Pharm. D. François Mullier

Elodie Lainey

Odile Fenneteau

Lydie Da Costa

Françoise Schillinger

Nicolas Bailly

Yvan Cornet

Christian Chatelain

Jean-Michel Dogne

Bernard Chatelain

Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: Results of a multicentre study Running short title: NEW DIAGNOSTIC PARAMETERS FOR HEREDITARY SPHEROCYTOSIS

Keywords: hereditary spherocytosis, reticulocyte, microparticles, ankyrin

Background: Hereditary Spherocytosis (HS) is characterized by weakened vertical linkages between the membrane skeleton and the red blood cell's lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. Design and methods: The levels of Hb, MCV, MCHC, Reticulocytes (Ret), Immature Reticulocytes Fraction (IRF), Hypochromic Erythrocytes (Hypo-He) and Microcytic Erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n: 1488). Results: HS is characterized by a high Ret count without an equally elevated IRF. All 45 HS have Ret > 80000/µl and Ret(/µl)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/ IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave Predictive Positive Value and Negative Predictive Value of respectively 75% and 100% (n: 1488), which is much greater than single parameters or existing rules.

Conclusions: This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates, ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

Introduction

Hereditary Spherocytosis (HS) [START_REF] King | Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia[END_REF][START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Perrotta | Hereditary spherocytosis[END_REF] is the most common cause of inherited chronic haemolysis in northern Europe and North America where it affects one person in 2000. HS refers to a group of heterogeneous regenerative haemolytic anaemias characterised by a loss of membrane surface area, leading to reduced deformability due to defects in proteins in the erythrocytic membrane (ankyrin, band 3 protein, β-spectrin, α-spectrin, or protein 4.2).

Osmotic resistance, hypertonic cryohaemolysis test, eosin-5-maleimide (EMA) binding in flow cytometry, Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) and ektacytometry are the reference tests for the diagnosis of HS. However, all tests suffer from specific limitations. Osmotic fragility test lacks of sensitivity and specificity [START_REF] Perrotta | Hereditary spherocytosis[END_REF].

Hypertonic cryohaemolysis test and EMA test are not specific and can also detect red cells with rare membrane disorders, such as aberrant band 3 proteins, a change in intracellular viscosity and temperature-sensitive monovalent cation transport [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]. Moreover, in mild or atypical cases, difficult interpretation is likely to occur. SDS-PAGE lacks sensitivity to very mild or asymptomatic HS carriers [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF], and a high reticulocyte count might mask a reduction in ankyrin-1 in SDS-PAGE [START_REF] Del Giudice | Apparently normal ankyrin content in unsplenectomized hereditary spherocytosis patients with the inactivation of one ankyrin (ank1) allele[END_REF] whereas an ankyrin-1 defect represents 40-65% of HS in the USA and Europe [START_REF] Perrotta | Hereditary spherocytosis[END_REF]. A key test for HS, osmotic gradient ektacytometry, is only available in specialised laboratories. Consequently, first, when selecting an appropriate test for HS, the sensitivity and specificity of the test for HS, the complexity of the protocol, and the total cost of instrument(s) and its maintenance should be taken into consideration [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]. Secondly, diagnosis of HS is currently based on a combination of clinical and family histories, physical examination (for splenomegaly and jaundice) and laboratory data including blood count, especially red cell indices, reticulocyte count and red cell morphology. Other causes of haemolytic anaemia should be excluded, particularly autoimmune haemolytic anaemia caused by warm (IgG) or cold (IgM) autoantibodies and also alloimmune haemolytic anaemia in the event of ABO incompatibility in neonates. Autoimmune haemolysis (AIHA) can usually be excluded by a negative direct anti-globulin test (DAT). Sometimes, there are difficulties in interpreting DAT-negative haemolytic anaemias in individuals with no family history, particularly in adults. In some cases, the density of attached autoantibody may be too low for detection by DAT. Polyspecific DAT reagents may also fail to detect some autoantibodies, particularly IgA autoantibodies. Moreover, diagnosis is often more difficult during the neonatal period than later in life, for many reasons [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Perrotta | Hereditary spherocytosis[END_REF].

In conclusion, the development of simple, fast, accurate, sensitive, and specific diagnostic laboratory tests for hereditary spherocytosis is a real challenge [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Perrotta | Hereditary spherocytosis[END_REF]. Some authors recommend automated red cell indices to predict or identify HS. Increased parameters such as reticulocytosis, % microcytes [START_REF] An | Disorders of red cell membrane[END_REF][START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Cynober | Red cell abnormalities in hereditary spherocytosis: Relevance to diagnosis and understanding of the variable expression of clinical severity[END_REF], combination of increased Mean Corpuscular Haemoglobin Concentration (MCHC) with increased red distribution width (RDW) [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Michaels | Screening for hereditary spherocytosis by use of automated erythrocyte indexes[END_REF] , combination of increased RDW with hyperdense cells (Hyperchromic cells) [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Kutter | Demonstration and quantification of "Hyperchromic" Erythrocytes by haematological analysers. Application to screening for hereditary and acquired spherocytosis[END_REF], combination of increased MCHC with increased hyperdense cells [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF], decreased red cell and reticulocyte surface area [START_REF] Costa | Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia[END_REF], discordance between the Cell Haemoglobin Concentration Mean (CHCM) and MCHC [START_REF] Kutter | Demonstration and quantification of "Hyperchromic" Erythrocytes by haematological analysers. Application to screening for hereditary and acquired spherocytosis[END_REF] or Mean Spherized Corpuscular Volume (MSCV) lower than Mean Cell Volume (MCV) [START_REF] Chiron | The gen.S: A fortuitous finding of a routine screening test for hereditary spherocytosis[END_REF] are used for HS screening. However, most of these parameters are mean parameters and none of the tests is efficient for HS screening, especially in mild forms (without anaemia, reticulocytes <6% and bilirubin 17.4-34.2µM) or carriers of an asymptomatic trait (without anaemia, slight reticulocytosis (1.5-3.0%) and slightly reduced haptoglobin) [START_REF] Perrotta | Hereditary spherocytosis[END_REF]. Although the main molecular defects in hereditary spherocytosis are heterogeneous, one common feature of the erythrocytes in this disorder is weakened vertical linkages between the membrane skeleton and the lipid bilayer and its integral proteins [START_REF] Perrotta | Hereditary spherocytosis[END_REF].

When these interactions are compromised, loss of cohesion between bilayer and membrane skeleton occurs, leading to destabilisation of the lipid bilayer and the release of skeleton-free lipid vesicles (or microvesicles or microparticles). Furthermore, one previous study [START_REF] Costa | Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia[END_REF] showed that different mechanisms lead to reduced membrane surface area in hereditary spherocytosis and some forms of AIHA. Indeed, in HS, but not in AIHA, the surface area loss is already present at the circulating reticulocyte stage. As a consequence of the release of such microparticles, the percentage of microcytes should be higher in HS. Since HS affects reticulocytes, the total reticulocyte count and Immature Reticulocytes Fraction could be of great interest. Therefore, we decided to study reticulocyte parameters to develop a diagnostic tool on 2 haematological analysers: XE-2100 and XE-5000 (Sysmex, Kobe, Japan).

The study objectives were i) to characterize a cohort of confirmed HS patients in terms of reticulocytosis and IRF ratio, and MicroR/Hypo-He ratio; ii) to study the efficiency of the combination of those 2 rules to screen confirmed HS cases and to differentiate HS from other haemolytic disorders, microcytic red cell disorders, healthy subjects and a routine haematological database; iii) to compare this tool with current existing rules, and iv) to assess the new method in neonates and in trait and mild forms.

Design and methods

Subjects

Between January 2008 and May 2009, the diagnosis of 45 HS patients (22 women, 23 men, mean age 13.1, from 1 day to 76 years old) was confirmed by clinical data and laboratory diagnosis; flow cytometry (eosin-5'-maleimide assay) and/or ektacytometry and/or SDS-PAGE electrophoresis as shown in Table 1. They were classified according to the haemoglobin level as severe (n= 6, Hb <8g/dl), moderate (n= 27, Hb 8-12 g/dl) and mild (n= 12, Hb > 12g/dl). Three of the 45 cases of HS were splenectomised before analysis (patients 1, 11 and 12 in Table 1).

The HS group was compared with a group of 108 patients suffering from various haemolytical disorders, such as ABO incompatibility (n=4), Thrombotic Thrombocytopenic Purpura (TTP, n=4), HUS (Haemolytic Uremic Syndrome, n=3), Drepanocytosis (n=5), PNH (Paroxysmal Nocturnal Haemoglobinuria, n=4), Glucose-6-PhosphateDeHydrogenase (G6PDH) deficiency (n=1), Pyruvate kinase (PK) deficiency (n=1), HbE + β-thalassemia (n=3), β-thalassemia (n=3) , haemolytic anaemia due to leukemias (n=7), lymphomas (n=12), myelomas (n=4), cold agglutinins (n=4) and others haemolytical disorders (n=53). Furthermore, we compared the HS group with a group of 93 microcytic anaemia patients (64 with iron deficiency and 29 with functional iron deficiency), with a control group of 61 healthy individuals and 1230 samples from the routine haematological database.

Finally, one membranopathie other than hereditary spherocytosis (ie a xerocytosis) was included.

Methods

HS diagnostic tests

In addition to the clinical data, serum bilirubin, hypertonic cryohaemolysis test [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF], flow cytometry (Eosin-5' maleimide assay) [START_REF] Girodon | Usefulness of the eosin-5'-maleimide cytometric method as a first-line screening test for the diagnosis of hereditary spherocytosis: Comparison with ektacytometry and protein electrophoresis[END_REF][START_REF] King | Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia[END_REF][START_REF] King | Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis[END_REF], SDS-PAGE electrophoresis [START_REF] King | Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia[END_REF] and ektacytometry [START_REF] Cynober | Red cell abnormalities in hereditary spherocytosis: Relevance to diagnosis and understanding of the variable expression of clinical severity[END_REF] were performed on HS patients. Osmotic fragility tests were not performed routinely since gradient ektacytometry is available in our laboratory which is considered superior to tests of osmotic fragility.

Haematological measurements (XE-2100 and XE-5000)

Blood was drawn in Venosafe ® terephthalate polyethylene tubes (Terumo Europe, Leuven, Belgium) containing dipotassium ethylenediaminetetraacetic acid (EDTA K2). Samples were taken during normal diagnostic follow-up. No additional sampling was performed. All the measurements were performed within 24 hours of blood sampling. Red cell morphology on the blood smears was evaluated by optical microscopy. Red cell and reticulocyte counts and Hyper-H e , MicroR and MacroR [START_REF] Urrechaga | Potential utility of the new sysmex xe 5000 red blood cell extended parameters in the study of disorders of iron metabolism[END_REF]. In addition, we calculated systematically the 2 following parameters: the MicroR/Hypo-He ratio and the Ret/IRF ratio ((10 9 )/ (l* %)).

%HYPO-H e and %HYPER-H e are unique parameters analysed in the reticulocyte channel of the XE-5000. The basis for analysis of these parameters is the mean haemoglobin content of all the measured red blood cells analysed in the reticulocyte channel. Red blood cells with decreased haemoglobin content (< 17pg) are classified as hypo-haemoglobinized cells, whereas the hyper-haemoglobinized red blood cell population contains cells with an increased haemoglobin content (>49pg) (Figure 1a)). The RBC cell size distribution curve shows an almost Gaussian distribution in healthy human blood. By applying two discriminators, one in the lower (<60fl) and one in the upper area (>120fl), a microcytic and a macrocytic population are analysed.

These parameters are sensitive for a small percentage of abnormal cells and therefore more sensitive than mean values.

Statistical methods

Sensitivity, specificity, ROC curves, and predictive values were calculated using Medcalc version 11.1.1.0 (Gent, Belgium). Comparison of Ret-IRF ratios (Figure 2), MicroR-HypoHe ratio (Figure 3) and MicroR (Figure 4) among trait or mild HS (n=12), moderate HS (n=27), severe HS (n=6), haemolytic disorders (n=108), iron deficiency (n=93), healthy subjects (n=61) and routine haematological database (n=1230) was performed using GraphPad Prism ® software Version 4. Results were presented as mean +/-standard error of the mean (SEM).

Results

All HS showed a high reticulocyte count without an equally elevated Immature Reticulocytes Fraction (Figure 1). The 45 confirmed cases of HS have reticulocytes > 80 10 9 /l and a Reticulocytes (10 9 /l) / Immature Reticulocytes Fraction (%) (Ret/IRF) ratio higher than 7.7 (Figure 2). As shown in Table 2, this limit is used as a precondition for the screening of all the cases of HS. The combination of reticulocytosis with index reticulocytosis/IRF is more discriminating than the combination of reticulocytosis with low IRF (data not shown).

Moreover, all trait and mild cases (Hb >12g/dl, n= 12) of HS have a Ret/ IRF ratio higher than 19 (Figure 2). Consequently, as shown in Table 2, this cut-off is used as rule 2 for the screening of trait and mild HS.

For moderate and severe cases of HS, MicroR was included since this parameter reflects the severity of the disease [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]. Furthermore, for those patients, MicroR and HypoHe were combined. Indeed, a preliminary study showed that combining MicroR and the MicroR/Hypo-He ratio led to the elimination of more non-HS cases than MicroR alone. ROC Curve analysis showed that the optimal cut-offs for MicroR and MicroR/Hypo-He in cases of Hb between 8 and 12 g/dl, were 3.5% and 2.5, respectively. When Hb was lower than 8 g/dl, the optimal cut-offs for MicroR and MicroR/Hypo-He were 3.5% and 2.0, respectively.

We then evaluated the efficiency of our diagnostic tool to screen confirmed HS cases to differentiate HS from other haemolytic disorders, iron deficiencies, healthy individuals and controls. Figure 2 showed that Ret/IRF ratio is highly efficient to discriminate confirmed mild and even moderate HS cases from patients suffering from various haemolytical disorders (n=108) , patients with microcytic anaemia (n= 93 whose 64 with iron deficiency and 29 with functional iron deficiency) , healthy individuals (n=61) and samples from the routine haemotological database (n=1230). The HS prevalence was 1.98% in our population.

However, as illustrated in Figures 234, addition of MicroR/Hypo-He (Figure 3) and MicroR (Figure 4) is required to discriminate severe HS from some haemolytical disorders and some healthy individuals, respectively.

The performances of the HS diagnostic tool were compared with single parameters and existing rules, as shown in Table 3. The Area Under the Curve (AUC), sensitivity, specificity, predictive positive value and negative predictive value were respectively 0.997 (95% Confidence Interval: 0.992-0.999), 100%, 99.3%, 75% and 100%. This diagnostic tool is therefore much more efficient than single parameters (Ret/IRF index) or the existing rules (reticulocytosis, % microcytes [START_REF] An | Disorders of red cell membrane[END_REF][START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Cynober | Red cell abnormalities in hereditary spherocytosis: Relevance to diagnosis and understanding of the variable expression of clinical severity[END_REF], combination of increased MCHC with RDW [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Michaels | Screening for hereditary spherocytosis by use of automated erythrocyte indexes[END_REF] , combination of increased RDW with hyperdense cells (Hyperchromic cells) [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Kutter | Demonstration and quantification of "Hyperchromic" Erythrocytes by haematological analysers. Application to screening for hereditary and acquired spherocytosis[END_REF], combination of increased MCHC with increased hyperdense cells [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]). The negative predictive value is excellent (>98.5 %) for all parameters or rules. However, the second best rule in term of positive predictive value is the combination of MCHC and Hyper-He with a PPV of only 32.5% (Table 3).

Finally, regarding ABO incompatibilities (n=4), all had Ret/IRF ratio higher than 7,7 but none had Ret/IRF ratio higher than 19,9, MicroR higher than 3,5 or MicroR/Hypo-He higher than 2.

Discussion

The diagnostic tool proposed in the present study is based on the physiopathology of HS. Indeed, one common feature of the red blood cells in this disorder is weakened vertical linkages between the membrane skeleton and the lipid bilayer leading to the release of skeleton-free lipid vesicles. In HS, the loss of surface area is already present at the circulating reticulocyte stage. We thus decided to study reticulocyte parameters to develop a diagnostic tool on 2 haematological analysers: XE-2100 and XE-5000 (Sysmex, Kobe, Japan).

As shown in Table 2, the diagnostic method includes a precondition to screen all cases of HS, and a second rule (rule 2) taking into account the severity reflected by the degree of anaemia.

Rule 1: Ret and Ret/IRF ratio. All 45 confirmed cases of HS have reticulocytes > 80 10 9 /l and a Reticulocytes (10 9 /l) / Immature Reticulocytes Fraction (%) (Ret/IRF) ratio higher than 7.7. This limit is used as a precondition for the screening of all the cases of HS.

Rule 2: Ret/IRF ratio or MicroR/Hypo-He ratio. The severity of the disease shown by Hb level is due to the intensity of release of microparticles, which is reflected by MicroR, the best indicator of HS severity [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]. Therefore, MicroR was included in rule 2, only for moderate and severe cases of HS.

Trait and mild HS

All trait and mild cases (Hb >12g/dl, n= 12) of HS have a Ret/ IRF ratio higher than 19. The screening of trait and mild HS based on this method is certainly a major advance. Indeed, interpretation of hypertonic cryohaemolysis test and EMA binding in flow cytometry is often difficult in mild cases. Moreover, SDS-PAGE suffers from many limitations: it lacks of sensitivity to very mild or asymptomatic HS carriers [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF], and a high reticulocyte count might mask a reduction in ankyrin-1 in SDS-PAGE [START_REF] Del Giudice | Apparently normal ankyrin content in unsplenectomized hereditary spherocytosis patients with the inactivation of one ankyrin (ank1) allele[END_REF][START_REF] Perrotta | Hereditary spherocytosis[END_REF]. Patients 3, 4 and 6 (Table 1), for whom HS was confirmed by clinical data and flow cytometry, illustrate the lack of sensitivity of SDS-PAGE.

Moderate and severe cases of HS

For moderate and severe cases of HS, MicroR and MicroR/Hypo-He were combined. The optimal cut-offs in cases of Hb between 8 and 12 g/dl, were 3.5% and 2.5, respectively. When Hb was lower than 8 g/dl, the optimal cut-offs were 3.5% and 2.0, respectively.

Neonates and ABO incompatibilities

Interestingly, the method was also valid for the 4 neonates (2 severe, 1 moderate and 1 mild) and the 4 cases of ABO incompatibility patients. The screening of HS in such individuals is another major advance of this method. Indeed, the diagnosis is often difficult during the neonatal period for several reasons, including infrequent splenomegaly, variable and usually not severe reticulocytosis, few spherocytes in their peripheral blood smears (whereas spherocytes (≤ 1%) are commonly seen in neonatal blood films in the absence of disease), and the fact that neonatal red blood cells are more osmotically resistant than adult cells. Furthermore, at this age, the difference between an ABO incompatibility patient and an HS patient is very difficult to detect [START_REF] Saada | Incidence of hereditary spherocytosis in a population of jaundiced neonates[END_REF][START_REF] Sgro | Incidence and causes of severe neonatal hyperbilirubinemia in canada[END_REF].

We then evaluated the efficiency of our diagnostic tool to screen confirmed HS cases, healthy individuals and to differentiate HS from other haemolytic disorders and iron deficiencies.

The Area Under the Curve (AUC), sensitivity, specificity, predictive positive value and negative predictive value were respectively 0.997, 100%, 99.3%, 75% and 100%. This diagnostic tool is therefore much more efficient than single parameters (Ret/IRF index) or the existing rules (reticulocytosis, % microcytes [START_REF] An | Disorders of red cell membrane[END_REF][START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Cynober | Red cell abnormalities in hereditary spherocytosis: Relevance to diagnosis and understanding of the variable expression of clinical severity[END_REF], combination of increased MCHC with RDW [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Michaels | Screening for hereditary spherocytosis by use of automated erythrocyte indexes[END_REF] , combination of increased RDW with hyperdense cells (Hyperchromic cells) [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF][START_REF] Kutter | Demonstration and quantification of "Hyperchromic" Erythrocytes by haematological analysers. Application to screening for hereditary and acquired spherocytosis[END_REF], combination of increased MCHC with increased hyperdense cells [START_REF] Bolton-Maggs | Guidelines for the diagnosis and management of hereditary spherocytosis[END_REF]).

As HS is a low prevalence disease, predictive values are more relevant parameters than sensitivity and specificity. The negative predictive value is excellent (>98,5%) for all parameters or rules. However, the second best rule in term of positive predictive value of MCHC and Hyper-He with a PPV of only 32.5% (Table 3). Besides its very good performances, this diagnostic method is also inexpensive, corresponding to a Cell Blood Count (CBC) and parameters linked to reticulocytes and red blood cell parameters (Hb, MCV, MCHC, RET, IRF, Hypo-He, MicroR).

From a technological point of view, the method contains a combination of reticulocytosis with a decrease in IRF, due to lower fluorescence intensity (Figure 1). To perform the IRF measurement, reticulocytes are labeled with a fluorescent dye (polymethine and oxazine) in a Ret-Search (II) reagent that results in the cell entry and staining of mRNA reticulocytes.

Several hypotheses could explain a high reticulocyte count without an equally elevated Immature Reticulocytes Fraction observed in HS. The first hypothesis is an insufficient entry of the fluorescence dye into the defective cells, leading to an abnormal ratio between the two parameters. Indeed, the loss of red cell membrane proteins (band-3 protein, ankyrin, spectrin) could disturb the response of spherocytic cells to permeabilisation. The staining reaction is thus abnormal at the time of measurement. This results in decreased staining of RNA and normal IRF as seen in the RET scattergram shown in Figure 1. A lower RNA concentration in the RBC implies increased maturity, so that immature reticulocytes will be falsely classified as a more mature fraction (MFR or LFR). Consequently, the highly fluorescent immature reticulocytes containing the most RNA are decreased. The second hypothesis is the early loss of cell surface area during HS. The maturation of reticulocytes into erythrocytes is associated with a loss of intracellular organelles, including mitochondria, endoplasmic reticulum, golgi apparatus and endocytic vesicles. There is also extensive remodeling of the plasma membrane, with progressive loss of various membrane proteins through the release of exosomes. These lost proteins include the transferrin receptor, flotillin, Glut-4, CD47, actin, Hsc70, aquaporin-1 (AQP-1) and adhesion receptors such as β1-integrin [START_REF] Blanc | The water channel aquaporin-1 partitions into exosomes during reticulocyte maturation: Implication for the regulation of cell volume[END_REF]. In HS, the loss of cell surface area at the reticulocyte stage [START_REF] Costa | Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia[END_REF] could explain that one or multiple of these compounds normally stained by the fluorescent dye in the reticulocyte channel are less marked in cases of HS.

Limitations of this study are that first, these results have been only validated on XE-2100 and XE-5000 instruments. In a near future, a similar method will be developed for other haematological analysers. Detection of abnormalities in the reticulocyte maturation could improve the current performances on Advia 2120 (Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA) and LH750/DXH800 (Beckman Coulter Inc, Miami, FL, USA) based respectively on the discordance between MCHC and CHCM [START_REF] Harris | Performance evaluation of the advia 2120 hematology analyzer: An international multicenter clinical trial[END_REF][START_REF] Kutter | Demonstration and quantification of "Hyperchromic" Erythrocytes by haematological analysers. Application to screening for hereditary and acquired spherocytosis[END_REF] and the difference between MCV and MSCV [START_REF] Banfi | Reticulocyte count, mean reticulocyte volume, immature reticulocyte fraction, and mean sphered cell volume in elite athletes: Reference values and comparison with the general population[END_REF][START_REF] Chiron | The gen.S: A fortuitous finding of a routine screening test for hereditary spherocytosis[END_REF][START_REF] Rodrigues | Clinical utility of beckman-coulter gen's reticulocyte analysis in the study of anemia of chronic disease (acd)[END_REF][START_REF] Urrechaga | Clinical utility of the new beckman-coulter parameter red blood cell size factor in the study of erithropoiesis[END_REF]. The second limitation is that, at present it is unknown whether the same phenomena concerning Ret-IRF-ratio and Micro-Hypo-ratio can be observed in other types of membranopathy like xerocytosis and related cryohydrocytosis. This differential diagnosis is particularly important since splenectomy is virtually contraindicated in xerocytosis [START_REF] Bruce | Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, ae1[END_REF]. However, the only included xerocytosis was positively screened by the precondition (rule 1) since the Ret-IRF ratio was higher than 7,7 [START_REF] King | Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis[END_REF][START_REF] Cynober | Red cell abnormalities in hereditary spherocytosis: Relevance to diagnosis and understanding of the variable expression of clinical severity[END_REF]. But, a Hb of 14,4g/dl allowed to exclude the diagnosis of HS with the rule 2 since the Ret-IRF ratio was lower than 19.

Conclusions

We have developed and validated of an original diagnostic tool of hereditary spherocytosis based on reticulocyte-derived microparticles release. This diagnostic tool could be used routinely as an excellent screening method for the diagnosis of HS. This is in line with recommendations proposed by some authors in recent reviews [START_REF] Perrotta | Hereditary spherocytosis[END_REF]. This rapid method also works on mild SH and in neonates, and overcomes the lack of sensitivity of SDS-PAGE in ankyrin deficiencies. This proposed tool in combination with clinical data could make the diagnosis of HS easier by reducing the number of expensive and time-consuming confirmation tests.

  Figure count: 4

  indices and haemoglobin values were determined using XE-2100 (n= 15) and XE-5000 (n= 30) (Sysmex, Kobe, Japan). These two automated blood cell analysers combine a red semiconductor laser technique with different polymethine dyes to produce a 5-part differential WBC count and reticulocyte measurement. The minimal volumes required for Cell Blood Count (CBC) and reticulocyte measurements are 40 µl (capillary mode), 130µl (manual mode) or 200 µl (automatic mode). Parameters of interest provided by both haematological analysers are: Haemoglobin (Hb, (g/dl)), Mean Corpuscular Volume (MCV, fl), Mean Corpuscular Haemoglobin Concentration (MCHC, (g/dl)), Reticulocytes (RET, (giga/l)) and Immature Reticulocytes Fraction (IRF, (%)) and the specific XE-5000 parameters Hypo-H e ,

Figure 1 .Figure 2 :

 12 Figure 1. Reticulocytes channel on Sysmex XE-5000 CM(a) Hereditary Spherocytosis: reticulocytosis with decreased Immature Reticulocytes Fraction (IRF). Definition of Hypo-He: Hypochromic erythrocytes (%) and Hyper-He: Hyperchromic erythrocytes, is also shown. The basis for analysis of these parameters is the mean haemoglobin content of all the measured red blood cells (RBC-He) analysed in the reticulocyte channel. Red blood cells with a mean haemoglobin content lower than 17pg, corresponding to the low discriminator for RBC-H e (RBC-He -LD), are classified as hypo-haemoglobinized cells, whereas the hyper-haemoglobinized red blood cell population contains cells with a haemoglobin content higher than to 49pg, the high discriminator for RBC-H e (RBC-H e -HD).

Figure 3 :

 3 Figure 3: Distribution of microR/hypoHe ratios (mean+/-SEM) among trait or mild HS (n=11), moderate HS (n=15), severe HS (n=4), haemolytic disorders (n=108), iron deficiency (n=93), healthy subjects (n=61) and routine haematological database (n=1230). The cut-offs defined in the diagnostic tool are also shown: 2,5 for moderate HS (continuous line) and 2,0 for severe HS (dotted line).

Figure 4 :

 4 Figure 4: Distribution of MicroR (mean+/-SEM) among trait or mild HS (n=11), moderate HS (n=15), severe HS (n=4), haemolytic disorders (n=108), iron deficiency (n=93), healthy subjects (n=61) and routine haematological database (n=1230). The cutoff defined in the diagnostic tool is also shown: 3,5 for moderate and severe HS (dotted line).

Table count

 count

	: 3
	Reference count: 21

Table 1 : Red Cell Indices, serum bilirubin, cryohaemolysis, flow cytometry, SDS-PAGE and ektacytometry for HS patients

 1

	Patient (sex)	Age	Hb	MCV	MCHC	RET	IRF	Ret/	MicroR	Hypo-He	MicroR/Hypo-	Indirect Bilirubin	Cryohaemolysis	Flow cytometry	SDS-PAGE H	Ektacytometry
			(g/dl) A	(fL) B	(g/dl) C	(10 9 /l) D	(%) E	IRF	(%) F	(%) G	He	(mg/dl)	(%)	(%)
	1(F) I	53y K	14,4	93,3	36,3	124,7	3,2	39	1,1	0,7	1,6	0,99	14,5	19,6	ND L	ND
	2(F)	5mon M	8,4	71,1	35,1	252	8,6	29,3	23,9	4,8	5	1,16	13,8	33,3	Spectrin and ankyrin deficiency	ND
	3(F)	2mon	8,7	79,6	33,9	228,7	21,2	10,8	9,2	2	4,6	ND	16,3	21,8	-N	ND
	4(M) J	3mon	8,7	75,2	35,4	199	8,8	22,6	16	3,8	4,2	ND	19,7	33,6	-	ND
	5(M)	15y	16,1	84,9	38,2	426,6	2,8	152,4	4,1	0,3	13,7	10,22	19,8	27,6	ND	ND
	6(M)	8y	11,4	74,9	36,8	303	4,2	72,1	16,1	1,3	12,4	1,3	48,9	43,1	-	ND
	7(F)	2y 10mon	11,2	79,5	34,8	202	4,8	42,1	13,7	2	6,9	2,7	51	39,9	Spectrin deficiency	ND
	8(M)	21y	13,6	95	33,9	514	10,7	48	5	1,2	4,2	8,12	56,3	34,3	Spectrin deficiency	ND
	9(M)	27y	12	82,3	36,5	538	10,9	49,4	10	1,8	5,6	ND	ND	34	ND	ND
	10 (F)	17y	12,5	87,6	37,7	125,1	0,4	312,8	1,6	0,2	8	4,94	ND	ND	Band 3 deficiency	ND
	11 (M)	42y	17,2	82,7	35,5	81,9	1,7	48,2	3,3	0,2	16,5	0,48	ND	ND	ND	ND
	12 (M)	57y	16,1	89,6	35,1	83,5	0,3	278,3	1,4	0,2	7	0,63	ND	ND	ND	ND
	13 (M)	14y	12,5	87,4	37,7	288,8	4,4	65,6	3,2	0,3	10,7	3,81	ND	ND	Band 3 deficiency	ND
	14 (M)	31y	13	93,2	33,8	178	4,7	37,9	1	0,3	3,3	8,4	ND	27	ND	ND
	15 (M)	UA O	9,5	71,3	35,1	113,6	5,6	20,3	21,3	2,7	7,9	ND	13,1	34,9	ND	ND
	16 (M)	34y	16,9	88,3	32,4	85,7	6,2	13,8	4,8	1,4	3,4	0,43	ND	32,8	ND	ND
	17 (M)	52y	13,3	82,8	35,9	290	4,7	61,7	7,6	1,2	6,3	2,72	19,3	18,8	Band 3 deficiency	ND
	18 (M)	8y 8mon	11,4	81,7	32,8	293,5	9,7	30,3	12,4	3,8	4,4	ND	ND	38	ND	ND
	19 (M)	5y 10mon	12,5	84,7	38,2	84,9	4,1	20,7	1,8	0,1	18	0,6	ND	ND	ND	+ P
	20 (M)	2mon	7,6	79,2	35,7	181	11	16,5	10,2	2,6	3,9	4	ND	34	ND	ND
	21 (M)	11y 5mon	9,2	75,3	35,1	378,6	13,8	27,4	20,8	2,9	7,2	4,3	ND	ND	ND	+
	22 (F)	8y 11mon	11,8	81,2	36,5	345,1	5,5	62,7	11	1,7	6,5	4,9	ND	ND	ND	+
	23 (F)	8y	11,4	86,8	32	584,3	8,4	69,6	10,6	4,1	2,6	ND	ND	ND	ND	+
	24 (F)	1mon	6,6	81,7	36,9	158,1	15,4	10,3	6,1	1,4	4,4	3,1	ND	41,9	ND	ND
	25 (M)	3y 10mon	5,1	65,8	32,3	209,5	21,7	9,7	40	15	2,7	2	ND	30,5	ND	ND
	26 (M)	76y	10,1	96,3	35,7	285,2	15,8	18,1				2,6	ND	47,7	Band 3 and protein 4.2	+
									50	25	2,0				deficiency
	27 (M)	11mon	10,8	69,4	34,8	280,7	13,6	20,6	3,5	1,4	2,5	ND	ND	ND	ND	ND
	28 (M)	2y 1mon	5,9	88,5	30,7	287,5	37,4	7,7	23,9	5,9	4,1	2	11,3	25,2	-	ND
	29 (F)	37y	10,9	88,4	32,4	370,5	7,1	52,2	7,9	1,5	5,3	18,9	ND	33,9	ND	ND
	30 (M)	17y	11,1	80,8	35,2	175,5	11,2	15,7	23,5	3,2	7,3	ND	ND	ND	Band 3 deficiency	ND
	31 (M)	8d Q	10,8	112,3	32	363,6	1,5	242,4	ND	ND	ND	29,5	ND	ND	ND	+
	32 (F)	2mon	6,6	77,9	34,7	279,4	33,4	8,4	ND	ND	ND	2,2	ND	ND	ND	+
	33 (F)	1mon	5,7	81,9	37	211,9	24,2	8,8	ND	ND	ND	3,4	ND	ND	ND	+
	34 (F)	2y 4mon	10,4	73,6	35,3	469,2	15	31,3	ND	ND	ND	3	ND	ND	ND	+
	35 (F)	4y	10,6	78,8	37,6	222,3	14,8	15	ND	ND	ND	3	ND	ND	ND	+
	36 (F)	7y 3mon	8,4	73,2	33,7	461,7	23,2	19,9	ND	ND	ND	4,8	ND	ND	ND	+
	37 (F)	11mon	10,9	78,9	33,4	432,4	24,5	17,6	ND	ND	ND	ND	ND	ND	ND	+
	38 (F)	6mon	9,3	73,6	33,8	471,1	23,5	20	ND	ND	ND	1,8	ND	ND	ND	+
	39 (F)	5y 4mon	10,4	77,7	35	308,7	19,3	16	ND	ND	ND	0,7	ND	ND	ND	+
	40 (F)	7mon	10,2	78,1	34,5	457,1	20,5	22,3	ND	ND	ND	ND	ND	ND	ND	+
	41 (F)	3y 11mon	11,5	82	35	277,1	11,5	24,1	ND	ND	ND	2,7	ND	ND	ND	+
	42 (F)	7mon	10,2	79,4	34	396,9	18,8	21,1	ND	ND	ND	ND	ND	ND	ND	+
	43 (M)	1d	17,3	97,8	36,2	419,1	37	11,3	ND	ND	ND	21,8	ND	ND	ND	+
	44 (F)	1y 10mon	9,8	78	32,6	584	28,5	20,5	ND	ND	ND	ND	ND	ND	ND	+
	45 (F)	5mon	9,9	80,9	32,9	508,5	31	16,4	ND	ND	ND	ND	ND	ND	ND	+

Table 2 . Hereditary spherocytosis diagnostic tool

 2

	Rule		Parameters
	Rule 1	Precondition	Ret ≥ 80000/µl and Ret/IRF > 7.7
	Rule 2	Severity	Trait or mild HS	Moderate HS	Severe HS
			Hb > 12g/dl	8 g/dl ≥ Hb ≤	Hb < 8 g/dl
				12g/dl
			Ret/IRF ≥ 19	MicroR ≥ 3,5%	MicroR ≥ 3,5%
				and	and
				MicroR/Hypo-	MicroR/Hypo-
				He ≥ 2,5	He ≥2
	Footnote:
	Ret indicates Reticulocytes (/µl); IRF, Immature Reticulocytes Fraction (%); HS, Hereditary
	Spherocytosis; Hb, Haemoglobin; MicroR, Microcytic erythrocytes (%); Hypo-He,
	Hypochromic erythrocytes (%)

Table 3 : Efficiency of the HS diagnostic tool and comparison with single parameters and existing rules.

 3 AUC indicates Area Under the Curve; 95% CI, 95% Confidence Interval; PPV, Predictive Positive Value; NPV, Negative Predictive Value; MCHC, Mean Corpuscular Hemoglobin Concentration (g/dl); MicroR, Microcytic erythrocytes (%); RDW-CV (%), Red Blood Cells Distribution Width-Coefficient of Variation; Hyper-He, Hyperchromic erythrocytes (%); Hypo-He, Hypochromic erythrocytes (%); MicroR/Hypo-He, Microcytic erythrocytes/ Hypochromic erythrocytes; Ret/IRF, Reticulocytes/ Immature Reticulocytes Fraction (/µl*%); HS, Hereditary Spherocytosis

	Parameter	AUC	Cut-off Sensitivity	Specificity	PPV (%)	NPV (%)
		(95% CI)		(%)	(%)
	MCHC	0.735	34.7	73.3	72.6	5.1	99.3
	(g/dl)	(0.711-0.758)
	MicroR (%)	0.744	7.8	56.7	84.8	7.0	99.0
		(0.721-0.766)
	RDW-CV	0.684	18.1	55.2	80.6	5.6	98.9
	(%)	(0.659-0.708)
	MCHC and	0.678	positive	37.9	97.6	24.4	98.7
	RDW-CV	(0.653-0.702)
	Hyper-He	0.750	0.5	55.2	82.1	6.0	98.9
	(%)	(0.726-0.772)
	MCHC and	0.714	positive	44.8	98.1	32 .5	98.8
	Hyper-He	(0.690-0.738)
	RDW-CV	0.642	positive	34.5	94.0	10.6	98.6
	and Hyper-	(0.617-0.667)
	He
	MicroR/Hy	0.743	4.0	76.7	65.6	4.3	99.3
	po-He ratio	(0.720-0.764)
	Ret (10 3 /µl)	0.938	103.5	93.3	83.6	10.3	99.8
		(0.925-0.950)
	Ret/IRF	0.976	9.7	96.7	89.6	15.9	99.9
	ratio	(0.967-0.983)
	HS	0.997	positive	100.0	99.3	75.0	100.0
	diagnostic	(0.992-0.999)
	tool
	Footnote:

Acknowledgments

François Mullier is a FRIA researcher. The authors wish to thank all participating haematological centers : Catherine Bardiau (Huy, Belgium), Christophe Chantrain (Bruxelles and Mont-Godinne, Belgium), Andre Delattre (Verviers, Belgium), Andre Gothot (Liege, Belgium), Jean-Sebastien Goffinet (Bruxelles, Belgium), Vincent Hennaux (Gilly, Belgium), Olivier Ketelslegers (Liege, Belgium), Salah-eddine lali (Verviers, Belgium), Jacques Mairesse (Ottignies, Belgium), Jean-Marc Minon (Liège, Belgium) and David Tuerlinckx (Mont-Godinne, Belgium) Lydie Da Costa (Paris, France), Françoise Schillinger (Besançon, France).


Francesca Palandri

Nicola Polverelli

Lucia Catani

Emanuela Ottaviani

Michele Baccarani

MD Nicola Vianelli
email: nicola.vianelli@unibo.it

Impact of leukocytosis on thrombotic risk and survival in 532 patients with Essential Thrombocythemia: a retrospective study

Keywords: Essential Thrombocythemia, leukocytosis, thrombotic risk

Established risk factors for thrombosis in Essential Thrombocythemia (ET) include age (≥60 years) and previous vascular events. Recently, also leukocytosis has been proposed in risk stratification of ET patients. We report a retrospective study on 532 ET patients followed for a median of 7.6 years. Sixty-four patients (12%) developed 95 thrombotic events during follow-up. Together with the high risk condition, a WBC value above 11x10 9 /L, corresponding to the fourth percentile value, significantly correlated with a higher thrombotic risk (p=0.033) by Cox proportional hazards. Moreover, the cumulative risk of thrombosis was significantly higher in high-risk patients with WBC>11x10 9 /L. JAK2 V617F mutation did not correlate with thrombosis. Overall, 123 (23%) patients died. Three independent parameters were noted as prognostic factors for survival in multivariate analysis: age>60 years, leukocytosis>11x10 9 /L and haemoglobin level below normal values. Based on these parameters, three groups of risk were defined, with significantly different survivals. Baseline leukocytosis correlated with a higher thrombotic risk in high risk patients and identified a cohort of patients with worse survival.

INTRODUCTION

Essential Thrombocythemia (ET) is a Philadelphia-negative chronic myeloproliferative neoplasm (CMPN) characterized by the abnormal proliferation of a malignant megakaryocytic clone, responsible for persistent thrombocytosis [START_REF] Harrison | Essential thrombocythaemia: challenges and evidence-based management[END_REF][START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF].

The clinical course is burdened by an increased incidence of vascular complications, whose pathogenesis is multifactorial. In the last years, many retrospective studies have failed to show a clear correlation between platelet count and thrombotic events [START_REF] Palandri | Long-term follow-up of 386 consecutive patients with essential thrombocythemia: safety of cytoreductive therapy[END_REF][START_REF] Passamonti | Prognostic factors for thrombosis, myelofibrosis, and leukemia in essential thrombocythemia: a study of 605 patients[END_REF][START_REF] Tefferi | Thrombosis in myeloproliferative disorders: prevalence, prognostic factors, and the role of leukocytes and JAK2V617F[END_REF] and the role of thrombocytosis in the assessment of vascular risk has been progressively minimized. Conversely, age (≥60 years) and previous vascular events remain the main established risk factors for thrombosis and on this basis patients are currently stratified in low and high risk.

Recently, also the presence of JAK2V617F mutation [START_REF] Vannucchi | Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia[END_REF][START_REF] Stefano | Leukocytosis is a risk factor for recurrent arterial thrombosis in young patients with polycythemia vera and essential thrombocythemia[END_REF][START_REF] Lussana | Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: a systematic review[END_REF] and baseline leukocyte (WBC) count have been considered as disease-related cardiovascular risk factors of ET patients. This latter has been found to play a biological role in determining thrombosis, mainly acute coronary syndromes, in CMPN and particularly in ET, although it is presently not completely defined whether elevated WBC levels actually contribute directly to causing thrombosis [START_REF] Marchetti | Leukocytosis, JAK2V617F mutation, and hemostasis in myeloproliferative disorders[END_REF]. Consequently, several clinical studies have investigated the association between leukocytosis at diagnosis and risk of thrombosis, with not univocal results. Indeed, some Authors, including our group, have reported that leukocyte count at presentation did not predict subsequent vascular events [START_REF] Palandri | Long-term follow-up of 386 consecutive patients with essential thrombocythemia: safety of cytoreductive therapy[END_REF][START_REF] Tefferi | The interaction between leukocytosis and other risk factors for thrombosis in essential thrombocythemia[END_REF][START_REF] Passamonti | Prognostic factors for thrombosis, myelofibrosis, and leukemia in essential thrombocythemia: a study of 605 patients[END_REF][START_REF] Girodon | Leukocytosis is associated with poor survival but not with increased risk of thrombosis in essential thrombocythemia: a populationbased study of 311 patients[END_REF], although none of these analyses investigated possible differences among low and high-risk patients. On the contrary, some other studies concluded that leukocytosis was associated with thrombosis, particularly in low-risk patients [START_REF] Carobbio | Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status[END_REF][START_REF] Carobbio | Leukocytosis and risk stratification assessment in essential thrombocythemia[END_REF][START_REF] Wolanskyj | Essential thrombocythemia beyond the first decade: life expectancy, long-term complication rates, and prognostic factors[END_REF], but no consensus was found on the numerical cut-off which should be utilized for the definition of leukocytosis. Furthermore, the implications of baseline WBC count on long-term survival remain to be determined.

The aim of this article is to contribute to these issues by reporting a long-term retrospective study on 532 ET patients followed for a median of 7.6 years, with particular focus on the evaluation of the additional risk factors for thrombosis and survival.

METHODS

Definitions

The diagnosis of ET was made according to the criteria proposed by the Polycythemia Vera Study Group (PVSG) [START_REF] Murphy | Experience of the Polycythemia Vera Study Group with essential thrombocythemia: a final report on diagnostic criteria, survival, and leukemic transition by treatment[END_REF] and/or, since 2002, the WHO criteria [START_REF] Vardiman | The World Health Organization (WHO) classification of the myeloid neoplasms[END_REF]. Additional risk factors for thrombosis (blood pressure >140/90 mmHg, overweight, smoking habit, mellitus diabetes, and hypercholesterolemia, defined as fasting serum total cholesterol concentration >200mg/dL) and renal and liver function were evaluated at diagnosis and at 6-months interval thereafter.

The definition of acute leukemia and myelodisplastic syndrome were made according to the French-American-British criteria [START_REF] Bennett | Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group[END_REF]; diagnosis of myelofibrosis was based on Italian Consensus Conference criteria [START_REF] Barosi | The Italian Consensus Conference on Diagnostic Criteria for Myelofibrosis with Myeloid Metaplasia[END_REF]. Thrombotic episodes were objectively documented by: venography or ultrasonography (deep venous thrombosis); arteriography (peripheral arterial thrombosis); computed tomography scanning, magnetic resonance or angiography (intracerebral vessels thrombosis). Acute myocardial infarction required acute clinical presentation, typical electrocardiograph features and elevated creatine-kinase MB fraction.

Cerebral transient ischemic attack (TIA) required neurological symptoms and/or signs lasting less than 24 hours.

Assessment of JAK2 V617F mutational status

Total RNA was extracted from peripheral blood mononuclear cells using RNAeasy MiniKit (QIAGEN). RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using 5µM of random examers and 200 U of M-MLV Reverse Transcriptase (Invitrogen, Paisley, UK) according to the manufacturer's recommendations. The cDNA was amplified using forward and reverse primers (5'-gatgagcaagctttctcacaagc-3' and 5'aaacagttggcatgggccatgc-3'); the amplified product (185bp) was digested with Bsa XI (5U) (New England Biolabs, Germany) over-night at 37°C [START_REF] Quentmeier | JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders[END_REF]. After digestion, 20 µl of amplified PCR products was electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Statistical Methods

Risk factors for thrombosis were tested using a Cox proportional hazards model. All multivariable models have been fitted after adjusting for sex, standard risk factors (age>60 years and/or previous thrombotic event), and quartile distributions of laboratory parameters measured at diagnosis (hemoglobin, hematocrit, leukocytes and platelet count). Overall survival (OS) was calculated by the product-limits method of Kaplan and Meier from the date of ET diagnosis to the date of death or last contact, whichever came first, with 95% confidence interval (95% CI). All p values were two-sided and the significance level for all statistical tests was 0.05.

RESULTS

Patients

From January 1978 to December 2008, 532 consecutive patients received a diagnosis of ET at the Institute of Hematology and Oncology "L. and A. Seràgnoli", Bologna, Italy.

General characteristic of the patients are summarized in Table I. Median age at diagnosis was 64 years (range, 16-95); 307 were 60 years or older (58%). In 350 (66%) patients, one or more additional risk factors for thrombosis were present at diagnosis.

According to standard risk factors (age≥60 years and/or history of vascular events), patients were classified as low risk (n=186; 35%) and high risk (n=346; 65%). However, during the years, indications for cytotoxic and antiplatelet therapy were also represented by extreme thrombocytosis (platelet count higher than 1000x10 9 /L), presence of additional cardiovascular risk factors and/or symptoms due to microvessel disturbances. Consequently, 86% of patients received cytotoxic therapy (mainly, Hydroxyurea) and 92% received an antiplatelet agent (mainly, aspirin). More specifically, during up to 32.5 years of follow-up (median, 7.6 years), 130 out of 186 low-risk patients (70%), and 330 out of 346 high-risk patients (95%) received at least one cytoreductive drug.

Overall, 64 (12%) patients (15/186 low-risk patients, 8%, and 49/346 high-risk patients, 14%) developed 95 thrombotic complications. Arterial events (including stroke, TIA, acute coronary syndromes and peripheral arterial occlusions) constituted the majority (61%) of events.

Risk factors for thrombosis

Multivariable analysis of risk factors for thrombosis confirmed the value of the high-risk condition. By dividing patients in quartile distribution of leukocytes and platelet number, hemoglobin concentration and hematocrite, also a WBC value above 11x10 9 /L, corresponding to the fourth percentile value, significantly correlated with a higher thrombotic risk, together with the high risk condition (p=0.033 and p=0.003, respectively) (Table II). When arterial thrombosis were analyzed separately, only high risk condition correlated significantly with a higher risk of such events (HR=2.2, 95% CI 1.07 -4.85, p=0.032). The presence of the JAK2 V617F mutation was not found to be an independent risk factor for thrombotic events (11 thrombotic episodes in 121 patients without the mutation versus 17 thrombotic episodes in 202 mutated patients, p=0.84).

Cumulative risk of thrombosis by baseline leukocytosis

Previous reports have shown that the cumulative risk of thrombosis was significantly higher in low-risk patients with leukocyte number over a median value (8.7x10 9 /L) [START_REF] Carobbio | Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status[END_REF], and also identified as best leukocyte cut-off for predicting thrombotic events the value of 9.4 x10 9 /L [START_REF] Carobbio | Leukocytosis and risk stratification assessment in essential thrombocythemia[END_REF]. We performed an equivalent analysis by using both the median cut-off value of our cohort of patients (8.9x10 9 /L) (Figure 1A) and the cut-off value of 9.4 x10 9 /L (Figure 1B). While the latter cut-off value did not allow to recognize different incidences of thrombosis between risk categories, when the median cut-off value was utilized, the presence of leukocytosis >8.9x10 9 /L identified a category of high risk patients with a thrombotic risk significantly higher than all the other patients (52% vs 17% at 20 years, p<0.0001), while high-risk patients with low WBC count and low-risk patients had the same cumulative thrombotic risk.

We also tested the WBC value of 11x10 9 /L (corresponding to the fourth percentile value).

As shown in Figure 1C, the cumulative risk of thrombosis was significantly higher in highrisk patients with WBC>11x10 9 /L ("very" high risk category) as compared to the group with lower leukocytes count (p=0.002) ("standard" high risk category). The cumulative risk of thrombosis in low-risk patients was comparable to high-risk patients with lower leukocyte count (18% vs 21.5% at 20 years from diagnosis, p=0.24); also, low-risk patients with leukocytosis had the same thrombotic risk than low-risk patients without leukocytosis (18% vs 13% at 20 yrs, p=0.92).

Overall survival

One hundred and twenty-three (23%) patients died at a median age of 81 years (range 51-100); the median survival was 24 years. Causes of death were: thrombotic/haemorrhagic events (8 patients, 6.5%), cardiac disease (13 patients, 10.5%), disease transformation (21 patients, 17%), second neoplasia (12 patients, 10%), senility (26 patients, 14.5%) or other causes unrelated to the haematological disease (not including cardiovascular events and development of second neoplasia) (43 patients, 27%). Thrombocytosis (>1500x10 9 /L) and leukocyte count above the median value (8.9x10 9 /L) resulted associated with higher mortality, but only in univariate analysis (p=0.01 and p=0.04, respectively). Conversely, neither a history of thrombosis nor the presence of the JAK2 V617F mutation were significantly associated with poorer survival (p=0.45 and p=0.57, respectively). Three independent parameters were noted as prognostic factors for survival also in multivariate analysis: age≥60 year, leukocytosis >11x10 9 /L and haemoglobin level below normal values (<12.5 g/dL in females and <13.5 g/dL in males). Taking into account these parameters, a model for survival was constructed in order to define three groups of risk: high (2 or 3 risk factors, 130 patients), medium (1 risk factor, 265 patients) and low (no risk factors, 137 patients). Median survivals were 13 years, 28 years and yet undefined in the three groups, respectively (P<0.0001) (Figure 2).

DISCUSSION

Many recent papers have investigated the possible association between leukocytosis and

thrombosis in patients with myeloproliferative disorders, and particularly in ET, with controversial results [START_REF] Palandri | Long-term follow-up of 386 consecutive patients with essential thrombocythemia: safety of cytoreductive therapy[END_REF][START_REF] Passamonti | Prognostic factors for thrombosis, myelofibrosis, and leukemia in essential thrombocythemia: a study of 605 patients[END_REF][START_REF] Girodon | Leukocytosis is associated with poor survival but not with increased risk of thrombosis in essential thrombocythemia: a populationbased study of 311 patients[END_REF][START_REF] Carobbio | Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status[END_REF][START_REF] Carobbio | Leukocytosis and risk stratification assessment in essential thrombocythemia[END_REF][START_REF] Wolanskyj | Essential thrombocythemia beyond the first decade: life expectancy, long-term complication rates, and prognostic factors[END_REF]. In the current study, we used the database from a cohort of 532 ET patients, homogeneously followed at a single Institution for a median time of 7.6 years, with the purpose to investigate the prognostic relevance of baseline leukocytosis for subsequent thrombotic events and survival. This analysis confirms the importance of the classical high-risk profile (age>60years and/or previous thrombosis), which resulted strongly associated with the thrombotic risk. Conversely, extreme thrombocytosis was not found to correlate with thrombotic complications, and rather showed a paradoxical trend towards a protection from thrombosis (HR 0.67). These findings are consistent with previous retrospective studies [START_REF] Carobbio | Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status[END_REF], suggesting an association between thrombocytosis and bleeding diathesis, partly explained by the occurrence of AVWS in some patients.

In addition to older age and previous thrombosis, also leukocytosis (cut-off, 11x10 9 /L) had a prognostic impact on thrombosis. After patients' stratification in high and low-risk according to the standard definition, baseline leukocytosis identified a cohort of "very" high risk group with leukocytosis (Figure 1c). Indeed, "standard" high-risk patients without leukocytosis presented a thrombotic risk fully comparable to low risk patients carrying leukocytosis. This result is in keeping with a recent analysis by Carobbio et al [START_REF] Carobbio | Leukocytosis and risk stratification assessment in essential thrombocythemia[END_REF], who reported that leukocytosis (median value) per se could discriminate, within the low-risk category, two subgroups of patients at different risk of future vascular complications. On the other hand, in our analysis the thrombotic risk was not significantly different among the two subgroups of low-risk patients (p=0.92) and the WBC cut-off was higher (4 th percentile). The role of baseline leukocytosis in the low risk category might have been masked by the fact that as many as 70% of these low-risk patients received cytotoxic therapy (plus aspirin), configuring them as high-risk from the point of view of prevention of thrombosis. The administration of cytotoxic therapy might have prevented an increase in leukocyte count over time, which has been reported to correlate with a higher thrombotic risk [START_REF] Passamonti | Increase in leukocyte count over time predicts thrombosis in patients with low-risk essential thrombocythemia[END_REF]. Moreover, the impact of leukocytosis might have been confounded by several other variables, including the co-existence or the appearance over the time of other morbidities, influencing both the thrombotic risk and long-term survival.

Our analysis also confirmed a direct correlation between survival and baseline leukocytosis and haemoglobin level below normal values, as previously reported [START_REF] Palandri | Long-term follow-up of 386 consecutive patients with essential thrombocythemia: safety of cytoreductive therapy[END_REF][START_REF] Girodon | Leukocytosis is associated with poor survival but not with increased risk of thrombosis in essential thrombocythemia: a populationbased study of 311 patients[END_REF][START_REF] Carobbio | Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status[END_REF][START_REF] Gangat | Risk stratification for survival and leukemic transformation in essential thrombocythemia: a single institutional study of 605 patients[END_REF].

Patients were accordingly stratified in three categories, projected to a significantly different survival (Figure 2). To build this model, we verified that the proportion of treated patients was the same in the three groups (P>0.05) and that there were no myelodysplatic or fibrotic features at bone marrow examination, in the 115 patients with low haemoglobin level at diagnosis. In addition, baseline leukocytosis (quartile distribution) did not correlate with disease evolution (p=0.81), which occurred in 28 patients (acute leukemia in 6 cases and myelofibrosis in 22 cases). However, the possibility that some of these patients with "ET" characterized by older age, anemia and leukocytosis may actually have an early stage Primary Myelofibrosis (PMF-0) [START_REF] Tefferi | Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms[END_REF] cannot be entirely ruled out.

In conclusion, with the limitations in the interpretation of the data due to the fact that this is a retrospective study, baseline leukocytosis seemed to be associated with higher thrombotic risk, especially in high-risk category, and worse survival.

Prospective studies are needed to confirm or to rule out the accuracy of these stratifying models and to identify a WBC value which could be a widely reliable cut-off to define a significant leukocytosis. 1A), 9.4 x10 9 /L (Figure 1B), 11x10 9 /L (4th percentile) (Figure 1C). High risk: age older than 60 yr and/or history of vascular events

Figure 2 .

 2 Figure 2. Overall survival curves for ET patients stratified according to the risk including the three risk-factors: age>60yr, leukocytosis >11x10 9 /L and haemoglobin level below normal values. The risk was classified as low (no risk factors, line a, 137 patients), intermediate (1 risk factor, line b, 265 patients), high (2-3 risk factors; line c, 130 patients, with 23 patients having all the 3 risk factors). Median survivals were yet undefined, 28 yrs and 13 yrs in the three groups, respectively (P<0.0001).

Table II . Multivariate analysis of risk factors for thrombosis in the follow-up

 II Cumulative probability of thrombosis in the follow-up according to standard risk factors and categories of WBC count at diagnosis. WBC cut-off values are as follows: 8.9x10 9 /L (median WBC count) (Figure

	Figure 1.
	Factor	HR	95% CI	P
	Sex (male vs female)	0.9	0.59 to 1.70	0.99
	High risk patients	2.45	1.35 to 4.43	0.003
	Haemoglobin, g/dL (<13)	1 (ref)
	13 -14.1	0.79	0.39 to 1.59	0.50
	14.1-15.1	0.81	0.39 to 1.70	0.58
	>15.1	1.17	0.61 to 2.25	0.63
	Hematocrite, % (<39.4%)	1 (ref)
	39.5 -42.8	1.66	0.79 to 3.54	0.18
	42.8 -45.9	1.47	0.65 to 3.18	0.36
	>45.9	1.36	0.62 to 3.03	0.46
	PLT, x 10 9 / L (<653)	1 (ref)
	653 -774	1.17	0.58 to 2.38	0.67
	774 -954	1.11	0.54 to 2.25	0.77
	>954	0.67	0.30 to 1.51	0.34
	WBC, x 10 9 / L (<7.4)	1 (ref)
	7.4 -8.9	0.85	0.40 to 1.77	0.65
	8.9 -11	0.89	0.39 to 1.76	0.77
	>11	1.76	1.05 to 2.97	0.033

The research was supported by the University of Bologna (Funds for selected topics), by the EuropeanLeukemiaNet and by BolognAIL. We thank dr Mauro Fiacchini for his helpful assistance.


Jean-Luc Bertrand-Krajewski
email: jean-luc.bertrand-krajewski@insa-lyon.fr

C Becouze

A Dembélé

Marina Coquery

Cren Olivé

B Barillon

P Dauthuille

J Chapgier

M F Grenier Loustalot

Petrescu Marin

Jean-Luc Bertrand-Krajewski

Prior- C Cren Olivé

J.-L Bertrand-Krajewski

C Cren-Olivé

M.-F Grenier-Loustalot

Priority pollutants in stormwater: the ESPRIT project

Keywords: Combined sewer, monitoring, priority pollutants, priority substances, separate sewer, stormwater

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

INTRODUCTION

The European Water Framework Directive (WFD) n° 2000/60/EC (EC, 2000) requires, in its article 16, a progressive reduction of discharges of priority substances into water bodies and the cessation of discharges of priority hazardous substances. In a global and integrated urban water management approach, all sources should be analysed (diffuse agricultural pollution, urban and industrial pollution, emissions from wastewater treatment plants, from separate and combined sewer systems, etc.) and quantified, in order to define priorities for action.

Considering that only limited information is available regarding most priority substances in stormwater, the ESPRIT collaborative research project aims to contribute to the identification, evaluation and characterisation of priority substances transported by stormwater, in both combined and separate sewer systems. It is part of the research cluster Rhodanos and has links with other French research projects (e.g. AMPERES, DEMA…) dealing with priority substances in i) wastewater treatment plant influent, effluent and sludge and ii) in agricultural Priority pollutants in stormwater: the ESPRIT project discharges. All together, these projects aim to quantify the main sources and pathways of priority substances, to suggest reduction strategies and to contribute to reach the WFD goals. ESPRIT is carried out by a consortium of six partners (INSA-Lyon, Cemagref Lyon, SCA-CNRS, Suez-Environnement, Lyonnaise des Eaux and Grand Lyon). It is funded by various institutions through the Chemistry and Environment Pole of Competitiveness "Axelera" in Lyon, France. ESPRIT started in March 2007 for a period of 3 years.

OBJECTIVES

The main objective of ESPRIT consists to evaluate the concentrations and loads of 41 priority substances transported in stormwater in two experimental sites in Lyon, France. Monitoring campaigns will be carried out at the outlet of both catchments for a large number of storm events, and also for some dry weather periods. Most campaigns will focus on event mean concentrations (EMC), and a limited number of pollutographs will also be measured. In addition, at both sites, specific prototypes have been built to collect both dry atmospheric fallout and rain water. They allow estimating the dry and wet contributions of the atmosphere to stormwater pollutant loads. In a first phase, screenings of the 41 substances will be performed. In a second phase, more accurate specific analyses for some substances will be carried out if necessary and/or appropriate. A special effort will be devoted to evaluate the event variability of concentrations and loads. Models will be developed to extrapolate results to annual and multi-annual time scales and, if possible, to un-monitored catchments with similar characteristics.

SELECTION OF SUBSTANCES MEASURED IN STORMWATER

According to the provisions of article 16 of the WFD, a list of 33 priority substances, which represent a significant risk to or via the aquatic environment at the EU level, has been established by the European Decision n° 2001/2455/EC (EC, 2001). The list of priority substances regulated at European level also includes 8 additional dangerous substances, which are part of the List I in the European Directive on Dangerous Substances [START_REF] Eec | Directive 76/464/EEC on pollution caused by certain dangerous substances discharged into the aquatic environment of the Community[END_REF]. The resulting main list of 41 substances to be monitored in the ESPRIT project is given in Table 1. An international literature review was carried out to identify other possible substances of interest to be monitored in stormwater; it includes for instance several metals listed in the dangerous substances directive (Becouze and Dembélé, 2007a).

EXPERIMENTAL SITES

Monitoring campaigns are carried out at the outlet of two experimental catchments in Lyon, France: Ecully and Chassieu, which are part of the OTHU (Field Observatory on Urban Hydrology). Ecully (Figure 1, left) is a 245 ha residential area in the Western hilly suburbs of Lyon, equipped with a combined sewer system. The mean slope of the catchment is 2 %, with a coefficient of imperviousness of 42 %. Chassieu (Figure 1, right) is a 185 ha industrial area in the Eastern flat suburbs of Lyon, equipped with a stormwater separate sewer system. The mean slope of the catchment is 0.4 %, with a coefficient of imperviousness of 75 %. and the 1 L bottles are used for pollutographs. All sampler components in contact with stormwater are made of or covered with Teflon to avoid any interactions between sampling devices and pollutants to be measured. The sampler is controlled by means of a Sofrel S50 data logger and control system, in order to collect flow proportional samples. The discharge is calculated from 2 minutes time step simultaneous measurements of water level and flow velocity in the outlet sewer pipe (Nivus ultrasonic Nivumaster P06 probe for water level and Nivus Doppler OCM Pro probe for flow velocity). Two seasons (winter and summer) are distinguished for the flow proportional sampling to account for the significantly different storm event characteristics (duration, intensities, antecedent dry periods) observed in past rainfall data sets.

In addition to the automatic sampler, other physico-chemical parameters and pollutant concentrations and loads are measured or estimated by means of 2 minutes time step continuous monitoring of pH, electrical conductivity, turbidity, temperature and UV-visible spectrometry. All sensors (except flow meters) are located in a shelter where a transit flume is continuously supplied with effluent from the outlet sewer by means of a 1 L.s -1 and 1 m.s -1 peristaltic pump (Figure 2). They are initially and then periodically calibrated to ensure data quality and reliability. In order to estimate both the dry and wet atmospheric contributions to loads of priority substances, specific prototypes for atmospheric sampling have been developed and installed in each catchment. Each prototype is composed of two parallel compartments built with plastic boxes (800×600×220 mm) entirely covered with Teflon (Figure 3). Each compartment is equipped with its own electric shutter. The dry compartment is open during dry weather to collect dry fallout whereas the other one is closed by the shutter. During storm events, the dry compartment is closed and the storm compartment is opened to collect rain water. Shutters opening and closing are controlled according to rainfall intensities measured in situ and online by means of OTT Pluvio weighing rain gauges. A specific algorithm has been developed and validated based on past storm data in order to ensure an appropriate control of the shutters [START_REF] Dembélé | Sampling of dry and humid atmospheric fallout: description of the sampling device and of its control[END_REF]. Dry fallout is collected monthly and rain water is collected after each storm event: the pollutants to be measured depend on the available mass (for dust) or volume (for rainwater) of collected material. Analyses of organics and metals in both dry fallout and rain water are made with the same protocols as those used for stormwater samples as described in the following section.

SAMPLING PROTOCOLS AND ANALYTICAL METHODS

Detailed protocols for sampling and samples handling, preservation and conditioning have been established and validated [START_REF] Becouze | Sampling and analytical protocols for the measurement of priority substances in stormwater[END_REF]. Immediately after sampling, samples are conditioned and split in four subsamples:

-one for the analyses of organic compounds; -one for the analyses of metals; -one for the analyses of total suspended solids (TSS) and chemical oxygen demand (COD), which give an additional characterisation of the samples; -one for the analyses of other compounds usually measured in stormwater: total organic carbon (TOC), total Kjeldahl nitrogen (TKN), ammonia, total phosphorous, orthophosphates.

Analyses of organic compounds

A multi-residue method by gas and liquid chromatography coupled to mass spectrometry has been developed and validated for the analysis of 36 priority organic substances listed in the WFD in various water matrices [START_REF] Barrek | Multi-residue analysis and ultra-trace quantification of 36 priority substances from the European Water Framework Directive by gas chromatography mass spectrometry and liquid chromatography coupled to mass spectrometry and fluorescence detector in surface and ground waters[END_REF]. More precisely, the method was developed in three steps, as illustrated in Figure 4. First, automated off-line solid-phase extraction was optimized to trap simultaneously the 36 studied compounds in the filtered water samples. Second, the 20 more volatile compounds were analysed by gas chromatography coupled to mass spectrometry with electron impact ionisation in selected ion monitoring mode (SIM). Third, the last 20 compounds were detected and quantified, in one run, by liquid chromatography coupled to fluorescence detector and mass spectrometry. The compounds measured by GS-MS and LC-FLD-M/MS are marked with * and ** respectively in Table 1.

Recoveries obtained were generally higher than 56 % for all tested waters. Extraction recoveries of substances analysed by GC-MS varied between 59.1 and 100 % with relative Priority pollutants in stormwater: the ESPRIT project standard deviations (RSD) below 4.5 %. Recoveries of substances analysed by LC-MS varied from 56 % for octylphenol to 100 % for pentachlorophenol. RSD varied from 2.1 % for chlorfenvinphos to 16.8 % for fluoranthene. Limits of detection (LOD) varied between 4 ng/L (pentachlorophenol) to 125 ng/L (octylphenol) for LC-FLD-MS/MS analysis. The limits of quantification (LOQ) for GC-MS analyses were between 6 and 41 ng/L for hexachlorobutadiene and endosulfan, respectively. During the ESPRIT project, the protocols will be adapted and validated for the analysis of organic substances in the suspended particulate fraction of stormwater samples.

Analyses of metals

Thanks to the techniques used, more metals than the four ones listed in Table 1 (Cd, Pd, Ni and Hg) are systematically analysed. All laboratory ware including filters, filtration units and polyethylene bottles are previously washed with 10 % (v/v) HNO 3 and rinsed with ultrapure water (Milli-Q, Millipore). An additional HCl 10 % (v/v) cleaning step is included for all equipment used for Hg analyses.

Water samples are filtered in the laboratory under a clean bench on acid-cleaned and preweighed filters (0.45 µm pore size; PVDF for metals or Teflon for Hg). The filtrate is immediately acidified with ultrapure acids (Suprapur, Merck): 0.2 % v/v HNO 3 for metals and 0.5 % v/v HCl for Hg analyses. Then they are stored at 4°C in darkness until analyses. Filters are put in plastic petri dish, dried for 24 h at 50°C, transferred in a dessiccator and reweighed. They are stored in plastic bags before analyses. Similar manipulations are performed with ultrapure water (Milli-Q, Millipore) to control the cleanness of the sampling and filtration procedures.

Metals are measured in both dissolved and particulate fractions. Filters are first mineralized using aqua regia (HNO 3 :HCl, 3:1) in a microwave oven. Samples are analyzed by inductively coupled plasma mass spectrometry (ICP-MS, Thermo X7 series II) according to ISO-CEN standard methods (ISO, 2003). The LOQs for the dissolved phase vary between 0.01 µg/L (for Cd) and 5 µg/L (for Al and Ba) (see Figure 7). Typical precisions (evaluated as expanded uncertainties) vary between 8 and 20 % depending on the element.

Total dissolved mercury analyses in water samples are performed according to the US Environmental Protection Agency standard method (EPA, 2002). Samples are first oxidized with bromine monochloride (BrCl), then hydroxylamine hydrochloride (NH 2 OH, HCl) is added to neutralise the remaining BrCl and Hg II+ is reduced to Hg 0 using a stannous chloride (SnCl 2 ) solution. Mercury is detected after preconcentration on a gold trap using an automated atomic fluorescence spectrophotometer (AFS, Mercur Analytikjena). The LOQ of the method is 0.5 ng/L. Precision, evaluated by replicate analyses, was between 5 and 10 %. The analytical method for Hg in particulate samples is under validation. Particles collected on filters are first mineralized using a mixture of (HNO 3 :HCl, 9:1) for 3 h at 90°C, then Hg is measured using the same analytical procedure as described above.

Analyses of TSS and COD

TSS concentration is measured according to the standard method EN 12880 (2000). COD concentration is measured with the small tube test (STT) standard method (ISO 15705, 2002) with Hach -Dr Lange tubes LCI 500 [0-150 mg/L] and LCI 400 [0-1000 mg/L], after homogeneisation with an Ultra-Turrax ® mixer.

Analyses of other compounds

All other compounds are measured in an external subcontracting certified laboratory according to French and international standard methods.

MONITORING CAMPAIGNS

The planned monitoring campaigns include initial pollutant screenings for both sites, field blank samples for sampling devices, storm event mean concentrations (EMC), dry weather daily mean concentrations (DMC) and a few pollutographs. Samples for EMC measurements are obtained by direct mixing of individual flow proportional sub-samples pumped in the 25 L vessel. Samples for DMC measurements are obtained by off-line flow proportional mixing of hourly sub-samples taken in 1 L bottles.

After cleanup of all sampling devices according to a suitable validated protocol [START_REF] Becouze | Sampling and analytical protocols for the measurement of priority substances in stormwater[END_REF], field blank samples are obtained by passing off benchmark water samples (BWS) into all sampling devices. BWS must be typical i) of urban wet weather effluent or of dry weather effluent for the automatic sampler and ii) of rainwater for the atmospheric sampler. In the later case, Evian water, which has a pH similar to that of rainwater, is used as BWS. The campaign aims to evaluate possible adsorption of or contamination by priority substances during sampling. Results obtained upstream and downstream sampling devices are compared to detect possible differences. In case significant differences are detected, they will have to be identified for correction in the following campaigns. Preliminary screening analyses have been carried out in 2007, to check if expected substances are effectively present in wet weather effluents in both catchments. Figure 7 shows the results obtained for dissolved metals for one EMC in Chassieu and one DMC in Ecully.

Most metals are present at both sites with concentrations higher than LOQs. However, some metals were absent or present with concentrations lower than their LOQ: selenium (Se) and titanium (Ti) in both sites, silver (Ag) in Ecully and tin (Sn) in Chassieu. These are only preliminary results that should not be used to draw general conclusions.

CONCLUSION

The ESPRIT project started in March 2007. All sampling equipments are installed since February 2008 on two sites in Ecully (combined sewer system) and Chassieu (separate sewer system) in Lyon, France. Sampling and analytical protocols for a list of 41 priority substances have been validated. Effective monitoring campaigns have started in 2008: their results will be shown and discussed on the poster to be presented during the 11 th International Conference on Urban Drainage.

Figure 1 .

 1 Figure 1. Aerial views and borders of the Ecully (left) and Chassieu (right) catchments.

Figure 2 .

 2 Figure 2. Left: scheme of the monitoring shelter installed on each inlet; Right: photo of the peristaltic pump and of the transit flume inside the shelter (photo JLBK).

Figure 3 .

 3 Figure 3. Two compartments sampler for atmospheric dry fallout and rain water in Chassieu, with the rain gauge at the forefront (photo AD).

Figure 4 .

 4 Figure 4. Flow chart of the analytical strategy developed for the sample extraction process and analysis.

Figure 5 .Figure 6 .

 56 Figure 5. Flow chart of the analytical protocol for metals analysis in dissolved and particulate fractions (except mercury).

Figure 7 .

 7 Figure 7. Results of preliminary screening of dissolved metals for both sites. LOQ = limit of quantification. Arrows above some bars indicate concentration lower than LOQ.

Table 1 .

 1 List of the 41 priority substances measured in stormwater.

th International Conference on Urban Drainage, Edinburgh, Scotland, UK, 2008

ACKNOWLEDGEMENTS

This work is carried out in the research action ESPRIT of the RHODANOS project within the pole of competitiveness AXELERA "Chemistry and Environment". The partners are: INSA-Lyon (coord.), Cemagref-Lyon, SCA-CNRS, Suez-Environnement CIRSEE, Lyonnaise des Eaux SDEI, Grand Lyon. The action is funded by the Rhône-Alpes Regional Council, the Grand Lyon, the FCE -Entreprises Competitiveness Funds, Suez-Environnement CIRSEE and ANRT. It also benefits from the OTHU (Field Observatory on Urban Hydrology) organisation and maintenance (www.othu.org). We thank L. Dherret of Cemagref-Lyon for metal analyses.

More information

ESPRIT website at http://esprit-rhodanos.fr