BONE-MARROW REACTIONS

11. THE BLOOD COUNT IN THE ALBINO RAT: BLOOD PLATELETS

FRANCIS D. GUNN AND STUART L. VAUGHAN Department of Pathology, Nortlawestern University Vedical School, and

Wesley Memorial Hospital

METHOD

That there is no entirely satisfactory method for the enu- meration of blood platelets is evident from the number of new methods or modifications of old methods appearing every year in the literature. Methods that are suitable for clinical work are frequently not applicable to experimental work, especially on small animals, either because they require too large amounts of blood or because they are not sufficiently accurate. Various modifications of the direct method devised by Oluf Thomsen ('20) seem to have given the most con- sistently reliable results in different hands. In the experi- ence of the writers, the modification described by Reimann ('24) is the most satisfactory fo r counts in the human and in laboratory animals where venepuncture can easily be exe- cuted. The impracticability of this method in experiments on rats where repeated heart puncture would have to be performed is obvious.

Consequently, an indirect method was used, similar to that described by Gunn ( '26) for platelet counts in rabbits. It is essentially that use by Cramer, Drew, and Mottram ('23), Bedson and Zilva ('23), Sooy and Laurens ('24), and others, with the difference that instead of counting the wet prepara- tion under a cover-slip, we make the counts on dried, stained films similar to the technique of Fonio ( '12).

59

THE ANATOMICAL RECORD, VOL. 45, KO. 1

60 FRANCIS D. GUNR A X D STUART L. VAUGHAN

2 ’ e c k ~ k p e The diluting fluid used was isotonic (3.8 per cent) sodium

citrate with the addition of 0.5 per cent neutralized commer- cial formalin. (The sodium chloride used in 1926 (Gunii, ’26) has been found to be superfluous.)

The fluid is drawn into a leucocyte diluting pipette until the bulb is half filled and theii emptied into a shallow depres- sion iii a clean paraffin block. The tail is prepared and clipped off as described in the previous paper and immedi- ately dipped into the diluting fluid, one large drop being allowed t o flow into the fluid. (A second dilution may be prepared by quickly transferring the eiid of the tail to I I

second depression in the paraffin block containing a similar quantity of fluid, aiid used as a check on the first.) This mixture is then drawn into tlie pipette previously wet with the fluid aiid f o l l o ~ ~ d 11)- enough diluting fluid to fill the bulb. After slinking for two or three minutes, the first few drops are discarded aiid thin films are made on scrnpulously clean slides. The firms are dried, stained with Wright’s blood stain, covered with oil and large cover-slips, and counts made under the 4-mm. objective axid lox ocular of a binocular miciwscoye. To aitl in tlic counting, an ocular net (ruled glass disc diiiali lics 011 the diapliragm of one ocular) w i h

uscd. 111 these preparations tlict crytlirocytcs appear bluish aiid

t l i ~ platelets, brick red or purplish red. Wheii platelets lie abovc~ or M o w tlic cir.ytlwoc?-tes, tlic latter are sufficieiitl? traiispiii*ciit so that the platelets, much more licavily stained, t i i ~ c~lci-lrly visible tlirongli them. If tlie films are allowed to dry for at least fifteen minutes in the air, thc caells do not was11 off the slide. T h e n the latter does occur, it is clue to im1)erfect cleaning of the slide and round, clear spots on the: film indicate tlie places wliere cells have washed awaj-.

first, that tlw plat e ld s are clearly s ta i l id aiid caniiot be confnsed viitli (Inst particles, lieinoconia, prccipitntccl stain, or ot l i tv cs- t ixticoiis mtitci-ial a i d , secoiitll~--, tliat tlie sliclcs ;iix’ pcrrniiii(>ii~

The caliief advantages of this metliocl arc :

BONE-MARROW REACTIONS. I1 61

and the counts can be made at any convenient time. A homo- geneous mixture of cells is insured by thorough agitation of the diluted blood in the pipette. The chief source of error is that inherent in every other method for platelet enumera- tion, namely, clumping of platelets at the time the blood is taken due to contact of the blood with tissue juice or foreign objects to which they adhere. This danger has been reduced to a minimum by allowing the blood to flow from the severed vessels directly into the anticoagulant fluid, and rarely has it been necessary to discard a slide on account of the appear- ance of clumping.

When the blood was taken from the femoral vein, the ani- mal was prepared as described in the previous paper (eryth- rocytes and hemoglobin), and after exposing the vein, three or four drops of the diluting fluid were dropped into the site and the vein snipped through the fluid so that the blood mixed with the anticoagulant as it flowed from the vein. This mix- ture was taken up immediately into a pipette, shaken thor- oughly, and films prepared iu the usual way. This technique was not so satisfactory as the tail method, chiefly because oi' the greater danger of admixture with tissue juice aid because of the difficulty of controlling the flow of blood. Coil- sequently, certain discrepancies occurred in the first groul, that did riot appear in tlie secoiid (table 1).

In determining tlie erythrocyte-platelet ratio, about 2000 erythrocytes were counted (1000 011 each of two slides) for each estimation and the results expressed in terms of platc- lets per one hundred erythrocytes (table 2) . In a film of average thicknoss about twenty visiial fields will give this number.

It has been thought, by workcrs who accey)t TTright 's thcor\- of the origin of platelets that the elongate forms, those with long processes, and especially the slender, spiiidle-sli2tpctl platelets represent a young or immature form a i d that thcii. appearance iii the blood stream is due to rapid prodnctioii, with in-eplar pincliing off of tlie processes of tlic megalraryo- cyteh. If this is true, we might expect a larger proportion

62 FRANCIS D. GUNN AND STUART L. VAUGHAN

TABLE 1

Platelets in thousands; counts at one-week intervals

Group I Rat c 3 781 1388 C8 1300 1005 c 5 966 1213 c7 1239 1012 c 2 .... 1589 c4 1202 1711 C6 591 1349

Average, 1013 1324 _ _ ~ ~ -

Rat C15 C14 c10 c11 c 9 C13 c12

1190 1406 1530 1222 1361 1366 1517 .... 1212 1605 1468 1509 1300 1469 1264 1565 1454 1383 1352 1264

1395 1464 1315 1475 1264

Group II

1372 1514 1431 1509 1732 1418 1422 1430 1469 1269 1812 1544 1223 . .. . 1154 1213 1561 1538 1425 1487 1257 1185 1507 1793 1470 1547 1354 1027 1019 1349 1213 998 1490 .... 1558

Average,

Rat c 3 C8 c 5 c 7 c2 c4 C6

Average, -. -.

Rat C15 c s 4 c10 c11 c 9 C13 c12

1275 1382 1521 1447 1298 1508 1354 1558

TABLE 2

Platelets per one hundred erythrocytes

Group I

8.9 17.1 12.9 10.9 8.4 13.4

11.9 13.6 . . . 17.0

10.8 19.1 7.0 16.7

10.0 15.4 __ ~ -

12.7 15.8 16.8 14.0 14.3 14.5 16.6 . . . 13.2 16.1 16.7 16.1 13.0 15.6 14.3 17.8 15.6 14.9 14.6 14.0 14.9

15.6 15.7 13.9 15.4 14.2 14.9 __ -___ ____

Group II

13.6 15.4 15.4 16.0 17.8 14.5 13.7 14.9 14.4 13.5 19.5 17.1 14.3 . . . 12.1 12.8 17.9 17.7 16.7 18.1 12.4 12.8 17.4 25.0 15.6 16.8 14.7 11.2 10.7 15.4 14.1 12.5 16.2 . . . 17.5

~ ~~

Average, 13.2 14.1 16.8 17.1 14.9 17.0 14.7 17.5

B0NE:MARROW REACTIONS. II 63

of these forms in conditions favoring rapid increase in their numbers. Experimental support for this idea has been fur- nished by results obtained in rabbits exhibiting increase in blood platelets due to bacterial infection (Gunn, ’27). In those experiments the spindle forms were increased from a level of 2.5 to 8.5 per cent before injection to a maximum of about 10 to 15 per cent after injection. Consequently, in our

TABLE 3 Platelet spindle forms in per cent

Rat c 3 2.8 C8 2.3 c5 3.3 C7 5.6 c 2 . .. c 4 5.9 C6 . ..

Average, 4.0 - -~ .-

Group average, 5.6.

Rat C15 8.4 C14 3.0 c10 4.3 c11 4.3 c 9 4.8 C13 6.8 c12 6.2 ___ __ -

Average, 5.4 Group average, 6.1.

Group I

3.5 3.9 5.7 2.7 5.5 5.1 . .. 4.8 6.2 3.1 4.7 6.3 . .. 3.9 5.8 5.6 5.2 4.3 4.1 6.3 3.1 . .. 6.5 10.8 8.9 5.2 5.8 9.2

4.1 5.7 6.6 5.0 5.8 4.5 9.2 __ __ - _ _ _

Croup II

7.0 4.0 5.1 7.0 5.6 3.3 5.9 7.4 4.8 . .. 5.4 7.1 6.3 6.9 4.8 3.0 4.8 5.1 7.8 8.8 8.9 7.1 5.2 3.7 7.0 3.7 . .. 5.7

5.6 5.9 4.6 7.2 5.8 8.9 5.7 - - - - _____

estimations, in the majority of cases we counted the slender, spindle forms and expressed their number in terms of per cent of the total platelet count. The results on these two groups of ‘normal’ rats have been tabulated for comparison in table 3. The normal values lie between 2.3 and 10.8 per cent (average, 5.6 in group I and 6.1 in group 11).

In order to determine the probable error in the method, five separate determinations on the same animal were made

64 FRANCIS D. G U N N AND STUART L. VAUGHAN

on blood from the tail, one after the other as rapidly as possible, and the results tabulated (table 4). The erythro- cyte count was 9.7 million. The maximum variation from the mean in these counts is 128,000, or about 8.7 per cent. The possible error in the estimation of spindle forms is neces- sarily greater, because the count is based upon a smaller total number (about 300 to 350) of cells (platelets). These counts mere made with usual care, and we consider that in our hands the error in technique is within 10 per cent. Need- less to say, this does not iiiclude the error in estimating the absolute number of erythrocytes, which must he taken into coiisideratioii in any indirect method.

TABLE 4

Reyen fed counts on the Same vat; dry-film metliod

E rytkrocytes Platelet8 counted p e r 1 0 0

2105 15.1 2241 16.8 2313 15.1 0064 14.1 2228 15.8

Average, 15.38 . . .

Platelrt-ervthrocyte Plntektn Rpindle forms, ratao in tlwusandg per cent 1-6.6 1430 9.5 1-5.9 1591 9.3 1-6.6 1430 7.5 1-7.1 1335 9.3 1-6.3 1496 8.3

1-6.5 1456 8.i8 -

L4i~otlier qnestioii which arose in connection with the use of this method was whethcr o r not the results so obtained were similar to those obtained by the more commonly used method of counting in wet-film preparations. To answer this, we have made counts on the same animal at the same time by both methods, each technique executed in duplicate. The diluting fluid used for the wet preparations was that of Rees and Ecker (sodium citrate, 3.8 per cent ; commercial formalin, 0.2 per cent; brilliant cresyl blue, 0.1 per cent). The dihhions were made in the usual manner, but instead of preparing dry films, w-e placed a standard drop of the mixture 011 a slide, covered with a thin cover-slip, sealed with melted paraffin, and made the count as usual. One of these preparations wis counted under the oil-immersion objective (Koristka 2 mm., N.A. 1.30) and the other under the 4-mm. dry lens. Matched

BONE-MARROW REACTIONS. I1 65

compensating lox oculars and achromatic coiideiiser N.A. 130 were used throughout.

The figures tabulated in table 5 show tlic results of this procedure. All four counts are well within the limits of ex- perimental error, and we may conclude that in our hands the resnlts obtained by the two methods are practically identical.

TABLE 5

Comparative counts on the same yat; ilrJ1- and wet-jiltit method8 Erytkrocytes Platelets Plnteletlr YPthocl Nicrorcope

counted per 100 i n thowanda used ohjrctice 2268 14.9 1343 Wet film Oil iiuiimrsion, 2 min. 2056 14.5 1306 Wet fihn Dry lens, 4 mm. 2145 14.7 1324 Dry-stained Dry lens, 4 mm. 2143 14.1 1270 Dry-stained Dry leiis, 4 mm.

COMAIEST

As will be seen by reference to the tables, there is a distinct teiidency toward elevation of the platelet iiumber after the initial count. This is true both of the percentage figure uiicl of the calculated iiumber and occurs a t the time the erythro- cytes are showing the greatest diminution, clearly clemoii- strating that this rise is not dependent upon fluctuations in the red-cell number. Whether this change is due to the anos- thetic, to the loss of blood, or to both is of little importance for the present purposes, as these factors will he practicall!- constant in all fnture experimental procedures.

I n spite of a few apparent cliscrepancies in individual counts in group I (initial counts in C3, C.5, aiid C6), the aver- ages by weeks in the two groups are fairly close to each other, the counts in the second gronp being somewhat higher. The averages of tlie thirty-three coniits in each group are 1,314,000 in the first and 1,418,000 in the second-a difference of slightly less than 8 per cent. I n the second group tlie masi- mum variations in individual counts are found in rats C11 and C13, the greatest variation from tlie initial couiit in each of these amounting to about 43 per cent. Rat C13, as was men- tioned in the previous paper, had a subcutaneous abscess which was noticed at the time the third count was made and

66 FRANCIS D. GUNN AND STUART L. VAUGH.43

which apparently had healed by the time the fourth count was made. Rat C l l appeared perfectly well and showed no evidence of infection throughout the period.

Comparing these figures with those obtained by others on normal rats, we find that the platelet percentage and the cal- culated platelet number are considerable higher than those found by other workers, except Bedson and Zilva ('23). These authors, using blood from the tail and the indirect method of counting, obtained in seven normal, young rats an average of 1,208,857 platelets and 7.8 million erythrocytes ; ratio about 1: 6.5. The exact age of their animals was not given, but they were much smaller than those used by us (about 50 grams). Taylor ( '15)' the first to publish results of platelet counts in rats, obtained in eighteen healthy albino rats an average of 1 million platelets and 10 million red cells. Cramer, Drew, and Mottram ( '23) found about 800,000 with 9 to 10 million red cells in animals weighing from 50 to 150 grams. Laurens and Sooy ( '24), in six-month-old rats kept in room light, found by the indirect technique 762,000 platelets and 7.3 million erythrocytes (ratio, 1 : 10). Stammers ( '25) states that in his colony of normal rats the level is slightly under 900,000. Direct methods in which measured dilutions are made in an erythrocyte pipette usually yield much lower results ( Shulman and Mendel, '24 ; Laurens and Sooy, '24). The results obtained by us in group 11, the animals. in which we used the tail blood for counts, are practically identical with those of Bedson and Zilva, the average in the initial counts being 1,275,000 platelets with 9.6 million erythrocytes ; ratio, 1: 7.5. It seems probable that the differences between our results and those of most other workers are to be ex- plained by differences in the strains of rats used, and not to differences in technique, since we have shown that the differ- ent methods commonly employed are capable of giving identi- cal results.

BONE-MARROW EEACTIOKS. I1 67

LITERATURE CITED

BEDSON, S. P., AND ZILVA, S. S. 1923 Platelet count in rats suffering fror!i vitamiiie A deficiency. Brit. J. Exper. Path., vol. 4, pp. 305-310.

CRAXER, W., DREW, A. H., AND MOTTRAM, J. C. On the behavior of plate- lets iii vitamin A dcficiency aiid on the technique of countiiig them. Brit. J. Exper. Path., vol. 4, pp. 37-44.

FONIO, A. 1912 Gber eiii iieues Verfahren der Blutplittehendhlung. Deutsche Ztschr. f . Chir., Bd. 117, 5. 176-194.

GUNN, F, D. 1926 The influence of ultraviolet light upoii blood platelets in young rabbits. Proc. Soc. Exp. Biol. and Med., vol. 24, pp. 120-123.

1927 Increase of blood platelets in rabbits by a specific baeterial infection. Proc. SOC. Exp. Biol. aiid Med., vol. 24, pp. 857-859.

LAURENS, H., AND SOOY, J. w. The effect of light and of darkness o n the blood'cell number of the albino rat. Proc. Soe. Exp. Biol. aid Med., vol. 22, pp. 114-115.

REIMANN, H. A. 1924 The blood platelets in piieumococcus infections. J. Ex- per. Med., vol. 40, pp. 553-565.

SHULMAN, D. N., AND MENDEL, L. B. The blood platelets in rats on ade- quate and inadequate diets. Proc. SOC. Exp. Biol. and Med., vol. 21,

SOOY, J. W., AND LAURENS, H. 1924 A method for counting blood platelets in the rat. Proc. SOC. Exp. Biol. and Med., vol. 22, pp. 116-117.

STAIINERS, A. D. 1925 011 the effect of deficiency of vitamin A 011 the blood platelet count in rats. Brit. J. Exper. Path., vol. 6, pp. 312-313.

TAYLOR, K. 1915 Studies on the blood of the albino rat. Proc. Soe. Exp. Biol. and Med., vol. 13, pp. 131-134.

THOMSEN, 0. 1920 Direct count of blood platelets. Acta Med. Scaiidiiiav., vol. 53, p. 507.

1923

1924

1924

pp. 435-436.