Image- and Fluorescence-Based Test Shows Oxidant-Dependent Damages in Red Blood Cells and Enables Screening of Potential Protective Molecules.

Manon Bardyn,David Crettaz,Michel Prudent,Benjamin Rappaz,Jérôme Allard,Gerardo Turcatti,Jean-Daniel Tissot

doi:10.3390/ijms22084293

Abstract

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape “Tirocytes”. The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.

Highlights

Red blood cells (RBCs) work in a high O2 content environment and possess a robust system to fight against oxidative stress
The imbalance between oxidants and antioxidants that generates this stress is counteracted by chemical-based antioxidant systems, such as lipophilic tocopherols, ubiquinol, carotenoids and flavonoids, hydrophilic ascorbic acid (AA), uric acid (UA) and thiols, as well as enzyme-based systems such as glutathione peroxidase or reductase, superoxide dismutase
The RBCs were pre-incubated for one hour with AA, UA, trolox or resveratrol antioxidants

Summary

Introduction

Red blood cells (RBCs) work in a high O2 content environment and possess a robust system to fight against oxidative stress. The imbalance between oxidants and antioxidants that generates this stress is counteracted by chemical-based antioxidant systems, such as lipophilic tocopherols, ubiquinol, carotenoids and flavonoids, hydrophilic ascorbic acid (AA), uric acid (UA) and thiols, as well as enzyme-based systems such as glutathione peroxidase or reductase, superoxide dismutase. In the context of transfusion medicine, the storage of RBCs as RBC concentrates (RCCs) induces an oxidative stress that plays a major role in the apparition of the so-called storage lesions [1,2]. Previous studies have explored the opportunity to compensate the apparition of oxidative stress by adding antioxidants (or precursors of them) in the preservative solution. The addition of AA, UA, precursors of glutathione or components such as vitamin E provided limited improvements of the storage lesions, whatever the parameters followed [1,3,4,5,6].

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Results

Discussion

Conclusion