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Measurement of Microsphere Concentration Using a Flow Cytometer with Volumetric Sample Delivery



Lili Wang, Steven J. Choquette, Yu-Zhong Zhang


Microsphere concentrations are needed to assign ERF units to microspheres used in quantitative flow cytometry. An Attune® acoustic focusing flow cytometer was evaluated for the measurement of the concentration of microspheres contained in a Trucount® (TC) vial. The TC vial was certified to contain 50,600±3000 microspheres. The concentration was measured by counting the number of microspheres contained in the volume delivered by the Attune® and dividing the number by the volume. The Attune® syringe volume was calibrated both in the delivery and draw modes, and the results of the volume calibration were summarized by two calibration lines. The delivered volume was obtained by dividing the number of recorded events by the concentration of TC in the sample tube. (Use TC as a volume calibrator). The draw volume was obtained by weighting the sample tube before and after the draw. The slope of the draw volume calibration line was equal to 1.00 with an offset of -13 µL. The slope of the delivered volume calibration with TC beads was 0.93 suggesting a systematic volume dependent bias. The reduced slope of the TC calibration can be rationalized as an effect of suspension flow in capillaries. When the sample volume was set to values between 150 µL and 300 µL, both calibration curves gave similar results suggesting that a good estimate of the true delivered volume can be obtained by subtracting 13 µL from the delivered volume indicated by the Attune® software. The number of microspheres in the volume was obtained by passing the suspension contained in the volume through a laser beam and counting the number of events in which the signals from the scattering and fluorescence detectors exceeded threshold values.
Journal of Research (NIST JRES) - 119.027
Report Number


Attune flow cytometer, concentration, microspheres


Wang, L. , Choquette, S. and Zhang, Y. (2014), Measurement of Microsphere Concentration Using a Flow Cytometer with Volumetric Sample Delivery, Journal of Research (NIST JRES), National Institute of Standards and Technology, Gaithersburg, MD, [online], (Accessed May 29, 2024)


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Created December 29, 2014, Updated November 10, 2018