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respiratory movements before death or had just ceased breathing, a matter of a few seconds, when the blood sample was withdrawn.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.