Human granulocyte isolation by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation (CFCL/CCE)

Abstract

Human granulocytes (PMNL) isolated by continuous flow centrifugation leukapheresis (CFCL) were further purified by counterflow centrifugation elutriation (CCE). Studies were conducted to determine the maximal granulocyte capacity of the 4.5-ml elutriation chamber and to determine the efficiency of granulocyte recovery from CFCL concentrates. The maximal capacity of the elutriation chamber was determined to be 1.30 x 10(9) granulocytes with a 98.8 percent granulocyte purity and a 51:1 PMNL/RBC ratio. The efficiency of recovery was 82.2 percent with a 95.1 percent granulocyte purity and a 68:1 PMNL/RBC ratio. The CCE flow system was designed to allow continuous elutriation runs without need of breakdown time between runs. In four to five hours, approximately 4 to 5 x 10(9) granulocytes can be isolated with purity in excess of 95 percent. In vitro analysis of the viability and function of CFCL/CCE-isolated granulocytes indicates that the morphologic integrity and physiologic function were not comprised as a result of the combined isolation procedure relative to granulocytes isolated by CCE from whole blood samples. Granulocytes were held at 4 to 6 C for 16 to 24 hours in granulocyte storage medium (GSM) plus 20 percent autologous plasma at concentrations of about 6 x 10(6) PMNL/ml prior to in vitro analysis.