PART TWO
Diagnostic Cytology
Even a few milliliters of blood drawn up into the syringe
barrel can result in the rapid formation of a clot. Clotting of
blood greatly interferes with good smear preparation. Whatever
cellular material may be present becomes entrapped within
the clotted blood. The cells cannot be spread satisfactorily on
the slide, often destroying any useful diagnostic pattern to the
smear. The presence of the blood with entrapped cells also
results in heavy and uneven staining, making cells appear much
more hyperchromatic than they would be if spread evenly.
Samples for cell blocks may be handled by a variety of
methods: (1) simple centrifugation and the addition of 10%
neutral buffered formalin to the resulting cell button, (2) addi-
tion of thromboplastin or bacterial agar to the cell button, or
(3) the commercially available cytoblock system. The author
has found the cytoblock system to be most satisfactory (see
Appendix). With any cell-block method, it is important to
work closely with the histotechnologist so that the blocks are
trimmed carefully to cut ribbons of thin sections of the block.
Some of these histologic slides should be stained initially
(e.g. slides that are odd numbered), while saving others to be
stained later if needed or for the application of special stains.
Cell-block sections are also more consistent for the applica-
tion of immunohistochemistry, with the exception of cases
of lymphoproliferative diseases, for which cytospins provide
good consistent preparations if enough cells are obtained from
the FNA.
The tip design of the Franseen-style needle seems to result in
the procurement of microcores from the aspiration procedure. It
is usually necessary to dislodge these very small cores by insert-
ing the stylus into the needle, then pushing them out onto a
plane glass slide. Do not attempt to smear these small cores.
That will only result in distortion of the cells and poor qual-
ity material from which to make an interpretation. To prepare
some smears, at least in an effort to provide a provisional inter-
pretation, gently roll the microcore over a small area of the slide.
Then the core should be placed very quickly in suitable fixative,
10% neutral buffered formalin, and processed as a small biopsy.
Because these tissue cores may be quite slender, it is equally
imperative to work closely with a knowledgeable histotech-
nologist to obtain good tissue sections and not destroy much
of the tissue when trimming the paraffin block. Some of these
microcores can be difficult to see when embedded in a paraf-
fin block. Use of a stain, such as mercurochrome, may help the
histotechnologist see them more clearly within the block. These
microcores require even more skill in histology than handling
the traditional somewhat larger needle core biopsies of liver or
kidney.
Fixatives and Stains
Smears that are air-dried are stained by a Romanowsky method
or one of a number of variations. The author prefers the com-
mercially available Diff-Quik stain. This stain is a three-step
process composed of methyl alcohol, which is the fixative, fol-
lowed by eosin Y and then azure A. Smears spray-fixed with one
of a number of commercial products available or wet-fixed in
95% ethyl, methyl, or isopropyl alcohol are stained with Papan-
icolaou stain or its modifications. As noted for the rapid Papani-
colaou stain all smears may be air-dried.46 When using spray
fixatives, it is important to allow those smears to dry at least
1 hour prior to staining.
Smears to be stained by a rapid hematoxylin and eosin benefit
from immediate fixation by immersion for a few seconds in
equal parts of 50% ethyl alcohol and 10% neutral buffered for-
malin. This brief fixation also improves staining of routine fro-
zen sections. When staining with a quick hematoxylin and eosin
method, in the rinsing steps use hot (tap, not boiling) water.
This improves the overall quality of the slides and sharpens cell
detail.
Cytospin preparations for immunohistochemistry are pre-
pared by rinsing either a separate aspirate from the lesion or a
portion of the aspiration biopsy into balanced salt solution. A
separate aspirate for cytospins is preferable. Cytospins may be
either air-dried or fixed in 95% ethyl alcohol. The author prefers
air-drying. Direct smears or cytospins either air-dried or fixed
to be used for immunohistochemistry may be stored in a deep
freezer for up to 4 weeks. Aspiration samples either fixed or deep
frozen are also suitable for molecular diagnostic methods and
may be stored as described above.
Both personal preference and experience seem to dictate
which stain or stains will be used on aspiration biopsy smears.
Cytopathologists with an orientation toward surgical pathol-
ogy may prefer a rapid hematoxylin and eosin stain. Those with
experience in cytopathology most often prefer the Papanicolaou
stain. Individuals influenced by a hematologic background or
trained in the aspiration biopsy clinic at the Karolinska or in
other Scandinavian countries seem to like the Romanowsky
stains, May-Grunwald-Giemsa, straight Giemsa Wright's stain,
or, in the United States, Diff-Quik.80,81 The basic stain discov-
ered by Romanowsky is azure A. This compound is unstable and
is continually oxidized to azure B, which is the actual staining
substance. This type of stain results in a metachromasia of any
stromal elements present with epithelial or other types of cells
seen in contrast to that stroma. In addition to this sharply dis-
tinguishing metachromasia, the major advantage of this stain
is its rapidity, which can lead to an immediate interpretation
following the aspiration biopsy. The quality of the aspirate and
the smears can also be checked. Repeat aspiration biopsies, if
necessary, can be obtained for both diagnosis and special stud-
ies as required from the preliminary evaluation.
The Diff-Quik stain is a three-step procedure. It takes less
than 20 seconds to obtain a stained aspiration smear with this
stain. Table 20.1 is a general comparison of the properties of
air-dried versus wet-fixed smears. Table 20.2 presents the fea-
tures emphasized by the Romanowsky stains compared with the
conventional Papanicolaou stain as outlined originally by Orell
and colleagues.80
It has been the author's experience that the conventional
Papanicolaou stain results in some significant loss of cells from
smears. Part of this occurs during fixation, even with spray
fixatives, and additional cells are lost during the staining proce-
dure. In 1995 a very rapid Papanicolaou stain was developed and
reported by Yang.46 For this staining method, aspiration biopsy
smears are allowed to air-dry. The smears are then rehydrated
with normal saline as the first step of the staining procedure. Next
is fixation by alcoholic formalin, a mixture that is 65% ethanol
and 4% formalin. Details of this staining method are provided
in the Appendix. Total fixation and staining time is usually only
90 seconds. It has been helpful in some cases to have the rapid
Papanicolaou-stained smears to compare cytologic features
with air-dried Diff-Quik-stained smears (Figs. 20.3 and 20.4).
Problem cases may be more quickly resolved. An added advan-
tage is that clinicians performing their own aspiration biopsies
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