Huian Liu

Jérôme Randon
email: randon@univ-lyon1.fr

Behavior of micro pillar array column in high pressure gas 1 chromatography. 2

Keywords: High pressure gas chromatography Micro pillar array column Intrinsic column efficiency Gases, VOC, gasoline samples

Micro pillar array column with interpillar distance of 2.5 µm for pillars diameter of 5 µm has been introduced in high pressure gas chromatographic systems for online industrial analysis. Separation of gas mixtures have been performed under carrier gas pressure as high as 60 bar using rotating valve for gas injection without sample decompression stage prior to injection. A very low intrinsic height equivalent to a theoretical plate value of 14 µm has been obtained in few seconds. Instead of conventional gas chromatography, carrier gas nature such as helium, argon and carbon dioxide and pressure can be used to tune the selectivity. Liquid hydrocarbon samples have been successfully introduced in the column using a septum based split/splitless injector modified to work up to 40 bar. Separations of VOCs and gasoline samples have been successfully performed.

Introduction

Since 1960's, gas chromatography (GC) plays a significant role in industry owing to its exceptional separation capabilities, and more specifically, online GC has become a very popular option due to several advantages such as automatic sampling, fast response, minimized contamination and security of the staff. However, several industrial processes are performed under very high pressure conditions and require a sample depressurization stage in order to later introduce the sample into the analyzer. Unfortunately, a pressure reduction stage can modify the composition of the sample and then the analytical method trueness is not anymore guaranteed. To avoid the pressure reduction stage, a GC system operated under high carrier gas pressure conditions could be a powerful tool because the sample can then be transferred directly from the process to the analytical column. Such high pressure gas chromatography (HPGC) approach involves two conditions to be fulfilled: the GC instrument must be able to control the carrier gas pressure above 7 bar (conventional gas chromatography instruments usually operate under an inlet pressure lower than 7 bar), and the GC column must have a flow resistance leading to a mobile phase velocity in agreement with the chromatographic requirements. If pressure regulation can be managed with home-made design, column choice is still a challenging task.

The concept of HPGC showed up in 1960s, when Myers and Giddings carried out the separation of light hydrocarbons on packed columns as long as 4000 feet (1200 m) at an inlet pressure up to 2500 psi (172 bar) [START_REF] Myers | High Column Efficiency in Gas Liquid Chromatography at Inlet Pressures to 2500 p.s[END_REF]. Later, several research groups have performed GC on packed capillary columns [START_REF] Myers | High Inlet Pressure Micro Column System for Use in Gas Chromatography[END_REF][START_REF] Takeuchi | New approach to the GC separation of hydrocarbons by using LC-like microcolumns[END_REF][START_REF] Berezkin | Chromatographic characteristics of capillary packed columns with increased pressure drop[END_REF][START_REF] Lauer | Application of high-pressure gas chromatography with columns packed with small particles[END_REF][START_REF] Huber | Reduction of theoretical plate height in gas chromatography by increase of pressure and decrease of particle size[END_REF][START_REF] Takeuchi | Gc with Lc-Like Packed Capillary Columns[END_REF][START_REF] Jonker | Improvement of speed of separation in packed column gas chromatography[END_REF][START_REF] Peichang | High-performance gas chromatographic columns packed with micro-particles[END_REF] with two distinct goals: decrease analysis time or increase efficiency. In 1970's, monolithic columns appeared in the field of the GC [START_REF] Schnecko | Foam filled columns in gas chromatography[END_REF][START_REF] Hileman | In situ preparation and evaluation of open pore polyurethane chromatographic columns[END_REF] but did not attract too much attention because open capillary columns have emerged at the same time leading to highly efficient separations which can be performed with low carrier gas pressure. However, for HPGC, monolithic columns still have specific advantages over packed columns: simplicity of preparation, good mechanical integrity, and higher permeability [START_REF] Kozin | The Influence of the Natures of the Carrier Gas and the Stationary Phase on the Separating Properties of Monolithic Capillary Columns in Gas Adsorption Chromatography[END_REF]. Column length can then be increased and so, for the same Height Equivalent to a Theoretical Plate value, monolithic column was able to produce higher efficiency. A.A. Kurganov and his team has deeply investigated silica monolithic column in gas chromatography [START_REF] Korolev | Fast separation of light hydrocarbons by gas chromatography on monolithic capillary columns based on silica gel[END_REF][START_REF] Korolev | High productivity chromatographic separations on monolithic capillary columns[END_REF][START_REF] Korolev | High-performance high-pressure gas chromatography on monolithic capillary columns[END_REF][START_REF] Korolev | Polar monolithic capillary columns: Analysis of light hydrocarbons[END_REF]. They studied the effects of several parameters like the carrier gas nature and the nature of stationary phase, but only few reviews have been dedicated later to monolithic columns in GC [START_REF] Kurganov | Monolithic column in gas chromatography[END_REF][START_REF] Svec | Less common applications of monoliths: III. Gas chromatography[END_REF][START_REF] Zajickova | Applications of monolithic columns in gas chromatography and supercritical fluid chromatography[END_REF]. Only recently, Maniquet et al. have investigated the behavior of short silica and vinyl silica monolithic column in HPGC for light hydrocarbon separations [START_REF] Maniquet | Behavior of short silica monolithic columns in high pressure gas chromatography[END_REF][START_REF] Maniquet | Behavior of macroporous vinyl silica and silica monolithic columns in high pressure gas chromatography[END_REF].

Since the early work of Terry et al. in 1979 [22], silicon based technology leading to microfabricated open tubular columns have been applied to GC, and microchip gas chromatography review has been recently published [START_REF] Ghosha | Microchip gas chromatography columns, interfacing and performance[END_REF]. In 2009, a new type of chromatographic design based on micropillar array has been proposed by Nakai et al. [START_REF] Nakai | Micro-fabricated semi-packed column for gas chromatography by using functionalized parylene as a stationary phase[END_REF] and Ali et al. [START_REF] Ali | MEMS-based semi-packed gas chromatography columns[END_REF]. These columns, also called semi-packed columns, have been used with conventional injection systems (for gas and liquid) and with such strategy the interpillar distance was in the range of 50 µm in order to be able to use electronic pressure controller working below 7 bar. Recently, a commercial silicon pillar array column grafted with C18 groups, µPAC column [START_REF] De Malsche | Capillary liquid chromatography separations using non-porous pillar array columns[END_REF][START_REF]Pharmafluidics[END_REF], has been introduced for nanoscale liquid chromatography and used for separation of peptides in complex samples [START_REF] Sandra | Evaluation of Micro-Pillar Array Columns (µPAC) combined with high resolution mass spectrometry for lipidomics[END_REF][START_REF] Tóth | Pillar array columns for peptide separations in nanoscale reversed-phase chromatography[END_REF][START_REF] Nys | Multidimensional performance assessment of micro pillar array column chromatography combined to ion mobility-mass spectrometry for proteome research[END_REF]. µPAC separation columns are formed by etching away the interstitial volumes out of a solid silicon wafer, leaving an array of pillars. The resulting channels are folded onto a small footprint by concatenating bed segments with optimized flow distributors that limit peak dispersion (fig. 1).

The pillar bed is of a width of 315 μm and a height of 18 μm, where pillars diameter of 5 μm are successively arrayed with an inter-pillar distance of 2.5 µm. Such an organization creates an internal column volume of 4 μL and the column was designed to be able to bear an inlet pressure up to 360 bar [31]. If this type of column has been developed for nanoLC, up to now no attempt has been done to use them in GC probably because, with an interpillar distance of 2.5 μm, the inlet carrier gas pressure to be used has to be higher than 10 bar. This paper describes a first study of μPAC in HPGC system with two high pressure injection adaptations for both gas and liquid analysis.

Experimental

2,1 High pressure gas chromatography system for gas and liquid injection Because conventional electronic pressure controllers for gas chromatography only allow 7 bar of carrier gas pressure, GC chromatographs from Perkin-Elmer (Autosystem XL and Clarus 590) have been modified to high pressure configuration for gas and liquid injection.

For gas injection (fig. 2-a), the injection system has been fully described in [START_REF] Maniquet | Behavior of short silica monolithic columns in high pressure gas chromatography[END_REF].

The gas sample was brought from a sample bottle using a four port Vici valve with a maximum carrier gas pressure at 69 bar (1000 psi) equipped with an internal sample loop of 5 µL. The carrier gas pressure was set by a pressure reducing regulator (single or double stage) linked to a manometer to monitor the applied pressure. A T-connection with a split capillary ( = 75μm, L = 15 cm) was introduced to control the injected sample amount and so to avoid column overload.

For liquid injection (fig. 2-b), the sample was injected with an autosampler using a syringe (Vsyr=0.5 µL; Vinj=0.1 µL) to a split/splitless GC injector on an Autosystem XL instrument. However, because of the limited carrier gas pressure regulation systems of such conventional GC instrument, the carrier gas line and the split line of the injector have been disassembled. A high pressure carrier gas line with pressure regulation was installed and a fused silica capillary was connected directly to the split line of injector. Due to commercial fittings of μPAC column, capillaries have been added at both ends to connect the column to the split injector and to the FID (20 μm I.D. and 20 cm length). With such configuration, a carrier gas pressure up to 40 bar can be used with a commercial septum based split/splitless injector.

2,2 Materials and reagents

The µPAC column was from PharmaFluidics (Gent, Belgium). An ODS-4 packed column was purchased from Shimadzu. Fused silica capillaries with 10 μm, 20 μm, and 75 μm I.D., 375 µm O.D. were purchased from Cluzeau (France). CO2, He and Ar cylinders were from Air Liquide (Paris, France). The sample bottle coded ME was from Air Liquide (Paris, France) and contained two alkanes at high concentration: methane (15 mol%) and ethane (15 mol%) with nitrogen as balance gas. Sample bottle 8lh was from Air Liquide (Paris, France) and contained 8 hydrocarbon compounds with low concentration: methane (0.1 mol%), ethane (0.1 mol%), ethylene (0.1 mol%), acetylene (0.1 mol%), propane (0.1 mol%), cyclopropane (0.1 mol%), n-butane (0.1 mol%), 1-butene (0.1 mol%), with nitrogen as balance gas. Sample bottle MEP7lh from Air Liquide (Paris, France) contains 10 compounds: methane (77.6 mol%), ethane (4 mol%), ethylene (0.05%), propane (15 mol%), propylene (0.1 mol%), n-butane (0.5 mol%), isobutene (0.5 mol%), pentane (0.1 mol%), isopentane (0.1 mol%) and hexane (0.05 mol%). The pressure for the three sample cylinders was in the range from 1 to 11 bar. The organic volatile compounds including hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane and tetradecane were from Sigma-Aldrich (St Quentin Falvier, France). A real gasoline #98 sample was collected at a local petrol station (Villeurbanne, France).

Results and Discussion

3,1 Gas separation μPAC

High Pressure Gas Chromatography (carrier gas pressure 40 bar) has been performed on micro-pillar (5 µm diameter, 2,5 µm interpillar distance) array column (500 mm long, 315µm width, 18 µm height) from Pharmafluidics connected to a gas injection valve. Two light hydrocarbon mixtures form sample cylinders (sample ME 6 bar, sample 8lh 11 bar) were directly injected into the µPAC column without any sample decompression stage, using carbon dioxide as carrier gas.

The mixture of methane (15 mol%) and ethane (15 mol%) (ME sample) was separated in 10 seconds (fig. 3-a) and the C1-C4 sample (fig. 3-b) was analyzed within 20 seconds at a mean carrier gas linear velocity of 5.2 cm.s -1 .

The column permeability was in the range of 10 -13 m 2 and require such high carrier gas pressure to work in the carrier gas velocity range for gas chromatography applications. Quantitatively, in terms of peak intensity, the methane (C1) peak for ME was expected to be around 82 times higher than methane peak for 8hl considering the injected amount in both conditions (ME: 15 mol%, 6 bar, 8lh: 0.1 mol% ,11 bar). The observed peak area ratio was equal to 83 for methane (ME 16.2 mV.s , 8lh 0.195 mV.s) in agreement with the previous assumption. Regarding the separation, µPAC C18 stationary phase was able to separate C3 isomers as well as saturated and unsaturated C4 alkanes at room temperature (on other silica based columns, cyclopropane was eluted after propane, and butene was eluted after butane [START_REF] Maniquet | Behavior of macroporous vinyl silica and silica monolithic columns in high pressure gas chromatography[END_REF]). C2 isomers are still coeluted at 25 °C and a lower temperature should be required to separate them. All these first results demonstrated the µPAC ability for light organic compounds separation with a modified gas injection HPGC system. (1

)
where L is the column length. , . is the observed total volume variance which includes the extra-column volume variance , and the contribution of the column , to the recorded band width according to:

, = , + , = , + ! " (2)
where VR is the retention volume and Nintrinsic is the intrinsic column efficiency.

In equation ( 1), the retention volume VR is given by:

# $ = # + # % . &1 + () (3)
where V0 is the column void volume, Vext is the extra-column volume (0.4 μL)

which is negligible compared to the parameter V0 • &1 + () for retention factors higher than 1 with μPAC column volume V0 equal to 4 μL.

From Eq. ( 2) and Eq. ( 1), a relationship between observed Hobs value and Hintrinsic can be obtained.

&*), = • ,+, + - ! " = • ,+, . • / &/0*) + 12 312 1 (4)
So, as long as is significant compared to 452 , the observed HETP values for a set of several injected compounds in a single run depend on retention factor k. The plot of observed HETP (Hobs) values versus 1/(1+k) 2 is expected to be linear. The intercept of the linear line is then an estimation of the intrinsic HETP value of the column for the applied inlet pressure.

As an illustration (fig. 4), the sample mixture MEP7lh has been used to estimate Hintrinsic at two different mobile phase velocities corresponding to inlet pressure of 28 bar and 60 bar.

The slope of each curve is related to the external band broadening at the corresponding velocity and so a higher mobile phase velocity brings a higher . Moreover, 12 312 1 can be determined for different carrier gas velocities and the corresponding Van Deemter curve of the intrinsic efficiency of μPAC has been plotted (fig 5). The Hintrinsic, value reached a minimum of 14 μm within the pressure limit (60 bar) applied in the HPGC system and this value was obtained for mobile phase velocity higher than 5 cm.s -1 . Extra column band broadening is still a significant issue which decreases observed efficiency. Detector was setup to avoid band broadening, connection contribution could probably be reduced but not with the commercialized design used in these experiments.

3,2 Liquid injection on μPAC

A septum based split/splitless injector has been modified in order to perform liquid injection using the autosampler syringe with inlet pressure as high as 40 bar. A mixture of alkanes (C6 to C14) in solution in CH2Cl2 was injected under isothermal condition with oven temperature from 100 to 160 °C using argon as carrier gas and as an illustration the chromatogram obtained at 130 ℃ is shown in figure 6.

For compounds from C8 to C14, the logarithm of retention factor k has been plotted as function of 1/T. For each compound, a linear trend was observed which is in agreement with ideal thermodynamic equilibrium relationships (temperature independence of enthalpy and entropy changes). As expected, the effect of temperature on the retention of heavier compounds was more significant than on lighter compounds as observed by the slope of the linear relations log(k)=f(1/T) reported on table 1.

The enthalpy change depends on the number of carbon atoms in the carbon chain and every -7 -group contributes to the enthalpy (the slope increased by 200 for each -7 -group added in the molecule). Such model can be used to predict the retention factor of different alkanes at different temperature on μPAC.

3,2,1, Effect of carrier gas nature on retention of μPAC column

The effect of carrier gas nature (helium, argon and carbon dioxide) was studied at temperature of 100 ℃ and 150 °C. Using helium as carrier gas, the inlet pressure did not have any influence on compound retention factors, so helium was considered as a reference gas and relative retention factors were used for argon and carbon dioxide in figure 7.

For both carrier gases, Ar and CO2, regardless of the temperature, higher pressure always led to a lower retention factor. Such behavior could be linked to adsorption of carrier gas molecules on stationary phase, or condensation in its porous structure, which was more significant under higher pressure conditions leading to a decrease of the number of accessible sites for the analytes. Similarly, for a selected carrier gas, carrier gas molecules were more adsorbed onto the stationary phase at lower temperature, thus a lower retention factor was observed at lower temperature for both argon and carbon dioxide.

Separation of gasoline on μPAC

Gasoline 98 is a light petroleum mixture which contains a large set of light hydrocarbons including paraffins, iso-paraffins, cycloalkanes, and aromatics from C4 to C12. Such real sample from a gas station has been injected under temperature programming conditions: 50 °C -175 °C, 25 °C/min at inlet pressure of 40 bar for two different carrier gas argon and carbon dioxide (fig. 8).

The separation was performed faster with CO2 (3.75 min) compared to Ar (around 5 min) and a selectivity effect related to carrier gas nature was observed. The three main compounds between octane and nonane which were co-eluted with Ar as carrier gas, are fully separated when CO2 was used.

Conversely, co-elutions happened less seriously for lighter compounds (peaks around hexane) with Ar than CO2. Such behavior could be linked to the carrier gas absorption onto the stationary phase, different for both argon and carbon dioxide, reducing the accessible sites on stationary phase for analytes but to different extent.

Conclusion

µPAC column has been used in high pressure gas chromatographic systems.

Compared to other silica based columns investigated on HPGC, µPAC column showed very good performance in terms of column efficiency and very fast separation can be carried out: HETP value as low as 14 µm has been observed with argon as carrier gas, with a hold-up time of only 5 second. Not only for gas injection, liquid injection on modified HPGC system with μPAC column has also been performed. Analysis of complex real samples has demonstrated that such approach using µPAC column is a promising tool for industrial gas chromatography. In addition, μPAC column is based on 50 cm separation bed arranged into several parallel channels on a micro-chip in a 3 mm thin pad, which gives the column an excellent compatibility to a future miniaturized on-line HPGC system.

32. Gritti, F., Guiochon, G., 2014. Accurate measurements of the true column efficiency and of the instrument band broadening contributions in the presence of a chromatographic column. Journal of Chromatography A 1327, 49-56. https://doi.org/10.1016/j.chroma.2013.12.003 Table Table 1: Slope and intercept of linear relationships for alkanes from C8 to C14.. 1

Fig. 4: Apparent HETPs vs 1/(1+k2) 2 of µPAC column for light hydrocarbon compounds at 28 bar (orange) and 60 bar (blue) using argon as carrier gas, room temperature.

1 1

3. 2

 2 Evaluation of µPAC efficiency in HPGC In any chromatographic system, when the column size is reduced, the relative contribution of extra-column band broadening is consequently enlarged, and the observed efficiency will no longer display the true performance of the column. For an estimation of true column efficiency of μPAC, intrinsic column efficiency and instrumental band broadening contributions were evaluated according to Gritti et al. [32] after adaptation to GC. The theorical basis for calculation of intrinsic HETP is based on observed HETP values for several compounds with retention factors in the range from 1 to 10. For each compound: = • , .

Fig 1 .

 1 Fig 1. The structure of the micro-pillar array column. Left: top view of two parallel separation channels (315 µm wide) interconnected with flow distributor structures. Middle: transverse section with 5 µm diameter cylindrical pillars. Right: 300 nm porous shell layer into a 5 µm pillar. Reprinted from Koen Sandra's paper http://www.chromatographyonline.com/evaluation-micro-pillar-array-columns-pac-combined-high-res olution-mass-spectrometry-lipidomics?pageID=3 Authorization necessary

Fig. 2 Fig 3 :Fig. 4 :

 234 Fig.2 Configuration of Injection and pressure regulation for High Pressure Gas Chromatography.

Fig. 5

 5 Fig.5 Van Deemter curve of intrinsic column efficiency for µPAC column for light hydrocarbons separation carried out at room temperature with argon as carrier gas.

Fig. 6 :

 6 Fig.6: Chromatogram of a separation of a mixture of alkanes from C6 to C14 in CH2Cl2, temperature at 130 ℃, inlet pressure 30 bar, argon as carrier gas.

Fig 7 .Figure 8 :

 78 Fig 7. Relative retention factor of decane with argon and carbon dioxide as carrier gases as a function of carrier gas pressure, oven temperature of 100 °C and 150 °C. Helium was used as a reference (retention factor did not change with helium pressure)

Fig 1 .

 1 Fig 1. The structure of the micro-pillar array column. Left: top view of two parallel separation channels (315 µm wide) interconnected with flow distributor structures. Middle: transverse section with 5 µm diameter cylindrical pillars. Right: 300 nm porous shell layer into a 5 µm pillar. Reprinted from Koen Sandra's paper http://www.chromatographyonline.com/evaluation-micro-pillar-array-columns-pac-combined-high-resolution-mass-spectrometry -lipidomics?pageID=3 Authorization necessary

Figure 2 ( 1 Figure 3 Fig 3 :

 2133 Figure 2

Fig. 5 1 Figure 6 Fig. 6 :

 5166 Fig.5 Van Deemter curve of intrinsic column efficiency for µPAC column for light hydrocarbons separation carried out at room temperature with argon as carrier gas.

Fig 7 .

 7 Fig 7. Relative retention factor of decane with argon and carbon dioxide as carrier gases as a function of carrier gas pressure, oven temperature of 100 °C and 150 °C. Helium was used as a reference (retention factor did not change with helium pressure)

Table 1

Table 1: Slope and intercept of linear relationships for alkanes from C8 to C14..


Elise Guillaume

Maria Zacharopoulou

Brice Reynolds

Luca Aresu

Lhorane Lobjois

Céline Bleuart

Nathalie Bourgès-Abella

Maxence Delverdier

Marie-Noëlle Lucas

Rachel Lavoué

Nathalie Bourges-Abella

Marie- Noëlle Lucas

Nicolas Gaide
email: nicolas.gaide@envt.fr

Additional Value of Second Harmonic Generation Microscopy in the Diagnosis of Feline Collagen Type III Glomerulopathy

Keywords: feline, kidney disease, second harmonic generation microscopy, transmission

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

electron microscopy

Collagen type III glomerulopathy (Col3GP), also called collagenofibrotic glomerulopathy, is a rare renal disease that has been described in humans, dogs, pigs, non-human primates and two cats [START_REF] Shirota | Renal glomerular fibrosis in two pigs[END_REF][START_REF] Nakamura | Renal glomerular fibrosis in a cat[END_REF][START_REF] Adachi | Collagenofibrotic glomerulonephropathy in a Cynomolgus macaque (Macaca fascicularis)[END_REF][START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF][START_REF] Davis | Diagnosis of collagen type III glomerulopathy using Picrosirius red and PASH/Masson's trichrome stain[END_REF][START_REF] Hilbe | Collagenofibrotic glomerulopathy in a young cat[END_REF]. In dogs, no sex or breed predilection has been established, although geographical distribution and familial onset are suggestive of an autosomal recessive inheritance [START_REF] Rørtveit | A canine autosomal recessive model of collagen type III glomerulopathy[END_REF]. Histological hallmarks of Col3GP include non-congophilic fibrillary glomerular deposition with positive staining by aniline blue of Masson's trichrome (MT) stain, positive immunolabelling with anti-type III collagen antibodies and observation of the atypical fibrillary material by transmission electron microscopy (TEM). More recently, additional techniques, such as staining with a combination of periodic acid-Schiff (PAS) and MT, and Picrosirius red (PR), have been proposed to complete the histological diagnosis of this condition [START_REF] Brodsky | When one plus one equals more than two -a novel stain for renal biopsies is a combination of two classical stains[END_REF][START_REF] Davis | Diagnosis of collagen type III glomerulopathy using Picrosirius red and PASH/Masson's trichrome stain[END_REF].

Using a combination of different methods, including histopathology, immunohistochemistry (IHC), TEM and second harmonic generation (SHG) microscopy, this investigation provides a complete description of a case of feline Col3GP. Urinalysis revealed inappropriately low specific gravity (1.024), suggesting renal azotaemia, and severe proteinuria with a high urine protein-to-creatinine ratio (4.2 [RI <0.4]) with inactive sediment and negative uroculture. On abdominal ultrasound examination, the kidneys were mildly and bilaterally hypertrophic and hyperechoic with irregular bumpy contours.

Overall, these clinical data were suggestive of an end-stage glomerular kidney disease. The cat received intravenous (IV) fluid therapy consisting of Ringer's lactate solution (2 ml/kg/h) with potassium supplementation. Antimicrobial (amoxicillin and clavulanic acid, 60 mg IV, three times a day) was initiated while waiting for the result of uroculture. The cat progressively gained weight (+500 g) and, on the third day of hospitalization, developed peripheral pitting oedema and concomitant systemic hypertension (210 mmHg). Fundus examination revealed retinal oedema and vascular tortuosity. Antihypertensive medication (amlodipine, 0.625 mg once a day) was added and fluid therapy was decreased. Given the lack of clinical and biological improvement, humane euthanasia was performed on the sixth day of hospitalization.

A complete post-mortem examination, performed a few hours after death, revealed poor body condition and generalized mucosal pallor. Both kidneys were enlarged, pale, firm with a cortical granular appearance (Fig. 1). Other lesions included subcutaneous abdominal and limb oedema, 600 ml of peritoneal transudate and acute diffuse pulmonary oedema.

Subacute bilateral symmetrical ventrolateral ulcerative glossitis and cheilitis, and multifocal moderate subacute peripancreatic steatonecrosis were also observed. The pathological findings were consistent with severe chronic bilateral glomerulopathy associated with nephrotic and uraemic syndromes. Tissues samples of selected organs were collected at necropsy, fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 3 µm and stained with haematoxylin and eosin (HE). In addition, kidney sections were stained with PAS, MT, Congo red (CR), PR and a combination of PAS and MT [START_REF] Brodsky | When one plus one equals more than two -a novel stain for renal biopsies is a combination of two classical stains[END_REF][START_REF] Davis | Diagnosis of collagen type III glomerulopathy using Picrosirius red and PASH/Masson's trichrome stain[END_REF].

Histopathological examination revealed bilateral diffuse global enlargement of glomeruli due to massive deposition of an extracellular pale, eosinophilic, amorphous to delicately fibrillary material (Fig. 2A). This material was deposited within the mesangium and capillary loops, and appeared occasionally associated with mild mesangial hypercellularity, multifocal hyperplasia of podocytes, synechiae and periglomerular fibrosis. Abundant proteinaceous material was found in ectatic tubules, with flattened epithelium, suggesting proteinuria, as well as multifocal moderate tubular mineralization, moderate interstitial fibrosis and mild multifocal lymphoplasmacytic infiltration. Multifocal mild lesions of acute tubular injury were also found, including tubular epithelial sloughing, simplification and necrosis, with occasional granular cast formation. The overall lesions were consistent with acute kidney injury following chronic kidney disease.

The biochemical nature of the glomerular deposition was first investigated with special stains, resulting in non-congophilic, deep blue and fibrillary staining with MT, and negative to weakly positive reactivity with PAS. The combined PAS/MT stain enabled differentiation of the blue material of the mesangium from purple-stained glomerular basement membranes (Fig. 2B). The material was bright red with PR stain (Fig. 2C) and had a yellow-red and green birefringence under polarized light (Fig. 2D).

To confirm the presence of type III collagen, immunohistochemistry was performed on kidney sections using rabbit polyclonal antibody against type III collagen (1:100, overnight incubation at 4°C; NB600-594SS, Novus Biologicals, Centennial, Colorado, USA), enzymatic antigen retrieval (pronase 0.05% 10 min at 37°C; Sigma-Aldrich, St. Louis, Missouri, USA ), EnVision+ Dual Link System-HRP (Agilent, Santa Clara, California, USA) and 3,3'diaminobenzidine chromogenic reaction (Diagomics, Blagnac, France) according to manufacturer's recommendations. Negative controls included a section incubated without specific primary antibody and a section incubated with normal rabbit serum. Human skin was used as positive control tissue. The homogeneous glomerular material stained positively with antibody against type III collagen (Fig. 2E).

In order to better characterize the presence of collagen within the glomeruli, we used two different direct microscopic methods: ultrastructural examination and SHG microscopy.

The SHG method is based on a second-order, non-linear optical process produced by noncentrosymmetric structures such as fibrillary collagen [START_REF] Chen | Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure[END_REF][START_REF] Bueno | Second harmonic generation microscopy: a tool for quantitative analysis of tissues[END_REF]. SHG microscopy has emerged as a label-free method for detecting and imaging fibrillary collagen in normal and pathological tissues (Vuillemin et al, 2016). For our case, paraffin-embedded tissues were sectioned at 10 µm, rehydrated, stored in phosphate buffered saline solution and then submitted to SHG analysis using an Ultima 2Pplus biphotonic microscope (Bruker Corporation, Billerica, Massachusetts, USA). A positive heterogeneous SHG signal was observed within the interstitium and the glomerular mesangium, the latter being slightly weaker and more diffuse (Fig. 2F). To compare the SHG signal, sections from a case of feline normal kidney, as well as cases of canine renal amyloidosis and feline end-stage kidney, were concurrently analysed. No glomerular SHG signal was seen in the normal tissue nor in the dog with amyloidosis. However, a SHG signal was observed in the cat with end-stage kidney, specifically within the fibrotic lesions of renal interstitium, capsule and synechiae of obsolescent atrophic glomeruli (Supplementary Fig. 1). For TEM the paraffin-embedded renal tissue was deparaffinized in xylene, stained in a 0.01% toluidine blue/absolute ethanol solution, infiltrated in a propylene oxide/resin mixture and embedded in epoxy resin. Ultrathin sections were double-stained with uranyl acetate and lead citrate and observed under a transmission electron microscope. By TEM, randomly arranged amorphous to fibrillary material was observed in the mesangial and subendothelial areas, with expansion of the mesangium and subendothelial space. On longitudinal sections, the fibrils had a periodicity characteristic of collagen fibrils. Podocyte foot process effacement was occasionally observed (Fig. 3).

This report provides a complete description of a case of Col3GP in a cat based on a combination of histopathology, IHC, TEM and SHG microscopy. Col3GP is a rare disease in humans, with less than 50 reported cases worldwide, mostly originating from Asian countries, especially in Japan, which possibly suggests an aetiological role for ethnic or genetic factors [START_REF] Alchi | Collagenofibrotic glomerulopathy: clinicopathologic overview of a rare glomerular disease[END_REF][START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF]. This disease can be divided into two different types depending on the age of onset: an adult-onset type and a paediatric type [START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF], with most reported paediatric cases originating from France [START_REF] Gubler | Collagen type III glomerulopathy: a new type of hereditary nephropathy[END_REF]. In dogs, the disease is clinically and morphologically similar to the paediatric human type. Almost all affected dogs developed signs of chronic kidney disease within a year of birth and, in some cases, several puppies originating from the same litter developed signs early in life, suggesting a hereditary basis [START_REF] Rørtveit | A clinical study of canine collagen type III glomerulopathy[END_REF][START_REF] Rørtveit | Glomerular collagen V codeposition and hepatic perisinusoidal collagen III accumulation in canine collagen type III glomerulopathy[END_REF][START_REF] Davis | Diagnosis of collagen type III glomerulopathy using Picrosirius red and PASH/Masson's trichrome stain[END_REF]. In cats, only two cases have been documented: a 1-year-old neutered female Japanese cat [START_REF] Nakamura | Renal glomerular fibrosis in a cat[END_REF] and an 11-month-old neutered male European Shorthair cat with concurrent angiostrongylosis [START_REF] Hilbe | Collagenofibrotic glomerulopathy in a young cat[END_REF]. No information regarding siblings and ancestors was available for the present case; however, the paediatric onset and the absence of major concurrent diseases could presumably suggest a genetic basis, similar to canine cases.

The morphological hallmark of Col3GP is the accumulation of atypical type III collagen within mesangial and subendothelial areas. Definitive diagnosis relies on TEM [START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF] or immunohistochemical detection of type III collagen deposition [START_REF] Rørtveit | Glomerular collagen V codeposition and hepatic perisinusoidal collagen III accumulation in canine collagen type III glomerulopathy[END_REF]. In our case, renal amyloidosis, which is the main differential diagnosis, was excluded through CR staining, while collagen deposition was confirmed on the basis of the results of special staining and the characteristic appearance of fibres on TEM and IHC. In addition to those methods, we report the possibility of directly confirming atypical collagen deposition through biphotonic microscopy using SHG. When available, this technique is label-free, relatively fast and only requires rehydrated unstained slides to directly image fibrillary collagen. In our case, SHG signal was detected in the expanded mesangium and was not detected in any glomeruli of control cases. Interestingly, the signal intensity was slightly weaker and more diffuse compared with that found in the intertubular interstitium.

Considering that type I collagen is known to produce a stronger SHG signal than type III collagen and that, in our case, glomerular collagen deposition had the morphological appearance of a combination of amorphous and fibrillary collagen, this could presumably explain the difference in terms of intensity and distribution pattern compared with the tubular interstitium [START_REF] Cox | Second-harmonic imaging of collagen[END_REF].

The pathogenesis of Col3GP is unknown. Two hypotheses are currently being considered concerning the origin of the type III collagen: local synthesis in mesangial cells that are able to produce both type III and type IV collagen fibres, particularly in the case of myofibroblastic transformation indicated by a α-smooth muscle actin (α-SMA)-positive phenotype [START_REF] Naruse | Mesangial cell activation in the collagenofibrotic glomerulonephropathy. Case report and review of the literature[END_REF][START_REF] Alchi | Collagenofibrotic glomerulopathy: clinicopathologic overview of a rare glomerular disease[END_REF], or a systemic origin [START_REF] Alchi | Collagenofibrotic glomerulopathy: clinicopathologic overview of a rare glomerular disease[END_REF][START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF]. Evidence of local collagen type III synthesis by mesangial cells has been described in affected dogs using in-situ hybridization analysis [START_REF] Rørtveit | A canine autosomal recessive model of collagen type III glomerulopathy[END_REF]. Another possible mechanism is the occurrence of systemic alterations in collagen metabolism, leading to the release of type III collagen precursors into the circulation; this hypothesis is supported by the fact that, in humans, elevated serum concentrations of amino-terminal pro-collagen type III peptide (PIIINP), a type III collagen precursor, could be considered as a suitable marker for this disease in the appropriate clinicopathological context [START_REF] Alchi | Collagenofibrotic glomerulopathy: clinicopathologic overview of a rare glomerular disease[END_REF][START_REF] Duggal | Collagenofibrotic glomerulopathy: a review[END_REF]. Only one study has been conducted (in seven affected puppies) and it showed significantly higher serum PIIINP concentrations in these animals compared with healthy puppies. However, those results require confirmation through further investigations, considering the use of a human test kit that has not been validated in domestic animals, the fact that serum PIIINP concentration is naturally higher in young animals, and the much more subtle increase in PIIINP than in affected humans [START_REF] Rørtveit | A clinical study of canine collagen type III glomerulopathy[END_REF].

In our case, α-SMA IHC did not reveal any substantial increase in intensity or cell density of α-SMA-positive cells within affected glomeruli (data not shown), but the accumulated collagen fibrils were located both in the mesangium and the subendothelial space, as described by [START_REF] Rørtveit | A canine autosomal recessive model of collagen type III glomerulopathy[END_REF]. Therefore, a definitive conclusion about the origin of the fibrils cannot be made in the present case.

CRediT Statement

Legends to Figures

  A 1.5-year-old neutered female Domestic Shorthair cat was admitted to the Teaching Hospital of the Veterinary School of Toulouse with a vague complaint of listlessness. The animal was calm and responsive but in poor body condition (body condition score of 3/9). Physical examination revealed mild hypothermia (37.4°C), pale mucous membranes and a grade 3/6 left sternal systolic murmur. The cat was normotensive at admission, with a mean systolic blood pressure of 140 mmHg, obtained with the Doppler technique. Haematological analysis revealed: moderate normocytic, normochromic, non-regenerative anaemia (haematocrit 20.5%, reference interval [RI] 29-48; haemoglobin concentration 5.8 g/dl, RI 9.3-15.9); corrected reticulocyte index 0.18%; severe azotaemia (913 µmol/l [RI 80-229]); hyperphosphataemia (9.8 mmol/l [RI 0.8-3.0]); ionized hypocalcaemia (0.7 mmol/l [RI 1.1-1.4]); mild hypokalaemia (3.3 mmol/l [RI 3.5-5.1]); normal total CO2 (18 mmol/l [RI 16-25]); moderate hypoproteinaemia (50 g/l [RI 55-71]); and hypoalbuminaemia (21 g/l [RI 27-9]).

Fig. 1 .

 1 Fig. 1. Collagen type III glomerulopathy, kidneys, cat. Diffuse pallor, swelling and granularity on cortical surface and a sagittal section.

Fig. 2 .

 2 Fig. 2. Collagen type III glomerulopathy, kidney, cat. (A) Glomeruli markedly expanded by pale eosinophilic, homogeneous to fibrillary material. HE. (B) Material stains deep blue with MT and is demarcated from basement membranes (purple). Combined PAS/MT stain. (C) Material stains bright red with PR. (D) Material has orange-yellow and green birefringence under polarized light. (E) Immunohistochemical labelling of type III collagen in mesangium. IHC. (F) Material (red) and endogenous two-photon excited fluorescence (dark green).Second harmonic generation microscopic imaging. Bar, 50 µm.

Fig. 3 .

 3 Fig. 3. Collagen type III glomerulopathy, kidney, cat. Deposition of amorphous to fibrillary electronlucent material, composed of bundles of atypical collagen fibres ( ) within mesangium in upper part of electron micrograph and in subendothelial space. Narrowed capillary lumen (L). Partially fused podocyte foot processes (arrowheads). Inset: details of collagen fibrils with distinctive periodicity. Bar, 7 µm.

Scientific Reports, 6, 1-13.

All authors reviewed and approved the manuscript.

Conflict of Interest Statement

The authors declared no potential conflict of interest with respect to the research, authorship or publication of this article.


Kursat Erdogan

François Lefebvre

Fred Xavier

Sybille Facca

Philippe Liverneaux

of digital flexor tendon sheath phlegmon: Exsanguination by mechanical compression versus simple elevation [Risque de dissémination septique au cours de la prise en charge chirurgicale des phlegmons de la gaine des tendons fléchisseurs: Exsanguination par compression mécanique versus surélévation simple]

Keywords: Infection, sepsis, Phlegmon, Flexor tendon, Tourniquet Mots-clés : Infection, Sepsis, Phlegmon, Tendons fléchisseurs, Garrot

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

Abstract

This study aimed to demonstrate that there was no risk of extension of infection in performing mechanical exsanguination before inflating the tourniquet for surgical treatment of digital flexor tendon sheath phlegmon. The series comprised 96 patients, with a mean age of 47 years (range, 18-87 years) and 37 women. Group I included 47 patients in whom exsanguination was performed with a Velpeau band before inflating the pneumatic tourniquet at the root of the limb. In Group II, which included 49 patients, the tourniquet was inflated after simple elevation of the limb. Six patients underwent revision surgery for recurrence or osteoarticular complications: 4 (8.5%) in Group I and 2 (4.1%) in Group II, the difference between two groups being non-significant (p = 0.6378). In conclusion, mechanical exsanguination before inflating the tourniquet did not incur risk of complications in surgical management of digital flexor tendon sheath phlegmon.

Résumé

Le but de ce travail était de démontrer qu'il n'y avait aucun risque d'extension de l'infection à procéder à une exsanguination mécanique avant de gonfler le garrot au cours du traitement chirurgical des phlegmons de la gaine des tendons fléchisseurs des doigts. Notre série comprenait 96 patients âgés en moyenne de 47 ans (18-87) dont 37 femmes. Le groupe I comprenait 47 patients chez qui une exsanguination a été réalisée avec une bande Velpeau avant de gonfler le garrot pneumatique à la racine du membre. Dans le groupe II qui comprenait 49 patients, le garrot pneumatique a été gonflé après surélévation du membre

Introduction

There is a dogma in surgical treatment of all infected limbs and phlegmons of the digital flexor tendon sheaths: one should not perform mechanical exsanguination of the upper limb before inflating the pneumatic tourniquet at the risk of causing proximal septic spread [START_REF] Stevanovic | Acute infections[END_REF][START_REF] Klenerman | The tourniquet manual: principles and practice[END_REF].

However, simple elevation of the limb makes the surgical procedure more difficult because of the persistence of a certain volume of blood in the limb, which can, despite the tourniquet, cause blood to ooze continuously [START_REF] Colville | Exsanguination of the upper limb in hand surgery comparison of four methods[END_REF][START_REF] Blønd | Clinical consequences of different exsanguination methods in hand surgery. A double-blind randomised study[END_REF].

The present study aimed to demonstrate that there is no risk in performing mechanical exsanguination before inflating the tourniquet during surgical treatment of digital flexor tendon sheath phlegmon.

The main hypothesis was that the rate of revision surgery after lavage with or without synovectomy of the flexor tendon sheaths was not greater when mechanical exsanguination was performed before inflating the tourniquet.

Patients and methods

The local review board approved this retrospective study (Reference CE-2020-204).

One hundred and eight patients were treated in our department from January 2018 to September 2020 for phlegmon of the flexor tendon sheath. Diagnosis was founded on presence of at least one of Kanavel's cardinal signs [START_REF] Kanavel | An anatomical and clinical study of acute phlegmons of the hand[END_REF] (pain on palpation along the flexor tendon path, digital flexion contracture, pain on passive extension of the finger, finger edema), supplemented by ultrasound scan of the diseased finger screening for ≥20% increase in sheath diameter compared to the contralateral side [START_REF] Prunières | Ultrasound for the diagnosis of pyogenic flexor tenosynovitis[END_REF]. Patients under the age of 18, pregnant women, with associated osteoarticular disorder, and with <2 weeks' follow-up were excluded. The series included 96 patients, with a mean age of 47 years (range, 18-87 years), including 37 women. Comorbidities, mechanism, injury agent and affected finger are shown in Tables 1 and2.

All the patients were treated in an outpatient hand trauma center under locoregional anesthesia by 8 level-3 surgeons and 2 level-4 surgeons [START_REF] Tang | Why and how to report surgeons' levels of expertise[END_REF]. Surgical treatment consisted in proximal to distal saline lavage of the flexor tendon sheath until the liquid flow became perfectly clear. Depending on phlegmon stage (stage 1: cloudy fluid, no synovitis; stage 2: suspicious or purulent fluid, synovitis but intact tendon; stage 3: tendon necrosis) [START_REF] Michon | Phlegmon of the tendon sheaths[END_REF], a synovectomy was performed in some cases.

After bacteriological sampling, a broad-spectrum antibiotic loading dose (amoxicillin / clavulanic acid or clindamycin) was delivered intraoperatively and continued postoperatively.

The antibiotic treatment was then adapted to the results of the antibiogram after consulting an infectious diseases specialist.

In the first 47 cases (group I), mechanical exsanguination was performed with a Velpeau band from the hand to the upper arm before inflating the pneumatic tourniquet at the root of the limb. In the following 49 cases (group II), the upper limb was kept elevated until the superficial veins appeared empty before inflating the pneumatic tourniquet.

Revision surgery was indicated whenever local, regional and/or general infectious signs did not decrease or reappeared within 8 days after primary surgery. Data collection consisted in reviewing medical files for surgical revision for recurrence and/or extension of the infection.

Statistical analysis first checked normal distribution in the 2 groups on Shapiro-Wilk test. Quantitative variables were compared between groups by Student test (when the variable of interest was Gaussian), with a possible correction for the heterogeneity of variances on Welsh test, or else by non-parametric Mann-Whitney-Wilcoxon test. Number of surgical revisions for recurrence and/or extension of the infection (qualitative variable) was compared between groups by Fisher exact test, with alpha risk of 5%. Analyses used R software version 4.0.2 (RStudio, Inc.) and all requisite software packages.

Results

Results are reported in Tables 1, 2 and 3, expressed considering missing data. No systemic or general complications of the infection were observed.

The majority of infections implicated wounds (45%), foreign bodies (23%) or animal bites (19%). The most frequently injured fingers were the middle (29%) and the index (27%).

Mean follow-up was 4.65 weeks (95% CI = [3.53; 5.77]). 57% of patients presented no comorbidities.

Phlegmon was mostly stage I (62.5%). Six patients were treated for recurrence or osteoarticular extension: 4 (8.5%) in Group I and 2 (4.1%) in Group II, the difference being non-significant (p = 0.6378).

Discussion

Esmarch bandages should no longer be used alone as a tourniquet in limb surgery to limit intraoperative bleeding and facilitate the comfort of the surgical procedure. It has been shown experimentally that pressure with an Esmarch bandage can reach 1,000 mmHg [START_REF] Mclaren | The pressure distribution under tourniquets[END_REF]; in clinical practice, it is not possible to control this pressure, which increases the risk of neurovascular injury [START_REF] Klenerman | Tourniquet paralysis[END_REF].

Instead, pneumatic tourniquets are used [START_REF] Colville | Exsanguination of the upper limb in hand surgery comparison of four methods[END_REF][START_REF] Blønd | Clinical consequences of different exsanguination methods in hand surgery. A double-blind randomised study[END_REF]. To enhance effectiveness, exsanguination is usually performed before inflation [START_REF] Klenerman | The tourniquet manual: principles and practice[END_REF][START_REF] Oragui | Tourniquet use in upper limb surgery[END_REF]. Exsanguination techniques include simple elevation of the limb or elevation associated with mechanical compression by manual maneuver, Velpeau stretchy band, Esmarch bandage, Rhys-Davies device or sterile Hemaclear® device. [START_REF] Pereira | Evaluation of a new tourniquet for hand surgery: Comparison of 76 carpal tunnel syndrome cases operated using a Hemaclear® Model-F tourniquet versus a pneumatic tourniquet[END_REF]. The effectiveness of various exsanguination techniques was measured by organ perfusion scintigraphy with 99mTc-labeled red blood cells in 10 volunteers [START_REF] Blond | Exsanguination of the upper limb in healthy young volunteers[END_REF]. The rate of exsanguination varied depending on the technique: 46% at 1 min with simple elevation, 63% with Velpeau band, and 69% with Esmarch bandage. Other authors measured the effectiveness of several exsanguination techniques using the water displacement method [START_REF] Alshawi | The crepe bandage as an alternative to the Esmarch bandage for upper limb exsanguination: A volumetric comparison study[END_REF]. Exsanguination volume varied: 28mL with Rhys-Davies device, 59mL with Velpeau band, and 63mL with Esmarch bandage. Whatever the assessment technique, the best exsanguination method is the Esmarch bandage, closely followed by the Velpeau band. However, complications have been reported using the Esmarch bandage: skin shear lesions, especially in fragile patients [START_REF] Alshawi | The crepe bandage as an alternative to the Esmarch bandage for upper limb exsanguination: A volumetric comparison study[END_REF], pulmonary embolism in patients operated on in the lower limb [START_REF] Boogaerts | Lower limb exsanguination and embolism[END_REF][START_REF] Darmanis | Fatal intra-operative pulmonary embolism following application of an Esmarch bandage[END_REF][START_REF] Lu | Massive pulmonary embolism after application of an Esmarch bandage[END_REF][START_REF] Desai | Fatal pulmonary embolism subsequent to the use of Esmarch bandage and tourniquet: A case report and review of literature[END_REF]. Although no studies have been reported, it is clear that the best rate of blood loss is obtained with Hemaclear®, although some authors object to the cost [START_REF] Pereira | Evaluation of a new tourniquet for hand surgery: Comparison of 76 carpal tunnel syndrome cases operated using a Hemaclear® Model-F tourniquet versus a pneumatic tourniquet[END_REF]; the best cost/efficacy/safety trade-off seems to be obtained with the Velpeau band [START_REF] Alshawi | The crepe bandage as an alternative to the Esmarch bandage for upper limb exsanguination: A volumetric comparison study[END_REF].

The method of exsanguination is controversial in hand surgery in infectious contexts. Some consider that mechanical exsanguination increases the risk of septic spread [START_REF] Stevanovic | Acute infections[END_REF][START_REF] Klenerman | The tourniquet manual: principles and practice[END_REF], although this dogma is not shared by everyone; there is no scientific evidence for or against the hypothesis of a risk of septic spread [START_REF] Farner | Potential for spread of hand infection after elastic exsanguination[END_REF]. Others performed mechanical exsanguination for all patients operated on for infection during 25 years of practice, without notable complications [START_REF] Green | General principles[END_REF]. A cadaver study on 8 upper limbs found no spread to the forearm on injection of a radiopaque fluid into the thenar and mid-palmar compartments after exsanguination of the limbs by elastic bandaging [START_REF] Farner | Potential for spread of hand infection after elastic exsanguination[END_REF]. Although the present clinical series was small, with short follow-up, this was the only study refuting the dogma prohibiting mechanical exsanguination in infection.

The main study hypothesis was confirmed: the rate of revision surgery after lavage of flexor tendon sheath phlegmon with or without synovectomy was not significantly greater when mechanical exsanguination was performed before inflating the tourniquet.

Conclusion

To improve the comfort of the operator during the surgical treatment of digital flexor tendon sheath phlegmon, we recommend mechanical exsanguination before inflating the tourniquet to avoid continuous bleeding during the procedure.

Conflicts of interest:

Philippe Liverneaux has conflicts of interest with Newclip technics, Caresyntax and Argomedical.

None of the other authors have a conflict of interest

1

  Risk of septic spread during surgical management of digital flexor tendon sheath phlegmon: Exsanguination by mechanical compression versus simple elevationRisque de dissémination septique au cours de la prise en charge chirurgicale des phlegmons de la gaine des tendons fléchisseurs : Exsanguination par compression mécanique versus surélévation simple

Table titles Table 1 .

 titles1 Characteristics of 47 patients treated surgically for phlegmon of the digital flexor tendon sheath with mechanical exsanguination before inflating the tourniquet (Group I)

Table 2 .

 2 Characteristics of 49 patients treated surgically for phlegmon of the digital flexor tendon sheath with simple elevation before inflating the tourniquet (Group II)

Table 3 .

 3 Results of 96 patients treated surgically for digital flexor tendon sheath phlegmon with mechanical exsanguination versus simple elevation before tourniquet inflation

Table 1 .

 1 Characteristics of 47 patients treated surgically for phlegmon of the digital flexor tendon sheath with mechanical exsanguination before inflating the tourniquet (Group I)

	Patient Gender Age	Comorbidity	Mechanism	Agent	Finger Stage Revision	Diagnosis	Follow up
	(N)	(F/M) (year)				(1-5) (I-IV)	(Y/N)		(weeks)
	1	F	23	Smoking	Bite	Cat	4	2	N		3
	2	M	44	0	Wound	Knife	4	1	N		2
	3	M	71	Diabetes	Iatrogenic	Postoperative	4	2	N		6
	4	F	30	0	Bite	Cat	2	2	N		2
	5	M	48	0	Bite	Cat	2	2	N		2
	6	M	38	0	Wound	Glass	3	1	N		2
	7	M	27	0	Wound	Crush injury	3	1	N		2
	8	M	78	Diabetes	?	?	4	3	N		3
	9	M	25	0	Wound	Knife	4	2	N		2
	10	F	48	0	Wound	Knife	3	2	N		12
	11	F	63	0	Wound	Metal	3	2	N		2
	12	M	41	0	Foreign body	Plant	3	1	N		10
	13	M	49	Drug addiction	?	?	2	1	N		4
	14	M	33	0	Foreign body	?	3	1	N		2
	15	F	62	Alcoholism	Wound	Crush injury	3	1	Y	Recurrence	2
	16	F	53	0	Bite	Cat	1	1	N		3
	17	F	46	0	?	?	4	1	N		2
	18	F	72	0	Bite	Dog	2	1	N		12
	19	M	50	0	Bite	Cat	2	1	N		5
	20	M	35	Smoking	Bite	Cat	5	1	Y	Osteoarthritis	6
	21	M	46	0	Wound	Glass	4	1	N		2
	22	M	46	0	Foreign body	?	2	1	N		4
	23	M	38	0	Wound	Metal	2	1	N		2
	24	M	50	0	Wound	Metal	2	1	N		2
	25	F	71	Rheumatoid Arthritis Foreign body	Plant	2	1	Y	Arthritis	2
	26	M	52	0	Foreign body	Plant	2	1	N		2
	27	F	41	Smoking	Wound	Knife	1	1	N		2
	28	F	47	0	Iatrogenic	Postoperative	1	1	N		24
	29	M	61	0	Bite	Goat	1	1	N		2
	30	F	35	0	?	?	1	1	N		2

F: female ; M: male ; Y: yes ; N: no

Table 3 .

 3 Results of 96 patients surgically treated for digital flexor tendon sheath phlegmon with mechanical exsanguination versus simple elevation before tourniquet inflation

	Group I Group II Difference Significance


Lara Mroueh

Dicranouhie Ekmekdjian

Elen Aghekyan

Samvel Sukiasyan

Margarit Tadevosyan

Vahan Simonyan

Armen Soghoyan

Clotilde Vincent

Pierre-Emile Bruand

Thomas Jamieson-Craig

Driss Moussaoui

Pierre-Marie Preux

Farid Boumédiène
email: farid.boumediene@unilim.fr

Can a brief training intervention on schizophrenia and depression improve knowledge, attitudes and practices of primary healthcare workers? The experience in Armenia

Keywords: Armenia, depression, primary healthcare workers, schizophrenia, training

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

INTRODUCTION

Although mental disorders contribute to a significant proportion of the global disease burden, they remain vastly under-diagnosed and under-treated. In particular, in low-and middleincome countries (LMICs), where it is estimated that 76-85% of people with mental disorders receive no treatment [World Health Organisation, 2019]. In LMICs, mental health professionals are in small number, and although the World Health Organisation (WHO) has recommended for mental health services to be integrated into primary care [World Health Organisation, 2019], mental health training is grossly inadequate in the curriculum of primary healthcare professionals.

Armenia is a small landlocked country in the Southern Caucasus region of Eurasia, of which Yerevan is the capital. It is a former republic of the Soviet Union, with a population of approximately 3 million people, which belongs to the upper-middle income countries group, according to the World Bank classification.

There is limited published evidence regarding the prevalence of mental disorders in Armenia with data from WHO estimating the prevalence of depressive disorders at 5.0% and of anxiety disorders at 3.5% [World Health Organisation, 2017a]. Data from Médecins Sans Frontières (MSF) suggest that, on average, 38% of Armenians would have mental health issues [START_REF] Van Baelen | Mental health problems in Armenia: low demand, high needs[END_REF]. What is obvious though, is that the country's history has been marked by tragic events, such as the 1915 Genocide, the 1988 earthquake and the armed conflict in Nagorno Karabagh, which have left deep and painful scars in the population. Through direct experience, or intergenerational effects related to their parents', grandparents' or great-grandparents' histories, these events have had a profound impact on the mental health of many Armenians. [START_REF] Gasparyan | The Armenian Genocide and Its Intergenerational Effects. An International Perspective on Disasters and Children's Mental Health[END_REF] With only 27.4 mental health professionals per 100,000 population (vs 271.3 in the US and 318.9 in the UK), the country lacks specialized workforce [World Health Organisation, 2017b, World Health Organisation, 2017c, World Health Organisation, 2014], and most mental health professionals are located in Yerevan. This scarcity is exacerbated by the fact that many trained psychiatrists emigrate to other countries within 5 years of completing their training. [START_REF] Soghoyan | Mental Health in Armenia[END_REF] Moreover, as is the case in most LMICs, mental health training for primary healthcare professionals is inadequate, leaving them ill-equipped to diagnose and manage people with mental disorders [START_REF] Soghoyan | Mental Health in Armenia[END_REF]. Furthermore, discrimination against people with mental disorders is high in the population, with data suggesting that 53% of people feel that patients with mental illness should be kept in hospital and 63% think that they are usually violent and dangerous. [START_REF] Van Baelen | Mental health problems in Armenia: low demand, high needs[END_REF] Our intervention aimed to provide training on schizophrenia and depression to general practitioners (GPs) and nurses, in several regions of Armenia, and to evaluate its effect on their knowledge, attitudes and practices.

METHODS

The training interventions were one-day, face-to-face, interactive workshops, including didactic presentations, as well as discussions of case studies using videos from the WHO mhGAP package and some involving local patients and psychiatrists. These workshops were conducted by Armenian psychiatrists. Schizophrenia training workshops were held in three regions (Armavir, Lori and Shirak) between October and December 2018 with 111 GPs and 167 nurses who participated. Depression training workshops were conducted in five regions (Armavir, Lori, Shirak, Syunik, and Yerevan) between June and November 2019, with 459 GPs and 197 nurses taking part.

Our evaluation had a quasi-experimental design of the before/after type: it compared data on knowledge, attitudes and practices collected before the training sessions, with data collected immediately after. Healthcare professionals taking part in the training workshops had to complete the same questionnaire just before the training and immediately after. There were however differences between the questionnaires for nurses and GPs, to reflect the differences in the content of the training. All scores have been converted to 100.

A comparison between the mean of pre-and post-training scores was conducted for nurses and GPs using Wilcoxon paired test. It was done for the total score nationally, as well as for each region and for each sub-score.

RESULTS

For schizophrenia, overall, the mean scores increased from 52.7% to 89.7% (p<0.001) for nurses (70.2% relative increase) and from 47.3% to 83.2% (p<0.001) for GPs (75.9% relative increase) (Figure 1). Differences between end-line and baseline were significant in all regions for both nurses and GPs (p<0.001). Whereas similar scores and increases were achieved in Armavir and Lori (nurses: 53.9% to 92.2% and 48.4% to 93.9% respectively; GPs: 45.5% to 79.3% and 41.5% to 89.4% respectively), in Tavush for both nurses and GPs, the baseline scores were higher and the change of a smaller magnitude (nurses: 55.8% to 80.8%; GPs: 57.8% to 82.4%). (Figure 1) Sub-scores were calculated for schizophrenia knowledge, screening/diagnosis and patient management, and the improvement between end-line and baseline were significant (p<0.001) for each subcategory for both nurses and GPs. (Figure 2)

For depression overall, mean scores increased from 29.3% to 74.8% (p<0.001) for nurses (155.3% relative increase), and from 39.7% to 78.7% (p<0.001) for GPs (98.2% relative increase) (Figure 3). Differences between end-line and baseline were significant in all regions for both nurses and GPs (p<0.001) with similar baseline scores observed across all regions. The highest post-training score was reached in Lori for both nurses (86.0%) and GPs (88.0%). (Figure 3) Sub-scores were also calculated for depression knowledge, screening/diagnosis and patient management, and the improvement between end-line and baseline were all significant (p<0.001) for each subcategory for both nurses and GPs. (Figure 4) 4. DISCUSSION Our analysis has shown low baseline scores for both depression (nurses: 29.3%, GPs: 39.7%) and schizophrenia (albeit slightly better; nurses: 52.7%, GPs: 47.3%). This confirms that in Armenia, primary healthcare workers might not have adequate skills to identify patients with depression or schizophrenia and manage them, as has also been shown in other LMICs [START_REF] Poudiougou | Mental health capacity building in Mali by training rural general practitioners and raising community awareness[END_REF][START_REF] Praharso | Mental health literacy of Indonesian health practitioners and implications for mental health system development[END_REF][START_REF] Reddy | Mental health literacy and familiarity with the term schizophrenia in a community health care setting in North India[END_REF][START_REF] Sawadogo | Knowledge, attitudes and practices in mental health of health professionals at the end of their curriculum[END_REF][START_REF] Shahif | Knowledge and attitude towards mental illness among primary healthcare nurses in Brunei: A cross-sectional study[END_REF]. This is particularly concerning as GPs or nurses would be, in most cases, the first port of call for these patients. In particular for depression, where patients might only complain about physical symptoms when they seek care from their GP: in a study conducted in 14 countries on 5 continents, 69% of patients with major depression reported only physical symptoms as the reason for their primary care physician visit. [START_REF] Simon | An international study of the relation between somatic symptoms and depression[END_REF] Our study suggests that a brief training intervention on schizophrenia and depression for GPs and nurses can result in significant improvements in knowledge, attitudes and practices.

Interventions to improve mental health literacy of primary health care workers in other LMICs have achieved similarly positive results [START_REF] Nimgaonkar | A task shifting mental health program for an impoverished rural Indian community[END_REF][START_REF] Poudiougou | Mental health capacity building in Mali by training rural general practitioners and raising community awareness[END_REF][START_REF] Shiva | A virtual course in perinatal mental health for healthcare professionals[END_REF].

Whether the improvements in knowledge, attitudes and practices, assessed immediately after the training in our study, were sustained over time remains to be seen. A 6-18 months post-intervention survey was planned to evaluate this specific point. The plan was also to survey local psychiatrists and assess whether a greater number of patients with schizophrenia or depression had been referred to them since the training. Unfortunately, due to the Covid 19 pandemic, and subsequently to the reignited war in Nagorno Karabagh, these surveys couldn't be conducted. Anecdotally though, local psychiatrists have reported that following the training, they have seen a greater number of patients with schizophrenia or depression being referred to them, suggesting that the training received by GPs and nurses has been put into practice.

  mental health treatment gap in LMICs, brief mental health training interventions for primary healthcare workers, based on the WHO mhGAP package, would help improve mental health services, and result in better diagnosis and management of mental disorders.

Figure 1 :

 1 Figure 1: Mean scores pre-and post-training for schizophrenia -Overall and in each region

Figure 2 :

 2 Figure 2: Sub scores pre-and post-training for schizophrenia

Figure 4 :

 4 Figure 4: Sub scores pre-and post-training for depression