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We read with interest the remarks of Crowston et al.  on our
article entitled "Value of two mortality assessment techniques for organ
cultured corneal endothelium: trypan blue versus TUNEL technique". 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:
1. The trypan blue staining procedure is identical to that used in all
European cornea banks that use organ culture during endothelial
examination(s) of grafts. Neither the low concentration of trypan blue
(0.4%) nor the short exposure time (about 1 minute) nor the short
incubation in the presence of 0.9% NaCl has ever been incriminated in ECs
over-mortality in routine practice. Moreover, the innocuity of
injections of trypan blue into the anterior chamber, a common feature
during cataract surgery, has been well demonstrated.
2. The time spent outside the organ-culture medium before fixing in
formaldehyde, a period required for vital staining and microscopic
examination of the endothelium, lasts only a few minutes. The cornea
remains under the microscope for about one minute only, the time needed
for image acquisition. Such rapidity is possible by using a prototype
automatic analyser of the endothelium, which we developed and have
recently published. This is very probably insufficient time for DNA
fragmentation to occur in the proportion we observed. Moreover, the fixing
of the endothelial layer in 10% formaldehyde is immediate, and prevents
any continuation of fragmentation phenomena. On balance, it is highly
unlikely that the succession of markings is responsible for the
discrepancy between the positivity percentages of the two techniques. In
addition, we chose not to perform the two techniques simultaneously on
paired corneas or on the halves of one cornea because we wanted to
superimpose the two stains on the same cornea and thus obtain a double
The second remark by Crowston et al. is particularly interesting. We
too were surprised by the high percentage of TUNEL-positive ECs (mean
12.7%, SD 16.4). This may imply that all the cells died within 8 days,
which was evidently not the case. We believe this apparent contradiction
can be explained by the following theory. The TUNEL staining is positive
during a relatively long window (24-48 hours ). The TUNEL index,
measured at a given moment, provides a global view of all the cells with
fragmented DNA. However, the DNA fragmentation may be at different stages,
and the cells very likely spread according to a Gaussian distribution.
Therefore the cells, which are TUNEL-positive at a given moment, will not
all die instantaneously and simultaneously. Only the cells furthest to the
right on the curve will die in the very short term, and it is probably
these that are liable to be revealed by trypan blue. If it were possible
to perform TUNEL on two consecutive days, the percentage of positive cells
revealed would probably be very similar, but a large majority of the
positive cells recorded on the second day would have already been counted
on day one... It is, however, undeniable that the cells TUNEL-positive at a
given moment will all die eventually. In other words, we believe that, at
the end of storage, corneas contain a number of ECs engaged in an
irreversible cell-death process far more extensive than the highly
unreliable trypan blue staining technique suggests.
(1) Crowston JG, Healey PR, Maloof A, et al . Quantifying corneal
endothelial cell death. Br J Ophthalmol 2002;86:1068.
(2) Gain P, Thuret G, Chiquet C, et al . Value of two mortality assessment
techniques for organ cultured corneal endothelium: trypan blue versus
TUNEL technique. Br J Ophthalmol 2002;86:306-10.
(3) Sperling S. Evaluation of the endothelium of human donor corneas by
induced dilation of intercellular spaces and trypan blue. Graefes Arch
Clin Exp Ophthalmol 1986;224:428-34.
(4) Norn MS. Per operative trypan blue vital staining of corneal
endothelium. Eight years' follow up. Acta Ophthalmol 1980;58:550-5.
(5) Gain P, Thuret G, Kodjikian L, et al. Automated tri-image analysis of
stored corneal endothelium. Br J Ophthalmol 2002;86:801-8.
(6) Mesner PW, Epting CL, Hegarty JL, et al. A timetable of events during
programmed cell death induced by trophic factor withdrawal from neuronal
PC12 cells. J Neurosci 1995;15:7357-66.