Jonathan P. Greenberg

Quantitative measurements of autofluorescence with the scanning laser ophthalmoscope.

PURPOSE To evaluate the feasibility and reliability of a standardized approach for quantitative measurements of fundus autofluorescence (AF) in images obtained with a confocal scanning laser ophthalmoscope (cSLO). METHODS AF images (30°) were acquired in 34 normal subjects (age range, 20-55 years) with two different cSLOs (488-nm excitation) equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, the zero GL, and the magnification, to give quantified autofluorescence (qAF). Images from subjects and fixed patterns were used to test detector linearity with respect to fluorescence intensity, the stability of qAF with change in detector gain, field uniformity, effect of refractive error, and repeatability. RESULTS qAF was independent of detector gain and laser power over clinically relevant ranges, provided that detector gain was adjusted to maintain exposures within the linear detection range (GL < 175). Field uniformity was better than 5% in a central 20°-diameter circle but decreased more peripherally. The theoretical inverse square magnification correction was experimentally verified. Photoreceptor bleaching for at least 20 seconds was performed. Repeatability (95% confidence interval) for same day and different-day retests of qAF was ±6% to ±14%. Agreement (95% confidence interval) between the two instruments was <11%. CONCLUSIONS Quantitative AF imaging appears feasible. It may enhance understanding of retinal degeneration, serve as a diagnostic aid and as a sensitive marker of disease progression, and provide a tool to monitor the effects of therapeutic interventions.

The Inner Segment/Outer Segment Border Seen on Optical Coherence Tomography Is Less Intense in Patients with Diminished Cone Function

UNLABELLED PURPOSE; The integrity of the inner segment ellipsoid (ISe) band, previously called the inner segment/outer segment (IS/OS) border, seen on optical coherence tomography (OCT) scans is of clinical significance. To better understand the influence of cones on the appearance of this band, the intensity of its signal in patients with diminished cone function was examined. METHODS Horizontal line scans through the fovea of 30 healthy controls, 10 patients with achromatopsia (A), and six with cone dystrophy (CD) were obtained with frequency domain (fd) OCT. The fdOCT borders were segmented with a computer-aided manual technique. The ISe was divided into regions 60.1 μm wide and 19.5 μm deep. The relative ISe intensity of each region was defined as its intensity divided by the intensity of a local region, which extended in depth from the choroid to the retinal ganglion cell/retinal nerve fiber layer. RESULTS Except for the central fovea, all patients had a clear ISe band across the region studied, ± 3 mm from the foveal center. However, the relative ISe intensity was significantly lower (P < 0.0001) in patients (A: 1.14 ± 0.14; CD: 1.27 ± 0.14), than in controls (1.61 ± 0.16). There were no differences in the relative intensity of the other retinal layers. CONCLUSIONS Although present, the intensity of this ISe band is lower in patients with diminished cone function than it is in healthy controls. This is consistent with the hypothesis that both rod and cone receptors must be absent or damaged for the ISe band to be missing.

Quantitative fundus autofluorescence in recessive Stargardt disease.

PURPOSE To quantify fundus autofluorescence (qAF) in patients with recessive Stargardt disease (STGD1). METHODS A total of 42 STGD1 patients (ages: 7-52 years) with at least one confirmed disease-associated ABCA4 mutation were studied. Fundus AF images (488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density to yield qAF. Texture factor (TF) was calculated to characterize inhomogeneities in the AF image and patients were assigned to the phenotypes of Fishman I through III. RESULTS Quantified fundus autofluorescence in 36 of 42 patients and TF in 27 of 42 patients were above normal limits for age. Young patients exhibited the relatively highest qAF, with levels up to 8-fold higher than healthy eyes. Quantified fundus autofluorescence and TF were higher in Fishman II and III than Fishman I, who had higher qAF and TF than healthy eyes. Patients carrying the G1916E mutation had lower qAF and TF than most other patients, even in the presence of a second allele associated with severe disease. CONCLUSIONS Quantified fundus autofluorescence is an indirect approach to measuring RPE lipofuscin in vivo. We report that ABCA4 mutations cause significantly elevated qAF, consistent with previous reports indicating that increased RPE lipofuscin is a hallmark of STGD1. Even when qualitative differences in fundus AF images are not evident, qAF can elucidate phenotypic variation. Quantified fundus autofluorescence will serve to establish genotype-phenotype correlations and as an outcome measure in clinical trials.

Quantitative Fundus Autofluorescence in Healthy Eyes

PURPOSE Fundus autofluorescence was quantified (qAF) in subjects with healthy retinae using a standardized approach. The objective was to establish normative data and identify factors that influence the accumulation of RPE lipofuscin and/or modulate the observed AF signal in fundus images. METHODS AF images were acquired from 277 healthy subjects (age range: 5-60 years) by employing a Spectralis confocal scanning laser ophthalmoscope (cSLO; 488-nm excitation; 30°) equipped with an internal fluorescent reference. For each image, mean gray level was calculated as the average of eight preset regions, and was calibrated to the reference, zero-laser light, magnification, and optical media density from normative data on lens transmission spectra. Relationships between qAF and age, sex, race/ethnicity, eye color, refraction/axial length, and smoking status were evaluated as was measurement repeatability and the qAF spatial distribution. RESULTS qAF levels exhibited a significant increase with age. qAF increased with increasing eccentricity up to 10° to 15° from the fovea and was highest superotemporally. qAF values were significantly greater in females, and, compared with Hispanics, qAF was significantly higher in whites and lower in blacks and Asians. No associations with axial length and smoking were observed. For two operators, between-session repeatability was ± 9% and ± 12%. Agreement between the operators was ± 13%. CONCLUSIONS Normative qAF data are a reference tool essential to the interpretation of qAF measurements in ocular disease.

Outer Retinal Tubulation in Degenerative Retinal Disorders

Purpose: To demonstrate outer retinal tubulation (ORT) in various degenerative retinal disorders. Methods: This was a retrospective review of the multimodal imaging of 29 eyes of 15 patients with various retinal dystrophies and inflammatory maculopathies manifesting ORT. The morphologic features of ORT and its evolution over time were analyzed using spectral-domain optical coherence tomography data. Results: Outer retinal tubulation was identified as round or ovoid structures with hyperreflective borders in pattern dystrophy (six eyes), acute zonal occult outer retinopathy (five eyes), retinitis pigmentosa (four eyes), Stargardt disease (four eyes), gyrate atrophy (two eyes), choroideremia (two eyes), and various other degenerative conditions. These structures appeared to develop from the invagination of photoreceptors at the junction of intact and atrophic outer retina. During follow-up, the number and distribution of ORT largely remained stable. As zones of atrophy enlarged, the frequency of ORT appeared to increase. The ORT structures were found in <10% of patients with retinitis pigmentosa, Stargardt disease, or pattern dystrophy. Conclusion: Outer retinal tubulation is found in various degenerative retinal disorders that share in common damage to the outer retina and/or retinal pigment epithelium. The presence of ORT may be an indicator of underlying disease stage and severity.

Quantitative Fundus Autofluorescence and Optical Coherence Tomography in Best Vitelliform Macular Dystrophy

PURPOSE Quantitative fundus autofluorescence (qAF), spectral domain optical coherence tomography (SD-OCT) segmentation, and multimodal imaging were performed to elucidate the pathogenesis of Best vitelliform macular dystrophy (BVMD) and to identify abnormalities in lesion versus nonlesion fundus areas. METHODS Sixteen patients with a clinical diagnosis of BVMD were studied. Autofluorescence images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density, to yield qAF. Horizontal SD-OCT scans were obtained and retinal layers manually segmented. Additionally, color and near-infrared reflectance (NIR-R) images were registered to AF images. All patients were screened for mutations in BEST1. In three additional BVMD patients, in vivo spectrofluorometric measurements were obtained within the vitelliform lesion. RESULTS Mean nonlesion qAF was within normal limits for age. Maximum qAF within the lesion was markedly increased compared with controls. By SD-OCT segmentation, outer segment equivalent thickness was increased and outer nuclear layer thickness decreased in the lesion. Changes were also present in a transition zone beyond the lesion border. In subclinical patients, no abnormalities in retinal layer thickness were identified. Fluorescence spectra recorded from the vitelliform lesion were consistent with those of retinal pigment epithelial cell lipofuscin. CONCLUSIONS Based on qAF, mutations in BEST1 do not cause increased lipofuscin levels in nonlesion fundus areas.

Quantitative Fundus Autofluorescence in Mice: Correlation With HPLC Quantitation of RPE Lipofuscin and Measurement of Retina Outer Nuclear Layer Thickness

PURPOSE Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age. METHODS Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4(-/-), Abca4(+/-), and Abca4(+/+) mice (ages 2-12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed. RESULTS The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2-12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4(-/-) mice than in Abca4(+/+) mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4(-/-) and wild-type mice was more pronounced (approximately 3-4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4(-/-) mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age. CONCLUSIONS The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.

Structural and Functional Changes Associated with Normal and Abnormal Fundus Autofluorescence in Patients with Retinitis Pigmentosa

Purpose To analyze the structure and visual function of regions bordering the hyperautofluorescent ring/arcs in retinitis pigmentosa. Methods Twenty-one retinitis pigmentosa patients (21 eyes) with rings/arcs and 21 normal individuals (21 eyes) were studied. Visual sensitivity in the central 10° was measured with microperimetry. Retinal structure was evaluated with spectral-domain optical coherence tomography. The distance from the fovea to disruption/loss of the inner outer segment (IS/OS) junction and thicknesses of the total receptor plus retinal pigment epithelial complex and outer segment plus retinal pigment epithelial complex layers were measured. Results were compared with measurements of the distance from the fovea to the inner and outer borders of the ring/arc seen on fundus autofluorescence. Results Disruption/loss of the inner outer segment junction occurred closer to the inner border of the ring/arc and it was closer to the fovea in eight eyes. For 19 eyes, outer segment plus and receptor plus RPE complex thicknesses were significantly decreased at locations closer to the fovea than the appearance of the inner border of hyperautofluorescence. Mean visual sensitivity was decreased inside, across, and outside the ring/arc by 3.5 ± 3.8, 8.9 ± 4.8, and 17.0 ± 2.4 dB, respectively. Conclusion Structural and functional changes can occur inside the hyperfluorescent ring/arc in retinitis pigmentosa.

Hyperautofluorescent ring in autoimmune retinopathy.

Purpose: To report the presence of a hyperautofluorescent ring and corresponding spectral-domain optical coherence tomography (SD-OCT) features seen in patients with autoimmune retinopathy. Methods: All eyes were evaluated by funduscopic examination, full-field electroretinography, fundus autofluorescence, and SD-OCT. Further confirmation of the diagnosis was obtained with immunoblot and immunohistochemistry testing of the patients serum. Humphrey visual fields and microperimetry were also performed. Results: Funduscopic examination showed atrophic retinal pigment epithelium (RPE) associated with retinal artery narrowing but without pigment deposits. The scotopic and photopic full-field electroretinograms were nondetectable in three patients and showed a cone–rod pattern of dysfunction in one patient. Fundus autofluorescence revealed a hyperautofluorescent ring in the parafoveal region, and the corresponding SD-OCT demonstrated loss of the photoreceptor inner segment–outer segment junction with thinning of the outer nuclear layer from the region of the hyperautofluorescent ring toward the retinal periphery. The retinal layers were generally intact within the hyperautofluorescent ring, although the inner segment–outer segment junction was disrupted, and the outer nuclear layer and photoreceptor outer segment layer were thinned. Conclusion: This case series revealed the structure of the hyperautofluorescent ring in autoimmune retinopathy using SD-OCT. Fundus autofluorescence and SD-OCT may aid in the diagnosis of autoimmune retinopathy and may serve as a tool to monitor its progression.

Spectral-Domain Optical Coherence Tomography Staging and Autofluorescence Imaging in Achromatopsia

IMPORTANCE Evidence is mounting that achromatopsia is a progressive retinal degeneration, and treatments for this condition are on the horizon. OBJECTIVES To categorize achromatopsia into clinically identifiable stages using spectral-domain optical coherence tomography and to describe fundus autofluorescence imaging in this condition. DESIGN, SETTING, AND PARTICIPANTS A prospective observational study was performed between 2010 and 2012 at the Edward S. Harkness Eye Institute, New York-Presbyterian Hospital. Participants included 17 patients (aged 10-62 years) with full-field electroretinography-confirmed achromatopsia. MAIN OUTCOMES AND MEASURES Spectral-domain optical coherence tomography features and staging system, fundus autofluorescence and near-infrared reflectance features and their correlation to optical coherence tomography, and genetic mutations served as the outcomes and measures. RESULTS Achromatopsia was categorized into 5 stages on spectral-domain optical coherence tomography: stage 1 (2 patients [12%]), intact outer retina; stage 2 (2 patients [12%]), inner segment ellipsoid line disruption; stage 3 (5 patients [29%]), presence of an optically empty space; stage 4 (5 patients [29%]), optically empty space with partial retinal pigment epithelium disruption; and stage 5 (3 patients [18%]), complete retinal pigment epithelium disruption and/or loss of the outer nuclear layer. Stage 1 patients showed isolated hyperreflectivity of the external limiting membrane in the fovea, and the external limiting membrane was hyperreflective above each optically empty space. On near infrared reflectance imaging, the fovea was normal, hyporeflective, or showed both hyporeflective and hyperreflective features. All patients demonstrated autofluorescence abnormalities in the fovea and/or parafovea: 9 participants (53%) had reduced or absent autofluorescence surrounded by increased autofluorescence, 4 individuals (24%) showed only reduced or absent autofluorescence, 3 patients (18%) displayed only increased autofluorescence, and 1 individual (6%) exhibited decreased macular pigment contrast. Inner segment ellipsoid line loss generally correlated with the area of reduced autofluorescence, but hyperautofluorescence extended into this region in 2 patients (12%). Bilateral coloboma-like atrophic macular lesions were observed in 1 patient (6%). Five novel mutations were identified (4 in the CNGA3 gene and 1 in the CNGB3 gene). CONCLUSIONS AND RELEVANCE Achromatopsia often demonstrates hyperautofluorescence suggestive of progressive retinal degeneration. The proposed staging system facilitates classification of the disease into different phases of progression and may have therapeutic implications.