Dried blood spots (DBS) collected on filter paper such as Guthrie cards are stored for years at room temperature. The supposition is that once dried, the samples remain stable and quantifiable indefinitely since the metabolites these were initially designed to measure, are known for their extended stability. The concentration of other blood proteins such as cytokines, however, are known to vary with storage even in liquid samples stored at -80 °C for extended periods of time. Thus, we sought to determine if cytokines are stable for up to 5 months when stored as a dried blood spot.

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To test this, blood was collected from 4 healthy participants, spiked with recombinant cytokines, and collected into 30 µL volumetric adsorptive microsampling (VAMS) devices. These prepared DBS were stored at room temperature, 4 °C, or -20 °C for up to 5 months and matching DBS liquid extracts were stored at -80 °C for the same period of time. At each timepoint, the DBS were extracted from the VAMS and the extracts were analysed by Luminex® for quantification of up to 31 cytokines.

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Cytokine analysis revealed that room temperature, the current standard for DBS storage, performed the poorest out of all storage temperatures with 9/21 significantly different to baseline by 5 months compared with 4/21 for the 4 °C comparator. Storage at 4 °C or colder performed well for the majority of analytes tested, however out of those, the optimal storage temperature differed for each analyte. For example, IL-21 was highly variable at 4 °C and more reproducible at -80 °C, whilst the opposite was true for IL-13. There were a small number of analytes that performed poorly regardless of storage conditions and for fractalkine, this was found to be caused by inefficient recovery during extraction.

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Our results highlight the need to understand the stability of analytes of interest before committing to longitudinal collection and storage of DBS.  These data give confidence that with storage at 4 °C or colder, 27/31 of the cytokines assessed in this study are quantifiably stable over for at least 5 months.