Dynamic Behavior of Polymer Microbubbles During Long Ultrasound Tone-Burst Excitation and Its Application for Sonoreperfusion Therapy.

OBJECTIVE: Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC)-mediated therapies have been found to restore perfusion and enhance drug/gene delivery. Because of the potentially longer circulation time and relative ease of storage and reconstitution of polymer-shelled MBs compared with lipid MBs, we investigated the dynamic behavior of polymer microbubbles and their therapeutic potential for sonoreperfusion (SRP) therapy. METHODS: The fate of polymer MBs during a single long tone-burst exposure (1 MHz, 5 ms) at various acoustic pressures and MB concentrations was recorded via high-speed microscopy and passive cavitation detection (PCD). SRP efficacy of the polymer MBs was investigated in an in vitro flow system and compared with that of lipid MBs. DISCUSSION: Microscopy videos indicated that polymer MBs formed gas-filled clusters that continued to oscillate, fragment and form new gas-filled clusters during the single US burst. PCD confirmed continued acoustic activity throughout the 5-ms US excitation. SRP efficacy with polymer MBs increased with pulse duration and acoustic pressure similarly to that with lipid MBs but no significant differences were found between polymer and lipid MBs. CONCLUSION: These data suggest that persistent cavitation activity from polymer MBs during long tone-burst US excitation confers excellent reperfusion efficacy.

Effect of Ultrasound Pulse Length on Sonoreperfusion Therapy.

In recent years, long- and short-pulse ultrasound (US)-targeted microbubble cavitation (UTMC) has been found to increase perfusion in healthy and ischemic skeletal muscle, in pre-clinical animal models of microvascular obstruction and in the myocardium of patients presenting with acute myocardial infarction. There is evidence that the observed microvascular vasodilation is driven by the nitric oxide pathway and purinergic signaling, but the time course of the response and the dependency on US pulse length are not well elucidated. Because our prior data supported that sonoreperfusion efficacy is enhanced by long-pulse US versus short-pulse US, in this study, we sought to compare long-pulse (5000 cycles) and short-pulse (500 × 10 cycles) US at a pressure of 1.5 MPa with an equivalent total number of acoustical cycles, hence constant acoustic energy, and at the same frequency (1 MHz), in a rodent hind limb model with and without microvascular obstruction (MVO). In quantifying perfusion using burst replenishment contrast-enhanced US imaging, we made three findings: (i) Long and short pulses result in different vasodilation kinetics in an intact hind limb model. The long pulse causes an initial spasmic reduction in flow that spontaneously resolved at 4 min, followed by sustained higher flow rates (approximately twofold) compared with baseline, starting 10 min after therapy (p < 0.05). The short pulse caused a short-lived approximately twofold increase in flow rate that peaked at 4 min (p < 0.05), but without the initial spasm. (ii) The sustained increased response with the long pulse is not simply reactive hyperemia. (iii) Both pulses are effective in reperfusion of MVO in our hindlimb model by restoring blood volume, but only the long pulse caused an increase in flow rate after treatment ii, compared with MVO (p < 0.05). Histological analysis of hind limb muscle post-UTMC with either pulse configuration indicates no evidence of tissue damage or hemorrhage. Our findings indicate that the microbubble oscillation induces vasodilation, and therapeutic efficacy for the treatment of MVO can be tuned by varying pulse length; relative to short-pulse US, longer pulses drive greater microbubble cavitation and more rapid microvascular flow rate restoration after MVO, warranting further optimization of the pulse length for sonoreperfusion therapy.

Targeted Anti-Cancer Provascular Therapy Using Ultrasound, Microbubbles, and Nitrite to Increase Radiotherapy Efficacy.

Hypoxia is an important mechanism of resistance to radiation therapy in many human malignancies including prostate cancer. It has been recently shown that ultrasound targeted microbubble cavitation (UTMC) can increase blood perfusion in skeletal muscle by triggering nitric oxide signaling. Interestingly, this effect was amplified with a sodium nitrite coinjection. Since sodium nitrite has been shown to synergize with radiotherapy (RT), we hypothesized that UTMC with a sodium nitrite coinjection could further radiosensitize solid tumors by increasing blood perfusion and thus reduce tumor hypoxia. We evaluated (1) the ability of UTMC with and without nitrite to increase perfusion in muscle (mouse hindlimbs) and human prostate tumors using different pulse lengths and pressure; (2) the efficacy of this approach as a provascular therapy given directly before RT in the human prostate subcutaneous xenografts PC3 tumor model. Using long pulses with various pressures, in muscle, the provascular response following UTMC was strong (6.61 ± 4.41-fold increase in perfusion post-treatment). In tumors, long pulses caused an increase in perfusion (2.42 ± 1.38-fold) at lower mechanical index (MI = 0.25) but not at higher MI (0.375, 0.5, and 0.750) when compared to control (no UTMC). However, when combined with RT, UTMC with long pulses (MI = 0.25) did not improve tumor growth inhibition. With short pulses, in muscle, the provascular response following UTMC (SONOS) + nitrite was strong (13.74 ± 8.60-fold increase in perfusion post-treatment). In tumors, UTMC (SONOS) + nitrite also caused a provascular response (1.94 ± 1.20-fold increase in perfusion post-treatment) that lasted for at least 10 min, but not with nitrite alone. Interestingly, the blunted provascular response observed for long pulses at higher MI without nitrite was reversed with the addition of nitrite. UTMC (SONOS) with and without nitrite caused an increase in perfusion in tumors. The provascular response observed for UTMC (SONOS) + nitrite was confirmed by histology. Finally, there was an improved growth inhibition for the 8 Gy RT dose + nitrite + UTMC group vs 8 Gy RT + nitrite alone. This effect was not significant with mice treated by UTMC + nitrite and receiving doses of 0 or 2 Gy RT. In conclusion, UTMC + nitrite increased blood flow leading to an increased efficacy of higher doses of RT in our tumor model, warranting further study of this strategy.

The effect of ultrasound pulse length on microbubble cavitation induced antibody accumulation and distribution in a mouse model of breast cancer.

In solid tumors, the limited diffusion of therapeutic molecules in the perivascular space is a known limitation impacting treatment efficacy. Ultrasound Targeted Microbubble Cavitation (UTMC) has been shown to increase vascular permeability and improve the delivery of therapeutic compounds including small molecules, antibodies (mAb), nanoparticles and even cells, notably across the blood-brain-barrier (BBB). In this study, we hypothesized that UTMC could improve the accumulation and biodistribution of mAb targeting the adenosinergic pathway (i.e. CD73) in mice bearing bilateral subcutaneous 4T1 mammary carcinoma. METHODS: A bolus of fluorescently labeled mAb was given intravenously, followed by a slow infusion of microbubbles. UTMC therapy (1 MHz, 850 kPa) was given under ultrasound image guidance for 5 minutes to the right side tumor only, using three different pulse lengths with identical ultrasound energy (5000cyc "long", 125x40cyc "mid" and 500x10cyc "short"), and leaving the left tumor as a paired control. Longitudinal accumulation at 0 h, 4 h and 24 h was measured using whole-body biofluorescence and confocal microscopy. RESULTS: Our data support an increase in antibody accumulation and extravasation (# extravasated vessels and extravasated signal intensity) at 0 h for all pulses and at 4 h for the mid and short pulses when compared to the control non treated side. However, this difference was not found at 24 h post UTMC, indicative of the transient nature of UTMC. Interestingly, confocal data supported that the highest extravasation range was obtained at 0 h with the long pulse and that the short pulse caused no increase in the extravasation range. Overall, the mid pulse was the only pulse to increase all our metrics (biofluorescence, fraction of extravasated vessels, amount of extravasated Ab, and extravasation range) at 0 h and 4 h time points. CONCLUSIONS: Our results support that UTMC can enhance antibody accumulation in solid tumors at the macroscopic and microscopic levels. This preferential accumulation was evident at early time points (0 h and 4 h) but had started to fade by 24 h, a time dependence that is consistent with the ultrasound blood brain barrier opening literature. Further development and optimization of this theranostic platform, such as repeated UTMC, could help improve antibody based therapies against solid cancer.

Sonoreperfusion therapy for microvascular obstruction: A step toward clinical translation.

Sonoreperfusion therapy is being developed as an intervention for the treatment of microvascular obstruction. We investigated the reperfusion efficacy of two clinical ultrasound systems (a modified Philips EPIQ and a Philips Sonos 7500) in a rat hindlimb microvascular obstruction model. Four ultrasound conditions were tested using 20 min treatments: Sonos single frame, Sonos multi-frame, EPIQ low pressure and EPIQ high pressure. Contrast-enhanced perfusion imaging of the microvasculature was conducted at baseline and after treatment to calculate microvascular blood volume (MBV). EPIQ high pressure treatment resulted in significant recovery of MBV from microvascular obstruction, returning to baseline levels after treatment. EPIQ low pressure and Sonos multi-frame treatment resulted in significantly improved MBV after treatment but below baseline levels. Sonos single-frame and control groups showed no improvement post-treatment. This study demonstrates that the most effective sonoreperfusion therapy occurs at high acoustic pressure coupled with high acoustic intensity. Moreover, a clinically available ultrasound system is readily capable of delivering these effective therapeutic pulses.

The Role of Nitric Oxide during Sonoreperfusion of Microvascular Obstruction.

Rationale: Microembolization during PCI for acute myocardial infarction can cause microvascular obstruction (MVO). MVO severely limits the success of reperfusion therapies, is associated with additional myonecrosis, and is linked to worse prognosis, including death. We have shown, both in in vitro and in vivo models, that ultrasound (US) and microbubble (MB) therapy (termed "sonoreperfusion" or "SRP") is a theranostic approach that relieves MVO and restores perfusion, but the underlying mechanisms remain to be established. Objective: In this study, we investigated the role of nitric oxide (NO) during SRP. Methods and results: We first demonstrated in plated cells that US-stimulated MB oscillations induced a 6-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation in vitro. We then monitored the kinetics of intramuscular NO and perfusion flow rate responses following 2-min of SRP therapy in the rat hindlimb muscle, with and without blockade of eNOS with LNAME. Following SRP, we found that starting at 6 minutes, intramuscular NO increased significantly over 30 min and was higher than baseline after 13 min. Concomitant contrast enhanced burst reperfusion imaging confirmed that there was a marked increase in perfusion flow rate at 6 and 10 min post SRP compared to baseline (>2.5 fold). The increases in intramuscular NO and perfusion rate were blunted with LNAME. Finally, we tested the hypothesis that NO plays a role in SRP by assessing reperfusion efficacy in a previously described rat hindlimb model of MVO during blockade of eNOS. After US treatment 1, microvascular blood volume was restored to baseline in the MB+US group, but remained low in the LNAME group. Perfusion rates increased in the MB+US group after US treatment 2 but not in the MB+US+LNAME group. Conclusions: These data strongly support that MB oscillations can activate the eNOS pathway leading to increased blood perfusion and that NO plays a significant role in SRP efficacy.

Influence of erythrocyte aggregation on radial migration of platelet-sized spherical particles in shear flow.

Blood platelets when activated are involved in the mechanisms of hemostasis and thrombosis, and their migration toward injured vascular endothelium necessitates interaction with red blood cells (RBCs). Rheology co-factors such as a high hematocrit and a high shear rate are known to promote platelet mass transport toward the vessel wall. Hemodynamic conditions promoting RBC aggregation may also favor platelet migration, particularly in the venous system at low shear rates. The aim of this study was to confirm experimentally the impact of RBC aggregation on platelet-sized micro particle migration in a Couette flow apparatus. Biotin coated micro particles were mixed with saline or blood with different aggregation tendencies, at two shear rates of 2 and 10s-1 and three hematocrits ranging from 20 to 60%. Streptavidin membranes were respectively positioned on the Couette static and rotating cylinders upon which the number of adhered fluorescent particles was quantified. The platelet-sized particle adhesion on both walls was progressively enhanced by increasing the hematocrit (p<0.001), reducing the shear rate (p<0.001), and rising the aggregation of RBCs (p<0.001). Particle count was minimum on the stationary cylinder when suspended in saline at 2s-1 (57±33), and maximum on the rotating cylinder at 60% hematocrit, 2s-1 and the maximum dextran-induced RBC aggregation (2840±152). This fundamental study is confirming recent hypotheses on the role of RBC aggregation on venous thrombosis, and may guide molecular imaging protocols requiring injecting active labeled micro particles in the venous flow system to probe human diseases.

Inertial Cavitation Ultrasound with Microbubbles Improves Reperfusion Efficacy When Combined with Tissue Plasminogen Activator in an In Vitro Model of Microvascular Obstruction.

We have previously reported that long-tone-burst, high-mechanical-index ultrasound (US) and microbubble (MB) therapy can restore perfusion in both in vitro and in vivo models of microvascular obstruction (MVO). Addition of MBs to US has been found to potentiate the efficacy of thrombolytics on large venous thrombi; however, the optimal US parameters for achieving microvascular reperfusion of MVO caused by microthrombi, when combined with tissue plasminogen activator (tPA), are unknown. We sought to elucidate the specific effects of US, with and without tPA, for effective reperfusion of MVO in an in vitro model using both venous and arterial microthrombi. Venous- and arterial-type microthrombi were infused onto a mesh with 40-µm pores to simulate MVO. Pulsed US (1 MHz) was delivered with inertial cavitation (IC) (1.0 MPa, 1000 cycles, 0.33 Hz) and stable cavitation (SC) US (0.23 MPa, 20% duty cycle, 0.33 Hz) regimes while MB suspension (2 × 106 MBs/mL) was infused. The efficacy of sonoreperfusion with these parameters was tested with and without tPA. Sonoreperfusion efficacy was significantly greater for IC + tPA compared with tPA alone, IC, SC and SC + tPA, suggesting lytic synergism between tPA and US for both venous- and arterial-type microthrombi. In contrast to our previous in vitro studies using 1.5 MPa at 5000 US cycles without tPA, the IC regime employed herein used 90% less US energy. These findings suggest an IC regime can be used with tPA synergistically to achieve a high degree of fibrinolysis for both thrombus types.

Effect of Thrombus Composition and Viscosity on Sonoreperfusion Efficacy in a Model of Micro-Vascular Obstruction.

Distal embolization of micro-thrombi during stenting for myocardial infarction causes micro-vascular obstruction (MVO). We have previously shown that sonoreperfusion (SRP), a microbubble (MB)-mediated ultrasound (US) therapy, resolves MVO from venous micro-thrombi in vitro in saline. However, blood is more viscous than saline, and arterial thrombi that embolize during stenting are mechanically distinct from venous clot. Therefore, we tested the hypothesis that MVO created with arterial micro-thrombi are more resistant to SRP therapy compared with venous micro-thrombi, and higher viscosity further increases the US requirement for effective SRP in an in vitro model of MVO. Lipid MBs suspended in plasma with adjusted viscosity (1.1 cP or 4.0 cP) were passed through tubing bearing a mesh with 40-µm pores to simulate a micro-vascular cross-section; upstream pressure reflected thrombus burden. To simulate MVO, the mesh was occluded with either arterial or venous micro-thrombi to increase upstream pressure to 40 mmHg ± 5 mmHg. Therapeutic long-tone-burst US was delivered to the occluded area for 20 min. MB activity was recorded with a passive cavitation detector. MVO caused by arterial micro-thrombi at either blood or plasma viscosity resulted in less effective SRP therapy compared to venous thrombi. Higher viscosity further reduced the effectiveness of SRP therapy. The passive cavitation detector showed a decrease in inertial cavitation when viscosity was increased, while stable cavitation was affected in a more complex manner. Overall, these data suggest that arterial thrombi may require higher acoustic pressure US than venous thrombi to achieve similar SRP efficacy; increased viscosity decreases SRP efficacy; and both inertial and stable cavitation are implicated in observed SRP efficacy.

Low Intensity Ultrasound Mediated Liposomal Doxorubicin Delivery Using Polymer Microbubbles.

Cardiotoxicity is the major dose-limiting factor in the chemotherapeutic use of doxorubicin (Dox). A delivery vehicle that can be triggered to release its payload in the tumoral microvasculature but not in healthy tissue would help improve the therapeutic window of the drug. Delivery strategies combining liposomal encapsulated Dox (LDox), microbubbles (MBs), and ultrasound (US) have been shown to improve therapeutic efficacy of LDox, but much remains to be known about the mechanisms and the US conditions that maximize cytotoxicity using this approach. In this study, we compared different US pulses in terms of drug release and acute toxicity. Drug uptake and proliferation rates using low-intensity US were measured in squamous cell carcinoma cells exposed to LDox conjugated to or coinjected with polymer MBs. The aims of this study were: (1) to compare the effects of low- and high-pressure US on Dox release kinetics; (2) to evaluate whether conjugating the liposome to the MB surface (DoxLPX) is an important factor for drug release and cytotoxicity; and (3) to determine which US parameters most inhibit cell proliferation and whether this inhibition is mediated by drug release or the MB/US interaction with cells. Low-pressure US (170 kPa) at high duty cycle (stable cavitation) released up to ∼ 70% of the encapsulated Dox from the DoxLPX, thus improving Dox bioavailability and cellular uptake and leading to a significant reduction in cell proliferation at 48 h. Flow cytometry showed that US generating stable oscillations of DoxLPX significantly increased cellular Dox uptake at 4 h after US exposure compared to LDox. Drug uptake was correlated with cytotoxicity at 48 h. Our results demonstrate that Dox-containing liposomes conjugated to polymer MBs can be triggered to release ∼ 70% of their payload using noninertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to nonreleased encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.

Unifying Concepts of Statistical and Spectral Quantitative Ultrasound Techniques.

Quantitative ultrasound (QUS) techniques using radiofrequency (RF) backscattered signals have been used for tissue characterization of numerous organ systems. One approach is to use the magnitude and frequency dependence of backscatter echoes to quantify tissue structures. Another approach is to use first-order statistical properties of the echo envelope as a signature of the tissue microstructure. We propose a unification of these QUS concepts. For this purpose, a mixture of homodyned K-distributions is introduced to model the echo envelope, together with an estimation method and a physical interpretation of its parameters based on the echo signal spectrum. In particular, the total, coherent and diffuse signal powers related to the proposed mixture model are expressed explicitly in terms of the structure factor previously studied to describe the backscatter coefficient (BSC). Then, this approach is illustrated in the context of red blood cell (RBC) aggregation. It is experimentally shown that the total, coherent and diffuse signal powers are determined by a structural parameter of the spectral Structure Factor Size and Attenuation Estimator. A two-way repeated measures ANOVA test showed that attenuation (p-value of 0.077) and attenuation compensation (p-value of 0.527) had no significant effect on the diffuse to total power ratio. These results constitute a further step in understanding the physical meaning of first-order statistics of ultrasound images and their relations to QUS techniques. The proposed unifying concepts should be applicable to other biological tissues than blood considering that the structure factor can theoretically model any spatial distribution of scatterers.

Radial modulation contrast imaging using a 20-MHz single-element intravascular ultrasound catheter.

Contrast-enhanced intravascular ultrasound imaging is a promising tool for the characterization of coronary vasa vasorum proliferation, which has been identified as a marker of, and possible etiologic factor in, the development of high-risk atherosclerotic plaques. Resonance-based nonlinear detection methods have required the development of prototype catheters which are not commercially available, thus limiting clinical translation. In this study, we investigated the performances of a radial modulation imaging approach (25/3 MHz combination) using simulations, implemented it on a clinical 20-MHz rotating catheter, and tested it in a wall-less tissue-mimicking flow phantom perfused with lipid-encapsulated microbubbles (MBs). The effects of the phase lag, low-frequency pressure, and MB concentration on the envelope subtracted radial modulation signals were investigated as a function of depth. Our dual-pulse dual-frequency approach produced contrast- specific images with contrast-to-tissue improvements over B-mode of 15.1 ± 2.1 dB at 2 mm and 6.8 ± 0.1 dB at 4 mm depths. Using this imaging strategy, 200-µm-diameter cellulose tubing perfused with MBs could be resolved while surrounding tissue scattering was suppressed. These results raise promise for the detection of coronary vasa vasorum and may ultimately facilitate the detection of plaque at risk for rupture.

Flexible integration of high-imaging-resolution and high-power arrays for ultrasound-induced thermal strain imaging (US-TSI).

Ultrasound-induced thermal strain imaging (USTSI) for carotid artery plaque detection requires both high imaging resolution (<100 µm) and sufficient US-induced heating to elevate the tissue temperature (~1°C to 3°C within 1 to 3 cardiac cycles) to produce a noticeable change in sound speed in the targeted tissues. Because the optimization of both imaging and heating in a monolithic array design is particularly expensive and inflexible, a new integrated approach is presented which utilizes independent ultrasound arrays to meet the requirements for this particular application. This work demonstrates a new approach in dual-array construction. A 3-D printed manifold was built to support both a high-resolution 20 MHz commercial imaging array and 6 custom heating elements operating in the 3.5 to 4 MHz range. For the application of US-TSI in carotid plaque characterization, the tissue target site is 20 to 30 mm deep, with a typical target volume of 2 mm (elevation) × 8 mm (azimuthal) × 5 mm (depth). The custom heating array performance was fully characterized for two design variants (flat and spherical apertures), and can easily deliver 30 W of total acoustic power to produce intensities greater than 15 W/cm(2) in the tissue target region.

Volumetric quantification of in vitro sonothrombolysis with microbubbles using high-resolution optical coherence tomography.

Several in vitro and in vivo studies have established accelerated thrombolysis using ultrasound (US) induced microbubble (MB) cavitation. However, the mechanisms underlying MB mediated sonothrombolysis are still not completely elucidated. We performed three-dimensional (3-D) volumetric optical coherence tomography (OCT) imaging before and after the application of contrast US to thrombus. The most dramatic reduction in clot volume was observed with US + MB + recombinant tissue plasminogen activator (rt-PA). Thrombus surface erosion in this group on the side of the thrombus exposed to MB and ultrasound was evident on the OCT images. This technique may assist in clarifying the mechanisms underlying sonothrombolysis, especially regarding the importance of US transducer orientation on lytic efficacy and the effects of MB cavitation on thrombus structure.

Effect of acoustic conditions on microbubble-mediated microvascular sonothrombolysis.

Ultrasound (US) mediated microbubble (MB) destruction facilitates thrombolysis of the epicardial coronary artery in acute myocardial infarction (AMI) but its effect on microvascular thromboemboli remains largely unexplored. We sought to define the acoustic requirements for effective microvascular sonothrombolysis. To model microembolization, microthrombi were injected and entrapped in a 40 µm pore mesh, increasing upstream pressure, which was measured as an index of thrombus burden. MBs (2.0 × 10(6) MBs/mL) were then infused while pulsed US (1 MHz) was delivered to induce MB destruction immediately adjacent to the thrombus. Upstream pressure decreased progressively during US delivery, indicating a reduction in thrombus burden. More rapid and complete lysis occurred with increasing peak negative acoustic pressure (1.5 MPa > 0.6 MPa) and increasing pulse length (5000 cycles > 100 cycles). Additionally, similar lytic efficacy was achieved at 1.5 MPa without tPA as was at 1.0 MPa with tPA. This model uniquely provides a means to systematically evaluate multiple acoustic and microbubble parameters for the optimization of microvascular sonothrombolysis. This treatment approach for thrombotic microvascular obstruction may obviate the need for adjunctive rt-PA and could have important clinical cost and safety benefits.

Ultrasound characterization of red blood cell aggregation with intervening attenuating tissue-mimicking phantoms.

The analysis of the ultrasonic frequency-dependent backscatter coefficient of aggregating red blood cells reveals information about blood structural properties. The difficulty in applying this technique in vivo is due to the frequency-dependent attenuation caused by intervening tissue layers that distorts the spectral content of signals backscattered by blood. An optimization method is proposed to simultaneously estimate tissue attenuation and blood structure properties, and was termed the structure factor size and attenuation estimator (SFSAE). An ultrasound scanner equipped with a wide-band 25 MHz probe was used to insonify porcine blood sheared in both Couette and tubular flow devices. Since skin is one of the most attenuating tissue layers during in vivo scanning, four skin-mimicking phantoms with different attenuation coefficients were introduced between the transducer and the blood flow. The SFSAE gave estimates with relative errors below 25% for attenuations between 0.115 and 0.411 dBMHz and kR<2.08 (k being the wave number and R the aggregate radius). The SFSAE can be useful to examine in vivo and in situ abnormal blood conditions suspected to promote pathophysiological cardiovascular consequences.

Ultrasonic parametric imaging of erythrocyte aggregation using the structure factor size estimator.

Ultrasound characterization of erythrocyte aggregation (EA) is attractive because it is a non-invasive imaging modality that can be applied in vivo and in situ. An experimental validation of the Structure Factor Size Estimator (SFSE), a non-Rayleigh scattering model adapted for dense suspensions, was performed on 4 erythrocyte preparations with different aggregation tendencies. Erythrocyte preparations were circulated in Couette and tube flows while acoustically imaged over a bandwidth of 9-28 MHz. Two acoustically derived parameters, the packing factor (W) and ensemble averaged aggregate size (D), predictably increased with increasing EA, a finding corroborated by bulk viscosity measurements. In tube flow, a "black hole" reflecting the absence of aggregates was observed in the center stream of some parametric images. The SFSE clearly allowed quantifying the EA spatial distribution with larger aggregates closer to the tube walls as the aggregation tendency was increased. In Couette flow, W and D were uniformly distributed across the shear field. Assuming that the viscosity increase at low shear is mainly determined by EA, viscosity maps were computed in tube flow. Interestingly, erythrocyte suspensions with high aggregabilities resulted in homogeneous viscosity distributions, whereas a "normal" aggregability promoted the formation of concentric rings with varying viscosities.

Increased shear rate resistance and fastest kinetics of erythrocyte aggregation in diabetes measured with ultrasound.

OBJECTIVE: To measure with ultrasound the increased erythrocyte aggregation (EA) kinetics and adhesion energy between erythrocytes in patients with type 2 diabetes and poor metabolic control. RESEARCH DESIGN AND METHODS: Blood samples were analyzed in a Couette rheometer at 32 MHz following shear rate reductions from 500 s(-1) to residual shears of 0 (stasis), 1, 2, 10, 50, 100, and 200 s(-1). The increase in EA was determined with the integrated backscatter coefficient as a function of time and shear rate. RESULTS: The time required to form aggregates was shorter in diabetic patients at shear rates below 200 s(-1) (P < 0.01). Erythrocytes formed larger aggregates in diabetic patients than in control subjects (P < 0.05 at 2 to 100 s(-1)). CONCLUSIONS: Ultrasound can potentially noninvasively demonstrate, in vivo and in situ, the impact of local abnormal EA on arteriovenous flow disorders in diabetes.

Simultaneous estimation of attenuation and structure parameters of aggregated red blood cells from backscatter measurements.

The analysis of the ultrasonic frequency-dependent backscatter coefficient of aggregating red blood cells reveals information about blood structural properties. The difficulty in applying this technique in vivo is due to the frequency-dependent attenuation caused by intervening tissue layers that distorts the spectral content of backscattering properties from blood microstructures. An optimization method is proposed to simultaneously estimate tissue attenuation and blood structure factor. With in vitro experiments, the method gave satisfactory estimates with relative errors below 22% for attenuations between 0.101 and 0.317 dBcmMHz, signal-to-noise ratios>28 dB and kR<2.7 (k being the wave number and R the aggregate radius).

Cyclic changes in blood echogenicity under pulsatile flow are frequency dependent.

Previous in vivo and in vitro studies have demonstrated that blood echogenicity varies under pulsatile flow, but such changes could not always be measured at physiological stroke rates. The apparent contradiction between these studies could be a result of the use of different ultrasound frequencies. Backscattered signals from porcine blood were measured in a pulsatile Couette flow apparatus. Cyclic changes in shear rate for stroke rates of 20 to 70 beats per minute (BPM) were applied to the Couette system, and different blood samples were analyzed (normal blood and blood with hyperaggregating erythrocytes promoted with dextran). To confirm that cyclic echogenicity variations were observable, spectral analysis was performed to verify if changes in echo-amplitude corresponded to the stroke rate applied to the flow. Echogenicity was measured with two single-element transducers at 10 and 35 MHz. At 35 MHz, cyclic variations in backscatter were observed from 20 to 70 BPM. However at 10 MHz, they were detected only at 20 BPM. For all cases except for hyperaggregating red blood cells (RBCs) at 20 BPM, the magnitude of the cyclic variations were higher at 35 MHz. We conclude that cyclic variations in RBC aggregation exist at physiological stroke rates, unlike what has been demonstrated in previous in-vitro studies at frequencies of 10 MHz. The increased sensitivity at 35 MHz to small changes in aggregate size might be the explanation for the better characterization of RBC aggregation at high stroke rates. Our results corroborate in-vivo observations of cyclic blood echogenicity variations in patients using a 30-MHz intravascular ultrasound catheter.