24
Lymph Nodes: Cytomorphology and Flow Cytometry
Fluidics
Red
diode
laser
635 nm
Laser light
488 nm
Fig. 24.1 Fluidics and electronics of the four-color flow cytometer.
markers (CD5, CD4, CD8). The majority of the mature T cells
are either CD4+ or CD8+. Thymocytes, however, and aberrant
neoplastic T cells may coexpress CD4 and CD8 as well as
immature T-cell markers TdT and CD1a.
Cells of the myeloid lineage may be identified by the expres-
sion of CD13 and CD33, while CD117 and CD34 are expressed
in immature and neoplastic myeloid precursors. The CD14 and
CD11c are markers of monocytes. The most specific granulocytic
marker is cytoplasmic myeloperoxidase.
All the above-discussed hematopoietic cells express the panhe-
matopoietic marker CD45, but it is usually randomly expressed
or negative in immature precursors, and plasma cells are CD45-.
Erythroid precursors also have down-regulated CD45, but they
have a high expression of CD71 (transferrin receptors) and glyco-
phorin A. CD56 is associated with natural killer (NK) lineage
but may be expressed in some myeloid leukemias as well as mul-
tiple myelomas and plasmacytomas. Plasma cells, in addition,
express cytoplasmic light chains, bright CD38, and CD138.
Sam ple Preparation and Processing
In our experience, effusions have to be washed several times,
because many of them have nonspecific proteins attached to the
cells. FNAs submitted in RPMI solutions can be washed only
once or twice and then incubated with antibodies. However, if
the aspirate is bloody then a brief red blood cell lysis may be
indicated. Following the red cell lysis (if needed), the cell yield
is determined by automated or manual cell counts. Cytospins
are made to serve as morphologic control and to ensure that the
critical cells have been retained for analysis. They may also serve
for future cytochemical or molecular genetic studies. The viabil-
ity of cells can be assessed by FCM with 7-immuno-actinomycin
D (7-AAD) or manually. A specimen with low viability can be
tested with 7-AAD dead cell exclusion. However, our usual cut-
off is 80% viability to perform dead cell exclusion.
Test CD Panel Selection and Staining
Samples should be stained as soon as possible, because any
delay would reduce viability and induce clumping. The anti-
body panels chosen should be flexible and tailored according
to the cytomorphology in the Diff-Quik examination and the
patient's history. As will be seen in later discussion, the most
important is to assess for clonality, because B-cell lymphomas
are the most common lymphoid neoplasm. So if we have a
small number of cells, 10 000 to around 200 000, we usually
select one tube that contains CD19, CD20 as B-cell markers
and kappa lambda light chains. If T-cell lymphoma is sus-
pected, then we use CD3, CD4, CD8, CD5, CD2, and CD7
to start with. When the morphology is blastoid, then we use
CD34 as well as the intracellular antigen TdT in addition to
CD3, CD19, and CD10. Permalizing agents are available com-
mercially and can be used according to the manufacturers'
instructions. In some laboratories, DNA ploidy and S phase
are performed and can be useful in estimating the aggressive-
ness of lymphomas.52
Data Acquisition
The light scatter measurement reflects the physical properties
of the cells (cell size or internal complexities). The fluorescence
data give information on the membrane and intracellular mol-
ecules, depending on the antibodies and dyes used for labeling
the cells. The fluorescence signals are collected and amplified by
photodetectors. The data for each of the cells are collected and
stored in a list mode. The immunophenotypic data as well as the
physical characteristics (forward scatter, which reflects the size,
and side scatter, which reflects the cytoplasmic complexity) are
usually given as a dot plot pattern and analyzed. The analysis
can be performed using the isotype control to exclude non-
specific fluorescence.
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