Retinal Vasculature

The AngioVue provides a noninvasive OCT-based method for visualizing the vascular structures of the retina. It uses an 840-nm light source and has an A-scan rate of 70,000 scans/s and a bandwidth of 50 nm. Each volume contains 304 × 304 A-scans with two consecutive B-scans captured at each fixed position. Each volume scan is acquired in 3 seconds and consists of two orthogonal volumes that are used to minimize motion artifacts arising from microsaccades and fixation changes. The split-spectrum amplitude-decorrelation angiography (SSADA) method was used to capture the dynamic motion of the red blood cells and provide a high-resolution 3D visualization of perfused retinal vasculature.^{30 (link)}The AngioVue characterizes vascular information at various user-defined retinal layers as a vessel density map and quantitatively as vessel density (%) (Fig. 1). Vessel density was automatically calculated as the proportion of measured area occupied by flowing blood vessels defined as pixels having decorrelation values acquired by the SSADA algorithm above the threshold level.
For this report, we analyzed vessel density in the peripapillary RNFL in images with a 4.5 × 4.5-mm field of view centered on the optic disc. Vessel density within the RNFL was measured from internal limiting membrane (ILM) to RNFL posterior boundary using standard AngioVue software (version 2015.1.0.90). Measurements were obtained in two areas. Whole enface image vessel density (wiVD) was measured in the entire 4.5 × 4.5-mm image, and circumpapillary vessel density (cpVD) was calculated in the region defined as a 750-μm-wide elliptical annulus extending from the optic disc boundary (Fig. 1).
Image quality review was completed on all scans according to a standard protocol established by the University of California, San Diego Imaging Data Evaluation and Analysis (IDEA) Reading Center. Trained graders reviewed scans and excluded poor quality images, defined as images with (1) a signal strength index of less than 48, (2) poor clarity, (3) residual motion artifacts visible as irregular vessel pattern or disc boundary on the enface angiogram, (4) local weak signal, or (5) RNFL segmentation errors. The location of the disc margin was reviewed for accuracy and adjusted manually if required.

Macular pigment imaging by dual wavelength AFI was performed on a Heidelberg MultiColor Spectralis as previously described.^{11 (link)} After pupil dilation, the subject's macula was raster scanned over 30° centered on the fovea by alternating blue and green laser light (485.6 and 516.7 nm, respectively) for approximately 30 seconds while AFIs of RPE lipofuscin for each excitation wavelength were collected and averaged. Autofluorescence detection was restricted to wavelengths above 530 nm with the help of a barrier filter. Specialized software then performed digital subtraction of the green excitation AFI from the blue excitation AFI using a correction factor to account for the fact that the blue excitation is not quite at the peak of macular pigment absorption (460 nm) and that there is still a substantial amount of macular pigment absorbance with the green excitation. The instrument's effective extinction coefficients, K_mp(Λ), are 0.789 for 485.6 nm and 0.205 for 516.7 nm, and the correction factor is: 1/[K_mp(485.6) − K_mp(516.7)] = 1.71, based on the image processing method described by Delori and colleagues^{32 (link)} using the macular pigment extinction coefficients calculated by Stockman and Sharp.^{33 (link)} In order to compensate for background signal, an offset parameter (“OFF”) is subtracted. This value is recorded by the system internally during the acquisition of the blue/green AFI with the lasers turned off.
A subtracted macular pigment autofluorescence attenuation image is produced showing a white region centered on the fovea corresponding to the macular carotenoid pigment (Fig. 1). The instrument calculates the average MPOD, SD, and range of MPOD levels along a series of concentric one-pixel width circles. The results are then plotted on a graph from 0° to 15° with a red curve corresponding to the average MPOD at each eccentricity, a green region corresponding to the SD of the average MPOD, and a blue region corresponding to the high and low range of MPOD. The user must choose a reference eccentricity where the MPOD is defined as zero. We chose 9.0° because the vast majority of subjects had near baseline measurements at this distance from the fovea, and readings beyond this eccentricity would likely be affected by retinal vasculature or the optic nerve, typically manifested as an increase of SD and range at eccentricities beyond 9°. For the instrument's automated results table, the user not only selects the zero point radius (green vertical line; 233 pixels at 9°; “plateau” column on the report) but can also choose two other analysis eccentricities. We routinely used 0.5° (red vertical line; 12 pixel radii at 0.47°) and 2° (blue vertical line; 51 pixel radii at 1.99°). The most important parameters from the report that we used for our analyses were the “average OD on radius,” which we report as “MPOD X°” (macular pigment optical density at X°) corresponding to the 360° averaged MPOD at that particular radius/eccentricity and “OD sum of volumes,” which we report as “macular pigment volume under the curve at X°” (MPVUCX°), which is the integral of the total MPOD within X° of the fovea and should correspond to the total macular pigment within that particular region always using 9° as the reference eccentricity.