Our methodology has several limitations. Study participants self-reported their past medical histories. To ensure a healthy retina, clinical fundus photographs and OCT scans of the macula and retinal nerve fiber layer surrounding the optic nerve head were obtained. No vital sign or laboratory testing was performed. An important limitation to the study was the relatively low number of individuals older than 70 years. Future studies should develop a larger normative database with evenly distributed sample size and wider age range that also control for sex- and race-related differences in vascular metrics as prior studies have shown sex- or race-bias in controls and disease eyes.
19,32,38,39 It is important to note that most previous studies did not correct for axial length magnification, as females have shorter eyes than males, the conclusions must be assessed carefully. Another limitation was that we used full layer scans containing the superficial and deep microvascular layers, and did not analyze them separately. This was performed to avoid the projection artifact of the superficial capillary layer onto the deeper capillary layers, which makes it difficult to study the deeper capillary layers separately. With the recent development of projection-resolved OCT-A,
40,41 future assessment and characterization of individual OCT-A vascular layers using our deviation mapping technique may further benefit diseases with layer-specific abnormalities. Newer studies using a 3D volumetric analysis of different ophthalmic structures, including the retinal vasculature, could prove to be the most accurate assessment yet.
42,43 The method in which our study generated deviation maps is another potential source of error. Since our normative database was constructed by measuring the parafoveal capillary density within multiple equidistant annuli around the FAZ margin, deviation maps generated with FAZ area outside the age group-specific normative range (
Fig. 3A) should be interpreted with caution. Also, the demarcation of the FAZ border was performed on the OCT-A image, and not on the original structural FAZ that may contain nonperfused blood vessels at its true anatomic border. This source of error can potentially be solved by delineating the FAZ borders based on the respective en face reflectance OCT images. Since prior studies have shown that averaged en face reflectance images can be used to reveal the original FAZ border before disease onset,
44,45 generating the eight annuli and the subsequent deviation map based on this original FAZ border may, therefore, provide more accurate assessment of disease severity. This is especially important when analyzing eyes with retinopathy where the FAZ area is significantly larger than normal.
44 In addition, our deviation mapping approach was performed under the assumption that capillary density is evenly distributed around the FAZ in normal eyes, which may limit its sensitivity for detecting subtle deviation at the parafoveal region where nonuniform distribution of vessel density has been reported previously.
33,46