Program #2785 Abstract

Technology giving researchers the ability to quickly and precisely determine the absolute number or concentration of cells with a given phenotype is of great interest in diverse fields including clinical research and diagnostics, drug discovery, and cell biology. The recent advent of small, fully digital flow cytometers combines the advantages of hydrodynamically focused cell sampling, high data acquisition rates and excellent light scatter resolution with the ability to calculate absolute counts for any identified population in a sample. In the study presented here, several different cell counting applications are explored: viability determination for cultured cell lines, platelet counts in whole, unlysed human peripheral blood samples, and B- and T-cell concentrations in human peripheral blood. Three cell counting methods are compared: the hemacytometer, counting bead frequency in flow cytometric samples, and direct sample volume measurement by a flow cytometer. No significant difference was found in the average cell count per L of sample determined by the three counting methods. However, significant differences were found when the precision of the cell count data for the hemacytometer and counting bead methods was compared to direct volume measurement by a flow cytometer. Not surprisingly, hemacytometer counts, including trypan blue for viability assessment, had the largest variability between quadruplicate counts on the same sample (average CV = 19.5%). The combined use of counting beads and the flow cytometer improved counting precision a great deal (average CV = 2.94%) over the hemacytometer method. However, the most precise measurement was obtained by direct volume measurement with the flow cytometer (average CV = 2.1%), with p values of 0.002 and 0.010 when compared to the hemacytometer and counting bead methods, respectively. This study shows that a benchtop flow cytometer with traditional laminar flow fluidics and direct volume measurement capabilities, is equally accurate and more precise, than either hemacytometer or counting bead methods for determining cell concentration for applications as diverse as cultured cell line viability or human platelet counts.

Comparison of Three Methods for the Assessment of Cell Phenotype, Viability, and Concentration in Cultures and Peripheral Blood
Clare Rogers, Maria Dinkelmann, Nathan Bair, Collin Rich, Grant Howes, Brett Eckert Accuri Cytometers, Inc. Ann Arbor, MI
Figure 1 Peristaltic pumps allow direct cell concentration measurements and high data acquisition rates. Figure 2 Validation of direct volume cell concentration measurements using counting beads.
Counting Bead: cell count per mL
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Figure 3 Viable cell concentration measurements are similar using direct volume, counting bead or hemacytometer methods.
Cell viability was measured in high and low density cultures of three cell lines: U937 (human lymphoma), Jurkat (human T-cell leukemia) and NIH/3T3 (mouse fibroblast) using three different methods. 7-AAD (Cayman Chemicals) and trypan blue (HyClone) exclusion were used to determine viability according to manufacturers instructions.

Results

. The development of a flow cytometer with a unique peristaltic pump fluidics system allows direct measurement of the volume sampled from cell suspensions.

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y = 1.116x r = 0.9774 n = 75

. The system operates with standard laminar flow crucial to

accurate fluorescence and light scatter measurements, and has no sample volume limit.

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. Cell concentration measurements determined by the direct

volume method gave comparable results to those obtained with standard methods (hemacytometer and counting beads).

A. An example of viable cell gate (P5) placement using un10,000

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Viable Cell Number

Viable Cell Number

Development of a unique peristaltic pump-driven laminar flow fluidics system combines the advantages of hydrodynamically focused cell sampling (high acquisition rates, good light scatter and fluorescence resolution) with the ability to report absolute cell counts for any identified population in a sample.

Direct Volume: cell count per mL sample

Comparison of cell concentration measurements by two methods. The x axis represents cell concentration calculated by absolute cell counts in relation to volume sampled. The y axis represents cell concentration as calculated by the absolute cell counts in relation to the number of SPHEROTM AccuCount beads measured. 75 different samples were analyzed in five independently run experiments. Cell types analyzed included human peripheral blood samples and mammalian cell lines.

Comparison of Viable Cell Counting Methods D 2500000

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Comparison of Viable Cell Counting Methods

volume counting beads volume

stained control cells. B. An example of dead cell gate (P6) placement using 7-AAD positive cells. 1.0 L 7-AAD was added to 500 L cell suspension and incubated in the dark at room temperature for 15 minutes. 7-AAD is a fluorescent dye which is excluded from viable cells, but taken up by cells when the outer membrane is compromised (as in cell death). C. Viable and dead cell concentrations for high density cell samples of three cell types as measured by gated cell counts relative to volume sampled.

. The direct volume measurement method was significantly

more precise for triplicate counts of the same cell sample than either the hemacytometer (p=0.002) or bead count (p=0.010) methods.

Conclusions

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D. Comparison of mean viable cell counts (and standard

deviation) for three methods of cell counting using high and low density cultures of three different cell lines.

. Hemacytometer counts can be highly imprecise (average CV

for triplicate counts = 19.51%).

counting beads
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hemacytometer

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. In contrast, flow cytometers can function as precision cell

counting tools and offer the additional advantage of multiparametric sample analysis.

hemacytometer
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. Flow cytometers offer a fast, precise alternative to hemacyed ity ity ed ns ed ra t ns ity

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tometers for viable cell counting.

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Coefficient of Variation

CD19-PE FL2

Historically, the combination of a light microscope and hemacytometer have been the tools of choice for performing cell counts in life science research laboratories. However, this method is slow and prone to error. When hemacytometer counts and flow cytometric (FC) phenotypic data are combined to determine cell subset numbers, errors are multiplied. Digital flow cytometers, utilizing laminar-flow fluidics, allow fast, phenotypic data collection (at rates up to 10,000 events per second) on a wide range of cell types (sub-micron sized bacteria through large mammalian cell lines) but still require the addition of counting beads to each sample to calculate cell-subset concentration. On the other hand, flow cytometers with syringe driven fluidics can deliver absolute count measurements without the addition of counting beads to samples, but are often limited by lower data acquisition rates (< 1,000 events per second) and the total volume of sample that can be analyzed from each sample tube. We have developed a fully-digital flow cytometer with a unique peristaltic pump driven, laminar-flow fluidics system (Figure 1). This combines the advantages of hydrodynamically focused cell sampling (high data acquisition rates and good light scatter and fluorescence resolution) with the ability to calculate absolute counts for any identified population in a sample. Several applications of direct cell concentration determination based on volume measurement are presented here: viability assessment of cultured cell lines, platelet counts for whole, human peripheral blood samples, and determination of T- and B-cell numbers in human peripheral blood. All methods make use of the fluorescence and light scatter measurements possible with a flow cytometer to identify sub-populations of interest. Cell counting data collected by at least one other traditional method (counting beads or hemacytometer) is included for comparison.

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Introduction Figure 4 Concentrations of B- and T-cell sub-populations in human peripheral blood are measured using a flow cytometer.
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Light Scatter Gate = Lymphocytes + Beads
Counting Beads

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. Direct identification and

Figure 6 The direct volume method is more precise for measuring cell concentration than counting bead or hemacytometer methods.
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counting of T- and B- cell populations is quickly and easily achieved by flow cytometry. signal triggering on a flow cytometer. Small particles, such as platelets cannot be effectively counted using a hemacytometer.

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Figure 5 Platelet concentrations in human blood samples are obtained using fluorescence triggering on a flow cytometer.

. Accurate platelet counting is enabled through fluorescence

. The addition of counting beads to every flow cytometric

sample saved time and improved counting precision compared to hemacytometer counts, but added expense to the analysis.

B
donor 1 donor 2 donor 3

average T-cell counts/ul (%CV) vol method bead method 113 (0.9%) 112 (2.1%) 297 (1.7%) 281 (3.3%) 126 (2.1%) 121 (2.2%)

average B-cell counts/ul (%CV) vol method bead method 24 (4.8%) 24 (5.3%) 69 (1.5%) 65 (3.2%) 19 (7.2%) 18 (4.7%)

Hemacytometer

p=0.002

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. The direct-volume measurement method was the most precise and least time-consuming approach for cell counting.

13.6%

58.8%

50 L of human peripheral blood from three donors was labeled with 5 L each of FITC-CD3 (eBioscience) and PE-CD19 (Biolegend) for 20 minutes at room temperature in the dark. Subsequently, red blood cells were lysed with BD FACS Lyse according to manufacturers instructions. Cells were pelleted, and resuspended in 500 L PBS + 5% BSA, and 50 L of SPHEROTM AccuCount counting beads were added to each tube. Triplicate readings were taken of each sample.

Beads

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direct-volume (C6 cytometer)

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counting beads (C6 cytometer)

p=0.010

0 1 2 3

A. B- and T-cell populations were identified by CD19 and CD3 positive

CD3-FITC FL1

Cell Counting Method

cells respectively. B. B- and T-cell concentrations were calculated using volume and count bead methods.

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. All data was collected using CD41-PE FL2 Trigger. Aliquots of whole blood were diluted 1:10 into HEPES-buffered saline with 1% formaldehyde. 20 L aliquots of diluted blood were incubated with 20 l CD41-PE antibody (DAKO clone 5B12) for 20 minutes in the dark at room temperature. Samples were then diluted with 1 mL HEPES-buffered saline with 1% formaldehyde. 5.0 L of RFP-50-5 beads (Spherotech) were added to allow comparison of two counting methods.

The average coefficient of variation for replicate cell counts using three different counting methods on the same samples. A paired students T test was used to determine p values (95% confidence, N = 23). The direct-volume measurement method provides the least variability between replicate cell counts (average CV values: direct-volume measurement =2.10%; counting bead method =2.94%; hemacytometer =19.51%).

2009