IMAGING

Practical and Diagnostic Considerations for Gated Myocardial Perfusion Tomography Using Sestamibi

M.G. Morgan and F. Mannting

Nuclear Section, Department of Clinical Physiology, University Hospital, Uppsala, Sweden

photon energy less appropriate for gamma cameras. Tcch­ Technetium-99m f9mTc) sestamibi makes gated single-photon netium-99m's energy, shorter half-life, and improved count­ emission computed tomography (SPECT) perfusion imaging pos­ ing statistics arc suitable for the gated SPEer technique. sible because of99mTc•s energy and counting statistics. The aim Computer systems in many departments of this study was to analyze the practical and diagnostic value of do not have computation capacity and software able to ac­ gated SPECT imaging while considering the extra investment in quire and process gated SPEer data effectively. The new time and effort. We studied 73 subjects (20 nonnals. 53 patients) RISC-typc computers, faster processors (CPU), parallel pro­ using a 1-day rest/stress protocol (6 mCi at rest/24 mCi at peak cessing, and improved software arc being introduced in nu­ stress). Camera time was -50% longer than a standard nongated clear medicine and arc capable of effectively handling data SPECT study; filtering and reconstruction took 2.5 times longer. Processing of diastolic studies took -8 min. Acquisition data. from gated SPECT studies. reconstructed studies. and formatted diastolic and dynamic stud­ Some investigators have applied gated planar principles to ies occupied 5.6 times more storage space. High quality dynamic 20 tTI studies (4-8) and have shown that end-diastolic data and perfusion studies were obtained in 70!73 patients. The right improve definition of myocardial borders. In selected cases, ventricle (RV) appeared more distinct on diastolic studies than in end-diastolic 20tTI images have been used for assisting in the nongated studies (p < 0.01). The left ventricle (LV) cavity was conformation of 20 tTI redistribution (8). Gated planar sesta­ larger in diastolic studies than in nongated studies (p < 0.001). mibi imaging has been described (9-l/) and used for evalu­ leading to more useful coronal slices with cavity (p < 0.001 ). A ation of myocardial wall motion (9-11). Gated SPEer has significant inverse relation between LV size and increase in been applied in a few studies using 20tTI (12) or sestamibi number of useful coronal slices with ventricular cavity in dia­ (13-17). These studies found advantages of gated SPECT stolic studies was found (r = -0.71. p < 0.001). The extra with respect to high-contrast perfusion images and additional effort. time. and storage space required by gated SPECT was balanced by the additional diagnostic information gained from information of regional function in the form of systolic wall dynamic and diastolic images and by clearer RV and LV cavity thickening (12-14,16) or regional wall motion (12-15). visualization. Gated SPECT is particularly useful in patients In a preliminary study (18), we showed that sestamibi with smaU or in patients with LV hypertrophy. gated SPECT was possible and realistic even when using conventional computers systems. The aim of this investiga­ J Nucl Med Techno/1993; 21:13-19 tion was to further evaluate the cost versus benefit of gated SPEer when applied to a larger patient population.

Use of the new radiopharmaceutical, tcchnctium-99m 99 ( mTc) 2-mcthoxy-isobutyl-isonitrile (scstamibi), makes MATERIALS AND METHODS gated single-photon emission computed tomography A normal database consisting of 20 subjects (10 normal (SPEer) of myocardial perfusion realistic. Technctium-99m volunteers approved by the University Ethics Committee sestamibi has been reported to have minimal redistribution, and 10 patients, determined to be free of cardiac disease by high myocardial extraction, and high photon flux (1-J). Prior coronary ), II men and 9 women, mean age 51.2 to the introduction of sestamibi, the usc of thallium-201 yr ± 8.1, range 37 to 68, served as a reference population. eotTI) for imaging myocardial perfusion was restricted The lower limits of the normal uptake distribution (for rest mainly to traditional, nongatcd tomography, due to the lower and stress) were established for each myocardial level (bas­ administered dose necessitated by its longer half-life and a al, mid, and apical) and segments by computing the mean uptake and subtracting two s.d. For reprints comact: Finn Mannting, MD, PhD. Nuclear Medicine Normal databases were established and defined for the Section, Department of Clinical Physiology, University Hospital. S-751 X Uppsala. Sweden. following protocols: traditional nongatcd rest/stress MIBI

VOLUME 21, NUMBER 1, MARCH 1993 13 GATED TOMOGRAPHY

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_() 180" Rotation, 32 Angles '1': -·· 8 Frames I Angle 32 S I Frame LPO 45" +---4-­ FIG. 1. Schematic representation of gated tomography technique. Each of the 32 ac­ quisition frames is divided into eight tempo­ ·-·L ral frames (squares), each representing 1/8 Crcle n•• of an R to R interval. (R to R Interval) for 1-day or 2-day protocols and gated sestamibi SPECT the MSE operating system (equal to the TSX operating sys­ (diastolic) studies. tem) on a PDP 11/73 DEC computer (Digital Equipment Stress and rest gated SPECT was attempted in 53 consec­ Corp, Maynard MA), controlled the gated SPECT acquisi­ utive patients referred for signs or symptoms of coronary tion. The patients were connected to an ECG monitor that artery disease (CAD). One patient was excluded due to high was interfaced to the computer. Special care was taken to subdiaphragmatic activity and we were unable to obtain establish a stable ECG for each patient. Gate tolerance was gated SPECT in two patients due to arrhythmias. The study set at 15o/c. Once a stable ECG pattern was obtained, gate patient population consisted of 50 patients (31 men and 19 interval time was calculated. This calculation was based on women) with signs or symptoms of CAD. Mean age for the a 16 beat average. Data were acquired over a 180° rotation patient population was 55.3 yr ± 12.3, range 13 to 74. for a total of 32 angles, 32 sec, and 8 temporal frames/angle (Fig. 1), in a 64 x 64 matrix. All studies were acquired on the Stress Testing same (SX 300, Picker International, High­ Symptom limited, upright, bicycle ergometry was per­ land Heights, OH) using a high resolution, hexagonal, par­ formed according to a standard protocol. The patients were allel-hole collimator. asked not to take any cardiovascular medication 24 hr prior Immediately following the rest SPECT, the patients were to examination. Baseline ECG, heart rate, blood pressure, stressed according to the protocol described earlier. At peak and respiration rate were recorded at rest, immediately be­ exercise, 24 mCi of YYmTc-sestamibi was injected. Fifteen fore start with the patient in position on the bicycle, and min after completion of stress testing, all patients ingested a every 2 min during exercise. Workload started at 20 Wand high fat snack. Stress sestamibi SPECT imaging began was increased by 20 W, every 2 min. The test was terminated 1-hour postinjection, using the same acquisition protocol at maximal exertion or dyspnea, or at maximal tolerable used during the rest SPECT sestamibi acquisition. chest pain, or if significant ventricular arrhythmia devel­ When interfering subdiaphragmatic activity was observed oped. during patient positioning, start of data acquisition was de­ layed for 10 to 15 min. These patients were taken off of the Gated SPECT Imaging Protocol imaging table and instructed to walk in the corridor. This All patients were examined according to a one-day rest/ was done in an attempt to allow high, interfering hepatic or stress protocol (19). Six mCi of YYmTc-sestamibi was injected bowel activity to move away from the cardiac region. at rest. The patients were asked to ingest a high fat meal -15 All studies were filtered with a 2-D, count-adaptive, FFT, min after injection. Rest SPECT imaging began 1-hr postin­ Metz filter (20) using an array processor (AP 400, Analogic jection. A 20o/c symmetrical window was centered at 140 Corp., Wakefield, MA). Reconstruction was performed on keY. A commercially available software, Gated SPETS (Nu­ the PDP 11/73 DEC computer using the reconstruction part clear Diagnostics AB, Stockholm, Sweden), running under of the Gated SPETS software. All gated sestamibi SPECT

14 JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY studies were reconstructed using eight temporal frames. At­ anterior wall endocardium to the inferior wall endocardium tenuation correction was performed after body outline defi­ at the widest dimension of the LV cavity. Also, a line was nition (21). Software zoom (x2) was applied during the re­ drawn from the septal endocardium to the lateral wall endo­ construction. Reconstruction was performed by filtered cardium. The mean of the two ventricular cavity measure­ backprojection (ramp filter). Standard transverse and true ments was calculated for determination of both the nongated cardiac sagittal (long-axis) and coronal (short-axis) slices and diastolic LV cavity size in nongated and diastolic stud­ were created after individual determination of the heart axis. ies. The number of useful coronal slices, defined as those Formatting slices containing LV cavity, was determined for the non­ The 256-frame, reconstructed coronal and sagittal studies gated and diastolic studies. The coronal studies were dis­ (each study, 32 spatial frames x 8 temporal frames) were played on a high resolution color monitor. Nongated and formatted into various types of studies. The formatted data diastolic studies were viewed separately. The same color were saved as new, additional patient studies. For this in­ table was used for viewing both nongated and diastolic stud­ vestigation, the reconstructed sagittal and coronal data for ies. The number of coronal slices with identifiable LV cavity stress and rest studies were formatted as follows. was determined. Traditional nongated study. The eight temporal frames All values are given as a mean ± s.d. unless otherwise of the R to R interval for the coronal studies were summa­ indicated. Relationships between variables were analyzed by rized to form "compressed" coronal slices. This was done linear or nonlinear regression analysis, a p value <0.05 was for each of the 32 slices contained in the coronal study. A considered significant. The paired t-test was used to com­ traditional, nongated, 32-frame coronal study was created pare differences between traditional nongated and diastolic and saved. studies. A p value <0.05 was considered significant. Diastolic studies. A study consisting of "pure" diastolic coronal frames was extracted. Diastolic frames were defined RESULTS as frames with constant or fixed LV cavity size. A mid­ Approximate imaging and processing times for gated ventricular coronal slice was selected and the corresponding SPECT studies are compared to times for standard SPECT eight temporal frames were displayed. The temporal frames studies and summarized in Table 1. Imaging and filtering/ with constant LV cavity were selected. A computer algo­ reconstruction takes -1.5 and 2.5 times longer, respectively. rithm automatically did this for the remainder of the 32 Overall, it takes about twice as long to acquire and process coronal reconstructed slices and saved this as a new study, a gated sestamibi SPECT study as it takes to perform a consisting of coronal slices representing the diastolic phase standard nongated sestamibi SPECT study. These times will of the heart cycle. vary depending upon the type of camera and computer used. Dynamic studies. Spatial and temporal coronal data The times reported for gated SPECT reflect filtering and were added together to form a dynamic (cine) study. Cines reconstruction performed using an array processor. were produced by adding a number of spatial coronal slices The space requirements for gated SPECT and traditional to evenly divide the myocardium into three portions, repre­ SPECT are summarized in Table 2. Each gated SPECT senting basal, mid, and apical thirds. Also, a midsagittal acquisition file is about four times the length of a standard long-axis cine was created by selecting the midventricular nongated SPECT file, thus occupying four times as much slice and adding one slice on either side. These four new storage space. Each gated SPECT reconstructed study oc­ dynamic studies were saved and later displayed to assess cupies approximately two megabytes, which is about six wall motion. times more computer storage media than standard SPECT RV target-to-background ratios were calculated from cor­ reconstructed studies. The nongated and diastolic formatted responding midventricular coronal slices selected from the studies used in this investigation are 256 kilobytes each and nongated and diastolic stress studies. A 2 x 2 pixel region of interest (ROI) was drawn over the area of the RV containing the maximum counts. The same size ROI was used for back­ TABLE 1. Summary of Imaging and Processing ground determination. The background ROI was carefully Times for Traditional SPECT and Gated SPECT placed away from the myocardial area, 3 to 5 pixels laterally Techniques from the RV. Mean counts per pixel for the RV and back­ ground ROis were recorded and RV/background ratios com­ Gated % puted. Similarly, a ROI was drawn around the pixels con­ SPECT SPECT Incr. taining the maximum activity in the LV. LV/RV ratios and Imaging Time 20 min 30 min 50 LV/background ratios were calculated for nongated and di­ Filtering/Reconstruction 10 min 25 min 150 astolic stress studies. All ratios were calculated for normal Formatting N/A 8 min Total 30 min 63 min 110 subjects and for patients with CAD. Corresponding midventricular slices from the nongated SPECT = traditional sestamibi SPECT; % lncr. = percent increase in time from SPECT to gated SPECT; N/A = not applicable to and diastolic studies were selected for determination of LV traditional SPECT studies. cavity size. A line was drawn across the cavity from the

VOLUME 21, NUMBER 1, MARCH 1993 15 TABLE 2. Summary of the Number of Frames and Diastolic images appeared sharper and more distinct than Lengths for Traditional SPECT and Gated SPECT nongated images (Fig. 2). Better contrast was noted on the Studies diastolic images. This was confirmed by the results for ven­ tricular target-to-background analysis (Fig. 3). RV/back­ SPECT Gated SPECT ground ratios were significantly higher in diastolic than in frames length frames length nongated studies when all subjects were evaluated (p < 0.01) (Fig. 3A). Significant differences were also found when the Acquisition 64 0.50 MB 256 2MB Reconstructed 32 each 256 each group was divided into two groups: normal subjects and slices (C+S) 0.50 MB (c+s) 4MB patients with CAD, p < 0.01 and p < 0.05, respectively. 64 256 There were no significant differences between nongated (trans) 0.50 MB (trans) 2MB and diastolic LV/background ratios (all p > 0.05) (Fig. 3B). Formatted N/A 32 0.25 MB RV !LV ratios were significantly different between non­ Dynamic N/A 8 0.06MB Total 1.5MB 8.31 MB gated and diastolic studies, p < 0.001, p < 0.01, p < 0.05 for all subjects, normals subjects, and patients with CAD, re­ SPECT = traditional sestamibi SPECT; c, s, t = coronal, sagittal, and transversal, respectively; MB = megabyte; N/A = not applicable spectively (Fig. 3C). to traditional SPECT studies. The LV cavity was larger in diastolic studies than in non­ gated studies (p < 0.001) and extended further in the apical direction, yielding more useful coronal slices with ventricu­ lar cavity (p < 0.001). A significant inverse nonlinear relation the dynamic studies are 64 kilobytes each. However, these between LV cavity size in nongated studies and increase in figures will vary depending upon the individual department's number of useful coronal slices with ventricular cavity in formatting needs. diastolic studies was found (r = -0.71, p < 0.001) (Fig. 4A). Overall, a gated SPEer study (acquisition, reconstruc­ This was particularly evident in patients with small hearts tion, diastolic study, and four dynamic studies) requires ap­ (Fig. 4B). proximately 5.6 times more storage media than a standard SPEer study. DISCUSSION Three of the seventy-three subjects (4%) were not in­ The results of this investigation indicate that the gated cluded in this investigation. We were unable to perform SPEer technique may be easily and routinely applied to gated SPEer on two of the patients due to arrhythmias sestamibi imaging using conventional computers found in during the acquisition. One patient was excluded due to many nuclear medicine departments. Sestamibi's high myo­ extreme and persistent, interfering subdiaphragmatic activ­ cardial extraction fraction and minimal redistribution (1-3) ity, despite delaying the start of acquisition for 15 min. Two combined with technetium's energy and counting statistics patients had high subdiaphragmatic activity. Imaging of make 99mTc-sestamibi well suited for tomography. Several these patients was delayed 15 min, after which subdiaphrag­ investigators have used gated sestamibi and planar imaging matic activity was considerably less and did not interfere (9-11). The data from these investigations yielded clinically with analysis. diagnostic information about wall motion (9-11). One study

FIG. 2. Corresponding midventricular coronal slices from (A) traditional nongated study and (B) diastolic frames from gated study in a normal subject. The RV is clearly seen on the diastolic images (arrow). Also, the LV cavity appears larger on the DIA image .

16 .JOURNAL OF NUCLIIAR M•DICIN• nCHNOLOGY A 8 RV TARGET TO BKG IUTIO LV TARGET TO BKG RATIO

10 NS NS ~·~------~p < 0.01 p < 0.01 p <0.05 TN; .... w---...1-----T-T....--1 ••• ~ l _l ~ ~~ ~ l .• w--..1-+-d+----r..L------l:J::.i--:::j:~l::-1 ••• ~ .... r- = r-- r-- :z.t ~ &ft - I - ... : - ,_ ~ ~--..j l.t r- r-- ~

I- 'I._ 0 .... ·-NON DIA NON DU NO."tl DIA NON DIA NON DIA - NON DIA ALL SUBJ. NL CAD ALL SUBJ. NL CAD - c RV I LV IUTIO

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t.l

o.z

t.l FIG. 3. Mean and standard error bars for ventricle ratios for (A) RV/background (BKG) and (B) LV/BKG and (C) RV/LV for all sub­ jects (ALL SUBJ.) and subdivided into normal subjects (NL) and "m'o DIA NON DIA NON DIA patients with coronary artery disease (CAD). Significance is noted ALL SUBJ. NL CAD above standard error bars. NS = not significant. has even eluded to the possibility that the gated technique Imaging time for gated SPECT was approximately 1.5 applied to myocardial perfusion imaging may detect smaller times longer than for a traditional SPECT study (Table 1). myocardial perfusion defects (9). Few studies have explored Part of this extra time is devoted to establishing a proper the possibility of performing gated SPECT with either 201 TI ECG signal. Proper placement of electrodes and strict con­ 99 or mTc-sestamibi (12-17), but have reported that handling trol of the ECG signal are necessary. This is very important the data was cumbersome and required long processing because no data are accepted during periods of cardiac ar­ times. One recent paper (17) applies three-dimensional mo­ rhythmia or disturbances in the ECG signal. Such interrup­ tion and perfusion quantification techniques to gated SPECT tion of data acceptance prolongs the acquisition time. The studies and the study's preliminary results indicate that im­ computer is programmed to accept a specified time per angle portant clinical information may be obtained from three­ (in our case 32 sec/angle). However this is 32 sec of ECG dimensional images obtained from gated SPECT. However, data that fit the established gate interval time. Once a stable the authors used dedicated computers and multidetector heart rhythm is obtained on the ECG gating system, acqui­ gamma cameras for acquisition and processing of gated sition of data proceeds. SPECT data. We performed gated SPECT using standard Gated SPECT filtering and reconstruction takes -2.5 nuclear medicine computers and a single detector camera. times longer than a standard SPECT study (Table 2). Gated Gated SPECT was applied to 50/53 consecutive patients referred for signs or symptoms of CAD with a success rate of SPECT filtering and reconstruction of transversals was per­ greater than 95%. We were unable to perform gated sesta­ formed with the assistance of an array processor. Our tradi­ mibi in two patients due to arrhythmias. One patient was tional SPECT filtering uses the array processor, but the excluded because of extremely high subdiaphragmatic activ­ reconstruction software does not. ity that interfered with the evaluation of myocardial perfu­ Formatting of gated SPECT reconstructed studies was sion. This is a consequence of in vivo kinetics of sestamibi fast. For the purpose of this investigation, we formatted the and not related to the gated SPECT technique. We have stress and rest reconstructed coronal studies to form dia­ learned that high interfering liver or bowel activity may be stolic and nongated studies and dynamic studies at three circumvented or lessened by delaying the start of data ac­ myocardial levels. Formatting of all data was easily accom­ quisition for 10 to 15 min. This was successful in two of the plished in -5 to 8 min. This time could be even faster three patients where bowel activity was considered high. depending upon individual department formatting needs.

VOLUME 21, NUMBER 1, MARCH 1993 17 A that high RV/background ratios found in diastolic studies were not due to the physiologic effect of . One could 88 (f) n 70 theorize that because the blood content in the lungs is lower LLI = during diastole, there would be less counts in the surround­ (.) I" = -0.71 ing tissue on diastolic images, and this may account for the ....J68 p < 0.001 increased RV/background ratios found in diastolic images. If -(f) this theory was true, the LV /background ratios on diastolic ....J :::::> studies should also be higher. This was not the case, we LL. found no significant differences between LV/background ra­ LLJ48 (f) tios for diastolic and nongated studies (p > 0.05) (Fig. 38). :::::> One possible explanation for the clearer visualization of the RV in the diastolic studies is that vigorous RV move­ •(.)28 ments during the cardiac cycle lead to a blurred RV appear­ :z: ance on traditional images, while in gated SPEer, diastolic frames are extracted, thus minimizing the blurring effect. -;-.: The moving object (heart) is formatted and presented as a e 0 fixed image representing the myocardium during the phase of e 4 9 12 16 28 the heart cycle with the least movement. LV CAVITY SIZE

18 .JOURNAL OF NUCLUR M.DICIN. nCHNOLOQY technetium-99m-hexakis-2-methoxy-2-methylpropyl-isonitrile. Circula­ WN. Assessment of systolic thickening with thallium-201 ECG-gated tion 1988;77:491-498. single-photon emission computed tomography: a parameter for local left 3. Wackers FJT, Berman OS, Maddahi J, et al. Technetium-99m hexakis ventricular function. J Nucl Med 1'191 ;32: 1496-1500. 2-methoxyisobutyl isonitrile: human biodistribution, dosimetry, safety, 13. Kahn JK. Henderson EB, Akers MS, et al. Gated or ungated tomographic and preliminary comparison to thallium-201 for myocardial perfusion im­ perfusion imaging with technetium-99m RP-30A: comparison with Tl-201 aging. J Nucl Med 1989;30:301-311. tomography in coronary artery disease. (Abstract.) JAm Col/ Cardiol 4. Alderson PO, Wagner HN, Gomez-Moeiras JJ, et al. Simultaneous de­ 1988;11:31A. tection of myocardial perfusion and wall motion abnormalities by cine­ 14. Larock MP, Cantineau R, Legrand V, Kulbertus H, Rigo P. 99mTc-MIBI matic Tl-201 imaging. Radiology 1978;127:531-533. (RP-30) to define the extent of myocardial ischemia and evaluate ventric­ 5. Garty I, Kardontchik A. Study of ECG-gated thallium-201 myocardial ular function. Eur J Nucl Med 1990;16:223-230. ; is imaging time a limiting factor' Eur J Nucl Med 1984;9: 15. Sochor H, Huber K, Probst P, et al. Assessment of myocardial perfusion 173-176. and wall motion by the new perfusion agent Tc-99m MIBI and SPECf. 6. McKillop JH, Fawcett HD, Baumert JE, et al. ECG gating of thallium-201 (Abstract.) Eur Heart J 1'188;'1:1-364. myocardial images: effect on detection of ischemic heart disease. J Nucl 16. Grucker D. Florentz P. Oswald T, Chambron J. Myocardial gated tomo­ Med 1981;22:219-225. scintigraphy with 99Tc m-methoxy-isobutyl-isonitrile (MIBI): regional 7. Hamilton GW, Narahara KA, Trobraugh GB, Ritchie JL. Williams DL. and temporal activity curve analysis. Nucl Med Commun 1989;10:723- Thallium-201 myocardial imaging: characterization of the ECG-synchro­ 732. nized images. J Nucl Med 1978;19:1103-1110. 17. Faber TL, Akers M. Peshock R, Corbett JR. Three-dimensional motion 8. Hurwitz G, Schwab M, MacDonald AC, Driedger A. Quantitative anal­ and perfusion quantification in gated single-photon emission computed ysis of myocardial ischaemia on end-diastolic thallium 201 perfusion im­ tomograms. J Nucl Med 1991;32:2311-2317. ages. EurJ Nucl Med 1990;17:257-263. 18. Mannting F, Morgan MG, Maripuu E, lsraelsson A. Gated vs non-gated 9. Maisey MN. Mistry R, Sowton E. Planar imaging techniques used with SPECf for quantitative assessment of myocardial perfusion with 99m-Tc technetium-99m sestamibi to evaluate chronic myocardial ischemia. Am J sestamibi. (Abstract.) r:urJ Nucl Med 1'191;1S:652. Cardiol 1990;66:47E-54E. 19. Taillifer R, Laflamme L, Dupras G, et al. Myocardial perfusion imaging HI. Najm YC. Timmis AD, Maisey MN. et al. The evaluation of ventricular with 'l'lm-Tc-methoxy-isobutyl-isonitrile (MIBI): Comparison of short function using gated myocardial imaging with Tc-'Nm sestamibi. Eur and long time intervals between rest and stress injections. Eur J Nucl Med Heart J 1989;10:142-14R. I 'ISS; 13:515-522. II. Marcassa C, Marzullo P, Parodi 0, Sambuceti G. L ·Abbate A. A new 20. King M. Doherty PW. Schwinger RB. Fast count-dependent digital filter­ method for noninvasive quantitation of segmental myocardial wall thick­ ing of nuclear medicine images: concise communication. J Nucl Med ening using technetium-'19m 2-methoxy-isobutyl-isonotrile 'cintigraphy­ 1'183;24: 1039-1045. results in normal subjects. J Nucl Med 1'1'10;31: 173-177. 21. Larson SA. Gamma camera emission tomography. Acta Radiologica 12. Mochizuki T. Murase K. Fujiwara Y. Tanada S, Hamamoto K, Tauxe 1'180;(suppl )363:30-32.

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