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Management of Patient Samples and Specimens
Chapter 35
Management of Patient Samples and Specimens Karen E. Pitt* and Amy P.N. Skubitzy *
Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota
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Chapter Outline Successful Research Rests On A Foundation of Careful Planning The Role of Pre-Analytic Variables in Research Using Patient Specimens The Importance of Good Record Keeping Specimen Tracking Specimen Collection Specimen Handling
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For many years, most of the attention in performing clinical research has been placed in three general areas: (1) the identification of biological markers or “biomarkers” that may indicate the onset of disease, (2) identification of pharmaceuticals that may reverse disease states, and most importantly, (3) develop approaches to prevent the disease state from evolving in the first place. While these aims continue to serve as the guiding forces for conducting clinical research, new attention has been placed on the importance of the biological specimen or “biospecimen” as the source material for scientific investigation. The handling of biological samples has relied upon assumptions such as “colder is better.” This relates to the idea that biological specimens are preserved best when handled and stored at colder temperatures or that fixation times may not be critical as long as the specimens are exposed to the appropriate chemicals and reagents. While these generalizations may hold true for many material types and cellular components, they do not hold true for all. A new field of science has emerged that is called “Biospecimen science” that aims to investigate these assumptions and clarify the optimal conditions for specimen collection, processing, storage, and dissemination. In recognition of the importance of improving the state of the science as to how biospecimens are collected and handled, several governmental and non-profit organizations
Principles and Practice of Clinical Research. DOI: 10.1016/B978-0-12-382167-6.00035-7 2012 Published by Elsevier Inc.
Specimen Transit Specimen Storage Access To Patient Samples Specimen Culling, Transfer of Collections, and Repository Closings Summary Questions References
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have emerged to exchange information on effective practices and research findings. In 1999, a small group of professionals working with biorepositories (i.e., centers for biospecimen processing, storage, and dissemination) gathered to share their experience and offer each other assistance to improve and enhance consistency in biospecimen handling. Since its inception, the International Society for Biological and Environmental Repositories (ISBER) has brought together professionals associated with biorepositories from all over the world and has produced two editions of its “Best Practices for Repositories”1e3 (also available in PDF form at www.isber.org), with a third edition in press as this book goes to press. In addition, ISBER’s working group on Biospecimen Science has compiled a comprehensive list of research findings on the field of biospecimen research that provides important insights regarding the research performed to date on specimen collection and handling practices (www.isber.org). The U.S. National Cancer Institute (NCI) has long recognized that the lack of standardized, high-quality biospecimens has resulted in one of the most significant roadblocks to the progress of cancer research. In 2002, the NCI initiated a due diligence process to understand the state of its funded biospecimen resources and the quality of biospecimens used in cancer research. To achieve this aim, the NCI conducted surveys and created community forums that led to 509
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the formation of a trans-divisional Biorepository Coordinating Committee. This was followed by the establishment of the Office of Biorepositories and Biospecimen Research (OBBR) to address issues pertinent to the development of practices that promote the creation of and utilization of highquality specimens in cancer research. The OBBR developed its “First-Generation Guidelines for NCI-Supported Research” (www.biospecimens.cancer.gov) in 2006 that was subsequently revised based on public comment and input from experts and was retitled, “NCI Best Practices for Biospecimens Resources.” or “NCI Best Practices.” The current version of the NCI Best Practices was released in 2011 and can be found at www.biospecimens.cancer.gov. Other important OBBR initiatives include the creation of the “Biospecimen Research Network” (BRN) which sponsors an annual symposium on the state of the art of Biospecimens Science and its creation of a “Biospecimen Research Database” that allows investigators a quick glance at important scientific publications in support of this new field. This chapter will focus on practices relevant to the management of patient samples in clinical research. The reader is encouraged to review the documents included as references to learn more about the issues pertaining to effective handling of biological specimens.
Principles and Practice of Clinical Research
Important scientific findings result from the use of a wellplanned research strategy and require the development of protocols that support the isolation and preservation of patient samples so that they closely resemble their status prior to their removal from the patient. The optimal situation is one in which the analytes to be measured appropriate have been well characterized, and specimen collection and processing practices are well-documented in the scientific literature. However, since new methods and technologies continuously are being developed, it may not be possible at the time of biospecimen collection to determine what downstream assays may be available in the future. When specific target analytes are known, it is important that the specimens used are “fit for purpose” or, in other words, have been demonstrated to support the integrity of the particular analyte as the biospecimen makes its way through the collection and processing pathway. When specific targets are not known at the outset of a study, general practices must be implemented in a timely manner according to information that is available in the literature.
Evidence-based protocols are defined as protocols that have been scientifically optimized for a specific result through the use of quality control measures with standards that allow for the reproducibility of the assay under defined conditions. In addition to having evidence-based protocols for specimen collection and processing, it is important for the investigator to understand that the molecular profile of a tissue or other biological material can be influenced by a variety of factors. These factors may include exposures of the patient to certain medications, food or beverages consumed prior to the collection of the biospecimen4,5 or certain environmental exposures.6 The time of day at which a specimen is collected may also influence the molecular profiles of some analytes under certain circumstances.7 Additional influences that should be considered are: the time that elapses between when the tissue is separated from the patient’s blood supply and when the tissue is removed (the ischemic time); the time that elapses between when specimens are removed from the patient and when they are processed8; and the temperature at which the specimen is kept prior to processing. The combined effects of these types of exposures or events prior to the time at which a biospecimen is used in an assay have been described as “preanalytic” variables (Figure 35-1). These factors are often some of the most difficult to capture and yet can have significant effects on research outcomes.9 The reader is directed to reviews of the burgeoning field of literature on pre-analytic variables that have been summarized by ISBER and OBBR. As much information as is possible about the condition of the patient from whom the specimen has been taken (e.g., disease status, drug profile, timing of the collection, and food exposures) should be recorded and tracked throughout the life cycle of the specimen. This information may become critical when evaluating the future suitability of the biospecimen for a particular investigation. Managing patient samples to ensure their suitability for specific tests can be achieved best when these pre-analytic variables can be traced. A system for documenting preanalytic variables was described by Betsou and colleagues10 in which a Sample PREanalytic Code (SPREC) is assigned to each biospecimen. The code is equivalent to a “specimen barcode” that provides details about pre-analytic processing for that specimen. The code is comprised of seven elements that contain information such as the material type, processing steps, and storage conditions that may be important in understanding a sample’s “fit for purpose” for a particular assay.
THE ROLE OF PRE-ANALYTIC VARIABLES IN RESEARCH USING PATIENT SPECIMENS
THE IMPORTANCE OF GOOD RECORD KEEPING
In recent years, the scientific community has come to the realization that all patient samples are not created equal.
The National Institutes of Health (NIH) has stated in its publication, “Guidelines for Scientific Record Keeping in
SUCCESSFUL RESEARCH RESTS ON A FOUNDATION OF CAREFUL PLANNING
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FIGURE 35-1 Variables affecting the molecular integrity of biospecimens. Both the pre- and post-acquisitions variables listed here are covered under the category of “pre-analytic variables.”
the Intramural Research Program at the NIH” (http:// sourcebook.od.nih.gov/ethic-conduct/RECORDKEEPING. pdf), that “the progress and excellence of NIH research are dependent on our vigilance in maintaining the highest quality of records for every aspect of the science conducted here.” Good record keeping is a requirement of all scientific endeavors and contributes to the successful analyses of research results, as well as for the review of those results once submitted for publication. It provides the basis for which results can be reproduced by others, to establish the scientific validity of findings. Careful record keeping may contribute to the establishment of intellectual property claims or assist with the defense against allegations of research misconduct. Records should be kept in a manner that is legible and would allow others to understand and reproduce the steps taken to obtain the same findings. Records should capture: l l l
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the name of the person making the record; the purpose of the research; the names of all individuals involved in the collection and processing efforts; lists of Standard Operating Procedures (SOPs);
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information about the participant (name, age, appropriate health statistics); and the time and date on which the specimens were collected and all subsequent processing steps.
Records can be kept in bound or unbound notebooks, or in electronic formats such as those found associated with ordinary software or specialized software packages for the specific purpose of recording laboratory and other aspects integral to specimen collection, processing, storage, and dissemination. All records should be kept in a secure manner to ensure that access to the records is limited to authorized users and that personal health information is kept confidential. Records should be kept for a minimum of two years following the close of a protocol, but may be kept indefinitely as records may become invaluable resources for future research endeavors, and in the documentation and reporting that may be performed for the existing work at an unspecified future date.
SPECIMEN TRACKING The ability to effectively manage patient samples requires that the original specimen that is collected be tracked, as
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well as all derivatives of the original specimen generated through various handling and processing protocols. Each specimen should be given a unique identifier (ID) that can be followed throughout its life cycle. These numbers should allow for traceability to an ID linked to a particular patient, unless the linkage between the patient ID and specimen ID has been irreversibly interrupted, such that the identity of the patient has been anonymized. Derivatives of the original specimen (e.g., DNA, RNA, or protein extractions) should always be traceable to the original specimen. All specimens should have a printed label that contains the biospecimen ID that is encoded in either a onedimensional (1D) or two-dimensional (2D) scannable barcode. One-dimensional barcodes have an advantage in that they contain a high degree of redundancy, which means that the barcode can be read even with considerable degradation. However, 1D barcodes are limited in the amount of information they contain, since all of the data is encoded in the horizontal width. Two-dimensional barcodes have the advantage that they contain information that is read in a vertical direction as well as a horizontal direction and so the amount of information in the code is greater (Figure 35-2). There are advantages to including human readable information on the label (i.e., the specimen ID) to facilitate manual processing, but this may not always be necessary. Many specimen containers are now manufactured with 2D barcodes already printed on the container, which can facilitate automated specimen handling without the need or expense of adding a physical
FIGURE 35-2 The images on the top and bottom represent a one-dimensional (1D) barcode and a two-dimensional (2D) barcode, respectively. The 1D barcode is comprised of a single row of bars and the data encoded is presented across the horizontal width of the label. Data encoded in the 2D label is contained in both the vertical and horizontal directions and is able to contain a greater amount of information.
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label. Effective use of these containers without additional printed labels requires that a scanner is always available should the identity of a specimen need to be verified. Selection of the best labels for a particular study and purpose should be performed well in advance of the commencement of specimen collection. Integral to the effective management of patient samples is having an inventory system that allows for tracking the specimens as they move from collection through processing, storage, and dissemination. Computer-based systems allow for the most accurate, dependable tracking of samples. Although this may be accomplished by using simple spreadsheet programs, the best way to track specimens and characteristics of those specimens is to use an inventory system that is created specifically for this purpose. Many inventory system software programs are commercially available and can be used easily without significant customization. These programs have the ability to track personal health information, subject IDs, specimen IDs, specimen processing events, and specimen location. In addition, many programs allow for the uploading and downloading of batch records, which facilitates specimen accession, distribution, and the sharing of data under carefully defined conditions. All inventory management systems or other computerbased systems that contain personal health information must be operated under strict security practices. Regardless of whether records are electronic or paper, access to patient information and information about patient samples
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must be tightly controlled. Access to view and modify information should be allowed according to defined privilege levels. Changes or modifications to records should be traceable to the time, date, and the identity of the individual making the changes. Records should be backed up daily to a format and location that allows for their secure maintenance according to a defined management plan that includes consideration of a wide variety of emergency situations.
SPECIMEN COLLECTION The effective collection of patient samples begins with the receipt of a signed consent form from the patient (or appropriate guardian) that documents permission to use the samples under defined conditions (see Chapter 2). Consent parameters must be tracked throughout the life cycle of the sample and its derivatives to ensure that any testing performed falls within the parameters to which the patient has agreed. Study design and the use of patient samples are governed by institutional review boards (IRBs, Chapter 5). Investigators are responsible for ensuring that appropriate approvals are in place for the use of biospecimens and biospecimen-related data and should allow sufficient time for appropriate review when planning for the implementation of research activities. The actual collection of the patient samples should be well orchestrated as it may involve a number of individuals and circumstances around which careful planning must be executed in order to ensure that the highest quality specimens are available for downstream processing. In addition to the biorepository technicians, some of the personnel involved in this process on a daily basis may include: the study coordinator, admissions nurse, anesthesiologist, operating room nurse, surgeon, pathologist, and/or pathologist assistant. All individuals involved in the collection process should have the appropriate expertise and be well trained in the tasks that they are expected to perform in the collection activities. Where possible and appropriate, the success of research efforts can be enhanced by having a joint meeting(s) among those involved in the collection of patient samples to ensure a smooth transition from one phase of the collection effort to another. When multiple collections sites are involved, it is even more critical that meetings are held in advance of specimen collection in order to ensure that protocols are implemented in a uniform manner. If collections take place over an extended period of time, there also should be opportunities to resolve questions rapidly and to find solutions for unanticipated situations. In order to ensure that biospecimens for clinical trials are collected and processed correctly, it is important that detailed SOPs are provided to the personnel involved in the collection effort and that they have a clear understanding of
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what needs to be done. As a first step, a research nurse or study coordinator should contact the biorepository staff in advance to apprise them of any necessary information pertaining to the patient who has been enrolled in the clinical trial and the specific date and time that the surgery is scheduled. The SOP should outline which consent form should be used as well as the type, number, and processing steps for each biospecimen to be collected. For a typical clinical trial study, a biorepository technician should review the operating room schedule a week in advance, then again one day in advance, and finally on the day of surgery. The technician should notify the operating room nurses of the cases that are expected to be collected that day and which tissues are to be obtained so that those specimens will be handled properly. This technician is responsible for ensuring that each patient who is scheduled for surgery and who will be contributing specimens signs appropriate consent forms. Separate IRBapproved patient consent forms may need to be designed for the acquisition of waste tissue following surgery, as well as for saliva, urine, ascites fluid, and blood. In addition, the patient consent form should be translated into languages other than English, as needed, to make sure that the patient can understand the information contained therein. A translator should be present if the patient does not understand the language in which the consent form has been written. A research study that is linked to a clinical trial may involve the collection of pre-surgical specimens or ones that are collected during the surgery itself. Samples collected prior to surgery may include blood, urine, saliva or a buccal smear. Samples collected during surgery may be resected from the patient or may be collected through the use of a catheter. Prior to surgery, a biorepository technician may post signs in the operating room to remind the operating room staff that tissues from a specific patient are required for the clinical trial study. All specimens should be handled in as sterile a manner as possible. Surgeons should be requested to place specimens designated for the clinical trial study into a sterile container (provided by the biorepository) or in a sterile basin covered with sterile towels. Sterile instruments for this purpose should be maintained by the biorepository. The technician should be paged when a requested specimen has been surgically removed from a patient. The technician should then transport the specimen to the accessioning area and wait until the appropriate staff member has taken the portion of the specimen that they require for a complete diagnosis. The surgical pathologist then will give the technician the waste tissue, and sign a “specimen inventory” form indicating their approval for the biorepository to procure the waste tissue. The technician should process the tissue under the guidance of the surgical pathologist according to the specifications of the clinical trial.
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Tissues collected may be snap frozen (either on dry ice or in liquid nitrogen), frozen in OCT (Optimal Cutting Temperature), or fixed in buffered formalin and then embedded in paraffin. Sections can be cut onto slides from either the OCT-embedded tissue or the formalin-fixed paraffin-embedded blocks, and then used for pathology review. Some clinical trials may require that touch preps be made from fresh wet tissues, while in other cases fresh tissues may need to be placed in a special tissue culture media or buffered saline solution so that the cells can be tested in functional studies. Blood taken from patients for clinical trial studies may be requested as whole blood, or may need to be processed into sera or plasma. Some studies may require further processing of the blood such that white blood cells or red blood cells need to be isolated. Sometimes the availability of patient samples may be realized with short notice for the collection staff (e.g., transplant, death/autopsy material). Planning efforts should anticipate the need for trained personnel, materials and supplies (e.g., chilled specimen containers, sterile instruments, etc.) under these conditions without the need for last minute arrangements that may compromise specimen integrity. Most collections will likely be performed under more controlled conditions, but preparation for all possible scenarios will increase the likelihood of successful specimen collections.
SPECIMEN HANDLING As has been underscored throughout this chapter, handling specimens according to evidence-based protocols for a particular research design is always the best approach to take. However, it is not always possible to know exactly which assays will be performed. In those cases, specimens should be collected according to protocols that are most likely to support the effective maintenance of the biospecimen to most closely resemble its state prior to removal from the patient. Having the correct cryovials, vacuutainers, and other containers available at the collection site is critical, and those containers should support anticipated downstream uses for the samples. Agents that stabilize biological samples such as anti-coagulants may be essential for some downstream applications while having a deleterious effect on others. In addition, some supplies may carry expiration dates that set a defined period of acceptable use (e.g., vacuutainers, tissue culture media, and antibiotics). Every effort should be made to track expiration dates and make sure that supplies and reagents are used within the allowable time frame. Container selection is another important area of consideration in the management of patient samples. Containers should be composed of materials that will not interfere with the downstream assays; this is especially
Principles and Practice of Clinical Research
important if testing will include measurements of trace compounds. In addition, containers must be stable under subsequent storage environments and be of a size appropriate to the volume of the material stored. In general, vials with threaded caps do better when specimens are stored at ultra-cold temperatures (i.e., 80 C and cooler). Selecting the appropriate containers may reduce sublimation of the specimen over time and will facilitate subsequent storage and handling. Smaller tubes take up less storage volume and thus allow for efficient use of environmental storage containers (e.g., refrigerators and freezers). Even when automated specimen handling is not anticipated in the immediate life of the study, the use of automated specimen handling equipment in clinical research is on the rise. Vials and other containers that are amenable to automated handling (e.g., liquid handling equipment for aliquot preparation or plates used in immunofluorescence or other multi-plex assays) can save labor and improve the accuracy and quality of specimen handling activities down the road. Finally, investigators should be sure that containers sent to outside laboratories for testing are appropriate for the protocols utilized by those laboratories. Another area that is important to consider when processing biological specimens is the amount of the specimen that is being stored. Frequently it is necessary to divide up, or prepare aliquots of, the material collected to allow for different uses for the specimens in question, or to be able to repeat tests when necessary. Increasing the number of aliquots is likely to increase the amount of storage room needed, adding to the costs of implementing the research. Certain molecular species, such as proteins, may be irreversibly modified by exposure to cycles of freezing and thawing, whereas other species such as DNA are thought to be relatively resistant to such temperature cycles. When sensitivity to cycles of freezing and thawing is anticipated, aliquots should be made that will most likely reflect the volume of material needed for anticipated assays. In contrast, source vials containing molecular species that are not affected by freezing and warming cycles can be stored and aliquots made according to the schedule of the testing to be performed. Delaying the preparation of aliquots offers the advantage that as new testing platforms are developed, the amount of material required can be specifically removed and the specimen can be used in the most efficient manner possible.
SPECIMEN TRANSIT Whether a sample is being transferred down the hall or across the globe, there are particular considerations that should be made to ensure specimen integrity. Even for short transfers, care should be taken to ensure that the temperature and other environmental conditions needed for the stabilization of the biospecimen are met. Sometimes
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Management of Patient Samples and Specimens
specimens must be immediately cooled by storage on frozen cold packs (approximating 4 C storage), dry ice (approximating storage at 80 C) or immersed into liquid nitrogen (196 C) to stop molecular and/or enzymatic activity. Sometimes the rate at which a biospecimen is cooled plays an important role in the subsequent viability of the cells contained therein or the structural integrity of the proteins. Other times, cooling the specimen to temperatures any lower than ambient may have a deleterious effect on some molecular components. This is where it can be advantageous if the analytes of interest can be measured as a function of different handling conditions during the specimen collection and processing before the study is initiated, to be sure that transit temperatures are appropriate. In cases where specimens are transported over periods longer than an hour, a monitoring device may be included within the shipment to allow investigators to document whether deleterious temperature excursions occurred during transit. Transport outside of an institution or over greater distances carries additional requirements for adherence not only to good practices that will ensure the stable transport of the sample, but also to national and international regulations. Transfer of specimens and/or data that is associated with a patient sample to a research entity outside of the one responsible for its collection may require the use of a Material Transfer Agreement (MTA). MTAs document the allowed uses for the samples and/or data, ensure that uses are consistent with permissions afforded in patient consent documents, and may address issues pertaining to intellectual property rights associated with the material. Depending upon institutional policies, MTAs may require approval by IRBs or other bodies that govern the use of human subjects in research. Consequently, efforts to have MTAs established and obtain all needed approvals should be initiated as early as possible in the research plan. All specimens transferred from one research institution to another must be packaged according to national and international regulations. Most couriers require that the standards created by the International Air Transportation Association (IATA) be met even when the transit may not involve transportation by air. Shippers begin by determining which regulatory requirements must be met and the physical requirements needed to ensure proper shipping. The shipper must determine how the specimens should be classified (e.g., infectious substance, Category A or Category B, patient specimen, or genetically modified organisms). In addition, the shipper must understand what preservatives may have been used that could be considered gaseous, toxic, flammable or corrosive; all of these are “dangerous goods.” Strict adherence to packaging and documentation requirements as outlined in the standards for each dangerous good being transported must be met. Shippers are required to receive training in this area before
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they perform any shipping activities. This training must include general familiarization with the regulations, on the specifics relating to the type and number of specimens being transported, and concerning safety matters relating to the preparation and implementation of the shipment. The shipper has sole responsibility for adhering to local, national, and international regulations governing the shipment of biological specimens.
SPECIMEN STORAGE Issues pertaining to specimen storage begin when a sample is received in the laboratory setting or specimen repository in which the specimen will be stored. SOPs should be written that govern the tracking of a specimen upon its arrival to document the time it arrives and its condition. Receipts (as well as shipments out of the laboratory or repository) should be recorded in a log that also records the name of the person who receives the shipment and any interim storage location used. Specimens should always arrive accompanied by a specimen manifest that documents the specimen IDs contained within the shipment. Any damage to the specimens should be documented and communicated to the shipper. Under some adverse situations, specimens may need to be replaced, and initiating this process as early as possible can minimize the effect of the occurrence on the overall implementation of the research effort. Once receipt of the specimens is recorded in the log, the specimens should be evaluated to ensure that all of the specimens in the container match what is included with the manifest. Any discrepancies should be immediately communicated to the shipper. If the samples have been labeled with barcodes, inventory of the specimens can be made rapidly using a barcode scanner. If specimen labels do not meet the specifications of the protocols for the study, appropriate labels should be generated and applied so that subsequent specimen handling can be expedited. After confirmation of the manifest has been completed, specimens should be transferred to the appropriate storage locations and the locations should be entered into the specimen inventory system. In addition to ensuring that specimens stored have proper labels, consideration should be given to the strategy used to place the specimens into their storage environments. Specimens should be stored according to a plan that reflects how they are likely to be removed at a later time for testing. Specimens are usually retrieved from storage for a particular study using a defined material type (e.g., plasma, serum, or urine). Storing specimens according to anticipated retrieval patterns will expedite the retrieval process and minimize the labor needed to retrieve specimens. When selecting the appropriate storage environment, factors to consider should include: temperature, humidity, and sensitivity to light, as appropriate. In the absence of
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data to the contrary, one may assume that the colder the storage environment, the greater the stability of the biological material. This is certainly true for the cryopreservation of cell suspensions, wherein the maintenance of viability is required. Intact cells are generally stored in liquid nitrogen vapor temperatures (<150 C) that are below what is referred to as the Tg or glass transition temperature (<135 C) where all molecular motion is believed to cease. Many biological materials are stored at what are considered “ultra-low” temperatures that range from 70 C to 80 C. In contrast, specimens that have been fixed with formalin or other fixatives may be stored at room temperature but under conditions that control for humidity. Tissue blocks that have been fixed and then embedded in paraffin may be vacuum sealed in plastic packets, then stored at 4 C in the dark.11 The storage conditions selected should reflect practices reported in the scientific literature for similar types of specimens, and for the performance of assays similar to those to be included in the planned study. Recently, new storage matrices have been developed that allow for room temperature storage of RNA and DNA. Use of these products may facilitate storage in situations where freezers or power supplies may not be available or when long-term storage is needed at a low cost. If biospecimens are to be stored in mechanical refrigerators or freezers, investigators should evaluate new products that are currently available. New designs include equipment that requires less energy and can significantly reduce facility costs for specimen storage. All equipment, whether mechanical or cooled by liquid nitrogen or other coolants, should be tested before specimens are added to ensure that the equipment performs under the manufacturer’s specifications. Emergency power (or reserves of liquid nitrogen or other coolant) should be available to allow for interruptions in power and should be maintained to allow at least 48 to 72 hours of emergency power supply. In addition, should individual units fail, some cold, empty back-up storage units should be available to hold specimens from the failing unit. In large biorepositories with dozens of storage units, a rule of thumb is to maintain approximately 10% of the total volume needed for ultra-cold and approximately 3% back-up for liquid nitrogen. All equipment in specimen repositories should be included in preventive maintenance programs to extend the timeeffective operation of the equipment for as long as possible. Monitoring systems should be implemented to alert appropriate staff on a 24 hour basis to any temperature excursions that might affect specimen integrity.
Principles and Practice of Clinical Research
implemented according to the scientific goals for the study. While custodians are generally individuals named as contacts for the biospecimen resource, decisions on use and requests to use the biospecimens for other allowable research may be made by a group of experts, and may include participation from lay persons with interests in the particular area of research. Guidelines on how specimen resources should be used need to be well defined and clearly articulated, and be processed in a timely manner. Requests should be based on sound scientific research questions and must fall within parameters established in patient consent documentation. At the time of consent, patients should be given contact information and guidance as to what steps should be taken if they later wish to have their specimens or data withdrawn from a study. Repositories should have procedures in place to identify the specimens and ensure that the specimens and any associated data are not used for subsequent research activity.
SPECIMEN CULLING, TRANSFER OF COLLECTIONS, AND REPOSITORY CLOSINGS Because of the expense of maintaining collections of patients’ samples, it should be a regular practice to make sure that the specimens stored are ones that are suitable for research. Sometimes conditions arise when the integrity of a specimen has been compromised (e.g., when its label has been lost or cannot be read or because of environmental problems such as a freezer failure). Periodic reviews of collections should be performed to make sure that the specimens stored are appropriate and are “fit for purpose” for the anticipated research. There are other situations, such as when research funds are no longer available, that collections must be destroyed or “culled” because they can no longer be maintained by the original biospecimen resource. Under these conditions, it may be possible to have the specimens transferred to another research entity that may be able to use the specimens in new research activities (which are consistent with consent documentation). All culling activities, whether by destruction or transfer to a new institution, should be well documented. Under conditions wherein specimen collections are transferred to a new institution, all requirements mentioned earlier regarding the establishment of MTAs, ensuring that other approvals are in place, and that specimen transport regulations have been met, must be considered prior to the transfer of the specimens.
SUMMARY QUESTIONS ACCESS TO PATIENT SAMPLES Use of patient samples for a particular study is under the guardianship of a custodian, who ensures that those uses are
1. What is a biorepository? a. A storage center for biological specimens b. An educational center for high school biology majors
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Management of Patient Samples and Specimens
c. A library of medical records d. A storage site for repossessed vehicles What does “fit for purpose” mean with respect to the development of scientific protocols? a. That residual specimens from prior testing efforts are used in the development of specimen handling protocols b. That the type of biospecimen used has been optimized for a particular type of analysis c. That individuals performing the testing are trained appropriately to perform the assays needed d. That multiple laboratories will perform testing simultaneously to assess consistency in the results What are “evidence-based” protocols? a. Protocols that have been developed within the past year b. Protocols that yield reproducible results c. Protocols that have been used consistently in a laboratory setting d. Protocols that have been optimized using highquality specimens and other reference standards to ensure that the procedures developed will yield scientifically significant results Which of the following would be considered “preanalytic variables”? a. Medications taken prior to specimen collection b. Time at which the specimen is collected c. The type of specimen fixative used and the time the specimen spent in the fixative d. All of the above Which of the following types of records should be maintained? a. The name of the person making the record b. The list of Standard Operating Procedures (SOPs) c. The time and date when the specimen was collected d. All of the above Which of the following statements are true regarding two-dimensional (2D) barcodes? a. They contain information stored in a vertical as well as a horizontal direction on the label b. They can contain less information than a onedimensional barcode c. They usually take up a smaller space on a label d. They may be printed on the bottom of a vial What is an MTA and what is its purpose? a. An MTA is a Management Transfer Agreement that describes what type of shipping must be used to send biospecimens to their next destination b. An MTA is a Material Transfer Agreement that it is used to document how a biospecimen may be used once it is transferred to its next destination
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c. An MTA is a Missing Tariff Analysis that is used to investigate biospecimens detained in customs during international shipments d. An MTA is a Manual Temperature Analysis that is performed when an environmental temperature chamber exceeds its allowable range of operation
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Pitt KE, Campbell LD, Skubitz APN, Aamodt RL, Anouna A, et al. Best practices for repositories. I: Collection, storage, and retrieval of human biological materials for research. Cell Preserv Technol 2005;3(1):5e48. Pitt KE, Campbell LD, Skubitz APN, Somiari SB, Sexton KC, Pugh RS. Best practices for repositories: collection, storage, retrieval, and distribution of biological materials for research. Second edition. Cell Preserv Technol 2008;6(1):3e58. Campbell LD, Betson F, Gariua DL, Grini JG, Pitt KE, Pugh RS, et al. Best practices for repositories: Collection, Storage, reterival and distribution of biological materials for research. Third edition. Biopresentation and Biobanking 2012;10(2)(inpress). So¨derholm PP, Alfthan G, Koskela AH, Adlercreutz H, Tikkanen MJ. The effect of high-fiber rye bread enriched with nonesterified plant sterols on major serum lipids and apolipoproteins in normocholesterolemic individuals. Nutr Metab Cardiovasc Dis; January 5, 2011 [Epub ahead of print]. Lippi G, Lima-Oliveira G, Salvagno GL, Montagnana M, Gelati M, Picheth G, et al. Influence of a light meal on routine haematological tests. Blood Transfus 2010;8(2):94e9. Hernandez ML, Harris B, Lay JC, Bromberg PA, Diaz-Sanchez D, Devlin RB, et al. Comparative airway inflammatory response of normal volunteers to ozone and lipopolysaccharide challenge. Inhal Toxicol 2010;22(8):648e56. Mazzoccoli G, De Cata A, Greco A, Carughi S, Giuliani F, Tarquini R. Circadian rhythmicity of lymphocyte subpopulations and relationship with neuro-endocrine system. J Biol Regul Homeost Agents 2010;24(3):341e50. Spruessel A, Steimann G, Jung M, Lee SA, Carr T, Fentz A-K, et al. Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision. Biotechniques 2004;36:1030. Betsou F, Barnes R, Burke T, Coppola D, DeSouza Y, Eliason J, et al. Human biospecimen research: experimental protocol and quality control tools [International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science]. Cancer Epidemiol Biomarkers Prevention 2009;18:1017e25. Betsou F, Lehmann S, Ashton G, Barnes M, Benson EE, Coppola D, et al. Standard preanalytical coding for biospecimens: defining the sample PREanalytical Code (SPREC). Cancer Epidemiol Biomarkers Prevention 2010;19(4):1004e11. Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Amer J Pathol 2002;161(6):1961e71.
Management of Patient Samples and Specimens Karen E. Pitt* and Amy P.N. Skubitzy *
Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota
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Chapter Outline Successful Research Rests On A Foundation of Careful Planning The Role of Pre-Analytic Variables in Research Using Patient Specimens The Importance of Good Record Keeping Specimen Tracking Specimen Collection Specimen Handling
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For many years, most of the attention in performing clinical research has been placed in three general areas: (1) the identification of biological markers or “biomarkers” that may indicate the onset of disease, (2) identification of pharmaceuticals that may reverse disease states, and most importantly, (3) develop approaches to prevent the disease state from evolving in the first place. While these aims continue to serve as the guiding forces for conducting clinical research, new attention has been placed on the importance of the biological specimen or “biospecimen” as the source material for scientific investigation. The handling of biological samples has relied upon assumptions such as “colder is better.” This relates to the idea that biological specimens are preserved best when handled and stored at colder temperatures or that fixation times may not be critical as long as the specimens are exposed to the appropriate chemicals and reagents. While these generalizations may hold true for many material types and cellular components, they do not hold true for all. A new field of science has emerged that is called “Biospecimen science” that aims to investigate these assumptions and clarify the optimal conditions for specimen collection, processing, storage, and dissemination. In recognition of the importance of improving the state of the science as to how biospecimens are collected and handled, several governmental and non-profit organizations
Principles and Practice of Clinical Research. DOI: 10.1016/B978-0-12-382167-6.00035-7 2012 Published by Elsevier Inc.
Specimen Transit Specimen Storage Access To Patient Samples Specimen Culling, Transfer of Collections, and Repository Closings Summary Questions References
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have emerged to exchange information on effective practices and research findings. In 1999, a small group of professionals working with biorepositories (i.e., centers for biospecimen processing, storage, and dissemination) gathered to share their experience and offer each other assistance to improve and enhance consistency in biospecimen handling. Since its inception, the International Society for Biological and Environmental Repositories (ISBER) has brought together professionals associated with biorepositories from all over the world and has produced two editions of its “Best Practices for Repositories”1e3 (also available in PDF form at www.isber.org), with a third edition in press as this book goes to press. In addition, ISBER’s working group on Biospecimen Science has compiled a comprehensive list of research findings on the field of biospecimen research that provides important insights regarding the research performed to date on specimen collection and handling practices (www.isber.org). The U.S. National Cancer Institute (NCI) has long recognized that the lack of standardized, high-quality biospecimens has resulted in one of the most significant roadblocks to the progress of cancer research. In 2002, the NCI initiated a due diligence process to understand the state of its funded biospecimen resources and the quality of biospecimens used in cancer research. To achieve this aim, the NCI conducted surveys and created community forums that led to 509
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the formation of a trans-divisional Biorepository Coordinating Committee. This was followed by the establishment of the Office of Biorepositories and Biospecimen Research (OBBR) to address issues pertinent to the development of practices that promote the creation of and utilization of highquality specimens in cancer research. The OBBR developed its “First-Generation Guidelines for NCI-Supported Research” (www.biospecimens.cancer.gov) in 2006 that was subsequently revised based on public comment and input from experts and was retitled, “NCI Best Practices for Biospecimens Resources.” or “NCI Best Practices.” The current version of the NCI Best Practices was released in 2011 and can be found at www.biospecimens.cancer.gov. Other important OBBR initiatives include the creation of the “Biospecimen Research Network” (BRN) which sponsors an annual symposium on the state of the art of Biospecimens Science and its creation of a “Biospecimen Research Database” that allows investigators a quick glance at important scientific publications in support of this new field. This chapter will focus on practices relevant to the management of patient samples in clinical research. The reader is encouraged to review the documents included as references to learn more about the issues pertaining to effective handling of biological specimens.
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Important scientific findings result from the use of a wellplanned research strategy and require the development of protocols that support the isolation and preservation of patient samples so that they closely resemble their status prior to their removal from the patient. The optimal situation is one in which the analytes to be measured appropriate have been well characterized, and specimen collection and processing practices are well-documented in the scientific literature. However, since new methods and technologies continuously are being developed, it may not be possible at the time of biospecimen collection to determine what downstream assays may be available in the future. When specific target analytes are known, it is important that the specimens used are “fit for purpose” or, in other words, have been demonstrated to support the integrity of the particular analyte as the biospecimen makes its way through the collection and processing pathway. When specific targets are not known at the outset of a study, general practices must be implemented in a timely manner according to information that is available in the literature.
Evidence-based protocols are defined as protocols that have been scientifically optimized for a specific result through the use of quality control measures with standards that allow for the reproducibility of the assay under defined conditions. In addition to having evidence-based protocols for specimen collection and processing, it is important for the investigator to understand that the molecular profile of a tissue or other biological material can be influenced by a variety of factors. These factors may include exposures of the patient to certain medications, food or beverages consumed prior to the collection of the biospecimen4,5 or certain environmental exposures.6 The time of day at which a specimen is collected may also influence the molecular profiles of some analytes under certain circumstances.7 Additional influences that should be considered are: the time that elapses between when the tissue is separated from the patient’s blood supply and when the tissue is removed (the ischemic time); the time that elapses between when specimens are removed from the patient and when they are processed8; and the temperature at which the specimen is kept prior to processing. The combined effects of these types of exposures or events prior to the time at which a biospecimen is used in an assay have been described as “preanalytic” variables (Figure 35-1). These factors are often some of the most difficult to capture and yet can have significant effects on research outcomes.9 The reader is directed to reviews of the burgeoning field of literature on pre-analytic variables that have been summarized by ISBER and OBBR. As much information as is possible about the condition of the patient from whom the specimen has been taken (e.g., disease status, drug profile, timing of the collection, and food exposures) should be recorded and tracked throughout the life cycle of the specimen. This information may become critical when evaluating the future suitability of the biospecimen for a particular investigation. Managing patient samples to ensure their suitability for specific tests can be achieved best when these pre-analytic variables can be traced. A system for documenting preanalytic variables was described by Betsou and colleagues10 in which a Sample PREanalytic Code (SPREC) is assigned to each biospecimen. The code is equivalent to a “specimen barcode” that provides details about pre-analytic processing for that specimen. The code is comprised of seven elements that contain information such as the material type, processing steps, and storage conditions that may be important in understanding a sample’s “fit for purpose” for a particular assay.
THE ROLE OF PRE-ANALYTIC VARIABLES IN RESEARCH USING PATIENT SPECIMENS
THE IMPORTANCE OF GOOD RECORD KEEPING
In recent years, the scientific community has come to the realization that all patient samples are not created equal.
The National Institutes of Health (NIH) has stated in its publication, “Guidelines for Scientific Record Keeping in
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FIGURE 35-1 Variables affecting the molecular integrity of biospecimens. Both the pre- and post-acquisitions variables listed here are covered under the category of “pre-analytic variables.”
the Intramural Research Program at the NIH” (http:// sourcebook.od.nih.gov/ethic-conduct/RECORDKEEPING. pdf), that “the progress and excellence of NIH research are dependent on our vigilance in maintaining the highest quality of records for every aspect of the science conducted here.” Good record keeping is a requirement of all scientific endeavors and contributes to the successful analyses of research results, as well as for the review of those results once submitted for publication. It provides the basis for which results can be reproduced by others, to establish the scientific validity of findings. Careful record keeping may contribute to the establishment of intellectual property claims or assist with the defense against allegations of research misconduct. Records should be kept in a manner that is legible and would allow others to understand and reproduce the steps taken to obtain the same findings. Records should capture: l l l
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information about the participant (name, age, appropriate health statistics); and the time and date on which the specimens were collected and all subsequent processing steps.
Records can be kept in bound or unbound notebooks, or in electronic formats such as those found associated with ordinary software or specialized software packages for the specific purpose of recording laboratory and other aspects integral to specimen collection, processing, storage, and dissemination. All records should be kept in a secure manner to ensure that access to the records is limited to authorized users and that personal health information is kept confidential. Records should be kept for a minimum of two years following the close of a protocol, but may be kept indefinitely as records may become invaluable resources for future research endeavors, and in the documentation and reporting that may be performed for the existing work at an unspecified future date.
SPECIMEN TRACKING The ability to effectively manage patient samples requires that the original specimen that is collected be tracked, as
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well as all derivatives of the original specimen generated through various handling and processing protocols. Each specimen should be given a unique identifier (ID) that can be followed throughout its life cycle. These numbers should allow for traceability to an ID linked to a particular patient, unless the linkage between the patient ID and specimen ID has been irreversibly interrupted, such that the identity of the patient has been anonymized. Derivatives of the original specimen (e.g., DNA, RNA, or protein extractions) should always be traceable to the original specimen. All specimens should have a printed label that contains the biospecimen ID that is encoded in either a onedimensional (1D) or two-dimensional (2D) scannable barcode. One-dimensional barcodes have an advantage in that they contain a high degree of redundancy, which means that the barcode can be read even with considerable degradation. However, 1D barcodes are limited in the amount of information they contain, since all of the data is encoded in the horizontal width. Two-dimensional barcodes have the advantage that they contain information that is read in a vertical direction as well as a horizontal direction and so the amount of information in the code is greater (Figure 35-2). There are advantages to including human readable information on the label (i.e., the specimen ID) to facilitate manual processing, but this may not always be necessary. Many specimen containers are now manufactured with 2D barcodes already printed on the container, which can facilitate automated specimen handling without the need or expense of adding a physical
FIGURE 35-2 The images on the top and bottom represent a one-dimensional (1D) barcode and a two-dimensional (2D) barcode, respectively. The 1D barcode is comprised of a single row of bars and the data encoded is presented across the horizontal width of the label. Data encoded in the 2D label is contained in both the vertical and horizontal directions and is able to contain a greater amount of information.
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label. Effective use of these containers without additional printed labels requires that a scanner is always available should the identity of a specimen need to be verified. Selection of the best labels for a particular study and purpose should be performed well in advance of the commencement of specimen collection. Integral to the effective management of patient samples is having an inventory system that allows for tracking the specimens as they move from collection through processing, storage, and dissemination. Computer-based systems allow for the most accurate, dependable tracking of samples. Although this may be accomplished by using simple spreadsheet programs, the best way to track specimens and characteristics of those specimens is to use an inventory system that is created specifically for this purpose. Many inventory system software programs are commercially available and can be used easily without significant customization. These programs have the ability to track personal health information, subject IDs, specimen IDs, specimen processing events, and specimen location. In addition, many programs allow for the uploading and downloading of batch records, which facilitates specimen accession, distribution, and the sharing of data under carefully defined conditions. All inventory management systems or other computerbased systems that contain personal health information must be operated under strict security practices. Regardless of whether records are electronic or paper, access to patient information and information about patient samples
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must be tightly controlled. Access to view and modify information should be allowed according to defined privilege levels. Changes or modifications to records should be traceable to the time, date, and the identity of the individual making the changes. Records should be backed up daily to a format and location that allows for their secure maintenance according to a defined management plan that includes consideration of a wide variety of emergency situations.
SPECIMEN COLLECTION The effective collection of patient samples begins with the receipt of a signed consent form from the patient (or appropriate guardian) that documents permission to use the samples under defined conditions (see Chapter 2). Consent parameters must be tracked throughout the life cycle of the sample and its derivatives to ensure that any testing performed falls within the parameters to which the patient has agreed. Study design and the use of patient samples are governed by institutional review boards (IRBs, Chapter 5). Investigators are responsible for ensuring that appropriate approvals are in place for the use of biospecimens and biospecimen-related data and should allow sufficient time for appropriate review when planning for the implementation of research activities. The actual collection of the patient samples should be well orchestrated as it may involve a number of individuals and circumstances around which careful planning must be executed in order to ensure that the highest quality specimens are available for downstream processing. In addition to the biorepository technicians, some of the personnel involved in this process on a daily basis may include: the study coordinator, admissions nurse, anesthesiologist, operating room nurse, surgeon, pathologist, and/or pathologist assistant. All individuals involved in the collection process should have the appropriate expertise and be well trained in the tasks that they are expected to perform in the collection activities. Where possible and appropriate, the success of research efforts can be enhanced by having a joint meeting(s) among those involved in the collection of patient samples to ensure a smooth transition from one phase of the collection effort to another. When multiple collections sites are involved, it is even more critical that meetings are held in advance of specimen collection in order to ensure that protocols are implemented in a uniform manner. If collections take place over an extended period of time, there also should be opportunities to resolve questions rapidly and to find solutions for unanticipated situations. In order to ensure that biospecimens for clinical trials are collected and processed correctly, it is important that detailed SOPs are provided to the personnel involved in the collection effort and that they have a clear understanding of
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what needs to be done. As a first step, a research nurse or study coordinator should contact the biorepository staff in advance to apprise them of any necessary information pertaining to the patient who has been enrolled in the clinical trial and the specific date and time that the surgery is scheduled. The SOP should outline which consent form should be used as well as the type, number, and processing steps for each biospecimen to be collected. For a typical clinical trial study, a biorepository technician should review the operating room schedule a week in advance, then again one day in advance, and finally on the day of surgery. The technician should notify the operating room nurses of the cases that are expected to be collected that day and which tissues are to be obtained so that those specimens will be handled properly. This technician is responsible for ensuring that each patient who is scheduled for surgery and who will be contributing specimens signs appropriate consent forms. Separate IRBapproved patient consent forms may need to be designed for the acquisition of waste tissue following surgery, as well as for saliva, urine, ascites fluid, and blood. In addition, the patient consent form should be translated into languages other than English, as needed, to make sure that the patient can understand the information contained therein. A translator should be present if the patient does not understand the language in which the consent form has been written. A research study that is linked to a clinical trial may involve the collection of pre-surgical specimens or ones that are collected during the surgery itself. Samples collected prior to surgery may include blood, urine, saliva or a buccal smear. Samples collected during surgery may be resected from the patient or may be collected through the use of a catheter. Prior to surgery, a biorepository technician may post signs in the operating room to remind the operating room staff that tissues from a specific patient are required for the clinical trial study. All specimens should be handled in as sterile a manner as possible. Surgeons should be requested to place specimens designated for the clinical trial study into a sterile container (provided by the biorepository) or in a sterile basin covered with sterile towels. Sterile instruments for this purpose should be maintained by the biorepository. The technician should be paged when a requested specimen has been surgically removed from a patient. The technician should then transport the specimen to the accessioning area and wait until the appropriate staff member has taken the portion of the specimen that they require for a complete diagnosis. The surgical pathologist then will give the technician the waste tissue, and sign a “specimen inventory” form indicating their approval for the biorepository to procure the waste tissue. The technician should process the tissue under the guidance of the surgical pathologist according to the specifications of the clinical trial.
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Tissues collected may be snap frozen (either on dry ice or in liquid nitrogen), frozen in OCT (Optimal Cutting Temperature), or fixed in buffered formalin and then embedded in paraffin. Sections can be cut onto slides from either the OCT-embedded tissue or the formalin-fixed paraffin-embedded blocks, and then used for pathology review. Some clinical trials may require that touch preps be made from fresh wet tissues, while in other cases fresh tissues may need to be placed in a special tissue culture media or buffered saline solution so that the cells can be tested in functional studies. Blood taken from patients for clinical trial studies may be requested as whole blood, or may need to be processed into sera or plasma. Some studies may require further processing of the blood such that white blood cells or red blood cells need to be isolated. Sometimes the availability of patient samples may be realized with short notice for the collection staff (e.g., transplant, death/autopsy material). Planning efforts should anticipate the need for trained personnel, materials and supplies (e.g., chilled specimen containers, sterile instruments, etc.) under these conditions without the need for last minute arrangements that may compromise specimen integrity. Most collections will likely be performed under more controlled conditions, but preparation for all possible scenarios will increase the likelihood of successful specimen collections.
SPECIMEN HANDLING As has been underscored throughout this chapter, handling specimens according to evidence-based protocols for a particular research design is always the best approach to take. However, it is not always possible to know exactly which assays will be performed. In those cases, specimens should be collected according to protocols that are most likely to support the effective maintenance of the biospecimen to most closely resemble its state prior to removal from the patient. Having the correct cryovials, vacuutainers, and other containers available at the collection site is critical, and those containers should support anticipated downstream uses for the samples. Agents that stabilize biological samples such as anti-coagulants may be essential for some downstream applications while having a deleterious effect on others. In addition, some supplies may carry expiration dates that set a defined period of acceptable use (e.g., vacuutainers, tissue culture media, and antibiotics). Every effort should be made to track expiration dates and make sure that supplies and reagents are used within the allowable time frame. Container selection is another important area of consideration in the management of patient samples. Containers should be composed of materials that will not interfere with the downstream assays; this is especially
Principles and Practice of Clinical Research
important if testing will include measurements of trace compounds. In addition, containers must be stable under subsequent storage environments and be of a size appropriate to the volume of the material stored. In general, vials with threaded caps do better when specimens are stored at ultra-cold temperatures (i.e., 80 C and cooler). Selecting the appropriate containers may reduce sublimation of the specimen over time and will facilitate subsequent storage and handling. Smaller tubes take up less storage volume and thus allow for efficient use of environmental storage containers (e.g., refrigerators and freezers). Even when automated specimen handling is not anticipated in the immediate life of the study, the use of automated specimen handling equipment in clinical research is on the rise. Vials and other containers that are amenable to automated handling (e.g., liquid handling equipment for aliquot preparation or plates used in immunofluorescence or other multi-plex assays) can save labor and improve the accuracy and quality of specimen handling activities down the road. Finally, investigators should be sure that containers sent to outside laboratories for testing are appropriate for the protocols utilized by those laboratories. Another area that is important to consider when processing biological specimens is the amount of the specimen that is being stored. Frequently it is necessary to divide up, or prepare aliquots of, the material collected to allow for different uses for the specimens in question, or to be able to repeat tests when necessary. Increasing the number of aliquots is likely to increase the amount of storage room needed, adding to the costs of implementing the research. Certain molecular species, such as proteins, may be irreversibly modified by exposure to cycles of freezing and thawing, whereas other species such as DNA are thought to be relatively resistant to such temperature cycles. When sensitivity to cycles of freezing and thawing is anticipated, aliquots should be made that will most likely reflect the volume of material needed for anticipated assays. In contrast, source vials containing molecular species that are not affected by freezing and warming cycles can be stored and aliquots made according to the schedule of the testing to be performed. Delaying the preparation of aliquots offers the advantage that as new testing platforms are developed, the amount of material required can be specifically removed and the specimen can be used in the most efficient manner possible.
SPECIMEN TRANSIT Whether a sample is being transferred down the hall or across the globe, there are particular considerations that should be made to ensure specimen integrity. Even for short transfers, care should be taken to ensure that the temperature and other environmental conditions needed for the stabilization of the biospecimen are met. Sometimes
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specimens must be immediately cooled by storage on frozen cold packs (approximating 4 C storage), dry ice (approximating storage at 80 C) or immersed into liquid nitrogen (196 C) to stop molecular and/or enzymatic activity. Sometimes the rate at which a biospecimen is cooled plays an important role in the subsequent viability of the cells contained therein or the structural integrity of the proteins. Other times, cooling the specimen to temperatures any lower than ambient may have a deleterious effect on some molecular components. This is where it can be advantageous if the analytes of interest can be measured as a function of different handling conditions during the specimen collection and processing before the study is initiated, to be sure that transit temperatures are appropriate. In cases where specimens are transported over periods longer than an hour, a monitoring device may be included within the shipment to allow investigators to document whether deleterious temperature excursions occurred during transit. Transport outside of an institution or over greater distances carries additional requirements for adherence not only to good practices that will ensure the stable transport of the sample, but also to national and international regulations. Transfer of specimens and/or data that is associated with a patient sample to a research entity outside of the one responsible for its collection may require the use of a Material Transfer Agreement (MTA). MTAs document the allowed uses for the samples and/or data, ensure that uses are consistent with permissions afforded in patient consent documents, and may address issues pertaining to intellectual property rights associated with the material. Depending upon institutional policies, MTAs may require approval by IRBs or other bodies that govern the use of human subjects in research. Consequently, efforts to have MTAs established and obtain all needed approvals should be initiated as early as possible in the research plan. All specimens transferred from one research institution to another must be packaged according to national and international regulations. Most couriers require that the standards created by the International Air Transportation Association (IATA) be met even when the transit may not involve transportation by air. Shippers begin by determining which regulatory requirements must be met and the physical requirements needed to ensure proper shipping. The shipper must determine how the specimens should be classified (e.g., infectious substance, Category A or Category B, patient specimen, or genetically modified organisms). In addition, the shipper must understand what preservatives may have been used that could be considered gaseous, toxic, flammable or corrosive; all of these are “dangerous goods.” Strict adherence to packaging and documentation requirements as outlined in the standards for each dangerous good being transported must be met. Shippers are required to receive training in this area before
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they perform any shipping activities. This training must include general familiarization with the regulations, on the specifics relating to the type and number of specimens being transported, and concerning safety matters relating to the preparation and implementation of the shipment. The shipper has sole responsibility for adhering to local, national, and international regulations governing the shipment of biological specimens.
SPECIMEN STORAGE Issues pertaining to specimen storage begin when a sample is received in the laboratory setting or specimen repository in which the specimen will be stored. SOPs should be written that govern the tracking of a specimen upon its arrival to document the time it arrives and its condition. Receipts (as well as shipments out of the laboratory or repository) should be recorded in a log that also records the name of the person who receives the shipment and any interim storage location used. Specimens should always arrive accompanied by a specimen manifest that documents the specimen IDs contained within the shipment. Any damage to the specimens should be documented and communicated to the shipper. Under some adverse situations, specimens may need to be replaced, and initiating this process as early as possible can minimize the effect of the occurrence on the overall implementation of the research effort. Once receipt of the specimens is recorded in the log, the specimens should be evaluated to ensure that all of the specimens in the container match what is included with the manifest. Any discrepancies should be immediately communicated to the shipper. If the samples have been labeled with barcodes, inventory of the specimens can be made rapidly using a barcode scanner. If specimen labels do not meet the specifications of the protocols for the study, appropriate labels should be generated and applied so that subsequent specimen handling can be expedited. After confirmation of the manifest has been completed, specimens should be transferred to the appropriate storage locations and the locations should be entered into the specimen inventory system. In addition to ensuring that specimens stored have proper labels, consideration should be given to the strategy used to place the specimens into their storage environments. Specimens should be stored according to a plan that reflects how they are likely to be removed at a later time for testing. Specimens are usually retrieved from storage for a particular study using a defined material type (e.g., plasma, serum, or urine). Storing specimens according to anticipated retrieval patterns will expedite the retrieval process and minimize the labor needed to retrieve specimens. When selecting the appropriate storage environment, factors to consider should include: temperature, humidity, and sensitivity to light, as appropriate. In the absence of
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data to the contrary, one may assume that the colder the storage environment, the greater the stability of the biological material. This is certainly true for the cryopreservation of cell suspensions, wherein the maintenance of viability is required. Intact cells are generally stored in liquid nitrogen vapor temperatures (<150 C) that are below what is referred to as the Tg or glass transition temperature (<135 C) where all molecular motion is believed to cease. Many biological materials are stored at what are considered “ultra-low” temperatures that range from 70 C to 80 C. In contrast, specimens that have been fixed with formalin or other fixatives may be stored at room temperature but under conditions that control for humidity. Tissue blocks that have been fixed and then embedded in paraffin may be vacuum sealed in plastic packets, then stored at 4 C in the dark.11 The storage conditions selected should reflect practices reported in the scientific literature for similar types of specimens, and for the performance of assays similar to those to be included in the planned study. Recently, new storage matrices have been developed that allow for room temperature storage of RNA and DNA. Use of these products may facilitate storage in situations where freezers or power supplies may not be available or when long-term storage is needed at a low cost. If biospecimens are to be stored in mechanical refrigerators or freezers, investigators should evaluate new products that are currently available. New designs include equipment that requires less energy and can significantly reduce facility costs for specimen storage. All equipment, whether mechanical or cooled by liquid nitrogen or other coolants, should be tested before specimens are added to ensure that the equipment performs under the manufacturer’s specifications. Emergency power (or reserves of liquid nitrogen or other coolant) should be available to allow for interruptions in power and should be maintained to allow at least 48 to 72 hours of emergency power supply. In addition, should individual units fail, some cold, empty back-up storage units should be available to hold specimens from the failing unit. In large biorepositories with dozens of storage units, a rule of thumb is to maintain approximately 10% of the total volume needed for ultra-cold and approximately 3% back-up for liquid nitrogen. All equipment in specimen repositories should be included in preventive maintenance programs to extend the timeeffective operation of the equipment for as long as possible. Monitoring systems should be implemented to alert appropriate staff on a 24 hour basis to any temperature excursions that might affect specimen integrity.
Principles and Practice of Clinical Research
implemented according to the scientific goals for the study. While custodians are generally individuals named as contacts for the biospecimen resource, decisions on use and requests to use the biospecimens for other allowable research may be made by a group of experts, and may include participation from lay persons with interests in the particular area of research. Guidelines on how specimen resources should be used need to be well defined and clearly articulated, and be processed in a timely manner. Requests should be based on sound scientific research questions and must fall within parameters established in patient consent documentation. At the time of consent, patients should be given contact information and guidance as to what steps should be taken if they later wish to have their specimens or data withdrawn from a study. Repositories should have procedures in place to identify the specimens and ensure that the specimens and any associated data are not used for subsequent research activity.
SPECIMEN CULLING, TRANSFER OF COLLECTIONS, AND REPOSITORY CLOSINGS Because of the expense of maintaining collections of patients’ samples, it should be a regular practice to make sure that the specimens stored are ones that are suitable for research. Sometimes conditions arise when the integrity of a specimen has been compromised (e.g., when its label has been lost or cannot be read or because of environmental problems such as a freezer failure). Periodic reviews of collections should be performed to make sure that the specimens stored are appropriate and are “fit for purpose” for the anticipated research. There are other situations, such as when research funds are no longer available, that collections must be destroyed or “culled” because they can no longer be maintained by the original biospecimen resource. Under these conditions, it may be possible to have the specimens transferred to another research entity that may be able to use the specimens in new research activities (which are consistent with consent documentation). All culling activities, whether by destruction or transfer to a new institution, should be well documented. Under conditions wherein specimen collections are transferred to a new institution, all requirements mentioned earlier regarding the establishment of MTAs, ensuring that other approvals are in place, and that specimen transport regulations have been met, must be considered prior to the transfer of the specimens.
SUMMARY QUESTIONS ACCESS TO PATIENT SAMPLES Use of patient samples for a particular study is under the guardianship of a custodian, who ensures that those uses are
1. What is a biorepository? a. A storage center for biological specimens b. An educational center for high school biology majors
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Management of Patient Samples and Specimens
c. A library of medical records d. A storage site for repossessed vehicles What does “fit for purpose” mean with respect to the development of scientific protocols? a. That residual specimens from prior testing efforts are used in the development of specimen handling protocols b. That the type of biospecimen used has been optimized for a particular type of analysis c. That individuals performing the testing are trained appropriately to perform the assays needed d. That multiple laboratories will perform testing simultaneously to assess consistency in the results What are “evidence-based” protocols? a. Protocols that have been developed within the past year b. Protocols that yield reproducible results c. Protocols that have been used consistently in a laboratory setting d. Protocols that have been optimized using highquality specimens and other reference standards to ensure that the procedures developed will yield scientifically significant results Which of the following would be considered “preanalytic variables”? a. Medications taken prior to specimen collection b. Time at which the specimen is collected c. The type of specimen fixative used and the time the specimen spent in the fixative d. All of the above Which of the following types of records should be maintained? a. The name of the person making the record b. The list of Standard Operating Procedures (SOPs) c. The time and date when the specimen was collected d. All of the above Which of the following statements are true regarding two-dimensional (2D) barcodes? a. They contain information stored in a vertical as well as a horizontal direction on the label b. They can contain less information than a onedimensional barcode c. They usually take up a smaller space on a label d. They may be printed on the bottom of a vial What is an MTA and what is its purpose? a. An MTA is a Management Transfer Agreement that describes what type of shipping must be used to send biospecimens to their next destination b. An MTA is a Material Transfer Agreement that it is used to document how a biospecimen may be used once it is transferred to its next destination
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c. An MTA is a Missing Tariff Analysis that is used to investigate biospecimens detained in customs during international shipments d. An MTA is a Manual Temperature Analysis that is performed when an environmental temperature chamber exceeds its allowable range of operation
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