FLOW CYTOMETRIC ANALYSIS USING DIGITAL SIGNAL-PROCESSING

Current commercial now cytometers employ analog circuits to produce th e feature values of the pulse waveforms that result from particle anal ysis, The use of analog pulse processing Limits the features that can be measured to pulse integral, pulse height, and pulse width, and a la rge amount of potentially relevant information about the shape of the pulse waveform is lost. Direct digitizing of the waveform provides a m eans for the extraction of additional features, for example, pulse ske wness and kurtosis, as well as the Fourier properties of the pulse, He re we describe a digital pulse waveform processing system that is comp atible both with a commercial now cytometer, and with a readily availa ble computational platform, The performance of the digital and analog systems were compared through analysis of synthetic waveforms, and the waveforms produced by standard fluorescence microspheres and biologic al particles, The digital waveform processing system was found to be a ccurate and flexible, and the value of several of its unique attribute s was demonstrated using biological cells, A protocol was designed in which digital pulse processing provided a means for the quantitative m onitoring of the optical alignment of the now cytometer, It was shown that digital pulse processing could be used to discriminate between pa rticle classes which produce feature values indistinguishable through analog pulse processing, and to discriminate accurately single cells f rom doublets and larger aggregates. (C) 1995 Wiley-Lss, Inc.