2.4 Automatic quantification of vascular depth

As discussed in Sec. 1, the capillary loop depth (CLD) and superficial plexus depth (SPD) within skin could potentially be used as a robust measure of acanthosis (epidermal hypertrophy). In our previous work, measurements of CLD and SPD were acquired qualitatively through visual observation of an en-face flythrough of the corresponding angiographic data [32]. Here, the skeletonization procedure outlined in Sec. 2.3 is used to automate this step. Firstly, a pseudo-3D skeleton was generated over the entire visible depth range (0-1mm) by applying the skeletonization methodology to each z-depth in turn. With OCT angiography, forward scattering of photons by the red blood cells result in a shadowing artefact beneath detected vasculature [33]. This effect was utilized to improve the skeletonization further, as signal derived from the vasculature was present over a range of depths within the tissue, while noise-derived signal was inconsistent with respect to depth. A simple median filter of 3-pixel window size in the z-direction was used to remove skeleton points which were inconsistent with depth. The total number of independent skeleton segments was then calculated for each z-depth in the volume. In this context, an independent skeleton section was defined as a section of skeleton that was disconnected from other sections of the skeleton. As there is no vasculature present in the upper sections of the epidermis, there were no independent skeleton segments detected for superficial depths. Once the tips of capillary loops start entering the field-of-view (typically around 40-100μm in depth) the number of independent skeleton segments increased rapidly until reaching a local maximum. This maximum value represents the point at which the maximum number of unconnected capillary loops are visible, and is thus defined as the CLD. Typically at the CLD depth, between 200 and 500 independent skeleton sections were observed (corresponding to 13-31 capillaries per mm²), this is in agreement with known measurements of the capillary density within skin, which typically range from 10 to 70 capillaries per mm² of skin [34]. Following the CLD, one might expect the number of independent skeleton segments to plateau as subsequent depths are simply following each loop along its axis. Instead, the value gradually reduces, suggesting that the vessel network almost immediately begins to interconnect. This is potentially a direct result of the ascending and descending limbs of each capillary loop spreading slightly apart with depth, as OCT lacks the resolution to clearly discriminate between each limb. The result is a dilation of the visible loops, with neighboring loops potentially merging together though not physically connected. At sufficient depth, the gradient of the curve of skeleton segment number vs depth reaches 0, suggesting that the network is fully connected; this point is defined as the SPD. The SPD depth was more challenging to automatically quantify, owing to an increase in noise and a lack of OCT signal at deeper depths in the tissue. To improve reliability of this detection, the curve was smoothed with a moving averaging filter which spanned ~20μm and each detection was manually checked for reliability. In future, SPD detection can potentially be improved through use of an alternate metric which would peak at the SPD depth, such as the total vessel signal or length, however this was not explored in the context of this study. Typically, past the SPD depth, the number of independent skeleton sections fluctuate slightly due to inconsistencies in the shadowing artefact of the vasculature, until reaching the noise floor, at which point a final reduction in value is observed. The noise floor in this context is the point at which the entire en-face angiogram shows detected speckle-variance due to random noise in the corresponding structural images, resulting in an extremely interconnected skeleton and a low number of independent skeleton segments. Figure 4 illustrates this process for both healthy and AD skin, with healthy skin typically exhibiting a thin peak at superficial depths and AD skin typically exhibiting a much wider peak at comparatively deeper depths in the tissue.

Automatic measurement of CLD and SPD through consideration of the number of independent (non-connected) skeleton segments as a function of tissue depth. The top row shows the results for a healthy participant at the left cubital fossa: the local maximum (CLD) is located at 42.9μm beneath the skin surface, while the following local minima (SPD) is located at 132.6μm beneath the surface. The bottom row shows the results for a participant with AD (Local EASI = 5.25) at the left cubital fossa: the local maximum (CLD) is located at 89.4μm beneath the skin surface, while the following local minima (SPD) is located at 304.2μm beneath the surface.