FISH analysis of PBMC from healthy donors after G-CSF mobilization. (A) Twenty-four healthy consenting donors (9 males, 15 females; mean age 44.2 ± 2.5 years; range, 23.3-57.9 years) were recruited in 3 transplantation centers (Besançon, Nancy, and Lyon, France), after approval by the Besançon University Hospital ethical committee. Blood samples harvested at baseline (2 control samples harvested before G-CSF administration, at day −30 ± 6 and −5 ± 1, respectively), just after G-CSF (Granocyte, Lenograstim, Chugai Pharma, Paris, France, n = 6 or Neupogen, Filgrastim, Amgen, Neuilly sur Seine, France, n = 18) mobilization (day 0, ie, before the first cytapheresis), after the last cytapheresis (day 0.7 ± 0.2) and at 1 (day 32. ± 1), 3 (day 92 ± 1), 6 (day 184 ± 1), and 12 (day 366 ± 2) months after mobilization were analyzed for aneuploidy, quantified after FISH of chromosomes 8 (gray diamonds, dashed line) and 17 (▲, full line) specific centromeric probes on PHA-stimulated whole-blood samples. For clarity, data are arbitrarily set at −40, −20, 0, 10, 30, 90, 180, and 360 days, respectively. Each time point is compared with the mean of baseline values using a paired t test; n = 24 except at days 180 (n = 20) and 360 (n = 17). *P < .05; **P < .005; ***P < .001. (B) CD34⁺ cells were purified to 96% and 89%, respectively, from 2 donors' peripheral blood mononuclear cells (PBMCs) harvested at day 0 (D21, ▲; D22, ◊) by positive immunomagnetic sorting (Miltenyi Biotec) according to the manufacturer's recommendations. The CD34⁺ fraction was either analyzed immediately after sorting, without stimulation (no), or was incubated in 200 μL of Iscove modified Dulbecco media (IMDM) in the presence of 20% FCS, 1% essential amino acid and stimulated with the hemopoietic growth factors Fms-like tyrosine kinase 3 ligand (Flt-3L; 300 ng/mL), stem cell factor (SCF; 300 ng/mL), IL-3 (10 ng/mL), IL-6 (10 ng/mL), and G-CSF (50 ng/mL) for 7 days at 37°C in a 5% CO₂ environment (CK) before hybridization with a chromosome 17–specific centromeric probe. The CD34⁻ fraction was either analyzed immediately after sorting, without stimulation (no) or stimulated by PHA (10 μg/mL), before hybridization. The CD34⁺ and CD34⁻ fractions were compared with their unsorted PHA-stimulated PBMC counterpart or to PHA-stimulated whole blood (WB) harvested from the same donors before G-CSF mobilization (control samples) and at day 0. Similar results were obtained, although at lower frequencies, when cells were hybridized with chromosome 8–specific centromeric probes.