NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Microscopic diffusion and hydrodynamic interactions of hemoglobin in red blood cells

The cytoplasm of red blood cells is congested with the oxygen storage protein hemoglobin occupying a quarter of the cell volume. The high protein concentration leads to a reduced mobility; the self-diffusion coefficient of hemoglobin in blood cells is six times lower than in dilute solution. This effect is generally assigned to excluded volume effects in crowded media. However, the collective or gradient diffusion coefficient of hemoglobin is only weakly dependent on concentration, suggesting the compensation of osmotic and friction forces. This would exclude hydrodynamic interactions, which are of dynamic origin and do not contribute to the osmotic pressure. Hydrodynamic coupling between protein molecules is dominant at short time- and length scales before direct interactions are fully established. Employing neutron spin-echo-spectroscopy, we study hemoglobin diffusion on a nanosecond timescale and protein displacements on the scale of a few nanometers. A time- and wave-vector dependent diffusion coefficient is found, suggesting the crossover of self- and collective diffusion. Moreover, a wave-vector dependent friction function is derived, which is a characteristic feature of hydrodynamic interactions. The wave-vector and concentration dependence of the long-time self-diffusion coefficient of hemoglobin agree qualitatively with theoretical results on hydrodynamics in hard spheres suspensions. Quantitative agreement requires us to adjust the volume fraction by including part of the hydration shell: Proteins exhibit a larger surface/volume ratio compared to standard colloids of much larger size. It is concluded that hydrodynamic and not direct interactions dominate long-range molecular transport at high concentration.     

NMR relaxation studies of intracellular Na in red blood cells

The state of intracellular Na⁺ in human and dog erythrocytes was characterized by ²³Na-NMR using dysprosium complexes as shift reagents. Intracellular Na⁺ concentrations were determined using integration of the inner Na⁺ NMR signals and measurements of the intracellular volume using ⁵⁹Co-NMR of extracellular Co(CN)³⁻₆. T₂ was found to be significantly shorter than T₁, indicating some binding to macromolecules. While the longitudinal magnetization decay follows a single exponential the transverse magnetization could be fitted with a double-exponential function. It was shown that neither the binding to the inner side of the membrane nor binding to hemoglobin contributes to the relaxation enhancement.     

The dynamics of shear disaggregation of red blood cells in a flow channel

Red blood cell (RBC) rouleaux were formed in a flow channel in the presence of 2 g/dl dextran (molecular weight 76,000). The partial separation of RBC rouleau doublets adhering to the floor of the flow channel in response to small oscillatory shear stresses was observed experimentally. Theoretical analyses on displacement and drag force were performed to determine whether the motion of the cell involves membrane rotation (i.e., rolling) or sliding. From the experimental data and the results of theoretical analyses, it is concluded that, under the conditions of the experiments, the RBCs in a doublet separate from each other by rolling, rather than sliding of the sheared cell.     

The bulk rheology of close-packed red blood cells in shear flow

A theoretical analysis is made of the dynamical behavior and bulk rheology of close-packed red blood cell suspensions subjected to simple shear flow. The model for the polyhedral cell shapes and tank-treading membrane motion developed in the companion paper (1) is used. The flow in the thin lubricating plasma layers between cells is analyzed taking into account the mechanical properties of the membrane at the corner regions of sharp membrane curvature. This leads to predictions for the apparent viscosity as a function of hematocrit and shear rate. Good agreement with experimental results is obtained at moderate and high shear rates (above 20 s-1). At lower shear rates, a rapid rise in apparent viscosity has been found experimentally, and the mechanisms leading to this behavior are examined.     

Tank treading of optically trapped red blood cells in shear flow

Tank-treading (TT) motion is established in optically trapped, live red blood cells (RBCs) held in shear flow and is systematically investigated under varying shear rates, temperature (affecting membrane viscosity), osmolarity (resulting in changes in RBC shape and cytoplasmic viscosity), and viscosity of the suspending medium. TT frequency is measured as a function of membrane and cytoplasmic viscosity, the former being four times more effective in altering TT frequency. TT frequency increases as membrane viscosity decreases, by as much as 10% over temperature changes of only 4°C at a shear rate of ∼43 s⁻¹. A threshold shear rate (1.5 ± 0.3 s⁻¹) is observed below which the TT frequency drops to zero. TT motion is also observed in shape-engineered (spherical) RBCs and those with cholesterol-depleted membranes. The TT threshold is less evident in both cases but the TT frequency increases in the latter cells. Our findings indicate that TT motion is pervasive even in folded and deformed erythrocytes, conditions that occur when such erythrocytes flow through narrow capillaries.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.     

E-wave deceleration time may not provide an accurate determination of LV chamber stiffness if LV relaxation/viscoelasticity is unknown

Average left ventricular (LV) chamber stiffness (Delta P(avg)/Delta V(avg)) is an important diastolic function index. An E-wave-based determination of Delta P(avg)/Delta V(avg) (Little WC, Ohno M, Kitzman DW, Thomas JD, Cheng CP. Circulation 92: 1933-1939, 1995) predicted that deceleration time (DT) determines stiffness as follows: Delta P(avg)/Delta V(avg) = N(pi/DT)(2) (where N is constant), which implies that if the DTs of two LVs are indistinguishable, their stiffness is indistinguishable as well. We observed that LVs with indistinguishable DTs may have markedly different Delta P(avg)/Delta V(avg) values determined by simultaneous echocardiography-catheterization. To elucidate the mechanism by which LVs with indistinguishable DTs manifest distinguishable chamber stiffness, we use a validated, kinematic E-wave model (Kovács SJ, Barzilai B, Perez JE. Am J Physiol Heart Circ Physiol 252: H178-H187, 1987) with stiffness (k) and relaxation/viscoelasticity (c) parameters. Because the predicted linear relation between k and Delta P(avg)/Delta V(avg) has been validated, we reexpress the DT-stiffness (Delta P(avg)/Delta V(avg)) relation of Little et al. as follows: DT(k) approximately pi/(2k). Using the kinematic model, we derive the general DT-chamber stiffness/viscoelasticity relation as follows: DT(k,c) = pi/(2skrt[k])+c/(2k)(where c and k are determined directly from the E-wave), which reduces to DT(k) when c < k. Validation involved analysis of 400 E-waves by determination of five-beat averaged k and c from 80 subjects undergoing simultaneous echocardiography-catheterization. Clinical E-wave DTs were compared with model-predicted DT(k) and DT(k,c). Clinical DT was better predicted by stiffness and relaxation/viscoelasticity (r(2) = 0.84, DT vs. DT(k,c)) jointly rather than by stiffness alone (r(2) = 0.60, DT vs. DT(k)). Thus LVs can have indistinguishable DTs but significantly different Delta P(avg)/Delta V(avg) if chamber relaxation/viscoelasticity differs. We conclude that DT is a function of both chamber stiffness and chamber relaxation viscoelasticity. Quantitative diastolic function assessment warrants consideration of simultaneous stiffness and relaxation/viscoelastic effects.     

Deformation and fragmentation of human red blood cells in turbulent shear flow.

By means of glutaraldehyde fixation, human erythrocytes are "frozen" while suspended in turbulent shear flow. As the shearing is increased in steps from 100 to 2,500 dyn/cm2, the deformed cells evolve gradually toward a smooth ellipsoidal shape. At stresses above 2,500 dyn/cm2, approximately, fragmentation of the cells occurs with a concomitant increase in free hemoglobin content of the suspending medium. The photographic evidence suggests that the cells rupture in tension in the bulk flow.     

Influence of gravity on flow distribution of red blood cells in microcirculation

The influence of the gravity on flow distribution of erythrocytes in microcirculation was examined. We developed a new centrifuge system with a rotation disc. An observation system of blood flow in a micro-flow channel was arranged on the disc. Erythrocyte flow in the micro-flow tube was displaced under the gravity. This study suggests that the gravity affects the transfer of substances from blood vessels to tissues.     

Ultrasound palpation for fast in-situ quantification of articular cartilage stiffness,thickness and relaxation capacity

Most current cartilage testing devices require the preparation of excised samples and therefore do not allow intra-operative application for diagnostic purposes. The gold standard during open or arthroscopic surgery is still the subjective perception of manual palpation. This work presents a new diagnostic method of ultrasound palpation (USP) to acquire applied stress and strain data during manual palpation of articular cartilage. With the proposed method, we obtain cartilage thickness and stiffness. Moreover, repeated palpations allow the quantification of relaxation effects. USP measurements on elastomer phantoms demonstrated very good repeatability for both, stage-guided (97.2%) and handheld (96.0%) applications. The USP measurements were compared with conventional indentation experiments and revealed very good agreement on elastomer phantoms (\(r = 0.98\)) and good agreement on porcine cartilage samples (\(r = 0.76\)). Artificially degenerated cartilage samples showed reduced stiffness, weak capacity to relax after palpation and an increase of stiffness of approximately 50% with each single palpation. Intact cartilage was measured by USP directly at the patella (in situ) and after excision and removal of the subchondral bone (ex situ), leading to stiffness values of \(12.1\pm 5.5\) and \(8.5\pm 5.9\,\hbox {MPa}\) (\(p<0.05\)), respectively. The results demonstrate the potential of the USP system for cartilage testing, its sensitivity to degenerative changes and as a method for quantifying relaxation processes by means of repeated palpations. Furthermore, the differences in the results of in-situ and ex-situ measurements are of general interest, since such comparison has not been reported previously. We point out the limited comparability of ex-situ cartilage with its in-situ biomechanical behavior.     

Simplified flow cytometric method for fetal hemoglobin containing red blood cells

Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.     

Rheological discrimination between native, rigid and aggregated red blood cells in oscillatory flow

The viscoelastic behaviour of hardened or aggregated red blood cells is compared with the flow pattern of native red blood cells, all suspended in buffer solution at a hematocrit of 45%. The rheological properties are investigated under oscillatory shear at the constant frequency of 2Hz. Variation of the amplitude covers a range of shear-rates from 0.5/s to 200/s. It can be seen that rigidification of the red cells by treatment with glutardialdehyde leads to changes of the flow properties in the range of shear-rates above 10/s, whereas aggregate formation due to addition of dextran distinctly alters the flow properties in the range of shear-rates below 10/s.     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Abnormal blood flow and red blood cell deformability in severe malaria

Obstruction of the microcirculation plays a central role in the pathophysiology of severe malaria. Here, Arjen Dondorp and colleagues describe the various contributors to impaired microcirculatory flow in falciparum malaria: sequestration, rosetting and recent findings regarding impaired red blood cell deformability. The correlation with clinical findings and possible therapeutic consequences are discussed.     

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).