Isolation of Mononuclear Leukocytes in a Plastic Bag System Using Ficoll‐Hypaque1

Abstract

Mononuclear cells, present in bone marrow and peripheral blood, have been isolated from red cells and granulocytes using a ficoll‐hypaque density centrifugation process. Cells isolated by this process which uses centrifuge tubes may become contaminated. In 19 studies in our laboratory we used Ficoll‐Hypaque treatment to isolate mononuclear cells from cellular residues obtained during plateletpheresis using a modified 600‐ml polyvinylchloride (PL‐146) plastic bag with the Haemonetics blood processor V‐50 or the Fenwal CS‐3000 blood processor. The 600‐ml PVC plastic bag was modified by sealing its vertical edges using radio frequency to form a narrow bag with a volume of approximately 200 ml. A 125‐volume of diluted apheresis cellular residue was collected, and the mononuclear cells were isolated as follows: the diluted cellular residue was layered onto 75 ml of Ficoll‐Hypaque with a specific gravity of 1.077 and was centrifuged at 260 g for 30 min at 22°C. The supernatant plasma was removed. The mononuclear cell layer was transferred to a sterile 600‐ml transfer bag, and the cells were washed with saline. Of the 4.24d±0.9 times 10⁹ mononuclear cells applied to the gradient, approximately 3.73±0.8times10⁹ cells were recovered. The recovered cells consisted of 77.3±8% lymphocytes, 19.0±7% monocytes, and 3.6±3% granulocytes. There was no significant difference in tissue culture growth in the CFU‐GEMM assay of mononuclear cells whether the plastic tube or the plastic bag system was used. Aerobic bacteriologic cultures were negative. The PL‐146 plastic bag system used in this study proved to be a significant aid in isolating mononuclear cells from plateletpheresis residue.