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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/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.

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0-33.6), 38.3 (24.3-75.4) and 57.9 (33.9-90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3-10.6), 10.5 (8.2-11.5), and 11.1 (9.9-12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.