FFPE Samples

What is Formalin Fixed Paraffin Embedded Tissue?

Formalin fixed paraffin embedded or FFPE tissues are valuable for both therapeutic applications and research. FFPE is a specific technique used to prepare and preserve tissue specimens utilized in research, examination, diagnostics, and drug development. Tissues are first collected from both diseased and non-diseased donors. The tissue specimen is first preserved through a process called formalin fixing. This step helps to preserve the vital structures and protein within the tissue. It is then embedded into a paraffin wax block and sliced into the required slices, mounted on a microscopic slide, and examined.

 

The FFPE Process

The process starts by a specimen being selected and then excised from a donor or patient. Samples can also be obtained from other animals such as snakes, mice, or many others. After excision, the tissue is immersed for approximately eighteen to twenty-four hours in a 10% neutral buffered formalin. The tissue is then dehydrated using increasing concentrates of ethanol. Next, the tissue is embedded into paraffin to become FFPE blocks. The methods utilized are dependent on the requirements of the researcher or physician who is requesting the FFPE samples. Specifications about how the issue is cut, size, and purpose of the tissue are all important. Once the procedure is complete a certified pathologist will  evaluate the quality of the specimen.

 

Storage of FFPE Tissue

FFPE samples can be stored in hospitals, biobanks, and research centers. Storage facilities often keep records of how the tissue was collected, the preservation procedures, and demographic information (such as, but not limited too: the origin, duration, age, ethnicity, gender, and stage of disease) of the donor. The demographic information is an important factor in research and in clinical trials. FFPE samples that are properly preserved are very valuable and can be stored at room temperature for a long period of time.

 

Applications

FFPE samples are important as they are often used in:

a)       Immunohistochemistry

The sectioned FFPE specimens are mounted on a slide, bathed in a solution containing antibodies, and then stained so that they can be more clearly seen. This method is important for physicians and researchers looking for pathology in the tissue such as Alzheimer’s or cancer.

b)      Oncology

FFPE samples are vital in the field of oncology as tumor tissues have characteristic morphologies allowing researchers to look for certain proteins. These proteins are then used to help in the assessment of treatment and diagnosis.

c)       Hematology

In the study of blood and its disorders, FFPE samples are important in determining the anomalies and discovery of cures. The specimens can be used in studies related to tissue regeneration, genetics, and toxicology.

d)      Immunology

FFPE samples from a donor with autoimmune disease helps in determining the cause and development of therapy for the condition.

 

Complications or Limitations

One of the possible limitations of the fixation process using formalin is the potential denaturation of the proteins that are present in the tissue making them undetectable to antibodies. To compensate for this issue, antigen retrieval techniques were developed. The antigen retrieval technique specifically recovers DNA, RNA, and proteins from FFPE samples. For this method to work, the quality of FFPE samples are critical. There is also the issue that there is no standard procedure to be used in the preanalytical processing such as fixation and DNA isolation. This means that minor differences such as the different use of instruments, sample handling, and methodology can result in variation that affects the quality of DNA and study results. Some of the factors that have been found to affect study results from FFPE samples are:

  1. Inaccurate logging of fixation protocol
  2. Variation in fixation time
  3. Temperature during fixation
  4. Storage conditions of FFPE samples

 

Quality Control

To ensure the highest quality of FFPE samples, those who collect and store these samples should:

  1. Follow ethical and legal standards.
  2. Keep a clear and accurate record of donors.
  3. Provide information regarding the sampling and collection process
  4. Be supervised by a licensed pathologist during the collection of samples
  5. Have a complete chain of custody
  6. Work only with a carefully selected network of distributors that consistently provide high quality and accurate samples

Fresh Frozen Tissue

Fresh frozen tissues are specimens that are preserved using liquid nitrogen through a method known as “flash freezing”. These specimens are then stored in a freezer that is set at a temperature of less than -80 degrees Celsius. Fresh frozen tissue has different applications than  FFPE samples as they can be used in native morphology studies or molecular analysis as well.

 

FFPE Samples Vs. Fresh Frozen Tissue

FFPE and fresh frozen tissue have their pros and cons. They are two different types of samples that have different uses dependent on the requirements of the research or clinical study. 

  1. FFPE blocks are very hard and can be easily stored at room temperature for decades without the need of special equipment making the type of tissue sample very cost efficient.
  2. There is a large archive of FFPE samples available for researchers due to the easiness associated with it storage.
  3. FFPE specimens have been used for decades making it incredibly familiar to pathologists.
  4. Fresh frozen tissue is much more suitable for the analysis of native proteins, polymerase chain reaction, and next generation DNA sequencing.
  5. Fresh frozen tissue ensures the preservation of DNA, RNA, and native proteins.
  6. Fresh frozen tissues require specialized equipment for storage. This means mechanical failure, power outages, and carelessness can affect the quality of the samples.

 

References:

1)      FFPE tissue samples / quality control. Horizon. Accessed 6/19/2018. https://www.horizondiscovery.com/reference-standards/our-formats/ffpe/ffpe-tissue-samples-quality-control4

2)      Ward T. The importance of proper formalin fixation of FFPE samples. Personalis. Accessed 6/19/2018. https://www.personalis.com/importance-proper-formalin-fixation-ffpe-specimens/

3)      Doiron L. 5 quality control rules for cancer tissue banks. Folio Conversant. Accessed 6/19/2018. http://www.conversantbio.com/blog/bid/339034/5-Quality-Control-Rules-for-Cancer-Tissue-Banks

4)      FFPE vs frozen tissue samples. BioChain. Accessed 6/19/2018. https://www.biochain.com/general/ffpe-vs-frozen-tissue-samples/

5)      What is FFPE tissue and what are its uses. BioChain. Accessed 6/19/2018. https://www.biochain.com/general/what-is-ffpe-tissue/

 

Cancer Therapy

Thanks to extended research from human tissue samples we have been able to make major breakthroughs in cancer research. In the twenty-first century, evidence, both epidemiologically and clinically, have supported that the changes in whole-body metabolism can affect oncogenesis, the progression of tumors, and the response of tumor to therapy. It has been observed that metabolic conditions such as hyperglycemia, obesity, hyperlipidemia, and insulin resistance are associated higher with risk of cancer development, accelerated progression of tumors, and poor clinical outcome. Due to these findings, many clinical studies indicate that statins and metformin may help in decreasing cancer-related mortality and morbidity. Phenformin is another drug used to treat diabetics that can help with anticancer effects. However, phenformin was discontinued in the late 1970s due to a high incidence of lactic acidosis. Metformin is the most commonly used antihyperglycemic agent globally. It has an optimal pharmacokinetic profile with:

·         50 – 60% of absolute oral bioavailability

·         Slow absorption

·         Negligible binding to plasma protein

·         Broad tissue distribution

·         No hepatic metabolism

·         Limited drug interactions

·         Rapid urinary interaction

It also has an exceptional safety profile as there is a low number of individuals who have side effects. Statins also have a great safety profile and is currently used by a large population.

Cancer and Cellular Metabolism

The accumulation of evidence has suggested that malignant transformation is linked to changes that affect several factors of metabolism. Metabolic rearrangements associated with cancer have been linked with the inactivation of tumor suppressor genes and activation of proto-oncogenes. However, the accumulation of metabolites such as fumarate, succinate, and 2-hydroxyglutarate (2-HG) drives oncogenesis through the signal transduction cascades. Conclusively, these observations support the notion that signal transduction and intermediate metabolism are associated.

 

a)       Oncogenes and Metabolism

The signaling pathways from oncogenic drivers are linked to metabolic alterations due to cancer. For example, the expression of the PKM2 (an M2 isoform of pyruvate kinase) encourages the alteration of glycolytic intermediates in the direction of anabolic metabolism while regulating both transcriptional and post-transcriptional program that leads to the addiction of glutamine.

 

b)      Oncosuppressors and Metabolism

There are some oncosuppressor proteins that can regulate cellular metabolism. The inactivation of tumor suppressor p53 happens in more than 50% of all neoplasms causes a variety of metabolic consequences that could potentially stimulate the Warburg effect. P53 can possibly suppress the transcription of GLUT4 and GLUT1 and stimulate the expression of apoptosis regulator (TIGAR), TP53 induced glycolysis, SCO2, glutaminase 2 (GLS2) and many other pro-autophagic factors. It also interacts physically with glucose-6-phosphate-dehydrogenase (G6PD) with RB1-inducible coiled-coil 1 (RB1CC1).

c)       Oncometabolites and Oncoenzymes

It was found that metabolites can contribute to oncogenesis when mutations such as fumarate hydratase (FH) and succinate dehydrogenase (SDH) was linked to sporadic and familial types of cancer including pheochromocytoma, leiomyoma, renal cell carcinoma, and paraganglioma. once the enzymatic activity of SDH and FH is disrupted, succinate and fumarate accumulate resulting in oncogenesis.

Targeting Cancer Metabolism

The metabolic targets for cancer therapy rewiring of cancer cells is seen as a promising source for new drug targets. Some different approaches have resulted in the identification of agents that can help with targeting glucose metabolism for cancer therapy. However, the low number of metabolic inhibitors reflect the recent rediscovery of the field. There are also some concerns about the uniformity between malignant cells and non-transformed cells that are undergoing proliferation.

 

a)       Targeting Bioenergetic Metabolism

Some cancer-associated alterations such as the Krebs cycle, glycolysis, glutaminolysis, mitochondrial respiration, and fatty acid oxidation have been studied as potential sites for drug therapy.

 

b)      Targeting Anabolic Metabolism

The anabolic metabolism in cancer cells increases the output from nucleotide, protein, and protein biosynthesis pathways to help with the generation of new biomass in rapidly proliferating cells (includes both normal and malignant). A high metabolic flux through the pentose phosphate pathway is vital to cancer cells as it generates ribose-5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH).

 

c)       Targeting Other Metabolic Pathways

Other pathways involved in the adaptation to metabolic stress may provide drug targets for cancer therapy. This applies to autophagy, hypoxia-inducible factors 1, and nicotinamide adenine dinucleotide metabolism. A competitor of nicotinamide phosphoribosyltransferase (NAMPT) known as FK866 has been observed to have antineoplastic effects in murine tumor models.

 

Conclusion

The extensive metabolic rewiring in malignant cells provides a large number of possible drug targets. Many agents that target metabolic enzymes are used for decades while others are being developed. Therefore, the use of metabolic modulators that could be complicated by the similarities of highly proliferating normal cells and metabolism of malignant cells, there might be a chance to harness the antineoplastic activity of these drugs clinically. While many efforts were focused on merging metabolic modulators and targeted anticancer drugs, there may be a common view that metabolism and signal transduction are mostly independent if not separate entities. More research is needed to study the extent of how the metabolic functions of oncosuppressive and oncogenic systems contribute to the biological activity.

References:

Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nature Reviews Drug Discovery. 2013; 12: 829-846.

The importance of Biorepositories

Biobanks & Biorepositories

A biorepository is a storage facility for biological materials that includes animal and human tissue samples. A biobank is similar but it is not the same thing as a biorepository. A bio bank is a collection of similar types of samples, that are grouped together based on population, disease type etc. A biobank collects, stores, and processes bio specimens for use in both research and clinical studies. There are countless parties involved in the successful operation of a biorepository like, Geneticist Inc.. a strong support system is required for one to function, these can include, but are not limited to, patients, regulators, investors, governments, healthcare workers etc.

A biobank functions as a biorepository that gathers, processes, stores, and provides specimens and data that is used in research and clinical studies. The biobanking field has changed greatly over the last three decades starting with a small university-based repository developed for the needs of particular projects. It then gradually evolved to include institutional repositories, government repositories, commercial repositories, population biobanks, and virtual biobanks. The data gathered provides information that demonstrates participant or patient phenotype which extends in both genetics and proteomics. Population-wide biobanks have been developed in many countries globally to collect, analyze, and store information that represents samples of their population source. As for virtual biobanks, they function using a special software or web portals that help to connect biobanks and investigators globally.

Biobanking: Responsibilities and Benefits

Biobanking is a process where tissues (both plants and animal) and bodily fluids are collected as samples for the purpose of research to improve the understanding of disease and health. Information that may affect the sample such as height, weight, lifestyle, and family history will also be recorded to provide some background information for the samples. The collected samples can be kept indefinitely or over a period of several years depending on the type of research. Researchers will then track the health of study participants by observing and recording their past, present, and future medical records if they have consent. There are specific biobank projects that specialize in specific conditions. While this may be the case, both healthy volunteers and individuals with the condition will be required for the participation of the study. Samples that are collected for a specific research can also be kept for future use in other research. In genetic conditions, family members of participants can also be recruited to compare their medical history to others who also suffer from the same condition.

Informed Consent

Before participants agree to participate in biobanking, they are usually informed in writing about what to expect and should understand that they can always refuse to be involved further if they feel uncomfortable at some point during the research. The data, information, and samples gathered can be shared with other scientists and researchers such as those in universities, private institutes, or government institutes in other parts of the world. However, it is made clear that samples collected cannot be sold for profit. The sharing of information allows research to be conducted on a larger scale leading to a better understanding of health, advancements, and faster development of new treatments.

Legal and ethical issues

Despite government and institutional involvement in biobanks there has been a lot of legal and ethical gray area attributed to bio banking. External regulatory pressure has led the industry to take much greater care in the execution of their collection and storage. One of the biggest issues that faces the industry, despite laws varying from country to country, is that donors are not getting financially rewarded even though their body parts are being sold for thousands of dollars.        

History of Bio banks

Biobanks have been around in one form or another for over a century. Back then they were shells of what they have become today. Similar to today’s biobanks they too were hosted at universities where scientists tend to conjugate. They were small and developed with specific research and studies in mind. Interestingly enough, mentions of histology have been found in literature as early as 1817. As time has passed and as the importance of biobanks has become more widely understood and appreciated they have grown to become much larger. Eventually governments and institutions alike have become involved and we now even have population wide biobanks.

Advancement

In the field of biorepository, it has evolved according to the changing needs of investigators and studies that utilize specimen banking while adhering to regulatory and related guidelines and pressures. The changing environment can be attributed to fields such as genomics, proteomics, and personalized medicine that increases the precision of science. It has increased the demand for high-quality specimens that are reliable, accurate, and has standardized laboratory and clinical data. This is why the process of collection, storage, tracking, and shipment are vital to the outcome of studies. Regulatory requirements such as the Health Insurance Portability and Accountability Act (HIPAA), and Institutional Review Board (IRB)have been developed to address consent, ethical, and legal issues.

Evolution of the Biobank and its Diverse Activities

In the United States, specimens have been collected and stored for more than a century. Banks have expanded their activities from small operations based on small studies to become a much more complex enterprise. Advances such as procedure automation and computerization have transformed the management of these biobanks. Specimens can now be logged onto a computerized database. Biobanks with sufficient funding can now invest in robotics to accelerate processing and sampling. The internet has enabled communication with clients and companies now exist to support biobanks in terms of inventory tracking, consent documentation, and handling of laboratory and clinical data. Robotic devices can handle specimen processing and national biorepositories have made it possible to study large populations throughout the entire lifespan. For example:

  1. UK Biobank – Created after 10 years of planning aiming to improve prevention, diagnosis, and treatment of life-threatening illnesses. They have reported a recruitment of half a million participants between the ages of 40 to 69 during 2006 to 2010.
  2. The University of California, San Francisco AIDS Specimen Bank (ASB) – Started in 1982 as a response to the challenges of AIDS epidemic.
  3. National Cancer Institute announced the establishment of US National Cancer Human Biobank (caHUB) – Created due to concerning needs for human biospecimens and aims to improve and modernize the field of biobanking through standard operating procedures and standards.
  4. Virtual biobank -  Created as an electronic database that contains information of biological specimens regardless where the specimens are stored. There is one in University College London Virtual Biobank that collects information of existing and new biospecimens. It will eventually become a data repository for all health science centers. Its founders are currently attempting to develop a software system that houses sample and phenotype data, so all researchers can view information on all collections.

Virtual biobanks are the future of bio banking. Technological advances in AI software and robotics is changing the way we manage and operate biobanks. The most modern of biobanks are using computerized databases of specimens accessible by a google-like search engine. Software companies have developed tracking, processing and documentation systems specific to the bio banking business. The future of bio banking looks very bright, so long that standard operating procedures are abided by.

 

References:

1)     Biobanking. Healthtalk. Accessed 5/30/2018. http://www.healthtalk.org/peoples-experiences/medical-research/biobanking/what-biobanking-and-why-it-important

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

Application of Tissue Microarrays in Genomic Research

Application of Tissue Microarrays in Genomic Research

Many current literatures have demonstrated TMAs using paraffin medium and FFPE blocks for most studies due to the ease of specimen availability, long term storage, and cost-effectiveness for specimens. The TMA platform is an unparalleled tool to optimize assay and adapt novel molecular assays to archival paraffin tissues which are still a large and relatively untapped molecular repository. The remarkable value of TMA applications has been the efficiency and accuracy in the detection of clinicopathologic associations in a wide variety of diseases. The portability of this technique has also played a vital role in the widespread use of it and will continue to drive TMA applications.  

DNA / RNA Extraction & Quality Control

DNA / RNA Extraction & Quality Control

DNA and RNA extraction has played important and crucial roles in helping researchers and scientists to manipulate molecular biology analysis to have a better understanding in the biology of the earth. Due to the rapid advancement of technology, DNA and RNA extraction has improved vastly however, weaknesses of the instruments should be bettered constantly by conducting quality control as it affects all subsequent results.