Ultrasonic backscatter from flowing whole blood. I: Dependence on shear rate and hematocrit

Previous results show that ultrasonic backscatter from red blood cells (RBCs) suspended in saline is a function of hematocrit and frequency and that it can be affected by flow disturbance. The experimental data agree well with the theories. In the present article, results on ultrasonic backscatter from flowing whole blood are reported. Studies have been conducted on porcine, bovine, and human blood. Ultrasonic backscatter of flowing whole blood differs from that of RBC suspensions in that it is shear-rate dependent, which means that it is a function of spatial position of the blood in the flow conduit. Moreover, the results indicate that it is also species dependent. This behavior can be readily understood when red cell aggregation is considered.     

High-frequency ultrasound backscattering by blood: analytical and semianalytical models of the erythrocyte cross section

This paper proposes analytical and semianalytical models of the ultrasonic backscattering cross section (BCS) of various geometrical shapes mimicking a red blood cell (RBC) for frequencies varying from 0 to 90 MHz. By assuming the first-order Born approximation and by modeling the shape of a RBC by a realistic biconcave volume, different scattering behaviors were identified for increasing frequencies. For frequencies below 18 MHz, a RBC can be considered a Rayleigh scatterer. For frequencies less than 39 MHz, the general concept of acoustic inertia tensor is introduced to describe the variation of the BCS with the frequency and the incidence direction. For frequencies below 90 MHz, ultrasound backscattering by a RBC is equivalent to backscattering by a cylinder of height 2 microm and diameter 7.8 microm. These results lay the basis of ultrasonic characterization of RBC aggregation by proposing a method that distinguishes the contribution of the individual RBC acoustical characteristics from collective effects, on the global blood backscattering coefficient. A new method of data reduction that models the frequency dependence of the ultrasonic BCS of micron-sized weak scatterers is also proposed. Applications of this method are in tissue characterization as well as in hematology.     

Modeling the frequency dependence (5-120 MHz) of ultrasound backscattering by red cell aggregates in shear flow at a normal hematocrit

The frequency dependence of the ultrasound signal backscattered by blood in shear flow was studied using a simulation model. The ultrasound backscattered signal was computed with a linear model that considers the characteristics of the ultrasound system and tissue acoustic properties. The tissue scattering properties were related to the position and shape of the red blood cells (RBCs). A 2D microrheological model simulated the RBC dynamics in a Couette shear flow system. This iterative model, described earlier [Biophys. J. 82, 1696-1710 (2002)], integrates the hydrodynamic effect of the flow, as well as adhesive and repulsive forces between RBCs. RBC aggregation was simulated at 40% hematocrit and shear rates of 0.05-2 s(-1). The RBC aggregate sizes ranged, on average, from 3.3 RBCs at 2 s(-1) to 33.5 cells at 0.05 s(-1). The ultrasound backscattered power was studied at frequencies between 5-120 MHz and insonification angles between 0-180 degrees. At frequencies below approximately 30 MHz, the ultrasound backscattered power increased as the shear rate was decreased and the size of the aggregates was raised. A totally different scattering behavior was noted above 30 MHz. Typical spectral slopes of the backscattered power (log-log scale) between 5-25 MHz equaled 3.8, whereas slopes down to 0.6 were measured at 0.05 s(-1), between 40-60 MHz. The ultrasound backscattered power was shown to be angle dependent at low frequencies (5-25 MHz). The anisotropy persisted at high frequencies (>25 MHz) for small aggregates (at 2 s(-1)). In conclusion, this study sheds some light on the blood backscattering behavior with an emphasis on the non-Rayleigh regime. Additional experimental studies may be necessary to validate the simulation results, and to fully understand the relation between the ultrasound backscattered power, level of RBC aggregation, shear rate, frequency, and insonification angle.     

Small-angle X-ray scattering probe of intermolecular interaction in red blood cells

With high concentrations of hemoglobin(Hb) in red blood cells, self-interactions among these molecules could increase the propensities of their polymerization and aggregation. In the present work, high concentration Hb in solution and red blood cells were analyzed by small-angle X-ray scattering. Calculation of the effective structure factor indicates that the interaction of Hb molecules is the same when they are crowded together in both the cell and physiological saline. The Hb molecules stay individual without the formation of aggregates and clusters in cells.     

Boundary element simulation of backscattering properties for red blood with high frequency ultrasonic transducers

High frequency ultrasonic imaging (e.g. >30 MHz) from blood is difficult due to its tenuous backscattered pressure and the interference from adjacent tissues as well. To increase the sensitivity focused transducer has to be used, thus raising the complexity of interpreting the received signals. A numerical simulation of the ultrasonic scattering property from erythrocyte and rouleaux based on boundary element method was performed with experimental results based on a modified substitution method. The results (proportional relationship between backscattered pressure and frequency and the frequency limit for Rayleigh scattering) closely coincide with experimental data for erythrocyte. Rouleaux model results also show the dependence of degree of red cell aggregation on backscattering properties. The boundary element method serves as a good means to calculate the acoustic scattering from blood cells under arbitrary incident waves.     

Ultrasonic backscatter coefficient quantitative estimates from high-concentration Chinese Hamster Ovary cell pellet biophantoms

Previous work estimated the ultrasonic backscatter coefficient (BSC) from low-concentration (volume density <3%) Chinese Hamster Ovary (CHO, 6.7-μm cell radius) cell pellets. This study extends the work to higher cell concentrations (volume densities: 9.6% to 63%). At low concentration, BSC magnitude is proportional to the cell concentration and BSC frequency dependency is independent of cell concentration. At high cell concentration, BSC magnitude is not proportional to cell concentration and BSC frequency dependency is dependent on cell concentration. This transition occurs when the volume density reaches between 10% and 30%. Under high cell concentration conditions, the BSC magnitude increases slower than proportionally with the number density at low frequencies (ka<1), as observed by others. However, what is new is that the BSC magnitude can increase either slower or faster than proportionally with number density at high frequencies (ka>1). The concentric sphere model least squares estimates show a decrease in estimated cell radius with number density, suggesting that the concentric spheres model is becoming less applicable as concentration increases because the estimated cell radius becomes smaller than that measured. The critical volume density, starting from when the model becomes less applicable, is estimated to be between 10% and 30% cell volume density.     

Experimental ultrasound characterization of red blood cell aggregation using the structure factor size estimator

The frequency dependence of the ultrasonic backscattering coefficient (BSC) was studied to assess the level of red blood cell (RBC) aggregation. Three monoelement focused wideband transducers were used to insonify porcine blood sheared in a Couette flow from 9 to 30 MHz. A high shear rate was first applied to promote disaggregation. Different residual shear rates were then used to promote formation of RBC aggregates. The structure factor size estimator (SFSE), a second-order data reduction model based on the structure factor, was applied to the frequency-dependent BSC. Two parameters were extracted from the model to describe the level of aggregation at 6% and 40% hematocrits: W, the packing factor, and D the aggregate diameter, expressed in number of RBCs. Both parameters closely matched theoretical values for nonaggregated RBCs. W and D increased during aggregation with stabilized values modulated by the applied residual shear rate. Furthermore, parameter D during the kinetics of aggregation at 6% hematocrit under static conditions correlated with an optical RBC aggregate size estimation from microscopic images (r(2)=0.76). To conclude, the SFSE presents an interesting framework for tissue characterization of partially correlated dense tissues such as aggregated RBCs.     

Frequency dependence of ultrasonic backscatter from human trabecular bone: theory and experiment

A model describing the frequency dependence of backscatter coefficient from trabecular bone is presented. Scattering is assumed to originate from the surfaces of trabeculae, which are modeled as long thin cylinders with radii small compared with the ultrasonic wavelength. Experimental ultrasonic measurements at 500 kHz, 1 MHz, and 2.25 MHz from a wire target and from trabecular bone samples from human calcaneus in vitro are reported. In both cases, measurements are in good agreement with theory. For mediolateral insonification of calcaneus at low frequencies, including the typical diagnostic range (near 500 kHz), backscatter coefficient is proportional to frequency cubed. At higher frequencies, the frequency response flattens out. The data also suggest that at diagnostic frequencies, multiple scattering effects on the average are relatively small for the samples investigated. Finally, at diagnostic frequencies, the data suggest that absorption is likely to be a larger component of attenuation than scattering.     

Modeling and analysis of multiple scattering of acoustic waves in complex media: application to the trabecular bone

The integral equations that describe scattering in the media with step-rise changing parameters have been numerically solved for the trabecular bone model. The model consists of several hundred discrete randomly distributed elements. The spectral distribution of scattering coefficients in subsequent orders of scattering has been presented. Calculations were carried on for the ultrasonic frequency ranging from 0.5 to 3 MHz. Evaluation of the contribution of the first, second, and higher scattering orders to total scattering of the ultrasounds in trabecular bone was done. Contrary to the approaches that use the μCT images of trabecular structure to modeling of the ultrasonic wave propagation condition, the 3D numerical model consisting of cylindrical elements mimicking the spatial matrix of trabeculae, was applied. The scattering, due to interconnections between thick trabeculae, usually neglected in trabecular bone models, has been included in calculations when the structure backscatter was evaluated. Influence of the absorption in subsequent orders of scattering is also addressed. Results show that up to 1.5 MHz, the influence of higher scattering orders on the total scattered field characteristic can be neglected while for the higher frequencies, the relatively high amplitude interference peaks in higher scattering orders clearly occur.     

Ultrasonic visualization of dynamic behavior of red blood cells in flowing blood

It is well known that the scatter of ultrasound by blood is mainly attributed to red blood cells (RBCs) and RBC aggregation. In the present review, researches of hemodynamic influence on RBC aggregation and ultrasound backscatter from blood were overviewed. A mock flow loop and a cylindrical chamber were employed to produce various blood flows, such as pulsatile, oscillatory, and rotational flow. The “black hole” (BLH), a dark hole at the tube center surrounded by bright zone in the cross sectional B-mode image and “bright collapsing ring” (BRCR) phenomena, appearance of bright ring at the periphery and collapse of it at the center during a pulsatile cycle, were observed under pulsatile flow. The combined effects of shear rate and flow acceleration on RBC aggregation were suggested as a possible mechanism for these phenomena. The stroke volume-dependence of the “bright ring” phenomenon under oscillatory flow could also be explained by flow acceleration. The enveloped echo images from rotational flow in a compact blood chamber showed the spatial and temporal variations of RBC aggregation, which varied with the mammalian species. In the stenotic model, it was found that the echogenic variation increased locally at a distance of three tube diameters downstream from the stenosis during decelerating period, which was proposed to be mainly due to flow turbulence. The similar ‘bright ring’ was also observed fromin vivo human carotid artery in harmonic imaging.     

Stress-dependent changes in the diffuse ultrasonic backscatter coefficient in steel: experimental results

In this article, the effects of uniaxial compressive loading on the ultrasonic scattering from polycrystalline grains are shown for 10 MHz ultrasound in annealed, 1018 steel. The results show a decreasing value of the stress-dependent backscatter coefficient for normal incident ultrasound when the compression loading is perpendicular to the scattering direction. The change due to scattering is about 2 orders of magnitude greater than changes observed by others using ultrasonic wavespeed measurements. It is anticipated that this research can serve as the basis for many methods associated with nondestructive determination of stress in structural materials.     

The extracellular matrix is an important source of ultrasound backscatter from myocardium

Ultrasound tissue characterization with measurement of backscatter has been employed in numerous experimental and clinical studies of cardiac pathology, yet the cellular components responsible for scattering from cardiac tissues have not been unequivocally identified. This laboratory has proposed a mathematical model for myocardial backscatter that postulates the fibrous extracellular matrix (ECM) as a significant determinant of backscatter. To demonstrate the importance of ECM, this group sought to determine whether measurements of backscatter from the isolated ECM could reproduce the known directional dependence, or anisotropy of backscatter, from intact cardiac tissues in vitro. Segments of left ventricular free wall from ten formalin fixed porcine hearts were insonified at 50 MHz, traversing the heart wall from endo- to epicardium to measure the anisotropy of myocardial backscatter, defined as the difference between peak (perpendicular to fibers) and trough (parallel to fibers) backscatter amplitude. The tissue segments were then treated with 10% NaOH to dissolve all of the cellular components, leaving only the intact ECM. Scanning electron micrographs (SEM) were obtained of tissue sections to reveal complete digestion of the cellular elements. The dimensions of the residual voids resulting from cell digestion were approximately the diameter of the intact myocytes (10-30 microm). These samples were reinsonified after seven days of treatment to compare the anisotropy of integrated backscatter. The magnitude of anisotropy of backscatter changed from 15.4 +/- 0.8 to 12.6 +/- 1.1dB for intact as compared with digested specimens. Because digestion of the myocardium leaves only extracellular sources of ultrasonic scattering, and because the isolated ECM exhibits similar ultrasonic anisotropy as does the intact myocardium, it is concluded that there is a direct association between the ECM and the anisotropy of backscatter within intact tissue. Thus, it is suggested that ultrasonic tissue characterization represents a potentially clinically applicable method for delineating the structure and function of the ECM.     

Numerical simulation of the red blood cell aggregation and deformation behaviors in ultrasonic field

The objective of this paper is to propose an immersed boundary lattice Boltzmann method (IB-LBM) considering the ultrasonic effect to simulate red blood cell (RBC) aggregation and deformation in ultrasonic field. Numerical examples involving the typical streamline, normalized out-of-plane vorticity contours and vector fields in pure plasma under three different ultrasound intensities are presented. Meanwhile, the corresponding transient aggregation behavior of RBCs, with special emphasis on the detailed process of RBC deformation, is shown. The numerical results reveal that the ultrasound wave acted on the pure plasma can lead to recirculation flow, which contributes to the RBCs aggregation and deformation in microvessel. Furthermore, increasing the intensity of the ultrasound wave can significantly enhance the aggregation and deformation of the RBCs. And the formation of the RBCs aggregation leads to the fluctuated and dropped vorticity value of plasma in return.     

Wigner distribution of a transducer beam pattern within a multiple scattering formalism for heterogeneous solids

Diffuse ultrasonic backscatter measurements have been especially useful for extracting microstructural information and for detecting flaws in materials. Accurate interpretation of experimental data requires robust scattering models. Quantitative ultrasonic scattering models include components of transducer beam patterns as well as microstructural scattering information. Here, the Wigner distribution is used in conjunction with the stochastic wave equation to model this scattering problem. The Wigner distribution represents a distribution in space and time of spectral energy density as a function of wave vector and frequency. The scattered response is derived within the context of the Wigner distribution of the beam pattern of a Gaussian transducer. The source and receiver distributions are included in the analysis in a rigorous fashion. The resulting scattered response is then simplified in the single-scattering limit typical of many diffuse backscatter experiments. Such experiments, usually done using a modified pulse-echo technique, utilize the variance of the signals in space as the primary measure of microstructure. The derivation presented forms a rigorous foundation for the multiple scattering process associated with ultrasonic experiments in heterogeneous media. These results are anticipated to be relevant to ultrasonic nondestructive evaluation of polycrystalline and other heterogeneous solids.     

Microwave backscatter from mesoscale breaking waves on the sea surface

Radar backscatter from mesoscale breaking waves on the sea surface is considered. Breaking waves are shown to be responsible for sea spikes and high Doppler shift with horizontal polarization observed at low grazing angles. The backscatter cross sections for scattering from a single breaking wave are computed for both orthogonal polarizations. An estimate is obtained of the backscatter cross section averaged over the sea surface. It is shown that the main scattering mechanisms are specular backscatter from the steep front of the breaking wave, and backscatter enhancement due to double-bounce scattering from the wave itself and from the foot of the breaking wave. Horizontally polarized backscatter is shown to be considerably higher than vertically polarized backscatter when the angle of incidence is close to the Brewster angle.     

Fibrinogen-β-Estradiol Binding Studied by Fluorescence Spectroscopy: Denaturation and pH Effects

Fibrinogen is a blood plasma protein that plays a crucial role in hemostasis. It is known that erythrocyte aggregation increases in the presence of fibrinogen, and that β-estradiol decreases erythrocyte aggregation with a constant fibrinogen concentration. In this work, we have used intrinsic tryptophan fluorescence to obtain information on the conformational changes of fibrinogen upon the recently proposed interaction with β-estradiol. To evaluate the effect on the conformational changes during fibrinogen-β-estradiol binding, fluorescence experiments were performed using guanidine hydrochloride (0–6 M) as denaturant, at different pH values. The results obtained for pH 6.5 and 8.0 showed no effect during the binding. The main differences were observed between pH 4.2 and 7.4, in the absence and in the presence of two different denaturant concentrations (1 and 5 M). A red shift of the fluorescence emission from 344 to 354 nm is observed when denaturant concentration is above 3 M for all studied pH values. This phenomenon may be explained by the loss of compact structure of the protein in the presence of denaturant, with tryptophan residues exposure to the aqueous environment and alteration of fibrinogen-β-estradiol binding. These results demonstrate that the binding sites of fibrinogen are strongly dependent on the conformational state of the protein.     

Acoustic characterization in whole blood and plasma of site-targeted nanoparticle ultrasound contrast agent for molecular imaging

The ability to enhance specific molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent [Lanza et al., Invest. Radiol. 35, 227-234 (2000); Ultrasound Med. Biol. 23, 863-870 (1997)]. One of the advantages of this agent is very low echogenicity in the blood pool that allows increased contrast between the blood pool and the bound, site-targeted agent. We measured acoustic backscatter and attenuation coefficient as a function of the contrast agent concentration, ambient pressure, peak acoustic pressure, and as an effect of duty cycle and wave form shape. Measurements were performed while the nanoparticles were suspended in either whole porcine blood or plasma. The nanoparticles were only detectable when insonified within plasma devoid of red blood cells and were shown to exhibit backscatter levels more than 30 dB below the backscatter from whole blood. Attenuation of nanoparticles in whole porcine blood was not measurably different from that of whole blood alone over a range of concentrations up to eight times the maximum in vivo dose. The resulting data provide upper bounds on blood pool attenuation coefficient and backscatter and will be needed to more precisely define levels of molecular contrast enhancement that may be obtained in vivo.     

表面粗糙度对固体内部超声背散射的影响

超声背散射法可通过多晶体金属内部的空间方差信号,实现微观结构参数的无损评价,但表面粗糙度对评价模型的精度及实用性存在显著影响.基于高斯声束理论推导垂直入射粗糙界面的纵波声场,以此研究声能的Wigner分布规律;在超声的波长远大于粗糙度的前提下,构造表面粗糙度修正系数,并建立粗糙界面的单次散射响应模型,揭示粗糙度对超声波背向散射的影响规律.用304不锈钢制备轮廓均方根值为0.159μm的光滑试块和25.722μm的粗糙试块开展超声背散射实验,结果表明模型在粗糙度修正前后均可实现光滑试块的晶粒尺寸有效评价,但未经修正的传统模型对粗糙试块的晶粒尺寸评价结果与金相法结果的相对误差高达-21.35%,而本模型的评价结果与金相法结果符合得很好,相对误差仅为1.35%.可见,本模型能有效补偿粗糙度引起的超声背散射信号衰减,从而提高晶粒尺寸无损评价的精度.     

光谱法研究聚赖氨酸与纤维蛋白原的相互作用

聚赖氨酸是一种重要的聚阳离子,在生物医药领域具有广泛的应用前景。但是,目前其血液相容性的相关报道较少,特别是通过光谱法研究其与血液中重要蛋白的相互作用。因此,通过多种光谱法研究聚赖氨酸与纤维蛋白原的相互作用,进一步评价其血液相容性具有一定的创新性。本实验通过荧光、紫外和圆二色谱研究聚赖氨酸对纤维蛋白原结构的影响。其中,聚赖氨酸的正电性随着浓度增大而增大;复合实验显示,0.01 mg·mL-1的聚赖氨酸对纤维蛋白原的功能影响较小,随着浓度增大,相互作用增强;荧光光谱显示,纤维蛋白原在λ_em=341 nm处出现浓度依赖性的荧光猝灭;紫外光谱显示,聚赖氨酸对纤维蛋白原吸收强度(200～240和278 nm处)的影响在0.025 mg·mL-1时较小,并出现浓度依赖性的减少;圆二色光谱显示,随着聚赖氨酸浓度增大,纤维蛋白原的α-螺旋含量减少,β-折叠、β-转角和无规卷曲含量增加。结果表明,聚赖氨酸会与纤维蛋白原发生静电相互作用,对其结构造成浓度依赖性的影响。当浓度为0.01和0.025 mg·mL-1时,聚赖氨酸对纤维蛋白原结构的影响较小;而浓度过大时,影响较大,势必破坏纤维蛋白原的生理功能。因此,研发和应用聚赖氨酸时必须充分考虑浓度因素。本实验提供了一种简便而系统的方法来研究材料与蛋白的作用,有利于充分评价材料的血液相容性。此外,上述研究结果对聚赖氨酸的生物医学应用具有重要的指导意义。