Surreal illustration of blood cells in a frozen landscape, emphasizing the impact of temperature on sample integrity.

Unlocking Complement Biomarkers: How Storage Temperatures Impact C3a and C4a Levels

Rhea Morgan in Science & Nature September 2025 • 4 min read.

"Discover the critical impact of sample storage on the reliability of complement biomarker testing and learn best practices for maintaining sample integrity."

In clinical laboratories, the accuracy of diagnostic tests hinges on numerous factors, with sample handling and storage conditions playing a pivotal role. When it comes to complement biomarkers, such as C3a and C4a, the stakes are particularly high. These biomarkers are key indicators of immune system activity, and their accurate measurement is essential for diagnosing and monitoring various diseases.

A recent study has shed light on the impact of storage temperatures on the stability of C3a and C4a levels in blood samples. Researchers investigated how storing samples at -80°C compared to -20°C affects the integrity of these biomarkers over time. The findings underscore the critical importance of adhering to best practices for sample storage to ensure reliable test results.

The study highlights the challenges faced by clinical laboratories, especially those lacking ultra-low temperature storage capabilities. While short-term storage at -20°C is often accepted, the long-term effects on complement biomarker stability remain a concern. This article delves into the study's findings, offering insights into optimal storage practices and their implications for clinical diagnostics.

Why Does Temperature Matter for C3a and C4a Stability?

Surreal illustration of blood cells in a frozen landscape, emphasizing the impact of temperature on sample integrity.

C3a and C4a are complement components generated during the activation of the complement system, a crucial part of the immune response. These biomarkers are used to assess immune system activity in various conditions, including autoimmune diseases, infections, and inflammatory disorders. However, their inherent instability makes them susceptible to degradation if not stored properly.

Temperature plays a significant role in the degradation process. Higher temperatures accelerate enzymatic activity and other biochemical reactions that can break down C3a and C4a. This degradation can lead to inaccurate measurements, potentially affecting clinical decisions. To investigate the impact of storage temperature, researchers conducted a study comparing C3a and C4a levels in samples stored at -80°C versus -20°C.

Sample Collection: EDTA plasma was collected from five healthy donors, and each sample was divided into 12 aliquots.
Storage Conditions: Ten aliquots were immediately stored at -20°C, while two aliquots were stored at -80°C as controls.
Analysis: Aliquots were analyzed by radioimmunoassay (RIA) for C3a and C4a the next day and once per week for four weeks. The percent difference relative to the -80°C aliquot (24h) was calculated.

The study revealed significant differences in C3a levels for samples stored at -20°C compared to those stored at -80°C. C3a levels increased over the four-week period, with changes ranging from 8% to a staggering 159%. Notably, the clinical interpretation of C3a levels shifted from 'normal' to 'elevated' in several subjects, highlighting the potential for misdiagnosis due to improper storage. Changes in C4a levels were less pronounced, ranging from 1% to 45%, with no changes in clinical interpretation. However, the variability in C4a levels still raises concerns about the reliability of measurements.

Ensuring Accuracy in Complement Biomarker Testing

The findings from this study emphasize the critical need for strict adherence to sample storage guidelines in clinical laboratories. Storing samples at -20°C for extended periods can lead to significant alterations in C3a levels, potentially compromising the accuracy of diagnostic tests. The observation of unfrozen or partially frozen samples after just 18 hours at -20°C further underscores the instability of these biomarkers.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

Everything You Need To Know

Why is proper storage of C3a and C4a samples critical?

Proper storage of C3a and C4a samples is critical because these are complement biomarkers indicative of immune system activity. Their stability is significantly affected by temperature, with higher temperatures accelerating degradation. This can lead to inaccurate measurements, potentially leading to misdiagnosis in conditions such as autoimmune diseases, infections, and inflammatory disorders. Specifically, the study highlights that storing samples at -20°C can cause significant changes in C3a levels, affecting clinical interpretation and thus the accuracy of diagnostic tests.

What is the impact of storing samples at -20°C versus -80°C on C3a and C4a levels?

A recent study investigated the impact of different storage temperatures on C3a and C4a levels. Samples stored at -20°C showed significant changes in C3a levels over time, ranging from 8% to 159%. In some cases, this shift in C3a levels altered the clinical interpretation from 'normal' to 'elevated', potentially leading to misdiagnosis. In contrast, changes in C4a levels were less pronounced, ranging from 1% to 45%, but still presented concerns regarding the reliability of measurements. The -80°C storage served as the control, showing better stability.

How do C3a and C4a function within the immune system, and why are they measured?

C3a and C4a are complement components generated during the activation of the complement system, a crucial part of the immune response. This system plays a vital role in recognizing and eliminating pathogens, as well as modulating inflammation. These biomarkers are measured to assess the level of immune system activity in various conditions, including autoimmune diseases, infections, and inflammatory disorders. By measuring C3a and C4a, clinicians can gain insights into the activity of the complement system and help diagnose and monitor these conditions.

What are the specific storage guidelines that clinical laboratories should follow to ensure the accuracy of C3a and C4a tests?

Clinical laboratories should adhere to strict sample storage guidelines, especially when dealing with C3a and C4a. The findings indicate that long-term storage at -20°C can compromise the accuracy of these tests. Ideally, samples should be stored at -80°C to maintain the integrity of C3a and C4a levels. This study's methodology also recommends collecting EDTA plasma and storing them immediately after collection. Laboratories should also ensure samples are fully frozen, as unfrozen or partially frozen samples can affect the reliability of the test results. These practices are essential for ensuring accurate and reliable diagnostic results.

What are the practical implications of the study's findings for clinical laboratories that lack ultra-low temperature storage?

The study's findings present challenges for clinical laboratories lacking -80°C storage capabilities. The instability of C3a and C4a at -20°C necessitates a careful consideration of storage protocols. Laboratories may need to implement stricter short-term storage practices, potentially limiting the duration samples are stored at -20°C. They may also need to consider alternative testing strategies or collaborate with other facilities that have -80°C storage. Moreover, the findings highlight the need for the laboratories to validate their existing protocols and perform regular quality control checks to ensure the reliability of their results when using -20°C storage. These implications underscore the need for laboratories to be aware of the limitations of their storage capabilities and adapt their practices accordingly.