Biospecimen Collection, Processing, Storage, Retrieval, and Dissemination
The aim of every biospecimen resource should be to collect, maintain, and disseminate the highest quality biospecimens, based on the intended research use. High-quality biospecimens are defined as those whose biology most closely resembles the biology of the biospecimen prior to its removal from the human research participant. Once the biospecimen is collected (and sometimes prior to its removal) the biospecimen may begin to take on new characteristics based on changes to the biospecimen’s environment; e.g., changes in exposure to certain nutritional, chemical, or other environmental factors that may occur during a surgical or collection procedure [4-11]. Such changes may result in inaccurate determinations of the molecular and physical characteristics of the biospecimen during subsequent analysis. Every attempt should be made to minimize the effects of biospecimen handling on biospecimen integrity.
Note that guidance provided in this section is intended for application when planning for biospecimen collection, processing, and storage, prior to the initiation of the collection efforts. Note that study design will also inform whether certain pre-analytical factors can be controlled and data collected as described below.
B.2.1. Pre-Analytic and Analytic Variables
A variety of factors may affect biospecimen quality and research results; these may be divided into two general categories designated “pre-analytic factors” and “analytic factors.” Pre-analytic factors refer to collection, processing and storage variations that influence biospecimen integrity prior to its removal from the human research participant and carry through to the point at which a biological specimen is ready for testing. Analytic factors refer to those variations that affect performance of a particular testing procedure [8, 27, 28].
The scientific study of biospecimen pre-analytic factors and their effects on molecular integrity is known as Biospecimen Science. Systematic studies in Biospecimen Science have been sponsored and conducted by programs such as the NCI’s BRN [3], and the Standardisation and improvement of generic Pre-analytical tools and procedures for In-vitro DIAgnostics project (SPIDIA) [29], a consortium that was funded by the European Union and coordinated by QIAGEN in Germany. The aim of these programs is to identify the major questions of biospecimen methodology and pre-analytic factors, conduct original research to address these questions, and develop evidence-based practices to guide new biospecimen collections and mitigate pre-analytic effects when collecting and utilizing stored biospecimens. Both the BRN and SPIDIA initiatives have resulted in significant findings which have advanced the field of biospecimen science and promoted best practices for biospecimen use in clinical and basic research programs [4-11, 30-32].
B.2.1.1. Pre-Analytic Factors
Pre-analytic variables may be divided into three general areas:
- The physiology of the human research participant prior to biospecimen collection;
- Biopecimen collection practices; and
- Biospecimen handling practices prior to their inclusion in downstream testing (Appendix 5, Example of Biospecimen Evidence-based Practice); [4-11]
B.2.1.1.1. Physiology of the Human Research Participant. Research has demonstrated that analyte levels may be affected by a variety of factors such as the overall health of the human research participant, the type of anesthesia used, food and beverages consumed prior to biospecimen collection, the medication status of the patient, and the time of day at which the biospecimen was collected [33, 34]. Additionally, the menstrual cycle in females may affect some downstream analyses. Efforts should be made to collect and record information pertaining to these factors to decrease or adjust for the variability of these contributing factors. The issue of medications is particularly important since many over-the-counter medicines may not be remembered as such by the patient (antacids, non-steroidal anti-inflammatory medications, for example).
B.2.1.1.2. Uniformity in Biospecimen Collection Practices. The methods used to remove and collect biospecimens from human research participants may influence the quality of the biospecimens collected. Significant research has indicated that during surgical removal of biospecimens the amount of time following the cessation of blood flow to an organ can affect both levels and molecular profiles of target analytes [5, 35-37]. The biospecimen should be preserved as quickly as possible after removal from the patient; e.g., appropriately sized tissue sections snap frozen and/or placed into 10 percent phosphate-buffered formalin, as appropriate. In recent years alternative fixatives have been developed and validated, such as PaxGene Tissue®, which allows for the preservation of tissue for molecular analysis as well as histological analysis [38]. As appropriate, consideration should be given to the use of newer preservation methods. Expected as well as unforeseen future uses of biospecimens should be considered when deciding on preservation methods. When biospecimens are collected from research participants, the organ site at which the biospecimen is removed (tumor or non-tumor, as well as location within the tumor), any anesthetic being used, warm ischemia time (the length of time the specimen is only partially perfused due to vessel ligation during surgery, before complete removal), any stabilizing agents used to preserve the biospecimen following its removal, the type of fixatives used and the length of time the tissues are exposed to fixatives, any further processing, and the temperature at which biospecimens are maintained following collection (as well as duration i.e. cold ischemia time) may all affect molecular stability and degradation. The NCI maintains a publicly available, online database that collates the published literature on these and other pre-analytical factors: the Biospecimen Research Database [14].
Prior to the collection or removal of biospecimens, a plan should be in place to allow for the appropriate annotation of the biospecimens. This annotation should include information about the human research participant as noted in Section B.2.1.1.1 above, and timing of collection and processing activities; e.g., the type of clearing agent, the type and temperature of paraffin used to process the biospecimen, etc. [15]. These data should be maintained in a database that can be linked to the biospecimen at all times (see Section B.5, Collecting and Managing Clinical Data, and Section B.6, Biospecimen Resource Informatics). Details about particular biospecimen collection, processing, and storage procedures can be important supporting information for scientific publications on research utilizing the biospecimens [39]. NCI, with an international committee, published a set of recommendations for reporting the conditions of biospecimen collection, processing, and storage procedures. These recommendations, known as BRISQ (Biospecimen Reporting for Improved Study Quality), have been adopted by several scientific journals and are mentioned in the Nature guidelines for authors [39-42].
B.2.1.1.3. Biospecimen Handling Procedures. Every attempt should be made to optimize the handling of biospecimens to minimize molecular changes that may result from the processing activities, most critically by reducing cold ischemia time (the time the biospecimen spends after complete removal from the patient but before being placed into fixative). Other critical factors include the temperature and timing of biospecimen processing, the size and volume of the biospecimens that will be stored for future use, and the number of aliquots to be prepared from each biospecimen. Multiple small aliquots allow for analysis or experimentation on one specimen, while minimizing freeze-thaw degradation of other stored samples ([43] CAP Biorepository Accreditation Program Requirement (Appendix 6, BAP.01900). When samples are stored in a frozen state, the rate at which they are cooled to the storage temperature can influence the rate at which molecular degradation occurs, and subsequent freeze-thaw cycles can further degrade the molecular integrity of the biospecimens. Tracking of temperature excursions is recommended, and required by accrediting organizations ([43] BAP.02000).
B.2.1.2. Analytic Factors
When pre-analytical variables are introduced they lead to differences in the performance of a particular assay. To minimize errors in assay reproducibility, the following considerations should be applied:
- Use of validated assays, where possible;
- Use of SOPs in which the technical staff are well-trained;
- Lot uniformity of reagents;
- Inclusion of appropriate type and number of quality control (reference) samples;
- Randomization, when possible; and
- Standardized methods for documenting and interpreting testing results.
B.2.2. Determining Which Biospecimens to Collect
The specific mission and goals of a biospecimen resource will influence the type of biospecimens collected. The biospecimens collected should be appropriate and feasible for the clinical setting, as well as appropriate for the downstream applications anticipated for the biospecimen. If tissue specimens are being collected, they should be reviewed histologically for accuracy by a qualified histopathologist; this is a requirement for some accrediting agencies ([43] BAP.02500).
B.2.3. Defining Reference Ranges
Aside from pre- and analytic factors, research dictates that values for particular cellular analytes are more accurately represented by a normal biological range of values (or reference range), even among individuals characterized as “normal” or “healthy.” Disease is defined as a distinct deviation from the range of normal variation, and diagnosis of disease depends on knowing the scope of boundaries of normal variation. Where possible, efforts should be made to characterize reference ranges for the analyte of interest in the biospecimen of interest to ensure the likelihood of accurately detecting any deviation from the reference range.
B.2.4. Requirement for Evidence-Based Standard Operating Procedures
To have confidence in research results, it is critical that all reagents be fit-for-purpose and quality-controlled for use in the assay. SOPs should be reproducible with standard reference material (where possible), and control biospecimens that provide a range of anticipated assay values should be utilized; this is a requirement for accrediting organizations ([43] BAP.01000, BAP.01500). Biospecimens that have been poorly handled are likely to provide erroneous test results because of the molecular changes resulting from the handling process.
A model for constructing and annotating biospecimen evidence-based practices has been published by the NCI [12] and is publicly available at the BBRB web site [44].
It is impractical and currently not possible to consider the development of assays to measure the stability of every cellular component within a biospecimen. To that effect, protocols that optimize the general stability of biomolecules under certain environmental conditions are recommended [15]. Should a particular biomolecule be of interest, it is important to perform some type of analysis to ensure that the storage and handling conditions implemented will allow for accurate determinations of that biomolecule.
B.2.5. Methods Research
All research endeavors should be based on well-characterized and validated assays, where possible [45]. Even assays that are developmental in nature should be tested to ensure that they are reproducible over time. “Proof of Performance” tests [15] allow for testing replicate samples over time to allow for measurement of standard deviations in the assays performed.
Where possible, research should be performed to ensure that the storage and handling procedures implemented are ones that will be conducive to stabilization of the molecular components and particular targets of interest within the biospecimen.
B.2.6. Biospecimen Storage
The following general best practices apply to all types of biospecimens, such as wet tissue, frozen tissue, paraffin-embedded tissue, glass slides, blood, serum, and urine. Individual types of biospecimens should be handled according to SOPs specific to the biospecimen type and the biomolecules to be analyzed — e.g., ribonucleic acid (RNA), deoxyribonucleic acid (DNA), protein, and lipid — when possible, recognizing that collection in the context of a clinical trial might be constrained by trial-specific protocols. Although most of the practices in this section assume freezing or formalin fixation of samples, dry or ambient temperature storage procedures may be appropriate for many samples [38, 46].
B.2.6.1.
Standardized protocols should be applied consistently in storing biospecimens to ensure quality and to avoid introducing variables into research studies. Biospecimen resource personnel should record storage conditions along with any deviations from SOPs, including information about temperature, thaw/refreeze episodes, and equipment failures [15]. Validation of storage equipment, for example, identifying “hot spots” in a freezer and ensuring that back-up equipment and temperature monitoring systems are functional, is essential.
B.2.6.2.
Biospecimens should be stored in a stabilized state. As discussed in previous sections, unnecessary thawing and refreezing of frozen biospecimens or frozen samples of biomolecules extracted from the biospecimens should be avoided, and appropriate size for aliquots and samples should be determined in advance to avoid thawing and refreezing of biospecimens. When thawing/refreezing is necessary, a biospecimen resource should follow consistent and validated protocols to ensure continued stability of the analytes of interest. Methods such as inventory tracking should be established to minimize disruption of the stable environment during sample retrieval; this is a requirement for accrediting agencies ([43, 47] BAP.02900).
In selecting biospecimen storage temperature, consideration should be given to the biospecimen type, the anticipated length of storage, the biomolecules of interest, and whether study goals include preserving viable cells [15, 48]; these are required by accrediting agencies ([43] BAP.04200, 04300). Paraffin blocks should be stored at temperatures below 80 °F (27 °C) in an area with pest and humidity control. Blocks stored in areas above these ‘room temperatures,’ duration of fixation, and most importantly humidity fluctuations have been shown to seriously compromise the expression of certain antigens when evaluated by immunohistochemistry [49].
In the case of liquid biospecimens, such as blood, consideration should be given to produce components such as plasma or serum, which should be separated before storage to preserve each constituent under its optimal condition. Whole blood (rather than fractionated blood) cryopreservation may be an efficient and cost-effective option for processing viable cells in large-scale studies [48, 50]. When in doubt as to possible future e uses, tissues should be stored in the vapor phase of liquid nitrogen freezers or frozen at -80 °C to ensure long-term viability. Lower storage temperatures and cryoprotectant (such as dimethyl sulfoxide) may be used to maintain viable cells for long periods of time [15]. The difference in temperature between the bottom and top of a liquid nitrogen freezer should be measured and taken into consideration in planned analyses; the temperature at the top of a liquid nitrogen freezer is consistently below -140 °C. Regular temperature mapping of the interior of freezers is recommended to insure uniform temperature through the storage unit, and is required by accrediting agencies ([43] BAP.08100).
B.2.6.3.
Storage vessels should be stable under planned storage conditions [15, 51]. Biospecimen containers should be chosen with analytical goals in mind and evaluated prior to use to ensure that contamination or chemical leaching into the biospecimen does not occur. Vial size and number should be suitable for typical aliquots and anticipated investigator uses. Optimal volume and type of containers may prevent sample loss and minimize the costs of collection, storage, and retrieval. Screw-cap cryovials may be used for long-term, low-temperature storage; glass vials or vials with popup tops are unsuitable for long-term storage [15]. Snap-frozen biospecimens should be wrapped in aluminum foil or placed in commercial storage containers to minimize desiccation [51]. Labeling and printing systems should be chosen for stability under the long-term storage conditions appropriate for the biospecimen. Face shields and appropriate gloves should be worn for worker protection (see Section B.4, Biosafety).
For optimal preservation, formalin-fixed, paraffin-embedded tissue should be stored as a block and not sliced until analysis is imminent because degradation will occur under even the best storage conditions (for a review see [52]). However, when slide-mounted cut sections must be stored prior to analysis several steps may be taken to minimize degradation, including thorough dehydration and processing prior to storage, and storing the slides frozen and protected from exposure to moisture [52]. Optimal storage conditions might vary according to the final use to which stored sections will be put ([52], and empirically determining the optimal storage conditions is recommended.
B.2.6.4.
Each biospecimen should have a unique identifier or combination of identifiers that are firmly affixed to the container, clearly and legibly marked, and able to endure storage conditions. All other relevant information should be tied to this identifier, bearing in mind research participant confidentiality, security, and informed consent provisions. Inventory systems should relate the presence of each aliquot to its position in a specific box, freezer, refrigerator, or shelf. Consideration should be given to the location of biospecimens within storage containers to allow for the most efficient strategies for subsequent retrieval; i.e., by study and by material type within studies, as appropriate. Additional information related to biospecimen resource informatics best practices can be found in Section B.6, Biospecimen Resource Informatics. A well-defined tracking system is a requirement by accrediting agencies ([43] BAP.02800, 02900).
B.2.6.5.
Automated security alarm systems should be in place to continuously monitor the function of storage equipment and should have the capability to warn resource personnel when equipment failure has occurred. Backup equipment, such as an alternative power source, should be set to activate automatically when necessary and should be tested regularly. Alternate cooling sources also might be needed in some cases. Written SOPs that are tested on a routine basis should be in place to respond to freezer failures, weather emergencies, and other disaster recovery/emergency situations ([15, 43] BAP.09200, 09300, 09400).
B.2.6.6.
Specimens should be stored in a secure location with limited access only by authorized personnel.
B.2.7. Biospecimen Retrieval
Samples should be retrieved from storage according to biospecimen resource SOPs that safeguard sample quality.
B.2.8. Shipping Samples
B.2.8.1. Shipping Conditions
B.2.8.1.1. When seeking to regulate sample temperature during shipping, the shipping time, distance, climate, season, method of transportation, and regulations as well as the type of samples and their intended use should be considered [15, 53]. To maintain proper temperature during shipping, appropriate insulation, gel packs, dry ice, or liquid nitrogen (dry shipper) should be used and these materials should be qualified for their intended use. To maintain refrigerated temperatures (2° C to 8° C), gel packs conditioned at -15° C or phase-change material rated for refrigerated transport may be used. To maintain frozen temperatures, gel packs conditioned at or below -20° C should be used. For frozen temperatures at -70° C, dry ice pellets or sheets should be used; dry ice is considered a hazardous substance for shipping purposes. For maintaining temperatures at or below -150° C, a liquid nitrogen dry shipper should be used [15]. Insulated packaging may be used to protect biospecimens from extremely hot or cold ambient conditions. Whenever intending to maintain samples below ambient temperature, enough refrigerant should be included to allow for at least a 24-hour delay in transport[15]. Temperature-sensitive material should be handled by a courier with resources to replenish the refrigerant in case of a shipping delay [15]. A simple colorimetric or other constant temperature-measuring device should be included with biospecimen shipments to indicate the minimum and/or maximum temperature within the shipping container.
B.2.8.1.2. Paraffin blocks and slides may be shipped at room temperature in an insulated package via overnight carrier. The use of insulated packages is considered important to minimize the effect of temperature fluctuations and to protect the blocks from temperatures higher than 27° C. There is convincing research that tissues stored in FFPE blocks may rapidly lose antigen expression for certain immunomarkers even when maintained in hospital storage areas at ‘room temperature’, which may fluctuate widely in non-temperature controlled areas [49]. Flat biospecimens, such as dried blood samples on absorbent pads or cards, may be enclosed in watertight plastic bags and shipped in a sturdy outer package or commercial envelope. Samples on glass or plastic slides should be cushioned and shipped inside a sturdy (not flexible) outer package. Inclusion of a simple maximum temperature indicator in each package and documentation of the maximum temperature upon receipt are recommended.
B.2.8.1.3. The number of biospecimens per package also affects whether the appropriate temperature can be maintained for all biospecimens in the shipment. A test shipment (e.g., frozen water samples) should be made before shipping extremely valuable samples to check the adequacy of coolants and any potential obstacles to a successful shipment. In addition, conditions throughout a critical shipment should be monitored by enclosing a device that records temperature during transport.
B.2.8.2. Shipping Documentation
B.2.8.2.1. Upon planned shipment of a package, documentation of the transfer in the form of a material transfer agreement (MTA) and requisition from the resource inventory is needed. An MTA or similar agreement (Appendix 4) governs the transfer of research materials and any associated data between two organizations. The MTA governs the rights and obligations of the provider and recipient with respect to the materials, and it should be consistent with all applicable laws, regulations, policies, and terms for transfer of those particular materials. The MTA also governs any timelines, commercialization, or third-party transfer of the materials and data ([15], Section M2.600).
B.2.8.2.2. Biospecimens should be shipped from an attended shipping facility or picked up for shipping by an appropriately authorized person. The biospecimen resource should notify the recipient before shipping to confirm that someone will be present to accept the package and properly store the samples. Shipments from and to the biospecimen resource should be tracked in a written or computerized shipping log [15], which should include shipment/invoice number, recipient (or source), date shipped (or received), courier name and package tracking number, sample description, number of samples shipped (or received), condition on arrival, study name and number (if available), key investigator’s name, and signature of biospecimen recipient [15].
Standardized paperwork should accompany shipments. Biospecimen resource personnel should electronically send a shipping manifest, a list of sample identification numbers, and descriptions of samples to the biospecimen recipient and should include a hard copy of the manifest inside the shipment. Identifying data should be available for the use of shipping or customs agents as well; some shipping agents require an itemized list of contents between the inner and outer packaging of diagnostic biospecimens.
Upon receipt, biospecimen resource personnel should verify biospecimen labels and any other documents or data shipped with the biospecimens against the packing list for consistency and correctness. A questionnaire requesting feedback about the quality of samples received may be enclosed in each shipment for quality management purposes [15].
In general the concept and procedures for chain of custody should be applied when shipping biospecimens ([15], Section G, Records Management).
B.2.8.3. Regulatory Considerations
B.2.8.3.1. All applicable laws and regulations for shipment should be satisfied. For example, ISBER Best Practices and International Air Transport Association (IATA) regulations [15, 53] should be consulted for information concerning international transport regulations and classifying samples for shipment. Variation in national and regional standards regarding biospecimen transport should be considered when shipping biospecimens to or from an international location.
B.2.8.3.2. Additionally, Occupational Safety and Health Administration (OSHA) regulations on toxic and hazardous substances [54] (29 CFR 1910 Subpart Z) should be consulted to determine whether a substance requires a biohazard label. Additional safety considerations are enumerated in Section B.4, Biosafety.
B.2.8.4. Training
Biospecimen resource personnel should be trained to ship samples appropriately. Periodic retraining according to governing regulations should be conducted and documented [15].