Measurement and quality control issues in multiplex protein assays: a case study

Area of Science:

• Biomarker Discovery
• Assay Development
• Proteomics

Background:

• Multiplex arrays offer efficient protein biomarker measurement, conserving specimens and reducing costs.
• However, challenges exist in assay format optimization, dilution factor selection, and quality control (QC).
• This study examines processing, analytic, and QC issues in multiplexed immunoassays using 15 protein biomarkers.

Purpose of the Study:

• To illustrate processing, analytic, and quality control challenges encountered with multiplexed immunoassays.
• To analyze the impact of protocol deviations and sample handling on measurement accuracy.
• To explore potential alternative quality control algorithms for multiplexed data.

Main Methods:

• Duplicate measurements of 15 proteins were performed on 2322 participants using custom planar microarrays.
• Standardized protocols for sample processing, storage, and freeze-thaw cycles were applied.
• Data were analyzed for effects of processing deviations, precision, and bias, including clustering of duplicate measurements.

Main Results:

• While measurements were within reportable ranges, 7 of 15 proteins lacked centered dose-response curves.
• Additional freeze-thaw cycles and dilution errors significantly altered results.
• Duplicate measurement differences clustered by analyte, plate, and participant, challenging conventional QC algorithms.

Conclusions:

• Multiplexed immunoassay measurements present substantial analytical and statistical challenges.
• Robust quality control algorithms are crucial for reliable data interpretation.
• Further development is needed to overcome these hurdles in biomarker analysis.