Newborn Genetic Screening Program

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

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

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

 

What is the Newborn Genetic Screening Test?

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

 

Duration and Location of DNA Storage

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

 

How are These Samples Used?

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

a. Research purposes such as:

  • Retesting the samples to confirm the screening results
  • Developing new screening tests
  • Developing new techniques for forensic studies
  • Identification of new diseases
  • Quality control purposes
  • Access for those who are not biorepository lab technicians (such as those people in law enforcement)

b. Law enforcement purposes such as:

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

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

 

Controversies

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

a)     Consent

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

b)      Ethics

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

c)       De-identification

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

 

Conclusion

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

Types of Biorepositories

Introduction

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

 

Operations

  • Upon delivery the tissue samples are recorded all information regarding the sample details are keyed into the laboratory information management system.
  • Biospecimens are then processed to ensure consistency of the samples. Proper tissue preservation methodology is absolutely crucial to a biorepository.
  • These specimens are then stored and held in their appropriate conditions. Sample holding boxes and freezers are sometimes used, however it depends on the storage requirements. For example Formalin-fixed paraffin-embedded tissue blocks can be stored at room temperature.
  • Distribution involves retrieving the required samples from the inventory.

Standard Operating Procedures

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

  • Reduce problematic variables within the samples,
  • Ensure that biospecimens resemble specimens in their natural state,
  • Provide standards on how operations should be conducted.

Types and Uses of Biorepositories

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

Examples

  1. The Alzheimer’s Disease Neuroimaging Initiative – is a disease-focused biorepository and biomarker validation program that uses the samples and data collected from Alzheimer’s disease patients and patients with other forms of memory impairment.
  2. The Health Outreach Program for the Elderly (HOPE) is a biorepository at Boston University that supports multiple studies. The HOPE registry follows up with their Alzheimer’s patients annually.
  3. The United Kingdom Biobank is a biorepository with a broad focus. They aim to improve the diagnosis, treatment, and prevention of various diseases such as cancer, stroke, diabetes, heart disease, eye disorders, depression, dementia, and arthritis. In between 2006 to 2010, they managed to recruit half a million individuals between the ages of 40 to 69. Samples such as blood, saliva, and urine have been donated for analysis. These participants have also provided detailed personal information and consented to future follow up for many years to help researchers discover how various diseases develop.
  4. The Autism Research Resource is sponsored by the state of New Jersey to research autism in families where more than one child is affected.
  5. The Centers for Disease Control (CDC) Cell and DNA Repository use samples from transformed cell lines available through the Genetic Testing Reference Material Coordination Program. Some of the samples obtained are from diseases such as Cystic Fibrosis, Huntington Disease, Alpha Thalassemia, Fragile X syndrome, and Muenke syndrome.
  6. The National Institute of Neurological Disorders and Stroke (NINDS) Human Genetics DNA and Cell Line Repository focuses on the identification of new genes that causes or contributes to conditions such as Parkinson’s disease, Tourettes syndrome, epilepsy, motor neuron disease, and cerebrovascular disease.
  7. The National Institute of Aging (NIA) Aging Cell Repository utilizes cellular and molecular research to determine the degenerative mechanisms and causes of aging. They have strict diagnostic criteria with cells collected over a span of thirty years. Scientists are using these cultures to study diseases such as Alzheimer’s disease, Parkinson’s disease, Progeria, and Werner Syndrome.
  8. The National Human Genome Research Institute (NHGRI) Sample Repository for Human Genetic Research successfully completed the sequencing of the human genome. They now aim to participate in a variety of studies that focuses on the understanding of the structure and function of the genome and the role it plays in disease and health.
  9. The National Eye Institute Age-Related Eye Disease Study (NEI-AREDS) Genetic Repository was founded to identify how macular degeneration and cataracts develop and progress. This is important as these two conditions are two main causes of vision loss among older patients.

Conclusion

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

 

References:

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

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

CRO Services

Introduction

A contract research organization or CRO refers to a company that provides support in the form of research. The research conducted through CRO services can be in one of the following fields:

  • Biotechnology
  • Pharmaceutical
  • Medical device industry

In full a CRO is a company contracted by another organization to help lead and manage their trials, responsibilities, roles, and their function.

 

Services and Advantages

Some CRO services executed include such things as, but not limited to:

  • Biologic assay development
  • Biopharmaceutical development
  • Commercialization
  • Preclinical and clinical research
  • Management of clinical trials
  • Database design and building
  • Data entry and validation
  • Medicine and disease coding
  • Quality and metric reporting
  • Statistical
  • Pharmacovigilance (the identification, detection, assessment, observation, and prevention of side effects of pharmaceutical products)

CROs are useful for companies when developing new drugs and medications as they reduce costs. CROs are able to simplify the development of new drugs and entry into drug markets. They also support governmental organizations, foundations, universities, and research institutions. CROs can range from small specialty groups to large international organizations. They aim to provide support for clinical studies and trials. Those that specialize in clinical trials services of a new drug are present from its conception until it is approved by the Food and Drug Administration (FDA) or by the  European Medicines Agency (EMA). Evidently CROs play a crucial role and pharmaceutical companies are continually outsourcing critical functions such as research and manufacturing to CROs.

The number of major corporations that are using CROs in clinical trials and the development of new drugs is increasing. Companies that establish contract with CROs aim to acquire the required expertise without having to hire permanent staff, keeping overhead low. Some CRO trade groups have claimed that contracting with CROs has helped reduce the cost by decreasing the time it takes to conduct a trial. This also means that the company that hires a CRO will not need the required infrastructure, manpower, and office space to conduct these trials. Some CROs can even manage all the aspects in a clinical trial starting from the site and patient selection all the way up until the final regulatory approval.

 

Regulatory Aspects

The International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) defined CROs as “a person or organization contracted by the sponsor to perform trial-related duties and functions”. Their guidelines highlight the following:

  • A sponsor can transfer all their duties and function to a CRO. However, the hiring company will be responsible for the integrity of data acquired from the CRO conducted study. It remains the hiring company’s responsibility to ensure that all the data is factual and backed by science.
  • CROs should ensure quality control and quality assurance.
  • All duties and functions transferred to a CRO should be in writing. The hiring company should oversee the duties and functions that are carried out on their behalf.
  • Duties and functions that are not transferred to a CRO will remain with the sponsor.

 

Market Size and Growth

The Association of Clinical Research Organizations has estimated that more than half of clinical studies conducted by the pharmaceutical industry have been outsourced to CROs. The most popular field for CROs is therapeutic work including infectious disease, oncology, central nervous system, cardiovascular disease, and metabolic disorders. A further 27% work for within the biotechnology field while the remainder work for governments, foundations, and the medical device industry. The CRO sector is doing extremely well for an industry that just started a decade ago. There is increasing pressure facing medical devices organizations and pharmaceutical companies for the high cost of drugs and they are trying to lower costs without decreasing their profits. One of the best and most common solutions is to outsource clinical trial management as it results in significantly lower overhead costs.

By the year 2013, there were more than 1,100 CROs globally. However, there are many CROs that have gone out of business or that have been acquired. In 2008, it was estimated that the top 10 companies control 56% of the market. A 2007 estimate showed that the market would reach $24 billion in 2010 with a growth rate of 8.5% from 2009 to 2015. In 2016, the research and development spending increased by 15.5% from 2015 to 2020.

 

Conclusion

CROs are an effective solution as it provides an affordable option for companies to pursue the development and approval of new medication. Before the existence of CROs, this was a hugely expensive endeavor which was only embarked on when the likely hood of regulatory approval was high. With CROs, companies are now able to develop drugs for specific markets.

 

References:

  1. Contract research organization. Wikipedia. Accessed 7/30/2018. https://en.wikipedia.org/wiki/Contract_research_organization
  2. Stone K. Contract research organizations (CRO) definition. The Balance. Accessed 7/30/2018. https://www.thebalance.com/contract-research-organizations-cro-2663066

What is a Geneticist?

Introduction

Geneticists are leaders in biology and are directly involved in unlocking some of the secrets of life. A geneticist has many roles such as putting together the puzzles of heredity and DNA. They spend most of their life looking for the answers to one to several specific questions and are incredibly dedicated to their work. With this devotion, the field of genetics has thrived, advanced, and progressed throughout the years.

 

What Does a Geneticist Do?

a)       There are several applications of genetics in a variety of fields. More are expected to become prevalent with the progression of technology and new research. The major fields involved in genetics are crime, medicine, and agriculture. Geneticists working at pharmaceutical companies help to uncover birth defects, the origin of diseases, developing prevention techniques, and even therapy.

 

b)      With the population increase, there are no more people to feed in the world. This also means that it is important for the supply to meet the demand of the people. Geneticists in the agricultural specialty strive to develop and improve crops that can grow in harsh conditions, yield more produce, or increase the size of the produce itself.

 

c)       Geneticists with the advancement of technology now have a better understanding of the DNA from tissue samples and are now applying it to their knowledge of solving crimes. Geneticists are able to be laboratory detectives with DNA sampling to ensure that the right perpetrator is convicted of the crime.

 

Types of Geneticists:

most geneticists are drawn to the fields of medicine, agriculture, and crime. With these three fields geneticists have a good chance of finding employment in government, universities, biorepository or biobanks, and major pharmaceutical companies. These three fields can be closely related in terms of research. This means that geneticists can make a lot of useful contacts within the industry regardless of specialization. Generally, there are two types of geneticists:

a)       Laboratory geneticist – the field that most geneticists choose to enter. This role involves the application of genetic technologies.

b)      Genetic counselor – a field where geneticists work as consultants or as a nurse. This role involves working closely with parents who are at risk of conceiving children with birth defects. They also play a crucial part in consulting with healthcare and insurance companies regarding new medical technologies.

 

Qualifications of a Geneticist:

To be a geneticist, extensive study at bachelor level is most often required. Most commonly  a Bachelor of Science in chemistry or biology is sought. However, any physical science will be accepted as long as it is paired with a minor in biology. There are very few positions available to those with only a Bachelor of Science. Most of these are lab assistant positions which lack the same upward mobility. A master’s in the field of genetics would be helpful but those looking for authority in research and development should acquire a Ph.D. or M.D. Generally, four to six years after the completion of an undergraduate degree is spent taking advanced science classes and conducting personal research projects. These are done with grants from pharmaceutical companies, universities, or the government. This project will be one of the main points in the resume and will play a major role in the hiring decision making process. Fresh graduates usually enter the company as a lab or research assistant. However, those with more advanced degrees will move faster through the ranks to develop new technologies and methods.

 

CRO Services

CRO services are also known as contract research organization (CRO) services. A CRO is a company that provides support to biotechnology, pharmaceutical, and medical device industries in the form of research. CRO services include services such as:

·         Biologic assay development

·         Biopharmaceutical development

·         Commercialization

·         Clinical research

·         Pre-clinical research

·         Pharmacovigilance

·         Clinical trials management

CROs help to lower costs for companies that are trying to develop new drugs and medication in niche markets. Their goal is to simplify drug development and facilitate entry into markets. They also aim to support research institutions, foundations, universities, and governmental organizations. Many CRO's provide clinical trials or clinical study support for medical devices and drugs. CROs can range in size from small and niche specialty groups to large and international organizations. CROs that specialize in clinical trials help their clients by offering their expertise of creating a new medical device or drug from conception until it has been  marketed and approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA). If you are interested in any CRO Services fill out our request form

 

References:

1)      Geneticist: a day in the life of a geneticist. The Princeton Review. Accessed 7/24/2018. https://www.princetonreview.com/careers/202/geneticist

2)      Contract research organization. Wikipedia. Accessed 7/24/2018. https://en.wikipedia.org/wiki/Contract_research_organization

3)      What is a geneticist? Environmental Science. Accessed 7/24/2018. https://www.environmentalscience.org/career/geneticist

4)      What does a geneticist do? Sokanu. Accessed 7/24/2018. https://www.sokanu.com/careers/geneticist/

Ethics in Biobanking

Introduction

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Biobanking ethics are important and one of the most debated issues in public health and bioethics. Biorepositories carry the potential to advance disease research in unprecedented ways. There are however concerns about donor privacy and not all biorepositories make sure that they follow ethical standards. People’s DNA and tissue sample has been used without respect for their rights. Regardless of peoples differing views and perspectives, one thing can be agreed upon among most experts, that biobanks are revolutionary. Biobanking ethics include issues such as, but not limited to:

  • Controversies and key challenges faced in biobanking ethics
  • Issues of informed consent
  • Withdrawal from participants
  • Broad consent
  • Ethics of re-contact
  • Confidentiality issues
  • Ownership, property and commercialization problems

 

Issues in Brief

1)      Informed Consent

Informed consent is crucial in ensuring that ethical standards are followed in both research and therapy. It ensures that the participant understands “the nature, duration, and purpose of the experiment; the method and means it is to be conducted; all inconveniences and hazards reasonable is to be expected; and the effects upon the individual’s health may be due to the participation in the experiment." (Excerpt from Springer Article) One of the major issues of informed consent in biobanking is that it only applies to the donor and not those who are connected to the donor. Next, since biospecimens can be used in future studies, participants cannot be “informed” at the time their tissue is obtained as the nature of future researches are not yet known.

 

2)      Broad Consent

There are some experts who believe that broad consent can be a potential solution to the issues of informed consent in biobanking. However, there are some who disagree as it offers minimal protection and minimal guarantees. Broad consent is the permission given by the donor for the biobank, so the biorepository can do what they see fit with the genetic material. While some individuals argue that broad consent is a means of maximizing autonomy, some see it as the opposite where it is an abuse for autonomy. Ethicists worry that broad consent causes donors to relinquish their rights regarding how their genetic material is used in the future.

 

3)      Confidentiality

One of the main features of genetic information is that it can be used to identify the donor and those related to them. While ethicists argue that identification can be discouraged through various methods of anonymization, there is always the possibility that identification is possible. The risk of identification increases as databases grow.

 

4)      Property and Profit

There is also the issue that participants or donors do not own their tissue samples. This is based on the traditional understanding that body parts are res nullius which means that they do not belong to anyone once detached. Ethicists have argued that there are valid reasons for following the “no property” rule for biospecimens. Allowing property would restrain studies and research to the point where it would become untenable. Another issue is that commercial companies may look to make very large profits from donated samples.

 

5)      Feedback to Participants

Another ethical issue is whether or not to tell participants regarding incidental findings from their donated tissue samples. Incidental findings can be defined as “observations of potential clinical significance that have been discovered unexpectedly in a healthy subject unrelated to the purpose and variables of the study.”(Excerpt from Springer Article)

 

6)      Participation, Representation, Maintenance of Trust

Biorepositories are also worried about the mass withdrawal of participants as it will ultimately result in the loss of set-up costs. The maintaining of trust between the public and biobanks are crucial to prevent participant withdrawal and biobank failure. Currently, there is still no one way that is viewed as the best method of practice in this area.

 

7)      Re-contact

Re-contact is becoming an increasingly crucial issue as there is very little industry conformity on how re-contact should be managed. There needs to be a balance between what donors are informed and what's included without overburdening them. Biorepositories and research teams should view the ability to re-contact as a limited resource. Currently, there is no standard that biobanks can look to adopt for this issue.

 

Conclusion

The problems and challenges in biobanking ethics mean that there is a need for alternative models to address the issues. Biobanking presents important and significant ethical challenges. It is important for those involved to be aware of the advancements and developments in the debates surrounding these issues. By raising awareness of these issues, public interest of will increase and as a result biobanking can continue to change the medical research landscape.

 

References:

Widdows H, Cordell S. The ethics of biobanking: key issues and controversies. Health Care Anal. 2011; 19:207-219.

Tissue Procurement Problems

Introduction

Formalin-fixed paraffin embedded (FFPE) tissue blocks are a valuable resource for many significant research programs as researchers tend to select this type of biospecimen as frozen or fresh tissue blocks may be harder to acquire. There is also the challenge that the number of fresh frozen tissue samples may not be able to fulfill the requirements of the research protocol. The FFPE preservation technique demands a fast and rapid fixation after the process of resection of the target biospecimen in a neutral buffered formalin. Once fixed, the specimen is then embedded in paraffin wax. FFPE is now the commonest tissue preparation method used to archive research biospecimens. Currently, almost all surgeries in this day generate FFPE tissue samples. FFPE tissue blocks are crucial as they offer the potential for the discovery of significant information especially in biomedical research programs and drug discovery. Other research applications that utilize FFPE biospecimens include:

  • genetic studies
  • biomedical identification or authentication
  • visualization of tissue structure

This, therefore, makes FFPE tissue blocks ideal for:

  • the study of autoimmune diseases – rheumatoid arthritis, systemic lupus erythematosus
  • the study of long-term cancers – colon cancer, lung cancer, breast cancer

 

Challenges

However, there are several challenges that researchers face when it comes to obtaining FFPE tissue samples. Some of the challenges include:

 

1)     Oversight of the Pathologist

In some cases, the FFPE samples are not obtained or processed appropriately as the certified pathologist is not on site to supervise and ensure that the proper procedures are followed during the procurement of the specimen. Biorepositories should ensure that they hire enough licensed pathologists to ensure that there is no manpower shortage as it could impact the quality of their tissue specimens.

 

2)     Difficulty Acquiring Samples

There are certain situations where a research team or company requires access to FFPE tissue samples that are hard to come by. For example, FFPE samples from patients with metastatic melanoma might present a challenge to biorepositories. Research teams should partner with a biorepository that has a vast network for tissue procurement as it can help tremendously in the collection of special samples needed in specific research protocols.

 

3)     Rapid Turnaround

Studies or research that needs a quick procurement of FFPE tissue blocks may pose a challenge to many biorepositories. These research teams that are looking for a rapid turnaround of samples should ask biorepositories about their accelerated procurement method which may then be able to provide the required biospecimens within the time frame.

 

4)     Transparency

Research teams looking to procure tissue samples need to keep in mind about transparency as studies rely on well-annotated tissue blocks that go through the proper fixation and preservation technique. Before obtaining samples from the biorepository, be sure to ask regarding their specifics and standard of procedure for the fixation and quality assurance protocols. This can impact the result and credibility of the entire study.

 

5)     Review of FFPE Biospecimens

Some biorepositories obtain their tissue samples from local sources such as the local hospitals. However, not all providers take the time to ensure that the specimens are of the highest quality. Biorepositories are responsible to ensure that a gross and microscopic examination of the FFPE biospecimens to ensure that the samples obtained are of the highest quality.

 

6)     Sourcing

With the growing number of organizations and biorepositories, research teams that are looking to source biospecimens should consider companies that follow the best practices and have high standards when it comes to their collection and fixation protocols. Unreliable sources may not be able to provide high-quality FFPE tissue samples. Before procuring the required biospecimens, enquire where the organization obtains their samples and the reliability of it.

 

7)     Patient Information

The patient information from the samples can be valuable and crucial in a research. The more information you can obtain regarding the patient from their data, it can help with some of the results from the research especially in terms of demographics and risk factors of a disease. Patient information also helps you to find the correct patient cohort as some study designs exclude those below or above a certain age. It is therefore important to procure the biospecimens from a biorepository that can provide the necessary data such as the details or a refractory disease, metastatic diseases, or newly diagnosed disease. A new case of cancer or a relapse can also affect the results of a study greatly.

 

Conclusion

In conclusion, there are many factors to consider when it comes to selecting a new biorepository or organization to partner with to obtain tissue samples. It is important as these specimens ultimately determine the results and credibility of the study. Choosing a credible company that provides the highest quality FFPE tissue samples is one of the most crucial steps in the early stages of a study. Low-quality biospecimens result in wasted hours of study, effort, and decreases the overall morale of the research team.

 

References:

Doiron L. Typical problems with FFPE tissue samples – and how to solve them. 2014. Folio Conversant. Accessed 7/11/2018. 

https://www.conversantbio.com/blog/bid/387449/Typical-Problems-with-FFPE-Tissue-Samples-And-How-to-Solve-Them

Tissue Microarray

Introduction

The recent advances that have occurred in the human molecular genetics field have found that there are gene-based disease mechanisms in various areas of medicine. Studies regarding diagnostic and prognostic markers in many clinical specimens is vital in the translation of new findings from basic science to applications in clinical practice. With the increased use and advancements of new molecular biology methods, the research of progression and pathogenesis of diseases such as cancer are now revolutionized. Understanding the basic molecular mechanisms in the progression of normal tissues to cancerous or malignant tumors is crucial in the knowledge of the disease as it can lead to improved treatment, diagnosis, and cures. Some clinical studies have discovered various novel markers at the gene level where validation of these markers is necessary. However, it can be a time consuming, costly, and labor-intensive process especially if tested on several specimens.

 

Tissue Microarray

Tissue microarray is a method used in the field of pathology to overcome issues where the validation of markers is:

  • Time-consuming
  • Costly
  • Labor-intensive

It can be used to organize small amounts of tissue samples on a solid support. It is a method designed to allow the:

  • Simultaneous assessment of gene expression on hundreds on tissue samples
  • Parallel molecular profiling of tissue samples at DNA, RNA, and protein level
  • Analysis of samples using fluorescence in situ hybridization (FISH), immunohistochemistry, and RNA in situ hybridization at lower costs and less time

 

Tissue Microarray Construction

Tissue microarrays can be constructed using composite paraffin blocks through the extraction of cylindrical core biopsies from donor blocks which are them embedded into a microarray or recipient block at specific array coordinates. Donor blocks are first retrieved and sectioned to produce the standard slides. These slides are then stained with hematoxylin and eosin. Once ready, the slides are examined by a certified pathologist who then marks the area of interest (usually an area with pathology such as cancer). Next, the samples can be arrayed. A tissue core can be acquired from the donor block using a tissue microarray instrument. This tissue core is then inserted into an empty recipient or paraffin block at a specific coordinate which is recorded on a spreadsheet. The sampling process is then repeated as many times as necessary from various donor blocks until many cores are placed in one recipient block. This results in the final tissue microarray block. A microtome is utilized to cut 5-micrometer sections from the blocks to produce slides necessary for immunohistochemical and molecular analyses.

 

Applications and Advantages

Tissue microarrays have many advantages over other techniques. Some of it include:

  1. Amplification of a scarce resource – After a standard histological section that is approximately 3 to 5 millimeters thick is used in primary diagnosis, the sections can further be cut 50 to 100 times yielding a total of 100 assays. In tissue microarrays, instead of 50 to 100 samples, it can produce material enough for 500,000 assays.
  2. Simultaneous analysis – Tissue microarrays allows the simultaneous analysis of many specimens as it provides high throughput data acquisition.
  3. Uniformity – In tissue microarrays, every tissue sample is treated uniformly. It can also be used in a variety of techniques such as fluorescent or chromogenic visualization, histochemical stains, tissue microdissection techniques, and more. Tissue microarray enables the analysis of the entire cohort on one slide standardizing the variables such as incubation times, antigen retrieval, washing procedure, reagent concentration, and temperature.
  4. Time and cost efficient – The tissue microarray method require small amounts of reagents for analysis. It is a method that is both time and cost efficient.
  5. Conservation of tissue samples – Tissue microarray is a technique that does not destroy the original block of the tissue sample.

 

Tissue Microarrays from Fresh Frozen Tissue

In tissue microarray, the method uses tissue samples from paraffin-embedded tissue donor blocks that are then placed into a recipient block. One of the challenges with paraffin-embedded tissue is the antigenic changes seen in proteins and degradation of mRNA due to the fixation and embedding process. Some researchers have modified the tissue microarray technique by using fresh frozen tissue that is embedded in optimal cutting temperature (OCT) compound. It is then arrayed into a recipient OCT block. Tissue samples are not fixed before the embedding process and the arrayed sections are assessed without fixation. The advantage of tissue microarrays from fresh frozen tissue is that:

  • It works well for DNA, RNA, and protein analyses.
  • Paraffin-embedded tissue arrays can be challenging for RNA in situ hybridization and immunohistochemistry analyses but tissue microarrays from fresh frozen tissue allow the optimal assessment by each technique.
  • It has uniform fixation throughout the whole array panel.
  • It is a technique that may have significant advantages in the assessment of certain genes and proteins as it improves both quantitative and qualitative results.

 

References:

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

2)      Fezjo MS, Slamon DJ. Tissue microarrays from frozen tissues – OCT technique. Methods Mol Biol. 2010; 664: 73-80.

 

What are 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)      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/

2)      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

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

4)      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.