Nuffield Department of Orthopaedics, Rheumatology & Musculoskeletal Sciences, University of Oxford, UK.
J Immunol Methods. 2012 Dec 14;386(1-2):22-30. doi: 10.1016/j.jim.2012.08.012. Epub 2012 Aug 28.
Synovial fluid (SF) is a difficult biological matrix to analyse due to its complex non-Newtonian nature. This can result in poor assay repeatability and potentially inefficient use of precious samples. This study assessed the impact of SF treatment by hyaluronidase and/or dilution on intra-assay precision using the Luminex and Meso Scale Discovery (MSD) multiplex platforms. SF was obtained from patients with knee osteoarthritis at the time of joint replacement surgery. Aliquots derived from the same sample were left untreated (neat), 2-fold diluted, 4-fold diluted or treated with 2mg/ml testicular hyaluronidase (with 2-fold dilution). Preparation methods were compared in a polysterene-bead Luminex 10-plex (N=16), magnetic-bead Luminex singleplex (N=7) and MSD 4-plex (N=7). Each method was assessed for coefficient of variation (CV) of replicate measurements, number of bead events (for Luminex assays) and dilution-adjusted analyte concentration. Percentage recovery was calculated for dilutions and HAse treatment. Hyaluronidase treatment significantly increased the number of wells with satisfactory bead events/region (95%) compared to neat (48%, p<0.001) in the polystyrene-bead Luminex assay, but the magnetic-bead Luminex assay achieved ≥50 bead events irrespective of treatment method. Hyaluronidase treatment resulted in lower intra-assay CVs for detectable ligands (group average CV<10%) than neat, 2-fold and 4-fold dilution (CV~25% for all, p<0.05) in both polystyrene- and magnetic-bead Luminex assays. In addition, measured sample concentrations were higher and recovery was poor (elevated) after hyaluronidase treatment. In the MSD 4-plex, within-group comparison of the intra-assay CV or concentration was not conclusively influenced by SF preparation. However, only hyaluronidase treatment resulted in CV<25% for all samples for TNF-α. There was no effect on analyte concentrations or recovery. Hyaluronidase treatment can improve intra-assay precision and assay signal of SF analysis by multiplex immunoassays and should be recommended for SF biomarker research, particularly using the Luminex platform.
滑液（SF）是一种难以分析的生物基质，因为其具有复杂的非牛顿特性。这可能导致分析重复性差，并可能对珍贵样本的利用效率低下。本研究评估了使用 Luminex 和 Meso Scale Discovery（MSD）多重平台，通过透明质酸酶和/或稀释对关节液内分析精度的影响。SF 是从接受膝关节置换手术的骨关节炎患者的关节中获得的。从同一样本中提取的等分试样未经处理（原样）、2 倍稀释、4 倍稀释或用 2mg/ml 睾丸透明质酸酶处理（2 倍稀释）。在聚苯乙烯珠 Luminex 10 plex（N=16）、磁性珠 Luminex 单plex（N=7）和 MSD 4-plex（N=7）中比较了不同的制备方法。评估了每种方法的重复性测量的变异系数（CV）、珠事件数（用于 Luminex 测定）和稀释调整后的分析物浓度。计算了稀释和 HAse 处理的回收率。与原样（48%，p<0.001）相比，透明质酸酶处理显着增加了具有满意珠事件/区域的孔数（95%），在聚苯乙烯珠 Luminex 测定中，但磁性珠 Luminex 测定法无论处理方法如何，都能获得≥50 个珠事件。透明质酸酶处理导致可检测配体的内分析 CV 降低（组平均 CV<10%），而原样、2 倍稀释和 4 倍稀释的 CV 均为~25%（所有均为 p<0.05），在聚苯乙烯珠和磁性珠 Luminex 测定中。此外，透明质酸酶处理后，测量的样品浓度较高，回收率差（升高）。在 MSD 4-plex 中，SF 制备对内分析 CV 或浓度的组内比较没有明显影响。然而，只有透明质酸酶处理导致所有样品的 TNF-α的 CV<25%。对分析物浓度或回收率没有影响。透明质酸酶处理可改善多重免疫分析中 SF 分析的内分析精度和分析物信号，应推荐用于 SF 生物标志物研究，特别是使用 Luminex 平台。
