Evaluation of the Role of Multiparametric Ultrasound Study in Discriminating Among Intestinal Complications After Hematopoietic Stem Cell Transplantation. A Pilot Study.
Phase: NA
Conditions: Intestinal GVHD
Sponsor: Fondazione Policlinico Universitario Agostino Gemelli IRCCS

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 1 di 17 
Evaluation of the role of multiparametric ultrasound study in discriminating among 
intestinal complications after hematopoietic stem cell transplantation. A pilot study. 
 
ECO-STEM 
 
Principal investigator: Dott.ssa Elisabetta Metafuni 
 
Sub-investigators: Dott. Fabrizio Pizzolante, Prof.ssa Simona Sica, Prof.ssa Patrizia 
Chiusolo, Dott.ssa Nicoletta de Matthaeis, Dott.ssa Laura Riccardi 
 
Funding: No Profit 
 
Version: 1.0 
 
Date: 03.06.2022 
 
________________________________ 
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 2 di 17 
INTRODUCTION 
Allogeneic stem cell transplantation (HSCT) represents a fundamental therapeutic option 
for patients affected by malignant hematological diseases. One of the major life-threatening 
complication after HSCT is acute graft versus host disease (aGvHD), especially when 
gastrointestinal tract is extensively involved. All grade aGvHD occurred in 40-80% of HSCT 
recipients, depending on donor choice, stem cell source, conditioning regimen and GvHD 
prophylaxis. Among patients with aGvHD, intestinal involvement accounts for 60% of the 
cases (1). Approximatively one third of patients with intestinal aGvHD reached a grade III-
IV stage with an increased risk of mortality and morbilities. The most common symptom of 
GI involvement of acute GVHD is diarrhea, secretory and usually voluminous, often 
reaching more than 1.5 liters per day. In severe cases, patients presented abdominal pain 
and gastrointestinal bleeding may occur, reflecting the mucosal ulceration (2). These 
symptoms are nonspecific given the fact they might be frequently observed after allo-SCT 
in Clostridium difficile colitis, bacterial colonization, cytomegalovirus or adenovirus 
infection, drug toxicity, or neutropenic enterocolitis (3). Weber et al (4) reported the use of 
contrast enhancement ultrasound imaging (CEUS) in diagnosing gastrointestinal aGvHD. 
The penetration of intravenous injected contrast microbubbles into the gut lumen showed a 
sensitivity of 92.9% and specificity of 94.4% for intestinal aGvHD diagnosis, but the 
specificity was only of 39.6% for advanced stage. On the other hand, sonographic criteria 

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 3 di 17 
such as free fluid, bowel wall thickness, color-coded Doppler sonography (CCDS), and 
compound elastography showed additional modifications in severe gastrointestinal GvHD. 
Also Schreyer et al (5), reported transmural penetration of intravenous applied 
microbubbles during CEUS as feasible diagnostic feature for intestinal aGvHD 
involvement. Contrary to nonspecific bowel wall thickening or free fluid observation, 
transmural penetration of contrast microbubbles allows to discriminate between aGvHD 
and chronic inflammatory bowel diseases, that were used as comparing group. Benedetti et 
al (6) were inspired by the findings of neovascularization detected in aGvHD intestinal 
biopsies. They observed abnormal bowel wall thickening in multiple bowel segments, due 
to mucosal edema, as documented also in intestinal infectious conditions. The addition of 
CEUS imaging revealed microcirculation enhancement of the bowel walls during arterial 
phase, followed by a prolonged venous phase washout. They also monitored patients’ 
response to treatment and noted that in patients with complete response, the picture return 
to be normal, both with conventional ultrasound and CEUS. In patients obtaining a partial 
response, ultrasound showed normal bowel wall, while CEUS still detects microvascular 
changes at a capillary level suggesting persistent inflammatory activity.  
Ultrasound evaluation on bed-side may represent a reliable tool for aGvHD diagnosis in 
patients with intestinal symptoms. Even if histological features is considered the gold 
standard for aGvHD diagnosis, a very probable diagnosis of aGvHD could be made quickly 
with ultrasound method allowing to start steroid treatment with more safety if compared 
to the only clinical suspicion. Patients with gastrointestinal aGvHD need to be promptly 

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 4 di 17 
diagnosed and treated given the high rate of mortality due to advanced staged disease. 
Ultrasound method might reduce time spent awaiting endoscopic and histological 
diagnosis, especially if will be confirmed a discriminating power of ultrasound for aGvHD 
rather than others intestinal complications.  
In the concept of multiparametric ultrasound study of the intestine of these patients we 
would also like to introduce the use of shear wave elastography (SWE). 
Shear wave velocity US or shear wave elastography is another ultrasonographic method for 
measuring tissue stiffness. Instead of direct pressure, it applies energy to the tissues through 
a pulse wave generated by the US probe. These vibrations displace tissue across multiple 
geometrical planes instead of only deforming it perpendicularly. They are propagated 
through tissues as a shear wave, which travels faster in denser and stiffer materials. 
The literature reports various experiences on the use of SWE in the study of the intestine, 
especially in patients with chronic inflammatory bowel diseases (7-9). But no data are 
reported on the use of the method in patients with graft versus host disease. Considering 
the simplicity of the method, we think it can be applied to an ultrasound study of the 
intestine to try to understand the potential of SWE to further define the characteristics of the 
intestinal segments involved. 
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 5 di 17 
OBJECTIVES 
 
Primary objective 
Primary objective of the study is to evaluate the use of bed-side multiparametric ultrasound 
study in the diagnosis of gastrointestinal aGvHD in patients underwent to allogenic 
transplant. 
  
Secondary objectives 
Secondary objective of the study is to evaluate the use of bed-side multiparametric 
ultrasound study in distinguishing complications different from aGvHD (i.e., infectious, 
drug-related o neutropenic colitis) in patients underwent to allogenic transplant as 
compared to auto-transplant. 
 
 
 
 
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 6 di 17 
METHODS 
 
Study design 
This is an ambispective interventional study with device.  
 
Setting 
The study will be realized at Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
in Rome.  
 
Study duration 
The enrollment will be start after ethic committee acceptance and will enroll during a period 
of 12 months. Patients will be enrolled consecutively. Data will be collected prospectively 
during the study course and until all patients will reached 100 days after HSCT. Data will 
be analyzed subsequently throughout a period of six months.  
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 7 di 17 
Population 
Study population will include all patients submitted to allogenic stem cell transplantation 
who developed diarrhea > 500 ml per day suggestive for aGvHD until 100 days from 
transplant date. Patients submitted to autologous stem cell transplant who developed 
diarrhea between transplant date and engraftment achievement will constitute control 
population as for other infectious complications assessment. 
 
Inclusion criteria 
 Age ≥18 years; 
 Submitted to first allogeneic stem cell transplant during the 12 months period of 
enrollment of the study; 
 Diarrhea onset until day +100 from transplant ; 
 Signed informed consent. 
 
Exclusion criteria 
 Previous allogeneic stem cell transplantation; 
 Patients refusing to sign informed consent for the study; 
 Patients affected by severe cardiomyopathy and respiratory distress/insufficiency; 
 Patients unable to fully understand and accept study protocol for various problems. 

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 8 di 17 
 
Variables and Procedures 
Within 24-48 hours from symptoms, onset full panel of stool cultures will be performed 
together with Clostridium toxin assessment. Multiparametric ultrasound study will be 
performed at the onset of the symptoms and then weekly to monitoring intestinal changes 
accordingly to symptoms evolution during treatment. In the case with high suspicion for 
aGvHD (stool colture negative, persistent or progressive diarrhea, ultrasound imaging 
closely suggestive for aGvHD), as per common clinical practice, colonoscopy will be 
required to evaluate internal macroscopy features of the bowel and to obtain histological 
samples for conclusive diagnosis. In the meantime steroid therapy will be started, as per 
common clinical practice. The only modification of this protocol to common clinical practice 
is represented by the addition of a non-invasive bed-side method of evaluation of the 
patients with diarrhea. None of the routinely performed diagnostic and therapeutic process 
will be modified.  
All patients submitted to autologous stem cell transplant who developed diarrhea between 
transplant date and engraftment achievement will constitute control population. Also in this 
case full panel of stool culture and clostridium toxin assessment will be performed at the 
onset of the symptoms. Multiparametric ultrasound study will be made al symptoms onset 
and then weekly until symptom resolution.  
Multiparametric ultrasound study will be performed by expert qualified staff as following: 

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 9 di 17 
The examinations will be performed at patient’s bed, in supine position, with a MyLab™9 
Platform (Esaote, Italy) system. Fasting is required. 
US for bowel requires both low-frequency convex probes (3.3-5 MHz) and high-frequency 
(5–17 MHz) linear assay probes to increase the spatial resolution of the intestinal wall and 
to assess the wall diameter and wall layer.  
The following ultrasound parameters will be collected: 
 Intestinal wall thickening (normal values < 3 mm; a mean of two measurements for 
section will be taken, in longitudinal and transverse sections). 
 Intestinal wall stratification; 
 Intestinal wall flow at colorDoppler;  
 Strictures;  
 Free fluid (FF) within the peritoneal cavity; 
 Enlarged mesenteric lymph nodes; 
 Mesenteric hypertrophy. 
 
The contrast agent is administered intravenously and excreted through the lungs without 
risk of nephrotoxicity or ionizing radiation as in the case of computed tomography contrast 
agent; it does not remain in the body longer than 15 min. It consists of microscopic bubbles 
of gas (size 3–5 lm), which are encapsulated in a shell of flexible and stiff phospholipids: 
they enhance the backscatter signal from blood cells, oscillating when exposed to a low-
intensity US field, thereby demonstrating tissue perfusion in real-time ‘‘blood-pool’’ 

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 10 di 17 
imaging. SonoVue (SV, Bracco, Italy) is actually the second-generation US contrast agent 
used for CEUS, consisting of phospholipid-stabilized microbubbles filled with sulphur 
hexafluoride. The recommended dose of SonoVue is 2.4 mL. Every injection should be 
followed by a flush with 5-10 mL of 9 mg/mL (0.9%) sodium chloride solution. 
Approximately, 10–15 s after intravenous injection, the SonoVue arrives to the intestinal 
wall, thereby achieving the maximum concentration (peak intensity) after approximately 30 
s. This stage is followed by a venous phase, and it is finally excreted by the lungs. No 
adverse event was reported with SonoVue use: the only contraindication is severe 
cardiomyopathy (7-9). 
The following CEUS parameters will be collected: 
 Time-intensity curve (TIC) included basic intensity (B), wash-out slope and/or decent 
slope (K), wash-in slope or rise slope (C), time to peak (TTP), area under the gamma curve 
(Area), arrive time (ATM), peak intensity (PI), change of peak intensity (I). 
 Any exam will be recorded; 
 Acquisition time: 5’ 
Moreover, each single evaluation will be completed with the use of SWE in order to further 
evaluate the characteristics of the inflammatory bowel. 
SWE of the bowel wall will be performed according to manufacturer guidelines 
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 11 di 17 
ENDPOINTS 
 
Primary endpoint 
Primary endpoint of the study is to evaluate the accuracy of bed-side multiparametric 
ultrasound study in the diagnosis of gastrointestinal aGvHD in patients underwent to 
allogenic transplant in terms of sensitivity, specificity, positive predictive value (PPV) and 
negative predictive value (NPV). 
 
Secondary endpoints 
 To assess potential associations between bed-side multiparametric ultrasound 
measurements weekly performed and aGvHD diagnosis in patients underwent to 
allogenic transplant. 
 To assess whether bed-side multiparametric ultrasound measurements at baseline differ 
in distinguishing complications different from aGvHD (i.e., infectious, drug-related o 
neutropenic colitis) in patients underwent to allogenic transplant as compared to auto-
transplant. 
 

 
 
 
 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
Dipartimento diagnostica per immagini, radioterapia oncologica ed ematologia  
Area Ematologica 
Divisione di Ematologia 
Largo Agostino Gemelli 8, 00168 Roma  
T +39 0630156244 +39 06 30155300 F +39 063017319  
www.policlinicogemelli.it – ematologia@policlinicogemelli.it 
Sede Legale  
Largo Francesco Vito 1, 00168 Roma 
Sede Operativa 
Largo Agostino Gemelli 8, 00168 Roma 
 
Codice Fiscale e Partita IVA 13109681000 
 
 
Pagina 12 di 17 
STATISTICAL ANALYSIS PLAN 
 
Sample Size calculation 
The literature reports various experiences on the use of SWE in the study of the intestine, 
especially in patients with chronic inflammatory bowel diseases. But no data are reported 
on the use of the method in patients with graft versus host disease. Hence, we consider this 
as a pilot study. As such, no formal sample size calculation is needed. However, based on 
common rules on pilot studies [10], the minimum sample size required would be of 20 
subjects. In our department, given the median annual number of allogenic stem cell 
transplant, we plan to enroll 75 patients. As for the secondary endpoints, an equal number 
of age-matched patients undergone to autologous transplant will be enrolled. 
 
Statistical Analysis 
All variables included in the study will be first analyzed by descriptive statistic techniques. 
In depth, qualitative variables will be described as absolute and relative percentage 
frequencies. The Shapiro Wilk’s test will be used to assess the distribution of quantitative 
variables. Data will be then expressed either as mean and standard deviation (SD), whether 
normally distributed, or as median and interquartile range (IQR), otherwise.  

 
 
 
Fondazione Policlinico Universitario Agostino Gemelli IRCCS 
Università Cattolica del Sacro Cuore 
 
 
Pagina 13 di 17 
Between groups differences (allogenic vs. autologous transplant) will be assessed by the 
Fisher exact test and the Chi-square test, with Yates correction, as appropriate, in the case 
of qualitative variables. Quantitative data, indeed, will be assessed either by the Student’s t 
test or the non-parametric Mann Whitney U test, as appropriate. 
Receiver Operating Curve (ROC) analysis will be used to assess the accuracy of 
multiparametric bedside ultrasound in aGvHD diagnosis as compared to the istologic 
diagnosis (gold-standard, used as reference). Positive (PPV) and Negative predictive values 
(NPV), sensitivity and specificity will be computed, as well as the area under the ROC curve 
(AUROC). 
To 
assess 
potential 
associations 
between 
bed-side 
multiparametric 
ultrasound 
measurements and aGvHD diagnosis in patients underwent to allogenic transplant uni- and 
multivariable logistic regression models will be used. 
Logistic regression models will be also applied to assess whether bed-side multiparametric 
ultrasound (US) measurements differ in distinguishing complications different from 
aGvHD (i.e., infectious, drug-related o neutropenic colitis) in patients underwent to 
allogenic transplant as compared to auto-transplant. More in depth, to evaluate the 
combined effects between US measurements and transplant group, multivariable 
interaction models will be fitted, and the interaction odd ratios (IOR) evaluated. In 
particular, for each US measurement, one interaction logistic regression model will be fitted. 
In summary, the coefficients of the main effects (in exponential terms) will be interpreted as 
ORs of the outcome by considering the allo-transplant group from control (ORautologous), by 

 
 
 
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Università Cattolica del Sacro Cuore 
 
 
Pagina 14 di 17 
fixing US measurements values to 0 (null), and ORs of the outcome by considering a unit 
increase of the US measurements in the auto-transplant group (ORUS_measurements). The 
interaction parameters (IOR) will be interpreted as difference (in OR terms) of the US 
measurements variations between transplant groups (autologous transplant as reference 
category). 
Statistical significance will be set at P value < 0.05. P values between 0.05 and 0.10 will be 
further reported as suggestive. All analyses will be performed by using R software (v. 4.1.2, 
R Core Team, 2021) [11]. 
 
Data Collection 
Each patient transplanted at Hematology and stem transplant Unit of the Fondazione 
Policlinico Universitario Agostino Gemelli IRCCS had to provide informed consent for 
enrollment. Data on patients and transplant characteristics will be recorded prospectively: 
age, underlying disease, conditioning regimen, donor type, stem cell source, GvHD 
prophylaxis, diarrhea features, treatment for diarrhea, microbiological and radiological 
findings, ultrasound findings, endoscopic finding and histological report.  
Adverse events definition and management 
Adverse events, or any harmful clinical event that occurs in subjects involved in a clinical 
study, regardless of the event is or is not related to the product under study, will be recorded 
and communicated to the Competent Authority and the Ethics Committee in accordance to 
what required by current legislation for medical devices (EU Regulation 2017/745 and 

 
 
 
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Università Cattolica del Sacro Cuore 
 
 
Pagina 15 di 17 
related texts), and for medicinal products, (DM 30 April 2015 and GVP-EMA module VI) 
according to the flow of pharmacovigilance postmarketing.  
In the case of a medical device, we mean • defect, any deficiency in the identity, quality, 
durability, reliability, safety or performance of a device under investigation, including 
malfunction, errors in its use or inadequacy of the information provided by the 
manufacturer; • accident, any malfunction or alteration of the characteristics or 
performance of a device made available on the market, including the error in using caused 
by the ergonomic characteristics, as well as any inadequacy in the information provided by 
the manufacturer and any side effect. A serious accident is defined as any accident which, 
directly or indirectly, has caused, may have caused or may cause one of the following 
consequences: a) the death of a patient, user or another person; b) the serious deterioration, 
temporary or permanently, of the health conditions of the patient, the user or another 
person; c) a serious threat to public health. In the case of a medicinal product, a suspected 
adverse reaction means a harmful and adverse effect resulting from the use of a drug, 
compliant or not compliant with the indications contained in the marketing authorization, 
including overdose, misuse, abuse, therapeutic error and events resulting from exposure for 
professional reasons. A suspected adverse reaction is defined as serious when it has resulted 
in death or life threatening, hospitalization or prolonged hospitalization, severe or 
prolonged disability or incapacity, a congenital anomaly or a birth defect, the onset of 
another clinically relevant condition "  

 
 
 
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Università Cattolica del Sacro Cuore 
 
 
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The reference documents for the study are among others: Summary of the product 
characteristics of the SonoVue and technical data sheets and user manual for MyLabTM9 
Esoate. The diagnostic investigation with contrast medium sulfur hexafluoride 
microbubbles, Sonovue is of common / consolidated use in clinical practice for 
gastrointestinal diagnostics diagnostics. 
References  
1. Harris AC, Young R, Devine S, et al. International, multicenter standardization of acute 
graft-versus-host disease clinical data collection: a report from the Mount Sinai Acute 
GVHD International Consortium. Biol Blood Marrow Transplant 2016; 22:4–10. 
2. Zeiser R, Blazar BR. Acute Graft-versus-Host Disease-Biologic Process, Prevention, and 
Therapy. N Engl J Med 2017; 377:2167–79. 
3. Malard F, Mothy M. New Insight for the Diagnosis of Gastrointestinal Acute Graft-
versus-Host Disease. Mediators Inflamm 2014; 2014:701013. 
4. Weber D, Weber M, Hippe K, et al. Non-invasive diagnosis of acute intestinal graft-
versus-host disease by a new scoring system using ultrasound morphology, compound 
elastography, and contrast-enhanced ultrasound. Bone Marrow Transplant 2019; 
54:1038–48. 
5. Schreyer AG, Landfried K, Zorger N, et al. Transmural penetration of intravenously 
applied microbubbles during contrast-enhanced ultrasound as a new diagnostic feature 
in patients with GVHD of the bowel. Bone Marrow Transplant 2011; 46:1006-11.  

 
 
 
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6. Benedetti E, Bruno B, McDonald G, et al. Prospective qualitative and quantitative non-
invasive evaluation of intestinal acute GVHD by contrast-enhanced ultrasound 
sonography. Bone Marrow Transplant 2013; 48:1421-28. 
7. Quaia E. Contrast-specific ultrasound techniques. Radiol Med 2007; 112(4):473-90. 
8. Wilson SR, Burns PN. Microbubble-enhanced US in body imaging: what role? 
Radiology 2010; 257(1):24-39. 
9. Piscaglia F, Bolondi L. Italian society for ultrasound in medicine and biology (SIUMB) 
study group on ultrasound contrast agents. The safety of Sonovue in abdominal 
applications: retrospective analysis of 23188 investigations. Ultrasound Med Biol 2006; 
32(9):1369-75. 
10. Birkett MA, Day SJ. Internal pilot studies for estimating sample size. Stat Med. 
1994;13(23-24):2455-2463. doi:10.1002/sim.4780132309 
11. R Core Team (2021). R: A language and environment for statistical computing. R 
Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-
project.org/.