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R. K. Sharma, A. Rogojina, K. V. Chalam; Use of Multiplex Technology for Analyzing Human Aqueous Samples for a Battery of Analytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):361.
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Aqueous is intimately related to the cells of anterior and posterior chambers which effect its composition. Aqueous analysis can provide useful information regarding the ocular environment including physiological and pathophysiological processes in the associated tissues. One major limitation of testing aqueous is that only small sample volumes can be obtained from human eyes which are insufficient to test large number of cytokines by traditional ELISA techniques.
In this study we have used a new technology developed by Luminex xMAP® that can analyze hundreds of analytes in a small volume of sample. In this pilot experiment, approximately 100 microL of aqueous humor was obtained from 3 patients undergoing cataract surgery and analyzed for a battery of 89 analytes.
All the analytes could be examined at least in one sample. Of 89 analytes 48 were detectable in the aqueous. To place these results in biological context, we analyzed the list of detectable analytes using Ingenuity Pathways Analysis software (Ingenuity® Systems). These analyses represented 32 functional categories with a significant representation from complement and coagulation cascades, chemokine signaling, hypoxia signaling in the cardiovascular system, leukocyte extravasation signaling, IL-6 signaling, and PPAR/RXR activation pathways. Ingenuity Pathway analysis revealed that detected analytes robustly represented three networks of genes involved in basal cell machinery. The aqueous values of detectable analytes were correlated with serum values by plotting a z-score of the fraction serum value detected in the aqueous. It was previously believed that the aqueous contains approximately 1% of the serum value of various proteins. However, our results showed that a significant number of analytes were present in concentrations higher than 1% of the serum values.
These results demonstrate Multiplex technology can assess levels of large number of analytes in small volumes of patient aqueous samples. It can be a useful tool for screening ocular environment for pathophysiological changes.
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