ASTM F2149-01(2007)
(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
This assay is used in university tissue culture laboratories, government research, and hospital, biomedical, and pharmaceutical laboratories to automate cell counting and sizing. This instrumentation provides very rapid, accurate, and precise results for any tissue culture facility. In addition, as noted, since the cell sizes to be analyzed by the instrument are set by the user, the analyses may be done on virtually any species of cells and cell type; it is not restricted to human cells or blood cells.
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’ease of use. Among these are the elimination of the mercury manometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.
This instrumentation offers a rapid result as contrasted to the manual counting of cells using the standard counting chamber, hemocytometer. The counting chamber is known to have an error of 10 to 30 %, as well as being very time consuming (10). In addition, when counting and sizing porcine hepatocytes, Stegemann et al concluded that the automated, electrical sensing zone method provided significantly 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, hence, this test method is not restricted to specific cell types. The method is appropriate for suspension as well as adherent cell cultures (). 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 () routinely are analyzed. Examples are chondrocytes (), fibroblasts (), and keratinocytes (). Szabo et al used the method for both pancreatic islet number and volume measurements (). 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 (), and others (, ), 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 ()). 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...
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Designation: F2149 − 01(Reapproved 2007)
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 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 liquid, traversing through a small aperture (see Fig. 1(9)).
When cells are suspended in a conductive liquid, phosphate-
1.1 This test method, provided the limitations are
buffered saline for instance, they function as discrete insula-
understood, covers a procedure for both the enumeration and
tors. When the cell suspension is drawn through a small
measurement of size distribution of most all cell types. The
cylindrical aperture, the passage of each cell changes the
instrumentation allows for user-selectable cell size settings,
impedance of the electrical path between two submerged
hence, this test method is not restricted to specific cell types.
electrodes located on each side of the aperture. An electrical
The method is appropriate for suspension as well as adherent
2 pulse, suitable for both counting and sizing, results from the
cell cultures (1). This is a quantitative laboratory method not
passageofeachcellthroughtheaperture.Thepaththroughthe
intended for on-line or field use. Results may be reported as
aperture, in which the cell is detected, is known as the
number of cells per millilitre or total number of cells per
“electronic sensing zone.” This test method permits the selec-
volume of cell suspension analyzed. Both count and size
tive counting of cells within very narrow size distribution
distribution may be expressed in cell micron diameter or
ranges by electronic selection of the generated pulses. While
volume, femtolitres.
the number of pulses indicates cell count, the amplitude of the
1.2 Cells commonly used in tissue-engineered medical
electrical pulse produced depends on the cell’s volume. The
products (2) routinely are analyzed. Examples are chondro-
baseline resistance between the electrodes is due to the
cytes (3),fibroblasts (4),andkeratinocytes (5).Szaboetalused
resistanceoftheconductiveliquidwithintheboundariesofthe
the method for both pancreatic islet number and volume
aperture. The presence of cells within the “electronic sensing
measurements (6). In addition, instrumentation using the elec-
zone” raises the resistance of the conductive pathway that
tricalsensingzonetechnologywasusedforbothcountandsize
depends on the volume of the cell.Analyses of the behavior of
distributionanalysesofporcinehepatocytesplacedintohollow
cells within the aperture demonstrates that the height of the
fiber cartridge extracorporeal liver assist systems. In this study
pulse produced by the cell is the parameter that most nearly
(7), and others (6, 8), the automated electrical sensing zone
shows proportionality to the cell volume.
method was clearly validated for superior accuracy and preci-
1.4 Limitations are discussed as follows:
sion when compared to the conventional manual method,
visual cell counting under a microscope using a hemocytom- 1.4.1 Coincidence—Occasionally, more than a single cell
transverses the aperture simultaneously. Only a single larger
eter.Thisvalidationhasbeendemonstratedoverawidevariety
of cell types. In addition, the automated procedure is rapid, pulse, as opposed to two individual pulses, is generated. The
result is a lower cell count and higher cell volume measure-
rugged, and cost effective; it also minimizes operator-to-
operator variability inherent in manual techniques. ment. The frequency of coincidence is a statistically predict-
able function of cell concentration that is corrected by the
1.3 This instrumentation is manufactured by a variety of
instrument. This is called coincidence correction (8). This
companies; however, the principle used in all is electrical
phenomenon may be minimized, thus ensuring greater result
impedance. This test method, for cell counting and sizing, is
accuracy, by using relatively low cell concentrations, around
based on the detection and measurement of changes in electri-
the 5% level.
cal resistance produced by a cell, suspended in a conductive
1.4.2 Viability—Automated cell counting enumerates both
viable and nonviable cells. It does not measure percent cell
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
viability.Tomeasurethepercentcellviability,eitheravitaldye
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
or nonvital dye, such as trypan blue, procedure must be
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved Oct. 1, 2007. Published October 2007. Originally performed.
approved in 2001. Last previous edition approved in 2001 as F2149–01. DOI:
1.4.3 Size Variation of the Cell Sample—Up to 30 to 1 by
10.1520/F2149-01R07.
cell diameter in microns; 27 000 to 1 by cell volume. This is
The boldface numbers in parentheses refers to the list of references at the end
of this standard. simply a function of the size range capability of the particular
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2149 − 01 (2007)
FIG. 1 Cell, Suspended in a Conductive Fluid, Traversing Through a Small Aperture
aperture size selected. Using this technology, measurements 2.1.7 raw count, n—the enumeration of the cell population
may be made in the range of about 0.6 to 1200 µm. The lower not corrected for coincidence.
size limit is restricted only by thermal and electronic noise.
2.1.8 ruggedness, n—the degree of reproducibility of the
1.4.4 Size Range of the Aperture—The size range for a
same sample under a variety of normal conditions; for
single aperture is proportional to its diameter, D.The response
example, different operators.
hasbeenfoundtodependlinearlyon Doverarangefrom0.02
2.1.9 size thresholds, n—the instrument’s lower and upper
D to 0.80 D; however, the aperture tube may become prone to
size settings for the particular cell population; adjustable “size
blockage at levels greater than 0.60 D. The practical operating
gate.” Cells or fragments outside the size settings are excluded
range, therefore, of the aperture is considered to be 2 to 60%
from the analyses.
of the diameter.
1.4.5 Humidity—10 to 85%.
3. Significance and Use
1.4.6 Temperature—10 to 35°C.
3.1 This assay is used in university tissue culture
1.4.7 Electrolyte Solution—The diluent for cell suspension
laboratories, government research, and hospital, biomedical,
must provide conductivity and have no effect on cell size. The
and pharmaceutical laboratories to automate cell counting and
electrolyte of choice is most often physiologic phosphate
sizing. This instrumentation provides very rapid, accurate, and
buffered saline.
precise results for any tissue culture facility. In addition, as
2. Terminology noted, since the cell sizes to be analyzed by the instrument are
set by the user, the analyses may be done on virtually any
2.1 Definitions:
species of cells and cell type; it is not restricted to human cells
2.1.1 channelyzer, n—a pulse height analyzer; places volt-
or blood cells.
age pulses into appropriate size bins for the size distribution
data. 3.2 The electrical sensing zone methodology was intro-
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).Inaddition,whencountingandsizingporcine
tre; measurement of cell diameter. hepatocytes, Stegemann et al c
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