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International audience; Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATAI ratio and decreased alpha/beta-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STATS and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

Wilson disease is a rare inherited disorder of copper metabolism that affects liver and brain due to copper tissue accumulation. The mechanism involved is based on mutations of the ATP7B gene. Children have predominant hepatic manifestations while adult are more often diagnosed by neurological and psychiatric symptoms. However, others features are tubulopathy, articular disorders and hemolytic anemia. We report the diagnostic of Wilson disease in a 14 years old girl and her sibling after investigation of hemolytic anemia, hepatic insufficiency, and hypophosphatemia.

We report two members of a French family who are carriers of a rare hemoglobin (Hb) variant leading to erythrocytosis: Hb Saint Nazaire [β103(G5)Phe→Ile

International audience; We describe the clinical, hematologic and genetic characteristics of a retrospective series of 126 subjects from 64 families with hereditary xerocytosis. Twelve patients from six families carried a KCNN4 mutation, five had the recurrent p.Arg352His mutation and one had a new deletion at the exon 7-intron 7 junction. Forty-nine families carried a PIEZO1 mutation, which was a known recurrent mutation in only one-third of the cases and private sequence variation in others; 12 new probably pathogenic missense mutations were identified. The two dominant features leading to diagnosis were hemolysis that persisted after splenectomy and hyperferritinemia, with an inconstant correlation with liver iron content assessed by magnetic resonance imaging. PIEZO1-hereditary xerocytosis was characterized by compensated hemolysis in most cases, perinatal edema of heterogeneous severity in more than 20% of families and a major risk of post-splenectomy thrombotic events, including a high frequency of portal thrombosis. In KCNN4-related disease, the main symptoms were more severe anemia, hemolysis and iron overload, with no clear sign of red cell dehydration; therefore, this disorder would be better described as a ‘Gardos channelopathy’. These data on the largest series to date indicate that PIEZO1-hereditary xerocytosis and Gardos channelopathy are not the same disease although they share hemolysis, a high rate of iron overload and inefficient splenectomy. They demonstrate the high variability in clinical expression as well as genetic bases of PIEZO1-hereditary xerocytosis. These results will help to improve the diagnosis of hereditary xerocytosis and to provide recommendations on the clinical management in terms of splenectomy, iron overload and pregnancy follow-up.

INTRODUCTION Development of additional parameters for complete blood count has emerged in recent hematology analyzers, leading to many publications. However, few studies have been conducted on advanced RBC parameters and hemolytic anemias. We investigated the interest of Sysmex unique parameters, MicroR and HypoHe, as well as the immature fraction of reticulocytes (IRF) in combination with complete blood and reticulocyte count, for screening hereditary spherocytosis (HS) and pyruvate kinase deficiency. METHODS We analyzed 182 samples using Sysmex XE-5000 analyzers from a cohort of red cell disorder patients from the Rouen University Hospital. These included 47 HS, 17 pyruvate kinase deficiencies, sickle cell diseases and trait, β-thalassemia minor, iron deficiencies, and 489 samples from a routine group. RESULTS Combining five parameters (hemoglobin level, reticulocyte count, IRF, MicroR, and %HypoHe), we developed a specific screening tool for HS allowing a sensitivity of 100% and a specificity of 92.1% and a specific screening tool for pyruvate kinase deficiencies allowing a sensitivity of 100% and a specificity of 96.5%. These parameters were also found accurate in infants and in HS without anemia. CONCLUSION We propose a costless, easy-to-use, and efficient approach to detect HS and pyruvate kinase deficiencies using Sysmex analyzers. These screening tools may help diagnosis of these disorders, help prevent complications, and result in a better management of these patients.

We report two members of a French family who are carriers of a rare hemoglobin (Hb) variant leading to erythrocytosis: Hb Saint Nazaire [β103(G5)Phe→Ile; HBB: c.310T>A]. The proband is a 38-year-old woman referred to our institution for a moderate but persistent polycythemia without any clinical consequence. As her mother had a similar blood count, a diagnosis of a Hb variant with high oxygen affinity was proposed. The variant was difficult to detect by capillary electrophoresis (CE) and not distinguishable by high performance liquid chromatography (HPLC) and isoelectric focusing. Finally, a heterozygous mutation on the HBB gene corresponding to Hb Saint Nazaire was identified. This case report illustrates that this rare cause of erythrocytosis can be easily under or misdiagnosed unless several Hb separation techniques are used.

Resume Introduction Le recours a une transfusion de culots de globules rouges chez un patient drepanocytaire majeur est parfois indispensable. L’hemolyse post-transfusionnelle retardee est une complication rare mais potentiellement grave. Cette manifestation peut survenir a tout âge. Elle est probablement sous-diagnostiquee du fait du peu de signes specifiques. Observations Nous decrivons dans cet article, les signes cliniques, biologiques et les aleas de la prise en charge therapeutique de trois cas d’hemolyse post-transfusionnelle retardee. La chromatographie haute performance evaluant le pourcentage d’HbA1 est l’examen permettant d’etablir le diagnostic. La physiopathologie de cet evenement est encore mal connue. Plusieurs traitements ont ete utilises, cependant, la prise en charge therapeutique reste discutee a l’heure actuelle. Conclusion La transfusion de tout patient susceptible d’avoir une hemolyse post-transfusionnelle retardee est fortement deconseillee car elle risque d’aggraver l’hemolyse. La prevention d’une recidive est primordiale.

The risk and clinical significance of cardiac iron overload due to chronic transfusion varies with the underlying disease. Cardiac iron overload shortens the life expectancy of patients with thalassemia, whereas its effect is unclear in those with myelodysplastic syndromes (MDS). In patients with sickle cell anemia (SCA), iron does not seem to deposit quickly in the heart. Our primary objective was to assess through a multicentric study the prevalence of cardiac iron overload, defined as a cardiovascular magnetic resonance T2*8 ECs in the past year, and age older than 6 years. We included from 9 centers 20 patients with thalassemia, 41 with SCA, and 25 with MDS in 2012-2014. Erythrocytapharesis did not consistently prevent iron overload in patients with SCA. Cardiac iron overload was found in 3 (15%) patients with thalassemia, none with SCA, and 4 (16%) with MDS. The liver iron content (LIC) ranged from 10.4 to 15.2 mg/g dry weight, with no significant differences across groups (P = 0.29). Abnormal T2* was not significantly associated with any of the measures of transfusion or chelation. Ferritin levels showed a strong association with LIC. Non-transferrin-bound iron was high in the thalassemia and MDS groups but low in the SCA group (P

Although dehydroepiandrosterone (DHEA) is widely used in the elderly to prevent some adverse effects of ageing, possible deleterious side effects have not been fully assessed. We evaluated the direct actions of DHEA and DHEA sulphate on angiogenesis, a critical event in pathologies that are common in the elderly (cancer, atherosclerosis, inflammation… etc.). At physiological concentrations found in human plasma following DHEA therapy (1–50 nM), DHEA had no action on angiogenesis in vitro. In contrast, higher concentrations of DHEA (10–100 μM), which can be found in tissues after local administration or storage, inhibited in vitro endothelial cell proliferation (blockage in G2/M), migration and capillary tube formation and in vivo angiogenesis in the Matrigel plug assay. This inhibition might be due to a decreased glucose-6-phosphate dehydrogenase activity and to a modification of the tubulin network involved in cell proliferation and migration. The sulphate ester form of DHEA had no effect on angiogenesis.

Dear Editor, Reports on the analytical evaluation of capillary electrophoresis (CE) for glycated hemoglobin (HbA1c) measurement have already been published [1]. There have been several recent reports concerning the impact of the presence of hemoglobin variants on HbA1c measurement with the CE method. One study focused on the analytical interference of some hemoglobin variants [2], demonstrating that hemoglobin variants S, C, D, and E do not interfere either with HbA1c measurement or the glycation rate of hemoglobin variants, which is equal to the glycation rate of HbA. In a second report, analytical evaluation of CE showed that hemoglobin variant E and β-thalassemia do not affect HbA1c results. Moreover, most hemoglobin variants common to China can be detected by using a CE analyzer, and HbA1c values do not seem to be affected [3]. We report a case of hemoglobin Camperdown (HbCa) detected by using a Capillarys 2 Flex Piercing instrument (C2FP, Sebia, Lisses, France) during HbA1c measurement. A 76-yr-old man receiving follow-up care for type 2 insulin-dependent diabetes was admitted to our hospital for a necrotic wound on the heel. To manage the diabetes, HbA1c was measured on a C2FP instrument in our clinical biology laboratory at Rouen University Hospital, France. The CE software flagged an alarm stating "Atypical profile" with an HbA1c value of 4.2% (22 mmol/mol). We observed an abnormality on the electropherogram (Fig. 1), with a peak of unknown hemoglobin quantified at 3.0% and named fraction 1 by the CE software. There was no other abnormality on the electropherogram. The sample was also tested with a Bio-Rad Variant II analyzer (Bio-Rad, Marnes-la-Coquette, France) in the clinical biology laboratory at Le Havre General Hospital, France, which measured the HbA1c value at 52.2% (547 mmol/mol) because of migration of a probable hemoglobin variant in the HbA1c zone. Concentration of fructosamine (261 µmol/L; 205-260 µmol/L) was just above the reference values, which was not consistent with the two HbA1c values obtained. The patient's last HbA1c measurement was performed in our laboratory 10 yr previously. At that time, we observed a peak of unknown hemoglobin on HPLC (Variant, Bio-Rad), and we were not able to quantify HbA1c. The unknown hemoglobin was identified in the National Hemoglobinopathy Reference Laboratory (Creteil, France) as HbCa [β104Arg→Ser]. Ten years ago, the percentage of HbCa was estimated at 51.5%, which was consistent with a heterozygote patient. We supposed that the unknown fraction 1 on the electropherogram was the glycated fraction of HbCa. We observed that both glycated fractions were clearly separate, unlike HbCa and HbA0, which exactly coeluate, explaining the abnormal value of HbA1c calculated by the CE software. A manual off-line recalculation of the global glycated fractions percentage was performed, by integrating the sum of both peaks instead of HbA1c with the formula for percentage of glycated fractions=(HbA1c+HbCa1c)/(Total HbA0 peak)×100. We then applied the calibration equation of the software, thereby obtaining a value of 6.3% (45 mmol/mol) for global glycated fractions. Fig. 1 Abnormal electropherogram obtained by Capillarys 2 Flex Piercing instrument. Abnormal electropherogram with software alarm "atypical profile" showing a peak of unknown hemoglobin (HbCa1c), which is the glycated form of hemoglobin Camperdown (HbCa). HbCa ... HbCa is a rare variant, first described in 1974 [4], which is not associated with hematological abnormalities. Hemoglobin variants can affect the accuracy of HbA1c measurement, resulting in interference. This interference depends on the type of hemoglobin variant and the method used. Recently, Gaborit et al. [5] published the effects of this variant on HbA1c measurement using HPLC and immunoassay methods. They concluded that it was not possible to measure the exact HbA1c value with the affinity chromatography method. Moreover, immunoassay methods are blind to detecting such variants and probably underestimate HbA1c values. Herein we describe for the first time a case of HbCa interfering with CE HbA1c measurement. The C2FP HbA1c method is not able to differentiate HbA0 from HbCa. The presence of an unknown peak must alert the pathologist. The CE software value is affected by negative bias due to the co-elution of HbCa and HbA0. Manual recalculation seems to yield good results for global glycated fractions, consistent with fructosamine. However, the threshold for HbA1c values must be applied with great care. In fact, the glycated rate of this rare variant has not yet been validated by any publication in the literature. Fructosamine, glycated albumin, or daily capillary blood glucose remain good markers to manage such patients.

Background. Cardiac iron overload is the major cause for death in regularly transfused thalassemic (Thal) patients. Its impact in myelodysplastic syndrome (MDS) patients is debated. Heart seems to be spared in regularly transfused patients with sickle cell anemia (SCA), which is supposed to be related to a later onset on transfusions and to the use of erythrocytapheresis rather than simple transfusion. Our aim was to assess the prevalence of cardiac iron overload, defined as a T2*cardiovascular magnetic resonance (MR) Patients and methods. Patients were enrolled if they were regularly followed in a center where the exact number of erythrocyte concentrates (EC) could be obtained, had received in the previous year more than 8 EC, were older than 6 years (limit for MRI without sedation), had no known heart disease related to another pathology, and had given informed consent. All patients underwent 1.5T myocardial T2*MR imaging after validation of the procedures in the different sites. Assessment of Liver Iron Content (LIC) used two signal intensity ratio of gradient echo imaging (R2*) MRI protocols. Serum Non-Transferrin Bound Iron (NTBI) was measured by the FeROSTM eLPI kit, and serum hepcidin by LC-MSMS. Results. 20 Thal, 41 SCA and 25 MDS patients were evaluable. We divided SCA in 2 groups, according to the procedure recorded at the time of the study, manual exchange transfusion (G1, N = 30 patients), or erythrocytapheresis (G2, N=11 patients). We found cardiac overload in 0, 3 (15%), and 4 (16%) of SCA, Thal, and MDS patients respectively. Serum ferritins at beginning of chelation were not statistically different in all categories of patients, as well as Ferritin and LIC at the time of the study. Increased LIC and abnormal T2* were associated in Thal and MDS patients (p=0.04), with no correlation between abnormal T2* and parameters of transfusion and chelation. Plasmatic iron level was increased in Thal and MDS patients but remained at normal range in SCA patients. NTBI level was high in Thal and MDS but completely absent in SCA groups. The major discrepancy was in the values of hepcidin, which were collapsed in Thal, at normal range in SCA, and highly elevated in MDS patients. Discussion and conclusion. We confirm that SCA patients are relatively protected from cardiac iron overload. This results probably from massive consummation of iron through effective erythropoiesis, making toxic free iron (NTBI) less available in the circulation. In addition, since iron overload in SCA results from a massive outflow of hemoglobin (Hb) due to intravascular hemolysis and transfusion, the heme/Hb-bound iron must be efficiently handled in liver macrophages, limiting its release in the bloodstream. In Thal patients, underlying defects in erythropoietic processes, together with low hepcidin that stimulates intestinal iron absorption and increases NTBI, must provoke more organ damages. Hepcidin levels were high in MDS patients, suggesting that transfusion-dependent iron overload was a more effective regulator of hepcidin production than dyserythropoiesis. The % of T2* Finally, we observe that, in opposition with previous reports, SCA patients undergoing eythrocytapheresis may experience severe iron overload and need iron chelation. Table 1. Thal SCA G1 SCA G2 MDS p Age at beginning of transfusion (yrs) 8.5[0-45] 7[0-45] 16.5[1-55] 66[38-83] Disclosures De Montalembert: Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau. Guerci-Bresler:ARIAD: Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau; PFIZER: Speakers Bureau.

Whether NO is involved or not in sustained conduit artery flow-mediated dilatation in humans remains unclear. Moreover, the role of endothelium-derived hyperpolarizing factor (EDHF), synthesized by cytochrome epoxygenases and acting through calcium-activated potassium channels, and its relationship with NO during flow-mediated dilatation have never been investigated previously. In 12 healthy subjects we measured radial artery diameter (echotracking) and blood flow (Doppler) during flow-mediated dilatation induced by gradual distal hand skin heating (34 to 44°C), during the local infusion of saline and inhibitors of NO synthase ( N G -monomethyl- l -arginine [ l -NMMA]: 8 to 20 μmol/min per liter), calcium-activated potassium channels (tetraethylammonium chloride: 9 μmol/min per liter), and cytochrome epoxygenases (fluconazole: 0.4 to 1.6 μmol/min per liter), alone and in combination. Mean wall shear stress, the flow-mediated dilatation stimulus, was calculated at each level of flow, and the diameter–wall shear stress relationship was constructed. During heating, compared with saline, the diameter–shear stress relationship was shifted downward by l -NMMA, tetraethylammonium, fluconazole, and, in a more pronounced manner, by the combinations of l -NMMA with tetraethylammonium or with fluconazole. Therefore, maximal radial artery flow-mediated dilatation, compared with saline (0.62±0.03 mm), was decreased under our experimental conditions by l -NMMA (−39±4%), tetraethylammonium chloride (−14±4%), fluconazole (−18±6%), and to a greater extent, by the combinations of l -NMMA with tetraethylammonium (−64±4%) or with fluconazole (−71±3%). This study demonstrates that NO and a cytochrome-related EDHF are involved in peripheral conduit artery flow-mediated dilatation in humans during sustained flow conditions. Moreover, the synergistic effects of the inhibitors strongly suggest a functional interaction between NO and EDHF pathways.

To assess the influence of initial diameter on the gender difference in flow-dependent dilatation (FDD) of the conduit artery, we measured radial artery internal diameter (echotracking), flow (Doppler) and total blood viscosity in 24 healthy (25 ± 0.8 yr) men and women during reactive hyperemia (RH) and during a gradual hand skin heating (SH). At baseline, mean diameter (men, 2.76 ± 0.09 vs. women, 2.32 ± 0.07 mm, P < 0.05), flow (men, 21 ± 4 vs. women, 10 ± 1 ml/min, P < 0.05), and blood viscosity (men, 4.13 ± 0.07 vs. women, 3.92 ± 0.13 cP, P < 0.05) were higher in men but mean shear stress (MSS) was not different between groups. During RH, the percent increase in diameter was lower in men (men, 9 ± 1 vs. women, 13 ± 1%, P< 0.05). This difference was suppressed after correction for baseline diameter. During SH, the increase in diameter with flow was higher in women ( P < 0.01). However, the increase in MSS was higher in women because of their smaller diameter at each level of flow ( P < 0.01) and there was no difference between groups for the increase in diameter at each level of MSS. These results demonstrate in a direct manner that initial diameter influences the magnitude of FDD of conduit arteries in humans by modifying the value of the arterial wall shear stress at each level of flow and support the interest of the heating method in presence of heterogeneous groups.

Dear Editor,Hydroxyurea (HU) is a chemotherapeutic agent used inadults and children severely affected with sickle cellanaemia (SCA). Current indications are: recurrent painfulcrises, more than two acute chest syndromes, early chronicorgan damage (respiratory, renal, hepatic, myocardialinsufficiencies), association with an auto-immune diseaseor Hb level below 6 g/dL [4, 5].HU is the only drug that has been proven to modify thedisease at short- or middle-term with an acceptable short-term toxicity [1]. However, few data are available about thepotential risk of carcinogenesis. We report the case of aHodgkin lymphoma that occurred after 4 years of HUtherapy.Our patient was diagnosed at the age of 12 months ascompound heterozygote for HbS and