3.0 Material and Methods : 3.1 Study Design A cross sectional analytical study was carried out. 3.

2 Place of study The study was carried out at the then Institute of Nuclear Medicine & Ultrasound (INMU), which is currently known as National Institute of Nuclear Medicine & Allied Sciences (NINMAS), Bangladesh Atomic Energy Commission (BAEC), Bangabandhu Sheikh Mujib Medical University (BSMMU) campus, Shahbag, Dhaka. 3.4 Period of study The study was carried out over a period of one year from July 2011 to June 2012. 3.5 Study Subjects The study population consisted of patients of CAD referred for GSMPI. 3.6 Sample size calculation The sample size was calculated for discordance rate () of 0.10 and tolerance probability () of 95% for agreement of two measurement methods assuming no discordant pair of measurement allowed (k=0). Appropriate formula was used (Liao JJZ 2009) which is given in appendix C. 3.7 Sample size The calculated sample size was 29 but a total of 27 patients could be included.

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3.8

Sampling technique The sampling technique was non random sampling which was used as per selection criteria.

3.9

Selection criteria

A. Inclusion criteria Adult male and female with known CAD under medical treatment. Adult male and female with known CAD after coronary revascularization.

B. Exclusion criteria History of cardiogenic shock Left ventricular failure Unorganized thrombus in LV cavity Recurrent chest pain unresponsive to anti ischemic medication Concomitant non cardiac illness that would require hospital admission Valvular heart disease Presence of intracardiac shunt Cardiac arrhythmia Implanted pacemaker or defibrillator device Renal failure Intervening cardiac events in between two radionuclide studies Pregnancy Inability to give informed consent.

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3.10

Ethical implication Prior to commencement of this study the Academic Committee of the Institute of Nuclear

Medicine & Ultrasound (INMU) approved the research protocol. Informed written consent (Appendix A) in Bangla language was obtained from all patients after full explanation of the nature, purpose, benefit and potential risk of procedure needed for the study. The participants preserved the right to withdraw themselves from the study at any point of time. 3.11 Some important variables

Dependent variable LVEF

Independent variable 3.12 EDV ESV Infarct size Wall motion abnormality Study procedure

Twenty seven patients with CAD referred to INMU for GSMPI at various stages of clinical workup constituted the study population. All patients underwent GSMPI and GSBPI within a span of three to seven days. Left ventricular parameters were measured in rest phase of one day stress-rest GSMPI and compared against those obtained from GSBPI performed at rest on a separate day.

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3.12.1 Pre procedure counseling & evaluation of patients: Face to face interview of patients was a part of data collection. All patients were clinically evaluated and following information pertinent to performing the procedure were noted: specific areas to be reviewed including the indication(s) for testing, symptomatology, cardiac risk factors, prior diagnostic or therapeutic cardiac procedures and medications. These were done to find the clinical objective for referral and to assess appropriateness of the referral by checking patients clinical history, previous pertinent investigations, coronary intervention note, and hospital discharge note. The purpose was also to check for confounding factors that may produce misinterpretation, to check for other associated pathology that may require some modification of the requested protocol and finally to select an optimum stress protocol. The session also included discussion on possible risk and symptoms, assurance, patient preparation and role of patient and attendant during the procedure. Cardiac rhythm was noted in all patients to exclude arrhythmia and chest auscultation for lung crepitations to exclude possibility of LV failure was done. Consultation of clinical cardiologist was necessary in few instances. 3.12.2 Supervision of quality control steps of imaging: The nuclear cardiology procedures GSMPI and GSBPI consist of multiple steps that involve incorporation of multiple level skills and requires adherence to quality control recommendations. Supervision of quality control in every steps of performing GSMPI & GSBPI was done by:

ensuring preparation of adequate amount of dose for the patients considering the length of time gap between injections, decay time and loss of activity in syringe.

supervision of quality control of prepared radiotracer.

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ensuring adequate time interval between injection of pyrophosphate and free pertechnatate in patients who would undergo GSBPI.

minimizing patient waiting time in echo lab and treadmill lab in department of cardiology during stress phase of GSMPI.

monitoring preparation of patients at stress lab which included placement of i.v. channel, chest electrodes, calculation of target heart rate, calculation of dose of dobutamine or adenosine according to patients weight and preparation of dobutamine in proper dilution with normal saline.

monitoring of patients during stress which included monitoring of symptoms, heart rate, blood pressure, ECG changes (ST, T changes and appearance of PVC)

deciding end point of treadmill or pharmacological stress according to symptoms, heart rate, blood pressure, and ECG changes.

administration of radiotracer at peak heart rate with monitoring for tracer stagnation in i.v. channel and contamination.

deciding management of patients during an emergency which included administration of atropine, adrenaline, oxygen inhalation and shifting of patients to cardiac emergency room or to CCU.

deciding repetition of acquisition by monitoring quality of image which included: monitoring of motion artifact, extra-cardiac activity, inferior wall artifact, breast attenuation and accuracy of gating.

deciding attempts for correction of bowel artifact when necessary. deciding dose of sublingual glycerine trinitrate and monitoring of patients after stress acquisition and before rest acquisition for symptoms, heart rate and blood pressure

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monitoring ECG changes (ST, T changes and appearance of PVC) during gated acquisition of stress & rest images. Image acquisition for both the GSMPI and GSBPI were conducted with Siemens E cam dual head gamma camera (Milwaukee, Wisconsin, USA). Best possible attempts were made to maintain adherence with the guidelines for performing radionuclide imaging of cardiac function by renowned authorities. Quality control checks for radiopharmaceuticals and periodic quality control of gamma

camera were done under supervision of an experience medical physicist. All hot lab procedures, acquisition and processing of raw images were done with help of three experienced nuclear medicine technologist. All interpretations were done under supervision of two experienced nuclear medicine specialist physicians.

3.12.3 Protocols for performing GSMPI For this study, the LV parameters obtained from rest phase images of GSMPI of the routine one day stress-rest protocol were considered. An outline of protocol that was followed is presented here: Patient preparation for GSMPI: Patients fasted for 4 hours. No cardiovascular medication was discontinued before the study. Morning dose insulin or oral hypoglycemic agent (OHA) in case of diabetic patients was discontinued on the day of study. An intravenous line was placed in a forearm vein using a 20 gauge intravenous channel attached to a three channel adapter. This was used for spillage free administration of radiotracer while continuing infusion of isotonic normal saline during treadmill exercise stress or during injection of dobutamine with a syringe pump in cases of inotropic stress. 32

Radiotracer for GSMPI: Tracer was injected as a compact bolus with a high specific activity.
99m

Tc labeled sestamibi

was used. Five milliliters of freshly eluted

99m

Tc pertechnetate was injected into a vial of

Sestamibi kit. Each vial contained 0.12mg (Cu/MIBI/4BF4) Methoxy-isobutyl-isonitrile-Cu(II)tetrafluoroborate as active substance and stannous (II) chloride dehydrate, tetrasodium

pyrophosphate decahydrate, L-cysteine hydrochloride monohydrate, glycine & sodium chloride as excipient. Vial was then heated in a boiling water bath for 10 minutes and was allowed to be cooled in room temperature for another 10 minutes. Optimum tagging of kit was confirmed with chromatography by a radiochemist before administration to patient.

Image acquisition parameters for GSMPI: SPECT acquisition with ECG gating at rest phase was done 45-60 minutes after rest injection

of 25 mCi tracer on the same day following the post-stress scan (done 15-30 minutes after stress injection of 10mCi of tracer). Acquisition of SPECT images were done with the double-headed SPECT scintillation camera (Siemens E cam) with detectors placed 76 to each other. Low energy all purpose (LEAP) collimators with parallel holes were used with 1.45 zoom. A symmetric 15% energy window around the 140 keV
99m

Tc photo-peak and was set. Data was

stored in 64 x 64 matrices (pixel size 6.59cmm, 21-27 slices in short axis). ECG gating was done with three limb leads and acquisition was set to eight frames per R-R interval (about 153ms/frame). Framing method configuration was with 60% width and forward backwards by thirds. The auto-centre set to average of 10 beats with pick bin of 10 bits and auto-tracking on. Reject PVC (Premature Ventricular Contraction) beat mode was set with PVC threshold of 300msec. Rotation of camera heads were set to counter clock wise with starting angle at 52. The

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detectors took 32 views over 104 of rotation in a non circular orbit using a step-and-shoot method, progressing from 45 right anterior oblique to 45 left posterior oblique projections. During acquisition the patient was in supine position with head out orientation. Acquisition time was 20 seconds per projection (about 450-750 kcounts/detector/view) requiring about 14 minutes per patient per scan. Butterworth filtering with cutoff of 0.5 cycles/sec and order 7 was used. 3.12.4 Protocols for performing GSBPI Patients who underwent GSMPI and met the selection criteria were scheduled for rest only GSBPI to obtain LV parameters from rest phase images of GSBPI. An outline of protocol that was followed to perform GSBPI is presented here: Patient preparation for GSBPI: Preparation of patients was similar to GSMPI preparation with fasting for 4 hours without discontinuing cardiovascular medication. Patients were refrained from the use of morning dose insulin or OHA in case of diabetic patients on the day of study to avoid hypoglycemia. Radiotracer for GSBPI: For in vivo labeling
99m

Tc stannous pyrophosphate in vivo kit was used. Each vial of kit

contained sodium pyrophosphate 60 mg, stannous chloride dehydrate 4.5mg and L-acidum ascorbicum 0.1mg. After 20 minutes of intravenous injection of 15-20 mg pyrophosphate, 15-20 millicurie of
99m

Tc-pertechnatate was injected in a different vein of contra-lateral arm.

Immediately thereafter gated blood pool SPECT images was acquired.

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Image acquisition parameters for GSBPI: Standard protocol was followed for image acquisition which was same in all aspects with the

GSMPI acquisition protocol as mentioned above, except in the frame rate with ECG gating. For three limb leads ECG gating the acquisition was set to 16 frames per R-R interval that took about 45-48 msec/frame and 300-350 kcounts/detector/view and the time of scan took 12 minute per patient per scan. Butterworth filtering with cutoff of 0.5 and order 7 was used. 3.13 Measurement of left ventricular ejection fraction (LVEF) Measurements of LVEF were fully automated and software generated. Raw SPECT images needed to be processed first. Image processing and interpretation of processed images with software were done at dedicated work station (Siemens e.soft). All image data were stored in Dicom format in Syngo medical imaging system provided by Siemens medical systems. Final result pages were saved both in Dicom format and jpeg format. 3.13.1 Processing and interpretation of GSMPI images: During processing a second check for quality of images were done. Adequacy of target to background ratio, motion artifact, extracardiac activity, inferior wall artifact, breast attenuation and accuracy of gating were checked once again. Processing of GSMPI images included checking for slice orientation, concordance of stress & rest slices of LV, definition of mid LV cavity & mid ventricular slices in short axis (SA), vertical long axis (VLA) & horizontal long axis (HLA) sections, masking of area outside LV, centering axis of LV and finally a visual check if any part of LV image is getting truncated by falling outside the region of interest (ROI) in gated images.

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For quantitative assessment of LVEF, EDV & ESV, 4D-MSPECT v4.2 software (Invia, LLC 2007) provided by Siemens medical solutions, Inc and Toshiba Corporation was applied to process & interpret raw GSMPI images. Perfusion data, measurements of LV volumes, LVEF and LV wall motion were obtained from GSMPI images. 3.13.2 Processing and interpretation of GSBPI images: Assignment of ROI for LV and right ventricle (RV) over raw image slices were done during processing of GSBPI image data. Processing was automated. GSBPI data was quantified to determine LVEF, EDV and ESV with QBS 2007 (Quantitative Blood-pool SPECT) Cedars cardiac quantification software 6.5.9.1 (Berman et al. 2007) provided by Siemens medical systems. Measurements of LV volumes, LVEF and LV wall motion were obtained from GSBPI images. 3.14 Data collection

Data were collected according to pre formatted data collection sheet. The data collection sheet was developed to obtain relevant information regarding clinical history, ongoing medications, pertinent investigations and LV parameters as measured by GSMPI & GSBPI. 3.15 Data processing and analysis

After the data were collected, editing, coding and entry were done manually. Statistical analysis of data was done using IBM SPSS statistics (Statistical Package for Social Sciences) version 20 (IBM corporation 2011) for windows. In SPSS, data were analyzed by two stages. Initially descriptive statistics such as frequency, percentage, mean, SD, range were calculated for the basic demographic characteristics and LVEF of the study patients. For analysis of agreement

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between GSMPI and GSBPI for measurements of LVEF at rest, Bland-Altman plots were constructed to observe if the differences of LVEF plotted against respective geometric means of LVEF falls within two standard deviations (SD) of difference. In this study, in addition to an overall agreement analysis, further assessment of agreement were done in the study patients among different ranges of LVEF, EDV, ESV, infarct size and wall motion. Since both GSMPI and GSBPI could measure LVEF, EDV and ESV, categorization was done twice according to measurement of each technique. Cut offs for LVEF ranges and cut offs for LV dimensions on basis of EDV & ESV limits for SPECT measurements were adopted from available published literature. LVEF was categorized as less than 35%, 35 to less than 55% and 55% or greater. EDV was categorized as below 85ml, 85 to 140 ml and above 140ml. ESV was categorized as below 15ml, 15 to 60 ml and above 60 ml. While assessing agreement in absence or presence of infarct, quantitative estimation of fixed defect on 17 LV segments were assigned in to qualitative categories. For qualitative infarct size assumption, fixed defect involving up to five out of 17 LV myocardial segments was considered as small LV infarct. Fixed perfusion defect size of more than five segments was considered as large infarct. For assessment of agreement in absence or presence of RWMA, summed motion score (SMS) at rest obtained from GSMPI images were qualitatively assigned in to three categories. A SMS at rest of up to 10 were considered as normal if there was no RWMA that is visually detectable in cine views of 4D reconstruction images. A resting SMS of 10 to 25 was considered as mild to moderate RWMA while SMS at rest more than 25 was considered as gross RWMA. Statistical interpretations were taken into account after consultation with an experienced statistician.

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3.16

Operational definitions

Coronary artery disease: Coronary artery disease (CAD) is a complex disease that causes reduced or absent blood flow in one or more of the epicardial coronary arteries that encircle and supply the heart (Pearlman 2012). Disease of coronary arteries is almost always due to atheroma and its complications, particularly thrombosis. Occasionally, the coronary arteries are involved in other disorders such as aortitis, polyarteritis and other connective tissue disorders.It is estimated that coronary heart disease (CHD), a result of CAD, will be the major cause of death in all regions of the world by 2020. The disease may be focal or diffuse. Apart from rare congenital anomalies, CAD is usually a degenerative disease, uncommon as a clinical problem before the age of 30 years and common by the age of 60 years (Boudi & Ahsan 2012). One in four people will have a heart attack. The first recognized symptom may be death. The term coronary is derived from crown, referring to the way these arteries sit on the heart. (Pearlman 2012)

End diastolic volume: During diastole, normal filling of the ventricles increases the volume of each ventricle to about 110 to 120 ml. This volume is called the end-diastolic volume. when large amounts of blood flow into the ventricles during diastole, the ventricular end-diastolic volumes can become as great as 150 to 180 ml in the healthy heart.

End systolic volume: As the ventricles empty during systole, the volume decreases about 70 ml, which is called the stroke volume output. The remaining volume in each ventricle, about 40 to 50 ml, is called the end-systolic volume. When the heart contracts strongly, the endsystolic volume can be decreased to as little as 10 to 20 ml in the healthy heart.

Left ventricular ejection fraction: The fraction of the end-diastolic volume that is ejected is called the ejection fractionusually equal to about 60 percent. 38

Myocardial Infarction (MI): Myocardial infarction is the irreversible necrosis of heart muscle secondary to prolonged ischemia (Zafari 2013). It is caused by acute occlusion of a coronary artery due to plaque rupture or erosion with superimposed thrombosis.

Heart failure: Heart failure develops when the heart, via an abnormality of cardiac function (detectable or not), fails to pump blood at a rate commensurate with the requirements of the metabolizing tissues or is able to do so only with an elevated diastolic filling pressure (Dumitro 2013). Myocardial dysfunction occurs due to ischemia or infarction.

Cardiomyopathy: Cardiomyopathy is the measurable deterioration of the function of the myocardium for any reason, usually leading to heart failure. The most common form of cardiomyopathy is dilated cardiomyopathy which is a progressive disease of heart muscle that is characterized by ventricular chamber enlargement and contractile dysfunction with normal left ventricular (LV) wall thickness.. Dilated cardiomyopathy is the third most common cause of heart failure and the most frequent reason for heart transplantation (Goswami et al. 2013).

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