Intraoperative liver ultrasound represents an essential component in the hepatobiliary surgery arsenal, having an essential role in describing liver lesions, their topography, and loco-regional extension. It also has an important role in establishing surgical strategy, in modulating the surgeon decisions, and thus in preventing postoperative complications. This chapter tries to make a synthetic review of principal indications for using ultrasound in liver surgical treatment, underlining the liver’s lesions characteristics and advantages brought by this method. Also, we wanted to underline the importance that ultrasound has for guiding the surgeon in interventional intraoperative techniques or in any anatomical liver resection. The role of enhanced contrast intraoperative ultrasound is put in front by the better diagnostic results obtained for both primary and metastatic tumors of the liver.
- intraoperative ultrasound
- liver tumors
- contrast enhanced ultrasound
1. Introduction: brief history
The main advantage of the ultrasound imaging method is the real-time visualization of the anatomy and structure of the liver lesions, allowing for the adaptation of the therapeutic decision during surgery.
The concept of intraoperative ultrasound (IOUS) was first introduced in the 60s and was used to evaluate renal lithiasis when doing nephrolithotomy. Due to the limitations of A-mode ultrasonography (difficulty in interpreting images), IOUS began to be more applicable in the surgical sphere later, in the early 80s , when high-frequency real-time B mode-ultrasound was introduced . The use of IOUS in hepato-bilio-pancreatic surgery was emphasized for the first time in the literature in the mid-80s ; later, it became an exploratory technique routinely performed in specialized centers for staging liver disease and guiding surgical procedures on patients diagnosed with hepatocarcinoma on cirrhotic liver [4, 5, 6, 7]. Studies in the 90s showed that the information provided by IOUS may modify the initial therapeutic plan in up to 53% of cases [8, 9].
Although first reports related to laparoscopic transducers used in A-mode date back to early 1964, the laparoscopic IOUS technique has been developed relatively recent .
2. General aspects
Currently, there is a wide range of equipment for IOUS, probes of different types and shapes, adapted according to the type and localization of the lesion. Standard transductors for transabdominal ultrasound can also be used, but there may be some limitations on image resolution and on the large size of the transducer that do not offer optimal maneuverability . Conventional transducers can be used at the beginning of the liver examination to obtain an overview of the organ anatomy [1, 11]. The transducers used in IOUS usually operate at high frequency: 7.5–10 MHz . There are different shapes: linear T-shaped probes, interdigital probes, microconvex probes and more recently, T-shaped probes with trapezoidal scanning window . In case of liver surgery, the ideal transducer should be a small one that can be easily manipulated in narrow spaces, with a special design to allow the probe to be held in the palm between two fingers, thus allowing the operator to have permanent contact with the surface of the liver, without omitting to scan some areas [11, 14] (Figure 1).
When necessary, IOUS can also be used in laparoscopic surgery, with special transducers suitable for this type of approach. Transducers used during laparoscopic surgery are either linear or curved, mounted at the end of a long, thin articulated arm, with a design that allows insertion and manipulation inside the trocar (Figure 2) .
The possibility of performing intra-operative contrast ultrasound (CE-IOUS) is an important factor in choosing the ultrasound equipment. Nowadays, the most commonly used contrast agents are SonoVue (Gaseous sulfur hexafluoride, Bracco, Milan Italy) and Sonazoid (Gaseous perflutane, GE Healthcare, Norway/DaiichiSankyo, Japan) [11, 16, 17, 18, 19].
In order to ensure a good examination, the ultrasound machine should be positioned in front of the main operator, the patient (the organ to be examined) being located between the surgeon and the monitor (a collinearity between operator, organ and monitor) in order to view simultaneously the ultrasound monitor and the surgical field. The ultrasound monitor should have size and resolution large enough to allow optimal remote viewing. Examination must always begin with the inspection and palpation of the liver and of the entire peritoneal cavity. These steps should not be avoided in favor of IOUS . Mobilization of the liver begins with the sectioning of suspensory ligaments, thus creating enough space to manipulate the ultrasound transducer. Worth mentioning some of the artifacts that may appear on the examination of the VIIIth and IVa liver segments after the sectioning of the cavo-hepatic adhesions. Therefore, in the case of suspected lesions located in these areas (adjacent to the cavo-hepatic region), dissection at this level should be performed only after ultrasound exploration.
3. IOUS of the liver: benign tumors
Benign tumors can develop on a normal or steatotic liver, may be solitary or multiple, with increased echogenity (hemangiomas, focal nodular hyperplasia) or anechogenic, with posterior acoustic strengthening (serous cysts) and distinct contours (hydatid cysts), with no vascularization or characteristic circulatory pattern; may have a mass effect on liver structures or even adjacent organs. A characteristic for benign tumors is the fact that they have elastic consistency and do not invade vascular elements [20, 21].
Hemangiomas are benign tumors, mostly asymptomatic, incidentally discovered. These tumors can present themselves under various echographic aspects; most commonly, are well-defined, round, hyperecogenic, homogeneous, usually small (<3 cm), and may present the posterior acoustic strengthening effect . As hemangiomas grow in size, they can change their echogenicity, from homogeneous to heterogeneous, with their edges becoming irregular. These features make them more difficult to differentiate from malignant tumor formations. When surgery is indicated, IOUS has the role to localize and visualize the relationships of the hemangioma with the intrahepatic structures. The surgeon can trace the hepatic resection line outside the hemangioma, minimizing hemorrhagic risk, and preserving the healthy hepatic parenchyma to its full potential. The CE-IOUS can be useful, capturing the contrast agent by the hemangioma being most of the time characteristic. Differentiation from malignant tumor formation becomes difficult for arterial hemangiomas or for those with arterio-venous shunts .
For focal nodular hyperplasia, the central location of a fibrous scar is characteristic. This tumor appears as a well-defined lesions with variable size, usually unique, of solid consistency and inhomogeneous structure. Rarely, the central scar can be distinguished when using simple ultrasound, without contrast agent. When using CE-IOUS, in the arterial phase, there is a central filling followed by a complete capture in the venous phase. At this stage, the center of the tumor becomes hypoechoic. In the late phase, the tumor remains isogenic together with the hepatic parenchyma, which strengthens the diagnosis of benign lesion .
Hepatic adenoma appears ecographically as a well-defined solid tumor lesion; it may have an inhomogeneous structure in the presence of intratumoral hemorrhage. Doppler ultrasound does not detect a vascular signal. When using CE-IOUS, in the arterial phase, there is a centripetal and inconsistent capture; in the venous phase, a moderate washout may be noted. In the late phase, the appearance is isoechoic or hyperechoic .
Differentiation between focal nodular hyperplasia and hepatic adenoma is important for establishing the therapeutic indication, surgery being indicated for large adenomas, due to the risk of rupture and hemorrhage as well as due to its malignant potential.
Because sometimes it is difficult to make a benign-malignant US differentiation, intraoperatory, when the situation imposes, might by necessary to make a bioptic puncture for establishing a correct diagnosis . IOUS has an important guiding role, especially in the case of lesions located in the depth of the liver parenchyma, hard to reach when palpating.
Simple hepatic cysts (biliary cysts) are benign tumors with no malignant potential, usually asymptomatic, that can be easily diagnosed with ultrasound imaging. They are described by ultrasound as well-defined lesions with very thin walls, no Doppler signal, anechoic, with transonic content due to the liquid composition. Simple hepatic cysts have therapeutic indications only when they become symptomatic, often due to symptoms related to the mass effect they have on neighboring structures.
Percutaneous ultrasound guided treatment with cyst evacuation is often possible, but is followed by an increased risk of relapse, with the rebound of collection. In this idea, the laparoscopic surgical resection of the cystic dome is indicated. This technique is easy if the lesions are located superficially, in segments II, III, IVB, V, VI (after Couinaud) . The lesions localized intraparenchymatous can be approached safely only when using IOUS .
Depending on the evolutionary stage, hydatid cysts may appear as single or multiple lesions, anechoic, with membranes and sediment inside, with thin or calcified walls. They may be multilocular or may contain multiple fluid compartments (daughter vesicles). IOUS helps the surgeon in finding the cysts and in some situations it can detect bile duct communication. These lesions can compress the intrahepatic vessels with mass effect, signs of invasion, or embedding of these structures being absent . IOUS has the same indications as in the case of simple cysts, being a real help for the surgeon, for establishing surgical tactics and for checking the radicality of the treatment (the content of the remaining cavity, residual content, multilocular abscesses, etc.) .
4. IOUS of the liver: malignant tumor
IOUS finds its usefulness in liver surgery for both primary and secondary malignant lesions facilitating the detection, characterization of lesions and guiding the surgical procedure [26, 27]. Most studies have evaluated the role of IOUS for treatment of hepatocarcinomas and hepatic metastases due to colorectal cancer, these pathologies being considered the most common liver malignant lesions. Intraoperative detection and local treatment of these lesions may have a major impact in choosing surgical strategy [28, 29].
4.1. Hepatocarcinoma (HCC)
Ecographically, this tumor has the appearance of a solid tumor with irregular contours, heterogeneous, uni-, or multilocular (“encephaloid form”). Typically, it invades the liver vessels, primarily the portal branches, but also the suprahepatic veins. Doppler screening usually highlights a high-speed arterial flow. Vessel distribution is irregular, disordered. CE-US shows hypercaptation in the arterial phase with a specific “washout” of contrast substance in the venous phase. In the late phase, the tumor appears as hypoechoic. This behavior is usually described in tumor nodules larger than 2 cm .
In the case of HCC, IOUS is superior in detecting lesions measuring less than 1 cm, preoperative MRI having a lower sensitivity and specificity for these lesions [11, 32]. It has also been shown in several studies that CE-IOUS can modify in 19–29% of the cases the initial treatment plan [33, 34]. CE-IOUS finds its usefulness especially in cirrhotic patients when it comes to differential diagnosis between malignant lesions and regenerative nodules [29, 35]. It has been demonstrated that neoangiogenesis of tumor nodules is a specific criteria for distinguishing hepatocarcinomas from dysplastic or regenerative nodules .
CE-IOUS has a sensitivity of 100%, a specificity between 69 and 100% and can modify the surgical strategy in up to 79% of patients [36, 37, 38], most frequently by detecting new lesions. The literature emphasizes that the filling pattern of the contrast agent in nodules found by IOUS can guide surgical resection . It has also been shown that the vascular pattern of HCC visualized by using CE-IOUS has been associated with the expression of some genetic profiles, suggesting that CE-IOUS images can be used as an indicator for predicting prognosis of patients .
During hepatic resection, which is the standard treatment for HCC, particular attention should be paid in preserving as much hepatic parenchyma as we can, the remaining hepatic volume being an important prognostic factor for the short outcome [37, 39, 40]. Thus, local resection of the tumor formation or its ablation under IOUS guidance may be chosen to minimize the volume of resected liver parenchyma, respecting the oncological resection margin. Also, in order to minimize the risk of postoperative complications (hemorrhage, necrosis of the liver parenchyma) and remote relapse (by satellite micrometastases, specific for HCC), the use of IOUS is vital in guiding anatomical resections. These involve the ultrasound identification of vascular pedicles corresponding to the affected hepatic segments and through various associated maneuvers (digital compression, injection of contrast agents) an exact delimitation of the targeted resection area can be obtained (Figure 3). More details will be given in the following rows, in the sub-section dedicated to the role of IOUS in guiding hepatic resections.
4.2. Hepatic metastases
Despite significant advances in preoperative staging diagnostic procedures (conventional CE-US, multi-sliced CT, CE-MRI, and PET-CT), studies have shown that 10–30% of the patients with colo-rectal cancer remain with undiagnosed hepatic metastases during primary tumor surgery [41, 42, 43, 44, 45, 46].
In this respect, IOUS and CE-IOUS have a special role in completing the diagnosis, in addition to the liver’s palpation technique. IOUS is considered the “gold standard” in open surgery for colorectal cancer since 1980, being able to detect liver metastases that cannot be palpated intraoperatively and that have not been visualized with preoperative imaging techniques [8, 47, 48, 49, 50].
Liver metastases have a non-characteristic echographic appearance, being circumscribed lesions with imprecise or halo delineation, with a homogeneous or heterogeneous pattern. They may be solitary (usually liver metastases from colonic neoplasms) or multiple. Their echogenicity is variable. When they are large, they can compress the bile ducts (which may appear to be dilated) and the liver vessels. As for their vascularization, they may be hypovascular (in gastric, colon, pancreatic, or ovarian cancers) with hypoechoic pattern in arterial phase and similar in the venous and late phases or hypervascular (neuroendocrine tumors, malignant melanomas, sarcomas, renal tumors, breast, or thyroids), with a hyperechoic appearance during the arterial phase, with wash out during the venous phase and hypoechoic pattern at about 30 s after the injection of the contrast substance .
Several studies in the literature have shown that after the surgical treatment of the primary tumor, the ultrasound of metastasis after colorectal cancer can be correlated with prognosis. Thus, Gruenberger et al.  demonstrated that in patients with hyperechoic ultrasound liver metastases, survival is longer than in those with the hypoechoic aspect of the lesions. This suggests that the role of IOUS is more than a diagnostic one and can be useful in establishing prognosis .
The CE-IOUS applied for colorectal liver metastases has an 96% accuracy, in contrast to 74 and 79%, percentages associated with pre-operative CT and MRI [34, 54]. The fact that undetected preoperative liver metastases represent the main cause of recurrent neoplasia  highlights the important role that IOUS has in the management of patients diagnosed with colorectal cancer. This is why routine IOUS is recommended in these patients .
Chemotherapy is an important, standardized element in regard with the adjuvant and neoadjuvant therapy in colorectal cancer patients. [57, 58] Regarding hepatic metastases, good results of cytostatic treatment mean either stagnation or regression of these lesions [59, 60]. A particular situation is when liver metastases are no longer visible in CT and/or MRI performed after chemotherapy. Literature indicates that the complete, real response is found in up to 66% of cases [61, 62]. For the rest of the cases (34%), chemotherapy can affect the echogenity of the metastases making them difficult to be identified with preoperative imaging (CT, MRI, even IOUS) [13, 33]. In these situations, CE-IOUS allows the surgeon to check areas where hepatic lesions have been described before chemotherapy . The role of this technique is highlighted in many studies that have shown that only be confirmation given by the CE-IOUS in regard with the lack of lesions can be associated with a complete therapeutic response [59, 62].
Resection or ablation of all lesions is the gold standard in the treatment of colorectal liver metastases . Even in patients with unresectable metastases, local ablation or combination between ablation and surgical resection of the lesions has been shown to be able to locally control the disease . It is obvious that IOUS plays a major role in liver surgery for the detection and localization of metastatic lesions .
5. The role of laparoscopic approach
The laparoscopic approach and minimally invasive surgery have more and more indications and thus the role of IOUS in laparoscopic surgery has become increasingly important. Of course, laparoscopic surgery has some disadvantages in assessing the liver because the surgeon loses the advantage of palpating the structures and lesions. IOUS manages to compensate for most of these laparoscopic minuses by providing intraoperative high utility imaging with greater sensitivity in detecting liver lesions than most preoperative imaging techniques [65, 66, 67, 68, 69]. Intraoperative laparoscopic ultrasound (LIOUS) has a sensitivity and specificity similar to that in open surgery . Several authors have suggested routine use of LIOUS in laparoscopic colorectal surgery  and prior to planned laparotomies for liver resections . In cases where hepatic disease is known, with the help of LIOUS data, around 64% of cases could be exempted from laparotomy [71, 72].
The success of the laparoscopic approach depends primarily on the location of the lesions [73, 74]. Guiding surgical maneuvers by the use of LIOUS is possible especially in superficial tumors on the left lobe or on the anterior segments of the right lobe (hepatic segments II, III, IVb, V, and VI). Direct visualization and LIOUS should be used to compensate for the impossibility of liver palpation in laparoscopic surgery [75, 76]. In the case of laparoscopically treated malignant lesions, it is important to mark by IOUS imaging the oncological resection margins, this way ensuring their tracing by minimally invasive approach. Furthermore, the completion of the treatment is possible using ablative techniques (radiofrequency, microwave). The laparoscopic approach finds its indications especially for higher-risk cirrhotic patients (altered hepatic markers, clotting disorders) with subcapsular neoplastic lesions (Figure 4).
With the evolution of technology and the experience of surgical teams, laparoscopic approaches to hepatectomies have become more and more used in centers of excellence. Several studies have shown that laparoscopic hepatectomy is a safe procedure and could have advantages over open surgery, translated by reduced blood loss and a shorter hospitalization stays [77, 78]. As for LIOUS, it should guarantee the same performance as the ultrasound used in conventional liver surgery. Although, LIOUS has been introduced since 1981, few studies have addressed this subject. Although reported to be a safe and accurate method , it is currently not routinely used in laparoscopic surgery , although the reliability of LIOUS in the staging of liver disease has been demonstrated to be similar to conventional IOUS . Moreover, although many articles mention LIOUS as an important technique, few scientific papers described this technique [82, 83, 84, 85].
6. Ultrasound-guided techniques
It has been demonstrated that making biopsies under IOUS guidance, laparoscopic or “classic,” have a high diagnostic accuracy and are considered safe procedures with possible impact on surgical management [86, 87]. For example, liver metastases detected intra-operatively and confirmed by histopathological examination as having pancreatic origin could be a contraindication for pancreatic radical surgery .
In terms of non-excisional treatment of hepatic tumor formations, this can also be achieved by ablative techniques, such as ethanol injection , RFA (coagulation necrosis induced by high-frequency alternating currents-thermal energy)  and MWA (same as RFA, although MWA uses different parts of the electromagnetic spectrum) . Although the elective treatment is by percutaneous approach, there are situations when both classical or laparoscopic method are indicated.
Laparoscopic approach is particularly preferred on patients who are on the waiting list for liver transplantation or for those who cannot benefit from liver resection due to comorbidities, liver cirrhosis, or hepatic dysfunction due to chemotherapy, especially when percutaneous procedures are not possible [91, 92, 93]. Indications are subcapsular lesions located in the immediate vicinity of important structures (diaphragm, stomach, and gallbladder) or difficult to approach (caudal lobe). [84, 94, 95, 96]. Moreover, these ablation techniques can be combined with hepatic resections or can be performed serially after surgical resections, improving the oncological outcome and prognosis [97, 98, 99]. In the majority of cases treated by these procedures, IOUS is used as a guidance tool and for evaluation the efficacy of the treatment and appearance of complications .
Multiple studies have demonstrated that IOUS-guided ablations are a safe and an effective treatment option that provides excellent local control of both primary and secondary hepatic tumor lesions [64, 94, 100, 101, 102]. Recent studies have also reported that intraoperative RFA has a local recurrence rate equivalent to that obtained from low-grade HCC surgery [11, 96] and colorectal hepatic metastases [64, 100].
7. Guiding liver resections
Localization of liver lesions is related to portal branches and suprahepatic veins, which are used to define segmental boundaries. Without the use of IOUS, it would probably be impossible to define correctly, anatomically, the hepatic segments and often the limits of the tumors, especially due to the existence of multiple anatomical variants .
Hepatic resections are known to be the standard treatment for malignant liver tumor formations, being the only procedure that provides oncological radicality . Preservation of hepatic parenchyma should be a goal of the surgical team, especially in patients with cirrhotic liver, whose liver function and prognostic could be influenced by extensive resection. In these situations, IOUS plays an essential role because it allows the evaluation of the intrahepatic tumors, facilitating a limited but oncological liver resection. Thus, in modern hepatic surgery, whether HCC or colorectal liver metastases, the use of IOUS allows the realization of the so-called “radical but conservative surgery.” Thus, obtaining continuous information on the relationship between liver lesions and intrahepatic bilio-vascular structures, the surgeon can guide his resection line, respecting the Glisson pedicles, and suprahepatic veins, with the ultimate goal of preserving as much functional hepatic parenchyma as possible [11, 12, 103, 104].
IOUS is also a real help for anatomical resections. This technique involves the compression of segmental portal branches between the transducer and the operator’s fingers, resulting in a transient ischemia of the target parenchyma. This area can be marked with the electrocautery, and then the resection is made along the demarcation line [105, 106, 107, 108, 109, 110].
Starting from the use of IOUS, Torzilli introduces new types of resection, such as mini-mesohepatectomy, for tumor formations located at the confluence of the cave vein with superhepatic veins [11, 12, 111]. These resections are based on the ultrasound study of the relationship between the tumor and the suprahepatic veins and the analysis of the blood flow at this level after clamping the proposed vein for resection. Evidence of an inverse flow in the peripheral portion of the compressed vein or of a collateral shunt between the clamped vein and the other superhepatic vein or cava vein will allow the ligation and segregation of the tumor-affected suprahepatic vein and the achievement of a limited resection, while maintaining the principles of oncological radicality [11, 12].
Summarizing, the use of IOUS allows the extension of surgical indications for certain liver lesions that were either considered unresectable or required major surgery .
8. Future perspectives
IOUS is still characterized by several drawbacks: it cannot detect lesions smaller than 3 mm, its accuracy is dependent on the surgeon’s skill and experience, the images are 2D and there is a “blind area “of about 1 cm below the surface of the liver, which is particularly problematic in the case of small hepatic metastases due to colorectal cancer that are mainly located on the surface of the liver. Of course, associating contrast agents has greatly improved IOUS accuracy; however, the disadvantage of visualization of the lesions for a too short period of time makes this technique to be of limited applicability in guiding hepatic resections that may last between 2 and 6 h .
Recently, a new fluorescent approach, using indocyanine green (ICG), has been proposed to improve the intraoperative detection of neoplastic lesions [113, 114]. ICG is a non-specific molecule that allows detection of tumor tissue, but with limited specificity. The main advantage of its use is its safety and its commercial availability as a contrast substance. The imaging technique of intraoperative fluorescence using ICG was initially used for the detection of sentinel lymph nodes in patients with gastric, colon, and breast cancer [115, 116]. Several studies have shown that malign liver tumors show strong fluorescence when preoperative ICG administration is made [117, 118]. This technique is based on the fact that ICG binds to plasma proteins and together emit light with a peak wavelength of approximately 830 nm when illuminated with infrared light .
Initially, ICG-fluorescence imaging was limited to open surgery alone. After year 2010, as laparoscopic and robotic imaging systems with fluorescence have developed, ICG-fluorescence imaging has been extended to minimally invasive abdominal surgery, especially for the visualization of extrahepatic biliary tract anatomy (during laparoscopic/robotic cholecystectomies) , an approach known as fluorescence cholangiography . In 2014, the use of ICG-fluorescence imaging was reported for the identification of subcapsular hepatic tumors before liver transection . A new laparoscopic imaging system is starting to be used, this system overlapping pseudo-color fluorescence images with white color-light images in real-time (fusion ICG-fluorescence imaging) with the proposal to identify segmental hepatic margins and localization of liver tumors . Thus, ICG has the ability to “label” bile ducts [121, 124, 125, 126], hepatic tumors [118, 127, 128, 129, 130], edges of liver segments [117, 131, 132, 133], this being due both to ICG fluorescence , and to its property to be excreted into the bile . Due to the property of being eliminated for more than 6 h after intravenous injection [126, 135], ICG-fluorescence imaging can also be used to identify small biliary fistulas after hepatectomy .
As for ICG-fluorescence imaging sensitivity in detecting liver metastases, it varies between 69 and 100%. However, sensitivity is limited because the examination does not have the ability to detect hepatic lesions at a depth greater than 8 mm in the hepatic parenchyma. It has also been shown that this method can detect new metastatic lesions in up to 43% of cases . In fact, it has been reported that ICG-fluorescence imaging can detect superficial lesions of up to 2 mm in both HCC and metastases liver disease due to colorectal cancers [127, 129].
Currently, a combination of a fluorophore, such as ICG, with an anti-tumor antibody is evaluated in preclinical studies. These new molecules could present a major advantage in the future for clinical applications that would allow the detection of tumor lesions with a higher TBR (tumor-to-background ratio between the intensity of fluorescence in tumor tissue and normal surrounding tissue). Recently, Harlaar et al. reported the first clinical trial using IRD-800CW-labeled bevacizumab for the detection of peritoneal metastases of colorectal origin .
9. Key points
The IOUS has applications in both open or laparoscopic abdominal surgery.
For benign hepatic tumors, IOUS has the role to localize and to visualize the relationships with the intrahepatic structures.
For intraoperative interventional maneuvers (biopsies, ablative techniques), IOUS guidance is mandatory.
In the case of HCC, IOUS is superior in detecting lesions measuring less than 1 cm.
In the case of HCC, CE-IOUS finds its usefulness especially in cirrhotic patients for the differential diagnosis between malignant lesions and regenerative nodules.
IOUS is considered the “gold standard” in open surgery for colorectal cancer.
CE-IOUS allows the surgeon to check areas where hepatic metastasis have been described before chemotherapy.
IOUS is mandatory for anatomic resections and for limited but radical hepatectomy.
Bartoș Adrian is the coordinator of this chapter.
Conflict of interest
The authors have no conflict of interest.