Pan-Endothelial Viability Assessment With The Triple HEC Staining of Organ-Cultured Precut DSAEK vs Full Thickness Corneas

Abstract

Purpose: : To accurately assess the viable endothelial cell density (ECD) of posterior lamellar grafts, precut by eye bank technicians, and sent to a distant center

Methods: : Paired organ cultured (OC) corneas with an ECD >2200 cells/mm2 at the standard assessment with the SambaCornea analyser, were randomly assigned as full-thickness grafts (Group I) or as precut posterior transplants for DSAEK (Group II), using a microkeratome (Moria, France). Both corneas were stored in CorneaMax (Eurobio, France) and deswelled in CorneaJet (5% Dextran T500) for 6 to 24 hours prior to pre-cutting. The paired cornea was deswelled simultaneously, but remained untreated. Then, both corneas were sent to the distant center in CorneaJet. The ECD and viability was assessed by using triple staining (Hoechst/Ethidium/Calcein (HEC)) and fluorescent image analysis of the entire posterior surface, as previously described (IOVS 2011.52:6018-2). Calculation of the area covered by living cells (H+/C+/E-) coupled with ECD in these areas allowed to determine the ‘viable ECD’, i.e. the useful cell pool. DSAEK and controls data were compared with non-parametric tests

Results: : In both groups, the viable ECD was lower than the standard ECD determined by the eye bank, because living cells never completely covered the entire posterior surface. Number of dead cells (E+) did not differ between both groups

Conclusions: : The HEC triple staining combined with image analysis, provides a unique accurate assessment of the endothelial quality by giving the "viable ECD". This laboratory technique allows for reliable assessment of the endothelial quality of tissues supplied to surgeons by new developments in eye banking

Keywords: cornea: endothelium • cornea: storage • microscopy: light/fluorescence/immunohistochemistry