ASTM F2149-01
(Test Method)Standard Test Method for Automated Analyses of Cells—the Electrical Sensing Zone Method of Enumerating and Sizing Single Cell Suspensions
Standard Test Method for Automated Analyses of Cells—the Electrical Sensing Zone Method of Enumerating and Sizing Single Cell Suspensions
SCOPE
1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings, hence, this test method is not restricted to specific cell types. The method is appropriate for suspension as well as adherent cell cultures (1). This is a quantitative laboratory method not intended for on-line or field use. Results may be reported as number of cells per millilitre or total number of cells per volume of cell suspension analyzed. Both count and size distribution may be expressed in cell micron diameter or volume, femtolitres.
1.2 Cells commonly used in tissue-engineered medical products (2) routinely are analyzed. Examples are chondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al used the method for both pancreatic islet number and volume measurements (6). In addition, instrumentation using the electrical sensing zone technology was used for both count and size distribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (7), and others (6, 8), the automated electrical sensing zone method was clearly validated for superior accuracy and precision when compared to the conventional manual method, visual cell counting under a microscope using a hemocytometer. This validation has been demonstrated over a wide variety of cell types. In addition, the automated procedure is rapid, rugged, and cost effective; it also minimizes operator-to-operator variability inherent in manual techniques.
1.3 This instrumentation is manufactured by a variety of companies; however, the principle used in all is electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see (Fig. 1)). When cells are suspended in a conductive liquid, phosphate-buffered saline for instance, they function as discrete insulators. When the cell suspension is drawn through a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submerged electrodes located on each side of the aperture. An electrical pulse, suitable for both counting and sizing, results from the passage of each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the "electronic sensing zone." This test method permits the selective counting of cells within very narrow size distribution ranges by electronic selection of the generated pulses. While the number of pulses indicates cell count, the amplitude of the electrical pulse produced depends on the cell's volume. The baseline resistance between the electrodes is due to the resistance of the conductive liquid within the boundaries of the aperture. The presence of cells within the "electronic sensing zone" raises the resistance of the conductive pathway that depends on the volume of the cell. Analyses of the behavior of cells within the aperture demonstrates that the height of the pulse produced by the cell is the parameter that most nearly shows proportionality to the cell volume.
1.4 Limitations are discussed as follows:
1.4.1 Coincidence—Occasionally, more than a single cell transverses the aperture simultaneously. Only a single larger pulse, as opposed to two individual pulses, is generated. The result is a lower cell count and higher cell volume measurement. The frequency of coincidence is a statistically predictable function of cell concentration that is corrected by the instrument. This is called coincidence correction (8). This phenomenon may be minimized, thus ensuring greater result accuracy, by using relatively low cell concentrations, around the 5 % level.
1.4.2 Viability—Automated cell counting enumerates both viable ...
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:F2149–01
Standard Test Method for
Automated Analyses of Cells—the Electrical Sensing Zone
Method of Enumerating and Sizing Single Cell Suspensions
This standard is issued under the fixed designation F 2149; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope buffered saline for instance, they function as discrete insula-
tors. When the cell suspension is drawn through a small
1.1 This test method, provided the limitations are under-
cylindrical aperture, the passage of each cell changes the
stood, covers a procedure for both the enumeration and
impedance of the electrical path between two submerged
measurement of size distribution of most all cell types. The
electrodes located on each side of the aperture. An electrical
instrumentation allows for user-selectable cell size settings,
pulse, suitable for both counting and sizing, results from the
hence, this test method is not restricted to specific cell types.
passage of each cell through the aperture.The path through the
The method is appropriate for suspension as well as adherent
2 aperture, in which the cell is detected, is known as the
cell cultures (1). This is a quantitative laboratory method not
“electronic sensing zone.” This test method permits the selec-
intended for on-line or field use. Results may be reported as
tive counting of cells within very narrow size distribution
number of cells per millilitre or total number of cells per
ranges by electronic selection of the generated pulses. While
volume of cell suspension analyzed. Both count and size
the number of pulses indicates cell count, the amplitude of the
distribution may be expressed in cell micron diameter or
electrical pulse produced depends on the cell’s volume. The
volume, femtolitres.
baseline resistance between the electrodes is due to the
1.2 Cells commonly used in tissue-engineered medical
resistance of the conductive liquid within the boundaries of the
products (2) routinely are analyzed. Examples are chondro-
aperture. The presence of cells within the “electronic sensing
cytes(3),fibroblasts(4),andkeratinocytes(5).Szaboetalused
zone” raises the resistance of the conductive pathway that
the method for both pancreatic islet number and volume
depends on the volume of the cell.Analyses of the behavior of
measurements (6). In addition, instrumentation using the elec-
cells within the aperture demonstrates that the height of the
tricalsensingzonetechnologywasusedforbothcountandsize
pulse produced by the cell is the parameter that most nearly
distribution analyses of porcine hepatocytes placed into hollow
shows proportionality to the cell volume.
fiber cartridge extracorporeal liver assist systems. In this study
1.4 Limitations are discussed as follows:
(7), and others (6, 8), the automated electrical sensing zone
1.4.1 Coincidence—Occasionally, more than a single cell
method was clearly validated for superior accuracy and preci-
transverses the aperture simultaneously. Only a single larger
sion when compared to the conventional manual method,
pulse, as opposed to two individual pulses, is generated. The
visual cell counting under a microscope using a hemocytom-
result is a lower cell count and higher cell volume measure-
eter.This validation has been demonstrated over a wide variety
ment. The frequency of coincidence is a statistically predict-
of cell types. In addition, the automated procedure is rapid,
able function of cell concentration that is corrected by the
rugged, and cost effective; it also minimizes operator-to-
instrument. This is called coincidence correction (8). This
operator variability inherent in manual techniques.
phenomenon may be minimized, thus ensuring greater result
1.3 This instrumentation is manufactured by a variety of
accuracy, by using relatively low cell concentrations, around
companies; however, the principle used in all is electrical
the 5 % level.
impedance. This test method, for cell counting and sizing, is
1.4.2 Viability—Automated cell counting enumerates both
based on the detection and measurement of changes in electri-
viable and nonviable cells. It does not measure percent cell
cal resistance produced by a cell, suspended in a conductive
viability.Tomeasurethepercentcellviability,eitheravitaldye
liquid, traversing through a small aperture (see Fig. 1 (9)).
or nonvital dye, such as trypan blue, procedure must be
When cells are suspended in a conductive liquid, phosphate-
performed.
1.4.3 Size Variation of the Cell Sample—Upto30to1by
This test method is under the jurisdiction ofASTM Committee F04 on Medical cell diameter in microns; 27 000 to 1 by cell volume. This is
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
simply a function of the size range capability of the particular
F04.42 on Tissue Characterization.
aperture size selected. Using this technology, measurements
Current edition approved Oct. 10, 2001. Published February 2002.
The boldface numbers in parentheses refers to the list of references at the end
of this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2149
may be made in the range of about 0.6 to 1200 µm. The lower 2.1.8 ruggedness, n—the degree of reproducibility of the
size limit is restricted only by thermal and electronic noise. same sample under a variety of normal conditions; for ex-
1.4.4 Size Range of the Aperture—The size range for a
ample, different operators.
single aperture is proportional to its diameter, D. The response
2.1.9 size thresholds, n—the instrument’s lower and upper
has been found to depend linearly on D over a range from 0.02
size settings for the particular cell population; adjustable “size
D to 0.80 D; however, the aperture tube may become prone to
gate.” Cells or fragments outside the size settings are excluded
blockage at levels greater than 0.60 D. The practical operating
from the analyses.
range, therefore, of the aperture is considered to be 2 to 60 %
of the diameter.
3. Significance and Use
1.4.5 Humidity—10to85%.
1.4.6 Temperature—10 to 35°C. 3.1 This assay is used in university tissue culture laborato-
1.4.7 Electrolyte Solution—The diluent for cell suspension ries, government research, and hospital, biomedical, and phar-
must provide conductivity and have no effect on cell size. The
maceutical laboratories to automate cell counting and sizing.
electrolyte of choice is most often physiologic phosphate
This instrumentation provides very rapid, accurate, and precise
buffered saline.
results for any tissue culture facility. In addition, as noted,
since the cell sizes to be analyzed by the instrument are set by
2. Terminology
the user, the analyses may be done on virtually any species of
2.1 Definitions:
cells and cell type; it is not restricted to human cells or blood
2.1.1 channelyzer, n—a pulse height analyzer; places volt-
cells.
age pulses into appropriate size bins for the size distribution
3.2 The electrical sensing zone methodology was intro-
data.
duced in the mid 1950s (9). Since this time, there have been
2.1.2 coincidence, n—more than one cell transversing the
substantial improvements which have enhanced the operator’s
aperture at the same time.
ease of use. Among these are the elimination of the mercury
2.1.3 corrected count, n—the cell count corrected for coin-
manometer, reduced size, greater automation, and availability
cidence.
of comprehensive statistical computer programs.
2.1.4 electrolyte, n—diluent, offering slight conductivity, in
3.3 This instrumentation offers a rapid result as contrasted
which cells are suspended.
to the manual counting of cells using the standard counting
2.1.5 femtolitre, n—a cubic micron; a measurement of cell
chamber, hemocytometer. The counting chamber is known to
volume.
have an error of 10 to 30 %, as well as being very time
2.1.6 micron (µ), n—0.001 mm, also known as a microme-
consuming (10). In addition, when counting and sizing porcine
tre; measurement of cell diameter.
hepatocytes, Stegemann et al concluded that the automated,
2.1.7 raw count, n—the enumeration of the cell population
electrical sensing zone method provid
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.