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Adults: Liver Function Test: For Determination of Plasma Elimination Rate and Blood Retention Rate: 0.5 mg/kg of body weight as indocyanine green is diluted to a concentration of 5 mg/mL with water for injection and is injected IV gradually into the cubitus vein within 30 sec with careful observation of the symptoms.
Principles: Determination of Plasma Elimination Rate: After IV injection, indocyanine green is uniformly distributed in the blood within 2-3 min and its blood concentration then decreases in an exponential manner for about 20 min. Therefore, for a period of 5-15 min after injection, the blood samples should be collected twice or more. After plasma separation, the concentration of indocyanine green is determined to obtain the plasma indocyanine green elimination rate constant (K).
Constant K, which represents the blood pigment uptake and excretory function in the liver, is lower in patients with hepatic diseases (liver cirrhosis, liver cancer, jaundice, hepatitis, gallstone, cholecystitis, Banti’s syndrome, portal disorder) than in healthy individuals.
Determination of Blood Retention Rate: This simple method is sufficiently effective and can be used in place of determination of plasma elimination rate method for measuring the plasma elimination rate in routine examinations. Fifteen (15) min after IV injection of indocyanine green, the blood sample is collected and the retention rate is calculated. The retention rate is higher in patients with various liver diseases than in healthy individuals.
Adults: Chorioretinal Angiography: 25 mg is dissolved in 2 mL of water for injection and immediately injected usually via the cubital vein.
Principle and Findings of the Test: Indocyanine green has maximum blood absorption and fluorescence wavelengths in the near infrared region. Wavelengths in the near infrared range easily reach retinal pigment epithelium and then choroid membrane, and indocyanine green in the choroid membrane is excited to cause fluorescence. Therefore, indocyanine green has high permeability not only to retinal pigment epithelium and macular xanthophyll but to subretinal serous fluid, haemorrhage and exudate.
In addition, since indocyanine green fundus angiography has excitation light and fluorescence in the near infrared region, a special fundus camera is required for test. The following symptoms are observed from early to later period of angiography depending on the elapsed time after the administration.
Choroidal Arterial Phase: In choroidal angiography of the posterior fundus, imaging begins when indocyanine green is flown into short posterior ciliary artery, and the starting times of choroidal arteriography in each controlled area are slightly different. Thereafter, indocyanine green is immediately transferred to choroidal capillaries via small choroidal artery.
Choroidal Arteriovenous Phase: Then, indocyanine green rapidly appears in the choroidal venous system, and choroidal fluorescence reaches maximum intensity in the medium and large choroidal veins within 3-5 sec after injection.
Choroidal Venous Phase: Thereafter, fluorescence in the choroidal artery is decreased, and that in the choroidal venous vessels predominates for 10-15 min after injection.
Elimination Phase in Choroid Membrane: Eventually the pigment disappears from the medium and large choroidal veins, and diffuse background fluorescence is found in the choroidal membrane. At this time, low fluorescence is observed in large choroidal vessels and retinal vessels.
Positioning of Indocyanine Green Fundus Angiography Compared with Fluorescein Fundus Angiography: For the diagnosis of chorioretinal diseases, clinical conditions and affected areas should be clarified in order to confirm the diagnosis and decide therapeutic policy, and thus, fundus angiography is required. In cases where chorioretinal disease is suspected from ophthalmoscopic findings, fluorescein fundus angiography is usually conducted to clarify the affected areas first, then followed by indocyanine green fundus angiography if needed. During a follow-up period after the 1st test, however, only indocyanine green fundus angiography may be conducted.
Determination of Plasma Elimination Rate Method: Preparation of a Calibration Curve: Dissolve 25 mg or 1 vial of Diagnogreen in distilled water and adjust the volume accurately to 250 mL. Quickly take 1, 2, 3, 5 and 10 mL of this solution in five 100 mL measuring flasks, respectively. Quickly add about 0.5 mL of normal serum to them for stabilization. After mixing, dilute the mixed solutions with distilled water and adjust the total volume of the respective solutions to 100 mL. Take 1 mL of each of the 5 mixed solutions in the 5 test tubes (A, B, C, D, and E). Add 1 mL of plasma (serum) and 1 mL of physiological saline to each tube and mix sufficiently. (The solutions prepared as mentioned are equivalent to the blood (plasma/serum) specimens containing indocyanine green at the rates of 0.1, 0.2, 0.3, 0.5 and 1 mg/dL, respectively). Add 1 mL of physiological saline and 1 mL of plasma (serum) to 1 mL of distilled water. Use this solution as a blank and measure the absorbance of the solutions in the above concentrations on a wavelength of 805 nanometer. Use graph paper to plot the actual measurement values by taking the absorbance along the vertical axis and the concentration along the horizontal axis. Connect these points with a straight line and draw a calibration curve. Update the calibration curve at least every 3 months.
Operation: Collect 3 mL of blood in a spit tube treated with sodium bisulfite-free heparin to obtain the plasma for the blank test in advance. 5, 10 and 15 min after IV injection of Diagnogreen, collect 3 mL of blood from 1 side of cubital vein in a spit tube treated with sodium bisulfite-free heparin. Centrifuge these blood samples along with the blood samples for the blank test. Separate 1 mL of plasma and mix with 2 mL of physiological saline to the plasma. Use the plasma for the blank test as a blank and measure the absorbance of the plasma samples on a wavelength of 805 nanometer. Use the calibration curve to obtain the concentration.
Calculation: Use semilog graph paper to plot the actual measurement values at the specified time points (5, 10 and 15 min after injection) by taking the concentration along the vertical axis (logarithmic scale) and the time along the horizontal axis. Draw a straight line that connects these 3 points. Use this line to obtain the t½ of concentration. Calculate the plasma elimination rate (K) using the following equation: K=0.693/t½.
Determination of Blood Retention Rate Method: Preparation of a Calibration Curve: Follow the procedure mentioned in Determination of Plasma Elimination Rate Method: Preparation of Calibration Curve as previously mentioned.
Operation: Follow the procedure mentioned in Determination of Plasma Elimination Rate: Operation as previously mentioned. After IV injection of Diagnogreen, the blood sample should be collected only once, 15 min later.
Calculation: Calculate the mean rate of retention of Diagnogreen Retention rate15 using the following formula: Retention rate15= C15/1×100 (%).
C15: Concentration of indocyanine green in the plasma 15 min after injection (mg/dL).
1: Mean concentration of indocyanine green in the plasma at the time of injection (mg/dL)
Reference: Normal Results of Liver Function Test: Plasma Elimination Rate: (K) ≥0.195±0.037.
Mean Retention Rate: R15 ≤10%.
