Flow cytometry in impression cytology specimens. A new method for evaluation of conjunctival inflammation

To investigate feasibility and potential uses of flow cytometry in impression cytology as a new procedure to assess and quantify conjunctival inflammation.

Specimens for cytology were collected by impression from 30 patients with various chronic ocular surface disorders and from 10 normal subjects. Two specimens were obtained in each eye: One was transferred onto a glass slide and processed by immunofluorescence with antibodies to human leukocyte antigen (HLA)-DR antigens; cells from the other were suspended in phosphate-buffered saline for flow cytometry. Monoclonal antibodies to HLA-DR antigens and CD23, the low affinity receptor to immunoglobulin E, were used.

Abnormal expression of HLA-DR and CD23 by conjunctival cells was found in 13 of 18 dry eyes and in 20 of 22 eyes with chronic conjunctivitis, whereas specimens remained almost negative (less than 10% of cells were positive) in normal eyes. Percentages of positive cells ranged between 20% and 98% of all conjunctival cells. Correlation between the two methods, immunocytology and flow cytometry, was highly significant (coefficient of correlation 0.77, P = 0.0001). Moreover, HLA-DR positivity, at its strongest intensity, was observed in a minority of cells (1% to 12%), most of which were resident class II-expressing dendritic cells. Percentages of those cells expressing high levels of HLA-DR were 3 +/- 1.2% in normal eyes, 5.8 +/- 4% in dry eyes (P = 0.05), and 5.9 +/- 3.5% in eyes with chronic conjunctivitis (P = 0.02).

Results of this preliminary study confirm that conjunctival epithelial cells may abnormally express inflammatory markers in chronic ocular surface disorders. Development of flow cytometry in analysis of cytologic specimens provides a new, sensitive, and objective tool for exploring conjunctival pathology.