Immunoscintigraphy of Inflammatory Processes with a Technetium-99m-Labeled Monoclonal Antigranulocyte

(MAb BW 250/183)

Peter Lind, Werner Langsteger, Peter KOltringer, Hans Peter Dimai, Rainer Passi, Otto Eber

Internal Department, Barmherzige BrüderEggenberg Hospital and Department ofAccident Surgery, A UKH, Graz, Austria

95 (nonspecific cross-reactingantigen) on the surfaceof Antigranulocyte immunoscintigraphy with a technetium neutrophil granulocytes (7). In spite of its strong bind 99m- (@“Tc)labeled monoclonal antigranulocyte antibody ing to the granulocyte membrane, no antibody- or (MAb BW 250/i 83) was performed in 34 in-patients of the complement-dependent lysis ofthe cells occurs (8). The departments of accident surgery and internal medicine in vitality ofgranulocytes is not inhibited by the antibody. order to prove or exclude inflammatory processes. After In contrast to labeling of leukocytes with 99mTc@HM@ labelingwith @“Tc,555 MBq, @“Tc-MAb(0.5mg antibody) PAO, diffusion of free activity into the intestines is were slowly injected intravenously over a period of 5 mm. prevented due to the high stability of 99mTcMAb bind A whole-bodyscan was done 4—6hr postinjection,and planar or SPECT images were performed 6, i 8, and 24 hr ing and the high affinity of MAb to the epitope. postinjection. Leukocyte immunoscmntigraphyproved in The purpose of this study, which included patients flammatory suppurating processes in 20 cases (true-posi hospitalized at the departments of accident surgery and tive)and excludedthem in 11 cases(true-negative).The internal medicine, was to investigate the clinical value findings were false-positive in two patients (hematoma ofantigranulocyte immunoscintigraphy with MAb BW without signs of infection, pseudoarthrosis) and false-nag 250/ 183. ative in one patient(encapsulated lung abscess with pleural fibrosis). Anti-idiotypic human anti-mouse (HAMA)werefoundonlyinoneout of 20 patients.Accord PATIENTS AND METHODS ing to our experiences, immunoscmntigraphywith @Tc Antigranulocyte immunoscintigraphy was performed in a MAbBW250/183 hasa sensitivityof 95%,andis, there total of 34 patients. Twenty-three patients of the department fore, well suited for the identification of leukocytic inflam of accident surgery were investigated in order to determine mations. whether inflammatory soft-tissue processes (abscesses, infected hematomas) or osteomyelitis were present; the method was J NucI Med 1990; 31:417—423 indicated in 11 patients for clarification of inflammatory, suppurating processes of inner organs (lung abscess, colitis ulcerosa, pyonephrosis) or leukocytosis of unknown origin. The antibody (1 mg) was labeled with 1, 100 MBq @mTc ith the introduction of technetium-99m- (99mTc) according to the method developed by Schwarz et al. (9). labeled antigranulocyte antibodies, leukocyte scintigra Five to ten minutes after labeling, 555 MBq 99mTc@MAb phy has become a method that can be performed at any BW250/183 (0.5 mg antibody)wereslowlyinjectedintrave time in any laboratory. Compared to nously. Antihistaminic or corticoid drugs were not adminis tered prior to injection of the monoclonal antibody; the in the time-consuming and elaborate in vitro labeling with dwelling cannula was left in place until the first examination indium-i 11- (“In) oxine or 99mTc-HM-PAO, in vivo 4—6hr postinjection. labeling of leukocytes permits easy and quick visuali The scintigram was made with an Elscint Apex 409 AG zation of inflammatory, suppurating processes (1—6). (Haifa, Israel)rotating gamma camera with whole-bodyop. The monoclonal antibody (MAb BW 250/183, Behr tion. At first, a whole-body scan was performed 5 hr postin ing Werke Marburg, FRG) is an intact murine mono jection followed by SPECT (single-photon emission computed clonal antibody, which reacts with an epitope of NCA ) or planar scans of the regions of interest 6, 18, and 24 hr postinjection. In the meantime, SPECT or planar ReceivedAug.2, 1989;revisionaccepted Nov.16, 1989. images of the pathological region 18 hr postinjection have Forreprintscontact:PeterLind,MD,InternalDepartment,Barmherzige proved to be sufficient, especially for the clarification of spe BrUderEggenbergHospital,Bergstrasse27,8020Graz,Austria. cific postoperative events. The SPECT images were taken in

Technetium-LabeledMAbBW250/i 83 •Lindat al 417 a 64 x 64 matrix in steps of 6°by continuous 360-degree rotation. The 60 planar projections were reconstructed to

@ transverse slices with the use of a Hanning filter. Using these .@. . transverse slices, coronal and sagittal slices were interpolated. The imageswereinterpretedby two independentobservers. HumanAnti-MouseAntibodies In 20 patients human anti-mouse antibodies (HAMAs) weredetermined2—4mo afteranti-granulocyteimmunoscin .4 tigraphy(EnzygnostHAMAmicro,BehringWerkeAG, Mar I burg, FRG). The sera were tested against the specific antibody used for immunoscintigraphy (anti-idiotypic and anti-isotypic response) as well as against unspecific antibodies (anti-isotypic response).Aswedid not measureHAMAsbeforethe admin istration of MAb BW 250/183, HAMA response was rated positive if the HAMA factor in the patient serum was twice as high as in the control serum.

RESULTS Soft-tissue abscesses had been suspected in 13 of the patients referred to us following surgical intervention after accidents and osteomyelitis in 10 of them. !m munoscintigraphy proved suspected inflammatory soft tissue processes and localized the foci of inflammation, FIGURE 1 which were confirmed by second-look operations in 10 A 70-yr-old female patient after left collum femons fracture; clearuptakeof @“Tc-labeledantigranulocyteantibodiesin the cases (Table 1, Figs. 1 and 2). A negative immunoscin soft tissuearoundthe left proximalshaft of the femur(anterior tigram excluded an inflammatory process in two cases projection);second-lookoperation: infected hematoma;bac (after total prosthetic replacement of the hip). In one terium:Staph.aureus. patient with slight positive immunoscintigram, merely hematoma fluid was evacuated after surgical interven tion; neither the macroscopic finding nor microbial• determination furnished evidence of an infection. In cases of suspected osteomyelitis, positive immu noscintigraphic findings were confirmed by surgery in four patients; the immunoscintigram was true-negative in five cases and false-positive in one patient with

TABLE I Suspected Soft-Tissue Abscesses Patientno. IS SO BSR LEUKO MIC LOC 1000+knee2+++11+++130/1401 08/13312300+thigh3——42/7089000hip4++++91/1288300+thigh5+++77/1247100—hip6+++94/1208300—knee7(+)+5/1

56600—knee8+++93/1 219300+tibia9——20/4763000hip1

077900—thigh10+++80/1 1++++108/1306400—thigh12+070/9892000tarsus1

3+058/9276300tibia FIGURE 2 Antigranulocyte immunoscintigraphy (IS), sonography (SO), A 60-yr-oldfemalepatient after open supra- and diacondylar fracture of the left femur; clear uptake of 99―Tc-labeledanti blood sedimentation rate (BSR), leukocytes (LEUKO),microbial granulocyte antibodies in the soft tissue around the left knee determination (MIC), and localization (LOC) of postoperative in joint (anteriorprojection);second-lookoperation;infectedhe flammatorysuppuratingsoft-tissueprocesses. matoma;bacterium:Vibriofiuvialis.

418 The Journalof NuclearMedicine•Vol. 31 •No. 4 •April1990 pseudoarthrosis (Table 2 and Fig. 3). In the search for inflammatory abscesses of inner organs or of inflam mations associated with internal diseases, the immu noscintigram was true-positive in eight cases, true-neg ative in two cases, and false-negative in one case of lung abscess with pleural fibrosis (Table 3, Figs. 4A—B,5A— B, 6A—C).The investigation of 34 patients produced 20 true-positive findings, 11true-negative, 2 false-positive, and 1 false-negative. Thus, the sensitivity of antigran ulocyte immunoscintigraphy with 99mTc@MAbBW 250/ 183 was 95% in the patients studied by us; its specificity was 85%. Side effects, such as allergic reactions, were not observed in any of our patients.

HAMAResults In 20 patients, the antibody response to the first administration of the murine monoclonal antibody (MAb BW 250/183) was measured 2—4mo after in vivo application. Only one patient developed HAMAs (IgG type), which were able to bind to MAb BW 250/ 183 at its variable region (anti-idiotypic antibodies). In two patients, the response was anti-isotypic (IgG and/ FIGURE3 or 1gM). However, in all three patients the calculated A 26-yr-oldmalepatientafterrighttibiafracture;suspected HAMA factor (patient serum —background/negative osteomyelitis;circumscribeduptakeof@rc-labeIed antigran serum —background) was very low (Fig. 7). ulocyteantibodiesintheareaof the rightdistaltibia(anterior projection—left upper image; right lateral projection—right upperimage;posteriorprojection—leftlower image);second DISCUSSION lookoperation:removalof theosteomyeliticfocus; no bacteria detected. Visualization of inflammatory suppurating processes via nuclear medicine techniques has become an ac knowledged method since the introduction of leukocyte In 1984 Buchegger and Mach defined a monoclonal with ‘‘‘In-oxine(10—13). Since in vitro antibody, which is directed against the nonspecific labeling is rather elaborate and cells are sometimes cross-reacting antigen (NCA 95), an epitope of the damaged during leukocytes separation, possibilities of carcinoembryonic antigen (CEA). This antigen is also in vivo labeling have been investigated intensively in present on the surface of neutrophil granulocytes (15). the past few years (14). In vivo experiments and pharmacokinetic data have

TABLE 2 TABLE 3 SuspectedOsteomyelitis SuspectedAbscessesof InnerOrgans Patientno.ISX-RAYBSRLEUKOLOCSURG1—+1 Patientno. IS Diagnosis BSR LEUKO 1/351 3900tibia0 abscess121/126125002+++Colitis1++Lung 2+++87/1237800tibia+3++(+)46/817800tibia—4+++50/9318/13164003++Colitis ulcerosa1 1 ulcerosa88/107111004+Pylonephrosis93/123164005+++Infarction 2300tibia+5++++60/919300tibia+6—(+)49/975700hip07——8/214600tibia08+++57/815600tibia+9——107/1204900thigh010—(+)70/1087800tibia0 12/12650006—Lungpneumonia1 abscess146/15085007—Suspected abscess54/102185008—Suspected discitis43/7581009+Appendicitis120/14212700I

gangrene70/102930010++Diabetes 1++Diabetes gangrene69/9810400 Antigranulocyteimmunoscintigraphy(IS), x-ray findings(X RAY),bloodsedimentationrate (BSR),leukocytes(LEUKO),10- Antigranukcyteimmunosantigraphy(IS)and bloodsedimen calization(LOC),andresultsof second-lookoperation(SURG)for tationrate(BSR),leukocytes(LEUKO)forsuspectedinflammatory questionableosteomyeliticevents. suppurating processes of inner organs.

Technetium-Labeled MAb BW 250/i 83 •Lind et al 419 A “@ The 99mTclabeled MAb BW 250/ 1 83 defined by Bosslet is also directed against NCA 95, an epitope of CEA and does not lead to antibody-dependent or com plement-dependent lysis ofgranulocytes (8). Schorlem mer showed that MAb BW 250/183 does not inhibit cell-specific functions such as endocytosis, lysosomal enzyme secretion, and superoxide anion production

.@“ (19). Bosslet et al. could demonstrate that no changes in physiologic granulocyte function, such as lysosomal enzyme secretion or oxidative bursts, occur (20). The specificity on human tissue was reported by Bosslet et al. (7). MAb BW 250/183 binds to the membrane of neutrophil granulocytes by more than 90%. When this 99mTclabeled monoclonal antibody is used, radiation exposure amounts to only 15% of the dose of67Ga or ‘‘‘In.Other than with 99mTc@HM@PAO, no radioactivity passes into the intestines so that inflam matory processes such as colitis ulcerosa or Crohn's disease can be visualized with @mTc@MAbBW 250/183 without the uncertainty of a false-positive unspecific uptake. Kroiss et al. have demonstrated in patients with inflammatory bowel disease that the sensitivity of leu kocyte immunoscintigraphy with 99mTc@labeledmono clonal antibodies to be 77% for planar imaging and 91% for SPECT (21). HAMA response to murine monoclonal antibodies depends on the route of application and the amount and type ofthe antibody (22). In our investigations (0.5 mg antibody), only one out of twenty patients showed .@‘& an anti-idiotypic, two an anti-isotypic response. How ever, no side effects occurred in our patients. Second administration of the antibody in two cases with ulcer ative colitis neither lead to a diminished image quality FIGURE 4 nor to allergic reactions. (A)An 80-yr-oldmalepatientwith suspectedinfarctionpneu Apart from its merits in the search for abscesses of moniaon the right side;circumscribeduptakeof @“Tc-Iabeled antigranulocyte antibodies in the right lower area of the lung. inner organs, leukocyte immunoscintigraphy can be (Whole-body scan 5.5 hr p.i. in posterior and anterior projec very helpful in the decision for or against second-look tion). (B) SPECT 6 hr postinjection (coronal slices) shows operations ofpatients with inflammatory soft-tissue and massiveantibodyuptake in the right lower pulmonaryarea. bone processes following orthopedic surgery. However, it also must be borne in mind that all noninflammatory processes accompanied by leukocytosis, such as hema shown that the function of granulocytes is not influ tomas or peripheral bone marrow expansion (e.g. pseu enced by in vivo labeling with monoclonal antibodies doarthrosis) will produce positive results. The sensitivity (16,17). ofleukocyte immunoscintigraphy was very high in our First clinical results with iodine- 123-labeled mono study (95%). On the other hand, precisely these leuko clonal antibodies (‘23I-MAb47)were reported by Locher cytes not associated with inflammatory events produced in 1986 and Seybold in 1988 (14,18). They confirmed false-positive results in the clarification of inflammatory the high sensitivity of leukocyte immunoscintigraphy suppurating processes, thus reducing the specificity to and its clinical relevance. However, the disadvantages 85%. Therefore, a differentiation between infected and of immunoscintigraphy with 1231are the high costs of noninfected postoperative hematomas by leukocyte 123j and the fact that the is not always scintigraphy does not seem to be possible. Yet this available in routine nuclear medicine laboratories. In qualification applies to the visualization of leukocytes 1987, Schwarz et al. succeeded in achieving stable label by nuclear medicine techniques in general and is not ing of monoclonal antibodies with 99mTc; thus, the limited to leukocyte immunoscintigraphy. Perhaps a utilization of leukocyte immunoscintigraphy has be differentiation between infected and noninfected he come possible in routine nuclear medicine (9). matoma is possible by a recently described new method

420 TheJournalof NuclearMedicine•Vol.31 •No.4 •April1990 A B@ ,iI@

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FIGURE 5 (A)A 31-yr-oldmalepatientwith hyperacuteulcerativecolitis.The whole-bodyscanshows a positiveuptakein the transverse, descending,andpelviccolon.(B)SPECTimaging6 hr postinjection(coronalslices)circumscnbedantibodyuptakeintransverse, descending,and pelviccolon.

@ UA@' SCAN 13 L E PC 217 F ME 38.0/ ST 15.1 RM 1.' AR 3.@

PC 2 ME 26 ST 11@ RM 0@ AR 3.@

LI 175 Co 70 DI 2.0 C • FIGURE 6 (A) A 75-yr-oldfemalepatientwith suspectedrightpulmonaryabscess.SPECT 6 hr postinjection(upperrow) and 24 hr postinjection(lowerrow):circumscribed,primarilymarginalantibodyuptake inthe rightlowerdorsal area. (B)SPECTimaging 24 hr postinjection (transaxial slice) demonstrates uptake of the @“Tc-labeledMAb in the area of the pulmonary abscess. (C) TCT(transaxialslice)showshyperdenselesionintherightlowerdorsalpulmonaryarea. @ .

HAMAfactor IgG (2-4 months p.i.) 20 -

15H H

10 @1

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@ 0 iiIiiiiiii_i i@ I I@iiiiiii@I I i 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 23 @ A anti-isotypic @.

HAMAfactor gM (2-4 months p.i.) 16 14 -

12

10 8—@ FIGURE 7 (A) Anti-isotypic and/or anti-idiotypic 6- HAMA response (lgG) in twenty pa tients(1—20patients,23 controlserum) 4@- after antigranulocyte immunoscintig raphy with 0.5 mg MAb BW 250/183. 2— (B) Anti-isotypic and/or anti-idiotypic HAMA response (1gM)in twenty pa 0 tients(1—20patients,23 controlserum) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 23 afterantigranulocyteimmunoscintigra phy with 0.5 mg MAb BW 250/183. —anti-isotypic anti-iso/idiotypic

with an ‘‘‘In-labelednonspecific immunoglobulin (23). quality SPECT images offer considerable advantages False-negative findings, on the other hand, must be compared to in vitro labeling of leukocytes with ‘‘‘In expected in the demonstration of older encapsulated oxine. abscesses, in which exchange or migration of neutrophil REFERENCES granulocytes have been reduced. A consecutive scan (24 hr postinjection) is indispen 1. Faird NA, White SM, Heck LL. @@mTclabeled leucocytes: sible in these cases, since quite frequently, at least preparation and use in identification of abscessesand tissue marginal leukocytosis is proved in the delayed image rejection.Radiology1983;148:827—831. 2. Laue A, Schulz-Heinken D, Heinken U. Blutzellmarkierung even though the 6-hr scan has been negative. The mit Indiumoxin (In-l 11), Medi:inische Monographie 15. sensitivity in cranial, thoracic, abdominal, and hip re Braunschweig:Amersham-Buchler 1986. gions can be increased significantly by use ofthe SPECT 3. ThakurML,LavenderJP,ArnotRN,SilvesterDJ,SegalAW. technique (24,25), while planar imaging (especially Indium-i 11-labeled autologous leukocytes in man. J Nz,cl when the x-ray findings are known) is sufficient for the Med 1977;18:1014—1019. 4. Joseph K, HOffkenH, BossletK, Schorlemmer HU. In vivo identification of inflammatory processes in the extrem labeling of granulocytes with Tc-99m anti-NCA monoclonal ities. antibodies for imaging inflammation. Eur J Nucl Med 1988: The introduction of 99mTc@labeledantigranulocyte 14:367—373. antibodies allows a further clinically relevant in vivo 5. Peters AM, Osman 5, Henderson BL, et al. Clinical experience application of monoclonal antibodies in addition to with Tc-99m hexamethylpropyleneamineoxime for labeling leucocyles and imaging inflammation. Lancet 1986: 2:946— anti-CEA immunoscintigraphy for the diagnosis of co 949. lorectal tumor recurrences (26). Easy labeling, the op 6. Joseph K, Damann V. EngeroffG, Gruner KR. Markierung timum gamma energy of 99mTcand the resulting high von Leukozytenmit Tc-99m HMPAO: ErsteklinischeErgeb

422 The Journal of Nuclear Medicine •Vol. 31 •No. 4 •April 1990 nisse. Nuc Compact 1986; 17:277—283. 17. Hasler PH, Seybold K, Andres RY, Locher JT, Schubiger PA. 7. Bosslet K, LUben G, Schwarz A, Ctal. Immunohistochemical Immunoscintigraphic localization of inflammatory lesions: localization and molecular characteristics of three mono pharmacokinetics and estimated absorbed radiation dose in clonal antibody-defined epitopes detectable on carcinoem man.EurfNuclMed 1988;13:594—597. bryonic antigen (CEA). mt J Cancer 1985; 36:75—84. I8. Locher iT, Seybold K, Andres RY, Schubiger PA, Mach JP, 8. Bosslet K, Schorlemmer HU, Steinstraesser A, Schwarz A, BucheggerF. Imagingof inflammatory lesionsafter injection Sedlacek HH. Molecular and functional properties of the of radioiodinated monoclonal antigranulocytes antibodies. granulocyte specific MAB BW 250/183 suited for the im NuclMedCommun 1986;7:659—670. munoscintigraphic localization ofinflammatory processes. In: 19. Schorlemmer HU. Inhibition of functions related to human HäferR, ed. Radioactive isotopes in clinical medicine and granulocytes by specific murine monocional antibodies. NucI research. New York: Schattauer; 1988:15—20. Med 1989;28:150. 9. Schwarz A, Steinstraesser A. A novel approach to @mTc@20. Bossiet K, Steinstrasser A, Schwarz A, Schoriemmer HU, labeled monoclonal antibodies [Abstract].J Nucl Med 1987; Krumwich D, Sedlacek HH. Generation and functional char 28:721. acteristics of the granulocyte selective monoclonal antibody 10. Coleman RE, Black RE, Welch DM, Maxwell JG. In-i 11 BW 250/183.NuclMed 1989;28:151. labeled leukocytes in the evaluation of suspected abdominal 21. KroissA, KöiblCh, WeissW, Neumayr A. Immunszintigra abscess. Am J Surg 1980; 139:99—104. phie mit J-123-und Tc-99m markierten Granulozytenanti 11. Segal AW, Enseli J, Munro JM, Sarner M. Indium-i 11tagged korpern beim M. Crohn und Colitis ulcerosa [Abstract]. Nucl leucocytes in the diagnosis of inflammatory bowel disease. Med 1989;2:72. Lancet1981:2:230—232. 22. BossletK, Auerbach B, HäflkenH, Joseph K. Frequencyand 12. Raptopoulos V. Doherty PW, Gross TP. Acute osteomyelitis: relevance of the human anti-mouse immunoglobulin advantage of white cell scans in early detection. Am J Roent (HAMA) response in immunoscintigraphy. NucI Med 1989; genol1982:139:1077—1082. 28:149. 13. Seabold JE, Wilson DC, Lieberman LM, Boyel CM. Unsus 23. Rubin RH, Fishman AJ, Callahan Ri, et al. “In-labeled pected extraabdominai sites of infection: scintigraphic detec nonspecific immunoglobulin scanning in the detection of tion with indium-i 1I-labeled leukocytes. 1984; focal infections. NEnglJMed 1989; 321:535. 151:213—217. 24. KroissA, KälblCh, Tuchmann A, et al. Immunszintigraphie 14. Seybold K, Locher iT, Coosemans C, Andres RY, Schubiger (IS) bei entzündlichenErkrankungen mit Tc-99m MAK BW PA, BläuensteinP. Immunoscintigraphic localization of in 250/183. In: Eber 0, Lind P. LangstegerW, eds. Workshop flammatory lesions: clinical experience. EurfNuclMed 1988; immunszintigraphie. Souderheft: Acta Med Austriaca; 1989; 13:587—593. 11—16. 15. Buchegger F, Schreyer M, Carrel S. Mach JP. Monoclonal 25. SeyboidK. ImmunszintigraphischerNachweisentzUndlicher antibodies identify a CEA crossreacting antigen of 95 kD Prozesse mit einem J-l23-AntigranulozytenantikOrper. In: (NCA-95) distinct in antigenicity and tissuedistribution from Eber 0, Lind P, Langsteger W, eds. Workshop immunszinti the previously described NCA of 55 kD. mt i Cancer 1984; graphic. Souderheft: Acta Med Austriaca; 1989; 5—11. 33:643—649. 26. Lind P, LangstegerW, Koltringer P. et al. Tc-99m labeled 16. Andres RY. Seybold K, Tiefenauer L, Schubiger PA, Locher monoclonal anti-CEAantibody (BW431/26): clinicalresults iT, Mach JP. Radioimmunoscintigraphic localization of in in the detection of colorectal carcinomas and recurrences. flammatory lesions: concept, radiolabelingand in vitro testing Scand J Gastroenterol I989; 24:1205—12 11. of a granulocyte antibody. Eur J NucI Med 1988; 13:582— 586.

Technetium-Labeled MAb BW 250/i 83 •Lind et aI 423