lll-Indium labelled canine platelets

T’HROMBOSIS K&SEARCH vol. 13, so. 2. pp. iTi .?: Peqzmon Press Ltd. 19%. Printed in Great Entin.

ill-INDIL? LABELLED CANINE PLATELETS

A.R.Wilkinson, R.J.Hawker and Linda M.Hawker, Department of Surqery, University of Birmingham, England

(Received

3.5.1978; in revised form 31.5.1976. Accepted by Editor A.L. Bloom)

ABSTRACT

This paper describes a method of separating and labellinq canine platelets with Ill-In-oxine with full retention of aggregation responses to adenosine diphosphate. The technique required only 22.5 ml of blood and the labelled platelets were reinjected within 45 minutes cf initial venepuncture. The platelet survival in beagle dogs was shown to be linear with 13.6% loss per day from the intravascular space, giving a total survival time of 7 days. The mean labellinq efficiency was 85% and elution of the isotope from the platelets was reproducibly less than 2%.

INTRODUCTION Isotopically labelled platelets have been used over many years for monitoring platelet kinetics in vivo, the most commonly used isotope being sodium 51chromate t51Cr) (1,2,3). Chromium is an unsatisfactory label in several respects, having a poor labelling efficiency, high in vivo elution, and low (8%) gamma emission.

This limits the in vivo visualisation of platelet accumulations using a gamma camera. Most reported methods require relatively large volumes of blood. Bj(Jrnson (4) has shown that under certain conditions platelets labelled with 51Cr

175

IN-LXBELLED PLATELETS

176

vo1.13,xo.2

retain aggregation responses to Adenosine diphosphate (ADP) and collagen similar to those seen with platelet rich plasma (PRP) 51 although other reported methods of Cr platelet labelling do not mention the effect of the labelling procedure on platelet function. In view of these disadvantages with 51Cr an alternative isotope for labelling platelets has been sought. Recently Thakur and colleagues (5) described the use of 111 Indium chloride (111In) complexed to 8-hydroxyquinoline (8HQ) for labelling both human and canine platelets. This method yielded a high labelling efficiency and low elution, the aggregation responses to collagen (of the washed platelet preparation) were unaltered by the labelling procedure. In addition,111In has a half-life of 2.8 days and has the advantage of gamma photon energies of 173 KeV (84%) and 247 Kev (96%). Platelet function, as measured by aggregation studies, is readily altered by many factors including pH, temperature, physical damage and storage time (6). For both kinetic measurements and the study of platelet deposition on prosthetic sur faces it is important to minimise these known harmful effects. This paper describes a method of separating and labelling canine platelets with I,'1In-oxine using minimal centrifugation in the presence of a potent aggregation inhibitor, prostaglandin El (PGKl) (7). MATERIALS AND METHODS Preparation of 111In-oxine Three mCi of 1llIn Cl2 in 1.0 ml 0.04M HCl obtained from the Radiochemical Centre (Amersham) was neutralised with 0.2M NaOH and buffered to pH 5.4 by addition of 200 ~1 0.3M sodium acetate-acetic acid buffer pH 5.4. One hundred kg 8HQ in redistilled ethanol (1 mg/ml) was added to the buffered 11lIn Cl2 and incubated at room temperature for five minutes. The resulting lipid soluble complex of 111 In-oxine was extracted twice with 1.5 ml dichloromethane (Merck) (8) and evaporated to The complex was redissolved in

dryness at room temperature.

150 ~1 ethanol and stored at 4OC until required.

i-.01.

1j

)

50.

-i>-- i

IS-LABELLED PLATEL.E?S

2

Ringer's Citrate Dextrose (306 mm01 pH 7.0) Made up freshly as two litres of aqueous solution containing 12.6g sodium chloride, 588 mg potassium chloride, 12.89 trisodium citrate.2H20, log D-glucose, 6.1 ml of 1M calcium chloride and 56 ml of 0.59 % w/v sodium hydrogen carbonate freshly saturated with carbon dioxide. Platelet Labelling Procedures Twenty-two point five ml of blood were withdrawn: by careful clean venepuncture from the jugular vein of dogs and transferred to a plastic universal specimen container (Sterilin, 128B) containing 2.5 ml 3.8% trisodium citrate.

After .!ixing,

the anticoagulated blood was centrifuged at 200 q for 10 minutes to produce PRP.

The PRP was gently transferred to a round

bottomed polystyrene tube using a siliconised pipette and 100 ng/ ml PGE1 was added.

The PRP was centrifuged at 640 g for 10

minutes, the platelet poor plasma (PPP) containing PGEl was removed and retained, and the platelets were resuspended in Ringer's citrate dextrose (RCD) containing PGE1 (100 ng/ml) * to one half the original volume. Two point five ~1 of the 111 In-oxlne was added to the resuspended platelets and prepared incubated at 37OC for varying time Lntervals.

The original

volume was restored at the end of this time by the addition of the retained PPP containing PGEl. The platelets were centrifuged as previously described, and after removal of the diluted PPP, the platelets were resuspended in autoloqous PPP (obtained by centrifugation,at lOOCg, of the anticoagulated blood after removal of PRP). The count rates of the 111In-oxine labelled platelets after resuspension and the diluted PPP were compared and labelling efficiency calculated.

Radioactive measurements were made in a well

crystal detector (NaI) linked to a counter (J & P Engineering Ltd. MS 310).

Experiments were carried out to show the effects of PGEl on the labelling efficiency of platelets.

Aggregation responses of the labelled platelets were compared with those of PRP using 2.2 x 1O'5 M ADP as the aggregating agent (Payton aggregation module). After completion of these in vitro tests the radio-labelled platelets were re-

IN-LABELLED PL.iTELETS

178

v01.13,50.2

injected into the donor animal. The in vivo elution of isotope from the labelled platelets was measured by withdrawing 5 ml of blood one hour after the labelled platelets had been re-injected, the cells and plasma being separated by centrifugation.

In vitro

elution was measured by diluting a platelet sample 1 in 20 in RCD and after centrifugation, the pellet and supernatant were counted. For survival studies, 5.0 ml of blood were withdrawn daily for 7 days.

The radioactivity in each sample was

expressed as a percentage of that taken one hour after the injection of labelled platelets.

Survival of labelled plate-

lets was calculated by a least squares linear regression plot of percentage circulating with time. RESULTS The optimal labelling time for retention of aggregation 111 In-oxine was found to be 7.5 minutes (Fig. 1)

and uptake of

This time interval was used for all future experiments involving in vivo studies. FIG. 1

-*-‘-* f”-

A comparison of the effects

oP O

\... ,I’ \ *, /

of time on the uptake of

\

lllIn-oxine by platelets (0) and the loss of platelet

’

0

I

function as measured by the

0

aggregation response to ADP (2.2 x 1C'5M3). The aggre-

gation responses (0) are expressed as a percentage

??

of that achieved prior to the 111In-oxine. addition of

10

20 llME lMlNZ.1

30

IX-LXSELLED PLATELETS

i’01.13,x0.2

I79

Figure 2 demonstrates that the addition of PGEl to washed canine platelet preparations does not greatly alter the uptake of 111In-oxine. 80

?? ---•_z=--0 -O-

0-O

c-

0

t

5I

I 10

1

15 TIME

1

20

IMINSI

FIG.2 Uptake of 111 In-oxine by canine platelets. of time (0) and PGEI (0).

Effect

The mean (+ one standard deviation) labelling efficiency 111In-oxine labelled canine platelets was in 17 preparations of 35.0 ,+ 12.7% (range 63.3 - 98.9%). The in vivo elution of isotope into the plasma at one hour was 1.7 + 0.9% (range 0.6 3.2%) and this corresponded to the results of in vitro elution. Figure 3 shows a typical comparison of aggregation responses between PRP and labelled canine platelets. An example of canine platelet survival is given in Figure 4.

180

IN-LABELLED PLiTELETS

RESPONSE

TO 2.2 x10-%

V01.13,x0.2

ALU?

‘I# Ill

In Oxine Platelets

Pluteiet Rich P&m FIG 3.

Comparison of aggregation responses to 2.2 x 10T5M ADP 100

??

. 80

-

\

60

.

40

.

to

.

.

\

.

0 2

4

6

DAYS

FIG 4 Typical platelet survival 6.9 days. (a) = 97.14% (b) = 14.01%

Vol.l3,Xo.2

IS-LXBELLED

The intercept gives value platelets

of the regression

(a) for the calculated

circulating

by equating

value of

181

line with

percentage

is estimated

(b) or loss percentage/day The mean values

(a).

in four normal beagle

dogs are given TABLE

Calculated

values

Loss following

the ordinate

of labelled

The survival

at zero time.

the slope

calculated

PLATELETS

with

the

of these parameters

in Table

1.

1

for labelled

injection

canine

platelets

4.70 2 3.12%

(100-a) =

Loss per day

(b)

= 13.62 2 0.674

Survival

(a/b)

=

7.01 f 0.24 days

DISCUSSION The present use in different factory method Thakur platelets

with

interest

aspects

of monitoring

all of which

stages

are known

technique

deficiencies

and prolonged

the number

Furthermore

time with

platelet

is comparable

platelets

as in the previously The survival

to that reported

using

imately ten percent circulation. to small amounts

with isotope,

to the platelet

(6). The

these

of Indium 51Cr

labelled

and expected

to

and labelling

after

the labelling

than to unlabelled

described

method

washed

(5).

platelets

is comparable

(5) and is linear until approx-

this value

radioactivity

remains

it is curvilinear,

of label attached

significant.

responses

function

of the injected

Below

of centrifugation stages 111 In-oxine is limited

to PRP rather

to other blood

Extrapolation

line to zero time shows a minimal observed

incubation

to counteract

aggregation

procedure

becoming

a satis-

in that the total ex vivo time for the platelets

to give optimal

efficiency.

drugs and their requires

kinetics.

to be injurious

and the incubating

7.5 min.

platelet

has been developed

is less than 45 minutes, reduced

disease

et a1.(5) have described a method for labelling 111 In-oxine which is time consuming, requires

many centrifugation

present

in antiplatelet

of arterial

(100%) value

in

probably

of the linear regression

difference

due

constituents

between

of the circulating

the radio-

IX-LABELLED

152

labelled platelets.

PLATELETS

v01.13,x0.2

This finding implies that only a small

number of the radiolabelled platelets are damaged although the differences might equally be explained by elution of isotope. This rapid and reliable method provides a useful technique for the study of platelet kinetics and may be employed to investigate platelet deposition on prosthetic surfaces. REFERENCES Reclassification of thrombocytopenias by the Cr-51 labelling method for measuring platelet life span. N.Engl. J.Med. 264, 1294, 1961.

1. COHEN, P., GARDNER, F.H. and BARNETT, G.O.

2. ABPAHAMSEN, A.F. A modification of platelet 51-Cr labelling. Stand. J. Haemat. 5, 53, 1968. 3. HARKER, L.A. and FINCH, C.A. Thrombokinetics in man. J.Clin. Invest. 48, 963, 1969. 4. BJBRNSON, J. The effect of the 51-Cr labelling procedure on platelet aggregability. Stand. J. Haemat. 13, 252, 1974. 5. THAKUR, M-L., WELCH, M.J., JOIST, J.H. and COLEMAN, R.E. Indium-111 labelled platelets: Studies on preparation and evaluation of in vitro and in vivo function. Thromb. Res. 2, 345, 1976. 6. HAN, P. and ARDLIE, N.G. The influence of pH, temperature and calcium on platelet aggregation: maintenance of environmental pH and platelet function for in vitko studies in plasma at 37oC. Brit. J. Haemat. 5,373, 1974. 7. SHIO, H. and RAMWELL, P.W. Prostaglandin E in platelet harvesting. An in vitro study. sci. 175,5 36, 1972. 8. SCHEFFEL, U., MCINTYRE, P.A., EVITT, B., DVORNICKY, J.A.Jr. BOLLING, D.R. and MURPHY, E.A. Evaluation NATARAJAN, T.K. , of Indium-111 as a.new high photon yield gamma emitting 'physiological' platelet label. John Hopkin Med. J. 140, 285, 1977.