aline is used in all other parts of the catheterization procedure. this matter of rinsing the dye syringe with distilled water may not be critical, since it is known that an amount of sodium chloride sufficient to make an isotonic solution may be added to dye that has first been dissolved in distilled water . this procedure has been used for constant-rate injection techniques without precipitation of the dye. the usual doses of indocyanine green which have been used for dilution curves are as follows: adults—5 mg children—2.5 mg infants—1.25 mg these doses of the dye are usually injected in a 1-ml volume. an average of five dilution curves is required in the performance of a diagnostic cardiac catheterization. the total dose of dye injected should be kept below 2 mg/kg. calibrating dye curves to quantitate the dilution curves, standard dilutions of indocyanine green in whole blood are made as follows: it is strongly recommended that the same dye that was used for the injections be used in the preparation of these standard dilutions. the most concentrated dye solution is made by accurately diluting 1 ml of the 5-mg/ml dye with 7 ml of distilled water. this concentration is then successively halved by diluting 4 ml of the previous concentration with 4 ml of distilled water. (if a 2.5 mg/ml concentration was used for the dilution curves, 1 ml of the 2.5 mg/ml dye is added to 3 ml of distilled water to make the most concentrated "standard" solution. this concentration is then successively halved by diluting 2 ml of the previous concentration with 2 ml of distilled water.) then 0.2-ml portions (accurately measured from a calibrated syringe) of these dye solutions are added to 5-ml aliquots of the subject's blood, giving final concentrations of the dye in blood beginning with 24 mg/liter, approximately (actual concentration depends on the exact volume of dye added). this concentration is, of course, successively halved in the succeeding aliquots of the subject's blood. these aliquots of blood containing known amounts of dye, as well as a blank sample to which 0.2 ml of saline containing no dye has been added, are then passed through the detecting instrument and a calibration curve is constructed from the deflections recorded. hepatic function studies due to its absorption spectrum, changing concentrations of indocyanine green in the blood can be monitored by ear densitometry or by obtaining blood specimens at timed intervals. the technique for both methods is as follows. the patient should be studied in a fasting, basal state. the patient should be weighed and the dosage calculated on the basis of 0.5 mg/kg of body weight. under sterile conditions, the indocyanine green for injection usp powder should be dissolved with sterile water for injection, usp . exactly 5 ml of sterile water for injection, usp , should be added to the 25-mg vial, giving 5 mg of dye per ml of solution. inject the correct amount of dye into the lumen of an arm vein as rapidly as possible, without allowing the dye to escape outside the vein. (if the photometric method is used, prior to injecting indocyanine green, withdraw 6 ml of venous blood from the patient's arm for serum blank and standard curve construction, and through the same needle, inject the correct amount of dye.) ear densitometry ear oximetry has also been used and makes it possible to monitor the appearance and disappearance of indocyanine green without the necessity of withdrawal and spectrophotometric analysis of blood samples for calibration. an ear densitometer which has a compensatory photo-electric cell to correct for changes in blood volume and hematocrit, and a detection photocell which registers levels has been described. this device permits simultaneous measurement of cardiac output, blood volume and hepatic clearance of indocyanine green* and was found to provide a reliable index of plasma removal kinetics after single injections or continuous infusions of indocyanine green. this technique was employed in newborn infants, healthy adults and in children and adults with liver disease. the normal subject has a removal rate of 18-24% per minute. due to the absence of extra-hepatic removal, indocyanine green was found to be ideally suited for serial study of severe chronic liver disease and to provide a stable measurement of hepatic blood flow. in larger doses, indocyanine green has proven to be particularly valuable in detecting drug-induced alterations of hepatic function and in the detection of mild liver injury. using the ear densitometer, a dosage of 0.5 mg/kg in normal subjects gives the following clearance pattern. *dichromatic earpiece densitometer supplied by the waters company, rochester, minnesota. using the ear densitometer, a dosage of 0.5 mg/kg in normal subjects gives the following clearance pattern photometric method determination using percentage retention of dye a typical curve obtained by plotting dye concentration versus optical density is shown opposite. percent retention can be read from this plot. if more accurate results are desired, a curve using the patient's blood and the vial of indocyanine green being used in the determination can be constructed as follows: 1. take 6 ml of non-dye-containing venous blood from the patient's arm. place in a test tube and allow the blood to clot. the serum is separated by centrifugation. 2. pipette l ml of the serum into a microcuvette. 3. add 1 lambda (λ) of the 5-mg/ml aqueous indocyanine green solution to the serum, giving a dilution of 5 mg/liter, the standard for 50% retention. (the addition of 2 lambda (λ) of the 5-mg/ml indocyanine green solution would give 100% retention; however, this concentration cannot be read on the spectrophotometer.) 4. the optical density of this solution is read at 805 nm, using normal serum as the blank. 5. plot the 50% figure obtained in step 4, and draw a line connecting this point with the zero coordinates. sterile indocyanine green, usp concentrations on serum following injection of 0.5 mg/kg dose percentage retention a single 20-minute sample (withdrawn from a vein in the opposite arm to that injected) is allowed to clot, centrifuged, and its optical density is determined at 805 nm using the patient's normal serum as the blank. dye concentration is read from the curve above. a single 20-minute sample of serum in healthy subjects should contain no more than 4% of the initial concentration of the dye. the use of percentage retention is less accurate than percentage disappearance rate but provides reproducible results. hemolysis does not interfere with a reading. determination using disappearance rate of dye to calculate the percentage disappearance rate, obtain samples at 5, 10, 15 and 20 minutes after injecting the dye. prepare the sample as in the previous section and measure the optical densities at 805 nm, using the patient's normal serum as the blank. the indocyanine green concentration in each timed specimen can be determined by using the concentration curve illustrated. plot values on semilogarithmic paper. specimens containing indocyanine green should be read at the same temperature since its optical density is influenced by temperature variations. normal values: percentage disappearance rate in healthy subjects is 18-24% per minute. normal biological half-time is 2.5-3.0 minutes. ophthalmic angiography studies the excitation and emission spectra (figure l) and the absorption spectra (figure 2) of indocyanine green make it useful in ophthalmic angiography. the peak absorption and emission of indocyanine green lie in a region (800-850 nm) where transmission of energy by the pigment epithelium is more efficient than in the region of visible light energy. indocyanine green also has the property of being nearly 98% bound to blood protein, and therefore, excessive dye extravasation does not take place in the highly fenestrated choroidal vasculature. it is, therefore, useful in both absorption and fluorescence infrared angiography of the choroidal vasculature when using appropriate filters and film in a fundus camera. dosages up to 40 mg indocyanine green dye in 2 ml of sterile water for injection, usp , have been found to give optimal angiograms, depending on the imaging equipment and technique used. the antecubital vein injected indocyanine green dye bolus should immediately be followed by a 5-ml bolus of normal saline. clinically, angiograms of uniformly good quality can be assured only after taking care to optimize the contributions of all possible factors, such as patient cooperation and dye injection. the foregoing injection regimen is designed to provide delivery of a spatially limited dye bolus of optimal concentration to the choroidal vasculature following intravenous injection. excitation and emission spectra of whole blood containing .05 mg/ml of sterile indocyanine green absorption spectrum spectrophotometric curve of normal serum containing 5 mg/l of sterile indocyanine green

fference in the calculated value for clearance.