Flow Cytometry

Flow cytometry is a technology that has impacted both basic cell biology and clinical medicine in a very significant manner. The essential principle of flow cytometry is that single particles suspended within a stream of liquid are interrogated individually in a very short time as they pass through a light source focused at a very small region. The optical signals generated are mostly spectral bands of light in the visible spectrum, which represent the detection of various chemical or biological components, mostly fluorescence. A key aspect of flow cytometers is that because they can analyze single particles/cells, it is possible to separate particles/cells into populations based upon a statistical difference of any of 10 to 20 variables that can be measured on each particle/ cell. Using these statistical analyses, it is possible to separate these populations electronically and identify them using multivariate analysis techniques. The most common detection system in flow cytometry uses fluorescent molecules that are attached by one means or another to the particle of interest. If the particle is a cell, such as a white blood cell, for example, the fluorescent probe might be membrane bound, cytoplasmic, or attached to nuclear material. It is a common practice to use monoclonal or polyclonal antibodies that recognize specific receptors on cells. By conjugating fluorescent molecules to these antibodies, it is possible to monitor both the location and number of these conjugated antibodies as they bind to cell receptors. Particles of almost any nature can be evaluated by flow cytometry. They can be very small, even below the resolution limits of visible light, because they can be detected by their fluorescent signatures. Similarly, depending on the structure of the flow cell and fluidics, particles as large as several thousand microns can be evaluated. The key advantage of flow cytometry is that a very large number of particles can be evaluated in a very short time; some systems can run particles at rates approaching 100,000 particles per second while collecting 10 to 20 parameters from each particle. Finally, the principle of cell sorting in flow cytometry allows this technology to separate single particles/cells physically from mixed populations. Thus single particles can be physically placed into a defined location for further analysis and, if necessary, this process can be performed under sterile conditions. This capability makes flow cytometry a valuable tool for rare event (1:100,000 or even 1:1,000,000) analysis. In 1983 Shapiro noted that multiparameter flow cytometry was now a reality in the field because of the availability of commercial instruments. Since that time, the field has expanded well beyond anything that was then considered possible. Today’s instruments have the capacity to measure 10–15 spectral bands simultaneously together with a variety of scatter signals. With modern computers it is possible to perform complex multiparametric analyses virtually instantaneously, allowing time to make sorting decisions after measurements are made. The result of this technology is that it is now possible to generate clinical diagnostic information rapidly from complex heterogeneous mixtures of samples such as human blood and to perform this in real time.