title: Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks.
abstract: The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm(2) by steps of 400 cells/mm(2)) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 μm(2) (incremental steps of 25 μm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 μm(2) grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 μm(2) grid counts. Digital image analysis with a variable frame counting method would be the best alternative.
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA 2521, SFR143, Faculty of Medicine, Jean Monnet University, 15, Rue Ambroise Paré, 42023, Saint-Étienne Cedex 2, France.
authors.forename: Nelly
authors.initials: N
authors.lastname: Campolmi
authors.forename: Sophie
authors.initials: S
authors.lastname: Acquart
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: He
authors.forename: Yann
authors.initials: Y
authors.lastname: Gavet
authors.forename: Remy
authors.initials: R
authors.lastname: Jullienne
authors.forename: Nicolas
authors.initials: N
authors.lastname: Naigeon
authors.forename: Aurelien
authors.initials: A
authors.lastname: Bernard
authors.forename: Fabien
authors.initials: F
authors.lastname: Forest
authors.forename: Michel
authors.initials: M
authors.lastname: Péoc'h
authors.forename: Gilles
authors.initials: G
authors.lastname: Thuret
authors.forename: Philippe
authors.initials: P
authors.lastname: Gain
mesh_terms: D002452:Cell Count / Q000379:methods*; D061827:Dimensional Measurement Accuracy; D042783:Endothelial Cells / Q000166:cytology*; D004728:Endothelium, Corneal / Q000166:cytology*; D005125:Eye Banks; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D012984:Software