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Experimental Studies |
1 From the Depts of Surgery (Y.O., S.M.K., O.M.M., C.O.E.), Radiology/Div of Nuclear Medicine (S.K., H.W.S., F.G.B.), and Pathology (J.P.T.H.), Stanford University School of Medicine, Lucile Salter Packard Children's Hospital, 725 Welch Rd, Palo Alto, CA 94304; and Dept of Laboratory Medicine, University of Washington, Seattle (J.F.T.). Received Apr 14, 1999; revision requested May 10; final revision received Aug 6; accepted Aug 30. F.G.B. and H.W.S. supported in part by Children's Health Research Fund, Lucile Salter Packard Children's Hospital and by National Institutes of Health grant HL61717. J.F.T., O.M.M., and S.M.K. supported in part by National Institutes of Health grants HL-47151, DK47810, and AI35994, respectively. Y.O. supported in part by Stanford University School of Medicine Dean's Postdoctoral Fellowship. Address reprint requests to F.G.B. (e-mail: ma.frb@forsythe.standford.edu).
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Abstract |
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 TOP Abstract IntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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MATERIALS AND METHODS: 99mTc-radiolabeled annexin V was intravenously administered to six allografted (immunologically mismatched) and five isografted (immunologically matched) recipient rats on days 2, 4, and 7 after orthotopic liver transplantation. Animals were imaged 1 hour after injection of 0.2–2.0 mCi (8.0–74.0 MBq) of radiolabeled annexin V by use of clinical nuclear scintigraphic equipment.
RESULTS: All animals in the allografted group demonstrated marked increases of 55% and 97% above the activity in the isografted group in hepatic uptake of annexin V on days 4 and 7, respectively. Severe acute rejection was histologically detected in all allografted livers on day 7. There was no histologic evidence of acute rejection in isografted animals. Dynamic hepatobiliary imaging with 99mTc and mebrofenin, an iminodiacetic acid derivative, demonstrated no correlation with the presence or absence of acute rejection or with annexin V uptake.
CONCLUSION: Noninvasive imaging with radiolabeled annexin V is more sensitive and specific than imaging with 99mTc-mebrofenin in the diagnosis of acute rejection of a liver transplant.
Index terms: Animals • Annexin V • Liver, radionuclide studies, 761.12172, 761.458 • Liver, transplantation, 761.12172, 761.458 • Mebrofenin • Radionuclide imaging, experimental studies, 761.12172, 761.458
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Introduction |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The diagnosis of rejection usually requires histologic evaluation of a hepatic sample obtained at biopsy (6,7). A variety of noninvasive modalities have been suggested for use in the assessment of hepatic allografts in the postoperative period. These modalities include gray-scale and Doppler ultrasonography (US) and hepatobiliary scintigraphy with use of the technetium 99m–iminodiacetic acid family of radiopharmaceuticals (8–15).
US is primarily useful in the detection of vascular complications. The most common of these is hepatic arterial thrombosis, which can be associated with hepatic graft failure, bile leakage, hemorrhage, or septicemia (16–22). Unfortunately, US is not reliable in the diagnosis or exclusion of acute rejection.
Hepatobiliary scintigraphy has been shown to be useful in the prediction of graft survival. Uptake and rapid excretion of 99mTc–iminodiacetic acid compounds in a hepatic graft virtually excludes the presence of higher grades of acute rejection (8–15). Unfortunately, 99mTc–iminodiacetic acid scintigraphy is nonspecific. Depressed hepatic function manifested by delayed hepatic uptake and excretion is found in acute rejection, viral hepatitis, ascending cholangitis, cholestasis, and biliary stenosis and/or obstruction. Therefore, the cause of an abnormal 99mTc–iminodiacetic acid scintigram usually must be confirmed at biopsy (10). Even histologic analysis may give equivocal results because other hepatic diseases may mimic acute rejection (10).
The pathophysiologic development of rejection involves injury to the graft hepatocytes caused by host immune cells. One type of injury is the initiation of apoptosis in the graft hepatocytes. Recently, 99mTc-radiolabeled annexin V, a human protein–based radiopharmaceutical, has been shown to bind specifically to apoptotic cells in a rodent model of acute rejection of a heterotopic cardiac allograft transplant (23–27). The uptake of annexin V observed in the analysis of region-of-interest images directly correlated with the severity of acute rejection, as seen at histologic evaluation (24). Annexin V imaging was also able to depict the response of allografted hearts to antirejection therapy.
In the present study, we used 99mTc–annexin V in the imaging of acute hepatic allograft rejection in a well-characterized rodent model of orthotopic liver transplantation (28). We compared the results of hepatobiliary scintigraphy with use of 99mTc and mebrofenin, an iminodiacetic acid derivative, with the results of scintigraphy with use of 99mTc–annexin V and with the results of histologic analysis of acute rejection of the liver transplant.
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MATERIALS AND METHODS |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Lewis rats (n = 6) were grafted with livers from Dark Agouti rats to create an allogeneic combination (allografts, immunologically mismatched). Lewis rats (n = 5) were also grafted with livers from Lewis rats to make a syngeneic combination (isografts, immunologically matched). Additional groups of allografted (n = 3) and isografted animals (n = 2) developed postoperative complications of hepatic infarction (n = 2), portal venous thrombosis (n = 2), or biliary obstruction (n = 1) and were excluded from the imaging analysis of acute rejection. These animals, however, did undergo serial imaging prior to the diagnosis of these complications. They also underwent autopsy and histologic analysis in the same fashion as did the study group animals, as described later.
Transplantation Procedure
Liver transplantation was performed according to Kamada and Calne's technique (28), with the exception of the bile duct anastomosis. Donor and recipient surgery was performed aseptically; the animals were anesthetized with use of isoflurane. After laparotomy, a polyethylene tube was inserted into the common bile duct. Immediately before hepatectomy, the donor liver was perfused in situ via the portal vein with 10 mL of a lactated Ringer and heparin solution, which was cooled to 4°C. Then, plastic cuffs were attached to both the portal vein and the infrahepatic inferior vena cava. The liver was excised and stored at 4°C in a sterile container filled with normal saline solution until transplantation.
Orthotopic liver transplantation was performed in hepatectomized recipient rats, without reconstruction of the hepatic artery. The suprahepatic inferior vena cava of the graft was anastomosed to that of the recipient. The cuffed portal vein and infrahepatic inferior vena cava of the donor were inserted into those of the recipient. Finally, the tube that was already secured in the bile duct of the donor was inserted into the bile duct of the recipient. No immunosuppressive therapy was given to the recipient rats in this study.
Preparation of Radiopharmaceuticals
99mTc hydrazinonicotinamide (HYNIC)–annexin V was prepared as previously described (23,26). HYNIC, an analogue of nicotinic acid, is a bifunctional molecule that is capable of bonding to the lysine residues of proteins on one moiety and to the conjugates of 99mTc on the other (27). This agent forms stable complexes without affecting protein bioreactivity (function).
In brief, human annexin V produced by means of expression in Escherichia coli was derivatized with HYNIC. The conjugate of HYNIC and annexin V had an ability to bind phosphatidylserine that was equivalent to that of native annexin V. (Binding activity was determined with fluorescein-labeled annexin V by use of a modified competitive-inhibition fluorometric assay [23]. The 50%-inhibitory concentration of HYNIC–annexin V was 12.7 nmol/L; that of native annexin V was 8.3 nmol/L).
HYNIC-derivatized annexin V was produced by gently mixing annexin V with 6–HYNIC. To bind 99mTc to the HYNIC–annexin V conjugate, a reduced tin (stannous ion) and tricine solution was added to 99mTc pertechnetate with an aliquot of HYNIC–annexin V under anoxic conditions according to the methods described by Abrams et al (27). A 92%–97% radiopurity was routinely achieved, as confirmed at instant thin-layer chromatography with use of a 0.9% saline solution as a solvent. Specific activity was varied from 10 to 100 µCi per microgram of protein (0.4 to 3.7 MBq/µg) depending on the desired activity.
For hepatobiliary scintigraphy, 2 mCi (74 MBq) of 99mTc-mebrofenin (Choletec; Bracco Diagnostics, Princeton, NJ) was administrated per animal.
Radionuclide Imaging
A model 420 mobile camera (Technicare, Solon, Ohio) equipped with a low-energy high-resolution parallel-hole collimator was used to record the radionuclide distribution. Data were recorded by use of a 20% window centered on the photopeak of 99mTc onto a 256 x 256 matrix on a computer system that was dedicated for digital display and analysis (Icon; Siemens, Hoffman Estates, Ill).
Once the animals were placed under anesthesia by means of methoxyflurane inhalation, they were imaged in the prone-anterior projection in 10-minute static acquisitions. Imaging was performed 1 hour after the intravenous injection of 150–250 µCi (5.5–9.2 MBq, 5–20 µg of protein) of radiolabeled annexin V prior to hepatobiliary scintigraphy or after the injection of 1–2 mCi (37–74 MBq) of 99mTc-annexin V in animals not undergoing hepatobiliary scintigraphy. One milliliter of a 100-mL dilution of the injected 99mTc–annexin V activity (1% of the injected dose) was placed into a 3-mL plastic sample tube; the tube was placed at the right side of each animal during the acquisition of each image.
A subset of animals was arbitrarily selected to undergo hepatobiliary imaging immediately after annexin V imaging with an intravenous injection of 2 mCi (74 MBq) of 99mTc-mebrofenin. One-minute dynamic images (256 x 256 matrix, high sensitivity, high-resolution parallel-hole collimation) were initially obtained for 30 minutes immediately after the administration of a bolus injection of 99mTc-mebrofenin. A 30-minute–delayed static image was also obtained. One milliliter of a 100-mL dilution of the total dose of injected 99mTc-mebrofenin was also placed in a tube at the side of each of these animals during the acquisition of each hepatobiliary image. No corrections were made for 99mTc–annexin V hepatic background activity, as it was assumed that the 10:1 ratio of 99mTc-mebrofenin activity to 99mTc–annexin V activity made this correction unnecessary.
Image Analysis
Static annexin V images were analyzed for total graft activity (counts per minute). This was expressed as a percentage of the injected dose (counts per minute) as determined from the 1-mL aliquots of the injected dose that were placed at the right side of each animal at imaging. Values for each variable were expressed as the mean ± SEM. Time-decay curves were generated for graft activity from the dynamic 1-minute serial hepatobiliary images. The results of 99mTc-mebrofenin uptake measurements were expressed as the ratio of observed hepatic activity at 5 minutes to observed hepatic activity at 30 minutes.
Analysis of imaging data was performed by use of an Icon system (Siemens). Regions of interest were obtained by one of the authors (Y.O.), who took care to not include any renal uptake on the annexin V or hepatobiliary images. Regions of interest for hepatobiliary images were placed by another author (S.K.) at the center of the liver to avoid inclusion of any activity excreted into the bowel.
Histologic Analysis
The recipients rats were sacrificed on day 7 after transplantation. Samples were fixed in 10% neutral buffered formalin before processing. Tissue samples for histologic evaluation were embedded in paraffin, sectioned, and stained with hematoxylin-eosin. All specimens were examined by a pathologist (J.P.T.H.) who was blinded to the imaging results for the presence and severity of acute rejection.
Statistical Analysis
Mean values and SEMs were calculated. Differences in mean values were determined by use of a two-tailed Student t test for the null hypothesis. P values less than .05 were considered to indicate significant differences.
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RESULTS |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Of the five animals that were excluded from the quantitative analysis (n = 11) because of postoperative complications, two (one isografted and one allografted) developed postoperative thrombosis of the portal vein. At the time of sacrifice, both animals demonstrated petechiae of the mesentery and venous congestion of the small and large bowels, which were also seen at histologic evaluation (Fig 3). These animals also had markedly increased annexin V uptake in the spleen and lung, with variable uptake in the liver.
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DISCUSSION |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Most of the observed biologic activities of annexin V center on its ability to bind to phosphatidylserine on the surfaces of cells or platelets. Phosphatidylserine is a constituent, anionic, membrane-bound phospholipid that is restricted to the inner leaflet of the plasma membrane by an adenosine triphosphate–dependent aminotransferase called translocase (31). However, cells (and platelets that are activated as part of the coagulation cascade) that undergo apoptosis rapidly redistribute phosphatidylserine from the inner leaflet of the lipid bilayer to the outer leaflet by activating a normally quiescent enzyme called scramblase. The externalization of phosphatidylserine is a general feature of apoptosis and occurs before the morphologically observable events that are classically associated with apoptosis occur. Such events include cytoplasmic and nuclear condensation, membrane bleb formation, and DNA degradation (32).
Apoptosis plays an important role in the pathophysiologic development of acute rejection of transplants of the liver, heart, kidney, and small bowel (2,23,24,33–37). Apoptotic nuclei have been identified in biopsy samples of acutely rejected transplants by use of terminal deoxynucleotidyl transferase–mediated deoxyuridine 5–triphosphate nick-end labeling, or TUNEL, staining.
Alternative noninvasive methods for the detection of acute rejection of a liver transplant have largely focused on the use of compounds related to 99mTc–iminodiacetic acid in the imaging of hepatocytic function (8–15). However, Gelfand et al (10), in their study of 30 children receiving liver transplants, were unable to derive useful criteria for the identification of acute rejection that is reversible with immunosuppressive therapy. In a porcine model, qualitative analysis of the half-life, the time for half of the activity to clear from the liver, increased with rejection (38).
Hawkins et al (9) observed in humans that 99mTc-disofenin uptake and excretion were prolonged in acute rejection of a liver transplant. In a study of 73 liver transplant recipients, Loken et al (8) found that liver uptake and excretion rates of hepatobiliary agents did not permit differentiation between the causes of depressed graft function; thus, biopsy was required to establish a specific diagnosis.
We were able to detect a marked increase in the uptake of 99mTc–annexin V in hepatic allografts, compared with that of the isografts, as early as 4 days after transplantation. Previous studies with this model showed clear histologic evidence of mononuclear infiltration that was associated with apoptotic hepatic nuclei in allografts; mononuclear infiltration occurred between 3 and 4 days after transplantation with a marked elevation in the level of serum glutathione S-transferase, compared with that of the isografts (2). Annexin V imaging qualitatively and quantitatively reflected this early stage of rejection in our animal model.
Annexin V imaging also demonstrated stereotypical patterns of regional uptake in the group of five animals that had postoperative complications. Lobar or segmental hepatic infarction manifested as photopenic regions at both 99mTc–annexin V and 99mTc-mebrofenin imaging presumably due to a lack of (or decrease in) delivery of the radiopharmaceutical to these regions by means of blood flow.
Portal venous thrombosis correlated with marked uptake values in the spleen and lung and with variable (increased or decreased) hepatic uptake. The reason for this pattern is unclear. Splenic uptake may be due to portal venous congestion, which allows for greater binding of annexin V over time, as compared with binding in the control animals. Pulmonary uptake was accompanied by segmental interstitial pneumonitis, as seen at histologic evaluation (data not shown).
In the setting of pulmonary infection, phosphatidylserine expression may be increased because of increased apoptotic cell death or local hypercoagulability. The variability of hepatic uptake with portal venous thrombosis may be due to a series of related factors, such as increased hepatic hypercoagulability or apoptosis. These can be associated with ischemia, which is balanced against decreased portal venous flow due to portal venous thrombosis and/or congestion. In the clinical setting, portal venous thrombosis can be readily diagnosed at US, computed tomography, or magnetic resonance imaging. Therefore, possible confusion of portal venous thrombosis with acute rejection as a cause of abnormal increases in hepatic uptake of annexin V could be avoided.
Annexin V labeled with the N2S2 method has been used in previous studies to image pulmonary emboli and deep venous thrombosis in both animals (39) and humans (Tait JF, unpublished data, 1998). In the prior models of fulminant hepatitis used by Blankenberg et al (23,25), interstitial hemorrhage was a potential confounding variable in the analysis of the uptake of radiolabeled annexin V. Of note, we found no evidence of interstitial hemorrhage in the rejected allografts in our current study.
The application of radiolabeled annexin V in the clinical monitoring of liver transplant recipients would have several complementary advantages over the current scintigraphic and biopsy techniques. Annexin V imaging appears to be sensitive and specific for the screening of acute rejection, even in the early postoperative period. 99mTc also has a physical half-life of only 6 hours, and intravenously administered annexin V is extremely safe. (Annexin V has only a transient anticoagulant effect at doses of greater than 30 times the dose needed for imaging, that is, 
300 µg/kg [40].)
The serum clearance of annexin V is also rapid (3–7 minutes), permitting imaging 30 minutes after injection (38). Patients could be monitored with serial studies for the severity of acute rejection and for a response to therapy by use of standard clinical portable or fixed radionuclide imaging cameras, with acquisition times of 10 minutes or less.
One major difficulty in the use of radiolabeled annexin V is the relatively high nonspecific uptake in the renal cortex (25). As previously noted, this uptake is most likely due to nonspecific uptake of low-molecular-weight proteins by the proximal renal tubules. Fortunately, for clinical use, the N2S2 labeling method has less renal uptake (16.5% of injected dose) in pigs and in humans (39).
In conclusion, 99mTc–annexin V radionuclide imaging in an experimental rodent model of orthotopic liver transplantation appears to be sensitive and specific for the diagnosis of acute rejection. Further, studies in humans with use of the currently available annexin V preparations need to be undertaken to confirm these findings.Practical application: In the near future, it may be possible to use radiolabeled annexin V to routinely and noninvasively screen asymptomatic liver transplant recipients for acute (or low-grade) rejection. Once identified, these patients could be monitored with serial studies to assess the efficacy of immunosuppressive therapy.
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Footnotes |
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Author contributions: Guarantors of integrity of entire study, Y.O., S.M.K., F.G.B.; study concepts, Y.O., S.M.K., O.M.M., J.F.T., F.G.B.; study design and definition of intellectual content, Y.O., S.M.K., O.M.M., H.W.S., C.O.E., F.G.B.; literature research, Y.O., S.M.K., F.G.B.; experimental studies, Y.O., F.G.B.; data acquisition, Y.O., S.K., F.G.B.; data analysis, Y.O., S.K., J.P.T.H., F.G.B.; statistical analysis, Y.O., F.G.B.; manuscript preparation, Y.O., F.G.B.; manuscript editing, Y.O., S.M.K., O.M.M., J.P.T.H., H.W.S., F.G.B.; manuscript review, C.O.E.
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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