Grading of FAF and MP Exams

Atrophic lesions on FAF images were graded by RC using a semiautomated software tool (RegionFinder; Heidelberg Engineering) according to previously established grading protocols [13, 14]. Two distinct types of areas of decreased autofluorescence were quantified. Herein, the level of darkness was used to define an area of decreased autofluorescence qualitatively as being definitely or questionably reduced. Blood vessels and the optic nerve head served as the reference point for 100% level of darkness. Definitely decreased autofluorescence (DDAF) describes areas in which the level of darkness was close to 100% (at least 90%; Fig. 2). Regions with levels approximately between 50 and 90% darkness were defined as questionably decreased autofluorescence (QDAF; Fig. 2) [15].

Results from mesopic (a, c) and scotopic (b, d) microperimetric exams super-imposed onto the corresponding fundus autofluorescence images of corresponding eyes. The eye shown in a and b shows a lesion of definitely decreased autofluorescence (this type of lesion has at least 90% darkness level compared to the optic nerve head [OHN]). The eye in c and d shows a lesion of questionably decreased autofluorescence (this lesion has darkness levels between 50– and 90% compared to OHN). Sensitivity values for the individual locations (range 0–20 dB) are shown.

A scale of 0–20 dB served to determine the sensitivity for each test location. The term “deep scotoma” was defined for test locations with 0 dB (i.e., retinal locations where only the brightest stimulus was detected or no stimulus at all was detected), and the term “relative scotoma” for test locations with sensitivity better than 0 dB but less than 12 dB [16]. Mean sensitivity across all tested locations, and number of absolute and relative scotoma were calculated.

Fixation stability was recorded (dynamic testing), which created a cloud of fixation events for each test session [17]. Fixation stability was then quantified as a continuous variable, the BCEA. The BCEA offers clear advantages over categorical biomarkers of fixation stability in STGD1 [18]. The global BCEAs for 1, 2 and 3 standard deviations were calculated using the following equation:

σ_H and σ_V are the standard deviations of horizontal and vertical eye movements, ρ is the Pearson product-moment correlation co-efficient of fixation positions in the horizontal and the vertical meridian, k is a constant dependent on the chosen probability area which is given by the equation:

P is the probability area and e is the base of the natural logarithm. P is the chosen probability for the SD that the BCEA is based on, and the equation is solved for k [16–18]: