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Travenol Laboratories, Inc., Morton Grove, Illinois,. U.S.A.. MATERIALS AND METHODS. The experiments were performed on 21 purebred Holstein calves with ...
The Piromen Test as an Assay of Bone Marrow Granulocyte Reserves in the Calf 1. Studies on Bone Marrow and Peripheral Blood Leukocytes J. H. Lumsden, V. E.

0.

Valli, B. J. McSherry and R. A. Willoughby*

ABSTRACT The use of Piromen, a polysaccharide complex of Pseudomonas aeroginosa, has been investigated in 21 calves by a test of marrow granulocyte reserves. The maximal increase in peripheral granulocytes (G) was determined following multiple intravenous and subcutaneous injections of Piromen at various time intervals and was correlated with the mature marrow graxulocytes on bone marrow smear differentials. Five ug Piromen per kg body weight, by the subcutaneous route, was found to give a mean A\G of 5200/mm', very similar to the A\G of 5300/min obtained in man with 0.1 ug/kg intravenous Piromen injections. Clinical effects in calves were minimal with the subcutaneous route as compared to the response following intravenous Piromen injections.

(AG) au moyen de plusieurs injections intraveineuses et sous-cutanees de Piromen, i differents intervalles, et ils la comparerent au nombre de granulocytes matures observe's dans des frottis de moelle osseuse. Ils constaterent que l'injection sous-cutanee de 5.0 ug/kg de Piromen donnait une AG moyenne de 5,209/mm-, comparable i la AG de 5,300/mm' obtenue chez l'homme, i la suite d'injections intra-veineuses de 0.1 ag/kg de ce polysaccharide. Les injections sous-cutanees de Piromen ne provoquerent chez los veaux que peu d'effets cliniques, comparativement aux injections intra-veineuses.

INTRODUCTION

Granulocytes perform a major role in protection of the body against disease, especially in the initial defense mechanisms (13). The availability of granulocytes in RkSUMA the face of a sudden demand is depedent upon a readily accessible reserve. Tie rate Les auteurs ont injecte it 21 veaux du Piro- of marrow granulocyte release which is men, un polysaccharide complexe de Pseu- independent of the cellular production time, domonas aeruginosa, dans le but de connaitre can be increased upon demand to the limits les reserves de la moelle osseuse en granulo- of the marrow granulocyte reserves (MGR) cytes. Ils determinerent la plus grande aug- (3). The ability of the cow to respond to mentation en granulocytes du sang circulant diseases of a septicemic or endotoxemic nature, such as coliform mastitis, Pasteurella pneumonia or acute metritis, appears to correlate closely with the ability to *Department of Pathology .. (Lumsden, Valli and McSherry) and Clinical Studies (Willoughby), Ontario Veterinary College, University of Guelph, Guelph, On- maintain an adequate circulating granulotario, Canada. cyte pool (17). If the MGR is directly related to the steady state level of granuloSubmitted April 4, 1973. 56

Can. J. comp. Med.

cytes in the peripheral blood, as has been indicated in the dog (14), then the normally lower peripheral leukocyte count and decreased granulocyte to lymphocyte ratio in the cow (16) would suggest a decreased MGR in this species. Peripheral granulocytosis was observed to be a characteristic reaction, after the initial leukopenia, following an injection of endotoxin (9). The early phase of granulocytosis was shown, by means of leukopheresis, to be due to granulocyte release from a reserve of maturing marrow granulocytes (4). Using similar techniques of leukopheresis, the presence of a MGR was further confirmed and measured in the dog (3, 14), in man (1) and in the calf (18). The peripheral blood neutrophilic response in dogs, after an injection of typhoid bacilli, was quite similar to that following leukopheresis and this response occurred only if there was an adequate preformed MGR (15). The magnitude of a post endotoxin peripheral granulocyte response was suggested to be related to the size of the MGR (9) and was therefore examined as a quantitative measure of the MGR. Heilmeyer (7) and Fink et al (6) demonstrated the efficacy of the Salmonella endotoxin, Pyrexal', to measure the functional marrow reserve in human patients. Since Pyrexal was no longer available, Korbitz et al (8) used Piromen2, a crude trypsinized

polysaccharide complex from Pseuadomonas aeruginosa, to determine the functional bone marrow granulocyte reserves in over 100 human patients. There was a close correlation observed in the peripheral blood quantitative granulocyte response in human patients following Pyrexal (12) and Piromen injections (8). Because of (a) the frequent occurrence of clinical neutropenia encountered in cattle, (b) the need to evaluate the MGR in studies involving leukokinetics and the response to hemoprovocatives and (c) the physical limitations of leukopheresis, the present study was undertaken to determine if the maximum increment in peripheral blood granulocytes (AG) following the administration of Piromen (Piromen test) would be an adequate assay of the MGR in cattle.

MATERIALS AND METHODS The experiments were performed on 21 purebred Holstein calves with initial weights from 95.5 to 200 kg and aged three to six months. The calves were fed commercial calf grower pellets according to directions and had free access to hay, water and salt. Each calf was clinically examined and blood samples were taken for the two weeks prior to each experiment. Piromen was given by intravenous (IV) and subcutaneous (SC) routes at dosages ranging from 0.1 to 25 ug/kg and at varying time intervals. Each injection was given at 0900 hours. The peripheral blood granulocyte response was determined after each injection of Piromen. Blood samples were collected in vials containing ethylenediamine tetraacetate3. Up to 17 blood samples and four bone marrow aspiration biopsies were obtained in the first 24 hours post injection, and up to six blood samples and three bone marrow aspiration biopsies in the second 24 hours. Daily blood samples were taken until the subsequent Piromen injection or until the experiment was completed. Temperature, pulse and respiration rates were determined at the time of each blood sampling. Leukocyte counts were determined using a Fisher Autocytometer4. Blood and bone marrow smears were Wright's stained using an Ames Hema-tek slide stainer4. A

total of 200 leukocytes were differentially counted' on each peripheral blood smear. Bone marrow aspiration biopsies were made using sterile 16 ga 1.5 inch needles6 and new sterile 12 ml disposable plastic syringes7. Bone marrow smears were prepared as described by Dacie (6). Five hundred consecutive marrow myeloid cells and all other nucleated cells encountered, including bare reticulum cell nuclei, were differentially counted on each marrow sample. GROUP I (Calves E, F, G, H and I) Calves A, B, C and D were used in pilot projects to examine the effect of low dos3Vacutainer, Becton Dickinson, Canlab Supplies, Toronto, Ontario.

'Difco Laboratories, Detroit, Michigan. 2BaXter

Laboratories,

Travenol Laboratories, U.S.A.

Malton, Ontario, Division of Inc., Morton Grove, Illinois,

Vol. 38 - January, 1974

4Fisher Scientific Co. Ltd., Toronto, Ontario. 5Clay Adams Inc. Laboratory Counter, Canlab Supplies, Toronto, Ontario. 6Yale Biopsv Needle, Becton Dickinson, Canlab Supplies, Toronto, Ontario 7Roehr Monoject Canlab Supplie3, Toronto, Ontario.

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age, 0.1 to 1 ug/kg, and high dosage, 5 to 20 ug/kg, IV injections of Piromen and the effect of various time intervals between injections. Four calves, E, F, G and H, were injected IV using 25 ug Piromen/kg body weight. The maximum peripheral granulocyte increase (AG) was determined for the immediate post injection interval. The available peripheral granulocytes were correlated with those observed in the bone marrow smear differentials. Fourteen days later an IV injection of 15 ug Salmonella endotoxin8 was given to calves E, F and G in an attempt to stimulate hematopoiesis, while calf H (hematoprovocative control) was given an injection of saline. After another ten days, the four calves were given a second IV injection of 25 ug/kg Piromen to compare the peripheral granulocyte response (AG) to the first and second injections of Piromen. Calf I was given three IV injections of saline and the blood and bone marrow were examined at the same time intervals as above. GROUP II (Calves J, K, L and M) Four calves were used, of which two

calves, J and K, were injected by the IV and two calves, L and M, by the SC route with 15 ug/kg Piromen on three consecutive days to compare the effect of the route of administration and daily injection upon the peripheral granulocyte response. GROUP III (Calves N, 0, P, Q, R and S)

The four calves, N, 0, P and Q were each given five SC injections of Piromen in 5, 10, 15 or 20 ug/kg dosages at ten day intervals arranged in an extra-period latinsquare change-over design (11) to examine the effect of dosage and repeated injections upon the measured AG. Calf R was given 15 ug/kg Piromen intramuscularly. Calf S (control) was similarly given five subcutaneous saline injections. GROUP IV (Calves T, U, V, W, X and Y) Four calves, T, U, V and W were given five SC injections of Piromen in 0.0 (saline), 0.1, 0.5 or 2.5 ug/kg dosages at ten day intervals in an extra-period latinsquare change-over design to further examine the relationship of dosage to the re-

8S. typ}iosa 00,1, Difco Lab., Detroit, Michigan.

TABLE I. The Maximum Increment in Granulocytes Endotoxinh Injection

Time (Days) After Initial Injection

Period 1..........

0

2.......... 3.........

14 24

a25

ug

Mean of Calves E, F, G (± 1 S.D.) A G

Dosage

(ug/kg)

(/mm3) 25, 4000 ±fi 600 0.15', 3800 ± 700 25a

1200

4

900

(ALG)

Following Piromen- and Salmonella

Control Calf H

Control Calf I

AG (/mm3) 6300 200 3000

Dosage

(ug/kg) 25a

Saline 25a

(ug/kg)

AG (/mm3)

Saline Saline Saline

2100 1100 1500

Dosage

IV Piromen/kg body weight

b20 ug IV Salmonella endotoxin S.D. = Standard Deviation of a distribution of individual observations

TABLE II. The Mean Granulocyte Response Following Three Daily Piromen Injections (15 ug/ kg) Given I.V. (Calves J and K) and SC (Calves L and M)

Period (Days) 1

2 3

58

Route I.V.

S/C I.V.

S/C I.V. S/C

AG (/mm3) 5300 7300 -3400 1500 -1500 1800

Absolute Granulocyte Values + 1 S.D. Minimum Maximum Preinjection Post Injection Post Injection 3400 450 3800 + 1200 8100 700 4700 1150 4400 i 100 3300 650

100 50 3200 + 650 80 i 10 2900 400 160 i 10 900 600

Can. J.

8700

400 350 4700 i 1000 6200 + 550 2900 200 5100 50

11100

4

comp.

Med.

TABLE III. The Mean Granulocyte Response in Calves N, 0, P and Q After Five SC Piromen Injections and Calf S After Saline Injections, Repeated at Ten Day Intervals

Dosage (ug/kg) 5 10

15 20 Saline Control

A G

5200 5400 6600 6000 1100

(/mm3) ±t 1100 -+- 750 4 2000 ± 900 + 150

Absolute Granulocyte Values + 1 S.D. Minimum Maximum Preinjection Post Injection Post Injection 4100 ±= 1050 3500 =t 800 9300 it 1300 3M00 it 900 3200 i 950 8700 i 1650 6000 4 1000 5400 it 1100 12600 i 1950 5700 -- 850 5000 ±t 800 11700 i 1600 3900 -- 7C0 3800 -- 9C0 5C00 + 850

TABLE IV. The Mean Granulocyte Response in Calves T, U, V and W After Five SC Piromen Injections Repeated at Ten Day Intervals

Dosage (ug/kg') 0.1 0.5 2.5

Saline Controls

AG 1600 1700 4200 1000

(/mm3) + 450 ± 750 ± 11C0 ± 350

Absolute Granulocyte Values + 1 S.D. Minimum Maximum Preinjection Post Injection Post Injection 45C0 + 850 3700 i 750 6100 i 700 4200 i 750 35C0 i 400 6500 + 800 + 3700 ± 650 33C0 500 7900 + 1550 3500 ± 7C0 2600 ± 4C0 45C0 ± 450

sulting AG. Calves X and Y were given saline injections and used as additional controls.

the second injection (Table I, F'ig. 1). The peripheral blood and bone marrow granulocyte response to the lV Salmonelba endotoxin was very similar to the response observed following the initial Piromen in-

RESULTS GROUP I

When 25 ug/kg Piromen was admin istered IV, a marked leukopenia and granulocytopenia immediately developed in the peripheral blood with the greatest decrease occurring at approximately one hour postinjection (Fig. 1). Peripheral leukocyte and granulocyte counts then rapidly increased peaking between 12 to 16 hours postinjection (Table I). The counts returned to prinjection values two days postinjection, then gradually increased peaking again at seven to eight days postinjection before returning to preinjection values (Fig. 1). Immature granulocytes were observed in the peripheral blood within four hours and were present until at least 24 hours postinjection. The percentage of bone marrow granulocytes decreased sharply within 12 hours after each 25 ug/kg IV Piromen injections but the AG was greatly decreased af ter

Vol. 38 - January, 1974

jection. Marked changes in the temperature, pulse and respiration occurred immediately after each Piromen or Salmonella endotoxin injection although the severity was less marked in response to the final Piromen

injection. GROUP II A marked granulocytopenia and leukopenia occurred in the peripheral blood after each daily IV injection of 15 ug/kg Piromen. The postinjection granulocyte increase after the initial injtction, e.g. AG of 5300 granulocytes/mm3, did not attain preinjection counts after either of the two subsequent daily injections (Table II). The same dosage of Piromen administered subcutaneously resulted in a mild granulocytopenia which was followed by a granulocytosis peaking at six to eight hours (Table II, Fig. 4). The AG was markedly decreased but present following the next two daily SC injections. Peripheral granulocyte immaturity occurred consistently after the

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hour post injection. Temperature elevation peaked at three to five hours postinjection. The clinical signs of depression decreased markedly after the second and third daily IV injections. When the same dosage of Piromen was given by SC injection, the initial clinical response was considerably less marked and had almost disappeared following the second and third daily injections.

IV but not after the SC injections. The marked peripheral granulocytosis which occurred approximately seven days after the last IV injection (Fig. 3) was minimal with the subcutaneous Piromen injections

(Fig. 4).

The bone marrow granulocyte reserves, as determined by differential counts on serial marrow aspiration biopsies, were decreased to a much greater extent with the IV as compared to the SC route of administration (10). The pulse and respiration rates of calves J and K increased immediately following 15 ug/kg IV Piromen injections with the maximum clinical depression occurring at one

GROUP III

A conisistent AG developed after each SC injection of Piromen (Table III). During the 24 hour post injection interval the

Cl)

cO

15

oC# E 10 C,

TIME (DAYS)

(Total leukocytes-, total granulocytes.--, band granulocytes... Fig. 1. The mean peripheral' blood leukocyte changes of calves E, F and G following intravenous injections of 25 ug/kg Piromen* or 0.15 uz/kg Salmonella endotoxin.**

I 96 40

20 E ..

0

"'

4

.

.....

....A

8

12

16

..

..

20

24

28

32

I1

.~~~~~~-

38

TIME (DAYS)

(segmented granulocytes-, band granulocytes.-., metamyelocytic granulocytes .). Fig. 2. The mean changes in maturation phase bone marrow granulocytes from calves E, F and G following intravenous injections of 25 ug/kg Piromen* or 0.15 jug/kg Salmonella endotoxin.

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Can. J. comp. Med.

Cv)

i

1i

0 5-

x

E E

14

cn

w

le

0 w

-J

2

TIME (DAYS)

(Total leucocytes -,total granulocytes.-., band granulocytes.-). Fig. 3. The mean peripheral blood leukocyte changes of calves J and K following three daily intravenous injections of 15 gg/kg Piromen.*

C#,

0 T-

15,

15

15

15

1

l

l

x

CIO

E E 10

a\0{

co w

0

"ae

:'

PU*I

51

\.%V*.~~

2e

0

1

N.

2

-."'

".

'

TIME (DAYS)

(Total Leucocytes-, Total Granulocytes.*-, Band Granulocytes.) Fig. 4. The mean peripheral blood leukocyte changes of calves L and M following three daily subcutaneous injections of 15 ug/kg Piromen.*

Vol. 38 - January, 1974

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percentage of bone marrow granulocytes observed on smears decreased as the dosage administered increased. The changes in temperature, pulse, respiration rate and depression increased directly with the dosage administered. The clinical response to subsequent injections at ten-day intervals was noticably decreased from the initial response.

GROUP IV A consistent AG did not occur after the 0.1 or 0.5 ug/kg SC Piromen injections (Table IV). The mean AG was similar to that observed after the saline injections. The mean AG after 2.5 ug/kg injections was not statistically significant (P-.0.05) but did greatly exceed the AG response obtained after the control saline injections (Table IV). The percentage of bone marrow granulocytes did not alter detectably in the 24 hour period after SC injection of 0.1-2.5 ug/kg of Piromen nor was there a significant change in the temperature, pulse or respiration rates when compared to the control calves.

DISCUSSION In our preliminary studies using calves, the IV injections of 0.1 ug/kg Piromen as used in man (8) produced no consistent or significant increase in peripheral granulocytes over a postinjection interval of 12 hours. Higher IV dosages of Piromen in calves, e.g. 5 to 25 ug/kg, resulted in an immediate severe leukopenia followed by a leukocytosis and granulocytosis which peaked about 12 to 16 hours postinjection. The variations in the interval between the IV injections and the peak granulocytosis necessitated frequent sampling to ensure that the maximum AG was observed. When the changes in peripheral leukocytes and granulocytes were monitored after an IV Piromen injection of 5 ug/kg or greater, the initial peak granulocytosis at 12 to 16 hours postinjection was followed in each case by a second period of granulocytopenia reaching a low at three to five days but followed by a marked increase peaking at five to seven days post-

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injection (Fig. 1). The granulocytopenia occurring at three to five days postinjection substantiates the importance of an adequate MGR as the maturation rate of committed granulocyte precursors can be altered to a very limited extent (2). The granulocytosis occurring at five to seven days postinjection would indicate the results of increased myelopoietic differentiation and maturation (19) initiated by the IV Promen injections and corresponds closely to the response which occurred after the depletion of granulocytes by leukopheresis (18). Both these observations concur with the reported granulocyte generation time of seven days in the calf (19). Because of the occurrence and the periodicity of this granulocytosis peaking at seven days following an endotoxin injection, all Piromen injections to the same calf were repeated at ten day intervals. When Piromen was administered subcutaneously to calves, instead of the immediate profound leukopenia observed after IV Piromen injections, a leukocytosis and granulocytosis occurred peaking at six to eight hours postinjection (Fig. 4). The leukocyte changes in calves after SC Piromen administration were similar to that observed in man after 0.1 ug/kg IV Piromen injections (8). Calves J and K were given IV injections while calves L and M were given SC injections of 15 ug/kg Piromen to demonstrate the effect of route of administration on the peripheral leukocyte and granulocyte response (Figs. 3 and 4). Injections of the same dosage and by the same route were repeated on each of the following two days to further compare the effect of the route of administration on the increment change in granulocytes (AG) and the bone marrow granulocytes. A marked decrease in the availability of marrow granulocytes w1is observed 24 and 48 hours after the initial IV Piromen injections. This change was suspected by the absence of a peripheral blood AG following the second and third (Table IV) injections (Fig. 3) and confirmed by bone marrow biopsy examinations which showed a depleted MGR. In comparison, following SC Piromen injections a minimal decrease in mature marrow granulocytes was observed and the peripheral granulocyte response, even though decreased (Fig. 4), was still greater than in most controls (Table V). When it was observed that a consistent response could be elicited in peripheral

Can. J. comp. Med.

TABLE V. The Mean Increment in Granulocytes ( AG), Direct and Permanent Effects, After Five Subcutaneous Piromen Injections at Seven Dosage Levels and Placebos of Saline

AG Direct

LDosT go

Salirne Controls (N =20) 0.1 05 2.5

10 15

20

Effcct (± S.D.)

(/mm3)

AG Permrnent Effects (/Ml:3)

900 ± 700

473

1600 1700 4200 5200 5400 6600 6000

+ +

±

± ± +

±

900

1500 2200 2200 1500 4000 1800

1333)

2252 4342 4557 5136

53l21

7720

granulocytes following SC Piromen injections, dosage levels of 5, 10, 15 and 20 ug/kg body weight were repeated five times in calves N, 0, P and Q at ten day intervals in an extra-period latin-square change-over design (11). Analysis of variance revealed no statistically significant difference in granulocyte response between injection periods (1 to 5), between dosages (5, 10, 15 or 20 ug/kg) or between calves (N, 0, P and Q). The mean AG of 5200 granulocytes/mm3 following 5 ug Piromen/ kg B.W., was clinically significant when compared to the control calf S or the mean AG of all the control calves. The statistical significance could not be confirmed because the control calf S was not included within the latin square even though it was used as a control throughout the five injection periods. The mean AG from calves N, 0, P and Q (5560/mm3) was only slightly lower than the mean AG of 5767/ mm3 from normal men given 0.1 ug/kg Piromen IV (8). The AG response following SC Piromen injections appeared to be greater at 15 ug/kg than 5, 10 or 20 ug/ kg B.W. (Table IV) but this relationship was not significant on analysis of variance. The second balanced square experiment was designed to further investigate the possibility of a dose-response relationship with lower SC dosages of 0.1, 0.5 and 2.5 ug Piromen/kg B.W., using saline injections as a control within the balanced square design. Statistical treatment by analysis of variance, calculation of confidence levels and comparison of the AG response after SC Piromen injections demonstrated that the 2.5 ug/kg dosage was well above the threshold response level

Vol. 38 - January, 1974

(Table V) but was not statistically different from the response in the control calves. Because the error mean squares for the two experiments was approximately equal (10), there is license to compare the direct effect means (A\G) of the various dosage levels and saline controls used in Group III and Group IV (Table V). The AG permanent effects are the direct effects plus any residual effects determined by analyses to be due to previous Piromen injections. A broad dose response relationship appears to be more evident when correction is made for the permanent effects on the AG (Table V). The presence of permanent AG effects after repeated SC Piromen injections in the calf, as shown by analysis of variance would appear to differ from the findings in man (8) where repeated IV endotoxin administration resulted in tolerance to the febrile effects but not to the hematological effects of endotoxin. The bone marrow aspiration biopsy examinations revealed striking differences in the percentage changes of maturing granulocytes after the IV and SC Piromen injections. As seen in Fig. 2, the IV Piromen injection of 25 ug/kg resulted in a marked decrease in the mature and band neutrophils with replenishment of the maturation pool over the next several days. In comparison the immediate decrease in marrow granulocytes after SC Piromen injections was much less marked (10). When 2.5 and 5 ug/kg Piromen were given SC, a significant AG response was seen peripherally while minimal or no concurrent drop in marrow granulocyte reserves was observed. The consistent peripheral AG response following SC Piromen injections with little change in marrow granulocytes, as compared to the marked drop in marrow granulocytes after the IV Piromen injections where an erratic and relatively decreased AG response was observed, would appear to indicate that different mechanisms are involved in AG production. As shown by Boggs (2), corticosteroids produce a blood granulocytosis by increasing the rate of release of marrow granulocytes as well as increasing the length of time the granulocytes remain in circulation. The plasma cortisol levels rose after each IV but not SC Piromen injection (10) and there was evidence of an increased percentage of immature blood granulocytes as well as a prolonged granulonytosis (Figs. 1 and 3). y

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Determination of the peripheral granulocyte circulating and marginated pool sizes may lead to further understanding of the mechanisms involved in post endotoxin granulocytosi s. VVhen Piromen injections greater than 5 ug/kg were given by the IV route, the clinical signs of shock were always evident and varied proportionally with the dosage administered. Some elevation in the respiratory and heart rate and temperature were present after the SC Piromen injections but they were minimal with dosage levels of 5 ug/kg or less. With the IV route of Piromen injection at the low dosages used in man (8), no consistent peripheral granulocyte response (AG) was detected and at the higher dosages examined, granulocytosis only occurred following the induction of severe leukopenia and clinical shock. When Piromen is injected subcutaneously using approximately 5 ug/kg body weight, measurement of the maximum increase in circulating granulocytes (AG) would appear to be an effective, simple method of measuring the presence of marroN grantulocvte reserves in the calf.

ACKNOWLEDGMENTS

This work was given financial support by the National Research Council, Ottawa and by the Ontario Ministry of Agriculture and Food, Toronto. The authors wish to thank Dr. J. Huang, Department of Mathematics and Statistics for advice on statistical evaluations and the technologists in Clinical Pathology for their willing assistance.

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tivity of bone marrow. Germ. med. Mon. 2: 300-303. 195'{. 8. KORBITZ, B. C., F. A. TORIN, H. L. DAVIS, G. RAMIREZ and F. J. ANSFIELD. The Piromen test: a useful assay of bone marrow granulocyte reserves Corr Therap. Ros. 11: 491-505. 1969. 9. LAWRENCE, J. S. Leukemia. A discussion of its various modes of production. J. Am. med. Ass. 116: 478-484. 1941. 10. LUMSDEN, J. H. The Piromen assay of marrow granulocyte reserves in the calf. Thesis, University of Guelph. 1971. II. LUCAS, H. L. Extra-per iod latin-square changeover designs. J. Dairy Sci. 40: 225-239. 1957. 12. MECHANIC, R. C., E. FREI, M. LANDRY and W. W. SMITH. Quantitative studies of human leucocyte and febirile response to single and repeated doses of purified bacteriial endotoxin. J. clin. Invest. 41: 162-172. 1962. 13. PAGE, A. R. and R. A. GOOD. A clinical and experimental study of the function of neutrophils in the inflammatory response. Am. J. Path. 34: 645-669. 1958. 14. PATT, H. M., M. A. MALONEY and E. M. JACKSON. Recovery of blood neutrophils after acute depletion. Am. J. Physiol. 188: 585-592. 1957. 15. PERRY, S., I. M. WEINSTEIN, C. G. CRADDOCK and J. S. LAWRENCE. The combined use of typhoid vaccine and p32 labelling to assess myelopoiesis. Blood 12: 549-558. 1957. 16. SCHALM, 0. W. Veterinalry Hematology. 2nd Ed. Philadelphia: Lea and Febiger. 1965. 17. STRAUB, 0. C., 0. W. SCHALM, J. P. HUGHES and G. H. THEILEN. Bovine hematology. II. Effect of partur ition and retention of fetal membr anes on blood mor phology. J. Am. vet. med. Ass. 15: 613-622. 1959. 18. VALLI, V. E., B. J. McSHERRY, G. A. ROBINSON and R. A. WILLOUGHBY. Leukopheresis in calves and dogs by extracorpoireal cir culation. Res. vet. Sci. 10: 267-278. 1969. 19. VALLI, V. E. O., T. J. IIULLAND, B. J. McSHERRY, G. A. ROBINSON and J. P. W. GILMAN. The kinetics of haematopoieAis in the calf. I. An autoradiographical study of myelopoiesis in normal, anaemic and endotoxin tr eated calves. Res. vet. Sci. 12: 535-550. 1971.

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