A novel application of flow cytometry - Quantification of cell surface molecule density and its clinical application

到目前为止，使用流式细胞术(FCM)的细胞免疫表型分型主要集中在对携带特定分子组合的细胞的比例进行量化。本研究的目的是利用单个细胞的信息来探索FCM在临床医学中的有效应用。为了限定细胞表面的分子密度，将FSM获得的单个细胞的前向光散射(FSC)和荧光强度(FI)绘制成散点图，其中x轴和y轴分别表示FSC和FI。在FI和FSC之间可以看到成比例的关系时，可以使用回归直线的斜率(或其绝对值)作为分子密度的指示器，这被称为FF指数。通过这种方式，FI VS FSC散点图能够评估细胞大小和所表达的抗原量之间的关系。在cd4阳性淋巴细胞中，cd4-fi实际上是…。与FSC或其细胞大小成正比。这表明，无论细胞大小，单位大小(或FSC)中的CD4分子数量是不变的。因此，得到的回归线的斜率，或称FF指数，可以作为分子密度的指示器。在实际应用中，使用新开发的软件在个人计算机上对FACScan流式细胞仪中单个细胞的FI(FI1、FI2和FI3)、FSC和SSC(侧向散射)五个参数进行了流式细胞仪测量，并在几个模型系统上测试了FF指数法在临床应用中的适用性。根据CD45亚型的表达将CD4+T淋巴细胞分为两个亚群，并比较这两个亚群之间的CD4分子密度，FF指数法显示CD45-RO阳性记忆细胞上的CD4密度明显高于CD45-RA初始细胞上的密度。这可能表明，不仅每个细胞的CD4分子总量，而且细胞表面的分子密度也受到严格的调节，这可能取决于细胞的功能性质。由于FF指数是分子密度的指示器，因此可以想象，这项技术在研究细胞激活方面可能有很大的价值，例如，细胞大小的变化往往与细胞分子表达的变化有关。为了验证这一可能性，分析了ConA刺激前后CD4+T淋巴细胞表面分子、CD4和CD25的表达。虽然在ConA刺激下，这两种分子的表达都显著增加，但使用这一技术可以看到CD4和CD25分子密度增加的程度有显著差异。因此，FF指数法可用于细胞表面分子的定量分析，特别是在涉及细胞大小变化的情况下。FF指数法是临床流式细胞术中定量分析细胞表面分子表达的一种简单方便的方法，从而能够评估具有重要功能的细胞表面分子数量的变化如何影响各种生理和病理条件。较少

Until now, cytometric immunophenotyping using flow cytometry (FCM) has focused mainly on the quantification of the proportion of cells bearing certain combinations of molecules. The purpose of this study is to explore an effective application of FCM to clinical medicine by using the information of individual cells.For the qualification of cell surface molecule density, the forward light scatter (FSC) and fluorescence intensity (FI), obtained by FSM on an individual cell basis were plotted in a scattergram where x-and y-axis show FSC and FI, respectively. Where a proportional relationship between FI and FSC can been seen, the slope of the line of regression (or its absolute value) may be used as an indicator of the molecule density, which is referred to as the FF index. In this way, the FI vs FSC scattergram enables the evaluation of the relationship between the size of the cell and the amount of antigen expressed. In the case of CD4 positive lymphocytes, CD4-FI was actually found to be … More positively proportional to FSC, or their cell size. This indicates that the amount of CD4 molecules per size (or FSC) unit is invariable regardless of cell size. Accordingly, the slope of the resultant regression line, or FF index, could be an indicator of the molecule density. In practice, flow cytometric measurements on five parameters-FI (FI1, FI2 and FI3), FSC and SSC (side scatter) for individual cells taken from the FACScan flow cytometer were processed on a personal computer using newly developed software.The applicability of the FF index method to clinical flow cytometry was tested on a few model systems. When CD4 positive T lymphocytes were divided into two subpopulations on the basis of the expression of CD45 isoforms and the density of CD4 molecule was compared between these two subpopulations, the FF index method clearly demonstrated that CD4 density on CD45-RO positive memory cells was significantly higher than that on CD45-RA naive cells. This may indicate that not only the total amount of CD4 molecules per cell, but also the density of the molecule on the surface of cell is under strict regulation, possibly depending on the functional nature of the cells.Since the FF index is the indicator of molecule density, it is conceivable that this technique could be of great value in the study of, for instance, cellular activation, where changes in cell size are often associated with the alteration in the expression of cell molecules. To verify this possibility, the expression of cell surface molecules, CD4 and CD25 on CD4 positive T lymphocytes were analyzed before and after stimulation by ConA. Although both molecules showed a remarkable increase in their expression upon ConA stimulation, a significant difference in the extent of the increase in density could be seen between CD4 and CD25 molecules by using this technique. Thus, the FF index method could be useful in quantitative analysis of cell surface molecules, particularly where changes in cell size are involved.The FF index method is a simple and convenient means for quantitative analysis of the expression of cell surface molecules in clinical flow cytometry, thereby enabling the evaluation of how alterations in the quantity of functionally important cell surface molecules underlie various physiological and pathological conditions. Less