1.1如果理解局限性，本试验方法涵盖了对大多数所有细胞类型的大小分布进行计数和测量的程序。仪器允许用户选择电池大小设置，因此，该测试方法不限于特定电池类型。该方法适用于悬浮和粘附细胞培养 () . 这是一种定量实验室方法，不适用于在线或现场使用。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。计数和大小分布可以用细胞微米直径或体积（毫微特）表示。 1.2组织工程医疗产品中常用的细胞 () 例行分析。例如软骨细胞 () ，成纤维细胞 () 和角质形成细胞 () . Szabo等人使用该方法测量胰岛数量和体积 () . 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 () ，和其他 (, ) 与使用血液细胞仪在显微镜下进行视觉细胞计数的传统手动方法相比，自动电感应区方法具有更高的准确性和精密度。 这种验证已在多种细胞类型上得到证明。此外，自动化程序快速、坚固且具有成本效益；它还最小化了手动技术中固有的操作员之间的可变性。 1.3本仪器由多家公司制造；然而，全部使用的原理是电阻抗。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 () ). 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，在非常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限制讨论如下: 1.4.1重合有时，多个电池同时穿过孔径。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 () . 通过使用相对较低的细胞浓度（约为5%），可以将这种现象降至最低，从而确保更高的结果准确性。 1.4.2可行性自动细胞计数枚举活细胞和非活细胞。它不测量细胞活力百分比。为了测量细胞活力百分比，必须进行活性染料或非活性染料（如台盼蓝）的程序。 1.4.3电池样品的尺寸变化，以微米为单位，最大为30至1； 按细胞体积计算为27000:1。这只是所选特定孔径的尺寸范围能力的函数。使用该技术，可在约0.6至1200 m的范围内进行测量。尺寸下限仅受热噪声和电子噪声的限制。 1.4.4孔径的尺寸范围单个孔径的尺寸范围与其直径成比例， D . 已发现响应与 D 在0.02范围内 D 至0.80 D ; 然而，孔径管在高于0.60的水平时可能容易堵塞 D . 因此，孔径的实际工作范围被认为是直径的2%到60%。 1.4.5湿度10%至85%。 1.4.6温度10至35℃。 1.4.7电解质溶液电池悬浮液的稀释剂必须具有导电性，并且对电池尺寸没有影响。电解质的选择通常是生理磷酸盐缓冲盐水。 ====意义和用途====== 该检测方法用于大学组织培养实验室、政府研究、医院、生物医学和制药实验室，以实现细胞计数和大小的自动化。该仪器为任何组织培养设施提供了非常快速、准确和精确的结果。此外，如前所述，由于仪器要分析的细胞大小由用户设定，因此可以对几乎任何种类的细胞和细胞类型进行分析； 它不仅限于人类细胞或血细胞。 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。 与使用标准计数室血细胞仪手动计数细胞相比，该仪器具有快速的结果。已知计数室的误差为10%到30%，并且非常耗时 (10) . 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，与传统的显微镜或细胞质量相比，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- 基于方法 (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 () . 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 method permits the selective counting of cells within very 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 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 () . This phenomenon may be minimized, thus ensuring greater result accuracy, by using relatively low cell concentrations, around the 5 % level. 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. ====== 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) .