Origin Biorepository Protocols Biorepository Biological Resource Centres: Underpinning the Future of Life Sciences and Biotechnology European Human Frozen Tissue Tumor Bank – TUBAFROST Human Tissue and Biological Samples for Operational Ethical and Research: in use Guidelines I: Repositories for Practices Best of Retrieval and Storage, Collection, BiologicalHuman Research Materials for UN Recommendations on the Transport of Dangerous Goods. Model Regulations and Preparation, Collection, Specimen Handling First-Generation NCI- Guidelines for Resources Supported Biospecimen Title Tissue Banking for Biomedical for Tissue Banking Research National Cancer Centre/Singapore Existing guidelines and best practices for biorepositories for practices best Appendix and guidelines Existing 3.1. Several organizations have published guidelines and best practices relevant to the discussion in this chapter (54). ThisRecommendations table is adapted from on Common the IARC Minimal publication Technical Standards International (2). Network of Biological Resource Centres for Cancer Research:

24 These terms reflect the fact that performed under strictly controlled the intended analyses? Will it be specimen management takes place procedures. As the sensitivity and necessary to store smaller volumes in an environment that includes a specificity of analytic techniques in aliquots for future unplanned use wide range of policies concerning the have increased to an extraordinary to avoid thawing a larger aliquot? specimens and data, as well as the degree in recent years (see Chapter For example, it is important to physical structure, the biorepository. 4), it has become even more important consider that new technologies have Biological resource centres are to assure that biospecimens are of resulted in more sensitive analytical engaged in many activities beyond the highest quality. In addition, from techniques to apply to older samples storage, such as acquiring, the point in time that the specimens (see also Chapters 4 and 7), or older processing (e.g. aliquoting, DNA are collected until laboratory results samples may become sources of extraction) and distributing biological are analysed and reported, all of information to study the natural materials. The practices and the relevant information concerning history of a seemingly ‘new’ disease. policies that have been organized the specimen, as well as data • What quality standards do the

into formal documents testify to the concerning the study participant specimens need to meet for valid Unit 2 importance of following proper steps and laboratory analyses, must be laboratory analyses? Have such that will result in the highest quality properly stored in interoperable quality measures been validated? 3 Chapter specimens for research purposes. information management systems. • Have specimen collection, The use of proper procedures to This could mean multiple systems or processing and storage protocols produce biological specimens of multiple databases interconnected been standardized and validated in the appropriate quality, as well as in a single system. All of these pilot studies? the collection of relevant clinical, steps must be performed under • If the specimens will be stored epidemiologic and quality control a well-planned quality assurance for some period of time before data, gives the biospecimens their programme, and according to analysis, has the stability of the value in research. relevant legal and ethical standards intended biomarker, or other analyte, (discussed in Chapter 2). been determined for the planned Context and public health storage conditions? significance Examples/case studies • Will specimens need to be shipped to distant locations for Biological specimens (or Prior to initiating a study that involves analysis? If so, have packaging and biospecimens), such as blood, specimen collection, several key shipping protocols been validated to urine, saliva, and many other points must be considered. The assure the stability and safety of the types, are collected for a variety answers to these questions will be specimens and personnel who will of reasons, for normal patient important in determining whether the handle them? monitoring and care as well as for appropriate materials, equipment • Have all other logistical issues basic, clinical and epidemiologic and procedures are in place: been resolved, such as proper research studies. Many medical • What are the goals of the coding, labelling and identifying the advances, including studies of study? types of storage vessels? heart disease, AIDS and cancer, • What laboratory analyses will • What data will be collected have resulted from preliminary be needed to accomplish the study with the sample and the study, and developmental studies that have goals? is an appropriate informatics system relied on access to and proper use • What type of biospecimens available to collect and process this of the appropriate biospecimens. will be necessary for the intended information? The sources of biospecimens for laboratory analyses? • Have all appropriate informed these studies have been varied, as • How many specimens will consent, privacy and other ethical has their quality (1–4). be collected? If necessary, a and legal rules and regulations been For molecular epidemiology biostatistician should be consulted reviewed and adhered to in the studies, the ultimate success of a to assist in determining the number study planning? study depends on reliable laboratory required to achieve statistical • Are funding and other analyses of these specimens. In significance. resources for the proposed study’s order for laboratory analyses to be • What volume or size will specimen collection adequate? Will reliable, the collection, processing be required for each specimen it be necessary to consider lower and storage of specimens must be to assure that it is adequate for cost alternate methodologies?

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 25 If there is a significant amount of to planning the most efficient to plan biospecimen collections uncertainty in answering the above specimen collection and processing with careful attention to the costs of questions, then additional thought to maintain the stability of the analyses and storage, especially if and planning will be needed before resulting sample aliquots, which are long-term storage will be necessary. beginning the study. For example, expected to number approximately For example, if a study requires before initiating the collection of 15 000 000 (5). only nanogram quantities of DNA blood and urine from 500 000 Among the issues outlined for genotyping purposes, one study participants in 2007, the above, cost is a major consideration, should consider collecting small United Kingdom Biobank conducted especially when designing a study amounts of blood or saliva on filter a series of sample processing that will include a large collection cards, instead of a large volume validation studies (5–7). These of biospecimens. Often the costs of blood that will yield hundreds of studies showed the effects of of collecting, processing and micrograms of DNA and incur larger sample processing delays, as well storing biospecimens are not well processing and storage costs. Other as storage conditions, on the results understood or estimated before alternate processing and storage of the wide variety of assays to be starting a study. The design approaches that may result in conducted on samples that will be and operation of the physical cost savings are considered in the collected over a four-year period, but biorepository also needs to be well Specimen collection section. may be used for studies for 20 years thought out. Baird and Frome (8) As shown in Figure 3.1, specimen or more. The long-term success have outlined the major elements collection, processing and storage of such a large and costly project of cost and design for a large are components of a series of steps depends on this careful approach biorepository. It is also important that are used in any study involving

Figure 3.1. The lifecycle of biospecimens in biological resource centres. Used with permission from (2).

26 the collection of biospecimens. Of particular interest for molecular in a specific order to avoid cross- Each of these steps is discussed in epidemiology studies are those contamination of additives (10,11) turn in the following sections. specimen types that can be collected (also see Chapter 12). most conveniently and efficiently, As shown in Table 3.1, blood Specimen collection and at the lowest cost for large is often fractionated before population-based studies. The most being analysed or stored (10,11). Specimen types common specimen types collected Fractionation of blood results in the for these studies are discussed in following components: A wide variety of specimen types the following sections: blood, tissue, • Mononuclear leukocytes may be collected for storage, and urine and saliva. (peripheral blood mononuclear in many molecular epidemiology cells, PBMCs) are the only cell type studies more than one of the Collection procedures in blood that can be maintained in a following (discussed in detail) viable state.

may be necessary, depending on Collection procedures will vary • Neutrophils (the most abundant Unit 2 the study goals (2,3). Additional according to specimen type and the type of granulocytes) are also collection, processing and storage intended analyses, but all procedures nucleated and another source of 3 Chapter guidance can be found in the should be carefully designed DNA. International Society for Biological and documented. It is normally • Erythrocytes can be used to and Environmental Repositories a good practice to perform pilot study adducts of haemoglobin. (ISBER), National Cancer Institute studies to validate new specimen • Plasma is obtained from (NCI) and International Agency collection methods and protocols an anticoagulated blood sample for Research on Cancer (IARC) (4). The discussion in this section by separating out the cellular documents (2–4). focuses on the specimens most components. • Blood and blood fractions commonly collected for molecular Serum isolation requires no (plasma, serum, buffy coat, red epidemiology studies. Additional anticoagulants. To reduce blood cells) information and collection protocols contamination, serum should • Tissue (from surgery, autopsy, may be found in several references be separated from other blood transplant) (2–4). Also see Chapter 12, Table components as soon as possible. • Urine 12.2 for additional information Serum allows for improved analyses • Saliva/buccal cells about specimen types collected of antibodies, nutrients, lipids and Many other types of specimens for epidemiologic studies, and their lipoproteins. Either serum or plasma may be collected, depending on advantages and disadvantages. may be used for proteomic analyses, availability and study goals, for although according to recent Human example: Blood collection Proteome Organization (HUPO) • Placental tissue, meconium, guidelines there are advantages and cord blood Collection of blood specimens (9) disadvantages in the use of either • Bone marrow should be carried out by trained specimen (12). For studies intended • Breast milk phlebotomists to avoid causing to investigate the broadest array of • Bronchoalveolar lavage study participant discomfort, or proteins and peptides, plasma is • Cell lines compromising the quality or quantity the better choice, as the process of • Exhaled air of the sample. Standard protocols blood coagulation results in the loss • Feces recommended by well-established of many proteins. Some differences • Fluids from cytology (ascites, organizations should be used. in endogenous hormone analytical pleural fluid, synovial fluid, etc.) An evacuated tube system (e.g. results have been found between • Hair Becton-Dickenson Vacutainer®) serum and plasma, but as noted in • Nail clippings with interchangeable glass or Chapter 12, both are acceptable as • Semen plastic tubes is commonly used to specimens for such analyses. Each of these specimen types collect blood. The tubes, some with Depending on the intended should be collected, processed, additives appropriate to a specific laboratory analyses, blood should and stored under conditions that application, are differentiated by be collected anticoagulated preserve their stability with respect their colour-coded stoppers. Blood (consisting of plasma, buffy coat to the intended future analyses. collection tubes should be drawn and red blood cells) or coagulated

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 27

Table 3.1. General guidelines for blood collection and processing

Blood Fraction Collection additive Preferred uses Limitations/problems

Whole Blood Anticoagulant (ACD, heparin, Genomics studies; Source of DNA, Anticoagulant effects need to be EDTA); protease inhibitor for RNA considered proteomics

Buffy Coat Anticoagulant DNA extraction; source of Limited yield if blood not properly lymphocytes, cell lines as unlimited processed. DNA source As a source of DNA, whole blood collection is generally more economical Serum None Proteomics; Source of DNA; DNA yield low (nanograms) but Multiple analytes suitable for genomics applications

Plasma Anticoagulant, possibly protease Proteomics (preferred sample) DNA yield low (nanograms) but inhibitor suitable for genomics applications.

Source of DNA, multiple analytes Analytical results may differ in serum and plasma.

Blood Clot None Source of DNA Extraction difficult, costly

(consisting of serum and red blood • Anticoagulants used in blood their integrity during collection cell clot) (9). There are several types collection, as described above and and processing. The addition of of anticoagulants which need to be in Table 3.1. commercially available RNase chosen carefully to avoid problems • Stabilizing agents are inhibitors preserves RNA integrity. with certain laboratory applications necessary to preserve some • Special collection systems (9,11). Other special collection tubes analytes, and should be included (ex. PAX DNA® Blood Collection such as Serum Separator Tubes® in the collection device or added as System by PreAnalytiX®) allow for and Cell Preparation Tubes® (SST, soon as possible after collection. the collection, shipping, and short- CPT, Becton-Dickenson) allow • The time elapsed between blood term storage of blood at room for more convenient separation of collection or removal from a storage temperature, and for subsequent blood fractions, but some problems unit and subsequent processing extraction of DNA according to a have been encountered in their may be important, depending on the single-tube protocol (14). use (9). Special collection tubes intended analyses. See the United with protease inhibitors have been Kingdom Biobank validation study Tissue collection developed, which preserve proteins summary for examples of such for proteomics analyses (9,12). The effects (summarized in reference 7). The primary sources of tissues for analysis of trace metals in blood • The temperatures at which research are biopsy, surgery and also requires caution, as they blood specimens are processed and autopsy. As noted in the ISBER may be present in the evacuated stored may be important, depending Best Practices and IARC Biological collection tubes. Lot-to-lot variation on the intended analyses (13). Resource Centre Guidelines (2,4), in the quality of collection tubes is • Thaw/refreeze cycles should tissues must be collected under also a potential source of spurious generally be avoided due to the strict ethical and legal guidelines, laboratory results. potential for instability of some and the collection of samples for There is no fixed time period that analytes. However, thaw/refreeze research must never compromise can be recommended for collecting effects are not well documented the diagnostic integrity of a and processing blood. However, for all analytes and may need to be specimen. Generally it is preferable depending on the intended analyses, evaluated through pilot tests (13). for a trained pathologist to be the stability of blood with respect to • Enzymatic degradation involved in the actual procurement various laboratory analyses may affects many biochemical markers. of the tissue specimen during a be affected or controlled as follows RNA and proteins are particularly surgical or autopsy procedure. (9,11): susceptible to this and require Other important considerations special procedures to maintain in collecting tissue are (adapted

28 from ISBER Best Practices and can be frozen in a Dewar flask of remains suitable for PCR-based IARC Biological Resource Centre liquid nitrogen or on dry ice at the analysis of short DNA fragments. Guidelines (2,4)): time of collection. Otherwise, it is Due to degradation issues, • Timing. In general, it is recommended that samples be formalin-fixed, paraffin-embedded important to minimize the time transported in saline on wet ice tissues are of limited use as a source between collection and stabilization to the repository or laboratory for of RNA. However, RNAlater® (16) is and processing of tissue specimens. additional processing. a commercial aqueous, non-toxic This time will vary according to • Autopsy specimens. It is tissue storage reagent that rapidly the intended use, since different important to know the time interval permeates tissues to stabilize and biomolecules degrade at different between death and collection and protect cellular RNA and eliminates rates. The effects of collection processing of the specimen, as the need to immediately freeze or timing on tissue and macromolecule specimens may degrade quickly otherwise stabilize tissue samples. preservation have not been well after death. Autopsy procedures Tissue samples can be harvested ®

studied. The best approach is may yield “normal” tissues (i.e. and submerged in RNAlater for Unit 2 to collect, stabilize (freezing normal lung), or large quantities storage for specific periods without or fixing) and process tissue of a specimen that would not jeopardizing the quality or quantity of 3 Chapter specimens as rapidly as possible. otherwise be available from surgical RNA extracted at a later time or date. It is recommended that surgical or procedures. Tissue specimens However, specimens processed in biopsy specimens be preserved collected at autopsy should be RNAlater® cannot be further used for within 1 hour (or less if possible) of appropriately labelled as to the histomorphopathological analyses. excision; however, tissue subject to organ site, tissue type, and time of Alternatives to formalin fixation a delay up to two hours should still resection, and then immediately include ethanol, Optimal Cutting be collected (15). Detailed records placed in a container of saline on Temperature (OCT) media, of the timing of events from excision wet ice for transport to the tissue methacarn, and Carnoy’s solution, to fixation or freezing should be repository for processing. among others. To achieve an kept. Tissue banking staff must be • Transplant tissue and organs acceptable balance between the present in pathology to freeze or fix that are inappropriate for transplant preservation of tissue morphology the tissue as quickly as possible. may sometimes be made available and nucleic acid integrity, it may Tissues must be snap frozen either for research. Often transplant tissue be necessary to alter fixation directly or enclosed in a container is of a higher quality than either methodology to achieve a study’s immersed in the freezing medium surgical or autopsy specimens, goals. Several studies have explored (e.g. precooled isopentane). Liquid due to the special efforts made the effects of the above standard nitrogen is not recommended as a to preserve the integrity of the fixatives, as well as newer ones suitable freezing medium for direct transplant organs. for special applications (17–20). snap freezing, due to the potential Although formalin-fixation remains formation of cryo-artefacts. When Tissue fixation the standard tissue preservation dry ice or liquid nitrogen are not method, these alternatives readily available, tissue collection Formalin- or alcohol-fixation and should be considered for special into RNAlater® (16) may be a paraffin embedding may be used research applications that require good alternative, provided that the to preserve tissues at relatively the preservation of particular tissue is not required for diagnostic low cost when adequate freezing macromolecules or morphological purposes and permission is given by procedures and storage facilities features. the pathologist. are not available (2). Formalin- • Surgical specimens. Remnant fixation is also the standard practice Urine collection samples may be collected from for preservation of tissues collected (see also Chapter 12) diagnostic procedures or, with during surgery or autopsy. Fixed proper IRB approval, specimens paraffin blocks may be stored Many analytes, such as steroid may be resected specifically in light- and humidity-controlled hormones, pesticides and a for research. Depending on the facilities at room temperature wide variety of drugs and their intended use, specimens may be (18–22°C). Formalin-fixed tissues metabolites, can be measured in transported or frozen immediately. may be used for DNA extraction. urine for molecular epidemiology Samples requiring snap freezing The DNA is usually fragmented but studies (11), making it a convenient

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 29 specimen for a variety of studies. buccal cells, including swabs, programme (24). The US Armed Urine collection can performed cytobrushes and a mouthwash Forces collect blood spot cards from under several conditions, depending protocol. The mouthwash protocol all service members and stores on the study design and analytical has been successfully used in large them for possible identification goals (4,11): population-based studies and has purposes, as well as research and • First morning. Collected been shown to yield DNA of good clinical purposes. DNA can be immediately upon rising in the quality and quantity for genetic easily extracted from blood spots morning, recommended for analytes analyses (21). However there are in amounts more than sufficient requiring concentration for detection limitations to buccal cell DNA, as for genetic studies. This process in laboratory assays. described below. has been automated, especially for • Random urine specimens are New methods are being forensic applications (27). appropriate for drug monitoring and developed for saliva collection. One In addition to standard filter cytology studies. such method has been developed cards, new technologies for dry- • Fractional specimens. The by DNAGenotek (22). A proprietary state specimen collection have study participant fasts after the reagent, Oragene, preserves saliva been developed. GenVault (28) last evening meal, and the second (and DNA) at room temperature. uses small elements of treated filter morning urine is collected. These The method has been successfully paper in 384-well plates for storage specimens are used to compare used in epidemiologic studies (23). of blood, DNA, plasma and serum urine analyte levels with their The yield and quality of DNA from specimens at room temperature. concentrations in blood. the Oragene collection is similar to DNA and protein can be eluted • Timed urine collections (e.g. that for the mouthwash method. from the elements by relatively 12 and 24 hour) are used to allow straightforward methods. comparisons of excretion patterns. Collection of blood, Urine collections should be saliva on treated cards Preserving specimen stability maintained on ice or refrigerated during collection for the duration of the collection. New technologies, such as whole- Collection vessels are generally genome amplification methods As noted above for tissue larger than for other liquid to increase genomic DNA yields, biospecimens, the elapsed time specimens, and may range from and the high cost of collecting and for collection, and between 50 to 3000 mL. Depending on processing blood or mouthwash collection and stabilization, should the analyte to be measured, a samples, have led to renewed be minimized, and the tissue preservative may be needed. The consideration of treated filter paper temperature should be reduced as type of preservative may differ cards as a method to collect DNA soon as possible after collection. according to test methodologies, from blood (24) and buccal swabs This is especially important if time delay, and transport conditions. (25) (also discussed in Chapter 12). freezing is the stabilization endpoint. EDTA and sodium metabisulfite Filter paper cards have been pre- If fixation is the stabilization endpoint, are examples of preservatives treated to retard bacterial growth, control of processing time between commonly used in urine collections inhibit nuclease activity, and release maximum and minimum durations (11). DNA during processing (26). The may be required. Rapid processing cards may be easier to use in may not be as critical for other types Saliva/buccal cell collection paediatric and elderly populations to of biospecimens, such as blood. collect specimens, and can be mailed Optimal processing times vary Saliva, with exfoliated buccal cells, in an envelope with a desiccant at a depending on the analysis method is an excellent source of DNA for nominal cost. for which a biospecimen is used. genetic studies (21). Self-collection Blood collected on filter cards Biorepositories should use the of buccal cells is a safe, convenient is well established as a source of processing method that preserves method that can be used to reduce DNA for genetic studies, as well as the greatest number of analytes. The the cost of specimen collection and is for a variety of other research and best scheme to preserve analytes often preferred over blood collection clinical applications. The US Centers is to divide specimens into aliquots by study participants (discussed for Disease Control and Prevention or fractions of appropriate size or in Chapter 12). Several methods (CDC) uses blood spot cards in volume and/or preserve them by have been developed for collecting its nationwide neonatal screening multiple processing methods.

30 Specimen processing Tissue – processing after for DNA extraction is generally surgery, autopsy considered to be phenolchloroform Specimens are processed according extraction, but other standard to the study design and the methods Specimens resected specifically for methods that are more efficient, less most appropriate for preserving the research may be either processed expensive, and that utilize less toxic analytes of interest. For a particular in the operating room or pathology chemicals provide similar yields and specimen type and analysis, suite, shortly after the time of DNA of similar molecular weight. several processing methods may collection, or may be transported Companies such as Gentra and be appropriate. The IARC standards to the repository for processing, Qiagen have collected DNA stability (2) list some of the more routine depending upon the requirements data of over 12 years’ duration (32). processing protocols. The general of the specific protocol. Additional Techniques for measuring the guidelines in this section outline details are discussed above, and quality and quantity of DNA range some of the important considerations may also be found in the ISBER and from absorbance at 260nm and

when choosing processing methods IARC Guidelines (2,4). 280nm, to fluorescence methods, Unit 2 for specimens most commonly to real-time PCR for detection collected for molecular epidemiology Urine of less than 25 picograms DNA. 3 Chapter studies. Additional issues concerning The A260/A280 ratio is a rough the processing and analyses Processing of urine before storage measure of DNA purity and protein of specimens for proteomic, is fairly straightforward. The primary contamination. Additional methods metabolomic, physical, chemical, decision is the size of the aliquots of measuring DNA quality include and immunologic applications are to be stored and is based on the gel electrophoresis. The accuracy discussed in Chapters 4 and 7. expected analyses. If the analytes of DNA quantitation by these are stable to thaw/refreeze cycles methods can vary widely and can Blood – separation into then larger aliquots can be stored. affect the quality of downstream fractions (e.g. plasma, serum, genomic analyses. Genomic buffy coat, red blood cells) Saliva/buccal cell processing assays may be very sensitive to from mouthwash protocol the quantity of DNA. A study by the The processing method used specimens US National Institute of Standards for blood specimens depends and Technology found a great on the laboratory analyses to be Buccal cells collected using the deal of variability between various performed. Cryopreservation is a mouthwash protocol (21) are methods and among laboratories cost-effective way of preserving processed by centrifugation of participating in a DNA quantitation viable lymphocytes for subsequent the cell suspension, resuspension study (33). Great care must be taken recovery of DNA, or for Epstein- in a buffer, and either processed to assure that DNA concentration is Barr Virus (EBV) transformation to immediately or frozen for future accurately measured before use in create lymphoblastoid cell lines as a use. Usually, additional processing any assay, especially PCR-based source of unlimited amounts of DNA involves DNA extraction. Note genomic applications that require (29). Cryopreservation typically that a special consideration in precise quantities of DNA. involves the use of a cryoprotectant, processing buccal cell DNA is the RNA is less stable than DNA such as dimethyl sulfoxide high percentage of bacterial DNA and is more difficult to extract (DMSO). However, commercial present in these specimens, which intact. However, special methods cryoprotectants that are less toxic requires special quantitation by real- and reagents have been developed have been developed (30). Whole time PCR. that allow for preservation of RNA blood may also be cryopreserved in blood and other specimens, as as an efficient and cost-effective DNA extraction noted in the discussion of tissue approach to centralized processing fixation. and storage of viable cells in large- DNA extraction methodology is well Saliva or blood collected on scale epidemiological studies (29). established for a variety of specimen treated paper cards is available, types, including whole blood, blood for example, from Whatman® for fractions, buccal cells, fresh and laboratory applications. Enough frozen tissues, and paraffin tissue DNA can be obtained from a 2mm blocks (31). The gold standard punch of a paper card for about 500

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 31 single nucleotide polymorphism hormones (discussed in Chapter suspensions. For smaller samples, (SNP) genotypes. The extraction of 12), are stable, other analyses may in the volume range of 50 uL to 1 DNA from blood spot cards can be be affected by one or more thaw- mL, the Qiagen EZ-1 and M-48 automated as noted above (27). freeze cycles (2,3). systems are available (32). Other Table 3.2 summarizes source commercial and custom systems material for nucleic acid extraction, Automated systems have been developed for specialized and some of the procedural and for specimen processing automated applications. methodological issues encountered The other major biorepository with each specimen type. Automated systems have been activity that is amenable to developed for specimen processing, automation is aliquoting. DNA in Aliquoting and several of these systems are solution, as well as for example useful in processing specimens for serum and plasma, must be stored Dividing specimens into smaller molecular epidemiology studies. in volumes suitable for downstream sample aliquots is usually Generally automation is most laboratory analyses. If standard necessary to preserve them in applicable to DNA extraction and collection and storage vessels are volumes useful for routine analyses. specimen aliquoting. used, and a standard aliquoting The aliquoting protocol should be For DNA extraction several protocol can be developed, then designed only to store the number of automated systems are available, aliquoting can be automated. An aliquots necessary for the intended depending on the specimen type example of a system for automated analyses, plus additional long- and volume. For blood specimens, aliquoting is from TECAN (34). term archival samples that will be and other blood fractions and available for unforeseen uses. In suspensions of buccal cells up Storage developing an aliquoting protocol, to 10 mL, the Gentra AutoPure is the consequences of repeated one of the preferred systems (32). Depending on the intended thawing and refreezing cycles The AutoPure has been validated laboratory analyses, and other should be considered. Although for use with plasma, serum, buffy considerations, specimens and many analytes, such as steroid coat, buccal cell and other cell their aliquots may be stored under

Table 3.2. Common DNA sources and extraction issues

Specimen source Collection method Extraction method DNA yield Advantages Challenges

Whole Blood Evacuated tube with Manual or automated 100s of micrograms High yield, minimal Refusal to participate anticoagulant processing

Blood -Buffy Coat Processing of anti- Manual or automated 100s of micrograms High yield, minimal Variable yield and coagulated blood (with some storage volume quality of buffy coat processing) cellular material Blood - Plasma, Processing of blood, Manual or automated Nanograms Good use of samples Low yield Serum with or without collected for other anticoagulant purposes Saliva Mouthwash, Oragene Manual or automated 10-50 micrograms High compliance rate Bacterial DNA

Blood clot Evacuated tube, no Manual (special Variable Good use of ‘extra’ Extractions anticoagulant processing samples expensive, necessary) DNA fragmented None Source of DNA Extraction difficult, costly

Tissue – Fresh or Surgery, autopsy Manual Variable Most appropriate DNA fragmented, Frozen sample for some RNA quality low studies Paraffin Embedded Tissue sections from Manual Variable Easily stored DNA fragmented, Tissue surgery, autopsy RNA quality low

32 a variety of conditions as shown in the equipment space. In addition, supply of liquid nitrogen must be Table 3.3. Most common specimens liquid nitrogen freezers are less maintained. Vapour phase liquid such as plasma, serum or DNA may susceptible to mechanical failure nitrogen storage is preferred over be securely stored in mechanical and can withstand power outages liquid phase storage, where cross- freezers at −80 °C. However, for long periods with no temperature contamination of specimens may lymphocytes, or other cellular deviations. occur. Cryovials must be capable specimens, should be stored in the In situations where freezer of withstanding liquid nitrogen vapour phase of liquid nitrogen at systems may not be available, a temperatures. Screw cap vials that −150 °C or lower, when long-term lower-cost option is collection of will not leak are necessary. A good cellular viability is necessary. Other saliva or blood spots on filter cards storage container in liquid phase storage conditions that are optimal and storage at room temperature. nitrogen is the CryoBio Systems for the preservation of specimen Below are some general storage plastic straw (35). stability should be considered, for considerations (1,2,4): • Alarm systems should be in place to monitor the temperature of example for endogenous hormones, • Adequate back-up storage Unit 2 as discussed in Chapter 12. Although capacity for low temperature mechanical freezers, or in the case generally not necessary in terms of units should be maintained. The of liquid nitrogen freezers, the liquid 3 Chapter sample and analyte stability, storage power supply must be connected nitrogen level and temperature. in the liquid phase of a liquid nitrogen to a back-up generator system • Dry ice is frequently used tank at −196 °C is an excellent that immediately provides power as a refrigerant for shipping and option. Although thorough cost during an electrical outage. emergency back-up for mechanical analyses have not been performed, Standard operating procedures and freezers. it is generally accepted that over the techniques for rapidly transferring • A system for maintenance and long term, liquid nitrogen freezers material to back-up units during repair of storage equipment, support are less expensive to maintain such emergencies should be systems and facilities should be in than mechanical freezers, due to documented. place. lower electrical requirements for the • Where liquid nitrogen • All equipment should be equipment and less need to cool freezers are used, an adequate validated before use, or following

Table 3.3. General specimen storage guidelines

Temperature in °C Preservation method Recommended for

+18 to +20 Room temperature Slides, tissue blocks

0 to +4 Refrigerator Processing fresh specimens

−0.5 to −27 Freezer Short-term DNA stability

−27 to −40 Freezer DNA stability

−40 to −80 Freezer DNA/RNA stability

−80 to −130 Freezer Recommended for urine, blood, blood fractions (plasma, serum etc)

−130 to −150 Liquid nitrogen vapour Recommended for storage of tissues, preservation of cellular viability

−196 Liquid nitrogen liquid phase Storage of living cells

Adapted from (2).

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 33 repairs that affect the instrument’s Special procedures should management is critical to the accuracy or other capabilities. be developed to assure that molecular epidemiology research • Labels for storage vessels freezers are properly validated, enterprise (38). Collation and must be capable of withstanding the in terms of maintaining their analysis of the data associated required storage conditions, i.e. the optimal temperatures, during with the collected specimens that label material must not deteriorate initial installation and at regular support biomedical research require and printing must be readable or intervals. As noted in the ISBER robust interoperability to allow scanable after long-term storage. best practices: “…any device that maximum usage of the collections provides a readout, data, or has a (3,4). Information management Automated freezer systems meter movement, is considered an and analysis tools across the instrument, and requires calibration.” spectrum of biomedical research Automated freezer systems are (4). are challenged to provide high available for convenient storage and performance, scalability and user- retrieval of samples. Commercial Freezer temperature friendly interfaces. Also, as data automated freezer systems include monitoring sharing and collaboration between a custom system built for ARUP global investigators increases, Laboratories (36) and systems Freezer temperatures must be secure interfaces for data transfer developed by REMP (37). Generally continuously monitored to assure among institutions is paramount. automated systems are developed proper storage conditions for To manage the vast amounts for storage at −80 °C, although some samples. For mechanical freezers of data in a variety of formats and liquid nitrogen systems are available. (−20° to −80 °C), temperatures environments, robust, flexible and Automation is most useful for are displayed on each freezer. extensible informatics systems studies and facilities that are focused For small biorepositories, regular are required (38). Too often, initial on one or a few specimen types that (twice daily) manual logging of research plans do not include a well- will be collected in large numbers temperatures may be adequate. thought-out approach to handle the and processed and stored in a However, larger biorepositories results of an investigation. Deliberate systematic way. If samples can be should have additional automated planning for data management is stored in microplates (for example, systems for remote monitoring of far less costly and time consuming 96- or 384-well), then automated temperatures to efficiently respond compared with ad hoc efforts that storage and retrieval systems should to malfunctions (4). occur post-collection. A plan for the be considered. However, due to Liquid nitrogen freezers require various disparate data types and the wide variety of specimen types monitoring of both temperature and formats should be included with and processing methods used in liquid nitrogen levels. Temperature special considerations for multisite molecular epidemiology studies, it monitoring is performed as for collection protocols. A major part of is often difficult to justify expensive mechanical freezers. Liquid nitrogen the integrated informatics system for automated storage and retrieval levels should be recorded manually, molecular epidemiology is support systems. on a regular basis, with a stick to for biospecimen collection, shipping, assure that normal levels (usually processing, storage, inventory and Storage system maintenance 8–10 cm) are maintained. It is retrieval processes. possible for liquid nitrogen freezers Freezers and other storage to overfill, which is detrimental Specimen tracking equipment should be validated to samples. Automated systems and maintained according to the should be used that can detect and Today, biospecimen collections are manufacturer’s recommendations. sound alarms for levels of liquid documented and tracked by many In addition, the biorepository should nitrogen that are either too low or forms of data management tools, develop additional protocols to too high. spanning from laboratory notebooks assure that equipment functions for a few hundred sample vials properly (3,4). A preventive Information management to real-time, multiuser software maintenance programme should implementations, which support be in place, with maintenance Driven by advances in molecular collections with millions of vials. performed at regularly established technologies, including genomics Clearly, there is a need for automated intervals. and proteomics, information information systems, but the level of

34 informatics sophistication needed type, and notations from hand-written of use varies. Many of the currently for a collection is limited by the vial labels. Standard operating available biorepository inventory availability of funding. In addition, it procedures for the development of systems include web-based access is incumbent upon the custodian of identifiers should be maintained with portals to make the systems easier human biospecimens to adhere to the system and updated to include to deploy and navigate. ethical standards to protect and use all labelling paradigms used in the the samples (3,4). Documentation repository. Informatics system security of the study protocol number and Bar code scanning technologies the informed consent for the study have become faster and more The size and scale of the subject should be easily linked back accurate in recent years. There are informatics needs of the molecular to the biospecimen to guarantee that several varieties of software solutions epidemiology group will determine the specific use of the specimens to generate bar codes, from stand- if the biorepository information has been verified before distribution. alone programs to those embedded system should include the subjects’ within other applications. Bar code Information technology software demographic and study annotation, Unit 2 for specimen tracking features printing options are recommended or whether these data can be held secure, validated environments based on the volume of labels within another database. Robust 3 Chapter that adhere to ethical practices. being printed. For high-volume label biorepository management systems As more and more collections are printing, thermal transfer or direct provide controlled user access for shared among investigators all over thermal bar code printers are the system security (39). The system the world, information on patient/ instruments of choice (39). When should include role-based security subject consent, sample collection choosing a device, the conditions for all repository staff, study techniques and processes, and under which the scanner will be coordinators and scientists with a annotation of the sample must be used, the frequency of use, the type need to access the biospecimens easily retrievable, exportable, and of bar code (linear or 2-D), and the inventory. If the study annotation is traceable through time. distance from which the scanning will held within the same data system, Biorepository information be performed should be considered security measures should be systems should support inventory (39). Cost considerations may enacted to protect the subjects’ functions by tracking all phases of influence the selection of the bar personal health information (PHI) sample acquisition, processing, code scanning technology employed from disclosure to unauthorized handling, quality control and by the biospecimen resource (4). users of the data. Regulations distribution from collection site Biorepository information governing the protection of (patient/subject) to utilization systems can report available individual identifying information (researcher) (3,4). The inventory space in the repository and assign vary from country to country, so tracking should include significant and reserve space for incoming it is important to reference the events, such as thaws, loss, depletion specimens. The location of a guidelines for the specific locations and destruction of specimens, specimen should be tracked, but of study and analysis in the study whether intentional or accidental. should not be used as part of the planning process (4). Restocking of returned, unused identifier naming convention, as If the biospecimen inventory is samples from the researcher, if locations of specimens may change physically separated from the study allowed per protocol, must also be in time. annotation, these systems should be documented. Current guidelines for The user interface of the system designed to interoperate and easily biorepository information systems must provide tools to search link the full study data, to maximize recommend the use of electronic the inventory based on various the ability to mine and analyse the (linear or two-dimensional) labels or specimen characteristics, as data. If the links between systems barcodes to document and associate well as support the requisition of are unstructured, the result can a unique identification number to the samples to use in research studies. be an extraordinarily challenging, samples. No identifying information Query interfaces should be easy expensive and time-consuming about the specimen should be to navigate by experienced and effort to produce scientific findings encoded as part of the identifier inexperienced users. Standard from the study. (3,4). The system should also be able and customizable queries are The system security architecture to track any pre-existing, external available in all commercially for information systems can be biospecimen identifiers, such as vial available systems, although ease two- or three-tiered, depending on

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 35 the separation of the user interface to study the environmental and for query and analysis in a secure client (tier one) from the application genetic causes of disease. It is collaborative environment. Efforts server (tier two), and then optionally important to be able to maintain tight to provide interoperability across from the data storage (tier three). integration of the demographic and many institutions and tools based Three-tiered systems are more clinical annotation of biospecimens, on grid computing are ongoing. flexible and scalable for groups that whether the data resides within the Grid technology can be viewed as have large concurrent user needs same data system or in physically an extension or application of the with heavy data load requirements distinct systems. Some study internet framework to create a more (39). collections may include data-use generic resource-sharing context agreements that require specimens (43). Cloud computing is a newer Inventory control to be de-identified before release delivery model for large, hosted from the biorepository for analysis. datacentres which offers various “Inventory control starts with an During the study planning process, computational and data access on an understanding of the conditions the rules that govern specimen as-needed, “utility company” model under which errors occur and ends access are key factors when over the internet. It typically involves with error-resistant processes, considering the use of pre-collected the provision of dynamically scalable intelligent use of technology, a biospecimens in a study (41). and often virtualized resources, thus well-trained and highly motivated The goals of each molecular avoiding the capital expenditure workforce, and an ongoing process epidemiology study will determine for purchase and maintenance of of continuous improvement” (40). the specific clinical annotation that infrastructure at each bioresource Inventory controls for should be maintained. Discussions centre location (44). biorepository management systems are ongoing across the international Whether the study data is include the creation and storage biomedical community to provide housed within one central data of audit trails to track data history, guidelines for minimal clinical system or in a federated, grid or data verification routines to assure annotation for various study types cloud framework, interoperability data quality, and process tracking (2–4,42), to facilitate data pooling is essential for the analysis of the to assure the integrity of the of studies across common research data and the publishing of results. sample data (39). The audit trails areas. The cohort, case–control, Efficient electronic data exchange will include any changes/additions/ and family-based consortia will or sharing between interoperable deletions of data identifying the benefit from the comparison and systems is based on shared common user that made the modifications. harmonization of their study data data element (CDE) definitions (45). The system should have the ability elements and definitions, and this When combining data from systems to generate configurable reports will allow faster mining to detect that do not share CDEs, mapping of and data files to provide the most underlying patterns across their the data to a shared set of elements complete information on the combined data sets. is required. Often, these mapping specimen. Inventory controls should efforts are labour-intensive and can include complete documentation System interoperability result in a loss of information, as of the information management local CDEs are fit into exchangeable system, updated standard operating Epidemiologists are employing definitions. It is possible that small procedures for the biorepository newer genomic technologies within differences in the way questions processes, security measures, and studies, which have resulted in and responses are worded or on-going training for those who exponentially larger data sets. presented in epidemiology survey access the data system (4). Legacy databases, however, that instruments can lead to significant were functional with smaller data sets (potentially unrecognized) Specimen annotation and do not communicate with other differences in interpretation. The systems, may need to be replaced or goal of developing CDEs is to The recognized value of molecular modified. Large data management enable semantic interoperability— epidemiology studies is the challenges require the integration of the ability to represent information collection of appropriate amounts heterogeneous data and tools in a precisely enough that it may pass of data, that when combined with scalable, high-performance system. between humans and electronic the study subject’s specimens and These systems can manage vast representations precisely without laboratory analyses, can be used quantities of data, and provide tools requiring absolute central control

36 of data systems or external the National Cancer Institute, The custom BioSpecimen human expertise (38). Semantic Office of Biorepository and Inventory System-II (BSI-II) was interoperability is a key component Biospecimen Research (NCI, initially developed on contract to speed data pooling efforts across OBBR) have focused on creating for the NCI’s Division of Cancer epidemiologic studies to replicate recommendations for best practices Epidemiology and Genetics to and validate study findings. associated with biorepository data support their large biospecimen systems, and the minimal clinical inventory from hundreds of molecular Informatics at the US National data set that should accompany all epidemiology studies (39). The Cancer Institute NCI-funded specimen collections BSI-II is flexible, extensible, and is (3). ISBER is focusing on the creation currently storing data associated Biomedical informatics systems are of best practices for biorepository with more than 10 million specimens evolving as the technology becomes management data systems. This will in storage across several contract available to “personalize medicine” foster the development of worldwide repositories. The NCI’s caBIGTM

for each patient. Towards this end, standardized methods for collection, project has developed an open- Unit 2 the NCI Center for Bioinformatics long-term storage, retrieval and source, modular caTissue Suite has begun the development of the distribution of specimens that will tool set for biospecimen inventory 3 Chapter cancer Biomedical Informatics enable their future use (4). management, tracking, and Grid or caBIGTM (45). This is a There is a large variety of highly annotation. This software permits voluntary network or grid connecting sophisticated, off-the-shelf, open users to enter and retrieve data individuals and institutions to enable source, and/or custom software concerning the collection, storage, the sharing of biomedical data and applications for biorepository quality assurance, and distribution tools, with a goal of creating a World information management (e.g. of biospecimens (47). Wide Web of cancer research. http://www.isber.org/ims-products. The focus is to speed the delivery html). Specific needs of the Additional issues of innovative approaches for the biorepository and the available prevention and treatment of cancer. funding will help guide the selection Although the issues discussed in The infrastructure and tools created of the system employed. One highly- the previous sections are critical by caBIGTM should have broad focused custom system serves to the successful collection and utility outside the cancer community. the CASPIRTM (US Centers for preservation of biospecimens, there An integral part of the caBIGTM Disease Control and Prevention- are other important considerations, plan is the cancer data standards ATSDR (Agency for Toxic concerning the control of specimen repository (caDSR) that will be used Substances and Disease Registry) quality, as well as the safety and to build and maintain a repository of Specimen Packaging, Inventory, security of personnel and facilities, CDEs for standardization of terms and Repository) biorepository that are equally important. and data storage practices. Tools (46). CASPIR is a central facility to for many aspects of biomedical store biological and environmental Quality assurance research are becoming available on biospecimens that the CDC-ATSDR and quality control the caGrid. began to develop in 1995. The mission of this biorepository is “… A Quality Management System Information management to provide storage for valuable, (QMS) is an essential element of systems from the US National mostly human, biological samples biospecimen management (3,4). Cancer Institute and Centers that have been collected from CDC The key to an effective QMS is the for Disease Control and and ATSDR diagnostic studies, development and adherence to Prevention epidemiologic outbreaks, and Standard Operating Procedures research studies for possible future (SOPs). SOPs should guide the Several organizations and use.” It has a storage capacity of collection, processing, storage companies around the US and more than six million biospecimens and equipment maintenance the world are creating solutions and is managed through customized processes described in this chapter. to address the information data management software called Biorepository staff should be trained management challenges presented the Archival Specimen Tracking and to adhere to all relevant quality by molecular epidemiology Retrieval Operations (ASTROTM) systems and SOPs. Additional studies. Informatics activities at system. elements that are important for a

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 37 QMS include: appropriate security exposed to human blood, body by the International Air Transport systems, computerized inventory and fluids and tissues, or other Association (52). specimen quality tracking systems, potentially infectious materials. Specimens are often exposed and a facility disaster plan (4). Biorepository work practices to temperature fluctuations during Several formal quality programs should be based on universal transit. The required shipping are appropriate for a specimen precautions similar to those used temperature depends on the QMS, including current Good in laboratories and clinical settings. intended analyses (3,4). Packaging Manufacturing Practices (cGMP) Good general laboratory work materials and equipment are and International Organization practices are outlined by Grizzle and available to preserve specimens for Standardization (ISO) (48) Fredenburgh (49). The CDC/NIH under ambient, refrigerated and certification. cGMP certification is booklet Biosafety in Microbiological frozen conditions, including liquid used in the USA to maintain quality and Biomedical Laboratories nitrogen dry shippers that can standards that are appropriate (51) outlines general biosafety preserve specimens frozen at or for Food and Drug Administration guidelines. All biorepositories that below –150 °C for up to several inspection of laboratories and handle human biospecimens should weeks (3,4). Devices are available biorepositories that process and store operate under the OSHA (or similar) to monitor temperature trends specimens for clinical applications. blood-borne pathogens standards during shipment, either by recording For research biorepositories, ISO and develop an exposure control temperatures precisely at certain certification, in general, is more plan. time intervals, or by changing colour appropriate for organizations that will In addition to biosafety, if a certain temperature is exceeded be collaborating with international biorepositories should follow strict during shipment. partners, and wish to assure that general safety regulations and they are operating under a common procedures regarding chemical, Security systems for set of recognized international electrical, fire, physical and biospecimen facilities standards. Both cGMP and ISO radiological safety (3,4,50). require extensive documentation of The use of liquid nitrogen poses Due to the irreplaceable nature the sources, quality and performance unique safety problems that are of many specimens collected for of materials, equipment, and not usually noted in laboratory molecular epidemiology studies, procedures. safety documentation. With a liquid it is critical to protect them from temperature of −196 °C, flesh destruction due to electrical Safety in the laboratory freezes almost instantly if it comes outages, equipment failures, and biorepository in direct contact with the liquid. and similar problems. The most Both face and eye protections are important systems to have in place Laboratories and biorepositories required. Oxygen level sensors are electrical back-up generators should assume that all human should always be employed, since and equipment alarms (4). biospecimens are potentially oxygen deprivation is a serious Generators should be available infective and biohazardous. A hazard in the event of a liquid to provide electrical service to predictable, small percentage of nitrogen leak. all freezers and any other critical biospecimens will pose a risk to equipment immediately upon the the biorepository workers who Proper packaging and loss of general electric service to the process them. All biospecimens shipping facility. They should be maintained should be treated as biohazards in good working order and started (49). In addition to taking biosafety Depending on whether they on a regular basis to assure that precautions, biorepositories should are known to contain infectious they are functioning properly (4). adhere to key principles of general agents, and the intended analyses, The appropriate fuel should be in laboratory safety. specimen shipments may be adequate supply for up to three In the United States, the regulated as infectious substances days of electrical outage during an Occupational Safety and Health or as diagnostic specimens. To emergency situation. Administration (OSHA) regulations properly classify the specimens Alarm systems should be (50) require that appropriate to be included in a shipment, provided in specimen storage areas vaccinations be offered to all consult references provided in to alert the staff when a freezer or personnel who may be potentially the ISBER Best Practices (4) and other equipment is malfunctioning.

38 They should be designed to and embedded in paraffin blocks • High temperature and humidity automatically (for example, by cell for low-cost storage and transport. during the day are common phone or paging device) notify If possible, given local conditions, conditions that may require special biorepository staff and other “cool packs” and other supplies shipping containers, such as coolers appropriate facilities maintenance can be provided from a central with cold packs. personnel during non-working coordinating centre and used to • Permits for importation and hours. Procedures should be in transport specimens at refrigerated exportation of human-derived place to immediately respond to temperatures. Note that any such substances and repository such equipment emergencies, and procedures that deviate from operation permits must be obtained to either move the specimens to a documented best practices must be before starting operations. Policies functioning back-up freezer, or take validated in a preliminary pilot study and procedures will vary according other appropriate action to preserve before full-scale adoption. to the country of origin and the their integrity. A specific example of working destination. • Laboratory equipment and In general, these measures under such conditions is the Costa Unit 2 should be part of a broader disaster Rica HPV Vaccine Trial conducted reagents may have to be imported, response plan that is designed by the US NCI in collaboration which will require a variable time for 3 Chapter to protect personnel as well as with the Fundacion Inciensa (53). customs and regulatory issues or specimens (4). Given the conditions under which the delivery time policy of the local specimens had to be collected in or international dealer. Because of Future directions Costa Rica, the following factors this, inventory management must and challenges were considered and accounted for: be highly coordinated to account for • Bad road conditions increase potential delays. Specimen management under shipment time and specimen adverse or low-resource shaking. Road conditions change Alternate collection conditions from the dry to rainy season every technologies year, and affect access to some In general, the methods, equipment communities. In addition to dry-state collection and supplies described in this • Liquid nitrogen may be hard and storage on treated cards, other chapter are practices that should to find in some countries, but not special collection and storage be adopted under the conditions impossible. For example, Nicaragua systems have been developed found in developed countries. does not produce any gases, but that are beginning to be used in However, it is not always possible has hospitals and factories that population-based studies. These in some developing countries with require oxygen and liquid nitrogen, approaches, mentioned briefly fewer resources to have access so oxygen is imported from Costa in other sections of this chapter, to liquid nitrogen or mechanical Rica. may gain more widespread use in ultra-low freezers, for example, or • The cost of liquid nitrogen, studies that require the collection of even electricity in some situations. equipment and reagents are large numbers of specimens that will These special circumstances need generally higher in Central America need purified DNA as the analytical to be carefully considered before than in developed countries, and in derivative. Some examples are: specimen collection is initiated. some cases, dealers for a particular • Oragene, developed by Some of the materials described in country are regional. For example, DNAGenotek (22). Oragene is a other sections of this chapter may a particular product produced in the reagent used for saliva collection. be useful. For example, if extreme USA may have to be acquired from The reagent saliva mixture is stable temperatures with little or no local a Mexican dealer that represents at room temperature. DNA can be refrigeration is an issue, then blood that product for Mexico and Central readily extracted either by using the or saliva can be collected on filter America. company’s manual procedure or an cards and shipped and stored at • In some countries the power automated procedure, such as the ambient temperature. Blood can supply may be regulated and/or in Gentra AutoPure. At least one large also be collected and shipped poor condition. If possible, a back- epidemiology study, performed by at ambient temperature using up power supply should be provided the Karolinska Institute, has had the PaxGene® collection tubes. or alternate storage methods should success with this protocol (23). Tissues can be fixed in formalin be considered.

Unit 2 • Chapter 3. Biological sample collection, processing, storage, and information management 39 • GenVault (28) has developed a • Ownership. It is often unclear have commercial value. Institutions small cellulose element, based on who ‘owns’ biospecimens once they should have clear intellectual the Whatman treated card, that can have been donated for research. property guidelines, and use be used in a 384-well microplate Court cases in the USA have ruled material transfer agreements to format to store DNA and other that the study participant does not assure that the sharing of specimens samples in the dry-state. DNA can have any ownership rights after and data are well controlled. The be eluted from the elements using donating a specimen for research. final disposition of specimens and a simple protocol, and adequate The NCI Best Practices (3) uses the data should be understood before amounts (up to 200 nanograms) of term “custodianship” to reflect the initiating a transfer. DNA can be extracted from each need for a biospecimen resource to In summary, the issues element, making this a convenient develop a plan for long-term care of surrounding the use of biospecimens system for long-term economical biospecimens. in research are complex and must archiving of DNA. • Specimen and data access. be approached with attention to Biospecimen resources should the many technical factors that may Biospecimen ethical, legal have clear rules for outside access affect the quality of the specimens. and policy issues to specimens and collected data (3). In addition, it is important to • Privacy protection. Study recognize that the quality of The ethical, legal and policy aspects participants need to be assured biospecimens is enhanced by the of biospecimen collection are as that their identity will be protected, collection and proper control of complex, if not more so, than the with respect to use of specimens various types of data. Finally, many technical matters outlined in this they have donated and any resulting issues discussed in this chapter are chapter. The following are some data. Privacy regulations are in subject to strict local and national of the issues that have not been place for this purpose (3). Due to policies and regulations concerning fully resolved in the international advances in genomic technologies, privacy and informed consent. community: it is becoming increasingly difficult • Informed consent. Formats to guarantee the protection of an and details vary greatly among individual’s identity. institutions. Policies for handling of • Intellectual property. Inventions biospecimens after withdrawal of and data arising from research consent are not well defined. using annotated biospecimens may

40 References

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