THE BLOOD COUNT O F NORMAL WHITE RATS'

ETHEL W. THEWLIS AND OVID 0. MEYER Department of Mrdicine, University of Wisconsaia Hedical School, Madison

FOUR CHARTS

Rats are becoming increasingly popular as small laboratory animals for hematologic studies, for the blood is easily ob- tained in adequate quantities for routine counts. There is considerable fluctuation in the leukocytes particularly, but the instability seems to be no greater than that of guinea pigs or rabbits. Rats are less expensive than either, and blood is more easily obtainable from them than from guinea pigs.

The standards of normalcy for formed elements of the blood of rats are not as definite as one might wish. Some figures are obtainable, Donaldson ( '24), Jordan ( '38) and Scarborough ('30), but these are in the main based upon tho accumul a t' 1011

from numerous sources of several rather small groups of animals. I n the course of 6 years' extensive and careful studies upon rat's blood, counts have been made upon about 450 rats. Control counts were always made whatever the subsequent experiments, and these preliminary observations furnish the data for this report.

METHODS

The white rats used were all of one pure inbred strain obtained from the Sprague-Dawley Company of BIadisoii and raised under ideal conditions. All rats were of course healthy. Since male rats were used almost exclusively, the counts reported here are for this sex alone ; the counts made 011 the few females that we obtained were, however, similar to those

'Thia study has been sided in part by a grant from the Wisconsin Aluinni Research Foundation.

115

116 E. W. THEWLIS AXD 0. 0. XEPEE

1.014

of the males in all respects. The rats, which ranged in weight from 125 to 450 gm., were arbitrarily divided into groups, the first weighing from 125 to 200 and tlie sccoiid, smaller, group, relatively mature animals, from 201 to 450 gm. Rats of the former weight are about 45 t o 60 days old, aiid those of the latter group over 60 days. All the animals were in our

8,553,000 1.055 2.02 1.157 18,819 7.327 27.13 (113) (11.2) (116) (100)

HGH.'

14.13

No. of rats

13.78 No. of rats (92)

4 . i2 4.09 0.18 0.39 62.81 13.11 3.74 3.85 0.92 1.26 0.30 0.84 . . . . . . . . . . . . . . . . . . . . I . I . .

TABLE 1

Avrmges and s t a n d a d deviations.

A -Rats of 125 t o 200 gin.

(307) (307)

B -Rats of 201 t o 450 gin.

- N. - 26.61 (153)

A -- Rats of 185 t o 200 gm.

- ux - 11.71

10.22

- -

R - R& Of 201 to 450 gm.

laboratory from 2 to 10 days before counts were made. They mere fed principally upon Steenbock ration ('23) while in our possession and on a similar ration before me purchased them. They mere not fasted before counts were made.

For the actual counting a rat was placed in a perforated cylinclrical cardboard box and the tail was allowed to protrude through a hole cut in the edge of c7 large cork stopper. The tail

BLOOD COUNT OF EATS 11i

was shaved over the vein after being cleansed with soap and water, and alcohol was wiped over the shaved area before a sharp cut was made across a vein. Bleeding was free, and usually an adequate quantity of blood was readily obtained

DISTRIBUTION CURVES

?! 3 L

I

IIC-IN m SRAIIS ILI (uLOcIT0 nn our L I ) * l m C v l ~ , I* WLLIO*S

Chart A The distribution of rcticulocytes, orythrocytcs and licmoglobin f o r two groups of rats.

for duplicate erythrocyte and leukocyte counts, for single hemoglobin determinations, and for cover slip blood smears.

Hemoglobin determinations were made on tlie Haden- Hausser hemoglobinometer so standardized that 15.4 gm. per

118 E. W. THIEN’LIS AN11 0. 0. MEYER

BLOOD COUNT OF BATS 1.19

100 cc. of blood equaled 100%. The pipettes fo r drawing the blood for this determination and for the erythrocyte and leukocyte counts were standardized by the U. S. Bureau of Standards. The erythrocyte counts were done in duplicate, one count being made from each of two pipettes, and the same

LEUKOCYfES PER GENT

The distribution of various cells in thc differeiitial leukocyte counts ( ‘ h u t C of 100 r a t s weighing 201 t o 450 gni.

procedure was carried out f o r the leukocyte counts. When there were variations greater than 200,000 in the two erptliro- cyte counts or variations greater than 500 (regardless of the height of the count) in the leukocyte count, the counts nTere repeated. The diluent for the erythrocyte counts was Hayem’s

120 E. W. THEWLIS AKD 0. 0. JfEYElt

solution; for the leukocyte counts, 1% acetic acid. Counts were made on the new bright-line improved Neubauer count- ing chamber, which had also been standardized by the U. s. Bureau of Standards. Differential leukocyte counts were made

C’lxirt U The distribution of various cells in the differential leukocyte eouiits of 133 rats weighing 125 to 200 gm.

011 cover slip smears stained with Wright’s stain, and the reported counts a re all based on a minimal count of 200 cells. Some of the differential counts were of 400 cells. Reticulocyte counts mere made on the same smears, the cover slips having

BLOOD COURT O F RATS 121

been previously prepared by staining with cresyl blue, and the percentages were obtained by examining 1000 erythrocytes.

All the counts were made by two technicians during the 6-year period. Both had been thoroughly trained, and com- parison of the counts of each for different groups of rats showed no sigiiificant variations.

RESULTS

The results are given in table 1 to show the mean and standard deviation for the various elements, and the charts depict the distribution curves.

DISCUSSION

The results here recorded are in many respects similar to those previously reported, but in other respects they are quite different. Our data furnish no information regarding the platelets, since they were not counted, but examination of the smears makes it obvious that normally the platelets are numerous, more numerous than in man. This is in accord with Gunn and Vaughan's ( '30) and Scarborough's ( '30) findings.

HEMOGLORJX

The average hemoglobin levels are similar to those previ- ously reported, and there are no significant differences in levels for the smaller and larger rats. I n 206 of the 309 smaller rats the hemoglobin levels were between 12 and 15 gm.; a larger proportion, 79 of 92, of the larger rats had hemo- globin levels within this range. The range for the younger rats, 9 to 20 gm., is wide, wider than for the more mature rats. This greater range for the younger rats might be anticipated, perhaps, since their stability is less. Blood loss, hemocon- centration, and perhaps other factors affect the younger animals more significantly.

ER,YTHROCTTES

The average erythrocyte counts are similar to these previ- ously reported, although that of the younger rats is somewhat

122 E. W. THEWLIS AND 0. 0. MEYER

lower, and is significantly lower than that of the older rats in our series. In 97 of 113 older rats the range was from 7 to 10 million cells, and in 288 of the 307 younger rats the erythrocyte levels were between 6 and 9 million. The range between the minimal and maximal erythrocyte counts was less marked than the variations in hemoglobin, and was of the same degree in the two groups of animals.

RETICCLOCYTES

Figures for the normal reticulocyte counts in rats were not available elsewhere. The average normal reticulocyte count for the younger rats was 4.82% and for the older rats, as might be expected, distinctly lower, 2.0276. For 82 of the 112 old rats the reticulocyte counts ranged between 1 and 4% : in 23 they were less than 1%). Few rats weighing from 201 to 450 gm. have reticulocyte counts above 4%. The younger rats, on the other hand, again evidenced their lesser stability in that a relatively great number had reticulocyte counts well above the average for the entire group. Only 7 of the 306 animals had counts below l%, but 54 had counts above 7%. Two huiidred and forty-five of the total number of younger rats had reticulocyte counts between 1 and 7%.

LEUKOCYTES

The average leukocyte counts of 18,819 for the older rats and 23,075 for the younger rats are about twice as high as the average levels previously reported. Furthermore, the standard deviations and distribution curves indicate the wide range of counts that are obtained in normal rats. This wide variation is demonstrable in both young and more matiire animals. No attempt was made to do counts in rats under so-called basal bnt the counts were done with care and under the usual experimental environments. The most labile elements of the blood of man are the leukocytes, and we founcl this to be true of rats to an even greater extent.

a E. J. F:irris in Am. J. Anat., 1938, has reported upon leukocyte counts obtained in normal rats under basal conditions.

BLOOD COUNT O F RATS 123

It has been generally recognized that the leukocyte counts in small laboratory animals-rats, guinea pigs, and rabbits- are unstable, but perhaps not to the extent that these results reveal. It will be recalled that all the rats used were fine healthy animals raised uiider ideal hygienic conditions.

The ranges for hemoglobin, erythrocytes, and reticulocytes in rats are narrow enough f o r all practical experimental purposes, but the ranges for the normal leukocyte count are so wide that experimentalists must use extreme caution in drawing conclusions from increases o r decreases in the lcuko- cyte counts unless they are marked.

DIFFERER’TIAL COUNTS

In the differential counts, in addition to the usual cells, neutrophiles, eosinophiles, basophiles, lymphocytes, large monoiiuclears (moiiocytes) and unclassified cells, there was also made a separate division, the so-called large young cells. This division seemed practical to us inasmuch as the cells, although apparently of the lymphocyte series, were young and not definitely classifiable as such, having some of the char- acteristics of large monocytes. These cells were usually about 10 to 12 ~1 in diameter: some were smaller, possessed a deep blue rough appearing cytoplasm; those older have a paler smoother cytoplasm, with a rather clear peri-nuclear zone. The nucleus was usually round, occasionally indented, most often centrally placed. The nuclear chromatin was coarse, moderately deep-staining, with two or three or more central depressions which resembled but were not nucleoli. In the youngest cells the surrounding nuclear membrane was indis- tinct; in the older cells the membrane was heavier and ap- peared to be complete. These cells averaged less than 1% of the total number.

The principal leukocytes of the rat are, as in man, the neutrophilcs and lymphocytes, but in the rat the lymphocyte is the predominant cell. The average percentage of iieutro- philes in the younger mid older rats was similar, 26.61 and

124 E. TV. THEWLIS AND 0. 0. MEYER

27.13%. The lymphocyte counts were also similar, although the younger rats had a higher percentage-68.35 as com- pared with 62.81. These figures for the neutropile and lympho- cyte counts agree with those reported by Scarborough ('30) hut differ from those reported by Donaldson ('24) who recorded the neutrophile as the predominant cell.

The distribution curves indicate that although there is a wide range for the normal count of both neutrophiles and lymphocytes, the large majority of both the older and younger animals fall within reasonable limits. Especially is this true for the neutrophiles of both groups and the lymphocytes of the younger rats.

The remaining cells, the minority, were similarly numerous in the older and younger rats save for the eosinophiles, which averaged 4.72% in the older rats and but 1.39% in the younger. There was a less distinct difference in the same direction in the monocytes, the average being 3.74 and 2.34% respectively. It is these differences principally that account for the deficit in the lymphocytes of the older groups of rats. The explanation for the distinctly higher eosinophile count in the older rats was not evident. The average figures for the total rats of both groups for the eosinopliiles was similar to those given by Scarborough ( '30), 2.176, but our figures for monocytes were lower, his reported average from several groups being

The basophiles were as scanty as reported by Scarborough ( '30). The large young cells averaged less than 1% in either group and the cells which were not classifiable averaged 0.5% or less.

The distribution curves indicate that there is a fairly wide spread for eosinophiles from less than 1% to 10% in each group, and for monocytes from less than 1% to 20 to 25% in one rat of the older group and to 10 to 15% in one rat of the younger group.

5.3%.

BLOOD COUNT OF RATS 1%

No attempt is made to give a description of tlie staiiiecl blood smear nor to picture the morphology of the various types of leukocytes, for this has been well done elsewhere (Jordan, ’38 ; Loewenthal, ’33).

S UMNARY

The average figures for blood counts of rats weighing 125 to 200 gm. are : hemoglobin 14.13 grn., erythrocytes 7,630,000, rcticulocytes 4.82% ; leukocytes 23,075 with 26.61% nontro- philes; the average figures for rats weighing 201 to 450 gm. are : hemoglobin 13.78 gm. ; erythrocytes 8,553,000 ; reticulo- cytes 2.027% ; leukocytes 18,819 with 27.13% neutrophiles.

We wish to gratefully acknowledge tlie help of Mrs. Nancy Lee Jolinson in the statistical analysis of the data hcrc I> resented .

LITERATURE CITED

DONALDSON, 1%. H. 1924 The Rat. Mem. of Wistar Inst. of Anat. and Biol.,

GUNN, I”. D., AND S. L. VAUGHAN 1930 Bone-marrow reactions; blood count

JORDAK, H. E. 1938 Comparative Hematology. Handbook of Hematology. H.

LOEWENTIIAL, N. 1933 Observations sur les globules blancs du sang des mam-

SCARBOROUGH, R. A. 1930 The blood picture of normal laboratory animals.

STEESBOCK, H. 1923 A satisfactory ration for stoek rats. Science, col. 58,

Philadelphia.

in albino rat; blood platelets. Ailat. Rec., vol. 45, pp. 59-67.

Downey, Editor. Hoeber, Xew York. Vol. 11, p. 703.

mi8res. Arch. d’Anat., d’histol. ct d’enibryol., rol. 17, pp. 249-37.

The Rat. Yale Jour. of Biol. and Med., vol. 3, pp. 267-272.

pp. 449-450.