Trypan-Assisted Automated Endothelial Cell Loss Measurements Compared With Specular Microscopy

PURPOSE: The aims of this study were 1) to compare area of cell loss (ACL) on trypan staining with ACL on specular imaging and 2) to evaluate the use of automated software for measuring ACL on trypan staining.

METHODS: Donor corneas with transplant-grade endothelium were mechanically injured with an 18-gauge cannula and a Fogla deep anterior lamellar keratoplasty dissector tip to create an easily identifiable "bullseye" pattern of cell death. Each cornea was then stained with trypan blue 0.06% for 90 seconds and imaged at 2× magnification. ACL on staining was measured using manual (ImageJ, National Institute of Health, Bethesda, MD) versus automated software tools (custom-built Aphelion macro, ADCIS, S.A., Saint-Contest, France). The bullseye was then imaged using specular microscopy, and ACL was measured by tracing the dead cell borders. ACL was then compared between both modalities.

RESULTS: Eleven donor corneas were evaluated. Both manual (0.42 mm2) and automated (0.45 mm2) measurements of ACL after trypan staining underestimated mean ACL on specular imaging (0.54 mm2) (P < 0.01). However, on regression analysis, there was a good predictive correlation between automated trypan measurements and specular imaging (R2 = 0.99, residual SE = 0.0044, P < 0.01). When ACL on specular imaging was measured by tracing cell nuclei along the margin of injury (rather than cell borders) (0.45 mm2), there was no statistically significant difference between specular and automated trypan measurements (P = 0.95).

CONCLUSIONS: Trypan-assisted automated measurements of ACL correlated well with ACL on specular imaging, suggesting that automated software may be a useful tool for evaluating endothelium in donor corneas.