Sequential gating strategy for CD160 detection on malignant CD19⁺ B cells. (A) Total B cells were identified using forward and side scatter to gate the lymphoid region and exclude any apoptotic cells and debris. (B) CD19⁺ B cells were compared with side scatter to exclude any nonspecific binding. (C) CD19⁺ B cells were further isolated by gating the CD2⁺ events and generating a “NOT” (exclusion) gate. (D) The malignant B cells were separated from the normal residual B cells using a CD2⁻CD5⁺CD19⁺ gate. (E) CD23 expression was calculated from the malignant population and used to generate the mini-CLL score and CD23r. (F) In the context of CLL, CD160 positivity defined on the CD2⁻CD5⁺CD19⁺CD23⁺ population was then calculated from this “pure” malignant B-cell population.