ASTM F2149-16
(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
SIGNIFICANCE AND USE
3.1 The electrical sensing zone method for cell counting is used in tissue culture, government research, and hospital, biomedical, and pharmaceutical laboratories for counting and sizing cells. The method may be applicable to a wide range of cells sizes and cell types, with appropriate validation (10).
3.2 The electrical sensing zone methodology was introduced in the mid-1950s (9). Since this time, there have been substantial improvements which have enhanced the operator's ease of use. Among these are the elimination of the mercury manometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.
3.3 This instrumentation offers a rapid result as contrasted to the manual counting of cells using the hemocytometer standard counting chamber. The counting chamber is known to have an error of 10 to 30 %, as well as being time-consuming (11). In addition, when counting and sizing porcine hepatocytes, Stegemann et al concluded that the automated, electrical sensing zone method provided greater accuracy, precision, and speed, for both counts and size, compared to the conventional microscopic or the cell mass-based method (7).
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 and is applicable to a wide range of cell types. The method works best for spherical cells, and may be less accurate if cells are not spherical, such as for discoid cells or budding yeast. The method is appropriate for suspension as well as adherent cell cultures (1).2 Results may be reported as number of cells per milliliter or total number of cells per volume of cell suspension analyzed. Size distribution may be expressed in cell diameter or volume.
1.2 Cells commonly used in tissue-engineered medical products (2) are analyzed routinely. 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 validated for precision when compared to the conventional visual cell counting under a microscope using a hemocytometer. Currently, it is not possible to validate cell counting devices for accuracy, since there not a way to produce a reference sample that has a known number of cells. The electrical sensing zone method shall be validated each time it is implemented in a new laboratory, it is used on a new cell type, or the cell counting procedure is modified.
1.3 Electrical sensing zone instrumentation (commonly referred to as a Coulter counter) is manufactured by a variety of companies and is based upon 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(9)). 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 narrow size distrib...
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This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F2149 − 16
Standard Test Method for
Automated Analyses of Cells—the Electrical Sensing Zone
1
Method of Enumerating and Sizing Single Cell Suspensions
This standard is issued under the fixed designation F2149; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope and measurement of changes in electrical resistance produced
by a cell, suspended in a conductive liquid, traversing through
1.1 This test method, provided the limitations are
a small aperture (see Fig. 1(9)). When cells are suspended in a
understood, covers a procedure for both the enumeration and
conductive liquid, phosphate-buffered saline for instance, they
measurement of size distribution of most all cell types. The
function as discrete insulators. When the cell suspension is
instrumentationallowsforuser-selectablecellsizesettingsand
drawnthroughasmallcylindricalaperture,thepassageofeach
is applicable to a wide range of cell types. The method works
cell changes the impedance of the electrical path between two
bestforsphericalcells,andmaybelessaccurateifcellsarenot
submerged electrodes located on each side of the aperture.An
spherical, such as for discoid cells or budding yeast. The
electrical pulse, suitable for both counting and sizing, results
method is appropriate for suspension as well as adherent cell
2 from the passage of each cell through the aperture. The path
cultures (1). Results may be reported as number of cells per
through the aperture, in which the cell is detected, is known as
milliliterortotalnumberofcellspervolumeofcellsuspension
the “electronic sensing zone.” This test method permits the
analyzed. Size distribution may be expressed in cell diameter
selective counting of cells within narrow size distribution
or volume.
ranges by electronic selection of the generated pulses. While
1.2 Cells commonly used in tissue-engineered medical
the number of pulses indicates cell count, the amplitude of the
products (2) are analyzed routinely. Examples are chondro-
electrical pulse produced depends on the cell’s volume. The
cytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al.
baseline resistance between the electrodes is due to the
used the method for both pancreatic islet number and volume
resistanceoftheconductiveliquidwithintheboundariesofthe
measurements (6). In addition, instrumentation using the elec-
aperture. The presence of cells within the “electronic sensing
tricalsensingzonetechnologywasusedforbothcountandsize
zone” raises the resistance of the conductive pathway that
distributionanalysesofporcinehepatocytesplacedintohollow
depends on the volume of the cell.Analyses of the behavior of
fiber cartridge extracorporeal liver assist systems. In this study
cells within the aperture demonstrates that the height of the
(7), and others (6, 8), the automated electrical sensing zone
pulse produced by the cell is the parameter that most nearly
method was validated for precision when compared to the
shows proportionality to the cell volume.
conventional visual cell counting under a microscope using a
1.4 Limitations are discussed as follows:
hemocytometer. Currently, it is not possible to validate cell
1.4.1 Coincidence—Occasionally, more than a single cell
countingdevicesforaccuracy,sincetherenotawaytoproduce
a reference sample that has a known number of cells. The transverses the aperture simultaneously. Only a single larger
pulse, as opposed to two individual pulses, is generated. The
electrical sensing zone method shall be validated each time it
is implemented in a new laboratory, it is used on a new cell result is a lower cell count and higher cell volume measure-
ment. The frequency of coincidence is a statistically predict-
type, or the cell counting procedure is modified.
able function of cell concentration that is corrected by the
1.3 Electrical sensing zone instrumentation (commonly re-
instrument. This is called coincidence correction (8). This
ferred to as a Coulter counter) is manufactured by a variety of
phenomenon may be reduced by using lower cell concentra-
companies and is based upon electrical impedance. This test
tions.
method, for cell counting and sizing, is based on the detection
1.4.2 Viability—Electrical sensing zone cell counting enu-
merates both viable and nonviable cells and cannot determine
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
percent viable cells. A separate test, such as Trypan blue, is
andSurgica
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F2149 − 01 (Reapproved 2007) F2149 − 16
Standard Test Method for
Automated Analyses of Cells—the Electrical Sensing Zone
1
Method of Enumerating and Sizing Single Cell Suspensions
This standard is issued under the fixed designation F2149; 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 (´) indicates an editorial change since the last revision or reapproval.
1. 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 settings and is applicable to a wide range of cell types. The method works best for spherical cells, and
may be less accurate if cells are not spherical, such as for discoid cells or budding yeast. The method is appropriate for suspension
2
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 millilitremilliliter or total number of cells per volume of cell suspension analyzed. Both count
and size Size distribution may be expressed in cell micron diameter or volume, femtolitres.volume.
1.2 Cells commonly used in tissue-engineered medical products (2) routinely are analyzed. analyzed routinely. Examples are
chondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et alal. 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.Currently, it is not possible to validate cell counting
devices for accuracy, since there not a way to produce a reference sample that has a known number of cells. The electrical sensing
zone method shall be validated each time it is implemented in a new laboratory, it is used on a new cell type, or the cell counting
procedure is modified.
1.3 This instrumentation Electrical sensing zone instrumentation (commonly referred to as a Coulter counter) is manufactured
by a variety of companies; however, the principle used in all is companies and is based upon 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(9)). 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 volu
...
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