Ann Nucl Med (2016) 30:263–271
DOI 10.1007/s12149-015-1055-6

ORIGINAL ARTICLE

Simultaneous acquisition of 99mTc- and 123I-labeled radiotracers
using a preclinical SPECT scanner with CZT detectors
Masato Kobayashi1 • Ichiro Matsunari2,3 • Kodai Nishi4 • Asuka Mizutani5 •Yoshiharu Miyazaki2 • Kazuhiro Ogai1 • Jyunko Sugama1 • Kazuhiro Shiba6 •Keiichi Kawai7 • Seigo Kinuya8

Received: 16 October 2015 / Accepted: 23 December 2015 / Published online: 8 January 2016Ó The Japanese Society of Nuclear Medicine 2016

Abstract
Objective Simultaneous acquisition of 99mTc and 123I was
evaluated using a preclinical SPECT scanner with cadmium zinc telluride (CZT)-based detectors.
Methods 10-ml cylindrical syringes contained about
37 MBq 99mTc-tetrofosmin (99mTc-TF) or 37 MBq 123I-15(p-iodophenyl)-3R,S-methyl pentadecanoic acid (123IBMIPP) were used to assess the relationship between these
SPECT radioactive counts and radioactivity. Two 10-ml
syringes contained 100 or 300 MBq 99mTc-TF and
100 MBq 123I-BMIPP to assess the influence of 99mTc
upscatter and 123I downscatter, respectively. A rat-sized
cylindrical phantom also contained both 100 or 300 MBq& Masato Kobayashi
kobayasi@mhs.mp.kanazawa-u.ac.jp
1

Wellness Promotion Science Center, Institute of Medical,
Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-0942, Japan

2

Clinical Research Department, The Medical andPharmacological Research Center Foundation, Hakui, Japan

3

Division of Nuclear Medicine, Department of Radiology,
Saitama Medical University Hospital, Saitama, Japan

4

Department of Radioisotope Medicine, Atomic Bomb
Disease Institute, Nagasaki University, Nagasaki, Japan

5

Graduate School of Medicine, Division of Health Sciences,
Osaka University, Osaka, Japan

6

Division of Tracer Kinetics, Advanced Science ResearchCenter, Kanazawa University, Kanazawa, Japan

7

School of Health Sciences, College of Medical,
Pharmaceutical and Health Sciences, Kanazawa University,
Kanazawa, Japan

8

School of Medical Sciences, College of Medical,
Pharmaceutical and Health Sciences, Kanazawa University,
Kanazawa, Japan99m

Tc-TF and 100 MBq 123I-BMIPP. The two 10-ml syringes and phantom were scanned using a pinhole collimator for rats. Myocardial infarction model rats were
examined using 300 MBq 99mTc-TF and 100 MBq 123IBMIPP. Two 1-ml syringes contained 105 MBq 99mTclabeled hexamethylpropyleneamine oxime (99mTcHMPAO) and 35 MBq 123I-labeled N-x-fluoropropyl-2bcarbomethoxy-3b-(4-iodophenyl) nortropane (123I-FPCIT). The two 1-ml syringes were scanned using a pinhole
collimator for mice. Normal mice were examined using
105 MBq 99mTc-HMPAO and 35 MBq 123I-FP-CIT.
Results The relationship between SPECT radioactive
counts and radioactivity was excellent. Downscatter contamination of 123I-BMIPP exhibited fewer radioactive
counts for 300 MBq 99mTc-TF without scatter correction(SC) in 125–150 keV. There was no upscatter contamination of 99mTc-TF in 150–175 keV. In the rat-sized phantom, the radioactive count ratio decreased to 4.0 % for
300 MBq 99mTc-TF without SC in 125–150 keV. In therats, myocardial images and radioactive counts of 99mTcTF with the dual tracer were identical to those of the99m
Tc-TF single injection. Downscatter contamination of123
I-FP-CIT was 4.2 % without SC in 125–150 keV. In thefirst injection of 99mTc-HMPAO and second injection of123
I-FP-CIT, brain images and radioactive counts of 99mTcHMPAO with the dual tracer in normal mice also were thesimilar to those of the 99mTc-HMPAO single injection. In
the first injection of 123I-FP-CIT and second injection of99m
Tc-HMPAO, the brain images and radioactive counts
with the dual tracer were not much different from those of
the 123I-FP-CIT single injection.
Conclusions Dual-tracer imaging of 99mTc- and 123I-labeled radiotracers is feasible in a preclinical SPECT
scanner with CZT detector. When higher radioactivity of99m
Tc-labeled radiotracers relative to 123I-labeled

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radiotracers is applied, correction methods are not necessarily required for the quantification of 99mTc- and 123Ilabeled radiotracers when using a preclinical SPECT
scanner with CZT detector.

Ann Nucl Med (2016) 30:263–271

HMPAO) and 123I-labeled N-x-fluoropropyl-2b-carbomethoxy-3b-(4-iodophenyl)nortropane (123I-FP-CIT).

Materials and methods
Keywords Dual tracer  99mTc  123I  Small animal
SPECT scanner  Cadmium zinc telluride

Introduction
Preclinical SPECT scanners are an important tool formolecular imaging in biomedical research and the development of new medicine [1]. Several recent scanners
incorporate semiconductor materials such as cadmium zinctelluride (CZT) [2–6]. The CZT-based scanners improveenergy and spatial resolution, as compared with sodium
iodide (NaI) scintillation detectors, which are used inconventional SPECT scanners, in not only preclinicalanimal imaging but also clinical imaging.
With conventional SPECT scanners, it is difficult toseparate 99mTc (140 keV) and 123I (159 keV) images
because the emission energies of these radiotracers areclose. Accurate quantification of dual-isotope imaging is
also difficult because the images are affected by scatter,cross-talk, attenuation and distance-dependent collimator
response. Especially, there is substantial influence on not
only downscattered 123I photons in the energy window of99m
Tc, but also cross-talk from primary photons of eachradionuclide. Attempts have already been made to discriminate downscatter contamination and cross-talk using
an energy window correction method in clinical SPECT
scanners with NaI scintillation detectors [6–12]. Constrained spectral factor analysis and artificial neural networks are two promising approaches to compensate forscatter, cross-talk, and high-energy septal penetration insimultaneous 99mTc and 123I imaging [7, 8]. Anothermethod, a Monte Carlo-based joint ordered-subset expectation maximization iterative reconstruction algorithm, wasapplied to 99mTc/123I brain or cardiac SPECT imaging [6,9–11]. However, these methods are generally complicatedand require many energy windows.
In this study, simultaneous acquisition of 99mTc- and123
I-labeled radiotracers was evaluated on a preclinical
SPECT scanner with a CZT-based semiconductor detector.
To assess each image and the quantification, we usedcylindrical syringes, cylindrical phantoms and myocardial
infarction model rats injected with 99mTc-labeled tetrofosmin (99mTc-TF) [13] and 123I-labeled 15-(p-iodophenyl)-3R,S-methyl pentadecanoic acid (123I-BMIPP) [14–16], and syringes and normal mice injected with 99mTclabeled hexamethylpropyleneamine oxime (99mTc-

123

Phantoms
Two kinds of phantoms for the rat study were created:
10-ml cylindrical syringes (diameter 15.8 mm) and 250-ml
rat-sized cylindrical phantoms (diameter 80 mm). For
evaluation of relationship between SPECT radioactive
counts (1 min acquisition) and radioactivity of 99mTc and123
I, 10-ml cylindrical syringes contained about 37 MBq
99m
Tc-TF or 123I-BMIPP were prepared and then attenuated in accordance with the radioactive decay. The
radioactivity was measured using dose calibrator (Capintec, Inc.)
In two experiments with the two 10-ml cylindrical syringes, one syringe contained 100 MBq 99mTc-TF (Nihon
Medi-physics Co., Ltd.) and the other contained 100 MBq
123
I-BMIPP (Nihon Medi-physics Co., Ltd.) to evaluate
equal doses of 99mTc-TF and 123I-BMIPP. In anotherexperiment, one syringe contained 300 MBq 99mTc-TF andthe other contained 100 MBq 123I-BMIPP to evaluate highdoses of 99mTc-TF and 123I-BMIPP. For the rat-sized
cylindrical phantoms, we evaluated two different versions.
The first contained both 100 MBq 99mTc-TF and 100 MBq
123
I-BMIPP and second had both 300 MBq 99mTc-TF and
100 MBq 123I-BMIPP.
For the mice study, one 1-ml cylindrical syringe (diameter 4.7 mm) contained 105 MBq 99mTc-HMPAO (Nihon Medi-physics Co., Ltd.) and the other contained
35 MBq 123I-FP-CIT (Nihon Medi-physics Co., Ltd.).
Animals
All animal procedures were approved by the institutional
committee at the Medical and Pharmacological ResearchCenter Foundation and were conducted in compliance with
the American Heart Association requirements regarding
the use of research animals. Eight male Wister rats
(8–11 weeks old, 250–320 g) and 10 male normal mice
(6–8 weeks old, 31–35 g) were purchased from Japan SLC
Inc. and were housed for 1 week under a 12-h light/12-hdark cycle with free access to food and water. The rats
were anesthetized with an intraperitoneal injection ofpentobarbital (30 mg/kg) and maintained with 2 % isoflurane (Abbott Laboratories), and then intubated and ventilated using a small animal ventilator (SN-480-7 9 2T,
Shinano). After left thoracotomy and exposure of the heart,a 7–0 polypropylene suture on a small curved needle wasAnn Nucl Med (2016) 30:263–271

passed through the myocardium beneath the proximal
portion of the left coronary artery (LCA), and both ends ofthe suture were passed through a small vinyl tube to make asnare. The suture material was pulled tightly against thevinyl tube to occlude the LCA. Myocardial ischemia wasconfirmed by ST-segment elevation on the electrocardiogram and regional cyanosis of the myocardial surface. The
LCA was occluded for 30 min, and reperfusion was
obtained by release of the snare and confirmed by amyocardial blush over the area at risk.
Description of preclinical SPECT/CT camera
The performance and features of the eXplore speCZT (GEHealthcare) have been reported previously [4]. Briefly, thissystem has a stationary detector with interchangeablerotating collimators. The detector consists of 10 CZT-based
detector panels surrounding the field of view (FOV). EachCZT detector panel consists of 32 9 32 arrays with a pixel
size of 2.46 mm 9 2.46 mm.
We used a 5-pinhole collimator for rat imaging because
the pinhole collimators are designed for high-resolution
images with small regions of interest [17, 18]. The main
characteristics of the pinhole collimators are: diameter of
pinhole 1 mm, FOV diameter of single-bed position
76 mm, axial FOV of single-bed position 38 mm, bore
diameter 89 mm and radius of rotation/focal length
50/70 mm. The system saves each event of the acquired
data in a list-mode format including its energy, and gatedelectrocardiogram (ECG)/respiratory motion trigger forlater, specialized analysis. Performance tests with the pinhole collimator showed that the transaxial and axial system
resolution with resolution correction, and sensitivity of thescanner were, respectively, 1.20/2.18 mm [full-width half
maximum (FWHM)/full-width tenth maximum (FWTM)]
and 1.11/2.02 mm (FWHM/FWTM), and 138.5 cpm/MBq
using a 99mTc radiotracer.
SPECT/CT scan with a pinhole collimator for rats
Experiments with cylindrical syringes, cylindrical phantoms and rats were performed using the eXplore speCZT
SPECT/CT scanner with a 5-pinhole collimator for rat
imaging. The voxel size of the image matrix was
0.5 mm 9 0.5 mm 9 0.5 mm because the voxel size ofthe single-orbit SPECT scan was fixed to 0.5 mm, which
was determined by the step size of the table motion. Theenergy window for SPECT acquisition was set to50–180 keV by default.
For the syringe and phantom experiments, two 10-ml
cylindrical syringes or 250-ml rat-sized cylindrical phantoms were placed on the scanner table in the center of the265

FOV and scanned for approximately 30 min using thepinhole collimator.
In rat experiments, the helical scan mode was used toimage the myocardium of the rats. Eight rats with myocardial
infarction were fasted, with water supplied ad libitum and no
food, at least 4 h before the SPECT experiments. The rats
were anesthetized with 1.5–2.0 % isoflurane (Abbott Laboratories) and placed in a supine position on the scanner table.
Limbs were fixed using surgical tape. Stainless steel electrodes were subsequently inserted into the skin of the rightand left forelimbs to monitor and record the ECG data.
Before SPECT scanning, the orientation of the myocardium
was determined using a laser beam and CT imaging on thescanner. For CT images, the tube voltage and current of the
X-ray tube were 60 kVp and 40 mA, respectively. The rats
were injected with approximately 300 MBq of 99mTc-TF via
the tail vein. Rats were scanned from 5 min after the injection using imaging parameters of 25 s/view and 72 views/
pinhole (total 360 view) in 1° increments. Immediately after
the first scan of 99mTc-TF, the same rats were injected with
100 MBq of 123I-BMIPP and scanned again using the samescanning protocols and parameters.
SPECT/CT scan with a pinhole collimator for mice
For the 1-ml cylindrical syringe and mice study, experiments were performed using a 7-pinhole collimator formice imaging. The voxel size of the image matrix was
0.5 mm 9 0.5 mm 9 0.5 mm, which was determined by
the step size of the table motion. The energy window for
SPECT acquisition was set to 50–180 keV by default in thelist mode.
The setup for the syringes and 10 normal mice was thesame as that for the rat study. Before SPECT scanning, theorientation of the brain was determined using a laser beam
and CT imaging on the scanner. For CT images, the tube
voltage and current of the X-ray tube were 60 kVp and
40 mA, respectively. Five of the 10 mice were injectedwith approximately 105 MBq of 99mTc-HMPAO via thetail vein. Mice were scanned from 5 min after the injection
using imaging parameters of 36 s/view and 50 views/pinhole (total 350 view) in 1.06° increments. Immediately
after the first scan of 99mTc-HMPAO, the same rats were
injected with approximately 35 MBq of 123I-FP-CIT andscanned again using the same scanning protocols andparameters from approximately 3 h after the injection.
The other five mice were injected with approximately
35 MBq of 123I-FP-CIT via the tail vein and then scanned
from approximately 3 h after the injection. Immediately
after the first scan of 123I-FP-CIT, the same rats were
injected with approximately 105 MBq of 99mTc-HMPAO
and scanned again from 5 min after the injection.

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Ann Nucl Med (2016) 30:263–271

Reconstruction settings

cylindrical phantoms. We placed spherical VOIs overnormal and myocardial infarction areas in the rat study andstriatum and whole brain in the mice study. In experimentswith the rat-sized cylindrical phantoms and 1-ml syringes
for the mice study, radioactive counts of 99mTc at
125–150 keV in the dual-tracer images were divided bythose of the 99mTc single-tracer images in 125–150 keV. In
addition, radioactive counts of 123I at 150–175 keV in the
dual-tracer images were divided by those of the 123I singletracer images in 150–175 keV, respectively.

All SPECT data were reconstructed using the three-dimensional maximum-likelihood expectation maximization
algorithm with 50 iterations, with distance-dependent
collimator response correction (DCC) [4]. For theparameters, the energy window was set to 125–150 keV
for 99mTc imaging and 150–175 keV for 123I imaging
using acquisition data of 50–180 keV. Attenuation correction (AC) was not applied because AC provided low
uniformity in the phantom study, and radioactive counts
with and without AC were not that much different in the
rat myocardial [19] mice brain studies (data not shown)in this study. Post-reconstruction filtering was also notapplied because the filter affects the radioactive counts in
the SPECT images. The scatter correction (SC) uses thetriple-energy window (TEW) method for experimentswith a single tracer of 99mTc [20]. Specifically, the TEW
method was applied to the experiments with 10-ml
cylindrical syringes and rat-sized phantom, and rats inthe experiments with a single tracer of 99mTc-TF, and we
set up to 3 keV in width for the left and right scatter as
sub-windows by default. In the experiments with thedual tracers, the TEW method was not available because
we could not set up the sub-window. AC, SC and DCC
can be selected for the reconstruction method in ourscanner, but DCC is always applied to improve image
resolution.

Statistical analyses
A statistical software package (JMPÒ version 9, SAS
Institute Inc.) was used for statistical analysis. We applieda paired t test, and statistically significant differences were
defined as P \ 0.01.

Results
In 10-ml cylindrical syringes including 100 MBq 99mTc or
100 MBq 123I, the relationship between SPECT radioactive
counts and radioactivity was excellent (Fig. 1).
In two syringes containing 100 MBq of 99mTc-TF or
123
I-BMIPP, the peaks of the energy spectrum were at thesame levels (Fig. 2a). Accumulation of 99mTc-TF images
in the 125–150 keV energy window were slightly higher
than that of 123I-BMIPP images in the 150–175 keV energy
window (Fig. 2b). Mean radioactive counts per voxel of99m
Tc-TF in 125–150 keV were 7 % higher than those of123
I-BMIPP in 150–175 keV (Table 1). When SC wasapplied, downscatter contamination of 123I-BMIPP wasapproximately 3.5 % of the counts per voxel of 99mTc-TF
in 125–150 keV, whereas in the case without SC, thedownscatter contamination was approximately 13.2 % of
the counts per voxel of 99mTc-TF in 125–150 keV. Withand without SC, no upscatter contamination of 99mTc-TF
was observed in 150–175 keV.

Image analysis
After reconstruction of SPECT data, radioactive counts
were obtained using the AMIDE medical imaging data
examiner and OsiriX (http://homepage.mac.com/rossetantoine/osirix). The voxels of interest (VOIs) were locatedin the center of the syringes and phantoms. The spherical
VOIs were approximately 5 mm in diameter for theexperiments with 1-ml syringes, and approximately 10 mm
in diameter for the 10-ml syringes and 250-ml rat-sized30

20

Y = 1.22X + 0.01
R2 = 1.0

10

0

40

30

20

Y = 1.18X + 0.02
R2 = 1.0

10

0
0

10

20

30

Radioactivity (MBq)123

Radioactive counts ( 103)

b

40

103)

a

Radioactive counts (Fig. 1 The relationshipbetween SPECT radioactive
counts and 100 MBq 99mTc(a) and 100 MBq 123I(b) images

40

0

10

20

30

Radioactivity (MBq)40

Ann Nucl Med (2016) 30:263–271

a

267

a
Radioactive counts

Radioactive counts

15000

10000

5000

30000
20000
10000
0
100

150

200

Energy (keV)

0
100

150

b

200

Energy (keV)bFig. 3 Energy spectrum (a) and SPECT images without SC (b) of
two cylindrical syringes with 300 MBq 99mTc-TF or 100 MBq 123IBMIPP. Energy peak of 99mTc-TF was approximately 3-fold higher
than that of 123I-BMIPP. 99mTc-TF image in 125–150 keV wassignificantly different from that of 123I-BMIPP in 150–175 keV.
Downscatter contaminations of 123I-BMIPP in 125–150 keV were,respectively approximately 0.9 and 4.1 % of the radioactive counts
for 99mTc-TF with and without SC

Fig. 2 Energy spectrum (a) and SPECT images without SC (b) of
two cylindrical syringes with 100 MBq 99mTc-TF or 100 MBq 123IBMIPP. Energy peak of 99mTc-TF was almost the same as that of 123IBMIPP. Accumulation of 99mTc-TF images in the 125–150 keVenergy window were slightly higher than that of 123I-BMIPP images
in the 150–175 keV energy window. Downscatter contaminations of123
I-BMIPP in 125–150 keV were approximately 3.5 and 13.2 % of
the radioactive counts for 99mTc-TF with and without SC, respectively. Upscatter contamination of 99mTc-TF was not observed in
150–175 keV

the SC of the TEW method was not applied because we
could not increase the number of energy windows in the
TEW method for dual-tracer imaging. The ratios ofradioactive counts of the dual tracer without SC to single
tracer of 99mTc-TF in 125–150 keV became 1.21. When
300 MBq 99mTc-TF and 100 MBq 123I-BMIPP were used,
the ratios were 1.08. There was almost no upscatter contamination of 99mTc-TF in 150–175 keV.
In the rat model of myocardial infarction (Fig. 4), the99m
Tc-TF images in 125–150 keV for the dual tracer
without SC were not different from those of 99mTc-TF forthe single tracer with SC. 123I-BMIPP images showed noaccumulation in the infarction area compared to 99mTc-TF.
Mean radioactive counts of 99mTc-TF in 125–150 keV forthe dual tracer were not much different from those of99m
Tc-TF for the single tracer in the normal and infarction

In two syringes including 300 MBq 99mTc-TF and
100 MBq 123I-BMIPP, the peak of the energy spectrum of99m
Tc-TF was almost 3-fold higher than that of 123I-BMIPP(Fig. 3a). Mean radioactive counts per voxel of 99mTc-TF
in 125–150 keV were also 3.3-fold higher than that of 123IBMIPP in 150–175 keV without SC (Table 2). Downscatter contaminations of 123I-BMIPP were approximately
0.9 and 4.0 % of the radioactive counts for 99mTc-TF in
125–150 keV with and without SC, while there was no
upscatter contamination of 99mTc-TF in 150–175 keV.
In the rat-sized cylindrical phantom including 100 MBq
of 99mTc-TF and 123I-BMIPP as the dual tracer (Table 3),

Table 1 Radioactive counts per voxel in the 10-ml cylindrical syringes including 100 MBq 99mTc-TF and 100 MBq 123I-BMIPP
SC–

SC?

Energy window (keV)

125–150

Radiotracers

99m

123

99m

Counts/voxel

413.3 ± 23.2

54.4 ± 9.5

1.5 ± 0.5

Tc-TF

150–175
I-BMIPP

Tc-TF

125–150

150–175

123

99m

123

99m

382.6 ± 19.8

361.1 ± 21.2

12.6 ± 7.5

0.5 ± 0.5

I-BMIPP

Tc-TF

I-BMIPP

Tc-TF

123

I-BMIPP335.3 ± 20.2

SC scatter correction, 99mTc-TF 99mTc-labeled tetrofosmin, 123I-BMIPP 123I-labeled 15-(p-iodophenyl)-3R,S-methyl pentadecanoic acid

123

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Ann Nucl Med (2016) 30:263–271

Table 2 Radioactive counts per voxel in the 10-ml cylindrical syringes including 300 MBq 99mTc-TF and 100 MBq 123I-BMIPP
SC–

SC?

Energy window (keV)

125–150

Radiotracers

99m

150–175

Counts/voxel

1294.7 ± 50.6

Tc-TF

125–150

150–175

123

99m

123

99m

123

99m

123

52.1 ± 9.1

1.3 ± 0.3

392.5 ± 21.4

1144.1 ± 53.1

10.6 ± 3.8

0.6 ± 0.5

332.5 ± 18.1

I-BMIPP

Tc-TF

I-BMIPP

Tc-TF

I-BMIPP

Tc-TF

I-BMIPPSC scatter correction, 99mTc-TF 99mTc-labeled tetrofosmin, 123I-BMIPP 123I-labeled 15-(p-iodophenyl)-3R,S-methyl pentadecanoic acid

Table 3 Ratios of radioactive counts in dual tracer without SC tosingle tracer with SC on 99mTc-TF images in 125–150 keV and 123IBMIPP images in 150–175 keV in the rat-sized cylindrical phantom
Energy window (keV)
99m

100 MBq Tc-TF
300 MBq 99mTc-TF
99m

125–150

150–175

1.21 ± 1.5
1.08 ± 1.4

1.01 ± 0.5
0.98 ± 0.4

Tc-TF 99mTc-labeled tetrofosmin

Single tracer
99m

Tc-TF

Discussion

Dual tracer
99m

123

Tc-TF

Downscatter contaminations of 123I-FP-CIT were approximately 1.4 % and 4.2 % of the radioactive counts for
99m
Tc-HMPAO in 125–150 keV with and without SC,
while there was no upscatter contamination of 99mTcHMPAO in 150–175 keV.
In the mice study, the 99mTc-HMPAO images for thedual tracer in 125–150 keV without SC were not different
from those of 99mTc-HMPAO for the single tracer with SC
in the first injection of 99mTc-HMPAO and second injectionof 123I-FP-CIT (Fig. 6). In the first injection of 123I-FP-CIT
and second injection of 99mTc-HMPAO, the brain images
and mean radioactive counts were also not much different
from those of the 123I-FP-CIT single injection (Fig. 7;
Table 6).

I-BMIPP

Fig. 4 SPECT images in a rat model of myocardial infarction using
300 MBq 99mTc-TF in the single tracer and 300 MBq 99mTc-TF and
100 MBq 123I-BMIPP in the dual tracer. The myocardial SPECT
images of 99mTc-TF in the dual tracer without SC were similar to
those of 99mTc-TF in the single tracer with SC. 123I-BMIPP images
showed a slight mismatch with 99mTc-TF images in the posterior wallat risk of acute myocardial infarction as memory imaging

areas when SC was applied for the single tracer (Table 4).
In the two syringes including high 99mTc-HMPAO doseand 123I-FP-CIT, the peak of the energy spectrum of 99mTcHMPAO was almost three times that of 123I-FP-CIT(Fig. 5). Mean radioactive counts per voxel of 99mTcHMPAO in 125–150 keV were 2.9-fold higher than that of123
I-FP-CIT in 150–175 keV without SC (Table 5).

The simultaneous acquisition of 99mTc-TF and 123I-BMIPP
yields myocardial blood flow and metabolism of rats underidentical conditions. To detect cardiac disease moreclearly, it is important for cardiac studies of rats to achieve
more accurate quantification and anatomical registration
between the two images of 99mTc-TF and 123I-BMIPP. The
quantification and anatomical registration are also crucial
for brain studies of mice using 99mTc-HMPAO and 123I-FPCIT. This dual-tracer imaging accordingly reduces theexperiment time and burden for research staff [6–12]. The
radioactive counts and each image of simultaneously
acquired 99mTc and 123I were evaluated using a preclinical
SPECT scanner with a CZT-based semiconductor detector.
Although we can select if AC, SC and DCC are applied in
the reconstruction method on this SPECT scanner, AC is
not applied because AC provided low uniformity and there

Table 4 Radioactive counts per voxel of 99mTc-TF images in single tracer with SC and in dual tracer without SC for the myocardial infarction
model rats
Radiotracers

Single tracer of 99mTc-TF

RegionNormalCounts/voxel
99m

Tc-TF

289.2 ± 54.8

99m

Tc-labeled tetrofosmin,* P \ 0.01 vs. normal area

123

123

I-BMIPP

123

Dual tracer
Infarction

NormalInfarction

27.9 ± 13.5*

297.1 ± 82.5

28.3 ± 17.2*

I-labeled 15-(p-iodophenyl)-3R,S-methyl pentadecanoic acid

Ann Nucl Med (2016) 30:263–271

a269

Radioactive counts

20000

10000

Single tracer
99mTc-HMPAO

0
100

150

200

b

99mTc

150 175 keV

123I

99mTc

123I-FP-CIT

Fig. 6 Fusion images of SPECT and CT in normal mice using asingle tracer of 105 MBq 99mTc-HMPAO, and dual tracers of the first
injection with 105 MBq 99mTc-HMPAO and second injection with
35 MBq 123I-FP-CIT. 99mTc-HMPAO images in the dual tracer
without SC were similar to those of 99mTc-HMPAO in the single
tracer with SC

Energy (keV)

125 150 keV

Dual tracer
99mTc-HMPAO

123I

Fig. 5 Energy spectrum (a) and SPECT images without SC (b) of
two 1-ml cylindrical syringes with 105 MBq 99mTc-HMPAO or
35 MBq 123I-FP-CIT. Energy peak of 99mTc-HMPAO was approximately 3-fold higher than that of 123I-FP-CIT. 99mTc-HMPAO image
in 125–150 keV was significantly different from that of 123I-FP-CIT
in 150–175 keV. Downscatter contaminations of 123I-FP-CIT in
125–150 keV were, respectively, approximately 1.4 and 4.2 % of theradioactive counts for 99mTc-HMPAO with and without SC

Single tracer

Dual tracer
99mTc-HMPAO

123I-FP-CIT

123I-FP-CIT

Fig. 7 Fusion images of SPECT and CT in normal mice using
35 MBq 123I-FP-CIT in the single tracer, and the first injection with
105 MBq 99mTc-HMPAO and second injection with 35 MBq 123I-FPCIT in the dual tracer. 123I-FP-CIT images in the dual tracer without
SC were not different from those of 123I-FP-CIT in the single tracer
with SCis no hard substance such as bone in the chest region
including the heart and scull of the mice (0.2 mm) which ismuch smaller thickness than that of rats (0.5–1.0 mm) andhuman (about 10 mm) [21], but, DCC is always applied to
improve significant SPECT images. Scatter and cross-talkeffects of 99mTc and 123I mainly hamper the simultaneous
acquisition of dual tracers whose photon energies are close.
The relationship between SPECT radioactive counts andradioactivity of 99mTc or 123I was excellent (Fig. 1).
However, each radioactivity of 99mTc and 123I in dual
tracers could not be accurately separated and measured bydose calibrator and ex vivo autoradiography. Therefore, weused SPECT radioactive counts for evaluation of dual
tracers in our study.

In the experiment with the cylindrical syringe, upscatter
contamination of 99mTc-TF was not incorporated in the
150–175 keV energy window of 123I-BMIPP without AC
and SC (Tables 1, 2, 5). The ratio of downscatter contamination from 123I-BMIPP in 125–150 keV divided by
300 MBq 99mTc-TF without SC (4.0 %) produces results
similar to when divided by 100 MBq 99mTc-TF with SC in
125–150 keV (3.5 %) because downscatter contaminationof 123I-BMIPP exhibited fewer radioactive counts of
300 MBq 99mTc-TF.
In experiments with a rat-sized cylinder phantom(Table 3), when 300 MBq 99mTc-TF and 100 MBq 123I-

Table 5 Radioactive counts per voxel in the 1-ml cylindrical syringes including high 99mTc-HMPAO dose and 123I-FP-CIT
SC–

SC?

Energy window(keV)

125–150

Radiotracers

99m

123

99m

123

99m

Counts/voxel

407.5 ± 20.6

17.3 ± 6.1

1.1 ± 0.3

140.4 ± 8.9

368.2 ± 17.1

99m

99m

TcHMPAO

150–175
I-FPCIT

Tc-HMPAO
SC scatter correction,
bomethoxy-3b-(4-iodophenyl)nortropane

TcHMPAO

125–150
I-FP-CIT

150–175

TcHMPAO

Tc-labeled hexamethylpropyleneamine oxime,

123

123

I-FPCIT

5.3 ± 4.6

I-FP-CIT

123

99m

123

0.5 ± 0.3

118.4 ± 6.8

TcHMPAO

I-FP-CIT

I-labeled N-x-fluoropropyl-2b-car-

123

270

Ann Nucl Med (2016) 30:263–271

Table 6 Radioactive counts per voxel of 99mTc-HMPAO images in 125–150 keV and 123I-FP-CIT images in 150–175 keV on normal mice
injected single tracer with SC and dual tracer without SC
First injection

99m

123

Radiotracers

Single tracer of 99mTc-HMPAO

Tc-HMPAO

I-FP-CIT

Single tracer of 123I-FP-CIT

Dual tracer

Dual tracer

RegionWB

Striatum

WB

Striatum

Striatum

Striatum

Counts/voxel

101.8 ± 5.3

105.1 ± 8.5

106.1 ± 7.1

108.3 ± 10.2

26.9 ± 9.3

28.3 ± 8.6

99m

Tc-HMPAO 99mTc-labeled hexamethylpropyleneamine oxime, 123I-FP-CIT 123I-labeled N-x-fluoropropyl-2b-carbomethoxy-3b-(4-iodophenyl)nortropane, WB Whole brain

BMIPP were used, there was little influence from thedownscatter contamination of 123I-BMIPP in 125–150 keV
despite SC as the radioactivity of 99mTc-TF was higher
than 123I-BMIPP. CZT detectors and pinhole collimators
yield a narrow energy spectrum [2–6], and preclinical
SPECT images are typically much less degraded by photon
scattering than clinical SPECT images because of smaller
body dimensions. Kao et al. has also reported that downscatter contamination of 123I-ADAM was less affected byradioactive counts of 99mTc-TRODAT in dual-tracer
imaging of 99mTc-TRODAT and 123I-ADAM when theradioactivity of 99mTc-TRODAT was approximately 3.5fold higher than that of 123I-ADAM [12]. In addition, there
was no upscatter contamination of 99mTc-TF in
150–175 keV. Therefore, each accurate radioactive countof 99mTc and 123I will be provided by the narrow energy
spectrum of radionuclides using a CZT detector with pinhole collimator and/or SC and DCC if the radioactivity of
the injected 99mTc-TF tracer is increased. Although thesimultaneous acquisition of 99mTc and 201Tl has alreadybeen reported using a clinical SPECT scanner with a CZT
detector, AC and DCC were not applied to measureaccurate radioactive counts [6]. Especially, DCC should beapplied to the simultaneous acquisition of 99mTc and 123I in
preclinical SPECT imaging with CZT detector. If thecorrection methods are unavailable, we should increase theradioactivity of 99mTc to decrease the downscatter contamination of 123I.
In the rat model of myocardial infarction using
300 MBq 99mTc-TF and 100 MBq 123I-BMIPP, the 99mTcTF images in 125–150 keV without SC of the TEW
method in the dual tracer were not significantly different
from those of the single tracer with SC of the TEW method
when DCC was applied (Fig. 3). The uptake of 99mTc-TF
in the myocardial infarction significantly decreased incomparison with that in the normal area (Table 5). In 123IBMIPP images, the infarct areas showed no accumulationcompared to those in 99mTc-TF images because 123IBMIPP images yielded memory imaging of the infarction
area [14, 15]. In the 125–150 keV energy window,
radioactive counts in dual tracers were not significantly
different from those of 99mTc-TF in the single tracer in123

normal and infarction areas when DCC were applied(Table 5).
Since 123I-FP-CIT specifically accumulates in striatum[23], we evaluated the accumulation in both 99mTcHMPAO and 123I-FP-CIT images. In striatum and wholebrain of normal mice, the 99mTc-HMPAO images andradioactive counts in 125–150 keV for the dual tracer
without SC were not different from those for the single
tracer with SC in the first injection of 99mTc-HMPAO(Fig. 6; Table 6) because, in human study, accumulation of99m
Tc-HMPAO maintains steady-state from early phase of
5 min to late phase of 8 h after the injection [22]. Brain
images and radioactive counts of 123I-FP-CIT in striatum
were also similar between the single tracer of 123I-FP-CIT(Fig. 7; Table 6). To decrease radioactivity of 99mTc-labeled radiotracers for dual-tracer imaging, we will develop
a program for the TEW method for dual-tracer imaging in
the future. In addition, we should consider the cardiac
motion of rats in this study. More accurate quantification of
the dual tracers may be achieved using gated SPECT.

Conclusion
Dual-tracer imaging of 99mTc- and 123I-labeled radiotracers
is feasible in small animal studies using a preclinical
SPECT scanner with CZT detectors. Radioactivity of theinjected 99mTc-labeled radiotracers should be increased asmuch as possible to improve quantification of 99mTc-labeled radiotracers in dual-tracer imaging if the correction
methods cannot be appropriately applied. In this case,correction methods are not necessarily required for thequantification of 99mTc- and 123I-labeled radiotracers whenusing a preclinical SPECT scanner with CZT detector.
Acknowledgments The author would like to thank Akiko Hayashi
and Ryoko Komatsu, Takafumi Tsujiuchi, Masatoshi Sakashita andmembers of the medical staff at the Ishikawa prefectural government
and Kanazawa University.
Compliance with ethical standards
Conflict of interestof interest.

The authors declare that they have no conflict

Ann Nucl Med (2016) 30:263–271
Sources of funding This study was partly funded by Grants-in-Aid
for Scientific Research from the Japan Society for the Promotion of
Science (24601008, 24659558, 25293260 and 15K09949), the Societyof Nuclear Medicine Technology, Japanese Society of RadiologicalTechnology and Ishikawa Prefecture Commission Research.

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