Diagnogreen

Indocyanine green.

Diagnogreen (Indocyanine green; ICG) is a dark, bluish-green dye introduced by Fox et al. (U.S.A.) in 1957. When injected intravenously, it binds itself immediately with serum protein and it is distributed in the blood of the whole body, taken up selectively by the liver and finally excreted from the liver into the bile in free form. As Diagnogreen is practically excreted neither from entero-hepatic circulation nor from the kidney and it is practically not seen in urine, it is a suitable dye to perform examinations of hepatic and circulatory functions.
Composition and Description: (See Table 1.)

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Diagnogreen Injection: Each vial contains 25mg of indocyanine green (ICG).
Attached solvent: 10ml (one ampoule) of J. P. water for injection per vial.
Generic name: indocyanine green.
Abbre.: ICG
Description: ICG is a dark bluish-green tricarbocyanic dye. It is soluble in water (a concentration of 11.6 w/v% at 20 °C), but practically insoluble in physiological saline and salt solution. It is slightly soluble in methanol and practically insoluble in acetone.
Wave-length of the maximum absorbance: 780nm in aqueous solution, but 805nm in protein-containing solution.
Stability: Unstable in aqueous solution, but stable in protein-containing solution or in dry form.

ICG binds with serum protein (lipo-protein, albumin, etc.) and is taken selectively by the liver. It is not observed in entero-hepatic circulation and not excreted from the kidney, but is excreted from the liver into bile. So, it is suitable for liver function test by determinating the rate of retention of the dye in the blood, the rate of disappearance of the dye in plasma or by assessing hepatic blood flow.
ICG is rapidly bound to protein in blood and is optically stabilized. So, wave-length of its maximum absorption, which is 780nm in aqueous solution, changes to 805nm immediately. Since 805nm comprises isobestic point at which absorption curve of oxidized form of hemoglobin in the blood intersects that of reduced form of hemoglobin in the blood, absorption is scarcely affected by the fluctuations in oxygen saturation of the blood so long as it is determined by the wave-length, 805nm.
ICG neither deposits in the skin nor accumulates in the blood. About 97% of the amount is excreted within 20 minutes after intravenous injection.
In presence of hepatic disorders, ICG is excreted from the liver.
Toxicology: Toxicity: Acute toxicity: LD₅₀ of ICG by intravenous injection is 64.3mg/kg in mice and 87.1 mg/kg in rats.
Fetal test: No teratogenecity was noted in animal experiments using mice and rats. Any passage through placental barrier can't be seen in the pregnant women.

Liver function test (Determination of the elimination rate of the dye in the plasma, the retention rate of the dye in blood, and determination of liver blood flow).
For diagnosing and judging the prognostic cure of liver diseases.
Circulation function test (Determination of cardiac output, mean circulatory time or abnormal blood flow).
For diagnosing cardiovascular diseases.

Liver function test: Determination methods for the disappearance rate of the dye in the plasma and for rate of retention in blood: An amount equivalent to 0.5mg per kilogram of body weight as indocyanine green is diluted to a concentration of 5mg/ml with water for injection and is injected intravenously gradually into the cubitus vein within 30 seconds with careful observation of the symptoms.
Determination method for hepatic blood flow: After 25mg as indocyanine green is dissolved in as small amount of water for injections as possible, the solution is diluted to a concentration of 2.5 ~ 5mg/ml with physiological saline and an amount of this solution equivalent to 3mg as indocyanine green is injected intravenously. Then, intravenous drip infusion is performed at a constant speed until blood collection for about 50 minutes at a rate of 0.27 ~ 0.49mg/minute is completed.
Circulatory function test: Depending on the purpose, a solution of indocyanine green is injected at various vascular sites until it reaches the peripheral veins from inside the heart. Usually, the injection is via the forearm vein. The amount each time per adult is 5 ~ 10mg of indocyanine green, i.e. about 1 ~ 2ml. Doses for children are decreased in accordance with the body weight.
Principle: Liver function test: Determination method for the disappearance rate of the dye in the plasma: Indocyanine green distributes uniformly in the blood within 2 ~ 3 minutes following intravenous injection. Then, its blood level falls logarithmically for about 20 minutes thereafter. Therefore, by collecting sample of blood 2 times during the period of 5 ~ 15 minutes following injection and by determining indocyanine green content in plasma separated from the collected blood samples, disappearance rate of dye in the plasma (K) can be calculated, and the K value indicates uptake of dye in blood by the liver and the excretion function.
These values are lower in patients with liver diseases (hepato-cirrhosis, liver cancer, icterus, hepatitis, cholelithiasis, cholecystitis, the punch syndrome, portal vein disorders, etc.) than in normal persons.
Determination method for rate of retention in blood: This is a simplified method in place of the above plasma disappearance rate and is sufficient for routine tests. Indocyanine green is injected intravenously, blood is collected after 15 minutes and the retention rate
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is obtained. This
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value is higher in liver disease patients than in normal persons.
Determination method for hepatic blood flow: Indocyanine green is incorporated from the blood only by the liver and is excreted at a high rate in the bile; elimination in other tissues can be disregarded. Because there is no enterohepatic circulation of indocyanine green, measurement of the hepatic blood flow is ideal.
After indocyanine green is injected by intravenous drip infusion at a constant speed, venous blood is collected from a liver catheter and arterial blood is collected simultaneously from the artery. Various concentrations of indocyanine green in the plasma are obtained and the hepatic blood flow is measured.
Circulatory function test: Injection of indocyanine green into the blood stream and continuous recording of the changes in indocyanine green concentration in another sites will be provided a curve (an indicator dilution curve). Analysis of the curve will afford information on cardiac output, pressure of shunt and rate shunt formation so that the type and severity of the disease can be decided.
Measurement method: Liver function test: Determination method for the disappearance rate of the dye in the plasma: 1) Preparation of calibration curve: A 25mg (1 vial) of indocyanine green is diluted with distilled water to make exactly 250ml, from which 1, 2, 3, 5 and 10ml are withdrawn and are placed into 5 volumetric flasks, each with a capacity of 100ml separately. Add about 0.5ml of normal serum as stabilizer to each of the flasks, mix well and dilute with distilled water to make 100ml. A 1 ml withdrawn from each of the above solutions of varying concentrations is placed into 5 test tubes designated as A, B, C, D and E respectively.
Add 1ml of plasma (serum) and 1ml of physiological saline to each respective tube and mix well. Then, indocyanine green content in the above mixture comes to correspond to indocyanine green concentrations of 0.1, 0.2, 0.3, 0.5 and 1.0mg/dl in the blood (serum or plasma) respectively. By employing the mixture of 1ml of distilled water, 1ml of physiological saline and 1ml of plasma (serum) as blank, the absorbance of the above indocyanine green solutions of varying concentrations is determinded at wave-length of 805nm. Plot the measured values on a graph with vertical axis expressing absorbance and horizontal axis expressing concentration, and draw point-to-point linear lines to establish a calibration curve.
2) Procedures: To obtain plasma for the blank test beforehand, 3ml of blood is collected in a Spitzglasses treated with Heparin. Five, 10 and 15 minutes after intravenous injection, each 3ml of blood is collected in a Spitzglas treated with Heparin from the contralateral cubitus veins.
The blood samples are centrifuged together with the blood for the blank test, and 1 ml of plasma is separated in each case, 2.0ml of physiological saline is added and they are mixed well. Each plasma sample is measured at a wave-length of 805nm using a photoelectric spectrophotometer with plasma for the blank test as the blank. The concentrations are obtained from the calibration curves.
3) Calculation: On semilogarithmic paper with vertical axis showing concentration and horizontal axis showing time, plot 3 values measured, for instance, at 5, 10, and 15 min. and draw a line to connect these 3 plotted points. From this value, calculate the half life of indocyanine green concentration (t 1/2). Then, calculate the disappearance rate of the dye in the plasma (K) from the following equation: (See Equation 1.)

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Determination method for rate of retention in blood: 1) Preparation of calibration curves: The calibration curves are prepared in the same way as for the previously described determination method for the disappearance rate of the dye in the plasma.
2) Procedures: The procedures are the same as those for the previously described determination method for the disappearance rate of the dye in the plasma.
However, blood is collected only once 15 minutes after intravenous administration of the drug.
Calculation: Mean retention rate of indocyanine green (
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₁₅) can be calculated from the following equation: (See Equation 2.)

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Determination method for hepatic blood flow: 1) Blood sampling: 20 minutes after start of intravenous infusion, blood is withdrawn from the liver vein by means of hepatic vein catheter and also blood is withdrawn from the femoral artery. Both blood samples are placed in Spitzglasses pretreated with Heparin or Non clot-D respectively. Thus, blood collection is further repeated 5 times at 5 minutes intervals and each collected blood sample, 6 in all, is centrifuged for isolation of plasma.
2) Calculation: Indocyanine green concentration in the plasma obtained from the blood samples collected in the above manner is measured by a spectrophotometer and hepatic blood flow is obtained from the following equation. (See Equation 3.)

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Circulation function test: Preparation: When a cuvette is used, blood is aspirated from the specified location and recorded on a densitometer. When an ear piece is used, the ear is massaged well and ointment mixed with a vasodilator is applied to promote congestion. The recording is then made.
Procedures: 1) Preparation of the solution for injection: A 25mg (1 vial) of indocyanine green is to be dissolved with exactly 5ml of water for injection. The prepared solution should be used immediately and not be stored. Physiological saline is also used.
Note: When injecting indocyanine green solution into cardiac cavity, fill catheter preliminarily with the solution so that it can be pushed forward into the cavity by the success ive injection of 5 ∼ 10ml of physiological saline.
In the case of peripheral intravenous injection of indocyanine green solu tion, a thick vein should preferably be selected. As soon as the solution is injected, a respectable volume of physiological saline need to be injected to send out the indocyanine green solution to the central blood stream.
For injection of indocyanine green solution into the elbow vein, keep the upper arm fastened tightly for 10 ~ 20 seconds and then abruptly unfasten 1 ∼ 2 seconds before injection. The increased blood flow then produced will carry the dye without retardation to the central blood stream.
Injection into the femoral vein has the advantage that it allows the use of a thick injection needle and is also applicable to infants. Since blood stream at the femoral region is strong, it may be unnecessary to let indocyanine green solution to be pushed forward by the successive injection of physiological saline. However, it is necessary to inject indocyanine green solution instantly and to limit the volume of the solution to be injected to 2ml or less.
2) Record: Along with the start of indocyanine green injection, recording by ear-densitometer of the changes in light intensity as measured at wave-length of 805nm is also commenced and continued until plateau is reached to provide an indicator-dilution curve. (About 1 minute is required for recording.) When using a cuvette-densitometer, blood is withdrawn from the peripheral artery and led to the cuvette to allow similar recording.
3) Evaluation: 1. Nomenclature of various parts of indicator-dilution curves. (See Figure 1.)

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2. Determination of presence and types of diseases: a) Cardiac output: Cardiac output is calculated by the following equation: (See Equation 4.)

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b) Mean circulation time: Mean circulation time is calculated by the following equation: (See Equation 5.)

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c) Abnormal blood flow: In reviewing the dilution curve, the hump of indocyanine green concentration produced by abnormal blood flow, when compared with that by normal blood flow, will afford information on abnormal blood flow which is usually expressed in terms of rate of shunt formation (%).
Right to left shunt formation rate can be calculated from the following equation: (See Equation 6 and Figure 2.)

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Case of a left-right shunt: Left to right shunt formation rate can be calculated from the following equation.
a) Forward triangle ratio equation (tap·Cp). (See Equation 7.)

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b) Maximum indicated drug concentration ratio equation (Cp). (See Equation 8 and Figure 3.)

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Reference: (See Table 2.)

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General Precautions: ICG may rarely produce shock. It should be selected with prudence, taking into consideration the conditions of patients. For diagnosis, the following points should be noted with care.
1) Patient's condition should be sufficiently inquired to predict whether shock-like reactions will occur.
2) ICG should be dissolved completely in the attached solvent. No other solvent (physiological saline, etc.) should be used.
When incompletely dissolved ICG is injected, it may cause nausea, fever and shock­ like reactions. For good dissolution, the vial should be turned upside down several times and, in addition, it should be slightly shaked so that even a small portion of the agent adhering to the inner side of rubber stopper can be dissolved completely. Then, keep the vial horizontal and rotate it to observe the inner side of the vial and to confirm that no portions remain undissolved.
3) Prepare drugs and utensils for emergency use beforehand. (See Side Effects.)
4) Throughout the whole process of test, patients should be kept quiet, leaving them lying on his back so as to observe them sufficiently.
ICG should not be administered in the following patients: 1) Patients having antecedents of hypersensitivity to ICG.
2) Patients having iodine-hypersensitivity to iodine (As ICG contains iodine, it may produce iodine-hypersensitivity).
ICG should be administered prudently in the following patients: Patients having allergy-tendency.
Diagnostic interference: ICG may affect radioiodine uptake test of the thyroid.
Therefore, there should be, at least, over one week washout period between the two respective tests.
the ampoule with disinfectant cotton impregnated with ethanol and cut.
Cautions for diagnosis: There may be some errors in measured values in the following cases: 1) Effects of the physical condition of the patient.
Patients of chylous serum, or extremely turbid or hemolytic serum.
Edematous, emaciated or obese patients or patients who lost blood heavily [In these cases, it is advisable to perform measurement by means of methods permitting determination of disappearance rate (ex. K-method)].
2) Effects of the concomitant use of other drugs: Gall bladder contrast media (iopanoic acid, etc.), cholagogues, rifampicin, drugs against gout.
3) Effects of substances other than drugs: Effects of diet (lipemia, increased hepatic circulation, etc.)
Administration to pregnant women or nursing mothers: Since the safety of this drug for pregnant women or nursing mothers has not been established, administer to pregnant women , women who might be pregnant or nursing mothers only when the therapeutic benefits are considered to outweigh the risks.

Administration to pregnant women or nursing mothers: Since the safety of this drug for pregnant women or nursing mothers has not been established, administer to pregnant women, women who might be pregnant or nursing mothers only when the therapeutic benefits are considered to outweigh the risks.

Shock: ICG may rarely produce shock. So, sufficient observation should be exercised to check the appearance of shock symptoms. Against shock, the following counter­ measures should be taken.
1. Discontinue injection immediately when symptoms such as numbness of lips, vomit, anxiety of the breast, conjunctiva! congestion of the eye and edema of the eyelids appear.
2. In case of shock-like symptoms, emergency measures such as administrations of infusion, hypertensors, cardiotonics, adrenocortical hormones, airway maintenance, artificial respiration, oxygen inhalation, heart massage and relieving bodily posture should be made rapidly.
Iodine hypersensitivity: ICG may sometimes produce symptoms such as vomit, urticaria, nausea, and fever.

1) The solution for injection should be prepared each time it is required and should not be stored after once it is dissolved.
2) In order to avoid entry of foreign matter at the time of ampoule cutting, first clean the ampoule with disinfectant cotton impregnated with ethanol and cut.

Store below 25°C, avoiding light.
(The agent once dissolved should not be stored.)

Radiographic & Diagnostic Agents

V04CX01 - indocyanine green ; Belongs to the class of other diagnostic agents.

P₁S₁S₃

Inj (vial) 25 mg x 10's.