Adrien Marziale
email: adrien.marziale@gmail.com

Gael Picart
email: gael.picart@chu-brest.fr

Salma Nafai
email: salmanafai@hotmail.com

Hubert Galinat
email: hubert.galinat@chu-brest.fr

Pier Luigi Meroni
email: pierluigi.meroni@unimi.it

Johan Frostegard
email: johan.frostegard@ki.se

Marta Alarcon-Riquelme

Yves Renaudineau
email: yves.renaudineau@chu-brest.fr

Eléonore Bettacchioli
email: eleonore.bettacchioli@gmail.com

PRECISESADS Clinical Consortium Marta E Alarcon- Riquelme

Antiphospholipid autoantibody detection is important in all patients with systemic autoimmune diseases

Keywords: systemic autoimmune diseases, antiphospholipid autoantibodies, dRVVT, anticardiolipin, anti-β2GPI

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

Introduction

Two forms of antiphospholipid syndromes exist, primary antiphospholipid syndrome (APS-I) in half of cases, and secondary APS (APS-II) when associated with another systemic autoimmune disease (SAD) [START_REF] Schreiber | Antiphospholipid syndrome[END_REF]. In both cases, the persistent detection (>12 weeks) of antiphospholipid (aPL) autoantibodies is predominantly associated with arterial/venous thrombosis and/or pregnancy morbidity. The aPL spectrum includes the presence of lupus anticoagulant (LAC) and/or anti-cardiolipin (aCL) autoantibodies in association with anti-β2 glycoprotein-I (aβ2GPI) autoantibodies of IgG and IgM isotypes [START_REF] Chaturvedi | Diagnosis and management of the antiphospholipid syndrome[END_REF][START_REF] Gomez-Puerta | Diagnosis and classification of the antiphospholipid syndrome[END_REF]. The main pathogenic target for aPL has been characterized and is located within the N-terminal domain (Domain I) of β2GPI, a PL binding glycoprotein [START_REF] Meroni | Pathogenesis of antiphospholipid syndrome: understanding the antibodies[END_REF].

With an aPL prevalence ranging between 1% and 5% in the general population, the risk of thrombosis and/or pregnancy morbidity (mainly miscarriage) due to aPL remains scarce with 40-50 cases of thrombosis per 100,000 persons corresponding to an incidence of 5 new cases per 100,000 persons per year. Such risk significantly increases in those patients with SADs as observed in aPL positive SLE patients with a risk of thrombosis estimated at 50-70% during 20 years of follow-up [START_REF] Duarte-Garcia | The Epidemiology of Antiphospholipid Syndrome: A Population-Based Study[END_REF]. Accordingly, we took advantage of the PRECISESADS study to better characterize the risk factors associated with aPL positivity not only in SLE patients but also in non-SLE SAD patients.

Material and methods

Study design and subjects

A total of 1,818 individuals with SADs were included in the observational, European multicentric, PRECISESADS study between 2015 and 2018 (Table 1). The study encompassed 453 patients with systemic lupus erythematosus (SLE), 359 with rheumatoid arthritis (RA), 385 with systemic sclerosis (SSc), 367 with Sjögren's syndrome (SjS), 94 with mixed connective tissue disease (MCTD), and 160 with undifferentiated connective tissue disease (UCTD). Diagnostic criteria used for patient inclusion followed criteria for SLE [START_REF] Hochberg | Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus[END_REF], RA [START_REF] Aletaha | rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative[END_REF], SSc [START_REF] Van Den Hoogen | classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative[END_REF], SjS [START_REF] Vitali | Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group[END_REF]; and MCTD [START_REF] Alarcon-Segovia | Comparison between 3 diagnostic criteria for mixed connective tissue disease. Study of 593 patients[END_REF]. Patients with clinical features of SADs but not fulfilling any specific SADs criteria for at least 2 years were referred to as UCTD, and patients were excluded from this group if they fulfilled 3 out of the minimal 4 SLE classification criteria or fulfilled early Systemic Sclerosis criteria [START_REF] Leroy | Criteria for the classification of early systemic sclerosis[END_REF]. Primary APS was an exclusion criterion for this study while the 100 primary APS patients and the 592 healthy controls (HC) from the PRECISESADS study were used to characterize the aPL assays as reported in supplementary figure S1 [START_REF] Miyakis | International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)[END_REF]. The Ethical Review Boards of the 18 participating institutions approved the protocol of the study, which adhered to the standards set by the International Conference on Harmonization and Good Clinical Practice (ICH-GCP), and to the Declaration of Helsinki.

The study is registered with number NCT02890121 in ClinicalTrials.gov.

Of the 41 clinical parameters recorded on the PRECISESADS project, [START_REF] Capaldo | The active immunological profile in patients with primary Sjogren's syndrome is restricted to typically encountered autoantibodies[END_REF] were selected based on their information in the "Euro-Phospholipid-Project" [START_REF] Cervera | Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients[END_REF] such as peripheral arterial/venous thrombosis, miscarriage, neurological manifestations (stroke, cognitive dysfunction, seizure, chorea, and psychiatric disorders), pulmonary manifestations (worsening lung function, fibrosis), cardiac manifestations (pericarditis, valve lesions), renal manifestations (abnormal creatinine, proteinuria), cutaneous manifestations (photosensitivity, skin fibrosis, Raynaud's phenomenon), hematological manifestations (reduced platelet count), arthritis and hypertension. In addition information regarding a history of cancer and chronic infection were recorded based on their association with aPL positivity [START_REF] Schreiber | Antiphospholipid syndrome[END_REF].

LAC detection by dRVVT

The STA-Staclot dilute Russell viper venom time (dRVVT) Screen® and Confirm® (Stago, Asnières-sur-Seine, France) assays were used to test LAC according to the international guidelines [START_REF] Pengo | Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis[END_REF]. The assay is based on the use of a protease extracted from the venom of the Russell's viper that directly activates the coagulation factor X in the presence of calcium ions and phospholipids added at low (screen assay) or high (confirm assay) concentrations.

Confirmatory tests were performed in those samples with a screen ratio against reference plasma above 1.20 and the final result was expressed as screen ratio/confirm ratio according to the manufacturer's instructions. In those patients with a screen ratio <1.2 and for screen ratio/confirm ratio calculation, the confirmatory ratio was fixed at 1. ROC curves were used to fix the best pairing of sensitivity/specificity for LAC at ratio ≥ 1.4 (specificity 95%) for positivity and at ≥ 1.6 for medium/high positivity (specificity 98%) when comparing HC with APS-I and as described in Supplementary figure S1.

aPL assay : aCL (IgG and IgM) and aβ2GPI (IgG and IgM) detection by CLIA

The IDS-Isys (Boldon, UK) is a fully automated chemiluminescent immunoanalyzer (CLIA) using coated magnetic particles (solid phase) coupled with bovine aCL and/or purified human β2-GPI from Technogenetics (Milan, Italy), which was used for aCL IgG/IgM and aβ2GPI IgG/IgM isotype determination [START_REF] Salma | Thrombotic risk assessment and analytical performance of the chemiluminescent analyzer IDS-iSYS for the detection of anti-cardiolipin and anti-beta 2 glycoprotein I autoantibodies[END_REF]. In the presence of sera, the chemiluminescent signal, proportional to the autoantibody (Ab) tested, is expressed in Relative Light Units (RLU) and is then converted to GPL or MPL U/ml (U=units) using the "Harris" reference standard for aCL IgG and IgM, respectively. For aβ2-GPI IgG and IgM, results are expressed in AU/ml (Arbitrary Unit). Results were considered positive when values exceeded 20 units/mL [START_REF] Salma | Thrombotic risk assessment and analytical performance of the chemiluminescent analyzer IDS-iSYS for the detection of anti-cardiolipin and anti-beta 2 glycoprotein I autoantibodies[END_REF], and to study their significance, positive samples were further dichotomized as low and medium/high.

Other biological parameters

The presence of anti-dsDNA, anti-Sm, anti-U1-RNP, anti-SSA 52kDa, anti-SSA 60kDa, anti-SSB, anti-centromere, anti-Scl70, and anti-CCP2 autoantibodies were tested by CLIA (IDS-Isys) as previously described [START_REF] Capaldo | The active immunological profile in patients with primary Sjogren's syndrome is restricted to typically encountered autoantibodies[END_REF]. Turbidimetry (SpaPlus, Binding Site, UK) was used for rheumatoid factor (RF) and complement fractions C3c and C4 determination. The inflammation criteria were defined by the presence of an erythrocyte sedimentation rate (ESR) >30 mm/h and/or a C-reactive protein (CRP) above the laboratory norm in the absence of a concurrent infection. Hypergammaglobulinemia was defined by IgG levels above the laboratory norm and/or >20% gammaglobulin within the past 12 weeks that has not resolved.

Statistical analysis

Continuous data are reported as mean ± standard deviation and differences were analyzed by performing a T test. For categorical data, differences among groups were analyzed using the Fisher's exact test and odds ratio (OR) when necessary. Receiver operating curves (ROC)

were generated to determine the area under the curve (AUC) and the optimal cut-off values were chosen. Two-tailed p-values were reported and p-values less than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism version 7.0 (La Jolla, CA).

Results

aCL, aβ2GPI and dRVVT prevalence in systemic autoimmune diseases

In order to estimate the prevalence of aPL in SADs, we took advantage of the PRECISESADS study composed of 592 HCs and 1,818 patients with SADs (SLE, RA, SSc, SjS, MCTD and UCTD). Gender and sex-based differences were reported between HC and SADs (Table 1).

Regarding the presence of aPL Abs in SADs, a higher prevalence was reported in the SAD group for dRVVT (p<10 -4 ), aCL IgG (p<10 -4 ), aβ2GPI IgG (p<10 -4 ), while this was not significant for aCL and aβ2GPI IgM. For statistical purposes, the detection of aCL and/or aβ2GPI, referred to as aCL±β2GPI, was considered after making sure that aCL and aβ2GPI for each isotype were highly correlated (r≥0.93, p<10 -4 ) and with a very good concordance assessment rate (Cohen's Kappa≥0.85) when using CLIA as previously described [START_REF] Salma | Thrombotic risk assessment and analytical performance of the chemiluminescent analyzer IDS-iSYS for the detection of anti-cardiolipin and anti-beta 2 glycoprotein I autoantibodies[END_REF], data not shown.

Based on the observation that aPL prevalence was significantly different between SLE (57.6%) and non-SLE patients (13.7%), as described in Figure 1, we have further differentiated SLE patients from non-SLE SAD patients. Differences were retrieved when considering individual assays: dRVVT functional assay (21.2% in SLE versus 7.0% in non-SLE SADs, p<10 -4 ), aCL±aβ2GPI IgG Abs (10.8% in SLE versus 1.3% in non-SLE SADs, p<10 -4 ) and aCL±aβ2GPI IgM Abs (7.1% in SLE versus 3.2% in non-SLE SADs, p<10 -3 ); or double positivity between assays (0.05<p<10 -4 ). Based on the critical impact of aPL titers on clinical associations and as one may argue that aPL titers were reduce in non-SLE SADs, aPL were further ranked in low and medium/high titers showing a similar distribution between groups.

dRRVT in SLE and non-SLE SADs

The clinical picture of the dRVVT positivity spectrum (Figure 2) was characterized by venous and/or arterial thrombosis in both SLE (p<10 -4 ) and non-SLE SADs (p<10 -4 ) with specific associations that characterize medium/high LAC levels in SLE: miscarriage (p=0.05) and involvement of the central nervous system (p=0.02). In non-SLE-SADs, associations were retrieved between dRVVT positivity with pulmonary fibrosis (p=0.01), worsening lung functions (p=0.002), cardiac valve lesions (p=0.007), hypertension (p=0.04), and abnormal creatinemia (p=0.00002). A low level of dRRVT in the non-SLE group was associated with a chronic infection report (p<10 -4 ), pericarditis (p=0.03), arthritis (p=0.01) and Raynaud's phenomenon (p=0.00008).

Regarding biological parameters tested, an association between dRVVT positivity with the C4 complement fraction (p=0.03), inflammation (p=0.01), hypergammaglobulinemia (p=0.01), and anti-SSA(60kDa)/SSB positivity was observed in the non-SLE SAD subgroup. In contrast, no association was retrieved for SLE.

aCL±aβ2GPI IgG autoantibodies in SLE and non-SLE SADs

Next, aCL±aβ2GPI IgG positivity at low and medium/high levels was explored revealing an association with venous and/or arterial thrombosis in both SLE (p<10 -4 ) and non-SLE SADs (p<10 -4 ) at medium/high levels (Figure 3). Regarding specific associations, miscarriage anti-Sm (p=0.05), and anti-SSB Abs (p=0.03). Such effect on the biological parameters was not retrieved in the SLE subgroup.

(

aCL±aβ2GPI IgM autoantibodies in SLE and non-SLE SADs

In the SLE group (Figure 4), with regards to aCL±aβ2GPI IgM positivity at low and medium/high levels, an association was retrieved for medium/high levels with hypertension (p=0.02), fever (p=0.02), and a history of cancer (p=0.04). In contrast, the non-SLE SAD patients with aCL±aβ2GPI IgM Abs at low levels presented significantly more venous and/or arterial thrombosis (p=0.02), skin fibrosis (p=0.005), inflammation (p=0.005); and at medium/high levels miscarriages (p=0.003), and hypertension (p=0.01).

Odds ratio

Finally, the odds ratio (OR) for aPL association with thrombosis was explored at positive cutoff and for medium/high levels (Figure 5). In SLE patients, associations with thrombosis ranged for dRVVT from OR=6.1 (IC95: 3.5-10.

Discussion

In this study, performed with a European multi-center study of 1,818 patients with SADs, we observed: (i) that aPL detection characterizes patients with SAD; (ii) that aPL positivity is less frequent in non-SLE SADs such as SjS, RA, SSc, MCTD and UCTD than in SLE (13.7% versus 57.6%); (iii) that dRVVT and aCL±aβ2GPI IgG Abs perform similarly for association with thrombosis and at medium/high level with miscarriage in SAD, while aCL±aβ2GPI IgM Abs detection was associated with miscarriage in non-SLE SADs. In case of multiple positivity, the risk for thrombosis and miscarriage is increased as described in primary APS; (iv) the aPL risk for non-thrombotic events exists in SADs with differences observed between SLE and non-SLE SADs; and (v) the critical role of complement consumption, when associated with aPL positivity, is further supported.

Initially included in the classification criteria to discriminate SLE from other SADs [START_REF] Hochberg | Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus[END_REF], aPL detection is elevated in SLE with 11-30% of patients positive for the LAC test, 17-40% for aCL±β2GPI, supporting a total prevalence of aPL ranging from 30% to 45% [START_REF] Petri | Epidemiology of the antiphospholipid antibody syndrome[END_REF][START_REF] Reynaud | Risk of venous and arterial thrombosis according to type of antiphospholipid antibodies in adults without systemic lupus erythematosus: a systematic review and meta-analysis[END_REF][START_REF] Gustafsson | Cigarette smoking, antiphospholipid antibodies and vascular events in Systemic Lupus Erythematosus[END_REF], which is in agreement with our report. In contrast, in non-SLE SAD patients, results from the literature, usually performed in limited cohorts, are more contrasted and range from 0 to 57% as described in SSc [START_REF] Sobanski | Prevalence and Clinical Associations of Antiphospholipid Antibodies in Systemic Sclerosis: New Data From a French Cross-Sectional Study, Systematic Review, and Meta-Analysis[END_REF]. Similarly, conflicting results are reported in RA with an aPL prevalence of 28% according to a meta-analysis reported by Olech et al [START_REF] Olech | The prevalence and clinical significance of antiphospholipid antibodies in rheumatoid arthritis[END_REF], but to only 10.4% in the report of Kim et al who found LAC in a cohort of 376 RA patients as the most prevalent test (6.6%) followed by aCL±β2GPI in 3.2% of the cases [START_REF] Kim | Association between antiphospholipid antibodies and arterial thrombosis in patients with rheumatoid arthritis[END_REF]. Regarding SjS, again important differences are observed with an aPL prevalence ranging from 2% to 37% [START_REF] Asherson | Antiphospholipid antibodies and HLA associations in primary Sjogren's syndrome[END_REF][START_REF] Pennec | Serial measurements of anticardiolipin antibodies in primary Sjogren's syndrome[END_REF] From our knowledge, there is no data reporting aPL in UCTD and MCTD. This discrepancy in the literature for aPL detection in non-SLE patients, however, could be partially explained by differences regarding patient selection and recruitment, the diversity of diagnostic tests used for aPL detection, and the use of different cut off points. To circumvent these limitations, a large population of non-SLE SAD patients was selected (n=1365), primary APS patients were excluded from this study, and aPL were detected in a single center with robust techniques (Russel's clotting time and CLIA), the optimal cut-off for positivity was evaluated, and aPL were ranked in low and medium/high levels. Our study supports the concept that aPL are not restricted to SLE but can be detected in other SADs, at lower prevalence (9.75% in RA to 20.63 in UCTD), and that such detection is associated with clinical symptoms including thrombosis and miscarriage, which is an argument to test aPL Ab in non-SLE SAD patients.

The thrombotic risk associated with aCL±aβ2GPI of IgG and IgM isotypes and LAC has been studied in patients with SLE. There are consistent data demonstrating an association between aCL±aβ2GPI of IgG isotype at medium/high titers and an increased risk of venous and/or arterial thrombosis in SLE patients [START_REF] Domingues | Assessment of the independent associations of IgG, IgM and IgA isotypes of anticardiolipin with thrombosis in SLE[END_REF]. In contrast, when considering aCL±aβ2GPI

IgM detection such an association was not observed as reported in a study conducted in 796 patients with SLE [START_REF] Mehrani | IgM anti-beta2 glycoprotein I is protective against lupus nephritis and renal damage in systemic lupus erythematosus[END_REF]. This statement was confirmed by other authors who failed to associate aCL±aβ2GPI IgM positivity in patients with primary and secondary APS manifestations with thrombosis [START_REF] Lakos | Isotype distribution and clinical relevance of anti-beta2-glycoprotein I (beta2-GPI) antibodies: importance of IgA isotype[END_REF][START_REF] Brusch | Immunoglobulin G is the only anti-beta-2glycoprotein I isotype that associates with unprovoked thrombotic events among hospital patients[END_REF]. The two greatest risk factors for miscarriage are elevated levels of aPL and a history of previous fetal loss as these women have up to 80% risk of current pregnancy loss [START_REF] Lockshin | Antibody to cardiolipin, lupus anticoagulant, and fetal death[END_REF]. Both aCL±aβ2GPI IgG and IgM Ab detection are associated with an increased risk of miscarriage [START_REF] Petri | Update on anti-phospholipid antibodies in SLE: the Hopkins' Lupus Cohort[END_REF][START_REF] Lockshin | Antibody to cardiolipin, lupus anticoagulant, and fetal death[END_REF][START_REF] Opatrny | Association between antiphospholipid antibodies and recurrent fetal loss in women without autoimmune disease: a metaanalysis[END_REF][START_REF] De Laat | IgG antibodies that recognize epitope Gly40-Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with thrombosis[END_REF][START_REF] De Laat | The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study[END_REF][START_REF] Galli | Clinical significance of different antiphospholipid antibodies in the WAPS (warfarin in the antiphospholipid syndrome) study[END_REF]. Such associations were retrieved in our study, in SLE medium/high titer aCL±aβ2GPI

IgG and dRVVT were associated with miscarriages, while in the non-SLA SADs group it was the association between aCL±aβ2GPI IgM detection and miscarriage that was retrieved.

However, when the whole population is considered instead of a selected SAD population, the association between aCL±aβ2GPI IgM detection and miscarriage is lost, which supports the idea of restricting aCL±aβ2GPI IgM Ab research to SAD patients [START_REF] Rai | Antiphospholipid antibodies and beta 2-glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach[END_REF]. Compared to single positivity, patients with double positivity have a higher risk for miscarriage and it's of particular note that triple positivity is exceptional (n=10/1818) in our study, which is in contrast to primary APS patients [START_REF] De Carolis | Antiphospholipid syndrome: An update on risk factors for pregnancy outcome[END_REF].

What about other clinical associations with aPL in SADs? Association with pulmonary fibrosis was retrieved in the non-SLE SAD patient group (LAC) as described in patients with SSc [START_REF] Morrisroe | The association of antiphospholipid antibodies with cardiopulmonary manifestations of systemic sclerosis[END_REF],

while others did not have such an association [START_REF] Gupta | Clinical significance of antiphospholipid antibodies in Indian scleroderma patients[END_REF][START_REF] Enzenauer | Anticardiolipin antibodies in scleroderma[END_REF]. Regarding the risk of stroke and cerebral venous thrombosis, LAC when diagnosed with SLE was found to increase the risk by [START_REF] Kozora | Functional Magnetic Resonance Imaging of Working Memory and Executive Dysfunction in Systemic Lupus Erythematosus and Antiphospholipid Antibody-Positive Patients[END_REF][START_REF] Gerosa | Antiglutamate Receptor Antibodies and Cognitive Impairment in Primary Antiphospholipid Syndrome and Systemic Lupus Erythematosus[END_REF] . A risk for LAC was retrieved for central nervous involvement in the SLE group of our study (p=0.02). From our knowledge, our large study further highlights other rare clinical associations with aPL not previously described in SADs such as hypertension, an abnormal creatinemia not associated with proteinuria, arthritis and skin fibrosis. As a consequence, the heterogeneity retrieved in the literature may be explained in part by the fact that most of the reported studies tested a relatively small number of patients, which could be responsible for a lower statistical power, in contrast to our study. Another explanation is related to the existence of confounding factors that could present aPL positivity beside SADs, such as malignant tumors or infections. These confounding factors were limited in our study and, when present, associated with aCL at low levels.

Several studies have shown consumption of complement proteins through the classical and alternative pathways responsible for miscarriages, in association with inflammation, and vascular clots during primary APS [START_REF] Tedesco | Pathogenic Role of Complement in Antiphospholipid Syndrome and Therapeutic Implications[END_REF]. The critical role played by the complement system in aPL pathophysiology is further supported by the observations that an increase in complement activation (C4d) on platelets coated with aPL Ab is observed in patients with SLE both in vitro and in vivo [START_REF] Lonati | Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome[END_REF]; and that mice deficient in C3, C5 or C5a receptors are protected from fetal loss induced by the injection of aPL [START_REF] Salmon | Activation of complement mediates antiphospholipid antibody-induced pregnancy loss[END_REF]. Then it is not surprising that we observed in SAD patients with aPL significantly lower levels of complement fractions and thrombopenia compared to SAD patient without aPL. With the exception of aCL±β2GPI IgG in the non-SLE group with hypergammaglobulinemia and with anti-SSB Abs, association between aPL and autoantibodies was not reported in our study and in line with previous observations.

Regarding strengths of the study, the current work is one of the first studies exploring clinical and biological associations with aPL in a large and multicentric study of patients with SADs.

In order to minimize differences between centers and reagents, autoantibody detections, including aPL determination, were centralized in the PRECISESADS' study. This large study has further permitted us to better characterize the prevalence of aPL Abs in SAD as this information was contradictory in the non-SLE SAD patients. Assays for aPL determination were optimized with regards to the technology selected (dRRVT, CLIA), redefinition of the optimal cut-off for positivity, and the ranking of aPL in low and medium/high levels in order to take into account possible complicating factors such as a chronic infection or a cancer, factors that were limited in our study. However, the study has potential limitations such as information for each patient on the decision whether or not to take an anticoagulant treatment was missing as well as the facts that aPL determination was conducted at one time point and not repeated later as recommended by the APS criteria, and that all patients recruited were of European origin.

In conclusion, association between aPL profile and SAD from a large study has permitted better characterization of the prevalence of aPL in SADs. Furthermore, we found differences in the aPL profile, clinical and biological manifestations when SAD were dichotomized into

Figure 2 :

 2 Figure 2: Comparison of clinical and biological characteristics in SLE and non-SLE-patients according to their dRVVT status at low (screen ratio/confirm ratio = 1.4-1.6) and medium/high levels (screen ratio/confirm ratio ≥ 1.6). A : clinical and biological characteristics in SLE ; B : clinical and biological characteristics in non-SLE. Statistical values are indicated when significant (p<0.05, Fisher's exact test). Abbreviations: SLE : systemic lupus erythematosus ; dRVVT : dilute Russell's viper venom time ; . CNS : Central nervous system ; PNS : peripheral nervous system; DNA: anti-DNA; Sm: anti-Sm; U1 RNP: anti-U1 ribonucleoprotein; SSA/SSB: anti-sicca syndrome A/B; Centro: anti-centromere; RF: rheumatoid factor; Scl70: anti-topoisomerase I; CCP: anti-cyclic citrullinated peptide.

Figure 3 :

 3 Figure 3: Comparison of clinical and biological characteristics in SLE and non-SLE-patients according to their aCL±β2GPI IgG status at low (20-60 U/mL) and medium/high levels (≥60 U/mL). A : clinical and biological characteristics in SLE ; B : clinical and biological characteristics in non-SLE. The combination of aCL±aβ2GPI referred to the detection of aCL and/or aβ2GPI autoantibodies and statistical values are indicated when significant (p<0.05). See figure 2 for abbreviations.

Figure 4 : 2 Figure 5 :

 425 Figure 4: Comparison of clinical and biological characteristics in SLE and non-SLE-patients according to their aCL±aβ2GPI IgM status at low (20-40 U/mL) and medium/high levels (≥40 U/mL). A : clinical and biological characteristics in SLE ; B : clinical and biological characteristics in non-SLE. The combination of aCL±aβ2GPI referred to the detection of aCL and/or aβ2GPI autoantibodies and statistical values are indicated when significant (p<0.05). See figure 2 for abbreviations.

Figure 6 :

 6 Figure 6: Odds ratios (95% CI) for miscarriage in SLE and in non-SLE group (A/B and C/D, respectively) according to the antiphospholipid (aPL) profile at cut-off for positivity (upper panel) and at medium/high level (lower panel). Statistical values are indicated when significant (p<0.05). See figure 5 for abbreviations.

Figure 1 SLE

 1 Figure 1

  Figure 3

  Figure 5

  Figure 3

  Figure 5

  .84) increases the risk for arterial and/or venous thrombosis in patients with SLE and this risk was highest with LAC (OR 4.16) as 50% of LAC positive patients, in the following 20 years, have developed a venous thrombosis/pulmonary embolus[START_REF] Petri | Update on anti-phospholipid antibodies in SLE: the Hopkins' Lupus Cohort[END_REF]. This is in agreement with our study confirming the association between LAC in SLE (OR=6.1; IC953.5-10.3) and

	aCL/aβ2GPI IgG in non-SLE SADs (OR=6.6; IC95:2.4-18.4) with thrombosis, and the absence
	of association for thrombosis with regards to aCL±aβ2GPI IgM.

Horbach et al found, using a multivariate analysis approach, that LAC performed better than aCL±aβ2GPI of IgG in predicting venous thrombosis (OR 6.55; 95% CI 2.36-18.17) and arterial thrombosis (OR 9.77; 95% CI 1.74-31.15) in SLE

[START_REF] Horbach | Lupus anticoagulant is the strongest risk factor for both venous and arterial thrombosis in patients with systemic lupus erythematosus. Comparison between different assays for the detection of antiphospholipid antibodies[END_REF]

. According to the Hopkin's lupus cohort analysis, the presence of any aPL (OR 1

Table 1 :

 1 SLE and non-SLE SADs. The main message from this work is that aPL determination remains important in SADs patients and should not be restricted to SLE patients. Antiphospholipid (aPL) autoantibody prevalence in healthy controls (HC) and in patients with systemic autoimmune diseases (SADs).As aPL prevalence was higher in the SLE sub-group, the SAD group was further subdivided into patients with systemic lupus erythematosus (SLE, n=453) and into non-SLE patients with rheumatoid arthritis (RA, n=359), systemic sclerosis (SSc, n=385), Sjögren's syndrome (SjS, n=367), mixed connective tissue disease (MCTD, n=94), and undifferentiated connective tissue disease (UCTD, n=160). The cut-off for positivity and the cut-off for medium/high levels are indicated (see material and methods and supplementary figureS1). The combination of aCL±aβ2GPI referred to the detection of aCL and/or aβ2GPI autoantibodies.

	Authorship contribution statement: AM, PLM, EB, and YR: Formal analysis and writing
	original draft; EB, GP, SN, HG: Investigation; MAR, JF: Project administration; and MAR:
	Funding acquisition.

Declaration of Competing Interest: All authors declare that they have no conflict of interest. Acknowledgements: The work described has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115565, the resources for which are composed of a financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and the European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in-kind contribution. We thank all the members of PRECISESADS Consortium and INSERM U1227 for their effort in the sample recruitment, distribution and assessment of the samples used in this study. We are grateful to Dr Wesley H. Brooks (Tampa, USA) for editorial assistance and to Valerie Le Troadec for secretarial assistance.

B2 IgG aCL/B2 GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Thrombosis in non-SLE (medium/high)

	A				Thrombosis Miscarriage in SLE in	SLE				C	Thrombosis in non-SLE Thrombosis in non-SLE
		dRVVT drVVT	drVVT						p<10 -4		drVVT dRVVT drVVT				p<10 -4	p<10 -4
	aCL±aβ2GPI IgG aCL/B2 aCL/B2 GPI IgG GPI	IgG					p=0.0003 p=0.03	aCL/B2 GPI IgG aCL/B2 GPI aCL±aβ2GPI IgG IgG				p=0.001 p=0.001
	aCL±aβ2GPI IgM aCL/B2 aCL/B2 GPI IgM GPI	IgM									aCL/B2 GPI IgM aCL/B2 GPI aCL±aβ2GPI IgM IgM
	dRVVT + aCL±aβ2GPI IgG drVVT/aCL/B2 drVVT/aCL/B2 IgG	IgG						drVVT/aCL/B2 IgG drVVT/aCL/B2 dRVVT + aCL±aβ2GPI IgG p=0.0001 IgG				p=0.001 p=0.001
	dRVVT + aCL±aβ2GPI IgM drVVT/aCL/B2 drVVT/aCL/B2 IgM	IgM						p=0.03	drVVT/aCL/B2 IgM drVVT/aCL/B2 dRVVT + aCL±aβ2GPI IgM IgM
	aCL±aβ2GPI IgG+IgM aCL/B2 aCL/B2 IgG/IgM IgG/IgM					p=0.05			aCL/B2 IgG/IgM aCL/B2 IgG/IgM aCL±aβ2GPI IgG+IgM				p=0.0002 p=0.0002
		0.1	0.1	1	1	10	10	100	100		0.1	0.1	1	1	10	10	100	1000 100	1000
					Odds Odds Ratio Ratio							Odds Ratio Odds Ratio
	B	Thrombosis in SLE (medium/high) Thrombosis in SLE (medium/high)		D	Thrombosis	in	non-SLE	(medium/high)
	dRVVT aCL±aβ2GPI IgG aCL±aβ2GPI IgM aCL/B2 aCL/B2 dRVVT + aCL±aβ2GPI IgG drVVT/aCL/B2 GPI GPI drVVT IgM IgG IgG aCL/B2					p<10 -4 p<10 -4 p=0.008 p=0.0001 p<10 -4 p<10 -4 p=0.008 p=0.0001 dRVVT + aCL±aβ2GPI IgG aCL/B2 GPI IgM aCL/B2 GPI IgG drVVT dRVVT aCL±aβ2GPI IgG aCL±aβ2GPI IgM drVVT/aCL/p<10 -4 p<10 -4 p<10 -4 p<10 -4 drVVT/aCL/B2 IgG
	dRVVT + aCL±aβ2GPI IgM drVVT/aCL/B2 drVVT/aCL/B2 IgG	IgM						p=0.007 p=0.007 drVVT/aCL/B2 IgM drVVT/aCL/B2 dRVVT + aCL±aβ2GPI IgM IgM
	aCL±aβ2GPI IgG+IgM aCL/B2 drVVT/aCL/B2 IgM IgG/IgM					p=0.03	p=0.03	aCL/B2 IgG/IgM aCL/B2 IgG/IgM aCL±aβ2GPI IgG+IgM
		aCL/B2 IgG/IgM
				0.1		1		10		100	0.01	0.1 0.01	0.1	1	1	10	10	100	100
		0.1		1		10			100
						Odds	Ratio								Odds	Ratio

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in SLE (medium/high)

		Miscarriage in non-SLE
	drVVT
	aCL/B2 GPI IgG
	aCL/B2 GPI IgM			p=0.02
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM			p=0.04
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	p=0.01
	p=0.02
	aCL/B2
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in non-SLE (medium/high)

				Figure 6
	A	Miscarriage in SLE	C
	drVVT
				p=0.03
	aCL/B2 GPI IgG
	aCL/B2 GPI IgM
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	B			D
	aCL/B2			p=0.003
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in SLE (medium/high)

		Miscarriage in non-SLE
	drVVT
	aCL/B2 GPI IgG
				p=0.02
	aCL/B2 GPI IgM
	drVVT/aCL/B2 IgG
				p=0.04
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	p=0.01
	p=0.02
	aCL/B2
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in non-SLE (medium/high)

									Figure 1
	SLE (n=453)		non-SLE (n=1365)
	dRVVT					dRVVT
	aCL±aβ2GPI IgG dRVVT				aCL±aβ2GPI	aCL±aβ2GPI IgG dRVVT			aCL±aβ2GPI
	aCL±aβ2GPI IgM 21.2%	8.7%	10.8%	IgG	aCL±aβ2GPI IgM 7.0%	0.5%	1.3%	IgG
	dRVVT + aCL±aβ2GPI IgG dRVVT + aCL±aβ2GPI IgM	5.1%	1.6%	3.1%		dRVVT + aCL±aβ2GPI IgG dRVVT + aCL±aβ2GPI IgM 1.1%	0.2%	0.4%
	aCL±aβ2GPI IgG+IgM					aCL±aβ2GPI IgG+IgM
			7.1%				3.2%
	aCL±aβ2GPI		aCL±aβ2GPI
		IgM			IgM
	dRVVT					dRVVT
	aCL±aβ2GPI IgG					aCL±aβ2GPI IgG
	aCL±aβ2GPI IgM					aCL±aβ2GPI IgM			p=0.003
	dRVVT + aCL±aβ2GPI IgG					dRVVT + aCL±aβ2GPI IgG
	dRVVT + aCL±aβ2GPI IgM					dRVVT + aCL±aβ2GPI IgM
	aCL±aβ2GPI IgG+IgM					aCL±aβ2GPI IgG+IgM

B2 IgM drVVT/aCL/B2 IgG aCL/B2 GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Thrombosis in non-SLE (medium/high)

	A				Thrombosis Miscarriage in SLE in	SLE				C	Thrombosis in non-SLE Thrombosis in non-SLE
		dRVVT drVVT	drVVT						p<10 -4		drVVT dRVVT drVVT				p<10 -4	p<10 -4
	aCL±aβ2GPI IgG aCL/B2 aCL/B2 GPI IgG GPI	IgG					p=0.0003 p=0.03	aCL/B2 GPI IgG aCL/B2 GPI aCL±aβ2GPI IgG IgG				p=0.001 p=0.001
	aCL±aβ2GPI IgM aCL/B2 aCL/B2 GPI IgM GPI	IgM									aCL/B2 GPI IgM aCL/B2 GPI aCL±aβ2GPI IgM IgM
	dRVVT + aCL±aβ2GPI IgG drVVT/aCL/B2 drVVT/aCL/B2 IgG	IgG						drVVT/aCL/B2 IgG drVVT/aCL/B2 dRVVT + aCL±aβ2GPI IgG p=0.0001 IgG				p=0.001 p=0.001
	dRVVT + aCL±aβ2GPI IgM drVVT/aCL/B2 drVVT/aCL/B2 IgM	IgM						p=0.03	drVVT/aCL/B2 IgM drVVT/aCL/B2 dRVVT + aCL±aβ2GPI IgM IgM
	aCL±aβ2GPI IgG+IgM aCL/B2 aCL/B2 IgG/IgM IgG/IgM					p=0.05			aCL/B2 IgG/IgM aCL/B2 IgG/IgM aCL±aβ2GPI IgG+IgM				p=0.0002 p=0.0002
		0.1	0.1	1	1	10	10	100	100		0.1	0.1	1	1	10	10	100	1000 100	1000
					Odds Odds Ratio Ratio							Odds Ratio Odds Ratio
	B	Thrombosis in SLE (medium/high) Thrombosis in SLE (medium/high)		D	Thrombosis	in	non-SLE	(medium/high)
	dRVVT aCL±aβ2GPI IgG aCL±aβ2GPI IgM dRVVT + aCL±aβ2GPI IgM drVVT/aCL/B2 aCL/B2 GPI aCL/B2 GPI drVVT IgM IgM IgG drVVT/aCL/B2 IgG dRVVT + aCL±aβ2GPI IgG drVVT/aCL/B2 IgG aCL/B2					p<10 -4 p<10 -4 p=0.008 p=0.0001 p=0.007 p<10 -4 p<10 -4 p=0.008 p=0.007 dRVVT + aCL±aβ2GPI IgM drVVT/aCL/B2 IgM aCL/B2 GPI IgM aCL/B2 GPI IgG drVVT dRVVT aCL±aβ2GPI IgG aCL±aβ2GPI IgM drVVT/aCL/p<10 -4 p<10 -4 p<10 -4 p<10 -4 p=0.0001 drVVT/aCL/B2 IgG dRVVT + aCL±aβ2GPI IgG
	aCL±aβ2GPI IgG+IgM aCL/B2 drVVT/aCL/B2 IgM IgG/IgM					p=0.03	p=0.03	aCL/B2 IgG/IgM aCL/B2 IgG/IgM aCL±aβ2GPI IgG+IgM
		aCL/B2 IgG/IgM
				0.1		1		10		100	0.01	0.1 0.01	0.1	1	1	10	10	100	100
		0.1		1		10			100
						Odds	Ratio								Odds	Ratio

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in SLE (medium/high)

		Miscarriage in non-SLE
	drVVT
	aCL/B2 GPI IgG
	aCL/B2 GPI IgM			p=0.02
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM			p=0.04
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	p=0.01
	p=0.02
	aCL/B2
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in non-SLE (medium/high)

				Figure 6
	A	Miscarriage in SLE	C
	drVVT
				p=0.03
	aCL/B2 GPI IgG
	aCL/B2 GPI IgM
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	B			D
	aCL/B2			p=0.003
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in SLE (medium/high)

		Miscarriage in non-SLE
	drVVT
	aCL/B2 GPI IgG
				p=0.02
	aCL/B2 GPI IgM
	drVVT/aCL/B2 IgG
				p=0.04
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM			p=0.05
	0.1	1	10	100
		Odds Ratio
	p=0.01
	p=0.02
	aCL/B2
	drVVT/aCL/B2 IgG
	drVVT/aCL/B2 IgM
	aCL/B2 IgG/IgM
	0.1	1	10	100

GPI IgM aCL/B2 GPI IgG drVVT Odds Ratio Miscarriage in non-SLE (medium/high)

	dRVVT	dRVVT
	aCL±aβ2GPI IgG	aCL±aβ2GPI IgG
	aCL±aβ2GPI IgM	aCL±aβ2GPI IgM
	dRVVT + aCL±aβ2GPI IgG	dRVVT + aCL±aβ2GPI IgG
	dRVVT + aCL±aβ2GPI IgM	dRVVT + aCL±aβ2GPI IgM
	aCL±aβ2GPI IgG+IgM	aCL±aβ2GPI IgG+IgM
	dRVVT	dRVVT
	aCL±aβ2GPI IgG	aCL±aβ2GPI IgG
	aCL±aβ2GPI IgM	aCL±aβ2GPI IgM	p=0.003
	dRVVT + aCL±aβ2GPI IgG	dRVVT + aCL±aβ2GPI IgG
	dRVVT + aCL±aβ2GPI IgM	dRVVT + aCL±aβ2GPI IgM
	aCL±aβ2GPI IgG+IgM	aCL±aβ2GPI IgG+IgM