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

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.

Keywords: antioxidant; digital holographic microscopy; fluorescence; high-throughput screening; oxidant; red blood cell; transfusion.

Figure 1

Principle of the TSOX Assay. (A) Red blood cells (RBCs) seeded in a microplate are treated with harmful (i.e., oxidant) and/or potentially protective molecules. Two possible readouts giving complementary information are proposed. The first one is based on the detection of fluorescence emitted by the DCFH-DA probe, when exposed to oxidative stress [20]. The second consists in the analysis by digital holographic microscopy (DHM) of the morphological changes triggered by the treatment(s). (B) Plate map (96 wells) used in the present study.

Figure 2

Time-Lapse Analysis of the Effects of Different Oxidants (i.e., AAPH, Diamide and H₂O₂) on the Oxidative Stress and Morphology of Red Blood Cells (RBCs). (A) Fluorescence emission induced by AAPH, diamide, H₂O₂ or no-oxidant treatments. (B) Area under the curve (AUC) calculated from fluorescence curves. (C) Morphological changes induced by AAPH, diamide, H₂O₂ or no-oxidant treatments. Left: average of the optical path difference distribution (OPD AVG) parameter; right: normalized confluency. (D) AUC calculated from OPD AVG and confluency curves. For the AUC, two-way ANOVA was performed to compare the effect of the three oxidants at different concentrations with a “no-oxidant” control; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, and “ns” non-significant. (E) Evolution of the morphology of RBCs treated with oxidants. Illustrative phase images acquired by digital holographic microscopy (DHM) at seven time points.

Figure 3

Summary Panel of the Effects on Red Blood Cells (RBCs) of 0, 10, 100 and 1000 µM Ascorbic Acid (AA), Uric Acid (UA), Trolox and Resveratrol against AAPH, Diamide and H₂O₂ at Different Concentrations. Top: effect on ROS by fluorescence emission; and bottom: effect on RBC morphology (i.e., average of the optical path difference distribution [OPD AVG] parameter and normalized confluency). Each cell on the heat map represents the mean area under the curve (AUC) for three RBC concentrates (RCCs) under a particular treatment. Two-way ANOVA was performed to compare the effect of the 0 µM versus 10, 100 or 1000 µM antioxidant conditions; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001.

Figure 4

Assessment of the Protection Provided by Resveratrol against AAPH, Diamide and H₂O₂ Oxidants. (A) Time-lapse analysis of fluorescence emission for red blood cells (RBCs) treated with 1 mM AAPH, 1 mM diamide or 0.001% H₂O₂, and 0, 10, 100 and 1000 µM resveratrol. (C) Time-lapse analysis by digital holographic microscopy (DHM) of morphological changes triggered by 5 mM AAPH, 2 mM diamide or 0.005% H₂O₂, and 0, 10, 100 and 1000 µM resveratrol. top: average of the optical path difference distribution (OPD AVG) parameter; bottom: normalized confluency (B) and (D) area under the curve (AUC) calculated from fluorescence and OPD AVG and confluency curves. Of note: control samples were neither treated with oxidants nor antioxidants. “No-antioxidant” control corresponding to the 0 µM antioxidant samples are in darker color. For the AUC, two-way ANOVA was performed to compare the effect of resveratrol at different concentrations with the no-antioxidant condition; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, and “ns” non-significant.

Figure 5

Effect of Resveratrol on Red Blood Cells (RBCs). Formation of “Tirocytes”. (A) Time-lapse analysis and the calculated AUC of fluorescence emission and morphology (i.e., average of the optical path difference distribution [OPD AVG] parameter and normalized confluency) for RBCs treated with 0, 10, 100 and 1000 µM resveratrol only (no oxidants). * p-value < 0.05, *** p-value < 0.001, and “ns” non-significant. (B) Illustrative phase images showing the morphological changes leading to a diamond-shaped RBC (treated with 1000 µM resveratrol). (C) Morphology of RBCs after 13h50 incubation with 1000 µM resveratrol. Diamond-shaped “Tirocytes” are surrounded by a red circle.