Assessment of tissue perfusion by contrast-enhanced ultrasound
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Contrast-enhanced ultrasound (CEUS) with microbubble contrast agents is a new imaging technique for quantifying tissue perfusion. CEUS presents several advantages over other imaging techniques in assessing tissue perfusion, including the use of microbubbles as blood-pool agents, portability, availability and absence of exposure to radiation or nuclear tracers. Dedicated software packages are necessary to quantify the echo-signal intensity and allow the calculation of the degree of tissue contrast enhancement based on the accurate distinction between microbubble backscatter signals and native tissue background. The measurement of organ transit time after microbubble injection and the analysis of tissue reperfusion kinetics represent the two fundamental methods for the assessment of tissue perfusion by CEUS. Transit time measurement has been shown to be feasible and has started to become accepted as a clinical tool, especially in the liver. The loudness of audio signals from spectral Doppler analysis is used to generate time-intensity curves to follow the wash-in and wash-out of the microbubble bolus. Tissue perfusion may be quantified also by analysing the replenishment kinetics of the volume of microbubbles after their destruction in the imaged slice. This allows to obtain semiquantitative parameters related to local tissue perfusion, especially in the heart, brain, and kidneys.
KeywordsUltrasound Microbubble Contrast agents Contrast-enhanced ultrasound Tissue perfusion
The ability to accurately quantify tissue perfusion, blood flow equalised for the volume or weight of the perfused tissue (cm3/s/cm3 or grams), is essential for the assessment of the physiological functionality and viability of a tissue. Different parameters are related to tissue perfusion including: blood velocity (cm/s), the speed of red blood cells in the region analysed; blood flow (cm3/s), the volume of blood passing in a section of tissue per unit of time; the fractional vascular volume (cm3), the proportion of tissue volume occupied by blood.
Laser Doppler , single photon computed tomography (SPECT) , multidetector computed tomography (CT) , magnetic resonance (MR) imaging  and positron emission tomography (PET)  all quantify tissue perfusion accurately but they are expensive and present some inherent limitations such as limited availability and patient exposure to radiation or nuclear tracers. Colour and power Doppler are limited by the low sensitivity to low-velocity flow in smaller vessels (<2 mm in diameter). Contrast-enhanced ultrasound (CEUS) with microbubble contrast agents [6, 7, 8] has recently been proposed as a new imaging technique for quantifying tissue perfusion [9, 10]. CEUS presents several advantages including low cost, portability, availability, lack of restrictions in performing serial examinations at short intervals, and absence of exposure to radiation or nuclear tracers.
Microbubble contrast agents
Microbubble contrast agents classified according to the filling gas
- Definity (Lantheus Medical Imaging)
- SonoVue (Bracco)
- Sonazoid (GE Healthcare)
- Optison (GE Healthcare)a
At resonant frequency (f o ) the microbubble radial oscillation becomes efficient and exaggerated; the scattering cross section of a microbubble is no longer simply dependent on microbubble size, and can reach peak values a thousand times higher than values at off-resonance. The insonation power is usually expressed by the mechanical index (MI) defined as p–/√fc where p– is the largest peak negative pressure and fc is the centre frequency of the pulse. When acoustic pressures at or near the resonant frequency are sufficiently high, non-linear microbubble oscillation develops producing harmonic frequencies. These frequencies allow us to distinguish microbubble signal from tissue clutter by using specialised contrast-specific US techniques. Pulse Inversion is the best-known phase-modulation technique. Vascular Recognition Imaging combines Doppler information with phase analysis and involves the transmission of four, alternately inverted, pulses along each imaging line. Cadence Contrast Pulse Sequencing works by interrogating each imaging line a number of times with pulses with various amplitudes and phases. Both harmonic and non-linear fundamental signals from microbubbles are represented on a grey-scale or colour map suppressing the linear fundamental echoes from native tissues.
Transit time measurements
Tracking the transit of a bolus of microbubbles enables measurements of the physiology of organs. The loudness of audio signals from spectral Doppler analysis can be used to generate time-intensity curves to follow the wash-in and wash-out of the microbubble bolus. In the kidney, where the adjacent position of the artery and the vein enables spectral Doppler measurement, the signal rises as the microbubble bolus arrives first in the artery and then in the vein and the directionality of spectral Doppler allows these two signals to be separated. In the normal kidney the arterial venous transit is less than 4 s, while it is increased in acute renal allograft rejection . The true mean transit time can be calculated by plotting the arterial and venous time intensity curves, applying a gamma-variate fit to select the first pass of the contrast agent, discarding signals from recirculating contrast, and then calculating the difference between the centroids of the two curves. In the breast, many studies showed that both transit time  and arrival time [21, 22] in cancers is shorter than in benign masses.
Objective analysis of echo-signal intensity
Determination of the degree of tissue contrast enhancement relies on the accurate distinction between microbubble backscatter signals and stationary tissue background. The fundamental assumption is the linear relation between video-intensity and microbubble concentration up to the achievement of a plateau phase . After achievement of the plateau phase microbubble concentration increases while videointensity remains constant, and at even higher concentrations, the video intensity actually decreases because of attenuation of the US beam by the microbubbles themselves. Quantitative analysis of tissue perfusion using CEUS is still limited by acoustic shadowing owing to the inadequate compensation for microbubble attenuation, and tissue attenuation correction algorithms or mathematical models estimating microbubble attenuation have been proposed [30, 31].
The direct visual assessment of the degree of log-compressed video-intensity is the least accurate method because the background tissue signal varies widely within the US sector, because of heterogeneities of acoustic power and differences in the attenuation and absorption of US energy by tissue. A practical approach consists of collecting imaging-converted video data, log-compressed and palletised as grey-scale or colour-coded 8-bit data, in the form of DICOM files. In this case, however, proper linearisation needs to be applied before curve-fitting and analysis, in order to reverse the effects of log-compression and possibly non-linear palette rendering . The quantitation of echo-signal intensity after anti-logarithmic transformation is the most accurate method, and eliminates the influence of logarithmic compression, colour maps, postprocessing curves, and techniques for edge enhancement on the input signal mapping for video presentation. Software packages access the raw data before application of non-linear modifications and allow image alignment, signal averaging and background subtraction.
A parametric image is an image where each pixel/voxel value represents the value of given parameters (e.g. blood flow or velocity or tissue perfusion) derived from multiple images at that location. Parametric images may be obtained after background subtraction in a pixel-by-pixel evaluation from the analysis of harmonic grey-scale imaging data through the use of dedicated software packages for the automated colour-coded depiction of the different kinetic parameters [34, 35].
The achievement of a steady-state microbubble concentration in the peripheral circulation is preferable in tissue perfusion studies . When microbubbles are administered as a constant infusion, the steady state is achieved after 2–3 min. This is obtained by dedicated microbubble injectors usually equipped with a rotating syringe to avoid microbubble sedimentation. At steady state the inflow and outflow of microbubbles in any microcirculatory unit is constant, proportional to the fractional blood volume of that unit, and dependent only on the flow rate of microbubbles.
Mathematical models for perfusion quantification
Potdevin et al. [37, 38] developed a dual model with a cross-plane and an in-plane vascularity for both the renal medullary and cortical regions, in which the whole refilling process is a weighted average of all possible elemental refill curves. Krix et al.  proposed a multivessel model that neglects diffusion, the microbubble movement from an area of high concentration to an area of lower concentration, and does not present exponential features, but assumes and takes into account a particular geometry of the vessels in the ROI. In all these mathematical models the time-intensity curves frequently present a wide data dispersion during both the ascending and the second plateau phase of the curve . Lucidarme et al.  proposed a model described by a sigmoid function (Fig. 6b) which is based on the assumption that microbubble destruction actually occurs in the feeding vessels that reach the ROI. Another model  states that the refilling kinetics depends on the distribution of vessel transit times and flows in the kidney, resulting in a piecewise linear function where the transit times are the times that separate the linear tracts, and the slopes are directly related to the flows (Fig. 6c).
Clinical applications of organ perfusion
Tissue perfusion quantitation represents one of the most promising new fields of application for CEUS. Measurement of transit time has been shown to be feasible and has started to become accepted as a clinical tool, especially in the liver. Reperfusion kinetics represents a reliable technique for obtaining semiquantitative parameters related to local tissue perfusion.
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