The European League Against Rheumatism Scleroderma Trials and Research Group (EUSTAR) has established an online database with clinical data of currently more than 8200 patients with systemic sclerosis (SSc). In addition to clinical research, EUSTAR fosters biomolecular studies to develop novel biomarkers and therapies for SSc. High-quality biospecimens are the basis for successful biomolecular studies. The EUSTAR biobanking group has therefore developed recommendations to standardise the collection, storage and distribution of SSc biospecimens at EUSTAR centres. These recommendations consider the scientific challenges associated with biomolecular research in SSc and the organisational requirements of EUSTAR. They were approved by the EUSTAR executive committee as well as the EUSTAR board. Once they become effective, these recommendations will be the basis for international EUSTAR studies with large numbers of SSc biospecimens. These recommendations might also be followed by other SSc consortia to enable exchange of biosamples between different SSc initiatives and might serve as a template for biobanking initiatives in other rheumatic diseases.
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Systemic sclerosis (SSc) is an orphan disease with a prevalence of approximately 1 in 12 000.1 The course of the disease is highly variable and patients with SSc may experience spontaneous regression of fibrosis. This places biomolecular research in a dilemma: because of the variable course of the disease, researchers need large numbers of SSc biospecimens to identify predisposing genetic factors, establish biomarkers or develop novel treatment strategies. The low prevalence of SSc, however, limits the size of study cohorts at local and regional medical centres. Thus, researchers can only establish large study cohorts with high numbers of SSc biospecimens by nationwide and international cooperation. In this context, the European League Against Rheumatism Scleroderma Trials and Research group (EUSTAR) has initiated a unique biobanking platform for the collection, storage and distribution of biospecimens in an international network of SSc research centres.
The Organisation for Economic Co-operation and Development first defined the term ‘biobank’ as ‘a collection of biological material and the associated data and information stored in an organised system for a population or a large subset of a population’.2 3 Biological material from patients with SSc is the direct source of biomolecular research. Regarding the high sensitivity of new biomolecular technologies, the reliability of SSc research depends on the quality and consistency of the specimens. The lack of standardised high-quality biospecimens is a major obstacle in biomolecular research.4 The EUSTAR biobanking group has therefore developed recommendations and quality standards for collection, storage and distribution of SSc biospecimens (see online supplement). These recommendations consider scientific challenges associated with biomolecular research in SSc as well as organisational requirements of EUSTAR. They rely on state-of-the-science approaches and concepts of good laboratory practice and are reviewed on a regular basis. If all participating SSc research centres achieve these quality standards, EUSTAR biobanking will provide a framework for pivotal studies.
Collection and processing
In addition to documenting a minimal essential data set (MEDS), acquisition and storage of serum samples, whole blood samples and skin biopsies from patients with SSc are strongly encouraged in the EUSTAR cohort. Prior to data assessment and biospecimen collection, each patient must give informed consent. Local or national ethics committees approve informed consent forms for each EUSTAR centre. These forms independently address data assessment and biospecimen collection according to national laws. Apart from other types of biospecimens, the collection of DNA samples requires separate informed consent.
For all kinds of biospecimens, it is important to maintain a cold chain from the point of collection through processing and storage. Biosamples should be kept on ice and the centrifuge should be cooled. In addition, collection and processing should be performed under sterile conditions using sterile needles, pipettes and other instruments as well as a sterile hood to avoid contamination. Appropriate aliquoting of biospecimens helps to avoid repetitive freeze and thaw cycles, which can damage the integrity of samples.5 6 A detailed guide on processing of blood, serum and skin samples from patients with SSc is given in the online supplement.
In EUSTAR biobanking, specimens are stored in the repositories of the affiliated research centres. Standardisation of storing procedures and equipment among EUSTAR centres is therefore crucial to ensure the high quality and consistency of SSc biospecimens. In general, selection of storage equipment depends on the type of specimens, the anticipated storage time and the intended use of the samples.6 7
Serum and (whole) blood samples as well as tissue samples for protein and RNA analysis should be stored at −80°C as provided by special mechanical freezers. Temperatures of −80°C or lower may be necessary for the stability of certain cytokines such as tumour necrosis factor α.5 6 8 Furthermore, freezing temperatures of −80°C are adequate for successful preservation of tissues for extended periods of time.9 If a −80°C freezer is not available, EUSTAR serum and blood samples can be stored at −20°C but need to be labelled appropriately. Independent of the type of specimen, proteomic techniques require very low temperature storage conditions as provided by liquid nitrogen freezers. In general, screw-cap cryovials should be used for long-term low-temperature storage; glass vials or vials with pop-up tops are not suitable. Finally, paraffin blocks should be stored at room temperature (not exceeding 27°C) in an area with pest and humidity control.5 6 8 10
For any storage equipment, acceptable temperature ranges should be determined before the equipment is put into service. Temperature ranges allow for operating variations and provide some variation for warming when the biospecimens are accessed. Of note, temperature probes measure the temperature where the probes are located, so different locations in the equipment might exhibit different temperatures. Freezers that are full will probably display temperature readings that are different from readings of empty equipment.5 6 10,–,12
Loss of electrical power or failure of freezers can lead to warming and destruction of the biospecimens. The length of time that results in the significant warming of the stored material will vary according to the properties and temperature of the material (thermal loading), the ambient conditions and the design of the unit.5 13 To bridge transient loss of commercial utility power, EUSTAR repositories should be connected to an uninterruptible power supply (UPS) such as a ‘battery backup’. Apart from storage equipment, computers and electronic systems such as environmental monitoring systems, safety systems (eg, oxygen sensors, ventilations systems) or controllers for freezers should also be protected by a UPS. In addition, adequate backup capacity for low temperature units is necessary for possible failure of storage equipment. The total amount of backup storage required for EUSTAR repositories is determined empirically, but will typically be 10% for mechanical freezer storage.5 6 Distributing aliquots to different low-temperature units can also prevent the total loss of single batches upon equipment failure. Special issues must be addressed when using liquid nitrogen freezers (see online supplement).
In EUSTAR repositories it is critical to maintain ambient temperature. For optimal life of mechanical freezers, repository ambient temperatures should be maintained between 15°C and 22°C. In addition, sufficient air circulation prevents excess moisture and condensation. Left unchecked, excess humidity can lead to fungal growth, which may affect specimen integrity and cause health problems for staff. Sufficient space for air circulation prevents excess heat accumulation in areas where freezers and refrigerators are employed. Lighting in EUSTAR repositories should provide a safe working environment and allow materials to be accurately put away and retrieved.5 6
Repositories should employ basic security systems to ensure protection of the specimens. The systems should be monitored and alarms responded to 24 h/day. An individual should be available at all times to respond to an alarm in a time frame that minimises loss or damage to the stored materials.5 6 8 Storage units with defined environmental conditions have temperature monitoring devices that can be visually inspected on a regular basis. In addition, automatic temperature monitoring systems continually monitor temperatures of all critical equipment and generate alarms. The alarm notification system should call or page the individual ‘on call’ (or should activate the ‘on call’ list) rather than simply providing passive notification.5 6 8
A system for preventive maintenance and repair of storage equipment and facilities should be in place. EUSTAR repositories should perform system maintenance at regular intervals in accordance with the manufacturer's recommendation. Essentially, all equipment comprised of multiple components and exposed to various environmental conditions wears out with time. Routine assessments and maintenance may significantly extend the lifetime of equipment. Well-qualified personnel with expertise in monitoring and repairing repository equipment (especially freezers and refrigerators) should perform regular and emergency repairs. Finally, EUSTAR repositories should establish security systems that limit access to appropriate staff and protect against physical intrusion from unauthorised individuals.5
Biological material tracking
EUSTAR centres should install effective tracking systems to track specimens from the site of collection to their arrival in the repository as well as subsequent shipment to other centres. Critical components of these systems include unique specimen identifiers (ID), appropriate specimen labels and inventory systems for specimen tracking. Each specimen container should receive a label that tightly adheres under all projected storage conditions. Information encoded on labels should be resistant to all common laboratory solvents. If possible, labels should be computer printed. Human specimens should be labelled in a way that protects privacy as well as confidentiality and is in compliance with applicable laws and institutional policies.5 6 To link SSc biospecimens with the MEDS online database, EUSTAR recommends generating the specimen ID as follows: EUSTAR centre number, EUSTAR patient number, date of collection, aliquot number (eg, EUSTAR centre 001, patient number 075, collected on 10 May 2009, aliquot number 002: 001-075–100509-002).
In addition to sample tracking, EUSTAR has developed an online records management system that permits detailed records of the collection, processing and distribution of specimens. The system should also track significant events such as thaws, loss and destruction of serum samples. Furthermore, records should include training documents, protocols, informed consent documentation, procurement documentation, processing records, equipment maintenance, specimen storage location information, sample distribution and quality control (QC) activities. Security systems should ensure confidentiality and security of all stored records. Access to records should also be restricted on a ‘need to know’ basis. A policy should be in place for the destruction or return of records that no longer need to be retained.4,–,6
Retrieval of specimens for analysis or shipment requires strict adherence to protocols for proper specimen inventory and tracking. First, the location of specimens should be verified. A specimen requisition is generated and checked for accuracy before transmission to the repository. Specimens should be located and pulled from storage as documented on specimen requisition forms. When frozen biospecimens are shipped to other EUSTAR centres, a cold chain should be maintained to avoid warming and thawing of biospecimens.5 6
Packaging and shipping
Prior to shipment the specifications for the biological material should be determined; this includes regulatory and physical requirements necessary to ensure proper shipping conditions. Packaging and shipping should conform to all governing regulations. Air shipments should conform to the International Air Transport Association (IATA) standards, and ground shipments to applicable national standards. International Society for Biological and Environmental Repositories best practices and IATA regulations provide information on international transport regulations and sample classification for shipment.5 6 12 14 Detailed information on technical and regulatory issues of biosample packaging and shipping is given in the online supplement.
Legal and ethical issues
Appropriate annotation of biospecimens is crucial to the overall usefulness of EUSTAR biobanking as a tool for scientific research. In addition to technical considerations relating to the physical quality of a biospecimen, multiple ethical and legal issues relate to biospecimen collection. In this context, EUSTAR biobanking follows highest ethical standards and addresses concepts of good clinical and laboratory practice; this includes respecting the autonomy of research participants, protecting research participants from breaches of privacy and confidentiality, developing appropriate policies for biospecimen use and ensuring scientifically sound research.5 6
On an international level, the collection and use of biospecimens is regulated by an amalgam of differing and occasionally conflicting local and international laws and policies. Thus, EUSTAR repositories proceed carefully, not only in their daily work but also with respect to international exchange of samples and associated data. Each EUSTAR centre needs to operate with approval of the relevant ethics committee. Prior to approval, ethics committees evaluate the processes for collection, storage, distribution and use of SSc biospecimens in the repository.5 6
Informed consent is mandatory before patients can be included in the EUSTAR cohort and before biosamples can be collected. Informed consent is a process that offers subjects sufficient information to make an informed choice about whether to provide specimens and data to the EUSTAR centre and agree to future research use. Informed consent information should be as specific as possible and provide information about the scientific goals of EUSTAR. This should include future analysis of molecules and biomarkers that are still unknown at the time of collection, if required by the ethical committee.5 6 15 16 EUSTAR believes it is unethical not to use existing biomaterials for this purpose as long as the patient's will or well-being would not be jeopardised (see online supplement which addresses in detail the content of EUSTAR informed consent forms).
Internal requests (ie, by EUSTAR centres) as well as external requests (eg, by other research centres or pharmaceutical companies) for EUSTAR SSc biospecimens and MEDS data will undergo scientific review by the appropriate EUSTAR committee and the EUSTAR board. EUSTAR has published strict rules to apply for, review and decide on scientific projects (see online supplement). Once biospecimens and clinical data are distributed to the investigators, their use is restricted to the approved study. Further analysis requires new EUSTAR approval.
Finally, SSc biospecimens (including DNA) and clinical data are the property of the patient and the EUSTAR centre that has delivered it. In this context, EUSTAR repositories address formal and continuing responsibility for custodianship of collected biospecimens and associated data. The repositories have to ensure the physical integrity of biospecimens and clinical data. Thus, EUSTAR centres—as well as individual patients—may withdraw their biosamples and clinical data from a specific project at any time.6
Standardised protocols, QC and quality assurance
To achieve high levels of intrinsic quality and comparability in EUSTAR research, the definition and publication of biobanking recommendations are an essential starting point. Based on these recommendations, EUSTAR centres develop standardised protocols for collecting, storing and distributing SSc biospecimens adapted to local requirements. These protocols should be written by an individual or group of individuals with experience in successfully performing the processes described. Effective protocols are reviewed on a regular basis.4,–,6
Quality assurance (QA) and QC policies are developed by repositories to minimise errors that could adversely affect scientific results. QA and QC policies encompass equipment maintenance and repair, training records, data management, record keeping and adherence to protocols.4,–,6 In addition, QC examination of biospecimens designated for research should be performed according to research protocols; this includes molecular QC which characterises nucleic acids and proteins. Cost-effective approaches for tissue resources require simple methods of QC which can be expanded at the request of the investigator.5 17
EUSTAR repositories should be subjected to regular audits.5 6 EUSTAR prefers independent auditors from external institutions. Audits focus on adherence to the recommendations presented herein. At the end of each audit, centres receive a list of deficiencies which they have to remedy within 3 months. If not accomplished, these centres will (at least temporarily) be excluded from the EUSTAR cohort. Paying tribute to high costs of audits, approximately 10% of centres actively contributing to biobanking will be randomly chosen to receive audits each year. This does not apply to EUSTAR centres that are already subject to regular external audits covering all necessary procedures for EUSTAR biobanking.
To ensure the collection of high-quality SSc biospecimens, personnel are well qualified and trained to adhere to applicable protocols. Proper training promotes quality in specimen handling, good ethical practices and compliance with appropriate policies and regulations of the repository. All repository staff should be adequately trained to perform the tasks required by their particular position. Support for training is essential for implementation of certain tasks and, in some cases, might require additional resources or time off from regular responsibilities. To ensure quality in repository activities, employee performance should be routinely monitored to identify needs for additional training.5 6
Federal law mandates the training of medical personnel who obtain informed consent and collect SSc biospecimens as well as clinical data. Without interfering with federal regulations, EUSTAR recommends additional yearly instructions for medical personnel to emphasise the needs for sound and successful SSc research. Except for some areas of safety, training of personnel involved in the processing, storage and retrieval of SSc specimens and data is less tightly regulated by federal law. These personnel should receive theoretical and hands-on training at least once a year. Each EUSTAR centre develops written training plans that cover all important biobanking procedures and consider local circumstances. EUSTAR will provide templates for these training plans. At the end of each training course, participants should pass tests with the results being recorded. Independent of position and tasks, all new employees should receive training before they start working.
Each EUSTAR repository should have an individual responsible for training (IRT) who monitors and documents training and maintains records of employees. The IRT will coordinate safety training with the repository's safety officer as well as with other individuals responsible for specific areas of repository procedures (eg, shipping and handling). Finally, the IRT determines trainers for selected working areas. Trainers regularly perform the procedures in question, have completed the training programme previously and are skilled in explaining the elements of the task. If necessary, the IRT may hire external trainers for special training programmes or send staff to external training courses.5 6 In this context, EUSTAR organises a biobanking workshop at the educational EUSTAR course for young researchers and physicians every other year.
Issues related to safe operation of repositories are complex and depend on the particular activities. National, regional or local statutes typically cover regulations governing safety. Each EUSTAR repository should determine affected areas of safety and develop a safety programme to protect its employees. EUSTAR repositories should address biological, chemical, electrical and fire safety issues. All human specimens are potential biohazards and should be treated with precautions. Individuals should be trained in the possible hazards and should take precautionary measures. For example, staff members working with human patients are encouraged to be vaccinated against hepatitis. In addition, personal protective wear such as eye shades or gloves can lower the risk for contamination with hazardous material.5 6 8
The EUSTAR cohort already harbours clinical data on more than 8200 patients with SSc. This offers a unique chance to study the course of SSc and the various disease manifestations. With the implementation of the EUSTAR biobanking project, EUSTAR enters a new era of biomolecular and translational research in the field of SSc. The collection of biospecimens from a large number of patients with SSc will help to establish novel biomarkers and develop effective therapies. The EUSTAR recommendations for biobanking are the basis for successful biomolecular studies applying high-sensitivity techniques. Obviously, these recommendations should not be a barrier to the participation in EUSTAR if not all points are totally satisfied. Nevertheless, only if all participating EUSTAR centres adopt these recommendations in daily laboratory routine will EUSTAR biobanking pave the way for biomolecular landmark studies which can change our understanding of SSc. For this reason, EUSTAR has defined mandatory requirements to participate in EUSTAR biobanking which are summarised, together with other important recommendations, in the online supplement. Other SSc consortia might also follow these recommendations to enable exchange of biosamples between different SSc initiatives. Notably, there are no detailed recommendations published for establishing biorepositories in rheumatic diseases. Thus, the EUSTAR biobanking recommendations might also serve as a template for biobanking initiatives in other rheumatic diseases.
▶ Biobanking: The process of storing biomaterial or specimens for future use.
▶ Biobank/biorepository: An entity that receives, stores, processes and disseminates biological material.
▶ Biohazard: An organism or substance derived from an organism that poses a threat to human health. This includes medical waste, samples of a microorganism, virus or toxin from a biological source.
▶ Custodian: The individual responsible for the management of a biospecimen resource. The custodian works with other key stakeholders in the management of the resource including the tracking of all relevant documentation for the resource and for ensuring that policies regarding access to the resource are in place and implemented according to appropriate recommendations.
▶ Dry ice: Solid phase carbon dioxide (CO2). CO2 solidifies at −78.5°C.
▶ Liquid nitrogen: Coolant used to cool and store samples. Nitrogen becomes liquid at −196°C. Samples stored in the vapour phase of liquid nitrogen are −190°C and warmer, depending on the distance from the liquid phase.
▶ Quality: Conformance of a specimen or process with pre-established specifications or standards.
▶ Quality assurance (QA): An integrated system of management activities involving planning, implementation, documentation, assessment and improvement to ensure that a process or item is of the type and quality needed for the project. Same as quality management system.
▶ Quality control: Specific tests defined by the QA programme to be performed to monitor procurement, processing, preservation and storage; specimen quality; and test accuracy.
Competing interests EUSTAR is supported by EULAR. There are no other competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.