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Oct 1, 2009 (Vol. 29, No. 17)

Cell-Counting Technologies Are Evolving

Flow Cytometer Aims to Improve Upon Traditional Methodologies

  • Conclusion

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    Figure 4. The average coefficient of variation +/- 1 SD for replicate cell counts using three different counting methods on the same samples

    No statistically significant differences were found between the volumetric C6 cell concentration measurements and those obtained with standard methods (hemacytometer and counting beads) for two multiparametric analyses: viable-cell concentration for cell lines and platelet numbers per µL of original whole-blood sample.

    However, the precision of the cell-count data obtained by C6 volume measurement was significantly better than that obtained by hemacytometer (p<.004) or counting bead (p<.04) methods (Figure 4). The average CV for triplicate cell counts with the C6 was 1.2%. The average CV for counts using counting beads was twice as high (2.8%) and that for hemacytometer counts was 20 times higher (25%).

    The sources of error inherent in hemacytometer counts are well known and it is not surprising that variation between replicate sample counts would be high. The use of counting beads in flow-cytometry experiments has largely replaced combining hemacytometer counts with flow-cytometry data as a way to determine cell concentration of phenotypically diverse populations. But even this approach, as demonstrated here, is likely impacted by sources of error such as pipetting technique and calibration, inherent variability in bead-stock concentrations, and the subjective setting of a bead gate in the flow cytometer data file.

    An added benefit of obtaining event counts per µL directly from the C6 data is the comparative ease of use. No complicated back calculations to determine the volume sampled based on bead number collected are required, nor are subjective decisions about how to gate on the singlet bead population.

    The unique peristaltic pump fluidics system of the C6 allows direct measurement of the volume pulled from each sample tube, yet also provides standard laminar flow, which is crucial to accurate flow-cytometric fluorescence and light-scatter measurements.



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