29 Quantitative Analysis of Parenchymal Flow at Contrast-Enhanced US

29 Quantitative Analysis of Parenchymal Flow at Contrast-Enhanced US

David O. Cosgrove, Robert J. Eckersley, and Martin J.K. Blomley

D. O. Cosgrove, MD; R. J. Eckersley, PhD;

M. J. K. Blomley, MD

Department of Imaging Sciences, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0HS, UK

but they can be used as tracers to enable measure- ment of flow in novel ways (Cosgrove et al. 2001).

The fact that they are strictly intravascular agents, unlike the diffusible agents used in CT and MR, simplifies these calculations; fundamental to these is the ability to quantify the amount of contrast in a region of interest. There are two ways to quantify ultrasound data: work within the scanner with the raw data or use the processed video or audio out- puts. The former method requires special software from the manufacturer and many are now beginning to offer such packages; the latter, although less accu- rate, is more easily implemented on a wide range of scanners. In addition, care must be taken to account for non-linear machine processing of the data.

When using spectral Doppler the loudness of the audio output is proportional to the number of reflec- tors in the sample volume (Fig. 29.1). The audio sig- nals can be used to generate time–intensity curves to follow the wash-in and wash-out of the microbubble bolus (Fig. 29.2); however, in some cases the small sample volume is a disadvantage of using spectral Doppler.

Working with two-dimensional images reduces this problem and regions selected for measurement can be tailored to the shape of the structure of inter- est; in addition, two or more regions can be meas- ured simultaneously and tracking algorithms can be deployed to follow movement and keep the region of interest correctly centred on the image (Phillips and Gardner 2005). The image information can be analysed for grey-scale intensity changes, for exam- ple, using the software package NIH-Image (nih- image162_fat.hqz), and this is useful in vitro and in the heart and for parenchymal enhancement when using newer perfluoro-based contrast agents. The same approach can be used for colour Doppler by first removing the black-and-white portions of the image (segmentation) and then analysing the colour segment. For conventional velocity colour Doppler, the colour components can be reverse-mapped to retrieve the original frequency shift values if the look-up table that was used for colour coding is

29.1 Introduction 383

29.2 Transit-Time Measurements 384 29.3 Time–Intensity Curve Analysis 387 29.4 Reperfusion Kinetics 388

29.5 Conclusion 390 References 390

29.1

Introduction

Amongst the various means available for quantifica- tion of blood flow, Doppler is the most widely used because it is non-invasive (Cosgrove et al. 2001).

Volume flow is calculated as the product of the mean blood velocity and the cross-sectional area of the vessel. Unfortunately, estimating both of these is fraught with technical problems: the velocity is not constant, varying across the vessel lumen and over time with the cardiac rhythm; and the mean velocity calculations vary with the make of scanner. Measur- ing the area is also difficult, especially for small arter- ies, and this also changes through the cardiac cycle.

Taken together, these uncertainties can amount to errors of as much as 100% in estimating volume flow.

Transferring this to perfusion is difficult because the tissue volume being supplied is usually not known. In addition, Doppler suffers from a fundamental limita- tion when slow flow, such as in the microvasculature, is considered because it cannot distinguish blood flow from bulk tissue movement.

The introduction of microbubble contrast agents

has opened up new opportunities in this field

(Jacobsen 2001). Not only does the signal boost they

produce make Doppler measurements more robust,

Fig. 29.1a, b. Spectral Doppler for hepatic vein transit-time measurement. The spectral Doppler sample volume is placed on a hepatic vein before injection of the contrast agent, in this case Levovist (a), and the time when the signal increases (b) is noted.

The audio output is fed into a laptop computer and the moment the signal increases consistently above 10% over baseline is taken as the arrival time. The method measures porto-systemic shunting.

a b

Fig. 29.2 Time–intensity curve. This curve was calculated form power Doppler data form a cine loop after a bolus injection of a contrast agent. The raw data, shown as a black line, has a phasic variation caused by cardiac action, and the red line is the result of applying a nine-point smoothing algorithm.

Many temporal and quantitative features can be calculated from such curves.

available. In power Doppler, the colours display the signal intensity and provide a direct representation of the main variable that is increased by microbub- ble enhancing agents. A similar look-up table gives the decibel values throughout the region of inter- est. At present, these measurements are only avail- able on laboratory systems such as the CQ software (www.kineticimaging.com).

Built-in software packages are now offered on most high-end scanners and they allow the same measurements using either the cleaner, uncom- pressed and unprocessed data available within the scanner, or at least appropriately corrected post- processed data. They generate the time–intensity curves in real time or at least immediately after the wash-out phase of the transit and can quickly perform calculations such as temporal indices (e.g.

arrival time, time-to-peak enhancement) and inten- sity indices (e.g. peak enhancement, area under the curve; Fig. 29.3).

29.2

Transit-Time Measurements

Tracking the transit of a bolus of microbubbles ena- bles measurements of the physiology of organs. An example is the kidney where the adjacent position of the artery and the vein enables spectral Doppler measurement of the signal rise as the microbub- ble bolus arrives first in the artery and then in the vein (Cosgrove et al. 2000). The directionality of spectral Doppler allows these two signals to be sepa- rated. In the normal kidney the arterial venous tran- sit is approximately 4 s. 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, discard- ing signals from recirculating contrast, and then calculating the difference between the centroids of the two curves. The results correlate closely to those found by more invasive techniques such as magnetic flow meters.

This method has been applied to transplant kid- neys in an attempt to distinguish between acute tubular necrosis and rejection. In a preliminary study of 21 patients, a trend to longer arterio-venous transit times was observed in acute rejection, though there was overlap and the difference did not reach statistical significance (Harvey et al. 2001b).

Larger studies are needed to refine the method and

to establish its clinical significance, but the goal of avoiding biopsies remains attractive.

A similar method has been developed for the liver, the goal here being to evaluate arterio-venous shunting (a feature of cirrhosis and malignancies as well as of some rare conditions, such as hereditary haemorrhagic telangiectasia; Blomley et al. 1998).

Normally, the hepatic artery supplies approximately 25% of the liver’s blood, but this increases in these conditions because of shunts as well as the scarring that increases resistance to portal vein flow in cir- rhosis.

The first method developed used spectral Dop- pler to assess the arrival of Levovist in hepatic veins with the audio output of the scanner fed into a laptop computer (Fig. 29.1; Albrecht et al. 1999). Software to plot the time–intensity curves was developed and the arrival time was defined as a sustained increase in signal strength of 10% over baseline. The time from the bolus injection was measured and, in normal subjects, the multiple microvascular beds that the microbubbles have to traverse before they reach the hepatic veins (lung, splanchnic and hepatic) means that the arrival time is usually around 40 s. (The lower limit of normal was 25 s.) In patients with cir- rhosis the arrival could be as early as 5 s and was always <25 s. In addition, the amount of enhance- ment was also greater, perhaps because less contrast was trapped in the sinusoids of the cirrhotic liver. In a pilot study of 50 patients with diffuse liver diseases of various aetiologies, early arrival times were found

in those with cirrhosis but not in those with uncom- plicated chronic hepatitis who had arrival times in the normal range (Blomley et al. 2003). There was one exception: a woman with primary biliary cir- rhosis who, despite a biopsy that was negative for cirrhosis, had an early arrival time. This could rea- sonably be attributed to sampling error of the biopsy whereby frankly cirrhotic tissue might have been missed. It is a potential advantage of the arrival- time method that the haemodynamic status of the entire liver be assessed.

A more extensive study focussing on 61 patients with hepatitis C was set up and again showed a good correlation with early arrival times in those with extensive necrosis (fibrosis scores: 6 of 6; Ishak et al. 1995) and late, more normal, arrival in those with no inflammatory change and minimal fibrosis (Ishak et al. 1995: necroinflammatory scores ≤3 of 18; Lim et al. 2005a). Interestingly, in patients with moderately severe disease (Ishak et al. 1995) ≤4 of 18 had significantly early arrival times, suggesting that not only can the arrival time pick out the devel- opment of cirrhosis, but that it might also detect pre-cirrhotic changes and thus be of value in deci- sions about when to offer antiviral treatment.

As part of this study, measurements were repeated in a cohort of 10 patients after a course of treatment with interferon-α and ribavirin. Although still pre- liminary, there was a trend for normalisation of the arrival time in those patients whose viral load was successfully reduced.

Fig. 29.3a,b. A built-in quantifi cation package (ACQ) was used to calculate perfusion curves for this focal liver lesion (yellow region of interest and curve) and an adjacent region of normal liver (green) following a bolus injection of the microbubble contrast agent SonoVue (a). The software incor- porates a tracking mechanism which follows the movement of the liver through breathing and adjusts the position of the regions of interest to maintain their position. Several features from the exponential reperfusion curve are automatically cal- culated (b) including the rate of rise (?) and the maximum level of enhancement reached (A). These are two important numbers related to perfusion.

a

b

assesses the relative contribution of the artery to total liver flow (Leen et al. 2000). It depends on an accurate measurement of the volume flow in both the hepatic artery and portal vein and has been shown to be able to detect not only overt but, impor- tantly, also occult metastatic disease (i.e. disease not detectable at preoperative staging or at laparotomy, which becomes manifest on follow-up). In fact, the basis of this approach was a nuclear medicine find- ing that the time–activity curve for the liver fol- lowing a bolus injection of a radio-isotope had two components: an early steep rise, corresponding to the arterial supply; and a later, more gentle rise from the portal supply (Robinson 1991). The first component was found to dominate in patients with liver metastases. For reasons that are unclear, this nuclear medicine test was never widely adopted.

The Doppler Perfusion Index, though very prom- ising, was found to be too technically demanding for general use, so simpler alternatives were sought (Harvey et al. 2001a; Bernatik et al. 2002). Track- ing the arrival of a microbubble bolus in the hepatic veins seems to be a feasible alternative that is practi- cal to perform. The earliest technique used spectral Doppler with the sample volume placed on a hepatic vein and the delay from a peripheral intravenous injection to a measured increase in the hepatic vein signal of 10% above baseline was timed.

In a study of 48 patients with colorectal carci- noma, the preoperative hepatic vein transit time was abnormal in 12 who had no definite metastases on CT staging, and of these, 5 had developed overt metastases at their follow-up CT at 1 year (Harvey et al. 2001a). None of those patients with normal hepatic vein transit times developed metastases. If further trials support this finding, the transit time could be useful in selecting patients for adjuvant chemotherapy.

Performing these transit-time studies is cumber- some as it requires at least two operators and dedi- cated computing equipment. With the advent of per- fluoro-gas contrast agents and the development of contrast-specific scanning modes, the possibility of

this method is that uninterpretable results where the tracing is flat, which occurs in approximately 10%

of cases when using Levovist, are rare; however, the discrimination between the degrees of hepatitis and cirrhosis is a little less clear-cut than with Levovist, perhaps because liver trapping is less marked with SonoVue. Methods to allow the person performing the scan also to give the injection are being explored in a further attempt to simplify the test for routine clinical use, and the most promising is to use an extension tube of the appropriate capacity, preloaded with the contrast agent and the saline flush, such that a single stroke of the plunger pushes the contrast fol- lowed by the flush.

All of these studies have focussed on a particular set of large vessels, but it is also possible to meas- ure transit-time curves for regions of tissue. These work best using non-linear modes, such as harmon- ics, and low-transmit-power methods are preferred because they cause less bubble destruction.

The regional approach has been used mainly for malignancies seeking to improve differential diag- nosis by revealing features of neovascularisation, e.g. in the liver (Burns et al. 2000; Harvey and Albrecht 2001; Tanaka et al. 2001). Many stud- ies in the breast show that the arrival time in can- cers is earlier than in benign masses, although some cases of benign breast change have been confusing, especially those that have an inflammatory compo- nent at histology (Kedar et al. 1996; Albrecht et al. 1998; Baz et al. 2000). Some studies have shown longer persistence of enhancement in cancers, but this has been more variable. The lack of vascular- ity in mature scars in the breast in comparison to the marked vascularity of tumour recurrence has emerged as a proven clinical tool for this difficult problem (Winehouse et al. 1999).

In the liver, temporal patterns that are generally

similar to those seen on triple-phase CT have been

described. Malignancies, especially hepatocellular

carcinomas, fill from their margin during the arterial

phase a few seconds after the injection while regener-

ating nodules and haemangiomas fill slowly, the latter

typically from the periphery and usually incompletely.

Focal nodular hyperplasia shows similar temporal features to malignancies, although the flow tends to emanate from a central supply artery and their liver- specific phase discriminates them well.

The prostate has also been studied extensively with microbubbles: it has generally been disap- pointing in cancer detection, although more work is needed here, but the time patterns of treated cancers remains different from benign prostatic hyperpla- sia, even when the prostate-specific antigen has been reduced to unmeasurable levels, suggesting that the abnormal vascularity persists despite suppression of biochemical activity (Eckersley et al. 1998). Time–

intensity curves fall rapidly when anti-androgen treatment is administered, as early as day 1, and in a few cases, a rising time–intensity curves value pre- dates escape from hormonal control as judged bio- chemically and clinically, and this might allow early adjustment of treatment (Eckersley et al. 2002).

All of the many indices that can be extracted from these time–intensity curves can also be displayed as colour-coded true functional images which may highlight regional differences in haemodynamics in a useful way.

29.3

Time–Intensity Curve Analysis

The forms of the time–intensity curves contain haemodynamic information which can be revealed using the same mathematics as has classically been applied to radionuclide tracers. In some ways the anal- yses are simpler because microbubbles are confined to the blood pool and do not diffuse into the interstitial space; however, rigorous analyses are complicated by bubble destruction, non-linearities in the measure- ments and inhomogeneities in the acoustic fields.

The approaches relevant to microbubble time–inten- sity curves include bolus transit studies in which the true tissue perfusion (i.e. the volume flow per unit of tissue per unit time) is calculated as the ratio of the peak tissue enhancement to the area under the arte- rial curve (Wolfkiel and Brundage 1991). A variant where the peak gradient of the tissue curve is divided by the peak height of the arterial curve has been developed for CT (Miles and Blomley 1997). Pro- vided the necessary arterial and tissue curves could be obtained (and this depends on the particular ana- tomical arrangements on the organ or lesion), these approaches could be applied to ultrasound.

Fig. 29.4a–c. Using a microbubble-specifi c mode (contrast pulse sequences, in this example), the arrival of the con- trast in the hepatic artery can be detected (arrowheads in a). The timer (highlighted in red) is started as soon as this is observed and then attention is focussed on the selected hepatic vein (arrowheads in b), looking for the arrival of the contrast, which in this patient with hepatitis C occurred at 6 s (arrowheads in c). This is abnormally early, the normal lower limit being 12 s, and indicates that the liver has arterio- venous shunts which are typical of cirrhosis.

a b

c

Thus far, there are no reports of attempts to make use of these established methods as experimental or clinical tools using microbubbles.

29.4

Reperfusion Kinetics

The fragility of microbubbles is a feature that can be exploited to obtain a measure of tissue perfusion.

First developed for the myocardium (Wei et al. 1998), the method uses a high-power ultrasound beam to destroy the microbubbles within a slice of tissue that corresponds to the orthogonal beam profile.

The destructive pulse creates a negative bolus and “reperfusion kinetics” are used to study the way contrast flows back into the void at a rate that depends on the tissue’s perfusion. This refill phase can be assessed in two ways, both of which assume a steady state of blood microbubble concentration which is usually achieved with an infusion pump.

In the first method, developed by Wei et al. (1998), further high-transmit power frames are sent along the same path at progressively increasing intervals, and the signal strength from each is measured. The refill curve has a rising exponential form described by the equation:

I=A(1-e

),

where I=signal intensity and t is the pulsing inter- val. Its initial exponent (β) is proportional to the speed of microbubble/blood flow into the slice while the peak signal strength, A, is proportional to the fractional vascular volume. The product of these two is an indirect measure of tissue perfusion. In principle, this could be converted to true perfusion if the volume perfused is known, but this is likely to prove elusive since it corresponds to the beam thickness whose shape is complex and dependent on machine settings (e.g. the focus position) and patient variables (e.g. attenuation). The beam's edge

2002), and the resulting perfusion overlay images are impressive, though it has not yet become rou- tinely accepted.

The original method is time-consuming and this makes it more difficult to keep the probe in the same place over the tissue. A faster and easier method is to switch the scanner to a very low transmit power imaging mode after the destructive burst and observe the reperfusion in real time (Fig. 29.5). This requires a non-linear microbubble-specific mode but, as it only takes a few seconds for most tissues to replenish completely, it can be performed at the pla- teau of a slow bolus injection. The equation describ- ing the reperfusion and the information that can be extracted from it are identical to the Wei method, although the correspondence between the two has not been investigated systematically; however, this approach does assume that the monitoring mode does not destroy any microbubbles. Just how sig- nificant this is, and the fact that the probe needs to stay steadily over the same part of the organ (which applies to both methods), is not yet known. As well as its speed, the real-time reperfusion method has the advantage that in-plane tissue movement can be corrected for by deploying a speckle-tracking method to follow the movement of the region of interest, for example, through the respiratory cycle, provided that the movement is confined to the scan plane. (Out-of-plane movement cannot be tracked.) If three-dimensional acquisition could be achieved, complete tracking should be feasible and the reper- fusion could be measured and depicted three- dimensionally.

These reperfusion methods have been applied

mainly to the heart in an attempt to quantify myo-

cardial perfusion, and they seem very promising

here. In the abdomen, reperfusion has been tried

in the kidneys (Fig. 29.6) and liver where, at the

least, the method allows a second look at the arte-

rial phase of flow, which is helpful for characteris-

ing focal lesions. In the liver, the dual supply means

that the reperfusion is not identical to a first-pass

bolus because the portal vein supply arrives at the

Fig. 29.5 Diagram of pulse sequence for reperfusion kinet- ics. The high-transmit-power pulses are designed to remove the microbubbles from the scanned plane. The scanner then switches to a low-transmit power mode to follow the exponen- tial reperfusion of the slice from its margins. The curve can then be analysed for features that describe the rate of refi ll and the maximum signal achieved. (Courtesy of M. Monaghan)

Fig. 29.6a–d. Reperfusion of a renal transplant. The fi rst frame from this cine loop (a) shows the transplant immediately after the destructive frames: the green box indicates the area subjected to the high transmit power (mechanical index: 1.5). Thereafter, frames at 1 s (b), 2 s (c) and 5 s (d) show the progressive refi ll of the main and then the small vessels with eventual fi lling of the microvasculature throughout the renal cortex.

a b

c d

same time as the hepatic arterial supply without the normal 15- to 30-s delay.

In the kidney a small study using the destruction–

reperfusion method with Levovist infusion in normal volunteers showed a good correlation between corti- cal flow increases produced by administration of dopamine and total renal blood flow measured as

the clearance of radio-labelled paramino hippurate (Kishimoto et al. 2003). The fractional vascular volume did not change. Although this was a small study, it convincingly demonstrates that non-invasive renal blood flow measurements are feasible.

The opposite approach, known as “diminution

kinetics” (Metzler et al. 2003), can also be imple-

mented, i.e. observing the rate of decay of the micro- bubbles when exposed to a high intensity beam, but this cannot be performed in real time so it has had limited application. This method has been used in the brain to create functional images of time-to- peak enhancement or of relative perfusion, and these seem to be useful in stroke patients because they can be performed at the patient’s bedside and can be repeated as often as necessary (Meyer and Seidel 2002).

The methods used to create functional images from time–intensity curves can be extended to reperfusion curves in which the product of A and β is plotted to create a map proportional to true perfusion (Fig. 29.7). Although not yet applied clini- cally, in even small series, this approach is expected to highlight tissues where perfusion is heterogene- ous, such as in malignancies.

29.5 Conclusion

Quantification methods can be applied to microbub- bles, although the limitations of the non-uniformity of real ultrasound fields in the body must be borne in mind. Measurement of a variety of transit times has been shown to be feasible and they have started to become accepted as clinical tools, especially in the liver. Time–intensity curves can be generated and classical functional analyses applied to them.

Functional images can be made from indices derived from them. Perhaps the most interesting approach

a transrectal scan of a prostate gland.

It was obtained with a phase inversion technique (Aplio US scanner; Pulse Subtract, Toshiba, Tokyo, Japan) by pro- cessing the cine loop offl ine. Functional images may usefully depict the hetero- geneity of the circulation in malignan- cies. (Courtesy of N. Kamiyama, Toshiba, Nasu, Japan.)

exploits the fragility of microbubbles to create nega- tive boluses, an approach that is unique in radiology.

The subsequent refill of the cleared volume can be used to generate information on tissue perfusion.

References

Albrecht T, Patel N, Cosgrove DO et al (1998) Enhancement of power Doppler signals from breast lesions with the ultrasound contrast agent EchoGen emulsion: subjective and quantitative assessment. Acad Radiol 5 (Suppl 1):

S195–S198

Albrecht T, Blomley MJ, Cosgrove DO et al (1999) Non-invasive diagnosis of hepatic cirrhosis by transit-time analysis of an ultrasound contrast agent. Lancet 353:1579–1583 Baz E, Madjar H, Reuss C et al (2000) The role of enhanced

Doppler ultrasound in differentiation of benign vs malig- nant scar lesion after breast surgery for malignancy.

Ultrasound Obstet Gynecol 15:377–382

Bernatik T, Strobel D, Hausler J et al (2002) Hepatic transit time of an ultrasound echo enhancer indicating the pres- ence of liver metastases: first clinical results. Ultraschall Med 23:91–95

Blomley MJK, Albrecht T, Cosgrove DO et al (1998) Liver vas- cular transit time analyzed with dynamic hepatic venog- raphy with bolus injections of an US contrast agent: early experience in seven patients with metastases. Radiology 209:862–866

Blomley MJK, Lim AK, Harvey CJ et al (2003) Liver microbub- ble transit time compared with histology and Child-Pugh score in diffuse liver disease: a cross sectional study. Gut 52:1188–1193

Burns PN, Wilson SR, Simpson DH (2000) Pulse inversion imaging of liver blood flow: improved method for char- acterizing focal masses with microbubble contrast. Invest Radiol 35:58–71

Cosgrove DO, Kiely P, Williamson R et al (2000) Ultrasono-

graphic contrast media in the urinary tract. Br J Urol Int 86 (Suppl 1):11–17

Cosgrove D, Eckersley R, Blomley M, Harvey C (2001) Quanti- fication of blood flow. Eur Radiol 11:1338–1344

Eckersley R, Butler-Barnes J, Blomley MJK et al (1998) Quan- titative microbubble enhanced transrectal ultrasound (TRUS) as a tool for monitoring anti-androgen therapy in prostate carcinoma. Radiology 209:310

Eckersley RJ, Sedelaar JP, Blomley MJK et al (2002) Quanti- tative microbubble enhanced transrectal ultrasound as a tool for monitoring hormonal treatment of prostate car- cinoma. Prostate 51:256–267

Gobbel G, Cann C, Fike J (1991) Measurement of regional cer- ebral blood flow using ultrafast computed tomography:

theoretical aspects. Stroke 22:768–771

Harvey CJ, Albrecht T (2001) Ultrasound of focal liver lesions.

Eur Radiol 11:1578–1593

Harvey CJ, Blomley MJK, Cosgrove DO et al (2001a) Liver vascular transit time measured using the carotid delay time with bolus injections of the microbubble Levovist can predict the presence of occult metastases in colorectal cancer. RSNA 2001

Harvey CJ, Lynch M, Blomley MJK (2001b) Quantitation of real-time perfusion with the microbubble optison using power pulsee inversion mode in renal transplants. Eur Radiol 11:103

Ishak K, Baptista A, Bianchi L et al (1995) Histological grading and staging of chronic hepatitis. J Hepatol 22:696–699 Jakobsen JA (2001) Ultrasound contrast agents: clinical appli-

cations. Eur Radiol 11:1329–1337

Kedar RP, Cosgrove DO, McCready VR et al (1996) Microbub- ble contrast agent for color Doppler US: effect on breast masses. Work in progress. Radiology 198:679–686 Kishimoto N, Mori Y, Nishiue T et al (2003) Renal blood flow

measurement with contrast-enhanced harmonic ultra- sonography: evaluation of dopamine-induced changes in renal cortical perfusion in humans. Clin Nephrol 59:423–

428

Leen E, Goldberg JA, Angerson WJ, McArdle CS (2000) Poten- tial role of doppler perfusion index in selection of patients

with colorectal cancer for adjuvant chemotherapy. Lancet 355:34–37

Lim AK, Taylor-Robinson SD, Patel N et al (2005a) Hepatic vein transit times using a microbubble agent can predict disease severity non-invasively in patients with Hepatitis C. Gut 54(1):128–133

Lim A, Patel N, Eckersley R et al (2005b) Hepatic vein transit times using SonoVue: a comparative study with Levovist.

Hepatology (in press)

Maier P, Zierler K (1954) On the theory of indicator dilution method for the measurement of blood flow and volume.

J Appl Physiol 4:308–314

Metzler V, Seidel G, Wiesmann K et al (2003) Perfusion har- monic imaging of the human brain. SPIE 5035:337–348 Meyer K, Seidel G (2002) Transcranial contrast diminution

imaging of the human brain: a pilot study in healthy vol- unteers. Ultrasound Med Biol 28:1433–1437

Miles K, Blomley MJK (1997) Perfusion CT: normal organs. In:

Miles K, Dawson P, Blomley MJK (eds) Functional com- puted tomography. ISIS Medical Media, Oxford

Phillips P, Gardner E (2005) Contrast agent detection and quantification. Eur J Radiol (in press)

Robinson P (1991) Detection of occult hepatic metastases using the hepatic perfusion index. Nucl Med Commun 12:153–158

Seidel G, Meyer K, Metzler V et al (2002) Human cerebral per- fusion analysis with ultrasound contrast agent constant infusion: a pilot study on healthy volunteers. Ultrasound Med Biol 28:183–189

Tanaka S, Ioka T, Oshikawa O et al (2001) Dynamic sonography of hepatic tumors. Am J Roentgenol 177:799–805 Wei K, Jayaweera AR, Firoozan S et al (1998) Quantification of

myocardial blood flow with ultrasound-induced destruc- tion of microbubbles administered as a constant venous infusion. Circulation 97:473–483

Winehouse J, Douek M, Holz K et al (1999) Contrast-enhanced colour Doppler ultrasonography in suspected breast cancer recurrence. Br J Surg 86:1198–1201

Wolfkiel C, Brundage BH (1991) Measurement of myocardial blood flow by ultrafast computed tomography: towards clinical applicability. Int J Cardiac Imaging 7:89–100