Purpose

Quality of the endothelial transplant is a critical parameter in the success of Descemet Membrane Endothelial Keratoplasty (DMEK) and in the graft survival. The peeling techniques, preservation methods and operator’s skill level need to be experimentally assessed and validated, as they are key elements influencing graft quality. The most reliable method of quality evaluation of the endothelial transplant is the triple staining with Hoechst-Calcein AM-Ethidium (HEC) allowing to determine the total number of viable endothelial cells. However, the test HEC has defects: (1) Unspecific fluorescence of the same filter than that Calcein AM prevents an accurate viability analysis; (2) Incompatibility with immunofluorescence (IF) which could provide additional information. The aim of this study is to develop technical tips to overcome these defects.

Methods

Two strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the concentration of Calcein AM and diluent; 2. Decrease unspecific fluorescence by adding the fluorescence quencher Trypan Blue (TB). In order to combine IF after the test HEC, an extensive wash in PBS was performed. In total, 19 human corneas had been used.

Results

Calcein AM at 4µM diluted in OptiMEM increased fluorescence intensity by 3-fold (p = 0.0017, n = 5) compared to conventional staining at 2 µM in PBS. Trypan Blue visibly decreased the unspecific fluorescence of Calcein, reduced the inter-operator (n = 6) variability of cell count by 42% (p = 0.0027, n = 10) and reduce analysis time by 40% (p = 0.0002, n = 10). To perform IF after HEC test, prolonged washing in PBS was an effective method to remove residual Calcein fluorescence and allow imaging using FITC/Alexa 488 filter.

Conclusions

This study provides effective technical tips for optimising assessment of the endothelial viability and for being able to perform IF after the test HEC.