Phenotypic and genetic characterization of the clonotypic cellular architecture in myeloma. (A) Multiparameter flow cytometry gating strategy for identification and flow sorting of CD19⁺ fractions (immature, naive, memory B cells, and PBLs) in the BM of patients with myeloma. Each of the indicated fractions was flow-sorted to high purity and subjected to patient-specific clonotypic genomic DNA quantitative PCR. (B) Top panel: Strategy for characterization of CD19⁻ clonotypic populations. Sequential gating of patient BM CD200⁺CD319⁺ and CD45^lo/⁻CD56⁺ cells allows identification and flow sorting of an almost entirely clonotypic hierarchy of CD138⁺ (PC), CD138^low and CD138⁻ (Pre-PC) cells. Bottom panel: Overlays of the CD19⁻ clonotypic cells (red) over the total BM cells (gray) indicates that, if the traditional gating on CD138⁺CD38⁺ events was applied, it would exclude Pre-PC and CD138^low cells from further analysis. (C-F) Frequency of clonotypic fractions in BM mononuclear cells (n = 30 patients; C,E) and PBMCs (n = 21 patients; D,F) of patients with myeloma shown as a cohort (C-D) or in individual patients (E-F). Horizontal bars represent median values excluding cases with undetectable clonotypic cells. (G) Top: May-Grünwald-Giemsa staining of flow-sorted Pre-PCs and PCs (original magnification ×1000). Bottom: Histogram and median FSC-A values of Pre-PCs and PCs. (H) Interphase FISH analysis of chromosome 13 complement showing loss of 1 red (13q34) and 1 green signal (13q14), consistent with monosomy 13 in highly purified, flow-sorted PB PBLs, Pre-PCs, and PCs from the same patient, confirmed as enriched in clonotypic cells by quantitative PCR. Chromosome 13 ideogram and location of FISH probes are shown on the left. The upper threshold for normal results is 5%. A minimum of 50 interphase cells were scored by 2 independent analysts in a blinded fashion. The average number of total interphases examined and the number of those with 13q deletion are shown below the FISH image.