Distribution of erythrocyte in a model vessel exposed to inhomogeneous magnetic fields

In order to investigate magnetic field effects on blood flow, changes in the flow of erythrocytes in a model branched vessel were observed in an inhomogeneous magnetic field. The magnetic field was applied perpendicular to the straight vessel before branching. When the suspension containing paramagnetic erythrocytes with high spin methemoglobin or deoxygenated hemoglobin flowed in the model vessel, the erythrocytes were attracted towards the stronger magnetic field (i.e. to the side branch) and an excess flow of erythrocytes to the side branch was detected. This excess flow of erythrocytes to the side branch was the highest at a hematocrit of about 5% for the suspension containing erythrocytes with high spin methemoglobin. In the case of mixed suspensions containing erythrocytes with high spin methemoglobin and oxygenated erythrocytes, the excess flow of erythrocytes to the side branch reached its maximum at the "partial hematocrit" for the paramagnetic erythrocyte of around 5% and remained nearly constant with a further increase of the "partial hematocrit." The effect of magnetic field decreased as the flow velocity increased. These results are explained with the paramagnetism of erythrocytes and with the assumption of a hydrodynamic interaction among erythrocytes which are pulled in the direction of the magnetic field. It is suggested that a strong inhomogeneous magnetic field is not totally negligible to the blood circulation.     

Effects of an inhomogeneous magnetic field on flowing erythrocytes

Effects of an inhomogeneous magnetic field on narrow erythrocyte streams in a wide and transparent laminar buffer flow were studied. The stream line of erythrocytes containing paramagnetic hemoglobin showed distinct displacement toward the stronger magnetic field. The displacement increased in the order, oxygenated erythrocytes (no displacement), erythrocytes containing cyanomethemoglobin, deoxygenated erythrocytes, erythrocytes containing methemoglobin in the high spin state; more precisely the displacement was proportional to the square of the paramagnetic moment of hemoglobin contained in the erythrocytes. In addition, the displacement was proportional to the product of the magnetic flux density and its gradient, and approximately proportional to the hematocrit of the flowing-erythrocyte suspension, and was much larger than that calculated for a single erythrocyte. These phenomena could be successfully interpreted by the interaction of paramagnetic erythrocytes with the inhomogeneous magnetic field, the resistance force (Stokes Law) from the bulk water, and the hydrodynamic interaction between erythrocytes.     

Reduced oxygen release from erythrocytes by the acceleration-induced flow shift,observed in an oxygen-permeable narrow tube

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.     

Orientation of glutaraldehyde-fixed erythrocytes in strong static magnetic fields

In a uniform static magnetic field up to 8 Telsa, glutaraldehyde-fixed erythrocytes showed an orientation in which their disk plane was perpendicular to the magnetic field. The paramagnetism of membrane-bound hemoglobin was thought to contribute significantly to this orientation. The observation of magnetic orientation is directed toward understanding the fundamental microstructural aspects of the erythrocyte. © 1996 Wiley-Liss, Inc.     

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.     

Susceptibility of erythrocytes from several animal species to Vibrio vulnificus hemolysin

The hemolytic activity of Vibrio vulnificus hemolysin (VVH) against erythrocytes from several animal species (sheep, horse, cow, rabbit, chicken) was investigated. VVH was active against erythrocytes from all species, but the amount of VVH causing 50% hemolysis under identical conditions (hemolytic susceptibility to VVH) differed. The degree of 125I-labeled VVH (125I-VVH) binding to each erythrocyte species correlated with the susceptibility of the cells to hemolysis. However, marked differences in the binding ability of 125I-VVH were not observed against liposomes constructed with lipids from each erythrocyte membrane. On the other hand, release of hemoglobin (Hb) differed for each of the erythrocyte species despite administration of approximately the same hemolytic VVH concentration to each species. Furthermore, under hypotonic conditions, the stability of each erythrocyte species varied markedly; the more susceptible the erythrocyte to VVH, the more unstable it was under such conditions. These results, therefore, suggest that the susceptibility of erythrocytes to VVH may be closely associated with the binding ability of VVH and erythrocyte membrane stability.     

Visible spectroscopic technique for flowing erythrocytes in capillary

An optical spectroscopic system for determining the rate of oxygen release from flowing erythrocytes in microvessel is developed. The apparatus consists of following units attached to an inverted microscope. 1) A scanning spectrophotometer, equipped with a grating and a photon counter, was connected to an eyepiece of the microscope through a narrow light-guide, as to obtain the absorption spectrum (wave length range: 450-650 nm) of a focused spot (phi = 7 microns). 2) The velocity of erythrocyte flow was measured by dual-spots cross-correlation method, using two photomultipliers (connected to A/D converter and microcomputer) with two light-guides inserted into another eyepiece. 3) The diameter of vessel was estimated from digitized video-images, using a color image-processor. The ability of the apparatus was tested with (a) hemoglobin solution, (b) flowing erythrocyte suspension and (c) capillaries of rat mesentery. The rate of oxygen release through the vessel wall was calculated.     

The pressure-flow relation in resting rat skeletal muscle perfused with pure erythrocyte suspensions

In spite of numerous investigations of erythrocyte rheology, there is limited information about the influence of erythrocyte suspensions on whole organ pressure-flow relationships. In this study, we present whole organ pressure-flow curves for resting vasodilated gracilis muscle of the rat, in which the microanatomy and vessel properties have been determined previously. For pure erythrocyte suspensions from donor rats, the organ resistance increases only mildly with perfusion time (less than a 5% shift over a one-hour perfusion time), while in contrast, erythrocyte suspensions containing leukocytes show an increases of resistance near 100% over a period of 25 min. Variation in pressure-flow curves in the muscle at the same arterial hematocrit between different rats is less than 15%. The pressure-flow relation for pure erythrocyte suspensions depends on hematocrit. Shear thinning is exhibited at high hematocrits, while Newtonian behavior is approached at arterial hematocrits below 15%. The whole organ apparent viscosity for pure erythrocyte suspensions (normalized by cell-free plasma resistance) is a non-linear function of hematocrit; at physiological pressures, it reaches values comparable to those of apparent viscosities measured in rotational viscometers or in in vitro tube flow (diameters greater than 0.8 mm). The apparent viscosities estimated from the whole organ experiments tend to be higher than those measured in straight tubes under in vitro conditions. The pressure-flow curves for pure erythrocyte suspensions are shifted towards lower pressures than the curves for mixed suspensions of erythrocytes at the same hematocrit and with leukocytes at physiological cell counts. These acute experiments show that pure erythrocyte suspensions yield highly reproducible resistances in the skeletal muscle microcirculation with dilated arterioles. Relative apparent viscosities measured in vivo are higher than those measured in straight glass tubes of comparable dimesions.     

Measurement of the erythrocyte orientation in a flow by spin labeling

When a suspension of erythrocytes labeled in their membrane with a fatty acid paramagnetic molecule is allowed to flow in a flat quartz sample cell, the recorded electron paramagnetic spectra change as a function of the orientation of the cell in the magnetic field. This indicates that the red cells are themselves oriented in the flow. Such spectral variations have been reproduced by a numerical simulation procedure, which allowed us to quantify the proportion of oriented red blood cells by measuring the amplitude of some characteristic lines on the experimental spectra. Orientation rates were then measured as a function of various rheological parameters, such as shear rate, hematocrit and viscosity of the suspending medium. The kinetics of the disorientation process was determined by stopping the flow.     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

Optical characteristics of flowing blood

The transmitted light strength (TS) through a thin blood layer changes with variation in blood flow, such as positive streaming transparency for low hematocrits and negative streaming transparency for high hematocrits. These phenomena are examined theoretically and experimentally. Maxwell’s equations are solved assuming that erythrocytes are oblate spheroids to investigate these phenomena due to flowing blood. The theoretical results reveal that the scattering and absorption cross sections for flowing blood are larger than those for stagnant blood. Experimental results indicate that the TS for both oxygenated and deoxygenated flowing blood, with a hematocrit of up to approximately 20%, was stronger than that for stagnant blood. The TS decreased for flowing blood with a hematocrit of approximately 20% or greater. Applying the theoretical scattering and absorption cross sections to the absorption and multiple scattering theory of Victor Twersky, the changes in the TS due to flowing blood are obtained theoretically. From the theoretical and experimental results, the positive streaming transparency phenomenon of flowing blood with a low hematocrit and the negative streaming transparency phenomenon with a high hematocrit are found to result from increased scattering and absorption cross sections because of the orientation of flowing erythrocytes.     

Measurement of erythrocyte orientation in flow by spin labeling II--phenomenological models for erythrocyte orientation rate

We present two phenomenological models describing the flowing erythrocyte orientation rate. The first concerns the onset of a stable orientation in a very dilute erythrocyte suspension. It is based on a simple formula for erythrocyte elongation as a function of shear stress, and we assume that beyond a threshold of elongation, erythrocytes take on a stable orientation, while below this threshold, they have a flipping motion. We extend this model to high hematocrit values assuming that the effect of red cell collisions imposes a random moment to each erythrocyte, shifting it from its stable orientation. We obtain an approximate expression for erythrocyte orientation rate as a function of shear rate and then we compare these results to our experimental data in part III of this series.     

Red blood cell magnetophoresis

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.     

High-resolution NMR studies of transmembrane cation transport in uremic patients

Cation transport in erythrocytes of some uremic patients is impaired. Most studies have focused on the defect of the erythrocyte Na+/K+ pump in these diseased states. Herein, this cation transport defect was studied by using nuclear magnetic resonance spectroscopy (NMR) which is a non-invasive method permitting study on living erythrocytes. Firstly, we verified that the Na+ transport defect in uremic erythrocytes was not due to non-specific causes such as membrane alteration or a modification of the intracellular metabolism. The proton relaxation data, determined using a paramagnetic doping method, are consistent with a lack of erythrocytic membrane damage in uremic patients. Also, 31P-NMR results showed that in our experimental conditions, uremic and normal erythrocytes exhibit similar variations of ATP level over time. Lastly, the use of anionic paramagnetic shift reagent in 23Na-NMR revealed a defect in the Na+/K+ pump of erythrocytes from uremic patients with high Nain concentration. This defect seems to be due to a reduced number of pump units and to the presence of an endogenous inhibitor in uremic plasma.     

The mechanism of erythrocyte sedimentation in Westergren's examination.

The authors deduced the equation that describes the sedimentation of erythrocytes as the function of time, hematocrit, hemoglobin and some plasma protein concentrations and the citrate viscosity and density. This values served to describe plasma and erythrocyte density, plasma viscosity, erythrocyte aggregation and the influence of suspension concentration on the erythrocyte sedimentation rate. The influence of citrate on blood dilution (the reduction of hematocrit and plasma protein concentrations) was also considered. A good agreement between the observed and predicted values was obtained.     

Osmolality dependence of erythrocyte sedimentation and aggregation in a strong magnetic field

In order to specify the major determinant of the magnetic enhancement of erythrocyte sedimentation observed previously, the dependence of erythrocyte sedimentation rate (ESR) on osmolality was measured under a strong magnetic field. Even at hypotonic osmolality, an increase in ESR due to aggregation was observed in plasma solution as compared with that without aggregation in saline solution. However, the magnetic field did not enhance ESR at hypotonic osmolality, when the cell shape was an isotropic sphere (spherocyte). Thus, we narrowed our search to a mechanism that would explain the enhanced ESR found specifically in anisotropic erythrocytes. It was concluded that the major determinant can only work for anisotropic erythrocytes and is a magnetic field-induced increase in an intermembrane adhesive area due to magnetic orientation of anisotropic erythrocytes.     

Plasma expander viscosity effects on red cell-free layer thickness after moderate hemodilution

The objective of the study was to investigate the effects of plasma viscosity after hemodilution on the thickness of the erythrocyte cell free layer (CFL) and on the interface between the flowing column of erythrocytes and the vascular endothelium. The erythrocyte CFL thickness was measured in the rat cremaster muscle preparation. Plasma viscosity was modified in an isovolemic hemodilution, in which the systemic hematocrit (Hctsys) was lowered to 30%. The plasma expanders (PE) of similar nature and different viscosities were generated by glutaraldehyde polymerization of human serum albumin (HSA) at various molar ratios glutaraldehyde to HSA: (i) unpolymerized HSA; (ii) PolyHSA24:1, molar ratio = 24 and (iii) PolyHSA60:1, molar ratio = 60. The HSA viscosities determined at 200 s(-1) were 1.1, 4.2 and 6.0 dyn x cm(-2), respectively. CFL thickness, vessel diameter and blood flow velocity were measured, while volumetric flow, shear rate and stress were calculated. Hemodilution with PolyHSA60:1 increased plasma viscosity and the blood showed marked shear thinning behavior. CFL thickness decreased as plasma viscosity increased after hemodilution; thus the CFL thickness with HSA and PolyHSA24:1 increased compared to baseline. Conversely, the CFL thickness of PolyHSA60:1 was not different from baseline. Blood flow increased with both PolyHSA's compared to baseline. Wall shear rate and shear stress increased for PolyHSA60:1 compared to HSA and PolyHSA24:1, respectively. In conclusion, PE viscosity determined plasma viscosity after hemodilution and affected erythrocyte column hydrodynamics, changing the velocity profile, CFL thickness, and wall shear stress. This study relates the perfusion caused by PolyHSA60:1 to hemodynamic changes induced by the rheological properties of blood diluted with PolyHSA60:1.     

High gradient magnetic separation of erythrocytes

The high gradient magnetic separation technique has been applied to separate paramagnetic erythrocytes from a cell suspension that also contained diamagnetic cells. Paramagnetism was induced in the red blood cells by oxidizing the iron atoms in the cell hemoglobin to the ferric state (methemoglobin). Diamagnetic cells were either untreated erythrocytes, containing oxyferrohemoglobin, or leukocytes in a suspension of mouse spleen cells. Cell suspensions were passed through a column containing 40 micron diameter stainless steel wire in a high magnetic field (33 kG). The paramagnetic cells were retained on the surface of the wire while the diamagnetic cells passed through. Elution of the paramagnetic cells was accomplished by removing the column from the magnet, in effect turning off the field.     

Investigation of the magnetic susceptibility properties of fresh and fixed mouse heart,liver, skeletal muscle and brain tissue

PurposeSeveral magnetic resonance imaging (MRI) techniques exploit the difference in magnetic susceptibilities between tissues, but systematic measurements of tissue susceptibility are lacking. Furthermore, there is the question as to whether chemical fixation that is used for ex vivo MRI studies, affects the magnetic properties of the tissue. Here, we determined the magnetic susceptibility and water content of fresh and chemically fixed mouse tissue.MethodsMass susceptibility of brain, heart, liver and skeletal muscle samples were determined on a vibrating sample magnetometer at room temperature. Measurements at 50, 125, 200 and 295 K were performed to assess the temperature dependence of susceptibility. Moreover, we measured water content of fresh and fixed samples.ResultsAll samples show mass susceptibilities between −0.068 and −1.929 × 10⁻⁸ m³/kg, compared to −9.338 × 10⁻⁹ m³/kg of double distilled water. Heart tissue has a more diamagnetic susceptibility than the other tissues. Compared to fresh tissue, fixed tissue has a less diamagnetic susceptibility. Fixed tissue was not different in water content to fresh tissue and showed no consistent dependence of susceptibility with temperature, whereas fresh tissue shows a decrease to at least 125 K, indicative of a paramagnetic component.ConclusionsBiological tissues are diamagnetic in comparison to water, where the heart is more diamagnetic than the other tissues, with paramagnetic contributions. Fixation rendered tissue less diamagnetic compared to fresh tissue. Our measurements revealed differences in tissue susceptibility between VSM and QSM, inviting more research to compare susceptibility-based MRI methods with physical measurements of tissue susceptibility.     

Oscillating viscometer--evaluation of a new bedside test

A viscometer for bedside blood measurements was developed, consisting of an oscillating resonator probe mounted directly into a disposable vacutainer tube for blood withdrawal. It was tested in vitro on blood samples with variable hematocrits (20-60%), increasing fibrinogen concentrations (0-20 g/l), increasing concentrations of an admixed radiographic contrast medium and erythrocyte suspensions in dextran 40 and dextran 70. Results were compared with those obtained with a conventional Couette viscometer. Oscillating viscometry yielded generally higher values than Couette viscometry, and had a good sensitivity for changes in hematocrit with a good correlation between the two methods (r=0.96, p<0.0001). Oscillating viscosity depended on the resonator frequency, it was higher at 3900 Hz than at 215 Hz, suggesting a viscoelastic behavior of blood. Erythrocyte aggregation, induced by increasing fibrinogen concentrations or dextran 70, affected oscillating viscometry. At a high frequency, i.e. a smaller penetration depth of the shear wave, oscillating viscosity tended to decrease, which suggests a depletion of the boundary layer from erythrocytes when they aggregate. At low frequency with a deeper shear wave penetration (about 50 microm), erythrocyte aggregation increased oscillating viscosity. Bedside tests in 17 patients with coronary heart disease and 10 controls confirmed the easy practicability of the test and showed lower oscillating viscosity in these patients despite higher fibrinogen concentrations presumably due to increased erythrocyte aggregation. We conclude that oscillating viscometry is an interesting bedside test, which is capable of providing new information on the biorheology of the erythrocyte-poor boundary layer near the vessel wall.