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In vivo Studies of 111In-Labelled canine Lymphocytes
WorkinProgress hr. J. Nucl. Med. Biol. Vol. 11, No. 1, pp. 3X--41. 1984 Pergamon Press Ltd 1984. Printed in Great Britain. 0047-0740/84 $3.00 + 0.00
In Viuo Studies of “‘In-Labelled Canine Lymphocytes LAWRENCE W. ROBINSON, SAMUEL D. J. YEH* and RICHARD S. BENUA Memorial Sloan-Kettering Cancer Center, Nuclear Medicine Service, 1275 York Avenue, New York, NY 10021, U.S.A. (Received 6 June 1983)
Introduction The use of “‘In I-hydroxyquinolin complex ([“‘In]oxine) is a simple and efficient method of radiolabelling many different types of cells. [“‘In]oxine has been used to label polymorphonuclear leukocytes, lymphocytes, platelets and tumor cells of several species to study cellular kinetics, localization and functions in in vitro and in uiuo systems. Despite extensive work on the localization of “‘In labeled polymorphonuclear leukocytes in inflammatory lesions(‘,2’ and the accumulation of “‘In labeled platelets and leukocytes in newly developed infarcts,(3.4ithe distribution of “‘In labeled lymphocytes is less well documented. However, labeled lymphocytes have been successfully used to image regional lymph nodes in patients with Hodgkin’s disease”) and in normal sheep,.(o to localize cutaneous lymphoma;(‘) to assess the patterns of human lymphocyte distribution”’ and, to detect rejection in rat heart”) and in canine renal allografts.(“r For this study, the canine model was selected because lymphocytes can be separated from a relatively small volume of peripheral blood and because canine lymphoma simulates human lymphoma.
Material and Methods Indium labeled lymphocytes were prepared from three mongrel dogs; two animals were free of disease while one had breast carcinoma. A total of five studies were done to assess which lymph node groups were visualized and the reproducibility of the procedure. Lymphocytes were separated from 40mL of peripheral blood by a previously described technique using a 60mL disposable plastic syringe containing 4 mL (1000 I.U./mL) preservative free heparin.(“r Eight milliliters of a 6% (w/v) dextran of 500,000mo1. wt in 0.9% saline solution were added to the heparinized blood. The syringe was clamped upright and the red cells were allowed to sediment for 30 min in a 37°C incubator. The leukocyte rich plasma was expressed into a sterile tube and diluted with an equal volume of sterile saline. Seven milliliters of diluted leukocyte rich plasma were then carefully layered over 4mL of Ficoll-Paque R (Ficoll 400 and Diatrizoate Sodium) lymphocyte separation media in the 16mL conical centrifuge tubes. The tubes were centrifuged at 264g (1200 rpm) for
*Author dressed.
to whom all correspondence
30 min at room temperature. The mononuclear cells (lymphocytes and monocytes) were collected at the interface between the plasma and Ficoll-Pique using a sterile Pasteur pipette. The mononuclear cells were then washed twice with sterile saline at 183g for 5 min. The tinal cell pellet was suspended in saline at a concentration of 25 to 50 x IO6cells/ml. About 50 x 106cells can be collected from 40 mL of blood by this procedure. Viability was tested using the trypan blue dye exclusion test and was between 85 and 99% with an average of 95”/,. Sterile, apyrogenic [“‘In]oxine dissolved in 50 PL ethanol was obtained from a commercial source. The oxineethanol solution was diluted with 200 PL of saline and added to the cell suspension dropwise with gentle swirling of the suspension between additions (150 pCi for 50 x 10bcells). The cell suspension was allowed to stand at room temperature for 20 min, and then centrifuged at 183g for 5 min. The supernatant was removed and the cells washed twice with saline to remove excess oxine. The radioactivity in the pellet was counted and the cells were resuspended in 5-IOmL of saline for injection into the peripheral vein of the test animal. The labeling efficiency varied from 40 to 75%. Under pentobarbital anesthesia, y-camera images of the head, neck, thorax, abdomen and pelvis were obtained 2448 h after injection using 173 and 247 keV y-peaks in a triple peak camera with a 400 keV parallel hole collimator.
Results In the normal animals, the lymph nodes visualized included the submandibular, parotid, superficial cervical, axillary, medial femoral, and popliteal groups (Figs 1 and 2). The superficial inguinal node group was not visualized due to underlying bowel activity and pelvic bony structures obscuring the uptake. Liver, spleen and marrow activity in the sternum, rib cage, pelvis and ends of long bones were invariably visualized. In the animal with breast carcinoma, the axillary and medial femoral groups were visualized (Fig. 3). The lymph node groups of the head and neck were not well seen. Significant activity was also seen in the lung probably related to trapping of damaged lymphocytes by pulmonary reticuloendothelial cells or associated local pathology. In the soft tissues of the left axilla, there was a focus of uptake corresponding to a draining surgical wound created five days prior to the study. The breast tumor mass, which lay on the surface of the abdomen and pelvis was visualized but was overlapped by the bowel activity. Within the region of the tumor, there was a small area of decreased activity corresponding to the biopsy site. To ascertain that uptake in the lymph nodes was indeed due to labeled lymphocytes, and not to indium bound to protein, [“‘In]chloride was given to a normal dog which was previously used for a lymphocyte study. Uptake occurred in the lungs, liver, spleen, bowel and bone marrow spaces but not in the lymph nodes identified in the “‘In labeled lymphocyte studies.
Discussion Lymphocytes are known to be radiosensitive. The amount of “‘In used for lymphocyte labeling may alter the functions of the lymphocytes. Such alterations may be either radiation induced cytotoxicity causing sequestration of dead lymphocytes within the reticuloendothelial system of liver and spleen, or induced neoplasia. Viability tested by the trypan blue exclusion test may or may not detect minor loss of cell integrity during separation and labeling procedures. The demonstration of lymph node uptake with labeled lympho-
should be ad38
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Rt.
Rt.
Fig. 1
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Fig. 2
39
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Work in Progress cytes but not with indium chloride indicates preservation of certain functions of the labeled lymphocytes. Opinions in the literature concerning the optimal amount of “‘In are diverse. Lavender et a!.(‘) used between 200 and 500 @i to label human lymphocytes from 80mL blood and found good images without apparent adverse effects. In the sheep, Frost et uZ.@’ used 100 @i for 1 x IO*cells with satisfactory images. However, other investigators have reported altered lymphocyte function even with much lower doses. For example, Segal et ~1.“‘)showed that 1 x 108human lymphocytes labeled with 400 PCi exhibited decreased E, EA, and EAC rosetting characteristics and absent ADCC (antibody dependent cytotoxicity) as compared to the same number of cells labeled with 150 PCi. Using rat lymphocytes, Chisholm ef uI.(i3)labeled 1 x lo* cells with 150 &i and demonstrated decreased uptake in the lymph nodes with increased uptake in the liver and spleen when compared to the same number of cells labeled with only 5pCi. Although the optimal amount of “‘In which can be used without causing cellular damage is unknown, it appears that 300 PCi for 1 x 10s cells as used in our study is appropriate. Our studies show that indium labeled lymphocytes localize in superficial lymph nodes and also in the liver, spleen, and bone marrow. The images in the dog are quite similar to those obtained in the sheep and man, but different from the images following the injection of indium chloride. Although the visualization of breast tumor was faint, we believe that labeled lymphocytes did migrate to the region of the tumor and the associated draining sinus. These preliminary results suggest that the labeled lymphocytes are acting as immunologically competent cells and circulate to the lymph nodes, localize in regions with inflammation, and accumulate in and around a tumor mass. Further studies of the distribution, kinetics and migration patterns of labeled lymphocytes in various disease processes may be quite fruitful. Summary [“‘Injoxine was used to label canine periperal blood lymphocytes from two normal animals and one animal with breast carcinoma. Using 15OuCi of [“‘In]oxine to label
41
50 x IO6 cells yielded a labeling efficiency between 40 and 75% and a viability of about 95%. Gamma camera images obtained 24-44 h after injection showed uptake in the subcutaneous lymph nodes of the head, neck, axilla, pelvis and popliteal regions. Uptake in the liver, spleen, bowel, and bone marrow was also seen. In the animal bearing breast carcinoma, uptake occurred in the tumor, lungs and in a draining sinus in the left axilla. These results suggest that “‘In labeled lymphocytes are useful in visualizing regional lymph nodes and may be valuable in assessing tumor recognition by these cells in uivo. References 1. Thakur M. L., Coleman R. and Welch M. J. Lab. C/in. Med. 89, 217 (1977). 2. Thakur M. L., Lavender J. P., Amot R. N., Silvester D. J. and Seeal A. W. J. Nucl. Med. 18. 1012 (19771. 3. Davies RTA., Thakur M. L., Berger H. J., Wackers F. M. Th., Gottschalk A. and Zaret B. L. Circulation 63, 826 (1981). 4. Thakur M. L., Gottschalk A. and Zaret B. L. Circulation 60, 297 (1979). 5. Lavender J. P., Goldman J. M., Amot R. N. and Thakur M. L. Br. Med. J. 2, 797 (1977). 6. Frost H., Frost P., Wilcox C. and Thrall J. Int. J. Nucl. Med. Biol. 6, 60 ( 1979). 7. Miller R. A., Coleman C. N., Fawcett H. D., Hoppe R. T. and McDougall I. R. N. Ennl. J. Med. 303,89 (1980).
8. Wagstaff J., Gilson C., Thatcher N., Ford W. L., Sharma H., Benson W. and Crowther D. Clin. EXD. Immunol. 9. Oluwole
43, 435 (198 1). S., Wang T.,
Fawwaz R., Satake K., Nowygrod R., Refmtsma K. and Hardy M. A. Transplantation 31, 51 (1981). 10. Pontes J. E., Frost P., Pokorny M. and Smith J. Invest Ural. 17, 45 1 (1980). 11. Boyum A. Stand J. Immunol 5 (Suppl. 5) 9 (1976). 12. Segal A. W., Deteix P., Garcia R., Tooth G. D., Zanelh G. D. and Allison A. C. J. Nucl. Med. 19, 1238 (1978). 13. Chisholm P. M., Danpure H. J., Healey G. and Osman S. J. Nucl. Med. 20, 1308 (1979).
In Viuo Studies of “‘In-Labelled Canine Lymphocytes LAWRENCE W. ROBINSON, SAMUEL D. J. YEH* and RICHARD S. BENUA Memorial Sloan-Kettering Cancer Center, Nuclear Medicine Service, 1275 York Avenue, New York, NY 10021, U.S.A. (Received 6 June 1983)
Introduction The use of “‘In I-hydroxyquinolin complex ([“‘In]oxine) is a simple and efficient method of radiolabelling many different types of cells. [“‘In]oxine has been used to label polymorphonuclear leukocytes, lymphocytes, platelets and tumor cells of several species to study cellular kinetics, localization and functions in in vitro and in uiuo systems. Despite extensive work on the localization of “‘In labeled polymorphonuclear leukocytes in inflammatory lesions(‘,2’ and the accumulation of “‘In labeled platelets and leukocytes in newly developed infarcts,(3.4ithe distribution of “‘In labeled lymphocytes is less well documented. However, labeled lymphocytes have been successfully used to image regional lymph nodes in patients with Hodgkin’s disease”) and in normal sheep,.(o to localize cutaneous lymphoma;(‘) to assess the patterns of human lymphocyte distribution”’ and, to detect rejection in rat heart”) and in canine renal allografts.(“r For this study, the canine model was selected because lymphocytes can be separated from a relatively small volume of peripheral blood and because canine lymphoma simulates human lymphoma.
Material and Methods Indium labeled lymphocytes were prepared from three mongrel dogs; two animals were free of disease while one had breast carcinoma. A total of five studies were done to assess which lymph node groups were visualized and the reproducibility of the procedure. Lymphocytes were separated from 40mL of peripheral blood by a previously described technique using a 60mL disposable plastic syringe containing 4 mL (1000 I.U./mL) preservative free heparin.(“r Eight milliliters of a 6% (w/v) dextran of 500,000mo1. wt in 0.9% saline solution were added to the heparinized blood. The syringe was clamped upright and the red cells were allowed to sediment for 30 min in a 37°C incubator. The leukocyte rich plasma was expressed into a sterile tube and diluted with an equal volume of sterile saline. Seven milliliters of diluted leukocyte rich plasma were then carefully layered over 4mL of Ficoll-Paque R (Ficoll 400 and Diatrizoate Sodium) lymphocyte separation media in the 16mL conical centrifuge tubes. The tubes were centrifuged at 264g (1200 rpm) for
*Author dressed.
to whom all correspondence
30 min at room temperature. The mononuclear cells (lymphocytes and monocytes) were collected at the interface between the plasma and Ficoll-Pique using a sterile Pasteur pipette. The mononuclear cells were then washed twice with sterile saline at 183g for 5 min. The tinal cell pellet was suspended in saline at a concentration of 25 to 50 x IO6cells/ml. About 50 x 106cells can be collected from 40 mL of blood by this procedure. Viability was tested using the trypan blue dye exclusion test and was between 85 and 99% with an average of 95”/,. Sterile, apyrogenic [“‘In]oxine dissolved in 50 PL ethanol was obtained from a commercial source. The oxineethanol solution was diluted with 200 PL of saline and added to the cell suspension dropwise with gentle swirling of the suspension between additions (150 pCi for 50 x 10bcells). The cell suspension was allowed to stand at room temperature for 20 min, and then centrifuged at 183g for 5 min. The supernatant was removed and the cells washed twice with saline to remove excess oxine. The radioactivity in the pellet was counted and the cells were resuspended in 5-IOmL of saline for injection into the peripheral vein of the test animal. The labeling efficiency varied from 40 to 75%. Under pentobarbital anesthesia, y-camera images of the head, neck, thorax, abdomen and pelvis were obtained 2448 h after injection using 173 and 247 keV y-peaks in a triple peak camera with a 400 keV parallel hole collimator.
Results In the normal animals, the lymph nodes visualized included the submandibular, parotid, superficial cervical, axillary, medial femoral, and popliteal groups (Figs 1 and 2). The superficial inguinal node group was not visualized due to underlying bowel activity and pelvic bony structures obscuring the uptake. Liver, spleen and marrow activity in the sternum, rib cage, pelvis and ends of long bones were invariably visualized. In the animal with breast carcinoma, the axillary and medial femoral groups were visualized (Fig. 3). The lymph node groups of the head and neck were not well seen. Significant activity was also seen in the lung probably related to trapping of damaged lymphocytes by pulmonary reticuloendothelial cells or associated local pathology. In the soft tissues of the left axilla, there was a focus of uptake corresponding to a draining surgical wound created five days prior to the study. The breast tumor mass, which lay on the surface of the abdomen and pelvis was visualized but was overlapped by the bowel activity. Within the region of the tumor, there was a small area of decreased activity corresponding to the biopsy site. To ascertain that uptake in the lymph nodes was indeed due to labeled lymphocytes, and not to indium bound to protein, [“‘In]chloride was given to a normal dog which was previously used for a lymphocyte study. Uptake occurred in the lungs, liver, spleen, bowel and bone marrow spaces but not in the lymph nodes identified in the “‘In labeled lymphocyte studies.
Discussion Lymphocytes are known to be radiosensitive. The amount of “‘In used for lymphocyte labeling may alter the functions of the lymphocytes. Such alterations may be either radiation induced cytotoxicity causing sequestration of dead lymphocytes within the reticuloendothelial system of liver and spleen, or induced neoplasia. Viability tested by the trypan blue exclusion test may or may not detect minor loss of cell integrity during separation and labeling procedures. The demonstration of lymph node uptake with labeled lympho-
should be ad38
IIt.
vent.
Lt.
Lt.
ner8.
Rt.
Rt.
Fig. 1
Vent.
Fig. 2
39
Lt.
40
Work in Progress cytes but not with indium chloride indicates preservation of certain functions of the labeled lymphocytes. Opinions in the literature concerning the optimal amount of “‘In are diverse. Lavender et a!.(‘) used between 200 and 500 @i to label human lymphocytes from 80mL blood and found good images without apparent adverse effects. In the sheep, Frost et uZ.@’ used 100 @i for 1 x IO*cells with satisfactory images. However, other investigators have reported altered lymphocyte function even with much lower doses. For example, Segal et ~1.“‘)showed that 1 x 108human lymphocytes labeled with 400 PCi exhibited decreased E, EA, and EAC rosetting characteristics and absent ADCC (antibody dependent cytotoxicity) as compared to the same number of cells labeled with 150 PCi. Using rat lymphocytes, Chisholm ef uI.(i3)labeled 1 x lo* cells with 150 &i and demonstrated decreased uptake in the lymph nodes with increased uptake in the liver and spleen when compared to the same number of cells labeled with only 5pCi. Although the optimal amount of “‘In which can be used without causing cellular damage is unknown, it appears that 300 PCi for 1 x 10s cells as used in our study is appropriate. Our studies show that indium labeled lymphocytes localize in superficial lymph nodes and also in the liver, spleen, and bone marrow. The images in the dog are quite similar to those obtained in the sheep and man, but different from the images following the injection of indium chloride. Although the visualization of breast tumor was faint, we believe that labeled lymphocytes did migrate to the region of the tumor and the associated draining sinus. These preliminary results suggest that the labeled lymphocytes are acting as immunologically competent cells and circulate to the lymph nodes, localize in regions with inflammation, and accumulate in and around a tumor mass. Further studies of the distribution, kinetics and migration patterns of labeled lymphocytes in various disease processes may be quite fruitful. Summary [“‘Injoxine was used to label canine periperal blood lymphocytes from two normal animals and one animal with breast carcinoma. Using 15OuCi of [“‘In]oxine to label
41
50 x IO6 cells yielded a labeling efficiency between 40 and 75% and a viability of about 95%. Gamma camera images obtained 24-44 h after injection showed uptake in the subcutaneous lymph nodes of the head, neck, axilla, pelvis and popliteal regions. Uptake in the liver, spleen, bowel, and bone marrow was also seen. In the animal bearing breast carcinoma, uptake occurred in the tumor, lungs and in a draining sinus in the left axilla. These results suggest that “‘In labeled lymphocytes are useful in visualizing regional lymph nodes and may be valuable in assessing tumor recognition by these cells in uivo. References 1. Thakur M. L., Coleman R. and Welch M. J. Lab. C/in. Med. 89, 217 (1977). 2. Thakur M. L., Lavender J. P., Amot R. N., Silvester D. J. and Seeal A. W. J. Nucl. Med. 18. 1012 (19771. 3. Davies RTA., Thakur M. L., Berger H. J., Wackers F. M. Th., Gottschalk A. and Zaret B. L. Circulation 63, 826 (1981). 4. Thakur M. L., Gottschalk A. and Zaret B. L. Circulation 60, 297 (1979). 5. Lavender J. P., Goldman J. M., Amot R. N. and Thakur M. L. Br. Med. J. 2, 797 (1977). 6. Frost H., Frost P., Wilcox C. and Thrall J. Int. J. Nucl. Med. Biol. 6, 60 ( 1979). 7. Miller R. A., Coleman C. N., Fawcett H. D., Hoppe R. T. and McDougall I. R. N. Ennl. J. Med. 303,89 (1980).
8. Wagstaff J., Gilson C., Thatcher N., Ford W. L., Sharma H., Benson W. and Crowther D. Clin. EXD. Immunol. 9. Oluwole
43, 435 (198 1). S., Wang T.,
Fawwaz R., Satake K., Nowygrod R., Refmtsma K. and Hardy M. A. Transplantation 31, 51 (1981). 10. Pontes J. E., Frost P., Pokorny M. and Smith J. Invest Ural. 17, 45 1 (1980). 11. Boyum A. Stand J. Immunol 5 (Suppl. 5) 9 (1976). 12. Segal A. W., Deteix P., Garcia R., Tooth G. D., Zanelh G. D. and Allison A. C. J. Nucl. Med. 19, 1238 (1978). 13. Chisholm P. M., Danpure H. J., Healey G. and Osman S. J. Nucl. Med. 20, 1308 (1979).