The OV-TL 12/30 clone of anti-cytokeratin 7 antibody as a new marker of corneal conjunctivalization in patients with limbal stem cell deficiency

Abstract

Purpose: To present cytokeratin (CK)7 (OV-TL 12/30 clone) as a newly identified, reliable marker for distinguishing between the conjunctival and corneal surface epithelia, which will contribute to the precise diagnosis of limbal stem cell deficiency (LSCD).

Methods: Corneal and conjunctival epithelial imprints from 12 cadaveric bulbi and from 9 patients with clinically diagnosed LSCD were used for CK7 and CK19 immunocytochemistry. Specimens on nitroacetate cellulose filter papers obtained from the patients were stained with a combination of periodic acid-Schiff (PAS) and Gill's modified Papanicolaou stains, to assess the presence of goblet cells (GCs).

Results: CK7 was present in almost all superficial conjunctival epithelial cells from the cadaveric specimens. No immunostaining was observed on the corneal surface. A prominent sharp border of stain was found between the positive conjunctiva and the completely negative epithelium of the central cornea. A more gradual centrifugal decrease in the number of positive cells between the conjunctiva and cornea was observed for CK19. Several CK19-positive cells were detected in the central corneal epithelium. All corneal specimens from affected eyes (unilateral as well as bilateral LSCD patients) revealed strong positivity for CK7, and GCs were present in only 78% of patients.

Conclusions: In cases in which GCs are severely decreased or are absent from the conjunctival surface, the detection of CK7 (OV-TL 12/30 clone) clearly confirms the overgrowth of the conjunctival epithelium over the cornea. Moreover, CK7 is a more reliable marker for distinguishing between the corneal and conjunctival epithelia compared with CK19.

Figure 1.

Indirect fluorescence immunocytochemistry on membranes. Expression of CK7 and -19 (FITC, green) in the control samples. The nuclei were counterstained with propidium iodide (red). Scale bar, 10 μm.

Figure 2.

Differences between the localization of CK7 and -19 (FITC, green) in the superficial epithelium of a control central cornea and conjunctiva. A prominent, sharp border between the absence and presence of the CK7 signal was noted, compared with the gradual decrease in CK19 staining. The nuclei were counterstained with propidium iodide (red). Scale bar, 50 μm.

Figure 3.

Expression of CK7 in two representative samples of surface conjunctival epithelium (con) and in two samples of corneal surface epithelium (cor) determined by RT-PCR. GAPDH was used as an internal control. NCo, negative control (reaction without sample cDNA), a marker for internal contamination; L, 50-bp DNA ladder (25–1000 bp).

Figure 4.

Expression of CK7 protein (54 kDa) in three different samples of corneal (cor) and conjunctival surface epithelium (con) as determined by Western blot analysis. β-Actin (42 kDa) was used as an internal control.

Figure 5.

Detection of CK7 (FITC, green) on a radial cryosection of a corneoscleral button. Nuclei were counterstained with propidium iodide (red). Scale bar, 50 μm.

Figure 6.

Ocular surface of patients (P) with LSCD. No CK7 (FITC, green) was present in the unaffected corneas. CK7 was expressed by normal conjunctiva as well as conjunctival cells that overgrew the corneas of the LSCD eyes. Indirect fluorescence immunocytochemistry was performed on the membranes, and nuclei were counterstained with propidium iodide (red). RE, right eye; LE, left eye. Scale bar, 10 μm.