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HOW TO
PREPARE AND EXAMINE
BLOOD FILMS
HOW TO
M
any practices have in-house
haematology analysers. However,
these should always be used in
conjunction with a blood film examination
since analysers are not always accurate. In
particular, the white blood cell differential
count is not always reliable nor is the
platelet count always correct, especially in
cats which have relatively small red blood
cells and quite large platelets most
analysers differentiate these cell types by
their size. Furthermore, platelet clumping
also falsely lowers the platelet count.
Blood film examination will detect these
errors and provides a lot of information
which is not available from the analyser. For
white blood cells: a left shift, toxic
neutrophils, reactive lymphocytes and
neoplastic cells can all be identified. When
assessing anaemic patients, examination of
Elizabeth Villiers, clinical
pathologist at Dick White
Referrals describes this
simple and rewarding
in-house diagnostic
technique
red blood cell morphology is vital, both to
distinguish between regenerative and
non-regenerative anaemia and sometimes to
identify the cause of the anaemia (e.g.
spherocytes in immune-mediated haemolytic
anaemia). Finally, by counting platelets seen
on the blood film, the analyser count can be
verified or corrected.
Making a blood film
Polished glass slides with frosted ends are
preferable since pencil can be used for easy
labelling. Slides should be handled by their
edges/ends since grease from fingers can
result in poor smearing. If in doubt the slide
can be wiped clean using a tissue before
use. Blood sample smears should be
prepared soon after taking the blood
sample. The blood should be mixed by
gently inverting the sample several times.
A spreader slide is required to make
the smear: this is narrower than the
smear slide to avoid spreading the cells
over the edge of the slide. Spreader
slides can be made by breaking off a
corner of a normal slide having first
scored it with a blade or diamond
writer. Spreader slides should be
washed in water and dried regularly,
and should be replaced periodically as
the edge can become roughened.
The blood sample is mixed carefully and
then a sample harvested using a
micro haematocrit tube. A drop of
blood is placed on to the slide towards
the opposite end to the frosted area
(Figure 1).
The spreader slide is held between
the thumb and second finger, placing
the index finger on top of the
spreader slide (Figure 2). This helps to
apply even pressure on the spreader
slide when smearing.
The spreader slide is placed at an angle
of about 30 in front of the blood spot
and slid backwards until it comes into
contact with the blood, which then
spreads out along the width of the
spreader slide (Figure 3).
The spreader slide is advanced forwards
smoothly and quickly. The spreader
slide should not be lifted up from the
smear slide until after the feathered
edge has formed. Ideally the smear
should extend approximately
2
/3 of the
length of the slide, and should a have
fairly square feathered edge (Figure 4).
The smear is allowed to dry.
Figure 1 Figure 2 Figure 3 Figure 4
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HOW TO
Canine red blood cells have a pale area in
the centre of the cells (central pallor) which
is not obvious in feline red blood cells,
which are smaller. If the animal is anaemic,
blood film examination helps determine if
the anaemia is regenerative.
Features of regeneration
Polychromasia these are young red
blood cells which stain this purplelilac
colour due to the presence of ribosomes
in the cytoplasm (Figure 10). Occasional
polychromatophils (one every 24
1000X field) are seen in films from
healthy animals. Several per field would
indicate a robust regenerative response.
Anisocytosis with large (young) red
blood cells.
Howell-Jolly bodies these are small
but prominent dark inclusions which are
remnants of nuclear material (Figure 11).
Figure 8: A large platelet clump seen
at the feathered edge of a blood film
from a dog
Feathered
edge
Examination area
Figure 9: Diagrammatic
representation of a blood film
showing the area of the blood film in
which cell examination should be
performed
Figure 5: Trouble-shooting poor quality blood smears
Problem Cause Solution
Smear too long
(feathered edge has
disappeared off the
end)
Spreader slide speed too slow
Low viscosity blood (i.e. anaemia)
More rapid spreader slide speed
Excess blood applied to slide Apply smaller blood spot
Smear too short/
thick
Spreader slide speed too fast
High viscosity blood (i.e. high PCV)
Slower spreader slide speed
Feathered edge
consists
of long streaked
tails (Figure 6a)
Uneven contact of spreader slide
with smear slide
Apply even pressure using index finger on
top of spreader slide
Spreader slide has roughened edge Replace spreader slide
Smear has holes or
gaps (Figure 6b)
Grease on slide
Lipaemia
Clean slide before use
Smear thick at
feathered edge end
of film (Figure 6c)
Blood in front of spreader slide Ensure firm contact between spreader slide
and smear slide when pulling spreader slide
backwards towards blood spot
Stains
Rowmanowsky stains include Wrights-
Giemsa and rapid dunking kits (Diff-Quik
kits). The latter are more than adequate for
in-house use (Figure 7). These kits have a
three-stage staining procedure, which
incorporates a fixative pot (usually 5 dips),
an orangepink dye in the second pot
(usually 3 dips), and a bluepurple dye in the
third pot (usually 6 dips). The stain is stored
in glass jars which should be cleaned
regularly to avoid build up of stain
precipitate. Periodically they should be
scrubbed out using methanol to remove all
stain. Stain containing precipitate can be
filtered or discarded and replaced with
fresh stain. With time, the redorange dye
can be inadvertently carried over into the
bluepurple dye resulting in weak staining.
The only solution to this problem is to
replace the bluepurple dye.
Coverslips
It is helpful to add a coverslip to preserve
the slide and to improve visual acuity,
especially when using the dry 40X lens.
Mounting medium glue such as DPX can be
used to permanently mount the slide. NB if
the glue spreads out beyond the edge of
coverslip great care should be taken to
avoid getting it on the microscope lenses.
Preferably, wait for it to set.
Alternatively, immersion oil can be used.
A drop of the mounting medium/oil is placed
on the slide near the feathered edge and the
coverslip is slowly lowered on to the slide.
Film examination
To ensure all cell lines are examined
properly, a set procedure of blood film
examination should be routinely followed.
Initially, the smear is checked for large
platelet clumps (Figure 8) by examining the
feathered edge at low power (20X). The
smear is then examined at higher power
(40X or 100X) in the thin area in from the
feathered edge, where the cells are evenly
distributed in a monolayer (Figure 9). The
red blood cells, white blood cells and
platelets are examined in turn.
Examination of red cells
This should include an assessment of colour,
size and shape, and a search for inclusions.
PREPARE AND EXAMINE
BLOOD FILMS
Figure 10: Blood film from an
anaemic dog showing marked
polychromasia (arrowed)
Figure 7: A rapid dunking kit. The
stains are stored in clean glass or
plastic pots with lids
Figure 6: Slide e is a good quality
smear
a b c d e
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HOW TO
Figure 12: Spherocytes are smaller
and denser than normal cells and
lack central pallor
PREPARE AND EXAMINE
BLOOD FILMS
Nucleated red blood cells do not
confuse these with lymphocytes.
Nucleated red blood cells have smaller
nuclei and the cytoplasm is a similar
colour to polychromatic red blood cells
(Figure 11).
Evaluation for a possible cause
of anaemia
The blood film can help identify several
causes of anaemia, including immune-
mediated haemolytic anaemia, babesiosis,
feline infectious anaemia, oxidant injury,
microangiopathic haemolytic anaemia and
iron deficiency.
Immune-mediated haemolytic
anaemia
This disease usually leads to circulating
spherocytes. These appear smaller than
normal red blood cells with a darker/denser
cytoplasm lacking central pallor (Figure 12).
Care should be taken to look in the
examination area monolayer and avoid
looking at the tail of the smear where cells
are flattened and lose their normal central
pallor, giving the false impression of
spherocytes. Spherocytes are difficult to
see in cat blood because normal feline red
blood cells have minimal or no central
pallor. NB small numbers of spherocytes
may be seen with other causes of anaemia.
Babesiosis
Babesia canis appear as large pear-shaped
organisms, usually in pairs (Figure 13).
B. gibsoni organisms are much smaller,
circular bodies. These parasites are more
often seen in capillary blood (e.g. from an
ear prick) and most frequently found along
the edges of films. However, these
organisms are not always visualized and
serology or PCR are more sensitive
methods for diagnosis.
Figure 13: B. canis organisms are
light-staining pear-shaped bodies,
usually in pairs
Figure 17: Hypochromic red blood
cells with wide central pallor and a
thin rim of haemoglobin
Feline infectious anaemia
(haemotropic mycoplasma infection)
Provided that samples are obtained during an
episode of parasitaemia, organisms may be
identified using the Rowmanowsky stains.
The organisms stain bluegrey to pale purple
with Rowmanowsky stains and appear as
small cocci singly or in chains. Again PCR is a
more sensitive diagnostic technique.
Oxidant injury
Ingestion of oxidants such as onions or zinc
can lead to the formation of Heinz bodies
or eccentrocytes with resultant anaemia.
Heinz bodies are seen as non-staining round
bodies, usually protruding from the surface
of the cell (Figure 14). Eccentrocytes have a
pale area on one side of the cell which is
devoid of haemoglobin, but with a cell
membrane visible around this pale area
(Figure 15).
Figure 16: Schistocytes are
irregularly shaped red blood cell
fragments (black arrow).
Acanthocytes are roundish but
have several irregular projections
(white arrow)
Figure 14: Heinz bodies are round
non-staining or light-staining bodies
adjacent to the cell membrane or
protruding from it
Figure 11: A Howell-Jolly body
(black arrow) and a nucleated red
blood cell (white arrow) Figure 15: Eccentrocytes have a
clear area on one side of the cell
encased in the cell membrane
Microangiopathic haemolytic anaemia
Red blood cell fragmentation, e.g. due to
vascular neoplasms such as
haemangiosarcoma, results in the formation
of schistocytes and acanthocytes (Figure 16).
Schistocytes are irregular-shaped, often with
elongated fragments and an irregular spiked
outline. Acanthocytes also have irregular
spiky projections but are roundish in shape.
Iron deficiency anaemia
This leads to defective haemoglobin
synthesis, and so red blood cells are very
pale with a wide area of central pallor and a
thin rim of haemoglobin (Figure 17).
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HOW TO
PREPARE AND EXAMINE
BLOOD FILMS
Examination of leucocytes
The morphological examination and
differential count are important to validate
(or refute) the analyser differential count,
to identify a left shift and/or toxic changes
(which are important indicators of
inflammation) and to identify atypical,
possibly neoplastic cells. It is essential to
evaluate a blood film prior to
administration of chemotherapy, rather
than relying on the neutrophil count
produced by the in-house analyser.
A differential white blood cell count is
performed by counting leucocytes at both
the edges and in the middle of the smear in
the examination area because larger cells
tend to be pushed to the edges of the
smear, and smaller cells tend to be more
concentrated in the middle. An example of a
battlement meander method of counting is
shown in Figure 18. The percentage of each
cell type is then multiplied by the total white
blood cell count to determine an absolute
count for each cell type. Cell counters are
useful to speed up this process (Figure 19).
Figure 20: A neutrophil with
segmented nucleus and pale
bluegrey cytoplasm
Figure 21: A band neutrophil with a
U-shaped nucleus with parallel sides
Figure 19: A cell counter used for
performing a differential leucocyte
count. Each cell type is assigned a
button on the counter. A bell is
sounded when 100 cells have been
counted
Figure 23:
A toxic
neutrophil
with darker
blue
(basophilic)
cytoplasm
and small
indistinct
cytoplasmic
granules
Figure 24:
A toxic
neutrophil
with foamy
cytoplasm
Figure 25: A small lymphocyte with
a round nucleus and scant
cytoplasm visible only on one side
of the cell
Figure 18: Battlement meander track
for performing a white blood cell
differential count
Figure 22: A Dhle body (indistinct
bluegrey cytoplasmic inclusion) in
a neutrophil (arrowed)
Neutrophils
Neutrophils have an elongated, segmented
nucleus and light bluegrey cytoplasm
without visible granules (Figure 20). Band
neutrophils should be counted separately.
These have a U-shaped non-lobulated
nucleus with parallel sides (Figure 21).
Shallow indentations <50% of the width of
the nucleus may be present. Bands indicate
acute inflammation.
Toxic changes in neutrophils and bands
are seen in severe inflammation, especially
associated with bacterial infection. Toxic
changes are listed below:
Dhle bodies indistinct bluegrey
cytoplasmic inclusions (Figure 22)
Increased cytoplasmic basophilia (but
not as dark as monocytes) (Figure 23)
Foamy vacuolated cytoplasm (Figure 24)
Cytoplasmic granules (Figure 23)
Giant neutrophils
Nuclear swelling
Doughnut-shaped nuclei.
Lymphocytes
Lymphocytes have a round nucleus with
condensed, smudged chromatin and a
narrow rim of basophilic cytoplasm.
Lymphocytes vary in size but most are
small, only slightly larger than canine red
blood cells (Figure 25). They have sparse
cytoplasm, which is not visible all the way
round the nucleus. Medium-sized
lymphocytes may approach the size of
neutrophils and have more abundant
cytoplasm, often completely encircling the
nucleus (Figure 26). Reactive lymphocytes
are larger still with a nucleus approximately
1.5 times the diameter of a canine red
blood cell and abundant deeply basophilic
cytoplasm, often with a darker tinge at the
periphery. (Figure 27).
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HOW TO
Figure 28: A monocyte with a broad
U-shaped nuceus and basophilic
cytoplasm with clear vacuoles
Figure 29: A canine eosinophil with
many round pink cytoplasmic
granules. (These granules are
rod-shaped in cats)
Figure 30:
A canine
basophil with
dark purplish
cytoplasm
Figure 31:
A feline
basophil
with more
abundant
granules,
which are
greylavender
in colour
Figure 32: Large atypical blast cells
in a peripheral blood sample from a
dog with acute leukaemia. The cells
have large nuclei containing stippled
chromatin and nucleoli. Cytoplasmic
granules are seen in some cells
Figure 33: Platelets are small
circular cells without a nucleus and
with pink grainy cytoplasm
Monocytes
Monocytes are larger than neutrophils and
have abundant sky blue cytoplasm, often
containing clear discrete vacuoles and
sometimes fine pink dust-like granules. The
shape of the nucleus is very variable and can
be round, kidney bean-shaped, lobulated,
U-shaped (Figure 28) or S-shaped.
PREPARE AND EXAMINE
BLOOD FILMS
Figure 26: A medium-sized
lymphocyte with a slightly larger
nucleus and more abundant
cytoplasm
Figure 27: A markedly reactive
lymphocyte with an enlarged
nucleus and more abundant
cytoplasm, which is deeply
basophilic
Eosinophils
Eosinophils are slightly larger than
neutrophils, and are characterised by
numerous prominent pink cytoplasmic
granules (Figure 29).
Basophils
Basophils are rare in blood smears from
normal animals. They are a similar size to
eosinophils and have an elongated ribbon-
like segmented nucleus and variable
numbers of cytoplasmic granules. In dogs
these granules are sparse and dark purple
(Figure 30), in cats they are abundant and
pale lilac, sometimes with a few dark purple
granules (Figure 31).
Examination of platelets
These are small round structures with no
nucleus. They are
1
/4
1
/2 the diameter of red
blood cells with pink cytoplasm and fine
granules. Platelet numbers can be estimated
by counting the number of platelets seen in
a 1000X field (i.e. 10X eye piece and 100X
objective), having first determined that no
platelet clumps are present. 5 fields are
counted and a mean value is calculated. The
normal count is 1030 platelets per 1000X
field (Figure 33). Each platelet per 1000X
field equates to approximately 15 x 10
9
/l.
Thus if 10 platelets are seen per 1000X
field, the platelet count is approximately
10 x 15 = 150 x 10
9
/l. Animals with severe
thrombocytopenia (< 30 x 10
9
/l) have only
03 platelets per field. Large shift platelets
suggest active thrombopoiesis.
Atypical cells
Atypical cells suggest leukaemia or
overspill of lymphoma into the blood,
especially when such cells are present in
large numbers. Features which would
suggest leukaemia include large cells with
large round or convoluted nuclei,
containing coarse or stippled chromatin
and sometimes nucleoli (Figure 32).
Suspicious cases should be reviewed by an
experienced cytopathologist.
Acknowledgements
The Figures in this article have
been reproduced from the BSAVA
Manual of Canine and Feline Clinical
Pathology, 2nd edition.

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