Analysis of Cytokeratin 12 and Mucin-1 Expression in Impression Cytology Specimens Through Immunofluorescence for the Evaluation of Ocular Surface Disorders

Abstract

Purpose: : To provide an innovative and effective diagnostic tool in ophthalmology for evaluating and carefully diagnosing the disorders and the alterations of the ocular surfaces through a new impression cytology procedure based on the employment of more specific markers of the ocular epithelia and quantification of their expression by means of laser scanning confocal microscope.

Methods: : The expression of cytokeratin 12 (K12) and Mucin1 (MUC1) was compared to that of currently used markers cytokeratin 3 (K3) and 19 (K19) through (i) semiquantitative RT-PCR in cultured conjunctival, limbal and corneal epithelial cells and (ii) immunofluorescence staining on both histological sections of conjunctiva, limbus and cornea and cytologic specimens collected by impression from normal subjects and patients with various chronic ocular surface disorders.

Results: : K12 and MUC1 showed to be expressed exclusively in the limbus/cornea and conjunctiva areas, respectively. In human ocular surface sections comprising corneal, limbus and conjunctival epithelia K12 stained all layers of corneal epithelium and the suprabasal layers of limbus but did not stain the conjunctiva, whereas MUC1 stained the superficial layers of the conjunctival epithelium but was absent in limbus and cornea. Impression cytology specimens from normal and diseased ocular surfaces showed distinct expression patterns for K12 and MUC1: healthy corneas expressed only K12 (but not MUC1) while conjunctivalized corneas from patients with limbal stem cell (LSC) deficiency were characterized by the presence of MUC1 and the disappearance of K12. Similar clear-cut results were not seen with the traditional pair of markers K3/K19, which showed lack of specificity and overlapping signals in cornea and conjunctiva specimens.

Conclusions: : Conjunctivalization is the hallmark of LSC deficiency. The ability of K12 and MUC1 to discriminate between limbus/cornea and conjunctiva in impression cytology specimens (unlike K3 and K19) could become a powerful diagnostic and clinical monitoring tool for ophthalmologists in order to evaluate alterations of the ocular surface and the grading of LSC deficiency.

Keywords: immunohistochemistry • cytology • cornea: epithelium