Effect of Age and Signal Strength Index on Retinal and Choriocapillaris Vessel Density Measured with Optical Coherence Tomography Angiography Andrew Sill, Oregon Health & Science University (OHSU) Co-Authors: Simon Gao, Yali Jia, Christina Flaxel, Andreas Lauer, Thomas Hwang, Phoebe Lin, David Huang, Steven Bailey Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 1

Background – Vessel Density Prior histologic analyses → retinal and choriocapillaris vascular networks’ macular vessel density decreases with age. Choriocapillaris capillary density diminished in a linear fashion throughout all decades (r = 0.56, P < 0.001). Ramrattan. IOVS, 1994. Mean number of retinal capillaries decreased in older (age > 66) than younger donors (age < 22) (P < 0.001). Cavallotti. CJO, 2004. Optical coherence tomography angiography (OCTA) provides a rapid, in vivo method for quantifying macular vessel density. Jia. PNAS, 2015. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 2

Background – Signal Strength Index (SSI) Signal strength index (SSI) – based on reflectance signal intensity of scan. Higher signal strength scores = higher scan quality. Influenced by patient-related factors (e.g. ocular media opacities, dry eyes, blinking, floaters, pupil size) and operator-related factors (e.g. scan focus, alignment). Studies with structural OCT have shown that signal strength may affect OCT quantification metrics. Zhang. TVST, 2015; Mwanza. J Glaucoma, 2011; Vizzeri. AJO, 2009. Many studies use SSI cutoffs (ranging from 30 to 50) to exclude low quality scans. Few studies have adjusted for the influence of SSI above a cutoff value. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 3

Background – Vessel Density & SSI Positive correlation between SSI and retinal vessel density has been observed. Analysis of vessel densities should consider the influence of SSI during interpretation. Gao. IOVS, 2016. Rao. J Glaucoma, 2017. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 4

Purpose To determine the effects of age and signal strength index on macular retinal vessel density and macular choriocapillaris vessel density derived with OCTA. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 5

Methods – Overview Observational cross-sectional study. Study participants (n=40) from Casey Eye Institute (CEI) Retina Clinics. Normal ocular exams and normal structural OCT. One study eye per participant was selected at random. Two consecutive 3 x 3-mm OCTA scans centered on the macula were obtained for each participant using the RTVue spectral domain OCT system (Avanti, Optovue, Inc). Exclusion criteria – SSI < 50 or evidence of poor fixation (e.g. motion artifact). Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 6

Methods – Segmentation Semi-automated segmentation using custom MATLAB software developed at Center for Ophthalmic Optics and Lasers (COOL) lab. Monitored automated cross-sectional segmentation and manually corrected any segmentation errors. Retinal slab – internal limiting membrane to outer border of outer plexiform layer. Choriocapillaris slab – 7-μm to 21-μm deep to Bruch’s membrane. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 7

Methods – Image Processing Retinal OCT Angiogram A – Excluded foveal avascular zone. B – Created binary image based on threshold value. A B Choriocapillaris OCT Angiogram A – Excluded projection artifact from retinal vessels. B – Created binary image based on threshold value. A B Vessel density – percentage (%) area occupied by pixels above threshold value (white) divided by total pixels in binary OCT angiogram. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 8

Results – Overview Two participants excluded due to poor image quality. Analyzed 38 eyes of 38 participants (16 males, 22 females). Mean age ± standard deviation was 63.6 ± 11.5 years (range 40-85). Within-visit repeatability (pooled standard deviation). Retinal vessel density – 4.72% Choriocapillaris vessel density – 1.10% Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 9

Retinal Vessel Density and SSI Simple linear regression. Positive correlation between retinal vessel density and SSI (R2 = 0.68, P < 0.001). This trend is consistent even at high SSI values (70 – 85). Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 10

Retinal Vessel Density and Age Simple linear regression. Negative correlation between retinal vessel density and age (R2 = 0.11, P = 0.04). Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 11

Results – Simple Linear Regression Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 12

Results – Multivariable Linear Regression Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 13

Results – Subgroup Analysis No difference in significant correlations with subgroup analyses. Limited to patients with no cataract or who were pseudophakic (n = 23). Limited to patients with no history of diabetes mellitus, hypertension, or smoking (n = 19). Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 14

Signal Strength Index and Age Simple linear regression. Negative correlation between signal strength index and age (R2 = 0.19, P < 0.01). Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 15

Signal Strength Index and Age Simple linear regression. Negative correlation between signal strength index and age (R2 = 0.33, P < 0.01). – All controls – Excluding cataracts Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 16

Results – Simple Linear Regression Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 17

Similar Studies Four studies have found a negative correlation between retinal vessel density and age. (Shahlaee. AJO, 2016; Wang. AJO, 2016; Yu. IOVS, 2015; Iafe. IOVS, 2016.) Only one of these studies accounted for SSI. (Shahlaee. AJO, 2016.) Two studies found no correlation between retinal vessel density and age when accounting for SSI. (Rao. J Glaucoma, 2017; Gadde. IOVS, 2016.) SSI is negatively correlated with age. (Shahlaee. AJO, 2016.) Choriocapillaris vessel density is not correlated with age. (Wang. AJO, 2016.) Did not evaluate relationship of choriocapillaris vessel density with SSI. ✓ ✗ ✓ ✓ ✓ Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 18

Limitations Small sample size with limited patient age range (40 – 85 years old). Unable to make conclusions about uncorrelated results. Unable to perform a detailed multivariable model to include diabetes, hypertension, and smoking as predictor variables. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 19

Conclusions Choriocapillaris vessel density was not associated with age or SSI using OCTA. The apparent relationship between retinal vessel density and age was not due to vascular aging changes but was an artifact due to lower OCT signal strength in older participants. The influence of SSI should be accounted for during quantitative analysis of retinal vessel density using OCTA. Methods may include reflectance compensation, as described by Gao et al., or statistical methods. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 20

Acknowledgements Casey Eye Institute – Retina Division Faculty Steven Bailey, Christina Flaxel, Andreas Lauer, Thomas Hwang, Phoebe Lin, and David Huang. Casey Eye Institute – COOL lab Simon Gao, Yali Jia, Liang Liu, David Poole, Kathleen Torok, Denzil Romfh, and the remainder of the COOL lab. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 21

References (In Order of Appearance) Ramrattan RS, Van der schaft TL, Mooy CM, De bruijn WC, Mulder PG, De jong PT. Morphometric analysis of Bruch's membrane, the choriocapillaris, and the choroid in aging. Invest Ophthalmol Vis Sci. 1994;35(6):2857-64. Cavallotti C, Artico M, Pescosolido N, Leali FM, Feher J. Age-related changes in the human retina. Can J Ophthalmol. 2004;39(1):61-8. Jia Y, Bailey ST, Hwang TS, et al. Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye. Proc Natl Acad Sci USA. 2015;112(18):E2395- 402. Zhang X, Iverson SM, Tan O, Huang D. Effect of Signal Intensity on Measurement of Ganglion Cell Complex and Retinal Nerve Fiber Layer Scans in Fourier-Domain Optical Coherence Tomography. Transl Vis Sci Technol. 2015;4(5):7. Mwanza JC, Bhorade AM, Sekhon N, et al. Effect of cataract and its removal on signal strength and peripapillary retinal nerve fiber layer optical coherence tomography measurements. J Glaucoma. 2011;20(1):37-43. Vizzeri G, Bowd C, Medeiros FA, Weinreb RN, Zangwill LM. Effect of signal strength and improper alignment on the variability of stratus optical coherence tomography retinal nerve fiber layer thickness measurements. Am J Ophthalmol. 2009;148(2):249-255.e1. Gao SS, Jia Y, Liu L, et al. Compensation for Reflectance Variation in Vessel Density Quantification by Optical Coherence Tomography Angiography. Invest Ophthalmol Vis Sci. 2016;57(10):4485-92. Rao HL, Pradhan ZS, Weinreb RN, et al. Determinants of Peripapillary and Macular Vessel Densities Measured by Optical Coherence Tomography Angiography in Normal Eyes. J Glaucoma. 2017. Shahlaee A, Samara WA, Hsu J, et al. In Vivo Assessment of Macular Vascular Density in Healthy Human Eyes Using Optical Coherence Tomography Angiography. Am J Ophthalmol. 2016;165:39-46. Wang Q, Chan S, Yang JY, et al. Vascular Density in Retina and Choriocapillaris as Measured by Optical Coherence Tomography Angiography. Am J Ophthalmol. 2016;168:95-109. Yu J, Jiang C, Wang X, et al. Macular perfusion in healthy Chinese: an optical coherence tomography angiogram study. Invest Ophthalmol Vis Sci. 2015;56(5):3212-7. Iafe NA, Phasukkijwatana N, Chen X, Sarraf D. Retinal Capillary Density and Foveal Avascular Zone Area Are Age-Dependent: Quantitative Analysis Using Optical Coherence Tomography Angiography. Invest Ophthalmol Vis Sci. 2016;57(13):5780-5787. Gadde SG, Anegondi N, Bhanushali D, et al. Quantification of Vessel Density in Retinal Optical Coherence Tomography Angiography Images Using Local Fractal Dimension. Invest Ophthalmol Vis Sci. 2016;57(1):246-52. Wednesday, May 10, 2017 Effect of Age and SSI on Retinal and Choriocapillaris VD with OCTA 22