t least a week following the use of ic-green ® .

he performance of dye dilution curves, a known amount of dye is injected as a single bolus as rapidly as possible via a cardiac catheter into selected sites in the vascular system. a recording instrument (oximeter or densitometer) is attached to a needle or catheter for sampling of the dye-blood mixture from a systemic arterial sampling site. under sterile conditions, the ic-green ® powder should be dissolved with the sterile water for injection, usp provided for this product, and the solution used within 6 hours after it is prepared. if a precipitate is present, discard the solution. the usual doses of ic-green ® for dilution curves are as follows: adults - 5.0 mg children - 2.5 mg infants - 1.25 mg these doses of the dye are usually injected in 1 ml volume. an average of five dilution curves are recommended in the performance of a diagnostic cardiac catheterization. the total dose of dye injected should be kept below 2 mg/kg. while sterile water for injection may be used to rinse the syringe, isotonic saline should be used to flush the residual dye from the cardiac catheter into the circulation so as to avoid hemolysis. with the exception of the rinsing of the dye injection syringe, saline should be used in all other parts of the catheterization procedure. calibrating dye curves: to quantitate the dilution curves, standard dilutions of ic-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 24.0 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. 2.2 hepatic function studies due to its absorption spectrum, changing concentrations of ic-green ® (indocyanine green for injection) 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 ic-green ® powder should be dissolved with the sterile water for injection, usp provided. 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 calculated amount of dye (0.5 mg/kg of body weight) 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 ic-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 ic-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 photo cell which registers levels should be used. this device permits simultaneous measurement of cardiac output, blood volume and hepatic clearance of ic-green ® *. this technique has been employed in newborn infants, healthy adults and in children and adults with liver disease. the normal subject has a removal rate of 18 to 24% per minute. due to the absence of extra-hepatic removal, ic-green ® was found to be suited for serial study of severe chronic liver disease and to provide a stable measurement of hepatic blood flow. in larger doses, ic-green ® can be used 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. figure figure photometric method - determination using percentage retention of dye: a typical curve obtained by plotting dye concentration versus optical density is shown. the 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 ic-green ® being used in the determination can be constructed as follows: 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 should be separated by centrifugation. pipette 1 ml of the serum into a microcuvette. add 1 lambda (λ) of the 5 mg/ml aqueous ic-green ® (sterile 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 ic-green ® solution would give 100% retention; however, this concentration cannot be read on the spectrophotometer.) the optical density of this solution should be read at 805 nm, using normal serum as the blank. using graph paper similar to that used in the illustration, plot the 50% figure obtained in step 4, and draw a line connecting this point with the zero coordinates. percentage retention: a single 20-minute sample (withdrawn from a vein in the opposite arm to that injected) should be collected and allowed to clot, centrifuged and its optical density determined at 805 nm using the patient's normal serum as the blank. the dye concentration can be 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. hemolysis is not expected to 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 ic-green ® concentration in each timed specimen should be determined by using the concentration curve illustrated. values should be plotted on semilogarithmic paper. specimens containing ic-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 to 24% per minute. normal biological half-time is 2.5 to 3.0 minutes. 2.3 ophthalmic angiography studies the excitation and emission spectra (figure 1) and the absorption spectra (figure 2) of ic-green ® make it useful in ophthalmic angiography. dosages up to 40 mg ic-green ® dye in 2 ml of sterile water for injection, usp should be used, depending on the imaging equipment and technique used. the antecubital vein can be injected with an ic-green ® dye bolus and should immediately be followed by a 5 ml bolus of normal saline. figure 1 figure 2

of ic-green ® lie in a region (800 to 850 nm) where transmission of energy by the pigment epithelium is more efficient than in the region of visible light energy. ic-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. the plasma fractional disappearance rate at the recommended 0.5 mg/kg dose has been reported to be significantly greater in women than in men, although there was no significant difference in the calculated value for clearance.