1、Designation: F 2149 01 (Reapproved 2007)Standard Test Method forAutomated Analyses of Cellsthe Electrical Sensing ZoneMethod of Enumerating and Sizing Single Cell Suspensions1This standard is issued under the fixed designation F 2149; the number immediately following the designation indicates the ye
2、ar oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method, provided the limitations are under-
3、stood, covers a procedure for both the enumeration andmeasurement of size distribution of most all cell types. Theinstrumentation 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 adheren
4、tcell cultures (1).2This is a quantitative laboratory method notintended for on-line or field use. Results may be reported asnumber of cells per millilitre or total number of cells pervolume of cell suspension analyzed. Both count and sizedistribution may be expressed in cell micron diameter orvolum
5、e, femtolitres.1.2 Cells commonly used in tissue-engineered medicalproducts (2) routinely are analyzed. Examples are chondro-cytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al usedthe method for both pancreatic islet number and volumemeasurements (6). In addition, instrumentation using t
6、he elec-trical sensing zone technology was used for both count and sizedistribution analyses of porcine hepatocytes placed into hollowfiber cartridge extracorporeal liver assist systems. In this study(7), and others (6, 8), the automated electrical sensing zonemethod was clearly validated for superi
7、or accuracy and preci-sion when compared to the conventional manual method,visual cell counting under a microscope using a hemocytom-eter. This validation has been demonstrated over a wide varietyof cell types. In addition, the automated procedure is rapid,rugged, and cost effective; it also minimiz
8、es operator-to-operator variability inherent in manual techniques.1.3 This instrumentation is manufactured by a variety ofcompanies; however, the principle used in all is electricalimpedance. This test method, for cell counting and sizing, isbased on the detection and measurement of changes in elect
9、ri-cal resistance produced by a cell, suspended in a conductiveliquid, 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 insula-tors. When the cell suspension is drawn through a small
10、cylindrical aperture, the passage of each cell changes theimpedance of the electrical path between two submergedelectrodes located on each side of the aperture. An electricalpulse, suitable for both counting and sizing, results from thepassage of each cell through the aperture. The path through thea
11、perture, in which the cell is detected, is known as the“electronic sensing zone.” This test method permits the selec-tive counting of cells within very narrow size distributionranges by electronic selection of the generated pulses. Whilethe number of pulses indicates cell count, the amplitude of the
12、electrical pulse produced depends on the cells volume. Thebaseline resistance between the electrodes is due to theresistance of the conductive liquid within the boundaries of theaperture. The presence of cells within the “electronic sensingzone” raises the resistance of the conductive pathway thatde
13、pends on the volume of the cell. Analyses of the behavior ofcells within the aperture demonstrates that the height of thepulse produced by the cell is the parameter that most nearlyshows proportionality to the cell volume.1.4 Limitations are discussed as follows:1.4.1 CoincidenceOccasionally, more t
14、han a single celltransverses the aperture simultaneously. Only a single largerpulse, as opposed to two individual pulses, is generated. Theresult is a lower cell count and higher cell volume measure-ment. The frequency of coincidence is a statistically predict-able function of cell concentration tha
15、t is corrected by theinstrument. This is called coincidence correction (8). Thisphenomenon may be minimized, thus ensuring greater resultaccuracy, by using relatively low cell concentrations, aroundthe 5 % level.1.4.2 ViabilityAutomated cell counting enumerates bothviable and nonviable cells. It doe
16、s not measure percent cellviability. To measure the percent cell viability, either a vital dyeor nonvital dye, such as trypan blue, procedure must beperformed.1.4.3 Size Variation of the Cell SampleUpto30to1bycell diameter in microns; 27 000 to 1 by cell volume. This issimply a function of the size
17、range capability of the particular1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Oct. 1, 2007. Published
18、 October 2007. Originallyapproved in 2001. Last previous edition approved in 2001 as F 2149 01.2The boldface numbers in parentheses refers to the list of references at the endof this standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United
19、States.aperture size selected. Using this technology, measurementsmay be made in the range of about 0.6 to 1200 m. The lowersize limit is restricted only by thermal and electronic noise.1.4.4 Size Range of the ApertureThe size range for asingle aperture is proportional to its diameter, D. The respon
20、sehas been found to depend linearly on D over a range from 0.02D to 0.80 D; however, the aperture tube may become prone toblockage at levels greater than 0.60 D. The practical operatingrange, therefore, of the aperture is considered to be 2 to 60 %of the diameter.1.4.5 Humidity10to85%.1.4.6 Temperat
21、ure10 to 35C.1.4.7 Electrolyte SolutionThe diluent for cell suspensionmust provide conductivity and have no effect on cell size. Theelectrolyte of choice is most often physiologic phosphatebuffered saline.2. Terminology2.1 Definitions:2.1.1 channelyzer, na pulse height analyzer; places volt-age puls
22、es into appropriate size bins for the size distributiondata.2.1.2 coincidence, nmore than one cell transversing theaperture at the same time.2.1.3 corrected count, nthe cell count corrected for coin-cidence.2.1.4 electrolyte, ndiluent, offering slight conductivity, inwhich cells are suspended.2.1.5
23、femtolitre, na cubic micron; a measurement of cellvolume.2.1.6 micron (), n0.001 mm, also known as a microme-tre; measurement of cell diameter.2.1.7 raw count, nthe enumeration of the cell populationnot corrected for coincidence.2.1.8 ruggedness, nthe degree of reproducibility of thesame sample unde
24、r a variety of normal conditions; for ex-ample, different operators.2.1.9 size thresholds, nthe instruments lower and uppersize settings for the particular cell population; adjustable “sizegate.” Cells or fragments outside the size settings are excludedfrom the analyses.3. Significance and Use3.1 Th
25、is assay is used in university tissue culture laborato-ries, government research, and hospital, biomedical, and phar-maceutical laboratories to automate cell counting and sizing.This instrumentation provides very rapid, accurate, and preciseresults for any tissue culture facility. In addition, as no
26、ted,since the cell sizes to be analyzed by the instrument are set bythe user, the analyses may be done on virtually any species ofcells and cell type; it is not restricted to human cells or bloodcells.3.2 The electrical sensing zone methodology was intro-duced in the mid 1950s (9). Since this time,
27、there have beensubstantial improvements which have enhanced the operatorsease of use. Among these are the elimination of the mercurymanometer, reduced size, greater automation, and availabilityof comprehensive statistical computer programs.3.3 This instrumentation offers a rapid result as contrasted
28、to the manual counting of cells using the standard countingchamber, hemocytometer. The counting chamber is known tohave an error of 10 to 30 %, as well as being very timeconsuming (10). In addition, when counting and sizing porcinehepatocytes, Stegemann et al concluded that the automated,electrical
29、sensing zone method provided significantly greateraccuracy, precision, and speed, for both counts and size,compared to the conventional microscopic or the cell mass-based method (7).FIG. 1 Cell, Suspended in a Conductive Fluid, Traversing Through a Small ApertureF 2149 01 (2007)24. Interferences4.1
30、Debris and Cellular FragmentsWhen these are in thecellular size ranges, they will be analyzed. Correct cell sizethreshold settings help to correct inclusion of debris or frag-ments in the analyses.4.2 Cellular AggregationAggregates will be enumeratedas a single cell. They will be sized larger than t
31、heir individualcell components. Commercially available enzyme solutions aidin the preparation of single cell suspensions.4.3 SettlingCell sample preparations may exhibit settlingresulting in decreased counts. Inverting the sample containerseveral times will produce more accurate and precise results.
32、4.4 AdherenceCells may adhere to the sides of the samplevessel, thus affecting count accuracy.4.5 Line NoiseInstruments must be located on a benchaway from flickering lights and other equipment such ascentrifuges.4.6 Excessive current across the aperture may damagecellular membranes. The optimum cur
33、rent is set by the instru-ment.5. Hazards5.1 Warning (Chemical)Do not use nonaqueous electro-lyte solutions (that in which the cells are suspended). Care shallbe taken when mixing some electrolyte solutions. Violentreactions may occur. Azide shall not be used in acid solutions.Flammable electrolyte
34、solutions and organic solvents, as notedabove, must not be used.5.2 Warning (Electrical)High voltages are present insidethe instrument. Instrument shall be sited on a firm, dry workbench and must be grounded correctly.5.3 Warning (Biological)Institutional, state, and OSHAapproved safety action plans
35、 shall be followed.6. Procedures6.1 The procedure for this test method is similar regardlessof the brand of commercial instrumentation used. Commercialinstrumentation among manufacturers may vary as to theinstruments size range, number of aperture diameters avail-able, and data acquisition capabilit
36、ies using various printers orcomputer data acquisition. Certain manufacturers provide in-strumentation in which both instrument function control anddata analyses are computer controlled. In addition, somemodels will provide cell counts only; however, other modelscontain a channelyzer enabling the in
37、strument to determine cellsize distribution in addition to count results.6.2 Calibrate the instrument following the instrument manu-facturers procedure. Instrument calibration should be per-formed monthly or after the unit has been serviced. For all celltypes, a commercial calibrator of known MCV sh
38、ould be used.6.3 Dilute the cell suspension in the electrolyte, typicallyphysiologic phosphate-buffered saline. Gently mix the sampleby inverting it seven or eight times. Perform the analyseswithin 20 min after sample preparation.6.4 Set the size range on the instrument for the particularcell popula
39、tion being analyzed. Choose the correct aperturesize. The manufacturers operator manual will contain appro-priate protocols.6.5 Select the volume of the suspension to be analyzed.Generally, this should be 0.5 mL. Place the cell sample onto thesample stand.6.6 Press Start. To determine aperture block
40、age, one canmonitor the analyses time for the volume of cells to beanalyzed. Typically, for 500 L, this would be 13 s; however,most instrumentation contains an aperture viewing screen onwhich any blockages may easily be seen. In addition, mostcontemporary instrumentation now provide an automatic ape
41、r-ture unblock function.6.7 Record the results of the analysis. Account for both thedilution factor and sample volume to determine the concentra-tion of the original cell sample. For instance, 10 000 cells in500 L would be 20 000 cells/mL. For a 1:500 dilution, theoriginal sample concentration is 1
42、3 107cells/mL. Mostinstrumentation now automatically provide this calculation.6.8 To ensure result quality, commercial controls in avariety of concentrations should be run periodically. It is up toeach laboratory to determine control protocols. Regulatoryagencies may require certain laboratories to
43、analyze controlson a more frequent basis.7. Precision and Bias7.1 Reproducibility on replicate samples of porcine hepato-cytes is 65 % (average 63%)(7). Lehmann reported a CV of3.0 % on pancreatic islets (6). Evaluation of this methodology,using red blood cells, by the International Committee forSta
44、ndardization in Hematology resulted in a CV in the range of0.24 to 0.72 % (8). The instrument operators manual, fromcertain manufacturers, lists precision on representative celltypes. Instructions for instrument installation qualification,operational qualification, and performance qualification area
45、vailable. A statement of bias is not available; however, thistechnology is the reference method for the enumeration of bothred blood cells (RBCs) and white blood cells (WBCs) as setforth by the International Council for Standardization inHematology in 1988 (11).8. Keywords8.1 automated cell counting
46、; electrical sensing zone; sizedistributionF 2149 01 (2007)3REFERENCES(1) Li, R., Chen, J., Hammonds, G., et al, “Identification of Gas as aGrowth Factor for Human Schwann Cells,” The Journal of Neuro-science, Vol 16(6), 1996, pp. 20122019.(2) Picciolo, G.L., Hellman, K.B., and Johnson, P.C., “Meeti
47、ng Report:Tissue Engineered Medical Products Standards: The Time is Ripe,”Tissue Engineering, Vol 4(1), 1998, pp. 57.(3) Lamo, S., Ronot, X., and Lechay, P., “Effects of Sodium Butyrate onGrowth and Cell-Cycle Kinetics of Cultured Rabbit Articular Chon-drocytes,” J. Cell Physiol., Vol 120, 1984, pp.
48、 384390.(4) Ryan, J.M., Sharf, B.R., and Cristofalo, V.J., “The Influence of CultureMedium Volume on Cell Density and Lifespan of Human DiploidFibroblasts,” Exptl. Cell Res. Vol 91, 1975, pp. 389392.(5) Betz, N.A., Fattaey, H.K., and Johnson, T.C., “Growth Inhibition ofBALB/c Mouse Keratinocytes by
49、TGF-B1 and CeReS-18 Appears toAct through Distinct Mechanisms,” Exptl. Cell Res., Vol 227, 1996,pp 4754.(6) Lehman, R., Fernanandez, L.A., Bottino, R., Szabo, S., et al, “Evalu-ation of Islet Isolation by a New Automated Method (Coulter Multi-sizer 11e) and Manual Counting,” Transp. Proc, Vol 30, 1998, pp.373374.(7) Stegemann, J. P., Santosh, R., Nicholson, D.T., et al, “Comparison ofAnalytical Methods for Quantitation of Isolated Porcine HepatocyteYields,” Tissue Engineering, Vol 6(3), 2000, pp. 253264.(8) Lewis, S.M., England, J.M., an
