ZAP-70 protein expression can distinguish CLL subtypes. (A) ZAP-70 protein expression was assessed by Western blotting in whole cell lysates of normal PBMCs, or CD19⁺-purified leukemic cells from blood of patients with Ig-unmutated and Ig-mutated CLL. The data are representative of Western blot analysis of 60 patient samples analyzed. Equal loading is demonstrated by probing for β-tubulin. (B) ZAP-70 can be detected by immunohistochemistry in clinical samples. PBMCs (top 2 rows; original magnification, × 200) were embedded in a fibrin clot, fixed, and processed by standard techniques. PBMCs and routine bone marrow trephine biopsies (bottom 2 rows; original magnification, × 100-200) were stained with CD20, demonstrating involvement by B-CLL, and CD3, which stains interspersed T cells. Insets show areas of the respective sample at an original magnification of × 1000. ZAP-70 was positive in T cells and Ig-unmutated CLL cells.