Preanalytical Variables: Long-Term Storage and Retrieval of Biospecimens

Introduction

The week before last we talked about how pre-analytical variables affect the integrity of human biospecimens, and this week we’ll be following up on this article by discussing the long term storage and retrieval of biospecimens.

The term “storage” comprises of both short and long-term storage of all biospecimens consistent with the study design and planned future use. Depending on the details of their future use, the specimens are either locally or centrally stored. It can also be stored in both locations. The decision will be made depending on the:

• Sample size of biospecimens

• Complexity of collection

• The accrual rate of biospecimens

• Processing procedures

• Logistics

• Cost of storage and retrieval

• Quality issues

• Biorepository governance factors

If the biospecimens are stored for various uses, biorepositories should have duplicates that are close in proximity to the main laboratory. Samples that are to be stored for more than a year should be stored centrally. Duplicates should also be stored on different power supplies or different locations as insurance against natural disasters or equipment failure. Biorepositories are also recommended to have approximately 10 percent of the total mechanical freezers as empty backup freezers to protect against freezer failure. Different storage conditions may be required based on the downstream analyses. Some of the pre-analytical variables that affect long-term storage include:

• The time involved from processing to storage

• Duration of storage

• Temperature

• Facility

• Environmental impact (such as moisture, sunlight, dehydration, humidity, oxidation, evaporation, and desiccation)

• Freeze-thaw cycles

• Some emergencies include: encapsulation of biospecimens in ice after refreezing and microbiological contamination

• Destroyed or no labeling

• Missing or misplaced aliquots

Since biobank material is valuable and hard to replace, the use of systems such as the laboratory information management system (LIMS) should be utilized as it helps allow traceability, confirm chain of custody, and manage biospecimens to improve data reliability and retrieval. Once the integrity of a biospecimen is compromised, it is no longer valuable and becomes useless. It is therefore important to retrieve only those biospecimens that are required. As previously mentioned, duplicate collections of biospecimens are ideal to prevent the destruction of samples.

Blood Sample

The study of the stability of analytes compared to the fresh sample, taking into account the recovery rates, are vital to determine the effects of long-term storage. After long-term storage, the recovery rates may decrease or increase resulting in increased or attenuated risk ratios. It is recommended that hormone, chemistry, and protein analytes are much more stable and stored at -80⁰C for up to 13 months. However, various studies have shown that there are different patterns of stability based on the analyte, time, and temperature of storage. There has been no systematic influence regarding omics analyses observed in samples collected in citrate, heparin, or ethylenediamine triacetic acid (EDTA) if stored at -80⁰C in liquid nitrogen. Long term storage in room temperature and repeated freeze cycles must be avoided. At room, low, and ultra-low temperatures, the extraction of DNA from whole blood samples using bio stabilization technology yielded samples that are pure and that have integrity. Although live cells are stable at room temperature for as long as 48 hours, it should be cryopreserved or cultured in liquid nitrogen to ensure its viability. The recovery of sufficient DNA or those that are of acceptable quality for microarray studies involves the transfer of thawed buffy coat or EDTA whole blood into RNA preservative. Serum or plasma that will be used for miRNA analysis must be extracted immediately or maintained at -80 in RNA free cryotubes.

Urine Sample Protocol

For urine samples, long term storage at temperatures less than -80⁰C without additives is ideal unless it has been specified for certain downstream analyses. Urine samples have been stored at -22⁰C for 12 to 15 years without the use of preservatives while ensuring the stability and measurement validity. Urine used for metabolome and proteome analyses will go through progressive protein degradation if stored at room temperature. While freeze-thaw cycles have minimal impact on the protein profiles, repeated cycles should ideally be avoided.

Saliva Sample Protocol

The protocols for saliva storage are ultimately dependent on the expected downstream analyses. There seems to be minimal impact of protein profile changes despite freeze-thaw cycles. It is recommended that it is stored at -80⁰C. If the saliva samples were divided into aliquots and frozen immediately at -80⁰C, there does not seem to be any differences in cortisol, C-reactive protein, mRNA, or cytokines.

Extracted DNA Sample protocol

The most common method of storage for DNA is still freezing it at -80⁰C. It should be noted that DNA degradation increases with repeated freeze-thaw cycles, higher storage temperature, dilution, and multiple suspensions. Special technologies allow the minimization of storage space and the reduction of shipping and electrical costs. This can be beneficial especially when cryogenic or mechanical equipment is unavailable. It can also be an alternative method for backup storage. Using this technology, there is no degradation or accelerated aging of DNA at room temperature or higher temperatures (50-70⁰C) throughout the 8-month storage duration.

RNA sample protocol

Some of the pre-analytical storage factors that can affect the quality and quantity of analyte or gene expression include the concentration of RNA, temperature, storage time, and repeated thaws. New technology for the dry storage of RNA at room temperature has been developed. This is a technology comparable to RNA that is cryopreserved for up to a year for downstream analyses such as RNA sequencing and real-time polymerase chain reaction.

References:

Ellervik C, Vaught J. Preanalytical variables affecting the integrity of human biospecimens in biobanking. Clinical Chemistry. 2015; 61(7): 913-934. http://clinchem.aaccjnls.org/content/clinchem/61/7/914.full.pdf

Pre-analytical Variables Affecting the Integrity of Human Biospecimens

Introduction

Biorepositories or biobanks function to collect, process, transport, and store biospecimens. The integrity of these biospecimens is crucial for the success of clinical trials and research. There are many factors that can influence the results within research such as:

• Pre-analytical environmental or biological variables

• Pre-analytical technical variables

• Analytical variables

• Post-analytical variables

Pre-analytical variables are defined as factors that can have an impact before the start of the analytical phase. It not only affects the integrity of the tissue samples, but also the results of the analysis. Pre-analytical variables are critical as the analytical integrity of the research can be jeopardized. Seeing as most errors in the laboratory can be attributed to pre-analytical errors this stage is of upmost importance.

Pre-analytical Factors in the Collection of Biospecimens

It is important to adhere to guidelines for general laboratory safety. The collection of biospecimens require a balance of:

• Accrual rate

• Types of biospecimens

• Sample size

• Costs

• Location

• Storage requirements

• Transport logistics

The biospecimens collected can be either invasive or noninvasive. Biospecimens that are collected through non-invasive methods may lead to an increase in sample size due to easiness of collection, reduced costs, and willingness of donor to participate. This method is especially important when dealing with pediatric biobanking. It is important that biological and environmental factors are standardized and documented when interpreting results as it can affect the downstream analysis. It is also vital to take measurements to observe the effects of intervention and the changes over time.

Pre-Analytical Factors That Affect the Collection of Blood Samples and Its Derivatives

The collection of blood samples should be performed by trained staff. Those that are involved in collecting samples from children should specialize in pediatric phlebotomy. The staff needs to be highly trained as this ensures the highest quality of specimens and prevents the donors from experiencing any kind of discomfort. Depending on the research requirements different additives may be required. Different types of additives are coded using different colored collection tubes. Some of the important pre-analytical factors to take note of include:

1. Using the same tube brand and the same lot number throughout the study. This would be ideal as different brands can have different anticoagulants, additives, and may introduce bias.

2. Another important factor is the expiration dates on the tubes as the vacuum in these tubes can decrease with age and negatively impact the blood draw and filling of the tube.

3. Using the same posture such as supine, standing, or supine as these can cause plasma volume changes that may lead to increased analyte levels.

4. Using the recommended needle gauge as a needle that is too thin can lead to hemolysis that distorts the potassium concentrations and hematological cell counts.

5. Using the recommended and same duration of tourniquet use as prolonged use can cause changes in analyte concentrations and hemoconcentration.

6. Avoid inadequate filling as this can result in inaccurate results due to the decrease in blood and additive ratio.

A general rule for common analyses is to use ethylenediaminetetraacetic acid for hematology, DNA, hemoglobin A1C, and a range of proteins. For plasma glucose, it is recommended to use sodium-fluoride tubes while lithium heparin plasma can be used for assays such as kidney function, iron parameters, liver enzymes, thyroid hormones, C-reactive protein, and more.

In remote sites that are resource-poor, capillary dry blood spot (DBS) are easy biospecimens that can be collected. Small volumes of capillary blood from the peripheries can be deposited onto specific paper cards and dried at room temperature for three to four hours. DBS can be used in many analyses. However, some of the pre-analytical variables to note are:

·         Type of collection paper used

·         Type of chemical used in the manufacturing of the paper

·         Thickness of paper

·         The volume of blood deposited

·         Environmental factors such as heat, humidity, sunlight, and moisture

In DNA and RNA collection, there are also biological factors that can affect the biospecimens. These include the donor’s:

·         Gender

·         Age

·         Body mass index

·         Tobacco consumption

Since RNA is more vulnerable to degradation, some of the preanalytical collection factors that can affect the integrity are:

• Tube additive

• Tube type

• Tube sterility

• Type of biospecimen

• The volume of blood collected

• Short-term storage temperature

• Lag time until extraction

In microRNA’s, the pre-analytical variables include:

• Diet

• Age

• Race

• Exercise

• Drugs

• Altitude

• Tobacco use

• Chemicals

• Hemolysis

• Coagulation times

• Temperature

Pre-analytical Factors That Affect the Collection of Urine and Saliva

Urine can be collected in many different ways as it can be used for measurements of many analytes. In urine biospecimens, the preanalytical requirements can be conflicting.  This may result in the requirement of multiple biospecimens. Some of the preanalytical variables for urine collection include:

• Collection method

• Environmental exposure

• Urine dilution

• Dipstick components

• Preservatives or additives used

For saliva, these biospecimens have many advantages as they are easy to collect and can be used in many situations especially if donors are afraid of needles. The preanalytical variables for this biospecimen include:

• The time of collection

• The temperature the specimen is stored

• The collection method

Conclusion

The factors mentioned are pre-analytical variables that affect the biospecimens during the collection phase. However, it is important to note that there are many more pre-analytical variables that can affect the integrity of the biospecimens during the processing, transport, and storage phase.

The Incredible MicroRNA's

What is microRNA?

MicroRNAs (miRNAs) are a group of small non-coding RNAs that are found in tissue samples of animals, plants, and some viruses. Since the discovery of circulating and extracellular miRNAs, there has been a rapid expansion of studies on miRNAs in biofluids like cerebrospinal fluid, plasma, serum, and urine. miRNAs are similar to small interfering RNAs (siRNAs). However, miRNAs originate from RNA transcripts forming short hairpins while siRNAs are from longer parts of double-stranded RNA. miRNAs are plentiful in mammalian cells and seem to target approximately 60% of these genes. miRNAs are thought to have important biological functions as they are evolutionary conserved. A good example is where there is conservation of 90 families of miRNAs as seen in the common ancestor of fish and mammals. The majority of these conserved miRNAs have been shown to play important roles.

Roles of miRNA

miRNA plays a role in RNA silencing and gene expression as post-transcriptional regulators. Within mRNAs, miRNAs function by base-pairing with the complementary molecules causing mRNA molecules to be silenced through cleavage of mRNA strand, destabilization of mRNA, or reduced efficiency of mRNA translation by ribosomes. Despite the low numbers of miRNA, it is estimated that the miRNAs regulate approximately more than 33% of the cellular transcriptome. Therefore, it should be no surprise that the miRNAs have crucial functions in the developmental and cellular processes that have been thought to be involved in many human diseases.

Since miRNAs are relatively stable in biofluids and have a wide range of biological potential, these molecules are suited to be used as non-invasive biomarkers in diagnosis, drug safety, and pre-clinical toxicity. Many studies have proven that secreted miRNAs are involved in conditions such as organ damage, cancers, and coronary heart disease. While the exact role of circulating miRNAs is mostly unknown, they have been observed to be protected from RNAse degradation through the inclusion in membranous particles or protein complexes.

Disease

Since miRNA plays a role in the normal functioning of eukaryotic cells, miRNA dysregulation is therefore linked to disease. For example:

a)       Hereditary Diseases

• It has been found that a mutation in the region of miR-96 results in progressive hearing loss.

• Hereditary keratoconus with anterior polar cataract is seen in mutation in the region of miR-184.

• Growth and skeletal defects can be seen in those with deletion of miR-17 to 92 cluster.

b)      Cancer

• Chronic lymphocytic leukemia was one of the first human diseases that has been associated with miRNA dysregulation. Other miRNAs that have also been linked to cancer are called “oncomirs”. miRNAs are involved in pathways that are crucial in B-cell development such as B-cell receptor signaling, cell-cell interaction, B-cell migration or adhesion, and production or class-switching of immunoglobulins. They also influence the generation of marginal zone, follicular, plasma, B1, and memory B cells.

• Another clinical trial is using miRNA as a screening assay for the detection of colorectal cancer in the early stages.

• miRNAs can also be used to determine prognosis in cancers based on their expression level. For example, a study on non-small-cell lung carcinoma determined that a low level of miR-324a levels is an indication of poor prognosis. In colorectal cancer, a low level of miR-133b and high level of miR-185 is linked with metastasis resulting in poor prognosis.

c)       Heart Disease

• Studies have found that there are specific changes in the expression levels of miRNAs in diseased hearts which points to the involvement of miRNAs in cardiomyopathies.

• miRNAs can also be used to determine the prognosis and risk stratification of cardiovascular diseases.

• In animal models, miRNAs have been associated with the regulation and metabolism of cholesterol.

d)      Nervous System

• miRNAs are thought to be involved in the function and development of the nervous system.

• Neural miRNAs such as miR-124, miR-132 and miR-134 are involved in dendritogenesis.

• Other neural miRNAs are involved in the formation of the synapses. miR-134 and miR-138 are thought to be included in the process of synapse maturation.

• Studies have found that conditions such as bipolar disorder, schizophrenia, anxiety disorders, and major depression have altered miRNA expression.

e)      Obesity

• miRNAs have a vital role in the differentiation of stem cell progenitors into adipocytes. Studies have found that the expression of miRNAs 155, 221, and 222 can inhibit adipogenesis paving a possible genetic treatment for obesity.

• miRNAs of the let-7 family was found to accumulate in tissues as aging occurs. Based on animal models, the excessive expression of let-7 class miRNAs resulted in accelerated aging, insulin resistance, and therefore increases the risk of obesity and diabetes. When let-7 was inhibited, it resulted in an increase in insulin sensitivity and resistance to high-fat-diet-induced obesity. This means the inhibition of let-7 may prevent, reverse, and cure obesity and diabetes.

Conclusion

Although miRNAs have great promise in the screening, diagnosis, treatment, and prevention of various pathological conditions, more research, and clinical trials will be needed in the study of miRNAs to further explore and discover the potential of miRNAs.

References:

1. microRNA. Wikipedia. Accessed 8/22/2018. https://en.wikipedia.org/wiki/MicroRNA#Disease

2. Blondal T, Nielsen SJ, Baker A, et al. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods. 2013; 59(1): S1-S6.

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.