Measurement and quality control issues in multiplex protein assays: a case study.
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.
Ellington
Allison A
AA
Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN, USA.
Kullo
Iftikhar J
IJ
Bailey
Kent R
KR
Klee
George G
GG
D015331:Cohort Studies; D006801:Humans; D011506:Proteins / Q000737:chemistry*; D011786:Quality Control
D011506:Proteins