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.