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Meeting Abstracts  |   April 2005
Transcutaneous Fluorescence Dilution Cardiac Output and Circulating Blood Volume during Hemorrhagic Hypovolemia
Author Affiliations & Notes
  • Jean-Michel I. Maarek, Dr. Eng.
    *
  • Daniel P. Holschneider, M.D.
  • Jun Yang, Ph.D.
  • Sarah N. Pniak, B.S.
    §
  • Eduardo H. Rubinstein, M.D., Ph.D.
  • * Senior Lecturer, § Research Assistant, Department of Biomedical Engineering, † Associate Professor, Departments of Cell and Neurobiology, Psychiatry, and Neurology, ‡ Research Associate, Department of Psychiatry, University of Southern California. ∥ Professor, Department of Anesthesiology and Physiology, University of California, Los Angeles, California.
Article Information
Meeting Abstracts   |   April 2005
Transcutaneous Fluorescence Dilution Cardiac Output and Circulating Blood Volume during Hemorrhagic Hypovolemia
Anesthesiology 4 2005, Vol.102, 774-782. doi:
Anesthesiology 4 2005, Vol.102, 774-782. doi:
DETERMINATION of cardiac output is standard clinical practice in the intensive care setting and in patients undergoing cardiac surgery.1,2 The thermodilution technique using a pulmonary artery catheter is considered the reference method for clinical measurement of cardiac output.3 However, the invasive nature of the pulmonary artery catheter has motivated numerous investigations of alternative, less invasive methods for determining cardiac output.4,5 We recently validated in laboratory animals a minimally invasive method for measuring cardiac output using dye dilution, in which a fluorescent signal, elicited after intravenous injection of the dye indocyanine green (ICG) is detected transcutaneously.6 Optical transcutaneous measurement of circulating ICG requires stable perfusion at the site of measurement on the body surface,7 which may be compromised during severe blood loss accompanied by peripheral vasoconstriction.8,9 Vasodilation of the skin produced by local heating counteracts, in part, the reduction in peripheral and cutaneous perfusion.10 Therefore, in principle, measurement of cardiac output by fluorescence dilution could remain reliable, even in moderately hypovolemic conditions, if local perfusion at the site of measurement was enhanced by localized heating.
In patients undergoing surgery as well as in intensive care settings, monitoring of cardiac output is an important aspect of clinical care, as is knowledge of the patient’s intravascular volume status.11 Without a practical clinical method to measure blood volume directly, surrogate measures such as the central venous pressure or the pulmonary capillary occlusion pressure have been used, but these parameters are also affected by factors unrelated to volume status.12 The analysis of ICG concentration traces to derive the circulating blood volume has been described by several authors.13–15 Although this method was initially validated using repeated16 or continuous blood sampling,13 more recent reports have used indwelling fiberoptic catheters17 or external sensors placed on the skin.15 In principle, the latter approach could also be implemented for transcutaneous sensing of ICG fluorescence.
In this study, we measured fluorescence dilution cardiac output in an animal model during baseline normovolemic conditions and during moderate hemorrhagic hypovolemia, while perfusion of the optical measurement site was accentuated by local heating. The cardiac output values were compared to reference thermodilution cardiac output estimates. In addition, we measured circulating blood volume using transcutaneous sensing of ICG fluorescence after injections of ICG. Results were compared to estimates of blood volume derived with the standard Evans Blue dilution technique.13,18 The transcutaneous sensing technique was then used to quantify changes in the circulating blood volume associated with both hypovolemia and blood reinfusion. Results of this study extend the range of applicability and usefulness of the fluorescence dilution technique.
Materials and Methods
Animal Preparation and Instrumentation
The study was approved by the Institutional Animal Care and Use Committee (University of Southern California, Los Angeles, California). Appropriate guidelines for the use of animals were observed throughout.
The animal preparation has been previously described in detail.6 Briefly, seven adult New Zealand White rabbits were maintained anesthetized with isoflurane (concentration: 1.5% during surgery, 0.8–1.0% during measurements), paralyzed with pancuronium bromide (0.1 mg · kg−1· h−1intravenous), and mechanically ventilated with pure oxygen through a tracheostomy. End-tidal partial pressure of carbon dioxide (Pco2) monitored with a capnometer (model 254; Datex, Andover, MA) was maintained at 32–36 mmHg. An 18-gauge catheter was inserted in the left axillary artery for continuous blood pressure monitoring and blood sampling. A venous catheter placed in the left axillary vein was used for fluid injections. A 4-French thermodilution balloon catheter (AI-07044; Arrow, Reading, PA) was inserted into the right femoral vein and guided until the thermistor at the catheter tip was inside the main pulmonary artery. Correct placement of the catheter was verified visually through a median sternotomy. The thermodilution catheter was connected to a cardiac output computer (Sat 2; Baxter, Irvine, CA) to measure thermodilution cardiac output (COTD). Core temperature monitored on the cardiac output computer was maintained at 40°C with heat lamps. After the surgery, the animal was turned to the prone position. The right ear was secured with adhesive tape on top of a custom-designed heater shaped to accommodate a rabbit’s ear and was heated to 40°–42°C to induce maximal vasodilation of the ear vasculature.19 
Near-infrared light from a 782-nm laser diode (SRT-F785S; Micro Laser Systems, Garden Grove, CA) modulated at 7.7 kHz was guided to the blood vessels in the midline of the heated ear (fig. 1) through an excitation optic fiber (400-μm diameter) at the center of a bifurcated multifiber optic probe (R400.7; Ocean Optics, Dunedin, FL). The optic probe was held flush in gentle contact with the skin above the central ear artery with an articulated manipulator and stayed in the same position during the entire study. Six detection fibers (400-μm diameter) surrounding the excitation fiber in the probe forwarded the detected fluorescence light to an 830-nm interferential filter (079-2230; OptoSigma, Santa Ana, CA) and a photomultiplier tube (H7732-10; Hamamatsu, Bridgewater, NJ). The photomultiplier output was amplified and demodulated with a lock-in amplifier (SR 830; Stanford Research Systems, Sunnyvale, CA), which also generated the modulation signal for the laser diode output. The wavelengths of excitation (782 nm) and detection (830 nm) were selected to maximize the intensity of the ICG fluorescence.20,21 
Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
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A four-channel analog/digital converter module (Powerlab/4SP; AD Instruments, Colorado Springs, CO) provided continuous recording and display of the arterial blood pressure, heart rate, expired carbon dioxide concentration, and the fluorescence and thermodilution curves. A computer program written in LabWindows CVI (National Instruments, Austin, TX) processed the optical signal for on-line estimation of fluorescence dilution cardiac output (COICG).
Experimental Protocol
The animal preparation was allowed to stabilize for 15–20 min to let the ear vasculature reach a stable, fully vasodilated state. The transcutaneous fluorescence intensity measured at the level of the ear was calibrated as a function of circulating blood ICG concentration as described previously.6 Briefly, a standard calibration curve was established to relate blood ICG concentration (0–2.25 μg/ml) to the fluorescence of ICG in the animal’s blood measured in a calibration cell (fig. 1). The standard calibration curve was approximated with a quadratic polynomial equation whose second-order term was less than 0 to reflect the progressively reduced fluorescence yield as the ICG concentration increased.20,22 Thereafter, a 1,000-μg dose of ICG (1 ml of 1 mg/ml in 5% dextrose solution) was rapidly infused through the distal port of the thermodilution catheter. Five arterial blood samples (1.5 ml) were withdrawn between 1 and 5 min after the ICG infusion when the ICG was homogenously mixed in the circulating blood while the transcutaneous fluorescence intensity was recorded. The blood samples were placed in the calibration cell, and their fluorescence was measured. The relation between transcutaneous fluorescence intensity and ICG fluorescence in blood was linear and passed through the origin of the axes, yielding a proportionality factor between ICG fluorescence measured in vivo  and in vitro  fluorescence in the cell. Multiplication of the quadratic equation by this proportionality factor produced the equation that was used to convert the transcutaneous ICG fluorescence signal in terms of ICG concentration in circulating blood.
Five measurements of the baseline cardiac output were obtained with the thermodilution and fluorescence dilution techniques. For each measurement, 1.5 ml iced solution containing 45 μg ICG (0.03 mg/ml in 5% dextrose solution) was bolus injected through the proximal port of the thermodilution catheter while the ventilator was stopped at end-expiration. Analysis of the ICG dilution trace also yielded circulating blood volume (BVICG) as described below. We waited 3–5 min between measurements for the transcutaneous ICG fluorescence signal to return to 0.
After the baseline readings, blood (30–50 ml) was withdrawn from the arterial catheter in a heparinized syringe until exhaled Pco2began to decrease to 28–29 mmHg, indicating that cardiac output had diminished. Ventilation was readjusted to normocapnia. Then, calibration of the ICG transcutaneous fluorescence as a function of blood ICG concentration was repeated to determine whether the calibration factor had changed with hypovolemia. Thereafter, five measurements of COICG, COTD, and BVICGwere obtained in the hypovolemia condition. Last, the blood was reinfused in the animal through the venous catheter. After a 5-min equilibration period, the calibration factor was measured a third time, after which cardiac output and circulating blood volume were again measured in quintuplicate.
Validation of Circulating Blood Volume Measurement
The capability of the ICG fluorescence dilution technique to estimate circulating blood volume (BVICG) was tested in four additional rabbits in which BVICGwas compared to estimates derived from the Evans Blue dilution technique (BVEB).13,18 These animals, prepared and instrumented as the other preparations, were only studied in baseline conditions. Three 1.5-ml injections of chilled ICG solution (0.03 mg/ml ICG in 5% dextrose solution), equivalent to approximately 13 μg/kg, were performed for triplicate measurement of COICG, COTD, and BVICG. After return of the ICG signal to baseline, a 1.5-ml arterial blood sample was drawn for measurement of central blood hematocrit and to serve as a blank for spectrophotometric measurement (Spectronic 20; Bausch & Lomb, Rochester, NY) of the plasma optical density needed for estimation of BVEB. A dose of 6 mg Evans Blue dye (1 ml of 6 mg/ml solution in 0.9% saline) was bolus injected intravenously. The 1-ml syringe containing the Evans Blue dye was flushed five times with saline within 20 s after the first dose injection. Blood samples (1.5 ml) were drawn at 90 s, 3 min, and 5 min after the Evans Blue injection and centrifuged (3,000 rpm) for preparation of 0.5-ml plasma samples. The samples were diluted with 2.5 ml distilled water before measuring their optical density at 620 nm relative to that of the blank plasma sample. To rule out major shifts of the cardiac output and blood volume associated with the Evans Blue injection and blood withdrawals, three additional ICG injections were performed after the last blood sample was obtained for repeated calculation of BVICG.
Data Analysis
Estimation of Cardiac Output.
The transcutaneous fluorescence data were converted to blood ICG concentrations using the calibration technique described previously.6 The algorithm used in conventional cardiac output computers to process thermodilution curves23 was applied to the ICG dilution traces to derive COICG, as previously reported.6 All values of COICGand COTDwere computed from single injections, as opposed to the average of replicated injections as is common in clinical practice.
Estimation of Circulating Blood Volume from ICG Injections.
After the initial ICG concentration peak, which corresponds to the first passage of the indicator bolus at the site of measurement, the intravascular ICG concentration trace typically exhibits a slow decay as the liver extracts the injected dye from circulating blood (fig. 2). The slow decay phase can be represented by a decreasing exponential function when the amount of ICG is small (<< 1 mg/kg) and the dye is assumed to be distributed in a single vascular compartment cleared through a first-order process.24 Back-extrapolation of the slow decay phase to the instant of ICG injection (time = 0) yields the theoretical concentration ([ICG]0) that would be obtained if the dye was homogeneously mixed in the entire circulating intravascular volume. Circulating blood volume BVICGis calculated as the ratio of the mass of the injected ICG divided by [ICG]0.12 
Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
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An algorithm for computing [ICG]0was devised to dynamically determine the starting point of the exponential fit (Texp), the duration of the transcutaneous fluorescence signal on which to perform the fit, and the time of injection (T0) needed to back-extrapolate the fit and estimate [ICG]0(fig. 2). To choose instant Texp, the average duration of one circulation of the dye around the vascular network (ΔTcirc) was estimated as the time interval between the first-pass concentration peak and the peak of the recirculation hump. Data from our previous study6 showed that duration ΔTcircwas approximately 12 s in rabbits. We assumed that homogeneous mixing of the ICG would be observed after a number of circulations (Ncirc), such that the exponential approximation could start Ncirc×ΔTcircs after the appearance of the dye. The concentration trace reached a stable decay trend 40–60 s after the injection of dye, such that parameter Ncircwas set at 3. We verified that use of Ncirc= 3, 4, or 5 produced comparable estimates of BVICG.
The ICG concentration returned to near the baseline 0 level at 120–180 s after dye injection. To avoid the low fluorescence signal at the trailing end of the trace, the exponential fit was applied to a segment of data lasting 45 s. Last, the instant of dye injection (T0) was assumed to occur at a time point that preceded the first-pass peak by half the circulation time (1/2ΔTcirc). This assumption considered that half the circulation time ΔTcircwas required for the dye to travel from the site of injection near the right atrium to the small systemic blood vessels in the ear, where the fluorescence detection occurred.
Estimation of Evans Blue Blood Volume.
The plasma concentration at the instant of Evans Blue injection ([EB]0) was estimated using standard methods by back-extrapolation of the experimental plasma concentrations of Evans Blue.13 BVEBwas calculated with the following formula:
where mEBis the injected mass (6 mg) of Evans Blue dye, hematocrit is the central hematocrit, and constants 0.96 and 0.9 represent correction factors for the plasma volume trapped between erythrocytes after centrifugation and for the microvascular hematocrit/central hematocrit ratio.13,25 
Statistical Analysis
Estimates of COICG, COTD, and BVICGwere analyzed with repeated-measures analysis of variance to investigate the effect of the hypovolemia and reinfusion conditions on these variables. For each animal and each condition, the five measurements obtained in that condition were arranged in random order before performing the analysis of variance to preserve the information related to intraanimal variability, while avoiding any effect of the temporal order in which the five measurements were obtained. Different randomizations were used for reordering COICG, COTD, and BVICG. The Bonferroni correction was used in post hoc  pairwise comparisons of the values obtained in the three experimental conditions.
The relation between COICGand COTDwas analyzed by linear regression analysis. Agreement between the two techniques was investigated using the approach of Sakka et al.  ,1 which is better suited to experimental designs with multiple measurements obtained in the same subject than the more conventional Bland-Altman technique.26 Specifically, the difference COICGminus COTDwas approximated with a general linear model comprising a constant term (measuring bias), a subject term (measuring interanimal variability), a condition term (measuring variability between experimental conditions), and an error term (measuring intraanimal variability). Only the main effects were considered. The general linear model analysis of the difference COICGminus COTDwas also performed after combining the data obtained in the three experimental conditions, i.e.  , considering only the bias, subject, and error terms. For visual purposes, the results of the analysis were represented with a modified Bland-Altman plot26 comprising two 95% bands. The narrower band, which reflected the difference between cardiac output measurement techniques for the average of repeated measurements in an animal, was calculated using the interanimal root mean square difference. The wider band reflecting the difference between techniques for single measurements in an animal was calculated using the total (interanimal + intraanimal) root mean square difference.
The algorithm for dynamic analysis of ICG dilution traces and estimation of BVICGwas validated in the four test animals in which the average of six values of BVICGwere compared to the Evans Blue blood volume BVEBwith a paired t  test. Values are reported as mean (± SE). In all analyses, statistical significance was set at P  < 0.05.
Results
Cardiac Output Determined by Transcutaneous Fluorescence during Baseline, Hypovolemia, and Blood Reinfusion
Estimates of COICGand COTDaveraged 455 ± 16 and 450 ± 13 ml/min, respectively, in baseline conditions. Cardiac output decreased markedly during hypovolemia (COICG= 323 ± 15 ml/min; COTD= 330 ± 13 ml/min) and recovered partially, but not entirely, after blood reinfusion (COICG= 426 ± 16 ml/min; COTD= 414 ± 11 ml/min). Similarly, mean arterial pressure decreased from 57 ± 1 mmHg at baseline to 36 ± 1 mmHg during hypovolemia. Mean arterial blood pressure was to 48 ± 2 mmHg after the blood was reinfused.
Relation between COICGand COTDin Normovolemia and Hypovolemia
Cardiac output COICGwas linearly related to COTD(fig. 3). The slope of the linear relation (1.13 ± 0.05) was slightly but significantly greater than 1.0, whereas the ordinate (−50 ± 19 ml/min) was less than 0. The variability of the differences between cardiac output estimates obtained with the ICG and thermodilution techniques primarily resided in interanimal differences (table 1). Furthermore, near-identical partitioning of this variability was obtained when the effect of the experimental condition was included in the analysis and when the condition term was not considered. Accordingly, in the former analysis, the condition term, which would have suggested an effect of the experimental condition on the variability between measurement techniques, was not significant (P  = 0.05). The average bias between techniques was also not significant (P  = 0.12). The total root mean square difference between cardiac output estimates obtained with the two techniques (45 ml/min), which represented all sources of error and bias, was approximately 10% of the average baseline cardiac output.
Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
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Table 1. Partition of the Variance between Cardiac Output Values Estimated with the Fluorescence Dilution and Reference Thermodilution Techniques 
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Table 1. Partition of the Variance between Cardiac Output Values Estimated with the Fluorescence Dilution and Reference Thermodilution Techniques 
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Cardiac output COICGwas more likely to be less than COTDwhen the mean cardiac output was in the low range (< 350 ml/min). Accordingly, the density of points below the zero line was higher toward the left side of the horizontal axis on the modified Bland-Altman plot (fig. 4), in agreement with the greater than 1 slope of the linear regression line between COICGand COTD.
Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24 The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24 The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
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Blood Volume Determined by Transcutaneous Fluorescence (BVICG)
In the validation group of animals, the average BVICG(205 ± 10 ml) was not different from the circulating blood volume obtained with the Evans Blue technique (206 ± 6 ml). The root mean square difference between blood volume measurements obtained with the two techniques was 8 ml. The measurement of BVICGwas reproducible, with a coefficient of variation between repeated measurements in the same animal of 4% (range, 2.3–5.7%).
In the main experimental group, baseline BVICGaveraged 204 ± 5 ml or 59 ± 7 ml/kg (range, 51–68 ml/kg), slightly above the reported total blood volume values for the rabbit (54 ± 4 ml/kg).27 BVICGdecreased significantly during hypovolemia (174 ± 8 ml/min) and returned to near the baseline level after blood reinfusion (191 ± 6 ml). The blood volume decrease varied between animals during the hypovolemic phase for comparable amounts of withdrawn blood (table 2).
Table 2. Blood Volume Estimates with Transcutaneous Fluorescence Dilution Technique during Baseline, Bleeding, and Blood Reinfusion 
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Table 2. Blood Volume Estimates with Transcutaneous Fluorescence Dilution Technique during Baseline, Bleeding, and Blood Reinfusion 
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Discussion
The main findings of this study are as follows: (1) The transcutaneous fluorescence dilution technique can be applied in a hemorrhagic hypovolemia model and yields estimates of COICGthat are comparable to those measured with the thermodilution technique. (2) BVICGestimated from the analysis of the slow transcutaneous fluorescence decay is nearly identical to blood volume measured with the reference Evans Blue technique and reflects blood volume changes in an acute bleeding–reinfusion model.
Comparing Cardiac Output Measured by Fluorescence Dilution and Thermodilution during Hypovolemia
The fluorescence dilution technique yielded estimates of COICGthat were strongly correlated with and nearly equal to estimates of COTDduring normovolemic baseline conditions, during hemorrhagic hypovolemia, and after blood reinfusion. Small systematic differences between the estimates produced by the two techniques remained relatively constant within each animal and independent of the volume status. Thus, hypovolemia and the ensuing decrease in peripheral perfusion pressure did not prevent the fluorescence dilution technique from yielding accurate estimates of circulating blood ICG concentration and, therefore, calculation of cardiac output.
Blood flow in the rabbit ear is characterized by an active thermovasodilatory mechanism that is modulated by the baroreflex.19,28 The rabbit ear provides a close model to the human cutaneous circulation in that skin blood flow is reduced in response to moderate reductions of cardiac output or blood pressure.10,28 In an awake rabbit model of severe hemorrhagic hypovolemia, previous work has shown that when cardiac output and blood volume decreased by approximately 50%, skin perfusion diminished by 72%, and the resistance of the skin vascular bed increased markedly.8 One aim of the current work was to determine whether the fluorescence dilution technique, which requires a stable peripheral perfusion at the site of the optical probe, would be compromised during hemorrhage. In the animals we used, the volume bled was approximately 17% of the initial total blood volume, which resulted in a 27% reduction of cardiac output. Such conditions, although associated with some peripheral vasoconstriction, were insufficient to preempt transcutaneous detection of circulating ICG fluorescence. This may have been in part because the local vasoconstrictive response to hemorrhage was blunted by vasodilatory heating of the measurement site on the ear19 and by anesthesia. Pilot studies in our laboratory (data not shown) have suggested that more severe bleeding (> 30% of the total blood volume) resulted in marked peripheral vasoconstriction that nearly suppressed ear blood flow and could not be counteracted by local heating.
The agreement between cardiac output estimates produced by the fluorescence dilution and thermodilution techniques was likely improved by recalibrating the transcutaneous fluorescence intensity measurements in terms of blood ICG concentration for each experimental condition. However, the relative change of the calibration factor6 between experimental conditions was small (average, +8%; range, −8% to +21%) relative to baseline values. This change may have originated from small local blood flow alterations or from changes in the hematocrit or blood composition that are often encountered in hemorrhagic hypovolemia models.12,29 Because the estimated cardiac output is inversely proportional to the calibration factor, keeping the same calibration factor would have mildly affected (approximately −8%) the fluorescence dilution measurements of cardiac output and circulating blood volume.
The current study did not investigate the effect of vasopressors whose vasoconstrictive effect on the peripheral circulation could greatly reduce the amount of circulating ICG detectable with a fluorescence dilution probe placed on the skin surface. We also did not test the influence of aggressive fluid resuscitation with crystalloid solution, which could change the binding of ICG to more diluted plasma proteins and modify the optical absorption of incident light and emitted fluorescence by erythrocytes.21 Such alterations could affect the calibration of the fluorescence dilution signal in terms of circulating ICG concentration and lead to discrepancies between measurements of COICGand COTD.
The linear regression analysis between COICGand COTDhad a slope of greater than 1 and an intercept of less than 0 while concurrently a slight upward trend was observed in the modified Bland-Altman plot. These results suggested a tendency for the thermodilution and dye dilution techniques to produce diverging estimates when cardiac output decreased. In such conditions, the thermodilution technique tends to overestimate cardiac output because thermal losses around the catheter increase when venous flow is slower, resulting in an artificially reduced area under the temperature deflection curve.30 States of low cardiac output also result in recirculation of the dye through rapid circulatory pathways, with a concomitant slowing of the blood flow in slow circulatory pathways such as the skin. The net effect artificially increases the area under the delayed dye dilution first-pass curve, leading to an underestimation of cardiac output.30,31 In the animals we used, ΔTcircincreased significantly from 11.7 ± 0.5 s to 13.4 ± 0.7 s in response to hemorrhage, which was evidence that the circulation of the dye was on average slower when cardiac output was reduced. Our results suggest that measurement of the ICG dilution curve by transcutaneous fluorescent dye detection follows the previously described trend for increasing discrepancy between dye dilution and other measurement techniques at low cardiac outputs.31 
The original Bland-Altman analysis requires that the difference between techniques being compared does not show a systematic trend over the range of the measurement.12,26 This condition was not satisfied in our study as shown by the greater than 1 slope of the regression line between COICGand COTD. Although the conventional Bland-Altman analysis was not used on our data, the modified Bland-Altman plot provided a useful illustration to distinguish between single-measurement variability and variability for the averages of repeated measurements.1 The modified plot also helped in recognizing systematic trends between the two cardiac output measurement techniques.
The difference between COICGand COTDvaried from animal to animal but remained relatively independent of the experimental condition. Systematic differences between animals may have had multiple origins, including experimental error in the calibration parameters of the fluorescence dilution technique6 and systematic errors related to the thermodilution technique. In a few animals, we noticed that thermodilution cardiac output was overestimated when the tip of the thermodilution catheter was inserted deep in the pulmonary artery, possibly because the thermistor was in contact with the arterial wall. We cannot exclude that the tip of the thermodilution catheter would have been in close proximity to the vessel wall in a few animals leading to a systematic error in the thermodilution measurements. Variability of the difference between COICGand COTDon repeated trials in an animal estimated from the intraanimal root mean square difference (approximately 27 ml/min; table 1) was between 6 and 8% of the cardiac output, depending on the experimental condition. Comparable levels of agreement have been reported between conventional dye dilution, thermodilution, and the Fick method.31 This observation suggests that the precision of the transcutaneous fluorescence dilution technique relative to standard techniques for cardiac output measurement was in the same range as that of conventional dye dilution.
Estimation of Circulating Blood Volume with Fluorescence Dilution Technique
Measurement of the circulating blood volume based on the analysis of the slow concentration decay of bolus-injected ICG has been validated in humans13,15,16 and experimental animals.12,14 Our study differed from previous reports in two ways. First, the ICG concentration was calculated based on the fluorescence intensity of the circulating dye measured transcutaneously with an external probe. A substantial advantage of this sensing modality over optical absorption measurements is that living tissues do not fluoresce at the near-infrared wavelengths of peak ICG fluorescence, such that there is practically no background signal in the absence of ICG.6,32 Second, previous studies used ICG doses of approximately 200–300 μg/kg, possibly to create a substantial light absorption difference over that of blood hemoglobin, whereas we injected approximately 13 μg/kg ICG. Use of the lower ICG dose was possible because we measured ICG fluorescence rather than absorption and because we used a noise-resistant optical detection scheme centered on a lock-in amplifier. The low dose of ICG quickly distributed in a rapidly circulating blood compartment, as suggested by the fact that we obtained a close match between BVICGand the reference Evans Blue blood volume BVEB, even when the exponential approximation of the ICG decay curve started soon (approximately 35 s) after the first pass peak of the dye curve. In both animals12 and humans,15 ICG can still be detected for longer than 15 min after injection of a 200- to 300-μg/kg dose, whereas the ICG dose of the current study was almost completely extracted by the liver from circulating blood after 3 min. The low amount of ICG used in our preparations conveniently allowed for COICGand BVICGto be frequently measured without interference from the previous dye injections. Using low ICG doses would be advantageous in situations that require frequent assessment of cardiac output and blood volume.
The baseline circulating blood volume in our animals, 59 ± 7 ml/kg, slightly exceeded total blood volume estimates for the rabbit,27 possibly because of the repeated 1.5-ml injections of dextrose associated with the cardiac output and blood volume measurements. The actively circulating blood volume is thought to represent approximately 90% of the total blood volume, except in severe hypovolemia, where a larger percentage of the blood volume may reside in slow exchanging compartments.12 In our animals, the difference between baseline BVICGand bleeding BVICG(approximately 30 ml) was comparable to the volume bled (35 ml) only on the average. In most animals, BVICGdecreased more after bleeding than was accounted for by the volume bled. The difference likely reflects a shift in the blood volume partition from fast to slow exchanging compartments. In addition, BVICGremained slightly below its baseline value after blood was reinfused in the animal, in part to reflect this shift, as well as to account for blood removal (approximately 10 ml) associated with the recalibration of the fluorescence signals and for fluid losses in the extravascular space.12,29 Despite these factors, circulating blood volume measured with the fluorescence dilution technique provided a convenient estimate of the circulating volume available for perfusion of vital organs, such as the liver.29 
Clinical Significance
The pulmonary artery catheter used for clinical measurement of thermodilution cardiac output has been associated in a small number of cases with severe complications related to positioning or maintenance of the catheter, including severe arrhythmias, pulmonary embolism, and catheter-related sepsis.33–35 Such complications and the invasiveness of the pulmonary artery catheter have motivated abundant research into alternative and less invasive techniques for measuring cardiac output.4,5 Assessment of the circulating blood volume provides additional information related to cardiac preload not available with the thermodilution technique and poorly reflected by traditional central pressure monitoring.17,36 If the fluorescence dilution technique can be successfully applied to a practical clinical device, it could allow for simultaneous monitoring of cardiac output and blood volume with minimal invasiveness. Knowing these variables at the same time could assist clinicians in identifying root causes of low cardiac output in surgical and critically ill patients and in deciding on a therapeutic course.7,37 The fluorescence dilution technique as implemented in this study requires much smaller doses of ICG than comparable dye dilution techniques that use optical absorption to measure the ICG concentration.5 This could be advantageous for rapid monitoring of therapeutic intervention, especially in patients with compromised hepatic function, who metabolize the dye at a slower rate than healthy individuals do.
Pilot experiments in rabbits have shown that circulating ICG is clearly detected by fluorescence dilution from the ear tissue away from the central vessels and from the nasal mucosa just above the upper lip and that the optical signal can be calibrated in terms of circulating ICG concentration to compute cardiac output (personal verbal communication, J.-M. I. M., September 2004). Circulating ICG is detectable in humans by monitoring optical absorption at the level of the finger and the nasal wing,1,5 which suggests that transcutaneous detection of ICG fluorescence is also possible. Future studies will aim at validating the fluorescence dilution technique in human subjects and at identifying accessible measurement sites where adequate perfusion can be maintained for measuring transcutaneous fluorescence during surgery and critical illness.
The applicability of the fluorescence dilution technique would be enhanced if the ICG were injected through a peripheral vein catheter. Central injections of the dye were used in this study to facilitate the comparison between fluorescence dilution and the standard thermodilution cardiac output measurements. Peripheral and central injections of the indicator have been reported to yield identical estimates of the cardiac output in humans38 and in experimental animals.39 
The authors thank Susan McCord, B.S. (Director of Software Development), and Sumit Yadav, B.S. (Programmer), from the Alfred E. Mann Institute for Biomedical Engineering at the University of Southern California, Los Angeles, California, who assisted in the development of the software for on-line estimation of the fluorescence dilution cardiac output.
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Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
Fig. 1. Schematic of the experimental setup. The laser excitation light was divided with a beam splitter and guided to the animal preparation and the calibration cell with two fiberoptic probes. The detail shows the cross section of the excitation (Ex)–detection (D) probe placed in contact with the animal ear. Identical photomultipliers (PMTs) detected the fluorescence emitted by indocyanine green (ICG) in the animal circulation and in the calibration cell. COICG= fluorescence dilution cardiac output; COTD= thermodilution cardiac output. 
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Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
Fig. 2. Analysis of indocyanine green (ICG) dilution trace used to derive circulating blood volume (BVICG). Mixing of the ICG dye in the circulating blood volume was considered complete after three circulation times (3 ×ΔTcirc). ΔTcircwas estimated from the duration between the first pass peak and the maximum of the recirculation hump. Texpmarks the beginning of the exponential approximation. Estimated injection time T0precedes the first pass peak by ΔTcirc/2 s. 
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Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
Fig. 3. Comparison of cardiac output estimated with fluorescence dilution (COICG) and thermodilution (COTD) in all animals (n = 7) and all three experimental conditions (baseline, bleeding, reinfusion). Each  point  represents a pair of simultaneous measurements in one animal. The regression line (  solid line  ) is slightly tilted upward relative to the line of identity (  dashed line  ). 
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Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24 The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
Fig. 4. Illustration of agreement between cardiac output measured with fluorescence dilution (COICG) and thermodilution (COTD) techniques using a modified Bland-Altman plot.  1,24 The  solid horizontal line  represents the mean bias (3 ml/min). The  dotted lines  correspond to limits of agreement (mean bias ± 1.96 * precision) for the averages of five repeated measurements obtained with the two techniques in a subject. The  dashed lines  correspond to limits of agreement for single measurements obtained with the two techniques. Each  point  represents a pair of simultaneous measurements in one animal. 
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Table 1. Partition of the Variance between Cardiac Output Values Estimated with the Fluorescence Dilution and Reference Thermodilution Techniques 
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Table 1. Partition of the Variance between Cardiac Output Values Estimated with the Fluorescence Dilution and Reference Thermodilution Techniques 
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Table 2. Blood Volume Estimates with Transcutaneous Fluorescence Dilution Technique during Baseline, Bleeding, and Blood Reinfusion 
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Table 2. Blood Volume Estimates with Transcutaneous Fluorescence Dilution Technique during Baseline, Bleeding, and Blood Reinfusion 
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