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We read with interest the remarks of Crowston et al1 on our article.2 We showed that the TUNEL technique revealed a far higher percentage of endothelial cells (ECs) irreversibly engaged in a cell death process than that obtained by trypan blue staining.
The two techniques were performed sequentially: after observation of trypan blue staining, corneas were immediately fixed in formaldehyde for TUNEL. Crowston et al suggest that the trypan blue itself and/or the time spent outside the organ culture medium before fixing in formaldehyde may have caused an artefactual increase in the percentage of TUNEL positive ECs. Two arguments counter this remark.
The trypan blue staining procedure is identical to that used, during endothelial examination(s) of grafts, in all European cornea …