18 ANALYTICAL AND LABORATORY EQUIPMENT
blue cells were performed.
Te Accuri C6 Flow Cytometer requires only a single 2D density plot of forward light scatter (FSC-A) versus 7-AAD fluorescence (7-AAD FL3-A) for data analysis. A negative control sample for each cell type (no added 7-AAD) defined the viable cell gate. Tis gate included events with high FSC-A, and defined the FL3-A background fluorescence.
Te dead cell gate was set with a cell sample containing 7-AAD. Data was copied into a spreadsheet program to determine the standard deviation (SD) and coefficient of variation (CV) for triplicate measurements (Fig. 1 A-D).
Small particle measurements Whole blood aliquots (1-2µL), collected in sodium citrate tubes, were diluted 1:10 into HEPES buffered saline with 1 per cent formaldehyde.
Twenty microliter aliquots of diluted blood were incubated in 1.5mL tubes at RT, 20 minutes, with 20µL of CD41-PE antibody (DAKO clone 5B12).
Samples were then diluted with 1 mL HEPES-buffered saline with 1 per cent formaldehyde. 5µL of
RFP-50-5 beads (Spherotech) were added to allow counting method comparison. Samples were well mixed and read on the C6.
Data collection was triggered by the positive fluorescence signal of CD41-PE labeled platelets (FL2-H), in order to improve discrimination of platelets from debris and to increase counting accuracy.
Te FL2-H threshold channel was determined by first triggering on FSC-H, determining where CD41-PE+ events fell relative to the negatives, and setting this value (FL2-H channel=1000) as the primary threshold.
All samples were collected using this threshold. Platelet counts per µL of sample were copied into a spreadsheet program, and dilution correction factors were applied to determine the platelets per µL of original whole- blood sample (Fig. 2 A-C).
Conclusion No statistically significant differences were found between the volumetric C6 cell concentration measurements and those obtained with a hemacytometer and counting beads. However, the precision of the cell-count data obtained by C6 volume measurement was significantly better than that
Fig. 3. The average coefficient of variation ± 1 SD for replicate cell counts using three different counting methods on the same samples. The Student’s T test was used to derive p values. The C6 direct count method provides the least variability between replicate cell counts.
obtained by hemacytometer (p<.004) or counting bead (p<.04) methods (Fig. 3). Te average CV for triplicate cell counts with the C6 was 1.2 per cent, that for counting bead counts was double (2.8 per cent) and for hemacytometer counts 20 times higher (25 per cent).
Te sources of error inherent in hemacytometer counts are well known. Te use of counting beads in flow-cytometry experiments has largely replaced combining hemacytometer counts with flow-cytometry data. But even this approach is likely impacted by sources of error such as pipetting technique and calibration, variability in bead-stock concentrations, and the subjective setting of a bead gate in the flow cytometer data file.
Obtaining event counts per µL directly from the C6 data is easy to do. 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.
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Fig. 2. Platelet-count determination with the C6: (A) Triggering data collection on forward scatter. (B) Same sample triggered on fluorescence, showing improved discrimination of platelets. (C) Comparison of platelet counts obtained for two methods of counting.
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Clare Rogers and MaryAnn Labant are with Accuri Cytometers, Ann Arbor, MI, USA
www.accuricytometers.com
“Flow cytometers with syringe- driven fluidics can deliver absolute count measurements without the addition of counting beads to samples.”
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