Radiopharmaceuticals for thrombus detection

Radiopharmaceuticals for Thrombus Detection Linda C. Knight

Most of t h e c o m p o n e n t s of the t h r o m b o t i c and fibrinolytic systems have at some time been evaluated as a means of carrying a radiolabel specifically t o t h r o m b i , although v e r y f e w have been promising enough to e m e r g e from investigational status to routine clinical use. N e w approaches are being explored, including improved methods of labeling platelets, chemically modified forms of previously tested plasma proteins, and n e w biomolecules, including monoclonal antibodies specific for fibrin and platelets. The current goal is to find one or m o r e r a d i o t r a c ers that bind specifically and rapidly to thrombi, and t h a t also have a rapid blood disappearance rate, p e r m i t t i n g a clear diagnosis within a f e w hours after

injection. Because this test may be needed to assess the course of therapy in an anticoagulated patient, t h e ideal radiopharmaceutical should be able to locate t h r o m b i w i t h o u t interference by anticoagulants. Until a suitable thrombus-specific radiopharmaceutical becomes generally available, many hospitals will continue to a t t e m p t to make a diagnosis with nonspecific radiopharmacauticals that can at best provide blood pool images to indicate filling defects. Several of the n e w a p p r o a c h e s seem likely t o provide the radiopharmaceutical sought, although clinical trials are at an early stage. 9 1990 by W.B. Saunders Company.

H R O M B O E M B O L I C disease is a common

Contrast venography has long been considered the standard test for DVT, but its high incidence of side effects prevents it from being used repeatedly to assess the course of therapy. 2 Compression B-mode ultrasound is rapidly gaining popularity for diagnosing the presence of thrombi in the legs, 3'4 and it may soon be difficult for a nuclear medicine test to compete with this noninvasive modality. However, a specific radiotracer that binds to thrombi with adequate specificity may permit imaging of thrombi anywhere in the body, and such a thrombus-specific agent could be used to target therapeutic agents: There are two main approaches to selecting a radiopharmaceutical that will bind specifically to thrombi. Venous thrombi are composed primarily of polymers of fibrin with entrapped cells, alternating with layers of aggregated platelets held to together with fibrin. 6 Arterial thrombi are composed mainly of aggregated platelets, with a lesser amount of fibrin. 6 Thus, in either case, selecting agents that will bind to either fibrin deposits or to platelet deposits seem to be the obvious approaches to specific thrombus detection.

T disorder, with an estimated incidence of 2.5 million cases per year in the United States

alone.1 Even asymptomatic deep venous thrombosis (DVT) carries a significant risk of morbidity and mortality; thus, early detection and treatment is important. Unfortunately, clinical signs of thrombosis are highly unreliable, and an objective test is needed. Anticoagulant therapy is available to treat thrombosis, but it carries a significant risk and should not be used without prior diagnosis confirming the presence of thrombi. Nuclear medicine scientists have evaluated numerous thrombus-specific agents that could carry a radiotracer to a thrombus to permit its external detection by gamma camera imaging. To date, however, in nonuniversity settings the commonly performed nuclear medicine imaging tests for DVT are based on nonspecific radiopharmaceuticals used for radionuclide venography. Although specific probes have been investigated for imaging thrombi, they have not been widely available, and are not felt by many investigators to provide an adequate solution to the need for a thrombus-specific radiopharmaceutical for rapid imaging of thrombi.

FIBRIN-DIRECTED RADIOPHARMACEUTICALS

Fibrinogen From the Section of Nuclear Medicine, Thrombosis Research Center, Temple University School of Medicine and Hospital, Philadelphia, PA. Address reprint requests to Linda C. Knight, PhD, Nuclear Medicine, Temple University Hospital, 3401 N Broad St, Philadelphia, PA 19140. 9 1990 by W.B. Saunders Company. 0001-2998/90/2001-0005505.00/0 52

The matrix of a venous thrombus is composed largely of fibrin, a material that spontaneously polymerizes when formed from its soluble precursor, fibrinogen. In the search for radiolabeled compounds to use as probes for thrombi in vivo, many investigators have studied molecules that can bind to fibrin by fibrin's inherent self-polySeminars in Nuclear Medicine, Vol XX, No 1 (January), 1990: pp 52-67

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

merization sites. One of the compounds studied most extensively has been fibrinogen, which becomes converted to fibrin in vivo and is incorporated into the matrix of a thrombus as it forms. 7 Radioiodinated fibrinogen has provided the basis for a successful screening test for newly forming thrombi, the Fibrinogen Uptake Test (FUT), which does not provide images.

1251FUT There have been several good reviews on the subject of this test. s~~ The FUT involves injection of 100 #Ci of xzSI-human fibrinogen intravenously into patients at risk for developing DVT, before thrombi have formed. Nonradioactive sodium iodide should be given orally before the test to block thyroid uptake of ~25I. Using a handheld scintillation detector attached to a portable ratemeter, counts are taken at marked points along the patient's legs. The counts at each point along the legs are expressed as a percent of the counts obtained when the probe is placed over the precordial region. The study can be highly sensitive and specific8 if established criteria are used for interpreting the resultsll: a study is considered positive for thrombus if there is an increase of >_20% over 1) an adjacent point on the same leg; 2) the same point on the opposite leg; or 3) the same point on the previous day. The test is convenient because it can be performed at the patient's bedside.125I has a sufficiently long physical half-life (60 days) to permit the screening study to be carried out for one week, and the shelf life of the radiopharmaceutical is long. However, the low-energy gamma rays associated with 125I do not permit imaging of the thrombi, and the count data is unreliable in the upper thigh and pelvis, owing to scattered radiation from the bladder and attenuation by overlying tissue in the thigh. It is generally believed that most calf vein thrombi are clinically innocuous and that it is ileofemoral thrombi, not accurately detected by this test, that pose the greatest risk for significant pulmonary embolism. 9 Other limitations are that the test is insensitive to older thrombi, 12 and that anticoagulants are known to interfere with the incorporation of labeled fibrinogen into a fresh thrombi. ~3 In a recent report by Paiement et al, 14 537 patients undergoing hip replacement surgery were studied by both FUT and contrast venogra-

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phy. Compared with venography in the calf, the FUT had a sensitivity of 59.1% and a specificity of 95.7%. The sensitivity of the FUT in the thigh was only 13.7%. Despite its limitations, the FUT has become a standard throughout the world as a screening test for forming DVT. ~522 It has even been used to document the incidence of postvenographic thrombophlebitis. 2324 Although this nonimaging test has found a place in the diagnosis of DVT, ~25I human fibrinogen may not be commercially available much longer. The sole supplier of this radiopharmaceutical in the United States and Europe is Amersham (US: Arlington Heights, IL; Europe: Amersham, England), which plans to stop producing the product as soon as the present stock of human fibrinogen is exhausted. The company has stated that customers will be notified approximately 6 months before the expected termination of the product.

Fibrinogen Imaging Techniques When labeled with appropriate imaging radionuclides, the uptake of fibrinogen by forming thrombi can be imaged, and diagnosis can be extended from the calves to most of the body. Several approaches to labeling fibrinogen have been used. 12~I fibrinogen. Iodination of fibrinogen is easy to carry out and the product has been wellcharacterized as to its retention of biologic function25; in fact, ~31I was used as the radiolabel for the first clinical imaging study with fibrinogen reported in the literature. 26 Because of dosimetry considerations and image quality, however, interest in this approach was limited until ~23Ibecame available. The use of 1231 fibrinogen for imaging DVT has been previously reviewed. 27 Only one institution has consistently used 123I fibrinogen for imaging of thrombi, probably because it is not commercially available and investigators must prepare it themselves. DeNardo et a127-29reported high correlation between 24-hour images with their use of this agent and either contrast venography or 125I fibrinogen uptake test: sensitivity was 92% and specificity was 87%. 28 99mTCfibrinogen. Several investigators have attempted to develop ways to radiolabel fibrinogen with 99mTc. The short physical half-life of 99mTC,coupled with the long lifetime of fibrin-

54

ogen in the blood circulation, presents something of a dilemma: by the time the blood background has cleared sufficiently for the target-to-background ratio to be high enough to permit visualization of the thrombus, the count rate may be rather low from physical decay. Despite this, 99mTcis attractive for labeling because it is more readily available than 123I. An initial report of an electrolytic method for preparing 99mTcfibrinogen with satisfactory in vitro and in vivo properties 3~ did not inspire follow-up studies. Another labeling method reported by Jeghers et al, 31 involving stannous chloride reduction of Tc in the presence of fibrinogen in an alkaline medium, seemed to provide a stable label in vivo and a long lifetime in the blood. This method has become the basis for a kit that became available in Europe. Studies of this compound in animal models indicated that it had the expected ability to image fresh thrombi, but was less able to image older thrombi. 32 In clinical trials, the blood levels of 99mTc fibrinogen were still quite high at 18 hours post-injection, making visualization of thrombus over the blood background difficult. However, Jonckheer et a133developed criteria for interpreting the early images without dependence on complete reduction of blood background: 1) nonvisualization of a large vein or segment thereof; 2) visualization of veins that are not normally seen, such as the saphenous vein; 3) irregular, beadlike appearance of a vein; or 4) actual hot spots representing deposition of 99mTcfibrinogen. Jonckheer et al caution that the hot spots can be nonspecific and are a potential cause of falsepositive results. The remainder of the criteria are much like those for blood pool radionuclide venography with nonspecific radiotracers. Vorne et a134 more recently evaluated 99mTc fibrinogen in a group of 38 patients with suspected DVT. The 99mTcfibrinogen study was done in three phases: 1) radionuclide venography following injection of the fibrinogen in the dorsal veins of the foot; 2) blood pool imaging at 5 to 20 minutes; and 3) uptake imaging at 4 to 6 and 17 to 26 hours. It was concluded that the radionuclide venography portion of the study was not helpful and could be replaced with a simple antecubital injection. When compared with contrast venography, the three-phase 99mTcfibrinogen study had a sensitivity of 73% and a

LINDA C. KNIGHT

specificity of 100%. Vorne et al felt that the 17to 26-hour delayed images were necessary for making an accurate diagnosis. Positive results on the blood pool phase of the study coupled with negative results on the delayed uptake imaging was believed to represent old thrombi. 67Gafibrinogen. By using a bifunctional chelating agent, deferoxamine (DFO), as the metal chelating agent, Ohmomo et a135 successfully attached a 67Ga label to fibrinogen without significant loss of function. The ability of this radiopharmaceutical to image thrombi in a rabbit model was evaluated,35 but no clinical studies were reported. The in vivo behavior of 67Ga fibrinogen was recently improved further, however, when a water-soluble polymer spacer molecule, dialdehyde starch (DAS), was placed between the Ga and the DFO. Use of the spacer molecule resulted in labeled fibrinogen that has higher specific activity, clottability, and stability in vivo.36 This new tracer, called Ga-67 DASDFO-fibrinogen, has achieved widespread use in Japan where it is available as a kit. The radiopharmaceutical is prepared by adding standard 67Ga citrate to the DAS-DFO-fibrinogen and allowing it to react for 30 minutes. The usual patient dose is 2 mCi. Yamamoto et a136 reported that in a group of patients with DVT proven by radionuclide venography and/or contrast venography, 10 of 17 studies showed abnormal accumulations of 67Ga DAS-DFO-fibrinogen at 48 hours postinjection. In nine cases of pulmonary emboli in the study, two of the patients had hot spots in the lung field by the 67Ga DAS-DFO-fibrinogen. Scans of seven of eight patients on anticoagulant therapy showed positive results. A number of hospitals in Japan have been using this agent with reported moderate success in imaging DVT. 36"41 In a study of four patients, Matsuura et a137 noted the ability to image tumor (bronchogenic carcinoma) and microthrombi in the region of a hepatoma, but was not able to visualize DVT. By imaging out to 96 hours postinjection, Yamamoto et a136 reported abnormal accumulation of 67Ga DAS-DFO-fibrinogen in seven patients with either venous or arterial thrombi. Kusakabe et a139 performed a clinical study on 40 patients with thrombi. The optimum imaging time was found to be 48 to 96 hours post-injection. In the entire patient population, 21 of 40 had positive planar imaging studies;

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

however, in a subgroup of patients with aortic aneurysm, 83% had positive studies. Itoh et al 4~ found five positive studies that correlated with other examinations. It was noted that arterial thrombi may continue to take up the tracer for as much as 1 year after onset of symptoms, whereas venous thrombi had to be less than 3 weeks old to be visualized with the 67Ga DAS-DFO-fibrinogen. Suzuki et a141 evaluated the biodistribution and kinetics of 67Ga DAS-DFO-fibrinogen in normal volunteers as well as in patients. Liver and kidney uptake were noted in early images, and liver, lung, and heart activity had diminished by 48 hours. At 72 hours post-injection, fresh and aged arterial thrombi associated with abdominal aneurysms and occluded bypass grafts were clearly visualized. Fresh venous thrombi were also visualized.

Soluble Fibrin Because of its long residence time in the blood, fibrinogen is not suitable as the basis of a rapid imaging test, one in which hot spots would be visible over the background in under 4 hours. In addition, radiolabeled fibrinogen does not incorporate sufficiently into older thrombi in which fibrin deposition has slowed (although these thrombi are still at risk for eventual propagation and embolization). Soluble fibrin oligomers can be prepared that have binding affinity for preformed thrombi, 42 but maintaining the solubility of these forms can be a problem. A recent study has reported new clinical studies with soluble fibrin, 43 with apparent modifications in the method to stabilize the fibrin in a soluble form. The investigators of this latter study reported that the soluble fibrin cleared from the blood pool more rapidly than fibrinogen, as expected; however, its uptake in thrombi was no greater and did not offer any advantages in terms of earlier detection or increased sensitivity for older thrombi. This phenomenon can be explained by the theory of Olexa and Budzynski 44 that fibrin monomer has available binding sites for fibrinogen. It is likely that fibrin monomer, soon after injection, binds two molecules of fibrinogen and then circulates as a fibrin oligomer. Such a molecule would not h a v e increased affinity for thrombi compared with fibrinogen: the same binding sites would be operative.

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Antifibrin Monoclonal Antibodies In the 1960s, Spar et al 4s investigated the use of polyclonal antibodies to detect thrombi in vivo. The antibodies were reactive with fibrinogen as well as fibrin. As a result, the radiolabeled antibodies became bound to circulating fibrinogen promptly after injection, and the radiolabel then essentially traced the incorporation of fibrinogen into forming thrombi. This method was found to be effective in permitting detection of thrombi, but the radiolabeled antibody-fibrinogen complex had a long residence time in the blood pool, resulting in a long delay (24 to 48 hours) before positive images could be obtained. Some investigators are now studying monoclonal antibodies for imaging deposits of fibrin. Theoretically, monoclonal antibodies offer the possibility of tailoring a radiopharmaceutical to specifically detect individual molecular sites exposed on fibrin deposits in thrombi, but not exposed on species found in the circulating blood. If the antibodies can be induced to leave the blood pool rapidly, this approach may provide a more rapid test for pre-existing thrombi. Recently, a number of monoclonal antibodies reactive with human fibrin have been reported (Table 1), all of which recognize sites on fibrin that are not exposed on fibrinogen. Some of the antibodies recognize sites that are exposed on fibrin monomer by cleavage of fibrinopeptide A o r B, 46"48 while the others recognize the region of linkage between the D domains of two fibrin molecules in a fibrin polymer. 49"52 Antibody fragments can be prepared by treating whole immunoglobulin G with enzymes. It has been shown that fragments of antifibrin Table 1. Monoclonal Antibodies That Recognize Fibrin Antibody 59D8

T2Gls Anti-fbn 17 DD-3B6-22 DG-1 15C5 and 8D3 GC4

Epitope Recognized

Reference

First seven amino acids of amino terminus of/~ chain of fibrin Amino terminus of/~ chain of fibrin Amino terminus of
46

47 48 49 50 51 52

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antibody retain sufficient immunoreactivity to bind to clots in vitro, although a small amount of affinity may be lost. 53 It should be noted that Fab fragments of some antifibrin antibodies still have sufficient immunoreactivity to be useful in vivo, 54 while Fab fragments of other antifibrin antibodies are not sufficiently immunoreactive. 55 The use of antibody fragments would be advantageous for enhancing the blood disappearance rate so that blood background is removed quickly. Use of antibody fragments should also favor excretion by the kidneys rather than by the liver. 56 Minimizing liver uptake may be important if the antifibrin antibodies are to be used for imaging pulmonary emboli. Most of the in vivo work has been done with the antibodies 59D8 and T2Gls, both of which recognize the site on the beta chain of fibrin monomer that is exposed when fibrinopeptide B is cleaved by thrombin: 6'4v Rosebrough et a157 studied 13q-labeled T 2 G l s antifibrin IgG in a model of fresh thrombi created by placing an embolization coil in a dog's femoral vein. They found that positive gamma camera images were obtained within 4 to 48 hours following injection. Thrombus-to-blood and thrombus-to-muscle ratios averaged 8.4 and 228, respectively, at 48 hours post-injection. Similar results were obtained with a model of aged thrombi. 58 Knight et a154 studied rain-labeled Fab fragments of antibody 59D8 in animal models of thrombi and emboli. In a dog model, thrombus-to-blood ratios of -8:1 were found at 24 hours post-injection; positive images of fresh thrombi were achieved within 4 hours after injection. Knight et al also demonstrated that Fab' fragments of antifibrin T2G 1s labeled with 99roTewere capable of producing images of fresh thrombi in dogs within 1 to 2 hours post-injection. 59'6~ Data in animal models suggest that heparin interferes with the thrombus uptake of the antibeta chain antibodies, either by inhibition of thrombus propagation or by loss of antigenic sites by fibrinolysis.6~ Theoretically, antibodies that recognize the D-dimer region of fibrin may be more successful in imaging aged thrombi and may be less susceptible to interference by heparin. Rosebrough et a152 reported initial studies with a new monoclonal antibody, GC4, that recognizes a site on fibrin not exposed until after partial plasmin degradationl When labeled with

LINDA C. KNIGHT

131I and 1i~in, the GC4 antibody exhibited better localization in animal thrombi than antibody T2Gls, especially when the thrombi were aged and when heparin was present. Hashimoto et a151 studied the ability of an anti-D-dimer antibody to image thrombi in an animal model. The ratio of thrombus-containing vessel to normal vessel was -11:1 at 24 hours post-injection, but was visible by imaging at 2 hours. Alavi et a162'63 have begun clinical trials with lllln-59D8 Fab in patients with suspected deep venous thrombosis. In 28 patients, imaging of the legs was performed at 4 and 24 hours post injection. Of 53 thrombi sites identified by contrast venography, 41 were correctly identified by 1HIn-antifibrin imaging. Most of the missed sites were in patients who were on heparin therapy at the time of imaging. Further results of this and other clinical trials of antifibrin antibodies are eagerly awaited. One possible disadvantage of the murine monoclonal antibody approach is the potential for formation of human AntiMouse antibodies (HAMA). With a study such as thrombus imaging, which may need to be performed repeatedly to assess the course of therapy, the risk of anaphylaxis is a major concern. Although the use of antibody fragments may reduce this risk, recent reports suggest that repeated injections even of Fab fragments eventually result in a significant H A M A titer. 64 Fragment E 1

Fragment E 1 is a 60 kd fragment of human fibrin, produced by controlled plasmin digestion of cross-linked fibrin. 65'66 It comprises the amino terminal regions of all six polypeptide chains of fibrin. 67 It has been shown to contain a pair of dimeric binding sites that are complementary in binding to sites in the D domains of fibrin, which are formed when fibrin polymerizes. 44 Thus, fragment E1 binds to fibrin dimers and polymers, but not to fibrin monomer nor to fibrinogen. 65 It may be more able to bind to aged thrombi than fibrinogen, fibrin, or monoclonal antibodies, which recognize fibrin monomer. Fragment E 1 is readily iodinated, as it contains 16 tyrosine residues. 67 Radioiodinated fragment E 1 (labeled with 131I or 1231) was evaluated in a pig model of venous thrombi in comparison with 125I fibrinogen. 68 In fresh thrombi (1 to 6 hours

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

old), the thrombus-to-blood ratios for fragment E 1were not significantly different from thrombusto-blood ratios for fibrinogen. However, fragment E 1 had significantly better thrombus-toblood ratios than fibrinogen for aged thrombi (>20 hours old at the time of injection). Although some of fragment El'S advantage is due to its more rapid blood disappearance, absolute uptake (% injected dose/g) of the fragment E 1 was also better than fibrinogen for aged thrombi. The magnitude of the thrombus-to-blood ratios obtained in this study, ~ 100:1, suggested that the thrombi should be easily imaged over the blood background, even at times earlier than 24 hours. Fragment E 1 has been derivatized with metal chelating groups, diethylenetriaminepentaacetic acid (DTPA) and deferoxamine, to facilitate labeling with 111|n69 and 67Ga.7~ After injection into rabbits with induced venous thrombi, 67GaDFO-fragment El, 2 produced thrombus-to-blood ratios of 6.3 and 9.1 at 6 and 20 hours after injection, respectively. 7~ Although these labels appeared promising in animal models, they have not yet achieved success in humans. To date, only limited initial clinical trials of fragment E 1 have been performed. In a study with 123I-fragment E 1, patients received contrast venography as a comparative test. 71 One mCi of 123I-fragment E l (100 #g) was administered in a peripheral vein and imaging was begun immediately. Images of thrombi were positive within 20 to 30 minutes after injection. All patients (n = 5) with documented venous thrombi had rapid localization of the fragment E 1 in the area of thrombus. Patients without evidence ofthrombi (n = 5) showed no focal localization, although two of these patients showed diffuse uptake along the length of the veins due to superficial phlebitis. In a patient with venographically proven thrombi in the thigh, focal uptake was clearly observed by 30 minutes post-injection (Fig 1). The positive images were not affected by concurrent heparin therapy. Due to lack of availability of radiolabeling grade 123I, however, image quality suffered from the low specific activity (10 mCi/mg) and low injected dose (1 mCi 1231). In a recent study, fragment E 1 was labeled with 123I at higher specific activity (100 to 500 mCi/mg) using a closed-system kit. 72 Up to 10 mCi 123I-fragment E 1was injected intravenously, and imaging was performed at 0 to 60 minutes, 2

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to 4 hours, and in some cases at 24 hours. Marked uptake of tracer was consistently observed in areas corresponding to the abnormalities in the correlative tests (contrast venography or B-mode ultrasound). The uptake was visible within the first hour in 19 of 20 patients and persisted as long as imaging was carried out. The 10 mCi dose permitted initial flow images to be obtained within the first few minutes after injection, to outline the vascular system and indicate areas of occlusion. Later images after blood background had cleared showed residual binding to exposed thrombus. Heparin had no apparent effect on fragment E l binding to thrombus, but enhanced the rate of initial blood clearance: less than 20% of the dose was circulating at 30 minutes. These studies suggest that radiolabeled fragment E 1 is a potentially valuable agent for diagnosis of DVT. Plasmin

Plasmin is the enzyme in the blood primarily responsible for the dissolution of fresh thrombi. Plasmin and its inactive zymogen plasminogen have both been evaluated as markers for thrombi, based on the assumption that they should have greater affinity for aged thrombi than markers like fibrinogen. A study by Back et a173 indicated that plasmin might have the highest thrombus affinity of any of the components of the fibrinolytic system. The mechanism of action of plasmin is somewhat controversial, since Alkjaersig et a174 found evidence that trapped plasminogen appears to become activated to plasmin and dissolves fibrin from within a thrombus. This casts doubt on whether exogenous labeled plasmin would bind to a thrombus in sufficient amounts to permit external imaging. In addition, the blood contains high concentrations of plasmin inhibitors, which rapidly form complexes with plasmin once it is injected, and prevent it from binding to a thrombus. 75 Before 1976, radioisotopes of iodine were used to label plasminogen and plasmin. 76-78 More recently, interest has been focused on the use of a 99rnTc label for plasmin, enhanced by the availability in Europe of a kit containing porcine plasmin for labeling with 99mTc. Several clinical trials reported scintimetry (leg counting with a handheld probe) studies using 99mTc-plasmin. No explanations were offered as to why imaging was

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LINDA C. KNIGHT

Fig 1. (A) Anteroposterior radiograph of the right hip of one patient obtained during contrast venography. This patient had undergone a total hip replacement 5 days earlier, The venogram shows an intraluminal filling defect representing thrombus (arrows) in the femoral vein. (B) Anterior ~231-fragment E~ scintigraphic image of both hips in the same patient, acquired during the first 30 minutes post-injection. This image demonstrates diffuse radiotracer accumulation around the recently implanted hip prosthesis (curved arrow). In addition, there is focal tracer localization in the right thigh (straight arrows), which corresponds to the intraluminal thrombus seen in (A). The diffuse activity represents fibrin deposition at the surgical site, and the focal abnormality confirms the presence of venous thrombosis. The level of the knees is indicated (K). (Reprinted with permission. 71)

not attempted. The procedure for scintimetry with plasmin is similar to the FUT, except that the counts are taken at only two times: 5 to 15 minutes post-injection and again at 30 minutes post-injection. In 105 patients studied by Edenbrandt et al, 79 99mTc-plasmin scintimetry was found to have a sensitivity of 100% and a specificity of 51% when compared with contrast venography. In a study by Deacon et al, 8~ there was 100% agreement between 99mTc-plasmin scintimetry and the 125I FUT for patients at risk for developing thrombosis. However, 99mTcplasmin scintimetry produced several falsenegative and false-positive results in patients who had clinical signs of DVT before the tracer was administered. Adolfsson et a181 studied l l0 patients with contrast venography and found a sensitivity of 91% and a specificity of 33%. Other groups agree that plasmin is most useful as a

screening procedure, as it rarely misses thrombi, but has a tendency to produce false-positive results. 82,83 The usefulness of 99mTc-plasmin for imaging of thrombi was recently evaluated by Christensen et al. 84 Using contrast venography as a reference procedure, the sensitivity and specificity were 50% and 91% for scintigraphy and 33% and 75%, respectively, for scintimetry. Explanations given for the false-positive results were the presence of hematomas, edema, and superficial thrombophlebitis. False-negative resuits were attributed to bilateral thrombi, when a criterion for positivity is uptake in one leg higher than the contralateral limb. The investigators concluded that 99mTc-plasmin scintigraphy and scintimetry are of no value as screening tests for DVT. In order to improve the thrombus uptake of

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

99mTc-plasmin by minimizing the binding by inhibitors in plasma, Baker et a185 modified plasmin by acylation. It was found that modification with a variety of acylating agents effectively blocked binding by ot2-antiplasmin without affecting the fibrin affinity of the plasmin. In an animal model, 99mTc-acylplasmin was found to have absolute uptake in thrombi that was two to three times as great as 99mTc-plasmin. Thrombus-to-blood ratios at 3 hours post-injection were c o m p a r a b l e with thrombus-to-blood ratios obtained at 18 hours post-injection with 1251fibrinogen because of more rapid blood clearance, but the absolute uptake of the 99mTcacylplasmin in the thrombus was about one third that of fibrinogen.

Plasminogen Activators Radiolabeled urokinase and streptokinase were previously evaluated for their ability to image thrombi in vivo. These tracers were discussed in a previous review. 86 These agents are not believed to have any specificity for fibrin. Because of lack of success in imaging thrombi clinically with these agents, no further work has been reported since the last review. t-PA. It has been shown that tissue plasminogen activator (t-PA) has affinity for fibrin, in contrast to streptokinase and urokinase. Hnatowich et a187 radiolabeled recombinant t-PA with lllln for thrombus detection. Their results in animal models suggested that the blood clearance of the labeled t-PA was very rapid, with an estimated initial clearance half-time of 4.9 minutes. In the one animal studied, the thrombus-toblood ratio reached 6:1 only 31 minutes after injection. Uehara et a188 also performed animal experiments with 131I t-PA and concluded that the lesion-to-blood ratios, which were less than 2:1, were not high enough for the radiopharmaceutical to be useful for imaging preexisting thrombi clinically. It is not surprising that the uptake ratios were low, because plaminogen activator inhibitors are present in plasma, which can rapidly bind the diagnostic dose of t-PA that was administered in these studies. 89 Uehara et al may have experienced less success in imaging thrombi than Hnatowich because they administered a much smaller amount of t-PA per unit of body weight than did Hnatowich et al. Overcom-

59

ing the problem of inhibitors in plasma is crucial to the success of this approach.

Heparin Several investigators have observed 99mTcheparin to bind to damaged myocardium and blood vessels. 9~ In a clinical study to evaluate the usefulness of 99mTc-heparin for detection of DVT, Esquerre et a191 found that 12 of 16 patients with proven DVT had increased activity in thrombotic vessels. Utne et a192 used 99mTcheparin radionuclide venography by injecting the tracer in the dorsal veins of the patient's feet and performing both an initial flow study of filling patterns and a delayed study to look for hot spots. A high correlation with contrast venography was found; however, the radionuclide venogram portion of the study was found to be more valuable since only 50% of thrombi appeared as hot spots.

Fibronectin Fibronectin, a 450 kD plasma glycoprotein, is an adhesive protein that may be involved in linking of fibrin and cells. In this case, the presence of heparin, rather than inhibiting binding, may actually have an enhancing effect on the binding of fibronectin to fibrin. 93'94Zoghbi et a195 labeled human and canine fibronectin with 1311or 111In via DTPA bifunctional chelation. In a canine model of fresh pulmonary emboli (PE), labeled fibronectin was used to image the lesions. llqn uptake at sites of emboli were visualized in excised lungs, but could not be imaged in vivo despite rapid clearance of blood background. The absolute percent of injected dose per gram of embolus was low (<0.03% per gram at 24 hours), and administration of heparin did not improve the ability to visualize emboli in vivo. It was concluded that labeled fibronectin is not useful for imaging pulmonary emboli, possibly because of competition from endogenous, nonlabeled fibronectin in the blood. PLATELET-DIRECTED RADIOPHARMACEUTICALS

Labeled Platelets 111In oxine. Since the development of a technique for labeling intact platelets with 111In,96the usefulness of imaging platelet deposits in a variety of thrombosis-related disorders has been under investigation.

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Labeling of autologous platelets with 111In oxine requires isolation of the platelets from other cell types and from most of the plasma, because the weak, lipid-soluble 111In oxine complex can label other types of cells and metalbinding proteins in plasma. 97 Careful attention to detail is required to keep the cells sterile and pyrogen-free, and to avoid damaging them. There are differences of opinion as to the optimal procedure to use. Successful imaging of thrombi with platelets usually requires a delay of 24 to 72 hours for adequate platelet deposition to occur and for blood background to clear, as platelets have a long lifetime in the blood. Seabold et a198claimed that diagnosis of DVT could be made at 4 hours post-injection in many patients. There is a danger, however, in interpreting an image as positive before the blood background has cleared adequately. In addition to the detection of DVT, labeled platelets have been used for clinical imaging of thrombi and platelet deposition in areas other than the legs, including the coronary arteries, 99 left ventricle, 1~176176 left atrium, 1~ carotid arteries, 1~ vascular grafts, a~ and pulmonary emboli. 1~176 Imaging with platelets in areas of high blood pool has been aided by image subtraction of 99mTc-red blood cell blood pool images. 99 Some very striking images of thrombi have been obtained clinically. For the most part, however, the uptake of 111In-platelets in thrombi in human subjects has been less consistently dramatic than in animal models. The reason may be that by the time a patient with calf pain is admitted and the test is ordered, the thrombus may have slowed in its growth and therefore may not be depositing platelets actively enough for significant accumulation for imaging. There is general agreement that thrombi must be fresh to be imaged by labeled platelets. In a study in an animal model, Knight et al 1~ found that labeled fibrinogen and platelets both bound better to fresh thrombi than to thrombi greater than 1 day old, although platelets achieved a higher target-to-background ratio than labeled fibrinogen. Moser et al 1~ found that thrombi up to 10 hours old could be imaged. In addition, however, anticoagulants may interfere with the incorporation of labeled platelets into thrombi. Seabold et al 1~ observed that in patients who

LINDA C. KNIGHT

were receiving heparin or warfarin at the time of a labeled platelet study, false-negative results were obtained. In other patients, if anticoagulants were discontinued at least 4 hours before injection of labeled platelets, the platelet images eventually became positive; however, the time to obtain a positive image was longer than in patients who had not received anticoagulants. These investigators also noted that focal activity in the inguinal region was a common source of false-positive results. Ezekowitz et al 1~~performed a study of platelet scintigraphy in two groups. Group I was asymptomatic and 1.1 days post-major orthopedic surgery; imaging was carried out for several days. Group II was symptomatic and most were receiving anticoagulants; imaging was carried out for at most 48 hours. Compared with contrast venography, the sensitivity and specificity of lllln-platelet scintigraphy was 93% and 97% in group I, and 42% and 67% in group II. An image from group I is shown in Fig 2. This study suggests that anticoagulants indeed seem to adversely affect the deposition of platelets and that platelets deposit more avidly in acutely forming thrombi than in preexisting thrombi. The 2.8 day half-life of lllln coupled with the 8 to 10-day biological lifetime of platelets means that imaging can be carried out for 5 days: hence, platelet scintigraphy should perhaps be done as a surveillance procedure on post-surgical patients, to provide more complete information than is currently provided with the 125I FUT in the same patient population. A preliminary study by Clarke-Pearson et al 1~ suggests the same finding in immediate post-operative period. Other lipophilic lllln labels. Because of concerns that platelets may be damaged by exposure to an environment containing ethanol or that is free of plasma with its nutrients, as in the 111In-oxine method, alternative methods of preparing lipid-soluble indium complexes have been explored. Ulln tropolone is more soluble in water than is indium oxine, but yet has a higher oil/buffer partition coefficient than indium oxine. 111 The need for ethanol is avoided, but plasma seems to decrease labeling efficiency. Sinn et a1112used acetylacetone to complex ~11In for platelet labeling, with similar results. Another indium complex, 11lin mercaptopyridine-Noxide (Merc) has also been investigated. 113 Merc

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

61

Fig 2. (A) Contrast venogram of the right calf in one patient who had undergone total hip replacement. 1111noxine-labeled platalets were injected 48 hours after surgery. (B) Scintigraphic image of both calves acquired 24 hours after injection. (F) Focal uptake. (c) Filling defects caused by venous thrombosis. (Reprinted Ezakowitz MD, Pope CF, Sostman HD, et ah Indium-111 platelet scintigraphy for the diagnosis of acute venous thrombosis. Circulation 73:668-674, 1986, by permission of the American Heart Association, Inc)

seems to be better able to label platelets in a plasma environment than the other labels: Thakur et al 1~3 have achieved 80% labeling efficiency in human plasma. All of these labeling methods result in viable labeled platelets; however, manipulation of the cells to separate the platelets and the time involved are still major drawbacks to these techniques. 99mTc exametazime. Thisradiopharmaceutical, which is a lipid-soluble complex of 99mTc designed for cerebral perfusion imaging, can also be used to label cells. It has been used to label leukocytes as well as platelets, 114 so the platelets must be isolated from other cell types. A small amount of plasma in the labeling medium is tolerated fairly well. ll5 A drawback of this technique is that the label elutes from the cells more rapidly (8%/h) than from rain labeled platelets. In a preliminary clinical study, Becker et a1116 imaged thrombi in three patients, with positive

uptake visible over blood pool at 4 hours. A major advantage of a Tc label is the higher count rate; but if thrombi cannot be visualized over blood background initially, the short physical half-life will result in a poor count rate at 24 to 48 hours.

Antiplatelet Monoclonal Antibodies A radiolabeled antibody that specifically recognizes platelets would offer the opportunity to label platelets without the tedious isolation from other cells and the plasma that is required with the rain oxine labeling method. With such specificity, a labeled antibody could simply be injected intravenously, without having to draw blood for ex vivo labeling of platelets. Several monoclonal antibodies that bind to human platelets have been tested for their ability to image thrombi in vivo (Table 2). Antibody 7E3 recognizes the fibrinogen binding site, the glycoprotein IIb/IIIa complex, on the surface of

62

LINDA C. KNIGHT

Table 2. Monoclonal Antibodies That Recognize Platelets Antibody

7E3

5OH. 19 P256 PAC-1 B59.2 KC4

EpitopeRecognized

Reference

Glycoprotein lib/Ilia complex (fibrinogen binding region on platelet surface) Low molecular weight antigens on platelet surface Glycoprotein lib/Ilia complex

117

Glycoprotein IIb/Ula complex Glycoprotein lib/Ills complex GMP 140 (platelet-activation-dependent granule-external membrane protein

123 119 120 122 121 125

human platelets. 117 The antibody (as IgG) has been radiolabeled with 1231and lllln and used to radiolabel platelets either in citrated whole blood or in vivo. ~18 This technique represents a major advance over the laborious cell isolation and washing procedure required for the labeling of platelets by the Hqn-oxine technique. Platelets labeled in this fashion have been shown to localize in fresh thrombi in dogs. 118 However, because antibody 7E3 blocks the fibrinogen binding site on platelets and thus prevents platelet aggregation, the amount of antibody used for imaging without affecting the localizing ability of the platelets is critical. Another monoclonal antibody, P256, directed against the same antigenic site, has no adverse effect on platelet aggregation if used as an 111In-labeled Fab' fragment. H9 It was capable of producing images of thrombi induced in primates. 12~DVT were also imaged in patients.~ 19 Five minutes after injection of 111InDTPA-(Fab') 2 of P256, 75% of the injected dose was cell-bound.119 Monoclonal antibody B59.2 appears to have the same epitope as 7E3 and P256. In a study performed with ]]~In-labeled B59.2, a thrombus was visualized in a canine model within 2 hours after injection, achieving a thrombus-to-blood ratio of 15:8.12~ Monoclonal antibody PAC-1 may behave similarly, 122 but no in vivo studies have yet been published. Another monoclonal antibody that recognizes human platelets, antibody 50H.19, was raised against human melanoma cells, but was found to also recognize three low molecular weight antigens on the surface of human platelets. 123 It does not recognize human erythrocytes, peripheral

lymphocytes, fibroblasts, serum, or plasma, so it can be used to selectively label platelets in whole blood. In the form of a mixture of F(ab')2 and Fab' fragments, it was radiolabeled with 99mTc and used to radiolabel platelets by direct injection of the labeled antibody. ~23 Although only about half the injected antibody bound to circulating platelets in the dog, the unbound antibody cleared quite rapidly from the circulation. Fresh thrombi in dogs were visualized within 1 hour in the peripheral veins and arteries, and within 2 to 4 hours for thrombi in the trunk, including the lungs.~23 Thrombus-to-blood ratios averaged 15:1 at 3 hours postinjection. Heparin appears to interfere with the thrombus uptake of antibody 7E3.124 This is not surprising, since heparin also interferes with the uptake of ~llln-oxine labeled platelets, and will probably affect the thrombus deposition of all of the aforementioned antiplatelet antibodies. Although antibodies that recognize glycoprotein l i b / I l i a complex should not bind to circulating (resting) platelets, antibodies 7E3, P256, and B59.2 seem to do so. A newly reported antibody, KC4, recognizes a site on the surface of platelets that is exposed only after the platelets have been activated. 125 This labeled antibody could theoretically circulate in a non-cell bound form, and bind to activated platelets on the surface of a thrombus. Since the long lifetime of platelets in the circulation is one of the main sources of background activity in H]In platelet imaging, an antibody that does not bind to resting platelets in the circulation might possibly leave the blood faster and thus result in high target-to-background activity at an early time after injection. 123I-labeled KC4 antibody was tested in a primate model 12s and enabled visualization of thrombi in an extremity within 15 to 60 minutes after injection. Despite high uptake ratios ex vivo, the in vivo thrombus-to-blood ratios at 60 minutes were only 3:1. RADIONUCLIDE VENOGRAPHY

The technique of radionuclide venography involves the use of nonspecific tracers injected into the dorsal veins of the feet to look for abnormal flling patterns of the veins of the legs, and to look

RADIOPHARMACEUTICALS FOR THROMBUS DETECTION

63

Fig 3. Examples of unilaterally abnormal blood pool radionuclide venograms in two patients obtained with aamTc-red blood cells. (Left) Poor visualization of left lilac veins. (Right) Poor visualization of left popliteal and calf veins. Contrast venography confirmed these findings in both cases. (Reprinted with permission from Zorba J, Schier D, Posmituck G: Clinical value of blood pool radionuclide scintigraphy. A JR 146:1051-1055, 1986).

for residual hot spots caused by adherence of the tracer to thrombi. The test is usually performed with readily available radiopharmaceuticals such as 99mTc-macroaggregated albumin (MAA), sulfur colloid, xenon, and krypton. 126-13~It is a rapid test and permits ascending venography to be performed safely in patients with contrast allergies. If the test is done with MAA, a lung scan can be performed immediately after the legs have been imaged. It has been criticized for lack of specificity because hot spots often constitute false-positive results. 131'132 For this reason, the current trend is to interpret radionuclide venograms solely on the basis of the filling patterns, and not to base a diagnosis on residual activity. Recently, a number of investigators have evaluated equilibrium blood pool images obtained with 99mTc-labeled red blood ceils as a means of diagnosing DVT. 133"137In a study of 198 vessel segments in 50 patients, Zorba et a1134 found a high overall concordance between radionuclide venography and contrast venography (sensitivity 90%, specificity 93%). Figure 3 shows an image from this study. The accuracy of the test was even higher, however, if the calves were excluded

from the analysis, because of the relatively poor resolution of the test. Oster et a1135 found that helpful additional information was obtained when the cells were labeled in vitro and injected into the dorsal veins of the feet for an initial flow study. This enabled selective visualization of the deep veins. Leclerc et a1136compared equilibrium red cell images of the legs with impedance plethysmography in 100 patients. For proximal DVT, the red cell venography method had a sensitivity of 68% and a specificity of 88%. It was suggested that an abnormal red cell venogram should be confirmed with another diagnostic method. Silverstein and Turbiner 137 described the use of the test to demonstrate an obstruction in an upper extremity in a patient with pulmonary embolism. A number of institutions use radionuclide venography because it is the only imaging method for venous thrombi which uses easily prepared, approved radiopharmaceuticals and therefore is available to nonresearch institutions. It is hoped that more specific thrombus-detecting radiopharmaceuticals will soon be approved for clinical use to fill this need.

64

LINDA C. KNIGHT

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