Clinical validation of the avidin/indium-111 biotin approach for imaging infection/inflammation in orthopaedic patients

We report here the results of a validation study of the avidin/indium-111 biotin approach in patients with skeletal lesions. This study involved 54 patients with orthopaedic conditions: 20 patients with intermediate suspected osteomyelitis of the trunk, 19 patients with infection/inflammation of prosthetic joint replacements, and 15 patients with suspected osteomyelitis of appendicular bones. Avidin (3 mg) was injected as an i.v. bolus, followed 4 h later by ¹¹¹In-biotin; imaging was acquired 30 min and 16–18 h after administration of ¹¹¹In-biotin. Technetium-99m hexamethylpropylene amine oxime (^99mTc-HMPAO)-labelled leucocyte scintigraphy was performed in 39/54 patients. The overall sensitivity of the avidin/¹¹¹In-biotin scan was 97.7% (versus 88.9% for ^99mTc-HMPAO leucocyte scintigraphy). While the diagnostic performance of avidin/¹¹¹In-biotin scintigraphy was similar to that of ^99mTc-HMPAO leucocyte scintigraphy in patients with prosthetic joint replacements or osteomyelitis of appendicular bones, the avidin/¹¹¹In-biotin approach clearly performed better than ^99mTc-HMPAO leucocyte scintigraphy in patients with suspected osteomyelitis of the trunk (100% sensitivity, specificity and accuracy versus 50% sensitivity, 100% specificity and 66.7% accuracy for ^99mTc-HMPAO-leucocyte scintigraphy). These results demonstrate the feasibility of the avidin/¹¹¹In-biotin approach for imaging sites of infection/inflammation in the clinical setting. Although no systematic advantages of avidin/¹¹¹In-biotin scintigraphy were found versus ^99mTc-HMPAO leucocyte scintigraphy, the newer scintigraphic method is more practicable and involves lower biological risk for the operators. Received 9 November 1998 and in revised form 1 February 1999     

Clinical evaluation of indium-111-labeled leukocyte imaging in bone infection

In-111-oxine-labeled leukocyte imaging was performed on twenty-one patients suspected of having bone infection. Nine of eleven cases (82%) were diagnosed as having active infection as demonstrated by abnormal accumulation of In-111-labeled leukocytes at the site of infection. There are two false negative (18%) cases. Two cases without active infection showed abnormal uptake. Four cases revealed cold defects on the scintigraphy. Marked uptake of radiotracer was noted not only in the case of acute osteomyelitis with acute septicaemia but also in the case with persistent chronic active osteomyelitis. It was observed that for precise evaluation of the test results it was equally important to compare the imaging findings with physical signs and laboratory investigations. It is concluded that In-111-oxine-labeled leukocyte imaging is a useful tool for the evaluation of the progression of bone infection.     

Clinical role of indium-111 antimyosin imaging.

Myocyte necrosis occurs in ischaemic, inflammatory and toxic heart diseases and can be detected by indium-111 antimyosin imaging. This allows a non-invasive evaluation of the site, extent and quantitation of the severity of myocardial necrosis. Simultaneous imaging of perfusion in patients with myocardial infarction allows the differentiation of necrosed and perfused areas and the varying degrees of mismatch and overlap, which has prognostic significance. 111In-antimyosin imaging is useful in the assessment of patients with unstable angina and in those for whom the diagnosis of infarction or unstable angina is not clear. In suspected myocarditis, a positive scan indicates the necessity for endomyocardial biopsy to confirm inflammation, whereas a negative scan makes the diagnosis of myocarditis unlikely. Antimyosin imaging is not useful as a marker of rejection in the 1 year post-transplant, but uptake after this period is associated with an increased rejection rate and is therefore an important tool in planning management strategies. Most patients on anthracycline treatment have demonstrable uptake, which is related to the cumulative dose and to the ejection fraction. Its role in this situation is as yet unclear. The use of new ligands and radioisotopes (99mTc) is likely to allow earlier imaging and produce improved quality.     

Clinical role of indium-111 antimyosin imaging

Myocyte necrosis occurs in ischaemic, inflammatory and toxic heart diseases and can be detected by indium-111 antimyosin imaging. This allows a non-invasive evaluation of the site, extent and quantitation of the severity of myocardial necrosis. Simultaneous imaging of perfusion in patients with myocardial infarction allows the differentiation of necrosed and perfused areas and the varying degrees of mismatch and overlap, which has prognostic significance.¹¹¹In-antimyosin imaging is useful in the assessment of patients with unstable angina and in those for whom the diagnosis of infarction or unstable angina is not clear. In suspected myocarditis, a positive scan indicates the necessity for endomyocardial biopsy to confirm inflammation, whereas a negative scan makes the diagnosis of myocarditis unlikely. Anti-myosin imaging is not useful as a marker of rejection in the 1 year post-transplant, but uptake after this period is associated with an increased rejection rate and is therefore an important tool in planning management strategies. Most patients on anthracycline treatment have demonstrable uptake, which is related to the cumulative dose and to the ejection fraction. Its role in this situation is as yet unclear. The use of new ligands and radioisotopes (^99mTc) is likely to allow earlier imaging and produce improved quality.     

Investigations of avidin and biotin for imaging applications

The attractive properties of avidin (streptavidin) and biotin, in particular their strong affinities (Kd = 10(-15)M), may be used to advantage in imaging applications. These molecules have been used in this preliminary investigation to improve the targeting of 111In in animals. Antibodies have been conjugated with biotin and administered unlabeled while, at a later time, the radiolabel was administered attached to DTPA-coupled avidin or streptavidin. An alternative procedure was also considered whereby the antibodies were conjugated with avidin and administered before the administration of radiolabeled biotin. Using a model in which the target consisted of conjugated agarose beads deposited in the peritoneum of mice, it has been shown that the target/nontarget radioactivity ratios may be significantly improved with respect to the conventional procedures through the use of this approach.     

Comparison of purified indium-111 granulocytes and indium-111 mixed leukocytes for imaging of infections

Several methods have been proposed for the separation and labeling of white blood cells for the diagnosis of suspected infection. We retrospectively compared 105 patients imaged with 111In purified granulocytes (GRAN) to 106 patients imaged with 111In mixed leukocytes (MIX). We found that in acute infection the sensitivity of GRAN and MIX were both high and not statistically different. In chronic infections the sensitivities were lower than for acute infections. Again, there was no significant difference between GRAN and MIX with the borderline significant exception of MIX being superior to GRAN in chronic soft tissue infections (p = 0.06). We then had independent observers blindly grade the degree of lesion visualization. We found that delayed images visualized the lesions better than early images (p = 0.0001) and acute infection was better visualized than chronic infection (p = 0.03). We concluded that, in routine clinical practice, MIX is probably the agent of choice for three reasons: (a) easier preparation, (b) comparable sensitivity in acute infection and, (c) borderline superior sensitivity in chronic infection.     

Bone marrow island vs focal infection. An indium-111 leukocyte imaging dilemma

An island of bone marrow surrounded by the glue used in securing a hip prosthesis accumulated In-111 leukocytes in a patient being imaged for hip pain. This was shown to be normal marrow uptake of labeled cells and not an abscess by performing bone marrow imaging. The appearance of the leukocyte and bone marrow scans of the area were identical.     

Imaging of tumor in patients with indium-111-labeled biotin and streptavidin-conjugated antibodies: preliminary communication

Tumor localization in patients has been achieved through the in vivo use of streptavidin and biotin. In these preliminary studies, the monoclonal antibody HMFG1 was conjugated with streptavidin and 1 mg was administered intravenously to each of 10 patients with documented squamous cell carcinoma of the lung. Two to 3 days later, 111In-labeled biotin was also administered intravenously. No evidence of toxicity was observed. Background radioactivity levels were reduced in liver (1% ID at 24 hr) and kidneys (2%) and in all other normal tissues and blood. Images of lung tumor were obtained in as little as 2 hr following administration of labeled biotin. In eight patients, tumor was detected with labeled biotin alone without the previous administration of streptavidin-conjugated antibody but in three of these patients, the images were improved with the prior administration of conjugated antibody. These results suggest that this approach may improve the tumor-to-normal tissue radioactivity ratios in radioimmunotargeting.     

Effect of isoelectric point on biodistribution and inflammation imaging with indium-111-labelled IgG

Electrostatic effects play an important role in protein interactions and may alter the biodistribution of antibodies. To study the effect of molecular charge on the biodistribution and infection imaging properties of human polyclonal immunoglobulin G (IgG), its iso electric point was varied by changing the level of diethylene triamine penta-acetic acid (DTPA) substitution: 0.8, 0.9, 3.7, 5.1 and 5.9 DTPA/IgG. Biodistributions of the different IgG preparations were determined at 10 min, 1, 6, 24, and 48 h post injection in normal rats, and infection imaging properties were determined in rats withEscherichia coli thigh infections. The biodistribution was significantly affected by pl. The immunoglobulin preparations with 0.9 and 3.7 DTPA/IgG showed faster clearance from the circulation and generally lower accumulation in most organs. The images had a target-to-background ratio of approximately 1.3–2.3:1. These results suggest that even though targeting is not affected by the level of DTPA substitutions, preparations with 0.9 and 3.7 DTPA/IgG may be superior imaging agents because of reduced accumulation by background organs.     

Role of indium-111 chloride imaging in osteoid osteoma

Indium-111 chloride imaging plays an important role in differentiating intracortical osteoid osteoma from chronic cortical abscess. The study also may be useful in the detection of intramedullary osteoid osteoma. Four patients who greatly benefited from indium-111 chloride imaging are presented.     

New concepts in infection/inflammation imaging.

Although autologous leukocytes, labelled with 111In or 99mTc, is still considered the "gold standard" nuclear medicine technique to image infection and inflammation, there is a great need for a less cumbersome and less hazardous approach. Over the last few decades the range of radiopharmaceuticals to investigate infectious and non-microbial inflammatory disorders is vastly expanding. Radiolabelled monoclonal antibodies and antibody-fragments, radiolabelled chemotactic peptides and cytokines, and radiolabelled antibiotics are promising new approaches in the field of nuclear medicine. Recently, positron emission tomography (PET) with 18FJDG has been introduced and has been shown to delineate infectious and inflammatory foci with high sensitivity. Here, a survey is presented of the different approaches in use or under investigation.     

Evaluation of indium-111-labeled anti-fibrin antibody for imaging vascular thrombi

Monoclonal antibody 59D8 developed by Hui et al., binds to fibrin but not fibrinogen. An 111In-labeled Fab fragment of 59D8 was studied in vitro and in animal models to evaluate its potential for imaging thrombi and emboli in man. Rabbits and dogs were used as models for studying thrombus uptake in vivo. Thrombi and emboli up to 4 days old were successfully visualized at 4-24 hr postinjection in five of eight rabbits. In dogs, 0.5-hr-old and 24-hr-old thrombi were successfully imaged at 24 hr in six of eight animals, and 3/6 of these were positive at 3-4 hr postinjection. Thrombus-to-blood ratios in the dogs averaged 7.1 +/- 1.3. The findings suggest this antibody may be useful for imaging thrombi in man.     

Evaluation of the utility of indium-111 oxine platelet imaging in renal transplant patients on cyclosporine

Twenty five In-111 oxine platelet imaging procedures were performed in 21 renal transplant patients to assess the value of this study for diagnosis of renal transplant rejection in recipients receiving cyclosporine (CYS) for immunosuppression. Fourteen biopsies were performed, and an extensive, in-depth review of the clinical progress of each patient was obtained. There was no ideal "gold standard" to which our imaging results could be compared, but we used a combination of biopsy findings, clinical impressions, and changes in renal function after pulsing with steroids and/or decreasing CYS dosage as the basis for our diagnoses. We were unable to distinguish between renal rejection and CYS toxicity using platelet imaging. The sensitivity of the platelet procedure for diagnosing rejection or CYS toxicity was 100%. The specificity for rejection or CYS toxicity was only 76.5%. In view of the inability of this test to distinguish between rejection and CYS toxicity, its rather low specificity, and its relatively high cost, it is not a particularly helpful study for the diagnosis of renal transplant rejection in patients on CYS.     

Value of blood-pool subtraction in cardiac indium-111-labeled platelet imaging

Blood-pool subtraction has been proposed to enhance 111In-labeled platelet imaging of intracardiac thrombi. We tested the accuracy of labeled platelet imaging, with and without blood-pool subtraction, in ten subjects with cardiac thrombi of varying age, eight with endocarditis being treated with antimicrobial therapy and ten normal controls. Imaging was performed early after labeled platelet injection (24 hr or less) and late (48 hr or more). Blood-pool subtraction was carried out. All images were graded subjectively by four experienced, "blinded" readers. Detection accuracy was measured by the sensitivity at three fixed levels of specificity estimated from receiver operator characteristic curve analysis and tested by three-way analysis of variance. Detection accuracy was generally improved on delayed images. Blood-pool subtraction did not improve accuracy. Although blood-pool subtraction increased detection sensitivity, this was offset by decreased specificity. For this population studied, blood-pool subtraction did not improve subjective detection of abnormal platelet deposition by 111In platelet imaging.     

Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi

The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with ¹¹¹In. The rt-PA was coupled with the bicyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with ¹¹¹In bound exclusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5–1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot: blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with ¹¹¹In without destroying the ability of the protein to localize in clot and images of forming clot can be obtained with this agent within 1 h after administration.     

Characterization of indium-111 labeled recombinant tissue plasminogen activator for the imaging of thrombi

The in vitro functional properties of recombinant tissue plasminogen activator (rt-PA), its biodistribution in mice, and its pharmacokinetics and clot localization properties in dogs have been investigated after labeling rt-PA with 111In. The rt-PA was coupled with the bicyclic anhydride of DTPA using standard methodology. Amidolytic and fibrinolytic assays showed retention of protein activity when rt-PA was conjugated with an average of one DTPA group or less per molecule. Size exclusion HPLC showed each preparation to be radiochemically pure with 111In bound exclusively to the attached DTPA groups. Biodistribution in mice showed major accumulation of activity in the liver and kidneys. After administration of 0.5-1.0 mg of the labeled protein to dogs, blood activity decreased with a half time of approximately 5 min in agreement with previous reports of rapid blood clearance. Largely because of decreased blood levels, clot: blood ratios of labeled protein increased rapidly, in one study reaching 6.3 after 31 min, and satisfactory images of fibrin thrombi were obtained. The rt-PA may be labeled with 111In without destroying the ability of the protein to localize in clot and images of forming clot can be obtained with this agent within 1 h after administration.     

Pulmonary uptake in indium-111 leukocyte imaging: clinical significance in patients with suspected occult infections

A retrospective review was undertaken to evaluate the frequency and significance of pulmonary activity noted on 306 indium-111 leukocyte studies involving 232 patients with suspected occult infections. Forty-eight studies showed pulmonary activity in one of two patterns of uptake, focal or diffuse. Fourteen of 27 studies (52%) with focal uptake and two of 21 studies (10%) with diffuse uptake were associated with infectious processes. Lung uptake of indium-111-labeled leukocytes was a poor predictor of pulmonary infection in patients studied for occult infection, although the focal pattern was more likely than the diffuse pattern to be associated with infection.     

Diagnostic significance of indium-111 granulocyte scintigraphy in febrile patients

Sixty-eight patients with fever of unknown origin, 32 patients with postoperative fever, and 26 patients with therapy-resistant fever after bacteremia were investigated with [111In] granulocyte scintigraphy for the detection of abscesses. The results showed that the value of [111In]granulocyte scintigraphy in the detection of infectious foci vary in these three types of febrile conditions. The overall sensitivity and specificity were 86.5% and 87.8%, respectively. We observed, however, a relatively low predictive value of a positive result in the fever of unknown origin group (73.1%), and also a low predictive value of a negative result in the bacteremia group (66.7%). The C-reactive protein (CRP) levels in patients with a true-positive scintigram were significantly (p less than 0.001) higher than in patients with a true-negative scintigram. There was also a significant positive correlation (p less than 0.01) between the serum CRP concentration and the intensity of the granulocyte accumulations. There was no correlation between the peripheral leukocyte count or the erythrocyte sedimentation rate (ESR) and the intensity of the granulocyte uptake. Therefore CRP, but not the leukocyte count or ESR, appears useful for selecting the patients who benefit most from granulocyte scintigraphy.     

Clinical evaluation of a scintigraphic method for diagnosing inflammations/infections using indium-111-labeled nonspecific human IgG.

This study was undertaken as part of a Phase II study to assess the sensitivity and safety of 111In-DTPA-human IgG, an imaging agent for the detection of inflammations and/or infections. Forty patients with infection/inflammation on the basis of clinical findings, microbiologic results, and/or the basis of results from other imaging modalities were studied. For evaluation of sensitivity, whole-body images were obtained at 6-12 hr (early) and 20-28 hr (delayed) postinjection and occasionally at 48 hr. No adverse reactions were recorded in any of the 40 patients studied. Positive results were obtained in 37 of 37 evaluable subjects (100%). The test appears to be a promising method for the detection of inflammation and/or infection.