Tear Film Mediators After Corneal Cross-Linking: Methodology Validation

Abstract

Introduction: Currently, the gold-standard treatment of progressive keratoconus (KC) is corneal collagen cross-linking (CXL), in which riboflavin and ultraviolet-A radiation increase the corneal biomechanical rigidity, arresting progression. However, post-operatory results vary significantly among patients. Although it has been suggested that corneal thickness, cone location and patient’s age are important factors, their contribution is controversial. The reason for this variability remains obscure. This project aims to find biomarkers that could explain the variability within surgical results after corneal CXL by investigating the correlation between the local corneal inflammatory environment and these results. We hypothesize that better post-operatory results are related to a reduction in the inflammatory cytokine levels in the patients’ tear film. Ultimately, we aim to optimize the patients’ surgical outcomes according to their corneal inflammatory environment.
Methodology: The research protocol was established after testing different sample collection methods. Patients are interviewed to assess their medical history and relevant habits. Their corneal tomographic and best corrected visual acuity data are registered before and 6 months after surgery. The patient´s tear film is sampled twice 6 months apart using Schirmer strips, as the volume yield is larger compared to direct methods (microcapillary tubes or micropipettes), which are more difficult to perform and require stimulation or the instillation of saline into the cul-de-sac, originating reflex tearing. The samples are analyzed for their total protein content (BCA Protein Assay Kit) and cytokine concentration using Multiplex technology (Th1/Th2/Th9/Th17 Cytokine 18-Plex Human Procarta-Plex™ Panel) and not ELISA, as we tried before. Statistical analysis to assess sample differences and the correlation between cytokine concentration and tomographic indexes and visual acuity values for each group and subgroup is performed at 6 months.
Results: Twenty patients and eight control subjects have been enrolled in the study and the first collection of tears has been carried out. CXL surgeries for the twenty patients were per-formed from October 2020 to September 2021. Two samples were lost due to excessively length of dry Schirmer strip, which we improved by cutting the dry portion. Additionally, the tear fluid from the first eight samples from patients and from control participants was successfully processed for their total protein content.
Conclusion: Tear film analysis poses several challenges concerning the small amount of material available without reflex production. This fact precludes the use of conventional ELISA, requiring multiplex modified technology. When dealing with tears it is also important to avoid contamination by using sterilized material, to avoid dilution and to establish an efficient flow of samples from the clinic to the laboratory. This pilot study enabled to identify several methodological frailties and to establish a solid research protocol that can be replicated by other researchers. Understanding the correlation between inflammatory cytokines and surgical outcomes of CXL may allow for the optimization and personalization of CXL. In addition, the results of this research protocol are expected to establish prognostic factors for CXL-related surgical outcomes, ultimately improving the quality of life of KC patients.