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


Why is Tissue Important?

Introduction

Tissue is a term that refers to a cellular organization of cells and extracellular matrix that usually has a synergistic function. The functional grouping of multiple tissues then forms organs. The term “tissue” originates from a French word “tissu” which carries the meaning of “woven” or “to weave”. The study of tissues is known as histology while the study of the disease of tissues is known as histopathology. Tissue is important in research and is often referred to as a biological sample, biospecimen, tissue sample, or specimen. These terms refer to fluid or tissue such as saliva, fluid, feces, spinal fluid, brain, organ tissue, tumor tissue, bone marrow, etcetera. Once permission is given by the donor or patient to have a medical procedure performed, it also allows the doctor to take a tissue specimen to help achieve a diagnosis and propose a treatment plan. These specimens are also crucial to help in advancing the industry of medical science and research as it provides researchers study material. 

Applications

As previously mentioned, tissues can be used in the diagnosis and classification of diseases. For example, when a patient is suspected of cancer, a specimen is obtained and the cell morphology in the tissue is studied. It will help the healthcare team to decide if the tissue is cancerous, type of cancer, and characteristics of cancer. Tissue samples can also help determine if the patient is responding appropriately to treatment and the side effects that occur. Besides diagnosis and treatment, the tissues can also be used in research to retrospectively compare tissue characteristics and patient response to help the team understand the effectiveness of a drug as a treatment option. Prospectively, it can be used to determine if the theories about how a drug works are accurate. Archived tissue is essential in testing new discoveries, understanding possible causes of cancer, discovering new biomarkers that identify cancer, identifying targets for treatment, and developing new treatments that target a gene or the signaling process.

Study of Tissue and Cancer

Cancer is thought to arise when there are gene mutations in a cell. When the mutations affect the normal cell growth, it can contribute to the development of cancer. A gene mutation can be hereditary. While there are inherited mutations that result in a condition or disease, there are also other mutations that increase the likelihood or risk of developing a disease. Gene mutations can be caused by various factors such as radiation, chemicals (present in the environment or diet), viruses, bacteria, and more. It is also important to note that most cancer-causing mutations are due to spontaneous errors. Spontaneous errors occur when mistakes occur during normal DNA replications. When the mistake is left uncorrected, further cell division may result in the persistence of mistakes in the subsequent cell generations. To learn and understand more about cancer, tissue specimens are vital to enable further research and study. 

This can be done by studying cancer cells and individual genes. For example, the study of oncogenes and their role in cancer led to the identification of the HER2 gene. The presence of this gene in a normal cell causes the cell to make receptors for a growth factor. The identification of this gene led the researchers to discover that too many copies of the HER2 gene in breast cancer patients causes the cells to grow and divide faster. The HER2 gene was discovered through the study of tumor tissues obtained from patients with breast cancer. This also allowed the researchers to observe how treatment affects different patients and the researchers concluded that patients with multiple HER2 gene copies have poorer survival rates due to more aggressive cancers. 

Study of Tissue and Treatment Development

Through tissue donation by breast cancer patients, it was discovered that approximately 25 to 30 percent of women have the HER2 gene mutation. Since this mutation results in the production of more copies than normal, it is also known as over expression or gene amplification. Via these samples, the researchers found that some breast cancers have more growth factors compared to others. This led to the identification of a target for treatment for patients with HER2 positive breast cancer. The treatment blocks the receptors and may slow the growth and division of cancer cells

To develop a treatment that is specific for those with HER2 positive breast cancer, the researchers looked for a chemical that could block the growth factor receptors. They tried a monoclonal antibody that would target the extra receptors on the cancer cell surfaces. The researchers were able to identify a chemical known as Herceptin for this purpose. After various experiments using tissues and animal models, Herceptin was finally ready for clinical trials involving volunteer patients. The study found that Herceptin was indeed effective for some with HER2 positive breast cancer even when other treatments were no longer working. Since Herceptin is target specific, it also had fewer side effects compared to other drug treatments.

Biomarkers

The study of tissues has also led to the discovery of biomarkers. Biomarkers are biological substances in a cell that can help predict the disease progress, prognosis, and effectiveness of treatment. Without the development of biomarkers, doctors would not be able to tell which patients would benefit from Herceptin. Another example would be the identification of Epidermal Growth Factor Receptor (EGFR) mutations among patients with lung cancer. Those who test positive will benefit from an EGFR inhibitor drug known as gefitinib. 

Conclusion

Tissue is important as it helps the study of disease progression, determine prognosis, and identify the best treatments for different diseases. It has significantly contributed to the advancement of the medical industry. 

References

  1. Tissue (biology). Wiki. Accessed 4/11/2019. https://en.wikipedia.org/wiki/Tissue_(biology)

  2. What is tissue? Why is it important? Research Advocacy Network. Accessed 4/11/2019. https://www.roswellpark.org/sites/default/files/What_is_Tissue__amp__Why_is_it_Important.pdf

  3. Cell differentiation and tissue. Scitable. Accessed 4/11/2019. https://www.nature.com/scitable/topicpage/cell-differentiation-and-tissue-14046412

FFPE vs Frozen Tissue Samples

Introduction

Both formalin fixed paraffin embedded (FFPE) and frozen tissues are important in research. They can be used to study the cell biology, morphology, biochemistry, and disease in all living creatures. Both FFPE and frozen tissues have their own pros and cons as both have different applications. Some basic differences seen between FFPE and frozen tissue samples are:

FFPE Samples

FFPE is a method where tissue samples are preserved by using formalin to paralyze cell metabolism while paraffin is used to seal the tissue and decrease oxidation rates. There are many tissue samples that are stored using this method due to its cost efficiency as it can be stored at room temperature. FFPE tissue blocks are crucial as they play a key role in biotech research, drug discovery, and retrospective gene studies. Tissue samples preserved using the FFPE method can be stored for decades and is an invaluable source to allow correlation of clinical outcome, therapy, and molecular findings. It is also easier to section FFPE samples since they are embedded in wax and can be easily mounted on a microscope for examination. 

  • Uses – FFPE samples are important in fields such as immunology, hematology, and oncology. 

  • Advantages – Due to its cost efficiency for storage at room temperature, it is a great resource as a research material as it remains viable without requiring specialized equipment. 

  • Disadvantages – Some main disadvantages of this method include using formalin for fixation of the tissue sample, time consuming process for fixation and embedding, and non-standardized protocols in preparation of the tissue samples. Due to its preparation method, the proteins in FFPE samples become denatured limiting the use of FFPE samples to only certain studies. 

Frozen Tissue

Frozen tissue refers to tissue samples that are preserved and stored using ultra-low temperature freezers and liquid nitrogen.

  • Uses – Frozen tissue is important in areas where FFPE samples are not reliable such as molecular analysis. It is also important to help determine if the margins are clear for tumor removal in surgeries. It is also preferable compared to FFPE in next generation sequencing, western blotting, and mass spectrometry.

  • Advantages – This method is much faster compared to the FFPE process. It also preserves proteins in their native state. 

  • Disadvantages – Some disadvantages of this method include the rapid deterioration rate of the frozen tissue samples once it is in room temperature. Since the samples need to be frozen as fast as possible once the sample is collected, it can pose some difficulty as the equipment required will need to be available. Storage of frozen samples are also expensive as specialized equipment are required to keep the samples frozen. The samples are also vulnerable especially if there are mechanical failures or power outages. 

Molecular Analysis

Frozen samples are better than FFPE samples for molecular analysis. This includes work that involves post translational protein modifications (PTMs), DNA, and RNA. This is due to the non-standardized preparation methods used for FFPE sample preparation. Another reason is because of the involvement of formalin in FFPE preparation. The use of formalin often results in non-native configurations of phosphorylated proteins and degraded RNA.  Frozen samples are also a necessity for procedures such as Western blotting, next generation sequencing, mass spectrometry, and quantitative real time polymerase chain reaction (PCR) as they are considered the gold standard. 

While FFPE samples are not the best option for molecular analysis, it can be used when there are no frozen samples available from a deceased donor. However, it is important to note that the isolation of proteins and DNA from FFPE samples can be difficult with results that are not on par with results obtained from frozen specimens. It is a known issue that DNA obtained from FFPE samples can lead to the accumulation of sequence artifacts resulting in false results in sequencing experiments. This issue has not been encountered with frozen tissue.

Immunostaining and Morphology

FFPE samples are preferable compared to frozen samples for immunostaining and morphology purposes. This is due to the poor or mediocre histomorphological quality when frozen tissues are used. Tissues that are frozen incorrectly can lead to the formation of vacuoles in the tissue. When both immunostaining and tissue structure analyses of the tissue are required simultaneously, FFPE samples are also better.

Native Morphology

For native morphological studies, frozen tissue samples are much more desirable compared to FFPE samples. Frozen specimens allow the closest to physiological native morphology study. Immunohistochemistry can be performed on the native form of antigen, epitope, or protein since these components in frozen tissue are not crosslinked due to formalin fixation. The results from the immunohistochemistry are also repeatable and much more reliable when performed using frozen tissue when compared to those using FFPE samples. However, it is important that studies on native morphology uses specimens where the freezing protocols are done as soon as possible as the quality of the specimen highly depends on the ischemia time. A rapid freezing time allows the PTMs and biomolecules to stay close to the living state. 

References

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

  2. FFPE vs frozen tissue samples. BioChain. Accessed 4/4/2019. https://www.biochain.com/general/ffpe-vs-frozen-tissue-samples/

  3. FFPE. Horizon. Accessed 4/4/2019. https://www.horizondiscovery.com/reference-standards/format/ffpe

  4. Smith C. FFPE or frozen? Working with human clinical samples. Biocompare. Accessed 4/4/2019. https://www.biocompare.com/Editorial-Articles/168948-FFPE-or-Frozen-Working-with-Human-Clinical-Samples/

The Various Tissue Samples Stored in a Biorepository

Introduction

A biorepository or biobank is a center that collects, processes, stores, and distributes biological materials to help support research teams and other professionals in future scientific investigations. Biorepositories help to manage and contain biospecimens from various living organisms such as animals, plants, and humans. The main purpose or function of the biorepository is to retain biospecimens and their associated data for research purposes. They will ensure and manage the quality, accessibility, disposition, and distribution of all the specimens. The biorepository has four main operations:

  • Collection – This is where the samples are recorded in the biorepository’s system. Collection is done by assigning unique barcodes to each sample. This is then scanned, additional information about the sample recorded, and transferred into the laboratory information management system (LIMS). 

  • Processing – This involves the testing process for each biospecimen that has arrived at the biorepository. The quality testing process is performed in the same way to minimalize variations that may occur due to sample handling. After testing is done, the biospecimen is prepared for storage. Storage preparation may differ depending on the biospecimen. The process prepares the specimens for long-term storage to ensure the quality of the specimen.

  • Storage – All the biospecimens are held at the storage and inventory until it is requested to be distributed. The inventory and storage system have holding boxes and freezers that fulfill the sample storage requirements. Samples must be maintained so there is minimal deterioration with time. It must also be protected from both accidental and intentional damage using back up systems and standard operating procedures (SOPs). In some cases, the specimens can be stored at room temperature as it helps to lower maintenance costs and to avoid issues such as equipment failure. 

  • Distribution – This involves retrieving of samples from the inventory. Retrieving samples from the inventory should be rapid and easy as the biorepository’s system should be able to pinpoint the location of each sample. 

Types of Biorepositories

There are various biorepositories that exist. Most biorepositories are focused on a specific disease while others help in the identification of genetic clues that may guide therapeutic development. For example:

  • The United Kingdom Biobank – This is a biobank that has a broad focus with aims to improve diagnosis, treatment, and prevention of diseases such as arthritis, stroke, diabetes, cancer, eye disorders, heart disease, depression, and dementia. In four years between 2006 to 2010, this biorepository was able to recruit half a million participants ranging from the ages of 40 to 69 years old. These participants have donated various samples such as urine, blood, and saliva for analysis. They also provided personal information and consent for follow up to help researchers understand how certain diseases develop.

  • The Alzheimer’s Disease Neuroimaging Initiative – This is a biorepository that is focused on Alzheimer’s. Their biomarker validation program uses data and samples collected from affected patients to understand more about the condition. 

  • The Autism Research Resource – This biorepository is sponsored by the state of New Jersey to understand more about families who have more than one child with autism. 

  • The Health Outreach Program for the Elderly (HOPE) – This is a biorepository focused on researching multiple diseases affecting elderly patients. It is located at Boston University. The HOPE registry performs annual follow ups with their Alzheimer’s patients. 

Types of Tissue Samples

There are various types of tissue samples stored in a biorepository. However, the availability of some biospecimens will be specific to some biorepositories. Generally, a biorepository would have tissue samples such as serum, urine, saliva, blood, tissue from different parts of the body, diseased tissues, DNA, and RNA samples. This allows the biorepository to cater to various industry researchers and meet their research needs. The tissue samples can be categorized according to disease such as :

  • Arthritis

  • Breast cancer

  • Brain cancer

  • Cardiovascular disease

  • Colon cancer

  • Cervical cancer

  • Dementia

  • Diabetes

  • Head and Neck Cancer

  • Leukemia

  • Lupus

  • Lymphoma

  • Multiple Sclerosis

  • Normal Tissue

  • And more. 

The samples can then be categorized based on their preparation such as:

  • Frozen tissue – These samples are snap-frozen when they were collected. It is then stored at low temperatures in liquid nitrogen to ensure that the RNA and proteins are preserved. The specimens can include tissues from various organs, diseases, and normal tissue from the surrounding area.

  • Formalin fixed paraffin embedded (FFPE) tissue - As the name suggests, these tissues are fixed using formalin and embedded in paraffin.

  • Human DNA and RNA – There are also various DNA samples from various diseases. The disease-free specimens are also available for control purposes. RNA samples from various tumors and normal tissue are also available. 

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

Biorepository clients can request various types of specimens from the biorepository based on their needs. They can also obtain the data associated with the sample. 

References

  1. Biobank. Wikipedia. Accessed 3/28/2019. https://en.wikipedia.org/wiki/Biobank#Biological_specimens

  2. Biorepository. Wikipedia. Accessed 3/28/2019. https://en.wikipedia.org/wiki/Biorepository

  3. Types of biorepositories. Geneticist. Accessed 3/28/2019. https://www.geneticistinc.com/blog/types-of-biorepositories

  4. Tissue procurement and biorepository. Department of Pathology & Laboratory Medicine. University of California, Irvine. Accessed 3/28/2019. http://www.pathology.uci.edu/tissue-use-committee.asp

  5. Global biorepository of human tissue samples. Reprocell. Accessed 3/28/2019. https://www.reprocell.com/human-tissue-samples-i35

Tissue Microarray's and their impact in Medicine

Introduction

The field of human molecular genetics has advanced significantly revealing many gene-based disease mechanisms in various areas of medicine. This is why the study of both diagnostic and prognostic markers are important to help translate new findings in basic science and apply it to clinical practice. The increasing use of new techniques in molecular biology has revolutionized the investigation of pathogenesis and disease progression. It is important to understand the fundamental molecular mechanisms involved in the progression of normal to malignant tissue as it may lead to improved detection and treatment for cancer. Studies have found multiple novel markers which are mostly at the gene level. Authentication of these markers via standard techniques can be time-consuming, costly, and labor intensive. Tissue microarray is a technique used in the field of pathology to overcome these significant issues. Designed as a high-throughput molecular biology technique, it allows the simultaneous assessment of expression in interesting candidate disease-related genes on hundreds of tissue samples. It also allows molecular profiling at the DNA, RNA, and protein level. Tissue microarray is a technique that enables pathologists to perform large-scale analyses using RNA in situ hybridization, fluorescence in situ hybridization, and immunohistochemistry at lower costs and faster duration. 

History

The technique was first reported by Battifora who described a method where he wrapped 1millimeter rods of tissues in small intestine sheets which were subsequently embedded in a paraffin block. This was then cut and examined. In 1987, the array format was conceived by Wan and colleagues. While there is a significant advantage of being able to simultaneously examine multiple specimens under the same conditions, there is the inability to identify individual rods resulting in unmeaningful interpretation. However, in 1998, Kononen et al were able to address this issue by inventing a device that could rapidly and accurately construct tissue microarrays in a way that is accessible to most pathology labs. This subsequently led to a dramatic increase in the popularity of the tissue microarray technique. 

Tissue Microarray Technique

The tissue microarray technique is useful in the organization of minute amounts of biological samples on a solid support. They are composite paraffin blocks that are constructed through the extraction of cylindrical tissue core “biopsies” from other paraffin donor blocks which are then re-embedded into a single microarray block. The donor blocks are first retrieved and sectioned to produce standard microscopic slides which are then stained with hematoxylin and eosin. Next, it is examined by a pathologist to mark the area of interest. The samples are then arrayed. 

A tissue microarray instrument is used to obtain a tissue core from the donor block. It is then placed in the recipient block, an empty paraffin block. The core is then placed at a specific coordinate which is recorded on a spreadsheet. The sampling process is repeated using different donor blocks until they are all placed into one recipient block. This produces the final tissue microarray block. The tissue microarray is then sectioned using a microtome to generate tissue microarray slides for molecular and immunohistochemical analyses. This method allows an entire cohort of cases to be analyzed by staining one or two master array slides. 

Advantages and Applications of Tissue Microarrays

There are many advantages and applications of tissue microarrays compared to other standard techniques. This includes:

  • The amplification of a scarce resource

  • Experimental uniformity

  • Simultaneous analysis of large numbers of specimens

  • Conservation of valuable tissue as the technique does not destroy the original block

  • Decreases the time, cost, and assay volume

  • Facilitates the standardization of fluorescence in situ hybridization, immunohistochemical, and other molecular assays allowing results to be reproducible between laboratories. 

  • Can be used for internal quality control and optimization of diagnostic reagents.

  • Facilitates the translation of new molecular discoveries to clinical applications.

  • Clinical validation of newly identified genes in histopathological specimens. 

  • Screening for presence or absence of novel markers in multi tumor arrays. 

  • Assessment of molecular and morphological changes in tumor progression microarrays.

  • Assessment of prognosis or patient outcome in prognostic arrays. 

Tissue Heterogeneity and Tissue Microarray Disadvantages

One of the commonest criticisms of the tissue microarray technique is that the sampled cores may not represent the entire tumor especially in heterogeneous cancers such as Hodgkin lymphoma and prostate adenocarcinoma. However, there are many groups that have proven excellent concordance between the spots and whole sections in immunohistochemical studies involving multiple tumor types. Another minor disadvantage of the technique is the absence of some core sections on the immunostained slide. While this may occur, the statistical analysis of many other cases eliminates the effect of a single data point in the conclusion. 

Conclusion

The tissue microarray technique is practical and effective for high throughput molecular analysis of tissues in the identification of new diagnostic and prognostic markers in human cancers. With varying degrees of use and range of potential applications, this technique is anticipated to become a widely used tool for various types of tissue-based research. It is believed that this technique will lead to a significant acceleration in the process of translating basic research findings into clinical applications. 

References:

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

  2. Tissue Microarray: An Evolving Diagnostic and Research Tool. Accessed 3/13/2019. https://www.geneticistinc.com/blog/tissue-microarray-an-evolving-diagnostic-and-research-tool

  3. Galdiero, Maria, et al. “Potential Involvement of Neutrophils in Human Thyroid Cancer.” PLoS One, vol. 13, no. 6, Public Library of Science, June 2018, p. e0199740.

What is Pathology?

Introduction

The term pathology can be translated into the study (logos) of suffering (pathos). It is a discipline that is devoted to studying both structural and functional changes while bridging clinical practice and basic science. Through morphologic, molecular, immunologic, and microbiologic techniques, pathology tries to explain why some signs and symptoms occur along with providing a basic understanding of rational therapy and clinical care. Traditionally, pathology can be divided into general and systemic (special) pathology). General pathology involves the basic reactions of cells and tissues to stimuli that lead to disease. Systemic pathology revolves around the specific responses of certain tissues to well-defined stimuli. The core of pathology can be formed by aspects of a disease. This includes:

  • Etiology – the cause of disease. Generally, it can be divided into intrinsic (genetic) or acquired. 

  • Pathogenesis – the mechanism of disease. It refers to the sequence of events caused by the etiologic agent or stimulus. Pathogenesis does not only involve the study of the etiology but also the various events that lead to the development and progression of the disease.

  • Morphologic changes – the structural alterations. These alterations seen in tissues or cells are characteristic or diagnostic. Diagnostic pathology helps identify the nature and progression of the disease through the study of changes and chemical alterations. 

  • Clinical significance – the functional consequences of the morphologic changes. This results in the signs and symptoms experienced in the disease. It also influences the course and prognosis of the illness. 

Surgical Pathology

Surgical pathology is one of the most time consuming and significant branch of pathology. It involves the examination of tissues to achieve a diagnosis. Specimens that are surgically removed such as core biopsies from suspected cancers, skin biopsies, and specimens resected in the operating room are subjected to examination or analysis. The molecular properties of these specimens are evaluated through immunohistochemistry and various other tests. Tissue sections are processed for histological examination using either frozen section or chemical fixation. A frozen section involves freezing of the tissue to generate thin slices of specimens that are mounted on glass slides. The slides are then stained with antibodies or chemicals before viewed under a microscope. The pathologist also performs autopsies to evaluate diseases, injuries, and determine the cause of death. 

Cytopathology

Cytopathology is a branch studying and diagnosing diseases on the cellular level. It is often used to help in the diagnosis of cancer, infectious diseases, and inflammatory conditions. It can be performed on specimens that contain tissue fragments or free cells. These specimens can be collected through procedures such as fine needle aspiration, removed through abrasion, or spontaneous exfoliation. One good application of cytopathology is the screening tool (pap smear) used in the detection of precancerous cervical lesions. 

Molecular Pathology

This is a fairly recent branch of pathology that has made great progress in the past decade. It involves the diagnosis and study of diseases via molecular examination of tissues, organs, and bodily fluids. Diseases such as cancer have been found to be due to alterations or mutations of the genetic code. The identification of these changes helps clinicians to choose the best treatment for the individual. This has resulted in personalized medicine where molecular analysis is used to predict responses to different therapies based on each individual’s genetic component. Molecular pathology also includes studying the development of genetic and molecular approaches to both the diagnosis and classification of tumors. It has also allowed experts to design and authenticate biomarkers to assess the prognosis and likelihood of disease in individuals. Molecular assays have high levels of sensitivity allowing the detection of small tumors that are usually undetectable through other means. This will lead to earlier diagnosis, improved care, and better prognosis for patients. 

Laboratories and Staff

There are different pathology labs available. This includes:

  • Hospital labs – that support clinical services that are provided by the hospital. Most pathology labs at hospitals usually include cytopathology, surgical pathology, autopsy, and clinical pathology. 

  • Reference labs – are private and commercial labs that provide special laboratory testing. These tests are generally referred from hospitals and other patient care facilities. 

  • Public health labs – are managed by the local health departments or state to protect the general population from potential health threats. They perform tests to monitor diseases in the community. 

All laboratories require trained staff such as:

  • Pathologists – are physicians who specialize in the diagnosis of disease. They may be general pathologists or have a subspecialty such as hematopathology, cytopathology, nephropathology, dermatopathology, etcetera. They ensure accurate and timely reporting of tests while serving as a resource that aids in result interpretation.

  • Pathologists’ assistants – are individuals who assist with some responsibilities of the pathologist. This includes gross description and dissection. Pathologists’ assistants work closely with pathologists and can also assist in intraoperative assessment and tissue selection.

  • Cytotechnologists – are individuals who help in screening specimens composed of cells instead of whole sections. They screen specimens and refer abnormal cells to pathologists for further review. 

  • Histotechnologists – manage tissue processing in the lab. They also make slides (fixing, embedding, sectioning, staining) that will be evaluated by the pathologists. 

  • Medical laboratory technician – perform tests and analysis on specimens to determine the absence or presence of disease.

  • Phlebotomists – are individuals trained to draw blood from a patient for various purposes such as research, testing, donations, or transfusions. 

References:

  1. Introduction to the pathology laboratory. Healio Learn Genomics. Accessed 3/8/2019. https://www.healio.com/hematology-oncology/learn-genomics/pathology-assessment-of-tumor-tissue/introduction-to-the-pathology-laboratory

  2. What is pathology? McGill Department of Pathology. Accessed 3/8/2019. https://www.mcgill.ca/pathology/about/definition

  3. Cellular Adaptations, cell injury, and cell death. Robbins and Cotran Pathologic Basis of Disease; page 6: 7th Edition, 2005. 

Importance of Red & Yellow Bone Marrow

Introduction

Bone marrow refers to the semi-solid tissue located within cancellous or spongy portions of bones. In mammals, bone marrow is the main site where new blood cells are produced. It consists of marrow adipose tissue, hematopoietic cells, and supportive stromal cells. In adults, the bone marrow can be found in the ribs, sternum, vertebrae, and pelvic bones. On average, it constitutes about 4 percent of the total body mass. It produces an estimated 500 billion blood cells daily. These cells join the systemic circulation through the permeable vasculature sinusoids in the medullary cavity. All hematopoietic cells are created in the bone marrow. However, some cells will need to migrate to other parts of the body to complete the maturation process. The bone marrow composition is dynamic as it shifts with age due to various systemic factors. Bone marrow can be differentiated into red or yellow marrow. Based on the prevalence of hematopoietic vs fat cells. For example, a newborn baby exclusively has red marrow that are hematopoietic cells that gradually convert to become yellow marrow with age. In situations where chronic hypoxia occurs, yellow marrow can convert to red marrow to help increase the production of blood cells.

Red and Yellow Bone Marrow

The red bone marrow refers to the red colored tissue that contains the reticular networks that are crucial in the production and development of red blood cells, white blood cells, and platelets. The red color can be attributed to the hemoglobin. It can be found in the flat and long bones such as hip bones, vertebrae, ribs, shoulder blades, and skull. The red bone marrow has an important role in the production of red blood cells, white blood cells, and platelets. Red bone marrow is also known as medulla osium rubra.

The yellow bone marrow is yellow colored tissue that can be found in the hollow parts of compact bones. The yellowish color can be attributed to the presence of carotenoid in the fat droplets. They function in the production of blood cells in life-threatening situations and the storage of fat. The fat in the yellow bone marrow is also the body’s last source when there is extreme hunger. Yellow bone marrow is also known as medulla osium flava.

Hematopoietic Components and Stroma

The main component in the brain marrow are the progenitor cells that mature into lymphoid and blood cells. The marrow contains hematopoietic stem cells that result in three classes of blood cells:

  • Red blood cells (erythrocytes)

  • White blood cells (leukocytes)

  • Platelets (thrombocytes)

The stroma consists of tissue that is not primarily involved in the main function of hematopoiesis. Stromal cells provide a microenvironment that influences the differentiation and function of hematopoietic cells. Cells that are found in the stroma include:

  • Macrophages

  • Fibroblasts

  • Osteoblasts

  • Osteoclasts

  • Adipocytes

  • Endothelial cells

Function

  1. Mesenchymal Stem Cells – mesenchymal stem cells or marrow stromal cells are multipotent stem cells that can differentiate into various cells such as chondrocytes, osteoblasts, marrow adipocytes, myocytes, and beta-pancreatic islet cells.

  2. Lymphatic Role – the red bone marrow is a vital part of the lymphatic system as it is the main organ that generates lymphocytes from immature progenitor cells. The thymus and bone marrow contain primary lymphoid tissue that is involved in the selection and production of lymphocytes. The bone marrow also has a valve-like function that prevents lymphatic fluid to flow back into the lymphatic system.

  3. Bone Marrow Barrier – the blood vessels make up the bone marrow barrier which functions to prevent immature cells from leaving the marrow. This can be attributed to mature blood cells that contain membrane proteins that are required to pass through the blood vessels. Since hematopoietic stem cells can cross the bone marrow barrier, it can be harvested from blood.

  4. Compartmentalization – compartmentalization can be seen in the bone marrow as specific cell types aggregate in certain areas. For example, macrophages, erythrocytes, and their precursors usually gather around blood vessels while granulocytes gather at the bone marrow borders.

Transplant

A bone marrow transplant can be used to replace diseased and nonfunctioning bone marrow such as in diseases like sickle cell anemia, aplastic anemia, and leukemia. It can also be used to replace bone marrow function after chemotherapy or radiation. The transplant will lead to the regeneration of a new immune system that helps fight the existing conditions. Some examples of transplant types are a syngeneic transplant, autologous transplant, allogeneic transplant, and haploidentical transplant. To determine a match for bone marrow, the person will go through an HLA-typing test. Once matched, several other tests should be performed. This includes chest x-ray, computed tomography scans, pulmonary function tests, heart function tests, skeletal survey, and bone marrow biopsy.

References:

  1. Difference between red and yellow bone marrow. They Differ. Accessed 3/1/2019. https://theydiffer.com/difference-between-red-and-yellow-bone-marrow/

  2. Bone marrow. Wikipedia. Accessed 3/1/2019. https://en.wikipedia.org/wiki/Bone_marrow

  3. Nichols H. All you need to know about bone marrow. Medical News Today. Accessed 3/1/2019. https://www.medicalnewstoday.com/articles/285666.php

Different FFPE Methods and Their Impact in Medicine

Introduction

Formalin-fixed paraffin embedded (FFPE) is a method used globally for the preservation of tissue. However, the steps for FFPE has not been standardized. One particular study found 15 preanalytical factors for the processing of FFPE tissue that affects the efficacy of immunohistochemistry. The different processing regimens, extraction techniques, patient-related factors, antigen retrieval techniques, and other preanalytical variables have led to varying levels of success with the molecular analysis of these biospecimens.

Acceptable Conditions of FFPE for DNA Analysis

1. Prefixation:


Current literature has found that there are several factors that can affect the DNA analysis of FFPE specimens. It includes:

• Cold ischemia – the time between removal of biospecimen from host and preservation. The DNA extracted from biospecimens that has a cold ischemia time of 1 hour was found to have reduced fluorescent in situ hybridization (FISH) signals. However, a cold ischemia time of 24 hours was not observed to have altered polymerase chain reaction (PCR) amplification success rates. 


• Postmortem interval (PMI) – the time elapsed since the death of the donor. A PMI of 48 hours was also found to reduce FISH signals when compared to a PMI of 1 hour. However, PCR remained unaffected despite a PMI of 4 to 8 days. 


• Decalcification method – multiple reports showed that decalcification using ethylenediaminetetraacetic acid (EDTA) was better than acid-based methods as it allows superior determination of gain and loss of sequences for comparative genomic hybridization, amplification of longer PCR products, stronger FISH signals, and reduced background staining. When acid-based methods were compared, decalcification using 5% formic acid for 12 to 18 hours resulted in FISH signals while the signal was abolished when decalcification was performed using a 10% formic acid solution for 7 to 10 days. 


• Specimen size – researchers have found that PCR success rates were highest when DNA extraction was performed on specimens ranging from 3 to 10mm in diameter compared to other smaller or larger specimens. 


2. Fixation:


Some preanalytical factors that involve the fixation of the biospecimen have also been reported. These factors include:


• Time of fixation – most agree that fixation of fewer than 72 hours in formalin was superior compared to longer durations as it improved yield, DNA integrity, PCR, in situ hybridization (ISH), and single nucleotide polymorphism detection assay performance. However, long term fixation has no consequence on DNA yield, success rates for PCR amplification of nuclear DNA, or mitochondrial and viral DNA amplification. 


• Temperature of fixation – studies have found that fixation performed at ambient temperatures or higher led to reductions in DNA integrity and yield. PCR success also drops at elevated temperatures. However, it is still unclear if fixation should be performed at 4⁰C or ambient temperatures as fixation at 4⁰C increases PCR success and high molecular weight DNA yield while fixation at ambient temperature increases the efficiency of amplification. 


• Buffered or unbuffered formalin – current literature concluded that DNA that was extracted from neutral buffered formalin biospecimens led to greater yields and higher success rates for in situ hybridization (ISH) and genotype determination. PCR success rates were unclear if it was equivalent or superior for neutral buffered formalin biospecimens when compared to unbuffered formalin. 


• Formalin penetration of tissue expedited by ultrasound or microwave irradiation – there is little information available regarding the impact of acceleration methods on DNA. However, some evidence suggests that both microwave or ultrasound accelerated fixation may improve PCR success rates and high molecular weight DNA yield from FFPE tissue. 


3. Processing and Storage:


Some factors that may influence DNA endpoints have not been addressed. Evidence is limited to one study that highlighted the importance of using a paraffin mixture with beeswax versus pure paraffin. These include:

• Dehydration


• Clearing


• Paraffin reagents


Many studies have reported the benefit of paraffin blocks where it can be stored for many years at room temperature with only minor effects on DNA analysis. However, there are also other studies that show the decrease of amplifiable DNA length and whole genome amplified fragments despite using optimized DNA extraction procedures. One study that investigated the effect of storage duration of FFPE sections on downstream DNA analysis found that storage of FFPE sections for 10 years was detrimental to PCR success rates. Literature has also suggested that various paraffin block sizes or even fractions of a standard 5μm section can be successfully analyzed, better PCR success rates and higher yields from DNA extraction were obtained from FFPE sections. More intense FISH signals were also obtained from analysis of sections compared to isolated nuclei.


Conclusion

Based on current literature, when FFPE specimens will be used for DNA analysis, cold ischemia times and PMI should be limited to 1 hour and 48 hours respectively. FFPE biospecimens should range from 3 to 10mm3, use EDTA for decalcification, fixation in neutral buffered formalin for less than 72 hours at 4⁰C or ambient temperature, and embedded in pure paraffin. There is also some evidence that suggests ultrasound or microwave accelerated fixed specimens are suitable for DNA analysis. Although FFPE blocks can be stored at room temperature for many years, it is important for professionals to note that amplifiable gene fragment length may decrease with time. Therefore, the extraction of DNA from FFPE sections, isolated nuclei, or cores that have been stored for more than 10 years should be avoided.

References:

1. Bass BP, Engel KB, Greytak SR, Moore HM. A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Archives of Pathology & Laboratory Medicine. 2014; 138(11): 1520-1530. Accessed 2/24/2019. https://www.archivesofpathology.org/doi/full/10.5858/arpa.2013-0691-RA


2. TP Lau. Assessment of telomere length in archived formalin fixed paraffinized human tissue is confounded by chronological age and storage duration. PLoS One. 2016; 11(9): p.e0161720.

What is a Biobank?

Introduction

A biobank is a biorepository that stores biospecimens that are useful for research purposes. They can also be known as tissue banks or bioresources. The biobanks have become a crucial resource for medical research since the late 1990s as they support various researches such as those in the field of personalized medicine and genomics. Biobanks allow researchers to access data that represents a large number of people. The biospecimens and data acquired can be used by multiple researchers for cross purpose research studies. This is vital as many researchers face difficulties in acquiring sufficient tissue samples before the existence of biobanks. Some biobanks store and collect samples from a specific population or from those with certain diseases. Biobanks can be found in places such as universities, hospitals, pharmaceutical companies, charities, and more. they are also required to follow the local legislation and provisions.

A Brief History

Before the existence of biobanks, scientists collected all the biospecimens required for their experiments. However, they came to realize that while genetics may contribute to many diseases, very few diseases can be attributed to a single defective gene. This means that most diseases are due to multiple genetic factors. This led them to start collecting more genetic information whenever they could. At the same time, the advancement of technology also enabled the wide sharing of information. This led to the conclusion where access to data collected for any genetics work could be helpful in other genetic research. Scientists then started to store genetic data in single places to allow community sharing. As a result, many single nucleotide polymorphisms were discovered. While the new practice allowed the collection and sharing of genotype data, there was no system in place that gathers the related phenotype data. Genotype data can be obtained from biospecimens while phenotype data is obtained from interviewing, examination, and assessment of the donor. In cases where phenotype data were available, there were ethical issues about the extent of sharing this information. The biobank was then developed to store both genotypic and phenotypic data, allowing access to researchers who may need it. In the United States, researchers store about 270 million specimens in 2008 with an average new sample collection rate of 20 million specimens annually. However, there are some issues regarding the ethical, social, and legal issues of biobanks which are constantly debated and improved.

Types of Biobanks

Biobanks usually collect human biospecimens. However, there are also biobanks who have a collection of animals, plants, and other nonhuman specimens. Cryogenic storage facilities are usually available for storage of biospecimens. These facilities can range from individual refrigerators to warehouses that are maintained by universities, nonprofit organizations, hospitals, and pharmaceutical companies. Biobanks can be categorized based on design or purpose. Biobanks that are disease-oriented are generally affiliated with a hospital where samples are collected to represent a variety of diseases. Biobanks that are population-based does not usually have an affiliation with a hospital as they obtain samples from large groups of different individuals. Tissue banks function to harvest and store tissues for research and transplantation. Virtual biobanks integrate their epidemiological cohorts into the general population and allow biospecimens to meet local regulations. Population banks store biomaterial, environmental data, lifestyle information, and clinical data.

Biospecimens

Biospecimen types that are available include organ tissue, blood, saliva, urine, skin cells, and other tissues or fluids taken from the body. The specimens are kept in the appropriate condition until a researcher requires it for an experiment, test, or analysis. The commonest test performed is a genome-wide association study.

Storage

The samples are maintained to prevent deterioration and protected from both accidental and intentional damage. The sample is registered in the computer-based system. The physical location of the specific sample is also noted to enable the specimen to be easily located when required. Samples are de-identified to ensure donor privacy and allow blinding of researchers to the analysis. Room temperature storage may be used in some cases as it helps with cost efficiency and to avoid issues such as freezer failure.

Ethics

Some ethical issues regarding biobanking are the ownership of samples, ownership of derived data, right to privacy for donors, the extent of donor consent, and the extent to which the donor can share regarding the return of research results.

Governance

Ethical oversight from an independent reviewer is required to ensure that all parties are protected. In medical research, this is often done by the institutional review board which functions to enforce the standards that are set by the local government. While different countries may have different laws, the regulations used are often modeled based on internationally proposed biobank governance recommendations. Most biobanks adapt to the broad guidelines that are internationally accepted. Examples of organizations that have helped in creating the biobanking guidelines include:

Council for International Organizations of Medical Sciences

World Medical Association

World Health Organization

Human Genome Organization

United Nations Educational, Scientific, and Cultural Organization

References:

Biobank. Wikipedia. Accessed 2/21/2019. https://en.wikipedia.org/wiki/Biobank

Biobank Introduction. UK Clinical Research Collaboration: Tissue Directory and Coordination Centre. Accessed 2/21/2019. https://www.biobankinguk.org/introduction/

Basics. EGAN Patients Network for Medical Research and Health. Accessed 2/21/2019. https://egan.eu/biomedical-research/medical-data-and-biobanks/basics/

Immunohistochemistry Protocol

Immunohistochemistry is a technique that is commonly used in the localization and monitoring of proteins in tissue sections. This technique uses antibodies to analyze and detect proteins while maintaining the structure, cellular characteristics, and composition of native tissue. It is useful when used to assess and monitor treatment or progression of diseases. Also, the information that can be gathered through immunohistochemistry combined with microscopy helps provide an overall picture that allows researchers to make sense of the data they have obtained through other methods. In this technique, chemical fixation helps lock the molecular interactions in the cells. The tissue samples can be frozen, or paraffin-embedded before it is sectioned and mounted on slides for analysis. Depending on the sample of interest, the steps taken for collection, preservation, and fixation of the biospecimen can vary. 

Through specific antibody binding, immunohistochemistry helps to identify the pattern of protein expression. This bringing between antibody and epitope enables detection of particular amino acid sequences that are found within a protein. These antibodies can also be used to detect post-translational modifications. The tissue sample should be preserved and hardened to retain both form and structure so it can be sectioned. One crucial choice is deciding if immunohistochemistry should be carried out on fresh frozen samples or formalin fixed paraffin embedded (FFPE) samples. Immunohistochemistry on FFPE samples results in a superior cell or tissue morphology. However, one disadvantage would be the potential compromise of antigenicity by the fixation that is required. The antigenic site can be masked by the protein cross-links formed through formalin fixation. Antigen unmasking can be performed using enzymatic digestion (by pepsin, trypsin, or another protease) or heat (water bath, microwave, pressure cooker) with specific buffers (sodium citrate or ethylenediamine triacetic acid (EDTA)). 


Immunohistochemistry Protocol (for Formalin-Fixed Paraffin-Embedded Samples)

The basic steps include:

  • Fixation and embedding of the tissue

  • Cutting and mounting

  • Deparaffinization and rehydration

  • Antigen retrieval

  • Immunohistochemical staining

  • Counterstaining

  • Dehydration and stabilization

  • Examination of the staining 

A) Reagents and Solutions

  • Xylene

  • Ethanol – anhydrous denatured, histological grade (95% and 100%)

  • Hematoxylin

  • Wash buffer (10X Tris Buffered Saline with Tween (TBST)) – Prepare 1 liter of 1X TBST by adding 100ml 10 TBST and 900ml dH2O and mix

  • Antibody diluent options (SignalStain Antibody Diluent #8112, TBST 5% normal goat serum #5425, PBST 5% normal goat serum #5425)

  • Antigen unmasking options (Citrate: 10mM sodium citrate buffer, EDTA: 1mM EDTA, TE: 10mM Tris / 1mM EDTA, Pepsin)

  • 3% hydrogen peroxide

  • Blocking solution (TBST 5% normal goat serum)

  • Detection system (SignalStain Boost IHC Detection Reagents – Mouse #8125, Rabbit #8114)

  • Substrate (Signal Strain DAB Substrate Kit #8059)


B) Sample Preparation

  • Put the paraffin-embedded tissue in a mold along with a small amount of liquid paraffin. Cool momentarily to immobilize the tissue. Put the base of a cassette on the mold filling it with liquid paraffin. Cool. 

  • Cut the sample into thin sections (4 to 6μm) using a microtome and float the cut sections in a water bath. 

  • Mount the cut sections on charged slides and allow it to dry overnight. Charged slides are used as it helps the sections to adhere to the slide. 

C) Deparaffinization and Rehydration

Paraffin wax must first be removed from the sample followed by rehydration before antibody staining can be performed. It is important not to let the slides dry as it can result in inconsistent staining.

  • Remove the paraffin wax by placing the sections in 3 containers with xylene for about 5 minutes. Remember to use fresh xylene to avoid incomplete deparaffinization that can result in inconsistent staining. 

  • Rehydrate by placing the sections in 2 containers with 100% ethanol for 10 minutes. 

  • Place the sections in 2 containers that have 95% ethanol for 10 minutes.

  • Complete the rehydration process by washing the sections twice in dH2O for 5 minutes. 

D) Unmasking Antigens

Remove the cross-links formed during formalin fixation before performing antibody staining.

  • If using citrate – bring the slides to a boil in 10mM sodium citrate buffer at pH 6.0. Maintain it just below the boiling temperature for 10 minutes. Then, cool the slides on a bench top for approximately 30 minutes. 

  • If using EDTA – bring the slides to a boil in 1 mM EDTA at pH 8.0. Continue at a sub-boiling temperature for 15 minutes. The slides do not need to be cooled.

  • For TE – bring the slides to a boil in 10mM Tris and 1 mM EDTA at pH 9.0. Follow with a sub-boiling temperature for 18 minutes and cool for 30 minutes at room temperature. 

  • If using pepsin – digest at 37⁰C for 10 minutes.

E) Staining

The following protocol is for chromogenic staining. However, immunofluorescent staining can also be an option. 

  • First, wash the sections three times in dH2O for 5 minutes every time. 

  • Place the sections in 3% hydrogen peroxide for 10 minutes to stop endogenous peroxidase activity as this can cause high background staining. 

  • Again, wash the sections three times in dH2O for 5 minutes each. 

  • Then wash it in wash buffer for 5 minutes.

  • Taking care not to touch the sample, draw a large circle around the sample using a hydrophobic pen to create a hydrophobic boundary. This allows a smaller volume of antibody to be utilized while also allowing the staining using different antibodies on one slide.

  • Use 100 – 400 μl of blocking solution to block the sections in a humidified chamber for 1 hour at room temperature to avoid non-specific binding of the antibody to the tissues. 

  • Remove the blocking solution. Add 100 – 400 μl of primary antibody that has been diluted in the antibody diluent to each section. Incubate at 4⁰C overnight in a humidified chamber. Additional optimization may be necessary for shorter incubations.

  • Equilibrate the SignalStain Boost Detection Reagent until it reaches room temperature. 

  • Remove the antibody solution. Then, wash the sections in a wash buffer 3 times for 5 minutes each time.

  • Cover the sections with 1 to 3 drops of SignalStain Boost Detection Reagent and incubate these for 30 minutes in a humidified chamber at room temperature. 

  • Again, wash the sections 3 times in wash buffer for 5 minutes every time.

  • Add a drop of SignalStain DAB Chromogen Concentrate to 1 ml of SignalStain DAB Diluent. Mix before use.

  • Apply 100 – 400 μl of SignalStain DAB to every section and observe closely. Acceptable staining intensity is achieved in 1 to 10 minutes.  

  • Immerse the slides in dH2O.

  • An additional optional step is to counterstain the sections using hematoxylin. This will result in the staining of the cell nuclei and provide a contrast to the DAB chromogen to help with visualization of the tissue morphology.

  • Rewash the sections in dH2O twice for 5 minutes each. 

F) Dehydration and Mounting of Slides

SignalStain DAB chromogen is compatible with both nonaqueous and aqueous mounting mediums. If a nonaqueous medium is chosen, the sections have to be dehydrated again before mounting. 

  • Place the sections in 2 containers containing 95% ethanol for 10 seconds.

  • Then, place the sections in 2 containers with 100% ethanol for 10 seconds.

  • Next, place the sections in 2 containers containing xylene for 10 seconds. 

  • Mount the sections using coverslips with a mounting medium while avoiding the introduction of air bubbles. 

  • Allow the mounting medium to set.

  • View the slides on a microscope. 


References:

Crosby K, Simendinger J, Grange C, Ferrante M, Bernier T, Standen C. Immunohistochemistry protocol for paraffin-embedded tissue sections. Cell Signaling Technology. Accessed 1/18/2019. https://www.jove.com/video/5064/immunohistochemistry-protocol-for-paraffin-embedded-tissue-sections

What Does a Geneticist Do?

What Does a Geneticist Do?

A geneticist is a medical professional that works in the field of genetics. A geneticist can specialize in agriculture, biomedicine, forensics, archaeology and bioinformatics among others. A geneticist will perform tasks such as planning and conducting research. They also keep notes that record their methodology, procedures and results while results are analyzed via mathematical and statistical methods.

What is Cryosectioning Protocol

Cryosectioning Introduction

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

Reagents

Some of the reagents used in a cryosectioning protocol are:

·         Fixative (such as formaldehyde)

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

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

·         Fresh tissue sample

Cryosectioning Equipment

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

·         Brush (camel hair)

·         Container for storage of tissue sample

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

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

·         Plastic or metal tissue mold

·         Moistened tissues

cryosection Procedure

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

A)      Cryostat preparation

i)                    Use proper cleaning agents and clean the cryostat.

ii)                   Insert a new sterile blade for the cryostat.

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

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

B)      Cryosection preparation

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

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

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

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

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

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

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

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

Cryosection Troubleshooting

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

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

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

References

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

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


What is a Frozen Section?

What is a Frozen Section?


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


The Principle of Frozen Section

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

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

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

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

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

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

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

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

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

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


Cryostat Sectioning

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

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

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

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

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

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

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

  • Load the cutting knife at the proper alignment.

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

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

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


Staining


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


Conclusion


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

References


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





What is a Biopsy?

Biopsy 101

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


Why is a Biopsy Performed?


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


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


Types of Biopsies


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


Some examples of biopsy procedures include

Diagram of Biopsy.png

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


Image Guided Biopsy


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


Risks of Biopsy


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


For surgical biopsy, risks include


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


Biopsy Results


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


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


Who Performs Biopsies?


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


References:


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










Biobanking of Fresh Frozen Tissue from Clinical Surgical Specimens

Fresh Frozen Tissue and Clinical Surgical Specimens


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

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

Tissue Sample Collection and Biobanking


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


1) Surgical Theater

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

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

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

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

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

2) Pathology Department

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

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

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

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

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

3) Biobank

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

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

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

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


Conclusion

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



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


Quality Management in International Biobanking

Introduction to International Biobanking


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

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


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

T. Peakman:

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

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

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

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

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

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


P. Watson and L. Matske:

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

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

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

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


H. Moore:

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

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

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

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

              
A. Abayomi:

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

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

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


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


Conclusion


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


Reference


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