Flow cytometry limitations

Accordingly, the use of flow cytometry can improve the design of toxicity bioassays, as the detection limit of this apparatus includes cellular concentrations equal to those of microalgal populations found in natural conditions. Comparison of compositions utilised in some known toxicity tests for microalgaes are shown in Table 7.1.1. 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

Up to 5% of unstimulated CB mononuclear cells are CSF-1-receptor positive as measured by flow cytometry (data not shown). In the first week of coculture on transduced MMCF cells all CSF-1 isoforms promoted proliferation of CB CD34+ cells with comparable rates (Fig. 5a). However, within four weeks the stimulatory activity of wildtype CSF-1 declined dramatically. Obviously, the soluble isoform provided only a limited expansion of CB CD34+ cells. 

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

We should, however, always be aware of situations in which microscopists are better than cytometrists—certain types of classification are easy by eye but not at all easy by cytometer. For example, a microscopist would never confuse a dead lymphocyte with an erythrocyte, nor a chunk of debris with a viable cell such mistakes are all too frequent in cytometry. Furthermore, microscopists, if they were not so polite, would find it laughable that cytometrists have a great deal of difficulty in distinguishing clumps of small cells from single large cells. However, if we think back to the discussion in Chapter 3 about the origin of the FSC signal, we can see why these problems occur and why we need to be aware of the limits of flow cytometry (and why a microscope is an essential piece of equipment in a flow cytometry lab). 

There has been much discussion about the potential utility of flow cytometry of chromosomes for clinical diagnosis. As regards its sensitivity, this technique appears to stand somewhere between the technique of flow analysis of whole cells for DNA content and that of microscope analysis of banded chromosomes. It may be a useful intellectual exercise for readers to ask themselves which technique or techniques would be most appropriate for detecting the following types of chromosome abnormalities (1) tetraploidy, where the normal chromosome content of cells is exactly doubled because of failure of cytokinesis after mitosis (2) an inversion in an arm of one particular chromosome and (3) trisomy (the existence of cells with three instead of two) of one of the small chromosomes. In addition to these limitations, the use of flow cytometry to look for abnormal chromosomes has been confounded by the fact that several human chromosomes are highly polymorphic, and flow karyotypes, therefore, vary considerably among normal individuals. 

The second aspect of clinical practice that has led to a reassessment of the nature of flow cytometry is the occasional clinical requirement for rare-event analysis. Methods have been developed, particularly with the use of multiparameter gating, to lower background noise in order to provide increased sensitivity for detection of rare cells. In the clinic, this increased sensitivity translates, for example, into earlier diagnosis of relapse in leukemia, more sensitive detection of fetal-maternal hemorrhage, and better ability to screen leukocyte-reduced blood transfusion products for residual white blood cells. Outside the clinic, these methods for rare-event detection have begun to stretch the limits of research applications as well. 

Although books on flow cytometry abound and articles on flow cytometry can be found throughout a great range of publications, the following is a limited list of references that I have found particularly useful for general information on the theoretical basis of flow analysis and as routes into the literature on particular subjects and techniques. 

A third aspect of flow cytometry (known sometimes simply with the acronym for fluorescence-activated cell sorter, FACS, or even more familiarly as just flow) that distinguishes it from many other techniques is the way in which its wide and increasing usefulness has continued to surprise even those who consider themselves experts. What began as a clever technique for looking at a very limited range of problems is now being used in universities, in hospitals, within industry, at marine stations, on submersible buoys, and on board ships plans have existed for use on board space ships as well. The applications of flow cytometry have proliferated (and continue to proliferate) rapidly both in the direction of theoretical science, with... 

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