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Doppler quantification of echo-contrast injections in vivo
Ultrasound in Meal & BioL Vol. 19, No. 4, pp. 269-278, 1993 Printed in the USA
0301-5629/93 $6.00 + .00 © 1993 Pergamon Press Ltd.
OOriginal Contribution DOPPLER
QUANTIFICATION INJECTIONS
OF ECHO-CONTRAST IN VIVO
CRAIG J. HARTLEY, JORGE CHEIRIF, KEVIN R . COLLIER, J. STANLEY BRAVENEC a n d JUDITH K. MICKELSON Department of Medicine, Sections of Cardiovascular Sciences and Cardiology, Baylor College of Medicine and The Veterans Administration Hospital, Houston, TX 77030, USA (Received 1 September 1992; in final form 17 December 1992) Abstract--It is difficult to quantify myocardial perfusion using contrast echocardiography because the echogenicity of injected contrast is unknown. We propose that a measurement of Doppler amplitude from blood in a systemic artery during the passage of contrast could define the needed input function. Time-amplitude curves from pulsed Doppler cuffs on coronary and carotid arteries of 7 dogs were analyzed during aortic root and left atrial injections of Albunex ®. We found in individual animals that the areas under the Doppler time-amplitude curves were correlated to the amount of Albunex ® injected (R = 0.87-0.99), inversely correlated to cardiac output (R = 0.83), and uncorrelated to coronary flow (R = 0.18). Due to better mixing, the coronary and carotid response areas correlated better for left atrial injections (R = 0.96) than for aortic root injections (R = 0.56). We conclude that Doppler amplitude detection can be used to quantify the passage of echo-contrast agents, that the measurements comply with indicator-dilution principles, and that systemic measurements in the carotid artery could be used to predict the coronary input function for injection sites with good systemic mixing.
Key Words: Ultrasound, Doppler amplitude, Contrast echocardiography, Myocardial perfusion, Indicator dilution, Sonicated albumin, Pulsed Doppler, Scattering.
INTRODUCTION
the evaluation of myocardial perfusion both in animals (Cheirifet al. 1989; Christensen et al. 1988; Kaul 1989; Keller et al. 1990; Kemper et al. 1986; Shapiro et al. 1990) and in man (Cheirifet al. 1988; Keller et al. 1988; Lim et al. 1989). This application requires knowledge of the properties, reproducibility, and stability of the contrast agent in vivo (Feinstein et al. 1991; Kaul 1989; Quinones and Cheirif 1991; Shapiro et al. 1990). Although transvenous agents are under development (Bleeker et al. 1990a; Feinstein et al. 1991), most myocardial perfusion measurements are done with aortic root injections using a variety of hand-prepared agents including: hydrogen peroxide (Kemper et al. 1986), sonicated Renografin (Cheirifet al. 1988; Keller et al. 1988), sonicated dextrose (Ten Cate et al. 1984), and sonicated albumin (Keller et al. 1989a; Shapiro et al. 1990). In addition, there are several commercially produced agents including SHU454 and SHU-508 made by Schering AG (Rovai et al. 1987) and Albunex ® made by Molecular Biosystems (Bleeker et al. 1990a, 1990b; Cheirifet al. 1992; Feinstein et al. 1991; Powsner et al. 1986), which are potentially more stable and reproducible than the handprepared agents. The method for measuring myocar-
Since their first description in the 1960s (Gramiak et al. 1969), ultrasonic contrast agents have become increasingly important in echocardiography and ultrasonic imaging in general (Feinstein et al. 1991; Ophir and Parker 1989). They are used routinely in many laboratories to enhance visualization of blood flow either by standard imaging or by color Doppler, and have improved the quantitative assessment of structural defects such as intracardiac shunts (Valdez-Cruz and Sahn 1984). In all cases, the agent must be delivered to the region of interest via catheter into a vein, an artery, or the heart itself. In these applications, the acoustic properties and reproducibility of the contrast agent are not critical to the measurement as long as the blood flow can be visualized. Contrast agents have also been applied to the measurement of tissue blood flow and especially to
Address correspondence to: Craig J. Hartley, Ph.D., Department of Medicine (CVS), Baylor Collegeof Medicine, Houston, TX 77030, USA. 269
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dial perfusion appears promising in animal experiments, and although useful in estimating myocardial perfusion in man (Cheirif et al. 1988; Keller et al. 1988; Lim et al. 1989), much improvement in the methodology is needed (Feinstein et al. 1991; Quinones and Cheirif 1991). Most of the proposed algorithms for quantifying perfusion require knowledge of the amount, echogenicity, and rate of delivery of the contrast agent to the coronary artery (Kaul 1989; Keller et al. 1989b). This is sometimes referred to as the "input function" (Feinstein et al. 1991; Kaul 1989). This may be especially critical for aortic root injections where the amount of contrast delivered to the coronary artery for a given amount injected is dependent on the adequacy of mixing in the aorta. One potential method for measuring the input function is to place a Doppler sample volume in the input vessel supplying the region of interest and measure the signal enhancement in blood as the contrast is injected. The sampling site could be the coronary artery, the ascending aorta, or any systemic artery if there has been adequate mixing to insure uniform distribution of the agent. We wanted to test this method under well-controlled conditions in vivo. The goals were to determine: 1) if Doppler amplitude detection can quantify the echogenicity of a contrast injection, 2) how the measurement varies with cardiac output and regional blood flow, and 3) how consistent aortic root injections are as measured by the amount of contrast reaching the coronary artery vs. the amount reaching a systemic artery. METHODS We chose to use Albunex ® (Molecular Biosystems, Inc, San Diego, CA, USA) for this study because of its stability and uniformity. Albunex ® consists of sonicated 5% human albumin, is nontoxic, biodegradable, stable for up to 6 months, and requires no special preparation. Others have shown it to have minimal or no effects on coronary flow and hemodynamics in animals (Powsner et al. 1986) as compared to other agents, some of which significantly depress cardiac function (Christensen et al. 1988). The backscatter and attenuation coefficients of Albunex ®were measured in vitro by Bleeker et al. (1990a, 1990b) at 5 and 7.5 MHz, and both were found to increase linearly with the number of microspheres at low concentrations. Because of the availability of transducers and to minimize interference with simultaneous imaging at 3.5 MHz, we made our measurements at 20 MHz using cuff type transducers (Hartley and Cole 1974) applied directly to coronary and carotid arteries of open chest dogs. The relatively high frequency also
Volume 19, Number 4, 1993
allowed us to sense flow close to the ultrasonic crystal, to keep the sample volume entirely within the lumen, and to obtain measurable blood echo signals for normalization. Seven male mongrel dogs were used in this study, and the protocol was approved by the Animal Protocol Review Committee of Baylor College of Medicine. The animals were anesthetized with sodium pentobarbital (30 mg/kg of body weight) and xylazine (0.5 mg/kg) intravenously. They were intubated, and ventilated with a respirator (Harvard Apparatus, South Natick, MA, USA). The chest was opened through a left thoracotomy to expose the heart which was suspended in a pericardial cradle. Catheters were placed in the femoral artery to measure arterial pressure and in the aortic root and left atrium to inject Albunex ®. Epoxy cuff type 20 MHz pulsed Doppler probes, constructed in our laboratory (Hartley and Cole 1974), were placed on the left anterior descending (LAD) and left circumflex (LCX) coronary arteries, on the right carotid artery, and in one animal on the left femoral artery. A screw type vascular occluder was placed distal to the Doppler cuff on the LCX to vary coronary flow. In two dogs, an electromagnetic flow probe (Carolina Medical Electronics, Inc., King, NC, USA) was placed on the ascending aorta to measure cardiac output. In two animals, LCX flow was reduced in steps using the screw occluder, and in another animal cardiac output was increased by administration ofdobutamine and then decreased by hypovolemia. Albunex ®was injected into either the left atrium or the aortic root in amounts ranging from 0.1 to 1.0 mL using a power injector (Medrad, Inc., Pittsburgh, PA, USA). The dose of Albunex ® was first delivered by hand through a three way stopcock into a catheter which was long enough to contain the maximum 1 mL dose. The power injector was set to deliver a 4 mL (left atrium) or 5 mL (aortic root) bolus of normal saline at 4 mL/s which pushed all of the Albunex ® into the dog and cleared the catheter. This procedure insured that the entire dose was delivered to the dog in a well-controlled way, and that the total volume of each injection was the same. In some animals, doses were repeated under similar conditions resulting in duplicate points with some clustering of the data. For consistency and to avoid any bias, all injections which were technically correct were used in the data analysis. The Doppler probes ranging in size from 2.0 to 6.0 mm diameter were connected to a multichannel pulsed Doppler system designed and built in our laboratory (Hartley et al. 1978). The amplifiers in this system are linear with no log compression, limiting, or time-gain compensation. The sample-hold circuits
Doppler quantification of contrast • C. J. HARTLEYel aL
and the pulse repetition frequency (PRF) filters limit the high frequency response of the Doppler output above 20 kHz, and the wall motion filters limit the low frequency response below 50 Hz. Between 50 Hz and 20 kHz, the system is designed to be fiat within 2 dB. Thus, for velocities between 0.25 and 100 cm/s, the amplitude of the Doppler signal should be proportional to the backscattered echo amplitude received by the crystal at 20 MHz. The 1 mm 2 crystal used in the probes coupled with the 0.4 us transmitter burst length produce a sample volume of approximately 0.5 mm 3, the position of which is movable between 1 and 10 mm from the crystal face. The sample volume can thus be placed entirely within the smallest vessel used and for each experiment was positioned to sense the maximal centerline velocity as measured by the Doppler shift. No detectable wall motion signals were present in any of the recordings. Audio Doppler signals from two of the flow sensors were recorded on a Hi-Fi video cassette recorder (JVC model HR-D470U, Elmwood Park, N J, USA) for later analysis as shown in Fig. 1. The responses to 94 aortic root and 78 left atrial injections given in varying amounts and under a variety of conditions were recorded and analyzed. For each injection we noted the amount of contrast injected, the injection site, coronary flow, and in some animals, cardiac output. For analysis, the signals were played back into a zero-crossing counter to measure the Doppler shift
(Hartley and Cole 1974) and also into an amplitude detector consisting of a precision full-wave rectifier followed by a two-pole 2 Hz low-pass filter. The frequency and amplitude signals were sent to a chart recorder (Gould Model TA-2000, Cleveland, OH, USA) for hard copy output and to a Macintosh Ilcx computer for analysis. The amplitude signals were normalized to the blood echo level before each injection (arbitrarily defined as 1 V), the baseline was subtracted, and the area under each Doppler time-amplitude curve was calculated in V. s. Normalization was done to compensate for differences in attenuation along the acoustic path, transducer sensitivity, acoustic coupling of the transducer to the vessel wall, and the gains of the amplifiers in the signal path. This allows comparison of values from different transducers and between animals. The frequency response and linearity of each element of the system shown in Fig. 1 were measured by feeding audio signals into the input and measuring the output level. For the pulsed Doppler, signals were injected before the sampling gate and ahead of any audio filters. The frequency responses for the Doppler, VCR, amplitude detector, and the total system are shown in Fig. 2. The system was found to be linear up to a Doppler audio output level of 10 V peak-to-peak. The recorder input levels were manually adjusted (no automatic level control) to - 3 0 dB on the record level meter during normal flow so that the enhancement during the passage of contrast
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would not saturate the recorder. The dynamic range of the recorder itself was measured at over 80 dB. RESULTS Doppler signals recorded from carotid and coronary arteries were played back through frequency and amplitude measurement circuits during left atrial injections of Albunex ® in the amounts shown at the bottom of Fig. 3. The injections produce amplitude response curves which resemble indicator dilution curves. The passage of contrast has little effect on the measured Doppler shift although a slight increase in coronary flow was often seen as a response to temporarily turning offthe respirator during each injection. To determine the relationship between the area under the Doppler time-amplitude (T-A) response curve and the concentration of Albunex ®, we varied the amount of Albunex ® injected into the left atrium from 0.1 to 1.0 mL. Plots of carotid and coronary T-A areas vs. the amount ofAlbunex ®injected into the left atrium of two dogs denoted by closed and open symbols are shown in Fig. 4. Linear regression equations are shown with R-values ranging from 0.87 to 0.99. The dose response for Albunex ®is well modelled by a straight line, but the slope can vary considerably between animals. According to indicator dilution theory, the Doppler T-A area (ArA) should relate to cardiac output (Q) by the equation Q = M/ArA, where M is the total backscatter of the injected bolus of Albunex ®
(Bleeker et al. 1990b; Meier and Zierler 1954). To test the effect of cardiac output on the measured response, we varied cardiac output in one animal from 0.4 to 4.0 L/min and plotted the inverse of the T-A areas in response to 1.0 m L of Albunex ® injected into the left atrium as shown in Fig. 5a. The R-value for the linear regression is 0.83, but the slope is low and the intercept is far from zero. Speculating that the weak correlation could be due to a time-dependent loss of contrast effect following injection, we estimated the loss by subtracting the femoral T-A area from the coronary T-A area. The difference was inversely related to cardiac output (R = 0.92), and when used to correct the data in Fig. 5a, resulted in the curve shown in Fig. 5b. The correction for time-dependent loss of contrast raises the slope, lowers the intercept, and improves the correlation to R = 0.95. To test the adequacy of mixing with aortic injections, we compared coronary to carotid T-A response areas for both left atrial injections and aortic root injections of varying amounts of Albunex ®. A summary of data from 3 dogs is shown in Fig. 6. Linear regression equations and R-values are shown for each curve. The T-A areas are well correlated for left atrial (R = 0.96) injections, and poorly correlated for aortic root (R = 0.56) injections. Data for one of the dogs in which we also compared LCX and LAD Doppler T-A responses to aortic root injections are shown in Fig. 7. Linear regression equations are shown with R-values of 0.98 for LCX vs. carotid T-A areas with left atrial injections, 0.65
Doppler quantification of contrast • C. J. HARTLEYet al.
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Fig. 3. Example of processed data showing Doppler frequency and amplitude from carotid and coronary arteries during left atrial injections ofAlbunex ®in the amounts shown below. The responses are similar in each vessel and appear related to the amount of Albunex ®injected. In the computer, each amplitude response was normalized by dividing it by the level of the blood signal before each injection. The baseline was then subtracted and the area under each curve was calculated in V. s.
for L C X vs. carotid T-A areas with aortic root injections, and 0.99 for L C X vs. L A D T-A areas with aortic root injections. Thus, for an individual experiment, the contrast is well mixed within the left coronary circulation even with aortic root injections. To test the effect of regional flow on the Doppler T-A response to contrast injections, we varied coronary flow over a 15-1 range in two dogs. Figure 8 shows a plot of coronary T-A area normalized to carotid T-A area vs. coronary flow as estimated by the
Doppler shift (Hartley and Cole 1974) for aortic root injections o f Albunex ®. Normalization to carotid area was done to correct for differences in the a m o u n t s of contrast injected and for differences in the dose-response between animals and allows all the data to be plotted on the same graph. These data were taken before we realized the inherently poor correlation (R = 0.56) between coronary and carotid T-A response areas for aortic root injections. Even so, all the normalized values lie between 0.6 and 1.6. The linear regres25
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Left Atrial. Injections
sion equation is shown with an R-value of 0.18 indicating that the T-A response area is independent of coronary flow over this 15-1 range.
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The curves in Fig. 3 show classic indicator dilution responses with rapid onsets and exponential decays similar to those seen with thermodilution and green dye (Meier and Zierler 1954). The amplitude and frequency seem to be largely independent with little change in measured Doppler shift during the response to contrast and little modulation of the amplitude curves due to frequency variations. The small modulation that is seen could be caused by flow approaching zero or reversing, thus bringing the frequency shift below 100 Hz transiently during the cardiac cycle, or by cyclic changes in blood echogenicity (de Kxoon et al. 1991). Note that the Doppler shift measured from the recorded signals cannot show the actual reversals in flow seen during the experiment because only one of the quadrature audio channels was recorded from each Doppler cuff. Both quadrature signals are needed to display directional Doppler recordings (Hartley and Cole 1974). Since the Albunex ® alters the amplitude but not the shape of the Doppler spectrum during its passage through coronary and carotid arteries, its distribution and velocity are similar to those of red blood cells in those vessels. Keller et al. (1989a) have shown that sonicated albumin microbubbles also mimic red
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Fig. 7. Data and linear regression equations for left circumflex (LCX) vs. carotid time-amplitude areas (A) for left atrial (a) and aortic root (b) injections and LCX vs. left anterior descending (LAD) time-amplitude areas for aortic root injections of Albunex® (c) in one dog. blood cells in the microcirculation. Thus, Albunex ® is an ideal tracer of blood flow in both large and small arteries. According to indicator dilution theory, the area under the T-A response curve measured beyond the injection site should be proportional to the concentration of the indicator, inversely related to flow at the site of mixing between the injection and measurement sites (cardiac output), the same in all vessels downstream from the mixing site, and independent o f local flow at the measurement site (Meier and Zierler 1954). Bleeker et al. (1990a) have shown that at 5 and 7.5 M H z and at low concentrations (<0.002%) the scattering cross-section ofAlbunex ® is linearly related to the n u m b e r of microbubbles in vitro (R = 0.86). Our results shown in Fig. 4 indicate that the area under the T-A response curve (a measure ofbackscatter at 20 MHz) is proportional to the a m o u n t of A1bunex ®injected in each animal (R = 0.87-0.99). However, due to other factors, the curves have greatly different slopes for each animal. One of these factors is cardiac output. Indicator dilution theory predicts (and experiments have shown) that as cardiac output increases, the indicator becomes more diluted and the response area falls (DeMaria et al. 1984; Meier and Zierler 1954). Our data plotted in Fig. 5 show that 1/T-A area increases with cardiac output (R -- 0.83), but that T-A area would be a poor predictor of cardiac output. For a 10-fold change in cardiac output, we obtained only a two-fold change in T-A area. Bleeker et al. (1990b) also found a weak correlation in vitro between p u m p flow and flow estimated from backscatter (R = 0.38) but found a good correlation when flow was estimated from attenuation (R = 0.97). They attributed the poor correlation for backscatter measurements to the fact that they (like us) did not compensate for attenuation
losses. However, because of the excellent linearity of the dose-response curves (Fig. 4), attenuation is an unlikely cause for the poor correlation in our experiments. Another possible cause is loss of contrast between the injection site (left atrium) and the measurement site (coronary artery). We noted in Fig. 5a that carotid T-A areas were on the average 20% smaller than coronary T-A areas. If the loss of contrast was related to the relative transit-times between the injection and measurement sites, the difference should increase with longer transit-times or lower cardiac outputs. An analogous problem occurs with thermodilution cardiac output measurements due to thermal diffusion or "loss of cold" (Dow 1956; Hayes et al. 1984). When we corrected the T-A area measureAortic Root Injections
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Fig. 8. Data and linear regression equation for left circumflex (LCX) coronary time-amplitude areas (A) normalized to carotid areas vs. LCX flow measured by Doppler shift in two dogs. Normalization was done to compensate for differences in the amount of contrast injected and for differences in response between animals. For the 3 mm probes used, 8 kHz of Doppler shift corresponds to a volume flow of about 90 mL/min.
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ments for loss of contrast, the correlation to cardiac output improved markedly (Fig. 5b). Additional factors which could influence this measurement are the various agents and fluids we gave and the blood volume we withdrew to alter cardiac output over such a large range. We had noted during the experiment that the dog with the closed symbols in Fig. 4 had depressed cardiac function visually with low ejection fraction and probably low cardiac output (although it was not measured). Rovai et al. (1987) have described a method to estimate ejection fraction ( E F ) from the time constant of the exponential decay of the washout phase of a time-intensity curve from the LV. Since our signals show individual cardiac cycles (Fig. 3), the calculations can be simplified. If we measure the height of two consecutive peaks (ha and ha) in the Doppler time-amplitude curve during the washout phase, then E F = (h~ - h2)/hl. Using this method and averaging over several peaks, we estimated the ejection fraction to be 15% for the closed symbols (response curves shown in Fig. 3) and 65% for the open symbols (response curves much shorter but not shown). If there were similar differences in cardiac output, this could explain the differences in the slopes of the dose-response curves shown in Fig. 4. Indicator dilution theory predicts that the area under the T-A response curve is independent of flow in the vessel where the measurement is made (Meier and Zierler 1954). Figure 8 shows that the correlation between the normalized coronary T-A area and coronary flow is indeed poor (R = 0.18). The lack of dependence on coronary flow may seem counterintuitive considering that time-intensity curves taken from myocardium are related to perfusion and thus to flow in the inlet vessel. When the blood-contrast mixture in the artery is further diluted by the tissue, the result is a time-intensity response area related to the blood volume in the tissue and to the input function (Feinstein et al. 1991; Kaul 1989) but not to perfusion. Although tissue blood volume is usually related to tissue perfusion, the relationship is not absolute and does not hold under all conditions. If there was perfect agreement between the areas under coronary and carotid ToA responses, the ratio would always be one. Figure 6 shows that coronary and carotid T-A response areas agree well for left atrial injections (R = 0.96) but not as well for aortic root injections (R = 0.56). Left atrial injections are assumed to be well mixed by the time they reach the aorta and will be uniformly distributed systemically. Aortic root injections, however, do not guarantee adequate mixing, and thus the correlation between coro-
Volume19, Number4, 1993 nary and carotid measurements is not as good. Aortic root injections do distribute evenly in the left coronary circulation as shown in Fig. 7c (R = 0.99 between LCX and LAD T-A areas). Thus, systemic sampiing by Doppler amplitude detection in the carotid artery (or perhaps in other systemic vessels) can predict the coronary input function for left atrial or more upstream injections, but may not adequately predict the coronary input function for aortic root injections. We found that with left atrial injections, coronary T-A response areas were consistently about 20% higher than carotid T-A response areas as indicated by the slope of the regression equation in Fig. 6a. We noted even larger differences (>2-1) between coronary and femoral T-A areas in the dog in which we varied cardiac output. Part of the difference is likely due to time-dependent loss of contrast as previously discussed, but attenuation may be another factor since the carotid and femoral arteries were larger than the coronary arteries (4-6 mm vs. 2-3 mm), and thus the range gate was set about 1 mm deeper. Since A1bunex ® attenuates as well as reflects ultrasound (Bleeker et al. 1990a), smaller responses would be expected from more distant sampling sites. The slope of the dose-response curves for the two animals shown in Fig. 4 varies by more than 5-1 for the carotid arteries and 3-1 for the coronary arteries. However, the maximum difference in T-A area we could produce in one animal by varying cardiac output over a 10-1 range was 2-1 as shown in Fig. 5a. There may thus be other effects which can cause large differences in the echogenicity of contrast among subjects. We normalized the T-A response areas to the echo amplitude from blood which is affected by hematocrit (Shung 1982) and several other factors (Shung et al. 1976; Sigel et al. 1982; Yaun and Shung 1988). Other possible factors are: differences in the reflectivity ofAlbunex®(Bleeker et al. 1990a), the way the contrast is injected, or differences in the injection site. The f~ct that these factors cannot easily be determined or predicted demonstrates the need to measure the input function for each injection in each subject. The data here were taken at 20 MHz for reasons of convenience and accuracy in this evaluation, but it is known that attenuation and scattering from blood, tissue, and contrast agents is a function of frequency (Bleeker et al. 1990a; Ophir and Parker 1989; Shung et al. 1976; Yaun and Shung 1988). Shung et al. (1976) showed that scattering from blood between 5 and 15 MHz obeys Rayleigh scattering theory for small particles and is dependent on the fourth power of frequency. Bleeker et al. (1990a) showed that the attenuation coefficient of Albunex ® varies in a com-
Doppler quantification of contrast • C. J. HARTLEYel al. plex way with frequency from 2 to 10 M H z with a peak between 3 and 5 M H z and that the backscatter coefficient is actually lower at 7.5 M H z than at 5 MHz. They attribute these findings to resonance which was also suggested by Ophir and Parker (1989). Therefore, because of the strong frequency dependence, Doppler signals m u s t be acquired at the same frequency used for imaging to be useful in predicting the coronary input function. There is no reason to suspect that the results described here would not be equally valid at the lower frequencies used for imaging if sufficient care was taken in recording and analyzing the signals. Even with complete knowledge of the input function, there are other difficulties in quantifying perfusion using echo-contrast injections. These include: differences in attenuation by tissues along the sound beam; attenuation by contrast along the beam; nonlinearities in signal detection and processing; regional heterogeneity in tissue echogenicity; variations in tissue blood volume independent of perfusion; trapping of larger microbubbles in the capillaries; and the various algorithms used to extract perfusion from the measured time-intensity curves (Feinstein et al. 1991). The time-dependent losses we have noted in dogs will also limit the ability to quantify perfusion and need to be studied more fully. There is also a potential for contrast methods to be applied to myocardial perfusion measurements in the animal laboratory. On an oscilloscope display of signals from an epicardial wall thickening sensor (Hartley et al. 1983), we have been able to detect the passage of sonicated Renografin (Cheirif et al. 1988) through the underlying m y o c a r d i u m following intracoronary injections. In the current study, we often saw myocardial e n h a n c e m e n t with left atrial and aortic root injections of Albunex ® from epicardial thickening sensors operating at l0 MHz. With appropriate signal processing, myocardial T - A response curves could be produced from multiple sample volumes positioned anywhere along the sound b e a m from epicardium to endocardium (Hartley et al. 199 l). Multiple transducers placed over the epicardium would allow regional m e a s u r e m e n t s at selected sites. Unlike the radio-labelled microsphere method, the signals could be analyzed in near real-time, with no radiation, and with no limit to the n u m b e r o f perfusion determinations which could be m a d e in a given experiment. The coronary Doppler T-A response curves illustrated here, and their general agreement with indicator dilution theory, demonstrate the feasibility o f this m e t h o d under animal lab conditions and would provide the needed input function directly. Echo-contrast perfu-
277
sion measurements could augment, or in some applications replace, the radio-labelled microspheres currently used to assess regional myocardial perfusion in animal experiments. CONCLUSIONS We conclude that Doppler amplitude detection can be used to quantify the passage o f contrast through an artery, that the measurements comply with indicator dilution principles, and that systemic measurements in vessels such as the carotid artery could be used to predict the coronary input function for injection sites with good systemic mixing. In addition, with left atrial or ventricular injections, it m a y be possible to estimate ejection fraction from the washout phase of the curve. Acknowledgements--The authors wish to acknowledge Molecular
Biosystems, Inc., San Diego, CA, for generously providing AIbunex®(sonicated albumin), BettyWashington for constructing the transducers, David Brown for technical assistance, and James A. Brooks for reviewingthe manuscript. This paper was supported in part by NIH Grant No. HL22512, The Veterans Administration, and The DeBakey Heart Center.
REFERENCES Bleeker, H. J.; Shung, K. K.; Barnhart, J. L. Ultrasonic characterization of Albunex, a new contrast agent. J. Acoust. Soc. Am. 87:1792-1797; 1990a. Bleeker, H. J.; Shung, K. K.; Barnhart, J. L. On the application of ultrasonic contrast agents for blood flowmetry and assessment of cardiac perfusion. J. Ultrasound Med. 9:461--471; 1990b. Cheirif, J.; Zoghbi, W. A.; Raizner, A. E.; Minor, S. T.; Winters, W. L.; Klein, M. S.; DeBauche, T. L.; Lewis,J. M.; Roberts, R.; Quinones, M. A. Assessment of myocardial perfusion in humans by contrast echocardiagraphy. I. Evaluation of regional coronary reserve by peak contrast intensity. J. Am. Coll. Cardiol. 11:735-743; 1988. Cheirif, J.; Zoghbi, W. A.; Bolli, R.; O'Neill, P. G.; Hoyt, B. D.; Quinones, M. A. Assessment of regional myocardial perfusion by contrast echocardiography.II. Detection of changes in transmural and subendocardial perfusion during dypridamole-induced hyperemia in a model of critical coronary stenosis. J. Am. Coll. Cardiol. 14:1555-1565; 1989. Cheirif, J.; Desir, R. M.; Bolli, R.; Mahmarian, J. J.; Zoghbi, W. A.; Verani, M. S.; Quinones, M. A. Relation of perfusion defects observed with myocardial contrast echocardiographyto the severity of coronary stenosis: Correlation with thallium-201 single-photon emission tomography. J. Am. Coll. Cardiol. 19:1343-1349; 1992. Christensen, C. W.; Reeves,W. C.; Holt, G. W. Intracoronary echocontrast agents: Abnormalities in myocardial function in a normal and reduced coronary perfusion model in dogs. Ultrasound Med. Biol. 14:199-211; 1988. DeMaria, A. M.; Bommer, W.; Kwan, O. L.; Riggs, K.; Smith, M.; Waters, J. In vivo correlation of thermodilution cardiac output and videodensitometric indicator-dilution curves obtained from contrast two-dimensional echocardiograms. J. Am. Coll. Cardiol. 3:999-1004; 1984. Dow, P. Estimations of cardiac output and central blood volume by dye dilution. Physiol. Rev. 36:77-102; 1956.
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Feinstein, S. B.; Voci, P.; Segil, L. J.; Harper, P. V. Contrast echocardiography. In: Marcus, M. L.; Skorton, D. J.; Schelbert, H. R.; Wolf, G. L., eds. Cardiac imaging. Philadelphia: W. B. Saunders; 1991:557-574. Gramiak, R.; Shah, P. M.; Kramer, P. H. Ultrasonic cardiography: Contrast studies in anatomy and function. Radiology 92:939948; 1969. Hartley, C. J.; Cole, J. S. An ultrasonic pulsed Doppler system for measuring blood flow in small vessels. J. Appl. Physiol. 37:626629; 1974. Hartley, C. J.; Hanley, H. G.; Lewis, R. M.; Cole, J. S. Synchronized pulsed Doppler blood flow and ultrasonic dimension measurement in conscious dogs. Ultrasound Med. Biol. 4:99-110; 1978. Hartley, C. J.; Latson, L. A.; Michael, L. H.; Seidel, C. L.; Lewis, R. M.; Entman, M. L. Doppler measurement of myocardial thickening with a single epicardial transducer. Am. J. Physiol. 245:H1066-H1072; 1983. Hartley, C. J.; Litowitz, H.; Rabinovitz, R. S.; Zhu, W.-X.; Chelly, J. E.; Michael, L. H.; Bolli, R. An ultrasonic method for measuring tissue displacement: Technical details and validation for measuring myocardial thickening. IEEE Trans. Biomed. Eng. 38:735-747; 1991. Hayes, B. E.; Will, J. A.; Zarnstorff, W. C.; Bisard, G. E. Limita~.ionsofthermodilution cardiac output measurements in the rat. Am. J. Physiol. 246:H254-H760; 1984. Kaul, S. Quantitation of regional myocardial perfusion using myocardial contrast two-dimensional echocardiography. In: Meerbaum, S.; Meltzer, R. S., eds. Myocardial contrast two-dimensional echocardiography. Dortrecht: Kluwer; 1989:117-139. Keller, M. W.; Glasheen, W.; Smucker, M. L.; Burwell, L. R.; Watson, D. D.; Kaul, S. Myocardial contrast echocardiography in humans. I. Assessment of coronary blood flow reserve. J. Am. Coll. Cardiol. 12:925-934; 1988. Keller, M. W.; Segal, S. S.; Kaul, S.; Duling, B. The behavior of sonicated albumin microbubbleswithin the microcirculation: A basis for their use during myocardial contrast echocardiography. Circ. Res. 65:458-467; 1989a. Keller, M. W.; Glasheen, W. P.; Kaul, S. Albunex: A safe and effective commercially produced agent for myocardial contrast echocardiography. J. Am. Coll. Cardiol. 13:468-483; 1989b. Keller, M. W.; Spotnitz, W. D.; Matthew, T. L.; Glasheen, W. P.; Watson, D. D.; Kaul, S. Intraoperative assessment of regional myocardial perfusion using quantitative myocardial contrast echocardiography: An experimental evaluation. J. Am. Coll. Cardiol. 16:1267-1279; 1990. Kemper, A. J.; Force, T.; Perkins, L.; Gilfoil, M.; Parisi, A. F. In
Volume 19, Number 4, 1993 vivo prediction of the transmural extent of experimental acute myocardial infarction using contrast echocardiography. J. Am. Coll. Cardiol. 8:143-149; 1986. de Kroon, M. G. M.; Slager, C. J.; Gussenhoven, W. J.; Serruys, P. W.; Roelandt, J. R. T. C.; Bom, N. Cyclic changes of blood echogenicity in high-frequency ultrasound. Ultrasound Med. Biol. 17:723-728; 1991. Lim, Y.-J.; Nanto, S.; Masuyama, T.; Kodama, K.; Ikeda, T.; Kitabatake, A.; Kamada, T. Visualization of subendocardial myocardial ischemia with myocardial contrast echocardiography in humans. Circulation 79:233-243; 1989. Meier, P.; Zierler, K. L. On the theory of the indicator-dilution method for measurement of blood flow and volume. J. Appl. Physiol. 6:731-744; 1954. Ophir, J.; Parker, K. J. Contrast agents in diagnostic ultrasound. Ultrasound Med. Biol. 15:319-333; 1989. Powsner, S. M.; Meller, M. W.; Saniie, J.; Feinstein, S. B. Quantitation of echo-contrast effects. Am. J. Physiol. Imag. 1:124-128; 1986. Quinones, M. A.; Cheirif, J. New perspectives in perfusion imaging in echocardiography. Circulation 83(Suppl. III): 104-110; 199 I. Rovai, D.; Nissen, S. E.; Elion, J.; Smith, M.; L'Abbate, A.; Kwan, O. L.; DeMaria, A. M. Contrast echo washout curves from the left ventricle: Application of basic principles of indicator-dilution theory and calculation of ejection fraction. J. Am. Coll. Cardiol. 10:125-134; 1987. Shapiro, J. R.; Reisner, S. A.; Amico, A. F.; Kelly, P. F.; Meltzer, R. S. Reproducibility of quantitative myocardial contrast echocardiography. J. Am. Coll. Cardiol. 15:602-608; 1990. Shung, K. K.; Sigelmann, R. A.; Reid, J. M. Scattering of ultrasound by blood. IEEE Trans. Biomed. Eng. 23:460-467; 1976. Shung, K. K. On the ultrasound scattering from blood as a function of hemotocrit. IEEE Trans. Sonics Ultrason. 29:327-331; 1982. Sigel, B.; Coelho, J. C. U.; Schade, S. G.; Justin, J.; Spigos, D. G. Effect of plasma proteins and temperature on echogenicity of blood. Invest. Radiol. 17:29-33; 1982. Ten Care, F. J.; Drury, J. K.; Meerbaum, S.; Noordsy, J.; Feinstein, S.; Shah, P. M. Myocardial contrast two-dimensionalechocardiography: Experimental examination at different levels of coronary flow. J. Am. Coll. Cardiol. 3:1219-1226; 1984. Valdez-Cruz, L. M.; Sahn, D. J. Ultrasonic contrast studies for the detection of cardiac shunts. J. Am. Coll. Cardiol. 3:978-985; 1984. Yaun, Y. W.; Shung, K. K. Ultrasonic backscatter from flowing whole blood. II: Dependence on frequency and fibronogen concentration. J. Acoust. Soc. Am. 84:1195-1200; 1988.
0301-5629/93 $6.00 + .00 © 1993 Pergamon Press Ltd.
OOriginal Contribution DOPPLER
QUANTIFICATION INJECTIONS
OF ECHO-CONTRAST IN VIVO
CRAIG J. HARTLEY, JORGE CHEIRIF, KEVIN R . COLLIER, J. STANLEY BRAVENEC a n d JUDITH K. MICKELSON Department of Medicine, Sections of Cardiovascular Sciences and Cardiology, Baylor College of Medicine and The Veterans Administration Hospital, Houston, TX 77030, USA (Received 1 September 1992; in final form 17 December 1992) Abstract--It is difficult to quantify myocardial perfusion using contrast echocardiography because the echogenicity of injected contrast is unknown. We propose that a measurement of Doppler amplitude from blood in a systemic artery during the passage of contrast could define the needed input function. Time-amplitude curves from pulsed Doppler cuffs on coronary and carotid arteries of 7 dogs were analyzed during aortic root and left atrial injections of Albunex ®. We found in individual animals that the areas under the Doppler time-amplitude curves were correlated to the amount of Albunex ® injected (R = 0.87-0.99), inversely correlated to cardiac output (R = 0.83), and uncorrelated to coronary flow (R = 0.18). Due to better mixing, the coronary and carotid response areas correlated better for left atrial injections (R = 0.96) than for aortic root injections (R = 0.56). We conclude that Doppler amplitude detection can be used to quantify the passage of echo-contrast agents, that the measurements comply with indicator-dilution principles, and that systemic measurements in the carotid artery could be used to predict the coronary input function for injection sites with good systemic mixing.
Key Words: Ultrasound, Doppler amplitude, Contrast echocardiography, Myocardial perfusion, Indicator dilution, Sonicated albumin, Pulsed Doppler, Scattering.
INTRODUCTION
the evaluation of myocardial perfusion both in animals (Cheirifet al. 1989; Christensen et al. 1988; Kaul 1989; Keller et al. 1990; Kemper et al. 1986; Shapiro et al. 1990) and in man (Cheirifet al. 1988; Keller et al. 1988; Lim et al. 1989). This application requires knowledge of the properties, reproducibility, and stability of the contrast agent in vivo (Feinstein et al. 1991; Kaul 1989; Quinones and Cheirif 1991; Shapiro et al. 1990). Although transvenous agents are under development (Bleeker et al. 1990a; Feinstein et al. 1991), most myocardial perfusion measurements are done with aortic root injections using a variety of hand-prepared agents including: hydrogen peroxide (Kemper et al. 1986), sonicated Renografin (Cheirifet al. 1988; Keller et al. 1988), sonicated dextrose (Ten Cate et al. 1984), and sonicated albumin (Keller et al. 1989a; Shapiro et al. 1990). In addition, there are several commercially produced agents including SHU454 and SHU-508 made by Schering AG (Rovai et al. 1987) and Albunex ® made by Molecular Biosystems (Bleeker et al. 1990a, 1990b; Cheirifet al. 1992; Feinstein et al. 1991; Powsner et al. 1986), which are potentially more stable and reproducible than the handprepared agents. The method for measuring myocar-
Since their first description in the 1960s (Gramiak et al. 1969), ultrasonic contrast agents have become increasingly important in echocardiography and ultrasonic imaging in general (Feinstein et al. 1991; Ophir and Parker 1989). They are used routinely in many laboratories to enhance visualization of blood flow either by standard imaging or by color Doppler, and have improved the quantitative assessment of structural defects such as intracardiac shunts (Valdez-Cruz and Sahn 1984). In all cases, the agent must be delivered to the region of interest via catheter into a vein, an artery, or the heart itself. In these applications, the acoustic properties and reproducibility of the contrast agent are not critical to the measurement as long as the blood flow can be visualized. Contrast agents have also been applied to the measurement of tissue blood flow and especially to
Address correspondence to: Craig J. Hartley, Ph.D., Department of Medicine (CVS), Baylor Collegeof Medicine, Houston, TX 77030, USA. 269
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dial perfusion appears promising in animal experiments, and although useful in estimating myocardial perfusion in man (Cheirif et al. 1988; Keller et al. 1988; Lim et al. 1989), much improvement in the methodology is needed (Feinstein et al. 1991; Quinones and Cheirif 1991). Most of the proposed algorithms for quantifying perfusion require knowledge of the amount, echogenicity, and rate of delivery of the contrast agent to the coronary artery (Kaul 1989; Keller et al. 1989b). This is sometimes referred to as the "input function" (Feinstein et al. 1991; Kaul 1989). This may be especially critical for aortic root injections where the amount of contrast delivered to the coronary artery for a given amount injected is dependent on the adequacy of mixing in the aorta. One potential method for measuring the input function is to place a Doppler sample volume in the input vessel supplying the region of interest and measure the signal enhancement in blood as the contrast is injected. The sampling site could be the coronary artery, the ascending aorta, or any systemic artery if there has been adequate mixing to insure uniform distribution of the agent. We wanted to test this method under well-controlled conditions in vivo. The goals were to determine: 1) if Doppler amplitude detection can quantify the echogenicity of a contrast injection, 2) how the measurement varies with cardiac output and regional blood flow, and 3) how consistent aortic root injections are as measured by the amount of contrast reaching the coronary artery vs. the amount reaching a systemic artery. METHODS We chose to use Albunex ® (Molecular Biosystems, Inc, San Diego, CA, USA) for this study because of its stability and uniformity. Albunex ® consists of sonicated 5% human albumin, is nontoxic, biodegradable, stable for up to 6 months, and requires no special preparation. Others have shown it to have minimal or no effects on coronary flow and hemodynamics in animals (Powsner et al. 1986) as compared to other agents, some of which significantly depress cardiac function (Christensen et al. 1988). The backscatter and attenuation coefficients of Albunex ®were measured in vitro by Bleeker et al. (1990a, 1990b) at 5 and 7.5 MHz, and both were found to increase linearly with the number of microspheres at low concentrations. Because of the availability of transducers and to minimize interference with simultaneous imaging at 3.5 MHz, we made our measurements at 20 MHz using cuff type transducers (Hartley and Cole 1974) applied directly to coronary and carotid arteries of open chest dogs. The relatively high frequency also
Volume 19, Number 4, 1993
allowed us to sense flow close to the ultrasonic crystal, to keep the sample volume entirely within the lumen, and to obtain measurable blood echo signals for normalization. Seven male mongrel dogs were used in this study, and the protocol was approved by the Animal Protocol Review Committee of Baylor College of Medicine. The animals were anesthetized with sodium pentobarbital (30 mg/kg of body weight) and xylazine (0.5 mg/kg) intravenously. They were intubated, and ventilated with a respirator (Harvard Apparatus, South Natick, MA, USA). The chest was opened through a left thoracotomy to expose the heart which was suspended in a pericardial cradle. Catheters were placed in the femoral artery to measure arterial pressure and in the aortic root and left atrium to inject Albunex ®. Epoxy cuff type 20 MHz pulsed Doppler probes, constructed in our laboratory (Hartley and Cole 1974), were placed on the left anterior descending (LAD) and left circumflex (LCX) coronary arteries, on the right carotid artery, and in one animal on the left femoral artery. A screw type vascular occluder was placed distal to the Doppler cuff on the LCX to vary coronary flow. In two dogs, an electromagnetic flow probe (Carolina Medical Electronics, Inc., King, NC, USA) was placed on the ascending aorta to measure cardiac output. In two animals, LCX flow was reduced in steps using the screw occluder, and in another animal cardiac output was increased by administration ofdobutamine and then decreased by hypovolemia. Albunex ®was injected into either the left atrium or the aortic root in amounts ranging from 0.1 to 1.0 mL using a power injector (Medrad, Inc., Pittsburgh, PA, USA). The dose of Albunex ® was first delivered by hand through a three way stopcock into a catheter which was long enough to contain the maximum 1 mL dose. The power injector was set to deliver a 4 mL (left atrium) or 5 mL (aortic root) bolus of normal saline at 4 mL/s which pushed all of the Albunex ® into the dog and cleared the catheter. This procedure insured that the entire dose was delivered to the dog in a well-controlled way, and that the total volume of each injection was the same. In some animals, doses were repeated under similar conditions resulting in duplicate points with some clustering of the data. For consistency and to avoid any bias, all injections which were technically correct were used in the data analysis. The Doppler probes ranging in size from 2.0 to 6.0 mm diameter were connected to a multichannel pulsed Doppler system designed and built in our laboratory (Hartley et al. 1978). The amplifiers in this system are linear with no log compression, limiting, or time-gain compensation. The sample-hold circuits
Doppler quantification of contrast • C. J. HARTLEYel aL
and the pulse repetition frequency (PRF) filters limit the high frequency response of the Doppler output above 20 kHz, and the wall motion filters limit the low frequency response below 50 Hz. Between 50 Hz and 20 kHz, the system is designed to be fiat within 2 dB. Thus, for velocities between 0.25 and 100 cm/s, the amplitude of the Doppler signal should be proportional to the backscattered echo amplitude received by the crystal at 20 MHz. The 1 mm 2 crystal used in the probes coupled with the 0.4 us transmitter burst length produce a sample volume of approximately 0.5 mm 3, the position of which is movable between 1 and 10 mm from the crystal face. The sample volume can thus be placed entirely within the smallest vessel used and for each experiment was positioned to sense the maximal centerline velocity as measured by the Doppler shift. No detectable wall motion signals were present in any of the recordings. Audio Doppler signals from two of the flow sensors were recorded on a Hi-Fi video cassette recorder (JVC model HR-D470U, Elmwood Park, N J, USA) for later analysis as shown in Fig. 1. The responses to 94 aortic root and 78 left atrial injections given in varying amounts and under a variety of conditions were recorded and analyzed. For each injection we noted the amount of contrast injected, the injection site, coronary flow, and in some animals, cardiac output. For analysis, the signals were played back into a zero-crossing counter to measure the Doppler shift
(Hartley and Cole 1974) and also into an amplitude detector consisting of a precision full-wave rectifier followed by a two-pole 2 Hz low-pass filter. The frequency and amplitude signals were sent to a chart recorder (Gould Model TA-2000, Cleveland, OH, USA) for hard copy output and to a Macintosh Ilcx computer for analysis. The amplitude signals were normalized to the blood echo level before each injection (arbitrarily defined as 1 V), the baseline was subtracted, and the area under each Doppler time-amplitude curve was calculated in V. s. Normalization was done to compensate for differences in attenuation along the acoustic path, transducer sensitivity, acoustic coupling of the transducer to the vessel wall, and the gains of the amplifiers in the signal path. This allows comparison of values from different transducers and between animals. The frequency response and linearity of each element of the system shown in Fig. 1 were measured by feeding audio signals into the input and measuring the output level. For the pulsed Doppler, signals were injected before the sampling gate and ahead of any audio filters. The frequency responses for the Doppler, VCR, amplitude detector, and the total system are shown in Fig. 2. The system was found to be linear up to a Doppler audio output level of 10 V peak-to-peak. The recorder input levels were manually adjusted (no automatic level control) to - 3 0 dB on the record level meter during normal flow so that the enhancement during the passage of contrast
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Fig. 2. Frequency response curves for the pulsed Doppler, video cassette recorder (VCR), amplitude detector (Ampl Det), and the total system from the pulsed Doppler input to the amplitude detector output.
would not saturate the recorder. The dynamic range of the recorder itself was measured at over 80 dB. RESULTS Doppler signals recorded from carotid and coronary arteries were played back through frequency and amplitude measurement circuits during left atrial injections of Albunex ® in the amounts shown at the bottom of Fig. 3. The injections produce amplitude response curves which resemble indicator dilution curves. The passage of contrast has little effect on the measured Doppler shift although a slight increase in coronary flow was often seen as a response to temporarily turning offthe respirator during each injection. To determine the relationship between the area under the Doppler time-amplitude (T-A) response curve and the concentration of Albunex ®, we varied the amount of Albunex ® injected into the left atrium from 0.1 to 1.0 mL. Plots of carotid and coronary T-A areas vs. the amount ofAlbunex ®injected into the left atrium of two dogs denoted by closed and open symbols are shown in Fig. 4. Linear regression equations are shown with R-values ranging from 0.87 to 0.99. The dose response for Albunex ®is well modelled by a straight line, but the slope can vary considerably between animals. According to indicator dilution theory, the Doppler T-A area (ArA) should relate to cardiac output (Q) by the equation Q = M/ArA, where M is the total backscatter of the injected bolus of Albunex ®
(Bleeker et al. 1990b; Meier and Zierler 1954). To test the effect of cardiac output on the measured response, we varied cardiac output in one animal from 0.4 to 4.0 L/min and plotted the inverse of the T-A areas in response to 1.0 m L of Albunex ® injected into the left atrium as shown in Fig. 5a. The R-value for the linear regression is 0.83, but the slope is low and the intercept is far from zero. Speculating that the weak correlation could be due to a time-dependent loss of contrast effect following injection, we estimated the loss by subtracting the femoral T-A area from the coronary T-A area. The difference was inversely related to cardiac output (R = 0.92), and when used to correct the data in Fig. 5a, resulted in the curve shown in Fig. 5b. The correction for time-dependent loss of contrast raises the slope, lowers the intercept, and improves the correlation to R = 0.95. To test the adequacy of mixing with aortic injections, we compared coronary to carotid T-A response areas for both left atrial injections and aortic root injections of varying amounts of Albunex ®. A summary of data from 3 dogs is shown in Fig. 6. Linear regression equations and R-values are shown for each curve. The T-A areas are well correlated for left atrial (R = 0.96) injections, and poorly correlated for aortic root (R = 0.56) injections. Data for one of the dogs in which we also compared LCX and LAD Doppler T-A responses to aortic root injections are shown in Fig. 7. Linear regression equations are shown with R-values of 0.98 for LCX vs. carotid T-A areas with left atrial injections, 0.65
Doppler quantification of contrast • C. J. HARTLEYet al.
273
O...,.r Frequency to (KHz) 0
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io-
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Fig. 3. Example of processed data showing Doppler frequency and amplitude from carotid and coronary arteries during left atrial injections ofAlbunex ®in the amounts shown below. The responses are similar in each vessel and appear related to the amount of Albunex ®injected. In the computer, each amplitude response was normalized by dividing it by the level of the blood signal before each injection. The baseline was then subtracted and the area under each curve was calculated in V. s.
for L C X vs. carotid T-A areas with aortic root injections, and 0.99 for L C X vs. L A D T-A areas with aortic root injections. Thus, for an individual experiment, the contrast is well mixed within the left coronary circulation even with aortic root injections. To test the effect of regional flow on the Doppler T-A response to contrast injections, we varied coronary flow over a 15-1 range in two dogs. Figure 8 shows a plot of coronary T-A area normalized to carotid T-A area vs. coronary flow as estimated by the
Doppler shift (Hartley and Cole 1974) for aortic root injections o f Albunex ®. Normalization to carotid area was done to correct for differences in the a m o u n t s of contrast injected and for differences in the dose-response between animals and allows all the data to be plotted on the same graph. These data were taken before we realized the inherently poor correlation (R = 0.56) between coronary and carotid T-A response areas for aortic root injections. Even so, all the normalized values lie between 0.6 and 1.6. The linear regres25
25
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Fig. 4. Data and linear regression equations for carotid (a) and coronary (b) Doppler time-amplitude areas (A) vs. the amount of Albunex ®injected into the left atrium in two dogs denoted by the closed and open symbols.
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Volume19, Number 4, 1993
Left Atrial. Injections
sion equation is shown with an R-value of 0.18 indicating that the T-A response area is independent of coronary flow over this 15-1 range.
0.2
DISCUSSION
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Fig. 5. Data and linear regression equation for the inverse of the coronary time-amplitude area (l/A) vs. cardiac output in one dog (a). Below (b) is the same data after correcting for the time-dependent loss of contrast between the injection site and the measurement site. The correction factor was estimated by subtracting femoral T-A areas from coronary T-A areas.
The curves in Fig. 3 show classic indicator dilution responses with rapid onsets and exponential decays similar to those seen with thermodilution and green dye (Meier and Zierler 1954). The amplitude and frequency seem to be largely independent with little change in measured Doppler shift during the response to contrast and little modulation of the amplitude curves due to frequency variations. The small modulation that is seen could be caused by flow approaching zero or reversing, thus bringing the frequency shift below 100 Hz transiently during the cardiac cycle, or by cyclic changes in blood echogenicity (de Kxoon et al. 1991). Note that the Doppler shift measured from the recorded signals cannot show the actual reversals in flow seen during the experiment because only one of the quadrature audio channels was recorded from each Doppler cuff. Both quadrature signals are needed to display directional Doppler recordings (Hartley and Cole 1974). Since the Albunex ® alters the amplitude but not the shape of the Doppler spectrum during its passage through coronary and carotid arteries, its distribution and velocity are similar to those of red blood cells in those vessels. Keller et al. (1989a) have shown that sonicated albumin microbubbles also mimic red
Aortic Root Injections
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Fig. 6. Data and linear regression equations for coronary vs. carotid time-amplitude areas (A) for left atrial (a) and aortic root (b) injections of varying amounts of Albunex® in three dogs denoted by open, shaded, and closed symbols.
20
Doppler quantification of contrast • C. J. HARTLEYet al. Left Ahtal InJectlom;
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Fig. 7. Data and linear regression equations for left circumflex (LCX) vs. carotid time-amplitude areas (A) for left atrial (a) and aortic root (b) injections and LCX vs. left anterior descending (LAD) time-amplitude areas for aortic root injections of Albunex® (c) in one dog. blood cells in the microcirculation. Thus, Albunex ® is an ideal tracer of blood flow in both large and small arteries. According to indicator dilution theory, the area under the T-A response curve measured beyond the injection site should be proportional to the concentration of the indicator, inversely related to flow at the site of mixing between the injection and measurement sites (cardiac output), the same in all vessels downstream from the mixing site, and independent o f local flow at the measurement site (Meier and Zierler 1954). Bleeker et al. (1990a) have shown that at 5 and 7.5 M H z and at low concentrations (<0.002%) the scattering cross-section ofAlbunex ® is linearly related to the n u m b e r of microbubbles in vitro (R = 0.86). Our results shown in Fig. 4 indicate that the area under the T-A response curve (a measure ofbackscatter at 20 MHz) is proportional to the a m o u n t of A1bunex ®injected in each animal (R = 0.87-0.99). However, due to other factors, the curves have greatly different slopes for each animal. One of these factors is cardiac output. Indicator dilution theory predicts (and experiments have shown) that as cardiac output increases, the indicator becomes more diluted and the response area falls (DeMaria et al. 1984; Meier and Zierler 1954). Our data plotted in Fig. 5 show that 1/T-A area increases with cardiac output (R -- 0.83), but that T-A area would be a poor predictor of cardiac output. For a 10-fold change in cardiac output, we obtained only a two-fold change in T-A area. Bleeker et al. (1990b) also found a weak correlation in vitro between p u m p flow and flow estimated from backscatter (R = 0.38) but found a good correlation when flow was estimated from attenuation (R = 0.97). They attributed the poor correlation for backscatter measurements to the fact that they (like us) did not compensate for attenuation
losses. However, because of the excellent linearity of the dose-response curves (Fig. 4), attenuation is an unlikely cause for the poor correlation in our experiments. Another possible cause is loss of contrast between the injection site (left atrium) and the measurement site (coronary artery). We noted in Fig. 5a that carotid T-A areas were on the average 20% smaller than coronary T-A areas. If the loss of contrast was related to the relative transit-times between the injection and measurement sites, the difference should increase with longer transit-times or lower cardiac outputs. An analogous problem occurs with thermodilution cardiac output measurements due to thermal diffusion or "loss of cold" (Dow 1956; Hayes et al. 1984). When we corrected the T-A area measureAortic Root Injections
1,6 1,4 1.2
ALCx carotid
1 0,8 0.6 0.4 0.2 y=1.16 + -0.02x R=0.18 0
1
2
3
4
5
LCX Doppler Shift
6
7
8
(kHz)
Fig. 8. Data and linear regression equation for left circumflex (LCX) coronary time-amplitude areas (A) normalized to carotid areas vs. LCX flow measured by Doppler shift in two dogs. Normalization was done to compensate for differences in the amount of contrast injected and for differences in response between animals. For the 3 mm probes used, 8 kHz of Doppler shift corresponds to a volume flow of about 90 mL/min.
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ments for loss of contrast, the correlation to cardiac output improved markedly (Fig. 5b). Additional factors which could influence this measurement are the various agents and fluids we gave and the blood volume we withdrew to alter cardiac output over such a large range. We had noted during the experiment that the dog with the closed symbols in Fig. 4 had depressed cardiac function visually with low ejection fraction and probably low cardiac output (although it was not measured). Rovai et al. (1987) have described a method to estimate ejection fraction ( E F ) from the time constant of the exponential decay of the washout phase of a time-intensity curve from the LV. Since our signals show individual cardiac cycles (Fig. 3), the calculations can be simplified. If we measure the height of two consecutive peaks (ha and ha) in the Doppler time-amplitude curve during the washout phase, then E F = (h~ - h2)/hl. Using this method and averaging over several peaks, we estimated the ejection fraction to be 15% for the closed symbols (response curves shown in Fig. 3) and 65% for the open symbols (response curves much shorter but not shown). If there were similar differences in cardiac output, this could explain the differences in the slopes of the dose-response curves shown in Fig. 4. Indicator dilution theory predicts that the area under the T-A response curve is independent of flow in the vessel where the measurement is made (Meier and Zierler 1954). Figure 8 shows that the correlation between the normalized coronary T-A area and coronary flow is indeed poor (R = 0.18). The lack of dependence on coronary flow may seem counterintuitive considering that time-intensity curves taken from myocardium are related to perfusion and thus to flow in the inlet vessel. When the blood-contrast mixture in the artery is further diluted by the tissue, the result is a time-intensity response area related to the blood volume in the tissue and to the input function (Feinstein et al. 1991; Kaul 1989) but not to perfusion. Although tissue blood volume is usually related to tissue perfusion, the relationship is not absolute and does not hold under all conditions. If there was perfect agreement between the areas under coronary and carotid ToA responses, the ratio would always be one. Figure 6 shows that coronary and carotid T-A response areas agree well for left atrial injections (R = 0.96) but not as well for aortic root injections (R = 0.56). Left atrial injections are assumed to be well mixed by the time they reach the aorta and will be uniformly distributed systemically. Aortic root injections, however, do not guarantee adequate mixing, and thus the correlation between coro-
Volume19, Number4, 1993 nary and carotid measurements is not as good. Aortic root injections do distribute evenly in the left coronary circulation as shown in Fig. 7c (R = 0.99 between LCX and LAD T-A areas). Thus, systemic sampiing by Doppler amplitude detection in the carotid artery (or perhaps in other systemic vessels) can predict the coronary input function for left atrial or more upstream injections, but may not adequately predict the coronary input function for aortic root injections. We found that with left atrial injections, coronary T-A response areas were consistently about 20% higher than carotid T-A response areas as indicated by the slope of the regression equation in Fig. 6a. We noted even larger differences (>2-1) between coronary and femoral T-A areas in the dog in which we varied cardiac output. Part of the difference is likely due to time-dependent loss of contrast as previously discussed, but attenuation may be another factor since the carotid and femoral arteries were larger than the coronary arteries (4-6 mm vs. 2-3 mm), and thus the range gate was set about 1 mm deeper. Since A1bunex ® attenuates as well as reflects ultrasound (Bleeker et al. 1990a), smaller responses would be expected from more distant sampling sites. The slope of the dose-response curves for the two animals shown in Fig. 4 varies by more than 5-1 for the carotid arteries and 3-1 for the coronary arteries. However, the maximum difference in T-A area we could produce in one animal by varying cardiac output over a 10-1 range was 2-1 as shown in Fig. 5a. There may thus be other effects which can cause large differences in the echogenicity of contrast among subjects. We normalized the T-A response areas to the echo amplitude from blood which is affected by hematocrit (Shung 1982) and several other factors (Shung et al. 1976; Sigel et al. 1982; Yaun and Shung 1988). Other possible factors are: differences in the reflectivity ofAlbunex®(Bleeker et al. 1990a), the way the contrast is injected, or differences in the injection site. The f~ct that these factors cannot easily be determined or predicted demonstrates the need to measure the input function for each injection in each subject. The data here were taken at 20 MHz for reasons of convenience and accuracy in this evaluation, but it is known that attenuation and scattering from blood, tissue, and contrast agents is a function of frequency (Bleeker et al. 1990a; Ophir and Parker 1989; Shung et al. 1976; Yaun and Shung 1988). Shung et al. (1976) showed that scattering from blood between 5 and 15 MHz obeys Rayleigh scattering theory for small particles and is dependent on the fourth power of frequency. Bleeker et al. (1990a) showed that the attenuation coefficient of Albunex ® varies in a com-
Doppler quantification of contrast • C. J. HARTLEYel al. plex way with frequency from 2 to 10 M H z with a peak between 3 and 5 M H z and that the backscatter coefficient is actually lower at 7.5 M H z than at 5 MHz. They attribute these findings to resonance which was also suggested by Ophir and Parker (1989). Therefore, because of the strong frequency dependence, Doppler signals m u s t be acquired at the same frequency used for imaging to be useful in predicting the coronary input function. There is no reason to suspect that the results described here would not be equally valid at the lower frequencies used for imaging if sufficient care was taken in recording and analyzing the signals. Even with complete knowledge of the input function, there are other difficulties in quantifying perfusion using echo-contrast injections. These include: differences in attenuation by tissues along the sound beam; attenuation by contrast along the beam; nonlinearities in signal detection and processing; regional heterogeneity in tissue echogenicity; variations in tissue blood volume independent of perfusion; trapping of larger microbubbles in the capillaries; and the various algorithms used to extract perfusion from the measured time-intensity curves (Feinstein et al. 1991). The time-dependent losses we have noted in dogs will also limit the ability to quantify perfusion and need to be studied more fully. There is also a potential for contrast methods to be applied to myocardial perfusion measurements in the animal laboratory. On an oscilloscope display of signals from an epicardial wall thickening sensor (Hartley et al. 1983), we have been able to detect the passage of sonicated Renografin (Cheirif et al. 1988) through the underlying m y o c a r d i u m following intracoronary injections. In the current study, we often saw myocardial e n h a n c e m e n t with left atrial and aortic root injections of Albunex ® from epicardial thickening sensors operating at l0 MHz. With appropriate signal processing, myocardial T - A response curves could be produced from multiple sample volumes positioned anywhere along the sound b e a m from epicardium to endocardium (Hartley et al. 199 l). Multiple transducers placed over the epicardium would allow regional m e a s u r e m e n t s at selected sites. Unlike the radio-labelled microsphere method, the signals could be analyzed in near real-time, with no radiation, and with no limit to the n u m b e r o f perfusion determinations which could be m a d e in a given experiment. The coronary Doppler T-A response curves illustrated here, and their general agreement with indicator dilution theory, demonstrate the feasibility o f this m e t h o d under animal lab conditions and would provide the needed input function directly. Echo-contrast perfu-
277
sion measurements could augment, or in some applications replace, the radio-labelled microspheres currently used to assess regional myocardial perfusion in animal experiments. CONCLUSIONS We conclude that Doppler amplitude detection can be used to quantify the passage o f contrast through an artery, that the measurements comply with indicator dilution principles, and that systemic measurements in vessels such as the carotid artery could be used to predict the coronary input function for injection sites with good systemic mixing. In addition, with left atrial or ventricular injections, it m a y be possible to estimate ejection fraction from the washout phase of the curve. Acknowledgements--The authors wish to acknowledge Molecular
Biosystems, Inc., San Diego, CA, for generously providing AIbunex®(sonicated albumin), BettyWashington for constructing the transducers, David Brown for technical assistance, and James A. Brooks for reviewingthe manuscript. This paper was supported in part by NIH Grant No. HL22512, The Veterans Administration, and The DeBakey Heart Center.
REFERENCES Bleeker, H. J.; Shung, K. K.; Barnhart, J. L. Ultrasonic characterization of Albunex, a new contrast agent. J. Acoust. Soc. Am. 87:1792-1797; 1990a. Bleeker, H. J.; Shung, K. K.; Barnhart, J. L. On the application of ultrasonic contrast agents for blood flowmetry and assessment of cardiac perfusion. J. Ultrasound Med. 9:461--471; 1990b. Cheirif, J.; Zoghbi, W. A.; Raizner, A. E.; Minor, S. T.; Winters, W. L.; Klein, M. S.; DeBauche, T. L.; Lewis,J. M.; Roberts, R.; Quinones, M. A. Assessment of myocardial perfusion in humans by contrast echocardiagraphy. I. Evaluation of regional coronary reserve by peak contrast intensity. J. Am. Coll. Cardiol. 11:735-743; 1988. Cheirif, J.; Zoghbi, W. A.; Bolli, R.; O'Neill, P. G.; Hoyt, B. D.; Quinones, M. A. Assessment of regional myocardial perfusion by contrast echocardiography.II. Detection of changes in transmural and subendocardial perfusion during dypridamole-induced hyperemia in a model of critical coronary stenosis. J. Am. Coll. Cardiol. 14:1555-1565; 1989. Cheirif, J.; Desir, R. M.; Bolli, R.; Mahmarian, J. J.; Zoghbi, W. A.; Verani, M. S.; Quinones, M. A. Relation of perfusion defects observed with myocardial contrast echocardiographyto the severity of coronary stenosis: Correlation with thallium-201 single-photon emission tomography. J. Am. Coll. Cardiol. 19:1343-1349; 1992. Christensen, C. W.; Reeves,W. C.; Holt, G. W. Intracoronary echocontrast agents: Abnormalities in myocardial function in a normal and reduced coronary perfusion model in dogs. Ultrasound Med. Biol. 14:199-211; 1988. DeMaria, A. M.; Bommer, W.; Kwan, O. L.; Riggs, K.; Smith, M.; Waters, J. In vivo correlation of thermodilution cardiac output and videodensitometric indicator-dilution curves obtained from contrast two-dimensional echocardiograms. J. Am. Coll. Cardiol. 3:999-1004; 1984. Dow, P. Estimations of cardiac output and central blood volume by dye dilution. Physiol. Rev. 36:77-102; 1956.
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Feinstein, S. B.; Voci, P.; Segil, L. J.; Harper, P. V. Contrast echocardiography. In: Marcus, M. L.; Skorton, D. J.; Schelbert, H. R.; Wolf, G. L., eds. Cardiac imaging. Philadelphia: W. B. Saunders; 1991:557-574. Gramiak, R.; Shah, P. M.; Kramer, P. H. Ultrasonic cardiography: Contrast studies in anatomy and function. Radiology 92:939948; 1969. Hartley, C. J.; Cole, J. S. An ultrasonic pulsed Doppler system for measuring blood flow in small vessels. J. Appl. Physiol. 37:626629; 1974. Hartley, C. J.; Hanley, H. G.; Lewis, R. M.; Cole, J. S. Synchronized pulsed Doppler blood flow and ultrasonic dimension measurement in conscious dogs. Ultrasound Med. Biol. 4:99-110; 1978. Hartley, C. J.; Latson, L. A.; Michael, L. H.; Seidel, C. L.; Lewis, R. M.; Entman, M. L. Doppler measurement of myocardial thickening with a single epicardial transducer. Am. J. Physiol. 245:H1066-H1072; 1983. Hartley, C. J.; Litowitz, H.; Rabinovitz, R. S.; Zhu, W.-X.; Chelly, J. E.; Michael, L. H.; Bolli, R. An ultrasonic method for measuring tissue displacement: Technical details and validation for measuring myocardial thickening. IEEE Trans. Biomed. Eng. 38:735-747; 1991. Hayes, B. E.; Will, J. A.; Zarnstorff, W. C.; Bisard, G. E. Limita~.ionsofthermodilution cardiac output measurements in the rat. Am. J. Physiol. 246:H254-H760; 1984. Kaul, S. Quantitation of regional myocardial perfusion using myocardial contrast two-dimensional echocardiography. In: Meerbaum, S.; Meltzer, R. S., eds. Myocardial contrast two-dimensional echocardiography. Dortrecht: Kluwer; 1989:117-139. Keller, M. W.; Glasheen, W.; Smucker, M. L.; Burwell, L. R.; Watson, D. D.; Kaul, S. Myocardial contrast echocardiography in humans. I. Assessment of coronary blood flow reserve. J. Am. Coll. Cardiol. 12:925-934; 1988. Keller, M. W.; Segal, S. S.; Kaul, S.; Duling, B. The behavior of sonicated albumin microbubbleswithin the microcirculation: A basis for their use during myocardial contrast echocardiography. Circ. Res. 65:458-467; 1989a. Keller, M. W.; Glasheen, W. P.; Kaul, S. Albunex: A safe and effective commercially produced agent for myocardial contrast echocardiography. J. Am. Coll. Cardiol. 13:468-483; 1989b. Keller, M. W.; Spotnitz, W. D.; Matthew, T. L.; Glasheen, W. P.; Watson, D. D.; Kaul, S. Intraoperative assessment of regional myocardial perfusion using quantitative myocardial contrast echocardiography: An experimental evaluation. J. Am. Coll. Cardiol. 16:1267-1279; 1990. Kemper, A. J.; Force, T.; Perkins, L.; Gilfoil, M.; Parisi, A. F. In
Volume 19, Number 4, 1993 vivo prediction of the transmural extent of experimental acute myocardial infarction using contrast echocardiography. J. Am. Coll. Cardiol. 8:143-149; 1986. de Kroon, M. G. M.; Slager, C. J.; Gussenhoven, W. J.; Serruys, P. W.; Roelandt, J. R. T. C.; Bom, N. Cyclic changes of blood echogenicity in high-frequency ultrasound. Ultrasound Med. Biol. 17:723-728; 1991. Lim, Y.-J.; Nanto, S.; Masuyama, T.; Kodama, K.; Ikeda, T.; Kitabatake, A.; Kamada, T. Visualization of subendocardial myocardial ischemia with myocardial contrast echocardiography in humans. Circulation 79:233-243; 1989. Meier, P.; Zierler, K. L. On the theory of the indicator-dilution method for measurement of blood flow and volume. J. Appl. Physiol. 6:731-744; 1954. Ophir, J.; Parker, K. J. Contrast agents in diagnostic ultrasound. Ultrasound Med. Biol. 15:319-333; 1989. Powsner, S. M.; Meller, M. W.; Saniie, J.; Feinstein, S. B. Quantitation of echo-contrast effects. Am. J. Physiol. Imag. 1:124-128; 1986. Quinones, M. A.; Cheirif, J. New perspectives in perfusion imaging in echocardiography. Circulation 83(Suppl. III): 104-110; 199 I. Rovai, D.; Nissen, S. E.; Elion, J.; Smith, M.; L'Abbate, A.; Kwan, O. L.; DeMaria, A. M. Contrast echo washout curves from the left ventricle: Application of basic principles of indicator-dilution theory and calculation of ejection fraction. J. Am. Coll. Cardiol. 10:125-134; 1987. Shapiro, J. R.; Reisner, S. A.; Amico, A. F.; Kelly, P. F.; Meltzer, R. S. Reproducibility of quantitative myocardial contrast echocardiography. J. Am. Coll. Cardiol. 15:602-608; 1990. Shung, K. K.; Sigelmann, R. A.; Reid, J. M. Scattering of ultrasound by blood. IEEE Trans. Biomed. Eng. 23:460-467; 1976. Shung, K. K. On the ultrasound scattering from blood as a function of hemotocrit. IEEE Trans. Sonics Ultrason. 29:327-331; 1982. Sigel, B.; Coelho, J. C. U.; Schade, S. G.; Justin, J.; Spigos, D. G. Effect of plasma proteins and temperature on echogenicity of blood. Invest. Radiol. 17:29-33; 1982. Ten Care, F. J.; Drury, J. K.; Meerbaum, S.; Noordsy, J.; Feinstein, S.; Shah, P. M. Myocardial contrast two-dimensionalechocardiography: Experimental examination at different levels of coronary flow. J. Am. Coll. Cardiol. 3:1219-1226; 1984. Valdez-Cruz, L. M.; Sahn, D. J. Ultrasonic contrast studies for the detection of cardiac shunts. J. Am. Coll. Cardiol. 3:978-985; 1984. Yaun, Y. W.; Shung, K. K. Ultrasonic backscatter from flowing whole blood. II: Dependence on frequency and fibronogen concentration. J. Acoust. Soc. Am. 84:1195-1200; 1988.