PART TWO
Diagnostic Cytology
Techniques
Collection and Cytologic Preparation
The technique for aspiration biopsy of lymph nodes, both super-
ficial and deep, is similar to that of other organs. Lymph nodes
in every location within reach of the needle may be examined,
although the head and neck region is preferentially the most
frequently sampled.42 Computed tomographic-guided percuta-
neous FNA is a relatively noninvasive technique for sampling
deep-seated lymph nodes in the mediastinum, abdomen, pelvis,
and retroperitoneum. Flexible transbronchial aspiration may be
used to sample mediastinal lymph nodes, and more recently,
sampling of lymph nodes above and below the diaphragm by
means of endoscopic ultrasound-guided FNA has been proven
to be a safe and effective modality as well.43-46
On-site adequacy reading by a cytologist is an important step,
particularly for deep-seated lymph nodes, to determine whether
the sample is representative and adequate for diagnosis, or if the
FNA needs to be repeated. The quantity and appearance of the
aspirated material gives clues to the likely diagnosis and helps
direct the appropriate triage for ancillary studies such as culture
for acute or granulomatous inflammation and FCM for lymphoid
lesions. On average about three or four passes are performed,
although extra needle passes may be requested if needed.
One method to ensure the availability of both Papanico-
laou- and Romanowsky-stained material is to smear one drop
of aspirated fluid from each pass between two slides, fixing one
in 95% ethanol for Papanicolaou staining in the laboratory and
air drying the mirror image for on-site Diff-Quik (Romanowsky)
staining for adequacy evaluation and triage. Having representa-
tive smears from each pass can also be useful to indicate partial
involvement of the lymph node by a neoplastic process when
cytologic features vary from one pass to the other in an adequate
sample. Well-made smears are critical to analyzing the cell pop-
ulation. For lymphoma, the differential diagnosis is made on
the cell composition (monomorphous or polymorphous) and
cell size (small, medium, large). Depending on the cytologic
evaluation and the patient's clinical history, appropriate ancil-
lary studies can then be ordered.47
In our laboratory, the remaining material from each pass is
rinsed in 40 mL of a cell culture preservative such as RPMI. When
a lymphoid lesion is suspected, approximately 25 mL is sent for
FCM. On average we harvest at least 500 000 to 1 000 000 cells.
The number of cells can be quantified by using an automated
cell counter prior to labeling the cells for FCM. The cell block is
made from the remaining 15 mL and whatever flecks and pieces
of lymphoid tissue can be "fished out" of the liquid media prior
to centrifugation. Depending on the working diagnosis and the
amount of cells available, one may choose to triage all fluid for
cell block as for example when immunohistochemistry studies
are needed. Cell blocks are also a priority when architectural
clues are helpful, such as when confirming transformation of
a known grade 2 follicular lymphoma (FL) to large-cell lym-
phoma (LCL) by identifying sheets of large cells. Other times all
the fluid may need to be triaged for FCM, as for example when
there is insufficient material for both cell block and FCM and
the differential diagnosis is between reactive hyperplasia and a
small-cell NHL. A supplemental core needle biopsy performed
at the time of aspiration can also provide additional architec-
tural information and paraffin-embedded material for immuno-
histochemistry.
Flow Cytometry Overview
Flow cytometry represents the cornerstone in the diagnosis and
the classification of lymphoid lesions. Immunophenotyping
is an absolutely essential component of the diagnosis of lym-
phoma on the basis of FNA.48 FCM is a powerful technique but
well suited for FNA material and other cytologic preparations.
Because the number of cells obtained by FNA is usually small,
the flexibility of FCM allows the proper choice of combinations
of antibodies to examine several antigens (markers) on the cell
surface at the same time. FCM is a very sensitive technique; it can
identify aberrant cells in the frequency of 1:1000 to 1:10 000 in a
complex background. It allows semiquantitative estimates of the
intensity of expressions of each marker on the cell and the per-
centage of positive cells. FCM has generally a rapid turnaround
time (3-4 h in our hands but generally 1-2 working days).
Despite its widespread use, proper use of antibodies and
interpretation of flow cytometric data are challenging not only
for individuals with limited experience but also for flow tech-
nologists and pathologists well versed in this technique.49 FCM
immunophenotyping is a complex and demanding exercise that
requires a good understanding of cell lineages, developmental
pathways, and physiologic changes, as well as a good experience
in hematopathology.50 The actual FCM testing has three major
stages: preanalytic (specimen processing, antibody staining),
analytic (acquiring data on the flow cytometer), and postanalytic
(data analysis and interpretation).
Clinically, a flow cytometer is an instrument that is used to
evaluate the physical and/or clinical characteristics of single
cells as they pass individually through a measuring device and
sensing point in a fluid stream and are illuminated by incident
light.51
The flow cytometer detects the characteristics of the cells
by measuring the amount of incident light reflected by them
by detecting the fluorescence produced by fluorochromes con-
jugated either directly with cell components or conjugated to
antibodies directed against various cell components (Fig. 24.1).
Lineage-associated Markers
Antigenic markers of hematopoietic differentiation are called
CDs (for clusters of differentiation). There are more than 300
CDs, but the ones used in hematopoietic malignancies are rela-
tively limited. Still more limited is the number of CDs that can
be tested in FNAs, because of the limitation of the material. Acti-
vation markers used in hematopoietic malignancies are CD38
and HLA-DR. The noncommitted hematopoietic stem cells
express CD34, and some of them express HLA-DR and CD38
in later stages. Another marker of immature cells is the terminal
deoxynucleotidyl transferase (TdT) present predominately, but
not exclusively, among lymphoid precursors.
Early, the B-cell precursors express cytoplasmic CD22 and
CD79 while surface CD19 and CD10 appear later. With increased
differentiation there is gradual decrease in CD10 and gradual
gain in CD20. CD10, however, appears again in follicular center
cells (FCCs) in the lymph nodes and the spleen. Normal mature
B cells express surface immunoglobulin as well as CD19, CD20,
and CD22.
The lineage-specific marker for T cells is cytoplasmic CD3,
which appears in the earliest T cells as precursors in the
thymus. Early T-cell precursors also express CD2 and CD7.
During thymic maturation, the T-cell precursor undergoes
rearrangements of the T-cell receptor. This process is accom-
panied by the appearance of other T-cell lineage-associated
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