Chapter 4

Veterinary Biobank Facility: Development and Management

for Diagnostic and Research Purposes
Tina Lombardo, Silvia Dotti, Riccardo Villa, Stefano Cinotti,
and Maura Ferrari

Abstract
Biobanking is an essential tool for ensuring easy availability of high-quality biomaterial collections that
combine essential samples and epidemiological, clinical, and research data for the scientific community.
Specimen collection is an integral part of clinical research. Indeed, every year throughout the world, mil-
lions of biological samples are stored for diagnostics and research, but in many fields the lack of biological
material and models is a major hindrance for ongoing research. A biobank facility provides suitable samples
for large-scale screening studies and database repositories. Software dedicated to biological banks simplify
sample registration and identification, the cataloging of sample properties (type of sample/specimen, asso-
ciated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality
assurance, and specimen availability characterized by well-defined features. Biobank facilities must adopt
good laboratory practices (GLPs) and a stringent quality control system and also comply with ethical
issues, when required.
The creation of a veterinary network can be useful under different aspects: the first one is related to
the importance of animal sciences itself to improve research and strategies in the different branches of the
veterinary area, and the second aspect is related to the possibility of data management harmonization to
improve scientific cooperation.

Key words Biobanking, Biological resources, Quality controls, Cryopreservation, Veterinary medicine

1 Introduction

The word “biobank” is a little over a decade old. It has been used
since 1996 [1] to describe collections of various types of biological
samples. Biobanks have been defined in a variety of different ways
and this has been a major challenge [2]. As reported by Hallmans
and Vaught, a biobank may be defined as the long-term storage of
biological samples for research or clinical purposes [3]. In addition
to storage facilities, a biobank can comprise a complete organization,
including biological samples, data, personnel, policies, and proce-
dures, for handling specimens and performing other services, such

Mónica V. Cunha and João Inácio (eds.), Veterinary Infection Biology: Molecular Diagnostics and High-Throughput Strategies,
Methods in Molecular Biology, vol. 1247, DOI 10.1007/978-1-4939-2004-4_4, © Springer Science+Business Media New York 2015

43
44 Tina Lombardo et al.

as database management and scientific studies planning [3]. The

Organisation for Economic Co-operation and Development,
OECD, defines biobanks as “a collection of biological material, the
associated data and information stored in an organized system for
a population or a large subset of a population” [4]. Based on this
definition, these infrastructures can be seen as components of dedi-
cated research facilities, which enable the linkage of samples, their
storage locations, and their “quality assured history since their
original time of sampling” with associated, well-documented, clin-
ical data. For managing such a biobank, Yuille et al. [5] have
emphasized that an efficient information infrastructure is a critical
component in life-science research and have even created the term
“biobank informatics” as a discipline, with the purpose of identify-
ing the best coding systems available for storing and retrieving
biobank information.
In veterinary medicine, the activity of a biobank should not be
limited to the collection of biomaterials, but should involve the
processing, cataloging, and distribution of samples to the scientific
community for research purposes and therapeutic applications.
The field of “biobanking” has evolved from a simple collection
of frozen specimens to a virtual biobank in response to regulation
changes, the advances made in biological sciences, and the advent
of the computer chip [6].
Biobanks play a pivotal role in research, diagnosis, and pro-
duction, as they provide high-quality biomaterial that is otherwise
difficult or impossible to access for scientists of both human and
veterinary medicine; in fact, the availability of high-quality sam-
ples is a reported problem and samples stored in improper condi-
tions may lack the expected quality for their use [7]. Human
biological specimens have been collected and distributed for many
decades and have played a key role in the understanding and treat-
ment of human diseases; in fact, in human medicine, the network
of biobanks is advanced, but in veterinary medicine such network-
ing is at a very early stage. Human specimen collections vary
widely in biological resources and structure. Furthermore, mod-
ern cryopreservation techniques allow the storage of different
types of biological samples, ready to be used upon request. The
role of biobanks is gradually increasing as they have been identi-
fied as a key source of biological tissues to be used in transplanta-
tion, tissue engineering, regenerative medicine, pharmaceuticals,
and diagnostics. One of the major objectives in Europe is to define
concerted actions aimed at developing and sharing best practices
and standardizing the procedures [8]. Currently, there is consid-
erable variation in national laws and local practices applied to the
processing and storage of biological samples in the various coun-
tries throughout the world [2]. This variability complicates the
conditions for collaboration among scientists from different coun-
tries, reducing the future sharing of research data and samples and
 Biobanking of Biological Samples for Diagnosis and Research 45

the possibility of carrying out collaborative research if regulations

are not harmonized [9]. A process to reduce this variability has
been initiated, and several sets of standards and best practices,
helpful in improving quality of standards for biobank operations
and biospecimens, have been published [10].
The Istituto Zooprofilattico Sperimentale della Lombardia e
dell’Emilia Romagna (IZSLER) is a veterinarian public institution
of the Italian Ministry of Health that includes 16 off-site laborato-
ries and has its headquarters in Brescia. In recent decades, almost
all of these laboratories involved in different areas have stored sam-
ples of animal origin, resulting from routinely diagnostic proce-
dures and research. This activity has created several biological
resources and materials collected according to different quality cri-
teria and without harmonized procedures, depending on the main
competence areas of the departments. In order to enhance interop-
erability and ensure the availability of biological material with well-
defined quality features, a centralized infrastructure for storing
biological resources in Brescia has been developed. This procedure
allows the storage of samples that are controlled by standardized
processes. At the same time, within national and international bio-
bank collaborations, studies are performed using a large number of
samples from different sources, which could be used for epidemio-
logical researches. For such studies, it is of pivotal importance to
establish quality criteria regarding the type of samples, storage con-
ditions, and the availability of specific information. The subsequent
paragraphs will describe the experience of IZSLER with the devel-
opment of a biobank infrastructure, mainly based on biological
resources, to be used in the field of diagnosis, research, and micro-
organism production.

2 Development of a Veterinarian Biobank Infrastructure

The development of a veterinarian biobank infrastructure was

based on the following steps: sample census, data recording, qual-
ity management system and quality controls, safety and security of
the storage area, and personnel.

2.1 Sample Census The first step consisted in the inventory of the existing resources
located in all the different facilities, the purpose of their use and
storage, and the amounts of existing aliquots; this approach was
necessary to evaluate the space and equipment required for their
preservation. In particular, the main aspects were to define the qual-
ity standards required and the various biohazard levels, storage con-
ditions, and selection of specific tests to be performed in order to
ensure the identity, purity, and suitability of each specific sample.
The outcome of this investigation has allowed the detection of 17
different types of biological resources equivalent to 42,683 stored
46 Tina Lombardo et al.

samples. Biological resources from different animal species were

subdivided into the following sections: microbiology and parasitol-
ogy (bacteria, Chlamydiaceae, fungi, metazoans, mycoplasmas,
Prototheca algae, protozoans), virology and prions (viruses, viral
pathological materials, prions), biological products (cell cultures,
field sera, hybridomas, IgG anti-immunoglobulins, immune sera),
and others (chemical compounds, histological materials). On the
basis of these findings, it was decided to proceed with the creation
of a centralized infrastructure. Biological resources included in the
biobank were selected for their suitability, depending on the type of
the sample collected and according to specific quality criteria. Due
to the presence of unique sample types, for some kind of biological
resources (i.e., bacteria, viruses), three different aliquots were con-
sidered: the base matrix (master sample) to be used for the prepara-
tion of new batches, the working batches originated from the master
to be used, and finally the “backup sample” as a deposit rate
(usually equal to ¼ of the amount fitted). Storage in a different
remote infrastructure is aimed at preserving samples in the event of
a technical failure or calamity. This additional “mirror banking” of
samples ensures that, if samples are compromised for whatever rea-
son, replicate undamaged aliquots will still be available.

2.2 Data Recording In order to ensure the traceability of biological samples, a database
has been developed with different data recordings, each one spe-
cific for a biological resource. All samples are identified by a code
and labeled with a bidimensional bar code. Data regarding all indi-
vidual samples are collected in a software database that allows the
number and position of the sample to be traced. In particular, bar
codes ensure major reduction of errors in all specimen-handling
processes and the possibility of tracking information about manip-
ulation activities according to standard operating procedures
(SOPs), quality control results, and loading and unloading order
management.
Standard operating procedures mean documented procedures
which describe how to perform tests or activities normally not
specified in detail in study plans or test guidelines. The database for
managing a biobank includes a web-based catalog, which lists the
access terms and conditions and the resource characteristics. The
catalog is intended to be used as a reference for scientists seeking
information about biological samples and data suitable for their
research.

2.3 Quality Currently, biobanks must follow internationally accepted guide-

Management System lines and best practices issued by ISBER (International Society for
and Quality Controls Biological and Environmental Repositories) [11] and BBMRI
(Biobanking and Biomedical Resources Research Infrastructure).
In particular, BBMRI aims to improve biobanking accessibility
and interoperability by harmonizing similar biobanks in different
 Biobanking of Biological Samples for Diagnosis and Research 47

locations and enriching the genotypic and phenotypic data [12].

All biospecimens should be handled according to well-defined
protocols, and the whole clinical “biobanking” process should be
fully documented, from seeking informed consent from the donor
or patient to collecting, transporting, processing, storing, and
retrieving the biospecimens [13]. Careful documentation of the
methods and conditions used during collection and processing
will increase the future value of specimens [14]. Furthermore,
quality control programs must be established to check compliance
with the standard operating procedures for specimen collection,
handling, and analyses [15, 16].
With regard to “animal” biobanks, informed consent is not
required; however, all other parameters must be performed accord-
ing to the same criteria. Biobanking work, including associated
laboratory handling specimens, has been performed in a standard-
ized way. It is recommended the use of SOPs with the aim of con-
tinuously assessing the methods, materials, and equipment
employed. SOPs should consider the specific requirements of anal-
ysis platforms and the biological questions to be addressed.
However, SOPs can only be applied to prospectively collected sam-
ples, whereas for old archived samples, processing has often not
been documented in sufficient detail without following specific
SOPs, as in the past there was no application of quality manage-
ment system, and storage of biological materials was performed by
scientists with no standardized procedures. In the context of qual-
ity management, the scientific use of this material therefore requires
special analyses to assess the proper preservation of biomolecules
[17]. The recommendations for biobanking activities, quality
assurance (QA), and quality control (QC) programs must be fol-
lowed to ensure the high quality of samples. As described by
OECD [18], good laboratory practices (GLPs) are “a quality sys-
tem concerned with the organizational process and the conditions
under which non-clinical health and environmental safety studies
are planned, performed, monitored, recorded, archived and
reported.” The principles of GLPs represent a managerial concept
in order to cover the whole organization process, beginning from
the planning and arriving to the reporting of the applied proce-
dures. Furthermore, this way of management facilitates the
exchange of information and contributes to the protection of
human health and the environment. Stability of biological samples
depends on the procedures from the time of acquisition to the time
of processing and storage; in fact, the use of anticoagulants, stabi-
lizing agents, the time from collection to storage, and the sample
handling temperature must all be controlled.
Biological samples can be stored for up to 30 years, but specific
protocols are required to reduce the damage induced by preserva-
tion techniques [19]. This issue has been widely reported with
proposals for rigorous quality controls for each step of the working
flow, including collection, processing, and storage [20].
48 Tina Lombardo et al.

These procedures aimed to assess the preserving conditions

depend on the type of biospecimen. Appropriate controls can be
applied to cell suspensions (cell viability assays, contamination
assays), to DNA and RNA (quantification and purity, function of
DNA-modifying enzymes), to tissue morphology, and to viral
batches (viral contamination tests, standard microbiological testing
methods, and real-time PCR). All strains enrolled in IZSLER collec-
tion are submitted to different cultural and molecular biology con-
trol tests, in order to evaluate their features, purity, and identity.
However, no appropriate quality controls exist for liquid bio-
samples (serum, plasma, urine, saliva, cerebrospinal fluid, syno-
vial fluid).
At IZSLER, the activity of the laboratories involved in bio-
banking is carried out using a quality assurance system in accor-
dance with the requirements of UNI CEI EN ISO/IEC
17025:2005 as well as ISO 9001:2008 quality system for cell cul-
tures. Biological resources are prepared and stored according to
internal procedures.

2.4 Personnel Laboratory activity is based on certain fundamental rules of good

microbiological practice for the safe manipulation of biological
samples.
Each operator (technician) must follow internal training
courses, specific for their activities, positions, and competencies.
The operators should have basic knowledge on various bio-
logical topics. First of all, microbiology is one of the main subjects
to know, as well as the isolation and cultivation of pathogens.
Another important aspect is represented by the information on dis-
infection of work surfaces, equipment, and environment. Personnel
should be aware of the need to minimize the production of aero-
sols and control other potential risks of contamination between
biosamples and workers.

2.5 Control, Safety, This is one of the main points that must be monitored and con-
and Security trolled. In fact, it represents a potential risk in the whole storage
of the Storage Area process. Starting from this point of view, monitoring temperature
within mechanical freezers and refrigerators is an important quality
assurance measure in addition to general building safety and secu-
rity to protect against fire, unauthorized entrance, and other usual
hazards. Furthermore, all freezers must be protected by a real-time
temperature monitoring and alarm system. Alarm systems should
be in place to monitor the temperature conditions of mechanical
freezers or, for nitrogen freezers, the liquid nitrogen level (Fig. 1).
Furthermore, a well-designed biobank software system has been
developed to provide the possibility of monitoring the temperature
values with an alarm service. Such temperature logging informa-
tion must be automatically transferred via electronic interface into
the biorepository management system and be linked with the
 Biobanking of Biological Samples for Diagnosis and Research 49

Fig. 1 Illustration of the real-time monitoring and alarm system of the IZSLER Veterinary Biobank facility

samples stored in the corresponding freezer. Any interruption of

electrical power should be compensated for within minutes by an
independent system, such as a generator with locally controlled
production of electrical power. Automatic data logging into a data-
base and the alarm service are very important for quality manage-
ment of a biobank. If a measured value drifts outside inner or outer
thresholds, an alarm is sent to staff members.

3 Sample Types and Preparation

Pre-analytical conditions are a key factor in maintaining the high

quality of biospecimens. They are necessary to achieve the highest
specificity of the laboratory test used for clinical diagnosis as well as
for accurate reproducibility of experiments in the field of biomarker
discovery. Inappropriate collection, handling, and storage of sam-
ples, as well as errors in data analysis and documentation, may all
contribute to the generation of irreproducible and unreliable data.
Preservation and optimization of biosample integrity to foster rel-
evant research results and outcomes is a guiding principle of sam-
ple management.
The field of veterinarian research is rapidly evolving with new
technologies and new standards. Samples are collected and stored
also for a long period of time (years) before being used. For this
reason, sample handling procedures should be defined and appro-
priated in order to guarantee a suitable use for the technology of
tomorrow.
50 Tina Lombardo et al.

Biological samples have different storage requirements. For

this reason, various approaches can be defined in order to obtain
the most correct procedure.

3.1 Blood Samples The routine use and collection of blood samples for diagnosis has
provided general information on optimal methodologies and
potential pitfalls.
Although procedures for collecting, processing, storing, and
shipping blood components are generally standardized and well
documented, several important factors need to be considered.
An important early decision in blood collection is whether to
collect anticoagulated blood (consisting of plasma, buffy coat, and
red blood cells) or coagulated blood (consisting of serum and red
blood cell clot). Hemolysis of the specimen affects the accuracy of
laboratory tests, particularly chemical and serological tests; there-
fore, it should be prevented by employing careful handling tech-
niques according to optimum needle size, proper handling of the
tubes, and proper pipetting techniques; however, if hemolysis is
observed (pink to red tinge in sample), this information should be
recorded [21]. In fact, hemolyzed samples would not be used for
proteomics analysis, but destroying them may be unnecessary as
any sample is worth saving, unless storage space is constrained.
Annotation of all pertinent information about the samples allows
the identification of potential factors that could influence out-
comes. Serum and plasma specimens are of better quality for analy-
sis if smaller-volume aliquots are initially prepared rather than
larger ones that have to be thawed, handled, and refrozen, perhaps
several times. Indeed, the ability to provide ready-to-use (RTU)
aliquots without additional handling steps facilitates the sharing of
samples and provides multiple replicates handled in an identical
manner. It is also important to consider new or alternative meth-
ods and reagents that may offer longer-term stability or increased
efficiency in the collection and preservation of blood [22, 23].

3.2 Cell Cultures The ability to cryopreserve and successfully recover cell lines has
been critical to the conservation of these biological samples, in
particular the preservation of stem cells and the preparation of
well-characterized cell banks. Indeed, the systematic storage and
establishment of cryopreserved banks of cells for the stem cell
research community is essential to the promotion of standardiza-
tion in stem cell research and its use in clinical applications. Despite
the significant potential for the use of stem cells in research and
therapy, they are challenging to preserve and have been shown to
be unstable after prolonged culture, often resulting in permanent
alterations in their genetic background, which ultimately alters the
phenotype of the culture. The working process related to cell cul-
ture isolation, amplification, control, storage, and shipment should
be carried out in accordance with a quality management system
 Biobanking of Biological Samples for Diagnosis and Research 51

such as ISO 9001:2008. Preparation and propagation of cell cultures

are performed as indicated by international guidelines, and their
sensitivity and reproducibility have been evaluated during inter-
laboratory tests. Each cell culture process provides ideal conditions
for the growth of many organisms [24]; for this reason, particular
attention must be placed on quality controls that should be per-
formed on all samples with the purpose of assessing their purity
and safety. These tests are carried out using microbiology, virology,
serology, and molecular biology methods, as reported by the
European Pharmacopoeia or other international guidelines. These
assays are mainly based on in vitro tests. However, in vivo methods
are used on laboratory animals, in the absence of validated in vitro
systems or as indicated by international guidelines. In particular,
the tests usually performed are aimed at detecting contamination
from bacteria, fungi, yeasts, and mycoplasmas as well as animal and
human viruses for human cell lines. Furthermore, bacterial endo-
toxin level, tumorigenicity, and cross contamination are investi-
gated. Tests on cell cultures for detecting bacteria, fungi, and yeasts
must be performed in an isolation unit, according to the European
Pharmacopoeia.

3.3 Chlamydiaceae Serological and molecular screening focused on the identification

of chlamydial infections is routinely performed. Isolation of chla-
mydial species is carried out on cell culture.
Identification of the species belonging to the Chlamydiaceae is
carried out using molecular tests, i.e., real-time PCR and PCR-
RFLP analysis, which amplify specific genome sequences. The iden-
tification of new chlamydial species not yet classified is also
performed using new real-time PCR and specific gene sequencing.

3.4 Field The field sera include samples from different animal species, mainly
and Immune Sera farmed, pets, and wildlife animals. The samples are collected by
blood, sent to IZSLER to be tested in accordance with national or
regional control programs or for specific serological tests. All these
samples can be used as positive reference due to the presence of
antibodies toward a specific pathogen, turning them very useful for
retrospective serological surveys and validation of innovative sero-
logical tests. All serum samples collected and stored in the biobank
are tested toward a panel of pathogens with the aim to know the
presence/absence of antibodies.
Different serological techniques can be used and these are
reported in specific sheets. Titers of humoral antibodies of serum
samples are also registered and values are performed at preestab-
lished intervals. The immune sera consist in polyclonal sera obtained
from different animal species (rabbit, goat, mouse, rat, guinea pig)
and toward several bacterial and viral antigens. They have been pro-
duced with antigens mostly purified and have a specificity panel
available at the facility where they have been produced.
52 Tina Lombardo et al.

3.5 Hybridomas The hybridomas that produce monoclonal antibodies are selected
to be used in in-house diagnostic assays. They were generated to
produce monoclonal antibodies against a wide spectrum of viruses,
bacteria, and proteins, mainly of veterinary interest, and immuno-
globulin isotypes of various animal species that were shown to be
strategic tools for both research and diagnostic purposes.
Hybridoma cultures are usually collected for freezing during the
exponential growth phase and then are submitted to a double
series of cloning procedure to assure the stability of the hybridoma
and the clonality of the produced antibody. The monoclonal anti-
bodies expressed by each hybridoma are controlled through a
series of immunological assays aimed at identifying and character-
izing their reactivity profile.

3.6 Microbiological Bacterial/fungal strains are identified by either phenotypic or

and Parasitological genotypic tests. The former consists of microscopic observation
Samples and isolation in specific culture media. Identification is made
through biochemical tests performed using commercial tests like
the “API” or the “Vitek” systems. Genotypic tests, mainly PCR-
based, are carried out to detect genes of virulence. Moreover, the
16S rDNA gene sequence analysis can be used to confirm the iden-
tity of bacterial species.
Parasites are identified using either phenotypic or genotypic
tests. The former consists of microscopic observation and compari-
son with identification keys. Genotypic tests, mainly PCR-based,
are carried out to identify genus or species.

3.7 Prototheca Algae The identification of Prototheca algae (species and eventually sub-
species) is currently carried out through microscopic examination,
growth in specific culture media, and molecular assays (HMR-
PCR, end point PCR, or sequencing).

3.8 Tissue Samples Tissue samples can be stored in different ways, depending on their
intended purpose. Indeed, molecular tools for tissue profiling,
such as real-time PCR and expression microarrays, generally
require collection of fresh frozen tissues as sources of high-quality
RNA. Frozen tissue sections are made from high-quality tissues
immediately snap-frozen in liquid nitrogen after being excised and
identified by a pathologist. Available tissues include normal, dis-
eased, and tumor animal tissues. The tissues excised are immedi-
ately frozen by liquid nitrogen and then stored at −80 °C. Tissue
sections of 5–10 μm in thickness are mounted on positively charged
glass slides. Furthermore, 0.4–0.7 g samples of standard size could
be frozen in blocks into liquid nitrogen for 20–40 min after surgi-
cal excision. Otherwise, tissues could be fixed in 10 % neutral for-
malin for a minimum of 24 h and stored as paraffin blocks of
0.5 × 1 × 1 cm. Formalin fixation and paraffin embedding (FFPE)
 Biobanking of Biological Samples for Diagnosis and Research 53

preserve the morphology and cellular details of tissue samples.

Thus, it has become the standard preservation procedure for diag-
nostic surgical pathology [25]. Historically, the archived FFPE
blocks have been successfully used for immunohistochemistry
application. However, formalin-fixed archival samples are known
to be poor materials for molecular biology applications due to the
irreversible modifications caused by formalin fixation on macro-
molecules. In the last ten years, there has been an exponential
increase in the development of molecular assays using FFPE blocks.
At present, when sections from FFPE blocks are to be used for
molecular extraction, focus is placed on the time of fixation in for-
malin in order to avoid over-fixation. The advances in the field of
molecular biology techniques have attempted to overcome the
issue of formalin cross-linking and have successfully extracted
DNA, RNA, and proteins, although fragmented.

3.9 Viruses and Viral Cell-associated viruses that can be grown in adherent or suspen-
Pathological Materials sion cell cultures or chorioallantoic membranes of embryonated
hen’s eggs can be isolated from several types of samples. The main
principle of isolating viruses is to choose the most suitable cell line
and mechanically lyse infected cells and subsequently carry out sev-
eral amplification passages to increase the titer in order to produce
the master sample and then the working samples. Virus batches are
tested for potential microbiological and viral adventitious contami-
nations; the tests are performed using microbiology, virology,
serology, and molecular biology methods. The primary sources of
potential viral contamination come from infected animal tissues
used to prepare biological reagents and media and during labora-
tory manipulation. Virus detection and identification can be made
by employing several methods, mainly based on serology tests
using monoclonal antibodies and standard and real-time PCR.
These assays amplify specific viral genome sequences known to be
characteristics of a virus with a nucleotide sequence available in
database collections. Extraneous viral contaminations can be veri-
fied through tests based on molecular biology techniques that
allow the detection of viral DNA and RNA of other viruses.
Mycoplasma contamination can be detected using real-time PCR
methods. Bacterial contamination is determined through inocula-
tion of nonselective culture media. In addition, electron microscopy
is available for viral detection and identification using negative
staining methods. Such “catchall” methods (able to detect even
non-suspected/unknown viruses) benefit from “Airfuge” ultra-
centrifugation, increasing the sensitivity of the detection level.
Indeed, immune electron microscopy methods (IEM and IEM
gold) based on the use of hyperimmune sera and/or monoclonal
antibodies may help viral identification and classification.
54 Tina Lombardo et al.

Fig. 2 Cryogenic area for preservation of biological resources

4 Storage of Biological Resources

Proper storage requires the use of cryovials and labeling systems

that will withstand the intended storage conditions: vessels, labels,
and bar codes or other printing systems are chosen for extended
storage periods.
Samples have been deposited in freezers or other appropriate
storage containers according to specific storage systems in order to
preserve several parameters known to influence the condition of
biospecimens.
In accordance with features, intended use and estimated length
of storage, specimens may be stored at: room temperature, 4 °C,
−20 °C, −80 °C, or −196 °C (vapor phase nitrogen) (Figs. 2 and 3).
Temperature is a major variable in specimen management.
Moreover microorganisms and viruses can be submitted to lyophi-
lization process that allows preserving viability for a long time.
These freeze-dried samples can be stored at 4 °C or at −20 °C,
reducing the necessity to have freezers with lower temperature
(−80 °C); for these reasons, this is a practical and efficient method
for prolonged storage, less expensive, and more available in
emerging countries.

5 Application of Biological Resources

Two major formats of biobanks with several subtypes can be distin-

guished: the population-based biobank and the disease-oriented
biobanks, each with distinct and complementary scientific value.
 Biobanking of Biological Samples for Diagnosis and Research 55

Fig. 3 Freezing area for preservation of biological resources

The most common format is the longitudinal population-based

biobank, with biological samples and data from randomly selected
individuals of a general population, used as resources for future
unspecified research [26–28]. The specific strength of this format
is the assessment of the natural frequency of occurrence and pro-
gression of common diseases, with special emphasis on predispos-
ing genetic variants and environmental risk factors. In contrast, in
disease-oriented biobanks, which may contain tissue, isolated cells,
blood, or other body fluids, specimens are collected in the context
of medical diagnosis and treatment. These biosamples allow the
comparison of different disease stages and/or forms of treatment
at a molecular level, in order to evaluate biomarkers for the diag-
nosis of a disease or assessment of risk/predisposition and progno-
sis (research purpose), monitoring the recurrence of diseases,
prediction of mortality, and response to therapy [29]. The devel-
opment of precisely defined clinical data elements (CDEs) may
help to ensure that clinically relevant data are collected at each time
interval [12]. In veterinary medicine, the wide availability of bio-
logical samples stored in biobanks provides the basis for research,
leading to a better understanding of animal disease biology and the
development of new diagnostic tests that require the use of biosa-
mples with well-defined features [13]. The European Technology
Platform for Global Animal Health (ETPGAH) has identified the
lack of biological material as one of the main gaps in the develop-
ment of new effective tools for the control and prevention of ani-
mal diseases. Biobanks represent an important tool in improving
epidemiological research dependent on the availability and quality
of the biomaterials but also on the collection of associated data [6].
In particular, for retrospective studies and longitudinal designs for
56 Tina Lombardo et al.

evaluating the course of diseases, the requirements for obtaining

time-specific data are even stronger. Furthermore, biological mate-
rials are a critical resource for genetic research. Major research in
genomics is being pursued to improve the efficiency of selection
for healthier animals with disease resistance properties. Molecular
genetic tests have been developed to select farm animals with
improved traits, for example, removal of the porcine stress syn-
drome and selection for specific estrogen receptor alleles [30]. The
sequencing of the genome to identify new genes and unique regu-
latory elements holds great promise in providing new information
that can be used for livestock production. Currently, in vitro
embryo production and embryo transfer are being the preferred
means of implementing these new technologies to enhance effi-
ciency of farm animal production. Furthermore, the possibility of
developing an integrated approach of genomics and proteomics
using bioinformatics is essential for obtaining complete use of the
available molecular genetic information. The development of this
knowledge will benefit scientists, industry, and breeders consider-
ing that the efficiency and accuracy of traditional farm animal selec-
tion schemes will be improved by the implementation of molecular
data into breeding programs.

6 Role of Biobanks in Veterinary Medicine

A decade ago, biobanks were simply repositories of biospecimens.

Clinical data of limited quantity and quality had to be retrieved
from hospital information systems retrospectively [31]. Nowadays,
biobanks are much more than just collections of biospecimens; in
fact, the legal entity “biobank” refers to the management of the
samples stored under that label, the huge amount of either clinical
or experimental data, and the requirements for their cession [32].
In human medicine the concept of storing biological material rep-
resents a consolidated aspect that involved either ethical or scientific
tools with a particular regard to both individual and community
interests [33]. The recent advances in veterinary medicine have
allowed the creation of a biobank for storing animal samples. These
resources should be useful for several different aspects. The first is
related to the role of veterinary medicine itself, as the biobank
allows more detailed information to be obtained regarding all bio-
logical resources available, for improving knowledge in several vet-
erinary areas such as food safety, animal health, global climate
change, public health emergency, and genetic conservation of bio-
diversity. The second aspect is related to the possibility of crossing
traditional discipline boundaries for future interdisciplinary research,
pathogenetic studies on eubacteria and viruses, detection of emerg-
ing zoonoses, epidemiological surveys and phylogenetic trees,
and genetic evolution studies that play a key role in public health.
 Biobanking of Biological Samples for Diagnosis and Research 57

The use of biobanks for epidemiological research does not only

depend on the availability and quality of the biomaterials, but also
on the collection of associated information, such as anamnestic and
epidemiological data, purity, and identity of the samples, (testing
performed) depending on the biological resource, allowing large-
scale screening studies.
Furthermore, experimental investigations and the development
of diagnostic tests and vaccines could be transferred to national
and international industrial branches.
The BVR (Biobank of Veterinary Resources) of IZSLER infra-
structure integrates existing and newly established animal biological
samples and data collections, resources, technologies, and exper-
tise to facilitate high-quality medical research, improving stan-
dardization and international cooperation. Furthermore, this
infrastructure will provide the flexibility needed to facilitate growth
of the network with new members and partners. An additional ser-
vice includes the possibility of storing human and animal biosam-
ples, offering repository services, and ensuring governance systems
that provide quality assurance.

7 Remarks and Future Perspectives

The field of biobanks is very complex and diversified, as it deals with

the collection, treatment, storage, distribution, and computeriza-
tion of biological material, not only of human, animal, and plant
material, but also that from sources such as fossils and microbes.
Biobanks and biospecimens are essential components for several
areas of clinical and basic research, and, in fact, public awareness of
biospecimens and biobanking has grown significantly [19].
The primary purposes for improving biobank networks are to
harmonize and spread quality banking practices to distribute bio-
specimens with well-defined features and associated data. One of
the major obstacles for developing a uniform system of regulation
across Europe is the lack of an agreed definition of “biobank” [2].
Biological samples collected at the IZSLER Veterinary
Biobank infrastructure could be a key resource for the scientific
community. The possibility of creating a veterinary biobanking
and biological resource infrastructure in Italy, and then in Europe,
will allow the harmonization of standards for sample collection,
storage, and techniques of analysis, data management, and prepa-
ration of a specific database. One solution to address the issues of
standardization of quality and capacity should be the creation of
biobank networks [34].
The main challenge of an international biobank network is bal-
ancing the need to centralize specimens and resources with the
reality of delocalized collection activities, especially in a clinical
context. Currently, these networks could be considered as a trade
58 Tina Lombardo et al.

union between human and veterinary medicine, which could

significantly develop a multidisciplinary approach for discovering
new frontiers within life sciences and patient care.
Interoperability in biobanking represents the key for improv-
ing national and international research collaborations [35].
Furthermore, the integration of human and veterinary biobank
initiatives should harmonize databank diagnostic and typing meth-
ods. There is a considerable variation in national laws and local
practices regarding the processing and storage of biological sam-
ples, personal information, and data recorded among countries
around the world. This variability complicates the conditions for
collaboration between scientists from different countries, reducing
future sharing of research data and samples. Furthermore, if regu-
lations are not harmonized between nations, the possibility of car-
rying out collaborative research decreases [29].
Millions of biological samples are stored every year through-
out the world for diagnostic and research, but in many fields the
lack of biological material and models is a major hindrance for
ongoing research. Currently, variability regards the collection, pro-
cessing, storage, and relative data of the majority of biospecimens
available for research and diagnosis. Such heterogeneous practices
provide biospecimens of unknown molecular integrity and con-
tribute to irreproducible results, impeding the development of
more effective therapeutics and diagnostics [36].
Biobanking plays a crucial role in providing access to high-
quality biomaterial, essential not only for human medicine, but
also for veterinary medicine. Modern techniques of cryopreserva-
tion allow the long-term storage of biological samples for clinical
or research purposes comprising a complete organization includ-
ing biological samples, data, personnel, policies, and procedures
for handling specimens and performing other services, such as
managing the database and planning scientific studies.
Software dedicated to biological banks facilitate sample regis-
tration and identification, the cataloging of sample properties (type
of sample/specimen, associated diseases and/or therapeutic proto-
cols, environmental information, etc.), sample tracking, quality
assurance, and specimen availability. Biobank facilities must adopt
good laboratory practices and a stringent quality control system
and comply with ethical issues when required [8].
The necessity for accessing high-quality specimens has increased,
along with the necessity for standards to guide the proper collec-
tion, processing, storage, and distribution of specimens.
Perhaps more importantly, it is essential to know that the sam-
ple used for research and diagnosis is accurately characterized; once
the most critical points in a biospecimen processing method have
been identified, specific tests or markers to assess the quality of the
biospecimen are needed.
 Biobanking of Biological Samples for Diagnosis and Research 59

If appropriately collected, documented, and stored, biospecimens

are a valuable resource that can help answer current and future
scientific questions, to meet the needs of emerging technologies
[37]. The lack of concerted efforts, together with heterogeneous
policy approaches and practices, jeopardizes international collabo-
ration and the sharing of samples and data. A new attitude and
strategy for sharing data and promoting cooperation in the field of
biobanks may not only have a great impact on public health pro-
grams, but also in the development of new biomarkers and drugs.
The importance of biobank networking has been emphasized
by many authors, and there are several major biobank networking
initiatives worldwide. Currently, international initiatives are emerg-
ing in order to find ways to cooperate even in the context of het-
erogeneous ethical and legal frameworks in which the national
biobanks are being established.

Acknowledgments

The authors thank Dr. G. Bontempi, L.R. Scorrano, colleagues of

IZSLER, and the personnel of Biobank Infrastructure for practical
support and collaboration.

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