Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design.
Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design.
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DOI：
10.1002/cyto.a.22251
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发表时间：
2013-03
期刊：
影响因子：
3.7
通讯作者：
Roederer, Mario
中科院分区：
文献类型：
--
作者：
Nguyen, Richard;Perfetto, Stephen;Mahnke, Yolanda D.;Chattopadhyay, Pratip;Roederer, Mario
补偿后，由多个荧光溢出到每个检测器的测量误差变得明显，名义上负分布的蔓延。根据仪器配置和性能以及所用试剂的不同，这种“溢出扩散”会影响任何给定参数的灵敏度。理论上预测了外溢扩散程度随测量误差的增加而增加，即随荧光强度的平方根而增加，并与光谱重叠矩阵系数直接相关。我们设计了一个度量来量化任何一对探测器之间的溢出扩散。这个度量是固有的，因为它与荧光强度无关。一个工具的所有这些值的组合可以表示为溢出扩散矩阵（SSM）。单染色对照用于在多个仪器上随时间和各种信号质量条件下测定SSM。SSM值揭示了荧光光谱的相互作用，这种相互作用可以限制试剂在不同颜色的明亮染色细胞存在时的灵敏度。SSM具有很高的重现性；其非平凡值表明，在2个月的时间框架内，CV低于30%。此外，SSM在类似配置的仪器之间具有可比性；SSM中仪器特定的差异揭示了表现不佳的探测器。定量和监测SSM可以成为仪器质量控制的有用工具，以确保一致的灵敏度和性能。此外，SSM是预测多色免疫荧光面板的关键元素，这将有助于新面板的优化和开发。我们认为SSM是评价流式细胞仪性能的QA/QC的关键组成部分。
After compensation, the measurement errors arising from multiple fluorescences spilling into each detector become evident by the spreading of nominally negative distributions. Depending on the instrument configuration and performance, and reagents used, this “spillover spreading” affects sensitivity in any given parameter. The degree of spillover spreading had been predicted theoretically to increase with measurement error, i.e., by the square root of fluorescence intensity, as well as directly related to the spectral overlap matrix coefficients. We devised a metric to quantify spillover spreading between any pair of detectors. This metric is intrinsic, as it is independent of fluorescence intensity. The combination of all such values for one instrument can be represented as a spillover spreading matrix (SSM). Single-stained controls were used to determine the SSM on multiple instruments over time, and under various conditions of signal quality. SSM values reveal fluorescence spectrum interactions that can limit the sensitivity of a reagent in the presence of brightly-stained cells on a different color. The SSM was found to be highly reproducible; its non-trivial values show a CV of under 30% across a 2-month time frame. In addition, the SSM is comparable between similarly-configured instruments; instrument-specific differences in the SSM reveal underperforming detectors. Quantifying and monitoring the SSM can be a useful tool in instrument quality control to ensure consistent sensitivity and performance. In addition, the SSM is a key element for predicting multicolor immunofluorescence panels, which will aid in the optimization and development of new panels. We propose that the SSM is a critical component of QA/QC in evaluation of flow cytometer performance.
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影响因子：
1.7
作者：
Mahnke, Yolanda D.;Roederer, Mario
通讯作者：
Roederer, Mario
影响因子：
2.2
作者：
Baumgarth, N;Roederer, M
通讯作者：
Roederer, M
影响因子：
3.7
作者：
Mahnke, Yolanda D.;Beddall, Margaret H.;Roederer, Mario
通讯作者：
Roederer, Mario
影响因子：
14.8
作者：
Perfetto, Stephen P.;Ambrozak, David;Roederer, Mario
通讯作者：
Roederer, Mario
DOI：
10.1002/1097-0320(20011101)45:3
发表时间：
2001-11-01
期刊：
CYTOMETRY
影响因子：
--
作者：
Roederer, M
通讯作者：
Roederer, M