Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.