Platelet Labeling with 67Ga-MPO: A Comparison with 111In-MPO

ISSN 0969-8051/98/$19.00 1 0.00

Nuclear Medicine & Biology, Vol. 25, pp. 165–168, 1998 Copyright © 1998 Elsevier Science Inc.

TECHNICAL NOTE

Platelet Labeling with 67Ga-MPO: A Comparison with 111In-MPO Georgios Karanikas, Margarida Rodrigues, Susanne Granegger, Ernst Havlik and Helmut Sinzinger Department of Nuclear Medicine, University of Vienna, Vienna, Austria 68 Ga-labeled platelets could be useful for positron emission tomography (PET) studies. The labeling behaviour of human platelets labeled with 67Ga-MPO and the viability of 67Ga-MPO platelets were studied and compared, respectively, with 111In-MPO. The platelet-labeling yield of 67Ga-MPO increased with platelet concentration and with temperature and time of incubation [reaching a maximum (,20%) at 1 3 109 platelets/mL, 37°C, 30 min]. 111 In-MPO shows a similar behaviour, but labeling yield of platelets is significantly higher as compared to 67Ga-MPO. Viability of 67 Ga-MPO- and 111In-MPO-labeled platelets is maintained. The data suggest that 68Ga-MPO is not a promising PET radiopharmaceutical for potential studies with radiolabeled platelets in clinical practice.

INTRODUCTION Examinations after radiolabelling of platelets allow the study of platelet behavior in vitro and in vivo, particularly the evaluation of kinetics, measurement of platelet survival, biodistribution, sites of sequestration, and accumulation of platelets in thrombosis and hemostasis. Radiolabeled platelets have also been used successfully for the monitoring of the efficacy of platelet-inhibitory drugs. Ever since the first report in 1976 by Thakur, Welch, and Malech (6), 111In-oxine and later on other chelators have been extensively used for the radiolabelling of platelets (3). Gallium is chemically analogous to indium, and the stability and the methods of preparation of the gallium-labeled blood components parallel those previously observed for 111In-labeled blood components (7). However, 68Ga produces high-resolution tomographic images with improved statistics. Two encouraging reports of uptake of 68Galabeled platelets in de-endothelialized arteries by positron emission tomography (PET) in dogs (7) and in rabbits (8) suggested that the method might be successfully extended to humans. In a preliminary study performed in only 10 patients, Goodwin et al. (1) reported a 68 Ga-mercaptopyridine-N-oxide (MPO)-platelet labeling yield of 36 6 12%. We examined the labeling yield of human platelets obtained from healthy volunteers with a complex of the longerlived 67Ga and MPO and the viability of 67Ga-MPO-labeled human platelets to study the feasibility of using platelets radiolabeled with shorter-lived generator-produced 68Ga as an imaging radiopharmaceutical for PET. Both the labeling and the viability of platelets were compared using the same chelating agent radiolabeled with 111 In. Parameters such as platelet concentration, temperature, and time of incubation and amount of radioactivity were investigated.

MATERIALS AND METHODS Gallium-67-MPO synthesis followed the method of Thakur, McKenney, and Park (5) and Yano et al. (8). Indium-111-MPO was synthetized according to the method of Thakur et al. (5). For platelet separation and labeling procedure, blood from 57 healthy volunteers (31 females and 26 males), age range 20 to 45 years (mean age 32.5 years), without any risk factor for the development of atherosclerosis (smoking, hypertension, hyperlipidemia and no drugs, among others), was used and the technique described by Sinzinger et al. (4) was performed. Briefly, blood (7 mL) was drawn from the cubital vein without occlusion into a Monovette vial using 2 mL acid citrate dextrose (ACD) as anticoagulant. Ten minutes were allowed for sedimentation of the red blood cells at room temperature. The vial was centrifuged at 150 g for 5 min. The supernatant platelet-rich plasma (PRP) was transferred into a 10-mL vial using a butterfly needle. The PRPcontaining vial was centrifuged at 500 g for 10 min. The platelets were sedimented in a pellet at the bottom of the vial. The supernatant platelet-poor plasma (PPP) was withdrawn, carefully preserving the pellet. The pellet in the vial was gently resuspended in 1 mL Tyrode buffer by shaking the tube gently; then the tracer was added (final concentration ; 10 mg/mL). The vial was incubated in a water bath at a predetermined temperature. The incubation mixture containing the labeled platelets was resuspended with the PPP preserved for this purpose. The solutioncontaining vial (pellet, Tyrode buffer, tracer, PPP) was centrifuged at 4°C, at 500 g for 10 min. The platelets were sedimented again in a pellet at the bottom of the vial. The supernatant PPP was withdrawn, carefully preserving the pellet, and aspirated in another vial. The two vials (the first vial containing radiolabeled platelets and the second one the supernatant) were counted in a gamma counter. The last step was to calculate the labeling yield of platelets. The influence of the following parameters was examined: platelet concentration (1.107, 1.108, and 1.109 platelets/mL), temperature (4°C, 22°C, and 37°C), time of incubation (1, 5, 10, 20, and 30 min), and amount of radioactivity (0.01, 0.1, and 1 mCi 67Ga-MPO or 111In-MPO, respectively). Six tests of each combination of the above-mentioned conditions were studied. In vitro platelet function was evaluated by adenosine diphosphate (ADP)-induced platelet aggregation. Blood anticoagulated with 3.8% sodium citrate (10% v/v) was centrifuged (150 g, 10 min, 22°C) and the removed PRP was adjusted with autologous PPP (1500 g, 10 min) to a final concentration of 250 3 103/mL. Platelet aggregation was induced in a Born-type aggregometer at 37°C, with a stirring speed of 800 rpm, by adding 1 mM (100 mL) ADP (Boehringer, Mannheim, Germany) to 600 mL PRP. Aggregation response was recorded by changes in light transmission and characterized by initial slope (tangent a) and response (delta Tmaxmaximal amplitude and delta T4-amplitude 4 min after aggregationinduction) of aggregation.

Quality Control Address reprint requests to: Helmut Sinzinger, MD, Department of Nuclear Medicine, University of Vienna, Wa¨hringer Gu¨rtel 18 –20, A-1090 Vienna, Austria; E-mail: helmut.sinzinger@akh-wien. ac. at

As there are no reliable thin-layer chromatography systems available for the two tracers, radiochemical purity was assessed by a

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FIG. 1. The platelet-labeling yield of 67Ga-MPO and 111 In-MPO increases with the platelet concentration and the temperature of incubation.

FIG. 2. The platelet-labeling yield of 67Ga-MPO and 111In-MPO increases with the platelet concentration and the time of incubation.

FIG. 3. The platelet-labeling yield of 67Ga-MPO and 111 In-MPO is independent from the amount of radioactivity.

Technical Note

FIG. 4. No significant elution was demonstrated until 4 h of incubation with both tracers. back-extraction procedure (2). The amount of free tracer was always less than 3%. Results are presented as mean values 6 standard deviation; calculation for significance was carried out using Student’s t-test. A p ,0.01 was considered as being significant. RESULTS The platelet-labeling yield of 67Ga-MPO increased with platelet concentration (highest at 1 3 109 platelets/mL) (Figs. 1 and 2). The labeling yield was dependent on both temperature (highest at 37°C) (Fig. 1) and time of incubation (highest at 30 min) (Fig. 2), but independent of the amount of radioactivity (Fig. 3). For 111 In-MPO, the time-dependent increase in labeling yield was less pronounced (Fig. 2). The platelet-labeling yield of 111In-MPO under identical incubation conditions showed a similar behaviour, but it was significantly higher as compared to 67Ga-MPO (Figs. 1–3). No relevant elution was demonstrated until 4 h of incubation (Fig. 4). Platelet viability was not altered significantly by the labeling with 67Ga-MPO or 111In-MPO (Fig. 5). DISCUSSION Although platelet labeling is long established and several attempts to develop simple methods allowing wide use of radiola-

FIG. 5. Platelet viability was not altered significantly by the labeling; platelet aggregation response curves ADP do not differ significantly before and after labeling. T, transmission; b, before labeling testing; a, after labeling testing.

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beled platelets in clinical and experimental applications have been performed, the studies with radiolabeled platelets are not yet widely and routinely used in clinical practice. For platelet labeling, 111In has attracted considerable interest and wide application owing to its advantageous physical characteristics that allow external imaging as well as parallel kinetic monitoring. However, the dose limitation due to the 2.8-day half-life (t1/2) of 111In and the resulting high radiation dose to the spleen restricts the counts available, thus preventing good imaging (1). Also, the need to wait for 24 h before imaging and a variety of other methodological factors are limiting the general application of 111In-labeled platelet studies. The 68Ge/68Ga generator is an economical source of PET radiopharmaceuticals. The ability to obtain more counts using short-lived (t1/2-68 min) generatorproduced 68Ga and the high sensitivity and resolution of PET might be a faster and promising potential alternative approach for using radiolabeled platelets in clinical practice. As indium and gallium in ionic form do not cross the cell membrane, chelating agents have to be used in order to form a lipid-soluble complex for the labeling of platelets. Various lipophilic complexes of indium (3) and gallium (8) have been studied as carrier molecules for the intracellular radiolabeling of platelets in an attempt to achieve a high labeling yield without losing platelet viability. However, the radiolabeling of platelets with 68Ga has not been studied extensively with different lipophilic chelators. In this study we investigated the radiolabeling and viability of 67 Ga-MPO-labeled human platelets and compared them with 111InMPO-labeled human platelets. Because blood platelets were derived under well-controlled laboratory conditions, excluding potential damage, and because we did not want eventually to mask an effect on labeling yield, we did not use prostaglandin I2 for stabilization of platelets in this study. Platelet viability after radiolabeling with 67 Ga-MPO or 111In-MPO was good. Our in vitro findings indicate a higher platelet-labeling yield as the platelet concentration, temperature, and time of incubation increased. Our data provide a quite low platelet yield of 67Ga-MPO (,20% at optimal conditions, i.e., platelet concentration of 1 3 109 platelets/mL, 37°C, 30 min of incubation) as compared to 111In-MPO. Our labeling yield of 67 Ga-MPO platelets was lower than that reported with 68Ga-MPO platelets in the few patients studied by Goodwin et al. (1). However, they used a very high platelet concentration, in which platelets were separated from 85 mL blood and were suspended in 1 mL PRP. The radiolabeling of platelets with gallium requires careful attention to possible trace-metal contaminants. Metal impurities in

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gallium citrate solution can effectively compete with radioactive gallium for the complexing ligand, and trace-metal complexes will also compete with radioactive gallium for the intracellular binding sites within the platelets (8), thus reducing platelet-labeling yield. This may partially explain the low labeling yield obtained in our study and in the study of Goodwin et al. (1). Moreover, the presence of transferrin in plasma may reduce the platelet-labeling yield because of its high affinity for gallium (8) and the high-stability constant of gallium-transferrin (7). Goodwin et al. (1) argued that it is feasible to image platelet deposition within the initial 4 h after injection of 68Ga-MPOlabeled platelets. However, the blood background at early times (necessitated by the use of 68Ga) was too high for visualization of clots by PET imaging. These data, together with our findings, indicate that 68Ga-MPO is not a promising PET radiopharmaceutical for further imaging and for assessing areas of platelet accumulation in clinical practice.

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68 Ga-MPO-labeled human platelets. Nucl. Med. Commun. 14, 1023– 1029. Neumann I., Strobl-Ja¨ger E., Angelberger P., O’Grady J. and Sinzinger H. (1992) A comparison of 111-In-oxine with 111-In-oxinesulfate for human platelet labeling. Thromb. Haemorrh. Disorders 5(1), 5–10. Rodrigues M. and Sinzinger H. (1994) Platelet labeling and clinical applications. Thromb. Res. 76, 399 – 432. Sinzinger H., Kolbe H., Strobl-Ja¨ger E. and Ho¨fer R. (1984) A simple and safe technique for sterile autologous platelet labelling using ‘‘Monovette’’ vials. Eur. J. Nucl. Med. 9, 320 –322. Thakur M. L., McKenney S. L. and Park C. H. (1985) Simplified and efficient labeling of human platelets in plasma using indium-111-2mercaptopyridine-N-oxide: Preparation and evaluation. J. Nucl. Med. 26, 510 –517. Thakur M. L., Welch M. J. and Malech H. L. (1976) Indium-111-labelled human platelets: Studies on preparation and evaluation of in vitro and in vivo functions. Thromb. Res. 9, 345–357. Welch M. J., Thakur M. L. and Coleman R E. (1977) Gallium-68-labeled red cells and platelets: New agents for positron tomography. J. Nucl. Med. 18, 558 –562. Yano Y., Budinger T. F., Ebbe S. N., Mathis C. A., Singh M., Brennan K. M. et al. (1985) Gallium-68 lipophilic complexes for labeling platelets. J. Nucl. Med. 26, 1429 –1437.

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