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

Abstract

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.

Examination of trafficking of phagocytosed colloid particles in neutrophils using synchrotron-based X-ray fluorescence microscopy (XFM)

“…These particles contain Sn, but not significant amounts of Cl, as previously described using SEM and energy-dispersive X-ray analysis (EDAX) [9]. McClelland also reported that, in addition, the particles contain O but not F [9], suggesting they consist primarily of stannous oxide. The majority of the Cl found in the TcSn colloid solution residue after dehydration is in separate NaCl salt crystals [9].…”

“…Hence, the most likely explanation for the subcellular association of Sn with Fe found in our study is that the TcSn colloid-containing phagosomes fuse with neutrophil granules, and more specifically the gp91 PHOX -containing specific granules, within the first hour after phagocytosis. However, previous studies have shown that there is no evidence of neutrophils undergoing the respiratory burst as a result of TcSn colloid labelling [4,5,9,12]. Hence, despite the colocalisation of at least one of the components of the NADPH oxidase to the TcSn colloid-containing phagosomes, this system is not activated.…”

“…McClelland also reported that, in addition, the particles contain O but not F [9], suggesting they consist primarily of stannous oxide. The majority of the Cl found in the TcSn colloid solution residue after dehydration is in separate NaCl salt crystals [9]. Although XFM is capable of detecting very low concentrations of elements, in the current study we were not able to directly identify the presence of Tc in TcSn colloid.…”

“…When fresh colloid is examined immediately after manufacture it is found to consist of clumps of particles measuring on the order of several micrometers [9,10]. This compound does not label phagocytic cells at 4…”

“…However, phagocytosis is a complex process which can occur via a number of different mechanisms, and can have varying effects on the cell. TcSn colloid uptake by neutrophils is dependent on the I domain of Complement Receptor 3 (CR3) [12], and after TcSn colloid uptake neutrophils are viable, functional and not significantly activated or primed [4,5,9,12]. This resembles type II phagocytosis of opsonised particles which is mediated by CR3 and during which the cell is not activated, and there is no proinflammatory response [13,14].…”