Coincidence Detection in Flow Cytometry Using Pulse-Shape Analysis in Polydisperse Suspensions

Flow cytometers, based on an optical measurement principle, are widely used for cell characterization in a variety of medical applications. Especially in hematology, high counting accuracy is crucial. Therefore, it is essential to develop accurate but also fast coincidence correction methods. We developed a procedure for post-processing the time-dependent signals (pulses) captured by the detectors during the transition of particles or cells through the flow cytometer's laser beam. These pulses are characterized by their area, maximum intensity, standard deviation, and skewness. By using standard deviation versus area, we are able to detect significantly more coincidences in monodisperse suspensions than by using the common method of area or width versus height. This demonstrates the superior sensitivity and accuracy of our approach for coincidence detection. In polydisperse suspensions and whole blood, we successfully identify and distinguish coincidences between differently sized particles (e.g., 1 and 5 μm) and particles of the same size, as well as between thrombocytes and erythrocytes based on pulse skewness and standard deviation. Unlike the current state-of-the-art method for coincidence correction in cell concentration measurements, which relies on statistical evaluation through dilution series, the developed procedure identifies a specific number of coincidences for individual measurements.