Laser scanning cytometry distinguishes lymphocytes, monocytes, and granulocytes by differences in their chromatin...

By providing rapid measurements of cellular fluorescence in addition to morphometric analysis, the novel microscope-based multiparameter laser scanning cytometer (LSC) combines advantages of flow and image cytometry. Analysis of the integrated fluorescence intensity (IF) versus peak fluorescence intensity (maximal fluorescence per pixel; FP) versus fluorescence area (FA) of the cells stained with the DNA intercalating fluorochrome propidium iodide (PI) made it possible to discriminate lymphocytes, monocytes, and granulocytes in samples of peripheral blood of normal individuals. Lymphocytes, characterized by maximally condensed chromatin, had the highest FP and lowest values of FA. Granulocytes had the lowest FP and the highest FA. They also had increased IF compared to lymphocytes and monocytes. The difference in IF between granulocytes and monocytes/lymphocytes was abolished after exposure of cells to 0.1 M HCl at 0-4 degrees C, which is known to dissociate histones from DNA in chromatin. Monocytes were characterized by intermediate values of peak and area fluorescence intensity compared to lymphocytes and granulocytes. Thus, although all three classes of white blood cells have the same DNA content, they can be distinguished based on differences in structure of their chromatin after staining with PI. Discrimination of these cells by LSC is similar to that provided by flow cytometry based on differences in forward and side light scatter properties.