Lysosomal hydrolase staining of conjunctival impression cytology specimens in keratoconus

This study sought to assess the feasibility of impression cytology for the determination of conjunctival intracellular lysosomal hydrolase (acid esterase) levels in patients with keratoconus.

Twenty-two patients with keratoconus currently enrolled in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study and 22 age-and sex-similar controls underwent impression cytology. Samples were collected from each subject and control pair on the same day. The cells of the respective specimens were fixed immediately and were stained for acid esterase with use of identical batches of fixatives and stains. After staining, the specimens were cleared in xylene for mounting in synthetic resin on glass slides. The acid esterase staining intensity of each specimen was quantified as the percentage of light transmitted with use of an image analysis system (Zeiss). Multiple cells from each specimen were analyzed for each sample collected.

Mixed model analysis was used to account for the subject-control pairings and for the multiple cells from each sample. With this method, the mean light transmission for normal controls (mean = 63.0; standard error [SE] = 3.0) was highly statistically significantly different from that for the keratoconus subjects (mean = 52.4; SE = 3.0) (two-tailed p= 0.0032).

This study establishes the feasibility of adapting an acid esterase staining technique to conjunctival cells collected via impression cytology. Higher levels of lysosomal enzyme staining in patients with keratoconus have been previously reported by other investigators using full-thickness conjunctival specimens. We also demonstrate the value of using objective microspectrophotometry in measuring lysosomal enzyme staining with impression cytology specimens.