Conjunctival impression cytology in horses

Aim Conjunctival impression cytology (CIC) is a technique described both in human and veterinary medicine (1;2). Information about CIC in the horse are scant (3). Aim of the study was to evaluate feasibility, protocol standardization and normal cytologic pattern of CIC in healthy horses. Methods Samples were taken from the bulbar conjunctiva of 25 healthy horses. The first 10 horses were used to standardize the method. CIC smears were collected following Bolzan???s protocol (4), after the instillation of Oxybuprocaine hydrochloride. Impressions were taken applying a 5x7 mm strip of Millipore filter with a pore size of 0.45µm (Merck Millipore, Milan) on the temporal bulbar conjunctiva and fixed in 95% alcohol. Specimens were stained with Periodic Acid???Schiff (PAS) and with Hematoxilin???Eosin (H&E) stains. The Bolzan???s protocol was modified, reducing the time of immersion in Schiff???s reagent (3 min) and elongating the immersions in sodium metabisul???te (10 min). Stained samples were immersed in xylene and then mounted on slides cover-slipped. Conjunctival and inflammatory cells were differentiated with a light microscope, counting 200 cells for each eye, by scanning each sample in a sinuous and continuous pattern. Cellularity, cell distribution and damage were scored following the criteria of Bauer et al. (5). Results Cells??? count and quality scores were reported in table 1. A nose twitch was used in 5 cases. The technique allowed to collect a sufficient quantity of well-preserved cells. Goblet cells and erythrocytes were not observed. Epithelial and inflammatory cells, debris, naked nuclei and keratinized cells were found for both stains. Conclusions CIC can be considered a quite good technique for the study of the conjunctiva in the horse. Even if difficulties in the placement on the bulbar conjunctiva and the maintenance of the open eye were observed, the instillation of local anesthetic facilitate samples collection. CIC is non-invasive and well tolerated by the animal, as demonstrated by the absence of erythrocytes in the smears. It is important to assess the size of the strip, because smaller strips leaked from the biocassette. Samples had a good cellularity and cells were well distributed and preserved. The staining methods influenced the evaluation. PAS stain gave a strongly pink background, although the time of staining was reduced and the time of discoloration was elongated. The H&E stain gave less background and a better definition of morphologic cellular features and allowed the differentiation of inflammatory cells. Thus, H&E may be considered advisable when an inflammatory disease is suspected.