Robert J. Eckersley

Non-invasive diagnosis of hepatic cirrhosis by transit-time analysis of an ultrasound contrast agent

BACKGROUND Hepatic cirrhosis is accompanied by several haemodynamic changes including arterialisation of the liver, intrahepatic shunts, pulmonary arteriovenous shunts, and a hyperdynamic circulatory state. We postulated that the hepatic first pass of a bolus of an ultrasound contrast agent injected into a peripheral vein is accelerated in patients with cirrhosis. We investigated this first pass in patients with diffuse liver disease and in normal controls to assess whether it provides useful differential diagnostic information. METHODS We enrolled 15 patients with biopsy-proven cirrhosis, 12 patients with biopsy-proven non-cirrhotic diffuse liver disease, and 11 normal controls. We carried out continuous spectral doppler ultrasonography of a hepatic vein from 20 s before to 3 min after a peripheral intravenous bolus injection of 2.5 g Levovist. The intensity of the doppler signal was measured and used to plot time-intensity curves. FINDINGS Patients with cirrhosis showed a much earlier onset of enhancement (arrival time; mean 18.3 s) and peak enhancement (mean 55.5 s) than controls (49.8 s and 97.5 s) or patients with non-cirrhotic diffuse liver disease (35.8 s and 79.7 s). All patients with cirrhosis had an arrival time of the bolus of less than 24 s, whereas the arrival time was 24 s or more in 22 of the 23 other participants. Peak enhancement was higher in patients with cirrhosis (mean 48.7 units) than in the other two groups (12.5 and 12.3 units, respectively). We found highly significant differences between the patients with cirrhosis and each of the other two groups for all variables (p<0.005), whereas we found no significant differences between non-cirrhotic patients and controls. INTERPRETATION Our preliminary study suggests that analysis of liver transit time of a bolus of ultrasound contrast agent provides useful information about haemodynamic changes in patients with cirrhosis. Measurement of the arrival time of the bolus allows discrimination of patients with cirrhosis from controls and from patients with non-cirrhotic diffuse liver disease, and has potential as a non-invasive test for cirrhosis.

Developments in ultrasound contrast media

Abstract Ultrasound microbubble contrast agents are effective and safe echo enhancers. An ingenious array of methods are employed to achieve stability and provide a clinically useful enhancement period. Microbubbles enhance ultrasound signals by up to 25 dB (greater than 300-fold increase) due to resonant behaviour. This is used to rescue failed Doppler studies and may be extended to image the microcirculation of tumours and the myocardium using non-linear modes. Functional studies open up a whole range of applications by using a variety of active and passive quantitation techniques to derive indices from the transit of contrast through a tissue of interest. This has been especially successful in the detection of liver metastases and cirrhosis and shows great promise as a clinical tool. It also has great potential in measuring microcirculatory flow velocity. The demonstration that some microbubbles are not just pure blood pool agents but have a hepatosplenic specific phase has extended the versatility of ultrasound. Imaging of this stationary phase with non-linear modes such as phase inversion and stimulated acoustic emission, has improved the sensitivity and specificity of ultrasound in the detection and characterisation of focal liver lesions to rival that of CT and MR.

Hepatic vein transit times using a microbubble agent can predict disease severity non-invasively in patients with hepatitis C

Background and aims: A reliable non-invasive assessment of the severity of diffuse liver disease is much needed. We investigated the utility of hepatic vein transit times (HVTT) for grading and staging diffuse liver disease in a cohort of patients with hepatitis C virus (HCV) infection using an ultrasound microbubble contrast agent as a tracer. Materials and methods: Eighty five untreated patients with biopsy proven HCV induced liver disease were studied prospectively. All were HCV RNA positive on polymerase chain reaction testing. Based on their histological fibrosis (F) and necroinflammatory (NI) scores, untreated patients were divided into mild hepatitis (F ⩽2/6, NI ⩽3/18), moderate/severe hepatitis (3 ⩽F <6 or NI ⩾4), and cirrhosis (F = 6/6) groups. In addition, 20 age matched healthy volunteers were studied. After an overnight fast, a bolus of contrast agent (Levovist) was injected into an antecubital vein and spectral Doppler signals were recorded from both the right and middle hepatic veins for analysis. HVTTs were calculated as the time from injection to a sustained rise in Doppler signal >10% above baseline. The Doppler signals from the carotid artery were also measured in 60 patients and carotid delay times (CDT) calculated as the difference between carotid and hepatic vein arrival times. The earliest HVTT in each patient was used for analysis. Results: Mean (SEM) HVTT for the control, mild hepatitis, moderate/severe hepatitis, and cirrhosis groups showed a monotonic decrease of 38.1 (2.8), 38.8 (2.4), 26.0 (2.4), and 15.8 (0.8) seconds, respectively. Mean (SEM) CDT for the control, mild hepatitis, moderate/severe hepatitis, and cirrhosis patients again showed progressive shortening of 30.3 (2.6), 25.9 (2.6), 14.8 (2.1), and 5.6 (1.2) seconds, respectively. There were significant differences between the groups for HVTT (ANOVA, p<0.001) and CDT (ANOVA, p<0.001). There was 100% sensitivity and 80% specificity for diagnosing cirrhosis and 95% sensitivity and 86% specificity for differentiating mild hepatitis from more severe liver disease. Conclusion: We have shown, for the first time, that HVTT using an ultrasound microbubble contrast agent can assess HCV related liver disease with clear differentiation between mild hepatitis and cirrhosis. There were significant differences between these two groups and the moderate/severe hepatitis group. CDT offers no additional benefit or greater differentiation than HVTT and can be omitted, thus simplifying this technique. HVTT may complement liver biopsy and may also be a useful alternative for assessment of liver disease in patients who have contraindications to biopsy.

Quantitative contrast-enhanced ultrasound imaging: a review of sources of variability

Ultrasound provides a valuable tool for medical diagnosis offering real-time imaging with excellent spatial resolution and low cost. The advent of microbubble contrast agents has provided the additional ability to obtain essential quantitative information relating to tissue vascularity, tissue perfusion and even endothelial wall function. This technique has shown great promise for diagnosis and monitoring in a wide range of clinical conditions such as cardiovascular diseases and cancer, with considerable potential benefits in terms of patient care. A key challenge of this technique, however, is the existence of significant variations in the imaging results, and the lack of understanding regarding their origin. The aim of this paper is to review the potential sources of variability in the quantification of tissue perfusion based on microbubble contrast-enhanced ultrasound images. These are divided into the following three categories: (i) factors relating to the scanner setting, which include transmission power, transmission focal depth, dynamic range, signal gain and transmission frequency, (ii) factors relating to the patient, which include body physical differences, physiological interaction of body with bubbles, propagation and attenuation through tissue, and tissue motion, and (iii) factors relating to the microbubbles, which include the type of bubbles and their stability, preparation and injection and dosage. It has been shown that the factors in all the three categories can significantly affect the imaging results and contribute to the variations observed. How these factors influence quantitative imaging is explained and possible methods for reducing such variations are discussed.

Pulse-inversion mode imaging of liver specific microbubbles : improved detection of subcentimetre metastases

Pulse-inversion mode (a new ultrasound mode) can be used to image the late liver-specific parenchymal phase of the microbubble contrast-agent Levovist. Scanning in pulse-inversion mode after Levovist improves the detection of liver metastases and reveals more lesions of smaller size than conventional ultrasonography and computed tomography.

Liver microbubble transit time compared with histology and Child-Pugh score in diffuse liver disease: a cross sectional study.

Background: A previous pilot study showed that early arrival time of a microbubble in a hepatic vein is a sensitive indicator of cirrhosis. Aim: To see if this index can also grade diffuse liver disease. Patients: Thirty nine fasted patients with histologically characterised disease were studied prospectively. Nine patients had no evidence of liver fibrosis, 10 had fibrosis without cirrhosis, and 20 had cirrhosis (five Child’s A, seven Child’s B, and eight Child’s C). Methods: Bolus injections of a microbubble (Levovist; Schering, Berlin) were given intravenously, followed by a saline flush. Time intensity curves of hepatic vein and carotid artery spectral Doppler signals were analysed. Hepatic vein transit time (HVTT) was calculated as the time after injection at which a sustained signal increase >10% of baseline was seen. Carotid delay time (CDT) was calculated as the difference between carotid and hepatic vein enhancement. Results: Diagnostic studies were achieved in 38/39 subjects. Both HVTT and CDT became consistently shorter with worsening disease, as follows (means (SD)): HVTT: no fibrosis 44 (25) s, fibrosis 26 (8) s, Child’s A 21 (1) s, Child’s B 16 (3) s, and Child’s C 16 (2) s; CDT: no fibrosis 31 (29) s, fibrosis 14 (6) s, Child’s A 8 (1) s, Child’s B 4 (4) s, and Child’s C 3 (3) s. These differences were highly significant (p<0.001, ANOVA comparison). A HVTT <24 s and a CDT <10 s were 100% sensitive for cirrhosis (20/20 and 18/18, respectively) but not completely specific: 2/8 subjects with fibrosis had CDT values <10 s and 3/9 had HVTT <24 s. Conclusion: This minimally invasive test shows promise not only in diagnosing cirrhosis but also in assessing disease severity.

Quantification of blood flow

Abstract. Traditionally, Doppler ultrasound has been used to estimate blood flow as the mean velocity multiplied by the vessel area, but this is subject to significant errors and may be difficult to perform accurately. Microbubbles, developed as contrast agents for ultrasound, were initially envisaged as useful for increasing the intensity of echoes and thus rescuing Doppler studies that were technical failures because of attenuated signals or very slow flow. However, they can act as tracers and, by analogy with isotope techniques, can be used to measure blood flow with transit-time methods which exploit both arterial and venous time–intensity data. An acceptable compromise is to acquire both a tissue intensity curve and one from the feeding artery. The transit of microbubbles across an organ or tissue can be used to estimate haemodynamic alterations, e.g. the arterialisation of the supply to the liver in malignancies and cirrhosis and the delayed arterio-venous transit in the transplant kidney during rejection. The fragility of microbubbles can be turned to advantage by being exploited to create a negative bolus by exposing a tissue slice to a high power beam. The rate of refilling of this slice by circulating microbubbles can then be followed with a low-intensity monitoring beam and the resulting rising exponential curve analysed to extract indices of both the reperfusion rate (the slope) and the fractional vascular volume (the asymptote). The product of these is a measure of true tissue perfusion.

Stimulated acoustic emission to image a late liver and spleen-specific phase of Levovist® in normal volunteers and patients with and without liver disease

Quantitative studies were performed to investigate liver- specific uptake of the microbubble Levovist, using stimulated acoustic emission (SAE), which can detect microbubbles even when stationary or slow-moving. These comprised studies of biodistribution comparing the liver and kidney in five normal volunteers, reproducibility in 34 patients, comparison between cirrhotics and controls (n = 9 each) and maximal depth of effect at different frequencies (180 measurements in 31 patients). Stimulated acoustic emission lasted beyond 30 min, with strongly liver-specific properties in each volunteer and was highly reproducible. No difference in the amount of SAE in the superficial liver was seen between cirrhotic and normal livers, but attenuation was higher in cirrhotics. This demonstrates a frequency-dependent effect on liver SAE penetration. We conclude that the liver uptake of Levovist lasts over 30 min, is reproducible, occurs even where diffuse liver disease is present and can be used to assess tissue attenuation in a novel fashion.

Mapping microbubble viscosity using fluorescence lifetime imaging of molecular rotors

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a “molecular rotor” embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component.

3-D In Vitro Acoustic Super-Resolution and Super-Resolved Velocity Mapping Using Microbubbles

The structure of microvasculature cannot be resolved using standard clinical ultrasound (US) imaging frequencies due to the fundamental diffraction limit of US waves. In this work, we use a standard clinical US system to perform in vivo sub-diffraction imaging on a CD1, female mouse aged eight weeks by localizing isolated US signals from microbubbles flowing within the ear microvasculature, and compare our results to optical microscopy. Furthermore, we develop a new technique to map blood velocity at super-resolution by tracking individual bubbles through the vasculature. Resolution is improved from a measured lateral and axial resolution of 112 μm and 94 μm respectively in original US data, to super-resolved images of microvasculature where vessel features as fine as 19 μm are clearly visualized. Velocity maps clearly distinguish opposing flow direction and separated speed distributions in adjacent vessels, thereby enabling further differentiation between vessels otherwise not spatially separated in the image. This technique overcomes the diffraction limit to provide a noninvasive means of imaging the microvasculature at super-resolution, to depths of many centimeters. In the future, this method could noninvasively image pathological or therapeutic changes in the microvasculature at centimeter depths in vivo.