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

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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