180

Technical notes Conclusions

Use of scintillation camera and a collimated line source has made the measurements simple and quick as compared to rectilinear scanners commonly used for such measurements. Five mCi of 153Gd (T112 = 242 days) in a form of a line source with scintillation camera seems to be great advantage to us as compared to 100-300 mCi of 1251 ( T 112 = 60 days) point source commonly used with rectilinear scanner especially as 1251 is not available from local sources. 153Gd gives good sensitivity and reproducibility and we expect to detect I 0-20% calcium loss from long bones.

u. R. RAIKAR R. D. GANATRA Radiation Medicine Centre Biomedical Group Bhabha Atomic Research Centre cfo Tata Memorial Hospital Pare!, Bombay, India References

I. SoRRENSON J. A. and CAMERON J. R. J. Bone Jt Surg. 49A, 41 (1967). 2. DE PuEY E. G. and BuRDINE J. A. Radial. 105, 607 (1972).

International Journal of Nuclear Medicine and Biology, 1975, Vol. 2, pp. 180-184. Pergamon Press. Printed in Northern Ireland

Rapid and Reliable Preparation of Macroaggregated Albu:min Suitable for Lung Scintigraphy (Accepted 22 January 1975) Introduction

THE usE of labeled albumin as a biological scanning agent was first reported in 1956 by BENACERAFF, who used macroaggregated albumin labeled with iodine-131 to study reticuloendothelial function ;U> seven years later TAPLIN proposed the use of macroaggregated albumin (MAA) labeled by a similar t The procedure used by those workers to label albumin with 1311 took about three days to complete, (S) but within a decade faster labeling techniques had been developed, and 131J-MAA was being widely used to study the hemodynamics of the pulmonary circulation. In 1964, McAFFEE and STERN proposed the labeling

ofa1bumin with technetium 99 m.! 4 ) Their technique was used two years later by GWYTHER to label macroaggregated albumin for use as a lung scanning agent. !S) Since then, numerous procedures have been suggested for labeling not only macroaggregated albumin with 99 mTc, (6- 10 > but also ferric hydroxide particles and other types of aggregated materials with various radionuclides, such as indium-113 m!ll) and gallium-68. !12) In spite of their usefulness, these other types of aggregates have not displaced macroaggregated albumin labeled with 99mTc. Today the only alternative to labeled MAA for obtaining lung scans is the use of labeled albumin microspheres. The high cost of the microspheres, however, and the complicated method needed to prepare them for use have limited their application, at least in routine lung perfusion studies, in which highly uniform particle size is not essential. Furthermore, poor labeling yields make it necessary to wash the preparation to eliminate free pertechnetate. Finally, the microspheres have a longer biological half-life than MAA. Several problems arise in the preparation of macroaggregated human serum albumin for use in lung scans. The particle size must lie within the range of about 20-50 p,m so that the particles will become trapped in the capillary bed of the lungs without causing the formation of clinically significant emboli. The particles must not be too fragile, lest they break up before the lung scan can be performed, yet not too hard for the body to metabolize them after a few hours. In addition, the labeling with 99 mTc must be complete and firm so that a scan of the macroaggregates lodged in the lungs with minimum background can be made. For this same reason it is desirable that no unprecipitated albumin remain in the scanning agent, because unprecipitated albumin will also be labeled with 99 mTc and will produce a high background in the scan. We have reviewed the published methods for preparing macroaggregates of albumin and studied the effects of the variables in those methods. The purpose of this paper is to present the studies we have performed to develop a standardized technique that will produce a well defined radiopharamceutical suitable for lung perfusion studies. There are two alternatives in preparing 99 mTcMAA. One possibility is first to label the albumin with the radionuclide and then to aggregate it by heating. In the other, the albumin is first converted to macroaggregates, and these are labeled with 99 mTc just before use. We prefer the latter sequence because tagging of preformed particles of the proper size is very simple. In our study of the preparation ofmacroaggregated human serum albumin we determined the effects of

181

Technical notes varying the pH, the temperature, the stirring speed, and the heating time on the size and hardness of the particles.

Thermometer

Materials and Methods

Albumin solutions. Portions of 5 ml, commercially available* 25% salt-poor human serum albumin were diluted to 100 ml with physiological (0·9%) saline solution. The pH of these solutions was adjusted to the desired value by the addition ofO·l M phosphoric acid or sodium phosphate buffer (pH 6·0). If the macroaggregated albumin was to be labeled, stannous chloride solution (l,umolefmg albumin) was also added. If the MAA was to be used in biological tests, the solution was sterilized by Millipore filtration, using a 0·22 ,urn pore-size membrane. Aggregation procedure. The albumin was converted onto macroaggregates by placing the solution in a stoppered, sterile 250 ml flask containing a vinylcovered magnetic stirring bar (25 x 10 mm); the stopper had a needle air vent and held a thermometer reaching into the solution. The flask was positioned as shown in Fig. 1 in a heated water bath (controlled at the desired temperature) on a submersible magnetic stirrert set to the desired agitation rate. After the heating period the flask containing the aggregated albumin was cooled under running water for 2-4 min. In preliminary experiments, the aggregates were not washed free of unprecipitated albumin, but if the MAA was to be used in scanning, the particles were washed with 50 ml portions of sterile 0·9% saline solution until the washings contained less than 0·05% albumin, as determined by the biuret reaction (see below). Usually, two washings were sufficient. The size of the particles in each batch was measured with the aid of a hemacytometer. Labeling technique. If the MAA was to be labeled and used immediately, an aliquot of the suspension containing 5 mg of particles was mixed with the desired amount of 99 mTc (usually about 40 mCi), and the mixture was incubated at room temperature for 5-10 min. (la-l4) Tagging efficiency was tested by rapid column chromatography(lS) and instant thinlayer chromatography. If the MAA was not to be used immediately, the suspension was divided into portions, each containing 5 mg of particles; these portions were transferred in to individual vials and promptly lyophilized. The lyophilized MAA could be kept in a refrigerator (2-4°C) for periods as long as 6 months. The addition of a *Armour Pharmaceutical Co.- Phoenix, Arizona 85077, U.S.A. t Tri R Instruments Co. - Rockville Center, N.Y. 11570, U.S.A.

FIG. I. Apparatus used to prepare the macroaggregated albumin: (a) a 250 ml Erlenmeyer flask; (b) propeller to keep the temperature in the water bath uniform; (c) micro submersible magnetic stirring device; (d) water or oil bath; (e) heating system; (f) speed controlling device. surfactant, such as sodium Iaury! sulfate, or polyoxethylene sorbitan mono-oleate, will assist in dispersing the particles, when needed. To label a lyophilized sample ofMAA 2 to 5 ml of a saline solution containing no more than 40 mCi of 99 mTc0-4 was added. Biuret reaction. U nprecipitated albumin in the MAA wash liquors was assayed by mixing I ml of the supernatant with 4 ml of biuret reagent, (16) letting the mixture stand 30 min, and measuring its absorbance at 540 nm against a blank (1 ml of water plus 4 ml of reagent), using a Bausch and Lomb Spectronic 30 spectrophotomete r. Absorbance values were converted to albumin concentrations by the use of a calibration curve. Experbnental In the initial group of experiments, several sets of eight 1·25% solutions of albumin were prepared; within each set of eight solutions, the pH was adjusted to provide a series in which the pH ranged from 4·0 to 7·5 increments of 0·5. These solutions were subjected to aggregation at various temperatures and stirring rates and for various periods of time. Figure 2 shows typical preparations made at 75-78°C at pH values varying by half-unitsfrom4· 0 to 7·0. At pH 4·0 (Fig. 2a), a gel formed, while at pH 4·5 (Fig. 2b), large clots were produced. At pH 5·0 (Fig. 2c), smaller particles were obtained, and at pH 5·5 and 6·0 (Figs. 2d and 2e), the macroaggregates were of the size required for lung scanning. At pH 6·5 (Fig. 2f) or higher, gels again resulted.

Technical notes

182

TABLE 1. Distribution of radioactivity in a dog sacrificed 30 min post i.v. Notice that lung-liver ratio is excellent for scintigraphic studies 9 ~c-HAA DISTRIBUTION IN DOG ( Animal Euthanized 30' After Dose Was Administered )

ORGAN

WEIGHT (g.)

COUNTS/100 SEC. *

CORRECTED COUNTS/100 SEC.

CORRECTED CPM

DISTRIBUTION S (INJECTED DOSE)

STOMACH

190

18.098

8. 217

4. 930

0,444

LUNG

186

1. 530.062

1. 520.181

912.109

82,25>

CPM/g. TISSUE 26

57:1

4.) 158:1

LIVER

519

36.438

26.559

15.935

1,436

31

SPLEEN

58

11.363

1.482

889

0,08

15

KIDNEYS

35

21.979

12.098

7. 259

0,65

207

HEART

228

15.557

5.676

3.406

0,31

15

263.874

158.324

14,3

CARCASS

273.755

STANDARD (DOSE)

1.919.093

LEFT IN SYRINGE

70.782

BACKGROUND

9.881

--------------- ............................. ---------------------------------------------Injected Dose:

(Dose - Background) - (Syringe - Background) (1.919.093- 9.881)- (70.782- 9.881) = 1.848.311/100 sec 1.108. 987 /cpm

* All Counts Were Done In A.Whole Body Counter.

A second group of experiments was conducted keeping temperatures, heating time and stirring rate constant, to observe the effect of varying the pH in increments of 0•1 unit within the range of 5•5-6·0 selected from the results of the first group of tests. More than 10 batches of MAA were prepared at each pH; at pH 5·9 ± 0·1, more than 75% of the preparations yielded particles of the size shown in Fig. 2e. In a third set of experiments, the effect of varying the temperature was studied, using albumin solutions buffered at pH 6·0 and holding the stirring speed at 550-600 rev/min and the aggregation time at 15 min. Some of the macroaggregates formed in these trials are shown in Fig. 3. Particles formed at 66-68°C or at 71-73°C (Figs 3a and 3b, respectively) were not well formed and had a soft appearance. Two different batches of such particles were injected into anesthetized dogs, which were sacrificed 30 min later. The physiological distribution of the radiopharmaceutical was poor: only 6·8% was found in the lungs, while more than 4% was found in the stomach, more than 10% in the liver, and about 10% in the kidneys and urme. Particles typical of those formed in 24 of 32 experiments conducted at temperatures of 76-78°C are shown in Figs. 3c and 3d. At higher temperatures, larger particles were obtained, as shown in Fig. 3e (temperatures 83-85°C) and Fig. 3f (96-100°C).

Even higher temperatures produce large clots, as shown in Fig. 3g (about 110°C, obtained by using an oil bath). In a few experiments in this group, the heating time was increased to as much as 90 min; in most of these longer experiments, particles more than 50 p,m in dia. were formed, as shown in Fig. 3h. In a fourth series of experiments, the pH was held at 6·0, the temperature at 76-78°C, and the heating time at 15 min, while the stirring speed was varied. The results are shown in Fig. 4. At low speeds150 rev/min (Fig. 4a) and 350 rev/min (Fig. 4b)the particles formed were too large; at the highest speed tested (750 rev/min; Fig. 4e), they were to small. The desired particle size was obtained when the stirring speed was kept at 550 revfmin (Fig. 4c) to 650 rev/min (Fig. 4d). Under these conditions (pH 6·0, temperature 76-78°C, heating time 15 min and stirring speed 550-650 revfmin), and using sterile solutions and equipment, the particles prepared were, in most cases, suitable for lung perfusion studies. Labeling of these particles is accomplished by adding a solution of pertechnetate 99mTc. Tagging efficiency proved to be greater than 95%, and the tagged particles remained stable in vitro for at least 8 hr. Figure 5 shows chromatogram obtained with one of these preparations. The biological distribution and catabolism of MAA prepared as described in the preceding paragraph

~

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(f )

(e)

.,., r

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3 (a)

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FIG. 2. Effect of solution 's pH on the particles size. From a to f, the pH of the solutions wen~ 4·0 , 4·5, 5·0, 5·5, 6·0 and 6·5. Notice that pH lower than 4·5 and higher than 6·5 produced a gel. The best particles were formed when pH was kept at 5·5 to 6·0 (Figs. d and e). FIG. 3. Effect of temperature in the particle formation. At low temperature, below 72° to 73°C (a and b) the particles seemed soft and not well formed. Batches c and d were prepared with temperatures ranging from 76 to 78°C. Temperatures of83-85°C (Fig. e) and 96-100°C (Fig. f) produced larger particles. Higher temperatures, ranging between 100-110°C (Fig. g) produced large clots. Increasing heating time, up to 90 min, seemed to increase particle size (Fig. h).

182

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6 Fw. 4. Effect of mixing speed in the preparation of particles at 76-78°C and solutions of a pH = 6·0. The best particles were obtained at mixing speeds of 550 rev/min (Fig. c) to 650 rev/min (Fig. d). Slower agitation (a-150 revfmin and b-350 rev/min) seemed to produce larger size particles, while faster agitation (e--over 750 rev/min) produced smaller particles. Fw. 6. Serial scans done on a dog at, from left to right, 5, 30 and 70 min and 6·5 hr posti.v. Notice that at 70 min post i. v. a shade of the kidneys and stomach can already be seen (third image from left). 6·5 hr post i.v. (right of picture), the metabolization of the particles is quite advanced. Activity can be seen in the stomach, liver and kidneys.

183

Technical notes 3

4

2

Fm. 5. Radiochrom atograms done on the labeled macroaggregates at I and 2 hr post preparation , on top, and 3 and 4 hr on bottom of the figure. It seems that the labeling of 99 mTc is quite strong,

in vitro,

were studied in dogs. Table I shows the distribution of activity in a dog sacrificed 30 min after administration of the dose. The organs were separated and counted in a whole body counter. The high ratio

(57: 1) of the activity in the lungs to that in the liver is still conducive to the production of good lung scintigrams. Figure 6 shows serial scans of a dog after injection with a 4 mCi dose of 99 mTc-labeled MAA. At the left, is the first scan, started 5 min after the injection; second from left is the scan started 30 min later. Next, thescanstar ted 70 min after the injection; the kidneys and the stomach are beginning to show some accumulati on of activity, indicating that some technetium has gone back into the blood circulation. At right of the figure is the scan started 6·5 hr after injection; it shows that much of the activity has become localized in the stomach, and the kidneys; activity also appears in the liver, indicating that breakdown of the particles is far advanced. The activity of blood samples drawn from injected dogs keeps increasing for about one hour, as shown by the data in Table 2, which also shows how, with the passage of time, the activity diminishes in the lungs and increases in the liver and the bladder. This data demonstrates that as technetium is released from the lungs it accumulates in the liver, the bladder and the gastrointestinal tract. We did not estimate acute or subacute toxicity of our MAA preparation s because appropriate studies of similar preparation s have already been reported by other investigators. 117)

macroTABLE 2. Blood and organs counts on an anesthesized dog, injected with 3 mCi of labeled aggregates. Activity in blood samples keeps increasing up to about one hour and then starts decreasing. while Counts over the organs of the dog, showed that 20 min post i. v. counts of lung is already decreasing, the dog counts over liver and bladder increased. Counts over bladder at 4 hr couldn't be taken because voided 9 ORGAN ACTIVITY Ill A DOG INJECTED WITH 3nCi OF 9nlrc-MAA ORGAN*

BLOOD

TIME POST I. V.

CPM ML

TIME POST I. V.

LUNG

LIVER

BLADDER

5 min.

12.308

5 min.

80.000

5.000

3.000

10 min.

62.814

20 min.

60.000

6.000

8.000

20 min.

103.117

55 min.

48.000

s.ooo

23.000

30 min.

140.628

2 hr.

25.000

10.000

43.000

1 hr.

162.570

3 hr.

20.000

8.500

52.000

4 hr.

107.982

4 hr.

12.000

9.000

**

COUNTS, OVER HOTTEST AREA OF THE ORGAN, TAKEN WITH A 3" PROBE

Technical notes

184

Acknowledgement-This project was partially supported by the Michael Reese Research Institute. LELIO G. COLOMBETTI STEPHEN MoERLIEN STEVEN PINSKY

Pharmacology Department Loyola University Stritch School of Medicine and Michael Reese Hospital and Medical Center 29th Street and Ellis Avenue Chicago, Illinois 60616, U.S.A. References 1. BENACERRAF B., Bmzzi G., HALPERN B. and STIFFEL C. Res. Bull. 2, 19 (1956). 2. TAPLIN G. D., DoRE E. K., joHNSON D. E. and KAPLAN H. lOth Annual Meeting of the Nuclear Medicine Society, Montreal, Canada, (1963). 3. BIOZZI G., BENACERRAF B., HALPERN B., STIFFEL C. and HILLEMAND B. J. Lab. clin. Med. 51, 230 (1958). 4. McAFFEE J. G., STERN H. S., FUEGER G. F., BAGGISH M.S., HoLZMAN G. B. and ZoLLE I. J. nucl. Med. 5, 936 (1964). 5. GwYTHER M. M. and FIELD E. 0. Int. J. appl. Radiat. Isotopes, 17, 485 (1966). 6. DEUTSCH M. E. and REDMOND M. L. J. nucl. Med. 13, 426 (1972). 7. HoNDA T., KAZEM I., CROLL M. N. and BRADY L. W. J. nucl. Med. 11, 580 (1970). 8. TAPLIN G. V., GRISWOLD L. M., HuRWIT ]., and jOHNSON D. E. J. nucl. Med. 8, 303 (1967). 9. SuBRAMANIAN G., ARNOLD L. W., THoMAs F. D., and McAFFEE J. G. J. nucl. Med. 13, 790 ( 1972). 10. RoBINS P. ]., FoRTMAN D. L., and LEWIS J. T. J. nucl. Med. 13, 463 (1972). 11. CoLOMBETTI L. G., GooDWIN D. A. and HINKLEY R. L. J. nucl. Med. 10, 633 (1969). 12. CoLOMBETTIL. G.,GooDwiND.A.and ToGAMIE. J. nucl. Med. 11, 704 (1970). 13. CoLOMBETTI L. G., LoPEZ-MAJANO V. and KAPLAN E. Proc. Int. Symp. Nucl. Med., in press, Karlovy-Vary, 1973. a. CoLoMBETTIL. G.,LoPEz-MAJANo v.,KAPLANE. and FELLO M. Rev. Iner. de Radial. 8 (1973). 15. CoLOMBETTI L. G., PINSKY S. and MoERLIEN S. Radiochem. Radioanalyt. Lett., 20, 77 (1974). 16. WoLF P. L. Methods and Techniques in Clinical Chemistry, p. 353. Wiley-Interscience, New York ( 1972). 17. TAPLIN G. V., and MAcDoNALD N. S. Seminars in nucl. Med. 1, 132 (1971).

International Journal of Nuclear Medicine and Biology, 1975, Vol. 2 pp. 184-185. Pergamon Press. Printed in Northern Ireland

An Alternate Approach to the Preparation of 99mTc-Sulfur Colloid (Received 17 February 1975) Introduction 99mTc-suLFUR

colloid has gained wide acceptance as the choice radiopharmaceutical for scanning the reticuloendothelial system. The labelled colloid particulate matter is accumulated preferentially by the Kupffer cells of the RE system by phagocytosis. There are a number of procedures for the preparation of this radiopharmaceutical. The earlier methods employed the use of hydrogen sulfide gas 11 •2 > or sodium thiosulfate with or without the presence of sodium perrrhenate as carrier.W Gelatin,(l) dextran, polyvinyl pyrrolidone,< 6 > mannitol< 7 > and citric acid have been used as stabilizers of the colloid in these preparations, although the stabilizing function of mannitol for hydrophobic colloids has been questioned. Phosphate has generally been used as the buffering medium to maintain the pH conditions but sometimes citrate< 8 >has also been used. BoYD(lO) and CIFKA< 11 >have made a detailed study of the various parameters influencing the preparation of 99mTc-sulfur colloid. BRuuNI12 > has commented on the particle size generally encountered in the labelled colloidal material and its instability and hence its possible propensity to retain in the lungs. This study presents the use of minimal yet effective quantities of sodium thiosulfate in the preparation of the colloid, which is stabilized with the polyhydric alcohol mannitol and buffered with sodium citrate. The entire preparation is completed in the shortest possible time. The content of sulfur in the total preparation is less than 0·2 mg/ml. This method has been routinely followed at our Centre for the preparation of 99mTc-sulfur colloid radiopharmaceutical for use in humans for the purpose of scanning the RE system.

Materials and Methods Sodium pertechnetate (99 mTc0 4-) was obtained by the organic solvent (methyl ethyl ketone) extraction process from sodium molybdate (99 Mo) and purified. t13l All reagents were prepared in large quantities using water for i~ection for both dissolution and dilution, and sterilized by passing through a 0·22 micron Millipore membrane filter and distributed in single-dose vials ( 10 ml capacity) and subsequently