Flow cytometry and AS-PCR assays in HCL and HCL-like disorders. (Top panel) Flow cytometry dot plots of a whole-blood sample subjected to RBC lysis from a representative HCL patient (top graphs) and of purified peripheral blood leukemic cells from a representative patient with HCL-v (bottom graphs). HCL cells (CD19⁺/CD25⁺ red events in the top right panel) represent 2% of all nucleated cells (CD45⁺ black events in the top left panel). HCL-v cells (CD19⁺/CD25⁻ and CD19⁺/CD103⁺ red events in the bottom left and right panels, respectively) represent 92% of all cells. (Bottom panel) Conventional agarose-gel electrophoresis of samples from 13 HCL patients (top panels; 12 pretreatment, 1 with residual disease after treatment) and 16 HCL-like patients (bottom gels; 6 SLLU, 10 SMZL), after AS-PCR for the mutant allele (1st and 3rd gel from the top) and for the wild-type allele (2nd and 4th gel from the top). In the top-most gels, serial dilutions of mutated and wild-type alleles (from 3.1% to 0% muted alleles) are also included to show the analytical sensitivity of the mutant AS-PCR (≥ 0.1% mutated alleles). All HCL samples gave rise to a mutant BRAF-V600E band as opposed to none of the HCL-like samples. One of the latter (SMZL case 30), which did not give rise to the wild-type band either, was not evaluable in this particular experiment (shown on purpose), but on repetition turned out to be evaluable (ie, strong wild-type band) and negative for BRAF-V600E (ie, mutant band not visible). To facilitate the visualization of the results, the gel lane of HCL case 13 was repositioned in the 2 top gels and the gel lane of the 50-bp DNA ladder was repositioned in the 2 bottom gels.