Cell surface IgM levels as well as expression and global phosphorylation levels of Syk reflect CLL B-cell responsiveness. Presence of surface IgM (A), CD20 (B), or Syk (C) and Zap70 (D) kinase levels was assayed on normal (control) and CLL B cells using flow cytometry analysis. (A) Cell surface IgM levels were calculated and graphed relative to control isotype labeling for 11 controls, 20 nonresponder (NR) samples, and 20 responder (R) samples. MFI indicates mean fluorescence intensity. (B-D) Cell surface CD20 expression on 20 NR and 20 R patients and intracellular Syk and Zap70 on 22 NR and 24 R cases were evaluated and graphed relative to control B cells. Means (± SEM) and significant P values are indicated (ns denotes not significant). (E) Anti-phosphotyrosine immunoprecipitation ([IP]; α-pY) was performed on cellular extracts of NR (n = 6) and R (n = 6) CLL B cells. Cells were either left unstimulated (−) or stimulated (+) with anti-IgM (α-IgM) antibody. Precipitates were analyzed with anti-Syk or anti-Zap70 antibodies, allowing detection of pSyk and pZap70. Total extracts are analyzed as a control for Syk and Zap70 expression. Fold increase is calculated as a ratio between stimulated and unstimulated levels.