Multiparameter Flow Cytometry for CLL Diagnosis

For diagnosing CLL and other B-cell lymphomas we used a B-cell panel which consisted of two tubes with different fluorescence antibody panels. The first tube (T1) included fluorescence antibodies against B-cell antigens (CD19, CD20, FMC7, CD79b, CD23, light chains of kappa and lambda), T-cell antigens (CD3, CD5, CD2, CD7, CD4, CD8), and the activation marker CD38, which has been described to be prognostic for CLL [14 (link)]. The second tube (T2) contained B-cell antigens (CD19, CD20, IgM), markers of hairy-cell leukemia (CD103, CD11c, CD25), follicular lymphoma, and high-grade lymphoma (CD10), and additional markers to ensure the diagnosis of CLL (CD43, CD200). The complete antibody panel, clones, and fluorescence dyes are stated in the Supplementary Information (Table S1).
Two 5 mL polystyrene FACS tubes with fluorescence antibodies in a dried-down layer (DuraClone-Technology, Beckman Coulter, Krefeld, Germany) were incubated for 15 min at room temperature in 100 μL prewashed peripheral blood. After antibody staining, red cells were lysed in 2 mL of VersaLyse™ (Beckman Coulter, Krefeld, Germany) for 10 min, washed with 3 mL of buffered phosphate saline (PBS Biochrom, Berlin, Germany), and centrifuged with 300× g for 5 min. The cell pellet was resuspended in 500 μL PBS and measured on a Navios Flow Cytometer (Beckman Coulter, Krefeld, Germany). In total, up to 1 × 10⁵ cells were acquired.