Assay development and validation of inflammatory markers in impression cytology specimens

The principle of Impression Cytology (IC) utilizes a non-invasive technique to remove superficial layers of the ocular surface. The application of a filter to the surface of the bulbar conjunctiva allows for repeated collection of superficial cells for the analysis of ocular surface disorders. Evidence suggests expression of the HLA-DR antigen on the surface of conjunctival epithelial cells is associated with allergic conjunctivitis and dry eye syndrome. Performance characteristics of HLA-DR was evaluated through intra, inter and stability assays. Evaluation of the reproducibility and overall performance of the novel methodology would be useful in understanding the tolerance level of the assay. Utilizing established techniques, IC specimens were obtained from 12 donors and assayed over multiple days and timepoints. Conjunctival epithelial cells were extracted and assayed based on differential immunostaining of both conjunctival epithelial cells and leukocytes. The reproducibility across donors over time was consistent, demonstrating conjunctival cells maintained their integrity of cell surface expression of HLA-DR. In conclusion, the studies met all validation criteria, and support the utilization of HLA-DR inflammatory measurements in impression cytology specimens. Utilization of a flow cytometric platform to examine the expression of HLA-DR antigen may help predict clinical outcome, evaluate disease progression, and monitor the effects of treatment.