title: Overestimation of Corneal Endothelial Cell Density in Smaller Frame Sizes in In Vivo Confocal Microscopy.
abstract: OBJECTIVE
  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 μm), the CECD was calculated using the fixed-frame method by counting cells in the following frame sizes: 80,000 μm, 40,000 μm, 20,000 μm, 10,000 μm, 5000 μm, and 2500 μm. 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/mm), and the fixed-frame method using a 40,000-μm frame (2023 ± 810 cells/mm). On the other hand, the calculated CECD showed significant overestimations in frame sizes of 20,000 μm (2066 ± 820 cells/mm), 10,000 μm (2156 ± 785 cells/mm), 5000 μm (2352 ± 783 cells/mm), and 2500 μm (2715 ± 754 cells/mm), 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.
authors.affiliation: Cornea and Refractive Surgery Service, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Kheirkhah
authors.forename: Ujwala S
authors.initials: US
authors.lastname: Saboo
authors.forename: Anna
authors.initials: A
authors.lastname: Marmalidou
authors.forename: Reza
authors.initials: R
authors.lastname: Dana
mesh_terms: D002452:Cell Count / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology*; D004728:Endothelium, Corneal / Q000166:cytology*; D006801:Humans; D018613:Microscopy, Confocal; D012016:Reference Values