Marie-Claude Brindisi
email: marie-claude.brindisi@chu-dijon.fr

Audrey Noacco
email: audreynoaccoau.noacco@gmail.com

Amel Ait

Boudaoud Hansal

Clémentine Hugol-Gential
email: clementine.hugol-gential@u-bourgogne.fr

Ait Amel

Boudaoud Hansal

Delivery of oral nutrition supplement in hospital: Evaluation of professional practices in evaluation of nutritional status and representations of ONS by the caregivers and patients

Keywords: malnutrition, nutritional status, oral nutritional supplements, representation, hospitalization

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Patient malnutrition is currently a major concern for hospitals. It is estimated that 30% of hospitalized patients on average suffer from malnutrition [START_REF] Beau | Épidémiologie de la dénutrition chez le malade hospitalisé[END_REF], and figures range from 20% up to 60% in geriatrics departments. Malnutrition is associated with patient mortality and morbidity [START_REF] Hiesmayr | Decreased food intake is a risk factor for mortality in hospitalised patients: The NutritionDay survey 2006[END_REF], the length of hospital stays [START_REF] Lim | Malnutrition and its impact on cost of hospitalization, length of stay, readmission and 3-year mortality[END_REF] and incurs health care costs 45 to 102 % higher than for a non-malnourished patient [START_REF] Gastalver-Martín | Individualized measurement of disease-related malnutrition's costs[END_REF].Screening for malnutrition should be carried out systematically upon hospitalization in order to ensure that patients are managed in an adequate and appropriate manner. French National Authority for Health (HAS) has published diagnosis criteria in order to screen and take charge of malnutrition [5] (tables 1a and b). For screening, criteria used in France include BMI (Body mass Index), weight loss before hospital admission, albumin and prealbumin (these two values can be interpreted if the concentration of C-reactive Protein is below 15 mg/L), age and MNA for patients more than 70 years old (Mini Nutritional Assessment).

Table 1a : criteria to evoke the diagnosis of malnutrition from "simple" diagnostic tools (ANAES, 2003/ One criteria, among them, is necessary to classify the patients

Index Calculation

MNA-SF ≤ 11

Table 1b : Revised criteria to evoke the diagnosis of malnutrition from "simple" diagnostic tools in older patients (≥70 years old) (HAS 2007). One criteria, amoung them, is necessary to classify the patients

Moderate malnutrition

Severe malnutrition

• Weight loss: ≥ 5 % over a 1-month period, or ≥ 10 % over a 6-month period

• BMI < 21

• Albumin < 35 g/l

• MNA global < 17

• Weight loss: ≥10 % over a 1-month period or ≥ 15 % over a 6-month period

• BMI < 18

• Albumin < 30 g/l

We used the criteria of ANAES (2003) (Agence nationale d'accréditation et d'évaluation en santé, part of HAS) and HAS 2007 for older patients (≥ 70 years old) in order to classify patients in two groups: moderate malnutrition and severe malnutrition. These criteria are used to classify patients for their nutritional status, but also to declare this status to medical insurance in order to obtain refund for care given during hospital stay. ESPEN (European Society for Clinical Nutrition and Metabolism) criteria are not used in current care in France until now [START_REF] Cederholm | Diagnostic criteria for malnutrition -An ESPEN Consensus Statement[END_REF]; [START_REF] Fink | The New European Society for Clinical Nutrition and Metabolism Definition of Malnutrition[END_REF]. In order to conform to the hospital reality, we used the same assessment criteria as those used in the hospital in which the study was conducted (HAS/ANAES criteria): BMI (kg/m²), percentage of weight loss before hospitalization, albumin (g/L), prealbumin (mg/L), and C-reactive protein (CRP) (mg/L), and MNA for patients older than 70. In practice, many doctors do not evaluate nutritional status of their patients whether because of a lack of time, faulty assumptions or hyper-specialized medical interests [START_REF] Stratton | Encouraging appropriate, evidence-based use of oral nutritional supplements[END_REF]. For example, in a study led by Lennard-Jones et al. [START_REF] Arrowsmith | Screening by nurses and junior doctors to detect malnutrition when patients are first assessed in hospital[END_REF] on 454 nurses and 319 junior doctors, two-thirds questioned patients about their food intake, and only 50 % asked about weight loss.

The aim of this study was to observe, during the in-hospital period of patients receiving ONS, the evaluation of the nutritional status of these patients and to highlight the different representations of ONS by the caregivers because these two things are correlated. Indeed, representations can have implications for practice and allow us to better understand the status of screening, management of undernutrition and the representations of ONS.

This study is part of a more comprehensive study on the prescription and monitoring of ONS consumption among hospitalized patients in this health facility. According to Marine Fontas [START_REF] Fontas | Manger après le diagnostic d'un cancer. La mise en place de stratégies d'adaptation[END_REF], who is particularly interested in the nutritional management of patients with lung cancer:

"the disparity of nutritional management approaches observed in the literature can be observed…. We have seen changes from one department to another, going from preventive or therapeutic approaches to dietary disturbances to a purely therapeutic approach for weight loss, through the prescription of ONS".

Patients and Methods

This research protocol is part of a larger study on the overall consumption and wasting of ONS at the Dijon University Hospital Center. This is why only patients who already have an ONS prescription were included whatever their nutritional status. This protocol has received a positive opinion from the committee for the protection of the population. All patients received an information note.

The data were collected in two departments at the Dijon University Hospital, belonging to the same pole with geographic proximity and same interest in nutritional problems from caregivers: digestive surgery and diabetes-endocrinology departments.

Patients. During 11 weeks, from January to march 2017, all the patients with a prescription for at least one ONS per day, no cognitive disorders, and hospitalized in the department for at least 4 days were included.

Study Design. The following data were retrieved from the medical records of our patients at study entry of hospital admission: age, BMI (kg/m²), percentage of weight loss before hospitalization, albumin (g/L), prealbumin (mg/L), and C-reactive protein (CRP) (mg/L), and MNA for patients older than 70 to determine the nutritional status of the patients in our study [5]. If the patients could not be classified with one of the simple criterion, other criteria were considered in order to determine nutritional status (i.e. Nutritional Risk Index (NRI) and Mini Nutritional Assessment long-form (MNA) for patients older than 70).

Changes in body weight were taken into account during hospitalization according to SFNCM (Société Française de Nutrition Clinique et Métabolisme) recommendations [START_REF] Bouteloup | Arbre décisionnel du soin nutritionnel[END_REF]. We also observed the implication of the team responsible for patient in the management of patient nutrition. Finally, post-hospitalization data was analyzed (prescription of ONS, follow-up for albumin levels, evaluation of malnutrition on the final patient report).

Questionnaires and semi-structured interviews: prescription, ONS follow-up, and feelings about ONS. Semi-structured interviews were also conducted with 11 medicine residents working in the two departments, and 11 nursing auxiliaries were asked to complete our questionnaire. The themes included: meal management, modalities for prescription, and attitude toward ONS (appendices 1-2). A semi-structured interview was also conducted with the 61 patients receiving ONS included in the study, to discuss their feelings and attitudes towards them (appendix 3). The average length of the interview was 10 minutes. All the interviews were transcribed and a thematic and qualitative analysis was conducted.

Statistical analyses.

A statistical analysis compared the averages obtained in the groups by Student's test (comparison between malnourished and no malnourished during hospitalization and posthospitalization). The tests were performed bilaterally with a risk of the first species set at 5%.

A p <0.05 was considered significant.

Statistical analysis were conducted on XLSTAT, software version 2017.4, developed by ADDINSOFT France, operating system Windows 7 professional.

Results.

Study population.Sixty-one patients were included in the study (32 men and 29 women) ranging from 24 to 96 years old. Sixty percent of patients came from the endocrinology department and 40% from digestive surgery. The average age was 65.4 years ± 16 years and the average stay was 10 days ± 4 days. At their admission, 39.35% were defined as suffering from severe malnutrition, 21.31% were suffering from moderate malnutrition, and 39.34%

were not malnourished. All these patients received ONS because of their nutritional status (severe or moderate malnutrition) or because of their low food consumption during their hospitalization.

Evaluation of nutritional status upon hospitalization. On admission, BMI was measured for 87% of patients. Albumin levels were tested in 98.6% of patients, prealbumin levels were tested in 87.7% of patients, and CRP was tested in 97% of patients. Concerning percentage of weight loss upon hospitalization, these data were not collected on patient file.

Nutritional evaluation of patients during hospitalization.

Over the course of hospitalization, patients with moderate malnutrition had their weight checked less often than patients with severe malnutrition (38.46 % vs 80 %) (p = 0.025). Albumin levels were checked in 23.08 % of patients with moderate malnutrition, in 52% of patients with severe malnutrition and 25% of non-malnourished patients (severe malnutrition vs other groups, p<0.02). Prealbumin levels were checked more systematically in patients with severe malnutrition (56%) than in patients with moderate malnutrition (23%) and non-malnourished patients (16,7%) (p<0.02). CRP levels were checked in 54% of patients with moderate malnutrition, in 56% of patients with severe malnutrition and in 41.6% of non-malnourished patients (no significant differences between the three groups).

Representations around the status of ONS among caregivers and patients.

Medicine residents, nurse auxiliaries and patients were asked about their attitude and representations towards ONS.

The semi-executive interviews and questionnaires with caregivers conducted as part of our studies have enabled us to promote a heterogeneous discourse according to the parties (medicine residents, nursing auxiliaries, patients). We have made a thematic and qualitative analysis in order to underline the main representations. (Table 2).

For medicine residents, ONS are mainly seen as medication. For nursing auxiliaries, ONS is not considered as medication but as nutritional supplement. They harbor the impression that ONS are only functional and useful if they are taken regularly. For patients, ONS are described as a medication for some and as a food or a supplement for others:

A lack of information about ONS is also mentioned by residents medicine: "Honestly, we don't know much about ONS -what else is in there? Is it just protein and a bunch of calories? Why does it suppress the appetite?". We also questioned more precisely the nursing about their knowledge of the ONS. Globally, there is a real lack of knowledge on behalf of the nursing around the prescription and the observance of the ONS of patients. Besides, the prescriptions do not take into account the tastes and the preferences of the patients. The notions of cost and real efficiency are also little taken into account. The residents medicine have no knowledge concerning the modalities of costs and refund. No significant difference was found between the three groups for follow-up in the posthospitalization period.

Discussion:

The aim of this study was to investigate, during the in-hospital period of patients receiving ONS, the evaluation of the nutritional status of these patients and . it was also about having a better understanding of the representations of the different stakeholders: medicine residents, nursing auxiliaries, patients.

The initial evaluation of nutritional status can be considered acceptable seeing as BMI was measured in 87 % of patients included in the study at admission. Laboratory testing at admission was also carried out almost systematically with rates of 98,6 % for albumin, 87.7%

for prealbumin and 97 % for CRP. In a study by Renoud-Grappin et al. [START_REF] Renoud-Grappin | Utilisation des compléments nutritionnels oraux dans la population gériatrique : évaluation des pratiques professionnelles[END_REF], including 50 patients who were hospitalized for a short stay or in follow-up care and rehabilitation, and whose mean age was 84 ± 8 years, BMI was calculated in 94 % of cases, and albumin and prealbumin in 100 % and 96 %, respectively. Toze et al. [START_REF] Toze | État des lieux de l'évaluation de l'état nutritionnel dans des services de soins gériatriques du groupe hospitalier de la région de Mulhouse et Sud-Alsace[END_REF] studied the evaluation of nutritional status in 130 patients who were admitted to one of the geriatrics departments in the Mulhouse and Sud-Alsace region. They revealed that BMI was measured in 89.2 % of cases, whereas weight loss was only calculated in 8.1 % of cases during hospitalization. It was also because we don't get enough exercise and vitamins and such".

shown that albumin levels were among the most commonly used criteria, and that the MNA test was only used in 14.6% of cases. Our results were also similar to data exposed by Hasselmann 1 , from Strasbourg University Hospitals. In their study, patient weight was recorded and BMI or change in weight calculated for only 28% of cases in the Strasbourg University Hospitals and 25% of cases in other university hospitals. Otherwise, Henriksen et al. [START_REF] Henriksen | A low proportion of malnourished patients receive nutrition treatment -results from nutritionDay[END_REF] shown that data on weight or length were frequently missing in the patient records, and BMI could only be calculated in two-thirds of the patients in Norwegian hospitals.

Anthropomorphic and biological laboratory evaluation is essential for the precise evaluation of nutritional status, follow-up, diagnosis of possible complications, and the implementation of appropriate care ([14]; [5]).

The ESPEN criteria seem easier to use on a daily basis because they contain only anthropometric criteria and no biological criteria such as albumin, which seems to pose problems of interpretation [START_REF] Aussel | L'albuminémie est-elle un marqueur de l'état nutritionnel ?[END_REF]. At the end of this year, new French criteria for assessing the nutritional status of patients will be proposed.

Though the average length of hospital stays in our study was 10 days ± 4 days, albumin levels were tested two times or more in 52% of patients with severe malnutrition and 25 % of patients with no malnutrition. However, albumin half-life is 21 days, so regular testing has limited interest in the evaluation of nutritional status of patients in mid-length or short stays [START_REF] Aussel | L'albuminémie est-elle un marqueur de l'état nutritionnel ?[END_REF]. The methods used by health care teams to follow-up on malnutrition are not always optimal and can lead to increased spending.

Baron et al. [START_REF] Baron | Les diététiciens : acteurs de la qualité et du Programme de médicalisation des systèmes d'information[END_REF] implemented an online system to collect diet-related advice in order to meet the criteria of HAS health care quality and safety indicators in screening for nutritional issues and to enhance the work of dieticians. This online file had improved coding for malnutrition which encouraged better practices for recording of diet-related patient data [START_REF] Duval | P115 Intérêt du codage de la dénutrition par les diététiciennes[END_REF].

Finally, weight and BMI were recorded in 95.8% and 62.5% of patient files, respectively, versus 91.6% and 33.3% of files the year before.

The various representations of caregivers on ONS lead to confusion among patients. This study, in addition to a previous patient-centered study [START_REF] Hugol-Gential | Les traces alimentaires du patient hospitalisé[END_REF], raises the question of attitudes toward ONS. We investigated the attitudes of various different stakeholders: the medicine residents who prescribe the ONS, the nursing auxiliaries who administer them, and the patients who consume them. ONS have a special status -they are prescribed by doctors but managed by nursing auxiliaries -and the opinions expressed by the three parties revealed that ONS are seen partly as food, as supplement and as medication. This unstable image has an impact on the perceived importance of ONS, the effort dispensed to use them in the care of malnutrition (preventatively or therapeutically) and patient compliance and follow-up.

For medicine residents that we interviewed, the status of ONS is clear: it is a medicine. Nevertheless, medicine residents, who have a good understanding of malnutrition (e.g. causes, diagnosis, consequences), have a lack of specific knowledge about ONS (e.g. drug deliverance, compliance, post-hospitalization follow-up). The interviews as a whole underscored the lack of information and training regarding ONS, which would explain the gap between the HAS recommendations and the realities in the hospital context.

For nursing auxiliaries, ONS are first and foremost a food product which has the advantage of supplementing nutritional intake, but they are not perceived as a medication. ONS are not seen as 'medical', and the nursing auxiliaries we interviewed wonder about the utility of ONS prescription.

For patients, the situation seems even more complex. ONS are described as being somewhere between a medication, because it is prescribed, a food and a supplement. Uijl et al. [START_REF] Uijl | That's why I take my ONS. Means-end chain as a novel approach to elucidate the personally relevant factors driving ONS consumption in nutritionally frail elderly users[END_REF] also showed the ambiguity of ONS status by patients. For 80% of them, the ONS was a food rather than a medicine. The varying attitudes displayed by medicine residents and nursing auxiliaries mean that patients receive mixed messaged. Overall, patients have an uncertain attitude toward ONS, which is corroborated by the lack of knowledge displayed by medicine residents.

The variability in the status of ONS has led to a complex situation. Today, there are a number of obstacles which must be removed in order to ensure better patient assessment and optimal follow-up. This must be done in view of the different stages of malnutrition (Table 2). The led interviews and questionnaires allowed underlining the lack of knowledge and different representations relative to the status of the ONS. These differences lead an indistinctness as for the roles and the functions of the complementation, this one finding itself in the interface of the food and the medication. It seems then necessary that the medical profession have a holistic approach to think of the ONS in a global approach integrating the food habits of the patients, their tastes and their preferences to rethink the ONS prescription.

Each step, from the prescription to the consumption of ONS, has been rendered more difficult by vastly varying attitudes in the hospital setting. Norms, uses and perceptions differ according to whether the ONS is considered a medication, a food or a nutritional supplement.

Patients are exposed to many sources of information -from doctors, dietitians, or nursing auxiliaries -which result in an unclear message about ONS and the nutritional status of ONS.

It is therefore necessary to align the various sources so that the information transmitted by the all clinicians including physicians can be properly understood by the patient, and so that patient compliance for ONS is satisfactory.

Conclusion:

This study reveals that initial evaluation of nutrition status upon arrival is satisfactory for our patients, but the follow-up during the hospital stay and post-hospitalization were not optimal and differed according to the severity of patient malnutrition. The varying attitudes towards ONS, conveyed by medicine residents, nursing auxiliaries and patients, reveal their ambiguous status. An interdisciplinary approach between doctors, medicine residents, dietitians, nurses and nursing auxiliaries is needed to align their practices in the care of malnutrition and the messages they transmit to the patient. Lastly, evaluation and nutritional follow-up need to become a central axis in global patient management.

Clarifying the status of ONS for the health care team will lead to optimal practices in prescription and nutritional follow-up, as well as improvements in compliance thanks to the delivery of better quality information to patients and general practitioners.

Currently, nutritional status is not recorded on the final patient file at an optimal rate seeing as only 64% of patients had any type of recorded indication. The lack of information in patient records leads to a lack of information available to the general practitioner when the patient is discharged from the hospital.

Limitations of the study. We did not evaluate the effect of ONS on biological and anthropometric parameters. It was not the aim of our study. Our aim was to photography habits of departments concerning nutritional status evaluation, and its follow-up, and also opinion about ONS. Furthermore, we did not voluntary evaluate the role of dieticians in this study. This specific evaluation is a part of an another work we led. One another significant limitation is the definition of malnutrition used in the study methods. We used the we used the classification used in the hospital in which we conducted this study. But the classification criteria vary and the one used may not be the most common one.

Table 2 :

 2 Attitudes towards ONS and possible obstacles

	Parties concerned	Representations towards ONS	Obstacles
	medicine residents (responsible for	medication, but in conjunction with other	Lack of knowledge
	prescribing ONS)	care methods
		"Helpful when providing richer food is not
		enough to treat malnutrition";
		"Improves nutritional status when there are
		not issues with malabsorption and
		swallowing";
		"Used until malnutrition is gone and there
		is a return to an appropriate diet";
		"I don't always explain when I prescribe a
		treatment. It depends what it is, but it is
		still a treatment".
	Nursing auxiliaries (responsible for	Food, nutritional support	Not convinced of usefulness
	delivering ONS)
	Patients	Food, supplement , medication	Not enough information, confusion about
		"My doctor gave them to me, so they are	how ONS should be taken
		certainly beneficial";
		"That dietary thing that they bring us at
		3pm, I'm not used to it and after I'm not
		really hungry for my evening meal";

"It's a supplement, and so I'm categorical that it doesn't replace anything"; "It's to give us vitamins, to bring vitamins,

Thanks:

We would like to thank the Dijon University Hospital and in particular the staff of the digestive surgery and endocrinology departments. We would like to thank Marion Goguely and Corentin Jaillet for their help with data collection and we also thank Suzanne Rankin for translating the manuscript.

Conflicts of interest:

None. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Abstract:

Background: Nutritional evaluation and detection of malnutrition are based on criteria recommended by French health authorities. In practice, doctors do not always ensure strict implementation of the recommendations. The aim of this study is to evaluate professional practices in France regarding nutritional follow-up on arrival, during and after the hospitalization of inpatients who have oral nutritional supplements (ONS) prescribed and to discuss how ONS are seen by medical staff and patients.

Methods: A prospective study including patients consecutively admitted to digestive surgery and endocrinology units of the Dijon university hospital was conducted. Malnutrition risk at hospital admission was identified using anthropometric and biological criteria: Body Mass Index, percentage of weight loss, albumin, prealbumin, C-reactive protein and Mini Nutritional Assessment. Nutritional evaluation and monitoring of inpatients on arrival, during and after hospitalization was analyzed. Interviews were held with caregivers and patients to raise the question of their attitudes toward ONS.


Sylvain Verdier

Zeina Maroun

Rasmus Egeberg

Joan L Tiedje

Sara Sandersen

Per Zeuthen

Brice Bouyssière

Victor Garcia-Montoto

Sylvain Verdierc

Brice Bouyssiere

Understanding the removal of V, Ni and S in crude oil atmospheric residue hydrodemetallization and hydrodesulfurization

Keywords: Vanadium, nickel, sulfur, demetallization, desulfurization, catalysis, residue, GPC

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Hydroprocessing atmospheric residues using fixed-bed reactors is a mature technology that has been extensively studied, and close to 100 units are in operation across the globe. [START_REF] Raybaud | Catalysis by transition metal sulphides[END_REF] Most of these units are located in Asia and the Middle East, and the total catalyst demand is estimated to be approximately 50,000 tons/year. This technology is still relevant for many refineries as it can address key issues faced by the refining industry. For example, environmental specifications are getting stricter for bunker fuels as the revised MARPOL Annex VI will reduce the sulfur cap from 3.5 to 0.5 wt% from 2020 on. Furthermore, heavier crude oils are being processed, and those feedstocks require residue technologies such as atmospheric residue desulfurization (ARDS).

Among the numerous elements present in crude oil, the most abundant and undesirable elements are sulfur, vanadium and nickel, [START_REF] Ali | A review of methods for the demetallization of residual fuel oils[END_REF] which have negative effects, such as catalyst poisoning, fouling and equipment corrosion. [START_REF] Reynolds | Reaction sequence of metallopetroporphyrins during heavy residuum upgrading[END_REF][START_REF] Caumette | Element speciation analysis of petroleum and related materials[END_REF] The presence of sulfur compounds causes environmental and health problems as well, with the emission of toxic S species into the atmosphere. [START_REF] Yépez | Influence of different sulfur compounds on corrosion due to naphthenic acid[END_REF][START_REF] Vorapalawut | Application of TLC and LA ICP SF MS for speciation of S, Ni and V in petroleum samples[END_REF] With the purpose of reducing these undesirable effects, catalytic removal of these elements is currently carried out in the industry, especially for atmospheric and vacuum residue oils, whose content of these elements is much higher. The ARDS process will be briefly described below, but various detailed reviews and their respective references can be consulted for further information about the process and its evaluation. [START_REF] Raybaud | Catalysis by transition metal sulphides[END_REF][START_REF] Rana | A review of recent advances on process technologies for upgrading of heavy oils and residua[END_REF][START_REF] Sahu | A review of recent advances in catalytic hydrocracking of heavy residues[END_REF] The process consists of downflow trickle bed reactors in series with the following catalyst loading distributed across several reactors [START_REF] Bartholdy | Optimizing hydrotreater catalyst loadings for the upgradingof atmospheric residues[END_REF] : large-pore guard hydrodemetallization (HDM) catalyst (whose function is to trap V, Ni and As), a smaller-pore transition catalyst combining HDM and hydrodesulfurization (HDS) activities and a third catalyst with a higher HDS activity. The deactivation mechanisms of the catalysts in such a service have also been widely studied over the years. [START_REF] Bartholdy | The Interplay of Coke and Metals in Resid Deactivation[END_REF] The initial step, which is quite rapid, is mostly due to coke formation. [START_REF] Zeuthen | Formation of coke on hydrotreating catalysts and its effect on activity[END_REF] This step is followed by a slower deactivation phase during which metals gradually cover the catalyst surface. [START_REF] Bartholdy | Hydrodemetallization in resid hydroprocessing[END_REF] The last phase is a rapid loss of activity where the metals and coke create pore plugging or restrict access to the pore structure.

Many ARDS units are operated to obtain a fixed sulfur or Conradson carbon residue (CCR) content in the product. However, due to the difficult nature of the feedstock (high amount of metals and coke precursors), many challenges are encountered: short cycle length due to high deactivation rates, pressure-drop issues due to coking, demanding catalyst unloading process, long turnarounds (typically one month), etc. To minimize these issues, it is crucial that the optimal ratio between the HDM, HDM/HDS and HDS catalysts is determined for each specific ARDS unit.

The total amounts of Ni, V or S are an important piece of information here but do not provide any information about the nature of the molecules containing these elements. Therefore, speciation of the metals in the various feedstocks is an asset in regard to understanding the feed reactivity and the influence on the various deactivation mechanisms and ultimately in optimizing the catalyst loading. One option is to use inorganic speciation in crude oil that has been developed by using size exclusion chromatography in an organic solvent linked with a highresolution mass spectrometer working as the detector. This technique is referred to as gel permeation chromatography (GPC) and employs an organic solvent to perform the analysis, in this case tetrahydrofurane (THF). [START_REF] Caumette | Element speciation analysis of petroleum and related materials[END_REF] In this technique, the compounds are separated according to their hydrodynamic volumes, which in the case of these samples follow the molecular weights of the nanoaggregates and molecules that are present in the sample. By dissolving the sample in THF, it is possible to obtain a fingerprint of the molecular distribution of the species present in the sample and evaluate how the concentrations of each fraction increase or decrease as a function of the industrial process carried out.

Pohl et al. [START_REF] Pohl | Multielement molecular size fractionation in crude oil and oil residue by size exclusion microchromatography with high resolution inductively coupled plasma mass spectrometric detection (HR ICP MS)[END_REF] successfully used this method for two crude oils and two residue fractions and managed to map and fingerprint V, Ni, S, Fe, Co, Cr, Zn and Si. The distributions between high molecular weight (HMW) aggregates (possibly asphaltene structures), medium molecular weight (MMW) aggregates and low molecular weight (LMW) compounds (possibly free metal porphyrins and nonporphyrinic complexes) differ significantly between these samples, which will affect the HDM and HDS activities and their relative deactivation rates. Gascon et al. [START_REF] Gascon | Size distributions of sulfur, vanadium, and nickel compounds in crude oils, residues, and their saturate, aromatic, resin, and asphaltene fractions determined by gel permeation chromatography inductively coupled plasma high-resolution mass spectrometry[END_REF] used the same approach on the Saturate, Aromatic, Resin and Asphaltene (SARA) fractions of four crude oils, one atmospheric residue and one vacuum residue. The amount of each type of aggregate (HMW, MMW and LMW) was calculated, and significant variations can be observed among the four crude oils and their residue fractions. However, to understand the significance of these variations in the ARDS process, it is necessary to study not only the feedstock but also the hydrotreated product to understand the reactivity of the various types of aggregates.

With an increasing demand for low-sulfur fuel oil (LSFO), refineries are starting to consider alternative usages for their ARDS units, for example, by coprocessing vacuum residues, which are typically processed in ebullated bed reactors due to the high amount of metals and asphaltenes in this type of feed, which causes an extremely rapid deactivation of the catalyst. A feedstock change often requires changing the operating conditions in the unit (e.g., by lowering the feed rate to lower the liquid hourly space velocity) and redesigning the catalyst loading. The latter is not always straightforward, as it requires a very thorough understanding of the feed and its effect on the catalyst activity throughout the entire cycle to avoid untimely shutdowns. To this end, a better understanding of not only the feed properties (contents of sulfur, nitrogen, asphaltene, etc.) but also the molecular composition is invaluable. Thus, in this paper, two ARDS pilot plant studies are reported. In study A, the temperature was adjusted from 375 to 385 °C to evaluate its influence on the reactivity of the various sulfur, nickel and vanadium species. In study B, the fate of sulfur, nickel and vanadium nanoaggregates was monitored under constant operating conditions to examine the role of the initial coke laydown on the HDM and HDS reactions.

MATERIALS AND METHODS

Instrumentation

A quantitative analysis of all the feedstocks was carried out with the purpose of determining the mass variations of the target elements by using inductively coupled plasma mass spectrometry (ICP-MS). The principle of the method is to determine the total amount of xylenesoluble Ni and V compounds with inductively coupled plasma mass spectrometry (ICP-MS) after suitable dilution of the petroleum/petroleum product sample by weight with xylene. The vanadium and nickel contents are quantified by ICP-MS using a vanadium-isotope mass of 51 and a nickel-isotope mass of 60 through comparison with calibration curves obtained via 500 wt ppm Conostan organometallic V and Ni standards diluted with xylene. Scandium is used as an internal standard to correct for drift and matrix effects. The removal of molecular interferences in the ICP-MS analysis was performed with helium kinetic energy discrimination. EnviroMAT used-oil matrix reference material containing V and Ni was analyzed with each sample batch to monitor the analysis accuracy. The physico-chemical properties of the feeds and products were measured using the following standard methods: Nitrogen (ASTM D5762), Sulfur (ASTM D 4294, Hydrogen (ASTM D 7171 H), asphaltene (ASTM D6560), microcarbon residue (MCR) (ASTM D4530), specific gravity (SG) (ASTM D4052), and simulated distillation (ASTM D7169). Carbon (wt %) content was calculated by difference with 100% with N, S and H content.

All the speciation analyses were carried out with a Thermo Scientific Element XR double focusing sector field ICP-MS (Thermo Fisher, Germany) instrument accessing the spectrally interfered isotopes of 32 S, 51 V and 58 Ni at a resolution of 4,000 (medium resolution). The mass spectrometer was equipped with a quartz injector (1.0 mm inner diameter (i.d.)), a Pt sampler cone (1.1 mm orifice diameter) and a Pt skimmer cone (0.8 mm orifice diameter). An O2 gas flow of 0.08 mL/min was included to avoid carbon deposition. Both the instrument and mass calibration settings were optimized daily at resolutions of 300 (low resolution) and 4,000. A medium resolution avoids the spectral interference of O2 on the 32 S isotope. These optimizations were carried out using a tuning solution containing 1.0 ng g-1 of Ag, Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, In, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Sc, Si, Sn, Ti, V, Zn and Y in tetrahydrofuran (THF). A mass offset was also applied to compensate for the mass drift coming from the sector field magnet. [START_REF] Gutierrez Sama | Study of the aggregation of metal complexes with asphaltenes using gel permeation chromatography inductively coupled plasma highresolution mass spectrometry[END_REF][START_REF] Desprez | Study of the size distribution of sulfur, vanadium, and nickel compounds in four crude oils and their distillation cuts by gel permeation chromatography inductively ccoupled plasma highresolution mass spectrometry[END_REF] The mass spectrometer was equipped with a modified DS-5 microflow total consumption nebulizer (CETAC, Omaha, NE) mounted with a laboratory-made jacketed spray chamber heated to 60 °C by a water/glycol mixture working with a temperature-controller bath circulator Neslab RTE-111 (Thermo Fisher Scientific, Waltham, MA). [START_REF] Caumette | Sensitivity improvement in ICP MS analysis of fuels and light petroleum matrices using a microflow nebulizer and heated spray chamber sample introduction[END_REF] The chromatographic separation was carried out using three Waters (Waters Corporation, Milford, MA) styrene-divinylbenzene gel permeation columns (7.8 mm i.d. and 300 mm length).

These three columns included an HR4 column (particle size of 5 µm with an exclusion limit of 600,000 Da of polystyrene equivalent), an HR2 column (particle size of 5 µm with an exclusion limit of 20,000 Da) and an HR0.5 column (particle size of 5 µm and an exclusion limit of 1,000 Da of polystyrene equivalent). A Dionex high-performance liquid chromatography (HPLC) system fitted with an UltiMate 3,000 microflow pump, an UltiMate 3,000 autosampler and a low port-to-port dead-volume microinjection valve were used to deliver the carrier solution and mobile phase. To prevent possible damage to the GPC columns, a Styragel guard column (4.6 mm i.d. and 30 mm length) was also fitted between the columns and the pumping system.

Methodology.

One feed, which is a blend of 93% of atmospheric residue (ATM) and 7% vacuum residue (VAC), was hydrotreated under different conditions for the purpose of evaluating and understanding the removal of the target compounds by applying the method optimized by Desprez et al. [START_REF] Desprez | Study of the size distribution of sulfur, vanadium, and nickel compounds in four crude oils and their distillation cuts by gel permeation chromatography inductively ccoupled plasma highresolution mass spectrometry[END_REF] and Vargas et al. [START_REF] Vargas | Development of a chromatographic methodology for the separation and quantification of V, Ni and S compounds in petroleum products[END_REF] . These researchers studied the size distributions of V, Ni and S and established the integration intervals for HMW, MMW and LMW aggregates in crude oil as follows: 1,100-1,400 s (18 -23 mL of eluted sample) for HMW compounds, 1,400-1,600 s (23 -27 mL) for MMW compounds and above 1,600 s (< 27 mL) for LMW compounds. The comparison of the different sizes of species will provide useful information about the removal of target compounds.

In addition, with the purpose of monitoring the stability of the ICP HR MS intensities, analysis of known crude oil samples, whose composition had been studied previously, was periodically carried out.

Reagents, samples and solutions. Tetrahydrofuran, multisolvent GPC grade, ACS, stabilized with 250 ppm of butylated hydroxytoluene (BHT) (Scharlau, Spain) to prevent the formation of peroxides, was used both for sample preparation and for chromatographic elution.

All the atmospheric residue crude oil feeds were provided by Haldor Topsoe (Denmark), and the feeds were analyzed by diluting them in THF at dilution factors of 50 for the 93% ATM/7% VAC feed blend and of 5 for the products.

Pilot plant testing. The pilot tests were performed in a once-through downflow trickle bed pilot plant located in Haldor Topsoe's R&D facilities. The unit consists of two reactors in series.

The product was first separated in a high-pressure separator (HPS). The liquid product from the HPS was sent to a low-pressure separator (LPS) and to a stripper for removal of gases and other noncondensed light hydrocarbons. Nitrogen was used as the stripping agent, and once-through pure hydrogen was used. The catalyst was diluted with an inert material (mesh 60 carborundum) using a 25 vol% SiC/75 vol% catalyst ratio. The operating conditions are given in Table 1 for study A (influence of the temperature) and study B (influence of the initial deactivation). The feed properties are listed in Table 2. The results of study A are summarized in Table 3, and the results of study B are provided in Table 4. Note that the catalysts loaded were commercial catalysts used in residue service developed and produced by Haldor Topsoe A/S.

RESULTS AND DISCUSSION

Feedstock properties and fingerprints of the metal complexes in the feedstocks

The physico-chemical properties of the atmospheric and vacuum residues as well as those of the blends are listed in Table 2. The properties are in line with those of the typical atmospheric and vacuum residues reported in the literature. [START_REF] Raybaud | Catalysis by transition metal sulphides[END_REF] It is interesting to note that there is a factor of 2 difference between the two residues with respect to the Ni, V, asphaltene and MCR contents.

When two feeds are blended, it is important to check whether aggregation phenomena occur as these aggregates may influence the asphaltene stability and thereby the residue hydrotreating process. As shown in Figure 1, the theoretical blend (i.e., the calculated profile based on the analysis of the atmospheric and vacuum residues) and the physical blend show the same distribution, indicating that no nanoaggregation occurs. It should be noted that this finding could be a consequence of the fractions stemming from the same crude oil. Indeed, trimodal size distributions are observed for both the V and Ni compounds present in these feedstocks, while a less pronounced trimodal distribution, much richer in MMW aggregates, is observed for the sulfur compounds.

Figure 1. GPC ICP HR MS chromatograms of the V, Ni and S species in the atmospheric residue feedstock (ATM, green line), vacuum residue (VAC, blue line), blend of 93 vol% ATM and 7% VAC (physical blend, red line) and theoretically reconstructed blend of 93 vol% ATM and 7 vol% VAC (black dashed line) based on individual ATM and VAC chromatograms.

The size distributions of different crude oils and residues were studied elsewhere, [START_REF] Gascon | Size distributions of sulfur, vanadium, and nickel compounds in crude oils, residues, and their saturate, aromatic, resin, and asphaltene fractions determined by gel permeation chromatography inductively coupled plasma high-resolution mass spectrometry[END_REF] showing the presence of HMW, MMW and LMW nanoaggregates, and the HMW compounds were the Figure 2 shows that, as expected, at higher temperatures, more V, Ni and S species are removed. For V and Ni, the LMW and MMW aggregates are easily hydrotreated even at low temperatures, and as the temperature is increased, only the remaining HMW aggregates are hydrotreated. This phenomenon can be quantitatively observed in Figure 3, where the concentration of each nanoaggregate size is determined based on the areas of the peaks (details of the integration method are available in the open literature). [START_REF] Gutierrez Sama | Study of the aggregation of metal complexes with asphaltenes using gel permeation chromatography inductively coupled plasma highresolution mass spectrometry[END_REF] The concentration of LMW and MMW aggregates is negligibly small at all temperatures, while the concentration of HMW aggregates shows a linear decrease as a function of the temperature. The correlation coefficient of the slope is better for the regression of the HMW nanoaggregates compared to those of the MMW and LMW nanoaggregates, confirming that the temperature is driving the HMW hydrotreatment. From the slopes of these linear trends, as listed in Behavior of S. Interestingly, the fate of the S aggregates differs from that observed for the V and Ni aggregates. Figures 2 and3 show that the LMW and MMW S aggregates are not fully hydrotreated at the lowest temperatures. This phenomenon indicates that these LMW and MMW compounds are more refractory compared to the size-similar V and Ni compounds. It is also worth noting that a bimodal HMW-MMW S distribution is observed in the hydrotreated products, while this distribution was not clearly present in the feedstock. It is likely that a trimodal distribution occurs in the feedstock but is hidden due to the high concentration of aggregates eluting in the MMW range. These results show that S compounds/nanoaggregates with molecular weights between HMW and MMW are more easily removed, indicating that the catalyst possesses a higher efficiency for these kinds of nanoaggregates.

Study B -initial deactivation due to coke laydown

The initial and fast deactivation during ARDS tests is due to coking and, more specifically, the strong initial adsorption of polyaromatic compounds, including N-containing molecules onto the surface of the catalyst. [START_REF] Zeuthen | Formation of coke on hydrotreating catalysts and its effect on activity[END_REF] In commercial units, the impact of the initial coke laydown on HDS and HDM is not simple to evaluate. For some units, it is clear that the HDM activity is more affected than the HDS activity during the first hundred hours. For other units, the initial deactivations of the HDS and HDM activities are equal, which depends on the catalyst loading, feed properties and operating conditions. Therefore, the goal of this study was to evaluate how the initial deactivation (which is due to coke laydown) affected the hydrotreatment rates of the various aggregates and if certain differences between the initial deactivations of HDM and HDS could be observed, which could be related to the aggregate distribution. For this purpose, the temperature of the process was kept constant at 375 °C to evaluate the changes due to the initial catalyst deactivation (at 23, 52, 76 and 100 run hours).

Behavior of V and Ni. The evolution of the V and Ni species present in the feed after having been through the hydrotreating process at different times has been studied. As shown in Figure 4, initially, the concentration of V and Ni species is very low, but during the first hours of the test, a steady increase in the total concentration of V and N is observed. Figure 5 shows the quantitative description of this increase. This increase is almost entirely ascribed to an increase in the HMW fraction. It should be noted that most of the LMW and MMW aggregates are removed throughout the test despite the increasing deactivation (Table 6) of the catalyst, as indicated by the total product Ni and V contents listed in Table 4. Behavior of S. In Figure 4, it is observed that the sulfur species follow a different evolution. A bimodal distribution of the HMW and MMW peaks is observed, and its ratio has been calculated, resulting in an almost constant value. As shown more clearly in Figure 5, the increase in S species is similar for the three groups of hydrodynamic volume distributions, which could indicate either a higher preference of the catalyst for the HMW species (if we compare the behavior of S with the behavior of V and Ni) or a more refractory behavior from the S species with the catalyst. Ni and V aggregates, it was found that the HMW compounds are more refractory compared to the LMW and MMW compounds, which are removed at low temperatures or even after long run hours.

For the S aggregates, a slightly different behavior was observed whereby the LMW and MMW aggregates are not as easily hydrotreated, and in fact, the MMW nanoaggregates/compounds are slightly less easily hydrotreated compared to the HMW aggregates. This phenomenon illustrates the inherent difference in chemical composition of the metallo-organic compounds compared to the sulfur-containing compounds and their reactivity. In a previous study [START_REF] Gutierrez Sama | Study of the aggregation of metal complexes with asphaltenes using gel permeation chromatography inductively coupled plasma highresolution mass spectrometry[END_REF][START_REF] Gascon | Simplification of heavy matrices by liquid-liquid extraction: part I-how to separate LMW, MMW, and HMW compounds in maltene fractions of V, Ni, and S compounds[END_REF][START_REF] Gascon | Simplification of heavy matrices by liquid-solid extraction: part II-how to separate the LMW, MMW, and HMW compounds in asphaltene fractions for V, Ni, and S compounds[END_REF] free porphyrinic compounds were easily removed, possibly via easy access of the molecules to the catalyzer. Of the HMW nanoaggregate part, only a small portion can be easily removed, which can be the porphyrins linked at the surface of the nanoaggregates, [START_REF] Gutierrez Sama | Study of the aggregation of metal complexes with asphaltenes using gel permeation chromatography inductively coupled plasma highresolution mass spectrometry[END_REF] but the Ni and V compounds that are located deeper in the nanoaggregates are more difficult to remove as the shell of the nanoaggregates has to be removed first before the V and Ni compounds can access the catalyst.

In regard to the S compounds, the observed behavior seems to me driven more by the types of S compounds than by the aggregation states of the S compounds. Here, the molecular conformation of the S compounds needs to be studied. More in-depth molecular studies have to be conducted.

Nevertheless, this study provides insights into the catalyst preference towards these compounds/nanoaggregates, which is an important factor for future optimization of the balance between the HDM and HDS catalysts for those ARDS units processing different feedstocks with different V, Ni and S nanoaggregate distributions.

Figure 2 .

 2 Figure 2. GPC ICP HR MS chromatograms of the V, Ni and S species in the feed (black dashed line) and the product after having been hydrotreated at different temperatures (the hydrotreatments at 375 °C, 380 °C and 385 °C correspond to the blue, green and red lines, respectively).

Figure 3 .

 3 Figure 3. The concentrations of V, Ni and S (total in black, HMW in blue, MMW in orange and LMW in gray) in the products that were hydrotreated at different temperatures quantified based on the total concentration and the GPC ICP HR MS chromatograms.

Figure 4 .

 4 Figure 4. GPC ICP HR MS chromatograms of the V, Ni and S species in the feed (black dashed line) and the product for different run times after having been hydrotreated at the same temperature (the run times of 23 h, 52 h, 76 h and 100 h correspond tothe light blue, red, green and blue lines, respectively).

Figure 5 .

 5 Figure 5. The concentrations of V, Ni and S (total in black, HMW in blue, MMW in orange and LMW in gray) in the products that were hydrotreated for different run times quantified based on the total concentration and the GPC ICP HR MS chromatograms.

Figure 1 .

 1 Figure 1. GPC ICP HR MS chromatograms of the V, Ni and S species in the atmospheric

Figure 2 .

 2 Figure 2. GPC ICP HR MS chromatograms of the V, Ni and S species in the feed (black dashed

Figure 3 .

 3 Figure 3. The concentrations of V, Ni and S (total in black, HMW in blue, MMW in orange and

Figure 4 .

 4 Figure 4. GPC ICP HR MS chromatograms of the V, Ni and S species in the feed (black dashed

Figure 5 .

 5 Figure 5. The concentrations of V, Ni and S (total in black, HMW in blue, MMW in orange and

Table 1 .

 1 Operating Conditions of the Pilot Plant Tests

	Inlet pressure hydrogen (barg) 165
	H2/oil (mL/L)	550
	Overall liquid hourly space 0.2

Table 2 .

 2 Feed Properties calculated from the properties of the atmospheric residue and of the blend assuming that volume and weight fractions are equivalent as density could not be measured for the vaccum residue.

			Atmospheric	Vacuum
	Property	Method	residue	residue	Blend 93 vol% ATM and 7 vol% VAC
			(ATM)	(VAC)
	Nitrogen, wt ppm	D 5762	2800	4500	3100
	Sulfur, wt%	D 4294	3.985	5.00	4.061
	Hydrogen, wt%	D 7171 H	10.75	(11.18)*	10.78
	C, wt%	difference By	85.0	83.4	84.8
	H/C	-	0.13	0.13	0.13
	SG 60/60 °F	D 4052	0.9797	-	0.9847
	Density at 40 °C, g/ml	D 4052	0.9621	-	0.9672
	Nickel, wt ppm	ICP-MS	21.1	42.4	27.0
	Vanadium, wt ppm	ICP-MS	61.1	121	73.0
	Carbon Residue, wt%	D 4530	12.02	24.42	12.96
	Asphalthenes, wt%	D 6560	5.1	11.0	6.4
	Simulated Distilation	D 7169
	0.5 wt% (IBP), °C		221	415	235
	5 wt%, °C		341	515	353
	10 wt%, °C		378	542	388
	30 wt%, °C		454	600	467
	50 wt%, °C		535	649	546
	70 wt%,°C		614	709	627
	80 wt%, °C		665	-	676
	85 wt%, °C		698	-	705
	90 wt%, °C		736	-	-
	95 wt%, °C		-	-	-
	99.5 wt% (FBP), °C		-	-	-
	*: H content back-

Table 3 .

 3 Results of Study A (Influence of the Temperature)

	Sample type		Feed		Product	Product	Product
	Run time, hours				1,003	1,101	1,337
	Temperature	of	the
	reactor, °C				375	380	385
	Sulfur, wt%		ASTM D4294	4.061	0.446	0.349	0.291
	Nickel, wt ppm		ICP-MS	27	5.16	4.12	3.62
	Vanadium, wt ppm	ICP-MS	73	8.61	6.65	5.85
	Asphaltenes, wt%	ASTM D6560	6.4	1.4	1.2	1.6
	Simulated Distillation	ASTM D7169
	0.5 wt% (IBP), °C		235	142	142	128
	5 wt%, °C			353	258	244	234
	10 wt%, °C			388	306	295	285
	30 wt%, °C			467	399	391	394
	50 wt%, °C			546	466	456	464
	70 wt%,°C			627	541	532	542
	80 wt%, °C			676	579	572	586
	85 wt%, °C			705	603	595	612
	90 wt%, °C			-	630	623	641
	95 wt%, °C			-	665	658	678
	99.5 wt% (FBP), °C		-	721	721	717

Table 4 .

 4 Results of Study B (Influence of the Initial Deactivation Due to Coke Laydown)

	Sample type		Feed	Product	Product	Product	Product
	Run time, hours			23	52	76	100
	Temperature of the reactor, °C		375	375	375	375
	Sulfur, wt%	ASTM D4294	4.061	0.098	0.179	0.212	0.282
	Nickel, wt ppm	ICP-MS	27	1.18	1.74	2.08	2.89
	Vanadium, wt ppm	ICP-MS	73	2.02	2.84	3.25	4.32
	Simulated Distillation ASTM D7169
	0.5 wt% (IBP), °C		235	142	67	142	155
	5 wt%, °C		353	249	247	260	272
	10 wt%, °C		388	301	300	313	323
	30 wt%, °C		467	407	403	415	421
	50 wt%, °C		546	482	474	491	497
	70 wt%,°C		627	572	553	577	583
	80 wt%, °C		676	632	598	632	639
	85 wt%, °C		705	675	626	668	676
	90 wt%, °C		-	-	659	716	721
	95 wt%, °C		-	-	702	-	-
	99.5 wt% (FBP), °C		-	-	-	-	-

Table 5

 5

, it can be readily observed that the HMW V aggregates are hydrotreated faster than the Ni aggregates. This observation is in agreement with previous results in the open literature.

19

Table 5 .

 5 Slopes and Regression Coefficients for the Variation in V, Ni and S Concentrations vs. Temperature

		Total		HMW		MMW		LMW
		Slope	r 2	Slope	r 2	Slope	r 2	Slope	r 2
	V -3.82 E-02 0.998 -3.39 E-02 0.988 -0.25 E -02 0.733 -0.19 E -02 0,965
	Ni -2.28 E-02 0.998 -1.98 E -02 0.992 -0.19 E -02 0.957 -0.10 E -02 0,879
	S	-24.84	0.999	-9.955	0.997	-10.21	0.999	-4.678	0,999

Table 6 .

 6 Slopes and Regression Coefficients for the Variation in V, Ni and S Concentration vs. Run Time (Hours).

		Total		HMW		MMW		LMW
		Slope	r 2	Slope	r 2	Slope	r 2	Slope	r 2
	V 4.96 E-03 0.991 4.44 E-03 0.987 0.38 E -03 0.989 0.14 E -03 0,997
	Ni 3.66 E-03 0.993 2.98 E -03 0.989 0.46 E -03 0.997 0.22 E -03 0,966
	S	6,22	0.994	2.19	0.994	2.87	0.990	1.16	0,995

TABLE CAPTIONS Table 1 .

 CAPTIONS1 Operating Conditions of the Pilot Plant Tests

Table 2 .

 2 Feed Properties

Table 3 .

 3 Results of Study A (Influence of the Temperature)

Table 4 .

 4 Results of Study B (Influence of the Initial Deactivation Due to Coke Laydown)

Table 5 .

 5 Slopes and Regression Coefficients for the Variation in V, Ni and S Concentrations vs.

	Temperature

Table 6 .

 6 Slopes and Regression Coefficients for the Variation in V, Ni and S Concentration vs.

	Run Time (Hours)

Funding Sources

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

Financial support from the Conseil Régional d'Aquitaine (20071303002PFM) and FEDER (31486/08011464) is acknowledged

AUTHOR INFORMATION

Corresponding Author


Nicolas Mottet
email: ncmottet@gmail.com

Claire Cochet

Chrystelle Vidal

Jean Patrick Metz

Sébastien Aubry

Aude Bourtembourg

Astrid Eckman-Lacroix

Didier Riethmuller

Lionel Pazart

Rajeev Ramanah

Feasibility of two-dimensional ultrasound shear wave elastography of human fetal lungs and liver: A pilot study

Keywords: Elasticity imaging techniques, Sonoelastography, Shear wave, Observer variation, Fetal lung, Fetal liver

de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Prenatal evaluation of fetal lung maturity (FLM) is still controversial and different approaches have been described with invasive biological tools and fetal imaging [START_REF] Besnard | Lecithin/sphingomyelin ratio and lamellar body count for fetal lung maturity: a meta-analysis[END_REF][START_REF] Palacio | Prediction of neonatal respiratory morbidity by quantitative ultrasound lung texture analysis: a multicenter study[END_REF][START_REF] Bhagwanani | Prediction of neonatal respiratory distress by estimation of amniotic-fluid lecithin[END_REF][START_REF] Field | Current status of amniotic fluid tests of fetal maturity[END_REF][START_REF] Varner | Amniocentesis for fetal lung maturity: will it become obsolete?[END_REF][START_REF] Sohn | Diagnosis of fetal lung maturity by ultrasound: a new method and first results[END_REF][START_REF] Cobo | Feasibility and reproducibility of fetal lung texture analysis by Automatic Quantitative Ultrasound Analysis and correlation with gestational age[END_REF][START_REF] Duncan | Assessment of fetal lung growth in utero with echo-planar MR imaging[END_REF][START_REF] Moshiri | Fetal lung maturity assessment with MRI fetal lung-to-liver signal-intensity ratio[END_REF][START_REF] Gorincour | Lung signal on fetal MRI: normal values and usefulness for congenital diaphragmatic hernia[END_REF]. FLM is mainly correlated with surfactant level and different tests have been developed to quantify this production through detection of components in the amniotic fluid [START_REF] Besnard | Lecithin/sphingomyelin ratio and lamellar body count for fetal lung maturity: a meta-analysis[END_REF]. However, performances of these tests to predict neonatal respiratory morbidity are limited for a routine clinical application because they need invasive procedures and all have a positive predictive value ranging from 18 to 34% [START_REF] Palacio | Prediction of neonatal respiratory morbidity by quantitative ultrasound lung texture analysis: a multicenter study[END_REF][START_REF] Field | Current status of amniotic fluid tests of fetal maturity[END_REF][START_REF] Varner | Amniocentesis for fetal lung maturity: will it become obsolete?[END_REF][START_REF] Sohn | Diagnosis of fetal lung maturity by ultrasound: a new method and first results[END_REF]. Thus, development of new and non-invasive methods to assess FLM is challenging to predict respiratory distress syndrome (RDS). RDS complicates 1% of all births and the incidence increases with prematurity. RDS ranges from 26% to 30% in preterm neonates before 34 weeks of gestation (WG) and from 5% to 20% after 34 WG [START_REF] Palacio | Prediction of neonatal respiratory morbidity by quantitative ultrasound lung texture analysis: a multicenter study[END_REF][START_REF] Donda | Trends in epidemiology and outcomes of respiratory distress syndrome in the United States[END_REF].

Fetal lung and liver signal or echogenicity are often compared to assess FLM [START_REF] Cobo | Feasibility and reproducibility of fetal lung texture analysis by Automatic Quantitative Ultrasound Analysis and correlation with gestational age[END_REF][START_REF] Duncan | Assessment of fetal lung growth in utero with echo-planar MR imaging[END_REF][START_REF] Moshiri | Fetal lung maturity assessment with MRI fetal lung-to-liver signal-intensity ratio[END_REF][START_REF] Gorincour | Lung signal on fetal MRI: normal values and usefulness for congenital diaphragmatic hernia[END_REF][START_REF] Maeda | Echogenicity of fetal lung and liver quantified by the grey-level histogram width[END_REF]. Non-invasive assessment of FLM has been first attempted using ultrasonography quantified by gray level histogram [START_REF] Serizawa | Noninvasive fetal lung maturity prediction based on ultrasonic gray level histogram width[END_REF][START_REF] Prakash | Fetal lung maturity analysis using ultrasound image features[END_REF][START_REF] Cannie | Prenatal prediction of survival in isolated diaphragmatic hernia using observed to expected total fetal lung volume determined by magnetic resonance imaging based on either gestational age or fetal body volume[END_REF]. These studies demonstrated imaging changes associated with lung maturation but weak correlations between echogenicity and clinical outcomes at birth. Recently, a quantitative fetal lung texture analysis has been described and demonstrated a strong correlation with gestational age, biological test on amniotic fluid and risk of neonatal respiratory morbidity [START_REF] Palacio | Prediction of neonatal respiratory morbidity by quantitative ultrasound lung texture analysis: a multicenter study[END_REF][START_REF] Bonet-Carne | Quantitative ultrasound texture analysis of fetal lungs to predict neonatal respiratory morbidity[END_REF]. Prediction of the pulmonary function through quantitative fetal lung stiffness measurement seems innovative and could be complementary to quantitative ultrasound texture analysis. Two-dimensional ultrasound (2D) shear wave elastography (SWE) is the latest evolution of elastography techniques and enables assessment of tissue stiffness in real time [START_REF] Bercoff | Supersonic shear imaging: a new technique for soft tissue elasticity mapping[END_REF]. The aim of elastography techniques is to assess the hardness of a tissue, either by objective quantification or compared to an adjacent tissue [START_REF] Franchi-Abella | Ultrasound elastography: advantages, limitations and artefacts of the different techniques from a study on a phantom[END_REF].Quantitative evaluation of the stiffness is expressed either in terms of shear wave speed (m. s-1 ) or Young's modulus (kPa) [START_REF] Bercoff | Supersonic shear imaging: a new technique for soft tissue elasticity mapping[END_REF][START_REF] Gennisson | Ultrasound elastography: principles and techniques[END_REF][START_REF] Carlson | Estimation of shear wave speed in the human uterine cervix[END_REF]. This technique has been validated in several organs (liver, breast, placenta, cervix) [START_REF] Carlson | Estimation of shear wave speed in the human uterine cervix[END_REF][START_REF] Alison | Quantitative shear-wave elastography of the liver in preterm neonates with intra-uterine growth restriction[END_REF][START_REF] Abeysekera | SWAVE Imaging of Placental Elasticity and Viscosity: Proof of Concept[END_REF][START_REF] Davutoglu | The role of shear wave elastography in the assessment of placenta previa-accreta[END_REF][START_REF] Habibi | In vivo assessment of placental elasticity in intrauterine growth restriction by shearwave elastography[END_REF], and some studies reported interesting results about exploration of non human fetal organs with 2D-SWE [START_REF] Quarello | Shear wave elastography of fetal lungs in pregnant baboons[END_REF][START_REF] Quarello | Feasibility and reproducibility of ShearWave(TM) elastography of fetal baboon organs[END_REF]. 2D-SWE could be a promising tool in fetal medicine by providing a new approach to studying changes in fetal lung tissue properties during gestation.

A pilot study is necessary to clarify applicability of 2D-SWE for exploration of human fetal lungs and liver during gestation. It seemed relevant to compare fetal lung and liver stiffness evolution during pregnancy, and a new ratio could be modeled to provide an objective quantification: the lung to liver elastography (LLE) ratio.

The primary objective of this study was to evaluate feasibility and reproducibility of 2-D SWE of human fetal lungs and liver between 24 and 34 weeks of gestation. The secondary objective was to model fetal LLE ratio and to assess its variations according to gestational age and maternal administration of corticosteroids.

Methods

Study design

A prospective case-control study was performed at the University Hospital of Besançon, (Besançon, France), Department of Obstetrics and Gynecology from April to November 2016, in strict accordance with the ethical guidelines of the Declaration of Helsinki. The « ELASTOMAP study » was approved by human ethical research committee (Comité de Protection des Personnes EST II, process number 15/494) and the French National Agency for Medicines and Health Products Safety (process number 2015-A01575-44) [START_REF] Mottet | Feasibility of 2-D ultrasound shear wave elastography of fetal lungs in case of threatened preterm labour: a study protocol[END_REF].

The study was registered on clinical trial.gov with the following number: NCT02870608.All participants provided written informed consent. Details of the rationale and design of the trial have been published previously [START_REF] Mottet | Feasibility of 2-D ultrasound shear wave elastography of fetal lungs in case of threatened preterm labour: a study protocol[END_REF].

Fetal lungs and liver were studied by 2D-SWE between 24 and 34 weeks of gestation (WG) in two groups: women with an uncomplicated pregnancy composed the control group (group 1), and women with a threatened preterm labor requiring administration of corticosteroids were enrolled as cases (group 2). The inclusion criteria were common to both groups: pregnant women aged 18 years or older, singleton pregnancy, eutrophic fetus, signature of consent and affiliation to health insurance scheme. Exclusion criteria were common to both groups: fetal lung or liver pathologies, inclusion in another medical study, and patients under legal incapacity. Two 2D-SWE examinations were performed at "day 0", and "day 2" in group 1, separated for 48h. A 2D-SWE examination was performed in group 2 before and 24h after a complete course of corticosteroids indicated for threatened preterm labor, respectively at "day 0" and "day 2" (Fig. 1). Betamethasone course was prescribed by maternal intramuscular injection of 12 mg and repeated 24 h after the first injection. All variables (patient parameters, data items, data elements) were aggregated into electronic Case report forms

Prenatal variables and measuring technique

A Logic ® E9 system (General Electric Healthcare) with CE certification CE LNE/G-MED [CE0459] equipped with abdominal convex probe 1-6 MHz (C1-6-D probe) was used for this study. The shear wave acquisition measurement protocol was the same for each patient. Details of the protocol have been published previously [START_REF] Mottet | Feasibility of 2-D ultrasound shear wave elastography of fetal lungs in case of threatened preterm labour: a study protocol[END_REF].

Three operators participated in this study. Operator 1 was the reference operator (NM, senior clinical lecturer), and two others operators were trained: operator 2 (JPM, resident) and operator 3 (CC, intern). To assess repeatability of measurements, operators performed 2 measurements to obtain duplicates on each target regions while systematically repositioning the probe on the target organ: three regions for the proximal lung (anterior "region A1", medium "region A2" and posterior portion "region A3") and three regions for the liver (IV, V, VI) (Fig. 2). To test the inter-observer reproducibility, a second observer successively performed measurements on 30 fetuses. All measurements were carried out directly by ultrasound on the target organ. The second observer performed measurements just after the first one and was blinded to the feasibility and results obtained by the first one.

The fetal LLE ratio was initially defined as the value of the proximal lung stiffness divided by the value of the liver stiffness. Only repeatable and reproducible values of each ROI were considered to model the fetal LLE ratio. Measurements were performed on a homogeneous circular ROI of 5 mm diameter manually drawn. Technical failure was defined as failure to obtain a homogeneous elastogram in greater than 50% in the sampling area. At the end of the follow up, the operator 1 was blinded of the women's status and reviewed all the measurements. Measurements were considered invalid when the ROI did not match with the requested region or when important artifacts interfered with the ROI.

Postnatal variables

Two pediatricians examined all the infants at birth and were blinded to the prenatal exposure to 2D-SWE. The following data were collected: term of birth, weight, neonatal transfer, Apgar score and evaluation of respiratory distress by Silverman score. In the two days after birth, automated evaluation of auditory evoked potentials were recorded in case of premature delivery before 37 WG to despite cochlear damage in the exposed fetus. After 37 WG, auditory evoked potentials were performed if otoacoustic emissions were absent. At three months after birth, the women received a phone call to assess infant well-being and to collect the following data: medical history, health problems, respiratory diseases or symptoms, liver disease or symptoms and number of hospitalizations since birth.

Statistical analysis

Given the paucity of data regarding 2D-SWE on human fetal lung, the sample size calculation was based on measurements of fetal lungs in pregnant baboons. All calculation hypothesis were detailed in the published protocol [START_REF] Mottet | Feasibility of 2-D ultrasound shear wave elastography of fetal lungs in case of threatened preterm labour: a study protocol[END_REF]. Forty-two patients per group were needed to have 90% power to statistically demonstrate such a difference (0.2 versus 0.057) assuming an α risk of 0.05 and standard deviation of 0.2.

We used descriptive analysis to examine the population in the study. Qualitative variables were expressed as raw numbers, proportions and percentages and quantitative variables were expressed as means, SDs and ranges. Feasibility was evaluated by assessing the number of examinations performed and the number of examinations with interpretable results (invalid data). In order to investigate the repeatability of measurements over a short period of time, two measurements per subject were made under identical conditions by the same observer [START_REF] Bartlett | Reliability, repeatability and reproducibility: analysis of measurement errors in continuous variables[END_REF]. A paired Student's t-test was used to test repeatability of measurements. Results were expressed as mean difference with 95% confidence interval, standard deviation of the differences and P value. The intraclass correlation coefficient (ICC) was used to test inter observer reproducibility with 95% CI [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. Differences in fetal LLE ratio values between two examinations were calculated by using the Wilcoxon test. Simple linear regression was used to test correlation between stiffness values and gestational age: the Pearson correlation coefficient R and the coefficient of determination R2 were mentioned. The strength of the association was regarded as very weak for Pearson coefficient values of 0-0.19, as weak for 0.2-0.39, as moderate for 0.40-0.59, as strong for 0.6-0.79 and as very strong for 0.8-1.

Concerning postnatal data, a descriptive analysis at baseline and at each follow-up was performed. Comparisons between group 1 and group 2 were performed using Student's t test or Mann-Whitney's U test for quantitative variable and Chi-squared test or Fisher's exact test for qualitative variables, as appropriate.

Results

During the study period, 103 patients were included: 55 women with an uncomplicated pregnancy composed group 1, and 48 women with a threatened preterm labor requiring administration of corticosteroids were enrolled as cases in group 2 (Fig. 1). Maternal characteristics are summarized in Table 1.

A total of 540 measurements were performed on each ROI. For the proximal lung, 8.6% of measurements were considered invalid (21.1% for A1, 2.8% for A2, 2% for A3), and 6.9% were considered invalid for the liver (14.4% for IV, 3.3% for V, 2.9% for VI). The number of invalid measurements did not differ between the control group and cases for each ROI. These data were excluded for statistical analysis.

For the 3 operators, measurements on each ROI were repeatable except for region IV for both operator 1 (mean difference = 0.1; 95 % CI: 0.002 -0.206; SD = 0.65) (P= 0.04) and operator 2 (mean difference = 0.13, 95 % CI: 0.006-0.26; SD = 0.54) (P = 0.04] (Table 2).

Interobserver ICC values for stiffness values (average of duplicates) in the different ROI among the three operators are presented in Table 3.

Considering invalid data for ROI A1 and IV, absence of repeatability for region IV for two operators, and low ICC between operator 1 and 3 for ROI A2 (ICC = 0.28; 95% CI: -0.72 -0.69), these ROIs were excluded for fetal LLE ratio modeling. Only ROI A3 for the lung and ROI V and VI for the liver were considered eligible for statistical analysis. The stiffness value for each region was calculated from the average of duplicates performed by the operator. Three ratios were modeled and compared. There were: Fetal LLE ratio 1: A3/ [(V + VI)/2]; Fetal LLE ratio 2: A3/ V, and Fetal LEE ratio 3: A3/ VI.

The median values of these three indexes were compared at "day 0" and "day 2", and the relative differences (["day 0 -day 2"]/"day 0") were evaluated in all the cohorts (group 1 and group 2). No statistically significant differences were noted among the three indexes, and we decided to retain fetal LLE ratio 1 for the remainder of the statistical analysis. Mean stiffness values for the lung (A3) and the liver (V, VI) were respectively 2.21 +/-0.41 [1.44-3.06] KPa, 3.74 +/-0.86 [2.38-6.36] kPa and 3.99 +/-0.91 [2.63-6.20] kPa in the group 1.

Analysis of stiffness variations according to gestational age and corticosteroids were based on the reference operator's data. Operator 1 performed examinations in 87 singleton pregnancies at a median age of 30 WG (range: 24-33.5 WG). According to gestational age, linear correlation was significant for stiffness values of ROI A3, V and VI with positive correlation coefficients (R: 0.56, 0.46 and 0.44 respectively) (Fig. 3). However, if variation of the fetal LLE ratio was assessed according to gestational age, the linear trend correlation was not significant (P = 0.6) and correlation was very weak (R= 0.08) (Fig. 4).

The fetal LLE ratio values for the first examinations at "day 0" were not significantly different between controls and cases. Operator 1 performed 2D-SWE examinations in 42 controls and 30 cases. The fetal LLE ratio values at "day 2" decrease in group 2 exposed to corticosteroids (0.2; 95% CI: 0.07 to 0.34) and increase in controls (-0.04; 95% CI: -0.29 to 0.08) (P < 0.001) Differences in fetal LLE ratio values between two examinations are presented in Figure 4.

Pediatricians examined a total of 102 children exposed to 2D-SWE in the two days following birth. There was one case of in utero fetal death secondary to uterine rupture in a woman hospitalized for threatened preterm labor at 31 WG. Infant characteristics and respiratory status at birth are summarized in Table 1. All infants in the control group were born after 37 WG, and otoacoustic emissions were present for 53 infants. Auditory evoked potentials were controlled in one case and were normal. Otoacoustic emissions were not registered in the medical file for one case, but no additional control was mentioned. In the group 2, 23 infants were born before 37 WG, and the abnormal auditory evoked potentials were registered for one ear in one case of premature delivery at 31 WG. Otoacoustic emissions were normal for 22 infants born after 37 WG in the group 2. Otoacoustic emissions were not registered in 2 cases in the two days following birth but were normal during a secondary control after leaving the maternity hospital (Table 1). Health status was evaluated four months after birth for 78 children and is summarized in Table 1.

Discussion

Our study demonstrates that quantitative fetal lung and liver stiffness measurement with 2D-SWE is feasible with acceptable reproducibility considering average pairs of measurements. Some ROIs are more reproducible and repeatable on these organs.

We assess lung stiffness on the proximal lung to reduce presumed impact of depth on measurements and to improve the reliability of fetal LLE ratio. Indeed, Quarello et al reported variation of ICC between proximal and distal lung in pregnant baboons, respectively 0.65 (95% CI: 0.42-0.81) and 0.42 (95% CI: 0.11-0.65) [START_REF] Quarello | Shear wave elastography of fetal lungs in pregnant baboons[END_REF]. Our study indicates that region A3 was the most relevant given its feasibility and acceptable reproducibility considering average pairs of measurements (ICC between 0.70 and 0.78) (Table 3). Invalid measurements were also the lowest in this region compared to both other regions. This difference can be explained by acoustic shadowing generated by fetal ribs and disturbance of share wave speed induced by cardiac movements that are more important in A1 and A2 compared to A3.

Concerning the liver, the left extremity (region IV) was not repeatable and contained many invalid measurements (14,4%) compared with ROI V and VI. These results were probably related to the close anatomical relationships between liver, stomach and bowels in these regions. It is likely that operators confused these organs with incorrect placement of the focused ultrasounds, obtaining inaccurate results. Considering average pairs of measurements, reproducibility was acceptable for region V (ICC between 0.75 and 0.73) and region VI (ICC between 0.70 and 0.84). Thus, in the modeling process of the fetal LLE ratio, we decided to retain only A3, V and VI as the most repeatable and reproducible ROI to compare fetal lung and liver stiffness on average of two measurements. As described in the results, three indexes were modeled and compared without significant differences for evaluation of lung and liver stiffness in the whole cohort. Thus, ROI V and/or VI can used to assess fetal liver stiffness with the same performance.

One advantage of this study was to evaluate stiffness in three tissues with different supposed biomechanical properties: normal lung, lung with increased surfactant after corticosteroids course, and liver. We noticed that 2D-SWE revealed stiffness variations according to organs: mean stiffness values for the lung (A3) and the liver (V, VI) in the group 1 were 2.30 kPa [1.30-3.40], 3.90 kPa [2.38-6.40] and 3.86 kPa [2.23-6.34], respectively. A moderate positive correlation with gestational age was found for the fetal lung. Considering that gestational age is one of the most important factors determining fetal lung maturation, increase lung stiffness during the studied period could reflect lung development and maturity.

Similarly, there was a moderate positive correlation between fetal liver stiffness and gestational age. Thus, because lung and liver stiffness was increased in parallel through gestation in this study, the value of the fetal LLE ratio between 24 and 34 WG was constant with a mean value of 0.6. As a consequence, a very weak correlation was found for the fetal LLE and gestational age (R=0.08). By comparing liver and lung elasticity, gestational agerelated changes in fetal lung maturation could be underestimated and masked. However, the studied period was limited between 24 and 34 WG, and the fetal LLE ratio may evolve differently after 34 WG because of surfactant synthesis by alveolar type 2 cells.

In this study, the fetal LLE ratio was interesting to compare variations of the fetal lung elasticity after a course of corticosteroids. Between two examinations, fetal lung elasticity values decreased while those of the liver remained stable. Indeed, the relative difference of the fetal LLE ratio value was more important in group 2 exposed to corticosteroids (0.2; 95% CI: 0.07 to 0.34) compared to group 1 (-0.04; 95% CI: -0.29 -0.08) (P < 0.001). These results could be explained by dispersion of shear wave speed and attenuation relevant to increase viscosity in fetal lung after corticosteroids secondary to surfactant synthesis, especially phosphatidylcholine. As dispersion increases, shear wave will be attenuated [START_REF] Carlsen | A comparative study of strain and shear-wave elastography in an elasticity phantom[END_REF][START_REF] Carstensen | Elastography in the management of liver disease[END_REF][START_REF] Barry | Shear wave dispersion measures liver steatosis[END_REF].

The use of 2D-SWE in fetal medicine is actually not recommended in current practice and must be regulated in registered research protocols. Issaoui al reviewed the potential effect of 2D-SWE on fetal tissues in a quantitative heath risk assessment [START_REF] Issaoui | Shear wave elastography safety in fetus: A quantitative health risk assessment[END_REF]. To date, no health agency has published recommendations about widespread use of 2D-SWE in obstetrics due to lack of data concerning impact of acoustic radiation force on particular displacement in fetal tissues [START_REF] Issaoui | Shear wave elastography safety in fetus: A quantitative health risk assessment[END_REF]. However, observational and reassuring data are reported in the literature about use of 2D-SWE after 24 WG in preterm infants [START_REF] Alison | Quantitative shear-wave elastography of the liver in preterm neonates with intra-uterine growth restriction[END_REF][START_REF] Franchi-Abella | Feasibility and diagnostic accuracy of supersonic shear-wave elastography for the assessment of liver stiffness and liver fibrosis in children: a pilot study of 96 patients[END_REF][START_REF] Albayrak | Evaluation of neonatal brain parenchyma using 2-dimensional shear wave elastography[END_REF]. The "Elastomap study" is the first study to provide a collection of postnatal variables in all infants exposed in utero to 2D-SWE [START_REF] Mottet | Comment on "Shear wave elastography safety in fetus: A quantitative health risk assessment[END_REF]. Indeed, vigilance data were collected at birth and 4 months later to ensure the absence of clinical effects. We did not collect severe adverse outcomes in the whole cohort, and the unique case of stillbirth is secondary to uterine rupture. The cochlea may be a particularly sensitive structure as 2D-SWE provides a mechanical pulsatile vibration [START_REF] Massó | On the safety of elastography in fetal medicine: A preliminary study of hypoacusia[END_REF]. In this study, neonatal hypoacusia screening tests were normal for 100 infants exposed to 2D-SWE during the prenatal period. In one case, otoacoustic emissions were not registered in the medical file, and one infant presented abnormal auditory evoked potentials for one ear in one case of premature delivery at 31 WG. These new data (medical history, health problems, exploration of olivo-cochlear reflex at birth, respiratory diseases or symptoms, and number of hospitalizations since birth) will enrich current literature concerning the use of 2D-SWE in fetal medicine. These vigilance data are only descriptive but provide preliminary reassuring results. This pilot study is monocentric, and limitations include impact of depth on measurements and use of a single transducer SC6-1 without comparison with others probes and machines. On the one hand, stiffness increases in both organs between 24 and 34 WG could be related to tissue density increases throughout pregnancy. On the other hand, the correlation coefficient was low, and other variables, especially depth, could influence the stiffness value. Indeed, the more pregnancy progresses, the more the distance between the probe and target organ is reduced, improving detection of the shear wave's speed and estimation of stiffness value. Some pediatric studies reported the influence of the probe on stiffness evaluation related to the relationship between viscoelastic properties and transducer frequency. Stiffness should be interpreted according to the transmitted frequency, and the same transducer should be used for the follow-up [START_REF] Franchi-Abella | Feasibility and diagnostic accuracy of supersonic shear-wave elastography for the assessment of liver stiffness and liver fibrosis in children: a pilot study of 96 patients[END_REF][START_REF] Hanquinet | Acoustic radiation force impulse (ARFI) elastography for the noninvasive diagnosis of liver fibrosis in children[END_REF][START_REF] Engelmann | Feasibility study and control values of transient elastography in healthy children[END_REF]. Other studies are needed to confirm the feasibility of 2D-SWE for exploration of human fetal lungs and liver during gestation and to validate its reproducibility according to the probe and the machine. This preliminary study contributes to clarifying the applicability of 2D-SWE for fetal lung and liver. 2D-SWE of human fetal lung and liver is feasible with acceptable reproducibility. This technique could be a new complementary tool for reliable prediction of the status of pulmonary function based on biomechanical properties. Further studies are needed to confirm the feasibility of 2D-SWE for exploration of human fetal lungs and liver during gestation and to validate its reproducibility according to the probe and the machine. Measurements were performed on a homogeneous circular ROI of 5 mm diameter manually drawn. Three measurements were performed on the proximal lung (2a) in three regions (A1, A2, and A3) and 3 measurements on the liver (2b) in three regions (IV, V, and VI). Average elasticity (Kpa) was obtained in target regions of interest (ROI) with an automatic calculation. ROI were placed manually on the different target regions: Elastography measurements of the fetal lung on region A2 (2b) and Elastography measurements of the fetal liver on region V (2d).

Fig 3. Analysis of the fetal LLE ratio variation and stiffness evolution (regions A3

and V) according to gestational age. The figure shows the « FitPlot » consisting of a Scatter plots of the data overlaid with the regression line, and 95% confidence and prediction limits. a,b,c) According to gestational age, linear correlation was significant for stiffness values of regions A3, V and VI with positive correlation coefficients. d) The value of the fetal LLE ratio between 24 and 34 WG could be considered constant with a mean value of 0.6. The linear trend was not significant (P=0.6) and the correlation was very weak (R 2 = 0.006). R² : coefficient of determination

  All statistical tests were two-sided with p-values inferior to 0.05 denoting statistical significance. Statistical analysis was performed with SAS/STAT version 9.4 for Windows (The SAS Institute, Cary, NC).

Figure captions Fig 1 .Fig 2 .

 captions12 Figure captions

Fig 4 .

 4 Fig 4. Fetal LLE ratio values between group 1 (a) and group 2 (b). This graphic represents fetal LLE ratio values obtained by the operator 1 in both groups. The fetal LLE ratio values at "day 2" decrease in fetuses exposed to corticosteroids.

  the reference operator (senior clinical lecturer), operator 2 was resident and operator 3 was intern. Bold indicates significant differences.

Table 1 .

 1 Maternal and infant characteristics.

Table 2 .

 2 Mean difference between pairs of elastographic measurements made by the three operators in different region of interest (ROI).

Table 3 .

 3 Intraclass correlation coefficients (ICC) of elastographic measurements (average of pairs) in different region of interest (ROI).Operator 1 was the reference operator (senior clinical lecturer), operator 2 was resident and operator 3 was intern. Bold indicates significant differences.

		Sample size	Mean	95% CI	Standard	P
			difference		deviation
			(KPa)
	Region A1
	Operator 1	125	-0.05	-0.13 to 0.04	0.41	0.26
	Operator 2	51	0.07	-0.02 to 0.162	0.32	0.12
	Operator 3	12	0.13	-0.03 to 0.30	0.26	0.10
	Region A2
	Operator 1	158	0.01	-0.04 to 0.07	0.38	0.69
	Operator 2	71	0.04	-0.05 to 0.13	0.39	0.41
	Operator 3	28	-0.03	-0.26 to 0.20	0.60	0.79
	Region A3
	Operator 1	162	0.04	-0.02 to 0.09	0.38	0.19
	Operator 2	73	-0.02	-0.11 to 0.07	0.38	0.59
	Operator 3	26	-0.01	-0.20 to 0.18	0.48	0.93
	Region IV
	Operator 1	159	0.10	0.002 to 0.21	0.65	0.04
	Operator 2	72	0.13	0.01 to 0.26	0.54	0.04
	Operator 3	24	-0.13	-0.45 to 0.18	0.75	0.40
	Region V
	Operator 1	160	-0.03	-0.13 to 0.07	0.65	0.52
	Operator 2	72	-0.03	-0.16 to 0.11	0.56	0.66
	Operator 3	23	-0.08	-0.50 to 0.34	0.97	0.68
	Region VI
	Operator 1	127	-0.03	-0.14 to 0.09	0.66	0.61
	-Operator 2	63	-0.02	-0.16 to 0.11	0.54	0.74
	-Operator 3	24	0.11	-0.20 to 0.42	0.74	0.47

Funding sources

This research was supported by University Hospital of Besançon, through call for project proposals: "APICHU" Ref: API/2015/60.

Conflict of interest

The authors have no conflicts of interest to disclose in relation with this article.