Studies on white blood cell labelling: (99)Tc(m)-HMPAO preferentially labels granulocytes.

Mixed leucocytes have been labelled in small volumes of plasma using small aliquots of hexamethylpropylene amine oxime (HMPAO) from vials of Ceretec, which were fractionated after reconstitution and stored frozen under nitrogen. Labelling efficiency was found to be independent of HMPAO concentration >8.8 microg.ml-1 and independent of cell concentration at >8x10(7) ml-1. Results are presented from 145 patients referred for white cell scintigraphy. Labelling efficiency was 78.9+/-9.8%. The labelled mixed leucocytes were separated on a 3-step density gradient of Percoll/plasma which showed that (99)Tc(m)-HMPAO gave a granulocyte/lymphocyte specific activity ratio of 1.67; the ratio for (111)In-tropolone or oxine was 1.0, with no preference for granulocytes. It was noted that the labelling efficiency was reduced when there was a high proportion of lymphocytes present, and also that in one patient with non-Hodgkin's lymphoma the lymphocytes labelled as equally well as the granulocytes. The stability of the label was studied by fractionating the mixed cells after in vitro incubation and after 1 h in vivo; very little loss of label occurred.

Value of protein-bound radioactive iodine measurements in the management of differentiated thyroid cancer treated with (131)I.

Measurement of the protein-bound radioactive iodine level (PBI(131)) in the plasma of patients following (131)I-iodide administration for thyroid cancer has been re-examined in a retrospective study of 171 patient episodes. It is shown that whereas the previously used threshold value for the measurement at 6 days does not correlate well with the 3-day whole body scan, there is good agreement between the scan and the temporal changes in PBI(131) from 1-6 days: an increasing PBI(131) correlates with a positive scan, and a decreasing PBI(131) with a negative scan. The area under the curve (AUC) for the PBI(131)-time curve is related to the absorbed dose for the tumour. For a small group of 11 patients, dosimetry estimates were made from serial scans, quantified with phantoms; these absorbed doses correlated with the AUC and the 6-day PBI(131). Therefore, it is suggested that these parameters may be useful in predicting absorbed radiation dose in these patients.

Uptake of technetium-99m hexamethylpropylene amine oxime labelled white cells in lymph nodes involved in non-Hodgkin's lymphoma.

Radiolabelled white cell scanning is widely used to detect the presence of infection. We present a case of non-Hodgkin s lymphoma manifesting with signs and symptoms suggestive of infection, in which a technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO) white cell scan demonstrated high uptake in lymph nodes involved by lymphoma. Differential cell analysis showed preferential lymphocyte labelling. The classification and management of the disease were changed accordingly. Our findings suggest that a future role for 99mTc-HMPAO labelled white cells in the assessment of disease activity of lymphoma should be investigated.

99mTc-HMPAO as a tumour blood flow agent.

The cerebral blood flow agent, 99mTc-HMPAO ('CeretecTM') has been investigated to see if it can be used to estimate tumour blood flow. Its distribution in Balb/c mice bearing either a subcutaneously implanted sarcoma or a plasmacytoma has been shown to be similar to that of 86RbCl. The changes in peripheral blood flow caused by the beta-blocker, propranolol, and by Nembutal anaesthesia, are manifested equally by 99mTc-HMPAO and 86Rb. We conclude, therefore, that HMPAO may be useful in estimating tumour perfusion.

In vitro uptake of gallium and chlorpromazine by mouse tumour cells.

Primary cell suspensions were prepared from mouse sarcoma by enzymatic digestion with pronase. The cells were incubated with gallium citrate Ga 67 or the basic drug 14C-chlorpromazine (CPZ) for up to 1 h at 37 degrees C, and the label uptake was determined. The Ga uptake was proportional to time (0-30 min), whilst the CPZ uptake rapidly reached apparent saturation (less than 10 min). Metabolic inhibitors did not affect label uptake; however, disrupting the cell membrane with N-ethylmaleimide or heating at 56 degrees C significantly reduced CPZ accumulation but did not inhibit Ga uptake. Ga accumulation was decreased by adding human transferrin (0.5 mg/ml). Both gallium and chlorpromazine are fixed in the lysosomes of cells; however, in this system, they appeared to enter mouse sarcoma cells by different energy-independent mechanisms. The Ga uptake may reflect adsorption to cell components, whilst CPZ uptake required an intact cell and may be due to passive diffusion.

Relative tumour enhancement of 67Ga uptake by desferrioxamine.

The administration of the iron- and gallium-chelating agent, desferrioxamine (DFO), to mice after the injection of 67Ga has been shown to improve tumour to normal tissue ratios. Time and dose parameters have been studied. The optimal time was about 7 h after 67Ga, when the tumour to blood ratio was increased 16 times compared with controls. The effect was due to the greater retention of 67Ga in tumour compared with other tissue. Tumour retention after DFO was 78% compared with controls at 7 h, giving a tumour to blood ratio of 25.9. Tumour retention of 67Ga correlated well with the proportion of cell-bound activity which was localised in the lysosomes. Prior administration of iron increased lysosomal accumulation of 67Ga in tumour cells, and hence the retention of the nuclide after the use of DFO.

The mechanism of 67Ga uptake in animal and human tumours.

The subcellular distribution of 67Ga has been studied by differential centrifugation in 3 transplantable mouse tumours, 3 transplantable rat tumours, 1 dog tumour, 3 human tumour xenografts and 2 human tumours in situ at various times after injection of the citrate complex. From 24 h post injection of nuclide was located predominantly in lysosomal structures in all the tumours studied. Studies in two murine tumours showed marked differences in the rate of lysosomal accumulation of 67Ga. In the ADJ/PC6 plasmacytoma lysosomal uptake of 67Ga had reached a plateau within 15 min while in the S180 tumour lysosomal accumulation of the nuclide occurred over the first 24 h. Normal mouse liver showed a similar pattern to this latter tumour. It is postulated that these variations in the rate of lysosomal accumulation of 67Ga reflect differences in the permeability of the lysosomal membrane. While large amounts of 67Ga were found in the crude nuclear fraction of some tumours this was attributed to unbroken cells as studies with purified nuclei from 7 different tumours indicated that between 2 and 14% of the total tumour 67Ga was associated with the nuclei.

The effect of the administration of iron on gallium 67 citrate uptake in tumours.

Consideration has been given to the use of iron as an aid in 67Ga scanning. The effects of various doses and the time of injection have been measured quantitatively in four transplantable rodent tumours. It was found most effective to administer the iron, as ferric ammonium citrate, before the 67Ga injection. The experimental tumours respond to different extents, and the tumour:non-tumour ratios at four hours after 67Ga injection have been found to be from two to four times better than those obtained at 24 hours with 67Ga alone. The effects of administered iron on the rate of blood clearance and the plasma binding of 67Ga have been measured. The use of carrier gallium or scandium was found to be less advantageous than the use of iron.

The relationship of 67Ga uptake to citrate dose, compared with 67Ga-chloride and 67Ga-transferrin in rodent tissues and tumours.

The uptake of 67Ga-citrate in the tissues of the mouse and rat and in two mouse tumours, S180 and PC6, has been measured and shown to be independent of the citrate concentration of the injection dose. The tumour uptake and tissue distribution of 67Ga-chloride and 67Ga-transferrin were identical with those of the citrate complex. Whole-body retention data for 67Ga-citrate in the mouse for 14 days after injection are also presented.

The role of lysosomal enzyme activity in the localization of 67 gallium citrate.

The distribution of 67Ga citrate at 24 h post injection has been studied in the normal tissues of the mouse, rat and dog; 13 transplantable mouse tumours and 7 rat tumours have also been examined. The total activities of four lysosomal enzymes, aryl sulphatase, beta-glucuronidase, acid phosphatase and cathepsin-D were measured as well as the incorporation of thymidine-3H and leucine-14C ad indicators of DNA and protein synthesis. The results show a close correlation between 67Ga uptake and lysosomal enzyme activity in the tumours studied, which is an extension of the same relationship for normal tissues. It is suggested that the reported correlation between the uptake of 67Ga and the rate of cellular proliferation is secondary to the primary function of the lysosome in the localisation of the nuclide, lysosomal enzyme activity also being enhanced in situations of increased metabolic activity. A similar relationship appears to occur following administration of 111IN-Bleomycin and 99mTc-Citrate.

The mechanism of the localization of 67gallium citrate in experimental abscesses.

67Ga accumulation in turpentine-induced abscesses, from 5 h to 20 days old, in the rat has been investigated, and the results indicate that this is dependent on polymorphonuclear leukocyte (PMN) infiltration of the inflamed site. 67Ga concentration 24 h after injection correlates well with the activities of the lysosomal enzymes, aryl sulphatase and beta-glucuronidase, P less than 0.001, and with the DNA content of the tissue. There is no direct relationship with the rate of DNA synthesis. The mechanism of lysosomal incorporation into PMNs is consistent with the factors involved in 67Ga uptake into tumours and normal tissues.

Uptake of 67Ga in the regenerating rat liver and its relationship to lysosomal enzyme activity.

The uptake of 67Ga citrate has been studied in the regenerating rat liver over a period up to 72 hr after partial hepatectomy. The concentration of 67Ga was found to be maximal (four times that of controls) 42 hr after hepatectomy. This was shown to be related to lysosomal enzyme activity rather than to specific phases of the cell cycle, there being a highly significant correlation (p smaller than 0.001) with aryl sulfatase activity. In both regenerating and normal rat livers, it was shown that 67Ga uptake is reduced when protein synthesis is inhibited by cycloheximide but is unaffected by inhibition of DNA synthesis by cytosine arabinoside.