Numerous studies point to the utility of blood
cytokine measurements in the diagnosis and treatment of human disease. Advances in detection allow robust multiplex analysis. However,
cytokines are present at low levels and are produced and act in complex networks which can remain active in stored blood. A major barrier to the routine use of
cytokines as clinical
biomarkers is sample management prior to analysis. Studies on
cytokine stability under storage frequently use 'spiked' normal control plasma or serum to generate detectable levels of the
cytokines of interest. These conditions may oversimplify the reality of clinically complex samples and provide limited information regarding optimal management of whole blood samples prior to plasma separation.
Cytokine stability has also been addressed previously using plasma from normal individuals under different conditions of
anticoagulant use, storage time and temperature of storage. No studies have as yet been undertaken to address
cytokine stability in
critically ill patients which may differ from normal, healthy individuals due to underlying cofounders such as
inflammation. To address these issues, we subjected samples from five patients exhibiting an inflammatory disease state to three storage extremes which might be encountered in a clinical setting, prior to analysis of 40
cytokines. Blood drawn into
EDTA or ACD
anticoagulant was immediately separated and plasma stored at -80 °C. Matched samples were stored as follows; whole blood overnight at room temperature, or whole blood or plasma stored 10 days at 4 °C. We used equivalence testing to determine the similarity of stored
cytokine values to baseline values. In ACD plasma, Eotaxin,
IL-6,
IL-11,
IL-15, IP10, MDC, MCP-1 met equivalence to baseline in all storage conditions while for
EDTA plasma stored 10 days at 4 °C
EGF,
FGF2,
Fractalkine,
G-CSF, IL-1β,
IL-5,
IL-6,
IL-7,
IL-11, IP-10, TGFα and TNFα showed equivalence to baseline measurements. Intraclass Correlation Coefficients (ICC) were calculated and the following
cytokines showed good to excellent agreement across all 4 storage conditions: Eotaxin,
IL-5,
IL-6,
IL-11,
IL-13, IP-10, MCP-1 and TNFα when collected in
EDTA, and Eotaxin,
IL-5,
IL-6,
IL-11, IL12-p40,
IL-15, IP-10 and MCP-1 when collected in ACD. Five plasma
cytokines were identified as being the least stable in both ACD and
EDTA:
IL-7,
IL-9, IL12p70,
RANTES, sCD40L, while IL-1β was identified as unstable stored in ACD plasma. This study identified several clinically important
cytokines that are remarkably stable in blood and plasma, and some that stored poorly. To our knowledge, this is the first
cytokine storage study to use medically unwell patient samples and equivalence testing to evaluate the stability of measured
cytokine values after storage.