Diagnostics in Macular Disorders S Natarajan, Hitendra B Mehta, Sumita Sharma
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IntroductionCHAPTER 1

Fundus fluorescein angiography (FFA) is the study of retinal and choroidal vasculature using fluorescein dye.
Fluorescence is a physical property of certain substances that, on exposure to light of short wavelength, emit light of longer wavelength in a characteristics spectral range. Sodium fluorescein, a yellow-red substance, absorbs light between 485 and 490 nm in aqueous solution and exhibits a maximum emission between 525 and 530 nm.
A 5 ml bolus of (1 gm {20%} in 5 ml) sodium fluorescein dye is rapidly injected via the antecubital vein, and rapid retinal photographs are taken with a fundus camera containing an excitatory filter with maximum transmission between 485 nm and 500 nm and a barrier filter peaking close to the maximum of the fluorescein.
Eighty percent of the dye is bound to plasma protein and not available for fluorescence, remaining 20 percent of unbound fluorescein is responsible for the fluorescence. Angiography is based on the fact that fluorescein dye leaks freely from the normal choriocapillaries but does not penetrate healthy RPE and normal retinal capillaries because of the tight endothelial junction present in the latter. Under optimum conditions, the smallest retinal capillaries (5–10 µ in diameter) can be seen with this technique, a feat impossible by ophthalmoscopy or by color photography.
Adverse Reactions
The adverse reactions can be mild, moderate and severe extending from nausea, vomiting and pruritus 2to bronchospasm, laryngospasm, anaphylaxis, circulatory shocks and a tonic clonic seizure. The reaction might be true or pseudo-allergic, exact mechanism being poorly understood.
Normal Fluorescein Angiogram
  • Autofluorescence
  • Pseudofluorescence.
Early Choroidal and Cilioretinal Artery Filling
  • Choroidal flush is seen 3–5 sec after the first appearance of the dye in the choroid
  • 10–12 sec after injection, the dye is seen in the choroidal and cilioretinal artery
  • 12–15 sec after injection: in old patients.
Retinal Arterial Filling and Increase Choroidal Filling
  • 10–15 sec post-injection (1–3 sec postchoroidal filling)
  • After the central retinal artery fills, dye flows to retinal arterioles, precapillary arterioles, capillaries and then the retinal veins.
Retinal A-V Filling and Full Choroidal Filling
  • 15–20 sec post-injection (5–10 sec after choroid filling)
  • Dye flows from venules into veins resulting in Laminar flow. With time, the laminae become thicker and finally fuse.
Full Retinal AV Filling and Choroidal Filling (Fig 1.1)
  • 20–25 sec post-injection. Perifoveal capillary network is seen clearly. Fovea appears hypofluorescent
  • Dye completely fills the lumen of the blood vessels.
AV Recirculation Phase and Decrease of Retinal and Choroidal Fluorescence
  • 30 sec post-injection. Lasts for 2 1/2 min
  • Fluorescence within the vessels decreases.
Reduced Retinal and Choroidal Fluorescence
  • Late staining of disc and visible sclera. 3–5 min post-injection
  • Total emptying of major vessels seen in 10 min.
zoom view
Fig. 1.1: Mid AV phase of FFA showing normal filling of the retinal arterioles and venules. The fovea appears hypofluorescent
Elimination Phase
  • Removal (intravascular—30 min, extravascular—60 min)
  • Average time of the fluorescein angiogram:
    • Arterial phase: 10.9 sec
    • Early venous: 13.0 sec
    • Late venous: 17.6 sec.
AV Transit Time
Period from first appearance of dye in the retinal arteries to the complete filling of veins, lasts 8–12 sec.
Abnormal Fluorescence
Can be:
  1. Blocked fluorescence: due to hemorrhage, tumors blocking underlying fluorescence, choroidal fluorescence being blocked by lipofuschin like material in Stargardt's disease.
  2. Vascular filling defect: In areas of vascular non-filling, either retinal or choroidal.
  1. Transmitted: Due to atrophy or absence of RPE.4
  2. Leakage: Either from neovascularization or membranes, an increase in size and intensity of the hyperfluorescence in late phases.
  3. Pooling: Accumulation of dye in an anatomical space.
  4. Staining: Taking up of the dye by scar tissue or sclera.
The choroidal circulation accounts for a significant part of the intraocular blood flow, and the use of indocyanine green angiography has made a major contribution in adding to our knowledge of the three-dimensional choroidal circulation.
  1. The water soluble, tricarbocyanine dye absorbs infrared light at 805 nm and maximally fluoresces at 835 nm. These spectral properties make penetration through ocular pigment and media opacities possible.
  2. It is retained in the vascular space because of the 98 percent binding with globulin proteins, allowing imaging of choroid and any associated abnormalities, as the globulin-ICG complex cannot leak out of choroidal vessels.
Adverse Reactions
The tricarbocyanine dye contains about 5 percent iodine so it should be used with caution in patients with a history of allergy to iodides.
The adverse reactions can be mild, moderate and severe extending from nausea, vomiting and pruritus to bronchospasm, laryngospasm, anaphylaxis, circulatory shock and a tonic clonic seizure. The reaction might be true or pseudo-allergic, exact mechanism being poorly understood. The adverse reaction may be caused by sodium iodide or the ICG molecule itself.
Adverse reactions to ICG dye occur less frequently than with fluorescein (0.15–0.2% with ICG as compared to between 1 and 10% with fluorescein)
Patient Preparation
Possible iodine sensitivity needs to be ruled out, and a thorough medical history taken. Maximum pupillary dilatation is required.5
At an average, 25 mg of ICG dissolved in 2 ml of aqueous solvent provides high contrast images. If necessary, it is possible to perform ICG angiography simultaneously with or sequential to FFA.
Photographic Technique
Preinjection monochromatic photographs: using red free filters (560 nm) or green free filters (640 nm) which enhance retinal and choroidal vasculature respectively, photographs are taken.
With the excitation and barrier filter in place, a preinjection image to identify pseudo or auto-fluorescence is taken.
Early phase angiography: Timer is started when ICG injection is begun.
Rapid, sequential photographs (1/sec) are acquired, beginning 8 to 10 seconds after dye injection.
To get the earliest vascular filling phase, images should be acquired even before the appearance of fluorescence.
Mid phase study: All images should be reviewed as they are taken (Fig 1.2).
Images are taken one every 2–3 seconds.
Late phase study: Images are taken at 7 min, 10 min, 12 min and 20 min (Fig 1.3).
zoom view
Fig. 1.2: Mid phase of ICG angiography showing normal filling of the choroidal vasculature. The retinal arterioles and venules also show filling. Disc appears hypofluorescent
zoom view
Fig. 1.3: Late phase of ICG angiography showing uniform background choroidal fluorescence. The disc appears hypofluorescent
The Normal Angiogram
The much more complex choroidal vascular architecture produces a wide variation of normal circulation patterns. The landmarks to keep in mind are the optic disc and the choroidal watershed zone.
Interpretation of the ICG Angiogram
It is important to examine an entire study, not just a single frame.
The stages in the normal angiogram are:
Early phase (within 2–60 sec)
  • Prominent filling of the choroidal arteries
  • The choroidal lobules are well defined at the end of the early phase
  • Early filling of the choroidal veins
  • Well-defined watershed zone seen, with hypofluorescent optic disc
  • Dye appears in retinal arteries.
Mid phase (1–15 min)
  1. Early Midphase:
    • Watershed zone fills up7
    • Prominent choroidal veins
    • Dye seen in retinal arteries and veins.
  2. Late Midphase
    • Choroidal vessels are less prominent
    • Diffuse choroidal hyperfluorescence because of diffusion of dye from choriocapillaries
    • Dye present in retinal vasculature.
Late phase (15–30 min)
  • Choroidal vasculature seen as dark bands against the background hyperfluorescence of stained extravascular choroidal tissue
  • Retinal vasculature not seen
  • Dye remains in abnormal tissue and appears hyperfluorescent against the background.
The first step is to determine whether an area exhibits hyperfluorescence (increased fluorescence) or hypofluorescence (reduced fluorescence). Depending on the phase of the angiogram, the interpretation may vary.
Features in an Angiogram
Watershed zone, disc in late phase, choroidal arteries and veins in late phase; overlying haemorrhage.
Choroidal vessels in early phase, hot spot, plaque, polyps.
OCT is a non-contact, non-invasive diagnostic imaging modality that can perform high resolution cross-sectional imaging in biologic tissues using light waves.
Since retina is accessible to external light, OCT is especially suited for retinal disorders. It provides information regarding the retinal tomography/cross-sectional imaging that is akin to in vivo histopathology of the retina.
  1. Principle: The imaging is based on the principle of Low Coherence Interferometry
    A broad bandwidth near infrared light beam (820 nm) is projected on the retina. This light gets backscattered or reflected from the microstructures, with the degree of backscatter depending upon the refractive index of the structure. The echo time delay of the light reflected from the retina is compared with that from a reference mirror at a known distance, thus leading to the phenomenon 8of interference. This interference is then measured by a photodetector, and the range of time delays compared.
    A realtime tomogram is created by integrating data points over a depth of 2 mm, using a false color scale. Red and white colors represent the highest back scattering, whereas the lowest is represented by blue and black.
    It is important to note that the colouring of different structures represent different optical properties and not necessarily different tissue morphology.
  2. It provides < 10 µ axial resolution and 20–50 µ transverse resolution, depending upon the ocular structure being viewed.
  3. Different cross sectional planes in the anterior segment or fundus are measured. Also, quantitative information on dimensions of intraocular structures gives it the potential to stage disease progression or response to therapy.
  4. Because OCT images are acquired rapidly and the measurement beam is infrared, patient discomfort is minimized.
  1. It is preferable to dilate the pupil before examination.
  2. Based upon the visual acuity of the patient, the internal or external fixation target is used. The internal fixation target is reliable because of its reproducibility.
  3. The various scan acquisition protocols available in the current OCT machines are specific for the kind of information needed. They are either image processing protocols or quantitative analysis protocols.
  4. For macular diseases, the protocols useful are:
    1. Line scan: The default angle is 0°. The length of the scan is 5 mm. Multiple scans of different parameters can be obtained, by changing the length and angle of the line.
    2. Radial lines: It consists of 6 to 24 equally spaced parallel line scans that can be varied in parameters. Useful for macular scan and retinal thickness/volume analysis.
    3. Macular thickness map: Using radial lines, within a circle at a fixed diameter of 6 mm, the retinal thickness can be measured.
    4. Fast macular thickness map: It takes 1.92 seconds to acquire six scans of 6 mm length each. It can be used for bilateral comparative retinal thickness/volume analysis.
    5. Raster lines: A series of equally spaced parallel lines placed in a rectangular box, can be used to cover the entire area of pathology by altering the size of the box.
    6. Repeat protocol: It allows one to repeat any of the previously saved protocols using the same set of parameters. Helpful for a comparative assessment with the previous scan.
Features of the Normal Macula
The large field optical tomogram through the macula demarcates the optic nerve head and foveal architecture along with vitreoretinal interface.
The optic nerve head is identified by its contour, with the central depression corresponding to the optic disc cup.
The anterior and posterior margins of the retina are denoted as highly reflective layers corresponding to the nerve fiber layer and the RPE-choriocapillaries respectively. Above the RPE-choriocapillaries layer is the minimally reflective layer of photoreceptors, above which moderate backscattering is seen from the outer and inner plexiform layer. The nuclear layer, like the photoreceptors shows only minimal backscattering. The retinal blood vessels are defined by their increased backscatter and shadowing on the reflection below. The larger choroidal vessels are seen occasionally as minimally reflective dark lumens, below the RPE—choriocapillaris layer.
The fovea shows the characteristics thinning of the retinal layers, along with the angulation of retina anterior to the photoreceptor layer. The RPE appears distinct from the choriocapillaries directly beneath the fovea.
The vitreoretinal interface is demarcated by the contrast between the non-reflecting vitreous and the backscattering surface of the retina (Fig 1.4).
zoom view
Fig. 1.4: The normal retinal tomogram denoting the uniform reflectivity of the retinal layers with the increased reflectivity of the RPE—choriocapillaris layer
In an OCT scan, the reflectivity pattern of the scanned images is studied.
  1. Hard exudates: Hyperreflective shadows in neurosensory retina that completely block the reflections from underlying retina.10
  2. Scar tissue and neovascular membranes: varying hyperreflectivity.
  3. Blood: If thin layer, it is hyperreflective. If a thick layer, it blocks the underlying reflections.
  1. Serous fluid: optically empty space with absence of backscattering.
  2. Cystoid spaces in the retina.
  1. H Schatz, TC Burton, LA Yannuzzi, F Rabb. Interpretation of Fundus Fluorescein Angiography. CV Mosby,  St Louis  1978.
  1. Lawrence A Yannuzzi, Robert W Flower, Jason Slakter. Indocyanine Green Angiography. Mosby  St. Louis, Missouri,  1997.
  1. Puliafito CA, Hee MR, Schuman JS, Fujimoto JG. Optical Coherence Tomography of Ocular Diseases. Slack Inc.  1996.
  1. Vishali Gupta, Amod Gupta, Mangat R Dogra. Atlas: Optical Coherence Tomography of Macular Diseases. Jaypee Brothers Medical Publishers (P) Ltd.,  2004.