Flow cytometry

Flow cytometry (FC) is a "technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake."

Method

Flow cytometry characterizes the counts and size distribution of particles, such as blood cells, in a fluid. The original technique passed cells through pores in a membrane and measured the current produced; this was the Coulter Counter of 1953. Prior to this invention, blood cell counting was a laborious and variable manual procedure. In current laboratory medicine, it primarily is a laser-based fluorescence detection method used to characterize cell antigens on large numbers of hematopoetic or lymphoid cells in suspension typically derived from peripheral blood, bone marrow aspirate or lymph node tissue.

Laser-based flow cytometry instruments use a thin laminar flow of fluid to direct thousands of cells from a small diameter nozzle through a thin beam of laser light of a certain wavelength. The cells are categorized by size forward light scatter, cellular complexity or granularity side scatter. They may also measure the fluorescence of antigen-coupled dyes with which the cells have been treated, as with fluorescence intensity for each 'CD' clusters of differentiation marker. The antigen distribution for thousands of cells is printed in graphical histograms which a pathologist interprets individually and collectively to determine the phenotype of dominant cell populations in leukemias and lymphomas of various types. More recently, flow cytometery is used to classify and sort various nanoparticles and their complexes with proteins, antibodies, or aptamers.

Current advances

Today's flow cytometers are considerably more advanced than the simple cell sorters of early times. For example, some are equiped with a dozen or more, even 18, detectors that selectively detect different fluorescent dyes. Although many dozens of dyes are available, some of the more popularly used dyes include Cy3, Cy3.5, Cy5, Cy5.5, flourescein and FITC. When using appropriately dye-labeled antibodies or aptamers to selectively bind to a subset of particles or cells, a population of particles can be separated and quantified. Typical high end flow cytometers can sort 100,000 particles in about a minute. By using very small electric fields, the cytometers can deflect the flowing particles into a number of different particle containers, thus sorting them, based on the relative side scattering versus the forward scattering. When detecting labeled binding agents on the particles, the ratio of side scattering to forward scattering is used to normalize for differential particle sizes. Although dyes typically have a narrow maximum emission spectrum, they always emit at other lower frequencies, which necessitates that the arrangement of detectors must be optimized to reduce cross-talk between dyes, such as between an orange dye and a red dye.