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The existence of dead cells in donor corneal endothelium preserved with storage media
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  • Koji Kitazawa, Hidetoshi Tanioka, Chie Sotozono and Shigeru Kinoshita
    Published on:
  • Philippe Gain, Zhiguo He, Thibaud Garcin and Gilles Thuret
    Published on:
  • Published on:
    • Koji Kitazawa, M.D., Ph.D. Kyoto Prefectural University of Medicine
    • Other Contributors:
      • Hidetoshi Tanioka, Ph.D.
      • Chie Sotozono, M.D., Ph.D.
      • Shigeru Kinoshita, M.D., Ph.D.

    We read with interest the insightful comments in the e-letter submitted by Dr. Gain, Dr. He, Dr. Garcin, and Dr. Thuret on our recently published article.1 As stated in their letter, they previously reported that by using a similar triple staining (i.e., Hoechst 33342, ethidium homodimere and calcein-AM) on the endothelium of whole donor corneas stored in long-term organ culture, the endothelial cell (EC) density in the whole pool of viable ECs in the cornea is routinely, and quite substantially, overestimated.2,3 We completely agree with the authors regarding the importance of assessing the whole pool of viable ECs in corneal grafts.

    We first reported at the 2009 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) that triple-staining a donor graft with propidium iodide, calcein-AM, and Hoechst 33342 allowed for a distinct discrimination between living cells and dead cells. In that report, we hypothesized that the existence of dead cells on the endothelium of the donor cornea suggests an association with the rapid loss of corneal ECs, at least at the early phase, post keratoplasty. However, Gauthier and associates reported3 that there was no difference between the density of viable ECs at day 0 and at day 5 postoperative, thus suggesting that very early EC loss in the host recipient is almost negligible. However, similar to Gain and associates, we believed that it was quite important to measure viable ECD, not to calculate just ECD by...

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    Conflict of Interest:
    None declared.
  • Published on:
    • Philippe Gain, Ophthalmic surgeon and researcher, head of the ophthalmology dept Laboratory "Biology, engineering and imaging of the Corneal graft", EA2521, University of St-Etienne, France
    • Other Contributors:
      • Zhiguo He, Research Engineer
      • Thibaud Garcin, Resident in ophthalmology
      • Gilles Thuret, Ophthalmic surgeon and researcher

    We noticed the article entitled “The existence of dead cells in donor corneal endothelium preserved with storage media” by Kitazawa with interest (Br J Ophthalmol 2017. Oct 5).
    Authors clearly demonstrated, using a triple staining with Hoechst, Propidium Iodide and Calcein-AM, that corneas stored at 4°C in Optisol-GS for 3 to 7 days, the technic most used worldwide, bear a significant number of dead endothelial cells (ECs). They underlined that these non-viable ECs are not recognized by specular cell count done by the eye bank and that this could explain the “cell loss” inevitably noticed post graft. Our team applied, for the first time in 2011 [1] and again in 2016 [2], a similar triple staining on the endothelium of whole corneas stored in long-term organ culture at 31°C, the dominant technic in Europe. We made the same findings as Kitazawa et al. and defined the notion of viable ECD (vECD) as the number of viable ECs per surface unit. Areas without ECs (especially in Descemetic folds), dead and dying EC (that will not survive the storage) clearly explain the important discrepancy between the cell count done by the eye bank (unable to spot them) and the very early postoperative ECD. Our team also demonstrated long ago that vital Trypan blue staining used by certain eye bank is unable to spot all dying cells because its time window of positivity is very narrow, corresponding only to ECs near to desquamate [3]. Viable ECD determined by triple staining therefore appea...

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    Conflict of Interest:
    None declared.