Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats
Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats
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DOI：
10.1002/1097-0320(20011101)45:3
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发表时间：
2001-11-01
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影响因子：
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通讯作者：
Roederer, M
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作者：
Roederer, M
背景：在流式细胞仪分析中，几乎总是需要对光谱重叠进行补偿。在大多数情况下，这种补偿相对简单，产生所需的直线分布。然而，在荧光分析领域，补偿的可视化经常导致意外的分布，主要是在轴上出现大量事件，并且甚至更令人不安的是，不能将补偿数据的程度降低到“自发荧光”水平。具有可变光子强度、溢出参数、测量误差数据存储特性用于说明补偿数据中的表观误差的来源。免疫荧光染色的细胞收集条件下，限制光收集和高溢出检测器之间，以确认方面的model.Results：光子计数统计贡献的非线性误差补偿参数。测量误差和对数尺度分箱误差对补偿参数贡献线性误差。这些误差在使用红色或远红荧光染料（发射光强度较低）和检测器之间有较大溢出时最为明显。这样的错误可能会导致数据可视化的文物，很容易导致不正确的结论有关的数据，并占明显的“补偿不足”先前描述的reflectingstaining.Conclusions：有不可避免的错误所产生的不完善的测量，光子计数统计，甚至数据存储方法，有助于线性和非线性的“传播”的一个适当的补偿自体荧光分布。这种现象排除了使用“象限”统计或门来分析受影响的数据，它也排除了视觉调整的补偿。最重要的是，它是不可能正确地补偿数据使用标准的视觉图形界面（直方图或点图）。需要计算机辅助补偿，以及仔细的门控和实验设计，以确定积极和消极事件之间的区别。最后，使用除感兴趣的试剂之外的所有试剂的特殊染色对照（称为荧光减一，或“FMO”对照）的使用对于准确鉴定完全染色的样品中的表达细胞变得必要，Cytometry 45：194 - 205，2001。(C)2001 Wiley-Liss，Inc.
Background: In multicolor flow cytometric analysis, compensation for spectral overlap is nearly always necessary. For the most part, such compensation has been relatively simple, producing the desired rectilinear distributions. However, in the realm of multicolor analysis, visualization of compensated often results in unexpected distributions, principally the appearance of a large number of events on the axis, and even more disconcerting, an inability to bring the extent of compensated data down to "autofluorescence" levels.Materials and Methods: A mathematical model of detector measurements with variable photon intensities, spillover parameters, measurement errors, and data storage characteristics was used to illustrate sources of apparent error in compensated data. Immunofluorescently stained cells were collected under conditions of limiting light collection and high spillover between detectors to confirm aspects of the model.Results: Photon-counting statistics contribute a nonlinear error to compensated parameters. Measurement errors and log-scale binning error contribute linear errors to compensated parameters. These errors are most apparent with the use of red or far-red fluorochromes (where the emitted light is at low intensity) and with large spillover between detectors. Such errors can lead to data visualization artifacts that can easily lead to incorrect conclusions about data, and account for the apparent "undercompensation" previously described for multicolor staining.Conclusions: There are inescapable errors arising from imperfect measurements, photon-counting statistics, and even data storage methods that contribute both linearly and nonlinearly to a "spreading" of a properly compensated autofluorescence distribution. This phenomenon precludes the use of "quadrant" statistics or gates to analyze affected data; it also precludes visual adjustment of compensation. Most importantly, it is impossible to properly compensate data Using standard visual graphical interfaces (histograms or dot plots). Computer-assisted compensation is required, as well as careful gating and experimental design to determine the distinction between positive and negative events. Finally, the use of special staining controls that employ all reagents except for the one of interest (termed fluorescence minus one, or "FMO" controls) becomes necessary to accurately identify expressing cells in the fully stained sample, Cytometry 45: 194 - 205, 2001. (C) 2001 Wiley-Liss, Inc.