Aggregation of red blood cells studied by ultrasound backscattering

The erythrocyte aggregation phenomenon is an important factor in capillary circulation. This phenomenon can be evaluated by a number of methods (microscopic observations, viscometry, light measurements) which cannot be applied simply to in vivo measurements. In contrast, ultrasound which propagates through soft tissues allows measurement of the mechanical properties of red blood cell (RBC) suspensions which depend on the aggregation phenomenon. We devised an apparatus in order to measure in vitro the ultrasonic backscattering intensity of RBC suspensions. First, with latex particles of different sizes, the ultrasonic backscattering coefficient has been measured in order to evaluate the apparatus response. Then, the ultrasonic backscattering coefficient of different aggregated erythrocyte suspensions has been measured and correlated with the erythrocyte sedimentation rate. Finally, the size of RBC aggregates of different suspensions has been evaluated.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.     

Competitive role between fibrinogen and albumin on thixotropy of red cell suspensions

Plasmatic proteins, namely fibrinogen and globulins, play a major role in red blood cell (RBC) aggregation which is accountable for the three-dimensional structure of blood. Consequently, blood rheological properties linked to this structure must be modified when the protein plasma content changes. This paper gives results and related comments on thixotropic properties of RBC suspensions (0.45 hematocrit) in isotonic solutions containing various amount of fibrinogen to which albumin is added. Thixotropic behavior of these RBC suspensions is studied with a low inertia coaxial cylinders viscometer at a shear rate step of Y = 1 s-1. Rheograms are interpreted in term of thixotropy coefficient. The main conclusion is that albumin improves RBC disaggregability of whole blood, resulting probably from a competitive effect between fibrinogen and albumin in the RBC aggregation process.     

Venous ultrasound catheter-tip technique for evaluation of arterial hemodynamics.

A variety of devices has been used for measuring flow properties of deep-lying arteries, but many have limitations. This paper describes a relatively nontraumatic intravenous approach which uses a catheter in connection with a pulsed ultrasonic Doppler velocity meter (PUDVM) and an ultrasound echo track. The venous ultrasound catheter (VUC) has permitted measurements of local instantaneols blood velocity, flow, and wall motion in the abdominal aorta and iliac arteries of beagle dogs; evaluation studies have been conducted to compare the VUC recordings with an independent method for measuring blood flow and wall motion. Coupling of this catheter-tip device with the PUDVM and echo track provides chronic measurements of hemodynamic parameters in these deep vessels which were virtually impossible to obtain previously. This technique may prove useful in monitoring vessel pathology longitudinally as well as in basic experimental situations requiring flow and arterial wall mechanical properties.     

Micro-macro link in rheology of erythrocyte and vesicle suspensions

We report on the rheology of dilute suspensions of red blood cells (RBC) and vesicles. The viscosity of RBC suspensions reveals a previously unknown signature: it exhibits a pronounced minimum when the viscosity of the ambient medium is close to the value at which the transition from tank-treading to tumbling occurs. This bifurcation is triggered by varying the viscosity of the ambient fluid. It is found that the intrinsic viscosity of the suspension varies by about a factor of 4 in the explored parameter range. Surprisingly, this significant change of the intrinsic viscosity is revealed even at low hematocrit (5%). We suggest that this finding may be used to detect blood flow disorders linked to pathologies that affect RBC shape and mechanical properties. This opens future perspectives on setting up new diagnostic tools, with great efficiency even at very low hematocrit. Investigations are also performed on giant vesicle suspensions, and compared to RBCs.     

Cellular determinants of low-shear blood viscosity

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48°C, 5 min), glutaraldehyde (0.005–0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer (“M” and “Ml” indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses (i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.     

Role of erythrocyte deformability during capillary wetting

Deformability of erythrocyte was found to fundamentally alter the wetting dynamics of red blood cell (RBC) suspensions during their invasion into capillaries. Normal RBC suspensions failed to penetrate more than 1 cm into a glass capillary when the capillary radius was smaller than a critical value that is dependent on the erythrocyte concentration (about 50 microm for whole blood). In contrast, suspensions of rigidified RBCs, after cross-linking with different concentrations of glutaraldehyde or incubating with 100 ng/mL of an endotoxin, could penetrate any capillary larger than the erythrocyte dimension. The effect of RBC deformability on penetration was attributed to the enhanced shear-induced migration of normal deformable RBCs toward the capillary centreline, which imparted a higher average velocity to the RBCs than the average plasma velocity. As a result, the erythrocytes advanced into the capillary faster than the wetting meniscus, packing behind it to form a concentrated slug. This tightly packed slug had a high hydrodynamic resistance that could arrest the penetrating flow of concentrated suspensions into the small capillaries.     

Ultrasound imaging

Modern ultrasonic transducers mainly employ lead zirconate titanate (PZT) but vinylidene fluoride trifluoroethylene copolymer (P (VDF-TrPE)) is becoming more competitive. The static scanner is now largely replaced by mechanical or electronically controlled array real time systems; the speed of scanning is limited by the speed of sound and the resolution depends on the wavelength and so, ultimately, on the attenuation in tissue. Tissue inhomogeneities degrade the resolution. Intraoperative and intracavitary scanners have advantages in some anatomical situations and ultrasonic imaging can guide extracorporeal shock wave lithotripsy. Inexpensive battery powered scanners will soon become available. Duplex scanners are used to localize the acquisition of Doppler signals; blood flow volume rate can be estimated from measurements of blood velocity and vessel cross-sectional area, or by the attenuation-compensated technique which avoids the main sources of error. Colour flow mapping combines real time imaging with Doppler information, but has limited scanning speed. Computed tomography and acoustical microscopy are feasible. Speckle arises from the coherent nature of ultrasound and can be suppressed by summing uncorrelated images or by filtering. Image manipulation and display techniques are being developed to cope with three dimensional scan data and the approach is compatible with picture archiving and communication systems (PACS). Tissue characterization based on the measurement of properties has been disappointing but blood flow analysis and contrast agents are promising. Quality assurance programmes are crucial; ultrasonic diagnosis appears to be free from hazard and prudent use is determined by cost-benefit considerations.     

Conductometric study of shear-dependent processes in red cell suspensions. II. Transient cross-stream hematocrit distribution

A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel. The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance. Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level. The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate. A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening. It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks. For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.     

Capillary blood flow imaging within human finger cuticle using optical microangiography

We report non‐invasive 3D imaging of capillary blood flow within human finger cuticle by the use of Doppler optical microangiography (DOMAG) and ultra‐high sensitive optical microangiography (UHS‐OMAG) techniques. Wide velocity range DOMAG method is applied to provide red blood cell (RBC) axial velocity mapping in capillary loops with ranges of ±0.9 mm/s and ±0.3 mm/s. Additionally, UHS‐OMAG technique is engineered to acquire high resolution image of capillary morphology. The presented results are promising to facilitate clinical trials of treatment and diagnosis of various diseases such as diabetes, Raynaud's phenomenon, and connective tissue diseases by quantifying cutaneous blood flow changes within human finger cuticle. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Pulse-Doppler Ultrasound and Its Clinical Application

Doppler devices can provide clinically useful information about blood flow. This paper describes how a pulse-Doppler instrument has been linked to a B-scan machine so that flow patterns in any ultrasonically accessible vessel can be monitored from the skin surface. The same transducer is used for both modes of investigation to enable accurate and reliable positioning of the sample-volume. After briefly summarizing the principles of continuous-wave and pulse-Doppler operation, the concepts and relative advantages of coherent and non-coherent detection are explained. The potential clinical uses of the pulse-echo-Doppler hybrid and Doppler devices in general are then discussed.     

Study of the trajectory of erythrocyte movement in microvessels using a method of automatic image analysis

Many physiological and pathological processes in the circulation are related to changes of blood rheological properties. Since blood represents a specific suspension of cells in plasma the mentioned changes are to a great degree dependent on behavior and interaction of red blood cells (RBC). For investigation of blood flow structure in microvessels we developed an algorithm and worked out a program for automatic treatment of RBC movement on the basis of automatic image analysis system "Leitz-TAS" (Ernst Leitz, FRG). The program was based on computation of coordinates of blood cell centers. Further we calculated the following values: the vector of displacements (S), its projection on both axes (dx, dy), their relationship (dy/dx), the relationship of RBC radial coordinate to vessel radius (y/R), and the velocity of RBC movements (V). Proceeding from these data we obtained such parameters, as e.g., the velocity profile, the radial displacements of red cells, etc. by which we could judge on the blood flow regime, the flow structure and changes of blood rheological properties.     

In vivo photoacoustic and photothermal cytometry for monitoring multiple blood rheology parameters

Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo assessment of multiple hemorheological parameters over long periods of time has not been reported. Here, we review the capabilities of label-free photoacoustic (PA) and photothermal (PT) flow cytometry for dynamic monitoring of hemorhelogical parameters in vivo which we refer to as photoacoustic and photothermal blood rheology. Using phenomenological models, we analyze correlations between both PT and PA signal characteristics in the dynamic modes and following determinants of blood rheology: red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, hematocrit, and likely shear rate. We present ex vivo and in vivo experimental verifications involving high-speed PT imaging of RBCs, identification of sickle cells in a mouse model of human sickle cell disease and in vivo monitoring of complex hemorheological changes (e.g., RBC deformability, hematocrit and RBC aggregation). The multi-parameter platform that integrates PT, PA, and conventional optical techniques has potential for translation to clinical applications using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency.     

Conductometric study of shear-dependent processes in red cell suspensions. I. Effect of red blood cell aggregate morphology on blood conductance

The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz. The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks. It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates. Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis. These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.     

Osmotic behavior of red blood cell as seen with an ultrasonic method

We have measured the density and ultrasonic velocity (usv) of swine red blood cell (RBC) suspensions in the wide osmolarity range from 300 mOsm to 1400 mOsm in saline solution. The cellular density and compressibility of RBC at each osmolarity were obtained using the fact that the density and the compressibility are additive by volume. The osmolarity dependence of hematocrit was also measured at a constant number concentration of RBC in the range of 300 mOsm to 1700 mOsm. The cellular density and the cellular compressibility of RBC as well as the inverse of hematocrit were expressed well into one unique exponential type equation f (pi) = a [1 - b exp (-c pi)] with a common value for the coefficient c = 0.0025 against the osmolarity pi. The results were analyzed with a simple consideration based only upon the contribution of free water inside the erythrocyte through the volume concentration phi of the free water in it. According to this theoretical analysis, the density and the compressibility of the free water were found to be 0.990 g/cm3 and 4.59 x 10(-11) cm2/dyne which agree closely with 0.998 g/cm3 and 4.59 x 10(-11) cm2/dyn of pure water at 20 degrees C within the experimental error.     

Simulation of ultrasound backscattering by red cell aggregates: effect of shear rate and anisotropy

Tissue characterization using ultrasound (US) scattering allows extraction of relevant cellular biophysical information noninvasively. Characterization of the level of red blood cell (RBC) aggregation is one of the proposed application. In the current paper, it is hypothesized that the microstructure of the RBCs is a main determinant of the US backscattered power. A simulation model was developed to study the effect of various RBC configurations on the backscattered power. It is an iterative dynamical model that considers the effect of the adhesive and repulsive forces between RBCs, and the effect of the flow. The method is shown to be efficient to model polydispersity in size, shape, and orientation of the aggregates due to the flow, and to relate these variations to the US backscattering properties. Three levels of aggregability at shear rates varying between 0.05 and 10 s(-1) were modeled at 40% hematocrit. The simulated backscattered power increased with a decrease in the shear rate or an increase in the RBC aggregability. Angular dependence of the backscattered power was observed. It is the first attempt to model the US power backscattered by RBC aggregates polydisperse in size and shape due to the shearing of the flow.     

Nontraumatic aortic blood flow sensing by use of an ultrasonic esophageal probe.

The measurement of blood velocity fields, volume flow, and arterial wall motion in the descending thoracic aorta provides essential hemodynamic information for both research and clinical diagnosis. The close proximity of the esophagus to the aorta in the dog makes it possible to obtain such data nonsurgically using an ultrasonic esophageal probe; however, the accuracy of such a probe is limited if the angle between the sound beam and the flow axis, known as the Doppler angle, is not precisely known. By use of a pulsed Doppler velocity meter (PUDVM) and a triangulation procedure, accurate empirical measurement of the Doppler angle has been obtained, allowing quantification of blood velocity scans across the aorta. Volume flow is obtained by integration of blood velocity profiles and arterial wall motion is measured with an ultrasonic echo tracking device. Accuracy of the probe was substantiated by comparison with ultrasonic and electromagnetic implanted flow cuff measurements. Use of the probe in measurement of blood velocity, volume flow and arterial wall motion at various locations along the 8- and 10-cm length of the descending thoracic aorta in adult beagle dogs is detailed. The simplicity, accuracy, and nontraumatic aspect of the technique should allow increasing use of such a probe in numerous research and clinical applications.     

Spatiotemporal Dynamics of Dilute Red Blood Cell Suspensions in Low-Inertia Microchannel Flow

Microfluidic technologies are commonly used for the manipulation of red blood cell (RBC) suspensions and analyses of flow-mediated biomechanics. To enhance the performance of microfluidic devices, understanding the dynamics of the suspensions processed within is crucial. We report novel, to our knowledge, aspects of the spatiotemporal dynamics of RBC suspensions flowing through a typical microchannel at low Reynolds number. Through experiments with dilute RBC suspensions, we find an off-center two-peak (OCTP) profile of cells contrary to the centralized distribution commonly reported for low-inertia flows. This is reminiscent of the well-known “tubular pinch effect,” which arises from inertial effects. However, given the conditions of negligible inertia in our experiments, an alternative explanation is needed for this OCTP profile. Our massively parallel simulations of RBC flow in real-size microfluidic dimensions using the immersed-boundary-lattice-Boltzmann method confirm the experimental findings and elucidate the underlying mechanism for the counterintuitive RBC pattern. By analyzing the RBC migration and cell-free layer development within a high-aspect-ratio channel, we show that such a distribution is co-determined by the spatial decay of hydrodynamic lift and the global deficiency of cell dispersion in dilute suspensions. We find a cell-free layer development length greater than 46 and 28 hydraulic diameters in the experiment and simulation, respectively, exceeding typical lengths of microfluidic designs. Our work highlights the key role of transient cell distribution in dilute suspensions, which may negatively affect the reliability of experimental results if not taken into account.     

Graded alterations of RBC aggregation influence in vivo blood flow resistance

Although the effects of red blood cell (RBC) aggregation on low-shear rate blood viscosity are well known, the effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F-98) and then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating and was verified by microscopy and erythrocyte sedimentation rate (ESR), which increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hindlimb preparation while the arterial perfusion pressure was held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (using 10(-4) M papaverin), a significant change in flow resistance was observed in a RBC suspension with a 97% increase of ESR. Additional enhancements of RBC aggregation (i.e., 136 and 162% increases of ESR) decreased flow resistance almost to control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions with a 200% increase of ESR. This triphasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.     

Shear‐induced diffusion of red blood cells measured with dynamic light scattering‐optical coherence tomography

Quantitative measurements of intravascular microscopic dynamics, such as absolute blood flow velocity, shear stress and the diffusion coefficient of red blood cells (RBCs), are fundamental in understanding the blood flow behavior within the microcirculation, and for understanding why diffuse correlation spectroscopy (DCS) measurements of blood flow are dominantly sensitive to the diffusive motion of RBCs. Dynamic light scattering‐optical coherence tomography (DLS‐OCT) takes the advantages of using DLS to measure particle flow and diffusion within an OCT resolution‐constrained three‐dimensional volume, enabling the simultaneous measurements of absolute RBC velocity and diffusion coefficient with high spatial resolution. In this work, we applied DLS‐OCT to measure both RBC velocity and the shear‐induced diffusion coefficient within penetrating venules of the somatosensory cortex of anesthetized mice. Blood flow laminar profile measurements indicate a blunted laminar flow profile and the degree of blunting decreases with increasing vessel diameter. The measured shear‐induced diffusion coefficient was proportional to the flow shear rate with a magnitude of ~0.1 to 0.5 × 10^?6 mm². These results provide important experimental support for the recent theoretical explanation for why DCS is dominantly sensitive to RBC diffusive motion.