Leucocyte scanning: preparation and labelling of leucocytes with 111-Indium oxide and its clinical application

A method for the concentration of leucocytes from blood and labelling of the separated cells with 111-Indium oxine is described. This method guaranteed a good preparation. On average there were 64.8% of leucocytes from the blood in the concentrate. The yield of the labelling averaged 93%. Seventy-two patients from various departments were examined to test the clinical application of the labelled leucocytes in the diagnosis of inflammatory diseases. The results obtained led to the formulation of six indications for the appropriate application of leucocyte scanning in everyday clinical routine.     

Cell labelling with colloidal substances in whole blood

A method for the labelling of leucocytes with ^99mTc-colloid is described. The labelling can be done in samples of whole blood, because the colloid is only taken up by the phagocytosing cells, the monocytes and the granulocytes. The part of the colloid that is not phagocytosed is brought to a soluble state with Na-citrate, so that only the phagocytosed colloid is reinjected. The labelling efficiency with this method is between 80% and 90%. Measurements of the activity in the leucocytes 3 h after reinjection, have shown that at least 50% of the labelled cells are at this time still available in the blood pool.The clinical results on 32 patients with the tentative diagnosis of an abdominal abscess and on 42 patients with the tentative diagnosis of septic loosening of an endoprosthesis have shown that the labelled leucocytes are very well suited to show up local foci of inflammation.     

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.     

Radiochemistry and biostability of autologous leucocytes labelled with 99mTc-stannous colloid in whole blood

Autologous leucocytes were labelled in whole blood by phagocytic uptake of ^99mTc-stannous colloid. This colloid has a mean particle size of 1.5 m and labels leucocytes with 81% efficiency. Individual cell uptakes were: granulocytes 42%, monocytes 39%. Isotonic sodium citrate added after the labelling procedure did solubilise excess colloid but was not necessary for adequate removal of excess colloid. Labelled leucocytes were shown in vitro to be viable and maintain normal bactericidal and chemotactic capacity. Biodistribution, clearance rates and dosimetry are presented. These results indicate that autologous leucocytes can be efficiently labelled with ^99mTc-stannous colloid with good residual cell function.     

A rapid method for the preparation of 99Tcm hexametazime-labelled leucocytes

99Tcm (+/-)-hexamethylpropyleneamineoxime (HMPAO) (99Tcm Hexametazime) has been recently reported as an alternative for labelling leucocytes. For reasons of convenience, radiation dose and image quality, many workers have welcomed this novel approach except for its complicated labelling protocol and venesection of 100 ml. This technique has been modified to give a simpler routine in-house labelling technique. It has three advantages: only about 20 ml of blood is required, the labelling time is just under 1 h and high yields of labelled leucocytes are obtained (mean of 500 MBq per injection dose). The properties of labelled leucocytes using this modified method are; 80% granulocyte-bound radioactivity, a rapid lung transit and a blood granulocyte recovery of 40% at 30 min similar to those described previously. The viability of the labelled leucocytes was tested and confirmed in vitro using a migration technique and in vivo by showing no lung retention on early imaging and high splenic uptake. A rapid in-process chromatography assessment procedure for regulating the protocol has been developed. Successful abscess imaging by 4 h has been achieved in 21 patients with normal results in another 22 patients without abscesses. This simpler method should encourage a more widespread application of scintigraphy using radiolabelled granulocytes.     

Experience with the iodine-123 and technetium-99m labelled anti-granulocyte antibody MAb47: a comparison of labelling methods

Four different methods of radiolabelling the anti-granulocyte monoclonal antibody MAW were compared and their influence on diagnostic value studied. The best clinical images were obtained following labelling with iodine-123 by the Iodogen method and direct labelling with technetium-99m after tris-(carboxyethyl)-phosphine treatment of MAW to achieve disulphide bridge reduction.^99mTc labelling using a specific ligand (MAb47-mtp), or a second method involving direct reduction with mercaptoethanol, led to an increased background activity in clinical studies, thus impeding the diagnosis of chronic disease. Fresh infections were clearly localized by all four preparations. The elimination of the activity from the blood was slower in the case of the iodinated MAb47, while the collected urine samples showed an excrection of about 10% of the injected activity per day independent of the labelling method. The results in terms of sensitivity and specificity were rather similar for all labelling methods and ranged from 90% to 99%.     

Biological consequences of the heterogeneous irradiation of lymphocytes during technetium-99m hexamethylpropylene amine oxime white blood cell labelling

Technetium-99m hexamethylpropylene amine oxime (^99mTc-HMPAO) labelling of white blood cells, routinely used for the detection of infection, results in the incorporation of radioactivity by polymorphonuclear leucocytes and also lymphocytes and can induce cell lesions in the latter case. The aim of this study was therefore to acquire data on the morphological and functional status of labelled lymphocytes present in the ^99mTc-HMPAO leucocyte mixture and to determine the cellular consequences of labelling. The mean radioactivity associated with lymphocytes was 325±10.8 kBq/10⁶ lymphocytes under standard labelling conditions. Microautoradiographic studies showed that labelling was heterogeneous (4% intensely labelled cells), which prevented calculation of the mean absorbed dose. The frequency of chromosomal aberrations (dicentrics and rings) in the labelled lymphocytes for 380 kBq/10⁶ cells was 1.08±0.09 but no abnormality was observed in the unlabelled control lymphocytes. The plating efficiency of labelled lymphocytes was reduced, as compared with that for control cells, but some lymphocytes were still able to form clones and were still ”alive” by radiobiological definition. It is therefore suggested that lymphocytes should be removed from ^99mTc-HMPAO cell preparations before administration to patients. Received 9 March and in revised form 1 June 1998     

Evaluation of white cell scintigraphy using indium-111 and technetium-99m labelled leucocytes

Indium-111 oxine labelled leucocyte (¹¹¹In oxine leucocyte) scintigraphy is the test of choice in detecting occult infection and localising focal inflammation. ¹¹¹In oxine labelling is technically difficult and expensive and leucocyte labelling with technetium-99m stannous colloid (^99mTc Sn colloid) has been considered to be an alternative. Leucocytes from 40 cases referred for investigation of occult infection or localisation of inflammation were simultaneously labelled with ¹¹¹In oxine and ^99mTc Sn colloid with dual isotope acquisition performed at 1, 3 and 24 h. Twenty-four hour ^99mTc Sn colloid scans were corrected for ¹¹¹In downscatter. Each case was independently interpreted by two experienced observers. Twentyone patients demonstrated positive ¹¹¹In oxine leucocyte scans. Using ¹¹¹In oxine leucocyte scans as the gold standard, ^99mTc Sn colloid leucocyte scanning had an overall sensitivity of 86% and a specificity of 95%. Clinical follow-up verified that three patients had false negative ^99mTc Sn colloid leucocyte scans and one patient had a false positive. Further clinical evaluation of ^99mTc Sn colloid labelled leucocytes is required before they can become a reliable replacement for ¹¹¹In oxine leucocytes. Correspondence to: S. Boyd     

An improved method for the in vivo labelling of red blood cells with 99mTc in gated cardiac imaging

Red blood cells of 10 patients were labelled with ^99mTc by the in vivo method, 5 of the patients were orally administired with 400 mg potassium perchlorate at the time of stannous pyrophosphate injection and 5 ml blood was withdrawn at 10 min postinjection of pertechnetate to determine the labelling efficiency of RBC's. The RBC and plasma volumes were also determined in all patients by the ⁵¹Cr method. Our results indicated higher incorporation of injected radioactivity into RBC's in the perchlorate administered group (83.8%±9.2%) compared to the other (58.5%±22.0%) (P0.01). The thyroid and stomach were visualized in the control group, but not in the perchlorate administered group. In routine application, 50 MUGA studies have been done with perchlorate administration with excellent results. In 60 patients without perchlorate administration previous to the present study the EF could not be determined in 6 patients (10%). Our results indicated that potassium perchlorate may be used routinely for in vivo labelling of RBC's to improve the labelling efficiency and the quality of gated cardiac imaging.     

99Tcm-exametazime-labelled leucocytes: effect of volume and concentration of exametazime on labelling efficiency, and clinical protocol for high efficiency multi-dose radiolabelling.

We have investigated the effect of volume and concentration of exametazime on the labelling efficiency of 99Tcm-exametazime-labelled leucocytes. The first study examined the effect of varying the volume of exametazine solution whilst the concentration remained constant. A vial of Ceretec (Amersham Ltd plc) was reconstituted with sodium chloride injection BP. Aliquots of 0.25-2.0 ml were removed, added to sodium pertechnetate injection BP and incubated with 2 x 10(7)-5 x 10(7) leucocytes from 25 ml blood. The labelling efficiency decreased from 65 +/- 10% S.D. (0.25 ml) to 45 +/- 8% (2.0 ml) (n = 4). In a second study different concentrations of exametazime solution were used whilst the volume remained constant. A vial of Ceretec was reconstituted with sodium chloride injection BP. Aliquots of 25-200 micrograms were removed, made up to a fixed volume of 0.6 ml, added to pertechnetate and incubated with the plug of leucocytes as before. The labelling efficiency decreased as the concentration of ligand decreased. Thus for 200 micrograms/0.6 ml the labelling efficiency was 64 +/- 5% and for 25 micrograms/0.6 ml the labelling efficiency was 43 +/- 18% (n = 4). Clinical studies were performed using 50 ml blood from patients with a wide range of inflammatory disorders including inflammatory bowel disease, abdominal abscess and vasculitis. The concentration of ligand used was 83 micrograms/0.25 ml. The labelling efficiency was found to be 82 +/- 7% (n = 36).     

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.     

99mTc-SnF2 colloid "LLK": particle size,morphology and leucocyte labelling behaviour

99mTc-SnF2 colloid (Radpharm LLK) leucocyte labelling agent is used in whole blood, exploiting phagocytosis. The objectives of this work were to optimize leucocyte labelling in leucocyte-enriched plasma, and to investigate: (i) the effect of temperature and other factors on labelling efficiency; (ii) the selectivity for different leucocyte types; (iii) the viability of the labelled cells and efflux of the radiolabel; and (iv) the physical characteristics of the colloid. Density gradient centrifugation was used to investigate the labelling efficiency, cell selectivity and efflux, Trypan blue to study the viability, and laser scattering, electron microscopy and membrane filtration to investigate particle size and morphology. Particles appeared as loose, coiled, chain-like aggregates of much smaller particles (<0.05 microm). The aggregate diameter ranged from <0.1 to >5 microm and increased with time. The distribution of radioactivity amongst the particle sizes varied widely. The labelling efficiency in leucocyte-rich plasma was enhanced at 37 degrees C compared to room temperature, and by centrifuging during labelling. The selectivity for different leucocyte types varied markedly between batches and blood samples, in some cases showing preference for mononuclear cells and in others for granulocytes. Viability was excellent and comparable with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO)-labelled cells. A significant fraction of radiolabel, comparable to that observed with 99mTc-HMPAO, was lost from leucocytes during incubation in vitro over 4 h. Thus, 99mTc-SnF2 is a convenient, efficient labelling agent for leucocytes, but shows variable cell selectivity which may be linked to particle size variability, and there is significant efflux of radioactivity from labelled cells.     

Isolation and labelling of human leukocytes with 99mTc

The role of the leukocyte isolation procedure on cell labelling with ^99mTc has been evaluated. Separation of leukocytes was performed by two procedures: (1) sedimentation on methyl cellulose, followed by discontinuous gradient centrifugation; (2) methyl cellulose sedimentation and hypotonic haemolysis of residual red blood cells. After washing the cells in saline and incubation with a stannous pyrophosphate agent, the leukocytes were labelled with 5–10 mCi ^99mTc. Procedure 1 gave a higher purity but lower recovery of polymorphonuclear leukocytes, and a minor contamination of red blood cells. ^99mTc labelling of cells was slightly more efficient with this method, probably due to the presence of red blood cells. Procedure 1 is recommended for in vitro studies on cell kinetics and procedure 2 is recommended for clinical use.     

Radiochemistry and biostability of autologous leucocytes labelled with 99mTc-stannous colloid in whole blood

Autologous leucocytes were labelled in whole blood by phagocytic uptake of 99mTc-stannous colloid. This colloid has a mean particle size of 1.5 micron and labels leucocytes with 81% efficiency. Individual cell uptakes were: granulocytes 42%, monocytes 39%. Isotonic sodium citrate added after the labelling procedure did solubilise excess colloid but was not necessary for adequate removal of excess colloid. Labelled leucocytes were shown in vitro to be viable and maintain normal bactericidal and chemotactic capacity. Biodistribution, clearance rates and dosimetry are presented. These results indicate that autologous leucocytes can be efficiently labelled with 99mTc-stannous colloid with good residual cell function.     

Rapid measurement of blood leakage during regional chemotherapy

In order to avoid complications after regional chemotherapy (isolated hyperthermic perfusion) of the extremities, rapid measurement of blood leakage from the extracorporeal to the systemic circulation is important. A method using technetium-99m in vivo red blood cell (RBC) labelling is reported that provides results within 3 min. Blood samples drawn from the systemic and the extracorporeal circulation were measured for ^99mTc activity using a mobile well counter, and the leakage values calculated. The mean result was 7.6%±6.5%/15 min (n=209). The corresponding flow rate was 100.2±85.7 ml/15 min (mean ± SD). The values for isolation perfusion of the upper and the lower extremities are compared. The leakage results using ^99mTc RBC labelling were correlated with other blood pool markers. Iodine-125 human serum albumin and indium-113 m transferrin were administered in subgroups of 4 and 19 patients simultaneously. Using linear regression, the coefficient of correlation was 0.72 for ^99mTc/^113mIn and 0.58 for ^99mTc/¹²⁵I. Comparison with the alternatives suggests that the rapid method of leakage measurement after ^99mTc RBC labelling can be considered one of the most practicable and reliable methods available.This paper is dedicated to Prof. E. Oberhausen, Homburg/Saar, on the occasion of his 65th birthday Correspondence to: C. Alexander     

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.     

Labelling of leucocytes with colloidal tech netium-99m-SnF2: an investigation of the labelling process by autoradiography

Autoradiography of smears and frozen sections of labelled cell suspensions was used to study the distribution of radioactivity in and among blood cells labelled in either whole blood or leucocyte-rich plasma (LRP) with technetium-99m-SnF₂ colloid. The tracer proved selective for neutrophils: the labelling probability (relative to that for erythrocytes) for each cell type in LRP (mean of five samples) was: neutrophils, 9.4; lymphocytes, 3.7; monocytes, 3.0; eosinophils 1.4; erythrocytes, 1.0. When labelling was carried out in whole blood (five samples), 74.5%±8.3% of the cell-bound radioactivity was bound to erythrocytes, 13.6%±6.5% to neutrophils, and 11.9%±2.1% to lymphocytes, whereas in LRP (in which the leucocytes were only slightly out-numbered by erythrocytes), 76.5%±14.9% of radioactivity was neutrophil bound. Labelled cells in smear autoradiographs exhibited two distinct silver grain patterns, diffuse, consistent with an intracellular radioactive particle (in neutrophils), and focal, consistent with a cell surface-adhering particle in direct contact with the emulsion (in other leucocyte types and erythrocytes). The phagocytic inhibitor cytochalasin B neither reduced the proportion of labelled neutrophils nor altered the labelling pattern. Neutrophils were able to scavenge radioactivity from the surface of erythrocytes. It is concluded that neutrophils bind^99mTc-SnF₂ intracellularly by phagocytosis, with high affinity; other cells become labelled at the cell surface reversibly and with lower affinity. This selectivity is high enough to permit predominantly leucocyte labelling in LRP but not in whole blood.     

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.     

Simultaneous administration of111In-human immunoglobulin and99mTc-HMPAO labelled leucocytes in inflammatory bowel disease

Technetium-99m hexamethylpropylene amine oxime (HMPAO) labelled leucocytes and indium-111 polyclonal immunoglobulin (IgG) were simultaneously injected into a group of 27 patients routinely referred for the investigation of inflammatory bowel disease (IBD). Ten-minute anterior abdomen and tail on detector views were obtained at 30 min, 4 h and 24 h p.i. of both tracers. The diagnosis of IBD was obtained in all cases by endoscopy with biopsy and/or surgery. Images were blindly evaluated by two experienced observers who only knew of the clinical suspicion of IBD. IBD was confirmed in 20 patients (12 with Crohn's disease and eight with ulcerative colitis). Sensitivity, specificity and accuracy were 100%, 85% and 96% respectively for labelled leucocytes and 70%, 85% and 74% for IgG. Both IgG and leucocyte scans were normal in six out of seven patients in whom a diagnosis of IBD was excluded; the remaining patient, with ischaemic colitis, was falsely positive with both agents. As far as disease extension is concerned, the IgG study localized 27 diseased segments, whereas 49 were seen with the leucocyte study. Eighty-four segments were normal and 25 showed tracer uptake with both agents. Twenty-four were positive only with the leucocyte study and two were positive only with the IgG study. Agreement between the agents was 80.7%. These results confirm that¹¹¹In-human polyclonal scintigraphy is less sensitive than^99mTc-HMPAO scintigraphy both for the diagnosis of IBD and in the evaluation of disease extension. Nevertheless, if leucocyte labelling is not available, labelled IgG can be used only for diagnostic purposes.     

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.