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Quantitative studies on the blood and bone marrow of newborn mongrel puppies.

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QUANTITATIVE STUDIES ON THE BLOOD AND BONE
MARROW O F NEWBORN MONGREL PUPPIES
R. M. MULLlGAN
Department of Pathology, University o f Colorado, School of Medicine, D a v e r
I n a previous report (Mulligan, '41) the quantitative data on the. bone
marrow of thirty-five adult mongrel dogs and of four puppies were presented and a method of serial biopsy of the bone marrow of the
living dog by resection of segments of the seventh through the twelfth
ribs was described. Since then two other papers dealing with the bone
marrow of adult dogs have been published. Van Loon and Clark ( '43)
performed differential counts on smears of extruded rib marrow examined immediately following the sacrifice of eight normal mongrel adult
dogs. Meyer and Bloom ( '43) studied the cellular composition of bone
marrow obtained by needle puncture from the crest of the ilium in
ten normal living dogs (eight thoroughbred, two mongrel), 1 to 11 years
old. No quantitative study of either the bone marrow or the blood
of newborn puppies has been found in the literature. During the past
few years the blood and bone marrow of twenty-one mongrel puppies
have been investigated in this laboratory. These animals were born of
clinically healthy bitches pregnant at the time of reception into the
animal quarters. The five litters represented included five puppies
born on November 26, 1941; nine on July 17, 1942; three on November 5, 1942; one on September 15, 1943 and three on June 14, 1944.
Following a lethal intraperitoneal dose of nembutal 3 to 24 days after
birth, 1 cc. of blood was obtained by apical intracardiac puncture between the ribs adjacent to the left side of the sternum and rendered
fluid by gentle and thorough mixing with an isotonic quantity of potassium oxalate. Each animal was either in the last few respiratory movements before death or had just ceased breathing, a matter of a few
seconds, when the blood sample was withdrawn. In most instances, a
second sample of about 2 cc. of blood was allowed to clot to obtain
serum for preparation of the marrow smears. This serum was invariably light red tinged, probably due to intravascular hemolysis, for the
2-cc. syringes and the no. 22-gauge needles, stored after cleansing in
an antiseptic solution of one part 40% formaldehyde and 99 parts 95%
ethyl alcohol, were thoroughly dried before use. The hemoglobin was
calculated with a Duboscq colorimeter equipped with 1-mm. thick
161
162
R. M. MULLIGAN
Newcomer glass square. To further standardize the instrument, the
hemoglobin content of a sample of oxalated whole human blood was
determined by the method of Van Slyke ( '18) and Haldane's scale.
Into each of 5, 7.5,10,15, and 20 cc. quantities of 1% hydrochloric acid,
.01 cu. mm. of this blood was introduced, mixed, and allowed to stand
for 24 hours at room temperature. By the light of the southern sky,
an average of twelve readings for each sample was then taken to construct a curve, a standard for this investigation and for a study of the
blood of adult mongrel dogs (Mulligan, '41). On this curve the hemoglobin could be read in grams per 100 cc. of whole blood directly from
each colorimeter point. The values so obtained were 8% lower than
those found by the use of Newcomer's tables (Newcomer, '19). The
erythrocytes in millions and the leucocytes in thousands per cubic
millimeter were counted in a Neubauer bright line counting chamber.
The differential counts were made on 200 cells on slide films stained
with May-Griinwald-Giemsa stain. The few damaged cells present in
each smear were disregarded.
The values for the blood constitiients mentioned for the thirteen male
and eight female puppies of this series have been summarized in detail
in table 1.
Since the level of hemoglobin, the partition of the differential count
of the leucocytes, and possibly the total leucocyte count were not
stabilized in the first day of life, nor for certain by 2 i days, means
and standard deviations for none of the blood constituents have been
calculated pending the opportunity for serial hematopoietic studies in
relation to age on puppies less than 2 months old. However, the data in
table 1may be summarized.as follows insofar as ranges of the values
are concerned : hemoglobin 13.0 to 23.4 gm. ; erythrocytes, 4,170,000 to
6,160,000 per cubic millimeter ; leucocytes (uncorrected for normoblasts present), 5,200 to 28,150 per cubic millimeter ; stab neutrophiles,
8.0% to 47.0% ; segmented neutrophiles, 31.5% to 67.0% ; lymphocytes,
2.0% to 32.5% ; eosinophiles, 0.0% to 5.5% ; monocytes, 0.0% to 3.0% ;
and normoblasts, 0.5% to 14.0%. Myelocyte neutrophiles were found
in two counts, 0.5% and 1.0%. Metamyelocyte neutrophiles were observed in thirteen counts, 0.5% to 5.0%. Basophiles were never encountered in any of the counts. The unstable hemoglobin values as compared with the relatively constant level of the erythrocytes may be
the result of the relase of hemoglobin from the erythrocytes into the
plasma by diffusion during and after the abrupt transfer of the puppies
at birth from the intrauterine environment to that of the outside world.
I n the first day of life, the increase in the total neutrophilic ceHs and
+
2
2
2
2
2
23
21
23
It
3
f
f
3
&
f
3
1
f
f
f
3
(days)
AQE
F
M
M
F
F
F
M
M
M
M
M
M
F
M
M
M
M
F
F
F
M
SEX
19.2
15.8
17.6
16.6
16.4
14.2
16.6
16.0
18.2
15.4
17.2
23.4
19.0
14.4
14.6
15.2
13.0
15.2
13.4
13.0
14.6
HB
6.13
5.85
5.87
4.57
4.59
4.17
5.24
5.03
5.20
4.48
5.46
6.16
5.05
4.83
4.65
5.62
5.60
4.92
4.68
4.26
5.06
EBC
22650
28150
22800
12400
12250
20300
22100
13400
11050
13900
13150
18800
11300
10700
10450
9000
9400
5200
21400
16400
12550
WBO
..
..
..
..
..
1.5
0.5
..
0.5
0.5
..
..
1.5
4.5
1.0
1.0
1.5
3.0
2.0
5.0
3.0
METAN
34.5
47.0
23.5
26.5
30.0
30.0
29.0
26.0
26.0
23.0
18.0
25.0
24.5
15.0
8.0
15.0
14.0
9.0
11.0
10.5
16.5
BTN
-
49.5
31.5
63.5
60.5
40.0
53.0
60.0
60.0
51.5
57.5
67.0
63.0
52.5
51.5
48.5
43.0
53.5
62.0
63.5
62.5
62.0
SEO N
-
11.0
9.5
5.0
8.5
13.0
8.0
2.0
7.0
15.5
9.5
11.5
5.5
19.5
23.5
31.0
32.5
25.0
22.0
22.0
19.0
18.0
LYMPH0
rwcr&lcg-unenewborn mongrel pzlppies.
TABLE 1
UJ
0.5
3.5
4.5
1.0
1.0
1.5
1.0
0.0
1.5
0.0
0.0
0.0
2.0
5.0
5.5
3.0
4.0
2.0
2.0
3.0
1.5
EOB
-
2.5
0.0
0.0
0.5
0.5
3.0
0.0
0.0
2.0
0.5
1.0
0.0
0.0
0.0
1.0
0.0
0.5
0.0
1.0
1.0
0.5
MONO
2.5
3.0
1.5
2.0
14.0
3.5
5.0
2.0
2.5
7.5
3.5
6.0
0.5
4.5
2.5
6.0
3.5
.3.0
1.0
4.0
2.0
NORYO
-
-leucocytes
Key: NO.-number o f ; Hb -hemoglobin in grams per 100 cc. of wbole blood; RBC erythrocytes in millions per cubic millimeter; WBC
per cubic millimeter; Meta - metamyelocyte; N -neutrophiles; St -stab; Seg segmented; Lympho lymphocytee; Em
eosinophiles; Mono - monocytes; Normo -normoblasts.
P17-3
P16-3
P15-3
P5-1
P3-1
P4-1
P1-1
P2-1
P6-2
"7-2
pa2
P9-2
Plo-2
Pll-2
P12-2
P13-2
P14-2
P21-5
P22-5
P23-5
Pl8-4
NO.DW
Data on the peripheral blood
164
R. M. MULLIGAN
the shift to the left in their partition with the concomitant reduction in
lymphocytes may be a reflection of lingering estrogenic material transfered across the placenta from the blood stream of the mother to that
of the fetus. The papers containing the evidence that estrogens stimulate granulocytopoiesis in the dog have been quoted in a previous communication (Mulligan, Longwell, and Morrell, '43).
Slide smears of the marrow of one or more ribs were prepared with
serum and histologic sections were cut from at least one rib of each
animal by a technique already described (Mulligan, '41). By study of
the histologic sections, the normal cellularity of the marrow of the
puppies was different from that determined for the thirty-five adult
mongrel dogs mentioned; namely, a ratio of 65 to 35 of marrow cells
to fat tissue, for the marrow of the puppies uniformly revealed no fat
tissue whatsoever, but rather consisted entirely of marrow cells.
The organs of all twenty-one puppies were anatomically normal at
reasonably complete autopsy including examination of the central
nervous system.
The results of the differential counts of 500 cells each on suitable
smears stained with May-Griinwald-Giemsa stain have been listed for
the twenty-one puppies in table 2. The data for the thirty-five adult
mongrel dogs have been arranged for comparison. I n contrast to the
values for the blood of the puppies, the data for their bone marrow
were reasonably constant, so that it was possible to calculate means
and standard deviations for them. The factor, n - 1 , was substituted
- ~ M X ) Z, for the calculation of
for m in the usual equation, a =v'Z?
the sigma, a, or standard deviation. I n this formula EX2 is the sum
of the squares of the values in the series of twenty-one puppies, n is the
number of animals, and ( M x ) ~is the square of the mean for the values.
I n checking the data for the thirty-five adult dogs, errors in the calculation of the sigmas for myelocyte neutrophiles and for normoblasts
were discovered. The correct figures have been inserted in table 2.
I n the marrow of the puppies, stem cells did not occur with significant enough frequency, actually an average of one cell per 500 cells
counted, to include them in the table. Megakaryocytes were plentiful
in the sections, but were too few in the smears to figure in the differential counts. Reticuloendothelial cells, plasma cells, and monocytes
165
PUPPY BLOOD AND BONE MARROW
were present in insignificant numbers in a count of 500 cells. Macrophages, heterophil cells (Stasney and Higgins, '37) and basophiles
were not found in either smears or sections of the rib marrow. Cells
having basophilic granules, which have been observed occasionally in
the bone marrow of normal adult mongrel dogs, but never in the
peripheral blood, have been identified as mast cells. With the technique
employed, the preservation of the nucleated cells of the bone marrow
of the puppies as viewed in the smears was not so good as that in the
TABLE 2
Data on the bone marrow of twenty-one newborn mongrel puppies and thirty-five
adult moiigrel dogs.
Minimum
Eosinophiles
Promyelocyte
Neutrophiles
Myelocyte
Neutrophiles
Yetamyelocyte
Neutrophiles
Stab
Neutrophiles
Segmented
Neutrophiles
Proerythroblasts
Erythroblasts
Normoblasts
Lymphocytea
Unidentified
Cells
Myeloid/Erythroid
Ratio
21 PUPPIES
Maximum
Mean
Sigma
35 ADULT D W E
Mean
Sigma
0.0
5.2
2.4
1.50
3.7
1.49
0.0
2.2
0.8
0.69
1.5
0.91
2 .o
6.6
4.3
1.47
4.7
1.98
7.2
12.0
9.7
2.45
10.5
3.49
14.8
28.4
20.6
6.27
31.0
8.32
0.8
6.6
3.4
1.92
0.4
1.3
0.6s
3.9
0.5
2.26
0.29
1.6
3.2
9.4
56.6
6.0
0.8
3.0
33.0
0.48
5.8
2.26
1.5
0.87
45.1
12.76
11.49
3.3
1.45
38.1.
1.9
5.4
3.1
1.33
2.1
1.18
1.39
0.83
0.33
1.6
0.88
1.46
adult dogs, although the leucocytes in the peripheral blood of the
puppies were as well preserved and stained as those of the adults.
This is reflected in the higher percentage of unidentified cells in the
marrow of the puppies noted in table 2. Damaged cells were disregarded and areas of the smear where the cells were well spread out and
stained were chosen in the differential counting of the bone marrow.
By employing the formulas, S. E. diff. = .\/ (S. E . M , ) 2
(S. E . M , )2
M - M1
and S. S. = s.'E.
, in which S. E. is the standard error of the
+
d,ff.
166
R. M. MULLIGAN
difference of the means, (S. E.M,)2
is the square of the standard error
of the first mean, (S. E.,* ) 2 is the square of the standard error of the
second mean, S . S. is the statistical significance, MI is the first mean,
and M, is the second mean - the significant differences between the
data on the bone marrow of the newborn puppies as compared with
those on the bone marrow of the adult dogs were in the eosinophiles
(3.2), promyelocyte neutrophiles (3.5), stab neutrophiles (5.2),
proerythroblasts (5.0), erythroblasts (8.4), lymphocytes (3.4), and
myeloid/erythroid ratio (4.7). The figures in parentheses indicate
statistical significance, since they are all three standard deviations or
more. The differences in the normoblasts (2.0) and the unidentified
cells (2.8) are significant beyond two standard deviations. The differences in the myelocyte (0.87), metamyelocyte (l.O), and segmented
(0.88) neutrophiles were one or less, demonstrating no statistical
significance.
SUMMARY
1. The blood and bone marrow of twenty-one newborn mongrel
puppies, 4 to 24 days old, were quantitatively studied.
2. Since the level of hemoglobin, the partition of the differential
count of the leucocytes, and possibly the total leucocyte count were not
stabilized in the first 4 day of life, nor for certain by 24 days, statistical
analysis of the blood constituents of newborn and very young puppies
must await the gathering of data related to age for the first 2 months
of canine life.
3. The figures for the bone marrow of the twenty-one mongrel
puppies were reasonably constant and were compared statistically with
those for thirty-five adult mongrel dogs previously reported. The
most important finding was a preponderance of erythrocytopoiesis
over granulocytopoiesis in the marrow of the puppies. This was reflected in the peripheral blood by the presence of 0.5% to 14% of
normoblasts in a count of 200 nucleated cells.
LITERATURE CITED
M E I Y ~L., M., AND F. BLOOM 1943 The bone marrow of normal dogs. Am. J. Med. Sci.,
vol. 206, pp. 637-641.
MULLIQAN,
R. M. 1941 Quantitative studies on the bone marrow of the dog. Anat. Rec.,
V O ~79,
. pp. 101-108.
1941 Studies on the blood of mongrel dogs at high altitude. Am. J. Physiol.,
vol. 133, p. 394.
AND R. M. MORRELL 1943 The tissue changes produced
MULLIOAN,
R. M., B. B. LONQWELL
by estrone injected into female dogs with bile fistulas. Am. J. Path., vol. 19, pp.
861-871.
PUPPY BLOOD AND BONE MARROW
167
N m o ~ m H.
, 8. 1919 Absorption spectra of acid hematin, oxyhmoglobin, and carbon
monoxide hemoglobin - a w w hemoglobinometer. J. Biol. Chem., vol. 37, pp.
465-490.
STASNEY,
J., AND G. M. HIMINB 1937 Quantitative cytologic study of the bone marrow of
the adult dog. Am. J. Med. Bci., voL 193, pp. 462470.
VAN LOON, E. J., AND B. B. CLARK 1943 Hematology.of the peripheral blood and bone
marrow of the dog. J. Lab. and Clin. Med., vol. 28, pp. 1575-1579.
VAN BLYgIE, D. D. 1918 Gaaometric determination of the oxygen and hemoglobin of blood.
J. Biol. Chem., vol. 33, pp. 127-132.
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