Lamoureux Daniel, Wong David T, Felfeli Tina
Northern Ontario School of Medicine University, Thunder Bay, ON, Canada.
Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, ON, Canada.
Clin Ophthalmol. 2023 Sep 8;17:2653-2663. doi: 10.2147/OPTH.S417821. eCollection 2023.
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.
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 -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.
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%).
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.
当使用多重检测方法对眼生物流体样本进行重复检测时，诸如仪器和样本处理误差等某些因素可能导致样本变异性。关于多重检测中重复样本变异性的文献较少。本研究旨在评估多重检测的重复分析中是否存在显著变异性。
共收集了152份人眼生物流体样本（51份房水和101份玻璃体），并检测了27种细胞因子生物标志物浓度（pg/mL）。在四个不同时间点将样本作为重复样本进行评估（重复分析）。采用配对样本t检验、三因素方差分析、组内相关系数（ICC；<0.5 - 0.75为差 - 中等，0.75 -> 0.90为好 - 极好的可靠性）和变异系数（CV）等统计方法来评估统计学显著性，经Bonferroni校正后P = 0.002。
在对房水和玻璃体进行的4104次生物标志物重复检测中，两种分析物（血小板衍生生长因子 - BB和白细胞介素 - 7）在玻璃体样本的重复样本采样浓度之间存在统计学显著差异（平均（差值）= 2.05，P < 0.001；平均（差值）= 1.56，P < 0.001）。大多数ICC值落在好 - 极好范围内（86%的样本），少数落在差 - 中等范围内（14%的样本）。在玻璃体液中观察到更多变异性，有五种分析物（白细胞介素 - 2、白细胞介素 - 10、白细胞介素 - 12(p70)、白细胞介素 - 13、白细胞介素 - 17）的平均ICC小于0.5。为每组重复样本计算的CV表明，93%的重复样本具有可接受的定量检测变异性水平（CV < 20%）。
本研究表明，眼液中大多数生物标志物的分析可能不需要使用重复样本。然而，某些分析物如血小板衍生生长因子 - BB和白细胞介素 - 7可能需要使用重复样本以确保结果可靠。由于我们的研究是由一名经验丰富的技术人员按照标准化方案进行的，因此在将这些结果应用于其他实验室环境时应谨慎。在标准化程度较低的环境中，可能需要重复样本以确保结果的准确性。这些发现可为研究人员设计眼科生物标志物分析研究提供指导。
