gms | German Medical Science

102. Jahrestagung der DOG

Deutsche Ophthalmologische Gesellschaft e. V.

23. bis 26.09.2004, Berlin

Grading the corneal limbal stem cell deficiency

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Evidenzbasierte Medizin - Anspruch und Wirklichkeit. 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft. Berlin, 23.-26.09.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04dogSA.10.12

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Published: September 22, 2004

© 2004 Ponzin.
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The assessment of the function and viability of corneal limbal stem cells is essential in the diagnosis of ocular surface disease, and to predict the outcome of keratoplasty. Several studies addressing this issue have concluded that demonstration of goblet cells containing conjunctival epithelium on the corneal surface by impression cytology is diagnostic of limbal stem cell deficiency. However, it is difficult to distinguish ectopic conjunctival epithelium from corneal epithelium by conventional cytologic techniques (i.e. hematoxylin-eosin or PAS staining) if typical goblet cells are not present on the specimen.

Cytokeratins are the most complex group within the intermediate filaments family and are present in almost all epithelial cells of vertebrates. Human cytokeratins have been catalogued by Moll into type I (small, acidic, including CK9 to CK20) and II (large, neutral to basic, including CK1 to CK8). The keratins of the corneal epithelium have been shown to be composed of a major keratin pair, formed by the acid keratin CK12 and the basic keratin CK3, together with a minor keratin pair (acid CK14 and basic CK5). In humans, CK3 and CK19 have been demonstrated to discriminate between corneal and conjunctival epithelium: CK3 stains all layers of normal human corneal epithelium but does not stain the conjunctiva, whereas CK19 stains the conjunctival epithelium but not the cornea.

Impression cytology, first reported in 1997 by Egbert, have subsequently been modified by several authors. This minimally-invasive technique was rapidly adopted for studies of diseases involving the ocular surface. A modified technique combined with immunologic antigen detection can now be used as simple and reliable test for ocular surface immunophenotyping with the aim of investigating limbal stem cell deficiency.

Corneal cytology impressions are obtained by a sterile membrane (Millicell CM 0.4 mm, Millipore Corp., Bedford, MA). After topical anesthesia, the membrane is gently pressed on the ocular surface for few seconds. To increase the number of harvested cells, the ocular surface is slightly dried by keeping the eye open before sampling. The cells are then fixed by an alcoholic spray. The procedure is performed at least twice per eye. Immunoperoxidase staining is performed on the membrane. Primary monoclonal antibodies against CK19 and CK3, secondary biotinylated bridge antibody, and tertiary streptavidin-biotin-peroxidase complex are used. At the end of the procedure, the membrane can be cut with a blade and laid down on a glass slide.

Since the evaluation of limbal stem cell deficiency by means of impression cytology requires a high-quality impression with regard to cellularity and preservation of immunogenicity, only samples with cells covering more than 50% of the membrane area can be considered suitable for diagnostic purposes, while cellularity less than 50% must be considered with caution.

The presence of clusters or randomly scattered CK19-positive cells represent an expression of limbal stem cell damage with acquisition of conjunctival immunophenotype, and are recorded as diffuse limbal stem cell deficiency (we arbitrarily rate diffuse imbal stem cell deficiency as mild (<25% of CK19-positive cells), moderate (25-50% of CK19-positive cells), and severe (>50% of CK19-positive cells). When CK19-positive cells are concentrated in one sector of the impression, and the same sector lacks of CK3-positive cells, a diagnosis of sectorial limbal stem cell deficiency can be formulated.

A high percentage of CK19-positive cells (>60%) with a near absence of CK3-positive cells (<2%), when a good or discrete sample for the latter was available, was defined as absolute limbal stem cell deficiency. Finally, the presence of a well functioning corneal limbus can be assumed when C19-positive cells are absent or less than 2%.

The cytologyc evidences described above, together with a thorough ocular surface examination and ocular history, allow to formulate an ultimate diagnosis of ocular surface disease with or without corneal limbal stem cell deficiency.


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