Characterization of CLL B cells with ongoing IgG CSR. (A-D) Representative flow cytometric profile from a patient with UM AID⁺⁺ CLL. The 3 CLL B-cell subsets as well as the surface expression of CD5 and CD19 markers are depicted (IgM⁺, IgM⁺/IgG⁺, and IgG⁺). (E-G) Semiquantitative RT-PCR from the 3 cell sorter–isolated subpopulations. (E) Clonal isotype switch transcripts with tumor-related VH and Cμ or Cγ primers. (F) Amplification of CTs and subsequently hybridization with Cμ probe encompassing 1-180 nt of the first Cμ exon. (D) AID amplification from isolated CLL subsets. For all PCR analysis putative B-cell contamination between isolated subsets, moreover to cytometric purity analysis, was estimated by amplifying tumor-related VH-Cμ inside the IgG⁺ subset and with VH-Cγ inside the IgM⁺ subset. GAPDH was amplified in all cases as internal control.