Use of a continuous‐flow cell separator in density gradient isolation of lymphocytes

Abstract

A continuous‐flow technique was developed to isolate and concentrate lymphocytes over Ficoll‐Hypaque (FH) density gradients using an automated cell separator (Model CS‐3000, Fenwal). Lymphocyte concentrates (LC) containing 6.51 × 10(9) mononuclear cells were obtained by standard leukapheresis techniques. Disposable apheresis kits were modified to allow the LC to be pumped into a separation chamber along with a counter‐centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with 300 ml of FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2 percent (99.9% lymphocytes, n = 14). The final product contained 6.7 percent of the initial platelets and had a hematocrit of less than 1 percent. In paired studies using split leukocyte concentrates (n = 15), lymphocyte recovery obtained by the automated apheresis technique compared favorably with that obtained by standard manual FH gradients (59.8 +/− 3.4% versus 67.3±/− 4.2%, p greater than 0.05) and platelet contamination was significantly reduced (2.7±/− 0.5% versus 26.6±/− 5.7% residual platelets, p less than 0.001). Equivalent lymphokine‐ activated killer (LAK) activity was generated from cells isolated by both manual and automated techniques. An automated continuous‐flow cell separator can be used for rapid FH isolation of large numbers of lymphocytes, providing a sterile product suitable for human use.