Overestimation of Corneal Endothelial Cell Density in Smaller Frame Sizes in In Vivo Confocal Microscopy

Purpose: To evaluate the effect of frame size on the calculated corneal endothelial cell density (CECD) in images of laser scanning in vivo confocal microscopy (IVCM). Methods: Forty-nine corneal endothelial images acquired by laser scanning IVCM (Heidelberg Retina Tomograph 3 with Rostock Corneal Module) with different endothelial cell densities were analyzed. In each image (160,000 &mgr;m2), the CECD was calculated using the fixed-frame method by counting cells in the following frame sizes: 80,000 &mgr;m2, 40,000 &mgr;m2, 20,000 &mgr;m2, 10,000 &mgr;m2, 5000 &mgr;m2, and 2500 &mgr;m2. The calculated CECD was then compared with that of the variable-frame method as the reference value. Results: There was no significant difference in the calculated CECD between the variable-frame method (2004 ± 832 cells/mm2), and the fixed-frame method using a 40,000-&mgr;m2 frame (2023 ± 810 cells/mm2). On the other hand, the calculated CECD showed significant overestimations in frame sizes of 20,000 &mgr;m2 (2066 ± 820 cells/mm2), 10,000 &mgr;m2 (2156 ± 785 cells/mm2), 5000 &mgr;m2 (2352 ± 783 cells/mm2), and 2500 &mgr;m2 (2715 ± 754 cells/mm2), with P < 0.001 in all. This resulted in overestimations of 4.8 ± 9.8%, 11.9 ± 16.2%, 24.9 ± 23.1%, and 49.1 ± 38.8% for these frame sizes, respectively. Images with lower CECD demonstrated higher overestimations of cell density in smaller frame sizes. Conclusions: In laser scanning IVCM images, there is significant overestimation of CECD if the cells are counted in frames smaller than 25% of the image. Similar frame sizes should be used when monitoring CECD over time.

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