Comparison of pre-processing methods for multiplex bead-based immunoassays

Background: High throughput protein expression studies can be performed using bead-based protein immunoassays, such as the Luminex (R) xMAP (R) technology. Technical variability is inherent to these experiments and may lead to systematic bias and reduced power. To reduce technical variability, data pre-processing is performed. However, no recommendations exist for the pre-processing of Luminex (R) xMAP (R) data. Results: We compared 37 different data pre-processing combinations of transformation and normalization methods in 42 samples on 384 analytes obtained from a multiplex immunoassay based on the Luminex (R) xMAP (R) technology. We evaluated the performance of each pre-processing approach with 6 different performance criteria. Three performance criteria were plots. All plots were evaluated by 15 independent and blinded readers. Four different combinations of transformation and normalization methods performed well as pre-processing procedure for this bead-based protein immunoassay. Conclusions: The following combinations of transformation and normalization were suitable for pre-processing Luminex (R) xMAP (R) data in this study: weighted Box-Cox followed by quantile or robust spline normalization (rsn), asinh transformation followed by loess normalization and Box-Cox followed by rsn.