Isolation of mononuclear leukocytes in a plastic bag system using Ficoll-Hypaque

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 polyvinyl-chloride (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 degrees 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 4.24 +/- 0.9 X 10(9) mononuclear cells applied to the gradient, approximately 3.73 +/- 0.8 X 10(9) 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.