CD34+ lineage positive hemopoietic progenitors purified from peripheral blood. To obtain PB progenitor cells, 12 healthy donors received glycosylated recombinant human (rh) G-CSF (Lenograstim, Rhone-Poulenc Rorer, Milan, Italy) administered subcutaneously at 10 g/kg/d for 5 6 d. Leukaphereses were performed on days 5 and 6 as previously described (Lemoli et al, 1997; Lemoli et al, 2000). Haematopoietic progenitor purification and subsequent studies were always performed using day-5 collections at the peak time of PB CD34+ cells. The protocol was approved by the ethical committee of the University Hospital and each donor gave written informed consent. Mononuclear light density (<1·077 g/ml) cells (MNC) were enriched using Ficoll-Paque (Pharmacia, Uppsala, Sweden) and resuspended in 1% bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO, USA). CD34+ cells were highly purified by MiniMacs high-gradient magnetic separation column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. To assess the percentage of CD34+ elements, aliquots of the CD34+ target cells were restained with an antibody (HPCA-2; IgG1a-fluorescein isothiocyanate [FITC]; Becton Dickinson, San Jose, CA) directed at a different epitope of CD34 antigen than that (QBend10) used with the MiniMacs system. Briefly, CD34+ cells were incubated for 30 minutes in the dark at 4°C with HPCA-2-FITC. Propidium iodide (2 µg/mL) was added for the detection of nonviable cells, which were excluded from analysis. After two washes in PBS/BSA, flow cytometric analysis was performed on a gated population set on scatter properties by using FACScan equipment (Becton Dickinson). A minimum of 10,000 events was collected in list mode on FACScan software. (Lemoli et al, 1997). The percentages of CD34+ cells in PB samples were 1·6±1%, and 0·9±0·3% of the MNC fraction respectively. After magnetic separation, the percentages of CD34+ in PB samples were 95±5%.
