Quantitative fundus autofluorescence in healthy eyes

Abstract

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.

Keywords: lipofuscin; melanin; quantitative fundus autofluorescence; retina; retinal pigment epithelium; scanning laser ophthalmoscope.

Figure 1

qAF by image analysis. Measurement areas (outlined in white) consist of three concentric rings (outer, middle, and inner) divided into eight segments, and a circular foveal area. Values used in the current work were obtained in the middle ring marked by thickened lines. The positions of the segments are all dependent upon the horizontal distance FD between the fovea (+) and the temporal edge of the disc (white line). The radii of the centerlines expressed in pixels (or in degrees visual angle, for the average value of FD = 315 pixels), for the outer, middle, and inner, are 0.90 × FD (11.1°), 0.68 × FD (8.4°), and 0.46 × FD (5.7°), respectively. The thickness of the segments is 0.2 × FD pixels (2.5°) and the angles subtended by the segments are 40°. The central circle (fovea) has a radius of 0.1 × FD pixels (1.2°). The internal fluorescent reference (top) was recorded simultaneously with the image; the GL_R was measured in the rectangular area outlined in black. The area of highest AF in the image is indicated by a square and cross.

Figure 2

Contour plots describing the average distribution of the AF obtained from the 97 subjects with data for both eyes. The data is presented as a left eye. The axes indicate the retinal eccentricity in degrees relative to the center of the fovea. The numbers for each contour are relative qAF (qAF_i/qAF₈) where qAF₈ is the mean of the eight segments of the middle ring delineated by the two concentric interrupted black lines. The sampling areas were the 24 segments (+ sign; areas: 6600–12,400 pixels²) and additional locations (X sign; areas: 2000–10,000 pixels²) near the center and at the edges and corners of the image. Single dots are locations where linear or quadratic interpolations were used. The coefficients of variation for these relative data were 4% to 6% for the middle ring, 4% to 9% for the inner and outer rings (except near the optic disc: 9%–14%), and 15% to 25% inside the inner ring reflecting the variability in amount and extent of RPE melanin and of macular pigment. The gray rectangle marks the position of the internal fluorescent reference.

Figure 3

qAF in 277 subjects (aged 5–60 years) with healthy retinal status. Mean qAF₈ intensity units, obtained by averaging the eight segments in the middle ring shown in Figure 1, are plotted as a function of age and subjects are grouped according to race/ethnicity. The solid curve is an exponential fit to the full sample (374 eyes). The two brackets at the arbitrary qAF values (125 and 350 qAF-units) illustrate the ±95% confidence limits (twice the SD) corresponding to the measurement variability (±9.4% of qAF).

Figure 4

Average qAF (solid line) and 95% confidence limits (dashed lines) for individuals reporting a single race/ethnicity.

Figure 5

Comparison of the spatial distribution of the AF determined in the current study using the SLO (Exc.: 488 nm; thick lines, filled symbols) and by fluorophotometry (Exc.: 550 nm; interrupted lines, open symbols) in a previous study. The values are plotted as a function of distance from the fovea along the nasal (N,n) to temporal (T,t) and superior (S,s) to inferior (I,i) axes (linear scale). Data were normalized to the average value at approximately 8.5° from the fovea (center location of the middle ring) (qAF_i/qAF₈). The profiles measured in this study are affected by macular pigment absorption, whereas those measured previously were not. qAF values were determined within a 30° SLO field.

Figure 6

Age-related change in AF for data acquired with the cSLO in this study (Exc.: 488 nm) and with the fundus fluorophotometer (FFP) in a previous study (Exc.: 550 nm). The FFP data was normalized to the mean qAF at 20 years of age. The interrupted curves are fits through the original data, not accounting for media absorption. High absorption of the 488-nm excitation in older lenses causes the uncorrected cSLO data to decrease for ages older than 55 years. The continuous lines are the media corrected (subscript MC) qAF's obtained using the same algorithm (Equation 2) with a coefficient of 5.56 × 10⁻⁵ for the 488-nm excitation and 1.33 × 10⁻⁵ for 550-nm excitation. The thin line (FFP_MC*) is the FFP data corrected using media absorption derived from a reflectometry method.