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Reproducibility of Endothelial Assessment during Corneal Organ Culture: Comparison of a Computer-Assisted Analyzer with Manual Methods

¹From the Laboratory Biology, Engineering, and Imaging of Corneal Graft, Faculty of Medicine, Saint Étienne, France; ²Centre of Medical Engineering, École Nationale Supérieure des Mines de Saint-Etienne, Saint Étienne, France; ³French Blood Center/Eye Bank of Saint Étienne, Saint Étienne, France; and ⁴French Blood Centre/Eye Bank of Grenoble, Saint Ismier, France.

PURPOSE. To investigate the reproducibility of endothelial assessment of organ-cultured corneas with the computer-assisted Sambacornea analyzer in comparison with manual methods.

METHODS. Seven observers of two eye banks determined the endothelial cell density (ECD) of 30 corneas through a grid overlay placed on endothelial photographs using two manual modes, unaided (naked-eye) and pointing (point-out). ECD was measured with the analyzer, first in automated mode, where analysis was completely machine determined, and then in touched-up mode, where the observer selected the analysis zone and corrected poorly drawn cell borders. Interobserver variability of ECD for the different methods was compared. Reproducibility of morphometry parameters was determined for the touched-up mode.

RESULTS. Interobserver variability was ±19.2% (95% confidence interval [CI], 13.0–25.4) and ±17.6% (95% CI, 11.9–23.3) for the naked-eye and point-out mode, respectively, whereas the touched-up mode gave the least variability of ±9.6% (95% CI, 6.5–12.7), confirmed by the highest intraclass correlation coefficient of 0.95 (95% CI, 0.91–0.97). Interobserver variability increased with worsening image quality. Manual modes underestimated ECD (naked-eye by a mean 10.7% [SD, 2.9%]; point-out by a mean 6.9% [SD, 2.3%]), whereas the automated mode overestimated ECD by a mean 14.7% (SD, 24.3%). Reproducibility of morphometric parameters by the touched-up mode was acceptable but was influenced by endothelial pleomorphism.

CONCLUSIONS. Manual counting shows systematic underestimation of ECD with high interobserver variability. The analyzer in automated mode overestimates ECD and is absolutely unreliable. Detection of cell contours by the specific algorithm, combined with manual correction by a skilled technician, appears to be the most reliable method of ECD and morphometry determination.

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