AN AUTOMATED HIGH-CAPACITY DATA CAPTURE AND ANALYSIS SYSTEM FOR THE IN-VITRO ASSESSMENT OF LEUKOCYTE ADHESION UNDER SHEAR-STRESS CONDITIONS

Parallel-plate flow chambers have been used to model the vascular micr ocirculation and study the in vitro dynamic adhesive interactions of l eukocytes and human umbilical vein endothelial cells (HUVECs). We desc ribe here a high capacity system which can simultaneously monitor the adhesive interaction of neutrophils and HUVECs in ten flow chambers. A utomated data collection was achieved with an image analyzer controlli ng the autostage and autofocus attachments of an inverted microscope. Images from the flow chambers were captured via phase-contrast microsc opy using a video camera and laser videodisk recorder. The images were downloaded off-line into an image analyzer for automated counting of rolling and adherent cells, Neutrophils were detected by their 'phase bright' characteristics. An automated optimization procedure allowed t he computer to choose the best setting for the selective detection of neutrophils, In addition, a method which utilized image averaging was used to distinguish between rolling and adherent cells. A comparison o f the results obtained from the manual and automated counting methods revealed linear relationships for the counting of both adherent (r = 0 .98) and rolling cells (r = 0.96) with counting efficiencies of 59% an d 46%, respectively. The utility of the system was demonstrated by its ability to measure the adhesive interaction between neutrophils and H UVEC in response to stimulus such as interleukin-1 alpha (IL-1 alpha), histamine, or formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP). In conclusion, we have developed an automated assay which combines the ca pacity of ten flow chambers with a computerized data analysis system; the result is an efficient and reproducible assay which minimizes oper ator associated errors and biological variability.