A blood separation system for separating mono nuclear cells from whole blood is described. The system comprises a chamber (12) that is rotatable about a rotational axis (28). The chamber comprises first and second spaced apart walls (24, 26) that form a separation zone having a high-G side and a low-G side which is located closer to the rotational axis than the high-G side. The chamber also includes an inlet region (74) where whole blood enters the separation zone for separation into red blood cells (96), a plasma constituent carrying platelets (98), and an interface (58) carrying mono nuclear cells between the red blood cells and the plasma constituent. A plasma constituent collection region (76) is also provided in the chamber where the plasma constituent is collected for removal from the chamber. An outlet path (46, 206) communicates with the plasma constituent collection region (76) and provides for removal of the interface (58) from the chamber. The system is operated under the control of a controller (134) that is operable in first and second modes. In the first mode, whole blood is conveyed into the inlet region while red blood cells and the plasma constituent are removed from the chamber and while the interface (58) is maintained at a set location within the chamber. In the second mode, the controller operates to move the interface from the set location for removal from the chamber via the outlet path. Finally, the system is equipped with a sensing element (226) for detecting mono nuclear cells in the interface in the outlet path. A method of separating mono nuclear cells from whole blood is also described.