Active Standard ASTM F2149 | Developed by Subcommittee: F04.43
Book of Standards Volume: 13.01
Historical (view previous versions of standard)
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).
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
1.2 Cells commonly used in tissue-engineered medical products
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
1.4 Limitations are discussed as follows:
1.4.1 CoincidenceOccasionally, 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
1.4.2 ViabilityAutomated cell counting enumerates both viable and nonviable cells. It does not measure percent cell viability. To measure the percent cell viability, either a vital dye or nonvital dye, such as trypan blue, procedure must be performed.
1.4.3 Size Variation of the Cell Sample Up to 30 to 1 by cell diameter in microns; 27 000 to 1 by cell volume. This is simply a function of the size range capability of the particular aperture size selected. Using this technology, measurements may be made in the range of about 0.6 to 1200 m. The lower size limit is restricted only by thermal and electronic noise.
1.4.4 Size Range of the ApertureThe size range for a single aperture is proportional to its diameter, D. The response has been found to depend linearly on D over a range from 0.02 D to 0.80 D; however, the aperture tube may become prone to blockage at levels greater than 0.60 D. The practical operating range, therefore, of the aperture is considered to be 2 to 60 % of the diameter.
1.4.5 Humidity10 to 85 %.
1.4.6 Temperature10 to 35C.
1.4.7 Electrolyte SolutionThe diluent for cell suspension must provide conductivity and have no effect on cell size. The electrolyte of choice is most often physiologic phosphate buffered saline.
ICS Number Code 07.100.01 (Microbiology in general)