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S. Grover, S. Balaiya, V. S. Brar, K. V. Chalam; Measurement of Aqueous VEGF Levels in Patients With Retinal Diseases is Superior and Accurate in Fresh Than Stored Samples. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3343.
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Several studies have investigated the aqueous levels of vascular endothelial growth factor (VEGF) in establishing its role in the pathophysiology of various ocular diseases including diabetic retinopathy, exudative macular degeneration, retinal vein occlusions, etc. The aqueous samples are stored in -80 degrees freezer for variable periods of time till a set of samples are ready to be tested. The aim of the present study was to investigate if there is a significant change in the levels of VEGF in the stored aqueous samples.
More than 100 microliters of aqueous sample was collected from each of five patients with exudative age-related macular degeneration (AMD) just before an intravitreal injection of bevacizumab. The collected sample was aliquoted into two 50 microliters testing samples. VEGF levels were determined in one sample on the same day of collection while the other sample was stored in a freezer at -80 degrees. This stored sample was tested for VEGF levels after 3 weeks of storage by the same person. VEGF levels were quantified using singleplex bead immunoassay (Luminex X Map) in which each spectral bead conjugates with protein/analyte specific capture antibody at specific bead region. Each analyte binds to its specific antibody followed by the detector antibody and the analyte levels are quantified by monitoring the associated fluorescence after the addition of fluorescent tag. A known concentration of recombinant human VEGF is included as an internal control in addition to assay standards.
A graphical plot was constructed using standard concentration against standard fluorescence. Statistical analysis was performed to determine the VEGF levels in normal aqueous samples. The VEGF levels in 5 patients with AMD performed on the same day of collection of the aqueous samples were 212.14, 228.46, 132.02, 80.1 and 45.98 pg/mL. After 3 weeks of storage, the aqueous VEGF levels in corresponding patients were 90.09, 59.16, 45.72, 36.30 and 49.75 pg/mL (p<0.001).
This pilot study highlights that VEGF levels in aqueous may change significantly after storage for 3 weeks. Future studies need to be done on a larger cohort of samples to confirm the validity of this study.
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