Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks
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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/mm2 by steps of 400 cells/mm2) 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 μm2 (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 μm2 grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 μm2 grid counts. Digital image analysis with a variable frame counting method would be the best alternative.
KeywordsCorneal endothelium Cell count Eye bank Fixed-frame Quality control Reliability assessment
The authors thank Mr Florent Malassagne (formerly at Samba Technologies, Meylan, France) for help with computer programming. This work was partly founded by the French Agence Nationale pour la Recherche, TecSan 2012, CORRIMO 3D, and by the french Health Ministry (PHRC IR, n1108035) and was promoted by the University Hospital of Saint Etienne. The sponsor or funding organization had no role in the design or conduct of this research.
Conflict of interest
The authors have no proprietary or commercial interest in any materials discussed in this article.
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