Optimized Autologous Serum and Activated Leukocyte-Poor Platelet-Rich Plasma Eye Drops Production Protocol: Effect of Fasting, Filtering, and Complement Inactivation on the Concentration of Growth Factors and Lipids

Purpose: To analyze the effect of fasting on volume, growth factor, and lipid concentration in eye drops derived from autologous serum (AS) and leukocyte-poor platelet-rich plasma (LP-PRP). To assess changes in protein and growth factor concentration after filtration with three different syringe filters and after complement inactivation with heat. Methods: Blood samples from 10 volunteers after a fasting and nonfasting period were harvested on two separate visits. The total volume gained of AS and LP-PRP was measured. Also, the concentration of lipids, albumin, lysozyme, beta-nerve growth factor (β-NGF), transforming growth factor-β1 (TGF-β1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) were quantified in each preparation. The effect of microfiltration was measured in pooled AS and LP-PRP samples before and after filtration with polyethersulfone, polyvinylidene fluoride, or mixed cellulose esters (MCE) membrane syringe filters. The extent of complement inactivation was quantified after incubation at +56°C for 15-240 min. Results: The total volume of AS and LP-PRP and the protein and growth factor concentration remained similar after fasting. Increased triacyl glycerides and similar phospholipid levels were observed in nonfasting samples. Except for a minimal decrease in TGF-β1 after filtration with an MCE membrane filter, filtering did not reduce the concentration of growth factors in AS and LP-PRP. Heat inactivation considerably reduced levels of lysozyme, EGF, HGF, and β-NGF in AS and LP-PRP. Conclusions: AS should be produced after a fasting period to facilitate the filtration. However, nonfasting does not affect concentrations of relevant biologically active constituents and phospholipids.