3.1.1.1. Governance organization

The biobank should have a structure of committees and appropriately qualified personnel in relevant roles to oversee its governance. The size, type, and number of committees and their composition will vary depending on the size and purpose of the biobank. Careful consideration should be given when participants, patient groups, or public representatives are asked to serve on biobank committees. Their roles on the committee should be clearly communicated, and training should be provided. The following types of committee may be considered (Fig. 1).

Fig. 1

Sample committee structure for internal biobank governance.

3.1.1.2. Documentation: plans, policies, and procedures

Documentation requirements include plans, policies, and specific SOPs. The documents should be compatible with international standards such as the ISO standards for biobanking and the CEN norms that articulate how activities of the biobank are to be performed (see Section 3.4 for more details about ISO and CEN). Some general principles in relation to these documents should be followed:

•

Documents should be developed in the context of a QMS, which includes document version control.

•

Documents should be developed in the context of an up-to-date risk assessment undertaken alongside a procedure that takes into account risks to the health and safety of people.

•

Documents should include a time frame for review and revision; 2 years is a recommended time frame (NCI, 2016).

•

To facilitate cooperation between biobanks, documents should adhere to internationally accepted technological standards and norms, ensuring that these are clearly referenced (OECD, 2009).

•

High-level policies, including data and sample access policies and terms of reference of committees, should be publicly and freely available, for example on a website.

•

Biobanks should consider implementing monitoring strategies with scheduled audits to ensure that policies and procedures are followed.

•

Researchers should submit reports annually and at the end of their projects, including information on publications and patent applications (OECD, 2009).

•

The biobank should maintain a system for reporting adverse events, anomalies, and non-compliance with the QMS; this reporting system supports corrective and preventive actions and enables any relevant documents to be updated (CCB, 2014).

A critical document is the biobank programme document (or biobank protocol), which contains information about the scientific rationale, scope, design, and strategy for the biobank. Other biobank plans, policies, and procedures should be developed in line with this protocol. The biobank’s mission should be clearly outlined in terms of its purpose, the types of research or other users supported (scope), and the types of samples and data collected. Additional considerations include which services are provided (e.g. specific research assays, storage of samples) and whether legacy samples can be incorporated into the biobank. This protocol and associated key biobank documents should be approved by a research ethics committee, and renewed approval should be required if the documents are amended (OECD, 2009).

An annually updated business and continuity plan or model is essential, especially if the biobank is planning to charge for use of the resources (Vaught et al., 2011). The business plan should include a strategy for both medium-term and long-term sustainability. A budgeting or costing exercise will assist in the development of such a plan.

All biobanks should also develop a quality management policy and a policy on access to samples and data from the biobank (see Annex 1).

Additional policies to consider include:

•

a governance policy, containing information about the biobank’s governance structure and the responsibilities of management (OECD, 2009);

•

a retention policy, covering biospecimen availability and whether collections can be shared or destroyed;

•

a policy on storage options;

•

a safety policy for staff and visitors;

•

a policy on transportation of material;

•

a policy on disposal of material and biosafety and biosecurity;

•

policies covering ethical issues, including information on the protection of the confidentiality and privacy of participants (see Section 3.1.3), informed consent, return of results and incidental findings, and so on;

•

a policy on the intellectual property generated from the use of the resources and research results;

•

a publication policy, governing publications arising from the use of the biobank; and

•

policies on how the termination of the biobank would be handled.

Guidelines recommend that accompanying SOPs should be put in place to govern all biobank activities: recruitment; consent; staff training; biosafety; the collection, receipt, processing, and storage of samples; sample QC, laboratory QA; participant de-identification; data collection, recording, storage, and management; data protection; the monitoring, calibration, maintenance, backup, and repair of equipment; the procurement and monitoring of supplies (disposables and reagents); the distribution and tracking of samples; records and documentation; reporting of non-conformity and complaints; and disaster management.

All staff members should be trained in the procedures at the biobank, and this should be documented.

Box

Key points: biobank governance.

3.1.2.1. Types of consent

Many biobanks use a broad consent, which allows patients or research participants to consent to a broad range of uses of their data and samples. Although broad consent allows for a broad range of research activities, it is regarded by research ethics committees as specific enough to be considered “informed”, because guidance is provided on the nature of the future undetermined research uses (e.g. research on breast cancer and associated conditions). It is important to note that broad consent forms usually contain a series of statements specific to the biobank, and not statements related to specific research projects. Table 2 outlines the different types of consent associated with biobanks and sample collections, together with key points about each approach and notes on information to be provided to participants.

Table 2

Types of consent and key considerations.

Further guidance on designing and implementing a broad informed consent is provided in Annex 2.

3.1.2.2. What information to provide to potential participants

Information about the biobank and biobanking activities is usually provided to potential participants using a participant information sheet in conjunction with a consent form (in some cases, these two documents together are called the informed consent form). The information provided will vary depending on the nature of the biobank and the type of consent requested. Further details are provided in Table 2 and Annex 2.

3.1.2.3. Potentially ethically or legally challenging issues

The following potential uses of samples and data are examples of issues that may be considered ethically or legally challenging:

•

transfer of samples or data across national borders; it is important to be aware of regional and national regulations, such as the EU Data Protection Directive of 1995 (Article 26) (European Commission, 1995) and the EU-U.S. Privacy Shield adopted by the EU Commission on 12 July 2016 (http://ec​.europa.eu/justice​/data-protection​/international-transfers​/eu-us-privacy-shield/index_en​.htm);

•

use of samples in experiments involving animals;

•

creation of cell lines from the samples, including stem cell lines;

•

use of samples and data by commercial researchers;

•

research linked to reproduction, including use of embryos;

•

return of individual research results and incidental findings (this is an important topic that warrants extensive discussion, which is beyond the scope of this document); and

•

research into high-penetrance genes linked to disease.

When information sheets and consent forms are being developed, the biobank should consider whether it may be involved in any potentially challenging uses and include information about these in the information sheet and/or the consent form as appropriate, in addition to the core elements usually included as part of a consent form. The consent form should provide the participant with a means to opt out of uses that the participant feels are ethically questionable. The consent form may also require specific opt-in provisions if they are legally required by national or regional laws; an example is transfer of data outside of Europe, according to the EU Data Protection Directive (European Commission, 1995). A means to opt out of certain uses should be provided by the biobank only if this is recordable (i.e. in a database), actionable (i.e. when distributing the samples for research), and practicable (e.g. given the number of participants or the number of samples distributed).

3.1.2.4. What to consider during the process of requesting consent

It is important to consider the following aspects during the consent process.

•

Where possible, the information sheet should be distributed ahead of the meeting with the potential participant.

•

The potential participant must be given adequate time to read and consider the information sheet and should be offered the option to decide and give consent at a later visit if required.

•

The person administering the consent process must be convinced of the capacity of the potential participant to give consent. If they are not convinced, Section 3.1.2.6 (on participants without the legal capacity to consent) may be applicable. Alternatively, the potential participant may require assistance in reading the form.

•

The person requesting consent should not coerce the potential participant in any way.

•

The person requesting consent should encourage open discussion and give the potential participant the opportunity to ask questions.

•

The person requesting consent should ensure that the potential participant is informed about their right to withdraw consent, about the risks and benefits of participating in the project, and about any other important issues.

3.1.2.5. What to consider if the country or the research area would benefit from community engagement in relation to the consent process

The consent process needs to be appropriate for the local cultural context, and in some cases this means that wider community engagement is appropriate (H3Africa, 2013). In cases where wider community engagement is needed, consultation on the biobank’s consent processes and documents should take place with the wider community, local leaders, and professionals. The requirement for community involvement in the consent process may also be specified in local ethical or legal guidance. In some of these cases, prior consent, assent, or permission may need to be obtained from community, tribal, or family leaders (Nuffield Council on Bioethics, 2002). In all cases, the potential research participant themselves must be approached for consent and must have the right to refuse participation.

3.1.2.6. What to do if the potential participants do not have the legal capacity to consent for themselves

This category of participant is often called “vulnerable people”, and careful consideration must be given to how recruitment will be conducted (WMA, 2013). Recruitment of “vulnerable” participants must respect the requirements in the Declaration of Helsinki that all vulnerable groups and individuals should receive specifically considered protection, that the research is responsive to the health needs of this group and cannot be carried out in a non-vulnerable group, and that this group will benefit from the research.

Three types of participant commonly identified as vulnerable are:

•

mentally incapacitated adults,

•

adults in emergency care situations, and

•

minors/children.

In the case of mentally incapacitated adults, a legally authorized representative can provide consent on their behalf. If the participant made legally approved provisions about research participation before they were incapacitated or if they assigned a legal representative, these should be respected. The participant should be involved as much as possible in the decision to participate, and any resistance or objections should be respected.

In the case of research taking place in an emergency clinical situation where consent has not been obtained, requirements will differ by country and may include:

•

the local ethics committee explicitly approving the recruitment pathway;

•

another medical professional authorizing the involvement of the participant;

•

the known wishes or objections of the participant being respected; and

•

a maximum time limit being imposed for participant involvement without consent.

Consent should be requested from the participant if the participant regains legal capacity.

In the case of research involving children, they should take part in the consent process in accordance with their age and maturity, and assent to participate (rather than consent). Information and consent materials can be designed for different age groups to aid understanding of the research. Any objection from the child should be respected (Hens et al., 2011). The assent process is based on the age and maturity of the child and on any applicable local laws or ethical guidance on the matter. In addition to the assent of the child, the child’s parent(s) or an appropriate legal representative must provide consent on the child’s behalf. It is also good practice to re-contact child participants once they reach the local legal age of maturity, to request consent, if possible (CIOMS, 2002; Hens et al., 2011).

3.1.2.7. Considerations when including samples or data from deceased participants in the biobank

Consent requirements will vary depending on whether the biobank intends to request consent for samples and data from potential participants before their death (for example, for a brain biobank) or request the samples and data after the participants’ death. Local legislation will dictate the applicable consent and legal requirements. Some legal or practical constraints may exist for biobanks accessing medical records after the participants’ death. Uses of the samples and data, collected before or after death, that fall outside the scope of the original consent will require approval by a research ethics committee (Tassé, 2011).

Where samples are to be collected for research after death, local institutional, ethical, and legal guidelines to obtain consent must be followed, and the individual’s wishes expressed before death, if they are known, should be respected. The consent procedure may be built into existing procedures for postmortems or for clinical use of postmortem samples for organ or tissue transplants, which may differ markedly from country to country.

3.1.2.8. The continuing nature of consent

If the participant has given prior written consent, they should always be asked to confirm (verbally and/or tacitly, as appropriate) their agreement to donate samples to the biobank, and they should always have the opportunity to ask questions, before additional sampling (e.g. blood, biopsy, aspirate, bronchial brushings) or data collection is performed. Where possible, the verbal consent should be recorded electronically or noted and stored with the original consent form. The participant may decline to provide further samples or data at any time. This does not invalidate the consent to use any previous samples or data given to the biobank, unless notice of withdrawal of consent is given.

3.1.2.9. When participants might need to be re-contacted to request new or updated consent

Several situations may arise where the biobank may need to re-contact the participants to request new or updated consent, apart from contact to request additional data and/or samples for the purposes of the same project. These situations may include children reaching the age of maturity and temporarily incapacitated participants regaining legal capacity (see Section 3.1.2.6), and in such cases full informed consent should be requested (Burke and Diekema, 2006). Another situation is when the information provided in the initial consent form and information sheets is modified or updated (e.g. if the scope of the biobank changes). In this case, re-contact of participants to request an updated consent may be required, given the changing conditions of their participation (Wallace et al., 2016). An appropriate research ethics committee should decide whether re-consent is required or whether a waiver can be applied.

A general guideline is that before re-contact is established, a participant’s options with respect to re-contact should be checked, because participants should be given the option not to be re-contacted (see Annex 3).

3.1.2.10. How to approach withdrawal of consent

At any time, participants can withdraw consent for the biobanking and use of their samples or data without giving a reason. It is crucial for the biobank to present withdrawal options to participants in the consent form or information sheets. This may not mean guaranteeing to destroy all samples and data; for example, the withdrawal options may stipulate that samples and data already released or used in analyses are not retrievable. Examples of withdrawal options include the following.

•

“No further use” option: the biobank will destroy all samples and data from the participant and will not contact the participant again.

•

“No further contact” option: the biobank will no longer contact the participant directly by any means but can continue to use samples and data already collected and can continue to access the participant’s medical records if necessary.

•

“No further access” option: the biobank will not contact the participant or access the participant’s medical records but can continue to use samples and data already collected.

The biobank should also clearly communicate to participants when it is impossible to destroy parts of the samples or data. Examples include being unable to destroy:

•

samples and data already distributed for research or used in analyses, and

•

data needed for audit purposes or already archived.

Box

Key points: informed consent.

3.1.4.1. Generalized, non-individual study results

According to best practices, research results can be published on the biobank website or via a newsletter (CCB, 2014). These are summary results of research using samples and/or data from the biobank and cannot be connected back to a specific individual.

The participant should be given the option during the consent process to receive these publications, and researchers using samples and data should commit in the MTA to providing the report of research results to the biobank.

3.1.4.2. Individual results from tests conducted during sign-up or registration

Some biobank studies request participants, after they consent, to undergo initial testing (baseline assessments), such as blood pressure measurements, lung function tests, and vision tests. In addition, biological samples may be routinely tested for infectious diseases, such as HIV, hepatitis B, and hepatitis C, before storage in the biobank. Biobanks should consider whether they will return the results of these tests, and this should be clearly indicated in the participant consent materials. These tests should not be used as incentives for the participants to sign up, and it should be made clear that they are not part of health checks.

3.1.4.3. Individual research results

Individual research results fall into two categories.

•

Results that can be anticipated because they are in line with the aims of the research. The method for handling these can be considered during the evaluation of the sample request.

•

Incidental findings that are not linked to the aims of the research. The method for returning these to the biobank can be addressed in the MTA.

In both cases, the biobank will need procedures for evaluating the validity of such results, as well as the period of validity, and for returning these results to the participants, including re-identification and re-contact of the participant if the participant has indicated in the consent form that they wish to be re-contacted (UK Biobank Ethics and Governance Council, 2015). The scope of the results to be returned and how they are to be returned should be defined in advance with relevant experts and the ethics committee (Thorogood et al., 2014), taking into account that biobank participants have the right to choose not to know research results. The ideal time to ask about an individual’s preferences is during the consent process. The information sheet and consent form should provide potential study participants with information and give them the opportunity to choose whether they wish to receive individual research results. The protocol must provide a way for participants to later change their preferences.

3.1.4.4. Results of genetic tests and next-generation sequencing

Returning the results of genetic tests or next-generation sequencing should involve offering a clinical-grade validation and making available clinical expertise or genetic counselling. Recommendations about which genetic test results to return are provided, for example, in the American College of Medical Genetics and Genomics (ACMG) recommendations for reporting of incidental findings in clinical exome and genome sequencing (Green et al., 2013) and in the Genomics England project (https://www.genomicsengland.co.uk/taking-part/results/).

3.1.4.5. Results of imaging studies and scans

Imaging biobanks are defined by the European Society of Radiology as “organised databases of medical images and associated imaging biomarkers (radiology and beyond) shared among multiple researchers, and linked to other biorepositories” (European Society of Radiology, 2015). Specialized facilities and biobanks that also conduct imaging studies such as scans are particularly likely to discover incidental findings. The Royal College of Radiologists provides guidance on the management of incidental findings detected during research imaging (RCR, 2011).

3.1.4.6. Results about children

If the biobank includes biological samples from children, the biobank needs to evaluate whether it has a stronger responsibility to return results in this case because the participants are children (Hens et al., 2011). It must also consider what action to take if the results become available after the child comes of age, because the original consent was not the child’s, and whether the parents have the right to receive all information about the child (see Section 3.1.2.6).

Box

Key points: return of results.

3.1.5.1. Access to stored materials and data for research purposes

The biobank should develop an access policy and access procedures, in line with its protocol (Section 3.1.1). The policy and procedures should describe the administrative and approvals process for applying for and obtaining access to samples or data, comprising an overview of applicable restrictions and obligations. A procedure should exist to ensure that the applicants are bona fide researchers, and the policy should be publicly available (CCB, 2014). See Annex 1 for the IARC access policy; other examples include those of the National Cancer Research Institute (NCRI, 2009) and P3G (Harris et al., 2012).

Evaluation of requests should be based on the notion of proportionality, balancing risks against benefits, and ensuring that the intended use follows the biobank’s protocol and priorities and the consent provided by the participants (Mallette et al., 2013). In general, samples should be shared in a fair, transparent, and equitable manner (Chen and Pang, 2015). In the case of scarce samples, a decision could be made to provide samples to projects more closely aligned with the aims and strategy of the biobank. The researcher should sign an MTA/DTA (see Section 3.1.5.5), which will include the obligations of the researcher, before receiving the samples and/or data.

3.1.5.2. Principles for international specimen exchanges

The legal aspects of sample sharing vary between countries, and an assessment should be made to ensure that the relevant legal regimes are compatible with those of the biobank and the consent. Where applicable, the participant should also have given consent for the transfer of data between countries. Examples of legal requirements are the EU-U.S. Privacy Shield principles, which deal specifically with transfer of data from Europe to the USA (see Section 3.1.2.3), and the new EU General Data Protection Regulation (see Section 3.1.3).

Finally, if there are any doubts in relation to privacy implications when samples or data are to be transferred internationally, a data privacy impact assessment can be performed before such transfer (GA4GH, 2015b). For further information on the legal requirements related to international sample sharing, researchers can use the Human Sample Exchange Regulation Navigator (hSERN) tool, available at http://www.hsern.eu/.

Special attention should be given to the transfer of samples to or from countries with regulatory frameworks that are poor or absent (Chen and Pang, 2015).

3.1.5.3. Collaboration with the private sector

Collaboration with the private sector must adhere to the same requirements and obligations with respect to data and sample sharing. It is important for the possibility of sharing samples and data with the private sector to be specifically mentioned in the informed consent and information sheet (European Commission, 2012a).

3.1.5.4. Intellectual property and ownership

Intellectual property policies vary across institutions, but the biobank should define an intellectual property policy. Aspects of this policy should be defined in the MTA/DTA (see Section 3.1.5.5), as well as ownership of biological samples.

Box

Key points: data and sample sharing.

3.1.5.5. Material Transfer Agreement (MTA)/Data Transfer Agreement (DTA)

An MTA, a DTA, or a similar agreement should be put in place before the transfer of samples and/or data between organizations (ISBER, 2012). An MTA/DTA is a legally binding document that governs the conditions under which the samples and/or data can be used (see Annex 4).

The MTA/DTA outlines the type of samples and/or data to be transferred, the purpose of the transfer, and all restrictions or obligations that relate to the use of the samples and data (NCI, 2011; ISBER, 2012; NCI, 2016). These restrictions and obligations must be in line with the conditions of the informed consent, ethics approval, and biobank governance attached to the samples and/or data. The agreement may include a statement that the samples and data have received appropriate ethics approval and consent.

The agreements should include specific aspects relating to the biobank’s policies and provisions that bind the researcher to:

•

use the samples and data in line with the biobank access approval given;

•

adhere to applicable laws, regulations, and guidance;

•

not further distribute the samples or data;

•

dispose of, or return, the samples and data after use;

•

guarantee confidentiality and data protection;

•

not attempt to re-identify participants;

•

inform the biobank of any issues with the data or samples;

•

provide traceability of samples;

•

return research results in the form of individual results, raw data, an interim/final report, relevant publications, or patent applications;

•

cite or acknowledge the biobank in publications, patents, or other documents, or include a citation in any published work to a specific publication describing the biobank; and

•

respect intellectual property terms.

An example of an MTA is provided in Annex 4. Other examples include those of Knoppers et al. (2013) (online supplementary material), NCI (NCI, 2016), the National Cancer Research Institute (NCRI, 2009), the Association of Research Managers and Administrators (ARMA, 2016), and Belgian Co-ordinated Collections of Micro-organisms (BCCM, 2016).

3.3.1.1. Cryopreservation

Cryopreservation is the recommended standard for preservation of human biological samples for a wide range of research applications. The challenge of tissue preservation is to be able to block, or at least slow down, intracellular functions and enzymatic reactions while at the same time preserving the physicochemical structures on which these functions depend.

Cryopreservation is a process in which cells or whole tissues are preserved by cooling to ultra-low subzero temperatures, typically −80 °C (freezer) or −196 °C (LN₂ phase). At these low temperatures, most biological activity is effectively stopped, including the biochemical reactions that would lead to cell autolysis. However, due to the particular physical properties of water, the process of cryopreservation may damage cells and tissue by thermal stress, dehydration and increase in salt concentration, and formation of water crystals. Specific applications (e.g. proteomics or storage of primary cell cultures) may require more complex cryopreservation procedures. General information is available on the principles of cryopreservation (Cryo Bio System, 2013) and on the optimal temperature for selected biomarkers and metabolites (Hubel et al., 2014). The process of thawing may also influence cellular structure or metabolite analyses.

Specimen freezing can be performed, for example, by placing the specimen in a sealed (but not airtight) container and immersing the container in the freezing medium. The ideal medium for rapid freezing is isopentane that has been cooled to its freezing point (−160 °C). To achieve this, the vessel containing the isopentane should be placed in a container of LN₂. The freezing point approximately corresponds to the moment when opaque drops begin to appear in the isopentane. Direct contact of the specimen with LN₂ should be avoided because this can damage tissue structure.

3.3.1.2. Other fixation and preservation methods

Formalin or alcohol fixation and paraffin embedding is the best method for morphological analysis, morphology-related methods, and immunohistochemistry. It may also be used as an alternative method to preserve tissues at relatively low cost when adequate freezing procedures and storage facilities are not available. Paraffin blocks and histological slides may be stored in light- and humidity-controlled facilities at 22 °C (Figs. 3 and 4).

Fig. 3

Paraffin-embedded tissues.

Fig. 4

Histological slides.

Tissue fixed according to strict protocols may be used for DNA extraction. The DNA is usually fragmented but remains suitable for polymerase chain reaction (PCR)-based analysis of short DNA fragments. However, fixed tissue is of limited usefulness for RNA extraction.

RNAlater is a commercial aqueous, non-toxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA at room temperature. RNAlater eliminates the need to immediately process tissue samples or to freeze samples in LN₂ for later processing. Tissue pieces can be harvested and submerged in RNAlater for storage for specific periods without jeopardizing the quality or quantity of RNA obtained after subsequent RNA isolation.

PAXgene tissue fixation is increasingly used for tissue preservation. PAXgene tissue systems are formalin-free solutions for the simultaneous preservation of histomorphology and biomolecules and the purification of high-quality RNA, DNA, microRNA (miRNA), proteins, and phosphoproteins from the same sample. Tissue specimens are collected, fixed, and stabilized with the PAXgene tissue fixation and stabilization products. PAXgene-fixed tissue can be processed and embedded in paraffin similarly to formalin-fixed tissue, and biomolecules can be extracted (Gündisch et al., 2014).

3.3.2.1. Liquid nitrogen storage

LN₂ facilities contain LN₂ in liquid-phase tanks (Fig. 5) and vapour-phase containers (Fig. 6). Cryogenic storage using LN₂ is an effective long-term storage system, because its extreme ultra-low temperatures slow down most biological, chemical, and physical reactions that may cause biospecimens to deteriorate.

Fig. 5

Liquid nitrogen facility with LN₂ tanks.

Fig. 6

Tank for storage in vapour-phase liquid nitrogen.

LN₂ vapour-phase containers with LN₂ in the base of the tank can maintain samples below T_g (the critical glass-transition temperature, i.e. −132 °C), and submersion in LN₂ guarantees a stable −196 °C temperature environment for all samples. Vapour-phase storage is preferred over liquid-phase storage, because it avoids some of the safety hazards inherent in liquid-phase storage, including the risk of transmission of contaminating agents (Fig. 6). The design of the tank is critical to maintain a sufficient amount of LN₂ in the vapour phase.

Liquid-phase storage needs less frequent resupply of LN₂ and thus affords better security in case of a crisis in LN₂ supply. Closed LN₂ tanks can maintain samples at below −130 °C for several weeks without the need to refill the LN₂ tank. The initial investment and the availability and cost of LN₂ can be major drawbacks. Also, safety hazards inherent in the use of LN₂, such as burning or oxygen deficit risks, should be managed. When LN₂ tanks are used, oxygen-level sensors must be used, and they should be calibrated every few years. The use of protective equipment – in particular, face shields, cryogenic gloves, and individual oxygen detectors – should be mandatory, and this equipment should be easily accessible (Fig. 2). Appropriate training in the safe handling of LN₂ must be provided, and this should be included in an SOP describing the potential health hazards and required safety precautions.

3.3.2.2. Mechanical freezers

Mechanical freezers are used for a variety of storage systems with temperatures ranging from low-temperature to ultra-low-temperature conditions, including −20 °C, −40 °C, −70 °C to −80 °C, and −150 °C, and come in a wide range of sizes and configurations (Figs. 7 and 8).

Fig. 7

Freezer facility.

Fig. 8

Freezer racks.

Ice crystals may form in biological samples at temperatures between 0 °C and −40 °C, and protein activity may persist until −70 °C or −80 °C; therefore, freezer temperatures should preferably be below −80 °C. Cascade compressor technologies may produce temperatures as low as −140 °C. Mechanical freezers, which generally require a lower initial investment than LN₂ tanks and provide easy access to biospecimens, can be installed if appropriate electrical power is available. However, the compressor technology requires constant electrical power to maintain subzero temperatures, so a backup power system and an emergency response plan are needed. Whether samples warm up significantly during power outages or freezer breakdowns depends on the temperature, type, and volume of the stored biospecimen, the ambient conditions of the environment where the freezers are stored, and the design and maintenance of the freezer.

Ambient temperature and humidity influence temperature stability considerably if doors are left open for prolonged periods, for example for sample loading, or if frost forms in the freezer, racks, or samples. Overheating of compressors may shorten their lives. Mechanical freezers and refrigerators should be positioned with sufficient air flow around the units and preferably in rooms that are air-conditioned or have equipment for extraction of the hot air generated by the compressors. Regular cleaning and maintenance of freezers should be planned; this should consist, at a minimum, of cleaning filters and removing ice around the door and seals. Freezers should be equipped with alarms set at about 20 °C warmer than the nominal operating temperature of the unit. An independent temperature monitoring system should be in place (Figs. 9 and 10), to prevent freezer failure or breakdown and/or to alert personnel in case this happens. Some of the freezers (approximately 10%) should be kept empty and cool to be used as a backup system.

Fig. 9

Temperature monitoring system.

Fig. 10

Graph of temperature log obtained from monitoring system.

3.3.2.3. Refrigerators

Refrigerators are commonly used for samples that can be maintained at ambient temperature. However, the longevity of biospecimens being stored is enhanced if they are stored below ambient temperature, due to biomolecular degradation that can occur at high ambient temperatures. Storage at 4 °C can be a temporary storage solution as an intermediate step before preparation for ultra-low-temperature storage or before sample processing. For refrigerators, as for mechanical freezers, it is important to maintain and monitor the temperature in the required operating range and to have a backup power system.

3.3.2.4. Ambient-temperature storage

If a biobank does not have mechanical freezers or cryogenic storage equipment, because of practical or financial reasons, then specific biological storage matrices may be used for long-term maintenance of some biological components at room temperature. Formalin-, PAXgene-, or ethanol-fixed, paraffin-embedded tissues and lyophilized samples can be stored at ambient temperatures. Dried samples, such as blood spots on filter paper, can be stored at ambient temperature (Figs. 11 and 12). There are also some new techniques for storage of DNA at ambient temperature, for example in mini-capsules after dehydration. A mini-capsule consists of a glass vial containing the sample, enclosed in a stainless steel shell with a cap. The mini-capsule is sealed by a laser, which welds the junction between the shell and the cap under an anhydrous and anoxic inert atmosphere.

Fig. 11

Room-temperature storage facility.

Fig. 12

Box of samples stored at room temperature.

Biological storage matrices should be evaluated before use to ensure that they are appropriate for downstream applications. Temperature, humidity, and oxygen levels should be controlled to avoid mould growth and microbial contamination.

3.3.4.1. IT functionality

Biobank management software must guarantee the management of different functions and data related to biobanking activities (see Section 3.8). It is fundamentally important that there is a method to track each sample throughout the biobank process and to document the actions that have been carried out on the sample.

Documentation related to sample collection (informed consent, participant information sheet, sample collection protocol), sample processing, sample sharing (MTA and DTA), and shipment (proof of shipment and delivery) must be appropriately referenced in the IT system (see Section 3.6). The IT system requires a backup process to ensure that all data recorded in the IT system are correctly tracked and maintained, and are recoverable in the event of erroneous modifications.

Semantic interoperability, in particular, presents a significant challenge for biobanking and IT support.

The specific location of every stored aliquot relating to a sample should be tracked. The biobank database containing this information should be updated in real time as a biospecimen is moved within or out of the biobank.

In addition to IT software to record the information at each point of the biobanking process, there need to be software solutions to document information about monitoring of storage infrastructure and to report alarms about adverse events.

It is also recommended that the biobank software record information about operations and operators. This should include information about the standard and regular measures taken to calibrate and repair biobank instruments.

The management of these functions is fundamental to provide high-quality samples.

3.3.4.2. Software solutions

As biobanking evolves in terms of the types of samples that are collected, archived, and stored and the downstream use of the samples, there continues to be a need to develop informatics tools for the management of biobanks. Different options may be considered depending on the needs, financial resources, and IT resources of the specific biobank. For the rapidly growing field of biobanking, commercial software solutions are increasingly available. Recently, open-source systems have emerged, and some have been selected by European biobanks (Kersting et al., 2015). However, commercial and open-source solutions mainly cover particular aspects and require adaptation to respond to the requirements of the individual biobank.

An alternative to commercial and open-source systems may be the development of a dedicated in-house system, noting that the internal cost of maintaining a development team for modifications and maintenance can be considerable (Voegele et al., 2010). In a larger scope, a laboratory information management system (LIMS) enables the management not only of the biobank but also of the entire sample life-cycle workflow. Electronic laboratory notebooks are also a solution for the management of procedures performed in a laboratory, and can be used by scientists, engineers, and technicians (Voegele et al., 2013).

3.3.4.3. Data management and informatics security

Biobank management systems must permit access to sample data in order to stimulate collaboration, but they must also guarantee the confidentiality of sample records.

Data security systems should be adequate to ensure confidentiality and safety. Electronic records should be adequately protected through regular backups on appropriate media. Intrusion-proof management systems should include solutions such as dedicated servers, secure networks, firewalls, data encryption, and user authentication through verification of user names and passwords.

All computers used by biobank personnel should be password-protected and have automatic timeout mechanisms. The biobank management software should also be password-protected and should have different user profiles to permit different levels of access. Each biobank staff member should have an individual user ID, to provide complete traceability of all actions performed on biobank data.

The protection of personal information and individual data associated with specimen collection is a fundamental requirement of a biobank. This should be achieved through the use of safe, structured bioinformatics systems. Personal identifiers should be replaced by codes, and all individual data stored in the biobank management system should be protected with the same stringency as patient clinical files. This also applies to data that are considered to be sensitive. Communication to third parties of data files containing personal information and identifiers should be strictly prohibited unless it is required by law or explicit permission to do so was granted. Examples of methods of coding are provided in Appendix 2 of the privacy and security policy of GA4GH (GA4GH, 2015b).

3.3.4.4. Biobank networking infrastructure

The facilitation of scientific networking is an important aspect of IT infrastructure. Networking can increase biobank use and therefore is an important element of biobank sustainability. Publication of data on the Internet can greatly increase the visibility of the biobank and its ability to participate in biobank networks. It is recommended that a biobank develop a website to present its operations to the scientific community, in addition to an online catalogue with information on the nature, characteristics, and quality of its biological samples. Networking to facilitate exchange and access to an increased number of samples requires that biobanks adhere to standards for use of samples and data to ensure semantic interoperability between different systems and different biobanks, and this in particular presents a significant challenge for biobanking and IT support (see Section 3.8, Section 3.5, and Section 2.1.4)

Box

Key points: IT systems.

3.4.2.1. Quality control of tissue (e.g. frozen section)

QC should be done during sampling in the grossing room on surgical samples using the frozen section method. This is done by sampling the area of suspected cancer macroscopically, performing a routine frozen section, preparing a stained slide, and documenting the review data on the sample collection sheet. The following information should be provided, which defines the quality of the tissue sample:

•

frozen section performed (yes or no);

•

pathologist who performed frozen section review;

•

tumour confirmed;

•

percentage of tumour cells;

•

percentage of stromal and inflammatory cells;

•

percentage of surface occupied by necrosis; and

•

other comments.

3.4.2.2. Methods for quality control of tissue sections for DNA/RNA extraction

Regardless of whether a frozen section is performed at the time of sampling, microscopic pathology review should be performed on the tissue sections taken for nucleic acid extraction. It is recommended that this is performed every 20 sections of 5 µm, because of the potential heterogeneity in the sample. This is also recommended for sections taken from formalin-fixed, paraffin-embedded (FFPE) blocks.

3.4.3.1. Methods for evaluating quality of nucleic acids from tissue

Spectrophotometers can measure absorbance and provide values for wavelengths of 260 nm, 280 nm, and 230 nm. However, they lack the sensitivity to measure small quantities of DNA. All nucleic acids (dsDNA, RNA, and ssDNA) absorb at 260 nm, and this method cannot distinguish between the various forms of nucleic acid. For example, the amount of genomic DNA (gDNA) present in an RNA preparation or the amount of RNA present in a gDNA sample cannot be determined. These contaminants contribute to the absorbance value, resulting in an overestimation of nucleic acid concentration. In addition, if samples are degraded, single nucleotides will also contribute to the 260 nm reading, and thus the nucleic acid concentration will be overestimated.

Fluorescent dye-based quantification uses dyes that only fluoresce when bound to specific molecules, dsDNA, ssDNA, or RNA, and thus the concentration of the specific molecule can be measured. This makes the measurement more accurate for samples that contain nucleic acid contaminants or samples that are partially degraded. Although this method provides a more accurate concentration of the sample for the molecule of interest, it does not give an indication of the contamination of the sample.

Gel electrophoresis verifies the integrity of DNA and RNA molecules by separating their fragments based on size and charge and thus estimating the size of DNA and RNA fragments.

The RIN is an algorithm for assigning integrity values to RNA measurements. The integrity of RNA is a major concern for gene expression studies and traditionally has been evaluated using the 28S/18S ribosomal RNA (rRNA) ratio. The RIN algorithm is applied to electrophoretic RNA measurements and is based on a combination of different features that contribute information about the RNA integrity to provide a robust universal measure. If RNA is purified from FFPE samples, the 28S/18S rRNA ratio and the RIN value are not useful for assessing RNA quality.

The DIN determines the level of sample degradation using an algorithm to evaluate the entire electrophoretic trace. The higher the DIN value, the better the integrity of the gDNA sample.

qPCR can be a useful technique in the QC of gDNA for downstream sequencing, because it simultaneously assesses DNA concentration and suitability for PCR amplification. However, this technique is labour-intensive and has higher costs.

Sequential use of spectrophotometric and fluorescence-based methodologies permits the cost-effective assessment of DNA quality for high-throughput downstream applications. This combination also enables the detection of impurities, and thus their removal from samples. This is particularly useful for samples such as FFPE samples that are available in limited amounts.

3.7.1.1. Blood

The following general guidelines should be considered.

•

All blood should be treated as potentially infectious. Blood samples for research purposes should be collected at the same time as routine clinical blood samples, so as to limit discomfort to individuals. Blood should be collected from fasting individuals (i.e. after abstinence from food, alcohol, and caffeine-containing beverages for 8–12 hours).

•

Blood should not be collected after prolonged venous occlusion.

•

Tubes into which the blood is collected should be clearly labelled (Fig. 20).

•

For blood collection, the recommended order of draw is the following (Calam and Cooper, 1982; CLSI, 2007):

1.

blood culture tube;

2.

coagulation tube;

3.

serum tube with or without clot activator, with or without gel;

4.

heparin tube with or without plasma separating gel;

5.

ethylenediaminetetraacetic acid (EDTA) tube with or without separating gel;

6.

glycolytic inhibitor.

•

For the preparation of plasma, blood may be collected into an EDTA tube, an acid citrate dextrose (ACD) tube, or a lithium heparin tube.

•

Ideally, blood should be processed within 1 hour of collection. After that time, cell viability decreases rapidly, resulting in poor cell structure and degradation of proteins and nucleic acids.

•

Lithium heparin is generally used if cytology studies will be performed, but it is not recommended for proteomics work.

•

PCR was clearly interfered with when heparinized blood (heparin 16 U/mL blood) was used as a source of template DNA (Yokota et al., 1999).

•

Either EDTA or ACD tubes can be used if DNA will be extracted or lymphoblastoid cell lines will be derived. Lithium heparin is not recommended for establishment of lymphoblastoid cell lines.

•

EDTA tubes are recommended if protein studies will be performed. The use of EDTA tubes results in less proteolytic cleavage compared with the use of heparin tubes or ACD tubes.

•

For the preparation of plasma, the blood should be centrifuged as soon as possible. For the preparation of serum, the blood should be processed within 1 hour of collection.

•

The amount of blood collected should be justified when applying for ethical clearance.

•

A reduced volume of blood in a tube containing additives should be recorded to avoid confounding the results.

•

The time and date of blood collection and the time of freezing should be recorded, as well as any deviations from the standard processing protocol.

•

Blood should be transported at room temperature or on melting ice depending on the particular applications. Samples to be used for proteomics assays should be processed immediately at room temperature, because cool temperatures can activate platelets and release peptides into the sample ex vivo.

•

Blood spot collection should be considered as an alternative to whole blood for validated techniques when conditions necessitate easier collection and cheap room-temperature storage. Different types of collection cards are available (e.g. Guthrie cards, “fast transient analysis” [FTA] cards, IsoCode cards) (see Section 4).

Fig. 20

Blood sample.

3.7.1.2. Blood spots

Guthrie cards (Schleicher & Schuell 903 filter paper) are used to collect heel-stick blood from newborns for metabolic disease screening. The 903 paper is manufactured from 100% pure cotton linters with no wet-strength additives. The critical parameters for collection of newborn screening samples are blood absorbency, serum uptake, and circle size for a specified volume of blood. Blood spots archived for as long as 17 years, sometimes at room temperature, have also provided valuable sources of amplifiable DNA (Makowski et al., 1996) (Fig. 21).

Fig. 21

Card with dried blood spots.

Modified cards (IsoCode cards or FTA cards) have been developed. These consist of filter paper impregnated with a proprietary mix of chemicals that serves to lyse cells, to denature proteins, to prevent growth of bacteria and other microorganisms, and to protect nucleic acids from nucleases, oxidation, and UV damage. Room-temperature transportation of such cards in folders or envelopes (by hand or by mail) has been common for years. The papers protect DNA in the samples for some years under ambient conditions. The main variable is expected to be the quality of the storage atmosphere, particularly the content of acid gases and free-radical-generating pollutants, although FTA paper can protect against such conditions (Smith and Burgoyne, 2004). Sample integrity is optimized when FTA cards are stored in a multi-barrier pouch with a desiccant pack. Whatman Protein Saver Cards are commonly used to collect dried blood spots for molecular and genomic studies and for the isolation of viruses (Mendy et al., 2005) and bacteria (see Section 4).

3.7.1.3. Buffy coat

For DNA testing, if DNA cannot be extracted from blood within 3 days of collection, the buffy coat may be isolated and stored at −70 °C or below before DNA isolation. Buffy coat specimens that are being used for immortalization by Epstein–Barr virus should be transported frozen on dry ice (solid-phase CO₂). RNA should be isolated from buffy coat within 1–4 hours of specimen collection; alternatively, RNA stabilization solution (e.g. RNAlater) should be used (see Section 4).

3.7.3.1. Urine

Urine is easy to collect and is a suitable source of proteins, DNA, and metabolites. Urine should be routinely stored at −80 °C. Ambient-temperature storage before freezing should be kept to a minimum (see Section 4).

3.7.3.2. Buccal cells

The collection of buccal cells is not difficult and does not require highly trained staff. Buccal cell collection is considered when non-invasive, self-administered, or mailed collection protocols are required for DNA analysis (Steinberg et al., 2002). However, buccal cells will yield only limited amounts of DNA compared with blood. Different methods of self-collection are available, depending on the end-points and the analyses to be performed (Mulot et al., 2005).

3.7.3.3. Saliva

Saliva is used as a biological fluid for the detection of different biomarkers, such as proteins, drugs, and antibodies. Saliva meets the demand for a non-invasive, accessible, and highly efficient diagnostic medium. The collection of saliva is non-invasive (and thus not painful), and a sample can easily be collected without a need for various devices. Whole saliva is collected by expectoration into a provided tube, whereas for the collection of submandibular saliva and sublingual saliva, different ducts need to be blocked by cotton gauze. For the collection of parotid saliva, a parotid cup should be used (see Section 4).

3.7.3.4. Bronchoalveolar lavage

The airways, and particularly the alveoli, are covered with a thin layer of epithelial lining fluid, which is a rich source of many different cells and of soluble components of the lung that help protect the lung from infections and preserve its gas-exchange capacity. Bronchoalveolar lavage performed during fibre-optic bronchoscopy is the most common way to obtain samples of epithelial lining fluid (Reynolds, 2000). The cellular and protein composition of the epithelial lining fluid reflects the effects of the external factors that affect the lung, and changes in this composition are of primary importance in the early diagnosis, assessment, and characterization of lung disorders as well as in the search for disease markers (Griese, 1999).

Bronchoalveolar lavage is classically performed by instillation of buffered saline solution divided into three or four aliquots (typically a total volume of 100–150 mL) through a flexible fibre-optic bronchoscope, after local anaesthesia. The first 10 mL should be processed separately and is denoted as bronchial lavage. The rest of the lavage, denoted as bronchoalveolar lavage, should be pooled into a sterile siliconized bottle and immediately transported on ice to the laboratory. At the laboratory, the total volume of the lavage is measured, and cells and proteins are separated by centrifugation. The lavage fluid should be frozen and stored at −80 °C until use.

3.7.3.5. Bone marrow aspirate

The following paragraphs on bone marrow aspirate and cerebrospinal fluid are derived from the Australasian Biospecimen Network recommendations (see Table 1) and the publication Guidelines on Standard Operating Procedures for Microbiology (Kanagasabapathy and Kumari, 2000).

Bone marrow is the soft tissue found in the hollow interior of bones. In adults, the marrow in large bones produces new blood cells. There are two types of bone marrow: red marrow (also known as myeloid tissue) and yellow marrow. In cancer research, red bone marrow from the crest of the ilium is typically examined.

Bone marrow should be collected by a doctor who is well trained in this procedure. Bone marrow should be aspirated by sterile percutaneous aspiration into a syringe containing an EDTA anticoagulant, and the specimens should be chilled immediately. Heparin is not recommended as an anticoagulant for molecular testing. If a specimen contains erythrocytes, it should be processed to remove the erythrocytes before freezing. The bone marrow samples should be fresh frozen and stored at −80 °C.

3.7.3.6. Cerebrospinal fluid (CSF)

Cerebrospinal fluid (CSF) originates from the blood. The choroid plexuses in the first, second, and third ventricles of the brain are the sites of CSF production. CSF is formed from plasma by the filtering and secretory activities of the choroid plexus and the lateral ventricles. CSF circulates around the brain and the spinal cord. It nourishes the tissues of the central nervous system and helps to protect the brain and the spinal cord from injury. It primarily acts as a water shock absorber. It totally surrounds the brain and the spinal cord, and thus absorbs any blow to the brain. CSF also acts as a carrier of nutrients and waste products between the blood and the central nervous system.

CSF is a very delicate biological material. Often, only small volumes of CSF are available for analysis, because of the difficulty of collecting CSF, and therefore it should be handled with care. Only a physician or a specially trained nurse should collect the specimen. After collection, the specimen should be transferred into a clean penicillin vial containing about 8 mg of a mixture of EDTA and sodium fluoride in the ratio of 1:2. Centrifuging CSF is recommended before freezing if the sample contains red blood cells or particulate matter. The specimen should be frozen and stored at −80 °C or in LN₂. Do not delay freezing the CSF, because cells are rapidly lysed once the CSF is removed from the body.

3.7.3.7. Semen

Seminal fluid, which is the liquid component of sperm, provides a safe surrounding for spermatozoa. At pH 7.35–7.50, it has buffering properties, protecting spermatozoa from the acidic environment of the vagina. Seminal fluid contains a high concentration of fructose, which is a major nutriment source for spermatozoa during their journey in the female reproductive tract. The complex content of seminal plasma is designed to ensure the successful fertilization of the oocyte by one of the spermatozoa present in the ejaculate. Seminal plasma is a mixture of secretions from several male accessory glands, including the prostate gland, seminal vesicles, epididymis, and Cowper’s glands (Pilch and Mann, 2006).

After collection, the fresh ejaculate should immediately be spun down at 4 °C to separate the seminal fluid from the spermatozoa. Protease inhibitors should then be added to the sample, to avoid digestion by powerful proteases present in seminal fluid. To ensure complete separation of cell debris or occasional spermatozoa from seminal plasma, the sample can be centrifuged a second time. The sample should be stored at −80 °C.

3.7.3.8. Cervical and urethral swabs

The quality of collected cervical and urethral specimens depends on appropriate collection methods. Swabs, brushes, or other collection devices should be placed in a transport medium, or transported dry in a sealed tube and resuspended in the transport medium upon arrival. The transport fluid may either be stored at −70 °C or below or immediately centrifuged, and the pellet processed for DNA or RNA extraction (see Section 4).

3.7.3.9. Hair

Currently, hair analysis is used for purposes of assessing environmental exposures, such as exposure to mercury from eating fish. Hair analysis is also used to test for illegal drug use and to conduct criminal investigations (see Section 4). Hair should be kept in sealable plastic bag, stored in the dark at room temperature (Fig. 22).

Fig. 22

Hair sample.

3.7.3.10. Nails

Nail clippings may contain analytes of interest that were deposited during the growth of the nail. Nail specimens can be collected for drug, nutritional, poisons, and toxicity testing (see Section 4) (Fig. 23).

Fig. 23

Nail clippings.