Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas

Abstract

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. 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.

Significant differences between specular microscopy and corneal bank endothelial cell counts – a pilot study

“…; Jumelle et al. ). As we have used research corneas, our study may have shown wrongly high values in the automatic CBLM.…”

“…Addressing this, Jumelle et al. () implemented a 3D EC counting method to increase cell sampling and to correct overestimation by the conventional en face counting performed in eye banks. In addition, regularly training the skills of the staff and optimizing the quality of the images is the key to reliable data (Lass et al.…”

Specular Microscopy of Human Corneas Stored in an Active Storage Machine

“…To determine the ECD, cells were counted on each field, and the mean of the five was calculated. We used the ImageJ freeware ( (accessed on 1 November 2016)) with a customized plugin ECD3D [ 16 ]. The observer chose areas where EC were most clearly visible and avoided folds when present.…”

“…An accurate monitoring of endothelial cell density was possible at day (D) 2, 26, and 28. n = number of cells counted; ECD endothelial cell density in cells/mm 2 . At D2, ( A ) raw image with endothelial cells, ( B ) each center was pointed according to the center method, ( C ) cells borders and each center were marked thanks to ImageJ plugin ECD3D [ 16 ]. At D26, ( D ) comparative raw image showed larger cells, ( E ) ECD count was determined with the same method, ( F ) reconstruction allowed global view of the studied sample.…”