Avian Hemotology
This web page is meant for the
veterinary staff training at Chicago Exotics Animal
Hospital.
Avian Hematology
From
Rosalie
Lane
, AHT
The first step in critically
analyzing a blood smear or cytology is the quality of
the smear and stain.
These are techniques you can learn everyday in
the lab. You
need an even smear with no smudge cells.
The stain should be fresh (changed every 2 to 4
weeks) in order to provide the right cell colors.
Adequate staining techniques should be second
hand to you now. 20-30
dips in each diff quick stain cup are required.
Erythrocytes (Red Blood Cells)
Assessment of RBCs provides
information concerning possible anemia problems and / or
bone marrow response (or lack there of).
In Maturity, these cells are oval in shape, with
a centrally placed nucleus that is comprised of evenly
distributed chromatin clumping and a homogeneous stained
cytoplasm regardless of the stain used.
The majority of the representative RBCs on the
differential should be mature.
There should also be a small percentage of
immature erythrocytes noted when evaluating the blood
picture which denotes the “normal” replenishment of
the RBC line. Immature
erythrocytes seen serving this function are slightly
larger than the mature RBC, having lightly stippled
cytoplasm, and the chromatin clumping within the nucleus
appears more loosely constructed.
Degenerative RBCs appear smaller in size as does
their nucleus, and the chromatin clumping within the
nucleus is much more dense.
Polychromasia (color variation
between RBCs) and anisocytosis (size variation between
RBCs) are reported as follows:
1.
Slight – approx
5 to 10
polychrome cells per oil field
2.
Moderate – approx 10 to 20 polychrome
cells per field
3.
Heavy – approx 40 to 50 % of RBCs are at
least slightly polychrome
An accurate PCV and TP should be
obtained. Using
a standard microhematocrit tube, centrifuge at 12,000 G
for 5 minutes.
Thrombocytes
Avian thrombocytes, although their
appearance is much different from the mammal platelet,
they perform primarily the same function as their
mammalian counterpart.
Other thrombocyte functions have been speculated
on, such as their possessing the ability to phagocytize
foreign material in overwhelming situations, their
response (or reactivity) to outside stimulus, and
lastly, the possibility that they might be able to
replace RBCs in cases of extreme anemias.
The avian thrombocyte is oval in
shape with light blue to colorless cytoplasm containing
approximately two to four small reddish granules usually
at one end of the cell (called the pole).
The nucleus stains very darkly and rarely has
visible clumps. These
cells are usually clumped together on the blood smear in
groups of two, three, four, or more.
These cells can be of help to the lab technician
performing a differential in two ways:
1.
These cells, as stated above, have the
ability to become very reactive to chronic conditions
and stimuli, thus denoting a chronic condition.
2.
By observing the aggregates of
thrombocytes, one may be able to evaluate the value or
quality of the slide submitted and thus take into
consideration when estimates and counts of the
representative cells are performed.
Granulocytes
Heterophils
The avian heterophil which is most
commonly compared to that of the neutrophil in mammals
is the most frequently seen granulocyte in all species.
As with the neutrophil, the heterophils are
instrumental in body defense with incredibly large
numbers available as well as their ability to
phagocytize foreign bodies, bacteria, etc.
The avian Heterophil is generally
circular-shaped with a bilobed nucleus.
This may or may not be very visible through the
rod-shaped granules contained in the colorless to faint
blue cytoplasm. Depending
on the species, there can be quite a variation of rod
sizes and shapes and also whether the cytoplasm is
colorless or faint blue.
Using the recommended stain, the rods should
stain a muddy pink in most species, and the cytoplasm
will stain colorless to uneven variations of pink in
cases of moderate to heavy degranulation.
Many times in cases of high WBC counts,
heterophils with very young granules (blue in color) and
nuclei that are either band cells or cells with
band-like nuclei would indicate that in response to
outside insult or stimulus, the bone marrow is releasing
cells before they mature.
This is a very important sign and one to watch
for and duly note with each differential performed.
Eosinophils
Eosinophils are also circular in
shape and contain granules in their cytoplasm.
The staining characteristics of this cell appear
to hinge on the type of stain used and the species of
bird we’re dealing with.
In our lab, the granules of the cockatiels,
African Greys, lovebirds, and raptors, for example, do
frequently stain a faint pink color.
(This color is not comparable to the heterophil
which is good). With
these species, the cytoplasm will often be abundantly
filled with granules, causing difficulty with
identification. Other
species such as cockatoos, Amazons, macaws, etc., seem
to stain colorless to faint blue, but are recognizable
due to the shapes of their granules.
With both groups the nuclei stain much more
intensely than the heterophil nuclei.
Eosinophils are typically
associated with allergic reactions and parasitic
infections and are rarely seen in high numbers except in
raptor species.
Basophils
The easiest granulocyte to recognize, tend to be
basophils. When
stained, the granules of the basophil become a
reddish-purple color.
The basophilic granules are usually much smaller
than the granules of the eosinophil.
The cytoplasm stains a colorless to a light
purple-red color and the nucleus doesn’t stain as
intensely as the eosinophil.
In cases of extreme toxicity, heterophils have
been found to have very basophilic granules also.
To avoid counting a toxic heterophil as a
basophil, notice the darker staining nucleus of the
basophil and the absence of cytoplasmic rods.
Usually the cell with the very basophilic
granules will be a basophil.
Basophils are seen in high
percentages with finches, canaries, and smaller birds
and are most of the time associated with chronic, long
term illnesses.
Mononuclear
cells
Lymphocytes and monocytes comprise this group of
white blood cells in exotics as well as mammals.
Since these cells do not have granules, they can
be difficult to differentiate, especially if the stain
used does not give good clarity and definition to the
cell nucleus.
Lymphocytes
The most numerous of the white blood cells are
lymphocytes and heterophils. Some species are naturally
lymphocyte and there are some disease states that can
cause a severe lymphocytic increase meaning more lymphs
than hets would be seen.
In some cases especially in Amazons, lymphocyte
counts as high as 85% have been noted.
After treatment, high counts remained from
60-65%. There
are two different types of lymphocytes, small and large.
The small lymphocyte is most commonly seen.
They have the same characteristics with the round
nucleus and chromatin clumps.
The lymphocyte cytoplasm in generally light blue
or blue stippled; the small lymphocyte and the large
lymphocyte have a large nuclear/cytoplasmic ratio.
Sometimes you can see lymphocytes so small that they may
be difficult to identify from the thrombocytes.
Keep in mind the different cytoplasmic colors
(thrombo = usually clear to blue-gray; lymphocyte =
colorless to light blue), the small granules in the pole
of the thrombocyte, and the characteristic chromatin
clumping in the nucleus of the lymphocyte.
Lymphocytes can become so reactive that the
cytoplasm have faint pink tinges, cytoplasmic blebbing
can occur on the borders of the cell, and less chromatin
clumping is noted in the nucleus.
When these signs in lymphocytes are present, it
can be harder to identify them comparing them to
monocytes.
Bacterial infection can be correlated to slight
to moderately reactive lymphs where as moderate to
heavily reactive lymphs have been most often associated
with viral conditions including PBFD, neoplasia, such as
lymphoma or lymphosarcoma, as well as other viral
problems.
Monocytes
Most of the time monocytes are the least numerous
and the largest of white blood cells.
They appear to have no consistent shape to the
cytoplasm and are large in size and cytoplasmic nuclear
ratio. The
cytoplasm color ranges from a dull blue-gray (ground
glass appearance) to a blue-gray with some pink tinged
areas. The
monocyte nucleus is not round like the lymphocyte.
It can range from a bean-shape to a very
indefinite shape with a fine, lacy reticular chromatin
pattern. Criteria
used to determine lymphocytes from monocytes are:
1)
Nuclear chromatin clumping (or lack of)
2)
Appearance of the cytoplasm for color,
volume, and consistency.
For years the issue of monocyte vs.
lymphocyte has lived on and until some extensive EM
studies are done or a specialized training technique is
found the issue will be unlikely resolved.
Depending on the species, it can be very
difficult to show or describe one or two examples of
each type of white blood cell.
Heterophils and eosinophils can look very
different.
White Blood Cell Count Determinations
There are several methods for determining the
white blood cell count in birds.
The method chosen is highly dependant on the
method chosen as well as the facility.
Eosinophil
Unopette Method
Actual counts of the avian granuloctyes
(heterophils, eosinophils, and basophils) by means of an
Eosinophil Unopette manufactured by Becton-Dickinson
comprise this method.
A dilution of blood is prepared in the Unopette
containing phloxine dye and is allowed to sit for
approximately 5-10 minutes.
Both chambers of a hemacytometer are then charged
with the solution and the dark pink or red cells
(representative granuloctyes) are counted.
This number is then multiplied by 1.1 (+/- 10%);
then by 16 and the final number is referred to as the
granulocyte count. Since
this method allows only for the granulocytes being
counted, adjustments must be made for the mononuclear
cells (monocytes and lymphocytes) and is accounted for
from the differential; the granulocyte count in divided
by the total number referred to as the Corrected White
Blood Cell Count. This
method (although probably the best actual counting
method for avian blood), has several aspects which
require consideration.
1) Time limitations:
The blood and phloxine dilution should not sit
over ten minutes causing significant amounts of the dye
being taken up by cells other than granulocytes making
an accurate count almost impossible.
Time restrictions can be very limiting as well as
blood clotting in the hematocrit tubes.
Being able to draw blood from the toe nail and
counting it quickly could make this a very useful method
in most instances.
2) Differential Dependence:
This method can also be affected by the
differential count which is used to calculate for the
corrected white blood cell count.
The extent to which this method is affected is
dependant on the technician’s avian hematology
experience.
3)
Size and state of the Bird:
Other conditions affecting to varying degrees the
Accuracy of the test is the size
and state of the patient.
It is this author’s experience that the
durability of the granulocytes is directly proportionate
to the size and/or health (or lack of) of the bird.
Many times the granuloctyes of these two groups
rupture all too easily and will definitely affect the
accuracy of this test.
Given the
right circumstances, there are a few other methods for
counting actual
counts, but are also limited to
time taken collecting the sample due to clotting
factors.
Estimated
White Blood Cell Counts
This
method, for the author, has been found to be the most
consistently reliable.
To begin, simply scan the stained
blood smear on low power (10x) to check the distribution
of the cells and focus on high dry (40x) in an area
found to be evenly distributed. Then count the white
blood cells found in ten evenly distributed fields.
As a general rule, do not use fields where clumps
of white blood cells found in those ten fields.
Divide by 10, thus finding the average number per
field. Multiply
this number by 2,000 and report the number incorporating
a range value. For
example:
Total White Blood Cells Counted in Ten Fields
: 165.
Divided by 10 (# of fields counted)
: 16.5
Multiplied by
2
: 33
.
Estimated White Blood Cell Count w/o Range
: 33,000.
Cases with
a white blood cell count in the 140,000 and up range
were found
primarily with Mycobacteria sp.
Cases. In
the case of the example given above, the range would be
31,000 to 35,000. In
more normal numbers an estimated white blood cell count
of 17,000 would be expressed as 16-18 thousand.
This method has both positive attributes and
limitations associated with it.
1)
Many
times complaints have been raised that using this method
is very
difficult to duplicate between
technicians given slides from the client.
This may be due to a lack of experience on the
technician’s part and/or the quality of the smears
made. It
will take a novice technician a few weeks to a few
months to feel reasonably comfortable with estimating.
However, if the blood smears that are sent are
poor quality, it will take even longer for the novice
technician to learn.
Even the experienced technician will take longer
to read these types of slides and the accuracy may be
affected as well.
2)
There are many advantages to of this method.
First, sample preparation is easily performed
since most veterinarians and staff members routinely
make blood slides. This
is not to say that good quality, equally distributed
blood smears are routinely made but that it is quite
possible for anyone to make good blood smears given
adequate practice. Accuracy
of the results is not affected with the clotting of
blood in the hematocrit tubes as with the previous
methods, thus eliminating possible problems with
transfer of sample to outside laboratories.
So,
practice in lab. Ask lots of questions!
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