Assessment of Variability of Intraocular Inflammatory Biomarkers in Ocular Fluid Samples using Multiplex Assays

Author Block: Daniel Lamoureux 1, David Wong2, Tina Felfeli2. 1Northern Ontario School of Medicine, 2University of Toronto. 

Author Disclosure Block: D. Lamoureux: None. D. Wong: None. T. Felfeli: None.

Abstract Body: 

Purpose: The use of biomarkers in clinical decision making is becoming more prevalent in the field of ophthalmology for diagnostics, prognostication and personalized treatment. Prior to implementation in patient care, it is imperative to ensure that findings in biomarker analyses are accurate and reliable. There is a paucity of literature on the variability of ocular biofluid samples when they are run as duplicates with multiplex assays, and if certain factors such as storage time, type of analyte, and type of ocular sample, have an impact on the variability of the assay results. This study aims to evaluate if the aforementioned factors have a significant influence on the variability of multiplex assay results. 

Study Design: Laboratory study. 

Methods: A total of 152 human ocular biofluid samples (51 aqueous humor and 101 vitreous) were collected and assayed for 27 cytokine biomarker concentrations (pg/ml). All samples were analyzed in duplicates using the Bio-Plex Pro Human Cytokine 27-plex (multiplex) assay. Four sample storage durations following sample collection were evaluated (baseline, 3 months, 9 months, and 15 months). Statistical methods including paired samples t-test, 3-way ANOVA, intraclass correlation coefficient (ICC; <0.5-0.75=poor-moderate, 0.75->0.90 =good-excellent reliability), and coefficients of variation (CV) were employed to evaluate for statistical significance, with Bonferroni corrected P=.002. 

Results:Amongst the 4,104 biomarker duplicates assays for aqueous humor and vitreous, 2 analytes (PDGF-BB and IL-7) had a statistically significant difference between the sampled concentrations of the duplicates in vitreous samples (mean(diff)=2.05, P<.001, mean(diff)=1.56, P<.001, respectively). No statistically significant differences were identified using a 3-way ANOVA examining storage time, biofluid and analyte, suggesting that these factors do not have an influence on the variability of the multiplex assay results run as duplicates. Majority of the ICC values fell within the good-excellent range (86% of samples) with a minority falling in the poor-moderate range (14% of samples). The CV calculated for each set of duplicates suggested that 93% of duplicates had an acceptable level of quantitative assay variability (CV<20%). 

Conclusions: This study suggests that analysis of human ocular biofluid samples using multiplex assays for cytokine biomarkers as singles instead of duplicates will likely produce a favorable result. These findings may guide researchers with the design of their studies on ophthalmic biomarker analysis.