Flow cytometry for the diagnosis of primary immunodeficiencies
- James Verbsky, MD, PhD
James Verbsky, MD, PhD
- Associate Professor of Pediatrics and Microbiology and Molecular Genetics
- Medical College of Wisconsin
- John M Routes, MD
John M Routes, MD
- Professor of Pediatrics, Medicine, Microbiology and Molecular Genetics
- Department of Pediatrics
- Children's Hospital of Wisconsin
- Medical College of Wisconsin
Flow cytometry is a powerful technique for the measurement of multiple characteristics of individual cells within heterogeneous populations. This topic review gives an overview of the technical aspects of flow cytometry and highlights some of its uses in the diagnosis of primary immunodeficiencies (PIDs). 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 (through which cells flow), 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.
Theoretically, any biologic sample can be analyzed by flow cytometry, although peripheral blood is the most common sample analyzed. Depending upon the specific assay, whole blood may be used, or peripheral blood mononuclear cells (PBMCs) may be isolated and used for analysis. Bone marrow samples are used during the workup of suspected leukemia. Other biologic sources have been used for flow cytometry, such as cerebral spinal fluid or bronchoalveolar lavage, although the lack of published normals from these biologic sites can make interpretation difficult.
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, flow cytometers currently in use may have as many as five lasers of different wavelengths. Each laser may be paired with three to four different filters, allowing for an analysis of up to 20 different proteins simultaneously.
Data collection and analysis — Each cell is analyzed by the following parameters as the cell suspension passes through the flow cytometer:
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- TECHNICAL ASPECTS
- Data collection and analysis
- DIAGNOSIS OF PRIMARY IMMUNODEFICIENCIES
- Defects in lymphocyte numbers
- - T cells
- - Regulatory T cells
- - Follicular helper T cells
- - B cells
- Defects in T cell function
- - Hyperimmunoglobulin M syndrome
- - Autoimmune lymphoproliferative syndrome
- - Hyperimmunoglobulin E syndrome (HIES)
- - X-linked lymphoproliferative syndrome, types 1 and 2
- - Wiskott-Aldrich syndrome
- Defects in B cell function
- - Common variable immunodeficiency
- Cytotoxic lymphocyte defects
- - Cytotoxicity assays
- - Expression of cytotoxic molecules and testing for degranulation
- Innate immune system defects
- - Leukocyte adhesion deficiencies
- - Chronic granulomatous disease
- - Mendelian susceptibility to mycobacterial diseases
- - Toll-like receptor defects
- - Gain-of-function mutations in immune deficiency/immune dysregulation syndromes