Restricted accessResearch articleFirst published online 2026-1
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
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.
SangwanVS, TsengSC. New perspectives in ocular surface disorders. An integrated approach for diagnosis and management. Indian J Ophthalmol, 2001; 49(3):153–168.
2.
SternME, SchaumburgCS, DanaR, et al.Autoimmunity at the ocular surface: Pathogenesis and regulation. Mucosal Immunol, 2010; 3(5):425–442; doi: 10.1038/mi.2010.26
SheppardJD, NicholsKK. Dry eye disease associated with meibomian gland dysfunction: Focus on tear film characteristics and the therapeutic landscape. Ophthalmol Ther, 2023; 12(3):1397–1418; doi: 10.1007/s40123-023-00669-1
8.
KlenklerB, SheardownH, JonesL. Growth factors in the tear film: Role in tissue maintenance, wound healing, and ocular pathology. Ocul Surf, 2007; 5(3):228–239; doi: 10.1016/S1542-0124(12)70613-4
9.
FoxRI, ChanR, MichelsonJB, et al.Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis Rheum, 1984; 27(4):459–461.
10.
TsubotaK, GotoE, FujitaH, et al.Treatment of dry eye by autologous serum application in Sjogren’s syndrome. Br J Ophthalmol, 1999; 83(4):390–395.
TananuvatN, DaniellM, SullivanLJ, et al.Controlled study of the use of autologous serum in dry eye patients. Cornea, 2001; 20(8):802–806.
13.
TsubotaK, SatakeY, OhyamaM, et al.Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am J Ophthalmol, 1996; 122(1):38–52.
KangMJ, LeeJH, HwangJ, et al.Efficacy and safety of platelet-rich plasma and autologous-serum eye drops for dry eye in primary Sjogren’s syndrome: A randomized trial. Sci Rep, 2023; 13(1):19279; doi: 10.1038/s41598-023-46671-2
16.
WangL, CaoK, WeiZ, et al.Autologous serum eye drops versus artificial tear drops for dry eye disease: A systematic review and meta-analysis of randomized controlled trials. Ophthalmic Res, 2020; 63(5):443–451; doi: 10.1159/000505630
17.
HeCZ, ZengZJ, LiuJQ, et al.Autologous serum eye drops for patients with dry eye disease: A systematic review and meta-analysis of randomized controlled trials. Front Med (Lausanne), 2024; 11:1430785; doi: 10.3389/fmed.2024.1430785
18.
Lopez-PlandolitS, MoralesMC, FreireV, et al.Plasma rich in growth factors as a therapeutic agent for persistent corneal epithelial defects. Cornea, 2010; 29(8):843–848; doi: 10.1097/ICO.0b013e3181a81820
19.
Lopez-PlandolitS, MoralesMC, FreireV, et al.Efficacy of plasma rich in growth factors for the treatment of dry eye. Cornea, 2011; 30(12):1312–1317; doi: 10.1097/ICO.0b013e31820d86d6
20.
Sanchez-AvilaRM, Merayo-LlovesJ, FernandezML, et al.Plasma rich in growth factors for the treatment of dry eye after LASIK surgery. Ophthalmic Res, 2018; 60(2):80–86; doi: 10.1159/000487951
21.
SoiferM, TovarA, WangM, et al.A multicenter report of the use of plasma rich in growth factors (PRGF) for the treatment of patients with ocular surface diseases in North America. The Ocular Surface, 2022; 25:40–48; doi: 10.1016/j.jtos.2022.04.007
22.
AnituaE, AguirreJJ, AlgortaJ, et al.Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers. J Biomed Mater Res B Appl Biomater, 2008; 84(2):415–421; doi: 10.1002/jbm.b.30886
23.
TorresJ, TamimiF, MartinezPP, et al.Effect of platelet-rich plasma on sinus lifting: A randomized-controlled clinical trial. J Clin Periodontol, 2009; 36(8):677–687; doi: 10.1111/j.1600-051X.2009.01437.x
24.
Lozano-SanromaJ, BarrosA, AlcaldeI, et al.Impact of Plasma Rich in Growth Factors (PRGF) eye drops on ocular redness and symptomatology in patients with dry eye disease. Medicina (Kaunas), 2023; 59(5):928; doi: 10.3390/medicina59050928
25.
Trindade-SuedamIK, LeiteFR, de MoraisJA, et al.Avoiding leukocyte contamination and early platelet activation in platelet-rich plasma. J Oral Implantol, 2007; 33(6):334–339; doi: 10.1563/1548-1336(2007)33[334:Alcaep]2.0.Co;2
26.
AnituaE, SanchezM, ZalduendoMM, et al.Fibroblastic response to treatment with different preparations rich in growth factors. Cell Prolif, 2009; 42(2):162–170; doi: 10.1111/j.1365-2184.2009.00583.x
27.
LawsonJH. The clinical use and immunologic impact of thrombin in surgery. Semin Thromb Hemost, 2006; 32(Suppl 1):98–110; doi: 10.1055/s-2006-939559
28.
LiuL, HartwigD, HarloffS, et al.An optimised protocol for the production of autologous serum eyedrops. Graefes Arch Clin Exp Ophthalmol, 2005; 243(7):706–714; doi: 10.1007/s00417-004-1106-5
29.
BastaniA, RajabiS, KianimarkaniF. The effects of fasting during ramadan on the concentration of serotonin, dopamine, brain-derived neurotrophic factor and nerve growth factor. Neurol Int, 2017; 9(2):7043; doi: 10.4081/ni.2017.7043
30.
BradleyJC, SimoniJ, BradleyRH, et al.Time- and temperature-dependent stability of growth factor peptides in human autologous serum eye drops. Cornea, 2009; 28(2):200–205; doi: 10.1097/ICO.0b013e318186321e
31.
Lopez-GarciaJS, Garcia-LozanoI, RivasL, et al.Stability of growth factors in autologous serum eyedrops after long-term storage. Curr Eye Res, 2016; 41(3):292–298; doi: 10.3109/02713683.2015.1016180
32.
AnituaE, MuruzabalF, De la FuenteM, et al.Effects of heat-treatment on plasma rich in growth factors-derived autologous eye drop. Exp Eye Res, 2014; 119:27–34; doi: 10.1016/j.exer.2013.12.005
33.
LongQ, ChuR, ZhouX, et al.Correlation between TGF-beta1 in tears and corneal haze following LASEK and epi-LASIK. J Refract Surg, 2006; 22(7):708–712.
34.
World Medical Association. World Medical Association Declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA, 2013; 310(20):2191–2194; doi: 10.1001/jama.2013.281053
35.
SanakF, BaenningerP, KaufmannC, et al.The lucerne protocol for the production of autologous serum eyedrops. Klin Monbl Augenheilkd, 2021; 238(4):346–348.
36.
AnituaE, MuruzabalF, PinoA, et al.Biological stability of plasma rich in growth factors eye drops after storage of 3 months. Cornea, 2013; 32(10):1380–1386; doi: 10.1097/ICO.0b013e31829f7088
37.
FreireV, AndolloN, EtxebarriaJ, et al.In vitro effects of three blood derivatives on human corneal epithelial cells. Invest Ophthalmol Vis Sci, 2012; 53(9):5571–5578; doi: 10.1167/iovs.11-7340
38.
AldoP, MarusovG, SvancaraD, et al.Simple Plex(): A novel multi-analyte, automated microfluidic immunoassay platform for the detection of human and mouse cytokines and chemokines. American Journal of Reproductive Immunology (New York, NY: 1989), 2016; 75(6):678–693; doi: 10.1111/aji.12512
39.
RantamakiAH, HolopainenJM. The effect of phospholipids on tear film lipid layer surface activity. Invest Ophthalmol Vis Sci, 2017; 58(1):149–154; doi: 10.1167/iovs.16-20468
40.
LamSM, TongL, DuanX, et al.Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res, 2014; 55(2):289–298; doi: 10.1194/jlr.M044826
41.
SavilleJT, ZhaoZ, WillcoxMD, et al.Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry. Exp Eye Res, 2011; 92(3):238–240; doi: 10.1016/j.exer.2010.12.012
