Biological samples in test tubes with fluctuating temperatures in background.

Unlocking the Secrets of Sample Storage: How Temperature Impacts Research Integrity

Samir D’Costa in Science & Nature September 2025 • 4 min read.

"Is -80°C really necessary? Discover how temperature fluctuations affect C3a and C4a levels, influencing clinical interpretations and research outcomes."

In the realm of scientific research, the integrity of samples stands as a cornerstone of reliable results. A seemingly minor detail, such as storage temperature, can wield significant influence over the accuracy and validity of experimental outcomes. For clinical laboratories and research facilities, maintaining optimal sample storage conditions is not merely a procedural step; it is a fundamental requirement for ensuring the dependability of research findings and clinical interpretations.

The common practice of storing samples at -80°C has long been considered the gold standard, especially when dealing with sensitive biomarkers. However, the reality is that many collection sites lack the capacity for such ultra-low temperature storage, often resorting to temporary storage at -20°C. This discrepancy raises a crucial question: How does temporary storage at -20°C impact the stability and reliability of key biomarkers like C3a and C4a?

A study published in Molecular Immunology sought to address this critical gap in knowledge by investigating the effects of storing samples at -20°C versus the recommended -80°C. The research sheds light on the potential pitfalls of deviating from established storage protocols and underscores the importance of stringent temperature control in maintaining sample integrity.

The Chilling Truth: How Temperature Affects Biomarker Stability

Biological samples in test tubes with fluctuating temperatures in background.

The study focused on complement biomarkers C3a and C4a, which are vital components of the immune system. Researchers collected EDTA plasma from five healthy donors, dividing each sample into multiple aliquots. Ten aliquots were stored at -20°C, while two control aliquots were immediately stored at -80°C. The levels of C3a and C4a were then measured using radioimmunoassay (RIA) the next day and once per week for four weeks, with the percent difference from the -80°C aliquot calculated.

The findings revealed a concerning trend: storing samples at -20°C led to substantial changes in C3a levels. Specifically, the percent difference in C3a levels for samples stored at -20°C ranged from 8% to a staggering 159%. Over the four-week period, C3a levels tended to increase, leading to notable shifts in clinical interpretation. In fact, for two of the five subjects, C3a levels shifted from 'normal' to 'elevated' after just one week, and for four of the five subjects after four weeks.

C3a Instability: C3a levels significantly fluctuate when stored at -20°C, affecting clinical interpretations.
C4a Stability: C4a levels remain relatively stable at -20°C, showing minimal changes in clinical interpretation.
Freezing Concerns: Samples stored at -20°C may partially thaw, compromising sample integrity.

In contrast, changes in C4a levels were less pronounced, with percent differences ranging from 1% to 45%. More importantly, these changes did not result in altered clinical interpretations. However, a significant observation was that aliquots stored at -20°C were often found to be unfrozen or partially frozen after just 18 hours, indicating potential instability even within a short timeframe.

Preserving Research Integrity: Best Practices for Sample Storage

The study underscores the critical importance of adhering to recommended storage protocols to maintain the integrity of biological samples. While short-term storage at -20°C may be unavoidable in certain circumstances, the findings suggest that it can lead to significant alterations in biomarker levels, particularly for C3a. To minimize variability and ensure accurate results, samples should ideally be stored at -80°C whenever possible, and any deviations from this standard should be carefully considered and documented. Snap freezing samples prior to storage at either temperature might mitigate some of the observed variability.

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Everything You Need To Know

Why is -80°C considered the gold standard for sample storage, and what are the implications of not using it?

Storing samples at -80°C is considered the gold standard because it helps to preserve the integrity of sensitive biomarkers, ensuring the reliability of research findings. The implications of not using -80°C storage, such as temporary storage at -20°C, can be significant. For instance, the study mentioned shows that storage at -20°C can lead to substantial changes in C3a levels, potentially altering clinical interpretations. These changes could lead to inaccurate diagnoses or flawed research conclusions, emphasizing the importance of adhering to optimal storage conditions whenever possible.

How does the storage temperature impact the stability of C3a and C4a biomarkers?

The storage temperature significantly impacts the stability of C3a and C4a biomarkers differently. Research indicates that C3a levels are highly susceptible to temperature fluctuations, showing substantial changes when stored at -20°C. These changes can lead to misinterpretations in clinical settings. In contrast, C4a levels are relatively stable at -20°C, with less pronounced changes that do not typically alter clinical interpretations. However, even the temporary storage at -20°C can cause samples to partially thaw, potentially compromising the integrity of all biomarkers.

What specific findings from the Molecular Immunology study highlight the risks of storing samples at -20°C instead of -80°C?

The study revealed that storing samples at -20°C led to significant fluctuations in C3a levels, with the percent difference in C3a levels ranging from 8% to 159%. This variability could shift clinical interpretations, such as elevating C3a levels from 'normal' to 'elevated' within a week for some subjects. Furthermore, the study observed that samples stored at -20°C were often not fully frozen after just 18 hours, suggesting potential instability even within a short timeframe. These findings emphasize the need for stringent temperature control to ensure sample integrity and accurate research outcomes.

In the context of sample storage, what are the key best practices to ensure research integrity and minimize variability?

To preserve research integrity, samples should ideally be stored at -80°C whenever possible, following recommended storage protocols. If short-term storage at -20°C is unavoidable, any deviations from the standard should be carefully considered and documented. One best practice is to snap freeze samples before storage, regardless of the temperature, to potentially mitigate variability. It's crucial to understand the potential impacts of storage conditions on specific biomarkers like C3a and C4a and to adhere to protocols that minimize the risk of sample degradation to ensure the accuracy of research results and clinical interpretations.

Can you explain the differences in stability between C3a and C4a when stored at -20°C and what are the potential consequences?

When stored at -20°C, C3a and C4a exhibit different stability profiles. C3a levels show significant fluctuations, with the study reporting percent differences ranging from 8% to 159%. This instability can lead to altered clinical interpretations, potentially misclassifying patients' health status. Conversely, C4a levels remain relatively stable at -20°C, showing smaller changes that typically do not affect clinical interpretations. However, the risk of samples partially thawing at -20°C could still compromise the integrity of both biomarkers over time, highlighting the importance of maintaining optimal storage conditions to prevent inaccuracies in research and clinical diagnostics.