The use of 2-[18F]fluoro-2-deoxy-D-glucose as a potential in vitro agent for labelling human granulocytes for clinical studies by positron emission tomography.

In this study, 2-[18F]fluoro-2-deoxy-D-glucose, [( 18F]FDG) was used to radiolabel human granulocytes in vitro for possible clinical use by positron emission tomography (PET). Uptake of [18F]FDG was dependent on the amount of glucose in the labelling medium, e.g. when 1 x 10(7) granulocytes were incubated with [18F]FDG containing 15 micrograms/mL glucose 80% of [18F]FDG was incorporated within 30 min, but in the presence of 1 mg/mL of glucose it was reduced to 2%. Increasing the cell concentration and activating the granulocytes with Streptococcus pneumoniae, opsonized zymosan or phorbol myristate acetate all increased the uptake of [18F]FDG. Retention of the [18F]FDG by the cells as [18F]FDG-6-phosphate was also dependent on the extracellular glucose concentration, 9% was released within 60 min in the absence of glucose, but 27% in the presence of 1 mg/mL glucose.

Imaging of metastatic colorectal cancer with tumour-activated killer lymphocytes.

Adoptive immunotherapy may be useful for treating or visualising metastatic cancer. Lymphocytes were taken from 6 patients with metastatic colorectal cancer and cultured with cells from the patients primary tumour to produce tumour-activated killer (TAK) lymphocytes. We re-injected each patient with IIIIn-labelled TAK cells in order to visualise metastases. Images were taken with a gamma-camera for up to 48 h after injection. Metastases were revealed as early as 4 h in the lung and as late as 48 h in the abdomen. Liver images produced "cold" spots corresponding to metastatic lesions. Lymph nodes were not visualised. Re-injection of TAK cells raised against autologous colorectal tumours reveals the sites of metastases.

Labelling autologous platelets with 111In tropolonate for platelet kinetic studies: limitations imposed by thrombocytopenia.

An in vitro method of radiolabelling platelets with 111In tropolonate in plasma has been devised enabling imaging and cell kinetic studies to be performed in patients with thrombocytopenia (TP) using autologous, rather than donor, platelets. Platelets from 10 TP patients, with platelet counts ranging from 4-91 x 10(9)/l, were labelled in 50% plasma with 111In tropolonate, containing the optimum tropolone concentration of 2 x 10(-4) mol/l, at platelet concentrations ranging from 0.08-4.5 x 10(9)/ml resulting in labelling efficiencies (LE) between 51 and 86%. In 4 patients, red cells contaminated the platelets but this was corrected for by measuring the proportion of 111In on the platelets prior to injection and in the post-injection blood samples. Platelet recovery (PR), mean platelet life-span (MPLS) and platelet production rate (PPR) were calculated and splenic and hepatic images were taken. The results clearly show that useful clinical data can be obtained by this method even in patients with severe TP.

Osteomyelitis.

The use of 111In-labelled granulocyte scintigraphy is recognized as a reliable method for detecting osteomyelitis and has similar sensitivity and significantly increased specificity compared to bone scintigraphy and 67Ga studies. Recent published work using pure granulocytes labelled with 111In tropolonate to detect osteomyelitis resulted in sensitivity of 100% and specificity of 92%. 99Tcm as an alternative granulocyte label offers advantages of convenience, lower radiation dose and higher image resolution. We have scanned 20 patients with suspected osteomyelitis using autologous granulocytes labelled with 99Tcm hexamethylpropyleneamineoxime (HMPAO), 12 of whom had prosthetic joints. The scan results were correlated with clinical, radiographic, microbiological and histological findings. Sensitivity was 100% and specificity was 93% which compares favourably with results obtained using 111In-labelled granulocytes. We believe that labelled granulocyte scintigraphy is a useful investigation in the diagnosis of osteomyelitis and that 99Tcm HMPAO appears to be at least as useful as 111In as the labelling agent.

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.

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.

Investigations to determine the optimum conditions for radiolabelling human platelets with 99Tcm-hexamethyl propylene amine oxime (99Tcm-HM-PAO)

99Tcm-hexamethyl propylene amine oxime (99Tcm-HM-PAO) has been evaluated as an agent to radiolabel human platelets in vitro. The rate of uptake of this lipophilic complex, the effect of HM-PAO, plasma and platelet concentration were measured to determine the optimum conditions for radiolabelling platelets for short-term clinical investigations of thromboses. The complex was made according to the manufacturer's instructions and immediately added to isolated platelets in vitro. The rate of labelling was slower than for leucocytes, reaching a plateau after approximately 40 min at room temperature (RT). Increasing the temperature to 37 degrees C did not increase the labelling efficiency (LE). Addition of plasma to platelets at a cell concentration of 1 x 10(9) [corrected] ml-1 reduced the LE from 66% in saline to 52% in 20% ACD-plasma. However, increasing the platelet concentration from 5 x 10(8) to 2 x 10(9) ml-1, increased the LE from 9 to 76% for platelets labelled in 20% plasma for 30 min at RT. The in vitro stability of the 99Tcm in the labelled cells showed that 7% of the radioactivity were immediately released from the platelets and a further 13% were eluted during a 60 min incubation in plasma at 37 degrees C. It has been concluded that incubation of platelets at RT with 99Tcm-HM-PAO containing 80 micrograms ml-1 HM-PAO at a cell concentration of 1 x 10(9) ml-1 or greater, results in a high LE, with more than 85% of the 99Tcm being retained by the platelets during a 60 min incubation in plasma.

Inflammation: imaging with Tc-99m HMPAO-labeled leukocytes.

Leukocytes labeled with technetium-99m hexamethylpropyleneamine oxime (HMPAO) were used in 100 patients: 32 with suspected inflammatory bowel disease, 17 with fever of unknown origin, 21 with suspected abdominal sepsis, 20 with suspected bone sepsis, seven with bronchiectasis, and three with recent myocardial infarction. The distribution of activity in patients subsequently shown not to have inflammatory bowel disease was similar to that previously described for indium-111-labeled leukocytes. However, in this study, activity was also seen in the kidneys and bladder and occasionally the gallbladder on both early (1-3 hours) and late (24 hours) views, and in the colon in late views. Migration of Tc-99m-labeled granulocytes was seen in inflammatory disease as early as 30 minutes after injection, while normal bowel activity was not seen before 4 hours. The sensitivity of Tc99m-labeled leukocytes in the detection of inflammation was 100%, the specificity was 95%.

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.

A simple in vitro method of radiolabelling human erythrocytes in whole blood with 113mIn-tropolonate.

A simple and rapid in vitro procedure has been developed for selectively radiolabelling erythrocytes in whole blood using 113mIn-tropolonate. A maximum labelling efficiency of 97% was achieved, of which 95.5% was on the erythrocytes after only 5 min incubation of whole blood at room temperature. The optimum amount of tropolone for labelling whole blood was 10 micrograms of tropolone per ml of blood using acid-citrate dextrose (ACD) as the anticoagulant and 50 micrograms of tropolone per ml of blood using heparin. Under these optimum conditions, only 2.5% of the cell-bound 113mIn was released from the labelled cells during a 1 h in vitro incubation in cell-free plasma, irrespective of the anticoagulant used. These results suggest that 113mIn-tropolonate may prove to be a useful in vitro agent for labelling erythrocytes for short-term clinical investigations, especially at centres where 99mTc and 111In are unavailable.

Clinical experience with 99mTc-hexamethylpropylene-amineoxime for labelling leucocytes and imaging inflammation.

Hexamethylpropylene-amineoxime (HMPAO) forms a lipid-soluble neutral complex with 99mTc which is rapidly incorporated into leucocytes in vitro. In six patients with suspected or known inflammatory disease, a "mixed" leucocyte suspension isolated from 85 ml blood anticoagulated with acid-citrate-dextrose was labelled by 99mTc-HMPAO with a mean efficiency of 47% (SE2%), of which 78% (3) was taken up by granulocytes. Activity eluted more rapidly from other cell types in vitro than from granulocytes, which remained firmly labelled. Mean initial biodistribution of the label and granulocyte recovery in blood of 32% (8) at 30-40 min showed that the granulocytes were not significantly activated during labelling. All six patients were positive for inflammatory disease, as early as 30 min in five patients and at 3 h in the sixth; they all remained positive at 20-24 h. Four patients also received 111In-labelled "pure" granulocytes. In terms of detail, the 99mTc images were comparable or superior to the 111In images.

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.

PAC studies of 111In binding to transferrin, tropolone and acetylacetone in aqueous solutions.

Time integral and time-differential PAC measurements have been made over a wide temperature range in aqueous solutions of [111In]tropolonate and [111In]acetylacetonate. The quadrupole frequency in the latter is approximately 30% higher than that in the former and the molecular volumes derived from rotational correlation times show the expected differences. Apo-transferrin was separately added to the two 111In-chelates and the transfer of activity from chelate to transferrin followed as a function of relative molar concentrations. Very much larger molar ratios of transferrin to tropolone than of transferrin to acetylacetone were required before substantial transfer of 111In from chelate to transferrin took place. This difference in affinity for transferrin could be one significant factor in explaining the enhanced ability of [111In]tropolonate to label blood cells in the presence of plasma. The determination of PAC parameters in [111In]transferrin over a range of temperatures showed that the values of quadrupole frequency obtained depended on the number of binding sites assumed. For only one 111In site per molecule, the quadrupole frequency increases by over 50% as the temperature is reduced below the freezing point of the solution. If two 111In sites are assumed there appears to be a change in the percentage occupancy of the two sites on either side of the transition.

Lung transit of 111Indium-labelled granulocytes. Relationship to labelling techniques.

The early in vivo distribution of 111Indium-labelled granulocytes, recorded by dynamic imaging using a gamma camara and computer, varied according to the separation and labelling technique. Following i.v. bolus injection, 4 kinetic patterns could be identified: (A) rapid transit through the pulmonary vasculature, (B) delayed transit through the lung with clearance by about 30 min, (C) complete retention by the lung, for up to 10 min, followed by slow release over a period of 1 to 2 h, (D) delayed transit through the lung with a similar time course to (B) but with subsequent heavy liver uptake. Granulocytes labelled with 111In-tropolonate and maintained in plasma throughout the labelling procedure, whether injected as a 'pure' (separated by plasma-enriched density gradient centrifugation) or 'crude' (separated by differential centrifugation) preparation, displayed type A kinetics, thought to most closely represent the normal behaviour of granulocytes. 'Crude' cells labelled in saline with 111In-acetylacetonate displayed type B kinetics. 'Pure' cells isolated on Percoll-saline and labelled in saline with 111In-acetylacetonate displayed type C kinetics, thought to represent granulocyte 'stimulation' and/or damage, or type D kinetics, thought to represent severe damage. The importance is stressed of labelling granulocytes for kinetic studies with a technique that results in minimal alteration of cell behaviour.

Imaging of inflammation with indium-111 tropolonate labeled leukocytes.

Indium-111 tropolonate has recently been introduced as a new cell-labeling agent. It has the interesting property of labeling cells in plasma with high efficiency, and may therefore promote an improvement in viability of labeled cells. This paper describes our initial experience with In-111 tropolonate as a leukocyte label for abscess imaging. Pure populations of separated granulocytes, as well as crude leukocyte preparations, have been labeled. Of 101 studies performed, 51 were positive (no false positives) and 50 negatives, of which only two were false negatives. Localization in sites of inflammation was prominent and rapid. Of 36 positive studies, 27 were already positive at 40 min following injection and an additional nine at 3 hr. Of the other 15 positive studies, 11 were scanned for the first time at 3 hr, when they were positive. Granulocytes labeled with this agent in plasma showed minimal sequestration in lungs and liver, interpreted as indicating improved viability in comparison with cells displaying prolonged lung sequestration.