The healing mechanisms of the corneal epithelium require adequate cell proliferation, migration, intercellular and basement membrane adhesion , all these processes are mainly regulated by the epitheliotrophic factors, fibronectin, and vitamin A. The concentration of epitheliotrophic factors plays a preponderant role in the therapeutic effects of topical autologous serum , which are dose-dependent  and strongly influenced by the production technique [21, 22]. The results of this study show that concentrations of epitheliotrophic factors are modified by increasing coagulation times. This effect has been related to the role of platelet alpha granules as primary storage source of these epitheliotrophic factors , a longer coagulation time results in platelet disintegration as well as secondary aggregation [25, 26] and increased output of epitheliotrophic factors.
The present study shows that EGF concentrations increase in a directly proportional manner to coagulation time,. In contrast, TGF-β1 exhibits the opposite behavior, which is partially consistent with the results reported by Liu et al., who found an increase in EGF, TGF-β1 and HGF . The reason why TGF-β1 decreases are not clearly understood.
The therapeutic effect of EGF is dose-dependent and mediated by a Fibronectin-dependent system ; its major effect is in the epithelium and corneal endothelium, where there are high and low-affinity receptors. At the same time, in keratocytes, there are only low-affinity receptors .
The TGF-B is a polypeptide that has pro and anti-scar functions, as it stimulates skin healing . However, is also a potent inhibitor of epithelial and endothelial cell proliferation [7, 11–13], probably through the modulation of other epitheliotrophic factors such as EGF [23, 29].
Fibronectin is an indispensable component for the healing of the corneal epithelium promoting the proliferation, differentiation, and migration of the corneal epithelium as well as the production of collagen  , Like Liu et al. , in our study we did not find a statistically different fibronectin concentrations associated with increased clotting time, this is because plasma fibronectin is produced in the liver , so its concentration is not influenced by the time of clot formation .
Vitamin A is vital to keep the ocular surface healthy and helps repair processes , the lack of vitamin A causes xerophthalmia characterized by an abnormal differentiation of the ocular surface that results in corneal and conjunctival keratinization, ulceration, metaplasia squamous epithelial and loss of conjunctival goblet cells [33, 34] as well as increased synthesis of fibronectin ; The origin of this vitamin is in the diet , is stored in the liver and is transported in the blood by its specific receptor (retinol-binding protein) . In our study we did not find a statistically significant change in vitamin A concentrations, this agrees with what was reported by Mejia and collaborators who studied the changes in vitamin A concentration by modifying the separation time of serum and clot 2, 4, 6, 12 and 24 hours, finding that at room temperature (26 to 28 º C) the concentration was stable for up to 24 hours  this could be since the retinol-binding protein does not change with the formation time of the clot.
EGF, and Fibronectin promotes corneal epithelial cell migration and proliferation, and TGF-β1 inhibit epithelial proliferation in a dose-dependent manner so longer coagulation time increases EGF and decreases TGF-β1 could improve corneal epithelial cell migration and proliferation.
This study has some limitations; the first one is the sample size; however, our sample size is similar to that of other studies [10, 16, 17, 21, 39–42]; since, due to the cost of supplies, it is difficult to include more patients. In addition, our study population it´s mainly of young adults, and according to Liu et al. age of the donors has an impact on Fibronectin and TGF-β1 concentrations , we cannot ensure that the effect of coagulation time is similar in all age groups. Finally, the clinical impact or therapeutic effects of the changes in the concentrations found was not evaluated.
In conclusions, a prolonged clotting time (24 hours) have a significant impact on the composition and epitheliotrophic factors of the serum, increasing the EGF concentrations, and lowering the TGF-β1.