What is Cryosectioning Protocol

Cryosectioning Introduction

The cryosection procedure is also known as the frozen section procedure. It is a laboratory procedure used to perform microscopic analysis of a specimen. This procedure is most commonly used in oncological surgery. A pathologist is necessary for the intraoperative consultation. The pathologist evaluates and examines the specimen. The pathologist then reports if the specimen is benign or malignant. They are also responsible for informing if the resection margin is clear of cancer. The cryosectioning procedure practiced today is based on the technique described by Dr. Louis B. Wilson in 1905. Cryosections are a good way to visualize the fine details of the cell. Although less stable than resin and paraffin-embedded sections, cryosections are typically more superior for the preservations and detection of antigens through microscopy. Cryosection preparation can usually be done in a day. The rapid freezing helps decrease the formation of ice crystals and minimizes morphological damage. Cryosections can be used in various procedures such as in situ hybridization, immunohistochemistry, and enzymatic degradation.

Reagents

Some of the reagents used in a cryosectioning protocol are:

·         Fixative (such as formaldehyde)

·         Optimal cutting temperature (OCT) compound (such as Tissue-Tek; Sakura Finetek USA)

·         Staining solution (such as toluidine blue, hematoxylin, eosin, or others)

·         Fresh tissue sample

Cryosectioning Equipment

Important equipment that would be required in a cryosectioning protocol are:

·         Brush (camel hair)

·         Container for storage of tissue sample

·         Cryostat with metal grids (The cryostat can be likened to a microtome in a freezer. It is a machine capable of slicing thin sections. Although expensive, hospital pathology laboratories may have cryostats available for rental)

·         Microscope slides (The slides can be silanized or poly-L-lysine coated)

·         Plastic or metal tissue mold

·         Moistened tissues

cryosection Procedure

The procedure for cryosectioning can be done quickly as it is relatively simple.

A)      Cryostat preparation

i)                    Use proper cleaning agents and clean the cryostat.

ii)                   Insert a new sterile blade for the cryostat.

iii)                 When it comes to frozen embedded block or fresh frozen specimens, use some frozen embedding media to adhere the sample to the mount in the proper cutting position. Ensure that the cutting surface is parallel to the blade.

iv)                 Allow the specimen to equilibrate reaching cryostat temperature. This takes approximately 20 minutes.

B)      Cryosection preparation

i)                    Freeze a tissue sample up to 2.0 cm in diameter in OCT using a suitable tissue mold. Freeze the OCT with tissue onto the metal grids fitting the cryostat. At room temperature, OCT is viscous but freezes at -20⁰C. Depending on the type of tissue, optimal freezing temperature may differ. For example, brain tissues are optimally frozen at -3⁰C in M-1 medium.

ii)                   Cut sections that are approximately 5 to 15μm in the cryostat at a temperature of -20⁰C. the temperature of the cutting chamber can be adjusted based on the tissue under study. These sections can be moved with the help of toothpicks and brushes if necessary. A camel hair brush can be helpful in guiding the merging section over the blade.

iii)                 Remove the folds and wrinkles present on the cut sections.

iv)                 Within 1 minute of cutting the tissue section, transfer it to a room temperature slide by touching the slide to the tissue. This allows the cut section to adhere to the slide. Using your gloved finger, rub the underside of the slide to help transfer heat as this helps with the adhesion. All this should be accomplished within 1 minute of cutting the section. This avoids the freeze drying of the specimen. Using silanized or poly-L-lysine coated slides improve the adherence of the section.

v)                   An optional step is to use ultraviolet treatment of the slide to increase the adherence and with sterilization. This can be done by incubating the slide for 15 to 20 minutes under the ultraviolet light. It works best if an ultraviolet sterilization hood is utilized. The light breaks down the membrane slightly helping with adhesion and sterilization. It is important to not incubate it longer than 30 minutes as it risks damaging the membrane.

vi)                 To evaluate the preservation and orientation of the tissue, the first slide of each set can be stained using toluidine blue, eosin, hematoxylin, or various aqueous stain.

vii)               Immerse the slide immediately into a fixative. Some researchers air-dry the section onto the slide at air temperature to maximize adherence before fixation. However, this technique has a disadvantage where the surface tension forces distortion of the cells resulting in the loss of high-resolution detail. It can also lead to changes in the results of immunostaining.

viii)              Any unused tissue should be covered using a layer of OCT to avoid freeze drying and storing leftover samples at -70⁰C. For long-term storage, adding moistened tissue to the container helps prevent desiccation especially in a frost-free freezer.

Cryosection Troubleshooting

If the tissue is difficult to section, it is important to consider the following:

·         If the frozen tissue in OCT is not cut in a thin and smooth sheet, the knife could be dull.

·         The tissue can be difficult to section if the tissues have variable textures, water, or fatty.

References

1)      Fischer AH, Jacobson KA, Rose J, Zeller R. Cryosectioning tissues. CSH Protoc. 2008: pdb.prot4991.

2)      Protocol Cryosectioning. Molecular Machines & Industries. Accessed 1/8/2019. http://www.molecular-machines.com/libraries.files/Cryosectioning.pdf


What is a Frozen Section?

What is a Frozen Section?


A frozen section is a term referring to a section of tissue that has been rapidly cooled using cryostat. It is an important feature that is needed in hospitals to assist with the diagnoses of lesions and the extent of the lesion during surgery. The cryostat is an instrument used to freeze the human tissue samples and cut it for microscopic section. It is used to aid in the immediate diagnosis of lesions to help medical professionals plan the management for the relevant patient. Frozen sections are also helpful for immunofluorescence and enzyme immunochemistry studies. Another useful indication would be to stain certain carbohydrates and lipids present in the tissue.


The Principle of Frozen Section

When the tissue sample goes through rapid freezing, it converts water into ice which acts as an embedding media allowing the tissue to be sectioned. The tissue can become firmer if the temperature of the tissue sample is lowered while increasing the temperature softens the tissue. Some important factors to note are:

  • The temperature range in the cryostat machine usually ranges from 0⁰C to -35⁰C. The majority of the tissue can be appropriately sectioned between -15⁰C to -25⁰C. Tissue samples that contain water can be sectioned at a higher temperature while tissues that contain more fat will need to be cut at a lower temperature.

  • The knife inside the rotary microtome is fixed, and the tissue can be moved with the help of the rotary wheel.

  • A tissue shelf in one side of the microtome can be used to keep the tissue. This helps to keep the samples at a freezing temperature as the temperature in the tissue shelf is usually lower compared to the cabinet temperature.

  • There is a small place to place the knife and brush holder in front of the microtome machine.

  • To obtain an even pressure during sectioning of the tissue samples throughout the whole length, the blade should be fixed to the holder with the knife angle kept between 5⁰ to 7⁰.

  • An antiroll plate is available in front of the knife to prevent the rolling of the tissue during the cutting process. The antiroll plate is usually glass within a metal frame.

  • A cool sable hair brush is also available to obtain unrolled tissue.

  • Depending on the manufacturer, the specimen holder can be available in various shapes and sizes.

  • The optimal cutting temperature (OCT) compound such as resin and water-soluble glycols will be used as an embedding medium to hold the tissue.


Cryostat Sectioning

  • The tissue sample and requisition form should first be identified.

  • Look at the clinical information available as it can help achieve the possible differential diagnoses.

  • Observe the gross appearance of the tissue in terms of color, consistency, texture, and presence of sutures used to mark the anatomical position of the sample.

  • Identify the resection margins and planes. Use different ink colors for identification of medial and lateral margins.

  • When cutting the tissue, ensure that the tissue is fresh, preferably dry, and free from any gauze, sutures, or staples. The tissue is then cut into multiple small pieces to assist freezing. Multiple sections of the tissue should be obtained to help minimize error and understand the primary pathology. Use a sharp blade to cut the most crucial area using gentle pressure.

  • To embed tissue in the mold, keep a small piece in the center of the mold and pour OCT in excess over it. A tissue holder is then placed over the tissue.

  • Place the tissue in the frozen section chamber. To hasten the process, cold spray can be used.

  • Load the cutting knife at the proper alignment.

  • Once the tissue is frozen, it will appear whitish. Place the in frozen tissue the holder of the microtome and trim to remove the excess OCT. This prepares the tissue surface for sectioning.

  • Cut the tissue gently and spread it over the antiroll plate using a cooled brush.

  • Press a glass slide firmly over the section and fix immediately in methanol for 1 minute. 95 percent ethanol can also be used for tissue fixation (for a few seconds). Rapid fixation is a must as a delay results in swollen cells and hazy cell margins.


Staining


Staining is usually done using hematoxylin and eosin (H&E) and toluidine blue stain. The slide is first rinsed in tap water and put in hematoxylin for a minute. It is then rinsed in tap water for 5 seconds followed by another rinse in Scott's tap water for another 5 seconds. The slide is then dipped in eosin for 20 seconds and rapidly rinsed in tap water after.


Conclusion


The frozen section refers to the process where there is rapid tissue section cooled with a cryostat to provide an immediate report of the tissue sample. The cryostat freezes the tissue allowing it to be cut for a microscopic section. The conversion of water into ice acts as the embedding media for cutting the tissue. It is a technique that is mostly used for the rapid diagnosis of lesions during intraoperative management. This helps to determine the extent of the lesion, allows for immunofluorescence and immunohistochemistry study, and staining of specific carbohydrate and lipid in the tissue. This article has described the principle, techniques, indications, and instructions on how to make a good quality frozen tissue section.

References


Dey P. (2018) Frozen Section: Principle and Procedure. In: Basic and Advanced Laboratory Techniques in Histopathology and Cytology. Springer, Singapore.





What is a Biopsy?

Biopsy 101

A biopsy is a test that is used to help determine the composition and structure of the cells and tissues of the body. The test can be used to evaluate human tissue samples from part of the body to allow the examination of the tissue sample under a microscope. Since most biopsies are minor procedures, patients do not usually require sedation. Patients may only require local or no anesthesia. The term biopsy is of Greek origin “bios” and “opsis” where the word “biopsy” can be loosely translated to “appearance of life” or “view of the living”. Biopsy is a good way to evaluate for the presence of malignancy or for confirmation if the abnormality is benign. If there is cancer, a pathologist studies the tissue sample to determine the type and grade of malignancy.


Why is a Biopsy Performed?


A biopsy is usually performed when there are lesions that can be felt or seen on imaging such as ultrasound, X-ray, computed tomography (CT) scan, or magnetic resonance imaging (MRI) scan. The biopsy is used to help determine the nature of the suspected abnormality. In cases of suspected cancer, the biopsy helps determine if the area involved is malignant or benign. One good example is the biopsy of a breast lump for histologic examination to determine if it is cancerous. The laboratory analysis of the specimen is performed by a clinical pathologist. A definitive and correct diagnosis is usually arrived through histological and cytological examination.


When a tumor is malignant, biopsies of the lymph nodes and surrounding tissues are performed to determine if the cancer has metastasized. Biopsy also helps to determine the grade of cancer. Surgical biopsy is a procedure that removes the entire tumor and can be done with the guidance of endoscopy or imaging. The pathologist can usually tell if the cancer is a slow or aggressive form.


Types of Biopsies


There are many types of biopsies. The type of biopsy used for the patient will be determined based on several factors such as:
• Location, body part, or organ to be sampled
• Number of lesions
• How suspicious the lesion appears to be
• Characteristics such as shape and size of the lesion
• Existing comorbid and patient preference
• Facilities and systems available at the current healthcare facility


Some examples of biopsy procedures include

Diagram of Biopsy.png

a) Aspiration or fine needle aspiration (FNA) biopsy
b) Cone biopsy
c) Core needle biopsy
d) Endoscopic Biopsy
e) Surface biopsy
f) Vacuum assisted biopsy
g) Punch biopsy
h) Surgical biopsy or excisional biopsy


Image Guided Biopsy


There are many biopsy procedures that are done with the help of image guidance such as CT and ultrasound. There are many breast biopsies that have been done with the guidance of stereotactic mammography. CT is increasingly used to guide the biopsy of liver and lung lesions. Interventional CT now helps to allow real time CT imaging during biopsy increasing diagnostic accuracy and shortening procedure times. Ultrasound is also useful as it offers great flexibility to follow the path of the needle to the lesion, provide real time display, and allows professionals unlimited imaging. In stereotactic mammography, it helps to show images of two angled directions to guide the needle. MRI helps provide real time images that guides the trajectory of the needle approaching the lesion. It also provides a high contrast resolution helping radiologists to differentiate between abnormalities and organ structures.


Risks of Biopsy


Although biopsy is a minimally invasive and relatively safe procedure, there are still certain risks that are involved. Some examples of risks for needle biopsy include:
• Infection of the area being biopsied
• Hematoma
• Puncture of structures near the biopsy target
• Vasovagal response
• Hemorrhage


For surgical biopsy, risks include


• Scarring due to stitches after excision
• Mortality due to risks of anesthesia
• Possibility of infection, bleeding, and delayed wound healing
• Longer recuperation necessary compared to needle biopsy


Biopsy Results


Biopsy results can be negative or positive. A negative result usually means that there are no abnormal cells seen in the examined tissue sample while a positive result means that abnormal cells are seen in the cell sample. Abnormal results help to identify:


- A possible infectious agent, changes to the cells caused by infection or disease.
- The presence of a benign growth or process.
- The presence of abnormal cells where cancer cells are seen. With these findings, the pathologist may be able to determine the origin of cancer cells to see if it is from a primary tumor or from metastasis.


Who Performs Biopsies?


Biopsies can be performed as an outpatient or inpatient bases by medical doctors or doctors of osteopathy. Surgeons are individuals who perform excisional and open biopsy. More invasive percutaneous biopsy such as the liver or lungs will be performed under guidance of medical imaging usually by a radiologist .The biopsy specimen is then analyzed by a pathologist who then renders a medical diagnosis based on the tissue sample.


References:


1) Biopsy procedure – what is a biopsy. Imaginis. Accessed 12/11/2018. http://www.imaginis.com/biopsy/biopsy
2) What is biopsy: overview, benefits, and expected results. Docdoc. Accessed 12/11/2018. https://www.docdoc.com.sg/info/procedure/biopsy/










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.


The Difference Between Biobanks and Biorepositories

What is a Biorepository

A biorepository is a center that functions to:

  • Collect

  • Process

  • Store

  • Distribute

Biospecimens help support present and future research studies and investigations. It is a place where various specimens from many living organisms such as, animals and humans are contained and managed. Many life forms such as arthropods, vertebrates and invertebrates can be analyzed and studied through the preservation and storage of their tissue samples. Besides maintaining the relevant biological specimens, biorepositories also have a role to collect the associated information from these specimens for future use in research. One of the most crucial roles the biorepository plays is to ensure the quality of the collected samples. They also have to manage the accessibility of biospecimens while handling the disposition and distribution of their collection.

Biorepository Operations

As previously mentioned, the four main operations of a biorepository are collection, processing, storage and distribution. For elaboration purposes:

a)       Collection – This is the first step where biospecimens are obtained and recorded in the records. This can be done by scanning the sample’s barcode where the information regarding the sample is then transferred into the biorepository’s laboratory information management system. Examples of the information recorded would include the origin of the sample and the time the sample arrived.

b)      Processing – This phase involves the testing of the biospecimens to minimize variation and preparing them for storage. One example is the processing of DNA samples into a salt buffer to stabilize the DNA for long-term storage.

c)       Storage – After the biospecimens are collected and processed, they are stored accordingly based on the required temperature and environment. Some samples are stored in freezers while some can be stored at room temperature. This is where all biospecimens are held before distribution.

d)      Distribution – This occurs when the biorepository fills an order or request from a research team from the biorepository’s inventory system.

Biorepository Standard Operating Procedures

Standard operating procedures or SOPs are vital in a biorepository. It helps to:

·         Minimize variation between samples and reducing issues through standardized guidelines

·         Ensure that biospecimens closely resemble their natural state

·         Provide a framework of how operations should be conducted in a biorepository

·         Ensure reliable and seamless process during operations

·         Provide guidelines for backup during emergencies

An Overview of Biobanks

A biobank is a type of biorepository that stores biological samples that are usually human for research. Biobanks are an important resource for medical research as it helps support various types of contemporary research. It allows access to data for researchers that represent a large population. Samples and data available in biobanks can also be used by many different researchers for various studies. This is crucial as there are many researchers who have difficulty acquiring samples before biobanks existed.

Although many issues such as privacy, medical ethics, and research ethics have been raised, a consensus has been reached that operating biobanks should consider the policies and governing principles that protect the communities that participate in their programs. The term “biobank” can be defined as “an organized collection of human biological material and associated information stored for one or more research purposes”. While biospecimen collections from other living organisms can also be called biobanks, many prefer to reserve the term only for human biospecimens.

Types of Biobanks

Biobanks can be classified based on their purpose or design. Both the terms “biobanks” and “biorepositories” have been used interchangeably. In the United States, the National Cancer Institute thinks of biorepository as a place or organization where biospecimens are stored. The term “biobank” is also being used in the same context in the United States and European institutions. Biobanks can be classified based on different approaches such as:

  • Population-based biobanks

  • Hospital or academic based biobanks

  • Disease-oriented biobanks

  • Non-profit organizations or commercial companies

Biobanks can also vary in nature, contents, participants, and scale. For example, a human biobank can be classified based on the tissue type, their research purpose, or ownership of the biobank. The size of the biobank can be based on the disease group, national, statewide, or regional. Other experts classify biobanks into four different basic types:

  • Clinical or control based biospecimens from non-diseased donors and donors with specific diseases.

  • Biobanks that follow their participants over a long period of time, also known as longitudinal population-based biobanks.

  • Biobanks with twin registries that obtain samples from both dizygotic and monozygotic twins.

  • Population isolate biobanks that have a setup using homogenous genetic donors.

Despite the various classifications of biobanks from various experts, the currently accepted classification is from the pan-European Biobanking and Biomolecular Resources Research Infrastructure (BBMRI). They distinguish only two types of biobanks which are:

  • Disease-oriented biobanks where it contains clinical data and tissue samples

  • Population-based biobanks where the focus is on the study and development of complex and common diseases.

Conclusion

In conclusion, both the terms “biorepository” and “biobank” are often used interchangeably as the distinction is blurry. However, one of the most significant differences is that biobanks often refer to collections of human biological material while biorepositories can refer to collections of all living organisms.

References:

1)      Biorepository. Wikipedia. Accessed 11/8/2018. https://en.wikipedia.org/wiki/Biorepository

2)      Biobank. Wikipedia. Accessed 11/8/2018. https://en.wikipedia.org/wiki/Biobank

3)      Kinkorova J. Biobanks in the era of personalized medicine: objectives, challenges, and innovation. EPMA J. 2016; 7(1):4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762166/


Preanalytical Variables: Long-Term Storage and Retrieval of Biospecimens

Introduction

The week before last we talked about how pre-analytical variables affect the integrity of human biospecimens, and this week we’ll be following up on this article by discussing the long term storage and retrieval of biospecimens.

The term “storage” comprises of both short and long-term storage of all biospecimens consistent with the study design and planned future use. Depending on the details of their future use, the specimens are either locally or centrally stored. It can also be stored in both locations. The decision will be made depending on the:

  • Sample size of biospecimens

  • Complexity of collection

  • The accrual rate of biospecimens

  • Processing procedures

  • Logistics

  • Cost of storage and retrieval

  • Quality issues

  • Biorepository governance factors

If the biospecimens are stored for various uses, biorepositories should have duplicates that are close in proximity to the main laboratory. Samples that are to be stored for more than a year should be stored centrally. Duplicates should also be stored on different power supplies or different locations as insurance against natural disasters or equipment failure. Biorepositories are also recommended to have approximately 10 percent of the total mechanical freezers as empty backup freezers to protect against freezer failure. Different storage conditions may be required based on the downstream analyses. Some of the pre-analytical variables that affect long-term storage include:

  • The time involved from processing to storage

  • Duration of storage

  • Temperature

  • Facility

  • Environmental impact (such as moisture, sunlight, dehydration, humidity, oxidation, evaporation, and desiccation)

  • Freeze-thaw cycles

  • Some emergencies include: encapsulation of biospecimens in ice after refreezing and microbiological contamination

  • Destroyed or no labeling

  • Missing or misplaced aliquots

Since biobank material is valuable and hard to replace, the use of systems such as the laboratory information management system (LIMS) should be utilized as it helps allow traceability, confirm chain of custody, and manage biospecimens to improve data reliability and retrieval. Once the integrity of a biospecimen is compromised, it is no longer valuable and becomes useless. It is therefore important to retrieve only those biospecimens that are required. As previously mentioned, duplicate collections of biospecimens are ideal to prevent the destruction of samples.

Blood Sample

The study of the stability of analytes compared to the fresh sample, taking into account the recovery rates, are vital to determine the effects of long-term storage. After long-term storage, the recovery rates may decrease or increase resulting in increased or attenuated risk ratios. It is recommended that hormone, chemistry, and protein analytes are much more stable and stored at -80⁰C for up to 13 months. However, various studies have shown that there are different patterns of stability based on the analyte, time, and temperature of storage. There has been no systematic influence regarding omics analyses observed in samples collected in citrate, heparin, or ethylenediamine triacetic acid (EDTA) if stored at -80⁰C in liquid nitrogen. Long term storage in room temperature and repeated freeze cycles must be avoided. At room, low, and ultra-low temperatures, the extraction of DNA from whole blood samples using bio stabilization technology yielded samples that are pure and that have integrity. Although live cells are stable at room temperature for as long as 48 hours, it should be cryopreserved or cultured in liquid nitrogen to ensure its viability. The recovery of sufficient DNA or those that are of acceptable quality for microarray studies involves the transfer of thawed buffy coat or EDTA whole blood into RNA preservative. Serum or plasma that will be used for miRNA analysis must be extracted immediately or maintained at -80 in RNA free cryotubes.

Urine Sample Protocol

For urine samples, long term storage at temperatures less than -80⁰C without additives is ideal unless it has been specified for certain downstream analyses. Urine samples have been stored at -22⁰C for 12 to 15 years without the use of preservatives while ensuring the stability and measurement validity. Urine used for metabolome and proteome analyses will go through progressive protein degradation if stored at room temperature. While freeze-thaw cycles have minimal impact on the protein profiles, repeated cycles should ideally be avoided.

Saliva Sample Protocol

The protocols for saliva storage are ultimately dependent on the expected downstream analyses. There seems to be minimal impact of protein profile changes despite freeze-thaw cycles. It is recommended that it is stored at -80⁰C. If the saliva samples were divided into aliquots and frozen immediately at -80⁰C, there does not seem to be any differences in cortisol, C-reactive protein, mRNA, or cytokines.

Extracted DNA Sample protocol

The most common method of storage for DNA is still freezing it at -80⁰C. It should be noted that DNA degradation increases with repeated freeze-thaw cycles, higher storage temperature, dilution, and multiple suspensions. Special technologies allow the minimization of storage space and the reduction of shipping and electrical costs. This can be beneficial especially when cryogenic or mechanical equipment is unavailable. It can also be an alternative method for backup storage. Using this technology, there is no degradation or accelerated aging of DNA at room temperature or higher temperatures (50-70⁰C) throughout the 8-month storage duration.

RNA sample protocol

Some of the pre-analytical storage factors that can affect the quality and quantity of analyte or gene expression include the concentration of RNA, temperature, storage time, and repeated thaws. New technology for the dry storage of RNA at room temperature has been developed. This is a technology comparable to RNA that is cryopreserved for up to a year for downstream analyses such as RNA sequencing and real-time polymerase chain reaction.

References:

Ellervik C, Vaught J. Preanalytical variables affecting the integrity of human biospecimens in biobanking. Clinical Chemistry. 2015; 61(7): 913-934. http://clinchem.aaccjnls.org/content/clinchem/61/7/914.full.pdf


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


SOP’s of a Biorepository

Introduction to Standard Operating Procedures

Biobanks and biorepositories needs to develop and adopt standard operating procedures (SOPs) that describe the policies and processes of the biorepository in detail.
SOPs should be detailed, well structured, and should undergo a strict approval process. SOP’s need to be reviewed periodically to assess the necessity of updates. Once implemented, SOPs should be routinely followed. Copies of SOPs should always also be stored in designated locations accessible to all personnel at any given time. Biorepository and biobank personnel should review SOPs prior to implementation.

Contents of the SOP

The SOP manual can be very dense and detailed, but should at the very least contain the following points:

Informed Consent

All biorepositories need to keep all records and related documents of the informed consent status for every biospecimen. Additionally, the procedures or protocols followed to obtain informed consent must be specified. Along with this the protection of privacy for all participants and data confidentiality should also be described.

Equipment

This section of the SOP should include the monitoring and calibration of all of the equipment. SOPs regarding the maintenance and repair of equipment is also crucial. All biorepositories should have their own procedures that routinely monitor all the equipment that is involved in the preparation and storage of biospecimens. The accurate calibration of equipment is vital to avoid affecting the quality of biospecimens and their data. The operational settings of the equipment should always be recorded along with all of the repairs that have been performed.

Collection Supplies

All biorepositories should have high standards for their reagents and consumable supplies that are used in the collection, processing, and storage of biospecimens. This means that the supplies should be acquired from certified and approved vendors and should meet the material specifications. Personnel should ensure that these supplies are in good condition prior to their utilization.

Identification and Labeling

All biorepositories will need to have their own guidelines and protocols for the labeling and identification of biospecimens. This should be coupled with the linking of biospecimens to their records regarding donor information and informed consent, so that when required it will be readily available.

Collection and Processing Methods

In this section details are extremely important to allow for the accurate replication of collection and processing procedures. This means that detailed descriptions of the supplies and equipment used are necessary. Along with the methods and processes used in the division of biospecimens into respective aliquots. The collection and processing of biospecimens must include records of staff names, dates, and specific times so potential pre-analytical variables are all recorded.

Storage and Retrieval

All procedures for the storage and retrieval of biospecimens from the biobank should be well described. This should include the guidelines for the addition and withdrawal of biospecimens, response to requests, fulfilling requests, and the disposition of biospecimens.

Transport and Distribution

All biorepositories should have designated policies and protocols for the transport and distribution of biospecimens that ensures their integrity, quality, and safety. This means guidelines should include packaging specifications addressing temperature conditions, temperature monitoring, regulations for the transport of hazardous materials, shipment logs, notifications for delivery, delivery confirmation, and agreements that cover transfers.

Quality Control

All biobanks should have their own testing procedures that document the results which are then kept in the records. This should comprise of tests that assess and control the quality of biospecimens, confirmation of histopathology diagnosis, assessment of nucleic acid integrity, biomarker expression, and more.

Informatics

Policies and guidelines for the management of records and procedures that define data collection methods, access to data, reporting, and quality control of data should be available for all biobanks.

Biosafety

It would be best for all biorepositories to have policies and procedures that address biosafety issues such as the reporting of staff injuries, precautions that the staff should have for bloodborne pathogens, the use of personal protective equipment, handling of hazardous material, and the disposal of biohazardous material and medical waste.

Training

All centers should have their own policies and procedures when it comes to the training of their personnel. These training should be documented, corrective actions that are taken, steps taken to resolve discrepancies for inventory or shipment, manage power outages, monitor samples, and the handling of emergencies and natural disasters.

Security

Procedures for security concerning administration and information systems should be available for all biobanks and biorepositories. These SOPs should address the different points of contact and personnel that are involved in backup. Names and contact numbers for the designated personnel should also be available.

Conclusion

It is important for all biorepositories and/or biobanks to have detail-rich standard operating procedures as it helps personnel get organized and offers guidelines in times of emergency. SOPs can also help to provide detailed information about the processes and preservation methods that ensure biospecimens retain their integrity and are of the highest quality.

References:

Quality management: Technical and operational best practices. National Cancer Institute. Accessed 10/18/2018.https://biospecimens.cancer.gov/bestpractices/to/qac.asp#b-3-3

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

CTM Logistics

Introduction to CTM Logistics

Clinical trial materials (CTM) logistics is an important business that manages the transport and delivery of biospecimens and tissue samples. The business of tissue preservation, meeting demands, and shipping these specimens from places like a biorepository to their destinations does not come without challenges.

Challenges facing CTM Logistics

Besides the conventional logistics and supply-chain challenges, those involved in CTM logistics also face:

1)      Standards

Many countries do not have standard protocols for the entry of trial supplies. Due to the demands of clinical materials worldwide, the challenge of shipping CTMs include the extraordinary constraints of import and export.

2)      Notice

Those involved in CTM logistics have very little to no notice when it comes to knowing when the materials need to be shipped out. The issue of material suppliers being disconnected can also cause delays at trial sites due to a supply chain issue.

3)      Product Standards

There are many trials that need both comparator or placebo drugs along with the trial drug. However, these trials are usually “randomized” throughout the course of the trial. This means that different labels will be needed for different trial protocols or different locations.

4)      Handling and Transport

Since the quality of these materials can be affected during handling and transport, companies involved in CTM logistics need to have the equipment and manpower that are suited to deal with the supplies.

5)      End Users

There is also the issue where some end users (such as researchers and physicians who conduct trials at investigator sites) who are not trained supply chain managers.

6)      Cost

Companies providing CTM logistics services also have a high risk of overrunning their cost due to difficulty addressing all cost drivers, inefficiencies in the system, and potential expenses when corrective actions need to be taken.

7)      Communication

Just like any other field, poor communication and coordination of the supply chain can result in action gaps, duplication of efforts, and delays. Frequent plan revisions are required if there are errors.

8)      Circumstances

Many studies often face the risk of delays and the need to lengthen their timeline in situations where there is the unexpected expiration of products or out of stock situations.

The industry has adapted to some of the challenges and constraints over the years. Although the clinical trials can be affected by issues in the supply chain, the successful completion of these researches has led to the better diagnosis, therapy, and prevention of diseases.

Supply Chain Stages

The following are critical supply chain stages:

  • Planning: Those that are responsible forecast and come up with a strategy.

  • Sourcing: This is done to acquire the necessary materials such as ancillary supplies, drugs, and lab materials.

  • Manufacturing: Once ready, the necessary packaging and labels that are needed are manufactured.

  • Storage: The CTMs are stored in the appropriate condition using the proper equipment (if needed).

  • Distribution: The CTMs are imported or exported according to local authority regulations.

  • Site: The CTMs are stored, samples managed, and data processed.

  • Return: The company manages the shipment along with final CTM reconciliation.

  • Destruction: The CTMs are recycled or destroyed accordingly.

  • Analytics: All accumulated data are processed and analyzed.

Training

Individuals who are interested in positions such as clinical supply chain managers or supervisors and those interested or new to the CTM industry. Employees that go through special training pertaining to CTMs are more efficient in their duties in the company and industry. They learn about:

  • The overview of the supply chain from beginning to end

  • The appropriate packaging and labeling that suits the needs of a specific study design

  • Having a plan of action to prepare the materials

  • Implementing their plan and troubleshooting

  • The logistics involved for distribution of CTM to trial sites that are located worldwide

  • How to outsource vendors for the labeling and packaging purposes

  • Interactive Response Technology

  • The different roles of their team members and their expectations for the CTM group

  • Current Good Manufacturing Practices (cGMP)

  • The implementation of cGMP in the packaging process

Services

The company that is in charge of CTM logistics generally provides services for:

  • The receiving, storage, and distribution of the materials

  • Obtaining the necessary import and export licenses from the authorities

  • Acquiring custom clearance for the CTM

  • Data management (verification, validation, query, tracking, coding for diseases or medications, result presentation)

  • Biostatistics (Statistical analysis, writing, result presentation, and more)

  • Importing and exporting CTMs

  • Short or long-term storage of specimens

Conclusion

More clinical trials are conducted globally. This creates a significant logistical challenge as the management of CTMs and the supply chain require tactical knowledge and a vast commitment of resources to avoid delays. There are many who would find that it is much more efficient to hire CTM service providers to manage the logistics as there are many challenges in this industry.

References:

1)      Basta N. Clinical trial logistics capabilities are expanding. Pharmaceutical Commerce. Accessed 9/5/2018. http://pharmaceuticalcommerce.com/clinical-operations/clinical-trial-logistics-capabilities-are-expanding/

2)      CTM logistics. Geneticist. Accessed 9/5/2018. https://www.geneticistinc.com/ctm-logistics/

3)      Clinical research services: clinical logistics. Parexel. Accessed 9/5/2018. https://www.parexel.com/files/6714/1155/2827/CLS_Supply_Chain_Poster_Sept14.pdf

4)      Clinical trial materials training course. ISPE. Accessed 9/5/2018. https://ispe.org/training/classroom/clinical-trial-materials


The Incredible MicroRNA's

What is microRNA?

MicroRNAs (miRNAs) are a group of small non-coding RNAs that are found in tissue samples of animals, plants, and some viruses. Since the discovery of circulating and extracellular miRNAs, there has been a rapid expansion of studies on miRNAs in biofluids like cerebrospinal fluid, plasma, serum, and urine. miRNAs are similar to small interfering RNAs (siRNAs). However, miRNAs originate from RNA transcripts forming short hairpins while siRNAs are from longer parts of double-stranded RNA. miRNAs are plentiful in mammalian cells and seem to target approximately 60% of these genes. miRNAs are thought to have important biological functions as they are evolutionary conserved. A good example is where there is conservation of 90 families of miRNAs as seen in the common ancestor of fish and mammals. The majority of these conserved miRNAs have been shown to play important roles.

Roles of miRNA

miRNA plays a role in RNA silencing and gene expression as post-transcriptional regulators. Within mRNAs, miRNAs function by base-pairing with the complementary molecules causing mRNA molecules to be silenced through cleavage of mRNA strand, destabilization of mRNA, or reduced efficiency of mRNA translation by ribosomes. Despite the low numbers of miRNA, it is estimated that the miRNAs regulate approximately more than 33% of the cellular transcriptome. Therefore, it should be no surprise that the miRNAs have crucial functions in the developmental and cellular processes that have been thought to be involved in many human diseases.

Since miRNAs are relatively stable in biofluids and have a wide range of biological potential, these molecules are suited to be used as non-invasive biomarkers in diagnosis, drug safety, and pre-clinical toxicity. Many studies have proven that secreted miRNAs are involved in conditions such as organ damage, cancers, and coronary heart disease. While the exact role of circulating miRNAs is mostly unknown, they have been observed to be protected from RNAse degradation through the inclusion in membranous particles or protein complexes.

MicroRNA.png

Disease

Since miRNA plays a role in the normal functioning of eukaryotic cells, miRNA dysregulation is therefore linked to disease. For example:

 

a)       Hereditary Diseases

  • It has been found that a mutation in the region of miR-96 results in progressive hearing loss.

  • Hereditary keratoconus with anterior polar cataract is seen in mutation in the region of miR-184.

  • Growth and skeletal defects can be seen in those with deletion of miR-17 to 92 cluster.

 

b)      Cancer

  • Chronic lymphocytic leukemia was one of the first human diseases that has been associated with miRNA dysregulation. Other miRNAs that have also been linked to cancer are called “oncomirs”. miRNAs are involved in pathways that are crucial in B-cell development such as B-cell receptor signaling, cell-cell interaction, B-cell migration or adhesion, and production or class-switching of immunoglobulins. They also influence the generation of marginal zone, follicular, plasma, B1, and memory B cells.

  • Another clinical trial is using miRNA as a screening assay for the detection of colorectal cancer in the early stages.

  • miRNAs can also be used to determine prognosis in cancers based on their expression level. For example, a study on non-small-cell lung carcinoma determined that a low level of miR-324a levels is an indication of poor prognosis. In colorectal cancer, a low level of miR-133b and high level of miR-185 is linked with metastasis resulting in poor prognosis.

 

c)       Heart Disease

  • Studies have found that there are specific changes in the expression levels of miRNAs in diseased hearts which points to the involvement of miRNAs in cardiomyopathies.

  • miRNAs can also be used to determine the prognosis and risk stratification of cardiovascular diseases.

  • In animal models, miRNAs have been associated with the regulation and metabolism of cholesterol.

 

d)      Nervous System

  • miRNAs are thought to be involved in the function and development of the nervous system.

  • Neural miRNAs such as miR-124, miR-132 and miR-134 are involved in dendritogenesis.

  • Other neural miRNAs are involved in the formation of the synapses. miR-134 and miR-138 are thought to be included in the process of synapse maturation.

  • Studies have found that conditions such as bipolar disorder, schizophrenia, anxiety disorders, and major depression have altered miRNA expression.

 

e)      Obesity

  • miRNAs have a vital role in the differentiation of stem cell progenitors into adipocytes. Studies have found that the expression of miRNAs 155, 221, and 222 can inhibit adipogenesis paving a possible genetic treatment for obesity.

  • miRNAs of the let-7 family was found to accumulate in tissues as aging occurs. Based on animal models, the excessive expression of let-7 class miRNAs resulted in accelerated aging, insulin resistance, and therefore increases the risk of obesity and diabetes. When let-7 was inhibited, it resulted in an increase in insulin sensitivity and resistance to high-fat-diet-induced obesity. This means the inhibition of let-7 may prevent, reverse, and cure obesity and diabetes.

 

Conclusion

Although miRNAs have great promise in the screening, diagnosis, treatment, and prevention of various pathological conditions, more research, and clinical trials will be needed in the study of miRNAs to further explore and discover the potential of miRNAs.

 

References:

  1. microRNA. Wikipedia. Accessed 8/22/2018. https://en.wikipedia.org/wiki/MicroRNA#Disease

  2. Blondal T, Nielsen SJ, Baker A, et al. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods. 2013; 59(1): S1-S6.

Newborn Genetic Screening Program

What actually happens to newborn DNA samples once its been tested for genetic disorders?

In the last five decades, most babies born in the United States have unknowingly participated in a test called the Newborn Genetic Screening program. The test has been established to identify treatable genetic disorders in newborn infants. Early identification of these disorders is crucial in addressing symptoms and preventing a lifetime of disability. The test is a simple one: one small prick to the heel to collect a blood sample. With this sample doctors and nurses test for a variety of hereditary and congenital disorders. The controversy surrounding this program doesn't start until after the completion of the testing, whereby the samples are often stored in state-run biobanks.

Your or your child's DNA may have been stored and shared without your consent. Given that this has been going on since the 1960’s it is more likely than not that your samples are out there without your knowledge. Most people don't even know what the Newborn Genetic Screening test is or that they were a part of it. It’s importance and significance in identifying preventable disorders is not under question, but what happens to residual samples should be brought to light. State-run biobanks (or data repositories as the Association of Public Health Laboratories calls them) are established to store these samples and are shared with departments such as law enforcement for analysis and research. 

 

What is the Newborn Genetic Screening Test?

The Newborn Genetic Screening test began in the 1960’s. Back then it served to simply detect one genetic disorder, phenylketonuria. A condition that causes brain damage but, if caught early enough can be treated. Since then our knowledge of genetic disorders has improved immensely, largely due to the NGS Program. Collection of the blood sample must be completed within 12 to 48 hours after birth and can now detect between 30 and 50 genetic disorders. It is without a doubt an important and lifesaving program, and an estimated 12,500 newborns are diagnosed and saved annually. Participation in the NGS Program is a legal requirement. and therefore, parental consent is not required. However most states allow parents to “opt out” if there are religious or philosophical reasons. However hospitals do not usually inform the parents that the test will be conducted, making it challenging to opt out.

 

Duration and Location of DNA Storage

Your blood sample storage is different depending on state of birth. The most common practice is for it to be stored in state-run biobanks. Parental consent laws also differ for storage, in some states parental consent is necessary before storage of samples. In California for example, once tested the state retains the rights to store the samples. other states destroy the samples after six to twelve months whilst other store it much longer, ranging from 21 years to indefinitely.

 

How are These Samples Used?

Even though states might not use the samples, other researchers and government agencies still have access to them. It might be necessary for parents to find out what their or their children residual blood spots are used for. Residual blood spots storage can be used in the following.

a. Research purposes such as:

  • Retesting the samples to confirm the screening results

  • Developing new screening tests

  • Developing new techniques for forensic studies

  • Identification of new diseases

  • Quality control purposes

  • Access for those who are not biorepository lab technicians (such as those people in law enforcement)

b. Law enforcement purposes such as:

  • According to a Columbia Broadcasting System (CBS) report, they discovered that a minimum of four court orders and fie search warrants were obtained for identified blood spots. One of these cases involved a request to test the residual blood spot for drugs at birth. There are also cases where coroners use these samples to help in the identification of bodies or parents who request it to prove paternity.

Most famously the issue of storing these samples was brought to light during the trial of the Golden State Killer. The DNA from the crime scene was matched by law enforcement officials with DNA from a California state-run biobank. They used an open-source genetic database, called GEDmatch to identify the killer.

 

Controversies

As you can imagine the NGS Program presents several opportunities for abuse. These residual blood spots are easily accessed and many issues can be raised, including: 

a)     Consent

Parents of the children are not usually informed or asked for consent to the screening. Given the nature of the information collected during this test many people are concerned with the number of loopholes that exist. In the Genetic Information Nondiscrimination Act of 2008 that exists to prevent genetic discrimination from health insurance companies. Since the screening is paid for through health insurance companies. Many fear that a positive test could very well taint a child's record and that insurance companies could use it against people in the future.

b)      Ethics

There are ethical concerns surrounding residual bloodspots. Some are concerned that residual blood spot research is a way for the government to further control its citizens and have access to not only their records but also their genetic material.

c)       De-identification

While some believe that de-identification of DNA is possible by not storing the identifying information together with the blood samples, many argue that the DNA itself is an individual’s unique code and can always be used to identify individuals.

 

Conclusion

The laws for residual blood spots vary depending on the state one is born in. Those concerned should read up on the state’s procedures and policies. It is also important to note that policies and laws can change with time. This means individuals concerned with what happens to DNA samples should stay up-to-date with the new policies.

Types of Biorepositories

Introduction

Screen Shot 2018-08-20 at 10.54.20 AM.png

A biorepository is a center that functions to collect, process, store, and distribute tissue samples or specimens to support future research. These tissue samples can be sourced from humans or animals. The biorepository is responsible for maintaining the quality, accessibility, and distribution of these tissue samples.

 

Operations

  • Upon delivery the tissue samples are recorded all information regarding the sample details are keyed into the laboratory information management system.

  • Biospecimens are then processed to ensure consistency of the samples. Proper tissue preservation methodology is absolutely crucial to a biorepository.

  • These specimens are then stored and held in their appropriate conditions. Sample holding boxes and freezers are sometimes used, however it depends on the storage requirements. For example Formalin-fixed paraffin-embedded tissue blocks can be stored at room temperature.

  • Distribution involves retrieving the required samples from the inventory.

Standard Operating Procedures

Standard operating procedures (SOPs) are important for biorepositories as they help in the following:

  • Reduce problematic variables within the samples,

  • Ensure that biospecimens resemble specimens in their natural state,

  • Provide standards on how operations should be conducted.

Types and Uses of Biorepositories

There are many different types of biorepositories that exist. Some help with biomarker validation, and others are integrated with registries. Most biorepositories are focused on collecting biospecimens for specific diseases. Others function to identify genetic clues that can aid in the guidance of therapeutic development. Similair to disease-focused biorepositories there are those  focused on the understanding of practices and habits. Biorepository sponsors can also vary. While some are funded as part of research to aid in the collection of specimens from participants, some are sponsored by organizations or medical centers to collect, process, and store samples from a wide variety of patients. Some biorepositories are organized by patient advocacy organizations to help kick-start research of specific diseases.

Examples

  1. The Alzheimer’s Disease Neuroimaging Initiative – is a disease-focused biorepository and biomarker validation program that uses the samples and data collected from Alzheimer’s disease patients and patients with other forms of memory impairment.

  2. The Health Outreach Program for the Elderly (HOPE) is a biorepository at Boston University that supports multiple studies. The HOPE registry follows up with their Alzheimer’s patients annually.

  3. The United Kingdom Biobank is a biorepository with a broad focus. They aim to improve the diagnosis, treatment, and prevention of various diseases such as cancer, stroke, diabetes, heart disease, eye disorders, depression, dementia, and arthritis. In between 2006 to 2010, they managed to recruit half a million individuals between the ages of 40 to 69. Samples such as blood, saliva, and urine have been donated for analysis. These participants have also provided detailed personal information and consented to future follow up for many years to help researchers discover how various diseases develop.

  4. The Autism Research Resource is sponsored by the state of New Jersey to research autism in families where more than one child is affected.

  5. The Centers for Disease Control (CDC) Cell and DNA Repository use samples from transformed cell lines available through the Genetic Testing Reference Material Coordination Program. Some of the samples obtained are from diseases such as Cystic Fibrosis, Huntington Disease, Alpha Thalassemia, Fragile X syndrome, and Muenke syndrome.

  6. The National Institute of Neurological Disorders and Stroke (NINDS) Human Genetics DNA and Cell Line Repository focuses on the identification of new genes that causes or contributes to conditions such as Parkinson’s disease, Tourettes syndrome, epilepsy, motor neuron disease, and cerebrovascular disease.

  7. The National Institute of Aging (NIA) Aging Cell Repository utilizes cellular and molecular research to determine the degenerative mechanisms and causes of aging. They have strict diagnostic criteria with cells collected over a span of thirty years. Scientists are using these cultures to study diseases such as Alzheimer’s disease, Parkinson’s disease, Progeria, and Werner Syndrome.

  8. The National Human Genome Research Institute (NHGRI) Sample Repository for Human Genetic Research successfully completed the sequencing of the human genome. They now aim to participate in a variety of studies that focuses on the understanding of the structure and function of the genome and the role it plays in disease and health.

  9. The National Eye Institute Age-Related Eye Disease Study (NEI-AREDS) Genetic Repository was founded to identify how macular degeneration and cataracts develop and progress. This is important as these two conditions are two main causes of vision loss among older patients.

Conclusion

Biorepositories are crucial in supporting different areas of research such as those focused on a specific diseases, broadly focused population studies, identification of genetic mutations, and many more. These studies may have a specific length and purpose and are ongoing studies that follow up with their participants for many years.

 

References:

1)      Biorepository. Wikipedia. Accessed 8/9/2018. https://en.wikipedia.org/wiki/Biorepository

2)      Greenberg B, Christian J, Henry LM, et al. Biorepositories: Addendum to Registries for Evaluating Patient Outcomes: A User’s Guide, Third Edition [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US); 2018 Feb. Types and Uses of Biorepositories and Their Application to Registries. Accessed 8/9/2018. https://www.ncbi.nlm.nih.gov/books/NBK493635/