Immunoscintigraphic imaging of inflammatory lesions: Preliminary findings and future possibilities

Immunoseintigraphic Imaging of Inflammatory Lesions: Preliminary Findings and Future Possibilities M a t h e w L. Thakur

"'Remembrance is the sweet flower Of all this World is perfuming, For love doth guard it, Sun or Shower, And Friendship keeps it blooming"

Clifton Bingham

The long and special friendship I have enjoyed with the late Drs Lakshman Rao, PhD, and Shanta Chervu, AID, has offered many cherished memories that will remain with me for years to come. Mixed leukocytes separated in vitro from patients' blood and labeled with lipophilic chelates of 1111nand ~mTc are now used clinically for imaging inflammat o r y foci in many medical centers worldwide. Recent immunological advances have prompted investigations about using radiolabeled antibodies potentially specific for human neutrophils, labeling of these blood cells in vivo, and imaging inflammatory processes in patients. Early results are encouraging,

allowing abscesses to be imaged w i t h high specificity

N THE PAST FEW YEARS, we have wit-

mation is normally associated with local heat, redness, swelling, and pain. The heat and redness result from dilation of the microvasculation; the swelling is largely produced by the accumulation of fluid, plasma protein, and blood cells in the injured area. The edema causes tissue tension resulting in pain. When inflammation is associated with bacteremia, it induces fever. Fever is caused by endogenous pyrogens. Pyrogens are low molecular ( _+15,000 d) weight proteins. Pyrogens exist in an inactive form in monocytes, macrophages, and possibly in eosinophils. When these cells are activated by bacterial endotoxin, viruses, immune complexes, and pyrogens are released into the bloodstream. They then act on the thermoregulatory centers in the hypothalamus and lead to the elevation of body temperature. In this situation, chills and shaking can also occur. More than a century ago, a well-known Russian biologist, Metchnikoff, concluded that the purpose of inflammation was to stimulate migration of phagocytic cells to the injured area in order to facilitate the opsonization of invading bacteria. Thus, without inflammation bacterial infection may go unchecked. Inflammation is generally divided in two forms: acute and chronic. Acute inflammation is relatively short in duration, lasting from a few hours to 3 to 4 days. Chronic inflammation generally lasts much longer. The cell type present in inflammation varies according to the age of the

extraordinary advances in immunolIogy.nessed Antibodies have been prepared against various biomedically interesting antigens, including certain glycoproteins on blood cell surfaces. This has prompted investigators to evaluate the use of radiolabeled antibodies for scintigraphic imaging of numerous disorders where radiolabeled blood cells play a major role. Since blood cells are currently labeled almost universally in vitro, the goals of investigations into the use of radiolabeled antibodies for in vivo labeling of a specific type of blood cells are laudatory. This review outlines the pathophysiology of human neutrophils, which forms the basis for their use in scintigraphic imaging of inflammatory lesions, and describes the early experience of using radiolabeled antibodies. Also, areas of current weakness are discussed and future approaches outlined for seemingly feasible and promising application of radiolabeled antibodies. PHOTOPHYSIOLOGY

Inflammation may be best defined as a local reaction of vascularized tissue to injury. InflamFrom the Department of Nuclear Medicine, Thomas Jefferson University, Philadelphia, PA. Address reprint requests to Mathew L. Thakur, PhD, Thomas Jefferson University, llth and Walnut Sts, 804 Main Bldg, Philadelphia, PA 19107. 9 1990 by W.B. Saunders Company. 0001/2998/90/2001-0008505.00/0 92

and presenting only minimal (10% to 20%) adverse immunogenic responses. However, problems remain, including low immunospecificity and lengthy procedures for radiolabeling of antibiodies. These areas are currently subjects to further research that in t i m e will contribute to make this modality convenient, simple, and efficacious. 9 1990 by W.B. Saundars Company.

Seminars in Nuclear Medicine, Vol XX, No 1 (January), 1990: pp 92-98

IMMUNOSCINTIGRAPHY OF INFLAMMATORY LESIONS

inflammation. In most types of acute inflammation, infiltration of neutrophils within the first 6 to 24 hours predominates. However, in infection produced by certain organisms, neutrophils may predominate for 2 to 4 days. 1 Neutrophils are said to contain chemotactic factors for monocytes, which begin to appear 24 to 48 hours later. In viral infections, monocytes may be the first cells to arrive. Chronic inflammation mostly consists of mononuclear cells, primarily macrophages, lymphocytes, and plasma cells. In the lesions of osteomyelitis, neutrophils can persist for many months, but mononuclear cells may be predominant. Leukocytosis is a common feature of inflammatory response, especially when induced by bacterial infection. Leukocytosis occurs because of accelerated release of cells from bone marrow. Neutrophils are known to migrate by chemotaxis at a rate of 6 to 10 ~tm/min in the inflamed area. Chemotaxis is defined as the unidirectional migration of white cells toward a chemoattractant. Soluble bacterial factors act as chemoattractants and lead neutrophils to the inflamed area, forming an abscess caused by suppuration, which is confined to tissues or organs. IMAGING ABSCESSES WITH RADIOACTIVE NEUTROPHILS

Because of their relative abundance in circulating blood and their ability to migrate into inflammatory lesions in response to a variety of chemoattractants, polymorphonuclear leucocytes (PMNs) lend themselves as an obvious vehicle to transport to the infected area a radionuclide that may be associated with them. Among the gammaemitting radionuclides, 99mTc, lllln, 197Ru,2~ 123I,67Ga, and 52Fe lead the list. The survey of various compounds, prepared with some of the above radionuclides, led to the development of now commonly used radiopharmaceutical mln-oxine.2 This lipid-soluble compound passively diffuses through the cell membrane and labels blood cells, such as PMNs, 3'4 lymphocytes, s platelets, 6 and even live bacteria. 7 Once within the cell core, the radioactive tracer binds to cytoplasmic components 8'9 and becomes a tracer that allows investigators to monitor cell distribution in vivo. Labeling blood cells with mIn-oxine is now an established technique for routine clinical applications.

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However, the disadvantage of this technique is that the lipid soluble agents are nondiscriminating; that is, they label all blood cells, requiring one to perform the labeling in vitro by using isolated blood cells of the type that is chosen. An intracellular tracer like rain, with its Auger electrons, induces a high radiation dose to the cells. Mononuclear cells, such as lymphocytes, are more susceptible to radiation damage than polynucleated PMNs or non-nucleated platelets and erythrocytes. lalln is also a cyclotron-produced radionuclide; therefore it is expensive and must be ordered 24 to 48 hours before its use. The radiation dose received by the liver and spleen from 0.5 mCi of rain-labeled neutrophils or platelets is not negligible either. The other above mentioned radionuclides are attractive; however, for imaging abscesses, none are more so than 99mTc. 99mTC, as a short-lived and generator-produced radionuclide, eliminates many of the disadvantages of rain and the other radionuclides. The search therefore continues for a 99mTccompound that labels blood cells, preferably selectively and as effectively as In-oxine. Several agents have been evaluated and are discussed by Srivastava in this issue. It is not the purpose of this article to discuss the advantages and disadvantages of those 99mTc agents. However, one common factor among all 99mTCagents is that they do not obviate the need for drawing patients' blood and separating from it the required type of blood cells for in vitro labeling. A compound that chelates either rain or 99mTc, binds only to specific types of blood cells extracellularly, and prevents the radioactive tracer from coming off the cells in circulation would have much to offer in the field of radiolabeled blood cells. The work in this field is still in its infancy and limited. However, it is worthy of a review for the readers of Seminars in Nuclear Medicine. CHEMOTACTIC OLIGOPEPTIDES

Attempts to explore the feasibility of labeling human P M N s selectively with a gamma-emitting radionuclide for the purpose of scintigraphic imaging, began a long time ago 1~with the use of P M N chemotactic factor, N-formyl-methionylleucyl-phenyl-alinne (FMLP). This synthetic oligopeptide was covalently coupled with transfer-

94

MATHEW L. THAKUR

rin with carbodiimide and labeled with nlln. Up to 60% of the added radioactivity was PMNassociated. However, difficulties were encountered in increasing the yield, probably because of the low specific activity of the agent prepared. McAfee et a111 later observed that as little as 0.5 #g FMLP given intravenously to a rabbit produced marked transient leukopenia. However, Fischman et a112recently reported a renewed interest in a PMN chemotactic peptide, N-formyl-nle-leu, PheNle-Tyr-Lys. The amino group of the lysine was conjugated with cyclic anhydride of diethylenetriaminepentaacetic acid (DTPA) and was used as a chelating agent for labeling the peptide with rain. Modifying the peptide with a smaller DTPA molecule technique, not available at the time of the investigations with FLMP, is a better approach to preserve the chemotactic properties of the agent. Approximately 100/~Ci rain labeled to 5 to 10 #g of the peptide was given to rats with experimental infection of left thigh with Escherichia coli. One hour following the administration of the agent, target-to-background (abscess-tomuscle?) ratios of 4 were reported. Surprisingly, however, 2 hours postinjection "the level of radioactivity in abscess had decreased and by 24 hours the lesion was barely discernible. ''12 At 24 hours postadministration of mln-oxine-labeled autologous white blood cells in dogs, abscess-tomuscle ratios of 315 were observed. 3 If the peptide were to be truly bound to the rat neutrophil in vivo, the target-to-background radioactivity ratios should have continued to increase since the labeled cells continue to migrate into the lesion, or remained the same. The fact that the ratios decreased only 2 hours postadministration strongly suggest a mechanism other than the migration of radiolabeled white blood cells to the area of infection. Further work is necessary. NONSPECIFIC POLYCLONALGAMMA GLOBULIN G Before the chemotactic polypeptide publication, Fischman et a113 reported the use of mln. labeled nonspecific polyclonal immunoglobulin (Ig)G to detect lesions of acute inflammation in patients. Milligram quantities of Sandoz (Hanover, PA) gamma globulin were labeled with 1 to 1.5 #Ci rain via the cyclicanhydride of DTPA method and given intravenously to pa-

tients. Sequential imaging performed at 6 to 72 hours postinjection correctly identified 48 of 52 lesions as confirmed either by other diagnostic modalities, such as X ray, computed tomography (CT), and biopsy, or surgery. It seems that most images were positive at 24 hours postinjection. Although no quantification of radioactivity distributed in various organs is reported, the 48hour images showed intense blood pool. This is unparallel to rain-labeled white cell scans. Furthermore, the liver uptake appeared to be much higher, and spleen and bone marrow uptake lower than the normal 111Inleukocyte scans. This distribution suggests that a major proportion of radioactivity may not have been bound to neutrophils in vivo. However, speculations are that the IgG uptake may have resulted from the infiltration of the protein into the interstitial space in the lesion. The interstitial space in the inflamed lesion would normally be much larger than in the normal healthy tissue; also, it would provide the radioactivity gradient for images of diagnostic quality. Analysis of plasma and cellular components separated from patients' blood at various times after administering the compound may provide better insight into the possible mechanism of the radionuclide uptake. NEUTROPHIL-SPECIFIC MONOCLONAL ANTIBODIES

12~1-Labeled CEA-47 The recent advances in hybridoma technology have provided an opportunity to produce antibodies specific for human neutrophils. These agents offer the possibility of labeling circulating PMNs selectively in vivo. The first such application in patients was reported by Locher et al. 14'15 The antibody, anticarcinoma embryonic antigen (CEA), CEA-47 (IgG) was labeled with 1231.16 The in vivo dehalogenation of radioactive iodine is a well-observed phenomenon. In order to prevent the thyroid uptake of radiohalogen, thyroids were blocked by giving the patients 1 mL Lugol's solution per day for 3 postinjection days. Four mg of antihistamine were also given orally to each patient against any possible immunologic responses from the infusion of 120 ug radiolabeled antibody, which was diluted to 20 mL in isotonic saline, containing 1 mg of human serum albumin (HSA) as a carrier. Planar and single photon emission computed tomography (SPECT)

IMMUNOSCINTIGRAPHY OF INFLAMMATORY LESIONS

images were obtained 3 to 24 hours later, and results were compared with those of the llllnoxine white cell studies performed on the same patient within one week. Highly encouraging results have been reported. An example of a patient with Crohn's disease is given in Fig 1. Generally, images presented high bone marrow radioactivity uptake, occasionally obscuring the lesion of osteomyelitis. The granulocyte-bound radioactivity was determined to be between 8% and 17%.]7 These values are low and if true, will raise questions about the validity of results and the mechanism by which radioactivity is taken up in inflammatory lesions.

Fig 1. Anterior gamma camera images of the pelvic area of a 32-year-old man with Crohn's disease. Radiographic examinations had suggested an inflamed bowel loop, The upper image was obtained using 111In-labeled leukocytes and the lower image, 1 week earlier, using lz31-1abeled CEA-47. Both procedures detected the lesion shown by an arrow. Note relatively higher bone marrow uptake of the lz31-CEA-47 than that of ~lln-leukocytes. (Courtesy of Dr J.T.H. Locher, Switzerland. TM)

95

99mTc-Labeled B W 250/183

This agent was developed by Behringwerke AG (Marburg, FRG). The antibody has the specificity for nonspecific cross-reacting antigen (NCA-95). The NCA-95 antigens exist on human granulocyte surface and allow the antibody to interact with "about 95% of all granulocytes.''~s Joseph et al ~9 labeled 100 to 400 ~tg of the monoclonal antibodies with 8 mCi 99mTc using the Schwarz method.2~ The Schwarz method calls for the reduction of antibody disulfide bonds to sulfhydryl groups to which binds reduced 99mTc. The reduction of 99mTc04 was achieved via the use of SnC1z present in commercially available kits, such as methylene diphosphonate (MDP). The reduced 99mTc then binds to antibody. Since the reduction of the antibody disulfide groups can be achieved before 99roTe-labeling and the reduced antibody can be stored dry, this method allows one to label an antibody with 99mTc by a kit procedure. If the labeling is inefficient, ie, the percentage of 99mTc associated with antibody is not greater than 90%, a purification procedure may be necessary, which may make the kit less attractive. Forty-five patients, suspected on clinical grounds of having abscesses or other inflammatory foci, were studied. The patients were not given a skin test nor any antihistamine drugs. Five patients were reexamined within 10 days of the immunoscintigraphic procedure using 99mTc hexamethylpropyleneamineoxime (HMPAO)labeled autologous leukocytes. Generally, results in imaging inflammatory processes were encouraging, delineating the abscesses at 2 hours postinjection. Similarly to the CEA-47, the bone marrow uptake was very high and the liver and spleen uptakes significant. The lung uptake was also very high, amounting to 53% to 66% at 30 to 45 minutes postinjection. Unlike the 99mTc HMPAO-labeled leukocytes, there was no elimination of radioactivity in the intestine, perhaps indicating the stability of the tracer in vivo. Only two patients developed HAMA (human antibody against mouse antibody). At 6 hours postinjection, -25% of the circulating radioactivity was bound to granulocytes. This number is better than that of the CEA-47; it is encouraging, but not high enough to be consistent with an antibody that is highly specific for the blood cells.

MATHEW L. THAKUR

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Table 1. In Vitro Evaluation of Monoclonal Antibodies Specific for Human Neutrophils Separated Calls (%)

Whole Blood (%)

MAB

Class

PMN

Plasma

No.

PMN

RBC

Plasma

No.

Anti-SSEA-1

IgM

8 0 -+ 5

20 • 5

20

27 _+ 5

2 8 -+ 3

4 5 +- 2

12

B.37.2.1

IgM

72 • 6

2 8 -+ 7

6

3 8 _+ 9

25 • 4

37 + 5

6

MCA87

IgG2a

37 • 5

6 3 -+ 3

3

2 5 _+ 3

15 -+ 4

60-+ 4

3

MCA 149

IgGla

15 -+ 6

85 • 3

3

14 • 3

16 _+ 4

7 0 +_ 3

3

MCA 167

IgG2a

41 • 4

5 9 -+ 4

3

41 _+ 4

14 _+ 2

4 5 _+ 4

3

MCA 2 1 5

IgM

17 • 6

83 • 3

3

14 • 4

10 -+ 3

76 + 3

3

FMC11

IgG1

3 9 -+ 5

61 -+ 5

3

--

--

--

B.6.2

IgG

9 • 5

91 +_ 8

4

--

--

--

MCA 161

IgG1

13 • 4

87 -+ 7

3

11 _+ 2

13 _+ 4

76 + 4

3

NOTE. Antibodies were coupled with c-DTPA and labeled with 1111n.

Abbreviations: RBC, red blood cell. Data from Thakur et al. 21 l l l l n - o r 99m T c - L a b e l e d

MCA-480

We have evaluated nine monoclonal antibodies, all specific for human neutrophils (Table 1), as possible agents for imaging inflammatory foci. 21 All antibodies were labeled either with lllin or 99mTcvia the cyclical anhydride of DTPA technique originally developed by Eckelman et a122 and Hnatowich et al. 23 A submicrogram, but an accurately known quantity of the labeled product was then incubated with a known number of freshly prepared human neutrophils; next, quantity associated with the cells was determined. As seen in Table 2, antibodies B37.2.1 and MCA-40 (antistage-specific embryonic antigen- 1 or anti-SSEA- 1) gave the best results. Out of the two, MCA-480, 24'25 was further studied to determine its specificity to human neutrophils and used to estimate the number of antibodyspecific antigens, lacto-N-fucopentoase, on human neutrophil surface. Table 2 shows these characteristics of MCA-480 and compares them with those of anti-CEA-47 and BW 250/183. The specifications favor the use of MCA-480, and we are hopeful for equal or better results. Unfortunately, the antibody MCA-480 has no specificity for neutrophils of commonly used

animal species, such as the rat, rabbit, mouse, cat, sheep, pig, or guinea pig. This quality of the antibody has prevented us from performing animal research that could possibly have answered many of our questions. We await the use of the antibody in patients. MODIFIED ANTIBODY-LABELING TECHNIQUES

Ease in preparing radioactive agents promotes their growth in clinical applications. The use of 99mTc in diagnostic imaging is ubiquitous. Imaging abscesses with radiolabeled monoclonal antibodies should be no exception. To date, only two groups 2~ have attempted to develop a 99mTc antibody labeling method that can be adapted to a kit procedure. Both methods are aimed at labeling antibodies with 99roTe directly on the protein molecule without the need of a bifunctional chelating agent. Each IgG antibody molecule contains -70 cysteine residues, producing a maximum of ~35 disulfide groups. Under suitable conditions, these disulfide groups could be reduced to sulfhydryl groups that could bind reduced 99roTedirectly. If the conditions are right, a quantitative radiochemical yield is feasible, making the labeling proce-

Table 2. Some Characteristics of Neutrophil-Specific Anti-CEA-47, Anti-SSEA-1 ( M C A - 4 8 0 ) , and B W 2 . 6 0 / 1 8 3 Anti-CEA-47

Anti-SSEA- 1

BW 250/183

Antibody

(Locher et alTM)

(Thakur at a121)

(Joseph et al lg)

PMN specificity

IgG 6.8 • 10 -9 M

IgM 1.6 x 10 -11 M

IgG NG

GP-NCA-95 7,1 x 104 I-123

Lacto-N-fucopentoase 5.1 x 102 99mTc/111in

GP-NCA-95 NG ~mTc

Isotope PMN antigen

Antigen Number/PMN Tracer P M N LE In vitro (%)

40

60

NG

In vivo (%)

8-17

ND

24-26

Abbreviations: LE, labeling efficiency; NG, not given; ND, not done.

IMMUNOSCINTIGRAPHY OF INFLAMMATORY LESIONS

97

d u r e simple and convenient for r e a d y use in patients. In the first method, R h o d e s et a126 used SnC12 to r e d u c e the disulfide groups; in the second method, S c h w a r z et al 2~ used 2 - m e r c a p t o e t h a n o l ( 2 - M E ) as a reducing agent. W e had an opportunity to investigate two other reducing agents, dithiotrietol ( D T T ) a n d d i t h i o e r y t h r i t o l ( D T E ) , and to c o m p a r e t h e m with the SnC12 and 2 - M E . T h e details of these investigations a r e published elsewhere, 27 but T a b l e 3 s u m m a r i z e s our results and depicts that all four agents c o m p a r e favorably. However, in our use, none of t h e m reliably e l i m i n a t e d the purification step a n d fell short of a true kit p r o c e d u r e t h a t can be conveniently p e r f o r m e d in a n y nonresearch, routine clinical r a d i o p h a r m a c y . F u r t h e r investigation continues in our l a b o r a t o r y . FUTURE DIRECTIONS Using radiolabeled antibodies specific for blood

cells t h a t will label a given type of blood cell in vivo will m a k e the technique a t t r a c t i v e a n d simple enough, thus a c c e l e r a t i n g the n u m b e r of clinical procedures p e r f o r m e d . It is exciting t h a t in the relatively early d a y s of diagnostic applications of radiolabeled monoclonal antibodies, labeling blood cells for i m a g i n g i n f l a m m a t o r y lesions in patients has progressed and given rise to highly e n c o u r a g i n g results. T h e s e results have established a useful place for r a d i o l a b e l e d antibodies in the field of i m a g i n g i n f l a m m a t o r y foci. However, i m p r o v e m e n t is necessary in two

Table 3. Evaluation of Agents for Direct Labeling of Antibodies with ssmTc % 99mTcAssociation

Agent

Antibody

PMNs

c-DTPA (control)

60 65 55

64.5 66 56

44

63.5

55 58

62 62

SnCI 2

SnCI2 + GA 2-ME DTE

D'IT

NOTE. The numbers are the averages of 3 separate experiments in duplicate. Human neutrophil specific antibody MCA-480 was used. Abbreviation: GA, gentensic acid. Data from Thakur et a l Y

areas. First, we need to produce antibodies with higher cell specificity. Second, b e t t e r and more efficient techniques a r e required for labeling the antibodies with 99mTc. W h i l e the search proceeds in both areas, the use of a nonspecific, lUlnlabeled, polyclonal I g G continues to be successful. A l t h o u g h the m e c h a n i s m by which this agent localizes in i n f l a m m a t o r y processes is c u r r e n t l y subject to speculation and further studies are required, including the use of r a d i o l a b e l e d proteins of different m o l e c u l a r weights, the applications of these agents have c r e a t e d e x c i t e m e n t by allowing one to i m a g e abscesses without having to label leukocytes in vitro. ACKNOWLEDGMENT

The helpful comments of C.H. Park, MD, proofreading of the manuscript by J. DeFulvio, and typing of the manuscript by Renee King are gratefully appreciated.

REFERENCES

1. Robbins SL, Cotran RS: Pathophysiologic Basis of Disease, (ed 2) Philadelphia, PA, Saunders, 1979, p 66 2. McAfee JG, Thakur ML: Survey of radioactive agents for in vitro labeling of phagocytic leukocytes. I. Soluble agents. J Nucl Med 17:480-487, 1976 3. Thakur ML, Coleman RE, Welch MJ: Indium-illlabeled leukocytes for the localization of abscesses: Preparation, analysis, tissue distribution, and comparison with gallium-67 citrate in dogs. J Lab Clin Med 89:217-228, 1977 4. Thakur ML, Lavender JP, Arnot RN, et al: Indium- 11 ilabeled autologous leukocytes in man. J Nucl Med 18:10141021, 1977 5. 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 6. Thakur ML, Welch M J, Joist JH, et al: Indium-11 ilabeled platelets: Studies on Preparation and Evaluation of in vitro and in vivo functions. Thromb Res 9:345-357, 1976 7. Thakur ML: Live bacteria labeled with In-ll 1. Eur J Nucl Med 13:266, 1987

8. Thakur ML, Segal AW, Louis L, et al: Indium-Illlabeled cellular blood components: Mechanism of labeling and intracellular location in human neutrophils. J Nucl Med 18:1022-1026, 1977 9. Thakur ML: Radioisotopic labeling of platelets. A historical perspective. Semin Thromb Hemost 9:79-85, 1983 10. Zoghbi SS, Thakur ML, Gottschalk A: Unpublished data, 1979. 11. McAfee JG, Subramanian G, Gagne G: Techniques of leukocyte harvesting and labeling: Problems and perspectives. Semin Nucl Med XIV:83-106, 1984 12. Fischman AJ, Rubin RH, Fuccello AJ, et al: Rapid detection of focal sites of infection with chemotactic peptide labeled with In-111. J Nucl Med 30:805, 1989 (abstr) 13. Fischman A J, Rubin RH, Khaw BA, et al: Detection of acute inflammation with In- 11I-labeled nonspecific polyclonal IgG. Semin Nucl Med XVIII:335-344, 1988 14. Locher JTH, Seybold K, Anders RY, et al: Imaging of inflammatory and infectious lesions after injection of radiolabeled monoclonal anti-granulocytes antibodies. Nucl Med Commun 7:659-670, 1986

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15. Seybold K, Locher JTH, Coosemans C, et al: Immunoscintigraphic localization of inflammatory lesions: Clinical Experience. Eur J Nucl Med 13:587-593, 1988 16. Schubiger PA, Hasler PH, Novak-Hofer I, et al: Assessment of the binding properties of Granuloszint, in Schubiger PA, Hassler PH (eds): 1-123 Granuloszint: Immunoscintigraphic Localization of Inflammatory Lesions. Paul Scherrer Institute, Switzerland, 1988 17. Hassler PH, Seybold K, Andres RY: Immunoscintigraphic localization inflammatory lesions: Pharmacokinetics and estimated absorbed radiation dose in man. Eur J Nucl Med 13:594-597, 1988 18. Bosslet K, Luben G, Schwarz A, et al: Immunohistochemical localization and molecular characteristics of three monoclonal antibody-defined epitopes detectable on Carcinoembryonic antigen (CEA). Int J Cancer 36:75-84, 1985 19. Joseph K, Hoffken H, Bosslet K, et al: In vivo labeling of granulocytes with Tc-99m anti-CEA monoclonal antibodies for imaging inflammation. Eur J Nucl Med 14:367-373, 1988 20. Schwarz A, Steinstraesser A: A novel approach to Tc-99m-labeled monoclonal antibodies. J Nucl Med 28:721, 1987

MATHEW L. THAKUR

21. Thakur ML, Richard MD, White FW III: Monoclonal antibodies as agents for selective radiolabeling of human neutrophils. J Nucl Med 29:1817-1825, 1988 22. Eckelman WC, Karesh SM, Reba RC: New compounds: Fatty acid and long chain hydrocarbon derivatives containing a strong chelating agent. J Pharm Sci 64:704-706, 1975 23. Hnatowich D J, Layne WW, Childs RL: The preparation and labeling of DTPA coupled albumin. Int J Appl Rad Isot 33:327-332, 1982 24. Solter D, Knowels BV: Monoclonal antibodies defining a stage-specific mouse in ionic antigen (SSEA-1 ). Proc Natl Acad Sci USA 75:5565-5569, 1978 25. Ballou B, Jaffe R, Taylor R J, et al: Tumor radioimmunolocation: Differential antibody retention by antigenic normal tissue and tumor. J Immunol 132:2111-2116, 1984 26. Rhodes BA, Zamora PO, Newell KD, et al: Technetium-99m-labeling of murine monoclonal antibody fragments. J Nucl Med 27:685-693, 1986 27. Thakur ML, Richard MD, DeFulvio J, et al: Technetium-99m labeled monoclonal antibodies: Evaluation of"new" reducing agents. J Nucl Med (submitted) (in preparation)