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experimental animals. The assessment of the proportion were recorded according to the following criteria (12): a
of poikilocytosis forms of red blood cells was discussed, few (5%–10%), moderate (11%–30%), and a large num-
as well as morphological and degenerative changes in ber (>30%)
the neutrophils and lymphocytes. An analysis of the ob- 2.3. STATISTICAL DATA PROCESSING
tained leukogram percentage was also performed. Minimum and maximum values, mean and standard
deviation of the results were determined. Statistical sig-
2. MATERIAL AND METHODS nificance of the results was tested using T-test and Fish-
The procedure for obtaining of approval for experi- er’s test.
mental part of the study was described in the previous
paper (1). 12 adult crossbreed dogs, aged 8-10 years, with 3. RESULTS
body weight 10-15 kg, were used in the study. They were The results of the leukogram of the dogs in the first
placed in standard cages with ambient temperature of group, (7th day, Table 1) correspond to the norm-refer-
20-24°C, with a 12 hour light/dark cycle. enced physiological intervals of stray dogs (10), as well
They were divided into two equal groups. Before sur- as to leukogram values of other categories of dogs: dogs
gery, peripheral blood was taken from both groups of in shelters and fostered dogs (15). The exceptions were
dogs to be used as a control. Then a titanium and plastic monocytes; the percentage values measured from the
clip was placed within the neurocranium of each dog in blood smears taken beforehand were significantly higher
the experiment. After seven days, the peripheral blood of (15.33%) than the upper physiological limit (9%) for adult
the first group was analyzed, while the peripheral blood dogs (10). The value of monocytes on the seventh day
of the second group was analyzed after sixty postopera- was twice higher (22.83%) than the results of Khan et al,
tive days. (10). The results of T-test and Fisher’s test did not show
2.1. PREOPERATIVE AND OPERATIVE PROCEDURES statistically significant differences between the mean val-
A few days before the surgery, the dogs provided for ues (p>0.05) of the control results and the results of the
the experiment were clinically healthy. They were sub- first experimental group.
jected to appropriate ecto and endoantihelmintics. Gen- Analyzing the leukogram of the second experimental
eral anesthesia and surgical procedure for the dogs was group (60th day, Table 2), an increase in lymphocytes
carried out according to the protocol described in the (38.80%) in agranulated leukocytes was noticed when
work of Delibegović et al. (1). compared to the same in the first experimental group,
2.2. HEMATOLOGY PROCEDURES and it reaches the upper physiological limit in dogs (10).
Peripheral blood was taken and blood smears were Monocytosis was also noticed in the control of the sec-
made in accordance with the normal laboratory proce- ond experimental group (14.80%), and in the second ex-
dures (11). For each original stained smear, 2000 eryth- perimental group(16.20%).
rocytes and 1000 leukocytes were counted and analyzed, In the first experimental group (7th day), the number
using a Motic Type 102M binocular light microscope, of monocytes had a tendency to decline. The number of
with a magnification of 900 times. neutrophils in the control, as well as in the first experi-
Poikilocytes were defined on the basis of the standard mental group (7th day) was within normal limits, while
morphology (12), while counting was
Parameter
limited to a representative one-tier visu- Minimum Maximum Mean SD
(%)
al fields in which about half of the eryth-
(n=6) control 7 day control 7 thday control 7th day control 7th day
th
rocytes touched, but did not overlap (11,
Lymphocytes 20 16 33 37 26.33 26.17 4.72 8.56
13, 14). The most representative fields of
Monocytes 3 14 24 35 15.33 22.83 7.78. 8.21
vision were stored in electronic form us-
ing Motic Images Plus 2.0.computer-soft- Mature neutr. 49 38 67 68 56.33 50.83 6.31 12.86
ware. Band neutr 0 1 14 15 4.83 6.50 5.46 4.85
The numbers and types of poikilocytes Acidophils 0 0 30 2 5.17 0.50 12.20 0.83
were recorded and expressed as a percent- Basophils 0 0 3 0 1.00 0.00 1.55 0.00
age (%) of the red blood cells. Poikilocy- Table 1. Leukogram (%) of the first group of dogs after implantation of plastic and titanium
tosis was classified semi-quantitatively clips on the 7th postoperative day
according to similar studies, using the
following criteria: non-existent (0%), rare Parameter (%) Minimum Maximum Mean SD
(0.05-0.5%), mild (>0.5-3%), moderate (n=6) 60 th 60 th 60th 60th
control control control control
day day day day
(>3-10%), or expressed (>10%) (11).
Lymphocytes 14 26 32 67 23.20 38.80 6.76 16.36
The leukogram was determined, and the
values were expressed as percentages, af- Monocytes 3 1 29 44 14.80 16.20 10.85. 18.06
ter analysis of 1,000 such cells from each Mature neutr. 37 23 70 73 56.60 43.20 12.62 20.70
animal in the experiment. The assessment Band neutr 1 0 19 11 9.20 3.60 6.57 4.39
of semi-quantitative degenerative changes Acidophils 0 0 9 3 3.40 0.60 3.91 1.34
in leukocytes, such as toxic granulation in Basophils 1 0 2 1 1.40 0.20 0.54 0.45
the cytoplasm of neutrophils, and an in- Table 2. Leukogram (%) of the second group of dogs after implantation of titanium and
creased number of reactive lymphocytes, plastic clips on the 60th postoperative day
4. DISCUSSION
Figure 1. Morphological changes in neutrophils (A and B) and Automated hematology equipment does not detect
Figure 1. Morphological changes in neutrophils (A and B) and lymphocytes (C and D)
lymphocytes (C and D) immature erythroid precursors, orpoikilocytotic forms
Table 3. Atypical leukocytes (%) after implantation of titanium and plastic clips on the 60th of
dogsdifferentiated
(7th day and 60th day),red blood value
the percentage cellsofin the peripheral
echinocytes blood.
was 12%, which represents
postoperative day (Group 2.)
apparent echinocytosis (Chart 1 and Chart 2).
Dog 1 Dog 2 There Dog 3
are obvious differences Dog
in the4 percentage values
Dog 5
of spherocytes Dog
in the 6 blood
control
Dog 1 Dog 2 Dog 3 Dog 4 Dog 5 Dog 6
Cells
Cells typetype
A B A B Bsmears; they amounted to 13% (control of 7th day group), which corresponds to apparent
Degenerat. neutrophils (%) 0 0 0 0
A A
0
B
24,6
B0 A B
0
A
A0 B
0
A
B0 1,6
A B A B A B A B
Atypical lymphocytes (%) spherocytosis. In both experimental groups, spherocytes ranged from 4% to 6%, which
0 10,4 0 6,6 0 0 3,1 0 0 7,0 0 0
Degenerat. neutrophils
A -Control before treatment (%) 0 0 0 0 0
corresponds to24,6 0
moderate spherocytosis 0 1 and Chart
(Chart 0 2). 0 0 1,6
B-Values after 60th day
Atypical lymphocytes (%) 0 10,4 0 6,6 0
Acanthocytes 0 mild to3,1
were 0
moderately abundant, 0 2% to 6%.
from 7,0Dacrocytes
0 (Figure 20B),
anulocytes, schizocytes, elliptocytes, stomatocytes, and meta-cells had a negligible percentage
Table 3. Atypical
Morphological leukocytes
changes (%)cells
in red blood after implantation
were of titanium
noticed; the presence and plastic
of echinocytes clips
occurred on the 60th postoperative day (Group 2.). A -Control before
share, which ranged from 0-2% (Chart 1 and Chart 2), i.e. from rare to mild representation
treatment,
(Figure B-Values
2 A) in afterblood
the peripheral 60th smears
day taken for control in both experimental groups. (11).
In
the control samples of the first experimental group, the presence of echinocytes accounted for
the(Chart
3% results ofrepresents
1), which the second group
the boundary (60mildday),
between showed
and moderate
th
mild while
echinocytosis,
neutropenia.
in the control sampleAcidophils and basophils
of the second experimental were accounted
group echinocytes dormant in what
for 15%,
the second
represents apparentexperimental
echynocytosis (11).group (Table
In the blood smears2). Results
of both of the
experimental groups of
T-test and Fisher’s test also showed no statistically sig-
nificant difference between the mean values (p > 0.05) of
the control results and the results of the second group. 6
In addition, Fisher’s test showed no significant difference
between the results of the first and the second group.
Microscopic analysis revealed atypical neutrophils as
well as atypical lymphocytes in the observed groups, es-
pecially in the second experimental group (Table 3), with
obvious blue granules in the cytoplasm.
Figure 1 shows: A) a neutrophils in the center of the
blue granules distributed throughout most of the cy-
toplasm; B) two neutrophils with blue granules in the
cytoplasm; C) atypical lymphocytes with dark blue cy-
toplasm; D) a lymphocyte with wavy edges of the cyto-
Figure 2. Poikilocytotic forms of erythrocytes
plasm. According to the semi-quantitative assessment Figure 2. Poikilocytotic forms of erythrocytes
of degenerative changes, leukocytes were classified Apart from these, it also cannot identify immature and
in percentages as follows: a few (5% - 10%), moderate toxic changes in neutrophils, atypical lymphocytes and
(11% - 30%), a large number (>30%) (12). Morphologi- leukemic cells. Therefore, it is advisable to analyze blood
cal changes in red blood cells were noticed; the presence smears microscopically to obtain more accurate results 7
of echinocytes occurred (Figure 2 A) in the peripheral of leukogram and resized forms of red blood cells and
blood smears taken for control in both experimental platelets (16). Blood smears can provide insights into any
groups. In the control samples of the first experimen- possible anisocytosis, the presence of abnormal cells or
tal group, the presence of echinocytes accounted for 3% inclusion, as well as the possible presence of blood par-
(Chart 1), which represents the boundary between mild asites (17-19).
and moderate echinocytosis, while in the control sample The presence of monocytosis in the control results
of the second experimental group echinocytes account- in both groups of dogs (Table 1 and Table 2) can be ex-
ed for 15%, what represents apparent echynocytosis (11). plained by the effect of prolonged stress, pain, and limited
In the blood smears of both experimental groups of dogs space during the experiment, and during hospitalization,
(7th day and 60th day), the percentage value of echino- all under the influence of endogenous glucocorticoids,
Chart 1. and Chart 2. Poikilocytotic forms of erythrocytes after implantation of titanium and plastic clips on the 7th and 60th postoperative days
Chart
what 1. and
results Chart other
in, among 2. Poikilocytotic
things, monocytosisforms of erythrocytes
(19). tive changes due after implantation
to the of These
effect of toxins. titanium and
morpho-
Also, a possible reason for the monocytosis in the con- logical abnormalities develop in neutrophils in the bone
trol results is the age of theplastic clips
tested dogs, on amounted
which the 7th andmarrow,60th postoperative
before they reach days
the circulation (12). No de-
to 8-10 years (15, 20). The very apparent monocytosis in generative neutrophils were detected in the first experi-
the experimental dogs in the first group (22.83% -Table mental group. Neutropenia and the observed degenera-
1) and the second group (16.20% -Table 2) is a sign of tive neutrophils in the second experimental group, may
4.infectious
DISCUSSION or chronic inflammatory processes, or tissue be connected to the subsequent development of inflam-
damage (15, 20, 21, 22, 23). matory processes. Morphological changes to neutrophils
The functions of mononuclear phagocytes, amongst were found in the blood smears in two dogs (Table 3),
Automated
other things, arehematology
the secretion of equipment does not
mediators and regula- whichdetect
indicatesimmature
the necessity erythroid
for the conduct precursors,
of addi-
tors of the inflammatory response, interactions with an- tional, more complex research, with the use of electron
orpoikilocytotic forms of differentiated red blood cells in the peripheral blood. Apart from
tigens and lymphocytes, and removal of the tissue debris microscopy.
through phagocytosis. In order to perform these func- The lymphocytes of the second experimental group
these, it also cannot identify immature and toxic changes in neutrophils, atypical lymphocytes
tions, they are arranged in all the places where they could were on the upper physiological limit (Table 2). Also,
and
comeleukemic
into contactcells. Therefore,
with infectious it isagents
and other advisable
(21). to analyze
atypical bloodwere
lymphocytes smears microscopically
only determined in the sec-to
Dogs have a neutrophils blood count (10, 12, 15, 17); ond experimental group (Figure 1C and 1D), and they
obtain more trend
the dominant accurateof theresults of leukogram
percentage values of neu- and are
resized forms
associated withofthered blood
reactive cells and
lymphocytes platelets
(Figure 1C)
trophils in relation to other leukocitic cells was also re- with highly basophilic cytoplasm. Lymphocytes with
(16).
cordedBlood smearsThecan
in our research. provide
control insights
results from the first intowavy
anyedges
possible anisocytosis,
with generally the presence
scarce cytoplasm were alsoof
group did not exceed the physiological framework. The noticed (Figure 1D) (28). Atypical lymphocytes were
abnormal
number of cells or inclusion,
immature neutrophils aswaswell as the
slightly possible
elevated presence
identified on theofblood
blood parasites
smears of three(17-19).
dogs in the sec-
and amounted to 6.5% in the first group (Table 1), while ond experimental group (10.4%, 6.6% and 7%), which
The presence
the upper of monocytosis
physiological limit was 4%in(10),the which
controlindi-results
belongin to
both
the groups of dogs
“few” group. Their(Table
presence1 indicates
and Tablea
cates enhanced granulopoesis in the bone marrow. Neu- response to antigenic stimulation. Lymphocytes exhibit
2)tropenia
can be was explained
obvious in thebysecond
the experimental
effect of prolonged
group increased stress,activity
pain,through
and limited
magnifying space duringcy-the
the basophilic
(43.2% - Table 2), while the lower physiological limit in toplasm (12, 24).
experiment,
adult dogs is 51% and(10).
during hospitalization,
The immature neutrophils allwere
under the influence
Acidophils of endogenous
and basophils were in theglucocorticoids,
normal range in
dormant in physiological terms. The presence of neu- both experimental groups, indicating the absence of al-
what results in, among other things, monocytosis (19). Also, a possible reason for the
trophils with morphological changes was very indicative lergic reactions in the tested animals, and confirms the
(Figure 1A and Figure 1B), but only in the second ex- cleansing of parasites in the animals previously conduct-
monocytosis in the control results is the age of the tested dogs, which amounted to 8-10 years
perimental group. Degenerative changes in neutrophils ed.
(15,20). The very
were determined apparent
(Table 3) in twomonocytosis
out of six dogs in inthethe experimental dogs
Fisher’s test found no in the firstsignificant
statistically group (22.83%
difference -
second group of tested dogs. In dog number 3 of the sec- between the mean values of the control and experimental
Table 1) and group,
ond experimental the second groupof degenerative
the percentage (16.20% -Table 2) is the
results within a groups
sign (pof> 0.05%),
infectious
although ormonocy-
chronic
neutrophils amounted to 24.6%, which, according to the tosis, neutropenia and slight lymphocytosis were detect-
inflammatory
semi-quantitativeprocesses,
evaluation ofor tissue damage
degenerative changes(15,20,21,
on ed, with 22,the23).
evident presence of atypical lymphocytes
neutrophils (12), belongs to the “moderate” group. Dog and degenerative neutrophils. The results are fully in line
The
number functions
6 had 1.6%ofof degenerative
mononuclear phagocytes,
neutrophils, which amongst other
with the results things,et al.,
Delibegović are2016thea
. secretion of
belongs to the “few” group (Table 3). Healthy erythrocytes in dogs (Figure 3C) are larger in
mediators
The changes andwereregulators
manifested inof thethe inflammatory
presence of pro- diameter response, interactions
when compared with
to the red blood antigens and
cells of oth-
nounced blue granules, which filled more than 50% of er domesticated animals, and they have a biconcave disc
lymphocytes,
the cytoplasm ofand removal
neutrophils of the
(Figure 1 Atissue debris
and Figure 1 through
shape (21).phagocytosis.
The life span of Incanine
ordererythrocytes
to performvaries these
B). When the cytoplasm of neutrophils is filled with ba- from 110 to 120 days. They often change their shape as
functions, theyvacuolation,
sophilic sparkling are arranged in allbodies,
called Dohle the places where
it is a result they could
of biochemical and come intochanges,
pathological contactduewith
to
considered that the neutrophils have suffered degenera-
infectious and other agents (21).