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Labeled cells in patients with malignancy
Labeled Cells In Patients With Malignancy Janice P. Dutcher The use of radioisotopes for cell labeling has been a major tool in hematology laboratory research. Chromium-51-1abeling of hematologic cells and lymphocytes has been used for years t o study t h e migration and sequestration of these cells in the spleen and o t h e r sites. The substantial recirculation of lymphocytes f r o m blood into lymphoid tissue and back into blood is well described. Recently, n e w approaches for radiosotopic cell labeling have gained prominence in t h e investigation of various aspects of malignant diseases and in t h e clinical care of such patients. Isotopes such as i n d i u m - I l l can be visualized w i t h standard scanning techniques providing f u r t h e r information about the migration of normal and malignant cells has been discovered. In vivo studies have been p e r f o r m e d w i t h indium111 in animals and humans, including comparisons of the migration of abnormal cells (malignant} and of lymphocytes t o abnormal nodes. Evaluation and comparison of t h e migration of carcinoma cells, normal lymphoid cells, and malignant lymphoid cells in animals s h o w markedly different patterns of distribution, which could have bearing on investigations of mechanisms of metastasis. In vivo human studies also have evaluated t h e migration patterns of lymphoid cells f r o m patients w i t h chronic lymphocytic leukemia and well-differentiated lymphoma, showing v e r y different migrating behavior b e t w e e n these t w o polarities o f a similar disease. These types of studies, w h i l e initially phenomenonologic, may provide a basis for a b e t t e r understanding of these diseases. There are concerns about the use of an isotope such as indium-111 for t h e labeling of long-lived cells such as lymphocytes. Laboratory studies have demonstrated impaired cell function at high concentrations of radioactivity. Some w o r k e r s have expressed
HE USE OF labeled cells for the investiga-
tion of various aspects of malignancy and T for the care of such patients has recently taken on new clinical prominence. This is particularly with regard to the hematologic malignancies. Initial investigations have been primarily in the realm of studying the basic biology of the diseases, rather than as a diagnostic test to identify sites of disease since the cells do not necessarily localize to sites of tumor. Nevertheless, with the use of radionuclides such as indium- I 11, which can be visualized with standard scanning techniques, new information about many of the hematologic malignancies has been uncovered. Chromium-51 labeling of hematologic cells and lymphocytes has been used for years to study the Seminars in Nuclear Medicine, Vol. XIV, No. 3 (July), 1984
concern about long-term changes in cells t h a t recirculate. Others cite precedents of o t h e r long-term uses of isotopes, therapeutically, w i t h o u t detrimental effects. These concerns continue to be investigated. Finally, an area of much interest in the use of indium-111 is t h e labeling of granulocytes. This technique has been useful diagnostically, t o localize infections. The major value in patients w i t h malignancy, primarily w i t h hematologic malignancies, is to evaluate the potential benefit of granulocyte transfusions. M a n y of these patients develop prolonged granulocytopenia and become infected, and granuloc y t e transfusions may become a t h e r a p e u t i c consideration. H o w e v e r , these patients usually have been subjected to m a n y transfusions and may be immunized t o granulocytes. This is difficult t o assess clinically, but t h e use of i n d i u m - I l l - l a b e l e d donor granulocytes provides a visual d e m o n s t r a t i o n of w h e t h e r or n o t t h e cells migrate t o t h e site of infection. This technique also can be used t o assess g r a n u l o c y t e s e q u e s t r a t i o n . It a p p e a r s t h a t in patients w h o develop antibodies, both anti-HLA and granulocyte specific, the donor granulocytes do not migrate t o sites of infection and in fact may sequest e r in the lungs. These data can help guide the use of granulocyte transfusion in these patients. The investigation of labeled cells, both malignant and normal, in patients w i t h malignancy is giving us n e w insight into t h e diseases. These studies are of p a r t i c u l a r benefit using n e w e r techniques w i t h isotopes t h a t can be scanned such as i n d i u m - I l l . Studies of the migration patterns of Sezery cells, granulocytes in chronic myelogenous leukemia and lymphocytes in lymphoid leukemia are continuing and may provide i m p o r t a n t i n f o r m a t i o n regarding t h e disease process.
migration and sequestration of these cells in the spleen and other sites. However, the investigation of indium-ll 1 as a cell label has added new dimensions to studies of lymphoid and hematologic cells and tissues.
From the Division of Oncology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY. Dr. Dutcher is a special fellow of the Leukemia Society of America. Address reprint requests to Janice P. Dutcher, MD, Division of Oncology, Department of Medicine, Albert Einstein College of Medicine (Room 2C-14 Van Etten), 1300 Morris, Park Ave, Bronx, NY10461. 9 1984 by Grune & Stratton, Inc. 0001-2998/84/1403~9007505.00/0 251
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Another area in which cell labeling has been particularly helpful in patients with malignancy is in the evaluation of granulocyte transfusion supportive care. These transfusions are particularly difficult to quantitate and at times to determine benefit. The use of indium-Ill-labeled granulocytes has provided a visual means of evaluating granulocyte migration and localization to sites of infection in granulocytopenic patients. LYMPHOCYTE MIGRATION STUDIES: BENIGN AND MALIGNANT
Animal Studies
The study of normal lymphocyte migration has been an area of great interest to hematologists and immunologists for some time. The substantial recirculation of lymphocytes from blood into lymphoid tissue and back into blood is well described) '2 The existence of separate lymphocyte migration pathways through the gut and the peripheral lymph nodes has been elegantly demonstrated with the observation of blast cells from mesenteric lymph nodes homing preferentially to the gut wall and blast cells from peripheral nodes returning preferentially to the peripheral nodes) The mechanisms of selective migration and the elucidation of pathways has been possible primarily through the use of isotopic labeling of lymphocytes. Rannie and Donald compared the distribution of radioactivity at intervals following transfusion of thoracic duct lymphocytes labeled with five different isotopes in animal studies: They demonstrated that the rate of elution of isotope from the labeled cells and the subsequent fate of the eluted isotope were the most important factors limiting the usefulness of such isotopes. Comparison of labeling procedures using 3H- and 14C-uridine, 3Hand ~4C-leucine, L-75Se-selenomethionine, 99mTcsodium pertechnetate and S~Cr-sodium chromate in vitro and 3H-thymidine in vivo showed that 5~Cr had the fewest disadvantages for studying lymphocytes. Unfortunately, this isotope cannot be scanned, so that studies of migration must be done by assessing blood and tissue radioactivity. As can be seen, most lymphocyte labeling studies have been limited to work in experimental animals, primarily because of the limitations of the isotopes and methodology available. In
JANICE P. DUTCHER
animals the lymphocyte migration studies have required the sacrificing of animals, the scintillation counting of their organs and autoradiography of various tissues in order to determine the location and amount of cellular radioactivity. These studies, although laborious, have been done carefully and have been important in studying lymphocyte recirculation and the events of the immune response. These methods have limitations, however, and only provide a description of the terminal deposition of lymphocytes in lymphoid and nonlymphoid tissue. They do not provide the details of the early phases of lymphocyte migration. Thus, despite the many demonstrable advantages of 51Cr as a cellular label, it is not an isotope that has allowed external and dynamic imaging of reinjected labeled cells or of in vivo demonstration of early lymphocyte migration patterns. Lymphocyte labeling studies therefore until recently, have not been directly applicable to studies in humans. However, at least two groups have attempted human studies using 5~Cr labeling and counting over sites of accumulation in lymphoid tissue) '6 Nevertheless, this does not, again, provide dynamic data of migration; only of accumulation in tissue. Recently, a number of investigators have begun to use indium-111-oxine for evaluation of lymphocyte migration. As first described by McAfee and Thakur, 7 this radiochemical binds intracellularly with very little subsequent elution from the cell. It has a comparatively short halflife (2.7 days) versus the 27.8 days of 5~Cr. In addition, l lqn has two energy peaks, both within the visible range of the standard gamma camera, thus allowing external imaging. The potential for kinetic and early migration studies of reinjected cells exists with this radiocompound. Rannie et al reported a comparison of llllnand 99mTc-labeled lymphocytes in rats with the standard method of 5~Cr labeling: They concluded that for the potential study of lymphocyte migration in humans, labeling with l~qn-oxine was the most promising approach based on the findings of minimal elution and their findings of essentially normal lymphocyte migration in their animal studies. Technetium-99m was found to rapidly dissociate from lymphocytes and poor lymphocyte recirculation was observed after labeling with this isotope.
LABELED CELLS IN MALIGNANCY
Subsequently, both animal and human lymphocyte migration studies using indium-Ill labeling have been reported. The ability to use this isotope for in vivo studies and the high correlation of results between ruin and mCr animal studies of lymphocyte migration have led to a number of investigations of both normal and abnormal lymphocyte migration and distribution. These studies will be reviewed. Rannie et al, in their preliminary studies in rats, showed that indium-11 l-labeled lymphocytes recirculated from blood to lymph, analogous to lymphocyte behavior when labeled with 5~CrS (Fig 1). Recirculation suggests that these labeled cells can be followed as markers for normal lymphocyte circulation. Issekutz et al,
Fig 1. Gamma-camera image of a rat that had received lymphocytes labeled with 300 #Ci of 1+11n 24 hours previously. The large group of cervical lymph nodes (arrow) as well as other groups of lymph nodes in the thorax and abdomen are visible. The major concentration of radioactivity is present in the liver and spleen. The latter is responsible for the small area of intense activity indicated by the triangle. (From Rannie at al. C/in Exp Immuno129:509-514, 1977. Reproduced by permission, e)
253
investigated lymphocyte migration in sheep using ~ttln-labeled nodal lymphocytes in similar studies.9 They found that the migration patterns of indium-11 l-labeled lymphocytes were essentially identical to those seen using S~Cr in sheep. Frost et al also evaluated the indium-l 1 l labeled lymphocytes and tumor cells in rats with promising results. ~~ They concluded that l~lln was a potentially useful isotope for subsequent human studies. Subsequent studies have investigated the migration of abnormal cells a n d o f lymphocytes to abnormal nodes. Dettman et al evaluated in vivo migration of ~l~In-labeled mouse spleen lymphocytes and compared this to the migration of labeled mouse lymphoma and mouse mammary adenocarcinoma cells. ~ They found markedly different migration patterns between the three types of cells. They compared viability of labeled to unlabeled cells and determined that labeling itself was not a factor in this difference. The mouse mammary adenocarcinoma cells were initially trapped in the lungs, to a much greater extent than the splenic lymphocytes or the T cell lymphoma cells. The normal lymphocytes subsequently deposited more heavily in the spleen than the other two types of cells. Schroeder et al used rats to observe the ability of indium-11 l-labeled lymphocytes to migrate to lymph nodes involved with tumor (H-4-II-E hepatoma)J z Lymphocytes labeled with t~In exhibited normal migration from blood to nodes and both normal and tumor-bearing nodes were visualized (Fig 2). Because of the small size of rat lymph nodes, qualitative or quantitative differences between normal and tumor-bearing nodes could not be detected. Although at the moment such studies are phenomenologic, these types of studies suggest that this technique may be helpful in the investigation of differences between benign and malignant cells of the hematopoietic and lymphoid systems, and perhaps of disorders of immunity. This technique may also be applicable to studies of mechanisms of metastatic disease .and with refinement may be relevant to investigation of tumor-bearing nodes.
Human Lymphocyte Studies Wagstaff et al have evaluated indium-111oxine labeling as a means of observing human
254
JANICE P. DUTCHER
Fig 2. (a) A composite of t h e upper and l o w e r body images of a normal animal 22 hours a f t e r injection of 5.7 x 10 s l y m p h o c y t e s labeled w i t h 100/~Ci l ~ l n - o x i n e . The spleen, liver, and cervical, axillary, inguinal, and popliteal lymph nodes a r e identified by arrows. (b) A composite of t h e u p p e r and l o w e r body images of a t u m o r - b e a r i n g rat 22 hours a f t e r injection of 2.1 x 10 s H-4-11-E hepatoma cells w e r e injected into the left hind f o o t pad 44 days before imaging. Severe metastatic involvement is clearly visible in the left popliteal region. (From Schroeder et al, Invest Radio/18:87-93, 1983. Reproduced by permission. TM)
lymphocyte migration. 13,14Patients with chronic lymphocytic leukemia (CLL) and well-differentiated diffuse lymphocytic lymphoma (WDDLL) as well as two normal volunteers were studied with ~lqn_labeled autologous lymphocytes. The viability of the labeled cells was tested by trypan blue dye exclusion and by morphology evaluation using phase-contrast microscopy. The important features in the normal subjects' studies were the lack of lung uptake, minimal liver activity, and marked splenic activity. Interestingly, the blood disappearance of lymphocytes was much greater in the patients with CLL than in the other subjects. In the CLL patients, also, there was a much more sustained high level of counts over the spleen throughout the 48-hour evaluation period and the counts over the lymph nodes in these patients rose over the 48-hour period (Fig 3).
It was also pointed out in these studies that the migration patterns will vary depending upon the population of lymphocytes being studied. In animal studies, the tempo of recirculation of T and B lymphocytes is different. T cells generally recirculate much more rapidly than do B cells. ~s It is therefore necessary to pay attention to the nature of the lymphocyte suspension being used for study since the results may vary depending upon the proportion of B and T cells. In Wagstaff's study, the two patients with WDDLL had markedly different proportions of B and T cells, with patient DB having a monoclonal B cell population and patient ES having many more T cells than patient DB. This is consistent with the clinical variability of this disease. Patient ES had a blood lymphocyte disappearance curve similar to the normal subjects, but DB, with the monoclonal B cell population, showed a blood lympho-
LABELED CELLS IN MALIGNANCY
255
Normal a 400
WDDLL (D.B.)
CLL
~Spleen
Spleen
[
_j.
Uv.r
'- 200 ~ N e c k
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a
~ " ' e Groi n
I
I
i
I
0
20
40
60
~ 0
Grlin
Neck Groin
I
I
I
20 40 60 Hr post- injection
0
20
40
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"~ 5 0 - o .Q
._= .~ 40 .E 0
i
30 20
"O
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,
=o
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,
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I,, I I I J/ 12 24 36 48 Hr after reinjection
Fig 3. (a) Blood disappearance curves for indium-Ill oxine-labeled lymphocytes. WDDLL = well-differentiated diffuse lymphocytic lymphoma (E.B.), CLL-chronic lymphocytic leukemia (n-4). (b) Surfaceprobe counts, indium-III-oxine-labe|ed lymphocytes. (From Wagstaff et ah Clin Exp Immunol 4 3 : 4 4 3 - 4 4 9 , 1981. Reproduced by permission.TM)
cyte disappearance pattern similar to the patients with CLL (a monoclonal B cell disorder). Wagstaff et al also studied a patient with Hodgkin's disease of the mediastinum and noted accumulation of autologous ]]]In-labeled lymphocytes in the involved mediastinal nodes. ~4 They did not discuss the findings in other lymph node-bearing areas or the specificity of this technique. Lavender et al looked at the kinetics of indium-l 1 l-labeled lymphocytes in patients with Hodgkin's disease and in two normal subjects in an earlier report. ]6 Radioactivity began to appear in the lymph nodes four to 18 hours after injection. They found no difference between normal subjects and the patients with Hodgkin's disease, but they studied a very small number of patients (two in each group). They concluded that the pattern of clearance of lymphocytes from the blood was consistent with a normal
circulation between blood and lymphoid tissue. The data are far too few to reach any conclusions regarding the diagnostic role of l~lln-labeled lymphocytes in Hodgkin's disease, but to date the data show primarily lymphocyte migration, not intense accumulation in abnormal nodes. Further investigations may be of some interest. In summary, both animal and human studies have shown that lymphocytes, both normal and abnormal, can be successfully labeled with ~]In and their migration pattern and kinetics can be evaluated. This methodology clearly has potential for the investigation of normal and abnormal lymphocytes in a number of diseases.
Labeled Cells in Malignant Diseases In addition to studies of normal and abnormal lymphocytes in malignant diseases, studies are now being reported investigating the migration of the malignant cells in a number of hemato-
256
logic malignant diseases. Miller et al used l l lIn to label Sezary cells in a patient with Sezary syndrome, a cutaneous T cell lymphoma.~7 This lymphoma can have cutaneous, visceral, and peripheral blood involvement by Sezary cells. Originally it was thought that Sezary cells originated in the skin and previous studies have shown a very high labeling index (rate of cell turnover) in the dermis. In the case studied with Hqnlabeled peripheral Sezary cells, however, the pattern was highly suggestive of migration from the blood to the skin. The distribution of radioactivity in the skin began to be apparent at 24 hours after reinjection of labeled Sezary cells. The kinetics of the radioactively labeled cells was evaluated by measurement of activity in blood samples taken serially and showed an initial rapid fall in activity over six hours (interpreted as redistribution to lungs, liver, spleen, bone marrow), followed by a gradual decrease that continued downward ( t u 2 = 249 hours). There appeared to be a gradual, steady accumulation of activity in the skin. This approach to studying Sezary syndrome in one patient can certainly be adapted to investigations of a number of hematologic malignancies. Recently, Yamauchi utilized this ~ In labeling to study the kinetics of leukemic cells in four patients in the blast crisis of chronic myelocytic leukemia (CML). 18 This disease is associated with splenomegaly and with extramedullary deposits of leukemic cells. Cell migration was evaluated by both gamma camera and blood sampling. Labeled leukemic cells were observed to accumulate in the liver and spleen, primarily the latter. The initial activity in the liver subsequently decreased over three hours, and then there was a secondary rise. The splenic activity slowly decreased over 48 hours. They postulate that the trapping of leukemic cells prevented release from the splenic sinusoids and thus the slow decrease in activity. Clearly these are preliminary investigations but they have potential for increasing our understanding of many of these diseases.
Potential Problems with rain-Labeled Lymphocytes There are, however, certain problems which have been uncovered in the use of lllln for
JANICE P. OUTCHER
labeling and studying leukocytes, and lymphocytes in particular. These have been pointed out by a number of investigators. ~927 Whereas most workers have been encouraged by the potential advantages of labeling blood cells with Hlln, there appear to be certain dose-related effects on lymphocytes that may alter the outcome of some studies. Sparshott et al pointed out that simply impurities in the labeling complex may damage the lymphocytes and suggested additional measures to optimize the purity of the complex. ~9 Segal et al have described modifications of the labeling method, using Hanks' balanced salt solution, and designed to protect lymphocytes from the harmful effects of oxine. 2~Others, however, have commented upon the detrimental effect of moderate doses of radioactivity itself on lymphocyte function, particularly and importantly, on migration. 19"21-23 Therefore, since the goal of many of these studies is investigation of migration patterns, the labeling technique and radioactivity dose used must be evaluated carefully for effects on lymphocyte function. Studies in rats have evaluated the labeling concentration of ~qn optimal for producing normal lymphocyte migration and recirculation. Sparshott et al observed recirculation of labeled lymphocytes in the thoracic duct and found that a labeling concentration of 20 #Ci/10 s cells was the highest concentration compatible with survival of most lymphocytes for 24 hours after infusion.~9Different labeling concentrations produced different tissue values, with higher values in lymph nodes being seen unexpectedly at the lower Hqn concentrations. This was felt to be due to poor cellular labeling efficiency at low concentrations and thus not from cellularly bound ~ qn. By one week after the injection of lymphocytes labeled at 20 uCi/10 s cells, most of the rain had been transferred from the lymphocytes to noncirculating radioresistant cells within the spleen and lymph nodes. Chisholm et al and others also described a dose-response effect, with incrementally decreasing cell function seen at increasing doses of II1in.2~-23Cell viability as assessed by trypan blue dye exclusion was greater than 95%. However, lymphocyte recirculation was greatly impaired at lJ~In concentrations greater than 20 ~tCi/108 cells, with very little migration to the lymph
LABELED CELLS IN MALIGNANCY
nodes. At doses less than 20 tzCi/108 cells, labeled lymphocytes appeared to behave normally. Dettman et al evaluated other lymphocyte functions such as the ability to incorporate 3Huridine and 3H-thymidine into nucleic acid synthesis in mouse spleen lymphocytes after labeling with 111In.24 As the concentration of indium-1 1 1 per cell increased, cell viability in culture decreased as did 3H-uridine incorporation. The effects on 3H-thymidine incorporation were mixed, in that mitogen-stimulated cells showed decreased incorporation with increasing ~t~In concentration, but during the culture period, there was little decrease in response. This is consistent with increased radiosensitivity during the synthesis part of the cell cycle. The conclusion seems to be that a certain amount of radioactivity will be detrimental to active lymphocytes. TerBerge et al have described a similar problem in human lymphocytes labeled with ~In; again a dose-response effect with decreased proliferative capacity dependent upon increasing concentration of the isotopeY They reported variable proliferative responses to different antigens in the affected cells, depending perhaps upon the subpopulation of T lymphocytes affected. There are mixed reports on the effect of l JJIn labeling on lymphocyte cell surface markers. Some workers have reported the loss of cell surface markers, perhaps related to a toxic effect of oxine. 2~ Others have described retention of surface markers on lymphocytes labeled with ~JZln in a buffer or plasma solution. 26 These workers did, however, comment on a detrimental effect on antibody-dependent cytotoxicity as a result of l~In labeling at concentrations greater than 20 ~Ci/1 0g cells. In summary, tttln-labeled lymphocytes seem to demonstrate accurately the recirculation patterns of normal lymphocytes provided that technical features of labeling are attended to and that a low concentration of ~ In per cell is used. These migration studies seem to correlate well with older studies using 51Cr labeling. However, because these 111instudies are being done in vivo and in humans for kinetics as well as migration and using long-lived cells, the effect of radioactivity on lymphocyte function and survival for
257
two to three days after reinjection must be considered. Whether any longer term effects of this radiation exposure might occur cannot be answered at this time. One group has reported preliminary data describing chromosomal abnormalities in radiolabeled lymphocytes.25 On the other hand, as pointed out by Thakur in an editorial on the subject, there are numerous examples of localized exposure to radioisotopes at inflammatory sites without long-term effects described (intraarticular injections of 198Au, 9Oyr).z7 All of these factors must be taken into consideration in evaluating this radionuclide for human studies. Nevertheless, the potential usefulness of indium-1 11 labeling in investigating normal and diseased lymphocytes clearly makes additional studies warranted. GRANULOCYTE LABELING IN PATIENTS WITH MALIGNANT DISEASES
Granuiocytes have been labeled for the purpose of both studying granulocyte kinetics and for identification of sites of infection. Most of the kinetic studies have been done using leukocytes labeled with 3H-thymidine2~ and DF32p,28-3~ again isotopes that cannot be used for scanning. Studies with 5JCr have shown accumulation of labeled granulocytes at sites of infection, but this again is an isotope that is gamma emitting, but lends itself poorly to scanningfl -~ Technetium99m sulfur colloid 34'35 and 67Ga-citrate36 also have been investigated as potentially useful granulocyte labels, but this has not been the case clinically. McAfee and Thakur have shown this to be one of most promising isotopes for the reasons cited in the previous section in their evaluation of indium- 111-0xine. Thakur et al and others have demonstrated the ability of granulocytes labeled with indium-Ill to successfully migrate to and identify sites of infection in animal studies. 37-39 Clinical studies seem to confirm the value of this isotope as a granulocyte label for detection of foci of inflammation.4~ Studies of granulocyte kinetics using granulocytes labeled with indium-1 11 have also been successful:5 The labeling of neutrophils with indium-1 1 1 does not seem to be fraught with as many technical difficulties as is lymphocyte labeling. Granu-
258
locytes seem to be relatively radioresistant, they are not proliferating cells, and metabolic and phagocytic function appear to remain relatively intact with radiation exposure. 42'47 Segal et al have also looked at neutrophil function during the indium-Ill labeling procedure and note some impairment of chemotaxis that must be considered during kinetic studiesfl~ However, the use of J~qn-labeled granulocytes in most patients with malignant diseases (primarily hematologic malignancies) is usually not for the purposes of kinetic studies, but to attempt to localize occult infections, or to determine the ability of granulocytes to accumulate at sites of known infection. The use of autologous indium-11 l-labeled granulocytes to localize infections has been discussed elsewhere and in this S e m i n a r . 42 44 However, granulocyte labeling requires a different approach in patients with hematolgic malignancies since these patients are usually granulocytopenic (<1000 granulocytes) when they are infected. Therefore, the use of autologous granulocytes is not possible. Fortunately, the majority of infections in these patients is apparent, so that localization of infection is not usually a major concern, but occasionally a search for occult infection is warranted. If this becomes necessary, then donor granulocytes must be labeled and used for the scans. This has been done successfully by several workers. 48-5~In our own study, we showed that donor granulocytes migrated very rapidly to sites of infection in granulocytopenic patients. 49 They migrate perhaps more rapidly than is seen in the usual autologous granulocyte scans because they may be less of a dilutional effect from unlabeled cells. Granulocytopenic patients pose a major therapeutic problem when they are infected, particularly if recovery of their bone marrow is not imminent. They frequently develop serious infections that may or may not respond appropriately to antibiotics. If such response is inadequate, then the use of therapeutic granulocyte transfusions becomes a consideration. The use of granulocyte transfusions is a major therapeutic decision and is somewhat controversial. Patients may have reactions to granulocyte transfusions and this may indicate that through multiple other transfusions they have become immunized against granulocytes.5~-53 However, an assess-
JANICE P. DUTCHER
ment of such immunization is very difficult. If these patients are immunized to random donor platelets, no rise in posttransfusion platelet count is seen except with HLA-matched platelets. Unfortunately, even with compatible granulocyte transfusions there may be no appreciable rise in posttransfusion white blood cell count, due to dose and dilutional factors. Granulocyte transfusions are usually given very slowly, so that with rapid migration, the WBC content of the peripheral blood remains low even at the end of a several hour-long transfusion. In addition, even with our best technology, the dose of granulocytes obtained for transfusion is usually quite small, particularly when compared with the potential of a normal bone marrow. Thus, the dose given is often not enough to produce a count increment. Therefore, evaluation of the success of a granulocyte transfusion is often made on a clinical basis: response of infection, lack of reaction to the transfusion. This is hardly satisfactory. In general, patients who are refractory to random donor platelets are not exposed to granulocyte transfusions for fear of serious immunologic reactions. Most serologic crossmatching tests are time-consuming and cannot be done in time to influence a specific donation. The wide variety of types of granulocyte crossmatching tests reflects the difficulty in finding one with good clinical correlation. Other means of evaluating white blood cell transfusions are clearly needed. Indium-Ill-labeled granulocyte scanning provides a visual means of evaluating granuloeyte migration. This technique has been applied to the assessment of response to granuloeyte infusion and of the effect of histocompatibility factors on granulocyte migration.49'54 As previously discussed, this technique yields good results of localization to sites of infection in granulocytopenic patients using labeled donor granulocytes.48-5~In the study by Dutcher et al, sites of infection showed uptake as early as 30 minutes following injection of indium-l 1 l labeled granulocytes and this activity at the infected site persisted for 24 to 48 hours49 (Fig 4). None of the patients in this study were alloimmunized and all responded to random donor platelet transfusions. Thus histocompatibility factors were not a variable in these studies.
LABELED CELLS IN MALIGNANCY
259 Table 1. Scan and Crossmatch Results in Nonalloimmunized and AIIoimmunized Patients
Nonalloimmunized patients LA LCT Alloimmunized patients LA LCT
Scan Negative
Scan Positive
0
20 3/18" O/17 3 2/3 1/3
11 8/11 10/11
* Number of positive crossmatches/number tested. P, 0,00001 nonalloimmunized patients versus alloimmunized patients. LA. leukoagglutinin crossmatch; LCT, lymphocytotoxic crossmatch. Reproduced by permission, ss
Fig 4. (a) Clinical picture of gingivitis in e granulocytopanic patient with acute leukemia. (b} Indium-Ill-labeled granulocyte scan in this patient at 30 minutes following injection of labeled granulocytes. There is an uptake in the gingiva as well as in a draining lymph node that was clinically involved.
Subsequently, however, we evaluated the migration of a small number (10 s) of labeled donor granulocytes in granulocytopenic patients with similar known infections, but who were known to be alloimmunized with at least antiHLA antibodies and many were later determined to have positive leukoagglutinin crossmatches with the donor. 55 In 11 of 14 such patients, donor granulocytes failed to localize to known, comparable sites of infection in scans done at 30 minutes, 4 hours, or 24 hours (Table 1). All 11 patients had anti-HLA antibodies and 8 of 11 had granulocyte-specific antibodies. Thus, we concluded that random donor granulocyte transfusions in these highly alloimmunized patients would be of little to no benefit and could potentially be harmful, leading to serious reactions. Thus, indium-111 granulocyte scanning has provided a very useful clinical tool to evaluate empiric granulocyte transfusions and also may
provide a visual clinical correlation, using a very small number of cells, to investigate the sensitivity and specificity of new serologic tests of granulocyte compatibility. Finally, we evaluated the early kinetics of labeled granulocytes in alloimmunized and nonalloimmunized patients, with particular attention to pulmonary retention during the first 30 minutes following injection)6 This is of interest because pulmonary reactions of varying severity are a known complication of granulocyte transfusion therapy. There are clinical data to suggest that these reactions are immunologically mediated. It is therefore of some interest to evaluate the fate of transfused labeled granulocytes that pass through the lungs. All cell preparations were done identically and cell viability was 90% to 95% by trypan blue dye exclusion. In this evaluation of pulmonary retention of radioactivity, curves of activity in the lungs and heart (the latter used to represent the blood pool of labeled granulocytes) were drawn for the first 30 minutes of scanning. A computer interface was used to collect data, and the area under the curve was calculated and compared. 56 A ratio of lung to heart activity was then calculated for all patients studied. There was significantly greater retention of pulmonary activity in patients who were alloimmunized compared to nonalloimmunized patients (P<0.001) (Table 2). These data suggest that mismatched granulocytes, if used for transfusions, may be retained in the lungs and therefore could potentially lead to respiratory compromise. These data also support clinical suspicions of an immunological basis for pulmo-
JANICE P. DUTCHER
260 Table 2. Lung/Blood Pool Ratio of Radioactivity Granulocyte Crossmatch
AUoimmunized Nonalloimmunized Autologous Platelets
Mean Lung/Blood Pool Ratio
4.04 2.08 2.72 1.85
_+ 1 . 4 2 ~
p < 0.001 -+ 0.34 I . J " ~ _ ~ P > 0.5 _+0.17_1 / F -+ 1.06 J
From Dutcher et al, Blood (Suppl) 60:177a, Dec. 1982. Reproduced by permission.
n a r y reactions following granulocyte transfusions. 51-53'57The use of indium-11 l - l a b e l e d granulocytes helps to s u b s t a n t i a t e these impressions by providing q u a n t i t a t i o n a n d visualization of migration or nonmigration. Indium-1 1 l - l a b e l e d granulocytes will be of use in evaluating additional aspects of granulocyte transfusion t h e r a p y including the use of H L A - m a t c h e d granulocytes and the evaluation of new serologic tests of granulocyte crossmatching. In conclusion, indium-1 11 has proven to be a very helpful cellular label with potential use for
investigation of a n u m b e r of problems relevant to h e m a t o l o g i c m a l i g n a n c i e s . T h e r e a r e also n u m e r o u s questions p e r t a i n i n g to solid t u m o r s that have yet to be investigated. T h e potential for studying and visualizing metastasis r e m a i n s a plausible direction for the use of indium-11 I labeled cells. The possibility of using labeled lymphocytes to evaluate t u m o r - b e a r i n g l y m p h nodes needs to be further investigated. Clinically, the m i g r a t i o n and h a b i t a t of malign a n t hematologic cells has only b e g u n to be investigated. T h e n a t u r e of the behavior of the spleen in both lymphoid a n d myeloid leukemias continues to be a n i m p o r t a n t theoretical b u t also clinical question. Perhaps studies of leukemic cell m i g r a t i o n will provide some clues. F i n a l l y , i n d i u m - l 1 l - l a b e l e d cells are of proven benefit in evaluating histocompatibility problems in g r a n u l o c y t e transfusion. This technique will c o n t i n u e to be utilized for f u r t h e r investigations of g r a n u l o c y t e migration.
REFERENCES
1. Gowans JL: The recirculation of lymphocytes from blood to lymph in the rat. J Physiol 146:54-69, 1959 2. Ford WL: The kinetics of lymphocyte recirculation within the rat spleen. Cell Tissue Kinet 2:171-191, 1969 3. Griscelli C, Vassali P, McCluskey RT: The distribution of large dividing lymph node cells in syngeneic recipient rats after intravenous injection. J Exp Med 130:1427-1451, 1969 4. Rannie GH, Donald KJ: Estimation of the migration of thoracic duct lymphocytes to non-lymphoid tissues. Cell Tissue Kinet 10:523-541, 1977 5. Hershey P: The separation and 5'chromium labelling of human lymphocytes with in vivo studies of survival and migration. Blood 38:360-371, 1971 6. Scott JL, Davidson JG, Marind JV, et al: Leukocyte labelling with 5~ChromiumIII. The kinetics of normal lymphocytes. Blood 40:276-281, 1972 7. McAfee JG, Thakur ML: Survey of radioactive agents for in vitro labeling of phagocytic leukocytes II. Particulate agents. J Nucl Med 17:488-493, 1976 8. Rannie GH, Thakur ML, Ford WL: An experimental comparison of radioactive labels with potential application to lymphocyte migration studies in patients. Clin Exp Immunol 29:509-514, 1977 9. Issekutz T, Chin W, Hay JB: Measurement of lymphocyte traffic with indium-111. Clin Exp Immunol 39:215-221, 1980 10. Frost P, Smith J, Frost H: The radiolabeling of lymphocytes and tumor cells with mlndium. Proc Soc Exp Biol Med 157:61-65, 1978 11. Dettman GL, Stopeck AT, Meyer GW, et al: In vivo migration of rain radiolabeled mouse-spleen lymphocytes and tumor cells. Int J Nucl Med Biol 10:11-16, 1983
12. Schroeder S, Caughran M, Jochelson M, et ah Imaging of lymphoid structures with Indium-11I-labeled lymphocytes. Invest Radiol 18:87-93, 1983 13. Wagstaff J, Gibson C, Thatcher N, et ah A method for following human lymphocyte traffic using indium-1l 1 oxine labelling. Clin Exp Immuno143:435-442, 1981 14. Wagstaff J, Gibson C, Thatcher N, et al: Human lymphocyte traffic assessed by Indium-111 oxine labelling: clinical observations.Clin Exp Immunol 43:443-449, 1981 15. Ford WL: Lymphocyte migration and the immune responses. Prog Allergy 19:1-59, 1975 16. Lavender JP, Goldman JM, Arnot RN, et ah Kinetics of indium-111 labelled lymphocytes in normal subjects and patients with Hodgkin's disease. Br Med J 2:797-799, 1977 17. Miller RA, Coleman CN, Fawcett HD, et al: Sezary syndrome: a model for migration of T lymphocytesto skin. N Engl J Med 303:89-92,1980 18. Yamauchi K, Sugihara M, Nagao T, et al: Kinetics of "qn-labelled leukaemic cells in blast crisis of chronic myelocytic leukemia. Scand J Haematol 31:221-228, 1983 19. Sparshott SM, Sharma H, Kelly JD, et ah Factors influencing the fate of mlndium-labelled lymphocytes after transfer to syngeneic rats. J Immunol Methods 41:303-320, 1981 20. Segal AW, Deteix P, Garcia R, et al: Indium-Ill labeling of leukocytes:a detrimental effect on neutrophil and lymphocyte function and an improved method of cell labelling. J Nucl Med 19:1238-1244, 1978 21. Chisholm PM, Danpure HJ, Healy G, et al: Cell damage resulting from the labeling of rat lymphocytes and HeLa $3 cells with In-111 oxine. J Nucl Med 20:1308-1311, 1979 22. Chisholm PM, Peters AM: The effect of Indium-111
LABELED CELLS IN MALIGNANCY
labeling on the rearculation of rat lymphocytes in Thakur ML, Gottsehalk A (eds): Indium-111 Labeled Neutrophils, Platelets and Lymphocytes. New York, Trivirum, 1980, pp 205-209 23. Van Dinther-Janssen ACHM, Seheper R J: Restrictions to the use of m lndium-oxine as a radiolabel in lymphocyte migration studies. J Immunol Methods 46:353360,1981 24. Dettmann GL, losiphidis AH: Functional integrity of mouse spleen lymphocytes in vitro after radiolabeling with Indinm-I I 1. Radiat Res 92:95-104, 1982 25. tenBerge RJM, Natarajan AT, Hardeman MR, et al: Labeling with Indium-I 11 has detrimental effects on human lymphocytes: Concise Communication. J Nucl Med 24:615620, 1983 26. Signore A, Beales P, Sensi M, et ah Labelling of lymphocytes with Indium-111 oxine: Effect on cell surface phenotype and antibody-dependent cellular cytotoxicity. Immunol Lett 6:151-154, 1983 27. Thakur ML: Cell Labeling. Achievements, challenges and prospects. J Nucl Med 22:1011-1014, 1981 28. Dancey JT, Deubelfeiss KA, Marker LA, et al: Neutrophil kinetics in man. J Clin Invest 58:705-715, 1976 29. Athens JW, Mauer AM, Ashenbrucker M, et al: Leukokinetic studies. 1. A method for labelling leukocytes with diisopropyl fluorophosphate (DF32P). Blood 14:303333, 1959 30. Athens JW, Haab OP, Raab SO, et ah Leukokinetic studies. IV. The total blood cirulating and marginal granulocyte pools and the granalocyte turnover rate in normal subjects. J Clin Invest 40:989-995,1961 31. Winkelman J, Collica C J, Sandier SG: The delineation of abscesses by scintiphotography using 5~Cr labelled leukocytes. Am J Roentgenol 103:881-885, 1968 32. Deysine M, Robinson RG, Wilder JR: Abscess detection by radioactive chromium labelled autologous white blood cells. Surg Gynecol Obstet 131:216-220,1970 33. McMillan R, Scott JL: Leukocyte labelling with SIChromium. I. Technic and results in normal subjects. Blood 32:738-754, 1968 34. English D, Andersen BR: Labelling of phagocytes from human blood with 99roTe-sulfur colloid. J Nucl Med 16:5-10, 1975 35. Andersen BR, English D, Akalin HE, et al: Inflammatory lesions localized with 99mTc labelled leukoeytes. Arch Intern Med 135:1067-1071, 1975 36. Burleson RL, Holman BR, Tow DE: Scintigraphic demonstration of abscesses with radioactive gallium labelled leukocytes. Surg Gynecol Obstet 141:379-382, 1975 37. Thakur ML: lndium-111: a new radioactive traser for leukocytes. Exp Hematol 5 (Suppl):145:145-150, 1977 38. Thakur ML, Coleman RE, Mayhall CG, et al: Preparation and evaluation of rain-labeled leukocytes as an abscess imaging agent in dogs. Radiology 119:731-732, 1976 39. Thakur ML, Coleman RE, Welch M J: Indium-111 labelled leukocytes for the localization of abscesses: preparation, analysis, tissue distribution and comparison with 67Gallium citrate in dogs. J Lab Clin Med 89:217-228, 1977 40. Segal AW, Thakur ML, Arnot RN, et al: lndium-I 11
261
labelled leukocytes for localization of abscesses. Lancet 1:1056-1058, 1976 41. Doherty PW, Bushberg JT, Lipton M J, et al: The use of tttln labeled leukocytes for abscess detection. Clin Nucl Med 3:108-122, 1976 42. Thakur ML, Lavender JP, Arnot RN, et ah IndiumIll-labeled autologous leukocytes in man. J Nucl Med 18:1014-1021, 1977 43. Forstrom LA, Gomez L, Weebten B, et al: Clinical use of rain oxine labeled leukocytes in the detection of inflammation or abscess. J Nucl Med 19:672-678, 1978 44. Ascher NL, Ahrenholz DH, Simmons RL, et ah Indium-111 autologous tagged leukocytes in the diagnosis of intraperitoneal sepsis. Arch Surg 114:386-392, 1979 45. Goodwin DA, Doherty PW, McDougall LR: Clinical use of lndium-1 l l-labeled white cells: an analysis of 312 cases, in Thakur ML, Gottschalt A (eds): Indium-Ill Labeled Neutrophils, Platelets and Lymphocytes, New York, Trivirum, 1980, pp. 131-145. 46. Weiblen B, Forstrom L, McCullough J: Studies of the kinetics of rain labeled granulocytes. J Lab Clin Med 94:246-255, 1978 47. Selvaraj RJ, SbarraAJ: Effects of x-irradiation on the metabolic changes accompanying phagocytosis. Nature 210:158-161, 1966 48. Anstall HB, Coleman E: Donor-leukocyte imaging in granulocytopenic patients with suspected abscesses: Concise Communication. J Nucl Med 23:319-321, 1982 49. Dutcher JP, Schiffer CA, Johnston GS: Rapid migration of Nqndium-labelled granulocytes to sites of infection. N Engl J Med 304:586-589, 1981 50. Alavi JB, Staum MM, Alavi A: Indium-Ill for granulocyte labeling in neutropenic patients, in Thakur ML, Gottschalk A (eds): Indium-111 Labeled Neutrophils, Platelets and Lymphocytes, New York, Trivirum, 1980, pp. 119122 51. Goldstein IM, Eyre H J, Terasaki PI, et al: Leukocyte transfusions: Role of leukocyte alloantibodies in determining transfusion response. Transfusion 11:19-24, 1971 52. Schiffer CA, Aisner J, Daly PA, et ah Alloimmunization following prophylactic granulocyte transfusion. Blood 54:766-774,1979 53. Dahlke MB, Keashen M, Alavi JB, et al: Response to granulocyte transfusions in the alloimmunized patient. Transfusion 20:555-558,1980 54. McCullough J, Weiblen BJ, Clay ME, et al: Effect of leukocyte antibodies on the fate in vivo of indium- 11 I-labeled granulocytes. Blood 58:164--170,198l 55. Dutcher, JP, Schiffer CA, Johnston GS, et al: Alloimmunization prevents the migration of transfused Indium11 l-labelled granulocytes to sites of infection. Blood 62:354360, 1983 56. Dutcher JP, Riggs CE, Fox J J, et al: The effect of histocompatibility factors on pulmonary retention of Indium-111-1abelled granulocytes. Exp Hematol 11 (Suppl 14):171, 1983 57. Dahlke MB, Keashen M, Alavi JB, et a!: Granulocyte transfusions and outcome of alloimmunized patients with gram-negative sepsis. Transfusion 22,374-378, 1982
HE USE OF labeled cells for the investiga-
tion of various aspects of malignancy and T for the care of such patients has recently taken on new clinical prominence. This is particularly with regard to the hematologic malignancies. Initial investigations have been primarily in the realm of studying the basic biology of the diseases, rather than as a diagnostic test to identify sites of disease since the cells do not necessarily localize to sites of tumor. Nevertheless, with the use of radionuclides such as indium- I 11, which can be visualized with standard scanning techniques, new information about many of the hematologic malignancies has been uncovered. Chromium-51 labeling of hematologic cells and lymphocytes has been used for years to study the Seminars in Nuclear Medicine, Vol. XIV, No. 3 (July), 1984
concern about long-term changes in cells t h a t recirculate. Others cite precedents of o t h e r long-term uses of isotopes, therapeutically, w i t h o u t detrimental effects. These concerns continue to be investigated. Finally, an area of much interest in the use of indium-111 is t h e labeling of granulocytes. This technique has been useful diagnostically, t o localize infections. The major value in patients w i t h malignancy, primarily w i t h hematologic malignancies, is to evaluate the potential benefit of granulocyte transfusions. M a n y of these patients develop prolonged granulocytopenia and become infected, and granuloc y t e transfusions may become a t h e r a p e u t i c consideration. H o w e v e r , these patients usually have been subjected to m a n y transfusions and may be immunized t o granulocytes. This is difficult t o assess clinically, but t h e use of i n d i u m - I l l - l a b e l e d donor granulocytes provides a visual d e m o n s t r a t i o n of w h e t h e r or n o t t h e cells migrate t o t h e site of infection. This technique also can be used t o assess g r a n u l o c y t e s e q u e s t r a t i o n . It a p p e a r s t h a t in patients w h o develop antibodies, both anti-HLA and granulocyte specific, the donor granulocytes do not migrate t o sites of infection and in fact may sequest e r in the lungs. These data can help guide the use of granulocyte transfusion in these patients. The investigation of labeled cells, both malignant and normal, in patients w i t h malignancy is giving us n e w insight into t h e diseases. These studies are of p a r t i c u l a r benefit using n e w e r techniques w i t h isotopes t h a t can be scanned such as i n d i u m - I l l . Studies of the migration patterns of Sezery cells, granulocytes in chronic myelogenous leukemia and lymphocytes in lymphoid leukemia are continuing and may provide i m p o r t a n t i n f o r m a t i o n regarding t h e disease process.
migration and sequestration of these cells in the spleen and other sites. However, the investigation of indium-ll 1 as a cell label has added new dimensions to studies of lymphoid and hematologic cells and tissues.
From the Division of Oncology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY. Dr. Dutcher is a special fellow of the Leukemia Society of America. Address reprint requests to Janice P. Dutcher, MD, Division of Oncology, Department of Medicine, Albert Einstein College of Medicine (Room 2C-14 Van Etten), 1300 Morris, Park Ave, Bronx, NY10461. 9 1984 by Grune & Stratton, Inc. 0001-2998/84/1403~9007505.00/0 251
252
Another area in which cell labeling has been particularly helpful in patients with malignancy is in the evaluation of granulocyte transfusion supportive care. These transfusions are particularly difficult to quantitate and at times to determine benefit. The use of indium-Ill-labeled granulocytes has provided a visual means of evaluating granulocyte migration and localization to sites of infection in granulocytopenic patients. LYMPHOCYTE MIGRATION STUDIES: BENIGN AND MALIGNANT
Animal Studies
The study of normal lymphocyte migration has been an area of great interest to hematologists and immunologists for some time. The substantial recirculation of lymphocytes from blood into lymphoid tissue and back into blood is well described) '2 The existence of separate lymphocyte migration pathways through the gut and the peripheral lymph nodes has been elegantly demonstrated with the observation of blast cells from mesenteric lymph nodes homing preferentially to the gut wall and blast cells from peripheral nodes returning preferentially to the peripheral nodes) The mechanisms of selective migration and the elucidation of pathways has been possible primarily through the use of isotopic labeling of lymphocytes. Rannie and Donald compared the distribution of radioactivity at intervals following transfusion of thoracic duct lymphocytes labeled with five different isotopes in animal studies: They demonstrated that the rate of elution of isotope from the labeled cells and the subsequent fate of the eluted isotope were the most important factors limiting the usefulness of such isotopes. Comparison of labeling procedures using 3H- and 14C-uridine, 3Hand ~4C-leucine, L-75Se-selenomethionine, 99mTcsodium pertechnetate and S~Cr-sodium chromate in vitro and 3H-thymidine in vivo showed that 5~Cr had the fewest disadvantages for studying lymphocytes. Unfortunately, this isotope cannot be scanned, so that studies of migration must be done by assessing blood and tissue radioactivity. As can be seen, most lymphocyte labeling studies have been limited to work in experimental animals, primarily because of the limitations of the isotopes and methodology available. In
JANICE P. DUTCHER
animals the lymphocyte migration studies have required the sacrificing of animals, the scintillation counting of their organs and autoradiography of various tissues in order to determine the location and amount of cellular radioactivity. These studies, although laborious, have been done carefully and have been important in studying lymphocyte recirculation and the events of the immune response. These methods have limitations, however, and only provide a description of the terminal deposition of lymphocytes in lymphoid and nonlymphoid tissue. They do not provide the details of the early phases of lymphocyte migration. Thus, despite the many demonstrable advantages of 51Cr as a cellular label, it is not an isotope that has allowed external and dynamic imaging of reinjected labeled cells or of in vivo demonstration of early lymphocyte migration patterns. Lymphocyte labeling studies therefore until recently, have not been directly applicable to studies in humans. However, at least two groups have attempted human studies using 5~Cr labeling and counting over sites of accumulation in lymphoid tissue) '6 Nevertheless, this does not, again, provide dynamic data of migration; only of accumulation in tissue. Recently, a number of investigators have begun to use indium-111-oxine for evaluation of lymphocyte migration. As first described by McAfee and Thakur, 7 this radiochemical binds intracellularly with very little subsequent elution from the cell. It has a comparatively short halflife (2.7 days) versus the 27.8 days of 5~Cr. In addition, l lqn has two energy peaks, both within the visible range of the standard gamma camera, thus allowing external imaging. The potential for kinetic and early migration studies of reinjected cells exists with this radiocompound. Rannie et al reported a comparison of llllnand 99mTc-labeled lymphocytes in rats with the standard method of 5~Cr labeling: They concluded that for the potential study of lymphocyte migration in humans, labeling with l~qn-oxine was the most promising approach based on the findings of minimal elution and their findings of essentially normal lymphocyte migration in their animal studies. Technetium-99m was found to rapidly dissociate from lymphocytes and poor lymphocyte recirculation was observed after labeling with this isotope.
LABELED CELLS IN MALIGNANCY
Subsequently, both animal and human lymphocyte migration studies using indium-Ill labeling have been reported. The ability to use this isotope for in vivo studies and the high correlation of results between ruin and mCr animal studies of lymphocyte migration have led to a number of investigations of both normal and abnormal lymphocyte migration and distribution. These studies will be reviewed. Rannie et al, in their preliminary studies in rats, showed that indium-11 l-labeled lymphocytes recirculated from blood to lymph, analogous to lymphocyte behavior when labeled with 5~CrS (Fig 1). Recirculation suggests that these labeled cells can be followed as markers for normal lymphocyte circulation. Issekutz et al,
Fig 1. Gamma-camera image of a rat that had received lymphocytes labeled with 300 #Ci of 1+11n 24 hours previously. The large group of cervical lymph nodes (arrow) as well as other groups of lymph nodes in the thorax and abdomen are visible. The major concentration of radioactivity is present in the liver and spleen. The latter is responsible for the small area of intense activity indicated by the triangle. (From Rannie at al. C/in Exp Immuno129:509-514, 1977. Reproduced by permission, e)
253
investigated lymphocyte migration in sheep using ~ttln-labeled nodal lymphocytes in similar studies.9 They found that the migration patterns of indium-11 l-labeled lymphocytes were essentially identical to those seen using S~Cr in sheep. Frost et al also evaluated the indium-l 1 l labeled lymphocytes and tumor cells in rats with promising results. ~~ They concluded that l~lln was a potentially useful isotope for subsequent human studies. Subsequent studies have investigated the migration of abnormal cells a n d o f lymphocytes to abnormal nodes. Dettman et al evaluated in vivo migration of ~l~In-labeled mouse spleen lymphocytes and compared this to the migration of labeled mouse lymphoma and mouse mammary adenocarcinoma cells. ~ They found markedly different migration patterns between the three types of cells. They compared viability of labeled to unlabeled cells and determined that labeling itself was not a factor in this difference. The mouse mammary adenocarcinoma cells were initially trapped in the lungs, to a much greater extent than the splenic lymphocytes or the T cell lymphoma cells. The normal lymphocytes subsequently deposited more heavily in the spleen than the other two types of cells. Schroeder et al used rats to observe the ability of indium-11 l-labeled lymphocytes to migrate to lymph nodes involved with tumor (H-4-II-E hepatoma)J z Lymphocytes labeled with t~In exhibited normal migration from blood to nodes and both normal and tumor-bearing nodes were visualized (Fig 2). Because of the small size of rat lymph nodes, qualitative or quantitative differences between normal and tumor-bearing nodes could not be detected. Although at the moment such studies are phenomenologic, these types of studies suggest that this technique may be helpful in the investigation of differences between benign and malignant cells of the hematopoietic and lymphoid systems, and perhaps of disorders of immunity. This technique may also be applicable to studies of mechanisms of metastatic disease .and with refinement may be relevant to investigation of tumor-bearing nodes.
Human Lymphocyte Studies Wagstaff et al have evaluated indium-111oxine labeling as a means of observing human
254
JANICE P. DUTCHER
Fig 2. (a) A composite of t h e upper and l o w e r body images of a normal animal 22 hours a f t e r injection of 5.7 x 10 s l y m p h o c y t e s labeled w i t h 100/~Ci l ~ l n - o x i n e . The spleen, liver, and cervical, axillary, inguinal, and popliteal lymph nodes a r e identified by arrows. (b) A composite of t h e u p p e r and l o w e r body images of a t u m o r - b e a r i n g rat 22 hours a f t e r injection of 2.1 x 10 s H-4-11-E hepatoma cells w e r e injected into the left hind f o o t pad 44 days before imaging. Severe metastatic involvement is clearly visible in the left popliteal region. (From Schroeder et al, Invest Radio/18:87-93, 1983. Reproduced by permission. TM)
lymphocyte migration. 13,14Patients with chronic lymphocytic leukemia (CLL) and well-differentiated diffuse lymphocytic lymphoma (WDDLL) as well as two normal volunteers were studied with ~lqn_labeled autologous lymphocytes. The viability of the labeled cells was tested by trypan blue dye exclusion and by morphology evaluation using phase-contrast microscopy. The important features in the normal subjects' studies were the lack of lung uptake, minimal liver activity, and marked splenic activity. Interestingly, the blood disappearance of lymphocytes was much greater in the patients with CLL than in the other subjects. In the CLL patients, also, there was a much more sustained high level of counts over the spleen throughout the 48-hour evaluation period and the counts over the lymph nodes in these patients rose over the 48-hour period (Fig 3).
It was also pointed out in these studies that the migration patterns will vary depending upon the population of lymphocytes being studied. In animal studies, the tempo of recirculation of T and B lymphocytes is different. T cells generally recirculate much more rapidly than do B cells. ~s It is therefore necessary to pay attention to the nature of the lymphocyte suspension being used for study since the results may vary depending upon the proportion of B and T cells. In Wagstaff's study, the two patients with WDDLL had markedly different proportions of B and T cells, with patient DB having a monoclonal B cell population and patient ES having many more T cells than patient DB. This is consistent with the clinical variability of this disease. Patient ES had a blood lymphocyte disappearance curve similar to the normal subjects, but DB, with the monoclonal B cell population, showed a blood lympho-
LABELED CELLS IN MALIGNANCY
255
Normal a 400
WDDLL (D.B.)
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~Spleen
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Fig 3. (a) Blood disappearance curves for indium-Ill oxine-labeled lymphocytes. WDDLL = well-differentiated diffuse lymphocytic lymphoma (E.B.), CLL-chronic lymphocytic leukemia (n-4). (b) Surfaceprobe counts, indium-III-oxine-labe|ed lymphocytes. (From Wagstaff et ah Clin Exp Immunol 4 3 : 4 4 3 - 4 4 9 , 1981. Reproduced by permission.TM)
cyte disappearance pattern similar to the patients with CLL (a monoclonal B cell disorder). Wagstaff et al also studied a patient with Hodgkin's disease of the mediastinum and noted accumulation of autologous ]]]In-labeled lymphocytes in the involved mediastinal nodes. ~4 They did not discuss the findings in other lymph node-bearing areas or the specificity of this technique. Lavender et al looked at the kinetics of indium-l 1 l-labeled lymphocytes in patients with Hodgkin's disease and in two normal subjects in an earlier report. ]6 Radioactivity began to appear in the lymph nodes four to 18 hours after injection. They found no difference between normal subjects and the patients with Hodgkin's disease, but they studied a very small number of patients (two in each group). They concluded that the pattern of clearance of lymphocytes from the blood was consistent with a normal
circulation between blood and lymphoid tissue. The data are far too few to reach any conclusions regarding the diagnostic role of l~lln-labeled lymphocytes in Hodgkin's disease, but to date the data show primarily lymphocyte migration, not intense accumulation in abnormal nodes. Further investigations may be of some interest. In summary, both animal and human studies have shown that lymphocytes, both normal and abnormal, can be successfully labeled with ~]In and their migration pattern and kinetics can be evaluated. This methodology clearly has potential for the investigation of normal and abnormal lymphocytes in a number of diseases.
Labeled Cells in Malignant Diseases In addition to studies of normal and abnormal lymphocytes in malignant diseases, studies are now being reported investigating the migration of the malignant cells in a number of hemato-
256
logic malignant diseases. Miller et al used l l lIn to label Sezary cells in a patient with Sezary syndrome, a cutaneous T cell lymphoma.~7 This lymphoma can have cutaneous, visceral, and peripheral blood involvement by Sezary cells. Originally it was thought that Sezary cells originated in the skin and previous studies have shown a very high labeling index (rate of cell turnover) in the dermis. In the case studied with Hqnlabeled peripheral Sezary cells, however, the pattern was highly suggestive of migration from the blood to the skin. The distribution of radioactivity in the skin began to be apparent at 24 hours after reinjection of labeled Sezary cells. The kinetics of the radioactively labeled cells was evaluated by measurement of activity in blood samples taken serially and showed an initial rapid fall in activity over six hours (interpreted as redistribution to lungs, liver, spleen, bone marrow), followed by a gradual decrease that continued downward ( t u 2 = 249 hours). There appeared to be a gradual, steady accumulation of activity in the skin. This approach to studying Sezary syndrome in one patient can certainly be adapted to investigations of a number of hematologic malignancies. Recently, Yamauchi utilized this ~ In labeling to study the kinetics of leukemic cells in four patients in the blast crisis of chronic myelocytic leukemia (CML). 18 This disease is associated with splenomegaly and with extramedullary deposits of leukemic cells. Cell migration was evaluated by both gamma camera and blood sampling. Labeled leukemic cells were observed to accumulate in the liver and spleen, primarily the latter. The initial activity in the liver subsequently decreased over three hours, and then there was a secondary rise. The splenic activity slowly decreased over 48 hours. They postulate that the trapping of leukemic cells prevented release from the splenic sinusoids and thus the slow decrease in activity. Clearly these are preliminary investigations but they have potential for increasing our understanding of many of these diseases.
Potential Problems with rain-Labeled Lymphocytes There are, however, certain problems which have been uncovered in the use of lllln for
JANICE P. OUTCHER
labeling and studying leukocytes, and lymphocytes in particular. These have been pointed out by a number of investigators. ~927 Whereas most workers have been encouraged by the potential advantages of labeling blood cells with Hlln, there appear to be certain dose-related effects on lymphocytes that may alter the outcome of some studies. Sparshott et al pointed out that simply impurities in the labeling complex may damage the lymphocytes and suggested additional measures to optimize the purity of the complex. ~9 Segal et al have described modifications of the labeling method, using Hanks' balanced salt solution, and designed to protect lymphocytes from the harmful effects of oxine. 2~Others, however, have commented upon the detrimental effect of moderate doses of radioactivity itself on lymphocyte function, particularly and importantly, on migration. 19"21-23 Therefore, since the goal of many of these studies is investigation of migration patterns, the labeling technique and radioactivity dose used must be evaluated carefully for effects on lymphocyte function. Studies in rats have evaluated the labeling concentration of ~qn optimal for producing normal lymphocyte migration and recirculation. Sparshott et al observed recirculation of labeled lymphocytes in the thoracic duct and found that a labeling concentration of 20 #Ci/10 s cells was the highest concentration compatible with survival of most lymphocytes for 24 hours after infusion.~9Different labeling concentrations produced different tissue values, with higher values in lymph nodes being seen unexpectedly at the lower Hqn concentrations. This was felt to be due to poor cellular labeling efficiency at low concentrations and thus not from cellularly bound ~ qn. By one week after the injection of lymphocytes labeled at 20 uCi/10 s cells, most of the rain had been transferred from the lymphocytes to noncirculating radioresistant cells within the spleen and lymph nodes. Chisholm et al and others also described a dose-response effect, with incrementally decreasing cell function seen at increasing doses of II1in.2~-23Cell viability as assessed by trypan blue dye exclusion was greater than 95%. However, lymphocyte recirculation was greatly impaired at lJ~In concentrations greater than 20 ~tCi/108 cells, with very little migration to the lymph
LABELED CELLS IN MALIGNANCY
nodes. At doses less than 20 tzCi/108 cells, labeled lymphocytes appeared to behave normally. Dettman et al evaluated other lymphocyte functions such as the ability to incorporate 3Huridine and 3H-thymidine into nucleic acid synthesis in mouse spleen lymphocytes after labeling with 111In.24 As the concentration of indium-1 1 1 per cell increased, cell viability in culture decreased as did 3H-uridine incorporation. The effects on 3H-thymidine incorporation were mixed, in that mitogen-stimulated cells showed decreased incorporation with increasing ~t~In concentration, but during the culture period, there was little decrease in response. This is consistent with increased radiosensitivity during the synthesis part of the cell cycle. The conclusion seems to be that a certain amount of radioactivity will be detrimental to active lymphocytes. TerBerge et al have described a similar problem in human lymphocytes labeled with ~In; again a dose-response effect with decreased proliferative capacity dependent upon increasing concentration of the isotopeY They reported variable proliferative responses to different antigens in the affected cells, depending perhaps upon the subpopulation of T lymphocytes affected. There are mixed reports on the effect of l JJIn labeling on lymphocyte cell surface markers. Some workers have reported the loss of cell surface markers, perhaps related to a toxic effect of oxine. 2~ Others have described retention of surface markers on lymphocytes labeled with ~JZln in a buffer or plasma solution. 26 These workers did, however, comment on a detrimental effect on antibody-dependent cytotoxicity as a result of l~In labeling at concentrations greater than 20 ~Ci/1 0g cells. In summary, tttln-labeled lymphocytes seem to demonstrate accurately the recirculation patterns of normal lymphocytes provided that technical features of labeling are attended to and that a low concentration of ~ In per cell is used. These migration studies seem to correlate well with older studies using 51Cr labeling. However, because these 111instudies are being done in vivo and in humans for kinetics as well as migration and using long-lived cells, the effect of radioactivity on lymphocyte function and survival for
257
two to three days after reinjection must be considered. Whether any longer term effects of this radiation exposure might occur cannot be answered at this time. One group has reported preliminary data describing chromosomal abnormalities in radiolabeled lymphocytes.25 On the other hand, as pointed out by Thakur in an editorial on the subject, there are numerous examples of localized exposure to radioisotopes at inflammatory sites without long-term effects described (intraarticular injections of 198Au, 9Oyr).z7 All of these factors must be taken into consideration in evaluating this radionuclide for human studies. Nevertheless, the potential usefulness of indium-1 11 labeling in investigating normal and diseased lymphocytes clearly makes additional studies warranted. GRANULOCYTE LABELING IN PATIENTS WITH MALIGNANT DISEASES
Granuiocytes have been labeled for the purpose of both studying granulocyte kinetics and for identification of sites of infection. Most of the kinetic studies have been done using leukocytes labeled with 3H-thymidine2~ and DF32p,28-3~ again isotopes that cannot be used for scanning. Studies with 5JCr have shown accumulation of labeled granulocytes at sites of infection, but this again is an isotope that is gamma emitting, but lends itself poorly to scanningfl -~ Technetium99m sulfur colloid 34'35 and 67Ga-citrate36 also have been investigated as potentially useful granulocyte labels, but this has not been the case clinically. McAfee and Thakur have shown this to be one of most promising isotopes for the reasons cited in the previous section in their evaluation of indium- 111-0xine. Thakur et al and others have demonstrated the ability of granulocytes labeled with indium-Ill to successfully migrate to and identify sites of infection in animal studies. 37-39 Clinical studies seem to confirm the value of this isotope as a granulocyte label for detection of foci of inflammation.4~ Studies of granulocyte kinetics using granulocytes labeled with indium-1 11 have also been successful:5 The labeling of neutrophils with indium-1 1 1 does not seem to be fraught with as many technical difficulties as is lymphocyte labeling. Granu-
258
locytes seem to be relatively radioresistant, they are not proliferating cells, and metabolic and phagocytic function appear to remain relatively intact with radiation exposure. 42'47 Segal et al have also looked at neutrophil function during the indium-Ill labeling procedure and note some impairment of chemotaxis that must be considered during kinetic studiesfl~ However, the use of J~qn-labeled granulocytes in most patients with malignant diseases (primarily hematologic malignancies) is usually not for the purposes of kinetic studies, but to attempt to localize occult infections, or to determine the ability of granulocytes to accumulate at sites of known infection. The use of autologous indium-11 l-labeled granulocytes to localize infections has been discussed elsewhere and in this S e m i n a r . 42 44 However, granulocyte labeling requires a different approach in patients with hematolgic malignancies since these patients are usually granulocytopenic (<1000 granulocytes) when they are infected. Therefore, the use of autologous granulocytes is not possible. Fortunately, the majority of infections in these patients is apparent, so that localization of infection is not usually a major concern, but occasionally a search for occult infection is warranted. If this becomes necessary, then donor granulocytes must be labeled and used for the scans. This has been done successfully by several workers. 48-5~In our own study, we showed that donor granulocytes migrated very rapidly to sites of infection in granulocytopenic patients. 49 They migrate perhaps more rapidly than is seen in the usual autologous granulocyte scans because they may be less of a dilutional effect from unlabeled cells. Granulocytopenic patients pose a major therapeutic problem when they are infected, particularly if recovery of their bone marrow is not imminent. They frequently develop serious infections that may or may not respond appropriately to antibiotics. If such response is inadequate, then the use of therapeutic granulocyte transfusions becomes a consideration. The use of granulocyte transfusions is a major therapeutic decision and is somewhat controversial. Patients may have reactions to granulocyte transfusions and this may indicate that through multiple other transfusions they have become immunized against granulocytes.5~-53 However, an assess-
JANICE P. DUTCHER
ment of such immunization is very difficult. If these patients are immunized to random donor platelets, no rise in posttransfusion platelet count is seen except with HLA-matched platelets. Unfortunately, even with compatible granulocyte transfusions there may be no appreciable rise in posttransfusion white blood cell count, due to dose and dilutional factors. Granulocyte transfusions are usually given very slowly, so that with rapid migration, the WBC content of the peripheral blood remains low even at the end of a several hour-long transfusion. In addition, even with our best technology, the dose of granulocytes obtained for transfusion is usually quite small, particularly when compared with the potential of a normal bone marrow. Thus, the dose given is often not enough to produce a count increment. Therefore, evaluation of the success of a granulocyte transfusion is often made on a clinical basis: response of infection, lack of reaction to the transfusion. This is hardly satisfactory. In general, patients who are refractory to random donor platelets are not exposed to granulocyte transfusions for fear of serious immunologic reactions. Most serologic crossmatching tests are time-consuming and cannot be done in time to influence a specific donation. The wide variety of types of granulocyte crossmatching tests reflects the difficulty in finding one with good clinical correlation. Other means of evaluating white blood cell transfusions are clearly needed. Indium-Ill-labeled granulocyte scanning provides a visual means of evaluating granuloeyte migration. This technique has been applied to the assessment of response to granuloeyte infusion and of the effect of histocompatibility factors on granulocyte migration.49'54 As previously discussed, this technique yields good results of localization to sites of infection in granulocytopenic patients using labeled donor granulocytes.48-5~In the study by Dutcher et al, sites of infection showed uptake as early as 30 minutes following injection of indium-l 1 l labeled granulocytes and this activity at the infected site persisted for 24 to 48 hours49 (Fig 4). None of the patients in this study were alloimmunized and all responded to random donor platelet transfusions. Thus histocompatibility factors were not a variable in these studies.
LABELED CELLS IN MALIGNANCY
259 Table 1. Scan and Crossmatch Results in Nonalloimmunized and AIIoimmunized Patients
Nonalloimmunized patients LA LCT Alloimmunized patients LA LCT
Scan Negative
Scan Positive
0
20 3/18" O/17 3 2/3 1/3
11 8/11 10/11
* Number of positive crossmatches/number tested. P, 0,00001 nonalloimmunized patients versus alloimmunized patients. LA. leukoagglutinin crossmatch; LCT, lymphocytotoxic crossmatch. Reproduced by permission, ss
Fig 4. (a) Clinical picture of gingivitis in e granulocytopanic patient with acute leukemia. (b} Indium-Ill-labeled granulocyte scan in this patient at 30 minutes following injection of labeled granulocytes. There is an uptake in the gingiva as well as in a draining lymph node that was clinically involved.
Subsequently, however, we evaluated the migration of a small number (10 s) of labeled donor granulocytes in granulocytopenic patients with similar known infections, but who were known to be alloimmunized with at least antiHLA antibodies and many were later determined to have positive leukoagglutinin crossmatches with the donor. 55 In 11 of 14 such patients, donor granulocytes failed to localize to known, comparable sites of infection in scans done at 30 minutes, 4 hours, or 24 hours (Table 1). All 11 patients had anti-HLA antibodies and 8 of 11 had granulocyte-specific antibodies. Thus, we concluded that random donor granulocyte transfusions in these highly alloimmunized patients would be of little to no benefit and could potentially be harmful, leading to serious reactions. Thus, indium-111 granulocyte scanning has provided a very useful clinical tool to evaluate empiric granulocyte transfusions and also may
provide a visual clinical correlation, using a very small number of cells, to investigate the sensitivity and specificity of new serologic tests of granulocyte compatibility. Finally, we evaluated the early kinetics of labeled granulocytes in alloimmunized and nonalloimmunized patients, with particular attention to pulmonary retention during the first 30 minutes following injection)6 This is of interest because pulmonary reactions of varying severity are a known complication of granulocyte transfusion therapy. There are clinical data to suggest that these reactions are immunologically mediated. It is therefore of some interest to evaluate the fate of transfused labeled granulocytes that pass through the lungs. All cell preparations were done identically and cell viability was 90% to 95% by trypan blue dye exclusion. In this evaluation of pulmonary retention of radioactivity, curves of activity in the lungs and heart (the latter used to represent the blood pool of labeled granulocytes) were drawn for the first 30 minutes of scanning. A computer interface was used to collect data, and the area under the curve was calculated and compared. 56 A ratio of lung to heart activity was then calculated for all patients studied. There was significantly greater retention of pulmonary activity in patients who were alloimmunized compared to nonalloimmunized patients (P<0.001) (Table 2). These data suggest that mismatched granulocytes, if used for transfusions, may be retained in the lungs and therefore could potentially lead to respiratory compromise. These data also support clinical suspicions of an immunological basis for pulmo-
JANICE P. DUTCHER
260 Table 2. Lung/Blood Pool Ratio of Radioactivity Granulocyte Crossmatch
AUoimmunized Nonalloimmunized Autologous Platelets
Mean Lung/Blood Pool Ratio
4.04 2.08 2.72 1.85
_+ 1 . 4 2 ~
p < 0.001 -+ 0.34 I . J " ~ _ ~ P > 0.5 _+0.17_1 / F -+ 1.06 J
From Dutcher et al, Blood (Suppl) 60:177a, Dec. 1982. Reproduced by permission.
n a r y reactions following granulocyte transfusions. 51-53'57The use of indium-11 l - l a b e l e d granulocytes helps to s u b s t a n t i a t e these impressions by providing q u a n t i t a t i o n a n d visualization of migration or nonmigration. Indium-1 1 l - l a b e l e d granulocytes will be of use in evaluating additional aspects of granulocyte transfusion t h e r a p y including the use of H L A - m a t c h e d granulocytes and the evaluation of new serologic tests of granulocyte crossmatching. In conclusion, indium-1 11 has proven to be a very helpful cellular label with potential use for
investigation of a n u m b e r of problems relevant to h e m a t o l o g i c m a l i g n a n c i e s . T h e r e a r e also n u m e r o u s questions p e r t a i n i n g to solid t u m o r s that have yet to be investigated. T h e potential for studying and visualizing metastasis r e m a i n s a plausible direction for the use of indium-11 I labeled cells. The possibility of using labeled lymphocytes to evaluate t u m o r - b e a r i n g l y m p h nodes needs to be further investigated. Clinically, the m i g r a t i o n and h a b i t a t of malign a n t hematologic cells has only b e g u n to be investigated. T h e n a t u r e of the behavior of the spleen in both lymphoid a n d myeloid leukemias continues to be a n i m p o r t a n t theoretical b u t also clinical question. Perhaps studies of leukemic cell m i g r a t i o n will provide some clues. F i n a l l y , i n d i u m - l 1 l - l a b e l e d cells are of proven benefit in evaluating histocompatibility problems in g r a n u l o c y t e transfusion. This technique will c o n t i n u e to be utilized for f u r t h e r investigations of g r a n u l o c y t e migration.
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