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Sample handling for kinetics and molecular assembly in flow cytometry

Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. We have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, we used fast valves to overcome the disparity between mixing 100 (mu) ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 (mu) l/sec. Particles were detected within 100 msec after mixing, but unstable flow was created which lasted for 1 sec after injection of the sample into the flow cytometer. We used optical criteria to discriminate particles which were out of alignment due to the unstable flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system we were able to automate sample handling and delivery with intervals of a few seconds. We used a fluidic approach to defeat the instability caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of instability was reduced to approximately 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.