PEF selectively purges myeloma cells from primary multiple myeloma bone marrow. (A) Small volumes (2 mL to 4 mL) of BM from patients with MM, some of which contained more than 3% myeloma plasma cells, by CD38CD138 staining, were processed and PEF-treated at 0 kV/cm (left) or 1.4 kV/cm (right). Representative photographs of cells stained with trypan blue after PEF treatment are shown. Dead cells were not separated by centrifugation over ficoll (unlike Figure 1A) and lysed cells appear as trypan blue–positive, on the right, whereas small cells including progenitor cells are preserved. (B) After PEF treatment cells were stained with anti–CD38-PerCP, anti–CD138-PE, anti–CD45–fluorescein isothyocyanate (FITC), or anti–CD34-FITC, and the nuclear exclusion dye 7-AAD. Cells were analyzed by Cellquest software. Cells gated as 7-AAD–negative and CD45^low were analyzed for the presence of CD38^high, CD138⁺ myeloma plasma cells. This population is noticeably absent in the dot plot on the right for cells pulsed at 1.4 kV/cm group. (C) Percent survival of CD38^highCD45^lowCD138⁺ myeloma cells (blue bars), CD34⁺ cells (cream bars), and other small cells (red) after PEF treatment, at 0 kV/cm or 1.4 kV/cm, of 4 independent multiple myeloma patient bone marrow specimens. The limit of detection based on input bone marrow cell number, and percent myeloma cells within bone marrow specimens, is approximately 3 logs (or 0.1% of starting number). Cells staining positive for the CD38^highCD45^lowCD138⁺ phenotype constituted 2% to 7% of total bone marrow mononuclear cells (45% for sample 4) from MM samples analyzed. Primary myeloma cells do not proliferate reproducibly in vitro; therefore, serial dilution tumor regrowth assays cannot be routinely performed to enumerate patient myeloma cells after PEF.