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.
To cite this article: Yu FTH, Armstrong JK, Tripette J, Meiselman HJ, Cloutier G. A local increase in red blood cell aggregation can trigger deep vein thrombosis: evidence based on quantitative cellular ultrasound imaging. J Thromb Haemost 2011; 9: 481-8.Summary. Background: Recurrent deep vein thrombosis (DVT) risk factors include a first idiopathic DVT, strongly suggesting the existence of undiagnosed and/or unidentified prothrombotic abnormalities. Objectives: To evaluate the impact of locally increased red blood cell (RBC) aggregation on DVT pathogenesis in a rabbit model. Methods: DVT presence, flow and aggregation were measured in situ with ultrasound. Greatly enhanced aggregation was achieved by covalent linkage of Pluronic F98 to the RBC surface; coating with Pluronic F68, which very mildly enhances aggregation, was used as a coating control. On day 1, endothelial damage and a partial stenosis were surgically created on the left femoral vein whereas the right femoral vein was not manipulated. Results: A thrombus was formed within 30 min in six out of seven left femoral veins of animals receiving a 30% volume blood exchange with F98-coated RBC, whereas a thrombus occurred in only one out of seven veins in F68-transfused controls. In vivo imaging using quantitative ultrasound confirmed increased aggregation in the thrombosed veins of the F98 group compared with the F68 group and the contralateral vessel. For each group, five animals were followed for 2 weeks before being killed. In F98-transfused animals, lysis of clots occurred and the presence of chronic thrombi totally occluding the vein in three out of five animals was confirmed by histology. Conversely, in the F68 group, a single disorganized blood clot was observed in one out of five animals. Conclusions: A marked increase in RBC aggregation promotes thrombosis in rabbit femoral veins, confirming a pathophysiological role of locally altered hemorheology in the onset of DVT.
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 ͑SFSAE͒. 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 dB/ MHz and kR Ͻ 2.08 ͑k being the wave number and R the aggregate radius͒. The SFSAE can be useful to examine in vivo and in situ abnormal blood conditions suspected to promote pathophysiological cardiovascular consequences.
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 (MB) 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 co-injected with polymer MB. 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; (3) to determine which US parameters most inhibits 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 MB can be triggered to release ~70% of their payload using non-inertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to non-released encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.
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