Purpose:Certain factors such as instrumental and sample processing errors may contribute to variability of ocular biofluid samples when they are run as replicates with multiplex assays. There is a paucity of literature on the variability of replicates in multiplex assays. This study aims to evaluate whether there is significant variability in replicate analyses of multiplex assays.
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). Samples were evaluated as replicates (duplicate analysis) at four different time points. 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=0.002.
Results:Among the 4104 biomarker replicate assays for aqueous humor and vitreous, two analytes (PDGF-BB and IL-7) had a statistically significant difference between the sampled concentrations of the replicates in vitreous samples (mean (diff)=2.05, P<0.001, mean (diff)=1.56, P<0.001, respectively). 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). More variability was noted in the vitreous humour, with five analytes (IL-2, IL-10, IL-12(p70), IL-13, IL-17) demonstrating an average ICC of less than 0.5. The CV calculated for each set of replicates suggested that 93% of replicates had an acceptable level of quantitative assay variability (CV<20%).
Conclusion:This study demonstrates that the analysis of most biomarkers in ocular fluids may not require the use of replicates. However, certain analytes such as PDGF-BB and IL-7 may require the use of replicates to ensure reliable results. Caution should be taken when applying these findings to other laboratory settings as our study was conducted by an experienced technician using a standardized protocol. In less standardized settings, replicates may be required in order to ensure accuracy of results. These findings may guide researchers with the design of their studies on ophthalmic biomarker analysis.
Keywords:aqueous; assay; biomarkers; cytokines; vitreous.

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乐备实（上海优宁维生物科技股份有限公司旗下全资子公司），是国内专注于提供高质量蛋白检测以及组学分析服务的实验服务专家，自2018年成立以来，乐备实不断寻求突破，公司的服务技术平台已扩展到单细胞测序、空间多组学、流式检测、超敏电化学发光、Luminex多因子检测、抗体芯片、PCR Array、ELISA、Elispot、PLA蛋白互作、多色免疫组化、DSP空间多组学等30多个，建立起了一套涵盖基因、蛋白、细胞以及组织水平实验的完整检测体系。

 
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