ACTA 0 P H T H A L M O L O G ICA
S H O R T C O M M U N l CAT1O N
Vital staining by fluorescein diacetate (FDA).
A method for the estimation of corneal endothelium
F. Wilhelm’, M. Melzig2 and G. Franke’
Departments of Ophthalmology’ and Pharmacy*,Ernst-Moritz-Arndt-University, Greifswald,GDR
Abstract. The success of keratoplasty depends on the in-
tegrity of the graft’s endothelial cell layer. We perform a
staining method for testing the vitality which is practic-
able, safe, fast and inexpensive.
Key words: corneal endothelium - fluorescein diacetate
- vital staining.
Since the first successful keratoplasty Zirm in 1905
it has been well known that the quality of the
donor button is vital for the success of the oper-
ation. Because of the special importance of the en-
dothelium, many surgeons subject this sensitive
cell layer to a careful examinationbefore the trans-
plantation. In this procedure the most widely used
method is specular microscopy. It is fast and
simple, and the graft is saved from unneccessary
damage. However, this method always requires a
clear stroma. Even a small swellingleads to a loss of
transparity of the tissue, and specular microscopy
is not possible.
An alternative is vital staining of the endothe-
lium. Several surgeons prefer to stain with trypan
blue. In experimental studies we used FDA to stain
the endothelium.
Method
The staining was done from the endothelial side.
The disc’s diameter was always 6 mm. The corneal
button was trephined by a Francechetti trephine;
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68 (1990) 94-96
in a few cases we had to cut parts of Descemets
membrane with scissors. Then it was put with the
endothelial side up on a glass slide. The staining
solution was diluted from the stock solution (1 mg
FDA/l ml dimethylsulphoxide)by phosphat buf-
fered saline (PBS) at 1:lO. This solution was
dripped on the corneal endothelium and incu-
bated for about 5 to 10 sec. After that it was gently
rinsed with PBS. The observation and photo-
graphs were made using a fluorescence micro-
scope (Jenaval-fluorescence,VEB, Carl Zeiss, Jena)
with an excitation of 430 nm.
We tested the staining of endotheliumfrom rab-
bit (Fig. l),human, pig and sheep. In all species the
vital cells were stained and showed a yellow-green
fluorescence. Cell borders were clearly visible. Avi-
tal cells, for example after storage in a moist cham-
ber (Fig. 2) and storage solutions (Fig. 3), or on De-
scemet’s folds showed as black gaps (Fig. 4).
Discussion
A method for estimating the endothelial vitality
must be fast and inexpensive,as well as safe for the
graft.
Despite the method of specular microscopy, vital
staining was used. The non-toxicity of trypan blue
was described for the first time in 1970 (Stocker).
Because of damaging of the endothelial layer dur-
ing the staining procedure, it was suggested
 Fig. 1.
Intact endothelium of fresh rabbit cornea (cell density:
3328/mm2,80 X ) .
(Schrapel et al. 1982) to take only the peripheral
corneal rim after trephination. But there are
known differences in density between central and
peripheral endothelium (Schimmelpfennig 1982).
That is why we tested staining with FDA in ex-
perimental studies for more than 2 years. FDA is
utilized in the assessment of the integrity of iso-
lated cells of many types (Rotman & Papermaster
1966) and for determination of cytotoxicity (Taka-
Fig. 3.
Isolated damaged cells on rabbit endothelium after pres-
ervation for 5 days in Greifswalder storage medium
(80 X).
Fig. 2.
Rabbit corneal endothelium after storage in a moist
chamber for 48 h. The black gaps indicate damaged or
dead cells (80 X ) .
sugi 1971). The low toxicity of the substance was
tested by Melzig & Wilhelm (unpublished results).
Similar results with FDA-staining of the corneal
endotheliumwe reported by Madden (1987).In ad-
dition to the morphological design, the test gives a
functional result. The staining dye cannot go pas-
sively into the cells; it is an active process. This
means that a yellow-greenfluoresceiningcell must
be functionally intact. Non-fluoresceiningcells are
Fig. 4.
Typical Descemet’s folds on rabbit cornea after storage in
a moist chamber for 48 h. Nonfluoresceining cells indi-
cated by dark lines (80 X).
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not able to take up the dye. The fact of the irrever- Rotman B & Papermaster B W (1966):Membrane proper-
sibility of such damage must be discussed. Grafts ties of living mammalioan cells by enzymatic hydro-
with an acceptable number of fluoresceinig cells lysis of nuorogenic esters. Proc Natl Acad Sci USA 55:
can be transplanted. 134-141.
Takasugi M (1971): A improved fluorochromatic cyto-
We are of the opinion that this method is fast
toxid test. Transplantation 12: 148-151.
and of importance, and is suitable for estimating
Madden P W (1987):The evaluation of endothelial dam-
the donor material before transplantation or after ages following conleal storage: a comparison of stain-
presevation. ing methods and the value of scanning electron micro-
scopy. Cur eye Res 6: 1441-1451.

References
Zirm E (1906): Eine erfolgreiche totale Keratoplastik.
Graefes Arch Ophthalmol64: 580-593.
Stocker F W (1970):Clinical test for evaluation donor cor- Received on August 4th, 1989.
neas. Arch Ophthalmol84: 27-33.
Schrapel A, Letko G & Giessman H G (1982): Ein objek- Author’s address:
tiver Schnelltest zur Vitalitatsbeurteilung des Horn- MR Prof. Dr. sc. med. Gunter Franke,
hautendothels nach Konservierung. Klin Monatsbl Department of Ophthalmology,
Augenheilkd 180: 53-57. Ernst-Moritz-Amdt-University,
Schimmelpfennig B (1982): Inhomogene Verteilung des RubenowstraDe 2,
menschlichen Korneaendothels. Klin Monatsbl Greifswald, DDR-2200.
Augenheilkd 180 350-351.

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