Stability of cytokines, chemokines and soluble activation markers in unprocessed blood stored under different conditions

Abstract

Background: Biomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24h at room and refrigerator temperature.

Methods: Blood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20-25°C) and refrigerator temperature (4-8°C) for 0.5, 2, 4, 6, 8, and 24h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation.

Results: IFN-γ, sIL-2Rα, sTNF-RII and β2-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature.

Conclusion: All the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.

Keywords: Biomarker; Chemokines; Cytokines; Significant relative change; Soluble activation marker; Stability.

Fig. 1

Cytokines that were significantly altered (p < 0.05) when unprocessed blood was stored at either room or refrigerator temperature for 24 h. Unprocessed heparin plasma stored at room temperature (a); unprocessed serum stored in the refrigerator (b); unprocessed serum stored at room temperature (c); and unprocessed haparin plasma stored at room temperature (d). Dashed lines represent data for each individual subject and the solid line represents the predicated average change for all subjects.

Fig. 2

Increases in IL-1ra that occur over time when unprocessed EDTA and heparin blood was stored at either room or refrigerator temperature for 24 h. Unprocessed serum stored in refrigerator (a); unprocessed EDTA plasma stored at room temperature (b); unprocessed EDTA plasma stored in the refrigerator (c); and unprocessed heparin plasma stored at room temperature (d); dashed lines represent data for each individual subject and solid line represents the predicted average change for all subjects.

Fig. 3

Chemokines and soluble activation markers that were significantly altered (p < 0.05) when unprocessed blood was stored at room temperature for 24 h. Unprocessed serum (a); unprocessed heparin plasma (b); unprocessed heparin plasma (c); and unprocessed EDTA plasma (d); dashed lines represent data for each individual subject and the solid line represents the predicated average change for all subjecs.