Pan-Corneal Endothelial Viability Assessment: Application to Endothelial Grafts Predissected by Eye Banks

Abstract

To present an experimental method for determining the viable cell pool of corneal endothelia and its application to assessing predissected endothelial grafts. The endothelial cell density (ECD) of five pairs of human organ cultured corneas was determined using a standard counting method with a calibrated image analysis system. A thin posterior graft (30-50 μm) was manually predissected from a cornea chosen at random. Predissected and control corneas were shipped to the remote center, where standard ECD determination was repeated and was immediately followed by a triple Hoechst/ethidium/calcein labeling coupled with image analysis of the whole graft surface. Numeration of nuclei (H+), dead cells (E+), and total area covered by viable cells (C+) allowed the calculation of viable ECD corresponding to the cell density that the cornea may have after redistribution of viable cells over the whole Descemet surface. The median (range) viable ECD was lower than the standard ECD determined immediately earlier in predissected and control corneas: 1628 (1138-2379) and 2065 (1492-2876) cells/mm(2) (P = 0.043), corresponding to -20% (-1%-38%) and -12% (-3%-26%), respectively (P = 0.08). Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.