Recently, Wiener et al.1 reported a new echocardiographic sign in mitral regurgitation (MR) that they called "splay." We agree that this sign could be helpful when other methods have difficulties in grading the MR correctly and can help unmask a significant MR as also reported by Bertrand et al. in their editorial.2

Background: The authors describe a previously unreported Doppler signal associated with mitral regurgitation (MR) as imaged using transthoracic echocardiography. Horizontal "splay" of the color Doppler signal along the atrial surface of the valve may indicate significant regurgitation when the MR jet otherwise appears benign. Methods: Splay was defined as a nonphysiologic arc of color centered at the point at which the MR jet emerges into the left atrium. The authors present a series of 10 cases of clinically significant MR (moderately severe or severe as defined by transesophageal echocardiography) that were misclassified on transthoracic echocardiography as less than moderate. The splay signal was present on at least one standard transthoracic view in each case. To better characterize the splay signal, two groups were created from existing clinically driven transthoracic echocardiograms: 100 consecutive patients with severe MR and 100 with mild MR. Results: Splay was present in the majority of severe MR cases (81 %) regardless of vendor machine, ejection fraction, or MR etiology. Splay was particularly prevalent among patients with wall-hugging jets (28 of 30 [93%]). In patients with mild MR, splay was present less often (16%), on fewer frames per clip, and had smaller dimensions compared with severe MR. Color scale did not differ between subjects with and those without splay, but color gain was higher when splay was present (P = .04). Machine settings were further explored in a single subject with prominent splay: increasing transducer frequency reduced splay, while increasing color gain increased it. Conclusions: The authors describe a new transthoracic echocardiographic sign of MR. Horizontal splay may be a clue to the presence of severe MR when the main body of the jet is out of the imaging plane. Splay is likely generated as a side-lobe artifact due to a high-flux regurgitant jet.

The above paper was printed with an error. Subfigures 3c and 3d were transposed relative to their description in the figure caption. The corrected figure 3 and figure caption are provided in this erratum. The authors regret any inconvenience to the readers. Trans-Stent B-Mode Ultrasound and Passive Cavitation ImagingUltrasound in Medicine and BiologyVol. 42Issue 2PreviewAngioplasty and stenting of a stenosed artery enable acute restoration of blood flow. However, restenosis or a lack of re-endothelization can subsequently occur depending on the stent type. Cavitation-mediated drug delivery is a potential therapy for these conditions, but requires that particular types of cavitation be induced by ultrasound insonation. Because of the heterogeneity of tissue and stochastic nature of cavitation, feedback mechanisms are needed to determine whether the sustained bubble activity is induced. Full-Text PDF

Perfluorocarbon droplets that are capable of an ultrasound-mediated phase transition have applications in diagnostic and therapeutic ultrasound. Techniques to modify the droplet size distribution are of interest because of the size-dependent acoustic response of the droplets. Differential centrifugation has been used to isolate specific sizes of microbubbles. In this work, differential centrifugation was employed to isolate droplets with diameters between 1 and 3 μm and 2 and 5 μm from an initially polydisperse distribution. Further, an empirical model was developed for predicting the droplet size distribution following differential centrifugation and to facilitate the selection of centrifugation parameters for obtaining desired size distributions.

Acoustic droplet vaporization (ADV) is a process that enables the in situ production of microbubbles from an injected perfluorocarbon emulsion and it has been investigated for imaging and therapeutic applications. High-speed mechanical shaking rapidly produces a polydisperse emulsion (~1010 droplets/mL) with droplets ranging from less than 400 nm to greater than 15 μm. The ADV pressure amplitude threshold is higher and fraction of transitioned droplets lower for droplets smaller than approximately 2 μm in diameter. Droplets greater than approximately 8 μm in diameter are not suitable for systemic administration. Therefore, high-speed mechanical shaking produces many droplets of limited utility. Differential centrifugation has been used as a size isolation technique for polydisperse ultrasound contrast agents. By applying a similar technique to a perfluorocarbon emulsion, the volume-weighted fraction of droplets between 2 and 5 μm was increased from 29±2% to 93±3%. The transition efficiency of the droplets between 2and 5 μm was the same regardless of whether a polydisperse or monodisperse distribution was insonfied with 2 MHz ultrasound. The ADV pressure threshold of differentially centrifuged droplets was similar to non-centrifuged droplets. [Supported in part by NIH grant KL2 TR000078.]

High-speed mechanical agitation is commonly used to produce microbubbles and droplets for ultrasound imaging and therapy. This technique results in a high concentration (~1010 particles/mL) of polydisperse particles (less than 400 nm to greater than 15 μm in diameter). Differential centrifugation has been used to isolate microbubbles and droplets of specific sizes. In our prior work, we have isolated droplets between 2 μm and 5 μm. In the current work, we have isolated different sizes of droplets by adjusting centrifugation speeds. Our size-isolation protocol increased the fraction of droplets between 1 μm and 3 μm from 8% for non-centrifuged droplets to 87% for differentially centrifuged droplets. An empirical model for the size distribution after differential centrifugation was developed. The measured fraction of droplets in the supernatant and pellet for all sizes after a single centrifugation was used in the empirical model. There was a 3% difference in the volume-weighted mean diameter of the experimentally measured and empirically modeled size distributions. The coefficient of variations of the experimentally measured and empirically modeled size distributions were 24% and 22%, respectively. The empirical model allows for determining appropriate centrifugation parameters to obtain desired size distributions. [Work supported in part by NIH grant KL2 TR000078.]

Angioplasty and stenting of a stenosed artery enable acute restoration of blood flow. However, restenosis or a lack of re-endothelization can subsequently occur depending on the stent type. Cavitation-mediated drug delivery is a potential therapy for these conditions, but requires that particular types of cavitation be induced by ultrasound insonation. Because of the heterogeneity of tissue and stochastic nature of cavitation, feedback mechanisms are needed to determine whether the sustained bubble activity is induced. The objective of this study was to determine the feasibility of passive cavitation imaging through a metal stent in a flow phantom and an animal model. In this study, an endovascular stent was deployed in a flow phantom and in porcine femoral arteries. Fluorophore-labeled echogenic liposomes, a theragnostic ultrasound contrast agent, were injected proximal to the stent. Cavitation images were obtained by passively recording and beamforming the acoustic emissions from echogenic liposomes insonified with a low-frequency (500 kHz) transducer. In vitro experiments revealed that the signal-to-noise ratio for detecting stable cavitation activity through the stent was greater than 8 dB. The stent did not significantly reduce the signal-to-noise ratio. Trans-stent cavitation activity was also detected in vivo via passive cavitation imaging when echogenic liposomes were insonified by the 500-kHz transducer. When stable cavitation was detected, delivery of the fluorophore into the arterial wall was observed. Increased echogenicity within the stent was also observed when echogenic liposomes were administered. Thus, both B-mode ultrasound imaging and cavitation imaging are feasible in the presence of an endovascular stent in vivo. Demonstration of this capability supports future studies to monitor restenosis with contrast-enhanced ultrasound and pursue image-guided ultrasound-mediated drug delivery to inhibit restenosis.

The aim of this study was to characterize the frequency-dependent acoustic attenuation of three phospholipid-shelled ultrasound contrast agents (UCAs): Definity, MicroMarker and echogenic liposomes. A broadband through-transmission technique allowed for measurement over 2 to 25 MHz with a single pair of transducers. Viscoelastic shell parameters of the UCAs were estimated using a linearized model developed by N. de Jong, L. Hoff, T. Skotland and N. Bom (Ultrasonics 1992; 30: 95-103). The effect of diluent on the attenuation of these UCA suspensions was evaluated by performing attenuation measurements in 0.5% (w/v) bovine serum albumin and whole blood. Changes in attenuation and shell parameters of the UCAs were investigated at room temperature (25 degrees C) and physiologic temperature (37 degrees C). The attenuation of the UCAs diluted in 0.5% (w/v) bovine serum albumin was found to be identical to the attenuation of UCAs in whole blood. For each UCA, attenuation was higher at 37 degrees C than at 25 degrees C, underscoring the importance of conducting characterization studies at physiologic temperature. Echogenic liposomes exhibited a larger increase in attenuation at 37 degrees C versus 25 degrees C than either Definity or Micro-Marker. (E-mail: raymonjl@mail.uc.edu) (C) 2014 World Federation for Ultrasound in Medicine & Biology.

Echogenic liposomes (ELIP) are being developed for the early detection and treatment of atherosclerotic lesions. An 80% loss of echogenicity of ELIP has been found to be concomitant with the onset of stable and inertial cavitation. The ultrasound pressure amplitude at which this occurs is weakly dependent on pulse duration. It has been reported that the rapid fragmentation threshold of ELIP (based on changes in echogenicity) is dependent on the insonation pulse repetition frequency (PRF). The study described here evaluates the relationship between loss of echogenicity and cavitation emissions from ELIP insonified by duplex Doppler pulses at four PRFs (1.25, 2.5, 5 and 8.33 kHz). Loss of echogenicity was evaluated on B-mode images of ELIP. Cavitation emissions from ELIP were recorded passively on a focused single-element transducer and a linear array. Emissions recorded by the linear array were beamformed, and the spatial widths of stable and inertial cavitation emissions were compared with the calibrated azimuthal beamwidth of the Doppler pulse exceeding the stable and inertial cavitation thresholds. The inertial cavitation thresholds had a very weak dependence on PRF, and stable cavitation thresholds were independent of PRF. The spatial widths of the cavitation emissions recorded by the passive cavitation imaging system agreed with the calibrated Doppler beamwidths. The results also indicate that 64%-79% loss of echogenicity can be used to classify the presence or absence of cavitation emissions with greater than 80% accuracy.

Cardiovascular disease is the leading cause of death and disability in the United States and worldwide. Echogenic liposomes (ELIP) are theragonistic ultrasound contrast agents (UCAs) being developed for the early detection and treatment of cardiovascular disease. Stability of the echogenicity of ELIP in physiologic conditions is crucial to their successful translation to clinical use. The stability of ELIP echogenicity was determined in vitro under physiologic conditions of total dissolved gas concentration, temperature, and hydrodynamic pressure in porcine plasma and whole blood. Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation as a function of pulse duration and pulse repetition frequency (PRF). Previous studies have also demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of ELIP as a function of pulse duration and pulse repetition frequency. Determining the relationship between cavitation thresholds and loss of echogenicity of ELIP would enable monitoring of cavitation based upon the on-screen echogenicity in clinical applications. ELIP were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations and four PRFs in a static fluid and in a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a single-element passive cavitation detection (PCD) system and a passive cavitation

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration-dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of UCAs as a function of pulse duration. Determining the relationship between cavitation thresholds and loss of echogenicity of UCAs would enable monitoring of cavitation based upon the onscreen echogenicity in clinical applications. Two lipid-shelled UCAs, echogenic liposomes (ELIP) and Definity (R), were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations ('sample volumes') in both a static system and a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a passive cavitation detection system and stable and inertial cavitation thresholds ascertained. Loss of echogenicity from ELIP and Definity (R) was assessed within regions of interest on B-mode images. A numerical model based on UCA rupture predicted the functional form of the loss of echogenicity from ELIP and Definity (R). Stable and inertial cavitation thresholds were found to have a weak dependence on pulse duration. Stable cavitation thresholds were lower than inertial cavitation thresholds. The power of cavitation emissions was an exponential function of the loss of echogenicity over the investigated range of acoustic pressures. Both ELIP and Definity (R) lost more than 80% echogenicity before the onset of stable or inertial cavitation. Once this level of echogenicity loss occurred, both stable and inertial cavitation were detected in the physiologic flow phantom. These results imply that stable and inertial cavitation are necessary in order to trigger complete loss of echogenicity acoustically from UCAs and this finding can be used when planning diagnostic and therapeutic applications.

Echogenic liposomes (ELIP) are under development as an ultrasound contrast agent (UCA) for image-guided, targeted drug delivery. The aim of this study was to investigate the effect of the lipid formulation and type of encapsulated gas on the acoustic response of ELIP under physiologic conditions. The influence of temperature and diluent on the measured attenuation coefficients was investigated for ELIP as well as the commercial lipid-based UCA Definity. Broadband attenuation spectra were obtained for agents diluted either in whole blood or in saline solution containing 0.5% bovine serum albumin (BSA) at 25°C and 37°C. The spectra and particle size distributions obtained using a Coulter counter were compared with scattering theory to estimate the properties of the lipid shell stabilizing the microbubbles. Dilution of ELIP or Definity in either whole blood or BSA solution did not affect the measured attenuation coefficients. However, the temperature and gas saturation of the diluent were found to impact the attenuation spectra significantly. The attenuation spectra of UCAs are strongly impacted by temperature and gas saturation relative to physiologic values. Knowledge of the acoustic properties and size distribution of these agents under physiologic conditions will allow for their safe use in vivo.

Echogenic liposomes (ELIP) encapsulate gas bubbles and drugs within lipid vesicles, but the mechanisms of ultrasound-mediated drug release from ELIP are not well understood. The effect of cavitation activity on drug release from ELIP was investigated in flowing solutions using two fluorescent molecules: a lipophilic drug (rosiglitazone) and a hydrophilic drug substitute (calcein). ELIP samples were exposed to pulsed Doppler ultrasound from a clinical diagnostic ultrasound scanner at pressures above and below the inertial and stable cavitation thresholds. Control samples were exposed to a surfactant, Triton X-100 (positive control), or to flow alone (negative control). Fluorescence techniques were used to detect release. Encapsulated microbubbles reduced the measured fluorescence intensity and this effect should be considered when assessing drug release from ELIP. The origin of this effect is not specific to ELIP. Release of rosiglitazone or calcein compared to the negative control was only observed with detergent treatment, but not with ultrasound exposure, despite the presence of stable and inertial cavitation activity. Release of rosiglitazone or calcein from ELIP exposed to diagnostic ultrasound was not observed, even in the presence of cavitation activity. Ultrasound-mediated drug delivery strategies with ELIP will thus rely on passage of the drug-loaded liposomes to target tissues.

Acoustically induced loss of echogenicity (LOE) from ultrasound contrast agents (UCAs) has been exploited in imaging techniques to improve delineation of pathology. Determination of the type of cavitation that accompanies LOE can be experimentally difficult to determine due to the complex microbubble activity elicited. A theoretical model has been derived to predict the LOE originating from rupture of the UCA shell, from stable cavitation, or from inertial cavitation. The predictions of the model for each cavitation phenomena will be compared to recent experimental LOE measurements of the lipid-based UCA Definity® and echogenic liposomes insonified by Doppler pulses from a clinical scanner. The backscatter coefficient was calculated for a population of UCAs exposed to 6-MHz pulsed ultrasound of duration 1.67μs—8.33 μs. The change in the total backscatter coefficient was used to predict the LOE. The size distribution of UCAs was adjusted according to the specific type of cavitation triggered by the ultrasound exposure. Comparison of the theoretical predictions and experimental measurements suggest that shell rupture is the dominant mechanism for LOE for both Definity® and echogenic liposomes. These results will be discussed in conjunction with a recently developed cavitation index to predict the LOE of UCAs.

Acoustic cavitation has been correlated with a variety of ultrasound-mediated bioeffects. Recently developed passive cavitation imaging methods provide spatially resolved maps of cavitation activity with good azimuthal resolution but poor axial resolution. Here, inter-element apodization is investigated as a means of improving image quality. Cavitation was induced from echogenic liposomes in a flow phantom exposed to 6 MHz Doppler ultrasound (Philips HDI-5000). The resulting acoustic emissions were passively recorded on 64 elements of a linear array (L8-3 transducer, Zonare z.one ultra scanner). Amplitude scaling of each waveform by its root-mean-square value improved axial resolution at the expense of creating an ‘X-shaped’ artifact. Cosine amplitude apodization of the received waveforms across the array and centered about the azimuthal location of the beamformed image pixel was found to reduce grating lobe artifacts. Numerical time reversal of the received waveforms, using the Fresnel approximation for the acoustic field of each array element, resulted in an effective apodization due to element directivity and also reducing grating lobe artifacts. Applying apodization may be an effective means of increasing passive image quality for certain cavitation distributions, which will be discussed. [Supported in part by NIH grants F32HL104916, R01HL074002, R21EB008483, R01HL059586, and R01NS047603.]

Careful determination of stable and inertial cavitation thresholds of UCAs exposed to pulsed ultrasound is required for their safe use in diagnostic and therapeutic applications. Echogenic liposomes and Definity® were diluted in porcine plasma and pumped through a physiological flow phantom. UCAs were insonified with pulsed Doppler ultrasound at three pulse durations (3.33 µs, 5.83 µs and 8.33 µs) over a range of peak rarefactional pressure amplitudes (0.06-1.9 MPa). A 10-MHz focused passive cavitation detector (PCD) was used to record cavitation emissions. PCD signals and B-mode images of UCAs and degassed water were acquired during insonation. Thresholds of stable and inertial cavitation, and loss of echogenicity were determined by piecewise linear fits of the cavitation powers and mean gray scale values, respectively. The stable cavitation thresholds were found to be lower than the inertial cavitation thresholds at each pulse duration setting. The thresholds of loss of echogenicity and stable and inertial cavitation were found to be dependent on pulse duration. The relationship between loss of echogenicity and cavitation emissions will be discussed in the context of using onscreen echogenicity to indirectly monitor cavitation during ultrasound-mediated therapy with UCAs. [Supported by NIH R01 HL059586.]

Previously, passive cavitation imaging has been described in the context of continuous-wave high-intensity focused ultrasound thermal ablation. However, the technique has potential use as a feedback mechanism for pulsed-wave therapies, such as ultrasound-mediated drug delivery. In this paper, results of experiments and simulations are reported to demonstrate the feasibility of passive cavitation imaging using pulsed ultrasound insonations and how the images depend on pulsed ultrasound parameters. The passive cavitation images were formed from channel data that was beamformed in the frequency domain. Experiments were performed in an invitro flow phantom with an experimental echo contrast agent, echogenic liposomes, as cavitation nuclei. It was found that the pulse duration and envelope have minimal impact on the image resolution achieved. The passive cavitation image amplitude scales linearly with the cavitation emission energy. Cavitation images for both stable and inertial cavitation can be obtained from the same received data set.

Echogenic liposomes (ELIP) are multifunctional ultrasound contrast agents (UCAs) with a lipid shell encapsulating both air and an aqueous core. ELIP are being developed for molecular imaging and image-guided therapeutic delivery. Stability of the echogenicity of ELIP in physiologic conditions is crucial to their successful translation to clinical use. In this study, we determined the effects of the surrounding media's dissolved air concentration, temperature transition and hydrodynamic pressure on the echogenicity of a chemically modified formulation of ELIP to promote stability and echogenicity. ELIP samples were diluted in porcine plasma or whole blood and pumped through a pulsatile flow system with adjustable hydrodynamic pressures and temperature. B-mode images were acquired using a clinical diagnostic scanner every 5 s for a total duration of 75 s. Echogenicity in porcine plasma was assessed as a function of total dissolved gas saturation. ELIP were added to plasma at room temperature (22 °C) or body temperature (37 °C) and pumped through a system maintained at 22 °C or 37 °C to study the effect of temperature transitions on ELIP echogenicity. Echogenicity at normotensive (120/80 mmHg) and hypertensive pressures (145/90 mmHg) was measured. ELIP were echogenic in plasma and whole blood at body temperature under normotensive to hypertensive pressures. Warming of samples from room temperature to body temperature did not alter echogenicity. However, in plasma cooled rapidly from body temperature to room temperature or in degassed plasma, ELIP lost echogenicity within 20 s at 120/80 mmHg. The stability of echogenicity of a modified ELIP formulation was determined in vitro at body temperature, physiologic gas concentration and throughout the physiologic pressure range. However, proper care should be taken to ensure that ELIP are not cooled rapidly from body temperature to room temperature as they will lose their echogenic properties. Further in vivo investigations will be needed to evaluate the optimal usage of ELIP as blood pool contrast agents.

Microbubble activity has been linked to bioeffects in a variety of therapeutic ultrasound applications including thermal ablation and localized drug delivery. Passive cavitation imaging of microbubble activity has been demonstrated in vitro during continuous-wave ultrasound exposure [Salgaonkar et al. (2009), JASA 126, 3071–3083; Gyongy and Coussios 2010, IEEE Trans BME 57, 48–56]. Here, we perform passive cavitation imaging of echogenic liposomes (ELIPs) in a flow phantom exposed to 6 MHz pulsed Doppler ultrasound (3.1 μs pulse duration, 1250 Hz pulse repetition frequency) transmitted from a diagnostic scanner (Philips HDI 5000, CL15-7 transducer). Frames of received ultrasound signal were obtained passively on 64 parallel channels recording for 112 μs (L8-3 transducer, Zonare z.one ultra scanner). Received echoes were beamformed by frequency-domain phase shifts to produce cavitation images. Localization of cavitation activity was tested by moving the Doppler exposure volume to three different positions and observing a concurrent spatial shift in cavitation activity. At the highest insonation pressures a loss in echogenicity was observed. Cavitation activity coincided spatially with this loss. Subharmonic and broadband cavitation images for multiple insonation pressures were consistent with previously determined stable and inertial cavitation thresholds for ELIP. [Work supported in part by NIH grants F32HL104916, R01HL074002, R21EB008483, R01HL059586, and R01NS047603.]