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Clinical Pathology Laboratory - Sample Submission
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Samples for Hematology
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| In general, hematology testing is performed on EDTA- (lavender top
tube) anticoagulated blood. This is the only type of anticoagulant
that can be assayed with our hematology analyzer, therefore all hematology
tests performed with this analyzer (routine hemograms, red and white
cell counts, etc) will only be done from EDTA tubes. Heparin (green
top tube) is not recommended as an anticoagulant for cell counts,
because the cells clump in heparin, invalidating counts. Citrate (blue
top tube) is not recommended due to the dilution of the blood by the
liquid citrate. These guidelines should be followed for collecting
blood for hematology tests: |
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A full EDTA tube should be submitted. Partially filled EDTA tubes
affect the cells because EDTA is hypertonic (e.g. echinocytes will
form in underfilled EDTA tubes). EDTA tubes should be more than half
full. |
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Ensure that the blood is mixed promptly with the EDTA to avoid sample
clotting. This is especially pertinent with microtainers. This should
be done by rolling the tube between your palms or gentle inversion
several times. Do not shake the tube!! |
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Microtainers should be avoided at all costs. There is no excuse for
submitting a microtainer from a large dog. However, if only a small
amount of blood can be collected, e.g. from a young dog or cat, or
very sick animals in which multiple, sequential samples are going
to be collected, the blood should be collected into a microtainer.
The microtainer should be full. Full microtainers are required, because
if we have too little blood, we may not be able to perform other tests
that may be required, e.g. diluting the sample, checking counts etc. |
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The tubes should be labeled with the patient identification and owner
name at the minimum. A request form with pertinent history details
should be submitted concurrently with the sample, e.g. dog administered
oxyglobin. |
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If there is going to be a delay between sample collection and submission,
always make 2-3 blood smears from the sample and submit with the EDTA
tube (see mailed in samples below). |
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See below for advice on non-mammalian hemograms. |
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An example of a correctly filled out CUHA hematology request form
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| Some hematology samples, e.g. packed cell volume and total protein
by refractometer, can be performed on heparin or citrate anticoagulants.We
can also perform cell counts on these anticoagulants, however this
will only be done on specific research samples or on individual patients,
after consultation with the Clinical pathologist on duty. In these
cases, the Advia will not be used for counts; instead we will use
bench methods, including the impedance-based Coulter Z2 for white
and red cell counts and a hemocytometer for manual platelet counts.
Note that for a fecal occult blood, we need feces, not blood! |
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Mailed in samples or samples collected after hours |
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| When there is going to be a delay between sample collection and
submission, e.g. samples shipped to the laboratory or collected after
hours, always make 2-3 peripheral blood smears as shown in the image
below. |
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Illustration on how to make a peripheral blood smear
(wedge smear). A: A drop of blood is placed at one end of the
smear. The drop should not be too large, otherwise the smear will
be too long. Place a spreader slide in front of the drop. B:
Draw back the spreader slide until the edge contacts the drop. The
drop will spread along the edge of the slide. C: Push the slide
forward in a smooth motion, maintaining even contact between the 2
slides. D: Keep moving the spreader slide forward until it
comes off the smear slide. A feathered edge should be produced. This
should be at least 1 cm away from the edge of the slide.
When making a blood smear, the following are important:
1. The size of the drop: If the drop is too large, the smear
will be too long.
2. Speed: The speed at which the spreader slide is moved is
very important. If you move it too fast, the smear is too short and
all the cells are at the feathered edge. If you go too slow, the smear
is too long (lacks a feathered edge).
3. Angle of the spreader slide: The angle determines the length
of the smear. An angle of approximately 30° is optimal. If you
use a larger angle (45°), the smear is very short. If you use
a lower angle, the smear will be too long.
From Cowell and Tyler, Diagnostic Cytology of the Dog and Cat |
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| Smears should be submitted, unfixed, unstained, with the EDTA blood
for any hemogram or test involving counts or blood smear examination.
We do not charge any extra for these blood smears, and we always (provided
smear quality is sufficient) do our blood smear examination from the
submitted smears. This is because changes occur in cells when they
are stored for more than a few hours. Platelets begin to clump, white
cells become pyknotic and undergo nuclear swelling so that many neutrophils
look like bands when they actually are not. The red cells may lyse.
Red cells also consistently swell in vitro, such that old samples
(usually > 24 hours) have macrocytic hypochromic red blood cells. |
Note that EDTA blood should be kept refrigerated until submission
or mailing and should be mailed on a cold pack, but should be
kept out of direct contact with the pack (insert paper towels
between the blood and the icepack). Direct contact may cause
freezing of red cells, with subsequent hemolysis. Furthermore,
blood smears should not be refrigerated (causes cell lysis)
or exposed to formalin (alters staining characteristics).
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Clotted samples |
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| If blood has clotted in the EDTA tube, the sample will not be analyzed.
Clotting affects the Advia adversely and also invalidates cell counts
in an unpredictable fashion. We will make every effort to notify the
clinician/technician/student that a sample has clotted so that a new
sample can be drawn from that patient. Furthermore, as soon as we
know the sample is clotted, the test is cancelled in the computer.
For samples submitted through the Diagnostic Laboratory, we cancel
hemograms or tests involving counts if the sample is clotted. However,
if a blood smear is provided with the sample, we will add on a blood
smear examination, which will provide valuable information. |
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Non-mammalian samples |
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| Only small amounts of blood can be collected from these patients,
necessitating the use of microtainer tubes. Similar to mammals, EDTA
is the preferred anticoagulant for non-mammalian hematology. However,
there are certain species of birds, e.g. cranes, and reptiles, e.g.
turtles, whose blood hemolyzes on contact with EDTA. This hemolysis
invalidates the PCV and affects assessment of red blood cell morphology
during blood smear examination. For these species, blood can be collected
directly from the needle into a eosinopette for WBC counts, into a
heparinized microhematocrit tube for measurement of PCV and fresh
blood smears can be made directly. This can be quite complicated and
time-consuming, hence an alternative is to collect the blood into
citrate. However, the correct citrate to blood ratio must be maintained,
i.e. 1 part citrate to 9 parts blood. Ideally, the citrate should
be placed into the syringe and the appropriate volume of blood withdrawn
directly into anticoagulant. For example, to collect 1 ml blood, 0.1
ml citrate is placed into a syringe and 0.9 ml of blood is taken from
the patient (collect blood up to the 1 ml mark). If less blood is
collected, you will have to resample, hence make sure you can obtain
the correct amount of blood. We require at least 500 µL of blood
for performing a hemogram, hence you can collect only this amount
of blood, which is achievable in most non-mammalian patients. The
correct amount of citrate to blood must be maintained because citrate
dilutes the blood; this dilution must be corrected for when evaluating
the hemogram (i.e. each value should be multiplied by 1.1 for a 1:9
citrate:blood ratio). We do not make this correction in our reports.
Heparin is not recommended as an anticoagulant because leukocytes
and thrombocytes clump, invalidating WBC counts and differential cell
counts. |
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