Differential diagnosis between HCL and its mimickers. (A) A needle biopsy of a bulky abdominal mass between the liver and the spleen, featuring mature lymphoid cells that infiltrate pancreatic glandular structures (left panel; hematoxylin and eosin [H&E] staining); the immunohistochemical staining with Annexin-1 (ANXA1; red labeling in the right panel) confirms the HCL origin of this infiltrate in such an unusual anatomic site. (B) A BM biopsy involved in SMZL with its typical intrasinusoidal infiltration pattern, which is highlighted by the B-cell markers CD79a (red; left panel) and PAX5 (brown; right panel). SMZL cells do not express ANXA1 (blue, right panel, with ANXA1-positive myeloid and T cells serving as internal control). Pictures of immunohistochemical stainings shown in panels A and B were taken with the 40×/0.85 objective (U Plan Apo) of a BX61 microscope equipped with a DP71 digital camera, using cell^B x.y acquisition software (all from Olympus). (C) A genetic diagnosis of HCL (vs HCL-v) can be easily made in whole-blood samples through BRAF-mutant allele-specific DNA PCR followed by agarose gel electrophoresis. The BRAF V600E gel band is visible in the blood sample from an HCL patient (known to contain <1% of leukemic cells by flow cytometry) but not in the blood sample from an HCL-v patient (containing >40% leukemic cells), which shows only the BRAF wild-type amplicon. The gel lanes to the left of the HCL and HCL-v samples represent a serial dilution (from 100% to 0%) of BRAF-mutant alleles with wild-type ones, which sets the detection limit of this qualitative assay at 0.05% mutant alleles. Dashed lines separate lanes of the same gels that were repositioned for clarity.