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Methodology Guidelines
to be removed intact. These are most easily removed by
twirling a glass or wooden rod in the specimen. The fibrin
web is wound onto the rod, and in the process, many of the
trapped cells will float free. After winding on the fibrin, it
is pressed gently against the side of the container to squeeze
out as much of the trapped fluid and cells as possible. As
with all cell manipulation techniques, it is important to be
gentle to avoid excessive cell damage.
Cytological samples may contain considerable quantities of
mucus. This a very thick mass that is difficult to dilute or
concentrate, and becomes a rubbery mass on fixation. Such
specimens should be processed prior to fixation. A common
way to break up mucus is to mix with an equal part of
Saccomanno-type fixative (for example Cytospin Collection
Fluid) and immediately blend in a small blender. Three to
five seconds is usually sufficient. The blending procedure
should be done in a biological safety cabinet. After blending,
the sample should be homogenous and non-stringy, and
can be deposited using the Cytospin. Certain chemicals
also react with mucus to produce liquefaction, such as
acetylcysteine (Boccato, 1981). A commercial product of
this type is Mucolexx, which contains not only a mucolytic
agent but Saccomanno-type fixative as well.
Bloody or serosanguineous specimens may contain
so many erythrocytes that examination of cytological
preparations is difficult, and when the specimen is diluted
sufficiently to obtain monolayer preparations, the cells
of interest are difficult to locate. Red blood cells may be
removed by gradient centrifugation or by various lysing
procedures. Lysing techniques are commonly used for
leukocyte counting on cell counters that employ sensing
orifices. CytoRich Red collection fluid completely destroys
erythrocytes. The large amounts of haemoglobin released
from the red cells may interfere with subsequent staining.
It can be removed by several washing steps using a
conventional centrifuge.
Gradient centrifugation is based on a density difference
between red cells and other cells within a specimen.
Commercial gradients are available, and are based on
A78310250ES 5.1ª Edición
Manual de operaciones de la Cytospin 4
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