Fluorescein Angiography
Novotny and Alvis performed first successful fluorescein angiography in humans. With digital imaging the technique and our understanding of fluorescein angiography is still expanding.
Sodium fluorescein is yellow-red in color with a molecular weight of 376.67, a spectrum of absorption at 465 to 490 nm (blue), and excitation at 520 to 530 nm (yellow-green) wavelengths. Once injected, 80% of the dye binds with plasma proteins, particularly to albumin. It is metabolized by the liver and kidney and within 24 to 36 hours is eliminated in the urine, which is discolored (bright orange hue). Adverse reactions range from mild to severe.
After rapid injection into the antecubital vein, fluorescein dye enters the short posterior ciliary arteries and is visualized in the choroid and optic nerve head in 10 to 15 seconds. The choroidal flush is the hallmark of choroidal filling. The mottled fluorescence of the choriocapillaris is attributed to variable blockage by the retinal pigment epithelium. Patchy filling of the choroid anatomically represents perfusion of choriocapillaris lobules sequentially, rather than simultaneously. In the early angiogram, leakage of the dye from the choriocapillaris and staining of the Bruch's membrane, eclipse choroidal vessel detail. Retinal circulation filling begins at 10 to 15 seconds, approximately 1 to 3 seconds after the onset of choroidal filling. After the dye is seen in the central retinal arteries, the fluorescein travels into the precapillary arterioles, the capillaries, the post-capillary venules, and then exits the eye through veins in a laminar pattern. Laminar filling of the veins is caused by a preferential concentration of unbound fluorescein along the vessel walls. This has been attributed to the faster flow of blood as well as higher concentration of erythrocytes in the central vascular lumen.
The early arteriovenous phase is followed by the late arteriovenous phase, which is characterized by the maximal fluorescence of the arteries and the capillary bed, with early laminar filling of the veins. Juxtafoveal capillary network achieves maximal fluorescence at 20 to 25 seconds. A dark background in the macula, created by blockage of choroidal fluorescence by xanthophyll pigments and a high density of retinal pigment epithelium cells, enhances capillary detail. The normal foveal avascular zone is 300 to 500 microns. At 30 seconds, the first pass of fluorescein dye is complete. This is followed by the recirculation phases, in which there is intermittent mild fluorescence. At 10 minutes both circulations are generally devoid of fluorescein. The late angiogram is characterized by staining of Bruch's membrane, the choroid, and sclera, which is more visible in patients with light retinal pigment epithelium. Staining of the disc margin and the optic nerve head is independent of the degree of retinal pigment epithelium pigmentation.
Identification of abnormal areas of fluorescence and the identification of hypofluorescence and hyperfluorescence is crucial in interpretation of fluorescein angiograms. Hypofluorescence is a reduction or absence of normal fluorescence, while hyperfluorescence is increased or abnormal fluorescence.
Hypofluorescence can be categorized into blockage or vascular filling defects. Blocked fluorescence can give us clues to the level of the blocking material. Vascular filling defects cause hypofluorescence because of decreased or absent perfusion of tissues.
Hyperfluorescence is caused by an increase in normal fluorescence or abnormal presence of fluorescence. A window defect refers to choroidal fluorescence produced by a relative decrease or absence pigment in the retinal pigment epithelium. Hyperfluorescence attributed to the presence of abnormal blood vessels is seen in the retina, choroid and the optic disc. Leakage of dye into the extravascular space leads to hyperfluorescence.
Indocyanine Green Angiography
Digital indocyanine green angiography is useful in imaging the choroid and its associated pathology. Indocyanine green is a tricarbocyanine dye that has several special properties that give it advantage over sodium fluorescein as a dye for ophthalmic imaging. The dye has a molecular weight of 775 daltons. Eighty percent of the dye is bound 5to globulins. Indocyanine green is active between 790 and 805 nm. It is excreted via bile by the liver. The dye is relatively safe. Properties of indocyanine green dye make it useful in imaging occult or poorly defined choroidal neovascularization secondary to age-related macular degeneration. Three morphological types of choroidal neovascularization may be observed.
Focal spots (hot spots) are areas of clinical subretinal exudation that appear as occult CNV on fluorescein angiography. It is a hyperfluorescent lesion less than one disc area in size located outside the foveal avascular zone. Focal spots occur in 29% of occult CNV cases.
Plaques are the most common pattern of occult CNV. It is a hyperfluorescent lesion greater than 1 disc area and is subfoveal. They can be well defined or poorly defined. Plaques occur in 61% of occult CNV cases.
Combined lesions are the least common and consist of both a focal spot and a plaque. Three subtypes exist depending upon the relationship between the focal spot and plaque.
Marginal spot is focal spot at the edge of margin of a plaque of neovascularization. It occurs in 3% of occult CNV cases.
Combination spot is a focal spot that overlies the plaque of neovascularization. It occurs in 4% of occult CNV cases.
Remote spot is focal spot that is not contiguous with a plaque. It occurs in 1% of occult CNV cases.
Scanning Laser Ophthalmoscope-based Angiography
The confocal scanning laser ophthalmoscope has the property of providing optical sections through the use of confocal optics. It utilizes a combination of three lasers at appropriate wavelengths to form a color image, which is analogous to the formation of color television images using red, green, and blue phosphors. The lasers used are 790, 820 and 488 nm. Each of the three beams are aimed and detected through the same set of optics. These beams can be used individually or in combination. The beam is swept horizontally and line by line vertically across the retina using a rotating polygon mirror and galvanometer-driven mirror respectively. Thus, a 20 micron moving spot is swept across the retina to form a rectangular raster. Because only one point of the retina is illuminated and imaged at any time, scattering is avoided and contrast enhanced. The average power of laser light used is 2 mW. The scanning beam illuminates each area point (10μ × 10μ) for only 0.1 to 0.7 microseconds. The reflected light passes through the scanning optics until it is separated from the incident light path by a partially reflecting mirror. The reflected light then passes through a confocal barrier which consists of a pin-hole. Finally, the light is collected by the light detecting unit, Avalanche Photo Diode. A computer digitizes the signal from Avalanche photo diode, assigning each point a number. This assignment of brightness point by point goes on continuously as the instrument produces a stream of point brightness measurements. From these, a two-dimensional image is constructed electronically. This signal is digitized and stored. Main advantages of Heidelberg Retinal Angiography (HRA2), over conventional imaging are its image quality, dynamic capabilities, and efficiency. Compared to both the original HRA and fundus cameras, the HRA2 takes higher-resolution images, which means better definition and greater detail. With fluorescein, the HRA2 enables physicians to see details just 5μm in size (three times smaller than the 15μm details visible with a fundus camera image) and all in an image that is just 1536 × 1536 pixels with a 30° field of view. Fundus cameras with much larger image sizes cannot match this resolution. Because the HRA2 suppresses scattered light, it can resolve to 5μm per pixel, compared to the fundus camera's 15μm to 18μm per pixel. Indocyanine green angiography performed with the HRA2 provides greater chorioretinal detail than ordinary fundus camera indocyanine green angiography 6because of both reduced light scatter and the HRA2 laser's particular sensitivity to the indocyanine green dye, lacking the fuzzy, diffuse quality of indocyanine green angiography images from fundus cameras. In terms of field of view, the HRA2 is narrower than the conventional fundus camera. Fundus cameras are generally capable of 45° or 50°, 30° or 35°, and 20°, whereas the HRA2's fields of view are 30°, 20°, and 15°. One disadvantage of the HRA systems is their lack of color imaging, which facilitates angiogram interpretation.
The HRA systems offer still-frames and full-motion indocyanine green angiography, a significant advantage over the fundus camera's static-only indocyanine green angiography, which is not informative and fast enough. For choroidal studies, the first 6 seconds are important. Infrared imaging, with extremely high resolution, and real-time video of the vessels filling, imparts the detailed information for circulation studies. Simultaneous fluorescein angiography and indocyanine green angiography not only saves time, but aids the diagnosis by helping the correlation of the choroidal pathology on the indocyanine green angiogram with the fluorescein angiogram's retinal landmarks.
In addition to its fluorescein and indocyanine green angiography imaging capabilities, the HRA2 also offers several noninvasive imaging modes, including blue-reflectance and infrared-reflectance imaging. Blue-reflectance imaging may be valuable for viewing details of the nerve fiber layer, but it requires a clear media without cataracts. Noninvasive imaging with barely visible infrared reflectance light is suitable for viewing the fundus of an extremely light-sensitive patient such as a child, as well as for viewing through a cataract. The HRA2 also provides autofluorescence imaging. Real-time angiography helps location of feeder vessels and choroidal neovascularization (CNV) associated with macular degeneration. The resolution and dynamicity of images illustrates circulation in the large and small vessels that feed CNV complexes, including their filling up and then draining, which is a rapid process, using the rapid frame rate to capture that circulation. In comparison, a fundus camera requires physicians to compare still images captured as far as 1 or 2 seconds apart. Thus, it gives deep understanding into choroidal vasculature.
HRA2 is particularly useful for locating and treating feeder vessels. It also helps physicians differentiate retinal angiomatous proliferation from CNV, which promotes early diagnosis and treatment.
FIGURES 1-1A TO C: Fluorescein angiography in central serous chorioretinopathy (Sandeep Saxena, MS, India).
FIGURES 1-1D TO F: Fluorescein angiography in central serous chorioretinopathy (Sandeep Saxena, MS, India).
FIGURES 1-1G TO I: Fluorescein angiography in central serous chorioretinopathy (Sandeep Saxena, MS, India).
FIGURES 1-2A AND B: Hot spot.
A. Occult choroidal neovascularization on fluorescein angiography.
B. Hot spot on indocyanine green angiography (Sandeep Saxena, MS, India).
FIGURE 1-3: Plaque.
Fluorescein angiogram shows occult CNV, whereas, indocyanine green angiography shows a plaque (Sandeep Saxena, MS, India).
FIGURES 1-4A AND B: Polypoidal choroidal vasculopathy.
A. Fluorescein angiogram shows occult choroidal neovascularization.
B. Indocyanine green angiography reveals polypoidal choroidal vasculopathy (Sandeep Saxena, MS, India).
FIGURES 1-5A AND B: Peripapillary polypoidal choroidal vasculopathy.
A. Fluorescein angiogram shows peripapillary occult occult choroidal neovascularization.
B. Indocyanine green angiography reveals peripapillary polypoidal choroidal vasculopathy (Sandeep Saxena, MS, India).
FIGURE 1-6: Scanning laser ophthalmoscope-based angiography.
Simultaneous fluorescein angiography and indocyanine green angiography. Hyperfluorescent leaking spots, not apparent on fluorescein angiography are much better visualized in indocyanine green angiography (Manish Nagpal, MS, FRCS, India).
FIGURE 1-7: Scanning laser ophthalmoscope-based angiography.
Simultaneous fluorescein angiography and indocyanine green angiography in classic choroidal neovascularization. In such cases fluorescein angiography is sufficient, and indocyanine green angiography does not reveal any further information (Manish Nagpal, MS, FRCS, India).
FIGURE 1-8: Scanning laser ophthalmoscope-based angiography.
Simultaneous fluorescein angiography and indocyanine green angiography image revealing diffuse leak on fluorescein angiography shows a localized hot spot in the indocyanine green angiography (Manish Nagpal, MS, FRCS, India).
FIGURES 1-9A AND B: Scanning laser ophthalmoscope-based angiography.
A. Mid-phase of simultaneous fluorescein angiography and indocyanine green angiography in age-related macular degeneration. Fluorescein angiography shows classic choroidal neovascular membrane. Indocyanine green angiography shows the vascular network with central feeding vessel.
B. Feeder vessel is very well visualized (Manish Nagpal, MS, FRCS, India).
FIGURE 1-10: Scanning laser ophthalmoscope-based angiography.
Fluorescein angiography shows staining and leakage from choroidal neovascular membrane. Indocyanine green angiography reveals feeding arteriole and venule of the choroidal membrane (Manish Nagpal, MS, FRCS, India).
FIGURE 1-11: Scanning laser ophthalmoscope-based angiography.
Intense hot spot on the right indocyanine green angiography picture is a macroaneurysm which has lead to subretinal blood collection, resulting in blocked fluorescence on the fluorescein angiography on the left, as a differential diagnosis to choroidal neovascular membrane (Manish Nagpal, MS, FRCS, India).