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PurposeTo compare macular and peripapillary vessel density values calculated on optical coherence tomography angiography (OCT-A) images with different algorithms, elaborate conversion formula, and compare the ability to discriminate healthy from affected eyes.MethodsCross-sectional study of healthy subjects, patients with diabetic retinopathy, and glaucoma patients (44 eyes in each group). Vessel density in the macular superficial capillary plexus (SCP), deep capillary plexus (DCP), and the peripapillary radial capillary plexus (RCP) were calculated with seven previously published algorithms. Systemic differences, diagnostic properties, reliability, and agreement of the methods were investigated.ResultsHealthy eyes exhibited higher vessel density values in all plexuses compared to diseased eyes regardless of the algorithm used (p<0.01). The estimated vessel densities were significantly different at all the plexuses (p<0.0001) as a function of method used. Inter-method reliability and agreement was mostly poor to moderate. A conversion formula was available for every method, except for the conversion between multilevel and fixed at the DCP. Substantial systemic, non-constant biases were evident between many algorithms. No algorithm outperformed the others for discrimination of patients from healthy subjects in all the retinal plexuses, but the best performing algorithm varied with the selected plexus.ConclusionsAbsolute vessel density values calculated with different algorithms are not directly interchangeable. Differences between healthy and affected eyes could be appreciated with all methods with different discriminatory abilities as a function of the plexus analyzed. Longitudinal monitoring of vessel density should be performed with the same algorithm. Studies adopting vessel density as an outcome measure should not rely on external normative databases.