Wang Lili, Gaigalas Adolfas K, Marti Gerald, Abbasi Fatima, Hoffman Robert A
National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA.
Cytometry A. 2008 Apr;73(4):279-88. doi: 10.1002/cyto.a.20507.
A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two-step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to poses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation.
本文介绍了一种用于将多色流式细胞仪的输出校准为每个细胞结合抗体单位（ABC）的方法。该方法包括两个步骤。首先，使用具有指定参考荧光团等效数量（ERF）值的超彩虹微球对流式细胞仪的每个荧光通道进行校准。此步骤的目的是在多色流式细胞仪的给定荧光通道中的荧光信号与ERF值之间建立线性关系。第二步涉及一种生物标准物，例如具有已知数量抗体结合位点（例如CD4结合位点）的淋巴细胞。将该生物标准物与针对特定荧光通道用一种荧光团标记的抗体一起孵育，用于将ERF刻度转换为ABC刻度。两步校准程序的一个重要部分涉及为不同群体的超彩虹微球分配ERF值。ERF值的分配量化了每个微球群体中嵌入的荧光团混合物的相对量。确保在给定的荧光发射波长范围内的荧光信号与分配的ERF值呈线性关系至关重要。生物标准物必须具有针对给定抗体的已知数量的结合位点。此外，该抗体必须适合用与各种荧光通道相关的不同类型的荧光团进行标记。目前的工作表明，可以满足多色流式细胞仪在ABC值方面成功校准的所有要求。校准程序基于坚实的科学基础，因此很容易设想未来在准确性和实施便利性方面的改进。
