Leucocyte and contaminant cell-bound activities resulting from the labelling of leucocytes with 111In-oxine

A simple method is described for estimating the activities bound to leucocytes, erythrocytes, platelets and the free activity, as resulted from the preparation and labelling of leucocytes with 111In-oxine. Measurements are required only of 111In activity, suspension volumes, and platelet concentrations. The limitations of the method are discussed. When blood from a normal volunteer was labelled by an 111In-oxine manufacturer's recommended technique, the greatest proportion of activity was bound to leucocytes, and in addition, a significant proportion of the activity was found to be associated with platelets. If the number of centrifugations between sedimentation and labelling was reduced from two to one, the proportion of free activity increased at the expense of a reduction in leucocyte activity, but the platelet activity remained unchanged. The relative distribution of cell-bound and free activities was independent of the relative centrifugal force (85-450 g), and of the time (15-30 min) and the suspension volume (5-10 ml) used to incubate the cells with 111In-oxine.     

A comparison of two methods of labelling autologous platelets with 111In-oxine in five different species

Several different methods for labelling autologous platelets with ¹¹¹In-oxine have been described. However, no comparative study has been reported.In the present investigation two different labelling methods were compared in terms of labelling efficiency and platelet function in five species: human, dog, pig, rabbit and rat.One of the labelling methods, utilising among other things a serum albumin gradient separation of platelets and incubation of ¹¹¹In-oxine in a water bath at 37° C, was superior in all species with significantly higher labelling efficiency and unchanged platelet function.     

Evaluation of 111In labelled white blood cells by in vitro functional tests and electron microscopy

We have studied the influence of granulocyte labelling with commercially available ¹¹¹In-oxine, tropolone (trop) or home made ¹¹¹In-Mercapto pyridine (Merc) prepared by the method of Thakur (1985) on the cell structure by electron microscopy and on the cell function by enzymatic tests, random migration, chemotaxis, phagocytosis and bactericidal activity. The granulocytes were labelled with 400 Ci ¹¹¹In-oxine in saline or ¹¹¹In-trop or Merc in plasma. The effect of the chelating agents with and without addition of the tracer was studied (n=4) with varying concentrations: 5–10 g/ml oxine, 10–160 g/ml trop and 1–4 g/ml Merc. Chemotaxis and random migration were not affected by ¹¹¹In-trop and clearly supressed by ¹¹¹In-oxine and Merc; the other tests were normal. The cell structure was disturbed by Merc. The labelling efficiency was excellent with oxine (90%), acceptable with trop (30%–80%) and poor with Merc (10%–25%). Without ¹¹¹In, chemotaxis and random migration were normal up to a concentration of 80 g/ml trop, 8.5 g/ml oxine and 1 g/ml Merc. With addition of ¹¹¹In, chemotaxis and random migration were unaffected up to 80 gmg/ml by trop and markedly supressed by Merc and oxine. It is concluded that labelling with ¹¹¹In-trop assures intact cells.     

111In-tropolonate labelled platelets; studies in normals and in patients with thrombocytopenia

Human platelets were labelled with aqueous ¹¹¹In-tropolonate in comparison with ¹¹¹In-oxinate. In normals the labelling efficiency with ¹¹¹In-tropolonate was higher (93%±2%) than with ¹¹¹In-oxinate (67%±8%) (P<0.05). In cases of severe thrombocytopenia, lower labelling efficiencies were obtained. In six normals a mean platelet life of 9 days ±3 days and an initial recovery of 59%±15% were obtained. In twelve patients with trombocytopenia the mean platelet life was 4 days±4 days and the initial recovery was 58%±20%. The absolute uptake of radioactivity in spleen and in liver in both groups are reported.     

Comparison of three different methods for radiolabelling human activated T lymphocytes

One approach in the treatment of ovarian cancer MOv18/anti-CD3 (biMAb OC/TR), which recognizes a 38-kDa glycoprotein expressed on ovarian carcinomas and the CD3 T cell receptor. However, little is known about the in vivo biodistribution of injected activated lymphocytes, information that could be obtained by scintigraphic imaging of radiolabelled T cells in order to visualize the migratory pattern. We compared the efficiency, stability and toxicity of technetium-99m hexamethylpropylene amine oxime (HMPAO),indium-111 oxine and fluorine-18 2-fluoro-2-deoxy-d-glucose (FDG) in radiolabelling activated lymphocytes targeted with biMAb OC/TR. The mean labelling efficiencies of¹¹¹In-oxine and¹⁸F-FDG using 2.5×10⁸ lymphocytes (68% and 64%, respectively) were more than twice that of^99mTc-HMPAO (31%). Retention of the radionuclide in the cell was highest in the case of¹¹¹In-oxine labelling (less than 25% of the initial cell-bound activity released after 240 min, as compared with 44% of the^99mTc label in the same period and 45% of¹⁸F radionuclide released after 150 min). None of the three radiolabelling reagents induced any significant alteration in cell viability or immunophenotype. However, both¹¹¹In-oxine and¹⁸F-FDG induced a loss of cytotoxic activity of lymphocytes against the ovarian carcinoma cell line IGROV1, and all three radiolabelling reagents caused a significant reduction in the proliferative ability of labelled lymphocytes compared to controls, with cell death occurring after 8–9 days. Radiolabelling with the more stable¹¹¹In-oxine reagent using a higher number of lymphocytes (1.4x109) but the same total activity (around 55.5 MBq) resulted in improved labelled T cell viability and proliferative ability, although the mean labelling efficiency decreased (35.8%). Together the data suggest that¹¹¹In-oxine at low activity per cell is the most appropriate reagent for radiolabelling activated retargeted T lymphocytes useful for in vivo biodistribution studies.     

Optimum conditions for radiolabelling human granulocytes and mixed leucocytes with 111In-tropolonate

To determine the optimum conditions for the in vitro radiolabelling of human granulocytes with ¹¹¹In-tropolonate for clinical studies, the factors which affected the amount of ¹¹¹In bound to the cells, the labelling efficiency (LE), were measured. These included the tropolone concentration, labelling medium and cell concentration. The tropolone concentration was dependant on the amount of plasma in the labelling medium; with 90% ACD plasma it was 4×10^-4 M and with Hepes saline buffer it was 4×10^-5 M. Using these tropolone concentrations and a low granulocyte concentration of 1×10⁷ ml^-1, the LE in 90% ACD plasma was 29% and in buffer was 74%. However, increasing the cell concentration to 1×10⁸ ml^-1 gave a LE of 90% in buffer and plasma. The optimum conditions for clinical studies involved incubating granulocytes, or mixed leucocytes as a source of granulocytes, at a cell concentration of at least 5×10⁷ cell/ml in 1 ml ACD plasma, pH 7–7.6 with 0.1 ml tropolone at 4.4×10^-3 M mixed with no more than 100 l ¹¹¹InCl₃ for 15 min at room temperature. Under these conditions more than 96% of the ¹¹¹In was taken up by the granulocytes and only 3% of the ¹¹¹In was released from the labelled cells during a 30 min incubation in plasma. ¹¹¹In-tropolonate is therefore an efficient agent for stably radiolabelling human granulocytes in plasma for clinical studies.     

A comparison of two methods of labelling autologous platelets with 111In-oxine in five different species

Several different methods for labelling autologous platelets with 111In-oxine have been described. However, no comparative study has been reported. In the present investigation two different labelling methods were compared in terms of labelling efficiency and platelet function in five species: human, dog, pig, rabbit and rat. One of the labelling methods, utilising among other things a serum albumin gradient separation of platelets and incubation of 111In-oxine in a water bath at 37 degrees C, was superior in all species with significantly higher labelling efficiency and unchanged platelet function.     

A giant tumor thrombus in the right atrium clearly detected by 111In-oxine labeled platelet scintigraphy

A 54-year-old man was admitted to hospital with a 3-month history of progressive dyspnea with coughing. A giant right atrial mass, originating from a hepatocellular carcinoma, was visualized by computed tomography, and digital subtration angiography. The volume of the right atrial mass was increasing rapidly. It was therefore essential to determine whether this giant mass was a tumor thrombus or a multiplication of the hepatocellular carcinoma. ¹¹¹In-oxine labeled platelet scintigraphy revealed active accumulation in the right atrium caused by the presence of active platelet deposition, and slight accumulation in the lung fields probably due to embolic showers originating from the tumor thrombus in the right atrium. This is the first case report showing that ¹¹¹In-oxine labeled platelet scintigraphy can aid in confirming the nature of a giant tumor thrombus in the right atrium and can clarify the pathogenesis of the respiratory symptoms.     

A simple and safe technique for sterile autologous platelet labelling using “Monovette” vials

A simple technique of autologous platelet labelling is described, which allows labelling within 40 min, and has the advantage of low costs, as no laminar air flow is required. Blood (16 ml) was withdrawn into 4 ml ACD, 500 ng prostacyclin was added. After 10 min sedimentation the vials were centrifuged for 5 min at 150 g. The plateletrich plasma in the supernatant was centrifuged at 500 g for 10 min to obtain a platelet pellet. The platelet-poor plasma was preserved in a sterile syringe and the platelet pellet was resuspended in 1 ml tyrode buffer. The cell suspension was labelled at 37° C for 5 min with 100 Ci ¹¹¹In-oxine sulphate and reinjected after dilution with the plasma. Mean labelling efficiency was 90%±3%, mean recovery 2 h after reinjection 76%±3% (mean±SD).     

Functional and ultrastructural alterations of autologous platelets labeled with 111In-oxine

High activity labeled platelets could be useful for the detection and observation of small foci of thrombosis by gamma-camera imaging. Therefore platelets were labeled with 111In-oxine containing increasing activities of 111In to determine the elimiting amount of this tracer that did not cause cell damage. A labeling procedure was employed so that all the chemical parameters remained constant except the amount of 111In. Platelet damage was studied by ADP-induced aggregability according to the Born procedure and by scanning and ultrastructural electron microscopy. Platelets labeled with the lowest activity 2.2 MBq/ml of platelet suspension (10(9) cells/ml) showed no alterations. With the highest activity studied, (22 MBq/ml) aggregability decreased by two-thirds and great changes in the shape of the platelets were seen by electron microscopy. These modifications were attributed to the decay of 111In predominantly located in the platelet cytosol. Labeling of platelets with an activity higher than 7.4 MBq/ml is unsuitable for detection of thrombosis since normal platelet functions are not retained.     

99mTc-labelling of leucocytes with 99mTc-DPO: a complex developed for myocardial imaging

The lipophilic ^99mTc-DPO complex, developed as a myocardial imaging radiopharmaceutical, was used to label leucocytes. After an incubation of 0.1 ml ^99mTc-DPO (8 g DMPE*2HCl) with mixed leucocytes in plasma, the labelling efficiency was over 70%. During incubation in 5 ml plasma, a loss of activity was found between 20% (1 h) and 35% (3 h) caused by elution. Disturbances of cell viability could not be found with the help of the chemiluminescence test. The in vivo recovery was determined in three dogs and was 45%–50% (0.5 h), 30%–36% (1 h), and 18%–24% (3 h). Autologous ^99mTc-DPO-leucocytes were used on seven patients with suspected osteomyelitis, there were four true negative and three true positive results. The target/nontarget ratio determined by ROI in the positive cases was 1.8 to 2.5 at 3 h after injection.     

Comparison of oxine and tropolone methods for labeling human platelets with indium-111

The effect of the chelates oxine and tropolone, used to label platelets, on the kinetics of indium-111-(111In) labeled platelets was studied in twelve normal human subjects. Autologous platelets were labeled either in saline with 111In-oxine or in plasma with 111In-tropolone. Mean platelet lifespan was estimated by fitting the disappearance curve of platelets from the circulation to the multiple hit and other mathematical models. The in vivo distribution of platelets was quantitatively imaged with a scintillation camera. The in vivo recovery of 111In-oxine and 111In-tropolone did not differ, and the mean platelet lifespan was also similar (111In-oxine: 230 +/- 29 hr; 111In-tropolone: 226 +/- 13 hr). At equilibrium (90 min after reinjection of labeled platelets) and at the end of platelet lifespan, 111In-oxine and 111In-tropolone radioactivities in the spleen and liver were similar. These results demonstrate that the results of kinetics measured with 111In-oxine or 111In-tropolone do not differ significantly.     

111In-Oxine-labelled autologous leucocytes in inflammatory bowel disease: New scintigraphic activity index

The usefulness of scintigraphy with ¹¹¹In-oxinelabelled autologous leucocytes was investigated in 27 patients with inflamatory bowel disease (IBD): 16 with ulcerative colitis and 11 with Crohn's disease. Scans were performed 2–4 h (early scan) and at 24 h (late scan) after leucocyte reinjection. No false-negative results occurred in the early scan; however, in the late scan, 2 patients with Crohn's disease had a normal scintigram. A new index of activity (I _l) based on the number and relative activity of abnormal ¹¹¹In-leucocyte zones was used for scan quantitation. All patients with clinically active IBD had I _l2. The scintigraphic index showed a significant correlation with the Harvey clinical index, especially in patients with ulcerative colitis. Our results suggest that an early scan (2–4 h) provides useful information in cases of IBD, and that I _l2 is indicative of the degree of disease activity in such patients.     

99mTc-human immunoglobulin (HIG) — first results of a new agent for the localization of infection and inflammation

Technetium (^99mTc) labelled, polyclonal human immunoglobulin (HIG) is a new agent that detects focal infection and inflammation. This new agent was compared in 40 patients with the accepted standard, namely¹¹¹In-oxine-labelled leucocytes. This comparison resulted in a sensitivity of 94% and a specificity of 96% for^99mTc-HIG when¹¹¹In-oxine leucocytes were defined as giving the true result. The new agent was shown to localize both sepsis and active inflammatory bowel disease (IBD). There was 100% concordance in the 16 patients with IBD who were imaged with both^99mTc-HIG and¹¹¹In-oxine leucocytes. Discordant results were obtained in one case of suspected osteomyelitis, which was false-positive on the^99mTc-HIG scan, and one case of pyrexia of unkown origin when the^99mTc-HIG was false-negative and the¹¹¹In-oxine leucocyte scan demonstrated accumulation of tracer in the caecum at 24 h post-injection. Normal distribution for^99mTc-HIG demonstrated activity in the kidneys and bladder and that 50% of the tracer is cleared through the kidneys during the first 24 h post-injection. There were no major or minor side-effects.     

Isolation of granulocytes and labelling with indium 111-oxine sulphate

The isolation of granulocytes from whole blood and labelling with Indium 111-oxine sulphate are described in detail. To isolate the cells a two-step method was used: (1) removal of the red blood cells by methyl cellulose-Ronpacon and (2) separation of the leucocyte-rich plasma with a double gradient (1077, 1097) technique. ¹¹¹In-oxine sulphate was prepared by adding ¹¹¹In-chloride to a buffered solution of oxine sulphate. The labelling of the granulocytes with ¹¹¹In-oxine sulphate was done by incubation at room temperature for 5 min.     

99mTc-HM-PAO for leukocyte labeling —experimental comparison with 111In oxine in dogs

^99mTc-hexamethylpropylene amine oxime d,1 diastereoisomer (HM-PAO), developed as a diffusible brain imaging agent, labels leukocyte suspensions in saline with an efficiency of 80% using 1–200 g quantities. In dogs, the recovery and survival of reinjected cells in the bloodstream resemble those of ¹¹¹In-oxine labeled cells at least for several hours. Images in control animals at 18 h show the spleen, liver, marrow, and bladder, minimal pulmonary activity and some gastrointestinal activity. Induced E. coli abscesses and joint inflammatory lesions in dogs are shown on 18 h images. This complex appears promising as an agent for abscess detection in humans. However, strict quality control of this agent is necessary, and it must be used immediately after the ^99mTc complex is formed for labeling cells.     

Autologous 111In-oxine-labeled granulocytes in Yersinia infections

Autologous ¹¹¹In-oxine-labeled granulocytes have proved to be valuable for the localization of inflammatory bowel diseases, especially Crohn's disease and ulcerative colitis. Other rare inflammatory bowel diseases also yield positive ¹¹¹In scans. One case of Yersinia infection of the terminal ileum (Yersinia enterocolitica) showing an accumulation of ¹¹¹In-oxine-labeled granulocytes 0.5, 4, and 24 h after the reinjection of the labeled cells is described. The 4-day fecal excretion of ¹¹¹In-oxine granulocytes showed a slight inflammatory activity of the terminal ileum. One negative scan is reported in a cotrimoxazoletreated patient with Yersinia infection.     

Indium-111 oxine labelling affects the cellular integrity of haematopoietic progenitor cells

Purpose Cell-based therapy by transplantation of progenitor cells has emerged as a promising development for organ repair, but non-invasive imaging approaches are required to monitor the fate of transplanted cells. Radioactive labelling with ¹¹¹In-oxine has been used in preclinical trials. This study aimed to validate ¹¹¹In-oxine labelling and subsequent in vivo and ex vivo detection of haematopoietic progenitor cells. Methods Murine haematopoietic progenitor cells (10⁶, FDCPmix) were labelled with 0.1 MBq (low dose) or 1.0 MBq (high dose) ¹¹¹In-oxine and compared with unlabelled controls. Cellular retention of ¹¹¹In, viability and proliferation were determined up to 48 h after labelling. Labelled cells were injected into the cavity of the left or right cardiac ventricle in mice. Scintigraphic images were acquired 24 h later. Organ samples were harvested to determine the tissue-specific activity. Results Labelling efficiency was 75 ± 14%. Cellular retention of incorporated ¹¹¹In after 48 h was 18 ± 4%. Percentage viability after 48 h was 90 ± 1% (control), 58 ± 7% (low dose) and 48 ± 8% (high dose) (p<0.0001). Numbers of viable cells after 48 h (normalised to 0 h) were 249 ± 51% (control), 42 ± 8% (low dose) and 32 ± 5% (high dose) (p<0.0001). Cells accumulated in the spleen (86.6 ± 27.0% ID/g), bone marrow (59.1 ± 16.1% ID/g) and liver (30.3 ± 9.5% ID/g) after left ventricular injection, whereas most of the cells were detected in the lungs (42.4 ± 21.8% ID/g) after right ventricular injection. Conclusion Radiolabelling of haematopoietic progenitor cells with ¹¹¹In-oxine is feasible, with high labelling efficiency but restricted stability. The integrity of labelled cells is significantly affected, with substantially reduced viability and proliferation and limited migration after systemic transfusion.     

In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis

Background

Several animal and few human studies suggest the beneficial role of bone marrow mesenchymal stem cells (MSCs) in liver cirrhosis. However, little is known about the fate of MSCs after infusion in cirrhotic patients. We evaluated stem cell biodistribution after peripheral infusion of MSCs in four cirrhotic patients.

Methods

After three passages of MSCs, the patients received a total of 250–400×10⁶ cells, of which only 50% of the cells were labeled. Specific activities of 0.21–0.67 MBq/10⁶ cells were maintained for the injected labeled MSCs. Planar whole-body acquisitions (anterior/posterior projections) were acquired immediately following infusion as well as at 2 h, 4 h, 6 h, 24 h, 48 h, 7th and 10th days after cell infusion.

Results

After intravenous infusion, the radioactivity was first observed to accumulate in the lungs. During the following hours to days, the radioactivity gradually increased in the liver and spleen, with spleen uptake exceeding that in the liver in all patients. Region-of-interest analysis showed that the percentage of cells homing to the liver (following decay and background corrections and geometric mean calculation) increased from 0.0%-2.8% at immediately post-infusion images to 13.0–17.4% in 10th-day post-infusion. Similarly, the residual activities in the spleen increased from 2.0%-10.2% at immediately post-infusion images to 30.1%-42.2% in 10th-day post-infusion. During the same period, the residual activities in the lungs decreased from 27.0–33.5% to 2.0–5.4%.

Conclusion

The infusion of MSCs labeled with ¹¹¹In-oxine through a peripheral vein is safe in cirrhosis. Cell labeling with ¹¹¹In-oxine is a suitable method for tracking MSC distribution after infusion.     

Pre-therapeutic dosimetry and biodistribution of 86Y-DOTA-Phe1-Tyr3-octreotide versus 111In-pentetreotide in patients with advanced neuroendocrine tumours

Purpose For the internal radiotherapy of neuroendocrine tumours, the somatostatin analogue DOTATOC labelled with ⁹⁰Y is frequently used [⁹⁰Y-DOTA-Phe¹-Tyr³-octreotide (SMT487-OctreoTher)]. Radiation exposure to the kidneys is critical in this therapy as it may result in renal failure. The aim of this study was to compare cumulative organ and tumour doses based upon dosimetric data acquired with the chemically identical ⁸⁶Y-DOTA-Phe¹-Tyr³-octreotide (considered as the gold standard) and the commercially available ¹¹¹In-pentetreotide.Methods The cumulative organ and tumour doses for the therapeutic administration of 13.32 GBq ⁹⁰Y-DOTA-Phe¹-Tyr³-octreotide (three cycles, each of 4.44 GBq) were estimated based on the MIRD concept (MIRDOSE 3.1 and IMEDOSE). Patients with a cumulative kidney dose exceeding 27 Gy had to be excluded from subsequent therapy with ⁹⁰Y-DOTA-Phe¹-Tyr³-octreotide, in accordance with the directives of the German radiation protection authorities.Results The range of doses (mGy/MBq ⁹⁰Y-DOTA-Phe¹-Tyr³-octreotide) for kidneys, spleen, liver and tumour masses was 0.6–2.8, 1.5–4.2, 0.3–1.3 and 2.1–29.5 (⁸⁶Y-DOTA-Phe¹-Tyr³-octreotide), respectively, versus 1.3–3.0, 1.8–4.4, 0.2–0.8 and 1.4–19.7 (¹¹¹In-pentetreotide), with wide inter-subject variability. Despite renal protection with amino acid infusions, estimated cumulative kidney doses in two patients exceeded 27 Gy.Conclusion Compared with ⁸⁶Y-DOTA-Phe¹-Tyr³-octreotide, dosimetry with ¹¹¹In-pentetreotide overestimated doses to kidneys and spleen, whereas the radiation dose to the tumour-free liver was underestimated. However, both dosimetric approaches detected the two patients with an exceptionally high radiation burden to the kidneys that carried a potential risk of renal failure following radionuclide therapy.