Work in progress International Journal of Nuclear Medxine and Biology. pp. 201 203. Pergamon Press Prmtrd m Great Brik
1977. Vol.
4.
Quality Control of TechnetiumLabelled Lung Imaging Agents (Receiued 14 March 1977) Introducth of y9”Tc to a variable extent in the kidney blood pool has been observed after the i.v. administration of macroaggregated albumin particles for lung scanning. Previous studies have shown that the MAA particles used for lung scanning are slowly metabolized to microaggregated albumin particles in the lung. These particles are removed from the circulation by the liver. (lp3) It is surprising that sufficient activity would accumulate in the renal area to permit visualization of the kidneys at times noted. The study reported here has revealed that technetium-labelled MAA preparations may contain g9”Tctagged albumin, similar to human serum albumin in solution, which accounts for higher blood pool activity. The nature of labelling pattern of MAA and the albumin microspheres indicates that the macroaggregates and microspheres are not completely or uniformly labelled during a one-step addition of pertechnetate. ~CALIZATION
Materials and Methods The labelling efficiency and radiochemical nature of technetium-labelled MAA supernates were studied using three commercially available kits.* Similar experiments were carried out with albumin microspherest for comparison. MAA kits were supplied as vials containing sterile, pyrogen-free lyophilized powder composed of denatured human serum albumin and stannous chloride or, in the case of the MediPhysics product, with the MAA aggregates suspended, in solution (2.2 ml/glass vial). The amount of stannous chloride in these preparations varied from 50 to 35Opg. The Macrotec (Squibb) also contains 10mg of lyophilized normal serum albumin. Each albumin microsphere labelling via1 (3M Brand) contains 5 mg of albumin microspheres and lW25O~g of tin.
* Squibb-Aggregated Albumin (Human) Macrote@. Mallinckrodt-TechneSan@ MAA Kit, Medi-physicsLungaggregateTM Reagent. t 3M Company. 1 MINITEC-“9”-Generator, E. R. Squibb & Sons. Inc., U.S.A. $Gelman Instrument Company, Ann Arbor. MI.
201
Tagging eficiency
Labelling efficiency of MAA and microspheres was determined by three different methods. (a) Centrifugation method. MAA and albumin microspheres were labelled according to directions of the manufacturer, with y”mLc-pertechnetate eluted the same day from a molybdenum generator.$ An aliquot of the tagged preparation was pipetted into a smal1 plastic or glass tube and then centrifuged at 3500 rev! min for 10 min. Total radioactivity of the suspension. sediment and supernate were measured with a radioisotope dose calibrator (Squibb CRC-6A). (b) Minicolumn method. Chromatographic minicolumns were prepared from disposable Pasteur glass pipets plugged at the tapered end with cotton and loaded to a height of 4cm with Sephadex-Gz5 presoaked in saline ovemight. Approximately O.l-ml aliquot of the test preparation was added to each of the minicolumns which were then eluted with 3 ml of saline. The eluate and the minicolumns containing the tagged particles were assayed using a Na1 (Tl) gamma scintillation counter or the radioisotope dose calibrator depending on the amount of activity used. The percentage radioactivity remaining on the Sephadex column, represents the percentage tagging. (c) Millipore jltration. 9yTc-MAA or ““Tc-albumin microspheres were passed through a 0.22 Pm Millipore membrane filter. The filtrate was assayed for free pertechnetate activity and the percentage tagging calculated. The following analytical procedures were used to determine the chemistry and the radiochemical nature of the ““Tc-MAA supemates or eluates. (a) Column and thin layer chromatography. Elution volumes of HSA and 99”Tc-pertechnetate were determined on a Sephadex-G25 column with ‘251-RISA and 9y”Tc-pertechnetate. After washing the columns with saline, 0.5 ml of 99”Tc-MMA supemate or the “‘“Tc-microsphere supemate was added to the column and eluted with saline. The percentage activity in each fraction was calculated. The supernates were also spotted on ITLC silica gel strips and acetone was used as the elution medium. After IS min, the strips were dried and cut into l-cm pieces. and counted to determine the distribution of radioactivity. (b) Protein electrophoresis. In order to confirm the findings of the chromatographic data, protein electrophoresis was carried out on the supemates from ‘9”Tc-MAA preparation (Squibb) using Sephraphore 1115 electrophoresis strips. Electrophoresis was also done on pure HSA (3 mg/cm3) and ‘*“I-RISA for comparison. Organ distrihution. The total body distribution of the 99”Tc-MAA supemates was determined in four mice weighing up to 3Og, which were killed 30 min after tail-vein injection. The radioactivity of the organs and the blood was measured in a Na1 (TI)
Work in progress
202
TABLE 1. Percentage labelling of lung aggregates
Preparation 99”‘Tc Microspheres (3M) 99”‘Tc MAA (Squibb) 99”‘Tc MAA (Mallmckrodt) 99”Tc MAA (Mediphysics)
Centrifuging (%)
% labeling* Mini column (%)
Millipore filtration (%)
98.8 83.8 98.3 95.8
99.0 89.0 96.9 96.1
97.1 86.0 99.0 95.7
* Average of four experiments. TABLE2. Sephadex column chromatography
of supernates
% activity* Albumin fraction
Preparation 99”‘Tc-Microspheres 99”‘Tc-MAA (Squibb) 99Tc-MAA (Mallinckrodt) 99’Q-MAA (Medi Phys)
Pertechnetate
10.9 65.7 1.2 12.5
67.1 20.0 29.3 30.5
Total recovery 78.0 85.7 30.5 43.0
* Average of four experiments. TABLE3. ITLC data on supemates % activity distribution* Albumin fraction
Preparation
99”Tc-pertechnetate fraction 34.8 66.2
65.2 33.8
99”Tc-MAA (Squibb) 99”Tc-MAA (Mallinckrodt) * Average of four expetiments.
TABLE4. Labelling of fresh MAA with 99”‘Tc MAA supemates*
Preparation
% tagged
% free activity
15.5 90.3
84.5 9.7
99”Tc MAA (Squibb) 99”‘Tc MAA (Mallinckrodt) * Average of two experiments. TABLE5.
Preparation 99*Tc Microspheres 99”Tc MAA (Squibb) 99”Tc MAA (Mallinckrodt) 9911TcMAA (Mediphysics) * Average of four experiments.
% @ging with successive* additions of 99”‘Tc pertechnetate Step 1 Step 11 Step 111 Step IV 98.8 83.8 98.3 95.8
90.0 91.1 99.0 86.4
57.0 60.7 64.0 55.8
66.0 26.1 35.7 27.4
Work in progress
gamma scintillation counter and determined as percentages of the total amounr injected. Labelling eficiency. 99”Tc-pertechnetate was added in mCi amounts in repeated steps until further uptake of activity by the aggregates or microspheres was not significant. Labelled particles were washed twice with saline before the next aliquot addition of 99’“Tcpertechnetate. The following test was performed to further confirm that the supemate of 99Tc-MAA preparation in some cases contains higher percentage of labelled soluble albumin. A known amount of 99”‘Tc-pertechnetate was added to a via1 of MAA, the preparation was centrifuged and the amount of radioactivity in the supemate was determined. A fresh via1 of MAA was then labelled with 99”Tc-MAA supemate obtained from the previous preparation. The percentage tagging of MAA in the second preparation was determined as above by centrifugation.
Tagging efficiency determined by the methods described (Table 1) revealed that as much as 15% of free activity (non MAA-Tc) is present in 99”Tc MAA of Squibb preparation, whereas the other three preparations did not contain more than 2% free activity. The chromatographic data given in Tables 2 and 3 indicate that up to 65% of the radioactivity in the Squibb 99”Tc MAA supemate was due to labelled soluble albumin. Protein electrophoresis experiments confirm that the free activity in the Squibb supemate is not unbound 99”Tc-pertechnetate but labelled soluble albumin fraction present in the kit itself, as seen in the single band similar to ““1-RISA or human serum albumin. The data given in Table 4 show the extent of labelling of fresh MAA with 99”Tc-MAA supemates obtained from another preparation. The test was done on the assumption that the 99”Tc-MAA supemates would tag most of the fresh MAA if they contained only free 99”Tc-pertechnetate. The supernate labelled as much as 90% of fresh MAA in Mallinckrodt prep aration compared to 15.5% tagging in the Squibb preparation, confirming the differente in the nature of supemates between the two preparations. Limited organ distribution experiments in mice do not indicate any evidente for the presence of labelled microaggregates, since no large amounts of radioactivity were localized in liver or kidney, with most of the activity in blood and in gut.
203
tium to MAA (Table 5), indicating that the aggregates were either incompletely labelled in the first place or non-uniformly labelled with 99”‘Tc in any lung prep aration.
The normal serum albumin in the Squibb kit is also tagged along with MAA when 99”Tc-pertechnetate is added to the vial, though not quantitatively. MAA preparations with free activity in any form are potentially confusing in evaluation of right-to-left cardiovascular shunts.“) So, in such cases, some caution is required with regard to the nature of the 99”TcMAA preparation used. Our experiments have shown that the lung aggregates have large residual labelling capacity, as evidenced by the data presented in Table 5. It is always assumed that the albumin aggregates or microsphere particles are uniformly labelled with 99”‘Tc-pertechnetate: either (a) the binding sites on al1 the particles are labelled partially and incompletely or (b) some of the particles are completely and uniformly tagged saturating al1 the binding sites on them, leaving behind totally unlabelled particles or partially labelled particles. This variability in labelling between individual particles is of importante in determining the total number of labelled particles to be injected to produce a lung scan. Not only uniform distribution of tagged particles in lungs is requiredC5) but also uniform distribution of uniformly labelled particles is necessary to create a more authentic image of the lung perfusion. This aspect is further being investigated at this laboratory. Acknowledgement-This work was supported in part by National Institute of Health Grant No. ROI GM 21217-01 RAD.
Dept. of Nuclear Medicine, Albert Einstein College
L. RAO CHERW* S. R. VALLABHAIOSULA M. DONALDBLAUFOX
of Medicine, NY 10461, C’.S.A.
Bronx,
References
1.
CHANDRA
R.,
SHAMOUN
J., BRAUNSTEINP. et al.
Successive additions of fresh 99”Tc-pertechnetate to labelled MAA resulted in further tagging of techne-
J. nucl. Med. 14, 102 (1974). 2. DELANDF. H. J. nucl. Med. 7, 883 (1966). 3. GALT J. M. and TOIHILL P. Br. J. Radiol. 46,
*Reprint requests to: L. Rao Chervu, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
272 (1973). 4. TONG E. C. K., LIU L., POTIFR R. T. et al. Radiology 106, 585 (1973). 5. HECK, L. L. and DULEYJ. W. Radiology 133, 675 (1974).
1977. Vol.
4.
Quality Control of TechnetiumLabelled Lung Imaging Agents (Receiued 14 March 1977) Introducth of y9”Tc to a variable extent in the kidney blood pool has been observed after the i.v. administration of macroaggregated albumin particles for lung scanning. Previous studies have shown that the MAA particles used for lung scanning are slowly metabolized to microaggregated albumin particles in the lung. These particles are removed from the circulation by the liver. (lp3) It is surprising that sufficient activity would accumulate in the renal area to permit visualization of the kidneys at times noted. The study reported here has revealed that technetium-labelled MAA preparations may contain g9”Tctagged albumin, similar to human serum albumin in solution, which accounts for higher blood pool activity. The nature of labelling pattern of MAA and the albumin microspheres indicates that the macroaggregates and microspheres are not completely or uniformly labelled during a one-step addition of pertechnetate. ~CALIZATION
Materials and Methods The labelling efficiency and radiochemical nature of technetium-labelled MAA supernates were studied using three commercially available kits.* Similar experiments were carried out with albumin microspherest for comparison. MAA kits were supplied as vials containing sterile, pyrogen-free lyophilized powder composed of denatured human serum albumin and stannous chloride or, in the case of the MediPhysics product, with the MAA aggregates suspended, in solution (2.2 ml/glass vial). The amount of stannous chloride in these preparations varied from 50 to 35Opg. The Macrotec (Squibb) also contains 10mg of lyophilized normal serum albumin. Each albumin microsphere labelling via1 (3M Brand) contains 5 mg of albumin microspheres and lW25O~g of tin.
* Squibb-Aggregated Albumin (Human) Macrote@. Mallinckrodt-TechneSan@ MAA Kit, Medi-physicsLungaggregateTM Reagent. t 3M Company. 1 MINITEC-“9”-Generator, E. R. Squibb & Sons. Inc., U.S.A. $Gelman Instrument Company, Ann Arbor. MI.
201
Tagging eficiency
Labelling efficiency of MAA and microspheres was determined by three different methods. (a) Centrifugation method. MAA and albumin microspheres were labelled according to directions of the manufacturer, with y”mLc-pertechnetate eluted the same day from a molybdenum generator.$ An aliquot of the tagged preparation was pipetted into a smal1 plastic or glass tube and then centrifuged at 3500 rev! min for 10 min. Total radioactivity of the suspension. sediment and supernate were measured with a radioisotope dose calibrator (Squibb CRC-6A). (b) Minicolumn method. Chromatographic minicolumns were prepared from disposable Pasteur glass pipets plugged at the tapered end with cotton and loaded to a height of 4cm with Sephadex-Gz5 presoaked in saline ovemight. Approximately O.l-ml aliquot of the test preparation was added to each of the minicolumns which were then eluted with 3 ml of saline. The eluate and the minicolumns containing the tagged particles were assayed using a Na1 (Tl) gamma scintillation counter or the radioisotope dose calibrator depending on the amount of activity used. The percentage radioactivity remaining on the Sephadex column, represents the percentage tagging. (c) Millipore jltration. 9yTc-MAA or ““Tc-albumin microspheres were passed through a 0.22 Pm Millipore membrane filter. The filtrate was assayed for free pertechnetate activity and the percentage tagging calculated. The following analytical procedures were used to determine the chemistry and the radiochemical nature of the ““Tc-MAA supemates or eluates. (a) Column and thin layer chromatography. Elution volumes of HSA and 99”Tc-pertechnetate were determined on a Sephadex-G25 column with ‘251-RISA and 9y”Tc-pertechnetate. After washing the columns with saline, 0.5 ml of 99”Tc-MMA supemate or the “‘“Tc-microsphere supemate was added to the column and eluted with saline. The percentage activity in each fraction was calculated. The supernates were also spotted on ITLC silica gel strips and acetone was used as the elution medium. After IS min, the strips were dried and cut into l-cm pieces. and counted to determine the distribution of radioactivity. (b) Protein electrophoresis. In order to confirm the findings of the chromatographic data, protein electrophoresis was carried out on the supemates from ‘9”Tc-MAA preparation (Squibb) using Sephraphore 1115 electrophoresis strips. Electrophoresis was also done on pure HSA (3 mg/cm3) and ‘*“I-RISA for comparison. Organ distrihution. The total body distribution of the 99”Tc-MAA supemates was determined in four mice weighing up to 3Og, which were killed 30 min after tail-vein injection. The radioactivity of the organs and the blood was measured in a Na1 (TI)
Work in progress
202
TABLE 1. Percentage labelling of lung aggregates
Preparation 99”‘Tc Microspheres (3M) 99”‘Tc MAA (Squibb) 99”‘Tc MAA (Mallmckrodt) 99”Tc MAA (Mediphysics)
Centrifuging (%)
% labeling* Mini column (%)
Millipore filtration (%)
98.8 83.8 98.3 95.8
99.0 89.0 96.9 96.1
97.1 86.0 99.0 95.7
* Average of four experiments. TABLE2. Sephadex column chromatography
of supernates
% activity* Albumin fraction
Preparation 99”‘Tc-Microspheres 99”‘Tc-MAA (Squibb) 99Tc-MAA (Mallinckrodt) 99’Q-MAA (Medi Phys)
Pertechnetate
10.9 65.7 1.2 12.5
67.1 20.0 29.3 30.5
Total recovery 78.0 85.7 30.5 43.0
* Average of four experiments. TABLE3. ITLC data on supemates % activity distribution* Albumin fraction
Preparation
99”Tc-pertechnetate fraction 34.8 66.2
65.2 33.8
99”Tc-MAA (Squibb) 99”Tc-MAA (Mallinckrodt) * Average of four expetiments.
TABLE4. Labelling of fresh MAA with 99”‘Tc MAA supemates*
Preparation
% tagged
% free activity
15.5 90.3
84.5 9.7
99”Tc MAA (Squibb) 99”‘Tc MAA (Mallinckrodt) * Average of two experiments. TABLE5.
Preparation 99*Tc Microspheres 99”Tc MAA (Squibb) 99”Tc MAA (Mallinckrodt) 9911TcMAA (Mediphysics) * Average of four experiments.
% @ging with successive* additions of 99”‘Tc pertechnetate Step 1 Step 11 Step 111 Step IV 98.8 83.8 98.3 95.8
90.0 91.1 99.0 86.4
57.0 60.7 64.0 55.8
66.0 26.1 35.7 27.4
Work in progress
gamma scintillation counter and determined as percentages of the total amounr injected. Labelling eficiency. 99”Tc-pertechnetate was added in mCi amounts in repeated steps until further uptake of activity by the aggregates or microspheres was not significant. Labelled particles were washed twice with saline before the next aliquot addition of 99’“Tcpertechnetate. The following test was performed to further confirm that the supemate of 99Tc-MAA preparation in some cases contains higher percentage of labelled soluble albumin. A known amount of 99”‘Tc-pertechnetate was added to a via1 of MAA, the preparation was centrifuged and the amount of radioactivity in the supemate was determined. A fresh via1 of MAA was then labelled with 99”Tc-MAA supemate obtained from the previous preparation. The percentage tagging of MAA in the second preparation was determined as above by centrifugation.
Tagging efficiency determined by the methods described (Table 1) revealed that as much as 15% of free activity (non MAA-Tc) is present in 99”Tc MAA of Squibb preparation, whereas the other three preparations did not contain more than 2% free activity. The chromatographic data given in Tables 2 and 3 indicate that up to 65% of the radioactivity in the Squibb 99”Tc MAA supemate was due to labelled soluble albumin. Protein electrophoresis experiments confirm that the free activity in the Squibb supemate is not unbound 99”Tc-pertechnetate but labelled soluble albumin fraction present in the kit itself, as seen in the single band similar to ““1-RISA or human serum albumin. The data given in Table 4 show the extent of labelling of fresh MAA with 99”Tc-MAA supemates obtained from another preparation. The test was done on the assumption that the 99”Tc-MAA supemates would tag most of the fresh MAA if they contained only free 99”Tc-pertechnetate. The supernate labelled as much as 90% of fresh MAA in Mallinckrodt prep aration compared to 15.5% tagging in the Squibb preparation, confirming the differente in the nature of supemates between the two preparations. Limited organ distribution experiments in mice do not indicate any evidente for the presence of labelled microaggregates, since no large amounts of radioactivity were localized in liver or kidney, with most of the activity in blood and in gut.
203
tium to MAA (Table 5), indicating that the aggregates were either incompletely labelled in the first place or non-uniformly labelled with 99”‘Tc in any lung prep aration.
The normal serum albumin in the Squibb kit is also tagged along with MAA when 99”Tc-pertechnetate is added to the vial, though not quantitatively. MAA preparations with free activity in any form are potentially confusing in evaluation of right-to-left cardiovascular shunts.“) So, in such cases, some caution is required with regard to the nature of the 99”TcMAA preparation used. Our experiments have shown that the lung aggregates have large residual labelling capacity, as evidenced by the data presented in Table 5. It is always assumed that the albumin aggregates or microsphere particles are uniformly labelled with 99”‘Tc-pertechnetate: either (a) the binding sites on al1 the particles are labelled partially and incompletely or (b) some of the particles are completely and uniformly tagged saturating al1 the binding sites on them, leaving behind totally unlabelled particles or partially labelled particles. This variability in labelling between individual particles is of importante in determining the total number of labelled particles to be injected to produce a lung scan. Not only uniform distribution of tagged particles in lungs is requiredC5) but also uniform distribution of uniformly labelled particles is necessary to create a more authentic image of the lung perfusion. This aspect is further being investigated at this laboratory. Acknowledgement-This work was supported in part by National Institute of Health Grant No. ROI GM 21217-01 RAD.
Dept. of Nuclear Medicine, Albert Einstein College
L. RAO CHERW* S. R. VALLABHAIOSULA M. DONALDBLAUFOX
of Medicine, NY 10461, C’.S.A.
Bronx,
References
1.
CHANDRA
R.,
SHAMOUN
J., BRAUNSTEINP. et al.
Successive additions of fresh 99”Tc-pertechnetate to labelled MAA resulted in further tagging of techne-
J. nucl. Med. 14, 102 (1974). 2. DELANDF. H. J. nucl. Med. 7, 883 (1966). 3. GALT J. M. and TOIHILL P. Br. J. Radiol. 46,
*Reprint requests to: L. Rao Chervu, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
272 (1973). 4. TONG E. C. K., LIU L., POTIFR R. T. et al. Radiology 106, 585 (1973). 5. HECK, L. L. and DULEYJ. W. Radiology 133, 675 (1974).
