Purpose The Active Storage Machine (ASM), designed by Sincler (a company of group Laboratoires Théa) for eyebanks, will be used for long term donor corneas preservation at 31°C before transplantation. In this device, the endothelial cell density (ECD) counting is expected to be performed non-invasively throughout the storage, thus without changing the storage medium nor handling the cornea. To meet these constraints, specular microscopy (SM), also used for cold storage was selected, instead of the standard light transmission microscopy (LTM-NaCl) used in eye banks storing corneas in organ culture at 31-34°C. The purpose of this study is to compare both imaging methods for ECD measurement of corneas preserved in ASM.
Methods Five human corneas from body donation to Science were preserved in a prototype ASM with 35mmHg in the endothelial chamber, 2.5µl/min of Corneamax® (Eurobio, France) at 31°C for 5 days. The endothelium of the cornea was imaged through the ASM window using the CellChek® D+ SM (Konan Medical, California, United-States) equipped with an add on device at customized stage. The cornea was then immediately removed from the ASM and prepared for standard endothelial assessment (dilation of the intercellular spaces using 0.9% NaCl and light transmission imaging). Finally, the endothelium was stained with alizarine red and trypan blue and observed again with the same microscope, to determine ECD using the referenced method up to now. For each cornea and each observation method, 5 images were acquired: 1 central and 4 paracentral. The SM images were counted with the Konan software. The LTM-NaCl and ‘Alizarin Red’ counts were performed with a dedicated plugin of ImageJ after microscope calibration.
Results The means ± SD of the ECD calculated for SM, LTM-NaCl and ‘Alizarine’ images were respectively of 2314 ± 537, 2243 ± 506 and 2354 ± 543 cells/mm2. There was no significant difference between the 3 methods (ANOVA one-way, p-value = 0.1066). The percentage error was -1.7% +/- 3.3% for SM and -4.7 +/- 4.0% for light transmission microscopy.
Conclusion Quality control of the endothelium of corneas stored in ASM can be performed non-invasively with a standard eye bank SM. The ECD measured by SM does not differ from that measured by the conventional microscopy technique used until now in organoculture.
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