Dynamic contrast-enhanced ultrasound parametric maps to evaluate intratumoral vascularization.

OBJECTIVES: The purposes of this study were to assess the reliability of parametric maps from dynamic contrast-enhanced ultrasound (DCE-US) to reflect the heterogeneous distribution of intratumoral vascularization and to predict the tissue features linked to vasculature. This study was designed to compare DCE-US parametric maps with histologic vascularity measurements. MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound was performed on 17 melanoma-bearing nude mice after a 0.1-mL bolus injection of SonoVue (Bracco SPA, Milan, Italy). The parametric maps were developed from raw linear data to extract pixelwise 2 semiquantitative parameters related to perfusion and blood volume, namely, area under the curve (AUC) and peak intensity (PI). The mathematical method to fit the time-intensity curve for each pixel was a polynomial model used in clinical routine and patented by the team. Regions of interest (ROIs) were drawn on DCE-US parametric maps for whole tumors and for several local areas of 15 mm within each tumor (iROI), the latter reflecting the heterogeneity of intratumoral blood volume. As the criterion standard correlation, microvessel densities (MVDs) were determined for both ROI categories. In detail, for all iROI of 15 mm, MVD and maturity were divided separately for vessels of 0 to 10 µm, 10 to 40 µm, and greater than 40 µm in diameter, and the results were correlated with the ultrasound findings. RESULTS: Among the 17 studied mice, a total of 64 iROIs were analyzed. For the whole-tumor ROI set, AUC and PI values significantly correlated with MVD (rAUC = 0.52 [P = 0.0408] and rPI = 0.70 [P = 0.0026]). In the case of multiple iROI, a strong linear correlation was observed between the DCE-US parameters and the density of vessels ranging in their diameter from 0 to 10 µm (rAUC = 0.68 [P < 0.0001]; rPI = 0.63 [P < 0.0001]), 10 to 40 µm (rAUC = 0.98 [P = 0.0003]; rPI = 0.98 [P = 0.0004]), and greater than 40 µm (rAUC = 0.86 [P = 0.0120]; rPI = 0.92 [P = 0.0034]), respectively. However, the DCE-US parameter values of perfusion and blood volume were not significantly different according to the diameters (AUC: P = 0.1731; PI: P = 0.2918) and maturity of blood vessels. CONCLUSIONS: Parametric maps of DCE-US can be reliably established from raw linear data and reflect the heterogeneous histological measures of vascularization within tumors. In contrast, the values of DCE-US parametric maps (AUC, PI) do not allow deduction of heterogeneous tissue features such as the diameters and maturity of vascular networks.

Assessment of quantitative perfusion parameters by dynamic contrast-enhanced sonography using a deconvolution method: an in vitro and in vivo study.

OBJECTIVES: The purpose of this study was to investigate the impact of the arterial input on perfusion parameters measured using dynamic contrast-enhanced sonography combined with a deconvolution method after bolus injections of a contrast agent. METHODS: The in vitro experiments were conducted using a custom-made setup consisting of pumping a fluid through a phantom made of 3 intertwined silicone pipes, mimicking a complex structure akin to that of vessels in a tumor, combined with their feeding pipe, mimicking the arterial input. In the in vivo experiments, B16F10 melanoma cells were xenografted to 5 nude mice. An ultrasound scanner combined with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. A mathematical model developed by the Gustave Roussy Institute (patent WO/2008/053268) and based on the dye dilution theory was used to evaluate 7 semiquantitative perfusion parameters directly from time-intensity curves and 3 quantitative perfusion parameters from the residue function obtained after a deconvolution process developed in our laboratory based on the Tikhonov regularization method. We evaluated and compared the intraoperator variability values of perfusion parameters determined after these two signal-processing methods. RESULTS: In vitro, semiquantitative perfusion parameters exhibited intraoperator variability values ranging from 3.39% to 13.60%. Quantitative parameters derived after the deconvolution process ranged from 4.46% to 11.82%. In vivo, tumors exhibited perfusion parameter intraoperator variability values ranging from 3.74% to 29.34%, whereas quantitative ones varied from 5.00% to 12.43%. CONCLUSIONS: Taking into account the arterial input in evaluating perfusion parameters improves the intraoperator variability and may improve the dynamic contrast-enhanced sonographic technique.

In vitro evaluation of the impact of ultrasound scanner settings and contrast bolus volume on time-intensity curves.

The objective of this study was to assess in vitro the impact of ultrasound scanner settings and contrast bolus volume on time-intensity curves formed from dynamic contrast-enhanced ultrasound image loops. An indicator-dilution experiment was developed with an in vitro flow phantom setup used with SonoVue contrast agent (Bracco SpA, Milan, Italy). Imaging was performed with a Philips iU22 scanner and two transducers (L9-3 linear and C5-1 curvilinear). The following ultrasound scanner settings were investigated, along with contrast bolus volume: contrast-specific nonlinear pulse sequence, gain, mechanical index, focal zone depth, acoustic pulse center frequency and bandwidth. Four parameters (rise time, mean transit time, peak intensity, and area under the curve) were derived from time-intensity curves which were obtained after pixel by pixel linearization of log-compressed data (also referred to as video data) included in a region of interest. Rise time was found to be the parameter least impacted by changes to ultrasound scanner settings and contrast bolus volume; the associated coefficient of variation varied between 0.7% and 6.9% while it varied between 0.8% and 19%, 12% and 71%, and 9.2% and 66%, for mean transit time, peak intensity, and area under the curve, respectively. The present study assessed the impact of ultrasound scanner settings and contrast bolus volume on time-intensity curve analysis. One should be aware of these issues to standardize their technique in each specific organ of interest and to achieve accurate, sensitive, and reproducible data using dynamic contrast-enhanced ultrasound. One way to mitigate the impact of ultrasound scanner settings in longitudinal, multi-center quantitative dynamic contrast-enhanced ultrasound studies may be to prohibit any adjustments to those settings throughout a given study. Further clinical studies are warranted to confirm the reproducibility and diagnostic or prognostic value of time-intensity curve parameters measurements in a particular clinical scenario of interest, for example that of cancer patients undergoing vascular targeting therapies.

Combining functional imaging and interstitial pressure measurements to evaluate two anti-angiogenic treatments.

BACKGROUND: Interstitial hypertension is responsible for poor capillary blood flow and hampered drug delivery. The efficacy of combined sorafenib/bevacizumab treatment given according to different administration schedules has been evaluated by measuring both interstitial pressure (IP) and quantitative dynamic contrast-enhanced ultrasonography (DCE-US) parameters in melanoma-bearing mice. MATERIAL AND METHODS: [corrected] Sixty mice were xenografted with B16F10 melanoma. Animals received a daily administration over 4 days (D0 to D3) of either sorafenib at 30 mg/kg, bevacizumab at 2.5 mg/kg alone, or different schedules of combined treatments. Perfusion parameters determined using an Aplio® sonograph (Toshiba) with SonoVue® contrast agent (Bracco) were compared to IP measurements using fiberoptic probes (Samba®) at D0, D2, D4, D8. RESULTS: The mean baseline IP values ranged between 6.55 and 31.29 mmHg in all the groups. A transient IP decrease occurred at D2 in all treated groups, and especially in the concomitant group which exhibited a significant IP reduction compared to D0. A significant decrease in both the peak intensity and the area under the curve was observed at D4 in the group with concomitant administration of both molecules which yielded maximal inhibition of the tumor volume and the number of vessels. No correlation was found between IP values and volume or perfusion parameters, indicating complex relationships between IP and vascularization. No IP gradients were found between the center and the periphery but IP values in these two regions were significantly correlated (R = 0.93). CONCLUSION: The results suggest that IP variations could be predictive of vascular changes and that one single IP measurement is sufficient to fully characterize the whole tumor.

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US.

AIM: To investigate intra-operator variability of semi-quantitative perfusion parameters using dynamic contrast-enhanced ultrasonography (DCE-US), following bolus injections of SonoVue(®). METHODS: The in vitro experiments were conducted using three in-house sets up based on pumping a fluid through a phantom placed in a water tank. In the in vivo experiments, B16F10 melanoma cells were xenografted to five nude mice. Both in vitro and in vivo, images were acquired following bolus injections of the ultrasound contrast agent SonoVue(®) (Bracco, Milan, Italy) and using a Toshiba Aplio(®) ultrasound scanner connected to a 2.9-5.8 MHz linear transducer (PZT, PLT 604AT probe) (Toshiba, Japan) allowing harmonic imaging ("Vascular Recognition Imaging") involving linear raw data. A mathematical model based on the dye-dilution theory was developed by the Gustave Roussy Institute, Villejuif, France and used to evaluate seven perfusion parameters from time-intensity curves. Intra-operator variability analyses were based on determining perfusion parameter coefficients of variation (CV). RESULTS: In vitro, different volumes of SonoVue(®) were tested with the three phantoms: intra-operator variability was found to range from 2.33% to 23.72%. In vivo, experiments were performed on tumor tissues and perfusion parameters exhibited values ranging from 1.48% to 29.97%. In addition, the area under the curve (AUC) and the area under the wash-out (AUWO) were two of the parameters of great interest since throughout in vitro and in vivo experiments their variability was lower than 15.79%. CONCLUSION: AUC and AUWO appear to be the most reliable parameters for assessing tumor perfusion using DCE-US as they exhibited the lowest CV values.

Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography.

OBJECTIVES: To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. MATERIAL AND METHODS: AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (T PI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann-Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. RESULTS: In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). T PI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). CONCLUSION: DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.

Acoustic characterization of a new trisacryl contrast agent. Part I: In vitro study.

The objective of the study was to acoustically characterize trisacryl polymeric microparticles (TMP), which are derived from biocompatible embolic agents. With significant acoustic properties, these polymeric particles could be potentially used as targeted ultrasound contrast agents, directed towards a specific site, with ligands conjugation on the polymeric network surface. In the in vitro study, a pulser/receiver (PRF of 1 kHz), associated to different transducers (5, 10 and 15 MHz), was used to measure the acoustic properties of the TMP inserted in a Couette flow device. Acoustic characterization according to TMP concentration (0.12-15.63 mg/ml), frequency (4.5-17 MHz, defined by each transducer bandwidth), ultrasound pressure (137-378 kPa) and exposure time (0-30 min) was conducted. Particle attenuation was also evaluated according to TMP concentration and emission frequency. Backscattering increased non linearly with concentration and maximum enhancement was of 16.4 dB+/-0.89 dB above 7.8 mg/ml. This parameter was found non-linear with increasing applied pressure and no harmonic oscillation could be noticed. Attenuation reached approximately 1.4 dB/cm at 15 MHz and for the 15.6 mg/ml suspension. The TMP have revealed in vitro ultrasound properties comparable to those observed with known contrast agents, studied in similar in vitro systems. However, such set-ups combined with a rather aqueous suspending medium, have some limitations and further investigations need now to be conducted to approach in vivo conditions in terms of flow and blood environment.

Acoustic characterization of a new trisacryl contrast agent. Part II: Flow phantom study and in vivo quantification.

The biocompatible trisacryl particles (TMP) are made of a cross-linked acrylic copolymer. Their inherent acoustic properties, studied for a contrast agent application, have been previously demonstrated in a in vitro Couette device. To measure their acoustic behaviour under circulating blood conditions, the TMP backscatter enhancement was further evaluated on a home-made flow phantom at different TMP doses (0.12-15.6 mg/ml) suspended in aqueous and blood media, and in nude mice (aorta and B16 grafted melanoma). Integrated backscatter (IB) was measured by spectral analysis of the Doppler signals recorded from an ultrasound system (Aplio) combined with a 12-MHz probe. Doppler phantom experiments revealed a maximal IB of 17+/-0.88 dB and 7.5+/-0.7 dB in aqueous and blood media, respectively. IB measured on mice aorta, in pulsed Doppler mode, confirmed a constant maximal value of 7.29+/-1.72 dB over the first minutes after injection of a 7.8 mg/ml TMP suspension. Following the injection, a 60% enhancement of intratumoral vascularization detection was observed in power Doppler mode. A preliminary histological study revealed inert presence of some TMP in lungs 8 and 16 days after injection. Doppler phantom experiments on whole blood allowed to anticipate the in vivo acoustic behaviour. Both protocols demonstrated TMP effectiveness in significantly increasing Doppler signal intensity and intratumoral vascularization detection. However, it was also shown that blood conditions seemed to shadow the TMP contrast effect, as compared to in vitro observations. These results encourage further investigations on the specific TMP targeting and on their bio-distribution in the different tissues.

Methodology for quantifying interactions between perfusion evaluated by DCE-US and hypoxia throughout tumor growth.

The objective was to validate a combination of two new technologies to depict tumor physiology both temporally and spatially with dynamic contrast-enhanced sonography and an oximeter. Human cancer prostate tumors xenografted onto mice were followed for three weeks using dynamic contrast-enhanced ultrasonography (DCE-US) to detect tumor perfusion. Time intensity curves in linear data were quantified on four regions-of-interest (ROI, main tumor section and its anterior, central and posterior intra-tumoral areas) to extract three indices of perfusion. An oxygen sensor was guided by sonography to obtain accurate pO(2) measurements in the three predefined areas of tumors during their development. No impact on tumor growth of subsequent pO(2) probe insertion was detected. Among the four ROIs studied, the local central tumor showed significant perfusion and oxygenation variations throughout the experiment. A correlation was observed between local central tumor perfusion and pO(2), both of them decreasing through time (p = 0.0068; r = 0.66). The methodology which we developed demonstrated the potential of combining DCE-US with direct tissue pO(2) measurements, improving the description of complex intratumoral dynamic behavior.

[Imaging of melanoma: accuracy of ultrasonography before and after contrast injection for diagnostic and early evaluation of treatments]. / Imagerie des mélanomes: performances de l'échographie sans et avec injection de produit de contraste pour le diagnostic et l'évaluation des traitements.

INTEREST FOR DIAGNOSIS: High-frequency sonography (12-16 MHz) allows to visualize primary cutaneous melanomas. The maximal echographic thickness was measured and strongly correlated to the Breslow index (p< 10(-4)). Lesions were well defined with homogeneous hypoechoic echostructure. A study including 111 patients with a 5 years median follow-up period demonstrated that the detection of neo-vascularization by color Doppler was significantly correlated to occurrence of metastasis (p < 10(-4)). INTEREST FOR EARLY EVALUATION OF TREATMENTS: For patients with local recurrent melanomas treated by isolated limb perfusion, contrast enhanced ultrasonography allows early evaluation of therapeutic. The access to raw linear data associated to quantification software permits to estimate tumoral perfusion. Several perfusion parameters (area under the contrast tune intensity curve, maximal intensity peak, mean transit time,...) are obtained after curves modelization. A study of 40 patients with a follow-up by dynamic contrast enhanced ultrasonography at D-1, D+1, D+7 and D+30 demonstrated that means of the quantification using raw linear data was able to identify as early as D+1 patients with complete response at 3 months.

Dynamic contrast-enhanced ultrasonography (DCE-US) with quantification of tumor perfusion: a new diagnostic tool to evaluate the early effects of antiangiogenic treatment.

Dynamic contrast-enhanced ultrasonography (DCE-US) using the contrast agent Sonovue and vascular recognition imaging software is a novel technique that enables the detection of microvessels and quantitative assessment of solid tumor perfusion using raw linear data. Clinical trials have shown that DCE-US can be used to assess the anticancer efficacy of antiangiogenic treatment, for which conventional efficacy criteria based on size are unsuitable. Reduction in tumor vascularization can easily be detected in responders after 1-2 weeks and is correlated with progression-free survival and overall survival. DCE-US is supported by the French Cancer National Institut. This program is currently studying the technique in metastatic breast cancer, melanoma, colon cancer, gastrointestinal stromal tumor, and renal cell carcinoma, as well as in primary hepatocellular carcinoma, to establish the optimal perfusion parameters and timing for quantitative anticancer efficacy assessments.

[Current events about echography in 2006: position of the ultrasound functional imaging for the early evaluation of targeted therapeutics]. / Actualités sur l'échographie en 2006: place de l'imagerie fonctionnelle ultrasonore pour l'évaluation précoce des thérapeutiques ciblées.

The early and functional evaluation of new treatments in oncology is a main goal. At present, technical advances in Doppler ultrasonography allow the detection of neovascularization for superficial and deep malignant tumours in order to evaluate the efficiency of new treatments such as antiangiogenic molecules. Contrast agents injection improves the efficiency of this technique and developments of perfusion softwares optimize this detection. Slow flows in tumour microvessels can be detected. Treatment response can be early predicted based on changes in the vascularization before volume modification. The availability of quantification softwares operating from the raw data before their compression for video display affords one and objective quantification of the contrast agent uptake.

Combination of HIFU therapy with contrast-enhanced sonography for quantitative assessment of therapeutic efficiency on tumor grafted mice.

The objective was to evaluate treatment efficiency of a new high-intensity focused ultrasound (HIFU) prototype combining a therapeutic transducer with a sonographic probe. The optimal HIFU sequence was defined on ex vivo samples before in vivo evaluation of tumor ablation was performed by perfusion quantification after contrast agent injection. The original feature of this prototype is a 9-MHz sonographic probe in a HIFU device and connected to an Aplio (Toshiba) sonograph. Acoustical power and treatment time were determined on ex vivo livers to generate 1-cm-long lesions. Lesion reproducibility was assessed for the power and treatment time selected. The gap between lesions and HIFU displacement shot procedures were optimized to ablate a 1-cm3 volume. The optimized protocol was applied to five murine tumors in vivo. Tumor ablation was quantified according to (1) contrast uptake (CU) after HIFU using perfusion software (Toshiba) in "vascular recognition imaging" mode and Sonovue (Bracco) contrast agent, and (2) the percentage of necrosis quantified on histologic slides. Ex vivo results: optimized settings, at 442 W/cm2 applied during three cycles (3 s on/5 s off) generated 10 identical elementary lesions measuring 9.78 (+/-0.66) * 2.11 (+/-0.33) mm2. A 4-mm gap between adjacent lesions and a 2-min pause between shot lines were found optimal. In vivo results: 60 % (+/-22) mean reduction in CU after HIFU and tumor necrosis histologically estimated at 58 % (+/-5.7) were quantified for the five animals. The therapeutic potential of this HIFU prototype was demonstrated in vivo through objective quantification of tumor ablation based on CU.

In vitro echogenicity characterization of poly[lactide-coglycolide] (plga) microparticles and preliminary in vivo ultrasound enhancement study for ultrasound contrast agent application.

OBJECTIVES: This work includes (1) the characterization of a reproducible poly[lactide-coglycolide] (PLGA) microparticle preparation with an optimial mean diameter and size distribution and (2) the preliminary in vivo ultrasonographic investigation of PLGA microparticles. METHODS: A first series of PLGA microparticle preparations (1 to 15 mum) was acoustically characterized on a hydrodynamic device to select the most appropriate for ultrasound contrast agent application. Preparations of 3-microm microparticles were selected, characterized at different doses, and then injected into 20 melanoma grafted mice for contrast-enhanced power Doppler ultrasonography evaluation. RESULTS: The 3-microm microparticles (3.26-microm mean diameter with 0.41-microm standard deviation) led to in vitro enhancement of 18.3 dB at 0.62 mg/mL. In vivo experiments showed 47% enhancement of intratumoral vascularization detection after PLGA injection, significantly correlated (P < 0.0001) with preinjection intravascularization and tumoral volume. No toxicity was histologically observed. CONCLUSION: The 3-microm PLGA microparticles provided significant enhancement in vitro and in vivo without any toxicity.

Validation of a new method for quantifying in vivo murine tumor necrosis by sonography.

RATIONALE AND OBJECTIVES: At present, the gold standard to evaluate tumor necrosis is histology. We described here a new method to quantify the degree of tumor necrosis by ultrasonography. This technique combines ultrasound exploration of tissue and post-treatment of the numerical sequences using a dedicated software to evaluate backscattered power within the tumor. MATERIALS AND METHODS: In order to establish that the backscattered power could be considered as a relevant marker of tumor necrosis, we performed (1) intra- and interoperator reproducibility in estimation of tumor dimensions obtained on sonographic scans; and (2) intra- and interoperator reproducibility in quantification of backscattered power in postprocessing using the HDILab software. The third part of the study consisted of correlating the degree of tumor necrosis estimated by histology and the ultrasound backscattered power, both obtained on xenografted melanomas at different days after tumor transplantation. RESULTS: Results concerning tumor size estimations and quantification of echogenicity were reproducible (coefficient of variation < 4.33%). The degree of necrosis measured in histology and echogenicity were significantly negatively correlated (P < 0.003). CONCLUSION: In conclusion, backscattered power could be considered as a relevant parameter to quantify tumor necrosis in vivo.

Application of validated ultrasound indices to investigate erythrocyte aggregation in pigs. Preliminary in vivo results.

Although some studies concerning the ultrasound (US) characterization of erythrocyte aggregation reported in the literature have been conducted in vivo, none of them has led to quantitative indices. To achieve this objective, we first finalized a method on a hydrodynamic bench. Particularly, we define a kinetic protocol consisting of applying a 200 s(-1) shear rate followed up by a rapid decrease to reach a residual shear rate between 0 to 32 s(-1). From the backscattered intensity curve recorded all along the kinetic procedure, US dynamic parameters were defined and validated by correlation with reference laser indices obtained with the same model suspensions of erythrocytes (different concentrations of dextran 70 kD). A particular interesting behavior has been demonstrated when studying aggregation vs. the residual shear rate applied. The aim of the present study was to test the applicability of this aggregation kinetics protocol during in vivo investigations in pigs and possibly to recover the same aggregating behavior. The backscattered intensity was recorded all along the kinetic procedure as defined in vitro. Taking the derivative of the velocity profile recorded on 56 electronic windows, the shear rate was finely computed in the same measurement window where the backscattered intensity was calculated. Each US parameter could, therefore, be correlated with the residual shear rate corresponding to the same depth of measurement. We found that the blood aggregation behavior was identical to that observed in vitro. Apparently, a specific range of residual shear rates accelerates the activation of the aggregation process and the final aggregation level attained.

Comparison of new ultrasound index with laser reference and viscosity indexes for erythrocyte aggregation quantification.

We have previously established new ultrasonic indexes for erythrocyte aggregation using a Couette device, and validated them toward the Rayleigh's theory and reproducibility. Two hydrodynamic protocols were applied on various suspensions and their aggregation degrees were characterized by: 1. for the decreasing shear rates protocol: the power P(US) at the nominal frequency of the transducer used; 2. for the kinetic protocol: aggregation times (latency and half-rise times), variation between initial disaggregated state (Vo) and final aggregated state (V(inf)) and AI(US), which is the integral of the kinetic curve over time. The objective of the present study was to demonstrate the ability of these indexes to characterize the aggregation dynamics of suspensions with various levels of aggregation induced by concentrations of dextran 70 kD (Dx) of 10, 20 and 40 g/L added to washed red cells resuspended in saline solution. The results showed a maximum of backscattered power (P(US)) for Dx = 40 g/L with the decreasing shear rates protocol. We measured a final aggregation level (V(inf)), a minimal aggregation time (T(m)) and a maximal value of AI(US) for Dx = 40 g/L with the aggregation kinetics protocol. On the other hand, viscosity is increased with dextran concentration. These evolutions of the ultrasound (US) indexes and viscosity with dextran concentrations are consistent with literature reports. In addition, a particularly interesting phenomenon of US backscattering enhancement was observed for kinetics with no null final shear rate, which has never before been reported in such a precise manner. By another way, each of the dextran suspensions was tested on the laser erythroaggregometer that is presently considered as the "gold standard" method for erythrocyte characterization. The laser indexes (aggregation time T(a), aggregation indexes AI(10s) and AI(60s)), deduced from a kinetic protocol, have similar significance to the US ones. Statistical comparisons have been done between laser and ultrasonic indexes and significant correlations (0.001 < p < 0.01) were obtained. The set of results allowed us to conclude that ultrasonic indexes are suitable markers for the erythrocyte aggregation.

A new ultrasound principle for characterizing erythrocyte aggregation: in vitro reproducibility and validation.

RATIONALE AND OBJECTIVES: There is no method currently available to quantify erythrocyte aggregation in vivo. In this work, using a Couette system, we defined new ultrasound indexes potentially applicable for non-invasive investigations. METHODS: Two ultrasound protocols were developed: (1) a protocol in which decreasing shear rates ranging from 200 to 1 s-1 were applied to solutions; and (2) a protocol in which a 200 s-1 shear rate was initially applied followed by stoppage of flow (a kinetics protocol). New ultrasound indexes were defined as: the power PUS at the nominal frequency of each transducer, Rayleigh's slope (tangent of the curve PUS = f(log(F)) through the 3.5 to 15 MHz frequency bandwidth) and kinetic indexes characterizing the aggregation/aggregability of the suspension. RESULTS: Using washed erythrocytes resuspended in saline, it was shown that the ultrasound intensity is dependent at 3.54 +/- 5.9% (NS) to the power of the frequency (theoretical value = 4). Using 10 total blood samples extracted from a single pig, good reproducibility for all indexes (5%) was demonstrated. CONCLUSIONS: A suitable and reproducible methodology was developed and validated for studying erythrocyte aggregation in calibrated in vitro conditions.