Clinical Chemistry
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Clinical Chemistry 55: 1092-1099, 2009. First published April 16, 2009; 10.1373/clinchem.2008.120717
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(Clinical Chemistry. 2009;55:1092-1099.)
© 2009 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Measurement and Quality Control Issues in Multiplex Protein Assays: A Case Study

Allison A. Ellington1, Iftikhar J. Kullo1, Kent R. Bailey2 and George G. Klee3,a

1 Division of Cardiovascular Diseases, 2 Department of Biostatistics, and 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

aAddress correspondence to this author at: 200 1st St. SW, Rochester, MN, 55905. Fax: 507-266-5193; e-mail klee.george{at}mayo.edu.

Background: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays.

Methods: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias.

Results: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms.

Conclusions: Multiplex measurements present difficult challenges that require further analytical and statistical developments.




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Home page
 Clin. Chem.Home page
L. J. Kricka and S. R. Master
Quality Control and Protein Microarrays
Clin. Chem., June 1, 2009; 55(6): 1053 - 1055.
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