An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry

Abstract

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.