[Progress in the analysis of hemolysis and coagulation models for interventional micro-axial flow blood pumps].

Interventional micro-axial flow blood pump is widely used as an effective treatment for patients with cardiogenic shock. Hemolysis and coagulation are vital concerns in the clinical application of interventional micro-axial flow pumps. This paper reviewed hemolysis and coagulation models for micro-axial flow blood pumps. Firstly, the structural characteristics of commercial interventional micro-axial flow blood pumps and issues related to clinical applications were introduced. Then the basic mechanisms of hemolysis and coagulation were used to study the factors affecting erythrocyte damage and platelet activation in interventional micro-axial flow blood pumps, focusing on the current models of hemolysis and coagulation on different scales (macroscopic, mesoscopic, and microscopic). Since models at different scales have different perspectives on the study of hemolysis and coagulation, a comprehensive analysis combined with multi-scale models is required to fully consider the influence of complex factors of interventional pumps on hemolysis and coagulation.

Sourcebook update: RBC hemolysis studies using a simple modified blood film technique.

Water movement across the cell membrane is crucial, with red blood cells (RBCs) experiencing the flow of water in both directions at a rate of approximately 100 times their volume per second. This process typically results in no net water flow due to an equal balance of water movement in opposite directions, a phenomenon known as osmosis, driven by water potential or impermeant solute concentration. Understanding osmosis is essential for both physiology and medical practice, yet its complexity may not be effectively conveyed to the students through traditional teaching methods. This study presents a novel approach to observing the osmotic effect on RBCs using a simple, modified blood film technique. Aimed at enhancing educational understanding of cellular behavior in different osmotic environments, this method provides a practical hands-on learning experience. By applying various osmotic solutions to prepared blood films and observing the resultant morphological changes in RBCs under a microscope, this technique allows for direct visualization of osmosis in action.NEW & NOTEWORTHY This study presents an innovative teaching approach for understanding osmosis and its effects on red blood cells. Using a simple, modified blood film technique, students can visually observe and engage with the dynamic process of osmosis. This hands-on method enhances learning, making complex physiological concepts accessible and practical. Ideal for resource-limited settings, it bridges theoretical knowledge and practical application, transforming physiology education.

Viscoelastic phenotyping of red blood cells.

Red blood cells (RBCs) are the simplest cell types with complex dynamical and viscoelastic phenomenology. While the mechanical rigidity and the flickering noise of RBCs have been extensively investigated, an accurate determination of the constitutive equations of the relaxational kinetics is lacking. Here we measure the force relaxation of RBCs under different types of tensional and compressive extension-jump protocols by attaching an optically trapped bead to the RBC membrane. Relaxational kinetics follows linear response from 60 pN (tensional) to -20 pN (compressive) applied forces, exhibiting a triple exponential function with three well-separated timescales over four decades (0.01-100 s). While the fast timescale (τF∼0.02(1)s) corresponds to the relaxation of the membrane, the intermediate and slow timescales (τI=4(1)s; τS=70(8)s) likely arise from the cortex dynamics and the cytosol viscosity. Relaxation is highly heterogeneous across the RBC population, yet the three relaxation times are correlated, showing dynamical scaling. Finally, we find that glucose depletion and laser illumination of RBCs lead to faster triple exponential kinetics and RBC rigidification. Viscoelastic phenotyping is a promising dynamical biomarker applicable to other cell types and active systems.

Impact of stalling events on microcirculatory hemodynamics in the aged brain.

OBJECTIVE: The role of cerebral microvasculature in cognitive dysfunction can be investigated by identifying the impact of blood flow on cortical tissue oxygenation. In this paper, the impact of capillary stalls on microcirculatory characteristics such as flow and hematocrit (Ht) in the cortical angioarchitecture is studied. METHODS: Using a deterministic mathematical model to simulate blood flow in a realistic mouse cortex, hemodynamics parameters, including pressure, flow, vessel diameter-adjustable hematocrit, and transit time are calculated as a function of stalling events. RESULTS: Using a non-linear plasma skimming model, it is observed that Ht increases in the penetrating arteries from the pial vessels as a function of cortical depth. The incidence of stalling on Ht distribution along the blood network vessels shows reduction of RBCs around the tissue near occlusion sites and decreased Ht concentration downstream from the blockage points. Moreover, upstream of the occlusion, there is a noticeable increase of the Ht, leading to larger flow resistance due to higher blood viscosity. We predicted marked changes in transit time behavior due to stalls which match trends observed in mice in vivo. CONCLUSIONS: These changes to blood cell quantity and quality may be implicated in the development of Alzheimer's disease and contribute to the course of the illness.

Is post-hypertonic lysis of human red blood cells caused by excessive cell volume regulation?

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

Red Blood Cells' Area Deformation as the Origin of the Photoplethysmography Signal.

The origin of the photoplethysmography (PPG) signal is a debatable topic, despite plausible models being addressed. One concern revolves around the correlation between the mechanical waveform's pulsatile nature and the associated biomechanism. The interface between these domains requires a clear mathematical or physical model that can explain physiological behavior. Describing the correct origin of the recorded optical waveform not only benefits the development of the next generation of biosensors but also defines novel health markers. In this study, the assumption of a pulsatile nature is based on the mechanism of blood microcirculation. At this level, two interconnected phenomena occur: variation in blood flow velocity through the capillary network and red blood cell (RBC) shape deformation. The latter effect was qualitatively investigated in synthetic capillaries to assess the experimental data needed for PPG model development. Erythrocytes passed through 10 µm and 6 µm microchannel widths with imposed velocities between 50 µm/s and 2000 µm/s, according to real scenarios. As a result, the length and area deformation of RBCs followed a logarithmic law function of the achieved traveling speeds. Applying radiometric expertise on top, mechanical-optical insights are obtained regarding PPG's pulsatile nature. The mathematical equations derived from experimental data correlate microcirculation physiologic with waveform behavior at a high confidence level. The transfer function between the biomechanics and the optical signal is primarily influenced by the vasomotor state, capillary network orientation, concentration, and deformation performance of erythrocytes.

Pathophysiology of Red Blood Cell Trapping in Ischemic Acute Kidney Injury.

Red blood cell (RBC) trapping describes the accumulation of RBCs in the microvasculature of the kidney outer medulla that occurs following ischemic acute kidney injury (AKI). Despite its prominence in human kidneys following AKI, as well as evidence from experimental models demonstrating that the severity of RBC trapping is directly correlated with renal recovery, to date, RBC trapping has not been a primary focus in understanding the pathogenesis of ischemic kidney injury. New evidence from rodent models suggests that RBC trapping is responsible for much of the tubular injury occurring in the initial hours after kidney reperfusion from ischemia. This early injury appears to result from RBC cytotoxicity and closely reflects the injury profile observed in human kidneys, including sloughing of the medullary tubules and the formation of heme casts in the distal tubules. In this review, we discuss what is currently known about RBC trapping. We conclude that RBC trapping is likely avoidable. The primary causes of RBC trapping are thought to include rheologic alterations, blood coagulation, tubular cell swelling, and increased vascular permeability; however, new data indicate that a mismatch in blood flow between the cortex and medulla where medullary perfusion is maintained during cortical ischemia is also likely critical. The mechanism(s) by which RBC trapping contributes to renal functional decline require more investigation. We propose a renewed focus on the mechanisms mediating RBC trapping, and RBC trapping-associated injury is likely to provide important knowledge for improving AKI outcomes. © 2024 American Physiological Society. Compr Physiol 14:5325-5343, 2024.

Quantitative study of spatial and temporal variation in retinal capillary network perfusion in rat eye by in vivo confocal imaging.

Microvascular dysfunction is the underlying pathological process in many systemic diseases. However, investigation into its pathogenesis is impeded by the accessibility and complexity of the microvasculature within different organs, particularly for the central nervous system. The retina as an extension of the cerebrum provides a glimpse into the brain through which the microvasculature can be observed. Two major questions remain unanswered: How do the microvessels regulate spatial and temporal delivery to satisfy the varying cellular demands, and how can we quantify blood perfusion in the 3D capillary network? Here, quantitative measurements of red blood cell (RBC) speed in each vessel in the field were made in the in vivo rat retinal capillary network using an ultrafast confocal technique with fluorescently labelled RBCs. Retinal RBC speed and number were found to vary remarkably between microvessels ranging from 215 to 6641 microns per second with significant variations spatially and temporally. Overall, the RBC speed was significantly faster in the microvessels in the superficial retina than in the deep retina (estimated marginal means of 2405 ± 238.2 µm/s, 1641 ± 173.0 µm/s respectively). These observations point to a highly dynamic nature of microvasculature that is specific to its immediate cellular environment and is constantly changing.

The Impact of COVID-19 on Cellular Factors Influencing Red Blood Cell Aggregation Examined in Dextran: Possible Causes and Consequences.

Several studies have indicated that COVID-19 can lead to alterations in blood rheology, including an increase in red blood cell aggregation. The precise mechanisms behind this phenomenon are not yet fully comprehended. The latest findings suggest that erythrocyte aggregation significantly influences microcirculation, causes the formation of blood clots in blood vessels, and even damages the endothelial glycocalyx, leading to endothelial dysfunction. The focus of this research lies in investigating the cellular factors influencing these changes in aggregation and discussing potential causes and implications in the context of COVID-19 pathophysiology. For this purpose, the aggregation of erythrocytes in a group of 52 patients with COVID-19 pneumonia was examined in a 70 kDa Dextran solution, which eliminates the influence of plasma factors. Using image analysis, the velocities and sizes of the formed aggregates were investigated, determining their porosity. This study showed that the process of erythrocyte aggregation in COVID-19 patients, independent of plasma factors, leads to the formation of more compact, denser, three-dimensional aggregates. These aggregates may be less likely to disperse under circulatory shear stress, increasing the risk of thrombotic events. This study also suggests that cellular aggregation factors can be responsible for the thrombotic disorders observed long after infection, even when plasma factors have normalized. The results and subsequent broad discussion presented in this study can contribute to a better understanding of the potential complications associated with increased erythrocyte aggregation.

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells.

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the microcirculation. Reduced deformability is seen in physiologically aged RBCs. Existing techniques to measure cell deformability cannot easily be used for measuring fatigue, the gradual degradation in cell membranes caused by cyclic loads. We present a protocol to evaluate mechanical degradation in RBCs from cyclic shear stresses using amplitude shift keying (ASK) modulation-based electrodeformation in a microfluidic channel. Briefly, the interdigitated electrodes in the microfluidic channel are excited with a low voltage alternating current at radio frequencies using a signal generator. RBCs in suspension respond to the electric field and exhibit positive dielectrophoresis (DEP), which moves cells to the electrode edges. Cells are then stretched due to the electrical forces exerted on the two cell halves, resulting in uniaxial stretching, known as electrodeformation. The level of shear stress and the resultant deformation can be easily adjusted by changing the amplitude of the excitation wave. This enables quantifications of nonlinear deformability of RBCs in response to small and large deformations at high throughput. Modifying the excitation wave with the ASK strategy induces cyclic electrodeformation with programmable loading rates and frequencies. This provides a convenient way for the characterization of RBC fatigue. Our ASK-modulated electrodeformation approach enables, for the first time, a direct measurement of RBC fatigue from cyclic loads. It can be used as a tool for general biomechanical testing, for analyses of cell deformability and fatigue in other cell types and diseased conditions, and can also be combined with strategies to control the microenvironment of cells, such as oxygen tension and biological and chemical cues.

Deformability of mouse erythrocytes in different diluents measured using optical tweezers.

Optical tweezers are widely used to measure the mechanical properties of erythrocytes, which is crucial to the study of pathology and clinical diagnosis of disease. During the measurement, the blood sample is diluted and suspended in an exogenous physiological fluid, which may affect the elastic properties of the cells in vitro. Here, we investigate the effect of different diluents on the elastic properties of mouse erythrocytes by quantitatively evaluating their elastic constants using optical tweezers. The diluents are plasma extracted from mouse blood, veterinary blood diluent (V-52D), Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and normal saline (NS). To create an environment that closely resembles in vivo conditions, the experiment is performed at 36.5 °C. The results show that the spring constant of mouse erythrocytes in plasma is 6.23 ± 0.41 µN m-1. The elasticity of mouse erythrocytes in V-52D and DMEM is 8.21 ± 0.91 and 6.95 ± 0.85 µN m-1, which are higher than that in plasma extracted from blood, whereas, the elasticity in PBS and NS is 4.23 ± 0.85 and 4.68 ± 0.79 µN m-1, which are less than that in plasma extracted from blood. At last, we observe the size and circularity of erythrocytes in different diluents, and consider that the erythrocyte diameter and circularity may affect cell deformability. Our results provide a reference of the diluent choice for measuring the mechanical properties of erythrocytes in vitro.

Angiogenic Microvascular Wall Shear Stress Patterns Revealed Through Three-dimensional Red Blood Cell Resolved Modeling.

The wall shear stress (WSS) exerted by blood flowing through microvascular capillaries is an established driver of new blood vessel growth, or angiogenesis. Such adaptations are central to many physiological processes in both health and disease, yet three-dimensional (3D) WSS characteristics in real angiogenic microvascular networks are largely unknown. This marks a major knowledge gap because angiogenesis, naturally, is a 3D process. To advance current understanding, we model 3D red blood cells (RBCs) flowing through rat angiogenic microvascular networks using state-of-the-art simulation. The high-resolution fluid dynamics reveal 3D WSS patterns occurring at sub-endothelial cell (EC) scales that derive from distinct angiogenic morphologies, including microvascular loops and vessel tortuosity. We identify the existence of WSS hot and cold spots caused by angiogenic surface shapes and RBCs, and notably enhancement of low WSS regions by RBCs. Spatiotemporal characteristics further reveal how fluctuations follow timescales of RBC "footprints." Altogether, this work provides a new conceptual framework for understanding how shear stress might regulate EC dynamics in vivo.

An impedance flow cytometry with integrated dual microneedle for electrical properties characterization of single cell.

Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m-2, with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.

Cell-free layer development and spatial organization of healthy and rigid red blood cells in a microfluidic bifurcation.

Bifurcations and branches in the microcirculation dramatically affect blood flow as they determine the spatiotemporal organization of red blood cells (RBCs). Such changes in vessel geometries can further influence the formation of a cell-free layer (CFL) close to the vessel walls. Biophysical cell properties, such as their deformability, which is impaired in various diseases, are often thought to impact blood flow and affect the distribution of flowing RBCs. This study investigates the flow behavior of healthy and artificially hardened RBCs in a bifurcating microfluidic T-junction. We determine the RBC distribution across the channel width at multiple positions before and after the bifurcation. Thus, we reveal distinct focusing profiles in the feeding mother channel for rigid and healthy RBCs that dramatically impact the cell organization in the successive daughter channels. Moreover, we experimentally show how the characteristic asymmetric CFLs in the daughter vessels develop along their flow direction. Complimentary numerical simulations indicate that the buildup of the CFL is faster for healthy than for rigid RBCs. Our results provide fundamental knowledge to understand the partitioning of rigid RBC as a model of cells with pathologically impaired deformability in complex in vitro networks.

Flow Heterogeneity and Factors Contributing to the Variability in Retinal Capillary Blood Flow.

Purpose: Capillary flow plays an important role in the nourishment and maintenance of healthy neural tissue and can be observed directly and non-invasively in the living human retina. Despite their importance, patterns of normal capillary flow are not well understood due to limitations in spatial and temporal resolution of imaging data. Methods: Capillary flow characteristics were studied in the retina of three healthy young individuals using a high-resolution adaptive optics ophthalmoscope. Imaging with frame rates of 200 to 300 frames per second was sufficient to capture details of the single-file flow of red blood cells in capillaries over the course of about 3 seconds. Results: Erythrocyte velocities were measured from 72 neighboring vessels of the parafoveal capillary network for each subject. We observed strong variability among vessels within a given subject, and even within a given imaged field, across a range of capillary flow parameters including maximum and minimum velocities, pulsatility, abruptness of the systolic peak, and phase of the cardiac cycle. The observed variability was not well explained by "local" factors such as the vessel diameter, tortuosity, length, linear cell density, or hematocrit of the vessel. Within a vessel, a moderate relation between the velocities and hematocrit was noted, suggesting a redistribution of plasma between cells with changes in flow. Conclusions: These observations advance our fundamental understanding of normal capillary physiology and raise questions regarding the potential role of network-level effects in explaining the observed flow heterogeneity.

Influence of the vessel wall geometry on the wall-induced migration of red blood cells.

The geometry of the blood vessel wall plays a regulatory role on the motion of red blood cells (RBCs). The overall topography of the vessel wall depends on many features, among which the endothelial lining of the endothelial surface layer (ESL) is an important one. The endothelial lining of vessel walls presents a large surface area for exchanging materials between blood and tissues. The ESL plays a critical role in regulating vascular permeability, hindering leukocyte adhesion as well as inhibiting coagulation during inflammation. Changes in the ESL structure are believed to cause vascular hyperpermeability and entrap immune cells during sepsis, which could significantly alter the vessel wall geometry and disturb interactions between RBCs and the vessel wall, including the wall-induced migration of RBCs and the thickening of a cell-free layer. To investigate the influence of the vessel wall geometry particularly changed by the ESL under various pathological conditions, such as sepsis, on the motion of RBCs, we developed two models to represent the ESL using the immersed boundary method in two dimensions. In particular, we used simulations to study how the lift force and drag force on a RBC near the vessel wall vary with different wall thickness, spatial variation, and permeability associated with changes in the vessel wall geometry. We find that the spatial variation of the wall has a significant effect on the wall-induced migration of the RBC for a high permeability, and that the wall-induced migration is significantly inhibited as the vessel diameter is increased.

Red Blood Cell Sublethal Damage: Hemocompatibility Is not the Absence of Hemolysis.

Blood is a complex fluid owing to its two-phase suspension of formed cellular elements within a protein-rich plasma. Vital to its role in distributing nutrients throughout the circulatory system, the mechanical properties of blood - and particularly red blood cells (RBC)-primarily determine bulk flow characteristics and microcirculatory flux. Various factors impair the physical properties of RBC, including cellular senescence, many diseases, and exposure to mechanical forces. Indeed, the latter is increasingly relevant following the advent of modern life support, such as mechanical circulatory support (MCS), which induce unique interactions between blood and artificial environments that leave blood cells with the signature of aging, albeit accelerated, and crucially underlie various serious complications, including death. Accumulating evidence indicates that these complications appear to be associated with mechanical shear forces present within MCS that are not extreme enough to overtly rupture cells, yet may still induce "sublethal" injury and "fatigue" to vital blood constituents. Impaired RBC physical properties following elevated shear exposure-a hallmark of sublethal injury to blood-are notable and may explain, at least in part, systemic complications and premature mortality associated with MCS. Design of optimal next-generation MCS devices thus requires consideration of biocompatibility and blood-device interactions to minimize potential blood complications and promote clinical success. Presented herein is a contemporary understanding of "blood damage," with emphasis on shear exposures that alter microrheological function but do not overtly destroy cells (ie, sublethal damage). Identification of key cellular factors perturbed by supraphysiological shear exposure are examined, offering potential pathways to enhance design of MCS and blood-contacting medical devices.

Novel blood typing method by discrimination of hemagglutination and rouleaux using an erythrocyte aggregometer.

BACKGROUND: In pretransfusion blood typing, pretreatments such as centrifugation and suspension of red blood cells (RBCs) and mixing them with sufficient amounts of reagents are required, but these steps are time-consuming and costly. OBJECTIVE: Aiming to develop a new blood typing method that requires no dilution and only a small amount of reagent, we attempted to determine blood type using syllectometry, an easy-to-use and rapid optical method for measuring the RBC aggregation that occurs when blood flow is abruptly stopped in a flow channel. METHODS: Samples of whole blood from 20 healthy participants were mixed with antibody reagents for blood typing at mixing ratios of 2.5% to 10% and measured with a syllectometry device. RESULTS: Amplitude (AMP), one of the aggregation parameters, showed significant differences between agglutination and non-agglutination samples at mixing ratios from 2.5% to 10%. Although there were significant individual differences in aggregation parameters, calculation of AMP relative to that of blood before reagent mixing reduced the individual differences and enabled determination of blood type in all participants. CONCLUSIONS: This new method enables blood typing with a small amount of reagent, without the time-consuming and labor-intensive pretreatments such as centrifugation and suspension of RBCs.