NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads
NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads
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DOI：
10.1002/cyto.a.22086
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发表时间：
2012-09-01
期刊：
影响因子：
3.7
通讯作者：
Nolan, John P.
中科院分区：
文献类型：
--
作者：
Hoffman, Robert A.;Wang, Lili;Nolan, John P.
本文报道了由国际细胞计数促进学会（ISAC）和美国国家标准与技术研究所（NIST）共同赞助的标准化研究结果。本研究评价了微珠生产商为荧光团标准微珠分配强度值的变异性，以及使用不同流式细胞仪将标准微珠交叉校准为染色聚合物微珠（硬染色微珠）的变异性。硬染色珠通常与用于染色细胞的荧光团在光谱上不匹配，并且光谱响应在流式细胞仪之间变化。因此，如果使用硬染色珠作为荧光校准物，则期望对特定荧光团（例如，FITC或PE），以在不同的仪器之间变化。使用标准珠表面染色与特定的荧光团（FITC，PE，APC，和太平洋蓝（TM）），该研究比较了荧光团标准珠的测量强度，硬染色珠通过交叉校准133个不同的流式细胞仪。使用稳健CV作为变异性的量度，对于特定检测通道中的特定硬染色珠，交叉校准值的变异通常为20%或更多。不同仪器型号之间的变化通常大于特定仪器型号内的变化。作为研究的单独部分，NIST和四家微珠制造商使用NIST提供的方案和校准的荧光团溶液标准品为荧光团微珠分配强度值。在大多数情况下，不同组分配给参比微珠的值按数量级变化，反映了用于执行校准的仪器的差异。该研究得出结论，必须针对每个特定仪器型号验证和表征使用任何光谱不匹配的硬染色微珠作为通用荧光校准品。建议微珠制造商与NIST密切合作，以获得可靠且统一分配的荧光标准微珠。(C)2012年国际细胞计数促进学会
Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue (TM)), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. (C) 2012 International Society for Advancement of Cytometry