DEVELOPMENTAL EPIGENOMICS

The Polycomb landscape in mouse development Julien Richard Albert 1 and Maxim V.C. Greenberg 1* 1. Institut Jacques Monod, CNRS, Université de Paris, Paris, France * Correspondence: maxim.greenberg@ijm.fr Polycomb group proteins assemble into two primary complexes-Polycomb repressive complex (PRC) 1 and 2-that safeguard cell fate by repressing gene transcription. Two new studies explore the PRC1 landscape during the transition from gametes to embryogenesis in mice, providing insight into the intergenerational transmission of epigenetic information and gene regulation dynamics as embryos prepare for gastrulation.

PRC1 and PRC2 are two evolutionarily conserved complexes that impact cell identity and fate via an epigenetic mode of gene repression. In classical Polycomb-mediated regulation, PRC2 catalyzes histone H3 lysine 27 trimethylation (H3K27me3), a mark recognized by PRC1, which in turn deposits histone H2A lysine 119 monoubiquitination (H2AK119ub1). However, more recent data have indicated that in animals the relationship is not quite so straightforward. Variant forms of PRC1 (vPRC1) can act upstream of PRC2 and initiate the canonical pathway in flies and mice 1- 5 . Furthermore, genetic perturbation indicates that PRC2 mutant mice die during gastrulation 6 , whereas complete PRC1 mutants exhibit an even more severe arrest at the 2-cell stage [START_REF] Posfai | Polycomb function during oogenesis is required for mouse embryonic development[END_REF] . Thus, the relationship between the two complexes is not unidirectional. Genome-wide patterns of H3K27me3 in mouse gametes and embryos have been described 8 , however H2AK119ub1 maps have not been so readily achieved. In this issue, Mei et al. [START_REF] Mei | H2AK119ub1 guides maternal inheritance and zygotic installation of H3K27me3 in mouse embryos[END_REF] and Chen et al. [START_REF] Chen | Distinct dynamics and functions of H2AK119ub1 and H3K27me3 in mouse preimplantation embryos[END_REF] utilized emerging technology-as well as complementary genetic approaches-to shine light on the interplay between PRC1 and PRC2 during the earliest windows of mammalian development.

H2AK119ub1 dynamics

In order to assess allele-specific H2AK119ub1 profiles during embryogenesis, both groups employed Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a method for semi-quantitative profiling of chromatin-bound factors genome-wide [START_REF] Skene | An efficient targeted nuclease strategy for highresolution mapping of DNA binding sites[END_REF] , in F1 hybrid mice. The technique is particularly useful for samples that are difficult to harvest, as high signal-to-noise ratios reduce the number of cells required. These H2AK119ub1 maps add to a publicly available tapestry of histone modifications, DNA polymerase, DNA methylation and transcriptional profiles in the early mouse embryo [START_REF] Xia | Rebooting the Epigenomes during Mammalian Early Embryogenesis[END_REF] . It should be noted that neither group collected CUT&RUN samples derived from reciprocal crosses of F1 hybrids, which can confound parent-specific effects with strain-specific effects [START_REF] Wang | Using next-generation RNA sequencing to identify imprinted genes[END_REF] . Exploring these data, both groups showed widespread H2AK119ub1 establishment during oogenesis, resulting in large domains that overlap the promoters of genes (Fig. 1a). Integrated analysis with existing datasets 8 revealed a strong correlation between H2AK119ub1 and H3K27me3 in mature oocytes. Dynamics between PRC1 and PRC2 during oocyte progression were not sampled, thus it is difficult to ascertain the stepwise establishment of the Polycomb landscape in the female germline.

Conversely, both groups generated maps of H2AK119ub1 at key stages of early embryogenesis. Strikingly, following fertilization, the symmetry between H2AK119ub1 and H3K27me3 observed in oocytes is lost over gene deserts. While H2AK119ub1 is depleted, H3K27me3 is stably retained through the blastocyst stage. In contrast, a distinct set of gene-rich loci devoid of both marks in the oocyte show sequential gain of H2AK119ub1 in the early 2-cell embryo followed by H3K27me3 gain in the blastocyst and epiblast. The findings reported here provide noteworthy in vivo data consistent with the studies largely performed in cell culture that demonstrated that PRC1 can act upstream PRC2 [START_REF] Blackledge | Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and Polycomb domain formation[END_REF][START_REF] Almeida | PCGF3/5-PRC1 initiates Polycomb recruitment in X chromosome inactivation[END_REF] . In embryos, symmetry is restored following implantation, where H2AK119ub1 and H3K27me3 domains are restricted to their canonical targets: the promoters of developmentally important genes.

PRC1 impacts embryogenesis

To understand the biological significance underlying the H3K27me3 and H2AK119ub1 dynamics, it is paramount to interrogate the relationship between PRC1 and PRC2 through genetic means.

To circumvent the severe PRC1 mutant phenotype, the two groups undertook complementary approaches: Mei et al. genetically ablated PRC1 subunits (Pcgf1/6) in oocytes to assess the role of PRC1 in the female germline and embryo (Fig. 1b). Importantly, PCGF1 and PCGF6 are components of vPRC1. Chen et al. utilized a PRC2 mutant (Eed) to determine the role of H3K27me3 in H2AK119ub1 establishment in oocytes (Fig. 1c). Additionally, Chen et al. overexpressed Polycomb repressive deubiquitinase (PR-DUB) in zygotes, which efficiently removes H2AK119ub1 (Fig. 1d).

Pcgf1/6 knockout (KO) oocytes exhibit substantial loss of H2AK119ub1, but still retain the vast majority (~88%) of H3K27me3 (Fig. 1b). RNA-seq analysis of oocytes globally devoid of H2AK119ub1 show specific upregulation of the subset of genes that had co-incidentally lost H3K27me3. Previous work demonstrated that PRC2 controls a non-canonical form of transient maternal imprinting and imprinted X-inactivation [START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF][START_REF] Inoue | Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells[END_REF] . Pertinently, embryos generated by fertilizing Pcgf1/6 KO oocytes with wild-type sperm exhibit upregulation of a subset of this class imprinted genes, including Xist-the key upstream regulator of X-inactivation. Analysis of extra-embryonic tissues demonstrates a failure of imprinting, associated with increased placental size. In sum, vPRC1 plays a key role in establishing repressive modifications transmitted from oocyte to embryo.

Genetically deleting Eed (essential for PRC2 activity) revealed that while H3K27me3 is globally lost, H2AK119ub1 is largely unaffected in oocytes and morula-stage embryos (Fig. 1c). Exceptionally, H2AK119ub1 is lost over non-canonically imprinted regions and Xist [START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF][START_REF] Inoue | Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells[END_REF] . Hence, there appears to be notably strong link between PRC1 and PRC2 to regulate this class of genes.

Chen et al. interrogated the role of H2AK119ub1, per se, by directly injecting PR-DUB mRNA into fertilized zygotes (Fig. 1d). Interestingly, while H2AK119ub1 was lost, H3K27me3 was unaffected, and RNA-seq analysis showed no change in imprinted gene expression in 4C embryos. Taken together, it appears PRC1 regulation of imprints likely occurs in the oocyte, possibly through PRC2 recruitment, but is dispensable in the embryo. Crucially, however, depleting H2AK119ub1 results in premature activation of canonical target Polycomb-regulated genes and arrest at the 4-cell stage.

The PR-DUB overexpression experiment strongly indicates that H2AK119ub1 is immensely important for the earliest stages of embryo progression. Both groups make the striking finding that PRC1 regulation at this class of genes occurs seemingly independently of PRC2, perhaps explaining the milder PRC2 mutant phenotype. While formal confirmation awaits, this suggests that upstream vPRC1 activity is taking place to repress Polycomb-regulated genes. However, it begs the question of why PRC2 does not nucleate at these regions soon after fertilization. Are key factors that recruit PRC2 to vPRC1-marked regions absent, or perhaps antagonistic factors are present? Moreover, what is the biological rationale for limiting PRC2 activity at these stages? Finally, what is the mechanistic explanation for the sufficiency of vPRC1 for repression in this window? Future work will hopefully address these exciting questions, further elucidating Polycomb-mediated gene regulation in the context of in vivo mammalian development.

Fig. 1 :

 1 Fig. 1: Polycomb and transcriptional landscapes in normal and modified mouse embryos. H2AK119ub1 and H3K27me3 dynamics over Polycomb Group (PcG) target gene promoters, gene deserts and the maternal allele of non-canonically imprinted genes in ooyctes and embryos in a, wild-type b, Pcgf1/6 maternal knockouts c, Eed maternal knockouts d, PR-DUB overexpressers. PcG; Polycomb Group, NGO; non-growing oocyte, FGO; fully grown oocyte, MII-O; meiosis II oocyte, 1C; 1-cell, E2C; early 2-cell, ZGA; zygotic genome activation, L2C; late 2cell, 4C; 4-cell, moru.; morula, blasto., blastocyst; n.a., not available. Arrows represent ectopic transcription.


Marie Blanquet

Sophie Dreux

Stéphanie Léger

Corinne Sault

Charline Mourgues

Hélène Laurichesse

Didier Lémery

Françoise Vendittelli

Dr Anne Debost-Legrand

Françoise Muller

Cost-effectiveness threshold of first-trimester Down syndrome maternal serum screening for the use of cell-free DNA as a second-tier screening test

Keywords: test ADN libre circulant, Trisomie 21, dépistage prénatal, analyse économique, ratio cout-efficacité, Non-invasive prenatal testing, Down syndrome, prenatal screening, economic analysis, cost-effectiveness ratio

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

Résumé

Introduction : Notre objectif était d'identifier la valeur seuil la plus cout-efficace des marqueurs sériques du 1 er trimestre pour l'intégration du test ADN libre circulant en deuxième ligne dans le cadre du dépistage prénatal de la trisomie 21 en France.

Matériel et méthodes : Une analyse cout-efficacité a été menée sur 108 121 grossesses uniques en utilisant un modèle de simulation. Les valeurs seuils testées des marqueurs sériques du 1 er trimestre étaient comprises entre 1/51 et 1/1000 par pas de 1/50. La valeur seuil la plus coût-efficace a été déterminée par le calcul du ratio coût-efficacité (RCE, coûts= coûts directs médicaux après le dosage des marqueurs du 1 er trimestre, efficacité=nombre de cas de trisomie 21 dépisté).

Résultat : Dans notre échantillon, 161 cas de trisomie 21 ont été identifiés. Pour une valeur seuil ≥ 1/50, 47,2% des cas ont été diagnostiqués. Dans le modèle de simulation, pour un seuil ≥ 1/250, 73,9% des cas de trisomie 21 étaient identifiés, pour un seuil≥ 1/500, le pourcentage de détection s'élevait à 78,8% et pour un seuil ≥ 1/1000, 2 cas supplémentaires ont été identifiés. Le montant des coûts était stable pour un seuil ≥ 1/250 (978 634€), puis augmentait rapidement pour un seuil ≥ 1/500 (1 966 576€) et devenait exponentiel pour un seuil ≥ 1/1000 (3 980 216€). Le RCE pour le seuil ≥ 1/500 était de 38 560.

Conclusion : La valeur seuil la plus coût-efficace pour l'implémentation du test ADN libre circulant en deuxième ligne semble être ≥ 1/500.

Introduction

Over the last 50 years, prenatal diagnosis procedures and screening have become safer and the capabilities of diagnostic laboratories have expanded [START_REF] Evans | Prenatal diagnosis : genetics, reproductive risks, testing and management[END_REF][START_REF] Chiu | Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma[END_REF][START_REF] Ehrich | Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting[END_REF][START_REF] Evans | Meta-analysis of first trimester Down syndrome screening studies: free beta-human chorionic gonadotropin significantly outperforms intact human chorionic gonadotropin in a multimarker protocol[END_REF][START_REF] Bianchi | Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study[END_REF][START_REF] Wapner | Chromosomal microarray versus karyotyping for prenatal diagnosis[END_REF]. Recently, the use of cellfree DNA (cfDNA) has dramatically increased. While cfDNA is considered as a screening test and not a diagnostic test by the scientific community, patients and most physicians see cfDNA as a substitute for foetal karyotyping [START_REF] Benn | Obstetricians and gynecologists' practice and opinions of expanded carrier testing and noninvasive prenatal testing[END_REF][START_REF] Brewer | Survey of US obstetrician opinions regarding NIPT use in general practice: implementation and barriers[END_REF][START_REF] Chan | Obstetrical provider knowledge and attitudes towards cell-free DNA screening: results of a cross-sectional national survey[END_REF].

Recently, new guidelines drawn up by the French National Authority for Health (HAS;

Haute Autorité de Santé) recommend cfDNA for Down syndrome (DS) screening [START_REF]Place des tests ADN libre circulant dans le sang maternel dans le dépistage de la trisomie 21 foetale[END_REF]. These guidelines, based on a cost-effectiveness analysis, recommend cfDNA as a second-tier screening test in women with a risk between 1/51 and 1/1000 after first-trimester combined maternal serum screening (T21T1). Moreover, in high-risk women (T21T1 ≥ 1/50), invasive testing is proposed but, after clear information, women still have the choice of cfDNA.

However, these guidelines were based on simulated data.

CfDNA is a reliable technique that should be included in DS screening, but it remains contingent on maternal serum screening and the terms of application need to be carefully defined. It is necessary to identify the most relevant threshold of the calculated risk of DS based on maternal serum markers for the use of cfDNA. Few studies have tested different thresholds of T21T1 to identify the most cost-effective one [START_REF] Neyt | Introducing the non-invasive prenatal test for trisomy 21 in Belgium: a cost-consequences analysis[END_REF][START_REF] Morris | Model-Based Analysis of Costs and Outcomes of Non-Invasive Prenatal Testing for Down's Syndrome Using Cell Free Fetal DNA in the UK National Health Service[END_REF][START_REF] García-Pérez | Cost-effectiveness of cell-free DNA in maternal blood testing for prenatal detection of trisomy 21, 18 and 13: a systematic review[END_REF][START_REF] Chitty | Uptake, outcomes, and costs of implementing non-invasive prenatal testing for Down's syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units[END_REF]. Even though these studies reached similar conclusions, the number of thresholds tested was limited and there was no step-by-step analysis. The principal objective of this study was to identify the most relevant cost-effectiveness threshold of T21T1 screening for use of cfDNA as a second-tier test, by carrying out an economic analysis, in the French context.

Methods

French prenatal screening for Down syndrome

In France, prenatal DS screening has been regulated by the Ministry of Health since 1997 [START_REF]fixant les règles de bonnes pratiques en matière de dépistage et de diagnostic prénatals avec utilisation des marqueurs sériques maternels de la trisomie 21[END_REF]. The French DS screening policy is organized at a national scale and since 2010 has been based on three steps. The first consists of first-trimester nuchal translucency (NT) and crown rump length (CRL) measurements (T1US), a cut-off ≥ 3.5 mm leading to chorionic villus sampling (CVS) for karyotyping. The second step consists of first-trimester maternal serum screening (or second-trimester when NT is not performed or when maternal blood sampling is performed after 13 +6 weeks of gestation). First-trimester DS maternal serum screening (T21T1) is performed at 11-13 +6 weeks and combined maternal age, maternal serum markers (pregnancy-associated plasma protein A, PAPP-A, and free beta-human chorionic gonadotropin, hCGβ) and nuchal translucency (NT). Karyotyping is offered for a calculated risk ≥ 1/50. If women have a risk between 1/51 and 1/1000 after first-trimester combined maternal serum screening (T21T1), cfDNA is recommended as a second-tier screening test [START_REF]Place des tests ADN libre circulant dans le sang maternel dans le dépistage de la trisomie 21 foetale[END_REF]. The third step consists of two ultrasound scan examinations at 20-22 weeks and 30-32 weeks of gestation.

Study population

Our economic analysis was applied to data from 108 121 singleton pregnancies with T21T1 (Autodelfia, dual kit and LifeCycle 4.1 software, PerkinElmer, Turku, Finland) during the year 2015 in the Biomnis Eurofins Laboratory, one of 86 accredited laboratories. Maternal serum markers and NT were expressed as multiple of the median (MoM) for gestational age (expressed in weeks and days of amenorrhea) as estimated by CRL measurement performed at the same time as NT. MoMs were adjusted for three confounding factors (maternal weight, smoking status and ethnicity). In accordance with French law, written informed consent for biochemical testing was obtained from each woman.

If chorionic villous sampling or amniocentesis was performed, a second written consent was needed for foetal karyotyping. The database has been reported to the French Data Protection Authority (CNIL: Commission Nationale de l'Informatique et des Libertés) as CNIL no. 1839545 V1.

Economic analysis

Identification of DS in the database

Cases in which embryo reduction was performed and cases in which a vanishing twin was detected at ultrasound scan were excluded. Because of the French policy using NT as contingent screening, all cases with NT ≥ 3.5 mm were excluded from the analysis. DS cases were identified prenatally when women had T21T1 screening ≥ 1/250 (based on French policy of DS screening strategy in 2015) or when anomalies were detected at ultrasound scan or at birth when the patient did not opt for an invasive procedure or for termination of pregnancy or when T21T1 was < 1/250 or without ultrasound anomalies. To sum up, among 108 121 singleton pregnancies 161 cases of DS were diagnosed, 124 by T21T1 screening, 16 by means of abnormal ultrasound scans and 21 at birth. All karyotyping results for the cohort were collected by the accredited laboratory, which performed serum marker screening.

Simulation model

The simulation model was based on actual data regarding DS status (including overall cases) and T21T1 results. Three variables were simulated based on assumptions from the international literature or expertise. Those variables were the women's characteristics (body weight and ethnicity), cfDNA (sensitivity, specificity and failure rate) and karyotyping acceptance rate. Women whose T21T1 results indicated a risk ≥1/1000 were considered to be part of a high-risk group in the simulation model, so the acceptability of cfDNA testing and karyotyping was presumed to be high. Table 1 presents the percentages with the 95% confidence intervals of each variable included in the model.

Cost-effectiveness analysis

A cost-effectiveness analysis (CEA) was performed for different thresholds and calculated risk as a discrete variable of T21T1 screening was used to identify the most relevant threshold. Figure 1 depicted the decision tree model used in the analysis describing the clinical pathway where cfDNA was used as a second-tier test if T21T1 screening was positive starting from ≥ 1/50 to ≥ 1/1000 by step of 1/50.

The perspective chosen was that of the third-party payer, ie, the French national health insurance fund (Caisse Nationale d'Assurance Maladie). Expressed in euros, the costs included were the direct medical costs after T21T1 screening: the cost of karyotyping, the invasive technique and the procedure used (chorionic villus sampling or amniocentesis) and of karyotype analysis (Table 1). Costs were calculated for the sample of 108 121 women. The incremental cost per DS case diagnosed was also calculated. The medical and societal costs of an undetected DS-affected child were not taken into account. The costs and effectiveness criteria were based on data from a single year and have not been actualized [START_REF]Caisse Nationale d'Assurance Maladie[END_REF][START_REF]Caisse Nationale d'Assurance Maladie. Table Nationale de codage de Biologie[END_REF].

The most relevant threshold of T21T1 screening for use of cfDNA as a second-tier test was based on cost-effectiveness ratio (CER). The criterion of effectiveness considered was the number of DS cases diagnosed. The CER was calculated as follows: CER = total cost for each threshold/number of DS cases diagnosed at each threshold. Univariate sensitivity analysis was used to test the robustness of the results by varying the cost of cfDNA.

Statistical analysis

A descriptive analysis of the data base was done for quantitative variables with standard deviation and for qualitative variables with 95% confidence interval (95% CI). The cohort's advance through the decision tree was based on DS prevalence in the actual database, cfDNA (sensitivity and specificity) and screening test participation rates for karyotyping (Table 1) [START_REF]Caisse Nationale d'Assurance Maladie[END_REF][START_REF]Caisse Nationale d'Assurance Maladie. Table Nationale de codage de Biologie[END_REF][START_REF] Ashoor | Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: relation to maternal and fetal characteristics[END_REF][START_REF] Akolekar | Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis[END_REF][START_REF] Gil | Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis[END_REF][START_REF] Cuckle | Maternal cfDNA screening for Down syndrome--a cost sensitivity analysis[END_REF]. The variation in the failure rate of the cfDNA test as a function of body weight was taken into consideration as was ethnicity [START_REF] Ashoor | Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: relation to maternal and fetal characteristics[END_REF][START_REF] Tanguy | Description du poids corporel en fonction du sexe et de l'âge dans la population française[END_REF]. Foetal loss due to invasive screening was also considered for each threshold [START_REF] Akolekar | Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis[END_REF]. The threshold of T21T1 screening ranged from 1/51 to 1/1000 in steps of 1/50. We used R 3.0.2. Software for this modelling.

Results

Descriptive analysis of actual data

A total of 108 121 women were included in the study. The mean maternal age was 29.7

[standard deviation 4.9] years. Median measurement of NT was 1.4 [95% CI: 0.8; 2.1]. Mean gestational age at T21T1 was 12 +6 WG (11 +0 -13 +4 ). In the sample, DS was diagnosed in 161 cases, giving a 0.2% [95% CI: 0.1; 0.2] global prevalence.

At the threshold of ≥ 1/50, among 76 cases of DS diagnosed, 71 were confirmed by foetal karyotyping and 5 after birth when the parents refused prenatal karyotyping (Table 2).

Results of the simulation model

For thresholds at 1/51-1/250, 43 additional cases were diagnosed (119 cases, 73.9% of total DS cases). Then, for thresholds at 1/251-1/500, 8 additional cases were diagnosed (127 cases, 78.9% of total DS cases). Finally, for thresholds at 1/501-1/1000, 2 additional cases were diagnosed (129 cases, 80.1% of total DS cases). One foetal loss was observed for a threshold of 1/251 (Table 2).

Considering that a cfDNA test costs 330 €, the slope of the cost increase was slight from a threshold 1/51-1/250 (978 634 €), then steep up to a threshold 1/251-1/500 (1 966 576 €) and increased exponentially from 1/501 to 1/1000 (3 980 215 €) (Figure 2). From a threshold 1/750 to 1/1000, no additional DS case was diagnosed, whereas the cost still increased (Figure 2). The CER was 38 560 for threshold 1/51-1/500, 56 471 at 1/51-1/750 and 75 098 at 1/51-1/1000.The incremental cost per DS case diagnosed was 191 637 € for threshold 1/51-1/500, 406 014 € at 1/51-1/750 and 735 092 € at 1/51-1/1000. The results were robust irrespective of the cost of cfDNA (Figure 2). Consequently, the most cost-effective threshold seems to be 1/51-1/500 as 127 (79.9% of total DS cases) DS cases were identified for a total cost of 1 966 576 €, a CER of 38 560 and an incremental cost per DS diagnosed of 191 637 € for a cfDNA cost of 330 € (Table 3).

Discussion

In our study based on actual data, the most cost-effective threshold of cfDNA as a second-tier test was 1/51-1/500. For higher thresholds, costs increase dramatically for the diagnosis of only a few additional cases of DS. This is the first economic study based on actual data in the general population to assess by a step by step analysis the inclusion of cfDNA in the French strategy of DS screening, following new national guidelines. The study was based on a large sample: 20.4% of women who underwent T21T1 in 2015 [START_REF]Agence de la biomédecine -rapport médical et scientifique[END_REF]. Because of exclusion of high NT, the prevalence of DS in our sample is 0.2% whereas in France in 2012 the estimated prevalence was 0.3% [START_REF]Malformations congénitales et anomalies chromosomiques[END_REF]. However, even if the prevalence of DS in our sample is low, the percentage of DS cases identified by screening remains stable, especially at the threshold from 1/51-1/750 to 1/51-1/1000, where no more cases of DS were identified.

First-trimester screening and cfDNA (based on the 1/1000 threshold [START_REF]Place des tests ADN libre circulant dans le sang maternel dans le dépistage de la trisomie 21 foetale[END_REF]) identified up to 129 cases (corresponding to a threshold 1/51-1/750) and missed 11 cases, all of which were diagnosed by prenatal karyotyping. Considering the most cost-effective threshold of 1/51-1/500, only two more DS cases were missed, but they may be identified later by secondtrimester ultrasound scan as it has been demonstrated that more than 30% of foetuses with DS can be detected among women classified as low risk after T21T1 screening [START_REF] Bromley | What is the importance of second-trimester "soft markers" for trisomy 21 after an 11to 14-week aneuploidy screening scan?[END_REF]. Different costs for the cfDNA test were considered in a univariate sensitivity analysis. One limitation of our study is that non-medical indirect costs were not considered [START_REF] Verhoef | Time and travel costs incurred by women attending antenatal tests: A costing study[END_REF]. The modelling did not consider the parents' decision based on the screening results. The major ethical and legal principle governing screening and prenatal diagnosis in France is that the final decision to continue or terminate the pregnancy belongs to the parents [START_REF]Avis sur les problèmes posés par le diagnostic prénatal et périnatal[END_REF][START_REF]Code de la Santé Publique[END_REF]. Other chromosomal aberrations such as trisomy 13 or 18 and sex chromosome abnormalities can be detected by cfDNA [START_REF] Benachi | Performance and indications of noninvasive prenatal testing using cell free circulating fetal DNA (cffDNA) for the detection of fetal trisomy 21, 18 and 13 in France[END_REF], however only results for DS were considered as it is the only chromosomal anomaly implemented in the national screening programme.

In our study, only the cfDNA contingent strategy was considered since most studies have concluded that universal cfDNA is nowadays too expensive and so irrelevant [START_REF] García-Pérez | Cost-effectiveness of cell-free DNA in maternal blood testing for prenatal detection of trisomy 21, 18 and 13: a systematic review[END_REF][START_REF] Nshimyumukiza | Cell-free DNA noninvasive prenatal screening for aneuploidy versus conventional screening: A systematic review of economic evaluations[END_REF]. We are also in accordance with current national French guidelines which recommend cfDNA as a second-tier test [START_REF]Place des tests ADN libre circulant dans le sang maternel dans le dépistage de la trisomie 21 foetale[END_REF]. Few studies have tested several thresholds to identify the most costeffective cfDNA contingent strategy [START_REF] Neyt | Introducing the non-invasive prenatal test for trisomy 21 in Belgium: a cost-consequences analysis[END_REF][START_REF] Morris | Model-Based Analysis of Costs and Outcomes of Non-Invasive Prenatal Testing for Down's Syndrome Using Cell Free Fetal DNA in the UK National Health Service[END_REF][START_REF] Chitty | Uptake, outcomes, and costs of implementing non-invasive prenatal testing for Down's syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units[END_REF]. Chitty et al., Morris et al. and Nyet et al. demonstrated that the cfDNA contingent strategy remained cost-effective until a threshold of ≥ 1/150, ≥1/500 and ≥ 1/600, respectively [START_REF] Neyt | Introducing the non-invasive prenatal test for trisomy 21 in Belgium: a cost-consequences analysis[END_REF][START_REF] Morris | Model-Based Analysis of Costs and Outcomes of Non-Invasive Prenatal Testing for Down's Syndrome Using Cell Free Fetal DNA in the UK National Health Service[END_REF][START_REF] Chitty | Uptake, outcomes, and costs of implementing non-invasive prenatal testing for Down's syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units[END_REF]. This is in line with our results regarding the dramatic cost increase at <1/500. The marginal cost of the cfDNA contingent strategy is very sensitive to the cost of cfDNA. In our study, for high-risk women (T21T1 ≥1/50) karyotyping was systematically proposed. To our knowledge, no study has implemented this strategy in its model.

Based on a large series of actual data, the most cost-effective threshold for cfDNA as a second-tier test in first-trimester screening seems to be 1/51-1/500. In the literature, the optimal threshold is between ≥1/150 and ≥1/600 according to current national guidelines of each country, assuming an unchanging budget [START_REF] Neyt | Introducing the non-invasive prenatal test for trisomy 21 in Belgium: a cost-consequences analysis[END_REF][START_REF] Morris | Model-Based Analysis of Costs and Outcomes of Non-Invasive Prenatal Testing for Down's Syndrome Using Cell Free Fetal DNA in the UK National Health Service[END_REF][START_REF] Chitty | Uptake, outcomes, and costs of implementing non-invasive prenatal testing for Down's syndrome into NHS maternity care: prospective cohort study in eight diverse maternity units[END_REF][START_REF] Nshimyumukiza | Cell-Free DNA-Based Non-invasive Prenatal Screening for Common Aneuploidies in a Canadian Province: A Cost-Effectiveness Analysis[END_REF]. For higher thresholds, costs increase dramatically for only a few additional cases of DS identified. In a recent metaanalysis of economic evaluations of prenatal screening by cfDNA testing, the authors demonstrated that despite heterogeneity between studies mainly because of several time-horizon considerations and different outcomes, the contingent strategy provides the best value for money [START_REF] Nshimyumukiza | Cell-free DNA noninvasive prenatal screening for aneuploidy versus conventional screening: A systematic review of economic evaluations[END_REF]. On the other hand, the universal strategy appears cost-effective when cost of cfDNA decreases. In our study, the cost of cfDNA is the one used by the French National Table of Laboratory Codes and has not been yet actualized. As already underlined, costeffectiveness studies results depend mainly of the cost of cfDNA. Another difference is also that many studies with simulated data include controversial future costs for children born with disabilities. However, a lifetime perspective is not appropriate in evaluating the prenatal screening strategy, as it appears unethical to estimate the value of life [START_REF] Kostenko | Clinical and Economic Impact of Adopting Noninvasive Prenatal Testing as a Primary Screening Method for Fetal Aneuploidies in the General Pregnancy Population[END_REF]. In the same way, we did not evaluate the viability of each pregnancy, as our objective was to simulate the foetal loss induced by invasive procedures. Nonetheless, the psychosocial consequences for families of these test results can be considerable and should be evaluated in economic studies [START_REF] John | The role of information provision in economic evaluations of non-invasive prenatal testing: a systematic review[END_REF]. Our choice to evaluate the hospital, rather than societal, point of view of the costs generated by positive test results means that we could not assess the costs of their psychosocial consequences and their effects on pregnancy outcomes [START_REF] Grosse | Economic methods for valuing the outcomes of genetic testing: beyond cost-effectiveness analysis[END_REF]. Although these intangible costs are difficult to quantify, they can be approached by questionnaires, which should be used to show the full value of the DS screening strategy to health policy decision makers.

We have demonstrated in accordance with the literature that the contingent strategy is cost-effective when cfDNA testing is performed at a threshold ≥1/500. National guidelines should take into account these results, especially in terms of opportunity costs for French society. Sensitivity analyses may be performed to assess validity of the model when cost of cfDNA will decrease as it will be used in routine screening in many countries. New thresholds may be more accurate than those used. The new challenge is to ensure that health professionals have adequate prenatal screening understanding in order to protect women's autonomy when consenting to genetic testing.

Table 3 :

 3 Costs of the Down syndrome screening strategy considering different costs for cell-free DNA, CER and incremental cost.

Figure 1 :

 1 Figure 1: Decision tree model of Down syndrome screening using cell-free DNA (cfDNA)

Figure 2 :

 2 Figure 2: Cost of Down syndrome screening when performing cell-free DNA testing as a

Conflict of interest

The authors declare that they have no conflict of interest.


Julia Sliwa
email: julia.sliwa@icm-institute.org

Toward collective animal neuroscience

From humans to fish to ants, social animals coordinate their behavior when in groups, and their neural system might do so as well. Yet the individual brain, studied in isolation, has so far been the fundamental unit of analysis of most neuroscience research. Animal wisdom has been searched for in how neurons interact within individual brains. But what if embedding the brain in a group changed how it works? And even more boldly, what if the laws of wisdom were to be found in how brains interact in a group, rather than in how neurons interact in a single brain? On pages 422 and 421 of this issue, Rose et al. (1) and Báez-Mendoza et al. (2) show that brains of bats and rhesus macaques track group interactions by, respectively, coordinating their activity and representing the group in single neuron activity.

Group living enables species to come up with solutions that each individual would not have accomplished alone. These include reducing predation or accomplishing substantial tasks, such as ants' nests or human cities. These exceptional endeavors emerge from individuals' social interactions, which are sometimes as simple as copying local neighbors and sometimes more elaborate, involving memory of others and signaling (3)(4)(5). The study of social interactions in neuroscience has been carried out by recording neural activity in the brains of two individuals simultaneously and led to the discovery that brains in a group of two can couple their activity (6). Brain-to-brain coupling has been reported in various social interactions, including communication, cooperation, competition, and learning. It has been shown to affect neural activity at different spatiotemporal scales (including neuronal firing, neural oscillations, and hemodynamic responses) and in different brain regions. Further, the temporally coherent link between the activity of two brains was either synchronous or asynchronous and directed from one brain to the other. Brain-to-brain coupling has been demonstrated with increasingly sophisticated analysis methods and in different species, including bats, mice, and monkeys (7)(8)(9)).

Yet it is only when multiple social interactions are coupled together that the power of a collective dynamic emerges. The change of scale from studying one social interaction between two individuals to studying collective social interactions within a group of individuals must go hand in hand with innovative methods. In human studies, the strategy has been to choose devices that are easy to use in a real environment, such as a classroom or a music studio, while being well coordinated to simultaneously record brain activity within the group in real time (10)(11)(12). In their investigations with Egyptian fruit bats and rhesus macaques, Rose et al. and Báez-Mendoza et al., respectively, took another direction. They did not want to trade off the resolution of single-neuron activity. They focused on the frontal cortex, which is involved in making and pondering decisions; the medial region is a hub of social cognition. They recorded from close, but not homologous, brain areas in the two species, allowing them to identify the activity of neurons in the frontal cortex that is sensitive to the identity of the individuals that the bats and monkeys interacted with. This means that interactions were tailored to the specific interacting partner (see the figure). In bats, this neuronal sensitivity was even more precise for close partners within the group than for distant partners, which were less well differentiated by neurons. In monkeys, dorsomedial prefrontal cortex neurons were active only during interactions; looking at the partner did not activate these neurons. When the interacting partner was a monkey plush puppet or a bat playback sound, these neurons stopped being active altogether. This contrasts with neurons representing peers in the nonhuman primate temporal cortex that are active when their peers' faces are presented as pictures (13). In the frontal cortex, these neurons appear to be tuned for possible action from peers, representing them as communication partners, competitors, and collaborators.

When socially interacting, the brains of two bats will couple their activity by synchronizing their frontal cortex neural oscillations as well as neuronal spiking activity (7). However, Rose et al. found that when a bat emits a call, interbrain coupling applies to all listening bats. Calling not only widely distributes information within the group but also induces collective brain coupling. By providing shared auditory stimulation to all brains at once, it could be that a communication call is not more specific than a gong or a bell ring in inducing collective brain coupling. However, communicative calls were found to be very specific in how they affected collective brain coupling within the bat group. The authors trained bats to emit vocalization to receive food from humans in the presence of other bats, but these trained calls did not elicit the same collective brain activity coupling as real communication. The caller's brain was no longer influenced by activity from the listener's brain when emitting trained calls. Conversely, real communication relied on bidirectional interbrain coupling, where the caller's brain accounts for the listener's brain before they send a signal.

Should research now focus on groups? In social insects, such as ants or bees, individuals are unable to represent the collective outcome of their group. Conversely, individual humans and most nonhuman primates can recapitulate part of the knowledge of the group, including the structure of their society and its culture, and use it for their own purpose (5,14). This makes it interesting to study how a single brain accomplishes this advanced cognitive ability. Báez-Mendoza et al. show how a group of rhesus macaques derive economic laws from their trading interactions and represent this knowledge in their brain to guide future decisions. The authors used a simple, yet highly controlled, task that taps into the foundation of economic trade in humans: giving, receiving, and reciprocating [START_REF] Mauss | The Gift: Forms and Functions of Exchange in Archaic Societies[END_REF]. Monkeys sitting at a turntable were sequentially distributing pieces of apple to each other. Monkeys chose strategically to reciprocate gifts of apple to monkeys that gave them apple and to retaliate against those who did not give to them. In their decisions, monkeys not only considered the last gift received but also the history of giving and even the reputation of individuals to give to guide their own behavior. Such behavior was absent when monkeys could reward plush monkey totems.

During the economic trade, Báez-Mendoza et al. found that single-neuron activity from the dorsomedial prefrontal cortex reflects reward history in the group, monitoring the social outcomes of other monkeys' choices. Based on neuron activity, they were able to decode past interactions. Future reciprocation and retaliation were also predicted, even before the monkey decided to give a piece of apple. The neuronal activity that they discovered not only reflected monkeys' behavior but also was necessary for performing reciprocation behavior. When neuronal activity of the medial prefrontal cortex was temporarily disrupted-through reversible microstimulation-just before a monkey made their decision, reciprocation was diminished.

The findings of Rose et al. and Báez-Mendoza et al. are major steps in identifying the neural mechanisms for maneuvering in complex social structures, including those for interacting with others and for knowing the specific identity of the interactors and the history of those interactions. It remains unclear how neurons from the same area can be involved in these different processes at the same time. It will be important to understand which neurons' activity contributes to synchronization versus encoding social knowledge. Are there separate populations of neurons, or are the same neurons doing both tasks? The resolution with which brain activity is probed will be key to answering this question. These studies also emphasize that in the future, it will be necessary to navigate between two levels of investigation to fully grasp social intelligence: that of the group of brains and that of the individual brain. Further research should assess additional dynamical properties of animal and human collectives, including changes that may result from virtual collectives on the internet. J

ACKNOWLEDGMENTS

Funding is from the Agence Nationale de la Recherche (ANR-21-CE37-0016-02, ANR-19-CE37-0002-01, and ANR-10-IAIHU-06) to J.S.


Oscar Laurent

Marcel Guillong

Christoph A Heinrich

Ken Neubauer

Chady Stephan

Advantages of a fast-scanning quadrupole for LA-ICP-MS analysis of fluid inclusions

For the past 20 years, LA-ICP-MS has been the method of choice to quantify element concentrations in fluid inclusions hosted in geological samples. However, the ablation of fluid inclusions typically produces short, transient signals that are difficult to representatively sample with sequential analysis inherent to single collector ICP-MS instruments, especially considering small inclusions (<20 µm) and/or low-concentration elements (<10 µg.g -1 ). This issue can be overcome by significantly reducing quadrupole settling times, allowing faster cycling through a given element list at constant duty cycle and therefore, better temporal resolution of the signal. In this note, we present results from already well-characterized fluid inclusion assemblages performed with a "fast-scanning" quadrupole ICP-MS, i.e. characterized by extremely short settling times (default 0.2 ms and down to 0.065 ms). This capability, which does not impede basic instrument performance, improves the results of LA-ICP-MS fluid inclusion analyses in three ways. First, it extends the possiblity to accurately and reproducibly quantify element concentrations to fluid inclusions down to <10 µm in size, with absolute detection limits in the same range as using a conventional quadrupole. Second, it releases the need to compromise between full chemical characterization of the fluids and accurate determination of some key elements of interest, as short settling times offer the possibility to rapidly scan through extensive element lists (e.g. 52 elements cycled in 52 ms at 80% duty cycle in the tests presented here). Third, it improves accuracy and precision for analytes hosted in small daughter crystals in assemblages of complex, polyphase fluid inclusions.

Introduction

The ability to accurately measure elemental concentrations in fluid inclusions hosted in geological samples provides critical information about hydrothermal processes, most importantly those leading to the formation of economically important ore deposits [1][2][3][4] . Laser Ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) has been the technique of choice for analysing fluid inclusions for the past 30 years [5][6][7][8][9] . The high spatial resolution of the technique allows the precise targeting of the inclusion with the laser within its textural position, compared to other techniques which involve bulk sample leaching, resulting in the possible mixing of different fluid inclusion generations 1,[7][8][9] . The high sample throughput of LA-ICP-MS analyses allows rapid acquisition of data from an assemblage of co-genetic inclusions to characterize the average composition of the corresponding paleo-fluid [7][8][9][10] . Tests on natural and synthetic inclusions show that such compositions are accurately determined, typically with uncertainties of 20 to 30% relative 7,9 .

Despite being the preferred technique for the analysis of inclusions, a limitation of LA-ICP-MS is that single collector ICP-MS instruments are sequential analysers, meaning that one analyte (i.e. m/z) is measured at a time, typically with a quadrupole. Even though these measurements are rapid (on the order of milliseconds), information is missed for short transient signals, which typically result from the ablation of fluid inclusions due to their small size (commonly <50 μm) and volume 11 . The resulting signals may not accurately represent the composition of the inclusion. Sequential analysis indeed increases the probability of missing the signals resulting from smaller inclusions and/or elements present at low concentrations in the fluid 12 (typically <10 µg g -1 ). Moreover, although fluids are trapped as a single phase in their host crystal, they usually split upon cooling into a heterogeneous, multi-phase mixture 13 . The resulting signals must therefore representatively sample all components of the inclusions to obtain the composition of the original paleo-fluid 7,8 , which is not always straightforward. Notably, the economically important metals (Cu, Mo, Au, Ag etc.) tend to concentrate in very small crystals, prone to be overlooked during sequential analysis 14 . These issues likely result in poor reproducibility of populations of co-genetic fluid inclusions 1,10 and therefore, represent an important limitation to the chemical characterization of the corresponding geological fluid 10,14 .

These problems can be avoided by the simultaneous acquisition of intensities for several analytes of interest, using either multicollector (MC-) or time-of-flight (TOF-) ICP-MS 8,15 . However, neither of these have been used for routine fluid inclusion analyses. The analytical mass range to analyse is much more restricted with MC-ICP-MS than with quadrupole instruments, while TOF-ICP-MS has not been tested so far regarding the accuracy of multi-element analyses of fluid inclusions. In the latter case, the already challenging data processing, notably baseline estimation and subtraction 16 , is complicated by rapidly changing signal intensities as typically produced by the ablation of fluid inclusions.

Many of these limitations can be overcome by using an ICP-MS which includes not only the ability of the electronics to quickly move between masses, but also an extremely short settling time of the electronics once the new mass is reached. One ICP-MS instrument satisfying these criteria is the PerkinElmer NexION 2000, as shown with respect to imaging by LA-ICP-MS [START_REF]Advantages of Short Settling Times for Quadrupole Based ICP-MS Laser Ablation Imaging[END_REF] . Based on tests from fluid inclusions chemically well-characterized in previous studies [START_REF] Rauchenstein-Martinek | [END_REF]19 , this work will explore and discuss how the use of such a fast-scanning quadrupole MS improves the reliability of LA-ICP-MS analysis of fluid inclusions. In particular, we will show that this (1) reduces the lower size limit for the exhaustive chemical characterization of fluid inclusions, thereby extending the range of possible applications for the technique; and (2) improves the accuracy and precision of the results for complex, polyphase inclusions.

Experimental Sample preparation

We studied two sets of already well-documented fluid inclusion assemblages in two distinct ore-forming environments: 1. Low-salinity aqueous fluid inclusions from low-temperature, gold-bearing quartz veins from Tiefengletscher, Swiss Alps [START_REF] Rauchenstein-Martinek | [END_REF] . 2. High-salinity, multiphase brine inclusions from quartz-wolframite veins associated with the Mole Granite magmatichydrothermal tin-tungsten-fluorine deposit (Australia) 1,19,20 . We targeted fluid inclusions hosted in quartz crystals, representing the main gangue mineral in both cases. The crystals were prepared as double-polished ca. 300 μm-thick sections and cut in small chips (<0.5 cm across). We studied leftover chips from samples that were already studied for fluid inclusion micro-thermometry and LA-ICP-MS analyses, allowing a direct comparison of the results. These former analyses were performed using the same laser instrumental setup as reported here, attached to a conventional ("slow-scanning") quadrupole ICP-MS. The newly obtained results are available as Electronic Supplementary Information.

Instrumental parameters and conditions

All analyses were carried out with the ETH-prototype of the GeoLas excimer laser ablation system, equipped with a ComPex Pro 102F (Coherent Inc., Santa Clara, California, USA) ArF laser source (operating at 193 nm), coupled to a PerkinElmer NexION 2000 quadrupole ICP-MS. The laser and ICP-MS parameters used for the tests on fluid inclusions are listed in Table 1.

A 193 nm excimer laser was used because this wavelength more effectively couples into different matrices than longer wavelengths (especially into quartz which is partly transparent to UV radiation), thereby minimizing fractionation and improving reproducibility 7,8,21 . A full glass, small volume ablation chamber (< 6 cm 3 ) constructed in-house was chosen for its fast washout (99% of the maximum signal intensity washed out in ca. 1 second), meaning that although the duration of the aerosol produced from the inclusion ablation event is shorter, the concentration of the aerosol particles, hence the ICP-MS signal-to-noise ratio, are higher, increasing the probability to detect elements at low concentrations 8,10,15 . Ablation cells characterized by longer washouts may be used, but this would counteract the benefit of using a "fast-scanning" quadrupole ICP-MS (see Results and Discussion). The system was cleaned and optimized by standard procedures for the analysis of fluid inclusions 22 to avoid any contamination.

The ablation spot diameter was set as to fully ablate the targeted inclusion, and enlarged progressively using an opening iris aperture to minimize quartz breakout [START_REF] Guillong | 19 th Biennial Conference "European Current Research on Fluid Inclusions, ECROFI-XIX[END_REF] . Transport from the ablation chamber to the ICP-MS was facilitated with the use of helium (He) carrier gas for optimal uptake of aerosol particles produced upon ablation [START_REF] Günther | [END_REF]25 . Argon (Ar) make-up gas was mixed downstream of the ablation chamber using a T-connector, and a short (<0.5 m) transport tubing ensuring fast aerosol transport was connected directly to the base of the ICP-MS injector. A long element list was used (52 analytes; see Table 1) to test the potential of the fast scanning capability to provide an extensive chemical characterization of the tested fluid inclusions.

All quantitative results were obtained from classical external calibration applied to LA-ICP-MS analyses 26 , employing NIST SRM610 glass (NIST, Bethesda, Maryland, USA) as reference material for most analytes (using reference values from Jochum et al., 2011) 27 and the Sca-17 scapolite reference material 28 for Cl, S and Br. The average Na concentrations of fluid inclusion assemblages targeted here, calculated from the equivalent NaCl concentration (NaCleq.) determined by micro-thermometry in previous studies [START_REF] Rauchenstein-Martinek | [END_REF]19 , were used as internal standards for relative sensitivity correction. Data were processed using the Matlab-based SILLS program 29 with limits of detection calculated after Pettke et al. (2012) 10 . The full dataset is reported as Electronic Supplementary Information (Table S1).

Results and Discussion

Specificities of a fast scanning quadrupole and benefits to analyse transient signals

The sampling time of quadrupole ICP-MS is a combination of dwell times ti (the time spent by the quadrupole measuring analyte i), the settling time τ (the time for electronics to settle after the quadrupole moves from one mass to another) and the number of analytes n (ref. 26). The total sweep time Tn required to scan the whole mass range in a given method setup is hence defined as:

T n = ∑ t i n i = 1 + nτ (1)
Another consideration is the duty cycle F which describes the fraction of the sweep time spent by the instrument to actually detect ions, therefore defined as:

F = ∑ t i n i = 1 T n (2)
While ti and n can be defined by the user, the quadrupole settling time τ is limited by the electronics of the ICP-MS. Typical settling times for commercial quadrupoles are in the range of 1 to 10 ms. However, the instrument used here has a default quadrupole settling time of 0.2 ms and is therefore able to scan 5 to 50 times faster through a given analyte list at a given duty cycle. The value of 0.2 ms corresponds to the working range for the mass jump from Li to U, but it can be theoretically modified to lower values. In practice, the lowermost possible value for τ is 0.065 ms, roughly corresponding to the time required for the quadrupole to be cleared of ions from the previous m/z recording. Using shorter settling times hence increases the probability of inaccurate results as the measured intensity for a given analyte may be influenced by that of the previous one in the element list.

Fast scanning quadrupoles are particularly beneficial when analysing transient signals, to avoid spectral skew and ensure that the acquired intensity ratios are representative of the sample. At slower quadrupole scan speeds, fewer data points are acquired, meaning that some data is missed, and in turn, the acquired data is less representative of the sample 11,14,15,17 . A generic practical example is shown in Figure 1 for two LA-ICP-MS signals both obtained from single laser shot analyses on NIST SRM610 glass, using exactly the same laser ablation, aerosol transport and ICP-MS setup parameters, with the exception of ti and τ (longer vs. shorter respectively; see details in the figure caption). For a given duration of the transient signal (ca. 5 seconds), less than 15 data points (sweeps) are acquired with ti = 10 ms (Fig. 1a). However, about 17 times more data points are acquired with ti = 0.4 ms (Fig. 1b), resulting in a much more representative recording of the short transient signal. For all elements, signal count rates are comparable for both timing setups (Fig. 1), indicating no effect of the reduced times on the ion filtering and counting capacities. This includes abundance sensitivity as peak shapes are the same regardless of ti and τ and providing that the quadrupole is cleared of ions from the previous m/z recording (see above).

Importantly, the maximum signal intensities are lower for some elements in the case of longer dwell/settling times (Fig. 1). This is a consequence of spectral skew, as the probability to miss the peak is higher when the temporal resolution of the transient signal is lower 11,15 . Conversely, although noisier, the analyte signals are more representative with the short dwell/settling times (Fig. 1). Element ratios are therefore more accurately determined throughout the signal time window -notably during the first second that represents >95% of the total signal counts (Fig. 1), which is crucial for accurate quantification 7,10,11 .

In the case of fluid inclusions, this problem is particularly acute as multi-element documentation of the fluid composition requires high signal-to-noise ratios to lower limits of detection and, therefore, as short transient signals as possible 15 (typically achieved by using small-volume ablation cells 8 , see Experimental section). It has been estimated that a minimum of 15 to 30 sweeps (i.e. readings) per transient signal are required to acquire representative data from an individual fluid inclusion, within the inherent reproducibility of the LA-ICP-MS technique 8,11 . In the fluid inclusion tests applied here, we have measured 52 analytes with uniform ti of 0.8 ms and the default value of τ = 0.2 ms, corresponding to Tn = 52 ms (eq. ( 1)) and F = 80% (eq. ( 2)). This allows 15 to 30 sweeps to be scanned in only 0.78 to 1.56 s. For the same element setup, a quadrupole ICP-MS instrument with a settling time of 3 ms would require ti = 12 ms to keep the same duty cycle, which would result in 15 times longer sweep times (Tn = 780 ms). This difference is very significant considering that typical signals resulting from the ablation of fluid inclusions are in the order of a few, to a few tens of seconds 10 . A more quantitative assessment of the improvement permitted by short settling times are provided in the following two test applications.

Analysis of <20 µm aqueous inclusions in gold-bearing quartz veins

The fluid inclusions in the metamorphic quartz vein sample KMP-1 from Tiefengletscher in the Swiss Alps range in size from <5 µm up to 150 µm. Previous chemical investigation of these inclusions by LA-ICP-MS [START_REF] Rauchenstein-Martinek | [END_REF] focused mainly on inclusions larger than 20 µm, because of the longer settling time of the quadrupole instrument used. Additionally, the relatively low fluid salinity (ca. 9 wt.% NaCleq.) [START_REF] Rauchenstein-Martinek | [END_REF] and resulting moderate intensity of the signal prevented a representative recording of smaller inclusions. The fast settling time used here allows smaller fluid inclusions to be measured, providing a test of the capability of a fast scanning quadrupole to accurately sample the fluid chemistry in cases where only small inclusions are available. Figure 2a shows a transmitted light microphotograph of fluid inclusions from a representative quartz sample from Tiefengletscher, indicating the size range of the inclusions that were measured previously and in the current study.

Figure 2b shows the time-resolved transient signal obtained from an 8 µm inclusion. The total duration of the inclusion signal was about 2.3 seconds, yet this signal is recorded with a maximum of 42 sweeps for major elements (Na, K, Cl) and about 10 even for the least concentrated elements (e.g. Rb, Sr, Cs, Pb). By comparison, using the element setup applied during earlier analyses [START_REF] Rauchenstein-Martinek | [END_REF] , the same inclusions would have been recorded with <10 sweeps, despite a much shorter analyte list (i.e. 14 elements vs. 52 here).

With the short settling time, inclusions down to 9 µm in size yielded data for 13 different elements, and some as small as 4 µm yielded detectable signals for 6 elements (Fig. 3). The average concentrations are identical to those obtained earlier based on larger inclusions and the relative standard deviations (RSDs) are comparable or even smaller (10-40%; Fig. 3). Low-concentration elements (<10 µg g -1 ) such as W and Tl, which were not quantified using longer settling and dwell times, were detected here with reasonable RSD (ca. 40% ) (Fig. 3). Elemental concentrations show no clear trend against inclusion size (Fig. 3) that would be indicative of a systematic bias due to the non-representative recording of very short signals resulting from the ablation of small inclusions.

Besides accuracy and reproducibility, the limit of detection (LOD) is another important figure of merit. At given background and sample signal durations and intensities, as well as analyte sensitivity, the LOD is roughly inversely proportional to the dwell time 10 , such that using shorter dwell times may at first glance entail higher LODs. However, if the temporal resolution of the analysis is increased proportionally, the probability of analyte detection over a certain sample signal duration, hence the LOD, remains the same (see equation (6) in ref. 10 and related discussion). In the case of a fast-scanning quadrupole, the short settling times allow to reduce in concert the dwell and sweep times (see section "Specificities of a fast scanning quadrupole and benefits to analyse transient signals"). Therefore, the fast-scanning operation should have theoretically no impact on the LODs.

The case of Au in sample KMP-1 provides a useful illustration, as Au concentration in the corresponding fluid inclusions is very low (0.011 ± 0.004 µg g -1 ; ref. 18). The LODs for Au obtained here range from 0.1 to 29 ppm (median = 2.4 ppm; Fig. 4), which is on average higher than in the previous study using a conventional quadrupole (ca. 0.001-2 ppm; ref. 18). However, this only reflects the fact that we specifically targeted smaller inclusions (<30 µm) than in the previous study, as is clear from the negative correlation between Au LOD and inclusion size (Fig. 4). In fact, when normalised to the finite mass of fluid represented by each inclusion, the LODs for Au are in the same range (1-15 fg) as obtained previously on sample KMP-1 (ref. 18) and lower than those from inclusions of the same type measured with a slower scanning ICP-Q-MS 12 (Fig. 4). Note in addition that in contrast to our setup, these previous studies applied a specific strategy to lower the LOD for Au, notably the use of a long dwell time for Au (80 ms) and a short element menu 12,[START_REF] Rauchenstein-Martinek | [END_REF] . Therefore, the LODs obtained here can be considered better than using a conventional quadrupole, and are even approaching those obtained employing a high-sensitivity, sector-field ICP-MS 12 (Fig. 4).

These results show the short settling time capability used here allows the determination of fluid inclusion compositions from fluid volumes 1 to 3 orders of magnitude smaller than with other quadrupole instruments, with comparable or even better accuracy, reproducibility (Fig. 3) and LODs (Fig. 4).

Analysis of complex, polyphase brine inclusions

Sample Loww9 is from a quartz-wolframite vein of the Mole Granite (Australia) and contains brine inclusions (ca. 48 wt.% NaCleq.) 19 ranging in size from 10 to 200 µm (Fig. 5a). These are polyphase inclusions, systematically containing similar proportions of an aqueous solution, a vapor bubble and several solids. These are both large (5-50 µm) salt crystals (Na-, K-and Fe-chloride) and smaller birefringent and/or opaque minerals of uncertain nature 19 and generally very small size (<5 and down to ca. 0.1 µm) (Fig. 5b). Such complex phase association and the range of crystal sizes challenge the accurate determination of the fluid chemistry, insofar it relies on a controlled, layer-by-layer ablation of the inclusion and representative recording of all its internal components 7,8,10,21 . Figure 5c shows an example of time-resolved, transient signal from a 60 µm brine inclusion from the Mole Granite. The signal duration is ca. 20 seconds. The signal patterns for the different elements shows controlled ablation of all internal components: the initial peak followed by steady decrease in Na and K records the release of the aqueous fluid, while the bell-shaped Na signal reflects the ablation of the halite crystal. Importantly, some elements including the economic metals (Sn, W but also Cu) show intensities below the LOD for most of the ablation duration, with the notable exception of one or several very sharp peaks of short duration (<2 seconds; Fig. 5c). These correspond to the sampling of the small daughter crystals (likely cassiterite, wolframite / scheelite and chalcopyrite) and are a common feature of such polyphase inclusion ablation 10 .With longer dwell and settling times, such sharp peaks would be much less accurately recorded or simply missed. Some of them are indeed <0.5 s long, approaching the sweep time used in earlier LA-ICP-MS analyses of these inclusions (ca. 0.4 s) 1,19 . Accordingly, Figure 6a shows that the RSDs of the corresponding elements in the analysed inclusion population are improved when using the fast scanning quadrupole capability (Cu = 28%; Sn = 18%; Sb = 14%; W = 16%) compared to earlier analyses (Cu = 70%; Sn = 33%; Sb = 25%; W = 22%; ref. 19).

The concentrations obtained for all elements show good agreement with those obtained from previous measurements within uncertainties (Fig. 6b). Moreover, Figure 6c shows that the longer element list permitted by the fast-scanning capability resulted in the quantification of 8 additional elements across the mass spectrum (Be, B, Br, Cd, Ba, La, Tl, U) compared to earlier analyses. This notably included two elements at sub-ppm levels that are not routinely measured in fluid inclusions (La = 0.72 ± 0.21 µg g -1 ; U = 0.32 ± 0.14 µg g -1 ). This result highlights that short settling times enable a more complete chemical characterization of the paleo-fluids.

Summary and conclusions

This work has demonstrated that short quadrupole settling times are beneficial for the analysis of short transient signals, such as those resulting from the LA-ICP-MS analysis of fluid inclusions. This faster analysis time improves the trace element quantification of fluid inclusions in three complementary ways.

(1) This allows the analysis of smaller inclusions than usually targeted (<20 µm and down to 4 μm as tested here), without sacrificing data quality (accuracy, reproducibility and LODs are comparable with or better than using a slower-scanning quadrupole). This widens the spectrum of possible application of fluid inclusion analyses by LA-ICP-MS as such small inclusions are more the rule than the exception in natural samples.

(2) Regardless of inclusion size, faster cycling permits the use of more elements (e.g. 52 elements cycled in 52 ms at a duty cycle of 80% for the tests presented here) than before (maximum 15-20 elements at comparable duty cycles). This capability releases the usual need for a compromise between complete characterization of the fluid and accurate determination of some key elements of interest 10 .

(3) A reduced settling time also helps to representatively record analyte signals resulting from small crystals enclosed in a complex, polyphase fluid inclusion, thereby allowing to determinate more precisely and accurately their average concentration in a population of co-genetic inclusions.

Taken together, a fast-scanning quadrupole ICP-MS instrument allows for more accurate and representative data to be acquired when performing LA-ICP-MS analyses of fluid inclusions. Examples of recent applications using this technique can be found in the literature [30][31][32] . Importantly, the same benefits would be relevant to other applications demanding short, transient signals, such as high-resolution LA-ICP-MS element mapping using the next generation of low dispersion ablation cells 33 . ) and size. The dots correspond to analyses obtained here, and the coloured boxes depict the size and compositional range of inclusions analysed in a previous study using a conventional quadrupole [START_REF] Rauchenstein-Martinek | [END_REF] . Larger dots with error bars correspond to the average ± standard deviation of analyses obtained in this study.

Figure 1 .

 1 Figure 1. Transient time-resolved LA-ICP-MS signals of two single shot analyses (60 μm laser spot) in NIST SRM610 glass using two different timing setups: (a) long and (b) short settling (τ), dwell (ti) and sweep (Tn) times. Intensities for a total of 28 analytes were acquired in both cases (amu 7, 11, 23, 25, 27, 29, 34, 35, 39, 43, 55, 57, 63, 66, 75, 79, 85, 88, 95, 107, 121, 133, 137, 140, 197, 208, 209, 238), only 5 are shown.

Figure 2 .

 2 Figure 2. (a) Microphotograph showing representative aqueous, liquid (L) + vapor (V) inclusions in quartz sample KMP-1; empty arrows show the typical size of inclusions previously analyzed by LA-ICP-MS (≥20 μm) 18 ; filled arrows show the typical size of inclusions targeted here (≤20 μm, down to 4 μm). (b) Transient time-resolved LA-ICP-MS signals from a fluid inclusion ca. 8 µm in diameter in sample KMP-1, showing intensities for 10 out of 52 measured elements; Ts corresponds to the total transient signal duration.

Figure 3 .

 3 Figure 3. Concentrations obtained for 15 elements in fluid inclusions from sample KMP-1, as a function of fluid inclusion (FI)volume (vol.) and size. The dots correspond to analyses obtained here, and the coloured boxes depict the size and compositional range of inclusions analysed in a previous study using a conventional quadrupole[START_REF] Rauchenstein-Martinek | [END_REF] . Larger dots with error bars correspond to the average ± standard deviation of analyses obtained in this study.

Figure 4 .

 4 Figure 4. Limits of detection (LOD) for Au as a function of approximate fluid inclusion (FI) diameter in sample KMP-1, compared to existing data on the same fluid inclusion assemblage 18 and from inclusions of similar type in the same context (aqueous fluids with ca. 4 wt.% NaCl in quartz from an Alpine fissure vein near Thusis, Switzerland) measured with two different ICP-MS systems 12 . The solid black lines indicate absolute Au LODs of ca. 1 and 10 fg.

Figure 5 .

 5 Figure 5. (a) Photomicrograph showing representative polyphase brine inclusions from sample Loww9. (b) Detail of one inclusion showing the nature of internal phases: aqueous liquid (L), vapor bubble (V), halite (h), another chloride salt (s) and unidentified opaque (o) or birefregent (b) crystals. (c) Transient time-resolved LA-ICP-MS signal from a brine inclusion ca. 60 µm in diameter in sample Loww9, showing intensities for 6 out of 52 measured elements; note the short, sharp peaks displayed by the metals (Cu, Sn, W) compared to the K and Na signals recording aqueous liquid and/or salt crystals.

Figure 6 .

 6 Figure 6. Quantitative results from the analysis of brine inclusions in sample Loww9. (a) Comparison of the relative standard deviation (RSD) of element concentrations in fluid inclusions analysed in this study compared with published data 19 ; note the improvement for Cu, Sn, Sb and W due to more representative recording of very short signals resulting from the ablation of tiny, metal-rich daughter phases. (b) Comparison of average element concentrations (Conc.) obtained in the fluid inclusions analysed in this study compared with published data 19 . (c) Plot of standard deviation (SD) vs. average concentration for 8 elements newly quantified (quant.) in this study, which were not included in the element list in previous work owing to scanning time constraints.

Table 1 :

 1 General LA-ICP-MS instrumental settings and conditions for fluid inclusion tests performed in this study

	Laser make, model and type	Coherent COMPex Pro 102F
	Laser wavelength	193 nm (Excimer ArF)
	Laser pulse duration	25 ns
	Laser energy density	ca. 20 J/cm 2
	Laser repetition rate	10 to 15 Hz
	Laser spot diameter	ca. 5 to 120 μm, set as to fully ablate the targeted inclusion
	Ablation cell	Custom-made cylindric, glass, ca. 6 cm 3 volume
	He carrier gas flow	1.1 L min -1
	Gas blank acquisition time	ca. 30 s
	ICP-MS model and type	PerkinElmer NexION 2000 quadru-pole MS
	Ar plasma gas flow	15 L min -1
	Ar auxiliary gas flow	1.05 L min -1
	Ar makeup gas flow	0.98 L min -1
	Injector	2 mm i.d., quartz
	Torch depth/z-position	-0.4 mm
	RF Power	1550 W
	Cones	Sampler & skimmer: Ni ; hyperskimmer: Al
		7, 9, 11, 23, 25, 27, 29, 31, 34, 35,
		39, 43, 49, 55, 57, 63, 66, 75, 77, 79,
		85, 88, 95, 107, 111, 118, 121, 125,
	m/z monitored (amu)	133, 137, 139, 140, 141, 146, 147,
		153, 157, 159, 163, 165, 166, 169,
		173, 175, 182, 197, 202, 205, 208,
		209, 232, 238
	Dwell time (ti)	0.8 ms
	Quad. settling time (τ)	0.2 ms
	Total sweep time (Tn)

Acknowledgements

We thank P. Weigel from PerkinElmer Switzerland for discussions and assistance and two anonymous reviewers for their relevant and constructive comments.

Author Contributions

Conflicts of interest

There are no conflicts to declare.


Florent Noel

Phan Hoang

PhD Jennifer Truchot

PhD Youri Yordanov

MD Pierre-Clément Thiebaud
email: pierre-clement.thiebaud@aphp.fr

An educational intervention to reduce unjustified peripheral intravenous infusions in the emergency department

Keywords: Intravenous infusion, Peripheral venous catheterization, Prescription, Educational intervention, Organization, Emergency department Tables: 2, References: 8

3

Dear Editor, 4 5 Peripheral intravenous infusion (PIVI) is a frequent procedure, especially in emergency 6 departments (EDs). However, studies revealed that 33.8 to 60.7% of these procedure orders are 7 unjustified, for example "to keep vein open" (KVO), "just in case" or for intravenous drug 8 administration (even though an oral alternative is available) [START_REF] Guihard | Appropriateness and 88 complications of peripheral venous catheters placed in an emergency department[END_REF][START_REF] Vandenbos | Relevance and complications of intravenous infusion at 92 the emergency unit at Nice university hospital[END_REF][START_REF] Limm | Half of all peripheral 95 intravenous lines in an Australian tertiary emergency department are unused: pain with no gain? 96[END_REF][4]. PIVI are time-consuming for 9 nurses and may cause various adverse events such for patients as pain, hematoma, 10 thrombophlebitis or infection [START_REF] Marsh | 101 Peripheral intravenous catheter non-infectious complications in adults: A systematic review and 102 meta-analysis[END_REF][START_REF] Miliani | 104 Peripheral venous catheter-related adverse events: Evaluation from a multicentre 105 epidemiological study in France (the CATHEVAL project)[END_REF]. The aim of this study was to evaluate the efficacy of an 11 educational intervention in an ED to reduce the number of unjustified PIVI. 12 13 A single-center before and after study was conducted in an urban university hospital ED. The 14 "before" period lasted from June 13 to June 20, 2019. The educational intervention occurred in 15 October 2019. Hospital guidelines regarding PIVI order were presented to the ED medical and 16 nursing staff during one dedicated meeting and three posters were displayed in the ED [START_REF]Bon usage des dispositifs 108 médicaux de perfusion[END_REF]. The 17 guidelines specified when PIVI was justified (life threatening emergency, need for an exclusive 18 intravenous drug with no oral alternative, impossibility to administer oral medication, need to 19 maintain an empty stomach) or unjustified (KVO, "just in case", to collect a blood sample). 20

The "after" period took place from January 6 to January 13, 2020 and was extended from 21 February 6 to February 9, 2020 because of a slow inclusion rate. All patients receiving a PIVI 22 in the ED between 8 am and 12 am during both study periods were eligible. Patients admitted 23 to the resuscitation room were excluded. This study involved the reuse of routinely collected 24 data and fell within the scope of the reference methodology MR-004 of the French legislation. 25

The protocol was registered in the Assistance Publique-Hôpitaux de Paris studies registry 26 (number 20210423180128). 27 28 For each patient, the emergency physician was asked to specify the main reason for prescribing 29 a PIVI among the six following categories: life-threatening emergency, volume expansion or 30 hydration, KVO, exclusive intravenous drug, impossibility of oral administration or need to 31 maintain an empty stomach. Other data collected included age, sex, chief complaint, 32 intravenous drug administration, blood test, imaging with contrast agent, and visit outcome. 33 Three emergency physicians randomly selected and not involved in the study reviewed the classify the procedure as justified or unjustified. They followed the guidelines and were blinded 36 to the main reason PIVI order. A PIVI was considered unjustified if at least two evaluators 37 considered it as such. 38 the significant decrease in unjustified PIVI orders for each evaluator supports the validity of 70 our results. 71 72 This study has some limitations. First, a few eligible patients might not have been included by 73 their treating physician. This risk was reduced by the presence of a coordinating physician 74 during the daytime, who ensured that all eligible patients were included, and the choice of a 75 convenience sample (no patients were enrolled during night shifts). Second, the "after" period 76 had to be extended in order to obtain a sufficient sample size. Third, a Hawthorne effect may 77 have affected and enhanced the changes in practices, through careful attention to following 78 guidelines or a possible change in the reason chosen for ordering PIVI. Fourth, the typology of 79 patients might have been different between the two study periods. Fifth, evaluators were not 80 blinded to the inclusion period. 81 82 In conclusion, implementation of an educational intervention in an ED appeared to reduce the 83 number of unjustified PIVI This may result in reduced adverse events, improve patients' 84 comfort, reduce nurses' job strain, associated costs and environmental footprint. 85

Acknowledgments:

The authors would like to thank Dr Fabien Brigant, Dr Nicolas Cury and Dr Sofiane Yefsah for agreeing to participate in this study as evaluators. We would also like to thank the medical and paramedical staff of the emergency department of Saint-Antoine Hospital, Paris, France.

Availability of data and material: All data analyzed during this study are included in this article Code availability : Not applicable Presentation: The results of this study have been presented at the French National Congress of Emergency Medicine in June 2-4, 2021 (Urgences 2021 virtual congress)

Author contributions: Florent Noël, Pierre-Clément Thiebaud: Conceptualization, Methodology, Literature search, Data curation, Writing -Original Draft; Phan Hoang: Formal analysis; Anne-Sophie Bard, Youri Yordanov: Investigation, Writing -Review & Editing; Jennifer Truchot:

Writing -Review & Editing

Qualitative variables were expressed as numbers and percentage and compared using Chi-2 or 40 Fischer's exact test. Quantitative variables were expressed as mean and standard deviation and 41 compared using Student's t-test. Inter-evaluators agreement was analyzed using Fleiss' kappa. 42 Statistical analysis were performed with the R software (version 4.0.2). A p-value <0.05 was 43 considered statistically significant. 44 45 One hundred forty-five patients were included: 83 during the "before" period (mean of 10.4 per 46 day, 8.5% of all ED visits) and 62 during the "after" period (mean of 5.2 per day, 3.6% of all 47 ED visits). Patients' characteristics are presented in Table 1. During the "after" period, the 48 population was significantly older (60.2 years vs 51.7 years, p=0.021) and received more 49 intravenous medications (81% vs 63%, p=0.019). A significant difference was found between 50 the two periods regarding the reason for PIVI orders (p<0.001). KVO was the main reason 51 during the "before" period (57%) and was almost never the reason during the "after" period 52 (2%). 53 54 Unjustified PIVI significantly decreased in the "after" period compared to the "before" period 55 (26% vs 52%, p=0.002; Table 2) with a relative risk reduction of 0.50 (95% CI, 0.31-0.80). 56 Agreement between the three evaluators was moderate in the "before" period (kappa 0.419) and 57 low in the "after" period (kappa 0.293). Individual analysis for each evaluator showed a 58 significantly lower unjustified PIVI orders rate after the intervention. 59 60

The rate of unjustified PIVI prior to the intervention is consistent with the literature [START_REF] Guihard | Appropriateness and 88 complications of peripheral venous catheters placed in an emergency department[END_REF][START_REF] Vandenbos | Relevance and complications of intravenous infusion at 92 the emergency unit at Nice university hospital[END_REF][START_REF] Limm | Half of all peripheral 95 intravenous lines in an Australian tertiary emergency department are unused: pain with no gain? 96[END_REF][4]. 61

Implementation of an educational intervention in the ED resulted in a 50% relative risk 62 reduction of unjustified PIVI. There was a significant reduction of daily patient inclusions 63 between the two periods, which may be related to the reduction in PIVI orders as a result of the 64 intervention. The number of KVO orders strongly reduced in the "after" period. This result is 65 consistent with an improved application of guidelines. Due to the Covid-19 pandemic, we were 66 unable to conduct a third inclusion period to confirm the sustainability of practice improvement, 67 as it has already been shown in other countries [START_REF] Lim | Evaluating the sustained effectiveness of a multimodal intervention aimed at influencing 113 PIVC insertion practices in the emergency department[END_REF]. Inter-rater agreement was low to moderate,