A QUANTITATIVE ASSAY FOR INTERCELLULAR AGGREGATION

In an earlier communication (Munn et al., J Immunol. Methods 166:11-25 , 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital imag e processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is intro duced to characterize the three-dimensional morphology of the aggregat es. This index is based on temporal changes in the projected area of t he cells and cell aggregates during the course of the experiment. By d rawing an analogy with the kinetic theory of gases, we have also intro duced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm t o remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysi s procedure, thus minimizing user intervention and improving the repro ducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte ag gregation.