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K. Chalam, S. Grover, S. Gupta, V. S. Brar, R. K. Sharma; Evaluation of Vascular Endotheilal Growth Factor Levels After Intravitreal Bevacizumab in Retinal Disorders. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3459.
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VEGF levels in both the aqueous and vitreous are elevated in patients with ocular ischemia secondary to exudative age-related macular degeneration, diabetes mellitus, and other retinal vascular diseases. We describe a unique assay for quantifying VEGF levels, that combines ELISA and flow cytometry with minimal quantity of aqueous aspirated at the time of intravitreal injection. Utilizing this sensitive technique, we investigated the effect of intravitreal injections of bevacizumab on VEGF levels in a variety of retinal disorders.
Aqueous samples were collected prior to and at monthly intervals at the time of intravitreal injection of bevacizumab (1.25 mcg in 0.05ml) for a variety of retinal disorders (N=24). Aqueous samples obtained from normal patients at the time of cataract surgery were used as controls (N=10). Detection of VEGF in the aqueous samples was performed at Rule Based Medicine Inc. using multiplex analysis. In brief, sample was incubated with capture microsphere multiplexes of the Human Antigen MAP for one hour at room temperature. Multiplexed cocktails of biotinylated reporter antibodies for each multiplex were then added, mixture incubated for an additional hour and multiplexes developed using streptavidin-phycoerythrin solution. Analysis was performed in a Luminex 100 instrument and the data was interpreted using proprietary data analysis software (Qiagen Instruments).
Vascular endothelial growth factor concentration in the aqueous humor ranged from 326 to 12476 pg/ml (mean±SD, 1306±125 pg/ml) before intravitreal injection of bevacizumab and decreased to less than 180 pg/ml (P< 0.01) in all eyes one month after injection. The concentrations in the control group ranged from 120 to 218 pg/ml (mean±SD, 162±42 pg/ml).
This study was also unique in that the assay employed to determine aqueous VEGF levels, utilized a combination of ELISA and flow cytometry, which afforded increased sensitivity in detecting VEGF, with the lowest detected value of 1.5 pg/ml. Estimation of VEGF in aqueous samples with ELISA is difficult as it requires large volume for reliable estimation. Our method utilizes only 100 mcl. and overcomes this difficulty.Intracameral and intravitreal dose of bevacizumab is empirical at this time. Neutralization of all available VEGF is necessary for successful resolution of retinal pathology. Reliable estimation of VEGF and subsequent administration of appropriate dose of bevacizumab is ideal in treatment of proliferative retinovasculopathies.
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