Dr Camille Tron
email: camille.tron@chu-rennes.fr

Marie-José Ferrand-Sorre

Julie Querzerho-Raguideau

Jonathan Maurice

M Chemouny

Pauline Houssel-Debry

Marie-Clémence Verdier

Eric Bellissant

Florian Lemaitre

Jonathan M Chemouny

Volumetric absorptive microsampling for the quantification of tacrolimus in capillary blood by high performance liquid chromatography-tandem mass spectrometry

Keywords: tacrolimus, volumetric absorptive microsampling, liquid chromatography, mass spectrometry, therapeutic drug monitoring

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Introduction

Immunosuppressive drugs (ISDs) have revolutionized the management of solid organ transplant recipients, by considerably reducing the incidence of acute rejection and improving graft and patient survival [START_REF] Shrestha | Two Decades of Tacrolimus in Renal Transplant: Basic Science and Clinical Evidences[END_REF]. Tacrolimus is the most widely prescribed ISD in solid organ transplant recipients. However, tacrolimus has a narrow therapeutic window, with low drug exposure increasing the risk of acute rejection and high exposure increasing the risk of drug toxicity (e.g., nephrotoxicity, neurotoxicity, diabetes). Therapeutic drug monitoring (TDM) is therefore essential for tacrolimus, to ensure treatment efficacy and safety through ISD dose adjustments. TDM is usually based on the determination of drug concentrations in whole blood obtained by venipuncture, which is an invasive procedure particularly because it has to be frequently repeated throughout the follow-up of the transplanted patient. Furthermore, venipuncture may prove difficult in some patients (e.g., elderly patients with a loss of vein patency, or very young patients) or should be minimized in others (e.g., frail patients, immunocompromised patients on cancer treatment). Moreover, venipuncture requires trained staff, and must be performed within institutions, such as hospitals or external biomedical analysis laboratories. As a result, for practical reasons, TDM is usually limited to a single measurement of trough concentration (C 0 ) in whole blood. The usual therapeutic range of tacrolimus C 0 is 3-15 ng/mL. It is assumed that C 0 is a surrogate marker of drug exposure (i.e., the area under the curve of drug concentrations (AUC)) at the time point concerned. Unfortunately, the relationship between AUC and C 0 may be weak, with coefficients of determination varying considerably between studies. That for tacrolimus, for example, has been reported to range from 0.11 to 0.94 [START_REF] Wong | Abbreviated tacrolimus area-under-the-curve monitoring for renal transplant recipients[END_REF][START_REF] Alloway | Conversion from twice daily tacrolimus capsules to once daily extended-release tacrolimus (LCP-Tacro): phase 2 trial of stable liver transplant recipients[END_REF]. In some clinical situations, the adaptation of tacrolimus dose according to AUC may therefore be a valuable alternative to C 0 determination to optimize tacrolimus treatment [START_REF] Brunet | Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report[END_REF].

Microsampling approaches are therefore of increasing interest as simpler, less invasive alternative to venipuncture to perform TDM of tacrolimus. Indeed, microsamples can easily be collected by the patients themselves at home. In addition, due to the low level of invasiveness of the process, more samples can be collected than with venipuncture. Thus, microsampling could be very convenient to calculated tacrolimus AUC from concentrations measured at several sampling times within the dosing interval. Among microsampling strategies, Dried Blood Spot (DBS) raised many hope in bioanalysis. However, hurdles have been identified for quantitative applications of DBS. (e.g., small amounts of the drug for quantification, hematocrit effect, volcano effect or volume effect that can influence the repeatability of the measurement). Most of the pre-analytical and analytical issues relating to the use of DBS should be minimized by using a volumetric adsorptive microsampling (VAMS) device to collect peripheral blood, and by performing high-performance liquid-chromatography tandem-mass spectrometry (HPLC-MS/MS) to determine drug concentrations [START_REF] Protti | Tutorial: Volumetric absorptive microsampling (VAMS)[END_REF][START_REF] Velghe | Is the hematocrit still an issue in quantitative dried blood spot analysis?[END_REF].

The aims of this study were to develop and to validate a simple HPLC-MS/MS method for determining tacrolimus levels in blood collected with VAMS devices. The purpose of this assay is to be suitable for routine TDM, as a complementary tool to liquid venous sampling. In particular, the objectives are i) to allow home sampling to facilitate and to strengthen tacrolimus TDM based on C 0 in patient living far from the transplant centers or having intercurrent events (e.g. infection, drug-drug interaction), ii) to allows repeated sampling in hospital to monitor full AUC in specific clinical context (e.g. to assess the C 0 -AUC correlation, in case of intra-patient variability of C 0 , or in case of sides effects occurring despite C 0 in the therapeutic range). Hence, we applied the developed method to specimens obtained from transplant recipients, to demonstrate the feasibility and the suitability of the approach.

Materials and methods

Chemicals and materials

Tacrolimus was purchased from LGC standards (Folsheim, France). Ascomycin was purchased from Sigma-Aldrich (Saint-Quentin, France). LC/MS-grade methanol and acetonitrile were purchased from Carlo Erba Reagents (Val de Reuil, France). Ammonium sulfate, zinc sulfate heptahydrate and LC-MSgrade water were obtained from Fisher Chemicals (Waltham, USA). Calibrators and controls for lyophilized blood were purchased from Chromsystems (Gräfelfing, Germany). These samples were reconstituted in LC-MS-grade water. Blood matrix used to prepare additional quality controls and spiked samples for analytical validation experiments as well as samples used for the clinical validation were all collected on tubes with EDTA as anticoagulant. Drug-free whole blood spiked with tacrolimus for some experiments of the analytical validation came from discarded anonymized blood samples. Spiked blood samples were gently mixed on a tumble roller for at least 2h at 37°C before proceeding to the experiment.

It allowed to get binding equilibrium between blood cells that mimic in vivo conditions. Mitra ® VAMS TM devices were purchased from Neoteryx, LLC. (CA, USA).

HPLC-MS/MS equipment and instrumental conditions

2.2.1.Chromatographic conditions

A C18 Hypersil Gold column (30 x 2.1 mm, 3 μm) fitted with a guard column (10 x 2.1 mm, 3 μm) (Thermo Electron, Dreieich, Germany) was used and maintained at a constant temperature of 60 °C.

The autosampler was set to 10 °C during analysis. The mobile phase consisted of a mixture of 95% acetonitrile and 5% 10 mM ammonium acetate in water. An isocratic elution mode was used, at a flow rate of 100 µL to 600 µL/min, with programming as follows: equilibration in initial conditions with a flow rate of 100 µL/min, increasing to 200 µL/min over the first minute, and then to 600 µL/min from between 1 and 1.5 minutes, before decreasing to 200 µL/min from 1.5 to 3.5 minutes. The total run time was, therefore, 2.5 minutes. Tacrolimus had a retention time of 0.6 min, and ascomycin had a retention time of 0.56 min.

2.2.2.Mass spectrometry conditions

An HPLC-Ultimate 3000 system was interfaced with a triple quadrupole mass spectrometer ENDURA MD (Thermo Fisher, San Jose, CA, USA). Positive electrospray ionization interface parameters were as follows: spray voltage 4.5 kV, sheath gas and auxiliary gas (N 2 ) 35 and 5 arbitrary units, respectively, capillary heater temperature 250 °C. The MS run was performed in the multiple reaction monitoring (MRM) scanning mode. The adduct [M+NH4] + transitions m/z 821.42  m/z 768.53 (collision energy at 20 eV and tube lens value at 135 V), m/z 809.55  m/z 756.52 (collision energy at 27 eV and tube lens value at 129 V), were monitored for tacrolimus and ascomycin, respectively. Data were acquired and analyzed with Endura MD Tune (version 1.0.1050), LC-Quan (version 2.5.6 SP1) and TraceFinder (version 3.2.513.0) (Thermo Fisher, San Jose, CA, USA).

Preparation of standards solution, calibrators and quality control samples

A stock solution of ascomycin was prepared in acetonitrile at 500 ng/mL. This solution was stored at -80°C. The day of the analysis, a working solution was prepared at 25 ng/mL by dilution of the stock solution in water. Manufactured calibrators and controls were used. Exact whole blood concentrations of tacrolimus in these samples depended on the batch of the product. Values were provided by the manufacturer. After solubilization of the lyophilized blood in water, samples of calibrators and controls were aliquoted and stored at -20°C.

Sample preparation

Calibrators and control microsamples were prepared in blood containing tacrolimus at the desired concentration. Calibrators and controls tips were load by gently touching the blood surface with a Mitra® tip. In accordance with the instruction of the manufacturer, the tips were held in the blood samples until they turned red and then for a further two seconds. The same procedure was used to collect patient samples from a finger blood drop. Tips were dried at ambient room temperature (RT) for at least 2 h. The Mitra® tips were then cut and dispensed into polypropylene microtubes, in which they were mixed with 100 μL LC-MS-grade water and 100 µL internal standard (IS) (ascomycin, 25 ng/mL in water) and shaken for 15 minutes (1000 rpm). The samples were sonicated, with shaking, for 10 minutes. We then added 100 µL acetonitrile, and precipitation was performed by adding 400 µL of a mixture of acetonitrile/0.05 M zinc sulfate in water (1:1 v/v). Samples were vortexed for 1 min and 100 µL ammonium sulfate 40% was added (salting out). Samples were vortexed again for 1 min and centrifuged at 16260 x g for 5 min at 8 °C. We then injected 10 µL of the supernatant into the LC-MS/MS system.

Hematocrit adjustment

For some method validation experiments, preparation of drug-free whole blood samples with known hematocrit was needed to assess impact of a range of hematocrit values on analytical performances. Hematocrits were therefore adjusted to assess the hematocrit effect, recovery and matrix effect. We adjusted hematocrit as described by Koster et al. [START_REF] Koster | What is the right blood hematocrit preparation procedure for standards and quality control samples for dried blood spot analysis?[END_REF]. We first measured the hematocrit of the collected drug-free blood with a flow cytometer (Sysmex, Kobe, Japan). The blood was then centrifuged for 6 min at 3000 x g and the appropriate amount of plasma was removed or added to obtain the target hematocrit. The proportion of red blood cells was checked again, after adjustment, with the automated cytometer.

Method development

We investigated several sets of sample preparation conditions to determine the best procedure for extracting tacrolimus from blood collected by VAMS. When blood samples are collected by VAMS, the first step in the extraction process is the solubilization of the dried blood and the analyte in a solvent, for extraction. First, we optimized the step of blood desorption. The most appropriate solvent for blood desorption from tips was chosen by visual inspection of the tips and was based on the assessment of precision and accuracy of tacrolimus measurements. Secondly, we also tested several mixtures of solutions for the precipitation step, including combinations of methanol, acetonitrile and aqueous zinc sulfate solution. Thirdly, we aimed to improve the sensitivity of the assay by working on the step of extract concentration.

Method validation

The analytical method was validated according to international guidelines for bioanalytical method validation from the European Medicines Agency [START_REF]European medicines agency, Guideline on bioanalytical method validation[END_REF]. Additional experiments were performed in accordance with guidelines from the International Association for Therapeutic Drug Monitoring and Clinical Toxicology [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF].

Linearity

The linearity of the method was assessed by analyzing calibration samples at six concentrations, from 2.25 ng/mL to 42.9 ng/mL (the concentrations of the 6 calibrators provided by the supplier were: 2.25, 6.05, 12.1, 17.7, 24.3, 42.9 ng/mL). Calibration standards were analyzed on three different days (n=2 per day). Calibration curves were established by plotting the peak area ratio of the target compounds to the IS versus the nominal concentrations. A minimum of 75% of the standard calibration sample had to be in the ±15% range of the nominal value.

Accuracy and precision

Accuracy and precision were determined by replicate analysis of control samples containing known amounts of tacrolimus. Quality controls were used for four concentrations. Exact tacrolimus concentrations in blood were provided by the supplier (QC1: 2.61 ng/mL, QC2: 6.96 ng/mL, QC3: 14.6 ng/mL, QC4: 31.7 ng/mL). An additional QC was prepared by spiking drug-free blood at the concentration of 1 ng/mL to validate the LLOQ of the assay. Within-day accuracy and precision were determined by analyzing QCs in five replicates in a single validation batch. Between-day accuracy and precision were determined by repeating the analysis of five replicates over three different days. Precision was assessed by calculating the coefficient of variation (CV). Between-day precision data were analyzed by one-way ANOVA to get between-day variance according to [START_REF] Tiwari | Bioanalytical method validation: An updated review[END_REF]. Accuracy is expressed as the relative error (RE) or bias with respect to the theoretical value. The acceptance criteria for precision and accuracy were ±15% except for the LLOQ (±20%).

Carry-over

Carry-over was evaluated by injecting a high-concentration sample (highest calibration standard), immediately followed by an extracted blank sample. The peak area at the retention time of tacrolimus in the blank extract should be less than 20% that the area measured for the LLOQ sample. Carry-over should not be greater than 5% for the IS.

2.7.4.Selectivity

Selectivity was assessed with six independent sources of human blood. VAMS samples were prepared from drug-free blood and from blood spiked with tacrolimus at a concentration of 1 ng/mL (LLOQ).

The lack of endogenous interference was checked by carefully examining the chromatograms of blank samples in the retention time window containing the peaks of tacrolimus or ascomycin. The blank signal should be less than 20% of the LLOQ area for tacrolimus and and <5% for the internal standard (according to [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF]).

Hematocrit effect

We investigated the effect of hematocrit on quantification accuracy, at four concentrations and three hematocrit levels. Venous blood from a single donor was used to prepare several blood samples with adjusted hematocrits (20%, 40%, 60%). These blood samples were then spiked at low (2.5 and 5 ng/mL), intermediate (15 ng/mL) or high concentration (30 ng/mL) of tacrolimus. VAMS samples from each blood sample were prepared in quintuplicate. Concentration measured in blood with a hematocrit of 40% was used as the reference for calculating the bias of the concentrations measured in blood with other hematocrits. This bias must be less than ±15% in order the hematocrit effect to be considered acceptable [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF]. The reference hematocrit was chosen at 40% because it is the median value reported for transplanted population according to [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF].

Influence of drying time

The use of VAMS in clinical settings for outpatients attending medical consultations will require the rapid analysis of samples in the laboratory. We therefore investigated the effect of short tip drying times on the accuracy of tacrolimus quantification. Venous blood samples from treated patients with known tacrolimus concentrations, across the therapeutic range, were used to prepare sets of VAMS tips.

Concentrations were compared between tips prepared from the same source of blood dried for 30 min at 37 °C (n=8), 1 h at 37 °C (n=2) (heat chamber), 2 h at room temperature (n=2) or 24 h at room temperature (n=8). The reference drying time was 24h at 37°C.

Extraction recovery and matrix effect

We assessed the extraction recovery of tacrolimus from VAMS tips for three concentrations and four hematocrits. The hematocrit of human blood samples was adjusted to 25%, 30%, 40%, and 50%, and, for each hematocrit, the blood was spiked with tacrolimus at a target concentration of 5, 15 or 30 ng/mL. This resulted in 12 different blood samples from each source (4 hematocrits x 3 concentrations). The extraction procedure described above (in section 2.4) was applied to both dried blood samples on VAMS tips (10 µL) and to 10µL of the same blood sample in liquid form (without tips). For a given concentration x hematocrit combination, recovery from the tips was calculated as the ratio of the area of tacrolimus peak (normalized by IS) in VAMS extract to the area of tacrolimus peak (normalized by IS) in liquid whole blood extract. Recovery rates should exceed 80 % for all samples, and the CV of recovery between blood samples at the same tacrolimus concentration but different hematocrits should be below 15%.

We also calculated process efficiency and matrix effect at several tacrolimus concentrations, with a procedure adapted from that described by Matuszewski et al. [START_REF] Matuszewski | Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS[END_REF]. In a first set of experiments, relative matrix effect and process efficiency were determined in VAMS tips prepared with venous blood from six different subjects (unknown hematocrits). These blood samples were spiked with tacrolimus at two concentrations (5 and 30 ng/mL). In a second set of experiments (procedure adapted from that described by Capiau et al. [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF]), we assessed the effect of hematocrit on matrix effect, using VAMS tips prepared with venous blood from six different subjects with hematocrit adjusted to 40% and one subject with hematocrit adjusted to 20%, 40% and 50%. In this experiment, blood samples were spiked with tacrolimus at an intermediate concentration from the usual therapeutic range (6 ng/mL). The coefficient of variation for process efficiency and matrix effect (with correction by the IS) for different blood sources or different hematocrits, should be lower than 15%.

Stability

We investigated the stability of tacrolimus in dried whole blood collected by VAMS, using blood from QC samples or venous blood from patients. Stability was assessed in QC samples and patients samples to cover low and high concentration levels (for each set of storage conditions). The conditions for tip storage likely to be encountered in practice were mimicked by the following storage duration and temperature conditions (tips were placed in sealed bags with desiccant): 7 days at RT, 48 h at 4 °C and 48 h at 60 °C (to mimic extreme storage conditions during transport), 1 month at RT and 1 month at -80°C. Analysis of freeze/thaw stability was not relevant to assess here since contrary to liquid whole blood, the totality of dried blood is extracted in this assay and there is no samples left to store in the freezer for further reanalysis. All samples were analyzed in triplicate, with fresh QC, and a freshly prepared calibration curve. The concentrations measured for each sample were compared to the expected concentration. Bias within ±15% of the nominal values was considered acceptable, and was interpreted as meaning that the tacrolimus was stable in VAMS tips under the conditions studied.

Clinical validation

The tacrolimus concentrations measured in dried capillary blood collected by fingerprick on VAMS tips was compared to those obtained for whole blood collected by venipuncture into EDTA-containing tubes (reference method). Paired blood samples were collected simultaneously from volunteer liver and kidney transplant recipients, to determine trough concentrations of tacrolimus in liquid venous whole blood and VAMS dried blood. VAMS samples were collected by nurses who beforehand received training regarding microsampling procedure. In compliance with ethical standards, all patients were informed of the purpose of this sampling and signed an information letter. The study was approved by the local ethical committee (authorization n° 20.130).

The comparison of the tacrolimus concentrations obtained with the two types of blood sample was performed on 42 paired samples. Patient hematocrit was also recorded, to assess whether difference between the two assays could be influenced by hematocrit. Pearson linear correlation was used to assess association between these too variables.

The concentrations obtained with the VAMS method and with the reference method (venous blood samples) were compared by Passing-Bablok regression.

Agreement between the two sampling methods was assessed with a Bland-Altman plot. Consistently with previous report, a clinically limit of acceptance was set as follow: relative difference between tacrolimus concentrations measured in liquid whole blood samples vs VAMS samples should be lower than ± 20% for 80% of paired samples.

In addition, the predictive performance of the VAMS assay was assess according to guidelines and formula reported in a previous study from Veenhof et al. [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF][START_REF] Veenhof | Volumetric absorptive microsampling and dried blood spot microsampling vs. conventional venous sampling for tacrolimus trough concentration monitoring[END_REF]. In brief, the following parameters were calculated: Median Prediction Error (MPE), Median Percentage Prediction Error (MPPE), Root Median Squared Prediction Error (RMSE), and Median Absolute Percentage Prediction Error (MAPE). MPPE and MAPE and should be <15%.

Clinical application

Besides, the developed VAMS assay was an opportunity to estimate AUC thanks to repeated samples collections in the context of routine therapeutic drug monitoring in clinical practice. Indeed, because of low invasiveness of microsampling, patients and clinicians were keen on accepting many sample collections within a day to allow pharmacologist to accurately estimate tacrolimus exposure. We present AUC results from 10 adults kidney transplant recipients treated once daily with tacrolimus, for whom pharmacokinetic profile and individual AUC/C 0 ratio evaluations could help to tailor tacrolimus dosage.

Blood samples were collected with a VAMS device by trained nurses in patients admitted to the day hospital. All tips were visually inspected by the lab analyst before extraction to check the quality of sampling. Under-filled tips were discarded. Every patient was informed of the purpose of these sampling by clinicians. This part of the study was also approved by the local ethical committee (authorization n° 20.130). In practice, an AUC was determined for a patient upon request of the clinician, for instance in cases of adverse effects despite C 0 lying within the therapeutic range, when a drug-drug interaction was expected or when an atypical pharmacokinetic profile was suspected. Sampling times were scheduled within tacrolimus dosing interval, from morning trough (T 0 ) to 12h after the drug intake (usual sampling times were: pre-dose, and 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 12h post-drug intake). Noncompartmental pharmacokinetic analysis (PK Solver®) was performed to estimate the AUC (linear trapezoidal method). AUC/C 0 ratios were calculated using morning trough concentration and values were interpreted according to ranges reported in Brunet et al. article [START_REF] Brunet | Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report[END_REF].

Results

Method development

First, to optimize the step of tacrolimus desorption from dried blood, we tested sample desorption under agitation (vortex) in organic solvents, such as acetonitrile and methanol. We assumed these solvents were suitable to solubilize lipophilic drug such as tacrolimus (LogP of about 3). However, with these solvent, tips remained red meaning that blood desorption from tips was not good enough. Consistently, we observed that tacrolimus recovery from tips was highly variable when organic solvents were used for tips desorption. Better results were obtained when desorption was performed in 100% deionized water. Indeed, with an aqueous solution, the blood was seen to be completely solubilized (the tips went from red to colorless) and high and repeatable extraction recoveries were achieved for tacrolimus as well as satisfying accuracy and precision (n=3 replicate / QC concentration levels, data not shown).

Thus, tips desorption process consisted in vortexing tips in water followed by a step of sonication to maximized effectiveness of desorption. Secondly, we optimized volumes and compositions of the solution used for the extraction of analytes by protein precipitation. To be able to achieve the expected LLOQ (1 ng/mL), a compromise had to be found, to achieve the cleanest possible extract with the lowest dilution of sample. The following mixtures were tested for precipitation: 100% methanol, 100% acetonitrile, 50% acetonitrile/50% 0.05 M zinc sulfate in water and 25% MeOH/25% 0.05 M zinc sulfate in water/50% acetonitrile. Various volumes, from 150 μL to 500 μL, were tested. Finally, we found that a precipitant solution including a mixture of organic solvent and aqueous zinc sulfate solution was required to obtain a limpid extract. In addition, a minimum of 500 μL precipitant was needed in order the mixture was strong enough to precipitate all the extract (below this volume, clogging of the extract occurred in the HPLC system). Thirdly, we had to add a step of extract concentration to the process to compensate for the dilution of the extract by precipitant solution. We found that salting out with ammonium sulfate was a very efficient and fast option to cope with this issue. It has the advantage to be less cumbersome than liquid-liquid extraction. This technique only works if the analyte is solubilized in a mixture of water and an organic solvent of low polarity which was the case of our extract composition.

The analyte can then be concentrated in the organic supernatant, after the addition of the salt. Interestingly, we found that salting out was not compatible with precipitant solutions including methanol (the solvent remained miscible with water). At the end of this preliminary work of method development, we ultimately determined that the best precipitant solution was a mixture of 50% acetonitrile/50% 0.05 M zinc sulfate in water. The use of zinc sulfate increased the precipitation power of the solution, counterbalancing the high proportion of water in the mixture. After salting out in ammonium sulfate, tacrolimus was concentrated in 200 µL of organic (acetonitrile) upper-layer, ready for injection into the LC-MS/MS system without further time-consuming step of evaporation and resolubilization.

Analytical validation

Linearity

The method was linear over the calibration range. Heteroscedasticity was checked. The best fit was obtained with a weighing factor of 1/x. The back-calculated concentrations of the calibration standards satisfied the acceptance criteria (± 15%) and the coefficient of correlation was > 0.99 for all calibration curves (Table S1).

Accuracy and precision

Within-and between-day precision and accuracy were determined at four concentrations (table 1). The acceptance criteria were satisfied, because bias and CV did not exceed 15% for any of the QCs .

Carry-over

No carry-over was observed in the validation experiments, as the signal for a blank sample injected after the highest calibration concentration of tacrolimus was 11.9% that obtained for the LLOQ sample. No significant carry-over was observed for ascomycin (signal ratio = 0.4%)

3.2.4.Selectivity

We observed no interference at the retention times of tacrolimus and ascomycin in blank samples extracted from six sources of human bloods. The mean (range) ratio of area of the background noise at the retention time of tacrolimus / area of the peak of analyte in the LLOQ was 3.6% (1.3-5.8%) for tacrolimus and 0.2% (0.1-0.3%) for ascomycin.

Hematocrit effect

The influence of hematocrit on tacrolimus concentrations (at 4 concentration levels) is reported in table 2. No significant hematocrit effect was observed relative to the standard hematocrit of 40% (relative differences <15%) in a large range of concentration (2.5 to 30 ng/mL) and a large range of hematocrit (20% to 60%)

Influence of drying time

Consistent tacrolimus concentrations were obtained for VAMS samples dried in the four different sets of conditions (Figure 1). Relative differences compared to concentration measured in tips dried for 24h at RT were below ±15%. Consequently, a short drying time of blood (30 min at 37°C or 2h at RT) should provide similar results than in blood dried longer (24h at RT).

Extraction recovery and matrix effect

The recovery results of VAMS samples and liquid venous blood samples are reported in table 3. At each concentration and hematocrit, recovery exceeded 80% and was not affected by hematocrit, because the CV was below 10% at each concentration.

Process efficiency and matrix effect satisfied the acceptance criteria, as the coefficient of variation remained below 15% for all concentrations and hematocrits (table 4).

Stability

Tacrolimus was stable in VAMS tips stored for 7 days at room temperature, 48 h at 4 °C and 48 h at 60 °C. This stability indicates that VAMS is a suitable method for blood sampling at home and transport by regular mail. For long-term storage in the laboratory in case of delayed analysis, samples should not be stored at RT, but at -80 °C, as a loss of more than 20% was observed for VAMS blood analyzed after one month of storage at RT, whereas the samples remained stable at -80 °C. The stability results are reported in table 5.

Clinical validation

Concordant results were obtained when tacrolimus determinations in liquid whole blood samples collected by venipuncture were compared with determinations on blood collected simultaneously by VAMS. Passing-Bablok regression analysis is shown in figure 2 (slope =0.98 [0.91;1.11], intercept = -0.2 [-0.99;0.18]). In addition, a Bland-Altman plot showed a minor mean negative systematic bias of -0.4 ng/mL(or -5.4%) (95% CI -0.08 to -0.92) for VAMS concentrations compared to liquid whole blood concentration, which is not clinically significant (figure 3). We observed that 90.5% of the paired concentrations (liquid whole blood-VAMS) were within the limits of acceptance (relative difference of ±20%). It fulfilled the required threshold of at least 80%. Overall, the predictive performances of VAMS assay were satisfying with a MPE of -0.33 ng/mL, a MMPE of -4.1%, a RMSE of 0.57 ng/mL and a MAPE of 7.4%. Hematocrit of the cohort ranged from 25% to 50%. No association was found between hematocrit and absolute differences between concentrations measured in liquid whole blood or in VAMS blood samples (Pearson-test : r=0.08 p-value =0.6).

Clinical application

Tacrolimus exposure was successfully assessed in patients treated once daily with tacrolimus (Advagraf® or Envarsus®). Demographics of these patients are presented in table S2. Results of the pharmacokinetics analysis are presented in table 6. We observed that all C 0 concentrations were within the usual therapeutic range (3-15 ng/mL). However the range of AUC/C 0 ratios was large (30.3 to 66).

It suggests that for some patients, tacrolimus exposure is not well reflected by trough concentration.

Mean Tmax were consistent with values expected for both formulations of once-daily tacrolimus.

Discussion

The determination of tacrolimus concentrations in dried blood collected with microsampling devices is of great interest as a means of improving the follow-up of transplant recipients. The development and validation of a bioanalytical LC-MS/MS method for quantifying this cornerstone immunosuppressant in blood collected by VAMS is therefore of considerable pertinence, although several analytical challenges remain to be overcome. Many methods for the determination of tacrolimus concentrations in dried blood spots collected on paper cards have been published [START_REF] Gallant | Predictability of Capillary Blood Spot Toward Venous Whole Blood Sampling for Therapeutic Drug Monitoring of Tacrolimus in Solid Organ Transplant Recipients[END_REF], but very few strategies based on volumetric microsampling have been described [START_REF] Kita | Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition[END_REF][START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF][START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF][START_REF] Zwart | Therapeutic drug monitoring of tacrolimus and mycophenolic acid in outpatient renal transplant recipients using a volumetric dried blood spot sampling device[END_REF][START_REF] Gruzdys | Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device[END_REF].

The method reported here is accurate, precise and sensitive. The assay was linear over a large calibration.

However, dilution integrity remained to be validated in order to deal with quantification of rare higher tacrolimus concentration that could be observed in some patient (for instance at Tmax). We used a fast extraction process based on protein precipitation with minimal centrifugation times and with no evaporation to dryness, contrasting with the more time-consuming processes reported in previous studies [START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF][START_REF] Gruzdys | Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device[END_REF]. In our method, salting out with an ammonium salt was used to obtain high recovery rates and high sensitivity (low LLOQ) despite the small volume of blood collected (10 µL). Our method is also the only VAMS tacrolimus assay to date to make use of a precipitation solution without methanol.

Koster et al. hypothesized that methanol would increase analyte solubility and recovery [START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF]. However, we found that the use of an extraction solution consisting of a water-methanol mixture resulted in a cloudy supernatant, and imprecise and inaccurate results. We found that an extraction mixture consisting of a water-acetonitrile and zinc sulfate mixture, combined with our salting-out step, resulted in better tacrolimus extraction rates. Another feature of our method is the used of ascomycin as an IS. Even though we acknowledge that deuterated IS is usually a preferable option for method with mass spectrometry detection, results of our method validation were satisfying enough to consider that ascomycin was an acceptable IS. Ascomycin was eluted almost at the same retention time than tacrolimus and this IS appeared to compensate matrix effect likely because of similarity in chemical structures of both compounds.

The main expected issue with dry blood microsampling strategy is the hematocrit effect. It should be greatly reduced by the use of volumetric blood collection methods, such as the VAMS method used here. We showed that tacrolimus measurement was not influenced by hematocrit for concentrations covering the therapeutic range and at hematocrits ranging from 20% to 60%. Most patients are expected to have hematocrit within this window but for patients with even more extreme hematocrit value, the method should not be used. We also reported that recovery was not influenced by hematocrit. As explained by Velghe et al. it is a crucial point that could explain why in some case a hematocrit effect still occurs despite the use of volumetric microsampling to cope with it [START_REF] Velghe | Is the hematocrit still an issue in quantitative dried blood spot analysis?[END_REF]. The authors underlined that the extraction procedure of the analyte from VAMS may be a determinant factor to get reproducible recoveries. For instance, Veenhof et al. found that the agreement between tacrolimus concentrations in liquid whole blood and in DBS was better than the agreement between liquid whole blood and VAMS, showing extraction recovery can still be an issue for VAMS [START_REF] Veenhof | Volumetric absorptive microsampling and dried blood spot microsampling vs. conventional venous sampling for tacrolimus trough concentration monitoring[END_REF]. This is in accordance with our observations during the optimization of our extraction procedure since we had to tried several solvent mixtures and to mix and sonicate samples a sufficient time to get satisfying results.

One challenge in the use of dried microsamples in clinical practice concerns the compatibility of preanalytic stability with real life conditions of use. We, therefore, assessed the effect of drying time on the tacrolimus concentrations measured in blood tips. A minimum drying time of 24 h at room temperature has frequently been reported [START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF][START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF][START_REF] Zwart | Therapeutic drug monitoring of tacrolimus and mycophenolic acid in outpatient renal transplant recipients using a volumetric dried blood spot sampling device[END_REF][START_REF] Gruzdys | Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device[END_REF]. However, a shorter drying time (2-3 h at RT) did not seem to affect tacrolimus quantification in VAMS tips in the studies by Koster et al. and Kita et al. [START_REF] Kita | Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition[END_REF][START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF], whereas Vethe et al. reported lower recovery rates for VAMS tips dried for 3 h rather than 24 h at RT [START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF]. Nevertheless, short drying times may be an advantage in laboratory workflows, facilitating the daily preparation of QCs and standards. We, therefore, investigated the impact of short blood drying times on the tacrolimus concentrations obtained. We found that tip drying could be accelerated by heating to 37 °C for 30 min in a controlled-temperature chamber. Like Kita et al., we also found that tacrolimus could be extracted and accurately quantified from tips dried for only 2 h at RT. Besides, conflicting results for the medium-and long-term stability of tacrolimus in microsamples have been reported. Kita et al. reported that tacrolimus was stable for only 43 h at RT, with a decrease in drug concentration of more than 15% by day 3, whereas Koster et al. and Vethe et al. reported tacrolimus stability in VAMS blood stored at RT for 1 month and 2 months, respectively [START_REF] Kita | Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition[END_REF][START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF][START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF]. We found that samples were stable at RT for seven days, but that tacrolimus concentration had decreased considerably after one month of storage at RT. For TDM with sample collection by patients at home, sample stability is crucial, because the patients must send their samples by post for testing. Samples may not, therefore, be analyzed until several days after collection. Stability over a period of seven days may be sufficient for samples shipped by regular mail. The differences in stability reported by different authors may reflect differences in recovery. Indeed, each team uses a different mixture to extract tacrolimus from tips, potentially resulting in different recovery rates from blood samples dried over a period of days.

Discrepancies in long-term stability may also reflect differences in the sealing of the storage bag and the desiccant used, because these elements are not standardized between laboratories. Moreover, one of the strengths of our stability study is the confirmation that tacrolimus levels remain stable in samples stored in conditions mimicking the sample being left in a mail box for a few days under extreme weather conditions. Like Koster et al., we found that samples could tolerate high temperatures (i.e. 60 °C or 50 °C) for two days, and, like Kita et al., we observed no decrease in drug concentration in samples stored at 4 °C for two days [START_REF] Kita | Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition[END_REF][START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF]. The long-term stability of frozen samples was also confirmed after one month of storage, consistent with the findings of Koster et al. Thus, VAMS devices can be used for clinical studies in which the samples need to be stored in the laboratory before analysis. A limit of our stability study is the lack of data regarding medium-term stability at intermediate storage conditions (e.g. 1 month at +4°C at -20°C). Stability of tacrolimus in VAMS samples at this condition should be further evaluated.

We also conducted a clinical validation, in which we compared venous and capillary tacrolimus blood concentrations in kidney or liver transplant recipients. The correlation between these concentrations must be systematically assessed, to check that VAMS is a reliable method for tacrolimus TDM [START_REF] Capiau | Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring[END_REF][START_REF] Timmerman | EBF recommendation on the validation of bioanalytical methods for dried blood spots[END_REF].

In this study, venous and capillary concentrations appeared to be very well strongly correlated (r of 0.97), and Bland-Altman analysis showed that the usual TDM method (liquid samples of venous blood) and the VAMS method (capillary blood) provided similar results. This good concordance may be linked to the VAMS technology, which prevented a hematocrit effect and ensured that the samples were homogeneous [START_REF] Kok | Volumetric absorptive microsampling: Current advances and applications[END_REF]. Indeed, other teams using the same device have also reported consistency between tacrolimus determinations on venous and capillary blood for a large range of concentrations [START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF][START_REF] Gruzdys | Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device[END_REF][START_REF] Gustavsen | Tacrolimus area under the concentration versus time curve monitoring, using home-based volumetric absorptive capillary microsampling[END_REF].

Zwart et al. used another device to collect an accurate volume of blood for dried blood spots [START_REF] Zwart | Therapeutic drug monitoring of tacrolimus and mycophenolic acid in outpatient renal transplant recipients using a volumetric dried blood spot sampling device[END_REF]. They reported good agreement between the tacrolimus AUC obtained with microsamples of capillary blood and that obtained with tubes of venous blood, but, they found a bias between the two methods when the analysis focused exclusively on trough concentrations of the immunosuppressant. Contrary to some other teams [START_REF] Vethe | Tacrolimus Can Be Reliably Measured With Volumetric Absorptive Capillary Microsampling Throughout the Dose Interval in Renal Transplant Recipients[END_REF][START_REF] Zwart | Therapeutic drug monitoring of tacrolimus and mycophenolic acid in outpatient renal transplant recipients using a volumetric dried blood spot sampling device[END_REF][START_REF] Gustavsen | Tacrolimus area under the concentration versus time curve monitoring, using home-based volumetric absorptive capillary microsampling[END_REF], we did not report a comparison between tacrolimus AUC obtained from liquid venous blood and AUC obtained from VAMS samples since the study was not design for this purpose.

Based on satisfying results of the clinical validation of our assay and above mentioned literature data, we assumed that the benefit-risk balance was good enough to use VAMS for tacrolimus routine TDM. Indeed, one of the major advantages of microsampling devices for the TDM of tacrolimus is that they make it possible to obtain repeat samples from patients, in a minimally invasive manner. The estimation of tacrolimus AUC is, therefore, compatible with clinical settings. We emphasized this aspect through clinical application of the method described here in kidney transplant recipients. Trough concentration is known to be a poor surrogate marker of tacrolimus exposure, and the last consensus conference for tacrolimus TDM recommended determining individual AUC/C 0 ratios several times during patient follow-up [START_REF] Brunet | Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report[END_REF]. The patients included in the clinical application of our assay benefited from this approach and we observed a wide range of AUC 0-24h and AUC/C 0 ratios which confirms the suboptimal correlation between tacrolimus C 0 and AUC. Furthermore, in our study AUCs were determined for hospitalized patients. Interestingly, Gustavsen et al. reported that AUC could be accurately determined from samples collected at home, by the patients themselves. They also showed that the AUCs obtained with capillary microsamples were highly consistent with those obtained for venous blood samples [START_REF] Gustavsen | Tacrolimus area under the concentration versus time curve monitoring, using home-based volumetric absorptive capillary microsampling[END_REF]. Hence, tacrolimus determinations in VAMS microsamples could overcome the practical obstacles to the measurement of tacrolimus exposure (e.g. through AUC). Thanks to analytical methods such as that described here, it should be possible to improve tacrolimus TDM and tacrolimus response in transplant recipients and further studies should be performed to confirmed this statement.

Otherwise, unfortunately our study was not designed to provide objective data regarding practical use of VAMS in clinical setting. In particular, we did not quantify users (medical staff and patients) feedbacks, medico-economics aspects and sample quality (over or under filling of tips). This last point might be critical to assess the efficiency of person's training and to rely on measurements. An automated quality control process should be implemented whenever possible [START_REF] Veenhof | Volumetric absorptive microsampling and dried blood spot microsampling vs. conventional venous sampling for tacrolimus trough concentration monitoring[END_REF][START_REF] Veenhof | Performance of a web-based application measuring spot quality in dried blood spot sampling[END_REF]. Future study is still needed to address these questions to fully demonstrate the reliability of VAMS strategy in routine.

The ability of our method to determine the levels of only one immunosuppressant, tacrolimus, could be seen as a limitation. Indeed, transplant recipients are almost always treated with a combination of immunosuppressive drugs for which TDM is necessary. Tacrolimus is usually the cornerstone of immunosuppression, but it would be advantageous to be able to use the same microsample-based analytical method to determine the levels of cyclosporin, m-tor inhibitors (everolimus and sirolimus) or mycophenolic acid, as reported in other studies [START_REF] Koster | A volumetric absorptive microsampling LC-MS/MS method for five immunosuppressants and their hematocrit effects[END_REF][START_REF] Zwart | Therapeutic drug monitoring of tacrolimus and mycophenolic acid in outpatient renal transplant recipients using a volumetric dried blood spot sampling device[END_REF][START_REF] Gruzdys | Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device[END_REF]. We are currently working on the extension of our method to these drugs.

Conclusion

We developed a rapid, sensitive and accurate HPLC-MS/MS method, and validated its analytical performance. Comparison with the reference method of quantification of tacrolimus in liquid venous whole blood fulfilled clinical requirements. This assay, based on VAMS microsampling strategy, is promising to be used in the routine TDM of tacrolimus or for pharmacokinetic studies. The implementation of microsampling strategies should be encouraged for the assessment of tacrolimus exposure and the optimization of tacrolimus response in transplant recipients. This feasible approach may constitute a crucial step forward for tacrolimus TDM and for improvements in the quality of life of transplant recipients. Samples were prepared by spiking drug-free blood from 6 different sources with tacrolimus at several concentrations. Samples were extracted according to the procedure described above (samples A). For each source, blank blood samples were extracted and post-fortified with tacrolimus and IS to reach the same concentrations than the one expected in samples A for a recovery of 100% (samples B). Neat solutions without blood matrix were prepared in duplicate by spiking tacrolimus and IS at the same concentrations than the final concentration expected in sample B (samples C). Process efficiency (PE) was calculated as follow: PE = (peak area of sample A / peak area of sample C). Absolute matrix effects of each source were estimated for tacrolimus and the IS by matrix factors (MF) determined as MF= (peak area of sample B / peak area of sample C). IS corrected MF was calculated by dividing the MF of the tacrolimus by the MF of the IS.

Figure 1 :Figure 2 :

 12 Figure 1: Influence of drying time on tacrolimus concentration in blood collected by VAMS. RT:

Figure 3 :

 3 Figure 3: Bland-Altman analysis of tacrolimus (Tac) concentrations determined in liquid venous

Table 1 : Within day and between day accuracy and precision of tacrolimus assay in VAMS

 1

	CQ level	Within day precision (CV%) (n=5)	Between day precision (CV%) (n=15)	Within day accuracy Bias (%)(n=5)	Between day Precision Bias (%) (n=15)
	LLOQ	4.0	8.0	12.9	6.0
	QC1	4.7	7.4	2.7	1.2
	QC2	7.7	11.6	-1.3	-6.6
	QC3	5.9	6.9	-7.4	-6.5
	QC4	10.2	10.2	-4.2	-4.9
		QC: Quality control; CV: Coefficient of variation

Table 2 : Influence of hematocrit on tacrolimus quantification in VAMS

 2

			Concentration levels
		2.5 ng/mL	5 ng/mL	15 ng/mL	30 ng/mL
	Ht 20%	-2.5	-0.2	-9.6	-5.7
	Ht 60%	3.2	1.4	-3.0	-3.7
	Ht: hematocrit
	Results are expressed in % as relative difference compared to concentrations measured
	in sample at hematocrit 40% (n=5 per condition)

Table 3 : Recovery of tacrolimus in samples of blood collected with VAMS

 3 Recoveries are expressed as are ratio of (IS normalized) LC-MS signal of tacrolimus in VAMS/ tacrolimus signal in wet sample at the same hematocrit and concentration; CV: coefficient of variation; Ht: hematocrit

		Tacrolimus concentration level in whole blood (ng/mL)
		5	15	30
	Ht 25%	1.04	0.91	0.87
	Ht 30%	0.89	0.94	0.93
	Ht 40%	0.88	0.98	0.85
	Ht 50%	0.91	0.87	0.81
	CV (%)	7.6	8.9	6.9

Table 4 : Process efficiency and matrix effect of tacrolimus assay in VAMS Experiment 1

 4

			Process efficiency		Matrix factor
	Tacrolimus blood concentration	Mean PE	CV PE (%)	CV-ISc (%)	CV (%)
	5 ng/mL (n=6)	0.94	13.94	11.00	10.58
	30 ng/mL (n=6)	0.94	4.91	9.58	5.39
		Experiment 2 (tacrolimus at 6 ng/mL)
		Mean MF	CV MF (%)	Mean ISc MF	CV ISc MF (%)
	Ht 40% a	1.15	14.44	0.92	12.56
	Ht 20% b	1.03		0.95
	Ht 40% b	1.01	3.20	1.12	9.60
	Ht 50% b	0.97		0.97

MF: Matrix factor; PE: process efficiency; CV: coefficient of variation; IS: internal standard; ISc: IS corrected; Ht: hematocrit a : n=6 sources of blood from 6 different individuals, each sample was analyzed in triplicate b : 1 source of blood at adjusted hematocrit, each sample was analyzed in triplicate

Table 5 : Stability of tacrolimus in VAMS samples Storage condition Type of blood sample

 5 QC: manufactured quality control sample. The first 4 QC and the last 2 QC correspond to two different batches. The expected concentration is provided by the manufacturer according to the amount of tacrolimus weighed in the sample; Pat: patient sample; RT: room temperature; n.t.: not tested, n=3 replicate/ condition Results are reported as bias (%) to the expected concentration

		Expected
		concentration (ng/ml)	7 days RT	48h 4°C	48h 60°C	1 month RT	1 month -80°C
	QC	2.61	-7.1	n.t.	n.t.	n.t.	n.t.
	QC	6.96	-1.7	n.t.	n.t.	n.t.	n.t.
	QC	14.6	7.2	n.t.	n.t.	n.t.	n.t.
	QC	31.7	1.3	n.t.	n.t.	n.t.	n.t.
	Pat	3.3	n.t.	4.2	-11.0	n.t.	n.t.
	Pat	14.2	n.t.	-7.5	-3.3	n.t.	n.t.
	Pat	6.00	n.t.	n.t.	n.t.	-37.7	5.8
	Pat	2.20	n.t.	n.t.	n.t.	-20.9	-6.3
	QC	2.73	n.t.	n.t.	n.t.	-21.8	-11.7
	QC	15.40	n.t.	n.t.	n.t.	-25.0	-6.1

Table 6 : Clinical application: monitoring of tacrolimus exposure and non-compartmental pharmacokinetic analysis in kidney transplant recipients Tac formulation Parameter

 6 Tac: tacrolimus; AUC: area under the curve of concentration versus time; C 0 : trough concentration; C max : peak concentration; T max : time when C max is observed; D: tacrolimus dose

			Tac
			daily dose (D)	AUC 0-24h (ng.h/mL)	C 0 (ng/mL)	Ratio AUC/C 0 (h)	Ratio AUC/D (ng.h/mL.mg)	C max (ng/mL)	T max (h)
			(mg)
		median	15.3	320.2	8.1	44.9	25.5	28.8	1.5
	Advagraf® (n=4)	min	5	305.2	6.1	30.3	18.0	22.0	1.0
		max	20	526	11.5	50.4	61.3	46.1	2.0
		median	8.5	350.4	8.3	39.5	44.3	24.0	6.0
	Envarsus® (n=6)	min	2.0	222.0	5.4	28.7	21.0	11.7	4.0
		max	17.0	541.9	14.9	66.0	111.0	37.7	6.0

This research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Abbreviations

Volumetric absorptive microsampling for the quantification of tacrolimus in capillary blood by high performance liquid chromatography-tandem mass spectrometry

Tron at al.

Highlights:

 Volumetric absorptive micro-sampling is an innovative alternative to venipuncture  A method was developed to measure whole-blood tacrolimus in microsamples  The UHPLC-MS/MS method is fast, sensitive, precise and accurate  This analytical assay was clinically validated according to a reference method  The assay was applied to assess tacrolimus exposure in transplanted patients


Caroline Polet
email: caroline.polet@naturalsciences.be

Merlin Leunda Martiarena

Sébastien Villotte

Martine Vercauteren

Throwing Activities Among Neolithic Populations

Keywords: Neolithic, throwing activity, avulsion fracture, adolescent, Belgium

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Introduction

When practised intensively (at high velocity and force) and repetitively, overhead throwing activities are very stressful to the elbow, and more particularly, to the anterior band of the medial collateral ligament (MCL) [START_REF] Nakanishi | MR arthrography of elbow: evaluation of the ulnar collateral ligament of elbow[END_REF][START_REF] Miller | Sonography of injury of the ulnar collateral ligament of the elbow-initial experience[END_REF]Gregory and Nyland 2013). The MCL links the humerus with the ulna, arising from the medial humeral epicondyle and inserting on the ulnar coronoid process. It is the primary stabilizer against valgus stress at the elbow [START_REF] Labott | Understanding the medial ulnar collateral ligament of the elbow: Review of native ligament anatomy and function[END_REF].

If throwing activities occur near the end of skeletal maturation, when the medial epicondyle (ME) is not yet fully fused (12-18 years of age), traction forces can result in bony detachments in the area of the MCL insertion (Figure 1). In individuals younger than 15years-old, these detachments can be complete and involve the entire medial epicondylar ossification centre. In older teens, they can be partial, leading to the avulsion of one or more bony fragments from the medial epicondylar apophysis [START_REF] Klingele | Little league elbow: valgus overload injury in the paediatric athlete[END_REF][START_REF] Kijowski | Pediatric throwing injuries of the elbow[END_REF].

When the individual with an avulsion fracture reaches adulthood, the healed lesion takes the form of a bony outgrowth or lacunae depending on whether or not the avulsed fragment re-united with the condyle (Lewis 2017, 254-255). On dry archaeological bones, such lesions are mainly observed on adult humeri as scars from a traumatic event that occurred during adolescence [START_REF] Villotte | Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: Evidence from upper limb enthesopathies[END_REF]).

[Figure 1 near here] Today, these ME injuries are common pathological findings in young baseball pitchers and are known as Little Leaguer's elbow [START_REF] Klingele | Little league elbow: valgus overload injury in the paediatric athlete[END_REF]. They are also reported in javelin throwers [START_REF] Miller | Javelin thrower's elbow[END_REF], but the incidence is lower, because Javelin competitions begin laterduring high school in the United States (Gregory and Nyland 2013).

The aim of this study was to look for the presence of avulsion fractures at the attachment site of the anterior band of the MCL during the Neolithic period in Belgium and thus demonstrate the occurrence of overhead throwing activities during adolescence in these prehistoric populations. We then try to answer the following question: who was practising these throwing activities, and based on archaeological data, in what context were these activities performed?

Materials and methods

More than 200 karstic caves located in the Meuse River Basin of Belgium have yielded human remains [START_REF] Toussaint | Apports récents sur l'anthropologie des Mésolithiques et des Néolithiques mosans[END_REF]. The majority of these funerary sites date to the Middle and Late-Final Neolithic, as evidenced by over 100 available radiocarbon dates; only about a dozen sites belong to the Early Mesolithic, and some caves contained skeletons from the Protohistoric Period [START_REF] Toussaint | Les sépultures néolithiques du bassin mosan wallon et leurs relations avec les bassins de la Seine et du Rhin[END_REF]. Many of these sites were excavated as early as the 19 th century (Table 1). Unfortunately, the skeletal remains are commingled and disarticulated. This is, of course, due to taphonomy, but it is also due to human manipulation of the remains, beginning in Prehistoric times [START_REF] Toussaint | Apports récents sur l'anthropologie des Mésolithiques et des Néolithiques mosans[END_REF]. A great number of Neolithic burials include the remains of 5-15 individuals [START_REF] Polet | Les squelettes néolithiques découverts dans les grottes du bassin mosan[END_REF] belonging to adults of both sexes and children [START_REF] Toussaint | Apports récents sur l'anthropologie des Mésolithiques et des Néolithiques mosans[END_REF].

[Table 1 near here] For this study, we selected 196 left and right adult humeri (fully fused epiphyses) with preserved distal epiphyses from a collection housed at the Royal Belgian Institute of Natural Sciences. They derived from 16 cave burials and come from 9 sites in the Meuse Basin (Figure 2). Five caves are radiocarbon dated to the Middle Neolithic, nine to the Late-final Neolithic, and one (Grotte de la Cave in Maurenne) covers both periods (Table 1). One of the caves was not radiocarbon dated and is therefore indicated as 'supposed Neolithic'. The minimum number of individuals varies from 5 to 59 individuals (Table 1).

[Figure 2 near here] The presence or absence of changes at the humeral attachment site for the anterior band of the MCL (see fig. 5 in [START_REF] Villotte | Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: Evidence from upper limb enthesopathies[END_REF]) was macroscopically recorded. We looked for two types of changes (see [START_REF] Villotte | In Search of Consensus: Terminology for Entheseal Changes (EC)[END_REF] for definitions and illustrations): a clearly distinct protrusion from the surface of the enthesis (Figure 3), or alternatively, a large surface discontinuity affecting the cortical bone and underlying trabecular bone (Figure 4; also see fig. 4 in [START_REF] Villotte | Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: Evidence from upper limb enthesopathies[END_REF]).

[Figures 3 and4 near here]

We studied the relation between the MCL lesions, siding, and robusticity, which was used instead of sex because skeleton individualization was not possible for these collective burials.

Pelvic bones could not be associated with the humeri, and thus no reliable methods for a pelvis-based sex determination could be applied. Robusticity was evaluated using distal humerus measurements. From a series of six measurements (Table 2, Figure 5), two were selected because they could be taken on a maximum number of Neolithic humeri (Leunda Martiarena 2016, 40-41). These were the epicondylar breadth and the minimum trochlear depth. The epicondylar breadth (Measurement 4 in [START_REF] Martin | Lehrbuch der Anthropologie in Systematischer Darstellung mit Besonderer Berücksichtigung der Anthropologischen Methoden für Studierende, Ärtze und Forschungsreisende[END_REF]Martin , 1010) ) is the maximum breadth between the most extreme aspects of the medial and lateral epicondyles of the humerus perpendicular to the axis of the humeral diaphysis. The minimum trochlear depth is the minimum anteroposterior dimension of the trochlea (Trinkaus 1983, 227). The humeri were classified as either gracile or robust based on a bivariate plot, where specimens with values below the median for both measurements were considered gracile, and those above were considered robust (Figure 6).

[Table 2 andFigure 5 near here] We applied the Fisher Exact test (2 × 2 contingency table) to test whether there was a difference in the lesion frequency between the Middle and Late Neolithic individuals. The Fisher-Freeman-Halton test (2 × 4 contingency table) was used to test whether there was a difference in frequency among the four groups of humeri: left and right gracile, and left and right robust.

[Figure 6 near here]

Results

Of the two types of lesions at the attachment site of the MCL anterior band, only the lacunar form was observed (Figure 4).

Table 3 shows the prevalence of MCL lesions at the 16 Neolithic burial sites. From a total of 196 humeri, 10 (5.1%) displayed this kind of lesion, affecting 10.3% (7/68) of the Late Neolithic individuals and 7.7% (1/13) of the Middle Neolithic individuals (in the Grotte de la Cave in Maurenne dated to the Middle and Late Neolithic, 12.5% [2/16] of the individuals were affected). The probability derived from the Fisher Exact test (1.000) used to compare these two groups was greater than 5%, and thus we cannot reject the null hypothesis (the distribution of the MCL lesions is independent of the chronological groups). We conclude that the frequency of fracture avulsions does not vary among the Late and Middle Neolithic groups.

[Table 3 near here]

The selection of robust and gracile individuals unfortunately resulted in the 'loss' of 91 individuals (specimens where either both measurements could not be taken or those where both measurements were not above or below the median values). The discussion will thus mainly concern the 105 remaining humeri.

The MCL lesion only affected the right robust bones (33.3% [7/21]; Table 4). None of the left humeri (gracile and robust) or right gracile humeri showed lesions. The probability determined from the Fisher-Freeman-Halton test (0.0000749) used to compare these four groups was less than 5%, meaning we should reject the null hypothesis (the distribution of the MCL lesions is independent of the humeri groups). We conclude that the frequency of fracture avulsions varies among the four categories of humeri.

[Table 4 near here]

Discussion

Fracture avulsion scars at the insertion of the anterior band of the MCL on nine Neolithic individuals from the Meuse Basin indicate that severe valgus stress occurred at their elbows.

No significant difference in the prevalence of lesions was observed between the Middle and the Late Neolithic individuals, indicating that throwing activities were performed during both periods.

Only the right humeri were affected by the avulsion fractures. This result was expected, as the valgus extension of the elbow is usually only reached by the dominant upper limb, given its dynamic and complex properties. Today, the right arm is dominant for about 90% of the population [START_REF] Scharoun | Hand preference, performance abilities, and hand selection in children[END_REF], which and it was also likely the case in prehistoric times based on studies on tool processing [START_REF] Steele | Humans, tools and handedness[END_REF] and handling [START_REF] Phillipson | Edge modification as an indicator of function and handedness of Acheulian handaxes from Kariandusi, Kenya[END_REF], cut mark direction [START_REF] Bromage | Microscopic criteria for the determination of directionality of cutmarks on bone[END_REF][START_REF] Pickering | Cutmarks and hominid handedness[END_REF], humeral diaphysis asymmetry [START_REF] Trinkaus | Postcranial robusticity in Homo. II. humeral bilateral asymmetry and bone plasticity[END_REF][START_REF] Sparacello | Patterns of humeral asymmetry among Late Pleistocene humans[END_REF], entheseal changes [START_REF] Villotte | I sing of arms and of a man…": medial epicondylosis and the sexual division of labour in prehistoric Europe[END_REF], directional skew in dental wear (Bermùdez de Castro, [START_REF] Bermùdez De Castro | Buccal striations on fossil human anterior teeth: evidence of handedness in the middle and early Upper Pleistocene[END_REF]Frayer et al. 2012), cave art [START_REF] Coren | Fifty centuries of right-handedness: the historical record[END_REF], and endocast asymmetries (Holloway 2008; Poza-Rey, Lozano, and Arsuaga 2017).

In our Neolithic sample, only robust humeri were affected by the avulsion fractures. Can we go one step further and state that some specific activities that produce severe valgus stress concerned only male individuals? Several studies have shown a marked sexual dimorphism also exists at the humerus level that allows reliable sex determination when it is not possible to sex either the pelvis or skull (e.g. [START_REF] İşcan | Sexual dimorphism in the humerus: A comparative analysis of Chinese, Japanese and Thais[END_REF][START_REF] Steyn | Osteometric variation in the humerus: sexual dimorphism in South Africans[END_REF][START_REF] Kranioti | Sexual dimorphism of the humerus in contemporary Cretans -A population-specific study and a review of the literature[END_REF]. In addition, the application of our graphic method based on the median values of two distal humerus measurements to a modern identified collection (known sex) shows good sexual discrimination (Leunda Martiarena 2016, 40-41). Finally, the fact that we used parameters (medians) calculated from our data avoids the bias of transposing methods created on a given population to another with different body proportions [START_REF] Ruff | Variation in human body size and shape[END_REF]. All of the above indicates that humerus robusticity can be a good sex indicator in our Neolithic sample. Thus, most of the gracile humeri probably belonged to females, and most of the robust humeri to males. However, it is not impossible that some robust humeri belong to females and vice versa, and thus some avulsions actually occurred in females.

If we look at current sport medicine data, the lesions in our Neolithic sample were likely due to activities that involve a throwing motion [START_REF] Kijowski | Pediatric throwing injuries of the elbow[END_REF]. These lesions typically result from repetitive stress due to overuse, but they can also be secondary to acute localized trauma (Gore et al. 1980;Gregory and Nyland 2013). Our results are similar to those of previous studies that, on the basis of changes at the ME (including the attachment site of the MCL), have demonstrated that prehistoric males were engaged in activities inferring a throwing motion [START_REF] Dutour | Enthesopathies (lesions of muscular insertions) as indicators of the activities of Neolithic Saharan populations[END_REF][START_REF] Villotte | I sing of arms and of a man…": medial epicondylosis and the sexual division of labour in prehistoric Europe[END_REF][START_REF] Villotte | Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: Evidence from upper limb enthesopathies[END_REF].

As the lesions observed on the ME in our sample are scars from avulsions that occurred in adolescents whose skeletons had not fully matured, they indicate these activities began before biological adulthood. This is a major outcome, because adolescent activities in prehistoric periods are poorly documented. Information on their place in prehistoric society has been inferred by funerary anthropology by associating the study of skeletal remains with patterns in grave goods and burial rites [START_REF] Thomas | Unpacking burial and rank: the role of children in the first monumental cemeteries of Western Europe (4600-4300 BC)[END_REF]Leroy 2015). However, this provides little information on their actual activities during life. Based on the study of artefacts, child participation in craft activities, including stone-tool production, ceramic manufacture, and weaving, has already been demonstrated in prehistoric populations [START_REF] Baxter | The archaeology of childhood[END_REF]. Still, only a few of these studies were able to distinguish different age categories in non-adults (including adolescents) using the types of errors related to cognitive developmental stages [START_REF] Crown | Socialization in American southwest pottery decoration[END_REF][START_REF] Bagwell | Ceramic form and skill: attempting to identify child producers at Pecos Pueblo, New Mexico[END_REF]. Two anthropological studies have nevertheless demonstrated a strenuous lifestyle (in the broader sense) during adolescence in prehistoric populations: [START_REF] Lovejoy | The analysis of fractures in skeletal populations with an example from the Libben site, Ottowa County, Ohio[END_REF] Faunal remains analyses from Neolithic sites in Belgium show that breeding was the most important source of animal protein (Cauwe, Vander Linden, and Vanmonfort 2001).

However, hunting and fishing were still performed, as demonstrated by the faunal remains at some Neolithic sites (e.g. De Ceunynck, [START_REF] De Ceunynck | Neolithicum-Bronstijdvondsten aan de Donk te Oudenaarde[END_REF][START_REF] Léotard | L'abri du Pape. Bivouacs, enterrements et cachettes sur la Haute Meuse belge : du Mésolithique au Bas Empire Romain[END_REF] and by stable isotope analyses [START_REF] Bocherens | Palaeodiet of Mesolithic and Neolithic populations of Meuse Basin (Belgium): evidence from stable isotopes[END_REF].

The use of the bow, widely attested for the Neolithic period in Europe, does not seem to be stressful for the ME [START_REF] Villotte | Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: Evidence from upper limb enthesopathies[END_REF]. However, its frequent adoption does not exclude the use of more simple overhead throwing weapons such as the sling, harpoon, killing stick, or even the boomerang [START_REF] Ramseyer | Les harpons néolithiques d'Europe occidentale[END_REF][START_REF] Ramseyer | Les armes de chasse néolithiques des stations lacustres et palustres suisses[END_REF][START_REF] Thomas | Les boomerangs aux temps anciens[END_REF].

Interpersonal violence, which has been detected in the Middle Pleistocene [START_REF] Sala | Lethal interpersonal violence in the Middle Pleistocene[END_REF], increased during the development of the first agro-pastoral societies in the Neolithic [START_REF] Keeley | War Before Civilization: The Myth of the Peaceful Savage[END_REF]Guilaine and Zammit 2001;[START_REF] Beyneix | Aux origines de la guerre : actes de violence et massacres dans le Néolithique européen[END_REF][START_REF] Heath | Warfare in Neolithic Europe: An archaeological and anthropological analysis[END_REF]. Although the traumatic lesions observed on the skeletons from Belgian Neolithic sites generally attest to injuries by arrows [START_REF] Cauwe | La grotte Bibiche à Anseremme (Dinant). Une sépulture collective du Néolithique récent[END_REF][START_REF] Polet | A healed wound caused by a flint arrowhead in a Neolithic human hip bone of the "Trou Rosette" (Furfooz, Belgium)[END_REF][START_REF] Joppart | Etude anthropologique et taphonomique de la sépulture néolithique de Martouzin-Neuville (comm. de Beauraing, BE)[END_REF], it is easy to imagine that overhead throwing weapons, such as the spear in Denmark (Bennike 1985, 110-112), could also have been used.

Games and sports are seldom considered as an interpretative model in archaeology due to the belief that they were insignificant in prehistory, as well as the paucity of uncovered artefacts (Fox 1977;Sansone 1988, 5). Nevertheless, many scholars are convinced that play has been a part of human society since prehistoric times given its ubiquity among primates and the free time at the disposal of prehistoric people (Blanchard and Taylor Cheska 1985, 96-97). Throwing games could have been practiced by all the male members of the Neolithic society in the Meuse River Basin or only by adolescents as training for the accurate and coordinated throwing of weapons for killing prey (or enemies?) (Reid 2001, 3).

It should also be noted that the use of the axe and adze require very similar movements of the upper limb as those made during overhead throwing and could also lead to ME lesions [START_REF] Villotte | I sing of arms and of a man…": medial epicondylosis and the sexual division of labour in prehistoric Europe[END_REF]. It can be assumed that their use, observed since the Mesolithic [START_REF] Verhart | Contact in stone: adzes, Keile and Spitzhauen in the Lower Rhine Basin. Neolithic stone tools and the transition from Mesolithic to Neolithic in Belgium and the Netherlands, 5300-4000 cal BC[END_REF], was very frequent during the Neolithic period, when intense deforestation occurred to clear land for agriculture [START_REF] Roberts | Europe's lost forests: a pollen-based synthesis for the last 11,000 years[END_REF].

The participation of adolescents in any of the above activities should be considered. Parallels can, for example, be drawn with present-day traditional societies such as contemporary hunter-gatherers, where teenagers learn various complex skills such as big game hunting during apprenticeships and initiation rites [START_REF] Macdonald | Cross-cultural comparison of learning in human hunting: Implications for life history evolution[END_REF]Hewlett 2016;[START_REF] Lew-Levy | How Do Hunter-Gatherer Children Learn Subsistence Skills? A Meta-Ethnographic Review[END_REF].

Conclusions

Lesions at the attachment site of the anterior band of the MCL were present during the Neolithic period in the Meuse Basin of Belgium. We have demonstrated that activities involving a throwing motion began in adolescence, suggesting we re-examine the role of teenagers in these prehistoric societies. The fact that it is only the more robust individuals that were affected leads to the hypothesis that it was probably the male individuals that were involved in throwing activities. This, in turn, suggests the hypothesis of a form of social/gender division in throwing activities in the Meuse River Basin during the Neolithic.

Based on archaeological data, four main types of activities can be proposed to explain the presence of this enthesopathy related to throwing activities: hunting/fishing, warfare/conflict, games, and wood-cutting. However, it is difficult based on our present knowledge and the available data to determine which is the more probable. the skeleton. Tara Chapman (RBINS) is gratefully acknowledged for proof-reading and language revision. (1928,1010) 4 maximum distance between the most extreme aspects of the medial and the lateral epicondyles, perpendicular to the axis of the diaphysis distal articular breadth [START_REF] Martin | Lehrbuch der Anthropologie in Systematischer Darstellung mit Besonderer Berücksichtigung der Anthropologischen Methoden für Studierende, Ärtze und Forschungsreisende[END_REF]Martin ( , 1012) ) 12.a distance between the most medial point on the trochlea to the most lateral point on the capitellum. olecranon fossa breadth [START_REF] Martin | Lehrbuch der Anthropologie in Systematischer Darstellung mit Besonderer Berücksichtigung der Anthropologischen Methoden für Studierende, Ärtze und Forschungsreisende[END_REF]Martin ( , 1012) ) 14 distance between the most medial point and the most lateral point on margin the of the olecranon fossa trochlear depth [START_REF] Martin | Lehrbuch der Anthropologie in Systematischer Darstellung mit Besonderer Berücksichtigung der Anthropologischen Methoden für Studierende, Ärtze und Forschungsreisende[END_REF]Martin ( , 1012) ) 13 distance between the most anterior point and the most posterior point on the trochlea epiphysis thickness at the proximal limit of the olecranon fossa (Leunda Martiarena 2016, 29) distance between the most anterior point to the most posterior point of the epiphysis at the level of the proximal limit of the olecranon fossa, perpendicular to the axis of the diaphysis minimum trochlear depth (1983,227) minimum anteroposterior dimension of the trochlea

Trinkaus

  , based on fracture rates in a Late Woodland sample from Ohio in the United States; and Mays et al. (2018), looking at spondylosis (a fatigue fracture of the neural arch, usually occurring in late adolescence or early adulthood in archaeological groups) in one Middle Neolithic male individual from Stonehenge Based on archaeological and ethnographical data, we tried to reconstruct the activities which caused the ME lesions. They could belong to four main categories: hunting/fishing, warfare/conflict, games, and wood-cutting.

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Map of Belgium with the locations of the studied sites. 1, Sclaigneaux; 2,

Figure 3 .

 3 Figure 3. Example of a distinct bony protrusion from the enthesis surface resulting from an

Figure 4 .

 4 Figure 4. Example of bony defect resulting from an avulsion fracture in a right Neolithic

Figure 5 .

 5 Figure 5. Measurements taken on the distal humerus. 1, epicondylar breadth; 2, distal

Figure 6 .

 6 Figure 6. Bivariate plot with the epicondylar breadth and minimum trochlear depth for the

Tables Locality Name of the site excavation Date 14C BP Date Ref. Date BC 2 σ Cultural attribution

								Ref.	MNI N
	Sclaigneaux Grotte de Sclaigneaux 1872	4155 ± 35	GrA-32975	2880-2620	LN	De Paepe and Polet 2007	58 47
	Anseremme Abri du Grogneau declaration of interest statement: none 1946-47 4945 ± 55 Abri des Autours 1992 5300 ± 55	OxA-9089 OxA-5387	3780-3660 4320-3980	MN MN	Orban et al. 2000 Cauwe 1993	? 2 9 1
	Hastière	Caverne B	1869-1879	5180 ± 45	OxA-9021	4160-3930	MN	Orban et al. 2000	14 5
		Caverne L	1869-1879	5070 ± 60	OxA-9088	3980-3710	MN	Orban et al. 2000	5 6
		Caverne M	1873 ?	4345 ± 60	OxA-6852	3320-2870	LN	Orban et al. 2000	44 17
		Petite Caverne	1869-1879	4300 ± 50	OxA-6852	3090-2700	LN	Orban et al. 2000	20 1
		Le Cimetière	1869-1879	4280 ± 50	OxA-6851	3030-2690	LN	Orban et al. 2000	19 4
		Caverne C	1869-1879	4220 ± 45	OxA-6853	2910 -2620	LN	Orban et al. 2000	? 4
	Maurenne	Grotte de la Cave	1869-1879	4635 ± 45	OxA-9025	3630-3130	MN	Orban et al. 2000	59 34
				4360 ± 45	OxA-6854	2880-2590	LN	Orban et al. 2000
				3830 ± 30	Lv-1482	2600-1950	LN	Gilot 1985
				3830 ± 70	Lv-1483	2470-2030	LN	Gilot 1985
	Furfooz	Trou du Frontal	1864-65	4430 ± 80	OxA-8828	3350-2920	LN	Charles 1998	21 8
				4430 ± 30	GrN-10179	3330-2920	LN	Leguebe and Orban 1984
		Trou Rosette	1864-65	4165 ± 70	OxA-5041	3900-2500	MN	Polet et al. 1996	14 7
	Brunot	Bois Madame	1938	4110 ± 110	Lv-265	2950-2300	LN	Dumbruch 2007	57 17
				4090 ± 110	Lv-264	2950-2300	LN
	Beauraing	Martouzin-Neuville 1977	4230 ± 40	KIA-48286	2920-2670	LN	Joppart, Polet and	34 31
				4140 ± 40	KIA-48293	2880-2580	LN	Warmenbol 2017
				4135 ± 35	KIA-48292	2880-2580	LN
				3760 ± 35	KIA-48285	2290-2040	LN
				4070 ± 100	Fra-98	2870-2480	LN	Gilot 1986
				3790 ± 90	Lv-1243	2500-1950	LN	Gilot 1986
	Bouvignes	Trou Madame	1869-1879		Supposed Neolithic			? 1
	Humain	Abri sous-roche	1962	4150 ± 35	KIA-48288	2880-2620	LN	Polet et al. 2014	40 32
		d'Humain		4085 ± 45	KIA-48289	2780-2480	LN

Table 1 .

 1 General information on the 16 burial sites from which the Mosan human material originates: locality, name of the site, excavation date, radiocarbon dating, cultural attribution, bibliographic references, minimum number of individuals (MNI; estimated on all the available adult and non-adult human remains), and the number of left and right humeri selected for this study. MN, Middle Neolithic; LN, Late Neolithic.

	Name	Reference	No.	Definition
	epicondylar breadth	Martin

Table 2 .

 2 List of the six measurements taken on the distal humerus.

					MCL lesion
	humerus		N tot	N obs Freq. (%)
	right	gracile	28	0	0
		robust	21	7	33.33
	left	gracile	30	0	0
		robust	24	0	0

Table 4 .

 4 Number and frequency of medial collateral ligament (MCL) lesions on the Neolithic humeri according to side and robusticity.

Acknowledgements

This work was performed with the support of the Wallonia-Brussels International and the Scientific Research Fund, the French Ministry of Foreign and European Affairs, and the Ministry of Higher Education and Research in the framework of the Hubert Curien Partnerships (Biologie, pathologie et comportements des derniers chasseurs-cueilleurs d'Europe occidentale, SCO/AD/FR -SOR/2015/223020). We would like to express our sincere thanks to Quentin Goffette (RBINS) for helpful comments and discussions on Neolithic subsistence, fishing, and hunting. We are particularly indebted to Katrien Van de Vijver (RBINS) for providing information on the reconstitution of adolescent activities from

Fox, S.J. 1977. "A paleoanthropological approach to recreation and sporting behaviors." In Studies in the anthropology of play edited by Tindall, B.A., and P. Stevens, 65-70. West Point (N.Y.): Leisure Press. Frayer, D.W., M. Lozano, J.M. Bermúdez de Castro, E. Carbonell, J.L. Arsuaga, J. Radovčić, I. Fiore, and L. Bondioli. 2012. "More than 500,000 years of right-handedness in Europe." Laterality 17, 51-69. Gilot, E. 1985. "Le squelette de Salet dans la chronologie 14 C." Helinium 25: 190-193. Gilot, E. 1986. "Abri Masson à Sprimont : datation l4 C." In L'ossuaire néolithique de l'abri Masson (Sprimont) edited by Dewez, M., E. Gilot, and M. Toussaint, 43-44. Liège: Société wallonne de Palethnologie, mémoire 6. Gore, R.M., L.F. Rogers, J. Bowerman, J. Suker, and C.L. Compere. 1980. "Osseous manifestations of elbow stress associated with sports activities." American Journal of Roentgenology 134 (5): 971-917. doi: 10.2214/ajr.134.5.971. Gregory B., and J. Nyland. 2013. "Medial elbow injury in young throwing athletes." Muscles, Ligaments and Tendons Journal 3(2): 91-100. doi: 10.11138/mltj/2013.3.2.91 Guilaine, J., and J. Zammit. 2001. Le sentier de la guerre. Visages de la violence préhistorique. Paris: Éditions du Seuil. Hewlett, B.S. 2016. "Social Learning and Innovation in Hunter-Gatherers." In Social Learning and Innovation in Contemporary Hunter-Gatherers. Evolutionary and Ethnographic Perspectives edited by Terashima, H., and B.S. Hewlett, 1-15. Tokyo: Springer Japan. Holloway, R.L. 2008. "The human brain evolving: A personal retrospective." In The human brain evolving: Paleoneurological Studies in Honor of Ralph L. Holloway edited by Broadfield D., M. Yuan, K. D. Schick, and N. Toth, 1-14. Gosport: Stone Age Institute Press.


PhD Guy Lenaers
email: guy.lenaers@inserm.fr

PhD Salim Khiati

MD Dominique Bonneau

Are your mitochondria ready for a space odyssey?

Keywords: space odyssey, OMICS, mitochondria, genome space odyssey, OMICS, mitochondria, genome space odyssey, OMICS, mitochondria, genome

Anticipating very long space trips, NASA has performed pan-OMIC analyses on inflight samples from astronauts, mice and cells. Results revealed major mitochondrial dysfunctions responsible for alterations in metabolism, immunity and circadian rhythm, which should prompt the evaluation of countermeasures to reduce the risks of future space odyssey, especially towards Mars.

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Core text (800 words)

Over the next decades, space flight will increase dramatically, including for recreational purposes. The duration of space cruises will also increase, since it will take at least 400 to 500 days to make a round-trip flight to the planet Mars [START_REF] Nordeen | Engineering Human Stasis for Long-Duration Spaceflight[END_REF]. This odyssey will undoubtedly induce physiological changes on the passengers that must be anticipated to reduce health problems. In a surprising study, entitled "Comprehensive multi- The multi-OMICS approach including epigenomic, transcriptomic, proteomic and metabolomic analyses were performed on cultured (fibroblasts and endothelial) and primary (T cells and hair follicles) human cells, on multiple organs from the C57BL/6 and BALB/c inbred mouse strains, as well as on urine and blood samples from 59 astronauts, the latter including the NASA twin study data [START_REF] Garrett-Bakelman | The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight[END_REF].

OMICs analysis reveals mitochondrial stress as a central biological hub

The main results emerging from the study of this remarkable collection of data concerned changes in mitochondrial physiology occurring during space flights. Indeed, OMIC signatures revealed a drastic uncoupling of the nuclear and mitochondrial genome cross-talk, with a systemic down-regulation expression of nuclear genes linked to energy production (i.e. electron transport, oxidative phosphorylation (OXPHOS) and ATP synthesis), while a progressive 2 to 9 fold longitudinal overexpression of mitochondrial genes, 3 paralleling increased mtDNA copy number, was observed, in particular in the blood cell fraction of astronauts, proportional to the time spent in space. This nucleo-mitochondrial genome uncoupling is responsible for misassembly of respiratory chain complexes, decreased mitochondrial oxidative metabolism and increased production of reactive oxygen species (ROS). In parallel, a metabolic shift from glycolysis to OXPHOS respiration, as well as increased beta-oxidation of long-chain fatty acids and Krebs cycle were observed in mouse skeletal muscle, but not in liver. In addition, the imbalance of the OXPHOS complex subunits led to proteostasis failures triggering the mitochondrial unfolded protein response (UPR mt ), and eventually the activation of Gadd34 and of the integrated stress response (ISR) in glycolytic muscle, while ISR biomarkers were downregulated in oxidative muscles together with a downregulation of the FGF21 and GDF15 mitokines. Alterations of mitochondrial physiology were further associated to the enrichment of genes associated to DNA repair as well as excretion of 8OH-dG in urine, witnessing possible alterations of the mitochondrial genome, and resulting in increased secretion of damage-associated molecular patterns (DAMPS) and VEGF, IGF1, IL1, IL1 and IL-1ra in blood, and up-regulation of IFN-dependent innate immune pathways in internal organs. In addition, genes involved in cell cycle were upregulated in all mouse tissues except T-cells, most likely to compensate for a higher level of apoptosis, as were genes involved in circadian rhythm in all internal organs, except liver, although animals were maintained in 12h/12h-light/dark cycle. Finally, the decreased in the synthesis of 1.25 vitamin D and the increase in the metabolism of NAD are further associated to the worsening of mitochondrial functions and alterations of DNA integrity, respectively. Notably, epigenomic methylation signatures reflected the major transcriptomic modifications induced by space flight. Ultimately, most of these deleterious changes reverted to normal in the weeks following the end of flight.

4

Altogether, these cellular modifications are witnessing processes related to premature aging, which could originate from major alterations of the mitochondrial genome, as loss of mtDNA integrity is clearly responsible for aging in mice [START_REF] Trifunovic | Premature ageing in mice expressing defective mitochondrial DNA polymerase[END_REF][START_REF] Desdín-Micó | T-cells with dysfunctional mitochondria induce multimorbidity and premature senescence[END_REF] and humans [START_REF] Fakouri | Toward understanding genomic instability, mitochondrial dysfunction and aging[END_REF]. In this regard, future assessments of mtDNA integrity in space-exposed samples are warranted to further reveal the initial mechanism responsible for the molecular changes reported in the Da Silveira paper, as mtDNA instability could be triggered both by the exposure to low doses of irradiation [START_REF] Shimura | Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM-and NBS1-deficient cells[END_REF] and by the consequences of microgravity [START_REF] Michaletti | Effects of microgravity on osteoblast mitochondria: a proteomic and metabolomics profile[END_REF].

The effects of accelerated aging of space flight further result in somatic changes, including loss of bone density, reduction in muscle mass and strength. Stiffening of the arteries, overactivation of the immune response, increased cardiovascular risks and possibly thrombovascular events were also observed. Alopecia, a marker of premature aging in animal models, is also affecting astronauts.Therefore, if astronauts (or future space tourists) do not wish to land on Mars as "centenarians", countermeasures are needed to minimize the consequences of long-duration space flights. In addition to daily muscular exercise, nutritional and pharmacological supplementation targeting mitochondrial physiology are mandatory. The "Mitochondrial Space Odyssey Cocktail" should be composed of anti-oxidants such as CoQ10, to counteract the oxidative stress and promote mitochondrial electron transfer, vitamin B3 to sustain NAD-related mitochondrial activity and DNA repair, and vitamin D to maintain bone mineral density and arteries plasticity. And why not a bottle of red wine to promote mitochondrial turnover and rejuvenation [START_REF] Wu | Grape skin extract improves muscle function and extends lifespan of a Drosophila model of Parkinson's disease through activation of mitophagy[END_REF]

  for spaceflight impact", by Da Silveira et al. published in November 2020 in Cell, NASA and associated teams addressed the molecular and biochemical consequences of space flights on cellular, animal and human samples collected during previous missions. Their study revealed drastic alterations of mitochondrial physiology as the main origin of most secondary metabolic and physiological manifestations occurring during space flights.

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 6 Figure Legend

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