With a stabilizing agent (i.e., methylene blue and sodium phosphate buffer mixture), the in vitro stability of 99mTc-exametazime has been increased to 4-6 hr postreconstitution. However, it is not feasible to use the stabilized 99mTc-exametazime for leukocyte radiolabeling. This is due to the deep blue appearance of the mixture of stabilized 99mTc-exametazime and blood components, which makes it impossible to separate properly the supernatant from the leukocyte button. In our study, we have developed a practical methodology for overcoming this difficulty in order to use stabilized 99mTc-exametazime in leukocyte labeling.
Methods: The stabilized 99mTc-exametazime preparation used in our method consisted of 2 ml 7.4-8.0 GBq (200-215 mCi) 99mTc and 2 ml methylene blue/phosphate buffer solution. The separated leukocytes from 80-ml fresh venous blood were incubated with three different ages (i.e., 0-, 4-, or 6-hr postreconstitution) of stabilized 99mTc-exametazime (approximately 925 MBq, approximately 25 mCi; 0.5-1 ml) at room temperature for 15 min. After incubation, 3 ml of 12.6% ACD/NS solution (anticoagulant citrate dextrose, solution A, USP mixed with 0.9% NaCl, v/v) was added to the tube and centrifuged at 160 g for 5 min. Three milliliters of the dark blue supernatant were carefully removed, and the bottom 1 ml portion was resuspended with 9 ml of 12.6% ACD/NS solution. After centrifugation (160 g for 5 min), the supernatant was clear enough to be drawn off without disturbing the radiolabeled leukocyte button. The white cell button was then resuspended in 4 ml of platelet-poor plasma.
Results: The overall labeling efficiency (LE) of our new technique was 67.8%-91.9%, with the higher LE associated with fresher stabilized 99mTc-exametazime. During a 6-hr in vitro stability evaluation, radiolabeled leukocytes lost 1.2% +/- 0.3% (n = 24), 1.3% +/- 0.1% (n = 16) and 1.8% +/- 0.1% (n = 16) each hour of the cell-bound 0-, 4-, and 6-hr-old 99mTc-exametazime, respectively. The 99mTc-exametazime-labeled leukocytes examined by the trypan blue staining technique at 6-hr postradiolabeling yielded nonstained cells indicating viable leukocytes.
Conclusion: We concluded that with a small volume of 99mTc-exametazime and double dilution steps with 12.6% ACD/NS solution, stabilized 99mTc-exametazime can be used effectively for leukocyte radiolabeling with a high LE and long in vitro stability.