Hemacytometry is not capable of providing repeatable and linear cell quantification at low concentrations, as its repeatability is compromised by multiple operators. Flow microscopy provides accurate and repeatable cell quantification over a wide range of concentrations, from low to high, with coefficients of variance ranging from 2-7%. It also has advantages over hemacytometry such as being operator independent, providing statistically significant data sets and images of cells that offer morphological information.

1. Determination of cell concentration in biological samples is commonly
accomplished using a hemacytometer. However, when samples contain low
total concentrations, or rare subpopulations, the small number of cells
counted leads to inaccuracies. This can be of practical importance in
applications such as early stage cell proliferation and stem/progenitor cell
studies which have highlighted the need to accurately quantify low
concentration populations. In this study we addressed the linearity and
repeatability of hemacytometry for low cell concentration samples, and
investigated the use of flow microscopy as an alternative method. A 1:3
dilution series was performed on four sample types (IC-21 murine
macrophage cells, EL4 murine T-lymphoma cells, primary murine spleen
cells, and 10µm polystyrene beads), with starting concentrations of
approximately 300,000/ml. Counts were obtained in triplicate with each
method. R² values for hemacytometry ranged from 0.7705 to 0.9926 at
concentrations <16,000/ml, with CV ranging from 28-108%. Linearity and
repeatability were poor for the hemacytometer at low concentrations. R²
values for flow microscopy ranged from 0.9997 to 1.0 at concentrations
Introduction
Quantification of Low Cell Concentrations Using Hemacytometry and Flow Microscopy
Teresa Martin¹, Ahmed Zafer², David King¹, Lakshmi Krishnan²
¹Brightwell Technologies Inc., Ottawa, ON, Canada; ²National Research Council – Institute for Biological Sciences, Ottawa, ON, Canada
The broad range of instrumentation available for characterizing cell
populations reflects the importance of this task in many areas of
biotechnology. Automated devices commonly employed for this purpose,
require concentrations of >50,000/ml for optimal results (1).
Hemacytometers and hemacytometry-based technologies require even
higher concentrations to achieve adequate accuracy (2), and results are
often inconsistent due to inter-operator variability (3). Recent advances in
early stage cell proliferation and stem/progenitor cell studies have
highlighted the need to accurately quantify low/rare concentration cell
populations (4, 5). New technologies which provide a solution to this need
are required to facilitate further studies into these areas of research.
Micro-Flow Imaging (MFI) is a type of flow microscopy which is
designed to automatically measure the size distribution, concentration and
morphological properties of multi-component populations of sub-visible
particles in solution. It provides sensitive detection and imaging of
Hemacytometry is not capable of providing repeatable and linear cell
quantification at low concentrations
When using a hemacytometer with optimal cell concentration,
repeatability is compromised by multiple operators
Flow microscopy provides accurate and repeatable cell
Abstract
A
Conclusions
MFI Hemacytometer
Single Operator 0.5% 5%
Multiple Operators 1% 10%
Table 1: Average % coefficient of variance achieved by one
operator vs multiple (n=3) operators
Results
2303/B819
Hemacytometry Linearity at Low Concentrations
Flow Microscopy Linearity at Low Concentrations
values for flow microscopy ranged from 0.9997 to 1.0 at concentrations
<16,000/ml, with CV ranging from 2-7%. Flow microscopy showed a high
degree of linearity and repeatability at low and high concentrations. Also,
flow microscopy provided benefits including a statistically significant data
set for both cell count and size, removal of operator subjectivity, images and
morphological attributes of the cell populations as well as digital record-
keeping.
particles in solution. It provides sensitive detection and imaging of
transparent particles, and image filtering abilities which allow for
identification and differentiation of rare populations or subpopulations (6).
MFI is used extensively in biotherapeutic formulation development, and
has capabilities which complement existing methods employed in cell
biology. Because no instrument specific running buffer is required, cell
samples can be analyzed in their native state.
1. Johnston, G. 2010. Automated handheld instrument improves counting
precision across multiple cell lines. BioTechniques 48(4):325-327.
2. Strober, W. 1997. Common ImmunolTechniques - Monitoring Cell
Growth. Coligan et al. (Eds.) In Current Protocols in Immunology, pp.
A.3A.1-A.3A.2ogic. John Wiley & Sons, Inc.
3. Lu JC et al. 2007. Comparison of three sperm counting methods for the
determination of sperm concentration in human semen and sperm
suspensions. LabMedicine 38(4):232-236.
4. Mimeault, M. and S.K. Batra. 2006. Concise review: recent
advances and the significance of stem cells in tissue regeneration and
cancer therapies. 2006. Stem Cells 24:2319-2345.
5. Badders NM et al. 2008. Quantification of small cell numbers with a
microchannel device. BioTechniques 45:321-325.
6. Huang CT et al. 2009. Quantitation of protein particles in parenteral
solutions using Micro-Flow Imaging. JPS 98(9):3058-71.
Flow microscopy provides accurate and repeatable cell
quantification at low through high concentrations
Flow microscopy is an operator independent method for cell
enumeration and provides a statistically enhanced data set due to
increased cell count per analysis
Cell size, morphology data and images obtained using flow
microscopy provide immediate insight into cell population
characteristics
Bibliography
Measurement Process:
• Sample is drawn through a precision flow cell
• Images are acquired (multiple frames/second)
• All particles in frame are detected and measured
System Configuration:
• 5X Magnification, 475nm LED light source
• 10 bit grayscale sensor, 1280x1024 pixels
• Automatic illumination control and thresholding
• Small volume sampling apparatus (<0.5ml)
System Output:
• Particle size distributions (concentration vs. size)
• Particle images
• Morphological parameters (shape, intensity)
• Particle sub-population isolation
Flow Microscopy
Principle of Operation
Detection
Zone
MFI Flow MicroscopyTechnology
B
Figure 2: (A) Linearity of hemacytometer at low concentrations
(B) Linearity of flow microscope at low concentrations
(C) Comparison of % coefficient of variance for hemacytometer
and flow microscope at low through high cell concentrations
Figure 1: Typical histogram, scatter plot and images of IC-21 cell
population obtained using flow microscopy
C
% Coefficient of Variance