Multiplex bead array assay for detection of 25 soluble cytokines in blister fluid of patients with complex regional pain syndrome type 1

Abstract

Inflammatory processes are known to be involved at least in the early phase of complex regional pain syndrome type 1 (CRPS1). Blister fluid obtained from the involved extremities displayed increased amounts of proinflammatory cytokines IL-6 and TNFalpha compared with the noninvolved extremities. The aim of this paper is to investigate the involvement of mediators by measurement of several other cytokines using new detection techniques that enable multiple cytokine measurement in small samples. The use of a multiplex-25 bead array cytokine assay and Luminex technology enabled simultaneous measurement of representative (1) proinflammatory cytokines such as GM-CSF, IL-1beta, IL-1RA, IL-6, IL-8, and TNF-alpha; (2) Th1/Th2 distinguishing cytokines IFN-gamma, IL-2, IL-2R, IL-4, IL-5, and IL-10; (3) nonspecific acting cytokines IFN-alpha, IL-7, IL-12p40/p70, IL-13, IL-15, and IL-17; and (4) chemokines eotaxin, IP-10, MCP-1, MIP-1alpha, MIP-1beta, MIG, and RANTES. Although minimal detection levels are significantly higher in the bead array system than those in common ELISA assays, in blister fluid, IL-1RA, IL-6, IL-8, TNF-alpha, IL-12p40/p70, MCP-1, and MIP-1beta were detectable and increased in CRPS1 affected extremities. Levels of IL-6 and TNF-alpha simultaneously measured by ELISA (Sanquin Compact kit) and by multiplex-25 bead array assay (Biosource) were highly correlated (r = 0.85, P < .001 for IL-6 and r = 0.88, P < .001 for TNF-alpha). Furthermore, IP-10 and eotaxin were detectable but diminished in CRPS1, whereas detectable amounts of IL-10 were similar in involved and noninvolved extremities. Multiplex bead array assays are useful systems to establish the involvement of cytokines in inflammatory processes by measurements in blister fluids of CRPS1. Ten representative cytokines were detectable. However, detection levels and amounts measured are at least 3 times higher in the multiplex-25 array assay than in the ELISA assays used simultaneously for the measurement of cytokines.

Figure 1

Regression curves of 44 samples of blister fluid obtained from 22 CRPS1 patients, both from the involved and the noninvolved extremity. Values calculated in pg/mL were plotted on logarithmic scales. Regression lines were calculated taking into account the left-censored values due to detection limits as described in the statistical methods. Dotted lines indicate detection levels of the multiplex-25 cytokine assay. (a) Regression curve of IL-6 data from the multiplex-25 cytokine assay and the ELISA kit (r = 0.85, P < .001), (b) regression curve of TNF-α data from the multiplex-25 cytokine assay and the ELISA kit (r = 0.88, P < .001).

Figure 2

Example of a regression curve between concentrations of IL-1RA and IL-12 in 22 blister fluid samples taken from the CRPS1 extremity (correlation coefficient 0.97, P < .001), measured by the multiplex-25 bead array assay. Values are calculated in pg/mL and plotted on a logarithmic scale.