Quantifying Sample Collection and Processing Impacts on Fiber-Based Tear Fluid Chemical Analysis

PURPOSE: Noninvasive analyses of tear fluid from humans and animal models in clinical and research settings most commonly use absorbent material for collection and processing. Still, the impact of these analytical techniques on tear chemical analyses remains largely unknown. The purpose of this study was to quantify the impacts of phenol red thread fiber-based tear sample collection and processing on the primary amine content. METHODS: Human tears were collected by placing the folded end of phenol red thread on the palpebral conjunctiva of the right eye for 20 seconds. The wetted thread was then processed using elution or extraction, and capillary electrophoresis with light-emitting diode-induced fluorescence detection was used for analysis and quantitation. RESULTS: Distinct processing methods impacted tear analysis differently. Primary amines adsorbed onto the thread partitioned in a chromatographic manner and thus any single portion of the wetted thread might not be representative of the whole sample. Quantitative assessment of five small molecule standards after on-thread processing showed significant overestimation of the actual concentration, with increased accuracy for larger volume samples. Yet collection of larger tear volumes introduced error in volume determination owing to evaporation and reduced small molecule separation resolution. CONCLUSIONS: These results indicated that absorption-based tear fluid collection and processing significantly alter chemical content analysis, suggesting that the impacts of methods used should be regularly evaluated to standardize results drawn from different studies. TRANSLATIONAL RELEVANCE: This study identifies potential inconsistencies and inaccuracies in tear analyses that are widespread across the published literature and clinical care.