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Suzanne Hagan, Alan Tomlinson, Louise Madden, Anne Marie Clark, Katherine Oliver, Ocular Surface; Tear Film Biomarker Profiling of Subjects with Dry Eye Disease by Multiplex Analysis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):955.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease (DED) is a distressing disorder, commonly associated with ageing, contact lens wear and autoimmune syndromes. It affects 15%-30% of the over-50s in Western and Asian populations and is one of the fastest-growing eye problems in this demographic. DED is significantly underdiagnosed and no “gold standard” currently exists for its clinical diagnosis. Recent Multiplex studies of tear fluids from DED subjects have implicated inflammatory cytokines in this disorder. In this study, we investigated tear fluids from DED and normal subjects for a panel of cytokines using the multiplex bead assay.
This study comprised 15 DED subjects (3 males, 12 females) and 20 healthy controls (2 males, 18 females). All subjects provided informed consent and the study adhered to the Declaration of Helsinki tenets. Tear samples were collected from the external canthus of open eyes, avoiding additional tear reflex. Glass microcaps were used to collect 1μl tears. Samples were diluted 1:50 and stored at -80C until use. Tear cytokine levels were determined with a multiplex bead assay (R and D Systems) and quantified using a Luminex IS200. Briefly, tear samples were incubated with specific antibody-coated beads for 3h. Washed beads were then incubated with biotin-labelled secondary antibodies, followed by a streptavidin-PE incubation. Standard curves of known cytokine concentrations were used to calculate protein concentrations and data underwent analysis by an in-house statistician.
Detectable levels of IL-8 (> 4.05pg/ml) were observed in 12/15 DED subjects (mean 23.1pg/ml) and 16/19 normals (mean 20.6pg/ml). Some variation in IL-8 levels was noted in DED subjects (4.9-83.8pg/ml), but a trend for increased IL8 in DED subjects was observed, compared to normals. Moreover, detectable levels of all 7 inflammatory proteins (IL1β, IL2, IL6, IL8, IL17, TNF-α and IFN-γ) were observed in 2 DED subjects, a result not observed in normals.
Increased IL8 levels in DED suggests a function in ocular surface inflammation. This data confirms previous Multiplex bead studies, indicating a role for IL8 in DED. Further studies may also shed light on roles for the other 6 cytokines detected in 2 DED subjects. Moreover, this technology appears to be sensitive enough to detect low abundance proteins in minute sample sizes and therefore may be useful in screening tear fluids for potential biomarkers of DED.
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