Gating strategy for specific identification of CLL cells.

(A) Total B cells were identified using 2 regions: (i) CD19 and side scatter, to exclude granular cells showing nonspecific CD19 binding; and (ii) forward and side scatter, to exclude apoptotic cells and doublets. (B) CLL cells were separated from normal B cells according to their CD5, CD20, and CD79b. CLL cells could be separated from normal B cells using (i) CD5 versus CD20 in 82% of cases; (ii) CD79b versus CD20 in 35% of cases; and (iii) CD5 versus CD79b in 88% of cases. (C) To separate CLL cells from normal B cells, all 3 antigens must be assessed on gated B cells. (D) To separate CLL cells from normal B cells and normal B progenitors in the bone marrow in all cases, CD38 is included; this requires 2 tests: (i) CD19 versus CD5 versus CD38 versus CD20; and (ii) CD19 versus CD5 versus CD38 versus CD79b.