Red Blood Cell Storage with Xenon: Safe or Disruption?

Xenon, an inert gas commonly used in medicine, has been considered as a potential option for prolonged preservation of donor packed red blood cells (pRBCs) under hypoxic conditions. This study aimed to investigate how xenon affects erythrocyte parameters under prolonged storage. In vitro model experiments were performed using two methods to create hypoxic conditions. In the first method, xenon was introduced into bags of pRBCs which were then stored for 42 days, while in the second method, xenon was added to samples in glass tubes. The results of our experiment showed that the presence of xenon resulted in notable alterations in erythrocyte morphology, similar to those observed under standard storage conditions. For pRBC bags, hemolysis during storage with xenon exceeded the acceptable limit by a factor of six, whereas the closed-glass-tube experiment showed minimal hemolysis in samples exposed to xenon. Notably, the production of deoxyhemoglobin was specific to xenon exposure in both cell suspension and hemolysate. However, this study did not provide evidence for the purported protective properties of xenon.

Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study.

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.

Stages of NETosis Development upon Stimulation of Neutrophils with Activators of Different Types.

Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.

Atomic Force Microscopy and High-Resolution Spectrophotometry for Study of Anoxemia and Normoxemia in Model Experiment In Vitro.

The oxygen content in the blood may decrease under the influence of various physicochemical factors and different diseases. The state of hypoxemia is especially dangerous for critically ill patients. In this paper, we describe and analyze the changes in the characteristics of red blood cells (RBCs) with decreasing levels of oxygen in the RBC suspension from normoxemia to hypoxemia/anoxemia in an in vitro model experiment. The RBCs were stored in hypoxemia/anoxemia and normoxemia conditions in closed and open tubes correspondingly. For the quantitative study of RBC parameter changes, we used atomic force microscopy, digital spectrophotometry, and nonlinear curve fitting of the optical spectra. In both closed and open tubes, at the end of the storage period by day 29, only 2% of discocytes remained, and mainly irreversible types, such as microspherocytes and ghosts, were observed. RBC hemolysis occurred at a level of 25-30%. Addition of the storage solution, depending on the concentration, changed the influence of hypoxemia on RBCs. The reversibility of the change in hemoglobin derivatives was checked. Based on the experimental data and model approach, we assume that there is an optimal level of hypoxemia at which the imbalance between the oxidative and antioxidant systems, the rate of formation of reactive oxygen species, and, accordingly, the disturbances in RBCs, will be minimal.

Mechanochemical Synergism of Reactive Oxygen Species Influences on RBC Membrane.

The influences of various factors on blood lead to the formation of extra reactive oxygen species (ROS), resulting in the disruption of morphology and functions of red blood cells (RBCs). This study considers the mechanisms of the mechanochemical synergism of OH• free radicals, which are most active in the initiation of lipid peroxidation (LPO) in RBC membranes, and H2O2 molecules, the largest typical diffusion path. Using kinetic models of differential equations describing CH2O2t and COH•t, we discuss two levels of mechanochemical synergism that occur simultaneously: (1) synergism that ensures the delivery of highly active free radicals OH• to RBC membranes and (2) a positive feedback system between H2O2 and OH•, resulting in the partial restoration of spent molecules. As a result of these ROS synergisms, the efficiency of LPO in RBC membranes sharply increases. In blood, the appearance of OH• free radicals is due to the interaction of H2O2 molecules with free iron ions (Fe2+) which arise as a result of heme degradation. We experimentally established the quantitative dependences of COH• CH2O2 using the methods of spectrophotometry and nonlinear curve fitting. This study extends the analysis of the influence of ROS mechanisms in RBC suspensions.

Structural Configuration of Blood Cell Membranes Determines Their Nonlinear Deformation Properties.

The ability of neutrophils and red blood cells (RBCs) to undergo significant deformations is a key to their normal functioning. Disruptions of these processes can lead to pathologies. This work studied the influence of structural configuration rearrangements of membranes after exposure to external factors on the ability of native membranes of neutrophils and RBCs to undergo deep deformation. The rearrangement of the structural configuration of neutrophil and RBC membranes under the influence of cytological fixatives caused nonlinear deformation phenomena. There were an increase in Young's modulus, a decrease in the depth of homogeneous bending, and a change in the distance between cytoskeletal junctions. Based on the results of the analysis of experimental data, a mathematical model was proposed that describes the process of deep bending of RBСs and neutrophil membranes.

Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships "cytoskeleton-membrane nanosurface-cell morphology". Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells.

BACKGROUND: Storage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days). MATERIALS AND METHODS: Bags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging. RESULTS: When PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19-21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created. DISCUSSION: Two stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.

The relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs.

BACKGROUND AND OBJECTIVES: In clinical practice, it has been shown that transfusion of packed red blood cells (pRBCs) with late shelf life increases the risk of post-transfusion complications. OBJECTIVE: To study relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs. MATERIALS AND METHODS: pRBCs were processed and stored according to blood bank procedure, for 42 days, at +4°C; pRBC samples were taken on days 3, 12, 19, 21, 24, 28, 35 and 42. Cytoskeleton images and membrane stiffness were studied using atomic force microscope. RESULTS: In the course of the pRBC storage, the cytoskeleton network configuration underwent structural changes. Simultaneously, pRBC membrane stiffness was increasing, with the correlation coefficient 0·88. Until 19 days, the stiffness grew slowly, in 19-24 days there occurred a transition period, after which its growth rate was three times higher than the initial. A chain of pathological processes developed in pRBC during long storage: pH reduction (linked to increased oxidative stress), then cytoskeletal destruction and an associated increase in pRBC membrane stiffness. CONCLUSION: During prolonged storage of pRBCs and their acidification, there is a progression of pRBC cytoskeletal changes and associated increase of membrane stiffness, observed to increase in rate after days 19-24. Mutual measurements of cytoskeletal integrity and membrane stiffness may be useful quality assessment tool to study the molecular mechanisms of RBC structural degradation during storage.

Assessment of carboxyhemoglobin content in the blood with high accuracy: wavelength range optimization for nonlinear curve fitting of optical spectra.

The impact of carbon monoxide (CO) gas on the human organism is very dangerous. The affinity of CO to hemoglobin is considerably higher than that of oxygen. Thus, the interaction of CO with the blood results in a higher content of carboxyhemoglobin (HbCO) in red blood cells (RBCs) and correspondingly in tissue hypoxia. The disruption in the organism depends on the HbCO content in the blood. To assess any complications in the body at a given moment due to CO exposure and predict future consequences, it is necessary to measure the dynamics of hemoglobin derivative concentrations simultaneously. However, measuring HbCO and other derivatives in RBCs without hemolysis accurately is complicated due to the strong intercollinearity between the molar absorptivities of hemoglobin derivatives and superposition of absorption and scattering spectra. In the present study, to quantitatively assess the contents of the hemoglobin derivatives in the blood after exposure to CO, improved accuracy is achieved by optimizing the wavelength range used for the nonlinear curve fitting of optical spectra. Experimental spectra were measured in the wavelength range Δ λ = 500 - 700 nm . For each experimental curve, it was established the value of optimal interval Δ λ o p t for which the correlation coefficient between experimental data and corresponding points of the theoretical fitting curve was the maximum in the wavelength range Δ λ t y p = 535 - 580 n m , which contains the typical absorption peaks for H b O 2 , H b , and H b C O . The concentrations obtained based on such fitting curves were considered to be highly accurate. The quantitative assessment enabled the determination of the H b C O nonlinear increase with the time of CO exposure in the in vitro experiment and the study of the dynamics of hemoglobin derivative transformations during blood incubation.

Conformational Distortions of the Red Blood Cell Spectrin Matrix Nanostructure in Response to Temperature Changes In Vitro.

The spectrin matrix is a structural element of red blood cells (RBCs). As such, it affects RBC morphology, membrane deformability, nanostructure, stiffness, and, ultimately, the rheological properties of blood. However, little is known about how temperature affects the spectrin matrix. In this study, the nanostructure of the spectrin network was recorded by atomic force microscopy. We describe how the nanostructure of the RBC spectrin matrix changes from a regular network to a chaotic pattern following an increase in temperature from 20 to 50°C. At 20-37°Ð¡, the spectrin network formed a regular structure with dimensions of typically 150 ± 60 nm. At 42-43°Ð¡, 83% of the spectrin network assumed an irregular structure. Finally, at 49-50°Ð¡ the chaotic pattern was observed, and no quantitative estimates of the spectrin structure's parameters could be made. These results can be useful for biophysical studies on the destruction of the spectrin network under pathological conditions, as well as for investigating cell morphology and blood rheology in different diseases.

Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action.

The ability of membranes of native human red blood cells (RBCs) to bend into the cell to a depth comparable in size with physiological deformations was evaluated. For this, the methods of atomic force microscopy and atomic force spectroscopy were used. Nonlinear patterns of deep deformation (up to 600 nm) of RBC membranes were studied in normal state and under the action of modifiers: fixator (glutaraldehyde), natural oxidant (hemin), and exogenous intoxicator (zinc ions), in vitro. The experimental dependences of membrane bending for control RBC (normal) were approximated by the Hertz model to a depth up to 600 nm. The glutaraldehyde fixator and modifiers increased the absolute value of Young's modulus of membranes and changed the experimental dependences of probe indentation into the cells. Up to some depth h Hz, the force curves were approximated by the Hertz model, and for deeper indentations h > h Hz, the degree of the polynomial function was changed, the membrane stiffness increased, and the pattern of indentation became another and did not obey the Hertz model. Quantitative characteristics of nonlinear experimental dependences were calculated for deep bending of RBC membranes by approximating them by the degree polynomial function.