AAO

Fluorescein angiography (FA) allows study of the circulation of

the retina and choroid in normal and diseased states.
Photographs of the retina are taken after intravenous injection
of sodium fluorescein, an orange-red crystalline hydrocarbon
with a molecular weight of 376 daltons that diffuses through
most of the body fluids. It is available as 23 mL of 25%
concentration or 5 mL of 10% concentration in a sterile
aqueous solution. It is eliminated primarily through the liver
and kidneys within 2436 hours via the urine. Eighty percent
of the fluorescein is protein-bound, primarily to albumin, and
not available for fluorescence; the remaining 20% is unbound
and circulates in the vasculature and tissues of the retina and
choroid, where it can be visualized.
Fluorescence occurs when a molecule is excited by light of a
certain wavelength that raises the molecule to a higher energy
state and then allows it to release a photon of light to bring it
back to its original state. To image this fluorescence, special
excitation and barrier filters are required. Sodium fluorescein
fluoresces at a wavelength of 520530 nm (green) after
excitation by a light of 465490 nm (blue). To obtain a
fluorescein angiogram, white light from the camera flash unit
is passed through a blue (excitatory) filter, and blue light
enters the eye. The blue light, with its wavelength of 465490
nm, excites the unbound fluorescein molecules circulating in
the retinal and choroidal layers or that have leaked out of the
vasculature and stimulates them to emit a longer-wavelength
yellow-green light (520530 nm). Both the emitted yellow-
green fluorescence and some degree of reflected blue light
from structures that do not contain fluorescein exit the eye and
return to the camera. A yellow-green (barrier) filter on the
camera lens blocks the reflected blue light, permitting only the
yellow-green light, which has originated from the fluorescein
molecules, into the camera.
The image formed by the emitted fluorescence is recorded on
either black-and-white, high-contrast 35-mm film, videotape,
or a digital camera. The 35-mm film permits higher-resolution
images of the retinal vessels and choroid and is generally
easier to use than videotape for capturing stereoscopic frames
and stereoscopic viewing. Newer digital systems offer high-
resolution images rivaling those of 35-mm film and can adjust
contrast and brightness to highlight certain details; they can
also zoom in on areas of concern, which is not possible with
film-based images. Digital images are seen immediately by
the photographer, who can adjust for focus and problems
during the procedure. This is not possible with film. Finally,
digital imaging systems allow easy image archiving and
retrieval, thus offering the capability of quickly comparing
images over time for diagnosis and treatment.
To properly interpret a fluorescein angiogram, it is vital to
understand retinal anatomy. The retina has a dual blood
supply. The central retinal artery and retinal circulation serve
the inner half of the retina, and the endothelial cell tight
junctions provide the inner bloodretina barrier. Normally,
neither bound nor unbound fluorescein can pass through this
barrier. The choroidal circulation serves the outer half of the
retina, and the RPE provides the outer bloodretina barrier.
Fluorescein particles that are not bound to protein can pass
through the fenestrated walls of the choriocapillaris but do not
normally pass through the RPE or zonulae occludentes
between adjacent RPE cells to gain access into the subretinal
space. Therefore, fluorescein from the choroid cannot enter
the neurosensory retina unless the RPE has a defect.
Although the fluorescence in the choroid is partially blocked by
the pigment in the RPE, it is visible as deep, diffuse
background fluorescence.
Fluorescein is injected into a peripheral vein and enters the
ocular circulation via the ophthalmic artery 812 seconds
later, depending on the rate of injection and the patients age
and cardiovascular status. The retinal and choroidal vessels
fill during the transit phase, which ranges from 10 to 15
seconds. Choroidal filling is characterized by a patchy
choroidal flush, with the lobules often visible. Because the
retinal circulation has a longer anatomical course, these
vessels fill after the choroidal circulation. The arterial phase of
the angiogram occurs after the choroidal phase, with filling of
the retinal arteries. The arteriovenous phase begins with
complete filling of the retinal arteries and capillaries and
completes with laminar filling of the retinal veins. This phase,
which usually occurs approximately 1 minute after dye
injection, is considered the peak phase of fluorescence, where
the most detail is evident in the fovea. Over the next few
minutes, the dye recirculates, with a gradual decline in
fluorescence. In the late phases of the angiogram, the
choroid, Bruchs membrane, and the sclera stain. The larger
choroidal vessels are often seen as hypofluorescent areas
against this hyperfluorescent background.
Fluorescein can leak out of retinal capillaries into the retina
only when the capillary endothelium is damaged, as in
diabetic retinopathy. Similarly, fluorescein can leak from the
choriocapillaris through pigment epithelial cells into the
subretinal space and the retinal interstitium only when the
latter are abnormal, as in central serous chorioretinopathy.
Thus, patterns of hyperfluorescence and stereoscopic images
yield valuable information about leakage from retinal vessels
or through abnormal pigment epithelium. Abnormalities seen
with FA can be grouped into 3 categories, associated with one
of the following:
autofluorescence
hypofluorescence
hyperfluorescence
Autofluorescence is fluorescence that can be seen before the
fluorescein dye is injected; this is caused by naturally highly
reflective substances, such as optic disc drusen.
Hypofluorescence occurs when normal fluorescence is
reduced or absent; it is present in 2 major patterns:
vascular filling defect
blocked fluorescence
Vascular filling defects occur where the retinal or choroidal
vessels do not fill properly, as in nonperfusion of an artery,
vein, or capillary in the retina or choroid. These defects
produce either a delay or a complete absence in filling of the
involved vessels.
Blocked fluorescence occurs when the stimulation or
visualization of the fluorescein is blocked by fibrous tissue or
another barrier, such as pigment or blood, producing an
absence of normal retinal or choroidal fluorescence in the
area.
Blocked fluorescence is most easily differentiated from
hypofluorescence due to hypoperfusion by evaluating the
ophthalmoscopic view, where a lesion is usually visible that
corresponds to the area of blocked fluorescence. If no
corresponding area is visible clinically, then it is likely an area
of vascular filling defect and not blocked fluorescence. By
evaluating the level of the blocked fluorescence in relation to
the retinal circulation, one can determine how deep the lesion
resides. For example, when lesions block the choroidal
circulation but retinal vessels are present over this blocking
defect, then the lesions are above the choroid and below the
retinal vessels.
Hyperfluorescence occurs when there is an excess of normal
fluorescence; it is seen in several major patterns:
leakage
staining
pooling
transmission, or window, defect
autofluorescence
Leakage refers to the gradual, marked increase in
fluorescence throughout the angiogram when fluorescein
molecules seep through the pigment epithelium into the
subretinal space or neurosensory retina, out of retinal blood
vessels into the retinal interstitium, or from retinal
neovascularization into the vitreous. The borders of
hyperfluorescence become increasingly blurred, and the
greatest intensity of hyperfluorescence is found in the late
phases of the study, when the only significant fluorescein dye
remaining in the eye is extravascular. Leakage occurs, for
example, in CNV (Fig 2-1), in microaneurysms in
telangiectatic capillaries in diabetic macular edema, and in
neovascularization of the disc.
Staining refers to a pattern of hyperfluorescence where the
fluorescence gradually increases in intensity through transit
views and persists in late views, but its borders remain fixed
throughout the angiogram. Staining results from fluorescein
entry into a solid tissue or similar material that retains the
fluorescein, such as a scar, drusen, optic nerve tissue, or
sclera (see Fig 2-1B).
Pooling refers to the accumulation of fluorescein in a fluid-
filled space in the retina or choroid. At the beginning of the
angiogram, the fluid in the space contains no fluorescein and
is not visible. As fluorescein leaks into the space, the margins
of the space trap the fluorescein and appear distinct, as seen,
for example, in an RPE detachment in central serous
chorioretinopathy (Fig 2-2D). As more fluorescein enters the
space, the entire area fluoresces.
A transmission defect, or window defect, refers to a view of
the normal choroidal fluorescence through a defect in the
pigment or loss of pigment in the RPE, such as shown in
Figures 2-1A and 2-1B. In a transmission defect, the
hyperfluorescence occurs early, corresponding to filling of the
choroidal circulation, and reaches its greatest intensity with
the peak of choroidal filling. The fluorescence does not
increase in size or shape and usually fades in the late phases
of the angiogram, as the choroidal fluorescence becomes
diluted by blood that does not contain fluorescein. The
fluorescein remains in the choroid and does not enter the
retina.
Autofluorescence describes the appearance of fluorescence
from the fundus captured prior to intravenous fluorescein
injection. It is seen with structures that naturally fluoresce,
such as optic nerve drusen and lipofuscin.