To precisely characterize IEL, all subjects underwent IC of the superior tarsum and immunofluorescence staining with antibodies directed against lymphocyte CD3 and CD20 antigens.
The CD3 complex serves as a T-cell coreceptor that associates noncovalently with the T-cell receptor (TCR). Both TCR and the CD3 protein complex are defining features of lymphocytes belonging to the T-cell lineage and, therefore, can be used as T-cell markers (for both CD4 and CD8 lymphocytes).
22 CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-lymphocytes beginning at the pro-B phase (CD45R+, CD117+) and progressively increasing in concentration until maturity.
23 Impression cytology was performed from 28 to 32 hours after confocal microscopy to avoid misinterpretation due to the mechanical pressure during execution of IVCM. The central part of the superior tarsum, which was the largest and still marked with the methylene blue, was chosen for the examination and centered with the IC stripping membrane.
Briefly, after topical anesthesia with oxybuprocaine hydrochloride 0.4% (Novesin; Novartis, Basel, Switzerland), a strip was applied to the central part of the superior tarsal conjunctiva, within the borders of the marked site, and then pressed gently by a glass rod. Impression cytology samples were collected using Millicell-CM 0.4 mL (12 mm of diameter) (Millipore, Bedford, MA, USA); cells were fixed with cytology fixative (Biofix; Bio Optica, Milan, Italy).
For CD3 and CD20 immunofluorescence staining, the Millicell membranes were hydrated with distilled water and placed in 80% alcohol for 2 minutes. The membranes were washed in distilled water and PBS was added for 2 minutes, followed by two washes with Wash Buffer (Dako, Glostrup, Denmark) of 2 minutes each. Then the filters were incubated with ribonuclease A (Sigma-Aldrich Corp., St. Louis, MO, USA) diluted 1:300 in PBS for 20 minutes at room temperature. The specimens were washed and Protein block (Dako) was added for 10 minutes at room temperature. Finally, CD3 antibody (Dako) 1:50 or CD20 (Dako) 1:25, both diluted in antibody diluent (Dako), were incubated overnight at 4°C. Samples were washed and anti-rabbit Alexa fluor 488 (Invitrogen, San Giuliano Milanese, Italy) for CD3 or anti-mouse Alexa Fluor 488 (Invitrogen) for CD20, diluted 1:200, and propidium iodide at 1:150 (both in antibody diluent) (Dako), were added and incubated for 1 hour at room temperature. Membranes were mounted with a drop of Fluorescent Mounting Medium (Dako) and the cells visualized with a Zeiss confocal laser-scanning microscope (510; Carl Zeiss MicroImaging, GmbH, Vertrieb, Germany). Five different fields for each sample were evaluated and two masked expert observers (AM and LB) in concordance counted the positive-staining cells.
The LyD, FD, and FA, were the primary outcomes. Follicular reflectivity and FLyD with IVCM, and CD3 and CD20 positivity at IC were secondary outcomes. The modification of all these parameters with aging was also evaluated.