Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.     

Hawthorn special extract WS? 1442 increases red blood cell NO-formation without altering red blood cell deformability

WS(?) 1442 is a special extract of hawthorn leaves with flowers used for the treatment of mild cardiac failure. The activation of endothelial nitric oxide synthase (eNOS) has been shown to contribute to its vasodilating properties. Quite recently it has been demonstrated that red blood cells (RBCs) express a functional NO-synthase (rbcNOS) and rbcNOS activation has been associated with increased RBC deformability. The aim of the present study was to determine whether WS(?) 1442 is able to activate rbcNOS, to induce NO-formation in RBC and to alter RBC-deformability. Blood from healthy volunteers was incubated with WS(?) 1442 (25-100 μg/ml) for up to 30 min. RbcNOS activation was detected by immunohistochemical staining of phosphorylated rbcNOS and NO-formation was examined by diaminofluorescein (DAF) fluorescence. RBC deformability was measured by a laser assisted optical rotational cell analyzer. Serine 1177 of RbcNOS (rbcNOS Ser(1177)) was time- and concentration-dependently phosphorylated by WS(?) 1442. Rates of rbcNOS Ser(1177) phosphorylation were up to 149% higher in RBCs treated with WS(?) 1442 in comparison to control (DMSO 0.05%). WS(?) 1442 induced a time-dependent increase in NO-formation in RBCs which reached its maximum after 5 min. An increase in shear stress (0.3-50 Pa) caused an increase in RBC deformability. WS(?) 1442 did not change either basal or maximal RBC-deformability or shear stress sensitivity of RBC at normoxia. CONCLUSION: WS(?) 1442 activates rbcNOS and causes NO-formation in RBCs. WS(?) 1442-dependent NO-formation however does not affect RBC-deformability at normoxia.     

Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response. RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde. The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35%. The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation. On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension. RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters. Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05). Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate. The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates. In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability. Deformability was measured with the same filtration technique. There was no difference in the deformability of hypoxic compared with normoxic RBCs. We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.     

Validation and application of an automated rheoscope for measuring red blood cell deformability distributions in different species

BACKGROUND: The deformability of red blood cells (RBCs) is of great importance for the conservation of oxygen delivery in the microcirculation. Even a small fraction of rigid cells is considered to harm the exchange of respiratory gases. Techniques that measure RBC deformability often provide an indication of the mean deformability. It may not be possible, however, to assess whether this mean value is reduced by the presence of a small rigid cell fraction or by a slight overall reduction in RBC deformability. A technique that provides a deformability distribution would be of great value to study diseases that are marked by subpopulations with a reduced deformability. METHODS: This paper describes a rheoscope system that uses advanced image analysis techniques to quickly quantify the deformability of many individual cells in shear flow, in order to find the RBC-deformability distribution. Since variations in the shear stress are responsible for variations in cell elongation, and hence introduce an additional spread in the cell deformability distribution, we first determined the spread caused by instrumental error. We then utilized the technique to investigate the relation between cell deformability and cell size of single blood samples of different species (human, pig, rat and rabbit). RESULTS: The spread caused by instrumental error was small compared to the actual RBC-deformability spread in blood samples. The deformability distribution of human and pig cells are alike although their cell sizes are different. Rat and rabbit cells show comparable deformability and size distributions. With this technique no correlation was found between cell deformability and cell size in animal RBCs. In the human sample a minor correlation was found between cell deformability and cell size. CONCLUSIONS: The automated rheoscope enables us to study the mechanical properties of RBCs more thoroughly by their deformability distribution. These deformability distributions are hardly influenced by the technique or by cell size.     

Measurement temperature plays a pivotal role in the distribution of erythrocyte deformability after LPS

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.     

A new red blood cell filtration device with improved time resolution and its application to the impaired RBC deformability in the diabetic ob/ob mouse

A new filtration device and blood handling technique for the assessment of RBC deformability in small blood samples is described and used to study RBC deformability in adult obese-hyperglycemic ob/ob-mice and normoglycemic controls. The new filtration device was designed to improve the time resolution during RBC incubation. Test and control RBC suspensions were directly filtered from two identical incubation chambers under a constant pressure of 1200 Pa. Nuclepore filters (3 microns) were mounted on top of several standard test tubes into which the filtrate was subsequently collected and weighed. Because the RBCs were resuspended to a very low (0.01%) hematocrit, the average number of RBCs passing each pore was less than 10. Therefore, any detectable difference must reflect the physical properties of RBCs, e.g. shape or viscoelasticity, whereas the role of white blood cells is negligible. When ob/ob-mouse RBCs were studied with the new technique they showed impaired filtrability as compared with control RBCs, both when incubated without glucose and with glucose present at the same concentration as that recorded in the RBC donating mouse.     

Multiscale modeling of red blood cell mechanics and blood flow in malaria

Red blood cells (RBCs) infected by a Plasmodium parasite in malaria may lose their membrane deformability with a relative membrane stiffening more than ten-fold in comparison with healthy RBCs leading to potential capillary occlusions. Moreover, infected RBCs are able to adhere to other healthy and parasitized cells and to the vascular endothelium resulting in a substantial disruption of normal blood circulation. In the present work, we simulate infected RBCs in malaria using a multiscale RBC model based on the dissipative particle dynamics method, coupling scales at the sub-cellular level with scales at the vessel size. Our objective is to conduct a full validation of the RBC model with a diverse set of experimental data, including temperature dependence, and to identify the limitations of this purely mechanistic model. The simulated elastic deformations of parasitized RBCs match those obtained in optical-tweezers experiments for different stages of intra-erythrocytic parasite development. The rheological properties of RBCs in malaria are compared with those obtained by optical magnetic twisting cytometry and by monitoring membrane fluctuations at room, physiological, and febrile temperatures. We also study the dynamics of infected RBCs in Poiseuille flow in comparison with healthy cells and present validated bulk viscosity predictions of malaria-infected blood for a wide range of parasitemia levels (percentage of infected RBCs with respect to the total number of cells in a unit volume).     

Aggregation of erythrocytes and their membranes flexibility in patients with cancer-associated anemia: Mechanisms of changes under the influence of epoetin alfa

The relationships between the red blood cell (RBC) membrane elasticity and RBC aggregation in healthy individuals and in patients with anemia of malignant tumors treated with human erythropoietin drug epoetin alfa (EA) were analyzed. It was found that prior to the treatment of patients, incubation of RBCs with EA was accompanied by an increase of RBC deformability and the reduction of their aggregation (RBCA). In these circumstances the two characteristics of the RBC microrheology correlated negatively with each other (r =–0.734, p < 0.05). In contrast, aggregation and deformability of RBCs from healthy individuals increased under the influence of EA and positively correlated with each other (r = 0.580, p < 0.05). After a 4-week treatment of patients with EA, aggregation response of the patients’ RBCs was increased by 29% (p < 0.05) and was close to that of healthy RBCs. This change of the RBC aggregation response may be connected with an alteration of the sensitivity of the membrane cationic channel to EA and an increase of the cell deformability. This possibility was supported by experiments with the use of Ca²⁺-channel blocker verapamil and Ca²⁺-chelating agent EDTA. Under these conditions a decrease of the RBC aggregation varied from 40 to 50% (p < 0.05). It was suggested that the effectors of calcium regulatory cascade upon exposure to EA may be membrane integrin receptors of type IIb–IIIa. This assumption was confirmed by experiments employing the inhibitors of these receptors (tirofibam and integrelin) and a preparation of monoclonal antibodies against IIb–IIIa receptors (monafram), which produced a significant decrease (20–30%, p < 0.05) of the RBC aggregation. Thus, our findings suggest that the altered aggregation response of RBCs in anemic patients with malignant tumors can be restored by the correction of anemia with epoetin alfa.     

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell–cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm² areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.     

Microfluidic analysis of red blood cell deformability

A common indicator of rheological dysfunction is a measurable decrease in the deformability of red blood cells (RBCs). Decreased RBC deformability is associated with cellular stress or pathology and can impede the transit of these cells through the microvasculature, where RBCs play a central role in the oxygenation of tissues. Therefore, RBC deformability has been recognized as a sensitive biomarker for rheological disease. In the current study, we present a strategy to measure RBC cortical tension as an indicator of RBC deformability based on the critical pressure required for RBC transit through microscale funnel constrictions. By modeling RBCs as a Newtonian liquid drop, we were able to discriminate cells fixed with glutaraldehyde concentrations that vary as little as 0.001%. When RBCs were sampled from healthy donors on different days, the RBC cortical tension was found to be highly reproducible. Inter-individual variability was similarly reproducible, showing only slightly greater variability, which might reflect biological differences between normal individuals. Both the sensitivity and reproducibility of cortical tension, as an indicator of RBC deformability, make it well-suited for biological and clinical analysis of RBC microrheology.     

Optical and photoacoustic radiofrequency spectroscopic analysis for detecting red blood cell death

Under stress, red blood cells (RBCs) undergo programmed cell death (eryptosis). One of the signaling molecules for eryptosis, sphingomyelinase (SMase), plays an important role in monitoring the efficacy of vascular targeted cancer therapy. The high optical absorption of erythrocytes coupled with the changes of eryptotic RBCs makes RBCs ideal targets for the photoacoustic (PA) detection and characterization of vascular treatments. In this work, experiments characterizing eryptosis were performed: PA detection of high frequencies (>100 MHz) that enabled analysis at the single‐cell level and of low frequencies (21 MHz) that enabled analysis at the RBC ensemble level. Ultrasound spectral analysis was performed on control and SMase‐treated RBCs. Spectral unmixing was applied to quantify methemoglobin production as a by‐product of RBC death. Validation was performed using a blood gas analyzer and optical spectrometry. Our results indicate that PA radiofrequency spectra could be used to differentiate the biochemically induced morphological changes as RBCs lose their native biconcave shape, and release hemoglobin into the surroundings. Spectral unmixing revealed a 7% increase in methemoglobin content for SMase‐treated samples due to the oxidative stress on the RBCs. These findings suggest that PA spectral analysis of RBC death can potentially serve as a biomarker of the efficacy of vascular targeted cancer therapies.     

Computational Analysis of Dynamic Interaction of Two Red Blood Cells in a Capillary

The dynamic interaction of two red blood cells (RBCs) in a capillary is investigated computationally by the two-fluid model, including their deformable motion and interaction. For characterization of the deformation, the RBC membrane is treated as a curved two-dimensional shell with finite thickness by the shell model, and allowed to undergo the stretching strain and bending deformation. Moreover, a Morse potential is adopted to model the intercellular interaction for the aggregation behavior, which is characterized as the weak attraction at far distance and strong repulsion at near distance. For validation of the present technique, the dynamic interaction of two RBCs in static blood plasma is simulated firstly, where the RBCs aggregate slowly until a balanced configuration is achieved between the deformation and aggregation forces. The balanced configuration is in good agreement with the results reported previously. Three important effects on the dynamic behavior of RBCs are then analyzed, and they are the initial RBC shape, RBC deformability, and the intercellular interaction strength. It is found that the RBC is less deformed into a well-known parachute shape when the initial RBC shape is larger. Similarly, if the elastic shear modulus and bending stiffness of RBC membrane increase, the RBC resistance to deformation becomes higher, such that the RBC is less deformed. The simulation results also demonstrate that the RBC deformability strongly depends on the intercellular interaction strength. The RBCs deform more easily as the intercellular interaction strength increases.     

Effects of r—HuEPO on the biophysical characteristics of erythrocyte membrane in patients with anemia of chronic renal failure

Using electron spin resonance (ESR) spin labeling technique,we have studied the conformation of sulfhydryl groups(-SH) binding sites in membrane proteins and mem brane fluidity of red blood cells(RBCs) from two groups of patients with anemia of chronic renal failure(ACRF).One of the groups is composed of patients who were untreated with recombinant human erythropoietin(r-HuEPO),and the other is composed of patients who were treated with r-HuEPO.The results indicated:1)the conformation of SH group binding site in RBC membrane proteins from former group was different from those of healty people.2)the fluidity in the region near the surface of RBC membrane from former group was lower than those of healthy people.3)However,the above biophysical properties of RBC membrane from later group were normal.We concluded that RBC membrane in patients with ACRF was abnormal,and the treatment of r-HuEPO may promote the production of normal RBCs,thus ameliorate the biophysical properties of RBCs from the patients with ACRF.     

Sublethal Supraphysiological Shear Stress Alters Erythrocyte Dynamics in Subsequent Low-Shear Flows

Blood is a non-Newtonian, shear-thinning fluid owing to the physical properties and behaviors of red blood cells (RBCs). Under increased shear flow, pre-existing clusters of cells disaggregate, orientate with flow, and deform. These essential processes enhance fluidity of blood, although accumulating evidence suggests that sublethal blood trauma—induced by supraphysiological shear exposure—paradoxically increases the deformability of RBCs when examined under low-shear conditions, despite obvious decrement of cellular deformation at moderate-to-higher shear stresses. Some propose that rather than actual enhancement of cell mechanics, these observations are “pseudoimprovements” and possibly reflect altered flow and/or cell orientation, leading to methodological artifacts, although direct evidence is lacking. This study thus sought to explore RBC mechanical responses in shear flow using purpose-built laser diffractometry in tandem with direct optical visualization to address this problem. Freshly collected RBCs were exposed to a mechanical stimulus known to drastically alter cell deformability (i.e., prior shear exposure (PSE) to 100 Pa × 300 s). Samples were subsequently transferred to a custom-built slit-flow chamber that combined laser diffractometry with direct cell visualization. Cell suspensions were sheared in a stepwise manner (between 0.3 and 5.0 Pa), with each step being maintained for 15 s. Deformability and cell orientation indices were recorded for small-scatter Fraunhofer diffraction patterns and also visualized RBCs. PSE RBCs had significantly decreased visualized and laser-derived deformability at any given shear stress ≥1 Pa. Novel, to our knowledge, observations demonstrated that PSE RBCs had increased heterogeneity of direct visualized orientation with flow vector at any shear, which may be due to greater vorticity and thus instability in 5-Pa flow compared with unsheared control. These findings indicate that shear exposure and stress-strain history can alter subsequent RBC behavior in physiologically relevant low-shear flows. These findings may yield insight into microvascular disorders in recipients of mechanical circulatory support and individuals with hematological diseases that alter physical properties of blood.     

Passage time measurement of individual and blood cells through arrayed micropores on Si3N4 membrane

A new system has been developed for determining the deformability of individual red blood cells (RBCs), simulating the passage of RBCs in capillaries. The kernel of this system was the micropore array filter with an accurately defined pattern made by semiconductor microprocessing techniques. Individual microscopic RBC images were processed in parallel through a microcomputer and its interfacing circuit. An experiment with a normal RBC from a human donor demonstrated that it could pass the circular pore filter with a diameter as small as 1.0 μm at 2 cm H₂O pressure difference. Deformability of RBCs treated with diamide or acetylphenylhidralazine was also measured, showing that the system was sufficiently sensitive to detect the deformability loss due to membrane damage or to polymerization of the cytoplasma.     

Alteration of red blood cell microrheology by anti-tumor chemotherapy drugs

The aim of this study was to estimate effects of some chemotherapy drugs on the elasticity and deformability of the membrane of a red blood cell (RBC). It was found that incubation of red blood cells (RBCs) with cisplatin or epoetin alpha led to considerable (by 10–17%; p < 0.05) increase in the RBC deformability and that cisplatin could activate tyrosine protein kinases (TPKs). Preincubation of RBCs with a specific inhibitor of EGF-R and Src kinase, lavendustin A, almost completely prevented the cisplatin effect. Tyrosine phosphatase inhibitor, sodium orthovanadate, increased the RBC deformability (p < 0.05). This effect was also abandoned by lavendustin A. To test a hypothesis on the involvement of protein kinases of mature RBCs in control of their membrane elasticity, the cells were incubated with phorbol 12-myristate 13-acetate (PMA) activating protein kinase Cα (PKCα). PMA increased the RBC deformability only moderately (by 8%, p < 0.05) and the effect was canceled by nonselective and selective PKC inhibitors staurosporin and 4-(1-methylindol-3-yl)maleimide hydrochloride. Erythropoietin is known to inhibit the nonselective cation channels of the RBC membrane; however, preincubation of the cells with verapamil did not cancel the increase in their deformability. Hence, this increase in deformability could be a result of the action of tyrosine protein kinases, the more so that this effect was almost completely canceled by lavendustion A. The results suggest that the presence of functionally active protein kinases and phosphatases in the membranes of mature RBC makes them a target for the addressed effects of signal molecules, including some chemotherapy drugs, causing consecutive alterations in the RBC membrane elasticity, microrheological properties, and transport potential.     

Microscopic photometric quantification of stiffness and relaxation time of red blood cells in a flow chamber

The Microscopic Photometric Monolayer Technique provides a tool to measure red blood cell (RBC) stiffness (resistance to elongation) and relaxation time. It combines many of the advantages of flow channel studies of point-attached RBCs with the simplicity, sensitivity and accuracy of photometric light transmission measurement This technique allows the study of the effects of physicochemical factors on the elongation and relaxation time of the same cells within an average of four to five thousand cells adhered as a monolayer to glass. Further, the time course of physicochemical effects on cell membrane and wash-in/wash-out kinetics of interactions can be followed. An automated version of this technique was developed. A dense monolayer of point-attached RBCs was prepared at the bottom of a flow-chamber. A steady-state flow, with stepwise increases of flow rate, induced the RBC elongation. The light transmission perpendicular through the monolayer plane was measured photometrically. Photomicrographs compared with photometric results showed that the flow-induced bending and curvature change of RBC membrane was associated with the increase of light transmission. There was a linear correlation between the photometric index of elongation and the elongation taken from photomicrographs for shear stresses up to 0.75 Pa. A stiffness parameter, S (in Pa), was defined as the ratio of shear stress and elongation at a shear stress of 0.25 Pa. Following a sudden flow stoppage, the RBCs returned to their resting shape and the RBC relaxation time was measured. The stiffness-relaxation time product, V (in mPas), was calculated to provide an estimate of viscosity. Diamide treatment, known to stiffen RBCs, did result in dose-dependent decreases of elongation and relaxation time. With increasing temperature, the relaxation time decreased at a rate of −2.96 ms/K; the stiffness increased significantly at a rate of 0.0038 Pa/K, and the stiffness-relaxation time product decreased with −2.95 mPas/K, reflecting an inverse relationship between RBC viscosity and temperature. Using the automated version of this technique (Elias-c-) to test RBCs of 36 healthy subjects, we found the inter-individual coefficients of variation to be 8.6% for stiffness, 7.9% for relaxation time and 12.4% for stiffness-relaxation time product.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Measurement of the distribution of red blood cell deformability using an automated rheoscope

BACKGROUND: Red blood cells (RBCs) have to deform markedly to pass through the smallest capillaries of the microcirculation. Techniques for measuring RBC deformability often result in an indication of the mean value. A deformability distribution would be more useful for studying diseases that are marked by subpopulations of less deformable cells because even small fractions of rigid cells can cause circulatory problems. METHODS: We present an automated rheoscope that uses advanced image analysis techniques to determine a RBC deformability distribution (RBC-DD) by analyzing a large number of individual cells in shear flow. The sensitivity was measured from density-separated fractions of one blood sample and from cells rendered less deformable by heat treatment. A preliminary experiment included the RBC-DDs of a patient with sickle cell anemia, one on dialysis and being treated with erythropoietin, and one with elliptocytosis. RESULTS: Measurement of the RBC-DD was highly reproducible. The sensitivity test showed markedly different deformability distributions of density-separated cells and yielded distinct RBC-DDs after each additional minute of heat treatment. CONCLUSION: The automated rheoscope enabled the determination of RBC-DDs from which less deformable subpopulations can be established. The shape of an RBC-DD may be valuable in assessing cell fractions with normal and anomalous deformability within pathologic blood samples.