Red vs Yellow Bone Marrow in Biology


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


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

•    Macrophages 

•    Adipocytes

•    Fibroblasts

•    Osteoblasts

•    Endothelial cells

•    Osteoclasts

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

Red vs Yellow Bone Marrow

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

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

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

Clinical Significance

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

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

An Inside Look at The Duty of a Geneticist


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


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

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

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

Workplace and Salary

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

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

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

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

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

•    Counseling patients who have risks or predispositions to genetic mutations

•    Counseling patients who have abnormal test results and screenings

•    Help to determine the best course of action 

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

•    Assisting peers and colleagues with research endeavors

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

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


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


1)    Geneticist. Wikipedia. Accessed 7/2/2019.

2)    What is a geneticist? Environmental Science. Accessed 7/2/2019.

3)    Geneticists. Genes in Life. Accessed 7/2/2019.

Why Use Tissue Microarray's in Pathology?


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

•    Complexity of procedures

•    Limited availability of diagnostic reagents

•    Limited patient size

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

•    Reduction in the workload 

•    Reduction in assay volume

•    Reduction in the number of slides

•    Experimental uniformity

•    Compatibility with automated staining systems

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


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

Why TMA?

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

For example:

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

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

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

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

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

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

TMA in Research

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

Possible Limitations

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


1)    Tissue microarray. Wikipedia. Accessed 6/25/2019.

2)    Tissue microarray. Novus Biologicals. Accessed 6/25/2019.

3)    Why use tissue microarrays. Yale. Accessed 6/25/2019.

Tissue Freezing Techniques


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

Frozen Section Technique

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

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

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

Examples of Freezing Methods

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

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

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

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

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

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

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

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


1)    Emge DJ. Tissue freezing methods for cryostat sectioning. Accessed 6/20/2019.

2)    Peters S. A method for preparation of frozen sections. IHC World. Accessed 6/20/2019.

3)    Freezing tissues for cryosectioning. UAB Research. Accessed 6/20/2019.

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

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

The Effects of Cell Preservation


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

•    Personalized medicine

•    Diagnostic development

•    Stem cell research

•    Discovery science

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

What is Cryopreservation?

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

Physical Effects of Cryopreservation

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

Molecular Effects of Cryopreservation

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

•    Uncoupling of biochemical pathways

•    The decrease in metabolic activity and energy

•    Activation of stress response pathways

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

•    Free radical production

•    Metabolic uncoupling

•    Dysregulation of cellular ion balance

•    Cell membrane fluidity and structure changes

•    Cryoprotective agent exposure

•    Osmotic fluxes

•    Calcium release from intracellular stores

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

Effects of Sample Storage Conditions

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


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


1)    Cryopreservation. Wikipedia. Accessed 6/17/2019.

2)    Baust JM. Biopreservation: The impact of freezing and cold storage on sample quality. Eppendorf. 2016. Accessed 6/17/2019.

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

FFPE Tissue and it's Use in Biobanks


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


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

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

  • Normal tissue for FFPE

  • Diseased tissue for FFPE

  • Normal tissue for freezing 

  • Diseased tissue for freezing

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

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

FFPE Tissue Applications

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

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

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

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

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

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


  1. What is FFPE tissue and what are its uses. BioChain. Accessed 6/14/2019.

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

The Importance of Tissue Procurement


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

Organ / Tissue Procurement Procedure

This describes the steps for organ procurement:

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

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

  • Brain death is declared in accordance with the law. 

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

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

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

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

  • Once the surgical teams have arrived, surgery begins.

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

Organ Transplantation

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

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

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

  • Surgery is performed.

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

Tissue Procurement Processing and Preservation

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

Tissue Procurement Objectives

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

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

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

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

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

  • Providing pathological review and histological staining of biospecimens.

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

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


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


  1. What is human body tissue? – Definition, types, examples. Accessed 6/6/2019.

  2. Understanding the organ/tissue procurement process. National Kidney Foundation. Accessed 6/6/2019.

  3. Why human tissue procurement is so important. Geneticist. Accessed 6/6/2019.

  4. Tissue procurement. Cancer Institute. Accessed 6/6/2019.

Various Tissue Samples Stored in Biorepositories


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


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

Biobank Taxonomy and Activities

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

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

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

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

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

  • Genetic or DNA / RNA

  • Project driven

Types of Tissue Samples

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

  • Arthritis

  • Brain cancer

  • Breast cancer

  • Colon cancer

  • Cardiovascular disease

  • Cervical cancer

  • Diabetes

  • Dementia

  • Head and neck cancer

  • Lymphoma

  • Lupus

  • Leukemia

  • Multiple sclerosis

  • Normal tissue

  • Etcetera

It can also be categorized based on preparation:

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

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

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

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

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

Best Practices

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

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

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

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

Legalities and Ethics

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

  • Providing results to study participants

  • Responsibility to report incidental findings or individual research results

  • Ownership of biospecimens

  • Privacy of patients

  • Consent for one or all research

  • Etcetera 


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

  2. The difference between biobanks and biorepositories. Geneticist. Accessed 5/21/2019.

  3. The various tissue samples stored in a biorepository. Geneticist. Accessed 5/21/2019.

Paraffin Embedded Tissue Blocks


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.


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. 


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.


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. 


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.


  1. What is FFPE tissue and what are its uses. BioChain. Accessed 5/16/2019.

  2. Paraffin processing of tissue. Protocols Online. Accessed 5/16/2019.

What is a Liquid Biopsy?


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


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. 


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. 


  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.

  3. McDowell S. Liquid biopsies: past, present, future. American Cancer Society. Accessed 5/9/2019.

FFPE and Tissue Microarray Samples


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. 


  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.

  3. What is FFPE tissue and what are its uses. BioChain. Accessed 4/30/2019.

Human Tissue Samples Used in Biobanks


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. 


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 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. 


  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.

  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.

What is a Biorepository and What do they do?


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. 


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. 


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.


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. 


Foxman B. Human and animal subject protection, biorepositories, biosafety considerations, and professional ethics. Molecular Tools and Infectious Disease Epidemiology. 2012. Accessed 4/16/2019.

Biorepository. Wikipedia. Accessed 4/16/2019.

Why is Tissue Important?


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. 


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.


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. 


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. 


  1. Tissue (biology). Wiki. Accessed 4/11/2019.

  2. What is tissue? Why is it important? Research Advocacy Network. Accessed 4/11/2019.

  3. Cell differentiation and tissue. Scitable. Accessed 4/11/2019.

FFPE vs Frozen Tissue Samples


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. 


  1. The pros and cons of FFPE vs frozen tissue samples. Geneticist. Accessed 4/4/2019.

  2. FFPE vs frozen tissue samples. BioChain. Accessed 4/4/2019.

  3. FFPE. Horizon. Accessed 4/4/2019.

  4. Smith C. FFPE or frozen? Working with human clinical samples. Biocompare. Accessed 4/4/2019.

The Various Tissue Samples Stored in a Biorepository


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. 


  1. Biobank. Wikipedia. Accessed 3/28/2019.

  2. Biorepository. Wikipedia. Accessed 3/28/2019.

  3. Types of biorepositories. Geneticist. Accessed 3/28/2019.

  4. Tissue procurement and biorepository. Department of Pathology & Laboratory Medicine. University of California, Irvine. Accessed 3/28/2019.

  5. Global biorepository of human tissue samples. Reprocell. Accessed 3/28/2019.

Tissue Microarray's and their impact in Medicine


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. 


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. 


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. 


  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.

  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?


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 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. 


  1. Introduction to the pathology laboratory. Healio Learn Genomics. Accessed 3/8/2019.

  2. What is pathology? McGill Department of Pathology. Accessed 3/8/2019.

  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


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


  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.


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.


  1. Difference between red and yellow bone marrow. They Differ. Accessed 3/1/2019.

  2. Bone marrow. Wikipedia. Accessed 3/1/2019.

  3. Nichols H. All you need to know about bone marrow. Medical News Today. Accessed 3/1/2019.

Different FFPE Methods and Their Impact in Medicine


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.


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.


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.

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.