Abstract
The in vitro viability, chemotaxis, and labeling stability of leukocytes labeled using 99mTc-exametazime stabilized with methylene blue were evaluated and compared with those obtained using nonstabilized 99mTc-exametazime. Methods: Two identical leukocyte populations, from 30 healthy donors, were labeled simultaneously using freshly prepared and 2-h-old stabilized 99mTc-exametazime. The following quality control techniques were performed on each labeled leukocyte sample: eosin Y staining, chemotaxis radioassay, and labeling stability at 2 h after labeling. Results: Eosin Y staining showed a cell viability of at least 98% in all samples, without a significant statistical difference between the populations. Chemotactic indices obtained with leukocytes labeled with freshly prepared, unstabilized 99mTc-exametazime were statistically greater than those obtained using 99mTc-exametazime stabilized with methylene blue (z = 2.41; P < 0.02). Labeling stability at 2 h after labeling was the same for both populations. Conclusion: The use of 99mTc-exametazime stabilized with methylene blue for leukocyte radiolabeling does not affect either cell membrane integrity or labeling stability but can cause a decrease in the cell chemotactic capacity that discourages its clinical use.
A mixture of methylene blue and phosphate buffer was approved by the Food and Drug Administration as a stabilizing agent for 99mTc-exametazime in 1995. However, in the Ceretec (Amersham International, Buckinghamshire, U.K.) package insert, 99mTc-exametazime stabilized with methylene blue and phosphate buffer is indicated only for brain imaging studies and not for labeling white blood cells (WBCs) (1). The reason is probably the dark blue appearance of the mixture of stabilized 99mTc-exametazime–methylene blue and isolated leukocytes, which makes separation of the free 99mTc-exametazime from the labeled cells difficult or impossible. Moreover, other possible disadvantages are a decrease in the viability of WBCs and a negative effect on their function.
Recently, Hung et al. (2) described a method for labeling WBCs with 99mTc-exametazime stabilized with methylene blue and phosphate buffer. Hung et al. lightened the dark blue color of the dye by diluting the labeling medium with 12.6% acid citrate dextrose mixed with 0.8% NaCl, volume in volume. They also showed that WBCs can be labeled with a high labeling efficiency (LE), a long-lasting in vitro stability, and a preserved in vitro viability (trypan blue technique). We developed a WBC labeling method with stabilized 99mTc-exametazime and saline but using 18.75 μg methylene blue instead of the 250–500 μg used by Hung et al. and using a labeling medium with 50% leukocyte-poor plasma. We also achieved a reasonable LE and in vitro viability (eosin Y technique) (3,4). However, both studies have two serious drawbacks to recommending the use of methylene blue in WBC labeling: the limited number of experiments performed (6 in the series of Hung et al.; 8 and 10 in our series) and the lack of testing for WBC function.
The purpose of our study was to test the usefulness of 99mTc-exametazime stabilized with methylene blue in the labeling of leukocytes, using our own method and a large series of blood samples, and to study the chemotactic capacity of the labeled WBCs.
MATERIALS AND METHODS
Preparation of Stabilized and Nonstabilized 99mTc-Exametazime
Exametazime fractions were previously prepared as follows: each vial of Ceretec was reconstituted with 2.1 mL N2-purged 0.9% NaCl sterile solution and split into 0.5-mL portions in separate N2-filled sterile vials. These were stored at −40°C, for a maximum of 1 mo, until their use. Nonstabilized 99mTc-exametazime was obtained by adding 1.1 mL fresh 99mTc-pertechnetate to each thawed exametazime fraction. 99mTc-pertechnetate was obtained from a generator that had been eluted within the previous 24 h. Radioactivity was always approximately 1,110 MBq.
Stabilized 99mTc-exametazime was obtained from 99mTc-exametazime prepared in the same way but using 1.0 mL 99mTc-pertechnetate and approximately 1,400 MBq. Two minutes later, 0.1 mL methylene blue (0.6 mg/mL in saline) was added to the vial.
The osmolarity and pH of six samples of both stabilized and nonstabilized 99mTc-exametazime were analyzed using an osmometer (Krioscop 800; Slamed, Frankfurt, Germany) and a pH meter (model 125; Corning, Medfield, MA). Therefore, we analyzed the osmolarity and pH of six samples of nonstabilized 99mTc-exametazime, following the manufacturer’s instructions, from an entire vial of Ceretec. The lipophilic fraction of 99mTc-exametazime was analyzed in all cases using the chloroform extraction technique (5).
Blood Samples
Two 45-mL samples of venous blood collected over 6 mL acid citrate dextrose were obtained from 30 healthy blood donors. Written informed consent was obtained from all donors. The leukocyte-rich pellet was obtained from each blood–acid citrate dextrose sample using the hydroxy-ethyl-starch sedimentation technique according to the consensus protocol of the International Society of Radiolabelled Blood Elements (6).
Radiolabeling of Mixed Leukocytes with Stabilized and Nonstabilized 99mTc-Exametazime
Each leukocyte-rich pellet was gently resuspended in 0.5 mL cell-free plasma using a polypropylene Pasteur-type pipette. Afterward, 0.5 mL 99mTc-exametazime (∼340 MBq, freshly prepared or stabilized 2 h before) was added to each sample. The incubation medium was mixed by swirling each test tube and was incubated for 15 min at room temperature.
Immediately after incubation, 4 mL cell-free plasma were added to each test tube. Afterward, both tubes were centrifuged at 150g for 5 min. The pellet containing the leukocytes labeled with methylene blue and 99mTc-exametazime was clearly seen because the plasma was light blue. Therefore, the plasma supernatant containing the unbound 99mTc-exametazime was easily removed with the aid of a polypropylene Pasteur-type pipette. Finally, both 99mTc-labeled leukocyte samples were suspended in sufficient Hank’s solution (GIBCO, Life Technologies Ltd., Paisley, U.K.) to yield a cell suspension of 3 × 107 leukocytes per milliliter.
Quality Control of the Labeled Leukocytes
LE.
The LE was calculated by the equation:
In Vitro Viability Test.
Eosin Y staining was performed on each labeled leukocyte suspension.
Chemotaxis Radioassay.
A chemotaxis radioassay was performed by modifying the technique of Gallin et al. (7), using conventional Boyden chambers (Nucleopore Corp., Pleasanton, CA). The chemotactic factor was generated in two steps. First, 60 μg endotoxin (Escherichia coli 055:B5, L6529; Sigma, St. Louis, MO) dissolved in 20 μL saline were added to 100 μL donor-cell–free plasma. Second, 50 μL of this endotoxin-activated plasma were added to 1,000 μL Hank’s solution.
The lower compartment of the Boyden chamber was filled with chemotactic factor (∼200 μL). Two 3-μm polycarbonate filters (Millipore Corp., Molsheim, France) were placed so as to separate the lower compartment from the upper. The upper compartment was filled with 200 μL cell suspension. After exactly 3 h of incubation at 37°C, the cells of the upper compartment were gently suspended with the aid of a Pasteur-type glass pipette, and all the fluid was immediately removed. Both filters and the fluid from the lower chamber were also removed. Each filter and the upper and lower liquids were put into test tubes and counted in a γ counter (LKB-Wallac 1282 CompuGamma; Wallac, Turku, Finland) 48 h later.
The chemotactic index (CI) described by English and Clanton (8) was calculated as follows:
In all cases, chambers containing Hank’s solution, instead of chemotactic factor, were used to evaluate the random chemotaxis. The random index (RI) was calculated as described above. The CI/RI ratio was used to compare the chemotactic capacity of the leukocytes labeled with unstabilized 99mTc-exametazime and the leukocytes labeled with methylene blue–stabilized 99mTc-exametazime.
In Vitro Stability.
The remaining labeled leukocyte samples, suspended in Hank’s solution, were stored at 37°C for 2 h. The in vitro cell-labeling stability at this time was determined by centrifuging both unstabilized and stabilized 99mTc-exametazime–labeled leukocytes and measuring the amount of 99mTc activity bound to the cells.
Statistical Analysis
A paired-sample statistical test was used to analyze the results.
RESULTS
Radiochemical Purity of 99mTc-Exametazime Preparations
The 99mTc-exametazime prepared without a stabilizing agent (n = 30) had a mean radiochemical purity of 95.5% (SD, 3.3%; range, 92.7%–98.2%). The 2-h-old methylene blue–stabilized 99mTc-exametazime had a mean radiochemical purity of 86.7% (SD, 3.2%; range, 77.1%–91.0%).
Osmolarity and pH of Stabilized and Nonstabilized 99mTc-Exametazime Preparations
The osmolarity of stabilized 99mTc-exametazime preparations ranged from 305 to 309 mOsm/L (mean, 307 mOsm/L; SD, 1.4 mOsm/L; n = 6), and the pH ranged from 7.0 to 7.6 (mean, 7.3; SD, 0.2; n = 6). The osmolarity of unstabilized 99mTc-exametazime preparations ranged from 309 to 313 mOsm/L (mean, 311 mOsm/L; SD, 1.9 mOsm/L; n = 6), and the pH ranged from 7.0 to 8.0 (mean, 7.5; SD, 0.3; n = 6). The osmolarity of 99mTc-exametazime obtained from an original unfractionated Ceretec vial ranged from 313 to 317 mOsm/L (mean, 315 mOsm/L; SD, 1.5 mOsm/L; n = 6), and the pH ranged from 8.2 to 9.0 (mean, 8.7; SD, 0.2; n = 6).
Quality Control of 99mTc-Exametazime–Labeled Leukocytes
LE.
Leukocytes were labeled with unstabilized 99mTc-exametazime with a mean LE of 63.5% (SD, 13.6%; range, 40.1%–83.9%; n = 30). Leukocytes from the same donors were labeled with 2-h-old methylene blue–stabilized 99mTc-exametazime with a mean LE of 58.7% (SD, 12.6%; range, 36.3%–79.7%; n = 30).
In Vitro Stability.
The radioactivity that remained bound to the leukocytes labeled with unstabilized 99mTc-exametazime, after 2 h stored at 37°C, ranged from 92.5% to 98.4% (mean, 96.0%; SD, 1.5%; n = 30). The activity that remained bound to the leukocytes labeled with 2-h-old methylene blue–stabilized 99mTc-exametazime, after 2 h stored at 37°C, ranged from 92.9% to 98.5% (mean, 96.0%; SD, 1.4%; n = 30). The results for the two populations did not statistically differ.
Eosin Y Staining.
At least 98% of the mixed leukocyte samples, labeled with either stabilized or unstabilized 99mTc-exametazime, did not concentrate the dye.
Chemotaxis Radioassay.
Leukocytes labeled with unstabilized 99mTc-exametazime preparations (n = 30) had a mean CI/RI ratio of 10.4 (SD, 8.8; range, 1.4–38.4). Leukocytes from the same donors, labeled with 2-h-old stabilized 99mTc-exametazime, had a mean CI/RI ratio of 8.0 (SD, 7.3; range, 1.3–32.2). The CI/RI ratios from the two populations were statistically different (z = 2.41; P < 0.02). Variations in CI/RI ratios were derived from variations in the CI values (Table 1).
Chemotaxis Radioassay
DISCUSSION
Many agents have been described as stabilizing 99mTc-exametazime (9–13). But only methylene blue and cobalt chloride have been described with regard to their usefulness for leukocyte labeling (2,14–15). However, none has been approved for labeling WBCs in either the United States (1) or Europe (16).
We performed this study to clarify the possibility of using methylene blue as a stabilizing agent for labeling WBCs. Using 18.75 μg methylene blue in each labeling medium instead of the 250–500 μg used by Hung et al. (2), we avoided the problems associated with the dark blue color of the stabilizer; therefore, we can perform our labeling method with or without methylene blue. Moreover, using this amount of methylene blue achieved a fair stabilization: 2 h after the 99mTc-exametazime stabilization, we obtained a mean lipophilic fraction of 86.7%. The pH and osmolarity of this stabilized solution were in the physiologic range.
In our department, we label a maximum of two different leukocyte samples simultaneously, to avoid undesirable cross-contaminations. Two hours later, another two leukocyte samples can be labeled. This is the reason we chose 2 h as a practical time for investigating the usefulness of the stabilized 99mTc-exametazime.
Leukocytes are labeled in a 50% plasma medium. Other authors who use only a saline medium have obtained an LE approximately 20%–25% higher than ours (mean LE, 58.7%) (2,14). Nevertheless, we prefer to maintain the leukocytes in a more physiologic medium during the incubation step instead of increasing the LE. The LE that we achieved using 2-h-old stabilized 99mTc-exametazime was as we expected: approximately 90% of that obtained with unstabilized 99mTc-exametazime.
The in vitro stability of 99mTc-exametazime-labeled leukocytes was the same for both leukocyte populations. Our results were similar to those obtained by authors who labeled leukocytes in saline medium (17–19) or in 50% plasma–saline medium (20).
The results of the eosin Y test showed suitable cell viability for both leukocyte populations. The in vitro stability and the results of the eosin Y test were as expected, because we used significantly less methylene blue (18.75 μg instead of 250–500 μg) than did other authors (2), with a similar number of leukocytes. However, the chemotaxis radioassay showed that the leukocytes labeled with unstabilized 99mTc-exametazime had a better preserved chemotactic response than did those labeled with stabilized 99mTc-exametazime. The value of our results is based on our simultaneously performing the labeling method on two equal leukocyte samples belonging to the same donor and on our inclusion of a large enough number of donors (n = 30) to achieve valuable statistical results.
The presence of methylene blue in the labeling medium acts on the capacity of the leukocytes to move in response to a chemotactic stimulus. However, the magnitude of individual-to-individual variations in CI/RI ratio is large, compared with the magnitude of the stabilized-versus-nonstabilized differences for leukocytes from individual subjects. For this reason, we doubt whether the observed stabilized-versus-nonstabilized difference in CI/RI ratio has clinical significance. However, the existence of this possibility, based on our study using much less methylene blue than the manufacturers recommend to stabilize 99mTc-exametazime, discourages us from advising that methylene blue–stabilized 99mTc-exametazime be used for labeling leukocytes.
CONCLUSION
The use of 99mTc-HMPAO stabilized with methylene blue in the labeling of leukocytes does not affect either cell viability or labeling stability but can cause a decrease in the cell chemotactic capacity that discourages its clinical use.
Acknowledgments
This study was supported by grant FIS 98/0582 from the Spanish Ministry of Health. This study was presented in part at the ninth congress of the International Society of Radiolabeled Blood Elements, October 20–23, 1999, and was published as an abstract (Nucl Med Commun. 1999;20:967).
Footnotes
Received May 10, 2000; revision accepted Nov. 7, 2000.
For correspondence or reprints contact: Manuel Roca, PhD, Medicina Nuclear, Ciutat Sanitària Universitària de Bellvitge, c/Feixa Llarga s/n, 08907-L’Hospitalet de Llobregat, Barcelona, Spain.
