Radiolabelled mixed leukocytes and pure granulocytes with stabilized ⁹⁹tc^m-exametazime

Although the methylene blue stabilizer extends the shelf life of ⁹⁹Tc^m-exametazime to 4–6 h after reconstitution, the dark blue appearance of the mixture of stabilized ⁹⁹Tc^m-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 ⁹⁹Tc^m-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 ⁹⁹Tc^m-exametazime (∼925 MBq ⁹⁹Tc^m and 62.5 μ 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 ⁹⁹Tc^m 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the 1-ml bottom portion from the first dilution, the supernatant of the centrifuged preparation was clear enough to be withdrawn.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 ∼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 ∼2% (n = 48) ⁹⁹Tc^m 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 ⁹⁹Tc^m-exametazime with the use of the ‘double dilution’ technique.