Article Text

Download PDFPDF

P38-A108 Optimization of the triple HEC staining for laboratory assessment of DMEK graft quality
  1. Sandrine Ninotta1,2,
  2. Tomy Sagnial1,
  3. Paul Goin1,3,
  4. Sylvain Poinard1,4,
  5. Oliver Dorado1,4,
  6. Anne Sophie Gauthier1,3,
  7. Philippe Gain1,4,
  8. Gilles Thuret1,4,
  9. Zhiguo He1
  1. 1Biology, Engineering and Imaging in Ophtalmology (BiiO), EA2521, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France
  2. 2Cornea Bank, EFS Rhône-Alpes-Auvergne, Saint Etienne, France
  3. 3Department of Ophthalmology, University Hospital of Besançon, France
  4. 4Department of Ophthalmology, University Hospital of Saint Etienne, France


Introduction The quality of the endothelial graft is critical to the success of DMEK and to the survival time of the graft. The peeling technique, preservation method, and skill level of graft preparers need to be evaluated and validated. The most reliable method of evaluation is the viability test based on a triple staining of Hoechst- Ethidium-Calcein AM (H-E-C) which allows the determination of the total number of viable cells on the graft. However, this test has some shortcomings for DMEK grafts: 1) The undesirable fluorescence of the Calcein AM stain prevents accurate viability analysis, especially in cases where the graft is attached to the cornea for preservation; 2) Incompatibility with immunofluorescence (IF) that could provide additional information. The objective of this study is to develop technical tricks to overcome these drawbacks.

Methods Two strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the diluent and the concentration of Calcein AM; 2. Decrease undesired fluorescence from keratocytes by adding Trypan Blue (BT). In order to combine the IF after the HEC test, an extension wash in PBS was performed.

Results Calcein AM at 4µM diluted in OptiMEM increased fluorescence intensity 3-fold (p=0.0017, n=5) compared with conventional staining at 2µM in PBS. BT decreased the undesired fluorescence of Calcein and thus optimized count variability between different operators by 42% (p=0.0027, n=10) and saved 40% (p=0.0002, n=10) of count time. To perform IF after HEC, prolonged washing in PBS is an effective method to remove residual Calcein fluorescence and allows release of the FITC/Alexa 488 filter.

Conclusion This study provides effective technical tips for optimizing the endothelial viability assay using Calcein AM and for performing IF after the viability assay.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.