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

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.