1. Oxidative Balance of Autologous Serum Eyedrops
Felipe Fernandes Nicola(Presenting Author); Patrícia Ioschpe Gus, MD, phD; Samira Küllinger Zelanis; Ana Laura Fisher Kunzler; Diane R. Marinho, MD; Claudete Inês Locatelli
The authors have no financial interests to disclose

2. Introduction
Autologous serum eyedrops (AS) is widely recognized and recommended as a therapeutic option to severe dry eye, specially in patients with ocular surface diseases1,2.
It is known that substances as fibronectin, vitamin A, epithelial growth factor and cytokines are involved in its therapeutic benefit1,2.
Oxidative stress has become increasingly accepted as playing a role in corneal inflammation and dry eye3.
No description about AS antioxidant capacity or reactive oxygen species concentration were reported in the literature.
Tananuvat N, Daniell M, Sullivan LJ, Yi Q, McKelvie P, McCarty DJ, Taylor HR. Controlled study of the use of autologous serum in dry eye patients. Cornea Nov;20(8):802-6.
Kojima T, Ishida R, Dogru M, Goto E, Matsumoto Y, Kaido M, Tsubota K. The effect of autologous serum eyedrops in the treatment of severe dry eye disease: A prospective randomized case-control study. Am J Ophthalmol 2005 Feb;139(2):242-6.
Nakamura S, Shibuya M, Nakashima H, Hisamura R, Masuda N, Imagawa T, et al. Involvement of oxidative stress on corneal epithelial alterations in a blink-suppressed dry eye. Invest Ophthalmol Vis Sci. 2007;48(4):    

3. Purpose
To evaluate the total reactive antioxidant potential (TRAP) and the concentration of reactive oxygen species (ROS) in samples of 50% Autologous Serum Eyedrops (AS) from patients with severe ocular surface diseases and healthy controls.
Results were correlated with patient’s health, demographic characteristics and lifestyle habits.

4. Methods Study design: a case-control study.
Biochemical evaluation was performed to detected ROS (pmol/mg) and TRAP (UTrolox/μl) from patients’ samples and controls taking vitamin E as standard.
Samples from patients with ocular surface diseases and controls matched by gender and age were evaluated before freezing (0), and after 15 and 30 days in the freezer.
All patients answered a questionnaire on demographic characteristics(gender and age), behavioral habits(smoking, alcohol, exercise and fruits, vegetables, synthetic vitamins and cereals consumption), medical diagnoses and medication in use.
Analysis: Mann-Whitney, Spearman`s and Wald tests by IBM SPSS.

5. Methods Autologous Serum Preparation*
120ml peripheral venous blood was withdrawn from each patient, distributed in 4mL tubes and immediately centrifuged by rotations per minute during 10 minutes at room temperature.
In a laminar flow cabinet, 50mL of autologous serum were withdrawn from tubes and added to 50mL of methylcellulose 0,5% (1:1).
2mL were taken from the sterile tubes for microbiologic analisis.
*AS was produced as recommended by the Infection Committee of Hospital de Clínicas de Porto Alegre.

6. Methods
Patients with ocular suface diseases donate 3 samples from their usual AS preparation to this present study.
5ml peripheral venous blood were withdrawn from healthy controls to preparation of 3 samples to the study analysis.

7. Results
It was possible to detect the presence of ROS and TRAP at all moments evaluated (Graphics 1 and 2).
Cases and controls have no statistically significant difference when compared by ROS and TRAB means distribuition (p=1,0).
No statistically significant difference was found in the concentration of ROS at 0, 15 and 30 days (Table 1).
ROS difference between 0 vs 15 days (p=0.245) and 15 vs 30 days (p=1) was not statistically significant (Table 1).
The TRAP differed between 0 vs 15 days (p=0.03), which was not repeated when comparing 15 vs 30 days (p = 1.0) (Table 1)
The variables assessed in the questionnaire did not correlate with the values of TRAP (Table 2).

8. Results
p=1,0
p=1,0
p=1,0

9. Results
p=1,0
p=1,0
p=1,0

10. Results Day 0 vs. Day 15 Day 0 vs Day 30 Day 15 vs Day 30
Table 1. ROS and TRAB`s Means Pairwised Comparison Between Days 0, 15 and 30.
ROS
(pmol/mg) p
TRAP
(UTrolox/μl)
Day 0 vs. Day 15
14,47 vs. 6,52
p=0,245
148,28 vs. 91,84
p=0.003
Day 0 vs Day 30
14,47 vs 5,78
p=0,101
148,28 vs. 108,54
p=0,170
Day 15 vs Day 30
6,52 vs. 5,78
p=1,0
91,84 vs. 108,54

11. Results
Table 2. Correlation between Cases and Controls` TRAP (day 0) Distribuition by Demographic Characteristics and Lifestyle Habits.
TRAP
Day 0
(fresh)
Gender
Alcohol
Vegetables
Exercise
Vitamins
Cereals
Systemic Diseases
Age
Case
p=0,724
p=0,610
p=0,903
p=0,556
p=0,409
p=0,896
p=0,624
p=0,447
Control
p=0,865
p=0,644
p=0,390
p=0,234
p=0,734
p=0,405
p=0,465
p=0,914

12. Conclusion
The 50% AS oxidative balance remained stable before and after freezing for 30 days and there was no diference between patients with ocular surface diseases and normal controls.
None of the variables assessed in the questionnaire influenced the oxidative balance, suggesting that patients undergoing AS are source of the same antioxidant capacity than healthy subjects.
This is the first description about AS antioxidant capacity or reactive oxygen species concentration in the literature.