Martin Tetard
email: tetard@cerege.fr

Ross Marchant

Two new free-access dry splitter designs (1/2 to 1/32 aliquots) for sediment fractionation

Keywords: micropalaeontology, microfossil, splitting, subsampling 1. Introduction

This study presents two new dry sediment splitters for use in sample fractionation for micropalaeontological studies, designed so that the aliquot of interest can be recovered in a single operation. The first model (adjustable) provides a single aliquot, adjustable from 1/2 to 1/32 fraction. The second model (fixed) divides the sediment into six aliquots of different sizes. Both designs enable faster sediment splitting in comparison to the traditional sequential 1/2 fraction method. The average split accuracy and repeatability across the range of aliquot fractions were tested for each splitter, giving 94 % and 85 % for the adjustable design, and 92 % and 84 % for the fixed design, respectively. A comparison with the classic splitter method gave equal or better results. The splitters were developed at the CEREGE laboratory and can be 3D printed very cheaply, and the design files have been made freely accessible online (https://github.com/microfossil/tetard-marchant-splitter).

2

(multiple steps each introduce an error). As we are aiming to replace the classic "Otto" splitter in our workflow, both new splitters have been designed primarily for sediments, and more particularly, for foraminifera in the 63um -3 mm range.

Material and Method

2.1. Components The adjustable splitter is composed of eight parts (Fig 1a), from top to bottom: -a lid, to prevent dust entering the splitter when not used, -a sieve, to prevent particles larger than 3 mm becoming stuck into the funnel, -a funnel, to concentrate the sediment and drop it on to the centre of the cone, -a cone, to uniformly and randomly split and disperse the sediment, -a body, to hold the cone straight, -a separator, to separate the sediment into the aliquot of interest which then falls in the receptacle, while the remainder of the sediment is retained inside the system by the separator, -a selector to allow the manual choice of the aliquot fraction, from 1/2 to 1/32, -a receptacle, where the aliquot of interest is collected, as well as -three size M1.2 to M1.6, 5 -6 mm long screws to attach the separator to the selector, -three size M1.2 to M1.6, 5 -6 mm long screws to attach the cone to the body, -one size M2 to M2.5, 10 mm length screw to attach the separator to the cone. The fixed splitter is composed of five parts (Fig 1b), from top to bottom: -a lid, a sieve, and a funnel as described above, -a separator to split the sediment into six aliquots: 1/2, 1/4, 1/8, 1/16 and 2 x 1/32, -a base to hold the vials, as well as -one 10 mm M2 or M2.5 screw to secure the base to the separator. Some extra parts are also available to reduce the funnel hole size for smaller sized samples: -2.5 mm reducer adapter and 2 mm sieve -1.5 mm reduce adapter and 1 mm sieve 3 Figure 1: 3D Internal and exploded views of the adjustable (a) and fixed (b) splitters.

Operation

To use the adjustable splitter: 1) Remove the protective lid.

2) Choose the size of the aliquot of interest (between 1/2, 1/4, 1/8, 1/16, and 1/32) by turning the selector, which in turn adjusts the separator within the splitter (Fig. 2a).

3) Pour the sample in the funnel (Fig. 2b). 4) Gently tap the splitter to ensure every particle falls into the system. 5) Recover the aliquot of interest in the receptacle (Fig. 2c). 6) Recover the remaining sample by removing the funnel component and reconnecting it upside down, then flipping the whole device upside down to pour the rest of the sediment back into its vial. 3) Gently tap the splitter to ensure every particle falls into the system and the vials (Fig. 3a). 4) Recover the different sized aliquots from the different vials (Fig. 3b). Figure 3: Fixed splitter showing (a) internal view of the separation system, (b) recovering every fraction from 1/2 to 1/32, (c) disassembly.

Principle

A sample is poured into the funnel which has a small hole at the bottom. To prevent blockage, the sieve prevents particles that are too big for this hole from entering the funnel. The particles fall through the hole with a motion similar to that of an hourglass, hitting the conical section below and evenly dispersing in all angles. The conical section has fins with adjustable angular spacing (adjustable splitter) or fixed angular spacing (fixed splitter) which direct the particles into a receptacle (adjustable splitter) or vials (fixed splitter) located below.

Development

The splitters were designed using the online Autodesk, Inc. 3D design platform "Tinkercad" (https://www.tinkercad.com/), and the 3D model files corresponding to each part of the splitters can be freely downloaded from https://github.com/microfossil/tetard-marchantsplitter. The parts were printed on a Raise3D fused deposition layer type printer using 1.75 mm R3D Premium PLA filament. The overall material cost was 3 to 4 euros per splitter (if you do not own a 3D printer a commercial service can be used, but they may charge more) plus a few cents for the screws. Approximately 100 g of filament was used for each splitter, and a minimum of 17 and 23 hours were needed to print the adjustable and fixed splitters, respectively. These results were obtained using a standard resolution layer height of at least 0.20 mm for two parts, with the remainder needing to be printed at a high resolution of 0.10 mm to ensure the highest possible accuracy and quality for the system (see Table 1). Depending on the quality of the 3D printed parts, some sanding might be required. Reducer 1.5mm 0h17min 1.4 g High Quality (0.10 mm resolution) Reducer 2.5 mm 0h17min 1.4 g High Quality (0.10 mm resolution) Sieve 1 mm 1h32min 9.4 g High Quality (0.10 mm resolution) Sieve 2 mm 1h30min 9.1 g High Quality (0.10 mm resolution) Total 3h36min 21.3 g (0.63 €) 2.5. Advice Some particles may accumulate in the funnel due to static electricity if it is cleaned with a brush between splits. This issue can be prevented by rubbing the internal surfaces with a wet (distilled water) paper towel. It is also recommended to not use a brush to clean the splitter. A gentle tap after the sample is dropped into the funnel should be enough to make all the particles fall through the system, and compressed air should be sufficient for cleaning.

Finally, due to the way samples are typically poured into the funnel (usually by hand from the side), more regular results are obtained when this operation is done quickly in one shot towards the centre of the funnel, with the separator facing toward the user in the case of the adjustable model. This avoids uneven distribution of the sediment in the funnel. The tip of the cone can be marked with coloured paint to help give a visual indication that the cone is well centred within funnel on assembly. When working with small-volume samples (typically a smaller volume of a small size fraction) there may not be enough particles to create the "hourglass effect" and particles may instead fall preferentially to one side. To remedy this, the 1.5 mm or 2.5 mm reducer adapters are placed in the funnel to reduce the hole size and thus flow rate. Their associated 1 mm and 2 mm sieves, respectively, are also employed to prevent blockage. The default 3.5 mm hole works well for most sample types, and thus the reducers were not considered in the accuracy and repeatability tests later in this paper.

Statistical validation

Two statistical approaches were used to test the reliability and efficiency of the new splitters. The accuracy, how close the measured values are to the theoretical expected values, was measured along with the repeatability, how similar the measured values are to each other. Evaluation was performed for both splitter types and each aliquot size (see measurements data in supplementary material). In particular, we performed: 1) Statistical accuracy and repeatability measurements, based on a large (n = 30) number of repeated measured values of a fixed sample weight (1.9925 g of > 150 µm sieved coarse fraction of sediment from Core SO204-PC1, depth 288-289 cm). Core SO204-PC1 is a 5 m long piston core that was retrieved from the South Pacific Ocean (latitude: 25°40,41'S; longitude: 146°42,33'W) using a 10 m long piston corer at a depth of 2312 mbsl. This core mainly consists of a white foraminifera sand. Sample 288-289 cm is only composed of heterogeneous planktic foraminifera with different morphotypes, such spherical or flattened shapes, and represents an "ideal" sample.

2) An accuracy comparison between our new splitters and a classic micropaleontological splitter (the Otto splitter; Otto, 1933), based on a limited (n = 5) number of measurements of randomised weights from 0.38 g to 2.75 g of 63 -150 µm sieved fine fraction and > 150 µm sieved coarse fraction of sediments from Core SO204-PC1, depth 288-289 cm.

3.1. Accuracy The accuracy (Eq. 1) is defined as the difference between the expected aliquot weight (defined in Eq. 2) and the measured aliquot weight, divided by the expected aliquot weight, and expressed in %: Eq. 1 Accuracy % = 100 -|Expected Aliquot Weight -Measured Aliquot Weight| × !"" Expected Aliquot Weight Eq. 2 Expected aliquot weight = Total Sediment Weight × Investigated Aliquot This accuracy index is derived from the traditional percent error formula (Eq. 3):

Eq. 3 Percent error % = |Experimental -Theoretical| |Theoretical| × 100

Repeatability

For each aliquot size, the standard deviation (+) was investigated, according to Eq. 4, where x is a measurement, and m the average value of the n number of measurements:

Eq. 4 + = , -. /0 1 2 -1 /0 1 2⋯2 -4 /0 1 5/! The expanded uncertainty (U) is then calculated (Eq. 5) with k = 2 for a 95 % confidence level: Eq. 5 6 = k × + which finally allows the calculation of a mean repeatability index for each aliquot of each fraction (Eq. 6), based on the relative uncertainty formula:

Eq. 6 Repeatability % = 100 -: ; 0 × 100<

Results and discussion

Each of the large (n = 30) sets of repeated measured values for each splitter and aliquot size showed an overall normal distribution (see Appendix 1), allowing the computation of the defined accuracy and repeatability indices. When using the adjustable splitter, the mean measured aliquot weight for each aliquot size was close to the expected aliquot weight (Table 2), suggesting a good accuracy across all aliquot sizes. This result is reinforced by the accuracy index that averaged 94 % between aliquots, (95.27, 95.12, 92.69, 93.50, and 92.63 % for the 1/2, 1/4, 1/8, 1/16, 1/32 aliquots, respectively, Table 2). The mean standard deviation was relatively low (0.0488, 0.0325, 0.0212, 0.0103, 0.0053 g for expected aliquot weights of 0.9963, 0.4981, 0.2491, 0.1245, and 0.0623 g, respectively, Table 2), suggesting good repeatability of the measurements for each aliquot size. The mean repeatability across all aliquot sizes was about 85 % (89.92, 86.73, 83.13, 83.35, 82.70 % for the 1/2, 1/4, 1/8, 1/16, 1/32 aliquots, respectively, Table 2). The fixed splitter also showed satisfying results, with mean measured aliquot weight also very close to the expected aliquot weight (Table 2), again suggesting a good accuracy for each aliquot size. These measurements resulted in an accuracy index of approximately 92 % across all aliquot sizes, (95.38, 94.57, 91.09, 89.37, 89.60, and 91.08 % for the 1/2, 1/4, 1/8, 1/16, and both 1/32 aliquots, respectively). Even though the mean standard deviation was slightly higher than for the adjustable version, it was still relatively low (0.0723, 0.0445, 0.0286, 0.0177, 0.0061, and 0.0061 g for expected aliquot weights of 1.3001, 0.6304, 0.3044, 0.1757, 0.0725, and 0.0757 g, respectively, Table 2), again suggesting a good repeatability of the measurements for each aliquot size. The mean repeatability across all aliquot sizes was approximately 84 % (88.85, 85.80, 81.07, 79.66, 83.12, and 83.81 % for the 1/2, 1/4, 1/8, 1/16, and both 1/32 aliquot sizes, respectively, Table 2).

The repeatability for both designs is acceptable considering the low cost and small amount of material required to make the splitters. Overall, the efficiency and reliability of the splits were satisfactory. The adjustable and fixed splitters are both accurate, averaging around 94 % and 92 % across each aliquot size, respectively. Comparison of the accuracy of the new splitters and the classic "Otto" splitter, using random weights of fine and coarse fraction sediments (Table 3), showed that both new designs were more accurate than the classic design (90.44 % (adj.) and 90.52 % (fixed) vs 83.79 % (classic)) for both size fractions, averaged over every aliquot size. 83.79 Our new designs performed best for the > 150 µm size fraction, with most of the measured weights close to the expected ones, however, the classic splitter gave lower values than expected, especially for the bigger sample weights (Fig. 4). For the 63 -150 µm size fraction, the new adjustable and fixed splitters exhibited again measured values close to expected (Table 3; 85 % and 89 % accurate, respectively) while the accuracy of the classic splitter dropped significantly to 74 %. This is likely due to the propagation of a splitting errors in the classic design, as repeated splits are required to obtain the smaller aliquot sizes. For example, five splits are required to obtain an aliquot size of 1/32 for the classic model, whereas our new design only requires one. The classic splitter also showed more variation for the small weight measurements, corresponding to the smaller aliquot sizes (1/16 and 1/32). The individual measurements for each experiment can be found in Appendix 2. Figure 4: Relationship between the expected and measured random weights test, performed on the (a) fine (63 -150 µm) and (b) coarse (> 150 µm) fractions.

Conclusion

Two new splitter designs, one with a single adjustable aliquot size going into one receptacle, and another one with multiple fixed aliquot sizes going into separate vials, were presented. Several tests were conducted to test their efficiency and reliability, giving an average accuracy of 94 % and 92 % and a repeatability of 85 % and 84 %, for the adjustable and fixed splitters, respectively. Comparison with a classic splitter model has shown the new designs give equal or better results for the coarse > 150 µm fraction, and better results for the fine 63 -150 µm fraction. Furthermore, the new designs save a considerable amount of time when splitting, as they give the aliquot size of interest directly in one operation. Both designs are freely available online and each splitter can be easily 3D printed, enabling laboratories to be equipped with more tools for each user as the need arises. Finally, both splitters are currently being incorporated into the workflow here at CEREGE and we have successfully used it to process 191 samples from piston Core MD02-2519 (22°30.89′N; 106°39.00′W; 955 mbsl) retrieved off Mazatlan, Mexico (Beaufort, 2002). Sediment retrieved from this core is mostly composed of dark sediment grains, benthic and planktic foraminifera, radiolarians and other biogenic debris, and correspond to a typical case study (different from the ideal planktic foraminiferal sand used for tests). No issue linked with the nature of the sediments was encountered during the processing of the samples from this core. Acknowledgements Martin Tetard would like to thank IODP-France for financial support to MT for this project. This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the French platform called Nano-ID (EQUIPEX project ANR-10-EQPX-39-01). References Charrieau, L.M., Bryngemark, L., Hansson, I., and Filipsson, H.L., 2018. Improved wet splitter for micropalaeontological analysis, and assessment of uncertainty using data from splitters. J. Micropalaeontol. 37, 191-194. Dryden, A.L., 1931. Accuracy in percentage representation of heavy mineral frequencies. Nat. Acad. Sci. Proc., 17, 233-238. Elmgren, R., 1973. Methods of sampling sublittoral soft bottom meiofauna. Oikos suppl. 15, 112-120. Fatela, F., and Taborda, R., 2002. Confidence limits of species proportions in microfossil assemblages. Mar. Micropalaeontol. 45, 169-174. Herguera, J.C., and Berger, W., 1991. Paleoproductivity from benthonic foraminifera abundance: Glacial to postglacial change in the west-equatorial Pacific. Geology 19, 1173-1176. Jensen, P., 1982. A new meiofaunal splitter. Ann. Zool. Fennici 19, 233-236. Murray, J.W., 2006. Ecology and Applications of Benthic Foraminifera. Cambridge Univ. Press, Cambridge. Otto, G.H., 1933. Comparative Tests of Several Methods of Sampling Heavy Mineral Concentrates. J. Sediment. Res. 3,[30][31][32][33][34][35][36][37][38][39]

Figure 2 :

 2 Figure 2: Adjustable splitter, showing (a) view of the selector to choose the required aliquot, (b) sample poured in the sieve, over the funnel, (c) recovering the fraction of interest in the receptacle, (d) disassembly. To use the fixed splitter: 1) Remove the protective lid. 2) Pour the sample in the funnel.3) Gently tap the splitter to ensure every particle falls into the system and the vials (Fig.3a). 4) Recover the different sized aliquots from the different vials (Fig.3b).

Table 1 :

 1 Summary of the time, and amount of filament (and cost) used for each part of the two splitter designs (including printing support structure).

	Part		Time	Filament	Printing set-up
			Lid 0h55min	11.2 g Standard Quality (0.20 mm resolution)
	Adjustable splitter	Sieve 3 mm 1h24min Funnel 5h15min Body 4h43min Cone 1h30min Separator 0h38min Selector 2h03min	8.7 g High Quality (0.10 mm resolution) 27.5 g High Quality (0.10 mm resolution) 22.0 g High Quality (0.10 mm resolution) 7.8 g High Quality (0.10 mm resolution) 5.5 g High Quality (0.10 mm resolution) 10.9 g High Quality (0.10 mm resolution)
		Receptacle 1h06min	13.3 g Standard Quality (0.20 mm resolution)
	Total	17h34min 106.9 g (3.22 €)
		Lid	0h55min	11.2 g Standard Quality (0.20 mm resolution)
	Fixed splitter	Sieve 3 mm 1h24min Funnel 5h15min Separator 12h39min Selector 2h56min	8.7 g High Quality (0.10 mm resolution) 27.5 g High Quality (0.10 mm resolution) 50.4 g High Quality (0.10 mm resolution) 21.2 g Standard Quality (0.20 mm resolution)
	Total	23h09min 119.0 g (3.58 €)
	Accessories

Table 2 :

 2 Accuracy and repeatability measurements for each aliquot size for the adjustable and fixed splitters.

		Investigate d fraction	Investig ated aliquot	Number of measurem ents	Total sediment weight (g)	Expected aliquot weight (g)	Mean measured aliquot weight (g)	Mean accuracy (%)	Standard deviation	Mean relative uncertainty (%)	Mean repeatability (%)
	Adjustable	>150 µm >150 µm >150 µm	1/2 1/4 1/8	30 30 30	1.9925 1.9925 1.9925	0.9963 0.4981 0.2491	0.9711 0.4920 0.2519	95.27 95.12 92.69	0.0488 0.0325 0.0212	10.08 13.27 16.98	89.92 86.73 83.02
		>150 µm	1/16	30	1.9925	0.1245	0.1242	93.50	0.0103	16.75	83.25
		>150 µm	1/32	30	1.9925	0.0623	0.0618	92.63	0.0053	17.43	82.57
							Average	93.84	Average	14.90	85.10
		>150 µm	1/2	30	2.5742	1.2871	1.3001	95.38	0.0723	11.15	88.85
		>150 µm	1/4	30	2.5742	0.6436	0.6304	94.57	0.0445	14.20	85.80
	Fixed	>150 µm >150 µm >150 µm	1/8 1/16 1/32	30 30 30	2.5742 2.5742 2.5742	0.3218 0.1609 0.0804	0.3044 0.1757 0.0725	91.09 89.37 89.60	0.0286 0.0177 0.0061	18.93 20.34 16.88	81.07 79.66 83.12
		>150 µm	1/32	30	2.5742	0.0804	0.0757	91.08	0.0061	16.19	83.81
							Average	91.85	Average	16.28	83.72

Table 3 :

 3 Comparison of accuracy between the new splitters and the classic model.

	Investigated	Investigated	Number of	Adjustable splitter	Fixed splitter	Classic splitter
	fraction	aliquot	measurements	accuracy (%)	accuracy (%)	accuracy (%)
	>150 µm	1/2	5	98.67	94.66	96.29
	63 -150 µm	1/2	5	93.01	91.90	95.37
	>150 µm	1/4	5	95.07	95.37	94.63
	63 -150 µm	1/4	5	94.07	90.36	80.71
	>150 µm	1/8	5	95.86	91.42	94.99
	63 -150 µm	1/8	5	84.62	88.55	78.78
	>150 µm	1/16	5	95.29	91.88	91.50
	63 -150 µm	1/16	5	77.11	87.90	66.67
	>150 µm	1/32	5	92.82	90.38	89.32
	63 -150 µm	1/32	5	77.89	90.84	49.62
	>150 µm	1/32 (2)	5		86.61

Appendix 1: Whisker box plots for the normalized repeated measured values for each splitter and aliquot size. Numbers next to the boxes correspond to the median measured value (in % of the expected value).


Joao Pedro Ferreira
email: j.ferreira@chu-nancy.fr

MD, PhD Xavier Rossello

MD, PhD Romain Eschalier

MD John J V Mcmurray

PhD Stuart Pocock

MD, PhD Nicolas Girerd

MD, PhD Patrick Rossignol

MD Bertram Pitt

MD, PhD Faiez Zannad

MD, PhD João Pedro Ferreira

Dr João Pedro Ferreira

MRAs in Elderly HF Patients: Individual Patient-Data Meta-Analysis of RALES, EMPHASIS-HF, and TOPCAT

Keywords: mineralocorticoid receptor antagonists, elderly, treatment effects

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

Introduction

Heart failure (HF) is a common disease in older people with a population prevalence of 10-20% in individuals aged above 75 years [START_REF] Bleumink | Quantifying the heart failure epidemic: prevalence, incidence rate, lifetime risk and prognosis of heart failure The Rotterdam Study[END_REF] ; and with incidence increasing among the very old [START_REF] Zannad | Rising incidence of heart failure demands action[END_REF][START_REF] Conrad | Temporal trends and patterns in heart failure incidence: a population-based study of 4 million individuals[END_REF] . In many developed countries the age of patients hospitalised for HF is above 75 years [START_REF] Dharmarajan | Epidemiology, Pathophysiology, and Prognosis of Heart Failure in Older Adults[END_REF] .

These older patients are underrepresented in randomized clinical trials and the balance of benefits and risks for standard therapies could differ substantially from the young [START_REF] Mogensen | Clinical characteristics and major comorbidities in heart failure patients more than 85 years of age compared with younger age groups[END_REF] . Agerelated physiological changes, multi-comorbidity, frailty, and polypharmacy, all can alter drug pharmacokinetics and change the effect of treatments in elderly HF patients [START_REF] Lazzarini | Heart failure in elderly patients: distinctive features and unresolved issues[END_REF] . Moreover, the prevalence of HF with preserved ejection fraction (HFpEF), is proportionately far greater in the elderly, compared to HF with reduced ejection fraction (HFrEF) [START_REF] Owan | Trends in prevalence and outcome of heart failure with preserved ejection fraction[END_REF] .

In patients with HFrEF and severe symptoms enrolled in RALES (The Effect of Spironolactone on Morbidity and Mortality in Patients with Severe Heart Failure) , treatment with spironolactone (vs. placebo) reduced the relative rate of all-cause death by 30%, without evidence of a treatment by age subgroup (median age: <67 vs. ≥67yr) interaction [START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF] . In patients with HFrEF and mild symptoms enrolled in EMPHASIS-HF (Eplerenone in Patients with Systolic Heart Failure and Mild Symptoms), treatment with eplerenone (vs. placebo) reduced the relative rate of the time-to-first composite of cardiovascular death or HF hospitalization by 37% and all-cause death by 24%, without evidence of a treatment by age subgroup (<65 vs. ≥65yr and <75 vs. ≥75yr) interaction [START_REF] Zannad | Eplerenone in patients with systolic heart failure and mild symptoms[END_REF] . In TOPCAT (Spironolactone for Heart Failure with Preserved Ejection Fraction) trial, treatment with spironolactone did not reduce the incidence of death due to cardiovascular causes or HF hospitalizations in the overall trial, but the rate of HF hospitalizations was reduced. Due to important heterogeneity in event-rates and treatment effect by geographical region (that have been described in detail [START_REF] Pfeffer | Regional Variation in Patients and Outcomes in the Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist (TOPCAT) Trial[END_REF][START_REF] Ferreira | Geographic differences in heart failure trials[END_REF][START_REF] De Denus | Spironolactone Metabolites in TOPCAT -New Insights into Regional Variation[END_REF] ), subsequent analyses of the TOPCAT trial have been limited to patients from "the Americas". The 2017 update of the ACC/AHA/HFSA guidelines gave a class IIb indication to spironolactone for treating HFpEF [START_REF] Yancy | ACC/AHA/HFSA Focused Update on New Pharmacological Therapy for Heart Failure: An Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America[END_REF] . In TOPCAT, no treatment by age subgroup (<65 vs. 65-74 vs. ≥75yr) was found [START_REF] Pitt | Spironolactone for heart failure with preserved ejection fraction[END_REF] .

Despite the randomized evidence, MRAs continue largely underused, especially in elderly patients [START_REF] Pitt | Mineralocorticoid receptor antagonists in patients with heart failure: current experience and future perspectives[END_REF] .

The individual trials were underpowered to assess the treatment effect in the elderly, hence pooling the individual patient data (IPD) provides more statistical power to assess the efficacy and safety of MRA treatment in this subpopulation. We examined the effects of MRA therapy in older (≥75 years), compared with younger (<75 years), participants using IPD from the major randomized trials with morbidity-mortality outcomes: RALES, EMPHASIS-HF, and TOPCAT-Americas.

Methods

Study design, setting, and participants

In RALES, 1663 patients who had HF with severe symptoms and a left ventricular ejection fraction (LVEF) ≤35%, were randomly assigned to spironolactone (up to 50 mg/day) or matching placebo. The primary outcome was all-cause death. In EMPHASIS-HF, 2737 patients who had HF and mild symptoms and a LVEF ≤35% were randomly assigned to eplerenone (up to 50 mg/day) or matching placebo. The primary outcome was a composite of cardiovascular death or HF hospitalization. In TOPCAT, 3445 patients with symptomatic HF and a LVEF ≥45% were randomly assigned to spironolactone (15 to 45 mg daily) or matching placebo. The primary outcome was a composite of death from cardiovascular causes, aborted cardiac arrest, or hospitalization for the management of heart failure [START_REF] Pitt | Spironolactone for heart failure with preserved ejection fraction[END_REF] . As stated in the introduction, due to the major regional variations found in the TOPCAT trial, we report only data regarding patients enrolled in "the Americas" (n =1767) [START_REF] Pfeffer | Regional Variation in Patients and Outcomes in the Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist (TOPCAT) Trial[END_REF] .

Each individual randomized controlled trial was conducted in accordance with the Declaration of Helsinki and approved by the site ethics committees. All participants gave written informed consent to participate in the respective studies.

Study outcomes

For consistency across trials the primary outcome was a composite of cardiovascular death or HF hospitalization in the present analysis. Cardiovascular death alone and all-cause death were also assessed.

The major clinical outcomes were centrally adjudicated by endpoint committees and defined by the conventional criteria (definitions have been published in the respective studies).

Statistical analysis

A fixed-effect model for a one-stage IPD meta-analysis was conducted [START_REF] Da Costa | Systematic reviews and meta-analyses of randomized trials: principles and pitfalls[END_REF] . Baseline clinical characteristics of patients were summarised by treatment group with means and standard deviation for continuous variables, with frequencies and percentages for categorical variables, hazard ratios (HRs) with their respective 95% confidence interval (95% CI) for treatment effect estimates. Time-to-first-event curves were obtained using the Kaplan-Meier method and compared using the log-rank test. Univariate Cox proportional hazards' modelling was used to explore the association of the intervention and the outcomes. A Cox model stratified by study was used to obtain the overall HR for this individual patient meta-analysis (assuming equal effects across strata but with a baseline hazard unique to each study) [START_REF] Riley | Meta-analysis of individual participant data: rationale, conduct, and reporting[END_REF] .

Event-rates, absolute treatment effects and number needed-to-treat to benefit were also computed. Beyond the group of patients aged 75 or more, we also studied patients aged 80 or more as exploratory analysis.

Statistical analyses were performed using the STATA/SE software, version 15.1 (Stata Corp, College Station, TX, USA).

Statistical heterogeneity

The individual effect size for each trial and overall effect size estimations and their 95%CIs are depicted in the Central Illustration, and suggest the lack of substantial statistical heterogeneity. To further investigate the variation of primary outcome treatment effect estimates between trials, the Cochran's Q test and the Wald test of the overall interaction were computed.

Results

Patient characteristics

In the present analysis, 1756 patients were aged ≥75 years (853 allocated to placebo and 903 to MRA) and 4411 were aged <75 years (2242 randomized to placebo and 2169 to MRA).

The number (proportion) of patients aged ≥75 years in RALES was 352 (21%), in EMPHASIS-HF was 657 (24%), and in TOPCAT-Americas was 747 (42%) (Supplemental The baseline characteristics were well balanced between treatment arms. Patients with HFrEF (i.e. RALES plus EMPHASIS-HF trials) had some characteristics (beyond ejection fraction) quite different from those with HFpEF (i.e. TOPCAT-Americas). Patients with HFpEF were older, more often female, hypertensive, with diabetes, and with peripheral vascular disease; and had less often a previous MI, had lower hemoglobin, and were less often treated with ACEi/ARBs (Table 1).

The median (percentile25-75) follow-up time was 1.8 (0.8-2.6) years in the HFrEF population, 2.9 (1.9-4.4) years in the HFpEF population, which gave a median follow-up of 2.2 (1.2-3.1) years in the overall population.

Events and treatment effects

In patients aged ≥75 years that participated the HFrEF trials (RALES plus EMPHASIS-HF), the primary outcome of cardiovascular death or HF hospitalization occurred in 215 (43.2%) patients taking placebo and 170 (33.3%) taking a MRA, with a corresponding HR (95%CI) of 0.69 (0.56-0.84), heterogeneityp between trials =0.70. A similar beneficial MRA effect was observed for the outcomes of cardiovascular and all-cause death. In patients with ≥75 years and HFpEF from the TOPCAT-Americas trial, the primary outcome of cardiovascular death or HF hospitalization occurred in 116 (32.7%) patients taking placebo and 111 (28.1%) taking a MRA, with a corresponding HR (95%CI) of 0.83 (0.63-1.08). Cardiovascular and all-cause death were also numerically lower in MRA-treated patients, but also did not reach statistical significance (p-value ≥0.1 for all outcomes). In the combined (HFrEF plus HFpEF) population aged ≥75 years, the primary outcome of cardiovascular death or HF hospitalization occurred in 331 (38.8%) patients taking placebo and 281 (31.1%) taking a MRA, with a corresponding HR (95%CI) of 0.74 (0.63-0.83), heterogeneityp between trials =0.52. A similar and homogenous treatment effect was also observed for the outcomes of cardiovascular and all-cause death (Table 2 & Central Illustration).

The treatment effect was similar in patients aged <75 years (compared with those aged ≥75 years) without significant statistical interactions by age subgroups (interactionp >0.1 for all outcomes). The cumulative hazard curves (MRA vs. placebo) are presented in the Figure 1.

The adjusted predictions of the treatment effect across the continuum of age is represented in the Figure 2. For any given age, treatment with MRA reduces the risk of events associated with ageing. The adjusted predictions of the treatment effect across continuum of age and renal function is represented in the Figure 3. For any given age and eGFR, MRAs reduce the event rate.

The absolute event reduction and number needed to treat to benefit was also similar between younger and older patients, and ranged from 8 to 31 patients for the primary composite outcome of cardiovascular or HF hospitalization (Supplemental Table 3).

Patients with ≥80 years, represented less than 10% of the RALES and EMPHASIS populations, and 24% of the TOPCAT-Americas (Supplemental Table 2). The primary outcome HR (95%CI) of the treatment in patients aged 80 or more was 0.86 (0.68-1.08), translating the reduced power to assess the efficacy in this population (Supplemental Figure 1).

Event rates

The event rates (per 100 person-years) of the composite of cardiovascular or HF hospitalization and cardiovascular death were nearly 3-fold higher for placebo-treated patients with HFrEF compared to HFpEF: 30.7 (26.8-35.1) vs. 12.5 (10.4-15.0); and 2-fold higher for MRA treated patients with HFrEF compared to HFpEF: 21.1 (18.2-24.6) vs. 10.5 (8.7-12.6). In consequence, the absolute incidence rate difference was higher in HFrEF than in HFpEF: -9.6 (-14.7 to -4.3) vs. -2.0 (-5.0 to 1.0). The rate of CV death was 3-fold higher and all-cause death 2-fold higher in HFrEF compared with HFpEF (Supplemental Table 4).

The events rates (per 100 person-years) of the composite of cardiovascular or HF hospitalization and cardiovascular death were similar between patients ≥75 vs. <75 years (primary composite outcome =17.2 [16.3-18.1] in <75yr vs. 17.9 [16.5-19.4] in ≥75yr; p =0.41). The rate of all-cause death was higher in elderly patients derived by an increase rate of death from non-cardiovascular causes: 1.7 (1.5-2.0) in <75yr vs. 3.4 (2.9-4.1) in ≥75yr; p <0.001 (Supplemental Table 5).

Adverse events

Older and younger patients in the placebo group, experienced similar frequency of adverse events (6% for hyperkalemia and 24% for worsening renal function. Older patients (≥75 years) taking a MRA experienced more frequently worsening renal function (eGFR drop >30%) during the follow-up compared to those <75 years (39% vs. 31%; interactionp =0.017), but not hyperkalemia (16% vs. 14%; interactionp =0.30) (Table 3).

Discussion

Our IPD meta-analysis suggests that MRAs are equally beneficial in younger and older HF patients, a beneficial effect that is more marked in HFrEF but homogenous across HFrEF and HFpEF. Compared to the younger, older patients may experience more worsening renal function episodes (but not hyperkalemia ones), in consequence a closer treatment monitoring may be required in this subpopulation.

The great majority of patients with HF are aged above 65 years, and in many contemporary studies a significant proportion of patients are 75 or more. These older patients are often female and have multiple comorbidities [START_REF] Conrad | Temporal trends and patterns in heart failure incidence: a population-based study of 4 million individuals[END_REF][START_REF] Levy | Long-term trends in the incidence of and survival with heart failure[END_REF] . In contrast, the mean age of the patients participating in each of RALES, EMPHASIS-HF and TOPCAT-Americas was 65, 69 and 72 years and the majority were male, in keeping with most other trials. However, by pooling the three trials we had a sufficient number of patients and events to assess the efficacy and safety of MRAs in truly elderly individuals with HF.

In the elderly (≥75 years) HF patients the rate of death from non-cardiovascular causes was overall low (3.4 events per 100 person-years), but higher than in younger patients (1.7 events per 100 person-years), supporting the notion that elderly HF patients are more prone to non-cardiovascular competing-events, on which cardiovascular drugs may fail to act [START_REF] Vaduganathan | Mode of Death in Heart Failure With Preserved Ejection Fraction[END_REF] ; it should be however noted that the relative and absolute reduction in all-cause death with MRAs was marked and similar to that observed in younger patients. Given that MRAs are inexpensive, widely available and overall well-tolerated, they should be given more priority for treating HF patients around the world [START_REF] Ferreira | Tailoring mineralocorticoid receptor antagonist therapy in heart failure patients: are we moving towards a personalized approach?[END_REF] . Notwithstanding, a close monitoring and assessment of serum potassium and renal function are warranted in patients taking MRAs, particularly in the elderly and those with renal impairment [START_REF] Eschalier | Safety and efficacy of eplerenone in patients at high risk for hyperkalemia and/or worsening renal function: analyses of the EMPHASIS-HF study subgroups (Eplerenone in Mild Patients Hospitalization And SurvIval Study in Heart Failure)[END_REF] ; however, it should be stressed out that the beneficial effect of MRAs in reduced major cardiovascular events is preserved across the continuum of age and renal function. Careful dose adjustment may also be required [START_REF] Rossignol | Balancing Benefits and Risks of Spironolactone in HFpEF and Chronic Kidney Disease Patients: Mind the Gap of Biological Monitoring! JACC Heart Fail[END_REF] . For example, in EMPHASIS-HF eplerenone dose was stratified by renal function, whereby patients with an eGFR of 50 ml/min or above received 50 mg/day of eplerenone and those with eGFR below 50 ml/min received 25 mg/day. The effect of eplerenone was similar between the renal function/dose strata [START_REF] Ferreira | Renal function stratified dose comparisons of eplerenone versus placebo in the EMPHASIS-HF trial[END_REF] . Inn view of this, eplerenone should not be given at a dose above 25 mg/day in patients with renal impairment. Whether elderly patients also require lower MRA dose requires further study, but this possibility underlay the design of TOPCAT, where spironolactone was initiated at a dose of 15 mg/day, which could be increased to 45 mg/day over a period of 4 months.

The anti-fibrotic properties of MRAs may also be of interest in elderly populations, where organ fibrosis (e.g. myocardium, kidney, joints) contribute to the impairment of lifequality, progression of HF, arrhythmias and ultimately death [START_REF] Ferreira | Collagen biomarker bioprofiles predicting the antifibrotic response to eplerenone in myocardial infarction: findings from the REMINDER trial[END_REF][START_REF] Ferreira | Potential spironolactone effects on collagen metabolism biomarkers in patients with uncontrolled blood pressure[END_REF] .

Another note to highlight that the cardiovascular event rates were lower (about 3-fold) in HFpEF patients (i.e. TOPCAT-Americas) compared with HFrEF (RALES plus EMPHASIS-HF), which consistently provided an absolute treatment benefit of higher magnitude in HFrEF; without evidence of relative treatment effect differences. The differences in all-cause death were less marked, suggesting that patients with HFpEF may be at higher risk to die from non-cardiovascular causes. These findings are relevant for the design of future trials, where the cardiovascular and competing non-cardiovascular eventrates should be taken in account.

Overall, our findings strongly support the use of MRAs in elderly HF patients. We hope that this information may serve a "nudge" for a better prescription.

Limitations

The present study has limitations. Our results cannot be extrapolated to patients that were excluded from the RALES, EMPHASIS-HF and TOPCAT trials, particularly patients with "severe" renal impairment (i.e. eGFR <30 ml/min) and hyperkalemia (K + >5.5 mmol/L). Our results are based on the assumption that both spironolactone and eplerenone provide similar treatment effect; although spironolactone and eplerenone differ in their molecular structure, pharmacokinetics, and pharmacodynamics, it is generally accepted that the benefits of different MRAs represent a "class effect" [START_REF] Iqbal | Selection of a mineralocorticoid receptor antagonist for patients with hypertension or heart failure[END_REF] . As these are selected trial populations, the rate of safety events may be under-represented when compared to "real world" patients. These are three distinct patient-populations, especially patients with HFpEF, that have more co-morbid conditions and lower cardiovascular event rates. Nonetheless, the treatment effect in the elderly populations from these trials was homogeneous and strongly suggests a beneficial MRA effect. The present study was underpowered to assess the effect of the treatment in patients aged above 80 years, however no signal of harm was observed in this "very elderly" population. The doses and the treatments (spironolactone or eplerenone) could vary between trials and according to the respective dose-adjustment algorithms, but for the purpose of this analysis we assume that MRAs have a "class effect".

Conclusion

MRAs reduce morbidity and mortality in elderly patients (aged 75 or more) with HF, with a homogenous treatment effect between HFrEF and HFpEF (and between younger and older patients). Compared to the younger patients, the older ones may experience more frequent episodes of worsening renal function when treated with a MRA, warranting close surveillance and dose adaptations in this population.

Clinical Perspectives

The findings presented herein, strongly support the use of MRAs in older HF patients, as they benefit similarly to younger patients without excessive hyperkalemia.

Translational Outlook

Future trials with MRAs could focus on elderly populations, assessing the trade-off between the benefits and risks incorporating quality of life measures. Age continuous interaction p Hyperkalemia 0.30 0.51 Worsening renal function 0.017 0.049 Hyperkalemia was defined as any laboratory value of serum potassium above 5.5 mmol/L during the follow-up and worsening renal function was defined as an estimated glomerular filtration rate drop greater than 30% during the follow-up. P-value from the Chi-square test.

Figure 1 .

 1 Figure 1. Survival curves of the MRA treatment effect (primary outcome of CV death or HF hospitalization) (a) Age ≥75 years (b) Age <75 years Log-rank test p-value <0.001 for both age subgroups; p for interaction with age =0.39. Legend: MRA, mineralocorticoid receptor antagonist; CV, cardiovascular; HF, heart failure.

Figure 2 .

 2 Figure 2. Adjusted predictions of the MRA treatment effect (for the primary outcome of cardiovascular death or heart failure hospitalization) across the continuum of age Legend: MRA, mineralocorticoid receptor antagonist.For any given age, MRA treatment reduces the risk of events associated with ageing.

Figure 3 .

 3 Figure 3. Adjusted predictions of the MRA treatment effect (for the primary outcome of cardiovascular death or heart failure hospitalization) across the continuum of age and renal function Legend: MRA, mineralocorticoid receptor antagonist; eGFR, estimated glomerular filtration rate.For any given age and eGFR, MRA treatment reduces the risk of major cardiovascular events

Table 1 & Supplemental Table 2). The

 1 mean age in the older age group was 80±4 years and the mean age in the younger age group was 64±7 years.

Table 1 . Baseline characteristics of the study population.

 1

	Age ≥75 years
	Patient characteristics

Table 2 . MRA treatment effect on the study population Outcome/Events Placebo MRA HR (95%CI) p-value Heterogeneity p Age ≥75 years HFrEF (RALES+EMPHASIS-HF); Total n =1009; Placebo n =498; MRA n =511.

 2

	CV death or HF hosp.	215	170	0.69 (0.56-	<0.001	0.70
		(43.2%)	(33.3%)	0.84)
	CV death	137	113	0.74 (0.58-	0.020	0.062
		(27.5%)	(22.1%)	0.96)
	All-cause death	160	134	0.75 (0.60-	0.016	0.059
		(32.1%)	(26.2%)	0.95)
	HFpEF (TOPCAT-Americas); Total n =747; Placebo n =355; MRA n =392.
	CV death or HF hosp.	116	111	0.83 (0.63-	0.20	-
		(32.7%)	(28.3%)	1.08)
	CV death	61 (17.2%) 50 (12.8%)	0.73 (0.50-	0.10	-
				1.06)
	All-cause death	105	102	0.87 (0.66-	0.32	-
		(29.6%)	(26.0%)	1.14)
	Overall population (HFrEF+HFpEF combined); Total n =1756; Placebo n =853; MRA n
	=903.
	CV death or HF hosp.	331	281	0.74 (0.63-	<0.001	0.52
		(38.8%)	(31.1%)	0.86)
	CV death	198	163	0.74 (0.60-	0.005	0.17
		(23.2%)	(18.1%)	0.91)
	All-cause death	265	236	0.80 (0.66-	0.013	0.13
		(31.1%)	(26.1%)	0.95)

Age <75 years HFrEF (RALES+EMPHASIS-HF); Total n =3391; Placebo n =1716; MRA n =1675.

  MRA, mineralocorticoid receptor antagonist; CV, cardiovascular; Heterogeneity p, Cochran's Q p-value of treatment effect by study interaction.

	CV death or HF hosp.	797	572	0.67 (0.60-	<0.001	0.30
		(35.6%)	(26.4%)	0.75)
	CV death	452	328	0.71 (0.61-	<0.001	0.91
		(20.2%)	(15.1%)	0.82)
	All-cause death	539	403	0.74 (0.65-	<0.001	0.81
		(24.0%)	(18.6%)	0.84)
	Overall population treatment effect in Older vs. Younger patients
	Outcome/Interaction	Age dichotomic (<75 vs. ≥75) interaction	Age continuous interaction
	test	p			p
	CV death or HF hosp.		0.39		0.26
	CV death		0.80		0.37
	All-cause death		0.52		0.69
	Legend:
	CV death or HF hosp.	633	441	0.65 (0.57-	<0.001	0.75
		(36.9%)	(26.3%)	0.72)
	CV death	386	282	0.72 (0.62-	<0.001	0.90
		(22.5%)	(16.8%)	0.84)
	All-cause death	439	323	0.72 (0.63-	<0.001	0.94
		(25.6%)	(19.3%)	0.83)
	HFpEF (TOPCAT-Americas); Total n =1020; Placebo n =526; MRA n =494.
	CV death or HF hosp.	164	131	0.81 (0.64-	0.069	-
		(31.2%)	(26.5%)	1.02)
	CV death	66 (12.6%)	46 (9.3%)	0.72 (0.50-	0.094	-
				1.06)
	All-cause death	100	80 (16.2%)	0.83 (0.62-	0.21	-
		(19.0%)		1.11)
	Overall population (HFrEF+HFpEF combined); Total n =4411; Placebo n =2242; MRA n
	=2169.
						21

Table 3 . Side-effects during the follow-up Side-effects Placebo MRA P-value Age ≥75 years HFrEF (RALES+EMPHASIS-HF); Total n =1009; Placebo n =498; MRA n =511.

 3

	Hyperkalemia	35 (7.0%)	81 (15.9%)	<0.001
	Worsening renal function	104 (20.9%)	183 (35.8%)	<0.001
	HFpEF (TOPCAT-Americas); Total n =747; Placebo n =355; MRA n =392.
	Hyperkalemia	18 (5.1%)	67 (17.1%)	<0.001
	Worsening renal function	110 (31.0%)	172 (43.9%)	<0.001
	Overall population (HFrEF+HFpEF combined); Total n =1756; Placebo n =853; MRA n
	=903.
	Hyperkalemia	54 (6.3%)	148 (16.4%)	<0.001
	Worsening renal function	214 (25.1%)	355 (39.3%)	<0.001
	Age <75 years
	HFrEF (

RALES+EMPHASIS-HF); Total n =3391; Placebo n =1716; MRA n =1675.

	Hyperkalemia	117 (7.0%)	231 (14.1%)	<0.001
	Worsening renal function	330 (20.7%)	428 (27.4%)	<0.001
	HFpEF (TOPCAT

-Americas); Total n =1020; Placebo n =526; MRA n =494.

	Hyperkalemia	27 (5.2%)	74 (15.0%)	<0.001
	Worsening renal function	179 (34.0%)	217 (43.9%)	0.001
	Overall population (HFrEF+HFpEF combined); Total n =4411; Placebo n =2242; MRA n
	=2169.
	Hyperkalemia	144 (6.6%)	305 (14.3%)	<0.001
	Worsening renal function	509 (24.0%)	645 (31.3%)	<0.001
	Overall

population side effects in Older vs. Younger patients

	Side-effect/Interaction	Age dichotomic (<75 vs. ≥75) interaction
		p

JPF, PR, FZ are supported by the French National Research Agency Fighting Heart Failure (ANR-15-RHU-0004), by the French PIA project "Lorraine Université d'Excellence" GEENAGE (ANR-15-IDEX-04-LUE) programmes, and the Contrat de Plan Etat Région Lorraine and FEDER IT2MP. X.R. has received support from the SEC-CNIC CARDIOJOVEN fellowship program.

Funding

None.

Disclosures

None.


Victoria Teissier

Philippe Tresson
email: philippe.tresson@hotmail.fr

Julien Gaudric

Jean-Michel Davaine

Caroline Scemama

Mathieu Raux

Laurent Chiche

Fabien Koskas

for any proven or suspected knee dislocation. It shows the importance of timely management that conditions the functional prognosis

Background and Objective: Arterial injury secondary to acute knee dislocation (KD) is a rare but devastative complication. The aim of this study is to evaluate functional sequelae and factors of poor prognosis.

Methods:

A retrospective monocentric series of consecutive knee dislocation with acute ischemia by popliteal artery injury was analyzed between 2005 and 2017. Main outcome was amputation rate.

Results: Sixteen dislocations were included. Nine (56%) were secondary to public road accident, 5 (31%) were due to falls from height and 2 (13%) to sports injuries. Dislocation was posterior in 8 (50%) cases. Regarding arterial injury, there were 7 (44%) ruptures, 7

INTRODUCTION

Acute knee dislocations (KD) are uncommon orthopedic injuries, with approximated occurrence of 0.02% to 0.1% of all musculoskeletal injuries [START_REF] Rihn | The acutely dislocated knee: evaluation and management[END_REF]. They usually result from high energy traumas or low energy traumas in overweight patients [START_REF] Peltola | Knee dislocation in overweight patients[END_REF][START_REF] Henrichs | A Review of Knee Dislocations[END_REF]. Associated cutaneous lesions, compartment syndrome, irreducible dislocation and neurovascular injuries are often present and complicate management [START_REF] Moatshe | Demographics and Injuries Associated With Knee Dislocation A Prospective Review of 303 Patients[END_REF].

Popliteal injuries are associated with knee dislocations in 2% to 11% of the cases [START_REF] Rihn | The acutely dislocated knee: evaluation and management[END_REF][START_REF] Moatshe | Demographics and Injuries Associated With Knee Dislocation A Prospective Review of 303 Patients[END_REF][START_REF] Robertson | Dislocation of the knee[END_REF][START_REF] Sillanpää | Incidence of knee dislocation and concomitant vascular injury requiring surgery: A nationwide study[END_REF].

Anatomic location of popliteal artery places it at great risk for damage during KD, since it is tethered firmly around knee joint [START_REF] Kropman | Variations in anatomy of the popliteal artery and its side branches[END_REF].The antero-posterior tibial displacement leads to popliteal artery elongation with intimal tear, dissection and sometimes acute thrombosis [START_REF] Henrichs | A Review of Knee Dislocations[END_REF][START_REF] Kauvar | Vascular Trauma: Extremity[END_REF][START_REF] Witz | Isolated complete popliteal artery rupture associated with knee dislocation. Case reports[END_REF][START_REF] Kirby | Recanalization of an Occluded Popliteal Artery following Posterior Knee Dislocation[END_REF].

Incidence of vascular injury in dislocation vary between 4.8% in low-velocity injuries and 65% in high energy trauma [START_REF] Robertson | Dislocation of the knee[END_REF].

Popliteal artery lesion secondary to KD is a limb-threatening condition [START_REF] Sillanpää | Incidence of knee dislocation and concomitant vascular injury requiring surgery: A nationwide study[END_REF][START_REF] Levy | Editorial comment: Symposium: Management of the dislocated knee[END_REF] which mandates prompt diagnosis and emergency care. Comprehensive treatment involves resuscitation team, orthopedic and vascular surgeons. The aim of the present study was to report outcomes and prognosis factors of popliteal traumatic occlusion after KD in a referral trauma center.

METHODS

Patient selection

Patients presenting with KD associated to arterial injury, who underwent surgical treatment, between January 2005 and May 2017 were included in this retrospective single center study.

KD was defined as complete displacement of tibia with respect to the femur, assessed on radiographs or CT-scan [START_REF] Versier | La luxation du genou[END_REF]. Kennedy KD classification is based on tibia displacement direction [START_REF] Kennedy | Complete Dislocation of the Knee Joint[END_REF] (table I). Body CT-scan was systematically performed in case of severe trauma.

Standard limb radiographies were done for mono traumatism. Suspected KD spontaneously reduced underwent systematic angio-CT-scan. Vascular injury was considered when following clinical findings were present: distal pulse loss, lower limb skin pallor, pain, or distal neurological deficit [START_REF] Weinberg | Can Vascular Injury be Appropriately Assessed With Physical Examination After Knee Dislocation?[END_REF]. In these cases a enhanced CT-scan was systematically performed. Vascular injury was defined by popliteal artery trauma as intimal tear, arterial rupture, and dissection, with or without thrombosis. Abbreviated Injury Scale (AIS), Injury Severity Score (ISS), Revised Trauma Score (RTS) and Trauma Related Injury Severity Score (TRISS) were calculated at patients' arrival.

TRISS score is calculated using updated regression coefficients, AIS and ISS [START_REF] Boyd | Evaluating trauma care: the TRISS method. Trauma Score and the Injury Severity Score[END_REF][START_REF] Raux | Comparison of the Prognostic Significance of Initial Blood Lactate and Base Deficit in Trauma Patients[END_REF].

Surgery was performed in

Short term follow-up (day 30)

Early follow-up included length of stay and number of days in intensive care unit. Technical success rate and early complications were reported according to reporting standards of the Ad Hoc committee for Standardized Reporting Practices in Vascular Surgery/International Society for Cardiovascular Surgery. In this reporting, mortality was defined as "early" when it occurred before day 30. Three types of complications were defined (systemic, local nonvascular or vascular and implant-related). Early secondary amputation, orthopedic or vascular revision surgery were recorded (before day 30).

Long term follow-up and patient evolution

Curative anticoagulation was introduced for 6 weeks after surgery when thrombectomy was performed during surgery. Every patient underwent Doppler ultrasound at month 1, 3, 6 and yearly thereafter. Clinical and radiological follow-up were recorded. Vascular and orthopedic complications during follow-up were analyzed: bypass stenosis >60% or thrombosis, knee pain or stiffness, osteoarticular infection or revision surgery (including delayed amputation, osteosynthesis material removal, arthrolysis). Follow-up duration and methods were assessed for each patient. Knee functional outcome was also retrieved: Oxford Knee Score (OKS) [START_REF] Murray | The use of the Oxford hip and knee scores[END_REF] was calculated at last follow-up. Oxford Knee Score (OKS) is a Patient Reported Outcome questionnaire with twelve questions. It covers function and pain associated with the knee. This questionnaire is short, practical, reliable, valid and sensitive to clinically important changes over time.

Statistical analysis

Continuous variables were presented median (extreme values) according to the low sample size. Data were compared using non-parametric Mann-Whitney test. Categorical variables were presented as percentage and compared using Fisher-exact test. P-value <0.05 was considered statistically significant. Risk factors for amputation have been investigated sought (tobacco, diabetes, obesity, dislocation type, popliteal injury type). Non-parametric Wilcoxon-Mann Whitney test was used to compare pre and post-trauma OKS. Data were anonymously recorded in Excel 2016 (Microsoft, Richmond, WA, USA). Statistical analysis was performed with SPSS Advanced Statistics 20.0 software (IBM, Armonk, NY, USA).

RESULTS

Descriptive data

Among 141 KD handled in our trauma center during the study period, 16 (11%) were associated with vascular injuries in fifteen patients (69% of men). Mean age was 40.8 years (16-61 years).

Clinical, demographics and traumatism data are shown in table II and III. Median TRISS score was 1.4 % (1-48.8%).

According to the Kennedy classification (13), dislocation was posterior (type A) in 8 cases (50%), anterior (type B) in 4 cases (25%) (Figure 1a, 1b) and lateral (type C) in 4 cases (25%). There were 2 (13%) cases of associated multidirectional rotational instability. Fracture was associated in 7 cases (44%), with 1 floating knee (6%) (flail knee joint resulting from fractures of the shafts or adjacent metaphysis of the femur and ipsilateral tibia ( 18)).

The diagnosis of popliteal artery lesion was based on the presence of symptoms of ischemia in eleven cases and on systematic imaging in five cases.

Eleven (69%) KD with vascular trauma were associated with signs of acute ischemia: 8 (50%) had sensory-motor loss and 3 (19%) had an isolated sensitivity loss. For the 5 others KD, vascular injury was suspected on: 4 (25%) suggestive mechanism of trauma (2 anterior trauma during sport, one during a public road accident and one fall in an obese patient), 1 (6%) temperature asymmetry between the 2 limbs.

Pre-operative imaging and intraoperative findings revealed 2 popliteal occlusion, 7 ruptures with 3 occlusions and 7 dissections with 3 occlusions patent artery. Among the 8 patients with sensory-motor loss, popliteal lesion was 2 occlusions, 3 dissections and 3 ruptures. The dissections and ruptures were associated with arterial occlusion, the 8 occlusions of the serie. Four patients had in addition an associated nerve injury and 3 associated popliteal vein injury.

Median time-to-surgery (time between trauma and vascular repair) was 7 hours (3,25 -60,92 hours); time-to-surgery was 8,75 hours (3,25 -60,92 hours) for patients who presented signs of acute ischemia. One patient came to the emergency department 3 days after the trauma. his patient obese fell from her height and came late to the emergency in front of signs of ischemia. All patients underwent immediate surgical repair. Thirteen patients (81%) had primary revascularization, one of them in two stages, after a temporary shunt was inserted and 3 (19%) had orthopedic reduction and stabilization first.

Operative characteristics are summarized in table IV. Surgical repair included either venous bypasses in 14 cases (87%), resection with direct anastomosis in 1 case (6%) and isolated thrombectomy in 1 case (6%). Mechanical thrombectomy of at least one leg artery was associated in 11 cases (69%). For the patients who had severe ischemia, five had primary revascularization, one had a vascular shunt first, and 2 had orthopedic reduction and stabilization first. A post-operative fasciotomy was performed in all these cases (7 were immediate and1 was delayed). Pathways were anatomical excepted for one due to significant tissue decay.

Control arteriography (12 cases -75%) or Doppler ultrasound (4 cases -25%) assessed immediate revascularization patency in all cases. One patient (6%) had a femoral vein bypass with saphenous vein to treat a venous rupture. Two popliteal venous thrombosis were identified and treated by anticoagulation. Four patients (25%) were obese according to the Body Mass Index classification (BMI >30) and suffered a KD while falling from their height. Dislocation was anterior in 2 cases (13%), posterior in 1 case (6%) and rotational in 1 case (6%). There were 3 irreducible KD with popliteal rupture and 1 spontaneously reduced KD with thrombosis that presented to the ER 3 days after the trauma with irreversible limb ischemia.

Orthopedic treatment mainly comported temporary stabilization with external fixator in 13 cases (81 %). There was 1 open reduction and internal fixation (ORIF) (6%), 1 long-leg cast (6%) and 1 ligamentoplasty (6%).

Short term follow-up (day 30)

Median length of stay was 36 (6-87) days, with median 1 (0-18) days in intensive care unit.

No patient died for the first 30 postoperative days. Three (19%) patients required transfemoral amputation within 30 days: for sepsis from muscle gangrene. They all first presented ischemia with sensory-motor loss. For one patient, time to surgery was very long (>60 hours); for the other 2 patients, time to surgery was <9hours. One of these patients experienced an isolated thrombectomy first and was secondary treated with an above to below the knee bypass for short term thrombosis ( day 2).

One (6%) patient presented a short term thrombosis (day 1). Twelve (75%) patients had systematic associated fasciotomy and one patient required a secondary fasciotomy for post operative compartment syndrome.

Postoperative period data are summarized in table V. All patients underwent Doppler ultrasound in the month following surgery.

Delayed follow-up superior to 30 days and clinical outcomes

Median follow-up was 23 months . All bypasses remained patent during follow-up.

Survival rate was 100% at the end of the follow-up. No more amputation was noted. Patency rate was 100% during follow-up.

Patients were contacted, regarding the Oxford Knee Score (OKS), 2 (13%) were not evaluated and had only a vascular follow-up. Mean pre-trauma OKS was 45.7 ± 3.2 (39-48), median 47 and last OKS 27.4 ± 11.9 (6-40), median 30, (p=0.00028).

DISCUSSION

Functional outcomes

KD associated with vascular injury is a rare and serious condition responsible for a poor functional prognosis. Presence of arterial or nerve injury associated with traumatic KD is significantly correlated with poor outcome, high rate of limb loss, functional disability, mortality and associated to a lower range of motion.

Fanelli et al reported sequelae after a long follow-up for patients with severe knee dislocation requiring surgical repair. They concluded that stability was restored but arthritis was frequent (23%). In our series 5 (33%) patients had an OKS <29 that indicates severe knee disability.

Association of osteoarticular trauma and ischemic lesions worsens functional prognosis. Thus 50% of patients who had a KD with vascular injury had severe functional sequelae due to osteoarticular lesions.

A fight for limb salvage through an efficient multidisciplinary approach

Literature data tend to show that, in case of associated acute ischemia, amputation rate vary in proportion with ischemic time [START_REF] Lustig | Dislocations of the knee, complex knee ligament injury, what care?[END_REF]. Inadequate initial assessment and delay of vascular repair lead to an increased amputation rate, up to 60-80% [START_REF] Witz | Isolated complete popliteal artery rupture associated with knee dislocation. Case reports[END_REF]. Urgent above-knee amputation is also performed when patients become febrile septic [START_REF] Pourzand | Management of high-risk popliteal vascular blunt trauma: Clinical experience with 62 cases[END_REF]. Bonnevialle et al. [START_REF] Bonnevialle | Traumatismes des genoux associés à une rupture de l'artère poplitée -54 cas[END_REF] reported 9 amputations in their multicentric cohort of 54 knee trauma with popliteal artery injury (16,6%). Mean ischemic time seemed to be higher in the amputation group (9,6 hours vs 8,8 hours). We did not find a statistically significant injury-related element with a high risk of amputation but very long time to surgery (>60 hours) is probably the major factor for one patient. For the other 2 patients who underwent amputation, time to surgery was <9hours. On the other hand, they presented with severe ischemic clinical condition with signs of sensory and motor loss. Secondary necrosis of muscular compartments required amputation. Severity of initial clinical signs should lead to urgent revascularization. That means that despite a prompt revascularization in these young people, in the absence of any collateral network, ischemia can be extremely severe and irreversible. That is why it is very important to diagnose vascular trauma associated to KD.

Sensitive or motor deficit observed at arrival must be correlated with presence or absence of concomitant pulse by medical team. In case of suspicion, a CT-scan imaging must not be delayed. Doppler ultrasound easily controls popliteal artery and search a venous thrombosis provider of pulmonary embolism.

Unfortunately, no risk nor prognosis factor associated with limb amputation was statically highlighted in this study. It might be related to the small number of patients. Severity of the clinical symptoms with presence of a sensory motor loss seems to be the most pejorative element for the limb prognosis. A primary amputation can be discussed in front of those symptoms. Young age and context of trauma make this decision extremely difficult even in case of high TRISS score.

Since this is the largest monocentric series in medical literature to our knowledge, we believe that an optimized and standardized care of such patients, led to a shortened time-to-surgery and a reduced amputation rate. As an immediate care might be associated with lower amputation rates, a future multicentric cohort could identify the maximal acceptable delay before surgical care.

Technical points

Endovascular repair with implantation of a covered stent is not recommended due to the flexibility and bending that occurs at the knee joint [START_REF] Rastan | Stent placement versus balloon angioplasty for the treatment of obstructive lesions of the popliteal artery: a prospective, multicenter, randomized trial[END_REF]. Venous bypass is our gold standard for below knee revascularization mostly in case of trauma, vascular rupture or dissection.

The need for angiography in traumatic knee dislocation remains controversial; some recommend it in all cases of complete dislocation of the knee, and others use it only when there is a clinical doubt of artery injury [START_REF] Maitrias | Superficial femoral artery transposition repair for traumatic popliteal rupture[END_REF], or when specific site of injury is in doubt [START_REF] Yahya | Popliteal artery injury: Royal Perth experience and literature review[END_REF]. In our series as in our clinical practice, all acknowledged knee dislocations, suspected spontaneously reduced one and polytrauma patients underwent systematic angio-CT-scan.

When surgically repaired, completion arteriography, fasciotomy, and knee stabilization are important adjuncts to revascularization [START_REF] Kirby | Recanalization of an Occluded Popliteal Artery following Posterior Knee Dislocation[END_REF]. The patient who had a thrombectomy first may have an intimal tear that was not found during per-operative control angiography as it is challenging to assess.

Use of temporary vascular shunts is indicated when arterial injury is associated with extensive wounds with major loss of soft tissues and contamination [START_REF] Hornez | Temporary vascular shunt for damage control of extremity vascular injury: A toolbox for trauma surgeons[END_REF]. Temporary intravascular shunts allow rapid arterial flow restoration, avoidance of ligation, assessment of distal perfusion, and knee stabilization before definitive vascular reconstruction. There is no consensus on knee dislocations management with arterial involvement. Hossny et al. [START_REF] Hossny | Blunt popliteal artery injury with complete lower limb ischemia: is routine use of temporary intraluminal arterial shunt justified?[END_REF] demonstrated that temporary arterial shunting after blunt lower limb trauma significantly reduced total ischemic time, complications, repeat operations, amputation, and hospitalization. Xu and al. [START_REF] Xu | Early diagnosis and treatment of trauma in knee joints accompanied with popliteal vascular injury[END_REF] proposed to first perform orthopedic reduction and stabilization when limb ischemia was short (1 hour). Revascularization had to be done first when ischemia time was longer than 1 hour with a relatively stable knee. We think strategy is more complex and has to be adapted to ischemic clinical signs and knee instability. As a bypass can be compromised when a very unstable member is mobilized, temporary vascular shunts can be primarily done if ischemic signs are severe. A shunt can be used first to allow treatment of other trauma and vital lesion like usually done in damage control surgery. It was used first in one case in this series. Revascularization first was preferred to avoid complication of shunt setting up in case of severe ischemia. Three patients had knee stabilization first in front of knee instability and only distal sensibility loss. Presence of neurological deficit, whether systematized or not, must evoke severe ischemia and must make discuss of revascularization gesture first. Contralateral vein is usually used in case of vascular trauma. Ipsilateral saphenous vein can be used if deep veins remained patent and not affected especially in front of blunt trauma. It can be objectified on late phase CT scan.

Obesity

Mechanism of injury for a morbidly obese patient can be as simple as ambulating or rising from a seated position (ultralow-velocity trauma) like the 4 patients who had a BMI >30 in our series.

Morbid obesity is a risk factor for both anterior (n=2) and posterior (n=1) dislocations due to the tremendous stresses placed on the stabilizing muscles and joints during movement [START_REF] Folt | Low-velocity knee dislocation in the morbidly obese[END_REF] and the suspicion for occult knee dislocation should be heightened in any obese patient with knee pain after minor trauma [START_REF] Georgiadis | Changing presentation of knee dislocation and vascular injury from high-energy trauma to lowenergy falls in the morbidly obese[END_REF].

Three patients presented severe neurological loss with one external popliteal sciatic nerve injury. Azar et al. [START_REF] Azar | Ultra-low-velocity knee dislocations[END_REF] have shown that nerve injury, associated fracture or vascular injury are significantly increased in patients with a higher BMI. They reported a significant association between BMI and combined vascular and nerve injury in their series of 17 knee dislocations in obese patients. This higher rate of neurovascular injury can be explained by the relevant anatomy of the knee joint in combination with the mechanism of injury. Ultralow-velocity knee dislocations most commonly result in an anterior dislocation, with hyperextension mechanism. This type of dislocation leads to the rupture of the posterior capsule and tethering of the contents of the popliteal fossa, including popliteal artery. Although low-velocity, these injuries are actually high energy due to the high mass of the injured patients and neurovascular associated complications should be thoroughly sought [START_REF] Carr | Knee Dislocation in the Morbidly Obese Patient[END_REF]. Diagnosis may be difficult; clinical examination may be difficult especially in those patients. Vascular imaging has to be performed on case of trauma with suggestive mechanism.

Limits

The most important limitation of this study is based on clinical challenge in KD diagnosis [START_REF] Weinberg | Can Vascular Injury be Appropriately Assessed With Physical Examination After Knee Dislocation?[END_REF][START_REF] Bonnevialle | Traumatismes des genoux associés à une rupture de l'artère poplitée -54 cas[END_REF][START_REF] Xu | Early diagnosis and treatment of trauma in knee joints accompanied with popliteal vascular injury[END_REF]. It is always possible that, after spontaneous reduction, severity of ligamentous injuries may be underestimated and KD can thus be misdiagnosed as an isolated cruciate ligament injury or a knee sprain [START_REF] Peltola | Knee dislocation in overweight patients[END_REF][START_REF] Georgiadis | Changing presentation of knee dislocation and vascular injury from high-energy trauma to lowenergy falls in the morbidly obese[END_REF][START_REF] Washer | Knee dislocation: initial assessment and implications for treatment[END_REF]. Therefore, some patients may have been treated for isolated popliteal artery injury without initial assessment of the dislocation. In addition, it is possible that a proportion of ligament reconstructive surgery performed after acute phase were missed [START_REF] Sillanpää | Incidence of knee dislocation and concomitant vascular injury requiring surgery: A nationwide study[END_REF]. In contrast, all acute surgeries performed during the first hospitalization period, such as amputations and acute vascular surgery, were accurately recorded. Another limitation is that we were not able to assess less severe and non-surgical vascular injuries from our database.

Choice of the OKS functional score can be discussed. It was designed specifically for the assessment of joint replacement but has also been used for evaluating other interventions (pharmacological treatment, after conservative surgery or after fractures). Although it has not been validated for these uses, authors have assessed its high reproducibility and accuracy for such cases [START_REF] Murray | The use of the Oxford hip and knee scores[END_REF].

The small number of cases (n=16) reflects rarity and makes it difficult to draw significant conclusions but data retrieved from this series reflect figures from larger multicentric cohorts (incidence rate, sex ratio).

CONCLUSION

Vascular injury in KD is rare and sometimes difficult to diagnose. This must be sought systematically even in low-velocity trauma considering serious disabling functional consequences. Clinical examination is associated with imaging examination, at least Doppler ultrasound post reduction or CT if in doubt. This study shows poor functional outcomes due to gravity of vascular lesion in orthopedic trauma patient with healthy arteries. Collaboration between different surgery teams and resuscitation is the key element for a better efficiency.

Future large-scale and prospective studies should analyze factors that may predispose to vascular injuries and limb amputation after knee dislocation. • None

• Tibia

• Femur

• Fibula

• Floating knee

  a single referral center. Orthopedic and vascular management outcomes were analyzed. Demographic characteristics, cardiovascular risk factors as well as initial clinical presentation and preoperative workups were recorded. Orthopedic dislocation characteristics, arterial lesion, outflow, intraoperative data and postoperative course data were intentionally sought. Potential risk factors and prognostic factors for early and delayed amputation were analyzed. Local ethical committee board approved the study. Surgical approach Closed knee reduction was performed and post reduction stabilization was obtained either by conservative treatment if the joint felt stable or by surgical treatment if needed (external fixation, open reduction internal fixation). Resection with direct anastomosis was performed in case of short lesion. A venous bypass was used in case of long arterial occlusion after systemic heparinization (50UI/kg). Popliteal artery downstream of proximal anastomosis and upstream of distal anastomosis was ligated in case of rupture. Intraoperative angiography or Doppler ultrasound ascertained bypass patency and absence of distal emboli. Intraoperative fluoroscopy was used to assess knee reduction, post reduction testing and accurate placement of pins.

• Score 0 to 19 • Score 20 to 29 •

 1929 Score

Table I .

 I Knee dislocation classification[START_REF] Kennedy | Complete Dislocation of the Knee Joint[END_REF]

	Kennedy classification based on direction of displacement of the tibia
	Anterior	Arterial injury is generally an intimal tear due to traction
	Posterior	Highest rate of vascular injury (25%) with complete tear of popliteal artery
	Medial
	Lateral	Highest rate of peroneal nerve injury
	Rotational	Most commonly posterolateral dislocation. Usually irreductible.

Table II .

 II Demographics; cardiovascular risk factors and clinical characteristics 362

	366	Table III. Traumatism data Variable	n	%
		Variable Associated fracture	n	%
		Total	16
		Age (yr): mean ± SD (max-min)	40.8 ± 14.7 (16-61)
		Sex
		• Men	11
		• Women	5
		Hypertension	4
		Diabetes	1	6
		Dyslipidemia	2
		BMI: mean ± SD (min-max)	29,43 ± 11,17 (20,45-62,5)
		Smoker	5
		Coronary artery disease	0
		Cerebrovascular event	0
		Orthopedic history	0
	363
	364