Ultra-Low Temperature Storage of Tissues and Blood Specimens

Ultra-Low Temperature Storage of Tissues and Blood Specimens

Every year, millions of biospecimens, including human tissues and blood, are preserved around the world for disease diagnosis and research. Biological samples can be maintained for extended periods of time for future use or to allow transportation to different locations for analysis. However, specific measures are required to mitigate possible damage caused by temperatures and preservation conditions. While most scientific procedures may be performed on specimens stored in ultra-low temperature freezers (ULTs), the quality of the specimens can be impacted within the storage equipment. This article examines optimum storage conditions for the long-term preservation of human tissue and blood specimens, as well as infrastructural considerations and best practices for long-term sample storage.

Background

Human biospecimens can broadly be classified as tissue, blood, or their derivatives, which include various cell organelles, DNA, RNA, and proteins1. With the advent of next-generation technologies, it has become common for biospecimens to be preserved for future use or to allow their transportation for analysis. Because storage conditions can have significant effects on sample integrity, it is important that biospecimens are appropriately handled and stored at optimum temperatures to always maintain their critical properties. Some of the most commonly used techniques for maintaining biospecimens are ultra-low temperature freezing (at temperatures below −80°C) and formalin-fixation paraffin-embedding (FFPE), etc1,2,3,4. Ultra-low temperature frozen samples are commonly used because they yield higher quality nucleic acids and proteins5.

Furthermore, entire organisms in ultra-low temperature frozen samples can be preserved for many years, making future research on them possible1. This emphasizes the significance of a dependable method for maintaining correct storage temperatures, as it is a predictor of sample integrity and, by extension, of the dependability of the research results.

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The optimum temperature for preserving biospecimens

1. Tissue Specimen

Tissue samples can be used to diagnose disorders, to determine an appropriate therapy, for treatments themselves (as in the case of transplants), and to monitor how patients are responding to them. Nevertheless, one fundamental drawback of their utilization is that after their blood supply is cut off, tissues undergo rapid changes that impact important biomarkers.

However, with appropriate storage conditions, the biomarkers found in tissues can be kept stable after collection. An investigation into the DNA and RNA stability of frozen tissues obtained from organs such as the lungs, breasts, colon, ovary, and cervix revealed that freezing at -80°C for up to 12 months was effective in preserving DNA quality in 80% of the samples6.

Moreover, in separate studies, the quantity and quality of DNA and RNA in brain autopsy tissue were effectively maintained at storage temperatures below −70°C for 7 years7.

2. Blood Specimen

Blood consists of cell components and proteins with varying degrees of stability. For instance, while immunoglobulins can be kept at room temperature for a few days without losing their properties, blood proteins are easily altered and last significantly shorter times if not stored at cold temperatures8.

Furthermore, DNA can be recovered in good quantity and quality from blood samples stored at room temperature for up to a month. However, prolonged storage can cause lysis of erythrocytes and leukocytes, which results in DNA loss9.

Studies evaluating the effects of storing whole blood at different freezing temperatures on the yield of methemoglobin (Met-Hb) reported that Met-Hb remained unaffected for at least 30 days at storage temperatures of −80°C or −196°C irrespective of the initial concentrations. According to the studies, significant deterioration occurred only when samples were kept at -30°C, particularly in samples with low Met-Hb levels at the start of the study10.

Some studies have recorded the deterioration of RNA derivatives following storage for more than 5 years at temperatures between −70°C and −80°C1, but there is evidence that a high yield of microRNA (miRNA) could be maintained for up to 10 years by storing it in plasma at −80°C, confirming that not all RNA types are equally vulnerable to degradation11. This also indicates the potential of using miRNAs as biomarkers based on their stability under ultra-low temperature storage.

Choose the right freezer for storing the samples

The performance and reliability of an ultra-low temperature freezer are among the most critical considerations when choosing such a cold chain solution12. The reliable performance of a ULT is important because the primary goal of it is to achieve and maintain the required temperatures necessary for the preservation of the samples’ integrity. Proven field reliability is essential to ensure the safety of research samples and good returns on investment.

Therefore, models with a track record of dependability, and performance, and with the lowest energy usage should be selected13, especially when anticipating possible adverse conditions. Other considerations in an ultra-low temperature freezer are temperature homogeneity, recovery time, and delayed warm-up14.

The freezer of choice should be capable of maintaining uniform temperatures from top to bottom and front to back and recover quickly in the event of a door opening. It should also be able to maintain ultra-low temperatures in the presence of high ambient temperatures and delay warm-up in the event of power outages.

It is also important to have a mechanism for monitoring temperatures to ensure appropriate temperatures are maintained. Advanced mobile monitoring systems continuously check the function of freezers and provide real-time text messages or email notifications straight to smart devices allowing the tracking of freezer temperatures, door status, and more14.

Implement best practices for storing tissue and blood specimens

Biospecimens should be handled in accordance with standard operating procedures tailored to the kind of biospecimen and the biomolecules of interest15. Standard operating procedures clearly outline how specific tasks should be undertaken to preserve specimen consistency and quality.

One of the most critical aspects of correctly preserving human specimens is using the appropriate storage temperature selected based on the type of biospecimen being stored, the estimated length of such storage, the biomolecular products of interest, and their intended use1. When there is uncertainty regarding potential future applications of certain biospecimens, they should be kept at a temperature of −80°C for long-term preservation16.

For blood biospecimens, various components such as plasma, red blood cells, white blood cells, platelets, and serum should be separated, and the preservation procedures optimized for each component. In addition, appropriate cryoprotectants can be employed to help maintain the viability of cells for extended periods of time17.

For easy identification, biospecimens should be labelled using lab-grade markers and the labelling should include as much information as possible. There should also be backup freezers in the event that the freezer fails, and alternate power sources designed to activate automatically in the event of power outages17.

Conclusion

Appropriate preservation of biological samples is crucial for achieving reliable and reproducible results in disease diagnosis and biomedical research. Biospecimens should be handled in accordance with standard operating procedures tailored to the kind of biospecimen and the biomolecules of interest. The choice of labelling materials should be based on stability under the long-term storage conditions appropriate for the biospecimen.

Moreover, current data suggests that long-term storage of human tissue and blood specimens at temperatures below −80°C is satisfactory for the preservation of most biomarkers, thus putting a lot on emphasis on the importance of reliable medical cold chain solutions such as ultra-low freezers. Because of this, the performance and reliability of the storage equipment are important to ensure that appropriate storage conditions are continually maintained throughout the freezer.

To achieve this, on top of temperature uniformity, and general reliability of the ULT being used, there should also be a mechanism for monitoring the function of such storage equipment. Finally, there should also be plans in place to deploy backup ULTs in the event that the one being used fails, while also ensuring that all the cold chain equipment can be connected to alternate power sources if the need for such measures arises.

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References:

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