HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: clinchem.2008.120717v1 55/6/1092    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ellington, A. A. Articles by Klee, G. G. Search for Related Content PubMed PubMed Citation Articles by Ellington, A. A. Articles by Klee, G. G.

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

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.

The following articles in journals at HighWire Press have cited this article:

				A. A. Ellington, I. J. Kullo, K. R. Bailey, and G. G. Klee Antibody-Based Protein Multiplex Platforms: Technical and Operational Challenges Clin. Chem., February 1, 2010; 56(2): 186 - 193. [Abstract] [Full Text] [PDF]

				L. J. Kricka and S. R. Master Quality Control and Protein Microarrays Clin. Chem., June 1, 2009; 55(6): 1053 - 1055. [Full Text] [PDF]