A technique for rapid isolation of bone marrow mononuclear cells using Ficoll‐Metrizoate and the IBM 2991 blood cell processor

Abstract

Marrow from seven normal donors and patients has been layered onto Ficoll-Metrizoate (FM) under pressure in the IBM 2991 blood cell processor to isolate the mononuclear cell (MNC) population prior to allogeneic transplantation or cryopreservation. This separation method, which takes less than 90 min, is a further development since our previous report detailing the use of the IBM 2991 to produce a concentrated marrow 'buffy coat' for infusion (Gilmore & Prentice, 1981). By adding FM to the system, marrow stem cells are further concentrated in a small volume with removal of unwanted granulocytes and red blood cells. This facilitates in in vitro treatment of marrow with monoclonal antibodies (Granger et al. 1982) or drugs, for either the selective elimination of malignant cells prior to autologous bone marrow transplantation (BMT), or T lymphocytes in an attempt to prevent graft versus host disease (GvHD) in allogeneic BMT. Five of the seven marrows processed by this procedure have thus far been infused into lethally irradiated recipients with engraftment (allogeneic); the other two marrows have been cryopreserved.

Successful collection of peripheral blood progenitor cells in patients with acute myeloid leukaemia following early consolidation therapy with granulocyte colony‐stimulating factor‐supported high‐dose cytarabine and mitoxantrone

“…The BM was collected by multiple aspirations from the posterior iliac crest. The cell suspension was depleted of red blood cells and of most of the polymorphonuclear leucocytes by a two-step procedure: a buffy coat and a Ficoll-Metrizoate gradient centrifugation using the Cobe Blood Cell Processor 2991 as described previously (Gilmore et al, 1982). Mafosfamide was added in a dose-adapted concentration (Douay et al, 1989;Martin et al, 1993).…”

Vasculogenic mimicry of acute leukemic bone marrow stromal cells

“…Isolation and long-term culture of CD34 (or CD133) bone marrow cells Bone marrow-adherent mononuclear cells (BMMNCs) were obtained by Ficoll density gradient centrifugation as described by Gilmore et al 21 The monocytes present in the mononuclear cells were discarded by adhesion on plastic Petri plates for 30 min. The non-adherent cells (BMMNCs) were collected and used to isolate the CD34 þ or CD133 þ cell population (with immunomagnetic beads; MACS; Miltenyi Biotec, Paris, France) according to the manufacturer's instructions.…”

Long-term outcome in acute myelogenous leukemia autografted with mafosfamide-purged marrow in a single institution: adverse events and incidence of secondary myelodysplasia

“…They were then thawed at 42°C in a water-bath and immediately infused into the patient's central line without further manipulation. The marrow cell suspension was depleted of RBCs and neutrophils using a buffy-coat and a Ficoll-Metrizoate (FM) gradient centrifugation method as described by Gilmore et al 23 The final cell suspension was washed twice using minimal essential medium (S-MEM), supplemented with ABO compatible human plasma (50%), and adjusted to a concentration of 2 ϫ 10 7 WBC/ml. Mafosfamide was added at a median dose of 70 g/2 ϫ 10 7 WBC (range, 60-80) according to the individual sensitivity of granulocytemacrophage colony-forming units, as described previously.…”