Biobanking storage conditions can affect long-term sample quality and integrity1. A number of biomarkers are particularly sensitive to storage temperature and can degrade even when biobanked at -80°C. Furthermore, researchers often struggle with variable results from assays run in different labs or when using reagents from different suppliers. These variabilities can make it difficult to produce robust, reproducible data. Many government and biobanking industry organizations, including the US National Cancer Institute, the UK Medical Research Council, and the International Society for Biological and Environmental Repositories (ISBER), are pushing for standardization of sample handling and biobanking procedures to improve sample and data quality.
Researchers from the Netherlands, Germany and Norway recently tested the effects of both biobanking storage conditions and assay variability on inflammatory biomarkers in plasma samples2. The samples came from participants in the LifeLines Cohort Study, a population-based cohort study from the Netherlands. Participants ranged in age from 18 years to 90 years. The researchers tested 240 plasma samples that had been biobanked at -80°C for less than 2 years or for longer than 4 years. They used immunonephelometry and ELISA assays to measure the levels of the inflammatory biomarkers: C-reactive protein (CRP), interleukin-6 (IL6) and tumor necrosis factor alpha (TNFα). The ELISA assays were sourced from two different manufacturers and the tests were run in 3 different collaborating biobanks. This study design allowed the authors to compare how assay and lab variability affected biomarker levels.
CRP and IL6 are Stable When Biobanked at -80°C
Using immunonephelometry, the authors found that CRP levels remained stable in plasma samples biobanked at -80°C for over 4 years. Likewise, using ELISA assays, the researchers found that IL6 levels remained stable at -80°C over a 4-year period.
The researchers repeated ELISA assays to measure IL6 at 2 different biobanking locations. The majority of their results were similar in both locations. However, 38 samples were below the detection limit at location A but not at location C. This level of variability could significantly affect study results.
The researchers compared interassay variability by using both immunonephelometry and ELISA assays to measure CRP levels in biobanked samples. Immunonephelometry results showed consistently higher CRP levels than ELISA assays for the same samples.
The authors also used linear correlation to compare IL6 levels using ELISA assays from two different manufacturers. One ELISA assay showed consistently higher results than the other. These results highlight the need to test assay performance on small sample sets before using an assay to test a large number of samples.
The authors also compared TNFα levels using ELISAs from two different companies. One of the ELISA assays failed to detect adequate TNFα in the majority of the samples. In contrast, the other assay detected TNFα in the same samples. This result shows that all assays should be tested before being used in large studies of biobanked samples.
This study showed that the levels of two inflammatory biomarkers, CRP and IL6, remain stable in plasma samples biobanked at -80°C for over 4 years. It also showed that 2 different biobanks had significantly different results for 38 of the tested samples, even though they were using the sample protocol and the same reagents.
This interlab variability can be compounded if labs use different assays, or reagents from different suppliers. As can be seen from the results of this study, ELISA assays from different suppliers can give significantly different results.
Overall, the data from this study highlights the need to test the performance of all assays on small numbers of samples before conducting large studies. It also supports the growing body of literature showing that the biobanking industry should standardize protocols and procedures to ensure samples and data are of the highest possible quality.
- NCI Best Practices for Biospecimen Resources. National Cancer Institute. 2016
- Van Waateringe et al. Influence of Storage and Inter- and Intra-Assay Variability on the Measurement of Inflammatory Biomarkers in Population-Based Biobanking. Biopreservation and Biobanking. 2017