Gallium-GB Labeled Red Cells and Platelets

New Agents for Positron Tomography

Michael J. Welch, Mathew 1.Thakur,* R.Edward Coleman,tMohan Patel4 BarryA. Siegel, and MichelM.Ter-Pogossian

Mallinckrodt institute of Radiology, St. Louis, Missouri

Red cells and platelets are labeled with Ga-68 by preparing the Ga-MIhydroxy-quinoline (oxine) complex and mixing the complex with the separated cellular components. The complex is prepared by first breaking tIu@Ga-68 EDT4 chelate with concentrated hydrochloric acid, adsorption of thegallium on a strong anionic ion-exchange column, and elution of the galliumactivity with distilled water. Oxine is added, the oxine complex extractedinto chloroform, and—following evaporation and dissolution—the solution

is added to either red cells or platelets that have been centrifuged from

whole blood, washed, and suspended in normal saline. After 15 mm ofincubation, labeling efficiencies of >90% and yields of @.‘7mCi are obtamed from 18 mCi of Ga-68 EDTA eluted from the generator. After injec

tion of the gallium-labeled red cells in dogs, excellent blood pool imageswere obtained by positron tomography. The distribution corresponded tothat of ‘1C0-labeled red cells. When labeled platelets were injected intodogs with experimentally induced intimal injury of on carotid artery, thearea of injury was clearly visualized 40 mm after tracer injection.

JNudMed 18:558—562,1977

For the past decade the major advances in clinicalnuclear medicine have been strongly dominated bythe use of the scintillation camera and ftftmTc_labeledradiopharrnaceuticals.This approachsuffers fromthree limitations (1).

1. The scintillation camera compresses into atwo-dimensional image the distribution of activitywithin a three-dimensional object. Further, activitycontained in tissue overlying and underlying the region of interest is superimposed on the image. Thismay seriously interfere with the identification of thestructures of interest.

2. The gamma radiation detected by the camerais attenuated, in general unaccountably, by the tissues interposed between the region of interest andthe crystal of the camera. In most cases this makesquantitation difficult if not impossible.

3. The field of view, resolution, and sensitivitychange with distance.

These disadvantages can be eliminated by the utilization of emission tomography (2—4) with posi

tron-emitting radiopharmaceuticals, which permitsaccurate quantification of the radiopharmaceuticaldistribution.

One major limitation to the general applicabilityof positron tomography is the scarcity of suitableradiopharmaceuticals. To date, the applications ofsuch devices discussed in the literature (5—8) mainlyinvolve radiopharmaceuticals labeled with the veryshort-lived nuclides 11C, 13N, 150. Although cornpounds labeled with these emitters have great potential for the study of regional physiology,their

Received Oct. 8, 1976; revision accepted Dec. 28, 1976.For reprints contact: Michael J. Welch, Mallinckrodt In

stitute of Radiology, 510 South Kingshighway Blvd., St.Louis, MO 63110.

* Present address: MRC Cyclotron Unit, Hammersmith

Hospital, Ducane Road, London, England.t Present address : Div. of Nuclear Medicine, Dept. of

Radiology, University of Utah Medical Center, Salt LakeCity, Utah.

@ Present address: Radiation Medicine Centre, Tata Memorial Hospital, Bombay, India.

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RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

use is limited to institutions with an “in-house―biomedicalcyclotron.

As a result, more widespread application of positron tomography will probably require the use ofpositron-emitting radionuclides that can be producedwithout an in-house cyclotron. A leading candidateis Ga-68 (T112 68 mm), which is formed by the decay of 275-day 68Ge. The commercially availablegenerator system allows the elution of the gallium inthe form of Ga-68 EDTA (9). In spite of the cornplexity of procedures where other than Ga-68 EDTAis used, several radiopharmaceuticals have been labeledwith Ga-68after the gallium-EDTAcomplexhas been decomposed (9—19). In the present workwe prepared two new agents, Ga-68-labeled redblood cells and platelets, and have studied their invivo behavior in dogs utilizing the positron-emissiontransaxial tomographic ( PETT ) scanner.

METHODS

Gallium-68 is obtainedas Ga-EDTA by elutionof a 25-mCi Ga-68 generators with 10 ml of 0.05 MEDTA solution (pH 7.0). Ten milliliters of 12 Mhydrochloric acid is added and the solution is passedthrough a small column (2 cm long by 0.3-cm diam)containing a strong anion-exchange resint (9). Thisresin adsorbs the gallium as GaCI4 and the EDTAis washed off the column. After a rinse with 20 mlof 6 M hydrochloric acid, the gallium is eluted offthe ion-exchange column with 2 ml of distilledwater. This solution is still acidic; it is boiled todryness in a 30-mi beaker, the residue dissolved in2 ml of 0.02 M hydrochloric acid, and the pH adjusted by the addition of 200 @lof acetate buffer(0.5 M, pH 5.5). During this preparation of Ga3@,care must be taken to remove all traces of metalsfrom the solutions involved. All glassware must bethoroughly cleaned and all the hydrochloric acidsolution used must be purified by passage througha large column containing the strong anion-exchangeresin.

8-hydroxyquinoline ( I 50 @gin I 50 @lof ethanol)is added to the gallium solution and mixed thoroughly. The resulting complex is then extracted intwo equal volumes ( 1.5 ml each) of chloroform.Typically, from 50 to 80% of the activity is extracted. After evaporation of the chloroform todryness in a boiling-water bath, the residue is dissolvedin 0.5 ml of.20% (v/v) ethanol.

Cell separation and labeling. Red cells are separated from 20 ml of heparinized whole blood bycentrifugation at 180g for I 5 mm. The separatedcells are then washed twice with equal volumes ofisotonic saline solution. Following the final wash,the gallium-oxine complex is added to the cells and

the mixture is incubated at room temperature for 15mm.

For the platelet separation, 43 ml of whole bloodis collected in a 50-mi syringe containing 7 ml ofACD solution. The blood is carefully transferred toa 50-mi sterile plastic centrifuge tube and centrifuged at 180g for 15 mm. With siliconized Pasteurpipets, the platelet-rich plasma is separated into twoequal volumes in sterile conical tubes, and is furthercentrifuged at I SOOg for I 5 mm. The platelet-poorplasma is removed and the platelets gently resuspended in 10 ml isotonic saline solution. The platelets are again centrifuged at I 500g for I 5 mm, resuspended, and the centrifugation repeated. Finally,the washed platelets are suspended in ‘—‘7ml of isotonic saline and the solution containing the 68Gaactivity added. This platelet suspension is allowed toincubate for I S mm at room temperature. The labeling yield in each study was determined by centrifugation of an aliquot of the sample that was injected,the cellular component being separated from thesupernatant and each fraction counted.

Animal studies. Dogs anesthetized with sodiumpentobarbital (30 mg/kg) were positioned in thepositron-emission transaxial tomograph (PETT)scanner (2,3) on their backs so that the tomographic slice would be through the region of interest.In all cases transmission scans (2,3) were performedbefore injection of the radiopharmaceutical, using aring filled Cu-64 in order to obtain data for theattenuation correction of the emission image.

The labeled blood components (@ mCi) wereinjected intravenously. For comparison studies, insome cases 11C-labeled carbon monoxide was administered by inhalation to form [‘1C]-carboxyhemoglobin. Before administration, the 11C0 is collectedin an ambu bag and diluted with air. The 11CO wasproduced by the 10B(d,n)11C reaction using the deuteron beam University Medical School cyclotron,with boric oxide as the target material. The resultingmixture of 11C0 and 11C02 is swept out of the targetby helium gas and passed over zinc at 700°C toreduce 11C02 to 11C0.

The accumulation of platelets in areas of endothelial injury was studied in four dogs by damaging the endothelium of the carotid artery (19). Thefemoral artery was exposed and, under fluoroscopiccontrol, a 4F Fogarty arterial catheter was insertedinto the carotid artery. The labeled platelets wereadministered, the catheter balloon inflated to 700mm Hg pressure and pulled back into the aorticarch. In one of the dogs the catheter was simply inserted and left in place, uninflated, for approximatelyI 5 mm. The catheter was then removed, the femoralartery repaired, and the surgical incision closed.

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WELCH, THAKUR, COLEMAN, PATEL, SIEGEL, AND TER-P000SSIAN

8-hydroxyquinoline complex was synthesized duringthe last wash. Owing to the lower concentration ofplatelets, this exchange is a greater problem withthe labeling of platelets than with the labeling ofred cells. The similarity of indium and gallium isalso evident in the fact that the stability and themethods of preparation of these gallium-labeledblood components parallel those previously observedfor In-i 1 1-labeled blood components (21—25) . Thelabeling is thought to occur (26) by the diffusion ofthe gallium-8-hydroxyquinoline through the cellmembrane and the transfer of the gallium to intracellular components.

In vivo studies. As with indium-labeled bloodcomponents (21—26) the Ga-68 remained bound tothe components for several hours after injection,and the tracer thus remained in the blood pool. Inthe abdomen (Fig. 1) excellent tomographic sections of the liver (upper level) and spleen (lowerlevel) wereobtained.WhenC-i 1 wasadministered,sections taken, and gallium-labeled red cells administered after the decay of the 11C,very similar imageswere obtained with the two blood-pool agents. Figure 2 shows typical images of the two tracers at thelevels of the heart and abdomen. The slight difference observed between the two images of the heartis probably due to the difficulty in repositioning theanimals for cardiac tomography, where a differenceof 2 mm will produce a different image (unpublished data) . The images obtained show the potential of Ga-68-labeled red cells. It seems probable thatthey can be used as a substitute for C-i 1-labeled car

FIG. 2. Tomographicimagesof dog, showingsimilarityofimages obtained with Ga-68 red cells and (‘1C0Jcarboxyhemoglobin. Red cells were injected intravenously,whereas“COwasadministeredby inhalation.

A

I

p

68Ga@red cells

FIG. 1. Upper-leveltomographicimageof Ga-68-labeledredcells injected intravenously in dog, showing accumulation in liverand spleen. Lower-level tomogram reveals mainly spleen and onlysmall amount of activity in tip of liver. Images were taken within30 mm of injection, and time of study (including transmissionimages) was approximately 1 hr.

RESULTS AND DISCUSSION

Cell labeling. High yields of labeled red cells wereobtained in all cases. In five studies, yields of 93 ±5 % were obtained. The generator was eluted whenthe cells were separated and washing was almostcomplete, so that there was minimal delay betweenthe procedures involving the short-lived gallium.Typically the cells were ready for injection 35 mmfollowing generator elution; I 7 mCi of 68Ga waseluted from the generator, 10 mCi of the galliumchloride was formed, and @-..‘7mCi of cells was injected into the dogs.Analysis of the injectatebycentrifugation showed that >90% of the activitywas associated with the cells. Attempts to label theblood components with Ga-68 produced by thermaldecomposition of the Ga-68 EDTA (9) proved unsuccessful.

Labeling yields with platelets tended to be morevariable, and maximum yields were only obtainedwhen all materials used in labeling were purified asdescribed in the section on methods. Low yields canbe caused by incomplete removal of transferrin fromthe cells or platelets before labeling. Like indiumtransferrin (20), gallium-transferrin has a high stability constantandwill form preferentiallyif all thetransferrin is not removed. The overall labeling procedure takes @-..-70mm. The Ga-68 was eluted fromthe generator before the last platelet wash, and the

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560 JOURNAL OF NUCLEAR MEDICINE

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“Ga-platelets - “CO-red cells

RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

great potential for imaging blood pools and assessing areas of platelet accumulation. These two new

radiopharmaceuticals add to the Ga-68 radiopharmaceuticalsavailablefor usewith positrontomography.

ACKNOWLEDGMENTS

This work was supported by NIH SCOR in ThrombosisHL14147 and NIH 5 P01 HLl385l. The authors thankRobert Feldhaus, Joan Primeau, Barbara Asberry, MickeyClarke, and Carol Higgins for their assistance.

Mohan Patel is a World Health Organization Fellow.

FOOTNOTES

C New England Nuclear Corp., Boston, Mass.

t Bio-Rad AG I-X8, Richmond, Calif.

REFERENCES

1. TER-POGOSSIAN M : The challenge of computerizedaxial tomography to nuclear medicine imaging. In YearBook of Nuclear Medicine, Quinn JL, ed, Year Book Mcdical Publ., Chicago, 1976, pp 9—14

2. TER-POG0SSIAN MM, PHELPS ME, HOFFMAN EJ, Ct al.:

A positron emission transaxial tomograph for nuclear mcdicineimaging(PElT). Radiology114:89—98,1975

3. HOFFMAN EJ, PHELPS ME, MULLANI NA, et al. : Dcsign and performance characteristics of a whole body positron transaxial tomograph. I Nucl Med 17: 493—502,1976

4. CHESLERDA: Positron tomography and three-dimensional reconstruction technique in tomographic imaging innuclear medicine. In Tomographic Imaging in NuclearMedicine, Freedman, GS, ed, Society of Nuclear Medicine,New York, 1973, pp 176—183

5. TER-POGOSSIANM, HOFFMAN EJ, WEISS ES, et al.:Positron emission reconstruction tomography for the assessment of regional myocardial metabolism by the administration of substrates labeled with cyclotron-produced radionuclides. In Cardiovascular Imaging and image Processing.Theory and Practice, Harrison DC, Sandier H, Miller HA,eds, Society of Photo-optical Instrumentation Engineers,Palos Verdes Estates, Calif., 1975, pp 277—284

6. Hoop B, HNATOWICHDJ, BROWNELLGL, et at.: Techniques for positron scintigraphy of the brain. I Nucl Med17: 473—479, 1976

7. JONEST, CHESLERDA, TER-POGOSSIANMM: Thecontinuous inhalation of oxygen-I 5 for assessing regionaloxygen extraction in the brain of man. Br I Radio! 49:339—343,1976

8. PHELPSME, HOFFMANEJ, COLEMANRE, et al.:Tomographic images of blood pool and perfusion in brainandheart.I NucIMed 17:603—612,1976

9. YANOT: Preparationand control of e@Ga@radiopharma@ceuticals. In Radiopharmaceutica!s from Generator-Producedisotopes, IAEA, Vienna, 1971, pp 117—125

10. HAYES RL, CARLTON JE, BYRD BL: Bone scanningwith gallium-68: a carrier effect. I NucI Med 6: 605—609,1965

11. HAYESRL: Radioisotopes of gallium. In RadioactivePharmaceuticals, Andrews GA, Kniseley RM, Wagner HN,Jr, ed, USAEC Conf-651 I 1 1, 1966, pp 603—617

12. WEBER DA, GREENBERG EJ, DIMICH A, et al. : Kinetics of radionuclide used for bone studies. I Nuc! Med10:8—17,1969

13. EDWARDCL, HAYES RL, AHUMADAJ, et al. : Gallium-68 citrate: A clinically useful skeletal scanning agent.I Nucl Med 7: 363—364,1966

FIG. 3. Tomogramsof neckof dog viewedfrom belowwithinduced endothelial damage of one carotid, showing marked accumulation of Ga-68-labeled platelets in abnormal carotid. Red-cellimage with “COreveals only slight asymmetry of carotids, whichis related to residual Ga-68 activity in damaged carotid. Ga-68-labeled platelets were administered intravenously, ‘1C0by inhalation.

boxyhemoglobin in institutions having no cyclotron.Figure 3 shows a typical image obtained following

the administration of Ga-68-labeled platelets to a dogwith intimal injury in one carotid artery. The imagewas taken 30 mm after injection of the radiopharmaceutical. The digital printout of the actual activitypresent in the tomogram showed that in this imagethe ratio of activity in the damaged carotid to that inthe undamaged carotid was 15 : 1. In the four dogsstudied the ratios were all greater than I 2 : 1. In onedog the ratio could not be calculated because thenormal carotid was not observable over the background in the neck. We have previously shown histologically (23) that intimal damage is producedin this model, and that there is platelet accumulationin the damaged area. In the dog where the catheterwas not inflated, the ratio of activity in “damaged―carotid to the activity in “normalcarotid―was@ : I.The simple insertion of the catheter appears to damagethe arterysufficientlyfor plateletsto accumulatein amounts detectable by positron tomography.

Figure 3 also shows a tomogram taken followingadministration of “COto the animal by inhalation,with visualization of both carotids. The right carotidappears to contain the greater activity, but there isstill residual gallium activity in the left carotid. Whenthe digital printouts of the activity of the galliumplatelet scan and the 11CO scan were compared, andthe residual gallium activity was subtractedfromthe [11CO}-carboxyhemoglobin picture, the 11C activity was equal in both arteries. In the study where thecatheter was not inflated, the carboxyhemoglobinactivity was the same in both arteries.

CONCLUSION

The combination of positron tomography and theGa-68-labeled blood components appears to have

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WELCH, THAKUR, COLEMAN, PATEL, SIEGEL, AND TER-P000SSIAN

14. HAYES RL, CARLTON JE, RAFTER JJ : Scanning withhydrous ferrous oxide colloid labeled with @Ga.I NuclMed7: 335—336,1966

15. ANGHILERI L: A new @Gacompound for kidneyscanning. mt I App! Radial isot 19: 421—422,1968

16. ANGHILERI L, Piu'IC B: A new colloidal @Ga-compound for liver scanning. ml J App! Radial isot 18: 734—735, 1967

17. GOULET RT, SHYSH A, NOUJAIM AA, et al. : Investigation of “Ga-tripolyphosphate as a potential bone scanning agent. list I App! Radial isol 26: 195—199, 1975

18. DEWANJEE MK, BEH R, HNATOWICH DJ : New @Galabeled skeletal imaging agents for positron scintigraphy.INucIMed 17: 566, 1976

19. STEMERMAN MB, Ross R: Experimental arterioscierosis. I. Fibrous plaque formation in primates, an electronmicroscopic study. I Exp Med 136: 769—789,1972

20. WELCH MJ, WELCH TJ: Solution chemistry of carrierfree indium. In Radiopharmaceutica!s, Subramanian G,Rhodes BA, Cooper JF, Ct al., eds, Society of Nuclear Mcdicine, New York, 1975, pp 73—79

21. THAKUR ML, COLEMAN RE, MAYHALL CG, et al.:Preparation and evaluation of 111In-labeled leukocytes as an

abscess imaging agent in dogs. Radiology 119: 731—732,1976

22. THAKUR ML, COLEMAN RE, WELCH MJ: Indium-i 11labeled leukocytes for the localization of abscesses. Preparation, analysis, tissue distribution and comparison withgallium-67 citrate in dogs. I Lab Clin Med 89: 2 17—228,1977

23. THAKUR ML, WELCH MJ, JoIsT JH, et al. : IndiumI 11 labeled platelets: Studies on preparation and evaluationof in vitro and in vivo function. Thromb Res 9: 345—357,1976

24. THAKUR ML: Gallium-67 and indium-I 11 radiopharmaceuticals. mt I App! Radiat isot, in press

25. COLEMAN RE, WELCH MJ, THAICURML: Preparationand evaluation of leukocytes labeled with indium-I 11. IAEASymposium on Medical Radionuclide Imaging, Proceedings,in press

26. THAKUR ML, DEES D, HARWIG SSL, et al.: Labelingblood components with 8-hydroxyquinoline chelates: simplified procedure and mechanism of labeling. Presented atFirst International Symposium on RadiopharmaceuticalChemistry, Brookhaven National Laboratory, New York,I Lab Cpds Radiopharm

562 JOURNAL OF NUCLEAR MEDICINE

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1977;18:558-562.J Nucl Med.   Michael J. Welch, Mathew L. Thakur, R. Edward Coleman, Mohan Patel, Barry A. Siegel and Michel M. Ter-Pogossian  Gallium-68 Labeled Red Cells and Platelets New Agents for Positron Tomography

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