Flow cytometric measurement of NK cell immunoconjugates by pulse width processing

Pulse width analysis, in flow cytometry, has been widely used for optimal cell size resolution in cell kinetics analysis. Pulses, generated by scattered light or fluorescence of cells, are electronically analyzed for their height and width. The information generated from these two properties of the pulses is utilized to distinguish signals from single cells vs. signals from cell clumps or aggregates. Pulse width, unlike pulse height, is more sensitive to differences in cell diameter, and therefore can discriminate very small differences in it, which pulse height cannot. We have exploited this property of pulse widths to measure immunoconjugates between NK cells and their targets. Discrimination of the free target cells from the conjugated ones is possible by the pulse widths of only light scatter signals, both forward and/or orthogonal. This resolution was not obtained if pulse height of the same signals was visualized. Using this resolution it was possible to distinguish single cells from the aggregates between target and effector cells. We propose that this is a better method for distinguishing conjugates than the method in which prior vital staining of cells is used.