Biobank of Cells , Tissues and DNA from Patients with Neuromuscular Diseases : An Indispensable link between Clinical Centers and the Scientific Community

Marina Mora, Cinzia Bragato, Sara Gibertini, Simona Zanotti, Maurizio Curcio, Eleonora Canioni, Franco Salerno, Flavia Blasevich, Simona Saredi, Alessandra Ruggieri, Maria Barbara Pasanisi, Pia Bernasconi, Lorenzo Maggi, Renato Mantegazza and Francesca Andreetta Neuromuscular Diseases and Neuroimmunology Unit, Istituto Neurologico C. Besta, Milan, Italy Corresponding author: Marina Mora (marina.mora@istituto-besta.it)

Italian Ministry of Health.

Project description
The biobank of "Cells, tissues and DNA from patients with neuromuscular diseases" (NeuMD) was established in 1986 based on the routine diagnostic activity of the Neuromuscular Diseases and Neuroimmunology Unit of the Carlo Besta Neurological Institute in Milan.This is a leading center for the diagnosis and treatment of neurological disorders and for pre-clinical and clinical research.The biobank was started as collection of muscle tissue samples; muscle cells and DNA were subsequently added.Very recently the biobank has started collecting also serum, fibroblasts and lymphoblasts from selected cases.
The NeuMD biobank has received funds from the Italian Telethon almost uninterruptedly since 1999 and was founding partner of the Telethon network of genetic disease biobanks (TNGB) (http://biobanknetwork.telethon.it/Pages/View/Catalogue), a non-profit association of Italian repositories, created in 2008 with the aim of developing a virtually unique catalogue of biological samples and associated data [1].Such catalogue lists, at present, more than 750 rare genetic defects.
The biobank is founding partner of the EuroBioBank (EBB) network (www.eurobiobank.org)[2], the first operating network of biobanks in Europe, providing human biological samples as a service to the scientific community conducting research on rare diseases (RDs).
The involvement of the NeuMD biobank in the TNGB and EBB networks has been instrumental for improving the quality control process, for standardization of biobanking procedures and activities, for implementation of sample access policies and for compliance with ELSI requirements.
In 2014, the NeuMD biobank joined the Italian node of the BBMRI-ERIC consortium (http://www.bbmri.it/home), and, in 2015, the NeuMD ID card has been included in the biobank catalogue of the RD-Connect platform (http://catalogue.rd-connect.eu/web/bank-ofthe-muscle-cell-biology-laboratory-fondazione-i.r.c.c.sistituto-neurologico-carlo-besta/bb_home), a European global infrastructure project, funded under the FP7 program, whose main aim is to link up databases, registries, biobanks and clinical bioinformatics data for rare disease research (www.rd-connect.eu)[3].
To date, the contribution of the NeuMD biobank has been acknowledged in 68 scientific publications.

Classification (2)
Biological samples and associated data.

Temporal coverage
Active since 1986.

Steps Compliance with ELSI requirements
Samples are collected for diagnosis by the laboratory of the Unit of Neuromuscular Diseases and Neuroimmunology.After signing an informed consent form for minimal surgery, patients undergo a muscle biopsy, which can either be performed as open surgery or as needle biopsy [4].In addition, patients are informed about depositing samples in the biobank and about their possible use for research, and asked to sign a biobank specific consent form.

Sample Collection
For all biopsies: • two samples are snap frozen in isopentane precooled in liquid nitrogen for histology and biochemistry; for most biopsies: • a small tissue fragment is fixed in glutaraldehyde for electron microscopy; • another fragment is collected in sterile medium for myoblast culture and processed as described [5]; • a blood sample is also withdrawn for DNA extraction.
The samples are assigned a laboratory code and, those to be stored in liquid nitrogen, also a unique bar code.

Data Collection
Patient personal data, bar code and laboratory code, are registered in the biobank database.Data and sample codes are also recorded in a database of the Unit containing the muscle biopsy reports.The sample data set of this database includes personal data (name, date of birth, address, ethnic origin), sex, phenotype (affected/not affected), essential anamnestic data, presence of consanguinity, presence of familiarity, diagnostic tests performed on the muscle biopsy and, when available, DNA molecular tests and diagnosis, and internal unit or external center sending the patient.

Sample storage
DNA samples are stored in freezers with protection against electrical failure.Muscle biopsies and cell cultures, after receiving a bar code uniquely identifying each sample, are transferred to a company in charge for the management of all Institutional biosample collections that are to be stored in liquid nitrogen (www.sol.it)(certified UNI CEI EN ISO 13485:2002).
The company ensures sample storage conditions by continuous presence of personnel during working hours, alert systems during non-working hours, restricted access to the bank facility; and guarantees traceability of samples and security of data by use of dedicated software and computers, use of passwords enabling different levels of access to data, updated antivirus and security software.

Sample access
Applications for accessing biological samples normally reach the biobank director either through the TNGB website or the EuroBioBank website.In both cases the biobank manages all sample requests through the TNGB website, upon users' registration and description of the project.All submitted requests are collected in a unique Request Control Panel, which is visible to all members of the network.After evaluation and approval of the submitted project (see access criteria), a "Material Transfer Agreement" (MTA) form and an order form for invoicing the service costs, are sent to the researcher.The website keeps records of all requests for biological samples, including those requests refused for any reason.

Stabilization/preservation
Glycerol or DMSO (cryopreservation) for cells; none for tissues.

Type of long-term preservation
Liquid nitrogen for tissues and cells; freezers with protection against electrical failure for DNA.

Storage temperature
From -20 °C to -180 °C (liquid nitrogen).Flask of cell cultures and DNA samples are shipped at room temperature; frozen tissues, frozen cell lines, and RNA samples are shipped in dry ice.Tissue sampling for shipping is done inside a cryostat chamber at -25 °C using pre-cooled tools.

Quality assurance measures
The quality of the material is ensured by compliance with standard operating procedures (available for each step of the process), established in collaboration with the Eurobiobank (http://www.eurobiobank.org/en/documents/sops.htm)and TNGB (http://biobanknetwork.org/documents/0-TNGB_SOPs_Aug2012_online.pdf) networks.In addition, regular updating of the control quality process for the annual renewal of UNI EN ISO9000 certification, maintains good quality practices in the laboratories and in the biobank of the Neuromuscular Diseases and Neuroimmunology Unit.

Main measures
• Regular inspections to guarantee that the cell viability and absence of mycoplasma, bacteria, and fungi; • Systematic detection/exclusion of mycoplasma by appropriate tests before distribution service; • Assessment of DNA integrity by agarose gel electrophoresis; • Adoption of procedures for correct sample authentication, based on bar code; • Controlled access to the cryocontainer room and regular replenishment of liquid nitrogen by the assigned staff of SOL Company; • Alarm system in cell culture incubators to control variations in temperature and % of CO 2 ; • Periodic clinical revision of the patients' data chart by clinicians experts in the field.

Source of associated data
Most samples stored derive from the diagnostic activity of the Division of which the bank is part; therefore data derive from medical records, instrumental tests and laboratory reports.

Ethics statement
Muscle biopsies are obtained after informed consent of patients or parents (in the case of children

Bioresource type
Rare Disease and genetic biobank.

Type of sampling
Disease based, sampled in clinical care.

Disease status of patients/source
Patients affected (muscle tissue) and healthy/carrier relatives (DNA).

Clinical characteristics of patients/source
General data (age, gender, disease) and all patient related information that is stored in the clinical information system of the Institute (upon request).

Size of the bioresource
• To date: 5896 individuals/13,380 samples.
• Average of the incoming samples per year: approx.250 muscle samples, approx.100 cell lines and 300-400 DNA.• Average of the outgoing samples per year: approx.170 samples.

Vital state of patients/source
Alive at the sampling.

Pathology diagnosis
Histological for all patients, with immunochemical tests (immunohistochemistry and Western blot) and/or genetic diagnosis for most of them.

Control samples
Individuals that have undergone a muscle biopsy, but resulted unaffected by clinical and histological criteria.

Biospecimen type
Frozen muscle tissue; frozen stocks of myoblasts, fibroblasts, and lymphoblasts; DNA; resin-embedded muscle samples.Sera are being collected in a few cases.

Access criteria
To access the Biobank service, rules are applied to all researchers, including Biobank staff, according to criteria agreed among the TNGB directors, as follows: • The biobank must guarantee the storage of an adequate aliquot of patient samples in order to offer the possibility of retrospective analysis on the basis of more recent scientific acquisitions; • Sample distribution should be only to qualified professionals working at research or medical institutions involved in health-related research or health care; • Project employing the samples must be compliant with the biobank mission; • Justification for use must be convincing (description of the research project and expected results should be sound, the PI scientific track record should be adequate, and a grant sponsor be present); • Results obtained using the samples including results relevant for patients' health should be made available in confidence to the biobank director and the biobank should be acknowledged in the resulting scientific publication; • Shipping costs and partial coverage of the cost of some basic procedures related to the Biobank distribution service (cost recovery) should be paid to the biobank.

(4) Reuse potential
Reuse is possible for diagnosis and research in the field of the disease for which the samples have been acquired, with the permission of the biobank director, who must be contacted directly by the applicant.If necessary, a new specific consent is sought and a new MTA issued.
Mora et al: Biobank of Cells, Tissues and DNA from Patients with Neuromuscular Diseases Art. 1, p. 3 of 5 Shipping temperature from patient/source to preservation or research use Mora et al: Biobank of Cells, Tissues and DNA from Patients with Neuromuscular Diseases p. 2 of 5