Corsetti J P, Sotirchos S V, Cox C, Cowles J W, Leary J F, Blumburg N
Department of Pathology, University of Rochester Medical Center, New York 14642.
Cytometry. 1988 Nov;9(6):539-47. doi: 10.1002/cyto.990090606.
A method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary. Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.
本文提出了一种在流式细胞术中校正背景荧光的方法，该方法对于低水平细胞表面膜抗原的定量具有特殊意义。该方法基于背景荧光（自发荧光对照或无关抗体对照）和总荧光（阳性染色细胞）的细胞荧光分布的数学建模。基于两种模型且仅利用常规可得的背景和总荧光直方图开发的算法，并在计算机程序中实现。这些算法允许估计仅对应于感兴趣的细胞表面抗原免疫荧光染色的荧光直方图。因此，背景荧光的校正仅通过对常规可得数据的软件处理来实现；无需额外的硬件或参数测定。选择了两种在物理上合理且代表背景与探针荧光之间相关性极端情况的模型。选择极端情况是为了评估解决方案对模型的依赖性，并在没有相关性信息时为实际解决方案提供边界。给出了计算机模拟以及对人红细胞上CR1补体受体的实际测定的结果，以测试和说明该技术。或者，可以假设一个特定模型来测试数据，以探索特异性和非特异性荧光之间的关系（如果有的话）。
