Indigo carmine (IDG) is a dye used widely in urological examinations to identify ureteral orifices, because it is freely filterable by the kidneys after intravenous (i.v.) administration . We developed a pulse dye-densitometric device1 (IDG-densitometer) which can non-invasively monitor an arterial IDG concentration in the range of 0.01-40 mg L−1 indicator-dilution technique with a precision of 0.04 ± 0.16 mg L−1 indicator-dilution technique (r = 0.953) permitting analysis of the dye-dilution curve from the beginning to the late phase.
A pulse dye densitometer using indocyanine green (ICG-densitometer) has been accepted as a new monitor of cardiac output (CO)  and circulating blood volume (CBV) [3,4] at the bedside, with minimum invasion; i.e. blood sampling for haemoglobin determination and i.v. dye injection. CO and CBV values have been in good agreement with those obtained by the gold standard methods, the thermodilution technique and the RI-method, respectively. Since IDG is less expensive than ICG, and can be injected into patients with an iodine allergy, the IDG-densitometer also seems to be promising as an alternative monitor to measure CO and CBV at the bedside.
The objective of the current experimental study was thus to validate the IDG-densitometer for the measurement of CO and CBV. We chose the thermodilution technique and the 51Cr-labelled red blood cell (RBC) method as the gold standards for CO and CBV measurements, respectively.
This study was approved by the Animal Care and Research Committee of Kawasaki Medical School (No. 00-055). After premedication with ketamine 50 mg intramuscularly (i.m.), nine adult dogs (11.2 ± 0.7 kg) were anaesthetized with pentobarbital 150 mg i.v., and the trachea was intubated. The lungs were mechanically ventilated with 30% oxygen, 70% nitrous oxide and 1-1.5% halothane. Muscle paralysis was achieved with intermittent administration of vecuronium 2 mg every hour. An arterial catheter was placed into the right femoral artery for blood sampling and arterial pressure recording. Crystalloid solution was infused, 30 mL h−1, via a catheter in the femoral vein. A pulmonary catheter (TD1504, Biosensors, USA) was placed in the pulmonary artery via the right external jugular vein for CO (COtherm) measurement with the thermodilution technique. The electrocardiogram, arterial pressure and pulmonary artery pressure were monitored during the experiment.
The principle of the IDG-densitometer is the same as that of the ICG-densitometer, which was marketed in 1997 in Japan [4,5]. We adopted the three-wavelength (620, 730 and 870 nm) method to estimate the arterial IDG concentration (see Appendix), while the ICG-densitometer uses two waves (805 and 940 nm). An optical sensor was attached either to the tongue or the auricle to measure the intensity of the light transmitted through the glossal or auricular tissue. The haemoglobin concentration was entered manually into this apparatus before starting each measurement. The apparatus calculates the arterial IDG concentration from the signals via the sensor and the haemoglobin concentration, and displays it together with the oxygen saturation (pulse oximeter) continuously. CO (COidg) and CBV (CBVidg) were calculated from the arterial dye concentration vs. time curve as described elsewhere . Briefly, COidg was obtained as I0/AUC, where I0 is the injected dose of the dye and AUC is the area under the first circulation curve vs. time extrapolated to infinity. CBVidg was obtained as I0/Cd0, where Cd0 is the initial concentration calculated by the back extrapolation of dye densitometry for 5-15 min after injection to the mean transit time of the dye [2,3].
Comparison of pulse IDG-densitometry with the thermodilution technique and 51Cr-labelled RBC method
COidg and CBVidg values were compared with those obtained by the gold standards, the thermodilution technique (COtherm) and the 51Cr-labelled RBC method (CBVRBC), respectively. COtherm was determined as the mean of the three measurements performed by injecting glucose solution 5%, 5 mL, at 0°C into the central venous port of the thermodilution catheter.
The 51Cr-labelled RBC method was performed as recommended by the International Committee for Standardization in Haematology  with some modifications. We injected 1 mL of the labelled RBC suspension (10 μCi) into the femoral vein, and collected 1 mL of blood from the catheter in the femoral artery at 10, 20 and 30 min after the injection. The radioactivity of each blood sample was counted in a well-type scintillation detector (Aloka, Japan). CBVRBC was calculated as CBVRBC = As/Cs with As: injected 51Cr-labelled RBC radioactivity (dtm); Cs: radioactivity (dtm) linearly extrapolated to the injection point from the radioactivity of the collected blood samples. When measurement of CBVRBC was repeated, background corrections were applied by measuring the residual radioactivity of the circulating blood immediately before injection of the 51Cr-labelled RBC suspension.
Animals were allowed to enter normovolaemic, hypovolaemic and hypovolaemic states in a randomized sequence. Hypervolaemic and hypovolaemic states were induced by homologous blood transfusion and exsanguination to the amount of 100-200 mL, respectively. This took about 30 min. In each state the haemodynamics were stabilized for at least 30 min before starting measurements. Arterial blood was then collected for determination of haematocrit and haemoglobin concentration. COtherm was determined by the thermodilution technique, as already described. A mixture of 8.0 mg IDG and 1 mL of 51Cr-labelled RBC suspension was then injected and flushed with 5 mL saline for determination of COidg, CBVidg and CBVRBC. These measurements were repeated in normovolaemic, hypervolaemic and hypovolaemic states in each animal.
The removal of IDG by continuous veno-venous haemofiltration using a 0.6 m2 polyacrylonitrile haemofilter (APF-06S, Asahi Medical, Japan) was determined in two anaesthetized dogs. One catheter was inserted into the right femoral artery for serial blood sampling and another into the left external jugular vein for IDG injection. Vascular access was achieved by the placement of a double-lumen catheter into the right femoral vein. Heparin was administered initially at 3 mg kg−1 and later at 1 mg kg−1 every hour for anticoagulation. Blood flow was set to 60 mL min−1. The effluent collection and post-filter infusion of acetate-based fluid replacement solution were both made at 0.50 L h−1. Beginning at 1 min before the i.v. injection of IDG 8.0 mg, successive arterial blood (2-3 mL each) and effluent samples were collected every 30 s for 11 and 21 min, respectively. Blood loss was replaced by the same volume of 6% hydroxyethyl starch solution. After 1 h, when IDG had been excreted from the circulating blood, the procedure was repeated. Blood samples were centrifuged at 1800 rpm for 10 min at 4°C for plasma separation. IDG concentrations of the plasma and effluent were determined using a spectrophotometer. The sieving coefficient (SC) was calculated as SC = Cf/Ci, where Cf is the mean concentration of IDG in the effluent and Ci, the mean concentration of IDG in plasma.
All data are shown as mean ± SD. The agreements between COidg and COtherm, and between CBVidg and CBVRBC were analysed according to Bland and Altman's recommendations .
Study 1: validation for CO and CBV
The optical sensor was attached to the tongue in seven dogs and to the auricle in two dogs. In five events, since the signals from the tongue or the auricle were not strong enough to determine the IDG concentration for 15 min, the comparisons of CO and CBV were made in 22 pairs of data. Figure 1 shows representative IDG-densitograms of the early phase (a) and the late phase (b). The recirculation peak of IDG is visible in both densitograms. The arterial IDG concentration decreased exponentially after the first circulation and became less than 1.5 mg L−1 after 15 min, as shown in Figure 1b.
Figure 2 depicts COtherm plotted against COidg. COtherm and COidg, respectively, ranged from 1.99 to 5.83 L min−1 and from 2.13 to 6.20 L min−1. These data agreed well (correlation coefficient = 0.885, P < 0.001). The difference in COtherm and COidg plotted against COtherm showed a high degree of agreement (Fig. 3). The mean difference (bias) and limits of agreement (i.e., the range of ±2 SD of the mean difference) were 0.09 and ±1.07 L min−1, respectively. The limits of agreement corresponded to 30% of COidg. There was a weak correlation between CBVidg and CBVRBC (r = 0.587, P = 0.0274) (Fig. 4). CBVidg was twice as much as CBVRBC.
Study 2: removal of IDG by continuous veno-venous haemofiltration
Eight measurements were performed in two dogs. IDG concentrations in the arterial blood and effluent samples, respectively, reached peak values in the 30-s sample and in the 7- or 8-min sample after injection. The mean arterial and effluent IDG concentrations were 1.07 ± 0.36 and 0.16 ± 0.03 mgL−1, respectively. The SC of IDG through the haemofilter was 0.34 ± 0.06.
We tried to validate pulse IDG-densitometry for CO and CBV measurements against the values obtained with the gold standard methods in this experimental study. We found that COidg agreed well with COtherm, whereas pulse IDG-densitometry overestimated the CBV value when compared with the value obtained by the 51Cr-labelled RBC method. The reliability of CBV measurement by pulse IDG-densitometry is thus questioned.
We obtained effective signals for 22 measurements of COidg and for 15 measurements of CBV among 27 IDG injections. We applied optical sensors either to the tongue or the auricle in the animals instead of the fingertip. Failure to obtain measurements was related to inadequate pulse amplitude of those sites. Since the pulse amplitude of the fingertip in human beings is great enough in most cases, we believe the failure in measurement in this animal study would not occur in clinical application.
The characteristics of IDG as an indicator for dye-dilution curves were investigated by Lacy and colleagues  as early as 1955. IDG was completely recovered in the serum when it was added to whole blood or when dialysis against serum was made. Furthermore, IDG is thought to remain in the vascular space in the first circulation to the heart and lungs, since simultaneous radio-iodinated serum albumin and IDG-dilution curves have been identical. They concluded that these features of IDG along with rapid elimination and easy quantification in blood by a spectrophotometer make it suitable as an indicator for haemodynamic studies using the dye-dilution principle. The good agreement of COidg with COtherm in the current study has confirmed their findings and shown that IDG is also a useful indicator for CO measurement using the pulse spectrophotometric principle.
There may be some debate on the use of the thermodilution technique as the gold standard of CO measurement. It is a form of indicator-dilution technique, which measures pulmonary blood flow ejected from the right ventricle, while the dye-dilution method measures systemic blood flow ejected by the left ventricle. Furthermore, the thermodilution technique is known to overestimate low CO . Thus it is not the gold standard for CO measurement in the strict sense. We used it as a gold standard for CO measurement in this study because it has an excellent correlation (r = 0.994) with ICG dye dilution  and it is widely accepted as a standard method for CO measurement in clinical practice.
Although ICG-dye densitometry is capable of precisely estimating CBV , IDG-dye densitometry overestimated CBV by 30% in this study. Similar to ICG, the IDG concentrations decreased linearly on a semi-logarithmic scale, and the initial concentration of IDG was calculated by back extrapolation to the mean transit time of the first circulation in the same way as with ICG-densitometry. We speculate that IDG distributes to the extravascular space to some extent, resulting in overestimation of CBV, whereas ICG is confined to the vascular space, which is bound to αS1-lipoproteins (MW = 200 000) in blood . The SC of IDG (0.34 ± 0.06) in this study suggests that IDG binds to proteins with relatively small molecules around 28 000 Da in the blood, because the SCs of prolactin (MW = 23 000) and α1-microglobulin have been reported to be 0.43 ± 0.13 and 0.013 ± 0.04, respectively (data from the Asahi Medical Co.). Hirszel and colleagues  showed that diffusion of macromolecules across the capillary wall increases as molecular size decreases from 43 000. The SC of IDG thus supports the extravascular distribution of IDG in the systemic circulation.
Perspective of pulse IDG-densitometry as a haemodynamic monitor
The pulse oximeter is used widely as a standard monitor in both anaesthetic care and intensive care, because of a permanent built-in calibration structure, non-invasiveness and simplicity of use. The IDG-densitometer has inherited these features and functions non-invasively except for dye injection and blood sampling for the haemoglobin concentration. ICG-densitometry estimates effective liver blood flow from analysis of the excretion curve in addition to CO and CBV measurements. Likewise, the IDG-densitometer may provide information from the elimination half-life of the dye on renal function at the bedside, because the dye is solely excreted in the urine. It does not require urine collection or repeated blood sampling and takes 10-15 min after IDG injection. We are planning to validate IDG excretion with parameters of renal function, such as creatinine clearance and renal blood flow by using the IDG-densitometer.
There have been a few case reports describing hypotension or hypertension after IDG injection. These haemodynamic changes may be mediated by an anaphylactic reaction to the dye or direct vasoconstrictive effects, respectively [13,14]. This may suggest that haemodynamic measurements and monitoring with the IDG-densitometer should be performed only in operating rooms or intensive care units.
In conclusion, our findings indicate that pulse IDG-densitometry is clinically useful for estimation of CO, but not for estimation of CBV. Leakage of IDG from the vascular bed seems to be the source of overestimation of CBV. We hope that pulse IDG-densitometry may perform additional functions to those of the existing pulse oximetry in CO measurement, and possibly in the estimation of renal function.
This work was performed in the Departments of Anaesthesiology and ICM and Urology, Kawasaki Medical School, Japan. We thank Ms S. Kawai and M. Ishikawa, students of Kawasaki College of Allied Health Professions, for their technical assistance. Nihon Kohden Co. provided the prototype IDG-densitometer for this experimental study.
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The three-wavelength method for determination of arterial IDG concentration (Cd) is described. The IDG spectrum has peak absorption at 620 nm. The ratio of optical density change (Φ12) using light at two wavelengths (λ1 = 620 nm and λ2 = 870 nm) is expressed as follows : Equation (1)
where Eoi is the absorption coefficient of oxyhaemoglobin, Edi = the absorption coefficient of IDG, Hb = the concentration of haemoglobin, Exi = the effect of tissue thickness change, i = the index of the wavelength (i = 1 or 2) and F = the scattering coefficient.
Since the absorption of IDG is less than 1/5 of that of ICG, the time dependability of Exi cannot be disregarded. Exi can be determined by using light of the third wavelength (λ3 = 730 nm), if the relation between Exis is known. As Exis are independent of wavelength, we assumed the relation between Ex1, Ex2 and Ex3 as: Equation (2)
The ratios of optical densities are expressed as: Equation (3)
Ex1, Ex2 and Ex3 are then calculated from equations 2 and 3. Cd can be computed from equation (1) by substituting Ex1 and Ex2 along with Hb.