Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis
Session Type: Abstract Submissions (ACR)
Rheumatoid factor (RF) and other circulating heterophilic antibodies have been shown to interfere with antibody-based immunoassays, in particular with multiplex protein detection platforms. Previous studies have suggested that available blocking agents reduce such erroneous signal amplifications. The purpose of the present study was to develop a protocol for multiplex cytokine analyses using standardized blocking procedures in patients with rheumatoid arthritis (RA), and to test this protocol in samples derived from patients with active RA who were started on adalimumab therapy.
Analyses of cytokines in peripheral blood samples was performed using a custom made 12-plex kit (Bioplex®, Bio-Rad) . Samples were obtained from patients with known high levels of serum RF, as well as from healthy controls. Based on the literature (Todd et al. Arthritis Rheum 2011; 63: 894-903), various concentrations of Heteroblock®, an agent that is known to block heterophilic binding activity, were added. In controls, including sera from healthy controls spiked with a cytokine mix (Bio-Rad), the results were stable regardless of the concentration of Heteroblock®. The optimal blocking concentration was determined based on analyses of patients with high levels of RF.
Furthermore, we investigated cytokine levels in 14 patients with active RA [11 females; mean age 63.7 years; median RA duration 9.0 years ; 78 % RF positive; mean DAS28: 5.6], who were started on treatment with adalimumab 40 mg subcutaneously every two weeks. In the present pilot study, data on interleukin (IL)-6, IL-7 and IL-8 are reported.
In patients with RA and high RF levels, there was a major effect on the measured signal from adding the blocking agent, with some differences across analytes and across individuals. In analyses using multiple concentrations of Heteroblock®, a higher optimal concentration than previously described was found and used in subsequent investigations.
In the 14 patients treated with adalimumab, the median impact of blocking on the estimated cytokine concentration was 39 % of the unblocked value for IL-6, 13 % for IL-7 and 9 % for IL-8. In analyses of baseline samples from patients in the adalimumab study with no blocking agent added, there was no significant correlation between IL-6 and CRP (r=0.19; p=0.51). By contrast, in baseline samples exposed to optimal concentration of the blocking agent, there was a significant correlation between IL-6 and CRP (r=0.65; p=0.01). The decrease in IL-6 from baseline to three months after adalimumab initiation correlated with the decreases in ESR (r=0.89; p=0.02) and CRP (r=0.77; p=0.07), with a similar trend for DAS28 (r=0.32; NS). IL-7 and IL-8 did not correlate with disease severity parameters.
These results support the concept that agents blocking heterophilic antibody activity should be used and systematically evaluated in immunoassays involving multiplex protein detection platforms. In analyses using optimized concentrations of the blocking agent, IL-6 levels correlated with baseline markers of inflammation, and changes of IL-6 correlated with clinically important measures of reduced inflammation in patients treated with adalimumab.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multiplex-cytokine-analyses-in-patients-with-rheumatoid-arthritis-require-use-of-agents-blocking-heterophilic-antibody-activity/