Medical test tubes transitioning between frozen and unfrozen, symbolizing sample instability.

Unlocking the Secrets of Complement Biomarkers: How Storage Impacts C3a and C4a Levels

Nico Varela in Health & Wellness December 2025 • 4 min read.

"Discover the critical role of proper sample storage in maintaining the integrity of complement biomarkers C3a and C4a, and learn how temperature fluctuations can compromise test accuracy."

In the realm of medical diagnostics, accuracy is paramount. When it comes to complement biomarkers, such as C3a and C4a, even slight variations in handling can significantly impact test results. These biomarkers play a crucial role in assessing immune system activity, making their precise measurement essential for diagnosing and managing various health conditions.

A primary concern in clinical laboratories is maintaining the integrity of samples from the moment they are collected until they are analyzed. Best practices dictate that samples should be processed quickly, immediately aliquoted, and stored at -80°C until testing. However, not all facilities have the capability to store samples at such low temperatures, leading to questions about the stability of these biomarkers at more accessible temperatures like -20°C.

Recognizing this challenge, a study was conducted to investigate the impact of storing samples at -20°C on C3a and C4a levels compared to the recommended -80°C. The findings shed light on the stability of these biomarkers under non-ideal storage conditions, providing valuable insights for clinical laboratories and healthcare professionals.

The Chilling Effect: How Temperature Alters C3a and C4a Levels

Medical test tubes transitioning between frozen and unfrozen, symbolizing sample instability.

The study, detailed in Molecular Immunology, examined EDTA plasma collected from five healthy donors. Each sample was divided into twelve aliquots, with ten stored at -20°C and two at -80°C as controls. The levels of C3a and C4a were measured using radioimmunoassay (RIA) the next day and then weekly for four weeks. This setup allowed researchers to track changes in biomarker levels over time, comparing the effects of the two storage temperatures.

The results revealed a significant impact on C3a levels. The percent difference in C3a levels for samples stored at -20°C ranged dramatically, from 8% to a staggering 159%. What's more, these levels tended to increase over the four-week period. This increase wasn't just a statistical anomaly; it had real clinical implications.

Variability: The impact of storing samples at -20°C is highly variable between individual samples and across analytes.
Clinical Interpretation Changes: In two out of five subjects, C3a levels shifted from 'normal' to 'elevated' after just one week of storage at -20°C. By the end of the four weeks, this was the case for four out of the five subjects.
C4a Stability: Changes in C4a levels were less pronounced, with percent differences ranging from 1% to 45%, and without causing a shift in clinical interpretation.
Physical State: Aliquots stored at -20°C were observed to be either unfrozen or partially frozen after just 18 hours.

In contrast, C4a levels showed less dramatic changes, with percent differences ranging from 1% to 45%. Notably, these changes did not lead to alterations in clinical interpretation. However, one striking observation was that aliquots stored at -20°C were often found to be either unfrozen or partially frozen after just 18 hours, raising concerns about sample integrity.

Preserving Accuracy: Best Practices for Sample Storage

This research underscores the critical importance of proper sample storage in clinical laboratories. The significant variability in C3a levels, the potential for altered clinical interpretations, and the observation of unfrozen samples at -20°C all highlight the risks associated with non-ideal storage conditions. To ensure the reliability of complement biomarker testing, it is recommended that samples be stored at -80°C whenever possible. If temporary storage at -20°C is unavoidable, it should not exceed 24 hours, and snap freezing prior to storage may help minimize variability.

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Everything You Need To Know

What are complement biomarkers C3a and C4a and why are they important?

Complement biomarkers like C3a and C4a are molecules that can be measured to assess the activity of the complement system, a part of the immune system. The complement system helps the body fight infections and clear away damaged cells. C3a and C4a are produced during complement activation. Measuring the levels of C3a and C4a can provide insights into various health conditions, including autoimmune diseases, infections, and inflammatory disorders. Accurate measurement of these biomarkers is crucial for proper diagnosis and management of these conditions.

Why is maintaining the correct storage temperature important for C3a and C4a?

Maintaining the correct storage temperature is very important for C3a and C4a because temperature fluctuations can compromise their integrity and lead to inaccurate test results. Specifically, storing samples at -20°C can cause significant changes in C3a levels, with some samples showing dramatic increases over time. While C4a levels are less affected, storing samples at -20°C can still lead to partially thawed samples, raising concerns about overall sample integrity. These changes can lead to misinterpretation of clinical results, potentially affecting patient care.

What did the study find about the impact of storing samples at -20°C on C3a and C4a levels?

The study revealed that storing samples at -20°C can lead to significant variability in C3a levels. In some cases, C3a levels increased dramatically, leading to changes in clinical interpretation from 'normal' to 'elevated' in a substantial number of subjects. While changes in C4a levels were less pronounced, the observation of partially thawed samples at -20°C raises concerns about the reliability of these measurements as well. These findings highlight the importance of adhering to recommended storage temperatures to ensure accurate and reliable complement biomarker testing.

What are the best practices for sample storage to preserve the accuracy of C3a and C4a measurements?

To ensure the reliability of C3a and C4a measurements, clinical laboratories should adhere to best practices for sample storage. The recommendation is to store samples at -80°C whenever possible. If temporary storage at -20°C is unavoidable, it should be limited to a maximum of 24 hours. Snap freezing samples before storage at -20°C may also help to minimize variability in biomarker levels. Following these guidelines helps to preserve the integrity of the biomarkers and ensure accurate test results.

What are the implications of these findings for clinical laboratories and healthcare professionals?

The findings regarding the impact of storage temperature on C3a and C4a levels have implications for clinical laboratories and healthcare professionals. It emphasizes the need for strict adherence to recommended storage conditions to avoid compromising the accuracy of test results. Consistent and reliable measurements of C3a and C4a are essential for proper diagnosis and management of various health conditions. Laboratories may need to re-evaluate their storage protocols to ensure they meet the recommended guidelines. Education and training for lab personnel on proper sample handling and storage are also crucial to maintaining the integrity of complement biomarker testing.