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Jun 4, 2003 - are difficult to diagnose with currently available imaging tech- niques, and may ... Program and an unrestricted grant from Agenix Ltd. ... This article has an online supplement, which is accessible from this issue's table .... tee approved this study in accordance with federal and institutional ..... ultrasound.
Single Photon Emission Computed Tomography of Pulmonary Emboli and Venous Thrombi Using Anti–D-Dimer Timothy A. Morris, James J. Marsh, Peter G. Chiles, Ronald G. Konopka, Craig A. Pedersen, Peter F. Schmidt, and Michael Gerometta Division of Pulmonary/Critical Care Medicine, Department of Medicine, University of California San Diego, San Diego, California; and Agen Biomedical Ltd, Queensland, Australia

Previous attempts to diagnose thromboemboli using radiolabeled antibodies and nuclear medicine imaging have been disappointing. We present the results of experiments with intravenous technetium99m–labeled deimmunized antifibrin Fabⴕ fragments to diagnose thromboemboli using single photon emission computed tomography (SPECT), a highly sensitive scintigraphic imaging technique. Pulmonary emboli (PEs) and lower extremity deep vein thrombi (DVTs) were formed in five dogs, then technetium-99m–labeled Fabⴕ (ⵑ 400 mg, ⵑ 260 MBq) were injected via forelimb veins. Thoracic and lower extremity SPECT scans were performed at 2-hour intervals after antibody infusion to visualize the thromboemboli. Four hours after antibody infusion, all PEs and DVTs of mass 0.4 g or greater were clearly visualized on SPECT scans as ‘hot spots’ within the lungs and legs, respectively. PEs (0.48 ⫾ 0.09 g) were intensely radiolabeled, yielding clot/blood radioactivity ratios of 22.8 ⫾ 5.6. DVTs (0.45 ⫾ 0.31 g) also had high clot/blood ratios (11.7 ⫾ 2.6). Infusion of these radiolabeled antibody fragments, combined with SPECT, produces clear images of PEs and DVTs within a clinically feasible time frame. The technique reliably identified even peripheral thromboemboli of relatively small size, which are difficult to diagnose with currently available imaging techniques, and may enable imaging of PEs, DVTs, or both in the same patient.

Venous thromboembolism is a common disorder, with an annual incidence of diagnosed disease estimated at 117 per 100,000 (1). The true incidence is likely much higher because it can be challenging to diagnose. For example, ventilation/perfusion scanning is prone to a high number of nondiagnostic results (2), and the sensitivity of contrast-enhanced computed tomography scanning has been reported to be as low as 70% (3). These difficulties may contribute to the fact that most patients who succumb to pulmonary embolism (PE) die without the diagnosis being made antemortem (4–6). Limitations in the ability to diagnose PE and deep vein thrombi (DVT) are especially unfortunate in light of the excellent outcomes usually observed with anticoagulation (7). We report the results of a novel noninvasive method for accurately detecting PE and DVT, using modified antifibrin antibody fragments and advanced scintigraphic imaging techniques.

Previous attempts to diagnose pulmonary emboli using radiolabeled fibrin-specific antibodies and nuclear medicine imaging have been disappointing (8–10). One reason for this may have been that the fibrin ␤-chain epitope that was targeted was available for antibody binding only during active fibrin polymer elongation (11). Systemic anticoagulants, which are commonly used empirically during work-up for PE (12) decrease the accessibility of antibody-binding sites in the emboli and lower the probability of detection (13, 14). In contrast, the D-dimer region of polymerized fibrin remains accessible to antibody binding even after thrombus propagation has been suppressed with large doses of anticoagulants. Anti–D-dimer antibodies may be better suited for imaging DVT and PE in clinical situations than the antibodies previously tested. Even in patients not taking anticoagulants, previous trials using conventional planar nuclear medicine techniques were not able to distinguish (presumably) highly labeled emboli from the background level of radiolabel remaining in the thorax. This problem might be solved by using single photon emission computed tomography (SPECT) to spatially resolve PEs from the thoracic blood pool background. SPECT is a nuclear medicine imaging technique that, like X-ray computed tomography, creates two-dimensional or three-dimensional tomographic images of the body. Moreover, SPECT constructs the images by detecting radioisotope emissions and so can distinguish densely labeled targets (such as PEs) from less densely labeled adjacent background tissue (such as lung parenchyma). We used the combined technique of radiolabeled anti–Ddimer antibody fragments, which clear from the circulation more rapidly than intact IgG, plus SPECT scans to identify PEs and DVTs of various masses in a well established canine model of acute pulmonary thromboembolism. The aim of the study was to determine if scans could accurately detect thromboemboli in the lungs and legs, even after large doses of systemic anticoagulants were administered. High sensitivity and specificity in the animal model would support the performance of clinical trials using this technique. Preliminary data on these experiments were previously published in abstract form (15).

METHODS Materials (Received in original form June 4, 2003; accepted in final form February 7, 2004) Supported by a grant from The American Lung Association of California—Research Program and an unrestricted grant from Agenix Ltd. Correspondence and requests for reprints should be addressed to Timothy A. Morris, M.D., Associate Professor of Medicine, University of California San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8378. E-mail: t1morris@ ucsd.edu This article has an online supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 169. pp 987–993, 2004 Originally Published in Press as DOI: 10.1164/rccm.200306-735OC on February 12, 2004 Internet address: www.atsjournals.org

A murine antifibrin monoclonal IgG antibody specific for the D-dimer epitope (16) formed the basis of these experiments. Deimmunization of the murine IgG was achieved by modifying the Fv regions and then inserting them into a vector containing the constant regions of a human IgG1 subclass antibody using a proprietary technique (Biovation Ltd., Aberdeen, UK). F(ab)⬘2 fragments of the deimmunized antibody were generated as described (17) and concentrated to 2.4 mg/ml in phosphatebuffered saline (20 mM sodium dihydrogen phosphate, 0.15 M sodium chloride, pH 7.4). The particular clone used in these studies is designated DI3B6/22-80B3 and was provided by Agen Biomedical Ltd. (Queensland, Australia). Human fibrinogen (⬎ 95% clottable) was obtained from Calbiochem (La Jolla, CA). Tranexamic acid, d-gluconic acid

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004

(hemicalcium salt), dithiothreitol, and stannous chloride were purchased from Sigma (St. Louis, MO). Unfractionated heparin (porcine intestinal mucosa) and bovine thrombin were obtained from American Pharmaceutical (Los Angeles, CA) and Jones Pharma, Inc. (St. Louis, MO), respectively. Freshly generated sodium technetium-99m [99mTc]pertechnetate (3,700 MBq/ml in normal saline) was obtained from Syncor (San Diego, CA).

Antibody Labeling with

99m

Tc

Fab⬘ fragments of DI3B6/22-80B3 were labeled with 99mTc using a modified exchange procedure as described previously (18). Briefly, F(ab)⬘2 (500 ␮g) were reduced by incubation with excess dithiothreitol (128:1 molar ratio). The resulting Fab⬘ fragments were separated from excess dithiothreitol by centrifugal size-exclusion chromatography on Biogel PGD6 (Biorad, Richmond, CA). Meanwhile, a 99mTc–gluconate complex was prepared by dissolving 10 mg of d-gluconic acid in 0.5 ml of [99mTc]pertechnetate (3,700 MBq/ml). Tin chloride was added from a freshly prepared stock solution (5 mg/ml in 0.1 N hydrochloric acid) at 37 ␮g/ml final concentration. The 99mTc–gluconate complex (370 MBq) was added to the Fab⬘ and after incubation at 37⬚C for 30 minutes, the labeled antibody was purified by size-exclusion chromatography on Biogel PGD6. 99m Tc-labeled Fab⬘ protein concentration was determined by UV spectroscopy at 280 nm using an extinction coefficient of 1.16 (1 mg/ml). Incorporation of 99mTc into Fab⬘ was estimated using Centrifree centrifugal ultrafiltration devices (Amicon, Beverly, MA). The immunoreactivity of purified 99mTc-labeled Fab⬘ (i.e., the fraction of labeled Fab⬘ which could still bind to the D-dimer antigen) was estimated using D-dimer coupled to Sepharose beads as described previously (18, 19).

Animal Model of Acute Venous Thromboembolism The University of California San Diego Animal Care and Use Committee approved this study in accordance with federal and institutional policies concerning the humane care and use of laboratory animals. Healthy adult mongrel male dogs (23–28 kg) were anesthetized with pentobarbital (30 mg/kg, intravenously) and ventilated to maintain arterial blood gases within normal limits. Dogs received tranexamic acid (110 mg/kg intravenously) every 4 hours to inhibit fibrinolysis (20). In one dog, no DVTs or PEs were formed. In five other dogs, doubleballoon catheters were advanced via hind leg saphenous veins into the femoral veins bilaterally under fluoroscopic guidance. After balloon inflation, femoral vein thrombi were induced in each leg as follows: blood (5 ml) was drawn through a port between the inflated balloons into a syringe containing 18 mg (0.7 ml) of purified human fibrinogen. It was necessary to supplement blood with human fibrinogen because the antibody does not cross-react with canine fibrin. Thrombin (200 U, 0.2 ml) was introduced into the syringe via a three-way stopcock to initiate clotting. The contents of the syringe were mixed briefly and then immediately injected through the port between the balloons. After 1 hour, the balloons were deflated to restore blood flow, and the clots were aged in situ for an additional 3 hours. Before balloon deflation, heparin was given (300 U/kg intravenous bolus, 90 U/kg/hour by continuous infusion) to prevent accretion of canine fibrin onto the thrombi. After aging, venograms were performed to confirm the presence of thrombus in each leg and then one thrombus was randomly selected for embolization. Embolization was achieved by passive leg motion followed by removal of the double-balloon catheter. (Complete embolization was confirmed at the end of the experiment by visual inspection of the vessel lumen and ␥ counting of the entire femoral vein segment—see below.) The catheter in the contralateral leg was left in place to prevent embolization of that thrombus. Approximately 1 hour after embolization, 99mTc-labeled Fab⬘ antibody fragments (ⵑ 400 ␮g, ⵑ 260 MBq) were injected via a forelimb vein.

Antibody Clearance Blood samples (2 ml) were collected at defined time intervals after injection of 99mTc-labeled antibody and radioactivity was measured in a ␥ counter (Beckman Instruments, Fullerton, CA). Protein-associated 99m Tc was also determined in selected blood samples by measuring the percentage of radioactivity in plasma precipitated by ice-cold trichloroacetic acid (10% final concentration). Total urine was collected via a Foley catheter at hourly intervals. The volume collected was recorded,

and an aliquot (1 ml) from each time interval was subjected to ␥ counting.

SPECT Imaging At intervals between 2 and 8 hours after 99mTc-labeled antibody injection, dogs were placed in the supine position under a dual-head Varicam SPECT scanner (GE Medical Systems, Milwaukee, WI) set for 99mTc imaging. Scans of the lower extremities and thoracic region were performed using the following imaging parameters: 180⬚ angular range, 6⬚ angular step, and 60-seconds frame time (total scan time: 30 minutes) following a contoured path within 1 to 2 cm of each subject’s body surface. Raw images were processed using eNTEGRA software (GE Medical Systems), using a Hann filter with a critical frequency of 1.50, quantitative, and saved as DICOM files. The files were viewed with Osiris software (University Hospital of Geneva, Geneva, Switzerland) by a reader (T.M.) who was blinded as to the allocation of the dogs (laboratory dogs vs. control dogs) and to the location(s) of the clot(s), as discovered during autopsy (see below). Focal accumulations of 99mTc (‘hot spots’) were interpreted as indicative of DVTs and PEs, and their locations were compared with the locations determined by autopsy. Sensitivity and specificity of the scans were calculated for detection of DVTs (using all femoral veins as potential sites) and for detection of PEs (using all lung lobes as potential sites).

Tissue Biodistribution On completion of SPECT imaging, dogs were killed with an overdose of pentobarbital. PEs were recovered by careful dissection of all pulmonary arteries from the pulmonic valve through the subsegmental level, without knowledge of the scan results. The weight and anatomical location of each embolus was recorded. Lower extremity veins were dissected bilaterally from the iliac veins to the saphenous veins for recovery of DVTs. Samples (ⵑ 1 g, in triplicate) of the following tissues were also obtained: adrenal gland, bile, bladder, heart, kidney, liver, lung, muscle, skin, spleen, thyroid, and femoral veins. All tissue samples, including clots, were weighed and subjected to ␥ counting. Clot (or tissue)/blood isotope density ratios were calculated by dividing the counts per minute/g of clot (or tissue) by the counts per minute/g of the final blood sample. Label uptake by clots (or tissue) was also quantified as the percentage of the injected dose (ID)/g clot (or tissue).

RESULTS Characteristics of

99m

Tc-labeled DI3B6/22-80B3 Fabⴕ

The average labeling efficiency (incorporation of 99mTc into Fab⬘ before gel filtration) was 96.3 ⫾ 0.4%. After purification by P6DG chromatography, 98.7 ⫾ 0.3% of the 99mTc was associated with Fab⬘. The average immunoreactivity of the labeled antibody (the percent capable of binding to the D-dimer antigen) was 94.5 ⫾ 2.0% and the mean amount injected before SPECT imaging was 388 ⫾ 14 ␮g (252 ⫾ 7 MBq). Clearance of DI3B6/22-80B3 Fabⴕ

Clearance of radiolabel from the blood circulation after intravenous injection of 99mTc-labeled DI3B6/22-80B3 Fab⬘ is shown in Figure 1. From these data, a circulating half-life of 44.9 minutes was calculated for the 99mTc-labeled Fab⬘ (Figure 1A). A significant percentage of the radiolabel was cleared in the urine (Figure 1B). By 4 hours, 38.3 ⫾ 8.2% of the ID was found in the urine and only 6.7 ⫾ 0.9% remained in the circulation. At the conclusion of the study, 55.3 ⫾ 8.3% was accounted for in the urine, whereas only 2.2 ⫾ 0.4% remained in the circulation. Protein-associated radiolabel at 2, 4, and 8 hours after injection, as measured by radioactivity in plasma precipitable by trichloroacetic acid, was 92.6 ⫾ 1.0, 87.1 ⫾ 4.8, and 87.4 ⫾ 6.1%, respectively. SPECT Imaging

High-resolution SPECT images of the thoracic and femoral regions of each dog were obtained at approximately 2, 4, and 8 hours after injection with labeled antibody. Chest images from

Morris, Marsh, Chiles, et al.: New Diagnostic Method for PE and DVT

989 Figure 1. Clearance of technetium-99m (99mTc)– labeled DI3B6/22-80B3 Fab⬘. (A ) Clearance of radiolabel from the blood circulation after injection of 99m Tc-labeled antibody. Data are expressed as a percentage of the injected dose (ID) per gram of blood. The half-life was calculated from an exponential fit of the data. (B ) Clearance of radiolabel from the blood (solid bars) and the cumulative appearance of radiolabel in the urine (open bars) after injection of 99mTclabeled antibody. Data are expressed as a percentage of the total ID. All data are presented as mean (error bars, SD) of the five animals that underwent single photon emission computed tomography (SPECT) imaging clot studies.

one dog (Dog 3) are presented in Figure 2. As shown, a PE in the left lower lobe was clearly visible within 4 hours from antibody injection. The PE could be detected in multiple views of SPECT images (a posterior–anterior view from a volume-rendered SPECT reprojection “rotating” image and an axial view are shown in Figure 2). The scans were 100% sensitive for PEs greater than 0.4 g in mass (see Table 1). In all dogs, PEs of this mass were clearly visible on the 4-hour scans (see Figure 3). The scans were also 100% specific. In none of the scans, including those of the dog without clots (see online supplement, Figure E1), were there findings interpreted as PEs or DVTs that were not subsequently confirmed by autopsy (false positives). The optimal time for imaging PEs that the authors investigated appeared to be the 4-hour interval after antibody injection (see Figures 2 and 3). At the earlier time of 2 hours, the blood pool background was high relative to clot labeling. At later times, image resolution suffered due to an overall lack of counts. Images of the lower extremities from one dog (Dog 5) are presented in Figure 4. As shown, a femoral DVT in the left leg was clearly visible as early as 4 hours after antibody injection. All DVTs greater than 0.4 g in mass were clearly visible on the 4-hour scans (see Table 2, Figure 5). The images were suggestive at 2 hours but again the blood pool background was high, relative to clot labeling. By 8 hours, the DVTs were still visible but the

resolution of the image had deteriorated. Thus, optimal timing for the detection of femoral DVTs was similar to that of PEs (i.e., 4 hours after antibody injection). Clot Characteristics

PEs and femoral DVTs were recovered from all dogs at the conclusion of the experiments (Tables 1 and 2). In each case, the location of clots detected by SPECT imaging coincided with the location of the clots recovered postmortem (see Tables 1 and 2, Figures 2–5). The median mass of PEs was 0.50 g (range, 0.33–0.57 g). The median mass of DVTs was 0.60 g (range, 0.11–0.75 g). As a group, PEs and DVTs did not differ significantly in mass. The median clot/blood ratio of PEs was 25.2 (range, 13.1–26.8). The median clot/blood ratio of DVTs was 12.4 (range, 8.8–14.8). As a group, the clot/blood ratio of the PEs was approximately twofold greater than that of the DVTs. Similarly, label uptake, as measured by the percentage of ID per gram of clot, was approximately twofold greater for PEs as compared with femoral DVTs (Tables 1 and 2). The smallest PE (0.33 g discovered in a division of a subsegmental artery of the left lower lobe in Dog 4) was not detected by SPECT imaging under the current study parameters. This embolus also possessed the lowest antibody uptake in terms of clot/blood ratio (13.1) and %ID/g (0.031). Similarly, the two smallest DVTs (0.11 in Dog 3 and 0.12 g in Dog 4) were not

Figure 2. SPECT images of the thoracic region of a representative dog (Dog 3, Table 1) obtained 2, 4, and 8 hours after injection of 400 ␮g (252 MBq) 99mTc-labeled DI3B6/ 22-80B3 Fab⬘. At each time point, a posterior–anterior view from a rotating image (top) and an axial view (bottom) are shown. A pulmonary embolus (E ) was detected in the left lower lobe. Blood pool activity in the heart (H ) and liver (L ) was also visible in some images. The characteristics of the embolus that was recovered postmortem are given in Table 1.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004 TABLE 1. PULMONARY EMBOLI RECOVERED FROM DOGS AFTER INJECTION OF TECHNETIUM-99M–LABELED DI3B6/22-80B3 FABⴕ FRAGMENTS Dog

PE Location (Lobe)

1 2 3 4 5 Mean SD

Right lower Left lower Left lower Left lower Left lower

Mass (g )

Clot/blood Ratio

%ID/g

Identified on Scan?

0.50 0.57 0.44 0.33 0.53 0.48 0.09

25.7 23.0 26.8 13.1 25.2 22.8 5.6

0.030 0.023 0.025 0.012 0.032 0.024 0.007

Yes Yes Yes No Yes

Definition of abbreviations: %ID/g ⫽ percent injected dose per gram clot; PE ⫽ pulmonary emboli. All emboli at least 0.4 g in mass were identified on the 4-h scans. There were no false positives in any of the lung lobes (five per dog) evaluated on autopsy.

detected by SPECT imaging. The clot/blood ratios of these DVTs were 14.8 and 8.8, respectively. Tissue Biodistribution Studies

The distribution of radiolabel among various tissues is presented in Table 3. Besides clots, the kidney was the major site of radiolabel uptake (kidney/blood ratio 295.8 ⫾ 91.5, 0.309 ⫾ 0.078%ID/g),

which is consistent with the high level of urinary excretion. A considerable amount of radiolabel was also concentrated in the bile (bile/blood ratio 11.9 ⫾ 6.3, 0.012 ⫾ 0.006%ID/g). Lesser amounts were found in the liver (liver/blood ratio 3.0 ⫾ 1.0, 0.003 ⫾ 0.001%ID/g) and thyroid (thyroid/blood ratio 2.8 ⫾ 1.7, 0.003 ⫾ 0.002%ID/g). No significant uptake was noted in femoral vessels sampled from the region of clot induction (vessel/blood ratio 1.6 ⫾ 0.4, 0.002 ⫾ 0.001%ID/g) or in any of the other tissues tested. Whole-body scans performed on two dogs did not reveal any additional sites of radiolabel uptake (data not shown).

DISCUSSION

Figure 3. Thoracic SPECT images 4 hours after injection of approximately 400 ␮g (260 MBq) 99mTc-labeled DI3B6/22-80B3 Fab⬘. For each dog, a single frame of the rotating image and a corresponding axial image are shown. Pulmonary emboli (white arrows) are identified in each dog except Dog 4, which had an embolus mass of less than 0.4 g (see Table 1).

These experiments demonstrate the reliable scintigraphic detection of both PEs and DVTs using SPECT scanning after intravenous injection of radiolabeled deimmunized anti–D-dimer Fab⬘ fragments. The technique detected all clots (both PEs and DVTs) 0.4 g or larger. The smaller, undetected PE corresponded to a division of a subsegmental pulmonary artery in the (25 kg) dog. A PE this size would be very unlikely to have significant consequences in a human and is well below the limit of detection by clinically available imaging techniques, including angiography (21). The two undetected DVTs were even smaller and by venography caused no visible venous flow limitation. The diagnostic technique employed in these experiments has several advantages over previous attempts at thrombus-specific nuclear imaging. First, signal interference from the large blood pool in the thorax was minimized by the use of SPECT scanning. The tomographic images in axial, sagittal and ‘rotating’ threedimensional views provided by SPECT (see online supplement) dramatically increased the spatial resolution of labeled PEs from less intensely labeled tissues within the thorax, compared with planar scans. In these experiments, PEs, which were virtually undetectable by planar scanning (data not shown), were identified very clearly by SPECT. SPECT was especially advantageous for central emboli, which lie close enough to the blood-filled heart (and far enough from the scintigraphic camera) that detection by planar scans would be virtually impossible. In these experiments, the murine antihuman D-dimer monoclonal antibodies we used were “deimmunized,” a process whereby polypeptide sequences identified as potential T cell– stimulating epitopes are modified by amino acid substitutions, reducing the risk of human antimurine antibody formation. Human antimurine antibodies may cause toxicity during clinical use of murine antibodies, especially during repeated administration (22). Because venous thrombosis is common and potentially recurrent, it may be tested for repeatedly in the same patient. For this reason, human antimurine antibodies are particularly unwanted in this diagnostic test. The deimmunized antibody

Morris, Marsh, Chiles, et al.: New Diagnostic Method for PE and DVT

991 Figure 4. SPECT images of the lower extremities of a representative dog (Dog 5, Table 2) obtained 2, 4, and 8 hours after injection of 400 ␮g (260 MBq) 99mTc-labeled DI3B6/22-80B3 Fab⬘. At each time point, an anterior view from a SPECT image is shown. A femoral thrombus (arrow) was detected in the left leg. The characteristics of the thrombus that was recovered postmortem are given in Table 2. Additional views of the legs (available in the online supplement, Figure E2) disclosed that the intensely highlighted region between the legs was from urine within the Foley urethral catheter and the small collection of radiolabel in the proximal portion of the right leg vein was due to the orientation of the femoral vein into the plane of the anterior image.

fragments we used in these canine experiments are identical to the ones that will be used in future clinical trials. The radiolabeled antibody fragments used in the present study are well suited for the diagnosis of PE and DVT. They are specific for the D-dimer region of cross-linked fibrin and so will bind to thrombi whether or not they are actively propagating (i.e., in the presence or absence of anticoagulants). In contrast, antifibrin antibodies that had been unsuccessful in previous clinical trials (9) were specific for the ␤-chain amino terminus of fibrin, which is only accessible to antibody binding during thrombus propagation (11, 13). In addition, deimmunization of the antibody will prevent patients from developing antimouse antibodies if the technique is used clinically. Fab⬘ fragments are cleared from the circulation rapidly enough to allow diagnostic imaging by 4 hours. Although this time frame may be longer than other clinical tests for thromboembolism, the high degree of accuracy of the technique, added to its ability to detect thrombi in the legs and the lungs, suggests that it will have an important role in the clinical detection of thrombosis, which can be further defined in clinical trials. The accurate detection of PEs and DVTs was observed in eight additional subjects (see online supplement), using variations on the methods reported here (15). As shown in the online supplement, the SPECT technique consistently allowed PEs and DVTs to be imaged despite variations in the imaging agent with respect to the amount of 99mTc used for labeling antibody doses and even the selection of the antibody clones (generated by the deimmunization technique). The robust nature of this imaging technique suggests that it will have promising results when tested in clinical trials. This report is somewhat limited by the small number of sub-

jects used. However, the consistency within the group, and within the similar experiments documented in the online supplement, supports the conclusion that the diagnostic technique is capable of reliable DVT and PE detection. The specificity of the antibodies for human D-dimer makes testing in other species somewhat problematic. It is possible that “human fibrin” clots formed in the present model may have been partially composed of canine fibrin. However, the presence of canine fibrin would likely have decreased the amount of antibody binding in these experiments, underestimating the accuracy of the technique. If this phenomenon did occur, one would expect that the clot imaging may be even better when the technique is used clinically in humans. Because the antibodies are cleared in the urine, residual radioactivity in the bladder may make adjacent thrombi difficult to detect. For this reason, it may be necessary for patients to empty their bladders to visualize thrombi in the pelvis. This consideration emphasizes why SPECT scanning, which would allow the reader to detect “hot spots” behind the bladder, is an important aspect of the technique. This technique may complement the currently used methods for diagnosing thromboembolism. For example, compression ultrasound, the most popular imaging test for DVT, is limited in its ability to distinguish recurrent thrombi from thickened, scarred veins resulting from previous thrombi. Likewise, thrombi in the calves and noncompressible veins in the abdomen and pelvis might be more readily identified with this technique than with ultrasound. It is interesting to note that PEs were more intensely labeled than DVTs. This may be because PEs are exposed to a higher

TABLE 2. FEMORAL THROMBI (DEEP VEIN THROMBI) RECOVERED FROM DOGS AFTER INJECTION OF TECHNETIUM-99M–LABELED DI3B6/22-80B3 FABⴕ FRAGMENTS Dog 1 2 3 4 5 Mean SD

DVT Location (Femoral Vein)

Mass (g )

Clot/Blood Ratio

%ID/g

Right Right Left Right Left

0.60 0.69 0.11 0.12 0.75 0.45 0.31

13.2 9.4 14.8 8.8 12.4 11.7 2.6

0.015 0.009 0.018 0.009 0.016 0.013 0.004

Identified on Scan? Yes Yes No No Yes

Definition of abbreviations: DVT ⫽ deep vein thrombi; %ID/g ⫽ percent injected dose per gram clot. All thrombi at least 0.4 g in mass were identified on the 4-h scans. There were no false positives in any of the legs evaluated on autopsy.

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 169 2004 TABLE 3. TISSUE BIODISTRIBUTION OF RADIOLABEL APPROXIMATELY 8 H AFTER INJECTION OF TECHNETIUM-99M–LABELED DI3B6/22-80B3 FABⴕ Radiolabel Uptake Tissue

Figure 5. SPECT images of the lower extremities of each dog 4 hours after injection of approximately 400 ␮g (260 MBq) 99mTclabeled DI3B6/22-80B3 Fab⬘. Femoral vein thrombi (white arrows) were identified in each dog except Dogs 3 and 4, each of which had a thrombus mass of less than 0.4 g (see Table 2).

Adrenal gland Bile Bladder Emboli (pulmonary) Kidney Liver Lung Muscle (heart) Muscle (skeletal) Skin Spleen Thrombi (femoral) Thyroid Vein (femoral)

Tissue/Blood Ratio 1.7 11.9 0.8 22.8 295.8 3.0 1.2 0.5 0.2 0.5 0.4 11.7 2.8 1.6

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.3 6.3 0.4 5.6 91.5 1.0 0.3 0.1 0.0 0.1 0.2 2.6 1.7 0.4

%ID/g 0.002 0.012 0.001 0.024 0.309 0.003 0.001 0.001 0.000 0.000 0.000 0.012 0.003 0.002

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.000 0.006 0.000 0.007 0.078 0.001 0.000 0.000 0.000 0.000 0.000 0.003 0.002 0.001

Definition of abbreviations: %ID/g ⫽ percent injected dose per gram clot; SPECT ⫽ single photon emission computed tomography. Data are presented as mean ⫾ SD of the five dogs that underwent SPECT imaging clot studies.

clinical situations, the overall sensitivity for disease detection in humans (with one or more “hot spots”) may be better than what we observed in the dog model. Modern techniques of mathematically correcting for signal attenuation from lung tissue (23, 24) may also improve the detection of central PEs in clinical situations. Finally, this technique has the sensitivity advantage of enabling the routine detection of both PE and DVT, which often coexist in the same patient (25–27). Conflict of Interest Statement : T.A.M. is the principal investigator of the study which was supported by unrestricted grants from Alen and served as a consultant on an advisory board regarding the antibody mentioned in this manuscript; J.J.M. has no declared conflict of interest; P.G.C. has no declared conflict of interest; R.G.K. has no declared conflict of interest; C.A.P. has no declared conflict of interest; P.F.S. is a full-time employee of Agen Biomedical Ltd., a wholly owned subsidiary of Agemix Ltd., M.G. is a full-time employee of Alen Biomedical, a wholly owned subsidiary of Agemix Ltd. Acknowledgment : The authors express their appreciation to Dr. Carl Hoh from the Division of Nuclear Medicine for his technical advice and constant support throughout this project.

References

proportion of the Q and therefore come in contact with more antibody fragments over time than the DVTs. It is also possible that, unlike DVTs, PEs are not adherent to the venous wall and so have a larger proportion of their surface area exposed to circulating blood. Whatever the reason, the result is fortuitous, because the diagnosis of PE using currently available techniques is particularly problematic. Ventilation/perfusion scanning to detect PE can be difficult to interpret in regions of the lung that have nonthrombotic reasons for decreased perfusion, such as areas of diseased lung parenchyma that have decreased ventilation and perfusion. The technique we are reporting would be useful under these circumstances because one would not expect the antifibrin antibodies to accumulate in these regions. Likewise, problematic areas for contrast-enhanced helical computed tomography scanning, such as hilar lymph nodes or anatomical variations in small pulmonary artery branches would be unlikely to interfere with the accuracy of this thrombus-targeting technique. The experiments in this report suggest that it would be feasible to diagnose thromboembolic disease in humans using a similar technique. Furthermore, because PEs are often multiple in

1. Silverstein MD, Heit JA, Mohr DN, Petterson TM, O’Fallon WM, Melton LJR. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Arch Intern Med 1998; 158:585–593. 2. PIOPED Investigators. Value of the ventilation/perfusion scan in acute pulmonary embolism: results of the Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED). JAMA 1990;263:2753–2759. 3. Perrier A, Howarth N, Didier D, Loubeyre P, Unger PF, de Moerloose P, Slosman D, Junod A, Bounameaux H. Performance of helical computed tomography in unselected outpatients with suspected pulmonary embolism. Ann Intern Med 2001;135:88–97. 4. Gillies TE, Ruckley CV, Nixon SJ. Still missing the boat with fatal pulmonary embolism. Br J Surg 1996;83:1394–1395. 5. Morgenthaler TI, Ryu JH. Clinical characteristics of fatal pulmonary embolism in a referral hospital. Mayo Clin Proc 1995;70:417–424. 6. Rubinstein I, Murray D, Hoffstein V. Fatal pulmonary emboli in hospitalized patients: an autopsy study. Arch Intern Med 1988;148:1425–1426. 7. Douketis JD, Kearon C, Bates S, Duku EK, Ginsberg JS. Risk of fatal pulmonary embolism in patients with treated venous thromboembolism. JAMA 1998;279:458–462. 8. Lusiani L, Zanco P, Visona A, Breggion G, Pagnan A, Ferlin G. Immunoscintigraphic detection of venous thrombosis of the lower extremities by means of human anitfibrin monoclonal antibodies labeled with In-111. Angiology 1989;40:671–677.

Morris, Marsh, Chiles, et al.: New Diagnostic Method for PE and DVT

993

9. Jung M, Kletter K, Dudczak R, Koppensteiner R, Minar E, Kahls P, Stumpflen A, Pokieser P, Ehringer H. Deep vein thrombosis: scintigraphic diagnosis with In-111-labeled monoclonal antifibrin antibodies. Radiology 1989;173:469–475. 10. Schaible TF, Alavi A. Antifibrin scintigraphy in the diagnostic evaluation of acute deep venous thrombosis. Semin Nucl Med 1991;21:313–324. 11. Morris TA, Marsh JJ, Fagnani R, Hagan M, Moser KM. Degree of polymer organization decreases the binding of a monoclonal antibody raised against the beta-chain amino terminus of fibrin. Thromb Haemost 1997;77:704–709. 12. Hyers TM, Agnelli G, Hull RD, Morris TA, Samama M, Tapson V, Weg JG. Antithrombotic therapy for venous thromboembolic disease. Chest 2001;119(1 Suppl):176S–193S. 13. Morris TA, Marsh JJ, Konopka R, Pedersen CA, Chiles PG, Fagnani R, Hagan M, Moser KM. Antibodies against the fibrin beta-chain aminoterminus detect active canine venous thrombi. Circulation 1997;96: 3173–3179. 14. Morris TA, Marsh JJ, Konopka R, Pedersen CA, Chiles PG. Anti-thrombotic efficacies of enoxaparin, dalteparin, and unfractionated heparin in venous thrombo-embolism. Thromb Res 2000;100:185–194. 15. Morris TA, Pedersen CA, Konopka RG, Chiles PG, Marsh JJ. SPECT scan images of pulmonary emboli using radiolabeled antibodies [abstract]. Am J Respir Crit Care Med 2001;163:A198. 16. Rylatt DB, Blake AS, Cottis LE, Massingham DA, Fletcher WA, Masci PP, Whitaker AN, Elms M, Bunce I, Webber AJ. An immunoassay for human D dimer using monoclonal antibodies. Thromb Res 1983: 31:767–778. 17. Lamoyi E, Nisonoff A. Preparation of F(ab’)2 fragments from mouse IgG of various subclasses. J Immunol Methods 1983;56:235–243. 18. Lee FT, Milner LJ, Boniface GR, Bautovich GJ, Weedon AR, Bundesen PG, Rylatt DB, Walker KZ. Evaluation of thrombus detection in a

rabbit model using a technetium-99m–labelled anti-fibrin monoclonal antibody. Immunol Cell Biol 1992;70:173–179. Lee FT, Boniface GR, Lambrecht RM, Rylatt DB, Bundesen PG. Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab’ for thrombus imaging. Immunol Cell Biol 1993;71: 117–124. Marsh JJ, Konopka RG, Lang IM, Wang HY, Pedersen C, Chiles P, Reilly CF, Moser KM. Suppression of thrombolysis in a canine model of pulmonary embolism. Circulation 1994;90:3091–3097. Tapson VF, Carroll BA, Davidson BL, Elliott CG, Fedullo PF, Hales CA, Hull RD, Hyers TM, Leeper KJ, Morris TA, et al. The diagnostic approach to acute venous thromboembolism: clinical practice guideline: American Thoracic Society. Am J Respir Crit Care Med 1999; 160:1043–1066. Mountain A, Adair JR. Engineering antibodies for therapy. Biotechnol Genet Eng Rev 1992;10:1–142. Madsen MT, Kirchner PT, Grover-McKay M, Aktay R, Seabold JS, Rezai K, Kelly G. Emission-based attenuation correction of myocardial perfusion studies. J Nucl Cardiol 1997;4:477–486. Wallis JW, Miller TR, Koppel P. Attenuation correction in cardiac SPECT without a transmission measurement. J Nucl Med 1995;36: 506–512. Girard P, Decousus M, Laporte S, Buchmuller A, Herve P, Lamer C, Parent F, Tardy B. Diagnosis of pulmonary embolism in patients with proximal deep vein thrombosis: specificity of symptoms and perfusion defects at baseline and during anticoagulant therapy. Am J Respir Crit Care Med 2001;164:1033–1037. Hull RD, Raskob GE, Ginsberg JS, Panju AA, Brill-Edwards P, Coates G, Pineo GF. A noninvasive strategy for the treatment of patients with suspected pulmonary embolism. Arch Intern Med 1994;154:289–297. Moser KM, Fedullo PF, LitteJohn JK, Crawford R. Frequent asymptomatic pulmonary embolism in patients with deep venous thrombosis [published erratum appears in JAMA 1994;271:1908]. JAMA 1994;271: 223–225.

19.

20. 21.

22. 23. 24. 25.

26. 27.