The Presentation of Clinical Results of Radiolabelling of 99mTc-MIBI Complex Using Ultrasound Technique for Myocardial Perfusion Scanning (MPS)

Background The initial objective and opinion of this study is introducing the clinical results of the 99m Tc-MIBI complex radiolabelling utilizing ultrasound method as an approach for labeling MIBI kits with 99m Tc instead of applying a boiling water bath as a usual and standard method. were provided to be 21.09 and 3.48 min for 99m Tc-MIBI complex sample and 99m TcO 4 respectively. These results and explanations indicate that the radio-labeling of 99m Tc-MIBI complex samples applying the sonication irradiation approach is quite successful and high eciency. For intravenous injection of 99m Tc-MIBI complex samples to patients, the yield of 99m Tc-MIBI complex samples must be more than 90%. All sick people who referred to Department of Nuclear Medicine of Ahvaz Golestan Hospital for myocardial perfusion scanning, had acceptable and favorable situations. The patient volunteers were assessed for 2 months after investigation. During this time, none of the patients reported any side effects from injecting 99m Tc-MIBI complex samples like nausea, vomiting, diarrhea, bronchospasm, hypotension, hypertension, tachycardia, bradycardia, fever, chills did not have muscle cramps and allergic reactions. The scans provided for every patient indicate a proper and considerable absorption of Tc-MIBI complex samples in the heart. The standard of scans was the same in both groups A and B. For these reasons, it was proven to us that myocardial perfusion scan investigations were done for patients in group A, as in the other group (Fig. 3). The imaging of total body at rest step, 1–2 hours after 99m Tc-MIBI complex injection was intravenously done. The biodistribution of radio-tracer samples was handled to evaluate the distribution of 99m Tc-MIBI complex samples in patient volunteers. The radiolabelling methods did not result in a signicant difference in the model of biodistribution of complex samples. Outcome indicated that the technique of the reconstruction of radiotracer samples could not cause to make changes to the characteristics of radiotracer samples in bioanalysis research. The result of all research indicated that the features of all radiotracer samples which were prepared through ultrasound irradiation were completely alike to the features of 99m Tc-MIBI complex samples were reconstituted through the boiling water bath technique. The accumulation of radiopharmaceuticals reconstituted through both sonication method and boiling water bath technique was almost 2% in the hearts of volunteers. The biodistribution This study's results indicated that sonication irradiation approach can be proposed to provide radio-complex samples. The reaction time to provide radio-complex could be signicantly decreased compared to boiling water bath approach. This main advancement may be reduced potential risk to the patient to inhibit any delay in the emergency conditions like acute therapy particularly for myocardial infarction patients in clinical practice. Green chemistry is an alternative approach for MIBI kit radiolabelling with 99m Tc radioactive isotope.


Introduction
Myocardial perfusion single-photon emission computed tomography (MPS) is one of the most signi cant and usually handled non-invasive cardiac imaging tests. MPS has a signi cant role in diagnosing cardiovascular disease, establishing prognosis, evaluating the e ciency of therapy, and evaluating myocardial viability. The success of this imaging modality has, in large section, been regarding developed technology which continues to advance and develop the eld. These involve the progressive advancement of single-photon emission-computed tomography (SPECT) technologies, novel radiopharmaceuticals, and novel software [1]. Thalium-201 (in the early 1970s and kept in usual utilization until the mid-1980s) and technetium-99m ( 99m TC) sestamibi and tetrofosmin are commonly applied radiopharmaceuticals [2]. 99m Tc-labeled radiopharmaceuticals were improved and, for the most section, replaced thallium to assess the myocardial perfusion abnormalities [3]. Technetium, the 43rd element in the periodic table, belongs to the group of transient metals. Owing to its electron con guration of 4d 5 5s 2 , technetium prepares a handful of situations for complex formation with many different ligands, and its oxidation state (OS) can alter from + 1 up to + 7. OS is regarded to be a major parameter identifying the chemical feature of complexes. Technetium can form chemical bonds including both sigma and pi electrons, and the sigma bonds can be the colligative and coordinative kinds when spin compensation and electron pair donation take place, respectively. Moreover, the structure of technetium complexes can be signi ed by the coordination number (N), which can change from 4 to 7, permitting tetrahedral (N = 4), tetragonal pyramidal (N = 5), octahedral (N = 6), capped octahedral (N = 7) or pentagonal bipyramidal (N = 7) geometry. The third parameter for characterization of technetium complexes is the electric charge (Z) of the whole molecule that might prepare an anionic (Z = − 1), neutral (Z = 0) or cationic (Z = + 1) character [4].
A handful of technetium 99m-labeled myocardial perfusion agents were under survey to identify their utility in evaluating regional myocardial blood ow and cellular viability in recent years. Technetium 99m-2-methoxyisobutyl isonitrile ( 99m Tc-MIBI), one of the most signi cant agents is a lipophilic cation largely sequestered in the mitochondria by the large negative transmembrane potential. Experimental research indicated that 99m Tc-MIBI complex is taken up in the myocardium in proportion to blood ow, but as other diffusible radionuclides underestimates ow at high ow rates. The research revealed that this agent uptake after reperfusion, preceded by changing coronary occlusion periods, re ects the degree of myocardial salvage and viability. Research indicated that this complex has good features to handle myocardial perfusion scans [6][7][8][9]10]. In the last decade, 99m Tc-labelled lipophilic cations emerged as appropriate tools to trace speci c cellular processes and functions in various malignant tumors, involving breast cancer. Among these agents, the 99m Tc-MIBI complex is the most highly assessed tracer and might serve as a paradigm for this class compound class [11]. 99m Tc-Sestamibi moreover is applied in Parathyroid Scintigraphy for the Detection and Localization of Parathyroid Adenomas in Patients with Hyperparathyroidism [12] which indicated to be suitable to diagnose lung cancer [13] conditions. In 99 Mo/ 99m Tc generators, regarding the imperfections in producing the generator or possible occurrence of mechanical defects, 99 Mo may leak from the generator due to the elution process [14][15][16]. To resolve the contents of vial, the protected vial should be strenuously shaken due to safety conditions. Then, the vials were heated on a boiling water bath for 10 minutes. After heating, the vial is placed into the lead shield and cooled at room temperature for approximately 15 min [17]. This complex can be applied within 6 h after preparation. The 99m Tc-MIBI kit has the + 1 charge. The labeling time of MIBI kit with technetium is prolonged applying common and usual approaches like boiling water bath. One of the approaches introduced to replace boiling water bath technique for labeling the MIBI kit with technetium is the microwave oven heating technique. A study indicates that the microwave oven heating approach prepares a reproducible heating technique for labeling 99mTc-sestamibi. By a labeling e ciency over 96.4% after 13 sec of heating, this method simpli ed and shortened the work included in providing 99m Tc-MIBI complex. It is reliable and well suited for emergency conditions when the agent requires to be available almost immediately [15].
The microwave heating method has technical precautions which should be regarded in applying this approach to provide 99mTc-MIBI complex. Despite, the bene ts of this approach, these precautions are regarded disadvantages for this approach. These precautions involve the optimum heating time and wattage for the successful labeling of 99m Tc-MIBI with a particular microwave oven, which must be experimentally identi ed, the metal cap of the vial requires to be covered with Styrofoam to avoid electrical sparking and a vacuum situation should be obtained before the labeled vial is heated with a microwave oven, any residual gas left in the head space of vial could cause an ejection of the rubber stopper due to the excess steam pressure built up the vial [17]. Despite above factors, the labeled vial should always be heated in the same position inside the microwave oven, microwave ovens with digital control panel are more suitable for setting short heating time (i.e. 10 s) since they can be correctly set at the needed heating period and the loss or variation of microwave power output and frequency related to extended the utilization of the microwave oven must be assessed on a long-term basis and nally any technical error in setting the microwave heating time below or beyond the predetermined time which may result in the 99m Tc-MIBI solution being rendered unsuitable for clinical utilization [18]. Due to mentioned factors, this approach is not customarily actually applied in nuclear medicine. In the early 1990's, Green chemistry is de ned as the use of a set of principles which decreases or deletes the utilization of hazardous substances in the design, manufacture and application of chemical products [19].
One of the methods mentioned as the green value in chemical reactions is ultrasound irradiation [20]. Comparing to traditional approaches, ultrasound irradiation is more comfortable and simply monitored. Many chemical reactions were done in higher yield, shorter reaction time, and milder condition under ultrasound irradiation [21].Doroudi A et al.
explained labelining of MIBI kits with 99m Tc applying ultrasound irradiation approach [17]. Regarding the study, (37MBq) 99m Tc-MIBI complex samples were provided utilizing ultrasound irradiation technique or boiled water bath approach as a standard method. Qualitative and quantitative research were done. The accumulation of 99m Tc-MIBI complexes provided by two above noted modalities were approximately 3 ± 0.1 % in the rats' heart. The ultrasound irradiation technique is proposed to provide 99m Tc-MIBI complex in effectively value [17]. The initial objective and opinion of this study is the presentation of clinical results of radiolabelling of 99m Tc-MIBI complex applying ultrasound technique as an approach for labeling MIBI kits with 99m Tc instead of utilizing a boiling water bath as a common and standard method and indication of bio-distribution of 99m Tc-MIBI complex samples which are labeled by ultrasound irradiation method compared to boiling water bath method in the human heart.

Materials And Methods
The whole chemical materials which are utilized in this research were purchased from Broker, Merck, and Sigma-Aldrich companies. The chemicals and solvents were among the highest purity or analytical grade and were applied without more puri cation. Radioisotope Division of AEOI provided the freeze-dried MIBI kits and 99Mo/99mTc generators. 99m Tc as Na + TcO 4 − is received by 99 Mo/ 99m Tc generator by using sodium chloride 0.9%. Providing 99m Tc-MIBI complex samples were done by manufacturer's instructions as a standard approach or by freshly advanced method. The whole materials utilized in this research were located in the appropriate environment and standard situations. 0.9% NaCl was applied to identify 99m TcO 4 − , and methanol, for identifying decreased 99m Tc. The whole reactions were controlled by analytical RP-HPLC on a JASCO 880-PU intelligent pump HPLC system (Tokyo, Japan) equipped to a multiwavelength detector and a ow-through Raytest-Gabi γ-ray detector. CC 250/4.6 Nucleosil 120-5 C-18 column from Teknokroma was applied for HPLC. All research utilizing 99m Tc were done in laboratories con rmed for radioactivity utilization. Suitable shielding and radiation safety methods were investigated at each step by trained personnel. AEOI provided MIBI compound used for preparing lyophilized MIBI kits. MIBI kits were provided applying almost the same composition as that of Cardiolite (DuPont) kit. 19.5 mg of anhydrous SnCl2 was dissolved in 3.0 mL of 37% HCl to provide a solution designated as solution C. 10 mg of [Cu(MIBI)4]BF4 was dissolved with stirring in 8 mL of water. Furthermore, 10 mg of L-cysteine hydrochloride monohydrate, 25 mg of sodium citrate, and 220 mg of mannitol were added and dissolved with shaking. After purging with oxygen-free N 2 for 15 min, 0.1 mL of solution C was added. The nal solution was balanced to pH 5.4-5.9 with 1 M NaOH. The nal volume was balanced to 10 mL with sterile water for injection (SWFI). were used at a distance of approximately 1 cm from the bottom of ITLC strips, were permitted to dry at room temperature, and were then located in air-tight containers. When applying 0.9% NaCl as the mobile section, 99m Tc-MIBI complex and decreased 99m Tc remained at the point of spotting, whereas free 99m TcO 4 − turned with the solvent front.
By HPLC-grade methanol as the mobile phase, 99m Tc-MIBI complex and free 99m TcO 4 − turned with the solvent front, whereas decreased 99m Tc kept at the point of spotting. The strips were cut to 1/4 and 3/4 pieces and counted for 2 min under a single-head camera equipped by a low-energy all-propose collimator applying an energy peak centered at 140 keV. The content of reduced 99m Tc was identi ed as the percent ratio of the activity of lower 1/4 piece from the run with methanol to the total activity, and the content of free 99m TcO 4 − as the percent ratio of the activity of the upper 1/4 piece from the run with 0.9% NaCl to the total activity; the content of complex was identi ed from the difference.
Next day, the rest test was done by the similar approach, except in the rest section, myocardial perfusion imaging was done 1 hour after handling 99m Tc-MIBI complex samples. Every phase scans (stress and rest) were provided from one to two hours after the complex samples were injected into the patients. The patients were in a supine situation with the arms which was raised above the head and were supported for imaging applying a single-head gamma-camera

Orbit
Scintigraphy imaging was provided on 180° rotation orbits applying a single-head gamma camera. Single-detector systems were utilized. In these systems, rotation from 45° right anterior oblique (RAO) to 45° left posterior oblique (LPO) was utilized. Pixel size is 6.4 ± 0.4 mm for a 64×64 image matrix. Zoom was done as necessary for cameras with a large eld of view (x1.5). This provides a good balance between image resolution and image noise. Since Noncircular orbits minimize the distance between the patient and the camera via the scan, we applied non-circular (elliptical) rotation.

Number of projections
Due to the 18° rotation, 64 views were applied.

Centre of rotation
Centre of rotation (COR) calibration was done due to the manufacturer's recommendations. The validity of COR correction was periodically tested by estimating full-width at half-maximum (FWHM) spatial resolution from a 180°S PECT study of a point or line source of 99mTc. After reconstructing with a ramp lter, SPECT resolution was estimated. To quantify the spatial resolution reliably, a large matrix size and a zoom factor was used. To explain and describe MIBI scans, two indices were applied at rest phase. The rst index: By dividing the value of complex absorbed in the heart into its counts to whole body utilizing the software available in the nuclear medicine department of the hospital, the heart uptake to the whole body uptake was computed for every person. Hence, applying whole body scan, the region of interest (ROI) was made in all directions the heart uptake and next the latter ROI was designed around the whole body uptake in the anterior sight. The cumulation of 99m Tc-MIBI complex in the cardiac to allover of total body was measured (Fig. 2). No context deduction utilized. To understand and show the uptake of complex in the thyroid, lung, liver, gallbladder, stomach, colon, kidneys and bladder, visual scoring (0, *, **, ***) was used. Scores ** indicate equal absorption of the complex with the heart, scores *** reveal greater absorption from the heart, scores * show less absorption from the heart, and nally 0 reveals no absorption. The scintigraphic scans were expounded by three experienced and independent Doctors of nuclear medicine, and the terminal point of views of all three of these doctors was the same. Watchers were uninformed of the making ready method of complex samples.

Results
99m TcO 4 and decreased 99m Tc are two major impurities made due to the labeling of MIBI kit with 99m Tc. MIBI-99m Tc complex, 99m TcO 4 and reduced 99m Tc are simply determined and estimated quantitatively by exploration and analysis of instant thin layer chromatography (ITLC). The decreased 99m Tc impurity cannot be diagnosed applying R-HPLC approach. ITLC analysis indicates that the average e ciency of 99m Tc-MIBI complex samples that provided applying a boiling water bath as a conventional method was 94 ± 1.8 % (n = 20) and the mean e ciency of the labelled vials through the ultrasound approach as a proposed method was 92.5 ± 3 % (n = 20). Radio-HPLC analysis prepared from 99m Tc-MIBI complex samples and 99m TcO -4 ( Fig. 1) indicated that the reactions were resulted in a single result and their retention times were provided to be 21.09 and 3.48 min for 99m Tc-MIBI complex sample and 99m TcO -4 respectively. These results and explanations indicate that the radio-labeling of 99m Tc-MIBI complex samples applying the sonication irradiation approach is quite successful and high e ciency. For intravenous injection of 99m Tc-MIBI complex samples to patients, the yield of 99m Tc-MIBI complex samples must be more than 90%. All sick people who referred to Department of Nuclear Medicine of Ahvaz Golestan Hospital for myocardial perfusion scanning, had acceptable and favorable situations. The patient volunteers were assessed for 2 months after investigation. During this time, none of the patients reported any side effects from injecting 99m Tc-MIBI complex samples like nausea, vomiting, diarrhea, bronchospasm, hypotension, hypertension, tachycardia, bradycardia, fever, chills did not have muscle cramps and allergic reactions. The scans provided for every patient indicate a proper and considerable absorption of Tc-MIBI complex samples in the heart. The standard of scans was the same in both groups A and B. For these reasons, it was proven to us that myocardial perfusion scan investigations were done for patients in group A, as in the other group (Fig. 3). The imaging of total body at rest step, 1-2 hours after 99m Tc-MIBI complex injection was intravenously done. The biodistribution of radio-tracer samples was handled to evaluate the  (Table 1). We did not observe any unanticipated impacts of radiopharmaceuticals on sick volunteers who gone for MPS to the nuclear medicine department of Golestan Hospital in Ahvaz. which must be decomposed through labelling [23]. 99m Tc-MIBI is taken up by the cells of the myocardium in passive diffusion [24], and then seems in the cytosol which is localized in mitrochondria. The uptake is proportional to the myocardial perfusion, and washout is rather slow (involving signi cant redistribution). At stress, more than 3% of the injected dose is accumulated in the myocardium, while nonbound part is deleted through the hepatobiliary route. The proper situations to provide 99m Tc-MIBI complex samples under ultrasound irradiation method were reported [17]. In new advanced approach, which locally induced heating related to ultrasound irradiation without heating to 65 ℃ was not effective to provide the radiotracer in enough yield. Isonitrile as an electron donor group in sestamibi is coordinated to the technetium. The feature of this type of bonding is not strong as covalent bonding. Thus, elevated temperature above 65 ℃for more than 1 min could affect the feature of bonding or degrade the framework of 99mTc-MIBI complex under ultrasound irradiation situation. For this cause, yield of 99mTc-MIBI complex samples was considerably reduced. The past evaluation showed that the volume of freshly eluted 99mTc pertechnetate sodium, temperature and the period of ultrasound irradiation, due to the power of ultrasound instrument facility were the most signi cant factors to provide 99m Tc-MIBI complex samples with enough yields. The radio-HPLC and TLC analysis research have indicated successful reconstitution sestamibi kits by novel advanced modality. 99m Tc-sestamibi is a lipophilic, monovalent cation which localizes in myocardial cells by easy diffusion without active transport. It was suggested that sestamibi molecule binds to a small molecular weight cytosolic protein [25]. Unlike thallium 201, it does not redistribute during the time. Hence, isolated injections have to be intravenously administrated to patient for stress and resting studies. The survey of the patients did not indicate signi cant differences in biodistribution of radiotracer samples in stress and rest phases that were provided due to ultrasound or boiled water bath techniques in present method. Visual analysis of images indicated a good image quality in every item. The consequence of our survey has indicated that providing 99mTc-MIBI complex samples by ultrasound irradiation method is reliable and reproducible approach to simplify reconstitution 99mTc-sestamibi kits. Providing 99m Tc-MIBI complex by novel advanced approach has under here bene ts. The time labeling of sestamibi by pertechnetate sodium by ultrasound irradiation method versus water bath approach was considerably decreased. 99m Tc-MIBI complex samples can be provided in enough amounts with good yields. The samples geometry in ultrasound device was not signi cant element. No potential sparking risk is present for metal cap presence of the vial inside the ultrasound apparatus. In addition to above elements, the potential risk of absorbed ionization irradiation to nuclear medicine department personnel can be signi cantly reduced. The proposed method to provide 99mTc-MIBI complex could be done in any nuclear medicine centers which allowed a fast and reliable approach to make 99mTc-MIBI complex available for either routine or emergency utilization. To access it, it is required to set out this method in nuclear medicine departments to understand the proper situations from the case of temperature and the period of ultrasound irradiation due to instrument power.  Entirety body radioisotope images patients were done the rest step after the 555-740 MBq (15-20 mCi) 99mTc-MIBI intravenously handled. Applying accessible commercial software, the accumulation proportion of radiotracer in the patient's heart to complete body was calculated by isolating the action of heart to add up to body counts. 99mTc-MIBI uptakes within the heart to the whole body were roughly 2 %. The 99mTc-MIBI complex tests were given here (a) ultrasound irradiation, (b) boiled water bath conditions Figure 3 SPECT perfusion scans of the same patients were prepared with 99mTc-MIBI radiotracer tests. A 2-day stress/rest myocardial perfusion ltering convention was handled. The short-axis (beat 2 columns), vertical long-axis (center 2 lines) and even long-axis (foot 2 columns) cuts were revealed at push and rest. The images in to begin with push of brief pivot, vertical long pivot and at long pivot have a place to the stretch stage of myocardial perfusion imaging. The camera rotation around the patient's chest from 45°C right front angled (RAO) to 45°C cleared out back angled (LPO). The lters were given 1-2 h after the 555-740 MBq (15-20 mCi) 99mTc-sestamibi radiotracer tests organization intravenously, (a) radiopharmaceutical test arranged through sonication strategy, (b) 99mTc-MIBI complex test reconstituted by means of boiling water shower strategy.

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