Assessment for potential bias in multiplexed IL-10 and TNF-α from plex count

Area of Science:

• Biomarker discovery and validation
• Immunology and inflammation research
• High-throughput biological assays

Background:

• Multiplex arrays enable simultaneous quantification of multiple analytes, crucial for cost-effective, large-scale biomarker research.
• Potential variations exist between multiplex and single plex assays due to shared reaction environments, possibly affecting research outcomes.

Purpose of the Study:

• To investigate and quantify differences between multiplex (3-plex) and single plex assays for tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10).
• To assess the impact of these differences on data comparability in biomarker studies.

Main Methods:

• Utilized Luminex HS Cytokine Panel A to test 72 serum samples across multiple 3-plex and 1-plex assay batches for IL-10 and TNF-α.
• Employed paired t-tests and Bland-Altman plots to compare cytokine values within and between assay types (1-plex vs. 3-plex).

Main Results:

• Interleukin-10 (IL-10) values showed minimal differences between 1-plex and 3-plex assays (average 10.7% difference).
• Tumor necrosis factor-alpha (TNF-α) exhibited a greater difference (16.7%) in between-plex comparisons versus within-plex comparisons (12%).
• Variability was more pronounced at low analyte concentrations, particularly for IL-10.

Conclusions:

• Despite observed variations at low concentrations, multiplex assays may offer acceptable comparability with single plex assays for IL-10 and TNF-α.
• The impact of assay differences on comparability is likely minimal in datasets with predominantly low analyte concentrations.