AUTOMATED IMAGE CYTOMETRY FOR DETECTION OF RARE, VIRAL ANTIGEN-POSITIVE CELLS IN PERIPHERAL-BLOOD

A cell detection method based upon automated screening is described fo r recognition of low frequencies (1 in 100,000) of immuno-enzymaticall y labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide-field, fully autom ated microscope (Autoplan) and a modular image analysis computer (MIAC ), both from Leica, Wetzlar, Germany. The MIAC contains image boards f or optimum use of mathematical morphology algorithms. Communication wi th the MIAC is via a personal computer. Programs for automated cell an alysis have been written in C language. Main features of the system ar e fast analysis of large microscope fields including a count of all ce lls, selection of objects of interest (alarms), and display of digital ly stored images of these alarms, We tested this system for the detect ion of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno-enz ymatic (peroxidase) staining was performed with DAB and counterstainin g with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. T he lowest frequency detected was 1 antigen-positive cell / 3 x 10(5) a ntigen-negative cells. Screening time was about 60 min for one million cells. (C) 1994 Wiley-Liss, Inc.