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S.J. Bakri, T. Bonfield, P. Kaiser, V.L. Perez; Microsphere Luminex Technology for the Analysis of Microliters of Vitreous Fluid . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5115.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Microsphere Luminex technology allows simultaneous analysis of 22 cytokines, chemokines and biochemical factors in small 50ul volume samples. It has been previously used for analysis of other bodily fluids, but its application for vitreous analysis has not been described. The goal of this work is to determine the feasibility of analyzing microliters of vitreous fluid using microsphere luminex technology Methods: Vitreous samples were obtained from 6 patients during scheduled pars plana vitrectomy. Diagnoses were epiretinal membrane (2), posterior hyaloidal traction in a diabetic patient (1), retained lens material after cataract extraction (1), macular hole (1), and vitreous hemorrhage due to choroidal neovascularization from age–related macular degeneration (1). The vitreous samples were analyzed neat, diluted with phosphate buffered saline (PBS), and pretreated with testicular hyaluronidase to determine both the effect of dilution on the sample, and whether the hyaluronidase digestion was necessary to reduce the viscosity of the sample in the machine. Microsphere Luminex technology was used with a 22–plex kit which analyzed the following 22 cytokines: IL–1ß, IL–2, IL–4, IL–5, IL–6, IL–7, IL–8, IL–10, IL–12, IL–13, IL–15, IL–17, IFN–g, TNF–α, GMCSF, IL–1a, Eotaxin, G–CSF, IP–10, MCP–1, MIP–1a and RANTES. Results: Luminex was successfully used to detect different chemokines and cytokines in the vitreous fluid with a sensitivity comparable to ELISA. Pretreatment with hyaluronidase was not necessary for the vitreous analysis and undiluted vitreous can be used for this analysis. In these samples, the most dominant cytokines were IL–8, IP–10, and MCP–1. Not surprisingly, the patient with vitreous hemorrhage had the highest levels of cytokines and chemokines measured. Conclusions: This pilot study shows that Microsphere Luminex technology is feasible for the analysis of multiple inflammatory factors in a small volume of undiluted vitreous fluid. In this limited study, the chemokines and cytokines measured represent a population of factors consistent with the recruitment of inflammatory cells. The use of Luminex in the analysis of small volumes of intraocular fluid has great potential in elucidating new immune–mediated mechanisms in ocular diseases.
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