Hospital puerta de hierro
A sustainable alternative to deep-freezing for Plasma preservation.
Plasma is a complex biological fluid with a high protein content and significant clinical relevance. It is particularly sensitive to freeze-thaw cycles and storage conditions, which can affect the stability of its components and compromise downstream analyses.
Objective
To compare the quality and functionality of plasma samples stabilized and stored at room temperature versus those frozen at -80°C as part of a prospective biobank sample collection.
Materials and Methods
Samples used for the study
Plasma samples
30 samples
Fresh
Procedure
The samples collected will be evaluated according to the QC established in the following section and subsequently divided into 5 parts: 1 for initial control, 2 for lyophilization and 2 for freezing at 80ºC. Aliquots of 500 µL will be lyophilized immediately after collection and at the same time as freezing.
All lyophilized samples, once the lyophilization cycle is completed, and after verifying that the pellet has been formed correctly, will be stored at room temperature in the corresponding boxes.
After a period of one and six months during which the samples were stored in a desiccated state, a second evaluation of the freeze-dried samples was conducted, with a comparison made to the frozen samples.
Quality Assurance
IFN-gamma, TNF-alpha, IL-8, IL-10 and IP10
Flow cytometry detection in Luminex and quantification of inflamatory cytokines secreted in serum (IFN-gamma, TNF-alpha, IL-8, IL-10 and IP-10)
Results
The concentration of IFNγ, IL-8 and TNFα obtained after freeze-drying is more similar to that of fresh samples than frozensamples at both storage times.
Freeze-dried samples showed higher concentrations of IFNγ, IL-8, TNFα, and IL-10 compared to frozen controls, suggesting better protection against degradation. Freeze-dried cytokine levels more closely resembled fresh sample concentrations, indicating minimal alteration during the lyophilization process.
| Frozen vs Freeze-dried | After 1 month | After 6 months | ||
|---|---|---|---|---|
| Difference from the average | 95% Cl Bland-Altman limits | Difference from the average | 95% Cl Bland-Altman limits | |
| IFNγ | -9.2 | -63.2-44.8 | -31.4 | -129.6-66.8 |
| IL-8 | -0.3 | -4.5-3.9 | -1.0 | -9.5-7.5 |
| TNFα | -1.6 | -18.6-15.3 | -7.1 | -31.5-17.2 |
| IL-10 | 0.4 | -2.9-3.6 | 2.3 | -49.4-54.1 |
| IP-10 | 115.6 | -19.5-250.7 | 112.1 | -71.3-295.5 |
IP-10 showed lower concentrations after freeze-drying, suggesting marker-specific stability profiles that may require optimization.
Conclusion
These results demonstrate that freeze-dried plasma preserves functional inflammatory markers suitable for immunological monitoring and diagnostic applications. This capability addresses a critical challenge in immunology research and diagnostics where cytokine stability during sample handling significantly impacts data quality and reproducibility.
The observed IP-10 reduction prompted detailed investigation revealing that the effect is related to stabilization buffer composition and rehydration timing rather than the freeze-drying process itself. Follow-up studies using artificial plasma spiked with cytokines and analyzed by cytometric bead array (CBA) confirmed that IP-10 levels are influenced by rehydration kinetics, while other cytokines remain stable regardless of these parameters. Formulation
optimization studies are ongoing to address this marker-specific behaviour, demonstrating 300K Solutions’ commitment to refining protocols for comprehensive analyte coverage.
Preliminary results support freeze-drying as an alternative for sample storage. However, further studies with larger sample sizes and different markers would be necessary to validate the method.
Presentation of results in a poster at the III Conference of the ISCIII Platforms
As part of the collaboration with Hospital Puerta de Hierro, the results of the validation of the 300K Solutions technology were presented in a poster during the III Conference of the ISCIII Platforms in Biomedicine and Health Sciences.
You can access the full poster below:
