105 (NeuronJ19) and fully automatic analysis (ACCMetrics20) in a cohort of sarcoidosis patients, as well as NFA FIJI and ACCMetrics calculated NFA. To improve the clinical applicability of CCM, automatic imaging analysis is preferred. Although normative values for corneal nerve parameters are established using manual analysis methods12, clinical applicability would greatly benefit from an automatic approach. Our results showed that an automatic approach is indeed feasible, that the results of automatic analysis are similar to the results of manual methods, and that different automatic techniques are sufficiently correlated to allow direct comparison of results obtained with different methods. Although a good correlation was demonstrated, some differences between the analysis techniques should be discussed. First, there may be a selection bias between manual, semi-automatic and automatic analysis technique. For manual and semi-automatic analysis, only 6 images were selected for analysis, while for automatic methods, all images of good quality were selected. Manual analysis methods required selecting a limited number of images to maintain an acceptable workload. However, selecting all images of good quality would reduce the risk of selection bias if future protocols include automatic analysis. Furthermore, this is a suitable approach for automatic analysis because increasing the number of images does not increase workload. In order to make a good comparison between the different analysis programs, these differences in methods have already been taken into account. Second, the automatic analysis technique was prone to a small underestimation of CNFL compared to the manual and semi-automatic analysis technique (Figure 5). This was consistent with previous research22 that examined the same software programs. As discussed in the previous article, this can probably be explained by the human eye’s ability to detect more complicated lines as nerve fibers, while the automatic system was limited by strict criteria. Furthermore, the fully automatic analysis generated both false positive and false negative errors. Thin nerves and nerves out of focus were prone to false negative errors, and other structures such as dendritic cells were prone to false positive errors. Third, a small difference was the slightly higher values obtained with NeuronJ compared to CCMetrics. As mentioned earlier, CCMetrics calculated values by manual nerve tracing, while NeuronJ traced the nerves semi-automatically. An automatic algorithm calculated the optimal nerve path, guided by manual instructions. Therefore, manual instructions were only necessary when the algorithm had a tendency to choose a wrong path. Compared to CCMetrics, this method was much faster and drew lines more easily than the fully manual method. This method therefore resulted in slightly higher values compared to CCMetrics. Finally, although NFA FIJI and ACCMetrics NFA showed a highly significant correlation, they also showed the lowest inter-rater agreement. The only difference discovered between NFA FIJI and ACCMetrics NFA was that ACCMetrics calculates NFA in mm2/mm2, while NFA FIJI calculates in µm2/mm2. The lowest step size for ACCMetrics was 100 µm2/mm2, while NFA FIJI was more accurate up to single µm2/mm2. However, this does not necessarily lead to lower inter-rater agreement. A previous study21 was able to distinguish between patients with sarcoidosis and diabetes with NFA in a higher CNFL range (>9.8 mm/mm2). Patients with sarcoidosis showed lower NFA compared to patients with diabetes. For the same group, CNFL was not significantly different between patients with sarcoidosis and diabetes. Therefore, they concluded that NFA reveals additional information as a function of neuropathic severity. However, it remained unknown whether NFA could differentiate neuropathic severity within a group of sarcoidosis patients. Our data showed that sarcoidosis patients with and without SFN have no difference in NFA measured by CCM, see Figure 6. Therefore, our study does not support the potential value of NFA in the diagnosis of SFN in patients with sarcoidosis. One explanation could be that the nerve fiber width is significantly greater in subjects with sarcoidosis 6 110 6
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