Off-axis acquisition of spectral domain optical coherence tomography (SDOCT) images has been shown to increase total retinal thickness (TRT) measurements. We analyzed the reproducibility of TRT measurements obtained using either the retinal pigment epithelium (RPE) or Bruch's membrane as reference surfaces in off-axis scans intentionally acquired through multiple pupil positions. Five volumetric SDOCT scans of the macula were obtained from one eye of 25 normal subjects. One scan was acquired through a central pupil position, while subsequent scans were acquired through four peripheral pupil positions. The internal limiting membrane, the RPE, and Bruch's membrane were segmented using automated approaches. These volumes were registered to each other and the TRT was evaluated in 9 Early Treatment of Diabetic Retinopathy Study (ETDRS) regions. The reproducibility of the TRT obtained using the RPE was computed using the mean difference, coefficient of variation (CV), and the intraclass correlation coefficient (ICC), and compared to those obtained using Bruch's membrane as the reference surface. A secondary set of 1545 SDOCT scans was also analyzed in order to gauge the incidence of off-axis scans in a typical acquisition environment. The photoreceptor tips were dimmer in off-axis images, which affected the RPE segmentation. The overall mean TRT difference and CV obtained using the RPE were 7.04 ± 4.31 μm and 1.46%, respectively, whereas Bruch's membrane was 1.16 ± 1.00 μm and 0.32%, respectively. The ICCs at the subfoveal TRT were 0.982 and 0.999, respectively. Forty-one percent of the scans in the secondary set showed large tilts (> 6%). RPE segmentation is confounded by its proximity to the interdigitation zone, a structure strongly affected by the optical Stiles-Crawford effect. Bruch's membrane, however, is unaffected leading to a more robust segmentation that is less dependent upon pupil position. The way in which OCT images are acquired can independently affect the accuracy of automated retinal thickness measurements. Assessment of scan angle in a clinical dataset demonstrates that off-axis scans are common, which emphasizes the need for caution when relying on automated thickness parameters when this component of scan acquisition is not controlled for.
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