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 image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced 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 to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.