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R. Sack, S. Sathe, A. Beaton, D. Dartt, K. Bower, C. Coe, D. Sediq, J. Edwards, L. Peppers, P. Iserovich; Micro Well Plate Antibody Array Screening of the Pre and Post Refractive Surgical Tears for Biomarkers of Induced Dry Eye. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2547.
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To refine array technology to allow the quantitative screening of tears for biomarkers that might predict risk of dry eye (DE) and epithelialopathy induced by refractive surgery.
Commercial and custom array kits were extensively modified to increase sensitivity and eliminate a previously confounding tear matrix effect (Sack etal. Ex. Eye Res. 2007) and thereby allow the assay of 40 + low abundance protein (LAP) that modulate inflammation and wound healing. Tear samples were collected so far from 67 patients before and after refractive surgery (LASIK and PRK) with Schirmer strips (SS). Samples analyzed came from 9 individuals who developed bilateral decreases in SS values from ~30 to10-0 mm accompanied in 1 instance with epithelialopathy and matched sets of samples from 9 "controls" who exhibited negligible decrease in SS values. The bulb of each SS was extracted in a proprietary buffer and aliquots assayed with a 16 plex cytokine array and several 9 plex arrays designed to provide redundant assays for several proteins.
Sufficient levels of ~14-18 LAPs were present in most extracts to allow quantification. These include cytokines, chemokines, growth factors, angiogenic modulators and proteases and inhibitors. The concentrations of many of these proteins (both pro and anti-inflammatory) increased in the vast majority of the post surgical samples. 4/9 of the pre-surgical samples from the DE population exhibited an unique LAP profile indicative of markedly lower concentrations of 1, sometimes 2 LAPs that serve to down-regulate inflammation. This absence was most extreme in bi-lateral pre-surgical samples obtained from an individual who developed severe DE and epithelialopathy. This difference was not apparent in the post surgical samples. A similar profile has yet to be observed in the assay of comparable samples obtained from individuals with a wide range of active ocular surfaces diseases.
Micro well arrays can be used for quantitative assay of LAPs in tears. Results suggest that ~4 % of the population may have a LAP profile that is indicative of a reduced capacity to handle short-term inflammatory stress. This protein loss may be a risk factor contributing to post-surgical DE and epithelialopathy.
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