New HECplus protocol to further improve the accuracy of pan‐endothelial viability measurement

Abstract

AbstractPurposeThe HEC (Hoechst‐Ethidium‐Calcein) labeling, which we described in 2011, is a simple destructive test unanimously adopted to determine the pan‐endothelial viable endothelial cell density (vECD) using image analysis (e.g. using CorneaJ, ImageJ dedicated plugin). Sometimes, the endothelial surface folded by stromal edema remains difficult to analyze. We present here the HECplus technique, which, with an optimization of image acquisition and analysis, further increases the accuracy of vECD determination, particularly in endothelial folds.Methods10 pairs of corneas stored in organoculture at 31°C were analyzed. The endothelial side was incubated with 4 µM Calcein‐AM, 1 µM Ethidium and 11 µM Hoescht 33342 for 45 min at RT. Fluorescence was acquired in 3 channels first with a macroscope, in 3D with an image stacking of the entire graft then with a microscope after flat mounting. A 2 mm plastic pellet emitting constant fluorescence at 510 nm was imaged at the same time to normalize the images. Only one sharp image was reconstructed in ImageJ from the stacks of images. The final standardized images were then analyzed by CorneaJ (HEC group) and then manually by delimiting all viable surfaces on a graphic tablet (HECplus group)ResultsThe vECD was significantly higher by 8.2 ± 1.9% in the HECplus group versus HEC (2059 ± 127 versus 1989 ± 156 cells/mm2). Endothelial cells died preferentially in the top of the folds and were better preserved in the bottom of the folds. While HEC wrongly considered some folds as dead, HECplus allowed the error to be corrected. The folding pattern was similar between the 2 corneas of the same pair but differed from one donor to another. The normalization of images for calcein showed overall differences in the level of fluorescence between corneas, suggesting differences in metabolic activity.ConclusionsWe recommend applying the HECplus protocol when a very high degree of accuracy is required, for example when comparing storage media.