Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.
Removal of more than 99% of the erythrocytes and 74% of the nucleated cells from marrow grafts was achieved by density floatation separation in Percoli gradients with a density of 1.070 g/ml in eight 250-ml tubes, containing up to 3 x 10^9 nucleated cells per gradient. More than 90% of the myeloid and erythroid progenitor cells were recovered in the low density fraction. It appeared mandatory to use a centrifuge with the possibility of a gradual acceleration and deceleration. Twenty-five patients received a marrow graft from a histocompatible sibling after additional lymphocyte depletion by counterflow centrifugation, and 5 patients with T lymphoblastic malignancies received an autograft after in vitro purging with immunotoxins. All evaluable patients engrafted within normal limits, except 1 patient with an autoimmune pancytopenia who responded to steroids and 1 patient with a CMV infection. Four patients died too early for complete evaluation. The described separation method is easy, cheap and requires only 2 h for the complete processing of a marrow graft.
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