Objective: The complexity of chromatin (i.e., irregular geometry and distribution) is one of the important factors considered in the cytological diagnosis of cancer. Fractal analysis with Kirsch edge detection is a known technique to detect irregular geometry and distribution in an image. We examined the outer cutoff value for the box-counting (BC) method for fractal analysis of the complexity of chromatin using Kirsch edge detection. Materials: The following images were used for the analysis: (1) image of the nucleus for Kirsch edge detection measuring 97 × 122 pix (10.7 × 13.4 μm) with a Feret diameter of chromatin mesh (n = 50) measuring 17.3 ± 1.8 pix (1.9 ± 0.5 μm) and chromatin network distance (n = 50) measuring 4.4 ± 1.6 pix (0.49 ± 0.18 μm), and (2) sample images for Kirsch edge detection with varying diameters (10.4, 15.9, and 18.1 μm) and network width of 0.4 μm. Methods: Three types of bias that can affect the outcomes of fractal analysis in cytological diagnosis were defined. (1) Nuclear position bias: images of 9 different positions generated by shifting the original position of the nucleus in the middle of a 256 × 256 pix (28.1 μm) square frame in 8 compass directions. (2) Nuclear rotation bias: images of 8 different rotations obtained by rotating the original position of the nucleus in 45° increments (0°, 45°, 90°, 135°, 180°, 225°, 270°, and 315°). (3) Nuclear size bias: images of varying size (diameter: 190 pix [10.4 μm], 290 pix [15.9 μm], and 330 pix [18.1 μm]) with the same mesh pattern (network width: 8 pix [0.4 μm]) within a 512 × 512 pix square. Different outer cutoff values for the BC method (256, 128, 64, 32, 16, and 8 pix) were applied for each bias to assess the fractal dimension and to compare the coefficient of variation (CV). Results: The BC method with the outer cutoff value of 32 pix resulted in the least variation of fractal dimension. Specifically, with the cutoff value of 32 pix, the CV of nuclear position bias, nuclear rotation bias, and nuclear size bias were <1% (0.1, 0.4, and 0.3%, respectively), with no significant difference between the position and rotation bias (p = 0.19). Our study suggests that the BC method with the outer cutoff value of 32 pix is suitable for the analysis of the complexity of chromatin with chromatin mesh.

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