Comparative hematology: Rhesus monkeys (Macaca mulatta)

COMPARATIVE RHESUS MONKEYS

HEMATOLOGY: (MACACA MULATTA)”

JESSICAH. LEWIS Department of Medicine. University of Pittsburgh, Pittsburgh. PA 15261. U.S.A. and the Central Blood Bank of Pittsburgh, 812 Fifth Avenue. Pittsburgh, PA 15219. U.S.A.

Abstract-l. Coagulation and fibrinolytic tests on rhesus monkey compared to human blood showed long Russell Viper Venom and thrombin times and higher levels of factors VJJ and XIJJ. 2. Platelets were smaller than human but similar in ultrastructure. Monkey platclcts aggregated with ADP, collagen and ristocetin but not with epinephrine. 3. Streptokinase and urokinase activated monkey plasminogen. 4. Erythrocytes were similar to human in numbers. hemoglobin content. and appearance on scanning electron microscopy. 5. Biochemical tests showed calcium. sodium, BUN, alkaline phosphatase, CPK. LDH and SCOT to be higher than the upper limits for normal humans.

INTRODUCX’ION

RESULTS

The rhesus monkey (Mucacu tnularta) has been widely

used as an animal model in physioiogical and pathological research. No thorough coagulation study on normal animaIs has been published since one from this laboratory (Didisheim ct d.. 1959) which, of course, did not include assays for activities not known at the time. This present study was undertaken to update knowledge concerning coagulation and platelet parameters in monkey blood. Observations on the fibrinolytic enzyme system, on erythrocytes and leukocytes and on biochemistry and blood enzymes are included. MATERIALS

AND METHODS

Fourteen adult (>4 yearsold) monkeys were studied at the Primate Research Laboratory. Department of Physiology, University of Pittsburgh, School of Medicine, through the courtesy of Drs Ernst Knobil and George R. Fritz. These included X females (#4 to # 1I). Blood was obtained from femoral veins using a two syringe technique and non-wettable surfaces. Blood for coagulation and electron microscope methods used 1/l@ part of 0,l M sodium citrate as anticoagulant. All coagulation tests employed fresh plasma. Coagulation and electron microscope methods have been published (Lewis. 1976; Lewis, 1975). Proteolytic assays were done using radial di~Llsion plates (Worthington Biochemical Co.) prepared with casein incorporated in agarose gel containing TrisHCl buffered at pH 7.6. calcium ion and inhibitors of microbial growth. Streptokinase (SK) was VaridaseR (Lederle) at a final concentration of 50 urni. Urokinase (UK) (Leo Pharmaceutical) was used a final concentration 100 Ploug u/ml. Activation was accomplished by preparing mixtures of 200~1 plasma plus 50~1 activator and then applying 10~1 mixture to a plate well. Inhibition of trypsin was measured by incubating 200~1 plasma with 50~1 O.ly,; try@ (Sigma) for 1hr at room temperature and filling a well with IO ~1. After 24 hr, the diameters of the cleared zones were read in millimeters. *This study was supported by Grant HL-02254 from the National Institutes of Health.

Table 1 lists the results which are very similar to those obtained on humans. The shorter clotting times probably relate to some difficulties in obtaining blood samples. Russell Viper Venom (RVV) times and thrombin times were consistently 5% set longer than human. Skin bleeding times were 2; to 3qmin. Thromboplastin generation test yielded a substrate clotting’ time of 13 set on monkey and I2 set on human platelet poor plasma.

On the first four animals factors VII and X were assayed together. In the last 6 monkeys separate assays, using human deficient plasmas, showed VII to be just above the human range and X to be at the lower limit. Factor XIII also assayed above the human range in most of the animals.

Platelet counts ranged from 289.000 to 804,000 per ~1 with a mean of 478,000 (human range 150,00&450,000). Platelet-glass adhesive index was measured in 5 animals in all of whom it was > 25”,, which is within the normal human range. Platelet aggregations are shown in Table 3. Response to adenosine diphosphate (ADP), bovine collagen (insoluble and solubilized) and Ristocetir? was identical to human. Epinephrine caused minimal aggregation in only one monkey platelet rich plasma. Platelet ultrastructure Platelets were small, averaging 2 p dia on stained smear. On transmission electron microscopy of ultrathin sections internal structures (see Figs 1 and 2) were identical to those found in human platelets and included a prominent open canalicular system, circumferential microtubules, glycogen particles, o! granules, dense bodies and mitochondria.

JESSICA

Table

I. General

H.

LbWIS

coagulation

tests

Clo~tmg time Glass (mm) Clottmg time-Slbcone (nun] Clot retractlo” (0 to z+, Clot lysn SPT-Anmul substrate’ (set) SPT Human substrate’ (set) PT Simplastm’ (xc) PT-Russell Vlprr Venom (set) APTT (secl Recalclficatlon tome (SK) LjSlS Lysls monochloroacctic Thromhm Time bovine thrambm (seci Amthrumbm III Immedlatr (xc) 6 ml” mcubatmn (xc)

(I”,>

SD. = Standard

’ SPT-serum

Deviation prothrombin

= t ‘(I: d’,N ~ I). time used animal or human

Table

”0 human standard 1X XI XII

1 XIII'

III 92

375 II4 105

563 100 I13

194 123 76

107 75 75

I16 125 I30

II2 I20 I75

I34 IO0

64

* As reciprocal Standard human

2. Coagulation

of plasma dilution plasma was pooled

64

6.4

110

26X 107 II3 I98 59 125 95

90 95 i(

64

which stabilizes from 12 donors.

Erythrocyte Values (Table 4) and Scanning Microphotography

BaSO,

factor

237 IO3 I Oh I63 47

iin

100 I20 YO x

a purified

adsorbed

assays

plasma

for substrate.

on plasmas

293 I19 I OX I x9 56 105 90 I(X) 90 I6

31x 107 95 I50 s4 I on IO0 IO loo 32

human

Elrctrorl

Table 4 gives erythrocyte values in 13 monkeys. In some of the animals red cells were slightly but not significantly smaller and contained less hemoglobin than their human counterparts. On scanning electron microscopy (Fig. 3) erythrocytes were biconcave or doughnut-like and indistinguishable from human cells.

oxalated

147 I47 I23 225 72

22s 107 I?1 18’) 41

I6

K? 90 90 x

fibrinogen

Table

12'

ix0 113x I(15 I 1X5.6 54.x 4 lOI 1

III Ih 149’) 1394 2h 49 1065 1633

92 0 144

447 27 II

IO’)

,:9x

I000

I in 4?(1

I cn

I ill

x

I6

1125

clot in lo,, monochloroacetic

3. Platelet

3 x IO “M fpme;hrme Ristocctm 9 mg,ml

aggregation

0 I no

IO IO0

acid.

tests

0 IO0

3

Ior1

KU 100

x0 100

Aggregation system = 50~1 reagent concentration above + 400~1 platelet rich plasma (count adjusted

as to

250,000).

White cell counts were very variable--from 5,200 to 17,200 per cm in these presumably healthy monkeys. Differential counts and cell appearance were not different from human. Fihrinolytic

system (Table 6)

Caseinolytic activity of human and monkey plasmas were compared after mixture with streptokinase or urokinase. Monkey plasminogens were activated to approximately the same extent as was human. Trypsin was inhibited slightly more by monkey than by human plasma. No sex difference was noted.

Proteirz

ekctrophorrsis

(Table 7)

Results fell within the human slightly higher p globulins. Biochemical

range

except

for

tests (Table 8)

The last column indicates values which show statistically significant (high or low) differences from human. Note that calcium, sodium, BUN and enzyme

values were higher than human. were low.

Uric acid and CO,

Comparative

hematology:

rhesus

381

monkeys

Figs I. and 2. Monkey platelets. Note microtubules (MT), 2 granules. dense bodies in vacuoles (DB VAC) the open canalicular system (OCS) and glycogen. particles (GLY) (Top x7X.600; bottom x 56,840) Table 4. Erythrocyte

“,,

46.0

46 0

46.0

44.0

400

.3x.5

Hgb g/d1 RBC x 10h:pI MCV R MCH PB MCHC g/d1

15.1 52 XX0 29.0 33.0

14.3 5 55 83.0 260 310

13.9 5.7 XI 0 24.0 30.0

14.3 6.6 67 0 22.0 33.0

13.7 50 80 0 27 0 320

I2.0 4.75 XI 0 15 0 32.0

PVC

41 0 ill 4Y5 X.70 26 0 320

values

440 I76 51

‘70 231) 310

I

39 I24 54 731) 23.0 315

42 6 Ii I 515 740 23 0 31.0

Average diameter on Wright stained smear = RBC = 6~. Values on Monkeys on Monkeys 9-14 with the Coulter S. Human range is for Coulter S.

Table

5. Leukocyte

and differential

460 149 61 760 24 0 320

l-8

4Y 5 15X 6.7 710 23.0 320

46.0 145 63 720 23 0 32.0

43.75 13.90 5 67 11.54 2453 ?I 71

3 30 II2 0 62 5 80 I 99 0 X1

obtained

with manual

37 52 I? IX 42-6.2 79-97 27 ~31 32-w

methods;

counts

Monkey

WBC

x lO”/pl

Polys “4, Lymphs :, Mono “:, Eos “/,

1

2

3

5

6

7

X

Y

8.0

93

I5 15

172

56 39 1 4

41 44 0 Y

55 43 0 2

39 5x 0 3

III I7 76 0 7

I-l7 26 71 0 5

I4.Y5 59 3x 2 3

XI 41 53 0 6

IO

6X 40 51 0 3

II

I?

13

14

M~iIll

S.D

10.9 -lo 52 3 3

IO 5 30 64 ‘4 2

52 41 55 0 4

73 4X 47 0

I07 41 5 53 2 077 43

37 12.1 II 7

5

2.1

Human Range ~0-100 55~75 20 40 2 IO 0 3

382

JE~SSICAH. LEWIS

Fig. 3. Scanning

electron microphotograph of monkey crythrocytes. Note the central Iqmphocyte. ably a T lymphocyte. and the doughnut-shaped erythrocytes ( x 5420)

prob-

DISCUSSION

Rhesus monkey blood coagulation is remarkably similar to human. Of the general coagulation tests, only the Russel Viper Venom (RVV) and thrombin times were significantly longer than human. The RVV difference was also noted by Nageswara et al. (1970) in female monkeys and both differences by Abildgaard et (I/. (1971). In this study all coagulation factor levels fell within the human range except factors VII and XIII, each Table

7. Protein

electrophoresis

(results

Table

X. Biochemical

tests

in g/dl)

I/ T. Protc~n Alhurmn Calcium Phosphorus Cholcstrrol tilucosr Llr,c Aad

P dl a dl

C rrat,n,nc T Btbruhm BUN Chlonde CO, Potasstum Sodturn Alk. Photpl CPK LDH SGOT

mg dl mg dl g dl

*

mg dl mg’dl mg dl rng. dl mg dl

m-cqu,i m-equl\ m-rq,,,\ I”-cqu,\ mu ml mu ml mu ml mu ml

Compared to human.

I I

I I

HI II 10 7 hl IhX 0 ,?I 0 0 -1

Y0 II I? 41 ,250 ‘a 0 0 7

51 IX0 16 0 II.?

4x 1780 XSO 0 7

Ii 0 ? 24 0 IOh 0 lU2

Ih 0 1 I6 0 105,) I’) 0

I I 03 26 0 1020 10 3

I’ (1.4 25.0 I OS.0 20 0

1x ITI0

1’10 I90 0 365 0 770

I

Y.6 42

II ?

Yh 4x

I2

52 ,ii,, I’7 0

42 47 IV10 I510 ?KO.O 1120

RtlO0 529 0 73 0

69 0 5x0 0 69 0

> I200 5 I?.0 77.0

x4 30 107 37 ,410 84 0 0.3 I6 03 ?2 0 IO6 0 IX5

4.0 I45C ,750

2 15.1) 39x 0 58 0

X.9 4.2 II 5 4’) I47.0 940 0 3 15 03 27.0 IOSO 19.2 4.4

0 69 0.44 0 8 I 00 19.60 21 64 007 0.21 005 3.16 I 66 0.97 07x

204 0 447 0 477.0 71 0

56 9X

,500

I

100

91.27 7 89

60 80 3 5-5.0

x5-105

2.5-45 Is&300 65-115 2.5 8.0 07~1 4 0.18-I 3 IO 20 95-105 24-32 3.5 50 135 I45 3&l OS 25- 160 9s 203 IU 58

H H

L

H L H H H H H

3x3

Comparative hematology: rhesus monkeys

of which was higher than human. Our results are fairly similar to those of Abildgaard et al. (1971) except the levels of factors XII and VIII both of which they found to be higher (XII = 527, VIII = 237) than in this study. Seaman & Malinow (1968) measuring only fibrinogen and (VII + X) found these within the human range. Bimdorf et a/. (1971) studying the Java monkey, found coagulation factor levels very similar to human. Platelet counts varied considerably and the mean was slightly greater than human. Monkey platelets aggregated readily with ADP, collagen and ristocetin but not with epinephrine. The small monkey platelets were ultrastructurally identical to their human counterparts. Ablondi & Hagan (1958) showed that SK activated monkey, cat, dog and rabbit plasminogens, probably through a preliminary activation of proactivator. Our results confirm their findings and show that both SK and UK activate monkey plasminogen. The biochemical tests showed some marked differences from human. Calcium, sodium an BUN were significantly elevated as were the enzymes, alkaline phosphatase, CPK, LDH and SGOT. Elevated enzyme levels have been found in some other mammals (Lewis, 1974, 1975, 1976).

REFERENCES ABILDG~RD C. F.,HARRISONJ. & JOHNSONC. A. (1971) Comparative study of blood coagulation. f. A&. Physiof. 30, 400-405. ABLONDIF. B. & HAC,ANJ. J. (1958) Studies on specificity of streptokinase. Proc. Sot. r\-p. Bid. Mrd. 99, 769172. BIRNDORFN. I., PEARSOKJ. D. & WREDMANA. (1971) The ciotting system of monkeys: A comparison of coagulation factors and tests between Cvnomolgus monkeys (Mucnccl irus) and humans. Cuntp. B&cheat. Ph.~siol. 38A, 157-161. DIDISHEIMP.. HATTORIK. & LEWISJ. H. (1959) Hematologic and coagulation studies in various animal species. J.-L& Clirt. Med. 53, X66-875. LEWISJ. H. (1976) Comnarative hematolopv: Studies on goats. 4nt. ‘.I. I&. K