Clonal plasma cells from patients with systemic AL-amyloidosis can be highly enriched after FACS-sorting. (A) Flow cytometric plot of FACS-sorted cells is shown with CD138⁺/DAPI⁻ cells in the bottom right quadrant. CD138⁺/DAPI⁻ cells were used for gene-expression analysis in 9 cases. In the insert, an IFM image of enriched clonal plasma cells from the marrow aspirate of an AL-amyloidosis patient is shown. These cells were stained intracellularly for lambda light chains with a FITC-linked monoclonal antibody and were more than 95% pure. (B) Box-and-whiskers plots of the expression levels of lineage-specific genes are depicted for the 9 specimens of purified clonal plasma cells used in gene-expression studies. The box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median (50th percentile), and the whiskers extend down to the smallest and up to the largest value. The genes (and probe sets in parentheses) include genes for clonality, namely the λ and κ light-chain constant region genes (Affymetrix probe sets 221651 and 215121). Eight of the 9 specimens were λ and hence the nonclonal gene was κ in those cases. The plot of expression levels for the clonal and nonclonal genes clearly shows that the clonal genes were expressed at higher levels than the nonclonal constant region genes (paired t test, P = .005). The other lineage markers were CD38 (205692), XBP-1 (200670), CD138 (201286), CD4 (200670), CD14 (201743), CD16 (206398), CD19 (206398), CD33 (206120), CD45 (212588) and CD64 (214511). Myeloma cells can aberrantly express some of these markers.⁷⁰  Comparison of the CR and NR sets showed no significant differences in gene- expression levels by paired t test for clonal and nonclonal IgV_L constant region genes, CD38, XBP-1, CD138, or other lineage markers with expression levels more than 100.