Venous blood was collected in EDTA Vacutainer tubes (Becton, Dickinson and Co, Franklin Lakes, NJ) and processed immediately. One milliliter of blood was lysed for 5 minutes at 37°C with 50 mL of lysis buffer (0.83% ammonium chloride, 0.1% kalium bicarbonate, and 0.004% EDTA) and the leukocytes were washed once in phosphate-buffered saline (PBS) solution. The cells were counted manually and resuspended in PBS solution for a concentration of 2.4 × 106/mL. Antibody staining for immunophenotyping by laser scanning cytometry was performed according to the method published by Clatch and colleagues,19-22 with slight modifications. Briefly, 3 μL of the fluorescein isothiocyanate (FITC)–, phycoerythrin (PE)-, and phycoerythrin cyanogen 5 (PECy5)–labeled antibodies (Table 1) was added to 20 μL of cell suspension and the mixture was gently pipetted onto custom-made chamber slides assembled on standard microscope glass slides. The following antibody-labeling reactions were performed: (1) CD45,CD4,CD8; (2) CD3,CD4, CLA; (3) CD4, HLA-DR, CD134; (4) CD4, CD45RO, CD45RA; (5) CD4, CD25, CD56; (6) CD4, CD7, CD26; (7) CD4, CD7, CD27; (8) CD4, CD7, CD28; (9) CD4, CD7, CD29; (10) CD4, CD7, CD30; (11) CD4, TCRαβ, TCRγδ; and (12) isotype controls. After a 30-minute incubation at 4°C the cells were washed with PBS and scanned in a laser scanning cytometer (CompuCyte, Cambridge, Mass) using the 488-nm line of argon laser as an excitation source. Integrated fluorescence in the green (FITC), orange (RPE) and far red (PC5) channels were collected on a cell-to-cell basis and presented as dot-plot diagrams following off-line fluorescence compensation with the CompuCyte software. After scanning, the chamber slides were disassembled and the cells adhering to the bottom slide were fixed briefly in methanol, air-dried, and stained with Wright-Giemsa. The slides with stained cells were repositioned in the laser scanning cytometer and the cells with the required characteristics were re-found for visual inspection.