FFPE vs Tissue Microarray's

Introduction

Formalin-fixed paraffin-embedded (FFPE) samples are crucial for use in the tissue microarray (TMA) technique. FFPE samples are important for the study of morphology, cell biology, biochemistry, and disease in all living organisms. FFPE is a method that preserves tissue samples using formalin to paralyze cell metabolism. After fixation, paraffin helps to seal the tissue as it decreases the rates of oxidation. Various tissue samples are stored using the FFPE method as it is cost-effective due to low storage cost. FFPE biospecimens have played a vital role in drug discovery, biotech research, and retrospective gene studies over the last few decades. Samples that are preserved using this method enables it to be stored for decades making it an invaluable source in the discovery of new drugs, therapy, correlation of clinical outcome, and molecular findings. Since these specimens are embedded in paraffin wax, it can be sectioned easily to be mounted on a microscope for examination purposes. 

Uses, Advantages, and Disadvantages of FFPE

FFPE tissues are commonly used in immunohistochemistry where the tissue is mounted on a slide and bathed in a solution containing antibodies, so it binds to proteins and other structures. Staining helps visualize the antibodies to show where the proteins are on the specimen. This is vital in studies involving diseases such as Alzheimer’s disease and cancer. FFPE tissues are therefore crucial in immunology, oncology, and hematology. In the hospital, when a biopsy is obtained, a portion of the sample will be archived for second opinions. Donated samples or tissues from humans or animals can be preserved using the FFPE method and stored in archives for other studies. These archives are known as biobanks or biorepositories that may be found in universities, hospitals, or organizations that serve the research community. 

As previously mentioned, FFPE samples are cost-efficient due to its storage at room temperature. It is also a crucial source of research material as the specimen remains viable despite not requiring any specialized equipment. The disadvantages of this method would be the use of formalin for fixation, non-standardized procedures to prepare the specimen, and time-consuming process to fix and embed the tissue sample. This method can cause the proteins in the specimen to become denatured thus limiting the use of FFPE samples to only specific studies. A good example would be that FFPE samples cannot be used for molecular analysis as the results from it is not on par with results from frozen tissue samples. 

Tissue Microarray (TMA)

TMA is a technique that is invented to overcome issues where marker validation is costly, labor-intensive, and time-consuming. This method allows parallel molecular profiling of tissue samples at protein, DNA, and RNA level. It also allows the analysis of samples using RNA in situ hybridization, fluorescence in situ hybridization, and immunohistochemistry at a shorter time and lower costs. TMAs can be constructed using paraffin blocks and extracted cylindrical core biopsies from donor blocks. The core is then embedded into a recipient block or microarray with specific array coordinates. Obtained donor blocks are retrieved then sectioned to produce standard slides which are stained with hematoxylin and eosin. When ready, the slides are examined and arrayed. The core can be retrieved using a TMA instrument and inserted into an empty recipient block. The sampling process can be repeated numerous times using different cores until there are multiple cores in one recipient block. A microtome can be used to cut the sections to produce slides that are used in molecular and immunohistochemical analyses. 

Uses, Advantages, and Disadvantages of TMAs

TMAs are useful as it can be used to amplify a scarce resource. While a standard histological section which is about 3 to 5 millimeters thick yields about 100 assays, the TMA technique produced material enough for 500,000 assays. The TMA technique also allows simultaneous analysis of multiple specimens due to high throughput data acquisition. Since the TMA technique enables all tissue samples to be treated uniformly, there is less variability. With TMA, it enables the analysis of the entire cohort while standardizing variables such as reagent concentration, incubation times, temperature, washing procedure, and antigen retrieval. It is also a method that is both time and cost-efficient as it only requires small amounts of reagents for analysis. Another important advantage of the TMA technique would be the conservation of the original block of the tissue sample. 

One of the main disadvantages of the TMA technique that is commonly highlighted would be that the cores used in TMA may not represent the entire tumor. This is especially concerning for heterogeneous tumors such as prostate adenocarcinoma and Hodgkin lymphoma. However, this concern has been proven by many studies to be trivial. The studies show high concordance between TMA spots and whole sections of different tumors. Another minor critique regarding this technique is the absence of one or more core sections. This issue is easily addressed through the statistical power of analysis as a single data point out of hundreds to thousands of cases is easily eliminated. 

References:

1. The pros and cons of FFPE vs frozen tissue samples. Accessed 9/10/2019. https://www.geneticistinc.com/blog/the-pros-and-cons-of-ffpe-vs-frozen-tissue-samples

2. FFPE vs frozen tissue samples. Accessed 9/10/2019. https://www.geneticistinc.com/blog/ffpe-vs-frozen-tissue-samples

3. Tissue microarray. Accessed 9/10/2019. https://www.geneticistinc.com/blog/category/Tissue+Microarrays

4. FFPE and tissue microarray samples. Accessed 9/10/2019. https://www.geneticistinc.com/blog/ffpe-and-tissue-microarray-samples

5. What is FFPE and what are its uses. BioChain. Accessed 9/10/2019. https://www.biochain.com/general/what-is-ffpe-tissue/

What is Pathology?

Introduction

Pathology refers to the study of disease including the causes and effects of it. It also comprises of various bioscience research fields and clinical practices. When this term is used in modern medicine, it often has a narrower fashion used to refer to tests and processes in the contemporary medical field where there is disease diagnosis through the analysis of bodily fluid, tissues, and cells. The term “pathology” can also be used to refer to the progression of disease. An individual that practices pathology is known as a pathologist. Generally, pathology involves four components of disease:

• The cause of disease

• Pathogenesis – the mechanism of disease development

• Morphologic changes seen – the structural alterations that may be present during the disease

• Clinical manifestations – the consequences of the disease

Areas of study include inflammation, necrosis, injury, wound healing, neoplasia, etcetera. Pathologists study cellular patterns under a microscope to determine if a specimen is benign or malignant, employ genetic studies, and assess various diseases using gene markers. 

History of Pathology

The study of pathology dates back to antiquity. In most early societies, there is a rudimentary understanding of various conditions based on records from China, India, and the Middle East. In ancient Greece, the study of disease was ongoing with many notable early physicians such as Hippocrates. These practices were then continued by the Byzantines and Romans. Due to the many areas of scientific inquiry, the growth in this field stagnated after the Classical Eras and gradually developed throughout various cultures. The growth of this field languished until experimentation proliferated in the Renaissance, baroque, and Enlightenment eras due to the resurgence of empirical methods. By the 17th century, the examination of tissues through microscopy led British Royal Society member Robert Hooke to use the term “cell”. Modern pathology developed as a distinct field in the 19th century through physicians and philosophers who study diseases. Formally, it was not recognized as an area of specialty until the late 19th to early 20th centuries. In the 19th century, physicians start understanding disease-causing pathogens such as viruses, bacteria, prions, parasites, and fungi existed and has the capability to multiply. With new understanding, there was a comparison of symptoms leading to the realization of replication and varied effects each disease can have on the human host. In the late 1920s to early 1930s, pathology became a medical specialty. By the 20th century, pathology can be divided into rarefied fields.

Types of Pathology

Pathology can be divided into subdisciplines. 

1. Anatomical pathology – this medical specialty is concerned with disease diagnosis based on the gross, microscopic, immunologic, chemical, and molecular examination of tissues, organs, or whole bodies. It can again be divided into:

1. Surgical pathology – A significant and time-consuming specialty with a primary focus on examining tissues for a definitive diagnosis. Specimens are received as core biopsies, small skin biopsies, etcetera where gross and histologic tissue analysis are assessed by laboratory tests in immunohistochemistry. An autopsy is performed by a pathologist to determine the cause of death, state of health before death, and medical diagnosis and treatment before death.

2. Cytopathology – A branch of pathology studying diseases on the cellular level. It is mostly used in the diagnosis of cancer, but it can also aid in the diagnosis of infectious diseases and inflammatory conditions. It is usually used on free cells or fragments that exfoliate or can be removed through fine needle aspiration or abrasion.

3. Molecular pathology – This is a recent discipline that has made great progress in the last decade. It involves the diagnosis and study of disease via molecular examination of bodily fluids, tissues, and organs. Various diseases such as cancer can be due to alterations in the genetic code, mutations, and identification of specific hallmark mutations which allow professionals to diagnose and choose specific treatment. Molecular pathology is a discipline that paves the way for personalized medicine through the prediction of patient response to treatment based on genetic make-up. It also involves the development of genetic and molecular approaches to classification, diagnosis, design, and validation of predictive biomarkers for disease prognosis and susceptibility of cancer development. The high levels of sensitivity allow the detection of small tumors that are otherwise undetectable. This leads to improved care, earlier diagnosis, and better prognosis.

Training and Accreditation

The accreditation needed to be a pathologist varies based on country. In the United States, pathologists are required to complete a four-year undergraduate program, four years of medical school, and another three to four years of postgraduate training. Per the American Board of Pathology, training can be within two specialties: anatomical pathology and clinical pathology. The American Osteopathic Board of Pathology recognizes four pathologies: dermatopathology, forensic pathology, anatomic pathology, and laboratory medicine. In the United Kingdom, the training to be a pathologist is licensed by the UK General Medical Council under the oversight of the Royal College of Pathologists. After undergraduate medical study lasting four to six years, trainees go on to a two-year program. 

References:

1. Pathology. Wikipedia. Accessed 8/28/2019. https://en.wikipedia.org/wiki/Pathology

2. What is pathology. McGill. Accessed 8/28/2019. https://www.mcgill.ca/pathology/about/definition

Issues in Biobanking

Introduction

Throughout the years, there have been major changes in the research industry due to alterations to policies and regulations that affect biobanking. Additionally, there has been various evolving ethical issues and significant shifts in ethics. In the last 20 years, the International Society for Biological and Environmental Repositories (ISBER) made significant strides by publishing guidelines such as “Best Practices for Repositories I: Collection, Storage, and Retrieval of Human Biological Materials for Research” in 2005. Subsequently, the second and third editions were published in 2008 and 2012. These guidelines cover ethical and technical best practices for the collection, storage, distribution, and use of biospecimens in research. The United States National Cancer Institute (NCI) also released the NCI Best Practices for Biospecimen Resources (NCI Best Practices) with subsequent versions in 2011 and 2016. This document covers the ethical, operational, technical, legal, and policy best practices which are limited to human specimen resource collections. Other guidelines such as the Organization for Economic Cooperation and Development (OECD) Guidelines for Biological Resource Centers and OECD Guidelines for Human Biobanks and Human Genetic Database were also published. 

Ethical Guidelines

To address ongoing and new ethical issues that are related to human biospecimens in research, the Council of Europe adopted the Recommendation of the Committee of Ministers to Member States on Research on Biological Material of Human Origin in 2006, updated in 2016. The World Medical Association (WMA) also issued the WMA Declaration of Taipei on Ethical Considerations in 2016 regarding biobanks and health databases. 

Regulations

New privacy rules have also been established due to concerns about privacy in health records. In the United States, the Health Insurance Portability and Accountability Act (HIPAA) Privacy Rule was founded to help provide protect information from health providers, healthcare clearinghouses, and health plans. Although the rule may not apply to biospecimens, it applies to the disclosure and use of health information that is associated with the specimen. In 2016, the European Union General Data Protection Regulation was adopted to protect personal data. Throughout the years, more regulations have been added or modified to suit the changing times. 

Evolving Ethical Issues

Over the last two decades, new ethical issues and been highlighted. This includes important existing issues related to the biospecimens and their associated data. The increasing use of genetic technologies and their data led to increasing concerns regarding the identifiability of data, biospecimens, and how to protect the research. Ethical issues have led to the establishment of the Human Tissue Act of 2004. In 2010, a book was published highlighting the issue of Henrietta Lacks where biospecimens were obtained from her without her knowledge and used commercially. This has brought attention to issues such as informed consent and the use of biospecimens commercially. Other cases were focused on the ownership of biospecimens and claims of private ownership of the biospecimens in research. There were also several other court cases that have resulted in the destruction of newborn bloodspots that have been retained due to unobtained parental consent. Other issues to consider include cultural perspectives, transparency, accountability in research, and changes in policies and regulations. These changes suggest that the broad consent models can be acceptable under specific circumstances due to the recognition of the importance of the biospecimens. Recently, some have proposed the use of dynamic consent models where participants can decide if they want to provide a broad or case-by-case basis consent. These models are promising as it allows research participants more choices and a medium for ongoing communication along with possible access to results from the research. Limitations of dynamic consent models include decreased access for populations who are unable to access the necessary equipment or for those who choose not to adopt it due to higher costs required to implement. Another popular issue would be the return of individual research results to biobank participants as challenges include feasibility, costs, and participant responses when returning these data. 

The Future

As the industry progresses, increasing debate and dialogue regarding existing and new ethical issues become more important. A good example would be the necessity to rethink concepts of identifiability as more advanced technologies are developed and continued proliferation of databases that contain unique data to the participant. It is also important to clarify the rights participants may have regarding their specimens. This issue has prompted donors to call for some benefit such as financial compensation, control over how their specimens are used, and return of individual research results. Increasingly important will be the governance systems used in biobanks to ensure that the biospecimens and associated data are used appropriately. The ISBER will also need to continue ensuring that policies and regulations are followed and providing input to modify policies and regulations to help protect research participants while facilitating research. Last but not least, public education and participant engagement are also key to ensure the future of the industry. 

References:

1. Bledsoe MJ. The final common rule: implications for biobanks. Biopreservation and Biobanking. 2017; 15(4): 283-284.

2. Bledsoe MJ. Ethical legal and social issues of biobanking: past, present, and future. Biopreservation and Biobanking. 2017; 15(2): 142-147.

The Importance of a Biorepository

Introduction

A biorepository can be defined as a center that collects, processes, stores, and distributes biospecimens to help support clinical research and scientific investigations. A biorepository can contain biospecimens from various living organisms including animals, humans, arthropods, plants, and more. The general purpose of a biorepository is to maintain biospecimens and its associated information for possible use in the future. The biorepository is in charge of the quality and manages the distribution of the samples available in their collection. It is a physical structure where biospecimens are stored along with all the relevant policies and processes. 

Operations

As previously mentioned, the four main operations in a biorepository are:

• Collection – This phase is where samples are collected and recorded in the laboratory information management system. All samples are given a unique barcode or identification number and the data associated with the sample is also recorded in the system.

• Processing – Processing of samples include a standardized quality testing process to minimize variation such as sample handling. This phase also prepares the samples for storage.

• Storage – In this phase, the samples are held in the inventory before distributed as requested. The inventory system contains various storage requirements due to the different types of biospecimens available.

• Distribution – This stage includes retrieving samples that are requested from the biorepository inventory system

All the above operations ensure that the biospecimen is of the highest quality as it is collected, processed, stored, and distributed per regulations and standard operating procedures (SOPs). SOPs have a crucial role as it helps to reduce the incidences of issues, provide standardized guidelines for biospecimen storage, ensure biospecimens are high-quality and provide a framework of how to conduct seamless and reliable operations in a biorepository. 

Addressing Biorepository Status and Gaps

Biorepositories are crucial to the research field as it represents a credible microbial forensics infrastructure that spans testing, research, evaluation, development, verification, independent validation, analysis, and investigation. Currently, various existing biorepositories are highly heterogenous and populated with private collections, government collections, individual laboratory collections, user-group collections, organism-specific collections, geographical collections, commercial collections, and large-scale public collections. However, many of these biorepositories are not accessible to the public as companies and organizations can limit access to their collections. The processes and procedures for quality control, culture expansion, maintenance, preservation, quality assurance, biosecurity, staff training, and bioinformatics data can be variable and problematic. The access of meta-data such as the geographic location of isolation, taxonomic identification methods, phenotypic properties, and bibliographic references may be unavailable or incomplete. These data gaps and procedural variations can compromise the utility of samples in various programs. For these reasons, it would be best to adhere to standards and protocols that are uniform and implemented by the biorepository community. 

 Issues that a Biorepository Faces

Since biorepositories play a vital role in the advancement of research, they are also subjected to scrutiny and face some issues. For example:

• Expectations – A donor who donates a specimen such as tissue, blood, or urine expects the specimen to be valued, handled appropriately, and distributed accordingly to contribute to scientific knowledge. Furthermore, they also expect their privacy to be respected and used only in ways for which consent has been granted.

• Custodianship – Since specimens are irreplaceable because an individual is never the same as the point of time the specimen is obtained, the more a specimen is used, the more valuable it has more potential to examine the different causal pathways without having to test and collect parameters that are already determined. A thoughtfully collected collection that contains the sociodemographic information can lead to data and inventions that have commercial value. This means that investigators can become reluctant to share their specimens. In responsible custodianship, proper handling and storage is a necessity. This entails following the SOPs, having trained personnel, ensuring quality control, etcetera.

• Informed Consent – Since various biorepositories are specifically established for designated studies and subsequently maintained, the informed consent that was obtained then may differ and pose ethical issues when the samples are used again after that. If the tissues are linked to personal information, it is subjected to privacy regulations which have additional requirements. The Health Insurance Portability and Accountability Act (HIPAA) required that permission is obtained for each use. Although investigators can ask donors for permission to recontact them for permission to use the samples in future studies, there are also issues where some participants are lost to follow-up or dislike being contacted while data made anonymous destroys the linkage of the identifying information.

• Privacy – While specimens become more valuable with associated information such as medical history, clinical history, and sociodemographic information of the donor, this makes it more difficult to protect the privacy of the individual and may lead to future issues such as jeopardizing the ability of the individual to obtain health insurance. Clear policies should be in place to protect the privacy and confidentiality of both donors and data.

Conclusion

While many may view a biorepository as simply a physical structure that holds various biospecimens, there are many responsibilities and issues involved in operating a biorepository. Biorepository staff has to be adequately trained to ensure seamless operations and handle various ethical issues such as privacy and confidentiality. Ultimately, a biorepository plays a crucial role in guaranteeing the future of clinical research. 

References:

1. Biorepository. Wikipedia. Accessed 8/14/2019. https://en.wikipedia.org/wiki/Biorepository

2. Foxman B. Human and animal subject protection, biorepositories, biosafety considerations, and professional ethics. Molecular Tools and Infectious Disease Epidemiology. 2012.

3. Leighton T, Murch R. Biorepositories and their foundations – Microbial forensic considerations. Microbial Forensics (Second Edition). 2011.

FFPE vs Frozen Tissue Samples

Introduction

Both formalin-fixed paraffin-embedded (FFPE) tissue and frozen tissue are cornerstones in the research field. Medical advancements and research rely on the availability of biospecimens as research material. For example, studying a specific disease, how it progresses, and how it can be cured would require access to high-quality biospecimens that contain the diseased tissue. Since biospecimens are fragile and perish easily, professional care is necessary to prepare and preserve the tissue to ensure that it can be studied by researchers. The two major tissue preparation methods used for preservation are FFPE and frozen tissue. 

FFPE

FFPE tissue specimens are invaluable to the research industry and have had therapeutic applications for decades. It is a method of preservation that enables biospecimens to be used in experimental research, drug development, and diagnostics. In this method, the tissue is first saturated with formalin or formaldehyde to preserve the vital structures in the tissue. It is then embedded in paraffin wax to make sectioning easier so it can be mounted on a microscope for examination purposes. FFPE specimens can include both normal and diseased tissue as comparison studies are also important. In some cases, both normal and diseased tissue can also be obtained from the same individual. In the field of oncology, a primary tumor sample is often compared to tissues from distant metastatic tumors. 

FFPE Method

Once the tissue is identified and excised, it is immersed in a solution of 10 percent neutral-buffered formalin for 18 to 24 hours to harden the tissue so it can survive the following steps. Once ready, the tissue is dehydrated using increasing concentrates of ethanol. It is then embedded into immunohistochemistry grade paraffin. Fixation time is crucial in FFPE as tissues that are fixed too soon after excision can be useless for molecular studies. Quality control is important as biospecimens that are not maintained properly may exhibit unusual characteristics that result in misleading data and conclusions in research. The preparation method for FFPE may also depend on requests from the researcher as some may have specific instructions regarding the size, purpose, and cut of the tissue. Biospecimens that are completed are stored in biobanks established in research centers or hospitals. Records regarding the time of collection, preservation, demographic information, and legal documents should be kept as it may have a definitive impact on its usability in clinical trials or research. 

Frozen Tissue

Frozen tissue are biospecimens that are preserved using an ultra-low temperature freezer and liquid nitrogen. Compared to FFPE, frozen tissue is generally more important in areas where FFPE samples are not as reliable. One good example is molecular analysis. Tissue preserved using this method is dipped in liquid nitrogen (flash freezing) and stored in a freezer at less than -80 degrees Celsius. Frozen tissue is also important in pathology as it can be used to determine if the margins of a tissue section are clear during tumor removal in surgeries. Frozen tissue is also preferable compared to FFPE in mass spectrometry, next-generation sequencing, and western blotting. Compared to the FFPE method, it is much faster and preserves proteins in their native state. However, this method is disadvantageous as there is rapid deterioration of the samples once it is in room temperature. Tissues need to be frozen as fast as possible and may pose some difficulty as it requires the proper equipment. Once the frozen tissue is ready, storage of the biospecimens is also much more expensive as it requires specialized equipment. Any power outages or mechanical failures may cause the biospecimens to go bad. 

FFPE vs Frozen Tissue

Both FFPE tissue samples and frozen tissue have their pros and cons. Besides the preparation and storage method that was previously mentioned, both samples have different applications. These include:

• Molecular analysis – Frozen tissue is much more suitable for molecular analysis due to the non-standardized preparation methods for FFPE samples. The use of formalin in FFPE preparation may also result in degraded DNA and non-native configurations of phosphorylated proteins. Frozen tissue is also considered the gold standard for mass spectrometry, Western blotting, quantitative real-time polymerase chain reaction, and next-generation sequencing.

• Immunostaining and Morphology – In this instance, FFPE specimens are preferable due to the mediocre histomorphological quality when frozen specimens are used. When samples are frozen incorrectly, it can also lead to the formation of vacuoles. FFPE samples are preferable when both tissue structure analyses and immunostaining are simultaneously required.

• Native Morphology – For studies involving native morphology, frozen specimens are preferable as it has the closest physiological native morphology. This allows IHC to be performed on the native form of protein, epitope, and antigen since the components in frozen tissue are not crosslinked as seen in FFPE specimens due to formalin fixation. Frozen tissue specimens also allow the results from IHC to be repeatable and more reliable compared to using FFPE samples. However, native morphology studies should be done as soon as possible as the specimen quality is highly dependent on ischemia time.

References:

1. What is FFPE tissue and what are its uses. BioChain. Accessed 8/11/2019. https://www.biochain.com/general/what-is-ffpe-tissue/

2. FFPE vs frozen tissue samples. BioChain. Accessed 8/11/2019. https://www.biochain.com/general/ffpe-vs-frozen-tissue-samples/

3. FFPE vs frozen tissue samples. Geneticist. Accessed 8/11/2019. https://www.geneticistinc.com/blog/ffpe-vs-frozen-tissue-samples

4. The pros and cons of FFPE vs frozen tissue samples. Geneticist. Accessed 8/11/2019. https://www.geneticistinc.com/blog/the-pros-and-cons-of-ffpe-vs-frozen-tissue-samples

Quality Assessment of Tissue Samples

Biobanks and biorepositories play a crucial role in the development and advancement of the research and medical field. For example, in 2008, the Asklepios Clinic that specializes in thoracic diseases founded a biobank. 11 years later, the biobank contained both solid and liquid biospecimens from about 4,000 patients. Some of the biospecimens include peritumor tissue from various bronchial carcinomas, solid tumors, metastasized tissue, tissue from benign malignancies, cell pellets, pleural effusions, serum, bronchoalveolar lavage fluids, plasma, and more. This biobank is also integrated into the German Center for Lung Research where biobanks collect samples per the harmonized Standard Operation Procedures. This ensures the quality of the biospecimen. Informed consent is obtained from the patients. Despite the various factors and challenges, biobanks and biorepositories aim to produce high-quality specimens that are suitable for various scientific researches. Solid samples are often snap frozen using liquid nitrogen and stored at -80°C as this process guarantees the conservation of biological processes. 

Biobanks are an important part of research infrastructures where it provides samples for various scientific purposes. The random samples are compiled into cohorts that can be categorized into specific therapies or diseases. The characteristics of the samples must be preserved throughout the collection, processing, and storage phases. This ensures that there is as little alteration as possible. Stabilization of the expression pattern may also be necessary if the samples are to be used for the analysis of mRNA expression. In various post mortem or ex vivo conditions, some authors were also able to demonstrate overall DNA and RNA stability along with the preservation of the global expression profile. However, a fraction of mRNAs has been observed to have changed in the expression patterns. These changes are even more pronounced if the specimen is exposed to room temperature instead of being kept on ice. This is believed to be due to the degradation process and transcriptional changes such as lack of oxygen. 

To ensure the preservation of the in vivo expression profile, samples can be collected in a standardized way and ensuring that the duration between harvesting and freezing is kept short. Some methods to minimize inter-sample variability include standardizing the samples once it has arrived in the lab, reducing duration between sample acquisition to freezing, and using protective reagents. One of the reagents known as RNAlater is a high salt ammonium sulfate aqueous solution that is specifically used to stabilize RNA. Several studies showed the preservation of expression profiles using RNAlater versus shock frozen tissue. This is also seen when the biospecimens are analyzed using RNA expression microarray analysis and real-time polymerase chain reaction (PCR). In RNAlater stabilized tissue, DNA for PCR analysis can also be extracted. However, the possibility of analysis of protein from these samples remains uncertain. It has been proven that the quantitative proteomic analysis between samples that are preserved using RNAlater and snap-frozen yield comparable results. However, more experiments are necessary before recommending the widespread use of RNAlater for proteomic and protein research. 

Another reagent is known as ProtectAll also helps to stabilize DNA, RNA, and proteins. Compared to RNAlater, it has a major disadvantage as tissue preserved using ProtectAll cannot be cut on a microtome and stained for analysis. This is due to the reason where event at -80°C, ProtectAll remains gelatinous and not frozen making it impossible for samples to be sectioned. The use of RNAlater has several major advantages:

·     Direct placement of samples into the preserving agent at the site of extraction

·     Omission of liquid nitrogen handling as it can be dangerous and expensive

·     Stabilization of nucleic acids especially RNAs.

However, it is important to note that the preservation of tissue samples using RNAlater is not as abrupt compared to shock freezing in liquid nitrogen as it needs to diffuse into the samples. This means that the samples have to be incubated in RNAlater for a minimum duration of 24 hours to ensure that there is sufficient absorption of the reagent before it is frozen. The reagent will inhibit the enzymatic activities present in the tissue samples during this time.

RNA degradation that occurs after the tissue has been withdrawn from the body may occur due to the presence of RNAses that degrade RNA and the altered environment such as decreased oxygen supply. In both cases, changes in gene expression are expected. It is important to not allow the samples to thaw during the shipping and distribution process. This is another reason why RNAlater is greatly advantageous as the RNA in a sample that is snap frozen degrades immediately after thawing while it is maintained in samples preserved using RNAlater. 

Ultimately, RNAlater is a reagent that has many benefits in clinical biobanking. When compared to snap freezing using liquid nitrogen, the samples preserved using RNAlater is of the same quality in terms of mRNA expression and overall RNA quality. 

References:

Lindner M, Morresi-Hauf A, Stowasser A, Hapfelmeier A, Hatz RA, Koch I. Quality assessment of tissue samples stored in a specialized human lung biobank. PLoS One. 2019; 14(4): e0203977.

Tissue Procurement (The Collection Process)

Introduction

The advancement of modern research in the medical field is very much dependent on access to high-quality human tissue specimens. There are now various organizations that have the responsibility to collect, process, store, and distribute human tissues to help researchers involved in biomedical research. While there may be guidelines for best practices in tissue procurement, these guidelines are usually general and may not include all the details required by individuals or investigators who are new to using human tissues. 

Tissue Procurement Model

There are various approaches to procure human tissues and fluids for research. Examples include:

•    “Catch as catch can” model – This is one of the most disorganized methods often used. This method involves the cooperation of professionals such as pathologists and surgeons to provide tissue samples whenever they are available to researchers. These specimens are usually only collected when the request is remembered and when time permits. This also means that there is very little to no quality control as there is no standard protocol regarding the collection, processing, and storage of the specimen. Therefore, the quality of these specimens can be poor and may provide inaccurate results or data. Another issue regarding tissues that are obtained via this method is the oversight of Institutional Review Boards (IRBs) and may violate the regulations or requirements of the IRB or Health Insurance Portability and Accountability Act (HIPAA). 

•    Banking model - Another more organized approach would be to obtain specimens that are research-grade quality from organizations such as biorepositories or biobanks. These organizations usually have a standard operating procedure (SOP) when collecting, processing, and storing specimens. The collected tissues are usually frozen or stored in paraffin blocks. Fresh samples are generally not available. Therefore, the main disadvantage of obtaining specimens from organizations with the “banking model” would be not meeting custom requirements such as tissue size, percentage of tumor in the specimen, or requests for normal or fresh tissues. The advantages of using this approach would be the ability to obtain large numbers of specimens that have corresponding demographic and clinical information. 

•    “Clinical trial model” – This model is a subtype of the banking model. This method involves using the remnants of specimens that were collected from clinical trials. This main issue in this model would be that the original informed consent from the clinical trial may not allow the specimen to be used in other studies. Therefore, the IRB involved may limit the use of these specimens for different researches. The specimens from the clinical study may also be limited in both number and size. The method the tissues were processed for the clinical trial may not be suitable for the investigators. 

•    Prospective collection model – This method requires the investigators to specify the specimen they need and how they want it to be collected, processed, or stored. The main disadvantage of this model would be the unavailability of large numbers of specimens with its main advantage being the investigator acquiring what is requested. The investigator should realize that it may take a while for the specimens requested to become available.

•    Tissue repository model – This is a combination of the banking and prospective model. This model includes the advantages of both models. The term “repository” is defined as “an entity that collects, processes, stores, and distributes specimens”. However, one of the main issues that a repository may face is the complex administrative needs and the increased requirements for an advanced bioinformatics system. 

With more development of tissue repositories, the optimization of such operations is being monitored by the International Society of Biological and Environmental Repositories (ISBER) to help organizations that are operating biorepositories. Since there is little published information regarding operational support of biorepositories, ISBER published best practices guidelines to help maintain the expectations of the professionals in this industry. Best practices for tissue practices focusing on neoplastic diseases have also been published by the National Cancer Institute (NCI). These documents are crucial as they provide details for specific operations. 

Quality Assurance

Quality assurance is crucial in all biomedical research as it helps improve operations, procedures, and products. Quality control refers to the technical activities measuring the performance and attributes of the process or product against current standards. Individuals responsible for quality assurance help in the development of new guidelines and to ensure that SOPs are followed. They also lead the efforts to identify and correct problems related to the collection, processing, storage, and distribution process. The minimum quality control for a biorepository should involve the examination of the tissue by a certified pathologist. It can also include a molecular analysis where DNA, mRNA, and protein are extracted. In more specific projects, a more extensive quality control examination can be requested. However, depending on the biorepository, there may be an additional price charged to their clients as extensive quality control involves increased time, effort, and cost. 

References:

Grizzle WE, Bell WC, Sexton KC. Issues in collecting, processing, and storing human tissues and associated information to support biomedical research. Cancer Biomark. 2010; 9(1-6): 531-549. 

Red vs Yellow Bone Marrow in Biology

Introduction

Bone marrow refers to the semi-solid tissue found in cancellous or spongy parts of the bones. In humans, the bone marrow is the main site where hematopoiesis or production of blood cells occurs. The bone marrow consists of marrow adipose tissue, hematopoietic cells, and supportive stromal cells. In adults, the bone marrow can mainly be found in the pelvic bones, sternum, vertebrae, and ribs. On average, bone marrow comprises about 4 percent of the body mass. This means in an average adult who weighs 65 kilograms (143lbs), the bone marrow is about 2.6 kilograms (5.7lbs). The bone marrow is estimated to produce about 500 billion blood cells daily. These blood cells then join the circulation through permeable vasculature sinusoids that can be found in the medullary cavity. The hematopoietic stem cells in the bone marrow produce all three classes of blood cells: erythrocytes (red blood cells), leukocytes (white blood cells), and thrombocytes (platelets). 

Structure

In humans, the bone marrow can be divided into the red and yellow bone marrow. This depends on the prevalence of hematopoietic cells and fat cells. A newborn baby usually exclusively has red bone marrow. With age, there is a progressive conversion towards yellow marrow. In adults, red bone marrow can be found in the pelvic bones, cranium, sternum, ribs, scapulae, vertebrae, and proximal epiphyseal ends of long bones. In dire situations where there is chronic hypoxia, yellow marrow can be converted to red marrow to help increase the production of blood cells. The tissue in the bone marrow that is not directly involved in hematopoiesis is also known as the stroma. The stromal cells function to provide an environment that helps the functioning and differentiation of hematopoietic cells. Some of the cell types found in the bone marrow stroma include:

•    Macrophages 

•    Adipocytes

•    Fibroblasts

•    Osteoblasts

•    Endothelial cells

•    Osteoclasts

The mesenchymal stem cells in the stroma are multipotent stem cells that can differentiate into chondrocytes, osteoblasts, marrow adipocytes, myocytes, and beta-pancreatic islet cells. 

Red vs Yellow Bone Marrow

Red bone marrow refers to the red colored tissue where there are reticular networks that are critical in the production and development of blood cells. Red bone marrow is situated in the shoulder blades, long bones, and skull. With age, it can be predominantly found in flat and long bones such as the hip bones, skull, ribs, and vertebrae. Yellow bone marrow can be found in the hollow regions of compact bones of the axial skeleton. It is called the yellow bone marrow as it refers to the yellow-colored tissue that functions to produce blood cells and store fats during dire circumstances. 

Generally, as the number of red bone marrow increases, the yellow marrow decreases. This also means that when the number of red bone marrow decreases, the yellow marrow increases. During fetal development until the point of childbirth, there is only red bone marrow found in the bone cavities. Once the individual reaches approximately 5 years old, the red bone marrow present in the long bones of the body is gradually replaced with yellow bone marrow. While red bone marrow is rich with hematopoietic cells that function to produce red blood cells, white blood cells, and platelets, the yellow bone marrow instead stores the adipocytes that function as fat storage. In emergency situations where there is blood loss, the yellow bone marrow can also be converted into the red bone marrow to help with the production of blood cells. The fats stored in the yellow marrow can also be used as the body’s last reserve for energy when there is extreme hunger. It can also convert into cartilage and bone as needed. 

The red bone marrow can attribute its red color to the presence of hemoglobin while the yellow bone marrow has a distinct yellowish color due to the presence of carotenoid in the tissue’s fat droplets. The red bone marrow contains active cells that constantly multiplies to continuously produce blood cells while the yellow bone marrow contains inactive cells. 

Clinical Significance

The architecture in the bone marrow can be distorted by various conditions such as multiple myeloma, aplastic anemia, tuberculosis, leukemia, etcetera. Besides diseases, therapy such as radiation and chemotherapy can also affect cell division in the marrow resulting in a suppressed immune system. To aid the diagnosis of diseases that may involve the bone marrow, an aspiration of the bone marrow can be performed by using a hollow needle to obtain a sample under local or general anesthesia. 

Stem cells derived from the bone marrow also has a wide application in regenerative medicine. Hematopoietic stem cells can be removed from one individual and infused into another if both donor and recipient are compatible. The stem cells can be harvested from the red marrow under general anesthesia. It is considered to be a minimally invasive procedure with minimal scarring. Another harvesting option would be the administration of certain medications that stimulate stem cell release into the circulating blood. An intravenous catheter is then inserted into the arm and stem cells are filtered out of the blood. It is a similar procedure to platelet or blood donation. 

An Inside Look at The Duty of a Geneticist

Introduction

A geneticist is a biologist involved in the study of heredity and genes. This field of study is known as genetics. This includes aging, disease, and gene mutation. There is also environmental genetics where the geneticist examines how various environmental factors affect the genes to cause disease or helps with adaptation to the environment. Genetics include understanding how genes are activated, mutated, inherited, and inactivated. A geneticist can be employed as a lecturer or scientist. They can perform research on genetic processes or to develop new genetic technology that can benefit industries such as agriculture or medicine. There are also some geneticists who experiment in model organisms such as zebrafish, rodents, or humans to analyze the data and interpret the hereditary patterns for various biological traits. 

Function

When studying the inheritance of biological traits, geneticists can focus on the population, molecular, or organism level. There are some geneticists who specialize in genetic disorders. Environmental geneticists, however, deal with epigenetics. Epigenetics refers to the process where parts of the genome can be switched “on” or “off” depending on the external environmental factors. Although there are many traits that have been set in stone, there are also some that are more flexible. For example, although there are many individuals with a predisposition to a certain disease, not all of them will develop the disease. However, the development of the disease can be affected by diet, lifestyle factors, stress, and more. Environmental geneticists play a critical role in analyzing how these interactions work.

In ecological genetics, geneticists in this field try to understand how genetics help in adapting to changing environments. These geneticists use population genetics to help improve management, conservation, and genetic betterment of the species. A good example would be estimating the survival and reproduction rates of a community or species. Using their knowledge of genetics, they help identify species that are at risk and increase genetic diversity. There is also some research on genetically engineering plants that can cope with demands such as increasing population or food crises, and adaptation to disease and climate. 

Despite having various specialties, most geneticists perform many similar tasks such as conducting research on gene expression. Notes such as laboratory records on procedures, methodology, and results should also be kept. Lab results can be reviewed and analyzed using mathematical and statistical methods. Geneticists can keep up with new methods and scientific literature by learning about results, tools, and new procedures that are being developed. This new information and knowledge can then be utilized to help improve on their own research. To fund research projects, geneticists also attend fundraising events and write grants. Research results are often shared through professional conferences and publication in academic journals. 

Workplace and Salary

Geneticists usually find employment in government agencies, hospitals, and university laboratories. These jobs can be found nationwide. There are also employment opportunities in private sectors. Depending on the location and type of employment, the salary of a geneticist can range between an estimated $35,000 to $125,000. Based on the National Human Genome Research Institute, it has been reported that the average income for environmental geneticist is about $58,000 annually. 

Geneticists can also opt to teach in academic positions or go into theoretical or applied research based on their area of specialization. They can evaluate, test, and diagnose individuals who have genetic mutations, genetic risks, or hereditary conditions. They can also serve as a resource to help patients who have genetic complications by working together with other medical professionals. Although various jobs may have different tasks, some of the following responsibilities still fall under their scope of practice:

•    Assessing and consulting patients who have potential mutations or genetic risks

•    Testing for hereditary or genetic markers for various mutations and risks

•    Reviewing research and literature to stay up to date in their field

•    Counseling patients who have risks or predispositions to genetic mutations

•    Counseling patients who have abnormal test results and screenings

•    Help to determine the best course of action 

•    Consult with other professionals such as community partners, physicians, and advocates to help in increasing awareness and educating the public

•    Assisting peers and colleagues with research endeavors

•    Assisting with maintenance and support to meet the health and safety requirements

Senior geneticists tend to have more responsibilities such as managing a lab or team. It may also include meeting with policy-makers, advising other researchers or agencies, creation of scientific reports, engaging in the design of studies or analysis, organization of public outreach programs, overseeing team budgets, mentoring, and more.

Demand

It has been predicted that the demand for geneticists will have little to no change. This means that the competition for basic or entry-level research positions will be strong. Growth may occur if there are advances in hyper or data computing that allows the analysis of large ecological or genetical datasets. There may also be more opportunities for environmental geneticists if there is increased interest or expanded focus in the environment or medical aspects of genetics. 

References:

1)    Geneticist. Wikipedia. Accessed 7/2/2019. https://en.wikipedia.org/wiki/Geneticist

2)    What is a geneticist? Environmental Science. Accessed 7/2/2019. https://www.environmentalscience.org/career/geneticist

3)    Geneticists. Genes in Life. Accessed 7/2/2019. http://www.genesinlife.org/testing-services/working-healthcare-professionals/geneticists

Why Use Tissue Microarray's in Pathology?

Introduction

Tissue microarrays (TMAs) are paraffin blocks where there can be up to 1000 individual tissue cores that are compiled in array fashion and allowing multiplex histological analysis. The TMA technique was developed to address the main limitations in molecular clinical analysis of tissues. The limitations include:

•    Complexity of procedures

•    Limited availability of diagnostic reagents

•    Limited patient size

In 1986, H. Battifora first introduced multi-tissue blocks which were known as “multi-tumor (sausage) tissue block”. This was subsequently modified in 1990 to the “checkerboard tissue block”. J. Kononen and his team then developed the TMA technique in 1998 with a novel sampling approach to produce standardized sizes and shapes allowing it to be precisely arrayed. TMAs contain preserved biomolecules such as carbohydrates, proteins, DNA, RNA, or lipids. this procedure offers a high throughput platform for rapid analysis of molecular markers that are associated with the prognosis or diagnosis of physiological conditions and illnesses. The method is an effective tool with advantages such as:

•    Reduction in the workload 

•    Reduction in assay volume

•    Reduction in the number of slides

•    Experimental uniformity

•    Compatibility with automated staining systems

•    Shelf stability up to 12 months when stored at 4°Celsius

Procedure

The TMA technique involves using a hollow needle to remove the tissue cores from regions of interest. These tissue cores can be obtained from paraffin-embedded tissues such as tumor samples and clinical biopsies. These tissue cores are inserted in a recipient paraffin block in an array pattern. The block is then sectioned and cut using a microtome, mounted on a microscope, and analyzed using standard histological analysis. Each block can produce about 100 to 500 sections which can be used for various tests. The tests that commonly use TMA sections are fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). TMAs are also especially useful in the analysis of cancer samples. 

Why TMA?

In biology, one of the classic issues would be the validation of a new concept using human tissue. However, it leads to the problem of not having enough tissue in case number or amount to help complete the analysis. The tissue shortage can be attributed to the fact where the initial sampling is performed by a pathologist whose main goal is for diagnostic purposes and not for research. Working with human tissue is also complicated by ethical issues and guidelines where informed consent is required before use. Therefore, once the tissue is obtained, it should be managed properly to maximize its value. TMA is a tool that can help resolve this issue. 

For example:

•    While conventional slides produce one case per slide, TMA can produce 50 to 500 cases per slide. This maximizes the use of a scarce resource. In conventional slides, 150 sections of 1 case only yields 150 assays while in TMA, 150 sections with more than 500 cases can yield up to about 75,000 assays.

•    Conventional slides usually require multiple batches in a single study. Using TMA, the entire cohort can be ready in one experiment.

•    Conventional slides need large amounts of reagent while TMA only takes less than 1 ml of total reagent volume for the whole cohort.

•    Conventional slides are subject to differential antigen retrieval while TMA is not.

•    It is expensive and slow for large cohorts of conventional slides. Using TMA, it is rapid.

TMAs can also be used in various techniques such as immunologic stains (fluorescent visualization or chromogenic), histochemical stains, tissue microdissection, and in situ hybridization (mRNA FISH and in situ hybridization). Even after the tissue has been used for array-based studies, it can still be used to make a diagnosis. This means that the tissue block can be cored several times without destruction of the tissue block.

TMA in Research

TMA combined with IHC is a preferred method to analyze cancer biomarkers in various patient cohorts. The ability to assemble large samples of representative cancer tissue with a corresponding clinical database makes it a powerful resource to learn the correlation between different protein expression patterns and different clinical parameters. Since multiple samples are assembled into one block, the sections can be prepared using the same protocol to help avoid technical artifacts and variability. TMA sets can be used to study cancer biomarkers that play a role in diagnostic, prognostic, and therapy for patients. 

Possible Limitations

One of the main limitations of TMA as proposed by experts is the amount of tissue volume being too small and not being able to represent the entire tumor. However, numerous studies have proven that this issue can be compensated by the statistical power of the analysis of hundreds or thousands of cases. Ultimately, the conclusion is not affected as numerous amounts of cases helps to eliminate the variability of a single data point. In more than 95 percent of cases, the staining and analysis of 2 microarray disks are comparable to the analysis of a whole tissue section. The limiting factor is due to the number of sections obtained that depends on the thickness of the donor block. The standard blocks are about 5mm thick. Therefore, when the blocks have been extensively cut and are less than 2.5mm, the array construction is no longer recommended. 

References:

1)    Tissue microarray. Wikipedia. Accessed 6/25/2019. https://en.wikipedia.org/wiki/Tissue_microarray

2)    Tissue microarray. Novus Biologicals. Accessed 6/25/2019. https://www.novusbio.com/product-type/tissue-microarrays

3)    Why use tissue microarrays. Yale. Accessed 6/25/2019. https://medicine.yale.edu/pathology/ypts/tissuemicroarrayfacility/why.aspx

Tissue Freezing Techniques

Introduction

Modern technology advancements in proteomics and genomics have enabled the identification of disease mechanisms at molecular levels. Research data can be obtained through animal models and cell lines. However, to translate knowledge into clinical applications, numerous well documented high-quality human tissues are required. For these investigations, tissue collections from pathology institutes are necessary. For diagnosis, tissue samples can be formalin-fixed paraffin-embedded or frozen using the cooling chamber of a cryostat or solid carbon dioxide. 

Frozen Section Technique

Frozen section technique is a tool that can rapidly prepare slides from donor tissues for microscopic interpretation. This technique is used in various research and clinical settings. Frozen sections are commonly used in surgical pathology for rapid intra-operative diagnosis as it can be a valuable guiding tool for surgeons. For example, in Mohs micrographic surgery, the frozen sections help the surgeon determine the extent of excision required to remove a skin tumor. There are many research applications that depend on the frozen section technique in the preparation of microscopic slides that use molecular, immunohistochemical, and morphologic methods. The preparation of frozen section slides can be a complex process that requires an understanding of pathology, microanatomy, histology along with refined technical skills. Regardless of the use of the frozen section, the results depend on the quality of the specimen. The training for frozen tissue preparation may vary depending on the different practitioners. It is part of the curriculum in pathologist assistant and formal histology programs. However, there is also a lot of hands-on technique that is learned and passed along through experience at the workbench. Just like many research applications and pathology residency programs, most of the training is accomplished on the job. 

In intraoperative sections, tissue samples can be placed onto a metal chuck that has the optimal cutting temperature (OCT) compound. Slow freezing of the tissues can cause artifacts because of the accumulation of water molecules that become ice crystals. This issue can be resolved using liquid nitrogen. Due to the low temperature of liquid nitrogen, tissues that are extremely soft such as lymph node, brain, or spleen become rigid, brittle, and difficult to cut. Additionally, liquid nitrogen also evaporates rapidly, especially when it is in a small container. This means that it has to be refilled multiple times a day. 

Since there is limited FFPE tissue of quality, there are additional tissue pieces that are frozen for diagnostic and research purposes in various pathology institutes. However, the protocols for freezing tissues are not standardized and commonly differ. When it comes to tissue banking, specimens that are not needed can be frozen in cooled isopentane or liquid nitrogen. There are different strategies to preserve tissue specimens. Cold resistant cryomolds and cryovials are usually used as it does not require much space and can be labeled easily. To protect the tissue from desiccation, a freezing medium can be added or wrapped in aluminum foil or plastic bags. These methods can be disadvantageous as the tissues become harder to handle when further processing is required. Snap-frozen tissues can be transferred into plastic bags that are then placed into a box that contains other samples. However, as the number of samples in the cryo-container continues to grow, it may cause issues when trying to determine the specific location of the tissue. As the collection grows, it can become unmanageable. 

Examples of Freezing Methods

As previously mentioned, there are many freezing methods. Some examples include:

•    Dry ice – A block of dry ice can be placed in a Styrofoam container. This gives an estimated -70°C. The filled cryomold is then placed on the block to freeze it. This method is safe and simple but does not freeze the tissue as fast when compared to immersing it in a freezing medium. 

•    Pellet – Another method is to use dry ice that is in the pellet form. This method requires the placement of a small bowl that is stainless steel, pyrex, or a polypropylene beaker in a Styrofoam container and filling the surrounding space with dry ice pellets. Some pellets should also be placed in the bowl and isopentane or acetone added. This should be done in a fume hood. Care should also be taken as it is easily flammable. Once the pellets have stopped bubbling, fill the mold and orientate the tissue before immersing it in the liquid for freezing. 

•    Liquid nitrogen – Place the liquid nitrogen in a Dewar flask or Styrofoam container. Lower a container of isopentane into the liquid nitrogen using a pair of tongs. The isopentane will then begin to appear opaque once it nears freezing. Once ready, take the isopentane out and freeze the specimen. The isopentane can be chilled for subsequent uses. This method is advantageous due to its rapid freezing time. 

•    Fresh tissue freezing – The tissue is placed in OCT and flash frozen.

•    4% Paraformaldehyde (PFA) – This method uses 4% PFA and sucrose as a cryo-protectant. The tissue is placed in OCT and frozen using dry ice or flash frozen. 

•    Enzyme study – This method is often used for fresh muscle tissue. 

It is important to note that using just liquid nitrogen can cause cracking of tissue and OCT due to the unpredictable freezing pattern. Liquid nitrogen also boils and creates a vapor barrier that can cause the freezing to occur in a slow and unpredictable pattern. 

References:

1)    Emge DJ. Tissue freezing methods for cryostat sectioning. Accessed 6/20/2019. https://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf

2)    Peters S. A method for preparation of frozen sections. IHC World. Accessed 6/20/2019. http://www.ihcworld.com/_protocols/histology/frozen_section_technique_4.htm

3)    Freezing tissues for cryosectioning. UAB Research. Accessed 6/20/2019. https://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx

4)    Steu S, Baucamp M, von Dach G, et al. A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology. Virchows Arch. 2008; 452: 305-312.

5)    Peters SR. A practical guide to frozen section technique. 2010. 

The Effects of Cell Preservation

Introduction

Although cryopreservation is viewed as an “old school” discipline, modern cryopreservation is going through a growth face. Today’s cryopreservation involves processes and products that are at the forefront of research in various fields such as:

•    Personalized medicine

•    Diagnostic development

•    Stem cell research

•    Discovery science

As the need for cryopreserved cells increases, the demands placed on biobanks are evolving and rising at a rapid rate. Researchers now require samples with high cell recovery and products that are biochemically and physiologically identical to its original state at the structural, functional, proteomic, genomic, and reproductive levels. This has caused biobanks to rise to the challenge to adapt to new protocols and strategies to meet the new demands. There have also been studies concluding that the direction and control of the cell’s molecular response to cryopreservation have a great impact on the final outcome. 

What is Cryopreservation?

Cryopreservation is a process where cells, organelles, extracellular matrix, tissues, organs, and various biological constructs are preserved by cooling these biospecimens to extremely low temperatures. This can be done using solid carbon dioxide to reach -80°C or liquid nitrogen to reach 196°C. When the temperatures are low enough, any chemical or enzymatic activity that can cause damage or degeneration of the material can be stopped. The methods involved aim to reach the low temperatures without damaging the specimens caused by ice crystal formation when freezing. Traditionally, cryopreservation involves coating the material with cryoprotectants. However, due to the toxicity of many cryoprotectants, new techniques are constantly being developed. By default, cryopreservation should be considered to alter the structure and function of tissues. 

Physical Effects of Cryopreservation

Cryopreservation usually involves reducing the temperatures of samples from normal temperatures to a final temperature for storage. Immediately before the cooling phase, samples are placed into a medium with a cryoprotective agent and maintained at about 4°C for equilibration. For optimal preservation, sample cooling is done at a rate of -1°C per minute. This process also helps minimize volume excursions and enables water efflux from cell to extracellular environment. This can cause cellular dehydration. This continues until the temperature is lower than the glass transition temperature. Although beneficial to reduce the likelihood of intracellular ice formation, continued dehydration can cause structural damage to the organelles, membranes, nucleic acids, and proteins. The damage can be detrimental as it can cause activation of degradation pathways (DNA repair, necrosis, apoptosis, etcetera) and delayed molecular based repair. Severe damage can result in loss of the sample. 

Molecular Effects of Cryopreservation

Besides the physical effects of cryopreservation, there are also molecular effects due to factors such as:

•    Uncoupling of biochemical pathways

•    The decrease in metabolic activity and energy

•    Activation of stress response pathways

During storage, temperature reduction can cause the metabolism and biochemical pathways to slow down because of low levels of kinetic energy. It is important to note that low temperature only slows and does not stop cellular biochemical reactions. Biochemical reactions will continue regardless of temperature and cause damage to subcellular components, cells, protein, DNA, RNA, viruses, and bacteria. These reactions can lead to the formation and accumulation of toxic compounds like anaerobic metabolism byproducts, free radicals, and waste products. This can lead to the activation of molecular stress responses or direct damage after thawing. When a sample experience stressors, it can lead to biochemical degradation or molecular death once thawed. Some of these factors include:

•    Free radical production

•    Metabolic uncoupling

•    Dysregulation of cellular ion balance

•    Cell membrane fluidity and structure changes

•    Cryoprotective agent exposure

•    Osmotic fluxes

•    Calcium release from intracellular stores

When there are production and accumulation of free radicals, it can damage mitochondrial integrity, DNA, and protein in samples or cells. It can also activate apoptotic pathways and unfolded protein response that can cause further degradation. 

Effects of Sample Storage Conditions

During storage, the damage continues at both the physical and molecular level. Samples that go through freezing-induced dehydration can lead to continual damage. Biochemically, the reactions continue despite the low temperature that slows it down. Although highly reduced, during storage, energy and oxygen consumption in the sample continues. This also means sample degradation also continues with amplified stress events when the storage temperature increases. Although the specific responses of various processes and reactions are not well understood, it is widely accepted that the temperature during storage greatly affects the time samples before a significant compromise is apparent. Based on estimations, it would take thousands of years before an effect on cryopreserved cultures become noticeable. Storage intervals of 1 to 2 years for DNA, RNA, protein, bacteria, and viruses at -80°C have been reported to be successful. These hold times are for reference only. The repeated process of freeze-thawing will lead to accelerated sample degradation. 

Conclusion

Continual physical, molecular, and biochemical responses in the biospecimens have a significant impact on the quality of the biospecimens. 

References

1)    Cryopreservation. Wikipedia. Accessed 6/17/2019. https://en.wikipedia.org/wiki/Cryopreservation

2)    Baust JM. Biopreservation: The impact of freezing and cold storage on sample quality. Eppendorf. 2016. Accessed 6/17/2019. https://pdfs.semanticscholar.org/faaf/d05959d82a2b43c9e78a6b6124d6f454df20.pdf

3)    Baust JM, Corwin WL, VanBuskirk R, Baust JG. Biobanking: The future of cell preservation strategies. Adv Exp Med Biol. 2015; 864: 37-53.

FFPE Tissue and it's Use in Biobanks

Introduction

The process of fixation of tissue using formalin and embedding it in paraffin helps to preserve the morphology and cellular details of the biospecimens. Formalin-fixed paraffin-embedded (FFPE) tissues have now become the standard preservation procedure for tissue samples obtained for diagnostic surgical pathology. One of the main advantages of this method is the long-term storage at ambient temperature which helps it to be cost-effective compared to the requirements of frozen tissue storage at ultra-low temperatures due to labor costs, space, and maintenance. Vast numbers of FFPE blocks are routinely stored by pathology departments compared to frozen tissues due to its cost efficiency. It is a valuable untapped resource for translational clinical research.

Methodology

FFPE tissues are often used in experimental research, development of therapy, and diagnosis. 

The tissue is first obtained from the donor. It can be diseased or normal. In oncology, primary tumor samples and tissues from distant metastatic sites are often compared. In some cases, four samples are obtained:

• Normal tissue for FFPE

• Diseased tissue for FFPE

• Normal tissue for freezing 

• Diseased tissue for freezing

A block of tissue is first excised from the donor. Ideally, the excised sample measures only a few centimeters. It is then prepared by fixing the specimen in a solution of 10% neutral buffered formalin to help preserve the vital structures and proteins in the tissue. This process takes 18 to 24 hours. It aims to harden the tissue to ensure it survives the subsequent process. The tissue is then dehydrated and cleared using increasing concentrates of ethanol. It is then embedded in paraffin to make it easier for sectioning purposes. It is then mounted on a microscopic slide for examination. The fixation time is vital as tissues that are fixed too soon becomes useless for molecular biology studies. Samples that are acquired must be handled with care to ensure the quality of the specimen. This is important as low-quality samples can cause anomalies in research data leading to misleading deductions. 

The researcher requesting for the FFPE sample can also ask for samples that fit their specifications in terms of size, the way the tissue is cut, and purpose. In the preparation of the sample, a certified medical pathologist will be involved to ensure that the sample is prepared properly via accurate procedures. The pathologist will also assess the sample quality. Completed samples are stored in biobanks located in various research centers or hospitals. The biobank should also keep records or data associated with the sample such as when the tissue was collected, preserved, and donor information (age, gender, ethnicity, etcetera). 

FFPE Tissue Applications

FFPE tissues are often used in immunohistochemistry (IHC) where the tissue sections are mounted, bathed in an antibody solution that binds to proteins or other structures, and stained to help visualize the proteins and structures located in the tissue. This information can be crucial for physicians who are looking for signs of disease such as Alzheimer’s disease and cancer. The information gained from IHC is also important for various cancer research projects that are currently being performed. Some uses of FFPE tissue include:

• Immunology – FFPE tissue is used to help analysis of the immune system response in both the healthy and diseased state. The analysis of tissue samples from an individual suffering from an autoimmune disease can help researchers to determine the cause and develop a therapy for the condition.

• Hematology – FFPE tissue in hematology is vital to help researchers learn more regarding the blood and its related conditions. It also helps with the discovery of various cures for anomalies in the blood components. Besides blood-related conditions, it also aids in bone marrow studies regarding tissue regeneration, genetics, and toxicology.

• Oncology – FFPE tissues in oncology are important as tissue samples from donors with cancer may exhibit characteristic morphologies. Researchers often look for specific proteins in FFPE tissue as the presence of these proteins helps with diagnosis. It also helps to assess if a treatment would be beneficial for the patient. FFPE tumor tissues should have at least 60 percent of tumor content.

• Comparative – FFPE tissues in comparative studies compare diseased and healthy tissue. 

One of the main disadvantages of the FFPE method is the possible denaturation of proteins due to the formaldehyde fixation process. This can cause the proteins to be invisible to the antibodies used to detect them. To compensate for this issue, antigen retrieval techniques can be used. Newer techniques have also been developed to help recover proteins, DNA, and RNA from FFPE tissue samples. This leads to a trove of material that can be used in biochemistry studies and molecular biology. This improvement has led to more work that has started to use FFPE specimens as a source of proteins, DNA, and RNA from documented and archived materials. However, the FFPE tissue quality is crucial as even well-preserved tissue may contain partially denatured DNA, RNA, and inactive proteins. This limits the selection of healthy archived materials.

References:

1. What is FFPE tissue and what are its uses. BioChain. Accessed 6/14/2019. https://www.biochain.com/general/what-is-ffpe-tissue/

2. Kokkat TJ, Patel MS, McGarvey D, LiVolsi VA, Baloch ZW. Archived formalin-fixed paraffin-embedded (FFPE) blocks: a valuable underexploited resource for extraction of DNA, RNA, and protein. Biopreservation and Biobanking. 2013; 11(2): 101-106. 

The Importance of Tissue Procurement

Introduction

The human body is made up of billions of cells that are grouped together and organized based on structure and function. Examples of tissue types include epithelial tissue, muscle tissue, nervous tissue, and connective tissue. Organ or tissue procurement is a process where the specimens or organs are collected to help support research or to help save another life. The process is complex and often involves various medical professionals in different settings. Both state and federal legislation has been enacted to help maintain the efficiency and fairness of the process. This process helps ensure that the donated organs for transplant and tissue for research are equally distributed. The donation of organs can save lives. However, the need for donated organs has always far exceeded the number of organs donated. Therefore, to ensure that a fair and efficient process, the National Organ Procurement and Transplantation Network (OPTN) was established. This organization also helps to match donor organs to potential recipients. OPTN is managed by the United Network for Organ Sharing (UNOS). UNOS works closely with various organ procurement organizations to place the donated organs locally, regionally, and nationally. 

Organ / Tissue Procurement Procedure

This describes the steps for organ procurement:

• A potential donor is identified based on several criteria: brain death, age limits, and absence of systemic infections or extra-cerebral malignancies. 

• The local organ procurement organization confirms the suitability of the donor.

• Brain death is declared in accordance with the law. 

• The organ procurement organization evaluates the potential donor’s history and physical examination. 

• The donor’s next of kin will sign a consent form.

• The donor will then be maintained medically once brain death has been declared. 

• Surgical transplant teams will arrive and depart based on a schedule. These teams usually consist of operating room clinicians, surgeons, or an organ or tissue preservationist.

• Once the surgical teams have arrived, surgery begins.

• After the organs and tissues have been procured, the body is prepared according to hospital guidelines. There will be no change in the appearance of the body which allows the option for open casket funerals.

Organ Transplantation

• Donor organs are matched with tissue typing procedure taking about 6 hours. Besides tissue type, other criteria that have to be met include blood type, waiting time on the list, percent of reactive antibody, medical urgency, and distance of organs from the transplant center.

• Once the recipient is identified, pre-operative workups are required.

• The donated organs or tissues are distributed to the centers where preservation or transplant will occur.

• Surgery is performed.

• The procurement organization will also be responsible for follow-ups which may include letters to the donor's family, physician, and nurses about the donor organs and tissue.

Tissue Procurement Processing and Preservation

For tissues donated for research purposes, these biospecimens can be fixed using formalin or snap frozen to preserve the DNA, RNA, and proteins in the specimen. This minimizes the denaturation of the tissue. It is then sent to laboratories where it is processed and stored appropriately. For example, some biospecimens can be formalin-fixed paraffin-embedded samples. These samples can be stored at room temperature for many years. Another method is freezing the specimens using liquid nitrogen and storing it in specialized freezers which maintain the temperature required for storage. The quality of the specimens is reviewed by a certified pathologist. These specimens are also assigned a unique identification for easier retrieval when required. The information associated with the biospecimen is also recorded in the laboratory information management system. It usually includes information such as gender, age, and other clinical data.

Tissue Procurement Objectives

Human organs and tissues are critical in modern medicine as it is vital in the research process to ensure efficacy and safety of new procedures and treatment for everyone. Research involving these specimens can also help in the improvement of diagnosis and prediction of disease progression. For example, to develop a treatment for cystic fibrosis, researchers observed the movement of cilia in lung tissue when injected with different medication. In the United States, tissue procurement from donors is important as the supply from private research facilities can be finite and limited. Some of the services provided by various biomedical entrepreneurs include:

• The collection and storage of diseased or normal tissue from surgery or autopsy.

• Providing fresh tissue (such as tumor tissue) for cell studies.

• Processing and banking of biospecimens (such as blood) from cancer patients.

• The maintenance of a tissue database that has links to clinicopathological data. 

• Providing pathological review and histological staining of biospecimens.

• Coordination of consent from donors and assuring them regarding regulatory compliance. 

Human tissue procurement allows maximizing the access of human tissues to researchers. 

Conclusion

Tissue and organ procurement are both important as it helps in saving lives and advancement of the medical field. In research, tissue procurement helps researchers to develop new tests for diagnoses of diseases, treatment for existing diseases, and testing the safety and efficacy of drugs before it is distributed for public use. It leads to new discoveries and possible improvement of healthcare for everyone. 

References:

1. What is human body tissue? – Definition, types, examples. Study.com. Accessed 6/6/2019. https://study.com/academy/lesson/what-is-human-body-tissue-definition-types-examples.html

2. Understanding the organ/tissue procurement process. National Kidney Foundation. Accessed 6/6/2019. https://www.kidney.org/news/newsroom/fs_new/organ%26tissueprocprocess

3. Why human tissue procurement is so important. Geneticist. Accessed 6/6/2019. https://www.geneticistinc.com/blog/why-human-tissue-procurement-is-so-important

4. Tissue procurement. Cancer Institute. Accessed 6/6/2019. http://med.stanford.edu/cancer/research/shared-resources/tissue-procurement.html

Various Tissue Samples Stored in Biorepositories

Introduction

A biorepository is a word used to describe a center that collects, processes, stores, and distributes biospecimens. The term “biobank” is used to refer to a collection of biospecimens obtained from humans, plants, and animals. Although there is a slight difference between biorepositories and biobanks, these terms are often used interchangeably as the distinction is blurry. These centers usually specialize in the collection of tissue samples. For example, in this article, the focus will only be on human biobanks. 

Development

Over the last thirty years, the industry has changed greatly starting with small university-based biorepositories that were started to cater to the research needs of their projects. With time, these gradually evolved into government or institution supported biorepositories, virtual biobanks, commercial biorepositories, and more. The data stored with the biospecimens include diagnosis, date of biospecimen collection, patient phenotype, etcetera. Large scale biorepositories can be found in biobanks in countries such as Denmark, Sweden, Latvia, Iceland, United Kingdom (UK), Singapore, Japan, South Korea, Canada, Estonia, and the United States. Biorepository science has evolved due to the evolving requirements and needs of projects, researchers, and external regulatory pressures. These evolving requirements can be attributed to emerging fields such as personalized medicine, proteomics, genomics, and increasing precision of various science fields. This led to a higher demand for high-quality biospecimens that produces reliable, accurate, and standardized clinical data.  

Biobank Taxonomy and Activities

Human biospecimens have been collected and stored for more than a century. These biorepositories have advanced from simple record keeping in a laboratory notebook to computerization, procedure automation, specimen annotation, and more. The internet has also allowed the expansion of client communication and establishment of virtual biobanks. There are various types of biorepositories such as:

• Population-based - Large population biobanks such as the Danish National Biobank that opened in 2012 as a collaboration between both private and public sectors make it possible to study populations over the duration of a whole lifespan. After a decade of planning, the UK biobank was established with the goal of improving diagnosis, treatment, and prevention of various diseases such as depression, cancer, diabetes, cardiovascular disease, and dementia. 

• Disease centric – The University of California, San Francisco (UCSF) AIDS Specimen Bank (ASB) was established in 1982 due to the Acquired Immunodeficiency Syndrome (AIDS) epidemic at the time. Since the cause of AIDS was unknown at the time, various experts from different disciplines gathered together and developed a small biobank.

• Commercial – Commercial biobanks are biorepositories that are for profit. 

• Virtual – Virtual biobanks are electronic databases of biospecimens and related information. The University College London (UCL) biobank act as a physical repository for the collection of biospecimens and data from consented patients in hospitals who are at UCL partnered hospitals. 

• Genetic or DNA / RNA

• Project driven

Types of Tissue Samples

In a biorepository, there are various types of tissue samples. Typically, tissue samples that are available would include saliva, blood, serum, urine, tissue from different parts of the body, RNA, DNA, and diseased tissues. This enables the biorepository to cater to researchers in various fields to meet their research needs. In some biorepositories, the biospecimens can be classified based on disease such as:

• Arthritis

• Brain cancer

• Breast cancer

• Colon cancer

• Cardiovascular disease

• Cervical cancer

• Diabetes

• Dementia

• Head and neck cancer

• Lymphoma

• Lupus

• Leukemia

• Multiple sclerosis

• Normal tissue

• Etcetera

It can also be categorized based on preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissue – Tissue samples obtained from the donor are fixed in formalin and embedded in paraffin wax for preservation.

• Frozen tissue – Tissue samples are snap frozen and stored at low temperatures using liquid nitrogen to preserve DNA, RNA, and proteins.

• DNA / RNA – DNA and RNA samples are available based on diseases, tumors, or disease-free (for control).

• Human serum – Serum samples from normal and diseased tissues are also available.

Clients can always request the types of biospecimens they require for research and clinical trials from the biorepository based on their requirements. 

Best Practices

To ensure the quality of biospecimens, the biorepository needs to train their staff to follow “best practices”. 

• International Society of Biological and Environmental Repositories (ISBER) has published a handbook known as “Best Practices for Repositories” and involves topics such as equipment, cost recovery, facilities, quality assurance, safety, quality control, ethical issues, shipping, processing, specimen collection, specimen retrieval, specimen culling, training, and more. 

• National Cancer Institute (NCI) has developed the First-Generation Guidelines for NCI Best Practices for Biospecimen Resources. 

• The Rand Corporation published the Best Practices for a Biospecimen Resource for the Genomic and Proteomic Era. 

Legalities and Ethics

The evolving industry constantly affects biobanks. New standards and regulations are regularly established to set new standards and protect patients and other parties in terms of privacy, confidentiality, and consent. Some ongoing issues include:

• Providing results to study participants

• Responsibility to report incidental findings or individual research results

• Ownership of biospecimens

• Privacy of patients

• Consent for one or all research

• Etcetera 

References:

1. Souza YG, Greenspan JS. Biobanking past, present, and future: responsibilities and benefits. AIDS. 2013; 27(3): 303-312.

2. The difference between biobanks and biorepositories. Geneticist. Accessed 5/21/2019. https://www.geneticistinc.com/blog/the-difference-between-biobanks-and-biorepositories

3. The various tissue samples stored in a biorepository. Geneticist. Accessed 5/21/2019. https://www.geneticistinc.com/blog/the-various-tissue-samples-stored-in-a-biorepository

Paraffin Embedded Tissue Blocks

Introduction

Paraffin-embedded tissue blocks usually refer to formalin fixed paraffin embedded (FFPE) tissue specimens that have been used in various therapeutic applications and research for many decades. It is a method used to preserve and prepare biospecimens to be used in experimental research, examination, diagnosis, and therapeutic development. The tissue sample is first preserved through fixation using formalin or formaldehyde to ensure that the vital structures and proteins within the tissue stay as close as possible to the condition when it is still part of the host. After fixation, it is embedded in a paraffin wax block to make it easier for sectioning and mounting on microscopic slides for examination purposes.

Process

The tissues are first collected from both diseased and normal hosts. In cancer biospecimens, oncologist often compares the primary tumor with samples that are obtained from distant metastatic sites. The tissues obtained usually measure only a few centimeters depending on nature and source and tissue. Immediately after excision, the biospecimen is immersed in 10% neutral buffered formalin for 18 to 24 hours to harden the tissue. It is then dehydrated and cleared using increasing concentrates of ethanol. It is then embedded into immunohistochemistry grade paraffin that is specifically used for embedding formalin-fixed tissues. One of the most crucial factors is the time of fixation as tissues that are fixed too soon may be unusable for molecular biology studies. The duration of fixation has the be long enough to ensure preservation.

Once acquired, the samples are handled carefully to maintain quality. Failure of proper handling can lead to an exhibition of unusual characteristics that can affect research results or deductions. The preparation method usually depends on the research team's requirements. It can range from specifications regarding tissue purpose, size, or cut of the tissue. One good example would be the cut of muscle along the muscle fiber "grain" or across them. Throughout the sample preparation, a certified medical pathologist will be involved to ensure that the procedure is completed accurately and also for quality assessment.

Once the samples are completed, they are stored in tissue banks. Useful records such as demographic information and when the tissue was collected or preserved should also be kept as it can be useful for the research team. Other critical information that should be stored includes signed consent forms and legal documents affiliated with the biospecimen as it can impact the usability of the biospecimen in research and clinical trials. 

Applications

Paraffin-embedded tissue blocks have many applications in research. These tissue blocks are often used in immunohistochemistry where the tissue sections are mounted on a slide. These sections are then bathed using a solution that contains antibodies that bind to proteins and structures. Staining can also be performed to help visualization of the antibodies which shows the location of structures that are present in the sample. This information can be critical to aid diagnoses of diseases such as Alzheimer's disease or cancer. The information gained form immunohistochemistry is also vital to many cancer projects that are being performed in laboratories today. Some of the therapeutic areas where the tissue samples are commonly used are:

1. Immunology – responses of the immune system are analyzed in both the diseased and healthy state. The study of tissue samples from a patient with the autoimmune disease helps determine the cause and development of therapy for those affected.

2. Hematology – paraffin-embedded tissue blocks are vital in the study of various blood and related disorders. Hematology is a crucial field that has helped with the discovery of many cures to diseases related to the blood and its components. Some studies that are related to this field include bone marrow studies which can include genetics, toxicology, and tissue regeneration.

3. Oncology – paraffin-embedded tissue blocks are important in oncology as the preserved tumor tissues have characteristic morphologies that are not present in other tissue. Research teams often use these samples to look for proteins that can aid in the diagnosis and assessment of the disease. Formalin-fixed paraffin embedded (FFPE) tumor tissues generally should have 60% tumor content.

4. Comparative – there are also paraffin-embedded tissue blocks that are healthy tissue collected from healthy donors. These tissues are used for comparative purposes and are also important for research and development.

Disadvantage

In paraffin embedded tissue blocks, the fixation process usually requires the use of formalin or formaldehyde that denatures the proteins that are in the tissue sample. This can cause the proteins to be invisible to antibodies that are specifically developed to detect them. To compensate, antigen retrieval techniques have been developed to specifically recover proteins, DNA, and RNA from these tissue blocks. This helps open up a vast archive of preserved and annotated material for biochemistry and molecular biology studies. 

Conclusion

The quality of paraffin-embedded tissue blocks is crucial for work using these samples as a source of proteins, DNA, and RNA. This is due to the fact that even the best-preserved tissue will contain partially degraded inactive proteins, DNA, and RNA.

References:

1. What is FFPE tissue and what are its uses. BioChain. Accessed 5/16/2019. https://www.biochain.com/general/what-is-ffpe-tissue/

2. Paraffin processing of tissue. Protocols Online. Accessed 5/16/2019. https://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/

What is a Liquid Biopsy?

Introduction

A liquid biopsy, fluid phase biopsy, or fluid biopsy is a procedure where biological tissue is obtained for sampling and analysis. It is a revolutionary technique that allows new perspectives. It involves the isolation and detection of circulating tumor DNA, circulating tumor cells. And as a source of proteomic and genomic information for individuals with cancer. For many years, healthcare professionals and researchers have tried to find a fast and easy way to diagnose and monitor cancer. This is a technique that can be used to diagnose and monitor diseases such as cancer as it has a benefit of not being too invasive. It can, therefore, be used more frequently for the tracking of tumors, mutations, and validation of cancer treatment efficacy. It can also be used to monitor relapse among patients after their treatment. The liquid refers to samples such as blood or other bodily fluid. 

Types

Depending on the condition of the liquid biopsy, there are several types of liquid biopsy such as:

• Circulating endothelial cells (CECs) – This can be used in the diagnosis of a heart attack.

• Cell-free fetal DNA (cffDNA) – This is used for prenatal diagnosis where samples can be obtained from amniotic fluid or maternal blood.

• Circulating tumor DNA (ctDNA) – This is used for cancer studies. ctDNA are tiny DNA fragments present in the blood as they break away from the tumors. ctDNA can be used to monitor the treatment progress as ctDNA levels are expected to decrease once the tumor has been removed or shrinks. When ctDNA levels in the blood increase, it could signify a recurrence of cancer. Since ctDNA levels increase several months before the cancer recurs, it allows for faster treatment response. 

Various biomarkers can be studied for the detection of other diseases. A good example would be the isolation of protoporphyrin IX for the diagnosis of atherosclerosis. In the study of the central nervous system, both blood and cerebrospinal fluid can be sampled. 

Current Issues in Liquid Biopsy Research

Some ongoing issues related to liquid biopsy research include:

1. Cancer location identification

This can be done by using the cell free DNA present in the blood to detect the presence of cancer in the body before it becomes visible through other techniques such as mammography, x-rays, colonoscopy, or CT scans. Since the cancer is yet to be visible, physicians need a screening tool that offers guidance on where they can look for cancer. One recent study showed the combination of technologies where protein markers and cell free DNA are observed for different cancer types. With that combination, researchers were able to sort for ctDNA and link the available information to a protein marker which offers clues on where cancer may be located. 

1. Tiny amounts of ctDNA

Even with significant sized tumors, there is only a very small amount of ctDNA. There are various types of DNA circulating in the blood as seen in pregnant women or those who have suffered a stroke or heart attack. All these DNA fragments are known as cell free DNA. Due to the various DNA fragments that are present in the blood, the researchers should be able to accurately identify ctDNA from other DNA to avoid false positives 

Liquid Biopsy Future

The future of liquid biopsy largely depends on several factors such as:

1. Catching up to technology

Since technology is advancing rapidly, the ability to analyze the impact of the technology and the best way to use it is left behind. It can be difficult as it takes time, effort, and practice to understand how to use the data that has been collected through translational science. In some centers, artificial intelligence is being used to obtain answer or recognition from data. 

1. Translation for undiagnosed individuals

This is required to obtain more data regarding the efficacy of liquid biopsy as a screening procedure such as for individuals who have no evidence of cancer.

1. Safety

The liquid biopsy test is a safe procedure that provides accurate results. Ideally, a liquid biopsy should be able to be used in the detection of cancer in the body, to tell if the individual should be treated, location of cancer, and best treatment options for the most favorable prognosis.

1. Funding

Research regarding liquid biopsy is important as it helps advance this field of science. While a lot has been achieved, there is still a long way to go. 

Conclusion

Liquid biopsy is a technique that can address various issues. It involves the identification and isolation of circulating tumor cells, ctDNA, exosomes, and various information in patients with cancer. With new techniques, liquid biopsy now has a wide application such as in prognosis, diagnosis, screenings, prediction, and monitoring or treatment efficacy. Further research is necessary to help advance this field. 

References:

1. Palmirotta R, Lovero D, Cafforio P, et al. Liquid biopsy of cancer: a multimodal diagnostic tool in clinical oncology. Ther Adv Med Oncol. 2018;10:1758835918794630. Published 2018 Aug 29. doi:10.1177/1758835918794630

2. Liquid biopsy. Wikipedia. Accessed 5/9/2019. https://en.wikipedia.org/wiki/Liquid_biopsy

3. McDowell S. Liquid biopsies: past, present, future. American Cancer Society. Accessed 5/9/2019. https://www.cancer.org/latest-news/liquid-biopsies-past-present-future.html

FFPE and Tissue Microarray Samples

FFPE

Formalin-fixed paraffin embedded (FFPE) is a method of preparation and preservation for biospecimens that are a staple of therapeutic and research applications for many decades. It aids experimental research, examination, and drug or diagnostic development. A sample is preserved by fixing it in formalin or formaldehyde to help preserve the structures in the tissue. It is then embedded in paraffin wax, sectioned, and mounted on a microscopic slide for further examination. The biospecimens that can be subjected through the FFPE method may be obtained from both normal or diseased tissues and human or any other living organism. 

FFPE Process

Once the tissue sample is obtained from the host, the tissue is immediately immersed in 10 percent neutral buffered formalin for 18 to 24 hours. Once ready, dehydrate and clear the tissue using increasing concentrations of ethanol. It is then embedded into paraffin. Samples should be handled with care to ensure the maintenance of quality. A certified pathologist helps ensure the quality of the sample. Once the sample is completed, it is stored in tissue banks such as research centers, universities, and hospitals. It is also important to keep available data or information such as origin, stage of the disease, donor age, and etcetera that is associated with the biospecimen. Another crucial point is to store the legal documents and signed consent forms for the tissue samples as it can affect the usability of the samples in clinical trials or research.

FFPE Applications

FFPE tissues are commonly used in immunohistochemistry (IHC). The information obtained from IHC can be vital especially in the detection of disease. Some of the applications of FFPE include:

1. Hematology – FFPE tissues can be used for the study of blood and related disorders which can be important in genetics, tissue regeneration, and toxicology.

2. Oncology – FFPE tissues are key to cancer research as the presence of specific proteins can help with assessment and diagnosis of cancer. The minimum tumor content in FFPE tumor tissues is generally 60 percent. 

3. Immunology – FFPE tissues are useful in the analysis of the immune system response in both diseased and healthy states. It can help with the development of treatment. 

4. Comparative – FFPE tissues that are both healthy and diseases are necessary for comparative purposes.

Tissue Microarray

Tissue microarray (TMA) is an innovation that is expected to overcome issues where the validation of markers in standard histopathological techniques are:

• Costly

• Labor intensive

• Time-consuming

Especially when multiple markers are required to be tested on various specimens. This is due to the high-throughput molecular biology design where it allows simultaneous assessment of expression of interesting candidate-related genes and gene products on hundreds of biospecimens. The TMA technique also allows:

• Parallel molecular profiling of proteins, DNA, and RNA

• Large scale analyses using IHC, RNA in situ hybridization, and fluorescence in situ hybridization (FISH) at significantly lower costs and lesser time.

The TMA Technique

The technique uses composite paraffin blocks (such as FFPE specimens) that are constructed through the extraction of cylindrical cores from various paraffin blocks which are then re-embedded into a recipient or microarray block at specific array coordinates. 

1. A TMA instrument is used to obtain a tissue core from the donor block.

2. The core is placed in the recipient block at specifically assigned coordinates and recorded on a spreadsheet. 

3. This block is then sectioned using a microtome, mounted, and analyzed. 

4. Each block can be cut into 100 to 500 sections.

TMA Advantages and Applications

There are many advantages of TMA compared to other techniques. Using the TMA technique, analysis of an entire cohort of cases is made possible just by staining one to two master slides. Other advantages include:

• Amplification of a scarce resource 

• Experimental uniformity

• Simultaneous analysis of many specimens

• Preservation of original block and conservation of valuable tissue

• Decreased use of assay volume

• Shorter duration and more cost-effective

TMA has been proven to be an efficient and effective tool in the assessment of quality assurance programs. A TMA block can be created from various tissue specimens, sectioned, and distributed to different labs that perform molecular tests and immunostaining. Therefore, it can facilitate the standardization of FISH, IHC, and other molecular assays so results would be reproducible. Other applications include:

• Internal quality control

• Optimization of diagnostic reagents

• Facilitation of rapid translation of molecular discoveries to clinical applications

• Clinical validation of histopathological specimens

TMA Disadvantages

One main criticism of the technique is that the cores used for TMA may not be representative of the entire tumor especially in heterogeneous cancers like Hodgkin lymphoma and prostate adenocarcinoma. However, there are many studies that have shown high concordance between TMA spots and whole sections in IHC of multiple tumor types. Another minor criticism would be the absence of one or more core sections. This can be addressed by the statistical power of analysis ranging from hundreds to thousands of cases as it eliminates the variability of a single data point in the conclusion. 

References:

1. Jawhar NMT. Tissue microarray: a rapidly evolving diagnostic and research tool. Ann Saudi Med. 2009; 29(2): 123-127. 

2. Tissue microarray. Wikipedia. Accessed 4/30/2019. https://en.wikipedia.org/wiki/Tissue_microarray#Procedure

3. What is FFPE tissue and what are its uses. BioChain. Accessed 4/30/2019. https://www.biochain.com/general/what-is-ffpe-tissue/

Human Tissue Samples Used in Biobanks

Introduction

Biobanks are centers that process, organize, and maintain biospecimens that would be used in various clinical and research-based purposes. There are many different types of biobank. That is why before starting a biobank, the goals of the biobank should be delineated. Accreditation and standard operating procedures are also crucial to ensure the biospecimens are of the highest quality as compromised biospecimens can negatively affect the results of clinical studies or research. Other important factors include developing a budget and obtaining funding sources to warrant that the designated space, equipment, and personnel necessary are acquired. Another critical part of the biobank would be the laboratory information management system. Extra effort should be expended to guarantee the security and effectiveness of the system. 

Operations

Biobanks are part of the research infrastructure as they provide samples for various purposes. The samples are randomly collected and compiled into categories such as based on diseases or therapies. It is vital that the sample characteristics are preserved with minimal alterations during the collection, processing, and storage stage. Degradation processes begin shortly after the specimen is obtained from the host. There are some factors that may contribute to the degradation process such as the lack of oxygen supply. The gold standard during the collection process is to collect the samples in a standardized method and to minimize the duration from harvesting to freezing. Samples can be collected during routine clinical procedures or from the remains after diagnostic procedures. Since samples are collected randomly, complete standardization of the preanalytical procedures may be impossible. 

To minimize variability, some methods include:

• Ensure that the duration from sample acquisition to freezing is kept as short as possible

• Standardizing handling of samples once it has arrived at the biobank

• Standardize the use of protective reagents

RNAlater

RNAlater is a high salt ammonium sulfate aqueous solution that is used specifically to stabilize RNA in tissue. This solution precipitates the RNAses depending on the concentration and pH. Some studies have shown that the expression profiles are well preserved in RNAlater compared to shock frozen tissue. This is exceptionally true for some genes when measured using real-time polymerase chain reaction (PCR) and also for RNA expression microarray analysis. Through RNAlater stabilized tissue, DNA that is suitable for PCR can be extracted. Tissue specimens preserved using RNAlater can also be sectioned on a microtome and later, stained for histological analysis. Further major advantages of using RNAlater includes:

• Omitting the use of expensive and dangerous liquid nitrogen

• Direct placement of samples into the preserving agent right after extraction

• Stabilization of nucleic acids such as RNA

However, it is important to note that the preservation of samples is not as abrupt compared to shock freezing in liquid nitrogen since RNAlater will need to diffuse into the samples. The samples will need to be incubated in the reagent for a minimum of 24 hours to confirm that there is enough absorption before freezing. Another added benefit of RNAlater preserved specimens is that samples remain protected even after freezing and thawing while snap-frozen tissue samples that thaw lead to degradation of RNA as the process destroys intracellular compartmentalization. This benefit minimizes the effect of temperature changes and makes sample handling much easier.

Human Biospecimens and Ethical Issues

One of the most prominent ethical issues with human biospecimens is consent. In a case involving the biospecimen of a woman known as Henrietta Lacks from Virginia, some of her cells were taken without consent during her treatment for cervical cancer. Her biospecimen subsequently played a role in decades of research which led to gene mapping, the creation of the polio vaccine, and cloning. Despite having many parties involved making a lot of money from the cells, Henrietta Lack's family never received any compensation, nor did they have any knowledge about her role in it until 25 years later. This case highlights a problem that still persists today. Many individuals do not realize that when they are donating their samples such as tissue or blood to science. Most consent obtained is "broad consent” where participants agree that their materials and information would be used without being given any additional details. 

Another major issue is those involving ownership of the specimens. A study published in 2014 found that there was no consensus regarding who the samples belong to. While the participants believe that the biospecimens belong to themselves, researchers think that it belongs to the institution. It is a difficult situation as donors will lose trust in the system if biobanks continue to partner and commercialize with private companies. In 2009, a group of parents in Texas filed a lawsuit regarding the banking and use of samples obtained from their children as they never consented for the samples to be used for experimentation. When the court ruled in the parents’ favor, it led to the destruction of millions of research samples. 

References:

1. Lindner M, Moressi-Hauf A, Stowasser A, Hapfelmeier A, Hatz R, Koch I. Quality assessment of tissue samples stored in a specialized human lung biobank. PLOS ONE. 2018. Accessed 4/23/2019. https://www.biorxiv.org/content/10.1101/407411v1.article-info

2. Harati MD, Williams RR, Movassaghi M, Hojat A, Lucey GM, Yong WH. An introduction to starting a biobank. Methods Mol Biol. 2019; 1897: 7-16. 

3. Dube J. “Biobanks” that store human blood and tissue have a consent problem. Motherboard. Accessed 4/23/2019. https://motherboard.vice.com/en_us/article/bjx49w/biobanks-that-store-human-blood-and-tissue-have-a-consent-problem

What is a Biorepository and What do they do?

Introduction

A biorepository is a center that collects, processes, stores, and transports biospecimens or biological samples to aid research or scientific investigations. Biorepositories can contain human specimens, animal samples, and various other living organisms. Any life form can be studied through the preservation and storage of samples. Biorepositories that revolve around human specimens are essential for research regarding personalized medicine. This is crucial as it helps to advance the study of disease and health with time. Biorepositories function to maintain biospecimens and all the associated information for research purposes. The center helps to assure the quality while managing the accessibility and distribution of the biological materials in the collection. 

Operations

All biorepositories have four main operations:

1. Collection – This is where the arrival of the samples is recorded. These biospecimens are assigned a unique identification. This information along with the data associated with the specimen is then recorded in the laboratory information management system. 

2. Processing – The samples at this stage are tested to ensure that there is minimal variation in the handling and preparation stage. 

3. Storage – The samples are held in their proper storage environments such as room temperature or freezers depending on the requirement.

4. Distribution – The required samples can be retrieved and transported to their designated locations when requested by research teams. 

Safety

Biorepositories need to consider important safety issues such as electrical, biological, physical, chemical, fire, and radiologic hazards. All biorepositories should have their own protocols regarding safety issues and needs. This may vary depending on the specific goals and functions for different biorepositories. In the European Union and the United States, biorepositories should have regulations regarding general areas of safety. There are also certain regions where there are additional regulations for electrical, physical, and fire safety. Biorepositories should also continually adjust, review, and update their safety protocols. 

Issues Associated with Biorepositories

There are several issues regarding biospecimens that have been collected for research purposes. Biospecimens can be tissue, blood, urine, etcetera that will be used for research purposes to contribute to scientific knowledge. Donors expect that their privacy will be respected, and their donated specimens are used only in cases where they have consented. This means that the biorepository is considered to be responsible for the donor’s privacy. 

Another issue would be concerning responsible custodianship. The specimens are precious and the more the specimen is used, the higher its value as different causal pathways can be determined without testing or collecting determined parameters. However, a renewable resource will lead to issues of costs of sharing, packing, and shipping. There are also costs associated with intellectual property loss along with concerns that the data will be misused or misinterpreted. With the above mentioned factors and the fact that a well-maintained collection annotated with data can have great commercial value, it is not surprising that investigators are unwilling to share their biospecimens. Responsible custodianship also requires proper handling and storage of the specimens. This includes having the proper personnel, quality control, standard operating procedures, and quality assurance measures. 

The next issue is a well-known issue known as informed consent. There are many biorepositories that are established and maintained after used for studies. These biorepositories are kept and maintained for future research. Since the informed consent obtained was for the previous study, it poses as a major ethical issue when the biospecimens are used in future research. This is especially true if the specimens are linked to personal information such as medical history as it involves privacy regulations such as the Health Insurance Portability and Accountability (HIPAA) act. HIPAA required permission or consent to be obtained every time the specimen is used for a different purpose. However, it is being reviewed to how the regulations may be improved. Obtaining future consent is also problematic as donors can be lost to follow-up or resent being contacted. However, it is important that the use of the specimen be consistent with the consent. Donors should also be informed that there have a right to withdraw their consent or request for their specimen to be destroyed. 

Another significant issue is the privacy protection of donors. While the specimens are much more valuable if it has associated information such as medical history and sociodemographic information of the donor, it also becomes harder to protect the privacy of the individual. There is also a concern where the information can or cannot be released. For example, the information obtained from the biospecimen may jeopardize the ability to obtain health insurance. This is why that is crucial for biorepositories to have policies that protect the confidentiality and privacy of donors.

Conclusion

There is much to know and learn about the operations and management of a biorepository. These centers play a key role in the advancement of the medical industry as it helps determine the progression, prognosis, and therapy for various diseases. However, there are also various issues that should be addressed to ensure that all parties involved are protected. 

References

Foxman B. Human and animal subject protection, biorepositories, biosafety considerations, and professional ethics. Molecular Tools and Infectious Disease Epidemiology. 2012. Accessed 4/16/2019. https://www.sciencedirect.com/topics/medicine-and-dentistry/biorepository

Biorepository. Wikipedia. Accessed 4/16/2019. https://en.wikipedia.org/wiki/Biorepository