HISTORICAL PERSPECTIVE

Fluorescein dye was initially synthesized by Adolf von Baeryer in 1871 and soon found use in the diagnosis of various corneal disorders.^5–7 Later work by Paul Ehrlich in 1882 revealed the presence of intraocular fluorescein after parenteral administration in rabbits.⁸ Study of the fundus by angioscopy after intravenous injection of fluorescein in animals was carried out by multiple investigators until 1955, when MacClean and Maumenee used fluorescein dye in human subjects.⁹ Novotny and Alvis later developed a photographic system for the sequential documentation of fluorescein flow through the ocular fundus.^10,11 Their published reports created a revolution in the understanding and treatment of posterior segment disease.¹²

CHEMICAL AND PHYSICAL PROPERTIES

Sodium fluorescein (C₂₀H₁₀Na₂) is a highly water-soluble complex organic molecule with a molecular weight of 376.27.^6,13,14 Its clinical utility results from the physical property of luminescence—the emission of light by means other than incandescence. In the case of fluorescent material, the emission of light is a direct result of excitation by light of a shorter wavelength and higher energy level. Unlike phosphorescent material, the rate of light emission is so rapid as to appear instantaneous. Quantum theory states that the absorption and emission frequencies for a particular material are spectrum specific for that particular molecule (Fig. 1). Light energy is released when an excited fluorescent material spontaneously decays from a higher energy state to a lower level.

Excitation of fluorescein occurs when it is exposed to blue wavelengths between 465 and 490 nm, resulting in emission of yellow-green frequencies (520 to 530 nm).^13–17 In the bloodstream, fluorescein is excited by a wavelength of 465 nm and emits a wavelength of 525 nm.^17,18 This greater spread between excitation and emission frequencies is clinically helpful in their separation by photographic filters during angiography (Fig. 2; see Fig. 1).^17,18

Sodium fluorescein has several physical and chemical properties that make it an excellent diagnostic tool. It is an inexpensive, water-soluble and relatively inert organic plant resin that exhibits its maximum fluorescence at a near normal blood pH of 7.4.^6,13,14,16 Its excitation and emission wavelengths are in the visible spectrum, allowing use of standard photographic equipment and materials. In addition, the molecular size of fluorescein prevents its passage through the tight endothelial junctions of retinal blood vessels and zonula occludens in retinal pigment epithelium while allowing for rapid diffusion in fluid compartments.^13,14,16 Conversely, sodium fluorescein's high percentage of binding to albumin and other bloodborne proteins drastically reduces the amount of free fluorescein readily available for excitation.¹⁴

ADMINISTRATION

Five milliliters of 10% sodium fluorescein solution is routinely given intravenously at the start of the procedure. A 25% solution is also available and may be better tolerated by the patient. Evaluation of smaller, 2-ml injections of 10% solution by Nasrallah and associates have given equally satisfactory results with fewer side effects.¹⁹ A scalp needle attached to a small syringe is ideal for administration (Fig. 3). The angiographic study is initiated with the patient seated comfortably at the fundus camera. The fluorescein dye is injected rapidly into an antecubital vein. Blood should be refluxed into the fluorescein-containing syringe before injection to ensure entry into the vein. Administration is monitored carefully because extravasation of dye results in severe localized pain and even overlying skin necrosis.^20,21 Blood precautions should be observed with discarded needles and syringes, and gloves should be worn by those administering dye.

Within 3 to 5 minutes, the fluorescein is distributed equally throughout the blood. It is rapidly eliminated, predominantly by the kidneys^14,16,22; most of it is removed from the bloodstream within 1 hour. Patients should be alerted to expected changes in skin tone and urine color in the post-test period.

Oral administration of fluorescein is also possible in those individuals for whom intravenous injection is difficult or impossible. Oral administration allows assessment of the blood-retinal barrier, but it is inadequate in determining blood flow velocities or anatomic detail.^23–25 Ten percent sodium fluorescein is mixed with orange juice to give a 1% to 2% solution; 1 g of dye is given to patients weighing 50 kg or less. More dye, up to a maximum of 2 g, is given to heavier patients.²³ Serum concentrations of orally administered fluorescein peak at 30 minutes and remain relatively stable for 1 to 2 hours at levels similar to intravenously administered dye.²³ Although the reported incidence of side effects is reduced with this route, allergic reactions still may occur.²⁶

TOXICITY

Although numerous reports of side effects secondary to fluorescein administration are present in the literature, it remains a remarkably safe diagnostic compound. Nausea and emesis are the most commonly reported reactions, occurring with a frequency of 5% or less.²⁷ Syncope, thrombophlebitis, temperature elevation, nerve palsy, and local tissue necrosis are classified as moderate adverse reactions whose incidence is less than 1%.^20,26,27 Severe adverse reactions requiring aggressive intervention include laryngeal edema, bronchospasm, anaphylaxis, circulatory shock, and myocardial infarction. A survey by Yannuzzi and associates reported one related death in more than 220,000 angiographic studies of the ocular fundus.²⁷ With patients in whom toxicity is suspected, an intradermal skin test has been recommended.²⁵ An emergency tray and oxygen should be available whenever fluorescein is administered. Prophylaxis for possible adverse reactions is controversial. Although there have been no reports of human fetal complications secondary to fluorescein injection during pregnancy, many physicians avoid administration if possible in pregnant individuals.^27,28

ANATOMIC CONSIDERATIONS

Fluorescein angiography of the iris and fundus is possible because of the unique specialization of the eye as an optical instrument. Only in the eye is the vascular system open to close scrutiny through noninvasive means.

The ocular fundus has two distinctly separate vascular systems—retinal and choroidal—separated by a specialized pigmented monolayer, the retinal pigment epithelium (RPE). Embryologically, the sensory retina and RPE are derived from the inner and outer layers of the optic cup. The choroid and its vasculature lie posterior to the RPE. The fluorescein angiographic patterns of the posterior uvea are, therefore, always partially obscured by the RPE (Fig. 4). The degree of pigmentation and the pathologic changes in this pigmented layer markedly influence the choroidal angiographic appearance. In the interpretation of fundus fluorescein angiograms, the physician must keep in mind and be familiar with the anatomy and interaction of these three layers.

CHOROID

The choroid is a highly vascular connective tissue layer with an average width of 0.25 mm. It is made up of three distinct layers and is perfused by the long posterior, short posterior, and recurrent anterior ciliary arteries. Drainage is primarily through the vortex veins. Its capillary system, the choriocapillaris, is innermost and lies directly beneath Bruch's membrane. The choriocapillaris has a lobular pattern made up of a central arteriole, peripheral draining venules, and an intervening capillary bed. The capillary walls are extremely thin and contain multiple fenestrations, allowing a high degree of fluid transport (Fig. 5). These fenestrations are thought to be actual openings in the endothelial wall through which fluid can undergo passive exchange between the lumen of the vessel and the surrounding extravascular space. As fluorescein enters the choriocapillaris, it quickly passes into the extracellular space. The outer choroidal layers are nonfenestrated and, similar to other typical arteries and veins, do not normally leak fluorescein dye.

Under normal conditions, the choroid begins to fill with fluorescein a moment prior to the retinal vascular system. This filling is difficult to study by fluorescein angiography because of obscuration by the overlying retina and RPE, its rapid filling, and leakage of fluorescein from the choriocapillaris.⁶

RETINAL PIGMENT EPITHELIUM

The RPE is a specialized monolayer of pigmented epithelial cells separating the choroid and sensory retina. It functions physiologically as both a metabolic support platform for the overlying photoreceptors and as an effective barrier against passive molecular transport between the choroid and retina.^29,30 In its normal state, the RPE prevents leakage or transport of fluorescein into or out of the choroidal space.

The RPE also functions as an optical barrier because of the presence of pigmented melanosomes (Fig. 6). The density of this pigment varies with retinal location; it is greatest in the foveomacular region and least anterior to the equator. There are more pigmented epithelial cells per unit area in the foveomacular region than in the periphery. Posteriorly, the cells are tall and columnar, with several layers of melanosomes crowded together. Anteriorly, the cells gradually become flatter and more cuboidal, with a corresponding loss of relative pigment concentration (Fig. 7). In any normal eye, regardless of the comparative fundus color, the greater pigmentation in the foveomacular area gives this zone a darker clinical appearance when compared with the remainder of the posterior pole and periphery.

RETINA

The retina, except for its vasculature, may be regarded as a transparent tissue comprised of tightly packed layers of cells with essentially no extracellular space. The retina is perfused by the central retinal artery and cilioretinal vessels present in 32% of eyes.³¹ The capillary bed is nonfenestrated and fluorescein does not, therefore, leak from the retinal vessels into the surrounding tissue (Fig. 8). Functionally, the retinal vasculature represents a “closed” system, as opposed to the choroid, in which fluorescein is free to move between the intravascular and extravascular compartments. The retinal circulation normally fills with fluorescein after the choroidal flush; however, a cilioretinal artery fills at the same time as the choroid, preceding the rest of the retinal circulation.

The retina also serves as an optical filter because of its high content of xanthophyll pigment, especially in the macular region. This yellow pigment has its highest concentration in the outer nuclear and plexiform layers, and may selectively absorb blue excitation frequencies.¹⁶

IRIS

The iris stroma, like the choroid, is composed of vascular connective tissue. Although electron microscopy and tracer studies have demonstrated the absence of fenestrations in both animal and human eyes,^32–34 other reports have supported the presence of gaps in the junctions between endothelial cells in some animal iris tissue.^34–36

Similarly, portions of the eye may be capable of fluorescence in the absence of dye when illuminated with blue light. This phenomenon is known as autofluorescence.^14,16,37 Optic nerve head drusen, Best's vitelliform lesions, lipofuscin over choroidal melanomas, and flecks associated with fundus flavimaculatus are abnormal conditions that may autofluoresce (Fig. 10).³⁸ Normal autofluorescent ocular structures include sclera, retina, lens, and Descemet's membrane.¹⁴ Larsen and associates has used the autofluorescent properties of the lens as an accurate indicator of metabolic control in diabetics.³⁹

Interpretation of negatives or contact sheets should be undertaken in a sequential manner. Stereo viewing aids in localizing the anatomic depth of disease processes. The normal fluorescein study can be divided into four separate divisions: the prefilling, transit, recirculation, and late phases. The prefilling phase includes those frames taken before fluorescein dye enters the globe. It is useful in determining the presence of autofluorescence or pseudofluorescence, thus preventing incorrect interpretation. The transit phase represents the first complete passage of fluorescein-containing blood through the choroidal and retinal circulations. It occurs normally within the first 30 seconds of the study. It is subdivided into arterial and venous filling phases. The recirculation phase occurs as fluorescein becomes equally distributed throughout the blood and re-enters the eye. This phase is complete approximately 3 minutes after injection. Early leakage or staining with fluorescein usually occurs within the recirculation phase. The late phase, or elimination phase, is the result of removal of fluorescein from the circulation. For clinical purposes, this is observed by 30 minutes after injection; during this time, any late staining and residual hyperfluorescence is seen.

The normal sequential movement of fluorescein through these phases produces the total angiographic pattern. However, at any given moment, the fundus fluorescein pattern depends not only on the phase but also on the additive features and changes of the fluorescein dye as it travels superimposed in three distinct tissue layers (i.e., the retina, choroid, and RPE).

NORMAL ANGIOGRAPHIC PATTERN

Fluorescein enters the choroidal vascular system via the posterior ciliary arteries. In a very lightly pigmented fundus, these larger choroidal vessels can be seen to fill with fluorescein (Fig. 11). The flow is directed toward the choriocapillaris, the filling of which is called the background choroidal flush (Fig. 12A and B). Normally, this stage begins before the arterial phase of the retinal circulation. If a cilioretinal artery is present, it will fill at the same time (see Fig. 12C). The arm-to-retina circulation time varies, averaging 10 to 20 seconds. The filling of the choriocapillaris occurs in irregular patches, best seen in the posterior pole (see Fig. 12B).^41,42

Each patch appears to fill evenly and is the result of separate, irregular areas of choriocapillaris supplied by larger choroidal vessels at slightly different times. The background choroidal flush gradually intensifies and spreads anteriorly toward the ora serrata. The entire choroidal sequence is obscured in the foveomacular area because of the denser pigmentation in the overlying RPE, and possibly the increased amount of xanthophyll in the sensory retina (see Fig. 7).¹⁶ To observe the larger choroidal vessels fill with fluorescein, attention must be focused deep to the RPE very early in the study, before the choroidal flush quickly masks these feeding vessels (see Figs. 7, 11B, 12B, 13B, and 14B). As the fluorescein enters the intravascular system of the choroid, it immediately leaks into the extravascular space (see Fig. 12A). This leakage is related to the capillary fenestrations seen in the choriocapillaris. Leakage of fluorescein in the choroid is most intense directly below Bruch's membrane and in the inner choroidal layers (see Fig. 13A). Diffusion then occurs throughout the entire extravascular space to eventually involve the inner scleral fibers (see Fig. 14A). The concentration of fluorescein rapidly equilibrates throughout the inner choroidal layers in both the intravascular and extravascular compartments. The concentration in the extravascular stroma of the outer choroid is probably less. This occurs during the filling phases of the study, usually by the venous phase of the retinal circulation (see Fig. 14). As the study progresses, fluorescein continues to leak from the choroidal vessels. Concurrently, (the density of fluorescein within the vessels becomes less because of the extravascular leakage and dilution by equal distribution throughout the entire blood volume. Therefore, the concentration of fluorescein within the choroidal vessels rapidly becomes less than that in the extravascular choroidal tissue. This occurs in the inner choroidal layers first and can be recognized as the medium-sized choroidal vessels begin to appear as silhouettes against the more concentrated extravascular fluorescence (Fig. 15).

Fluorescein is removed from the choroid in the reverse manner. As vascular fluorescence becomes less because of recirculation of dye and removal by the kidneys, the extravascular fluorescein passes back into the vessels and is eliminated. Once the choroidal fluorescence has become more concentrated in the extravascular compartment, the relative contrast to vascular fluorescence remains throughout the entire study.

Fluorescein most likely leaves the extravascular tissue in the area of the choriocapillaris first and the outer choroid and inner scleral layers last. This dilution causes the extravascular fluorescein of the inner choroidal layers to become less concentrated than the outer extravascular choroidal fluorescein (Fig. 16). This is seen when the large choroidal vessels stand out in dark relief against the more concentrated extravascular fluorescence of the outer choroidal layers (see Fig. 16B). Even in the late phase, when fluorescein is no longer seen in the vasculature, patterns of choroidal fluorescence arising from extravascular fluorescein can be seen. This very late extravascular fluorescence arises from the outer choroidal and inner scleral layers (Fig. 17).

The RPE, with its tight junctions, acts as a barrier to the leakage of fluorescein from the choroid into the overlying outer retinal layers. Within 1 second of the choroidal flush, fluorescein dye is seen within the arterioles. In a healthy patient, with proper administration of fluorescein, this occurs rapidly, filling the arterioles completely. As bloodborne dye crosses the capillary network and enters the venous system, a columnar appearance to the veins initially is observed—the laminar venous phase. It occurs secondary to margination of fluorescein because of more rapid flow of blood in the vein's central lumen.¹⁶ As more fluorescein enters the veins, they (fill totally—full venous phase. Arteriovenous transit is dependent on the distance fluorescein dye must flow and is therefore delayed in the fundus periphery and more rapid within the macula.

HYPERFLUORESCENCE

Hyperfluorescence due to accumulation of fluorescein in an area where it is not normally seen is well portrayed in a focal detachment of the RPE (Fig. 18), in which the dye accumulates between the RPE and Bruch's membrane (see Fig. 18A). The RPE appears to be firmly attached to Bruch's membrane, and when fluid accumulates in this area, the RPE delineates itself with sharp, abrupt borders (see Fig. 18B-D).

Central serous chorioretinopathy causes an area of hyperfluorescence because of the presence of the dye between the retinal photoreceptor cells and the RPE (Fig. 19). This may be related to a detachment of the RPE and is associated in some way to a defect in the normal mechanism of fluid exchange across the RPE between the choroid and the sensory retina. The attachment of the photoreceptors to the RPE may not be as strong as the attachment of the RPE to Bruch's membrane. This may be why the borders of fluorescence in the secondary serous retinal detachments found in central serous chorioretinopathy are more diffuse (see Fig. 19B) than those in a detachment of the RPE (see Fig. 18B).

Ocular histoplasmosis may also show choroidal hyperfluorescence because of the abnormal presence of a choroidal neovascular membrane forming a nodular elevation of the RPE (Fig. 20A and B). In the early stage of this disorder, when a neovascular tuft is clearly seen, the very early phases of the fluorescein study clearly show the new vessels filling with fluorescein. In the later phases, dye leaks from the vessels to fill the surrounding tissue and may even cause staining of the RPE (see Fig. 20C-E). Hyperfluorescence is also caused by an increased concentration of fluorescein in an area of the uveal tract, where it is normally seen in the intravascular and extravascular components of the choroidal stroma. A choroidal hemangioma shows hyperfluorescence very early in the study (Fig. 21A and C). This vascular tumor and the choroidal vasculature fill with dye at the same time. For this reason, these lesions often reach a peak of hyperfluorescence in the early phases of the fluorescein study. Even though there is leakage and staining of the extravascular components, because these are highly vascular tumors, the peak of the hyperfluorescence may diminish before the late phases of the study (see Fig. 21D and E).

Hyperfluorescence from a solid tumor (e.g., a choroidal melanoma or metastatic lesion; Figs. 22 and 23) also is caused by an increased accumulation of fluorescein in the uveal stroma at the tumor site. These masses are usually associated with increased vascularity and therefore increased fluorescence. Fluorescein leaks rapidly into the extravascular space to fill the stroma of these tumors (see Figs. 22A and 23A). Because these are solid tumors of densely packed cells, it probably takes time for the fluorescein to completely penetrate the extravascular space of the tumor. Lesions of this nature may show a delay in the hyperfluorescent peaks as compared with choroidal hemangiomas, and for the same reason may fluoresce longer.

Hyperfluorescence, as seen with the previously mentioned tumors, also results from transmission through pigment epithelial defects and from the presence of fluorescein in cystic spaces within the retina.

Hyperfluorescence may also be caused by the transmission of normal background choroidal fluorescence through defects of the RPE secondary to various pathologic conditions. In albinism, a defect in production of melanosomes leads to increased transmission of choroidal fluorescence through the nonpigmented RPE (see Fig. 17). With degenerative disease affecting the RPE, focal areas lacking pigment are seen with adjacent areas of hyperpigmentation. In the early phases of a fluorescein study, the choroidal fluorescence is transmitted through these defects in the RPE,⁴³ whereas in the late phases, there is actual staining with fluorescein in these areas (Fig. 24). The adjacent focal areas of hyperpigmentation cause blockage of the background choroidal fluorescence. Drusen may act in the same manner because the RPE may be thinned over these lesions.⁴⁴ Transmission of the choroidal fluorescence occurs early, whereas actual staining with the dye is seen late (Fig. 25).

HYPOFLUORESCENCE

Hypofluorescence from absence of fluorescein in an area where it would normally be seen may be secondary to a lack of perfusion or to absence of the tissue. The hypofluorescence seen in the early frames of an individual with a coloboma results from the absence of the choriocapillaris; even though the RPE is absent, there is only transmission of the large choroidal fluorescein-filled vessels (Fig. 26A and B). In the very late stages of the study, the coloboma hyperfluoresces secondary to scleral staining in the area devoid of RPE (see Fig. 26C and D). Hypofluorescence can also be secondary to a relative decrease in the fluorescein concentration. This may partially explain why a choroidal nevus does not fluoresce (Fig. 27). In addition to the nevus cells occupying space where the dye would normally accumulate, hypofluorescence is also caused by obscuration of fluorescence by the heavy concentration of pigment (Fig. 28).

(Obscuration or blockage of choroidal fluorescence as a mechanism of hypofluorescence may be seen in several other conditions. A hemorrhage under the RPE causes a corresponding area of hypofluorescence due to a blockage of the underlying fluorescence (Fig. 29). For the same reason, an area of hypertrophy of the RPE will appear hypofluorescent (see Fig. 28). In addition, patients with Stargardt's disease often exhibit a silent choroid secondary to blockage of background choroidal fluorescence by lipofuscin accumulation in the RPE (Fig. 30).

In patients with choroidal folds, there are alternating areas of hypofluorescence and hyperfluorescence (Fig. 31).⁴⁵ The hypofluorescent lines correspond to the areas between the peaks of the folds where the RPE pigment concentration is greatest, causing a relative blockage of the underlying fluorescence. The alternating hyperfluorescent lines represent the peaks of the folds where the pigment concentration is less, allowing a greater transmission of the choroidal fluorescence.

The filling of iris vessels takes place approximately 10 to 15 seconds after injection of fluorescein. Iris transit is more sluggish than either the retinal or choroidal circulations.^34,47,48 Fluorescein fills the major arterial circle of the iris from the long posterior and anterior ciliary arteries.⁴⁹ Filling of stromal arterioles progress from the iris root and is variable and frequently segmental.^34,48 Van Nerom and associates found that 90% of their healthy subjects showed filling of the temporal quadrant last, whereas in 80%, the inferior quadrant filled first.⁴⁸ After arteriolar filling, the arteriovenous loops at the collarette fill, followed by the capillary network at the pupillary margin. Venous filling develops 2 to 4 seconds later.

Under normal conditions, leakage from the iris vasculature is rare but can occur at the pupillary margin in individuals older than 40 years of age. Leakage of dye occurs much more frequently in pathologic states, however, and therefore makes this technique valuable in the study of diabetes, uveitis, iris tumors, central retinal artery and vein occlusions, and hypertension.^48,50

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