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.     

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.     

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.     

Guidelines for the labelling of leucocytes with 111In-oxine

We describe here a protocol for labelling autologous white blood cells with ¹¹¹In-oxine based on previously published consensus papers and guidelines. This protocol includes quality control and safety procedures and is in accordance with current European Union regulations and International Atomic Energy Agency recommendations.     

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.     

Comparison of indium-111 oxinate labelled autologous granulocytes with indium-111 oxinate and indium-111 chloride as abscess scanning agents

Bacterial abscesses were evoked in goats. Imaging of these abscesses was obtained by means of labelling autologous granulocytes with ¹¹¹In oxinate, reinjection of the cells into the animal, and scintigraphy by gamma camera one day later. Comparable imaging results, however, were obtained after intravenous injection of ¹¹¹In oxinate or of ¹¹¹In chloride.The gamma camera images were supported by tissue distribution studies. In the case of administration of ¹¹¹In oxinate to the goats, the radioactivity accumulated in the cell fraction of the blood to a significant extent. This did not occur in the case of plain ¹¹¹In chloride. It remained unexplained why such different accumulation in cells did not result in differences in the scintigraphic studies.Blood clearance studies supplied conclusive evidence that the granulocytes stayed in the circulation for several days following labelling with ¹¹¹In oxinate and reinjection of the cells into the animals.     

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.     

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.     

Evaluation of 111In labelled white blood cells by in vitro functional tests and electron microscopy. Comparison of three labelling methods

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 microCi 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 micrograms/ml oxine, 10-160 micrograms/ml trop and 1-4 micrograms/ml Merc. Chemotaxis and random migration were not affected by 111In-trop and clearly suppressed 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 micrograms/ml trop, 8.5 micrograms/ml oxine and 1 microgram/ml Merc. With addition of 111In, chemotaxis and random migration were unaffected up to 80 micrograms/ml by trop and markedly suppressed by Merc and oxine. It is concluded that labelling with 111In-trop assures intact cells.     

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 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).     

Technetium-99m and indium-111 double labelling of granulocytes for kinetic and clinical studies

A new technique of labelling granulocytes with both technetium-99m hexamethylpropylene amine oxime (HMPAO) and indium-111 in a single protocol was developed in order to exploit the advantages of each radiolabel in clinical and investigative studies. Fourteen patients were included in this prospective study. Granulocytes were labelled with both¹¹¹In-tropolonate and^99mTc-HMPAO. In vitro shape change assay and in vivo distribution and recovery studies were performed to assess the activation of and damage to these cells due to the labelling procedure. The comparative kinetics of¹¹¹In and^99mTc in the blood, liver, spleen, and bone marrow were studied by blood sampling and dual radionuclide imaging early (1 h) and late (24 h) after injection. The functional integrity of the double-labelled granulocytes and the feasibility of the technique were investigated in 14 patients with a painful prosthetic hip due to causes other than infection. The efficiency of double labelling was 63% (SD 14%) for¹¹¹In and 39% (SD 12%) for^99mTc-HMPAO. In vitro granulocyte activation and ex vivo recovery values were comparable to those from single radionuclide labelling. No artefactual granulocyte sequestration was seen in the lungs or liver. The radioactivity was distributed between the liver, spleen and bone marrow and, to a lesser extent, the lung. Early^99mTc counts in the liver, spleen and bone marrow, in relation to background, were significantly higher than¹¹¹In counts while the reverse was seen in late images. Furthermore, circulating free^99mTc was significantly higher than free¹¹¹In at 24 h. Organ^99mTc counts, expressed in relation to the activity in early images, decreased in the spleen, increased in the liver and remained unchanged in bone marrow, whereas¹¹¹In counts increased in the bone marrow and liver, and decreased in the spleen. Granulocytes can be labelled with both¹¹¹In and^99mTc-HMPAO in a single protocol without crosschelation, cellular activation or damage. By favourably exploiting their kinetics for early and late imaging, double-labelled granulocytes may be useful in several clinical and investigative situations.     

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 study of leucocyte labelling efficiencies obtained with 111In-oxine

The range of leucocyte labelling efficiencies with 111In-oxine for a group of patients extended significantly (P = 0.05) below that obtained for a series of labellings of the same normal blood from a volunteer. A retrospective analysis was made of the results in the two groups to identify the cause of this difference in range. The labelling efficiency for patients did not vary with the volume of 111In-oxine, and was independent of the whole blood leucocyte concentration. The difference between the average labelling efficiencies obtained for a group of patients and the normal series labelled by the same operator was more significant than the difference in average labelling efficiencies obtained by different operators. It was concluded that biological variation in patients' blood, rather than operator technique, must have been a more important cause to the difference in the labelling efficiency range between patients and normal. It was also concluded that variations of contaminant platelet-bound activity and of plasma viscosity were greater in the patient group than the normal series, and contributed to this difference in labelling efficiency range.     

Comparative evaluation of red cell-labeling parameters of three lipid-soluble-111In-chelates: Effect of lipid solubility on membrane incorporation and stability constant on transchelation

A rabbit red cell model was used to determine the cell labeling properties of three lipid-soluble ¹¹¹In-complexes: ¹¹¹In-oxine, ¹¹¹In-acetylacetone, and ¹¹¹In-tropolone. Partition coefficients (olive oil/buffer) were measured to determine the lipid solubility and were 3.54, 7.93, and 18.18 for ¹¹¹In-oxine, ¹¹¹In-acetylacetone, and ¹¹¹In-tropolone respectively. The effect of the concentration of these three chelating agents on labeling efficiencies was studied.The factors influencing the labeling efficiencies of these complexes such as cell density, time of incubation, influence of temperature, pH, effect of plasma proteins, and citrate ion concentration in the cell-labeling medium were studied. Labeling yields as high as 95.15±4.15% were achieved with ¹¹¹In-tropolone after a 10-min incubation at 37° C. The optimum pH for cell labeling was 6.5. Excess citrate ion (>3.02 mg/ml) and small amounts of plasma proteins (>10 l/ml) decreased the labeling efficiencies in all three cases.Distribution of these ¹¹¹In-complexes in membrane, membrane fragments, and hemoglobin was studied after hemolysis. In spite of the higher lipid solubility of ¹¹¹In-tropolone, the transchclation capacity appears to be similar to that of ¹¹¹In-oxine. ¹¹¹In-acetylacetone had the highest transchelation capacity.     

Comparison of 99Tcm-HMPAO and 111In-oxine labelled granulocytes in man: first clinical results

The in vitro and in vivo behaviour of 99Tcm-HMPAO (hexamethylpropyleneamineoxime) (n = 12) and 111In-oxine (n = 11) labelled granulocytes, isolated by density-gradient centrifugation (Metrizamide/plasma gradients), was compared in patients with suspected inflammatory diseases. The in vitro elution of both labels and the viability of the labelled cells (99Tcm, 98.5%; 111In, 96.5%) was comparable but the labelling efficiency was different (99Tcm, 44 +/- 13%; 111In, 72.5 +/- 5.5%). In vivo, the lung (t1/2 max: 7.7 min), liver and spleen perfusion patterns were nearly identical; the image quality for detail in 99Tcm scans was superior to 111In images. The blood disappearance curves of 99Tcm and 111In were comparable. In the small number of patients examined all infections could be diagnosed correctly, without false-positive or false-negative results. Disadvantageous is the renal excretion of 99Tcm complexes (3+% over 20 h) with kidney and bladder activity from the beginning of the study. The biliary excretion in half of the patients (n = 6) with unspecific positive small and large bowel visualization and the late intestinal excretion also render the diagnosis more difficult. The recommended best imaging times for abdominal and retroperitoneal inflammations are 30 min to 2 h after injection. Late scans in septic prosthetic joints have disproportionate long acquisition times. As a potential cell labelling compound, 99Tcm-HMPAO has a promising future in comparison to 111In scans because of the good availability of 99Tcm, the image quality and the lower radiation exposure to the patient when lower activities for the early diagnosis of abdominal inflammatory diseases are reinjected.     

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.     

In-111 Pseudomonas aeruginosa: A simple method of labeling live bacteria with a gamma-emitting radioisotope

We describe a simple and reliable technique for labeling Pseudomonas aeruginosa with a readily available commercial preparation of indium-111 (¹¹¹In) oxine. Labeling of a heavy bacterial suspension with 500 Ci of commercially prepared ¹¹¹In-oxine resulted in a yield of 0.0004 Ci of cell-associated ¹¹¹In per 10⁶ colony-forming units (CFU). The label was 88% bacterially associated and did not effect viability of the organism. Radiolabeling a gram-negative organism with ¹¹¹In-oxine provides a nontoxic, stable gamma-emitting bacterial tracer.     

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 cotrimoxazole-treated patient with Yersinia infection.     

The preparation and in vivo behaviour of 111In-oxine labelled neutrophils separated from whole blood using mono-poly resolving medium

using a single step separation procedure, we have developed a method for labelling human neutrophils with 111In-oxine. This method allows a rapid separation of neutrophils from whole blood, with negligible mononuclear or red cell contamination. Preliminary studies using 111In-labelled neutrophils show minimal lung retention and early accumulation in the spleen consistent with viable cells. In addition, focal accumulation of 111In has been imaged in patients with localized inflammation or sepsis.