LRCs include drug-resistant progenitors able to generate clonotypic progeny. (A-D) Mononuclear cells were isolated from the mobilized blood preparations, labeled with CFSE, grown in 3-D for 14 days, and treated with melphalan (“Preclinical drug treatment studies”). (A) Flow cytometric profile of the LRCs. The gates for sorting were set to isolate cells with lymphocyte scatter properties (scatter plot shown is gated on the lymphocytes) and CFSE^high fluorescence, comparable with that in the stained population preculture, as expected for nonproliferating or slowly proliferating cells. Percentage of LRCs was calculated as the percentage of CFSE^high cells in the lymphocyte fraction of the 3-D culture. Representative FACS plots from 5 individually analyzed patients. (B) May-Grunwald-Giemsa (MGG) staining of the sorted LRCs (top) and immunohistochemical staining of sorted LRCs for CD20 (bottom; original magnification ×400); see “Immunohistochemistry.” Representative images from 3 individually analyzed patients. (C) Representative colonies from the CFU assay (original magnification 40×; n = 10 individually analyzed patients) as indicated in “Microscopy.” (D) Progeny generated from sorted LRCs from both untreated and melphalan-treated cultures were analyzed by RQ-PCR to quantify the number of clonotypic cells. These c(T) values, compared with control values, indicate that approximately 100% of the progeny were clonotypic. No RQ-PCR amplification was detectable for nonclonotypic colonies. Similar c(T) values were observed for clonotypic colonies from both treatment groups (n = 8 colonies from untreated group and n = 11 colonies from melphalan group). Horizontal lines represent medians. (E) Quantification of the LRCs as a percentage of total cells in the culture, CFU as a percentage of LRCs, and clonal CFU as a percentage of total colonies obtained in the CFU assay (mean ± SD of 8 individually analyzed patients).