### 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 IC-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 1 mL of the serum into a microcuvette. 
  3. 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.) 
  4. The optical density of this solution is read at 805 nm, using normal serum as the blank. 
  5. 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Percentage Retention: A single 20‑minute sample (withdrawn from a vein in the opposite arm to that injected) is allowed to clot, centrifuged and its optical density is determined at 805 nm using the patient's normal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 IC‑GREEN™ concentration in each timed specimen can be determined by using the concentration curve illustrated. Plot values 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 18‑24% per minute. Normal biological half‑time is 2.5 ‑ 3.0 minutes. 

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. The peak absorption and emission of IC‑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. 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. 

Dosages up to 40 mg IC‑GREEN™ dye in 2 mL of aqueous solvent have been found to give optimal angiograms, depending on the imaging equipment and technique used. The antecubital vein injected IC‑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.