S Rodosik

J.-P Poirot-Crouvezier

Y Bultel
email: yann.bultel@grenoble-inp.fr

Impact of Alternating Fuel Feeding on a PEMFC stack durability

Keywords: Hydrogen feed strategy, Anode system architecture, Local temperature, Current distribution, Degradation, Automotive conditions

The integration of PEMFC for transport applications requires cost as well as system complexity reduction. A novel anode circuit architecture named "Alternating Fuel Feeding" (AFF) was developed. It combines on one hand, the benefits of the hydrogen recirculation and on the other hand, the simpleness of Dead-End Anode (DEA). A previous work demonstrated the benefits on the costs and on the anodic line weight. In the present work, investigations on stack durability, from the system scale to the cell scale, are performed during operation in two anode feeding modes: AFF and recirculation. The performances of stacks are evaluated along ageing tests, coupled with local measurements. At the end of life, postmortem analyses are performed on the aged cells. The degradation rates of stack performances are quite similar in both cases while more heterogeneous cell degradation are observed in recirculation mode along with the ageing according to a Fuel Cell Dynamic Load Cycle (FC-DLC).

Introduction

Today, Europe through the Fuel Cells and Hydrogen Joint Undertaking (FCH JU) depicts a fuel cell system that is economically viable for transport in light of 2030, with a power density of the stack cells at 10 kW/L, a durability of 7000 h and a production cost of the system lower than 40 €/kW [START_REF]Addendum to the Multi -Annual Work Plan[END_REF].

To achieve these targets, two axes of work are identified: the first one on the reduction of stack volume and mass, and the second one on the systems complexity. According to the US Department of Energy [START_REF] Thompson | Direct hydrogen fuel cell electric vehicle cost analysis: System and high-volume manufacturing description, validation, and outlook[END_REF][START_REF]DOE Technical Targets for Polymer Electrolyte Membrane Fuel Cell Components[END_REF], the air and hydrogen supply lines influence strongly the complexity of the system and thus its cost. The main function of the anodic fluidic architecture is the control of the flow and the pressure of hydrogen entering the fuel cell, but rarely of its humidification.

This can be accomplished by different fluid management strategies and different architectures.

The Dead-End Anode (DEA) strategy is the simplest architecture [START_REF] Abbou | Time Evolution of Local Potentials during PEM Fuel Cell Operation with Dead-Ended Anode[END_REF][START_REF] Abbou | Impact of Water Management on Local Potential Evolutions during PEM Fuel Cell Operation with Dead-Ended Anode[END_REF][START_REF] Dubau | Carbon corrosion induced by membrane failure: The weak link of PEMFC long-term performance[END_REF][START_REF] Asghari | Investigation of self-humidified and deadended anode proton exchange membrane fuel cell performance using electrochemical impedance spectroscopy[END_REF][START_REF] Siegel | Measurement of Liquid Water Accumulation in a PEMFC with Dead-Ended Anode[END_REF][START_REF] Lee | An experimental study on water transport through the membrane of a PEFC operating in the dead-end mode[END_REF]. The anodic output valve is kept closed, opening only intermittently to evacuate any species accumulated at the exit of the cell. In DEA, the anode is therefore supplied with dry gas. This causes a drying out of the area close to the hydrogen inlet. This phenomenon, coupled with the accumulation of water and nitrogen at the outlet, induces performance losses [START_REF] Abbou | Time Evolution of Local Potentials during PEM Fuel Cell Operation with Dead-Ended Anode[END_REF][START_REF] Siegel | Measurement of Liquid Water Accumulation in a PEMFC with Dead-Ended Anode[END_REF][START_REF] Abbou | Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode -Part II: Aging mitigation strategies based on water management and nitrogen crossover[END_REF][START_REF] Kocha | Characterization of gas crossover and its implications in PEM fuel cells[END_REF][START_REF] Ahluwalia | Buildup of nitrogen in direct hydrogen polymer-electrolyte fuel cell stacks[END_REF][START_REF] Baik | Characterization of nitrogen gas crossover through the membrane in proton-exchange membrane fuel cells[END_REF][START_REF] Paganelli | Fuel Cell and Method for Stopping a Fuel Cell[END_REF][START_REF] Nikiforow | Optimization study of purge cycle in proton exchange membrane fuel cell system[END_REF][START_REF] Meyer | Nitrogen Blanketing and Hydrogen Starvation in Dead-Ended-Anode Polymer Electrolyte Fuel Cells Revealed by Hydro-Electro-Thermal Analysis[END_REF][START_REF] Abbou | Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode -Part I: Impact of water diffusion and nitrogen crossover[END_REF] as well as irreversible degradations [START_REF] Abbou | Time Evolution of Local Potentials during PEM Fuel Cell Operation with Dead-Ended Anode[END_REF][START_REF] Dubau | Carbon corrosion induced by membrane failure: The weak link of PEMFC long-term performance[END_REF][START_REF] Pozio | Nafion degradation in PEFCs from end plate iron contamination[END_REF][START_REF] Lee | Pinhole formation in PEMFC membrane after electrochemical degradation and wet/dry cycling test[END_REF][START_REF] Sanchez | PEMFC operation failure under severe dehydration[END_REF][START_REF] Guétaz | Study by electron microscopy of proton exchange membrane fuel cell membrane-electrode assembly degradation mechanisms: Influence of local conditions[END_REF][START_REF] Pei | Main factors affecting the lifetime of Proton Exchange Membrane fuel cells in vehicle applications: A review[END_REF][START_REF] Qiu | Effect of relative humidity cycles accompanied by intermittent start/stop switches on performance degradation of membrane electrode assembly components in proton exchange membrane fuel cells[END_REF][START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF][START_REF] Zhao | Experimental Observations of Microstructure Changes in the Catalyst Layers of Proton Exchange Membrane Fuel Cells under Wet-Dry Cycles[END_REF][START_REF] Sanchez | Local impact of humidification on degradation in polymer electrolyte fuel cells[END_REF]. So, anodic purge strategy is investigated [START_REF] Nikiforow | Optimization study of purge cycle in proton exchange membrane fuel cell system[END_REF][START_REF] Rabbani | Effect of nitrogen crossover on purging strategy in PEM fuel cell systems[END_REF][START_REF] Chen | Optimization of purge cycle for dead-ended anode fuel cell operation[END_REF][START_REF] Han | PEM fuel-cell stack design for improved fuel utilization[END_REF][START_REF] Strahl | Experimental study of hydrogen purge effects on performance and efficiency of an open-cathode Proton Exchange Membrane fuel cell system[END_REF][START_REF] Sasmito | A Factorial Study to Investigate the Purging Effect on the Performance of a Dead-End Anode PEM Fuel Cell Stack[END_REF][START_REF] Werner | The impact of operating parameters and system architecture on the water management of a multifunctional PEMFC system[END_REF][START_REF] Eskin | Anode bleeding experiments to improve the performance and durability of proton exchange membrane fuel cells[END_REF] and often, frequent purges are recommended, inducing hydrogen losses impeding the system efficiency [START_REF] Abbou | Time Evolution of Local Potentials during PEM Fuel Cell Operation with Dead-Ended Anode[END_REF][START_REF] Siegel | Measurement of Liquid Water Accumulation in a PEMFC with Dead-Ended Anode[END_REF][START_REF] Abbou | Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode -Part II: Aging mitigation strategies based on water management and nitrogen crossover[END_REF][START_REF] Kocha | Characterization of gas crossover and its implications in PEM fuel cells[END_REF][START_REF] Ahluwalia | Buildup of nitrogen in direct hydrogen polymer-electrolyte fuel cell stacks[END_REF][START_REF] Baik | Characterization of nitrogen gas crossover through the membrane in proton-exchange membrane fuel cells[END_REF][START_REF] Paganelli | Fuel Cell and Method for Stopping a Fuel Cell[END_REF][START_REF] Nikiforow | Optimization study of purge cycle in proton exchange membrane fuel cell system[END_REF][START_REF] Meyer | Nitrogen Blanketing and Hydrogen Starvation in Dead-Ended-Anode Polymer Electrolyte Fuel Cells Revealed by Hydro-Electro-Thermal Analysis[END_REF][START_REF] Abbou | Local potential evolutions during proton exchange membrane fuel cell operation with dead-ended anode -Part I: Impact of water diffusion and nitrogen crossover[END_REF][START_REF] Rabbani | Effect of nitrogen crossover on purging strategy in PEM fuel cell systems[END_REF][START_REF] Chen | Optimization of purge cycle for dead-ended anode fuel cell operation[END_REF][START_REF] Han | PEM fuel-cell stack design for improved fuel utilization[END_REF][START_REF] Strahl | Experimental study of hydrogen purge effects on performance and efficiency of an open-cathode Proton Exchange Membrane fuel cell system[END_REF][START_REF] Sasmito | A Factorial Study to Investigate the Purging Effect on the Performance of a Dead-End Anode PEM Fuel Cell Stack[END_REF][START_REF] Werner | The impact of operating parameters and system architecture on the water management of a multifunctional PEMFC system[END_REF][START_REF] Eskin | Anode bleeding experiments to improve the performance and durability of proton exchange membrane fuel cells[END_REF].

The most widespread architecture to our knowledge on systems for the mobility is the recirculation of hydrogen using ejectors [START_REF] Arboleda | New Hyundai NEXO FCV, European Hydrogen Energy Conference[END_REF][START_REF] Nikiforow | Discrete ejector control solution design, characterization, and verification in a 5 kW PEMFC system[END_REF][START_REF] Milewski | Off-design analysis of SOFC hybrid system[END_REF][START_REF] Marsano | Ejector performance influence on a solid oxide fuel cell anodic recirculation system[END_REF] or recirculation pumps [START_REF] Konno | Development of Compact and High-Performance Fuel Cell Stack[END_REF][START_REF] Hasegawa | Development of the Fuel Cell System in the Mirai FCV[END_REF][START_REF] Zhao | Study on voltage clamping and selfhumidification effects of pem fuel cell system with dual recirculation based on orthogonal test method[END_REF][START_REF] Toghyani | A parametric comparison of three fuel recirculation system in the closed loop fuel supply system of PEM fuel cell[END_REF]. This second configuration allows participating to water management at the anode inlet of the stack. Indeed, the water vapor is recirculated at the anode outlet in order to hydrate the membrane [START_REF] Ahluwalia | Buildup of nitrogen in direct hydrogen polymer-electrolyte fuel cell stacks[END_REF][START_REF] Uno | Reactant recirculation system utilizing pressure swing for proton exchange membrane fuel cell[END_REF][START_REF] Migliardini | Optimization of hydrogen feeding procedure in PEM fuel cell systems for transportation[END_REF][START_REF] Migliardini | Hydrogen purge and reactant feeding strategies in self-humidified PEM fuel cell systems[END_REF][START_REF] Jiang | Experimental study on dual recirculation of polymer electrolyte membrane fuel cell[END_REF][START_REF] Lee | A gas management strategy for anode recirculation in a proton exchange membrane fuel cell[END_REF][START_REF] Steinberger | Anode recirculation and purge strategies for PEM fuel cell operation with diluted hydrogen feed gas[END_REF]. A few authors investigated the feasibility to recirculate hydrogen through the stack based on a combination of a specific architecture and a pressure-swing concept [START_REF] Uno | Reactant recirculation system utilizing pressure swing for proton exchange membrane fuel cell[END_REF][START_REF] Grisard | Long Term Performance of the Pressure Swing Recirculation System[END_REF].

Lately, an upgraded anode fluidic architecture, called Alternating Fuel Feeding (AFF), has been proposed at the anode by Rodosik at al. [START_REF] Rodosik | Simplified anode architecture for PEMFC systems based on alternative fuel feeding: Experimental characterization and optimization for automotive applications[END_REF]. This latter only requires a new stack configuration and an additional solenoid valve. The stack is divided into two parts allowing a circulation of the hydrogen gas in the whole anode compartment between the two half-stacks.

AFF stack is as efficient as the current hydrogen recirculation reducing the complexity and volume of the fuel cell systems. Benefits have been demonstrated on the weight of the anodic line (25% compared to pulsed ejector, and 43% compared to blower architecture) and on the costs (6% compared to pulsed ejector, and 57% compared to blower architecture).

For vehicle application, the sudden changes in current induce the most important degradations [START_REF] Pei | Main factors affecting the lifetime of Proton Exchange Membrane fuel cells in vehicle applications: A review[END_REF][START_REF] Pei | A quick evaluating method for automotive fuel cell lifetime[END_REF]. Water management is a key factor in fuel cell operation and it is accepted that a good hydration of the membrane facilitates proton conductivity and therefore is mandatory to obtain good performance. The management of this hydration depends both on the chosen fluidic architecture and the operating conditions of the system. An inefficient water management leads to flooding or drying out phenomena, which has a direct impact on fuel cell performance and lifespan. The drying out can induce local degradation within MEAs, particularly on the membrane [START_REF] Sanchez | Local impact of humidification on degradation in polymer electrolyte fuel cells[END_REF]. Paradoxically, a cell supplied with dry gas can be flooded [START_REF] Sanchez | PEMFC operation failure under severe dehydration[END_REF][START_REF] Gößling | Analysis of liquid water formation in polymer electrolyte membrane (PEM) fuel cell flow fields with a dry cathode supply[END_REF]. The power profiles, for example, induce temperature and humidity cycles that can severely damage the stack, either at the membrane or at the electrode level [START_REF] Lee | Pinhole formation in PEMFC membrane after electrochemical degradation and wet/dry cycling test[END_REF][START_REF] García-Salaberri | Hydration and dehydration cycles in polymer electrolyte fuel cells operated with wet anode and dry cathode feed: A neutron imaging and modeling study[END_REF][START_REF] O'dea | Surface Morphology of Nafion at Hydrated and Dehydrated Conditions[END_REF]. Low current density operation has a tendency to flood the fuel cell due to a reduced flow rate and therefore poor fuel cell capacity to evacuate the water accompanied by a drop in temperature that accentuates the phenomenon. Several models for the formation of a water film on the catalytic layer have been developed to elucidate this phenomenon that occurs at low power [START_REF] Koz | Oxygen transport resistance at gas diffusion layer -Air channel interface with film flow of water in a proton exchange membrane fuel cell[END_REF][START_REF] Jo | Analysis of water film formation and low-humidity operation characteristics of a polymer electrolyte fuel cell (PEFC)[END_REF]. As both modes of hydrogen feeding, the recirculation mode or the alternating fuel feeding one, exhibit similar performance results [START_REF] Grisard | Long Term Performance of the Pressure Swing Recirculation System[END_REF], this study focuses on a correlation between the anode architecture and fuel cell durability. One ageing test is performed for both hydrogen-feeding architectures considering test representative operating conditions and load cycles. The experimental data are analyzed at different scales up to the post-mortem expertise of membrane-electrode assemblies.

Material and methods

Fuel Cell system architecture

In order to compare the Alternating Fuel Feeding operation to the hydrogen recirculation, two different test benches are used. The first one is an autonomous 5-kW system already presented in previous studies [START_REF] Rodosik | Simplified anode architecture for PEMFC systems based on alternative fuel feeding: Experimental characterization and optimization for automotive applications[END_REF][START_REF] Rodosik | Impact of humidification by cathode exhaust gases recirculation on a PEMFC system for automotive applications[END_REF]. The test bench has a classical configuration at the cathode, with an air filter, an air compressor and a passive membrane humidifier with flow through architecture. All the cells of the stack are connected in parallel to the cathodic line, and are equally fed with air. At the anode side, an AFF architecture is set up. With this new configuration, the stack is divided in two groups of cells. In this experiment, each group is located on one side of a central distribution plate. Hydrogen is supplied in one or the other halfstack, by means of two independent solenoid valves, labelled 1 and 2 on Figure S1.

In AFF operation, hydrogen is alternately supplied to each half-stack [START_REF] Rodosik | Simplified anode architecture for PEMFC systems based on alternative fuel feeding: Experimental characterization and optimization for automotive applications[END_REF]. To do so, the solenoid valves 1 and 2 open alternately. When valve 1 is opened, the half-stack on the right side is fed with hydrogen from the outlet of the other half-stack (Figure S1-a). Conversely, when valve 2 is opened, the half-stack on the left side is fed by hydrogen from the outlet of the half-stack on the right side (Figure S1-b). Finally, the hydrogen outlet being closed by valve 3 during the alternating hydrogen supply, liquid water is collected in the central distribution plate, and nitrogen concentration increases in the anodic gas mixture. Both species are evacuated periodically when valve 3 is opened together with valves 1 and 2, as represented on Figure S1-c. The complete sequence of AFF operation is provided in Rodosik et al. [START_REF] Rodosik | Simplified anode architecture for PEMFC systems based on alternative fuel feeding: Experimental characterization and optimization for automotive applications[END_REF].

The second test bench is a commercial Greenlight G100 fuel cell bench that can test fuel cell stacks of a few hundred watts. The flow rate, pressure, temperature and relative humidity are controlled on each line. This test bench is used in order to simulate an operation in hydrogen recirculation. To do so, in the absence of a recirculating device, a gas mixture composed of hydrogen and water vapor is injected at the inlet of the cell with a hydrogen stoichiometric ratio of 1.5, which is frequently used with this stack technology [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF]. Hydrogen stoichiometric ratio is often set in the range 1.5-2.0, especially for high power operating points [START_REF] Nikiforow | Discrete ejector control solution design, characterization, and verification in a 5 kW PEMFC system[END_REF][START_REF] Reshetenko | Systematic study of back pressure and anode stoichiometry effects on spatial PEMFC performance distribution[END_REF]. Greater stoichiometric ratios, like in the Toyota Mirai system [START_REF] Hasegawa | Development of the Fuel Cell System in the Mirai FCV[END_REF], would not be suitable here, due to the large thickness of our membranes (25 µm). Indeed, high stoichiometric ratios associated with thin membranes allow adequate humidification of the MEAs without cathode humidifier, but it can have a detrimental effect on the stack performance if the membranes are too thick [START_REF] Hasheminasab | A novel experimental based statistical study for water management in proton exchange membrane fuel cells[END_REF].

The anode relative humidity is set to 50%, regarding the stoichiometric ratio. Indeed, we can evaluate the relative humidity at the stack inlet from the mass balance of a recirculation loop.

If we consider that the exit valve of the loop is closed, and if we assume that the gas is saturated with water vapor at the stack outlet, the gas relative humidity at the stack inlet is linked to the stoichiometric ratio through the equation:

= 1 + -1 1 - (1)
Where is the total pressure and the saturation pressure for water vapor, both assumed constant in the hydrogen loop. With this simplified approach, a total pressure of 1.3 bar and a temperature of 80°C leads to a relative humidity of 45%. Thus, imposing a value of 50% during the test that simulates the operation with a recirculation loop is a valid approximation.

The presence of nitrogen in the actual mixture is neglected. The stack tested on this test bench will be called "Recirculation stack".

Test Protocol

Similar ageing tests are performed in this study for the two hydrogen feeding architectures.

The test is based on the Fuel Cell Dynamic Load Cycle (FC-DLC) [START_REF] Tsotridis | EU harmonised test protocols for PEMFC MEA testing in single cell configuration for automotive applications[END_REF], and similar operating conditions are applied to both stacks, except for hydrogen feeding. The duration of both tests is approximately 1000 hours.

The reference FC-DLC load cycle is simplified due to power limitations of the test bench, keeping a few different current density steps as illustrated in Figure S2. This adaptation of the cycle consists in a squaring of the current density pulses. As a consequence, some losses of accuracy are observed by comparison to the genuine dynamic load profile. However, the current density of 0.42 A cm -2 still represents approximately 50 % of the maximum current density achievable with this stack technology. In contrast, typical driving cycles tested with the Toyota Mirai by Argonne National Laboratory induce a low power demand. Thus, the maximum power provided by the Mirai stack to cover 90 % of the NEDC power demand is only 10% of its maximum power [62]. Even without high power peaks, the simplified FC-DLC cycle defined here remains dynamic and harsh.

The operating conditions for both tests are chosen according to automotive conditions. All parameters are gathered in Table 1. The temperature is maintained at a high level of 80°C.

The relative humidity at the cathode is quite low, due to the consideration of a volume constraint for the system, leading to a downsizing of the passive humidifier. The relative humidity is set to around 30% at the cathode side on both test benches with AFF and passive humidification.

Alternating Fuel Feeding introduces some specific operating parameters, such as the AFF frequency. The latter corresponds to the switching frequency of the two inlet valves (1 and 2).

The sensitivity analysis presented previously [START_REF] Rodosik | Simplified anode architecture for PEMFC systems based on alternative fuel feeding: Experimental characterization and optimization for automotive applications[END_REF] about the operating parameters that are specific to AFF shows that an AFF frequency of 0.7 Hz is optimal in this case. Furthermore, the outlet valve 3 is opened regularly during one second in order to purge the anode circuit. A purge period of 70 seconds is selected. The amount of hydrogen rejected during the purges is equivalent to 2% of the hydrogen consumption inside to stack, leading to an average stoichiometric ratio of 1.02 at the anode.

Caracterization methods

PEMFC degradations are characterized at the stack scale as well as at the cell one. At the stack scale, operating heterogeneities between cells may appear, while, at the cell scale, local operating heterogeneities on the surface of the MEAs can be detected.

In-situ diagnosis

The distribution of the current density and temperature over the cell area is measured using a Current Scan Lin S++ [START_REF][END_REF] device apparatus. This local in-situ diagnosis is a custom-built segmented printed circuit board inserted between two cells inside the stack. Local current is measured using ring cores of a magnetically soft material (ferrite). The size of the segments is approximately 7 x 7 mm. Therefore, for a 220 cm² active area, the current distribution is measured by using 480 measuring segments. The board is also equipped with 120 temperature sensors, based upon the dependency of the resistivity of metals such as copper upon temperature. The current density is measured with a precision of 0.02 A cm -², and the temperature with a precision of 1 °C. This sensor plate, placed inside the stack between two bipolar plates, replaces one membrane electrode assembly. The gas channels in contact with the sensor plate are not fed with reactants.

In addition to the current density distribution measurement, a set of analysis is performed throughout the ageing test [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF]. Punctual polarization curves are performed each 200 h. All cells in the Recirculation stack are monitored. This is not the case in AFF mode due to the size of the stack. The location of the tested cells is chosen either because of their singular position (extreme cells) in the stack, or because of their particular behavior. The LSV is used to study the evolution of the hydrogen permeation through the membrane for all the cells of the Recirculation stack and only for eight cells on the AFF stack.

Post-mortem analyses of the PEMFC stack

At the end of life, postmortem analyses are performed in order to investigate the MEA degradation using different characterization methods.

Potential cracks or pinholes in the aged MEA can be, first, located by using a hydrogen box associated to an infrared camera. In this device, diluted hydrogen flows in the box, on the back side of the aged MEA, while atmospheric air is present at the front-side of the MEA, namely outside of the box. Eventual pinholes or cracks in the membrane can be detected by hot spots.

Indeed, they form direct paths between both sides of the MEA, and induce a direct combustion of hydrogen in air. Images of the whole active area of a MEA obtained with an infrared camera (FLIR® E-Series Advanced Thermal Imaging Camera) provide mappings of the permeation rate of hydrogen through the membrane. High permeation rates are detected where the temperature is high and are associated with pinholes or cracks. This post-mortem analysis technique has already been used and described in previous studies [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF].

Moreover, mini-cell tests are performed in order to evaluate the local behavior of the MEAs after the tests. These cells have an active area of less than 2 cm², and are dedicated to the characterization of the electrochemical performance of catalytic layers and gas diffusion layers.

The effect of mass transports in the channels is neglected due to the use of high reactant flows, corresponding to stoichiometric ratios greater than 10 [START_REF] Fontana | Direct growth of carbon nanotubes forests on carbon fibers to replace microporous layers in proton exchange membrane fuel cells[END_REF]. Therefore, 1.8 cm² rectangular samples are cut in several aged MEA in different locations, such as near the gases inlets, in the cell center and near the gases outlets. Once inserted in the mini-cell device, their ECSA are measured using the Cyclic Voltammetry. The selection and location of the areas to be cut are made using current and temperature density maps (Figure S3). In total, six zones are cut out, in Air Inlet (AI), Air Outlet (AO), Hydrogen Inlet (HI), Hydrogen Outlet (HO) and in the middle of a Hot Spot (HS) supposedly drier, and of a Cold Spot (CS) supposedly wetter.

Different operating conditions are tested on each sample. These are grouped in Table 2. Each sample is first subjected to the so-called favorable conditions and then the sensitivity to moisture is analyzed by comparing behavior in dry and wet conditions.

Experimental results

In situ analysis

Stack scale

The following section presents an analysis at the stack scale for a global understanding of degradation phenomena.

The global performances of the two stacks are recorded on a characterization bench at the beginning and end of the test under system conditions of 80°C, 1.3 bar, with stoichiometric ratios (SR) at the anode and the cathode of respectively 1.5 and 1.8 and relative humidity (RH)

of respectively 50% and 30%. The polarization curves at the beginning and end of the test are shown in Figure 1. The general pattern of the curves between the beginning and end of the test shows similar performance.

At the beginning of the test, the two polarization curves of the 8-cell recirculating stack and of the 70-cell AFF stack are superimposed. A slight deviation can be observed at high current densities (> 0.5 A.cm -2 ) where a mass transport effect begins to build up on the AFF stack.

This difference might be due to the endplates design of the AFF stack, which are not optimized for flow-through mode used in the characterization process or to the test facilities, which have quite different power ranges. However, in the target operating range (cycle < 0.42 A cm -²), the performances obtained in both cases are comparable. This result confirms that the MEA batches are similar, and that the stack configuration and size have no impact on the initial performance if current density is lower than 0.5 A/cm². At the beginning of life, the maximum standard deviation between the cell voltages is less than 8 mV in recirculation, and less than 14 mV in AFF. At the end of life, the maximum standard deviation between cell voltages is higher and is about 50 mV for the recirculation test and only 22 mV for AFF.

Degradation rates are calculated for each cell and for different current levels, namely 0.1 and 0.42 A cm -2 obtained from the adapted FC-DLC speed cycle. They correspond to the slope of the linear regression plotted for all cell voltage measurements collected during the test at the same current. By comparing degradation rates of individual cells, heterogeneities in behavior between cells within the same stack can be observed. b. At the higher current density (0.42 A cm -2 ) on the plateau of the load cycle, the average degradation rate is 56 µV/h. This value is high but consistent with degradation rates obtained with this stack design in other tests [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF]. At lower current density (0.1 A cm -2 ), the heterogeneity of the degradation rates increase, with a standard deviation of 19 µV/h, and two groups of cells can be distinguished. Four of these, namely cells 2, 4, 6, and 8, have rates well above the calculated average of 35 µV/h. For instance, cell 2 even reaches a maximum of 70 µV/hour. The latter is generally colder than the other ones and seems to be flooded more easily, especially at the anode. This may explain its very high degradation rate. Avoiding this particular issue in future stacks is crucial. It might require an improvement of the flow fields inside the cell and the stack, and a better thermal management of the stack.

At low current (0.1 A cm -2 ), a few cells exhibit degradation rates well above the average of 25 µV/h. Cells with high degradation rates at low current also have high degradation rates at high current. Only cell 60 seems to be degraded at low current but presents a degradation rate in the average at high current. All the cells of the stack tested in recirculation mode undergo permeation current measurements every 200 hours during the durability test (Figure 3).

Among the cells of the stack in recirculation, four cells already identified in the previous section (cells 2, 4, 6 and 8) with a very low end-of-life OCV and high degradation rates show an early increase of their permeation current. It exceeds widely the nominal value of 1.8 mA cm -2 at 600 h of operation, and the following measurement points are out of range. Conversely, the permeation of the cells aged in AFF operation remains small even after 1000 hours of operation. Only the cell 36 located close to the central distribution plate exhibits a high permeation current after 500 hours of operation.

Cell scale

Current density distributions are measured in both stacks during ageing and at the beginning and end of the test. between the end and the beginning of the test for the two stacks tested. They are calculated for the maximum current density of the cycle, i.e. 0.42 A cm -2 and under the conditions of the durability test. The methodology consists in subtracting two current density distributions to obtain an image of the current density variation during a defined period. On the resulting mappings, the values are negative where the current density decreases over time.

The differential mapping obtained in recirculation mode has a less homogeneous distribution than in AFF. The evolution of the current densities shows a maximum decrease of 0.13 A cm -2 during the test near the hydrogen outlet and an increase of 0.17 A cm -2 close to the hydrogen inlet. This evolution is less noticeable in AFF with a maximum decrease of 0.12 A cm -2 on the central hot spot and an increase of 0.09 A cm -2 at the air outlet. This method shows that both stacks behave differently, because the differential mappings highlight distinct redistributions of the current with ageing.

Post-mortem ex-situ analysis

Local measurement

Electroactive surface area and polarization curves in different conditions are measured for each mini-cell. Figure 6 shows polarization curves of samples from MEAs extracted from the stack in AFF mode in two different regions: Hydrogen Outlet (HO) and the central Hot Spot (HS) (see Figure S3).

Polarization curves are recorded in dry and wet conditions for the Hydrogen Outlet (HO) as well as the Hot Spot (HS). The performances of the two latter zones drop in wet condition at high current density, probably due to water flooding. Nevertheless, this phenomenon is observed only for a few cells of the AFF stack, and is never recorded for the stack in recirculation. Loss of active surface area is measured on several cells for the stacks in recirculation as well as in AFF. Whatever the case, the drop is lower than 10% of the initial value, which does not explain the observed performance loss. This observation is in accordance with previous analyses conducted with the same MEA technology [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF][START_REF] Robin | Proton exchange membrane fuel cell model for aging predictions: Simulated equivalent active surface area loss and comparisons with durability tests[END_REF].

To go further, Figure 7 shows the SEM images taken in the Air Inlet zone (AI). In recirculation, as shown in Figure 7-a, the area seems very degraded. A compaction of the cathode catalytic layer is observed. Its thickness has been reduced to 4 µm instead of the 13 µm initially measured. The zone is very clear, highlighting a high platinum concentration. The decrease in thickness is attributed to corrosion of the electrode carbon support, which modifies its microstructure by reducing its porosity.

Discussion

The multi-scale analysis of the degradations in the two stacks, each subjected to a different operating mode, first shows similar behaviors. After a current cycling test of more than 1000h, the degradation rates calculated at 0.42 A cm -2 are equivalent: 56 µV/h in recirculation and 52 µV/h in AFF. However, there are local differences related to the anodic fluidic architectures.

In recirculation, the Hydrogen Outlet (HO) and Air Inlet (AI) are the most degraded areas. The performances recorded by the local current density measurement in this zone are the worst at the beginning of the test and they degrade with ageing. After the test, half of the MEAs in this stack are drilled in these areas. In AFF, a performance decay is observed close to the Air Inlet (AI) and attributed to the drying out during operation. Nevertheless, the highest current density decrease is recorded in zones in the middle of the cell where MEA degradation is the strongest during ageing. The performance decay observed in the central region is likely due to the specific thermal management of the bipolar plates design. Fredy et al. [START_REF] Nandjou | Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application[END_REF] well described the temperature distribution of this stack technology and its impact on water management as well as durability. A temperature hot spot is identified in the central area often leading to membrane drying and thus lower performance. The different current decrease distribution and degradation observed in the two tests indicates that each hydrogen feeding strategy leads to specific local degradations, while the global performance degradation is similar.

Figure 8 presents a summary of the observed local degradation for both operating modes. All identified degradations are superimposed on temperature distribution mapping obtained in the tested conditions at 0.42 A/cm² (like in Figure 4). It can be observed that the temperature difference between inlet and outlet is lower in recirculation than in AFF. This is due to the test facilities. The minimum cooling flow reached with test bench used for recirculation was slightly higher than the one reached on the system test bench. Therefore, cooling water temperature difference was lower in recirculation. A lower temperature difference would favor the durability with lower degradation rates. However, this was not observed during the test. During this test and in the target operation range, it seems that cooling temperature difference did not play a major role in degradation occurrence.

Among the observed degradations, membrane drilling, represented by red discs, is observed in both cases at the hydrogen outlet, but it is more frequent in recirculation than in AFF. Local cell flooding events are suspected to explain these degradations. They might be less frequent, or have less impact, in AFF compared to recirculation.

The decrease of the active area, represented by an orange disc, remains generally weak, except close to the hydrogen outlet in recirculation. It can be assumed that the membrane holes may have exacerbated these degradations in this zone. In AFF mode, this type of degradation is observed in a more moderate way in Hydrogen Outlet (HO) and in the Cold Spot (CS). The presence of liquid water in these areas could favor the loss of active surface.

Another difference observed between recirculation and AFF modes is the corrosion of the carbon support of the electrodes (orange squares), which can be also correlated to a decrease of the active area. This degradation mode is mainly associated with hydrogen starvation, which can occur locally where the anode is flooded with water, blocking the hydrogen access to the catalyst. This phenomenon is mainly observed, for the stack in recirculation, at the air inlet. A similar corrosion is recorded in the central hot zone of the stack but with a lower amplitude.

Conversely, carbon corrosion is not observed in AFF mode whatever the location on the electrode surface area.

Moreover, the distribution of water in the cell components is again questioned to explain the precipitation of Platinum in the membrane (symbolized by an orange triangle on Figure 8). This degradation phenomenon is generally related to important voltage variations, associated with wet conditions. In recirculation, the condensation of water at the anode outlet generates wetter conditions throughout the left half of the active surface, thus favoring the precipitation of platinum. In AFF, the wettest areas are the air outlet and the hydrogen inlet and outlet, as well as the cold environment where Platinum precipitation is observed in the membrane.

Finally, the last type of degradation relates to the loss of hydrophobicity of the electrodes (green squares). This is usually favored by a high temperature and strong variations in humidity. This is the case in AFF where the variations in relative humidity at the anode are very strong (from 0 to 100% RH at each purge cycle). The hypothesis of loss of hydrophobicity has not been validated by dedicated analyses. It will be the subject of a complementary study by measuring, for example, the contact angles of water on the GDL.

Conclusion

The AFF architecture allows to simplify the system complexity and to reduce significantly the weight of the anodic line maintaining similar performance levels in comparison to recirculation one. In this work, one ageing test has been performed for a classical mode of recirculation of hydrogen and for the alternating fuel feeding one, implementing the Fuel Cell Dynamic Load Cycle (FC-DLC). Overall, the degradation rates of stack performances are quite similar in both cases along with the ageing. Indeed, polarization curves highlighted only a slight deviation at OCV and at high currents densities.

At the cell scale, the hydrogen feeding mode induces heterogeneous ageing processes on the different components. Indeed, the most degraded areas are observed close to the hydrogen outlet and air inlet in recirculation mode while degraded areas are more uniformly distributed on the cell in AFF mode. Moreover, the degradation processes seem to be also different for both modes. On one hand, both architectures trigger the occurrence of membrane pinholes and micro-cracks at the hydrogen outlet responsible for a sudden loss of functionality of the cells. On the other hand, the recirculation hydrogen mode accelerates cathode catalyst layer corrosion close to the hydrogen outlet (HO) and air inlet (AI) while the AFF mode might favor loss of hydrophobicity of the electrodes in some areas.

This study highlights the fact that the hydrogen feeding strategy has a noticeable influence on the distribution of the local degradation along the cell, and on the current density distribution.

For durability improvement purposes, the study of Bipolar Plate designs could help in optimizing the gas flows and temperature distribution.

  Figure5), and the configuration of the serpentine channels. In AFF operation, the gas

  Moreover, Linear Sweep Voltammetry (LSV) and Cyclic Voltammetry (CV) are also performed for both stacks in order to characterize respectively the evolution of the hydrogen permeation through the membrane and the electrochemical active surface area (ECSA) loss at the cathode. During the electrochemical characterization test (CV), nitrogen flows over the working electrode while hydrogen flows over the counter-electrode. For the CV curves measurement, the cathode potential ranges from 0.05V to 0.80V vs. standard hydrogen electrode with a scan rate of 50 mV s -1 . The ECSA are estimated based on the calculation of the area of the desorption/adsorption peak of a monolayer of hydrogen adsorbed on the Pt catalyst surface and after subtracting the contribution of the double layer charge. Permeation measurement (Linear Sweep voltammetry) consists in applying a potential ramp to an electrochemical cell with a scanning rate close to 1 mV/s. The voltage applied to a single cell ranges from 0.05 V to a maximum of 0.7 V. The resulting current is measured and correspond to the quantity of hydrogen crossing the membrane. The stack in recirculation is characterized each 200 h while the one in AFF only each 500h for technical reason.

Figure 1 :

 1 Figure 1: Polarization curves of the two stacks tested in durability at the beginning and end of the test (80°C, 1.3 bar, SR=1.5/1.8, RH 50/30%). At the end of the test, again the polarization curves of both stacks are superimposed. The decrease in overall performance is almost the same for both configurations. At a current density of 0.42 A cm -2 , the drop in performance reaches 90 mV in recirculation and 89 mV in AFF. However, the voltage drop in open circuit is greater in recirculation than in AFF. This voltage is less than 900 mV at the end of the test in recirculation, whereas it reaches 930 mV on the AFF stack.

Figure 2 :

 2 Figure 2: Degradation rates of individual cell in both stacks and location of the S++® measurement board a) at 0.42 A cm -2 with recirculation, b) at 0.1 A cm -2 with recirculation, c) at 0.42 A cm -2 with AFF and d) at 0.1 A cm -2 with AFF.

Figure 2

 2 Figure 2 shows the degradation rates for each cell in both stacks at 0.42 A cm -2 and 0.1 A cm -2 . The values for the stack aged in recirculation are represented on Figure 2-a and 2-

Figure 2 -

 2 Figure 2-c and 2-d show the degradation rates obtained for the aged AFF stack at the same current densities as the recirculating stack. Figure 2-a shows the degradation rates for a current density of 0.42 A cm -2 while Figure 2-b corresponds to 0.1 A cm -2 . The S++® measurement boards are located in the middle of the stacks. The average degradation rate is 52 µV/h, which is almost similar to that measured in recirculation at the same current density.

Figure 3 :

 3 Figure 3: Permeation current for stacks aged in (a) recirculation mode and (b) AFF mode.

Figure 4

 4 presents for both stacks the current density mappings at the beginning of the test, and the differential current density mappings between the beginning and the end of the test. The current density distributions represented on Figure4-a and 4-b are quite similar. A low current density region is observed at the air inlet, which may be due to a drying effect induced by the automotive conditions.

Figure 4 :

 4 Figure 4: Current density distributions at the beginning of the test for (a) the stack in recirculation and (b) the AFF stack at 0.42 A cm -2 and conditions detailed in Table 1; differential current density mappings in the same conditions calculated between the beginning and the end of the test (c) in recirculation and (d) in AFF The differential current density mappings represented on Figure 4-c and 4-d are calculated

3. 2 . 1 .

 21 Figure 5.

Figure 5 :Figure 5 -

 55 Figure 5: Location of the pinholes or cracks in the MEAs carried out with the infrared camera on (a) the recirculating aged stack and (b) the aged stack in AFF Figure 5-a shows that four MEAs among the eight of the recirculating aged stack are drilled, while Figure 5-b highlights that only three drilled cells among the 70 aged in AFF mode. The pinholes are located close to the hydrogen outlet manifold in all cases. In recirculation, the pinholes in cells 2, 6 and 8 are very close to the hydrogen outlet manifold, while the pinhole in cell 4 is in the center of the last hydrogen channel.

Figure 6 :

 6 Figure 6: Local polarization curve recorded in dry and wet conditions (Stack in AFF)

Figure 7 -

 7 c and 7-d show SEM images obtained on the AFF stack at the Air Inlet zone (AI). The structure of the electrode does not appear to have changed compared to the reference sample. The thickness of the cathode at 14 µm is close to the reference. In the other locations on the MEAs (Air Outlet, Hot and Cold Spot, Hydrogen Inlet and Outlet), the cathodes are always thinner than initially with thicknesses around 11 µm in both recirculation and AFF modes.

Figure 7 :

 7 Figure 7: Cross-sectional SEM images of the air inlet area of an aged MEA and zoom on the membrane area near the cathode to visualize the precipitated Platinum deposition, a) and b) in recirculation, and c) and d) in AFF Finally, a platinum band is clearly visible in the membrane at a few µm from the cathode indicating that a mechanism for dissolving and then precipitating the platinum in the membrane has taken place. This phenomenon occurs at Hydrogen outlet, Air Inlet (Figure 7-c) and Hot Spot (HO, AI, HS) in recirculation mode, while it occurs in AFF mode at Hydrogen Inlet/Outlet, Cold Spot and Air Outlet (HI, HO, CS, AO).

Figure 8 :

 8 Figure 8: Graphical summary of the main degradations deduced from local postmortem analyses schematized on temperature maps a) in recirculation and b) in AFF

Table 1 : Experimental parameters for ageing tests

 1

	Anodic architecture	AFF (0.7Hz) Recirculation
	Cell number	70	8
	Relative humidity anode/cathode (%)	-/30 ± 2	50/30
	Stack stoichiometry H2/air	1.02/1.8	1.5/1.8
	Outlet temperature of the stack	80°C	80°C
	Inlet gas pressure	1.3 bar	1.3 bar

Table 2 : Operating conditions tested on differential cells

 2

		Polarization curve	ECSA measurement
	Condition	Favorable Dry	Wet	Ambient
	Temperature (°C)	80	80	60	25
	Pressure (bar)		1.5		1.5
	Relative Humidity (%)	80/80	50/50 80/80	100
	anode/cathode
	Gas		H2/Air		H2/N2

Finally, samples from a reference MEA and from the six zones of several aged MEAs are analyzed by Scanning Electron Microscopy (SEM), and compared.

Acknowledgements

The authors are grateful to the French Commissariat à l'Energie Atomique (CEA), which supported this work, and to F. Sauzedde, L. Rouillon, L. Jacqmin and S. Germe for their help during the realization of the test program.


Laure Ricard

Charlotte Laurent

Matthias Papo

Sophie Deriaz

Jennifer Catano

Sonia Alamowitch

Gilles Kayem

François Chasset

Claire De Moreuil

Jean Jacques Boffa

Jean Jacques Boffa

Grigorios Gerotziafas

Ismail Elalamy

Marie Bornes

François Maillot

Alexandra Audemard-Verger

Virginie Planche

Eric Ballot

Olivier Fain

Arsène Mekinian
email: arsene.mekinian@aphp.fr

Clinical and prognostic significance of antinuclear antibodies in primary antiphospholipid syndrome: A multicenter retrospective study

Keywords: antiphospholipid syndrome, antiphospholipid antibodies, antinuclear antibodies, outcome

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

1.Introduction

The antiphospholipid syndrome [START_REF] Rodriguez-Pinto | Catastrophic antiphospholipid syndrome (CAPS): Descriptive analysis of 500 patients from the International CAPS Registry[END_REF] is defined by vascular thrombosis and/or pregnancy morbidity associated to persistent antiphospholipid antibodies [START_REF] Rodriguez-Pinto | Catastrophic antiphospholipid syndrome (CAPS): Descriptive analysis of 500 patients from the International CAPS Registry[END_REF]. APS can be primary or in association with other autoimmune diseases (associated APS), especially systemic lupus erythematosus (SLE) [START_REF] Garcia | Diagnosis and Management of the Antiphospholipid Syndrome[END_REF]. Even if APL are frequent during SLE, the progression from primary APS to SLE occurs only in 4 to 10% of patients after a ten-year follow-up [START_REF] Gomez-Puerta | Long-term follow-up in 128 patients with primary antiphospholipid syndrome: do they develop lupus?[END_REF]. Otherwise, antinuclear antibodies (ANA) can be detected in primary APS patients without any clinical SLE and are sometimes called "lupus-like" APS [START_REF] Gomez-Puerta | Long-term follow-up in 128 patients with primary antiphospholipid syndrome: do they develop lupus?[END_REF][START_REF] Cervera | Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients[END_REF][START_REF] Mackworth-Young | Primary antiphospholipid syndrome: a distinct entity?[END_REF]. The presence of ANA could be associated with more pregnancy morbidity and recurrence thrombosis even in the absence of any well-defined associated autoimmune disease [START_REF] Natorska | High prevalence of antinuclear antibodies in patients following venous thromboembolism[END_REF][START_REF] Reimand | Autoantibody studies of female patients with reproductive failure[END_REF][START_REF] Taylor | Presence of autoantibodies in women with unexplained infertility[END_REF]. However, data about the impact of ANA positivity in primary APS remain scarce and clinical phenotype, outcomes and relapses in these patients remain to be determined.

The aims of this study were to evaluate characteristics, outcomes, and management of primary APS patients with positive ANA without any clinical autoimmune disease (ANApositive APS) in comparison with primary APS without ANA (ANA-negative APS) and SLEassociated APS (SLE-APS).

2.Methods

Data about patients with APS between January 2015 and June 2019 from Saint Antoine, Tenon University Hospitals (Paris) and Brest University Hospital, Tours University Hospital were retrospectively collected. Inclusion criteria were APS (Sydney criteria), without any other systemic auto-immune disease than SLE associated systemic disease [START_REF] Miyakis | International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)[END_REF]. APS patients were then classified into three groups: primary APS with positive antinuclear antibodies without any associated autoimmune disease (ANA-positive APS) and which not fulfilled SLE ACR/EULAR classification criteria; primary APS without antinuclear antibodies (ANAnegative APS) and APS patients with SLE (SLE-APS).

Clinical and laboratory data were retrospectively collected as follow: age, sex, cardiovascular risk factors (arterial hypertension, diabetes, overweight (BMI>25) (Table S1 and S2, See the supplementary material associated with this article online) , tobacco use (active smoking was defined as regular cigarette smoking (duration >6 months) , hypercholesterolemia), associated SLE [START_REF] Aringer | European League Against Rheumatism/American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus[END_REF], types (arterial or venous) and site of thrombosis, type of pregnancy adverse obstetrical events, non-criteria manifestations (such as immune thrombocytopenia, multiple sclerosis like disease, Libman Sacks endocarditis, APS nephropathy, etc.). Treatments were recorded as follows: steroids, hydroxychloroquine, anticoagulants, antiplatelets, and duration of each therapy.

APS relapse was defined as a new arterial or venous thrombosis or obstetrical adverse event occurring after the diagnosis of APS.

ANA were detected by indirect immunofluorescence. ANA test was considered to be positive when titers were superior or egal to the 1/80 dilution [START_REF] Aringer | European League Against Rheumatism/American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus[END_REF].

Lupus anticoagulant (LA) was detected using two tests: diluted Russell's viper venom (dRVVT) and diluted activated partial thromboplastin time in accordance to ISTH recommendations. Anticardiolipin (ACL) antibodies IgG/IgM and anti-β2GP1 antibodies IgG/IgM were measured by standard ELISA. ACL positivity was defined as a titer superior to 40 UGPL for IgG and 40 UMPL for IgM and anti-β2GP1 (IgM/IgG) as a value above 99 th percentile. Repeated laboratory testing had to be at least 12 weeks apart. Triple positivity was defined by the association of ACL ( IgG and/or IgM), anti-β2GP1( IgG and/or IgM) and LA test.

Statistical analysis

Variables with quantitative distributions were tested by Shapiro-Wilk test. Data were expressed according to their distributions as medians with ranges, as means with standard deviation and numbers with their frequencies. The Student t-test or the Mann-Whitney test was used to compare quantitative variables according to the distribution. Qualitative variables were compared by the Chi-2 test or the Fisher's exact test as appropriate. Relapses were compared by using log-rank test and expressed with Kaplan-Meier curves. A p value <0.05 was considered as statistically significative. All analyses were performed using JMP-SAS and GraphPad Prism softwares.

Results

Patients' characteristics

One hundred and ninety-five APS patients were included in the study : 40 patients (21%) with ANA-positive APS, 77 patients (39%) with ANA-negative APS and 78 patients (40%) with SLE-APS.

Among the 117 primary APS patients, the median age at APS diagnosis was at 47 years (IQR [36-58]), with 90 females (77%). Primary APS was purely thrombotic (n=66; 56%), purely obstetrical (n=22; 19%) or mixed (n=29; 19%). 49 patients (43%) had arterial thrombosis and 41 patients (40%) had veinous thrombosis. Triple positivity was noted in 37 cases (32%).

Catastrophic antiphospholipid syndrome occurred in three patients with primary APS.

Comparison of ANA-positive APS with ANA-negative APS

ANA-positive APS (n=40) had a median age at 46 years (IQR[36-55]) with female sex in 85% (Table 1). APS thrombotic features were arterial thrombosis in twenty cases (51%) and venous thrombosis in fourteen cases (41%). Adverse obstetrical events were noted in 19 cases (51%), with recurrent miscarriages in five cases, intrauterine deaths in nine cases, preeclampsia and placental insufficiency in seven cases. Non-criteria APS features were noted in nineteen cases (51%): autoimmune cytopenia in eight cases (21%) (thrombocytopenia in six cases and hemolytic anemia in two cases), APS nephropathy in two cases (5%), neurological involvement in five cases (multiple sclerosis like seizure, chorea or migraine), livedo reticularis (n=1) and Libman Sacks endocarditis (n=1). Antiphospholipid antibodies (aPL) were single-positive in seven cases, double-and triple-positive aPL in ten and twentythree (58%) cases, respectively. The most frequent isolated antibody was lupus anticoagulant (n=27; 87%). Median ANA titers were at 1/320e [IQR 1/320-1/640], 39 (98%) patients have ANA titers ≥ 160e and anti-DNA antibodies were > 20 UI in 3 cases (8%).

In comparison to ANA-negative APS, ANA-positive APS patients had significantly more frequent non-criteria features (48% versus 25%; p≤0.01), whereas other APS features and demographic data were not significantly different (Table 1). ANA-positive APS had more anticardiolipin IgG positivity (54% versus 33%; p<0.05), more anti-B2GP1 IgG positivity (54% versus 34%; p<0.05), and more lupus anticoagulant (87% versus 63%, p<0.05), with higher prevalence of triple positivity than ANA-negative APS (59% versus 18%; p<0.001) patients (Table 1).

Whereas there was no difference in frequencies of anticoagulant and antiplatelet therapies, ANA-positive APS patients received significantly more corticosteroids (32% versus 9%; p<0.01) and hydroxychloroquine (38% versus 17%; p≤0.01) than ANA-negative APS. ANApositive APS patients presented more relapses (63% versus 36%, p<0.01) whereas both groups had a similar follow-up (28 months (11-50) versus 18 months (7-39); non-significant).

During the follow-up no patient from ANA-positive APS group declared an associated SLE (ACR/EULAR criteria)(10).

Comparison of ANA-positive primary APS patients with SLE-associated APS

Clinical and laboratory data of primary ANA-positive APS (n=40) and SLE-APS (n=78) are described in Table 2. There was no difference in demographic data, nor in frequency of APS features. ANA-positive APS patients had significantly more frequent anticardiolipin antibodies positivity (54% versus 33%, p<0.05), lupus anticoagulant (87% versus 67%; p≤0.05) and triple-positivity than SLE-APS patients (59% vs 32%; p<0.01).

Whereas there was no difference for the frequencies of antiplatelet and anticoagulant therapies, ANA-positive APS patients received less corticosteroids (32% versus 90%; p<0.001), hydroxychloroquine (38% versus 97%, p<0.001) and other immunomodulatory drugs (28% versus 60%; p≤0.01). There was no difference in the relapse rates between the groups, and ANA-positive APS had a significantly increased mortality (13% vs 2%; p<0.05)(Table 2).

Relapses

Relapse-free survival rate in ANA-positive APS patients was significantly lower than in patients with ANA-negative APS (Hazard Ratio 1.88 [95% CI 1.01-3.48] p<0.05) (Figure 1), but was not significantly different from SLE-APS patients (non-significant). In ANA-positive APS patients, there was no difference of relapse-free survival in patients who received hydroxychloroquine and those without hydroxychloroquine (Hazard Ratio 2.14 [95% CI 0.84-5.45] ; non-significant)(Figure 2).

Discussion

In this study, we raise several important messages about the clinical value of ANA positivity in patients with primary APS. First, the presence of ANA seems to confer a particular clinical phenotype, characterized by more prevalent non-criteria features (notably cytopenia), whereas the usual APS features seem to be similar to those without ANA antibodies. Second, the presence of ANA is associated with increased frequencies of triple-positive APS, which are known to have a particular outcome and increased risk of relapses. Indeed, ANA-positive APS have a significantly increased relapse rates in comparison with ANA-negative APS patients, whereas similar frequencies of antithrombotic therapies have been used in these patients. Last, the use of hydroxychloroquine was not associated in our study with a better relapse-free survival, and studies are necessary to determine the better treatment strategies in ANA-positive APS. Despite increased non-criteria features, ANA and triple positivity, none of ANA-positive APS developed a SLE during the follow-up time.

ANA positivity was associated with more prevalent non-criteria features [START_REF] Cervera | Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients[END_REF][START_REF] Schreiber | Antiphospholipid syndrome[END_REF]. In Cervera et al study, SLE-APS had more livedo and more thrombocytopenia than primary APS [START_REF] Cervera | Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients[END_REF]. Even if the non-criteria are not specifically observed in APS patients, studies suggested that their presence conferred a particular « high risk » profile for future thrombotic events [START_REF] Udry | A high-risk laboratory profile of antiphospholipid antibodies and thrombosis is associated with a large number of extra-criteria manifestations in obstetric antiphospholipid syndrome[END_REF]. Some clinical extra-criteria manifestations like livedo reticularis and migraine have been shown to be associated with increased risk of thrombosis. Non-criteria manifestations could also correlate with APS prognosis and seem to confer a more risk of active persistent disease [START_REF] Toubi | Livedo reticularis as a criterion for antiphospholipid syndrome[END_REF][START_REF] Abreu | The relevance of "non-criteria" clinical manifestations of antiphospholipid syndrome: 14th International Congress on Antiphospholipid Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical Features[END_REF].

The presence of ANA was associated in our study with increased frequencies of triplepositive aPL, which are known to have a particular outcome and increased risk of relapses [START_REF] Pengo | Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study[END_REF][START_REF] Ruffatti | Laboratory and clinical features of pregnant women with antiphospholipid syndrome and neonatal outcome[END_REF][START_REF] Tektonidou | EULAR recommendations for the management of antiphospholipid syndrome in adults[END_REF]. The frequencies of triple positive aPL were more important in our patients than usually reported on APS patients, with 10-30% of triple positive according to the APS subtypes. Triple-positivity in asymptomatic patients is associated with first thrombosis and triple positivity in APS patients is associated with recurrent thrombosis [START_REF] Pengo | Clinical course of high-risk patients diagnosed with antiphospholipid syndrome[END_REF][START_REF] Mustonen | Persistent antiphospholipid antibody (aPL) in asymptomatic carriers as a risk factor for future thrombotic events: a nationwide prospective study[END_REF]. Indeed, our ANA-positive APS had a significantly increased relapse rates in comparison to ANA-negative primary APS patients, whereas similar frequencies of antithrombotic therapies have been used in these patients.

The increased risk of thrombosis relapses questions the value of additional therapies in this subset of APS patients. Several studies [START_REF] Mekinian | The efficacy of hydroxychloroquine for obstetrical outcome in anti-phospholipid syndrome: Data from a European multicenter retrospective study[END_REF][START_REF] Belizna | Hydroxychloroquine as an anti-thrombotic in antiphospholipid syndrome[END_REF] raised the potential benefit of hydroxychloroquine for the obstetrical and thrombotic APS. Studies suggested that hydroxychloroquine could be effective to prevent thrombosis in patients with SLE and persistently positive aPL antibodies [START_REF] Wang | What is the role of hydroxychloroquine in reducing thrombotic risk in patients with antiphospholipid antibodies?[END_REF][START_REF] Tektonidou | ) Livedo reticularis (%) 1/39 (2.5) 1/77 (1.3) Thrombocytopenia/ autoimmune hemolytic anemia (%) 8/39 (20.5) 6/77 (7.8)* Catastrophic APS (%) 2/39 (5.1) 1/77 (1.3) Laboratory data IgG anti-cardiolipin (%) 19/35 (54.3) 21/63 (33.3)* IgG anti-β2GP1 (%) 21/39 (53.8) 23/68 (33.8)* Lupus anticoagulant (%) 27/31[END_REF]. In obstetrical refractory APS, a retrospective European study showed a potential benefit of hydroxychloroquine to reduce the adverse obstetrical outcome [START_REF] Mekinian | The efficacy of hydroxychloroquine for obstetrical outcome in anti-phospholipid syndrome: Data from a European multicenter retrospective study[END_REF]. In thrombotic APS, adjunction of hydroxychloroquine to vitamin K antagonists reduced the recurrence of thrombosis in a small uncontrolled study [START_REF] Belizna | Hydroxychloroquine as an anti-thrombotic in antiphospholipid syndrome[END_REF]. Despite these data, we did not find any benefit of hydroxychloroquine in ANA-positive APS, even the low number of patients could limit the definite conclusion from this preliminary report.

Conflicts of interest and findings: none

Figure 1 Figure 2 .

 12 Figure 1 Comparison of relapse-free survival between ANA-positive APS , ANAnegative APS and SLE-APS patients (ANA-positive APS vs ANA-negative APS p<0.05; SLE-APS vs ANA-positive APS p=0.86)

Table 1 . Primary APS patients with antinuclear antibodies (ANA-positive APS) (n=40) and those without ANA positivity (ANA-negative APS) (n=77).

 1

	Parameters	ANA-positive APS (n=40)	ANA-negative APS (n=77)
		General characteristics

Acknowledgements : none

Online Material. Supplementary data Supplementary data (Table S1-S2) associated with this article can be found in the online version at …