Wang Lili, Gaigalas Adolfas K, Wood James
National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, MD, 20899, USA.
Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA.
Methods Mol Biol. 2018;1678:93-110. doi: 10.1007/978-1-4939-7346-0_6.
Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results independent of flow cytometers used. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure includes the following critical steps: (a) quality control (QC) and performance characterization of the multicolor flow cytometer, (b) fluorescence calibration using hard dyed microspheres assigned with fluorescence intensity values in equivalent number of reference fluorophores (ERF), (c) compensation for correction of fluorescence spillover, and (d) application of a biological reference standard for translating the ERF scale to the ABC scale. The chapter also points out current efforts for implementing quantification of biomarkers in a manner which is independent of instrument platforms and reagent differences.
多色流式细胞仪检测在临床实验室中常用于免疫表型分析、疾病和治疗监测以及确定预后因素。然而，现有的定量测量方法尚未能独立于所使用的流式细胞仪而产生令人满意的结果。本章详细介绍了一种通过将多色流式细胞仪的输出校准为每个细胞结合的抗体单位（ABC）来定量表面和细胞内蛋白质生物标志物的程序。该程序包括以下关键步骤：（a）多色流式细胞仪的质量控制（QC）和性能表征，（b）使用分配有等效参考荧光团数量（ERF）荧光强度值的硬染色微球进行荧光校准，（c）对荧光溢出进行校正的补偿，以及（d）应用生物参考标准将ERF尺度转换为ABC尺度。本章还指出了当前为以独立于仪器平台和试剂差异的方式实现生物标志物定量所做的努力。
