Flow cytometry is a powerful technique for the measurement of multiple characteristics of individual cells within heterogeneous populations. This review gives an overview of the technical aspects of flow cytometry and highlights some of its uses in the diagnosis of primary immunodeficiencies. Each of these immunodeficiencies is discussed in greater detail separately in specific topic reviews.
A basic flow cytometer consists of five main components: a flow cell, a laser, optical components, detectors to amplify signals, and an electronics/computer system. With these five components, the flow cytometer is capable of performing instantaneous measurements by passing thousands of cells per second through a laser beam and capturing the emerging light from each cell as it passes through the interrogation point. Any suspended cell or particle ranging from 0.2 to 150 micrometers in size is suitable for analysis. Analyses to determine cellular characteristics, such as size, granularity, viability, and immunophenotyping, are the most common types of studies done.
Immunophenotyping — Immunophenotyping is a technique used to characterize the makeup of cell populations by detecting cellular protein expression. Immunophenotyping uses an antibody specific for the antigen of interest that is conjugated to a fluorescent compound known as a fluorophore or fluorochrome (figure 1). These fluorescent compounds absorb energy from the laser source causing an electron to be raised to a higher energy level. The excited electron quickly returns to its ground state, emitting the excess energy as a photon of light of a characteristic wavelength that is detected by the flow cytometer. Different fluorochromes are excited by different wavelengths of light and emit light at different wavelengths. Thus, it is possible to simultaneously detect several different antigens on a cell by using lasers of different wavelengths and filters of specific wavelengths to detect the fluorescent emission. As an example, current flow cytometers typically have four lasers of different wavelengths, with each laser paired to three different filters to allow for up to 12 different analyses at once.
Data collection and analysis — Each cell is analyzed by the following parameters as the cell suspension passes through the flow cytometer:
- The intensity of the forward scatter (FSC) of light, which is a reflection of cell size
- The intensity of the side scatter of light (SSC), which is a reflection of cell granularity
- The intensity of light emitted by each fluorochrome, which reflects the level of expression of the antigen of interest