Biobanking of Fresh Frozen Tissue from Clinical Surgical Specimens

Fresh Frozen Tissue and Clinical Surgical Specimens

Since many pathology departments at hospitals have procedures for the reception and handling of fresh specimens, a biobanking manual based on the already established structure to enable the collection of unfixed tissue samples can be produced. This allows the collection of all types of surgical lesions. The procedures can be used for all specimens such as a tumor, rejected transplanted organs, atherosclerosis, inflammatory bowel disease, etcetera. Most surgical specimens are transported to the pathology department for potential biobanking. One of the most important concerns for all clinical biobanks is diagnostic security. The pathologist involved has the responsibility to report the diagnosis based on the fresh specimen.

One main limiting factor in frozen tissue biobanking is the well-understood hesitance of pathologists to remove abnormal tissue for biobanking purposes as it may jeopardize the appropriate diagnosis and treatment for the patient. A potential solution to this issue is to perform cryosection and histological examination of the specimen once it enters the biobank. Another important concern for biobanks is the possibility of tissue degradation during transport from the surgical theater to a facility. However, most tissues are usually stable for hours since it is transported on ice. It is also important for each research project to define their tissue quality criteria to ensure that the samples meet their standards.

Tissue Sample Collection and Biobanking

These are some of the methods that can be applied through different stages of biospecimen collection:

1) Surgical Theater

• Fresh specimen should only be handled in a designated area. Between each case, the area should be decontaminated by removing material from previous cases. Specimens should only be handled using gloves and instruments. Responsibilities of various staff members should be documented.

• The pathology chart should note the time when the specimen has been removed from the patient.

• Specimens should be placed in a clean surgical cloth, plastic bag, jars, or a test tube. It should also be immersed in a cold saline solution.

• The specimen should be transported at ±0⁰C (partly filled with wet ice). It is important to note that the specimen should not be in direct contact with the cooling agent (water or ice) during transport.

• Inform the technician at the pathology department for reception of the specimen. The communication routine between the theater and pathology department should be safe and clear. Ensure that the specimen is delivered.

2) Pathology Department

• Upon arrival at the pathology department, the time of arrival should be noted on the chart. It should be registered in the clinical laboratory management system and labeled with a case number.

• The pathologist on call should be notified regarding the arrival of the specimen. If a delay is inevitable, the specimen should be placed in the refrigerator.

• Once ready, the specimen is removed and placed on a clean sheet of filter paper. The macroscopic features of the specimen (weight, measurements, description) should be noted in the chart.

• Pieces of the specimen that represents the lesion and normal tissue should be cut out and placed into a cryomold for cryogel coverage. Storage of samples in cryogel prevents the lyophilization of the specimens. It also helps o keep the DNA and RNA intact. The mold is then snap-freeze in dry ice or isopentane. The time of freezing, biobank numbers, pathologist signature, and technician signatures are noted. These tissue blocks are then transferred to a low-temperature freezer.

• It should be noted that the thawing of a sample during the lifespan of a fresh frozen biobank sample is one of the most important risk factors for the degradation of tissue. A cryostat to avoid thawing during delivery can be used to slice sections for protein, DNA, or RNA extraction. The tissue lock can also be cracked on a cutting board that has been cooled on dry ice if a larger portion of the sample is needed.

3) Biobank

• The biobank technician then makes cryosections of the biobank samples.

• An adhesive tape helps support the section during the cutting and transfer process to prevent folds and tears. Once the section is crossed-linked onto a slide, the tape can be peeled off.

• Sections are fixed, stained, mounted, and lastly labeled with a case number. These slides are then delivered to the pathologist who is responsible for reporting the diagnosis. The biobank technician registers the case along with relevant information such as name, identification number, age, gender diagnosis, and more.

• Biobanking protocols concerning biospecimens should be integrated with both local and national established clinical or diagnostic procedures. The protocols should also be authorized by those relevant.

Conclusion

It is critical for research teams involved in molecular diagnostics and translational cancer to have access to quality fresh frozen tissue. This article helps to describe a workflow for the collection of frozen biospecimens after derived from patients after surgery. These routines are used at Uppsala University Hospital since 2001 where the team integrates cryosection and histopathologic examination of the samples in the manual this is to help procure small lesions while avoiding a diagnostic hazard due to the removal of abnormal tissue from the surgical specimen.

References
Botling J, Micke P. Biobanking of fresh frozen tissue from clinical surgical specimens: transport logistics, sample selection, and histologic characterization. Joakim Dillner (ed.). Methods in Biobanking, Methods in Molecular Biology, vol. 675: page 299-306.

Quality Management in International Biobanking

Introduction to International Biobanking

Biobanks and biorepositories fulfill clinical or research purposes through the collection, processing, storage, and distribution of various biospecimens or materials that are often required. With time, it has been recognized that biobanks and biorepositories should follow a complex array of regulatory or ethical considerations. The procedures and policies they follow are usually documented by the best practices that can either be voluntary or by rules and regulations reinforced by Institutional Review Boards (IRBs), governments, and organizations. Issues of concern include participant privacy, informed consent, quality control of biospecimens, and various other matters.

Since biobanking is now a global endeavor, international collaboration and national networks are more important than ever. It is also vital that standards and practices are coordinated and developed. Although biobanking may be a business endeavor to some, it is still important that formal plans are in place to ensure the survival of associated research programs. With increasing development of new technology to aid in diagnoses, treatment, and genetic evaluation of diseases, more patients are now aware of the importance of biobanking in research. As a result, donors who participate in studies are also interested in learning more about their sample and results from the research. The following question is addressed by various experts in their field:

What are the important issues that are related to quality management in the collection, processing, and storage of samples?

T. Peakman:

• Specimens should be collected in the form that is best for scientific research. This means that the specimen should resemble the biological environment as closely as possible. Variables should also be avoided as much as possible.

• Shipping of samples for processing can lead to loss of unstable markers due to the time delay.

• Local processing of samples can be challenging due to the maintenance of consistent intersite processing.

• In many studies, the pre-analytical stage is often the greatest source of variation. This can be managed through a proper quality program that helps to make the collection and processing as standard as possible.

• A quality management process should include documentation of the sample such as dates, temperatures, times, operator, location, and more. Other important factors include the use of standard operating procedures (SOPs), audits, training of staff, review of critical materials, etcetera.

• Barcodes are also important to reduce the risk of misidentification.
  Systems and processes should be established to ensure the stability of samples and analytes used.

P. Watson and L. Matske:

• Quality management is essential in the maintenance and operation of a biobank. Biobanks should be able to track each biospecimen as this helps to manage biospecimen quality and the effective use of the sample in the future.

• Quality systems should involve protocols, SOPs, verification, staff education, and training. A good reference point would be standards set by international organizations.

• Quality management can be time-consuming and costly. It is therefore important for the scale of the quality management program to be dictated by the scope of the biobank and researches it supports.

• Training and education of staff are important to ensure they are up-to-date on current role specific practices to ensure consistent quality control and assurance.

H. Moore:

• The meaning of quality can vary among different individuals. In biobanking, complex procedures regarding the collection, processing, annotation, storage, and transport of biospecimens are required for quality management.

• Some crucial elements involve well-documented SOPs that are easily understood and accepted by staff and personnel. Foundational training and annotation of SOP deviations should also be done.

• A quality management plan should be reasonable in scope with room for expected errors. A good example would be the unavoidable biospecimen degradation in certain circumstances. It is vital to be aware of this possibility with the ability to measure relative degradation.

• A good quality management plan can lead to higher quality biospecimens and reproducible research results.      

              
A. Abayomi:

• Attention to detail is critical especially in regions with extreme temperatures where samples may be required to travel large geographical distances.

• The key to ensuring sample integrity is clear and comprehensible SOPs with frequent training, especially at sample acquisition research sites.

• The minimization of preanalytical variables allows the biospecimen to stabilize and closely resemble the donor’s state. This can be possible with a team that is strategic, good with logistics, and can synchronize operations between biobanking staff and researchers. Communication is an essential factor in this process.

Harmonized operational activity and good training are essential in the development and dispatch of kits to collection sites. This is mandatory until the sample reaches the final storage site.
In some environments, the use of appropriate transportation technology and room temperature storage to stabilize biospecimens after collection or isolation of nucleic acids can be extremely useful.

Conclusion

This article addresses one main critical issue that many biobanks or biorepositories are currently facing. Quality management of the biospecimens is important to ensure the highest quality of samples and reproducible results from research.

Reference

Vaught J, Abayomi A, Peakman T, Watson P, Matzke L, Moore H. Critical issues in international biobanking. Clinical Chemistry. 2014; 60(11): 1368-1374.

The Use of Human Tissues in Research: A Summary

There have been several high-profile legal cases which have ended in the removal of human biospecimens from research teams. One case resulted in the destruction of more than 5 x 106 dried blood spots that were obtained from infants after a suit challenged the state of Texas’ right to store these blood spots for future research. All these cases have one thing in common: the argument whether participants were properly informed regarding how their samples will be used for research. Informed consent is being increasingly discussed not only by professionals but also by the public. These cases have also prompted discussions regarding the timing and type of consent required, use of samples, and more.

This article highlights the opinions of the following experts who all represent different viewpoints on informed consent:

• David S. Wendler (D): Advocate for rights of research donors

• Arthur L. Caplan (A): Bioethicist

• Michael Christman (M): President and Chief Executive Officer (CEO) for an independent non-profit biomedical research institution with a large biorepository

• Jack Moye Jr. (J): Researcher

Is consent necessary or do you prefer a presumed consent with an opt-out option with the idea that human biospecimens should be a common heritage that is used for the collective good?

David: The tissues are not just “a collective good”. Tissues are obtained from specific individuals with their use involving the interests of donors and participants. Some of the things to keep in mind include the risks involved when obtaining samples, privacy, contribution, and use of the sample in future research. The necessity of obtaining consent allows donors to decide if they are willing to face the risks involved, increase awareness of the possibility of new information, and the advantages of contributions.

Arthur: The efforts taken to obtain informed consent are doomed to failure as there are many programs that use open-ended informed consent forms that are incomplete and vague. There are also those that ask donors or participants to waive their commercial interest such as the use of blanket waivers. A more appropriate way would be through altruistic gifting. This means that the specimens are made a gift making it clear that commercial interest is forgone, the use of specimen is open-ended, and possession has been transferred to a third party. A presumed consent to gifting would make more sense as long as patients retain the ability to opt out of gifting.

Michael: Current specimens that are anonymous should be allowed for use by research without the need of consent as there identification of donors will not be required. While studies that use anonymous specimens are usually exempted from the institutional review board (IRB) review, proper regulations should be implemented to ensure that this is upheld. However, an exception should be made for the use of anonymous specimens in genomic research as there is a possibility of identification.

Jack: The concept of human biospecimens as a shared resource for the collective good is a fascinating idea that should be given more attention. A framework where human tissue is a common heritage of humanity that is to be used for the collective good can help prevent disputes about both specimens that are left over from clinical purposes and those obtained for research.
What type of informed consent is best: general permission, tiered consent, specific consent, or other?

David: Many studies have been conducted regarding individuals’ attitudes about consent. They have consistently observed that the majority of donors want to control if their samples are used for research. Most participants are also willing to contribute when asked. These studies have also found that most donors support one-time general consent with the understanding that future use will require a review and approval from the ethics review committee such as the IRB. A widespread support shows that the one-time general consent offers the choice most donors would make. It also offers an opportunity to decline from contributing for those unwilling to contribute or for those who want more specific control over their specimens.

Arthur: A tiered consent would outline the likely uses of the specimen, disposition of materials, policy regarding the sale of material to third parties, transfer of control, and availability of clinical findings that would be relevant to donors.

Michael: A consent menu that has multiple choices would be best. A study found that although 10 percent support the consenting menu, most prefer 48 percent of blanket consent while the rest (42 percent) prefer re-consenting when a new research project begins.

Jack: Specific consent would help provide assurances that both the participants and researchers are equal in the enterprise. It is also easily accomplished when samples are obtained for a specific project. However, it can become impractical if the samples are stored for long-term with undefined uses. Most participants that are based in the United States are willing to contribute their samples.

What about property rights to specimens? Should research participants share potential financial gain?

David: Generally, individuals should share the benefits to the project they contributed to. Failing to provide a fair level of benefit can be regarded as a case of exploitation. Since the samples are part of an important contribution, it would suggest that the participants should share the benefits such as financial gain from the research projects. However, this can become complicated in practice as it is unclear what is a fair level of benefit or how benefits can be provided.

Michael: As part of the consent process, participants should be informed about property rights, the potential for financial gain (for the investigator), and if they themselves will share any financial gain. Once the participant is aware before enrolled in the study, the allowing or disallowing of financial gain and property rights should be acceptable. Consent should not be waived in cases if there is expected financial gain for the investigator.

Jack: Unless the research is conducted with the objective of developing a commercial product, proprietary interest in research by the donors can be difficult. It can be hard to put a value on something that has yet to exist especially in cases where there is an assertion of property rights where there is litigation corresponding with perceived value.

References:
Gronowski AM, Moye J, Wendler DS, Caplan AL, Christman M. The use of human tissues in research: what do we owe the research subjects? Clinical Chemistry. 2011; 57 (4): 540-544.

Human Biospecimens: Ethics and Regulations

Overview of Human Biospecimens

The human body and its collection of tissues have been studied since the ancient Greek times. After the Roman Empire fell, anatomical studies slowed down considerably as the use of cadavers became illegal in many places. Researchers were prosecuted for many years if they performed postmortem dissections. In the 15th century, medical schools in Europe allowed their researchers to study the human body and tissues without prosecution. Since then, the study of the human body has advanced significantly. Today, human biospecimens and tissue samples are vital for genetic research. Human biospecimens can be collected from several different sources:

• Prospective tissue collection

• Excess tissue obtained from clinical samples

• Specimens from cadavers

• Tissues with reproductive potential

With the increasing use of human biospecimens in research and clinical trials, issues regarding the ethics and regulations of these specimens needs to continuously be observed.

Governing Treaties, Laws, and Regulations

It is important to understand laws and regulations concerning human biospecimens as it helps researchers with issues of biospecimen ownership and ethical principles about human experimentation. One of the first important efforts of the medical community to regulate this kind of research is the Declaration of Helsinki. While it is not an international legally binding instrument, it has significantly influenced many regulations and national legislations.

Originally adopted in 1964, it has gone through six revisions. In the United States, the Code of Federal Regulations established by the government addresses the protection of the donors. In the Code of Federal regulations is the Common Rule that details the function and role of institutional review boards (IRBs) in the protection of human participants during the research activities. It also outlines the requirements in obtaining informed consent and additional protection for vulnerable groups such as pregnant mothers, neonates, fetuses, children, and prisoners. Some states also have their own laws that govern research using human participants.

Informed Consent

For informed consent, researchers must provide an explanation to potential participants regarding the purposes of the research and expected duration of the study. It should be noted that the descriptions provided should not be general and must be specific to the study. Without being adequately informed about the intended purpose of the research, participants cannot give “informed” consent the key element in the consent process is transparency. Participants should also know all the intended uses of the specimens. If their specimen is required in future research, additional informed consent should be obtained from the donors. However, the IRB can waive the need for informed consent for the use in a secondary project. IRB waiver is more likely if the donor has consented to future research at the time of tissue collection.

The participant also has to be informed regarding the potential risks, benefits, alternatives to participation, and what may be required of them during the study. Additional information required includes compensation and medical treatments that could be available should injury occur to the participants. Participation must be voluntary, and participants should be allowed to withdraw at any time without risk of penalty. Participants should also be provided a contact if they have any questions or concerns regarding the research.

The Common Rule is only applicable to human participants. In some circumstances, it permits research without participant consent. If the research is conducted using anonymous samples without access to the participant’s private information, by definition, informed consent would not be required. The Common Rule also does not apply if the IRB exempts it as the information used does not involve the identification of the donor. Finally, the IRB can waive or change the requirements of the informed consent if:

• The research poses no more than minimal risk to participants

• The welfare and rights of participants are not affected

• The research cannot be conducted without alteration or waiver of informed consent

• The participants are provided with relevant information

Biospecimen Ownership

The ownership of biospecimens has been analyzed in many cases. It has been a question of whether the donor retains ownership rights of their tissue. It has also been debated as an issue of “guardianship” versus “ownership”. In most cases involving excised tissue, courts have concluded that donors do not retain ownership of their excised tissue. However, different rulings have been reached in cases where there has been a previous understanding that the patient would retain their ownership rights. With leftover materials, many are considered to be “abandoned” with patients no longer having any property rights. In tissue obtained postmortem, the Common Rule does not apply as it only applies to living individuals. The Uniform Anatomical Gift Act (UAGA) allows individuals to give their bodies for the study of science. Without the individual’s consent, their spouse or family can also make the gift.

Conclusion

The laws regarding human biospecimens are still evolving. There will be much effort and discussion needed to improve the efficiency of informed consent. With increasing studies using human biospecimens, the frequency of lawsuits may be higher. It is therefore important for new legislatures and regulations as it can help to protect or help both participants and researchers. It is crucial for researchers to strive for transparency and avoid using specimens not outlined in the consent form. The awareness of existing rules is also essential to avoid lawsuits and the destruction of valuable human biospecimens.

Reference:

Allen MJ, Powers MLE, Gronowski KS, Gronowski AM. Human tissue ownership and use in research: what laboratorians and researchers should know. Clinical Chemistry. 2010; 56 (11): 1675-1682.

Biospecimen Collection, Processing, Storage, and Information Management

Introduction To Biospecimen Management

Biospecimens have been collected for various uses such as clinical trials, molecular epidemiology, and other research. It is important for specimen management to occur in a controlled environment. An environment where there are strict policies and guidelines in place that help ensure the quality and integrity of the specimen and data. With proper procedures, biorepositories are able to produce high quality biospecimens that are needed for research. Biospecimens are collected from donors for patient monitoring, care, and research studies. They have helped many medical advances such as those for cancer, heart disease, and AIDs. Due to the increased sensitivity and specificity of analytic techniques over the years, it is crucial that biospecimens are of the highest quality.  

Biospecimen Collection

There are various types of specimens that are required based on different research goals. Some examples include:

• Whole blood and blood fractions (red blood cells, serum, plasma, buffy coat)

• Tissue obtained from transplants, surgery, or autopsy

• Urine

• Buccal cells and saliva

• Bone marrow

• Placental tissue, cord blood, or meconium

• Feces

• Hair

• Semen

• Nail clippings

• Etcetera

Specimens should be collected, processed, and stored according to guidelines that take into account future analyses. The collection procedures will differ for different biospecimens and intended analyses. However, all procedures should be accurately documented.

a)       Blood Collection

Blood sample collection should be performed by a trained phlebotomist to reduce donor discomfort and to avoid compromising the quality of the sample. These standard protocols should be followed.

• Glass or plastic tubes with appropriate additives should be used

• Blood should be drawn in an orderly manner to avoid cross-contamination of additives.

• Serum should be separated from other components as soon as possible to reduce contamination. This is important as serum can be used for the improved analyses of nutrients, lipids, antibodies, and lipoproteins.

• Follow guidelines and recommendations for time elapsed between blood collection or removal from the storage unit and temperature for processing of blood specimens depending on the intended analyses.

• Avoid thaw and refreeze cycles.

• Since RNA and proteins are vulnerable to enzymatic degradation, follow necessary protocols that help ensure their integrity during the collection and processing phase.

b)      Tissue Collection and Fixation

Most tissues are obtained through surgery, biopsy, or autopsy. Generally, it would be best if the procurement of the specimen is performed by a trained pathologist. The time between the collection and stabilization process should be minimal. This means that the best approach is to collect, stabilize, and process the specimens rapidly. Detailed records regarding the timing for excision, fixation, or freezing should be kept. For autopsy specimens, it is vital to know the interval between death, collection, and processing of the specimen as tissues degrade rapidly after death. Tissues can be fixed using formalin, alcohol, and paraffin embedding as it has a relatively low cost when freezing or when storage facilities are unavailable.

c)       Urine Collection

Urine collection can be performed in some study designs and to achieve certain analytical goals. Examples of this include:

• Urine collected upon waking up in the morning

• Random specimens used for drug monitoring or cytology studies

• Timed urine collections

• Etcetera

Urine specimens should be kept refrigerated or kept on ice with or without a preservative.

d)      Saliva or buccal cell collection

Saliva along with buccal cells are a great source of DNA for genetic studies. Samples are easily collected by asking donors for self-collection. Methods include using cytobrushes, swabs, and a mouthwash protocol.

Preservation of Biospecimen Stability

As previously mentioned, it is important to minimize the interval between collection and stabilization. The temperature of biospecimens should be reduced when freezing is the endpoint. Control of processing time is necessary if fixation is the stabilization endpoint. Biobanks should utilize the method that preserves the highest number of analytes.

Biospecimen Processing

Biospecimens should be processed using the methods that preserve the analytes of interest or following the study design. For blood specimens, the processing method used should be based on the laboratory analyses. Tissues can be processed in the pathology suite or operating room once the specimen is resected. Buccal cells can be processed via centrifugation. For DNA extraction, the gold standard method is phenol-chloroform extraction. However, other methods can be used.

Storage of Biospecimen

Based on the intended laboratory analyses and requirements, biospecimens can be stored in various conditions. Examples include mechanical freezers, liquid nitrogen tank, room temperature, among others. Backup and alarm systems are necessary in case of mechanical failure. Staff should be trained for maintenance and repair of equipment. The labels used for biospecimens should be capable of withstanding the storage conditions.

Information Management and Specimen Tracking

Information management involves the collation and analysis of data associated with biospecimens as it helps support research. Since there are vast amounts of data, extensible and flexible informatics systems will be required. Biospecimens are documented and tracked using various forms of data management tools such as notebooks, multi-user software, and various automated information systems.

References

Vaught JB, Henderson MK. Biological sample collection, processing, storage, and information management. IARC Sci. Publ. 2011; 163:23-42. Accessed 10/25/2018. https://publications.iarc.fr/_publications/media/download/1398/68b153f74693289ae66d767a8cbe1ca667df4f1b.pdf

Pre-analytical Variables Affecting the Integrity of Human Biospecimens

Introduction

Biorepositories or biobanks function to collect, process, transport, and store biospecimens. The integrity of these biospecimens is crucial for the success of clinical trials and research. There are many factors that can influence the results within research such as:

• Pre-analytical environmental or biological variables

• Pre-analytical technical variables

• Analytical variables

• Post-analytical variables

Pre-analytical variables are defined as factors that can have an impact before the start of the analytical phase. It not only affects the integrity of the tissue samples, but also the results of the analysis. Pre-analytical variables are critical as the analytical integrity of the research can be jeopardized. Seeing as most errors in the laboratory can be attributed to pre-analytical errors this stage is of upmost importance.

Pre-analytical Factors in the Collection of Biospecimens

It is important to adhere to guidelines for general laboratory safety. The collection of biospecimens require a balance of:

• Accrual rate

• Types of biospecimens

• Sample size

• Costs

• Location

• Storage requirements

• Transport logistics

The biospecimens collected can be either invasive or noninvasive. Biospecimens that are collected through non-invasive methods may lead to an increase in sample size due to easiness of collection, reduced costs, and willingness of donor to participate. This method is especially important when dealing with pediatric biobanking. It is important that biological and environmental factors are standardized and documented when interpreting results as it can affect the downstream analysis. It is also vital to take measurements to observe the effects of intervention and the changes over time.

Pre-Analytical Factors That Affect the Collection of Blood Samples and Its Derivatives

The collection of blood samples should be performed by trained staff. Those that are involved in collecting samples from children should specialize in pediatric phlebotomy. The staff needs to be highly trained as this ensures the highest quality of specimens and prevents the donors from experiencing any kind of discomfort. Depending on the research requirements different additives may be required. Different types of additives are coded using different colored collection tubes. Some of the important pre-analytical factors to take note of include:

1. Using the same tube brand and the same lot number throughout the study. This would be ideal as different brands can have different anticoagulants, additives, and may introduce bias.

2. Another important factor is the expiration dates on the tubes as the vacuum in these tubes can decrease with age and negatively impact the blood draw and filling of the tube.

3. Using the same posture such as supine, standing, or supine as these can cause plasma volume changes that may lead to increased analyte levels.

4. Using the recommended needle gauge as a needle that is too thin can lead to hemolysis that distorts the potassium concentrations and hematological cell counts.

5. Using the recommended and same duration of tourniquet use as prolonged use can cause changes in analyte concentrations and hemoconcentration.

6. Avoid inadequate filling as this can result in inaccurate results due to the decrease in blood and additive ratio.

A general rule for common analyses is to use ethylenediaminetetraacetic acid for hematology, DNA, hemoglobin A1C, and a range of proteins. For plasma glucose, it is recommended to use sodium-fluoride tubes while lithium heparin plasma can be used for assays such as kidney function, iron parameters, liver enzymes, thyroid hormones, C-reactive protein, and more.

In remote sites that are resource-poor, capillary dry blood spot (DBS) are easy biospecimens that can be collected. Small volumes of capillary blood from the peripheries can be deposited onto specific paper cards and dried at room temperature for three to four hours. DBS can be used in many analyses. However, some of the pre-analytical variables to note are:

·         Type of collection paper used

·         Type of chemical used in the manufacturing of the paper

·         Thickness of paper

·         The volume of blood deposited

·         Environmental factors such as heat, humidity, sunlight, and moisture

In DNA and RNA collection, there are also biological factors that can affect the biospecimens. These include the donor’s:

·         Gender

·         Age

·         Body mass index

·         Tobacco consumption

Since RNA is more vulnerable to degradation, some of the preanalytical collection factors that can affect the integrity are:

• Tube additive

• Tube type

• Tube sterility

• Type of biospecimen

• The volume of blood collected

• Short-term storage temperature

• Lag time until extraction

In microRNA’s, the pre-analytical variables include:

• Diet

• Age

• Race

• Exercise

• Drugs

• Altitude

• Tobacco use

• Chemicals

• Hemolysis

• Coagulation times

• Temperature

Pre-analytical Factors That Affect the Collection of Urine and Saliva

Urine can be collected in many different ways as it can be used for measurements of many analytes. In urine biospecimens, the preanalytical requirements can be conflicting.  This may result in the requirement of multiple biospecimens. Some of the preanalytical variables for urine collection include:

• Collection method

• Environmental exposure

• Urine dilution

• Dipstick components

• Preservatives or additives used

For saliva, these biospecimens have many advantages as they are easy to collect and can be used in many situations especially if donors are afraid of needles. The preanalytical variables for this biospecimen include:

• The time of collection

• The temperature the specimen is stored

• The collection method

Conclusion

The factors mentioned are pre-analytical variables that affect the biospecimens during the collection phase. However, it is important to note that there are many more pre-analytical variables that can affect the integrity of the biospecimens during the processing, transport, and storage phase.

Human Blood Samples in Biobanks

What are Blood Samples?

Blood samples are most commonly obtained from the antecubital fossa where the veins are closest to the surface. The blood sample can be taken by anyone from a doctor to a phlebotomist or a nurse. Most blood sample collections will occur at a clinic, hospital, or at a pathology collection center. A tourniquet is first wrapped around the upper arm to slow down the blood flow while the area where the insertion area for the needle is cleaned with an antiseptic cloth. The needle is inserted, and the blood sample is transferred into containers or tubes. Proper dressing of the wound is then administered to prevent infection and to keep it clean. These tubes are then labeled with a unique identification number along with other important information. These samples are then transported to their respective destinations, such as laboratories or biorepositories.

An Introduction to Biorepositories

Biobanks and biorepositories assist in providing the materials required in clinical trials and research. There is a growing number of biobanks that help to collect data and samples from the population as a response to the increased demand of these services. The services provided by various biobanks also mean that acquiring biological materials can be guaranteed. The existence of biobanks has allowed the accumulation of biological samples from various resources. 

Biospecimens in Biobanks

Some of the examples of human biospecimens available through biorepositories include both normal and diseased states such as:

• Purified DNA

• Hair tissue

• Nail

• Whole blood

• Plasma

• Serum

• Red blood cells

• Platelet concentrates

• Platelet-rich plasma

• Saliva

• Semen

• Breast milk

• Organ tissue

• Etcetera

All specimens should be collected and processed according to the proper guidelines and procedures. 

Functions of Biorepositories

While collecting biospecimens, biorepositories also collect demographic data such as medical history, lifestyle habits, medications, and family history to create an accurate scientific database. These samples are only labeled with a unique code for identification purposes. These specimens are then maintained in the proper environment and equipment to ensure the highest quality. Another crucial point in the management of any biobank is the privacy and rights of the donors. This means biobank managers need to train their staff regarding the policies and standard operating procedures (SOPs) of the biobank.

Whole Blood and Blood Cells 

In human biospecimens, the buffy coat and whole blood are essential for biorepositories. Whole blood refers to a sample that consists of red blood cells, white blood cells, platelets, and plasma. The buffy coat describes the white blood cells and platelets that form the anti-coagulated blood sample. Both these samples are essential as they are the main source for cellular nucleic acids, construction of a DNA biobank, and achieving the maximum quality and quantity of germline DNA. 

Storage for Human Blood Cells

Blood is one of the most common biospecimens collected in human biobanks as it is a source for DNA and RNA. This is why anti-coagulated blood is a prerequisite for plasma-derived cell-free circulating nucleic acid molecules and genomic or mitochondrial DNA and RNA. One of the most commonly used anticoagulants is ethylenediaminetetraacetic acid (EDTA) for various protein assays and DNA based studies. However, citrate is more appropriate for white blood cell cultures. Storage conditions and quality of biospecimens are of vital importance as it determines the yield of extracted DNA and RNA from buffy coat or whole blood samples. 

Since RNA is easily degradable, the World Health Organization – International Agency for Research on Cancer (WHO-IARC) has suggested that it be stored in nitrogen storage below -130⁰C. Samples stored at -140⁰C by liquid nitrogen have been observed to keep the RNA in a functional state and intact for more than 50 months. To maintain the biospecimen’s reliability and preventing the possibility of multiple freeze-thaw cycles, DNA protection and stabilization can be done at room temperature which eliminates the costs for freezer storage and lowering maintenance costs for biobanks. While purified DNA can be stored at -20⁰C for months, both purified DNA and RNA are much more stable at -80⁰C in nuclease-free water or aqueous buffers for long-term storage. 

For plasma, anticoagulants such as lithium-heparin and EDTA can be used. Storage of both serum and plasma at -80⁰C have shown that there is adequate stability in the different biomolecules. The cycle of freezing and thawing should be avoided as it leads to the degradation of nucleic acids and proteins. 

Conclusion

Due to the increasing scientific developments in the past few years, it has increased the need for biological material in clinical trials and research. Biorepositories play a crucial role in supporting the researcher’s access to samples that meet their scientific criteria. It is important for biobanks to play their role in the management of data, collection, processing, and storage of biospecimens.  

References:

Mohamadkhani A, Poustchi H. Repository of Human Blood Derivative Biospecimens in Biobank: Technical Implications. Middle East J Dig Dis 2015;7:61-8.

Custom Collection Services

What is a Biorepository?

A biorepository is an organization that collects, processes, stores, and distributes tissue samples for clinical research or other scientific investigations. They assist in maintaining and managing specimens such as tissue samples from humans, animals and other living organisms. A biorepository functions to maintain biospecimens, collect relevant information and assure the quality of the samples in their collection. They follow standard operating procedures (SOPs) that reduce anomalies in samples and which also provide guidelines for storage and maintenance. SOPs also ensure that biospecimens collected closely resemble that of their natural state. It helps biorepositories maintain a standardized framework for conducting operations and allows for the seamless implementation of processes.

What is Custom Procurement?

Some biorepositories, like Geneticist Inc., provide custom tissue procurement. This means that they are able to provide custom collection of biofluids, tissue samples, and blood samples in various specialties such as gastroenterology, oncology, rheumatology, neurology, and dermatology. Biorepositories that offer custom collection services have the ability to collect from a vast range of medical procedures such as elective skin biopsies, resections, autopsies, endoscopies, blood draws, and more. Due to the nature of the collection processes and procedures, biorepositories need to have a skilled logistics team and an expert medical courier network. The custom procurement of biospecimens is important especially if:

• Fresh collections are required

• There is a necessity for matched tissue pairs

• Rare indications or specific specimens

• The need for specific procedures during the collection and processing of samples

• Active cases and autopsies are needed

• The study or research is complicated

Examples of Biospecimens

• Biofluids – Examples of biofluids include stool, urine, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum, and swabbed material. Biofluids can be available frozen or fresh. Depending on client preference, it can be with or without additives.

• Tissue – Some examples of human tissue include fresh tissue, fixed tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE) blocks, whole tissue samples, stained slides, unstained slides, tissue microarrays, and more. These samples can be annotated with the proper genetic and molecular characterizations, outcomes data, pathology reports, and patient characteristics.

• Cells – Some research may require frozen or fresh cells that are still viable. These cells can be isolated from peripheral blood, cord blood, and bone marrow of normal donors and those with disease. Some examples include myeloid cells, pluripotent stem cells, mononuclear cells, and lymphoid cells.

What to Look For?

There are several factors that help decide which biorepository to go with when custom collection services are needed. Some of the factors that can help decide are:

a)       Procurement format options

Since prospective collection enable clients to decide which elements fit their needs, a custom procurement format can be designed with the help of our experienced team of scientists. Depending on the need of clients, custom procurement of biofluids, fresh tissue, frozen tissue, and more are available. Clients can also set the exclusion and inclusion criteria for specimens and donors.

b)      Partnerships

It is important to look for a biorepository that has a vast network of clinical partners to help ensure the highest quality of required biospecimen collection. It would also increase the access to more human tissue samples in various formats.

c)       Team members

A biorepository with experienced and certified team members would be the best choice as they would be well-trained with the ability to better understand the needs of researchers and to help find better solutions if necessary. With a great team on hand, specimens are more likely to be of the highest quality and fulfill the requirements of clients.

d)      Quality assurance

It is important for the biorepository to perform quality control checks to ensure that specimens are of the highest quality. This means that the staff should understand the appropriate storage or procurement procedures and ensure that the SOPs are adhered to strictly.

e)      Consent and privacy

Biorepositories should follow the procedures and guidelines during the procurement of human tissue samples. Informed consent and privacy of the donors should be of the highest priority to protect their interests.

Why Geneticist?

For custom collection services, the staff at Geneticist can design custom collections of a wide variety of biospecimens. Our staff are certified, experienced, and highly trained. Geneticist is a biorepository that is compliant with the Institutional Review Board (IRB) standards and provides the highest quality of collections. All our biospecimens and material adheres to the official protocols and is approved by the IRB and Independent Ethical Committee (IEC). Geneticist operates in accordance with current Federal Regulations, Health Insurance Privacy and Portability Act (HIPAA) and International Conference on Harmonisation – Good Clinical Practice (ICH-GCP) guidelines. With the proper inventory management process and extensive procurement formats, Geneticist strives to fulfill client requirements and satisfaction.

Specimen Extraction and Collection Process

Introduction to Specimen Extraction

The number of research and clinical studies being conducted to help improve screening procedures, diagnostic tests, therapies, and prognosis is rapidly increasing. This means tissue samples or biospecimens such as fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) blocks are of great value researchers. Obtaining high-quality samples is the first step towards credible data and testbale results. This is why suppliers of biospecimens should aim to collect, maintain, and disseminate the best biospecimens possible. High-quality samples are those that resemble the biology of the donor before its removal from the host. Once removed, the specimen may change based on the environment. Such things as exposure to different chemicals or environmental factors during the collection or storage procedure are all things that can negatively affect the sample.

Pre-Analytic Variables

Pre-analytic variables are factors that affect the collection, processing, and storage conditions that impact the integrity of the biospecimen before their removal from the host. Examples include, but are not limited to:

a)       Donor Physiology

This includes things such as the health of the donor, consumption of food, beverages or medications before the collection of the specimen, time of day the specimen was collected, and the type of anesthesia used. In female donors, even the time of their menstrual cycle can affect the downstream analysis. This stresses the importance of collecting this information from donors to decrease variability between samples.

b)      Collection Practices

It is important to maintain uniformity during the removal and collection of specimens from donors as different methods can affect the quality of the biospecimens. These specimens should also be preserved very quickly after removal from the donor. Newer preservation methods should be considered as they can allow for better and more accurate preservation of the biospecimen. Some of the factors that should be considered during biospecimen collection include, inter alia:

• The site of collection

• Type of anesthesia used

• Warm ischemia time

• Use of stabilizing agents

• Types of fixatives used

• Exposure time to fixatives

• The temperature for maintaining biospecimens.

All the above factors can affect the stability and degradation of molecules in the samples. Annotation of the biospecimens should include the donor’s information. This data should be recorded and maintained in a database. Handling of biospecimens should also be optimized to reduce molecular changes that may occur due to processing activities.

Analytic Variables

Analytic variables are factors that affect the performance of a testing procedure. To reduce errors, some of the following factors should be considered:

• Always use validated assays whenever possible.

• Train technical staff regarding the standard operating procedure.

• Use uniformed reagents.

• Use the proper type and number of control samples.

• If possible, randomization should be applied.

• Use standardized methods during the documentation and interpretation of results.

Biospecimen Collection and Reference Ranges

The specimens collected has to be appropriate for the clinical study and downstream applications that will be used in the research. Biospecimens should be examined by a qualified histopathologist to ensure quality and accuracy. Reference ranges should be used to ensure that any deviation from the reference range can be accurately detected. This is due to the reason where disease can be defined as a deviation from normal variation. This means the diagnosis of the disease depends on the scope of normal variation. All reagents should be quality controlled, so it is fit to be used in the assay. The standard operating procedures (SOPs) should be reproducible with control biospecimens that have a range of anticipated assay values. Poorly handled biospecimens tend to produce erroneous test results due to molecular changes.

Biospecimen Storage

The following practices should be applied to all types of biospecimens:

• Follow standard protocols when storing biospecimens to maintain quality. Personnel should record the conditions for storage, deviations from SOPs, temperature, equipment failures, and thaw / refreeze episodes. It is essential to validate storage equipment, maintain back up equipment, and identify “hot spots” in the freezer.

• Store specimens in a stabilized state.

• Avoid unnecessary thawing and refreezing.

• Follow protocols if thawing and refreezing are necessary.

• Inventory tracking is desirable as it helps reduce the disruption of the environment during retrieval of samples.

• Consider the length of storage, type of biospecimen, biomolecules of interest, and study goals when selecting storage temperature for samples.

• Use appropriate storage vessels and ensure stability under storage conditions. The proper storage vessel can prevent sample loss and reduce costs of storage and retrieval of biospecimens.

• Choice of labels and printing should include the consideration of long-term storage conditions.

• Protection for personnel such as face shields and gloves should be worn.

• Each specimen should have a unique identifier that is clear, affixed, and able to endure the storage conditions.

• Automated alarms that monitors the storage equipment with the capability to warn personnel when equipment failure occurs should be in place.

• An alternative power source and other backup equipment should activate automatically if there is equipment failure.

• SOPs to routinely test equipment failure, backup equipment, and other emergency situations should be available.

• Specimens should only be available to authorized personnel.

• Ensure appropriate shipping conditions and documentation.

References

Biospecimen collection, processing, storage, retrieval, and dissemination. National Cancer Institute. Accessed 9/18/2018.https://biospecimens.cancer.gov/bestpractices/to/bcpsrd.asp

Ethics in Biobanking

Introduction

Biobanking ethics are important and one of the most debated issues in public health and bioethics. Biorepositories carry the potential to advance disease research in unprecedented ways. There are however concerns about donor privacy and not all biorepositories make sure that they follow ethical standards. People’s DNA and tissue sample has been used without respect for their rights. Regardless of peoples differing views and perspectives, one thing can be agreed upon among most experts, that biobanks are revolutionary. Biobanking ethics include issues such as, but not limited to:

• Controversies and key challenges faced in biobanking ethics

• Issues of informed consent

• Withdrawal from participants

• Broad consent

• Ethics of re-contact

• Confidentiality issues

• Ownership, property and commercialization problems

Issues in Brief

1)      Informed Consent

Informed consent is crucial in ensuring that ethical standards are followed in both research and therapy. It ensures that the participant understands “the nature, duration, and purpose of the experiment; the method and means it is to be conducted; all inconveniences and hazards reasonable is to be expected; and the effects upon the individual’s health may be due to the participation in the experiment." (Excerpt from Springer Article) One of the major issues of informed consent in biobanking is that it only applies to the donor and not those who are connected to the donor. Next, since biospecimens can be used in future studies, participants cannot be “informed” at the time their tissue is obtained as the nature of future researches are not yet known.

2)      Broad Consent

There are some experts who believe that broad consent can be a potential solution to the issues of informed consent in biobanking. However, there are some who disagree as it offers minimal protection and minimal guarantees. Broad consent is the permission given by the donor for the biobank, so the biorepository can do what they see fit with the genetic material. While some individuals argue that broad consent is a means of maximizing autonomy, some see it as the opposite where it is an abuse for autonomy. Ethicists worry that broad consent causes donors to relinquish their rights regarding how their genetic material is used in the future.

3)      Confidentiality

One of the main features of genetic information is that it can be used to identify the donor and those related to them. While ethicists argue that identification can be discouraged through various methods of anonymization, there is always the possibility that identification is possible. The risk of identification increases as databases grow.

4)      Property and Profit

There is also the issue that participants or donors do not own their tissue samples. This is based on the traditional understanding that body parts are res nullius which means that they do not belong to anyone once detached. Ethicists have argued that there are valid reasons for following the “no property” rule for biospecimens. Allowing property would restrain studies and research to the point where it would become untenable. Another issue is that commercial companies may look to make very large profits from donated samples.

5)      Feedback to Participants

Another ethical issue is whether or not to tell participants regarding incidental findings from their donated tissue samples. Incidental findings can be defined as “observations of potential clinical significance that have been discovered unexpectedly in a healthy subject unrelated to the purpose and variables of the study.”(Excerpt from Springer Article)

6)      Participation, Representation, Maintenance of Trust

Biorepositories are also worried about the mass withdrawal of participants as it will ultimately result in the loss of set-up costs. The maintaining of trust between the public and biobanks are crucial to prevent participant withdrawal and biobank failure. Currently, there is still no one way that is viewed as the best method of practice in this area.

7)      Re-contact

Re-contact is becoming an increasingly crucial issue as there is very little industry conformity on how re-contact should be managed. There needs to be a balance between what donors are informed and what's included without overburdening them. Biorepositories and research teams should view the ability to re-contact as a limited resource. Currently, there is no standard that biobanks can look to adopt for this issue.

Conclusion

The problems and challenges in biobanking ethics mean that there is a need for alternative models to address the issues. Biobanking presents important and significant ethical challenges. It is important for those involved to be aware of the advancements and developments in the debates surrounding these issues. By raising awareness of these issues, public interest of will increase and as a result biobanking can continue to change the medical research landscape.

References:

Widdows H, Cordell S. The ethics of biobanking: key issues and controversies. Health Care Anal. 2011; 19:207-219.