The Toxic Influence of Excess Free Iron on Red Blood Cells in the Biophysical Experiment: An In Vitro Study.

Iron is needed for life-essential processes, but free iron overload causes dangerous clinical consequences. The study of the role of red blood cells (RBCs) in the influence of excess free iron in the blood on the pathological consequences in an organism is relevant. Here, in a direct biophysical experiment in vitro, we studied the action of free iron overload on the packed red blood cell (pRBC) characteristics. In experiments, we incubated pRBCs with the ferrous sulfate solution (Fe2+). Wе used free iron in a wide range of concentrations. High Fe2+ concentrations made us possible to establish the pattern of the toxic effect of excess iron on pRBCs during a reduced incubation time in a biophysical experiment in vitro. It was found that excess free iron causes changes in pRBC morphology, the appearance of bridges between cells, and the formation of clots, increasing the membrane stiffness and methemoglobin concentration. We created a kinetic model of changes in the hemoglobin derivatives. The complex of simultaneous distortions of pRBCs established in our experiments can be taken into account when studying the mechanism of the toxic influence of excess free iron in the blood on pathological changes in an organism.

Atomic force microscopy study of red blood cell membrane nanostructure during oxidation-reduction processes.

The morphology and functional state of red blood cells (RBCs) mainly depends on the configuration of the spectrin network, which can be broken under the influence of intoxication because of oxidation processes in the cells. Measurement of these processes is a complex problem. The most suitable and prospective method that resolves this problem is atomic force microscopy (AFM). We used AFM to study the changes in the spectrin matrix and RBC morphology during oxidation processes caused by ultraviolet (UV) irradiation in RBC suspension. The number of discocytes decreased from 98% (in control) to 12%. We obtained AFM images of the spectrin matrix in RBC ghosts. Atomic force microscopy allows for the direct observation and quantitative measurement of the disturbances in the structure of the spectrin matrix during oxidation processes in RBCs. The typical section size of the spectrin network changed from approximately 80 to 200 nm (in control) to 600 nm and even to 1000 nm after UV irradiation. An AFM study showed that incubation of RBCs with Cytoflavin® after UV irradiation preserved the forms of RBCs almost at control levels; 89% of the cells remained as discocytes. To quantify the intensity of the oxidation-reduction processes, the percentage of haemoglobin derivatives was measured. The content of methaemoglobin varied in the range of 1% to 70% during the experiments. These evidence-based studies are important for the fundamental research of interactions during redox processes in RBCs at the molecular level.

Morphology, membrane nanostructure and stiffness for quality assessment of packed red blood cells.

Transfusion of packed red blood cells (PRBC) to patients in critical states is often accompanied by post-transfusion complications. This may be related with disturbance of properties of PRBC and their membranes during long-term storage in the hemopreservative solution. The purpose of our work is the study of transformation of morphology, membranes stiffness and nanostructure for assessment of PRBC quality, in vitro. By atomic force microscopy we studied the transformation of cell morphology, the appearance of topological nanodefects of membranes and by atomic force spectroscopy studied the change of membrane stiffness during 40 days of storage of PRBC. It was shown that there is a transition period (20-26 days), in which we observed an increase in the Young's modulus of the membranes 1.6-2 times and transition of cells into irreversible forms. This process was preceded by the appearance of topological nanodefects of membranes. These parameters can be used for quality assessment of PRBC and for improvement of transfusion rules.

Disorders in the Morphology and Nanostructure of Erythrocyte Membranes after Long-term Storage of Erythrocyte Suspension: Atomic Force Microscopy Study.

Disorders in the erythrocyte morphology and structure of their membranes during long-term storage of erythrocyte suspension (30 days at 4°C) were studied by atomic force microscopy. The morphology and nanostructure of erythrocyte membranes, biochemical parameters, ion exchange parameters, and hemoglobin spectra were recorded. The transformation of erythrocyte morphology and destruction of their membranes were observed throughout the storage period. Irreversible forms of spheroechinocytes and their fragments formed by the end of storage. The concentrations of potassium ions and lactate in solution of the blood preservatiive increased, while pH value decreased. Hemolysis detected by the erythrocyte "leakage" effect was observed starting from days 16-23 of storage.

Reversible zinc-induced injuries to erythrocyte membrane nanostructure.

Zinc-induced injuries to red blood cell membrane nanostructures at different zinc concentrations were studied by atomic force microscopy. In order to distinguish the intrinsic characteristics of membrane nanostructures, the membrane surfaces were represented by three orders using 3D Fourier transform. Increasing the concentrations of zinc ions modified the pattern of induced injuries: their depths and diameters and their number on the membrane surface test area increased. The injuries and their distribution for each order of membrane surface were analyzed. Albumin restored membrane nanosurface.