In vitro human leukocyte labeling with 64Cu: an intraindividual comparison with 111In-oxine and 18F-FDG

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Abstract

We investigated labeling human leukocytes [white blood cells (WBCs)] in vitro with copper-64 (Cu) comparing labeling efficiency, viability and stability of Cu-WBCs with 111In-oxine (In) WBCs and 18F-FDG (FDG) WBCs.

Methods

Leukocytes from 10 volunteers were labeled with Cu, In and FDG. Forty milliliters of venous blood was collected and leukocyte separation was performed according to standard methods. In-WBCs and FDG-WBCs were labeled according to published methods. For Cu-WBCs, tropolone initially was used as a single chelating agent. Because of poor intracellular Cu retention (54±4% at 3 h and 24±5% at 24 h), the fluorinated, membrane-permeable divalent cation chelator quin-MF was added. WBCs were incubated in 5 ml saline containing 100 μl of 1mM quin-MF/AM in 2% dimethyl sulfoxide and 74–185 MBq Cu-tropolone for 45 min at 37°C. Labeling efficiencies; in vitro cellular viabilities at 1, 3 and 24 h; and in vitro stabilities at 1, 2, 3, 4 and 24 h (except FDG-WBCs) were determined.

Results

Mean Cu-WBCs (87±4%) and In-WBCs (86±4%) labeling efficiencies were comparable and were significantly higher than FDG-WBCs (60±19%, P<.001). Cell viabilities, similar at 1 h, were significantly higher for 64Cu-WBCs at 3 and 24 h. Intracellular retention of activity was always significantly higher for In-WBCs than for Cu-WBCs and FDG-WBCs. At 24 h, intracellular retention was 88±4% for In-WBCs and 79±6% for Cu-WBCs.

Conclusion

Cu-WBC labeling efficiency and viability were comparable or superior to In-WBCs and significantly higher than FDG-WBCs. Although significantly more activity eluted from Cu-WBCs than from In-WBCs, Cu-WBC probably is adequate for imaging. These data suggest that further investigation of in vitro copper-64-labeled leukocytes for PET imaging of infection is warranted.

Introduction

In vitro labeled leukocyte imaging, using white blood cells (WBCs) labeled either with 111In-oxine (In) or 99mTc-exametazime (Tc), is the radionuclide gold standard for evaluating infection and inflammation in the immunocompetent population [1], [2]. Positron emission tomography (PET) potentially has several important advantages over conventional gamma camera imaging using single-photon emitting agents, and several studies suggest that 18F-fluorodeoxyglucose (FDG) is useful for infection and inflammation imaging [3], [4], [5], [6]. Although FDG is exquisitely sensitive, it is not specific and accumulates in a variety of conditions, including malignant and benign neoplasms, and fractures, as well as infection and inflammation. The avidity of inflammatory cells for FDG has led to efforts at labeling leukocytes with FDG in vitro, in an attempt to combine the advantages of the labeled leukocyte study with those of PET [7], [8], [9], [10], [11], [12], [13], [14]. Although initial results obtained with FDG-WBCs were encouraging, there are significant disadvantages to the procedure. The labeling efficiency of FDG-WBCs is more variable and generally lower than that of In-WBCs [11], [12], [14]. Another disadvantage is label stability. Pellegrino et al. [13] reported that, in rats, more than 50% of the FDG eluted from leukocytes at 90 min and about 80% eluted by 6 h. The short physical half-life of fluorine-18 poses logistical challenges such as coordinating delivery of 18 F-FDG with patient arrival. It also precludes next day imaging. It is unlikely therefore that infection imaging with FDG-WBCs will ever be clinically practical [2].

The ideal PET tracer for labeling leukocytes should have a consistently high labeling efficiency while preserving cell viability. The radiolabel should be stable, with as little elution of radioactivity from the cells as possible, and the physical half-life of the radionuclide should be long enough to make in vitro labeling practical and to permit delayed imaging.

Copper-64 (Cu) is an intermediate half-lived positron-emitting radionuclide (T½=12.7 h, e.c. 45%, β 37.1%, β+ 17.9%) suitable for positron emission tomography. The chemistry and in vivo behavior of Cu are well understood, and it has been used both for imaging and therapy [15], [16]. The purpose of this investigation was to establish methodology to label WBCs in vitro with Cu and to compare the labeling efficiency, viability and stability of Cu-WBCs with those of 111In-WBCs and FDG-WBCs.

Section snippets

Leukocyte separation

Ten healthy volunteers, seven men and three women, 30 years to 57 years of age, were enrolled in this investigation. Leukocyte separation was performed in a manner similar to previously published methods [17], [18]. For all labelings, 40 ml of venous blood was collected in a syringe anticoagulated with heparin. Seven milliliters of a settling agent, hydroxyethyl starch (6% hetastarch in 0.9% NaCl solution), was added to the syringe, which was maintained in a vertical position in a laminar flow

Results

The mean labeling efficiency for Cu-T-WBCs (n=3) was 83±4%. At 3 h, 54±4% of the radioactivity was retained intracellularly. At 24 h, 24±5% of the radioactivity was retained intracellularly (Fig. 1). Because of the amount of Cu elution from the cells, Cu-T-WBCs were not investigated further.

Labeling efficiency, cell viability and label stability were normally distributed for Cu-WBCs, In-WBCs, and FDG-WBCs, for all time points (P>.05 in each case). Labeling efficiencies were significantly

Discussion

Positron emission tomography offers important advantages over conventional gamma camera imaging using single-photon emitting agents, and the role of FDG-PET for imaging infection and inflammation is increasing. Although it is exquisitely sensitive, FDG is not specific and accumulates in a variety of conditions in addition to infection and inflammation. The avidity of inflammatory cells for FDG has led to attempts at labeling leukocytes with FDG in vitro, and initial results obtained with

Conclusions

This investigation demonstrates the feasibility of labeling human leukocytes with the positron emitter 64Cu, which, in vitro, is superior to FDG-labeled leukocytes. These results suggest that the potential of copper-64-labeled leukocytes for imaging infection warrants further investigation.

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    This work was supported in part by NIH Grant #5RO1GM 071324-20.

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