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Clinical Pathology Laboratory - Sample Submission
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Samples for Cytology
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| All cytology requests must come with a completed request form (as
outlined in the introduction) and a complete and relevant history.
Give us all the information you have available, including nature of
the lesion aspirated, ultrasonographic or radiologic findings, differential
diagnoses. For example, for a pleural effusion, it is not enough to
tell us that the animal has a pleural effusion, tell us if you have
seen radiographic or ultrasonographic evidence of thoracic masses
and what their location is. This is especially important for mass
or tissue aspirates. Tell us what the nature of the mass is, if it
involves bone or joints, how large it is, etc. All this information
is vital to us when interpreting the cytologic results. |
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This is a representative image of a correctly filled out cytology
request form. Notice the provided history details. |
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Smears |
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| For all samples (e.g. aspirates from masses) submitted as smears,
do the following: |
Use clean glass slides with frosted ends.-
Place the aspirate near the frosted end such that the majority
of the aspirate is in the middle 2/3 of the slide. Our slide stainer
cannot stain the edges of the slides.
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Make gentle squash smears (see images below). Avoid making "splat"
smears (spraying the aspirate on the slide without any kind of
smearing). These are sub-optimal because they are very thick,
they dry slowly and the cells do not spread well. This markedly
hinders evaluation.
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Rapidly air-dry the slides. Blowing on the back of the slide with
a hair-dryer is best.
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Label the slide with the patient ID, date and owner name in pencil.
If more than one site is aspirated, e.g. submandibular and popliteal
lymph nodes, label each slide as to which site they represent.
This is extremely important!! If the slides are not identified
by site, and the cytologic results are different, we cannot tell
which site corresponds to which cytology.
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Submit promptly to the laboratory. Do not stain, oil or fix the
slides in anything. If the slides are stained and/or oiled, do
not coverslip, in case we need to restain them (as is often the
case)!!
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Ensure that the smears do not get moist (do not refrigerate) or
exposed to formalin at all!
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Fill out a request form with the appropriate patient information,
test requests and history.
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These images illustrate how to prepare a squash smear. A:
A small drop of fluid (or aspirate; in this case it is tracheal
wash fluid) is placed near the frosted end of the slide. B:
A spreader slide is placed over the drop and the drop is allowed
to spread between the 2 slides. C: The smears are gently
pulled apart (principally by moving the top spreader slide). Minimal
pressure is used during this procedure. D: Once the smear
has been made, it is rapidly air-dried, yielding the completed product.
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Centesis fluids (peritoneal, pericardial, pleural), joint fluids,
bronchoalveolar lavage fluid (with counts) |
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| For these fluids, we provide cell counts (total nucleated, red cell
counts), total protein by refractometer (excluding BALs), visual parameters
(eg. color, turbidity), and cytology smear evaluation. You only need
to check off the request for the specific fluid on the request form.
If you check off total protein, this will be an additional test (at
extra charge) from our chemistry analyzer. This request is not indicated
with these fluids. For these fluids, do the following: |
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Submit the fluid in EDTA (lavender-top tube). We perform all the
above evaluations from this fluid.
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Fluid should also be submitted in a red-top tube under the following
circumstances:
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If a need for culture is anticipated. Check off "culture
if" indicated and attach a filled out diagnostic laboratory
form.
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If any chemistry tests are to be performed from the fluid,
e.g. BUN, creatinine, triglycerides.
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If the fluid is very bloody. If the fluid clots in the tube,
this suggests blood contamination of the sample.
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If only a very small sample of fluid is aspirated (e.g. some joint
aspirates) that is too small to place in an EDTA tube, make some
direct smears of the fluid and rapidly air-dry the slides.
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If there is to be a delay between sample collection and submission
to the laboratory, refer to the delay section on this page.
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Submit promptly to the laboratory.
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Fill out a request form with the appropriate patient information,
test requests and history.
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CSF samples |
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| For CSFs, follow the same procedures as listed above for centesis
fluids. However, CSF is unstable and samples should be submitted promptly
after collection (i.e. ASAP)!! |
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Bone marrow aspirates |
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| For CUHA patients, bone marrow aspirates must be organized after
consultation with the Clinical Pathologist on duty. Bone marrow is
aspirated directly into a syringe containing anticoagulant and brought
promptly to Clin Path. Do not delay bringing the sample to Clin. Path.
as it may clot and morphology deteriorates rapidly. The Clinical Pathologist
will make smears directly from the aspirate. Always submit peripheral
blood in an EDTA (lavender-top tube) on the same day the marrow was
aspirated for a hemogram.
For Diagnostic Laboratory patients, please click here for more
information on how to collect a bone marrow
aspirate. We require the following with each bone marrow aspirate
submission:
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A minimum of 2 unstained, unoiled, air-dried (unfixed) bone marrow
aspirate smears. If cytochemistry is required, we need a minimum
of 4 unstained, unoiled, air-dried smears.
Always submit complete history details, including hemogram results
obtained from the same day as or the day before the bone marrow
aspirate.
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We prefer to examine peripheral blood ourselves with all bone
marrow aspirate submissions. There have been many occasions on
which we have detected something diagnostic in peripheral blood
smears, not specified in the hemogram report provided to us. Submit
either:
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Peripheral blood in EDTA (lavender-top tube) and 2 unstained,
unoiled, air-dried freshly made blood smears for a full hemogram
(preferred), OR
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2-3 freshly made air-dried, unstained smears of peripheral
blood (for blood smear examination).
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Urine cytology |
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| Submit urine in a red-top or sterile container. Cells in urine deteriorate
rapidly with time, therefore always submit 2-3 sediment smears concurrently
with the fluid. These smears should be rapidly air-dried, unfixed
and unstained. You can also make line smears of the sediment, the
technique of which is illustrated below. Line smears tend to better
preserve the morphology of cells in urine and concentrates the cells
in the "line" created. This is especially useful for fluid
samples (not only urine) that are poorly cellular. With these smears,
it is important to ensure the "line" is at least 1 cm from
the edge of the slide, otherwise it will not be stained in our automated
stainer. |
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Illustration on how to make a line smear. A: A drop of fluid
is placed near one end of the slide. B: The spreader slide
is drawn back to make contact with the drop which then spreads along
the edge of the slide. C: The smear is drawn forward, similar
to making a blood smear. D: Before you create an edge to the
smear, stop and lift the slide up, leaving a line of fluid. This line
should be at least 1 cm from the edge of the slide. The slide with
the smear can then be tilted gently to allow the fluid to run slightly
back towards the starting drop (this ensures the "line"
is not too thick).
From Cowell and Tyler, Diagnostic Cytology of the Dog and Cat |
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Tracheal wash, BALs (without counts), other fluids (fluids from cystic
masses, nasal flushes, bile etc) |
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| We do not provide cell counts, total protein by refactometer or
visual parameters from these fluids. All we perform is a cytologic
evaluation. These should be submitted in an EDTA (lavender-top) tube
or as rapidly air-dried, unstained, unfixed smears. As these smears
are often very thick and dry slowly, rapid air-drying is essential
to preserve cell detail. We always prefer to make our own slides so
if fluid is aspirated in sufficient quantities, always submit it in
an EDTA tube. If a need for culture is anticipated, a small amount
should be placed in a red-top tube as well. As with all cytology submissions,
provide an appropriate history. |
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Buffy coats |
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| We prefer to make our own buffy coat smears. For this test, peripheral
blood should be submitted in an EDTA (lavender-top) tube. |
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Delay in sample submission (after hour samples, Diagnostic Laboratory
samples) |
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Smears: These should be kept out of light and should not
be stained or oiled. If they are stained and/or oiled, do not
coverslip, in case we need to restain them!! Smears should be
kept dry and out of any kind of contact with formalin! Formalin
fumes affect the staining quality of the smear (everything is
green!), which hinders interpretation.
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Fluids: Fluids should be submitted in EDTA or non-anticoagulant
tubes, as described above. Centesis fluids are generally stable
for up to 24 hours after collection, if refrigerated. Some changes
will occur in vitro, such as phagocytosis of erythrocytes and
bacteria. This complicates result interpretation, so freshly made
smears, that are unfixed, rapidly air-dried and unstained, should
be provided (at least 2-3) with the fluid when sample submission
is delayed. Smears can be made from either concentrated fluid
(sediment smears) or unconcentrated fluid (direct smears). If
spinning down the fluid to make sediment smears, ensure a portion
of the fluid is left untouched for us to make our own smears and
do cell counts etc!! Please tell us what type of smears you have
made (direct or sediment).
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CSFs: If CSF samples are collected out of hours, they should
be refrigerated until analysis the next day. However, both white
cell counts and differential cell counts are affected by storage.
Ideally, to preserve cell morphology and optimize white counts
in stored samples, an equal volume of patient serum (non-hemolyzed)
can be added to the CSF after collection, if the sample is going
to be stored > 12 hours. However, a separate aliquot (to which
no serum has been added) must be maintained (at least 500 µL
or 1/2 ml) for measuring protein (which will obviously be affected
by addition of the serum).
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Bone marrows: These are not usually collected after hours.
Note that for animals that are recently deceased, it will be impossible
to aspirate marrow as soon as the animal is dead. A sample of
marrow will need to be taken directly from the marrow cavity and
gentle squash smears made as soon as possible. Marrow deteriorates
very rapidly after death and morphologic assessment can be affected
as soon as 30 minutes after death. The rest of the marrow can
be placed in formalin (keep out of contact with any smears made!!)
for histologic assessment (however, this is not as good as cytologic
evaluation).
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