A simple and rapid method to distinguish between rosettes and non-specific aggregation of erythrocytes in the rosette assay for human T-lymphocytes

Abstract

A method is described for rapid and simple visualization of lymphocytes in the rosette assay for human T-lymphocytes. A drop of acridine orange (pH 7.4) is added to the suspension of rosetted cells and allowed to incubate for 1 min on ice. Nucleic acids are then visualized in a fluorescence microscope by excitation at 430-500 micrometer. Simultaneous semi-illumination with light microscopy allows enumeration of total lymphocytes and the number of lymphocytes rosetted with sheep red blood cells. The central lymphocyte is clearly distinguishable even when it is crowded with red cells, and this makes the differentiation between rosettes and non-specific aggregation of cells easy.