Akkurt Arslan Murat, Rabut Ghislaine, Chardonnet Solenne, Pionneau Cédric, Kobal Alfred, Gratas Pelletier Marilyne, Harfouche Nouara, Réaux La Goazigo Annabelle, Baudouin Christophe, Brignole-Baudouin Françoise, Kessal Karima
Institut National de La Santé et de La Recherche Médicale INSERM UMRS 968, CNRS, UMR 7210, Institut de La Vision, IHU ForeSight, Sorbonne Université UM80, 75012, Paris, France.
Hôpital National de La Vision des 15-20, Service 3, 75012, Paris, France.
Exp Eye Res. 2023 Dec;237:109679. doi: 10.1016/j.exer.2023.109679. Epub 2023 Oct 18.
The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).
泪膜在眼表与外部环境之间形成一道保护屏障。尽管其体积微小，但近期在分析前和分析程序方面的进展使得能够采用多种方法对其进行深入分析。然而，泪膜采集方法的多样性以及分析前方法缺乏标准化是获得可重复结果以及不同研究之间进行比较的主要障碍。在本研究中，我们首先改进了从泪液滤纸条（ScS）中提取各种分子实体的分析前程序。随后，我们的研究重点是分析两种主要泪液采集方法——毛细管（CT）和ScS之间可能出现的分子差异。此外，我们检查了ScS的不同部位以强调这些差异，这可能是制定标准化、优化样本处理方案的关键因素。我们的结果表明，在提取过程中加入表面活性剂可有效裂解细胞并提高几种细胞内蛋白质的溶解度，从而提高蛋白质提取产量以及在ScS中鉴定出的蛋白质数量。除了蛋白质外，还可以回收核酸用于基因表达分析，特别是从置于结膜囊的ScS的球部区域回收。尽管提取物较为稀释，但由于采用了高灵敏度免疫测定法，ScS提取物仍是用于检索低至fg/mL水平的泪液蛋白质（如IL - 17A）的合适材料。采集方法会影响所测得的泪液蛋白质水平。与用CT采集的泪液相比，在使用ScS采集的泪液的毛细管电泳分析中发现乳铁蛋白的百分比更高（39.6 ± 4.8%对31 ± 4.4%）。
