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.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

“…Well characterized phenotypes of storage lesion, including membrane lipid peroxidation [ 73 ], GSSG generation [ 74 ], oxidatively-induced lysis, and defected Hb binding to the membrane [ 5 , 75 , 76 ], were ameliorated in the presence of antioxidants. Both AA and UA protect lipids from detectable peroxidative damage and quench ROS, either alone [ 21 , 77 , 78 ] or in combination [ 79 ]. Additionally, the role of GSH in lipid safeguarding becomes clear when considering that its depletion results in a lethal accumulation of lipid peroxides during ferroptosis [ 80 ].…”

The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation

“…Firstly, it seems that lysis after oxidative stimuli "belongs" to a different branch of parameters (mostly redox-related) than the ones attributed to corpuscular phenomena, as supported by a recent GWAS (Page et al, 2021). In addition, having in mind that (a) until middlestorage RBCs age in a more "ordered" way, at least in terms of energy and redox metabolism (Bordbar et al, 2016;Paglia et al, 2016;D'Alessandro et al, 2017b), and (b) beyond this time point RBCs accumulate more and more defects (Bardyn et al, 2017;Francis et al, 2020;Barshtein et al, 2021), it seems plausible to support that oxidative lysis becomes even more multiparametric at late storage resting its strong interconnection with other variables (including storage hemolysis) (Kanias et al, 2017) difficult. Osmotic and mechanical fragilities are not usually found intercorrelated even though they seem to affect one another (Tan et al, 2010).…”

Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes

“…DHM is a label free microscopy using a low intensive laser source limiting cell alteration. This technique is based on the quantitative measurement of the optical pathlength (OPL) delay (interferometric phase shift measurements) induced by the sample [37]. This parameter is linked to the cell volume and refractive index.…”

“…As before, RBC were suspended to 10 % hematocrit and treated with HgCl 2 (10, 20 and 40 µM). Subsequently, RBC were diluted in Krebs solution and 100 µL, containing 80,000 RBCs, were seeded per well, as quadruplicates, in a 96-well black imaging plate coated with poly-L-ornithine [37]. To accelerate cell sedimentation, the plate was centrifuged (140 g for 2 min at RT).…”