Anti cd5 pe cy5

Evaluation of ZAP-70 expression in CD19⁺/CD5⁺ leukemic cells in all tested samples was performed according to the previously described procedure (Hus et al. 2006 (link)) using monoclonal antibodies: anti-CD19 FITC, anti-ZAP-70 PE (clone 1E7.2) and anti-CD5 PE-Cy5 (BD Pharmingen, USA). Expression of CD38 on leukemic cells was evaluated using monoclonal antibodies anti-CD19 FITC, anti-CD38 PE and anti-CD5 PE-Cy5 (BD Pharmingen, USA). The cut-off point for leukemia cells with ZAP-70 expression was ≥ 20%, while for leukemia cells with CD38 expression it was ≥ 30%.

CLL cells were stained for ZAP-70 protein expression as previously described [8 (link)]. Briefly, ZAP-70-positive cells were evaluated in the PB via analysis of the surface expression of CD19 and CD5 antigens, as well as intracellular expression of ZAP-70 by flow cytometry. The percentage of CD19+CD5+ZAP-70+ cells was determined. MoAbs used for analyses included anti-ZAP-70 PE (Clone 1E7.2, IgG1, BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD19 FITC (Clone HIB19, IgG1) and anti-CD5 PE-Cy5 (Clone UCHT2, IgG1) (BD Pharmingen, San Diego, CA, USA). The cutoff point for ZAP-70 positivity in leukemic cells was ≥20 %.

As previously described [8 (link)], flow cytometry analysis of CD38 was performed on fresh PB samples stained with anti-CD38 FITC (Clone HIT2, IgG₁), anti-CD5 PE-Cy5, and anti-CD19 PE (BD Pharmingen, San Diego, CA, USA). A standard, whole-blood assay with erythrocyte cell lysis was used for preparing the PB specimens. The samples were analyzed by flow cytometry directly after preparation. CLL cells were considered CD38-positive when ≥20 % was expressed in the membrane antigen.