**Section 3**

**Creation of ASDs**

36 Atrial Septal Defect

Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, et al. (1999).

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Jacobs JP, Mavroudis C, Jacobs ML, et al. (2004). Lessons learned from the data analysis of

Loffredo C, Wilson P, Ferencz C. (2001). Maternal diabetes: An independent risk factor for

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Reller MD, Strickland MJ, Riehle-Colarusso T, et al. (2008). Prevalence of Congenital Heart

Riehle-Colarusso T, Strickland MJ, Reller MD, et al. (2007). Improving the quality of

Rosas M, Attie F, Sandoval J, et al. (2004). Atrial septal defect in adults > 40 years old: negative impact of low atrial oxygen saturation. *International Journal of Cardiology* 93:145-155. Schott JJ, Benson DW, Basson CT, et al. (1998). Congenital Heart Disease caused by mutations in the transcription factor NKX2-5. *Science* 2f81: 108-111. Wren C, Richmond S, Donaldsdon L. (2000). Temporal variability in birth prevalence of

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*Molecular Teratology* 79:714-728.

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39:1890-1900.

*Research* 11:1065-1071

Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac

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**4**

*Japan*

**Computer-Aided Automatic Delivery System of**

Hiromasa Yamashita, Gontaro Kitazumi, Keri Kim and Toshio Chiba

Several fetal cardiac malformations have been increasingly treated before birth. Fetal cardiac intervention targets an in utero correction of simple intracardiac abnormalities that potentially progress to complex heart diseases in utero, such as fetal critical aortic stenosis that might cause to hypoplastic left heart syndrome (HLHS) and HLHS with restrictive atrial septum leading to irreversible pulmonary vascular damages (Kohl et al., 2000; Marshall et al., 2004; Makikallio et al., 2006). Current operative procedures for correction of fetal restrictive atrial septum with an atrial decompression are still invasive because they, percutaneously and through both the uterine wall and fetal chest walls, require ultrasoundguided maternal puncture into the frequently-pulsating (120-180 counts per minute) cardiac cavity of tiny fetal hearts and to create interatrial communications. Accordingly, these procedures have been reportedly accompanied by serious complications including profound bradycardia, bleeding and hemopericardium, intracardiac thrombus formation, and

To establish fetal interatrial communications with minimal adverse effects, we developed an entirely new approach with the use of high intensity focused ultrasound (HIFU) (Fig. 1). HIFU is acoustic modality using ultrasound energy with cavitation and/or coagulation effect focused to operate on an internally targeted tissue without damaging overlying and/or underlying tissues. HIFU has been employed predominantly for non-touch treatment of tumors including prostate cancer, breast cancer and uterine fibroids (Rebillard et al., 2005; Chan et al., 2002; Hengst et al., 2004). Unlike an extensive ablation of stationary tumors, the HIFU ablation to a frequently-pulsating narrow area in the beating and tiny fetal heart requires highly accurate pinpoint delivery in real-time based on computer-aided auto-

We developed a new automatic delivery system of HIFU with real-time two dimensionalultrasound (2D-US) imaging analysis. Features of this system are 1) automatic detection of heartbeat rates, 2) automatic estimation of atrial septum position, 3) automatic generation of HIFU delivery timing and we did feasibility study for creation of an atrial septal defect

recurrent in utero closure of the created atrial septal defects.

using the beating heart of anesthetized adult rabbits.

**1. Introduction**

tracking of atrial septum.

**High-Intensity Focused Ultrasound**

*National Center for Child Health and Development,*

**for Creation of an Atrial Septal Defect**

## **Computer-Aided Automatic Delivery System of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect**

Hiromasa Yamashita, Gontaro Kitazumi, Keri Kim and Toshio Chiba *National Center for Child Health and Development, Japan*

#### **1. Introduction**

Several fetal cardiac malformations have been increasingly treated before birth. Fetal cardiac intervention targets an in utero correction of simple intracardiac abnormalities that potentially progress to complex heart diseases in utero, such as fetal critical aortic stenosis that might cause to hypoplastic left heart syndrome (HLHS) and HLHS with restrictive atrial septum leading to irreversible pulmonary vascular damages (Kohl et al., 2000; Marshall et al., 2004; Makikallio et al., 2006). Current operative procedures for correction of fetal restrictive atrial septum with an atrial decompression are still invasive because they, percutaneously and through both the uterine wall and fetal chest walls, require ultrasoundguided maternal puncture into the frequently-pulsating (120-180 counts per minute) cardiac cavity of tiny fetal hearts and to create interatrial communications. Accordingly, these procedures have been reportedly accompanied by serious complications including profound bradycardia, bleeding and hemopericardium, intracardiac thrombus formation, and recurrent in utero closure of the created atrial septal defects.

To establish fetal interatrial communications with minimal adverse effects, we developed an entirely new approach with the use of high intensity focused ultrasound (HIFU) (Fig. 1). HIFU is acoustic modality using ultrasound energy with cavitation and/or coagulation effect focused to operate on an internally targeted tissue without damaging overlying and/or underlying tissues. HIFU has been employed predominantly for non-touch treatment of tumors including prostate cancer, breast cancer and uterine fibroids (Rebillard et al., 2005; Chan et al., 2002; Hengst et al., 2004). Unlike an extensive ablation of stationary tumors, the HIFU ablation to a frequently-pulsating narrow area in the beating and tiny fetal heart requires highly accurate pinpoint delivery in real-time based on computer-aided autotracking of atrial septum.

We developed a new automatic delivery system of HIFU with real-time two dimensionalultrasound (2D-US) imaging analysis. Features of this system are 1) automatic detection of heartbeat rates, 2) automatic estimation of atrial septum position, 3) automatic generation of HIFU delivery timing and we did feasibility study for creation of an atrial septal defect using the beating heart of anesthetized adult rabbits.

Computer-Aided Automatic Delivery System

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 41

scale NTSC-video data (640x480 pixels) into the workstation with 30 frames/sec through a video capture (ADVC-55, Canopus, Kobe) and IEEE1394 cable. The third part is a workstation (HP xw8400, Windows XP x64, Intel Xeon 2.33 GHz, Quad Core, 16GB RAM,DirectX9.0c) to analyze an input 2D-US tomographic image of cardiac four-chamber with our original real-time HIFU delivery control algorithm. The workstation detects heartbeat rates, estimates atrial septum positions, and generates HIFU delivery timing by RS-232 interface into a signal transform box automatically. The fourth part is a function generator (Agilent 33220A, Agilent technologies, Santa Clara, CA) to output intermittent sine-wave with certain frequency and voltage into the fifth part, and RF power amplifier (AG1012, T&C power Conversion Inc., Rochester, NY). The timing to drive the transducer by amplified sine-wave is determined by TTL trigger from the signal transform box. Automatic HIFU delivery is accomplished by some adjusting with various parameters. This setting is done through a graphical user interface on the monitor by surgeons (Fig. 3).

Fig. 3. Graphical user interface for setting, adjusting and monitoring of automatic HIFU

delivery.

Fig. 1. New approach with HIFU to correct cardiac morphologic abnormalities less invasively.

#### **2. Methods**

#### **2.1 System configuration**

The system configuration of the computer-aided automatic delivery system of HIFU consists of predominantly 5 parts (Fig. 2). The first one is a HIFU delivery device which comprises a monocoque spherical shaped piezo transducer and a diagnostic 2D-US imaging probe mounted on the transducer. The tomographic images are taken by the probe including the focal point of the transducer. The curvature radius of the transducer is 40 mm, and the focal point is located 40-mm apart from the edge face of the transducer. The focal point is an elliptical in shape (0.6-mm wide, 5.0-mm long). The second part is a diagnostic 2D-US imaging equipment (En-Visor C HD, Philips, Andover. MA) to scan a cardiac four-chamber view by 2D-US imaging probe (s12, Philips, Andover. MA). The equipment outputs gray

Fig. 2. System configuration of the computer-aided automatic HIFU energy delivery.

Fig. 1. New approach with HIFU to correct cardiac morphologic abnormalities less invasively.

The system configuration of the computer-aided automatic delivery system of HIFU consists of predominantly 5 parts (Fig. 2). The first one is a HIFU delivery device which comprises a monocoque spherical shaped piezo transducer and a diagnostic 2D-US imaging probe mounted on the transducer. The tomographic images are taken by the probe including the focal point of the transducer. The curvature radius of the transducer is 40 mm, and the focal point is located 40-mm apart from the edge face of the transducer. The focal point is an elliptical in shape (0.6-mm wide, 5.0-mm long). The second part is a diagnostic 2D-US imaging equipment (En-Visor C HD, Philips, Andover. MA) to scan a cardiac four-chamber view by 2D-US imaging probe (s12, Philips, Andover. MA). The equipment outputs gray

Fig. 2. System configuration of the computer-aided automatic HIFU energy delivery.

**2. Methods**

**2.1 System configuration**

scale NTSC-video data (640x480 pixels) into the workstation with 30 frames/sec through a video capture (ADVC-55, Canopus, Kobe) and IEEE1394 cable. The third part is a workstation (HP xw8400, Windows XP x64, Intel Xeon 2.33 GHz, Quad Core, 16GB RAM,DirectX9.0c) to analyze an input 2D-US tomographic image of cardiac four-chamber with our original real-time HIFU delivery control algorithm. The workstation detects heartbeat rates, estimates atrial septum positions, and generates HIFU delivery timing by RS-232 interface into a signal transform box automatically. The fourth part is a function generator (Agilent 33220A, Agilent technologies, Santa Clara, CA) to output intermittent sine-wave with certain frequency and voltage into the fifth part, and RF power amplifier (AG1012, T&C power Conversion Inc., Rochester, NY). The timing to drive the transducer by amplified sine-wave is determined by TTL trigger from the signal transform box. Automatic HIFU delivery is accomplished by some adjusting with various parameters. This setting is done through a graphical user interface on the monitor by surgeons (Fig. 3).

Fig. 3. Graphical user interface for setting, adjusting and monitoring of automatic HIFU delivery.

Computer-Aided Automatic Delivery System

**2.3 Automatic estimation of atrial septum**

1. Position adjustment of HIFU transducer

timing when left ventricle shrinks most.

estimate the atrial septum position (Fig. 6(B)).

2. Making template information

4. Frequency analysis of the left ventricular area variation

conditional for HIFU delivery based on the allowable error.

small as possible.

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 43

calculation of its geometry described below. Overestimation or underestimation of the left ventricular area causes "undetectable". In our system the threshold value is adjusted empirically considering actual 2D-US image so that a ratio to be "undetectable" becomes as

It becomes possible to take samples about variation of the left ventricular area at frequency of 30 times per seconds when the area is extracted adequately. This is because the variation of areas includes heartbeats, low-frequency component by breathing and high-frequency component by noise when 2D-US image is acquired and transferred to the workstation. It is necessary to remove these components by filtering with a small calculation cost. Our system uses the moving average calculation with 8 past samples to remove the low frequency component and the simplicity mean calculation with 2 past samples to remove the highfrequency component. Using fast Fourier transformation the system calculates heartbeat period, frequency and period from 64 past samples without some noises automatically.

As mentioned previously, the atrial septum has less features in 2D-US image which is difficult to detect its position by image processing directly. Therefore the system is set to delivery HIFU only, when position of the atrial septum can be estimated from position and shape of the left ventricle, which is easy to be detected in 2D-US image, and its position corresponds to the focal point of HIFU delivery by a fixed transducer. In this study an automatic estimation of the atrial septum is realized according to following procedures. In addition, the positional information of estimated atrial septum is used as a safety

The transducer and 2D-US probe are fixed physically for one HIFU delivery device. At first, the focal point of HIFU delivery is confirmed precisely by 2D-US image when HIFU is delivering to a low decrement polymer-coated rubber with the acoustic impedance (Acoustic standoff, Eastek Corporation, Tokyo, Japan) that is approximately equivalent to a human body (Fig. 5). The resolution of this 2D-US image around the focus is about 0.5 mm in the direction that throws the beam and about 0.2 mm in the depth direction. We mark a circle to the focal position on this 2D-US image supported by orthogonally-crossed guide lines (Fig. 6(A)). The position and the posture of HIFU delivery device is fixed by manual so that position of the atrial septum corresponds to the focal point of HIFU transducer in the

As well as preceding clause, we mark a cross on the atrial septum of the timing when the left ventricle shrinks most by manual operation and define the coordinate with (XAt, YAt). In addition, we define the fillet diameter of the left ventricle shape approximated by a ellipse with (RXt, RYt) and the coordinate of center of gravity position with (XGt, YGt), and use these parameters for the image analysis in the next clause as template information to

#### **2.2 Automatic detection of heartbeat rates**

The atrial septum, which is a target of this system to be perforated, repeats expansion, shrinkage and movement with heartbeat. It is assumed that the best timing to perforate an atrial septum safely by HIFU delivery system is when the atrial septum is expanded most. However, because the atrial septum has less features in 2D-US image, it is difficult to detect its movement directly. Therefore, using the fact that atrial septum expands when ventricle contracts, we tried to detect the timing when the left ventricle contracts most, which is easy to detect by 2D-US image. The left ventricular movement of expansion and shrinkage are caused by heartbeats, and its variation can be associated with the change of the left ventricular area in 2D-US image. In this paper, we utilized the automatic detection of the heartbeat period by the following procedures.

#### 1. Setting of Region of Interest (ROI)

We set a rectangular ROI surrounding the left ventricle of 2D-US image on the display while confirming four chamber view of 2D-US image (Fig. 4(A)). ROI should be set so that the left ventricle, which repeats expansion, shrinkage and movement, doesn't protrude it, and ROI should be the smallest to minimize calculation cost in the image processing.

Fig. 4. (A) Setting of ROI and point of inside the left ventricle (LV). (B) Binary image of 2D-US image, extraction of the left ventricle area and approximate LV ellipse.

2. Pointing of inside the left ventricle

Using 2D-US binarized image by white and black pixels, and the central of inside the left ventricle is pointed by manual in ROI to set target region of the left ventricle to be tracked (Fig. 4(A)). The amount of black pixels which is communicating with the point in ROI is extracted automatically as a left ventricular area.

3. Adjustment of a threshold value in binarization of 2D-US image

2D-US image for the image processing is expressed in gray scale of 256 brightness levels. The threshold value for binarization should be adjusted between 0 and 255 levels so as to extract the left ventricular area adequately. When it is adjusted most adequately, a shape of the left ventricle is approximated an ellipse (Fig. 4(B)). However depending on threshold, the outside of the left ventricle is detected by error and, to the contrary, inside of the left ventricle is not detected. These errors influence precision of the left ventricular area and calculation of its geometry described below. Overestimation or underestimation of the left ventricular area causes "undetectable". In our system the threshold value is adjusted empirically considering actual 2D-US image so that a ratio to be "undetectable" becomes as small as possible.

4. Frequency analysis of the left ventricular area variation

It becomes possible to take samples about variation of the left ventricular area at frequency of 30 times per seconds when the area is extracted adequately. This is because the variation of areas includes heartbeats, low-frequency component by breathing and high-frequency component by noise when 2D-US image is acquired and transferred to the workstation. It is necessary to remove these components by filtering with a small calculation cost. Our system uses the moving average calculation with 8 past samples to remove the low frequency component and the simplicity mean calculation with 2 past samples to remove the highfrequency component. Using fast Fourier transformation the system calculates heartbeat period, frequency and period from 64 past samples without some noises automatically.

#### **2.3 Automatic estimation of atrial septum**

42 Atrial Septal Defect

The atrial septum, which is a target of this system to be perforated, repeats expansion, shrinkage and movement with heartbeat. It is assumed that the best timing to perforate an atrial septum safely by HIFU delivery system is when the atrial septum is expanded most. However, because the atrial septum has less features in 2D-US image, it is difficult to detect its movement directly. Therefore, using the fact that atrial septum expands when ventricle contracts, we tried to detect the timing when the left ventricle contracts most, which is easy to detect by 2D-US image. The left ventricular movement of expansion and shrinkage are caused by heartbeats, and its variation can be associated with the change of the left ventricular area in 2D-US image. In this paper, we utilized the automatic detection of the

We set a rectangular ROI surrounding the left ventricle of 2D-US image on the display while confirming four chamber view of 2D-US image (Fig. 4(A)). ROI should be set so that the left ventricle, which repeats expansion, shrinkage and movement, doesn't protrude it, and ROI

Fig. 4. (A) Setting of ROI and point of inside the left ventricle (LV). (B) Binary image of 2D-

Using 2D-US binarized image by white and black pixels, and the central of inside the left ventricle is pointed by manual in ROI to set target region of the left ventricle to be tracked (Fig. 4(A)). The amount of black pixels which is communicating with the point in ROI is

2D-US image for the image processing is expressed in gray scale of 256 brightness levels. The threshold value for binarization should be adjusted between 0 and 255 levels so as to extract the left ventricular area adequately. When it is adjusted most adequately, a shape of the left ventricle is approximated an ellipse (Fig. 4(B)). However depending on threshold, the outside of the left ventricle is detected by error and, to the contrary, inside of the left ventricle is not detected. These errors influence precision of the left ventricular area and

US image, extraction of the left ventricle area and approximate LV ellipse.

3. Adjustment of a threshold value in binarization of 2D-US image

should be the smallest to minimize calculation cost in the image processing.

**2.2 Automatic detection of heartbeat rates**

heartbeat period by the following procedures.

1. Setting of Region of Interest (ROI)

2. Pointing of inside the left ventricle

extracted automatically as a left ventricular area.

As mentioned previously, the atrial septum has less features in 2D-US image which is difficult to detect its position by image processing directly. Therefore the system is set to delivery HIFU only, when position of the atrial septum can be estimated from position and shape of the left ventricle, which is easy to be detected in 2D-US image, and its position corresponds to the focal point of HIFU delivery by a fixed transducer. In this study an automatic estimation of the atrial septum is realized according to following procedures. In addition, the positional information of estimated atrial septum is used as a safety conditional for HIFU delivery based on the allowable error.

1. Position adjustment of HIFU transducer

The transducer and 2D-US probe are fixed physically for one HIFU delivery device. At first, the focal point of HIFU delivery is confirmed precisely by 2D-US image when HIFU is delivering to a low decrement polymer-coated rubber with the acoustic impedance (Acoustic standoff, Eastek Corporation, Tokyo, Japan) that is approximately equivalent to a human body (Fig. 5). The resolution of this 2D-US image around the focus is about 0.5 mm in the direction that throws the beam and about 0.2 mm in the depth direction. We mark a circle to the focal position on this 2D-US image supported by orthogonally-crossed guide lines (Fig. 6(A)). The position and the posture of HIFU delivery device is fixed by manual so that position of the atrial septum corresponds to the focal point of HIFU transducer in the timing when left ventricle shrinks most.

2. Making template information

As well as preceding clause, we mark a cross on the atrial septum of the timing when the left ventricle shrinks most by manual operation and define the coordinate with (XAt, YAt). In addition, we define the fillet diameter of the left ventricle shape approximated by a ellipse with (RXt, RYt) and the coordinate of center of gravity position with (XGt, YGt), and use these parameters for the image analysis in the next clause as template information to estimate the atrial septum position (Fig. 6(B)).

Computer-Aided Automatic Delivery System

3. Geometric estimation of the atrial septum position

**2.4 Definition of HIFU delivery timing**

time that left ventricle shrinks most (TS) (Fig. 7).

be minimized locally.

HIFU delivery is allowed.

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 45

Using the left ventricular fillet diameter (RX, RY) and the geometric center of gravity position (XG, YG) detected in 30 frames per seconds, template fillet diameter (RXt, RYt) and template geometric center of gravity position (XGt, YGt). We estimate current atrial septum position (XA, YA) by formula (1) automatically (Fig. 6(C)). HIFU delivery is permitted only

> XA = XG + XAt + XGt × RX / RXt YA = YG + YAt + YGt × RY / RYt

It is determined the timing when HIFU should be delivered by analysis of the four chamber view in 2D-US image. However, there is the delay time (TD) constantly between acquisition of 2D-US image, image transformation by the video capture, image analysis with the workstation, trigger output to a function generator, sinusoidal amplification in the RF power amplifier and HIFU delivery by a transducer. It is about 33 ms in this system, which is just equal to the delay for 1 frame of 2D-US image. Therefore based on the image analysis results HIFU delivery is enabled the timing when the left ventricle shrinks most by the trigger of HIFU delivery at the time of "TS-TD", which deducted the delay time from the predicted

Fig. 7. Definition of HIFU delivery starting trigger timing with total delay time in the system. The timing is the delay time TD before the timing TS that the left ventricle area will

(1)

when the position (XA, YA) is in a circle around (XAt, YAt) for allowable error.

of approximate LV ellipse (RXt, RYt) and its center of gravity (XGt, YGt) when the left ventricle is minimum. (C) Automatic estimation of atrial septum position (XA, YA) with geometric ratio between current information (fillet radius and center of gravity position) and template parameters. If estimated position is in the error margin circle of HIFU focus,

Fig. 5. Confirmation of ablated focal point by HIFU delivery on 2D-US image. Ablated focal point is ellipsoid of 5-mm long and 2-mm wide.

Fig. 6. (A) Pointing focus of HIFU with X-Y guide lines on the 2D-US image. (B) Determination of template parameters by pointing of atrial septum and getting fillet radius

of approximate LV ellipse (RXt, RYt) and its center of gravity (XGt, YGt) when the left ventricle is minimum. (C) Automatic estimation of atrial septum position (XA, YA) with geometric ratio between current information (fillet radius and center of gravity position) and template parameters. If estimated position is in the error margin circle of HIFU focus, HIFU delivery is allowed.

3. Geometric estimation of the atrial septum position

Using the left ventricular fillet diameter (RX, RY) and the geometric center of gravity position (XG, YG) detected in 30 frames per seconds, template fillet diameter (RXt, RYt) and template geometric center of gravity position (XGt, YGt). We estimate current atrial septum position (XA, YA) by formula (1) automatically (Fig. 6(C)). HIFU delivery is permitted only when the position (XA, YA) is in a circle around (XAt, YAt) for allowable error.

$$\begin{cases} \text{XA} = \text{XG} + \text{(XAt} + \text{XGt}) \times \text{RX} / \text{RXt} \\ \text{YA} = \text{YG} + \text{(YAt} + \text{YGt}) \times \text{RY } / \text{RYt} \end{cases} \tag{1}$$

#### **2.4 Definition of HIFU delivery timing**

44 Atrial Septal Defect

Fig. 5. Confirmation of ablated focal point by HIFU delivery on 2D-US image. Ablated focal

Fig. 6. (A) Pointing focus of HIFU with X-Y guide lines on the 2D-US image. (B)

Determination of template parameters by pointing of atrial septum and getting fillet radius

point is ellipsoid of 5-mm long and 2-mm wide.

**(C)**

It is determined the timing when HIFU should be delivered by analysis of the four chamber view in 2D-US image. However, there is the delay time (TD) constantly between acquisition of 2D-US image, image transformation by the video capture, image analysis with the workstation, trigger output to a function generator, sinusoidal amplification in the RF power amplifier and HIFU delivery by a transducer. It is about 33 ms in this system, which is just equal to the delay for 1 frame of 2D-US image. Therefore based on the image analysis results HIFU delivery is enabled the timing when the left ventricle shrinks most by the trigger of HIFU delivery at the time of "TS-TD", which deducted the delay time from the predicted time that left ventricle shrinks most (TS) (Fig. 7).

Fig. 7. Definition of HIFU delivery starting trigger timing with total delay time in the system. The timing is the delay time TD before the timing TS that the left ventricle area will be minimized locally.

Computer-Aided Automatic Delivery System

possible.

**3.1 Purpose**

**3.2 Methods**

Development.

240 ml/cycle).

equipment.

septum of the beating heart.

mg/kg IM) and isoflurane by inhalation.

concentration were monitored intraoperatively in real time.

6. Various parameters for HIFU delivery were specified. 7. Automatic HIFU delivery was carried out with this system.

**3.** *In vivo* **experiment**

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 47

the conditions are easy. However, we have only a rough index for optimal parameters because individual difference is large by the delivered target of HIFU. Therefore, it is necessary to judge during securing safety as the trigger off for HIFU delivery while watching the temporal change in HIFU delivery condition on the display whether you can irradiate it more effectively, the parameters are appropriate, or more effective delivery is

In this experiment, we confirmed feasibility of our computer-aided automatic delivery system of HIFU to perform atrial septal defect creation of animals in vivo. We used four healthy animals (Japanese white rabbit, male, 2.8 kg) with cardiac pulsation under

We tested HIFU delivery to adult rabbits of which limbs were fixed to the operating table. This is because it was covered by flat breast bone, and HIFU reflected the heart of the rabbit. We tried HIFU delivery in a state exposed with rabbit's heart by a median section and under the environment that which heart moves during the pulmonary respiration by the respirator unlike intrauterine fetuses. In the case of practical intrauterine fetuses, their undeveloped breast bone does not damp HIFU and they don't breathe with lung. We performed this experiment in the following procedures strictly in accordance with the rule that the animal executive committee in National Center for Child Health and

1. Four adult rabbits (Japanese White, 2.8 kg, male) were anesthetized with xylazine (5

2. After endotracheal intubation or tracheostomy placement, anesthesia was maintained on the mechanical ventilation with isoflurane and oxygen inhalation (20 cycles/min,

3. ECG, arterial blood pressure/oxygen saturation, and end-tidal carbon dioxide

4. The animals underwent median sternotomy to expose the heart, chest cavities were filled with buffering gel on both sides. The heart beats was set in the direct contact with

5. A HIFU transducer combined with a diagnostic 2D-US imaging probe was fixed on a two-directional (X-Y) linear stage with a pivot hinge placed on the tank which was manually steered, so that the HIFU focal point could be roughly located on the atrial

8. After HIFU delivery, we confirm whether new blood flows is generated across the atrial septum using color Doppler echocardiography by the diagnostic 2D-US imaging

a silicone sheet-bottomed tank filled with degassed water (37◦C ) (Fig. 9).

anesthesia. Their bodies are slightly larger than our practical target fetus.

#### **2.5 Security condition for HIFU delivery**

HIFU delivery is conducted based on the timing when the left ventricle shrinks most in consideration of the delay time in the system. In order to avoid some issues, caused by various factors and disturbances, to deliver HIFU outside of the allowable circle The system is permitted to delivery HIFU as far as meeting following security conditions so as to avoid "shooting by mistake", which means that HIFU delivery is outside the allowable limit of error radius and ablated point is overshot the actual atrial septum, even if the point is inside the allowable limit of error radius (Fig. 8).

Fig. 8. Monitoring of HIFU delivery condition defined by focal accordance, area of LV, heartbeat synchronous and circularity of LV.

These conditional thresholds are settable individually, and we can select any conditions. Delivery frequency decreases if the conditions are strict. On the other hand, it increases if the conditions are easy. However, we have only a rough index for optimal parameters because individual difference is large by the delivered target of HIFU. Therefore, it is necessary to judge during securing safety as the trigger off for HIFU delivery while watching the temporal change in HIFU delivery condition on the display whether you can irradiate it more effectively, the parameters are appropriate, or more effective delivery is possible.

#### **3.** *In vivo* **experiment**

#### **3.1 Purpose**

46 Atrial Septal Defect

HIFU delivery is conducted based on the timing when the left ventricle shrinks most in consideration of the delay time in the system. In order to avoid some issues, caused by various factors and disturbances, to deliver HIFU outside of the allowable circle The system is permitted to delivery HIFU as far as meeting following security conditions so as to avoid "shooting by mistake", which means that HIFU delivery is outside the allowable limit of error radius and ablated point is overshot the actual atrial septum, even if the point is inside

Fig. 8. Monitoring of HIFU delivery condition defined by focal accordance, area of LV,

3. Success or failure of the heartbeat cycle detection by fast Fourier transformation

1. The upper limit of movement distance of the left ventricular geometric center of gravity

4. The minimum value of the ellipsoid roundness, which is approximate shape of the left

5. Whether an estimated position of the atrial septum is inside focal allowance limits of

These conditional thresholds are settable individually, and we can select any conditions. Delivery frequency decreases if the conditions are strict. On the other hand, it increases if

**2.5 Security condition for HIFU delivery**

the allowable limit of error radius (Fig. 8).

heartbeat synchronous and circularity of LV.

ventricle

error or not

2. The maximum value of the left ventricle area

In this experiment, we confirmed feasibility of our computer-aided automatic delivery system of HIFU to perform atrial septal defect creation of animals in vivo. We used four healthy animals (Japanese white rabbit, male, 2.8 kg) with cardiac pulsation under anesthesia. Their bodies are slightly larger than our practical target fetus.

#### **3.2 Methods**

We tested HIFU delivery to adult rabbits of which limbs were fixed to the operating table. This is because it was covered by flat breast bone, and HIFU reflected the heart of the rabbit. We tried HIFU delivery in a state exposed with rabbit's heart by a median section and under the environment that which heart moves during the pulmonary respiration by the respirator unlike intrauterine fetuses. In the case of practical intrauterine fetuses, their undeveloped breast bone does not damp HIFU and they don't breathe with lung. We performed this experiment in the following procedures strictly in accordance with the rule that the animal executive committee in National Center for Child Health and Development.

Computer-Aided Automatic Delivery System

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 49

(A)

(B)

(C)

(D) Fig. 10. Heart specimens ablated with HIFU delivery. Left images are gross appearances of the inadvertent ablation with opening. Right images are magnifications of the area ablated by HIFU deliveries. (A) A small opening was made in the posterior wall of the left atrium. (B) Atrial septum apparently remained non-penetrated with mere coagulation changes. (C) Non-penetrated small hole was made on the atrial septum with hematoma formation. (D)

Non-penetrated small hole was made on the atrial septum accurately.

9. After this experiment, we inject pentobarbital into a peripheral venous path and sacrifice the rabbits.

Fig. 9. Experimental setup using an adult rabbit. HIFU delivery was done through a silicone sheet at the bottom of the degassed water tank.

#### **3.3 HIFU delivery condition**

The waveform output from a function generator is observable with an oscilloscope, which is amplified by RF power amplifier and is input to a transducer. In this experiment, the input waveform was sine wave of 3.3 MHz, 160 Vpp and 6.5 kW/cm2.

For the total HIFU delivery time in one trial, we set three seconds from experiences of the prior pilot study. The allowable limit of error radius of HIFU delivery spot is set to the same value in all trial so that it was with the smallest circle which a focus shape of HIFU was fit into on a display.

9. After this experiment, we inject pentobarbital into a peripheral venous path and

Fig. 9. Experimental setup using an adult rabbit. HIFU delivery was done through a silicone

The waveform output from a function generator is observable with an oscilloscope, which is amplified by RF power amplifier and is input to a transducer. In this experiment, the input

For the total HIFU delivery time in one trial, we set three seconds from experiences of the prior pilot study. The allowable limit of error radius of HIFU delivery spot is set to the same value in all trial so that it was with the smallest circle which a focus shape of HIFU was fit

sheet at the bottom of the degassed water tank.

waveform was sine wave of 3.3 MHz, 160 Vpp and 6.5 kW/cm2.

**3.3 HIFU delivery condition**

into on a display.

sacrifice the rabbits.

Fig. 10. Heart specimens ablated with HIFU delivery. Left images are gross appearances of the inadvertent ablation with opening. Right images are magnifications of the area ablated by HIFU deliveries. (A) A small opening was made in the posterior wall of the left atrium. (B) Atrial septum apparently remained non-penetrated with mere coagulation changes. (C) Non-penetrated small hole was made on the atrial septum with hematoma formation. (D) Non-penetrated small hole was made on the atrial septum accurately.

Computer-Aided Automatic Delivery System

ECG together in near future.

future.

**4.3 In vivo experiment**

**4.2 Estimation of atrial septum**

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 51

is longer than ECG signal analysis. This is because arrhythmic development is expected, and heartbeat cycle collapses with a diseased heart. It is better that the delay time is short to detect arrhythmic development quickly. We had a plan to introduce the system with fetal

About the estimation of the atrial septum position, although the surgeon confirmed it by monitoring before HIFU delivery in vivo experimental qualitatively, ablated point was not so overshot the atrial septum, of which position was estimated from 2D-US image. However, when the 2D-US image was not clear, the left ventricular position and shape were not obtained precisely. There were some cases that estimation of the atrial septum was impossible. Consequently, HIFU delivery was not permitted by security conditions in this case. We were able to avoid shooting by mistake outside the allowable limit of error radius in 2D-US image. Also, we calculated geometric ratio between the left ventricular center of gravity position and the approximate fillet diameter for "correct answer" which a surgeon sets by manual operation as template information, however the procedure is limited on 2D plane. We don't consider that the heartbeat includes movement and transformation (expansion, shrinkage and distortion) not only in 2D plane (X-Y plane), but also in the depth direction (Z-axis direction). It depends on how detection of the heartbeat and the estimation of the atrial septum position are accurate what appropriate four chamber view is acquired. The detection of the heartbeat from the extraction of the left ventricular area on 2D-US image is no more than approximation. Even if the focal point is inside allowable limit of error radius in 2D-US image, the HIFU delivery results in shooting by mistake threedimensionally because prior confirmation of the HIFU focal size and shape, fixation of the HIFU delivery device, and setting of the parameters about HIFU delivery condition were performed on the basis of 2D-US image only. This is the major reason of the HIFU delivery position error. For the solution of this issue, at least when HIFU delivery device is fixed on target we should use a three-dimensional ultrasound (3D-US) probe to recognize detail of 3D positional relationship between the focal point and the actual atrial septum in beating heart for more accurate focal position alignment. It is expected that performance of the 3D-US diagnostic equipment improves to acquire and analyze 3D-US voxel data in real time directly, which leads drastically higher precision of HIFU delivery with our system in near

In addition, the real intrauterine fetus is floating in the amniotic fluid and is movable threedimensionally. During surgery, the fetus doesn't exercise spontaneously because both mother and fetus are under anesthesia, however we have to track not only movement of the atrial septum but also the fetal movement of oneself. That is to say in addition to 2D-US for four chamber view of the heart. We need 3D-US to track movement of intrauterine fetus three-dimensionally. At the same time it is important to move HIFU delivery device itself

We tried HIFU delivery to the animals which imitated intrauterine fetuses, however the results didn't achieved both delivery efficiency and delivery accuracy of HIFU. The only

mechanically to track fetal wide movement in utero (Koizumi et al., 2008).

The HIFU delivery began at the moment when a surgeon pushed the button for beginning of automatic delivery on a display and came back to the standby state when 3-seconds HIFU delivery was completed in total.

#### **3.4 Results**

Four rabbits were euthanized after HIFU delivery, and dissected their isolated hearts and observed around the atrial septum (Fig. 10(A) - (D)). In the first trial, it took 23 seconds from beginning of automatic HIFU delivery to the end (Fig. 10(A)). The penetration of the tissue in the chamber was completed, however the position was missed from the atrial septum toward posterior parietal side, which is the left atrium side. In the second trial, it took 57 seconds by the end (Fig. 10(B)). A cauterization spot was found on the atrial septum in pinpoint, however the HIFU delivery did not achieve a penetration. In the third trial, it took 44 seconds by the end (Fig. 10(C)). The perforation on the atrial septum was confirmed, however it did not achieved a penetration. In addition a hematoma-like change was found beside a perforation spot. In the last trial, it took 46 seconds by the end (Fig. 10(D)). The perforation on the atrial septum was confirmed, however it did not achieve a penetration. Unlike the third trial the hematoma was not found.

#### **4. Discussion**

In this study, we developed automatic delivery system of HIFU based on real-time 2D-US imaging analysis. In in vivo experiment we confirmed pinpoint delivery of HIFU to the pulsating atrial septum within beating hearts of anesthetized adult rabbits.

In the field of cardiology, HIFU was investigated as a promising device to treat arrhythmia, relieve valvular stenosis and ameliorate obstructive hypertrophic cardiomyopathy. HIFU also enables us to create defects in cardiac tissues such as ventricles or cusp of the aortic valve in vitro or ex vivo (Otsuka et al., 2005; Fujikura et al., 2006; Lee et al., 2000; Xu et al., 2004; Strickberger et al., 1999). Particularly in fetal cardiac intervention, less invasiveness of HIFU is effective for the mother as well. In addition, HIFU delivery can be a actual operative method with security, a short time and low cost. Our new system is useful for an approach into the heart chamber with more safety owing to detection of a heartbeat cycle only from 2D-US four chamber view without other measurement instruments and estimation of atrial septum position pulsating with heartbeats and breathing for more effective HIFU delivery.

#### **4.1 Detection of heartbeat**

In order to obtain heartbeat information, it is common to use an electrocardiogram (ECG). There is a reported study to try the treatment of arrhythmias using the HIFU delivery triggered by ECG (Strickberger et al., 1999). However, it is difficult to isolate only ECG of fetus from a maternal ECG definitely although fetal ECG monitoring is technically quite demanding because ECG of fetus is obtained only through the mother. Therefore, the method to detect the heartbeat cycle of fetus only from real-time 2D-US image in our system is thought to be appropriate for clinical application from convenient point of view. However, based on the image processing, the amount of calculation is larger and delay time is longer than ECG signal analysis. This is because arrhythmic development is expected, and heartbeat cycle collapses with a diseased heart. It is better that the delay time is short to detect arrhythmic development quickly. We had a plan to introduce the system with fetal ECG together in near future.

#### **4.2 Estimation of atrial septum**

50 Atrial Septal Defect

The HIFU delivery began at the moment when a surgeon pushed the button for beginning of automatic delivery on a display and came back to the standby state when 3-seconds HIFU

Four rabbits were euthanized after HIFU delivery, and dissected their isolated hearts and observed around the atrial septum (Fig. 10(A) - (D)). In the first trial, it took 23 seconds from beginning of automatic HIFU delivery to the end (Fig. 10(A)). The penetration of the tissue in the chamber was completed, however the position was missed from the atrial septum toward posterior parietal side, which is the left atrium side. In the second trial, it took 57 seconds by the end (Fig. 10(B)). A cauterization spot was found on the atrial septum in pinpoint, however the HIFU delivery did not achieve a penetration. In the third trial, it took 44 seconds by the end (Fig. 10(C)). The perforation on the atrial septum was confirmed, however it did not achieved a penetration. In addition a hematoma-like change was found beside a perforation spot. In the last trial, it took 46 seconds by the end (Fig. 10(D)). The perforation on the atrial septum was confirmed, however it did not achieve a penetration.

In this study, we developed automatic delivery system of HIFU based on real-time 2D-US imaging analysis. In in vivo experiment we confirmed pinpoint delivery of HIFU to the

In the field of cardiology, HIFU was investigated as a promising device to treat arrhythmia, relieve valvular stenosis and ameliorate obstructive hypertrophic cardiomyopathy. HIFU also enables us to create defects in cardiac tissues such as ventricles or cusp of the aortic valve in vitro or ex vivo (Otsuka et al., 2005; Fujikura et al., 2006; Lee et al., 2000; Xu et al., 2004; Strickberger et al., 1999). Particularly in fetal cardiac intervention, less invasiveness of HIFU is effective for the mother as well. In addition, HIFU delivery can be a actual operative method with security, a short time and low cost. Our new system is useful for an approach into the heart chamber with more safety owing to detection of a heartbeat cycle only from 2D-US four chamber view without other measurement instruments and estimation of atrial septum position pulsating with heartbeats and breathing for more

In order to obtain heartbeat information, it is common to use an electrocardiogram (ECG). There is a reported study to try the treatment of arrhythmias using the HIFU delivery triggered by ECG (Strickberger et al., 1999). However, it is difficult to isolate only ECG of fetus from a maternal ECG definitely although fetal ECG monitoring is technically quite demanding because ECG of fetus is obtained only through the mother. Therefore, the method to detect the heartbeat cycle of fetus only from real-time 2D-US image in our system is thought to be appropriate for clinical application from convenient point of view. However, based on the image processing, the amount of calculation is larger and delay time

pulsating atrial septum within beating hearts of anesthetized adult rabbits.

delivery was completed in total.

Unlike the third trial the hematoma was not found.

**3.4 Results**

**4. Discussion**

effective HIFU delivery.

**4.1 Detection of heartbeat**

About the estimation of the atrial septum position, although the surgeon confirmed it by monitoring before HIFU delivery in vivo experimental qualitatively, ablated point was not so overshot the atrial septum, of which position was estimated from 2D-US image. However, when the 2D-US image was not clear, the left ventricular position and shape were not obtained precisely. There were some cases that estimation of the atrial septum was impossible. Consequently, HIFU delivery was not permitted by security conditions in this case. We were able to avoid shooting by mistake outside the allowable limit of error radius in 2D-US image. Also, we calculated geometric ratio between the left ventricular center of gravity position and the approximate fillet diameter for "correct answer" which a surgeon sets by manual operation as template information, however the procedure is limited on 2D plane. We don't consider that the heartbeat includes movement and transformation (expansion, shrinkage and distortion) not only in 2D plane (X-Y plane), but also in the depth direction (Z-axis direction). It depends on how detection of the heartbeat and the estimation of the atrial septum position are accurate what appropriate four chamber view is acquired. The detection of the heartbeat from the extraction of the left ventricular area on 2D-US image is no more than approximation. Even if the focal point is inside allowable limit of error radius in 2D-US image, the HIFU delivery results in shooting by mistake threedimensionally because prior confirmation of the HIFU focal size and shape, fixation of the HIFU delivery device, and setting of the parameters about HIFU delivery condition were performed on the basis of 2D-US image only. This is the major reason of the HIFU delivery position error. For the solution of this issue, at least when HIFU delivery device is fixed on target we should use a three-dimensional ultrasound (3D-US) probe to recognize detail of 3D positional relationship between the focal point and the actual atrial septum in beating heart for more accurate focal position alignment. It is expected that performance of the 3D-US diagnostic equipment improves to acquire and analyze 3D-US voxel data in real time directly, which leads drastically higher precision of HIFU delivery with our system in near future.

In addition, the real intrauterine fetus is floating in the amniotic fluid and is movable threedimensionally. During surgery, the fetus doesn't exercise spontaneously because both mother and fetus are under anesthesia, however we have to track not only movement of the atrial septum but also the fetal movement of oneself. That is to say in addition to 2D-US for four chamber view of the heart. We need 3D-US to track movement of intrauterine fetus three-dimensionally. At the same time it is important to move HIFU delivery device itself mechanically to track fetal wide movement in utero (Koizumi et al., 2008).

#### **4.3 In vivo experiment**

We tried HIFU delivery to the animals which imitated intrauterine fetuses, however the results didn't achieved both delivery efficiency and delivery accuracy of HIFU. The only

Computer-Aided Automatic Delivery System

44, Issue 4, pp. 339–346

No. 1, pp. 89–97

pp. 693–701

Circulation, Vol. 113, pp. 1401–1405

& Biology, Vol. 31, Issue 1, pp. 109–114

ultrasound,Circulation, Vol. 100, pp. 203–208

Physics, Vol. 29, Issue 11, pp. 2611–2611

**7. References**

of High-Intensity Focused Ultrasound for Creation of an Atrial Septal Defect 53

Chan, AH., Fujimoto, VY., Moore, DE., Martin, RW. & Vaezy, S.(2002). An image-guided

Fujikura, K., Otsuka, R., Kalisz, A., Ketterling, JA., Jin, Z., Sciacca, RR., Marboe, CC.,Wang,

Hengst, SA., Ehrenstein, T., Herzog, H., Beck, A., Utz-Billing, I., David, M., Felix, R., Ricke,

Kohl, T., Sharland, G., Alllan, LD., Gembruch,U., Chaoui, R., Lopes, LM., Zielinsky, P.,

Lee, LA., Simon, C., Bove, LE., Mosca, RS., Ebbini, ES., Abrams, GD. & Ludomirsky,

Marshall, AC., van der Velde, ME., Tworetzky,W., Gomez, CA., Wilkins-Haung, L.,

Otsuka, R., Fujikura, K., Hirata, K., Pulerwitz, T., Oe, Y., Suzuki, T., Sciacca, R., Marboe, C.,

Rebillard, X., Gelet, A., Davin, JL., Soulie, M., Prapotnich, D., Cathelineau, X., Rozet, F.

Strickberger, SA., Tokano, T., Kluiwstra, JUA., Morady, F. & Cain, C.(1999). Extracardiac

obstruction, American Journal of Cardiology, Vol. 85, pp. 1230–1233 Koizumi, N., Ota, K., Lee, D., Yoshizawa, S., Ito, A., Kaneko, Y., Yoshinaka, K., Matsumoto,

Clinical application. Echocardiography, Vol. 17, Issue 6, pp. 563–566 Makikallio, K., McElhinney, DB., Levine, JC., Marx, GR., Colan, SD., Marshall, AC., Lock JE.,

restrictive atrial septum, Circulation, Vol. 110, pp. 253–258

Journal of Ultrasound in Medecine, Vol. 25, No. 11, pp. 1375–1386

high intensity focused ul-trasound device for uterine fibroids treatment, Medical

J., Muratore, R., Feleppa, EJ. & Homma, S.(2006). Effects of ultrasonic exposure parameters on myocardial lesions induced by high-intensity focused ultrasound,

J.(2004), Magnetic resonance tomography guided focused ultrasound surgery (MRgFUS) in tumor therapy--a new noninvasive therapy option, Radiology, Vol.

Huhta, J. & Silverman, NH.(2000). World experience of percutaneous ultrasoundguided balloon valvuloplasty in human fetuses with severe aortic valve

Y., & Mitsuishi, M.(2008). Feed-forward controller for the integrated non-invasive ultrasound diagnosis and treatment. Journal of Robotics and Mechatronics, Vol. 20,

A.(2000). High intensity focused ultrasound effect on cardiac tissues: Potential for

Marcus, EN. & Tworetzky, W.(2006). Fetal Aortic valve stenosis and the evolution of hypoplastic left heart syndrome patient selection for fetal intervention,

Benson,CB., Jennings, RW. & Lock, JE.(2004). Creation of an atrial septal defect in utero for fetuses with hypoplastic left heart syndrome and intact or highly

Wang, J., Burkhoff, D., Muratore, R., Lizzi, FL. & Homma, S.(2005). In vitro ablation of cardiac valves using high-intensity focused ultrasound, Ultrasound in Medicine

& Vallancien, G.(2005). Transrectal high-intensity focused ultrasound in the treatment of localized prostate cancer, Journal of Endourology, Vol. 2005, Issue 6,

ablation of the canine atrioventricular junction by use of high-intensity focused

one trial perforated tissue in the beating chamber, and the perforated point was on the adjacent tissue of the atrial septum. On the other hand, the three trials ablated just the atrial septum, and no perforations were generated. The HIFU delivery efficiency depends on the ratio of delivery time in one heartbeat and the total time to complete preset HIFU delivery in each trial. In all trials, we set the same delivery conditions on a respirator, size (weight) of the animal, the allowable limit of error radius around HIFU focal point, and the total HIFU delivery time. However, there were individual differences in animals' heartbeat cycle and 2D-US image of the left ventricle, and also, we found small differences in relative position relationship between animal body and HIFU delivery device because we made adjustments to delivery setting in each trial. Therefore, the variation occurred in the total time to complete preset HIFU delivery in each trial. To increase the safety, the shorter delivery ratio in one heartbeat and more strict security conditions are required. However, if the total time lead to longer to complete preset HIFU delivery, the one HIFU delivery interval gets longer, which results in no perforation of the tissue due to diffusion of converged HIFU energy into intracardiac blood flow. HIFU delivery frequency around 3.3 MHz may cause heating effect and/or mechanical (cavitation) effect. If the perforation of tissue is caused by only mechanical effect of cavitation, we need not consider the diffusion of the HIFU energy in to the blood flow. In order to avoid cooling effect by the blood flow, it is more useful to take advantege of mechanical effect of cavitation positively. However, it is necessary to progress clarification about which is dominant heating effect or mechanical effect of cavitation by detailed simulation and local observation around the focal point. When heating effect is dominant, we should lengthen the delivery ratio in one heartbeat to raise HIFU delivery efficiency and make continuous HIFU delivery by loose of security conditions. However, the security of HIFU delivery tuens down by contraries and the possibility is reduced to be delivered in the point that we aim at. Assurance of security is in a relation of the trade-off with HIFU delivery efficiency, and balancing both is not easy. For safe and secure HIFU delivery we will progress further validation to investigate optimal HIFU delivery conditions from both hardware and software point of view.

#### **5. Conclusion**

We developed computer-aided automatic delivery system of HIFU for creation of an atrial septal defect which perforates tissue in the intracardiac chamber for less invasive cardiac intervention of intrauterine fetuses. In vivo experiment using animals, by only 2D-US image of four chamber view, it was possible to detect heartbeat period, estimate the atrial septum position, and determine HIFU delivery timing in real-time under heartbeats.

In near future, we will aim at the clinical application as a minimal invasive surgical system in order to improve precision improvement of the focus positioning of HIFU delivery and improvement of HIFU energy efficiency to intracardiac tissue.

#### **6. Acknowledgement**

We wish to thank Akiko Suzuki for help in preparing the manuscript. And a part of this work was supported by Grant Program for Child Health and Development (16-3) administrated by Ministry of Health, Labour and Welfare of Japan.

#### **7. References**

52 Atrial Septal Defect

one trial perforated tissue in the beating chamber, and the perforated point was on the adjacent tissue of the atrial septum. On the other hand, the three trials ablated just the atrial septum, and no perforations were generated. The HIFU delivery efficiency depends on the ratio of delivery time in one heartbeat and the total time to complete preset HIFU delivery in each trial. In all trials, we set the same delivery conditions on a respirator, size (weight) of the animal, the allowable limit of error radius around HIFU focal point, and the total HIFU delivery time. However, there were individual differences in animals' heartbeat cycle and 2D-US image of the left ventricle, and also, we found small differences in relative position relationship between animal body and HIFU delivery device because we made adjustments to delivery setting in each trial. Therefore, the variation occurred in the total time to complete preset HIFU delivery in each trial. To increase the safety, the shorter delivery ratio in one heartbeat and more strict security conditions are required. However, if the total time lead to longer to complete preset HIFU delivery, the one HIFU delivery interval gets longer, which results in no perforation of the tissue due to diffusion of converged HIFU energy into intracardiac blood flow. HIFU delivery frequency around 3.3 MHz may cause heating effect and/or mechanical (cavitation) effect. If the perforation of tissue is caused by only mechanical effect of cavitation, we need not consider the diffusion of the HIFU energy in to the blood flow. In order to avoid cooling effect by the blood flow, it is more useful to take advantege of mechanical effect of cavitation positively. However, it is necessary to progress clarification about which is dominant heating effect or mechanical effect of cavitation by detailed simulation and local observation around the focal point. When heating effect is dominant, we should lengthen the delivery ratio in one heartbeat to raise HIFU delivery efficiency and make continuous HIFU delivery by loose of security conditions. However, the security of HIFU delivery tuens down by contraries and the possibility is reduced to be delivered in the point that we aim at. Assurance of security is in a relation of the trade-off with HIFU delivery efficiency, and balancing both is not easy. For safe and secure HIFU delivery we will progress further validation to investigate optimal HIFU delivery conditions from both

We developed computer-aided automatic delivery system of HIFU for creation of an atrial septal defect which perforates tissue in the intracardiac chamber for less invasive cardiac intervention of intrauterine fetuses. In vivo experiment using animals, by only 2D-US image of four chamber view, it was possible to detect heartbeat period, estimate the atrial septum

In near future, we will aim at the clinical application as a minimal invasive surgical system in order to improve precision improvement of the focus positioning of HIFU delivery and

We wish to thank Akiko Suzuki for help in preparing the manuscript. And a part of this work was supported by Grant Program for Child Health and Development (16-3)

position, and determine HIFU delivery timing in real-time under heartbeats.

improvement of HIFU energy efficiency to intracardiac tissue.

administrated by Ministry of Health, Labour and Welfare of Japan.

hardware and software point of view.

**5. Conclusion**

**6. Acknowledgement**

**Section 4**

**Transcatheter Closure of ASD**


**7. Future perspectives including economic aspects of genetic test for statin intolerance**

Implementation of a genetic test for statin intolerance into routine practice definitely requires analysing its benefits not only for patients but also for health care providers. In this context, pharmacoecomic data on genetic testing statin intolerance have been scarce. The existing literature on cost-effectiveness of pharmacogenetic testing has been either general [58] or described economic savings solely due to hypolipidemic effect of statins [59]. The first specific data for statin intolerance and its genetic testing appeared only very recently [60]–the authors estimated 356 Canadian dollars as the cost limit for economic feasibility and at the same time dominant health effect for cardiovascular prevention. In extension of this very first report [60], this topic should be, therefore, addressed more intensively and also from other angles in the future. This has been the case with other pharmacogenetic applications (e.g. [61]), it will be also innovative to use new approaches which utilise alternative parameters for assessing effectiveness (e.g. [62–64]).

Though important, inclusion of economic criteria is the only one part of the future priorities in the field of application of genetic variation for testing statin intolerance. Other avenues for future may address (1) further search for and verification of other genetic markers than *SLCO1B1* including providing pharmacokinetic data [65, 66], (2) reflection of ethnic differences in distribution of genetic markers between populations [64, 67], (3) inclusion of the results of genetic test into electronic medical records [68], (4) performing meta-analyses of studies reported so far, and last but not least, (5) performance of well-designed clinical studies implementing also other non-genetic criteria in order to propose a risk-score or clinically applicable algorithm. The existing examples from other pharmacogenetic applications (e.g. [69]) and above described initiatives such as U-PGX [55], RNPGX [56], or the recent idea to provide patients with their DNA (pharmacogenetic) passport [70], allow us to expect further developments targeted at patient benefit and innovation of medical care.

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College of Cardiology. 2017;**69**(11):1396-1398. DOI: 10.1016/j.jacc.2017.01.019


2.3.3. UPLC-MS/MS Analysis

The prepared samples were analyzed by ultra-performance liquid chromatography. The Nexera X2UPLC-MS/MS (Shimadzu Corp., Kyoto, Japan) contained two LC-30AD pumps, SiL-30AC autosampler, CTO-20AC column oven, CBM-20A communication bus module, and mass spectrometer LC-MS8050 with electro spray ionization with positive and negative ion mode (Table 1). Chromatographic separation was performed using an analytical column Kinetex 2.6 μm, Phenyl-Hexyl 100 Å 4.6 mm, 150 mm (Phenomenex, Torrance, CA, USA). The gradient program consisted of the following: 7-min sequence of linear gradient flows of solvent B (acetonitrile:methanol 1:1 *v*/*v*) balanced with solvent A (water with 0.1% formic acid) at a flow rate of 0.6 mL/min: 50–80% B over 1 min, 80–100% B over 2 min, isocratic 100% B for 3 min, and finally, 100–50% B over 1 min. The injection volume was 1 μL and column temperature was 40 ◦C.

The main parameters used to identify analytes were their retention times and multiple reaction monitoring (MRM) ratio, which were obtained at 0.25 μg/mL working standard solutions for most standards, and 2.5 μg/mL for AMP, IB, SPEC, STREP, SFC, SNA and TET. Chromatographic data processing was carried out using LabSolution® software (Shimadzu Corp., Kyoto, Japan).

**Table 1.** The analysis parameters for the monitored ion transitions and triple quadruple mass spectrometer (MS/MS) operation (CE- collision energy, Q1 and Q3Pre Bias—quadruple pre-rod bias voltage).

#### 2.3.4. Quality Assurance/Quality Control (QA/QC)

Ensuring the quality of the measurements results was carried out by:

The calibration step involves the preparation of calibration curves and calculation of response factor (RF) for internal standards. Eleven working standard solutions have been prepared. Limit of determination (LOD) has been calculated from the parameters of a calibration curve constructed on the basis of the three lowest concentrations of working standards solutions. The limit of determination has been calculated according to the formula:

$$LOD = \frac{3.3 \times s}{a} \tag{1}$$

where: a—slope of the calibration curve; s—the standard deviation.

For the calculation of the validation parameters, residual standard deviation (sxy) and standard deviation of the intercept (sa) were taken. Based on LOD(sxy) and LOD(sa) the mean values (LOD) were calculated (see Equation (1)). The coefficients of variation (CV) and uncertainty (U) were calculated based on standard deviations and the average area of the chromatographic peaks obtained during separation of standard mixtures.

To check the matrix effect, two samples were prepared using real soil samples with addition of standards, and using the procedure described in Section 2.3.2. Sample Pretreatment and additionally using solide phase extraction (SPE) cleaning. Based on peak areas, the recovery (%) for standard solutions was calculated.

#### *2.4. Antibiotic Resistance in Microorganisms Isolated from Soil*

The soil samples (1–6A; 1B and 1C, Figure 1) were plated on different growth media for evaluation of microbial growth (Luria Bertani Agar—LA, MacConkey Agar, Chapman Agar, Medium with cetrimide, Merck, Darmstadt, Germany). Briefly, 10 g of the sample was mixed with 90 mL of 0.85% NaCl and shaken for 0.5 h, and then serially diluted to 10−4. In addition, 100 μL of each dilution was plated on LA, while solely the dilution 10−1 was spread on MacConkey Agar, Chapman Agar and Medium with cetrimide. The plates were incubated at 30 ◦C for 24–72 h to obtain sufficient growth of the colonies. From each sample, several differently-looking colonies were picked and grown to pure cultures on LA, and then stored frozen with 20% glycerol at −40 ◦C.

The isolated bacteria were then subjected to antibiotic resistance disc diffusion test with different antibiotics (TMP 5 μg, SMZ 20 μg, TET 10 μg, SPEC 25 μg, and CIP 10 μg). TET, SPEC, and CIP discs were purchased from Oxoid, UK, while trimethoprim and sulfametazine discs were prepared in the laboratory. Briefly, the cotton discs were cut from MN85/220 paper filters (Macherey-Nagel, Germany) and autoclaved. The appropriate amount of the antibiotic stock solution was poured onto the paper disk with automatic pipette and let dry. For the antibiotic resistance disc test, the bacteria were cultured in LB (Luria Bertani boullion, Btl, Łód´z, Poland) with 150 rpm shaking at 30 ◦C, overnight. Then, the OD600 measurements of the bacterial cultures were taken with Spectroquant Prove 600 (Merck, Darmstadt, Germany) spectrophotometer and diluted in LB to achieve OD600 equal to 0.1. The dilution accurateness was verified with repeated spectrophotometer measurements. In addition, 100 μL of each bacterial suspension was spread on a plate with Muiller-Hinton medium (Graso, Owidz, Poland). Discs with antibiotics were then put on the surface of the plate and the plates were incubated for 24 h at 30 ◦C. Afterwards, the diameter of the growth inhibition zone around each antibiotic disc was measured.