1.1 如果理解了局限性，本试验方法涵盖了大多数所有细胞类型的大小分布计数和测量程序。该仪器允许用户选择单元格大小设置，并适用于多种单元格类型。该方法最适用于球形细胞，如果细胞不是球形，例如盘状细胞或芽胞酵母，则可能不太准确。该方法适用于悬浮和粘附细胞培养 ( 1. ) . 2. 结果可以报告为每毫升细胞数或分析的每体积细胞悬液的细胞总数。 尺寸分布可以用细胞直径或体积表示。 1.2 组织工程医疗产品中常用的细胞 ( 2. ) 常规分析。例如软骨细胞 ( 3. ) ，成纤维细胞 ( 4. ) 和角质形成细胞 ( 5. ) . Szabo等人将该方法用于胰岛数量和体积测量 ( 6. ) . 此外，使用电传感区技术的仪器用于对放置在中空纤维筒体外肝脏辅助系统中的猪肝细胞进行计数和大小分布分析。在本研究中 ( 7. ) ，和其他 ( 6. , 8. ) 与使用血液细胞仪在显微镜下进行的常规视觉细胞计数相比，自动电感应区方法的精度得到了验证。目前，不可能验证细胞计数设备的准确性，因为没有办法产生具有已知细胞数的参考样本。每次在新实验室中实施、在新电池类型上使用或修改电池计数程序时，应验证电感应区方法。 1.3 电气传感区仪器（通常称为库尔特计数器）由多家公司制造，并基于电阻。 本试验方法用于细胞计数和尺寸确定，基于检测和测量悬浮在导电液体中穿过小孔的细胞产生的电阻变化（见 图1 ( 9 ) ). 当细胞悬浮在导电液体（例如磷酸盐缓冲盐水）中时，它们起到离散绝缘体的作用。当细胞悬浮液通过一个小圆柱孔时，每个细胞的通道会改变位于孔两侧的两个浸入式电极之间的电路阻抗。 每个细胞通过孔径产生一个电脉冲，适用于计数和大小调整。通过孔径检测细胞的路径称为“电子传感区”该测试方法允许通过电子选择产生的脉冲，在狭窄的尺寸分布范围内选择性计数细胞。虽然脉冲数表示细胞计数，但产生的电脉冲的幅度取决于细胞的体积。电极之间的基线电阻是由于孔径边界内导电液体的电阻引起的。 “电子传感区”内细胞的存在增加了导电通路的电阻，该电阻取决于细胞的体积。对孔径内细胞行为的分析表明，细胞产生的脉冲高度是最接近细胞体积比例的参数。 1.4 局限性讨论如下: 1.4.1 巧合- 有时，不止一个细胞同时穿过孔径。只产生一个较大的脉冲，而不是两个单独的脉冲。 结果是较低的细胞计数和较高的细胞体积测量。符合频率是由仪器校正的细胞浓度的统计可预测函数。这称为重合校正 ( 8. ) . 通过使用较低的细胞浓度，可以减少这种现象。 1.4.2 生存能力- 电感应区细胞计数同时枚举活细胞和非活细胞，无法确定活细胞百分比。需要单独测试，如台盼蓝，以确定活细胞百分比。 1.4.3 电池直径- 这是所选孔径尺寸的尺寸范围能力的函数。可在0.6μm至1200μm的试管直径范围内进行测量。在仪器上设置计数尺寸范围可能会影响测试结果，尤其是当试管尺寸分布较大时，应仔细控制，以帮助实现重复性。 1.4.4 孔径大小范围- 单个孔径的尺寸范围与其直径成正比。在直径的2%-80%范围内，响应与直径呈线性关系。 然而，孔径管在大于直径60%的水平时可能容易堵塞。因此，孔径的实际工作范围被认为是2%-60% % 直径的。 1.4.5 湿度- 10%-85% %. 1.4.6 温度- 10°C至35°C。 1.4.7 电解质溶液- 细胞悬浮液的稀释剂应具有导电性，对细胞大小的影响最小。电解质的选择通常是磷酸盐缓冲盐水。 ====意义和用途====== 3.1 用于细胞计数的电感应区方法用于组织培养、政府研究以及医院、生物医学和制药实验室中的细胞计数和大小测定。 该方法可适用于多种细胞大小和细胞类型，并经过适当验证 ( 10 ) . 3.2 电气传感区方法于20世纪50年代中期引入 ( 9 ) . 从那时起，有了实质性的改进，提高了操作员的易用性。其中包括消除水银压力计、缩小尺寸、提高自动化程度以及提供全面的统计计算机程序。 3.3 与使用血细胞仪标准计数室手动计数细胞相比，该仪器提供了快速的结果。 已知计数室的误差为10到30 %, 而且很耗时 ( 11 ) . 此外，当对猪肝细胞进行计数和大小测定时，Stegemann等人得出结论，与传统的显微镜或基于细胞质量的方法相比，自动化的电感应区方法在计数和大小方面提供了更高的准确性、精密度和速度 ( 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 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 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 volume. 1.4 Limitations are discussed as follows: 1.4.1 Coincidence— Occasionally, 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 ( 8 ) . This phenomenon may be reduced by using lower cell concentrations. 1.4.2 Viability— Electrical sensing zone cell counting enumerates both viable and nonviable cells and cannot determine percent viable cells. A separate test, such as Trypan blue, is required to determine percent viable cells. 1.4.3 Cell Diameter— This is a function of the size range capability of the aperture size selected. Measurements may be made in the cell diameter range of 0.6 μm to 1200 μm. Setting the counting size range on the instrument can affect the test results, especially if the cell size has a large distribution, and should be carefully controlled to help achieve repeatability. 1.4.4 Size Range of the Aperture— The size range for a single aperture is proportional to its diameter. The response has been found to depend linearly on diameter over a range from 2 % to 80 % of the diameter. However, the aperture tube may become prone to blockage at levels greater than 60 % of diameter. Therefore, the practical operating range of the aperture is considered to be 2 % to 60 % of the diameter. 1.4.5 Humidity— 10 % to 85 %. 1.4.6 Temperature— 10 °C to 35 °C. 1.4.7 Electrolyte Solution— The diluent for cell suspension shall provide conductivity and have minimal effect on cell size. The electrolyte of choice is commonly phosphate-buffered saline. ====== 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 ) .