Impression Cytology of the Conjunctiva

IC was performed to evaluate the HLA-DR expression at the temporal bulbar conjunctiva, and was conducted 24 hours after the IVCM assessment to avoid misinterpretation due to the technical execution of the confocal microscopy. Samples were collected using Millicell-CM 0.4 µm (Millipore, Bedford, MA); the specimen acquiring procedure was performed as previously described.18^,19 Zeiss Confocal LSM 510 (Carl Zeiss MicroImaging GmbH, Vertrieb, Germany) was used to visualize cells. Five different fields for each sample were evaluated. Positive (red nucleus and green cytoplasm) and negative (red nucleus) cells were counted. Minor irregularities that may have been present after cutting the membranes were normalized by reporting the percentage of positive cells for HLA-DR.

All evaluations of impression cytology samples were performed by two independent observers (authors L.B. and R.D.A.). IVCM (author M.L.) and IC (authors L.B. and R.D.A.) operators were masked for grouping and subject history of patients. Both eyes for patients were evaluated in the study, but one eye per subject was randomly chosen (using a computer-generated random number list) for the statistical analysis.