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Pediatric Anesthesiology: Research Report

A Novel Method for Ultrasound-Guided Radial Arterial Catheterization in Pediatric Patients

Nakayama, Yoshinobu MD*; Nakajima, Yasufumi MD, PhD*; Sessler, Daniel I. MD; Ishii, Sachiyo MD*; Shibasaki, Masayuki MD, PhD*; Ogawa, Satoru MD*; Takeshita, Jun MD*; Shime, Nobuaki MD, PhD; Mizobe, Toshiki MD, PhD*

Author Information
doi: 10.1213/ANE.0000000000000164
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Percutaneous radial arterial catheterization is routinely used to monitor hemodynamic status and facilitate blood sampling, with catheter insertion typically guided by palpation. However, palpation alone is sometimes insufficient in patients who are hemodynamically unstable, obese, or edematous; furthermore, palpation alone often fails in pediatric patients. Even trained and experienced anesthesiologists thus often find pediatric percutaneous radial arterial catheterization difficult.

In adults, ultrasound-guided percutaneous radial arterial catheterization improves first-attempt and overall catheterization success and shortens insertion time, irrespective of experience.1,2 Although ultrasound guidance has been used in pediatric patients3 and reportedly improves first-attempt success as compared with Doppler-assisted technique,4 this procedure still requires considerable training.5–7 Factors other than operator experience may also markedly affect the success of real-time ultrasound-guided percutaneous radial arterial catheterization in pediatric patients.

We report here the results from a prospective observational study for determining the factors that best predict successful real-time ultrasound-guided percutaneous radial arterial catheterization in pediatric patients. We also report the results of a randomized trial that evaluated a novel modification to the ultrasound technique based on the strongest predictor of catheterization success from our initial observational study.


Our studies were approved by the Kyoto Prefecture University of Medicine IRB, Kyoto, Japan and registered at UMIN Clinical Trials Registry as UMIN000009803 (January 18, 2013). Written informed consent was obtained from the guardians of all patients. We enrolled pediatric patients (<3 years old), having elective surgery in the Kyoto Prefecture University of Medicine in 2013, who required radial arterial catheterization. We excluded patients with an abnormal Allen test, congenital aortic disease (e.g., coarctation of aorta, patent ductus arteriosus), and malformation of forearm arteries (hypoplastic or absent)8 during prescanning with ultrasound.

Two Japanese Society of Anesthesiologist Board-certified anesthesiologists performed the arterial catheterizations. Each had previously performed >50 ultrasound-guided percutaneous arterial catheterizations in pediatric patients using the out-of-plane approach where the needle is advanced based on the short axis of the ultrasound probe. Each anesthesiologist performed ultrasound-guided radial artery catheterization with the out-of-plane approach in 15 patients with similar clinical characteristics (diameter of the artery, subcutaneous arterial depth, catheter size, etc.), and the catheterization time was evaluated. The intraclass correlation coefficient was 0.90 (95% confidence interval [CI], 0.70–0.97; P < 0.001), indicating good reliability.

Arterial catheterization was attempted after induction of general anesthesia. A roll was placed under the puncture site, and the arm was taped to maintain optimal extension. The wrist was dorsiflexed at approximately 45°.9 The puncture site was cleaned with povidone-iodine, observing the standard precautions for percutaneous arterial catheterization.

We used Sonosite M-turbo Ultrasound System (FUJIFILM SonoSite Japan, Inc., Japan) for ultrasound examinations. A SLAx/13–6 MHz transducer (hockey-stick type) in a sterile cover was positioned at the puncture site such that the artery was visualized in the short axis. A 24-gauge catheter (Jelco Plus, Smiths Medical Japan Ltd., Japan) was inserted 1 to 2 mm distal to the transducer at a 10° to 30° puncture angle, adjusting the catheter tip toward the center of the anterior wall of the radial artery.

We advanced the catheter until the anterior wall was seen to collapse, and the catheter tip appeared on the display. The presence of blood was confirmed in the catheter hub, and the catheter was advanced slightly at a reduced angle in an effort to avoid the posterior wall of the artery. On removing the inner stylet, if blood flow continued, we replaced the inner stylet and advanced the catheter into the artery, threading it off the needle; if the blood flow disappeared or when the puncture pressure completely collapsed the artery, we used a through-and-through approach. Specifically, we withdrew the stylet and then slowly withdrew the catheter tip until a flash of blood was visible in the catheter or hub. After confirmation of blood flow, we partially reinserted the stylet to stiffen the cannula and then advanced the catheter into the artery. No other methods (e.g., bevel-down approach or in-plane approach) or materials (e.g., guidewires) were used. Catheterization was considered complete when arterial blood flow was confirmed after inserting the total length of the catheter.

We recorded the following clinical and demographic variables: height, weight, age, sex, ASA physical status, Risk Adjusted classification for Congenital Heart Surgery (RACHS-1 scoring system),10–12 presence of cyanosis (right-to-left shunt), presence of pulmonary hypertension (left-to-right shunt), trisomy 21, diameter of the artery, subcutaneous arterial depth, insertion time, number of attempts in each trial (until successful insertion or until catheterization was abandoned), and overall success or failure of catheterization. Arterial blood pressure was measured at the brachial artery using a blood pressure cuff immediately before the catheterization attempt.

Arterial diameter was considered as the distance in the short-axis plane between the trailing and leading edges of the artery, as recommended by the American Society of Echocardiography, during 2-dimensional imaging.13 Arterial depth was considered as the distance from the transducer to the near edge of the artery on 2-dimensional imaging.

All the patients’ clinical and demographic measurements, including insertion time and ultrasound measurements, were performed by other anesthesiologists, and the 2 operators of catheterization were not involved.

The insertion duration was measured as the time required from skin puncture by the catheter until catheterization was completed or failed. The upper limit for the insertion time was 10 minutes; any attempts requiring a longer duration were considered as catheterization failure. Insertion was again attempted at the same radial artery until catheterization was completed. Each distinct skin puncture was considered an attempt, whereas subcutaneous catheter tip adjustments were not counted. The number of attempts until catheterization was completed was recorded, and >3 attempts were considered catheterization failure.

To assess the consistency of catheterization time between these 2 anesthesiologists, we preliminarily quantified interoperator reliability using an intraclass correlation coefficient (> 0.75, adequate).14,15 Statistical analyses were performed with StatFlex ver. 6.0 (Artech Co., Ltd., Osaka, Japan) and SPSS 19.0 statistical package (SPSS, Chicago, IL). Sample size was calculated by PASS 11 (NCSS, LLC, Kaysville, UT) and Power and Sample Size Calculations ver. 3.0.43. (Vanderbilt Biostatistics, Nashville, TN). P < 0.05 was considered to be statistically significant. Values are expressed as median (quartile 1–quartile 3).

Assessment Phase

The success rate for ultrasound-guided radial artery catheterization in pediatric patients varies among previous reports.3–6 Based on these reports, we estimated that 92 patients would provide 80% power for detecting a 50% improvement in the success rate, from 60% to 90% at an α-level of 0.05. Allowing for dropouts and technical problems, we therefore enrolled 102 patients in our observational study.

Multivariable logistic regression analysis with forward selection was used. The independent variables used were as follows: weight (kg); sex (male/female); ASA PS; systolic blood pressure (mm·Hg); RACHS-1 category; cyanosis (yes/no); trisomy 21 (yes/no); pulmonary hypertension (yes/no); cardiac surgery (yes/no); diameter of the artery (mm); and location depth of the artery < 2, 2–4, and ≥4 mm. First-attempt and overall catheterization success were included as the dependent variables.

To assess the importance of arterial depth, we used Kaplan-Meier curve plots for the catheterization time to the success of catheterization in 102 patients divided into 3 groups (arterial depth <2, arterial depth = 2–4, and arterial depth ≥4.0 mm). The 3 groups were compared for time with log-rank tests and for success rates with Dunn tests. Two expert anesthesiologists punctured the radial artery at a site of their choosing in the distal half of the forearm.

Validation Phase

Based on results of the observational study, we tested the hypothesis that subcutaneous injection of saline to increase the arterial depth from <2 to 2–4 mm improves catheterization success rate and reduces the time required. Time to successful insertion was the primary outcome.

We enrolled 60 patients, 20 of whom had a subcutaneous radial arterial depth 2 to 4 mm and 40 of whom had a radial arterial depth <2 mm as determined by the short-axis echo view. The 40 patients with an initial depth <2 mm were randomly assigned to immediate catheterization or to catheterization after subcutaneous injection of a small amount (<2 mL) of saline solution using a 27-gauge needle until the artery was located 2 to 4 mm below the skin surface (Fig. 1).

Figure 1
Figure 1:
Diagram showing injection of saline to increase the subcutaneous depth of the radial artery from <2 to 2–4 mm. A = radial artery; NSS = normal saline solution.

Randomization was based on computer-generated permuted blocks that have no stratification. Allocation was concealed in sequentially numbered opaque envelopes that were opened shortly before induction of anesthesia. The 2 anesthesiologists who performed catheterization were not informed of the results of the Assessment Phase. In the saline-injection group, other anesthesiologists, excluding the operators of catheterization, measured the depth of artery, performed subcutaneous injection of saline, and marked the insertion point on the patient’s skin on the distal half of the forearm before the actual procedures. In the noninjection group (arterial depth <2 or 2–4 mm), anesthesiologists other than the 2 performing catheterization measured the arterial depth and marked the appropriate points of insertion on the patient’s skin on the distal half of the forearm, as in the injection group.

Sample size was estimated for detecting a reduction in the median arterial catheterization time from 145 (52–600) seconds (arterial depth <2 mm in the assessment phase) to 58 (39–132) seconds (arterial depth 2–4 mm in the assessment phase) on 80% power at an α-value of 0.05 for the log-rank test. Nineteen patients per group were required; therefore, we enrolled 20 patients per group.


Assessment Phase

Overall, 102 patients were included in multivariable logistic regression. Demographic characteristics of the patients are shown in Table 1. Age (months), weight (kg), and height (cm) were highly collinear (r > 0.80). We therefore selected weight as an independent variable to represent all 3 characteristics. Similarly, systolic, diastolic, and mean arterial blood pressure (mm·Hg) and pulse pressure (mm·Hg) were collinear (r > 0.80); we therefore used systolic blood pressure as the independent blood pressure variable in multiple logistic regression analysis.

Table 1
Table 1:
Demographic Characteristics of Patients Included in the Multiple Logistic Regression Analysis

The results of multiple logistic regression analyses are shown in Table 2. The location depth of the artery <2 and ≥4 mm were independent predictors of catheterization difficulty in both the first-attempt and overall success. Trisomy 21 was also an independent variable for difficulty, rather than success, with arterial catheterization in the overall. The relative risks16–18 in overall success are as follows: 0.58 (95% CI, 0.40-0.83) for depth of the artery ≥4.0 vs 2–4 mm, 0.69 (95% CI, 0.52-0.92) for depth of the artery <2 vs 2–4 mm, and 0.49 (95% CI, 0.36-0.68) for trisomy 21. The relative risks in the first-attempt success are as follows: 0.26 (95% CI, 0.18-0.37) for depth of the artery ≥4.0 vs 2–4 mm, 0.57 (95% CI, 0.47-0.76) for depth of the artery <2 vs 2–4 mm.

Table 2
Table 2:
Factors affecting the artery catheterization time extracted with multiple logistic regression analysis.

Kaplan-Meier curves for time to successful catheterization as a function of arterial depth are shown in Figure 2. Catheterization time was significantly shorter in pediatric patients with a subcutaneous arterial depth of 2 to 4 mm as compared with those with <2 or ≥4 mm in the log-rank test. (2–4 vs <2 mm group; P = 0.01, 2–4 vs ≥ 4 mm; P < 0.001). There was no significant difference in catheterization time between <2 and ≥4 mm group (P = 0.31).

Figure 2
Figure 2:
Kaplan-Meier curves for the catheterization success time as a function of subcutaneous arterial depth. The 2 to 4 mm depth group (n = 39) had the shorter catheterization success time compared with the <2 (n = 32) and the ≥4 mm group (n = 31) with statistically significant differences in the log-rank test (2–4 vs <2 mm group; P = 0.01, 2–4 vs ≥4 mm; P < 0.001). The <2 and the ≥4 mm group did not show significant difference in catheterization success time (P = 0.31). The median catheterization time of 3 groups were as follows: <2 (145 seconds), 2–4 (58 seconds), and ≥4 mm group (220 seconds).

Dunn test results demonstrated that the subcutaneous arterial depth of 2 to 4 mm had a statistically significant better success rate in the first attempt as follows: <2 mm (43.8%, N = 14/32, 95% CI, 26.6%–60.9%) vs 2–4 mm (76.9%, N = 30/39, 95% CI, 63.7%–90.1%), P = 0.02; <2 (43.8%) vs ≥4.0 mm (19.4%, N = 6/31, 95% CI, 5.7%–33.1%), P = 0.16; 2–4 (76.9%) vs ≥4.0 mm (19.4%), P < 0.001. Dunn test results also demonstrated the overall success rate as follows: <2 (62.5%, N = 20/32, 95% CI, 45.8%–79.2%) vs 2–4 mm (89.7%, N = 35/39, 95% CI, 80.2%–99.2%), P = 0.04; <2.0 (62.5%) vs ≥4.0 mm (51.6%, N = 16/31, 95% CI, 34.0%–69.2%), P > 0.99; 2–4 (89.7%) vs ≥4.0 mm (51.6%), P = 0.002.

Validation Phase

A flow diagram according to the consolidated standards of reporting trials statement shows patient selection in Figure 3. Four patients were excluded as per protocol. The clinical and demographic characteristics of the 60 participants are shown in Table 3. Kaplan-Meier curve plots for time to successful catheterization are shown in Figure 4. By increasing depth with injecting saline in the <2 mm group, catheterization time was significantly improved as compared with the nonintervention <2 mm group in the log-rank test (P = 0.002) and no longer differed significantly from patients who initially presented with arteries located at a 2 to 4 mm depth (P = 0.2, log-rank test).

Table 3
Table 3:
Demographic Characteristics of Patients Enrolled in the Prospective Randomized Trial
Figure 3
Figure 3:
A flow diagram of the consolidated standards of reporting trials statement.
Figure 4
Figure 4:
Kaplan-Meier curve plots for the catheterization time to the success of catheterization. Increasing the subcutaneous depth of the radial artery by saline injection shortened catheterization time to the level observed in patients in whom the arteries were initially located 2 to 4 mm below the skin surface (saline injection group vs 2–4 mm group;P = 0.2, saline injection group vs <2 mm group; P = 0.002, 2–4 vs <2 mm group, P = 0.012). The median catheterization time of 3 groups were as follows: saline injection group (38 seconds), 2–4 (55 seconds), and <2 mm group (149 seconds).

Dunn test comparisons of catheterization success rates in the first attempt were as follows: <2 (30.0%, N = 6/20, 95% CI, 10.0%–50.0%) vs saline injection (85.0%, N = 17/20, 95% CI, 70.1%–99.9%), P < 0.001; 2–4 mm (80.0%, N = 16/20, 95% CI, 62.5%–97.5%) versus saline injection (85.0%), P > 0.99; <2 (30.0%) vs 2–4 mm (80.0%), P = 0.03. Dunn test comparisons of catheterization success rates in the overall success rate as follows: <2 mm (55.0%, N = 11/20, 95% CI, 33.2%–76.8%) versus saline injection (90.0%, N = 18/20, 95% CI, 76.9%–100.0%), P = 0.02; 2–4 mm (85.0%, N = 17/20, 95% CI, 69.4%–100.0%) vs saline injection (90.0%), P > 0.99; <2 (55.0%) vs 2–4 mm (85.0%), P = 0.04.


In our observational analysis, a subcutaneous depth of the radial artery between 2 and 4 mm was associated with the most successful percutaneous ultrasound-guided radial artery catheterization in surgical patients <3 years old. Arteries positioned <2 and ≥4 mm below the skin surface were significantly harder to cannulate. In the subsequent prospective randomized trial, we show that by increasing subcutaneous depth from <2 to 2 to 4 mm by injection of saline, the catheterization time and success rate were both significantly improved.

The subcutaneous arterial depth probably influences the success rate because it determines the likelihood of puncturing the center of the artery. A central puncture is critical for successful catheterization because the outside diameter of a 24-gauge catheter (0.7 mm) is nearly the size of the radial artery in pediatric patients, the mean diameter being 1.0 mm in the present report (25th–75th percentile, 0.9–1.1 mm). This small difference is in marked contrast to adults wherein there is a sizable gap between the outer diameter of a 22-gauge catheter (1.1 mm) and the inner diameter of the radial artery (almost equal to 3 mm). Central arterial puncture is thus far more important in pediatric patients than in adults.

To puncture the artery at its center, the catheter is guided toward the center of the artery’s surface by varying the puncture angle. However, a depth <2 mm is too shallow to allow easy adjustment of the catheter. A depth ≥4 mm is also problematic because it is hard to direct the catheter tip to the center of the artery. An additional problem with deep arteries is that they often require a steep puncture angle, which then mandates more flexibility when sliding the catheter off the needle. Consequently, an arterial depth between 2 and 4 mm is best for ultrasound-guided catheter insertion.

Surprisingly, we did not observe a relationship between patient size (age), blood pressure, or severity of disease (ASA PS and RACHS-1 category) and the success of catheterization on multivariable analysis, although patients with RACHS-1 scores >3 included patients with a single ventricle and hypoplastic left heart syndrome. The lack of such association with age and disease severity indicates that arterial depth is an important independent determinant of successful insertion.

In our randomized trial, we tested a novel technique for improving catheterization speed and success for arteries located at a subcutaneous depth <2 mm. Specifically, we showed that increasing the depth to between 2 and 4 mm by subcutaneous injection of saline significantly improved the catheterization success rate and shortened the time required. In fact, after saline injection, the catheterization success rate and time required were essentially equivalent to those in patients with an initial subcutaneous radial arterial depth of 2 to 4 mm.

The most obvious benefit of saline injection is that it provides optimal arterial depth for ultrasound-guided catheter insertion. In addition, the enhancement artifact created by the saline injection improves imaging of the catheter tip and the artery’s surface.19,20 Specifically, the area beyond the anechoic (saline) deposit appears hyperechoic because of the mismatch between the echo amplitude and the penetration depth in the presence of anechoic or hypoechoic areas (heterogeneous tissues or presence of fluid or air). Injection of saline solution between the surface of the skin and the artery thus creates an anechoic area, which leads to the enhancement of ultrasound signals and improved visibility of the artery and the catheter tip (Fig. 5, A and B). Saline may also enhance stability of the artery in surrounding tissues or might reduce arterial collapse during puncture attempts.

Figure 5
Figure 5:
A, Radial artery before injection of saline solution. B, Radial artery after injection of saline injection to increase the subcutaneous arterial depth to 2 to 4 mm. (a), Catheter positioned in the pooling saline solution. (b), Enhancement artifact of the anterior wall of the radial artery. (c), The artery is flattened by probe pressure on the skin and puncture pressure.

A previous study validated our general strategy by showing that visualization was improved by inserting a commercially available 3-mm jelly pad between the skin surface and ultrasound probe.21 However, catheters cannot be inserted through pads, whereas saline administration in no way compromises insertion efforts.

Injected saline solution remains at the injection site for about 10 minutes but subsequently spreads outward into the surrounding tissues. These 10 minutes are normally sufficient for catheter insertion (we considered longer times to represent failed insertion). That the saline is thereafter absorbed into the surrounding tissues presumably reduces the risk of pressure-induced complications, unlikely as these are.

A previous study has reported that arterial catheterization is difficult in patients with trisomy 21 because of skin and subdermal abnormalities.22 Similarly, we observed catheterization to be difficult in these patients. For example, the odds ratio from our multivariate regression was 0.09 (95% CI, 0.01–0.76, P = 0.027). However, difficult catheterization in trisomy 21 patients did not result from differences in arterial depth. In fact, there were no statistically significant differences in depth: 3.0 mm (2.6–4.3 mm) in the trisomy patients vs 2.6 mm (1.7–4.2 mm) in the others, P = 0.45 by Mann-Whitney test. Furthermore, the diameter of the radial artery was only slightly smaller in trisomy 21 patients (0.8 mm [0.7–0.9 mm]) than in the other patients (1.0 mm [0.9–1.2 mm], P = 0.01 by Mann-Whitney test). Thus, skin and subdermal abnormalities may generate insertion resistance, accounting for difficult catheterization in trisomy 21 patients. In addition, higher plasma endothelin-1 levels in patients with trisomy 21 may be related to difficult catheterization as a result of arterial spasms induced by secreted endothelin-1.23–25

Whether the short axis or long axis is preferable for peripheral vascular catheterization remains controversial in both pediatric and adult patients.26–28 We used the short-axis view because the long axis is subject to slice-thickness artifacts.29 Specifically, the ultrasound machine assumes that the plane of image is extremely thin. However, the ultrasound beam does have a measureable thickness that varies with depth. Therefore, in pediatric patients with a thin radial artery, the 24-gauge catheter in the long axis can appear to be in the same plane as the artery even when the catheter has not successfully cannulated the artery (Fig. 6A).

Figure 6
Figure 6:
A, Diagram of a long-axis view. The artery and catheter appear to be in the same plane on the right; catheterization failure in the short axis is shown on the left. a = radial artery. B, Diagram of a short-axis view. The artery and catheter are shown in the same plane on the left; successful catheterization in the long axis is shown on the right. a = radial artery.

Slice-thickness artifacts can also occur in the short-axis method, but its influence on catheterization is minimal. In the short-axis method, if an image of the catheter tip in the center line of the artery is obtained, there is a strong probability of successful arterial puncture because the catheter tip is actually on the line that extends to the artery (Fig. 6B). Slice-thickness artifacts are presumably more common in pediatric patients since the radial artery in pediatric patients is thinner than that in adults, making it more likely to be encompassed in the beam width.

Direct measurement of arterial pressure is invasive and thus is typically used only for major surgery. Our pediatric study population primarily included cardiac surgical patients. However, multivariable analysis revealed no relationship between the success of catheterization and cardiac surgery, suggesting that our results are broadly applicable to relevant populations.

We were unable to accurately record the needle angle, which may be related to catheterization success. However, all punctures were performed by 2 pediatric anesthesiologists who used an approach that their experience suggested would be most successful. We maintained a 2 mm distance between the ultrasound probe and the insertion site; however, it is possible that success rates may differ with different distances, possibly as a function of the subcutaneous arterial depth. Furthermore, we used a 13-MHz hockey-stick type probe that is commonly used for catheterization in pediatric patients. It remains possible that higher-frequency probes (such as 18-MHz) would provide better guidance. However, a subcutaneous arterial depth of 2 to 4 mm appears likely to remain beneficial with any probe.

There are some limitations in our study. Puncturing an artery that is in spasm, for example after a first attempt at catheterization has failed, may affect the success of catheterization.30–33 Injecting local anesthetic drugs instead of saline solution might prevent arterial spasm but was not tested in this study. Second, we threaded catheters off the needle rather than using a through-and-through approach that is more traumatic. The benefits34–36 and difficulties37 associated with guidewire use in arterial catheterization are well established in adults, but there is little information in pediatric patients.35 Whether arterial depth is critical with a through-and-through approach remains unknown.

In summary, we demonstrated that a subcutaneous radial artery depth between 2 and 4 mm was associated with fastest ultrasound-guided catheterization and greatest overall catheterization success in pediatric patients aged <3 years. For patients in whom the radial artery was located at a depth <2 mm, increasing the depth to 2 to 4 mm by subcutaneous saline injection improved catheterization time and the success rate.


Name: Yoshinobu Nakayama, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Yoshinobu Nakayama has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Yasufumi Nakajima, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Yasufumi Nakajima has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Daniel I. Sessler, MD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Daniel I. Sessler reviewed the analysis of the data and approved the final manuscript.

Name: Sachiyo Ishii, MD.

Contribution: This author helped conduct the study.

Attestation: Sachiyo Ishii has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Masayuki Shibasaki, MD, PhD.

Contribution: This author helped design the study.

Attestation: Masayuki Shibasaki has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Satoru Ogawa, MD.

Contribution: This author helped conduct the study.

Attestation: Satoru Ogawa has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Jun Takeshita, MD.

Contribution: This author helped conduct the study.

Attestation: Jun Takeshita has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Nobuaki Shime, MD, PhD.

Contribution: This author helped design the study.

Attestation: Nobuaki Shime has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Toshiki Mizobe, MD, PhD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Toshiki Mizobe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Peter J. Davis, MD.


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