Diagnostic Accuracy in a New Ultrasound Software Compared to Histology for Non Alcoholic Fatty Liver Disease (NAFLD) and Non Alcoholic Steato Hepatitis (NASH) in Morbidly Obese Individuals Undergoing Bariatic Surgery and/or Cholecystectomy
Phase: NA
Conditions: Non-Alcoholic Fatty Liver Disease
Sponsor: University of Roma La Sapienza

8 january 2019 
 
Diagnostic accuracy of a new ultrasound software compared to liver biopsy for Non Alcoholic Fatty 
Liver Disease (NAFLD) and Non Alcoholic Steato Hepatitis  (NASH) in morbidly obese candidate to 
bariatric surgery and/or cholecystectomy. 
 
 

Diagnostic accuracy of a new ultrasound software compared to liver biopsy for Non Alcoholic Fatty 
Liver Disease (NAFLD) and Non Alcoholic Steato Hepatitis  (NASH) in morbidly obese candidate to 
bariatric surgery and/or cholecystectomy. Pilot study. 
 
PILOT PROSPECTIVE STUDY 
 
1. STUDY DIRECTOR  
Prof. Gianfranco Silecchia , Direttore UOC Chirurgia Generale e Centro di Eccellenza in Chirurgia 
Bariatrica e Metabolica Sicob – Dipartimento di Scienze e biotecnologie medico-chirurgiche, 
“Sapienza” Università di Roma – Polo Pontino – gianfranco.silecchia@uniroma1.it; 
 
RESEARCHERS  
2. Dott.ssa Anna Guida, resident in General Surgery, University of Rome “La Sapienza” – Polo Pontino 
– Dipartimento di Scienze e biotecnologie medico-chirurgiche – anna.guida@uniroma1-it 
3. Dr. Piero Maceroni, CADI (Centro Alta diagnostica Immagini) director ICOT – 
piero.maceroni@gmail.com 
4. Prof. Vincenzo Petrozza, Direttore UOC Anatomia Patologica- Dipartimento di Scienze e 
biotecnologie 
medico-chirurgiche, 
“Sapienza” Università di 
Roma 
– Polo Pontino 
– 
vincenzo.petrozza@uniroma1.it 
5. Prof.ssa Annarita Vestri, Dipartimento Sanita’ Pubblica e malattie infettive, Sapienza-  
  “Sapienza” Università di Roma – Polo Pontino - annarita.vestri@uniroma1.it  
The present ___ has been financed within the framework of “Progetti per Avvio alla Ricerca – Tipo 2”  
Year 2018, Sapienza University of Rome. 
Research manager: Dr. Anna Guida, 
Reference tutor: Professor Gianfranco Silecchia 
 
Introduction 
The non-alcoholic fatty liver disease (NAFLD) is a systematic disease characterized by the presence of at 
least 5% of liver tissue, in the absence of other known causes of hepatopathy. To the present day, the 
NAFLD represents the first chronic hepatopathy cause. (1) It is a benign clinical condition that might evolve 
in non-alcoholic steato hepatitis (NASH) if inflammation and fibrosis occur along with the hepatic steatosis, 
with possible evolution in hepatic cirrhosis (in 25-30% of cases) and, subsequently, in hepatocellular 
carcinoma (7%) (2, 3).  

The pathophysiological mechanisms that foster the progression from simple steatosis to NASH are complex 
and still not entirely clarified. It is certain that multiple genetic factors and acquired factors – such as insulin 
resistance, high ferritin and adiponectin reduction) are involved. NAFLD prevalence is directly proportional 
to obesity: it is estimated that 30% of the US population and 24% of the European population is affected by 
NAFLD and that within the obese population candidate to bariatric surgery the prevalence is at 90%. (4) In  
the western world, the prevalence of type 2 diabetes patients is at 60-70%. NAFLD shows a strong 
association with all the components of the metabolic syndrome, enough to be considered the hepatic 
manifestation of the metabolic syndrome (5).  
Key elements of the NAFLD pathogenesis are hyperinsulinemia and insulin resistance. Insulin has three 
main actions in the liver: it increases glycogen synthesis, inhibits gluconeogenesis, and promotes 
lipogenesis. In patients with NAFLD, insulin resistance determines: 
1) An increase in lipolysis in adipose tissue, resulting in increased circulating levels of free fatty 
acids, which convey to the liver level; 
2) A reduction in glycogen synthesis and an increase in gluconeogenesis in the liver. 
In response to insulin resistance, the body produces higher levels of insulin, which further increase liver 
lipogenesis, resulting in an intrahepatic accumulation of lipids and increased liver secretion of triglycerides 
in the form of VLDL lipoproteins. 
In hepatocytes, the infestation of lipids causes a lipotoxicity that further worsens insulin resistance, increases 
oxidative stress and promotes inflammation and fibrosis (6,7). 
The diagnosis of NAFLD is based on the combination of a number of clinical and radiological features. 
Hepatic enzymes are not a diagnostic criterion, as more than 60% of NAFLD patients and normal GPT 
values may have advanced degrees of steatohepatitis and 53% of NAFLD and increased GPT patients do not 
have NASH.Hepatic enzyme levels, therefore, are not reliably correlated with liver histology (8).  
Currently, validated methods for quantization of fibrosis, steatosis and hepatic necroinflammatory 
phenomena are: Elastography for the evaluation of hepatic fibrosis(9), contrastless MRI (10,11)or 
ecoguidated percutaneous biopsy for the evaluation of hepatic steatosis (12), ecoguidated percutaneous 
biopsy for the evaluation of necroinflammatory phenomena (13). 
 
The NAFLD Activity Score (NAS) is the most widely used validated classifications for staging the NAFLS 
from a histological point of view(14), which assigns a score based on the following parameters: 
- The steatosis level (with a score from 0 to 3) 
- The degree of lobular inflammation (with a score from 0 to 3) 
- The degree of balloning, i.e. hot air balloon hepatocytes (with a score from 0 to 2) 
According to the score, we can distinguish among: 
- NAS 0 patients: with NAS Score < 3, in which the diagnosis of steatohepatitis is excluded (NO NASH) 
- NAS 1 patients: with NAS Score between 3 and 4, indicative of probable pathology or borderline 
- NAS 2 patients: with NAS Score ≥ 5 where steatohepatitis (NASH) diagnosis is made 

Purpose of the pilot study: 
Evaluate the diagnostic accuracy of the new ultrasound software vs. liver histology, current reference 
standard for the diagnosis of NAFLD and NASH in obese patients (BMI > 30) candidates for bariatric 
laparoscopic surgery and/or cholecystectomy. 
 
Type of study: 
Monocentric, open, sequential enrollment pilot study of 20 patients. 
Datas will be analyzed with descriptive statistics, absolute values and percentages of concurrent and 
conflicting cases according to the two methods. 
The concordance between the new ultrasound method and the liver biopsy will be evaluated and tested by 
the Mcnemar test. 
As the pilot study is used to assess whether there is good agreement between the two 
methods and in case of clinically significant response a study will be set up 
diagnostic with formal calculation of the sample size, with adequate power 
statistics and level of significance. 
 
Inclusion criteria: 
1. 
Patients who are candidates for bariatric surgery and are between 18 and 65 years of age,  
- BMI > 40 kg/m2, in the absence of any other comorbidity 
- BMI > 35 kg/m2, in the presence of comorbidities among those classically considered as associated with 
obesity (tab,) (15) 
 
Metabolic disease 
Neoplasm 
 
Type 2 Diabetes Mellitus 
Dyslipidemia 
Hyperuricemia and Gout 
Female infertility 
Polycystic ovary syndrome 
Breast cancer                                Esophageal cancer 
Pancreas cancer                           Gallbladder cancer 
Colorectal cancer                         Renal cancer 
Prostate cancer                             Leukemia 
Endometrial cancer                      Ovarian cancer 
Liver cancer 
Cardiovascular Disease 
Others 
Arterial Hypertension 
Coronary heart disease 
Congestive heart failure 
Pulmonary embolism 
Stroke 
Bronchial asthma                                    Psychological disorders 
Obstructive sleep apnea syndrome         Gastroesophageal reflux 
Non alcoholic steatohepatitis                  Urinary incontinence 
Gallbladder disease                                 Osteoarthritis 
Intertrigo                                                 Plantar fasciitis 
 
- ICM > 30 kg/m2, in the presence of T2DM not in glycomethabolic compensation 
 
2. 
Patients who are candidates for laparoscopic cholecystectomy for symptomatic calculosis with BMI 
> 30 
 

Exclusion criteria: 
- Age ≤ 18 or ≥ 65 
 
- Any concomitant surgical procedure except cholecystectomy and repair of iatal hernia  
- Conversion into laparotomic surgery 
- Post-operative complications requiring new surgical treatment 
- Non-compliant patient in follow-up 
 
 
Methods: 
Pre- and post-operative evaluations: 
All patients will sign an informed consent ad hoc  
Patients will be enrolled according to the PDTA deposited at the Health Directorate of the ICOT 
Before surgical treatment all patients will be subjected to: 
- standard liver ultrasound, shear wave elastography, attenuation imaging, shear wave dispersion 
blood tests: coagulation, GOT, GPT, GGT, alkaline phosphatase, total and fractionated bilirubin, fractionated 
cholesterol, triglycerides, insulinemia, glycaemia, Cpeptide 
 
 
    Imaging: 
The software is designed to be used on the Aplio i800 TUS system. 
(already supplied by our structure) and detects the attenuation that the signal 
ultrasound meets crossing the hepatic parenchyma and expresses this attenuation 
in Decibels on cm of hepatic parenchyma as a function of frequency (dB/cm/MHz) in a 
ROI of analysis. 
Non-homogeneous zones are automatically excluded from the calculation. 
Being a newly introduced software, there is no reference standard with respect to 
the ultrasound systems currently used for the analysis of hepatic steatosis 
as the transient elastography (Fibroscan, Echosense Paris) or the 
CAP (Controlled AttenuationParameter). 
There are no studies carried out with this software to date. 
 
The methods that will be used are as follows: 
Shear Wave Elastography (SWE) 
It quantifies the degree of liver fibrosis by measuring the speed of propagation of SW 
A focused ultrasonic pulse generates a tissue movement that creates shear waves (SW). 
The speed of movement of the shear waves depends on the rigidity of the crossed tissues.  

Its tracking makes it possible to evaluate the rigidity of the tissue (the speed, expressed in m/s, is converted 
into "stiffness", expressed in kPa, using Young's equation). 
 
Attenuation Imaging (AI) 
Quantifies the ultrasound attenuation of the tissue as an indicator of liver steatosis 
Ultrasonic waves in the body are attenuated by sound loss, reflection and absorption (heat). Attenuation is 
frequency-dependent. 
Attenuation Imaging is based on the RF information generated by the probe during conventional B-mode 
examination. The system compares the amplitude of the signal transmitted by the transducer with the return 
signal from the tissues at different depths, removing from the return ultrasound information all those pre- and 
post-processing compensations that the system introduces precisely to avoid having an uneven image between 
surface and depth. This analysis is carried out in a specific region of interest that the operator places on the 
tissue that is being analyzed. The value obtained represents the attenuation in Decibels per cm as a function 
of the transmission frequency used (dB/cm/MHz). 
 
 
 
Shear Wave Dispersion (SWD) 
SWE has some limitations in the presence of steatosis and inflammation because it starts from the assumption 
that the liver is perfectly elastic. 
Increasing the viscosity of the liver (inflammation, steatosis, circulation disorders) increases the phenomena 
of dispersion. 
SWD measures the frequency dispersion of the shear wave propagation in the viscoelastic liver 
The dispersion quantification ((m/s)/kHz) allows estimation of liver viscosity 
SW signals are converted to SW frequencies with FFT (Fast Fourier Transform) processing 
Increasing the viscosity (Pa*s) with constant elasticity (kPa)-> the dispersion increases (steepest slope of the 
curve). 
 
 
Surgical technique: 
All patients will undergo bariatric surgery according to the standard procedures validated by SICOB (15) and 
wedge resection of 1.5 cm x 1.5 cm at the level of the left hepatic lobe. In routine surgical practice, a liver 
biopsy is indicated in cases of hepatomegaly and clinical radiological suspicion of steatosis (16). 
 
 
 
 

Histology: 
Histological examination of liver tissue will be performed on material from liver biopsy, fixed in formalin, 
included in paraffin and finally dissected. The reading of the set preparations will be performed by an expert 
pathologist who will not have access to the identity, clinical history and biochemical parameters of the patients.  
 
A minimum biopsy sample length of 1.5 x 0.8 cm. or at least 10 complete portal spaces will be required, it is 
strongly recommended whenever clinically possible to have a separate core for inclusion in cryomold and 
freezing at -80 C so as to have additional tissue available for possible molecular biology studies. 
For liver histology sections of 3-4 μm will be obtained by staining with Hematoxylin/Eosin, PAS, PERLS, 
Gomoric Reticulum and Masonic Trichrome. NASH will be diagnosed according to Brunt's criteria (17). The 
NAFLD activity score (NAS) and a staging score capable of quantifying the extent of fibrosis in order to 
perform statistical correlations will be calculated (17). In addition, patients will be stratified according to the 
SAF score into no NAFLD, NAFLD or NASH (18). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

          
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