Fluorescein and Indocyanine Green Angiography for Uveitis

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In recent years enormous progress has been achieved in investigational procedures for uveitis. Imaging is one such example with the advent of new methods such as indocyanine green angiography, ultrasound biomicroscopy and optical coherence tomography to cite only the most important. This tremendous increase in precision and accuracy in the assessment of the level and degree of inflammation and its monitoring comes in parallel with the development of extremely potent and efficacious therapies. In view of these developments, our whole attitude in the appraisal and investigation of the uveitis patient has to be adapted and correctly reoriented integrating the recent developments and this is no different for ocular angiography.

Since about 15 years, a second angiographic procedure is being used, using indocyanine green, a dye that fluoresces in the infrared wavelengths. It allows imaging of the poorly accessible choroid beforehand. This procedure, in contrast to FFA, often gives additional information undetected by clinical examination or FFA or OCT. Therefore, indocyanine green angiography (ICGA) is indispensable in the proper assessment of inflammatory involvement in uveitis as it gives information, which is otherwise lost. Very often, ICGA has a diagnostic value, rarely the case for FFA. For all these reasons, in most cases where angiographic work-up is required and choroidal involvement cannot be excluded, dual FFA and ICGA should be performed. 1 &#; 4

Fluorescein angiography (FFA) is being performed for over 40 years. As fluorescein sodium fluoresces in the wavelengths of the visible light, they mainly give information on the superficial structures of the fundus and therefore, mostly, only confirm signs already known to the clinician as in most instances OCT is already available when FFA is performed.

One of the essential procedures performed in uveitis to complement clinical appraisal of the patient with intraocular inflammation is angiographic investigation of the posterior segment. Angiography may be performed to confirm elements already revealed by clinical examination or other investigational methods such optical coherence tomography (OCT). A second reason to perform an angiography is for better grading of the inflammation of the fundus. A third reason is to make a good baseline inventory of inflammatory involvement to subsequently use it for follow-up purposes. In follow-up situations, angiography is usually performed to monitor disease intensity and impact of therapy [ ].

It is beyond the scope of this article to describe the equipment and instruments used. However, there are some adjustments of the angiographic timing and technique for uveitis to be observed. Early frames are important and should be taken up to two to three minutes from the posterior pole as information from the choriocapillaris is available only up to about 60 seconds and will indicate whether there is any choriocapillaris filling defect. Regarding the retinal circulation, early frames give us information on the arteriovenous circulation time, the presence of disc neovessels (NVD), and presence of diffuse capillary leakage. At five to seven minutes, full 360 degree panoramas are performed and thereafter (10&#;12 minutes), again, posterior pole frames are taken and frames of specific lesions identified during previous angiograsphic times.

Fluorescein angiographic concepts, classically described since more than 40 years, also apply to inflammatory diseases [ ]. Increased fluorescence can be due to three main mechanisms: (1) leakage producing pooling (in a space) or staining (in tissues); (2) increased transmission of fluorescence due to fundus atrophy with removal of the RPE producing larger hyperfluorescent areas or due to smaller window-defects produced by areas of RPE defects; (3) presence of abnormal vessels (retinal vessels or choroidal neovascular membranes). Decreased fluorescence can be either due to transmission decrease (blockage) or filling defect (vascular delayed perfusion or non perfusion).

Two crucial principles characterize FNa. Firstly, fluorescein has a micromolecular behavior. This means that fluorescein easily gets out, at the slightest breakdown of the hemato-retinal barrier, from the usually impermeable retinal vessels. It diffuses easily into tissues but is also quickly washed out. The other characteristic is that fluorescein fluoresces at 520&#;530 nanometers within the wavelengths of visible light and is, therefore, blocked by the retinal pigment epithelium (RPE), giving no useful information on choroidal circulation and compartment except on the choriocapillaris during the first 40&#;60&#; of angiography. These characteristics determine the use and limitations of fluorescein angiography.

In fluorescein angiography, a natural dye, fluorescein sodium (FNa) is injected intravenously to analyze the blood circulation of the ocular fundus, mainly the retina. Fluorescein sodium is a small hydrosoluble molécule of 354 daltons of which 80% is bound to proteins and 20% is free, the latter free form being responsible for the emission of fluorescing light.

FFA SYMPTOMATOLOGY AND USE FOR UVEITIS

The clinician should know what information he can expect from FFA and be aware that superficial structures such as the optic nerve head, retinal vessels, retina and sub retinal space will be highlighted by FFA. In addition, FFA is an ideal tool to analyze the RPE and atrophic areas that can be seen, thanks to the underlying fluorescence seen through window defects of the RPE or due to complete absence of the RPE screen in chorioretinal atrophy. FFA is an inadequate method to analyze the choroid as the RPE is a screen for the choroidal fluorescence. However, during early FFA frames (up to 60&#;), because early choroidal fluorescence is so strong, information on the choriocapillaris is also obtained. Moreover, choroidal fluorescence is at the base of the two main FFA concepts of &#;window defect&#; when choroidal fluorescence is seen through areas of missing RPE and the concept of &#;masking (blocking) effect&#; in which choroidal fluorescein background fluorescence is attenuated or lost due to decreased transmission, which can be seen in individuals with African ancestry.

The FFA signs are not analyzed according to angiographic principles exposed in , but according to the anatomical structure, going antero-posteriorly from optic disc to choriocapillaris.

Before analyzing the different structures, it should be specified that some posterior uveitis can cause retinal or preretinal hemorrhages producing hypofluorescence through a &#;blocking effect&#;.

Optic disc

FFA is an adequate and sensitive method to detect inflammation of the optic discs which appear hyperfluorescent and, in addition, which leak in case of severe inflammation. FFA is especially helpful when clinical examination does not clearly reveal inflammation of the optic discs. Subclinical papillitis (hyperfluorescence) can be detected by FFA and this is useful in conditions known to be bilateral when the controlateral, apparently uninvolved eye, shows angiographic papillitis. FFA usually helps differentiate papillitis from papilloedma. In both conditions there are early dilated capillaries and late hyperfluorescence. The papilla is, however, more swollen in papilloedema than in papillitis. Papillitis is more prone to produce leakage [ ].

Retinal vessels

FFA furnishes three types of information on the retinal circulation which include: (1) imaging of inflammatory damage to vessel walls in retinal vasculitis, (2) display occlusive vasculopathy of retinal arteries or arterioles and (3) detection of retinal neovessels situated at the disc (NVD) or elsewhere in the retina (NVE).

Retinal vasculitis or inflammatory vasculopathy

Retinal vessels normally are highly impermeable due to the tight junctions between endothelial cells and they usually don't leak. However, the slightest inflammation of retinal vessel walls allows the small fluorescein molecule (359 daltons) to leak out of vessels and FFA is therefore a very sensitive to detect retinal vessel inflammation mostly veins [ ]. Leakage is a characteristic of the disease for Behçet's uveitis, intermediate uveitis of the pars planitis type, intermediate uveitis related to multiple sclerosis, intermediate uveitis of unknown cause and birdshot chorioretinopahty [ ]. However, every inflammatory focus of the fundus will have areas of leaking vessels in its vicinity. A special situation is represented by the particular vasculopathy of frosted branch angiitis with thick perivascular sheathing not only visible on FFA but also on fundus examination. Frosted branch angiitis can be associated with herpetic or cytomegalovirus, especially in the context of AIDS, as well as with toxoplamic retinitis, SLE, Crohn's disease and leukemia and lymphomas.5

Occlusive retinal vasculopathy

Areas of non perfusion are best investigated with FFA that shows the extent of retina involved due to ischemia. Inflammatory conditions that often present retinal non perfusion include, without being exhaustive, Behçet's uveitis [ ], sarcoidosis, systemic lupus erythematosus (SLE), Eales' disease or more appropriately called tuberculous hypersensitivity retinal vasculitis, IRVAN (Idiopathic retinal vasculitis, aneurisma and neuro-retinitis) and Susac's syndrome.

Retinal neovascularization is best demonstrated by fundus fluorescein angiography (FFA) and can complicate any of the conditions that produce retinal non perfusion6 [ ].

Retina

Peripheral retina

FFA is the investigation of choice of retinal foci that appear hypofluorescent in the early phases and progressively become hyperfluorescent. Some conditions such as birdshot chorioretinopathy produce profuse capillary leakage that produces bright and diffuse hyperfluorescence of the whole retina [ ]. The capillary leakage is sometimes such that there is not enough dye to mark the large veins. Gass interpreted this angiographic finding as perfusion delay. Indocyanine green angiography has helped us understand that the arteriovenous perfusion time is normal7,8 [ ].

Macula

Macular ischemia is best detected using FFA and has to be looked for in case of severe fundus inflammation such as Behçet's uveitis9,10 [Figure and ].

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Cystoid macular edema (CMO)

CMO is visible by funduscopy if it is sufficiently pronounced. Until recently, FFA was, however, recommended to have a precise image of the extent of CMO. Two grading systems of CMO have been put forward by the groups of Miyake and Yanuzzi.11,12 More recently, optical coherence tomography (OCT) has been used routinely for the evaluation of CMO. In the field of inflammatory diseases, however, FFA still has its place and should at least be performed in parallel with OCT at presentation of the patient, the follow-up being performed by repeated OCTs. Nevertheless, some of the inflammatory CMOs may not have any translation on OCT and are well identified by FFA [Figure and ].

Sub retinal space

Retinal pooling of retinal origin

Inflammation of the choriocapillaris mostly manifests by non perfusion with the consequence that the outer retina becomes ischemic which in turn produces leakage from reacting inner retinal capillaries. In some of the inflammatory choriocapillaropathies (formerly classified under the term of white dot syndromes), the process can be very pronounced as in some cases of APMPPE (acute posterior multifocal placoid pigment epitheliopathy) causing massive intraretinal and sub retinal pooling of fluorescein. Any other disease causing choroidal ischemia can cause similar pooling [ ].

Exudative retinal detachment of choroidal origin

Sub retinal fluid can also originate directly from the choroid such as in VKH (Vogt-Koyanagi-Harada) disease, where massive primary choroidal inflammation spills over to the retina and causes profuse leakage of liquid through the retinal pigment epithelium (RPE) forming exudative retinal detachments (ERD). Other diseases that can produce ERD include sympathetic ophthalmia and posterior scleritis [Figure and ].

Retinal pigment epithelium

Inhomogeneity of background fluorescence (mottled RPE)

Exudative retinal detachments produce RPE changes characterized by loss of RPE cells causing small areas of window defects as well as clumping of cells causing masking effect. This alternation of loss and clumping of RPE gives a mottled aspect on early angiographic frames due to increased transmission of background choroidal fluorescence (window effect) in some areas together with decreased transmission of choroidal fluorescence (blocking effect). Very often, the limits of the ERDs are well identified on FFA by a demarcation line between the affected and normal RPE called high water marks [ ].

Hyperfluorescent pinpoints

The leaking points where fluid passes from the choroid into the subretinal space accumulate a large amount of fluorescein dye and appear brightly hyperfluorescent on FFA (as well as on ICGA), which is explained by the deposition of fluorescein at this transit points [Figure and ].

Chorioretinal atrophy

Chorioretinal atrophy can be limited to the absence of the choriocapillaris-RPE complex or be a complete atrophy including the choroidal stroma. In the first case, the area is characterized by early hypofluorescence with large stromal choroidal vessels that are hyperfluorescent and well visible on a dark background.

In case of complete atrophy, the whole area is dark on early frames becoming hyperfluorescent on late frames reflecting the bare sclera that is impregnated with fluorescein, which easily diffuses in tissues due to its small size. Atrophic areas on the reverse appear hypofluorescent on ICGA as the large ICG molecular complex is not diffusing and does not impregnate the bare sclera [ ].

Choriocapillaris (up to 60&#;)

Unless the RPE screen is very dark, as in patients of African ancestry, the choriocapillaris fluorescence of the fluorescein dye can be seen despite the presence of the RPE in the early frames of fluorescein angiography when fluorescence is massive. These early frames can show areas of hypofluorescence due to choriocapillaris non perfusion. Alert clinicians understood and adequately interpreted these signs as due to choriocapillaris inflammation causing non perfusion well before ICGA became available and Deutman clearly indicated that disease in APMPPE was primarily due to the choriocapillaris and not the RPE.12 Indeed, because the time of access to the choroid is limited with FFA, it is not possible to tell whether this hypofluorescence is due to perfusion delay or complete non perfusion of the choriocapillaris. This can only be shown by ICGA when choriocapillaris is accessible during the whole angiographic sequence; and the interpretation of FFA signs by Deutman for diseases such as APMPPE (and other white dot syndromes) were indeed confirmed by ICGA findings12,13 [ ].

Choroidal inflammatory neovascularization

Inflammatory choroidal neovessels are mostly type 2 membranes and, therefore, readily accessible to FFA, being above the RPE. Occult choroidal neovessels are also detected by FFA showing progressive diffuse leakage in the macular area. In both these situations, complementary ICGA is, however, recommended. On one hand it can delineate occult membranes and furthermore, in case of CNV associated with a chorioretinal scars, ICGA allows to differentiate between a recurrent inflammatory focus and CNV, the former being early hypofluorescent whereas the latter appears hyperfluorescent since early angiographic frames. ICGA is also mandatory in case of CNV associated with a choriocapillaritis such as multifocal choroiditis, where it shows the extent of occult choriocapillaris non perfusion (iceberg constellation or complex) and hence the danger for CNV development14 [ ].

Study population

In this prospective interventional case series, all patients with non-resolving CSCR who were referred to Farabi Eye Hospital from December to April were enrolled. Non-resolving CSCR was defined as persistent subretinal fluid involving foveal area for at least three months based on enhanced depth imaging optical coherence tomography (EDI-OCT) images.

History of steroid usage (either systemic or ocular), Cushing's syndrome, diabetic retinopathy, high myopia (6 D and above), previous PDT or focal laser photocoagulation, treatment with anti-VEGF agents, choroidal polypoid vasculopathy, congenital macular or retinal disease, pregnancy, and posterior uveitis were noted as exclusion criteria. Written informed consent letter was obtained from all participants. The ethics committee of Tehran University of Medical Sciences approved this study (Code: IR.TUMS.FARABIH.REC..025). The study adhered to the tenets of the Helsinki Declaration.

Demographic data was collected and a comprehensive ocular examination and dye-based angiography (Heidelberg Spectralis, Heidelberg Engineering, Germany) was performed for all patients. Additionally, patients underwent EDI-OCT (Heidelberg Engineering Inc., Heidelberg, Germany) and OCT angiography (OCTA) (AngioVue, Optovue, Inc., Fremont, CA, USA) at baseline. Patients were non-randomly allocated to receive either FA guided or ICGA guided PDT. As our allocation was not random, there was a concern for unequal distribution of FA leakage patterns among the two groups. Therefore, from both groups, we selected eyes having both FA and ICG available, and compared the patterns of FA leakage between them. These FA leakage patterns were classified into discrete, multiple discrete, and diffuse leakage. We also evaluated areas of abnormal leakage in both FA and ICGA images. We determined whether these areas are compatible with each other, in both treated and asymptomatic fellow eye.

In FA group, areas of active leakage and in ICGA group, areas of choroidal vascular hyperpermeability in middle and late phases were considered for laser application. Meticulous attention was paid to exclude areas of window defect and staining in FA group, from laser treatment. All patients received intravenous infusion of Verteporfin (3 mg/m2) over eight minutes and were treated with laser two minutes later. The standard dose of 50 J/cm2 laser was applied for 83 s. For those who underwent ICGA, an interval of at least three days from ICGA, was considered for PDT application.

Patients were re-examined four months after PDT when thorough ophthalmic examinations, EDI-OCT and OCTA were performed. In addition, the following data was collected at baseline and four months after half-dose PDT; the treated eye, BCVA, the status of foveal subretinal fluid (FSRF), SFCT, CVI, PED area, total PDT laser energy dose, area and number of spots applied.

Image acquisition and analysis protocol

EDI-OCT was performed using Heidelberg spectralis OCT. All EDI OCT images were performed between 9 and 12 AM. Patients were positioned appropriately and a 5&#;×&#;5 mm image centered at the fovea was obtained for each eye. Similarly, OCTA images were obtained from the fovea (6&#;×&#;6 mm) area. Images with poor quality or from eyes with media opacity precluding acceptable image acquisition, were excluded from the study. Segmentation errors were manually corrected by a blind expert investigator (A.M). Image analysis was conducted by FIJI (an expanded version of ImageJ software, version 1.51 h; National Institutes of Health, Bethesda, Maryland, available at http://imagej.nih.gov/Fiji/). Both EDI-OCT and OCTA images were exported into FIJI software. Imaging measurements were performed by two investigators who were blind to the treatment label (K.F, A.M). The measurement of retina and choroid was performed manually in horizontal B-scan EDI-OCT images centered at fovea; thickness of the choroid was considered as the distance between the outer border of the RPE and the choroid&#;sclera border, and the thickness of retina was considered as the distance between internal limiting membrane and RPE (Fig. 1a).

Measurement of retinal and choroidal thickness, choroidal vascular index (CVI) and pigment epithelial detachment (PED) area: Choroidal area is delineated followed by binarization of the region of interest (a). The image is then converted to RGB using color threshold. CVI is calculated by dividing areas without pixels as choroidal lumens to total choroidal area (b). En-face RPE elevation map, is generated by OCTA device. This map represents PED as hot regions. PED area is manually delineated, using FIJI software (c).

Full size image

In order to measure CVI, horizontal B-scan of EDI-OCT images centered at fovea were imported in FIJI were used. The borders of the choroid were selected using free hand tool of the software. The upper margin was RPE and the lower margin was the choroidoscleral border. The nasal margin was the temporal edge of the optic nerve head and the temporal margin was 8 mm from the temporal edge of the optic nerve head. To binarize choroidal area in OCT images, a modified Niblack method was used as previously described12. Briefly, three choroidal vessels with lumens larger than 100 \(\mu{m}\) were randomly selected by the oval selection tool of the toolbar, and the average reflectivity of these areas was determined by the software. The average brightness was set as the minimum value to reduce the noise in the OCT image. The ROI was selected and set by the ROI manager in the OCT image. Then, the image was converted to 8 bits and adjusted by the auto local threshold of Niblack. The binarized image was reconverted to an RGB image, and the luminal area was determined using the color threshold tool. The light pixels were described as the choroidal stroma or interstitial area and the dark pixels were defined as the luminal area (LA). TCA, LA, and stromal area (SA) was automatically calculated. (Fig. 1b) Herein, we refer to the ratio of LA to TCA as the choroidal vascular index (CVI). They were calculated for all patients at baseline and month four following PDT.

Measurement of PED area was performed as previously described13. Briefly, En-face RPE elevation maps, obtained from OCTA imaging were used to depict the total PED area. Segmentation errors were manually corrected, considering the corresponding OCT B-scan. The images were imported to FIJI and scales were set accordingly. In these images, areas of RPE elevation appears as hot colors in heat map image. Consequently, borders of PED could be manually selected using the free hand tool of FIJI (Fig. 1c). A blind expert investigator (A.M) corrected the segmentation error and delineated the PED borders. PED area at baseline and follow-up images were measured and used for analysis.

Statistical analysis

Data were entered into 'IBM SPSS Statistics for Windows, version 23 (IBM Corp., Armonk, N.Y., USA) and reported by descriptive statistics such as mean, standard deviation for quantitative values, and number and percentage for qualitative ones. The normality of the quantitative data was assessed using the Kolmogorov-Smirnov test. Inter- and intra-group analyses of normally-distributed data at baseline and four months after PDT were performed by independent t-test and paired t-test, respectively. Non-normally distributed data at baseline and four months after were compared by Mann-Whitney U test and Wilcoxon test. The analysis of covariance (ANCOVA) was used to adjust for baseline measures and to provide an unbiased estimate of the mean group difference of the 4 months results in two groups. Also, in this study, qualitative variables were compared using Fisher's exact test and Chi-square. A p-value less than 0.05 was determined as statistically significant. Statistical analysis was performed by a statistician blinded to group label (KF).