ASTM_F_2149_-_16.pdf

Designation:F214916Standard Test Method forAutomated Analyses of Cellsthe Electrical Sensing ZoneMethod of Enumerating and Sizing Single Cell Suspensions1This standard is issued under the fixed designation F2149;the number immediately following the designation indicates the year 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()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method,provided the limitations areunderstood,covers a procedure for both the enumeration andmeasurement of size distribution of most all cell types.Theinstrumentation allows for user-selectable cell size settings andis applicable to a wide range of cell types.The method worksbest for spherical cells,and may be less accurate if cells are notspherical,such as for discoid cells or budding yeast.Themethod is appropriate for suspension as well as adherent cellcultures(1).2Results may be reported as number of cells permilliliter or total number of cells per volume of cell suspensionanalyzed.Size distribution may be expressed in cell diameteror volume.1.2 Cells commonly used in tissue-engineered medicalproducts(2)are analyzed routinely.Examples are chondro-cytes(3),fibroblasts(4),and keratinocytes(5).Szabo et al.used the method for both pancreatic islet number and volumemeasurements(6).In addition,instrumentation using the 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 validated for precision when compared to theconventional visual cell counting under a microscope using ahemocytometer.Currently,it is not possible to validate cellcounting devices for accuracy,since there not a way to producea reference sample that has a known number of cells.Theelectrical sensing zone method shall be validated each time itis implemented in a new laboratory,it is used on a new celltype,or the cell counting procedure is modified.1.3 Electrical sensing zone instrumentation(commonly re-ferred to as a Coulter counter)is manufactured by a variety ofcompanies and is based upon electrical impedance.This testmethod,for cell counting and sizing,is based on the detectionand measurement of changes in electrical resistance producedby a cell,suspended in a conductive liquid,traversing througha small aperture(see Fig.1(9).When cells are suspended in aconductive liquid,phosphate-buffered saline for instance,theyfunction as discrete insulators.When the cell suspension isdrawn through a small cylindrical aperture,the passage of eachcell changes the impedance of the electrical path between twosubmerged electrodes located on each side of the aperture.Anelectrical pulse,suitable for both counting and sizing,resultsfrom the passage of each cell through the aperture.The paththrough the aperture,in which the cell is detected,is known asthe“electronic sensing zone.”This test method permits theselective counting of cells within narrow size distributionranges by electronic selection of the generated pulses.Whilethe number of pulses indicates cell count,the amplitude of theelectrical 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 thatdepends 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 than 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 that is corrected by theinstrument.This is called coincidence correction(8).Thisphenomenon may be reduced by using lower cell concentra-tions.1.4.2 ViabilityElectrical sensing zone cell counting enu-merates both viable and nonviable cells and cannot determinepercent viable cells.A separate test,such as Trypan blue,isrequired to determine percent viable cells.1.4.3 Cell DiameterThis is a function of the size rangecapability of the aperture size selected.Measurements may bemade in the cell diameter range of 0.6 m to 1200 m.Settingthe counting size range on the instrument can affect the test1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs f