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The posterior side of the cornea is covered by the endothelial monolayer, which governs corneal transparency but cannot proliferate. Determination of endothelial cell density (ECD) is therefore the minimal and mandatory quality control in all eye banks. It avoids primary graft failures caused by endothelial insufficiency, and allows allocation of corneas to surgical techniques requiring different numbers of endothelial cells (ECs). Corneas stored in organ culture (17% of grafts worldwide), are characterized by heavy stromal swelling and numerous deep endothelial folds, up to 200 µm high. During microscopic en face observation, flat surfaces are thus exceptional and EC counting is biased by parallax errors, resulting in overestimated eye bank ECD (ebECD). We used a motorized transmitted light microscope to acquire Z-stacks of images every 10 µm, and processed them to reconstruct the 3D surface of the folded endothelium. This method (3D-ECD) takes into account the local point-by-point slope in order to correct ECD. On a set of 30 corneas, we compared 3D-ECD and ebECD determined on five identical zones at the center of the cornea. 3D reconstruction allowed us to visualize twice as many cells, and ebECD was 8.1 ± 4.5% (95%CI 6.4-9.7) higher than 3D-ECD, with 1744 ± 488 versus 1606 ± 473 cells/mm 2 . 3D counting makes it possible to increase cell sampling and to correct overestimation by the conventional en face counting still routinely performed in eye banks.

Purpose: To determine the factors influencing endothelial morphometry by using image analysis of corneas stored in organ culture to determine the coefficient of variation (CV) in cell area and percentage of hexagonal cells., Methods: The endothelia of 505 of the 559 corneas consecutively stored at the eye bank were routinely analyzed with Sambacornea image-analysis software (ver. 1.2.10; Tribvn, Châtillon, France) on three large-field images of 750 x 1000 microm, obtained after osmotic dilation of the intercellular spaces with 0.9% sodium chloride. Analysis was performed on at least 300 cells. The quality of the three-image set was graded poor, average, or good by an independent observer. The studied parameters were donor age and sex, lens status, storage time, and intrinsic quality of captured images. Statistics were analyzed by nonparametric tests., Results: Image analysis was possible for 504 of the 505 assessed corneas. Donor age correlated significantly with endothelial cell density (ECD; r = -0.343), CV (r = 0.221), and hexagonality (r = -0.314; P < 0.001 for the three). Image quality significantly influenced these three parameters. ECD and hexagonality decreased parallel to image quality, whereas the CV increased. In the 258 corneas assessed twice (on average, at day [D] +4, then D +14) ECD, CV, and hexagonality decreased during storage., Conclusions: Despite the sometimes mediocre quality of the transmitted light microscopy images, endothelial parameters supplied by the analyzer were clinically reliable, since variations similar to those long known in specular microscopy were found. Endothelial morphometry (CV and hexagonality) is likely to provide further information on the endothelial function of the graft tissue, perhaps particularly for grafts of borderline ECD, close to the discard threshold.

Aim The human corneal endothelium is a monolayer of flat hexagonal cells. It is a nearly regular hexagonal tessellation during the first years of life, but with age, becomes less regular in shape and size. The aim is to evaluate geometrically the age of an endothelial mosaic. Material and methods Segmented endothelial mosaics of healthy subjects of different age groups are compared by morphological criteria. The mosaics are studied according to their age group (decades), their age and their location (center or mid-periphery of the cornea). The measures used are: the cell density, the Ripley's L function and the cell area and perimeter density. Results These measures point out the endothelial cell density decrease, the cell area, perimeter and diameter increase, the cell heterogeneity increase, and the differences between central and mid-peripheral cells increases with age. Conclusion These measures are able to characterize healthy mosaics. [ABSTRACT FROM AUTHOR]

Purpose Corneal endothelium of organ cultured cornea forms a 3D surface because of corneal curvature and of the Descemetic folds (of hundreds of μm depth) that appear during storage. Eye bank technicians have therefore a risk of endothelial cell density (ECD) overestimation when counting on an image projection. Our aim is to develop a 3D reconstruction from a Z stack of images in order to take account of the local slope and increase ECD reliability Methods Z stacks of 35 images were acquired with a motorized light microscope (BX41, Olympus) through a x10 objective. After routine eye bank preparation with saline, EC appeared with a clear cytoplasm and dark borders but could locally show a contrast reversal. In each section, the best‐focused pixels were determined using focus measurement algorithms. Depth map and texture image were then separately generated and the 3D reconstruction was obtained by projecting the texture image onto the depth map. A new algorithm, based on projections onto eigenbases, was developed and compared to 5 existing algorithms. Comparison was done using 1/ simulated images with additive noises to assess the reliability and noise robustness 2/ true endothelial image Results The new algorithm exhibited a more robust behaviour in presence of noise than the other algorithms. 3D endothelial reconstructions were also visually better, especially in case of local contrast reversal Conclusion Using a technology easily transposable into an eye bank, a reliable 3D reconstruction is possible from routine images that are often of average quality with poor contrast and high background noise. Our next step is to integrate the local slope information to correct local ECD [ABSTRACT FROM AUTHOR]