Purpose: To evaluate the effect of signal strength and improper scan alignment on retinal nerve fiber layer (RNFL) thickness measurement variability. Design: Retrospective, longitudinal clinical study. Methods: All eyes of healthy subjects with at least 2 fast RNFL scan sessions were selected from the Diagnostic Innovations in Glaucoma Study. The chronological first scan was considered to be the baseline. Absolute differences in signal strength and RNFL thickness measurements between baseline and subsequent scans were calculated. Regression analysis was conducted to assess whether signal strength and scan shifts along the horizontal (nasal-temporal) but not the vertical (superior-inferior) axis affect average RNFL thickness measurements. Results: Ninety-four eyes of 94 subjects were included. All eyes were tested twice or more on the same visit, whereas 30 eyes were followed up longitudinally for 32.4 ± 13.3 months (1 scan per annual follow-up). For quadrants, absolute differences from baseline were greater than for average RNFL thickness and were significantly larger for scans acquired on separate visits. Average RNFL thickness increased only when the difference between the nasal and temporal quadrants increased (R2 = 0.16; P < .0001), suggesting it may be affected by horizontal but not vertical scan shifts. Differences in signal strength were associated with differences in average RNFL thickness (R2 = 0.19; P < .0001). Conclusions: Even under optimal testing conditions, scan quality can adversely effect the ability to detect change over time. Therefore, caution is warranted when detecting glaucomatous progression using scan series of different quality. Careful overall assessment of quadrants and average RNFL thickness measurements is suggested to help identify scan misalignment.
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