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Gallium-67-labelled red blood cells as a blood-pool marker for dual-isotope imaging
Nucl. Med. Biol. Vol. 19, No. 1, pp. 7%81, 1992 Int. J. Radial. Appl. Inswum. Parr B Printed in Great Britain. All rights reserved
0883-2897/92 $5.00 + 0.00
Copyright 0 1991Pergamon Press plc
Gallium-67-labelled Red Blood Cells as a Blood-pool Marker for Dual-isotope Imaging JAMES R. BALLINGER
and I. BOXEN
Department of Nuclear Medicine, Princess Margaret Hospital, 500 Sherboume Street, Toronto, Ontario, Canada M4X 1K9 (Received 9 March 1991) Red blood cells were labelled with 67Ga oxine prepared from 67Ga citrate, a routinely available and relatively inexpensive radiopharmaceutical which is suitable for dual-isotope imaging with %Tc. Labelling efficiency was 88 k l%, 24-h in vitro stability was 93 + 5% in saline at room temperature and 68 + 4% in whole blood at 37”C, and the method could be performed within 2&30min.
Introduction
utions were prepared in absolute ethanol while tropolone and MPO were dissolved in distilled water. 67Ga citrate injection was manufactured by Nordion International Inc. and purchased from Frosst Radiopharmaceuticals; the radioactivity concentration was 81.4MBq/mL (2.2mCi/mL) at time of calibration.
It is sometimes necessary to correct nuclear images for the radioactivity which is solely due to blood volume. This can be done by injection of a second radiopharmaceutical and subtraction of images; for example, *Tc-labelled red blood cells (RBCs) have been used to correct images of “‘In platelets (Funke Kupper et al., 1986; Machac et al., 1989). Although this can be done sequentially, it is optimally performed by simultaneous dual-isotope imaging. This is especially true during dynamic imaging with pharmaceuticals which may affect relative blood volumes in organs, although even without such pharmaceuticals, relative blood volumes may change during imaging studies. However, when the primary radiopharmaceutical is labelled with 99mTc,correction cannot be performed by simultaneous injection of g9”Tc-RBCs and thus we looked for an alternative radioisotope with which to label RBCs. The requirements of such an agent were that it emit suitable photons for dual-isotope imaging with 99mT~,that it be readily available and relatively inexpensive, and that the labelling method be simple and efficient. We therefore investigated 67Ga for RBC labelling.
Preparation of lipophilic complexes
To OS-mL aliquots of 67Ga citrate (lo-40 MBq) was added the desired quantity of oxine, tropolone or MPO. After 5 min of incubation at room temperature, the samples were diluted to 1 mL with saline and extracted with 1 mL chloroform. The yield of 67Ga complex was calculated as the percent of the total radioactivity extracted into chloroform. From these experiments, the optimal quantity of each ligand to use in further studies was determined. Comparison of complexes for RBC labelling
67Ga oxine, tropolone, MPO or citrate was added to 1 mL of heparinized whole blood and allowed to stand 5 min at room temperature, after which 3 mL saline was added and the tubes were centrifuged (500 g, 2 min) to determine the percent activity associated with the RBC pellet. The pellet was washed once with 3 mL saline to determine the percent labelling after washing, then suspended in 3 mL saline, incubated 24 h at room temperature and centrifuged again to determine the stability of the label. To study the stability of the label under more physiological conditions, 0.5 mL labelled RBCs were added to 2 mL whole blood, incubated 24 h at 37”C, then diluted with 3 mL saline and centrifuged.
Materials and Methods Oxine (8-hydroxyquinoline) and mercaptopyridine-N-oxide (MPO; 1-hydroxypyridine-2-thione sodium salt) were purchased from Sigma Chemical Co. Tropolone (2-hydroxy-2,4,6-cycloheptatriene-lone) was purchased from Fluka Chemical Corp. All were used without further purification. Oxine sol79
80
JAMES R. BALLINGER and
67Ga oxine labelling of washed RBCs
Aliquots of 1, 2, 3 or 4 mL whole blood were diluted to 10 mL with saline and centrifuged (500 g, 2 min). The supematant was discarded and the RBCs were washed with 9 mL saline. After the wash supernatant had been discarded, 67Ga oxine (prepared from 0.5 mL 67Ga citrate and 50 p g oxine) was added and incubated for 5 min at room temperature, then processed as described above.
I.
BOXEN
Table 2. Comparison of labelling efficiency and 24-h stability of 6’Ga RBCs prepared from different 67Ga complexes and 1 mL whole blood Labelling efficiency (%I Initial After wash Stability
“‘Ga complex Oxine
Tropolone
MPO
Citrate
39.6 + 4.3 26.0 k 5.6 83.3 + 1.7
31.1 * 2.7 16.8 + 1.0 63.7 + 2.6
10.4 f 0.5 4.7 * 0.3 81.8 + 6.3
9.1 + 1.0 2.2 f 0.6 56.3 + 20.1
Each value is mean f SD for 3 determinations.
Discussion
Labelling protocol for clinical use
A 3-mL aliquot of heparinized whole blood is transferred into a sterile tube which contains 7 mL saline. The tube is centrifuged (5OOg, 2 min), then the supernatant is discarded, replaced with 9 mL saline, and the tube is centrifuged again. The washed RBC pellet is then transferred to a tube which contains 0.5 mL 67Ga citrate (-40 MBq) and 50 PL oxine solution, 1 mg/mL in ethanol. After 5 min, 3 mL saline is added and the tube is centrifuged. The supernatant is discarded, replaced with 3 mL saline, and the tube is again centrifuged. The RBC pellet is drawn into a syringe for reinjection. Assaying the labelled cells and the washes allows calculation of labelling yield.
Results 67Ga from the radiopharmaceutical citrate readily formed a lipophilic complex in high yield with oxine, tropolone and MPO, although the quantity of MPO required was much greater than the other two ligands (Table 1). All three complexes labelled RBCs from whole blood with higher efficiency and greater stability than did 67Ga citrate (Table 2). Because 67Ga oxine resulted in the highest labelling efficiency and greatest stability, it was investigated further using washed RBCs; larger volumes of whole blood alone did not improve labelling efficiency (data not shown). With washed RBCs, oxine remained superior to tropolone and MPO (data not shown). With 67Ga oxine, labelling efficiency and stability improved with larger volumes of washed RBCs, with an optimal value of 3 mL (Table 3). The 24-h in vitro stability of the label was 92.6 f 4.5% (n = 6) in saline at room temperature and 68.1 + 4.4% (n = 3) in whole blood at 37°C. Table
1.Effect of choice and quantity of ligand on formation of lipophilic complexes from 6’Ga citrate
peg Ligand per 0.5 mL 67Ga citrate
% Yield lipophilic complex Oxine
Tropolone
10 25 50 100 500 1000 2000
93.7 + 1.6 96.5+0.4 98.850.2 98.6 k 0.5 -
14.1 +0.3 44.55 16.3 97.5_+ 1.9 98.3 f 1.3 -
MPO 15.4 + 88.7 + 98.8 f 99.1 +
Each value is mean k SD for >5 determinations.
3.5 1.4 0.5 0.3
Srivastava and Chervu (1984) have reviewed the radiolabelling of RBCs. Of the gamma-emitting radioisotopes reviewed, 5’Cr and “Fe are unsuitable for imaging, leaving only 99mTcand “‘In. “3mIn has subsequently been added to this list (Morrissey et al., 1988). Although “‘In and “3mIn would be suitable for dual-isotope imaging with 49”‘Tc,both are expensive and not routinely on hand (“‘In) or not widely available ( 113mIn generators). Because of previous reports of cell labelling with positron-emitting 6*Ga chloride (Welch et al., 1977; Yano et al., 1985), we investigated 67Ga citrate, which is a routinely available and relatively inexpensive radiopharmaceutical. The suitability of 67Ga for dual-isotope imaging with 99”Tc has been demonstrated (Hartshorne et al., 1985; Malik, 1985). Lipophilic chelates of indium and gallium are able to cross the RBC membrane by passive diffusion. Once inside the cell, the complex dissociates and the metal ion is trapped by binding to intracellular components. Lipophilic 67Ga complexes were prepared from 67Ga citrate in high yield with 50 pg oxine or tropolone, whereas 1 mg MPO was required to obtain similar yields (Table 1). Similar results were reported by Yano et al. (1985) using 68Ga chloride. The three lipophilic 67Ga complexes differed in the efficiency with which they labelled RBCs from whole blood (Table 2). 67Ga citrate alone resulted in low labelling efficiency and poor stability of the label. 67Ga oxine gave the best labelling and stability, and further studies determined the optimal conditions in which the RBCs from 3 mL whole blood were separated and washed before labelling (Table 3). The lower yield obtained with 4 mL blood may be due to incomplete removal of plasma by this protocol. With this optimal technique, overall labelling efficiency was 88% and the 24-h in vitro stability of the label was 93% in saline at room temperature and 68% in whole blood at 37°C. Table 3. Effect of volume of whole blood sample on labelling efficiency of washed red blood cells with 6’Ga oxine Labelling efficiencv
(%I Initial After wash
mL whole blood 1
78.7kO.9 65.9k2.5
2 89.5kl.l 82.7 +0.9
3 93.3kO.7 88.2 f0.6
Each value is mean + SD for 3 determinations.
4 84.325.4 71.3 f 10.6
67Ga red blood cells
Welch et al. (1977) reported RBC labelling efficiencies with 68Ga oxine of 93 f 5%; however, that method started with 20mL whole blood, included an extra wash step, and had longer centrifugation and incubation times. In contrast, the present method requires only 3 mL blood and can be completed within 20-30 min, yet the yield is only slightly lower. There has been a previous report of RBC labelling with 67Ga by a different approach. Pant et al. (1983) treated washed RBCs with tannic acid prior to surface labelling with 67Ga chloride in 89% yield. However, their method did not work with 67Ga citrate and thus would not be readily applicable. The main disadvantage to the use of 67Ga is its long half-life, which limits the dose which can be administered. In addition, radioactivity remains in the circulation, making it impossible to repeat a quantitative study for a number of days. The IOO-min half-life of 1’3mInmakes it the most desirable of the currently available radionuclides for cell labelling. Increased use of “3mIn may make it more costeffective to supply in the future. It has also been suggested that one ‘13Yn generator could be shared among several institutions (Morrissey et al., 1988). In summary, we have reported a rapid and efficient method to label RBCs with a routinely available radiopharmaceutical which is suitable for dualisotope imaging with 99mTc.
81
References Funke Kupper A. J., Verheugt F. W. A., Jaarsma W., van der Wall E. E., van Eenige M. J., den Hollander W. and Roos J. P. (1986) Detection of ventricular thrombosis in acute myocardial infarction: value of indium-I 11 platelet scintigraphy in relation to two-dimensional echocardiography and clinical course. Eur. J. Nucl. Med. 12, 337-34 1. Hartshorne M. F., Graham G., Lancaster J. and Berger D. (1985) Gallium-67/technetium-99m methylene diphosphonate ratio imaging: early rabbit osteomyelitis and fracture. .I. Nucl. Med. 26, 272-277. Machac J., Vallabhajosula S., Goldman M. E., Goldsmith S. J.. Palestro C.. Strashun A..,.Vaouer R.. Phillios R. A. and Fuster V. (1989) Value of blood-pool subtraction in cardiac indium-1 1 l-labeled platelet imaging. J. Nucl. Med. 30, 14451455. Malik M. H. (1985) Simultaneous dual isotope studies in the diagnosis of infection. J. Nucl. Med. 26, 722-725. Morrissey G. J., Gravelle D., Dietz G., Driedger A. A., King M. and Cradduck T. D. (1988) In vitro red blood cell compatibility testing using indium-113m tropolonelabeled red blood cells. J. Nucl. Med. 29, 684-688.
Pant K. D., Coupal J. J., Shih W-J. and DeLand F. H. (1983) A new approach to gallium-67 labeling of human ervthrocvtesandnlate1ets.J. Nucl.Med.XP123(Abstract). Srivastava-S. C. and Chervu L. R. (1984) Radionuclidelabeled red blood cells: current status and future prospects. Semin. Nucl. Med. 14, 68-82. Welch M. J., Thakur M. L., Coleman R. E., Pate1 M., Siegel B. A. and Ter-Pogossian M. M. (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. and Moyer B. R. (1985) Gallium-68 lipophilic complexes for labeling platelets. J. Nucl. Med. 26, 1429-1437.
0883-2897/92 $5.00 + 0.00
Copyright 0 1991Pergamon Press plc
Gallium-67-labelled Red Blood Cells as a Blood-pool Marker for Dual-isotope Imaging JAMES R. BALLINGER
and I. BOXEN
Department of Nuclear Medicine, Princess Margaret Hospital, 500 Sherboume Street, Toronto, Ontario, Canada M4X 1K9 (Received 9 March 1991) Red blood cells were labelled with 67Ga oxine prepared from 67Ga citrate, a routinely available and relatively inexpensive radiopharmaceutical which is suitable for dual-isotope imaging with %Tc. Labelling efficiency was 88 k l%, 24-h in vitro stability was 93 + 5% in saline at room temperature and 68 + 4% in whole blood at 37”C, and the method could be performed within 2&30min.
Introduction
utions were prepared in absolute ethanol while tropolone and MPO were dissolved in distilled water. 67Ga citrate injection was manufactured by Nordion International Inc. and purchased from Frosst Radiopharmaceuticals; the radioactivity concentration was 81.4MBq/mL (2.2mCi/mL) at time of calibration.
It is sometimes necessary to correct nuclear images for the radioactivity which is solely due to blood volume. This can be done by injection of a second radiopharmaceutical and subtraction of images; for example, *Tc-labelled red blood cells (RBCs) have been used to correct images of “‘In platelets (Funke Kupper et al., 1986; Machac et al., 1989). Although this can be done sequentially, it is optimally performed by simultaneous dual-isotope imaging. This is especially true during dynamic imaging with pharmaceuticals which may affect relative blood volumes in organs, although even without such pharmaceuticals, relative blood volumes may change during imaging studies. However, when the primary radiopharmaceutical is labelled with 99mTc,correction cannot be performed by simultaneous injection of g9”Tc-RBCs and thus we looked for an alternative radioisotope with which to label RBCs. The requirements of such an agent were that it emit suitable photons for dual-isotope imaging with 99mT~,that it be readily available and relatively inexpensive, and that the labelling method be simple and efficient. We therefore investigated 67Ga for RBC labelling.
Preparation of lipophilic complexes
To OS-mL aliquots of 67Ga citrate (lo-40 MBq) was added the desired quantity of oxine, tropolone or MPO. After 5 min of incubation at room temperature, the samples were diluted to 1 mL with saline and extracted with 1 mL chloroform. The yield of 67Ga complex was calculated as the percent of the total radioactivity extracted into chloroform. From these experiments, the optimal quantity of each ligand to use in further studies was determined. Comparison of complexes for RBC labelling
67Ga oxine, tropolone, MPO or citrate was added to 1 mL of heparinized whole blood and allowed to stand 5 min at room temperature, after which 3 mL saline was added and the tubes were centrifuged (500 g, 2 min) to determine the percent activity associated with the RBC pellet. The pellet was washed once with 3 mL saline to determine the percent labelling after washing, then suspended in 3 mL saline, incubated 24 h at room temperature and centrifuged again to determine the stability of the label. To study the stability of the label under more physiological conditions, 0.5 mL labelled RBCs were added to 2 mL whole blood, incubated 24 h at 37”C, then diluted with 3 mL saline and centrifuged.
Materials and Methods Oxine (8-hydroxyquinoline) and mercaptopyridine-N-oxide (MPO; 1-hydroxypyridine-2-thione sodium salt) were purchased from Sigma Chemical Co. Tropolone (2-hydroxy-2,4,6-cycloheptatriene-lone) was purchased from Fluka Chemical Corp. All were used without further purification. Oxine sol79
80
JAMES R. BALLINGER and
67Ga oxine labelling of washed RBCs
Aliquots of 1, 2, 3 or 4 mL whole blood were diluted to 10 mL with saline and centrifuged (500 g, 2 min). The supematant was discarded and the RBCs were washed with 9 mL saline. After the wash supernatant had been discarded, 67Ga oxine (prepared from 0.5 mL 67Ga citrate and 50 p g oxine) was added and incubated for 5 min at room temperature, then processed as described above.
I.
BOXEN
Table 2. Comparison of labelling efficiency and 24-h stability of 6’Ga RBCs prepared from different 67Ga complexes and 1 mL whole blood Labelling efficiency (%I Initial After wash Stability
“‘Ga complex Oxine
Tropolone
MPO
Citrate
39.6 + 4.3 26.0 k 5.6 83.3 + 1.7
31.1 * 2.7 16.8 + 1.0 63.7 + 2.6
10.4 f 0.5 4.7 * 0.3 81.8 + 6.3
9.1 + 1.0 2.2 f 0.6 56.3 + 20.1
Each value is mean f SD for 3 determinations.
Discussion
Labelling protocol for clinical use
A 3-mL aliquot of heparinized whole blood is transferred into a sterile tube which contains 7 mL saline. The tube is centrifuged (5OOg, 2 min), then the supernatant is discarded, replaced with 9 mL saline, and the tube is centrifuged again. The washed RBC pellet is then transferred to a tube which contains 0.5 mL 67Ga citrate (-40 MBq) and 50 PL oxine solution, 1 mg/mL in ethanol. After 5 min, 3 mL saline is added and the tube is centrifuged. The supernatant is discarded, replaced with 3 mL saline, and the tube is again centrifuged. The RBC pellet is drawn into a syringe for reinjection. Assaying the labelled cells and the washes allows calculation of labelling yield.
Results 67Ga from the radiopharmaceutical citrate readily formed a lipophilic complex in high yield with oxine, tropolone and MPO, although the quantity of MPO required was much greater than the other two ligands (Table 1). All three complexes labelled RBCs from whole blood with higher efficiency and greater stability than did 67Ga citrate (Table 2). Because 67Ga oxine resulted in the highest labelling efficiency and greatest stability, it was investigated further using washed RBCs; larger volumes of whole blood alone did not improve labelling efficiency (data not shown). With washed RBCs, oxine remained superior to tropolone and MPO (data not shown). With 67Ga oxine, labelling efficiency and stability improved with larger volumes of washed RBCs, with an optimal value of 3 mL (Table 3). The 24-h in vitro stability of the label was 92.6 f 4.5% (n = 6) in saline at room temperature and 68.1 + 4.4% (n = 3) in whole blood at 37°C. Table
1.Effect of choice and quantity of ligand on formation of lipophilic complexes from 6’Ga citrate
peg Ligand per 0.5 mL 67Ga citrate
% Yield lipophilic complex Oxine
Tropolone
10 25 50 100 500 1000 2000
93.7 + 1.6 96.5+0.4 98.850.2 98.6 k 0.5 -
14.1 +0.3 44.55 16.3 97.5_+ 1.9 98.3 f 1.3 -
MPO 15.4 + 88.7 + 98.8 f 99.1 +
Each value is mean k SD for >5 determinations.
3.5 1.4 0.5 0.3
Srivastava and Chervu (1984) have reviewed the radiolabelling of RBCs. Of the gamma-emitting radioisotopes reviewed, 5’Cr and “Fe are unsuitable for imaging, leaving only 99mTcand “‘In. “3mIn has subsequently been added to this list (Morrissey et al., 1988). Although “‘In and “3mIn would be suitable for dual-isotope imaging with 49”‘Tc,both are expensive and not routinely on hand (“‘In) or not widely available ( 113mIn generators). Because of previous reports of cell labelling with positron-emitting 6*Ga chloride (Welch et al., 1977; Yano et al., 1985), we investigated 67Ga citrate, which is a routinely available and relatively inexpensive radiopharmaceutical. The suitability of 67Ga for dual-isotope imaging with 99”Tc has been demonstrated (Hartshorne et al., 1985; Malik, 1985). Lipophilic chelates of indium and gallium are able to cross the RBC membrane by passive diffusion. Once inside the cell, the complex dissociates and the metal ion is trapped by binding to intracellular components. Lipophilic 67Ga complexes were prepared from 67Ga citrate in high yield with 50 pg oxine or tropolone, whereas 1 mg MPO was required to obtain similar yields (Table 1). Similar results were reported by Yano et al. (1985) using 68Ga chloride. The three lipophilic 67Ga complexes differed in the efficiency with which they labelled RBCs from whole blood (Table 2). 67Ga citrate alone resulted in low labelling efficiency and poor stability of the label. 67Ga oxine gave the best labelling and stability, and further studies determined the optimal conditions in which the RBCs from 3 mL whole blood were separated and washed before labelling (Table 3). The lower yield obtained with 4 mL blood may be due to incomplete removal of plasma by this protocol. With this optimal technique, overall labelling efficiency was 88% and the 24-h in vitro stability of the label was 93% in saline at room temperature and 68% in whole blood at 37°C. Table 3. Effect of volume of whole blood sample on labelling efficiency of washed red blood cells with 6’Ga oxine Labelling efficiencv
(%I Initial After wash
mL whole blood 1
78.7kO.9 65.9k2.5
2 89.5kl.l 82.7 +0.9
3 93.3kO.7 88.2 f0.6
Each value is mean + SD for 3 determinations.
4 84.325.4 71.3 f 10.6
67Ga red blood cells
Welch et al. (1977) reported RBC labelling efficiencies with 68Ga oxine of 93 f 5%; however, that method started with 20mL whole blood, included an extra wash step, and had longer centrifugation and incubation times. In contrast, the present method requires only 3 mL blood and can be completed within 20-30 min, yet the yield is only slightly lower. There has been a previous report of RBC labelling with 67Ga by a different approach. Pant et al. (1983) treated washed RBCs with tannic acid prior to surface labelling with 67Ga chloride in 89% yield. However, their method did not work with 67Ga citrate and thus would not be readily applicable. The main disadvantage to the use of 67Ga is its long half-life, which limits the dose which can be administered. In addition, radioactivity remains in the circulation, making it impossible to repeat a quantitative study for a number of days. The IOO-min half-life of 1’3mInmakes it the most desirable of the currently available radionuclides for cell labelling. Increased use of “3mIn may make it more costeffective to supply in the future. It has also been suggested that one ‘13Yn generator could be shared among several institutions (Morrissey et al., 1988). In summary, we have reported a rapid and efficient method to label RBCs with a routinely available radiopharmaceutical which is suitable for dualisotope imaging with 99mTc.
81
References Funke Kupper A. J., Verheugt F. W. A., Jaarsma W., van der Wall E. E., van Eenige M. J., den Hollander W. and Roos J. P. (1986) Detection of ventricular thrombosis in acute myocardial infarction: value of indium-I 11 platelet scintigraphy in relation to two-dimensional echocardiography and clinical course. Eur. J. Nucl. Med. 12, 337-34 1. Hartshorne M. F., Graham G., Lancaster J. and Berger D. (1985) Gallium-67/technetium-99m methylene diphosphonate ratio imaging: early rabbit osteomyelitis and fracture. .I. Nucl. Med. 26, 272-277. Machac J., Vallabhajosula S., Goldman M. E., Goldsmith S. J.. Palestro C.. Strashun A..,.Vaouer R.. Phillios R. A. and Fuster V. (1989) Value of blood-pool subtraction in cardiac indium-1 1 l-labeled platelet imaging. J. Nucl. Med. 30, 14451455. Malik M. H. (1985) Simultaneous dual isotope studies in the diagnosis of infection. J. Nucl. Med. 26, 722-725. Morrissey G. J., Gravelle D., Dietz G., Driedger A. A., King M. and Cradduck T. D. (1988) In vitro red blood cell compatibility testing using indium-113m tropolonelabeled red blood cells. J. Nucl. Med. 29, 684-688.
Pant K. D., Coupal J. J., Shih W-J. and DeLand F. H. (1983) A new approach to gallium-67 labeling of human ervthrocvtesandnlate1ets.J. Nucl.Med.XP123(Abstract). Srivastava-S. C. and Chervu L. R. (1984) Radionuclidelabeled red blood cells: current status and future prospects. Semin. Nucl. Med. 14, 68-82. Welch M. J., Thakur M. L., Coleman R. E., Pate1 M., Siegel B. A. and Ter-Pogossian M. M. (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. and Moyer B. R. (1985) Gallium-68 lipophilic complexes for labeling platelets. J. Nucl. Med. 26, 1429-1437.