Functional and ultrastructural alterations of autologous platelets labeled with 111In-oxine

Eur J Nucl Med (1983) 8:172-178 European Nuclear Journal of

Medicine © Springer-Verlag 1983

Functional and Ultrastructural Alterations of Autologous Platelets Labeled with 111In-Oxine Pierre Bernard 1, Maurice Bazan a, Colette Foa 3, K6bir Mountaz ~, and Irene Juhan-Vague z Laboratory of Nuclear Hematology 1, and Laboratory of Hematology 2 (H6pital de la Timone Marseille) and INSERM Unit 1193 27 bd. Lei Roure, Marseille

Abstract. High activity labeled platelets could be useful for the detection and observation of small loci of thrombosis by gamma-camera imaging. Therefore platelets were labeled with 1 ~ lin_oxine containing increasing activities of 1 ~ ain to determine the elimiting amount of this tracer that did not cause cell damage. A labeling procedure was employed so that all the chemical parameters remained constant except the amount of l~XIn. Platelet damage was studied by ADP-induced aggregability according to the Born procedure and by scanning and ultrastructural electron microscopy.

Platelets labeled with the lowest activity 2.2 MBq/ml of platelet suspension (109 cells/ml) showed no alterations. With the highest activity studied, (22 MBq/ml) aggregability decreased by two-thirds and great changes in the shape of the platelets were seen by electron microscopy. These modifications were attributed to the decay of ~ 1 XIn predom- inantly located in the platelet cytosol. Labeling of platelets with an activity higher than 7.4 MBq/ml is unsuitable for detection of thrombosis since normal platelet functions are not retained.

Introduction

In 1976 Thakur et al. published a new method for labeling platelets using a lipophilic compound: 8-hydroxyquinoline complexed with l ~ I n or 11tIn-oxine. The in vivo survival of autologous l~lIn-oxine labeled platelets was similar to that measured with 51Cr-sodium chromate labeled platelets (Thakur et al. 1976; Vigneron et al. 1980; Schmidt et al. 1982) and the labeling efficiency was five to eight times greater with ~lXIn-oxine than with 5~Cr-chromate. Also, the physical properties of 1 ~ q n were compatible with detection by a gamma camera so it became possible to obtain imaging of platelet deposition on vessels or organs.

In the last 5 years many papers have been published on the correlation between platelet deposition and thrombus formation and detection of thrombi by x~-In-oxine platelets (Price et al. 1980; Moser et al. 1980; Stratton et al. 1981; Bernard et al. 1981, 1982a, b; Yui et al. 1982).

Owing to the high labeling efficiency of this radiopharmaceutical compound it appeared possible to label a small quantity of autologous platelets (2-5 x 109 cells) with a high activity, up to 100 MBq. These labeled platelets now appear

Offprint requests to: P. Bernard, Service d'H6matologie Isotopique, H6pital de la Timone, F-13385 Marseille Cedex 5, France

to be a very suitable tool for image recording in various pathological disorders. At the third World Congress of Nu- clear Medicine (Paris 1982) more than 20 lectures discussed this topic. Owing to the fact of the long delay in recording (1-6 days) and the relatively rapid decay of 111In, some authors have used a high activity of up to 55 MBq (Piek- erski et al. 1982).

We therefore tried to study radiation injury of platelets labeled by this method in relation to their functional properties tested by an aggregation method and the morphological changes detected by electron microscopy. The highest activity studied in the literature in respect to functional alterations was half that used in our work (Thakur et al. 1981).

Materials and Methods

Patients

Platelets were first collected from six healthy volunteers with normal platelet counts. Then, platelets were collected from sixteen cardiac patients to verify if differences appeared between normal and sick subjects. Informed consent was obtained from each patient. Labeled platelets were never reinjected into normal subjects.

Platelet Separation

Fifty milliliters of blood was collected with a 19 G needle into a plastic syringe containing 10 ml acid citrate dextrose anticoagulant (ACD) prepared by dissolving 1.78 g dehy- drated trisodium citrate, 0.63 g monohydrated citric acid and 3.00 g glucose in 100 ml distilled and apyrogenic water. All manipulations were then carried out in a laminar-flow hood.

After gentle mixing of the blood with ACD solution, the pH was nearly 7.05. Blood was transferred into two conical plastic tubes in equal volume (30 ml) and centrifuged 10 min at 180 g. The platelet-rich plasma (PRP) of both tubes was carefully removed and stored under U.V. light at room temperature p. The red cells buttons were centrifuged again for 10 rain at 200 g and the new PRPs were removed as completely as possible without red cell contamination and added to the previous PRP. About 30 ml PRP was generally obtained. ACD solution (1 ml) was added (pH dropped to 6.7) and 2 ml of this acidified PRP was kept for platelet count, aggregability determina-

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tion, and electron microscope study. The remaining PRP was then centrifuged for 10 min at 1,000 g, the platelet-poor plasma (PPP) was removed and stored at 37 ° C. The platelet button was washed once with acidified isotonic saline (1 vol ACD, 6 vol. saline: NaC1 9 g/l) and then finally suspended in i ml acidified saline. The pH of the platelet suspension was about 4.8.

Labeling of Oxine with 11 l in_Chlorid e

Commercially prepared x x lIn-oxine is not still available in France, and so the radiopharmaceutical compound was prepared in our laboratory.

An ethanol suspension of 8-hydroxyquinoline was desic- cated by lyophilisation and stored in dried form. For the labeling procedure the lyophilisate was resuspended in ab- solute ethanol (final concentration 1 mg/ml). Fifty micro- liters of an alcoholic solution of oxine were placed in a 2-ml bottle and 0.2 0.6 ml sodium citrate 0.1 M (pH 9.6) was added, according to the volume of xXlIn-chloride (11qn-S1 CEA Sclay-France) required for labeling. Oxine in sodium citrate was mixed 5 min on a vortex. ~X~Indium chloride solution (11, 37, or 110 MBq: 7(~200 MBq/ml) was then added and mixed for 5 rain. The final pH of 1 x 1-In-oxine in sodium citrate solution was 6.5.

A chromatographic control of complexation of oxine with ~X~In was systematically performed (Whatman paper No. 1, methanol-water 85+15 solvent). The labeling efficiency of oxine was generally greater than 90%.

In this preparation procedure the amount of oxine and ethanol was always the same and the indium activity was the only varying factor. This a ~ XIn-oxine was prepared daily so that fresh ~ ~ ~In-oxine was always used. (This is not the case with the commercially available compound).

Labeling of Platelets

The ~ ~ lIn-oxine solution was added dropwise to the platelet suspension and incubated at 37 ° C for 3 rain according to Sinzinger (1981). The suspension was gently mixed every minute. Then, 5 ml PPP was added and the incubation was continued for 3 min. The suspension was then centrifuged for 10 rain at 1,000 g and the supernatant was removed as completely as possible. The platelet button was washed twice with 2 ml PPP and then suspended in 5 ml PPP, the final pH was near 6.9.

The platelet suspension, supernatant, and contaminated washing PPP were counted in a precalibrated ionizing chamber to determine the labeling efficiency. Then 2 ml plasma suspension of labeled platelets (2.2-22 MBq/ml) was kept for functional and morphological examinations.

Platelet Count and Aggregability

The platelet count was performed with an automatic counter (Hemalog 8 Technicon). Platelet aggregability was studied with a turbidimetric aggregometer (coultronic). Ac- cording to the Born and Cross procedure (1963) the aggregability was measured after adding 0.1 ml (10 gM final concentration) ADP and calcium (10 mM final concentration) to 0.3 ml platelet suspension (diluted to 4 x l0 s cells/ml in PPP). The platelets were kept at 37 ° C before measurement. The aggregability of unlabeled and labeled platelets was measured at the same time, 2 h after the beginning of the

labeling procedure. The velocity of aggregability and the maximal amplitude of light transmission were measured. All results were expressed in percent of variation compared with unlabeled platelets.

Preparation for Electron Microscopy

For transmission electron microscopy, 200 gl platelet suspension of each group was fixed in 3% glutaraldehye in 0.2 M cacodylate buffer, pH 7.4, for 1 h at 4 ° C. After cen- trifugation, the platelets were resuspended in the same buffer, left overnight and postfixed the following day in 1% osmium tetroxide for I h at 4 ° C. Specimens were dehy- drated in increasing concentrations of ethanol and propylene oxide. After 24 h infiltration with a 50% Epon 50% propylene oxide solution, the platelets were embedded in Epon 812. Ultrathin sections obtained in an ultramicrotome (MT2 Reichert) were counterstained with uranyl acetate and lead citrate and observed in a Jeol 100 C.

For scanning microscopy, after fixation and dehydra- tion, the platelets were spread on a copper specimen holder, passed through acetate and critical-point-dried with liquid CO2 in a Balzers, coated with gold palladium in a Nanotech SEMREP 2 sputter coater and examined at 40 kV in the Jeol 100 C scanning electron microscope.

Results

Seventeen labeling procedures were performed on platelets of 6 normal subjects. In five cases each volunteer was bled weekly and their platelets were successively labeled with 11, 37, and 110 MBq of lZlIn-oxine, whereas, platelets of the sixth donor were labeled twice only: firstly with 37 MBq of 11 lin_oxine and secondly with ' unlabeled oxine' (all the reagents except indium chloride).

Platelets of cardiac patients were always labeled with 37 MBq 11~In-oxine which was the amount of ~a 1In generally employed by previous workers in surface imaging of thrombi (Stratton et al. 1981 ; Ezekowitz et al. 1981).

Platelet count

By withdrawing 50 ml blood, 10 ~° platelets were generally collected. During the platelet separation half the cells were lost so 5 x 10 9 platelets were labeled in final concentrations n e a r 10 9 cells/ml. There were very few other blood cells, less than 105 leucocytes and 10 v red blood cells/ml.

Labeling Efficiency

Labeling efficiency of platelets from normal subjects varied from one subject to another (35%-80%) and also from one labeling to another for the same subject. Average values and estimation of standard deviation were 56.8% _+ 12.3% (n= 16). There was no statistical difference between the three groups of labeled platelets 2.2, 7.4, and 22 MBq/lnl of platelet suspension (10 9 cells/ml).

On 13 occasions, the labeling efficiency of cardiac patients was greater than 45% (45%-82%) and the platelet- bound radioactivity was sufficient 18.8_+8.7MBq) to perform cardiac imaging. On three occasions, the labeling yield was too poor (17%, 21%, and 26%) and platelets were not reinjected.

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Table 1. Aggregability of t ~ lIn-oxine labeled platelets

2.2 MBq/ml 7.4 MBq/ml 22 MBq/ml Normal subjects Normal subjects Cardiac patients Normalsubjects n=5 n=6 n=16 n=5

Unlabeled oxine Normal subject n = l

Velocity of aggregability 104.6 -- 10.2 93.5 + 8.5 92.0 _ 9.8 32.0 +_ 20.9 105%

Maximum of light transmission 106.2_+ 9.6 87.8+_7.2 86.3__8.9 32.4_+20.2 97%

Results are mean _+ S.D. values, expressed as percentages of initial values before labeling

Table 2. Ultrastructural modifications in 11 ~In-labeled platelets

Ultrastructural Control 11XIn-labeling appearance platelets

2.2 MBq/ml pa 7.4 MBq/ml pa 22 MBq/ml pa

Scattered organelles 28 32 0.13 NS 32 0.43 NS 15 0.001 HS Clumped organelles 4 1 0.05 NS 2 0.29 NS 12 0.02 S Without granules 2 1 0.17 NS 3 0.19 NS 9 0.02 S

Total number of platelets 34 34 37 35

Each sample comes from the same normal subject

" P Fisher exact probability; HS =highly significant; S =significant; NS =non-significant

Platelet Aggregability

All results are summarized in Table 1. Great differences were found between the three groups of labeled platelets. With low activity (2.2 MBq/ml), the initial velocity and maximum aggregability were slightly enhanced compared with unlabeled platelets (105% and 106% respectively). When platelets were labeled with 7.4 MBq/ml, a decrease in the initial velocity and the maximum of aggregability was observed: there was no significant difference between normal subjects and cardiac patients. When the labeling procedure was performed with 22 MBq/ml, a marked decrease in the two studied parameters was recorded and, in one case, we observed no aggregability.

In the only case where platelets were labeled with oxine without 1ilIn there was no difference between aggregation before and after addition of oxine.

Electron Microscopy

It was found that platelet modifications were a function of the amount of 11 lIn-oxine used for labeling the platelets. Thus, at the lowest dose of 2.2 MBq/ml, no obvious changes in the platelets were observed, neither in the shape seen by scanning electron microscopy nor in ultrastructural mor- phology. Also, the general appearance of the open canicular system was normal and the c~ granules were scattered in the cytoplasm in a way similar to that of the c~ granule distribution of unlabeled platelets (Fig. 1 a-c). The microtubules were perfectly visible and numerous pseudopodia were present (Fig. 2 a-c) exactly as in normal platelets.

With 7.4 MBq/ml, the platelets showed some modifications. Firstly, slight changes in the general appearance were detected by scanning microscopy. Namely, the platelets ex- hibited very thin, long pseudopodia (Fig. 3 c, d). Secondly, the fine structure of the platelets appeared modified. Though there was no apparent alteration in the distribution of the different organelles, the contents o f the ~ granules

were less electron dense and the open canicular system seemed overdeveloped (Fig. 3 a and b).

When platelets were labeled with 22 MBq/ml the general shape visualized by scanning microscopy was modified. Sometimes the platelets were completely smooth (Fig. 4c) while sometimes they were completely covered with more or less elongated pseudopodia which gave them a spiky appearance (Fig. 4d). Fine structure observations showed that the organelles were clumped significantly in the cell center (Fig. 4a, b). Microtubules encircled the organelles. Complete degranulation was not detected and no platelet aggregates were observed. All these changes are listed in Table 2.

Discussion

By our platelet labeling procedure, the same amount of reagent was added in each group studied except 11 lin_chlo_ ride. Thus differences between groups could be only due to the effect of irradiation.

Indeed, various factors could alter platelet function. Joist et al. in 1978 and Thakur et al. in 1981 found no adverse effect of ethanol at concentrations less than 1.5% in the labeling solution (we used 1% concentration). These results were confirmed by Bengmark et al. (1981). Thakur (1981) found an inhibition of platelet aggregation with oxine concentrations higher than 20 gg/ml but we used only 10 gg/ml.

Other factors such as the relatively low pH of the medium (Djaletti et al. 1979) and protein-free medium (Graf et al. 1979) could damage the platelets. All these factors o f platelet alteration were found in the three groups studied. In the first group labeled with 2.2 MBq/ml 111-In-oxine we found only a slight enhancement of platelet aggregability (both velocity and maximum of light transmission). There were no morphological changes.

These findings were in complete agreement with those

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Fig. 1 a. Fine structure of non-labeled platelet. Numerous c~ granules (c~g) mitochondria (m) and vesicles of the open canalicular system (ocs) are randomly distributed in the cytoplasm. Some microfilaments (m J) are present, particularly visible in the pseudopodia, x 26,400. Figs. 1 b, c. Scanning electron microscopy of unlabeled platelets. The characteristic, irregular shape of the platelets exhibiting more or less numerous and elongated pseudopodia appear on both photographs, b x 8,480, e x 16,400. Fig. 2a. Fine structure ofa 111In-labeled platelet (2.2 MBq/ml). No significant modification can be observed, either in the c¢ granule (c~g) appearance and distribution or in the appearance of the vesicles of the open canalicular system (ocs). Some microtubules (rot) are visible on the peripheral area. x 20,000. Fig. 2b, c. Scanning electron micrograph of 111In-labeled platelets (2.2 MBq/ml). Both photographs show normal platelets with more or less elongated pseudopodia, b x 9,600, e x 11,200

of Joist et al. (1978) on platelet labeling and Zakhireh et al. (1979) or Chisholm et al. (1979) on morphopolynuclears and HeLa cells. They found no great functional or ultrastructural modifications in cells labeled with 111In-oxine with activity varying from 20 to 200 gCi/ml (0.74-7.4 MBq/

ml). As found by Danpure and Osman (1981) we noticed no functional modification of platelets ' labeled ' with oxine alone. So we could suppose that the nonradioactive reagents of the labeling procedure did not cause the observed modifications. In the 22 labeling procedures with 7.4 MBq/

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Fig. 3a, b. 111In-labeled platelets (7.4 MBq/ml). Firstly the ~ granules (c~g) appear with a low contrast and secondly the open canalicular system (ocs) appear overdevelopped. No pseudopodia are visible on these sections, a x 26,400, b x 17,600. Fig. 3c, d. 1lain-labeled platelets (7.4 MBq/ml). The general appearance of the platelets is preserved but some slight modifications in the appearance of the pseudopodia suggest alterations, e x 9,600, d x 10,800 Fig. 4a, b. 111-In-labeled platelets (22 MBq/ml). The picture shows clumping of the organelles in the central region of the cytoplasm. The granules are surrounded by some microtubules (mt). Moreover, some cytoplasmic zones seem altered (az) and may correspond to disruption of the open canalicular system, a x 20,000, b x 16,000. Fig. 4e, ft. These scanning pictures of the platelets show that high activity 11~in_labeling (22 MBq/ml) produces changes in the shape. The platelets exhibit either a very smooth surface (Fig. 4c) or numerous microvilli (Fig. 4d). e and fl x 11,200

ml there were only slight morphologica l al terat ions by scanning electron microscopy (Fig. 3 c and d) as Sinzinger et al. found in 1981, and a decrease of aggregabil i ty near 15% compared with unlabeled platelets. So we could consider that such platelets retained quas i -normal functions. Con-

versely, platelets labeled with high activity (22 MBq/ml) showed marked morphologica l a l terat ions: clumping of organelles, al terat ions in the open canalicular system, and disorders of villosity going as far as complete disappear- ance. I t must be stressed that the only difference with the

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previous group is the amount of radioact ivi ty. Thus we have to consider the absorbed dose of i r radia t ion during the labeling procedure.

111Indium decays by electron capture with a 2.8-day half-life. Two gamma rays 0.172 and 0.247 MeV are emitted but also conversion electrons, X-rays, and Auger electrons. Only the lat ter particles have a sufficiently short range to deliver their energy to these small cells, more especially if 11~In-oxine is located predominant ly in platelet cytosol (Hudson et al. 1981). Silvester in 1979 indicated that the radia t ion dose resulting f rom the decay of each t~ l Ind ium a tom was 0.145 Gy. When 5 x 109 cells were labeled with l l 0 M B q each platelet contained near 8 ,000a toms o f

~ aIndium, the integrated radia t ion dose per cell was thus 1,160 Gy. Hawker (cited by Thakur 1981) found an absorbed dose of 145 G y per cell for 3.7 MBq/109 platelets in 1 ml.

Platelets offer high radio-resistance to external i r radia- t ion; But ton et al. (1981) showed that 5 krads (50 Gy) did not alter the aggregabil i ty but such i r radia ted cells were less effective in s topping aspir in- induced hemorrhages.

Dur ing the labeling procedure and before measurements of aggregabil i ty or f ixation with glutaraldehyde, the absorbed dose per cell can be est imated as between 30 and 50 Gy. Such doses, which did not alter platelets in normal medium (their own plasma), could greatly damage cells in the labeling medium (low pH, ethanol, oxine, protein-free medium). Funct iona l al terat ions and morphologica l changes described in the high activity labeling procedure appeared to be the result of a react ion of platelets to mem- brane injury, especially due to the protein-free medium, (Gra f et al. 1979) and cytoplasmic i r radia t ion (Hudson et al. 1981).

Such functional and morphologica l injury was found with t reatments not involving i r radia t ion such as l idocaine and tetracaine (Nachmias et a l . 1977, 1979) or bacterial endotoxin (Ausprunk and Das 1978). White in 1979 and Drol ler in 1973, to a lesser extent, found such morphologi - cal modif icat ions by adding A D P (but in this case the procedure induced platelet aggregation) and especially clumping of organelles.

In view of all these functional and morphologica l results it appears that ~ l I n - o x i n e is a good radiopharmaceut ica l agent for platelet labeling provided that concentrat ions of less than 7.4 MBq/ml platelet suspension (109 cells/ml) are used.

This amount is ten times more than is necessary for evaluat ion of platelet life-span in hematological disorders. I t is also sufficient for gamma-camera imaging in the detec- t ion and observat ion of small thrombosis areas. This work shows that higher tracer doses damage cells and are not suitable for use, and in any case would be rather dangerous for pat ient (Bjurman et al. 1982).

Acknowledgment. We wish to thank Mr. J.R. Galindo and Mrs. M. Billery for excellent technical assitance and Mrs. C. Lipcey and Mr. M. Seagar for revision of the English text.

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Received October 16, 1982

Functional and ultrastructural alterations of autologous platelets labeled with 111In-oxine

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