Pathology and Biobanking
Canan KELTEN TALU, Muhammed Hasan TOPER, Yasemin ŞAHIN, İbrahim Halil ERDOĞDU
Department of Molecular Pathology, Graduate School of Health Sciences, Dokuz Eylul University, IZMIR, TURKEY
Keywords: Biobank, Human tissue biobank, Pathology-centered biobank, Quality control, Sustainability
Biobanks are units where high quality and long-term protection of biomaterials is maintained. This system, in which biological materials and
data are systematically recorded and stored, is a unique resource for the study of the pathophysiology of disease, the development of diagnostic
biomarkers, and working with human tissues for the potential discovery of targeted therapeutic agents. At this point, the pathology unit plays
a unifying and complementary role between the clinical and core disciplines and offers optimal management of the patients biomaterials for
diagnostic and research projects. The aim of this article is to present general information with regard to a biobank constructed for the storage
of tumor tissue and blood biospecimens.
Ethical issues (informed consent, protection of confidentiality and privacy, and secondary use of biospecimens) and the information technology
system (collection, systematic recording, backup and protection of clinical information) are important issues in biobanking. The selection of
freezers to be used in storage (mechanical freezers, liquid-vapor nitrogen tanks), and if mechanical freezers are preferred the establishment of
the relevant infrastructure and support team (such as additional power units for protection from power outages), the preservation of materials by
aliquoting in different freezers, ensuring financing so as to afford the cost of the infrastructure, and implementation of all these dynamics while
adhering to international guidelines are of the utmost importance.
It was during the mid-1990s that researchers began to
consider collections of biospecimens as a resource for their
projects (1). However, the value of biological materials
reached the biggest impetus with sequencing of the human
genome in 2001. Additional popularity of biobanks was
ensured by a Time Magazine article in 2009 where biobanks
were identified among the top 10 ideas changing the world
with regard to health and well-being (1).
A biobank is a valuable resource to access high-quality
human biospecimens while preserving them over the long
term. Those biospecimens may be used to elucidate the
pathophysiology, diagnoses, and finally the treatments
of diseases (2). Biobanks (also called Biorepositories)
are defined as the infrastructures that enable the
collection, handling, storage, retrieval, and distribution
of biospecimens (3). A high-quality biorepository should
adhere to standard operating procedures (SOPs) and
disseminate best practices for annotating, collecting,
processing, storing and retrieving biospecimens (4). The
term Biological Resource Centre (BRC) describes the
combination of infrastructure, facilities, and resources.
Therefore, tumor biobanks are basically BRCs. The
Organisation for Economic Cooperation and Development
(OECD) describes biobanks as service providers and repositories of living cells, organisms, cells and tissues, and
of information relating to these materials (5).
Biobanks comprise a wide range of specimen types and
sample collection formats, ranging from populationbased
biobanking of specimens from healthy individuals
to specific diseased tissue specimens obtained by surgical
interventions (6). Thus the main objective of biobanks
is to share their collections with national/international
communities in order to improve biomedical research
and health care (5, 7). The protection of the privacy of
sample donors and informed consent must be ensured
for biobanks (8). Furthermore, bioinformatics, long term
financial support and administrative sustainability are the
other important issues for maintaining biobanks (9).
Pathology is the fundamental component of hospital-based
tissue biobanking (9, 10). Pathologists provide essential
diagnostic information for the treatment of the patients and
can also make decisions on the sampling of tissues for the
biobank as well as optimal preservation of biospecimens.
Tissue biobanking is of particular importance for the use of
novel biomarkers in clinical trials and the application of new
technologies. Pathologists therefore act as a bridge between
the clinicians and the researchers. In this review, we aimed
to present information related to tumor biobanking.
ETHICAL AND LEGAL REGULATIONS FOR
The developments in science and technology in the last
half of the twentieth century and the vast accumulation
of knowledge in health services, medical applications and
biomedical sciences have resulted in new ethical problems
where traditional medical ethics principles are inadequate.
In the Medical Ethics Manual published by the World
Medical Association, it is stated that bioethics is a more
comprehensive field than medical ethics (11). The subject
of bioethics, which has especially developed following
the progress made in modern gene technologies, requires
a global approach to ethics. Gene technologies have first
aimed to add new perspectives to research, as well as
contributing to a better quality of life for human, animal
and plant species. However, they have been alarmingly
found over time to potentially lead to abuse of technology
and unpredictable damage.
In addition to the problems brought about by modern gene
technologies, the concept of value has been questioned
again in various conditions emerging in the field of
bioethics (such as research using human subjects, artificial
termination of pregnancy, prenatal diagnostic methods,
genetic counseling, assisted reproductive techniques,
definition of death, and organ transplantation) (12).
Physicians and health professionals interested in this issue
had to make health-related decisions in such an area where
values were being questioned. The Universal Declaration
on Bioethics and Human Rights was adopted at the
UNESCO General Conference assembled on October 19,
2005 in order to propose principles related to these issues
and the relevant decisions to be made, and an important
contribution was made to the field of bioethics (13). The
Declaration, although not binding, is an international
legal document that comprehensively emphasizes the
relationship between human rights and bioethics. This
document sets out the following bioethical principles;
Human Dignity and Human Rights
Benefit and Loss
Autonomy and Individual Responsibility
Consent of individuals who are not competent to give
Respect for the individual and respect for the integrity
of the individual
Privacy and Confidentiality
Equality, Justice, Equity
Respect for cultural differences and pluralism
Solidarity and Cooperation
Social responsibility and Health
Protecting next generations
Protecting the environment, biosphere and diversity of
the species (13).
The main objective of the Universal Declaration of Bioethics
and Human Rights is to bring together the fundamental
principles for biomedical research and clinical practice in
accordance with the international law on human rights
(13). In this respect, it is an important step in the quest
to develop bioethical standards worldwide. However, the
declaration has no intention of developing a new bioethical
Biobanking has become more popular with the completion
of the Human Genome Project. The conditions that make
biomarketing legally hazardous can be specifically listed as
Biospecimens can be easily obtained without the
consent of the person concerned,
Biospecimens can be easily misused,
The genetic data obtained are permanent personal
They contain very important data for the life of the
They contain information about the family and the
group of the person concerned;
The data carry the potential to be used for discrimination,
The use of the data by others creates scientific and
There is the possibility of neglecting personal rights in
case of misuse.
The predominant ethical issues related to biobanks include
informed consent, protection of confidentiality and privacy,
the secondary use of biospecimens, and profit sharing.
Informed consent is an important concept in research
as well as applications of therapeutic medicine. On the other hand, biobank-based research has led to the need
to reconsider traditional research ethics. In particular,
developments in research using human-based biological
materials have necessitated the identification of a new
informed consent model for biobank research (15). The
informed consent obtained from the subjects for biobank
research is more comprehensive and detailed than the
ones used in daily practice. Consent for biobank research
should therefore be obtained in written form and signed
by the subject. Various provisions have been established
by international institutions (such as the World Medical
Association, European Union and UNESCO) for the
realization of informed consent in daily life. The basis for
this sensitivity goes back to the Nuremberg Code (1947) and
the Declaration of Helsinki (1964), which prohibited the
inhumane use of individuals (16). Although the principles
of informed consent cannot protect the individual on their
own, they allow making a decision on how the materials
and data to be obtained in the research will be used (16). In
case of biological materials, it can be predicted that the data
obtained as a result of the research may pose a risk to the
The consent forms to be used in biobank-based studies
generally include the following items (15, 18, 19);
Respecting the autonomy of the participants, and
recognizing the benefits and risks, if any,
Type of biospecimens to be collected and the relevant
Research projects, objectives and research data,
The purpose of the research (the collection of samples
for a particular study requires a separate consent system.
In other words, the participants provide consent for a
single study. Biobanking requires a different consent in
order to provide a source where the long-term stored
samples can be used in many studies),
Storage period of the biospecimens,
Principles of sharing the data and biospecimens with
other research organizations,
Principles and procedures for biospecimen and data
access by the researchers,
Obtaining permission to collect health-related records
from other databases,
Procedures for re-contacting the participants;
Regulations on the privacy and confidentiality of the
Restrictions for anonymization procedures and reidentification
of the biospecimens;
Feedback for the research results and methods of
The right to withdraw from the research;
Arrangements in case of failure or death;
Regulations regarding sharing of gains;
Possible commercial agreements indicating that the
participants will not earn any business profits.
Although the consent process is implemented by many
countries, it may vary in terms of national regulations. This
may cause problems in international projects that require
the use of biobanks (15).
Biobank-based studies are divided into two groups as those
related to retrospective biobanks (previously collected
biospecimens) and prospective biobanks (newly collected
biospecimens) according to the collection time of the
biological material and data contained. Informed consent
indicates different conditions for studies on these two
groups of biobanks.
Informed Consent in Retrospective Biobank Research
Informed consent for retrospective biobanks containing
previously collected and archived biological materials in
medical care units and their data is a controversial issue
(15, 20). The general trend in legal regulations is to permit
the use of previously collected biological biospecimens and
related data without an approval if they are anonymous
and meet certain conditions (15, 20). However, consent
requirements may vary from country to country depending
on the current legal situation. This also affects the evaluation
period of the consent process.
Informed Consent in Prospective Biobank Research
A prospective biobank is established to create new
infrastructures at the national and international level so that
biospecimens can be used in projects that are scheduled at
the time of collection or later on. A large number of national
and international participation for each project requires all
parties to provide the same type of consent (if possible).
The relevant regulations include some rules that ensure the
autonomy of the participants such as volunteering by the
participants, comprehensibility of any information to be
obtained, and systematically providing such information
before starting any research project (20).
The informed consent forms for biobank research should
include the following concepts as identified by the
international literature and documents on ethics (15);
General consent: Consent allows the use of biological
biospecimens and related data at the time of their
acquisition and in the future.
Specific informed consent: Permits the use of biological
biospecimens and associated data only for the research
project at the time they are received.
Partially limited consent: Permits the use of biological
biospecimens and associated data for current specific and
potential future research.
Multilayered consent: Requires a detailed description of
the various options related to the research topic.
Dynamic consent: This is an internet-based interactive
consent model that has been developed in recent years
where the participants play an active role in the decisionmaking
Confidentiality and Privacy
The personal information contained in biobanks
is considered private information. The use of this
information is regulated by the consent of the individual
and the decisions of the researchers, ethics committees and
government bodies. Protecting the identities of biobank
data holders is important both to ensure the confidentiality
of the participants and to protect the privacy of the
biospecimens and related data (15).
There are four main sample types in biobanks as regards
protecting the privacy of the participants. These are:
Anonymous biospecimen: Biospecimens obtained
during community screening from individuals with
Anonymized biospecimen: Biospecimens that were
obtained from identified individuals but anonymized
before being studied
Identifiable biospecimen: Biospecimens belonging to
known individuals whose data are used as encrypted
and coded information.
Identified biospecimen: Biospecimens belonging
to known individuals whose personal information is
The most commonly used method is coding or
anonymization. The coding method allows reestablishing
contact with the owner of the data. This is advantageous
in terms of providing new data for prospective studies.
However, it is open to the improper use of information by
authorized persons and may not provide complete security
for participants (22). Anonymization ensures elimination of the link between biospecimens and their associated data
with the participants, either reversibly or irreversibly.
Secondary Use of Samples and Profit Sharing
Other important ethical issues include commercialization,
patenting of the collected biospecimens and related
data, and who will and in what terms benefit from the
biospecimens and research. Ethical and legal regulations
that protect the rights of both donors and researchers are
required for research using human biospecimens. Options
to participate, not to participate, or withdraw from the trial
should be offered to the participants (15).
Legislation in Turkey
Turkish legislation has no regulation specific to biobanks
yet. However, a legal infrastructure needs to be constructed
in order to establish biobanks in Turkey and to make
sure that they operate in accordance with international
standards. The relevant law should be enacted within the
framework of the European Union directives.
Although the Turkish Draft Law on a National DNA
Database and National DNA Databank prepared by the
Ministry of Justice on January 17, 2007 was submitted to the
Prime Ministers Office, the draft was returned for review
on April 14, 2008. No other draft bill has been submitted
since then (14, 23, 24). The draft justification states that
DNA is personal data that contains a lot of confidential
information about the person, and it is very important to
comply with the international conventions our country is
a part of as a third party regarding the use and protection
of this data, and to refrain from violating the personal
rights defined in our Constitution. The definitions of the
biological sample, DNA, DNA Analysis, DNA Profile and
DNA Database terms have been included in the second
article of the draft bill. The basic criteria related to DNA data
have been defined under the "Basic Principles"
section of the draft.
Accordingly, article 4 introduces the following regulations
related to DNA data (14):
Processing of DNA data in accordance with the law and
general principles of integrity,
Collecting DNA data only for legitimate purposes as
stated in the relevant articles of the law and refraining
from using and transmitting the data otherwise,
Ensuring that the DNA collection is relevant, sufficient
and appropriate for the purpose,
Keeping the data until the end of the period specified by
Ensuring accuracy of the data and providing updates as
The draft also addresses the subject of providing
information in the Principles of Volunteering and the
Obligation to Provide Information. However, the relevant
legislations, ethics committees and consent form have
not been developed for biobanks in Turkey yet and the
adaptation process will need to be realized quickly.
INFORMATION TECHNOLOGY IN BIOBANKING
An information technology (IT) system is a very important
part of quality management as the entire process for
each material collected in the biobank can be recorded
(7). First of all, it provides a rich and high-quality
information pool including the clinical information and
consent documentation related to solid tissue and liquid
biomaterials. In addition, it makes the journey of the
biomaterial easily traceable by recording relevant data
(information related to collection, processing, preservation
times, storage properties and localization of the material
in the repository, ambient temperature) (25). Although
most pathology reports provide systematic information on
the cases, it is recommended to use standard terminology
based on international nomenclature to optimize data in
The biobank document contains clinical, pathological
and other information of the subjects together with the
following parameters (5):
Gender and date of birth, patient identity, demographic
data, and status of vital signs.
Diagnostic data, clinical stage (cTNM)
Sample information, sampling date, and details of
sample quality, collection method, stabilization process
Lesion information; tumor information (primary or
metastatic), histological type, size, pathological stage
(pTNM), grade, and other important data specific to
the disease state
Type, number, size, characterization of sample data (for
tissue sampling; tumor focus, area adjacent to the tumor,
sampling away from the tumor), and preanalytical data.
DNA or RNA data such as concentration, purity,
Storage information; the type of storage used, the
localization of the material in the storage area and the
Information technology applications have been developed
to address biobanking processes, optimize workflow
efficiency, and provide quality assurance. This system can
manage data obtained from molecular research and provide
controlled access to data acquired from the research
community. In this respect, biobank system software
is designed on the basis of security and robustness (7).
Numerous software programs are available to assist in the
use of information technology in tumor biobanks (3). With
these software programs, all operations and procedures,
including information about the operator, the equipment
used, reagents and other consumables, can be recorded
with the time also specified (27). Similarly, the process of
sample collection, sample acceptance forms, processing and
anonymizing of samples can be performed electronically
to reduce user-related errors due to manual entry of data,
and to improve workflow efficiency and quality assurance
(7). Most software programs allow tracking the position
of biospecimens in the biobank storage units and the
procedures they are subjected to over time. Depending on
the total number of samples to be stored, these programs
can therefore propose sample storage space for new
material and optimize space utilization. The software
programs enable recording of all kinds of information on
the processing and storage of biomaterials. This recorded
information should include not only the process flow, but
also the non-conforming situations during the process,
whether corrective measures have been taken, and at which
stage if so (5).
Software programs can also be used to monitor and manage
donation approval. For example, a copy of the informed
consent can be saved to the system, allowing biospecimens
to be used only with the donors consent (5). Software
programs can also monitor the distribution of tissue
samples registered in the system and all processes related
to those biospecimens. For example, requests and the
transfer and return processes related to the biospecimens
can be recorded as of the date of their realization Material
transfer agreements related to the subject can also be
stored in the system. Furthermore, data obtained from
the research performed with the use of biobank materials
can be added to the biobank database, thus contributing
to the continuous development of data. Taking the above
issues into account, when deciding on the software system
to be used for the biobank one should be careful regarding
topics such as security, robustness, data to be recorded,
data quality, recovery of data, and the possibility to backup
stored data to alternative databases at regular intervals
(5, 28). In particular, it is very important to protect and
anonymize the identity data of the donors as the donors
should always be anonymized (3, 5).
COLLECTION AND PROCESSING OF
All the surgical excision specimens obtained from the
different types of Surgery Departments are gathered and
processed in the Pathology laboratory with the intention of
diagnosing the disease. Therefore, pathology laboratories
and the pathologists have a central role in tissue biobanking.
A pathologist can evaluate the specimen macroscopically
and then make appropriate sampling of the tissue for both
diagnostic purposes and for biobanking concurrently.
The medical and scientific expertise of the pathologist is
important in terms of making diagnostic decisions as well
as managing the procurement and preservation of the
surgical excision materials. Thus, the pathologist has an
essential role to provide continuity between medical care
and research (10).
Specimens collected during clinical practice include tissues,
fluid samples, and body secretions. Samples for diagnosis
and research samples should be taken from the materials
at the same stage if possible and collected in separate
containers for biobanking. A biobank has to evaluate all the
different uses of the samples in the future and maximize
the conditions for the survival and potential use of these
In molecular studies, frozen tissue (-80°C to -190°C) and
formalin-fixed, paraffin-embedded (FFPE) tissues are
known to have their own advantages and disadvantages
(30, 31). Although histological detail in frozen tissue
is microscopically lower than in FFPE tissue, frozen
biospecimens are ideal for DNA/RNA, genome
amplification, genome sequencing and cDNA microarray
analyses (32). Frozen tissue samples therefore often offer
sufficient quality for molecular studies. In addition,
proteins in frozen tissue specimens are protected from
degradation including enzymatic activity, while protein
loss is seen in their FFPE samples (33).
Accurate recording of preanalytical details is crucial
for immunoassay, molecular or proteomic analyses
of biospecimens containing tissues and fluids (29).
Information on the parameters affecting the preanalytical
processes regarding these materials will be given in more
Human tissues can be obtained from surgical materials
or autopsy samples. While delivering these tissues to the
pathologist, it is essential to minimize the duration of warm
ischemia (the time spent at room temperature until tissue is removed from the human body and treated with fixation
material) and cold ischemia. If possible, the container
containing the tissue samples should be delivered on wet
ice cubes and stored in ice or at 4°C in the refrigerator
until it is placed into fixing solution to limit changes at
the cellular level (29). The ischemia process has been
shown to affect the values of biomarkers detected using
molecular techniques or standard immunohistochemistry.
In one study, needle biopsy specimens of breast cancer
and excision materials sent after surgery have revealed
various PI3K pathway markers. It has been reported that
this difference may be due to the loss of phosphorylation in
the material during surgery or the process of cold ischemia
For solid tissue samples to be fixed, it is also necessary
to specify the type and duration of fixation and the
duration and conditions of storage. Optimum fixation of
biospecimens depends on variables such as the volume of
the fixative, tissue ratio, fixation time, temperature, and
tissue thickness (35). Formalin fixation of biospecimens
leads to fragmentation of nucleic acids (36, 37). Since the
length of fixation also affects nucleic acid quality, a fixation
time of 6-18 hours for biopsy specimens and 12-36 hours
for surgical specimens is recommended. However, 70%
ethanol or alcohol-based fixatives can yield higher quality
nucleic acids compared to formalin fixation and can be
more suitable for molecular studies (37). A commonly
preferred tissue preservative, RNALater, has recently been
shown to provide better quality RNA and gene expression
profiles compared to snap-frozen samples or formalinfixed
paraffin-embedded (FFPE) samples when used on
tissue samples (38, 39).
Liquid biospecimens (e.g., whole blood, plasma, serum,
urine, bronchoalveolar lavage, saliva, ascites, tear fluid and
seminal fluid) include cells, proteins, lipids, and metabolites
that can function as biomarkers. The preanalytical
information required for liquid materials includes the type
of primary collection tube, delay time and temperature
before centrifugation, centrifugation conditions, as well as
long-term storage time and temperature (29, 40).
The container used to collect a blood sample is itself an
important preanalytical variable. These tubes and their
content may contain stabilizing agents for anticoagulants
or nucleic acids and thus affect the analysis of biomarkers in
the blood sample. The tube content has been shown to affect
proteins, hormones and other biomarkers used in various
analytical techniques (41, 42). In addition, some studies investigating the waiting time before centrifugation have
shown that this duration may affect protein measurements
in blood samples. Ayache et al. reported significant changes
in plasma protein levels within 2 hours (43) Similarly, Banks
et al. reported the development of important changes in
low-molecular weight serum proteins within 30-60 minutes
(44). In another study, it was reported that proteins remain
stable by centrifugation of samples within 2 hours and
then freezing within 2 hours after centrifugation (45). The
temperature at which the samples are kept is another factor
affecting protein stability. Significant protein losses were
observed when the samples were kept at room temperature
for more than 4 hours or kept at 4°C for 24 hours (46).
Blood is one of the most widely used biomaterials and
contains various fractions such as plasma, serum, white
blood cells and red blood cells. Tube types compatible with
the intended test(s) should be used for blood collection
(47, 48). A collection tube containing silica or thrombinlike
coagulation accelerator is required for studies in which
serum will be used. Anticoagulated blood (consisting of
plasma, buffy coat, and RBCs) is preferred for DNA- or
Although there are several types of anticoagulants, blood
samples stabilized with citrate provide higher quality RNA
and DNA yield compared to other anticoagulants (47).
EDTA-coated collection tubes are suitable for various
DNA- and also protein-based assays; however, these tubes
are not sufficient for cytogenetic studies (47, 49).
The collected blood samples can be divided into fractions.
Serum and plasma can be used for the analysis of
protein, lipid, small molecules, and nucleic acid (48). Cell
concentrates can be used in functional studies and also
in flow cytometry as a source for culture experiments or
cellular nucleic acids (48, 50). Since blood components can
maintain their viability at room temperature for up to 48
hours, the time between collection and processing of the
samples should be kept short (less than 24 hours if possible).
A variety of Standard Operating Procedures (SOPs) of the
International Society for Biological and Environmental
Repositories (ISBER), the National Cancer Institute (NCI),
and other organizations exist and systematically describe
the process of collecting and processing the tissue and fluid
(blood) biospecimens described above (40, 51).
The liquid biopsy procedure is an important development
that has recently been introduced for the molecular
profiling of cancer patients. Various biomarkers reflecting
tumor-specific changes can be identified in cell-free DNA
(cfDNA) from patient blood samples using this method (52).
The method is therefore useful for molecular monitoring
of cancer treatment as well as detecting recurrence and
resistance (53). The FDA has recently approved the first
liquid biopsy test for EGFR mutations in patients with
non small cell lung cancer (NSCLC) (53).
STORAGE OF BIOSPECIMENS
Storage facilities are important factors in maintaining
sample quality. The establishment of a storage unit is
determined by the type of biobank to be established,
the type of samples to be stored, the storage period of
the samples, the intended use of the samples, and the
financial resources (4). Additionally, the presence of
infrastructure facilities such as an electrical power system,
backup systems, transport conditions, and on-site support
services should be considered. Compared with the modest
conditions required for the storage of FFPE blocks (such
as room temperature below -25°C, air conditioning,
space), a biobank storage unit requires more sophisticated
conditions and expensive equipment.
The sample freezing temperature is determined by the
amount of water and other tissue components within
the sample (4). Biospecimens can therefore freeze over a
range of temperatures. All biological specimens contain
degradative molecules affected by the temperature. As
the ambient storage temperature decreases, the activity
of the protein within the biospecimen also decreases. The
optimal storage temperature should therefore be below the
threshold temperature of protein activity (4, 54). Optimal
storage temperatures are below -132°C, which is the glass
transition temperature (Tg) of pure water. Most of the
chemical and physical reactions that cause deterioration of
the specimen slow down below this temperature.
It is possible to store specimens at low temperatures
using mechanical freezers or liquid nitrogen-based (LN2)
cryogenic storage (4, 40, 54). The LN2-based storage units
provide effective and long-term storage and are preferred
to mechanical freezers in areas where the power supply is
unreliable (4, 40). LN2-based storage units are composed of
two main groups as the smaller aluminum dewars and the
large storage units (4, 40). Both are double-walled, vacuuminsulated
storage units that efficiently hold LN2. The units
have different sizes and specimen capacities. Aluminum
Dewars are small and transportable containers that can be
installed conveniently in labs and they are readily accessible
(4, 40). They provide a stable storage temperature and low
LN2 usage but most of them require manual filling of the
LN2 to maintain the temperature. Dewars typically lack
full monitoring and LN2 level control options. The liquid storage units are medium to large storage areas that provide
long-term storage of specimens (4, 40). Due to their size,
they may be installed in a lab or require a special area.
The majority of these units have auto-fill capabilities for
temperature and LN2 level with a monitoring system. Even
so, manual verification at regular intervals is important to
ensure specimen integrity in these units. Biospecimens can
be stored in the liquid or vapor phase of liquid nitrogen (4,
40, 54). In vapor phase containers, the biospecimens are
stored above the liquid phase nitrogen but are surrounded
by the vapor (gaseous) phase. Storage in the LN2 vapor
phase (≤150°C) provides appropriately low temperatures
maintained below the Tg (-132°C) and protects biospecimens
from the risk of contamination and safety hazards related to
liquid phase storage. Vapor phase LN2 storage is therefore
usually preferred to liquid phase LN2 (-196°C). However,
one must consider that liquid phase LN2 provides a stable
temperature at -196°C. Oxygen level sensors should be
used and calibrated when LN2 freezers are used. Protective
goggles and gloves must be used. Appropriate training
should also be provided as a part of an SOP related to health
hazards and safety precautions (4, 40).
The container system is another important component of
the storage unit. There are three main types of containers:
screw-cap vials, bags and cryogenic straws (4, 40). Screwcap
vials are made from polypropylene and polystyrene in
capacities ranging from 0.2 to 5 mL and are recommended
for long-term, low temperature storage (4, 40, 55).
Covering of vials with a membrane is important to reduce
contamination in liquid nitrogen (4, 56). Cell freezing bags,
which are commonly used in blood banking, can also be a
container of choice for other types of cells or tissues (4, 40).
Storage of brain slices within these bags is well-known (57).
It is recommended to use vial systems for sample volumes
below 5 mL and bags for larger volumes. Cryogenic straws
are hermetically sealed and designed for the safe storage of
specimens within the liquid phase of nitrogen (4, 40). These
straws are made from chemically inert and biocompatible
material and show physical characteristics resistant to ultra
low temperatures and storage pressures (4, 40). They are
therefore stable at compelling circumstances such as snapfreezing,
exposure to low temperatures for a long time
(years), or several freeze-thaw cycles. Wrapping frozen
tissue slices in aluminum foil also helps to minimize tissue
When selecting storage containers for biospecimens, the
following issues should be considered (5)
Cooling and warming rates required
Potential risk of contamination of the sample or the
Storage temperature and conditions
Available space for storage
Frequency of access
Specimen identification requirements (identification
labels without personal identifiers that are compatible
with the storage temperature and medium, eye-readable
Specimen preparation and processing techniques
All human specimens should be treated as potential
biohazards. Taking precautions against the risk of
contamination of laboratory workers who handle the
specimens in the laboratory or during transportation is
therefore important and in fact a part of good laboratory
Mechanical freezers are available in varying sizes,
configurations, and electrical voltages (4, 54). Since these
are devices attached to power systems, a back-up power plan
and emergency response plan must be established. Lower
cost of initial investment and easier access to samples are
the main advantages of mechanical freezers (4, 54). Storing
biospecimens at low temperatures (-20°C) for the short
term or at ultra low (-80°C and -150°C) temperatures for
the long term is possible but the use of -20°C is gradually
decreasing as tissue degradation occurs at this level (4, 54).
Most of the centers prefer to use ultra low temperatures
(-80°C; -150°C), because ice crystals can develop within
the biospecimens at temperatures near -70°C (4, 54).
Cascade compressors might produce temperatures as
low as -140°C but require constant electrical power to
maintain these temperatures. The temperature stability of
freezers is influenced by several factors such as the ambient
temperature, humidity, open doors during sample loading,
and frost within the freezer. Freezers should therefore be
placed in rooms with proper air-conditioning and any frost
should be removed regularly (4, 54).
Refrigerators are used where the durability of the material
being stored is preserved by storage below ambient
temperature. Storage at 4°C could be an intermediate step
before preparation for ultra low temperature storage (4,
54). The temperature stability and back-up power plan are
important for refrigerators as for mechanical freezers (4,
Ambient Temperature Storage
Formalin-fixed paraffin-embedded tissue specimens can
be stored at room temperature in routine pathology lab
practice. Recently developed biological storage matrices
allow for the long-term maintenance of biological
components at room temperature (4, 54). These matrices
can be helpful in the absence of mechanical or cryogenic
equipment due to practical or financial issues and enable
the storage of many types of tissue specimens such as
formalin-fixed, PAXgene-fixed, ethanol-fixed, paraffinembedded
and lyophilized samples (58). Other studies have
suggested different methods to preserve nucleic acids in
FFPE tissue blocks. Baeane-Del Valle et al. have examined
the effects of tissue block age on FFPE tissues from radical
prostatectomy specimens from patients with prostate
cancer using a number of RNA in situ probes (59). They
found a decrease in signals after 5 years and a significant
decrease after 1 year. They also showed that storing
unstained slides in recent cases (< 1 year old) in the cold
(20°C) preserves RNA in situ hybridization signals, and
was superior to leaving the tissues in FFPE blocks stored
at room temperature. Therefore, the authors suggested this
simple solution (cold storage of unstained slides) to better
preserve invaluable RNA-based information currently
locked in massive FFPE archives (59).
The effects of various storage temperatures on the
molecular integrity of frozen tissues have been studied.
Some studies have demonstrated reduced RNA integrity in
specimens stored at −70°C or −80°C for 5 years or more
(60, 61). Whether the reduced RNA integrity was due to
the peculiar inherent characteristics of the tissue samples
or storage conditions was not clear in these studies.
Conversely, the integrity of RNA was assessed with the
reverse transcription polymerase chain reaction (RT-PCR)
after the storage of brain autopsy tissue for 15 years at −70°C
and no RNA deterioration was reported (62). In contrast to
the conflicting results for RNA, DNA integrity is usually
preserved with long-term storage at -80°C (61). Similarly,
the proteome may be preserved for years when tissue if
stored at or below -70°C (63). In another study, the activity
of epidermal growth factor receptor was investigated in
excision materials of patients with breast cancer and there
was no difference related to the storage temperatures
(liquid nitrogen, -70°C, -20°C) (64). It has been suggested
that a minimum temperature of -80°C should be preferred
for long-term storage. Although storage at -150°C may
reduce the effects of temperature fluctuations resulting
from opening freezer doors, additional data are needed
before these costly freezers are preferred.
The effects of different temperatures on the storage of
blood specimens have also been analysed. Collected
blood samples should be processed as soon as possible to
maintain biomolecule yield and to prevent degradation
(47). It has been demonstrated that DNA could be
extracted with admissible yield and quality from blood
samples that are stored at room temperature, at 4°C, and
at -20°C for a maximum period of 1 month (65). As the
storage term becomes prolonged, the erythrocytes and
some of the leukocytes will undergo lysis and consequent
loss in the amount of extracted DNA (65). In such
circumstances, freezing the blood samples at -80°C has
been recommended to avoid lysis and to improve the
DNA yield (66). Extracted DNA from blood samples
can maintain its stability at 4°C for weeks, at -20°C for
months, and at -80°C for years. However, RNA lability
and degradation begins at temperatures higher than -80°C
(67). Interestingly, miRNA, a species of RNA, can maintain
its integrity without prominent degradation for years in
plasma samples that are stored at -80°C(68).
Freeze-thaw cycles can be damaging to the biomolecules
and cells intended for analysis. Repeated cycles lead
to increased cell death via apoptosis and necrosis (4).
Therefore, it is important to aliquot the biospecimens to
the proper size in order to minimize the number of these
freeze-thaw cycles before they are used. If aliquoting
is not possible, placement of samples on dry or wet ice
during sampling will help to maintain the vitality of the
biospecimens (4, 54). As far as we know, preservation of the
sample integrity is possible below the level of Tg (-132°C)
where biochemical activity of the cell is almost stopped (4,
54). A specimen therefore experiences a micro-thaw event
each time it is warmed above Tg. There are several factors
that can cause temperature fluctuations such as power
outages, mechanical failure of the freezer, or frequent door
openings (4, 54).
Most of the studies investigating the freeze-thaw cycles
address the integrity of RNA since it is the most sensitive
biomolecule of unfixed tissue. Some of the studies have
shown that repeated freeze-thaw cycles (as few as 2 thaw
events) are sufficient to reduce RNA quality, particularly in
autopsy brain tissue (69, 70). However, other studies have
not found any alterations in RNA quality, gene expression
profile, or protein expression in frozen ovarian and brain
tissue samples (71, 72). Additionally, it has been reported
that at least 3 freeze-thaw cycles can be performed without
loss of RNA quality in ovarian tissue samples (72).
Similarly, minimal RNA degradation was reported after
6 freeze-thaw cycles in tonsillar tissue in another study
emphasizing the importance of short-term (i.e., 5 minute)
thaw cycles for RNA integrity (73). The reason for the
contradictory RNA integrity results reported from various
studies could be the lability of biomolecules in different
tissue types, the varying nuclease content, and the thawing
conditions. In fact, total thaw time at ambient temperature
has been considered to have a stronger effect on RNA
integrity than the number of freeze-thaw cycles (73, 74).
However, a total thaw time of less than 30 minutes at
ambient temperature has been reported to suitable for the
preservation of RNA integrity, regardless of the number of
freeze-thaw cycles (73, 74). In addition, aliquots stored in
RNAlater (Qiagen, Valencia, CA) have been reported to
have a higher quality RNA than snap-frozen samples and
also to be more resistant to freeze-thawing (69). Preserving
specimens in RNAlater mitigates the effect of thawing on
Freeze thaw cycles also influence DNA, RNA, and protein
stability in blood samples. The number of freeze-thaw
cycles of blood samples should be minimized as in frozen
tissue samples. Aliquoting as well as pre-extraction of stable
molecules (DNA) can be used for this purpose.
Finally, extra care is required regarding the aliquoting
and documentation of the biospecimens. Biospecimens
collected from the same patient should be aliquoted and
then preserved in two different repositories so as to protect
them from the adverse effects of undesirable conditions
such as power outage. Conditions for preventive
interventions in case of failure should be provided. For
example, mechanical freezers should be protected from
power outages particularly via an electrical backup system
(2 uninterruptible power supplies). The location of the biospecimens within the storage, the information regarding
the amount of tissue used for each assay, and the residual
tissue samples should also be recorded carefully.
QUALITY CONTROL PROGRAM IN BIOBANKS
Biobanks are very important centers for understanding the
mechanisms of diseases, developing clinical biomarkers
associated with diseases, and discovery of new drugs. These
repositories where the properties of the relevant material
are defined and preserved in high quality therefore require
the implementation of quality control and quality assurance
plans at each stage (Table I) (75).
Biobanks are resources that work as a library within the
medical sciences. It is therefore very important to record
and ensure the safety of biobank material data. During
biobanking, data processing and sample processing work
packages as well as quality control mechanisms should
be established. The aim of data processing is to record
the demographic and clinical data of the patients and the
identity information on the sample using a unique and
holistic approach (1). The use of standardized methods
during registration is important to ensure quality. For
example, the use of the BRISQ (Biospecimen Reporting for
Improved Study Quality) reporting system allows for better
storage, recording and comparison of the data records
(Table II) (76-78).
Click Here to Zoom
|Table II: Items included in the BRISQ (Biospecimen Reporting
for Improved Study Quality) reporting system.
Quality management and standardization in biobanks
is ensured by the necessary guidelines and standard
operating procedures (SOPs). There are standard operating
procedures for biobanking published by the International
Society for Biological and Environmental Repositories
(ISBER) and the National Cancer Institute Biorepository
and Biospecimen Research Branch of the National
Cancer Institute (NCI BBRB). These procedures, which include guidance on issues such as material collection,
processing, and use in addition to education and ethics can
be used by bringing them into conformity with the legal
framework of the biobank and the countrys legislation
(79). Operability of national or international networks that
have been already established or are likely to be established
for similar purposes, usually in close geographic regions,
should be ensured. The Molecular Pathology Working
Group of the European Society of Pathology (ESP) works
in collaboration with other European organizations (such
as the Organization of European Cancer Institutes, and the
European Biobanking Infrastructure), and continue their
research on pathology-centered biobanks (80).
The ISO 9001, ISO 17025 and ISO 15189 standards published
by the International Organization for Standardization (ISO)
are not specific to biobanking but have been previously in
use for this purpose (81). Standards specific to biobanking
(ISO/TC276) were determined with the publication in 2018
of ISO 20387 that includes the requirements for biobanking.
ISO 20387 includes standards for the collection, recording,
cataloging, classification, processing, reproduction,
packaging, storage, disposal, distribution, transportation of
materials, as well as security measures, risk management
and personnel-related issues. The Accreditation Institute
of Turkey, a member of the Accreditation working group
of the European Union Accreditation Agency, has taken
the ISO 20387 biobanking standard into consideration.
Studies for its validation in European Countries including
Turkey are still continuing (82, 83).
Within the framework of these standards, quality
assurance programs (QAPs) regarding educational and
infrastructure issues in diagnostic research should be
established in biobanks. Improvement of the quality levels
in the pathology-centered biobanks in accordance with
the accreditation programs of CAP (College of American
Pathologists) has been planned.
Quality assurance and quality control programs should
be defined in detail for the biobanks that make up a large
number of important organizations established to facilitate
diagnosis and research. The quality assurance program
aims to minimize the impact of preanalytical variables on
the biospecimens stored in biobanks. Standard operational
procedures (SOPs) which are understandable, clear and
easily monitored by the employees should be developed
and implemented. Quality assurance programs should
also be applied to determine the effect of the SOPs and
preanalytical variables on biospecimens stored in the
biobank (84, 85).
Quality Assurance Program in Biobanks
The components of the quality assurance program in
pathology-centered biobanks include technical and
operational elements, the role of the pathologist and the
employees, and the quality of the banked sample (Tables
III,IV). Since the quality assurance program in biobanks
cannot obtain data without a written record, everything
related to quality should be recorded. A quality assurance
program and a quality assurance committee should be
established for each biobank. Committee members may
consist of a biobank manager, quality officer, laboratory
officer pathologist, and a technician. In addition,
employment of personnel trained in the field of quality
control may be considered within the scope of possibilities
(84, 86, 87).
The Role of the Pathologist in Quality Processes
The pathologist is responsible for managing and regulating
a quality control program in biobanks. In addition to
macroscopic evaluation and identification of biospecimens
stored in the biobank, the role of the pathologist in the
differentiation of the lesion (tumor) tissue from control
tissue is very important (88). Histomorphological evaluation
during the quality control of the tissue specimens is
necessary for the confirmation of the pathological diagnosis
and determination of the disease status and tumor-necrosisstroma-
inflammation-normal tissue ratios. In addition, the
pathologist is responsible for detecting tumor specimens
with heterogeneous cell populations and performing
manual macrodissection or manual/laser microdissection if necessary. Tissue microarray (TMA) preparation can
facilitate the diagnostic procedures, conduct of research,
and the financial processes in some cases (84).
Reevaluation of stored tissue specimens by a pathologist
using Hematoxylin & Eosin-stained slides, and performing
comparisons between frozen and FFPE tissue specimens
can help to evaluate the quality of the stored material (89).
In addition, pathologists are responsible for the quality
control of molecular pathology procedures related to the
Calculating the cost and budget of a biobank requires a
detailed plan to be prepared for the installation, operation,
development and long-term sustainability of the relevant
biobank. Large material resources are therefore needed
(1, 90-92). To this end, public institutions, private
enterprises or associations can contribute to this financing. Consequently, financing may be provided by a public
model or the private financing model in general. Funds
for a pathology-centered biobank in our country can be
provided by the public, various state institutions (e.g.,
TUBITAK) or private universities.
Biobank expenditures are analyzed in two phases as initial
and operational (Table V). The initial stage involves the
planning and establishment of the biobank. The first
budget prepared should cover the determination of and
the necessary arrangements to be made for the biobank
area, establishment of an infrastructure suitable for the
workflow, the provision of the necessary devices and
equipment, establishment of appropriate recording and
monitoring software/hardware information systems,
identification and provision of preventive measures against
natural disasters and occupational risks, planning and
monitoring tissue safety, and the provision of ventilation and proper air conditioning. The size and scale of the
biobank, the size of the area required, and the quality of
the equipment and services are the factors that determine
the amount of initial funding (90). At the operational stage,
personnel costs (such as the biobank manager, pathologist,
technician, data assistant, quality control personnel)
are expected to take up most of the budget. The cost of
consumable materials should also be calculated, in addition
to utilities, electricity, water, natural gas, and medical waste
disposal. Consultancy services related to quality control
and assurance, and ethical or legal matters should also be
included in the financial plan. In addition, the financing of
educational meetings such as training programs, seminars
and congresses as well as certification and accreditation
costs should be included in the budget so that biobanks can
be established and developed and high quality services then
purchased (90). The organizational scheme of a biobank is
summarized in Figure 1.
Click Here to Zoom
|Table V: Some expenses related to the establishment and
operation of the biobank.
The cost-effectiveness of the biobank process should be
considered carefully. Weighing the sustainability costs
of each project in terms of duration and project outcome
is important, and costs should be delineated. Even if a
biobanks ability to recover costs is often limited, it can
generate income from the research budget, the donations,
and the user fees it will receive from the patients. After a
certain period of time, biobanks may become financially
Consequently, a pathology-centered biobank offers significant
advantages (Table VI). First of all, it ensures correct
use of tissue specimens in the diagnosis of diseases and in
the definition of targeted individualized therapies. Effective
selection of the necessary tissue specimens for research
projects can also be realized within the same process.
Thus, each of the tissue-related processes is realized in
the presence of experienced personnel and they can all
be gathered at a single center. This provides effective and
significant savings at both the tissue and cost levels (9, 93).
We would like to thank Prof. Dr. Sülen Sarıoğlu and Prof.
Dr. Kutsal Yörükoğlu for reviewing the article.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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