Separation and characterization of mobilized and unmobilized peripheral blood progenitor cells by counterflow centrifugal elutriation
Hematopoietic Stem Cells
Peripheral blood stem cell (PBSC) harvests are highly contaminated with lymphocytes. In allogeneic transplantations with PBSC, this might lead to an increased risk of graft-vs.-host disease (GVHD). Marrow graft T cells depletion (TCD) by counterflow centrifugal elutriation (CCE) has been performed to decrease the incidence of GVHD, but in some cases, it has also led to a reduction in the PBSC content of the graft due to the distinct elutriation properties of these cells. We studied the ability of CCE to deplete lymphocytes from recombinant human granulocyte colony-stimulating factor (rhG-CSF)-mobilized PB mononuclear cells (PBMNC) and from unmobilized PBMNC from healthy volunteers. We characterized the elutriation properties of circulating mobilized and unmobilized hematopoietic progenitors. With an experimental three-flow rate CCE procedure, originally described for separation of bone marrow, fractions 24, 28, and Rotor Off (RO) were obtained without impairing yield or viability. This CCE procedure can also be used for the separation of PBMNC harvests by reducing the T cell contamination to or below the range seen in bone marrow grafts; CD3+ cell contamination in the RO fractions of mobilized and unmobilized PBMNC was reduced to < 10% of the input. This could bring allogeneic PBSCT within the range of feasibility. In unmobilized PBMNC, 25% of the recovered CD34+ cells were found in fraction 24, where no colonies were detected, and 50% of the recovered CD34+ cells were found in fraction RO, where most of the colonies were detected. In mobilized PBMNC, 80% of the recovered CD34+ cells were found in fraction RO, together with 90% of the colonies, but almost no colonies or CD34+ cells were detected in fraction 24. CD34+ cells in fraction 24 expressed less CD13, and antigen found on more mature myeloid cells. Mobilization with rhG-CSF causes a shift of CD34+ cells from smaller cells (fraction 24) to larger cells (fractions 28 and RO).