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 dependent on the amount of plasma in the labelling medium; with 90% ACD plasma it was 4 x 10(-4) M and with Hepes saline buffer it was 4 x 10(-5) M. Using these tropolone concentrations and a low granulocyte concentration of 1 x 10(7) ml-1, the LE in 90% ACD plasma was 29% and in buffer was 74%. However, increasing the cell concentration to 1 x 10(8) 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 x 10(7) cell/ml in 1 ml ACD plasma, pH 7-7.6 with 0.1 ml tropolone at 4.4 x 10(-3) M mixed with no more than 100 microliter 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.     

The importance of radiolabelling human granulocytes with 111In-tropolonate or 111In-2-mercaptopyridine-N-oxide in plasma containing acid-citrate-dextrose

When blood cells are radiolabelled in plasma, for example with 111In-tropolonate, the plasma always contains an anticoagulant, usually acid-citrate-dextrose (ACD) or heparin. The effect of ACD and heparin on the labelling efficiency (LE) and optimum concentration of ligand required to radiolabel human granulocytes in plasma with 111In-tropolonate or 111In-2-mercaptopyridine-N-oxide (111In-merc) has been measured. The concentrations of ligand (tropolone or merc) required for maximum cell labelling in plasma containing ACD were 4 X 10(-4) M tropolone and 1 X 10(-4) M merc, whereas using plasma containing heparin, the optimum concentrations were 10-fold higher, at 4 X 10(-3) M and 1 X 10(-3) M respectively. At the optimum ligand concentrations, the LE for 1 X 10(8) granulocytes labelled in plasma containing ACD was 90% using 111In-tropolonate and 82% using 111In-merc, whereas using plasma containing heparin they were 68% and only 20%, respectively. Addition of ACD to heparinised plasma abolished the necessity for more ligand and increased the LE to the same values as those for plasma containing ACD alone. These results clearly demonstrate that to obtain a high LE using the lowest possible concentrations of tropolone or merc, the granulocytes must be labelled in plasma containing ACD.     

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.     

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).     

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.     

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 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 degrees C for 5 min with 100 muCi 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.     

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.     

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.     

The development of a clinical protocol for the radiolabelling of mixed leucocytes with 99Tcm-hexamethylpropyleneamine oxime

To devise a protocol for the radiolabelling of mixed leucocytes with 99Tcm-hexamethylpropyleneamine oxime (99Tcm-HMPAO), the effect of HMPAO concentration, cell concentration, plasma concentration and anticoagulant on the uptake of the lipophilic complex was measured, together with the stability of the 99Tcm on the labelled cells. It was found that cell uptake was rapid, independent of HMPAO concentration over the range 20-80 micrograms ml-1, but dependent on cell and plasma concentration. Incubation of mixed leucocytes from 85 ml blood with 1 ml ACD-plasma and 4 ml 99Tcm-HMPAO containing 400 MBq 99Tcm for 10 min at room temperature gave optimum results and was used in 90 patients. The mean labelling efficiency, which was the amount of added 99Tcm incorporated by the cells, was 55% (+/- 13 S.D.), of which 77% were incorporated by the granulocytes, 17% by the mononuclear cells and 6% by the platelets and erythrocytes. During a 1 h incubation in plasma 9% (+/- 4% S.D.) of the 99Tcm were released from the labelled mixed leucocytes. This occurred predominantly from the mononuclear cells.     

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.     

Significance of alteration in biodistribution of labeled lymphocytes exposed to stannous ion

The biodistribution patterns of ^99mTc (^99mTc-lymph) and ¹¹¹In-lymphocytes with [¹¹¹In-(Sn)-lymph] or without (¹¹¹In-lymph) stannous ion treatment was compared in Lewis rats. Syngeneic lymphocytes were labeled with either 125 Ci (4.63 MBq) ^99mTc or 5 uCi (1985 kBq) ¹¹¹In per 2×10⁷ cells. Mean labeling efficiency for ^99mTc and ¹¹¹In was 68.61%±3.90% (SEM) and 87.22%±2.01% (SEM) respectively. ^99mTc-lymph (n=4), ¹¹¹In-lymph (n=6) and ¹¹¹In-(Sn)-lymph (n=6) rats received 2x10⁷ cells and were killed 18 h later. While ^99mTc-lymph demonstrated significantly less localization in spleen, lymph nodes, and blood (P(F)0.01) as compared with ¹¹¹In-lymph, ¹¹¹In-(Sn)lymph also demonstrated a significant difference (P[F]=0.0001) in lymph node accumulation when compared to ¹¹¹In-lymph. As the activity levels utilized are not associated with cell radiation damage, these alterations in biodistribution do not reflect viability or chromosomal damage, but appear related to stannous ion exposure.Support from the American Heart Association-Virginia Affiliate     

Functional alterations of human platelets following indium-111 labelling using different incubation media and labelling agents

Human platelets were labelled in the absence or presence of plasma using indium-111 labelled oxine sulphate, tropolone or 2-mercaptopyridine-N-oxide (MPO). Under in vitro and in vivo conditions, platelet functions were evaluated by measuring their aggregability, survival, recovery and early distribution. High labelling efficiency was achieved in saline labelling, whereas with plasma labelling, it was necessary to concentrate the platelet-rich plasma to 4.8 × 10⁶ platelets/l. The aggregation of platelets labelled in plasma or saline was compared with that of controls; platelets labelled in saline showed lower aggregability in 2 M ADP but not in 5 M ADP nor with collagen. No significant differences in platelet survival and recovery were noted between platelets labelled in plasma and those labelled in saline. Our results indicate that partial loss of ADP aggregability in vitro does not influence the in vivo viability of platelets labelled in saline. Scintigraphic studies showed that platelets labelled in a saline medium were temporarily sequestrated in the liver but not in the spleen or heart. Thus, platelet labelling in saline does not affect platelet function adversely, but platelets labelled in plasma are more desirable for assessing the early distribution of platelets in the reticuloendothelial system.     

6-Alkoxymethyl-3-hydroxy-4H-pyranones: potential ligands for cell-labelling with indium

We have identified ligands for cell labelling with indium-111: 3-hydroxy-6-propoxymethyl-4H-pyran-4-one and 6-butoxymethyl-3-hydroxy-4H-pyran-4-one. The leucocyte labelling efficiencies of ¹¹¹In complexes of these ligands were higher and label stabilities were found to be similar compared with those obtained using ¹¹¹In-tropolonate. High labelling efficiencies of neutrophils and lymphocytes were achieved with ¹¹¹In complexes of pyranones. Tropolone was found to have a greater inhibitory effect on metalloenzymes and to cause greater impairment of platelet function than 3-hydroxy-6-propoxymethyl-4H-pyran-4-one. Thus 6-alkoxymethyl-3-hydroxy-4H-pyran-4-ones may have advantages over current ligands used in cell labelling with ¹¹¹In. Received 8 December 1998 and in revised form 25 June 1999     

99Tcm-HMPAO labelled leucocytes: comparison with 111In-tropolonate labelled granulocytes

The lipophilic complex, 99Tcm-hexamethylpropyleneamine oxime (HMPAO) is an efficient leucocyte label, and labels granulocytes with more stability than mononuclear leucocytes. The recovery of 99Tcm-HMPAO granulocytes, expressed as the percentage of injected granulocyte-associated activity circulating as granulocyte-associated activity 40-45 min after injection, was 37% (S.E. 3%), similar to the recovery of 111In-labelled granulocytes isolated and labelled in plasma using tropolone. The T1/2 of 99Tcm-HMPAO labelled granulocytes in blood was 4.4 h (S.E. 0.4 h), less than that of 111In-labelled granulocytes, although when a correction was made for 99Tcm elution, it was 6.4 h. The initial biodistribution of 99Tcm-labelled leucocytes was similar to 111In-labelled granulocytes, with a rapid initial lung transit, prominent splenic activity, bone marrow activity and minimal hepatic activity, although, unlike 111In, 99Tcm activity was also seen in urine, occasionally in the gallbladder, and, from about 4 h, consistently in the colon. Bone marrow activity was particularly prominent with 99Tcm. About 6% of 99Tcm was excreted in the faeces up to 48 h after injection, and about 17% in urine up to 24 h. The time-activity curves of reticuloendothelial activity up to 24 h were broadly similar for the two labelled cell preparations, and the differences that were observed can be explained on the basis of a higher rate of 99Tcm elution. Clinical information given by the two agents was similar in 27 of 30 patients who received both. Of the three who gave different information, one received 111In-labelled granulocytes which were considered to be functionally suboptimal and two, with inflammatory bowel disease, showed different distributions of abnormal bowel activity. We conclude that with respect to granulocyte kinetics and clinical data, 99Tcm-HMPAO labelled leucocytes are comparable with 111In-tropolonate labelled granulocytes.     

Radiolabelled mixed leukocytes and pure granulocytes with stabilized 99Tcm-exametazime

Although the methylene blue stabilizer extends the shelf life of 99Tcm-exametazime to 4-6 h after reconstitution, the dark blue appearance of the mixture of stabilized 99Tcm-exametazime and blood components makes it impossible to separate out the leukocyte button. The aim of this study was to assess the feasibility of using stabilized 99Tcm-exametazime to radiolabel mixed leukocytes separated by Volex sedimentation with hypotonic lysis (VL) and pure granulocytes isolated by a single-density Ficoll-Hypaque gradient with hypotonic lysis (FL). Isolated cells from 40-ml and 80-ml donor blood samples were mixed with 0.5 ml stabilized 99Tcm-exametazime (approximately 925 MBq 99Tcm and 62.5 micrograms exametazime) and incubated at room temperature for 15 min. After incubation, two dilution steps with 3 ml and 9 ml of 12.6% ACD/NS (anticoagulant citrate dextrose, solution A, USP, mixed with 0.9% NaCl, v/v) were conducted to dilute the dark blue mixture and to remove any unbound 99Tcm activity. With the addition of 9 ml of 12.6% ACD/NS solution to the 1-ml bottom portion from the first dilution, the supernatant of the centrifuged preparation was clear enough to be withdrawn. The overall labelling efficiency (LE) of labelled leukocytes and granulocytes was 87.1 +/- 4.9% and 87.7 +/- 6.2%, respectively (n = 12 each). Overall, radiolabelled cells (n = 12) from the 80-ml blood samples (LE = 90.3 +/- 2.8%) had an approximately 6% higher labelling efficiency than from the 40-ml blood samples (LE = 84.5 +/- 6.0%) and also had a slightly better in vitro stability compared to the 40-ml samples. The in vitro stability studies showed that only approximately 2% (n = 48) 99Tcm activity was eluted each hour from the radiolabelled leukocytes or granulocytes for the 40-ml or 80-ml blood samples during the 6-h evaluation period. Cell viability of all labelled leukocyte samples was confirmed by the trypan blue staining technique. In conclusion, mixed leukocytes separated by the VL method and pure granulocytes isolated by the FL method can be effectively labelled with stabilized 99Tcm-exametazime with the use of the 'double dilution' technique.     

<Superscript>68</Superscript>Ga-labelled DOTA-derivatised peptide ligands

⁶⁸Ge/⁶⁸Ga generators provide cyclotron-independent access to positron emission tomography (PET) radiopharmaceuticals. We describe a system which allows the safe and efficient handling of ⁶⁸Ge/⁶⁸Ga generator eluates for labelling of DOTA-derivatised peptide ligands. The system comprises concentration and purification of the ⁶⁸Ga eluate as well as labelling and purification steps for peptides, and can be used with different ⁶⁸Ge/⁶⁸Ga generator types. The suitability and efficiency were tested with two different DOTA-derivatised somatostatin derivatives and a DOTA-derivatised bombesin derivative. Amounts of 10–20 nmol of the peptides were sufficient and resulted in labelling yields of 50% for all peptides. The built-in safety precautions have proven to be appropriate in allowing use of the method for routine clinical applications. The system was set up and operated in a hot lab by personnel with no previous experience in the preparation of PET radiopharmaceuticals.     

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.