Development of a limbal stem cell deficiency diagnostic system for ophthalmic use. an example of the molecular genetics applications to clinical demands

Resumen

Limbal Stem Cell Deficiency (LSCD) is a pathological eye condition characterized by the loss/disfunction of the regenerative and/or barrier function of the limbal stem cells, deriving in the invasion of conjunctival epithelium onto the cornea (conjunctivalization).Goblet cells are responsible for the production of mucin 5ac, which is a gel-forming glycoprotein with important roles in tear film maintenance. Given that these cells are only found in the conjunctival epithelium in healthy conditions, MUC5AC expression in the corneal epithelium is an indicative of corneal conjunctivalization, which in turn is indicative of LSCD. The lack of a sufficiently sensitive and standardized method for LSCD diagnosis, encouraged us to assess the presence of the MUC5AC transcript in corneal impression cytologies for LSCD diagnosis.For that purpose, custom primers for MUC5AC mRNA detection were designed after sequence characterization, and comparatively tested with a set of previously published primers. The assay with best results was then selected for clinical validation. Based on these findings, a kit for use in hospital laboratories was further developed and validated in a large set of samples.The results obtained in this work showed that the molecular detection of the MUC5AC transcript in corneal epithelium is a more sensitive method for LSCD diagnosis than PAS-hematoxylin staining, and correlates better with clinical diagnosis. More importantly, this system may detect early stages of LSCD when a clinical suspicion exists. Moreover, the kit developed for use in hospital laboratories is slightly more sensitive than the standard method performed in the laboratory and, additionally, the PCR-strip system enhances user-friendliness, specificity, result visualization and diagnosis. Finally, the possibility of automating the result development with an automatic dispensing system commonly found in hospital laboratories, made the kit more easily implementable.This system has several potential applications, as it can help on diagnosing clinically uncertain samples and assist the ophthalmologist on surgery decision taking. In addition, this system can also be used to verify corneal restoration after treatment or surgery, and to monitor long-term contact lens users, among others.