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）的细胞免疫分型主要集中在定量具有特定分子组合的细胞比例上。本研究的目的是探索利用细胞个体信息的流式细胞仪在临床医学中的有效应用。为了确定细胞表面分子密度，将FSM在单个细胞上获得的前向光散射（FSC）和荧光强度（FI）绘制成散点图，其中x轴表示FSC， y轴表示FI。当FI和FSC之间可以看出成比例关系时，可以用回归曲线的斜率（或其绝对值）作为分子密度的指标，称为FF指数。通过这种方式，FI与FSC散点图可以评估细胞大小与抗原表达量之间的关系。在CD4阳性淋巴细胞的情况下，CD4- fi实际上被发现与FSC或其细胞大小成正比。这表明无论细胞大小如何，每个大小单位（或FSC）的CD4分子数量是不变的。因此，所得回归线的斜率，或FF指数，可以作为分子密度的一个指标。在实践中，流式细胞术测量的五个参数- fi (FI1， FI2和FI3)， FSC和SSC（侧散点）从FACScan流式细胞仪采集的单个细胞在个人计算机上使用新开发的软件进行处理。在几个模型系统上测试了FF指数法在临床流式细胞术中的适用性。根据CD45亚型的表达将CD4阳性T淋巴细胞分为两个亚群，并比较这两个亚群之间CD4分子密度，FF指数法清楚地显示CD45- ro阳性记忆细胞上的CD4密度明显高于CD45- ra初始细胞。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标，因此可以想象，该技术在诸如细胞活化等研究中具有重要价值，细胞大小的变化通常与细胞分子表达的改变有关。为了验证这一可能性，我们分析了ConA刺激前后CD4阳性T淋巴细胞表面分子、CD4和CD25的表达情况。虽然这两种分子在ConA刺激下的表达均显著增加，但使用该技术可以看到CD4和CD25分子之间密度增加的程度有显著差异。因此，FF指数方法可用于细胞表面分子的定量分析，特别是涉及细胞大小变化的情况。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便捷的方法，可以评估功能重要的细胞表面分子数量的变化如何影响各种生理和病理状况。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