Background/Purpose:  Cytokines and chemokines (CK) are central to RA pathogenesis, a fact underscored by the emergence of multiplex immunoassays for quantifying CK values in both research and clinical endeavors. Intrinsic factors to RA, namely rheumatoid factor (RF), may interfere with assay outcomes by nonspecifically binding detection analytes. Thus, we evaluated the performance of a commercially available multiplex platform using banked serum samples and assessed the impact of RF depletion on these measurements.

Methods:  Forty-five CK analytes were tested in a central laboratory using the Meso Scale Discovery V-PLEX^TM  immunoassays and serum from 40 RA and 40 OA patients. True serum duplicates were tested for 20 RA and OA samples, while 20 additional samples from seropositive RA patients were depleted of RF using a commercial binder and compared to duplicates spiked with equal volumes of saline. Intra-assay coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated for each analyte measured using true duplicates. The percent change in analyte concentrations were determined. Finally, rank sum tests were used to compare CK concentrations between RA and OA. Analytes were determined to be “high performers” if the CV was <10% and there was <15% change following RF depletion. Conversely, “low performers” were defined as those with a CV >20% or if RF depletion altered values >30%.

Results:  CVs and ICCs generated using true serum duplicates for the 45 analytes tested are shown in Table 1. Of the 45 analytes, 22 yielded CVs <10%; all but two were “high performers”. ICCs universally exceeded 0.85 with the exception of 7 analytes (6 were “low performers”). RF depletion altered CK values by <15% for 35 analytes with larger changes (>30%) seen only for IL-2 and TNF-α (Figure 1). IL6, IL8, IL10, TNFα, and TNFβ concentrations were increased in RA vs. OA (p<0.05); IL16 was decreased (p<0.05).

Conclusion:  In this study, a commercially available multiplex assay performed well in the context of RA. For most analytes, results were highly reproducible with minimal interference from RF. Additional testing will be needed to identify the source of variability observed for the analytes with higher CVs.

*Intraclass correlation coefficient (ICC) shown in parentheses

Figure 1.  Mean percent change in cytokine/chemokine analyte concentration following RF depletion.

*35 of 45 analytes exhibited <15% change; 8 analytes showed intermediate changes (15-30%) and two (IL-2 and TNF-α) demonstrated >30% change.