Page 1279 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPtEr 92 Flow Cytometry 1241

light has a longer wavelength (lower energy) than the wavelength subpopulation is an accurate measurement. Without gating, data
of the excitation beam. The number of commercially available can also be negatively impacted by coexpression of surface antigens
fluorochromes has increased dramatically with the routine use on different cell lineages (e.g., CD4 is found on lymphocytes
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of dye conjugates and instruments with three or more lasers. and monocytes at various densities). In addition, nonspecific
Commonly used fluorochromes in clinical immunophenotyping binding of monoclonal reagents through Fcγ receptors, and the
include the organic dyes fluorescein isothiocyanate (FITC), level of cytophilic human immunoglobulin (Ig), varies between
phycoerythrin (PE), peridin chlorophyll protein (PerCP), and cell types, making appropriate gating crucial to generate valid
allophycocyanin (APC). Conjugations of PE and APC to cyanines data. These techniques are also used to focus the evaluation on
(Cy5, Cy5.5, and Cy7) and Alexa Fluor dyes produce tandem other hematopoietic cells, including monocytes, granulocytes,
dyes with additional emission spectra, based on energy transfer eosinophils, erythrocytes, and platelets.
from one fluorochrome to the second fluorochrome serving as Initial gating to focus on a specific leukocyte population
the source of emitted light. This allows for the simultaneous typically involves the use of two nonfluorescent parameters,
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evaluation of 6–8 colors in most current clinical instruments forward scatter (FSC) and side scatter (SSC) (see Fig. 92.2A).
with only two or three lasers. FSC is a reflection of cellular cross-sectional area (direct relation-
One recent advance in the field was the development of a ship to cell size), whereas SSC is an indication of the cellular
new class of inorganic fluorescent semiconductor nanocrystals, granularity. The combination of these two nonfluorescent
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named quantum dots (QDs). These particles are perfectly suited parameters provides a three-part differential in red blood cell
for polychromatic flow cytometry, as they have broad excitation (RBC)–lysed whole blood that distinguishes between normal
spectra (525–800 nm) and sharp, discrete emission spectra that lymphocytes, monocytes, and granulocytes. As can be seen in
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varies depending on their core size. This means that QDs of Fig. 92.2A, among leukocytes, lymphocytes have the lowest FSC
different sizes (and consequently of different colors) can be excited and SSC, monocytes have higher FSC and SSC, and granulocytes
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by the same laser source, allowing simpler multiplexing. In have the greatest SSC. This method is effective in distinguishing
addition, QDs have high quantum yield, high molar extinction a relatively pure population of lymphocytes under most circum-
coefficients, and extraordinary resistance to photodegradation stances. However, the presence of nucleated RBCs, large platelets,
and chemical degradation. These qualities make them perfectly basophils, or other particulate debris can produce contaminating
suitable for use in biological studies, including intracellular in events (cells) within this “lymphocyte gate.” Furthermore,
vivo imaging, fluorescence resonance energy transfer (FRET) malignant or activated lymphoid cells may not fit into the previ-
analysis, and dynamic imaging of single proteins for longer ously outlined standard light scatter patterns.
periods. 9 A method for confirming the integrity of the light scatter–based
Additional dyes are available for functional studies and include lymphocyte gate uses the directly conjugated monoclonal “gating”
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calcium-sensitive dyes (e.g., fluo-3), glutathione-sensitive dyes reagents anti-CD45 and anti-CD14. These two mAbs more
(e.g., monochlorobimane), and hydrogen peroxide (H 2 O 2 )–respon- accurately identify the three-part differential. Lymphocytes have
sive dyes (e.g., dihydrorhodamine 123 [DHR123]). 10,11 Assessment the highest level of CD45 binding but are negative for CD14;
of DNA content can be performed with dyes that intercalate granulocytes have a lower level of CD45 binding and an intermedi-
double-stranded DNA and RNA, including propidium iodide ate level of CD14 expression; and monocytes have high levels
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(PI) and ethidium bromide. In addition, there are ultraviolet- of both CD45 and CD14 expression (see Fig. 92.2B). Importantly,
excited dyes that are highly specific for DNA, including Hoechst nonleukocytes, including erythrocytes and platelets, are negative
33258 and 4,6-diamidino-2-phenylindole (DAPI); acridine orange for these markers. However, malignant leukocytes that have
is used for simultaneous staining of DNA/RNA. 12 characteristics of early precursor cells often have altered CD45
and/or CD14 expression, which must be recognized when studying
DATA ANALYSIS hematological malignancies. Gating reagents provide a reliable
means of checking the light scatter–based lymphocyte gate for
Gating the frequency of nonlymphocytes within the gate as well as the
extent of lymphocyte exclusion from the gate. Guidelines for an
KEY COnCEPtS acceptable degree of contamination within the lymphocyte gate,
Gating as well as the level of lymphocyte exclusion, are contained in
the US Clinical and Laboratory Standards Institute guideline
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• Method for defining cell population of interest for lymphocyte immunophenotyping. With the expanded use
• Typically performed using forward scatter and side scatter and lineage- of polychromatic flow cytometry, some centers now include
specific antibodies anti-CD45 in every tube to refine the gate and prevent cell
contamination as described above.
Data Display
The proper assessment of specific cell types within a mixture
requires initial identification of lineage-specific cells, an approach KEY COnCEPtS
referred to as gating. In practical terms, immunophenotyping Data Presentation
focused on lymphocytes requires minimizing the nonlymphocytes
included in the evaluation, and this is accomplished by lymphocyte • Fluorescence intensity is plotted versus cell number.
gating. The standard sample for clinical studies is anticoagulated • Flow cytometry can present cumulative data on more than one
whole blood; directing the study to lymphocytes requires eliminat- parameter for each cell.
ing the great majority of nonlymphocytes from the collected • Multicolor data presentation can increase cell subpopulation
resolution.
data such that the expression of a percentage for a specific cell