PURPOSE: The purpose of our study was to investigate the potential pitfalls linked to different vessel segmentation methods when using the built-in software of the Retinal Function Imager (RFI) for the analysis of retinal blood flow velocities (BFVs). METHODS: Ten eyes of nine healthy subjects were enrolled in the study. Retinal blood flow measurements were obtained with the RFI device with a 20° field of view imaging. The same grader segmented the retinal vasculature using the RFI software in two sessions with segments ranging from 50 to 100 pixels (“short segments”) or 100-200 pixels long (“long segments”). The blood flow velocities for the arteriolar and venular system were calculated and the percentage of excluded vessel segments with high coefficients of variation (>45%) was recorded and compared by paired t-test. Spearman correlation was used to analyze the link between the two measurements by the two vessel segmentation methods. RESULTS: The number of analyzed vessel segments did not differ significantly in the two groups (28.6±2.6 short and 26.7±4.6 long segments, respectively), while the percent of acceptable segments was significantly higher in the long segment group (65.2±11.4% vs 85.2±5.87%, p=0.001). All subjects in the short segment group had more than 15% of vessel segments rejected, while in the long segment group only three subjects had a rejection rate of 15% (16.7%, 18.7% and 28%). Both arteriolar and venular velocities were lower in the short segment group, although it reached significance only in the case of the arteriolar velocities (3.93±0.55 vs 4.45±0.76 mm/s, p=0.036 and 2.95±0.56 vs. 3.17±0.84 mm/s, p=0.201 for arterioles and venules, respectively). Only the venular velocities showed significant correlation (p=0.003, R(2)=0.67) between the two groups. CONCLUSIONS: Our results suggest that BFV measurements by the RFI may be affected by the segment length and therefore care should be taken when choosing the vessel segment lengths used during the analysis of RFI data. Long segments of 100-200 pixels (400-800 μm) seem to provide more robust measurements which can be explained by the analysis methodology of the RFI device
