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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`(cid:58)(cid:83)(cid:78)(cid:91)(cid:74)(cid:87)(cid:88)(cid:78)(cid:89)(cid:168)(cid:89)(cid:88)(cid:82)(cid:74)(cid:73)(cid:78)(cid:95)(cid:78)(cid:83)(cid:5)(cid:73)(cid:74)(cid:87)(cid:5)(cid:47)(cid:84)(cid:77)(cid:70)(cid:83)(cid:83)(cid:74)(cid:88)(cid:5)(cid:44)(cid:90)(cid:89)(cid:74)(cid:83)(cid:71)(cid:74)(cid:87)(cid:76)(cid:18)(cid:58)(cid:83)(cid:78)(cid:91)(cid:74)(cid:87)(cid:88)(cid:78)(cid:89)(cid:168)(cid:89)(cid:5)(cid:50)(cid:70)(cid:78)(cid:83)(cid:95)
`
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`
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`
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`
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`
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`
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`ORIGINAL ARTICLES
`
`Aspects on radiolabeling of 177Lu-DOTA-TATE: After
`C18 purification re-addition of ascorbic acid is
`required to maintain radiochemical purity
`
`Stephan Maus1, Erik de Blois2, Stephan J. Ament1, Mathias Schreckenberger1, Wouter A. P.
`Breeman2
`
`1. Clinic of Nuclear Medicine, University Medical Centre Mainz, Langenbeckstr, Mainz, Germany. 2. Department of Nuclear
`Medicine, Erasmus MC Rotterdam, Rotterdam, Netherlands
`
`Correspondence: Stephan Maus. Address: Klinik und Poliklinik für NuklearmedizinUniversitätsmedizin der Johannes
`Gutenberg-Universität Mainz, Germany. Email: stephan.maus@unimedizin-mainz.de
`
`Received: January 24, 2014
`DOI: 10.5430/ijdi.v1n1p5
`
`Online Published: February 21, 2014
`
`Accepted: February 16, 2014
`URL: http://dx.doi.org/10.5430/ijdi.v1n1p5
`
`Abstract
`Purpose: Radiolabeled peptides like 177Lu-DOTA-TATE are vulnerable to radiolysis, which results in decreased
`radiochemical purity (RCP) of these radiopeptides. Gentisic acid (GA) and ascorbic acid (AA) are well known ingredients
`to reduce the effects of radiolysis. Currently, there is a trend to change the procedure from a manual to a cassette-based
`automated labeling and to introduce a C18 solid phase extraction (SPE) post-radiolabeling in order to remove
`non-incorporated 177Lu from the injection solution. However, with the introduction of SPE purification, GA and AA
`might effectively be removed from injection solution with a concordant dramatically drop of the RCP. Therefore we
`investigated the impact of tC18 SPE purification on the RCP of 177Lu-DOTA-TATE.
`Methods: We compared the manual radiolabeling procedure with the cassette-based automated radiolabeling procedure
`with/out tC18 SPE purification cartridge. The effect of tC18 purification on RCP of 177Lu-DOTA-TATE was
`investigated by HPLC as function of the post-radiolabeling time and the concentration of activity.
`Results: After tC18 SPE purification, GA and AA were effectively removed and resulted in volume-dependent decrease
`in RCP, e.g. <95% after 5h in 20 mL. Re-addition of AA directly after tC18 SPE purification resulted in a RCP (cid:149)95% at
`72h. In addition, with the cassette-based automated radiolabeling procedure we also found 28% of the original activity
`remaining in the activity-containing vial and tubing vs. < 1% with the manual procedure.
`Conclusion: Re-addition of AA post tC18 SPE purification is required to maintain RCP of 177Lu-DOTA-TATE.
`Keywords
`Lutetium-177, 177Lu-DOTA-TATE, Cassette-based, Automated radiolabeling, Radiochemical purity, Ascorbic acid,
`Quencher, Gentisic acid, Radiolysis
`
`1 Introduction
`Radiolabeled somatostatin analogues, such as [DOTA0,Tyr3]octreotate, further referred as DOTA-TATE have been
`subject of intensive research during the last 2 decades and play an important role in somatostatin receptor imaging and
`peptide receptor-targeted radionuclide therapy (PRRT) e.g. 177Lu-DOTA-TATE[1-7].
`Published by Sciedu Press
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`RCP of 177Lu-DOTA-TATE is an essential factor for successful PRRT. Because of the high doses of
`177Lu-DOTA-TATE (7.4 GBq 177Lu per PRRT administration), the peptide is subject to radiolysis. The degree of
`radiolysis is influenced by several factors like the amount of DOTA-TATE, temperature, time, the total activity, the
`volumic activity, quencher(s) et cetera [8-12].
`
`In the current publication we present a comparative study to investigate the effect of gentisic acid (GA) and ascorbic acid
`(AA) as quenchers during and after (manual and cassette-based automated) the radiolabeling 177Lu-DOTA-TATE
`procedures.
`
`There is currently a trend to move from manual radiolabeling to a cassette-based automated radiolabeling procedure;
`therefore we investigated these two different procedures in parallel. Sep-Pak Light tC18 SPE purification (further referred
`as tC18) is included as default in the cassette-based automated procedure. However, the tC18 purification of the reaction
`mixture after radiolabeling potentially removes GA and/or AA effectively. The aim of this study was to compare
`radiolabeling procedures (manual vs. cassette-based automated) with and without tC18 purification and to investigate the
`impact on the RCP of 177Lu-DOTA-TATE as function of time until the moment of administration to patient.
`
`Materials and chemicals
`Reagents and solvents were used in the highest quality grade without further purification.
`
`GA and water for trace analyses (Trace-SELECT®) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,
`Germany). Hydrochloric acid 30% (HCl) Ultrapur was obtained from Merck KGaA (Darmstadt, Germany). AA was
`purchased from WÖRWAG Pharma GmbH & Co. KG (Böblingen, Germany). DOTA-TATE as kit formulation [13] was
`provided by Erasmus MC Rotterdam (Rotterdam, The Netherlands). Ethanol (99.5%), Aqua ad iniectabilia and isotonic
`0.9% NaCl (further referred as saline) were purchased from B.Braun Melsungen AG (Melsungen, Germany). tC18
`cartridges were obtained from Waters GmbH (Eschborn, Germany). 177LuCl3 with specific activities in the range
`740-1000 GBq/mg was bought from IDB-Holland (Baarle Nassau, the Netherlands).
`
`2 General methods
`The manual radiolabeling procedure was performed in a temperature-controlled heating block from CardiRad (Lohja,
`Finland) and cassette-based automated radiolabeling procedure was performed in a Modular-Lab Pharm Tracer module
`(EZAG, Berlin, Germany) using the C4-Y90-00-standard synthesis and C1-PR-00 pressure test cassette (EZAG, Berlin,
`Germany).
`
`2.1 Cassette-based automated radiolabeling procedure
`The cassette was prepared according to the Y-Lu-INCASSETTE-TEST protocol (EZAG, Berlin, Germany). For standard
`patient radiolabeling, 240 μg DOTA-TATE in 0.6 mL (400 μg/mL) of the DOTA-TATE kit formulation [13] was
`transferred automatically and quantitatively in to a glass reaction vial (RV) and eventually 7.5 GBq 177LuCl3 (0.3 mL)
`was added in to RV. Subsequent program steps are consecutively executed and the radiolabeling was running
`automatically according to the LU-177-DOTA-PEPTIDES-PT-V.X.X protocol (according EZAG). RV was heated for 30
`min at 80°C. After 5 min cooling to ambient temperature the reaction mixture was transferred from the RV to the
`preconditioned tC18 cartridge. Pre-conditioning of the tC18 cartridge was performed with an ethanol/water mixture (5
`mL, 50:50% v/v). This tC18 procedure was introduced in order to remove non-incorporated 177Lu from the final product.
`The content of RV was transferred to the tC18 cartridge and rinsed with 3 mL saline. 177Lu-DOTA-TATE was desorbed
`from the tC18 cartridge with 2.5 mL of ethanol/water (50:50% v/v). An aliquot of the final mixture was taken and
`subjected to quality control by ITLC and HPLC. The eluate plus 17.5 mL saline solution (final volume 20 mL) were
`
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`filtered with means of a sterile Millex-GV 0.22 μm filter into a 25 mL glass vial (product vial or PV). Finally, a filter
`integrity test was performed. The cassette-based automated radiolabeling procedure process takes about 60 min.
`
`2.2 Manual radiolabeling procedure
`The manual radiolabeling procedure was performed directly in the activity-containing vial (AV), containing 7.5 GBq
`177LuCl3 in 0.3 mL. For standart patient radiolabeling 0.6 mL (240 μg DOTA-Tate) of the DOTA-TATE kit formul-
`ation [13] was added to the AV, and incubated for 30 min at 80 °C [13]. After cooling down to ambient temperature
`non-incorporated 177Lu was complexed by the addition of 0.25 mL DTPA-solution (4 mg/mL) to the reaction
`mixture [14-15]. An aliquot of the reaction mixture was taken and quality control was performed using ITLC and HPLC. The
`residual was diluted with 5 mL saline solution, filtered with means of a sterile Millex-GV 0.22 μm filter into PV and
`finally adjusted to a final volume of 20 mL. The manual radiolabeling procedure takes about 40 min.
`
`3 Studies on RCP of 177Lu-DOTA-TATE
`RCP is defined as the % of the activity of the radionuclide present in the desired radiopharmaceutical form of the total
`radioactivity. RCP of 177Lu-DOTA-TATE was investigated with/out tC18 purification and re-addition of quenchers as
`shown in experiments 1-4, see Table 1. In order to investigate the dilution of the quenchers two additional radiolabeling
`were performed (experiment 5-6, see Table 1). One radiolabeling included tC18 purification (experiment 5, see Table 1),
`while another radiolabeling (experiment 6) was performed without the tC18 purification. Both samples were diluted with
`saline up to a final volume of 20 mL, at constant concentration of ~ 0.5 GBq/mL. In addition, experiment 7 was performed
`without tC18 purification post radiolabeling and was diluted to a patient dose (7.4 GBq in 100 mL). RCP was determined
`by HPLC, as described below.
`
`Table 1. Different post radiolabeling procedures of 177Lu-DOTATATE
`Experiment
`1
`2
`3
`tC18 purification
`-
`+
`+
`Ethanol/H2O 50/50% (2.5 mL)
`-
`+
`+
`Re-addition AA or GA
`(100 mmol/L)
`Final volume (mL)
`Total activity (GBq)
`0.5 GBq/mL
`
`-
`
`5
`2.5
`+
`
`-
`
`5
`2.5
`+
`
`4
`+
`+
`
`GA
`
`5
`2.5
`+
`
`5
`-
`-
`
`-
`
`20
`10
`+
`
`6
`+
`+
`
`-
`
`20
`10
`+
`
`7
`-
`-
`
`-
`
`100
`7.4
`-
`
`AA
`
`5
`2.5
`+
`
`Labelings were performed using 7.5 GBq of 177LuCl3 (0.3 mL) and 0.6 mL of kit Erasmus MC matrix. Thereafter post
`radiolabeling procedures were performed either with/out tC18 purification, re-addition of AA or GA, and diluted in
`different final volumes (5, 20 and 100 mL) with saline. RCP of 177Lu-DOTA-TATE was monitored as function of time
`
`4 Analytical methods
`
`4.1 Incorporation by ITLC
`ITLC-SG glass fibre sheets were purchased from PALL Life Sciences (Port Washington, NY, USA). Small portions (1-3
`μL) were added on the ITLC-SG strips and sodium citrate buffer (0.1 M, pH 5) was used as mobile phase as
`described [14, 16]. Activity was recorded by Gina Star TLC and analyzed using Raytest miniGita software (Straubenhardt,
`Germany). Calculation of incorporation was performed as described [16].
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`4.2 HPLC
`HPLC methods were performed using a Dionex-3000 HPLC system with a variable wavelength Dionex GmbH detector
`(Idstein, Germany) containing a Waters 4.6 mm × 250 mm, 5μm Symmetry C18 column (Eschborn, Germany) . The
`gradient elution system utilized mobile phase A (methanol) and mobile phase B (0.06 M sodium acetate buffer, pH 5.5).
`Gradient was performed with a flow rate of 1 mL/min starting with 100 % B for 6.5 min; and was changed to 50% A and
`50% B within 0.5 min and increased to 60% A over 20 min.
`
`Thereafter, the mobile phase A was increased within 0.2 min to 100 % and was kept constant for 4.8 min. Finally, the
`gradient parameters returned to the initial starting conditions. The data were analyzed using Chromeleon Client Software
`Version 6.8.9. from Dionex GmbH (Idstein, Germany).
`
`5 Results
`
`5.1 Radiochemical yield
`With the cassette-based automated radiolabeling procedure we obtained radiochemical yields of 71 ± 18 % (n=12). The
`manual labeling achieved radiochemical yields (cid:149) 99% (n=3), independent of activity (range 2.5-10 GBq) or final volume
`(range 5-20 mL).
`
`5.2 Radiolabeling without tC18 Purification
`RCP of 177Lu-DOTA-TATE was measured by HPLC up to 168 h post-radiolabeling. Fig. 1A shows a typical HPLC
`radiochromatogram of 177Lu-DOTA-TATE which was prepared as described [13]. Fig. 1B shows the HPLC
`radiochromatogram of 177Lu-DOTA-TATE in the absence of quenchers. Fig. 2A clearly demonstrates that
`177Lu-DOTA-TATE without the tC18 purification post-radiolabeling remained stable (RCP (cid:149)95% at 72h post
`radiolabeling, experiment 1 and 2, see Table 1).
`
`
`
`Figure 1. Typical RP-HPLC radiochromatogram of 177Lu-DOTA-TATE
`(A) with a RCP of >95% and 177Lu-DOTA-TATE (B) with a RCP <95%. Peaks (I): 177Lu-DOTA-TATE, (II):177Lu-DTPA
`and 177Lu and (III): radiolysed fragments of 177Lu-DOTA-TATE. These fragments were not further characterised. X-axes
`are expressed in time (min) and Y-axes in mV.
`
`After increasing the volume up to 20 mL at constant volume activity (experiments 5, Table 1), 177Lu-DOTA-TATE
`remained stable (RCP (cid:149)95% ~ 24h post-radiolabeling) as shown in Fig. 2B, while the RCP of 177Lu-DOTA-TATE in a
`patient dose (7.4 GBq/100 mL) rapidly decreased below 95% within 12 h (Fig 2C, experiment 7, see Table 1).
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`5.3 Radiolabeling with tC18 purification
`tC18 purification of reaction mixture containing 177Lu-DOTA-TATE totally removed both AA and GA (>99%), as
`confirmed by HPLC (data not shown). The tC18 purified fraction without re-addition of quencher in total volume of 5 mL
`resulted in a 95% RCP after ~ 35h and 92% at 72h post radiolabeling (Fig. 2A, experiment 2, see Table 1). While RCP of
`177Lu-DOTA-TATE in 20 mL final volume decreased much more rapidly and resulted in a RCP of < 95% after ~5h and
`74% at 24h post radiolabeling (Fig. 2B, experiment 6, see Table 1).
`
`
`
`
`
`
`Figure 2. RCP of tC18 purified 177Lu-DOTA-TATE as function of time in a final volume
`5 mL (A, see experiments 1 and 2, Table 1) and 20 mL (B, see experiments 5 and 6, Table 1), both at 0.5 GBq/mL. (C):
`represents a patient dose (7.4 GBq/100 mL) without tC18 purification diluted with saline to a final volume of 100 mL
`(experiment 7, Table 1). X-axes are expressed in time (h) and Y-axes in RCP (%). Dotted lines represents 95% RCP and is
`taken as lowest level suitable for patient administration.
`
`5.4 Re-addition of AA or GA post tC18 purification
`RCP of 177Lu-DOTA-TATE with the re-addition of AA (100 mmol/L) post tC18 purification was (cid:149)95% at ~ 72h post
`radiolabeling (Fig. 3, experiment 3, see Table 1). Re-addition of GA (100 mmol/L, experiment 4, see Table 1) had only
`minor stabilizing properties, RCP of 177Lu-DOTA-TATE decreased below <95% within ~ 24h post radiolabeling (data
`not shown). Fragments observed in HPLC radiochromatogram as peaks prior to the main peak were caused by radiolysis
`of 177Lu-DOTA-TATE (Fig. 1B), were not further characterised. Fragments observed were not caused by formation of
`ionic 177Lu, 177Lu-DTPA or 177Lu-DOTA, since they have no retention on C18 column and are eluted from the HPLC
`system directly after void volume (Fig 1B, fragment II).
`
`5.5 Activity loss, localisation of activity
`Performing the manual radiolabeling including tC18 purification resulted in 5.2 ± 0.5 % (n=3) loss of activity in the tC18
`purification cartridge. Whereas with the cassette-based automated radiolabeling procedure, loss of activity in the cassette
`was 28 ± 18 % (n=12), mainly caused by adhesion in the RV, tubing and tC18 cartridge.
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`Figure 3. RCP of tC18 purified 177Lu-DOTA-TATE as a function of time in a total volume
`
`
`
`5 mL with/out re-addition of AA (see experiments 2 and 3, Table 1). X-axes are expressed in time (h) and Y-axes in RCP
`(%). The dotted line represents 95% RCP and is taken as lowest level suitable for patient administration.
`
`
`
`6 Discussion
`Liu et al. reported the addition of GA and AA as quenchers for radiolabeled DOTA-biomolecules in order to prevent
`radiolysis [8, 10]. They clearly demonstrated factors which influence RCP, factors such as presence and the relative amount
`of quencher, as well as the activity amount and the activity/quencher ratio. These data are in accordance with our studies.
`AA and GA-containing DOTA-TATE kit formulation was used and radiolabeled as described [13]. With regards to the
`production of radiopharmaceuticals, the existing GMP requirements as well as the reduction of the radiation exposure for
`the employees, Petrik et al.[18] reported radiolabeling of peptides for diagnostic and therapeutic purposes using a
`cassette-based automated radiolabeling procedure. They also showed radiochemical yields of 89% and RCP of (cid:149)95%,
`however, without presenting data on RCP as function of time post radiolabeling, whereas in daily practice radiolabeled
`peptides will be administered hours after radiolabeling. We used the same cassette-based automated radiolabeling
`procedure and a DOTA-TATE kit formulation and were also able to maintain RCP at (cid:149)95%. However, we observed 28 ±
`18 % loss of activity in the cassette-based procedure (n=12), and, in addition, a decrease of RCP after tC18 purification as
`function of the volumic activity.
`
`To investigate this phenomenon, we performed parallel manual and cassette-based automated radiolabeling syntheses. In
`the latter, tC18 purification is included as a safty-net system, to remove possible to free non-incorporated 177Lu ions from
`the reaction solution. Therefore, a tC18 purification step was also included in the manual radiolabeling. In two identical
`performed radiolabeling syntheses the initial RCP was (cid:149)95%, however, since tC18 purification of eluate after
`radiolabeling resulted in total elimination of AA and GA and resulted in a decreased RCP of < 95% (Fig. 2A, experiments
`1 and 2, Table 1). The experiments were repeated and after tC18 purification step GA or AA (final concentrations of 100
`mmol/L) were added directly in to 177Lu-DOTA-TATE containing labeling mixture (experiments 3 and 4, Table 1).
`
`Additional experiments were performed in 20 mL total volume, (experiments 5 and 6, see Table 1), while keeping the
`volume activity constant (0.5 GBq/mL). These experiments showed a much more rapidly decrease in RCP of
`177Lu-DOTA-TATE (<95% RCP after ~5h and 74% at ~ 24h post radiolabeling, Fig. 2B,). Further dilution (5(cid:314)20 mL)
`resulted in concordant dilution of quencher and might explain the decrease of RCP of 177Lu-DOTA-TATE.
`
`As shown in Fig. 2A and B, experiments 2 and 6, Table 1, both after C18 purification, we found a more rapid decrease in
`RCP in 20 mL vs 5 mL. Without C18 purification the difference in decrease of RCP was less clear, experiments 1 and 5,
`Table 1, Fig. 2A and B. However, there is a difference between experiments 2 and 6: after the C18 purification (and 2.5 mL
`(50:50 water:ethanol)) the residual is diluted with 2.5 mL saline solution (experiment 2) or 17.5 mL saline solution
`(experiment 6). In consequence, the final ethanol concentration is different, 0.25 mL ethanol per mL or 0.062 mL ethanol
`per mL (1 mmoles/L ethanol in experiment 2 (5 mL) and 0.25 mmoles/L) in experiment 6 (20 mL).
`
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`Based on these results we can conclude that the standart implementation of a tC18 purification, which are generally
`effective for eliminating non-incorporated 177Lu ions, needs the re-addition of a quencher to maintain RCP of
`177Lu-DOTA-TATE, although when ethanol is also known as an effective quencher in the mmoles/L range [17-18].
`
`In daily practice, performing a manual radiolabeling as described [13], without tC18 purification, patient doses are diluted
`to 100 mL with saline. However, without the re-addition of quenchers, the RCP decreases time-dependent, while the RCP
`could be maintained > 95% up to 12h after radiolabeling (Fig. 2C). Thus, the dilution to 100 mL after radiolabeling, even
`without using tC18 purification, is preferably performed by the addition of AA up to a final concentration of 100 mmol/L.
`The re-addition of GA under the same conditions showed only minor stabilizing properties. To our knowledge RCP data
`on GA addition on 177Lu-labeled DOTA-peptides are not available in literature, however, Liu et al.[8, 10] reported a
`beneficial effect on the addition of GA to 90Y-DOTA-biomolecules.
`
`When therapeutic doses of 177Lu-DOTA-TATE are administered (7.4 GBq for PRRT), radiolysed 177Lu-labeled
`fragments will most likely not bind to somatostatin receptor-positive tumor tissue. Here, however, it should be clarified,
`that 1% loss in RCP represents 74 MBq 177Lu non-characterised radiolysed peptide (and 370 MBq at 95% RCP), adding
`undesired radiation dose burden to the patient. As a consequence, we strongly advise re-addition of AA post tC18
`purification. In addition, dilution after radiolabeling, even without using tC18 purification, should be performed with
`addition of AA.
`
`Although not investigated here, the decrease in RCP of all 177Lu-labeled DOTA-biomolecules in the absence of quencher
`can be anticipated, and thus requires further study [17].
`
`7 Conclusion
`Re-addition of AA post tC18 SPE purification is required to maintain RCP of 177Lu-DOTA-TATE. RCP of tC18 purified
`177Lu-DOTA-TATE, either labeled manually or cassette-based automated, decreases time- and volume-dependent.
`
`Conflict of interests
`The authors declare no conflicts of interests.
`
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