Flow Cytometry Core, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland 20892-3015, USA.
Cytometry A. 2013 Mar;83(3):306-15. doi: 10.1002/cyto.a.22251. Epub 2013 Feb 6.
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" (SS) affects sensitivity in any given parameter. The degree of SS 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 SS 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 less than 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 the performance of 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.
补偿后，由于多个荧光信号溢出到每个探测器中，名义上的负分布会扩散，从而导致测量误差明显。根据仪器配置、性能和使用的试剂，这种“溢出扩散”（SS）会影响任何给定参数的灵敏度。SS 的程度已从理论上预测会随着测量误差（即荧光强度的平方根）而增加，并且与光谱重叠矩阵系数直接相关。我们设计了一种衡量任意两个探测器之间 SS 的指标。该指标是内在的，因为它独立于荧光强度。一台仪器的所有此类值的组合可以表示为溢出扩散矩阵（SSM）。使用单染对照品来确定多个仪器在不同信号质量条件下随时间的 SSM。SSM 值揭示了荧光光谱相互作用，这些相互作用会限制试剂在不同颜色的强染细胞存在下的灵敏度。SSM 值被发现具有高度可重复性；在 2 个月的时间内，其非平凡值的 CV 小于 30%。此外，SSM 在类似配置的仪器之间具有可比性；SSM 中的仪器特定差异揭示了性能不佳的探测器。量化和监测 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性能的关键因素，这将有助于优化和开发新面板。我们提出 SSM 是评估流式细胞仪性能的质量保证/质量控制的关键组成部分。
