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METHODS Systems Overview Dual energy x-ray absorptiometry (DEXA) measurements of bone mineral content (BMC) and bone mineral density (BMD) were performed with the Hologic Inc. (Waltham, MA) quantitative digital radiographic (QDR) 1000W and QDR 2000 bone densitometers. The QDR 1000W system employs a dual pulsed x-ray source mounted under the subject which produces low and high energy voltages of 70 kvp and 140 kvp, respectively. These energies are pre-selected and fixed to maximize the separation between bone and soft tissue compartment, and within the soft tissue compartment, between fat and lean tissue. The tightly collimated x-ray beam, generated from a tube in the under-table portion of the C-arm, filters through a rapidly rotating calibration wheel prior to patient entry. The photons, having passed through the patient and the calibration wheel, are picked up by a crystal detector located in the over-table arm. The calibration wheel is composed of alternating radiopaque segments equivalent to that of tissue, bone and air. At each pixel, the high and low energy photon signals passing through the patient are compared to known bone and tissue values derived from the calibration wheel. This information is received and interpreted by the detector which converts it into current. An analog to digital (A/D) converter translates the current into a digital signal directed to the computer. The system software integrates the signal information and creates a screen image. The values for area scanned in cm2, bone mineral content (BMC) in gms of calcium hydroxyapatite, and bone mineral areal density (BMD) in gms/cm2 are computed for specific regions of interest. The QDR 1000W employs a collimator assembly with three sizes of aperture which are software selected for specific applications. Imaging time is dependent upon the dimensions of the regions being scanned, the aperture setting and the line spacing. This, in turn, affects resolution and precision. Line spacing is the distance between each step of the scanning arm and resolution is defined as the distance between samples along each line. These parameters are fixed according to the application selected and cannot be altered. The QDR 2000 employs a fan-shaped x-ray beam instead of a single collimated pencil beam. The fan beam is directed at a 32 channel detector array behind a series of focused collimators. This enables the QDR 2000 to sample 32 times the data collected by the QDR 1000W, thereby significantly reducing scan times and improving spatial resolution. The characteristics of the x-ray source/detector technology are the same for both models. Consequently, with appropriate software upgrades, studies performed on one device can be incorporated into the database of the other. The QDR 2000 can be operated in either the fan beam or pencil beam mode. All QDR modes incorporate an Automatic Internal Reference System which adjusts for temperature change, power or tube flux, and provides the machine with a method of pixel by pixel calibration in each detector channel thereby eliminating the need for daily operator calibration.

Patient dose levels are significantly lower for digital radiography than for conventional radiography. Patient entry dose levels for whole body scans utilizing either the pencil beam or fast array fan beam mode are less than 1.5 mRem and other applications are also well below the exposure levels of traditional radiography.

Quality Control The QDR 1000W and 2000 are equipped with quality control (QC) software programs. QC phantom scans were performed daily in both single beam and array modes and QC plots for BMD were carefully reviewed to monitor instrument precision. The system software COMPARE function was used to simultaneously display baseline and current QC values in order to track instrument changes and control drift. Scan Protocol Two instruments, the QDR 1000W and the QDR 2000, were used to collect whole body (WB), BMC and BMD data of 228 children (115 females, 113 males) aged 8-15 years (means) in year one. The QDR 2000 bone densitometer was used to collect all data at the proximal femur in the fast array scanning mode in year one. Starting in year two the QDR 2000 was used exclusively for three scans: Whole Body, Proximal Femur and Anterior-Posterior Spine scans. All measurements were completed in the Department of Nuclear Medicine at the Royal University Hospital, Saskatoon, SK. The subjects wore plain, loose fitting shorts and a T-shirt and all metal objects (snaps, belts, jewelry), socks and footwear were removed. The Wole Body desitometric exam was performed with the subject positioned supine on the scanning table (Figure 1). The body was centered, and straightened along the midline within the solid longitudinal, whole body lines outlined on the scan mat. The shoulders were slightly depressed to below the level of the lower border of the jaw. The head was positioned so that the first scan line was in air but no more than six scan lines below the lateral line marked on the head end of the mat. For tall subjects the feet were dorsi-flexed during scanning below the knee. The subjects were requested bend their knees slightly if ankle flexion failed to accommodate the entire skeleton. The hands were placed prone and equidistant from the torso on either side of the body. A large subject was asked to tuck the thumbs slightly beneath the soft tissue of the thigh to accommodate both elbows within the scan zone. The toes were inverted to maximize femoral neck display during analysis. The feet (big toes) were taped together to immobilize this region and to provide a standardized position for repeat measures. The soft tissue calibration wedge was positioned approximately 2 inches from the right side of the subject at the lower leg. The step of maximum density is positioned toward the subject's head within the scan zone and without touching any portion of the subject. It is essential that the subject be precisely positioned to allow standardization of analysis and to compare subjects between measurement occasions. The table accomodates a maximum height of 190 cm and a body weight of 136 kg.

Figure 1. Above view of whole body scan positioning of subject including tissue bar. The pencil beam WB scan required approximately 12 minutes to complete for a child 160 centimeters tall. The scan was terminated by the operator and then saved when the entire skeletal image and soft tissue calibration wedge were displayed on the monitor. The array beam WB scan required approximately 6 minutes to complete for a 160 cm tall subject. The left proximal femur scans were performed with the subject supine and in a straight line on the table and with the iliac crest beneath the mid-vertical scan line on the mat (Figure 2). The left foot was inverted (approximately 10 degrees) and fixed with a nylon strap against the lucite positioning wedge provided by Hologic. The center of the fixture wedge was aligned with the midline of the subject and the leg was slightly abducted at the hip. The arms were positioned at the sides outside the scan zone. A "scout" view was taken to verify positioning and to ascertain that the femoral head, greater and lesser trochanter were included in the global region of interest (ROI). The greater trochanter must appear at least 5 scan lines from the outer edge of the ROI for analysis. Hologic software was used to reposition the hip as required with the global ROI.

Figure 2. Positioning of subject for left proximal femur scan including view of left foot strapped to hip scan positioning fixture. The AP lumbar spine scans were performed with the subject supine and positioned within the vertical lines marked at the head and the foot of the scan mat and with the lumbar spine wihtin the horizontal hip/spine center lines on the table mattressin. The spine (assumably at the subjects vertical midline) is positioned in a straight line along the hashed line marked to the right of the scan mat center line. The lumbar spine must be within one and a half inches of this brokent line. The subjects hands are placed beneath the head and both elbows are supported with small pillows. This removes the arms from within the scan zone and raises the ribs as high as possible ouside of the ROI. The subjects knees are bent towards the chest and a large square cushion is positioned under the subjects lower legs so both legs flexed to 90 degrees at the hip and knee (see Figure 1). The femora should be as vertical as possible to the line of the table without causing the subject discomfort. This position serves to reduce the natural lordotic curve of the lumbar spine and to place the spine in contact with the table. Initially a Turbo array spine scan is performed to ensure the subject is positioned correctly. Once positioned correctly, The laser dot is centered at the vertical midline of the subject at a level one to two inches below the level of the iliac crest. The tube arm is moved the length of the subject's's torso to confirm positioning and the laser dot should describe a straight line between the umbilicus and the xiphoid process of the sternum. The array spine mode is selected which utilizes the fan beam and takes approximately 1.5 minutes to complete along the length from L3 - L4 to T12-L1(approx. 6 inches). For heavier subjects the Hi-res array spine mode is selected in order to maximize scan resolution. The high resolution scan represents a longer scan time (approximately 3 minutes) and a greater patient radiation dose so is used only in extreme cases. The subject is instructed to remain motionless during scan acquisition. The L5 - S1 joint space should be (just) visible on the image and the vertebral column should be centered in the center of the global ROI and between the red lines on the image. The software enables the operator to reposition the scan starting point at the work station should this be necessary. The AP scan is terminated by the operator when one half of the T12 vertebral body is visible on the screen. Following acquisition of the AP lumbar spine the scan is analyzed (see Analysis protocol).

Figure 3. Positioning of subject for A-P lumbar spine scan including foam cushion and region of interest.

All data were archived to an optical disk from the instrument hard drive at the end of each data collection day, transported to a bone density work station at another location, restored to the work station hard disk drive and analyzed.

All analysis procedures were completed as outlined in the HOLOGIC QDR Operators Manual and Users Guide, Hologic Inc., MA., 1991. Hologic system software Version 7.0.01 was used for all anallyses. Supplementary notes are provided as an adjunct to the manual procedures. Several non-diagnostic values are displayed above every scan image. The abbreviation d0 refers to the attenuation of the beam within the bone segment of the calibration wheel and is a function of the thickness of the tissue through which the beam passes. A d0 less than 90 represents an abundance of overlying tissue and a less precise bone measurement as a result. k is the ratio of attenuation of tissue in both the low (70kvp) and high (150kvp) energy beam and is usually about 1.20. The coefficient of variation (CV) for the BMD for all of the regions included in the analysis is indicated on the report beneath the patient biography.

The QDR 1000W and 2000 were used to acquire scans of the entire skeleton. These scans were performed in the pencil beam mode on both instruments for the initial data collection session (Oct.-Dec. 1991) in the hospital. Subsequent sessions used only the QDR 2000 in array mode. Software version 5.40 was used for analysis of the whole body scan. The whole body procedure consists of the following steps: 1. Image adjustment 2. Region of Interest selection 3. Generation of a report When an image appears on the monitor the ALT Z command is used to adjust the magnified, right hand image to display either the upper or lower portion of the skeleton depending on the region being analyzed. The yellow lines on the image are adjusted to define ten region of interest.

1. Head: the horizontal line above the shoulders is adjusted to immediately below the chin. The head region is defined superiorly and laterally by the borders of the global ROI and inferiorly by the shoulder line.

2-3. Arm (Land R): the vertical lines at the shoulder are positioned to between the head of the humerus and the glenoid fossa of the scapula. All of the soft tissue region of the arms should be included in this region. The image adjust function is used to highlight the soft tissue image and the lines repositioned to maximize the amount of soft tissue included in each region. The arm regions (L Arm, R Arm) are defined superiorly by the horizontal shoulder line, medially by the vertical arm line bisecting the glenoid fossa and laterally by the border of the global ROI. 4-5. Spine [Thoracic (T) and Lumbar (L)]: the vertical lines on either side of the spine are moved close to but not touching the vertebral bodies. These lines should be bent to match the curvature of the spine as closely as possible. This is accomplished with the cursor in active point (blue) mode which is enabled with the (+) key. The (INS) key will add additional points to the line if more precise contouring is required. The small, transverse line is positioned at the T12 L1 interspace. The thoracic spine region (T spine) is defined superiorly by the horizontal shoulder line, laterally by the two vertical spine lines and inferiorly by the short transverse line at T12 - L1. The lumbar spine (L spine) region is defined superiorly by the small transverse line at T12 L1, laterally by the vertical spine lines and inferiorly by the line at the iliac crest. 6. Pelvis: the horizontal line that marks the superior border of the pelvic region is placed immediately above the iliac crest. This line is extended laterally to maximize the amount of soft tissue included in the chest and pelvic regions. It is important that soft tissue in the region of the arm not be included with the soft tissue from the chest region if at all possible. The horizontal, iliac crest line determines the angle at which the oblique segment lines bisect the neck of the femur to describe the lateral border of the pelvic region. This oblique line is directed medially and should bisect the neck of the femur at its anatomical midpoint. This positioning may, in some subjects, move the medial arm segmental lines into the soft tissue boundaries of the arm region. The bony, femoral neck, landmark is always maintained at the expense of the soft tissue if necessary. This problem is minimized by careful positioning of the subject with the arms slightly abducted from the torso during scan acquisition. The region of the pelvis is defined superiorly by the horizontal line at the iliac crest and laterally and inferiorly by the oblique lines passing through the center of the femoral neck. 7-8. Ribs (L and R): The region of the ribs (L Ribs, R Ribs) are defined superiorly by the shoulder line, laterally by the vertical arm lines, medially by the vertical spine lines and inferiorly by the line at the iliac crest. 9-10. Legs (L and R): the vertical line extending downward from the lowermost point of the pelvic region is positioned between the feet. The vertical lines on the lateral aspects of the legs are extended laterally to include as much of the soft tissue of the thigh as possible. The position of this line is most often limited by the bony hand which should not be included in the soft tissue

leg region. The vertical leg line may extend into the tissue bar image without affecting the analyses. The leg regions (L Leg, R Leg) are defined by the oblique femoral line superomedially, by the vertical leg line inferomedially and laterally by the vertical line on the lateral aspect of the leg. Figure 1 describes correct placement of the segmental lines for whole body analysis. The whole body scan data is processed and values for Area (cm2), BMC (gms), and BMD (gms/cm2) are calculated and reported for each region and for the entire skeleton (TOTAL). A report may be generated when the whole body analysis is complete by depressing the (print screen) key once the calculated values and the whole body image appear on the monitor.

II. PROXIMAL FEMUR The hip analysis procedure consists of the following steps: 1. 2. 3. 4. 5. 6. 7. 8. Image adjustment Region of interest (ROI) selection Bone map editing (inserting or deleting points) Confirming the femoral midline Outlining the femoral midline Confirming the position of Ward's triangle Confirming the position of the greater trochanter Generating a report

All analysis procedures were completed as outlined in the HOLOGIC QDR Operator's Manual and User's Guide, Hologic Inc., Waltham, MA., 1991. Software version 7.0.01 was used for analyses. Proximal femur version 4.42:1. Supplementary notes, specific to pediatric hips, are provided as an adjunct to the manual procedures. Global Region of Interest (ROI) The global ROI defines the total anatomical area to be included in the analysis. The upper ROI margin is adjusted to include all of the femoral head and describes the superomedial border of the acetabular rim (approximately 5 lines superior to the head of the femur; Figure 4). The upper left corner should be in the soft tissue region of the pelvic cavity. The lateral ROI margin should include all of the greater trochanter and approximately 5 lines outside of it. Optimally, the lateral ROI border remains in soft tissue with at least one line between the bone and the edge of the ROI. This is mandatory for accurate analysis. The medial ROI border

defines the medial, outermost, rim of the acetabulum and includes all of the femoral head and approximately 10 lines medially. The lower ROI margin is positioned along the long axis of the femur at a point twice the length of the greater trochanter or, 10 lines below the lesser trochanter if it is visible on the hip image.

Figure 4. Region of interest for either right or left proximal femur scan. Bone Map Editing The small size of the pediatric skeleton required bone to be deleted in those areas where the regions of interest overlapped. The Delete key changes the neutral (green) bone cursor color to an active (blue) mode which allows bone to be removed. The bone map cursor was used most frequently to delete that portion of the ischial rim which overlapped the femoral neck box. This step can only be performed after the entire analysis is completed and the positioning of the femoral neck box determined. The F10 function key prompts the program to return to the first step for re-anlysis. In those cases where the bone outline was incomplete the hip was re-analyzed with the vertical ROI extended. This served to complete the bone map and only infrequently was the operator required to manually outline the bone borders or insert bone. When edge outlines are incomplete the Pg Dn key is used to decrease the cursor size, which maximizes operator control and the edges of the bone are drawn in with the cursor in the active (yellow) mode. The system fills in these designated regions with the bone which is included in the BMD calculations. Sub-Regions of Interest The sub-regions of interest (femoral neck, Ward's triangle, trochanter, and intertrochanteric regions) are automatically defined following the bone map editing procedure. These regions are represented diagrammatically in Figure 4. The narrowest portion of the femoral neck is automatically determined and marked by the position of the femoral neck box. The femoral midline is drawn as a line perpendicular to the neck box and parallel to the main axis of the femur and describes the femoral neck axis of

symmetry. All sub-regions are computed based on the positioning of the femoral midline which should not be adjusted from the system determination. If the midline appears to be positioned incorrectly the bone map should be carefully studied to ascertain that the entire bony anatomy of the hip is outlined and filled in. Only in extreme cases are any adjustments to the femoral midline required. The femoral neck box is most often the sub-region requiring adjustment in the hip analysis. The corners of the 1.5 cm. by 5 cm. box cover only the femoral neck and the inferolateral border is positioned immediately adjacent to, and flush against, the medial aspect of the greater trochanter. The (F10) key allows re-analysis and the deletion of any bone from the ischium overlapping the region of the neck box. This is the critical standardization landmark of the femoral neck region. Altering the size of the femoral neck box should be avoided if possible. Ward's triangle is an area of geometric significance and describes a trabecular lattice structure which constitutes an area of minimal bone density within the proximal femur. A one centimeter square area marks Ward's triangle which is determined densitometrically following a search within the femoral neck region. Commonly in young subjects at variable stages of growth the least dense region of bone within the femoral neck is impossible to determine via densitometric analysis. In these subjects a default position at the intersection of the femoral midline and the bottom edge of the femoral neck box is identified. The coordinate location of the Ward's triangle box is identified just above the Hologic logo on the screen image. If the coordinates (-5,5) are shown this indicates that the region of least density was not found and the system has defaulted to the anatomical center of the hip. The QDR 2000 automatically identifies the base of the greater trochanter although this line may have to be adjusted manually to where the convex curvature of the trochanteric region becomes concave at the femoral shaft. The lateral, lowermost border of the neck box provides the uppermost border of the trochanteric region and the solid line extending from the midpoint of the femoral midline to the trochanteric base describes a triangle. This triangle is the region of the greater trochanter. The intertrochanteric region is the area of the proximal femur described by the medial, lowermost border to the neck box, the trochanter line and the bony edges of the femur itself. The area total is the sum of the neck, trochanteric and intertrochanteric sub-regions. III. A-P LUMBAR SPINE All analysis procedures were completed as outlined in the HOLOGIC QDR 2000 Operators Manual and Users Guide, Hologic Inc., Waltham, MA., 1991. Hologic system software version 7.0.01 was used for analyses. Software version 4.42:1 was used for analysis of the AP/Lat lumbar spine. Anteroposterior (AP) The AP spine analysis consists of the following steps:

1. Image adjustment 2. Region of interest (ROI) selection 3. Marking of intervertebral spaces 4. Labeling of lumbar vertebrae 5. Bone map editing 6. Tracking line confirmation 7. Lateral start adjustment 8. Generation of reports Contrast and brightness of the image is adjusted as required to optimize the visibility of all anatomical features and especially the intervertebral spaces. Global Region of Interest (ROI) A yellow box on the screen describes the area to be analyzed for bone mineral content (BMC). The size of this ROI is displayed numerically beneath the scan image and is important for comparison purposes. The width of the ROI is preset to 10 pixels within the scan area width and should not be adjusted. The length of the ROI is adjusted with the <T> key so that the superior border is at the level of the T12 - L1 interspace. The lower border is adjusted with the <B> key and is positioned to between L4 - L5. The second (L2) to the fourth (L4) lumbar vertebrae should be include in the global ROI for analysis. The borders of the ROI , as well as the intervertebral markers must be horizontal and should not be bent to follow the contour of the spine. This, again, is based on the analysis of the lateral scan which is determined from the AP. Marking of Intervertebral Spaces The active line is used to define the lowermost intervertebral space and is positioned slightly above the superior surface of the lowermost vertebrae. The third and fourth (L3 - L4) and the second and third (L2 - L3)vertebral spaces are marked (in that order) with the active cursor.The first and second (L1 - L2) and fourth and fifth (L4 - L5) lumbar vertebral interspaces are marked by the upper and lower borders of the global ROI, respectively. Labeling of Lumbar Vertebrae. The system automatically labels the vertebrae from L1 to L4 based on the placement of the intervertebral space lines. The operator confirms that the vertebrae are correctly labelled and identifies those vertebrae which must be excluded from the analysis. This is most often necessary at L2 if the twelfth rib is superimposed or at L4 if the iliac crest overlies the vertebral boundary. Pathological or crushed vertebrae are also excluded at this stage. Bone Map Editing Once the vertebrae are labeled the system processes the scanned image and outlines those regions that are bone (colored yellow) and those regions it identifies as soft tissue (colored blue). Although the system removes all recognizable artifacts it is sometimes (although rarely)

necessary to do further editing of the bone map. This procedure is described in the Proximal Femur section, Bone Map Editing Tracking Line Confirmation The system provides a vertical line which describes the center of the vertebrae from L2 to L4. This line is essential for analysis of the lumbar spine and should be no more than five degrees off of the vertical. An tracking angle of greater than five degrees requires that the patient be repositioned and that the AP scan repeated.

Report Generation A report is generated with the <Print Screen> command key. The estimated area (Est. Area, cm2) the estimated BMC (Est. BMC, grams) and the BMD (gms/cm2) is calculated and reported separately for each vertebrae.and as a the total (TOTAL) for the ROI on the first results page. Those vertebrae excluded at the Labeling of lumbar vertebrae step are noted with a black label on the scan image.

IV. COMPARISONS In order for longitudinally comparisons of BMC and BMD to be made all bone scans must be replicated as exactly as possible. This includes having the subject wear the same type of clothing between measurements, positioning the subject as precisely as possible for all measurements, using the same beam mode and utilizing the COMPARE function for analysis. The COMPARE function allows referencing of a previous scan of the same site for the same subject for comparison to a current scan. The baseline measurement is called up and displayed next to the current scan on the monitor. The regions of interest are matched as closely as possible on the reference and current scans This includes duplicating the size, shape and location of the global ROI and the sub-regions.(if applicable). The values indicating the size of the global ROI and the anatomical ROI must be identical on both scans. Similarly the shapes of the area being analyzed must be as exact as possible if precision is to be maximized. The system replicates the outlined image of the analyzed region of the baseline scan over the current scan. The operator manipulates the position of this outline until the anatomy of the current scan matches that of the previous scan as closely as possible. If bony anatomy exists in the most recent scan which was not present in the reference scan the reference scan should be reanalyzed to exclude this region. The analysis is carried out following those steps as outlined in the analysis procedures of each site. Software update

On March 2,1992 Hologic Inc., Waltham, MA. distributed a software update which accounted for measurement differences between the pencil beam and the array beam scan mode. The pencil beam values were approximately 4% lower than those obtained using the array beam for hips and 2% lower than for the lumbar spine. The old software (version 437A)was replaced with the new software version (V 4.42A:1). The updated version introduced a fan factor that would be applied to the pencil beam mode scan values automatically the first time array scan results were called up on the monitor.