Page 1277 - Clinical Immunology_ Principles and Practice ( PDFDrive )

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Flow Cytometry

Sergio D. Rosenzweig

Flow cytometry has become a standard laboratory tool in the activation and cell-mediated cytotoxicity studies, cell cycle analysis,
evaluation of hematopoietic cells, including the identification apoptosis detection, and multimer technology, focusing on the
of leukocyte populations and subpopulations, a method referred appropriate application of these approaches as well as their
to as immunophenotyping. The clinical application of this technol- limitations.
ogy has been facilitated by the development of instruments and
data analysis systems suitable for routine use in diagnostic labo- INSTRUMENTATION
ratories. In addition, the expanded range of monoclonal antibodies
(mAbs) specific for lymphocyte (and other hematopoietic cell) The basic components of a flow cytometer, as shown in Fig.
surface antigens directly conjugated to a number of different 92.1, include an illumination source, an optical bench, a fluidic
1
fluorescent indicators (fluorochromes) provide an extensive panel system, electronics, and a computer. Briefly, stained cells flow
of reagents that facilitate multicolor (polychromatic) studies. into single file by the fluidic system and are interrogated by one
The clinical needs that pushed this technology date back to or more light sources; these sources generate light signals, which
the emergence of absolute CD4 T-cell counts as a critical measure are directed by the optical system to the photodetectors, which,
for disease assessment and follow-up in managing patients infected in turn, convert light into electronic signals for storage and
with human immunodeficiency virus (HIV). Flow cytometry subsequent analysis. This process is discussed further in the
applied in the monitoring of HIV infection was followed by the section below.
routine application of cell characterization by flow cytometry The fluidic system lies at the heart of the flow cytometer and
in the evaluation of hematologic malignancies and, more recently, consists of isotonic sheath fluid that moves the sample stream
in the study of immunodeficiency disorders and other immune- containing the cells. This is accomplished by injecting the cell
mediated diseases. sample into flowing sheath fluid, establishing a hydrodynamically
Recent advances in instrumentation and fluorochrome focused single-file flow of cells (particles) that move through
chemistry now allow for routine polychromatic flow cytometry the analysis point while maintaining the cell stream in a constant,
2
studies, with concomitant assessment of cell surface markers central location. The centrally focused cell stream ensures that
and intracellular parameters, including intracellular proteins, the illumination of all cells is virtually equivalent. Thus the
phosphoproteins, and cytokines, as well as identification of difference in magnitude of the emission signal(s) generated from
changes linked to cellular activation and apoptosis. Intracellular each cell reflects biological differences between the cells (rather
flow cytometry also can be applied to evaluate cell cycle status than reflecting the variation in the illumination energy if the
(i.e., G 0-G 1, S, G 2 -M) based on DNA staining, which is useful in cells were not tightly focused). The use of hydrodynamic focusing
evaluating tumor cells and assessing the in vitro lymphocyte has the additional advantage of producing little or no change
response to various stimuli. Additionally, evaluation of lymphocyte in cell shape, although it may have an effect on cell orientation.
proliferation can be performed with cell tracking dyes that allow The consistency in maintaining cell shape facilitates distinguishing
quantitation of the rounds of cell division associated with cell “architectural” differences between specific leukocyte types (see
3
activation, and techniques to assess immune cell mediated Gating section). However, this method can generate single-file
cytotoxicity have also been developed. Finally, characterization cell rows with precision only up to a flow rate of 60 to 100 µL/
of antigen-specific T cells following immunization or associated min, which can lead to long acquisition times for the detection
with normal and/or abnormal immune responses in association of very rare events. To overcome this problem, recently introduced
with disease states can be accomplished by using multimer flow cytometry instruments utilize acoustic focusing, which align
technology as well as intracellular cytokine detection following cells through the use of sound waves, allowing sample flow rates
antigen exposure. of up to 1000 µL/min, without loss of signal quality. 4,5
This chapter focuses on the basic concepts of flow cytometry, Illumination in standard clinical instruments is gener-
including instrument characteristics, data management, lym- ated by two or more lasers, each of which provides a specific
phocyte gating, and directed use of test reagents. In addition, it monochromatic light source (e.g., a sapphire laser generates a
provides a brief overview of intracellular protein detection, cell 488-nm-wavelength [blue] beam). Modern lasers are small and
available in multiple wavelengths, including ultraviolet (350 nm),
violet (405 nm), blue (488 nm), green (532 nm), yellow (560 nm),
This work was supported in part by the Intramural Research Program orange (610 nm), and red (633 nm), permitting the simultane-
of the National Institutes of Health Clinical Center. ous use of multiple fluorochromes having different excitation

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