Figure 1

Flow cytometric analysis of ZAP-70 expression (test set). PB cells of CLL samples were analyzed after staining with anti-CD19-APC, anti-CD3-PE, anti-CD5-PECy7 and AlexaFluor488-conjugated isotype control or anti-ZAP-70 antibodies. Panel A shows the gating strategies used to select lymphocytes in the left plot, CLL cells (CD19+/CD5+/CD3-) or T cells (CD19-/CD5+/CD3+) in middle and right plots, upon gating on lymphocytes. Panel B contains plots showing a representative ZAP-70 negative (upper row) and a representative ZAP-70 positive (lower row) sample, both analyzed according to the three different approaches utilized to evaluated ZAP-70 expression. The ISO- T-, and T/B Ratio-method readouts are shown respectively in the left, middle and right panels. For the ISO-method marker was set to have <1% CLL positive cells with isotypic control. For the T-method, marker was set on the left edge of T cells cluster, to have about 98% of positive cells. For the T/B Ratio-method the ratio was calculated directly from MFI values as separately read from T cell and CLL cell gates defined in panel A.