Since the advent of the first commercially available ultrasound enhancers in the mid-1990s, researchers have made many advances in contrast-enhanced ultrasonography, most importantly in the detection and characterization of focal liver lesions.

Contrast-enhanced ultrasound commonly depends on color or power Doppler signals, but the discovery of the nonlinear behavior of contrast agents, as a reaction to the insonation of an ultrasound wave, has led to additional and different imaging techniques.^1,2

Basically, all ultrasound contrast agents consist of a solution of gas-filled microbubbles. The bubbles in these agents are protected by a stabilizing layer or shell consisting of various chemicals that allow the bubbles to withstand several passes through the pulmonary and peripheral capillary systems. After intravenous injection, they enhance the entire vascular system, including large and small arteries as well as peripheral veins and the portal venous systems. The duration of enhancement after a bolus injection varies from agent to agent but lasts from two to 10 minutes. This can be prolonged for as long as necessary by infusing the agents.³

The earliest agent to provide clinically useful enhancement for radiology was Levovist from Schering/Berlex, a galactose-based agent with air bubbles protected by a thin layer of palmitic acid. This layer ensures the capillary passage, while the agent provides marked enhancement of Doppler signals from large and small vessels of up to 20 dB.⁴

Several newer contrast agents in clinical development use gases other than air, such as perfluorocarbons (see table). The advantage of such gases is that they are stronger enhancers than air and are less soluble in water and plasma. They not only provide stronger Doppler enhancement but also have a longer duration.^5,6

A common characteristic of all ultrasound contrast agents is their size of about 1 to 7 microns, which ensures that capillaries are not embolized. Most contrast agents are essentially blood-pool agents and do not leave the intravascular space, making them fundamentally different from x-ray or MR agents. The reticuloendothelial system (RES) takes up some of the newer agents (Sonovist, for example), which have hepatosplenic-specific properties,^7,8 and the shells therefore must be stable enough to allow the bubbles to be phagocytosed by Kupffer cells. But even agents such as Levovist, which produce more fragile bubbles that would not survive cellular uptake, are shown to have a liver late phase. Depending on the agent, the phase varies from less than an hour to days.

The lungs exhale the gas contained by the agents over several minutes after injection. The shells or protective layers vary in their chemical constituency and undergo different metabolic pathways. Clinical trials have proved that all current echo-enhancing agents are safe, with profiles comparing favorably with iodinated contrast agents. Almost no substantial adverse events have been reported.

Contrast Agent Behavior

The actual behavior of microbubbles in a sound wave is highly complex. Apart from simple backscatter, a variety of other effects occur on insonation. At low sound pressures, microbubbles change rhythmically and oscillate, tending to be resonant. This greatly increases the backscatter signal at the same frequency as the insonated signal. Fortunately, the resonance frequency of microbubble contrast agents happens to be in the frequency range of diagnostic ultrasound. This is a linear effect of enhancement; other effects produce distorted nonlinear signals.^9-11

On modern ultrasound scanners, sound pressure can be increased by the mechanical index (MI), and the size of these rhythmic changes cease equivalency. When this occurs, harmonic and subharmonic signals at multiples and fractions are emitted.

Further increases of sound pressure, although still in the diagnostic range, cause the bubbles to disrupt and produce strongly enhanced signals in gray-scale harmonic imaging and pseudo-Doppler signals. This effect is called stimulated acoustic emission (SAE).

Because the bubbles are destroyed, the signals are transient and last for only a limited number of frames. This effect, initially thought to be produced by a strong ultrasound signal resulting from the bubble destruction, seems now to derive from lost correlation between trains of ultrasound pulses that depend on the sudden disappearance of the reflectors.¹² Terms other than SAE (e.g., loss of correlation, or LOC) have been proposed for this effect.

Imaging Techniques

Imaging techniques used with the application of ultrasound contrast agents are mostly “nonlinear” and therefore depend on the nonlinear effects produced by the contrast agent. These include harmonic resonance imaging techniques and techniques that uses the SAE/LOC effect.

Modern broad-bandwidth transducers can send signals at a lower frequency while listening for returning signals at higher harmonic frequencies. The echoes produced by a microbubble are thus preferentially received while echoes from tissue, which returns only the fundamental signal, are relatively suppressed. This technique is called harmonic imaging. It can be used both in gray-scale and Doppler ultrasound. In gray-scale, areas of tissue, including the parenchyma of solid organs such as the liver, kidneys and spleen, will appear relatively bright when enhanced with microbubbles. Vessels will be greatly enhanced (hyperechoic) against a relatively suppressed hypoechoic background. This is particularly useful for detecting contrast in microvessels even in the presence of highly echogenic tissue such as the liver. Under harmonic Doppler ultrasound, the amount of clutter (undesired artifactual signals from moving tissue and flash artifacts) can be greatly reduced. Thus the technique is potentially useful in Doppler studies of moving tissues, such as the left lobe of the liver.

Harmonic imaging, however, is limited. To ensure that the detected signals are due only to harmonics emitted by the bubbles, the transmitted and received signal has to be filtered. To avoid an overlap of the filtered frequencies and resulting loss in contrast, the bandwidth of the filters has to be reduced. The restriction then degrades the resolution of the image, which leads to a compromise between contrast and resolution. Furthermore, the second harmonic signal from many microbubble agents is usually weaker than the fundamental signal. For example, with Levovist the signal of the second harmonic peak is 13 dB weaker than that of the fundamental.¹³ To achieve a higher signal the scanning has to be performed with a higher sound pressure (high MI). This results in irreversible disruption of the bubbles and leads to a transient signal, which reduces the assessment of vessels in the scan plane.

To overcome the limitations of the contrast and resolution compromise, we use the pulse or phase inversion imaging (PII) technique. In PII, two pulses are sent in rapid succession into the tissue, and the second pulse undergoes a 180° phase change. The scanner then receives the echo from these two pulses and forms their sum. For tissue that behaves in a linear manner, the sum is zero, but for an echo with nonlinear components, such as that from a microbubble, the sum is not zero. The full frequency of sound emitted from the transducer can be detected. So no restriction of bandwidth occurs, and therefore no restriction in resolution. The effect of halving the effective frame rate can be overcome by using modern ultrasound scanners that can perform parallel processing and therefore double the frame rate without loss of image quality. If no vessel assessment is needed for the detection of focal liver lesions, this technique can also be used with high MI. Then the second pulse will not produce a response, since the bubble has been destroyed by the first pulse and the sum of the two pulses will yield a particularly strong SAE/LOC signal.

Clinical Applications

Before the introduction of harmonic imaging methods, the clinical use of contrast agents for the liver was limited to color and power Doppler ultrasound techniques. Although these methods are still useful, scientific interest grows in detecting and characterizing focal liver lesions while using the nonlinear capabilities of the microbubbles in B-mode imaging. The advantages are fewer blooming and clutter artifacts and a higher spatial and temporal resolution.

In our group, most research has been done with Levovist, originally developed to enhance Doppler signals during an early vascular phase. It also has a specific liver and spleen late phase. Early studies of this still used Doppler examination technique showed random Doppler signals in a mosaic pattern due to the SAE/LOC of disrupted bubbles on high MI.¹⁴ After about six minutes, the contrast agent was seen only in liver and spleen parenchyma of examined volunteers and patients.

This lasts up to 30 minutes. The lack of normal liver parenchyma in most benign and malignant lesions causes the lesions to be spared from the Doppler signals (Figure 1), thus these lesions compare better with conventional B-mode gray-scale imaging. To overcome the limitations of the SAE/LOC Doppler method, which depends on the transient signal character, the color box, and the focus depth, our recent studies used PII methods.^15-18

Phase inversion mode presents an opportunity to enhance liver and spleen in B-mode imaging, especially with high MI. In such a case, we also have a transient signal requiring a special examination technique—the sweep technique—which also has to be used in SAE/LOC studies (slow sweeps through the entire left or right liver lobe). This was done so that undestroyed bubbles would be imaged with each new frame. Afterwards, the result can be reviewed as often as necessary in a cine loop, and the procedure may be repeated with another focal zone to cover the whole liver. We compared the results with baseline conventional ultrasound and found more metastases confirmed on reference imaging in 45% of 62 examined patients and in 72% of 36 patients whose findings were positive on both sonographic techniques.¹⁸ This leads to increased conspicuity of liver metastases (Figure 2). In addition we showed an increase of the average sensitivity for detecting individual metastases, from 63% up to 91%.

PII Technique

In addition to the detection and characterization of focal liver lesions, we are also investigating the enhancement of hepatosplenic parenchyma and vessels (e. g., the hepatic artery) (Figure 3) without Doppler ultrasound.^15,16 Recent studies have shown that besides the enhancement of well-vascularized parenchymas such as the liver, the PII technique can also achieve a reproducible enhancement of vessels, especially in the early vascular phase. This may improve characterization of liver lesions because of better assessment of specific vascularization patterns.

The enhancement of the liver and spleen parenchyma with Levovist persists for about 30 minutes and is virtually independent of flow. Thus the PII technique has potential not only for vascular imaging but to give ultrasound contrast agents a role similar to that of contrast agents in CT and MRI. With newer agents like Sonavist, the hepatosplenic late phase can persist for days.

But we aim not only to detect focal liver lesions such as metastases but also to differentiate and characterize them. There are some promising results showing significant vascular patterns and characteristic enhancement features of different lesions like hemangiomas, hepatocellular carcinomas, and metastases (Figure 4).^19-21 The imaging techniques used are always PII methods, but differences in the MI produce different results. At low MI, continuous harmonic imaging reveals vessels that are not detectable on conventional Doppler ultrasound modes, with or without contrast agents. One reason seems to be the relative absence of motion artifacts with the harmonic approach. At high MI with the interval-delay imaging technique, a propagating veil is produced due to the bubble destruction. This veil shows a high SAE/LOC signal in a small band while descending through the liver parenchyma. The signal of the lesions in question differs greatly between baseline and the veil, compared with the liver parenchyma.¹⁹

Major Leap

The development of new nonlinear, bubble-specific harmonic imaging methods represents a major leap in the examination of focal liver lesions with contrast-enhanced ultrasound. The methods advance the assessment of specific patterns in gray-scale harmonic imaging with low and high MI and color or power Doppler ultrasound, as well as in evaluating tumor vascularity and/or blood flow changes in the liver due to the lesions. In addition, the new methods may enable better detection of vessels and promote a more detailed diagnostic process.

The newer contrast agents themselves hold promise with expanded features going beyond the early enhancers: They are more stable, produce stronger echoes, and have a very long late hepatosplenic phase. All this demonstrates the need for further investigation of ultrasound contrast agent imaging methods, which are in some aspects ahead of the state of enhanced CT and MRI.

References

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