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L. Liu, S. Harloff, P. Herminghaus, D. Hartwig, T. Wedel, G. Geerling; Influence of clotting time, diluent, systemic immune disease and immuosuppressive drugs on the epitheliotrophic capacity of serum eyedrops . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4890.
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Purpose: A number of clinical studies have shown variable efficacy of serum eyedrops to treat ocular surface disorders. In these studies the protocol to produce serum varied considerably. We previously could show that the concentration of growth factors in autologous serum and its epitheliotrophic capacity depend on the centrifugation force used to prepare the serum. However, other production parameters, such as the clotting time, the type of diluent, as well as the general health of the serum donor may determine the composition of the blood product. We therefore examined the effect of these factors in a human corneal epithelial cell culture model. Methods:Blood from 10 healthy donors and individuals suffering from rheumatoid arthritis treated with systemic immunosuppressive drugs were obtained, allowed to clot for 20, 60 and 120 minutes and diluted with BSS or 0.9% saline. All samples were centrifuged under standard conditions and the EGF, FGF, HGF, TGF–ß1, PDGF–AB, Fibronectin as well as Vitamin A and E were quantified by means of routine ELISA– or HPLC–technology. SV–40 immortalised human corneal epithelial cells were cultured in 96–well plates at 37°C, 5% CO2 with a fully defined culture medium. Proliferation of cell cultures was quantified by means of a luminescence–based ATP–Assay in dose–response experiments. A colony dispersion assay was used to examine the effect on cell migration. Results:With increasing clotting time, the serum contained significantly higher amounts of EGF and HGF. Serum from healthy donors contained significantly higher amounts of fibronectin than serum from immunosuppressed donors. Support of proliferation did not depend on clotting time, diluent and the presence of rheumatoid arthritis or systemic immunosuppressive medication. However, migration was better supported by serum with longer clotting times and serum from healthy donors. Conclusions:The amount of fibronectin in serum and its capacity to support migration is reduced in patients with rheumatoid arthritis and systemic immunosuppression. The use of quality controlled serum from healthy donors other than the patient should therefore be considered as an alternative treatment if autologous serum eyedrops failed to achieve epithelial wound healing. Complete clotting helps to optimise the epitheliotrophic capacity of serum. A standard protocol for the production of autologous serum eyedrops will be presented.
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