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(Stroke. 1997;28:1006-1008.)
© 1997 American Heart Association, Inc.

Articles

Characteristics of Transcranial Doppler Signal Enhancement Using a Phospholipid-Containing Echocontrast Agent

Manfred Kaps, MD; Peter Schaffer, MD; Klaus-Dieter Beller, MD; Eng; Günter Seidel, MD; Harald Bliesath, MD, MSc; Edgar Diletti; Eng

From the Department of Neurology, University at Lübeck (M.K., G.S.), and Clinical Pharmacology and Biometry, Byk Gulden Pharmaceuticals, Konstanz (P.S., K.-D.B., H.B., E.D.), FRG.

Correspondence to Professor Dr med M. Kaps, Klinik für Neurologie, Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, FRG.

	Abstract

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Background and Purpose Ultrasound attenuation caused by the skull is a major limitation of transcranial Doppler. Echocontrast agents (EAs) may solve this problem. The aim of the present study was to investigate the characteristics of a new echocontrast agent (BY963) containing air bubbles stabilized by phospholipids.

Methods Nine healthy volunteers received three different doses (2.5, 5.0, and 10 mL) of BY963 at an injection rate of 0.25 mL/s. The Doppler signal amplitude obtained from the middle cerebral artery was recorded with a 2-MHz pulsed-wave Doppler system. After complete decay of the signal enhancement, upward stroking of the veins of the upper arm was performed to evaluate the stability of the EA in the venous system.

Results A dose-dependent increase of at least 30 dB in the Doppler signal amplitude lasted 19 to 47, 35 to 64, and 48 to 126 heart cycles (68% range) after 2.5, 5.0, and 10 mL EA, respectively. In 6 cases, there was a biphasic increase in EA enhancement. Upward stroking of the forearm, in general 12 to 18 minutes after administration, caused a Doppler signal enhancement of at least 30 dB in 6 cases.

Conclusions Each injection of BY963 caused a diagnostically relevant Doppler signal enhancement. A considerable amount of EA remained stable in the venous system for at least 12 minutes. The biphasic dose-response fits to models of dilution-indicator theory and indicates free recirculation, as well as a nonlinear washout curve.

Key Words: cerebrovascular disorders • contrast media • ultrasonics

	Introduction

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The clinical usefulness of transcranial ultrasound diagnostics is limited by the fact that about 20% of cerebrovascular patients cannot be examined because of ultrasound attenuation by the temporal skull.^{1 2} A promising method of solving this problem is provided by echocontrast agents (EAs).^{3 4 5 6} Earlier investigations with the EA BY963 have demonstrated a diagnostically relevant enhancement of the intracranial Doppler signals⁷ and a good tolerability. The purpose of the present study was to analyze the course of the Doppler signal enhancement in more detail.

	Subjects and Methods

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The investigation was designed as an open, nonrandomized phase 1 study approved by an ethics committee and carried out in accordance with the guidelines of the Declaration of Helsinki (1964) with the revision supplements of Tokyo (1975), Venice (1983), and Hong Kong (1989).

Nine healthy volunteers in a supine position (mean age, 29±4.3 years; weight; 65±6.0 kg) subsequently received 2.5, 5, and 10 mL BY963. The BY963 suspension consists of spherosomes containing 0.04 mL air per milliliter. The size of the microbubbles is standardized, the mean value being 3.9 µm (95% <8 µm). The carrier substance used is a saturated stearic acid–containing phospholipid of plant origin.⁸ The individual injections, performed at intervals of more than 10 minutes, were given in an antecubital vein at a constant rate of 0.25 mL/s. Injection time lasted 40 seconds, corresponding to approximately 40 to 50 heart cycles. To determine how much EA adhered in the peripheral venous system of the arm after injection of 10 mL, the veins in the upper arm were stroked in the proximal direction from the injection site. The procedure was carried out about 1 minute after the primary contrast-agent effect had completely subsided (Figure).

View larger version (16K): [in this window] [in a new window]   Figure 1. Example of the course of Doppler signal enhancement after injection of a 10-mL dose of BY963 and after squeezing the venous system of the upper arm (U). The ordinate shows the signal intensity (in decibels) and the abscissa the duration (heart cycles). For technical reasons, signal enhancement above 42 dB could not be displayed. I indicates injection; A1, duration of signal enhancement to 30 dB after initial injection; A2, duration of signal enhancement to 30 dB after expressing the venous reservoir in the upper arm; P, start of the second enhancement peak; and E, complete subsidence of the echocontrast effect.

The enhancement of the Doppler signals was measured with a 2-MHz pulsed-wave Doppler system (Multi-Dop X, DWL). The maximal spatial peak–temporal averaged intensity was 100 mW/cm². Transcranial recording of the middle cerebral artery was carried out in accordance with generally accepted guidelines, with the ultrasound probe attached above the temporal calvaria with a headband for continuous registration. At the beginning of the examination, the Doppler sample volume was centered on the main branch of the middle cerebral artery, and the intensity of the baseline signal was reduced to 9 dB, so that the Doppler frequency spectrum just remained visible on the monitor. The contrast medium was administered with the instrument kept at this setting throughout.

The evaluation was carried out in a manner analogous to that of previous animal studies,⁹ with the maximum signal intensity being measured after every 2 heart cycles. The reference was a colored bar on the edge of the monitor display, showing the signal intensity in distinct shades of color in 3-dB steps. A signal intensity of 30 dB was chosen as the reference for the duration of the EA effect, a value distinctly higher than the signal strength required for diagnostic purposes. Because the baseline intensity before administration of the EA was set at 9 dB, the selected reference point of 30 dB corresponds to a 21-dB increase in intensity. The maximum signal intensity (in decibels) in the course of the measurement was recorded.

The units of the time axis were heart cycles. Any increase in signal intensity above 42 dB could not be quantified for technical reasons (limited dynamic range). The echocontrast effect was characterized on the basis of the parameters A₁ (duration of signal intensity 30 dB [in heart cycles] after the initial injection ) and A₂ (duration of signal intensity 30 dB [in heart cycles] after expression of the venous reservoir in the upper arm) (Figure).

The duration of signal intensity of 30 dB or above is characterized by the median and the distribution-free 68% range, which corresponds to the mean±SD in the case of a symmetrical normal distribution. The distribution-free Page test was used to check for dose dependence. In addition, the medium dose was compared with the low dose by the Wilcoxon matched-pairs signed-rank test; =0.05 (one-sided) was taken as the level of significance.

	Results

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Signal enhancement to at least 30 dB occurred in all 9 subjects even after the lowest dose of 2.5 mL BY963. The initial signal enhancement varied individually but was dose dependent (P<.001) (Table). The median values were 35, 45, and 79 heart cycles after doses of 2.5, 5, and 10 mL, respectively. In 6 subjects, subsidence of the initial contrast effect after the 10-mL dose was followed by a second signal enhancement of lower amplitude. The size of this second peak varied individually, and in 2 cases it was also observed after administration of the 5-mL dose. In 3 subjects, the signal reached more than 20 dB and in another 3, at least 30 dB. In 3 volunteers, no biphasic course was discernible after administration of 10 mL. The heart rate during the injection, the body weight, blood pressure, and sex did not have any recognizable correlation with the second intensity enhancement.

View this table: [in this window] [in a new window]   Table 1. Duration (Heart Cycles) of Doppler Signal Intensities 30 dB After IV Injection of BY963 in 9 Healthy Volunteers

In 6 volunteers, expressing remaining EA from the upper brachial venous pool after the subsidence of the initial EA effect led to a distinct contrasting of the cerebral arteries with signals of at least 30 dB (Figure, Table). In 1 further case (subject 6), the signal intensity after expression reached at least 20 dB.

	Discussion

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Within the foreseeable future, EA will become an important instrument for investigating intracranial hemodynamics, and clinical users should understand that the mode of action of EAs differs greatly from that of the familiar radiological contrast agents.

In accordance with previous studies⁷ and investigations of other organs,¹⁰ dose-dependent but individually variable echocontrast effects were observed. A biphasic increase of enhancement appeared in 6 volunteers after administration of 10 mL BY963. This differed from person to person and reached a maximum after more than 200 heart cycles. Very similar findings were observed in animal studies using a saccharide-based suspension of microbubbles.¹¹ This Doppler kinetic study showed a brief first-pass effect, which depended on dose, animal size, and cardiac output. The phase 2 kinetics were much longer and related to dose, animal size, and specific decay characteristics but were independent from cardiac output. The biphasic dose-response of BY963 in healthy volunteers follows a similar pattern and fits well to models of traditional dilution-indicator theory. According to this theory, BY963 may be understood as a blood-pool indicator with free recirculation between the arterial and venous circulation.

Characteristics of a contrast agent depend on specific properties of the compound, on individual physiology, and likely on a complex interaction of both. Properties of EAs include principles of bubble generation (ie, agitation or phase shift), solubility, pressure stability, composition of the bubble shell, and the application mode.^{12 13 14 15} Individual physiology depends on factors such as cardiac hemodynamics, resistance in the pulmonary circulation, and size of hemodynamic pools "entrapping" EA. Those pools delivering BY963 with some latency and in a more diluted concentration will contribute predominantly to phase 2 kinetics. From echocardiographic observations, it is known that it can take a fairly long time until all EA is washed out of the cardiac ventricular system. Moreover, stroking the brachial veins brought about a delivery of EA 12 to 18 minutes after the initial administration of EA. These findings prove that the cardiac chambers, as well as the venous walls and valves, may maintain a slow release additional to recirculation and provide diagnostically useful contrast effects in the cerebral circulation with longer latency.

The great variety of factors influencing the echocontrast effect may explain the individual variability seen in our volunteers. In 3 cases, a biphasic course of enhancement could not be depicted, probably because phase 1 and 2 merged into one another. There was no evident correlation with the body weight, sex, blood pressure, or the heart rate during injection. However, for more detailed analysis of these variables, a larger cohort is needed.

Our results contribute to a better understanding of the specific diagnostic effects of phospholipid-based EAs, which is a prerequisite for their possible future use in clinical neurosonology.

	Acknowledgments

 
We gratefully acknowledge the technical support of H. Frank (DWL; Sipplingen, FRG).

Received March 18, 1996; revision received July 25, 1996; accepted August 29, 1996.

	References

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